All ToxKits Algaltoxkit F Artoxkit M Ceriodaphtoxkit F Daphtoxkit F magna Daphtoxkit F pulex Marine Algaltoxkit Ostracodtoxkit F Phytotestkit Phytotoxkit Protoxkit F Rapidtoxkit Rotoxkit F Rotoxkit F acute Rotoxkit F short-chronic Rotoxkit M Spirodela Duckweed Toxkit Streptoxkit F Thamnotoxkit F Toxi-Screening Kit	All Organisms Artemia (salina) franciscana Brachionus calyciflorus Brachionus plicatilis Brachionus rubens Ceriodaphnia dubia Daphnia magna Daphnia pulex Heterocypris incongruens Lepidium sativum Phaeodactylum tricornutum Pseudokirchneriella subcapitata Raphidocelis subcapitata Selenastrum capricornutum Sinapis alba Sorghum saccharatum Spirodela polyrhiza Streptocephalus proboscideus Tetrahymena thermophila Thamnocephalus platyurus	All Topics air algal growth inhibition test antifouling paints ballast waters bathing waters behavioural tests bioremediation biotoxins chemicals - allelochemicals chemicals - biocides chemicals - disinfectants chemicals - drugs chemicals - general chemicals - household products chemicals - hydrocarbons chemicals - inorganic compounds chemicals - metabolites chemicals - metals chemicals - microplastics chemicals - nanoparticles chemicals - organic compounds chemicals - paints chemicals - pesticides chemicals - plant extracts chemicals - plastics chemicals - surfactants coastal water combination experiments composts confounding factors cyanobacterial blooms cyanotoxins data base decision support system drinking water duckweed tests effluents field tests fly ashes groundwaters interlaboratory testing landfill leachates landfill percolates leachates limit values mineral waters nanoparticles oil shale plant tests pulp and paper rainwater rapid tests regulatory testing review ring testing seawater sediment and soil contact tests sediment pore waters sediments shale oil sludges soil leachates soil runoff soils solid phase test solid waste leachates solid wastes stormwaters surface waters test development test selection test sensitivity test validation toxic interactions toxicity classification Toxicity Identification Evaluation (T.I.E.) Toxicity Index (T.I.) toxicity monitoring Toxicological Index (TOX) waste waters wood preservation
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Selected Publications about MicroBioTests : 708 publications

C. Blaise
Canadian Application of Microbiotests to assess the Toxic Potential of Complex Liquid and Solid Media
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 1 3-12
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : sediments

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Abstract : Microbiotesting activities comprise one important approach to address the issue of chemical contamination and its potential impacts on water quality and biota of receiving aquatic ecosystems. Under the second Saint-Lawrence River Action Plan (1993-1998), the Centre Saint-Laurent (Environment Canada, Québec Region, Montréal) continued its development and application of microbioanalytical tools and techniques to assess, control and prevent point source pollution of industrial origin to the freshwater portion of the Saint-Lawrence River ecosystem. In this review, some of the major studies conducted in the area of microbiotesting are highlighted. These include 1) adapting the Microtox® acute toxicity assay in a 96-well microplate format to markedly increase its sample throughput capabilities, 2) employing the SOS Chromotest™ to determine the genotoxic status of major industrial effluents discharging to the Saint-Lawrence River and their potential impact on downstream biota, 3) developing an alternative to whole fish acute (sub)lethal toxicity testing with the help of rainbow trout primary hepatocytes, 4) developing a simple microplate-based Hydra toxicity assay as an effective screen for chemicals and various environmental media, 5) developing a novel algal solid phase assay to predict the toxic potential of freshwater sediments, and 6) conducting a major investigation to develop a cost-effective multitrophic bioanalytical battery to assess the (geno)toxicity of freshwater sediments. The aforementioned studies are but a few examples of the diverse and bustling research endeavours presently ongoing internationally in microbiotesting and many important challenges still lie ahead for this field well into the third millennium.

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C.R. Janssen, M. Vangheluwe and P. Van Sprang
A Brief Review and Critical Evaluation of the Status of Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 3 27-37
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Protoxkit F^TM; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : regulatory testing; test selection; test validation

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Abstract : A review is given on some of the current trends in the field of alternative, small-scale aquatic toxicity tests. Examples of recently developed microbiotests with bacteria, algae and invertebrates are used to illustrate potential applications and short-comings of some of these assays. The major drawback of the majority of new microbiotests is the lack of published data on various aspects of their test development and application possibilities. Consequently many of these tests do not receive the regulatory recognition which they may deserve. It is concluded that for most microbiotests further research and test evaluation is needed before they will gain widespread acceptance as valid alternatives to the currently used conventional test procedures.

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S. Canna-Michaelidou, A.S. Nicolaou, E. Neopfytou and M. Christodoulidou
The Use of a Battery of Microbiotests as a Tool for Integrated Pollution Control : Evaluation and Perspectives in Cyprus
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 4 39-48
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Daphnia magna
Topics : regulatory testing; surface waters; toxicity monitoring; Toxicological Index (TOX)

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Abstract : The application of a battery of microbiotests i.e. Microtox test, Daphtoxkit F™, Algaltoxkit F™ and Mutatox test in parallel and in combination with chemical methods is discussed as a tool for the development of integrated monitoring and water pollution control. In order to get a more holistic and synthetic evaluation of the ecotoxicological quality of the aquatic environment, the results from different toxicity tests are integrated and expressed in one single toxicological index. As a first step the results from the acute toxicity tests are integrated into a sub-index, the PAF (%) i.e. the Potentially Affected Fraction of Species. This index is further integrated with results from the genotoxicity tests and eventually develops into one ecotoxicological index, the TOX. Finally, the TOX and 8 chemical and microbiological indices, form one Yardstick for pollution evaluation and hazard identification. The single toxicological index provides an overall view of the possible impacts on the ecosystem, and can therefore facilitate the integration of ecotoxicological results into the decision making process. The aim of the present work is to show how toxicity testing can be integrated with well-established chemical/microbiological investigations. This integration can provide the basis for a robust, monitoring system for surface waters, by which priorities for action can be clearly identified.

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F. Heinis, J.M. Brils, S.P. Klapwijk and L.R.M. De Poorter
From Microbiotest to Decision Support System : an Assessment Framework for Surface Water Toxicity
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 6 65-72
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM acute
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : decision support system; regulatory testing; surface waters

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Abstract : A decision framework for the site-specific detection of toxic effects of contaminants in surface waters is presented. Although the main objective of the development of this framework aimed at supporting water quality boards in their studies on polluted waters and sediments, it could also be of wider use.
The decision framework is illustrated by means of a case study on a ditch that is contaminated with heavy metals. From this study it appears that the designed framework considerably facilitates the choices to be made in the assessment of surface water toxicity. It also illustrates that biological techniques provide additional information on the biological availability and combined toxicity of all the contaminants present and not only of those which have been analyzed by chemical methods.

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I. Johnson
Criteria-Based Procedure for Selecting Test Methods for Effluent Testing and its Application to Toxkit Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 7 73-94
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - general; effluents; test selection; test validation; toxicity classification; toxicity monitoring

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Abstract : In recent years there has been increasing development of methods (particularly rapid tests) for testing environmental samples. If appropriate methods are to be used for a particular operational role then it is necessary to adopt a robust and objective selection mechanism. This paper describes the application of a criteria-based procedure to the selection of test batteries of methods for use in effluent testing. In the procedure tests are scored against a series of criteria with the exact criteria used depending on the operational role and form of test (for example rapid or conventional). Examples of criteria used include : previous application of a method to an operational role, discrimination between samples, influence of physico-chemical parameters, ease of use, test duration, test costs, test capability (sensitivity and spectrum of response) test precision (repeatability and reproducibility), availability of a standard operating procedure, availability of test organisms, test organism variability, the use of indigenous test organisms, importance of test organisms, the relationship between the data for rapid and conventional tests and the ecological relevance of test endpoints. To ensure each method is assessed in a consistent way test scores are assigned using defined guidelines and for certain criteria methods have to satisfy a threshold requirement otherwise they are not deemed to be suitable for the intended purpose. The use of the procedure has been demonstrated by considering the suitability of a series of Toxkits and, where appropriate, their conventional counterparts for effluent assessment.

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B. Marsalek and R. Rojickova-Padrtova
Selection of a Battery of Microbiotests for Various Purposes - the Czech Experience
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 8 95-101
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Protoxkit F^TM; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : test selection; toxicity monitoring

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Abstract : Microbiotests are a useful tool in acute toxicity investigations for pre-screening and screening of chemicals and wastes; tests based on enzymatic activity may be useful as a signal for chronic toxicity.
The composition of a battery of microbiotests is function of the problem(s) under consideration. A battery of microbioassays should in principle be composed of representatives of the producers (algae, higher plants), consumers (mostly zooplankton or zooedaphon invertebrates), and decomposers (bacteria or fungi). The selection of the test species and the design of the bioassay depends on the origin and the characteristics of the sample. Soil or sediment samples, coloured or turbid samples of surface and wastewaters, or totally transparent samples from deep-well monitoring systems, all require different approaches. Microbiotests for the detection of specific toxicants, such as screening for microphyte toxins is an another example of a situation where careful selection of the testing procedure is necessary, bearing in mind time and cost benefits, as well as ecological reliability. Well-experienced ecotoxicologists should be capable of selecting the right test organisms and design from a broad spectrum of microbiotests, in order to give a correct interpretation of the ecotoxicological results obtained for specific problems. This paper reviews and comments on the microbioassays available to date for toxicity screening of chemicals and wastes, based on their own experience in the Czech Republic.

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J.G. Ruck, M. Martin and M. Mabon
Evaluation of Toxkits as Methods for Monitoring Water Quality in New Zealand
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 9 103-119
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM acute; Rotoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : effluents; interlaboratory testing; landfill leachates; regulatory testing; test selection; waste waters

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Abstract : The Resource Management Act 1991, of New Zealand, requires local authorities and industry to apply for consent to discharge effluent to water or land. The requirement for whole effluent toxicity testing is now being included in these Resource Consents. The Ministry for the Environment (MfE) has encouraged research which evaluates technology used to monitor environmental parameters (e.g. effluent toxicity). Supported by a grant from the Sustainable Management Fund (SMF) of the MfE, Wellington Polytechnic undertook a project to evaluate Toxkits as methods for monitoring water quality in New Zealand. Comparative toxicity tests, on a variety of split effluent samples, were performed with new microbiotests (Toxkits) and with benchmark and native species used routinely by NIWA. Six Toxkit tests with a protozoan, micro-algae, rotifers and crustacean test species, and the IQ Toxicity Test™ (a rapid microbiotest using fluorescing enzyme split substrate) were used in the comparison. In addition a round-robin intercalibration exercise was carried out, using selected Toxkits, and involving representative diagnostic laboratories, throughout New Zealand. A range of criteria were used to evaluate the microbiotests : suitability for an operational role, methodology, test method variability and relevance of test data. Recommendations were made to the MfE about the potential role of Toxkits and the IQ Toxicity Test™ as screening and monitoring tests for use in Resource Consents. The Toxkit and IQ Toxicity Test™ organisms were, in general, not as sensitive as the native species used by NIWA. Daphtoxkit F™ magna, Thamnotoxkit F™, IQ Toxicity Test™ and Algaltoxkit F™ are recommended as being suitable, overall, for screening purposes, although the IQ Toxicity Test™ is somewhat subjective and too variable for more definitive testing. Artoxkit M™, Rotoxkit F™ and Rotoxkit M™, while very cost effective and with very good precision (repeatability), were not as sensitive as the others, and therefore are not recommended for whole effluent toxicity screening. Protoxkit F™ and Artoxkit M™ were used in the round-robin. Artoxkit M™ performed very well with 70-80% success rate and low interlaboratory coefficients of variation (32%). Protoxkit F™, performed badly, for reasons related to the inherent variability of the test itself (since greatly improved), and unfamiliarity of participants with ecotoxicity testing methodology. In spite of this the round-robin was extremely valuable in raising the level of awareness of staff in diagnostic laboratories throughout New Zealand, to principles of ecotoxicity testing.

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I. Aoyama, H. Okamura and L. Rong
Toxicity Testing in Japan and the Use of Toxkit Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 10 123-133
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - metals; chemicals - organic compounds; solid wastes; Toxicity Index (T.I.); toxicity monitoring

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Abstract : Management of toxic chemicals in the aquatic environment is performed in Japan by setting environmental standards by law. Forty-nine chemicals are regulated and measured once a month at specific sites in rivers and lakes. Many of these chemicals are detected at various places in Japan, but in concentrations below the standard values. Dioxins, which are not regulated, have been detected in leachates from a landfill area of industrial wastes and also in human mother milk.
In Japan, bioassays are not used to date as a monitoring tool. However, a manual of ecotoxicological test methods for chemicals is presently under examination by the Ministry of International Trade Industry, the Ministry of Agriculture, Forestry and Fisheries and the Environmental Agency. In most cases, the methods proposed are in accordance with OECD Guidelines for the testing of chemicals; they comprise algal, Daphnia and fish tests. Many toxicity test methods have already been described in Japanese scientific literature, based on various types of test organisms from different phylogenetic groups such as e.g. bacteria, yeasts, protozoans, micro- and macro-algae, crustaceans, molluscs, insects, amphibians, fish and birds.
Taking into account the difficulties associated with the culturing and/or maintenance of live stocks, the culture free Toxkit microbiotest technology appeared appealing to the authors and is now used for environmental monitoring purposes in their laboratory.
A new toxicity testing method using mobility analysis of Daphnias obtained from ephippia has recently been developed and will be reported. The test criterion is the change in mobility of Daphnia neonates, which is measured by computer analysis of videocamera-pictures (0.013 sec) during 5 seconds. The mobility per time unit is calculated subsequently. Toxicity is evaluated by comparing the change in mobility of Daphnias exposed to chemicals, versus that in the controls. The technology allows to detect the increase in mobility by stimulus at low concentration and the decrease at higher concentrations of toxicants. The method has a high sensitivity and allows for toxicity detection in less than 3 hours. Results with the new mobility analysis method with Daphnia neonates will be compared with those obtained with the conventional Daphnia method prescribed by the OECD.

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M.M. Moreira Dos Santos and G. Persoone
The Use of Daphnia magna Neonates hatched from Ephippia for Toxicity Testing
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 13 145-153
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - organic compounds; chemicals - inorganic compounds

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Abstract : The continuous increase in the number of new chemicals as well as the discharges of solid and liquid wastes potentially hazardous for the environment has in the past years triggered the need for simple and inexpensive bioassays for routine testing. Although Daphnia magna is a key test species in aquatic toxicology, toxicity testing with this species implies continuous culturing of live stocks of test organisms, in good health and in sufficient numbers. Besides the technological and financial burden, which this represents, conventional tests also often suffer from a poor precision of the test results. The use of resting stages of bioassay species, from which the test organisms can be hatched on demand, is an attractive alternative to the problems posed by the year-round culturing of live stocks. In this study, the suitability of D. magna neonates hatched from ephippia, for acute tests, was evaluated in comparison to neonates taken from live laboratory stock cultures. The analysis was based on test sensitivity as well as on test precision. Twenty four hour and 48-h EC50’s for one inorganic and 3 organic chemicals for both ex-ephippial and live stock neonates were determined according to the OECD Guidelines for Daphnia acute testing. For all the chemicals tested, neonates hatched from ephippia never differed in sensitivity by more than a factor 2 from those taken from live stock cultures. The precision of the EC50’s obtained with ex-ephippial neonates was found to be highly comparable to that of the values obtained with live stock neonates. Despite the low number of chemicals tested, the results of this study again confirm that an ephippium-based toxicity test is a valuable alternative to the conventional Daphnia acute test.

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W. Pauli and S. Berger
A New Toxkit Microbiotest with the Protozoan Ciliate Tetrahymena
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 16 169-176
Toxkits : Protoxkit F^TM
Test Species : Tetrahymena thermophila
Topics : chemicals - general; chemicals - inorganic compounds; chemicals - metals; chemicals - organic compounds; chemicals - pesticides; sludges

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Abstract : Culture free microbiotests with organisms from different trophic and functional levels, e.g. bacteria, algae, rotifers and crustaceans, are presently available. This paper presents the status quo and general design of a new, ready to use multi-generation microbiotest with the standard ciliate Tetrahymena, a representative from a further ecologically relevant group - the protozoa. The latter constitute a major link between bacteria and metazoa, playing a key role in the natural microbial loop and as consumers in the artificial system of waste water treatment.
Based on a heterogeneous set of chemicals, toxicological results with 2 Tetrahymena species, T. pyriformis and T. thermophila, reveal a highly significant correlation (r=0.928, n=52) and nearly equal susceptibility of species. Comparisons of toxicological literature data from the International Uniform ChemicaL Information Database (IUCLID) indicate a similar toxic response even for distant ciliate relatives. Data from IUCLID also reveal that ciliate toxicity data are supplementary to those of the standard activated sludge respiration inhibition. Ciliate tests could hence serve as a powerful tool for the prediction of possible hazards to sewage treatment processes.

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T.W. Snell
The Distribution of Endpoint Chronic Values for Freshwater Rotifers
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 18 185-190
Toxkits : Rotoxkit F^TM acute
Test Species : Brachionus calyciflorus
Topics : chemicals - metals; chemicals - organic compounds

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Abstract : Several types of toxicity tests have been described using the freshwater rotifer Brachionus calyciflorus. Variation among different endpoints has not been described and compared to variation in toxicant sensitivity among rotifer species. Data is presented demonstrating that endpoint chronic values for pentachlorophenol are log-normally distributed with a mean of 251 µg/l PCP and a coefficient of variation of 12%. This compares to the distribution of rotifer species sensitivities, which is also log-normally distributed with a mean of 29 µg/l PCP and a coefficient of variation of 53%. Since the breadth of rotifer species sensitivity distribution is much greater, which endpoint that is measured is less important than making toxicity measurements on several species. This suggests that toxicity should be assessed using the simplest, fastest, and most inexpensive tests available. Toxkits are well designed to meet these needs and should play an expanding role in toxicity assessments in the future.

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I. Blinova
Comparison of the Sensitivity of Aquatic Test Species for Toxicity Evaluation of Various Environmental Samples
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 22 217-220
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : effluents; surface waters

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Abstract : A sensitivity comparison has been made of test species belonging to different trophic levels of the aquatic food chain, exposed to water samples from polluted rivers and waste waters from 2 industries and a municipal water treatment plant. The samples, collected in North Estonia in 1997-1998, differed substantially in their content of (biodegradable) organic matter, sulphate, total nitrogen and phosphorus.
The test battery was composed of 4 Toxkit microbiotests (Algaltoxkit F™, Protoxkit F™, Daphtoxkit F™ and Thamnotoxkit F™) and one bioassay with a higher plant.
The percentage effect was determined on non-diluted, 50 % diluted and 75% diluted samples.
The results revealed that the 5 test species reacted quite differently, qualitatively as well as quantitatively, to the non-diluted and diluted samples to which they were exposed.
The growth of the green algae (Selenastrum capricornutum) was stimulated in all samples, (non-diluted and diluted); duckweed (Lemna minor) growth in turn was inhibited in most non diluted samples, but stimulated in several cases in diluted samples. The former primary producer is thus a good indicator of the eutrophication potential of wastes with high (biodegrable) organic load, whereas the latter reacts better to the toxicants present in such wastes.
The growth of the ciliate protozoan (Tetrahymena thermophila) was neither inhibited nor stimulated in any of the non-diluted or diluted samples.
Daphnids (Daphnia magna) in turn were inhibited (30 to 40%) in the non-diluted waste waters of the fishing industry and the treatment plant, but not in the other samples.
The Thamnotoxkit™ with the crustacean Thamnocephalus platyurus was the assay which reeacted most to the toxicants present in the samples, with up to 100% mortality in the 2 industrial waste waters.
This short study confirms the need for a mixed test battery for an ecologically realistic hazard evaluation of environmental samples, and the usefulness of practical and low cost microbiotests.

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B. Clement
The Use of Microbiotests for Assessing the Influence of the Dilution Medium Quality on the Acute Toxicity of Chemicals and Effluents
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 23 221-228
Toxkits : Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : chemicals - inorganic compounds; chemicals - metals; chemicals - organic compounds; effluents; landfill leachates; mineral waters; surface waters

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Abstract : The influence of the dilution medium quality on the ecotoxicological response of test organisms using microbiotests was studied. The toxicity of reference toxicants (potassium dichromate, zinc sulfate, phenol) and of a landfill leachate and a process water discharged from a metal finishing plant was assessed in various dilution media including standard test media, 3 mineral waters and 28 river waters. The tests used were the Rotoxkit™ with the freshwater rotifer Brachionus calyciflorus, the Thamnotoxkit™ with the crustacean Thamnocephalus platyurus, and the Lumistox® with Vibrio fischeri. The river and mineral waters generally induced a lower toxicity of reference toxicants compared to the standard media. Hardness associated with Mg and the Ca/Mg ratio might explain in most cases the attenuation of toxicity by natural dilution media (i.e. river waters). With the landfill leachate and the process water, the effects of dilution waters on the acute toxicity were significant but not consistent, i.e. increase or decrease of toxicity was observed for the same sample, depending on the type of dilution water used.

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S. Dyatlov
Comparison of Ukrainian Standard Methods and New Microbiotests for Water Toxicity Assessment
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 24 229-232
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : regulatory testing; surface waters; drinking water; waste waters

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Abstract : The incorporation of new microbiotests in Ukrainian environmental legislation for toxicity assessment of natural and waste waters is of great practical interest. In order to determine the usefulness of some of these new microbiotests, comparative experiments have been carried out on the sensitivity of 2 Toxkit tests with the crustaceans Thamnocephalus platyurus and Daphnia magna, versus that of the crustacean Ceriodaphnia affinis and the protozoan ciliate Paramecium caudatum which are the test species indicated in the Ukrainian Standard Methods for toxicity testing. The investigations were made on natural waters of the Dniester river, tap water of Odessa, waste waters of the Odessa Priport chemical plant and the Low Danube town Kiliya treatment plant, and drainage waters from Odessa beaches.
The results obtained indicated that all the samples analysed were acutely toxic to the test organisms. Although C. affinis was in most cases the most sensitive species, its sensitivity in many cases was not substantially different from that of the 2 other crustaceans used, in particular T. platyurus. The ciliate test organism in turn was in all cases much less sensitive than the 3 crustaceans. From this study it was concluded that Toxkits are a valuable and very convenient tool for a rapid toxicity evaluation of natural waters and waste waters in Ukraine. Approval of the Toxkit methodology is under consideration for the small rivers in South Ukraine and near Danube reservoirs where mass mortality of fish is observed.

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P. Fochtman
Acute Toxicity of Nine Pesticides as Determined with Conventional Assays and Alternative Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 25 233-241
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - pesticides

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Abstract : Conventional bioassays in aquatic toxicology are time and space consuming and depend on the continuous culture and maintenance of the test species. Since Toxkit microbiotests do not suffer from these handicaps, they seem to be a tempting alternative.
In order to find out the potential and the limitation of these new microbiotests the sensitivity of 4 Toxkit microbiotests has been compared to that of conventional bioassays. The battery of microbiotests was composed of 2 assays with crustaceans : the Daphtoxkit F™ magna with Daphnia magna and the Thamnotoxkit F™ with Thamnocephalus platyurus, one assay with microalgae : the Algaltoxkit F™ with Selenastrum capricornutum and one test with a ciliate protozoan : the Protoxkit F™ with Tetrahymena thermophila. The conventional assays comprised the following test species : the crustacean D. magna, the micro-algae Scenedesmus quadricauda and the fish Cyprinus carpio and Salmo gairdneri.
Nine commercial pesticides currently used in Poland were selected as test chemicals : 2 herbicides, 3 insecticides and 4 fungicides. The assays were also performed on the reference chemical potassium dichromate.
The outcome of the assays revealed that the sensitivity of the microbiotests was comparable to that of the conventional tests with the same or related species, with Toxkit microbiotests even being somewhat more sensitive than conventional assays
The present study confirms that, because of their culture free nature and rapid, user-friendly and space saving character, Toxkit microbiotests seem to be attractive alternatives to conventional bioassays.

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V. Lucivjanska, M. Lucivjanska and V. Cizek
Sensitivity Comparison of the ISO Daphnia and Algal Test Procedures with Toxkit Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 26 243-246
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - inorganic compounds; fly ashes

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Abstract : A short comparative study has been made on the sensitivity of 2 conventional bioassays (the ISO Daphnia magna immobilization test and the ISO algal growth inhibition test with Scenedesmus quadricauda) and 3 Toxkit microbiotests : the Daphtoxkit F™ magna, also with D. magna, the Thamnotoxkit F™ with the crustacean Thamnocephalus platyurus, and the Algaltoxkit F™ assay with Selenastrum capricornutum (presently renamed Raphidocelis subcapitata). The tests were performed on the reference chemical potassium dichromate and a real world sample : a leachate of fly ash. The results showed good correlation between the 2 algal test procedures for potassium dichromate, but not for the fly ash leachate, which may be due to the use of 2 different algal species in the test procedures… The tests with both D.magna tests on fly ash leachate gave quite comparable results, but diverging results were found for potassium dichromate. The latter differences can be explained by the (unintentional) lower water hardness which was discovered in the Toxkit assay. The anostracan crustacean T.platyurus appeared to be more sensitive than the cladoceran D.magna for potassium dichromate, but not for the fly ash leachate.
Despite some shortcomings, this short study shows that Toxkit microbiotests seem to be an attractive alternative to the (substantially more costly) conventional assays, especially when considering application in routine.

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L. Ulm, J. Vrzina, V. Schiesl, D. Puntaric and Z. Smit
Sensitivity Comparison of the Conventional Acute Daphnia magna Immobilization Test with the Daphtoxkit F^TM Microbiotest for Household Products
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 27 247-252
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - inorganic compounds; solid waste leachates; chemicals - household products

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Abstract : A comparative study has been made of the sensitivity of the acute Daphnia magna immobilization test and the Daphtoxkit F™ magna microbiotest for 30 household products of different categories (cleaning and degreasing products used in the industry, wash products for large laundries, household cleaning products and products for hygienic and personal care). The study revealed that the 48h EC50’s encompassed 3 orders of magnitude (from a few mg/l up to 1000 mg/l) with large sensitivity differences between products use category. Median toxicities were very similar for the conventional D. magna test and the Daphtoxkit F™ microbiotest. Regression analysis revealed the excellent correlation between the 2 test methods (r value of 0.971). This study confirms the analogous findings made in several other recent studies with a variety of chemicals, wastes and natural samples and further validates the usefulness of the new culture free microbiotest technology, not only from the point of view of sensitivity, but also practicality and cost-efficiency.

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M.A. Vaal and A.J. Folkerts
Suitability of Microscale Ecotoxicity Tests for Environmental Monitoring
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 28 253-260
Toxkits : Algaltoxkit F^TM; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - organic compounds; chemicals - pesticides; combination experiments; regulatory testing; toxicity monitoring

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Abstract : Aquatic microbiotests were selected and evaluated for their potential usefulness in measuring ecotoxicity in monitoring programs. In comparison with conventional aquatic ecotoxicity test methods they have the advantage of a shorter exposure period, a smaller test volume and (for many of them) the independence of culturing/maintenance of live stocks of test biota. The microbiotests evaluated are the Thamnotoxkit F™ the Rotoxkit F™, the Algaltoxkit F™, the Microtox® and the Daphnia IQ™ test. They were tested with 2 defined mixtures of chemicals. One mixture contained toxicants with a non-specific mode of action and the other pesticides. The sensitivity of the microbiotests was compared with the conventional short-term tests on daphnids and fish. Special attention was paid to concentration levels during the exposure period.
The loss of even moderately volatile organic compounds from the original microbiotest containers appeared to be considerable and could therefore result in a serious underestimation of toxicity especially when environmental samples of unknown composition are tested. Modification of the test containers of the Thamnotoxkit F™ and Daphnia IQ™ tests resulted in an improvement of the test performance. Due to their sensitivity, these modified tests together with the Microtox® test form a good base for a test battery. The Rotoxkit F™ test and the Algaltoxkit F™ in their present form, appeared to be less useful because of the loss of compounds from the test containers.

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M.C. Vandenbroele, D.G. Heijerick, M.L. Vangheluwe and C.R. Janssen
Comparison of the Conventional Algal Assay and the Algaltoxkit F^TM Microbiotest for Toxicity Evaluation of Sediment Pore Waters
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 29 261-268
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : sediment pore waters

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Abstract : Conventional algal tests have not been utilized to their full potential for routine toxicity assessments. This is mainly due to the complexicity of these assays and the fact that many operators are still encountering practical difficulties. In view of the need for a cost-effective alternative to standard algal assays for routine testing, a new culture/maintanance free algal test was recently developed at the Laboratory for Biological Research in Aquatic Pollution of the University of Ghent in Belgium : the Algaltoxkit F™. In the present study, the conventional algal assay (OECD 201) and the alternative algal microbiotest, both using the micro-algae Raphidocelis subcapitata, were applied in parallel to evaluate the toxicity of 43 sediment pore waters collected from different water courses in Flanders (Belgium). The aim of the study was to compare the sensitivity of the 2 assays, performed in 50 ml erlenmeyers flasks and in 25 ml long cells (disposable spectrophotometric cells with a 10 cm pathlength) respectively. Both the conventional assay and the microbiotest were started with micro-algae de-immobilized from algal beads. The 72h EbC50 values were calculated according to the ‘area under the curve’ method recommended by the OECD. The results of the toxicity tests on the pore waters revealed a comparable sensitivity of the Algaltoxkit F™ test and the conventional algal assay. Furthermore, the repeatability of the microbiotest proved to be as good as - and in most cases even better than - that of the standard algal test. On the basis of the results obtained, it can be concluded that the Algaltoxkit F™ microbiotest is a valid alternative to the conventional algal assay, not the least for routine toxicity evaluation of sediment samples.

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C. Van Der Wielen and I. Halluex
Shifting from the Conventional ISO 8692 Algal Growth Inhibition Test to the Algaltoxkit F^TM Microbiotest ?
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 30 269-272
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - organic compounds

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Abstract : Comparison is made between results of tests performed using the conventional algal growth inhibition test according to ISO 8692, in 250 ml Erlenmeyer flasks and based on laboratory algal cultures, with electronic counting of the algal cells (N), and those obtained with the Algaltoxkit-F™ tests using algal beads, long cells and Optical Density (OD) measurements. The exercise focuses on validation elements such as the variability of controls (C.V. %) and the conformity to ISO 8692 quality criteria. Adequate illumination conditions have been defined and OD measurements versus N-countings were examined. Based on the positive findings, ISSeP\'s practice has since shifted to the use of algal beads, 50 ml Erlenmeyer flasks, a 4400 lux illumination table and electronic counting of the algal cells. Reproducibility of toxicity results (n=7) of the microbiotest procedure, as determined with the reference chemical K2Cr2O7 , is very close to the ISO 8692 intercomparison (interlaboratory) exercise data.

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M. Zaleska-Radziwill
Toxicity Assessment of Chemicals using Conventional Acute Daphnia magna Tests, Toxkits and Fluotox Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 31 273-277
Toxkits : Artoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Thamnocephalus platyurus
Topics : chemicals - organic compounds

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Abstract : Simple, rapid and cost-effective testing procedures were developed over the last few years, mainly for application in routine toxicity screening. One of the most significant breakthroughs in this regard are the highly standardized Toxkit microbiotests, which make use of dormant or immobilized stages of selected invertebrate species as the biological material from which the test species can be obtained. Toxkits can also be used for Fluotox bioassays, in which sublethal (enzymatic) effects are detected after only one hour of exposure to toxicants. The prerequisite for the use of Fluotox tests as (rapid) alternatives to acute toxicity tests is a good correlation of the results of the former with those of the latter. The present study deals with the comparison of 24h toxicity tests performed with 2 Toxkit microbiotests (Artoxkit M™ and Thamnotoxkit F M™) and the conventional Daphnia magna assay, with 1h Fluotox tests on the same species.
Results are presented for 3 phenolic compounds : phenol, p-chlorophenol and 2,4-dichlorophenol, and revealed the very good correlation between the 2 methods and exposure times. The low variation coefficients of the Fluotox tests furthermore indicated the high intralaboratory repeatability of this rapid toxicity test.
Comparison of the magna Fluotox data with those of the Lumistox test showed comparable sensitivity of the crustacean and the bacterial test species for the phenolic compounds. This study indicates that the Fluotox bioassay may be a useful methodology for incorporation in a test battery for routine toxicity testing.

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A. Angelaki, M. Sakellariou, D. Pateras and A. Kungolos
Assessing the Quality of Natural Waters in Magnesia Prefecture in Greece using Toxkits
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 32 281-288
Toxkits : Algaltoxkit F™; Daphtoxkit F™ magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : surface waters

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Abstract : In this study, the results of water quality analyses in the reservoir of the former Lake Karla in Magnesia prefecture in Greece are presented. Physical, chemical and ecotoxicological parameters were examined, the latter by application of 2 Toxkit microbiotests with crustaceans (Daphnia magna) and micro-algae (Selenastrum capricornutum). According to the classification criteria used for assessing the suitability of water for irrigation purposes, the reservoir water quality was poor in terms of electrical conductivity and good in terms of boron concentration. Mortality of D. magna in reservoir waters was generally low, with occasional peaks of up to 30%. The results of the Algaltoxkit F^TM test appeared to indicate that the water was not suited for irrigation purposes since the growth of S. capricornutum, using natural water as the test medium was less than 20% of that in the controls. Addition of nutrients did, however, result in markedly better growth.

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M. Isidori, A. Parrella, C.M.L. Piazza and R. Strada
Toxicity Screening of Surface Waters in Southern Italy with Toxkit Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 33 289-293
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM pulex; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus ; Daphnia pulex ; Pseudokirchneriella subcapitata; Raphidocelis subcapitata ; Selenastrum capricornutum ; Thamnocephalus platyurus
Topics : surface waters; toxicity monitoring

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Abstract : This preliminary study reports on the toxicity of surface waters collected twice in 1997 (once in late spring and once in late autumn) at 19 sampling stations in 5 areas (Domitian, Flegrean, Regi Lagni, Vesuvian and Coast area) in the Province of Naples in Italy. The acute toxicity of the surface waters was determined with a battery composed of 4 microbiotests : the Algaltoxkit F™, the Rotoxkit F™, the Thamnotoxkit F™ and the Daphtoxkit F™ pulex. With the exception of the surface waters from 2 stations in the Vesuvian area (but only for the autumn samples) in which no acute toxicity was detected, all other samples were found to be slightly toxic or toxic according to the arbitrary (log) classification in toxic units of Persoone et al. (1993). In virtually all cases, the waters were toxic to several if not all the test biota used. This preliminary investigation points to the urgent need for surface water quality improvement in the areas investigated. It furthermore confirmed that a battery of Toxkit microbiotests is a practical and reliable low cost tool for the hazard assessment of polluted waters.

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C. Van Der Wielen and I. Halleux
Toxicity Monitoring of the Scheldt and Meuse Rivers in Wallonia (Belgium) by Conventional Tests and Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 34 295-303
Toxkits : Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : surface waters; toxicity monitoring

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Abstract : The Microtox® acute toxicity test using the luminescent bacterium Vibrio fischeri, was chosen as a complementary descriptive parameter of toxic pollution in the framework of monitoring surface water quality in Wallonia, Belgium. In order to examine the samples on a routine basis, ensure a cost-effective approach adapted to the dilution effects, and enhance the sensitivity of the system, a two-step strategy has been adopted and validated : a qualitative screening test, followed by a standard test for all the samples inducing a light inhibition ³ 20%.
For samples in which toxicity was detected, Microtox® responses were compared with the results of a battery of tests including conventional bioassays (algal growth inhibition, Daphnia magna acute test) and microbiotests (Rotoxkit F™and Thamnotoxkit F™). These additional bioassays generally confirmed the toxicity detected by the bacterial test, at least qualitatively. This battery of tests has also been used to characterise the toxicity of effluents upstream of toxic points, and to identify the assays appropriate for studying the toxic plume.
Ten % of the 1500 results from about 50 locations sampled every 4 weeks since 1994, appeared to be acutely toxic to Vibrio fischeri at different scales. Results can be negative, toxic or positive when showing a slight effect at high concentrations. The frequency of toxic events ranged from occasional to permanent. Analysis of the results at each toxic point or on the whole data set showed that toxicological and chemical parameters are complementary.
In view of the rapid evolution of materials and methods in toxicity testing, a critical analysis is made of the use of the acute bioassay with Vibrio fischeri for monitoring the quality of river waters and some new perspectives are presented.

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I. Czerniawska-Kusza and M. Ebis
Toxicity of Waste Dump Leachates and Sugar Factory Effluents and their Impact on Groundwater and Surface Water Quality in the Opole Province in Poland
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 36 319-322
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : effluents; groundwaters; landfill percolates; solid waste leachates; waste waters

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Abstract : A battery of Toxkit microbiotests with 4 aquatic species from different trophic levels has been used to evaluate the toxicity of 2 types of point source pollution in the Voivoidship of Opole in Poland : percolating waters from drainage systems of 4 municipal waste dumps and the effluents of a sugar factory. In addition, the toxicity of groundwaters from 2 wells near 2 waste dumps was also assessed.
All the drainage waters of the waste dumps and the groundwaters near one dump (an unprotected landfill) showed toxic effects on all the test species, for samples taken in spring as well as in autumn. The well water of the (more recent) waste dump of the City of Opole in turn did not show any toxicity. The sugar factory effluents were very toxic to the test biota, but a water sample taken in the River Nysa at 800 mf from the discharge outlet did not show any toxicity.
The findings of this preliminary study confirm the usefulness of cost-effective microbiotests for detection and quantification of the toxic hazards of contaminated discharges.

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U. Dmitruk and J. Dojlido
Application of Toxkit Microbiotests for Toxicity Evaluation of River Waters and Waste Waters in the Region of Warsaw in Poland
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 37 323-325
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM pulex; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : surface waters; waste waters

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Abstract : The toxicity of surface waters (Vistula river in Warsaw) and of raw and treated wastewaters (treatment plants in Grodzisk Mazowiecki and in Piaseczno) has been measured at 2 occasions with 4 Toxkit microbiotests : the Algaltoxkit F™, the Daphtoxkit F™ pulex, the Thamnotoxkit F™ and the Protoxkit F™.
Although the Vistula river is classified as being highly polluted, only the Thamnotoxkit F™ could detect toxicity; undiluted surface water induced 50% in the test organisms after 24h exposure.
The raw wastewaters of the 2 plants were toxic to all test organisms, but most of all to the microalgae, with 72h EC50’s below the 5% dilution level.
Mechanical treatment of the wastewater of the plant in Piaseczno only decreased the toxic impact on the test biota marginally. Biological treatment in turn had a more pronounced effect, but did not succeed either in making the treated wastewater harmless to the test biota, except for the micro-algae in the Grodzisk Mazowiecki plant.
This preliminary study indicates that the Toxkit microbiotests seem to be a very convenient tool for toxicity assessment of wastewaters in treatment plants, and in particular for monitoring the impact of biological treatment on the toxic hazard. In turn, at the exception of the Thamnotoxkit F™ assay, the other microbiotests were not enough sensitive to detect the presence of toxicants in the highly polluted Vistula river.

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M. Isidori
Toxicity Monitoring of Waste Waters from Tanneries with Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 39 339-345
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Rotoxkit F^TM acute; Rotoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Daphnia magna; Daphnia pulex; Thamnocephalus platyurus
Topics : waste waters

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Abstract : Waste waters from tanneries pose one of the most difficult problems for the treatment and subsequent discharge of such industrial effluents in surface waters. The biological breakdown of these wastes must be preceded by flocculation to prevent biomass inhibition from exposure to the toxicants present. Residual toxicity, however, persists in the supernatant after the chemical treatment, which makes biological cleanup difficult.
This paper reports on the ecotoxicological analyses of tannic wastes at different treatment stages, in particular after a new biological treatment based on non-stop inoculation of immobilized enzymes and bacterial mix. The bioassays were performed in complement to chemical analyses which do not always reflect the complex composition of these wastes. Toxkit microbiotests, based on dormant or immobilized biological material from which live test-organisms can easily be hatched, were selected for this study, complemented by the bacterial Toxichromotest on an E. coli mutant. The investigations with a battery of toxicity tests, composed of species from different trophic levels and taxa, clearly showed the substantial decrease in toxicity of the tannic wastes after each treatment step and the virtual absence of acute impact on the receiving surface water.

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A. Kahru, L. Pollumaa, R. Reiman and A. Ratsep
Microbiotests for the Evaluation of the Pollution from the Oil Shale Industry
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 41 357-365
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Protoxkit F^TM; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - organic compounds; surface waters; waste waters; oil shale; shale oil

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Abstract : In this study a comparative chemical and toxicological analysis of 8 phenolic river water and wastewater samples originating from the Kohtla-Järve oil shale industry region (north-western part of Estonia) was made. The total phenolic content of the samples ranged from 0.01 to 195 mg/l. For 5 wastewaters the corresponding synthetic phenolic analogues were prepared. A battery of biotests was applied to study the toxicity of environmental wastewaters and their synthetic phenolic analogues. The battery consisted of 3 crustacean tests (Daphtoxkit F™ magna, Daphtoxkit F™ pulex, Thamnotoxkit F™), one protozoan test (Protoxkit F™), one rotifer test (Rotoxkit F™), one algal test (Algaltoxkit F™) and 3 photobacterial tests (Microtox™, BioTox™ and Vibrio fischeri 1500).
The observed toxicity correlated with the amount of phenolic compounds in the samples. The most sensitive tests in the battery were the Daphtoxkit F™ magna and the photobacterial tests. The comparison of the toxicity of environmental wastewaters and their synthetic phenolic analogues showed that, in general, the environmental wastewaters exhibited higher toxicity than the analogues. For some samples and some tests the difference was as high as a factor 75. This study again shows that (exclusive) chemical analysis is inadequate for toxicity prediction and should be complemented by ecotoxicological testing.

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M. Latif and A. Zach
Toxicity Studies of Treated Residual Wastes in Austria using Different Types of Conventional Assays and Cost-Effective Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 42 367-383
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : composts; solid waste leachates; solid wastes

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Abstract : The toxicity and chemical quality of 12 mechanically-biologically treated residual wastes were studied during different treatment periods. Acute and chronic bioassays were carried out with a battery of test organisms. The acute toxicity was assessed using Daphnia magna (crustacea) and Vibrio fischeri (luminescent bacteria). The chronic toxicity was tested with Selenastrum capricornutum (green algae) and Lepidium sativum (a higher plant). In an effort to integrate less expensive methods of assessing waste toxicity, 2 cost-effective new microbiotests were used in addition to conventional bioassays. These new microbiotests were the Daphtoxkit F™ with Daphnia magna for acute toxicity and the Algaltoxkit F™ with Selenastrum capricornutum for chronic toxicity. Samples were analysed for various physico-chemical parameters (pH, conductivity, O2, BOD5, COD, TOC, NH4-N, NH3, NO3-N, NO2-N, PO4-P, SO4, Cl-, F-, and Fe). Toxicity of the samples was expressed as German regulatory G-values and Toxic units (TU). The results of these studies indicate that high toxicity was observed in samples with a short treatment period. In most cases toxicity decreased significantly when the duration of treatment increased. Marked differences were observed in the sensitivity among the test species. The toxicity was found to vary between 1 and 256 G-values and 0 and 58 TU, respectively. The results showed that Daphtoxkit F™ bioassays were as sensitive as the conventional Daphnia test. When comparing the data of Algaltoxkit F™ tests to standard algal bioassays, in most cases the same G-values and similar Toxic Units were found; differences, if any, were small (less than a factor 2). With regard to toxicity-chemistry relationships, it was found that high levels of ammonia explained a part of the acute toxicity and a good relationship was noted between the toxicity and conductivity, TOC, COD and BOD5 of the most loaded samples.
This study provides evidence that mechanical-biological treatment of waste lowers the amount of organic pollutants and decreases the toxicity. This study also demonstrates the potential of cost-effective microbiotests as alternatives to expensive conventional bioassays for routine detection of acute and chronic hazard of xenobiotics in complex wastes.

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J. Mala, E. Marsalkova and P. Rovnanikova
Toxicity Testing of Solidified Waste Leachates with Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 43 385-390
Toxkits : Algaltoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : fly ashes; solid waste leachates; solid wastes

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Abstract : Solidification is a process for treating contaminated wastes that reduces or entirely prevents the release of hazardous waste compounds into the environment. This process aims to make materials either not harmful nor toxic at all or at least less hazardous than the raw waste. The most widely used basic materials for solidification are cement, lime, fly ash, and blast-furnace slag. No toxicity data are available for these materials. This contribution describes the partial results of an experimental study dealing with the toxicity testing of 2 types of cement, fly ash, and blast furnace slag. Samples of the test materials were solidified and then pulverized by crushing. The specimens were leached before and after solidification by a 2-step procedure according to the CEN standard. Deionized water acidified to pH 3.5 was used as the leaching solution. All the leachates were tested for toxicity using microbiotests with crustaceans (Thamnotoxkit F™), algae (Algaltoxkit F™) and bacteria (Toxi-ChromoPad™). The heavy metals in the solid materials and leachates were measured. The study demonstrates the short- and long-term leachability of metals from some materials commonly used for solidification with the corresponding toxicity of the leachates; it also indicates the toxicity levels of the raw materials. The results of the leaching tests followed by ecotoxicological analyses provide information on the stability of the solidified waste and on the significance of the solidification technology for the protection of the environment.

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L. Manusadzianas, L. Balkelyte, K. Sadauskas and L. Stoskus
Microbiotests for the Toxicity Assessment of Various Types of Water Samples
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 44 391-399
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : effluents; Toxicity Index (T.I.)

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Abstract : A microbiotest battery consisting of 4 Toxkit microbiotests (Algaltoxkit F™, Daphtoxkit F™ magna, Protoxkit F™ and Thamnotoxkit F™) and one rapid electrophysiological test, based on membrane response of Nitellopsis obtusa cells (Charatox), was used for the assessment of the acute toxicity of complex effluents in Lithuania. According to the integral toxicity evaluation, i.e. the toxic print, the least toxic effluents were biologically treated municipal wastewater samples. For the majority of these effluents it was not possible to calculate EC50 end point values for the Daphtoxkit F™ magna, Protoxkit F™ and Algaltoxkit F™ tests. The Charatox and the Thamnotoxkit F™ test were the most sensitive in the battery, responding to almost all samples that were characterized by chemical analysis as being outside of the Lithuanian MAC requirements.
Among the chemical parameters available for the effluents, total nitrogen showed the highest correlation (r² = 0.93) with the toxic print values. The determination coefficient (r²) between the toxic print and an integral chemical characteristic (the sum of degrees of excess) was equal to 0.64 which implies a rather poor linear prediction of toxicity from chemical data.

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M. Michniewicz, G. Nalecz-Jawecki, J. Stufka-Olczyk and J. Sawicki
Comparison of Chemical Composition and Toxicity of Wastewaters from Pulp Industry
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 45 401-411
Toxkits : Protoxkit F^TM; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : waste waters

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Abstract : Wastewaters from different pulp-making processes contain various organic and inorganic compounds : wood-derived, process chemicals and compounds generated during the reactions between the chemicals and raw materials. Carbohydrates and lignin-derived compounds constitute the main groups of components that cause oxygen demand in wastewater (COD), but they have rather low toxicity. Among all wood-derived compounds, resin acids (e.g. abietic acid, dehydroabietic acid) are generally recognised as the main source of acute toxicity in pulp and paper industry effluents. A review of studies on toxicity of resin acids has shown that the acute lethal concentration of individual acids for fish (rainbow trout and salmon) is around 0.4-1 mg/l. The lowest sublethal concentration of dehydroabietic acid (DHAA) has been reported to be between 5 and 20 mg/l (rainbow trout - inhibition of enzyme conjugation and bile acid uptake). Unsaturated fatty acids are also present in wastewaters but their contribution to toxicity is less than that of resin acids.
In this study the concentration of toxic compounds (resin and fatty acids, chlororesin acids, phenolic and chlorophenolic compounds) in wastewaters from pulping, recovery of chemicals and bleaching processes have been determined. The chemical composition of influents and effluents of biological treatment plants were also studied. Five microbiotests : Microtox®, Spirotox, Protoxkit F™, Rotoxkit F™ and Thamnotoxkit F™ were used to assess the toxicity of the wastewater streams mentioned above. The Microtox® test was the most sensitive microbiotest from the battery applied, but the toxicity values were not correlated with the concentrations of the pollutants analyzed. The other tests in turn had L(E)C50s which correlated better with the toxicity equivalency units (TEUs).

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G. Persoone and M. Vangheluwe
Toxicity Determination of the Sediments of the River Seine in France by Application of a Battery of Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 47 427-439
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM acute; Rotoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : confounding factors; limit values; sediment pore waters; sediments; toxicity monitoring

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Abstract : This study reports on the potential of the Toxkit microbiotests and the bacterial luminescence test Microtox®, to detect and quantify the toxicity of the sediments of the river Seine and a few tributary rivers in France. The objective of the research was to try to identify a minimum test battery for routine toxicity screening of river sediments. Attempts were also made to correlate the ecotoxicological findings with their chemical cause(s) and to evaluate the suitability of the microbiotests as tools for hazard assessment of river sediments. During a 12 months period in 1996-1997, sediments were collected from 12 stations on the river Seine, from Paris to the estuary in Le Havre, and from 7 stations along the rivers Marne, Eure and Commerce. Pore waters were extracted by centrifugation for performance of the bioassays on the interstitial waters. The test battery comprised the following freshwater and estuarine/marine microbiotests : the Microtox® with Vibrio fischeri, the Algaltoxkit F™ with Selenastrum capricornutum, the Rotoxkit F™ and Rotoxkit M™ with the rotifers Brachionus calyciflorus and B. plicatilis respectively, the Daphtoxkit F™ magna with the cladoceran crustacean Daphnia magna, the Thamnotoxkit F™ and the Artoxkit M™ with the anostracan crustaceans Thamnocephalus platyurus and Artemia franciscana respectively, and the Spirostomum assay, an experimental new microbiotest with the ciliate protozoan Spirostomum ambiguum. From all the freshwater stations on the Seine, only one did not give a toxic signal with any of the microbiotests used. No toxicity was detected either in 2 out of the 7 stations on the tributary rivers, nor in the 3 estuarine pore waters. In most stations, toxic efects were observed with several if not all of the microbiotests of the battery, except the Microtox® assay for which only one pore water sample appeared to be toxic. The intensity of the toxicity signal varied substantially from one station to the other, but none of the Toxkit tests was either the most or the least sensitive for all stations. Principal component analysis (PCA) was applied on the data set which allowed to select a minimum battery of only 2 microbiotests, accounting for 90% of the observed variance in the data set : the Thamnotoxkit F™ and the Algaltoxkit F™. Attempts to correlate the chemical data with the ecotoxicolgical results were biased by the fact that the chemical analyses had been performed only on the sediments (total pollutant load) and not on the pore waters (“bioavailable” contaminants). Comparison of the outcome of the PCA analysis on both the ecotoxicological and chemical data sets revealed positive correlation between the toxic effects and the metal content of the sediments. The study corroborates analogous findings made by the same laboratory on a very large number of sediment pore waters from rivers in Flanders, Belgium. The investigations confirm that a small battery of Toxkit microbiotests is a suitable tool for low cost routine ecotoxicity evaluation of contaminated river sediments.

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M.L. Vangheluwe, C.R. Janssen and P.A. Van Sprang
Selection of Bioassays for Sediment Toxicity Screening
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 49 449-458
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : sediment pore waters; sediments; test selection

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Abstract : A large monitoring study of 80 freshwater sediments, using the TRIAD approach, was conducted in Flanders, Belgium using a battery of microbiotests and conventional assays. The test battery covered several phylogenenic groups and included acute or short-term chronic tests on the sediments’ porewater with bacteria (Vibrio fischeri), algae (Raphidocelis subcapitata), crustaceans (Thamnocephalus platyurus and Daphnia magna) and fish (Clarias gariepinus). Whole sediment tests with Hyalella azteca and Chironomus riparius were conducted concurrently. Uni- and multivariate statistical techniques were used to determine the discriminatory power, the toxicity detection capacity and the redundancy of the different assays. A battery composed of a minimal set of porewater and whole sediment assays is proposed for the initial toxicity screening of contaminated sediments.

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P.A. Van Sprang, M.L. Vangheluwe and C.R. Janssen
The Toxicity Identification of Inorganic Toxicants in Real World Samples
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 50 459-465
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : effluents; sediment pore waters; Toxicity Identification Evaluation (T.I.E.); waste waters

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Abstract : The toxicity of a sediment pore water and an effluent was characterized using Toxicity Identification Evaluation (TIE) procedures. Both samples exhibited acute toxicity to the freshwater crustacean Thamnocephalus platyurus. The TIE approach was conducted using an enlarged battery of fractionation tests. Toxicity of the pore water sample was removed or reduced significantly by the cation exchange procedure and the air-stripping test at pH 11. Additionally, reduced toxicity was found at a reduced pH. Based on this TIE study and chemical analysis, ammonia was identified as the main toxic agent. Toxicity of the effluent sample was reduced significantly by the anion exchange resin. Additionally, an increased toxicity was found at a decreased pH. It was concluded from this TIE study and chemical analysis of the effluent that metal chromium was the causative toxicant.

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K. Eleftheriadis, A. Angelaki, A. Kungolos, L. Nalbandian and G.P. Sakellaropoulos
Assessing the Impact of Atmospheric Wet and Dry Deposition using Chemical and Toxicological Analysis
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 51 469-473
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : air; chemicals - metals

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Abstract : Chemical analyses and toxicity test results for atmospheric wet and dry deposition from 5 sampling sites in Greece are presented in this study. In most of the samples examined, the heavy metals content of the filtered water samples was relatively low. There was quite a high concentration of amorphous sulphur and carbon in the solid particles of the deposition. Beta radiation level was generally low in all samples. About half of the samples exhibited toxicity to Daphnia magna, which exceeded the 50% effect level after 48h exposure.

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D. Drobne and J. Strus
Toxicity Determination of Zinc and Cadmium (Single and in Combination) with a Battery of Toxkit Microbiotests
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 53 481-486
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM pulex; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - metals; combination experiments;

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Abstract : Toxicity tests were performed with 4 Toxkit microbiotests, based on aquatic species of different phylogenetic groups and representing different trophic levels, on 2 heavy metals (zinc and cadmium) which are common contaminants of the aquatic environment. The metals were tested out single on micro-algae (Algaltoxkit F™ with Selenastrum capricornutum), a ciliate protozoan (Protoxkit F™ with Tetrahymena thermophila), and 2 crustaceans (Daphtoxkit F™ with Daphnia pulex and Thamnotoxkit F™ with Thamnocephalus platyurus); combination experiments were performed on the micro-algae and one of the 2 crustaceans (D. pulex).
The study showed that cadmium is substantially more toxic than zinc for all the test biota used, but also revealed that the ratios between the L(E)C50s for the 2 metals was nearly the same for each species, namely a factor 4. The factorial experiments with binary mixtures showed that for the micro-algae, both metals showed “additivity” in accordance with the concentration-addition model of Anderson and Weber (1975). This was, however, not the case for the metal combinations applied to D. pulex, the outcome of which seemed to be dependent on the relative contributions of the individual metals.
Besides bringing additional information on the toxicity of 2 important metal contaminants to aquatic biota, the study also showed the difficulty of general application of models for predicting toxic impacts, and the need for toxicity testing of mixtures to determine their real hazard.

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A. Kungolos, V. Batziaka, P. Samaras, G.P. Sakellaropoulos, A.M. Kipopoulou, A. Zoumboulis and Th. Kouimtzis
Using Toxkits for Calculating Interactive Effects of Chemicals
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 54 487-493
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - metals; chemicals - pesticides; combination experiments; toxic interactions

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Abstract : The effects of 3 common pesticides (lindane, methyl-parathion and atrazine) and of 2 metals (cadmium and chromium) are investigated in this study on a crustacean (Daphnia magna) and on micro-algae (Selenastrum capricornutum). Methyl-parathion was the most toxic compound towards both test species, while lindane was more toxic to D. magna than atrazine and atrazine was more toxic to S. capricornutum than lindane. The interactive effects of the pesticides were also investigated. The interactive effect between lindane and methyl-parathion on survival of D. magna was synergistic, while that between lindane and atrazine was generally additive. The combined effect of the 3 pesticides on the growth of S. capricornutum showed indications of antagonism. The interactive effect of cadmium and chromium on D. magna was also investigated using Toxkit microbiotests and the results were comparable with literature results using traditional D. magna test.

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T. Pereira, M.J. Cerejeira, M.F. Brito and M.A. Morbey
Laboratory Studies with Microbiotests to Evaluate the Acute Toxicity to Aquatic Biota of Herbicides used in Portugese Paddy Fields
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 55 495-500
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - pesticides; soil runoff

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Abstract : Rice crops constitute a potential risk of contamination of aquatic ecosystem since the water from paddy fields (which may still contain certain amounts of herbicides used to combat plant pests) is in most cases, discharged directly or indirectly into the surrounding water bodies. In order to evaluate the possible effects of this contamination, laboratory and field work is performed in the Sado Valley in Portugal. In the present study, laboratory results are presented on the effects of the main herbicides used in paddy fields to aquatic biota. Mcrobiotests have been performed with bacteria (Vibrio fischeri), micro-algae (Selenastrum capricornutum, renamed Raphidocelis subcapitata) and crustaceans (Daphnia magna, Thamnocephalus platyurus and Artemia franciscana). The experiments were performed with different dosages of the most used commercial products based on MCPA, molinate, propanil and quinclorac as active ingredients. The concentration ranges used in the laboratory are the same as those applied by the farmers in real field conditions.

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B. Marsalek and L. Blaha
Microbiotests for Cyanobacterial Toxins Screening
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 58 519-525
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Protoxkit F^TM; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : biotoxins; cyanobacterial blooms

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Abstract : The development of massive blooms of cyanobacteria (blue-green algae) in eutrophicated fresh and salt waters is an actual problem in many countries. Moreover, many strains of cyanobacteria produce toxins with a specific mode of action, the most studied being hepatotoxins (such as e.g. microcystin) and neurotoxins (e.g. anatoxin-a). Toxicity testing of crude extracts of cyanobacterial biomass often causes problems of interpretation of results because of the presence of many compounds interfering with the detection and the quantification of observed effects. A fractionation technique based on SPE C-18 cartridges was developed allowing to separate and recover fractions selectively containing cyanobacterial hepatotoxins. Two samples of cyanobacterial biomass (both crude extract and toxic fraction) were tested for toxicity with a battery of 7 different toxicity microbiotests with different test organisms : bacteria, ciliate protozoans, rotifers, crustanceans and nematodes. In addition, one mutagenic bacterial microbioassay was also applied. Samples were selected on the basis of the content of cyanobacterial hepatotoxin microcystin-LR (high content vs. absence). The toxic data of the crude extracts were compared with those of the water and toxin fractions.
The outcome of the study revealed that the crustacean microbiotests were by far the most discriminative for toxicity detection between non-toxic and toxic samples, and that the Thamnotoxkit F™ assay was the most sensitive bioassay within the battery. The study also showed that fractionation of the crude extract of cyanobacterial biomass allows to distinguish toxic effects of the hepatotoxins proper from those of other toxic compounds present in the crude extract.

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M. Tarczynska, G. Nalecz-Jawecki, M. Brzychcy, M. Zalewski and J. Sawicki
The Toxicity of Cyanobacterial Blooms as Determined by Microbiotests and Mouse Assays
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 59 527-532
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : biotoxins; cyanobacterial blooms; cyanotoxins

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Abstract : Cyanobactrial blooms are common throughout the world in eutrophic water bodies. Cyanobacterial hepato- and neurotoxins represent a health threat for humans as well as for animals in drinking water supplies. Mouse bioassays have been used extensively to detect cyanobacterial blooms toxicity but the use of warm-blooded vertebrates is limited to specialised laboratories and poses ethical problems. Hence, it is necessary to find alternative assays to detect and quantify cyanobacterial hazards.
This study reports on the toxicity of 7 cyanobacterial bloom samples from the Jeziorsko Reservoir in central Poland as determined with 4 microbiotests : the Thamnotoxkit F™, the Daphtoxkit F™ magna, the ProtoxkitF™ and the Spirotox. The phytoplankton samples were lyophilized, sonicated and passed through octadecyl cartridges. The results were compared to those obtained with mouse bioassays and HPLC analysis of microcystin-LR (MC/LR) in the bloom samples.
24h LC50s for the Thamnotoxkit F™ and the Spirotox tests showed statistically significant correlations with MC/LR concentrations in the samples. In turn, no statistically significant correlations were found between mouse 24h LD50s and microbiotest 24h L(E)C50s which indicates that cyanobacterial hepatotoxins may exert different effects in microbiotests with invertebrates and in mouse bioassays.

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A.K.Törökne
The Potential of the Thamnotoxkit Microbiotest for Routine Detection of Cyanobacterial Toxins
In : New Microbiotests for Routine Toxicity Screening and Biomonitoring (G. Persoone, C. Janssen and W. De Coen , eds. - Kluwer Academic / Plenum Publishers) 2000, chapter 60 533-539
Toxkits : Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : biotoxins; cyanobacterial blooms; interlaboratory testing; cyanotoxins

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Abstract : An interlaboratory trial with 8 laboratories was organised for evaluating the potential of the Thamnotoxkit F™ microbiotest for toxicity detection of 5 toxin producing cyanobacteria (Anabaena flos-aquae, Microcystis aeruginosa, Cylindrospermopsis raciborskii, Aphanizomenon flos-aquae, Tychonema bourrellyi) and the green alga Selenastrum capricornutum (Raphidocelis subcapitata) as a control. The Thamnotoxkit F™ test is a 24h bioassay using, as test organisms, larvae of the freshwater anostracan crustacean Thamnocephalus platyurus hatched from cysts. Dilution series were prepared from freeze-dried samples of microalgae which were submitted twice to freezing and melting before extraction of the water soluble cyanotoxins. The concentration series used for each microalgal extract ranged from 0.3 mg/ml to 5.0 mg/ml. All samples were tested twice at different time by each examiner, eventually yielding 16 independent results obtained from 3 parallels (48 data for each sample). The samples of C. raciborskii were only studied by 4 examiners and gave 8 results (24 data). The water extracts of An.flos-aquae and M. aeruginosa were the most toxic to T. platyurus inducing 100% mortality in all concentrations used, except the highest dilution in which the mortality was, however, still around 70-80%. The extracts of C. raciborskii also induced 100% mortality as from the 1.0 mg/ml concentration and Aph.flos-aquae killed all the test organisms at the 3.0 mg/ml dilution. Water extracts of T. bourrellyi only caused total mortality in the highest test concentration. The water extract of the control alga S. capricornutum only gave a significant toxicity effect (around 30%) on the crustaceans in the highest concentration.
From this study it can be concluded that the Thamnotoxkit F™ assay is a sensitive and convenient method for the detection of cyanotoxins and constitutes an interesting alternative for the mouse bioassay for routine hazard screening of water bodies for biotoxins.

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T.W. Snell and G. Persoone
Acute Toxicity Bioassays using Rotifers. - II. A Freshwater Test with Brachionus rubens
Aquatic Toxicology 1989 14 81-92
Toxkits : Rotoxkit F^TM acute
Test Species : Brachionus calyciflorus; Brachionus rubens
Topics : test development; chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : A 24 h acute toxicity test for freshwater is described using the rotifer Brachionus rubens hatched from cysts. Hatchlings are used in a simple protocol that provides for LC50 calculation and yields highly repeatable results. Hatching is initiated by transferring cysts to warmer temperatures and light. At 25 °C, hatching commences after 17 h and by 25 h, 40% of cysts have hatched. The average hatching percentage for B. rubens cysts was 53%. A reference test using sodium pentachlorophenate (NaPCP) yielded an 24hLC50 of 0.62 mg/l, with a coefficient of variation of 9.7%. The no observed effect concentration (NOEC) for NaPCP was 0.28 mg/l. Protocols for range-finding and definitive tests of unknown toxicants are also described. Six compounds were assayed and had the following toxicity rankings : copper > NaPCP > cadmium > SDS > free NH3 > malathion. B. rubens was at least twice as sensitive as Brachionus plicatilis to all toxicants except malathion. The precision of the B. rubens acute toxicity test is about 3 times better than that of Daphnia. Like its marine counterpart with B. plicatilis, the B. rubens test for fresh water has a major advantage over current aquatic tests in that it eliminates culturing and maintenance of live stocks. Test animals are obtained from dormant eggs which have a shelf life of at least 1 year. Moreover, the rotifer test proposed is fast, convenient, sensitive and repeatable, making it a useful new tool for routine assessment of aquatic toxicity of chemicals and effluents.

N.B.It was eventually found out that the controlled production of cysts of the species Brachionus calyciflorus was more easy than that of Brachionus rubens. For reasons of convenience the former rotifer species hence been given preference as the the test species for the Rotoxkit F microbiotest.

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G. Persoone, C.R. Janssen and W. De Coen
Cyst-Based Toxicity Tests : Comparison of the Sensitivity of the Acute Daphnia magna Test and Two Crustacean Microbiotests for Chemicals and Wastes
Chemosphere 1994 29, 12 2701-2710
Toxkits : Streptoxkit F^TM; Thamnotoxkit F^TM
Test Species : Streptocephalus proboscideus; Thamnocephalus platyurus
Topics : chemicals - inorganic compounds; chemicals - organic compounds; effluents; solid wastes; sediment pore waters; sludges

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Abstract : This paper statistically evaluates the sensitivity of the acute Daphnia magna bioassay in comparison to that of two crustacean microbiotests : the Streptoxkit F and the Thamnotoxkit F, which make use of larvae of the anostracans Streptocephalus proboscideus and Thamnocephalus platyurus respectively, hatched from cysts. Regression equations were calculated for 146 data pairs, taken from 5 different studies dealing with the acute toxicity of pure chemicals, effluents, river sediments, solid wastes and monitoring wells, and sludges. All comparisons show that there is a significant relationship (p< 0.05) between the acute effects found with the D.magna and the two crustacean microbiotests; correlation coefficients ranged from 0.84 to 0.92.
In the majority of cases, effect ratios between the conventional D.magna and the microbiotests were within a factor 2 for both pure chemicals and environmental samples.
Variation coefficients for repeated tests on the reference chemicals potassium dichromate indicate a good precision and hence a good degree of standardization of the microbiotest procedures. The evidence provided in this paper demonstrates that the new crustacean microbiotest can be used as low cost alternatives to conventional D.magna acute assay.

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M.D. Centeno, G. Persoone and M.P. Goyvaerts
Cyst-Based Toxicity Tests. - IX. The Potential of Thamnocephalus platyurus as Test Species in Comparison with Streptocephalus proboscideus (Crustacea / Branchipoda / Anostraca)
Environmental Toxicology and Water Quality 1995 10, 4 275-282
Toxkits : Streptoxkit F^TM; Thamnotoxkit F^TM
Test Species : Streptocephalus proboscideus; Thamnocephalus platyurus
Topics : test development; chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : The potential of the larvae of the freshwater anostracan crustacean Thamnocephalus platyurus has been evaluated as an alternative to those of the related species Streptocephalus proboscideus, used so far as test organism in the standard Streptoxkit F test. In line with the former cyst-based toxicity test method, the hatching of dried T.platyurus eggs, the molting of larvae, and the sensitivity of the latter to toxicants were assessed. The acute toxicity of 10 pure chemicals, 4 solid waste leaches and 5 sediment pore waters to T .platyurus nauplii was determined according to the standard Streptoxkit test protocol. Hatching success and reliability of T. platyurus cysts proved better than that of S. proboscideus cysts after 20 hours incubation in standard conditions; hatching percentage after 24 hours was slightly improved by diluting the EPA medium to 1:8 with deionised water. The sensitivity of T. platyurus nauplii to pure toxicants and wastes was at least equal, and in many cases even higher than that of the S. proboscideus larvae. Precision of the toxicity tests with T. platyurus was better than that of of the S. proboscideus assay. Consequently, the standard operational testing protocol of the Streptoxkit F has been slightly modified with replacement of S. proboscideus by T. platyurus as a test species. The Streptoxkit F has consequently been renamed into Thamnotoxkit F for use as a cost-effective routine acute toxicity testing of chemicals and wastes.

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C.R. Janssen, G. Persoone and T.W. Snell
Cyst Based Toxicity Tests. - VIII. Short-Chronic Toxicity Tests with the Freshwater Rotifer Brachionus calyciflorus
Aquatic Toxicology 1994 28 243-258
Toxkits : Rotoxkit F^TM chronic
Test Species : Brachionus calyciflorus
Topics : test development; chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : The development and potential use of a 4-day static renewal test [4-day Life Table (LT) test] and a 3-day static test [3-day Population Growth (PG) test] with the freshwater rotifers Brachionus calyciflorus are described. For both bioassays, test animals are obtained by hatching cysts wich eliminates the need for the culturing and the maintenance of the organisms. The toxicity of copper (Cu), pentachlorophenol (PCP), 3,4-dichloroaniline (DCA) and lindane was assessed using the developed methods. The NOEC’s, based on the test endpoint r_m, obtained with the 4-day LT test were 0.0025, 0.4, 5 and 20 mg/l for Cu, PCP, DCA and lindane, respectively. Similar results were obtained with the 3-day PG test for which NOEC’s of 0.005, 0.8, 20 and 10 mg/l, respectively, were recorded. The mean CV between replicated 3-day PG tests was 10%, indicating a good intra-laboratory reproducibility of the test results. For Cu and PCP, the sensitivity of B. calyciflorus compared favourably to chronic toxicity tests with Daphnia magna, while for the other two compounds B. calyciflorus proved to be rather insensitive. Considering the increasing need for relatively short toxicity tests, the two described short-chronic bioassays could be valuable new tools for routine toxicity evaluations. The major advantages associated with these tests are : they are less labour-intensive than existing chronic tests, they can be completed within one work week, and do not require stock culturing of test organisms.

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E.Q. Espiritu, C.R. Janssen and G. Persoone
Cyst-Based Toxicity Tests. - VII. Evaluation of the 1-h Enzymatic Inhibition Test (Fluotox) with Artemia nauplii
Environmental Toxicology and Water Quality 1995 10 25-34
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : test development; chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : The newly developed 1 h enzymatic inhibition bioassay (Fluotox) was applied to toxicity testing with the larvae of the brine shrimp Artemia. The method consists of visual observation of in vivo inhibition of an enzymatic process using a fluorigenic enzyme substrate. The 1 h Fluotox test and the conventional 24 h and 48 h LC50 toxicity tests were conducted in parallel on 8 different organic and inorganic chemicals. A good correlation was found between the 1 h EC50 and the 24 h LC50 data with r² s of 0.94 and 0.93 for each respective regression. Addition of the enzyme substrate did not influence the toxicity of the chemicals in the 24 h and 48 h acute test; coefficients of variation (CV) ranged from 8 to 34% and from 1 to 39%, respectively.
Investigations on the applicability of the Fluotox procedure for testing in brakish waters revealed that lowering the salinity from 35 to 15 ppt did not affect the fluorescence and mortality responses in the controls.
The same decrease in salinity had no significant influence on the 1-h EC50 except for NaLS and acetic acid. The ratio between the 1 h EC50s at 35 and 15 ppt varied from 0.46 to 2.18 depending on the chemical.
However, the same decrease in salinity significantly influenced (p<0.05) the 24 h LC50s (except NaPCP) and the 48 h LC50 (except formaldehyde). In general, the ratio of the LC50 values obtained at 35 and 15 ppt varied from 0.30 to 2.21 and from 0.48 to 2.53 for the 24 h and 48 h exposures, respectively.In view of several inherent advantages such as rapidity, experimental simplicity, low cost of performance, and the use of dry cysts as biological starting material (which eliminates the maintenance of live stock culture) the 1 h Artemia Fluotox test offers promising potential for numerous ecotoxicological applications.

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G. Persoone
Cyst-Based Toxicity Tests. - VI. : Toxkits and Fluotox Tests as Cost-Effective Tools for Routine Toxicity Screening
Schriften-Reihe Verein Wasser-Boden-Luft, Gustav-Fischer Verlag, Stuttgart 1992 89 563-575
Toxkits : Artoxkit M^TM; Rotoxkit F^TM acute; Rotoxkit M^TM; Streptoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Streptocephalus proboscideus
Topics : test development; test validation

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Abstract : During the last two decades microbiotests have been developed which are independent of recruitement, maintenance and/or culturing of live stocks of test organisms. Culture and maintenance free microbiotests have been worked out in the Laboratory for Biological Research in Aquatic Pollution at the University of Ghent, with selected aquatic invertebrates. The new approach is based on the use of resting stages (cysts) as inert biological material from which live test organisms can be hatched on demand. The cyst-based bioassays have recently been miniaturized in Toxkits. Four cyst-based screening tests have reached the stage of commercialisation : two freshwater 24hLC50 tests (Rotoxkit F and Streptoxkit F) and two estuarine/marine 24hLC50 bioassays (Rotoxkit M and Artoxkit M), based on cysts of rotifer and crustacean species, respectively.
Recently, the same laboratory has also been focusing on the development of a rapid (one hour) sublethal bioassay with aquatic invertebrates. The so called Fluotox screening test is based on the visual observation of in vivo inhibition of an enzymatic process, using a fluorescent substrate. Besides the advantages of rapidity and cost-effectiveness, the Fluotox assay also appears to have a remarkable predictive potential for mortality, as displayed by the remarkable correlation between the Fluotox 1hEC50’s and conventional 24hLC50’s with the same species.

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M. Van Steertegem and G. Persoone
Cyst-Based Toxicity Tests. - V. Development and Critical Evaluation of Standardized Toxicity Tests with the Brine Shrimp Artemia (Anostraca, Crustacea)
In : Progress in Standardization of Aquatic Toxicity Tests (A.M.V.M. Soares and P. Calow , eds. - Lewis Publishers) 1993 81-97
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : test development; chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : The use of cryptobiotic stages (called cysts of dormant eggs), from which live test organisms can be hatched at will, eliminates the need for continuous stock culturing and opens a promising perspective for better standardization of test methodologies in aquatic toxicology. Although there are several groups of aquatic invertebrates which produce resting stages, the brine shrimp Artemia (Crustacea, Anostraca) was the very first organism used in toxicity tests starting from cysts.
Considering the need for simple but at the same time reliable test methods for toxicity screening in estuarine and coastal environments, a number of standard methods have been developed, based on both lethal and sublethal endpoints, and with different life stages of the brine shrimp. Since there are no specific guidelines for the development of a standard toxicity test, a general framework was worked out for this specific goal. Next to tests with the conventional criteria involving mortality, growth and reproduction, standard protocols were worked out with less conventional test criteria, e.g., hatchability, phototactic behaviour, food uptake, and recovery.
Taking into account the importance of cost-effectiveness for routine testing, the standard acute mortality test with Artemia larvae has recently been modulated in a kit, containing all (disposable) material to perform a number of toxicity tests. Based on the results of a large intercalibration exercise in Europe, the U.S.A., and Canada, the test methodology has been further improved and the Artemia cyst-based toxicity test is now available under the name of Artoxkit for cost-effective routine screening of xenobiotics in estuarine and marine environments.

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M.C. Calleja and G. Persoone
Cyst-Based Toxicity Tests. - VI. The Potential of Ecotoxicological Tests for the Predicton of Acute Toxicity in Man as Evaluated on the First 10 Chemicals of the MEIC Programme
Alternatives To Laboratory Animals 1992 20 396-405
Toxkits : Artoxkit M^TM; Protoxkit F^TM; Rotoxkit F^TM acute; Streptoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Streptocephalus proboscideus
Topics : chemicals - organic compounds

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Abstract : In the framework of the multicentre evaluation of in vitro cytotoxicology (MEIC) programme, the first ten chemicals of the prescribed list were tested for acute toxicity in four standardised cyst-based aquatic invertebrate tests, consisting of two rotifer species (the estuarine Brachionus plicatilis and the freshwater Brachionus calyciflorus) and two crustacean species (the halophilic anostracan Artemia salina and the freshwater anostracan Streptocephalus proboscideus). Mortality was the test criterion and toxic effects, expressed as 24-hour LC50 values, were correlated with rodent and human acute oral toxicity data. Generally, a good correlation was obtained between any of the invertebrate tests and the rodent data. Likewise, the predictive screening potential of the aquatic invertebrate tests for acute oral toxicity in man was slightly better than the rat for eight (excluding diazepam and digoxin) and nine (including diazepam, excluding digoxin) of ten substances. The aquatic test systems, however, appear to be more suitable for compounds soluble in water.

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M.D. Centeno, L. Brendonck and G. Persoone
Cyst-Based Toxicity Tests. - III. Development and Standardization of an Acute Toxicity Test with the Freshwater Anostracan Crustacean Streptocephalus proboscideus
In : Progress in Standardization of Aquatic Toxicity Tests (A.M.V.M. Soares and P. Calow , eds. - Lewis Publishers) 1993 37-55 0
Toxkits : Artoxkit M^TM; Rotoxkit F^TM acute; Rotoxkit M^TM; Streptoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Streptocephalus proboscideus
Topics : test development; chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : A new method is presented for determination of the acute toxicity of chemicals and effluents to larvae of the freshwater anostracan Streptocephalus proboscideus (Frauenfeld), using resting eggs (cysts) as the source of test organisms. The larvae are obtained by controlled hatching of standard reference eggs. For the testing laboratory, this procedure eliminates all the difficulties inherent in recruitment or continuously culturing live stocks.Appropriate test conditions (temperature, dilution medium, photoperiod, and duration) have been selected and a protocol has been developed for the execution of a 24 h toxicity test, with scoring of larval mortality as the effect criterion. Standard Reference Water (SRW) is used as hatching and dilution medium for the bioassay, which is carried out in polystyrene multiwell plates, at 25 °C, in darkness. Since the sensitivity of the larvae changes with age, instar II-III nauplii obtained within 24 h from the start of hatching are used for the test. 24 h LC50s determined with the new test procedure on 8 chemical compounds gave the following ranking of decreasing sensitivity (mg/L) in : HgCl2 (0.03) – CuSO4 (0.21) – CdCl2 (0.46) – NaPCP (0.80) – trichloroacetic acid (1.2) – K2Cr2O7 (1.9) – ZnSO4 (4.0) – sodium lauryl sulfate (23).The Streptocephalus proboscideus acute toxicity test is a useful addition to the present limited battery of cyst-based toxicity tests (Toxkits). The sensitivity and reliability of the Streptoxkit test compare favorably with those of other commonly used aquatic invertebrate bioassays.

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T.W. Snell and C.R. Janssen
Microscale Toxicity Testing with Rotifers
In : Microscale Testing in Aquatic Toxicology (P.G. Wells, K. Lee and C. Blaise , eds. - CRC Press) 1998 chapter 28 409-422
Toxkits : Rotoxkit F^TM acute; Rotoxkit M^TM
Test Species : Brachionus calyciflorus; Brachionus plicatilis
Topics : review; test development; test validation; chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : Toxicity tests are one of the essential tools for evaluating the effects of anthropogenic stress in aquatic ecosystems. Methods using microscale organisms have improved the speed, simplicity, and reduced the cost of making toxicity measurements. The size, ecology, and life cycle of rotifers makes them well suited for microscale assays and toxicity assessment. In the last five years the use of rotifers in routine toxicity assessment has dramatically increased, due primarily to the widespread availability of rotifer cysts and standardized protocols for performing tests. Development of new endpoints for rapid toxicity measurements are described, as well as their application to evaluating effluents, sediment and soil elutriates, and sediment pore waters. Most work has used 24 h acute tests with brachionid rotifers, but a standardized 2 d population growth test and a 7 d full life cycle test are available for estimating chronic toxicity. The chronic toxicity of several compounds has been evaluated and acute/chronic ratios of 0.9 to 33 reported. Behavioral assays of swimming are described that are based on computer tracking of rotifer movement. Methods for measuring ingestion rates are described using fluorescent microspheres and image analysis of single rotifers. Several substrates that are metabolised to fluorescent products have been used to measure in vivo enzyme activity. The reduction of enzyme activity with increasing toxicant exposure has been quantified in single rotifers with image analysis or in populations with a fluorometer. Results from changes in rotifer stress protein gene expression as a result of toxicant exposure are summarized. The need to relate biological effects at one level of organization to those at higher levels is discussed. Understanding how effects in population level processes like growth, predation, and competition translate into changes in community structure is especially critical. Several areas where rotifers could contribute to the understanding of how toxicants modify aquatic ecosystems are described.

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G. Persoone, C. Blaise, T.W. Snell, C.R. Janssen and M. Vansteertegem
Cyst-Based Toxicity Tests. - II. Report of an International Intercalibration Exercise with 3 Cost-Effective Toxkits
Zeitschrift für angewandte Zoologie 1992/1993 79 (1) 17-36
Toxkits : Artoxkit M^TM; Rotoxkit F^TM acute; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis
Topics : interlaboratory testing; chemicals - inorganic compounds

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Abstract : What very likely constituted the largest bioanalytical round robin exercise ever conducted in modern times was undertaken in Europe, the United States, and Canada to appraise the interlaboratory reliability and reproducibility of three new cost-efficient cyst-based toxicity test procedures. Close to 500 pre-packaged toxicity kits (Toxkits) of either freshwater Brachionus calyciflorus (Rotoxkit F), marine Brachionus plicatilis (Rotoxkit M), or Artemia salina (Artoxkit M) were mailed out to 183 participating laboratories in this mammoth enterprise. The exercise consisted in undertaking a 24h LC50 bioassay with one or more of the three Toxkits using copper sulphate as reference toxicant. Toxkits contained everything required to conduct a complete assay as well as very explicit and illustrated Standard Operational Procedure so that all participants would be able to perform prescribed tests without extensive ecotoxicological knowledge.
Despite various uncontrollable / unexpected technical and biological problems which have since been resolved, 60, 80, and 87% of participants were nevertheless able to successfully complete Rotoxkit F, Rotoxkit M, and Artoxkit M testing, respectively. This statistic in itself, resulting from laboratory operators previously unfamiliar with the Toxkit concept, confirms the undeniable potential of this practical bioanalytical technology. Coefficients of variation from LC50 mean values for the three tests ranged from 25 (Rotoxkit, Canada) to 68% (Rotoxkit F, U.S.A.). This variability is quite acceptable in view of the formidable logistical complexity involved in the organization of this two-continent international ringtest. Overall, the organizers of this major initiative were very satisfied with its outcome and felt that it had accomplished its objectives of making known and of validating three novel tools now readily available for ecotoxicological screening.

In addition to the overwhelming success of this exercise in terms of the marked interest it generated, the toxicity data and participant feedback proved to be invaluable to later design new and optimised versions of three Toxkits. Since the holding of the international ringtest, all steps of the procedures have been thoroughly reviewed and many improvements have been made to the Toxkit bioassays. As a consequence, the original Toxkits have been modified to a large extent. Upgraded versions of the Toxkits, whose reliability has been validated by extensive intralaboratory tests, are now even more cost-effective as they each allow the conduct of 6 complete assays. A fourth Toxkit, based on cysts of the freshwater anostracan crustacean Streptocephalus proboscideus (Streptoxkit F), has recently joined the Toxkit family. All four newly improved Toxkits are presently available commercially to interested users to conduct various types of ecotoxicological screening applications.

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G. Persoone
Cyst-Based Toxicity Tests. - I. A Promising New Tool for Rapid and Cost Effective Toxicity Screening of Chemicals and Effluents
Zeitschrift für angewandte Zoologie 1991 78 (2) 235-241
Toxkits : Artoxkit M^TM; Rotoxkit F^TM acute; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis
Topics : interlaboratory testing

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Abstract : The determination of the toxicity of effluents and wastes at the aid of biological tests (bioassays) is hampered very seriously by the necessity of continuous culturing or maintenance of live stocks of test organisms in sufficient numbers and in a healthy state. Consequently such assays are (and can) only be performed in specialized laboratories and in limited number because of the high costs.
To date, however, it has become possible to do acute testing in routine at low cost and without any need for sophisticated equipment simply by using dormant eggs (cysts) of specific species of aquatic biota as starting biological material. Such cryptobiotic eggs can be stored for long periods without losing their viabiltity, and hatched at will in 24 hours of time. This new concept eliminates the (very expensive) continuous culturing or maintenance of live stocks.
Recently three of these \'cyst-based tests have been miniaturized into Toxkits which have been submitted to a very large intercalibration exercise (more than one hundred participants) in Europe, in the USA and in Canada, in order to determine their repeatability and reproducibility.

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T.W. Snell and G. Persoone
Acute Toxicity Bioassays using Rotifers. I. A Test for Brackish and Marine Environments with Brachionus plicatilis
Aquatic Toxicology 1989 14 65-80
Toxkits : Rotoxkit M^TM
Test Species : Brachionus plicatilis
Topics : test development; chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : A standardized 24-h acute toxicity test for the marine environment using the rotifer Brachionus plicatilis is described. Test animals are obtained by hatching cysts, thus eliminating the need for stock cultures. Since animals hatching from cysts are of similar age, genotype and physiological condition, test variability is greatly reduced. Controlled cyst hatching is achieved by transferring to lower salinity, warmer temperature and light. After 23h at 25 °C and 15 ppt salinity, hatching begins and proceeds rapidly. By 28 h, 90% of the cysts have hatched. A protocol is outlined to collect the neonates and use them in a simple acute toxicity test to calculate a 24 h LC50. A reference test using sodium pentachlorophenate (NaPCP) is described as well as a range-finding test and a definitive test for unknown toxicants. The toxicity of six compounds to B. plicatilis was examined with the following decreasing sensitivities : copper > NaPCP > SDS > free NH3 > cadmium > malathion. For 3 of the 6 compounds tested, salinity increase from 15 to 30 ppt resulted in higher sensitivity, whereas for the other 3 compounds there was no effect. In comparison with current test organisms B. plicatilis is either more equal or less sensitive depending on the compound, confirming the species-chemical specificity of mode of action of toxicants. The repeatability of the rotifer test is 5-6 times better than reported for Daphnia tests and twice as good as the Artemia nauplii bioassay. Like the standard brine shrimp nauplii acute test, the cyst-based rotifer test is an important advance in acute toxicity testing since it eliminates stock cultures, is rapid, sensitive, highly repeatable, easy to execute and cost effective.

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G. Persoone
Development and Validation of Toxkit Microbiotests with Invertebrates, in Particular Crustaceans
In : Microscale Testing in Aquatic Toxicology (P.G. Wells, K. Lee and C. Blaise , eds. - CRC Press) 1998 chapter 30 437-449
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Rotoxkit F^TM acute; Rotoxkit M^TM; Streptoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Daphnia magna; Daphnia pulex; Streptocephalus proboscideus; Thamnocephalus platyurus
Topics : review; test development; test validation; chemicals - inorganic compounds; chemicals - organic compounds; sludges; effluents; solid wastes; leachates; sediment pore waters; groundwaters

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Abstract : The review first addresses the major bottleneck of conventional toxicity tests with invertebrates, namely the need for continuous culturing and/or maintenance of stocks of live test-organisms. Subsequently the history of the development and the validation of the first cyst-based microbiotests are outlined. Details are given of the large intercalibration exercises organized concurrently in Europe, the USA and Canada as well as of the extensive microbiotest validation study on 350 samples from various sites in Flanders (Belgium) from waste dumps, groundwaters, river sediments, sludges and industrial effluents. The gradual incorporation during the early nineties, of Toxkit microbiotests in monitoring studies of wastewaters, industrial effluents, landfills and river sediments is reported as well as the predictive potential of these small-scale assays in human toxicology. Finally the development and the first validation of the Daphtoxkit microbiotests with the waterfleas Daphnia magna and Daphnia pulex are outlined in detail, and sensitivity comparisons and correlations with the conventional assays on Daphnids are given for inorganic and organic compounds and for effluents from the textile industry.

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G. Persoone and C.R. Janssen
Freshwater Invertebrate Toxicity Tests
In : Handbook of Ecotoxicology (P. Calow , ed. - Blackwell Scientific Publications) 1993 chapter 4 51-65
Toxkits : Artoxkit M^TM; Rotoxkit F^TM acute; Rotoxkit M^TM; Streptoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Streptocephalus proboscideus
Topics : regulatory testing; review; interlaboratory testing

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Abstract : The first part of the paper addresses the state of the art of standard freshwater invertebrate toxicity tests for regulatory purposes, with details of the characteristics and the test procedures of the acute and chronic tests with Daphnids (Daphnia magna, Daphnia pulex and Ceriodaphnia dubia).
Subsequently this review reports on standard tests used for non-regulatory assays, and in particular the development of cyst-based toxicity tests with their advantages over conventional bioassays based on stock-culturing of the test species.
Finally an overview is given of the numerous toxicity tests developed by individual laboratories with particular invertebrate species.
It is concluded that toxicity tests for screening purposes, regulatory requirements and predictive hazard assessment, each have a different set of requirements as to precision, test organism choice, exposure time and costs. Each type of bioassay can have its own merits when properly used in the correct context, but whoever develops new methods must be receptive to the needs of those who actually have to use bioassays to solve real problems.

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M.D.F. Centeno, L. Brendonck and G. Persoone
Acute Toxicity Tests with Streptocephalus Proboscideus (Crustacea / Branchipoda / Anostraca) : Influence of Selected Environmental Conditions
Chemosphere 1993 Vol.27, No. 11 2213-2224
Toxkits : Streptoxkit F^TM
Test Species : Streptocephalus proboscideus
Topics : chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : The influence of selected environmental conditions on the acute toxicity of 3 selected chemicals (K2Cr2O7, CuSO4 and Na pentachlorophenate) to instar II-III larvae of Streptocephalus proboscideus was evaluated. Mortality after a 24 h incubation was used as effect criterion. Factorial experiments were set up resulting to 25 different combinations of temperature and water hardness. The effects of pH of the dilution medium and light regime during toxicant exposure were also assessed. Results (24-h LC50’s) from nearly 200 tests revealed that the degree of variation is chemical- and test condition-dependent. Water hardness had the greatest effect on larval sensitivity, showing a significant (p<0.05) negative correlation. A significant effect (p<0.05) on sensitivity was furthermore detected only for the extreme values of temperature (30 °C) and pH (6.0) tested. Light conditions during acute exposure caused no change in larval sensitivity.

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M. Latif, G. Persoone, C.R. Janssen, W. De Coen and K. Svardal
Toxicity Evaluations of Wastewaters in Austria with Conventional and Cost-Effective Bioassays
Ecotoxicology and Environmental Safety 1995 32 139-146
Toxkits : Rotoxkit F^TM; Streptoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Streptocephalus proboscideus; Thamnocephalus platyurus
Topics : waste waters

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Abstract : The acute toxicity of 42 samples of different types of domestic and industrial discharges was assessed with a battery tests comprising the standard Daphnia magna bioassay and three cost-effective new microbiotests (cyst-based Toxkits) : the Rotoxkit F with the freshwater rotifer Brachionus calyciflorus and the Streptoxkit F and Thamnotoxkit F tests with the freshwater fairy shrimps Streptocephalus proboscideus and Thamnocephalus platyurus, respectively. Chemical analyses were performed for conventional water quality parameters such as chemical oxygen demand (COD), biological oxygen demand (BOD5), NO2, NH3, NH4⁺, O2 and pH. Toxicity of the samples, expressed as German regulatory G-values, was found to vary between 1 and 128. The results of these toxicity tests indicate that the Toxkits bioassays were as sensitive as the D. magna acute test. The crustacean T. platyurus was in 75 % of the toxic samples more sensitive than D. magna. Relationships between the chemical composition and the toxicity of the discharges could be established in some cases, but not in others, which confirms the difficulties of extrapolating toxic hazards of complex wastes from (mostly restricted) chemical analyses. This study demonstrates the potential of cost-effective bioassays (such as, e.g., cyst-based Toxkits) as attractive alternatives to (expensive) conventional bioassays for routine monitoring of effluents and wastes.

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A. Bispo, M.J. Jourdain and M. Jauzein
A Procedure to assess Contaminated Soil Ecotoxicity
Contaminated Soil '98 1998 335-364
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : soil leachates

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Abstract : Two polluted soil samples were assessed for both acute (Microtox®, Daphnia magna, Thamnocephalus platyurus) and chronic (Pseudokirchneriella subcapitata) toxicity, as well as for genotoxicity (Mutatox®). Bioassays were performed on soil water leachates and on soil solvent extracts to characterize not only the water soluble pollutants but also to evaluate less soluble pollutants or bound to soil fractions.Coupling water leaching and solvent extractions to acute, chronic and genotoxicity bioassays allows us to define a step by step procedure to characterise the ecotoxicological hazard of soils. This procedure helps in defining the mobility and identifying the nature of the pollutants responsible for the measured toxicity. It can be used (i) to indentify the hazard, (ii) to classify soil hazards and in so doing to map contaminated sites qualitatively (locating more polluted areas), (iii) to monitor and judge treatment success (iv) to control rehabilitated sites.

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G. Persoone
Ecotoxicology and Water Quality Standards
In : River Water Quality - Ecological Assessment and Control (P. Newman, A. Piavaux and R. Sweeting, eds.) - Published by the Commission of the European Communities 1992 III 751 p
Toxkits : Rotoxkit F^TM; Streptoxkit F^TM
Test Species : Brachionus calyciflorus; Streptocephalus proboscideus
Topics : surface waters

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Abstract : The establishment of water quality objectives (WQO) for individual chemicals is a complex process fraught with many uncertainties. WQO should (in principle) be derived from no-observed-effect-concentrations (NOEC\'s) determined experimentally on selected test species and even preferably on biological communities in field situations. In practice this is seldom the case. The number of species of different trophic levels, currently available for toxicity testing is extremely low and not representative for the biological communties in natural aquatic ecosystems; furthermore, the number of standardized test methods is very limited. Data bases on the effects of xenobiotics on aquatic biota mainly contain figures on acute tests with but a few species; worse, no information is available for approximately 90 % of all industrial chemicals.
WQO are derived from tests with (almost exclusively) pelagic species without consideration for the partitioning of pollutants to the sediments and the threat to benthic biota. WQO\'s for individual chemicals do not take into consideration either additive and/or synergistic effects of toxicants. Control of industrial emissions is mostly limited to chemical analyses imposed for a limited number of compounds; toxicity testing is avoided as much as possible for reasons of complexicity and costs.

In order to establish more realistic WQO\'s, toxicity data need to be generated with a battery of representative test species comprising pelagic as well as benthic organisms and additional (standard) test methods must therefore be worked out. Short-chronic tests should be developed and validated, in replacement of the present chronic bioassays which are too complex and too time consuming. New bioassay procedures should be user-friendly and cost-effective in order to allow large scale and routine application.
The control of the hazard of industrial emissions in the aquatic environment should be based on biomonitoring with a battery of cost-effective toxicity tests, rather than on exclusive chemical analyses. Newly developed screening bioassays such as e.g. the Microtox® test, the cyst-based 24 h LC50 Toxkit tests and the 1 hr EC50 Fluotox tests have a promising future in this regard, since they bypass the major bottleneck in ecotoxicological testing, namely the need for continuous culturing and maintenance of live stocks.

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M.C. Calleja, G. Persoone and P. Geladi
Comparative Acute Toxicity of the First 50 MEIC Chemicals to Aquatic Non-Vertebrates
Archives of Environmental Contamination and Toxicology 1994 26 69-78
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Streptoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Streptocephalus proboscideus
Topics : chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : The acute toxicity data of the first 50 chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme is compared for three cyst-based toxicity tests (Artoxkit M with Artemia salina, Streptoxkit F with Streptocephalus proboscideus and Rotoxkit F with Brachionus calyciflorus and two other tests (the Daphnia magna and the Photobacterium phosphoreum Microtox^TM tests) commonly used in ecotoxicology. The difference in sensivity for the 50 chemicals was as high as 9 orders of magnitude (on a molecular weight basis) between the most and least sensitive species. Generally, a similar toxicity ranking of the 5 test species was found for most of the chemicals and the interspecies correlations were high. Results from Principal Component Analysis (PCA) and cluster analysis indicated that the groupings are not related to a clear and defined chemical structure. However, the loading plot of the first two principal components may aid in selecting the minimum number and type of tests that have to be included in a battery which encompasses a broad spectrum of toxicity levels. Consequently, this study supports the use of a selected battery of tests to evaluate ecotoxicity and suggests its possible importance for screening of biologically-active compounds from natural sources.

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B. Clement, G. Persoone, C.R. Janssen and A. Le Du-Delepierre
Estimation of the Hazard of Landfills through Toxicity Testing of Leachates
Chemosphere 1996 Vol. 33, No. 11 2303-2320
Toxkits : Ceriodaphtoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Ceriodaphnia dubia; Daphnia magna; Thamnocephalus platyurus
Topics : landfill leachates

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Abstract : Twenty-seven landfill leachates were tested on a battery of conventional toxicity tests (microalgae, daphnids, duckweeds) and new microbiotests (rotifers, crustaceans, protozoans, luminescent bacteria).The toxicity varied substantially from one test species to the other, from one site to the other, as well as from one type of landfill to the other. Leachates of domestic wastes were significantly more toxic than those of pure industrial wastes; the most toxic leachates were found for landfills receiving hazardous industrial wastes mixed with domestic wastes.
The highest sensitivity was found for the protozoan assay, followed by the crustacean microbiotests. All other types of bioassays appeared to be substantially less sensitive to the toxicants present in the landfill leachates.The results of a Principal Component Analysis suggest that in approximately 90 % of the cases the toxicity of landfill leachates can be assessed by applying a test battery composed of a bacterial assay, a protozoan test and an assay with micro-algae, jointly with one of the following bioassays : higher plants, rotifers or crustaceans.
The application of a factor 100 to the highest toxicity figure for each landfill leachate to extrapolate a Predicted No-Effect Concentration (PNEC) revealed that in quite a number of cases, the leachates need to be diluted by more than 10.000 times to make them innocuous for environmental biota.

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M.C. Calleja, P. Geladi and G. Persoone
QSAR Models for Predicting the Acute Toxicity of Selected Organic Chemicals with Diverse Structures to Aquatic Non-Vertebrates and Humans
SAR and QSAR in Environmental Research 1994 Vol. 2 193-234
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Streptoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Streptocephalus proboscideus
Topics : chemicals - organic compounds

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Abstract : The linear and non-linear relationships of acute toxicity (as determined on five aquatic non-vertebrates and humans) to molecular structure have been investigated on 38 structurally-diverse chemicals. The compounds selected are the organic chemicals from the 50 priority chemicals prescribed by the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. The models used for the evaluations are the best combination of physico-chemical properties that could be obtained so far for each organism, using the partial least squares projection to latent structures (PLS) regression method and backpropagated neural networks (BPN). Non-linear models, whether derived from PLS regression or backpropagated neural networks, appear to be better than linear models for describing the relationship between acute toxicity and molecular structure. BPN models, in turn, outperform non-linear models obtained from PLS regression. The predictive power of BPN models for the crustacean test species are better than the models for humans (based on human lethal concentration). The physico-chemical properties found to be important to predict both human acute toxicity and the toxicity to aquatic non-vertebrates are the n-octanol water partition coefficient (Pow) and heat of formation (HF). Aside from the two former properties, the contribution of parameters that reflect size and electronic properties of the molecule to the model is also high, but the type of physico-chemical properties differs from one model to another. In all of the best BPN models, some of the principal component analysis (PCA) scores of the ¹³C-NMR spectrum, with electron withdrawing/accepting capacity (LUMO, HOMO and IP) are molecular size/volume (VDW or MS1) parameters are relevant. The chemical deviating from the QSAR models include non-pesticides as well as some of the pesticides tested. The latter type of chemical fits in a number of the QSAR models. Outliers from one species may be different from those of other test organisms.

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M.C. Calleja, P. Geladi and G. Persoone
Modelling of Human Acute Toxicity from Physicochemical Properties and Non-Vertebrate Acute Toxicity of the 38 Organic Chemicals of the MEIC Priority List by PLS Regression and Neural Network
Food and Chemical Toxicology 1994 32, 10 923-941
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Streptoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Streptocephalus proboscideus
Topics : chemicals - organic compounds

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Abstract : Linear and non-linear modelling of human acute toxicity (as human lethal concentrations; HLCs) of the 38 organic chemicals from the 50 priority compounds of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme was investigated. The models obtained were derived either from a set of 23 physicochemicals properties of the compounds of from their acute toxicities to five aquatic non-vertebrates together with the physicochemical properties. For the linear type, modelling was performed using a partial least square projection to latent structures (PLS) regression method; for the non-linear models both PLS regression and neural network were utilized. A neural network using a combination of backpropagation and cascade-correlation algorithms was applied in this study. The results generally reveal a slightly better predictive performance of the models obtained from PLS regression than those obtained from neural networks. However, the model composed of physicochemical properties (PC-model) from the trained neural network using a back propagation algorithm with pruning technique proved superior to that trained with a combination of backpropagation and cascade-correlation algorithms after leave-one-out cross-validation. The predictive power of the PC-models, whether linear or non-linear, was comparable with that of the corresponding models consisting of both structural descriptors and the ecotoxicological tests (ECOPC-models), except for the battery (ECOPC-model) from the neural works. The composition of the \'best\' PLS and neural network models points to the importance of the combination of physiochemical properties reflecting lipophilicity, size, volume, intermolecular binding forces and electronic properties of the molecule. All the aquatic non-vertebrate tests are shown to be essential in explaining human acute toxicity. However, the degree of contribution differed, with the crustacean (Artemia salina) and the bacterial (Microtox) bioassays being more important to the linear and non-linear PLS models, whereas the crustacean (Artemia salina and Streptocephalus proboscideus tests, and the rotifer (Brachionus calyciflorus) assay were important to the neural network models. The organochlorine (lindane) and bipyridinium (paraquat) pesticides were common outliers in all the models. Moreover, the latter two compounds and the organophosphate (malathion) pesticide were also common outliers in all ECOPC-models. Other types of pesticides, however, fit the models. The predicted HLCs of a number of non-pesticides, including some chlorinated compounds, also deviated from the observed HLCs by more than one order of magnitude.

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C. Blaise
Microbiotests in Aquatic Ecotoxicology : Characteristics, Utility and Prospects
Environmental Toxicology and Water Quality 1991 Vol. 6 145-155
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis
Topics : review

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Abstract : Small-scale biological tests (microbiotests) have steadily increased in development and application over the last 30 years in the field of aquatic ecotoxicology. Multitrophic level assessment requirements, attractive features of microbiotests, and the constant search for simplicity and cost efficiency of testing are reasons explaining the expanding use of microbiotests. In this article, the major characteristics that advantageously confer popularity on microbiotests are presented and 25 currently applied aquatic toxicity microbiotests are listed. Conducted with bacteria, protozoans, microalgae, small invertebrates and fish cell lines, these microbiotests represent a realistic cross section of thos that are becoming an essential part of ecotoxicological assessment. Microbiotests can be profitably employed for ranking and screening chemicals, for novel applications enabling rapid detection of ecotoxic effects in complex liquid samples, and for increasing the cost efficiency and diagnostic potential of hazard assessment schemes. Microbiotesting research, development and applications will continue to surge in the 1990s, driven, among other factors, by the imperative need for cost effectiveness in environmental programs. Research in the fields of ecotoxicology, biotechnology and immunochemistry should provide interesting breakthroughs to further enhance the specificity and diagnostic value of microbiotests.

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A. Kahru, L. Pollumaa, R. Rieman and A. Ratsep
Predicting the Toxicity of Oil-Shale Industry Wastewaters by its Phenolic Composition
Alternatives to Laboratory Animals 1999 27 359-366
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - organic compounds; waste waters

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Abstract : The chemical composition and toxicity of five phenolic wastewaters samples collected from the Kohlta-Järve (Estonia) oil-shale industry region were analysed. The total phenolic contents (HPLC data) of these samples ranged from 0.7 mg/l to 195 mg/l. A total of 11 phenol (up to 84 mg/l) and p-cresol (up to 74 mg/l). Artificial phenolic mixtures were also composed, to mimic the content of phenolic compounds in the wastewater samples. The theoretical toxicities of these artificial mixtures were calculated by using the toxicities of the individual phenolic constituents to photobacteria (the BioTox^TM test) and were assumed to have an additive mode of action. From the BioTox data, the additive toxic effects of phenolic compounds in the artificial mixtures were confirmed to be highly probable. The toxicities of the wastewater samples and their artificial phenolic analogues (mixtures) were studied by using a battery of Toxkit microbiotests (Daphtoxkit F^TM magna, Thamnotoxkit F^TM, Protoxkit F^TM and Rotoxkit F^TM) and three photobacterial tests (Microtox^TM, BioTox^TM and Vibrio fischeri 1500). The wastewaters were classified as toxic (two samples), very toxic (two samples) and extremely toxic (one sample). Comparison of the test battery responses showed that the industrial wastewaters were 2-28-fold more toxic than the respective artificial phenolic mixtures. The photobacterial tests proved to be the most appropriate for screening purposes. This was the first attempt to use a test battery approach in the toxicity testing of Estonian wastewaters. The study showed that the toxicity of oil-shale industry wastewaters could not be predicted solely on the basis of their phenolic composition, since only 7-50% of their toxicity was shown to be due to phenolic compounds. It is true, to a certain extent, that the majority of environmental samples are usually very complex and contain various types of pollutants. As even a full chemical analysis (which is very expensive) can easily miss the constituent(s) with the greatest toxic effect(s), the use of toxicity tests in parallel to chemical analysis should be encouraged.

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M.C. Calleja, G. Persoone and P. Geladi
The Predictive Potential of a Battery of Ecotoxicological Tests for Human Acute Toxicity, as Evaluated with the First 50 MEIC Chemicals
Alternatives to Laboratory Animals 1993 21 330-349
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Streptoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Streptocephalus proboscideus
Topics : chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : The acute toxicities of the first 50 chemicals (dextropropoxyphene hydrochloride excluded) of the multicentre evaluation of in vitro cytotoxicity (MEIC) programme were determined on four aquatic invertebrates and a bacterial strain (Photobacterium phosphoreum for the Microtox^TM test) commonly used in ecotoxicology testing. Three of the aquatic invertebrate tests consisted of cyst-based toxicity tests (Artoxkit M with Artemia salina, Streptoxkit F with Streptocephalus proboscideus, and Rotoxkit F with Brachionus calyciflorus) and the Daphnia magna test. Results of simple linear regression analyses indicated that the rodent tests (rat and/or mouse) were better than the ecotoxicological tests for predicting acute oral lethal doses in man. However, it appears that the batteries of ecotoxicological tests resulting from the partial least squares method appear to be better than the rodent tests for predicting human oral lethal doses.

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C.R. Janssen
Alternative Assays for Routine Toxicity Assessments : a Review
In : Ecotoxicology (G. Schuurman and B. Markert, eds. - J. Wiley and Sons) 1998 chapter 26 813-839
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Rotoxkit F^TM; Rotoxkit M^TM; Streptoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Daphnia magna; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Streptocephalus proboscideus; Thamnocephalus platyurus
Topics : review

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Abstract : The number of available aquatic toxicity tests is large and increases every year. For certain types of applications such as the routine screening of large numbers of environmental samples, standard conventional tests may be perceived as being unpractical. This has lead to the development of a (increasing) number of alternative small-scale toxicity tests also called microbiotests. The present paper reviews the potential use and limitations of a number of these alternative assays. For bacteria, algae and invertebrates, a brief description of conventional assays is given followed by an analysis of the sensitivity, reproducibility, cost-efficiency and apllication potential of freshwater microbiotests. In addition, several of case studies are discussed is which alternative toxicity tests were evaluated as routine environmental monitoring tools.
From this review it can be concluded that microbiotests have an important role to play in the screening and ranking of the hazards of chemicals and environmental wastes. However, except for a few bacterial and invertebrates tests, there is a lack of published information on the various aspects of test development and especially on the application of these alternative assays. For most microbiotests, further research and test evaluation is needed before these methods will gain widespread acceptance as valid alternatives to currently use ‘conventional’ test procedures routine environmental monitoring.

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R.F. Amparado Jr. and G. Persoone
The use of Algae in Ecotoxicological Testing : a Review
The Philippine Scientist 1996 Vol. 33 116-147
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : review

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Abstract : An intensive review on the use of algae in ecotoxicological testing is herein described. The state of the art in algal toxicity testing is given and the currently applied algal tests in hazard assessment schemes are emphasized. However, the need of an alternative, rapid and cost-effective algal tests is underscored considering the inequality between the plethora of new chemicals being produced each year and the need of toxicity data of these chemicals. The static algal tests still remain to date the workhorse in the tiered approach in the hazard assessment of chemicals as mandated in national and an international legislations. The limitations of the currently applied static algal tests are however, identified and an alternative procedure is proposed.

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C. Keil, A. Forchert, J. Fastner, U. Szewzyk, W. Rotard, I. Chorus and R. Kraetke
Toxicity and Microcystin Content of Extracts from a Planktothrix Bloom and Two Laboratory Strains
Water Research 2002 36, 8 2133-2139
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : biotoxins; cyanobacterial blooms; cyanotoxins

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Abstract : Toxic effects and microcystin content from various extracts of a Planktothrix agardhii bloom and two different strains of Planktothrix agardhii, HUB 076, and NIVA 34 were investigated. Extracts were obtained with solvents of different polarity such as hexane, dichloromethane, methanol, and water. Additionally, different pre-treatments were used to break the cells before extraction. Acute toxicity was determined with the fairy shrimp Thamnocephalus platyurus, subchronic effects were detected in embryos and larvae of the zebrafish Danio rerio. The extracts affected the test species to a different extent. Effects were severe in polar extracts (water and methanol) of all strains tested. Although the strain NIVA 34 did not contain any microcystins, aqueous extracts of this strain showed the highest acute toxicity to the crustacean species tested (LC50= 0.08 mg ml(-1)). In contrast aqueous extracts of the Planktothrix bloom containing high amounts of microcystins were less acutely toxic (LC50 = 0.46 mg ml(-1)). Our results indicate the existence of further toxic metabolites in different Plankorothrix agardhii strains.

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E.A. Cho, K. Tameda, M. Hanashima, T. Yamada and S. Higuchi
Toxicological Evaluation of the Chemical Oxidation Methods for Landfill Stabilization
Waste Management 2009 29 1006-1011
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : landfill leachates

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Abstract : As the stabilization criteria for landfill sites, only chemical criteria for the leachate discharges from the landfill sites have been used in Japan and many other countries. Recently, chemical oxidation has been developed as a method for the early-stabilization of landfills. However, by-products that are difficult to detect by chemical analysis can be produced by this method. Therefore, toxicity tests are useful tools for detecting the changes of leachate quality after application of this method. The heat source in the A landfill was analyzed by organic position inquiry technology, and ozone-treated leachate was sprayed back to the heat source in the landfill. Toxicity changes of the leachate after the spray were monitored using Microtox®, ToxScreen-II, and DaphTox tests. The hardly-degradable organic matter was efficiently removed and toxicities of the leachate in the heat source decreased after the application. These toxicity results were significantly related to chemical oxygen demand (COD) changes. Thus, it was concluded that the toxicity tests were effective for monitoring the leachate quality after applying the chemical oxidation method for landfill stabilization, and its incorporation to establish the criteria for early-stabilization of landfill sites needs to be considered.

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R. Rojickova, B. Marsalek, B. Dutka and R. Mcinnis
Bioassays used for Detection of Ecotoxicity at Contaminated Areas
In : Environmental Contamination and Remediation Practices at Former and Present Military Bases (F. Fonnum et al., eds - Kluwer Academic Publishers) 1998 227-232
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : soil leachates; soils

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Abstract : Ten bioassays were used for the evaluation of acute, (sub)chronic toxicity and genotoxicity of an unknown soil from a military base and six model soils that were contaminated at former military bases with the major pollutants : kerosene, petroleum and used motor oil. The test species represented algae and higher plants, invertebrates and bacteria. The ability to detect toxicants in test soils and their waste extracts was investigated and the sensitivity of each bioassay was compared with regard to the tested material (soil vs. water extract from oil) and other test species.
The most sensitive bacterial assays (Toxi-ChromoPad and ECHA Biocide Monitor) were able to detect acute toxicity in most samples. Another bacterial assay, MetPAD, responded positively to the presence of metals. The least sensitive test of the whole battery was the bacterial luminescence assay. The two phytotoxicity tests were similar in sensitivity. The crustacean test Thamnotoxkit F detected acute toxicity only in three samples. The maturation of nematodes was the most sensitive endpoint in nematode assay that was able to indicate potential genotoxicity in most extracts. The MutaChromoPlate identified the presence of mutagenic agents in all soil extracts, unlike the SOS-Chromotest Pad, which was positive for genotoxicity only for the most toxic soil with kerosene.
A minimised battery of bioassays for detecting toxicity in investigated soils could be composed of a bacterial assay (Toxi-ChromoPad or ECHA), a phytotoxicity assay (a seed germination and root elongation test) and an invertebrate test (nematode assay).
Basic information about genotoxicity could be given by the Muta-ChromoPlate.

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J G. Ruck
Toxkit Technology and New Zealand Water Quality Guidelines
Toxkit Technology and New Zealand Water Quality Guidelines 1998 3-38 42 pages
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM; Rotoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : effluents; interlaboratory testing; waste waters

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Abstract : The primary objective of this project was to evaluate six Toxkits and the IQ Toxicity Tests^TM, for suitability in the role of screening of whole effluents in DTA required by Resource Consents. As an extension, their role as \'up the pipe\' toxicity identification tests were also evaluated.
Screening tests must be cost effective (so that regular testing may be performed), easy to use (at site desirable but not essential), readily available, sensitive to a wide range of toxicants and therefore able to discriminate between effluents. Furthermore, they must be rapid enough to allow immediate modifications to effluent flow and quality, or herald the need for more definitive site specific testing, and (to some degree) be repeatable and reproducible. Based on these criteria, and consistent with other evaluatory research (see above sections), are the following conclusions and recommendations :
- The Toxkit and IQ Toxicity Test^TM organisms were, in general not as sensitive as the native species used by NIWA.
- Daphtoxkit F^TM magna, Thamnotoxkit F^TM, IQ Toxicity Test^TM (as a potentially rapid method) and Algaltoxkit F^TM are recommended as being suitable, overall, for screening purposes, and therefore may be included as DTA requirements for Resource Consents.
- Daphtoxkit F^TM magna, Thamnotoxkit F^TM, IQ Toxicity Test^TM (as a rapid method) and Algaltoxkit F^TM are also recommended as being suitable for toxicity identification evaluations (\'up the pipe\' toxicity) for industrial and municipal plants.
- Artoxkit M^TM, Rotoxkit F^TM and Rotoxkit M^TM, while very cost effective, and with very good precision (repeatability), were not as sensitive as the others, and therefore are not recommended for whole effluent toxicity screening in New Zealand. The rotifer tests may be useful in wastewater treatment plants where operators may wish to monitor influent toxicity to protect rotifer populations in their treatment ponds.
- Further research should be carried out with the more recently developed Toxkits for suitability as screening tests - Ceriodaphtoxkit F^TM (test species C. dubia is native to New Zealand), the modified Protoxkit F^TM and the new ostracod Toxkit, soon to be released.

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M.A. Vaal and A.J. Folkerts
Sensitivity of Microscale Ecotoxicity Tests and their Suitability to Measure Toxicity of Environmental Samples
National Institute of Public Health and the Environment - Report no. 607042 009 1998 8-31 44 pages
Toxkits : Algaltoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : Aquatic microbiotests were selected and evaluated for their potential usefulness in measuring ecotoxicity in monitoring programs. Microbiotests are tests with aquatic invertebrates that demand a shorter exposure period and a smaller test volume than conventional aquatic ecotoxicity test methods. Microbiotests evaluated were the Thamnotox F test, the Rotox F test, the Algaltoxkit F test, the Microtox test and the Daphnia IQ test. They were tested with a reference toxicant (a metal salt), and, except for the Algaltoxkit F, with two defined mixtures of chemicals. One mixture contained toxicants with a non-specific mode of action and the other pesticides. The sensitivity and reproducibility of the microbiotests was compared with the conventional short-term tests on Daphnia magna and a fish species. Since the loss of even moderately volatile organic compounds from the original microbiotest containers appeared to be considerable, it could therefore result in a serious underestimation of toxicity when used in monitoring programmes. Modification of the test containers of the Thamnotox F and Daphnia IQ tests resulted in an improvement of the test performance. Along with the Microtox test, they form a good base for a test battery. The Rotox F test in its present form, appeared to be less useful because of its low sensitivity. At present, no suitable algal microbiotest is operational. Because of their ecological function as primary producers, algae need to be included in the test battery.

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H. Okamura, R. Luo, I. Aoyama, D. Liu and G. Persoone
Ecotoxicity Assessment of Landfill Leachates using a Battery of Bioassay and Toxicity Characterization of the Leachates
(in Japanese - with English Abstract) Japanese Journal of Environmental Toxicology 1998 1 43-50
Toxkits : Daphtoxkit F^TM magna, Daphtoxkit F^TM pulex; Thamnotoxkit F^TM
Test Species : Daphnia magna, Daphnia pulex; Thamnocephalus platyurus
Topics : solid waste leachates; soil leachates, landfill leachates

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Abstract : The western part of Teshima Island in the Seto Inland Sea (Japan) had been used as an illegal dumping ground for more than 10 years and the accumulated waste was estimated to be about 510.000 tons. There has been no systematic toxicity assessment conducted on this waste. In this study, a battery of bioassays was applied to the collected leachates. Toxicity of the leachates was evaluated using the Microtox test, umu test (a mutagenicity assay), yeast inhibition zone test, acute lethality tests using crustaceans, and root elongation test using lettuce. The landfill leachates collected at 4 sites on November 7, 1996 were filtered through 0.45 and 0.2 µm filters sequentially. All samples were highly toxic to crustaceans and lettuce, but showed a weak or no toxicity to three kinds of test microorganisms.All samples were also found to have no mutagenic activity, but to have high concentrations of TOC, TN, NH4, Cl, SO4 and low levels of heavy metals. Four leachates retained their toxicity to lettuce, despite a 6-months storage at 4° C.
These samples were subject to further toxicity characterization. The results indicated that the toxicity of sample No. 1 could be reduced by aeration, solid phase extraction, autoclaving, and UV-C irradiation. This implies the causative toxicants being some volatile, heat- and photo-degradable nonpolar organics. The toxicity reduction in samples No. 2 and 3 could be only achieved through solid phase extraction. Our study has clearly showed that a systematic identification of toxicants in all leachates samples is needed in the management of toxicity reduction in landfill leachates.

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A. Kungolos, P. Samaras, V. Kimerogly, X. Dabou and G.P. Sakellaropoulos
Water Quality and Toxicity Assessment in Koronia Lake - Greece
Fresenius Envir. Bull. 1998 7 615-622
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : surface waters

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Abstract : Lake Koronia, located in Northern Greece, is an environmentally sensitive area protected by Ramsar Convention. However, COD values higher that 900 mg/l and nitrate-nitrogen concentration higher than 10 mg/l were measured in the lake water. Furthermore, toxicity bioassays based on Daphnia magna, resulted in mortality values of test species higher than 40% in the lake water. The poor water quality level has been attributed to the discharge of industrial and domestic wastewater into the lake and to the negative water balance due to intense water pumping for various uses.

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P. Samaras, G.P. Sakellaropoulos, A. Kungolos and S. Dermissi
Toxicity Assessment Assays in Greece
Fresenius Envir. Bull. 1998 7 623-630
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna
Test Species : Artemia (salina) franciscana; Daphnia magna
Topics : surface waters; waste waters

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Abstract : In this study the results of toxicity assessment assays in the area of Thessaloniki in Greece are presented. Daphnia magna tests were used for the assessment of freshwater and wastewater and Artemia salina tests for the assessment of seawater. There was increased toxicity in the seawater of inner Thermaikos gulf as well as in the freshwater of Koronia lake. Measures, taken to prevent toxic shock of the activated sludge microorganisms of Thessaloniki municipal wastewater treatment plant, are also presented in this study.

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A. Kungolos, P. Samaras, M. Kipopoulou, A. Zoumboulis and G.P. Sakallerapoulos
Interactive Toxic Effects of Agrochemicals on Aquatic Organisms
Water Science and Techology 1999 Vol. 40, No. 1 357-364
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - pesticides

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Abstract : The effects of three common agrochemicals, lindane, methyl parathion and atrazine, on crustacean Daphnia magna, alga Selenastrum capricornutum and marine bacterium Vibrio fischeri were investigated in this study. Methyl parathion was the most toxic compound towards all three organisms, while lindane was more toxic to Daphnia magna and Vibrio fischeri than atrazine, and atrazine was more toxic to Selenastrum capricornutum than lindane. Among the three aquatic organisms, Selenastrum capricornutum was most sensitive in detecting lindane and atrazine toxicity, while Daphnia magna was most sensitive in detecting methyl parathion toxicity. The interactive effects of the pesticides were also investigated. The interactive effect between lindane and methyl parathion on survival of Daphnia magna was synergetic, while the ones between lindane and atrazine and between methyl parathion and atrazine were generally additive. The interactive effect of the three pesticides on the growth of Selenastrum capricornutum was antagonistic with few cases of addition, while the effect of all the three pairs of pesticides on algal growth was also antagonistic. The interactive effect of lindane and methyl parathion on Vibrio fischeri was additive.

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G.A. Burton Jr., R. Baudo, M. Beltrami and C. Rowland
Assessing Sediment Contamination using Six Toxicity Assays
Journal of Limnology 2001 60 (2) 263-267
Toxkits : Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : sediments; sediment pore waters

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Abstract : An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment detphs. Lake Orta received excessive loadings of copper and ammonia during the 1900\'s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists in sediments at a depth of 5 cm and greater. Assays that detected the highest levels of toxicity were two whole sediment exposures (7 d) using Hyalella azteca and Ceriodaphnia dubia. The Microtox® assay using pore water was the third most sensitive assay. The Thamnotoxkit, Rotoxkit, Microtox solid phase, and Seed Germination-Root Elongation (pore and solid phase) assays showed occasional to no toxicity. Based on similarity of responses and assay sensitivity, the two most useful assays were the C. dubia (or H. azteca and Microtox pore water. These assays were effective at describing sediment toxicity in a weight-of-evidence approach.

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P.A. Van Sprang and C.R. Janssen
Identification and Confirmation of Ammonia Toxicity in Contaminated Sediments using a Modified Toxicity Identification Evaluation Approach
Environmental Toxicology and Chemistry 1997 Vol. 16, No. 12 2501-2507
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : sediments; chemicals - inorganic compounds; sediment pore waters; Toxicity Identification Evaluation (T.I.E.)

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Abstract : Toxicity identification of sediment pore waters from four sites in the upper Scheldt (Belgium) was assessed using a simplified and discriminative toxicity identification evaluation procedure. The samples from all locations exhibited acute toxicity toward the freshwater crustacean Thamnocephalus platyurus. Toxicity was removed or considerably reduced by the cation exchange resins and air stripping at pH 11. In addition, the toxicity of the pore waters was found to be highly pH dependent. Increased toxicity was observed at higher pH levels, whereas reduced toxicity was found at lower pH levels. Based on these results, ammonia was suggested as the main toxic agent. The presence of ammonia concentrations exceeding the 24 h median lethal concentration and comparison of the toxicity characterization profiles of the pore waters with those of the suspected toxicant supported this hypothesis. Furhtermore, a significant positive correlation between the observed toxicity of the pore waters and the expected toxicity (due to the presence of the suspected toxicant) confirmed ammonia as the true toxic agent. Finally, the ratio between the expected ammonia toxicity and the observed toxicity from the characterization tests was approx. 1, meaning that all or most of the observed toxicity was caused by the presence of one toxicant (i.e., ammonia). The developed toxicity identification evaluation procedure is suggested as a useful tool for the identification and confirmation of toxicants in contaminated sediments.

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C. Van der Wielen, G. Persoone, M.P. Goyvaerts and B. Neven
Toxicity of the effluents of Three Pharmaceutical Companies as Assessed with a Battery of Tests
Tribune de l'Eau 1993 July-August, N° 564/4 19-29
Toxkits : Rotoxkit F^TM; Streptoxkit F^TM
Test Species : Brachionus calyciflorus; Streptocephalus proboscideus
Topics : effluents

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Abstract : The toxicity of 27 samples of effluents of three pharmaceutical plants located in Belgium, has been determined with the aid of a battery of tests. Besides conventional bioassays representative for three different trophic levels (microalgae with Selenastrum capricornutum, acute crustacean test with Daphnia magna, acute fish test with Brachydanio rerio), cost-effective acute bioassays have been carried out with bacteria (TTC test and Microtox® test), a rotifer (Rotoxkit F test) and a crustacean (Streptoxkit F test). Chemical analysis of the effluents has been performed to quantify the toxic parameters considered by the Belgian legislation on industrial discharges.The toxicity of the effluents (expressed in toxic units - TU) ranged from 0 to over 1850, depending on the sample and the type of bioassay. No correlation could be established between the chemical composition of the wastes and their toxicity. Each of the three pharmaceutical plants had an own toxicity spectrum, reflecting the different types of chemicals produced as well as the different treatment procedures of the effluents. The information generated once again confirms that in addition to chemical monitoring toxicity testing is essential to control complex effluents. A battery of selected tests should be the backbone of every programme of toxicity reduction. A case-to-case determination of the toxicity spectrum of pharmaceutical effluents should allow to select the suite of tests which is most suited for the control of specific discharges. The present study also confirms the potential of cost-effective microbiotests for routine monitoring of industrial wastes.

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S. Hadjispyrou, A. Kungolos and A. Anagnostopoulos
Toxicity, Bioaccumulation and Interactive Effects of Organotin, Cadmium and Chromium on Artemia franciscana
Ecotoxicology and Environmental Safety 2001 49
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : The effects on three organotin compounds - trimethyltin chloride, dimethyltin dichloride and dibutyltin diacetate - and two heavy metals - cadmium and hexavalent chromium - on Artemia franciscana mortality are investigated in this study. Of all the compounds tested in this work, trimethyltin chloride was, by far, the most toxic. The toxicity order for the five compounds was trimethyltin chloride > potassium dichromate > dimethyltin dichloride > dibutyltin diacetate > cadmium chloride. The big difference in toxicity between dialkyltin and trialkyltin was not accompanied by an equally big difference in bioaccumulation. At a Sn concentration in water of 10 mg/L, the bioconcentration factor was 50 for dimethyltin dichloride and 75 for trimethyltin chloride. At a Sn concentration in water of 100 mg/L, the bioconcentration factor was 6 for dimethyltin dichloride and 9 for trimethyltin chloride. The interactive effect of trimethyltin chloride and cadmium, as well as that of trimethyltin chloride and chromium, was found to be synergistic. Also found to be synergistic was the interactive effect of trimethyltin chloride with cadmium and chromium applied together.

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A.K. Törökne, E. Laszlo, I. Chorus, J. Fastner, R. Heinze, J. Padisak and F.A.R. Barbosa
Water Quality Monitoring by Thamnotoxkit F^TM including Cyanobacterial Blooms
Water Science and Technology 2000 42, 1-2 381-385
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : biotoxins; cyanobacterial blooms; cyanotoxins

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Abstract : The Thamnotoxkit F^TM was evaluated for detecting cyanobacterial toxins as they may be hazardous to human health if they reach drinking water networks or if people are exposed through recreational activity.
This test kit is a 24-hour biosassay using larvae of the freshwater anostracan crustacean Thamnocephalus platyurus hatched from cysts. Nine freeze-dried Microcystis aeruginosa samples from freshwaters of Hungary, Germany and Brazil were tested with the Thamnotox test, rat hepatocyte test, mouse test and analysed for microcystins by high performance liquid chromatography (HPLC). It can be concluded that the Thamnotox test is an alternative simple, cost-effective method that may replace the mouse bioassay used previously for determination of cyanobacterial toxicity.

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A.K. Törökne
A new Culture-Free Microbiotest for Routine Detection of Cyanobacterial Toxins
Environmental Toxicology 1999 14, 5 466-472
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : biotoxins; cyanobacterial blooms; interlaboratory testing; cyanotoxins

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Abstract : The Thamnotoxkit F^TM was evaluated for detecting cyanobacterial toxins. This is a 24-hour bioassay using as test organisms larvae of the freshwater anostracan crustacean Thamnocephalus platyurus hatched from cysts. Freeze-dried Microcystis aeruginosa samples from freshwaters throughout Hungary were tested. Good correlation was obtained between the results of intraperitoneal mouse bioassays and Thamnotoxkit F^TM assays. The LC50’s for purified microcystins- LR, -RR and -YR ranged from 0.1 to 2.27 µg per mL respectively in the Thamnotoxkit assay. The LC50 of laboratory cultures of Cylindrospermopsis raciborskii isolated from samples from lake Balaton, was 0.574 mg lyophilized material per mL, in a four-laboratory trial. Altough not developed originally for detecting cyanobacterial toxkins, the Thamnotoxkit F^TM has good potential as an initial screening procedure for toxicity assessment of cyanobacterial scums and blooms in freshwaters.

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P. Fochtman, A. Raszka and E. Nierzedska
The use of Conventional Bioassays, Microbiotests and Some Rapid Methods in the Selection of an optimal Test Battery for the Assessment of Pesticides Toxicity
Environmental Toxicology 2000 15, 2 376-384
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - pesticides; test selection

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Abstract : Due to the increasing use of pesticides, as well as the introduction of new formulations of biologically active substances, there are significant effects of these preparations on the aquatic environment. The aim of this study was to select effective biotests for a test battery to assess the potential effects of pesticides on aquatic life. Thirteen widely used and registered pesticides in Poland, with potassium dichromate as a reference chemical, as well as seven different test species (ranging from protozoan to fish) were chosen for the study. Principal component analysis (PCA) and cluster analysis (CA) were used as helpful statistical methods for the selection of the test battery. PCA clearly isolated two components that explained almost 82 % of total variance. The first consists of all crustacean and fish tests while the second of all tests with algae and the protozoan Tetrahymena thermophila. In other words, PCA separated unicellular and multicellular organisms in regard to their sensitivity to pesticides. CA has confirmed the above results and has provided more detailed insight into the connections within the separated group of biotests. Regression analysis, PCA and CA allowed for the selection of the following test battery : Algaltoxkit F^TM (Raphidocelis subcapitata), Protoxkit F^TM (Tetrahymena thermophila), Daphnia magna (Fluotox) and an acute test with Oncorhynchus mykiss or, alternatively Thamnotoxkit F^TM (Thamnocephalus platyurus). This test battery is comprised of organisms that belong to four different phylogenic groups as well as three trophic levels of the aquatic ecosystem. Acute, sublethal, and chronic effects are taken into consideration.

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I. Blinova
The Perspective of Microbiotests Application of Surface Water Monitoring and Effluent Control in Estonia
Environmental Toxicology 2000 15, 5 385-389
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : surface waters; toxicity monitoring; effluents; test selection

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Abstract : In Estonia, traditional chemical monitoring of surface water and effluents is performed but bioassays are not used as monitoring tools. Furthermore, the expediency of application of toxicity tests to surface water monitoring is questionable. The main aim of this study was to test the feasibility of using microbiotests in the biomonitoring of surface waters and effluents. The sensitivity of five test species was also evaluated during the study. Water samples from rivers and wastewater treatment plants were collected over a period of 3 years and tested using bioassays with Selenastrum capricornutum, Thamnocephalus platyurus, Daphnia magna, Tetrahymena thermophila and Lemna minor. The species used in the battery of tests showed different sensitivities for a specific sample. This confirms the need for a battery of bioassays for the assessment of possible ecological consequences of wastewater discharge. The studied test species are sensitive enough to indicate negative changes in surface water quality, which can affect the aquatic biota. The battery of bioassays consisting of T. platyurus, L. minor, S. capricornutum and D. magna can be recommended for control of waste water quality and assessment of a potential risk for aquatic biota. The results of tests with plant species may give additional important information on the possible effect of effluent on the eutrophication process in receiving waters. From the results obtained during the present study it can be recommended that toxicity tests be included in the effluent and surface water monitoring programme in Estonia.

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T. Pereira, M.J. Cerejeira and J. Espirito-Santo
Use of Microbiotests to Compare the Toxicity of Water Samples Fortified with Active Ingredients and Formulated Pesticides
Environmental Toxicology 2000 15, 5 401-405
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - pesticides; chemicals - organic compounds; surface waters

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Abstract : In Portugal rice occupies a relevant position among other irrigated crops and, since treated water is frequently discharged to the surrounding water bodies, field studies to determine the influence of pesticides used in rice treatments to aquatic biota are being performed. In order to better understand the toxicity results obtained in field conditions, particularly the influence of the pesticides formulations in the toxic effecs, laboratory studies were performed using water samples fortified with the active ingredient and the respective formulated product (commercial product) of some pesticides (molinate, MCPA, propanil, endosulfan, chlorfenvinphos) usually applied in Portuguese paddies. The range of concentrations tested reflects the rates used by the farmers in real field conditions. The toxicity values observed in the laboratory during 1998 with the microbiotests Daphtoxkit F^TM magna , Thamnotoxkit F^TM, Artoxkit M^TM and Algaltoxkit F^TM are presented and discussed in this study. The results obtained until now suggests that organic solvents and surfactants present in some of the tested formulations may affect the toxicity of the sample. In fact, water samples fortified with formulated pesticides seemed to be, in the majority of the cases, more toxic than the respective active ingredients solutions, particularly in the case of the three tested crustaceans. The microbiotests used seem to be a useful tool to evaluate toxicity with relative low cost, rapidity and simplicity, compared with the traditional tests.

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A.K. Törökne, E. Laszlo, I. Chorus, J. Fastner, R. Heinze, J. Padisak and F.A.R. Barbosa
Cyanobacterial Toxins Detected by Thamnotoxkit (a Double Blind Experiment)
Environmental Toxicology 2000 15, 5 549-553
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : biotoxins; cyanobacterial blooms; cyanotoxins

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Abstract : The Thamnotoxkit F^TM was evaluated for detecting cyanobacterial toxins compared with the rat hepatocyte test and mouse test. This test kit is a 24-hour biosassay using larvae of the freshwater anostracan crustacean Thamnocephalus platyurus hatched from cysts. Thirteen freeze-dried cyanobacterial samples from freshwaters of Hungary, Germany and Brazil, and strains from the culture collection of the University of Helsinki were tested and analyzed for microcystins and anatoxin a by high performance liquid chromatography and in some cases, the enzyme-linked immunosorbent assay test. The samples were examined by a double blind approach. The Thamnotoxkit test reacts to pure microcystins, neurotoxins and crude extracts of cyanobacteria. It can be concluded that the Thamnotox test is an alternative simple, cost-effective method that may replace the mouse bioassay used previously for determination of cyanobacterial toxicity.

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M.N. Reskone and A.K. Törökne
Toxic Microcystis aeruginosa in Lake Velencei
Environmental Toxicology 2000 15, 5 554-557
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : biotoxins; cyanobacterial blooms; cyanotoxins

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Abstract : Lake Velencei is a shallow lake in Hungary. The first cyanobacterial bloom of Microcystis aeruginosa appeared in the lake in 1991. This cyanobacterial species was also found earlier but blooms were not recorded. The toxins of M. aeruginosa were studied in Lake Velencei in 1992. The present study deals with the toxicity of M. aeruginosa biomass of different origin detected by intraperitoneal (ip.) mouse assay, Thamnotoxkit F^TM microbiotest, and toxin analysis by high-performance liquid chromatogramy (HPLC). The biomass was collected by plankton net and the isolated strains were cultured from sediments and from bloom material. Minimal lethal doses (MLD) by ip. mouse assay ranged from 20 to 50 mg/kg ^-1. Comparing the results from 1992 and 1997-1998, no change in the toxicity of M. aeruginosa was detected. According to the HPLC analysis the combination of microcystins varied in a narrow range with microcystin-LR found in each sample. The cyanobacterial blooms caused by M. aeruginosa in Lake Velencei considerably decreased, though it remained in the sediment with the same toxicity. The Thamnotoxkit F^TM microbiotest was an alternative method for detecting the cyanotoxins.

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S. Galassi and V. Croce
Daphtoxkit F^TM magna Acute Test in Waste Water Monitoring
Biologia Ambientale 2000 14 (2) 21-28
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : waste waters; toxicity monitoring; effluents

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Abstract : Toxicity of a textile effluent and of its fractions, obtained by crhomatographic techniques, has been evaluated by Daphnia magna acute test. For the bioassays a commercial kit using Daphnia magna resting eggs was adopted after comparison of its sensitivity with the traditional test on parthenogenetic daphnids. Most toxicity was due to xenobiotics, recovered in the most hydrophobic HPLC fraction. The compound responsible of the observed toxicity was identified and quantified by GC-MS. The adopted procedures, notwithstanding their complexity, offer new useful tools to address effluent treatments and to replace dangerous chemicals in production cycles.

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B.Z. Chial and G. Persoone
Cyst-Based Toxicity Tests. - XI. Influence of the Type of Food on the Intrinsic Growth Rate of the Rotifer Brachionus calyciflorus in Short Chronic Toxicity Tests
Chemosphere 2002 50 (3) 365-372
Toxkits : Rotoxkit F^TM short-chronic
Test Species : Brachionus calyciflorus
Topics : test development

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Abstract : As important members of zooplankton communities worldwide, rotifers are used extensively in ecotoxicological research. Chronic rotifer tests are, however, dependent on live algal food which adds to the complexity, the variability and the costs of these bioassays.
To bypass the former problem, experiments have been undertaken with the freshwater rotifer Brachionus calyciflorus, to determine their intrinsic growth rate (r) when fed for 48h on a mixture of green algae (Raphidocelis subcapitata) obtained from algal beads stored for different periods of time, and other inert foods. All tests have been performed in disposable multiwells, in 1 ml cups each inoculated with 1 rotifer freshly hatched from dried cysts. The majority of the growth tests was performed in 8 duplicates.
The investigations revealed that micro-algae from algal beads stored for up to one year, in darkness, at 4°C, supplemented with dried blue-green algae (Spirulina) gave satisfactory rotifer reproduction. The intrinsic growth rates of the rotifers, were, however, dependent on the storage time of the algal beads; the highests r’s (0.7-0.8) were obtained with algae from beads not older than 4 months. Growth tests with combinations of R.subcapitata and other inert feeds revealed that the enrichment food Selco used in aquaculture, also gave the same reproductive output as the combination micro-algae/Spirulina.
A rotifer growth experiment with 18 replicates showed that the variation coefficient is below 20% when the tests comprise 8 parallels.
This study demonstrated that micro-algae from beads, supplemented by other inert food, open the door for a practical and cost-effective short-chronic rotifer test, which is totally independent of the culturing of both the test species and its live food.

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B.Z. Chial and G. Persoone
Cyst-Based Toxicity Tests. - XII. Development Of A Short Chronic Sediment Toxicity Test With The Ostracod Crustacean Heterocypris incongruens. Selection Of Test Parameters
Environmental Toxicology 2002 17 (6) 520-527
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : test development; sediments; sediment and soil contact tests; solid phase test

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Abstract : Experiments have been carried out with neonates of the freshwater ostracod Heterocypris incongruens hatched from cysts, to develop a new culture/maintenance free solid phase microbiotest for toxicity assessment of contaminated sediments.
Based on preliminary investigations, a number of test parameters were investigated for a short-chronic assay : the hatching time, the size of the test cups, the feeding of the test organisms prior to the test, the amount of supplemental algal food, the volume of sediment and the duration of the test. On the basis of the findings, a test protocol was formulated for a 6 days assay in 12 cup multiwell plates with 10 organisms per cup and 3 replicates. The test organisms are collected 52h after the start of the incubation of the cysts in moderately hard water (EPA formula) at 25° C under continuous illumination after 4h pre-feeding with 1.3 mg/l Spirulina. The test biota in the cups are exposed to 300 µL test sediment in 2 mL EPA water, with 3.107 live algal cells (Raphidocelis subcapitata) as food supplement. Calibrated sand is used as reference sediment. Mortality and growth of the ostracods are determined after 6 days incubation at 25°C in darkness. The selected test parameters for the new microbiotest were found adequate for toxicity determination of natural sediments, in comparison to the 10 days contact test with the amphipod Hyalella azteca.

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B.Z. Chial and G. Persoone
Cyst-Based Toxicity Tests. - XIII. Development Of A Short Chronic Sediment Toxicity Test With The Ostracod Crustacean Heterocypris incongruens. Methodology And Precision
Environmental Toxicology 2002 17 (6) 528-532
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : test development; sediments; sediment and soil contact tests; solid phase test

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Abstract : Experiments have been carried out with neonates of the freshwater ostracod Heterocypris incongruens to verify and complete former choices of test parameters for a new culture/maintenance free solid phase microbiotest for freshwater sediments. From the trials with increasing volumes of reference sediment, it was concluded that 300 µL was the most appropriate amount of sediment to be put in 12 cup multiwells with 2 ml moderately hard EPA water. Tests in 3 to 9 replicates eventually showed that 6 parallels are needed for a good repeatability (precision) of the assays. Application of the final test protocol to oil-contaminated sediments from the St Lawrence river in Canada revealed that the 6 days chronic ostracod microbiotest gave less variation in repeated tests than the 10 days contact assay with Hyalella azteca and can hence be considered as more precise. From the 95% confidence intervals for mortality and growth of the ostracods calculated for the controls in reference sediment carried out for the 56 tests of the Canadian project, a validity threshold of 20% for mortality was eventually selected, in analogy to the acceptability limits applied in many chronic bioassays. A minimum length of 600 µ in the control sediment after 6 days exposure was also taken as the threshold for good health of the test organisms and reliable test conditions. The new microbiotest has been tailored in a handy and user-friendly new Toxkit : the Ostracodtoxkit, which is particularly suited for cost-effective routine monitoring.

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B.Z. Chial and G. Persoone
Cyst-Based Toxicity Tests. - XIV Application Of The Ostracod Solid Phase Microbiotest For Toxicity Monitoring Of River Sediments In Flanders (Belgium)
Environmental Toxicology 2002 17 (6) 533-537
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments; sediment and soil contact tests; solid phase test; toxicity monitoring

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Abstract : In view of the need for rapid and low cost solid phase assays for contaminated sediments, a new 6 days “culture/maintenance free” microbiotest has been developed at the Laboratory for Biological Research in Aquatic Pollution at the Ghent University in Belgium, with the freshwater ostracod crustacean Heterocypris incongruens. The sensitivity of the Ostracodtoxkit F^TM has been compared with that of the 10 days sediment contact test with the amphipod crustacean Hyalella azteca and the 2 pore water assays (on the anostracan crustacean Thamnocephalus platyurus and the micro-algae Raphidocelis subcapitata) selected by the Flemish Environmental Agency for initial hazard assessment of contaminated sediments. The exercise was performed on 26 sediments collected at random from various rivers of the Flemish hydrographic basin in Belgium. The toxicity data ranged from non-toxic to highly toxic for the solid phase assays and showed that the mortality of the 2 test biota varied from “nearly identical” to “substantially different” depending of the sediment. The correlation coefficient for the data pairs was but weakly positive (r = 0.60) which is mainly due to 5 samples for which a low mortality had been noted with the ostracod, versus a much higher for the amphipod. For 4 out of the 5 concerned samples, a substantial growth inhibition had, however, been recorded for H.incongruens, showing that this assay was eventually as performing as the amphipod test in detecting the toxic character of the sediments. The comparison of the toxicity data of the 2 solid phase assays with those of the 2 pore water tests again confirmed that both types of bioassays give complementary (non-redundant) information. On the basis of its various advantages (independence of stock-culturing/maintenance, user-friendliness, precision, time and cost saving) the new ostracod microbiotest appears to be a promising new tool for routine toxicity assessment of contaminated sediments.

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M.W. Toussaint, T.R. Shedd, W.H. Van der Schalie and G.R. Leather
A Comparison Of Standard Acute Toxicity Tests With Rapid-Screening Toxicity Tests
Environmental Toxicology and Chemistry 1995 Vol. 14, No. 5 907-915
Toxkits : Rotoxkit F^TM; Artoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Daphnia magna; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - inorganic compounds; chemicals - organic compounds; chemicals - metals; chemicals - pesticides; sensitivity comparisons

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Abstract : This study compared the relative sensitivity of five inexpensive, rapid toxicity tests to the sensitivity of five standard aquatic acute toxicity tests through literature review and testing. The rapid toxicity tests utilized organisms that require little culturing or handling prior to testing : a freshwater rotifer (Brachnionus calyciflorus); brine shrimp (Artemia salina); lettuce (Lactuca sativa); and two microbial tests (Photobacterium phosphoreum - Microtox® test, and a mixture of bacterial species - the Polytox® test). Standard acute toxicity tests species included water fleas (Daphnia magna and Ceriodaphnia dubia), green algae (Selenastrum capricornutum, fathead minnows (Pimephales promelas), and mysid shrimp (Mysidopsis bahia). Sensitivity comparisons between rapid and standard acute toxicity tests were based on LC50/EC50 data from 11 test chemicals. Individually the lettuce and rotifer tests ranked most similar in sensitivity to the standard tests, while Microtox fell just outside the range of sensitivities represented by the group of standard acute toxicity tests. The brine shrimp and Polytox tests were one or more orders of magnitude different from the standard acute toxicity tests for most compounds. The lettuce, rotifer, and Microtox tests could be used as a battery for preliminary toxicity screening of chemicals. Further evaluation of complex real-world environmental samples is recommended.

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G. Persoone and B.Z. Chial
Low-Cost Microbiotests For Toxicity Monitoring During Bioremediation Of Contaminated Soils
The Utilization of Bioremediation to Reduce Soil Contamination : Problems and Solutions (V. Sasek et al, eds) - Kluwer Academic Publishers) 2003 155-163
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Ceriodaphtoxkit F^TM; Daphtoxkit F^TM magna and pulex; Protoxkit F^TM; Ostracodtoxkit F^TM; Rotoxkit F^TM acute; Rotoxkit F^TM short-chronic; Rotoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Ceriodaphnia dubia; Daphnia magna; Daphnia pulex; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : soils, solid phase test, toxicity monitoring, review

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Abstract : This paper addresses in the first place the need to apply toxicity tests with batteries of species from different trophic levels for an (ecologically meaningful) determination of the toxic hazard of contaminated soils. Furthermore, \'direct contact\' tests must be performed as well as assays on pore waters, leachates or percolates.Most toxicity tests available to date are dependent on the continuous maintenance of live stocks and their application is restricted to a limited number of highly specialized laboratories. Repeated toxicity testing in bioremediation programs is very expensive and there is an urgent need for cost-effective alternatives. Microbiotests with micro-algae, protozoans and various invertebrate test species have been developed over the last few years, which depart from dormant or immobilized stages of the test biota and are hence independent of the burden of continuous stock culturing/maintenance.
The sensivity of these new assays (named Toxkits) has been compared to that of \'conventional\' aquatic and direct-contact tests in a variety of studies on pure chemicals as well as on natural samples, and found to be equivalent, if not better.
Recently a direct contact microbiotest, the Ostracodtoxkit, was developed for sediments, which now also seems to be applicable to contaminated soils. Besides numerous applications in aquatic toxicology, the low-cost and user-friendly Toxkit microbiotests now also appear to be an attractive tool for routine toxicity monitoring and/or follow-up of detoxication in bioremediation programs of contaminated soils.

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A.P. Loibner, R. Braun, V. Boller and O.H.J. Szolar
Evaluation Of Toxic Effects Exhibited By Organic Pollutants Present In Soil
In : The Utilization of Bioremediation to Reduce Soil Contamination : Problems and Solutions (V. Sasek et al, eds) - Kluwer Academic Publishers) 2003 165-175
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Thamnocephalus platyurus
Topics : review, soils, chemicals - organic compounds

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Abstract : Bioassays are widely applied to measure toxic effects of aquatic samples such as surface water and groundwater, leachates and elutriates, as well as sediments, sludges, and soils. Contrary to (eco)toxicity tests, which translate single chemical concentrations into toxic effects (dose-response relationship giving NOEC, EC50, etc.), bioassays are applied to samples from contaminated sites without detailed knowledge of qualitative and quantitative extent of pollution. Depending on the type of test, bioassays may deliver fast and relevant information on adverse effects that contaminated samples pose to certain test organisms. Currently, bioassays are used to prioritise contaminated sites and to establish site specific remediation goals. However, as results cannot be linked to concentrations of single pollutants, the calculation of toxicity data (LC50, NOEC, etc.) for particular contaminants is not possible but needed for risk characterisation (linking exposure to effects). It might be appropriate, to calculate for instance an LC50 for an effluent of an industrial plant which emits into a river. Based on flow rates, the exposure for organisms present in the river can be modelled and the risk may be calculated. However, further research is necessary to develop similar applications for contaminated soil. Moreover, at most sites, pollutants are heterogeneously distributed in soil. Therefore, an increased number of samples has to be taken to meet statistical needs. This may increase costs for site characterisation dramatically, in particular, when applying test batteries for each sample. Nevertheless, when selecting appropriate test-organisms, bioassays provide useful data to screen for hazards present and to establish a priority ranking for contaminated sites.

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L. Pollumaa, A. Kahru, M. Trapido and A. Maloveryan
Ecotoxicological Hazard Assessment of Solid-Phase Samples
In : The Utilization of Bioremediation to Reduce Soil Contamination : Problems and Solutions (V. Sasek et al, eds) - Kluwer Academic Publishers) 2003 217-220
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM; Rotoxkit F^TM short-chronic; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : sediments; soil leachates; solid wastes

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Abstract : 14 samples originating from Estonia, collected from the oil-shale region (7), oil-polluted sites (4), presumably non-polluted areas (2) and 1 sediment sample from the harbor were studied for the toxicity and presence of the priority pollutants. The soil aqueous extracts were analyzed with a test battery of organisms of different trophic levels (photobacteria, crustacea, algae and protozoa). Particle-bound toxicity was evaluated using photobacterial Flash-Assay. The microbiological activity of the soils was evaluated by the total number of heterotrophic bacteria, dehydrogenase activity and ATP content in the soils.
The soils were extracted for 24 h (1 part of soil+3 parts of water) and extract/slurry exposed at 50% to the test organisms. The extract/slurry was considered toxic when its L(E)C20 <12.5% at least in one test in the battery (altogether 7 tests). Concentrations of heavy metals, oil products and PAHs in the soils were analyzed and compared to the Estonian permitted limit values. In general there was a correlation between the level of the pollutants and toxicity of the soils. The control soils chosen (one from the oil-shale region) proved clean both, chemically and toxicologically. Within 10 toxic samples in 2 soils the concentration of heavy metals/oil products/PAHs did not exceed the permitted level for the usage in the living zone.
The Flash-Assay indicated the presence of particle-bound highly adsorbed toxicants in 4 soil samples (all from the oil-shale region). For two of these soils the solid-phase Flash-Assay was the only test in the battery indicating the toxicity.
The test battery applied was sensitive, contained organisms of different trophic levels and yielded additional information (to the chemical data) on the toxic hazard of the polluted soils. The comparison of the toxicity of the soil slurries and extracts enabled to distinguish between the particle-bound and water-extractable toxicities that is important when the different exposure routes are assessed.

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A. Kahru, R. Reiman and A. Ratsep
The Efficiency Of Different Phenol-Degrading Bacteria And Activated Sludges In Detoxification Of Phenolic Leachates
Chemosphere 1998 37 301-318
Toxkits : Daphtoxkit F^TM pulex; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia pulex
Topics : solid waste leachates

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Abstract : Phenolic composition, toxicity and biodegradability of three different phenolic leachates/samples was studied. Samples A and C were the leachates from the oil-shale industry spent shale dumps at Kohtla-Järve, Estonia. Sample B was a laboratory-prepared synthetic mixture of 7 phenolic compounds mimicking the phenolic composition of the leachate A. Toxicity of these 3 samples was analyzed using two photobacterial test (BioTox™ and Microtox™), Daphnia test (Daphtoxkit F™ pulex) and rotifers’ test (Rotoxkit F™). All the LC50 values were in the range of 1-10%, leachate A being the most toxic.
The growth and detoxifying potential (toxicity of the growth medium was measured using photobacterial tests) of 3 different phenol-utilizing bacteria and acclimated activated sludges was studied in shake-flask cultures. 30% leachate A (altogether 0.6 mM total phenolic compounds) was too toxic to rhodococci and they did not grow. Cell number of Kurthia sp. and Pseudomonas sp. in 30% leachate A increased by 2 orders of magnitude but despite of the growth of bacteria the toxicity of the leachate did not decrease even by 7 weeks of cultivation. However, if the activated sludge was used instead of pure bacterial cultures the toxicity of the 30% leachate A was eliminated already after 3 days of incubation. 30% samples B and C were detoxified by activated sludge even more rapidly, within 2 days. As the biodegradable part of samples A and B should be identical, the detoxification of leachate A compared to that of sample B was most probably inhibited by inorganic (e.g., sulphuric) compounds present in the leachate A. Also, the presence of toxic recalcitrant organic compounds in the leachate A (missed by chemical analysis) that were not readily biodegradable even by activated sludge consortium should not be excluded.

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L. Pollumaa, A. Kahru, A. Eisentrager, R. Reiman, A. Maloveryan and A.Ratsep
Toxicological Investigation of Soil with the Solid-Phase Flash Assay : Comparison with other Ecotoxicological Tests
Alternatives to Laboratory Animals 2000 28 461-472
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : soils, soil leachates

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Abstract : In this work a new direct contact toxicity test solid-phase flash assay which utilises photobacteria in direct contact with soil particles during the exposure, was evaluated on 4 soil samples. Samples HTNT1 and HTNT2 originated from former military sites in Germany, and were highly contaminated with nitroaromatics (approximately 20 g/kg), lead and polycyclic aromatic hydrocarbons. Samples LMKW1 and LMKW2 were taken from the bioremediation stacks in Germany and were contaminated with mineral oils. The solid-phase flash assay was applied to soil-water slurries and the results were compared with the toxicity data for soil-water extracts obtained by using various conventional ecotoxicological tests, in which photobacteria, crustaceans, protozoa and algae were used as test organisms. The LMKW1 and LMKW2 samples were not toxic (EC20 > 12.5%) according to all the tests applied, except for the Photobacterium phosphoreum conventional luminescence-inhibition test for LMKW1 (15-min EC20=5.4%). The HTNT1 and HTNT2 samples were toxic according to all the tests applied, with the majority of EC20 values being lower than 1%. The solid-phase flash assay (1 minute of extraction and 30 seconds of exposure time) gave comparable results to the conventional tests. Therefore, the solid-phase flash assay could be applied as a fast screening test in parallel with conventional toxicity tests that use soil 24-h extracts. The flash assay results will be ready by the start of the conventional assays and could serve as range-finding for these slower and more expensive tests.

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A. Kahru, L. Pollumaa, R. Reiman, A. Ratsep, M. Liiders and A. Maloveryan
The Toxicity And Biodegradability Of Eight Main Phenolic Compounds Characteristic To The Oil-Shale Industry Wastewaters : A Test Battery Approach
Environmental Toxicology 2000 15(2) 431-442
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - organic compounds; wastewaters

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Abstract : The toxicity of eight phenolic compounds characteristic to the oil-shale industry wastewaters was analyzed using a battery of 7 microbiotests with species representing different trophic levels (photobacteria, protozoa, crustaceans, micro-algae). Altogether 5 monobasic phenolic compounds (phenol, p-cresol, 2,4-dimethylphenol, 2,3- dimethylphenol, 3,4-dimethylphenol) and 3 dibasic phenolic compounds (resorcinol, 5-methylresorcinol and 2,5-dimethylresorcinol) were studied.
Phenol, 2,4-dimethylphenol, 2,3-dimethylphenol and 5-methylresorcinol were classified as “toxic” (lowest L(E)C50 of the battery 1-10 mg/L). P-cresol, 3,4-dimethylphenol, resorcinol and 2,5-dimethylresorcinol were classified as “very toxic” (lowest L(E)C50 of the battery <1 mg/L). The most sensitive test organisms were crustaceans and/or photobacteria. The toxicity of 3 different equitoxic mixtures (mixture of all 8 phenols, mixture of 5 monobasic phenols and mixture of 3 dibasic phenols) was studied using Vibrio fisheri, Daphnia magna and Thamnocephalus platyurus. For all 3 test organisms the toxicities of individual phenols proved additive. The efficiency of biotreatment on the toxicity reduction of the individual phenolic compounds (2.5 mM) as well as the equimolar mixtures of all 8 phenols (1/8*2.5 mM each), 5 monobasic phenols (1/5*2.5 mM each) and 3 dibasic phenols (1/3*2.5 mM each) was studied using acclimated and non-acclimated activated sludges. In the case of both activated sludges phenol, p-cresol, resorcinol and 5-methylresorcinol were most rapidly detoxified. Dimethylphenols and the mixtures had moderate detoxification times, while the slowest was the detoxification of 2,5-dimethylresorcinol and the mixture of resorcinols. Thus, out of eight phenolic compounds studied 2,5-dimethylresorcinol was considered most environmentally hazardous as very toxic (LC50 for crustaceans 1-5 mg/L) and slowly biodegradabable.

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S. Leitao, M. Moreira dos santos, R. Ribeiro, M.J. Cerejeira, P. Van den Brink and J.P. Sousa
Using a Soil-Water Flume on Pesticide Ecological Risk Assessment under Mediterranean Exposure Scenarios
From : SETAC Europe 21st Annual Meeting, Milan - Italy, 15-19 May 2011
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - pesticides

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Abstract : An integrated chemical and biological approach for the quality assessment of freshwater resources in a vineyard area of the ‘Alentejo’ region (South Portugal) is presented. This includes analysis to 11 pesticide compounds and whole toxicity testing on algae and crustaceans. Simazine, terbuthylazine, terbutryn, desethylatrazine and chlorpyrifos were the most frequently detected pesticides in water collected from wells and drainage channels. Mixtures of up to three compounds in different qualitative combinations were also found. The quality standards for individual pesticides (0.1 µg L^-1) and pesticides-total (0.5 µg L^-1) were exceeded in some samples. However, their maximum concentrations were lower than the WHO guidelines, the USEPA health advisory values and the environmental quality standards for priority substances applicable to surface water. In five samples, the herbicides terbuthylazine and terbutryn and the insecticide chlorpyrifos did not pass the toxicity exposure ratio (TER) trigger values specified for aquatic organisms (algae, Daphnia and fish). Maximum toxic effects on Daphnia magna (100%) and Pseudokirchneriella subcapitata (82.56%) were determined in groundwater samples, while in surface water, no toxicity was observed. Concerning effects on Heterocypris incongruens in sediment samples collected at the drainage channels, mortality and growth inhibition values were below 38%. Pro-active management of the use of pesticides is recommended for implementing at the farm and catchment level to reduce inputs into ground- and surface water.
See corresponding Poster

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L. Manusadzianas, L. Balkelyte, K. Sadauskas, I. Blinova, L. Pollumaa and A. Kahru
Ecotoxicological Study Of Lithuanian And Estonian Wastewaters : Selection Of The Biotests, And Correspondence Between Toxicity And Chemical-Based Indices
Aquatic Toxicology 2003 63 27-41
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : waste waters

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Abstract : The toxicity of industrial and urban wastewater (WW) samples collected in Lithuania and Estonia was evaluated by using a suite of biological tests comprising the Algaltoxkit F™ with Selenastrum capricornutum, the Charatox with Nitellopsis obtusa, Daphtoxkit F™ with Daphnia magna, Thamnotoxkit F™ with Thamnocephalus platyurus, Protoxkit F™ with Tetrahymena thermophila and the Microtox® with Vibrio fischeri. The Charatox and Thamnotoxkit F™ tests showed highest relative sensitivity, responding to 80-90 % of samples, respectively, and both expressed good discrimination capacity between samples. Principal Component and pairwise correlation analysis allowed to select a test-battery consisting of Charatox, Thamnotoxkit™ and Microtox®. The wastewater (WW) toxicity was evaluated by means of cumulative indices such as Average Toxicity and two indices derived from the PEEP-index (Environ. Toxicol. Water Qual. 8 (1993) 115). In addition to these integrated evaluations of test-battery response, WW toxicity was evaluated according to the most sensitive test (MST) in the battery. The linear regression analysis between cumulative toxicity indices and chemical-based indices (derived from comparison of WW chemical concentrations and their respective maximum allowable concentration) revealed positive linear relationships (r²=0.7-0.8), while toxicity evaluation based on the MST was less positively related with chemical analysis data (r²=0.5-0.6). Although better coincidence between the toxicity and chemical-based assessments was achieved when information from all tests in the battery was assembled, the prediction of toxicity from chemical data was still limited. In search of a suitable test-battery for the screening of certain types of WWs, a preliminary study comprising an excessive suite of tests might be useful.

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A. Kungolos, P. Samaras, E. Koutseris and G.P. Sakellaropoulos
Using Bioassays For Testing Seawater Quality in Greece
Environmental Coastal Regions III (G.R. Rodriguez, C.A. Brebbia, E. Perez-Martell, Eds. - WIT Press) 2001 401-410
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : seawater; coastal water

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Abstract : The purpose of this study is to show the seawater quality in the Thermaikos Gulf, the Pagassitikos Gulf and Skiathos island in the Northern Aegean Sea. The assessment of coastal water quality presented here is based on two bioassays that use marine organisms as indicators of seawater quality, the invertebrate Artemia fransiscana and the marine bioluminescent bacterium Vibrio fischeri. Bioassays are necessary in water polution evaluations as physical and chemical tests alone are not sufficient to assess potential effects on aquatic organisms. According to our results, there was an improvement in costal water quality in the Termaikos Gulf between September 1997 and April-May 2000. In the Pagassitikos Gulf the coastal water quality was generally good in April-May 2000, while in October 1999 it was generally poor. Between the two bioassays that we used in this study, the Microtox test, which uses the marine bacterium Vibrio fischeri as a test organism, was more sensitive in detecting toxicity in seawater.

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C. Blaise
Microbiotesting : An Expanding Field in Aquatic Toxicology
Ecotoxicology and Environmental Safety 1998 40 115-119
Toxkits :
Test Species :
Topics : review

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Abstract : In terms of bioanalytical development and application, environmental evolution in this century can be perceived as having gone from the \'dark ages\' (time period preceding the 1960s and essentially characterized by nonuse of bioassays), to the \'beginning of enlightenment\' (use of fish bioassays during the 1960s as screening tools for effluents and specific chemicals), to the \'regulatory 1970s\', when newly created environment departments in developed countries began to sanction bioassays for regulatory purposes, to the \'ecotoxicological 1980s\', when suites of micro(bioassays) were incorporated in various hazard assessment schemes, right up to the present \'microbiotesting 1990s\', when an unprecedented upsurge in development and demand for cost-effective multilevel small-scale assays is manifest. This paper offers the view that microscale aquatic toxicology is a rapidly expanding field of ecotoxicology involving numerous bioanalytical techniques developed and applied at various levels of biological organization. This is demonstrated by recalling some of the major highlights that triggered the way for increased use of microbiotesting over the past decades. It is expected that the field of microbiotesting will sustain continuing growth in the future and contribute significant diagnostic power to environmental programs requiring ecotoxicology.

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C. Bernard, C.R. Janssen and A. Le Du-Delepierre
Estimation of the Hazard of Landfills through Toxicity Testing of Leachates
Chemosphere 1997 Vol. 35, No. 11 2783-2796
Toxkits : Ceriodaphtoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Ceriodaphnia dubia; Thamnocephalus platyurus
Topics : soil leachates

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Abstract : Twenty-two landfill leachates were tested on a battery of aquatic organisms (microalgae, daphnids, rotifers, curstaceans, protozoans, luminescent bacteria) and analysed for various physico-chemical parameters (pH, conductivity, alkalinity, chemical oxygen demand, dissolved organic carbon, organic nitrogen, total ammonia, K⁺, Na⁺, Cl^-, So4^2-, Ca²⁺, Mg²⁺, Fe, Zn, Cu). Principal Compontents Analyses (PCA) and regression analyses were performed on both types of variables in order to find possible explanations for the observed toxicity. The results of multivariate analyses showed a general relationship between both data sets, namely the most (or least) contaminated samples were also generally the most (or least) toxic. These analyses also suggested that ammonia, alkalinity and chemical oxygen demand (COD) were associated with increasing toxicity. Simple and multiple regression analyses allowed to confirm the importance of ammonia and alkalinity for causing toxicity to most organisms. Luminescent bacteria, however, were found to be more sensitive to the organic load of the leachates.The results are discussed in the context of risk assessment of various types of landfills.

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C. Cote, M. Douville and J.-R. Michaud
Evaluation Of Micro-Scale Bioassays As Toxicity Monitoring And Identification Tools For Pulp And Water Effluent
St. Lawrence Technologies 1999 October 1-4
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : Toxicity monitoring; Toxicity Identification Evaluation

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Abstract : The use of four micro-scale assays for toxicity monitoring and identification was investigated to evaluate their performance and usefulnuess compared to conventional assays specified in the Canadian Pulp and Paper Regulations (i.e. Daphnia magna and rainbow trout tests). The micro-scale assays (Daphtoxkit F^TM, Daphnia I.Q.^TM, Thamnotoxkit F^TM and Microtox) were first evaluated and compared to the standard Daphnia magna assay using effluent samples from five different mill operations from the J. Ford & Co. mill.The Thamnotoxkit F^TM and the Daphtoxkit F^TM were retained to pursue the evaluation and one type of effluent was selected to perform a Toxicity Identification Evaluation (TIE). The two micro-scale assays provided similar results to those of the Daphnia magna assay, indicating that they could prove useful for screening and monitoring toxicity of chemical products used in the mill.

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A. Willemsen, M.A. Vaal and D. de Zwart
Microbiotests As Tools For Environmental Monitoring
National Institute of Public Health and Environmental Protection - Report 607042005 1995 January 1-39
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Rotoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Thamnocephalus platyurus
Topics : review, test selection

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Abstract : In this study the usefulness of small-volume rapid aquatic toxicity tests (microbiotests) as environmental monitoring devices is reviewed. The individual tests are evaluated and a test battery is designed with the objective to be able to detect most classes of toxicants at low concentrations with a high reliability. Most of the discussed tests are bacterial luminometric or colorimetric assays for general toxicity determination. An algal microplate assay is also included as well as some miniaturized invertebrate LC50 tests (Toxkits). Additionally, invertebrate in vivo enzyme inhibition assays and a frog teratogenicity assay are evaluated. For the determination of genotoxicity, two bacterial assays are compared. For every test the following evaluation is made :- Convenience (ease of operation, test duration, required manpower capacity and skill)
- Completeness of documentation
- Costs
- Reproducibility
- Level of standardization
- Influences of experimental conditions
- Sensitivity to environmental samples
- Sensitivity to a variety of single compounds
The results indicate that most tests are insufficiently documented. The designed battery includes the Microtox assay, the algal microplate assay and the anostrocan Thamnotoxkit F. Genotoxicity may be measured reliably and cost-effectively by the Mutatox assay if the Microtox assay is also used.

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M. Cerejeira, T. Pereira and A. Silva-Fernandes
Use Of New Microbiotests With Daphnia magna And Selenastrum capricornutum Immobilized Forms
Chemosphere 1998 37 (14-15) 2949-2955
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - pesticides; surface waters

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Abstract : A preliminary assessment of toxicity to aquatic life of water from rice fields treated with pesticides, and particularly with the herbicides molinate and quinclorac, was performed from June to August 1996 in a rice paddy area integrated in a Natural Reserve. New rapid-screening microbiotests with Daphnia magna and Selenastrum capricornutum were used. Water samples were collected from : a water source for irrigating the rice fields, two rice plots, a drainage channel and the river into which the water is discharged. The results showed that while the water for irrigation was not toxic to D. magna, the samples collected from the river were very toxic causing 100% immobility in D. magna and substantial inhibition of Selenastrum growth. This, however, was due to the brackish nature of the river water. Water collected in the drainage channel of the rice fields were also found to be quite toxic. A greater effect was detected in samples from the rice plot treated with molinate than from that treated with quinclorac, suggesting that molinate treatment was more toxic to both species. The results indicate that the rapidity, simplicity and relatively low cost of the new microbiotests make them very practical for initial acute toxicity screening and offer an alternative to provide evidence for changes needed in agriculture practices for a better protection of the aquatic environment.

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M.J. Cerejeira, T. Pereira, A. Silva-Fernandes and F. Brito
Preliminary Toxicity Evaluation Of Water From Rice Fields using Cost-Effective Microbiotests
Toxicological and Environmental Chemistry 1999 69 373-380
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - pesticides; chemicals - organic compounds; surface waters

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Abstract : A study was performed in a Protected Area (Natural Reserve of Sado Estuary) in 1996, in order to evaluate the acute toxicity for aquatic life of surface water from rice fields treated with pesticides, and particularly with molinate and quinclorac. As a preliminary assessment, rapid toxicity tests were used, namely the “Rotoxkit F”, “Thamnotoxkit F” and the recently introduced “Daphtoxkit F magna” and “Algaltoxkit F”. Water samples were collected from : a water source for irrigating the rice fields, two rice plots, a drainage channel and from Sado river in which the water is discharged. The results showed that samples from the rice plot treated with molinate were more toxic than those obtained from rice plot treated with quinclorac. Water samples from the river were the most toxic, causing 100% of immobility in all organisms and a substantial inhibition in the algal growth. Water collected from the drainage channel in late July, was also found to be quite toxic. The results obtained until now indicate that some characteristics of these microbiotests, like their rapidity, simplicity and relatively low cost, make them a practical tool to evaluate acute effects of pesticides in freshwater ecosystems. They may also contribute to suggest some changes in pesticides use in order to promote agricultural practices with lower impact into aquatic life.

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T. Pereira, M.J. Cerejeira and J. Espirito-Santo
Toxicity Evaluation Of Pesticide Treated Waters From Rice Fields To Aquatic Organims In Sado Estuary During 1998
From : 9th Annual Meeting of Setac-Europe Book of Abstracts 133
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - pesticides

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Abstract : Pesticide use in rice crop may affect the quality of surface waters due to direct or indirect discharge of irrigation waters from paddy fields. Therefore, possible toxic effects to aquatic life should be evaluated, particularly in protected environments. Sado River Estuary, located in the Portuguese coast, is integrated in a Natural Reserve which includes an agricultural area where rice is one of the most important crop. A study was started to evaluate toxicity effects of the main pesticides introduced in that ecosystem to the aquatic biosphere through the use of microbiotests. The results obtained in water samples collected from rice plots, drainage channels and Sado Estuary during 1998 crop season, in two different sampling areas, are presented. The toxicity results of the different aquatic organisms used, namely those obtained with Daphnia magna, showed that insecticides usually applied by the farmers, like chlorfenvinphos and endosulfan, were the pesticides that caused the highest effects to the studied aquatic species. Toxicity results were also correlated with pesticides concentration levels detected in the same water samples. The toxicity information obtained along this study may contribute to suggest some changes in traditional agricultural practices in order to prevent contamination of estuary and coastal waters.

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M. DellaGreca, A. Fiorentino, M. Isidori, P. Monaco, A. Zarrelli
Antialgal Ent-Labdane Diterpenes From Ruppia Maritima
Phytochemistry 2000 55(8) 909-913
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - plant extracts

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Abstract : Seven ent-labdane diterpenes have been isolated from Ruppia maritima. The structures 15,16-epoxy-ent-labda-8(17),13(16),14-trien-19-ol acetate; methyl 15,16-epoxy-12-oxo-ent-labda-8(17),13(16),14-trien-19-oate, 15,16-epoxy-ent-labda-8(17),13E-dien15-ol and 13-oxo-15,16-bis-nor-ent-labda-8(17)-ene have been assigned to the five new compounds by spectroscopic means and chemical correlations. The phytotoxicity of the diterpenes has been assessed using the alga Selenastrum capricornutum as organism test.

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T. Cangiano, M. DellaGreca, A. Fiorentino, M. Isidori, P. Monaco E A. Zarrelli
Lactone Diterpenes From The Aquatic Plant Potamogeton Natans
Phytochemistry 2001 56 469-473
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - plant extracts

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Abstract : Four lactone diterpenes and two related glucosides with a labdane skeleton have been isolated from the aquatic plant Potamogeton natans. The structures 19-acetoxy-20-oxo-8(17),13-ent-labdadien-15→16 lactone, 8(17),13-ent-labdadien-15→16,19→20 dilactone and 6’-acetyl-19-glucopiranosiloxy-8(17),13-ent-labdadien-15→16 lactone have been assigned to the three new compounds by spectroscopic means. Antialgal assays showed inhibitory activity for some compounds.

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M. DellaGreca, A. Fiorentino, M. Isidori, A. Zarrelli
Toxicity Evaluation Of Phenanthrenes In Aquatic Systems
Environm. Toxicol. Chem. 2001 20(8) 1824-1830
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM pulex; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - plant extracts

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Abstract : Seven natural 9,10-dihydrophenanthrenes were isolated from the common reed Juncus effusus using chromatographic processes and identified by spectroscopic means. Furthermore, mimics of natural isolated compounds were synthesized to try and evaluate the influence of functional groups on the dihydrophenanthrene skeleton. Syntheses of compounds were based on the cross coupling of 1-(2-iodo-5-methoxy)phenyl-ethanol with variously substituted iodobenzenes by zerovalent nickel. All the chemicals were tested to evaluate their effects on fresh water organisms from different trophic levels. Toxicity tests were performed on reducers (the bacterium Escherichia coli), producers (the alga Raphidocelis subcapitata, previously known as Selenastrum capricornutum) and consumers including a rotifer (Brachionus calyciflorus), a cladoceran (Daphnia pulex) and an anostracan (Thamnocephalus platyurus). Results suggested no one organism was uniquely sensitive to the chemicals tested. Toxicity depended on the kind and on the position of substituents on the aromatic skeleton.

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M. DellaGreca, A. Fiorentino, M. Isidori, P. Monaco, F. Temussi E A. Zarrelli
Antialgal Furano-Diterpenes From Potamogeton Natans L.
Phytochemistry 2001 58(2) 299-304
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - plant extracts

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Abstract : Six new furano-ent-labdanes, 19-acetoxy-15,16-epoxy-8(17),13(16),14-ent-labdatrien-20-al (2), 15,16-epoxy-12-oxo-8(17),13(16),14-ent-labdatrien-20,19-olide (3), 12(S)-hydroxy-15,16-epoxy-8(17),13(16),14-ent-labdatrien-20,19-olide (4), 10a,19-dihydroxy-15,16-epoxy-8(17),13(16),14-nor-ent-labdatriene (5), 19,20-dihydroxy-15,16-epoxy-8(17),13(16),14-ent-labdatriene (6), 15,16-epoxy-12-oxo-8(17),13(16),14-ent-labdatrien-19,20-olide (7), were isolated, together with the known potamogetonin 1, from the aquatic plant Potamogeton natans. Their structures were determined on the basis of their chemical and spectral data. The compounds showed in vitro phytotoxicity against Raphidocelis subcapitata, a microalga used in aquatic tests. Compounds 4 and 5 showed the highest antialgal activity.

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T. Cangiano, M. DellaGreca, A. Fiorentino, M. Isidori, P. Monaco, and A. Zarrelli
Effect Of Ent-Labdane Diterpenes From Potamogetonaceae On Selenastrum Capricornutum And Other Aquatic Organisms
Journal of Chemical Ecology 2002 28(6) 1091-1102
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - plant extracts

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Abstract : Twenty ent-labdane diterpenes were isolated from the aquatic plants Ruppia maritima and Potamogeton natans by means of chromatographic processes and identified on the basis of their spectroscopic properties and by chiroptical methods. All the chemicals were tested to detect their effects on aquatic organisms from different trophic level. Toxicity tests were performed on aquatic producers (the alga Selenastrum capricornutum), and reducers including a rotifer (Brachionus calyciflorus), crustacean cladoceran (Daphnia magna), and crustacean anostracan (Thamnocephalus platyurus and Artemia salina). Furano-ent-labdanes exhibited high toxicity toward these organisms. 15,16-epoxy-12(S)-hydroxy-8(17),13(16),14-ent-labdatrien-20,19-olide had a high toxicity only toward the algae and the rotifers, while is inactive for the crustacean.

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M. DellaGreca, A. Fiorentino, M. Isidori, M. Lavorgna, P. Monaco, L. Previtera, A. Zarrelli
Phenanthrenoids From The Wetland Juncus Acutus
Phytochemistry 2002 60 633-638
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - plant extracts

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Abstract : Nine 9,10-dihydrophenanthrenes, three phenanthrenes and a related pyrene have been isolated from the wetland plant Juncus acutus. The structures have been attributed by means of their spectral data and chemical correlation. 5-(1-Ethoxy-ethyl)-2-hydroxy-7-methoxy-1,8-dimethyl-9,10-dihydrophenanthrene and 5-(1-phytoxy-ethyl)-2-hydroxy-7-methoxy-1,8-dimethyl-9,10-dihydrophenanthrene, 2,7-dihydroxy-1-methyl-5-vinylphenanthrene, 2,7-dimethoxy-1,6-dimethyl-5-vinylphenanthrene and 2,7-dihydroxy-1,6-dimethylpyrene are described for the first time. Many of the compounds showed in vitro phytotoxicity against Selenastrum capricornutum, a microalga used in aquatic tests.

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M. DellaGreca, A. Fiorentino, M. Isidori
Bioactive Compounds From Potamogetonaceae On Aquatic Organisms
In : Recent Advances in Allelopathy Vol II - A Science for the future (F.A. Macias, J.C.G. Galindo, J.M.G. Molinillo and H.G. Cuttler, eds. - Cadiz, Spain) 2003 21-41
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - plant extracts

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Abstract : Twenty ent-labdane diterpenes, isolated from the aquatic plants Ruppia maritima and Potamogeton natans, were tested to detect their effects on aquatic organisms from different trophic levels. Toxicity tests were performed on aquatic producers (the alga Selenastrum capricornutum), and consumers including a rotifer (Brachionus calyciflorus), a cladoceran crustacean (Daphnia magna), and two anostracan crustaceans (Thamnocephalus platyurus and Artemia salina). Furano-ent-labdanes exhibited high toxicity toward all of these organisms.15,16-Epoxy-12(S)-hydroxy-8(17),13(16),14-ent-labdatrien-20,19-olide had a high toxicity only toward the algae and the rotifers, while it was inactive for the crustaceans.

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A. Fiorentino, A. Gentili, M. Isidori, P. Monaco, A. Nardelli, A. Parrella, F. Temussi
Environmental Effects Caused By Olive Mill Wastewaters : Toxicity Comparison Of Low Molecular Weight Phenol Components
Journal of Agricultural and Food Chemistry 2003 51(4) 1005-1009
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : wastewaters

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Abstract : Olive oil mill wastewaters (OMW) show significant polluting properties due to their content of organic substances, and because of high toxicity towards several biological systems. Waste water toxicity has been attributed to their phenolic constituents. A chemical study of wastewaters from a Ligurian oil mill characterized phenolic products such as 1,2-dihydroxybenzene (catechol), derivatives of benzoic acid, phenyl acetic acid, phenyl ethanol, and cinnamic acid. The OMW were fractioned by ultrafiltration and reverse osmosis techniques and tested for toxicity on aquatic organisms from different trophic levels : the alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum); the rotifer Brachionus calyciflorus; and two crustaceans, the cladoceran Daphnia magna and the anostracan Thamnocephalus platyurus. The most toxic fraction for the utilized test organisms was that from reverse osmosis containing compounds with low molecular weight (<350 Da), and this was especially due to the presence of catechol and hydroxytyrosol, the most abundant components of the fraction.

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B. Ferrari, N. Paxéus, R. Lo Giudice, A. Pollio and J. Garric
Ecotoxicological Impact of Pharmaceuticals found in treated Wastewaters : Study of Carbamazepine, Clofibric Acid and Diclofenac
Ecotoxicology and Environmental Safety 2003 56, 3 359-370
Toxkits : Rotoxkit F^TM short-chronic
Test Species : Brachionus calyciflorus
Topics : chemicals - drugs; waste waters

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Abstract : In four countries (France, Greece, Italy and Sweden) occurrence in sewage treatment plant (STP) effluents and ecotoxicity of the pharmaceuticals carbamazepine, clofibric acid and diclofenac were investigated. Bioassays were performed on bacteria, algae, microcrustaceans and fish in order to calculate their predicted no-effect concentrations (PNEC) and to perform a first approach of risk characterization. For this aim, risk has been estimated by the predicted environmental concentration/PNEC ratio and the measured environmental concentration/PNEC ratio. First, regarding the PNEC, carbamazepine appears to be the more hazardous compound. Second, even though it is demonstrated that carbamazepine, clofibric acidand diclofenac have been detected in effluents, only carbamazepine have been detected in all sewage treatment plants with the greatest concentrations. Third, risk quotients greater than unity were calculated only for carbamazepine, suggesting that risk for the water compartment is expected.

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M. Isidori, M. Lavorgna, A. Nardelli, L. Pascarella and A. Parrella
Toxic and Genotoxic Evaluation of six Antibiotics on non-target Organisms
Science of The Total Environment 2005 346, 1-3 87-98
Toxkits : Algaltoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - drugs

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Abstract : The ecotoxicity of the following six antibiotics on aquatic organisms was investigated: Erythromycin, Oxytetracyclin, Sulfamethoxazole, Ofloxacin, Lincomycin and Clarithromycin. Bioassays were performed on bacteria, algae, rotifers, microcrustaceans and fish to assess acute and chronic toxicity, while SOS Chromotest and Ames test were used to detect the genotoxic potential of the investigated drugs. For risk assessment, the environmental impact was calculated by MEC/PNEC ratio using the available data from the literature regarding their occurrence in the aquatic environment and the toxicity data obtained from the bioassays performed. The ecotoxicological results showed that acute toxicity was in the order of mg/L while, for the chronic data the antibiotics were bioactive at concentrations in the order of µg/L, mainly for the algae. Drugs investigated were one or two order of magnitude less active against rotifers and crustaceans. Ofloxacin was the only genotoxic compound and Sulfamethoxazole, Ofloxacin and Lincomycin were mutagenic. As for environmental risk, the macrolides were found to be the most harmful for the aquatic environment.

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M. Isidori, A. Parrella, M. Lavorgna, and A. Nardelli
Toxicity Identification Evaluation (TIE) Of Leachates From Municipal Solid Waste Landfills : A Multispecies Approach
Chemosphere 2003 52(1) 85-94
Toxkits : Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Thamnocephalus platyurus
Topics : Toxicity Identification Evaluation; solid waste leachates

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Abstract : The toxicity of leachates from two municipal solid waste (MSW) landfills in Southern Italy was characterized using a Toxicity Identification Evaluation procedure. The chemical and physical fractionation techniques were : pH adjustment, pH adjustment/filtration, pH adjustment/C18 solid phase extraction, graduated pH and EDTA chelation. All the samples exhibited acute toxicity towards the bacterium Vibrio fischeri, the freshwater rotifer Brachionus calyciflorus and the freshwater crustaceans Thamnocephalus platyurus and Daphnia magna. Statistical techniques were used to determine the discriminatory power and the toxicity detection capacity of the different assays and to choose a minimal battery of tests for the toxicity identification of leachates. Toxicity was closely associated with pH, generally increasing at higher pH levels and decliming at lower ones. Furthermore, results showed that toxicants could be characterized as cations, basic chemicals, suspended solids and apolar compounds.

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C.R. Janssen and G. Persoone
Routine Aquatic Toxicity Testing : Some Problems And New Approaches
In : Biological Indicators For Environmental Monitoring (S. Bonotto, R. Nobili and R.P. Revoltella, eds. - Publisher : Ares-Serono Symposia, Rome, Italy) 1992 Serono Symposia Review 27 195-205
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Rotoxkit M^TM; Streptoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Streptocephalus proboscideus
Topics : review

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Abstract : Biological indicators can be used to assess either the actual or the potential impact of xenobiotic substances on the aquatic ecosystem and can thus be applied in two different ways : (a) a-posteriori assessment - to monitor the actual effects in nature, or (b) a-priori assessment - to predict the impact of a substance prior to its release (3).
In this paper we will discuss some of the problems associated with, and alternative approaches to the latter aspect of aquatic hazard assessment.
A bioassay can be defined as a procedure that uses living material to estimate the effects of pollutants. Ideally, bioassays are used to predict the levels of chemicals that produce observable effects on biological systems, e.g. populations, communities, and ecosystems, and to identify the biological resources at risk (2). However, because of the complexity of natural systems and the resulting poor understanding of the structure and function of these systems, aquatic toxicologists in practice mostly have to restrict their investigations to the testing of the chemical compounds under (very) limited conditions.
Most of the toxicity testing to date has been performed with single-species laboratory test systems. In a recent review on the applications of bioassay techniques, Maltby and Calow (10) reported that 90 % of all studies classified as predictive bioassays were single species laboratory tests. These authors also showed that the most commonly used test organisms were invertebrates (74.8 %) mostly cladoceran crustaceans. Fish with fathead minnow (Pimephales promelas) and rainbow trout (Salmo gairdneri) as the major representatives accounted for 23.9 % of the test species. Cladocerans and fish are in fact the only categories of aquatic animals which are prescribed by the various national and international regulatory and standardization organizations in their standard toxicity test methods. Compared to the complexity of natural systems these standard bioassays are, however, so surprisingly simple, that one can be concerned about the ecological relevance and predictive capacity of these types of bioassays. This issue will be briefly discussed hereunder.

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A. Lindgren, M. Sjostrom and S. Wold
QSAR Modelling Of The Toxicity Of Some Technical Non-Ionic Surfactants Towards Fairy Shrimps
Quant. Struct.-Act. Relat. 1996 15 208-218
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - surfactants

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Abstract : The toxicity of a number of non-ionic surfactants towards Thamnocephalus platyurus (sweet water fairy shrimp) has been examined. The measurements have been performed with the use of so-called Toxkits, and the results have been expressed both as a mean LC50 value for each of the surfactants, and with a relative scale estimated using of multi dimensional scaling (MDS). An algorithm is shown in matlab code, with which it is possible to perform MDS on a difference matrix with a lot of systematically missing values. The thus obtained results have been combined with the physico-chemical data for the surfactants, and QSAR models have been estimated for the toxicity (log(LC50)) of the surfactants. The physico-chemical properties with the main influence on the models were the hydrophilic-lipophilic-balance (HLB), the hydrophobicity (log P), the critical micellar concentrations (CMC) and the number of carbon atoms in the hydrophobic part (C) of the surfactant.

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M. Sjostrom, A. Lindgren and L.-L. Uppgard
Multivariate Structure-Property And Structure-Activity Relationships Of Technical Non-Ionic Surfactants
37th International Detergency Conference 1996 76-83
Toxkits : Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : chemicals - surfactants

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Abstract : A series of 36 non-ionic technical surfactants (ethoxylated fatty alcohols) was characterized by 18 different physico-chemical descriptor variables (e.g. critical micelle concentration, critical packing parameter, cloud point, etc.). The information content of the party correlated variables was summarized by principal component analysis (PCA). This analysis showed that the surfactants were divided into subgroups according to their structure. The conclusion was that the performance in water ought to be similar within each subgroup.
The detergency performance towards non-polar soil on textile was examined for a selection of the non-ionic surfactants with the use of statistical experimental design. In these experiments the surfactant concentration, the washing time and temperature was altered in a systematic way. It was shown that the detergency effects reached a plateau. It also turned out to be possible to impirically model the relationship between the physico-chemical properties and the surfactant concentration and washing temperature needed to reach the edge of this plateau. Emperical quantitative structure effect relationships (QSERs) were determined. The most important variables for the model were those which can be considered to be connected to the flexibility properties of the surfactants.
Further, the toxicity (LC50) of a number of the non-ionic surfactants was tested with cyst based toxicity test. Multivariate quantitative structure activity relationships (QSARs) with good predictive capabilities were possible to establish with PLS modelling. The results showed that in the QSAR models, variables that can be considered to measure equilibrum properties (e.g. critical micelle concentration, log P and hydrophobic-hydrophilic balance) were the most influential. A comparison of the results of the QSAR analysis showed that it was possible to find surfactants with high efficiency and low toxicity.

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I. Blinova
Advantages And Disadvantages Of Phytotoxicity Tests
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila
Topics : effluents; chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : In spite of the fact that aquatic plants are very important for aquatic ecosystems the phytotoxicity tests are conducted less frequently than acute toxicity tests with animal species since historically freshwater plants are considered less sensitive than animal species to pollution. Such problems as sensitivity of plants specie, choice of endpoint in the test procedure, possible fields of application are discussed in presentation.
Tests with freshwater microalgae Selenastrum capricornutum and duckweed Lemna minor were used for the toxicity evaluation of effluents, chemicals and hazardous waste leachates (130 samples). Received results reveal that sensitivity of both species depend on the nutrient content in the samples, however, in many cases Selenastrum capricornutum and/or Lemna minor were more sensitive than crustacean Daphnia magna and protozoa Tetrahymena thermophila. Comparatively high sensitivity of plant species to pollutants can explain our unsuccessful attempts to connect results of phytotests with nutrient content in the water samples from rivers. On the basis of analysis of our results and early reported data it was judged that use of the plant bioassays for evaluation of surface water pollution by nutrients or possible impact of effluents on trophic status of receiving water is inexpedient.
The effect of quality of receiving water on the evaluation of potential hazard of effluents to water ecosystem were investigated in parallel tests using for control and sample dilution both standard solution and water from receiving water body. Conducted tests showed very large difference between test results, therefore, to get more objective results it can be recommended to use natural water in the test procedure.

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L. Dubova and Dz. Zarina
Application Of Toxkit Microbiotests For Toxicity Assessment In Soil And Compost
Environmental Toxicology 2004 19 - 4 274-279
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Ostracodtoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : soils; composts

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Abstract : The potential of Toxkit microbiotests to detect and analyze pollution in agricultural soil and the quality of compost was studied. The toxicity tests used included seed germination biotests using cress salad (Lepidum sativum L.), tomato (Lycopersicum esculentum L.), and cucumber (Cucumis sativus L.), and the Toxkit microbiotests included those with microalgae (Selenastrum capricornutum), protozoa (Tetrahymena thermophila), crustaceans (Daphnia magna, Thamnocephalus platyurus and Heterocypris incongruens), and rotifers (Brachionus calyciflorus). Experiments on compost were undertaken in a modified solid-state fermentation system (SSF) and under field conditions (in a windrow). To promote the composting process, two strains of Trichoderma (Trichoderma lignorum and Trichoderma viride), as well as a nitrification association that regulated the nitrogen-ammonification and nitrification processes were applied.

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A. Kahru and L. Pollumaa
Ecotoxicological Evaluation Of The Hazard Caused By The Oil-Shale Industry-Related Phenolic Pollution In Estonia
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : solid wastes; solid waste leachates; soils; wastewaters

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Abstract : In Estonia there is the largest industrially-used oil-shale basin in the world. About 90% of the excavated oil-shale is used for fueling of the powerstations and 10% for the producing of oil and chemicals. One main source of the environmental pollution is leaching of toxicants from waste dumps (semi-coke, ash, waste rock) whereas phenolic compounds have been considered main hazardous pollutants in these leachates. The authors have been involved in the ecotoxicological hazard assessment of oil-shale industry pollution forabout eight years. In the presentation the overview of this work will be presented. The study concerning 8 main oil-shale industry phenolic pollutants showed that 3,4-dimethphenol and 2,5-dimethylresorcinol proved most environmentally hazardous as toxic (a battery of ecotoxicological tests was used) and slowly biodegradable in the activated sludge process (1). However, their concentration in semi-coke mountain toxic leachates (EC50 to photobacteria and Daphnia 1-5%; 2) was relatively low (<8 mg/L) compared to phenol and p-cresol that occurred in very high concentrations, both till 70 mg/L (2), and despite of their toxicity proved rapidly biogradable (1). As phenols make up 4-6% of the COD of the leachates (3) and their concentration explains only from 4-50% of the toxicity of the leachates (2), phenols are apparently not the biggest troublemakers. Moreover, while directed to the Gulf of Finland by the local rivers Kohtla and Purtse, the phenols get diluted and biodegraded by bacteria. However, accumulation of pollutants in the river sediments could pose a problem. The concentration of phenols in the soils surrounding the ditches was low, practically not exceeding the permitted concentrations for living areas. The heavy metals were present in very low levels but the concentration of oil-products was medium to high. The soils showed water-leachable toxicity that was not explained by their measured pollutant levels (4). Unidentified toxicants present in the oil-shale industry polluted soils, solid wastes and wastewaters (incl. potential mutagenic and hormone-system disrupting chemicals) have to be elucidated in the future.

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K. Kasemets, R. Reiman, L. Pollumaa, A. Ivask, M. Francois, H. C. Dubourguier, F. Douay, N. Ulitzur and A. Kahru
Application Of Different Toxicity Tests For The Detection Of Water-Extractable Toxicity Of Heavy Metal Polluted Soils
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila
Topics : soils

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Abstract : The water-extractable toxicity of 60 soils sampled around metal smelters in the North of France was studied. The total concentration of heavy metals in these soils reached ~1200 ppm for Zn, ~13 ppm for Cd and ~650 ppm for Pb. However, the toxicity of the water extracts of soils (1 part of soil was extracted with 9 parts of water) to protozoa, algae, Vibrio fischeri and crustaceans was practically absent as the concentrations of metals in these extracts (till ~0.6 ppm for Zn, till ~ 0.1 ppm for Pb and till ~0.006 ppm for Cd) were lower than the sensitivity of these tests.
In addition to above mentioned bioassays two new screening tests were used. One of them (Metal Detector^TM test; CheckLight, Israel) utilizes a highly sensitive recombinant luminescent Escherichia coli (strain K12 harbouring the luminescence system of Vibrio fischeri in pACYC184; Ulitzur & Kuhn, 1988) and allows the detection of ppb levels of heavy metals (Table) if tested in the special sensitizing buffer. The other test (ToxScreen^TM, CheckLight Ltd, Israel) is based on naturally luminescent marine bacterium Photobacterium leiognathi and is performed in two sensitizing buffers : one (pro-organic buffer) favours the detection of organic toxicants and the other (Pro-Metal buffer) cationic heavy metals (Ulitzur et al., 2002).
Out of 60 soil-water extracts tested 23 contained Zn more than 0.08 ppm (thus exceeding the EC50 value for Metal Detector assay; Table). However, only 9 samples of 23 showed toxic signal (inhibition of luminescence). Therefore, if could be supposed that the metals in the soil extracts were sorbed to humus particles and not bioavailable, or, the nutrients of the soil solution were masking the toxic influence of heavy metals. The levels of Cd and Pb were too low even for the Metal Detector assay. The ToxScreen assay did not show toxicity if tested in pro-metal buffer, as the metal concentration in the soil extracts was too low for this test. Surprisingly, 31 water extracts out of 60 showed toxicity in pro-organic buffer. Therefore, the presence of organic pollutants (PAHs, dioxins) could be presumed in these soils apart of heavy metals.

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A. Kungolos, Ch. Papadimitriou, M. Petala, P. Samaras and N. Christofi
Evaluation Of Toxic Properties Of Industrial Effluents
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Tetrahymena thermophila; Thamnocephalus platyurus
Topics : effluents

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Abstract : The objectives of this work were the assessment of toxic properties of industrial effluents by using various bioassays, the investigation of the relation between different test species used as indicator microorganisms, and the study of the correlation of toxicity results to physical-chemical properties for the identification of potential by toxic substances.
Effluent samples were collected from a number of industrial plants located in the Greater Industrial Area of Thessaloniki, in Northern Greece. The monitoring programme included several representative industrial plants such as food industries, tanneries, dying plants, metal processing units, etc. Samples were analyzed for their physical and chemical characteristics (organic loading, nitrogen, phosphorous and metal content, pH, salinity, etc). Toxicity of samples was determined by measurement of the bioluminescence inhibition of the saline bacterium Vibrio fischeri, using the Microtox® (Azur Environmental) and Lumismini® (Dr Lange) instruments. Toxic properties of samples were furthermore estimated by measuring the survival of two test species in contact with the samples : the freshwater crustacean Thamnocephalus platyurus and the freshwater protozoan Tetrahymena thermophila.
Poor correlation was found between physical-chemical characteristics of samples and bioassay results indicating that toxicity properties resulted from the combined action of a complex mixture of chemical substances usually found in industrial effluents. Toxicity results reaching up to 100% inhibition to bioluminescent bacteria were observed for some samples. Furthermore, the comparison of bioassays results showed that an almost linear correlation appeared between Microtox® Vibrio fischeri and the aquatic invertebrates, while the Lumismini® results were affected by several factors such as the temperature of the sample, the measurement time, etc. A stimulation effect to Vibrio fischeri, known as hormesis, was observed for certain samples (especially food industry effluents) associated to the presence of toxicants at low concentrations.

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M. Latif and E. Licek
Toxicity Assessment of Waste Waters, River Waters and Sediments in Austria using Cost- Effective Microbiotests
Environmental Toxicology 2004 19 (4) 302-309
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Ostracodtoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : wastewaters; surface waters; sediments; pore waters

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Abstract : The toxicity and chemical quality of surface water and sediment in the River Traun in Austria were studied because of recurrent fish mortality in some alpine rivers over the last few years. The analyses were carried out on samples collected during winter and summer upstream and downstream of two municipal wastewater treatment plants (WWTPs) and on effluents taken at the points of discharge of these two plants. Toxicity tests were performed on 20 samples of surface water, effluent, and sediment pore water. The test battery was composed of microbiotests with protozoans (Protoxkit F), microalgae (Algaltoxkit F), crustaceans (Daphtoxkit F magna and Thamnotoxkit F), and a higher plant (seed germination and root elongation assay on cress). Direct contact tests were performed on whole sediment with crustaceans (Ostracodtoxkit F). The physical-chemical characteristics of the surface water, effluent, and sediment pore water samples analyzed were conductivity, total hardness, pH, O(2), BOD(5), TOC, DOC, AOX, NH(4), NH(3), NO(2), PO(4)--P, Cd, Pb, Cu, and Zn. The toxicity data were expressed as percentage mortality or percentage inhibition, depending on the effect criterion of the respective assay. None of the water samples collected upstream and downstream of the WWTPs showed any significant (short-term) toxicity in either winter or in summer, but the effluents of the first municipal wastewater treatment plant were toxic to some of the test biota. All the sediment pore water samples induced serious inhibition of root growth of cress, and several pore waters were toxic to other test biota as well, particularly at the outlets of the WWTPs. The toxic character of some sediments was confirmed by direct contact tests with the ostracod crustacean. The chemical analyses did not reveal particularly high concentrations of any chemical that is very toxic. As a result no direct causal relationship could be established between the detected toxic effects and the chemical composition of the surface waters or sediment pore waters. The outcome of this preliminary study again highlights the need to complement chemical analyses with toxicity tests to determine the toxic hazard to aquatic environments that may be threatened by contamination. Furthermore, the investigations also confirmed the need to apply a battery of tests for an ecologically meaningful evaluation of the hazards of waters, sediments, and wastewaters. Finally, the results of the 360 bioassays performed show that culture-independent microbiotests are practical and reliable tools for low-cost toxicity monitoring of aquatic environments.

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B. Marsalek and L. Blaha
Comparison Of 19 Bioassays For Cyanotoxin Detection
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Ceriodaphtoxkit F^TM; Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Ceriodaphnia dubia; Daphnia magna; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : biotoxkins; cyanobacterial blooms; cyanotoxins; cyanotoxins

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Abstract : Comparison of literature results and the sensitivity of bioassays to cyanotoxins is impossible, because different authors use different cyanobacterial samples with uncomparable toxin composition, different methods for sample preparation, different design of bioassays, media, etc. A critical comparison of alternative bioassays for cyanotoxin detection, which is important for water management, is to date still missing. The aim of our study was to compare the sensitivity of 17 bioassays for cyanotoxin detection using the same cyanobacterial biomass, and the same sample preparation. An additional aim of this study was to find out which bioassay can discriminate the best the presence of microcystins. Results expressed as EC 50 and TU with 95% CI were compared by Principal Component Analysis and Discrimination Analysis. Bioassays were grouped into 3 groups (in order from the most sensitive to the least sensitive test of total biomass toxicity) i) Thamnocephalus platyurus 24h, Drosophilla melanogaster 24h, Spirostomum ambiguum 24h, Tetrahymena termophila 24h, ii) Tubifex tubifex 24h, Lepidium sativum 72h, Sinapis alba 72h, Daphnia pulex 24h, Hydra atenuata 96h, Artemia salina 48h, iii) Culex piiens 48h, Brachionus calycifloris 24h, Daphnia magna 24h, Tetrahymena pyriformis 24h, Ceriodaphnia dubia 24h, Panagrellus redivivus 96h and Artemia salina 24h. A different order can be derived, if we are looking for the ability of tests to discriminate microcystin content. The sequence from the test most able to discriminate microcystin to the least performing is : Thamnocephalus platyurus 24h, Drosophilla melanogaster 24h, Tubifex tubifex 24h, Tetrahymena termophyla 24h, Sinapis alba 72h, Daphnia pulex 24h, Hydra tenuata 96h, Artemia salina 48h, Spirostomum ambiguum 24h, Tetrahymena pyriformis 24h, Lepidium sativum 72h, Culex piiens 48h, Daphnia magna 24h, Ceriodaphnia dubia 24h, Brachionus calycifloris 24h and Panagrellus redivivus 96h. This means that for example, the protozoan Spirostomum ambiguum is sensitive to an extract from cyanobacterial biomass, but it has only a modest ability to detect microcystins. The crustacean Thamnocephalus platyurus has a good potential for cyanotoxin detection. The aquatic oligochaete Tubifex tubifex also has an interesting potential since it gives not false positive results and is sensitive to microcystin. The sources of variability can be decreased and the correlation between toxicity response and microcystin content improved by pre-purification of cyanobacterial samples for example by C-18 cartridges. Chronic and reproductive toxicity tests with Daphnia magna and FETAX (Frog Embryo Test with Xenopus laevis) also give non-lethal endpoints like malformations, teratogenic index, etc. Even if those tests are more difficult to perform than acute tests, chronic and reproduction bioassays give important information for ecological risk assessment of cyanobacterial toxins.

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S. Marvanova, V. Maresova, V. Koci, T. Ocelka, J. Huzlík, M. Sucmanova, K. Muzikantova, L. Kochankova, R. Grabic and S. Crhova
Ecotoxicological And Chemical Assessment Of Runoff Waters Originated From Motorways
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : soil runoff

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Abstract : The aim of this work is to evaluate an environmental risk caused by runoff waters originating from motorways. Water washed away from road surface and drainage water from the layers under the roads are conducted to receiving collectors, small settlers. An excess of runoff water is freely discharged from the settlers to a water stream or to the soil. It is expected that the water contains serious pollutants originating from traffic. Water of these receiving collectors was sampled with semipermeable membrane devices (SPMDs) and analyzed for its level of POPs contamination (PCBs, PAHs, PCDDs/Fs, OCPs). Together with chemical analysis, toxicity tests on extracts obtained from exposed SPMDs were performed : inhibition of bacterial bioluminescence, inhibition of algal growth and an acute toxicity test on the crustacean Daphnia magna. Values of obtained Vtox parameters were compared to values in main Czech rivers sampled with the same passive sampling method. Besides the SPMDs sampling, water and sediment of the receiving collectors was analyzed to determine the content of PAHs, nitro-PAHs, mineral oils and tested by ecotoxicological biotests : inhibition of bacterial bioluminescence, inhibition of algal growth and acute toxicity test to the crustacean Thamnocephalus platyurus. Samples with higher level of toxicity were analysed for their content of selected metals. The results of this work show that the runoff water from motorways could cause an important risk to the water and soil environment.

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G. Nalecz-Jawecki and J. Sawicki
Spirotox - Spirostomum Ambiguum Acute Toxicity Test - 10 Years Of Experience
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Artoxkit M^TM; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - inorganic compounds; chemicals - pesticides; chemicals - organic compounds; chemicals - surfactants; chemicals - drugs

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Abstract : Spirotox is a short-term acute toxicity test with a large ciliated protozoan Spirostomum ambiguum. The test was developed in the Department of Environmental Health Sciences, Medical University of Warsaw in Poland in the early 1990s. It was presented for the first time during the 6th ISTA in Berlin in 1993. During 10 years the sensitivity of S. ambiguum to many classes of toxicants was evaluated. We started with inorganic compounds, simple organic compounds, then we switched to pesticides, surfactants and drugs. Spirotox was also an important element of our battery of bioassays. The toxicity of a lot of various environmental samples was analyzed and Spirotox was always one of the most sensitive tests of all used.

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G. Persoone
Rapid Toxkit Microbiotests For Water Contamination Emergencies
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : rapid tests; drinking water; surface waters; behavioural tests

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Abstract : The determination of the potential hazard of environmental contaminants is to date mainly performed by chemical analyses. These investigations are, however, mostly restricted to a limited number of compounds or group parameters, and the outcome very often does not give a satisfactory answer to the question of the “real danger”.
The use of toxicity tests which reveal the “global” impact on the test biota, of all the chemicals present, is to date acknowledged as a complementary (if not even a much better tool) for environmental hazard assessment.
Whereas in investigations on the impact of chemicals or wastes that are discharged in aquatic or terrestrial environments, or in the biomonitoring of contaminated sites, there is mostly no need to have “immediate answers” with regard to the toxic impact, there is in turn an absolute need for “rapid signals” in cases of water contamination emergencies by accidental or deliberate inputs of toxicants in water supplies or reservoirs.
Since such situations can occur at any time and at any place, this means first of all that the live biological material to perform the assays must be available 7 days a week, year-round !
During the last three decades, toxicity tests have been developed with organisms belonging to different trophic levels (bacteria, micro-algae, protozoans, rotifers and crustaceans) which are based on “inactive, dormant or immobilised” stages of the biota, and hence bypass the need for continuous culturing/maintenance of live stocks. These assays have been miniaturised and were hence given the name “microbiotests”.
Bacterial microbiotests are undoubtedly the “most rapid” assays available to date since they give an effect-answer in less than one hour. These tests are, however, not very sensitive to particular categories of toxicants and like in the classic ecotoxicological approach, there is hence a need for additional tests with other biota, in order to detect possible contamination hazards and avoid “false negatives”.
The culture/maintenance independent “Toxkit” microbiotests at first sight appear to be good candidates to complement the rapid microbial assays. Unfortunately, the dormant eggs of the invertebrate test species require one to several days to hatch, and the Toxkit assays are based on exposure times of one to several days as well.
Thanks to extensive recent research the two former handicaps have now been solved.First of all “rapid” (one hour) Toxkit tests have been developed which are based on either enzymatic inhibition or on the inhibition of particle uptake by the test species. Comparisons of the 1h sublethal effect levels with the 24h mortality endpoints revealed that the rapid endpoints were as sensitive as the lethal ones.
In addition, an “automatic hatching incubator” was worked out, which makes fresh live biota available 7 days a week, to cope with “instantenous needs” for emergency testing.Details will be given during the presentation on the recent developments of the rapid Toxkit microbiotests.

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L. Põllumaa, K. Kasemets, H. C. Dubourguier, I. Kurvet, R. Reiman and A. Kahru
Ecotoxicological And Mutagenic Hazard Of PAH-Polluted Soils
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : soils; chemicals - organic compounds

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Abstract : Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental contaminants because some of them are toxic, mutagenic and resist biodegradation.
In this work the data on environmental hazard of bioremediated soils containing ~3800 mg PAH/kg before planting with different plants (e.g., flowers, trees, grass) are presented for two sequential samplings (after 3 and 15 months of phytoremediation). Also, non-polluted soil (negative control) and polluted but not remediated soil (positive control) were analyzed. In all soils 4-ring and 5 ring PAHs were dominant. By the 15th month of phytoremediation the 3-ring PAHs were reduced about 35% (16-57%).
Two different extraction techniques were applied for the preparation of samples for the toxicity testing : extraction of soil with water (to mimic the hazard via the soil-water path and to predict the leaching of the contaminants into the groundwater) and extraction of soil with methanol (to predict the potential hazard of less soluble and soil-bound pollutants).
In addition to aquatic toxicity tests (algae, crustaceans, protozoa and photobacteria) the Solid-Phase Flash-Assay and a novel bioluminescent ToxScreen test (CheckLight Ltd., Israel) were used. Mutagenicity was studied using the Ames assay (TA 98) with and without metabolic activation. The analysis of the soils sampled after 3 months of phytoremediation showed that the water-extractable toxicity for all test organisms was low (not exceeding 1.4 TU). Although some PAHs are known pro-mutagens, no water-extractable mutagenicity of soils was detected at 100 mg per plate level. The toxicity of methanol extracts exceeded the toxicity of respective water extracts for algae 20000-40000 fold (upper extreme in the test battery) and for photobacteria 200-1700 fold (lower extreme). The composite sample of planted soils proved mutagenic at 1-5 mg level per plate.
The data on the influence of bioremediation and phytoremediation on water- and methanol extractable ecotoxicity and mutagenicity of the PAH-polluted soil will be shown.

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M. Tarczynska, A. Drobniewska and T. Jurczak
Possible Ways To Increase The Sensitivity Of Microbiotests For The Determination Of Cyanobacterial Hepatotoxins
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Artoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Thamnocephalus platyurus
Topics : cyanobacterial blooms; cyanotoxins

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Abstract : Microbiotests with invertebrates are a new interesting approach for low cost and straightforward detection of the cyanobacterial bloom toxicity. The Thamnotoxkit F and the Artoxkit M with the crustacean test species Thamnocephalus platyurus and Artemia salina respectively, were applied on Microcystis bloom samples containing hepatotoxins : microcystin-LR, -RR and -YR. The following factors to increase microinvertebrates sensitivity for cyanobacterial extracts were tested : longer exposure time (48h), higher temperature (27°C) and 2h preincubation in 1% DMSO (a substance which increases the permeability of the cell membrane).
The sensitivity of the freshwater crustacean to hepatotoxic purified extract in standard condition was 2,9±0,8-fold higher than that of the marine crustacean. The most effective method to increase the sensitivity of organisms to cyanobacterial toxins was 2h preincubations in 1% DMSO followed by 24h incubation with cyanobacterial extract at 27°C. The gain in sensitivity obtained this way was 161±17% for the Thamnotoxkit and 46±22% for the Artoxkit in comparison to standard biotest conditions.
The prolongation of exposure time to 48h was only possible for the Artoxkit. This method increased the sensitivity of organisms by 50±32%. The correlation between the microbiotest results and microcystin concentration in samples was statistically significant, especially under the modified conditions (1%DMSO and 27°C).
Two kinds of cyanobacterial bloom sample preparations were tested : crude extracts (CE) and purified extracts (PE).
The correlation after incubation of the test organisms in 1% DMSO and at 27°C during 24h is r= -0,81, and after an additional 24h exposure r= -0,83. On the other hand, for Thamnocephalus platyurus increase of the incubation temperature was very effective : 44±12%.
The study also showed that the applied modifications increased correlation between toxic response of Artemia salina and the CE and the fraction with toxins in comparison to standard test conditions.
Higher toxicity values of crude cyanobacterial extracts compared to partially purified ones were obtained during our research for both test species which might be explained by the presence of additional or other toxic compounds in these samples. These compounds were detected in the eluate (PI), after passage of a crude extract (CE) through a C18 cartridge. No microcystins were detected in this eluate. These substances would be a possible source of interference in bioassays.
To increase the sensitivity of the test organisms to cyanobacterial extracts, especially the more hydrophobic types of microcystins, the use of a 1% (v/v) DMSO solution was found to be effective. Two - hour incubation of the test species in DMSO prior to the start of the tests appeared to be the best procedure to increase the sensitivity to cyanobacterial extracts. The gain in sensitivity obtained this way was about 50%.
Increase of the incubation temperature as a second way to increase test sensitivity also appeared quite effective. The best results were obtained at a temperature 5 oC higher than that of the standard test procedure. The increase in sensitivity obtained with this procedure was also about 50%.

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A. Törökne
Sensitivity Evaluation Of The Daphtoxkit And Thamnotoxkit Microbiotests On Blind Samples
Journal of Applied Toxicology 2004 24 - 5 323-326
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - inorganic compounds; chemicals - organic compounds; sediments

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Abstract : At the Oregon State University Workshop we utilized two culture/maintenance-free microbiotests, the Thamnotoxkit F with the anostracan crustacean Thamnocephalus platyurus and the Daphtoxkit F magna with the cladoceran crustacean Daphnia magna, to determine the toxicity of water samples in a blind format. The Thamnotoxkit was applied to 7 samples and the Daphtoxkit to 12 samples. The chemical composition of the water samples to which the Toxkit microbiotests had been applied was disclosed a posteriori by the organizers and appeared to contain chlordimeform, colchicine, phosdrin, mercuric chloride, sodium arsenite, metham sodium, sodium cyanide, trimethylol propane phosphate, p-chlorophenol and a natural sediment sample containing mercury. Three of the water samples were blanks that had not been spiked with chemicals. No false positives were obtained with the two Toxkits and all the toxic waters were earmarked as such by the microbiotests, except trimethylol propane phosphate, which was not found to induce acute effects in Daphnia magna in the non-diluted water sample containing 100 mg/l of this compound.

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L. Wolska, M. Michalska, M. Rawa-Adkonis and M. Bartoszewicz
Ecotests As A Tool In Water Quality Assessment
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : surface waters; sediments; wastewaters

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Abstract : Surface water contains thousands of substances of natural and anthropogenic origin. Taking into account that a great number of them are toxic, the presence of some of them may a have negative influence on aquatic ecosystems.
Although chemical analysis allows the identification and quantitation of organic pollutants, it does not provide sufficient information to assess the ecological risk. Furthermore, it is not possible to investigate all substances and their interactions in bodies of water.
Therefore, chemical analysis should be supported by measurements of toxicity.
This paper presents the results :
1) of ecotoxicological survey in surface waters, sediments and wastewaters within the Bug River catchment area.
The main purpose of the ecotoxicological surveys was to screen potential hot spots as a basis for subsequent hydrobiological and physico-chemical analyses thus verifying preliminary selected locations for the monitoring network. The toxicity assessment in waters and sediments were conducted with the use of a wide range of bioindicators, i.e. bacteria, rotatoria, crustaceans and higher plants. The test results obtained showed high correlation.
Based on the results obtained from toxicity measurements in surface waters within the Bug River catchment area, three locations, characterized by high toxicity, were distinguished : the Bug River at Terespol, the Bug River at Krzyczew and the Krzna River at Neple.
Water at the other sites can be considered as non-toxic to the test organisms used. Contrary to waters, all samples of sediments collected within the Bug River catchment area were highly toxic.
2) of ecotoxicological quality analyses of surface waters, which are the sources of drinking water for some Polish cities.
The toxicity of surface waters (being a source of drinking water) was measured on the basis of the following tests :
acute toxicity : ToxAlert 100 (Merck, Germany), Daphtoxkit F magna (Crustacean Toxicity Screening Test for Freshwater);
chronic toxicity : Daphnia magna.
Chemical quality of these waters was quite good. The results of acute toxicity tests on the samples of surface waters indicated that these waters are non-toxic. However, some water samples can be regarded as toxic based on chronic toxicity results.

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I. Czerniawska-Kusza, M. Duzynski and G. Kusza
Toxicity Assessment Of Salinated Soils By A Toxkit Microbiotest And By Chemical Analysis
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Protoxkit F^TM
Test Species : Tetrahymena thermophila
Topics : soils

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Abstract : In the last decades the use of simple bioassays such as cost-effective microbiotests for routine toxicity screening of various environmental samples has been growing. Due to an increasing number of chemicals released into the environment, an application of the ecotoxicological techniques gives useful information on the potential effects of contaminants in the ecosystem.
This paper reports on the toxicity assessment of snow removal agents and salinated soils from different locations in the town of Opole (Poland) using the ciliate Tetrahymena thermophila (Protoxkit F^TM) as the testorganism. To gather information on the impact of snow removal chemicals on urban land, toxicity testing has been combined with physical-chemical analysis of soil samples and plant matter (conductivity, sodium, chloride, and bicarbonate content, among others). The experimental procedures and field results of chemical and toxicological analysis of salinated soils are discussed and interpreted in an ecological perspective.

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M. Petala, V. Tsiridis, P. Samaras, A. Kungolos and G.P. Sakellaropoulos
The Use Of Bioassays For Environmental Monitoring
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM
Test Species : Daphnia magna; Tetrahymena thermophila
Topics : surface waters; wastewaters; solid wastes

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Abstract : The purpose of a large number of regulating acts, issued by national and international authorities regarding the management and disposal of liquid and solid wastes to the environment, is to protect both human health and the condition of biological organisms that live in an ecosystem. Most of these acts are relied on laboratory analysis tests for the determination of physical and chemical parameters of wastewaters which are going to be discharged in a recieving water body, or of solid wasted which are going to be disposed in a landfill. As a result, the assessment of water quality contaminated with chemicals has been based on chemical analyses and the compliance or not of the measured concentrations with limit concentrations imposed by the legislation. However, physical-chemical analysis alone is not enough to predict the direct environmental impact of wastes due to a number of reasons such as complicated analytical procedures, large number of compounds, possible synergistic-antagonistic effects. The direct effect to the environment due to disposal of wastes may be evaluated by the use of certain organisms-indicators (bioassays). Various test species can be used representing different levels of life forms, and various procedures can be applied in order to estimate toxic properties of a sample. As a result an integrated strategy for water quality monitoring should combine chemical and biological methods; chemical methods give an indication of the pollution content and source, while biological methods measure their real effect to the environment. The primary objective of this study is to present the main field of application of bioassays for environmental treatment plant operation, determination of the effect of solid wastes to the environment, and to present the results from environmental monitoring in Greece.

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P. Fochtman, A. Raszka and E. Nierzedska
Efficiency Of Chosen Preparations In Mosquito (Aedes Aegypti L.) Control And Their Side Effects For Aquatic Environment
From : 11th International Symposium on Toxicity Assessment, Vilnius, Lithuania, 1-6 June, 2003 Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - pesticides

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Abstract : Mosquitoes are of no epidemiological significance in Poland, however, a few species are able to reproduce into plague quantities and become quite strenuous. During the past years mosquito outbreaks were observed in Poland after floods as well as on recreational areas. Therefore, mosquito control, sometimes over large areas, was essential.
The aim of the study was to evaluate the effectiveness of preparations recommended and used in practice for mosquito control as well as to determine their side effects for organisms being representative of different trophic levels of the aquatic environment.
The larvicidal and insecticidal activity of 15 preparations to Egyptian mosquito (Aedes aegypti L.) was evaluated under laboratory conditions. Microbiological and chemical preparations as well as growth and development regulators preparations were chosen for the studies.
The side effects of the chosen preparations were evaluated for the test organisms representative of successive trophic levels of the aquatic environment, i.e. : algae, aquatic plants, protozoans, rotifers, crustaceans and fish. The short-term and long-term tests were performed under laboratory conditions in compliance with the respective methods (OECD, ISO, EPA or Toxkit SOPs).
The results obtained indicate differentiated activity of the tested preparations towards the successive trophic levels of the aquatic environment. The most harmful preparations for the aquatic organisms were pyrethroids while the least toxic were biological preparations. Taking into account the results of efficiency of the tested preparations in mosquito control and their side effects to the aquatic environment the best compromise would be the use of biological preparations.

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D. Sabaliunas, J. Lazutka, I. Sabaliuniene And A. Sodergren
Use Of Semipermeable Membrane Devices For Studying Effects Of Organic Pollutants : Comparison Of Pesticide Uptake By Semipermeable Membrane Devices And Mussels
Environmental Toxicology and Chemistry 1998 Vol. 17, No. 9 1815-1824
Toxkits : Daphtoxkit F^TM pulex; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia pulex
Topics : chemicals - pesticides

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Abstract : Uptake of four pesticides - the organochlorines chlordane and endosulfan and the synthetic pyrethroids fenvalerate and allethrin - by triolein-containing semipermeable membrane devices (SPMDs) and by the lake mussel Anodonta piscinalis was studied in a laboratory continuous-flow system. Uptake of the analytes by the SPMDs and mussels was linear during the exposure period of 20 d. These kinetic data were used to calculate the first-order uptake rate constants. On a SPMD-whole body basis, the uptake rates were 3.5 to 5.5 times higher in the membrane devices than in the organisms. The synthetic pyrethroids were sampled at lower rates than the organochlorines, and this difference may be attributed to the larger molecular dimensions of the pyrethroids rather than analyte molecular weight and lipophilicity, which were similar for all test compounds. Because of the disparate sampling rates, concentration factors of analytes differed between SPMDs and mussels. However, the percent composition (ratios) of analytes in SPMDs and in mussels was similar, which indicates that SPMDs may serve as good surrogates for aquatic organisms with respect to the discriminatory uptake of hydrophobic chemicals. Semipermeable membrane device dialysate, mussel extract, as well as two artificial mixtures of the four pesticides were tested with standard toxicity and genotoxicity tests, including Microtox® (inhibition of bacterial luminescence), Daphtoxkit^TM, and Rotoxkit^TM (toxicity tests with freshwater invertebrates Daphnia pulex and Brachionus calyciflorus, respectively), and sister chromatid exchange in human lymphocytes in in vitro assay. Results of these tests suggest that integration of the SPMD technique and bioassays may be a valuable approach for the assessment of levels and effects of bioavailable hydrophobic pollutants.

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D. Sabaliunas, J.R. Lazutka and I. Sabaliuniene
Acute Toxicity And Genotoxicity Of Aquatic Hydrophobic Pollutants Sampled With Semipermeable Membrane Devices
Environmental Pollution 2000 109 251-265
Toxkits : Daphtoxkit F^TM pulex
Test Species : Daphnia pulex
Topics : surface waters

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Abstract : Triolein-filled semipermeable membrane devices (SPMDs) were deployed for 4 weeks in polluted water sources in Lithuania. The mixtures of pollutants sampled by the SPMDs were fractionated by size-exclusion chromatography (SEC). The fraction containing average molecular weight compounds such as polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides was screened by gas chromatography and mass spectrometry. The whole (non-fractionated) samples and their SEC fractions were tested in bioassays including Microtox^TM, Mutatox^TM, Daphnia pulex immobilization assay and the sister chromatid exchange (SCE) in human lymphocytes in vitro test. The Microtox^TM test was most sensitive with the estimated EC50 values in the range of milligrams or even micrograms per milliliter based on the amount of the SPMD triolein. Part of the observed toxicity was caused by elemental sulfur co-sampled by the SPMDs from sediments. The sum of toxicity equivalents of the SEC fractions was smaller than the relative toxicity of the whole samples indicating the presence of synergistic interactions in the complex mixtures of chemical pollutants. The toxic or genotoxic response induced by the chemical mixtures and their fractions was smaller in the D. pulex, Mutatox^TM and SCE tests. In Mutatox^TM, a positive response was only detected without the S9 metabolic activation which indicates the presence of mainly direct-acting mutagens in the samples. Interpretation of the Mutatox^TM data was difficult due to the complexity of dose-response and time-response relationships. The study has demonstrated the potential as well as some limitations of SPMDs in the monitoring of biological e.ects of bioavailable organic pollutants in the aquatic environment.

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G. Van Eetvelde, M. Stevens, F. Mahieu, H.-W. Wegen and A. Platen
An Appraisal of Methods for Environmental Testing of Leachates from Salt-Treated Wood - Part 1
In : The International Research Group on Wood Preservation - Section 5 - Environmental Aspects 1998 IRG/WP 98-50115 1-12
Toxkits : Algaltoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - inorganic compounds; chemicals - organic compounds; wood preservation

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Abstract : The magnitude and diversity of leaching tests with salt-treated wood, performed by institutes as well as industry, strongly appeal for harmonisation towards one single method. That procedure should be well-defined, cost- and time-effective and moreover be extended with a clear detection and interpretation scheme.
The paper(s) presented here tend to evaluate 2 existing leaching methods, the ENv 1250.2 and the EN 84, for their potential use as an evironmental standard in high hazard class wood preservation. The leachates obtained are chemically examined for their patterns and levels of metal emission and are biologically screened for their releavance towards in service biomonitored samples.
Upon strenghtening the objectives and characteristics of the EN 84 ageing test, the results reported here justify the support and upgrading of this procedure as an early stage leaching method within the standard efficacy testing of new or renewed wood preservatives.

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G. Van Eetvelde, S. De Geyter, P. Marchal and M. Stevens
Aquatic Toxicity Research of Structural Materials
In : The International Research Group on Wood Preservation - Section 5 - Environmental Aspects 1998 IRG/WP 98-50114 1-10
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila
Topics : chemicals - inorganic compounds; chemicals - organic compounds; wood preservation

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Abstract : Continuing from preliminary results reported on the aquatic toxicity of some tropical hardwoods under high hazard conditions, a more comprehensive research study is set-up in order to assess the environmental toxicity of various structural materials in water applications. Hazard class 4 preservative treated wood is tested for its toxic response on a battery of aquatic test organisms, next to concrete and plastic and a selection of tropical hardwoods used in waterworks.
The results reveal a wide range of toxicity responses covering tropical and treated timbers, the alternative materials bearing a considerably lower toxic profile. Still, a general observation is found in the enhanced inhibition of algal growth for all structural materials.

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H.W. Wegen, A. Platen, G. Van Eetvelde and M. Stevens
An Appraisal of Methods for Environmental Testing of Leachates from Salt-Treated Wood - Part 2
In : The International Research Group on Wood Preservation - Section 5 - Environmental Aspects 1998 IRG/WP 98-50110 2-18
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - inorganic compounds; wood preservation

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Abstract : For wood preservatives fur use in hazard class 4 information on the ecotoxicity of preservatives and ingredients as well as on the effect of losses from impregnated timber is needed for a proper environmental assessment.
In the evaluation of a suitable test procedure the leaching behaviour of, copperbased formulations was studied using analytical and ecotoxicological test methodology. These studies included an analytical comparison of end grain sealed and not sealed wood blocks. Using sensitive bioindicators in ecotoxicological studies, real effects of the leachates gained from EN 84 were measured. The possibility to use a laboratory test procedure based on the leaching according to EN 84 is shown and discussed for the risk assessment of treated timber.

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A.P. Loibner, R. Braun, V. Boller, and O.H.J. Szolar
Application Of Bioassays For Site Specific Risk Assessment - Evaluation Of Toxic Effects Exhibited By Organic Pollutants Present In Soil
In: The Utilization of Bioremediation to Reduce Soil Contamination: Problems and Solutions, (Sasek, V., Glaser, J.A. & P. Baveye, eds., Kluwer Academic Publishers, Dordrecht, The Netherlands) IV. Earth and Environmental Sciences 2003 vol. 19 165-175
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Thamnocephalus platyurus
Topics : soils, soil leachates

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Abstract : Bioassays are widely applied to measure toxic effects of aquatic samples such as surface water and groundwater, leachates and elutriates, as well as sediments, sludges, and soils. Contrary to (eco)toxicity tests, which translate single chemical concentrations into toxic effects (dose-response relationships giving NOEC, EC50, etc.), bioassays are applied to samples from contaminated sites. When testing soil samples directly, organisms will only respond to the bioavailable part of toxicants, thus, enabling the assessment of the effective toxicity posed by the contaminated soil. Two model soils different in soil texture and organic matter content have been used to evaluate the influence of the soil composition on the toxicity of a non-aged soil contamination. It appears that a higher organic matter content reduces toxic effects as observed for worms and plants. Moreover, an industrially contaminated soil was directly tested with cress, millet, rape, earthworms, autochthonous nitrifying bacteria, and Bacillus cereus. To assess elutriate toxicity of the industrial soil, the luminescent bacteria test (Vibrio fischeri bioassay) as well as commercially available toxkits (using the rotifer Brachionus calyciflorus, and the crustaceans Thamnocephalus platyurus and Artemia salina as test organisms) were employed. These ready to use testkits include the biological material in a dormant or immobilised form and enable fast and convenient toxicity testing. Moreover, genotoxity of elutriates was investigated using Escherichia coli PQ37 (SOS-Chromotest). Despite a high PAH-contamination, no genotoxicity was detected. Apart from the nitrification test (the only test system using autochthonous organisms), only low toxicity has been observed for direct soil and elutriate tests. This lack in toxicity indicates low bioavailability of contaminants. Therefore, bioassays are not only suggested to screen for hazardous soil samples but may also be used to establish a priority ranking for contaminated sites that considers effective toxicity.

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A.P. Loibner, O.H.J. Szolar, R. Braun, and D. Hirmann
Ecological Assessment And Toxicity Screening In Contaminated Land Analysis
In: Chemical Analysis of Contaminated Land (Thompson, K.C. & Nathanail, P., eds., Blackwell Publishing, Oxford, UK) 2003 229-267
Toxkits : Algaltoxkit F^TM; Ceriodaphtoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Protoxkit F^TM; Rotoxkit F^TM acute; Rotoxkit F^TM short-chronic; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Ceriodaphnia dubia; Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : soils, soil leachates

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Abstract : Toxicity tests provide an integrated measure on toxicity of contaminated land. Test organisms are either applied to chemicals (ecotoxicity testing) or directly to potentially contaminated soil samples (bioassays). While ecotoxicity test results are used together with toxicant concentrations of contaminated sites to deduce a potential threat, the application of bioassays allow for a more direct determination of adverse effects. Major benefits of the application of bioassays in the course of a site specific risk assessment are the integrated consideration of undetected toxicants, limited bioavailability and combined toxic effects due to pollutant interactions.
The growing interest in applying bioassays for the evaluation of contaminated land has resulted in standardised procedures for soil testing. With the development of short-term bioassays that are commercially available as ready to use testkits, the application of biological test methods is facilitated, in particular because the biological material is included in a dormant or immobilised form. In order to obtain relevant and valuable information, appropriate biological tests have to be selected depending on the particular needs. If bioassays are used in the framework of an ecological risk assessment in order to enable a more reliable prediction of environmental threats, ecologically relevant criteria govern the selection of tests. Inexpensive screening tests however may be sufficient to either identify toxic samples that require a more comprehensive analysis or to indicate the performance of bioremediation measures. This chapter provides an overview on soil toxicity testing and describes fields of application for bioassays. A summary of tests that may be employed for the investigation of soil pollution is given at the end of the chapter. Bioassays that are available as testkits are particularly considered.

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G. Persoone
Development and First Validation of a Stock Culture Free Algal Microbiotest : the Algaltoxkit
In : Microscale Testing in Aquatic Toxicology (P.G. Wells, K. Lee and C. Blaise , eds. - CRC Press) 1998 chapter 20 311-320
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : test development; test validation; chemicals - inorganic compounds; chemicals - organic compounds; effluents

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Abstract : The paper first deals with the complexity and variability of algal toxicity tests and subsequently briefly reviews the development of microbiotests with algae, and in particular the stock-culture free algal microbiotest : the Algaltoxkit.
The first validation of this new microbiotest is described in detail with data comparisons and correlations with the conventional algal assay in flasks. The precision of the Algaltoxkit is highlighted with data on the reference chemical potassium dichromate and effluents from the textile industry.
Finally the potential and the limitations of the new algaltoxkit are forwarded and discussed shortly.

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M. Renkin
Environmental Profile of Sophorolipid and Rhamnolipid Biosurfactants
La Rivista Italiana delle Sostanze Grazze 2003 Vol. 80 249-252
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila
Topics : chemicals - surfactants

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Abstract : The two last decades have been marked by an increasing environmental awareness. In relationship to detergents, biodegradability and ecotoxicity have become important issues in the study, devolopment and application of surfactants. In this respect biosurfactants are an interesting new class of surfactants that gain more and more interest on the market. Two commercially available biosurfactants, sophorolipids and rhamnolipids have been examined for their biodegradability, presence of stable metabolites and ecotoxicity. Both biosurfactants are ready biodegradable without evidence of stable metabolites and show more favourable ecotoxicity to some commercial synthetic surfactants.

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M. Podimata, E. Koutseris and N. Tsiropoulos
Use of Ecotoxicological and Chemical Analyses for Testing Freshwater and Seawater Quality in Magnesia Prefecture, Greece
Proceedings of the International Conference 2002 795-802
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna
Test Species : Artemia (salina) franciscana; Daphnia magna
Topics : surface waters; toxicity monitoring; groundwaters; coastal waters

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Abstract : The purpose of this study is to investigate fresh- and seawater quality in several seas of Magnessia Prefecture, using two different approaches. The first one is assessment of water quality based on two different bioassays that use aquatic organisms as indicators; the crustacean Daphnia magna for environmental monitoring of freswater samples, and the invertebrate saline water shrimp Artemia franciscana for testing seawater quality of Pagassitikos Gulf. The second approach is the chemical analysis, in which several chemical and physicochemical parameters were measured.

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V. Tsiridis and G. Persoone
Toxkit Microbiotests : New Low Cost Tools for Hazard Detection/Monitoring in Environmental Toxicology
Proceedings of the International Conference 2002 809-816
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : test development; toxicity classification; toxicity monitoring; chemicals - pesticides

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Abstract : In recent years, attention has gradually focussed on the use of toxicity tests as a useful tool to estimate chemical contamination and its potential impact on terrestrial and aquatic environments. New microbiotests, called Toxkits, have been developed in the Laboratory for Environmental Toxicology and Aquatic Ecology at the Ghent University in Belgium. The new approach is based on the use of \'dormant, inactive or immobilized\' stages of selected aquatic biota as the biomaterial from which life test organism can easily be obtained on demand to perform the toxicity tests. Several highly standardized \'culture/maintenance free\' toxicity tests are now commercially available, with selected species of micro-algae, protozoans, rotifers and crustaceans. A brief review of Toxkit microbiotests and their applications is given in this study.

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A. Kungolos, S. Hajispirou, M. Petala, V. Tsiridis, P. Samaras and G.P. Sakellaropoulos
Toxic Properties of Metals and Organotin Compounds and their Interactions on Daphnia magna and Vibrio fischeri
Proceedings of the International Conference 2002 817-824
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - metals; chemicals - organic compounds

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Abstract : In the present study toxicity tests wee performed in order to determine the toxic properties of four organotin compounds and three heavy metals and to examine the interactive effects of binary metal mixtures on two different test species : the freshwater crustacean Daphnia magna and the marine bacterium Vibrio fischeri. Daphnia magna proved to be more sensitive to metals than Vibrio fischeri. Tributyltin chloride and copper were found to be the most toxic substances on both tests organisms. The interactive effects between trimethyltin chloride/copper and trimethyltin/chromium on both test species were also investigated and indicated synergistic, additive and antagonistic actions.

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B.Z. Chial, G. Persoone and C. Blaise
Cyst-Based Toxicity Tests. - XVIII. Application of Ostracodtoxkit Microbiotest in a Bioremediation Project of Oil-Contaminated Sediments : Sensitivity Comparison with Hyalella Azteca Solid-Phase Assay
Environmental Toxicology 2003 18, 5 279-283
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemical - hydrocarbons; sediments; sediment and soil contact tests; test validation; toxicity monitoring

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Abstract : To further validate the scope of use of the 6-day sediment contact microbiotest conducted with the ostracod Heterocypris incongruens, we compared the sensitivity of this small-scale culturel maintenance-free assay with the 14-day solid-phase Hyalella azteca test. The present study was undertaken within the framework of a Canadian bioremediation project on oil-contaminated freshwater sediments along an intertidal shoreline of the Saint-Lawrence River near the town of Sainte-Croix, Quebec, Canada. Sediment subsamples, collected during three sampling periods over 21 weeks from five plots (each with four replicates) contaminated with different treatments, were analyzed for their toxic effects on the two test species. Sediment samples taken immediately after the plots were spread with oil were very toxic to both crustaceans (mortality between 80% and 100%), but the mortality of the amphipods was substantially lower than that of the ostracods for samples collected after 6 weeks. Fifteen weeks after the onset of the controlled oil spill experiment, the sediments of all plots were still quite toxic to Heterocypris but not to Hyalella. Statistical analysis of the mortality figures was performed by Blaise et at. (2003) and revealed a statistically significant correlation (R = 0.584 at the P = 0.001 level) between data pairs. Analysis of the precision of the two assays showed a substantially higher uniformity (lower variation coefficients between the four replicas) of the ostracod results over that of the amphipod assay. This study corroborated the findings of two previous investigations conducted in Canada and in Belgium with the same test species. All three investigations concur in pointing out the potential of the new ostracod microbiotest as a reliable and sensitive ecotoxicological test for routine and low-cost monitoring of contaminated sediments.

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G. Persoone et al.
A Practical and User-Friendly Toxicity Classification System with Microbiotests for Natural Waters and Wastewaters
Environmental Toxicology 2003 18 395-402
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : wastewaters; effluents; surface waters; toxicity classification; toxicity monitoring

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Abstract : Various types of toxicity classification systems have been elaborated by scientists in different countries, with the aim of attributing a hazard score to polluted environments or toxic wastewaters or of ranking them in accordance with increasing levels of toxicity. All these systems are based on batteries of standard acute toxicity tests (several of them including chronic assays as well) and are therefore dependent on the culturing and maintenance of live stocks of test organisms. Most systems require performance of the bioassays on dilution series of the original samples, for subsequent calculation of L(E)C₅₀ or threshold toxicity values. Given the complexity and costs of these toxicity measurements, they can only be applied in well-equipped and highly specialized laboratories, and none of the classification methods so far has found general acceptance at the international level. The development of microbiotests that are independent of continuous culturing of live organisms has stimulated international collaboration. Coordinated at Ghent University, Belgium, collaboration by research groups from 10 countries in central and eastern Europe resulted in an alternative toxicity classification system that was easier to apply and substantially more cost effective than any of the earlier methods. This new system was developed and applied in the framework of a cooperation agreement between the Flemish community in Belgium and central and eastern Europe. The toxicity classification system is based on a battery of (culture-independent) microbiotests and is particularly suited for routine monitoring. It indeed only requires testing on undiluted samples of natural waters or wastewaters discharged into the aquatic environment, except for wastewaters that demonstrate more than 50% effect. The scoring system ranks the waters or wastewaters in 5 classes of increasing hazard/toxicity, with calculation of a weight factor for the concerned hazard/toxicity class. The new classification system was applied during 2000 by the participating laboratories on samples of river water, groundwaters, drinking waters, mine waters, sediment pore waters, industrial effluents, soil leachates, and waste dump leachates and was found to be easy to apply and reliable.

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B. Bartusevieiene and L. Manusadzianas
Comparison of the toxic responses of Nitellopsis obtusa, Lepidium sativum and Thamnocephalus platyurus treated by Kaunas city wastewaters
Botanica Lithuanica 2003 9 (1) 43-53
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : wastewaters; effluents; surface waters

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Abstract : Two plant and one invertebrate tests were used for an assessment of six wastewater and one natural surface water samples collected in Kaunas. The sensitivities of Charatox with Nitellopsis obtusa 45-min resting potential depolarisation, Thamnotoxkit F^TM with 24-h Thamnocephalus platyurus lethality, and Lepidium sativum 120-h root/shoot elongation inhibition tests were compared. Charatox showed good toxicity quantifying capacity (= 1.0 toxic units; TU) toward filtered and unfiltered samples, and the same results were revealed for Thamnotoxkit F^TM, when filtered effluents were tested. Lepidium sativum root elongation test was characterised as having good toxicity detection capacity (= 0.4 TU), while Lepidium sativum shoot elongation test was assessed as inadequately sensitive for the toxicological assessment of the studied effluents. The assessment of filtered and unfiltered samples showed that filtration significantly reduced toxicity of effluents tested by Nitellopsis obtusa and Lepidium sativum (roots). The relation of toxicity and chemical analysis data by using an extended data base for Charatox and Thamnotoxkit F^TM tests identified ammonium and total nitrogen as the main possible toxicants (r = 0.7–0.8).

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J. Lahr et al.
Responses in Sediment Bioassays used in the Netherlands : can observed Toxicity be epxlained by routinely monitored priority Pollutants ?
Water Research 2003 37 1691-1710
Toxkits : Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Thamnocephalus platyurus
Topics : sediments; sediment pore waters; confounding factors

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Abstract : In order to identify the cause of toxicity in sediments and suspended matter, a large number of samples with different degrees of contamination was taken at various locations in The Netherlands. Standard acute bioassays were carried out with the bacterium Vibrio fischeri, the rotifer Brachionus calyciflorus and the anostracan Thamnocephalus platyurus.
Chronic standard tests were performed using the water flea Daphnia magna and larvae of the midge Chironomus riparius. Some novel bioassays were performed as well. Most toxic effects observed in standard bioassays with sediments from polluted sediments (class 3 and 4 on a scale of 0–4 according to the Dutch criteria) could be partly explained by toxic concentrations of known persistent priority pollutants, mainly heavy metals and occasionally polycyclic aromatic hydrocarbons. In some of the samples, ammonia toxicity was a confounding factor during testing.
Suspended matter from the Meuse river at Eijsden, which may be considered as ‘new’ sediment (pollution class 2), was moderately to highly toxic in almost all bioassays. This could have been associated with a combination of heavy metals, PAHs and ammonia. At two locations from the Lake IJssel area with no apparent persistent pollution, moderate and strong effects were nonetheless observed in invertebrate tests. This might have been due to agricultural run-off of pesticides, which are not routinely measured in sediments. A few effects on V. fischeri in canals and a small stream could not be explained with standard chemical analysis, but seemed associated with the outlets of sewage water treatment plants and industrial effluents. Additional chemical analysis of pore water samples from five selected sediments yielded more identified substances such as phtalates, decanes, cosanes and fragrances, but it was estimated that their contribution to the effects observed on V. fischeri, D. magna and C. riparius was negligible.

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S. Waara, A.S. Allard, M. Ek and A. Svenson
Chemical and Toxicological Characterization of Landfill Leachate after Treatment in a Pilot Scale Plant using Different Treatment Methods
Ninth International Waste Management and Landfill Symposium Sardinia 2003
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : soil leachates; leachates

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Abstract : Two different leachates from the same landfill were treated in pilot scale equipment. The equipment comprised biological nitrification and denitrification that was used all the time, and ozone treatment with carbon filtration that was used during some periods. Broad chemical and toxicological characterisations were made before and after different treatment steps.
Just biological treatment decreased nitrogen, metals, oil, PAH, monocyclic aromatics, phenoxy acids and chlorobenzenes effectively. Acute toxicity against Microtox, nitrification and Daphnia magna was completely removed. Estrogenic hormonal effects of the leachates were also removed.
The combination of biological treatment with ozone treatment and carbon filter decreased organic material (COD, TOC) and colour more effectively. There might also have been an extra effect on toxicity against green algae and on androgenic hormonal effects.

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L. Guzzella
Toxicity Test with Microalgae for Freshwaters (Test di Tossicita con Microalghe per Acque Dolci)
Acqua & Aria 2003 9 80-82
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : surface waters; sediment pore waters; effluents

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Abstract : The Algaltoxkit F is a chronic test produced by Microbiotests and commercialised in Italy by Ecotox LDS, for toxicity assessment of aquatic samples (surface waters, waste waters and drinking waters) and leachates or extracts from sediments. This assay allows to determine in 72h the inhibition of the growth of the green microalga Selenastrum capricornutum. The microalgae are provided in an immobilized form in \'beads\' from which they can be reactivated to immediately perform the assay. All the materials needed are provided in the kit.

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B.Z. Chial and G. Persoone
Cyst-Based Toxicity Tests. - XV. Application of Ostracod Solid-Phase Microbiotest for Toxicity Monitoring of Contaminated Soils
Environmental Toxicology 2003 18, 5 347 - 352
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : soils, soil leachates

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Abstract : A new \'culture/maintenance-free\' microbiotest has recently been developed for \'direct-contact\' toxicity determination of contaminated sediments. The 6-day Ostracodtoxkit ^TM makes use of the neonates of the ostracod crustacean Heterocypris incongruens hatched from dormant eggs (cysts). The new low-cost assay has already been applied in three studies on river sediments from Flanders (Belgium) and Canada and was found to perform comparably to the 10-day Hyalella azteca and Chironomus riparius tests in detecting and quantifying sediment toxicity. Taking into account that sediments are in fact underwater soils, the ostracod microbiotest was tentatively applied on 15 contaminated soils from Flanders (Belgium) and its sensitivity (based on mortality as the test criterion) was evaluated in comparison to the 28-day reproduction inhibition assay with the springtail Folsomia candida. The results revealed that the ostracod test species was as sensitive as or, in several samples even more sensitive than the springtail. Leachate experiments on the same soil samples also showed that in most cases ostracod mortality was a result of the (nonsoluble) toxicants bound to the solid-phase particles, rather than of those that had dissolved in the water phase. Providing confirmation of these first findings through additional studies, the new culture/maintenance-free ostracod microbiotest seems to have good potential as a low-cost and user-friendly tool for routine toxicity monitoring of contaminated soils.

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B.Z. Chial, G. Persoone and C. Blaise
Cyst-Based Toxicity Tests. - XVI. Sensitivity Comparison of the solid Phase Heterocypris incongruens microbiotest with the Hyalella azteca and Chironomus riparius contact assays on freshwater sediments from Peninsula Harbour-Ontario
Chemosphere 2003 52 95-101
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments; sediment and soil contact tests; test validation; toxicity monitoring

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Abstract : In view of the complexity and costs of traditional whole sediment assays, a culture/maintenance free direct contact microbiotest has been developed with the freshwater ostracod Heterocypris incongruens. The new Toxkit assay (named Ostracodtoxkit) has been applied to 33 sediment samples from Peninsula Harbour, located in Lake Superior of the Great Lakes water basin in Ontario, Canada. The microbiotest was applied in parallel to direct contact tests with the amphipod Hyalella azteca and the midge larva Chironomus riparius, to compare its relative sensitivity with that of the two conventional assays. The study was undertaken in the framework of remediation action plans for specific areas of concern, to enable decision making by the Canadian authorities for the restoration of impacted aquatic environments. Most sediments were found non-toxic (<20% mortality) to both the conventional test species and the ostracod. For the large majority of samples, a very good correspondence was found between the two crustacean test species for the intensity of the toxic signal mortality, as reflected by a 0.71 (p < 0.05) correlation coefficient. Growth inhibition, which is determined in the ostracod microbiotest as a sublethal effect criterion, allowed the earmarking of some sediment samples, which were apparently more toxic for the amphipod than to the ostracod. For 20% of the samples, substantially higher mortality scores were noted with the ostracod assay than with the midge larvae tests and the overall correlation coefficient between these two tests was lower (r = 0.60, p < 0.05). The results obtained in the present study corroborate those of previous research on sediments collected from various rivers in Flanders, Belgium, and confirm the potential of the new ostracod microbiotest as a reliable and sensitive low cost alternative for traditional whole sediment assays.

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R. Baudo, A. Sbalchiero, M. Beltrami
Test di Tossicità Acuta con Daphnia magna
Igiene dell'Ambiente e del Territorio 2004 6 62-69
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : interlaboratory testing; chemicals - inorganic compounds; chemicals - metals

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Abstract : In order to verify whether toxicity tests with testorganisms obtained from cryptobiotic forms provide results comparable with those of traditional cultures, in 2003 APAT (Italian Agency for environmental protection and technical services) organized the 2nd National Intercomparison for the acute toxicity test with Daphnia magna with the reference toxicant potassium dichromate K2Cr2O7, by using the Daphtoxkit F^TM magna (in the following indicated as kit), produced by MicroBioTests Inc. (Belgium) and distributed in Italy by the company Ecotox LDS (Pregnana Milanese).
Participants in the 2nd Intercomparison wishing to use their own Daphnia culture were allowed to perform the test also with these animals, by using a method of their choice (but providing all details about rearing and test protocol adopted for performing the toxicity test).
For the kit users, the methodological protocol for hatching and testing proved to be fully adequate; the participants obtained a mean 24hEC50 of 1.077 mg K2Cr2O7/l, and quite low variation coefficients (intralaboratory 15.40 %; interlaboratory 20.36 %).
The corresponding data, for daphnids obtained from in-house cultures, are : mean 24hEC50 1.057 mg K2Cr2O7/l; intralaboratory coefficient of variation 13.55 %; interlaboratory coefficient of variation 27.50 %.

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B.Z. Chial and G. Persoone
Cyst-Based Toxicity Tests. - XVII. Prefeeding Advantages in short-chronic Rotifer Microbiotests
Ecotoxicology and Environmental Safety 2005 60 73-80
Toxkits : Rotoxkit F^TM short-chronic
Test Species : Brachionus calyciflorus; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : test development

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Abstract : Attemps were made to overcome too-low reproductive output in the 48h short-chronic microbiotest with the rotifer Brachionus calyciflorus fed with microalgae (Pseudokirchneriella subcapitata) from algal beads older than 4 months (Chemosphere 50 (2002a) 365). Prefeeding of freshly hatched rotifers prior to the start of the actual growth experiments resulted in a satisfactory reproduction (i.e., an intrinsic growth rate r>0.65), even with subsequent feeding of the rotifers with microalgae from beads stored up to 1 year. Rotirich is an attractive alternative inert food compared with the mixture of microalgae and Spirulina, originally used to prefeed the rotifers. When the rotifers were prefed for 2h with 30 µg/mL Rotirich, it was then not necessary to add Spirulina to the microalgal food from beads. The above findings are a substantial simplification of the testing procedure of the new short-chronic culture/maintenance-free Rotoxkit microbiotest.

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C. Blaise, F. Gagné, N. Chèvre, M. Harwood, K. Lee, J. Lappalainen, B.Z. Chial, G. Persoone and K. Doe
Toxicity Assessment of Oil-Contaminated Freshwater Sediments
Environmental Toxicology 2004 19, 4 267-273
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments; chemical-hydrocarbons; toxicity monitoring

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Abstract : The performance of four microscale toxicity bioassays conducted on whole sediments was evaluated during a bioremidation project undertaken in 1999-2000 on a crude oil-contaminated freshwater shoreline of the St. Lawrence River, Quebec, Canada. The toxicity tests assessed included : (1) the Microtox^® solid-phase assay (MSPT), (2) the Biotox^TM Flash solid-phase test (Flash), (3) the algal solid-phase assay (ASPA), and (4) the Ostracodtoxkit solid-phase assay. Data generated with these assays were compared with those obtained using the standard endobenthic amphipod (Hyalella azteca) bioassay.
Bioanalytical comparisons indicated that all five solid-phaxse tests were useful in detecting the toxicity of oiled sediments; however, statictical analyses distinguished a difference in response between the invertebrate (amphipod and Ostracodtoxkit) and bacterial luminescence tests (MSPT and Flash). Based on these results, it is recommended that careful selection of biotests be made in the design of the test battery for assessment of residual oil sediment toxicity. Time-series toxicity data generated with ASPA indicated that oil sediments in the freshwater wetlands of the St. Lawrence River remained toxic to phytoplankton for at least 65 weeks and that remediation treatment was able to accelerate detoxification by 16 weeks.

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M. Isidori, A. Parrella, A. Nardelli, M.R. Iesce
Environmental Toxicity of Three Fungicides and their Photoderivates on Freshwater Crustaceans
SETAC World Conference 2004, Portland, Oregon, U.S.A. 2004, Abstract Poster paper
Toxkits : Ceriodaphtoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Ceriodaphnia dubia; Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - pesticides; surface waters

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Abstract : The fungicides Carboxin, Oxycarboxin and Furalaxyl occur mainly on the soil, even if surface waters are subjected to contamination due to phenomena of run off with continuous exposure of non-target aquatic organisms. Carboxin and Furalaxyl undergo photooxidation and the photoproducts may constitute a further potential risk for aquatic biota. Here we report acute and chronic effects of the three fungicides and their derivates obtained by sunlight exposure in water, on freshwater crustaceans (Thamnocephalus platyurus, Daphnia magna and Ceriodaphnia dubia). The acute results showed that Furalaxyl was particularly active on Thamnocephalus platyurus (LC50=0.46 mg/l), while Carboxin and Oxycarboxin were less toxic. Chronic data demonstrated that Furalaxyl had a toxic potential in the same order found for acute toxicity towards Thamnocephalus platyurus while an EC50=0.02 was found for Oxycarboxin. A different behavior was found for the fotoderivates, in fact the 2(5H)-furanone (the main photoproduct obtained from Furalaxyl) was found more toxic than the parent compound while the main photoproduct obtained from Carboxin, the Sulfoxide, was found to be less toxic that the parent compound. Therefore the possible effects of the transformation products should be investigated too.

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M. Daniel, A. Sharpe, J. Driver, A.W. Knight, P.O. Keenan, R.M. Walmsley, A. Robinson, T. Zhang and D. Rawson
Results of a Technology Demonstration Project to compare Rapid Aquatic Toxicity Screening Tests in the Analysis of Industrial Effluents
Journal of Environmental Monitoring 2004 6, 11 855-865
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : effluents; test sensitivity; test validation

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Abstract : The results of a \'BioWise\' demonstration project to assess the comparative sensitivity and practicaly of seven new assays for the direct assessment of ecotoxicity in industrial effluents are presented. In addition the aim of the project was to validate the results of the new assays against benchmark data generated from non-proprietary, rapid, microplate screening assays using the regulatory species; freshwater crustacean Daphnia magna and green algae Selenastrum capricornutum, chosen in view of their environmental relevance. The new commercial test assays were : Daphnia magna, Selenastrum capricornutum and Thamnocephalus platyurus Toxkits®, containing dormant, immobilised life stages of the test species; GreenScreen® EM, a yeast based assay for genotoxicity and general acute toxicity and CellSence® a mediated, amperometric whole cell biosensor based on immobilised activated sludge and E. coli. 38 effluent samples were examined over a period of 13 months, in the project co-ordinated by the AstraZeneca Brixham Environmental Laboratory, and part funded by BioWise via the UK Government Department of Trade and Industry.
From the conclusions :
From statistical data and comparison of assay specifications it appears that each of the assays applied in this demonstration programme has its own strenghts and weaknesses.
The Daphtoxkit and Algaltoxkit assays have one clear advantage, in that they make use of the same test organisms as the benchmark assays, and as a result the data produced showed good correlation with those given by the benchmark tests. Like the other alternative tests they are independent of the culturing of live stock of test species, and provide organisms of uniform size and age, and in good physiological condition. They also have the advantage that they do not require costly equipment. Furthermore the Daphtoxkit and Algaltoxkit are applied internationally and validated by numerous studies. The Thamnotoxkit, despite having similar advantages to the Daphtoxkit and Algaltoxkit, has a lower degree of correlation with the Daphnia screen, and has not yet been validated for industrial effluents. It also requires the use of a dissection microscope.
On the basis of the objective of the demonstration programme in seeking alternative rapid screens which would give results indicative of the standard tests, and taking into account considerations on practicality and costs, the Daphtoxkit and the Algaltoxkit microbiotests have the best overall correlation to the standard tests of all the alternative methods applied, and are well suited for cost-effective routine monitoring of industrial effluents.

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T.W. Snell and C.R. Janssen
Rotifers in ecotoxicology : a review
Hydrobiologia 1995 313/314 231-247
Toxkits : Rotoxkit F^TM acute; Rotoxkit M^TM
Test Species : Brachionus calyciflorus; Brachionus plicatilis
Topics : review

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Abstract : In the past five years the use of rotifers in ecotoxicologial studies has substantially increased. This greater interest has been due to the central role of rotifers in freshwater planktonic communities, the ease and speed of making quantitative measurements of mortality and reproduction, their sensitivity to common pollutants, the commercial availability of cysts, and the existence of reliable, standardized protocols. The main endpoints used in ecotoxicology studies are reviewed, including mortality, reproduction, behavior, cellular biomarkers, mesocsms, and species diversity in natural populations. For each endpoint, published studies are cited, along with the compounds investigated, duration of exposure and the LC50s, EC50s or NOECs reported. Rotifers have been included as part of a standardized mesocosm and in several large-scale, outdoor mesocosm studies. A critique of rotifer use in ecotoxilogy is offered and it is concluded that the scientific basis for including rotifers as part of a battery of ecotoxicological tests is well established.

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I. Aoyama, P. Minguy and M. Tanaka
Hazard Evaluation for Intermediate Treatment Residues of Medical Solid Wastes
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 165
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : solid wastes; chemicals - drugs

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Abstract : Wastes released from medical facilities include domestic wastes and infectious wastes which is assigned as specially controlled wastes. As infectious wastes is at risk of infection, sufficient attention should be paid for treatment of the wastes, and intermediate treatment such as sterilization and/or incineration of the wastes are obligated before the final dumping. Until now, incineration is considered the most favored method of medical waste treatment in the variety of intermediate treatments because of excellence in detoxification and volume reduction. However, in Japan, Dioxins released from incinerators has become serious and social issue and small scales of incinerators are prohibited to operate by constitution of a strict legal system. Therefore, in non-incineration system for the intermediate treatment of medical wastes will increase in near future in Japan.
In this study the safety evaluation of intermediate treatment residues by the non-incineration treatment such as a steam sterilization under pressure and an electromagnetic waves sterilization were performed using a battery of bioassays. The experimental results showed that bacteria and fungi did not remain in the residues of the incineration treatment but remained in non-incineration treatment. These results suggest that the incineration method is the most excellent method in sterilization activity but toxic chemicals remain in the residues of the incineration treatment.

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C. Blaise, P. Pavlova, F. Gagné, R. Tardif and M. Harwood
Toxic effects of selected pharmaceuticals emitted to the St-Lawrence River from a major municipal wastewater source (Montreal, QUE., Canada)
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 111
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - drugs; waste waters

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Abstract : Municipal wastewaters are now well-recognized as sources of “emerging chemicals” comprising diverse household products, hormonally-active substances and pharmaceuticals. In particular, effects of the latter class on aquatic biota are poorly understood at present. Twelve such chemicals were recently detected in the final effluent of the City of Montreal’s wastewater treatment plant which treats close to three million cubic meters of combined sewage and industrial liquid waste per day. To gain a first insight on the effects they might have on aquatic organisms, their individual and combined (sub)lethal toxicity was reported via standard measurement endpoints using laboratory bioassays representative of different levels of biological organization. Toxicity testing was conducted with bacteria (Vibrio fischeri light inhibition assay), micro-algae (Selenastrum capricornutum growth inhibition assay), micro-crustaceans (Thamnocephalus platyurus lethality assay), cnidarians (Hydra attenuata sublethality and lethality assay) and fish cells (rainbow trout hepatocyte cytotoxicity assay). Additionally, with the cnidarian animal model, Hydra attenuata, a more subtle sub-lethal endpoint – prey ingestion inhibition – was also investigated. The relative toxicity of these 12 pharmaceuticals to bioassay organisms and their potential impact on freshwater receiving environments will be highlighted during this presentation.

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R. Kurmayer and F. Jüttner
Strategies for the Co-Existence of Zooplankton with the Toxic Cyanobacterium Planktothrix Rubescens in Lake Zurich
Journal of Plankton Research 1999 21, 4 659-683
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : biotoxins; cyanobacterial blooms; cyanotoxins

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Abstract : Since the cyanobacterium Planktothrix rubescens, which dominates the phytoplankton community in Lake Zurich, is generally considered toxic to zooplankton, we addressed the question whether co-occurring zooplankton species have developed adaptive responses. Artificially shortened filaments (<30 µm in length) of P. rubescens significantly reduced survival of Thamnocephalus platyurus (Crustacea, Branchiopoda, Anostraca) naturally occurring in temporary ponds. In contrast to Thamnocephalus, the survival of co-existing zooplankton was unaffected (Eudiaptomus gracilis (or enhanced (Daphnia hyalina and Cyclops abyssorum). High sensitivity to the microcystins of Planktothrix was coupled to strict food avoidance in Eudiaptomus, but not in Thamnocephalus. Daphnia and Cyclops exhibited higher physiological resistance to cyanobacterial toxins, and ingested Planktothrix. For the lake zooplankton species, the feeding rates on high-quality algae were not significantly reduced in the presence of Planktothrix. In order to separate the effects of mechanical interference (filament length) versus toxins, clearance rates on Planktothrix filaments were compared to clearance rates on filaments subjected to toxin extraction. The results show that microcystins are important feeding deterrents against grazing by Daphnia since feeding rates on Planktothrix increased significantly after an aqueous-methanolic extraction of the major part of microcystins. On the other hand, copepods persisted in food avoidance, but exhibited high clearance rates on Planktothrix after a more lipophilic extraction was applied. Both microcystins and a lipophilic, unidentified toxin may contribute to the avoidance behaviour of copepods. For both Daphnia and copepods, the grazing resistance of Planktothrix is mediated by chemical defences rather than by the large size and the rigidity of the filaments.

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P. Cojocaru
Phytotoxicity of Cadmium and Zinc on Brassica napus, Sinapsis alba and Spinacia oleracea using Two Soil Types
SGEM 2013 Conference Proceedings 2013 619-626
Toxkits : Phytotoxkit
Test Species : Sinapis alba
Topics : soils; chemicals - metals; plant tests

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Abstract : The paper presents studies concerning the toxicity of Cadmium (Cd) and Zinc (Zn), individual and in combination, on seed germination and root elongation of rape (Brassica napus), white mustard (Sinapis alba) and spinach (Spinacia oleracea) using two types of soils, a reference OECD soil and river sand, respectively. The soil was placed in the bottom of the Phytotoxkit plates and then saturated to the water holding capacity (WHC) with Cd(II) and Zn(II) solution. Stock cadmium and zinc solutions were prepared using 3CdSO4.8H2O and ZnSO4.7H2O, respectively. The concentrations in cadmium and zinc of stock solutions were 50 mg/L and 6000 mg/L, respectively. An artificial contamination of soil was achieved by using these solutions with different heavy metal content (adjusted by the following dilution factors: 1; 2; 4; 8; 16). Tap water was used as a dilution agent and it was considered the reference solution. After 3 days of incubation in the dark at a temperature of 25 ± 1 ºC a digital picture of the germinated plants was recorded. Each experimental trial was performed in duplicates while the reference in triplicates. The germination rate in each plate was calculated for each seed and the root length of each plant was measured using ImageTools3.0 software. A linear correlation coefficient (R2) and standard error estimations were used to characterize the linearity of the relationship between pollutant dose and the plant response. Statistica10 software was used to analyze the experimental results from the statistical point of view. The germination rate of the studied plant seeds and the root elongation decreased along with the increasing of the pollutant concentration, both of them depending on the soil and pollutant types used in this study. The most affected seeds were the ones of spinach, which did not germinated for a solution concentration of 10 mg of Cd(II) /L and of 1200 mg Zn(II)/L for OECD soil and respectively of 12.5 mg Cd(II) /L and 1500 mg Zn(II)/L for the river sand.

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G. Nalecz-Jawecki and J. Sawicki
Toxicity of metals to protozoa in the presence of selected pharmaceuticals
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 35
Toxkits : Protoxkit F^TM
Test Species : Tetrahymena thermophila
Topics : chemicals - metals; chemicals - drugs

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Abstract : Pharmaceuticals are by nature biologically active compounds. As they are highly soluble in water and hardly degradable, they have been observed not only in wastewaters, but also in rivers and lakes at concentrations above 1 µg/L. The mechanism of action of drugs in invertebrates is not known. The mechanism of action of some drugs in mammals is associated with cellular pumps. Thus they may influence on the toxicity of metals.
The goal of our study was the evaluation of the toxicity of metals to protozoa in the presence of selected pharmaceuticals: Thioridazine, Amitryptyline, Verapamil and Propranolol. Two ciliated protozoa were used as test-bionts: Spirostomum ambiguum in the acute toxicity test (Spirotox) and Tetrahymena termophila in the chronic toxicity test (Protoxkit F^TM). Two kinds of media were applied: soft and hard water.
All tested drugs increased the toxicity of lead to both species of the protozoa. The highest increase, by 2.5-folds was observed in the Protoxkit F treaded with Verapamil in soft water.On the other hand cadmium toxicity decreased in the presence of drugs, especially of Thioridazine and Verapamil in the Spirotox and Protoxkit F tests, respectively. Propranolol decreased the toxicity of zinc ions towards S. ambiguum, but increased the toxicity of zinc in the Protoxkit F.
The project was financed by Polish Ministry of Scientific Research (grant no 2P05F03226)

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A. Kuczynska, M. Michalska, L. Wolska, and J. Namiesnik
Toxicity Monitoring and Classification of Underground Waters in the Vicinity of the Municipal Landfills
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 153
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : groundwaters; landfill leachates

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Abstract : Waters contain thousands of substances of natural and anthropogenic origin. Taking into account that a great number of them are toxic, the presence of some of them may have negative influence on aquatic ecosystem.
_Although chemical analysis allow to identify and quantify the organic pollutants, they do not provide sufficient information for ecological risk assessment. Furthermore, it is not possible to investigate all substances and their interactions in bodies of water. Therefore, the chemical analysis should be supported by toxicity measurements.This paper reports on the toxicity assessment of underground water samples collected in the vicinity of the waste dumps placed in the Pomeranian voivodship: Bądki (Kwidzyn), Bierkowo (Słupsk), Linowiec (Starogard Gdański), Lucin (Lębork), Rokitki (Tczew), Rybska Karczma (Wejherowo), Swarzewo (Puck). These landfills were activated before 1990. They are not sealed and the amount of collected wastes exceedes 10 th. Mg.
The toxicity of underground waters was measured applying the following tests :
- acute toxicity tests :
* inhibition of bioluminescence of Liquid-dried >i>Vibrio fischeri bacteria - ToxAlert 100^® (Merck, Germany),
* immobilization - Daphtoxkit F^TM magna (Crustacean Toxicity Screening Test for Freshwater),
- chronic toxicity test :
* reproduction - Daphnia magna.
The leachates from two landfills - Lucin and Linowiec, pointed out to the lowest ecotoxicological quality. The leachates from other landfills may be recognized as not creating toxicological hazard.

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E. Mendonça, M.A. Morbey, S. Leitão, L. Silva, A. Picado, F. Brito, C. Cortez, and I. Johnson
Evaluation of Wastewater Toxicity in Trancão River Basin (Portugal)
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 152
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : effluents; waste waters

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Abstract : The integrated evaluation of the effects of industrial discharges in ecosystems is of most importance in the control of wastewaters quality. This approach is as more valuable as the effluent is complex, not only due to the difficulty to analyse all chemicals present in the sample but also because it allows a direct evaluation of potential biological effects.
In the global evaluation the toxicity of the effluent is assessed as a whole using bioassays as a complement to the chemical characterisation substance by-substance._The results presented in this work were obtained during the development of the Demonstration Project ECORIVER - Ecotoxicological Evaluation of Municipal and Industrial Wastewaters LIFE 02/ENV/P/000416 – (2002-2005). The adopted methodologies are supposed to be implemented in other geographical areas densely populated and heavily industrialised.
The main objectives of ECORIVER Project are the characterisation of municipal and industrial wastewaters and of the receiving waters, the selection of the methodologies more adequated for the monitoring of wastewaters and the establishment of ecotoxicological criteria to support decision enabling in a short term the implementation, in Portugal, of a toxicity global evaluation approach.
In this Project 17 industries from different industrial sectors and 2 WWTPs were characterised, after a screening of about 50 industries and 2 WWTPs in which two bioassays of acute toxicity were used. In the selected industries and WWTPs acute and chronic ecotoxicological tests were performed, using species from different trophic levels.
The ecotoxicological evaluation showed, in general, high toxicity values for the test-organisms in wastewater samples, even if in some cases, chemical parameters were under the limits established in the legislation.

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A. Soupilas, Ch. Papadimitriou, D. Papastergiou, P. Samaras, and G.P Sakellaropoulos
Monitoring of Industrial Effluent Ecotoxicity in the Greater Area of Thessaloniki
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 135-136
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM
Test Species : Daphnia magna; Tetrahymena thermophila
Topics : effluents; waste waters

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Abstract : Activated sludge is a common process used in wastewater treatment, based on the development of a complex community of microorganisms into the aeration tank of the treatment units. The maintenance of the activated sludge microbial community is of significant importance in wastewater treatment plants, in order to ensure a continuous and effective operation. The balance between the various microorganism populations is dependent on various functional parameters such as organic loading, detention time and especially on the presence of toxic substances in the influent. The occurrence of toxic substances may be attributed to industrial effluents which are fed to the treatment system and may result in the collapse of activated sludge process; recovery of the unit may take place in extended periods. As a result, during co-treatment of industrial and municipal wastewaters, and effective protection system should be installed based on the measurement of toxic substance content in influents and on the prediction of their effect to the activated sludge microorganisms. The influent quality and the performance of the activated sludge units are conventionally evaluated be the measurement of certain physical – chemical parameters. However these parameters alone may give limited information regarding the actual effects to the biota. Bioassays have been already used for the assessment of water and wastewater quality and may be used as an indication of potential toxic properties of industrial wastewaters.The objective of this work was the assessment of various industrial effluents toxicity in the Greater Area of Thessaloniki by a battery of bioassays in order to investigate their potential impact during co-treatment of industrial and municipal wastewaters. This work was conducted as a result of Water Supply and Sewerage Co efforts for the efficient treatment of industrial effluents in the existing Municipal Wastewater Treatment Plant of Thessaloniki. The battery test included three indicating test-species: the protozoan unicellular organism Tetrahymena thermophilla, the daphnid Daphnia magna and the marine photobacterium Vibrio fischeri. The results that were obtained from the ecotoxicological bioassays showed highly toxic effluents from metal coating, chemical and dying industries; Daphnia magna exhibited the highest sensitivity in all cases while Tetrahymena thermophilla appeared as the most tolerant species. A high variability observed in organism responses, ranging from hormesis up to 100% mortality, during the examination of food industry effluents. However, a specific relation between the chemical analysis data and the bioassay results was not observed, possibly due to the complex nature of industrial wastewaters containing a large number of contaminants, resulting to the development of synergistic and antagonistic toxic effects.

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Ch. Papadimitriou, P. Samaras, and G.P Sakellaropoulos
Toxicity reduction of certain industrial effluents by activated sludge
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 140
Toxkits : Protoxkit F^TM
Test Species : Tetrahymena thermophila
Topics : effluents; waste waters

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Abstract : The objective of this work was the examination of the performance of two activated sludge systems, a conventional continuous stirring tank reactor (CSTR) and a sequential batch reactor (SBR), for the treatment of high strength wastewaters and the assessment of the toxicity reduction potential by bioassays. Both units were fed by wastewaters containing ammonia nitrogen up to 290 mg/L, phenol with a concentration reaching 1000 mg/L and cyanides in concentration of about 300mg/L; these influents simulated certain industrial wastewaters. Both reactors were inoculated by activated sludge from a municipal wastewater treatment plant and the biomass was gradually acclimatized to the synthetic substrate. The operation of the reactors was monitored by measuring the following parameters: BOD5, COD, NH4, NO3, phenol, cyanide, MLSS and SVI. Furthermore detoxification potential of the systems was monitored by employing two bioassays, utilizing the marine photobecterium Vibrio fischeri and the typical activated sludge ciliate protozoan Tetrahymena thermophilla. In all cases, the influent was highly toxic to both organisms used, resulted in almost complete bioluminescence inhibition of V. fischeri and growth inhibition of the protozoan populations respectively. However activated sludge treatment resulted in the reduction of toxicity of the influent. Enhanced toxicity removal was observed in the SBR system, where almost complete degradation of phenol and cyanides was measured and correlated to negligible toxic effects to the test species; however the continuous systems resulted in lower pollutant removal capacities and slight toxicity effects. The continuous system effluent presented toxic properties that corresponded to about 70% bioluminescence inhibition and 60% growth inhibition; the respective values for the SBR system were 10% and 7%. Furthermore, the performance of both systems was enhanced by the addition of powdered activated carbon in the aeration tank. The presence of activated carbon resulted in the upgrade of system performance due to the simultaneous biological removal of pollutants and carbon adsorption; almost negligible values of phenol and cyanide were measured in the effluents while further toxicity reduction was observed in both systems.

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G. Persoone
Recent new microbiotests for cost-effective toxicity monitoring : the Rapidtoxkit and the Phytotoxkit
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 112
Toxkits : Rapidtoxkit^TM; Phytotoxkit
Test Species : Thamnocephalus platyurus; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : rapid tests; soils; plant tests; behavioural tests

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Abstract : During the last two decades, a whole battery of acute and short-chronic microbiotests with micro-algae, protozoans and aquatic invertebrates has been developed at the Laboratory for Biological Research in Aquatic Pollution (recently renamed Laboratory for Environmental Toxicology and Aquatic Ecology) at the Ghent University in Belgium.
The major characteristic of these user-friendly and low cost assays - which were given the generic name “Toxkits” - is their independence of the time consuming and expensive year- round culturing of live stocks of the test species.
All Toxkits indeed make use of “dormant” or “immobilised” stages of selected aquatic test species which can be stored for long periods of time and “hatched” or “deimmobilzed” at the time of performance of the assays to provide the live test organisms for performance of the toxicity tests.
Recently two new Toxkit microbiotests have been worked out in MicroBioTests Inc, the spin-off company of the laboratory at the Ghent University :
a) the Rapidtoxkit, a 1h sublethal assay with the aquatic crustacean Thamnocephalus platyurus, based on the inhibition of uptake of colored particles by the test organisms, upon exposure to toxic stress
b) the Phytotoxkit, a 3 days seed germination and early growth toxicity test with seeds of several higher plants.
The Rapidtoxkit was developed specifically to address the need for “very rapid” toxicity detection in cases of water contamination emergencies or bioterrorism.
The Phytotoxkit is a simple and practical assay for soil contamination which, contrary to all phytotoxicity tests in pots, allows for direct length measurements inside transparent test containers, by image analysis.br>Beyond a variety of “ecotoxicological” applications, the latter microbiotest also has a large potential in agriculture, e.g. for seed quality control and soil quality testing.
Examples of recent applications of these 2 new Toxkit microbiotests will be given and discussed.

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M.D. Petala, P. Samaras, A. Zouboulis, A. Kungolos and G.P. Sakellaropoulos
The effect of advanced treatment on the toxicity of municipal secondary effluents
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 137-138
Toxkits : Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Daphnia pulex; Thamnocephalus platyurus
Topics : effluents; waste waters

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Abstract : The potential of wastewater reuse is greatly determined by the quality of reclaimed water; chemical and physical parameters are currently employed for the characterization of wastewaters. However, the use of these parameters may not be enough for the prediction of the real environmental effect of reclaimed wastewaters in an ecosystem and additional tests may be required. Bioassays have been widely used for the assessment of the water and wastewater quality in combination to chemical analysis and may be applied for the evaluation of reclaimed wastewater toxic properties. In this study, the efficiency of coagulation and ozonation for the production of reclaimed wastewaters was investigated. Secondary effluents were treated with two different coagulants (alum and commercial coagulant PAC18) at metal ion concentrations 0.5 and 1 mmol/L, while the addition of a cationic polyelectrolyte was also investigated. Additionally, the secondary effluents were fed into a vertical ozone bubble column operated in semi-batch mode. The feed gas was introduced at a flowrate of 3 L/min and contained ozone at various concentrations, between 2 and 8 mg/L, while the reaction time was 2, 5, 15 and 30 min. The toxic effects of the advanced treated effluents were examined on test organisms, including the marine bacteria Vibrio fischeri, the freshwater crustaceans Daphnia magna, Daphnia pulex and Thamnocephalus platyurus and the rotifers Brachionus calyciflorus. In addition, chemical analysis of the samples was performed, including the determination of organic content, nitrates, total nitrogen and heavy metals. The removal of the organic content slightly exceeded 40% after the addition of alum at final concentration of 1 mmol Al³⁺/L. On the other hand, ozonation was more efficient and the organic content removal reached up to 60% for ozone dosage of about 6 mg/L, while the removal reached up to 70%, after ozonation at a dosage of 8.2 mg/L. Among the test organisms, Daphnia pulex was more sensitive and presented significant toxicity, mainly after exposure at the secondary effluents. The addition of alum decreased the toxic effect of reclaimed wastewaters on Daphnia pulex, from 90% to 60%, independently of the addition of the polyelectrolyte. Acute toxic effects after ozonation, were observed on Vibrio fischeri and were related to ozone concentration and contact time. At the highest ozone dosage, the toxicity reached almost 100% after 15 min of reaction time. However, the toxicity of the ozonated effluents decreased with storage time and was almost eliminated after storage for 48 h, indicating that the potential adverse effects of reclaimed wastewaters were reduced by storage of reclaimed water for a certain time prior to reuse.

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L. Põllumaa, L. Bityukova, J. E. Sorlie, and A. Kahru
Ecotoxicological Evaluation of Groundwater in Estonian Oil-Shale Region
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 128
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : groundwaters; solid wastes leachates

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Abstract : In Estonia there is biggest industrially used oil-shale basin in the world exploiting of which has caused several environmental problems, e.g., negative water balance and pollution of upper groundwater layers. The latter is very alarming as groundwater is the only source of drinking water in this region. In this study semicoke leachates and groundwaters (altogether 20 samples) were collected and analysed for toxicity using a multitrophic battery of biotests (crustaceans, protozoa, bacteria and algae). According to the most sensitive test / highest toxic signal (MaxTox) the samples were classified as not toxic (<1TU), toxic (1-10 TU), very toxic (10-100 TU) and extremely toxic (>100 TU). In parallel, PAHs, BTX, phenols, heavy metals, pH, conductivity, etc. were measured. The impact of unfavourable pH (alkalinity) and phenols - two major suspected “toxic factors” - was evaluated by Microtox assay.The semi coke dump leachates contained 2.6-195 mg phenols/l, were very alkaline (pH up to 13) and ′very toxic′ to ′extremely toxic′ (MaxTox 14-345 TU). The semi coke heap pore waters contained up to 93 mg/l phenols, up to 19 mg/l of BTX, up to 0.6 mg/l PAHs and were ′toxic′ to ′extremely toxic′ (4 -449 TU). 12 groundwater samples of the upper layer of aquifer from the vicinity of semi coke dumps were studied: 8 samples could be considered not hazardous (MaxTox about 1 TU). However, some groundwater samples contained up to 17 mg phenols/l and were very toxic (24 TU). We showed that the toxicity was caused not only by phenols and alkalinity but there must be other toxicants of concern.

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P. Rakimbei, Tzafeiropoulou K., Karagiannidis A., Tsiouvaras C., Tsiridis V., Samaras P., Keramidas V. , Nastis A.
Inventory of the Accumulated Pollution in the Uncontrolled Landfill of Eukarpia
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 179
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : landfill leachates

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Abstract : The objective of the present project (PENED 2001/01ED626) is the studying and implementation of an ecological and aesthetic restoration of a small uncontrolled landfill, located at the Thessaloniki’s suburbs. The afore-mentioned landfill was established in 1990 to provide final-disposal services for household and industrial solid wastes and continued to operate till 1998. Five years after the closing up of the uncontrolled landfill, various actions were taken within the project that aimed at the aesthetic restoration of the dump site. Taking into account the adjacent industries, their environmental impact and the accumulated pollution of household waste dumping, the following activities were implemented:a) Chemical analyses of sixty-up to 16m deep- excavated samples.b) Monitoring of landfill gas concentrations in and over the landfill body.c) Measuring of the plant growth-rate and physiology in relation to the waste layer underneath.The measurement of heavy metals’ concentration (Cd, Cu, Ni, Pb, Cr, and Zn) in the excavated samples took place after the treatment with HNO3 and according to the leaching test EN12457-2.Furthermore, the correlation between the type of the native soils of the dump, the encumbrance of the soils and the types of dumped wastes is currently estimated.In most of the cases, the concentration of heavy metals in the samples approximate to the corresponding mean value in native soils, however some samples adjacent to solid wastes, have high concentrations compared to the mean value of heavy metals in soils (e.g. the concentration of Cu in a sample, was found 7 times above the mean value in soils and the concentration of Cd, 9 times above). Furthermore, some samples went through the toxicity test of daphnia magna, without presenting any toxicity. As far as the methane concentration, it was not traceable.Finally the successful growth of the plants Festuca Arundinacea and Cynodon Dactylon, demonstrate their high capability in producing a rich and dense sod.

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A. Törökné
Rapid Test for Detecting Cyanobacterial Toxins
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 9
Toxkits : Rapidtoxkit^TM; Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : rapid tests; biotoxins; cyanobacterial blooms; cyanotoxins; behavioural tests

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Abstract : Cyanobacteria occur widely in lakes, reservoirs, ponds and slow flowing rivers. Many species are known to produce toxins, i.e., “cyanotoxins”, a number of which are of concern for health. Cyanotoxins vary in structure and may be found within cells or released into water. There is wide variation in the toxicity of recognized cyanotoxins (including different structural variants within a group: more than 70 variants in microcystins), and there are further toxins remain unrecognized. Chemical analysis of cyanotoxins is not the preferred focus of routine monitoring because of it requires time, equipment and expertise and expensive. It is a high need to detect cyanobacterial toxins with rapid tests in source water because of the safe drinking water supply and safe bathing in natural bathing waters burdened by cyanobacterial blooms or scums.
Previous examinations proved the sensitivity of larvae of the anostracan crustacean Thamnocephalus platyurus to cyanotoxins both to neurotoxins and hepatotoxins as well. This presentation reports on the sensitivity comparison of the (1 hour) Rapidtoxkit and the (24 hour) Thamnotoxkit microbiotest. Both assays make use of freshly hatched larvae of the crustacean. The Rapidtoxkit is a new microbiotest based on the decrease of ingestion of coloured particles by larvae stressed by a short exposure to toxicants.
Fifteen cyanobacterial samples were tested by both microbiotests. Strains and natural bloom samples were analyzed for microcystins and cylindrospermopsin by HPLC. Both toxicity tests were carried out for 15 samples and the correlation coefficient between the two tests is 0.93 representing the usefulness of the Rapidtoxkit and the comparable results.

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S. Vratolis, K. Eleftheriadis, and A. Kungolos
Chemical and Toxic Evaluation of Wet and Dry Deposition Samples from Urban and Industrial Areas
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 94
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : rainwater; air

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Abstract : Wet and dry precipitation samples from the region of Athens (Nea Filadelphia, Demokritos Environmental Laboratory) and the region of Aliveri were analyzed, to detect the concentration of heavy metals (zinc, cadmium, copper, nickel, chromium, molybdenum and arsenic) and there toxic effects.
The three sampling sites are part of the network of N.C.S.R. DEMOKRITOS. This sampling network collects wet and dry precipitation samples for the evaluation of radioactivity levels in Greece.
The first sampling site is situated in Aliveri, a small town in Evia island where the main pollution source is a lignite-fuelled power plant. The second sampling site is situated in Nea Filadelphia, in a park at the northern end of Athens valley and is effected by vehicle emissions due to its proximity to a main motorway. The third sampling site is within the area of N.C.S.R. DEMOKRITOS, in Agia Paraskevi, at the suburbs of Athens. Wet and dry precipitation samples were collected monthly in plastic gauges (diameter 0.32 m, depth 0.13 m). The gauges were inspected daily and a water layer was always maintained, by the addition of distilled water if necessary. The sample deposited is collected at the end of each month and is diluted to a total volume of 5 l.
The samples (filtrated wet and solid) were then analyzed by ICP spectrometry in order to determine there content in heavy metals. At the same time filtrated samples and wet samples including dry deposition were used in toxic assessment experiments (OECD guideline 202) using the crustacean Daphnia magna for time periods of 24 and 48 hours. The heavy metals concentration was too low to conduct the test for diluted samples.
The results showed that significant amounts of these metals were found in the Demokritos region (in wet and dry precipitation, especially copper) and in the Aliveri region (dry precipitation). The most toxic samples were those of Demokritos. That was expected, as in those samples the largest concentrations of pollutants were detected.
The results indicate that OECD guideline 202 test can not be used for the evaluation of wet and dry precipitation as the toxicity observed is very low.
Toxicity appears to be larger for filtrated than for not filtrated samples. This might be due to the fact that natural organic matter, such as humic and fulvic acids, are strong complexing agents that affect the bioavailable copper (Cu²⁺) or other metal concentration.

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K. Wadhia
Cost-Effective Ecotoxicity Testing of Environmental Samples
From : 12^th International Symposium on Toxicity Assessment - Book of Abstracts 2005 116
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Phytotoxkit; Protoxkit F^TM; Rapidtoxkit^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : review

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Abstract : The ecological status of both aquatic and terrestrial ecosystems has been greatly affected by the explosive increase in anthropogenic activity relating to industrialisation and urbanisation. This has been extensively researched and reported. And regulatory agencies in the recent decades have paid increasing amount of attention to the assessment of toxic contamination of the environment, in particular the aquatic ecosystem. The evaluation of toxic impact has seen the development of various methods.These methods include ecological indicators, bioassays and biosensors using microbial species, invertebrates and plants. Standardised protocols for toxicity testing of environmental samples have been issued by international organizations. Two bodies of significant prominence are the Organization for Economic Cooperation and Development (OECD) and the International Organization for Standardization (ISO).The focus on testing and monitoring in the past has been on selected groups of chemicals. Monitoring agencies have now become aware that the analysis of water, effluent, sediment and other solid-phase samples for extensive range of chemicals is both time consuming and expensive and fails to determine the interactive effects.A cost-effective solution has been the development of microbiotests. The Toxkits with microalgae, protozoa and various invertebrates have overcome the problem of dependency of the conventional tests in their reliance on the maintenance of continuous cultures of live stocks. Included in the range of tests in this significant innovation are a direct contact test (Ostracodtoxkit F^TM) and a seed germination/root elongation test (Phytotoxkit F^TM) designed to provide a rapid low-cost phytotoxic evaluation of sediments, soils and other solid-phase samples. The utilisation and benefits of the Toxkit microbiotests will be discussed.In relation to the assessment of potential toxic impact of industrial effluents on treatment processes, nitrification and respiration -inhibition tests provide a pertinent means of evaluating the toxicity implications. These test methods will be described.

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G.A. Plaza, G. Nalecz-Jawecki, K. Ulfig and R.L. Brigmon
The Application of Bioassays as Indicators of Petroleum-contaminated Soil Remediation
Chemosphere 2005 59 289-296
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum
Topics : soil leachates; chemicals - hydrocarbons; bioremidiation; plant tests; soils

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Abstract : Bioremediation has proven successful in numerous applications to petroleum contaminated soils. However, questions remain as to the efficiency of bioremediation in lowering long-term soil toxicity. In the present study, the bioassays Spirotox, Microtox^®, Ostracodtoxkit ^TM, umu-test with S-9 activation, and plant assays were applied, and compared to evaluate bioremediation processes in heavily petroleum contaminated soils. Six higher plants species (Secale cereale L., Lactuca sativa L., Zea mays L., Lepidium sativum L., Triticum vulgare L., Brassica oleracea L.) were used for bioassay tests based on seed germination and root elongation. The ecotoxicological analyses were made in DMSO/H2O and DCM/DMSO soil extracts. Soils were tested from two biopiles at the Czechowice oil refinery, Poland, that have been subjected to different bioremediation applications. In biopile 1 the active or engineered bioremediation process lasted four years, while biopile 2 was treated passively or non-engineered for eight months. The test species demonstrated varying sensitivity to soils from both biopiles. The effects on test organisms exposed to biopile 2 soils were several times higher compared to those in biopile 1 soils, which correlated with the soil contaminants concentration. Soil hydrocarbon concentrations indeed decreased an average of 81% in biopile 1, whereas in biopile 2 TPH/TPOC concentrations only decreased by 30% after eight months of bioremediation. The bioassays were presented to be sensitive indicators of soil quality with chemical monitoring for evaluation of the bioremediation effectiveness and assessing of the contaminated/remediated soils.

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G.A. Plaza, G. Nalecz-Jawecki, K. Ulfig, J. Sawicki and R.L. Brigmon
Evaluation of Leachates Toxicity to Assess the Bioremediation Progress
F.E.B. - Fresenius Environmental Bulletin 2004 13/6 532-536
Toxkits : Ostracodtoxkit F^TM; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Heterocypris incongruens; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : leachates; soil leachates

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Abstract : In this study the impact of bioremediation on heavily petroleum-contaminated soil leachates by measurements of toxicity and physico-chemical parameters was investigated. Leachates were collected from two biopiles located at a Polish oil refinery representing different bioremediation applications. To evaluate the toxicity of leachates the following bioassays were applied : Spirotox test, Protoxkit F^TM, Rotoxkit F^TM, Thamnotoxkit F^TM, Ostracodtoxkit F^TM, Microtox^®, and different plant tests. The measurements were carried out after 48 and 8 months of the bioremediation process for engineered biopile 1 and non-engineered biopile 2, respectively. The leachates from both biopiles were found to be not toxic for most of the test organisms. This was most likely due to reduction in hydrocarbon cnocentrations by bioremediation. The protozoa Spirostomum ambiguum and Tetrahymena thermophila were sensitive to the leachates from biopiles 1 and 2, and toxic effects were noted. The leachate from biopile 2 was 4 times more toxic than that from biopile 1. The seven plant species in the seed germination tests were also sensitive to hydrocarbon concentrations in both biopile leachates. The use of bioassays together with chemical analyses gives the most reliable results for risk analyses, and confirm the effect of active bioremediation reducing the toxicity of contaminated biopile leachates on several test organisms at different trophic levels.

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J. Mankiewicz-Boczek, K. Izydorczyk, M. Kaza and J. Sawicki
Ecotoxicological Analysis of Water Samples from four Different Rivers of Central Poland – a Pilot Study
Abstract - Second National Conference on Ecotoxicology - New Methods in Ecotoxicology - Warsaw, September 2005
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rapidtoxkit; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : surface waters

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Abstract : The goal of this pilot study is the adaptation and application to Polish conditions of a battery of microbiotests representing different taxonomic groups and trophic levels : bacteria (Vibrio fischeri - Microtox^®); protozoans Spirostomum ambiguum (Spirotox) and Tetrahymena thermophila (Protoxkit F^TM); rotifers Brachionus calyciflorus (Rotoxkit F^TM); crustaceans Thamnocephalus platyurus (Thamnotoxkit F^TM and Rapidtoxkit) and Daphnia magna (Daphtoxkit F magna^TM); micro-algae Selenastrum capricornutum (Algaltoxkit F^TM) and higher plants (the macrophyte Lemna minor). The investigations which will be continued in autumn 2005 and in spring 2006 can be helpful for classification and monitoring of the toxicity of rivers. Such studies are important and needed since part of the water of almost all Polish rivers is to date used for agriculture and recreation because of shortage of surface freshwater.
Four rivers of Central Poland were selected for the present study : Pilica River near Tomaszow city as a control, Bzura River, Ner River and Utrata River. Each of these rivers has been subjected to anthropogenic influences in different degrees for several years.
The first physico-chemical analyses showed only slight difference in water temperature, pH, dissolved oxygen and metal concentrations in the four rivers. Conductivity and nutrient concentration (TP, P–PO4, TN, N–NH4, N–NO3) in turn were more different. Tested samples were not toxic for the bacteria, protozoa and rotifers. Low toxicity (effects between 20% and 50%) was observed in a few samples for the crustacean test species, but only after prolongation of the exposure from 24h to 48h. The growth of the micro-algae and of the higher plants was determined on not diluted water samples. The assays were performed in two variants, namely with and without addition of nutrients to the medium. The two variants were applied to obtain information not only on the toxicity of the samples, but also on the potential stimulation of plant growth induced by the nutrient complexes in the medium. The samples with addition of nutrients did not show toxic effects above 20% for both primary producers. For water samples without nutrient spiking no growth inhibition was found for the duckweed, whereas for the micro-algae poor growth was recorded (less than 80% in comparison to the controls). This finding indicates that the content of natural nutrients in the water samples was insufficient to sustain good algal growth.

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I. Czerniawska-Kusza, T. Ciesielczuk, A Cichon and G. Kusza
The Phytotoxkit Microbiotest : Application to Sediment Toxicity Assessment
Abstract - Second National Conference on Ecotoxicology - New Methods in Ecotoxicology - Warsaw, September 2005
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; sediment and soil contact tests; plant tests

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Abstract : Toxkit microbiotests, based on different trophic organisms, have been successfully used for toxicity evaluation of contaminated soils and sediments. The objective of the present study was (1) to assess the toxicity of sediments through comparison of physical-chemical and phytotoxicological analyses, (2) to evaluate the potential of the Phytotoxkit microbiotest for routine sediment toxicity assessment. Sediment samples were collected from three urban canals (Kanał CO, Malina and Młynówka) in the town of Opole in southern Poland. The sediments differed in their granulometric composition and in organic matter content. The following physical-chemical parameters were analyzed with standard methods: granulometric composition, pH, electrical conductivity, organic matter content, oil derivatives, polycyclic aromatic hydrocarbons (PAHs) and heavy metals (Cd, Cr, Cu, Hg, Ni, Pb and Zn). Sediment toxicity was assessed using the standard Phytotoxkit microbiotest based on three species of higher plants: Lepidium sativum, Sinapis alba and Sorghum saccharatum. The two end-points of the Phytotoxkit microbiotest (seed germination and root elongation) were combined in a germination index to allow for a more comprehensive interpretation. The data from this study revealed that (1) the level of sediment contamination varied significantly between particular sampling stations and urban canals. (2) the Phytotoxkit microbiotest was effective in identifying toxic sediments, but the three plant species responded differently in the magnitude of the effects. The order of increasing plant sensitivity to contaminated sediments was L. sativum < S. alba < S. saccharatum. (3) although the highest concentrations of oil derivatives (1.9 - 8.0 g.kg^-1d.m.) and heavy metals (1200 - 1580 mg.kg^-1d.m.) were found in sediments collected from the Kanal CO, the sequence of increasing sediment toxicity was as follows: Kanal CO < Mlynowka < Malina. (4) plant responses and phytotoxic effects were modified by other factors than contaminant concentrations, such as organic matter content and sediment grain size composition.

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G. Nalecz-Jawecki
Ecotoxicological Tests for rapid Evaluation of Water Toxicity : Microtox^® and Rapidtoxkit
Abstract - Second National Conference on Ecotoxicology - New Methods in Ecotoxicology - Warsaw, September 2005
Toxkits : Rapidtoxkit
Test Species : Thamnocephalus platyurus
Topics : rapid tests; behavioural tests

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Abstract : The Microtox^® test was developed in the USA about 25 years ago. This test makes use of luminescent bacteria (Vibrio fischeri) and is the first assay based on test organisms in a cryptobiotic stage. The lyophylised bacteria can be indeed be stored in a refrigerator for several months and “revitalised” at the time of performance of the bioassay. In the presence of toxicants the bacterial metabolism is disturbed causing the decrease of light output. The Microtox^® equipment is composed of an incubator, a very sensitive luminometer and interfaces with software which immediately calculates the percentage effect in the toxicant concentrations used.
The Microtox^® is one of the fastest bioassay systems ever developed – the results are indeed available within 15 to 30 minutes. Bacteria are, however, procaryotic organisms which don’t have a nervous system and are hence not sensitive to some groups of toxicants such as e.g. neurotoxins. In addition, V. fischeri is a marine bacterium and the addition of 2% NaCl which is necessary to make the test medium salty may decrease the toxicity of some chemicals, for example metals.
The Rapidtoxkit is a newly developed rapid bioassay based on the visual observation of sublethal stress of an anostacean crustacean (Thamnocephalus platyurus). The larvae of this crustacean are hatched from dormant eggs (cysts) which can be stored for several months prior to performance of the test. The test bionts are incubated for 15-60 minutes in the suspected water sample after which an artificial “food” – special red, latex microspheres – is added. Intoxicated organisms do not ingest the food and their digestive tract is empty whereas that of the control organisms if colored red.
The Rapidtoxkit - which was developed specifically for rapid detection of water contamination – not only gives a very fast response but its sensitivity is “complementary” to that of the Microtox test. Although the Standard Operational Procedure of this new microbiotest only describes the screening method for application on not diluted water samples, the Rapidtoxkit can, however, - like all other bioassays also be applied with a dilution series for evaluation of EC50 values and toxicity thresholds. Such a study has recently been performed on 29 drugs with different modes of action, with comparison of the sensitivity to that of the 24h Thamnotoxkit microbiotest based on mortality as the effect criterion (Nalecz-Jawecki and Persoone, 2005). The latter study clearly showed that the (sublethal) Rapidtoxkit is a good predictor of important biological effects (mortality) after more prolonged exposure to toxicants.

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B. Sumorok, A. Drobniewska, G. Nalecz-Jawecki, J. Sawicki and A. Zawadzka
The Toxicity of River Sediments and Soil from Floodplains of four Rivers in Central Poland - a Case Study
Abstract - Second National Conference on Ecotoxicology - New Methods in Ecotoxicology - Warsaw, September 2005
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; ; plant tests

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Abstract : River sediments and soil in floodplain areas tend to accumulate various contaminants such as e.g. heavy metals and pesticides. Changes in physical and chemical characteristics in rivers during a flood may make contaminants bioavailable and hence capable of exerting their toxicity. Soils of floodplains constitute a buffer zone for pollutants and floodplains can, therefore, serve as a natural remediation system by reducing the loads of biogens such as phosphorus and nitrogen, and of pollutants discharged into aquatic ecosystems. In addition, the ecotons play an indirect but significant role in water quality management.
In Poland, monitoring programs are based on traditional methods which examine the physical, chemical and biological parameters of water. However, these monitoring programs do not include sediment and soil toxicity testing for environmental quality assessment. The aim of the present study was to evaluate various microbiotests for their potential use as sediment and/or soil quality assessment tools.
Four rivers of Central Poland were selected for the study : Pilica River near Tomaszow city as a control, Bzura River, Ner River and Utrata River. Each of these rivers has been subjected to anthropogenic influences in different degrees for several years.
Samples of sediments and soil were collected in April 2005 and the following microbiotests were used to evaluate their toxicity: Microtox^®-STP (solid phase test) with luminescent bacteria Vibrio fischeri, Spirotox-TBK with the ciliate Spirostomum ambiguum, direct contact Ostracodtoxkit F^TM with the crustacean Heterocypris incongruens and the Phytotoxkit with 3 higher plant species : the monocotyl Sorghum saccharatum and the dicotyls Lepidium sativum and Sinapis alba.
Our preliminary results indicate that the Spirotox-TBK and the Ostracodtoxkit F^TM were the most responsive of all the microbiotests applied. In the Phytotoxkit test the two dicotyl test species were found to be more sensitive than the monocotyl. It was also found that the sediments of the four rivers exhibited greater toxicity than the soils from the floodplain areas. This project will be continued in autumn 2005 and in the 2006.

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P. Fochtman
Toxkit Microbiotests in Effluent Toxicity Testing
Abstract - Second National Conference on Ecotoxicology - New Methods in Ecotoxicology - Warsaw, September 2005
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM short-chronic
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila
Topics : effluents; waste waters

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Abstract : Effluents of a municipal Wastewater Treatment Plant, sampled before and after treatment, were screened for physical-chemical parameters (BOD, COD, suspended solids, N/NH4, P) and for biological effects (Daphtoxkit F magna^TM, Thamnotoxkit F^TM, Algaltoxkit F^TM, Protoxkit F^TM and Rotoxkit F chronic^TM microbiotests). The two data sets (toxicity vs. physical - chemical parameters) were compared by regression analysis and Principal Component Analysis (PCA). Regression analysis revealed some statis-tically significant correlations within and between the groups. PCA, in turn, separated the two sets clearly into consecutive components. Taking into account that in PCA the components are orthogonal (i.e. there is no correlation between them) it can be concluded that physical - chemical parameters alone cannot totally explain potential harmful effects of effluents to the environment. In other words, bioindication (toxicity tests with aquatic organisms) and physi-cal - chemical parameters are complementary and do not give redundant information.
This study also showed that the use of a battery of microbiotests for testing the toxicity of effluents also allows to monitor the efficiency of treatment (effluent toxicity before and after treatment).
Last but not least the microbiotests also proved to be useful for toxicity classification of the effluents according to the Classification system for wastes discharged into the aquatic envi-ronment (Persoone et al., 2003).

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R. Baudo
Conventional Tests and Microbiotests in Ecotoxicology
Biologi Italiani 2005 XXXV 8 5-12
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Protoxkit F^TM; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Ceriodaphnia dubia; Daphnia magna; Daphnia pulex; Heterocypris incongruens; Phaeodactylum tricornutum; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Streptocephalus proboscideus; Thamnocephalus platyurus
Topics : review; test selection

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Abstract : Standardized methodological protocols for toxicity testing (ISO, OECD, ASTM, EPA, …) usually make use of organisms either cultured or maintained in the laboratory, according to some predefined procedure aimed at ensuring result comparability. Moreover, since no single organism is the most sensitive to all possible environmental toxicants, to warrant a proper ecological relevance different tests must be run simultaneously on more than one test organism, by using an appropriate battery of tests which encompasses bacteria, algae, invertebrates and vertebrates.
This means that the laboratory has to spend a lot of time and money to build the infrastructure and maintain all the required cultures.
Unfortunately this is not always possible, for instance for lack of facilities or personnel. It has therefore been suggested to make use, as an alternative, of so-called “microbiotests”.
According to Blaise (1991), a microbiotest can be defined as the exposure of a unicellular or small multicellular organism to a liquid sample in order to measure a specific effect.
Willemsen et al. (1995) specified that a microbiotest should:
- use a volume smaller than 100 mL;
- preferably be available as a kit;
- have a short duration (maximum a few days)
- be performed with the equipment usually available in a laboratory.
This paper briefly describes the most common kits and compares microbiotests with the corresponding “conventional” toxicity tests.

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G. Nalecz-Jawecki and G. Persoone
Toxicity of selected Pharmaceuticals to the anostracan crustacean Thamnocephalus platyurus : Comparison of sublethal and lethal Effect Levels with the 1h Rapidtoxkit and the 24h Thamnotoxkit microbiotest
Environmental Science and Pollution Research 2006 13, 1 22-27
Toxkits : Rapidtoxkit; Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - drugs; rapid tests; behavioural tests

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Abstract :
Background, Aims and Scope.
In view of the limited amount of information on the potential hazard of the ever increasing amounts of drugs in surface waters to aquatic biota, a study was undertaken to determine the effect levels of 28 selected pharmaceuticals to the crustacean test species Thamnocephalus platyurus. The drugs belong to 5 different groups: non steroidal anti-inflammatory agents, biocides, cardiovascular compounds, nervous system drugs and purine alkaloids.
Methods.
Toxicity tests were carried out with the 1h Rapidtoxkit and the 24h Thamnotoxkit microbiotests in order to make a comparison of sublethal effects (visible as stress through absence of feeding) measured after a very short time of exposure (1 h) and lethal effects after prolonged exposure (24 h). Dilution series starting at 200 mg l^-1were prepared and applied, and median effects levels were calculated and transformed into Toxic Units (TU) for easy data comparison.
Results and Discussion.
The toxic effects found have been ranked into 4 arbitrary toxicity classes: not toxic (TU<0.2), low toxicity (0.2 TU10). The toxicity levels noted ranged from virtually no effects for a few of the pharmaceuticals, at the highest concentration tested out, to LC50’s below 1 mg l(-1) (>100 TU) for 3 nervous system drugs (Amitryptiline, Thioridazine and Chlorpromazine). According to the toxicity classification, 17 of the 28 compounds (i.e. 67%), belong to the same class for the lethal and the sublethal tests. More pronounced differences in effect levels between the two assays were observed mainly for the pharmaceuticals which were either not toxic or only slightly toxic at the 200 mg l^-1 level. For 90% of the toxic drugs the ratio between the toxicity values for both tests is below 5.
Conclusion.
An overall correlation coefficient of 0.96 was found between the 2 microbiotests, confirming the good predictive potential of the 1h stress-based Rapidtoxkit in revealing important biological effects (mortality) after more prolonged exposure of the crustacean test species to chemical compounds.
Recommendation and Outlook.
The present study clearly shows that new microbiotests such as the 1h Rapidtoxkit and the 24h Thamnotoxkit are attractive tools for rapid cost-effective screening of \'new\' pollutants such as drugs which may threaten the biological communities of the aquatic environment.

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L. Wolska, A. Kuczyńska, M. Kursa-Mikołajczak and J. Namieśnik
Ecotoxicity of Waste Material from a Metallurgical Industry in Poland
Abstract - Second National Conference on Ecotoxicology - New Methods in Ecotoxicology - Warsaw, September 2005
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : solid wastes

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Abstract : The increasing amount of industrial and municipal wastes, and their illegal or improper storage, cause a serious hazard for aquatic ecosystems and for human health. In Poland, ninety percent of the total amount of industrial wastes originate from the metallurgical industry. One of the old industrial waste lagoons is located close to the bank of the Mała Panew river, near its mouth to the Lake Turawskie. For many years, toxic substances present in the slags, were eluated by rain water and then transported by river water, either dissolved or adsorbed on suspended matter, directly into Lake Turawskie.A preliminary screening evaluation of the hazard caused by the slag dump for the ecosystem of Lake Turawskie, was performed with a biotest battery composed of the acute toxicity test with luminescent bacteria Vibrio fischeri (Microtox^®) and the “direct contact” chronic toxicity test with the ostracod crustacean Heterocypris incongruens (Ostracodtoxkit F).The Ostracodtoxkit F microbiotest was carried out directly on the waste material, according to the Standard Operational Procedure. Microtox tests were performed on aqueous extracts prepared by shaking waste material with aerated distilled water (1:4, v:v) for 24 hours. After centrifugation and filtration (using fiberglass prefilters; pore size: 0.45 µm), the pH of the extracts was adjusted to 6.0 – 8.0 pH. Tests were carried out according to the Basic Test Protocol of the Microtox with four concentrations and one control in each test and measurement of the inhibition of bioluminescence of freeze-dried Vibrio fischeri bacteria after 30 min. Internal quality control tests using zinc sulphate (ZnSO4.7H2O) were run periodically during the study.The data were used to calculate the mean % mortality and growth inhibition of the ostracods, and the EC20 (NOEC) and EC50 for the bacterial bioluminescence assay.All the samples examined were found very toxic to the two types of test organisms, pointing to the serious hazard of the metallurgical wastes discharged into the Mala Panew river. NOEC values for the Microtox were even below 1%. The results obtained with the Ostracodtoxkit F generally confirmed the toxicity levels detected with the Microtox.

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I. Blinova
Use of Freshwater Algae and Duckweeds for Phytotoxicity Testing
Environmental Toxicology 2004 19, 4 425-428
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - metals; chemicals - organic compounds; surface waters

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Abstract : The toxicity of contaminated water of different origins and chemicals [Cr(III), Pb(II), CU(II), pyrene] were tested using four test species : the alga Selenastrum capricornutum (new name Raphidocelis subcapitata), the duckweed Lemna minor, and the crustaceans Thamnocephalus platyurus and Daphnia magna. On the basis of the results obtained, the sensitivity of plant species and problems concerning the interpretation of the phytotoxicity data are discussed. The data indicated that the sensitivities of crustaceans and plant species both to individual contaminants and to mixtures are unpredictable and that there is no reason to consider plant species less sensitive than animal species. Lemna minor is more sensitive than Selenastrum capricornutum. With colored samples, duckweed is preferable for toxicity testing. To raise the predictive utility of the phytoxocity data, it is recommended that natural water be used in the test procedure.

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A. Kahru, A. Ivask, K. Kasemets, L. Pollumaa, I. Kurvet, M. François and H.-C. Dubourguier
Biotests and Biosensors in Ecotoxicological Risk Assessment of Field Soils Polluted with Zinc, Lead and Cadmium
Environmental Toxicology and Chemistry 2005 24, 11 2973-2982
Toxkits : Algaltoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : soils; chemicals - metals; soil leachates

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Abstract : The combined chemical and ecotoxicological hazard evaluation study was conducted on 60 smelter-influenced soils containing 1 to 13, 50 to 653, and 100 to 1,198 mg/kg of Cd, Pb, and Zn, respectively. For these soils (liquid-to-soil ratio = 10), water extractability of Zn, Cd, and Pb was less than 0.19% (median values). Acetic acid (0.11 M) extracted 23, 9.7, and 0.7% of Cd, Zn, and Pb, respectively. Although heavy metal concentrations in the studied soils were high, the toxic effects of water extracts were observed only in few samples and in few biotests (algae Selenastrum capricornutum and metal detector assay). For most of the aquatic test organisms (e.g., crustaceans, photobacteria), the bioavailable concentrations of metals in soil–water extracts were either subtoxic, or the adverse effects were compensated by soil nutrients, etc. However, analysis of the soils with recombinant Cd sensor Bacillus subtilis (pTOO24) showed that about 65% of these apparently subtoxic samples contained bioavailable Cd when analyzed in the suspension assay (detection limit 1.5 mg Cd/kg soil), indicating the desorption of Cd induced by direct contact of bacteria with soil particles. The median bioavailable fraction of Cd (1%) was 23-fold lower than the fraction extracted by acetic acid. The Pb–Cd sensor Staphylococcus aureus (pT0024) detected bioavailable Pb only in the suspensions of five of the most lead-polluted soils (>417 mg Pb/kg): the median bioavailability of Pb was 0.42%. Consequently, the hazard assessment relying on total metal levels in soils should be revised by critical comparison with data obtained from bioassays. Development and use of biosensors (excellent tools for mechanistic studies and signaling hazard already at subtoxic level) should be encouraged.

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L. Põllumaa, A. Kahru and L. Manusadzianas
Biotest- and Chemistry-Based Hazard Assessment of Soils, Sediments and Solid Wastes
JSS - J Soils & Sediments, Vol. 4 2004 267-275
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : soils; sediments; solid wastes; chemicals - metals; chemical - organic compounds; chemicals - hydrocarbons

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Abstract :
Background and Objectives
The current environmental legislation regulating pollution issues is based on total levels of pollutants. This approach is not taking into account the bioavailability of pollutants (that is especially important for an analysis of soils and sediments as heavy metals and hydrophobic organic toxicants tend to sorb to solid matrix) and effects of toxicants in mixtures. Thus, toxicity-based criteria should be added to the currently existing chemical ones for the meaningful evaluation of the environmental hazard. The aim of the current study was 1) to compare the ecotoxicity and chemistry-based environmental evaluations for 27 solid-phase environmental samples (soils, sediments, solid wastes); 2) to suggest the battery of biotests for the screening of water-extracted toxicity.
Methods
14 soils, 9 sediments and 4 oil-shale industry solid waste samples from Estonia and Lithuania were analyzed for the concentration of total PAHs, heavy metals, oil products and water-extracted phenols. The pollution level was evaluated by comparison of measured concentrations with Estonian permitted limit values in residential (PLVr) and industrial (PLVi) areas for each key pollutant.
A battery of 8 aquatic toxicity tests was applied for the analysis of aqueous extracts (L/S=3) of samples : tests with microalgae Selenastrum capricornutum, macroalgae Nitellopsis obtusa, protozoa Tetrahymena thermophila, crustaceans Daphnia magna and Thamnocephalus platyurus, rotifers Brachionus calyciflorus and photobacteria Vibrio fischeri.Particle-bound bioavailable toxicity was evaluated using a kinetic photobacterial assay with Vibrio fischeri (Solid-Phase Flash-Assay). The toxicity data were evaluated by MaxTox index (highest toxic signal of the battery).
Results
Chemical evaluation and toxicological evaluation pointed to the same direction in half of the cases (13/27) : 5 samples (including 2 presumably clean control soils) proved harmless and 8 hazardous to environment in case of both evaluations. However, there was a disagreement between chemical and toxicological evaluations for the rest of the samples (14/27). In two mismatching cases (soils from the territory of former gasoline stations) the level of oil products exceeded the PLVr, but no toxicity was detected, most probably due to the low bioavailability of aged pollutants. It must be taken as a warning that a majority of mismatching cases (12/14) of the samples proved to be toxic or even very toxic despite the fact that the measured hazardous key pollutant levels were below the PLVr. Within these 12 samples were 2 soils from municipal dumping sites, 2 soils from the territory of military airport, 6 sediments from Curonian lagoon (Lithuania) and 2 oil-shale industry solid wastes (Estonia).
Conclusions
The results of this study show the necessity of biotesting in environmental risk assessment to avoid the false-negative results that may result in harmful effects for the ecosystems and also to human health. The following, reduced test battery was proposed for the ecotoxicological hazard assessment of water-extractable toxicity of solid-phase samples : Tetrahymena thermophila growth inhibition assay, Daphnia magna mortality assay and Selenastrum capricornutum growth inhibition assay. Also, the Microtox test (Vibrio fischeri luminescence inhibition assay) could be valuable for screening purposes. For the evaluation of particle-bound, bioavailable toxicity of soil suspensions, Solid-Phase Flash-Assay (test organism Vibrio fischeri) is suggested. Compared to the results obtained with the initial battery of 8 tests, the reduced battery detected the toxicity in 85% of the cases.

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S.B. Sefrioui, G. Persoone and H. Chergui
Toxkits : New Cyst-based Ecotoxicological Tests : Application to Industrial Effluents of The City of Fès (Morocco)
L'eau, l'industrie, les nuisances 1998 216 43-47
Toxkits : Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : effluents; waste waters

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Abstract : The evaluation of toxicity of wastewaters and pollutants with biological tests show one major inconvenient due to the necessity of continuous culturing of live stocks of test organisms in sufficient number and in a healthy state. These biotests as consequent, can only be performed in specialized laboratories because of their high cost. At this moment, it is possible to perform routine toxicity tests that are simple and at low cost by using dormant eggs or cysts of specific species of aquatic biota as starting biological material. These cysts can be stored and preserved for long periods without losing their viability, and hatched within 24 hours.
This study reports on the acute toxicity of industrial effluents (textile, brewery and oil industry) in the city of Fès, with a rotifer (Rotoxkit F^TM) and with a crustacean (Thamnotoxkit F^TM). The results show the potential and the sensitivity of these toxkits, especially the Thamnotoxkit F^TM to estimate the ecotoxicological impact.
From all industries, the oil industry is the most dangerous one as far as pollution is concerned with an LC50 between 0.21 and 1.02 % of effluent.
As a conclusion, we need urgent measures to prevent and control the discharge of industrial effluents.

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S.B. Sefrioui and H. Chergui
Application of (Toxkits) Microbiotests in Toxicity Characterization of Wastewaters of the City of Fès (Morocco)
Journal Européen d'Hydrobiologie 2002 33, 1 101-114
Toxkits : Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Thamnocephalus platyurus
Topics : effluents; waste waters

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Abstract : The assessment of the hazard to the environment from toxic pollutants present in wastewater discharges is in many countries still performed exclusively by chemical analysis. However, during recent years, it has become generaly accepted that chemical data by themselves do not allow to evaluate the global effect of pollutants. In fact, an exclusive chemical approach suffers from a number of shortcomings :
- the large number and the diversity of toxic substances potentially present in the wastes
- the limitation in practice of the number of chemical analyses performed
- the difficulty in extrapolating the real hazard of wastewaters, from the chemical data (different compounds present in different concentrations)
- the inability to predict the interactive effect of the toxicants.
As a result, for a realistic estimation of the hazard of waste discharches, increasing attention is to date focussed on the incorporation of biological tests. Bioassays are generally performed with aquatic organisms such as fish, crustacean or microalgae. Unfortunately, biological testing also suffers from a number of disadvantages, of which the major one is the need for continuous culturing and maintenance of live stocks of test organisms in sufficient numbers and in a healthy state. Consequently, such assays are only carried out in specialized laboratories and in limited numbers because of the high costs. To date, however, it has become possible to do acute testing in routine, at low costs and without any need for sophisticated equipment simply by using dormant eggs or cysts of specific species of aquatic biota as starting biological material. Such cryptobiotic eggs can be stored for long periods without losing their viability and hatched in 24-72 hours of time. This new concept eliminates the (very expensive) continuous culturing or maintenance of test organisms. Standardized tests procedures for acute screening tests have been developed with freshwater and estuarine/marine rotifers and crustaceans. These tests have recently been miniaturized in Toxkits to optimize their cost-effectiveness.
This study shows the acute toxicity of surface waters of 3 rivers in the city of Fès (Morocco). These rivers are receptors of several domestic and industrial effluents. 44 samples were collected from different points within the rivers, for several days and assessed with a battery of new microbiotests (cyst based Toxkits) comprising the Daphnia magna bioassay (Daphtoxkit magna), the Rotoxkit with the freshwater rotifer Brachionus calyciflorus and the Thamnotoxkit test with the freshwater fairy shrimp Thamnocephalus platyurus. Chemical analyses were performed for conventional water quality parameters such as chemical oxygen demand (COD), biological oxygen demand (BOD5), NH3, NH4+, O2 and pH. Toxicity of the samples expressed in toxic units (TU) ranged between 0 and 20. The majority of the examined samples can be classified as toxic for aquatic biota, which requires an emergency control and treatment of the waste waters.
Relationships between the chemical composition and the toxicity of the effluents could be established in some cases, but not in others, which confirms the difficulties of extrapolating toxic hazards of complex wastes from (mostly restricted) chemical analyses.
The present research demonstrates the potential and sensitivity of cost-effective microbiotests (cysts-based Toxkits) as attractive alternatives for routine monitoring of effluents and wastes.

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A. Törökné, B. Olah, M. Reskone, I. Baskay and J. Bercine
Utilization of Microbiotests to Assess the Contamination of Water-Bases
Central European Journal of Public Health 2000 8 Suppl. 97-99
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : leachates; solid waste leachates

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Abstract : In the present study 4 hazardous wastes originated from 4 different typical industrial activities (leachates of waste oil, waste stockpiles of aluminium factory, sewage of leather industry and of the chemical industry) were examined with several Toxkit microbiotests: the Algaltoxkit, the Protoxkit, the Daphtoxkit and the Thamnotoxkit. The data obtained with the microbiotests were compared to those obtained with the standard conventional acute toxicity tests. The goal of the study was to try out the reproducibility of the tests on the same samples, prepared in the same laboratory and provided to all the participants of the interlaboratory exercise. The first reproducibility evaluation of the tests was made with the reference toxicant potassium dichromate (K2Cr2O7). The outcome was that the lowest coefficients of variation were found at the Thamnotoxkit and highest with Algaltoxkit. The same findings were observed with the industrial waste samples. Different responses were obtained depending on the preparation and the character of the waste. For example, whereas a strong (and highest) toxic effect was found with the Thamnotoxkit, the algal cells in the Algaltoxkit test grew better than in the controls, when exposed to the same waste sample. The sensitivity of the Protoxkit ciliates was the lowest for all the wastes examined. In conclusion of this study, the experience gained and data obtained indicate that the Toxkit microbiotests are suited for routine monitoring after modification of some technical aspects.

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U. Viesturs, D. Zarina, S. Strikauska and A. Berzins
Utilisation of Food and Woodworking Production By-products by Composting
Bioautomation 2004 1 83-98
Toxkits : Ostracodtoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Heterocypris incongruens; Thamnocephalus platyurus
Topics : composts

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Abstract : The purpose of the study was to develop laboratory-scale technologies for composting milk/cheese whey, spent liquor, brewery yeast, fish processing by-products, etc., adding these by-products and special microorganism associations to the basic material - sawdust, bark, etc., also arranging different experimental composting sites.
Two Trichoderma strains (Tr. lignorum, Tr. viride) and a nitrification association for regulating the circulation of nitrogen-ammonification and nitrification processes were applied. Monitoring of the composting quality was realised by microbiological and chemical analyses, and biotests for compost quality (toxicity) assessment. For purifying the polluted air from the composting facilities, the biofiltration technique was realised in a modified SSF system. Biodegradation of ammonia was investigated in a two-stage system with the inert packing material - dolomite broken bricks, and hemoautotrophic microorganisms: DN-1 (Pseudomonas sp.), DN-2 (Nitrosomonas sp.), DN-3 (Nitrobacter sp.) and DN-13 (Sarcina sp.). For hydrogen sulphide biodegradation, Thiobacillus thioparus-3 was immobilised on glass bricks as the carrier material.
Biodegradation efficiency of hydrogen sulphide was 87%. Biodegradation of ammonia in the first step in the two-stage system reached 77%, degradation of the gas remaining in the second step was 75%. Compost’s quality was similar to black soil - brown-coloured, with good soil odour and without toxic compounds.

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M. DellaGreca, A. Fiorentino, M. R. Iesce, M. Isidori, A. Nardelli, L. Previtera and F. Temussi
Identification of Phototransformation Products of Prednisone by Sunlight : Toxicity of the Drug and its Derivatives on Aquatic Organisms
Environmental Toxicology and Chemistry 2003 22(3) 534-539
Toxkits : Rotoxkit F^TM; Thamnotoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus; Daphnia magna
Topics : chemicals - drugs

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Abstract : Solar simulator irradiation of an aqueous suspension of prednisone, a widely prescribed drug, produces seven photochemical derivatives. The compounds have been identified on the basis of their physical features. All the chemicals have been tested to evaluate their toxic effects on freshwater organisms from different trophic levels. The rotifer Brachionus calyciflorus and two crustaceans, the cladoceran Daphnia magna and the anostracan Thamnocephalus platyurus, were used to perform acute toxicity tests. Chronic toxicity tests have been performed on the alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) and the crustacean Ceriodaphnia dubia. The results showed low acute and chronic toxicity of prednisone. Some of the photoproducts had high toxic effects on C. dubia.

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E. Vindimian, J. Garric, P. Flammarion, E. Thybaud and M. Babut
An Index of Effluent Aquatic Toxicity designed by Partial Least Squares Regression, using Acute and Chronic Tests and Expert Judgements
Environmental Toxicology and Chemistry 1999 18 2386-2391
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : effluents; toxicity index (T.I.); toxicity classification; decision support system

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Abstract : The evaluation of the ecotoxicity of effluents requires a battery of biological tests on several species. In order to derive a summary parameter from such a battery, a single endpoint was calculated for all the tests: the EC10, obtained by nonlinear regression, with bootstrap evaluation of the confidence intervals. Principal component analysis was used to characterize and visualize the correlation between the tests. The table of the toxicity of the effluents was then submitted to a panel of experts, who classified the effluents according to the test results. Partial least squares (PLS) regression was used to fit the average value of the experts\' judgements to the toxicity data, using a simple equation. Furthermore, PLS regression on partial data sets and other considerations resulted in an optimum battery, with two chronic tests and one acute test. The index is intended to be used for the classification of effluents based on their toxicity to aquatic species.

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P. Radix, M. Léonard, Ch. Papantoniou, G. Roman, E. Saouter, S. Gallotti-Schmitt, H. Thiébaud and P. Vasseur
Comparison of Brachionus calyciflorus 2-d and Microtox® Chronic 22-h Tests with Daphnia magna 21-d Test for the Chronic Toxicity Assessment of Chemicals
Environmental Toxicology and Chemistry 1999 18(10) 2178-2185
Toxkits : Rotoxkit F^TM
Test Species : Brachionus calyciflorus
Topics : chemicals - inorganic compounds; chemicals - organic compounds; test sensitivity

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Abstract : The Daphnia magna 21-d test may be required by European authorities as a criterion for the assessment of aquatic chronic toxicity for the notification of new substances. However, this test has several drawbacks. It is labor-intensive, relatively expensive, and requires the breeding of test organisms. The Brachionous calyciflorus 2-d test and Microtox® chronic 22-h test do not suffer from these disadvantages and could be used as substitutes for the Daphnia 21-d test for screening assays. During this study, the toxicity of 25 chemicals was measured using both the Microtox chronic toxicity and B. calyciflorus 2-d tests, and the no-observed-effect concentrations (NOECs) were compared to the D. magna 21-d test. The Brachionus test was slightly less sensitive than the Daphnia test, but the correlation between the two tests was relatively good (r² = 0.88). On average, the Microtox chronic test presented the same sensitivity as the Daphnia test, but the results did not correlate as well (r² = 0.54). The B. calyciflorus 2-d test, and to a lesser extent the Microtox chronic 22-h test, were able to predict the chronic toxicity values of the Daphnia 21-d test. They constitute promising cost-effective tools for chronic toxicity screening.

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D.J. Versteeg, D.T. Stanton, M.A. Pence and C. Cowan
Effects of Surfactants on the Rotifer Brachionus Calyciflorus in a Chronic Toxicity Test and in the Development of Qsars
Environmental Toxicology and Chemistry 1997 16(5) 1051-1058
Toxkits : Rotoxkit F^TM
Test Species : Brachionus calyciflorus
Topics : chemicals - surfactants

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Abstract : The toxicity of a range of surfactant and surfactant-related compounds was investigated in the 2-d whole life cycle bioassay with the rotifer, Brachionus calyciflorus. Compounds were selected to gain an understanding of how structural components, especially the polar head groups, contribute to toxicity. Rotifers were exposed under static test conditions to the 22 compounds for 2d. Exposure concentrations were verified analytically in the test system. Results demonstrate a relationship between alkyl chain length and toxicity within a surfactant class. Between classes, N-containing amines and quaternary ammonium compounds had greatest toxicity, in general, followed by the nonionic compounds. Anionic compounds were typically least toxic. A good quality (R² = 0.86), three-variable, parametric QSAR model was developed using the ADAPT software package. The model contains one variable to account for the contribution of the hydrophobic tail group to observed toxicity (the number of sp³-hybridized carbons bonded to two other carbons [2SP3]) and two descriptors, the valence-corrected, fourth-order cluster index (⁴X^vC) and a count of the number of nitrogens in the molecule (NN), to account for the contribution of the polar head group.

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B. Ferrari, R. Mons, B. Vollat, B. Fraysse, N. Paxéus, R. Lo Giudice, A. Pollio and J. Garric
Environmental Risk Assessment of six Human Pharmaceuticals : are the current Environmental Risk Assessment Procedures Sufficient for the Protection of the Aquatic Environment ?
Environmental Toxicology and Chemistry 2004 23 (5) 1344-1354
Toxkits : Rotoxkit F^TM short-chronic
Test Species : Brachionus calyciflorus
Topics : chemicals - drugs; regulatory testing

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Abstract : In this study, exposure and ecotoxicity data of six human pharmaceuticals (carbamazepine, clofibric acid, diclofenac, ofloxacin, propranolol, and sulfamethoxazole) were collected, including our own experimental data and literature data. From this data collection, the two-tiered European draft guideline on the environmental risk assessment of human pharmaceuticals was tested. Measured environmental concentrations in effluents from France and in effluents and surface waters from Germany were compared to the predicted environmental concentrations (PECs) in both countries. In a similar manner, predicted no-effect concentrations (PNECs) derived from acute data and PNECs derived from chronic data were estimated for each pharmaceutical and corresponding PEC/PNEC ratios then were compared in both countries. Globally, results demonstrated that all environmental concentrations (predicted or measured) for each considered pharmaceutical exceeded the 10-ng/L cutoff value, which requires the implementation of the second-tier assessment based on ecotoxicity data. Moreover, the six pharmaceuticals showed a relatively limited acute toxicity, and carbamazepine and propranolol were inaccurately identified as having negligible risks under the current European draft procedure. Such results lead to discussion of the actual procedure on pharmaceuticals, especially on the need of appropriate ecotoxicity tests.

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M. DellaGreca, A. Fiorentino, M. Isidori and A. Zarrelli
Toxicity Evaluation of Natural and Synthetic Phenanthrenes in Aquatic Systems
Environmental Toxicology and Chemistry 2001 20 (8) 1824-1830
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM pulex; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - plant extracts

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Abstract : Seven natural 9,10-dihydrophenanthrenes were isolated from the common reed Juncus effusus by means of chromatographic processes and identified by spectroscopic means. Furthermore, mimics of natural isolated compounds were synthesized to try to evaluate the influence of functional groups on the dihydrophenanthrene skeleton. Syntheses of compounds were based on the cross-coupling of 1-(2-iodo-5-methoxy)phenyl-ethanol with variously substituted iodobenzenes by zerovalent nickel. All the chemicals were tested to evaluate their effects on freshwater organisms from different trophic levels. Toxicity tests were performed on reducers (the bacterium Escherichia coli); producers (the alga Raphidocelis subcapitata, previously known as Selenastrum capricornutum); and consumers including a rotifer (Brachionus calyciflorus), a cladoceran (Daphnia pulex), and an anostracan (Thamnocephalus platyurus). Results suggested no one organism was uniquely sensitive to the chemicals tested. Toxicity depended on the kind and position of substituents on the aromatic skeleton.

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A.E. Girling, D. Pascoe, C.R. Janssen, A. Peither, A. Wenzel, H. Schafer, B. Neumeier, G.C. Mitchell, E.J. Taylor, S.J. Maund, J.P. Lay, I. Jüttner, N.O. Crossland, R.R. Stephenson and G. Persoone
Development of Methods for Evaluating Toxicity to Freshwater Ecosystems
Ecotoxicology and Environmental Safety 2000 45 (2) 148-176
Toxkits : Protoxkit F^TM; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Tetrahymena thermophila
Topics : review; surface waters; test validation; tests sensitivity; chemicals - inorganic compounds; chemicals - organic compounds; chemicals - pesticides

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Abstract : This article presents a summary of a collaborative research program involving five European research groups, that was partly funded by the European Commission under its Environmental Research Program. The objective of the program was to develop aquatic toxicity tests that could be used to obtain data for inclusion at Level 2 of the Risk Evaluation Scheme for the Notification of Substances as required by the 7th Amendment to EC Directive 79/831/EEC. Currently only a very limited number of test methods have been described that can be used for this purpose and these are based on an even smaller number of test species. Tests based upon algae (Chlamydomonas reinhardi, Scenedesmus subspicatus, and Euglena gracilis), protozoa (Tetrahymena pyriformis), rotifera (Brachionus calyciflorus), crustacea (Gammarus pulex), and diptera (Chironomus riparius) were developed. The tests encompassed a range of end points and were evaluated against four reference chemicals: lindane, 3, 4-dichloroaniline (DCA), atrazine, and copper. The capacity of the tests to identify concentrations that are chronically toxic in the field was addressed by comparing the effects threshold concentrations determined in the laboratory tests with those determined for similar and/or related species and end points in stream and pond mesocosm studies. The lowest no-observed-effect concentrations (NOEC), ECx, or LCx values obtained for lindane, atrazine, and copper were comparable with the lowest values obtained in the mesocosms. The lowest chronic NOEC determined for DCA using the laboratory tests was approximately 200 times higher than the lowest NOEC in the mesocosms.

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P. Radix, M. Leonard, Ch. Papantoniou, G. Roman, E. Saouter, S. Gallotti-Schmitt, H. Thiebaud and P. Vasseur
Comparison of four chronic Toxicity Tests using Algae, Bacteria and Invertebrates assessed with Sixteen Chemicals
Ecotoxicology and Environmental Safety 2000 47 (2) 186-194
Toxkits : Rotoxkit F^TM short-chronic
Test Species : Brachionus calyciflorus
Topics : chemicals - inorganic compounds; chemicals - organic compounds

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Abstract : The performances of four chronic toxicity tests, comprising the Daphnia magna 21-day (d) (crustacean), Brachionus calyciflorus 2-d (rotifer), Pseudokirchneriella subcapitata 72-h (green algae), and the Microtox chronic 22-h (bacteria) tests, were compared. Sixteen chemicals with toxicity covering 6 orders of magnitude were studied. Very high correlations were found between the NOEC/EC10 Pseudokirchneriella 72-h, NOEC/EC10 Brachionus 2-d, and the NOEC Daphnia 21-d tests. The toxicological response of rotifers and microalgae were within the same order of magnitude as the response of Daphnia in 80% of cases (13/16 chemicals). The Microtox chronic test also anticipated the overall results of the Daphnia 21-d test, but the prediction was rather imprecise, compared with microalgae and rotifers. The test measuring the algal growth inhibition of P. subcapitata after 72h was the most sensitive bioassay. Toxicity on microalgae after 72h could be estimated after 5h by measuring either the direct fluorescence of either photosynthetic pigments or fluorescein diacetate in 56 and 43% of cases, respectively. The median value of the ratio between EC10 and EC50 was 3.75, 2, and 1.5 with the algae, the rotifers, and the bacteria, respectively.

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A. Rouvalis, J. Iliopoulou-Georgudaki and G. Lyberatos
Application of two Microbiotests for acute Toxicity Evaluation of Olive Mill Wastewaters
Parlar Scientific Publications 2004
Toxkits : Daphtoxkit F^TM pulex; Thamnotoxkit F^TM
Test Species : Daphnia pulex; Thamnocephalus platyurus
Topics : waste waters; effluents

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Abstract : Two microbiotests (Thamnotoxkit F^TM and Daphtoxkit F^TM pulex) were used to evaluate the acute toxicity of olive mill wastewaters (traditional and continuous processes) in the Achaia prefecture in Greece. Acute toxicity values of olive mill wastewater (OMW) ranged from 3.3-8.9 % in Thamnotoxkit F^TM and 1.7-12.4 % in Daphtoxkit F^TM pulex, expressed as LC50 and EC50 values, respectively. According to the values obtained, all samples were evaluated from toxic to very toxic for both species tested. Significant correlation was obtained between LC(E)50 and total phenols, total suspended solids and density, while lignin appears to influence only Daphtoxkit F^TM pulex. Despite the detected significant correlation between the two microbiotests, this study compares the sensitivity of both toxkits, which was found to be higher for Daphtoxkit F^TM. But for an accurate routine biomonitoring of OMW toxicity the use of both toxkits can be suggested.

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S. Jurkoniene, G. Maksimov, J. Darginaviciene, K. Sadauskas, R. Vitkus and L. Manusadzianas
Leachate Toxicity Assessment by Responses of algae Nitellopsis obtusa Membrane ATPase and Cell Resting Potential, and with Daphtoxkit F magna Test
Environmental Toxicology 2004 19-4 403-408
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : solid waste leachates; toxicity monitoring

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Abstract : A microscale bioassay based on 50% inhibition of K(+), Mg(2+)-ATPase activity in a microsomal fraction isolated from Nitellopsis obtusa cells was developed. Compared to that for a plasma membrane fraction purified in a sucrose gradient, the preparation procedure for a microsomal fraction is less time consuming and the yield is substantially higher. Characteristics of the microsomal preparation proved to be similar to those of the highly purified plasma membrane preparation (Manusadzianas et al., 2002), at least for heavy metals. Sensitivity to CuSO(4) of the frozen (-8 degrees C) microsomal fraction [49 + 17 (SD) microM; n = 8] did not significantly differ from that of the freshly isolated one (52 + 30, n = 8), at least for 40 days. Toxicity of leachate water from Kairiai (northern Lithuania) solid waste landfill was assessed by taking samples from various points including temporary reservoirs and analyzing them immediately after spillage (summer 2002) and after storage for almost 2 years at 4 - 6 degrees C. Two tests with the macrophytic alga Nitellopsis obtusa (Charatox, 45-min EC(50) of resting potential depolarization, and ATPase assay, IC(50) of membrane ATPase activity) and one test with the crustacean Daphnia magna (Daphtoxkit F, 48-h 50% immobilization) tests were used. In general, all three tests showed successively decreasing values of landfill leachate toxicity with an increasing degree of dilution with surface waters. The possibility of employing preserved algal preparations on demand in test batteries seems to be promising, especially in emergencies.

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K.A.C. De Schampelaere, D.G. Heijerick and C.R. Janssen
Cross-phylum Comparison of a Chronic Biotic Ligand Model to Predict Chronic Toxicity of copper to a Freshwater Rotifer, Brachionus calyciflorus (Pallas)
Ecotoxicology and Environmental Safety 2006 63 - 2 189-195
Toxkits : Rotoxkit F^TM short-chronic
Test Species : Brachionus calyciflorus
Topics : chemicals - metals

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Abstract : Short chronic 48-h toxicity tests with the freshwater rotifer Brachionus calyciflorus (Pallas) were conducted to assess the modifying effects of pH and natural dissolved organic carbon (DOC) concentration on reproductive toxicity of Cu. Toxicity tests were carried out in four test waters according to a 2×2 design, in which pH (6 and 7.8) and DOC (5 and 15 mg C/L) were the test variables. Concentrations of dissolved Cu with no observed effect at 48 h (NOEC) varied 12-fold between 8.2 and 103 ug/L. Higher DOC and higher pH resulted in a reduction of toxicity, which is in line with the concepts of the biotic ligand model (BLM). A chronic Cu-BLM, originally developed for the cladoceran Daphnia magna, was calibrated to the rotifer dataset and was found to be able to predict all rotifer NOECs with an error factor of less than 1.6. This finding may be of great interest for risk assessment and the establishment of water quality criteria, as it suggests that chronic Cu-BLMs are comparable across phyla (i.e., arthropoda to rotifera).

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M.R. Iesce, F. Cermola, M.L. Graziano, S. Montella, L. Di Gioia and M. Isidori
Sensitized Photooxygenation of the Fungicide furalaxyl
Environmental Science and Pollution Research 2004 11 - 4 222-226
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - pesticides; toxicity monitoring

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Abstract :
BACKGROUND :
The photolysis of pesticides is of high current interest since light is one of the most important abiotic factors which are responsible for the environmental fate of these substances and may induce their conversion into noxious products. The action of light can also be mediated by oxygen and synthetic or naturally occurring substances which act as sensitizers. Our objective in this study was to investigate the photochemical behaviour of the systemic fungicide furalaxyl in the presence of oxygen and various sensitizers, and to compare the toxicity of the main photoproduct(s) to that of the parent compound. Previous reports on the direct photolysis of the pesticide demonstrated a very slow degradation and the only identified photoproducts were N-2,6-xylyl-D,L-alaninare and 2,6-dimethylaniline.
METHODS :
Solutions of furalaxyl in CH3CN were photooxygenate using a 500W high-pressure mercury lamp (through a Pyrex glass filter, lambda>300 nm) or a 650W halogen lamp or sunlight and the proper sensitizer. When sunlight was used, aqueous solutions were employed. The photodegradation was checked by NMR and/or GC-MS. The photoproducts were spectroscopically evidenced and, when possible, isolated chromatographically. Acute toxicity tests were performed on the rotifer Brachionus calyciflorus, the crustacean cladoceran Daphnia magna and the anostracan Thamnocephalus platyurus, while chronic toxicity tests (sublethal endpoints) comprised a producer, the alga Pseudokirchneriella subcapitata and the crustacean Ceriodaphnia dubia, as a consumer.
RESULTS AND DISCUSSION :
In the presence of both oxygen and sensitizer, furalaxyl underwent rapid photochemical transformations mainly to N-disubstituted formamide, maleic anhydride and a 2(5H)-furanone derivative. The formation of these products was rationalized in terms of a furan endoperoxide intermediate derived from the reaction of furalaxyl with active dioxygenated species (singlet oxygen, superoxide anion or ground state oxygen). The 2(5H)-furanone exhibited a higher toxicity than the parent compound.
CONCLUSION :
This work reports the first data on the photosensitized oxygenation of furalaxyl with evidence of the high tendency of the pesticide to undergo photodegradation under these conditions leading, among other things, to a 2(5H)-furanone, which is more toxic than the starting furalaxyl towards aquatic organisms.
RECOMMENDATIONS AND OUTLOOK :
Investigation highlights that the photolytic fate of a pesticide, although quite stable to direct photoreaction due to its low absorption of solar radiation at ground level, can be significantly influenced in the environment by the presence of substances with energy or electron-transfer properties as natural dyes, e.g. chlorophyll, or synthetic pollutants, e.g. polycyclic aromatic hydrocarbons (PAH).

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M. Isidori, M. Lavorgna, A. Nardelli and A. Parrella
Chemical and Toxic Evaluation of a Biological Treatment for Olive-Oil Mill Wastewater using Commercial Microbial Formulations
Applied Microbiology and Biotechnology 2004 64 - 5 735-739
Toxkits : Daphtoxkit F^TM magna; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna
Topics : wastewater; effluents; toxicity monitoring

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Abstract : Olive-oil-mill wastewater (OMW) has significant polluting properties due to its high levels of chemical oxygen demand (COD), biochemical oxygen demand (BOD), and phenols. In the present study, different commercial bacterial formulations were used in the biological treatment of OMW. COD and toxicity testing using primary consumers of the aquatic food chain (the rotifer Brachionus calyciflorus and the crustacean Daphnia magna) were employed to evaluate abatement of the organic load and reduction of the toxic potential. In addition, the four most active formulations were tested mixed pair-wise on the basis of their unique characteristics in order to evaluate the improvement of treatment. The effect of treatment was assessed by measuring COD removal, reduction of total phenols, and decreased toxicity. The results obtained with the mixed formulations showed that the maximum removal of the organic load was about 85%, whereas phenols were reduced by about 67%. The toxicity for rotifers decreased by 43% and for crustaceans by about 83%.

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B. Marsalek and L. Blaha
Comparison of 17 Biotests for Detection of Cyanobacterial Toxicity
Environmental Toxicology 2004 19 - 4 310-317
Toxkits : Daphtoxkit F^TM pulex; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia pulex; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : biotoxins

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Abstract : The aim of the present study was to compare the sensitivity of 17 acute bioassays of cyanobacterial toxicity by assessment of crude extracts of three cyanobacterial samples (all dominated by Microcystis sp. but substantially differing in microcystin-LR content). Toxicity of the fractions prepared by solid-phase extraction (SPE) for microcystins was also determined. The most sensitive bioassay was the 24-h test with crustacean Thamnocephalus platyurus, which elicited high lethality in the samples and also in fractions without microcystins. The fruit fly Drosophila melanogaster, protozoans Spirostomum ambiguum and Tetrahymena termophyla, and the crustacean Daphnia pulex formed the second group of sensitive bioassays. Good selective toxicity response to microcystins also was observed in the weakly sensitive biotests with the oligochaete Tubifex tubifex and the rotifer Brachionus calyciflorus. Preconcentration of microcystins by SPE substantially decreased variation of the results in bioassays and improved the discriminating potential of most assays employed.

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S.M. Paixão, E. Mendonça, A. Picado and A.M. Anselmo
Acute Toxicity Evaluation of Olive Oil Mill Wastewaters : A Comparative Study of Three Aquatic Organisms
Environmental Toxicology 1999 14, 2 263-269
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : wastewaters; effluents

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Abstract : Acute toxicity of olive mill wastewaters (traditional and continuous processes) collected from different regions of Portugal was evaluated using three test species (Vibrio fischeri formerly Photobacterium phosphoreum, Thamnocephalus platyurus, and Daphnia magna) and correlated with several physical and chemical parameters. Acute toxicity of these effluents, expressed in LC50 or EC50, ranged from: 0.16 to 1.24% in Microtox test, 0.73 to 12.54% in Thamnotoxkit F test, and 1.08 to 6.83% in Daphnia test. These values reflect the high toxicity of the olive mill wastewaters to all test species. Statistical analysis of the results shows a high correlation between the two microcrustacean bioassays. Microtox test did not correlate significantly with the other bioassays used. A significative correlation (p 0.05) could also be established between L(E)C50 obtained in the microcrustacean tests and some physicochemical parameters of the effluent.

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M. Bartoszewicz, H. Mazur-Marzec, M. Michalska, and J. Kobos
Ecotoxicological Testing in Studies on Toxicity of Cyanobacterial Blooms
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Artoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Thamnocephalus platyurus
Topics : biotoxins; cyanobacterial blooms; toxicity monitoring; cyanotoxins

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Abstract : Cyanobacterial blooms, which is a natural phenomenon occurring in seawater as well as freshwater, have intensified alarmingly in recent years; this is due to significant eutrophication and overall pollution of waters. Nodularia spumigena and Microcistis aeruginosa are the two toxic species that predominate in algal blooms. The presence of these blue-green algae in water may be hazardous to the health of humans and animals alike, particularly in freshwater reservoirs used for the abstraction of drinking water, and in bathing waters adjacent to the shores of inland and marine water bodies. Nodularins and microcystins are detected and identified by means of chromatography, namely HPLC. However, cyanobacteria do not always produce toxic substances, therefore at the preliminary stage of surface waters monitoring it is more important to determine whether they do; the identification of toxins is of secondary importance. Moreover, the results of chemical analyses do not define the toxicity of the detected substances sufficiently.
The aim of the presented study was to investigate the possible application of specific ecotoxicological tests for rapid evaluation of water toxicity during cyanobacterial blooms. The parallel studies were conducted which employed ecotoxicological and chemical methodology; it was assumed that such complementary application of different methods should allow the adequate assessment of a negative impact from harmful metabolic products of blue-green algae. The toxicity level in water samples was evaluated by ecotesting while chemical methods were used for identifying the cyanobacterial toxins. The applicability of ecotesting was also assessed based on a number of laboratory experiments. Moreover, the collected data allowed the selection of most useful tests (in reference to rapidity and properly chosen test organisms) for evaluating the risk from cyanobacterial toxins.The results obtained prove that the Thamnotoxkit and Artoxkit microbiotests are useful tools in measuring ecotoxicity in monitoring programmes.
See corresponding Poster

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C. Blaise and J.F. Férard
Microbiotests in Aquatic Toxicology: the Way Forward
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits :
Test Species :
Topics : review

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Abstract : The industrial revolution has driven the need for ecotoxicology and shaped its evolution. Indeed, increased use and transformation of (non)renewable resources for over one century to benefit mankind have had a downside and created a plethora of contaminants harmful to receiving environments. With time, we have gone from an age of darkness in the 1950s (i.e., diagnostic ignorance in terms of recognizing and dealing with contamination) to one of enlightenment as the 21rst century unfolds (i.e., use of tools and strategies to identify and correct environmental pollutions). Effects measurements, reflected by toxicity testing conducted at different levels of biological organization, have especially proven useful to achieve proper hazard/risk assessments of contaminants. Knowing why toxicity testing has been conducted over the past decades to protect and conserve freshwater environments is also essential to grasp the importance and breadth of this field. For this purpose, we have recently reviewed a substantial number of articles describing numerous bioanalytical endeavours undertaken to comprehend toxic effects associated with the discharge of xenobiotics to aquatic environments. Scrutiny of publications identified in our literature search has enabled us to uncover the various ways in which laboratory toxicity tests have been applied, many of which are small-scale in nature. In essence, freshwater toxicity testing has significantly focussed on liquid (complex environmental samples, chemical and biological contaminants) and solid media (sediments) assessment. For both media, miscellaneous studies/initiatives linked to toxicity testing applications have again promoted the development, validation, refinement and use of toxicity testing procedures. Bioassays are clearly an essential component of environmental management programs and several small-scale tests (microbiotests) can be employed to generate cost-effective toxicity data that assist decision-making.

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I. Czerniawska-Kusza, T. Ciesielczuk and G. Kusza
Ecotoxicological and Chemical Assessment of Contaminated Sediments
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Algaltoxkit F^TM; Phytotoxkit
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; sediment and soil contact tests; sediment pore waters; plant tests

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Abstract : Bioassays provide useful tools for ecotoxicological risk assessment of contaminated sediments. In contrast to chemical analyses, which mainly focus on the qualitative and quantitative determination of contaminants in environmental samples, biological tests detect combined effects of different pollutants, their mobility and bioavailability.
The objective of the present study was to evaluate the extent of environmental contamination of sediments using combined chemical analyses and toxicity bioassay techniques. The research also aimed at estimation of the suitability of the recently developed Phytotoxkit microbiotest as an adequate tool for hazard assessment ofriver sediments. Additionally, the influence of organic matter and sediment grain size composition on phytotoxicity was investigated. For general classification scenery of pollution, sediment samples of nine sites in three canals and rivers located in Opole region (southern Poland) were collected to determine the concentrations of heavy metals (Cd, Cr, Cu, Zn, Ni, Pb, and Hg), oil derivatives and PAH’s. The toxicity of both pore water and whole sediments was assessed using two microbiotests, respectively, Algaltoxkit F^TM with Selenastrum capricornutum and Phytotoxkit with Lepidium sativum, Sinapis alba and Sorghum saccharatum.
The outcome of this study revealed that (1) the level of sediment contamination varied significantly among particular sampling sites, (2) absence of pore water toxicity was not always consistent with the results of the whole sediment toxicity test performed with higher plants, (3) the Phytotoxkit microbiotest was effective in identifying toxic sediments, but the three plant species responded differently in the magnitude of the effects, (4) phytotoxic effects were modified by organic matter content and sediment grain size composition.
See corresponding Poster

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A. Drobniewska, B. Sumorok, G. Nalecz-Jawecki and J. Sawicki
The Seasonal Changes of the Toxicity of Sediments and Soil from Rivers and Floodplains in Central Poland and the Sewage Applied on the Willow Plantation - a Case Study
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : toxicity monitoring; sediments; sediment and soil contact tests; sludges; plant tests

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Abstract : Development of agriculture and urbanization contribute to the decrease of freshwater ecosystems quality e.g., by the accumulation of contaminants in rivers’ sediments.
During flooding periods, sediments are also deposited on floodplains, contributing to the contamination of the rivers\' valleys. These areas serve as natural remediation systems, reducing loads of suspended matter, nutrients and pollutants in rivers. That makes them an excellent tool for the protection of freshwater ecosystems against eutrophication and pollution. The remediation system can be also used for the utilization of sewage. The wastewater treatment plant produces a lot of sewage sludge, utilization of which causes economic and ecological issue. The composition of sewage makes it possible to use it for non-food agriculture, e.g., willow plantation, if the heavy metal kept within limits according to the legislation.Remediation processes depend on biological processes, especially on the uptake by plant tissues and the degradation by microbial activity in the rhizosfere. Exposition of microbial communities to pollution may result to temporary decrease in their species diversity and change their metabolic functions. Therefore they may be used as bioindicators for the changes in the soil. On the other hand, many microorganisms demonstrate increased resistance to presence of metals in the environment, thus remain unaffected until high concentrations of pollutants are reached. Considering those metabolic adaptations and characteristics, the efficiency of natural remediation processes in the floodplain and also in the willow plantation may be difficult to assess. The assessment can be performed by applying ecotoxicological bioassays. The aim of the presented study is to indicate application of biotests battery and respiration test as tools for assessment of the efficiency of natural remediation in degraded rivers\' valleys.
Samples were collected in autumn (2005) and spring (2005, 2006) and the following microbiotests were used to evaluate their toxicity: Microtox^®-SPT, Spirotox-SPT, Ostracodtoxkit F^TM and the Phytotoxkit. Our preliminary results indicated that the tested organisms react differently in the same sediments’ and soils’ samples. Seasonal changes of toxicity sediments and soils showed the higher toxicity of autumnal samples compared to spring samples. This study will be continued.

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M. Kaza, J. Mankiewicz-Boczek, K. Izydorczyk and J. Sawicki
The Use of Vascular Plant Lemna Minor in the Battery of Microbiotests for the Toxicity Assessment of Water from Rivers in Central Poland
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : surface waters; toxicity monitoring

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Abstract : The conventional classification of surface water based is primarily on chemical and physical analyses. An evaluation of wastewater hazards to the aquatic environment with biotests can support the traditional monitoring. However, next to short-term tests with animal species there should be space for chronic test with plants. Plant communities are important to the functioning of freshwater, estuarine and marine ecosystems. In many studies the relative sensitivities of plants and animals were found to be species and chemical specific and, therefore, unpredictable. Thus, phytotoxicity tests are as necessary as tests with animals.
The toxicity of samples from 4 rivers located in Central Poland was studied in the research. Rivers Pilica, Bzura, Ner and Utrata, which are polluted to a different degree, were monitored with a battery of 9 biotests composed of representatives of producers (micro-algae - Pseudokirchneriella subcapitata, duckweed - Lemna minor), consumers (rotifer - Brachionus calyciflorus, crustaceans - Daphnia magna, Thamnocephalus platyurus) and decomposers (bacteria - Vibrio fisheri, protozoans - Spirostomum ambiguum, Tetrahymena thermophila).
Lemna test with the duckweed Lemna minor was performed according to the standard ISO/WD 20079 with modification of the type of test container. Instead of beakers or flasks six well plastic plates with covers were used. After 7 days exposure images of the test plates were taken for analysis with the digital image analysis program ImageTool. No permanent and high toxic effects were observed in the study. The most toxic effects found in spring were not confirmed in studies in autumn and vice versa. Most test organisms gave a response. The long-term tests such as e.g. the Lemna test seem to be a useful tool in this kind of studies because chronic data are here mostly needed. However, it is too early to evaluate the sensitivity of the biotests and their usefulness in a monitoring system of rivers. A minimum additional study of one year is needed.

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M. Kaza, G. Nalecz-Jawecki and J. Sawicki
Lemna Growth Inhibition Test as a Part of Microbiotests Battery in the Toxicity Assessment of Selected Pharmaceuticals
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Rapidtoxkit; Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - drugs

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Abstract : Pharmaceuticals are an integral part of our civilization. They are released into aquatic ecosystems with the urine and faces in municipal waste as well as with components of industrial and agricultural discharges in large amounts. The occurrence of pharmaceuticals in the aquatic environment has been recognized in recent years as one of the most important issues in environmental sciences.
Environmental risk assessment protocols require using organisms from various taxonomic groups considered key elements for each ecosystem. The evaluation of the toxic effects of chemicals, including pharmaceuticals, on primary producers is based on tests with green micro algae. However, large physiological differences between unicellular algae and vascular plants preclude extrapolating from algae studies to the whole plant family.
To confirm the suitability of the Lemna growth inhibition test the effects of thirteen pharmaceuticals from three therapeutic groups were analyzed : nervous system drugs, non steroidal anti-inflammatory drugs, and cardiovascular drugs. Two endpoints were studied and compared. Sensitivity of L. minor to thirteen selected drugs was compared with findings achieved in previous studies in our laboratory with short-term microbiotests: 24h-Thamnotoxkit F^TM and 1h-Rapidtoxkit with the crustacean Thamnocephalus platyurus, 24h-Spirotox with the protozoan Spirostomum amibiguum, 24h-Protoxkit F^TM with the ciliate Tetrahymena termophila and 15min-Microtox with bacteria Vibrio fisheri.

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A. Kontana, C.A. Papadimitriou, P. Samaras and M. Yiangou
Application of Bioassays and Potential New Molecular Biomarkers for the Evaluation of Municipal Wastewater Treatment Processes
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Daphtoxkit F^TM magna; Phytotoxkit
Test Species : Daphnia magna; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : waste waters; effluents; plant tests

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Abstract : The objective of this work was to examine the efficiency of various wastewater disinfection methods combined with coagulation treatment technologies for toxicity reduction of municipal secondary treated effluents, with respect to their reuse potential. The secondary treated samples were subjected to ozonation followed by coagulation, chlorination followed by coagulation and coagulation followed by chlorination. The effects of both secondary treated effluents as well as of disinfected and tertiary treated samples to various organisms were evaluated by the use of toxicity bioassays and molecular biomarkers. The battery of bioassays used included the measurement of bioluminescence inhibition to Vibrio fischeri, the percentage of immobilization mortality to Daphnia magna and the inhibition of root growth and seed germination to three plant species. For the molecular biomarkers experiments were conducted with rats that were given water samples for 2 weeks. Stress protein genes were analyzed in isolates from blood leucocytes and blood serum. Semi quantitative estimations of a1-acid glycoprotein (AGP), a1-antitrypsin (AT), C Reactive Protein (CRP), Albumin (ALB) and Hsp 70 proteins were made. The results showed increase of AGP and ALB while AT levels were unaffected. Additionally, intracellular AGP levels, isolated from blood leucocytes, were increased significantly, while Hsp 70 levels remained unaffected. Furthermore, ferric chloride coagulation of secondary treated wastewater resulted in significant reduction of serum and intracellular AGP levels. The results of the battery of toxicity bioassays showed decreased toxicity of tertiary treated disinfected samples. Both bioassays and biomarkers exhibited comparable results; however it was evident that the combination of both is required in the monitoring of reclaimed wastewater intended for reuse.
See corresponding Poster

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A. Koutsaftis and I. Aoyama
Evaluation of the Joint-Action Toxicity of Mixtures of Antifouling Biocides on Artemia salina
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : antifouling paints

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Abstract : Zinc pyrithione, copper pyrithione, chlorothalonil and diuron belong to the around 18 worldwide currently used alternative organic booster biocides. There are still many uncertainties about the environmental parameters associated with all booster biocides such as the joint toxic effects between them. Mixture toxicity studies in binary and tertiary mixtures for the above mentioned four antifouling organic biocides, and one experiment using all chemicals were performed using the brine shrimp Artemia salina as the test organism, (ARTOXKIT M, 24 h acute toxicity test). The binary mixtures were prepared in predefined ratios of 4:1, 3:2, 1:1, 2:3, and 1:4. Combination toxicity was evaluated using models such as isobole representations, concentration addition, and a mixture toxicity index. The isobole representations were not applied for the tertiary mixtures and the mixture of all chemicals. All evaluations were conducted as equitoxic mixtures. The results of all the models applied were in agreement. In most of the test combinations of the binary mixtures, chlorothalonil was found to consistently reduce the toxic effect of all the other compounds when tested jointly against Artemia salina. The Zn pyrithione-Cu pyrithione combination had a strictly synergistic effect, despite the similar structure of the two compounds which is a factor that usually leads to additive effects, while the binary mixtures of diuron with the two pyrithiones exhibited interactive effects that varied for different ratios. The four tertiary mixtures tested exhibited several effects on Artemia salina, with diuron and chlorothalonil combined with each of the two pyrithiones having less than additive effects, and the two pyrithiones combined with diuron and chlorothalonil having an additive and a synergistic effect respectively. The mixture of all four chemicals exhibited synergism. The isobole representations’ results were in agreement with the mixture toxicity index results, giving a clear view of the toxicity classification of the binary mixtures.

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G. Nalecz-Jawecki, L. Szczesny, T. Wójcik and J. Sawicki
Evaluation of the Toxicity of Propranolol Metabolites with a Battery of Bioassays
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : chemicals - drugs; chemicals - metabolites

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Abstract : Pharmaceuticals and their metabolites have been emitted in large amounts to the aquatic environment for many years, mostly in the effluents of sewage treatment plants. Chemical and ecotoxicological analyses of drugs have been performed in many countries for more than a decade. However, there have been no studies dealing with the metabolites. The main reason for it is the complexity of metabolism in human beings and the lack of chemical standards.
One of the most important group of prescription drugs are beta-blockers used for the therapy of cardiovascular diseases. Propranolol was introduced for therapy almost 50 years ago and it is still one of the most popular beta-blockers.
The goal of the present study was to evaluate the toxicity of propranolol metabolites with a battery of bioassays comprising the protozoan Spirostomum ambiguum, the rotifer Brachionus calyciflorus and the crustacean Thamnocephalus platyurus.
The mixture of metabolites was obtained in vitro with the use of S9 rat liver fraction. The chemical composition of the mixture was evaluated with HPLC-MS/MS, and the concentration of the propranolol was monitored with HPLC equipped with two detectors DAD and RF.

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P. Oleszczuk
Evaluation of Compost Toxicity by Direct Contact Methods (Phytotoxkit and Ostracodtoxkit)
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : composts; sludges; plant tests

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Abstract : Limitations related to permissible standards of undesirable substance in sewage sludges make it necessary to optimize sludge properties. One of the methods to achieve the above goal is a composting process. The aim of the study was to determine the toxicity of composts obtained from sewage sludges. Sewage sludges were composted for 76 days. A Phytotoxkit test and Ostracodtoxkit were used for toxicity determination. The results obtained showed differentiation in toxicity of sewage sludge in relation to the sludge dose and the bioassays applied. The influence of the composting on toxicity of biosolids also showed various trends depending on the sludge type. Inhibition of root growth proved to be the best parameter for the evaluation of sludge toxicity. The lack of significant influence of the composting process on toxicity was more often noted during the evaluation of phytotoxicity than with the use of ostracods.

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P. Oleszczuk, S. Baran and E. Baranowska
Application of some Chemical, Biological and Enzymatic Tests for Evaluation of Soil Reclamation
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : bioremediation; sludges; plant tests

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Abstract : A visual evaluation and analysis of chemical soil properties do not always fully reflect how efficient re-cultivating treatments are. Application of natural indices (in a form of living organisms) can be used as a supplement for such analyses in order to determine (ir)regularities in the functioning of the eco-system. The aim of the present study was to evaluate reclamation treatments using chemical analysis (heavy metals and polycyclic aromatic hydrocarbons) and biological analysis (plants, crustaceans and enzymatic activity). Polycyclic aromatic hydrocarbons and heavy metals content were characterized by a very low level. Soil pH after reclamation was in the range 6-7. Biological tests using crustaceans (Heterocypris incongruens) showed malfunctions in the re-cultivated soil. Unfavourable conditions for the crustacean development were noted both in the soil re-cultivated with after-float lime and NPK (mortality, body length) as well as sewage sludge with addition of mineral wool (mortality).
Tests carried out with plants confirmed unfavourable conditions in the soil recultivated with after-float lime and NPK and additionally also with sewage sludge alone and sewage sludge with mineral wool in an amount of 400 Mg• ha^-1. Enzymatic activity (phosphatases, protease, dehydrogenaze and urease) was significantly different dependent on soil treatment.

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C.A. Papadimitriou, P. Samaras, K. Gudulas, E. Bagavou, A.I. Zouboulis, G.P. Sakellaropoulos
Stabilization of Assessment of Leaching and Ecotoxicological Properties of Fly Ash and Lime Stabilized Sewage Sludge
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : fly ashes; sludges; solid wastes; leachates; plant tests

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Abstract : The sewage sludge that is generated from wastewater treatment plants and the fly ash generated from thermoelectric plants for energy production are two types of solid wastes for which an efficient management is a worldwide problem. From the total quantities of fly ash only a small percentage is utilized – reused in applications such as road construction, production of new materials (e.g zeolites) and wastewater treatment. Similar to fly ash, only a small percentage of activated sludge can be used in agriculture, while a negligible percentage can be incinerated in order to utilize the energy content. As a result stabilization of sludge by fly ash has to be examined as an alternative method for the management of these solid wastes; however this method has to be thoroughly evaluated prior to each implementation in order to determine the optimum mixing conditions. In this work, different ratios of fly ash and sewage sludge, as well as lime and sewage sludge were mixed and the mixtures were maintained under experimental conditions for up to 90 days. Samples of these mixtures were collected at 1,5,15,30 and 90 days after the mixing. The samples were analysed for total coliforms and E.coli, and for their ecotoxicological properties with 3 higher plants (one monocotyl and two dicotyls). The leaching properties of the mixtures were analyzed with the TCLP. Following the application of TCLP procedure the leachates were analyzed for their metal content, by AAS, while their ecotoxicological characteristics were determined by the use of the marine photobacterium Vibrio fischeri.

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G. Persoone
Ring-Tests and Reference Tests in Ecotoxicology as a Must for Credibility and Acceptability
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits :
Test Species :
Topics : review; test validation; ring testing

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Abstract : A variety of toxicity tests for aquatic and terrestrial environments have been developed during the last 50 years, the operational protocols of which are described with more or less details in scientific literature. A limited number of these tests have gradually been endorsed by national and international organisations for application in a regulatory framework.
Inter-laboratory ring-tests with the selected bioassays, on reference chemicals or natural samples, are in this regard of utmost importance since their outcome will determine whether or not the test method is described with sufficient accuracy, and applicable from the point of view of practicality and precision.
Unfortunately the organisation of ring-tests is laborious and time consuming both for the organisers and the participants, and therefore often restricted to a small number of laboratories. A critical analysis shows that the outcome of many round robin exercises is questionable with regard to the credibility of the mean results on which quality control (QC) tests are subsequently based.
In house quality control tests with one or more reference toxicants are indeed recommended in virtually all endorsed test protocols but they are mostly not mandatory and hence in practice not applied on a regular basis. Yet, QC tests are the burden of the proof that a laboratory is able to apply a specific test method with sufficient accuracy. Ring-tests on standard bioassays should therefore be organised on a much more frequent basis, and especially each time their operational protocol has undergone revision. In addition no new toxicity test should be taken into consideration by organisations dealing with the endorsement of bioassay methodologies before a (serious) ring-test has proven its credibility.
This presentation will highlight a number of ring-tests performed over the last few years with Toxkit microbiotests at the national and international level, with emphasis on sensitivity comparison of the culture/maintenance free Dapthoxkit F magna and the Algaltoxkit F microbiotests with the conventional standard assays based on laboratory cultures of the same test species.

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B. Slomczynska, T. Slomczynski and M. Lebkowska
Toxicological Evaluation of MSW Landfill Leachates after their Biological Treatment
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Daphnia magna; Thamnocephalus platyurus
Topics : solid wastes; landfill leachates

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Abstract : Leachates from municipal solid waste landfills should be included in the category of strongly contaminated industrial wastewaters, because they contain various organic and inorganic compounds in high concentration, which frequently have toxic properties. Proper purification of the leachates prior to their discharging to the environment is therefore of great importance.
The main methods for landfill leachate treatment are : biological degradation, coagulation, adsorption, membrane filtration or oxidation (Lema et al. 1988, Baumgarten and Seyfried 1996, Marttinen et al. 2002). Toxicological assays, besides studies on the physico-chemical characteristics of the leachates, allow to estimate the harmfulness of leachates for organisms of the food chains in various ecosystems as well as for human health. Relatively few toxicity data are available for MSW leachates after their treatment.
This study presents an evaluation of the harmfulness of leachates from municipal landfill for Warsaw (Poland) and after their biological treatment in the hybrid reactor based on chemical and toxicological determinations. Measurements of 8 basic parameters of leachate quality were conducted by standard analytical procedures. Raw leachate waters as well as effluents from the reactor were tested using algal growth inhibition test with Scenedesmus quadricauda, crustacean microbiotests (Artoxkit, Daphtoxkit, Thamnotoxkit), survival tests with fish Lebistes reticulatus and bacterial luminescence inhibition tests (LUMIStox).
Bioassays revealed the high toxicity of raw leachate waters: the number of toxic units (TUs) ranged from 5,1 to 111,1 depending on the test organism and the leachates. High (that is over 90 %) reduction in the values of the chemical parameters of the effluent from the hybrid reactor resulted in significant decrease of its toxicity. The battery of tests used in the present studies proved their usefulness for evaluation of the toxicity of leachates with various degrees of contamination as well as after their purification.

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B. Sosak-Swiderska, P. Bienkowski and I. Kostrzewska-Szlakowska
Microscale Testing in Field Ecotoxicology
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila
Topics : cyanobacterial blooms; toxicity monitoring; cyanotoxins

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Abstract : Eutrophication is the most common and most dangerous anthropogenic disturbance in freshwater ecosystems. Toxicity testing in the field has an important role to play in environmental monitoring. The most undesirable effect of eutrophication is a mass development of toxic cyanobacteria blooms. During three summer seasons we observed in the Sulejowskie reservoir (Central Poland) a mass cyanobacteria blooms with dominant Microcystis genera, producing toxic microcystins. The reservoir fulfils retention, recreational and electricity production functions and is the main source of drinking water for Lódz City Agglomeration. The aim of this study was to test the utility of using artificial pond microcosms for aquatic toxicity testing with natural water from the Sulejowskie reservoir.
The artificial substrate microcosm (AS-M) was composed of standard test organisms, such as microalgae (Selenastrum capricornutum), Protozoa (Tetrahymena termophila) and small multicellular zooplankton (Brachionus calyciflorus and Daphnia magna). The organisms originated from Toxkit microbiotests and conventional laboratory cultures. In the AS-M, microbial communities were obtained by colonizing small plates (diameter 10 cm x 5cm) for 1 to 21 days in a reference system or in a control location water. Communities were observed in the field and then collected and transported to the laboratory. Growth, reproduction and mortality were chosen as endpoints for the microbial communities in toxicity tests with cyanobacteria blooms. We analyzed the size of the organisms, their generation time and their physiological activity. Similarity indices were used for measuring changes in the stressed microcosm. pH, conductivity, oxygen levels, ammonia, nitrate, ortophosphate and temperature were also determinated in the AS-M tests and in a natural water of the Sulejowskie reservoir.
The AS-M system coordinated the hierarchical trophic structure of the microbial communities and their ecotoxicological endpoints in relative receptor scale for the cyanobacteria bloom stress. We therefor suggest that correlations of field impairment and laboratory toxicity tests are necessary. The AS-M system can be helpful to extrapolate data of laboratory microbiotests and methods used in aquatic ecotoxicology. Integration of ecotoxicity testing with microbiological and chemical analysis, can result into a robust monitoring system for the quality of drinking water.

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B. Sumorok, A. Trojanowska, A. Drobniewska,B. Pawlicki, G. Nalecz-Jawecki and J. Sawicki
Determination of Sediment Toxicity in the Sulejow Reservoir of the Pilica River in Central Poland by Means of the Phytotoxkit Test
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; cyanobacterial blooms; toxicity monitoring; plant tests; cyanotoxins

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Abstract : Sediments of lakes and dam reservoirs are collectors of substances transported by water. Sediments in several water bodies contain harmful substances such as trace metals, PCBs, cyanotoxins etc., which might have a toxic effect on benthic organisms and cause a hazard for people. The aim of this study was to evaluate the potential toxicity of bottom sediments of the shallow freshwater Sulejow Reservoir in Poland. The reservoir is 17 km long with an average width of 2 km and a maximum depth of 8 m near the dam. In the nicely developed lacustrine zone of the reservoir, intensive cyanobacterial blooms are appearing every summer. Blooms with prevalence of Microcystis spp., commonly occurring in this reservoir, are usually hepatotoxic This suggest that the cumulated, highly organic sediments (up to 18 % organic matter) are also potentially endangered by retention of cyanotoxins. Occurrence of toxic substances in sediments of this reservoir is highly undesirable as soon as these sediments rich in nutrients are used as a fertiliser on energetic plantations. That is why the research was focused on estimation of the potential toxicity of sediments coming from different parts of the Sulejow Reservoir on seed germination and plant growth.
Pilot studies indicated that the most sensitive tests for this study on sediment samples were Phytotoxkit assays with L. sativum and S. alba which were applied. The three days germination and root growth inhibition test with seeds of three higher plants (monocotyl Sorghum saccharatum, dicotyls Lepidium sativum and Sinapis alba) were performed according to the standard operational procedure of the Phytotoxkit. Toxic effects were measured in comparison to the effects on a control soil sample. For this study two control soils were used: a reference soil provided in the Toxkit and a river sand. The sand was water-washed and sieved to eliminate contaminations and debris, and then air-dried at 105°C. Estimation of root growth inhibition was made using a Nikon® digital camera and the image analysis software package ImageTool.
The analyses of seed germination and root growth made in 2005-2006 revealed insignificant toxic effect or lack of effect in a few samples, but with the reference soil the effect was even weaker. With regard to seed germination of Sorgum toxic effects were noted for all the sediment samples as compared to only weak effects on Sinapis. In case of Sinapis, weak inhibition of root growth was observed as compared to even less effects on Lepidium.
All tested sediment samples either show weak effect or no toxic effect for the 3 plants applied as indicators. Seasonal variations of toxic effect in sediment samples were, however, observed, which points to the necessity of continuation of the studies in parallel with analyses of concentrations of potentially toxic substances present in the sediments.

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J. Zielinski, J. Zakrzewski, M. Lewandowska and M. Szyderska
Environmentally Friendly Ionic Liquids : Biodegradation and Ecotoxicity Studies
From : International Conference on Ecotoxicology - Trends and Perspectives, 17-20 September 2006, Wisla, Poland Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rapidtoxkit; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila
Topics : chemicals - general

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Abstract : The use of ionic liquids is of increasing interest due to their properties which seem to be environmentally friendly. Unknown toxicological and ecotoxicological risk potentials of ionic liquids should, however, be assessed and minimized.
The aim of the present study was to investigate and assess the biodegradability and ecotoxicity of some classes of these compounds : ammonium (9 compounds), quaternary ammonium (12 alkyl and alkoxy compounds) and phosphonium salts (12 compounds)..
The set of examinations consisted of :
determination of n-octanol/water partition coefficient (a measure of hydrophobicity) evaluation of biodegradability : by manometric respirometry (EEC C.4-D), closed bottle test (EEC C.4-E) and aerobic mineralisation in surface water (OECD 309), assessment of acute toxicity for Vibrio fisheri (PN-EN ISO 11348-3) testing of toxicity to the protozoan ciliate Tetrahymena thermophila (PROTOXKIT F), the rotifer Brachionus calyciflorus (ROTOXKIT F), the micro-algae Selenastrum capricornutum ALGALTOXKIT F) and the crustacean Daphnia magna (DAPHTOXKIT F MAGNA).
Some efforts have been made to correlate the obtained results of biological activity to some calculated (using a Cache program) parameters of chemical structure (molecular size, E_HOMO , E_LUMO, heat of formation) and measured hydrophobicity of examined compounds.
A relationship has been found between n-octanol/water partition coefficient and the length of the carbon chain for quaternary ammonium salts. The incorporation of an ester in the side chain led to a greater biodegradability of the compounds for ammonium salts. Longer alkyl side chains of ammonium and quaternary ammonium salts resulted in a decrease of toxicity.
The anion effect on the bioactivity of ionic liquids has also been evaluated.
The revealed biological properties of the investigated ionic liquids can be helpful for introducing some structural modifications leading to compounds with enhanced biodegradability and minimmum toxicity for the aquatic environment.

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A. Drobniewska, M. Tarczynska, J. Mankiewicz, T. Jurczak and M. Zalewski
Increase of Crustacean Sensitivity to Purified Hepatotoxic Cyanobacterial Extracts by Manipulation of Experimental Conditions
Environmental Toxicology 2004 19 - 4 416-420
Toxkits : Artoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Thamnocephalus platyurus
Topics : cyanobacterial blooms; biotoxins; cyanotoxins

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Abstract : Toxic cyanobacterial blooms are one of the most common consequences of water eutrophication. Microbiotests with crustaceans are not expensive and are easy to prepare for screening tests. They can be applied in the determination of bioactivity and interaction between toxic substances in water, including hepatotoxins. The principal aim of this study was to modify the standard conditions in the Thamnotoxkit F^TM and Artoxkit M ^TMin order to increase crustacean sensitivity to purified cyanobacterial extracts containing microcystins. The results reported show that exposure time, higher temperature, and presence of DMSO can increase the sensitivity of microbiotests to microcystins. The best sensitivity with the Artemia salina test was achieved after a 48-h exposure at 25°C. The tests using a 24-h exposure at 27°C were the most sensitive for Thamnocephalus platyurus. The test without preincubation with DMSO provided the best correlation of microcystin concentration and LC50 for Thamnocephalus platyurus and is recommended.

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I. Czerniawska-Kusza, T. Ciesielczuk, G. Kusza and A. Cichon
Comparison of The Phytotoxkit Microbiotest and Chemical Variables for Toxicity Evaluation of Sediments
Environmental Toxicology 2006 21 - 4 367-372
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; plant tests

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Abstract : The main objective of the research was to evaluate the suitability of the Phytotoxkit microbiotest as a tool for hazard assessment of sediments. The concentrations of oil derivatives, polycyclic aromatic hydrocarbons (PAHs), and heavy metals (Cd, Cr, Cu, Hg, Ni, Pb, and Zn) were determined in sediment samples collected from the urban canal in Opole (Poland), in order to obtain a general insight of the level of sediment contamination. Phytotoxicity of sediments was estimated on the basis of seed germination and root elongation measurements, combined into an overall germination index (GI). The results revealed spatial and vertical differentiation in sediment contamination. A good correlation was obtained between organic matter content and the concentrations of particular sediment pollutants. Values of correlation coefficients at P < 0.05 ranged from 0.3246 for oil derivatives to 0.8929 for PAHs. Phytotoxicity tests, carried out on the monocotyl Sorghum saccharatum and the dicotyls Sinapis alba and Lepidium sativum, showed different responses of the three plant species to sediment samples ranging from growth inhibition to growth stimulation. The GI values revealed the following increasing order of plant sensitivity to contaminated sediments: L. sativum < S. alba < S. saccharatum. The study demonstrated that the Phytotoxkit microbiotest was effective in identifying toxic samples. However, sediment organic matter content and grain-size distribution had a significant impact on both sediment contamination and higher plantresponses to contaminated samples. The implication of these findings are discussed.

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M. Petala, P. Samaras, A. Zouboulis, A. Kungolos and G. Sakellaropoulos
Ecotoxicological Properties of Wastewater Treated using Tertiary Methods
Environmental Toxicology 2006 21 - 4 417-424
Toxkits : Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Daphnia pulex; Thamnocephalus platyurus
Topics : waste waters; effluents

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Abstract : The objective of this work was the examination of the efficiency of coagulation and ozonation processes for the production of reclaimed wastewater with low toxicity. Municipal secondary effluents were treated by FeCl3, Al2(SO4)3 (alum), and a commercial substance at metal ion concentrations of 0.5 and 1 mmol/L. Alternatively, the effluents were treated by ozonation in a semibatch ozone reactor. The feed gas was introduced at a flowrate of 3 L/min containing ozone at various concentrations, ranging between 2.5 and 8 mg/L; ozone residence times were 2, 5, 15, and 30 min. The toxic effects of the advanced treated effluents were examined by a battery of tests using the marine bacteria Vibrio fischeri, the freshwater crustaceans Daphnia magna, Daphnia pulex, and Thamnocephalus platyurus, and the rotifers Brachionus calyciflorus. The addition of alum decreased the toxic effect of reclaimed wastewater on immobilization of D. pulex, from 90 to 60%. Ozonation was also effective for toxicity removal, which decreased to 25% effect on D. pulex after treatment by 2.5 mg O3/L for 2 min.
However, acute toxic effects after ozonation, were observed on V. fischeri and were related to ozone gas concentration and contact time. At the highest ozone dosage, the toxicity reached almost 100% inhibition of bioluminescence after 15 min. The toxicity of the ozonated effluents to bacteria decreased with sample storage time and was almost negligible after 48 h, indicating that the potential adverse effect of reclaimed wastewaters on receiving waters might be reduced by storage for a certain time.

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V. Tsiridis, P. Samaras, A. Kungolos and G. P. Sakellaropoulos
Application of leaching Tests for Toxicity Evaluation of Coal Fly Ash
Environmental Toxicology 2006 21 - 4 409-416
Toxkits : Daphtoxkit F^TM magna; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna
Topics : fly ashes; leachates

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Abstract : The toxic properties of coal fly ash samples obtained from various coal combustion power plants were evaluated in this work using physicochemical analyses and bioassays. Physicochemical analyses showed that heavy metals present in solid samples included Cr, Cu, Mn, Ni, Pb, and Zn. The results of the chemical analysis of eluates deduced by the application of standard leaching tests according to EN 12457-2 and Toxicity Characteristic Leaching Procedure (TCLP) methods indicated that the compounds contained in fly ashes could potentially be transferred to the liquid phase depending upon the leaching method used. Heavy metal concentrations were higher in TCLP eluates, indicating that the initial pH value of the leaching medium significantly affected the transfer of these elements to the liquid phase. Tests conducted with the photobacterium Vibrio fischeri (Microtox test), the crustacean Daphnia magna, and the rotifer Brachionus calyciflorus were used to assess toxicity of eluates obtained by both leaching tests. Daphnia magna was the most sensitive test organism. The EN 12457-2 method proved to be more reliable for toxicity evaluation of eluates. In contrast, the TCLP method showed some interference owing to acetic acid toxicity, and precipitation occurred after pH adjustment of eluates from acid to neutral range. The toxicity of both fly ashes and the corresponding solid leaching residues of EN 12457-2 and TCLP leaching tests was also measured using the Microtox Basic Solid phase Test. The results generated with this bioassay indicated that toxicity was greatly influenced by the pH status of the solid samples.

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O.S. Okay, L. Tolun, V. Tüfekçi, B. Karacik, A. Kungolos, P. Samaras, C. Papadimitriou, M. Petala and V. Tsiridis
Comparison of Several Toxicity Tests Applied to Complex Wastewaters and Mussel Biomarkers in Receiving Waters
Journal of Environmental Science and Health 2005 40, 8 1525-1541
Toxkits : Artoxkit M^TM; Marine Algaltoxkit; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis; Phaeodactylum tricornutum
Topics : waste waters

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Abstract : In this study, the complex wastewaters/effluents discharged to coastal regions of Turkey and Greece were sampled, and various toxicity tests were applied. The bioassays used included the assessment of the luminescence inhibition of the bacterium Vibrio fisheri, the photosynthesis inhibition (14C uptake rate) and growth inhibition of the algal species Phaeodactylum tricornutum, and the mortality of the crustacean Artemia franciscana and rotifer Brachionus plicatilis. Additionally, neutral red retention and filtration rate sublethal biomarker techniques were performed by utilizing the mussel species Mytilus galloproviancialis, inhabiting the points of discharges in Turkey. All discharges tested were found to be acutely toxic to P. tricornutum and slightly toxic to V. fisheri, A. franciscana and B. plicatilis. Test results showed that the 14C uptake rate toxicity test was the most sensitive one among the other bioassays. The biomarker results showed that the health status of mussels in the coastal areas decreased significantly, indicating the pollution of the receiving waters in Turkey. The conclusions drawn highlight the necessity for an intensive ecotoxicological monitoring scheme that will incorporate the most suitable bioassays and biomarkers to adequately contribute to the upgrading and maintenance of the ecological quality of the coastal waters in Greece and in Turkey.

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B. Panagoula, M. Panayiota and J. Iliopoulou-Georgudaki
Acute Toxicity of TBT and IRGAROL in Artemia salina
International Journal of Toxicology 2002 21, 3 231-233
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : chemicals - organic compounds

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Abstract : A 24-hour LC50 bioassay method was carried out to study acute toxicity of Tributyltin (TBT) and IRGAROL (C11H19N5S) in Artemia salina. Five graded levels of both biocides were tested. According to the range test, the doses of TBT were 11.6 ng•l^-1, 21.0 ng•l^-1, 37.3 ng•l^-1, 65.2 ng•l^-1, and 116.5 ng•l^-1, and for IRGAROL were 1.0 mg•l^-1, 1.8 mg•l^-1, 3.2 mg•l^-1, 5.6 mg•l^-1, and 10 mg•l^-1. The determined LC50 values were 41.41 ng•l^-1 and 1.62 mg•l^-1 respectively. These results indicate that in this system TBT is acutely more toxic than IRGAROL; however, both are proven environmentally toxic substances.

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M. Brilly, M. Mikos, G. Petkovsek, M. Sraj, J. Kogovsek, D. Drobne and L. Stravs
The Experimental Monitoring of Water Regime in the Reka River
Acta Carsologica 2002 31, 1 65-74
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : surface waters

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Abstract : The river Reka, with 422 square kilometres of drainage area sinks into the Skocijan Cave system, which was proclaimed by UNESCO as a World Heritage Site in 1986. In the seventies, the Reka river was one of most polluted rivers in Slovenia. During floods in 1999 and 2000, experimental measurements of velocity, water level, suspended sediment transport, chemical parameters and toxicity tests were conducted. The main tasks in the first stage of the investigation: check the equipment in field conditions and test the toxicity of water in particular cross sections. In the paper, the measurements and some discussion of the results and applicability of equipment are presented.

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A. Kahru, M. Drews, L. Pollumaa, K. Kasemets, T. Veidebaum and P. Kogerman
Toxicity of Nanoscale Cationic Polymers in vitro and in vivo
In : Altex (Special Issue) 2005 22 902
Toxkits : Protoxkit F^TM
Test Species : Tetrahymena thermophila
Topics : chemicals - organic compounds; nanoparticles

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Abstract : The toxicity issues concerning nano-scale compounds are constantly gaining more attention. Lack of inherent toxicity is of strategic importance if nanomaterials are planned for medicine. Nanoparticles (e.g. dendrimeric macromolecules, cationic polymers, functionalised fullerenes) are widely sudied for their use in targeted drug delivery, particularly drugs based on proteins, DNA and RNA. We have analyzed the toxicity of two cationic polymers: poly(amidoamine) PAMAM G5 dendrimer (Mwt ca 25,000) and branched polyethyleneimine (PEI, Mwt ca 25,000) to biological systems of different complexity (a test battery). Different acute toxicity endpoints: the 30-minute inhibition of light output of photobacteria Vibrio fischeri, 24 h impairment of growth of protozoa Tetrahymena thermophila, 24 h viability of human cell line K562 (trypan blue exclusion) and mortality of mice (i.p.) were evaluated. In all tests dendrimer was 2...10-fold less toxic than PEI. For the in vitro test the average acute toxicity of PEI and dendrimer were 18 and 180 mg/l, respectively. The acute toxicity of PEI to mice was 74 and that of dendrimer 150 mg/kg. The 24 h IC50 for K562 cells and 30 minute EC50 for photobacteria were practically similar: 25 mg/l for PEI and about 270 mg/l for dendrimer, showing the potential of rapid ecotoxicological tests in toxicity screening of nanoparticles. In addition, the study of the adverse effects on different biological organizational levels helps to discover the mechanisms of toxicity of these emerging chemicals and to predict their hazard to ecosystems as the production of some nanomaterials is already in high production volume scale.

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North Carolina DWQ Aquatic Toxicoly Unit
Toxicity Assessment of Little Lick Creek Stormwaters
Final Report 2005
Toxkits : Algaltoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : stormwaters; surface waters

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Abstract : The DWQ Aquatic Toxicology Unit (ATU) performed toxicity assessments on five storm flow surface waters collected in Little Lick Creek and its tributaries. Sampling sites were in the Neuse River Basin area of Durham County, NC. The DWQ Watershed Assessment Team (WAT) collected samples on June 2, 2005. Stormwaters were evaluated using five discrete toxicity assays, utilizing bacteria, yeast, green algae, and two crustacean (fairy shrimp and Cladoceran) test organisms. All but the fairy shrimp assay provided sub-lethal, as well as lethality, endpoints.
Quantifiable levels of inhibition exceeding 20.0% were observed in at least two assays for each site. Adverse responses were observed in all assays for at least one site except the acute Crustacean assay. Sites were evaluated for cumulative toxicity across all 5 assays, resulting in a toxicity ranking (IC50 data, high to low toxicity) of: Little Creek LL06 > LL05 > UT01 > UT03 > LL02. Levels and combinations of observed inhibitions across the 5 assays indicate that stormwaters from each of these sites could potentially induce significant ecological impacts to at least one critical biotic component of the aquatic ecosystem. Assays included assessment of genotoxic potential to eukaryotic cells. All sites were classified as not significantly genotoxic, although site LLCLL06’s calculated DNA-repair mechanism induction value was 1.49, with values greater than or equal to 1.50 receiving a significantly genotoxic classification.
Recommendations for further assessment of sites exhibiting significant toxicity in this study include evaluation of comprehensive analytical chemistry scans with comparison to ecological screening values and study aquatic toxicity data. Further temporal and spatial delineation of toxicity is also recommended, including sediment toxicity assessment at sites with observed significant water-column toxicity.

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A.R. Fernandez-Alba, M.D. Hernando Guil, G. Diaz Lopez and Y. Chisti Yusuf
Comparative Evaluation of the Effects of Pesticides in Acute Toxicity Luminescence Bioassays
Analytica chimica acta 2002 451, 2 195-202
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - pesticides

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Abstract : Acute toxicity of pesticides in water was assessed singly and in mixtures using the responses of the luminescent bacterium Vibrio fischeri (BioTox), the aquatic invertebrate Daphnia magna (Daphtoxkit), and the MitoScan assay. The latter utilized fragmented mitochondria to enzymatically convert B-nicotinamide adenine dinucleotide (NADH) to its oxidized form, NAD[+]. The rate of the conversion being sensitive to type and concentration of toxicants. The pesticides tested were Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate), Cyromazine (N-cyclopropyl-1,3,5-triazine-2,4,6-triamine), Fe-namiphos (ethyl 4-methylthio-m-tolyl isopropylphosphoramidate), and Formetanate (3-dimethylaminomethyleneiminophenyl methylcarbamate). The toxicity bioassays were characterized in terms of relative sensitivity, reproducibility, range of the linear response, and the ability to reveal synergistic/antagonistic interactions among toxicants. The D. magna assay was the most sensitive and best able to detect toxic interactions of mixtures. Also, unlike the other assays used, the response of the daphnid system was linear over a 10-fold change in pesticide concentration. Relative to the BioTox, the MitoScan was 2- to 11-fold more sensitive for the compounds and mixtures tested. The EC[50] reproducibility of all tests was within ±20% coefficient of variation; however, the lowest observable effect concentration (LOEC) were only reproducible to ±35% on average. Cyromazine was the least toxic of the pesticides tested. To test the predictive value of the concept of concentration addition, toxicities of binary and quaternary mixtures of four different pesticides were analyzed. Synergistic/antagonistic responses were most frequently observed in testing with D. magna. Synergistic/antagonistic effects were seen only in 25 and 50% of the cases with the BioTox and the MitoScan assays, respectively.

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M. Davoren and A.M. Fogarty
A Test Battery for the Ecotoxicological evaluation of the Agri-Chemical Environ
Ecotoxicology and Environmental Safety 2004 59 (1) 116-122
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - pesticides

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Abstract : The ecotoxicological effects of the agri-chemical Environ were evaluated using a test battery comprising organisms representing three trophic levels of the aquatic ecosystem. The sensitivities of the test species to Environ were as follows: Microtox > Daphnia magna > Pseudokirchneriella subcapitata = Thamnocephalus platyurus = Oncorhynchus mykiss > Artemia salina > Tetrahymena thermophilia. An order of magnitude sensitivity between the test species was observed which emphasizes the importance of a test battery approach in the assessment of possible ecological consequences of agri-chemicals. In addition, the aquatic bioassays were found to be more sensitive (e.g., greater than three orders of magnitude for D. magna) than previously reported mammalian toxicity data for Environ. Toxicity of Environ was also investigated using fish (RTG-2) and human fibroblast cell lines (HepG2 cells) and juvenile O. mykiss. Environ was shown to have greater toxicity in the acute lethality test than with the fish cell line. However, in vivo/in vitro comparisons in this instance we feel would be premature and imprecise owing to valid concerns regarding fish loading rates for the in vivo test, and exposure duration with the in vitro test.

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E.J. Joner, D. Hirmann, O.H.J. Szolar, D. Todorovic, C. Leyval and A.P. Loibner
Priming Effects on PAH Degradation and Ecotoxicity during a Phytoremediation Experiment
Environmental Pollution 2004 128 429-435
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : soils; chemicals - hydrocarbons; bioremediation

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Abstract : An experiment was conducted to distinguish priming effects from the effects of phytoremediation of a creosote-polluted soil. The concentration of 13 polycyclic aromatic hydrocarbons (PAHs), and their combined soil toxicity (using four bioassays), was determined on recently excavated, homogenized soil and on such soil subjected to a time-course phytoremediation experiment with lucerne. The results showed a high priming effect, with minor positive and synergistic effects of planting and fertilization on PAH degradation rates. At the end of the experiment, PAH degradation reached 86% of the initial 519 mg PAHs kg^-1. Two of the four toxicity tests (bioluminescence inhibition and ostracod growth inhibition) corroborated the chemical data for residual PAHs, and indicated a significant reduction in soil toxicity. We conclude that priming effects can easily surpass treatment effects, and that an unintentional pre-incubation that ignores these effects can jeopardize the full quantitative assessment of in situ bioremediation of contaminated soil.

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H. Hamdi, S. Benzarti, L. Manusadzianas, I. Aoyama and N. Jedidi
Bioaugmentation and Biostimulation Effects on PAH Dissipation and Soil Ecotoxicity under Controlled Conditions
Soil Biology and Biochemistry 2007 39, 8 1926-1935
Toxkits : Ostracodtoxkit F™
Test Species : Heterocypris incongruens
Topics : soils; chemicals - hydrocarbons; bioremediation; sludges; composts

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Abstract : The degradation of spiked anthracene (ANT), pyrene (PYR) and benzo[a]pyrene (B[a]P) in soil (3000 mg Σ 3 PAHs kg^-1 dry soil) was studied in aerobically incubated microcosms for 120 d. The applied treatments aimed at enhancing PAH removal from the heavily contaminated soils are: (i) bioaugmentation by adding aged PAH-contaminated soil (ACS) containing activated indigenous degraders; and (ii) combined bioaugmentation/biostimulation by incorporating sewage sludge compost (SSC) and decaying rice straw (DRS). The adopted treatments produced higher PAH dissipation rates than those observed in unamended PAH-spiked soils, especially for ANT and PYR in the presence of DRS or ACS (>96%). However, B[a]P was the most recalcitrant hydrocarbon to biodegradation. Extracellular enzyme investigation revealed the existence of ligninolytic activities in all soil treatments, including control but no relationship could be found with PAH dissipation. The ecotoxicological assessment indicated that regardless of applied treatment, PAH-spiked soils were chronically lethal to ostracod Heterocypris incongruens and confirmed the sensitivity of the microcrustacean to the concomitant presence of these three hydrocarbons. Lettuce root elongation inhibition was correlated with PAH level but the presence of SSC conferred a strong phytotoxic capacity to PAH-spiked soils. DRS amendment may constitute a cost-effective alternative for hydrocarbon bioremediation as it has impacted positively on soil microbial activity and enhanced PAH removal with no apparent changes in soil physico-chemical properties.

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H. Hamdi, L. Manusadzianas, I. Aoyama and N. Jedidi
Effects of Anthracene, Pyrene and Benzo[a]Pyrene Spiking and Sewage Sludge Compost Amendment on Soil Ecotoxicity During a Bioremediation Process
Chemosphere 2006 65, 7 1153-1162
Toxkits : Daphtoxkit F^TM magna; Ostracodtoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens
Topics : soils; chemicals - hydrocarbons; bioremediation; sludges; composts

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Abstract : The fate of spiked anthracene, pyrene and benzo[a]pyrene in soil with or without sewage sludge compost was assessed during a 6-month bioremediation process simulating landfarming. Bioassays and physico-chemical analyses were employed to monitor toxicity change in soil samples and elutriates through ten sampling campaigns. Pearson product–moment correlation coefficient was determined to measure the strength of relationship between bioassays and physico-chemical analyses. The PAH dissipation in soil was enhanced after the first water addition, and the remaining amounts at the end of the experiment were positively correlated to the number of benzene rings and the presence of sewage sludge compost. Toxicity of soil elutriates to Daphnia magna was evident at early stages, originating exclusively from sewage sludge compost amendment. The lettuce root elongation was continuously inhibited by elutriates for all the treatments including control soil, probably due to high salinity or to unaddressed leachable phytotoxic compounds that were present in the experimental soil. The newly developed direct solid-phase chronic toxicity test using ostracod (Heterocypris incongruens) succeeded in evaluating the soil-bound PAH toxicity, as PAHs could not be detected in elutriates.

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V. Tsiridis, C. Emmanouil, A. Kungolos and G. P. Sakellaropoulos
The Use of Physicochemical and Ecotoxicological Analyses for Hazard Classification of Coal Fly Ash
Proceedings of Secotox Conference and International Conference on Environmental Management, Engineering, Planning and Economics, Skiathos, Greece, June 24-28, 2007 289
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : solid waste leachates

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Abstract : The classification of solid wastes is one of the major concerns of waste management in Europe. The European Directive 2003/33/EC for landfill of wastes recognizes different types of waste and of classes of landfill depending on the type of waste to be disposed off. The wastes are classified in four categories, from inert waste to waste acceptable at landfills for hazardous waste, setting leaching limit values for various constituents. Furthermore, there is a growing concern in predicting the overall environmental hazard of wastes, which has led to the development of appropriate methods for assessment of the harmful properties of leachates, using a combination of physicochemical and ecotoxicological analyses.
The chemical and ecotoxicological properties of leachates of coal fly ashes collected from different coal incineration plants were examined in the present work. The two-stage batch leaching test EN 12457-3 and the percolation test NEN 7343 for characterization of wastes as suggested by the European Council decision 2003/33/EC, were applied in conjunction with a battery of bioassays. The test organisms that were used for the toxicity evaluation of fly ash leachates included the photobacterium Vibrio fischeri (Microtox test), the algae Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) and the crustacean Daphnia magna.
The physicochemical parameters derived through the application of the two-stage batch leaching test EN 12457-3 and the percolation test NEN 7343, indicated that the fly ash samples may be classified as waste acceptable at landfills for non hazardous waste. However, the results of the ecotoxicity tests showed that the leachates of fly ash samples caused adverse effects on the test organisms. D. magna and S. capricornutum proved to be the most sensitive test organisms, exhibiting up to 100 % immobilization and growth inhibition, respectively. The low alkaline fly ash showed high leaching capacity as well as high toxic effects on the test organism, indicating that pH of the wastes is a significant factor controlling the contamination and toxicity of their leachates. The combination of physicochemical and ecotoxicological analyses for hazard classification of coal fly ash indicated that for an integrated approach, both physicochemical and ecotoxicological criteria should be evaluated.

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A. Koutsaftis and I. Aoyama
Temperature and Salinity Effects on the Toxicity of Diuron and Copper Pyrithione on the Brine Shrimp, Artemia franciscana
Proceedings of Secotox Conference and International Conference on Environmental Management, Engineering, Planning and Economics, Skiathos, Greece, June 24-28, 2007 2007 107-112
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : chemicals - pesticides; chemicals - paints; combination experiments

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Abstract : Diuron and Copper pyrithione (CPT) are two substances which have been used worldwide as alternatives to tributyl tin (TBT) in antifouling paints for the protection of ship hulls. In this study their toxicity against the brine shrimp Artemia franciscana is examined under several combinations of salinity and temperature using the LC20, LC50 and LC80 values found for the 25oC and 35‰ standard conditions. A significant interaction between temperature and salinity effects was observed for both chemicals. Decreasing temperature almost eliminated Diuron’s toxicity, while a toxicity reduction was also observed for CPT. Decreasing salinity decreased Diuron’s toxicity, while for CPT the effect of salinity was more complicated. Due to the significant effects of the two abiotic factors on the toxicity of the two chemicals, it was concluded that when conducting toxicity assessment, environmentally realistic conditions should be always taken in consideration.

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A. Kahru, M. Heinlaan, A. Ivask and I. Blinova
Ecotoxicity and Bioavailability of Metal Oxide Nanoparticles
Proceedings of Secotox Conference and International Conference on Environmental Management, Engineering, Planning and Economics, Skiathos, Greece, June 24-28, 2007 264
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - nanoparticles

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Abstract : As by today there is increasing scientific evidence that many physical and chemical effects of manufactured nanoparticles (NPs) (compared to their bulk forms, if existing) lead to an increase of toxicity and bioavailability. The existing (eco)toxicological methods may not be sufficient to address all of the issues arising with NPs. In addition, nanotoxicity can not be predicted by QSARs – (Quantitative Structure-Activity Relationships).
The aim of our research is i) to work out cost-effective, predictive and ethical testing strategies for evaluation of biological effects of NPs applicable also for risk-assessment and ii) to obtain comparative information on toxicity and mechanism of action of NPs of various type and size for cells/organisms of different biological complexity (e.g., bacteria, yeasts, protozoa, algae and invertebrate animals).
In the current presentation, toxicity of commercially available ZnO, TiO2, and CuO NPs (size range of 20–70 nm according to the information of the manufacturer) was studied to luminescent bacteria Vibrio fischeri (30-minute Flash Assay where inhibition of luminescence of test bacteria is used as a toxicity endpoint) and crustaceans Daphnia magna and Thamnocephalus platyurus (48 and 24 h mortality, respectively, was used as a toxicity endpoint). These NPs were chosen as TiO2 and ZnO are already used in different consumer products (as UV filters, etc). Bulk forms of oxides and respective metal salts were used as controls.
There was no direct relationship between the nominal size of metal oxide particles and toxicity, probably due to aggregation of particles in aqueous environments. In case of all particles the toxicity to crustaceans (ingesting particles) Thamnocephalus platyurus and Daphnia magna (where 24h & 48h mortality, respectively) was higher than to bacteria Vibrio fischeri (30 minute inhibition of luminescence).
However, the order of toxicity of tested metal oxides and metal ions was practically the same:
V. fischeri: Zn²⁺< Cu²⁺< (nano) ZnO < nano CuO < CuO< (nano) TiO2;
D. magna: Cu²⁺< Zn²⁺< (nano) ZnO < nano CuO < CuO < CuO < (nano)TiO2;T. platyurus: Cu²⁺< Zn²⁺< (nano) ZnO < nano CuO < CuO< (nano) TiO2.
TiO2 aqueous dispersions did not show adverse effects even at 20 000 mg/l level if tested in standard conditions. However, the use of specific recombinant metal sensor bacteria showed that the toxicity of ZnO and CuO was partially caused by dissolving of metal ions that was enhanced by direct contact of test organisms and particles. Thus, combined in vitro toxicity and bioavailability tests will be further investigated in order to propose valuable QAAR (Quantitative Activity-Activity Relationships) models for environmental and health risk assessments of NPs.

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I. Czerniawska-Kusza, G. Kusza and K. Borecka
The Triad Approach for Assessment of Industrial Contamination in Freshwater Sediments, Opole Region, Poland
Proceedings of Secotox Conference and International Conference on Environmental Management, Engineering, Planning and Economics, Skiathos, Greece, June 24-28, 2007 2007 96-100
Toxkits : Daphtoxkit F^TM magna; Phytotoxkit
Test Species : Daphnia magna; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; sediment and soil contact tests; plant tests

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Abstract : The sediment quality triad (SQT), integrating data on the chemistry, toxicity and benthic community structure, was used to evaluate the extent of sediment contamination. To assess potential environmental impacts of various industrial branches, sediment samples were collected from streams located in the county town of Opole (southern Poland). The SQT demonstrated that the level of site degradation varied significantly, which was attributed to the elevated concentrations of heavy metals, oil derivatives, and polycyclic aromatic hydrocarbons. The distribution and condition of the benthic community were found to vary in response to natural and anthropogenic factors, and apparent associations between degraded infaunal conditions and sediment contamination/toxicity were observed over half of the sampled stations.

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M. Sakellariou, Makrantonaki and A. Angelaki
Toxicity tests on reclaimed municipal wastewaters
Proceedings of Secotox Conference and International Conference on Environmental Management, Engineering, Planning and Economics, Skiathos, Greece, June 24-28, 2007 265-270
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : waste waters

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Abstract : One of the most important problems of humanity is water shortage. Wastewater reuse is one possible solution that is being implemented, especially in agricultural and landscape irrigation. The main aim of the present work is to evaluate the quality and the toxicity of reclaimed wastewaters compared to freshwater. For this purpose samples of reclaimed municipal wastewaters of the city of Volos (in Magnesia – Greece), which were used for irrigation purposes at the farm of the University of Thessaly, were examined for toxicity at the laboratory. Toxicity of wastewaters was measured using microbiotests, Daphnia magna and Selenastrum capricornutum. Discussion on the effect of wastewater on these organisms was also performed and interesting conclusions were obtained from this study.

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G. Persoone
Field Testing in Ecotoxicology : a Black Hole ?
Proceedings of Secotox Conference and International Conference on Environmental Management, Engineering, Planning and Economics, Skiathos, Greece, June 24-28, 2007 131-132
Toxkits : Toxi-Screening Kit
Test Species :
Topics :

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Abstract : Toxicity testing is to date well established with a variety of acute and chronic test methods on test species belonging to the different trophic levels of aquatic and terrestrial environments.
Over the last two decades toxicity testing with micro-algae and invertebrates has been very substantially simplified by the development of Toxkit microbiotests which are independent of the costly and time consuming year-round culturing/maintenance of live stocks of test biota.
Toxicity testing is nowadays, however, still performed virtually exclusively in the laboratory, with assays which mostly take one to several days to obtain the evaluation of the toxic hazard (and its degree of intensity) of the analysed samples.
The same situation also applies to the examination of the degree of bacterial contamination of suspected water samples, which is also only performed in the laboratory involving duration spanning one to several days.
Despite the need for rapid and practical methods for in situ determination of the toxicity and/or bacterial contamination of suspected waters, very little attention has so far been paid to this important matter. This is particularly remarkable since a variety of field kits are now available for determination of the concentrations of (mostly individual) chemical pollutants.
Unfortunately these chemical kits cannot convey the overall hazard of polluted waters, since their toxicity is very often (if not predominantly) caused by a mixture of different compounds.
Efforts have been made to address the black hole in field toxicity testing, which have recently led to the development of a microbiotest for ultra-rapid in situ determination of the toxicity of suspected waters. A second field microbiotest which evaluates the degree of contamination by bacteria and/or biological residues (which ultimately result in bacterial infiltration) was developed in parallel with the field toxicity test, and can be applied concurrently with the former.
Both new microbiotests are based on the measurement of luminescence in a small portable luminometer, and have been packaged in user-friendly kits.
The Toxicity Detection Kit is a practical and low cost field variant of the bacterial luminescence inhibition tests which are in contemporary use in the laboratory.
The Water Contamination Detection Kit for bacteria and biological residues makes use of the (well-known) ATP-AMP chemical luminescence measurement principle.
A portable luminometer and various accessories contained in a compact case allow for the combined application of toxicity and bacterial contamination screening using the two kits. The field microbiotests can be applied anytime and anywhere, taking less than 30 minutes of time, and without the need for any additional equipment or materials.
The two ultra-rapid field microbiotests are well suited for various applications in different domains such as e.g. toxicity/contamination monitoring of surface and groundwaters, routine screening of drinking water, water contamination emergencies and in situ follow up of the efficiency of various kinds of water and waste water treatments.
The presentation will outline the principles of the new microbiotests and give details on the practical aspects of their performance.

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L. Manusadzianas, K. Sadauskas and R. Vitkus
Comparative Study of Currently Used Toxicity Indices Based on the Evaluation of Lithuanian Urban Wastewater Toxicity
Proceedings of Secotox Conference and International Conference on Environmental Management, Engineering, Planning and Economics, Skiathos, Greece, June 24-28, 2007 238
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : waste waters; toxicity classification; toxicity index; toxicological index

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Abstract : The necessity to incorporate biologically valid assessment of water pollution into existing environmental protection system has inspired, during the last decades, the origin of various toxicity indices. Based on numerical value of certain index the classification of samples might be accomplished. Part of these indices is limited to the toxicity of aquatic sample per se, such as Toxic Print [Costan et al., 1993], pT-scale [Krebs, 2005] or EDAR [Ronco et al., 2005], while other ones, in attempting to extend the laboratory-based evaluation to real environmental conditions, include flow characteristics of discharges [Costan et al., 1993] or propose to incorporate median river flow [Vindimian, 2005]. In order to have realistic evaluation of the potential effects, the information generated by toxicity test-battery should be ecologically relevant, however, to validate this, the in-depth and long-term surveys that take into account ecotoxicological impact of the effluent to biological communities in its natural environment are necessary, e.g. Scroggins et al. (2005). Such investigations are difficult to fulfill. In some countries, the environmental authorities demand for effluent classification system based on toxicity testing, which could be suitable for taxation. To do this, it seems that there is no need to accomplish costly ecological surveys, since as stated in US EPA (1999) review on a predictive power of single species toxicity tests to envisage aquatic community responses, there are no empirical data which demonstrate that these tests fail to render reliable extrapolations to instream biological responses. In this study, we examine various indices currently used (inter)nationally by applying them for the evaluation of 25 urban effluents discharged into aquatic environment of Lithuania. Test-battery consisted of four tests with a measuring effect at 50%, i.e. 30-min photobacteria bioluminescence inhibition assay (Microtox), 24-h microinvertebrate Thamnocephalus platyurus lethality test (^TM), and two macrophytic algae Nitellopsis obtusa cell tests, 90-min depolarization (Charatox) and 96-h lethality (Niteltox). In search of an index suitable for regulatory purposes of effluents we considered that it should fulfill, at least, these criteria: (1) information obtained from test-battery has to be integrated into numerical value; (2) it does not depend analytically on the number of tests applied in the battery; (3) it has to be related with the effluent flow rate and/or characteristics of receiving waters (in Lithuania, effluent to river flow ratios vary from 20 to 20000); (4) the dynamic range gradually covers effluent toxicity from practically equal to natural background to accidental spills. We discuss pro and contra of each index, such as principles of its formation, backgrounds of classification and discriminative power to classify effluents.

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M. Petala, P. Samaras, A. Kungolos, A. Zouboulis, E. Kalogianni and G.P. Sakellaropoulos
The Cytotoxic and Mutagenic Potential of Advanced Treated Wastewater Effluents : Effect of Certain Treatment Conditions
Proceedings of Secotox Conference and International Conference on Environmental Management, Engineering, Planning and Economics, Skiathos, Greece, June 24-28, 2007 227-228
Toxkits : Daphtoxkit F^TM magna; Daphtoxkit F^TM pulex; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Daphnia pulex; Thamnocephalus platyurus
Topics : waste waters

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Abstract : Water resources are going to deteriorate during the forthcoming years, because of increased water demand; in addition long-term pollution is expected to affect an ever-increasing number of water bodies. Consequently, planning, management and optimization of water resources represent an essential element of environmental protection. Wastewater reclamation and reuse may play an important role on the development of strategies for the utilization of water resources, generating supplementary water sources. However, wastewater reuse depends greatly on issues related to public health and environmental protection, social acceptance and legislation. So far, national or regional legislative guidelines determine the framework of wastewater reuse; the application of reclaimed wastewater depends on the effluent quality, according to physical-chemical and microbiological characteristics. However, the use of physical-chemical control parameters provides only partial information of effluent quality, since they cannot be directly related to the environmental impact of effluents. The latter can be deduced from additional assessment of water and wastewater quality by the application of specific bioassays, i.e. tests using living microorganisms or aquatic organisms as indicators. Such bioassays are proposed by Directive 2000/60/EC.
The objective of this work was the investigation of the effect of coagulation and ozonation on the toxicity of secondary municipal effluents. Secondary effluents from the wastewater treatment plant of Thessaloniki were submitted to coagulation or ozonation and the acute toxicity of the original and treated effluents was evaluated using the organisms V. fischeri, D, magna, D. pulex, T. platyurus and B. calyciflorus. Chronic toxic effects were evaluated by exposing bacteria V. fischeri to pre-concentrated wastewater samples. Mutagenicity was assessed by using the strains TA100 and TA98 of bacteria S. typhimurium. Along with the above, several physical-chemical parameters were also determined. Examination of the effect of coagulation on secondary effluents included the type of coagulant (aluminum sulfate, ferric chloride and pre-polymerized product poly-aluminum chloride, PAC-18), the dose (0.5 and 1.0 mmol Me³⁺/L) and the addition of a flocculant agent (Magnafloc LT-25). On the other hand, the examination of ozonation on the quality of secondary effluents included the effect of ozone dose (2.5, 5.0, 6.5, 7.3, 8.0 mg O3/L) and reaction time (2, 5, 15, 30 min).
Secondary effluents were quite toxic to D. pulex with the immobilization of daphnids reaching 90%. On the contrary, negligible acute toxic effects were observed for B. calyciflorus, D. magna and T. platyurus. Furthermore, pre-concentrated samples of secondary effluent caused significant toxic effects on V. fischeri, possibly due to the transformation of certain heavy metals species to more bioavailable ones, during the concentration procedure. The mutagenic activity of secondary effluent on strain TA98 was also significant and was attributed to the organic content of samples.
Coagulation changed the toxic and physico-chemical properties of secondary effluents. In particular, coagulation with 0.5 mM Al³⁺ (PAC-18) decreased the toxicity on D. pulex roughly 40%. To the rest of the bio-indicators no acute toxic effect was observed similarly to the secondary effluents. Coagulation with 1 mM Al³⁺ (PAC-18) decreased significantly the chronic toxicity of pre-concentrated samples. This was ascribed to the observed decline of zinc and organic matter concentration. Mutagenicity increased by coagulation with ferric chloride but decreased with alum and PAC-18. The effect of ferric chloride was probably due to release of organic pollutants that were initially adsorbed on the organic matter of effluents or to the formation of free radicals as a result of the oxidizing action of ferric chloride.
In the study of ozonation, the toxicity test on V. fischeri proved a sensitive indicator for the appraisal of toxicity of the reclaimed waste. Acute toxicity depended chiefly on ozone dosage, most likely due to intermediate side-products, as well as on the very low residual ozone concentration. Toxicity of pre-concentrated samples was influenced by both ozone concentration and reaction time. In general, it was found that treatment with an ozone dosage of 2.5 mg O3/L required prolonged reaction times to reduce appreciably the toxicity of pre-concentrated samples whereas for higher ozone dosages this was only a matter of 2 minutes. Mutagenicity was affected mostly by ozone dosage while contact time was found to play a role only for the lowest dosages (2.5 and 5 mg O3/L).
This study provided evidence that solely the determination of physico-chemical parameters, as mentioned in directives for water reuse, can not lead to accurate and safe conclusions regarding the quality of water. For instance, despite the reduction of organic load by coagulation, mutagenicity was found to rise in some cases. To this end, it appears that ecotoxicity tests combined with conventional chemical tests comprise an integrated approach for the appraisal of water quality. In this respect, toxicity tests conducted with the microorganisms D. pulex, D. magna, V. fischeri (in pre-concentrated or not, samples) could serve as the basis for the development of a toxicity analysis protocol which could be employed for assessing the quality of secondary and tertiary municipal effluents.

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N. Irha and I. Blinova
The Use of Bioassays for the Risk Assessment of Toxic Leachates : An Experimental Study
ATLA - Alternatives to Laboratory Animals 2007 35, 1 111-118
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : solid waste leachates; chemicals - metals; chemicals - hydrocarbons

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Abstract : Solid wastes from the oil-shale industry produce leachates containing toxic compounds such as heavy metals and persistent polycyclic aromatic hydrocarbons (PAH). The hazard to the environment represented by waste leachates depends not only on their chemical composition, but also on the mobility and bioavailability of toxic contaminants in soils. We evaluated the applicability of bioassays for toxicity assessment of the bioavailable fraction of heavy metals and PAH in soils, in experiments with samples of four different soil types (Rendzina, Brown pseudopodzolic, Typical brown, Sodpodzolic), the pH of which ranged from 6.2 to 7.2. The toxicity of the bioavailable fraction of the soil contaminants was assessed with the dehydrogenase enzyme activity assay, and with a Toxkit microbiotest with the crustacean, Thamnocephalus platyurus, after treatment of the soil samples with an artificial solution containing chromium (III), lead (II), copper (II), cadmium (II) and pyrene. The test results confirm those of earlier experiments, which characterised the sorption potential of investigated soils for the same compounds. Both tests turned out to be sufficiently sensitive, and hence can be recommended as effective and useful tools for the assessment of the bioavailable fraction of soil contaminants.

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L. Kanarbik, I. Blinova and A. Kahru
Hazard Evaluation of Shale Oil to the Environment
22nd Annual Meeting SETAC EUROPE, Berlin, Germany; 20-24 May 2012 - Book of Abstracts 334
Toxkits : Daphtoxkit F^TM magna; Phytotoxkit
Test Species : Daphnia magna; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : oil shale; chemicals-hydrocarbons; shale oil; plant tests; soils

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Abstract : Estonia is the country with the largest commercially used oil shale basin. Oil shale is used for the firing of local power stations but also for the production of shale oil.
The latter is used in boiler houses, industrial furnaces but also for heating of houses, etc. The current annual production volume of shale oil is about 0.5 million tons and that will be increased up to one million tons per year in the near future. Increased production, transportation and use of shale oil in Estonia entail risks of environmental contamination. Different shale oil fractions contain various hydrocarbons, phenols, sulfur- and nitrogen-containing compounds, etc. The knowledge of shale oil toxic properties and behaviour in the soil and water ecosystems is limited. This current study aims to contribute to filling of these data gaps. In order to study the mobility and fate of the shale oil, the spiked soil samples were incubated in laboratory (i.e., controlled test conditions) and outside (natural climatic conditions) for several months. The dynamics of chemical composition of spiked soils was followed using different analytical methods. Potential hazard of shale oil to terrestrial (soil microbial community, higher plants) and aquatic organisms (bacteria Vibrio fischeri and crustaceans Daphnia magna) was investigated using various test formats.
The assessment of risks related with contamination of soils and surface waters by different fractions of shale oil based on the obtained results will be presented. This research is supported by Central Baltic INTERREG IV A Finnish-Estonian project: Risk Management and Remediation of Chemical Accidents.

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V. Aruoja, I. Kurvet, H.-Ch. Dubourguier and A. Kahru
Toxicity Testing of Heavy-Metal-Polluted Soils with Algae Selenastrum capricornutum:a Soil Suspension Assay
Wiley Periodicals 2004 396-402
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals-metals; soils

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Abstract : A small-scale Selenastrum capricornutum (Rhapidocelis subcapitata) growth inhibition assay was applied to the toxicity testing of suspensions of heavy-metal-polluted soils. The OECD 201 standard test procedure was followed, and algal biomass was measured by the fluorescence of extracted chlorophyll.The soils, which contained up to (per kilogram) 1390 mg of Zn, 20 mg of Cd, and 1050 mg of Pb were sampled around lead and zinc smelters in northern France. The water extractability of the metals in suspensions (1 part soil/99 parts water w/v) was not proportional to the pollution level, as extractability was lower for soil samples that were more polluted. Thus, the same amount of metals could be leached out of soils of different levels of pollution, showing that total concentrations of heavy metals in soil (currently used for risk assessment purposes) are poor predictors of the real environmental risk via the soil-water path. Despite high concentrations of water-extracted zinc (0.6 - 1.4 mg/L of Zn in the test), exceeding by approximately 10-fold the EC50 value for S. capricornutum (0.1 mg Zn/L), 72-h algal growth in the soil extracts was comparable or better than growth in the standard control OECD mineral medium. The soil suspension stimulated the growth of algae up to eightfold greater than growth using the OECD control medium. Growth stimulation of algae was observed even when soil suspensions contained up to 12.5 mg Zn/L and could not be explained by supplementary nitrogen, phosphorous, and carbonate leached from the soil. However, if the growth of algae in suspensions of clean and polluted soils was compared, a dose-dependent inhibitory effect of metals on algal growth was demonstrated. Thus, as soil contains nutrients/supplements that mask the adverse effect of heavy metals, a clean soil that has properties similar to the polluted soils should be used instead of mineral salt solution as a control for analysis of the ecotoxicity of soils.

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M. Heinlaan, A. Ivask, I. Blinova, H.-Ch. Dubourguier and A. Kahru
Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus
Chemospere 2008 71 1308-1316
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : nanoparticles; chemicals-metals

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Abstract : As the production of nanoparticles of ZnO, TiO2 and CuO is increasing, their (eco)toxicity to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus was studied with a special emphasis on product formulations (nano or bulk oxides) and solubilization of particles. Our innovative approach based on the combination of traditional ecotoxicology methods and metal-specific recombinant biosensors allowed to clearly differentiate the toxic effects of metal oxides per se and solubilized metal ions.Suspensions of nano and bulk TiO2 were not toxic even at 20 g l^_1. All Zn formulations were very toxic: L(E)C50 (mg l^_1) for bulk ZnO, nanoZnO and ZnSO4.7H2O: 1.8, 1.9, 1.1 (V. fischeri); 8.8, 3.2, 6.1 (D. magna) and 0.24, 0.18, 0.98 (T. platyurus), respectively. The toxicity was due to solubilized Zn ions as proved with recombinant Zn-sensor bacteria. Differently from Zn compounds, Cu compounds had different toxicities: L(E)C50 (mg l_1) for bulk CuO, nano CuO and CuSO4: 3811, 79, 1.6 (V. fischeri), 165, 3.2, 0,17 (D. magna) and 95, 2.1, 0.11 (T. platyurus), respectively. Cu-sensor bacteria showed that toxicity to V. fischeri and T. platyurus was largely explained by soluble Cu ions. However, for Daphnia magna, nano and bulk CuO proved less bioavailable than for bacterial Cu-sensor.This is the first evaluation of ZnO, CuO and TiO2 toxicity to V. fischeri and T. platyurus. For nano ZnO and nano CuO this is also a first study for D. magna.

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A. Kahru, H.C. Dubourguier, I. Blinova, A. Ivask and K. Kasemets
Biotests and Biosensors for Ecotoxicology of Metal Oxide Nanoparticles: A Minireview
Sensors 2008 8 5153-5170
Toxkits : Algaltoxkit F^TM; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : nanoparticles; chemicals-metals

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Abstract : Nanotechnologies have become a significant priority worldwide. Several manufactured nanoparticles - particles with one dimension less than 100 nm - are increasingly used in consumer products. At nanosize range, the properties of materials differ substantially from bulk materials of the same composition, mostly due to the increased specific surface area and reactivity, which may lead to increased bioavailability and toxicity.Thus, for the assessment of sustainability of nanotechnologies, hazards of manufactured nanoparticles have to be studied. Despite all the above mentioned, the data on the potential environmental effects of nanoparticles are rare. This mini-review is summarizing the emerging information on different aspects of ecotoxicological hazard of metal oxide nanoparticles, focusing on TiO2, ZnO and CuO. Various biotests that have been successfully used for evaluation of ecotoxic properties of pollutants to invertebrates, algae and bacteria and now increasingly applied for evaluation of hazard of nanoparticles at different levels of the aquatic food-web are discussed. Knowing the benefits and potential drawbacks of these systems, a suite of tests for evaluation of environmental hazard of nanoparticles is proposed.Special attention is paid to the influence of particle solubility and to recombinant metal-sensing bacteria as powerful tools for quantification of metal bioavailability. Using recombinant metal-specific bacterial biosensors and multitrophic ecotoxicity assays in tandem will create new scientific knowledge on the respective role of ionic species and of particles in toxicity of metal oxide nanoparticles. (Full article)

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M.Z. Vosyliene
Review of the Methods for Acute and Chronic Toxicity Assessment of Single Substances, Effluents and Industrial Waters
Acta Zoologica Lituanica 2007 17 3-15
Toxkits :
Test Species :
Topics : review

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Abstract : Over the last decades the necessity for the development of new methods for the assessment of water quality, or effluent toxicity was recognized by environment protection organisations. In countries of Western and Eastern Europe as well as in North America several approaches were applied. In the former SU special recommendations were also put forward for the assessment of effluent toxicity and the estimation of their .safe. concentrations. All approaches recommendedtesting aquatic organisms at different phylogenetic levels and different stages of their development. What is more, various behavioural, physiological and biochemical parameters were proposed to be determined. A large number of bioassays are available at present in EU, the USA and Canada. The recommended methods can be divided into: short-term, acute toxicity bioassays used for the rapid assessment of effluent discharge and chronic tests which are applied for the evaluation of long-term effects on growth, development, and reproduction of aquatic organisms. Standard methods were described and recommended by the International Standardization Organization (ISO), the Organization for Economic Cooperation and Development (OECD), the U.S. Environmental Protection Agency (USEPA), the American Society for Testing and Materials (ASTM), Environment Canada and other organizations. EU countries mainly used methods proposed by ISO, while OECD proposed to implement both standard and national methods. The performance of the majority of these standard methods, their reproducibility, validity of the obtained results, ranges of LC50, (or EC50) of reference chemicals were evaluated during inter-laboratory tests.ISO acute toxicity standards are very close to OECD guidelines. Therefore this review deals mainly with ISO standards.

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K. Wadhia, T. Dando and K. C. Thompson
Intra-laboratory evaluation of Microbial Assay for Risk Assessment (MARA) for potential application in the implementation of the Water Framework Directive (WFD)
Journal of Environmental Monitoring 2007 9 953-958
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - metals; test sensitivity; test validation

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Abstract : The Microbial Assay for Risk Assessment (MARA) is an innovative system based on an array of 11 different microbial species freeze-dried in a 96-well micro-titre plate format. Developed for testing the toxicity of chemicals, mixtures and environmental samples, the assay employs species of a taxonomically diverse range. In addition to ten prokaryotic species, a eukaryote (yeast) is included in the range. The MARA\'s innate scope of a multi-dimensional test allows determination of toxicity based on a unique assay fingerprint or index, numerically expressed as the mean Microbial Toxic Concentration (MTC). The most significant potential of the test is in the additional inference that can be conveyed to the toxicity evaluation because of the presence of each of the constituent species. In view of the fact that conventional aquatic bioassays, like fish or cladoceran tests, are expensive and impractical, the MARA could provide a cost-effective solution for routine ecotoxicological testing. The performance of the MARA was evaluated to ascertain its capability and potential scope. Sensitivity to toxicants and different environmental samples was assessed. Evaluation included comparison with other tests: namely Microtox^®, invertebrate (Daphnia magna and Thamnocephalus platyurus) microbiotests, and respiration-inhibition and nitrification-inhibition tests. The most sensitive invertebrate test was found to be the T. platyurus microbiotest for three of the four metals tested. The LC50 values for this test for Cd(ii), Cr(vi) and As(iii) were 0.2, 0.018 and 0.3 mg l(^-1), respectively; and the corresponding most sensitive MARA species MTC values were 4.4, 2.8 and 17 mg l(^-1), respectively.

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N. Venetsaneas and J. Iliopoulou-Georgudaki
A Preliminary Study of Toxicity by Bioassay of the Wastes of Pulp and Paper Production Units
Ecotoxicology, Ecological Risk Assessment and Multiple Stressors (NATO Security through Science Series) 2006 307-316
Toxkits : Daphtoxkit F^TM pulex; Thamnotoxkit F^TM
Test Species : Daphnia pulex; Thamnocephalus platyurus
Topics : waste waters; pulp and paper

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Abstract : The wastewaters from pulp and paper production units are considered significant pollutants because of their physicochemical characteristics (color, suspended solids, high organic load, chlorinated compounds, tannins, lignins, etc.).The aim of this study is to evaluate the toxicity of two paper mills’ wastewaters, one of which recycles paper. The toxicity test of these wastewaters was done with the use of bioindicators, using the microbiotests Thamnotoxkit F^TM, Daphtoxkit F^TM pulex and rainbow trouts (Oncorhynchus mykiss). In all the samples, the most important physicochemical parameters were measured (BOD5, COD, pH, total suspended solids, volatile solids, lignins, tannins, phenols, phosphates, nitrates, nitrites, chlorine) in order to correlate them with the results of the toxicity tests.The wastewaters from the recycling paper mill (PM1) proved “toxic” for Daphtoxkit F^TM pulex and the fish bioassay, while the wastewaters from the paper mill (PM2) were “non toxic” for the trouts and the microbiotest Thamnotoxkit F^TM. Also, according to the results from the biochemical analysis, the wastewaters from PM1 are estimated as more toxic compared to the effluents from PM2.

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N. Lapa, R. Barbosa, M.H. Lopes, B. Mendes, P. Abelha, I. Gulyurtlu and J. Santos Oliveira
Chemical and Ecotoxicological Characterization of Ashes Obtained from Sewage Sludge Combustion in a Fluidised-Bed Reactor
Journal of hazardous materials 2007 147 175-183
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : fly ashes; sludges

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Abstract : In 1999, the DEECA/INETI and the UBiA/FCT/UNL started a researching project on the partition of heavy metals during the combustion of stabilised sewage sludge (Biogran^®), in a fluidised-bed reactor, and on the quality of the bottom ashes and fly ashes produced. This project was entitled Bimetal and was funded by the Portuguese Foundation for Science and Technology. In this paper only the results on the combustion of Biogran^® are reported. The combustion process was performed in two different trials, in which different amounts of sewage sludge and time of combustion were applied. Several ash samples were collected from the bed (bottom ashes) and from two cyclones (first cyclone and second cyclone ashes). Sewage sludge, bed material (sand) and ash samples were submitted to the leaching process defined in the European leaching standard EN 12457-2. The eluates were characterized for a set of inorganic chemical species. The ecotoxicological levels of the eluates were determined for two biological indicators (Vibrio fischeri and Daphnia magna). The results were compared with the limit values of the CEMWE French Regulation. The samples were also ranked according to an index based on the chemical characterization of the eluates. It was observed an increase of the concentration of metals along the combustion system. The ashes trapped in the second cyclone, for both combustion trials, showed the highest concentration of metals in the eluates. Chemically, the ashes of the second cyclone were the most different ones. In the ecotoxicological point of view, the ecotoxicity levels of the eluates of the ashes, for both combustion cycles, did not follow the same pattern as observed for the chemical characterization. The ashes of the first cyclone showed the highest ecotoxicity levels for V. fischeri and D. magna. This difference on chemical and ecotoxicological results proves the need for performing both chemical and ecotoxicological characterizations of the sub-products of such type of thermal processes.

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P. Samaras, C.A. Papadimitriou, I. Haritou and A.I. Zouboulis
Investigation of Sewage Sludge Stabilization Potential by the Addition of Fly Ash and Lime
Journal of Hazardous Materials 2008 154, 1-3 1052-1059
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : fly ashes; sludges; plant tests

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Abstract : The aim of this work was the examination of stabilization potential of sewage sludge by the addition of fly ash and/or lime and the investigation of the effect of stabilization time on the properties of produced mixtures. Five samples were prepared by mixing fly ash, sewage sludge and lime in various ratios and the mixtures were stabilized for a period of 35 d. The addition of alkaline agents resulted in the increase of sample pH up to 12, the increase of total solids content to about 50% and the reduction of the organic fraction of the solids. The produced samples presented inhibition effects to seed germination and root length growth of three higher plants (one monocotyl and two dicotyls); however, samples with high sludge content resulted in negligible seed germination inhibition at prolonged stabilization times. The standard TCLP leaching procedure was applied in all the produced samples in order to evaluate the extraction potential of certain metallic elements; the content of metals in the eluates was varied, depending upon their speciation and form. Eluates presented significant inhibition to the marine photobacterium Vibrio fischeri bioluminescence, while the lowest inhibition was detected for the samples containing higher sludge content. These samples potentially could be applied as soil amendment, offering an efficient method for the combined utilization of two different solid wastes; however, low dosages of fly ash should be used for the production of a stabilized material presenting negligible effects with respect to its phytotoxic and ecotoxic properties.

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C.A. Papadimitriou, I. Haritou, P. Samaras and A. I. Zouboulis
Evaluation of Leaching and Ecotoxicological Properties of Sewage Sludge-Fly Ash Mixtures
Environmental Research 2008 106, 3 340-348
Toxkits : Daphtoxkit F^TM magna; Phytotoxkit
Test Species : Daphnia magna; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : leachates; fly ashes; sludges; plant tests

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Abstract : The objectives of this work were the evaluation of sewage sludge stabilization by mixing with fly ash, the examination of the physicochemical properties of the produced materials and their leachates and the assessment of their environmental impact by the evaluation of the ecotoxic characteristics. Different ratios of fly ash and sewage sludge (1:1, 1:2, 1:3, 1:6, and 1:9) were mixed for 48 and 72 h. After mixing, the liquid phase of the produced materials was analyzed for total coliforms and Escherichia coli, while the solid residue was dried and tested for the leaching characteristics by the application of TCLP and EN 12457-2 standard leaching methods. Furthermore, the produced leachates were analyzed for their content of specific metals, while their ecotoxicological characteristics were determined by the use of toxicity bioassays, using the marine photobacterium Vibrio fischeri and the crustacean Daphnia magna. The phytotoxicity of sewage sludge-fly ash mixtures was also determined by utilizing seeds of three higher plants (one monocotyl and two dicotyls). The mixtures exhibited low metal leaching in all cases, while the ecotoxic properties increased with the increase of fly ash/sewage sludge ratio. The phytotoxicity testing showed increased root length growth inhibition.

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P. Oleszczuk
Forms of Polycyclic Aromatic Hydrocarbon in the Formation of Sewage Sludge Toxicity to Heterocypris incongruens
The Science of the Total Environment 2008 404, 1 94-102
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sludges; chemicals-hydrocarbons

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Abstract : The aim of the present study was to evaluate to what degree polycyclic aromatic hydrocarbon (PAH) determines sewage sludge toxicity in relation to Heterocypris incongruens. Six differing sewage sludges with increasing contents of polycyclic aromatic hydrocarbons were selected for the present study. As well as total PAH content, the content of the potentially bioavailable fraction was also determined in the sewage sludges using a method of mild-solvent extraction (with n-butanol). The PAH content was also calculated in the sewage sludge pore water by the equilibrium partitioning method. The total PAH content in the sewage sludges studied were in the range 3.60 to 27.95 mg kg(-1). The contribution of the n-butanol extracted fraction was in the range 38.7 to 75.4%. In the group of individual PAHs, 4- and 5-ring compounds had the highest content in the potentially bioavailable group. H. incongruens mortality in the range 6.7 to 100%, depending both on the sewage sludge and the dose applied. An increase of the sewage sludge dose usually resulted in an increase in toxicity. At the highest dose, a 100% mortality of H. incongruens was found in half of the sludges. The lowest dose, irrespective of the sludge type, caused over 40% growth inhibition. However, the results obtained did not allow for the establishing of an unambiguous relationship between various sludge toxicity levels and the content of potentially bio-available PAHs. In some cases only, the extraction using n-butanol explained the high difference in toxicity despite a slight differentiation in the PAH content.

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P. Oleszczuk
Heterocypris incongruens as a Tool to estimate Sewage Sludge Toxicity
Environmental Toxicology and Chemistry 2008 27, 4 864-872
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sludges; waste waters

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Abstract : The toxicity of 13 sewage sludges was evaluated in relation to the crustacean Heterocypris incongruens. The bioassay used was a commercial, direct contact test that allows evaluation of solid-phase toxicity. Evaluation of sewage sludges included physicochemical properties (pH, total organic C, total N, cation-exchange capacity, total exchangeable bases, available forms of P and K, and electrical conductivity), heavy metal content (cadmium, chromium, copper, lead, nickel, and zinc), and content of polycyclic aromatic hydrocarbons (16 from the U.S. Environmental Protection Agency list). The evaluated parameters were then compared with bioassay results (mortality and growth inhibition). Sewage sludges were characterized by optimal physicochemical properties; low K (<5.64 mg/kg) and total organic C (177–253 g/kg) levels were noted. Contaminant content (heavy metals and polycyclic aromatic hydrocarbons) clearly differed between individual sewage sludges. Polycyclic aromatic hydrocarbon contents ranged from 3.8 to 36.4 mg/kg. Total concentrations of heavy metals in all sewage sludges (with the exception of one sludge) were lower than the pollutant concentration limits for land application. Heterocypris incongruens mortality when sludge was applied at doses of 6, 12, or 24% ranged from 0 to 70%, from 4.7 to 72.1%, and from 0 to 100%, respectively. Average growth inhibition was 39.7 to 46.6% (depending on the sludge dose applied). Any stimulating influence of two sewage sludges on the test organism was recorded. The median lethal concentrations calculated for individual sewage sludges ranged from 88 to 956 g sewage sludge/ kg.

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P. Oleszczuk
The Toxicity of Composts from Sewage Sludges Evaluated by the Direct Contact Tests Phytotoxkit and Ostracodtoxkit
Waste Management 2008 28, 9 1645-1653
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum
Topics : composts; sludges; waste waters; plant tests

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Abstract : Limitations relating to permissible standards of undesirable substances in sewage sludges make it necessary to optimize sludge properties. One of the methods to achieve the above goal is the use of a composting process. The aim of this study was to determine the toxicity of composts obtained from sewage sludges composted for 76 days. Dewatered sewage sludges were collected from the four wastewater treatment plants located in the south-eastern part of Poland (Kraśnik, Lublin, Biłgoraj and Zamość). The sludges were mixed with standard OECD soil at doses of 6% and 24%. Phytotoxkit (with Lepidium sativum) and ostracodtoxkit (with Heterocypris incongruens) tests were used to evaluate toxicity. The results obtained showed different toxicity of sewage sludge depending on the sludge dose and bioassay used. H. incongruens mortality ranged from 0% to 90% and depended on the sewage sludge. The greatest inhibition of test organism growth was noted at a level of 55%. In the case of the Phytotoxkit test, a clearly negative influence of the sewage sludges on seed germination was observed at a dose of 24%. Root growth inhibition was noted in the case of most sewage sludges and was at a level of 20-100%. The influence of the composting on the toxicity of biosolids also showed various trends depending on the sludge type. Sludge composting often resulted in a toxicity increase in relation to H. incongruens. In the case of plants (Phytotoxkit test) and most sewage sludges, however, the composting process influenced both the seed germination and root growth in a positive way.

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P. Oleszczuk
The Evaluation of Sewage Sludge and Compost Toxicity to Heterocypris incongruens in Relation to Inorganic and Organic Contaminants Content
Environmental Toxicology 2007 22 587-596
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : composts; sludges; waste waters

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Abstract : Sewage sludges and the composts were evaluated chemically and ecotoxicologically to determine their suitability as fertilizers for land application. Four municipal sewage sludges with various properties and pollutant contents were composted in aerobic conditions for 76 days. The content of heavy metals (Cd, Cu, Cr, Pb, Ni, Zn) and polycyclic aromatic hydrocarbons (14 from US EPA List) was analyzed in the sewage sludges and composts as well as together with their physico-chemical properties (total organic carbon, total nitrogen, pH, available forms of magnesium, phosphorus and potassium, cations content, cation exchange capacity, total of exchangeable bases). To evaluate the toxicity of the materials, bioassays with crustacean Heterocypris incongruens (Ostracodtoxkit test) were performed. Neither physical-chemical properties nor heavy metal content underwent any significant changes during the composting process. However, composting significantly influenced the range of losses of polycyclic aromatic hydrocarbons. The magnitude and the ranges of changes in the toxicity H. incongruens were found to be dependent on the tested parameter as well as on sewage sludge type and its dose. It was noted that composting could even increase the toxicity of sewage sludges. Sewage sludge toxicity was mainly determined by the presence of heavy metals, although for some sludges the contribution of organic pollutants as well as the synergistic activity of several factors could not be excluded. According to proposed classification (on the basis of Ostracodtoxkit test) only one sewage sludge/compost was suitable for land application. The studies conducted provide interesting information pointing to the necessity to supplement chemical studies with biological assays.

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L. Vojtova, M. Vavrova, K. Bebnarik, E. Sucman, J. David and J. Jancar
Preparation and Ecotoxicity Assessment of New Biodegradable Polyurethane Foams
Journal of Environmental Science and Health 2007 42A, 5 677-683
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : leachates; chemicals; organic compounds

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Abstract : Flexible biodegradable polyurethane foams (BIO-PUR) were prepared by a one-shot process using commercially available coreactants and modified by substituting biodegradable additive based on the acetylated starch (AS), acetylcellulose (AC), 2-hydroxyethylcellulose (HEC) and carboxymethylcellulose sodium salt (CMC) for 5 or 10 wt% of commercial polyether polyol. The BIO-PUR foams were characterized by FTIR, TGA and polarization microscopy. Ecotoxicity of BIO-PUR foams freshwater extracts was evaluated using the alternative crustacean toxicity test Thamnotoxkit F. Values of toxicity were expressed as percentage mortality of the instars II-III larvae of freshwater fairy shrimps (Thamnocephalus platyurus) dependence on the effect criterion of the respective assay. The leaches of BIO-PUR foams modified with 5 or 10 wt% of HEC showed higher toxicity then other BIO-PUR foams, whereas leaches of BIO-PUR with 5wt% of AS and 10wt% of AC were even less toxic than REF. PUR foam.

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J. Mankiewicz-Boczek, G. Nalecz-Jawecki, A. Drobniewska, M. Kaza, B. Sumorok, K. Izydorczyk, M. Zalewski and J. Sawicki
Application of a Microbiotests Battery for Complete Toxicity Assessment of Rivers
Ecotoxicology and Environmental Safety 2008 71, 3 830-836
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Protoxkit F^TM; Rapidtoxkit; Rotoxkit F^TM acute; Rotoxkit F^TM short-chronic; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : surface waters; soils; sediments; toxicity classification

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Abstract : Acute hazard classification based on selected microbiotests was proposed to assess and compare the toxicity of rivers including surface-water, sediment and soil from floodplains. No direct relationship between the classification of pollution for surface-water based on physical-chemical parameters and proposed acute hazard classification based on organisms’ sensitivity was observed. The quality of water according to hazard classification was better than in the mandatory classification of pollution, with the domination of Class II (slight acute hazard). The samples of sediment and soil were more toxic and represented Class II or Class III (acute hazard). The results indicated a need to complete the mandatory monitoring of surface-water in rivers with biological monitoring with toxicity assessment of rivers including water, sediment and soil from floodplains based on acute hazard classification. This integral approach enables a complete evaluation of the toxicity of aquatic life together with an estimation of negative changes in river systems.

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A.R. Fernandez-Alba, L. Piedra, M. Mezcua, and M.D. Hernando
Toxicity of Single and Mixed Contaminants in Seawater Measured with Acute Toxicity Bioassays
The Scientific World Journal 2002 2 1115-1120
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : seawater; chemicals - organic compounds

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Abstract : Different types of organic pollutants commonly detected in seawater have been evaluated by acute toxicity bioassays. Vibrio fischeri, Daphnia magna and Selenastrum capricornutum were selected to test toxic effects of individual compounds and mixtures of these compounds, obtaining EC50 values in the range of 0.001 to 28.9 mg/l. In the case of mixtures, synergistic toxic responses were seen for a clear majority of the cases (>60%). Mixtures containing methyl-tertiary-butyl ether (MTBE) exhibit accelerated processes that result in a change in concentration required to produce a toxic effect; for example, in the case of mixtures containing MTBE and Diuron and Dichlofluanid.

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A. Drobniewska, B. Sumorok, G. Nalecz-Jawecki and J. Sawicki
Toxicity Assessment of Sediments and Soil from Rivers and Floodplains in Central Poland using a Battery of Microbiotests - A Case Study
Fresenius Environmental Bulletin 2007 16, 2 109
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; soils; plant tests

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Abstract : In Poland, programmes of river-monitoring are based on traditional methods which examine the physico-chemical and biological parameters of water quality. However, these programmes do not include sediments and soil toxicity testing for environmental quality assessment. Rivers` sediments and soil from floodplain areas tend to accumulate various contaminants. Changes in physical and chemical characteristics in rivers may make contaminants bio-available and hence capable of exerting their toxicity. Soils of floodplains serve as a natural remediation system by reducing the loads of pollutants. The aim of this study was to indicate application of a battery of micro-biotests as tools for assessing the quality of sediments and/or soil from rivers and floodplains. Four rivers of Central Poland were selected for the study. Each of these rivers has been subjected to anthropogenic influences for several years. Samples were collected in spring and autumn 2005. The following microbiotests were used to evaluate the toxicity: Microtox^®SPT, Spirotox-SPT, Ostracodtoxkit F and Phytotoxkit. The physico-chemical characteristics and respiration of soil and sediment samples have also been performed. To summarize, for the studied samples it was found that sediments exhibited higher toxicity than the soils. Furthermore, seasonal changes of toxicity and respiration of sediments’ and soils’ samples were observed. This project will be continued in 2006 and 2007.

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J.A.K. Mitchell, J.E. Burgess and R.M. Stuetz
Developments in Ecotoxicity Testing
Re/Views in Environmental Science & Bio/Technology 2002 1, 2 169-198
Toxkits : Algaltoxkit F^TM; Ceriodaphtoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Protoxkit F^TM; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Ceriodaphnia dubia; Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : toxic interactions; review; regulatory testing

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Abstract : Effluent discharges from industrial facilities are complex, comprising of many different components and vary continuously in quantity and quality. Traditionally aquatic ecosystems have been protected from anthropogenic impacts by controlling the input of wastewaters and other sources of pollution, which reduce dissolved oxygen concentrations in receiving waters. Recent attention has focused on the direct effects that toxic pollutants can impact on biological systems. Consequently, the use of whole effluent testing has increased as a rapidly and cost-effective means of evaluating and controlling the environmental impact of such emissions. However, unless the causes of the toxicity can be identified, the toxicity of the effluent cannot be effectively managed. The traditional way of identifying the toxic elements is to conduct a Toxicity Identification Evaluation (TIE). However, this provides little information on the toxicity of defined mixtures of chemicals. This paper reviews the current status of aquatic toxicity testing in relation to test species, methodology and media and compares the results of exposure of individual compounds to that of chemical mixtures. It discusses the application of using mathematical models to predict the toxicity of chemical mixtures in order to reduce the need to generate large amounts of experimental data. The different regulatory approaches between North America and Europe are introduced for testing the toxicity of discharges to the aquatic environment as well as the need for future research.

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L. De Vetter, G. Depraetere, C. Janssen, . Stevens, and J. Van Acker
Methodology to Assess both the Efficacy and Ecotoxicology of Preservative-Treated and Modified Wood
Annals of Forest Science 2008 65, 5 504
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : wood preservation

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Abstract : Wood used in outdoor conditions out of ground contact is susceptible to weathering, inducing both fungal decay and leaching of components to the environment.
This paper presents a methodology to determine these two parameters for untreated, preservative treated and modified wood. Therefore the wood was first leached and subsequently exposed to fungal decay of the most prominent wood rotting fungi. The crustacean Daphnia magna was exposed to the leachates to provide information on their impact on the environment.
Combining both parameters reveals that preservative treated wood and modified wood are capable of protecting the wood adequately for application under use class 3 conditions without putting a threat to the environment.
This proves the suitability of the concept of combining efficacy and ecotoxicology for the evaluation of new type wood treatments.

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P. Palma, V. Palma, R. Fernandes, A. Soares and I. Barbosa
Acute Toxicity of Atrazine, Endosulfan Sulphate and Chlorpyrifos to Vibrio fischeri, Thamnocephalus platyurus and Daphnia magna, Relative to their Concentrations in Surface Waters from the Alentejo Region of Portugal
Bulletin of Environmental Contamination and Toxicology 2008 81, 5 458-489
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - pesticides; surface waters

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Abstract : Ecotoxicological effects of the herbicide atrazine and the insecticides endosulfan sulphate and chlorpyrifos were evaluated using a test battery comprising aquatic organisms from different trophic levels. According to the categories established in the EU legislation, atrazine can be considered non-harmful for the species tested, while the insecticides can be considered very toxic for the crustaceans. The results of acute toxicity tests showed that the sensitivity of organisms were as follows : Thamnocephalus platyurus > Daphnia magna > Vibrio fischeri. Chlorpyrifos may act as a toxic compound in the aquatic environment of Guadiana River, as it may be detected in water at levels that promote toxic effects.

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P.G. Becher, S. Keller, G. Jung, R. D. Süssmuth and F. Jüttner
Insecticidal activity of 12-epi-hapalindole J isonitrile
Phytochemistry 2007 68 2493-2497
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - pesticides

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Abstract : 12-epi-Hapalindole J isonitrile (1) and three previously described hapalindoles, 12-epi-hapalindole C isonitrile (2), hapalindole L (3) and 12-epi-hapalindole E isonitrile (4) were isolated and identified as insecticidal alkaloids of the biofilm-forming freshwater cyanobacterium Fischerella ATCC 43239 (Stigonematales). The structures of the purified compounds were elucidated by ESI-FTICR-MS, GC-EI-MS and various 2D NMR experiments. At 26 µM hapalindole 1 killed 100% of the larvae of the dipteran Chironomus riparius within 48 h. Insecticidal activities were also found at similar concentration for the hapalindoles 2-4. The bioactivity of hapalindoles demonstrates that cyanobacterial biofilms can be considered as promising sources of insecticidal metabolites which might be useful for the biocontrol of dipterans.

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M. Riepsaite and A. Stankevicius
Toxic Effects of Some Oil Dispersants
Environmental research, engineering and management 2005 1, 31 27-33
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - surfactants; seawater; surface waters

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Abstract : Unavoidable possibility of oil spills, when they are polluting environment increases because of the increasing growth of oil industry (building of mechanisms for oil extraction, growing number of petrol stations, motor transport, navigation) and rising usability of oil and i ts products in various ways. It makes a lot of problems: financial damage, impacts on wildlife or loses of aesthetic view. Various clean–up technologies are used for recovering oil spills. Dispersion is one of them. Dispersive measures (dispersants) are the substances that break oil slick into small droplets. Though modern dispersants are less toxic for environment than oil, however, they can cause danger to organisms. These pollutants (the dispersants and the oil) can affect live organisms synergetically with bigger harm if they work separately. So, the necessity of the real assessment of the environmental condition determines near direct (physical and chemical) methods to use such methods that are based on responsive reactions of the organisms to the pollution factors. The influence of oil and the dispersing agents ‘Simple green’, ‘Hydro Break Plus’, ‘Degradoilas’, ‘BR’, and ‘BEC 400’ on organisms in fresh (test - organisms Daphnia magna and Thamnocephalus platyurus) and the Baltic sea (test - organism Artemia salina) water determined using TOXKIT microbiotests. LC50’s were calculated to compare the toxicity of these pollutants. The LC50 of petroleum hydrocarbons and the dispersing agents ‘Simple green’, ‘Hydro Break Plus’, ‘Degradoilas’, ‘BR’, and ‘BEC 400’ on water organisms are different though there is no doubt about their toxicity.

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Z. Prokop; M.L. Vangheluwe; P.A. Van Sprang; C.R. Janssen and I. Holoubek
Mobility and Toxicity of Metals in Sandy Sediments Deposited on Land
Ecotoxicology and Environmental Safety 2003 54 65-73
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : sediments; chemicals - metals

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Abstract : A times series of laboratory experiments were conducted to investigate the effect of land deposition of contaminated sediments on the bioavailability and mobility of metals. Four sandy sediments were sampled at sites expected to have elevated levels of cadmium and zinc. The physical and chemical characteristics and ecotoxicity of sediments, pore waters, and leachates were evaluated after periods ranging from 1 to 45 days of land deposition. Cd and Zn retardation and leaching potential were calculated and this simulation gave good predictions of subsequently observed Cd and Zn mobility. The mobility and leaching of Cd and Zn in the sediments increased with decreasing pH and with decreasing content of organic matter. During the deposition an increase in sediment toxicity to plants and an increase in eluate toxicity to invertebrates were observed. A high rate of water flow through the sediment resulted in a lower toxicity enhancement of the sediments and a higher toxicity enhancement of the eluates. This result suggests that water flow through the sediment reduces the actual toxicity of the upper layer of deposited sediment but at the same time intensifies the risk of groundwater contamination.

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P. van Beelen, M. Wouterse, J.J. Bogte, D. de Zwart, B. van Dijk, A.C. de Groot, J.L. Maas and A. Espeldoorn
Is the Amount of Pesticides in Dutch Regional Surface Waters Correlated with Toxic Effects ?
RIVM Rapport 860701001 2004
Toxkits : Algaltoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : surface waters

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Abstract : In the summer of 2002, Dutch regional surface waters were sampled for analysis of 53 different pesticides and for toxicity measurements. The hydrophobic chemicals, including many pesticides, were concentrated by sorption to synthetic resins before the toxicity measurements. The concentrates were tested with the PAM test using the green algae Selenastrum capricornutum, the Microtox test using the bacterium Vibrio fisheri, the IQ test using the water flea Daphnia magna, a crustacean test using Thamnocephalus platyurus and a rotifer test using Brachionus calyciflorus. In order to obtain 50% inhibition, the samples had to be concentrated over 100 times for the rotifer test and over 10 times for the other tests. In 44 out of 45 samples the observed toxicity was too high to be explained by the low concentrations of the measured pesticides. This implies that the contribution of these pesticides to the total toxicity is probably very low in most samples, except for one sample that contained 3.1 ug/liter of parathion. This is close to the parathion concentration that, according to the scientific literature, can reduce the mobility of Daphnia magna for 50%. In our toxicity tests however, this sample had to be concentrated 20 times in order to inhibit 50% of the Daphnia magna. At the moment, we do not have a good explanation for this discrepancy. The standard Daphnia magna test might differ from the rapid Daphnia IQ test performed here. Further research is needed to determine whether pesticides can really pose an acute threat for aquatic ecosystems in Dutch regional surface waters.

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M. Patra, K. Rogers, X. Ma, D. C. Bouchard, H. Poynton and J. Lazorchak
Measurement of the Bio-Accumulation of Nano-Fullerenes into Thamnocephalus platuyrus
From : 42nd Western Regional Meeting - September 2008 - Las Vegas, NV, U.S.A. 2008 2008
Toxkits : Rapidtoxkit
Test Species : Thamnocephalus platyurus
Topics : chemicals; organic compounds; chemicals - nanoparticles

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Abstract : Nanotechnology is a rapidly growing industrial enterprise encompassing a diverse array of engineered nanomaterials, that are developed and applied in fields such as medicine, plastics, energy, electronics, and cosmetics. Nanomaterials possess unique properties often dissimilar from macro-scale particles with the same elemental composition. It is likely that the prevalence of these materials in various microenvironments will increase, along with the risk of exposure to humans and ecosystems. Because aquatic invertebrates may provide useful information with respect to how nanomaterials (specifically fullerenes) interact with the ecosystem, we are investigating the exposure of indicator species Thamnocephalus platyurus to stable aqueous suspensions of the fullerenes C60, C70, and PCBM ([6,6]-Phenyl C61-butyric acid methyl ester). Suspensions were prepared by stirring fullerenes in water for approximately 100 days. Our results indicate that each of these fullerenes aggregate in the digestive track of T. platyurus forming dark masses visible under a stereo microscope. When T. platyurus that hatched from cysts (30-45 hr, Rapidtoxkit) were exposed to C60, C70, and PCBM, the accumulated fullerene masses displaced both polystyrene beads (5 ìm, observed as red masses and used to assess chemically-induced metabolic shock) as well as algae (observed as green masses in the digestive track). The relative volume and displacement of beads and algae by aggregate fullerenes was concentration-dependent over initial fullerene concentrations of 0.9 to 60 ppm. We are further investigating these observations with respect to bioaccumulation. More specifically, we are investigating chemical analysis of the accumulated material, as well as potential long-term detrimental effects on these organisms.

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G. Persoone and K. Wadhia
Comparison Between Toxkit Microbiotests and Standard Tests
Ecotoxicological Characterization of Waste. Results and Experiences of an International Ring Test 2009 Chapter 23 213-221
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Phytotoxkit; Rotoxkit F^TM short-chronic; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : solid wastes; solid waste leachates; ring testing; plant tests; soils

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Abstract : In addition to their practicality and user-friendliness, Toxkit microbiotests have the major advantage over “standard” bioassays in that they are all independent of the (costly and time consuming) year round culturing of live stocks of the test species, which makes them very popular for routine applications in aquatic and terrestrial ecotoxicology. In the framework of the International ring test for the characterisation of waste, several laboratories from 8 European countries performed assays with various Toxkits. A detailed discussion of the results obtained with these microbiotests, with specific considerations on their interlaboratory variability, is given in Chapter 13 of this volume. In the present chapter, the Toxkit data are compared with the results obtained with “standard tests”. The findings confirmed those obtained in recent years from similar intra-lab and inter-lab comparative studies on pure chemicals and on mixed environmental samples, namely that the sensitivity of the Toxkit microbiotests is identical to that of the standard tests with the same test species. The present study furthermore revealed that the precision (in terms of variation coefficients) of the Algaltoxkit and Daphtoxkit microbiotests was better than that of the standard tests. Based on the previous findings and taking into account the advantages of microbiotests, it is recommended that the Algaltoxkit and Daphtoxkit should be taken into consideration as valid alternatives to the standard algal growth inhibition assay with Pseudokirchneriella subcapitata and the acute Daphnia magna test for the evaluation of the toxicity of solid waste eluates.

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T. Sekutowski
Application of Bioassays in Studies on Phytotoxic Herbicide Residues in the Soil Environment
In : Herbicide and Environment (Ed. A. Kortekamp) InTech Publisher 2011 Chapter 12 253-272
Toxkits : Phytotoxkit
Test Species : Sinapis alba
Topics : soils; chemicals - pesticides; plant tests

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Abstract : Bioassays are methods commonly applied in ecotoxicology in the determination of the levels of bioavailable phytotoxic residue of herbicide active ingredients in soil. Tests with the use of rapidly germinating seeds have several very important advantages, as they are cheap and easy to perform, they do not require expensive laboratory equipment and they yield reproducible results. The phytotoxic effect of herbicide active ingredient may be stated on the basis of the dynamics of germination, seedling growth, reduction of dry or fresh weight of roots or aboveground parts (stems, leaves) of test plants. On the basis of selected parameters, such as the reduction of root length, the toxic effect of herbicide active ingredients may be determined already after approx. 24 h, while the dynamics of root growth - after 3-5 days from the onset of the test (Phytotoxkit). In turn, the reduction in fresh or dry weight of aboveground parts of plants may be established after approx. 10-14 days (a conventional biotest).
Unfortunately, drawbacks of such a method include first of all the fact that it is impossible to identify the tested active ingredient. This problem may be solved by using different biological factors forming a set of biotests (Phytotoxkit --> ELISA --> HPLC), which will make it possible to precisely determine the herbicide active ingredient. It also needs to be stressed that biotests with the application of rapidly germinating seeds of selected plant species may be a good supplementation or even an alternative to classical instrumental measurements, used in the detection of phytotoxic residue of herbicide active ingredients in soil.
Probably the scope of bioassay application within the next few years will be increasing and thus collected information will constitute the basis for the initiation of analyses using classical analytical methods.

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K. Wadhia and G. Persoone
Toxkit Tests
Ecotoxicological Characterization of Waste. Results and Experiences of an International Ring Test 2009 Chapter 13 145-152
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Phytotoxkit; Rotoxkit F^TM short-chronic; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : solid wastes; solid waste leachates; ring testing; plant tests; soils

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Abstract : Research on the controlled production and storage of dormant (or immobilised) stages of selected test species resulted in the development of the Toxkit microbiotests which bypass the need for continuous culturing/maintenance of live stocks of test species. These culture/maintenance free assays are used today worldwide for a variety of applications in aquatic and terrestrial ecotoxicology. In the framework of the international ring test for the characterisation of waste, several laboratories from 8 European countries performed assays with the Algaltoxkit and the Daphtoxkit (both of which abide to the ISO norms for algal tests and acute Daphnia tests) ; some of the participants also submitted results for the Thamnotoxkit, the chronic Rotoxkit and the Phytotoxkit. In all, 72 Toxkit test data were submitted, 63 of which (87%) can be considered acceptable. In view of the limited number of data for the Thamnotoxkit and the Phytotoxkit, the statistical evaluation of the EC50s was limited to the Algaltoxkit, Daphtoxkit and chronic Rotoxkit results, which revealed that for these assays all EC50s were within the warning limits of 2 standard deviations from the mean. Variation coefficients, however, varied substantially from one test to another and for different types of waste ranging from 16% to as high as 92 %. A comparison of these latest figures with those of national and international ring tests performed over the previous years with pure chemicals using the Algaltoxkit and the Daphtoxkit indicated that the CVs in the ring test reported here were much lower than those obtained with the solid waste eluates. This may be possibly attributed to the homogeneity of the waste samples and/or the preparation of the eluates. In view of the advantages pertaining to the Toxkit microbiotests compared with the conventional assays, and the associated robustness exemplified in the ring test discussed here, it is recommended that the microbiotests should be considered valid alternatives to the standard bioassays for the evaluation of toxicity of solid wastes.

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C. Karadima and J. Lliopoulou-Georgudaki
Application of three Bioassays for Acute Toxicity Evaluation of Raw Dairy Effluents
Fresenius Environmental Bulletin 2006 16, 6 497-502
Toxkits : Daphtoxkit F^TM pulex; Thamnotoxkit F^TM
Test Species : Daphnia pulex; Thamnocephalus platyurus
Topics : effluents

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Abstract : The toxicity of raw dairy effluent was evaluated by two microbiotests, using Thamnotoxkit F and Daphtoxkit F pulex^TM, and the trout Oncorhynchus mykiss. The 24-h LC50 for Thamnotoxkit F was 3.43-14.13 %, the 48-h L(E)C50 for Daphtoxkit F pulex^TM was 1.79-5.93 %, and the 96-h LC50 for Oncorhynchus mykiss ranged from 2.59 to 13.44 %, in proportion to the effluent concentration. The correlation of the physicochemical parameters with the L(E)C50 values revealed that the Daphtoxkit F pulex^TM and the trout toxicity test demonstrated better relation in comparison with the Thamnotoxkit F. The slight predominance of Daphtoxkit F pulex^TM correlation values (ranging from 0.44 to 0.99) supports its reliability and usefulness for the monitoring of these specific effluents. BOD5 and COD are proven to be the major contributors to the toxicity of the raw dairy effluents.

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M. Cotman, A. Drolc and Tatjana Tisler
Interlaboratory studies on wastewater toxicity using Daphnia magna
Accreditation and Quality Assurance 2009 14 319-327
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : waste waters; interlaboratory testing

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Abstract : In order to verify the quality of biological measurements, the National Institute of Chemistry in Slovenia is regularly organizing interlaboratory comparisons to estimate the analytical precision of different laboratories using the same test and to determine if statistically significant differences exist between results. Several interlaboratory trials named ‘‘ILC-Waste Water’’ (ILCWW) were organized in the last 7 years. Acute toxicity testing with Daphnia magna mobility inhibition assay (ISO 6341) was included in eight successive rounds and about 20 laboratories from Slovenia and a few adjacent countries took part in each study with two samples (T1 and T2) at two different concentration levels. Variation coefficients of the mean 24 h EC50s were determined for each exercise and the evaluation of the interlaboratory variability was analysed. Comparisons were also made of the results and the variability laboratories using organisms taken from laboratory cultures versus those who used daphnias hatched from dormant eggs (Daphtoxkits).

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L. Migliore, C. Civitareale, G. Brambilla and G. Dojmi Di Delupis
Toxicity of Several Important Agricultural Antibiotics to Artemia
Water Research 1997 31, 7 1801-1806
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : chemicals - drugs

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Abstract : The possible effects of antibiotic drug contamination in marine and brackish systems were evaluated using a new methodological approach. Five drugs, namely aminosidine (A), bacitracin (B), erythromycin (E), flumequine (F) and lincomycin (L), were subjected to toxicity tests using nauplii and cysts of Artemia as a model of drug contamination from intensive farming. Tests on nauplii were performed by the Artoxkit-M test (Persoone and Van Haecke, 1981), test on cysts by our experimental protocol (Migliore et al., 1993a, b). The lethal effect on nauplii were evaluated between 24 and 120 h: the sequence of decreasing toxicity was B> F> L> A> E. A high toxicity of B was recorded. In addition, B and F were tested on cysts. B significantly lowered hatching; this depended on the interference of B with normal development. F did not depress hatching, but it deeply altered nauplii pigmentation. The interest in assessing the possible environmental risks due to drugs used in intensive farming and the need for suitable standards to improve sea-water quality is discussed.

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R.E. Dewhurst, A. Callaghan, R. Connon, M. Crane, J.D. Mather and R. Wood
Toxicity Testing of Groundwater Quality
Journal of the Chartered Institution of Water and Environmental Management 2005 19, 1 17-24
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : groundwaters

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Abstract : Groundwater is an important resource in the UK, with 45% of public water supplies in the Thames Water region derived from subterranean sources. In urban areas, groundwater has been affected by anthropogenic activities over a long period of time and from a multitude of sources. At present, groundwater quality is assessed using a range of chemical species to determine the extent of contamination. However, analysing a complex mixture of chemicals is time-consuming and expensive, whereas the use of an ecotoxicity test provides information on (a) the degree of pollution present in the groundwater and (b) the potential effect of that pollution. Microtox, Eclox and Daphnia magna microtests were used in conjunction with standard chemical protocols to assess the contamination of groundwaters from sites throughout the London Borough of Hounslow and nearby Heathrow Airport. Because of their precision, range of responses and ease of use, Daphnia magna and Microtox tests are the bioassays that appear to be most effective for assessing groundwater toxicity However neither test is ideal because it is also essential to monitor water hardness. Eclox does not appear to be suitable for use in groundwater-qualify assessment in this area, because it is adversely affected by high total dissolved solids and electrical conductivity.

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L. Pollumaa, A. Maloveryan, M. Trapido, H. Sillak and A. Kahru
Evaluation of Ecotoxicological Effects Related to Oil Shale Industry
PhD Thesis 2005
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : solid wastes; chemical - hydrocarbons; chemicals - organic compounds; oil shale; shale oil

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Abstract : In Estonia there is a largest industrially-used oil-shale basin in the world. This PhD Thesis addresses the environmental hazard of oil shale industry solid waste (semi coke) deposition, mainly focusing on solid waste-water path, i.e. spreading of the toxicants to surrounding soils and groundwater. This is the first comprehensive ecotoxicological analysis of oil shale industry pollution in Estonia.

The toxicity of eight pure phenolic compounds (phenol, cresols, dimethylphenols, resorcinols) characteristic to oil shale industry waste products was analyzed using a multitrophic ecotoxicological test battery (microalgae Selenastrum capricornutum, protozoa Tetrahymena thermophila, crustaceans Daphnia magna and Thamnocephalus platyurus, photobacteria Vibrio fischeri and Photobacterium phosphoreum). The most abundant phenols in oil shale industry leachates phenol and p-cresol (up to 85 mg/l) were classified as ‘toxic’ and ‘very toxic’, respectively but they proved rapidly biodegradable. 3,4-dimethylphenol and 2,5-dimethylresorcinol were classified ‘very toxic’ and of slow biodegradability in the activated sludge test, but their concentration in most of the semi coke leachates was relatively low (<8 mg/l).

The battery of aquatic toxicity tests was used for the analysis of aquatic samples (e.g., wastewaters) as well as aquatic leachates of semi coke and soils. Solid-Phase Flash-Assay (SPFA) was used for the detection of particle-bound toxicity. Chemical analysis covered main key pollutants (PAHs, phenols, heavy metals, oil), pH etc. Fresh semi coke was classified as hazardous waste e.g., due to its water-leachable toxicity. Comparison of pollutant levels and their toxicities (Microtox model) showed that toxicity of semi coke leachates and wastewaters as well as polluted groundwater was caused by phenols and often also by alkalinity, but there must be also other (so far not identified) toxicants present. SPFA was the most sensitive test for majority of oil shale region solid samples indicating the hazard via contact exposure. The following (reduced) test battery for the ecotoxicological hazard assessment of solid-phase samples via soil-water path was proposed: Tetrahymena thermophila growth inhibition assay, Daphnia magna mortality assay, Selenastrum capricornutum growth inhibition assay and Vibrio fischeri luminescence inhibition assay (Microtox test). For the evaluation of particle-bound toxicity SPFA (Vibrio fischeri) was suggested.

Outlook: as the toxicological and chemical evaluations in case of oil shale industry polluted environmental samples analyzed often did not match, unknown/so far not identified toxicants present in the oil shale industry solid wastes and wastewaters have to be elucidated in the future. Also, potential genotoxic hazard of semi coke and sediments of local rivers due to aged particle-bound toxicants should be analyzed.

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L. Pollumaa, A. Maloveryan, M. Trapido, H. Sillak and A. Kahru
Study of the Environmental Hazard Caused by the Oil Shale Industry Solid Waste
Alternatives to Laboratory Animals 2001 29, 3 259-267
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM acute; Rotoxkit F^TM short-chronic; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : solid wastes; chemicals - hydrocarbons; oil shale; shale oil

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Abstract : The environmental hazard was studied of eight soil and solid waste samples originating from a region of Estonia heavily polluted by the oil shale industry. The samples were contaminated mainly with oil products (up to 7231mg/kg) and polycyclic aromatic hydrocarbons (PAHs; up to 434mg/kg). Concentrations of heavy metals and water-extractable phenols were low. The toxicities of the aqueous extracts of solid-phase samples were evaluated by using a battery of Toxkit tests (involving crustaceans, protozoa, rotifers and algae). Waste rock and fresh semi-coke were classified as of high acute toxic hazard, whereas aged semi-coke and most of the polluted soils were classified as of acute toxic hazard. Analysis of the soil slurries by using the photobacterial solid-phase flash assay showed the presence of particle-bound toxicity in most samples. In the case of four samples out of the eight, chemical and toxicological evaluations both showed that the levels of PAHs, oil products or both exceeded their respective permitted limit values for the living zone (20mg PAHs/kg and 500mg oil products/kg); the toxicity tests showed a toxic hazard. However, in the case of three samples, the chemical and toxicological hazard predictions differed markedly: polluted soil from the Erra River bank contained 2334mg oil/kg, but did not show any water-extractable toxicity. In contrast, spent rock and aged semi-coke that contained none of the pollutants in hazardous concentrations, showed adverse effects in toxicity tests. The environmental hazard of solid waste deposits from the oil shale industry needs further assessment.

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A. Kahru and L. Pollumaa
Environmental Hazard of the Waste Streams of Estonian Oil Shale Industry : an Ecotoxicological Review
Oil Shale 2006 23, 1 53-93
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM acute; Rotoxkit F^TM short-chronic; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : waste waters; chemicals - hydrocarbons; review; soils; solid wastes; oil shale; shale oil

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Abstract : In Estonia there is the largest industrially-used oil-shale basin in the world.
This review addresses the environmental hazard of the waste streams of oilshale industry via solid waste- and water-path, mainly focusing on ecotoxicological risk due to open semicoke deposition inducing hazard to surrounding soils and groundwater. This is the first comprehensive ecotoxicological review of available data on oil shale industry pollution in Estonia as well as world-wide.

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R. Rojickova-Padrtova, B. Marsalek and I. Holoubek
Evaluation of Alternative and Standard Toxicity Assays for Screening of Environmental Samples : Selection of an Optimal Test Battery
Chemosphere 1998 37, 3 495-507
Toxkits : Algaltoxkit F^TM; Ceriodaphtoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Ceriodaphnia dubia; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : test sensitivity; waste waters; groundwaters; solid wastes; solid waste leachates; sludges; sediments; sediment pore waters; soils; soil leachates

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Abstract : Six muniaturized alternative assays (called microbiotests) and three standard toxicity tests were used for a comparative study based on the evaluation of acute toxicity of fifty environmental samples. The test species used in the alternative assays were microalga Raphidocelis subcapitata, crustaceans Thamnocephalus platyurus and Ceriodaphnia dubia, rotifer Brachionus calyciflorus, protozoan Spirostomum ambiguum and bacterium Vibrio fischeri. The standard toxicity tests utilized microalga Raphidocelis subcapitata, crustacean Daphnia magna, and fish Poecilia reticulata as the test organisms. The study compared the ability of bioassays to detect acute toxicity, relative sensitivity of the six microbiotests with regard to three standard toxicity tests, and similarity in their sensitivity to fifty samples. Algal bioassays were the most sensitive tests. Ceriodaphnia dubia and Spirostomum ambiguum detected acute toxicity in the majority of samples (in 62–72%). Vibrio fischeri showed a specific sensitivity pattern that was associated neither with algal nor with animal tests. The other species formed the least sensitive organisms, having similar sensitivities. A battery of three to four alternative assays was selected on the basis of the statistical analyses, sensitivity comparisons and general conditions for the selection of a test battery member like incorporation of different trophic levels or complementation of assays in a battery. Therefore, miniaturized algal assay, rotifer or crustacean microbiotest, bacterial test and possibly protozoan microbiotest could represent an optimal battery of alternative assays for the toxicity evaluation of fifty environmental samples presented in this study.

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A.J. Schoutten, J.J. Bogte, E.M. Dirven-van Breemen and M. Rutgers
Site Specific Ecological Risk Assessment, Research with the TRIAD Approach : Part 2
RIVM rapport 711701032
Toxkits : Ceriodaphtoxkit F^TM; Ostracodtoxkit F^TM; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Ceriodaphnia dubia; Heterocypris incongruens; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : decission support system; soils; review; test selection

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Abstract : This report describes the results of a research programme on the application of the so-called TRIAD approach for site specific ecological risk assessment of soil pollution. The method means to optimise the estimation of actual ecological effects in a specific area or ecosystem, by gathering multiple evidence along various ways. The TRIAD includes three pillars consisting of chemical, toxicological and ecological measurements. Each pillar of the TRIAD can be extended over three tiers, representing low, middle and high levels of sophistication in research methods. In the second phase of the field research, three locations were investigated with emphasis on the selection of a local reference site and ecological measurements. The chemical, toxicological and ecological measurements were integrated into an effect estimation per TRIAD-unit and one overall assessment. Effects were scaled from 0 to 1 and divided into 3 colour-categories to obtain a better overview. Although most bioassays and ecological indicators were able to characterise the samples, they showed much lower effects than predicted from the measured concentrations of pollutants. The (chemical) Toxic Pressure method, based on Species Sensitivity Distributions, seems to give an overestimation of ecological effects. It is also less discriminating in situations of multiple substance pollution exceeding Serious Risk Levels.

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M. Zaleska-Radziwill and D. Wojewodka
The Assessment of Effluent Toxicity by Ecotoxicological Tests
Environmental Engineering 2007 101-108
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : efflluents; chemicals - drugs

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Abstract : Ecotoxicity tests of petrochemical effluents and effluents from the production of chrythromycin have been performed. The battery of tests used encompassed Toxkits (Daphtoxkit F, Thamnotoxkit F), growth test on the alga Scenedesmus quadricauda, survival test on the fish Lebistes reticulatus, enzymatic inhibition test on the bacteria Vibrio fischeri (Lumistox), genotoxicity test on the bacteria Escherichia coli (SOS Chromotest).
Moreover, for the tested effluent samples, the values of the toxicity index PEEP have been determined. Among the bioindicators used, crustaceans, algae and the bacteria Vibrio fischeri proved to be the most sensitive. In no case was genotoxic activity of the tested effluents observed. The values of the PEEP index for petrochemical effluents exceeded the level of 5 (toxic effluents), while for effluents from the production of chrythromycin they exceeded the level of 2 (medium toxic effluents). Considerable negative correlations between the results of toxicological tests and COD values for petrochemical effluents were found.

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J. Castritsi-Catharios; N. Bourdaniotis and G. Persoone
A New Simple Method with High Precision for Determining the Toxicity of Antifouling Paints on Brine Shrimp Larvae (Artemia): First Results
Chemosphere 2007 67, 6 1127-1132
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : antifauling paints

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Abstract : The use of antifouling paints is the only truly effective method for the protection of underwater structures from the development of fouling organisms. In the present study, the surface to volume concept constitutes the basis for the development of a new and improved method for determining the toxicity of antifouling paints on marine organisms. Particular emphasis is placed on the attainment of a standardized uniformity of coated surfaces. Failure to control the thickness of the coat of paint in previous studies of this type, has led to inaccurate evaluation of the relative toxicity of samples. Herein, an attempt is made to solve this problem using a simple technique which gives completely uniform and smooth surfaces. The effectiveness of this technique is assessed through two series of experiments using two different types of test containers: 50 ml modified syringes and 7 ml multiwells. The results of the toxicity experiments follow a normal distribution around the average value which allows to consider these values as reliable for comparison of the level of toxic effect detected with the two types of test containers. The mean lethal concentration L(S/V)50 in the test series conducted in the multiwells (20.38 mm2 ml^-1) does not differ significantly from that obtained in the test series using modified syringes (20.065 mm2 ml^-1). It can thus be concluded from this preliminary study that the new method and the two different ways of exposing the test organisms to the antifouling paints and their leachates gave reliable and replicable results.

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A.C. Sneekes and N.H.B.M. Kaag
Comparing the Sensivity of four Bioassays for Acrolein
IMARES, Wageningen UR 2009
Toxkits : Artoxkit M^TM; Marine Algaltoxkit; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis; Phaeodactylum tricornutum
Topics : ballast waters; chemicals - organic compounds; test sensitivity

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Abstract : Introduction of non-indigenous species is a risk associated with discharge of ballast water from ships transporting cargo between regions. The IMO has set out a mandatory framework for ballast water management on board ships.

EnvioMar GmbH has developed a Ballast Water Treatment System (BWTS) using acrolein as active substance. The toxicity of the active substance, as well as the residual toxicity of the treated ballast water has to be assessed, according to IMO regulations on BWTS using active ingredients (G9). EnvioMar asked IMARES to conduct bioassays in order to assess the toxicity of the treated ballast water and the active ingredient, acrolein.

As a first step in the toxicity assessment, the sensitivity of four marine test species was assessed for acrolein. These species are canditates for use in experiments in which the toxicity of treated ballast water is followed in time, in order to establish the most efficient treatment concentration : Algae (Phaeodactylum tricornutum - Algae are required for approval), Crustacean (Artemia franciscana - Crustaceans are required for approval), Rotifer (Brachionus plicatilis - Easy in use) and Bacteria (Vibrio fischeri (Microtox) - Fastest and cheapest option. Helgoland results may be used for comparison).

Based on literature data, effect concentrations were expected in a range between 0.020 and 0.500 mg/l. Test concentrations were, therefore, established in a logarithmic range from 0.010 to 1.000 mg/l acrolein, except for the Microtox tests, where a starting concentration of 0.100 mg/l was diluted 50% in each step.

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J.W. Kim, H. Ishibashi, R. Yamauchi, N. Ichikawa, Y. Takao, M. Hirano, M. Koga and K. Arizono
Acute Toxicity of Pharmaceutical and Personal Care Products on Freshwater Crustacean (Thamnocephalus platyurus) and Fish (Oryzias latipes)
The Journal of Toxicological Sciences 2009 34, 2 227-232
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - drugs; chemical - organic compounds

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Abstract : Pharmaceutical and personal care products (PPCPs) enter aquatic environments via sewage treatment facilities and their potentially toxic effects on biota, particularly aquatic organisms, are of considerable concern. In this study, we investigated the acute toxicity of selected PPCPs on a freshwater crustacean (Thamnocephalus platyurus) and a fish species (Oryzias latipes). The 24-hr median lethal concentration (LC50) values of ibuprofen, mefenamic acid, indometacin, carbamazepine, propranolol, ifenprodil, clarithromycin and triclosan for T. platyurus were estimated to be 19.59, 3.95, 16.14, > 100, 10.31, 4.43, 94.23 and 0.47 mg/l respectively. Conversely, the 96-hr LC50 values for these PPCPs were estimated at > 100, 8.04, 81.92, 45.87, 11.40, 8.71, > 100 and 0.60 mg/l for O. latipes, respectively. The toxic sensitivity of T. platyurus to these PPCPs, except for carbamazepine, was therefore higher than for O. latipes. No acute toxicity effects were associated with PPCPs, such as atenolol, disopyramide, famotidine, fluconazole, erythromycin and levofloxacin, in the two aquatic organisms at the concentrations tested in this study (> 100 mg/l). These findings may help us to understand the potential biological effects and risks associated with PPCP exposure in aquatic organisms. Further long-term studies are required to fully assess the growth and reproduction of these compounds on aquatic biota.

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C. Höckelmann, P. G. Becher, S. H. von Reuß and F. Jüttner
Sesquiterpenes of the Geosmin-Producing Cyanobacterium Calothrix PCC 7507 and their Toxicity to Invertebrates
Zeitung für Naturforschung 2009 64c 49-55
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - organic compounds

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Abstract : The occurrence of sesquiterpenes was investigated with the geosmin-producing cyanobacterium Calothrix PCC 7507. The essential oil obtained by vacuum destillation was studied in more detail by GC-MS methods and superposition with authentic compounds. Geosmin was the dominating compound while the other sesquiterpenes were minor components. Sesquiterpenes that have not been described before in cyanobacteria were isodihydroagarofuran, eremophilone and 6,11-epoxyisodaucane. Closed-loop stripping analysis revealed that most of the sesquiterpenes were found in the biomass of Calothrix, while eremophilone was mainly observed in the medium of the axenic culture. Eremophilone showed acute toxicity (LC50) against Chironomus riparius (insecta) at 29 µM and against Thamnocephalus platyurus (crustacea) at 22 µM. The compound was not toxic for Plectus cirratus (nematoda). 6,11-Epoxyisodaucane and isodihydroagarofuran exhibited no toxicity to invertebrates when applied in concentrations up to 100 µM.

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P. Bakoulia; C. Karadima; A. Rouvalis and J. Iliopoulou-Georgudaki
Acute Toxicity Evaluation of an Insecticide used in Potato Cultures with the Use of Bioassays
Fresenius Environmental Bulletin 2008 17
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - pesticides; chemicals - organic compounds

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Abstract : A toxicity assessment for the anostracan Thamnocephalus platyurus and the fish, trout (Oncorhynchus mykiss) and tilapia (Oreochromis niloticus), exposed to the insecticide Lannate and its main substance methomyl was conducted based on LC50 estimation. The bioindicators were exposed to Lannate concentrations of 0.1-10 mg/1 (Thamnocephalus platyurus) and 1.5-15 mg/l (fish).The crustacean was also exposed to concentrations of 0.01mg/l -lmg/l of methomyl. The results define that the LC50 values for Lannate were 3.73 mg/1 for the trout(Oncorhynchus mykiss), 1.89 mg/1 for the tilapia (Oreochromis niloticus) and 0.12 mg/l for the Thamnocephalus platyurus. The LC(50) values for methomyl, which was tested only with Thamnocephalus platyurus was 0.13 mg/l. These values classify both substances from very toxic to exceptionally toxic. Among the test organisms used, Thamnocephalus platyurus is appointed as a particularly sensitive and easily applicable bioindicator, especially in research on the completed management of rural products, in order to minimize the environmental problems by the sustainable use of plant-protection products.

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I.G. Osojnik Crnivec and R. Marinsek-Logar
Evaluation of Toxic and Genotoxic Potential in Lake Water Samples by Bioassays
Acta agriculturae Slovenica 2007 90, 2 115-124
Toxkits : Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Tetrahymena thermophila; Thamnocephalus platyurus
Topics : surface waters

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Abstract : Preservation of the natural resources, valuable natural features and spatial characteristics plays an important role in the environmental management and is based on permanent environmental monitoring. For the time being, the current legislation regarding environmental monitoring in Slovenia is based mostly upon physicochemical analyses. Since the physicochemical analyses do not provide information about biological effects, interactions between sample compounds and bioactivation, bioassays have been considered for environmental monitoring supplementation. In the present study the adequacy of a commercial toxicity screening test Thamnotoxkit F^TM, which includes a freshwater crustacean Thamnocephalus platyurus and two genotoxicity determination test, a standard microbial Ames plate incoroporation test with Salmonella typhimurium TA97, TA98 and TA100 strains and the comet assay with the eukaryotic microorganism Tetrahymena thermophila has been evaluated. With the selected biotests toxic and genotoxic effects of lake water samples from Šalek valley were evaluated and compared with the measured physicochemical values.

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P.C. Naha, A. Casey, T. Tenuta, I. Lynch, K. A. Dawson, H. J. Byrne and M. Davoren
Preparation, Characterization of NIPAM and NIPAM/BAM Copolymer Nanoparticles and their Acute Toxicity Testing using an Aquatic Test Battery
Aquatic Toxicology 2009 92, 3 146-154
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - organic compounds; chemicals - nanoparticles; nanoparticles

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Abstract : Poly N-isopropylacrylamide and N-isopropylacrylamide/N-tert-butylacrylamide copolymer nanoparticles of 50-70 nm were prepared by free radical polymerisation. The particle sizes of the copolymer nanoparticles were measured in the test media Milli-Q water, Algae Media, Daphnia Media and Microtox Diluent as a function of temperature. Whereas in Milli-Q water the particle size was seen to decrease above the lower critical solution temperature of the thermoresponsive polymer, in the test media it was seen to increase significantly, indicative of aggregation. At the temperatures employed for the ecotoxicological studies all particles, with the exception of the 50:50 copolymer existed as nanoparticles. The zeta potential of Poly N-isopropylacrylamide and N-isopropylacrylamide/N-tert-butylacrylamide copolymer particles measured in the different media was seen to correlate well with the ratio of N-tert-butylacrylamide monomer and therefore the hydrophobicity of the particles. Ecotoxicological studies of the copolymer nanoparticles was performed using four test species Vibrio fischeri, Pseudokirchneriella subcapitata, Daphnia magna, Thamnocephalus platyurus and the cytotoxicity of the 100% Poly N-isopropylacrylamide and 85:15 N-isopropylacrylamide/N-tert-butylacrylamide copolymer nanoparticles was evaluated using a salmonid cell line. Although no significant cytotoxicological response was recorded, significant ecotoxicological response was observed at particle concentrations of up to 1000 mg l^-1. The ecotoxicological response was seen to correlate well with the ratio of N-tert-butylacrylamide monomer and therefore with the zeta potential of the nanoparticles. The toxic response in Daphnia magna was seen to further correlate with the reduction in zeta potential pointing towards a contribution of secondary effects due to modification of the medium. No correlation with particle size was observed. The sensitivity of the test species was seen to vary depending on copolymer composition. The relevance of the derived structure-activity relationships is discussed.

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E. Silva, S. Batista, L. Caetano, M.J. Cerejeira, M. Chaves and S.-E. Jacobsen
Integrated Approach for the Quality Assessment of Freshwater Resources in a Vineyard Area (South Portugal)
Environmental Monitoring and Assessment 2011 176 331-341
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - pesticides

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Abstract : An integrated chemical and biological approach for the quality assessment of freshwater resources in a vineyard area of the ‘Alentejo’ region (South Portugal) is presented. This includes analysis to 11 pesticide compounds and whole toxicity testing on algae and crustaceans. Simazine, terbuthylazine, terbutryn, desethylatrazine and chlorpyrifos were the most frequently detected pesticides in water collected from wells and drainage channels. Mixtures of up to three compounds in different qualitative combinations were also found. The quality standards for individual pesticides (0.1 µg L^-1) and pesticides-total (0.5 µg L^-1) were exceeded in some samples. However, their maximum concentrations were lower than the WHO guidelines, the USEPA health advisory values and the environmental quality standards for priority substances applicable to surface water. In five samples, the herbicides terbuthylazine and terbutryn and the insecticide chlorpyrifos did not pass the toxicity exposure ratio (TER) trigger values specified for aquatic organisms (algae, Daphnia and fish). Maximum toxic effects on Daphnia magna (100%) and Pseudokirchneriella subcapitata (82.56%) were determined in groundwater samples, while in surface water, no toxicity was observed. Concerning effects on Heterocypris incongruens in sediment samples collected at the drainage channels, mortality and growth inhibition values were below 38%. Pro-active management of the use of pesticides is recommended for implementing at the farm and catchment level to reduce inputs into ground- and surface water.

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M. Kaza, J. Mankiewicz-Boczek, K. Izydorczyk and J. Sawicki
Toxicity Assessment of Water Samples from Rivers in Central Poland using a Battery of Microbiotests - a Pilot Study
Polish Journal of Environmental Studies 2007 16, 1 81-89
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : surface waters

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Abstract : An evaluation of wastewater hazards to aquatic environments with biotests can support traditional monitoring. The conventional classification of surface water is based primarily on chemical and physical analyses. The toxicity of samples from 4 Polish rivers - Pilica, Bzura, Ner and Utrata, which are polluted to different degrees, has been assessed with a battery of biotests composed of representatives of producers (micro-algae - Pseudokirchneriella subcapitata, duckweed - Lemna minor), consumers (rotifer - Brachionus calyciflorus, crustaceans - Daphnia magna, Thamnocephalus platyurus) and decomposers (bacteria - Vibrio fisheri, protozoans - Spirostomum ambiguum, Tetrahymena thermophila). The physicochemical characteristics of water also have been performed. No permanent and highly toxic effects were observed. The most toxic effects in spring did not find a confirmation in studies in autumn and vice versa. Most test organisms gave responses. However, it is too early to evaluate the sensitivity of biotests and their usefulness in a monitoring system for rivers. A minimum of one more year of study is needed.

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M. Antonelli, V. Mezzanotte and M. Panouillères
Assessment of Peracetic Acid Disinfected Effluents by Microbiotests
Environmental Science & Technology 2009 43, 17 6579-6584
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : effluents; chemicals - inorganic compounds

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Abstract : Bioassays were performed by commercially available kits on peracetic acid (PAA) solutions, at different concentrations, and on secondary effluents (from two different wastewater treatment plants) after disinfection at bench-scale, considering both samples containing residual active PAA and the same samples where residual PAA was quenched. Four indicator organisms were used : Vibrio fischeri, Thamnocephalus platyurus, Daphnia magna and Selenastrum capricornutum. The experiments lead to conclude that Thamnocephalus platyurus is a very sensitive organism, probably not adequate to perform a reliable toxicity assessment of effluents for monitoring purposes. The presence of specific organic compounds deriving from human metabolism and urban pollution, even at very low concentrations, can affect the results of bioassays, especially those performed on Vibrio fischeri. PAA is toxic for bacteria and crustaceans even at concentrations lower than the ones commonly used in wastewater disinfection (2-5 mg/L), while its effect on algae is smaller. The toxic effect on bacteria was expected, as PAA is used for disinfection, but its possible influence on biological processes in the receiving aquatic environment should be considered. Toxicity on crustaceans would confirm the fact that discharging disinfected effluents could raise some environmental problems.

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A. Törökné, R. Vasdinnyei and B.M. Asztalos
A Rapid Microbiotest for the Detection of Cyanobacterial Toxins
Environmental Toxicology 2007 22, 1 64-68
Toxkits : Rapidtoxkit; Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : cyanobacterial blooms; biotoxins; cyanotoxins

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Abstract : Cyanobacteria occur widely in lakes, reservoirs, ponds, and slow flowing rivers. Many species are known to produce toxins (cyanotoxins), a number of which are of concern for health. Cyanotoxins vary in chemical structure and may be found intracellular or released into water. There is not only a wide variation in the toxicity of known cyanotoxins but a substantial number of toxins have to date not been identified chemically. Chemical analysis of cyanotoxins is nowadays not used for routine monitoring because it is time consuming, it requires specialized equipment and expertise, and is hence expensive. There is hence an urgent need for rapid tests in surface waters to detect cyanobacterial toxins because of the need for safe drinking water and safe natural bathing waters, which may be burdened by cyanobacterial blooms or scums. Previous investigations have already shown that larvae of the anostracan crustacean Thamnocephalus platyurus are quite sensitive to neurotoxic and hepatotoxic cyanotoxins. The present paper reports on the sensitivity comparison of the (1 h) Rapidtoxkit (based on a sublethal endpoint) and the (24 h) Thamnotoxkit microbiotest (based on mortality). Both assays make use of larvae of T. platyurus. The Rapidtoxkit is a new microbiotest that determines the decrease of ingestion of colored particles by the crustacean larvae, which are stressed by a short exposure to toxicants. Fifteen cyanobacterial samples composed of laboratory strains and natural bloom samples were tested by both microbiotests. All samples were also analyzed concurrently by HPLC for microcystins and cylindrospermopsin. The correlation coefficient between the two microbiotests (r = 0.82) showed the very good correspondence between the sublethal and the lethal effects. No known toxins could be detected in some samples, although the latter were found highly toxic to the test organisms in both bioassays. These results point to the presence of unknown toxin(s) produced by some cyanobacteria such as e.g., the Cylindrospermopsis raciborskii strain isolated from Lake Balaton in Hungary. This comparative study clearly showed that the 1 h Rapidtoxkit is an attractive rapid alternative to the Thamnotoxkit microbiotest.

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Y. Marneffe, C. Chalon, A. Claessens, M. Hémart, P. Naport, V. Rollin, C. Verbruggen and V. Wrona
Bioassays for Investigative Monitoring in the Context of the WFD : Assessment of Waste Effluents and Receiving Water Bodies
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : effluents; regulatory testing; waste waters

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Abstract : Bioassays are especially suitable for investigative monitoring in the context of the Water Framework Directive. Indeed, they are important tools to ascertain the causes of a water body failing to achieve the environmental objectives. They integrate the effects of all the contaminants including unknown substances and breakdown products contained in environmental samples and wastewaters. They also take into account any additive and synergistic effects. These properties show that they can be good diagnostic tools which enable the causes of poor ecological quality to be determined and traced back to source. They can also help to early assess the effectiveness of management measures. In the Walloon Region of Belgium, an effect directed active monitoring using bioassays is being carried out for many years. It combines ecotoxicological measurements at emission and immission and physico-chemical measurements. We use a battery of short term and chronic bioassays with the bacteria Vibrio fischeri, the alga Pseudokirchneriella subcapitata, the rotifer Brachionus calyciflorus and the microcrustacea Daphnia magna. The river capacity to dilute toxic loads is estimated on the basis of the maximum toxicity for species, the maximum permitted discharge flow and the measured minimum river flow. Results show that bioassays are particularly useful for investigative monitoring to help to determine the reasons for restricted ecological status i.e. to separate effects due to toxicity and other problems such as dissolved oxygen, suspended solids or habitat constraints (e.g. heavily modified water bodies).
See corresponding Poster

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M. Sihtmäe, I. Blinova, M. Mortimer and A. Kahru
Toxicity of five Anilines to Crustaceans, Protozoa and Bacteria
Journal of the Servian Chemical Society 2010 75, 9 1291-1302
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - organic compounds

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Abstract : Aromatic amines (anilines and related derivates) are an important class of environmental pollutants. These compounds can be released to the environment as industrial effluents or as break-down products of pesticides and dyes. We investigated the toxicity of aniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline and 3,5-dichloroaniline to a multitrophic test battery comprising of bacteria (Aliivibrio fischeri, formerly Vibrio fischeri), a ciliated protozoan (Tetrahymena thermophila) and two crustaceans (Daphnia magna and Thamnocephalus platyurus). The toxicity of anilines notably varied among test species, depending on the degree of chlorosubstitution as well as on the position of chloro-substituents. The bacteria and protozoa were much less sensitive towards anilines than crustaceans: EC50 values 5 - 600 mg/l versus 0.07 - 3.6 mg/l. The results showed that toxicity to bacteria and protozoa (both unicellular organisms) increased with increasing chlorosubstitution, i.e. aniline was the least toxic chemical and 3,5-dichloroaniline was the most toxic. For crustaceans, however, the opposite trend was observed: 3,5-dichloroaniline was the least toxic. The replacement of the artificial test medium by river water remarkably decreased the toxicity of anilines to crustaceans but not to protozoa. This research is part of the EU 6th Framework Integrated Project OSIRIS where also the ecotoxicogenomic studies of anilines will be performed that may help to clarify the mechanisms of the toxicity of anilines.

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B. Beck, P. Spanoghe, N. Duponchelle, W. Steurbaut and G. Persoone
Determination of the Phytotoxicity of Pesticide Adjuvants with the Phytotestkit Microbiotest
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts
Toxkits : Phytotestkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemical - pesticides; plant tests; soils

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Abstract : The aim of the study was to determine if and to which extent adjuvants which enhance the effectiveness of pesticides used for crop protection, exert a phytotoxic influence on plants.Experiments have been carried out with five adjuvants which were applied either separately or in combination with the herbicide propyzamid (trade name : Kerb).The effects were determined with the Phytotestkit microbiotest by measurement of the seed germination and the root growth of three plant species exposed for 3 days to the adjuvants or the adjuvant/pesticide mixture : the dicotyls : Sinapis alba (mustard) and Lepidium sativum (garden cress) and the monocotyl Sorghum saccharatum (Sorghum).No significant differences in the germination percentage of the seeds were recorded over the different treatments.Statistically significant differences were found between the root lengths of the plants treated with different adjuvants, but no synergistical phytotoxic effects between adjuvants and the herbicide could be noted. One adjuvant showed additive phytotoxic effects in two plant species when combined with propyzamid, and some adjuvants even lowered the toxicity of the herbicide.The Phytotestkit microbiotest technology proved to be a suitable tool for rapid detection of the phytotoxicity of pesticide adjuvants.
See corresponding Poster

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C. Blok, G. Persoone and G. Wever
A Practical and Low Cost Microbiotest to Assess the Phytotoxic Potential of Growing Media and Soil
ISHS - Acta Horticulturae 2008 779 367-374
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; plant tests; composts

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Abstract : For routine toxicity testing of composts and growing media, two different types of tests are used : contact tests and extract tests. In contact tests, plants are grown in containers with various mixtures of peat and the material under investigation. In extract-based tests, seeds are germinated and exposed to an exudate from the material to be tested.A novel extract based method, the Phytotoxkit microbiotest, was compared to the standard phytotoxicity test as used by the RHP foundation in Holland. This was done by comparing two bark samples of which one was known to be phytotoxic. In the Phytotoxkit test two dicotyls (Lepidium sativum and Sinapis alba) and one monocotyl (Sorghum saccharatum) were used. In the standard phytotoxicity test lettuce (Lactuca sativa) was used. In the Phytotoxkit a direct measurements of root and shoot length of the seedlings was performed by means of image analysis. In the standard test the weight of the above ground plant parts was measured.The Phytotoxkit showed a seedling length inhibition of 60 - 80% for the dicotyls and less than 20% for the monocotyl after three days. The standard phytotoxicity test showed 30% weight inhibition of the lettuce after two weeks of plant growth. This preliminary comparison showed that the Phytotoxkit microbiotest is a quick and practical bioassay for phytotoxicity testing, with a high resolution.
See corresponding Poster

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J. Mankiewicz-Boczek, R. Osiecki and K. Rydzynski
Effect of Ceria and Silica Nanoparticles on Pelagic (Thamnocephalus platyurus) and Benthic (Heterocypris incongruens) Crustaceans
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts
Toxkits : Ostracodtoxkit F^TM; Thamnotoxkit F^TM
Test Species : Heterocypris incongruens; Thamnocephalus platyurus
Topics : chemicals - nanoparticles; nanoparticles

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Abstract : The potential toxic effect of cerium dioxide nanoparticles (CeO2 NPs) and two types of silicon dioxide nanoparticles: positively charged silica coated by Al2O3 (LUDOX® CL) and negatively charged silica (LUDOX® CL-X) was investigated using an acute toxicity test with Thamnocephalus platyurus and a chronic toxicity test with Heterocypris incongruens. CeO2 NPs had a slight or low effect (< 50% effect) on the viability of T. platyurus or mortality and growth of H. incongruens exposed to a maximum test concentration of 5,000 mg/l.Positively charged silica NPs coated by alumina were toxic for the pelagic (T. platyurus) and the benthic (H. incongruens) aquatic crustaceans with an LC50 of 673 mg/l and an EC50 of 652 mg/l, respectively. The second type of silica LUDOX® CL-X was not toxic to both tested crustaceans at the maximum tested concentration of 1,000 mg/l. An uptake of CeO2 and SiO2 nanoparticles into the gastrointestinal tract of both tested organisms was noted. Additionally, CeO2 NPs adhered to the T. platyurus exoskeleton.These results point to the sensitivity of the tested pelagic and benthic crustaceans for silicon dioxide nanoparticles. However, only positively charged silica NPs coated by alumina caused a toxic effect at the concentrations tested out. The results obtained could indicate on important rule of the charged and the Al ions in silica NPs activity.
See corresponding Poster

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I. Czerniawska-Kusza
The Potential of the Phytotoxkit Microbiotest for Toxicity Eveluation of Sediments in Eutrophicated Freshwater Ecosystems
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts
Toxkits : Phytotoxkit
Test Species : Heterocypris incongruens; Thamnocephalus platyurus
Topics : sediments; sediment and soil contact tests; plant tests

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Abstract : Toxicity tests provide an integrated measure on the toxicity of contaminated environments. Major benefits of the application of bioassays in the course of the site specific risk assessment are the integrated consideration of various toxicants, their bioavailability, and combined harmful effects due to pollutant interactions. So far, various approaches, methods, and techniques have been tested in contaminated aquatic environments, referring to waters and sediments. Much research is being conducted with vascular plants, both terestrial and aquatic. Recent works, however, showed the opposite effects of contaminated sediments on seed germination and early seedling growth due to the possible impact of nutrients. Higher plants, used in the Phytotoxkit microbiotest (Lepidium sativum L., Sinapis alba L., Sorghum saccharatum L.), were investigated by exposure to sediments collected from the eutrophicated dam-reservoir of Turawa (south-western Poland). Results of phytotoxic effects and their changes due to the integrated impact of nutrients and other pollutants as well as the different sample treatment are demonstrated.
See corresponding Poster

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G. Nalecz-Jawecki, M. Olszewska and J. Sawicki
Effects of the Parasiticides Avermectins on the Ostracod Heterocypris incongruens
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - pesticides

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Abstract : Avermectins are insecticidal and anthelmintic compounds widely used in the world due to their high potency and wide spectrum of activity against endo- and ectoparasites in livestock and pets. Avermectins are excreted through faeces either unchanged and as active metabolites. They were also applied directly to water in the salmon farms. Due to their strong affinity to organic matter they can persist several months in sediments. Ostracodtoxkit F is a direct contact toxicity test for freshwater sediments. As test biont it utilises a freshwater benthic ostracod crustacean Heterocypris incongruens freshly hatched from cysts. Three avermectins were tested: abamectin, ivermectin and doramectin. All compounds were very toxic to the ostracod with LC50 values in a range 3.4-12.2 µg/l. However, the toxicity depended on the level of the organic carbon in the sediment. A relationship between the toxicity and the composition of the sediment is discussed.
See corresponding Poster

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L. Manusadzianas, R. Grigutyte, S. Jurkoniene, R. Karitonas, K. Sadauskas, J.-F. Férard, S. Cotelle and L. Foucaud
Toxicity of Various Size Zinc Oxide Nanoparticle Suspensions to Aquatic Biota
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts
Toxkits : Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : chemicals - nanoparticles; chemicals - metals; nanoparticles

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Abstract : Toxicity effects induced by ZnO nano-suspensions of various particle size to different aquatic organisms, macrophytic algae cells of Nitellopsis obtusa, invertebrate Thamnocephalus platyurus and rotifer Brachionus calyciflorus were investigated. Generally, both unsonicated and sonicated ZnO-formulations of 10 nm (Alfa/Aesar) particle size were less toxic than 20-30 nm (Alfa/Aesar) ones to charophyte cells (96 h and 192 h LC50s, and electrophysiological response expressed as percentage membrane depolarization after 18 h) and rotifers (24-h LC50s), however, no differences were found in the case of shrimps (24-h LC50s). No toxic Zn ionic concentrations were measured by capillary electrophoresis and AAS in supernatants of centrifuged zinc oxide suspensions both unsonicated and sonicated, thus confirming toxicity effects to algae cells being induced by nanoforms of suspensions. Among three test organisms, macrophytic alga was substantially less susceptible to nano-ZnO particle toxicity (effect concentrations ranging 500-1000 mg/l) than shrimps (0.09-0.21 mg/l) and rotifers (0.34-2.11 mg/l). Titanium oxide nanoparticle suspensions (anatase, average size 29 nm, Alfa/Aesar) either sonicated or unsonicated showed no lethal effect to algae within 12 day exposure period in the range 1-1000 mg/L. The data will be discussed in comparison to CuO nanoparticle toxicity to above test-organisms.
See corresponding Poster

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A. Picado, F. Brito, E. Mendonça, M.A. Morbey, A. Fernandes, S. Leitão, L. Silva, C. Cortez, A.R. Trancoso and R. Neves
Direct Toxicity Assessment and Modelling in Trancão River Basin Management
From : 14th Annual Meeting SETAC EUROPE, Prague, the Czech Republic; 18-22 April 2004 - Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : decision support system; waste waters; test selection; toxicity classification

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Abstract : Direct Toxicity Assessment (DTA) of wastewaters is an added value in evaluating the vulnerability of ecosystems to complex discharges. However legislation in most countries is still based on chemical parameters for which thresholds have been defined. A major requirement for inclusion of DTA into environmental legislation is the selection of evaluation methods and the ability of assessing the fate of toxic discharges in the receiving waters. EcoRiver (LIFE02/ENV/P/000416) aims to demonstrate the technical advantages of DTA for environmental control and the benefits of including ecotoxicity into environmental legislation. This project uses bioassays with different test species in the assessment of ecotoxicity to ensure the detection of biological effects and includes a model for the fate of toxic compounds in Trancão River Basin, a heavily industrialised area, north of Lisbon, Portugal. After a screening phase of 50 industrial discharges and 2 WWTP, 15 industries from different sectors (Chemical, Food, Printing, Repair, Surface Treatment) and the WWTP were selected according to Daphnia and Microtox test results, the potential for biodegradation and site of discharge. A full characterisation has been performed using species from different trophic levels: V. fischeri, D. magna, T. platyurus, P. subcapitata, L. minor, both in the selected industries and in the river basin. Results will be presented relating chemical and toxicity values, selecting the more sensitive species and the more toxic discharges. A model was developed for the whole basin to simulate the flow, the sediment transport and toxicity. Simulations considering interaction between toxicity and suspended matter allow the evaluation of the importance of suspended matter for river water toxicity.
See corresponding Poster

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W. De Cooman and G. Persoone
The Ostracodtoxkit Microbiotest as an Alternative Screening Solid Phase Assay to the Conventional Sediment Contact Test with Hyalella azteca
From : Aquatic Toxicity Workshop 2009, La Malbae, Quebec, Canada, September 27-30, 2009 - Book of Abstracts
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments; sediment and soil contact tests; decision support system; test sensitivity; test validation

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Abstract : Sediments are the centre of intense biological activity which co-determine the good ecological functioning of all aquatic ecosystems, and which are affected by pollutants that accumulate in the sediments. The toxicological impact of sediment contaminants in freshwater bodies is to date also determined regularly with solid phase tests based on selected benthic species such as e.g. the amphipod crustacean Hyalella azteca. In analogy to other culture/maintenance free Toxkit microbiotests which are now used extensively for toxicity testing of contaminated waters, a direct contact microbiotest has recently been developed for sediment toxicity testing with the ostracod crustacean Heterocypris incongruens.Over the last 8 years river sediments of the hydrographic basin in Flanders, Belgium, have been monitored annually for their toxicity with both the H.azteca assay and the alternative Ostracodtoxkit microbiotest. Comparison of more than 700 data pairs from a network of several hundreds sampling sites revealed that the Ostracodtoxkit is a good alternative for the conventional contact test with H.azteca for routine toxicity monitoring of river sediments in Flanders. The converted respons of Heterocypis in an ecotoxicological class - used in the triad approach to rank the ecological status of sediments in Flanders - is indeed identical to the class calculated with the conventional test for half of the samples or only differs by one class (i.e. either a higher or a lower toxic signal) for most other samples. The good correspondence in sensitivity and the practicality and the cost-efficiency of the Ostracodtoxkit microbiotest are certainly worth further consideration of this attractive alternative screening solid phase assay for routine toxicity monitoring of contaminated sediments.
See corresponding Poster

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G. Persoone
Sensitivity and Precision of Acute Daphnia magna Tests Performed with Organisms from Laboratory Cultures or Hatched from Dormant Eggs
From : Aquatic Toxicity Workshop 2009, La Malbae, Quebec, Canada, September 27-30, 2009 - Book of Abstracts
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : test sensitivity; test validation; test development

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Abstract : The presentation forwards and discusses the major facts and the figures of an extensive recent review elaborated by nine ecotoxicologists from different countries.First the history of the acute Daphnia magna test is outlined, with data on intra- and interlaboratory studies on chemicals and effluents and of quality control tests with reference compounds, performed with organisms from laboratory stock cultures. The development of culture/maintenance free microbiotests, and in particular the Daphnia microbiotest which makes use of organisms hatched from dormant eggs is then outlined.Figures on intra- and interlaboratory comparisons of quality control tests performed with lab cultured Daphnias and with Daphnia microbiotests are given, as well as those of ringtests performed at national and international level, with both types of Daphnia magna neonates.The conclusions of the review summarised in the presentation, is that the sensitivity of the Daphnias hatched from the dormant eggs is similar to that of the test organisms taken from laboratory cultures and that the precision of the Daphnia microbiotest is in most cases greater than that of the assays based on lab cultures. The review therefore recommends that the use of Daphnias hatched from dormant eggs should also be incorporated in national and international standards, as an alternative to organisms taken from laboratory cultures.

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J. de Jonge, J.M. Brils, A.J. Hendriks and W.C. Ma
Ecological and Ecotoxicological Surveys Of Moderately Contaminated Floodplain Ecosystems in the Netherlands
Aquatic Ecosystem Health and Management 1999 2, 3 9-18
Toxkits : Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : toxicity monitoring

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Abstract : Floodplains of the rivers Rhine and Meuse in the Netherlands are moderately polluted with heavy metals and toxic compounds. In these floodplain areas a number of nature rehabilitation programs are being planned and executed. The question arises whether or not the contaminants of concern pose a risk to organisms exposed, and, if so, whether this will hinder the expected ecosystem recovery. This study focuses on the effects of contaminants on aquatic and terrestrial macro-invertebrates in the field situation. In three shallow lakes in the floodplains of the river Rhine, showing different degrees of contamination, chemical analyses and laboratory bioassays have been carried out on sediment samples together with intensive biological field surveys (a so called TRIAD-approach). A nearly identical set-up was chosen to assess 18 terrestrial floodplain sites. The concentrations of several contaminants exceed generic critical risk levels. Risk assessment models show that actual effects on top predators cannot be excluded. Species of lower trophic status might be at risk as well. In the aquatic compartments chemical, ecotoxicological and ecological results point in the same direction. They can be summarized as moderate biological effects, which can mainly be attributed to the elevated levels of a mixture of priority pollutants (metals and poly-aromatic hydrocarbons). No large differences were found among the three lakes. At terrestrial sites, however, fewer effects were found in field surveys and bioassays, while the chemical analyses revealed the same levels of pollution as in the lakes study. This suggests a lower bioavailability of the contaminants than expected. In conclusion, no extreme effects have been found. This conclusion only partly supports the predictions made by present day-risk assessment models. The advantages of nature rehabilitation, that is, improvement of ecological quality through habitat diversity, seem to be larger than the disadvantages of increasing toxic stress by exposing a larger diversity of species to floodplain contaminants. To confirm this statement rehabilitation programs should include overall as well as in-depth monitoring studies.

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L. Szczesny, G. Nalecz-Jawecki and J. Sawicki
The Toxicity of Selected Sunscreens to Brachionus calyciflorus
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts
Toxkits : Rotoxkit F^TM short-chronic
Test Species : Brachionus calyciflorus
Topics : chemicals - general

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Abstract : Sunscreens are one of the most commonly used personal care products. They absorb solar radiation (UVA 320-400 nm and UVB 290-320 nm) and protect the human body mainly against UVB radiation. They can reach the aquatic environment through two principal input pathways: from recreational activities (release from skin during swimming and bathing) and via wastewater treatment plants, for instance, rubbed off by towels, washed off during showering, or even from renal excretion after percutaneous or oral (lipsticks) uptake. They have been detected in environmental samples, freshwaters but also in soils, sediments and marine waters.
There are no data about the influence of UV screens to the freshwater rotifer Brachionus calyciflorus.The toxicity of 7 sunscreens to the latter test species was assessed with the Rotoxkit F chronic and data are presented.

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L. Szczesny, D. Solecka, G. Nalecz-Jawecki and J. Sawicki
A Comparison of Acute Rotifer Toxicity Tests with two Endpoints : Lethality and Inhibition of Ingestion
From : The First Joint PSE-SETAC Conference on Ecotoxicology, Krakow, Poland; 14-16 September 2009 - Book of Abstracts
Toxkits : Rotoxkit F^TM acute
Test Species : Brachionus calyciflorus
Topics : chemicals - pesticides; rapid tests; test sensitivity; behavioural tests

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Abstract : Introduction : As toxicants were used macrolactones - avermectins. It’s a group of anthelmintic and insecticidal compounds. They are derived from Streptomyces avermitilis. The mechanism of their action is bound up with decreasing nerve transmission. They are potential agonists of gamma amino butyric acid receptor and glutamate gated chloride channels. The influx of chloride into the nervous cell cause nerve hyperexcitation. Their mode of action isn’t species specific. So when they reach the aquatic environment, they may affect non-target organisms. There are three reasons why this compounds are widely used in veterinary medicine: broad activity spectrum, high efficiency and high safety margin to mammals. The influence of this group onto aquatic invertebrates isn’t known well that’s why ecotoxicity data are needed.
Methods : the freshwater rotifer Brachionus calyciflorus was used as bioindicator. Organisms were hatched from cysts (MicroBioTests, Belgium). The toxicity of samples were assessed with two acute toxicity tests: Rotoxkit F^TM and RotoxRapid. The first one is a 24 hour lethality test and was performed according to the standard operational procedure of microbiotest (Rotoxkit F^TM). The procedure was insignificantly modified. RotoxRapid is a fast 1 hour toxicity test with inhibition of ingestion of carmine suspension endpoint. The test is similar to the procedure proposed by Snell with some modifications in the kind of artificial food and the volume of samples.
Results and conclusions : Ivermectin was the most toxic from the assessed avermectins. Doramectin was less toxic than ivermectin, and the lowest toxicity was caused by abamectin. The same order of toxicity was observed in Rotoxkit F and RotoxRapid.There were no statistical differences between the toxicity of samples in both tests. The Rotoxkit F can therefore be replaced by the RotoxRapid test for evaluation of the toxicity of this group of veterinary drugs. When using ingestion as test endpoint, the time of analysis is shortened from 24h to 1h.

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A. Baran, C. Jasiewicz and J. Antonkiewicz
Testing Toxicity of Oily Grounds using Phytotoxkit Tests
From : The First Joint PSE-SETAC Conference on Ecotoxicology, Krakow, Poland; 14-16 September 2009 - Book of Abstracts
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - hydrocarbons; soils; soil runoff; plant tests

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Abstract : The investigations were conducted to assess toxicity of grounds susceptible to runoffs from the second hand car market in Miedziana Góra village near Kielce. According to the regulation of the Minister of the Natural Environment dated 9 September 2002 on the soil quality standards and earth quality standards [Journal of Laws No. 165, item 1359] the analyzed grounds were classified into group B. Ten soil samples were collected from the depth of 0-30 cm for analyses. The toxicity test Phytotoxkit for germination and root growth was performed on the soil samples using the plants mustard (Sinapis alba), watercress (Lepidium sativum) and sorghum (Sorghum saccharatum). Chemical analysis of the soil samples comprised determination of the heavy metal contents (Cr, Zn, Pb, Cu, Cd and Ni) and carbohydrates C6-C12 (petrol - total) and C12-C35 (mineral oil). Total heavy metal contents in the grounds were assessed after hot mineralization in a mixture of HNO3 and HClO3 acids using ISP-EAS method, while hydrocarbons content using gas chromatography.
On the basis of the conducted Phytotoxkit test it was found that sorghum was the plant which most strongly responded to the soil pollution. The degree of germination and growth inhibition in the plant roots was the greatest and reached respectively about 30% and 52% in relation to the reference soil. Weaker inhibition of germination and root lengths, respectively about 20% and 25% was noted in mustard. Watercress was the plant most resistant to phytotoxic effect of oil-derivatives and heavy metals. Inhibition of the analyzed parameters in this plant was slight, reaching ca. 10% for germination and ca. 17% for root growth.
The analyzed samples contained from 3.95 to 24.15 mg Cr, from 36.80 to 395 mg Zn, from 3.85 to 157.50 mg Pb, from 2.08 - 36.13 mg Cu, 0.20 - 3.68 mg Cd and from 4.70 to 19.08mg Ni . kg^-1 of soil. The results of chemical analysis conducted on the soil samples revealed excessive contents of lead in three samples and zinc in one soil [Journal of Laws, No. 165, Item 1359]. The contents of the other heavy metals: chromium, copper, cadmium and nickel, remained within the norm. Also exceeded in the analyzed soil samples were permissible concentrations of C6-C12 carbohydrates, as stated in the Regulation of the Minister of the Natural Environment [Journal of Laws, No. 165, Item 1359] were registered. Chemical pollution of soil with hydrocarbons testifying petrol presence was between 3.5 and 11 times higher that the permissible norm and reached from 3.32 to 11.1mg . kg^-1. Exceeding the permissible contents of C12-C35 hydrocarbons evidencing mineral oil presence were assessed only in one sample and the content of these compounds fluctuated from 2.21 to 59.4 mg . kg^-1.
Summing up the obtained results it was found that the examined soils were polluted chemically with oil derivatives and heavy metals which was confirmed by the observed phytotoxic effect, i.e. a considerable reduction of the test plant root growth.
See corresponding Poster

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C. Karadima and J. Iliopoulou - Georgudaki
Toxicity Estimation of Cheese Whey Effluents after Treatment for Methane Production
From : The First Joint PSE-SETAC Conference on Ecotoxicology, Krakow, Poland; 14-16 September 2009 - Book of Abstracts
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : effluents; surface waters; soils

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Abstract : Introduction : Disposal of cheese whey effluents into surface water and soil recipients, without any treatment, provoke major environmental problem in Greece because of their high toxicants concentrations. In this study, the toxicity of the cheese whey effluents, after treatment in an anaerobic fermentation system for methane production, is evaluated by using three biotests : the zebrafish Danio rerio embryo test and the freshwater crustaceans Thamnocephalus platyurus and Daphnia magna as Thamnotoxkit F and Daphtoxkit FTM respectively. The goal was to evaluate the acute toxicity of treated cheese whey effluents after anaerobic fermentation and to assess the possibility of this specific treatment to detoxify these effluents and render them environmentally safe for disposal.
Methods : For evaluating the toxicity of treated cheese whey effluents, 25 samples were collected from a continuous stirred-tank reactor for methane production (CH4-CSTR) which was operated for a period of 12 months. Toxicity results were expressed as 24h and 48h lethal concentration (LC50) for the Thamnocephalus platyurus and Daphnia magna tests, while certain toxicological endpoints were assessed for the zebrafish toxicity test. The observations were performed using a stereoscope and lethal endpoints were determined after 24 and 48 hours. A number of physicochemical parameters (SO4, PO4, NO3, NO2, NH3-N, Cl, TDS, pH) were assessed for testing possible correlation with the toxicity values.
Results and conclusions : All samples (25) were classified from toxic to extremely toxic according to the bioassay tests. The average toxicity values were 1.99% (24h) and 0.73% (48h) for zebrafish, 1.40% (24h) for i>Thamnocephalus and 5.85% (24h) and 2.53% (48h) for Daphnia which seems to be the least sensitive test species for these effluents. According to the correlation values, cheese whey effluents’ toxicity is mainly caused by phosphates and nitrites for zebrafish and TDS for T. platyurus. Zebrafish, is testified as the most indicative organism for the monitoring of these effluents; T. platyurus and Daphnia magna which are characterized, in this case, as less sensitive. Further treatment is necessary to reduce the nutrients’ concentrations and resulting to an effluent with fewer repercussions when discharged to the ecosystem.

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A. Rouvalis, Ch. Theodoropoulos and J. Iliopoulou-Georgudaki
Ecological Risk Assessment of Olive Oil Mill Wastewaters Discharged in a Medium-Sized River in Western Greece
From : The First Joint PSE-SETAC Conference on Ecotoxicology, Krakow, Poland; 14-16 September 2009 - Book of Abstracts
Toxkits : Daphtoxkit F^TM pulex; Thamnotoxkit F^TM
Test Species : Daphnia pulex; Thamnocephalus platyurus
Topics : waste waters; surface waters

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Abstract :
Introduction : Olive oil production is of great economic importance in the countries of the Mediterranean region. Olive mill wastewaters (OMW) are a major environmental problem due to the high organic load and toxic effects of specific constituents, such as polyphenols. The additional high content of TSS, NO3-, NO2-, NH4⁺ and PO4^3- of these effluents constitute a threat to river ecological quality. Ecological risk assessment is a function of environmental exposure and ecotoxicological effects. It is described on the basis of the risk (hazard) quotient, which is the ratio between predicted no effect concentration (PNEC) and predicted environmental concentration (PEC). PNEC is based on toxicity data (E/LC50) from short-term lethal studies or long-term sublethal studies, with safety factors of usually between 10-1000. PEC is estimated from direct measurement, or models of varying complexity. The downstream route of Selinountas river (Western Greece), that receives wastewaters from an adjacent olive oil mill, was selected for such a risk assessment.
Methods : In order to assess the risk posed on the aquatic ecosystem, an impacted site located downstream of an olive oil mill was selected for chemical, biological and toxicological analyses during autumn and winter of the year 2008. Physicochemical analyses were carried out using a HACH Lange - DR2800 Spectrophotometer, tannins and lignins were analyzed according to Standard Methods for the Examination of Water and Wastewater and phenolic compounds using the Folin - Ciocalteu method. Toxicity tests were conducting by using two crustaceans (Daphnia pulex, Thanmocephalus platyurus) and a zebrafish embryo test (Danio rerio). Finally, river quality was assessed according to the STAR AQEM methodology and samples were classified into one of five quality classes, as indicated by the WFD 2000/60/EU.
Results and conclusions : The toxicity tests classified the OMW as extremely toxic against all organisms (LC50 0.14%-5.28%). The invertebrate toxicity tests indicate a heavily impacted status of this particular site due to the olive oil mill wastewaters. The ecological quality was found moderate during autumn and shifted to poor during winter. Risk Quotient (RQ=PEC/PNEC) exceeded 1 in both seasons (9.93 in autumn and 12.24 in winter), revealing the highly impacted status of the sampling site. The above, in combination with the ecological classification using WFD 2000/60/EU criteria, are a good evaluation of the risk status. The integrated analysis confirms the heavy degradation of the sampling site due to the OMW discharge. Finally, it may be underlined that this is the first attempt for a risk assessment of these complex effluents for surface waters is attempted.

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A. Törökne
The EU Bathing Water Directive (2006/7/EC) Including a Bathing Water Profile, with Emphasis on Cyanobacterial Proliferation
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : regulatory testing; cyanobacterial blooms; decision support system; bathing waters; cyanotoxins

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Abstract : It is an obligatory issue for member states of the European Union to build up by 2011 a bathing water profile as part of the Bathing Water Directive (2006/7/EC). The main concept of the Directive is to protect human health against environmental contaminants such as bacteria, cyanobacteria etc. The European Commission therefore has established a working group composed of 6 subgroups which are in charge of producing a guidance document with instructions on how to build up a bathing water profile.
An overview is given in this presentation on the potential for proliferation of cyanobacteria, macroalgae and phytoplankton as a part of this guidance document, with description of the succession process in the environment among different genera of diatoms, green algae and cyanobacteria, and the factors which favour their respective proliferation. Besides bacteriological contamination, toxin production by cyanobacteria may affect the quality of bathing water and it is important to collect historical data and to monitor on site to generate data on toxin production. Cyanobacterial blooms influence the colour, odour and texture of bathing water quality, and through the toxin production they are also harmful agents for fishes, birds, pets and animals drinking from the water containing toxic secondary products of metabolism. The main reason to include toxin producing cyanobacteria into the bathing water profile is to take into consideration their effects on human health caused by irritative and/or allergenic and hepatotoxic/neurotoxic symptoms. The presentation shows mitigation measures and gives practical advice for monitoring the algal proliferation. Decision trees are suggested as well as modelling methods for decision makers.
See corresponding Poster

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C. Karadima, Ch. Theodoropoulos and J. Iliopoulou-Georgudaki
Environmental Hazard Assessment of Cheese Manufacturing Effluent Treated for Hydrogen Production
Bulletin of Environmental Contamination and Toxicology 2009 83,3 428-434
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : effluents

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Abstract : Toxicity of effluents after treatment in an anaerobic fermentation system for hydrogen production is evaluated with three biotests: The zebrafish Danio rerio embryo test, the Thamnotoxkit F and the Daphtoxkit F^TM magna. Samples were classified from ‘‘very’’ to ‘‘extremely toxic’’. Average toxicity values for zebrafish were 1.55% (24 h) and 0.75% (48 h), for Thamnocephalus 0.69% (24 h) and for Daphnia 2.51% (24 h) and 1.82% (48 h).Statistical analysis between physicochemical parameters and LC50 values revealed that PO4^-3, SO4^-2, NH3N and NO3^- have the major contribution to toxicity. Based on results, this treatment is considered an environmentally ineffective way of managing the specific wastes.

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G. Persoone, R. Baudo, M. Cotman, C. Blaise, K.Cl. Thompson, M. Moreira-Santos, B. Vollat, A. Törökne and T. Han
Review on the Acute Daphnia magna Toxicity Test - Evaluation of the Sensitivity and the Precision of Assays Performed with Organisms from Laboratory Cultures or Hatched from Dormant Eggs
Knowledge and Management of Aquatic Ecosystems 2009 393, 1
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : review

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Abstract : Executive summary : One of the most internationally used bioassays for toxicity screening of chemicals and for toxicity monitoring of effluents and contaminated waters is the acute toxicity test with daphnid crustaceans, and in particular that performed with Daphnia magna.
Standard methods have been developed for this assay that were gradually endorsed by national and international organisations dealing with toxicity testing procedures, in view of its application within a regulatory framework. As for all toxicity tests, the organisms used for the acute D. magna assay have to be obtained from live stocks which are cultured in the laboratory on live food (micro-algae).
Unsurprisingly the various standard protocols of this particular assay differ - at least to a certain extent - with regard to the test organism culturing conditions. In addition, some technical aspects of the toxicity test such as the effect criterion (mortality of immobility), the exposure time, the type of dilution water, etc., also vary from one standard to another.
Although this particular assay is currently used in many countries, the technical and biological problems inherent in year-round culturing and availability of the biological material and the culturing/maintenance costs of live stocks restrict its application to a limited number of highly specialised laboratories.
This fundamental bottleneck in toxicity testing triggered investigations which brought forward the concept of “microbiotests” or “small-scale” toxicity tests. “Culture/maintenance free” aquatic microbiotests with species of different phylogenetic groups were developed in the early 1990s at the Laboratory for Environmental Toxicology and Aquatic Ecology at the Ghent University in Belgium.
These assays which were given the generic name “Toxkits”, are unique in that they employ dormant stages (“cryptobiotic eggs”) of the test species, which can be stored for long periods of time and “hatched” at the time of performance of the assays.
One of these microbiotests is the Daphtoxkit F magna, which is currently used in many laboratories worldwide for research as well as for toxicity monitoring purposes.
The microbiotest technology has several advantages in comparison to the “traditional” tests based on laboratory cultures, especially its independence of the stock culturing burden. However, the acceptance (or possible non-acceptance) of performing assays with test organisms obtained from “dormant eggs” should be clearly dictated by the “sensitivity” and “precision” criteria of the former assays in comparison to the latter.
The first part of this review therefore thoroughly reviews the scientific literature and of data obtained from various laboratories for assays performed with either D. magna test organisms obtained from lab cultures or hatched from dormant eggs.
Attention has focused on data of quality control tests performed on reference chemicals, and in particular on potassium dichromate (K2Cr2O7) for which an acceptability range of 0.6-2.1 mg.L^-1 has been set in ISO standard 6341 for the 24 h EC50 of the acute D. magna assay.
Mean EC50s, standard deviations and variation coefficients were calculated from the collected data, all of which are presented in tables and figures and discussed in detail.
The major conclusions drawn from the analysis of the large number of quality control (QC) data on the acute D. magna toxicity test are that :
(1) Virtually all results from assays performed with Daphnias taken from lab cultures or with Daphnia microbiotests are within the acceptability range set by ISO standard 6341 for the reference chemical potassium dichromate.
(2) The mean 24 h EC50s of the Daphnia microbiotests performed in different laboratories are within the range of the mean EC50s of the assays based on lab cultures, and the variation coefficients (20 to 30%) are similar.
(3) The precision – in terms of the long term in house variability - of the quality control Daphnia microbiotests is as good as that of the QC tests based on lab cultures.
The review further reports on intra-laboratory sensitivity comparison studies performed during the last 15 years on pure chemicals and on natural samples, with both laboratory cultured organisms and Daphnias hatched from dormant eggs. These studies carried out in different laboratories showed EC50 correlation coefficients of 0.86 to 0.98, corroborating a similar sensitivity of the two types of test organisms.
The third part of the review reports and analyses data on proficiency ringtests on the acute D. magna assay which have been organised in different countries since 2002 with either reference chemicals or with natural samples, and in which part of the laboratories performed their assays with Daphnia microbiotests and others with lab cultured Daphnias.
The conclusions drawn from all the ringtests indicate that the sensitivity of Daphnia neonates hatched from dormant eggs is similar to that of test organisms taken from lab cultures and that in most cases the precision of the Daphnia microbiotest is superior to that of the assays based on lab cultures.
The review finally addresses the issue of possible sensitivity differences of Daphnias hatched from dormant eggs which are produced by different D. magna strains.
From these investigations it appeared that the EC50 s from assays performed with Daphnias hatched from dormant eggs of different strains did not differ significantly from those from assays undertaken with daphnids from lab cultures.
The obvious advantages of Daphnia microbiotests over tests with Daphnias stemming from lab cultures have led to the worldwide use of these culture/maintenance free and low cost small-scale assays in both research and toxicity monitoring.Daphnia microbiotest is in current use in several countries for toxicity testing in a regulatory framework, and recent calculations indicate that about 10 000 acute D. magna assays are now performed annually with neonates hatched from dormant eggs.
The use of dormant eggs to obtain test organisms independently of stock culturing has recently also been accepted in international standards for toxicity testing. ISO standard 20665 (2008) related to the determination of chronic toxicity with Ceriodaphnia dubia, and ISO standard 20666 (2008) for the determination of the chronic toxicity with Brachionus calyciflorus in 48 h, both indicate that the assays can be conducted with organisms hatched from dormant eggs.
On the basis of the extensive scientific evidence provided in this review that is justifiably supported by the two ISO methods mentioned above, the authors therefore recommend that the use of Daphnias hatched from dormant eggs should also be incorporated in national and international standards, as an alternative to the use of Daphnias taken from laboratory cultures.
Full article (from open access periodical : Knowledge & Management of Aquatic Ecosystems - KMAE)

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S. Waara, B. Hyerta Buitrago, P. Ferrer Munoz, V. Ribé, M. Larsson and M. Engwall
Hazard Assessment of Sediment from a Constructed Wetland for Treatment of Landfill Leachate
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments; landfill leachates

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Abstract : Many landfills in Sweden have recently been forced to shift treatment options from municipal sewage treatment to on-site treatment. Biological methods combined with other treatment technologies have often been seen as the preferred options. At the Atleverket landfill in Orebro, a constructed surface flow wetland covering 8 ha consisting of 10 ponds of various sizes in series has been operated since 2001. Previously assessment of water quality data from various points in the wetland has shown that most of the heavy metals are removed already during the first third of the system. However, not much is known about the toxicity of the sediment and what hazard it poses to sediment living organisms and to those organisms that feed upon them. Another unresolved issue is how to best manage the wetland in order to minimize the risk of spreading the accumulated contaminated sediment in case of high flows and/or rupture of the pond. In this study, sediment has been recovered from 8 different points in the wetland system. A toxicity test battery was selected to cover different endpoints, test organisms and sediment fractions. The test organisms chosen were Vibrio fischeri, Heterocypris incongruens and Salmonella typhimurium (Umu test). The presence of dioxin-like compound was analysed using a bioassay with a genetically modified rat hepatoma cell line (H4IIE cells). The results from this ecotoxicological evaluation will be presented and discussed in terms of possible ecological risk of the sediment and the best management option for the wetland.

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E. Minagh, R. Hernan, K. O’Rourke, F. M. Lyng and M. Davoren
Aquatic Ecotoxicity of the Selective Serotonin Reuptake Inhibitor Sertraline Hydrochloride in a Battery of Freshwater Test Species
Ecotoxicolgy and Environmental Safety 2009 72 434-440
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemical - drugs

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Abstract : Sertraline hydrochloride is a selective serotonin reuptake inhibitor (SSRI) widely prescribed to patients suffering from psychiatric disorders. Pharmaceutical products such as sertraline have been identified in environmental waters. This study describes the evaluation of sertraline using a battery of freshwater species representing four trophic levels. The species most sensitive to sertraline were Daphnia magna 21 d reproduction test, Pseudokirchneriella subcapitata 72 h growth inhibition, and Oncorhynchus mykiss 96 h mortality, with the Microtox assay being the least sensitive assay. The D. magna 21 d reproduction test was approximately two orders of magnitude more sensitive than the other bioassays. These results show the advantages of having a tiered approach within a test battery. The presented results indicate that sertraline hydrochloride adversely affects aquatic organisms at levels several orders of magnitude higher than that reported in municipal effluent concentrations, however adverse effects may result from lower concentration exposures, further research into chronic toxicity is therefore advocated.

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B.J. Ripley, K.C. Davis, B.J. Carter and M.A. Simovich
Toxicity of Malathion and Roundup® to the San Diego Fairy Shrimp
Transactions of the Western Section of the Wildlife Society 2002-2003 38/39 13-21
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - pesticides

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Abstract : The purpose of this study was to measure the toxicity of malathion and glyphosate (the active ingredient in Roundup®, to San Diego fairy shrimp (Branchinecta sandiegonensis). The endemic San Diego fairy shrimp has been listed as endangered due to habitat loss and degradation of southern California vernal pools. The risk to fairy shrimp from the potential impact of herbicides and insecticides sprayed near pools has not been assessed. The LC50 for malathion was 24.5 mg/L (estimated active ingredient in Dexol Home Insect Killer). Based on possible environmental concentrations of malathion, it is unlikely to pose a threat to individual San Diego fairy shrimp. The LC50 for glyphosate was 11.8 µg/L (estimated active ingredient in Monsanto Ready-To-Use Weed & Grass Killer). Environmental concentrations of glyphosate are usually well below concentrations that show acute toxicity to other crustaceans, but assuming a 1/20 safety margin as recommended by the U.S. Environmental Protection Agency, they may be above safe concentrations estimated from toxicity data measured here for B. sandiegonensis. However, concentrations in pools are not known, so caution is recommended when making management decisions about these pesticides. We recommend that spraying be avoided in pool watersheds in the rainy season, and that future testing be done on different chemicals, other organisms, and possible sublethal effects on shrimp.

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J.M. Brausch and P.N. Smith
Toxicity of three Polyethoxylated Tallowamine Surfactant Formulations to Laboratory and Field Collected Fairy Shrimp, Thamnocephalus platyurus
Archives of Environmental Contamination and Toxicology 2007 52 217-221
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - pesticides; chemicals - surfactants

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Abstract : Polyethoxylated tallowamine (POEA) is a non-ionic surfactant used in herbicide formulations to increase the efficacy of active ingredients. POEA promotes penetration of herbicide active ingredients into plant cuticles, and in animal species is known to cause alterations in respiratory surfaces. POEA use has increased recently with the advent of “Roundup-Ready” crops; however, its potential effects on aquatic invertebrates are relatively unknown. The aquatic macroinvertebrate Thamnocephalus platyurus (Crustacea, Anostraca) was used to assess the acute toxicity of POEA. Three formulations of POEA consisting of a 5:1, 10:1, and 15:1 average oxide:tallowamine were used in this study. All POEA formulations were found to be extremely toxic to T. platyurus with 48-h LC50 concentrations as low as 2.01 µg/L for 15:1. POEA toxicity increased as the tallowamine chain length was reduced, whereas the oxide chain length appeared to only slightly increase toxicity. Based on these results, POEA has the potential to adversely affect aquatic organisms in areas in which it is used.

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J.F. Blom,B. Bister, D. Bischoff, G. Nicholson, G. Jung, R.D. Süssmuth and F. Jüttner
Oscillapeptin J, a New Grazer Toxin of the Freshwater Cyanobacterium Planktothrix rubescens
Journal of Natural Products 2003 66, 3 431-434
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : biotoxins

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Abstract : Oscillapeptin J (1), a new and highly potent crustacean grazer toxin, was isolated from the axenic cyanobacterium Planktothrix rubescens, which frequently forms blooms in freshwater lakes. Chemical and spectroscopic analyses, including high resolving MS and two-dimensional NMR, were used to elucidate the compound\'s structure as a depsiheptapeptide of the oscillapeptin type. Strict application of a bioassay-guided isolation procedure proved this compound to be one of the major causative agents (besides [D-Asp³,(E)-Dhb⁷]microcystin-RR) of the acute grazer toxicity of P. rubescens from Lake Zürich. The LC50 value of oscillapeptin J as determined for the freshwater crustacean Thamnocephalus platyurus was 15.6 µM.

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M. DellaGreca, M.R. Iesce, M. Isidori, A. Nardelli, L. Previtera and M. Rubino
Phototransformation Products of Tamoxifen by Sunlight in Water. Toxicity of the Drug and its Derivatives on Aquatic Organisms
Chemospere 2007 67, 10 1933-1939
Toxkits : Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - drugs

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Abstract : Transformation of tamoxifen has been observed in water by prolonged sunlight irradiation. The main photoproducts, isolated by chromatographic techniques, have been identified by spectroscopic means. Photoisomerization, photocyclization and, to a lesser extent, photooxygenation appear to be involved in the degradation of the drug. The acute and chronic toxicity of the parent drug and its photoproducts were tested on non-target aquatic organisms (Brachionus calyciflorus, Thamnocephalus platyurus, Daphnia magna and Ceriodaphnia dubia). Exposure to all the compounds induced mainly chronic effects without significant differences among the parental and derivative compounds.

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J. Struijs and D. de Zwart
Evaluation of PT - Determination of toxic pressure in water
RIVM rapport 860703001 2003
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - metals; chemicals - organic compounds; toxicity classification; toxicity index; toxicity monitoring

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Abstract : The project the development of pT is completed with this report. The name pT refers to toxic potency of surface water due to the presence of toxic substances, of which the identity and concentrations are unknown. The multitude and diversity of potentially occurring chemicals put strong limitations to information conventional chemical monitoring may provide. Even if it were doable to analyse most chemicals, it would be impossible to interpret such information in terms of toxic stress because toxicity data are available for only a limited number of environmental pollutants.

In environmental policy a need was expressed to develop an instrument to monitor toxic stress in ecosystems. There is a need to keep track of the efficacy of environmental measures as well as for weighting toxic stress in association with respect to other environmental stressors, such as acidification and eutrophication (how serious is the problem of toxic substances for the environment ?).
A method was developed to 1.) extract and concentrate the unknown cocktail of toxic substances from a sample of surface water, 2.) to determine the acute toxicity of concentrated water samples using a test battery of miniaturised in vivo-bioassays (toxkits) and 3.) to compute, from the observed interspecies variation of acute toxicity in the toxkits, impacts at a higher ecological level, i.e. toxic stress in terms of the potentially affected fraction of species (PAF) that occur in the field.

The concentration technique was optimised by means of different test mixtures, consisting of chemicals with a non-specific (narcotic) mode of action, pesticides and surfactants. Toxkits were employed to test these mixtures. The test battery consisted of Daphnia IQ, PAM (an algae test), Microtox and Thamnotox F. The test mixtures were made of more than 30 chemicals with varying physico-chemical and toxic properties.

The recovery of the finally adopted concentration technique was higher than 60 % and should be considered as the maximum achievable, because the solid phase extraction on which it relies is specific with respect to physico-chemical properties of chemicals.
For metals an alternative technique was developed, based on multi-element analyses of a water sample in combination with ecotoxicity data of 16 metals.

Monitoring programs were executed to sample, concentrate and test the toxicity of surface waters in the Netherlands. Both for organic micropollutants and metals it was shown that toxic pollution order is Scheldt > Meuse > Rhine. The pT method, also known as measured msPAF (ms means multi substance), was applied to varying test mixtures in a first attempt to compare the calculation rules for msPAF which is computed for complex mixtures with pT (measured msPAF) derived from measured toxicity distributions.

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F. Gagné, C. Blaise, J. Auclair and J.F. Férard
Ecotoxicity of Drug Nano-Formulations (Pamam-Dendrimers and Minocycline)
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts 2009
Toxkits : Algaltoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - nanoparticles; nanoparticles

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Abstract : Among the diverse applications foreseen for nanotechnology, formulations employing nano-based polymers as drug delivery systems are presently being developed. As this may eventually lead to the release of such nano-formulations into aquatic environments most likely via urban point sources of pollution, gathering information on their ecotoxicity is certainly justified. We thus conducted an initial investigation of the potential hazard that a specific type of intended formulation (PAMAM-dendrimers coupled with the antibiotic minocycline) might have on receiving water biota by assessing their toxicity with a suite of small-scale biological tests reflecting a large spectrum of taxa. Minocycline (a tetracycline antibiotic) and 2^nd, 4^th and 5^th generation PAMAM-dendrimers were appraised to determine their singular and interactive effects on decomposers (Microtox Vibrio fischeri bacterial luminescence inhibition assay; MARA, or Microbial Array for Risk Assessment, assay), primary producers (Pseudokirchneriella subcapitata growth inhibition assay; Luminotox photosynthesis efficiency inhibition assay), primary consumers (micro-crustacean Thamnocephalus platyurus lethality assay) and secondary consumers (Hydra attenuata assay measuring lethality and sublethality endpoints; rainbow trout primary hepatocytes cytotoxicity assay). Results of this preliminary ecotoxicity assessment undertaken at different levels of biological organization will be presented and implications that nano-formulations might have on aquatic (micro)organisms will be discussed.

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G. Nalecz-Jawecki, A. Hajnas and J. Sawicki
Photodegradation and Phototoxicity of Thioridazine and Chlorpromazine Evaluated with Chemical Analysis and Aquatic Organisms
Ecotoxicology 2008 17, 1 13-20
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - drugs

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Abstract : The photochemical behaviour of chlorpromazine (CPZ) and thioridazine (THR) incubated under VIS light and a UV-A lamp was investigated with a high-performance liquid chromatography photodiode array detector (HPLC-PAD) and two bioassays. VIS light caused the decrease of CPZ and THR to 25% and 34% of the initial level, respectively, while UV-A degraded the drugs almost totally. CPZ and THR were very toxic to the protozoan Spirostomum ambiguum (Spirotox) and anostracan crustacean Thamnocephalus platyurus (Thamnotoxkit F^TM) with 24-h LC50 values of around 0.5 mg l^-1. In spite of the drastic decrease of the concentration of the drugs, the irradiated samples were toxic to the protozoan, especially when a sublethal end-point was taken into consideration. Contrary to the protozoan the crustacean was not sensitive to the products of photodegradation. Mass spectrometry analysis showed the presence of dimers and trimers of the CPZ and mono-, di-, and tri-oxygenated derivatives of THR. The presented data give a strong indication of the importance of the investigation of the environmental fate of drugs, especially those known to be phototoxic.

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A. Dell'Aquila, A. Diodati, M. Isidori, M. Lavorgna and A. Mancini
Assessment of Toxicity and Genotoxicity of Industrial Effluents in Southern Italy
From : 31st Annual Meeting of the European Environmental Mutagen Society, September 2001 - Ghent, Belgium 2001
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : effluents

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Abstract : The increase of industrial sites triggered the need to assess the release of countless man-made chemicals to the biospere and the potentially harmful impact they can determine alone or in combination toward biological integrity.
Effluents from the factories of the Industrial Development Area of Caserta (Southern Italy) were tested to evaluate their toxic and genotoxic effect on biota of receiving environments.
Toxicity tests were perfromed on reducers (the bacterium Vibrio fischeri), producers (the alga Selenastrum capricornutum) and consumers including a rotifer (Brachonionus calyciflorus), a cladoceran (Daphnia magna) and an anostracan (Thamnocephalus platyurus) to evaluate effects on freshwater organisms from different throphic levels.
Furthermore SOS Chromotest, a bacterial colometric assay with E. Coli PQ37, was carried out to assess genotoxic activity of these industrial effluents, detecting DNA-damaging agents.
Results showed that some industrial effluents are only toxic for organisms of the aquatic chain whereas others are highly genotoxic for E. coli PQ37.
The present study suggests that different kinds of bioassays such as toxic and genotoxic tests offer complementary tools and approaches that can be applied to measure hazard and risk of xenobiotics on the environment.

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S. Waara and C. Färm
An Assessment of the Potential Toxicity of Runoff from an Urban Roadscape during Rain Events
Environmental Science and Pollution Research 2008 15, 3 205-210
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : rainwater

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Abstract : Goal, Scope and Background The potential negative impact of urban storm water on aquatic freshwater ecosystems has been demonstrated in various studies with different types of biological methods. There are a number of factors that influence the amount and bioavailability of contaminants in storm water even if it is derived from an area with a fairly homogenous land use such as a roadscape where a variation in toxicity during rain events might be expected. There are only a few previous investigations on the toxicity of highway runoff and they have not explored these issues extensively. The main objective of this study is therefore to characterize the potential toxicity of highway runoff during several rain events before it enters a detention pond in VästerOas, Sweden, using laboratory bioassays with test organisms representing various functional groups in an aquatic ecosystem. The results are to be used for developing a monitoring program, including biological methods.
Materials and Methods The storm water was sampled before the entrance to a detention pond, which receives run-off from a highway with approximately 20,000 vehicles a day. The drainage area, including the roadscape and vegetated areas, is 4.3 ha in size. Samples for toxicity tests were taken with an automatic sampler or manually during storm events. In total, the potential toxicity of 65 samples representing 15 different storm events was determined. The toxicity was assessed with 4 different test organisms; Vibrio fischeri using the Microtox^® comparison test, Daphnia magna using Daphtoxkit-F^TMmagna, Thamnocephalus platyurus using the ThamnotoxkitF^TM and Lemna minor, duckweed using SS 028313.
Results and Discussion Of the 65 samples, 58 samples were tested with DaphniatoxkitF^TMmagna, 57 samples with the Microtox^® comparison test, 48 samples with ThamnotoxkitF^TM and 20 samples with Lemna minor, duckweed. None of the storm water samples were toxic.
No toxicity was detected with the Lemna minor test, but in 5 of the 23 samples tested in comparison to the control a growth stimulation of 22-46% was observed. This is in accordance with the chemical analysis of the storm water, which indicated rather large concentrations of tot-N and tot-P. In addition to the growth stimulation, morphological changes were observed in all the 5 samples from the winter event that was sampled. The lack of toxicity observed in our study might be due to a lower traffic intensity (20,000 vehicles/day) at the site and the trapping of pollutants in the vegetated areas of the roadscape, resulting in much smaller loads of pollutants in the storm water than in some previous studies.
Conclusions Ecotoxicological evaluations of storm water including run off from rain events from urban roadscape studies clearly reveal that toxicity may or may not be detected depending upon site, storm condition and the test organism chosen. However, storm water might not be as polluted as previously reported nor may the first flush be such a widespread phenomenon as we originally expected. In this study, there was also a good correlation between pollutant load measured and the lack of toxicity. The test organisms chosen in this study are commonly used in effluent control programs in Sweden and other countries, which makes it possible to compare the results with those from other effluents. In this study, only acute toxicity tests were used and further studies using chronic toxicity tests, assays for genotoxic compounds or in situ bioassays might reveal biological effects at this site. Furthermore, most of the samples were taken in spring, summer or fall and it is possible that winter conditions might alter the constituents in the storm water and, thus, the toxicity of the samples.
Recommendations and Perspectives Considering the complex nature of run off from urban roadscapes, it will be virtually impossible to evaluate properly the potential hazard of particular storm water and the efficiency of a particular treatment strategy from only physical and chemical characterizations of the effluent. Therefore, despite the lack of toxicity detected in this study, it is recommended that toxicity tests or other biological methods should be included in evaluations of the effects of runoff from roadscapes.

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E. Nehrenheim, S. Waara and L. Johansson Westholm
Metal Retention on Pine Bark and Blast Furnace Slag - On-Site Experiment for Treatment of Low Strength Landfill Leachate
Bioresource Technology 2008 99,5 998-1005
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : landfill leachates; leachtates: solid wastes

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Abstract : Treatment of landfill leachate using blast furnace slag and pine bark as reactive sorbents was studied in an in situ column experiment at the Lilla Nyby landfill site in Eskilstuna, Sweden. The columns were filled with approximately 10 l of each sorbent and leachate was supplied at three different flow rates during a period of 4 months. Samples of inflow and outflow were collected three times a week and were analyzed for physical and chemical parameters, including concentrations of some metals, and toxicity. It was found that pine bark removed metals more efficiently than did the blast furnace slags; that Zn was most efficiently retained in the filters and that both retention time and initial concentration played an important role in the sorption process. It was also observed that the pine bark column did not release COD. No toxicity of the untreated or the treated leachate was found with the test organisms and test responses used.

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V. Ribé, E. Nehrenheim, M. Odlare and S. Waara
Leaching of Contaminants from Untreated Pine Bark in a Batch Study : Chemical Analysis and Ecotoxicological Evaluation
Journal of Hazardous Materials 2009 163, 2-3 1096-1100
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : leachates

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Abstract : Low cost sorbents have been widely studied in recent years in the search for filter materials that retain contaminants from water. One promising, low cost material is pine bark, a by-product from the forest industry. Many studies have shown that pine bark has great potential for the treatment of metals and organic substances, as a replacement for other commercial sorbents such as active carbon. However, some potential problems are introduced through the use of natural materials and by-products. One such problem that must be addressed is the possibility of leaching of contaminants from the filter material, especially in the initial filtration step or during flushes of lightly contaminated water, e.g. during rainfall for on-site treatment of storm water or landfill leachate. The aim of this preliminary study was therefore to identify potential risks and limitations of using pine bark as a filter material. Leachate from a standardized batch test was analysed for metals, dissolved organic carbon (DOC) and phenols. In addition to these chemical analyses, an ecotoxicological test was conducted using the test organism Daphnia magna. The results showed significant leaching of DOC and some metals. Only a small fraction of the DOC was present as phenols. The leachate was however found to be toxic to the test organism without pH adjustment, and the EC50 was established at an approximate leachate concentration of 40%. This was concluded to be related to the low pH in the eluate, since no toxicity was observed after pH adjustment before the toxicity tests.

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S. Waara, M. Ek, Å. Forsberg, M. Fridolfson and K.-O. Waara
Hazard Assessment of Untreated and Biologically Treated Landfill Leachate using Toxicity Tests - Lessons Learnt from some Swedish Case Studies
From : Conference on Ecological Engineering, Polish Academy of Sciences, Gdansk University of Technology, Gdansk, Poland, 6-8 September 2009 - Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : landfill leachates

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Abstract : On-site treatment of landfill leachate has lately been introduced in Sweden and other countries. In Sweden, the preferred treatment technology has often been based upon biological methods alone or in combination with other techniques. As several studies indicate that untreated landfill leachate may contain many different persistent pollutants and is often causing toxicity in laboratory toxicity tests, the monitoring program of the treatment efficiency should also include analytical methods to determine the toxicity of the leachates before and after different treatment steps.
In this study the toxicity of four different landfill leachates have been studied before and after treatment in three different in situ treatment plants and one pilot study. In general the studies revealed that the biological treatment was a highly effective technique for removing toxicity in landfill leachate. In some cases toxicity was observed even though the treated water fulfilled issued discharge limits. In the majority of samples high concentrations of unionized ammonia and/or salts may have caused the toxicity but the presence of persistent toxic compounds cannot be ruled out. The study shows that toxicity tests are a valuable tool, preferentially in combination with extensive chemical analysis, in order to properly evaluate and optimize treatment technology and to protect the recipient aquatic ecosystem from toxic discharges. A battery of selected test method based upon these studies is presented and discussed.

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S. Waara, K.O. Waara, Å. Forsberg and M. Fridolfsson
Performance of a Constructed Wetland System for Treatment of Landfill Leachate
From : Waste and Resource Managment - a Shared Responsibility, Stratford-upon-Avon, Warwickshire, England, 16-17 September 2008 - Proceedings Waste 2008
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM acute
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : landfill leachates; leachates

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Abstract : The performance of a constructed wetland for the treatment of landfill leachate has been evaluated based upon data obtained during 4 years (2003-2006). It consists of a series of 10 ponds with a total capacity of 52,000 m³ covering 8 ha. Using univariate and multivariate statistics (PCA) the reduction pattern of a large number of chemical parameters including heavy metals have been investigated in 3 parts of the wetland with equal volume. Analyses show that many parameters are removed to the greatest extent in the first part of the system (e.g. many heavy metals, total suspended solids) or the second part of the system (N-NH⁴) while other parameters such as total nitrogen are more gradualy reduced (10 ton/year removed). Toxicity testing with 5 bioassays showed that toxicity was sometimes observed at the inlet but no toxicity was observed at the outlet for 4 of the test species. The data presented will be used for optimizing the treatment process as well as to improve the monitoring program.

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L. Molander, M. Ågerstrand and C. Rudén
Wikipharma - a Freely Available, Easily Accessible, Interactive and Comprehensive Database for Environmental Effect Data for Pharmaceuticals
Regulatory Toxicology and Pharmacology 2009 55 367-371
Toxkits :
Test Species :
Topics : toxicity classification, review, data base

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Abstract : A significant number of Active Pharmaceutical Ingredients (APIs) have been identified in the environment and in surface waters. Data on the environmental hazards associated with these substances are emerging but are still scarce. We have compiled publicly available ecotoxicity data for APIs into a database called WikiPharma. The use of the database is free of charge. It can be accessed and updated continuously as a “wiki”. The aim of WikiPharma is to provide an easily accessible, comprehensive and up-to-date overview of effects caused by pharmaceuticals on non-target organisms. The database currently contains basic information, i.e. substance name, ATC code(s) and pharmaceutical group(s), for 831 APIs representing 35 different drug classes. Effect data have been identified and included for 116 of these substances. These ecotoxicity test data have been extracted from 156 different sources. The development of a comprehensive database on ecological hazard of APIs can facilitate identification of data gaps and promote environmental risk assessment of these substances. The database is available at www.wikipharma.org .

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P. Oleszczuk
The Tenax Fraction of PAHs Relates to Effects in Sewage Sludges
Ecotoxicilogy and Environmental Safety 2009 72, 4 1320-1325
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - hydrocarbons; sediments; sediment and soil contact tests

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Abstract : In the present study a toxicity evaluation (Heterocypris incongruens, OstracodToxKit F^TM Test) was carried out for ten municipal sewage sludges in relation to polycyclic aromatic hydrocarbon (PAH) contents in these sludges. PAHs content in the sewage sludges was determined by a vigorous extraction method and solid phase extraction with Tenax-TA. The main aim of the evaluation was establishing whether it was possible to determine the negative influence of PAHs in the sewage sludges on H. incongruens mortality and growth inhibition using the Tenax-TA method. H. incongruens showed increased growth inhibition and mortality when exposed to all sewage sludges. Significant relationships were noted between the content of some Tenax-TA PAH fractions and the total organic carbon content, cation exchange capacity and concentration of Ca²⁺ ions. The toxicity was proportional to Tenax-TA concentrations and was not governed by the total concentration found in the sewage sludge.

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J. Kalka, A. Oslislok, J. Surmacz-Gorska, K. Krajewska, D. Marchiocha and A. Raszka
Evaluation of Landfill Leachate Treatment and Toxicity
From : The First Joint PSE-SETAC Conference on Ecotoxicology, Krakow, Poland; 14-16 September 2009 - Book of Abstracts 2009
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : landfill leachates; leachates

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Abstract : Combined treatment of landfill leachate and municipal wastewater were performed in order to investigate the toxicity of leachate changes during biological treatment. Treatment was performed with A2O activated sludge systems and the share of leachate in the influent was calculated at the base of “worst-case” scenario. Landfill leachate increased initial toxicity of wastewater. During biological treatment significant decline of toxicity was observed. Toxicity identification procedure allowed to conclude, that toxicity of samples was connected with pollutants unionized form, easily stripped or oxidized during the aeration.
See corresponding Poster

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L. Wolska, A. Sagajdakow and J. Namiesnik
Indicators for Underground Water Quality
From : 14^th International Symposium on Toxicity Assessment, Metz, France, 30 August - 4 September 2009 - Book of Abstracts
Toxkits : Daphtoxkit F^TM magna; Phytotoxkit; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus, Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : groundwaters; solid wastes; landfill leachates; solid waste leachates; soils; plant tests

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Abstract : In Poland, until 1997, there was no obligation to monitor the composition of waste stored in municipal landfills. Large amounts of hazardous wastes got together with municipal refuses at these landfills. The wastes mainly contain batteries, fluorescent lamps, varnish manufacture’s wastes, old drugs and pesticides, and also used oil and lubricants. Nowadays, it is hard to say what kind of compounds (and of which toxicity) will permeate from municipal landfills to underground waters. There is a high probability of negative effects of waste dumps on the quality of underground water, especially those dumped prior to 1990, and especially in the waste dumps that were not closed off watertight.
The main purpose of this study is to compare the chemical quality of the underground waters around one of the oldest and largest municipal landfills of the Pomeranian province (Poland) with the results of ecotoxicological screening (Fig. 1). Ecotoxicological assessment of the examined underground waters was performed on the basis of the classification system of Persoone et al.
See corresponding Poster

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G. Nalecz-Jawecki and J. Sawicki
Influence of Water Hardness on the Toxicity of Selected Pharmaceuticals and Metals to the Protozoa Spirostum ambiguum and Tetrahymena thermophila
Fresenius Environmental Bulletin 2005 14 873-877
Toxkits : Protoxkit F^TM
Test Species : Tetrahymena thermophila
Topics : chemicals - drugs; chemicals - metals

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Abstract : The goal of the study was the evaluation of the toxicity of selected metals and pharmaceuticals to ciliated protozoa in two media with different hardness: very soft and hard water. Two ciliates were used as test-bionts: Spirostomum ambiguum in the acute toxicity test (Spirotox) and Tetrahymena termophila in the chronic toxicity test (Protoxkit F^TM). Thioridazine and Amitriptyline were the most toxic drugs to S. ambiguum with an EC50 below 0.5 ppm. Pro-pranolol and Verapamil were 3-6 folds and 6-10 folds less toxic, respectively. Spirotox was much more sensitive to tested drugs than Protoxkit F^TM, from 25-folds for Thioridazine and Verapamil to 100-folds for Amitriptyline. Hardness of water did not influence significantly the toxicity of the drugs to very sensitive S. ambiguum, whereas it increased the sensitivity of lower sensible protozoan T. termophila. In case of metals the sensitivity of the test depended on the metal, water hardness and the procedure of the test. Spirotox comparing to Protoxkit F was more sensitive to zinc, similar sensitive to cadmium in soft water, less sen-sitive to lead and cadmium in hard water.

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A. Kungolos, C. Emmanouil, V. Tsiridis and N. Tsiropoulos
Evaluation of Toxic and Interactive Toxic Effects of three Agrochemicals and Copper using a Battery of Microbiotests
Science of the Total Environment 2009 407 4610-4615
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - metals; chemicals - pesticides; toxic interactions

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Abstract : Three commonly used test organisms of different trophic levels (Vibrio fischeri, Pseudokirchneriella subcapitata and Daphnia magna) were exposed to selected agrochemicals (fosthiazate, metalaxyl-M, imidacloprid) and copper, in single doses or in binary mixtures. The toxicity of each single compound varied up to two orders of magnitude, depending on the test species examined. V. fischeri was the most sensitive test organism regarding fosthiazate and metalaxyl-M, indicating an IC50 value of 0.20 mg/L (0.17-0.25 mg/L) and 0.88 mg/L (0.35-1.57 mg/L), respectively. Imidacloprid was the least toxic compound, indicating an EC50 value on D. magna of 64.6 mg/L (43.3-122.5 mg/L) and an IC50 value on V. fischeri of 226 mg/L (159-322 mg/L), while for imidacloprid at a concentration of 1000 mg/L the effect on P. subcapitata was lower than 50%. Copper was the most toxic compound towards all test organisms exhibiting the highest toxic effect on P. subcapitata, with an IC50 value of 0.05 mg/L (0.003-0.008 mg/L). The toxic effects of the binary mixtures have been compared to the theoretically expected effect, resulting from a simple mathematical model based on the theory of probabilities. The independent action model was used in order to predict the theoretically expected effect. The interactive effects were mostly antagonistic or additive, while in few cases (interactive effects of metalaxyl-M and copper on V. fischeri) a synergistic mode of action was observed for some concentration combinations. Experiments showed that interactive effects of chemicals may vary depending on the test species used as well as on the chemicals and their respective concentrations. Although most of the concentrations of chemicals tested in this study are higher than the ones usually found in natural environment, the evaluation of their interactive toxic effects using a battery of bioassays may comprise a useful tool for the estimation of the environmental hazard of chemicals.

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A. Kungolos
Application of Microbiotests and Activated Sludge Respirometry for the Evaluation of Industrial Wastewater Toxicity
Bulletin of Environmental Contamination and Toxicology 2005 74 801-808
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM
Test Species : Daphnia magna; Tetrahymena thermophila
Topics : waste waters; test sensitivity

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Abstract : A lot of different bioassays have been used in order to investigate the effect of toxicants on aquatic ecosystems. Among the bioassays that have been used for the estimation of the toxic effects of chemicals on aquatic organisms, microscale procedures, now commonly referred to as microbiotests, have emerged as cost-effective and simple to operate methods (Blaise, 2000). On the other hand, different techniques have been used in order to monitor toxicants present in industrial sewage that reduce the effeciency of biological wastewater treatment due to intoxication phenomena. Most of toxicity directed approaches for the evaluation of industrial effluent toxicity mainly utilize a respirometric biosensor (Vanrolleghem et al., 1994). Moreover, the majority of studies correlating respirometry with bioassays deal wit pure substances (Kong et al., 1993). There has not been an indication whether the respirometric techniques can be correlated with microbiotests in order to monitor industrial effluents. It is worthwile to investigate such a correlation, since respirometric techniques involve great costs both for the acquisition of the equipment and for its maintenance and operation, while microbiotests are inexpensive. In this study, three microbiotests, the crustacean Daphnia magna test, the marine bacterium Vibrio fischeri test and protozoan Tetrahymena thermophila test were used in order to investigate their potential correlation with a respirometric technique (Vanrolleghem et al., 1994).

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I. Blinova, A. Ivask, M. Heinlaan, M. Mortimer and A. Kahru
Ecotoxicity of Nanoparticles of CuO and ZnO in Natural Water.
Environmental Pollution 2010 158 41-47
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - nanoparticles; chemicals - metals

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Abstract : The acute toxicity of CuO and ZnO nanoparticles in artificial freshwater (AFW) and in natural waters to crustaceans Daphnia magna and Thamnocephalus platyurus and protozoan Tetrahymena thermophila was compared. The L(E)C(50) values of nanoCuO for both crustaceans in natural water ranged from 90 to 224 mg Cu/l and were about 10-fold lower than L(E)C(50) values of bulk CuO. In all test media, the L(E)C(50) values for both bulk and nanoZnO (1.1-16 mg Zn/l) were considerably lower than those of nanoCuO. The natural waters remarkably (up to 140-fold) decreased the toxicity of nanoCuO (but not that of nanoZnO) to crustaceans depending mainly on the concentration of dissolved organic carbon (DOC). The toxicity of both nanoCuO and nanoZnO was mostly due to the solubilised ions as determined by specific metal-sensing bacteria.

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A. Rouvalis, C. Karadima, I. V. Zioris, V. A. Sakkas, T. Albanis and J. Iliopoulou-Georgudaki
Determination of Pesticides and Toxic Potency of Rainwater Samples in Western Greece
Ecotoxicology and Environmental Safety 2009 72,3 828-833
Toxkits : Daphtoxkit F^TM pulex
Test Species : Daphnia pulex
Topics : chemicals - pesticides; rainwater

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Abstract : Rainwater samples from four municipalities located in Achaia Prefecture, Greece, were collected from March to September 2006. The toxic potency of pollutants present in 36 rainwater samples was tested using Daphnia pulex. The pesticide determination was conducted with GC-MS. Only phosphamidon was detected, which appeared in 52% and 13% of the rural and urban areas, respectively. The toxicity of rainwater was determined in 52% and 46.7% of the rural and urban area samples, respectively. Chemical analyses showed that in rural areas, the PO43- ions had higher concentrations than in urban areas. On the other hand, the SO42-, NO-3, and NO-2 anions are more highly concentrated in urban areas. Correlation analysis proved that the toxicity of the rainwater samples is moderate, affected by the presence of the insecticide only in the rural areas. The results indicated that toxicity can be directly assessed via bioassays, even when unknown pollutants are present.

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C. Blok, M. Aguilera and E.A. van Os
Validation of a New Phytotoxicity Test (Phytotoxkit) against an Established Four-Week Growing Test with Pre-Grown Plant Plugs
Acta Horticulturae 2009 819 209-214
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - organic compounds; soils; composts; plant tests

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Abstract : The aim of the studies was to correlate results of a fast extract-based toxicity test with results of an established growing test with mostly pre-grown plants. A standard white peat was contaminated with four levels of TCA (trichloroacetic acid), a known toxic substance. A range was composed of five levels of TCA, including zero, 0.0013, 0.013, 0.13, and 1.3 g/L of peat. Part of the material was entered in Phytotoxkit containers and covered with a filter paper. At the other side of the filter paper two dicotyledonous species, garden cress (Lepidium sativum) and mustard (Sinapis alba) and one monocotyledon, Sorghum (Sorghum saccharatum) were allowed to germinate for a three day period on the extract from the substrate in a climate-controlled cabinet. The same range of amended peats was used in the standard test to grow transplanted lettuce (Lactuca sativa), Chinese cabbage (Brassica rapa subsp. pekinensis) and directly sown barley (Hordeum vulgare). For the Phytotoxkit the above ground length and root length were recorded, and for the standard test the fresh weight and dry weight of the above ground parts were recorded. Results showed a growth reduction for all levels of TCA when using the standard method. When using the Phytotoxkit only the two highest concentrations of TCA reduced growth. For screenings of product alternatives or dissolution series the Phytotoxkit is a faster and cheaper alternative.

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A. Kahru and H.C. Dubourguier
From Ecotoxicology to Nanoecotoxicology
Toxicology 2010 10 105-119
Toxkits : Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - nanoparticles; chemicals - metals

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Abstract : For hazard assessment of NPs quantitative nanoecotoxicological data are required. The objective of this review was to evaluate the currently existing literature data on toxicity (L(E)C50 values) of synthetic NPs in environmentally relevant species in order to: (i) identify tentatively most harmful NPs and most sensitive organism groups, and (ii) to provide relevant ecotoxicological information for further risk assessment. The focus was set on selected synthetic NPs (nano TiO2, nano ZnO, nano CuO, nano Ag, SWCNTs, MWCNs and C60-fullerenes) and organism groups representing main food-chain levels (bacteria, algae, crustaceans, ciliates, fish, yeasts and nematodes).

Altogether 77 effect values were found, mostly for nano iO2 (31%), C60 (18%), nano ZnO (17%), nano Ag (13%), SWCNTs and nano CuO (both 9%). Only 3% of the available quantitative ecotoxicological information concerned MWCNTs. Organism-wise, 33% of the data concerned crustaceans, 27% bacteria, 14% algae and 13% fish. For all organism groups studied, solubility of CuO- and ZnO-NPs was a key factor in their aquatic toxicity.

On the basis of the 34 median L(E)C50 values derived from 77 individual values, NPs were ranked according to their lowest median L(E)C50 value for the above described organism groups: the most harmful were nano Ag and nano ZnO that were classified extremely toxic, (L(E)C50 < 0.1 mg/l), followed by C60 fullerenes and nano CuO that were classified very toxic, (L(E)C50 0.1-1 mg/l). SWCNTs and MWCNTs were classified toxic (L(E)C50 1-10 mg/l). Nano TiO2 was classified as harmful, (L(E)C50 10-100 mg/l). Throughout, algae and crustaceans (daphnids) were most sensitive and thus probably most vulnerable organism groups in aquatic exposure to NPs. Very low L(E)C50 values should deserve thorough attention of environmental risk assessors for evaluation of the potential adverse effects of synthetic NPs on ecosystems. As the quantitative nanoecotoxicological data are still rare, further studies are needed.

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M. Mortimer, K. Kasemets and A. Kahru
Toxicity of ZnO and CuO Nanoparticles to Ciliated Protozoa Tetrahymena thermophila
Toxicology 2010 269, 2-3 182-189
Toxkits : Protoxkit F^TM
Test Species : Tetrahymena thermophila
Topics : chemicals - nanoparticles; chemicals - metals

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Abstract : The toxic effects of nanoparticles (NPs) of ZnO and CuO to particle-ingesting model organism protozoa Tetrahymena thermophila were evaluated. Nano-ZnO was remarkably more toxic than nano-CuO (EC50 values ~ 5 mg metal/l versus 128 mg metal/l). Toxic effect of CuO depended on particle size: nano-CuO was about 10-20 times more toxic than bulk CuO. However, when calculated on basis of bioavailable copper (quantified using recombinant Cu-sensor bacteria) the 4-h EC50 values of nano- and bulk formulations were comparable (2.7 and 1.9 mg bioavailable Cu/l, respectively), and statistically different from the EC50 value of Cu²⁺ (1.1 mg/l). Differently from CuO particles, bulk and nanosized ZnO as well as Zn²⁺ were of similar toxicity (4-h EC50 values 3.7 and 3.9 mg bioavailable Zn/l, respectively, and 4.9 mg Zn²⁺/l). Thus, the toxic effect of both, CuO and ZnO (nano)particles to protozoa was caused by their solubilised fraction. The toxic effects of the copper compounds were not dependent on exposure time (4 and 24 h), whereas the toxicity of zinc compounds was about 1.5 times lower after 24 h of exposure than after 4 h, probably due to adaptation. In summary, we recommend T. thermophila as a simple eukaryotic particle-ingesting model organism for the toxicity screening of NPs. For the high throughput testing we suggest to use the 4-h assay on microplates using ATP and/or propidium iodide for the evaluation of cell viability.

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B. Biega and K. Nowak
The Effect of Cyanobacterial Blooms on the Water Toxicity in Turawa Dam Reservoir
Report from the Scientific Circle of students of Environmental Protection (SCSEP), University of Wroclaw, Poland 2010
Toxkits : Rapidtoxkit
Test Species : Thamnocephalus platyurus
Topics : biotoxins, cyanobacterial blooms; cyanotoxins

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Abstract : One of the most dangerous effects of eutrophication, which appear both in lakes and dam reservoirs, are cyanobacterial blooms. Cyanobacteria can cause many unfavorable changes in physicochemical characteristics of water. Repeated occurrence of cyanobacteria in water contributes to deterioration of light conditions, depletion of oxygen content and changes of pH and Eh. Cyanobacterial blooms also cause an increase in the amount of suspended organic matter, and in consequence an increase of the biological oxygen demand and changes in the color and the odor of water. Moreover, the appearance of cyanobacterial blooms in water has many economic and social consequences such as the decrease in the usefulness of water and the increase in the cost of water treatment. Furthermore, in European waters 30 - 90 % of cyanobacterial blooms are toxic. The most dangerous substances produced by cyanobacteria are toxins with an oligopeptide structure (hepatotoxins) and an alkaloid structure (neurotoxins). These toxins are characterised by high toxicity, carcinogenicity, and high chemical stability and they therefore constitute a dangerous threat to human health.
Biotests are useful tools for toxicity assessment. A quick and simple biotest is the RAPIDTOXKIT microbiotest for rapid detection of water toxicity with the aid of larvae of the freshwater crustacean Thamnocephalus platyurus. This assay is also particularly sensitive to toxic cyanobacteria.
The present study aimed at determining the water toxicity in Turawa Dam Reservoir, situated near Opole (SW Poland) with the RAPIDTOXKIT microbiotest. In addition physical and chemical parameters, such as: temperature, pH, conductivity, phosphate and ammonia concentration were also measured.
Samples for the experiments were collected during the most intense cyanobacterial blooming of water (August 2009) from 5 stations, situated in different parts of Turawa Dam Reservoir. The water samples were filtered through GF/C filters and transported to the laboratory in 1.1 dm3 plastic bottles. All the analyses were performed in the Laboratory of Isotope Geology and Geoecology at the University of Wroclaw.
In order to assess the toxicity in the water samples, the total number of larvae in the sample and the number of larvae with a clearly colored digestive tract were counted (Table 1). Subsequently the average percentage inhibition of red particles uptake was calculated (Table 2). Test organisms exposed to contaminated samples showed a decrease or total absence of ingestion of red microspheres.CONCLUSIONS
The RAPIDTOXKIT microbiotest for rapid detection of water contamination revealed that the water in Turawa Dam Reservoir was toxic.
The water toxicity was associated with the presence of cyanobacterial blooms.

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G. Arru, A.M. Congiu, E. Burdino and G. Ugazio
Toxicity of atrazine and its metabolite deethylatrazine in Thamnocephalus platyurus and Dugesia gonocephala
Giornale Italiano di Medicina del Lavore ed Ergonomia (in Italian) 1997 19, 1 17-19
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals-pesticides

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Abstract : The toxicity of Atrazine and that of its Desethylatrazine metabolite has been defined employing two organisms already used in our laboratory : the flatworm Dugesia gonocephala, from a scissiparous strain coming from the island of Tavolara (Sardinia) and the anostracod crustacean Thamnocephalus platyurus, produced under the form of dormant cysts by the company MicroBioTests Inc. (Mariakerke - Gent, Belgium).
The lethal concentrations at 50% of Atrazine and of Desethylatrazine have been determined, as well as the relationship dose-effect in the comparisons of the rectilinear motility. The results highlight that in the comparisons with T. platyurus Atrazine has a toxicity of around three times that of its Metabolite. This is not the case with the Planarians which are equally sensitive to both compounds. The use of these two experimental models for the evaluation of the contamination of water bodies is shown to be particularly useful given their great sensitivity to pollutants.

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P.C. Naha, L. Davoren, A.Casey and H.J. Byrne
An ecotoxicological study of poly(amidoamine) dendrimers-toward quantitative structure activity relationships
Environmental Science and Technology 2009 43, 17 6864-6869
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - organics

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Abstract : Polyamidoamine (PAMAM) dendrimers of generation G-4, G-5 and G-6 were evaluated for their aquatic toxicity using the test models Vibrio fischeri, Daphnia magna, Thamnocephalus platyurus, and two fish cell lines.
Physico-chemical characterization of the particles in each of the different test media was performed. A significant eco- and cytotoxicological response was recorded at concentrations from 0.129 µM (7.4 mg L^-1) to 16.30 µM (231.5 mg L^-1). Daphnia magna was found to be the most sensitive test model, the RTG-2 fish cell line the least. The toxicological response correlated well with the dendrimer generation and therefore with the particle surface area, increasing surface area leading to increased toxic response. The response also correlated well with changes to the 4 potential in the different media, rather than the actual 4 potential, indicating a potential contribution of changes to the effective composition of the medium. For the cell lines, although spectroscopic studies indicated an interaction with the serum supplement, trends for this interaction do not correlate to those observed for the toxic response. The clear correlations of the observed toxic response with the measured physicochemical properties point toward underlying structure activity relationships.

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M. Bahadir and M. E. Aydin
Evaluation of Suitable Toxicity Tests for Industrial Wastewaters, 2005-2008
Final Report to the International Bureau of German Ministry for Education and Research
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Phytotoxkit; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus; Lepidium sativum
Topics : waste waters; leachates; solid wastes; waste waters, soils; plant tests

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Abstract : The fish test with Lebistes reticulatus is the only officially accepted toxicity test for the evaluation of the toxic hazard of industrial wastewaters in Turkey. Fish toxicity tests have several disadvantages since they are laborious, require a lot of test organisms, holding tanks and aeration equipment, are time consuming and require feeding the fishes. Moreover, fish assays depend on only one type of test organisms. The most important disadvantage of the fish toxicity test is its cost. For these reasons, despite the regulatory obligations the fish toxicity test is often avoided. In this project, in order to overcome the disadvantages of fish toxicity tests, rapid, low cost, easy and correct and repeatable alternative toxicity tests were evaluated, which could be applied in Turkey instead of fish toxicity tests. Besides industrial wastewaters, the applicability of these toxicity tests was also determined on leachate and solid waste eluates. The alternative tests were carried out in parallel with fish toxicity tests and the results were compared.
In this study, the aquatic plant Lemna minor, the micro-algae Selesnastrum capricornutum and the terrestrial plant Lepidium sativum were used for the producers trophic level. The protozoan Tetrahymena thermophila, the fish Lebistes reticulatus, and the crustaceans Daphnia magna and Thamnocephalus platyurus were applied for the consumers trophic level. For the decomposers trophic level bacteria Vibrio fischeri were used. The Daphtoxkit, Thamnotoxkit, Vibrio fischeri, Lemna minor and Lepidium sativum toxicity tests were applied successfully for the evaluation of the toxicity of wastewaters, lechates and eluates. It was found that especially the Daphtoxkit and Thamnotoxkit toxicity tests could be used as alternatives to fish toxicity test prescribed in regulations. Comparing these two alternative toxicity tests with the fish toxicity test, the former are quite advantageous in terms of sample volume, labor, test time and cost. Moreover, these microbiotests were applied quite successfully to different types of domestic and industrial wastewaters. As a result of this work it is suggested that the Daphtoxkit and the Thamnotoxkit microbiotests should be included in the water pollution control regulation in Turkey as toxicity prescreening tests.

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M.E. Aydin, S. Özcan and S. Yildiz
Toxicity evaluations of solid waster employing various methods
Turkish Toxicology Journal (in Turkish) 2008 6, 1-2 4-13
Toxkits : Phytotoxkit; Protoxkit F^TM
Test Species : Tetrahymena thermophila; Lepidium sativum
Topics : solid wastes; solid waste leachates; plant tests; soils

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Abstract : In this work, toxicities of solid waste samples taken from Konya sanitary landfill were evaluated using Lemna minor, Lepidium sativum, Tetrahymena thermophila (Protoxkit) and Vibrio fischeri as toxicity indicator organisms. Reference tests were carried out in parallel to check the test performance and the sensitivity of the test organisms, using different dilutions of K2Cr2O7 solutions for all test methods. Inhibitions as % and EC50 values were calculated comparing the test results with the results of the control tests. For the eluates prepared from solid waste samples the highest sensitivities of the toxicity tests were observed with Lemna minor and Tetrahymena thermophila. Solid waste samples taken from Konya landfill were not found toxic in the tests with Lepidium sativum and Vibrio fischeri toxicity tests whereas they were found toxic with the Lemna minor and the Protoxkit toxicity tests.

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M.E. Aydin, S. Yildiz, S. Özcan and G. Kara
Application of Different Biotests in Determination of Acute Toxicity of Wastewaters
From : VII. National Enviromental Engineering Congress, 693-700, 24-27 October 2007, Izmir, Turkey 2007
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : waste waters

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Abstract : Wastewater samples were collected from various sampling points of Konya sewerage system and seven different toxicity tests were carried out on wastewater samples. Wastewater samples were also analyzed for pH, suspended solids (SS), electrical conductivity (EC), temperature, chemical oxygen demand (COD), total organic (TOC) and inorganic carbon (TIC). Correlations between the toxicity tests and physical and chemical parameters were determined. Correlation between the Daphtoxkit F magna, TOC, TIC, TC and pH. For the Thamnotoxkit F correlations were also noted with TOC, TC, pH, conductivity and Lemna minor. The results of the Fish toxicity test did not correlate with any parameter.
Results of the Thamnotoxkit and the Fish toxicity tests toxicity were determined as LC50 values; for the other tests toxicity was determined as EC50 values. For checking the correct performance of the biotests, reference tests were carried out with K2Cr2O7 solution for each type of test concurrently with the assays on the wastewater samples. Samples toxicities were expressed as toxic unit (TU). TU’s ranged between 2,12 and 6,01 for the Fish toxicity test, between between 2,12 and 10,08 for the Thamnotoxkit, between 1,34 and 6,11 for the Daphtoxkit and between 1,62 and 2,67 for Lemna minor. The results revealed that each type of tests shows different sensitivities for different wastewater characteristics. The Daphtoxkit F magna was the most sensitive test for wastewater samples of industrial and domestic nature while the Thamnotoxkit test showed more sensitive results for industrial wastewater samples.

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M.E. Aydin, S. Özcan, S. Yildiz and G. Kara
Toxicity evaluation of by different biotest methods
From : Environmental Problems Symposium, 695-704, Turkey, Book of Abstracts 2008
Toxkits : Phytotoxkit; Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus; Lepidium sativum
Topics : soil leachates; plant tests; soils

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Abstract : Toxicity of Konya landfill leachate was determined using different biotest methods. For the evaluation of phytotoxicity Lemna minor and Lepidium sativum toxicity tests were used. Toxic effect of landfill leachates on aquatic life was determined using Vibrio fisheri and Thamnotoxkit toxicity tests. With regard to the sensitivities of the toxicity test employed, the test organisms could be ranked as follows for the landfill leachates : Vibrio fischeri, Thamnocephalus platyurus (Thamnotoxkit), Lepidium sativum and Lemna minor. 24h LC50 values ranged between 0.36% and 1.16% for Thamnocephalus platyurus, and 30 min EC50’s for Vibrio fischeri ranged between 0.43% and 24.34%.
After 72 hours incubation of Lepidium sativum and 7 days incubation of Lemna minor EC50 values were obtained between 0.25% and 4.6% (root length) and 2.28% and 7.95%, respectively.

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R. Baudo, G. Persoone and C.Janssen
Ring-testing and Quality Control Testing in Ecotoxicology : State of the art with Toxkit microbiotests
From : 13^th International Symposium on Toxicity Assessment - Toyama, Japan - 2007 - Book of Abstracts
Toxkits :
Test Species :
Topics : review

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Abstract : A variety of toxicity tests for assessing the toxicity of chemicals and wastes to aquatic and terrestrial environments have been developed during the last 50 years.A limited number of these tests - mostly after extensive inter-laboratory testing - have been endorsed by national and international organisations for application in regulatory frameworks. Ring-tests are of utmost importance for the credibility of any method since their outcome will determine not only whether or not a test procedure is described with sufficient accuracy, but also whether the method is applicable from the point of view of practicality, reproducibility and precision.
Although intra-laboratory (in house) quality control (QC) testing with one or more reference toxicants is recommended; in practice, this is rarely implemented on a routine basis.
During the past decades a number of microbiotests have been developed which are independent of the expensive and time-consuming culturing of live test organisms.
These so-called Toxkit microbiotests - available with various test species - are presently used worldwide for toxicity monitoring of contaminated environments as well as for ecotoxicological research.
This presentation will highlight the results of 4 large-scale ring-tests which have been performed over the last few years with Toxkit microbiotests, with particular emphasis on the precision of the microbiotests in comparison to conventional standard assays.

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V. Tsiridis and G. Persoone
Toxkit microbiotests : new low cost tools for hazard detection/monitoring in environmental toxicology
From : Proceedings of the International Conference Protection and Restoration of the Environment VI - Skiathos, Greece 2002
Toxkits :
Test Species :
Topics : review

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Abstract : In recent years, attention has gradually focussed on the use of toxicity tests as a useful tool to estimate chemical contamination and its potential impact on terrestrial and aquatic environments. New microbiotests, called Toxkits, have been developed in the Laboratory for Environmental Toxicology and Aquatic Ecology at the Ghent University in Belgium. The new approach is based on the use of “dormant, inactive of immobilized” stages of selected aquatic biota as the biomaterial from which live test organisms can easily be obtained on demand to perform the toxicity tests. Several highly standardized “culture/maintenance free” toxicity tests are now commercially available with selected species of micro-algae, protozoans, rotifers and crustaceans. A brief review of Toxkit microbiotests and their applications is given in this study.

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G. Persoone
Field testing in ecotoxicology
From : 13^th International Symposium on Toxicity Assessment - Toyama, Japan - 2007 - Book of Abstracts
Toxkits : Toxi-Screening Kit
Test Species :
Topics : rapid tests; field tests; behavioural tests

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Abstract : Over the last two decades toxicity testing with micro-algae and invertebrates has been simplified very substantially by the development of Toxkit microbiotests which are independent of the costly and time consuming year-round culturing/maintenance of live stocks of test biota.
Yet, toxicity tests are to date still performed virtually exclusively in the laboratory, and the assays mostly take one to several days to obtain a first answer of the toxic hazard of the analysed samples.
The same situation also applies to the examination of the degree of bacterial contamination of suspected water samples, which is also only performed in the laboratory in a time span of one to several days.
Despite the need for rapid and practical methods for in situ determination of the toxicity and/or bacterial contamination of suspected waters, very little attention has so far been paid to this important matter. Contrary to chemical analysis, for which a variety of field kits is now available for determination of the concentrations of (mostly individual) chemical pollutants, there are to date only a few field tests for the rapid analysis of the toxicity and/or the bacterial contamination of freshwaters and marine waters.
Recently two new very practical and low cost microbiotests have been developed for ultra-rapid in situ toxicity detection and analysis of bacterial contamination. Both microbiotests are based on the measurement of luminescence in a small portable luminometer These field microbiotests which have been tailored in user-friendly kits can be applied anytime and anywhere, in less than 30 minutes of time, and without any need for additional equipment nor materials. These new assays are well suited for various applications such as e.g. toxicity/contamination monitoring of surface and groundwaters, routine screening of drinking water, water contamination emergencies and in situ follow up of the efficiency of various kinds of water treatments.
The presentation will outline the principles of the new field microbiotests and give details on the practical aspects of their application.

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V. Tsiridis, C. Emmanouil, A. Kungolos and G.P. Sakellaropoulos
Bioavailability of heavy metals contained in coal fly ash
From : 13th International Symposium on Toxicity Assessment - Toyama, Japan - 2007 - Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - metals; fly ashes; leachates

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Abstract : The presence of heavy metals in coal fly ash is an important issue related to the pollution of the environment. It is generally accepted that the solubility, bioavailability and toxicity of heavy metals are dependent on various physicochemical parameters such as pH, hardness, interactive effects, and presence of natural organic matter. The availability and toxicity of heavy metals leached from fly ash samples were examined using the leaching test NEN 7371 (availability test) and bioassays. The effects of humic acids on the leaching capacity of heavy metals as well as on the toxicity of fly as leachates were also evaluated by the addition of 25 and 50 mg/L humic acids in the leaching medium and in the fly ash leachates. The test organisms that were used for the toxicity evaluation of fly ash leachates included the photobacterium Vibrio fischeri (Microtox test) the algae Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) and the crustacean Daphnia magna. Physicochemical analyses showed that heavy metals present in fly ash leachates included Cr, Cu, Ni and Zn. The toxic effect of leachates was high on D.magna and P.subcapitata indicating an immobilization and growth inhibition respectively, up to 100%, while the bioluminescence inhibition of V.fischeri was not higher than 70%. The leaching tests with the presence of humic acids in the leaching medium dit not affect the leaching capacity of heavy metals and the toxicity of the leachates. However, the addition of humic acids in the leachates caused a decrease in the concentration of heavy metals in the leachates as wel as of their toxic effect on the test organisms.

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H. Hamdi, L. Manusadzianas, I. Aoyama and N. Jedidi
Ecotoxicological and analytical assessment of effects of phytoremediation on physicochemically-degraded soils contained aged PAHs

Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - organic compounds; soils

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Abstract : The biodegradation of concomitantly spiked anthracene (ANT), pyrene (PYR) and benzo[a]pyrene (B[a]P) in soil (15, 75, 150, 300 mg Σ 3 PAHs kg-1 soil) was studied for two years at pot-scale under greenhouse conditions. The initial bioremediation strategy consisted of soil landfarming based on weekly moderate waterings and soil turnover in the presence or absence of sewage sludge compost (SSC). Changes in soil properties and ecotoxicity throughout the experimental period were evaluated using physico-chemical analyses, soil enzymatic activities and solid-phase bioassays (lettuce and ostracod biotests). After 15 months, recalcitrant B[a]P was still persisting, most apparently in SSC-amended soils but no noticeable ecotoxic effects of the aged PAHs could be detected as compared to early stages of bioremediation. However, long-term SSC mineralization caused both soil salinization and acidification at the end of the bioremediation process simulating landfarming. Consequently, degraded SSC-amended soils regained toxicity that surpassed PAH deleterious effects. Phytoremediation using Lucerne (Medicago sativa L.) was then adopted in an attempt to achieve complete B[a]P removal and repair degraded SSC-amended soils. After six months, the presence of strong root system along with adequate waterings had more amerliorating effects on soil physico-chemical and biological properties than on weathered PAH dissipation. SSC-amended soils lost their reinduced ecotoxicity as a result of neutral pH reestablishment and significant salinity decrease by the end of the phytoremediation process. Therefore, lettuce germination, ostracod viability and soil microbial activities were all improved. However, B[a]P was apparently bound irreversibly to the solid-phase matrix.

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C. Blaise, F. Gagné, J.F. Férard and P. Eullaffroy
Ecotoxicity of selected nano-materials to aquatic organisms
From : 13^th International Symposium on Toxicity Assessment - Toyama, Japan - 2007 - Book of Abstracts
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - nanoparticles

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Abstract : Nano-materials are chemical structures with novel properties owing to their small sizes (1-100 nm). Because of the expected surge in nanotechnology products in economical areas such as the bioi-medical (e.g. metal imaging, drug delivery), electronics (e.g. semi-conductors) and environmental (e.g. solar/hydrogen fuel cells) fields, release of nano-articles into aquatic systems is likely to occur, particularly via municipal treatment plants of cities supporting nano-industries. Present knowledge concerning the ecotoxic effects of nano-materials is very limited and merits to be documented more fully. For this purpose, we recently appraised the toxicity of 11 nano-materials whose pending commercial usages leave little doubt : copper zinc iron oxide, nickel zinc iron oxide, yttrium iron oxide, titanium (IV) oxide, fullerene-C60, strontium ferrite, indium tin oxide, samarium (III) oxide, erbium (III) oxide, holmium (III) oxide, carbon nanotube (single-walled). Our bioassay results show some of these to display toxic effects across several taxonomic groups including decomposers (bacteria), primary producers (micro-algae), as well as primary and secondary consumers (micro-invertebrates and fish). This preliminary investigation suggests that chemicals emerging from nanotechnology may pose a risk to aquatic life and further studies on their adverse effects are to be encouraged.

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G. Kusza, I. Czerniawska-Kusza and T. Ciesielczuk
Vulnerability of soils to ecotoxic effects of oil derivatives
From : 13^th International Symposium on Toxicity Assessment - Toyama, Japan - 2007 - Book of Abstracts
Toxkits : Phytotoxkit; Protoxkit F^TM
Test Species : Tetrahymena thermophila; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; chemicals - hydrocarbons

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Abstract : Oil entering the terrestrial environment is subjected to a variety of natural processes, such as dispersion, dissolution, evaporation and burial. Under normal conditions, oil will be absorbed in the organic rich surface layer of oil, impeding downward migration and influencing the biotic components of soil. This study investigated the effect of oil derivatives on soil properties in order to evaluate soil vulnerability to the the ecotoxic influence of those contaminants. Soil samples (0-20 cm layer) of different properties were collected from uncontaminated areas in Opole region (Poland) and then artificially contaminated with fuel oil. Ecotoxicity testing was performed with two microbiotests : Phytotoxkit (with Sorghum saccharatum, Sinapis alba, Lepidium sativum) and Protoxkit F (with Tetrahymena thermophila). The toxicity levels were determined by measuring respectively seed germination/root elongation of plant species and growth inhibition of ciliates. Germination response varied greatly with plant species, type of the soil and contaminated soil treatment. The experimental procedures, including agrotechnical measures, and results of chemical and toxicological analysis of soils are discussed and interpreted in an ecological perspective.

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P.J. den Besten, J.F. Postma, J.W.M. Wegener, H. Keidel, A. Klink, J. Mol and C. van de Guchte
Biological and Chemical Monitoring after Pilot Remediations in the Delta of the Rivers Rhine and Meuse
Aquatic Ecosystem Health and Management 2000 3 317-334
Toxkits : Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : toxicity monitoring

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Abstract : Two pilot projects were carried out to investigate the effects of sediment remediation. One project was situated in a groyne section of the Nieuwe Merwede, a watercourse in the Rhine delta. The second was situated in a creek named Spijkerboor, which receives water from the river Meuse. For both sites it was concluded earlier that sediment pollution posed a high risk to the ecosystem. The remediations consisted of partial excavation of the contaminated sediments, followed by application of a clean layer of sandy material on top of the remaining contaminated sediment. Before and at various times after the remediation, the following investigations were carried out: physical-chemical analyses of sediment, benthic community structure observations, bioaccumulation measurements and sediment bioassays. After the remediation, a new silty sediment top layer was formed with lower contaminant levels exhibiting a lower toxicity. In the remediated site in the Nieuwe Merwede, chironomids, oligochaetes and nematodes reappeared within 3–7 months at normal densities, while in the Spijkerboor recolonization by chironomids and nematodes proceeded more slowly. After 2 years, in both the remediated sites chironomids and nematodes were present in densities much higher than before the remediation. Bivalves showed a low recolonization rate in both sites. In non-remediated, polluted reference sites also a lower sediment toxicity and a nearly comparable recovery of the benthic community was observed, probably the result of natural sedimentation of material with lower contaminant levels. Because of the natural improvement of sediment quality, the net effects of remediation were negligible in the Spijkboor. For the Nieuwe Merwede, after 2 years still lower contaminant levels were observed in the remediated site compared to the non-remediated site, resulting in lower bioaccumulation in oligochaetes. However, the contaminant levels in sediment and biota still do not meet all environmental quality objectives. Based on the outcame of this study it is recommended that the priority for remedial action is made dependent on the rate of natural covering with sediments from the rivers Rhine and Meuse.

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F. Wang, A.O. Leung, S.C. Wu, M.S. Yang and M.H. Wong
Chemical and ecotoxicological analyses of sediments and elutriates of contaminated rivers due to e-waste recycling activities using a diverse battery of bioassays
Environmental Pollution 2009 157, 7 2082-2090
Toxkits : Algaltoxkit F^TM; Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : sediments, sediment and soil contact tests, leachates

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Abstract : A multi-trophic, multi-exposure phase assessment approach was applied to characterize the toxicity of sediments collected from two rivers in Guiyu, China, an e-waste recycling centre. Elutriate toxicity tests (bacterium Vibrio fischeri and microalga Selenastrum capricornutum) and whole sediment toxicity test (crustacean Heterocypris incongruens) showed that most sediments exhibited acute toxicity, due to elevated heavy metals and PAHs levels, and low pH caused by uncontrolled acid discharge. The survival rates of crustaceans were negatively (p < 0.05) correlated with total PAHs in sediments (411-1755 mg kg(-1)); EC50s of V. fischeri on the elutriates were significantly correlated with elutriate pH (p < 0.01). Significant (p < 0.05) correlations between the induction of hepatic metallothionein in tilapia (Oreochromis mossambicus) and metal concentrations (Cu, Zn, Pb) in sediments were also observed, when fish were fed with diets containing sediment. The results showed that uncontrolled e-waste recycling activities may bring adverse effects to local aquatic ecosystem.

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B. Kudlak, L. Wolska and J. Namiesnik
Determination of EC50 toxicity data of selected heavy metals toward Heterocypris incongruens and their comparison to direct-contact and microbiotests
Environmental Monitoring and Assessment 2010 DOI10.1007/s10661-070-1474-8
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - metals

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Abstract : The sensitivity of Heterocypris incongruens to selected heavy metal ions is discussed. Although the Ostracodtoxkit has been present on the market for a few years, data on its selectivity and sensitivity to toxicants is scarce; such data is indispensable when interpreting the results of sediment toxicity. The LC50 and EC50 results with the Ostracodtoxkit are compared with those obtained with other commercially available direct-contact tests (utilizing Hyalella azteca and Chironomus riparius) and microbiotests for assessing the toxicity of aqueous samples (Vibrio fischeri, Daphnia magna and Selenastrum capricornatum). The sensitivity of H. incongruens to metal ions (Cd²⁺>>Hg²⁺>Cu²⁺>Cr⁶⁺>Ni²⁺≈Mn⁷⁺>Zn²⁺>Pb²⁺>Li¹⁺> Fe³⁺) was found to be similar to that of H. azteca and of C. riparius. The Ostracodtoxkit has shown itself to be an efficient and reliable element of test batteries for toxicity determination.

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E. Mendonça, A. Picado, S.M. Paixão, L. Silva, M.A. Cunha, S. Leitão, I. Moura, C. Cortez and F. Brito
Ecotoxicity tests in the environmental analysis of wastewater treatment plants : Case study in Portugal
Journal of Hazardous Materials 2009 163, 2-3 665-670
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : waste waters

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Abstract : A global evaluation of wastewaters should include ecotoxicological tests to complement the chemical characterization, with advantages especially in the case of complex wastewaters. A European project developed in Trancão River Basin (Portugal), integrated the ecotoxicological and physicochemical studies of wastewater samples from two municipal sewer networks and respective wastewater treatment plants. Wastewater samples were analysed for physicochemical parameters, ecotoxicological acute and chronic tests performed and the potential for endocrine disruption evaluated. Organic load parameters and total suspended solids showed significant correlations with Microtox and Thamnotoxkit test results. Data analysis showed that treated treatment plant effluent samples are associated with less organic contamination and less toxicity in Thamnotoxkit test. Chronic toxicity test and endocrine disruption assay of treatment plant effluent samples indicated that, in a long term, potential population effects could arise in the receiving waters. A test battery to monitor this type of wastewaters is proposed, including tests with a bacterium, an alga and a crustacean. In a screening phase the most sensitive test, Microtox, can be used. The use of an ecotoxicological approach can have added value to hazard and risk assessment of discharges to the receiving waters and can contribute to the environmental management of the treatment plant.

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L. Manusadzianas, K. Sadauskas, and R. Vitkus
Comparative study of indices used in toxicity evaluation of effluents
Desalination 2010 250, 1 383-389
Toxkits : Algaltoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : effluents; toxicity index; toxicological indes

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Abstract : Several toxicological indices currently proposed (inter)nationally to aid decision-making tasks in effluent control were compared. To evaluate effluent ranking differences between the indices an inversion distance as a similarity measure was proposed. The major factor influencing effluent ranking were found to be a composition of test-battery. Differences in mathematical operator of the indices such as, e.g. arithmetic mean (used in averaging indices) or maximum one (used in an approach of the most sensitive test in a battery), affect the ranking similarity noticeably lesser than the modification of the percentage effect level of the measurement endpoints. Effluent and/or river flow parameters incorporated into the indices substantially modify ranking results of the effluent set. For the analysis, the toxicological information on 23 effluents from municipal wastewater treatment plants operating in cities and small towns of Lithuania as well as test-battery data on industrial effluents published elsewhere were used. It seems that the general statements achieved in the current study can be applied to any set of effluents.

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M. Rubino
Xenobiotics in the environment : abiotic transformation and toxicity
Universita degli studi di Napoli Federico II - Open Archive 2006
Toxkits : Algaltoxkit F^TM; Ceriodaphtoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TMThamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Ceriodaphnia dubia; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - pesticides; chemicals - drugs

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Abstract : Given the potential human and wildlife health risks associated with toxic chemicals (pesticides, pharmaceuticals, etc.), it is important to have considerable information on their persistence in surface waters and/or in the soil by considering their reaction mechanisms under typical environmental conditions. Xenobiotics can be subjected to biotic (biotransformation by aquatic organisms such as algae, bacteria) and abiotic (hydrolysis, oxidation, photodegradation) processes in the environment giving derivatives that can be more persistent and more toxic than the parent compounds. The aims of this thesis have been to study the photolitic and hydrolitic processes of certain xenobiotics and evaluate their toxicity as well as that of their degradation products. Investigation has been devoted on some pesticides, in particular on carboxin and carbammates, and on different groups of pharmaceuticals, among these, steroidal anti-inflammatory drugs (prednisolone and dexamethasone) and non-steroidal antinflammatory drugs (naproxen sodium salt), diuretics (furosemide and hydrochlorothiazide), fibrate drugs (bezafibrate, fenofibrate and gemfibrozil), and proton pump inhibitors (lansoprazole and omeprazole). These chemicals have been selected on the basis of their sale and/or their presence into the aquatic environment. Their abiotic degradation has been studied as close to natural conditions as possible. They have been dissolved (for analytical purposes) or dispersed (for preparative purposes) in aqueous media, using distilled water, distilled water with added nitrates or humic acids, which are considered natural photosensitizers, in sewage treatment plant water, and irradiated by a solar simulator or with solar light. In certain cases, photolysis and hydrolysis have also been examined at the different pHs that are possible in polluted aquatic environments. Degradation products have been isolated by chromatographic techniques (silica gel chomatography, TLC, HPLC) and characterized by spectrometric means (one and two dimensional NMR, IR, EI-MS, UV). The potential environmental impact of the selected xenobiotics and their derivatives has been evaluated, in collaboration with the Seconda Università degli Studi di Napoli, by performing acute and chronic toxicity tests on different organisms of the aquatic chain. For the acute toxicity, the bacterium Vibrio fisheri, the rotifer Brachionus calyciflorus, the anostracan crustacean Thamnocephalus platyurus and the cladoceran crustaceans Daphnia magna and Ceriodaphnia dubia have been used. The chronic toxicity has been evaluated on producers such as algae (Selenastrum capricornutum), and primary consumers (Brachionus calyciflorus, Ceriodaphnia dubia).

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A. Rouvalis and J. Iliopoulou-Georgudaki
Comparative Ecological Quality Assessment of Olive Oil Mill Effluents from three-phase and two-phase Systems, Treated for Hydrogen Production
Bulletin of Environmental Contamination and Toxicology 2010 85, 4 432-436
Toxkits : Daphtoxkit F^TM pulex; Thamnotoxkit F^TM
Test Species : Daphnia pulex; Thamnocephalus platyurus
Topics : effluents

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Abstract : By-products of a two-phase and a three-phase olive oil mill process treated in an anaerobic fermentation system for hydrogen production, were evaluated by three bioassays: the zebrafish Danio rerio embryo test and two microbiotests, Thamnotoxkit F and Daphtoxkit F™ pulex. Samples from both processes were classified as very toxic with LC50 values ranging from 1.52% (T. platyurus 24 h test) to 4.48% (/i>D. pulex 48 h-LC50). Toxicity values were differently correlated to physicochemical parameters showing different degree of influence. The treated effluents of both process systems remained very toxic showing the necessity for further treatment, aiming to environmentally safe discharges.

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H. Watanabe, F. Nakajima, I. Kasuga And H. Furumai
Toxicity Characterization of Urban River Sediments using Bioassay with Ostracod
Journal of Japan Society on Water Environment 2008 31, 11 671-676
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments

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Abstract : To protect the aquatic ecosystem, it is important to evaluate the toxicity of contaminated sediments. Moreover, the identification of chemical compounds responsible for the toxicity is needed for effective remediation. In this study, we aimed to identify the principal toxicants of urban river sediments, applying Toxicity Identification Evaluation (TIE) methods to the toxicity test with an ostracod (a benthic crustacean). The toxicity test was conducted using sediments amended with three adsorbents: Zeolite, Chelex-100 and Ambersorb-572. Four of the nine urban river sediment samples showed lethal toxicity to the ostracod, but none of the three adsorbents reduced the mortality of the ostracod. Growth inhibition was observed in all the samples; however, the addition of the carbonaceous adsorbent Ambersorb-572 suppressed the growth inhibition in the 6 samples. This indicates that hydrophobic substances in the sediments inhibit the growth of the ostracod.

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A.M. Coccia, P.M.B. Gucci, I. Lacchetti, E. Beccaloni, R. Paradiso, M. Beccaloni and L. Musmeci
Hydrocarbon Contaminated Soil Treated by Bioremediation Technology : Microbiological and Toxicological Preliminary Findings
Environmental Biotechnology 2009 5, 2 61-72
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - hydrocarbons; soils; bioremediation; plant tests

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Abstract : Soil contamination is a significant factor in the general degradation of the environment. Remedial intervention on contaminated soils may be carried out by means of specific and different technologies, including chemical, physical and biological methodologies. Of these, the latter are the least invasive and bioremediation intervention can be carried out both in situ and ex situ. The object of this study was a clay soil highly contaminated by heavy hydrocarbons and polycyclic aromatic hydrocarbons (PAHs). The soil was subjected to remediation treatment using a bioremediation technique, applied by inoculating a nutritive and enzymatic-bacterial mixture. A qualitative-quantitative microbial characterization of the soil matrix was undertaken, both before, and thirty days after, the treatment. The percentage abatement in hydrocarbon concentration and the overall microbial concentration present in the soil were measured, and the eco-toxicological impact was estimated. The eco-toxicological aspect was assessed by applying the ostracod test and the phytotoxicity test. The microbial component was estimated by the Direct Count method on plates : this method highlighted the vital countable cells of telluric and degrader mesophilic bacteria, of the Pseudomonadaceae, of the actinomycetes and total mycetes (moulds and yeasts). This study was focused on evaluating biological remedial technology combining chemical analysis with microbiological assessment and bioassays, because chemical analysis alone cannot provide a full picture of the bioremediation process and its effectiveness. The data obtained indicated that the bioremediation technique applied gave optimal results in terms of percentage abatement of the hydrocarbon concentration, microbial enrichment of the soil matrix and reduction of toxicity found in the soil before the clean-up treatment.

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J. David, L. Vojtova, K. Bednarik, J. Kucerik, M. Vavrova, and J. Jancar
Development of Novel Environmental Friendly Polyurethane Foams
Environmental Chemistry Letters 2010 8, 4 381-385
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - general

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Abstract : To prepare materials with improved recycling capability, new flexible biodegradable polyurethane foams, in which non-degradable polyether polyol was partly substituted by the bio-polyols based on cellulose or starch derivatives were synthesized. The incorporation of bio-polyols into the foams’ structures as well as their influence on the foam thermal stability was assessed by Fourier-transformed infrared spectroscopy and thermogravimetric analysis analyses. The ecotoxicological aspects of the newly synthesized foams were investigated by extracting the samples using freshwater as a solvent followed by applying the microbiotest screening toxkit under trade name “Thamnotoxkit F™” with larvae of freshwater shrimps Thamnocephalus platyurus.

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M. Petala, L. Kokokiris, P. Samaras, A. Papadopoulos and A. Zouboulis
Toxicological and Ecotoxic Impact of Secondary and Tertiary Treated Sewage Effluents
Water Research 2009 43, 20 5063-5074
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : effluents

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Abstract : Secondary sewage effluents are discharged in significant quantities in aquatic environments delivering pollutants that were not removed during treatment; yet advanced treated effluents are not lacking of contaminants. In this study, biochemical biomarkers were measured in liver and kidney of rainbow trout (Oncorynchus mykiss) exposed to unchlorinated, chlorinated and tertiary treated secondary sewage effluents. In addition, organic matter, nitrogen and suspended solids were assayed, while a common bioassay, Daphnia magna 21d reproduction test was also applied in order to examine potential relation between the performed bioassay and the biomarkers. Processes using oxidative conditions, such as ozonation and chlorination, resulted in significantly increased breeding rate (up to 74%) of the organism. Biomarkers measurements incorporated the determination of total glutathione (GSH), glutathione S-transferases (GST), glutathione peroxidase (GPX), lipid peroxidation (LPO) and an innovative biomarker in such applications, haem peroxidase. In general, the response of biomarkers was dependent upon the treatment method and it was tissue specific. Secondary effluents inhibited liver GST and haem peroxidase, while GSH levels and LPO were significantly provoked in liver. Ozonation provoked hepatic peroxidation, in terms of haem peroxidase and LPO, and GST; while the protective (to Reactive Oxidant Species – ROS) GSH was depleted, suggesting extended ROS attack to the organism. Similar response of biomarkers (but to a lesser extend) was observed after exposure of trout to effluents submitted to both coagulation and ozonation, emphasizing the significance of removing the residual organic matter by other methods than oxidative ones. Ozonation also enhanced renal LPO and GPX; however the former employment of coagulation limited the peroxidation phenomena. Chlorination mainly affected the levels of total GSH in both tissues.

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A. Giannis, G. Makripodis, F. Simantiraki, M. Somara and E. Gidarakos
Monitoring Operational and Leachate Characteristics af an Aerobic Simulated Landfill Bioreactor
Waste Management 2008 28, 8 1346-1354
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : leachates; landfill leachates; composts

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Abstract : Long-term biodegradation of MSW in an aerobic landfill bioreactor was monitored as a function of time during 510 days of operation. Operational characteristics such as air importation, temperature and leachate recirculation were monitored. The oxygen utilization rates and biodegradation of organic matter rates showed that aerobic biodegradation was feasible and appropriate to proceed in aerobic landfill bioreactor. Leachate analyses showed that the aerobic bioreactor could remove above 90% of chemical oxygen demand (COD) and close to 100% of biochemical oxygen demand (BOD5) from leachate. Ammonium, nitrate and sulphate concentrations of leachate samples were regularly measured. Results suggest that nitrification and denitrification occurred simultaneously, and the increase in nitrate did not reach the levels predicted stoichiometrically, suggesting that other processes were occurring. Leachate recirculation reduced the concentrations of heavy metals because of the effect of the high pH of the leachate, causing heavy metals to be retained by processes such as sorption on MSW, carbonate precipitation, and hydroxide precipitation. Furthermore, the compost derived from the aerobic biodegradation of the organic matter of MSW may be considered as soil improvement in the agricultural plant production. Bio-essays indicated that the ecotoxicity of leachate from the aerobic bioreactor was not toxic at the end of the experiment. Finally, after 510 days of degradation, waste settlement reached 26% mainly due to the compost of the organic matter.

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A. Giannis, A. Nikolaou, D. Pentari and E. Gidarakos
Chelating Agent-Assisted Electrokinetic Removal of Cadmium, Lead and Copper from Contaminated Soils
Environmental Pollution 2009 157, 12 3379-3386
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; chemicals - metals; plant tests

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Abstract : An integrated experimental program was conducted to remove Cd, Pb and Cu from contaminated soil. The chelate agents nitrilotriacetic acid (NTA), diethylenetriamine pentaacetic acid (DTPA) and ethyleneglycol tetraacetic acid (EGTA) were used as washing solutions under different pH conditions and concentrations. Results showed that the extraction efficiency for Cd in decreasing order was NTA > EGTA > DTPA, while for Pb and Cu it was DTPA > NTA > EGTA. The use of higher chelate concentrations did not necessarily result in greater extraction efficiency. Electrokinetic remediation was applied by conditioning anolyte–catholyte pH to neutral values in order to avoid any potential alterations to the physicochemical soil properties. The removal efficiency for Cd was 65–95%, for Cu 15–60%, but for Pb was less than 20%. The phytotoxicity of the treated soil showed that the soil samples from the anode section were less phytotoxic than the untreated soil, but the phytotoxicity was increased in the samples from the cathode section.

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I. Czerniawska-Kusza and G. Kusza
The Potential of the Phytotoxkit Microbiotest for Hazard Evaluation of Sediments in Eutrophic Freshwater Ecosystems
Environmental Monitoring and Assessment 2011 179 113-121
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sorgum saccharatum; Sinapis alba
Topics : sediments; plant tests

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Abstract : The applicability of the Phytotoxkit microbiotest for toxicity assessment of sediments in eutrophic freshwater ecosystems was evaluated. Sediments were collected from Turawa dam reservoir (southwestern Poland) which, for years, has been subjected to a marked nutrient enrichment and heavy metal contamination. The test plant species were exposed to whole sediments, solid phases of sediments, and pore waters. Phytotoxicity was estimated on the basis of seed germination and root elongation measurements, combined into an overall germination index (GI). For pore waters, the majority of GI values were not statistically different from the controls, which was consistent with chemical data. For solid phases and whole sediments, GI values showed diversified effects ranging from growth stimulation to growth inhibition. The results obtained vary depending on the plant species and the type of sediment samples. Generally, tests with solid phases of sediments showed phytostimulation, suggesting that higher amount of nutrients adsorbed on organic matter-rich sediments might conceal the inhibitory impact of heavy metals (Cd, Cr, Cu, Mn, Ni, Pb, and Zn). However, this beneficial impact is indicative of a significant nutrient load and, with respect to aquatic ecosystems, its potential resuspention that might accelerate the reservoir eutrophication. Under appropriate conditions heavy metals exerted stronger negative impact on plants. Tests with whole sediments had a higher acidity (pH 5.85) and showed adverse effects, though plant responses vary from inhibition (Lepidium sativum) to stimulation (Sorghum saccharatum). The study demonstrated variability in toxicity of contaminated nutrient-rich sediments as well as the effectiveness and usefulness of the Phytotoxkit microbiotest as a practical and reliable tool for evaluation of the hazard of eutrophic ecosystems to higher plants.

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Y. Murakami, K. Nakayama, S.I. Kitamura, H. Iwata and S. Tanabe
Perspective of Ecotoxicological Conduction for Water Quality Monitoring in Thailand
Interdisciplinary Studies on Environmental Chemistry - Biological Responses to Chemical Pollutants 2008 31-35
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Thamnocephalus platyurus
Topics :

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Abstract : As one of the industrialized countries, Thailand uses many exotic chemicals in a wide range of fields and that has resulted in chemical discharges from agricultural activities, industrial sources, and human dwelling into the natural watercourses.
In general, both basic and advance analytical chemical instruments such as ICP, GC-MS and HPLC-MS are used for water quality analysis. However, it is difficult to distinguish the diverse and complex exotic chemicals accurately even when using those advance chemical instruments. Furthermore, it is also almost impossible to detect the impact on living organisms in the receiving environment due to their bioavailability and the interaction caused by the synergistic and antagonistic effect of different chemicals delivered into the natural watercourses which produce an impact on the exposed living organisms.
Therefore a new approach of identifying viable and ecologically relevant invertebrate toxicity testing models seems very promising to assess the biological effects and ecological risk of exotic chemicals when released into the environment as a battery of single species bioassay. This also meets the mandate of the Thai Department of Health to promote and protect human health by ensuring that people live in a healthy environment.
The Department of Health set up the ecotoxicological testing facilities for the conduction of a battery of single species bioassay in 1994 to determine the effects of the endpoints which are expressed by EC50, LC50 and IC50 of the Microtox test, the algal inhibition test, the acute toxicity tests of Brachionus calyciflorus, Artemia nauplii, Thamnocephalus platyurus and local fish as test indicators for the determination of the toxicity of water pollutants, effluents, and solid waste leachates. The advantages of the tests are that they are rapid, cost-effective, reproducible, ecological relevant, easy to conduct, and available when needed. There are still difficulties to be overcome in carrying out such tests in Thailand for the betterment of the environment and human health. The difficulties are as follows. (1) Lack of integration of policy and regulation to support and enforce the need of the tests. (2) Lack of comprehension the of the ecotoxicological impact from related organizations. (3) Lack of advocacy from both private sectors and government organizations. (4) Lack of human resources for ecotoxicology and ecotoxicological testing. (5) Lack of adequate financial support from administrators for setting up ecotoxicological bioassay. (6) Lack of environmental standards of ecotoxicological effects of the endpoint data of complex chemicals and effluents to control toxic discharges from industries.

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G. Persoone
Toxicity Analysis of Water and Wastewater with Practical and Low Cost Microbiotests
Proceedings of the International Sustainable Water and Wastewater Management Symposium, 26-28 October 2010, Konya/Turkey
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Phytotoxkit; Thamnotoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : review; waste waters; surface waters; sediments; soils; plant tests

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Abstract : The evaluation of the degree of pollution of waters and wastewaters is to date mainly performed by chemical analyses. Polluted waters and wastewaters, however, mostly contain many chemicals and it is economically not possible to make detailed chemical analyses of each individual polluting compound. Consequently it is in most cases not possible to predict the overall toxicity of water and wastewaters from the (restricted) chemical analyses. In many countries toxicity tests with selected test organisms are therefore now used in complement of chemical analyses, to determine the real toxic hazard of polluted waters and wastewaters.Toxicity tests are, however, dependent on the availability of live test organisms, and the need for the culturing of live stocks of test organisms make their application expensive and restricts their use to a small number of specialised laboratories.This bottle-neck has now been solved by the development of microbiotests which make use of dormant or immobilised stages of the test organisms which can be stored for a long period of time. Contrary to the conventional bioassays, the microbiotests are totally independent of the culturing and the year-round maintenance of live stocks of test organisms. Toxkit microbiotests with several types of test organisms are to date available for practical and low cost toxicity testing of polluted waters and wastewaters. Extensive studies and ringtests have shown that the sensitivity and the precision of the Toxkit microbiotests is as good as that of bioassays performed with organisms from laboratory cultures. Since the commercial availability of the Toxkit microbiotests, more than 150.000 bioassays have already been performed with these culture independent bioassays, in more than 40 countries. Because of their independence of culturing of live stocks of test organisms and their cost-effectiveness , Toxkit microbiotests are to date used worldwide for a variety of applications in research as well as for toxicity biomonitoring. The battery of Toxkit microbiotests has also been enlarged recently with low cost field tests for rapid biological, microbiological and toxicological screening of contaminated waters and wastewaters.

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M. Kolb, S. Ozcan, S. Yildiz, G. Kara, M.E. Aydin And M. Bahadir
Biotesting of untreated Wastewaters and Landfill Leachates of the City Konya
International Sustainable Water and Wastewater Management Symposium, Konya - Turkey - 26-28 October 2010
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM; Phytotoxkit
Test Species : Daphnia magna; Thamnocephalus platyurus; Lepidium sativum
Topics : waste waters; landfill leachates; plant tests; soils

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Abstract : Wastewater samples and landfill leachates of Konya City were assessed with biotests from different trophic levels. For this purpose, cost effective and relatively easy to run biotests were selected. The aim was to enable frequent control of pollutants that are assumed to be toxic for the environment also in regions where high sophisticated and elaborate chemical and/or ecotoxicological analyses cannot be performed regularly.
In case of wastewater samples, the tests were compared with the fish test (Poecilia reticulata) that is officially prescribed by the Turkish Water Pollution Control Regulation for industrial wastewater. The results of the microbiotests Daphtox F^TM with Daphnia magna and Thamnotox F^TM with Thamnocephalus platyurus were comparable with the fish test. The L(E)C50 values of these tests were in the range of 10 to 81% wastewater concentration (v/v). Thamnocephalus platyurus was in all tests slightly more sensitive as Poecilia reticulata except of one sample. The reproduction rate of Lemna minor was inhibited in a similar extent only by some samples. The luminescence bacteria test (Lumitox^®) with Vibrio fischeri showed only in single cases effects and the germination test with Lepidium sativum nearly no effect after incubation with the wastewater samples. In contrast, the landfill leachates inhibited all test organisms in a similar range. The L(E)C50 values were mainly in a range of 1 to 5% of leachate concentration. For single samples the germination test with Lepidium sativum and especially the luminescence test with Vibrio fischeri were more sensitive as the other tests. The results show that each of both tested crustacea microbiotests could be a cost effective tool to screen the hazard of wastewater samples regularly and would be also a progress with regard to animal welfare. For the controlling of the landfill leachates the germination test with Lepidium sativum offers a cost effective and especially easy to handle and low tech possibility for regular toxicity screenings. The other tests could be sensitive alternatives but need more time or in case of the luminescence bacteria more technical equipment and infrastructure.

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P. Mayorga, K.R. Pérez, S.M. Cruz and A. Cáceres
Comparison of Bioassays using the Anostracan Crustaceans Artemia Salina and Thamnocephalus Platyurus for Plant Extract Toxicity Screening
Brazilian Journal of Pharmacognosy 2010 20, 6 897-903
Toxkits : Artoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Thamnocephalus platyurus
Topics :

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Abstract : Three lethality bioassays, using the salt-water crustacean Artemia salina Leach, Artemiidae, (conventional 96 microwell plate test and the Artoxkit M microbiotest) and the freshwater crustacean Thamnocephalus platyurus Packard, Thamnocephalidae, (Thamnotoxkit F microbiotest), were compared using extracts of ten Guatemalan plant species. It was previously observed that five of them have anti-Artemia activity. These were: Solanum americanum Mill., Solanaceae, Gliricidia sepium (Jacq.) Kunth ex Walp., Fabaceae, Neurolaena lobata (L.) Cass., Asteraceae, Petiveria alliacea L., Phytolaccaceae, and Ocimum campechianum Mill., Lamiaceae. The five others: Curatella americana L., Dilleniaceae, Prunus barbata Koehne, Rosaceae, Quercus crispifolia Trel., Fagaceae, Rhizophora mangle L., Rhizophoraceae, and Smilax domingensis Willd., Smilacaceae, do not. All plants without anti-Artemia activity had no lethal effects in both assays with A. salina. For the plants with anti-Artemia activity the Artoxkit M was not sensitive to G. sepium and the conventional Artemia test was not sensitive to S. americanum, G. sepium and N. lobata. All the plant extracts, except for that of C. americana, had lethal effects on T. platyurus and the lethal median concentration (LC50) levels for this organism were in all cases substantially lower than those of the salt-water test species. This study revealed that T. platyurus is a promising test species worth further in depth investigation for toxicity screening of plant extracts with potential medicinal properties.

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M. DellaGreca, M.R. Iesce, F. Cermola, M. Rubino and M. Isidori
Phototransformation of Carboxin in Water. Toxicity of the Pesticide and its Sulfoxide to Aquatic Organisms
Journal of Agricultural and Food Chemistry 2004 52 (20) 6228-6232
Toxkits : Algaltoxkit F^TM; Ceriodaphtoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Ceriodaphnia dubia; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - pesticides

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Abstract : Sunlight exposure of aqueous suspensions of carboxin (1) causes its phototransformation to sulfoxide 2 and minor components. Similar effects are observed in the presence of humic acid or nitrate or at different pH values. Photoproducts 2-9 were isolated by chromatographic techniques and/or identified by spectroscopic means. Carboxin 1 and its main photoproduct sulfoxide 2 were tested to evaluate acute toxicity to primary consumers typical of the aquatic environment: the rotifer Brachionus calyciflorus and two crustaceans, Daphnia magna and Thamnocephalus platyurus. Chronic tests comprised a producer, the alga Pseudokirchneriella subcapitata and a consumer, the crustacean Ceriodaphnia dubia.

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M. Davoren and A.M. Fogarty
Ecotoxicological Evaluation of the Biocidal Agents sodium o-phenylphenol, sodium o-benzyl-p-chlorophenol, and sodium p-tertiary amylphenol
Ecotoxicological and Environmental Safety 2005 60 203-212
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; i>Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : chemicals - organic comounds; composts; chemicals - biocides; chemicals - disinfectants

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Abstract : The ecotoxicological effects of three biocidal agents frequently employed as active ingredients in phenolic-based disinfectants were evaluated using a test battery comprising of organisms representing three aquatic trophic levels. Phenolic-based disinfectants are commonly used by mushroom growers to disinfect spent mushroom compost. In general, the most sensitive assay used in this study was the Microtox test. In the case of the fish lethality assay, sodium o-benzyl-p-chlorophenol was found to be slightly more sensitive than the bacterial test system. The freshwater alga and invertebrate tests were also among the most sensitive test species employed. The active ingredient, sodium o-benzyl-p-chlorophenol (with the exception of the Microtox assay), was the most toxic chemical tested on each species. The majority of ecotoxicity data obtained in this research has not been previously reported and may therefore assist in the management and planning decisions regarding the application of pesticides and utilisation of SMC.

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M.E. Aydin, M. Bahadir, M. Kolb, S. Ozcan, S. Yildiz and G. Kara
Assessment of Landfill Leachates Toxicity using Different Bioassays
Proceedings of the International Sustainable Water and Wastewater Management Symposium, 26-28 October 2010, Konya/Turkey
Toxkits : Daphtoxkit F^TM magna; Phytotoxkit; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus; Lepidium sativum
Topics : leachates; landfill leachates; solid waste leachates; plant tests; soils

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Abstract : 350000 tonnes of waste are produced each year, the problem of waste disposal is recognized as one of the most serious environmental problems in Konya. These wastes largely are household waste but also include some commercial and industrial wastes. There are strong national concerns about the possible adverse health effects in the vicinity of municipal waste landfills.
In the study, the acute toxicity of the leachate from a municipal landfill in Konya city, relating toxicity to chemical compostion was evaluated. Leachate samples from the landfill of Konya city were collected considering different seasons and analyzed for their toxiciy by using five different toxicity test methods (Daphtox, Thamnotox, Vibrio fischeri, Lemna minor, Lepidium sativum). Leachate samples were also characterized measuring some physical and chemical parameters. Relationships between composition and toxicity of Konya landfill leachate samples were establised. EC50 values for Thamnotox, Lemna minor and Lepidium sativum were negatively correlated with COD. There are significant associations between TC, TOC with EC50 values of Vibrio fischeri and Lemna minor toxicity tests. Vibrio fischeri was the most sensitive in the investigated test organisms for Konya landfill leachate.
Considering toxic unit values, leachate samples were vey toxic or extremely toxic according to all the tests carried out. Therefore, discharging leachate without treatment will cause hazardous consequences on animal and plant life in the environment.

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A. Bispo, M. J. Jourdain and M. Jauzein
Toxicity and Genotoxicity of Industrial Soils Polluted by Polycyclic Aromatic Hydrocarbons (PAHs)
Organic Geochemistry 1999 30, 8, 2 947-952
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : soil leachates

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Abstract : Three soil samples polluted by PAH were assessed for both acute (Vibrio fischeri [Microtox® test], Daphnia magna, Thamnocephalus platyurus) and chronic (Pseudokirchneriella subcapitata) toxicity, as well as for genotoxicity (Vibrio fischeri M169, [Mutatox® test]). Bioassays were performed on soil water leachates and on soil solvent extracts to characterise not only the fraction of water soluble pollutants but also to evaluate less soluble and soil-bound pollutants. The toxicity of the water leachates was low to moderate (from 0 to 30 toxic units) whereas the toxicity of the methanol extracts was about 100 to 10,000 toxic units. Although only slightly toxic, the genotoxicity of water leachates was clearly demonstrated. This finding shows that acute and chronic toxicity assays alone may not be sufficient to characterize soil hazards. A step procedure to characterise soil ecotoxicity is therefore proposed which allows one to study the pollutant mobility, toxicity and genotoxicity. It can be used to identify the hazard, to classify soil hazards and thus map contaminated sites, to assess the success of treatment and finally to monitor rehabilitated sites.

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G. Nalecz-Jawecki
In Vitro Biotransformation of Amitriptyline and Imipramine with Rat Hepatic S9 Fraction : Evaluation of the Toxicity with Spirotox and Thamnotoxkit F Tests
Archives of Environmental Contamination and Toxicology 2008 54, 2 266-273
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - drugs; chemicals - metabolites

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Abstract : Pharmaceutical products, as well as their related metabolites, end up in the aquatic environment after use. Little is known about the effects and the hazard of exposure to drugs for aquatic organisms. This study was designed to assess the ecotoxicity of amitriptyline (AMI), imipramine (IMI), and their metabolites. The tested drugs were very toxic to the protozoan Spirostomum ambiguum and the crustacean Thamnocephalus platyurus with the LC50 values around 1 mg l(-1). Moreover, simple additivity occurs between the drugs and their N-desmethyl metabolites. Tested compounds were incubated with S9 rat hepatocyte fraction at 37 degrees C for 4 hours. Unchanged drugs and metabolites were determined using high-pressure liquid chromatography-photodiode array detector. AMI and IMI were biotransformed almost completely. Three AMI and IMI metabolites were detected: desmethyl-, didesmethyl-, and N-oxide. The toxicity of the deproteinated reaction mixtures (TU) was compared to the toxicity equivalency units (TEU) calculated based on the concentrations of the drugs and their LC50 values. It has been demonstrated that the toxicity of mixture of metabolites to Spirotox and Thamnotoxkit F is higher than the predicted value calculated from the concentrations of the drugs and their N-desmethylated derivatives in the sample. The results indicate that the harmfulness of the drug metabolites should be taken into consideration in the ecotoxicological studies.

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N. Kováts, L. Füle, I. Magyar, T. Szalay and I. Kiss
Sensitivity of Ecotoxicological Tests in Analysis of Superfund Sites
Acta Biologica Hungarica 2006 57, 2 211-220
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : groundwaters; soil leachates; solid waste leachates

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Abstract : During the analysis of environmental risk posed by hazardous waste disposal sites, ecological impact on whole ecosystems should be assessed. It requires a complex testing scheme where surrogate species represent key elements of the ecosystem. However, different organisms are exposed to a differing degree, also, their sensitivity to the same contaminant may vary. A possible way to determine which test reflects most the actual toxic conditions, correlation can be calculated between the measured ecological parameter (such as growth inhibition, mortality, etc.) and a contaminant gradient. The basic aim of this study was to determine which ecotoxicological test shows the best correlation with the measured analytical parameters. The selected tests were Lemna minor (representing primary producers), Thamnocephalus platyurus (a primary consumer organism) and Vibrio fischeri (decomposer). When testing soil samples, the Thamnocephalus test showed excellent consistency with most contaminants but was oversensitive in the case of groundwater samples. The Vibrio fischeri bioluminescence inhibition test (ToxAlert) behaved in a different way, reflecting well the distribution of most contaminants in groundwater samples. Finally, Lemna test proved to be completely inadequate.

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B. Pushparaj, E. Pelosi and F. Jüttner
Toxicological Analysis of the Marine Cyanobacterium Nodularia harveyana
Journal of Applied Phycology 1999 10 527-530
Toxkits : Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : chemicals - metabolites; chemicals - allelochemicals

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Abstract : Nodularia harveyana, a dinitrogen-fixing cyanobacterial isolate from the Mediteranean Sea, grown in an outdoor photobioreactor, was assayed for its bioactive compounds. The active substance(s) were lypophilic and showed strong allelopathic activity against other axenic cyanobacteria, antibiotic activity against Gram-positive pathogenic bacteria and antifungal activity against two plant pathogens. The extracts were toxic (LC50 at 30 µL) for grazers such as a rotifer (Brachionus calyciflorus) and a crustacean (Thamnocephalus platyurus).

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I.-Z. Chiang, W.-Y. Huang and J.-T. Wu
Allelochemicals of Botryococcus Braunii (Chlorophyceae)
Journal of Phycology 2004 40 474-480
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - metabolites; chemicals - allelochemicals

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Abstract : The alga Botryococcus braunii Kützing (Chlorophyceae) present in Liyu Lake (Huanlien County, Taiwan) has toxic effects on a variety of aquatic organisms. Blooms of this alga, which typically occur in autumn, are associated with fish deaths in this lake. Experiments using 15 phytoplankton and 5 zooplankton isolated from Liyu Lake indicate that these plankton exhibit various susceptibilities to B. braunii. A close correlation between the degree of susceptibility tested in the laboratory and the absence of certain phytoplankton during B. braunii blooms in the lake was observed, suggesting allelopathic effects. Isolation, identification, and verification with authentic compounds indicated that allelochemicals were a mixture of free fatty acids, including α-linolenic, oleic, linolic, and palmitic acids. Compared with other phytoplankton isolates, B. braunii produced significantly higher amounts of free fatty acids, particularly of oleic and α-linolenic acids. The role of these fatty acids in favoring dominance of B. braunii in the natural environment was elucidated.

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M. Davoren, S. N. Shúilleabháin, J.O’Halloran, M.G.J. Hartl, D. Sheehan, N.M. O’Brien, F.N.A.M. van Pelt and C. Mothersill
A Test Battery Approach for the Ecotoxicological Evaluation of Estuarine Sediments
Ecotoxicology 2005 14,7 741-755
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : sediments; sediment pore waters

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Abstract : The purpose of this study was to evaluate the overall sensitivity and applicability of a number of bioassays representing multiple trophic levels, for the preliminary ecotoxicological screening (Tier I) of estuarine sediments. Chemical analyses were conducted on sediments from all sampling sites to assist in interpreting results. As sediment is an inherently complex, heterogeneous geological matrix, the toxicity associated with different exposure routes (solid, porewater and elutriate phases) was also assessed. A stimulatory response was detected following exposure of some sediment phases to both the Microtox® and algal bioassays. Of the bioassays and endpoints employed in this study, the algal test was the most responsive to both elutriates and porewaters. Salinity controls, which corresponded to the salinity of the neat porewater samples, were found to have significant effects on the growth of the algae. To our knowledge, this is the first report of the inclusion of a salinity control in algal toxicity tests, the results of which emphasise the importance of incorporating appropriate controls in experimental design. While differential responses were observed, the site characterised as the most polluted on the basis of chemical analysis was consistently ranked the most toxic with all test species and all test phases. In terms of identifying appropriate Tier I screening tests for sediments, this study demonstrated both the Microtox® and algal bioassays to be more sensitive than the bacterial enzyme assays and the invertebrate lethality assay employing Artemia salina. The findings of this study highlight that salinity effects and geophysical properties need to be taken into account when interpreting the results of the bioassays.

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J.F. Blom and F. Jüttner
High Crustacean Toxicity of Microcystin Congeners Does Not Correlate with High Protein phosphatase inhibitory activity
Toxicon 2005 46, 4 465-470
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : cyanobacterial blooms; cyanotoxins

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Abstract : Microcystins are strong toxins and efficient inhibitors of eukaryotic protein phosphatases. To determine structure related properties of six different microcystin congeners, we applied standardized inhibition assays for the protein phosphatases 1 and 2A, and an acute toxicity assay with Thamnocephalus platyurus. Protein phosphatase inhibition and acute toxicity did not correlate with each other. While the inhibition of the protein phosphatases 1 and 2A was much weaker for [D-Asp³,( E)-Dhb⁷]microcystin-RR than for the other congeners, the toxicity was one of the highest. [D-Asp³]microcystin-LR exhibited only small differences to microcystin-LR. The data show that mechanisms other than the inhibition of protein phosphatases, such as uptake, transport, detoxification or other target sites may have a strong modulating effect on the toxicity of a microcystin congener for a particular animal. Structural changes can offset or even reverse the specific toxicity of microcystin congeners.

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J.F. Blom, J.A. Robinson and F. Jüttner
High Grazer Toxicity of [D-Asp³,(E)-Dhb⁷]microcystin-RR of Planktothrix rubescens as Compared to Different Microcystins
Toxicon 2001 39, 12 1923-1932
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : cyanobacterial blooms; cyanotoxins

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Abstract : Planktothrix rubescens, the dominant cyanobacterium in Lake Zürich, is generally considered to be toxic to zooplankton. The major toxin was determined by NMR spectroscopy and chemical analysis to be [D-Asp³,(E)-Dhb⁷]microcystin-RR. The compound was isolated in high purity, and its 24-h acute grazer toxicity was compared with microcystin-LR, microcystin-RR, microcystin-YR, and nodularin using a Thamnocephalus platyurus bioassay. Based on LC50 values D-Asp³,(E)-Dhb⁷]microcystin-RR was the most toxic microcystin tested. Nodularin was slightly more toxic under the conditions of the assay. The large number of individuals available for the grazer bioassay allowed the determination of dose-response curves of the different microcystins. These curves showed marked differences in their steepness. Microcystin-RR, which had nearly the same LC50 as microcystin-LR and microcystin-YR, exhibited a very flat dose-response curve. This flat curve indicates that, for some individuals, lower concentrations of this microcystin are much more toxic than are the other two microcystins. Mortality of 100% requires much higher concentrations of microcystin-RR, indicating the resistance of some animals to the toxin. The purified D-Asp³,(E)-Dhb⁷]microcystin-RR exhibited a higher molar absorption coefficient determined by quantitative amino acid analysis than the coefficients generally used for other microcystins. This observation has consequences for the risk assessment for microcystins and makes a structural determination of microcystins an absolute requirement. The presence of the dehydrobutyrine residue may be the reason for the higher specific toxicity of D-Asp³,(E)-Dhb⁷]microcystin-RR when compared to the N-methyldehydroalanine-containing microcystins.

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E. Grabinska-Sota and J. Kalka
An Assessment of the Toxicity of Pyridinium Chlorides and Their Biodegradation Intermediates
Environment International 2003 28, 8 687-690
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - surfactants

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Abstract : Toxicity investigations were conducted for four pyridinium chlorides belonging to cationic surface-active substances (CSAS), which differed from each other in the numbers of methyl groups (CH(3)) in pyridinium ring.
The crustacean Daphnia magna, the fish Lebistes reticulatus and the alga Scenedesmus quadricauda were chosen as biotests. Toxicity of examined preparations appeared to be very high but did not depend on their chemical structure. S. quadricauda was the most sensitive organism. Toxicity of intermediate products obtained in biological oxidation process was also examined. Biodegradation was conducted according to the river water test. It was found that only partial degradation took place while pyridinium chlorides constituted main energy and carbon source. Presence of biodegradation intermediate products was shown on the basis of ¹H NMR analysis. Intermediates were not toxic to any biotests.

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E. Liwarska-Bizukojc, K. Miksch, A. Malachowska-Jutsz and J. Kalka
Acute Toxicity and Genotoxicity of five Selected Anionic and Nonionic Surfactants
Chemosphere 2005 58, 9 1249-1253
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM
Test Species : Artemia (salina) franciscana; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - surfactants

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Abstract : The results of four toxicity bioassays of selected anionic and nonionic surface active agents were presented. Three widely used anionic surfactants that belong to alkyl sulphates (AS), alkylbenzene sulphonates (LAS) and alkylpolyoxyethylene sulphates (AES) as well as nonionic surfactants: polyoxyethylene alkyl ethers (AE) and polyoxylethylene alkylphenyl ethers (APE) were tested. Three different toxicity assays to aquatic organisms: Physa acuta Draparnaud, Artemia salina and Raphidocelis subcapitata were applied. Additionally, the genotoxicity test with Bacillus subtilis M45 Rec- and H17 Rec+ strains was performed. The obtained results showed that none of the surfactants studied was genotoxic at the concentration 1000 mg l^-1. On the basis of toxicity tests to aquatic organisms all tested anionic surfactants were harmful (LC50 between 10 and 100 mg l^-1), whereas nonionic ones were toxic (LC50 between 1 and 10 mg l^-1) or even highly toxic (LC50 below 1 mg l^-1). Moreover, the bigger was the molecular weight of the tested compound, the higher toxicity was observed.

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F. Brito; M.A. Morbey; S. Leitão; C. Cortez; F. Ferreira and R. Hernan
Test Organisms Behaviour on Municipal and Industrial Wastewater Quality Assessment
From : SETAC Europe 15th Annual Meeting, Lille, France, 22-26 May 2005 2005
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : effluents; waste waters

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Abstract : Industrial wastewaters quality became a target for the environmental toxicity assessment nowadays. When there are issues with complex effluents for which many chemicals cannot be quantified and/or interactive effects are likely to be significant, the use of bioassays with different test species becomes a more reliable way in the assessment of toxicity. Ecotoxicity evaluation of the several samples shows different behaviours among the groups tested, e.g. bacteria, algae, microcrustaceans and macrophyte. In some cases for the same sample the highest toxicity is shown by one of the test organisms while the remaining ones show no toxicity. The opposite case also occurs.

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I. Blinova, P. Kajankari, L. Kanarbik, M. Heinlaan and A. Kahru
Evaluation of the Potential Hazard of Silver Nanoparticles to Crustaceans
From : 15th International Symposium on Toxicity Assessment, Hong Kong, Hong Kong, 3-8 July 2011
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - nanoparticles

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Abstract : It is known that silver ions are very toxic to many aquatic species, however, information concerning potential hazard of silver nanoparticles to aquatic ecosystems in still limited. Acute and chronic toxicity of collargol (protein-coated colloidal silver), PVP (polyvinylpyrrolidone)-coated nanosilver and silver nitrate (ionic control) to particle-ingesting aquatic crustaceans Daphnia magna and Thamnocephalus platyurus was studied. The results revealed that D. magna was more sensitive to silver compounds than T. platyurus. The silver compounds proved less toxic to the test organisms in natural water than in the OECD test medium.
Long-term effects of the collargol and PVP-coated nanosilver on D. magna were investigated in microcosms which also included duckweed Lemna minor and algae Pseudokirchneriella subcapitata. Data on test organisms` mortality, reproduction and physiological changes (histological study) will be presented.

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L. Szczesny, G. Nalecz-Jawecki and J. Sawicki
The Toxicity of UV Screens in Selected Microbiotests
From : 15th International Symposium on Toxicity Assessment, Hong Kong, Hong Kong, 3-8 July 2011
Toxkits : Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : chemicals - organic

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Abstract : Ultraviolet filters are substances reflecting, scattering and absorbing UV radiation. The European Commission has authorized for use in cosmetics 27 UV screens. The main source of UV filters in the aquatic environment is recreational activity. They are washed off the skin during bathing or swimming, and they may be transferred to towels or clothes and rubbed off during showering ultimately reaching sewage treatment plants. The highest concentrations of ultraviolet filters in water samples are in the summer time.
There are no ecotoxicological studies for UV screens. Thus the aim of this study was the assessment of the toxicity of sunscreens using microbiotests with luminescent bacteria - Vibrio fisheri, protozoan - Spirostomum ambiguum, crustacean - Thamnocephalus platyurus and rotifer - Brachionus calyciflorus.
A group of 16 chemicals with photoprotection properties was tested. Most of them were toxic to bacteria and rotifers. Several were highly toxic to the rotifers with an EC50 of less than 1 mg/l.
High toxicity to B. calyciflorus was shown by: octocrylene (OC), octyl salicylate (OS), octyl methoxycinnamate (OMC) and octyldimethyl PABA (ODP). ODP and OMC also showed high toxicity to bacteria. For the crustacean T. platyurus OMC was very toxic while ODP was toxic with EC50 over 1 mg/l. S. ambiguum wasn’t sensitive to these two sunscreens.
Toxic for all organisms used in this study were 4-methylbenzylidene camphor (4-MBC) and benzophenone - 3 (BP - 3). All are approved for use in cosmetics in the European Union countries and are detected in environmental samples.

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G. Kusza, I. Czerniawska-Kusza, Cz. Rosik-Dulewska and T. Ciesielczuk
Using Microbiotests to Define their Potential for Soil Pollution Assessment of Ethylbenzene
From : 15th International Symposium on Toxicity Assessment, Hong Kong, Hong Kong, 3-8 July 2011
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit; Thamnotoxkit F^TM
Test Species : Heterocypris incongruens; Thamnocephalus platyurus; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; groundwaters; chemicals - organic; plant tests; soils

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Abstract : According to the EU Dangerous Substances Directive ethylbenzene is currently not classified as hazardous for the environment, as it degrades rapidly and does not significantly bio-accumulate. However, due to accidental spills a high amount of ethylbenzene may penetrate into soil and water compartments, leading to environmental pollution. We investigated soil and ground water samples to evaluate (1) the pollution level remained three years after accidental spill of ethylbenzene (47 Mg/400 m² ) in Opole region (Poland), (2) the potential of toxicity tests for soil risk assessment of volatile chemicals. Basic physical-chemical properties of soil samples and the concentration of ethylbenzene were analyzed according to standard methods. For toxicity testing we used Toxkit microbiotests with crustaceans Thamnocephalus platyurus (Thamnotoxkit F^TM) and Heterocypris incongruens (Ostracodtoxkit F) as well as higher plants Lepidium sativum, Sorghum saccharatum, and Sinapis alba (Phytotoxkit). In spite of removal mechanisms, such as volatilization from the soil surface and biodegradation processes in soils, ethylbenzene content was still very high: 67 - 2865 mg/kg d.w. in soil samples, 536 - 1313 mg/l in pore waters, 24 - 75 mg/l in ground waters. The toxicity results vary significantly. The distinct inhibition in seed germination and root elongation of higher plants was obtained almost in all cases, and significant correlations were found between them. No mortality occurred in the acute 24-h lethality test with T. platyurus, only narcotic symptoms were observed. On the contrary, results reaching up to 100% mortality were obtained for the chronic toxicity test with H. incongruens. However, ostracods showed more sensitivity to humic substances than phytotoxicity assays, which might affect their response to contaminated soil samples.

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M. Cieszynska, G. Galezowska, K. Wilma and L. Wolska
How to Assess Toxicity of VOC’s Emissions from Building and Furniture Materials ?
The Pilot Studies of Biotests Application
From : 15th International Symposium on Toxicity Assessment, Hong Kong, Hong Kong, 3-8 July 2011
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - organic compounds; air

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Abstract : Biotests are commonly applied to assess toxicity of various components of the environment such as water (tap water, drinking water, surface water, underground water, etc.) or sediments. Their application for the indoor air and toxicity assessment of various emissions from buildings and selected furniture materials is rather scarce. One of the most dangerous pollutants for human health emitted from building and fitting materials are VOCs.

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G. Nalecz Jawecki, L. Szczesny, D. Solecka and J. Sawicki
Short Ingestion Tests as Alternative Proposal for Conventional Range Finding Assays with Thamnocephalus platyurus and Brachionus calyciflorus
International Journal of Environmental Science and Technology 2011 8, 4 687-694
Toxkits : Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : rapid tests; test development; behavioural tests

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Abstract : The goal of this study was to evaluate whether short 1 h sublethal assays may predict the results of 24h lethality assays with rotifers Brachionus calyciflorus and anostracan crustaceans Thamnocephalus platyurus. The test bionts were hatched from cysts. Inhibition of ingestion was observed after 15 min of incubation of rotifers and crustaceans with the suspension of carmine and latex beads, respectively. Nine compounds with different modes of action were used as toxicants zinc ions, sodium dodecyl sulphate, p-nitrophenol, 3, 5-dichlorophenol and pharmaceuticals propranolol, fluoxetine, abamectin, doramectin and ivermectin. The toxicity values observed in the ingestion tests were very close to the mortality values over a wide range of toxicity from a low toxic surfactant to very toxic avermectins.
The ratio between the 1 h EC50’s in the ingestion test and the 24 h LC50’s in the lethality test was below 2 in all cases for rotifers, and 7 in 9 cases for crustaceans. The toxicity of zinc and 3,5-dichlorophenol in the Thamnotoxkit F^TM was 15-fold higher and 10 fold lower than in the ingestion test, respectively. The 24 h LC50 values are within the range of 25-400 % of the 1 h EC50 values for almost all toxicants tested with the exception of p-nitrophenol for B. calyciflorus and zinc and 3, 5-dichlorophenol for T. platyurus. Short, 1 h ingestion assays Rotoxrapid and Rapidtoxkit are good predictors of the mortality over the next 24 h and can be used as a range finding tests for representatives of pharmaceuticals and surfactants.

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I. Putna, M. Balode, S. Purvina, S. Strake and M. Pfeifere
Biologicaly treated Latvian municipal and industrial waste water toxicity to crustaceans D.magna and A.salina
From : SETAC Europe 21st Annual Meeting, Milan - Italy, 15-19 May 2011
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna;
Test Species : Artemia (salina) franciscana; Daphnia magna
Topics : waste waters

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Abstract : One of the main reasons of the Baltic Sea pollution is leaching waste water from the waste water treatment plants. With HELCOM Baltic Sea Action Plan the Baltic Sea countries have committed themselves to achieve Baltic Sea with life undisturbed by hazardous substances hence reaching good environmental quality status.
The aim of this study was to evaluate Latvian municipal and industrial waste water toxicity after their treatment in the waste water biological treatment plants using freshwater (D. magna) and saltwater (A. salina) crustacean tests.
Tested biologically treated waste water samples were collected in the time period from May 2009 until August 2010 from two industrial and two municipal waste water treatment plants. Toxicity was determined by using two different acute toxicity standard methods: freshwater - ISO Daphnia magna immobilization test (EN ISO 6341:1996) and saltwater - Artemia salina test (ArtoxKit M standard method).
The acquired results showed that A. salina is more sensitive against biologically treated municipal and industrial waste water than D. magna. Both test results indicates that Latvian industrial waste water toxicity has a seasonal character showing higher toxicity in autumn and winter while municipal waste water is none or slightly toxic during all seasons. According to test results success of treatment process of Latvian municipal waste waters depend from inhabitant amount in the area the waste water is collected. Study revealed that biological waste water treatment is not sufficient for Latvian industrial waste waters.
This study was performed in the frame of project INTERREG COHIBA (Control of hazardous substances in the Baltic Sea region).

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R. Oliveira, S. Mcdonough, F. Pitanga, A.M. Moura, R. Bhujel, M.V.M. Soares, J.A. Nogueira, and I. Domingues
Toxicity Assessment of Oxytetracycline at Different Trophic Levels
From : SETAC Europe 21st Annual Meeting, Milan - Italy, 15-19 May 2011
Toxkits : Artoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Thamnocephalus platyurus
Topics : chemicals - organic; chemicals - drugs

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Abstract : Use of antibiotics in the aquatic environment has become an important concern. Oxytetracycline (OTC) is one of the most common veterinary antibiotics used worldwide. OTC is utilized in fish and shrimp farming, and for other animal intensive farming systems, as a growth promoter or for bacterial disease treatment. Several field studies have showed that OTC can persist in the sediment of aquatic environments for more than180 days. Considering the extensive use and high environmental persistence of OTC, this study aims at assessing the lethal and sublethal toxicity of OTC for species of different trophic levels. To achieve these objectives a battery of acute toxicity tests with algae, rotifers, crustaceans and fish were performed following OECD and ToxKit protocols. Following the acute toxicity assessment, different assays were performed for sublethal effect assessment. Representative biomarkers from different metabolic pathways were chosen (catalase-CAT, lactate dehydrogenase-LDH, cholinesterase-ChE, and glutathione-S-tranferase-GST) and teratogenic endpoints were determined using Danio rerio embryos. The results of OTC acute toxicity show Thamnocephalus platyurus, a freshwater species, as the most sensitive species with a 24h-LC50 of 0.042 mg/l (CI: 0.039-0.044) and Artemia salina, a saltwater species, as the most resistant with a 24h-LC50 870 mg/l (CI: 797-987). No difference was found on morphological development of zebrafish early life-stages but a significant (p<0.05) delay in hatching time was found for embryos exposed to concentrations above 150 mg/l. The enzymatic markers analyzed showed inhibition of oxidative stress enzyme (96h-LOECCAT = 5mg/L) and effects of OTC on detoxification phase II (96h-LOECGST =10 mg/L) pathways. In comparison with the 96h-LC50 of 330 mg/l (CI: 314-345) for zebrafish embryos the sublethal endpoints analyzed seem to be more accurate in effects assessment of OTC. Linkages between the sublethal (enzymatic and developmental parameters) effects found in this study indicates that with parameters at higher levels of organization are essential for better understanding of the effect of OTC on organisms and the environment. Moreover, studies on environmental risks of OTC, even at lower concentrations focusing on long term effects applying other methods such as chronic toxicity tests, mesocosm assays, and mixture exposures should be carried out.

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F. Pitanga, R. Oliveira, I. Domingues, M. Moura, R. Bicho and A.M.V.M. Soares
Effects of Sodium Hypochlorite at Different Trophic Levels
From : SETAC Europe 21st Annual Meeting, Milan - Italy, 15-19 May 2011
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - disinfectants

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Abstract : Sodium hypochlorite (SH) is a chemical compound used on a large scale, frequently as a disinfectant or a bleaching agent. It is present in agriculture, chemical, paper, pharmaceutical and waste disposal industries, among others. Chlorine disinfectants in wastewater react with organic matter, giving rise to organic chlorine compounds which are toxic for aquatic organisms and are persistent environmental contaminants.
In this work we assessed the toxicity of SH to organisms belonging to different trophic levels. Effects of SH were evaluated in the growth of the algae Pseudokirchneriella subcapitata and Chlorella vulgaris (concentrations ranging from 0,1 to 100 mg/L), in the survival of Artemia fransciscana and Thamnocephalus platyurus (concentrations ranging from 0,05 to 10 mg/L) and in Danio rerio (zebrafish). Zebrafish assays include acute (concentrations ranging from 0,7 to 10,1 mg/L) and chronic (concentrations ranging from 5 to 500 µg/L) exposure of adult organisms and early life stages test in which embryo development was monitored. The biomarkers lactate dehydrogenase (LDH); GST, glutathione-S-tranferase (GST); Cholinesterase (Che) and catalase (CAT) were measured after zebrafish early life stages and adults exposure to SH.
Most sensitive species was T. platyurus with a LOEC of of 0,5 mg/L, followed by P. subcapitata (LOEC =1 mg/L), C. vulgaris (3,2 mg/L), A. fransciscana (10 mg/L) and D. rerio (adult) (5,5 mg/L). Moreover, it was observed that SH induced an early hatching in D. rerio embryos exposed to concentrations above 6,4 mg/L. Preliminary results show that D. rerio embryos exposed to concentrations above 7.4 mg/L also presented a higher GST activity.

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S. Steudte, P. Stepnowski, A. Igartua and S. Stolte
Eco Design of Environmental Benign Ionic Lubricants : Assessment of Toxicity and Biodegradability
From : SETAC Europe 21st Annual Meeting, Milan - Italy, 15-19 May 2011
Toxkits : Algaltoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - organic

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Abstract : The philosophy of green or sustainable chemistry is the development of high efficient technical processes and applications using chemicals with a reduced or eliminated hazard potential for man and the environment. Therefore the development of chemicals with optimised technical properties should run in parallel with the minimisation of (eco)toxicological hazard potentials.
In the field of designing chemicals, ionic liquids represent an excellent model substance class.
Ionic liquids are salts with melting points below 100°C or even room temperature. Their physicochemical properties like high thermal stability or low vapour pressure raised the interest of this substance class for their application as lubricants in resent years. Additional the high structural variety is an advantage of these compounds. By changing the anion or modifying the fine structure of the cation it is possible to define attributes like viscosity or density, which allows for an optimized application-related design of ionic liquids. Beside the optimization for application also the environmental impact should be considered. Therefore studies on toxicity and biodegradability have been performed for selected ionic liquids. The compounds, mainly ammonium based cations, were chosen according to structure-activity-relationships and their potential applicability as lubricants. Toxicity was tested with several model organisms varying from isolated enzymes, mammalian cells, marine bacteria and algae to higher organisms like crustacean. Data about biodegradation are discussed as well. The present study shows that the design of environmental benign ionic lubricants is - with limitations - feasible.

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P. Stepnowski, S. Steudte, M. Skup and S. Stolte
Prospective Hazard Assessment of New Industrial Chemicals : The Case of Ionic Liquids as High Performance Lubricants
SETAC Europe 21st Annual Meeting, Milan - Italy, 15-19 May 2011
Toxkits : Algaltoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - organic

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Abstract : The interest in ionic liquids for their potential in different chemical processes is increasing, as they are claimed to be environmentally benign and very good non-volatile solvents for a wide range of applications. Responsible product design however should always take into consideration not only technological demands but also the risks arising out of possible (eco)toxicity. This contribution presents our strategy where technical properties of ionic liquids, potentially used as lubricants, are investigated in parallel with the examinations of (eco)toxicological hazard potentials.
Toxicological studies of imidazolium, ammonium and choline based ionic liquids presented here using test systems at different level of biological complexity, such as isolated enzymes, mammalian cell lines or algae. In addition data about biodegradation and sorption of selected ionic liquids to different types of soil and marine sediment are also discussed. A set of instrumental methods for analyzing ionic liquids in environmental and biological samples is presented as well.

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L. Guzzella, L. Ghislanzoni, F. Pozzoni and D. Copetti
Dissolved and Intracellular Microcystins in Lake Waters during a Planktothrix rubescens Algal Bloom : HPLC Quantification and Crustacean Acute Toxicity Test
SETAC Europe 21st Annual Meeting, Milan - Italy, 15-19 May 2011
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : cyanobacterial blooms; cyanotoxins

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Abstract : Microcystins, highly toxic cyclic peptides, are a group of hepatotoxins produced by a number of aquatic species of cyanobacteria, such as Microcystis, Anabaena and Planktothrix. Worldwide contamination in water has prompted the development of detection methods for their identification and quantification. A massive seasonal development of Planktothrix rubescens in a reservoir destined for crop irrigation located in Southern Italy has lead to quantify algal toxin content in the lake water to verify the possible health risk. Microcystins dissolved into the water were separated from intracellular ones by filtering raw samples. Filters were extracted by methanol/water solutions after frozen/defrozen treatment over night. Water samples were concentrated and extracted by SPE-C18 cartridges. Toxin content was detected and quantified using high performance liquid chromatography (HPLC-DAD). The only microcystin detected was [d-Asp3] microcystin-RR. It was identified by retention time and spectrum comparing with a certified standard. Quantification was made by means of a calibration curve obtained at 238 nm. Microcystin extracellular concentration was never above the WHO limits for drinking waters (1 µg/L).
Maximum level as dissolved microcystin was 0.7 µg/L on April 2009 sample. In the same sample the highest endocellular concentration (30.8 µg/L) of [d-Asp3] microcystin-RR was measured.
As predictable, endocellular toxin was 90-95% of the total microcystin content; the endocellular content ranged from 10 to 40 times greater that the dissolved content. Probably it depends on the physiological state of the alga; for example the extracellular content of microcystin is different, if the samples are collected from a fresh or aged population of P. rubescens; the lysis of dead cells can release endocellular toxins into water environment. Acute toxicity tests (24 h exposure) with Thamnocephalus platyurus were carried on using endocellular and extracellular water extracts.
Only the endocellular extract, 10 times concentrated, obtained from April 2009 sample, showed a measurable inhibition (12.5%); it means that the acute toxicity effects on T. platyurus appeared starting from a concentration of about 300 µg/L of [d-Asp3] microcystin-RR. The 24 h doseresponse curve showed that the EC50 of desmetyl-microcystin-RR to T. platyurus was 845 µg/L.
Comparing with literature results, this microcystin is less toxic than microcystin-LR, but 3 times more toxic than microcystin-RR.
See corresponding Poster

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A. Picado, E. Mendonça, L. Silva, S.M. Paixão, F. Brito, M.A. Cunha, S. Leitão, I. Moura and R. Hernan
Ecotoxicological Assessment of Industrial Wastewaters in Trancão River Basin (Portugal)
Environmental Toxicology 2008 23, 4 446-472
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : waste waters; toxicity monitoring

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Abstract : It is important to assess the toxicity of complex effluents, since chemical evaluation alone is insufficient to protect the environment. Direct Toxicity Assessment is valuable in the decision process regarding the final disposal of complex wastewaters as it measures the total effects of the discharge, because of its known and unknown chemicals, additionally having some degree of ecological relevance. In Portugal, ecotoxicity tests are not used on a regular basis to control wastewaters. So, an integrated ecotoxicological, physical, and chemical study of wastewaters from 17 industries, in the Trancão River Basin, was carried out viewing proposing a test battery to be used in wastewater evaluation. An approach which does not include an ecotoxicological characterization may not properly evaluate the potential risks of effluent discharges, especially when they are complex. From the study carried out the use of a battery of assays to apply in the monitoring of complex wastewaters was proposed, including Microtox test, Daphnia test, and an algal test. Moreover, the added value of the ecotoxicological assessment of industrial wastewaters was demonstrated and could support the implementation of EU Directives (e.g. IPPC, WFD) within the Portuguese situation.

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E. Silva and M.J. Cerejeira
Site-Specific Risk Assessment of Pesticide Mixtures for Freshwater Ecosystems
20th Annual Meeting SETAC EUROPE, Seville, Spain; 23-27 May 2010 - Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - pesticides

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Abstract : Most regulatory methods for the management of pesticides are based on single-substance risk evaluations. However, when used in conjunction with some basic toxicological non-interaction models may also be used to predict the joint effect of pesticide mixtures.
Taking into account the pesticides used on specific crops in an agriculture scenario of the Tagus river basin district (Central Portugal) and their environmental fate through screening models, ‘priority’ compounds were selected to be found in freshwater resources. A multi-residue methodology was optimised and developed to assess water exposure to these compounds. The potential risk posed by the pesticide mixtures for the ‘base set’ of aquatic taxa (TUs for algae, Daphnia, and fish) and for the whole aquatic ecosystem (PRISW-1 values) was calculated considering the Concentration Addition model as a ‘reasonable’ worst case for predicting mixture response. This methodological approach allows for the ecological risk assessment of environmentally relevant pesticide mixtures that occur in surface waters of application scenarios of these compounds within a river basin. The data generated are of importance for the development of decision support systems on land management practices which ensure compliance to EU legislation with regard to water quality and sustainable use of pesticides.

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E. Silva, S. Batista, L. Caetano and M.J. Cerejeira
Ecotoxicological Studies in Vineyard Freshwaters Towards a Sustainable Use of Pesticides
19th Annual Meeting SETAC EUROPE, Göteborg, Sweden; 31 May-4 June 2009 - Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - pesticides

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Abstract : This study was performed in the scope of an International Project entitled Waterweb “Water Strategies and Drought Alleviation in Western Balkan Agriculture”. Wine production exerts a considerable number of environmental pressures, including soil degradation, intensive use of plant production products (in particular fungicides) and disposal of waste/by-products from vine-growing and wine-making. Several national studies have detected the presence of agrochemicals in Portuguese groundwater and surface waters and side-effects on aquatic biota have been demonstrated. However, no detailed studies had been carried out in the Portuguese vineyard environment before. In the present study, environmental hazards associated with the use of pesticides in a vineyard located in the Alentejo region (south-central region of Portugal) were evaluated by an integrated approach of ecotoxicological characterisation, modelling, field and laboratory work. Freshwater resources were particularly studied in order to define strategies for their protection and management, based on an evaluation of the exposure of surface water and groundwater to pesticides and their toxic effects on aquatic biota. Mitigation strategies to reduce pesticide inputs into ground- and surface water at the farm scale are discussed.
See corresponding Poster

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P. Palma, M. Köck, P. Alvarenga, L. Ledo, M.J. López de Alda, D. Barceló and I.R. Barbosa
Integrative Approach for the Risk Assessment of Freshwater Reservoirs Influenced by Intensive Agricultural Activities : a Case-Study of Alqueva Reservoir (South Of Portugal)
22nd Annual Meeting SETAC EUROPE, Berlin, Germany; 20-24 May 2012 - Book of Abstracts 406
Toxkits : Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Thamnocephalus platyurus
Topics : chemicals - pesticides; chemicals - general; surface waters

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Abstract : Freshwater reservoirs located in intensive agricultural areas are more vulnerable to chemical ‘stressors’, such as pesticides. So, the characterization of these water bodies for prevalent pesticides is extremely important, once most of these compounds are used in an indiscriminate way by farmers and induce toxic effects in species of aquatic ecosystems and benthic communities.
The purpose of this study was to evaluate the dynamic of pesticides and its correlation with the toxicity values obtained in the Alqueva reservoir, an important source of water irrigation and supply (South of Portugal).
Water and sediments samples from the Alqueva reservoir were analysed considering: the chemical profile (pH, temperature, dissolved oxygen, conductivity, chloride, calcium, magnesium, sodium, phosphorus, Kjeldahl nitrogen , ammonia, nitrite, nitrate, biochemical oxygen demand, chemical oxygen demand, organic matter and trace elements), the pesticides analysis by on-line solid phase extraction-liquid chromatography-electrospray-tandem mass spectrometry (online SPE-LC-ESI-MS/MS), and the ecotoxicological effects (luminescence inhibition of Vibrio fischeri, 24-h mortality test with Thamnocephalus platyurus, 48-h immobilization/mortality assay of Daphnia magna; 6-day mortality/ growth inhibition of the crustacean Heterocypris incongruens).
The pesticides more frequently detected in the water column were atrazine, bentazone, MCPA, mecoprop, metolachlor and terbuthylazine, and in sediments diuron, terbuthylazine and chlortoluron. The highest levels of these pesticides were quantified in the water column. The areas (sampling stations) most polluted by pesticides were Sra. Ajuda, Lucefecit and Álamos, located in the northern and in the middle of the reservoir, respectively. Considering the toxicological results we can conclude that the species most sensitive to the environmental samples from the Alqueva reservoir were the bacteria Vibrio fisheri and the crustacean Heterocypris incongruens. Further, the results showed that some locations at the Alqueva reservoir, namely, Alcarrache, Lucefécit and Álamos induce acute and chronic toxic effect in the bioindicators used. Taking into consideration the pesticides concentrations found and the toxic effects observed in each location of the Alqueva reservoir, we can establish a possible relation-ship between this environmental risk factor and the observed stress in the bioindicators used.
See corresponding Poster

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Y. Marneffe, C. Chalon, A. Claessens, C. Corin, M. Hémart, P. Naport, V. Rollin, S. Sior and V. Wrona
Multi-Bioassay Approach for Assessing the Impact of Industrial Discharges on the Water Quality in Wallonia, Belgium
22nd Annual Meeting SETAC EUROPE, Berlin, Germany; 20-24 May 2012 - Book of Abstracts 289
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : effluents; toxicity monitoring

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Abstract : Bioassays offer an opportunity for a more holistic and meaningful way of assessing effects of environmental samples and wastes on ecosystems than what is possible by using chemical-based monitoring alone. They can provide predictions of environmental impacts whereas ecological community measures only determine impacts after they have occurred. Therefore, bioassays are useful in helping to implement the Water Framework Directive (WFD). In Wallonia (Belgium), an effect directed active monitoring using bioassays is being carried out for many years. It combines ecotoxicological measurements at emission and immission and physico-chemical measurements. We use a battery of short term and chronic bioassays with the bacteria Vibrio fischeri, the alga Pseudokirchneriella subcapitata, the rotifer Brachionus calyciflorus and the microcrustacea Daphnia magna. Moreover, a yeast estrogen screen (YES) assay was conducted as an assessment tool to detect the presence of endocrine disrupting compounds. During 2011, 14 major industrial discharges and 26 sampling points in the receiving waters (upstream and downstream of the effluent) were monitored. The effluents were sampled 6 times a year whereas receiving waters were sampled 4 times a year. Priority List substances of the WFD and other pollutants discharged in significant quantities were also measured. More detailed studies were carried out with complementary tools: upstream and downstream of a discharge of a metallurgical industry, bioassays were conducted on both water samples and sediments and bioaccumulation of metals was assessed using encaged bryophytes; upstream and downstream of a pharmaceutical industry, YES assay was conducted as potential assessment tools in combination with passive samplers (POCIS). The results of these monitorings show that bioassays are good diagnostic tools to determine the causes of poor ecological quality and to trace back to the source of contamination. They are an important “tool in the toolbox” for environmental management. They add value and provide complementary information to that supplied by the chemical and ecological community measures and could help to design appropriate management measures.
See corresponding Poster

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M. Hémart, Y. Marneffe, R. Pirotte, V. Wrona, C. Chalon, P. Naport, V. Rollin, C. Corin and A. Claessens
Bioassays in Sediment Assessment for Investigative Monitoring in the Context of the WFD
22nd Annual Meeting SETAC EUROPE, Berlin, Germany; 20-24 May 2012 - Book of Abstracts 323
Toxkits : Algaltoxkit F^TM; Ostracodtoxkit F^TM; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : sediments; sediment pore waters; sediment and soil contact tests; toxicity monitoring

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Abstract : An intense industrial activity has been taking place in Wallonia (Belgium) since the Middles Ages. The proximity to major waterways has enhanced the industrial development and has facilitated pollution toward aquatic environment. Sediments are known for their ability to trap pollutants and release these substances years or even decades after pollution has ended. In order to assess the current status of sediment pollution, bioassays can be used as suitable monitoring tools to answer the Water Framework Directive (WFD) requests and ascertain the causes of a water body failing to achieve the environmental objectives. They integrate all contaminants effects including additive and synergistic effects of unknown substances and breakdown products.
A sediment quality recurrent monitoring throughout the Walloon Region has been set up, taking into consideration physico-chemical parameters (including Prioritary List substances from the WFD) and ecotoxicological effects of collected environmental samples in a triad-type approach. Different species (Chironomus riparius, Heterocypris incongruens for whole samples, Vibrio fischeri, Pseudokirchneriella subcapitata and Brachionus calyciflorus for pore waters) are used in this approach because they provide different information due to the way of exposure (ingestion, tegumentary contact) and because they represent different trophic levels (decomposer, producer or consumer).
Among the sediments of 10 stations studied in 2010, four showed a relatively low toxicity toward several organisms of the bioassays battery (the Wiltz, the Sure, the Gueule and the Rhosnes), 3 showed low toxicity toward one of the organisms (the Oise,the Molignée and the Eau d’Heure) and 3 showed no signs of toxicity (the Biesmes the Masblette and Salm) even if 2 non-toxics stations exceed the maximum acceptable levels as defined by decree of the Walloon government.
Results of the 2011 monitoring program will also be presented.
See corresponding Poster

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B.S. Nunes, F.D. Carvalho, L.M. Guilhermino and G. Van Stappen
Use of the Genus Artemia in Ecotoxicity Testing
Environmental Pollution 2006 144 453-462
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : review

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Abstract : Information related to varied uses of several species of the genus Artemia (commonly known as brine shrimp), is dispersed among literature from several scientific areas, such as Ecology, Physiology, Ecotoxicology, Aquaculture and Genetics. The present paper reviews information related to Artemia that may be considered relevant for ecotoxicity testing. Integration of different areas of scientific knowledge concerning biology, life cycle and environmental needs of Artemia is of crucial importance when considering the interpretation of results drawn from tests involving this genus. Furthermore, this paper provides suggestions to overcome problems related to toxicity assessment with the use of Artemia as test organism in bioassays, under the scope of estuarine, marine and hypersaline environments. Aspects related to variability in results, adoptable toxicity end-points, culture conditions, characteristics of species and strains, influence of geographical origins over physiological features and responses to exposure to chemical agents are considered.

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A. Koutsaftis and I. Aoyama
Toxicity of four Antifouling Biocides and their Mixtures on the Brine Shrimp Artemia salina
Science of the Total Environment 2007 387 166-174
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : antifouling paints; toxic interactions; chemicals - paints

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Abstract : Zinc pyrithione (ZPT), Copper pyrihione (CPT), Chlorothalonil and Diuron are four of the most widely used as alternative to tributlytin (TBT) antifouling biocides in boat paints. As most previous laboratory bioassays for these biocides have been conducted solely based on acute tests with a single compound, information on the possible combined toxicity of these common biocides to marine organisms are limited. In this study, the toxicity of binary (in several proportions), ternary and quaternary mixtures were evaluated using the brine shrimp Artemia salina as test organism. Mixture toxicities were studied using the concentration addition model (isobolograms and toxic unit summation), and the mixture toxicity index (MTI). The ZPT-CPT combination had a strictly synergistic effect which requires attention because the coexistence of ZPT and CPT in the marine environment, due to transchelation of ZPT, may occur. The binary mixtures of Diuron with the metal pyrithiones exhibited various interactive effects (synergistic, antagonistic or additive) depending on concentration ratios, whereas all binary mixtures that contained Chlorothalonil exhibited antagonistic effects. The different types of combined effects subsequent to proportion variation of binary mixtures underline the importance of the combined toxicity characterization for various ratios of concentrations. The four ternary mixtures tested, also exhibited various interactive effects, and the quaternary mixture exhibited synergism. The models applied were in agreement in most cases. The observed synergistic interactions underline the requirement to review water quality guidelines, which are likely underestimating the adverse combined effects of these chemicals.

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F. Garaventa, C. Gambardella, A. Di Fino, M. Pittore and M. Faimali
Swimming Speed Alteration of Artemia Sp. and Brachionus plicatilis as a Sub-Lethal Behavioural End-Point for Ecotoxicological Surveys
Ecotoxicology 2010 19 512-519
Toxkits : Artoxkit M^TM; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis
Topics : rapid tests; test development; test sensitivity; chemicals - organic compounds; behavioural tests

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Abstract : In this study, we investigated the possibility to improve a new behavioural bioassay (Swimming Speed Alteration test-SSA test) using larvae of marine cyst-forming organisms: e.g. the brine shrimp Artemia sp. and the rotifer Brachionus plicatilis. Swimming speed was investigated as a behavioural end-point for application in ecotoxicology studies. A first experiment to analyse the linear swimming speed of the two organisms was performed to verify the applicability of the video-camera tracking system, here referred to as Swimming Behavioural Recorder (SBR). A second experiment was performed, exposing organisms to different toxic compounds (zinc pyrithione, Macrotrol MT-200, and Eserine). Swimming speed alteration was analyzed together with mortality. The results of the first experiment indicate that SBR is a suitable tool to detect linear swimming speed of the two organisms, since the values have been obtained in accordance with other studies using the same organisms (3.05 mm s(-1) for Artemia sp. and 0.62 mm s(-1) for B. plicatilis). Toxicity test results clearly indicate that swimming speed of Artemia sp. and B. plicatilis is a valid behavioural end-point to detect stress at sub-lethal toxic substance concentrations. Indeed, alterations in swimming speed have been detected at toxic compound concentrations as low as less then 0.1-5% of their LC(50) values. In conclusion, the SSA test with B. plicatilis and Artemia sp. can be a good behavioural integrated output for application in marine ecotoxicology and environmental monitoring programs.

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M. Tarczynska, G. Nalecz-Jawecki, Z. Romanowska-Duda, J. Sawicki, K. Beattie, G. Codd and M. Zalewski
Tests for the Toxicity Assessment of Cyanobacterial Bloom Samples
Environmental Toxicology 2001 16, 5 383-390
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : cyanobacterial blooms; cyanotoxins

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Abstract : Cyanobacterial (blue-green algal) blooms are one of the common consequences of the increasing eutrophication of surface waters. The production of cyanobacterial toxins and their presence in drinking and recreational waters represents a growing danger to human and animal health. Due to a lack of toxin standards and to resource limitations on the wide-scale use of analytical methods (e.g., high-performance liquid chromatography, enzyme-linked immunosorbent assay (ELISA)) in cyanobacterial toxin monitoring, it is necessary to assess and to develop additional methods for their detection and estimation. Microbiotests using invertebrates offer a possible approach for the inexpensive and straightforward detection and assessment of cyanobacterial bloom toxicity. Three microbiotests with: Thamnocephalus platyurus, Daphnia magna, and Spirostomum ambiguum were examined with bloom samples containing hepatotoxic microcystin-LR and up to five additional microcystin variants. Two kinds of cyanobacterial bloom sample preparations were tested: crude extracts (CE) and purified extracts (PE). The highest toxicity was found when CE was used for microbiotests. The sensitivity of microorganisms decreased from S. ambiguum to T. platyurus and to D. magna. A statistically significant correlation was found between microcystin concentration and T. platyurus biotest, and between mouse bioassay and S. ambiguum results. Addition of Me2SO (1%, v/v) is a possible method to increase the sensitivity of the microorganisms for microcystin-LR.

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R. Guerra
Ecotoxicological and Chemical evaluation of Phenolic Compounds in Industrial Effluents
Chemosphere 2001 44, 8 1737-1747
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis; Daphnia magna
Topics : chemicals - organic compounds

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Abstract : The aim of this paper was to evaluate the ecotoxicological response of industrial effluents containing phenolic compounds. All complex effluents collected from a chemical plant and then after both a chemical–physical and biological treatment were characterised with chemical analysis, biodegradability tests and four ecotoxicological tests (Daphnia magna, Artemia salina, Brachionus plicatilis and Vibrio fisheri with Microtox®). The evaluation of the chemical and ecotoxicological data was useful for predicting the effect of the raw effluent on the treatment plant and the impact of the final treated effluent on the receiving water. Besides the toxicity of the effluent from the chemical plants, the acute toxicity of its main components was also determined. The results of the tests and toxicity data from literature were transformed in Toxic Units (TUs). Effluent toxicity was under- or over-estimated by calculating the sum of the TUs of the individual components, depending on which toxicity data and test organisms were used.

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V. Tsiridis, P. Samaras, A. Kungolos and G.P. Sakellaropoulos
Characterization of Coal Fly Ashes using Leaching and Ecotoxicity Tests
Book of Abstracts of Second International Symposium on Green Chemistry for Environment and Health, September 27-29, Mykonos, Island, Greece 50
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : fly ashes; leachates

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Abstract : The chemical and ecotoxicological properties of eluates of six coal fly ash samples collected from different coal incineration plants were examined in the present work. The batch leaching tests EN 12457-2 and EN 12457-3 and the percolation test NEN 7343 for classification of waste as suggested by the European Council decision 2003/33/EC, were applied in conjunction with a battery of bioassays. The test organisms that were used for the toxicity evaluation of fly ash eluates included the photobacterium Vibrio fischeri, the algae Pseudokirchneriella subcapitata and the crustacean Daphnia magna. Results indicated that the concentration of Ba, Cr, Mo, sulphates and TDS exceeded the limit values for inert or for nonhazardous waste. The toxicity of undiluted eluates obtained from batch leaching tests towards algae P. subcapitata and crustacean D. magna was generally high, reaching up to 100 % effect, while low toxic response was observed for photobacterium V. fischeri. The ecotoxicological index of three fly ash samples was in agreement with physicochemical index, as samples yielding high ecotoxicological index had elevated number of constituents exceeding the limit values for inert or non-hazardous waste (high physicochemical index). However, for the other three fly ash samples, the ecotoxicological index was high, while the physicochemical index was specifically low, indicating that physicochemical analyses may underestimate the environmental hazard of these samples.
See corresponding Poster

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V. Tsiridis, M. Petala, P. Samaras, A. Kungolos and G.P. Sakellaropoulos
Environmental Hazard Assessment of Coal Fly Ashes using Leaching and Ecotoxicity Tests
Ecotoxicology and Environmental Safety 2012 84 212-220
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : fly ashes; leachates

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Abstract : The environmental hazard of six coal fly ash samples collected from various coal incineration plants were examined in the present study, using various leaching tests along with physicochemical and ecotoxicological analyses. Physicochemical analyses indicated that the concentration of Ba, Cr, Mo, Se, sulfates and total dissolved solids (TDS) exceeded the threshold values, according to the European Council decision 2003/33/EC for inert, non-hazardous or hazardous waste. The highest sensitivity, among examined organisms, was presented by Daphnia magna and was ascribed to the lowest tolerance of the organism to Cr. The toxic effect profile of the leachates toward Vibrio fischeri revealed that its bioluminescence was significantly altered by the presence of heavy metals in the leachates (such as Cu, Ni and Zn) in low or sub-lethal doses. Although the toxicity of the leachates toward Pseudokirchneriella subcapitata was relatively high, the correlation between the toxicity and heavy metals concentration was rather poor, indicating the significance of the bioavailability of constituents and the importance of toxicity testing. Based on the classification of the samples resulted by physicochemical analyses of the leachates and the results of ecotoxicity tests, Physicochemical and Ecotoxicological indices were also defined, for an overall evaluation of the environmental impact assessment of fly ashes. The suggested indices showed that ecotoxicity tests were in some cases inconsistent with the outcome of waste classification based on physicochemical analyses. Moreover, the examination of leaching protocols and obtained results showed that one stage leaching test (L/S=10 L/kg) underestimated the fly ash classification, when compared with the two stage test EN 12457-3 at the cumulative ratio L/S=10 L/kg. In addition, the application of availability test NEN 7341 provided useful results about the ecotoxicity wastes. Thus, NEN 7341 is strongly suggested for the implementation of such studies, since the bioavailability of constituents may not be altered by pH adjustment tactics and pH-originated toxicity effects may be avoided.

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A. Kungolos, V. Tsiridis, M. Petala, C. Emmanouil, S. Kipouros, D. Antoniadis, G. Drakopoulos, P. Dimakos and K. Perakis
Ecotoxicological Assays as a Means of Monitoring Vulnerable Aquatic Ecosystems
From : 3rd International Symposium on Green Chemistry for Environment, Health and Development - October 3-5, 2012 - Skiathos, Greece
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : toxicity monitoring

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Abstract : Until the recent past, the Hellenic policy of monitoring and evaluation of aquatic reservoirs focused on physicochemical and microbiological analyses of relevant samples, in order to test whether legislation limits were met. This strategy, however, presents some disadvantages which can be summarized in the following points: 1) detection and quantification of pollutants in small quantities is often cumbersome and expensive, 2) combined action of these pollutants in aquatic organisms cannot be highlighted, 3) bioaccumulation, bioavailability and transport through food chains are also not quantified. Conducting ecotoxicological assays in parallel to chemical analyses, answers some of these questions, since these tests assess the effect of polluted aquatic specimens on survival, growth and other basic functions on organisms of different trophic levels. Results are then compared to organisms not exposed to the polluted aquatic specimens or to EU-set levels of toxicity (e.g. 67/548/EC) so that useful deductions on the toxic potential of said specimens can be made.

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V. Tsarpali and S. Dailianis
Landfill Leachate Composition and Toxic Potency in Semi-Arid Areas : an Integrated Approach with the Use of Physicochemical and Toxicological Data
From : 3rd International Symposium on Green Chemistry for Environment, Health and Development - October 3-5, 2012 - Skiathos, Greece
Toxkits : Artoxkit M^TM; Rotoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis; Thamnocephalus platyurus
Topics : landfill leachates

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Abstract : Landfills are among the most widely practiced methods for the disposal of municipal solid residues. During this process, a complex mixture of liquid effluent, commonly called leachate, is generated by the precipitation and penetration of water into the mass of residues undergoing biodegradation. Leachate is characterized by high concentrations of organic and inorganic compounds. The contact of such a complex mixture with the surrounding water bodies, could lead to environmental alterations, thus it is considered as an environmental matter of concern. Different biotests have shown significant toxic effects of leachate in various organisms. One of the known parameters to affect leachate strength and toxic potency is the local rainfall regime, but little it is known about the way that happens, especially in semi-arid climatic conditions, such as the most of the Mediterranean countries. Since the knowledge of leachate composition is necessary in order to manage the long-term impacts of a landfill, the aim of the present study was to monitor leachate composition and toxic potency alterations with time on different aquatic organisms, such as the crustaceans Artemia franciscana and Thamnocephalus platyurus, the estuarine rotifer Brachionus plicatilis and the unicellular algal Dunaliella tertiolecta as well as to what extent the local rainfall regime could mediate leachate strength and toxicity.

Analysis of leachate samples regularly collected from the municipal landfill site of Aigeira (Peloponissos, Greece) during the year 2011, showed seasonal alterations of its physicochemical parameters and its toxic effects, following seasonal changes of the amount of rainfall. In all cases, leachates appeared to have toxic effects on all species tested, showing type- and time-dependent alterations. In addition, certain rainfall-related leachate parameters, such as nitrates (NO3-), total nitrogen (TN), ammonium (NH4-N), total dissolved solids (TDS) and the BOD5/NH4-N ratio, could merely reflect on the leachate’s strength and toxicity, as verified by the significant correlations occurred among each of them with the 24hLC50 and/or 72h IC50 values observed in all species tested. This fact indicates that the above parameters could be used independently, or in combination for investigating and predicting seasonal leachate toxic potential, at least in the case of semi-arid areas, such as the most of the Mediterranean countries.
See corresponding Poster

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G. Persoone
Toxkit Microbiotests : Practical and Low Cost Tools for Research and Toxicity Monitoring
From : 3rd International Symposium on Green Chemistry for Environment, Health and Development - October 3-5, 2012 - Skiathos, Greece
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Phytotoxkit; Thamnotoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : test development; toxicity monitoring; test validation; plant tests; soils

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Abstract : Toxicity testing of chemicals and toxicity monitoring of contaminated aquatic and terrestrial environments is expensive and is seldom applied in routine because of the necessity of year-round availability, and hence continuous culturing and maintenance of live stocks of test species. As a result, toxicity testing is to date in every country only performed in a small number of specialized laboratories.

Extensive research during the last decades at the Laboratory for Environmental Toxicology and Aquatic Ecology at the Ghent University in Belgium, and subsequently in the spin-off company MicroBioTests Inc. has led to the development of a new generation of bioassays which, contrary to “conventional” toxicity tests, are independent of the culturing and maintenance of live stocks of test species. The new assays are based on “dormant or immobilized” stages of the test biota which can be stored for long periods of time, and from which the live test organisms can easily be obtained “on demand”, i.e. at the time of performance of the assays, and without any need for culturing or maintenance of live stocks.

The “culture/maintenance free” bioassays have been miniaturized in user-friendly and low cost kits (called Toxkits) which allow to perform toxicity testing for research or toxicity monitoring in any laboratory, without the need for specialized know how or infrastructure.

As of to date a whole battery of acute and short chronic Toxkit microbiotests has been developed with freshwater, marine and terrestrial test species which are now available commercially. Several Toxkits make use of the same test species as “conventional” toxicity tests (e.g. the Daphnia and the algae microbiotest) and they totally follow the same test procedures.

Six Toxkit microbiotests have already become ISO standards and can hence be used in a regulatory framework. The microbiotests are used for a variety of applications encompassing toxicity ranking of pure chemicals, toxicity testing of surface and groundwaters, effluents, sediments, solid wastes and soil.

The availability of these practical and low cost tools for toxicity screening and toxicity biomonitoring has gradually led to their worldwide use, and Toxkit microbiotests are now applied in more than 40 countries for research and toxicity monitoring, and more than 400 reports and publications are already listed on the website www.microbiotests.be.

The presentation will give an overview of the Toxkit microbiotests and their test procedures, and will highlight a number of specific applications.

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V. Tsarpali, M. Kamilari and S. Dailianis
Seasonal Alterations of Landfill Leachate Composition and Toxic Potency in Semi-Arid Regions
Journal of Hazardous Materials 2012 233-234 163-171
Toxkits : Artoxkit M^TM; Rotoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis; Thamnocephalus platyurus
Topics : landfill leachates

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Abstract : The present study investigates seasonal variations of leachate composition and its toxic potency on different species, such as the brine shrimp Artemia franciscana (formerly Artemia salina), the fairy shrimp Thamnocephalus platyurus, the estuarine rotifer Brachionus plicatilis and the microalgal flagellate Dunaliella tertiolecta. In specific, leachate regularly collected from the municipal landfill site of Aigeira (Peloponissos, Greece) during the year 2011, showed significant alterations of almost all its physicochemical parameters with time. Further analysis showed that seasonal alterations of leachate composition are related with the amount of rainfall obtained throughout the year. In fact, rainfall-related parameters, such as conductivity (Cond), nitrates (NO3-), total nitrogen (TN), ammonium (NH4-N), total dissolved solids (TDS) and the BOD5/NH4-N ratio could merely reflect the leachate strength and toxicity, as verified by the significant correlations occurred among each of them with the toxic endpoints, 24 h LC50 and/or 72 h IC50, obtained in all species tested. According to the result of the present study, it could be suggested that the aforementioned leachate parameters could be used independently, or in combination as a low-cost effective tools for estimating leachate strength and toxic potency, at least in the case of semi-arid areas such as the most of the Mediterranean countries.

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J. Gonçalves, M. Moreira-Santos, I. Lopes and R. Ribeiro
Efficacy of Recent Remediation Measures in a Protected Coastal Lagoon (Paramos, Portugal) : Toxicity of Surface Versus Subsurface Sediments
22nd Annual Meeting SETAC EUROPE, Berlin, Germany; 20-24 May 2012 - Book of Abstracts 336
Toxkits : Algaltoxkit F^TM; Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : sediments

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Abstract : Wetlands, including coastal lagoons, are among the richest ecosystems concerning productivity and are responsible for processes such as biomass production, water replacement, sediment and nutrient retention, and floods control. However, coastal areas are also regions supporting large urban and industrial areas, as well as tourism, all leading to the deterioration of these ecosystems. This is the case of the Paramos lagoon (NE Portugal), which despite its recognized ecological significance has a long contamination history (e.g. domestic sewage, agricultural and industrial effluents) that resulted in high levels of sediment contamination. The present study aimed to assess the efficacy of the remediation measures already implemented in the Paramos lagoon, by comparing the toxicity of surface versus subsurface sediments, as depth profiles in sediments provide information about the temporal contaminant inputs. Tests with the following battery of species, representative of different taxonomic groups, throphic levels and key ecosystem functions, were conducted: bacteria Vibrio fischeri (decomposer), microalgae Pseudokirchneriella subcapitata (primary producer), crustacean ostracod Heterocypris incongruens (epibenthic omnivorous), and the midge Chironomus riparius (benthic insect larvae; deposit feeder). Four sites were selected within the wet area of the lagoon to cover not only the whole lagoon area but also the regions close to the two tributaries (likely to be the most contaminated) and to the non-permanent channel (likely to be the least contaminated). Overall, two sites presented higher organism responses, the one located next to the non-permanent channel, which endures some water renewal, and the one close to the area where remediation measures first took place. The other two sites, one located close to a contaminated tributary and the other close to the centre of the lagoon, were those with lower organism responses and higher uncertainties in what regards risk. Also, for all except one site (one of the most contaminated), organism performances were generally best with surface sediments.
Although the selected test battery proved to have potential to map toxicity levels within the Paramos lagoon and areas where remediation measures are being successful, the uncertainties associated to the test results at some sites point to the need for a more detailed sediment toxicity assessment (e.g. additional tests and sites, chemical analysis).
See corresponding Poster

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M.L. García-Lorenzo, M.J. Martínez-Sánchez, C. Pérez-Sirvent and J. Molina
Ecotoxicological Evaluation for the Screening of Areas Polluted by Mining Activities
Ecotoxicology 2009 18 (8) 1077-1086
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; sediment and soil contact tests; plant tests

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Abstract : The aim of this study was to evaluate the applicability of three bioassays representing multiple trophic levels, for the preliminary ecotoxicological screening of sediments from sites contaminated by mining activities. Of the bioassays used in this study, the ostracod test was the most responsive. Vibrio fischeri luminescence inhibition assay was less sensitive to the toxicants in the sediments than the phytotoxicity assays. The general trend observed was an increase in toxicity values measured by the bioassays with increasing metal mobilization in sediment samples. Therefore, the test battery can be used as a rapid and sensitive tool to evaluate the heavy metal contamination in sediments.

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L. Van der Vliet, J. Velicogna, J. Princz and R. Scroggins
Phytotoxkit : a Critical Look at a Rapid Assessment Tool
Environmental Toxicology and Chemistry 2012 31 (2) 316-323
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : plant tests; regulatory testing; soils; chemicals - inorganic compounds

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Abstract : Terrestrial plant toxicity testing contributes critical information to many site risk assessments, but standardized tests can be labor-intensive, use large amounts of soil, and employ long test durations. The Phytotoxkit minimizes the time and cost associated with terrestrial plant testing with a unique test setup, a shorter test duration, and less soil. However, the sensitivity of the test remains an open question. In this research, the Phytotoxkit and the standardized Environment Canada terrestrial plant toxicity test (definitive test) are compared using a parallel testing approach. Three different scenarios were examined: a multiconcentration test, in which an inhibiting concentration (ICp) was derived from chemically amended soils; a soil remediation test, in which plant growth in a remediated soil was compared to the original contaminated soil; and a site soil test, in which plant growth in a contaminated soil was compared to a reference soil. The contaminants tested were boric acid, Cr(VI) with cyclodextrin as a remediation agent, and petroleum hydrocarbons. Trifolium pratense (red clover) was used in the first and second scenarios, and six different plant species were used in the third scenario. In the first scenario, the Phytotoxkit results compared well with the definitive test results after 5 and 7 d of exposure. In the second scenario, the Phytotoxkit results agreed with the definitive test when evaluating the effectiveness of remediation. In the third scenario, the Phytotoxkit results were often not in agreement with the results from the definitive test. The reduced sensitivity of the Phytotoxkit in the third scenario may be driven by test unit design, as plant roots are separated from soil by filter paper.

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M.L. Garcia-Lorenzo, M.L. Martinez-Sanchez, J. Molina and M. Hernandez-Cordoba
Phytotoxkit and Ostracodtoxkit Tests for Assessing the Toxicity of Sediment Samples With High Concentration of Heavy Metals
From : EGU General Assembly, Vienna-Austria, 2-7 May 2010 1014
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - metals; sediments; sediment and soil contact tests; plant tests

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Abstract : To estimate the risk of contaminants, chemical methods need to be complemented with biological methods. Ecotoxicological testing may be a useful approach for assessing the toxicity as a complement to chemical analysis. The aim of this study was to evaluate the sensitivity and applicability of two bioassays representing multiple trophic levels, for the preliminary ecotoxicological screening of sediments from sites contaminated by mining activities : a chronic toxicity test with the ostracod Heterocypris incongruens and a phytotoxicity test using Lepidium sativum, Sorghum saccharatum and Sinapis alba seeds. For this study, 30 soils samples were collected from the Sierra Minera (Murcia, SE Spain) for general analytical determinations and the total metal content (Pb, Zn, Cd and As) was determined. The Phytotoxkit test measures the decrease in (or the absence of) seed germination and of the growth of the young roots after 3 days of exposure of seeds of selected higher plants to a contaminated matrix compared with the controls germinated in a reference soil. The plants selected for the Phytotoxkit microbiotest were: the monocotyl Sorghum saccharatum (Sorgho) and the dicotyls Lepidium sativum (Garden cress) and Sinapis alba (mustard). The percent inhibition of seed germination (SG) and root growth inhibition (RI) for each plant were calculated. Ostracodtoxkit test consists of placing freshly hatched ostracod neonates in multiwell cups in 2 ml synthetic freshwater, with 300 µl sediment and 3x10⁷ algal cells (Selenastrum capricornutum). After 6 days, incubation at 25 °C in darkness, the mortality of test organisms was determined and growth inhibition was calculated. Soil samples showed a mean pH value of 6.0 in water and 5.7 in KCl. The EC varied from 1.0 dS m-1 to 56.2 dS m-1, with a mean value of 17.7 dS m-1. The mean value for Pb was 0.84 mg kg-1, 10593 mg kg-1 for Zn, 23.18 mg kg-1 for Cd and 0.16 mg kg-1 for As. As regards the phytotoxicity test, an influence on seed germination was observed. Correlation analysis between heavy metal concentration, soil characteristics and plant test results showed that growth inhibition in the plant species was negatively correlated with pH and calcium carbonate content and was positively correlated with Eh, EC and total arsenic content in soil samples. All three species chosen for this assay showed greater sensitivity to Cd than As and Pb (in that order). Correlation analysis showed that the mortality of ostracods was positively correlated with Eh and EC and negatively correlated with pH and calcium carbonate content. As in the case of plants, the sensitivity of ostracods was greatest for Cd, followed by As and Pb.

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M.J. Martínez-Sánchez, M.L. García-Lorenzo, C. Pérez-Sirvent, J. Molina, M.L. Tudela and M. Hernández-Córdoba
Use of Bioassay Test for the Environmental Evaluation of Mining Residues and their Leachates : the Singular Case of the Portman Bay (SE, Spain)
From : EGU General Assembly, Vienna-Austria, 2-7 May 2010 1024
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : leachates; landfill leachates; sediment and soil contact tests; plant tests; soils

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Abstract : The aim of the present study was to evaluate the toxicity of sediments and their pore-water extracts from sites contaminated by mining activities using two assays: bacteria and plants. The acute toxicity in pore-waters was determined using the Microtox bioassay, which uses the naturally luminescent marine bacterium Vibrio fischeri. Phytotoxicity in soil samples was tested by way of the seed germination and root elongation technique in three plant species, Sorghum saccharatum, Sinapis alba and Lepidium sativum. The aim of applying these assays is to establish a method for evaluating the real risks within a risk analysis process, considering both present and future risks, bearing in mind that the uses to which soil is put (urban, recreational or industrial) may change. In the zone studied, mining activities have led to heavy metal contamination with the risk of runoff and wind dispersion of the contaminated material. For this study, 6 sediment samples were collected from Portman Bay (Murcia, SE Spain). The soil extract was prepared by saturation with distilled water and allowing it to stand for four hours. Then, the soil was subjected to a vacuum pressure to extract the soil solution through filter paper. The Zn and Fe content was determined by flame atomic absorption spectrometry (FAAS). The Pb, Cd and Cu content was determined by electrothermal atomization atomic absorption spectrometry (ETAAS). The As content was analysed by atomic fluorescence spectrometry using an automated continuous flow hydride generation (As-AFS) spectrometer. Total Pb concentration varied from 600 to 2500 ppm, with a mean value of 1200 ppm. The average content of Zn was 5300 ppm. The mean concentration of Cd and Cu was 23 and 59 ppm, respectively. Total As concentrations varied from 180 to 470 ppm, with an average value 280 ppm. Finally, the total Fe content ranged from 37% to 47%, with an average value of 40%. Pore-water samples showed neutral pH values and average electrical conductivity was 8.4 ds m-1. Mean heavy metal content in leachates from Portman Bay was 6.8 ppm for Pb, 0.1 ppm for Zn, 17 ppb for Cd, 5.6 ppb for Cu, 3.7 ppb for As and 0.6 ppm for Fe. The bioassays showed different sensitivities to the target metals. The Vibrio fischeri luminescence inhibition assay showed less sensitivity to the toxicants in the sediments than phytotoxicity assay. According to our results it is highly advisable to complement chemical analyses with environmental toxicity testing to characterise the risks presented by contaminated soils. Finally, these methods satisfy the requirements of environmental toxicology in their reliability, sensitivity, reproducibility, rapidity and low cost.

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M. Matejczyk, G.A. Plaza, G. Nalecz-Jawecki, K. Ulfig and A. Markowska-Szczupak
Estimation of the Environmental Risk Posed by Landfills using Chemical, Microbiological and Ecotoxicological Testing of Leachates
Chemosphere 2011 82, 7 1017-1023
Toxkits : Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Thamnocephalus platyurus
Topics : landfill leachates

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Abstract : The leachates from 22 municipal solid waste (MSW) landfill sites in Southern Poland were characterized by evaluation of chemical, microbiological and ecotoxicological parameters. Chemical analyses were mainly focused on the identification of the priority hazardous substances according to Directive on Priority Substances, 2008/105/EC (a daughter directive of the WFD) in leachates. As showed, only five substances (Cd, Hg, hexachlorobutadiene, pentachlorobenzene and PAHs) were detected in the leachates. The compounds tested were absent or present at very low concentrations. Among them, only PAHs were found in all samples in the range from 0.057 to 77.2 10^-6g/l. The leachates were contaminated with bacteria, including aerobic, psychrophilic and mesophilic bacteria, coliform and fecal coliforms, and spore-forming-bacteria, including Clostridium perfringens, and with filamentous fungi. From the analysis of specific microorganism groups (indicators of environmental pollution by pathogenic or opportunistic pathogenic organisms) it can be concluded that the landfill leachates showed sanitary and epidemiological hazard. In the ecotoxicological study, a battery of tests comprised of 5 bioassays, i.e. Microtox(®), Spirotox, Rotoxkit F^TM, Thamnotoxkit F^TM and Daphtoxkit F^TM magna was applied. The leachate samples were classified as toxic in 13.6%, highly toxic in 54.6% and very highly toxic in 31.8%. The Spirotox test was the most sensitive bioassay used. The percentage of class weight score was very high - above 60%; these samples could definitely be considered seriously hazardous and acutely toxic to the fauna and microflora. No correlations were found between the toxicity values and chemical parameters. The toxicity of leachate samples cannot be explained by low levels of the priority pollutants. It seems that other kinds of xenobiotics present in the samples at subacute levels gave the high aggregate toxic effect. The chemical, ecotoxicological and microbiological parameters of the landfill leachates should be analyzed together to assess the environmental risk posed by landfill emissions.

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P. Palma, P. Alvarenga, V. Palma, C. Matos, R.M. Fernandes, A. Soares A and I.R. Barbosa
Evaluation of Surface Water Quality using an Ecotoxicological Approach : a Case Study of the Alqueva Reservoir (Portugal)
Environmental Science and Pollution Research International 2010 17, 3 703-716
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : surface waters

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Abstract : Freshwater reservoirs can be impacted by several hazardous substances through inputs from agricultural activity, sewage discharges, and groundwater leaching and runoff. The water quality assessment is very important for implementation of the monitoring and remediation programs to minimize the risk promoted by hazardous substances in aquatic ecosystems. Evaluation of the degree of contamination of aquatic environments must not take in account only its chemical characterization but it must be complemented with biological assays, which determine potential toxic effects and allows an integrated evaluation of its effects in populations and aquatic ecosystem communities. The application of this type of strategy has clear advantages allowing a general evaluation of the effects from all the water components, including those due to unknown substances and synergic, antagonistic, or additive effects. There are only a few studies that reported ecotoxicological acute end points, for the assessment of surface water quality, and the relationship among toxicity results and the anthropogenic pollution sources and the seasonal period. The aim of this study was to assess the ecotoxicological characterization of the surface water from Alqueva reservoir (South of Portugal) and to evaluate the influence of anthropogenic sources of pollution and their seasonal variation in its toxicity. The construction of Alqueva reservoir was recently finished (2002) and, to our knowledge, an ecotoxicological assessment of its surface water has not been performed. Because of that, no information is available on the possible impact of pollutants on the biota. The surface water toxicity was assessed using acute and chronic bioassays. The results are to be used for developing a monitoring program, including biological methods.

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A. Harada, K. Komori, N. Nakada, K. Kitamura and Y. Suzuki
Biological Effects of PPCPs on Aquatic Lives and Evaluation of River Waters Affected by Different Wastewater Treatment Levels
Water Science and Technology 2008 58, 8 1541-1546
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - drugs; chemicals - general; chemicals - desinfectants

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Abstract : The existence of pharmaceuticals and personal care products (PPCPs) in the water environment is an emerging problem. In this study, we investigated the toxicity of eleven PPCPs through bioassays on bacteria, algae, crustaceans, amphibians and protozoa, and compared the toxicology indexes with the concentration of PPCPs in river water for ecotoxiclogical risk evaluation. Toxicity of the eleven PPCPs was observed and the values of EC50 or LC50 were in the order of mg/L. A distinctive finding is that antibacterial triclosan affected all aquatic lives tested. The effects of PPCPs varied according to species of lives. Contamination from PPCPs was detected at observation stations on the river, and the range of concentration was in the order of ng/L far lower than the values of toxicity indexes EC50 or LC50. Ecotoxicological risks posed by PPCPs at the observation stations was evaluated using the concentration in the river water and the NOEC examined by AGI tests. The results revealed that three PPCPs, triclosan, clarithromycin, and azithromycin, posed an ecotoxiclogical risk in rivers where wastewater treatment systems are not yet well developed.

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P. Palma, C. Matos, M. Kuster, L. Ledo, I. Simoes, M. Lopez de Alda and D. Barcelo
Ecological and Ecotoxicological Responses to Water Pollution : a Case-study of the Temporary River Brejo do Carragão (South of Portugal)
From : SETAC Europe - 23rd Annual Meeting, Glasgow, Scotland, May 2013 - Book of Abstracts 2013
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : surface waters

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Abstract : Temporary rivers occur throughout the arid and semiarid regions that cover approximately a third of the world’s surface. They are defined as rivers that are normally dry, at least during an extended part of the year. They expand during wet period, through floods events, and contract and fragment during dry periods. These events connected to several environmental factors of the drainage basin (climatic, geological, and topographical) and anthropogenic actions induce the disruption of the dynamics of the river, with effects on the physical and chemical composition of the water, on the aquatic communities, and on the amount of material transferred along the basin. The temporary riverof Brejodo Carragão is located in MiraRiver Basin(South of Portugal), in a semi-arid region where the agricultural is one of the main activities. Water samples were analysed considering: the chemical profile (pH, temperature, electrical conductivity, chloride, phosphorus, kjeldahl nitrogen, ammonia, nitrite, nitrate, alkalinity, hardness, biochemical oxygen demand, chemical oxygen demand, iron, manganese and arsenic); the occurrence of medium to polar pesticides; the evaluation of the benthic macroinvertebrates communities; and the ecotoxicity assessment through bioindicators of different trophic levels (luminescence inhibition of Vibrio fischeri, 24-hmortality test with Thamnocephalus platyurus, 48-h mortality and reproduction assays with Daphnia magna). Considering the physical-chemical analysis the results showed high levels of organic matter and nutrients, mainly in the dry period. The main pesticides detected were terbuthylazine, 2-methyl-chlorophenoxyacetic (MCPA), bentazone, mecoprop and metolachlor. The benthic macroinvertebrates analysis showed low levels of communities’ diversity, with populations dominated by groups resistant to the organic pollution, mainly the Chironomidae family. On the other hand, the results from the ecotoxicological assessment showed that there was a marked decreased of the D. magna reproduction when exposed to water samples; this fact may be correlated to the amounts of pesticides quantified. Thus, this integrative approach highlights the importance of a biological evaluation that covers the interactions between the ecosystem´s species, as well as the detection of different types of pollution.

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G.A. Plaza, G. Nalecz-Jawecki, O. Pinyakong, P. Illmer and R. Margesin
Ecotoxicological and Microbiological Characterization of Soils from Heavy-Metal- and Hydrocarbon-Contaminated Sites
Environmental Monitoring and Assessment 2010 163, 1-4 477-488
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - metals; chemicals - hydrocarbons; soils

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Abstract : The aims of this study were to characterize soils from industrial sites by combining physicochemical, microbiological, and ecotoxicological parameters and to assess the suitability of these assays for evaluation of contaminated sites and ecological risk assessment. The soil samples were taken from long-term contaminated sites containing high amounts of heavy metals (sites 1 and 2) or petroleum hydrocarbons (site 3) located in the upper Silesia Industrial Region in southern Poland. Due to soil heterogeneity, large differences between all investigated parameters were measured. Microbiological properties revealed the presence of high numbers of viable hetrotrophic microorganisms. Soil enzyme activities were considerably reduced or could not be detected in contaminated soils. Activities involved in N turnover (N mineralization and nitrification) were significantly (P < 0.05) higher in samples from the metal-contaminated sites than in samples from the hydrocarbon-contaminated site, whereas the opposite was observed for phosphatase activity. The Microtox test system appeared to be the most appropriate to detect toxicity and significant differences in toxicity between the three sites. The Ostracodtoxkit test was the most appropriate test system to detect toxicity in the hydrocarbon-contaminated soil samples. Correlation analysis between principal components (obtained from factor analysis) determined for physicochemical, microbiological, and ecotoxicological soil properties demonstrated the impact of total and water-extractable contents of heavy metals on toxicity.

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I. Blinova, L. Bityukova, K. Kasemets, A. Ivask, A. Käkinen, I. Kurvet, O. Bondarenko, L. Kanarbik, M. Sihtmäe, V. Aruoja, H. Schvede and A. Kahru
Environmental Hazard of Oil Shale Combustion Fly Ash
Journal of Hazardous Materials 2012 229-230 192-200
Toxkits : Daphtoxkit F^TM magna; Phytotoxkit
Test Species : Daphnia magna; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : oil shale; shale oil; fly ashes; plant tests; soils

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Abstract : The combined chemical and ecotoxicological characterization of oil shale combustion fly ash was performed. Ash was sampled from the most distant point of the ash-separation systems of the Balti and Eesti Thermal Power Plants in North-Eastern Estonia. The fly ash proved potentially hazardous for tested aquatic organisms and high alkalinity of the leachates (pH>10) is apparently the key factor determining its toxicity. The leachates were not genotoxic in the Ames assay. Also, the analysis showed that despite long-term intensive oil-shale combustion accompanied by considerable fly ash emissions has not led to significant soil contamination by hazardous trace elements in North-Eastern Estonia. Comparative study of the fly ash originating from the ‘new’ circulating fluidized bed (CFB) combustion technology and the ‘old’ pulverized-fired (PF) one showed that CFB fly ash was less toxic than PF fly ash. Thus, complete transfer to the ‘new’ technology will reduce (i) atmospheric emission of hazardous trace elements and (ii) fly ash toxicity to aquatic organisms as compared with the ‘old’ technology.

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I. Blinova, J. Niskanen, P. Kajankari, L. Kanarbik, A. Käkinen, H. Tenhu, O.-P. Penttinen and A. Kahru
Toxicity of two Types of Silver Nanoparticles to Aquatic Crustaceans Daphnia magna and Thamnocephalus platyurus
Environmental Science and Pollution Research International 2013 20,5 3456-3463
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics :

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Abstract : Although silver nanoparticles (NPs) are increasingly used in various consumer products and produced in industrial scale, information on harmful effects of nanosilver to environmentally relevant organisms is still scarce. This paper studies the adverse effects of silver NPs to two aquatic crustaceans, Daphnia magna and Thamnocephalus platyurus. For that, silver NPs were synthesized where Ag is covalently attached to poly(vinylpyrrolidone) (PVP). In parallel, the toxicity of collargol (protein-coated nanosilver) and AgNO3 was analyzed. Both types of silver NPs were highly toxic to both crustaceans: the EC50 values in artificial freshwater were 15–17 ppb for D. magna and 20–27 ppb for T. platyurus. The natural water (five different waters with dissolved organic carbon from 5 to 35 mg C/L were studied) mitigated the toxic effect of studied silver compounds up to 8-fold compared with artificial freshwater. The toxicity of silver NPs in all test media was up to 10-fold lower than that of soluble silver salt, AgNO3. The pattern of the toxic response of both crustacean species to the silver compounds was almost similar in artificial freshwater and in natural waters. The chronic 21-day toxicity of silver NPs to D. magna in natural water was at the part-per-billion level, and adult mortality was more sensitive toxicity test endpoint than the reproduction (the number of offspring per adult).

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Y. Mamindy-Pajany, B. Hamer, M. Roméo, F. Géret, F. Galgani, E. Durmiši, C. Hurel and N. Marmie
The Toxicity of Composted Sediments from Mediterranean Ports Evaluated by Several Bioassays
Chemosphere 2011 82, 3 362-369
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; plant tests

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Abstract : This work investigates the ecotoxicological evaluation of contaminated dredged sediments from French Mediterranean navy harbour (A), commercial port (B) and two composite specimens (C) and (D) coming from the mixture of A and B with other port sediments. The toxicity of elutriates from these sediments is estimated using embryo-toxicity test, Microtox® solid phase test, LuminoTox, phytotoxicity tests and genotoxicity test. Bioassay responses are not clearly correlated with chemical contamination in the whole sediment and vary as a function of tested organisms. The highest contaminated samples (A and C) are almost always more toxic than the less contaminated samples (B and D). Among composite sediments, the mixture effect with other sediments is not efficient to decrease toxicity in sample C, suggesting that other parameters influence toxicity level such as particle size or organic matter content. These parameters should be taken into consideration in order to improve the efficiency of the mixture process and produce composite sediments with low toxicity.

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J. Kalka, D. Marciocha, J. Turek-Szytow and J. Surmacz-Górska
Subacute Effects of Selected Antiparasitic Drugs
16 th Biennial International Symposium for Toxicity Assessment, February 2013, Cape Town South Africa
Toxkits :
Test Species :
Topics :

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Abstract :
Introduction : Exposure of living organisms to xenobiotic factors can lead to the changes including one generation damages or to the long-term effects. Veterinary pharmaceuticals are micropollutants with increasing importance to the wildlife protection and conservation. The aim of the study was to investigate the influence of two veterinary pharmaceuticals on stability of biological membranes in a beneficial organism, Eisenia fetida

Methods : Acute toxicity test of Albendazole (ALB) and Levamisole (LEV) was performed according to OECD 207 This allowed to select subacute range of drugs concentrations for further testing.
Eisenia fetida (30 worms) was exposed in the laboratory to a range of concentrations. The lysosomal membrane stability was determined as the number of cells exhibiting dye leakage from the lysosomes to the cytosol, compared with the number of cells with unstained cytosols (no leakage).

Results : The lysosomal membrane stability of coelomocytes, measured as neutral-red retention time (NRR-time), was reduced at soil pharmaceutical concentrations lower than those affecting survival and biomass growth and demonstrated a dose–response relationship. After the recovery period of 7 and 14 days membrane stability of lysosomes was increased, but it was still significantly reduced compared to the control.

Conclusions/ Discussion : The NRR-time reflects the bioactive fraction of pharmaceuticals in the soil. The bioactive fraction is a part of xenobiotic that interacts with metabolic processes. For organic compounds, the bioactive fraction may also be a metabolic product of the original compound.

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I. Blinova, L. Kanarbik, M. Sihtmäe and A. Kahru
Evaluation of the Potential Hazard of Shale Oils for Aquatic Community
16 th Biennial International Symposium for Toxicity Assessment, February 2013, Cape Town South Africa
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : shale oil, oil shale

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Abstract :
Background, Gap and Aims : Estonia is the leading producer of synthetic crude oil (shale fuel oil) derivable from oil shale by retorting. The main goal of the current investigation was to evaluate the potential hazard of Estonian shale fuel oils to aquatic communities. However, it is very difficult to extrapolate laboratory toxicity data to real water ecosystem. In case of hazard assessment of oil, the protocol used for preparation of Water Accommodated Fractions (WAF) may significantly influence the results of toxicity testing.

Methods : Two most widely used shale fuel oil fractions (“VKG sweet” and “VKG D”) were studied using a combined chemical and ecotoxicological approach. Different leaching ratios (1:10 and 1:10000) were applied for the WAF preparation. The WAF was prepared from pure oils and from soils spiked with shale oils. Toxicity assays involved two crustaceans (Daphnia magna and Thamnocephalus platyurus) and bacteria Vibrio fischeri. The concentrations of the hydrocarbons and PAHs in the eluates were measured in parallel.

Results : The shale fuel oil with lower viscosity (“VKG sweet”) was slightly more toxic than the shale fuel oil with higher viscosity (“VKG D”). Toxicity of (1:10) eluates was nearly 100-fold higher than that of (1:10000) eluates. The toxicity of WAFs prepared from the soil samples spiked with the same volume of shale oils depended on the soil type.

Conclusions/ Discussion : Both investigated shale oil fractions proved very hazardous to aquatic organisms. However, the different results obtained with different WAF protocols once more raises the question on interpretation of the laboratory toxicity data for the prediction of ecological effects.

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A. Kungolos, V. Tsiridis, M. Petala and E. Darakas
Ecotoxicity of Hexavalent Chromium Contained in the Leachates of a Lignite Fly Ash
16 th Biennial International Symposium for Toxicity Assessment, February 2013, Cape Town South Africa
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - metals; fly ashes

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Abstract :
Background, Gap and Aims : Fly ash is produced in huge amounts by coal incineration process for power production. Although there are several ways for fly ash utilization, the vast majority of generated fly ash is disposed as a solid waste. The release of toxic compounds from solid to liquid phase, when fly ash comes into contact with water during its disposal or utilization, is of major environmental concern. Hexavalent chromium [Cr(VI)] can be included in the constituents that may leach out from fly ash during leaching.

Methods : In the present work the ecotoxicity of Cr(VI) released from a lignite fly ash was evaluated using various leaching procedures [leaching under various liquid to solid ratios (L/S), leaching stages, and pH values] and a battery of ecotoxicity tests (acute Daphnia magna and Vibrio fischeri tests).

Results : The concentration of Cr(VI) in the leachates obtained under leaching of fly ash at pH 7 for various L/S ratios decreased with the increase of L/S, from 0.712 mg/L for L/S= 10 L/kg to 0.096 mg/L for L/S= 200 mg/L, while leaching at various pH values showed a maximum release of Cr(IV) from fly ash (14.87 mg/kg fly ash) under leaching at pH 8. The leachates obtained under leaching at pH 7 for various L/S ratios exhibited a relatively high toxic effect toward D. magna, having a TU= 28.6 (23.8–35.7) at L/S= 10 L/kg, while V. fischeri was less sensitive to the action of the leachates. Furthermore, a significant correlation was observed between Cr(VI) concentration and the toxicity of the leachates toward D. magna (r= 0.961, p<0.01).

Conclusions/Discussion : The study showed that L/S ratios and pH affect Cr(VI) leaching from fly ash and that these parameters can affect the ecotoxicity of the leachate.

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C. Blaise and F. Gagné
Aquatic Ecotoxicology: What Has Been Accomplished and What Lies Ahead ?
16 th Biennial International Symposium for Toxicity Assessment, February 2013, Cape Town South Africa
Toxkits :
Test Species :
Topics :

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Abstract : Background : Our recent history shows that degradation of aquatic ecosystems essentially stems from industrialization, urbanization and increasing human populations. While environmental problems began to appear as a result of the first industrial boom of our era in the late 19th century, contamination of receiving waters appears to be a continual process owing to new economies and technologies developing at the planetary scale. Given the diversity of issues, problems and challenges facing water quality today because of complex waste and chemical discharges into waterways, ecotoxicology has blossomed with time into a more mature discipline of the environmental sciences. Its two fundamental pillars, bioassays and biomarkers, have become essential tools that allow the determination of numerous and versatile effects measurements. In this ISTA 16 presentation, we demonstrate some of the ways in which they have been applied and how they have evolved over the past decades to appraise the ecotoxicity of contaminants impacting aquatic systems. Examples discussed are largely reflective of work conducted in the Saint-Lawrence Centre, a research institute of Environment Canada located in Montréal, Canada.
From their early single use in a target species to measure one effect, bioassays and biomarkers are now employed in batteries across several taxonomic groups to determine multiple endpoints. Indices that integrate and reduce toxicity data into single numerical values have also proven useful to assess water and sediment quality. In terms of accomplishments, development and application of bioassays and biomarkers have enabled overall acquired knowledge to contribute markedly to the protection and conservation of water and biodiversity. Indeed, success stories include improvement of industrial effluent quality leading to beluga whale population recovery in the Saint-Lawrence River, enhanced sample throughput via small-scale technologies, increased discernment on the hazard potential posed by emerging classes of chemicals, as well as others. While several battles related to aquatic pollution have been won in the past, the war being waged against ecotoxicity rages on. Ecotoxicology is notably confronted with diverse issues some of which entail toxicity of mixtures, contaminant/climate change as well as contaminant/parasite relationships, and bioaccumulation of chemicals up the food chain. To cope with such challenges, ecotoxicology will have to bank on new tools (e.g., toxicogenomics, bio-informatics, modeling) and become more interdisciplinary by taking into account knowledge provided by other disciplines (e.g., ecology, chemistry, climatology, microbiology) in order to more fully understand and adequately interpret hazard stemming from a myriad of chemical contaminants continually released to aquatic environments worldwide. This will be paramount to supply regulators and legislators with the sound and scientifically valid information needed in order to mitigate environmental degradation.

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L. Manusadžianas, J. Darginaviciene, B. Gylyte, S. Jurkoniene, A. Krevš, A. Kucinskiene, R. Mackinaite, K. Sadauskas, J. Sendžikaite, R. Pakalnis, and R. Vitkus
Ecotoxicity Effects Triggered in Aquatic Organisms by Invasive Acer negundo and Native Alnus glutinosa Leaf Leachates Obtained During Their Aerobic Decomposition
16 th Biennial International Symposium for Toxicity Assessment, February 2013, Cape Town South Africa
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : leachates

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Abstract : Background, Gap and Aims : Interrelation between terrestrial and aquatic ecosystems has recently been investigated in terms of DOM influence originated from tree leaves litter to hydrobiota, however, a little data, if any, is available on invasive species. Considering coastal and littoral zones, we explored the impacts of invasive to Lithuania boxelder maple (Acer negundo) and native black alder (Alnus glutinosa) fallen leaf leachates during their biodegradation by evaluating toxicity responses of selected organisms.

Methods : The leachates were obtained during 90-day decomposition of leaves in lake water (100g dw/L) at constant O2 and 13–17°C. Leachate physical-chemical and microbiological analyses were performed. Toxicological endpoints (50% effects) comprised charophyte N.obtusa 8 day lethality, 90-min cell depolarization, 60-min in vitro microsomal H+-ATPase activity inhibition, 48-h D.magna and 24-h T.platyurus lethality and 15-min V.fischeri luminescence inhibition.

Results : Under oxygen conditions, boxelder leaves decomposed faster than those of alder due to mineralization driven by bacteria and fungi. For boxelder leaf leachate, toxicity to target organisms was evident within 12–60 days, while for alder leaf leachates within 30–60 days. On 30th day, both leachates were more toxic to algae than to invertebrates and not toxic to V.fischeri. Toxicity of the leachates to all test-organisms disappeared after 90 days. A biomarker of H+-ATPase activity showed slightly higher toxicity of A. glutinosa leaf extracts than those of A.negundo, IC50 = 231 ± 27 and 366 ± 60 mg/L DOC.

Conclusions/ Discussion : Under aerobic conditions, biodegradation of leaves was accelerated by fungi, contrary to anaerobic conditions when bacteria played a key role (Krevš et al., 2013). In both cases decomposition of boxelder leaves was faster than that of alder, and substances leached out from the leaves into water body, in particular invasive A.negundo, can be an important environmental factor affecting chemical and microbiological situation as well as aquatic organisms in the littoral zone.

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M.J. Villarroel, E. Andreu-Moliner, E. Sancho and M.D. Ferrando
Toxicological Assessment of Prochloraz by Comparing Acute and Subacute Endpoints of Toxicity in Brachionus calyciflorus and Daphnia magna
CICTA 2013 - Valencia, Spain - 1-4 July, 2013
Toxkits : Daphtoxkit F^TM magna; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna
Topics : chemicals - pesticides

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Abstract : Prochloraz is an imidazole fungicide with a high bioaccumulation potential (BCF 200). It is widely used for controlling Pyricularia oryzae in transplanted and direct-seed rice fields. Cladocerans, especially Daphnia magna, are among the most favorable test animals in aquatic toxicology. It has historically been used in toxicity testing establishing a large data base of this species.
Rotifers are ideal bioassay animals for acute and subacute toxicity tests because of their sensitivity, short generation time and genetic uniformity. The main purpose of the current study was to assess firstly, the acute effects of Prochloraz on the rotifer (Brachionus calyciflorus) and the cladoceran (Daphnia magna) to compare their relative sensitivities to this fungicide. Secondly, to assess the impact of the toxicant on feeding behavior of both species as earlier indicator of fungicide toxicity. Prochloraz LC50 values for D. magna in our experimental conditions were 18.78 mg L-1(15.45-22.90 mg L-1) and 9.29 mg L-1(8.67-9.82 mg L-1) at 24 and 48 h, respectively. An estimation of the LOEC value at 24 h would be 10.0 mg L-1. Prochloraz LC50 value for B. calyciflorus in our experimental conditions was 7.54 mg L-1(4.98-12.01 mg L-1) at 24h of exposure. NOEC and LOEC values for this parameter were 1.25 and 2.50 mg L- 1, respectively.
Subacute short-term tests (Feeding study) was assessed during 5 h exposure to the fungicide.
Test concentrations were selected from acute toxicity results. Filtration rates of D. magna were significantly (p<0.001) reduced from 274.22±18.17 µL per individual per h (control value) to 194.51±24.41 and 107.05±23.44 µL per individual per h at 0.23 and 0.38 mg L-1, respectively.
Ingestion rates were similarly reduced above a 40% and a 65% respect to the controls (109.39±4.46 x103 cell per individual per h) at the same concentrations. Filtration and ingestion rates of rotifer control animals were 6.06±0.54 µL per animal per h and 2.35±0.11 x103 cell per animal per h, respectively. Concentrations above 0.054 mg L-1 caused significant decreases in the filtration and ingestion rates. Filtration rate was reduced from 6.06 µL per animal per h (control) to 3.84, 2.63 and 1.04 which is a reduction of 36, 57 and 83% at Prochloraz concentrations of 0.069, 0.094 and 0.377 mg L-1, respectively. Similarly, the ingestion rates were reduced from 2.35 x103 cells per animal per h in the controls to 1.63, 1.18 and 0.56 cell per animal per h at the same Prochloraz concentrations. Acute/subacute/chronic ratios play an important role in the development of water quality standards. Regarding on the acute/subacute short-term study done in the present study, B. calyciflorus was earlier affected by Prochloraz than D. magna.

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M.M. Gladkova and V.A. Terekhova
Phytotoxicity of Nano-TiO2 and Effect of Humus Preparation
22nd Annual Meeting SETAC EUROPE, Berlin, Germany; 20-24 May 2012 - Book of Abstracts 269-270
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - nanoparticles; plant tests; soils

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Abstract : The objective of this study was to determine the bioactivity of nano-TiO2 and to assess their toxicity in presence of humic substances (HS) which are natural components of soil necessary for higher plants growth.
In this work we applied metal-containing nanomaterials - nanodioxide titanium (nano-TiO2), which are characterized <75 nm in size of particles in 10 weights % of water suspension, manufactured in the company “Sigma-Aldrich”, USA. Nano-TiO2 represents oxide the titanium (IV), the mix of two known crystal modifications of dioxide titanium - anatase and rutile.
Among HS we have chosen “Pow-Humus”(Le-PhK) (K-humate, originated from leonardite), manufactured by German firm ‘Humintech’.
Concentration of nano-TiO2 was varied in range 5-500 mg/l; concentration of humate Le-PhK was 5 mg/l in water medium.
Bioactivity of preparations was examined by early seed root elongation assessment related to Brassica juncea L. as a test-organism. We have used a modification of the method “Phytotoxkit - Seed germination and early root growth”,developed by Belgian scientists (http://www.microbiotests.be). The probe is considered toxic if the deviation from the control of average roots length is 20 and more %.
Depressing effect of nano-TiO2 on early root growth (4 days of germination) was marked. The inhibition of root growth was showed already at the low concentration (5 mg/l).The most effect was shown at the higher concentration: probe with nano-TiO2 500 mg/l was toxic because the deviation of average roots length was more than 25 % from control.
Inhibitory effect of nano-TiO2 was reduced in presence of humate Le-PhK 5 mg/l. Samples with nano-TiO2 500 mg/l after humate application was qualified as nontoxic because the deviation of average roots length was only 14 % from control.
The obtained data has shown that: 1) higher plants are sensitive to the nano-TiO2 presence in growing medium 2); the degree toxicity of nano-TiO2 depends on the concentration of nanoparticles; 3) humus substances eliminate the toxic effect of nano-TiO2 in water suspension which inhibits root growth.
Further research is required for the evaluation of humus substances influence on the nanomaterials which are more and more widely distributed in the environments including the natural soils [1; 2].

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I. Nogueira, I. Lopes, T. Rocha-Santos, A.C. Duarte, M. Rasteiro, F. Goncalves, A. Soares and R. Pereira
Ecotoxicological Effects of the Nanoparticles NiO (100nm and 10-20nm), Fe2O3 and TiO2 on Aquatic Organisms
22nd Annual Meeting SETAC EUROPE, Berlin, Germany; 20-24 May 2012 - Book of Abstracts 428
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - nanoparticles

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Abstract : The rapid development of nanotechnology and the increasing use of nanomaterials (NM) have been raising concerns about their fate and potential effects in the environment, since some of them could be used for remediation purposes. Despite emerging literature addressing the biological effects of NMs to aquatic organisms the existing information is still scarce and contradictory. Therefore, aimed in selecting NMs for the treatment of organic effluents, we assessed the potential toxicity of four NMs NiO (100nm and 10-20nm), Fe2O3 (d=40-130nm, c=250-600nm) and TiO2 (<25nm), on a battery of assays using aquatic organisms from different trophic levels : the bacterium Vibrio fisheri (Microtox^®) the microalgae Pseudokirchneriella subcapitata (growth inhibition test), the aquatic macrophyte Lemna minor (growth inhibition test), the cladoceran Daphnia magna (acute and chronic test) and two additional marine organisms, rotifer Brachionus plicatilis (Rotoxkit) and crustacean Artemia salina (Artoxkit). Suspensions of each NM, prepared on the different test mediums and at different concentrations tested, were characterized by Dynamic Light Scattering (DLS). For the assays with marine species (V. fischeri included) no toxicity was observed for any of the compounds tested. In what concerns the growth inhibition test with P. subcapitata no significant inhibitory effects were observed for Fe2O3, contrary to the outcome for the other NMs, being both nano-NiO the most toxic, with EC50 values of 8.24 mg L-1 for NiO 100nm and 15.15 mg L-1 for NiO 10-20nm. Regarding the results with L. minor no significant effects were observed for TiO2 and Fe2O3, whilst for NiO 100nm and 10-20nm the LOEC values were 5.24 and 8.19 mg L-1, respectively. In the acute test with D. magna the EC50 ranged from 9.74 mg L-1 (24h) and 14.59 mg L-1 (48h) for both nano-NiO of 100nm and 10-20nm. In the 21-day reproduction test the LOEC values determined for the same NM ranged from 0.045 to 0.14 mg L-1. Regarding, nano-TiO2 no acute toxic effects were observed but in the chronic test a LOEC value of 20 mg L-1 was recorded. According to all the results the nano-NiO (100nm) was the most toxic for the aquatic species tested. From the results of this study we can establish a decreasing order of toxicity NiO(100)>NiO(10-20)>TiO2>Fe2O3 for the tested NMs and the most sensitive organism was D. magna. According to the results, Fe2O3 NM seems to be the one with less risks to the receiving aquatic systems.

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O. Andreu-Sánchez, E. Sancho and E. Andreu-Moliner
Comparative Toxicity of the Fungicide Prochloraz between Estuarine and Freshwater Organisms
CICTA 2013, Valencia, Spain; 1-4 July 2013
Toxkits : Artoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Thamnocephalus platyurus
Topics : chemicals - pesticides

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Abstract : Albufera Lake (Eastern Spain) presents two differentiated aquatic environments: estuarine and freshwater. Estuarine area is due to influence of seawater intrusion and freshwater area is fed by from irrigation channels and ditches. Prochloraz is a fungicide widely used as alternative to tryicyclazole and carbendazim treatments in paddy fields from Eastern Spain among the Spanish Mediterranean wetlands to avoid and treat rice blast disease caused by Pyricularia oryzae during July and early August. During this period spraying operations with commercial Octagon E® (45% a.i.) are conducted by farmers. Its water contamination varies significantly with the season and year; however, the type of water seems not to influence its distribution within the watershed. Information about the environmental fate of this fungicide is scarce. Laboratory studies indicate that it is quite persistent in the soil-water system (approximately 6 months in the field) it does not readily hydrolyze in the environment and it is stable at pH=7 @ 20 ºC without volatilization.

To assess the effect of pesticides on estuarine and freshwater environments, it is necessary to compare representative organisms for both environments, marine/estuarine and freshwater. For this reason, we have selected Artoxkit M^TM with Artemia franciscana as marine-estuarine organism and Thamnotoxkit F^TM with Thamnocephalus platyurus as freshwater organism. Toxkits™ microbiotests are based in dormant or immobilized eggs (cryptoforms), which are hatched to obtain live organisms to perform the tests. All bioassays were performed according to the Standard Operational Procedure (SOP) provided in each Toxkit^TM.

Two commercial microbiotests, Artoxkit M^TM and Thamnotoxkit F^TM based in 2 species from different aquatic environment (estuarine and freshwater respectively) has been used to evaluate the toxicity of Prochloraz, which is widely used as fungicide in rice cropping areas like Albufera Natural Park (Valencia, Spain).

The organisms were exposed to different concentrations of a.i. Prochloraz (Purity >99.5%, purchased from Riedel-de-Haën) for a period of 24 hours to evaluate acute toxicity. Final assayed concentrations of Prochloraz were: 100, 50, 25, 12.5 and 6.25 mg/L to Thamnoxkit F™ and 50, 25, 12.5, 6.25 and 3.12 mg/L to Artoxkit M^TM, respectively. LC50-24h values were obtained using a Probit regression included in SPSS+ v.15 Statistical Software.

Results show that Thamnocephalus platyurus (LC50-24h = 12.9 mg/L. I.C.95%= 11.5 - 14.3 mg/L) was more sensitive than Artemia franciscana (LC50-24h = 40.7 mg/L. I.C.95% = 36.4 – 45.1 mg/L). The difference of sensibility justifies the need to use a battery of microbiotest. According to 93/67/EEC European Directive, this compound is classified like Harmful (H) (Harmful: EC50=10-100mg/L). The use of a dual battery with Artemia franciscana and Thamnocephalus platyurus is a simple tool to know the potential toxicity of pesticides used in coastal irrigated areas.
See corresponding Poster

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N. Roig, Ö Kiliçoglu, J. Sierra, M. Nadal, M. Schuhmacher and J.L. Domingo
An Integrated Study of Metals Behaviour in Low Ebro River.
CICTA 2013, Valencia, Spain; 1-4 July 2013
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : sediments, sediment pore waters; sediment and soil contact test

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Abstract : Metals are good indicators of anthropogenic pollution of aquatic ecosystems. These elements have the capability to be present in water and can be accumulated for years in sediments, being a potential sink and source of contaminants to the water column. Although all the legislation refers to the analysis of total concentrations of contaminants in freshwater environment, the real risk for the organisms is more related to the bioavailable fractions. The study of the bioavailable fraction of the pollutants is still a controversy subject. The aims of the present study are to (1) assess the bioavailability of some potentially inorganic toxic elements in the low Ebro river (NE Catalonia, Spain), and (2) evaluate the relationship between stream metal concentrations, sediments ecotoxicity and the structure of the macroinvertebrate community. For this purpose, five sampling sites within the low Ebro river near to a chlor-alkali plant factory and a nuclear power plant were selected. In each sampling reach, composite samples of sediment and aquatic macroivnertebrates were collected. Regarding sediments, the concentration of total heavy metals was analyzed and metal bioavailability was calculated by a sequential extraction according to the Community Bureau of Reference (BCR) method. Aquatic macroinvertebrates were sampled in order to estimate the IBMWP index, Shannon diversity and the EPT index at family level. The ecotoxicity of sediment samples was evaluated in Vibrio fischeri by Microtox Acute Test and Daphnia magna toxicity screening test in aqueous and organic extracts, pore water, as well as solid phase test. Moreover, to distinguish the potentially toxic fraction associated to heavy metal burden of sediments, an analysis of acid-volatile sulphide (AVS) and simultaneously extracted metals (SEM) was performed. This study demonstrates that the integration of metal analysis (total metals and potential bioavailable fraction), ecotoxicity assays and biological indices could be crucial to understand the hazard of metals in aquatic ecosystems, especially, in freshwater sediments.
See corresponding Poster

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S. Magrisso and S. Belkin
Assessment of River Health by Combined Microscale Toxicity Testing and Chemical Analysis
Atmospheric and Biological Environmental Monitoring 2009 241-249
Toxkits : Daphtoxkit F^TM pulex; Thamnotoxkit F^TM
Test Species : Daphnia pulex; Thamnocephalus platyurus
Topics : surface waters; waste waters

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Abstract : A battery of commercially available toxicity bioassays was applied to assess the quality status of two polluted coastal rivers in Israel: the Yarkon River, polluted with treated domestic wastewater, and the Kishon River, that at the time of sampling served as a conduit of industrial wastewaters. Samples from the latter displayed a much higher toxicity; in both cases, sediment toxicity was considerably higher than that of the water. Highest sensitivities were exhibited by assays based on the micro-crustaceans Daphnia pulex and Thamnocephalus platyurus. A new index (total relative toxicity) is presented for river health assessment, which integrates and normalizes the toxicity of all identified pollutants. Calculated total relative toxicity values were in excellent agreement with the biological toxicity tests. We highlight the significance of toxicity bioassays as an essential component of any river monitoring program, both for the assessment of river health and for following the progress of remediation schemes. For this purpose, it is proposed that a river-specific panel of toxicity bioassays is selected, representing different trophic levels and taxonomic complexity.

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D. Veneiri, A. Rouvalis and J. Iliopoulou-Georgudaki
Microbial and Toxic Evaluation of Raw and Treated Olive Oil Mill Wastewaters
Journal of Chemical Technology and Biotechnology 2010 85, 10 1380-1388
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics :

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Abstract : BACKGROUND : Olive oil mill wastewaters (OMW) from a three-phase system were treated in a laboratory-scale continuous stirred tank reactor (CSTR) for hydrogen production. Microbiological and physicochemical characterizations of raw and anaerobically treated olive oil mill effluents were performed. Toxicity assessment was also conducted in order to determine the impact of residual waters on the aquatic environment.

RESULTS : Pseudomonas aeruginosa that has the ability to utilize phenolic compounds was isolated in raw OMW, along with several gram-negative bacteria (Serratia odorifera, Enterobacter cloacae and Aeromonas hydrophila). The dominant species in the untreated effluent appears to be Enterobacter cloacae, whereas in the treated effluent it is Citrobacter braakii. Phenols, pH, NO^-2, total dissolved solids (TDS) and conductivity were the main parameters related to the observed toxicity.

CONCLUSION : A significant differentiation in the microbiological flora was detected between treated and untreated effluents whereas the microbial communities were correlated to the toxicity test results, pointing to the need for further research regarding identification of the microbial flora. This can improve the treatment processes to optimize management of OMW for environmentally safe discharge.

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I. Czerniawska-Kusza, G. Kusza and M. Duzynski
Effect of Deicing Salts on Urban Soils and Health Status of Roadside Trees in the Opole Region
Environmental Toxicology 2004 19 296-304
Toxkits : Protoxkit F^TM
Test Species : Tetrahymena thermophila
Topics : soils

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Abstract : This article reports on a study whose aim was to evaluate the impact of snow removal salts on urban soil properties and the health of roadside trees. The evaluation was done by chemical analyses of soil samples and plant matter combined with toxicity testing, performed with a Protoxkit F^TM, a protozoan microbiotest. Samples were collected at 45 locations on three main roads in the town of Opole (Poland). The roads differed in the snow removal technology and amount of chemical substances (mostly NaCl) used on them during the winter. The study showed that when soil was exposed to a high level of NaCl, it tended to be more alkaline and also exhibited increased content of Na(+) and Cl(-). The toxic effects of the soil extract on protozoa appeared at 26.0 mg Na(+)/100 g soil dry mass (s.d.m.) and 12.0 mg Cl(-)/100 g s.d.m., whereas salt injury symptoms (chlorosis and necrosis of the edge of leaf blades) appeared at 13.2 mg Na(+)/100 g s.d.m. and 3.9 mg Cl(-)/100 g s.d.m., becoming more severe at 26.0 mg Na(+)/100 g s.d.m. and 12.0 mg Cl(-)/100 g s.d.m. because of extensive necrosis and defoliation. The lysimetric experiment, which was used to test soil samples collected from the city park area, indicated that salt plays a significant role in the pollution of soil in urban areas, with the least toxic salt being CaCl(2).

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M. Kopec, K. Gondek and A. Baran
Assessment of Respiration Activity and Ecotoxicity of Composts Containing Biopolymers
Ecotoxicology and Environmental Safety 2013 89 137-142
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : composts; chemicals - organic compounds; plant tests; soils

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Abstract : The research was conducted to determine if introducing biodegradable polymer materials to the composting process would affect selected biological properties of mature compost. Determination of biological properties of composts composed of testing their respiration activity and toxicity. Respiration activity was measured in material from the composting process by means of OxiTop Control measuring system. The ecotoxicity of composts was estimated by means of a set of biotests composed of three microbiotests using five test organisms. Introduction of polymer materials caused a decrease in respiration activity of mature compost. Similar dependencies as in the case of mass loss were registered. Compost to which a biodegradable polymer with the highest content of starch was added revealed the smallest difference in comparison with organic material composted without polymers. Lower content of starch in a polymer caused lower respiration activity of composts, whereas microorganism vaccine might have accelerated maturing of composts, thus contributing to the smallest respiration of compost. In composts containing biopolymers the following were observed : an increase in germination inhibition--2.5 times, roots growth inhibition--1.8 times, growth inhibition of Heterocypris incongruens--four times and luminescence inhibition of Vibrio fischeri--1.6 times in comparison with the control (compost K1). Composts containing biopolymers were classified as toxicity class III, whereas the compost without polymer addition as class II.

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G. Maisto, S. Manzo, F. De Nicola, R. Carotenuto, A. Rocco and A. Alfani A
Assessment of the Effects of Cr, Cu, Ni and Pb Soil Contamination by Ecotoxicological Tests
Journal of Environmental Monitoring 2011 13, 11 3049-3056
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Phytotestkit; Phytotoxkit
Test Species : Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; chemicals - metals; plant tests

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Abstract : This study aimed to assess soil quality by chemical and ecotoxicological investigations and to check the correspondence between soil metal concentrations and ecotoxicity. For these purposes, surface soils collected at four adjacent roadside urban parks and at a former industrial area were characterized for C/N, organic matter content, texture, and pH. Cr, Cu, Ni and Pb, chosen among the most representative soil metal contaminants, were measured as total content and as available and water soluble fractions. In addition, the total concentrations of the investigated metals were used to calculate two chemical indices : the contamination and the potential ecological risk factors. The toxicity of the investigated soils was evaluated by an ecotoxicity test battery carried out on both soil samples (Vibrio fischeri, Heterocypris incongruens and Sinapis alba) and elutriates (Vibrio fischeri, Daphnia magna and Selenastrum capricornutum). The findings, both by the chemical and ecotoxicological approaches, would suggest that the soils with high metal contamination pose ecological risks. On the other hand, moderately metal contaminated soils did not exclude soil ecotoxicity. In fact, toxic effects were also highlighted in soils with low metal content, toxicity being affected by metal availability and soil characteristics. Moreover, the results suggest the importance of using a battery of tests to assess soil ecotoxicity.

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B. Adomas and A. I. Piotrowicz-Cieslak
Yellow Lupin is a Good Bioindicator of Soil Contamination with Sulfamethazine
Proceedings of the 12th International Lupin Conference, 14-18 Sept. 2008, Fremantle, Western Australia 2008 362-367
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - drugs; plant tests; soils

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Abstract : Sulfamethazine is an antimicrobial sulphur drug used to treat big animals suffering from lung and digestive track diseases. The aim of the current study was to evaluate the suitability of selected leguminous plants as a bioindicators of sulfamethazine contamination in the soil. Pea, lentil, soybean, adzuki bean, alfalfa and yellow lupin seeds were germinated for six days using PHYTOTOXKIT (MicroBioTests Inc., Belgium). The soil was watered with distilled water supplemented with different concentrations of sulfamethazine: 10, 100 µM and 1, 5, 15, 20 mM. No-observed-effect-concentration (NOEC) for all germinated seeds was equal 20mM. Effective concentration causing a 50% (EC50) reduction of the root growth after 3, 6 or 9 days was the same for lupin, pea and lentil and equalled 10 µM. The EC50 determined for soybean after 3 and 6 days was higher and amounted 0.25mM and 1.0 mM after 9 days It was shown that root elongation was a better indicator of sulfamethazine phytotoxicity than the seed germination. Increasing concentrations of sulfamethazine led to decrease of the fresh mass and slight increase of the dry mass and electroconductivity. Yellow lupin proved to be the best bioindicator of soil contamination with sulfamethazine among all tested plants.

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S. Bakopoulou, C. Emmanouil and A. Kungolos
Assessment of Wastewater Effluent Quality in Thessaly Region, Greece, for Determining its Irrigation Reuse Potential
Ecotoxicology and Environmental Safety 2011 74, 2 188-194
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna;Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : waste waters; soils

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Abstract : The objective of the present study is to assess wastewater effluent quality in Thessaly region, Greece, in relation to its physicochemical and microbiological burden as well as its toxic potential on a number of organisms. Wastewater may be used for agricultural as well as for landscape irrigation purposes; therefore, its toxicity potential is quite important. Thessaly region has been chosen since this region suffers from a distinct water shortage in summer period necessitating alternative water resources. During our research, treated effluents from four wastewater treatment plants operating in the region (Larissa, Volos, Karditsa, and Tirnavos) were tested for specific physicochemical and microbiological parameters [biochemical oxygen demand (BOD(5)), chemical oxygen demand (COD), total suspended solids (TSS), pH, electrical conductivity, selected metals presence (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn, As), and fecal coliforms’ (FC) number]. The effluents were also tested for their toxicity using two different bioassays (Daphnia magna immobilization test and Phytotoxkit microbiotest). The findings were compared to relative regulations and guidelines regarding wastewater reuse for irrigation. The results overall show that secondary effluents in Thessaly region are generally acceptable for reuse for irrigation purposes according to limits set by legislation, if effective advanced treatment methods are applied prior to reuse. However, their potential toxicity should be closely monitored, since it was found that it may vary significantly in relation to season and location, when indicator plant and zooplankton organisms are used.

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T. Banaszkiewicz, J. Szarek and K. Wysocki
Biological Evaluation of Soil Contamination around a Non-Operating Pesticide Tomb
Polish Journal of Environmental Studies 2011 20,2 485-488
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; plant tests

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Abstract : Our study was a biological evaluation of soil contamination near a non-operating pesticide tomb. The soil contaminated with pesticides had a negative effect on the growth of crustaceans such as Heterocypris incogruens (Ostracodtoxkit test) and the development of plants such as Lepidium sativum, Sinapis alba, and Sorghum saccharatum (Phytotoxkit test). This adverse effect persisted up to 5 years following the liquidation of the facility. The crustaceans under study were considered more useful in the evaluation of chronic toxicity than the root elongation test.

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A. Baran
Assessment of Zea mays Sensitivity to Toxic Content of Zinc in Soil
Polish Journal of Environmental Studies 2013 22,1 77-83
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - metals; soils; plant tests

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Abstract : Our work aims to assess Zea mays sensitivity to soil pollution with zinc. The aim was realized on the basis of studies on zinc’s effect on the germination and intial growth of Zea mays, determining toxicity thresholds (PT) and estimating the toxic zinc content in zinc, at which a significant inhibition of germination and growth of roots occurs and a significant decline in this plant yields. On the basis of estimated values of EC10, EC20 and EC50 toxicity indicators and zinc phytotoxicity tresholds (PT), it was demonstrated that Zea mays is a plant little sensitive to over-the-norm zinc concentrations in soils and should not be used as an indicator plant.

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I. Josko, P. Oleszczuk, J. Pranagal, J. Lehmann, B. Xing and G. Cornelissen
Effect of Biochars, Activated Carbon and Multiwalled Carbon Nanotubes on Phytotoxicity of Sediment Contaminated by Inorganic and Organic Pollutants
Ecological Engineering 2013 60 50-59
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : sediments; chemicals - general; chemicals - nanoparticles; plant tests

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Abstract : To reduce the risk related with the presence of contaminants in sediments, methods based on the use of adsorbents (mainly activated carbon) are applied. Adsorbents neutralise the contaminants by reducing their mobility, bioavailability and toxicity. The objective of this study was to determine the toxicity of sediment contaminated with organic compounds and heavy metals with and without the addition of selected adsorbents. In the study three carbonaceous materials (CM) potentially useful in remediation were applied: activated carbon (AC), biochars (BC1, BC2), and multi-walled carbon nanotubes (CNTs). The effect of the dose, particle diameter and time of contact between sediment and CM on the effectiveness of the detoxification were estimated. Ecotoxicological assessment was made based on the Phytotoxkit F test, using Lepidium sativum as the test plant. The materials tested reduced the negative effect of sediment on L. sativum. The highest effectiveness was achieved after the application of AC (70% reduction of seed germination inhibition, 27.5% reduction of root growth inhibition). The reduction of phytotoxicity of the sediment as a result of addition of BC1, BC2 and CNT varied within the range from 30 to 40% (reduction of seed germination inhibition) and from 17.7 to 28.9% (reduction of root growth inhibition). The reduction of sediment toxicity decreased with decreasing diameter of the biochars applied. Probably because of increasing fouling extension of the time of contact between CM and sediment had an unfavourable effect on the reduction of root growth inhibition in the case of all materials tested.

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P. Oleszczuk, I. Josko, M. Kusmierz, B. Futa, E. Wielgosz, S. Ligeza and J. Pranagal
Microbiological, Biochemical and Ecotoxicological Evaluation of Soils in the Area of Biochar Production in Relation to Polycyclic Aromatic Hydrocarbon Content
Geoderma 2014 213 502-511
Toxkits : Daphtoxkit F^TM magna; Phytotoxkit
Test Species : Daphnia magna; Lepidium sativum
Topics : soils; chemicals - hydrocarbons; plant tests

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Abstract : The objective of the study was the estimation of the effect of polycyclic aromatic hydrocarbons (PAH) on the biological properties of soils in the vicinity of biochar production sites. Assays performed in the soils included the content of PAHs, as well as the microbiological (counts of bacteria and fungi), biochemical (enzymatic activity: dehydrogenases, acidic and alkalic phosphatase, protease and urease) and ecotoxicological properties of the soils (phytotoxicity to Lepidium sativum, Microtox^®-Vibrio fischeri, MARA-11 different microorganisms, Daphtoxkit F^TM-Daphnia magna). In spite of the low level of industrialisation of the area studied, the content of PAHs in the soils was very high. Depending on the soil sampling site, the content of total PAHs varied from 1796.8 to 101,282.7 µg/kg. The highest levels in the soils studied were characteristic of fluoranthene and pyrene. The counts of bacteria and fungi, as well as the enzymatic activity, were clearly related with the sampling sites. In most of the objects studied those parameters had high values, which indicated correct runs of the biological processes in most of the soils studied. Depending on the ecotoxicological assay applied, and on the sampling sites, the toxicity of the soils was varied, but only in a few cases the phytotoxicity was correlated with the content of PAHs.

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P. Oleszczuk, A. Malara, I. Josko and A. Lesiuk
The Phytotoxicity Changes of Sewage Sludge-Amended Soils
Water, Air and Soil Pollution 2012 223, 8 4937-4948
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : plant tests; sludges; plant tests

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Abstract : The aim of the present study was the estimation of changes in the phytotoxicity of soils amended with sewage sludge with relation to Lepidium sativum, Sinapis alba and Sorghum saccharatum. The study was realised in the system of a plot experiment for a period of 29 months. Samples for analyses were taken at the beginning of the experiment, and then after 5, 17 and 29 months. Two kinds of sewage sludge, with varying properties, were added to a sandy soil (soil S) or a loamy soil (soil L) at the dose of 90 t/ha. The addition of sewage sludge to the soils at the start of the experiment caused a significant reduction of both seed germination capacity and root length of the test plants, the toxic effect being distinctly related to the test plant species. With the passage of time the negative effect of sewage sludge weakened, the extent of its reduction depending both of the kind of sewage sludge applied and on the type of soil. Phytotoxicity of the soils amended with the sewage sludges was significantly lower at the end of the experiment than at the beginning. The species of the plants grown on the soils also had a significant effect on their phytotoxicity. The greatest reduction of toxicity was observed in the soil on which no plants were grown (sandy soil) and in the soil under a culture of willow (loamy soil). Solid phase of sewage sludge-amended soils was characterised by higher toxicity than their extracts.

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G.A. Plaza, W. Lukasik, G. Nalecz-Jawecki, and R. Margesin
Application of the Root Elongation Test as Indicator to Assess Phytotoxicity of Contaminated Soils
Fresenius Environmental Bulletin 2009 18, 3 353-359
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : soils; plant tests

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Abstract : The goal of this study was to apply and to compare two /i>Lepidium root elongation tests, the traditional test and the commercial kit - Phytotoxkit microbiotest, to evaluate the phytotoxicity of long-term contaminated soils and the suitability of root elongation tests as phytotoxicity endpoint. The soil samples were collected from three long-term contaminated industrial sites containing heavy metals (two sites, named TG-site 1 and P-site 2) or petroleum hydrocarbons (named R-site 3). Statistically significant correlations (p<0.05) between the results obtained from the two investigated tests were not found. One-way ANOVA analysis showed that the results obtained with the standard Lepidium test and the Phytotoxkit microbiotest were statistically significantly (p<0.05) different. The soils from all three sites could be classified as toxic. The results obtained showed large differences between all investigated parameters in the investigated soils, which were attributed to soil heterogeneity. This paper promotes the application of the root elongation method for ecological risk assessment of contaminated sites. Our results could represent a step toward the selection of a proper phytotoxicity test to characterize the soil ecotoxicological risk.

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Z.B. Romanowska-Duda,M. Grzesik and H.M. Kalaji
Phytotoxkit Test in Growth Assessment of Corn as an Energy Plant Fertilized with Sewage Sludge
Environment Protection Engineering 2010 36, 1 73-81
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sludges; plant tests

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Abstract : The aim of the research was to analyze and evaluate both growth and physiological features of corn, cultivated in various soils and fertilised with sewage sludge. The present study investigates the effect of manuring on energy plant development, especially those cultivated on low-quality soils, and the usefulness of Phytotoxkit test in predicting the efficiency of such a treatment. Sand as well as various types of soil, e.g. sandy, peat or clay, were enriched with 10–15% doses of sewage sludge which, to a large extent, influenced the growth of plants. Moreover, their biomass and chlorophyll a and b content increased, and photosynthesis, membrane stability and RNase activity in leaves were considerably improved. As far as Phytotoxkit is concerned, it appears that it might be useful when predicting the development of fertilized plants.

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T. Sekutowski and J. Sadowski
Phytotoxkit Microbiotest Used in Detecting Herbicide Residue in Soil
Environment Protection Engineering 2009 35, 1 105-110
Toxkits : Phytotoxkit
Test Species : Sinapis alba
Topics : soils; chemicals - pesticides; plant tests

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Abstract : The studies on the phytotoxic residue of herbicide active substances in the soil were conducted using a germination and early growth microbiotest Phytotoxkit by Tigret. Three plant species Sinapis alba, Fagopyrum esculentum and Cucumis sativus, were used as bioindicators. The herbicide active substances tested included chlorsulfuron, nicosulfuron, 2,4 DP and dicamba. A plant which responded most strongly to chlorsulfuron and nicosulfuron was Sinapis alba. Cucumis sativus responded most quickly to 2,4 DP residue, while Fagopyrum esculentum was most suitable for the detection of dicamba residue.

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O. Sestinova, L. Findorakova and J. Hanculak
Toxicity Testing of Sediments
Nova Biotechnologica et Chimida 2012 11, 2 111-116
Toxkits : Phytotoxkit
Test Species : Sinapis alba
Topics : sediments; chemicals - metals; plant tests

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Abstract : This study presents the results of the testing toxicity of the contaminated sediments from the water reservoir of Ružín No.I deposit (Slovak Republic) by using Phytotoxkit tests (MicroBioTests Inc., Belgium). The Phytotoxkit system is a screening tool used for a variety of toxicity testing applications. The advantages of this toxicity bioassay are its speed, relative simplicity and low cost compared to chemical analysis and many other biotests. Evaluation of sediments phytotoxicity was based on the testing of seed germination and the assesment of the root growth decrease of the plant Sinapis alba which allows to complete the assays after only 3 days of incubacion. Chemical analysis of the sediment samples involved determination of heavy metal (Cu, Zn, Ni, As, Sb and Hg) concentration. No potential phytotoxic effect of heavy metals in contaminated sediments was observed in the majority of tested seeds of Sinapis alba.

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D. Wieczorek, E. Kwapisz, O. Marchut-Mikolajczyk And S. Bielecki
Phytotests as Tools for Monitoring the Bioremediation Process of Soil Contaminated with Diesel Oil
BioTechnologia 2012 93, 4 431-439
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; chemicals - hydrocarbons; plant tests

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Abstract : The aim of the study was to determine changes in phytotoxicity levels during bioremediation of soil contaminated with 5% (v/w) diesel oil and find their correlation with the effectiveness of hydrocarbon degradation. Bioremediation trials were performed using a Gordonia alkanivorans S7 strain. The clean-up process was enhanced through the addition of dried fungal enzymatic preparations obtained from fungi Phanerohaete chrysosporium and Aspergillus niger. After 110 days of bioremediation the decrease in soil pollution ranged between 68 and 77% depending on treatment conditions. Toxicological tests using marker plants revealed significant differences in the phytotoxicity levels of soil during bioremediation, depending on the treatment variant. The addition of an enzymatic fungal preparation to soil was found to accelerate the rate of contaminant degradation. The rate of hydrocarbon depletion in subsequent phases of the remediation process was found to be correlated with the phytotoxicity level. The obtained results provide evidence that phytotests can be useful tools for monitoring toxicity changes during bioremediation either mediated only by bacteria or additionally stimulated by fungal enzymatic preparations.

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A.L. Witcher, E.K. Blythe, G.B. Fain, K.J. Curry and C.T. Pounders
Assessing Phytotoxicity in Fresh and Aged Whole Pine Tree Substrates^©
In : Combined Proceedings - International Plant Propagators Society 2011 61 477-482
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; plant tests

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Abstract : Reduced plant growth in wood-based substrates has been attributed to a variety of factors,including phytotoxity. A detailed method for evaluating the phytotoxic potential of woodbased substrates has not been identified. Two biological assays (Phytotoxkit and seedling growth test) were conducted for identifying phytotoxicity in WPT, while examining the potential of such methods for testing other alternative substrates. Substrates evaluated in the Phytotoxkit included a reference soil (RS), aged (WPTA) and fresh (WPTF) whole pine tree, aged (PNA) and fresh (PNF) pine needles, pine bark (PB), peat moss (PM), and a 2 saline pine bark (SPB) substrate. Substrates evaluated in the seedling growth test included WPTA, WPTF, PB, and a peat-lite (PL) substrate. The Phytotoxkit revealed some plant species may be sensitive to compounds present in PNF. The greatest germination/emergence rate and root length varied by species in regard to WPTA and WPTF; therefore, factors other than phytotoxicity affected seedling development in WPT.
In the seedling growth test, total root length was greatest in the PL substrate for all three species, while substrate air space was lowest in the PL substrate.

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N.H.B.M. Kaag and A.C. Sneekes
The Effect of Low Temperatures on the Toxicity of PERACLEAN^® Ocean
IMARES, Wageningen UR 2013
Toxkits : Rotoxkit F^TM; Rotoxkit M^TM
Test Species : Brachionus calyciflorus; Brachionus plicatilis
Topics : ballast waters; chemicals - biocides; chemicals - disinfectants

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Abstract : Ballast water treatment systems are routinely tested for efficacy and residual toxicity at spring and summer temperatures in temperate areas, as that is the period when biological production is sufficiently high to match IMO testing requirements (guideline G8). Shipping, however, continues throughout the year and, as polar ice rapidly disappears, also shipping into the Arctic rapidly increases. This makes assessment of the performance of ballast water treatment technology at low temperatures urgent, especially when biocides are used.
In order to assess the influence of temperature on the efficacy (toxicity) of PERACLEAN^® Ocean (PO), IMARES has tested the toxicity of (PO) to various organisms at standard test temperatures and at low temperatures.
It appeared very difficult to adapt standard test species to the extremely low temperatures needed (<5°C), and the results of the test with these species are unambiguous.
More progress was made using cultures of natural communities samples at the desired temperatures. These showed that at higher temperatures, the direct toxicity of PO increases by the addition of catalase, but that at low temperatures PO alone is just as toxic. An interesting observation is that at higher temperatures, the natural communities show some recovery (regrowth) after prolonged exposure (5 days), which was not observed at temperatures <5°C. The recovery was less when catalase was added to the treatment.

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A. Baran and M. Tarnawski
Phytotoxkit/Phytotestkit and Microtox^® as Tools for Toxicity Assessment of Sediments
Ecotoxicology and Environmental Safety 2013 98, 1 19-27
Toxkits : Phytotoxkit; Phytotestkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; sediment pore waters; plant tests

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Abstract : The aim of the study was to use bioassays to evaluate the toxicity of pore water, sediments and sediments elutriates. Furthermore, a possible relationship between observed toxicity and results of chemical analysis was examined. Sediment (0–10 cm) samples were collected from 21 locations in Zeslawice reservoir, Southern Poland using an Ekman sampler. Toxicity assessment of the sediment and pore water samples was performed using direct-contact tests Phytotoxkit/Phytotestkit and Microtox^®. Inhibition of seed germination in the test plants was from -25 to 38 percent for sediment and from 0 to 50 percent for pore water, whereas inhibition of root growth was within a range from -42 to 37 percent for sediment and from -49 to 37 percent for pore water. Depending on the sediments, Vibrio fischeri luminescence inhibition was from -18 to 40 percent for sediments elutriates and from -12 to 28 percent for pore water. The toxicity tests showed a positive correlation between metals and the root growth inhibition in Lepidium sativum and Sinapis alba as well as the luminescence inhibition in V. fischeri. No significant correlations were found between the inhibition of luminescence and the phytotoxicity assays, so these analyses do not show a similar sensitivity to toxicants in the sediments. While estimating the sensitivity of the performed biotests, the highest number of toxic responses was recorded in the Microtox^® test towards V. fischeri. Among the plant species, Sorghum saccharatum appears to be the most sensitive plant species. Most of the examined sediment samples (67 percent) were classified as class II (low-toxic samples, low acute hazard) and 33 percent of the examined samples were class I (no essential toxic effect, non-toxic sample, no acute hazard) in terms of toxicity. Most of the pore water samples (71 percent) were also classified as class II.

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V. Verma, R. Rico-Martinez, N. Kotra, C. Rennolds, J. Liu, T.W. Snell and R.J. Weber
Estimating the Toxicity of Ambient Fine Aerosols using Freshwater Rotifer Brachionus calyciflorus (Rotifera : Monogononta)
Environmental Pollution 2013 182 379-384
Toxkits : Rotoxkit F^TM
Test Species : Brachionus calyciflorus
Topics : air

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Abstract : The toxicity of atmospheric fine particulate matter (PM2.5) in Atlanta is assessed using freshwater rotifers (Brachionus calyciflorus). The PM-laden quartz filters were extracted in both water and methanol. Aerosol extracts were passed through a C-18 column to separate the PM components into hydrophobic and hydrophilic fractions. Toxicity data reported in the units of LC50 (concentration that kills 50% of the test population in 24 h) shows that ambient particles are toxic to the rotifers with LC50 values ranging from 5 to 400 µg of PM. The methanol extract of the aerosols was substantially more toxic (8 + 6 times) to the rotifers compared to the water extracts. A sizeable fraction (>70%) of toxicity was found to be associated with the hydrophobic fraction of PM. However, none of the bulk aerosol species was strongly correlated with the LC50 values suggesting a complicated mechanism of toxicity probably involving synergistic interactions of various PM components.

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R. Rico-Martínez, T.W. Snell and T.L. Shearer
Synergistic Toxicity of Macondo Crude Oil and Dispersant Corexit 9500A® to the Brachionus plicatilis Species Complex (Rotifera)
Environmental Pollution 2013 173 5-10
Toxkits : Rotoxkit M^TM
Test Species : Brachionus plicatilis
Topics : chemicals - hydrocarbons; chemicals - biocides; chemicals - dissinfectants

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Abstract : Using the marine rotifer Brachionus plicatilis acute toxicity tests, we estimated the toxicity of Corexit 9500A®, propylene glycol and Macondo oil. Ratios of 1:10, 1:50 and 1:130 for Corexit 9500A®:Macondo oil mixture represent : maximum exposure concentrations, recommended ratios for deploying Corexit (1:10-1:50), 1:130 the actual dispersant:oil ratio used in the Deep Water Horizon spill. Corexit 9500A® and oil are similar in their toxicity. However, when Corexit 9500A® and oil are mixed, toxicity to B. manjavacas increases up to 52-fold. Extrapolating these results to the oil released by the Macondo well, suggests underestimation of increased toxicity from Corexit application. We found small differences in sensitivity among species of the B. plicatilis species complex, likely reflecting phylogenetic similarity. Just 2.6% of the water-accommodated fraction of oil inhibited rotifer cyst hatching by 50%, an ecologically significant result because rotifer cyst in sediments are critical resources for the recolonization of populations each Spring.

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B. Huerta Buitrago, P. Ferrer Muñoz, V. Ribé, M. Larsson, M. Engwall, E. Wojciechowska and S. Waara
Hazard Assessment of Sediments from a Wetland System for Treatment of Landfill Leachate using Bioassays
Ecotoxicology and Environmental Safety 2013 97 255-262
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments; landfill leachates

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Abstract : Four bioassays were used in this study for the hazard assessment of sediments from sediment traps and several ponds in a treatment wetland for landfill leachate at Atleverket, Sweden. In the 6-day solid phase microbiotest with the sediment-dwelling crustacean Heterocypris incongruens both acute and chronic effects were observed with a gradual decrease and loss of toxicity with treatment in the wetland system. Some samples showed a low toxicity in pore water and only one sample was weakly toxic in the whole sediment test when assessed with Aliivibrio fischeri (Vibro fischeri). No genotoxicity was detected in the umu test. The toxicity response in the H4IIE-luc test evaluating the presence of dioxin-like compounds was considerably higher in the samples from the sediment traps. The hazard of the sediment therefore appears to be highest in the sediment traps and pond 1 with the methods employed. The result indicates that the wetland system has a design supporting the concentration and sequestration of toxic substances in the first part of the wetland. Based upon the results we suggest that hazard assessment of sediments from other treatment wetlands for landfill leachate should be conducted.

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Y. Marneffe and C. Chalon
Effect Monitoring of Selected Effluents and Receiving Waterbodies in the Meuse and Scheldt Basins : Overall Toxicity and Endocrine Disruption Assessment
From 24th Annual Meeting SETAC Europe, Basel, Switserland, 11-15 May 2014 - Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Selenastrum capricornutum
Topics : effluents; surface waters

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Abstract : Bioassays are a holistic and meaningful way of assessing effects of environmental samples and wastes on ecosystems. A study was carried out with complementary tools in different rivers of the Meuse and Scheldt basins, upstream, downstream and in the effluent of selected industries and urban wastewater treatment plants. An effect active monitoring using bioassays was carried out, combining ecotoxicological and physico-chemical measurements. A yeast estrogen screen (YES) assay was conducted as effective first-tier screening tool to assess the estrogenic potential of surface water, effluent or sediment samples. Moreover, we used a battery of short term and chronic bioassays with the bacteria Vibrio fischeri, the alga Pseudokirchneriella subcapitata, the rotifer Brachionus calyciflorus and the microcrustacea Daphnia magna to assess their overall toxicity. Polar organic chemical integrative samplers (POCIS) were also used at some locations to sample hydrophilic compounds such as pharmaceuticals, personal care products, hormones, and pesticides having low log n-octanol-water partition coefficents (Kows < 3). POCIS extracts were screened for the presence of estrogenic chemicals using the yeast estrogen screen (YES) assay and analyzed by gas chromatography/mass spectrometry (GC/MS) and we used AMDIS (Automated Mass Spectral Deconvolution and Identification software) and the NIST 2.0 mass spectral library for the identification of the compounds. Results from the YES indicated important estrogenicity in different small rivers such as River Haine and River Hain (>15 ng eq E2/l), whereas samples collected in more important rivers like the River Meuse showed lower estrogenic activities (<2 ng eq E2/l). High estrogenic activities were also measured in water extracts of selected industrial and urban effluents. As far as the chemical screening is concerned, several chemical classes known to produce a positive response in the YES (i.e., estrogen receptor agonists) were identified in POCIS extracts at every selected location. Although mostly qualitative, the results confirmed the observations based on the effect parameters (YES test).
See corresponding Poster

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G. Persoone, M. Foudoulakis, G. Arapis and R. Baudo
Development and Validation of a Stock Culture Independent Duckweed Microbiotest with Spirodela polyrhiza
From 24th Annual Meeting SETAC Europe, Basel, Switserland, 11-15 May 2014 - Book of Abstracts
Toxkits : Spirodela duckweed Toxkit
Test Species : Spirodela polyrhiza
Topics : duckweed tests; test validation; test development; plant tests; ring testing; interlaboratory testing

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Abstract : There are relatively few toxicity tests with macrophytes to assess the negative effects of pollutants on aquatic plants, and of these the most practiced is the standard duckweed assay with Lemna minor or Lemna gibba. Similarly to most toxicity tests, Lemna assays are, however, also dependent on the (year-round) culturing and maintenance of live stocks of the test species; the tests furthermore require substantial bench and incubation space and they are hence quite expensive. Efforts have been made to develop a “stock culture free” duckweed microbiotest based on the use of “vegetative dormant stages”; the latter (called turions) are only produced by a few duckweed species but these dormant stages can be stored and germinated at the time of performance of the bioassay. The test species selected for this alternative duckweed test is Spirodela polyrhiza, a species closely related to Lemna, and of which the turions germinate in a few days of time and then immediately develop a first frond. A simple and practical 3 days microbiotest in multiwell test plates has been worked out based on the measurement of the area of the first frond of the germinated turions in the negative controls and in the toxicant concentrations as the effect criterion. The area measurements are made by image analysis on a photo of the test plate taken at the end of the exposure time. The sensitivity of the Spirodela polyrhiza microbiotest has been determined on a number of inorganic and organic chemicals, and the 72h EC50’s for 16 compounds have been compared with the 7 days EC50’s of the Lemna toxicity test. The data pair comparison revealed that the alternative duckweed microbiotest has basically the same sensitivity as the conventional Lemna bioassay (R2 = 0.97). A preliminary ringtest on the Spirodela polyrhiza microbiotest has recently been organized with 6 participants (from 6 different countries), for performance of the new assay on the reference chemical KCl. The preliminary ringtest revealed that all 6 participants obtained very similar 72h EC50’s (ranging from 5.855 mg/l KCl to 7.116 mg/l KCl) with a mean value of 6.593 mg KCl/l and a variation coefficient of only 6.19 %. To assess further the robustness and the interlaboratory precision of this promising practical and low cost new microbiotest, an International Interlaboratory Comparison is now in progress, with many participants from a large number of countries. It is intended subsequently to propose the stock culture independent duckweed test to the ISO as a new standard toxicity test with Spirodela polyrhiza, for toxicity analysis of chemicals and wastewaters.
See corresponding Poster

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M. Wawryniuk, K. Czaplicka and G. Nalecz-Jawecki
Assessment of Direct and Indirect Photodegradation of Selected Drugs Acting on the Central Nervous System
From 24th Annual Meeting SETAC Europe, Basel, Switserland, 11-15 May 2014 - Book of Abstracts
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - drugs

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Abstract : Drugs acting on the central nervous system (CNS), especially antidepressants have a very high growth dynamics of consumption. The widespread use of these compounds has lead to their detection in surface waters and ground waters. In recent years, the occurrence and fate of pharmaceutically active compounds (PhACs) in the aquatic environment has been recognized as one of the emerging issues in environmental chemistry. Solar radiation is the main abiotic factor influencing the distribution of chemical compounds present in the environment. Many drug substances and drug products are found to be decomposed under exposure to light, but the practical consequences will not necessarily be the same in all cases. The aim of the research was a comprehensive evaluation of photodegradation of selected drugs acting on the central nervous system. Aqueous solutions of drugs were irradiated in sunlight simulator without (direct photodegradation) and with the addition of humic acid (indirect photodegradation). The samples were tested using the bioassay and physicochemical methods. Toxicity of the samples were evaluated with standard organisms, from bacteria (Microtox), through protozoa (Spirotox) to crustaceans (Daphtoxkit F™ Magna, Daphnid, Thamnotoxkit F). At the same time the concentration of the parent substances was monitored using HPLC. Mass spectrometry was used to identify the photoproducts formed. Most of the pharmaceuticals tested were toxic in the bioassays. Their photoproducts were also toxic to test-bionts, as the toxicity of the samples decreased slower than the concentration of the parent compounds. Moreover, the sensitivity of the assays applied depended not only on the drug, but also on the photodegradation process. The addition of humic acid in the indirect photodegradation influenced the photodegradation ratio evaluated with chemical and biology analysis.
See corresponding Poster

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L. Manusadzianas, R. Karitonas, R. Vitkus, K. Sadauskas, R. Juknys and J. Zaltauskaite
Implementation of the Test-Battery Approach into Routine Effluent Control in Lithuania
From 24th Annual Meeting SETAC Europe, Basel, Switserland, 11-15 May 2014 - Book of Abstracts
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : effluents; test selection; regulatory testing

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Abstract : Recently, HELCOM, expressing a concern on the status of the Baltic Sea, has initiated several international programmes such as Cohiba, BaltHaz, etc., particularly targeted to promote the usage of Whole Effluent Assessment (WEA) methods in routine regulation. Contrary to the control of individual substances, which is based on knowledge of a single concentration, municipal and industrial effluents can be regulated directly on basis of harmful effects that occur in testing organisms. Various countries use laboratory toxicity tests to monitor effluent discharges into surface waters in various degrees. For example, acute and chronic toxicity tests are used in the USA and Sweden, whereas acute tests are used in Canada and several European countries. Up to date, the majority of countries provide chemical-based effluent controls, however, the progressive increase of chemicals under control and difficulties in measuring them make individual-substance approach problematic. In some countries, toxicity data are included for taxation purposes, in parallel to chemical-based taxation. For example, toxicity data obtained from D. magna acute and fish embryos tests used in France and Germany, respectively.
In 2011, the Ministry of Environment of Lithuania put forward a national programme with the aim to elaborate a system of effluent control by implementing toxicity tests. The outcome of this programme includes appearance of necessary environmental juristic documents and/or their updates, selection of test-batteries, selection of the cumulative index for toxicity evaluation and performance of initial stage of effluent inventory monitoring. The evaluation of potential harm for aquatic environment is limited to the application of end-of-pipe principle, the assessment of effluent by acute and/or chronic toxicity test-batteries as well as effluent-and stream-flow rates. The polluters will be grouped according to the criteria of the type of economical activity and discharge rate. During one-year inventory period, respective test-battery(ies) will be applied and the most sensitive test(s) will be prescribed for routine effluent control. In this study, a system for municipal and industrial effluents control as well as simultaneous steps of its implementation into routine will be overviewed.
See corresponding Poster

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A. Klimkowicz-Pawlas, B. Smreczak, B. Maliszewska-Kordybach and A. Ukalska-Jaruga
Ecotoxicity Characterization of Soils Exposed to Carbon Derivative Pollutants from the Coking Area
IV National Bioindication Conference, Olsztyn - Poland - May 2014
Toxkits : Phytotoxkit; Protoxkit Rapidtoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorghum saccharatum; Thamnocephalus platyurus
Topics : soils; chemicals - hydrocarbons; plant tests

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Abstract : Protection of soil environment in the European Union has become a priority in recent years. The basic document of the EU Thematic Strategy for Soil Protection (COM (2006) 231 final 2006) points to the need to preserve soil functions and their protection against environmental factors causing soil degradation. Among the major threats to the proper functioning of soil, pollution play an important role. The majority of contaminants present in the environment comes from anthropogenic sources such as industrial emissions, transport and use of sewage sludge and pesticides in agriculture. Pollutants emitted into the atmosphere are transported over long distances and ultimately most of them accumulate in the soil, where even at low concentrations may result in adverse effects, not only to human health but also for the growth and development of many groups of organisms. Assessment of the risks associated with soil contamination is often based on the measurement of the total content of the chemicals. However, chemical analysis does not provide information on a wide range of toxic substances and their synergistic or antagonistic interactions. Therefore, to evaluate the current risk associated with contaminants, the chemical analyses have to be complemented with the results of ecotoxicological test.

The aim of the study was to evaluate the ecotoxicity of soils exposed to long-term impact of hydrocarbon emissions. The research area (about 100 km2) was located in the south-western part of Upper Silesia region in the vicinity of the coking plant operating for 100 years. 25 sampling points were collected with respect to different soil conditions and direct influence of local and trans boundary PAHs emission sources. The basic physicochemical and biological properties and the content of selected pollutants (metals and PAHs) were determined in the soil samples.

For detailed characteristic of soils ecotoxicity, the battery of tests was applied: the test of bioluminescence inhibition (Vibrio fischeri) with application of Microtox toxicity test system; phytotoxicity test Phytotestkit for the evaluation the direct impact the chemicals contained in the soil solution on the plant growth; the Rapidtoxkit using crustaceans Thamnocephalus platyurus; a screening test for emergence of lettuce seedlings (ISO 17126), substrate induced respiration (ISO 14240-1) and potential nitrification (ISO 15685).

The results showed, that the significant threat at the study area is the presence of organic pollutants (PAHs). Application the classification of samples toxicity led to include 57% of the soil samples to the class III and IV; sampling points with the highest toxicity were located in the area directly exposed to the impact of hydrocarbon contamination. Additionally the risk factors (IR) were determined; calculation of IR values allowed to subsequent elimination areas with low risk and the final delineation of an area where the risk is not acceptable.

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W. Ratajczyk, M. Cieszynska and L. Wolska
Sewage Quality According to Chemical and Bioanalytical Index
IV National Bioindication Conference, Olsztyn - Poland - May 2014
Toxkits : Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Phytotestkit; Thamnotoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Thamnocephalus platyurus; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : effluents; waste waters; plant tests

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Abstract : According to Polish legislation, the control of the quality of sewage is based on chemical parameters. Wastewater discharged into municipal facilities can pose a serious threat to the environment, living organisms , and can be a potential factor dangerous to human health. The quality of water flowing into the municipal sewage treatment plant is very variable in composition and concentration levels of toxic substances. That is why, there is a need of introducing and developing effective indicators allowing to assess the total sample load of hazardous substances. In the bio-analytical methods, standard living organisms are exposed to a sewage sample with complex mixture of pollution. In the present study as a measure of the toxicity of the samples was mainly used indicator of cell growth, mortality and a decrease in bioluminescence.

There were analyzed samples of raw sewage, samples following mechanical treatment and samples following biological treatment. The samples were taken in two seasons, warm and cool from the wastewater treatment plants located in area of Gulf of Gdansk. The toxicity of wastewater samples were tested against several test organisms used in standard, commercially available acute and chronic bioassays. The samples of raw sewage collected from wastewater treatment plants gave a very diverse biological response in standard tests towards acute and chronic toxicity. In each case, the same results were obtained after mechanical treatment plant and effluent treatment level. It suggests that mechanical treatment does not remove the compounds responsible for the toxicity of the wastewater. The strongest response was observed in the case of Thamnotoxkit F and Daphtoxkit F bioassays. Raw sewage gave 100% toxic effect resulting in complete mortality of the test organisms. The dilution of raw sewage by 50% caused 100% mortality of Thamnocephalus platyurus crustacean. In such dilution of raw sewage, the reaction of Daphnia magna crustacean was different - from total lack of biological response to 100% toxic effect. The studies with Vibrio fischeri bacteria showed high toxicity of wastewater flowing into the treatment plant and the lack of a toxic effect due to treated wastewater. In both cases of plant tests (Phytotoxkit F test) and tests using Heterocypris incongruens (Ostracodtoxkit F test), observed biological response is lower. These organisms tend to be less sensitive to contaminants contained in the raw wastewater delivered to treatment plants. Furthermore, it was observed that the substances present in the sewage stimulate the growth and evolution of the organisms. This may be due to the nutritional properties of sewage which provide biogenic substances to organisms and are also responsible for the eutrophication or excessive biomass growth of organisms and depletion of aquatic environment in dissolved oxygen. All samples with treated sewage in different dilutions were not toxic to all test organisms which were used, indicating that the wastewater treatment process during the period from leading to effectively removal of substances responsible for the high toxicity towards the organisms living in the environment.

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P. Alvarenga, C. Mourinha, M. Farto and P. Palma
The Use of Aquatic Bioassays in the Risk Assessment to Surface and Groundwater from the Application of Organic Wastes as Soil Improvers
From 24th Annual Meeting SETAC Europe, Basel, Switserland, 11-15 May 2014 - Book of Abstracts
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : regulatory testing; soils; solid wastes; leachates; sludges; composts

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Abstract : The Proposal for a European Soil Framework Directive recognized that ‘soil degradation or soil improvers have a major impact on other areas, (…) such as surface waters and groundwater, human health, climate change, protection of nature and biodiversity, and food safety’. This study aims to assess the potential impact on surface and ground water from the application of different organic wastes as soil improvers. For this purpose, sewage sludge, composted sewage sludge with agricultural wastes, agro-industrial sludge, mixed municipal solid waste compost (MMSWC), compost produced from agricultural wastes, pig slurry digestate (PSD), municipal slaughterhouse sludge, and paper mill wastes (sludge and ashes), were analyzed considering their main physicochemical characteristics, and their trace elements (TEs) total concentrations (Cd, Cr, Cu, Hg, Ni, Pb and Zn). In order to assess their potential impact on surface and groundwater, a leachate was obtained using DIN 384|4-S4 method (1984). Several aquatic bioassays were used in order to assess the leachate ecotoxicity : luminescence inhibition of the marine bacteria Vibrio fischeri, Daphnia magna immobilization, Thamnocephalus platyurus survival, and seed germination (Lolium perenne and Lactuca sativa. Considering the total TEs concentrations in the organic wastes, and their organic contaminants concentrations, the sludge samples characterized in the study can be used for agricultural purposes, in accordance with the European Community Directive 86/278/EEC and with the Portuguese legislation. As for the compost samples, they can also be land applied considering the DG Env.A.2. Working document on ‘Biological Treatment of Biowaste – 2_nd draft’ : mixed municipal sewage sludge with agricultural wastes is a Class I compost.
However, compost produced from agricultural wastes and pig slurry digestate cannot be land applied, due to their Ni and Zn content, respectively. Despite the fact that some organic wastes are allowed to be land applied according to the existing regulations, their leachates can be very toxic towards some organisms. That toxicity was difficult to be predicted from the total TEs concentrations, which are one of the main criterions to be accomplished when evaluating organic wastes risk – PSD and MMSWC were the organic wastes with overall higher total TE concentrations and were not identified as having higher toxicity. See corresponding Poster

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M. Renzi, L. Roselli, A. Giovani, S.E. Focardi and A. Basset
Early Warning Tools for Ecotoxicity Assessment Based on Phaeodactylum tricornutum.
Ecotoxicology 2014 23 (6) 1055-1072
Toxkits : Marine Algaltoxkit
Test Species : Phaeodactylum tricornutum
Topics : chemicals - metals; chemicals - organic compounds

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Abstract : Phaeodactylum tricornutum was exposed to various toxic substances (zinc, copper or dodecylbenzenesulfonic acid sodium salt) in accordance with the Marine AlgalToxkit protocol based on the UNI EN ISO 10253 method in order to quantitatively compare the responses obtained by traditional growth-rate inhibition tests with morphological (biovolume) and physiological (chlorophyll-a, phaeophytin ratio) endpoints. A novel approach is proposed for detecting early and sub-lethal effects based on biovolume quantification using confocal microscopy coupled with an image analysis system. The results showed that effects on both biovolume and the photosynthetic complex are sensitive and powerful early warning tools for evaluating sub-lethal effects of exposure. Specifically, biovolume showed significant sensitive and early responses for the tested surfactant. Qualitatively, we also observed structural anomalies and effects on natural auto-fluorescence in exposed cells that also represent potentially useful tools for ecotoxicological studies.

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A. Acs, A.W. Kovacs, J.Z. Csepregi, N. Töro, G. Kiss, J. Gyori , A. Vehovszky, N. Kovats and A. Farkas
The Ecotoxicological Evaluation of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) Employing a Battery of Bioassays and Chemical Screening.
Toxicon 2013 70 98-106
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : biotoxins

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Abstract : Ecotoxicity of four Cylindrospermopsis raciborskii strains (ACT 9502, ACT 9503, ACT 9504, ACT 9505) isolated from Lake Balaton (Hungary) was evaluated in four aquatic bioassays including the Thamnocephalus platyurus acute lethality test; Daphnia magna acute immobilization assay; D. magna feeding inhibition assay and Danio rerio embryo developmental toxicity assay, assisted by chemical screening for known toxins by HPLC-MS. For reference, we analyzed in parallel the toxin content and toxic effects of two previously characterized toxin-producing strains: the Australian cylindrospermopsin producer AQS C. raciborskii and the anatoxins producer Oscillatoria sp. PCC 6506. Bioassays were used to evaluate the overall toxicity of the hydrophilic bioactive metabolites pool synthesized by the selected cyanobacteria. Chemical screening has proven that the ACT C. raciborskii extracts investigated did not contained cylindrospermopsins and anatoxins. The relative toxicity of the ACT C. raciborskii aqueous extracts observed in each bioassay was comparable to the effects recorded for the anatoxins producer PCC 6506 strain while toxicity values (EC50/LC50) calculated for the AQS extract were in general one order of magnitude lower. Concerning sublethal effects of ACT C. raciborskii extracts to the D. rerio embryogenesis, the general morphological abnormality observed was a significant retardation of development. Overall, our results suggest that C. raciborskii populating Lake Balaton produce metabolites with significant bioactive potencies. Therefore, continued investigation of these unknown compounds is required.

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M. Isidori, E. Coppola, M. R. Iesce, F. Cermola, G. Papa and A. Parrella
Comparative Abiotic or Biotic Degradation of Carboxin by Two Entisols with Different Surface Properties Orpseudomonas Aeruginosa Strain: a Toxicity Study using the Crustacean Thamnocephalus Platyurus
Journal of Environmental Science and Health, Part B : Pesticides, Food Contaminants, and Agricultural Wastes 2012 47, 9 891-900
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - pesticides

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Abstract : The microcrustacean Thamnocephalus platyurus was used to detect the toxicity reduction of carboxin in abiotic degradation compared to biotic degradation. The abiotic degradation was obtained using two sterilized Entisols with different surface properties while the biotic degradation by Pseudomonas aeruginosawas obtained using the fungicide as the only C source. The results showed that the highest toxicity reduction rates for the abiotic degradation were achieved in 20 days with 49.2% for the coarser soil, 60.7% for the soil with a finer texture, whereas for the biotic degradation, 60.6%. Analysis (¹H NMR) showed that the soils transformed carboxin to produce sulfoxide and enol in different concentrations depending on the soil properties, while P. aeruginosa metabolized the fungicide to produce inorganic compounds such as ammonium and nitrite, minor degradation pathways were oxidized to sulfoxide and hydrolytic ring-opening to 2-[(2-hydroxyethyl)thio]acetoacetanilide enol. These results indicated that the degradation of carboxin occurred via abiotic catalytic processes as well as via biotic transformation leading to less toxic derivatives and such phenomena are caused by exchange/surface features of soils, rather than by the mere content of clay or organic matter fractions.

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M. Patra M, X. Ma , C. Isaacson , D. Bouchard , H. Poynton , J.M. Lazorchak and K.R. Rogers
Changes in Agglomeration of Fullerenes During Ingestion and Excretion in Thamnocephalus platyurus
Environmental Toxicology and Chemistry 2011 30, 4 828-835
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - general; chemicals - inorganic compounds

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Abstract : The crustacean Thamnocephalus platyurus was exposed to aqueous suspensions of fullerenes C(60) and C(70) . Aqueous fullerene suspensions were formed by stirring C(60) and C(70) as received from a commercial vendor in deionized water (termed aqu/C(60) and aqu/C(70)) for approximately 100 d. The Z-average (mean hydrodynamic) diameters of aqu/C(60) and aqu/C(70) aggregates as measured by dynamic light scattering were 517 + 21 nm and 656 + 39 nm (mean + 95% confidence limit), respectively. Exposure of T. platyurus to fullerene suspensions resulted in the formation of dark masses in the digestive track visible under a stereo microscope (×40 magnification). Fullerene ingestion over 1 h of exposure was quantitatively determined after extraction and analysis by high-performance liquid chromatography-mass spectrometry (HPLC-MS). One-hour exposures (at 3 mg/L and 6 mg/L) resulted in aqu/C(60) burdens of 2.7 + 0.4 µg/mg and 6.8 + 1.5 µg/mg wet weight, respectively. Thin-section transmission electron microscopy (TEM) images of aqu/C(60) -exposed T. platyurus showed the formation in the gut of fullerene agglomerates (5-10 µm) that were an order of magnitude larger than the suspended fullerene agglomerates. Upon excretion, the observed fullerene agglomerates were in the 10- to 70-µm size range and settled to the bottom of the incubation wells. In contrast to the control polystyrene microspheres, which dispersed after depuration, the aqu/C(60) agglomerates (greater than two orders of magnitude larger than the suspended fullerenes) remained agglomerated for up to six months. When exposed to fullerenes, T. platyurus shows the potential to influence agglomerate size and may facilitate movement of these nanoparticles from the water column into sediment.

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A.M. Sulej, Z. Polkowska , L. Wolska , M. Cieszynska and J. Namiesnik
Toxicity and Chemical Analyses of Airport Runoff Waters in Poland
Environmental Science. Processes & Impacts 2014 16, 5 1083-1093
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : effluents

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Abstract : The aim of this study was to assess the ecotoxicological effects of various compounds in complex airport effluents using a chemical and ecotoxicological integrated strategy. The present work deals with the determination of sum of PCBs, PAHs, pesticides, cations, anions, phenols, anionic, cationic, non-ionic detergents, formaldehyde and metals - as well as TOC and conductivity - in runoff water samples collected from 2009 to 2011 at several locations on two Polish international airports. Two microbiotests (Vibrio fischeri bacteria and the crustacean Thamnocephalus platyurus) have been used to determine the ecotoxicity of airport runoff waters. The levels of many compounds exceeded several or even several tens of times the maximum permissible levels. Analysis of the obtained data shows that samples that displayed maximum toxicity towards the bioindicators Vibrio fischeri were not toxic towards Thamnocephalus platyurus. Levels of toxicity towards T. platyurus are strongly correlated with pollutants that originate from the technological operations related to the maintenance of airport infrastructure. The integrated (chemical-ecotoxicological) approach to environmental contamination assessment in and around airports yields extensive information on the quality of the environment. These methodologies can be then used as tools for tracking the environmental fate of these compounds and for assessing the environmental effect of airports. Subsequently, these data will provide a basis for airport infrastructure management.

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P.M. Rowinski and M.M. Chrzanowski
Influence of Selected Fluorescent Dyes on Small Aquatic Organisms
Acta Geophysica 2011 59, 1 91-109
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - paints; chemicals - general

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Abstract : Rhodamine B and Rhodamine WT are fluorescent dyes commonly used as tracers in hydrological investigations. Since introducing intensely red substances into rivers raises understandable doubts of ecological nature, the authors aimed at examining the influence of these dyes on small water fauna using bioindication methods. Quantitative results, calculated with the use of Bliss-Weber probit statistical method, were achieved by means of standardized ecotoxicological tests containing ready-to-hatch resting forms of fairy shrimp (Thamnocephalus platyurus). Qualitative studies included observation of water flea crustacean (Daphnia magna) and horned planorbis snail (Planorbiscorneus), both typically present in rivers and representative for temperate climate, as well as guppy fish (Poecilla reticulata), paramecium protozoan (Paramaecium caudatum) and the above-mentioned fairy shrimp. The investigation revealed that both dyes in concentrations used for hydrological purposes are low enough to exert almost no toxic impact on water fauna considered.

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M. Passananti, M. Lavorgna, M.R. Iesce, M. DellaGreca , E. Criscuolo , A. Parrella , M. Isidori and F. Temussi
Chlorpropham and Phenisopham : Phototransformation and Ecotoxicity of Carbamates in the Aquatic Environment
Environmental Science. Processes & Impacts 2014 16, 4 823-831
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - pesticides

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Abstract : In this study, a comparison of two carbamic pesticides, chlorpropham and phenisopham, was carried out in terms of both photodegradability and ecotoxicity. The photochemical behaviour of the two pesticides was investigated under environmental-like conditions (aqueous media, UVB or solar irradiation). The photochemical kinetic parameters were calculated by irradiating 5 × 10(-5) M solutions (H2O-CH3CN, 9 : 1 v/v) using UVB lamps. For chlorpropham and phenisopham similar half-life times (39.0 and 55.0 min) were determined. Irradiation by sunlight leads to longer degradation half-life times (about 3 months), while it is possible to observe the formation of the same photoproducts. The well-known dechlorination reaction to a hydroxyphenylcarbamate was observed for chlorpropham. Phenisopham undergoes photo-Fries reaction to give rearranged products (hydroxybenzamides) and fragmentation products (hydroxyphenylcarbamate and N-ethylaniline). Acute and chronic toxicity tests of pesticides and their photoproducts were performed on organisms from two levels of the freshwater aquatic chain, the anostraca crustacean Thamnocephalus platyurus, the rotifer Brachionus calyciflorus and the alga Pseudokirchneriella subcapitata. The acute results showed that chlorpropham had median lethal concentrations for the crustacean T. platyurus and the rotifer B. calyciflorus of 10.16 and 35.19 mg L(-1), respectively, and phenisopham did not show any acute toxicity as the derivatives up to 10 mg L(-1). The only exception was N-ethylaniline which exhibited an acute LC50 value of 0.46 mg L(-1). Phenisopham was the most toxic in the long term exposure while its five derivatives showed lower chronic potential for rotifers and algae. The same trend was observed for chlorpropham except for rotifers.

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C. Karadima, C. Theodoropoulos, A. Rouvalis and J. Iliopoulou-Georgudaki
Ecological Risk Assessment of Cheese Whey Effluents Along a Medium-Sized River in Southwest Greece
Journal of Environmental Science and Health. Part A : Toxic/Hazard Substances & Environmental Engineering 2010 45, 6 775-781
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : effluents

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Abstract : An ecological risk assessment of cheese whey effluents was applied in three critical sampling sites located in Vouraikos river (southwest Greece), while ecological classification using Water Framework Directive 2000/60/EU criteria allowed a direct comparison of toxicological and ecological data. Two invertebrates (Daphnia magna and Thamnocephalus platyurus) and the zebra fish Danio rerio were used for toxicological analyses, while the aquatic risk was calculated on the basis of the risk quotient (RQ = PEC/PNEC). Chemical classification of sites was carried out using the Nutrient Classification System, while benthic invertebrates were collected and analyzed for biological classification. Toxicological results revealed the heavy pollution load of the two sites, nearest to the point pollution source, as the PEC/PNEC ratio exceeded 1.0, while unexpectedly, no risk was detected for the most downstream site, due to the consequent interference of the riparian flora. These toxicological results were in agreement with the ecological analysis: the ecological quality of the two heavily impacted sites ranged from moderate to bad, whereas it was found good for the most downstream site. The results of the study indicate major ecological risk for almost 15 km downstream of the point pollution source and the potentiality of the water quality remediation by the riparian vegetation, proving the significance of its maintenance.

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K. Gondek, A. Baran and Michal Kopec
The Effect of Low-Temperature Transformation of Mixtures of Sewage Sludge and Plant Materials on Content, Leachability and Toxicity of Heavy Metals
Chemosphere 2014 117 33-39
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : sludges; plant tests

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Abstract : The aim of the study was to determine the influence of the process of low-temperature transformation and the addition of plant material to sewage sludge diversifying the content of mobile forms of heavy metals and their ecotoxicity. The experimental design included: sewage sludge + rape straw, sewage sludge + wheat straw, sewage sludge + sawdust, sewage sludge + bark and sewage sludge with no addition. The mixtures were subjected to thermal transformation in a chamber furnace, under conditions without air. The procedure consisted of two stages: the first stage (130 °C for 40 min) focused on drying the material, whereas in the second stage (200 °C for 30 min) proper thermal transformation of materials took place. Thermal transformation of the materials, caused an increase in total contents of heavy metals in comparison to the material before transformation. From among elements, the cadmium content changed the most in materials after thermal transformation. As a result of thermal transformation, the content of water soluble form of the heavy metals decreased significantly in all the prepared mixtures. Low toxicity of the extracts from materials for Vibrio fischeri and Lepidium sativum was found in the research, regardless of transformation process. L. sativum showed higher sensitivity to heavy metals occurring in the studied extracts from materials than V. fischeri, evidence of which are the positive significant correlations between the content of metals and the inhibition of root growth of L. sativum.

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A. Baran, T. Czech and J. Wieczorek
Chemical Properties and Toxicity of Soils Contaminated by Mining Activity
Ecotoxicology 2014 23 1234-1244
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; chemicals - metals; plant tests

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Abstract : This research is aimed at assessing the total content and soluble forms of metals (zinc, lead and cadmium) and toxicity of soils subjected to strong human pressure associated with mining of zinc and lead ores. The research area lay in the neighborhood of the Boleslaw Mine and Metallurgical Plant in Bukowno (Poland). The study obtained total cadmium concentration between 0.29 and 51.91 mg, zinc between 7.90 and 3,614 mg, and that of lead between 28.4 and 6844 mg kg- 1 of soil d.m. The solubility of the heavy metals in 1 mol dm- 3 NH4NO3 was 1–49 % for zinc, 5–45 % for cadmium, and \\ 1–10 % for lead. In 1 mol HCl dm- 3, the solubility of the studied metals was much higher and obtained values depending on the collection site, from 45 to 92 % for zinc, from 74 to 99 %, and from 79 to 99 % for lead. The lower solubility of the heavy metals in 1 mol dm- 3 NH4NO3 than 1 mol HCl dm- 3 is connected with that, the ammonium nitrate has low extraction power, and it is used in determining the bioavailable (active) form of heavy metals. Toxicity assessment of the soil samples was performed using two tests, Phytotoxkit and Microtox_. Germination index values were between 22 and 75 % for Sinapis alba, between 28 and 100 % for Lepidium sativum, and between 10 and 28 % for Sorghum saccharatum. Depending on the studied soil sample, Vibrio fischeri luminescence inhibition was 20–96 %. The sensitivity of the test organisms formed the following series : S. saccharatum [S. alba = V. fischeri [L. sativum. Significant positive correlations (p B 0.05) of the total and soluble contents of the metals with luminescence inhibition in V. fischeri and root growth inhibition in S. saccharatum were found. The general trend observed was an increase in metal toxicity measured by the biotest with increasing available metal contents in soils. All the soil samples were classified into toxicity class III, which means that they are toxic and present severe danger. Biotest are a good complement to chemical analyses in the assessment of quality of soils as well as in properly managing them.

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P. Palma, L. Ledo, S. Soares, I.R. Barbosa and P. Alvarenga
Integrated Environmental Assessment of Freshwater Sediments : a Chemical and Ecotoxicological Approach at the Alqueva Reservoir
Environmental Geochemistry and Health 2014 36, 2 209-223
Toxkits : Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Thamnocephalus platyurus
Topics : sediments

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Abstract : In order to study the pollution of an aquatic ecosystem, it is necessary to analyze not only the levels of chemical pollutants in water, but also those accumulated in the sediment matrix, as well as to assess its ecotoxicological status. The Alqueva reservoir, the largest artificial lake in Europe, was chosen as case study as it constitutes the most important water supply source in southern Portugal. It is located in the Guadiana River Basin, in a semi-arid region with high levels of water scarcity and where agriculture is one of the main activities. The evaluation of sediments comprised: (1) physical and chemical analysis (grain size, pH, organic matter, nitrogen, phosphorus); (2) potentially toxic trace elements (Cu, As, Pb, Cr, Cd, Zn and Ni); and (3) ecotoxicological evaluation with Vibrio fischeri, Thamnocephalus platyurus, Daphnia magna, and Heterocypris incongruens. Total trace element concentrations indicated that As, Cd, and Pb surpassed the Canadian levels for the protection of aquatic life, in most of Alqueva\'s sites. The results of the toxicity assessment showed that some locations induced acute and chronic toxicity in the species used. Further, the H. incongruens was the most sensitive species as far as the contamination found in the sediment is concerned, followed by the bacteria V. fischeri. This integrative approach, together with the water column quality assessment, allowed a comprehensive evaluation of the environmental quality of this strongly modified water body and will allow the implementation of remediation strategies to obtain a good ecological potential as proposed in the Water Framework Directive.

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M.E. Aydin, S. Tongur and S. Ozcan
Determination of Acute Toxicity of Wastes and Wastewaters
Proceedings of the 10th International Multidisciplinary Scientific GeoConference 2010 2 455-460
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : waste waters

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Abstract : Toxicities of waste samples taken from landfill site and waste water samples taken from outlet of sewerage system were investigated. Eluates were prepared for the waste samples taken from landfill. In order to determine toxicities of waste eluate and wastewater three different biotests namely Thamnotoxkit F^TM , Daphtoxkit F^TM, Protoxkit F^TM were employed. Test results were expressed as LC50 for Thamnotoxkit EC50 for Daphtoxkit and Protoxkit. Physical and chemical characteristics of wastes and wastewater samples were also determined and relation between physical and chemical parameters of the waste and waste water sample with toxicity results were evaluated.
Reference tests were carried out with K2Cr2O7 to check the test procedures prior to the analyses with samples. Sample toxicity results were reported as Toxic Units (TU) and sensitivities of each test for each type of samples were determined.

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K.F. Burga-Perez, H. Toumi, S. Cotelle, J.F. Ferard and D.M. Radetski
Sensitivity of Different Aquatic Bioassays in the Assessment of a New Natural Formicide
Journal of Environmental Science and Health. Part B, Pesticides, Food Contaminants and Agricultural Wastes 2013 48, 1 57-62
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - organic compounds; chemicals - pesticides; chemicals - biocides

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Abstract : Agrochemicals have the potential to cause deleterious effects on living organisms and therefore they must be subjected to various (eco)toxicological studies and monitoring programs in order to protect human health and the environment. The aim of this study was to assess the ecotoxicity of a new natural formicide with a battery of three classical and three ecotox-kit tests. The former tests were performed with Aliivibrio fischeri bacteria (Lumistox test), the cladoceran Daphnia magna and Pseudokirchneriella subcapitata algae, and the latter with Thamnotoxkit F^TM (Thamnocephalus platyurus), Ostracodtoxkit F (Heterocypris incongruens) and LuminoTox (photosynthetic enzyme complexes). In the range of formicide concentrations tested (from 0.06 to 2.0 g L(-1)), the measurement endpoint values varied from 0.79 g L(-1) for the algal test to > 2 g L(-1) for the LuminoTox and Ostracodtoxkit F® tests. Hierarchical sensitivity ranking based on the no-observed effect concentration (NOEC) values established to assess the formicide ecotoxicity was as follows: algal growth inhibition test ~ daphnid immobilization test ~ bacterial luminescence inhibition test > Thamnotoxkit F^TM > LuminoTox > Ostracodtoxkit F. Overall, results from the battery of bioassays showed that this formicide preparation presents low ecotoxicity as compared to the aquatic ecotoxicity of presently commercialized formicides. In conclusion, classical aquatic bioassays are more sensitive than ecotox-kit tests in the assessment and monitoring of the new natural formicide.

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H. Dolezalova-Weissmannova, I. Stepankova, M. Vavrova, and A. Lapcikova
Determination of Toxicity of Musk Compounds using Alternative Tests of Ecotoxicity
Chemické Listy 2013 107 172-177
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - organic compounds

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Abstract : Alternative ecotoxicity tests Daphtoxkit F^TM, Thamnotoxkit F^TM, Rotoxkit F^TM and Artemia salina were used for determination of ecotoxicity of the musk compounds galaxolide, tonalide, musk ketone, musk xylene. The results confirmed a higher toxicity of polycyclic musk compounds than that of nitromusk compounds. The highest toxicity was observed for galaxolide. Polycyclic musk compounds showed high toxicity for aquatic invertebrates. The freshwater crustacean Daphnia magna was confirmed as the most sensitive whereas Brachionus calyciflorus the least sensitive organism. As to the toxicity of nitromusk compounds, the freshwater crustaceans Thamnocephalus platyurus and Daphnia magna were the most sensitive organisms. By comparing the ratio of 24 h/48 h LC50 and 24 h/48 h EC50, a significant effect of exposure was found especially in the case of tonalide. All the musk compounds pose risk for aquatic ecosystems and can be considered toxic for aquatic invertebrates.

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A. Zgorska, A. Arendarczyk and E. Grabinska-Sota
Toxicity Assessment of Hospital Wastewater by the Use of a Biotest Battery
Archives of Environmental Protection 2011 37, 3 55-61
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : waste waters

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Abstract : In the paper toxicity assessment of hospital wastewaters samples was performed using direct-contact tests consisting of five species, which represent three different trophic levels of the food chain. IC50 or EC50 values were estimated for each tested organism : Pseudokirchneriella subcapitata IC50/72h 18.77%, Daphnia magna EC50/48h 20.76%, Thamnocephalus platyurus EC50/24h 22.62%, Artemia salina EC50/24h 59.87% and Vibrio fisheri EC50/15min 46.17%. Toxic potential of hospital wastewater was described using a system of wastewater toxicity classification. The toxic units (TU) values estimated for each test indicate that hospital wastewaters are toxic (Class III). The variable results of the tests\' sensitivity confirmed the need of application of microbiotests battery with organisms of different trophic levels.

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G. Nalecz-Jawecki, L. Szczesny, B. Adomas, A.I. Piotrowicz-Cieslak and J. Sawicki
Evaluation of Toxicity of Anthelmintic Pharmaceuticals with Culture-Free Microbiotests
Fresenius Environmental Bulletin 2011 20, 4a 1093-1096
Toxkits : Ostracodtoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Heterocypris incongruens; Thamnocephalus platyurus
Topics : chemicals - drugs

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Abstract : Anthelmintic compounds are widely used in the world against endo- and ecto-parasites in medicine and veterinary medicine. Several groups of antiparasites are used comprising avermectins and benzimidazoles. Avermectins due to their high potency and wide spectrum of activity are some of the most potent compounds and they are also used as insecticides.

The goal of the present study was the evaluation of the sensitivity of culture-free microbiotests to selected anthelmintic pharmaceuticals. Four tests were applied with aquatic test biota: bacteria, protozoa, rotifera and crustacea. Moreover, a direct contact test for freshwater sediments (Ostracodtoxkit F^TM) was utilized. In this assay a freshwater benthic ostracod crustacean Heterocypris incongruens freshly hatched from cysts is used as a test biota. Seven pharmaceuticals were tested: avermectins (abamectin, ivermectin, doramectin), benzimidazoles (albendazole, mebendazole, thiabendazole) and morantel.

Avermectins were not toxic to luminescent bacteria Vibrio fisheri and protozoan Spirostomum ambiguum, but they were toxic to rotifer Brachionus calyciflorus, with 24h-LC50 values from 1.57 to 2.38 mg l^-1. The highest toxicity was observed for crustaceans, both planktonic Thamnocephalus platyurus and benthic Heterocypris incongruens. LC50 values ranged from 5.7 to 25.2 µg l^-1. Morantel did not cause adverse effects in any microbiotests at concentrations up to 10 mg l^-1.

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M. Riepsaite
Data of Petroleum Hydrocarbons and the Dispersing Agents 'Simple Green' Toxicity Assessment
European Directory of Marine Environmental Data (EDMED) 2002
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - hydrocarbons; chemicals - surfactants

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Abstract : The aim of the experiment was to determine the influence of petroleum hydrocarbons and the dispersing agent ‘Simple green’ on organisms in fresh and sea water using TOXKIT microbiotests and to define which toxic substance - the petroleum hydrocarbons or the dispersing agents are more harmful for water biota. In order to achieve the results bioresearches with microbiotests (Daphtoxkit F^TM (Daphnia magna), Thamnotoxkit F^TM (Thamnocephalus platyurus), Artoxkit M^TM (Artemia salina), which analyze the influence of the dispersing agents and the petroleum hydrocarbons on organisms, were performed, the sensibility of the test - organisms was analyzed and determined, and the toxicity of the investigated substances was evaluated. After the bioresearches with the mentioned microbiotests was performed, the toxicity of the investigated substances for test organisms was determined : LC50 (LC50 - lethal concentration) of the emulsion on the test organism D. magna 48h LC50 =0.3 mg/l; the LC50 of the dispersing agent ‘Simple green’ on : D. magna - 24h LC50=109.4 mg/l and 48h LC50=75.7 mg/l; on Th. platyurus - 24h LC50=108.3 mg/l, A. salina - 24h LC50=843.4 mg/l and 48h LC50=702.4 mg/l; LC50 of the dispersing agent ‘Simple green’ in the mixture of the emulsion : D. magna - 48h LC50=164 mg/l ‘Simple green’, Th. platyurus - 24h LC50=183 mg/l ‘Simple green’, A. salina - 24h LC50=698 mg/l ‘Simple green’ and 48h LC50=440 mg/l ‘Simple green’. The LC50 of petroleum hydrocarbons and the dispersing agent ‘Simple green’ on water organisms is different though there is no doubt about their toxicity. After the evaluation of the effects produced by the dispersing agent, the emulsion and the mixture of the emulsion and the dispersing agent ‘Simple green’ those substances according to the toxicity can be lined up this way : Emulsion > the mixture of the emulsion and the dispersing agent ‘Simple green’ > the dispersing agent. In conclusion, it is possible to state that the oil and its products are more toxic than modern dispersing agents. However, it is necessary to perform repeated analyses in order to receive more precise information.

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A. Rouvalis, Ch. Theodoropoulos and J. Iliopoulou-Georgudaki
Assessment of Toxicity of the Untreated and Pleurotus ostreatus Treated Olive Mill Wastewater by using Microbiotests
International Journal of Environmental Engineering 2013 5, 4 373-386
Toxkits : Daphtoxkit F^TM pulex; Thamnotoxkit F^TM
Test Species : Daphnia pulex; Thamnocephalus platyurus
Topics : waste waters

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Abstract : Toxicity of raw and biotreated olive mill wastewater (OMW) was tested using three organisms, Daphnia pulex, Thamnocephalus platyurus and zebrafish (Danio rerio). Untreated samples demonstrated LC50 values of 1.27%, 0.96% and 0.48% v/v against the aforementioned organisms respectively. After treatment with the white rot fungi Pleurotus ostreatus, 50% and 56% reduction in toxicity values were observed for Thamnocephalus platyurus and zebrafish respectively, but samples were categorised as ‘very toxic’. A significant reduction in phenols (53%), lignin (74%) and tannin (73%) as well as a differentiation in the impact of the physicochemical parameters on toxicity between the two groups of samples was detected. Toxicity values of untreated samples against the first two aforementioned tests were highly related to phenols and tannins, whereas of treated samples, only to ammonium and phenols. According to the present study, OMW treatment with Pleurotus ostreatus, despite the significant reduction in the toxic contents, does not produce effluents environmentally acceptable for safe disposal.

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E.P.C. Lai
Bioassay Kits for Ecotoxicological Testing of Wastewaters with Nanoparticles
Biochemistry & Analytical Biochemistry 2013 2, 3
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : waste waters; nanoparticles

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Abstract : Evaluation of complex wastewaters should include acute/chronic ecotoxicological tests and the potential for endocrine disruption to complement the physicochemical characterization. The advantages of bioassay (with a bacterium, alga or crustacean) are simplicity, speed and low cost for monitoring toxicity over time. Its strongest attribute is usefulness as a primary screening test for a broad spectrum of toxicants. Several bioassay kits are commercially available, including the Lux-fluoro, Polytox, Microtox and Thamnotox kits that require no specialized equipment. A combined approach using instrumental methods for chemical analysis and bioassays for ecotoxicological testing would be extremely important to hazard/risk assessment of wastewater treatment plant discharges.

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H. Heida and R. van der Oost
Sediment Pore Water Toxicity Testing
Water Science and Technology 1996 34, 7-8 109-116
Toxkits : Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : sediments; sediment pore waters

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Abstract : The toxicity of the pore water from the sediments of nine fresh water systems in and around Amsterdam City was studied with the aid of four acute toxicity tests. Based on preliminary chemical analysis, the sediments of three of the tested systems had to be considered strongly contaminated, according to the quality standards derived from the Dutch Law on Soil Pollution Control. In addition, the acute toxicity of groundwater from a heavily contaminated industrial building site was also examined. The following toxicity tests were used.
1. Microtox bioluminescence assay, or Microtox test. This test is based on the inhibition of bioluminescence of Photobacterium phosphoreum; 2. Rotoxkit F, based on the LC50 of Brachionus calyciflorus; 3. Thamnotoxkit F, based on the LC50 of Thamnocephalus platyurus; 4. Toxichromotest, based on the inhibition of ß-galactosidase formation in Escherichia coli. The objective of the study was to test the feasibility of the toxicity tests as a tool for the risk evaluation of contaminated sites, both in water and on land. The results indicated that the sediments of three of the eight sites studied could be classified as toxic and one site even as very toxic. The outcome of the toxicity testing confirmed the results of the chemical analysis for only one site. It appeared that the Microtox and the Thamnotoxkit F tests were the most sensitive acute toxicity tests. It could be concluded that the resolution of the toxicity tests scrutinized was insufficient to reliably discriminate the environmental quality of the sediments at the locations studied. The bioassays, however, are useful in determining remediation priorities of contaminated sites.

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Z. Holubova, L. Sommer, M. Moos and J. Kuta
Simultaneous Determination of Metal Traces using SPE Preconcentration on Amberlite XAD -7 by ICP -MS in Environmental Water and Sample Ecotoxicology Assessment
Parlar Scientific Publications 2012 21, 10A 3127-3135
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - metals

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Abstract : This study has been focused on the determination of nine trace elements Be-9, V-51, Co-59, Ni-60, Y-89, led, (208)pb, Th-232 and U-238 by inductively coupled plasma mass spectrometry (ICP-MS) in natural waters and water samples after mining and processing of uranium. An ICP-MS spectrometer Agilent 7500ce, with a plasma generator of 27.12 MHz, power output of 1500 W and concentric nebulizer MicroMist (TM) with a cooled Scott chamber was used. The nonspectral interferences were eliminated by using a solution of internal standards Li-6 for Be-9, Sc-45 (V-51), Ge-72 (Co-59, Ni-60, (89)y), Rh-103 (Cd-111), Bi-209 ((208)pb, Th-232, U-238) The spectral interferences were eliminated by using the Helium collision cell during measurement. Monitored interfering elements were K+, Na+, NH4+ Ca2+, Mg2+, Al3+, Fe3+ and Cl-, HCO3-, SO42-, PO43-. For the preconcentration polymeric sorbent Amberlite XAD-7 was used. Elements were retained as their chelates with different organic reagents: 4-(2-pyridylazo) resorcinol (PAR), 3,4-Dihydroxy-9,10-dioxo-2-anthracenesulfonic acid sodium salt (ALS), 8-Hydroxyquinoline-5-sulphonic acid (8HQS) and Ammonium pyrrolidinedithiocarbamate (APDC). The sorption process was studied under various conditions with respect to pH. Sorbents were also conditioned with different surfactants: Benzyldimethyltetradecylammonium chloride (Zephyramine), 1-etoxycarbonyl-pentadecyl-trimethyl-ammonium bromide (Septonex (R)), Brij 35 or Sodium Dodecylsulphate (SDS). Various elution mixtures were also tested, the best results were obtained for the mixture of acetone and ethanol (1:1) in the presence of 1 mol.l(-1) HCl. The excess of acid and organic solvents were evaporated in Teflon (R) dishes prior to the determination by ICP-MS. The Thamnocephalus platyurus as Thamnotoxkit F^TM, Daphnia magna as a Daphtoxkit F^TM and Artemia salina as Artoxkit M^TM are organisms, which were used for ecotoxicology assessment of water samples after mining of uranium.

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C. Blaise, F. Gagné, J. Auclair, D. Maysinger and P. Sutthivaiyakit
Ecotoxicity of a Potential Drug Nano-Formulation : PAMAM-dendrimer and Minocycline
Journal of Xenobiotics 2014 4, 4902 85-87
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - drugs; chemicals - nanoparticles

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Abstract : The varied composition, size, and shape of nanomaterials (1-100 nm size range) offer numerous exciting possibilities for the development of new industrial, biomedical, electronic products and have the potential to stimulate the global economy. On the darker side, the marked research efforts presently deployed to develop novel applications with\r\nnanomaterials will eventually lead to some releases in the aquatic environment likely via urban industrial/municipal point sources of pollution. This is clearly cause for concern as recent studies suggest that adverse health and environmental effects are linked to nanoproduct pollutants.
In the biomedical field, drug delivery systems combining nano-dendrimers (as a platform for delivery) and specific guest molecules (e.g. pharmaceuticals) are being investigated for efficient treatment of diseases (e.g., cancer, inflammation, cardiac and microbial problems). While characteristics (molecular size, shape, dimension, density, polarity, flexibility, solubility, drug carrying capacity, etc.) of dendrimers will vary based on their construction, cationic dendrimers are among the most common candidates in terms of pharmaceutical development and they are being considered for\r\nthe drug delivery of anti-microbials such as minocycline. In this study, we investigated the ecotoxicity of three PAMAM dendrimers (artificial macromolecules with tree-like structures, described in the methods section, and of minocycline, individually and in combination by conducting toxicity tests with microorganisms representing different levels of biological organization. Our objectives are to obtain preliminary information on the potential hazard of PAMAM dendrimers and minocycline.

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H. Watanabe, F. Nakajima I. Kasuga and H. Furumai
Toxicity Evaluation of Road Dust in the Runoff Process using a Benthic Ostracod Heterocypris Incongruens
Science of the Total Environment 2011 409, 12 2366-2372
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : solid wastes; solid waste leachates

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Abstract : Road dust is considered an important source of sediment contamination; however, there are few studies on the toxicity of road dust on benthic organisms. This study evaluates the toxicity of road dust on the benthic ostracod, Heterocypris incongruens, through a 6-day direct exposure experiment. All six road dust samples collected in heavy traffic areas caused high mortality of the ostracod, whereas the road dust collected in a residential area did not show toxicity to the ostracod. After maintaining the mixture of road dust and water for 24h, the toxicity of the road dust decreased significantly for three of the six samples in the heavy traffic areas, suggesting these road dust samples become less toxic in the surface runoff process. In addition, we conducted the same toxicity test on manipulated road dust using different solid/liquid ratios and holding times to evaluate the change in toxicity caused by the runoff process. Wet road dust that had been separated from a dust-water mixture after a holding time of 1h or 24h did not show lethal toxicity, while the water-soluble fraction of the mixture caused high mortality of the ostracod at a solid/liquid ratio of 1:2 and 1:4. However, after a 7-day holding time of the dust-water mixture, the wet road dust and the water-soluble fraction showed lethal toxicity to the ostracod. These results suggest that toxicants of road dust seemed to exist mainly in water soluble fraction eluted off by rain water; however, particle-bound fraction again showed lethal toxicity after 7 days of incubation.

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T. Steliga
The Use of Biotests in Estimation of Weathered Drilling Waste Bioremediation
Archives of Environmental Protection 2011 37, 2 61-79
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : solid wastes; chemicals - hydrocarbons; bioremediation; plant tests; soils

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Abstract : Petroleum products are complex mixture of compounds of varied biological properties. They can cause harmful changes in contaminated ecosystems and threaten humans and living organisms as well. Bioremediation (including bioremediation stimulated by biogenic substances and inoculation with biopreparations from autochthonous bacteria and fungi) can result in creation of metabolites of a varied structure and biological activeness, which has been partly recognised. Some of them are more toxic than an initial substrate. Besides, they might have mutagenic features and be responsible for cancer. Estimation of bioremediation effectiveness in waste pits was completed with toxicological monitoring. It was led with the use of living organisms as biomarkers representing all trophic levels of a chosen ecosystem : producers, consumers and reducers. This process enables total estimation of natural environment conditions. The aim of the research was to determine the influence of petroleum contaminants and indirect metabolites (produced during bioremediation) on soil biocenose. The results of biotests (toxicity, phytotoxicity and genotoxity) have been taken into account. The following biotests, prepared and produced by Microbiotest, have been applied: Phytotoxkit, Ostracodtoxkit F^TM, acute toxicity tests Microtox^© Solid Phase and Ames mutagenicity tests. The obtained results enabled observation of changes in toxic properties during purification of the soil taken from waste pits. In addition, it can be claimed whether the areas are suitable for forest usage.

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D. Sheahan and T. Fisher
Review and Comparison of Available Testing Approaches and Protocols for Testing Effects of Chemicals on Sediment-Dwelling Organisms with Potential Applicability to Pesticides
European Food Safety Authority - External Scientific Report 2012
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : review; chemicals - pesticides; test selection; sediment and soil contact tests

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Abstract : Risk assessment of pesticides aims to ensure they cause no harm to humans, domestic animals or the environment. This report reviews the current position of sediment toxicity testing with potential applicability to pesticide assessment. A literature search identified relevant literature on protocols to test the effects of chemicals and in particular pesticides upon sediment dwelling freshwater and marine organisms. An assessment was made of scientific literature, previous reviews and reports from Government and industry websites. Information was obtained for oligochaete and polychaete worms, crustacea, the branchipoda (which includes Daphnia magna), copepoda, ostracoda amphipoda and isopoda, insects, molluscs and the group including sea urchins. A brief note is given on the ecology of each its tolerance of sediment type, test methods and endpoints. The report considers factors such as sediment quality and feeding of test organisms that may affect the chemical availability, quality of test water and the final interpretation of the tests. The statistical analysis of test data is also considered including the preferred use by some authors of regression based techniques with derivation of predicted low percentage effects e.g. 10% considered as nominal ‘no observed effect concentrations’ for use in risk assessment. It is recommended that studies should make use of all data availble in time-based assessments and that biologically/ecologically relevant endpoints should be used. Existing freshwater sediment tests recommend use of Chironomid species, oligochaetes and crustacea to cover relevant uptake and exposure routes. Existing marine tests that are ready for adoption for pesticide testing include those for several polychaete, amphipod and copepod species that allow assessment of reproduction and for some, full life-cycle tests. Bivalve mollusc species are used mostly for studies of contaminant bioaccumulation from sediments but studies with marine gastropod molluscs offer potential for a first tier of chemical assessment that includes reproduction.

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H. Watanabe, F. Nakajima, I. Kasuga and H. Furumai
Application of Whole Sediment Toxicity Identification Evaluation Procedures to Road Dust using a Benthic Ostracod Heterocypris Incongruens
Ecotoxicology and Environmental Safety 2013 89 245-251
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : solid wastes; solid waste leachates

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Abstract : Road dust is considered to be an important source of sediment contamination in receiving water bodies; however, few studies have evaluated the toxicity of road dust to benthic organisms. This study evaluated the toxicity of road dust to a benthic ostracod, Heterocypris incongruens, using a six-day direct exposure experiment. We applied whole sediment toxicity identification evaluation (TIE) methods to identify the primary group of toxicants in road dust. Three road dust samples from Tokyo caused high ostracod mortality. The addition of hydrophobic adsorbents, Ambersorb and XAD, eliminated toxicity in all samples, suggesting that hydrophobic compounds were the main toxicants in road dust samples. A cation exchange resin, Chelex, also reduced the toxicity of two samples, although the measured concentrations of dissolved heavy metals in the test solution did not exceed the LC(50) values in the literature. In addition, the sum of toxic unit (TU=measured concentration/LC(50)) of each individual metal which predicts the toxicity of the metal mixtures did not exceed 1.0 in all samples, suggesting that heavy metal mixtures did not have additive effects. We hypothesized that the toxicity reduction by XAD and Chelex was due to the removal of hydrophobic compounds, rather than heavy metals. Thus, a toxicity test was conducted on fractions eluted with organic solvents from the XAD and Chelex recovered from one of the road dust samples. Methanol-eluted fractions of XAD and Chelex showed 100 percent ostracod mortality, indicating that the hydrophobic organic compounds removed by these adsorbents were the principal toxicants in the road dust sample.

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F. Ruiz, M. Abad, A. M. Bodergat, P. Carbonel, J. Rodriguez-Lazaro, M.L. Gonzalez-Regalado, A. Toscano, E.X. Garcia and J. Prenda
Freshwater Ostracods as Environmental Tracers
International Journal of Environmental Science and Technology 2013 10 1115-1128
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : review

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Abstract : This paper revises the response of freshwater ostracods to different environmental conditions and anthropogenic impacts, with a worldwide overview of the potential use of these microcrustaceans as bioindicators and several examples of applications in different scenarios. The development of either a single species or an ostracod assemblage is influenced by physical–chemical properties of waters (salinity, temperature, pH, dissolved oxygen), hydraulic conditions, bottom grain sizes or sedimentation rates. In addition to population and community changes, morphological and geochemical changes can also be detected in the ostracod carapace, which serves as a tracer of the water quality. All these features permit to delimit the spatial effects of urban sewages, mining effluents, agricultural wastes, watershed deforestation or road building. These data are the basis for the palaeoenvironmental reconstruction of cores, with an interesting application to archaeology. In addition, favourable results of recently developed bioassays, coupled with an important variability of local assemblages under changing conditions in both waters and sediments, suggest that these microcrustaceans may included between the most promising sentinels groups in freshwater areas. These microcrustaceans show high sensitivity to pesticides, herbicides, heavy metal pollution and oil inputs.

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R. Khanal, H. Furumai and F. Nakajima
Toxicity Assessment of Size-Fractionated Urban Road Dust using Ostracod Heterocypris incongruens Direct Contact Test
Journal of Hazardous Materials 2014 264 53-64
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : solid wastes; solid waste leachates

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Abstract : Urban road dusts (URDs, n=10) were collected from arterial, residential, parking area in highway and highway roads in and around Tokyo, Japan, to characterize toxicity of size-fractions by the ostracod Heterocypris incongruens direct contact test. The URDs were collected with vacuum cleaner and highway sweeping vehicles, dried and size-fractionated before conducting toxicity test. The LC20 and LC50 of URDs varied (v/v) from 1.6 to 49%, and 3.8 to 67% respectively. Cluster analysis of URDs based on the concentration of heavy metal and PAHs standardized with the organic matter content was able to differentiate URDs into two groups, one group of higher toxicity and the other group of medium and lower toxicity. Mortality of ostracod decreased for some of the URDs when holding time of URD-water mixture was changed from 1h to 24h prior to the toxicity test. Fraction of fine particles was not always more toxic than the other fractions of coarse and medium particles. Site specific differences in toxicity of size-fractionated URDs indicated the complexity in defining URD toxicity as there could be co-existence of various non-targeted toxicants.

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J.B. Sevilla, F. Nakajima and I. Kasuga
Dose-Response Relationship of Solid-Phase Metals to Benthic Ostracod Heterocypris incongruens
From 13th International Conference on Urban Drainage, Sarawad, Malaysia, 7-12 September 2014
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics :

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Abstract : Urban road runoff contains toxic substances, which may contaminate the sediment in the receiving water bodies. Ostracod Heterocypris incongruens is one of the suitable test organisms for sediment toxicity evaluation, however, fundamental information on dose-response relationship of solid-phase toxicants is still limited. The objectives of this study were to obtain dose-response curves for solid-phase heavy metals (Cd, Cu and Zn) to ostracod mortality and to compare two exposure routes (aqueous and dietary exposures). For the preparation of solid-phase heavy metals, microgreen algae Scenedesmus acutus were cultivated in C medium with different metal concentrations for 10 days. In the dietary exposure tests, the ostracod was exposed to the metal-contaminated algae in clean standard freshwater. Mortality of ostracod increased with increasing total accumulated Cd in the food, up to 57% at the highest Cd content. Copper- and Zn-contaminated algae affected the mortality of ostracod, 12 to 100% and 32 to 90% respectively. The aqueous exposure route was more important in Cd toxicity while dietary Cu was more toxic than dissolved form in this study. The dose-response relationship of solid-phase metals obtained in this study would be useful for users of the standardized ostracod toxicity test to estimate the metal contribution to the ostracod mortality.

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A. Melnyk, K. Kuklinska, L. Wolska and J. Namiesnik
Chemical pollution and toxicity of water samples from stream receiving leachate from controlled municipal solid waste (MSW) landfill
Environmental Research 2014 135 253-261
Toxkits : Phytotoxkit; Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : landfill leachates; plant tests; soils

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Abstract : The present study was aimed to determine the impact of municipal waste landfill on the pollution level of surface waters, and to investigate whether the choice and number of physical and chemical parameters monitored are sufficient for determining the actual risk related to bioavailability and mobility of contaminants.
In 2007–2012, water samples were collected from the stream flowing through the site at two sampling locations, i.e. before the stream’s entry to the landfill, and at the stream outlet from the landfill. The impact of leachate on the quality of stream water was observed in all samples. In 2007–2010, high values of TOC and conductivity in samples collected down the stream from the landfill were observed; the toxicity of these samples was much greater than that of samples collected up the stream from the landfill. In 2010–2012, a significant decrease of conductivity and TOC was observed, which may be related to the modernization of the landfill. Three tests were used to evaluate the toxicity of sampled water. As a novelty the application of Phytotoxkit for determining water toxicity should be considered. Microtox^® showed the lowest sensitivity of evaluating the toxicity of water samples, while Phytotoxkit showed the highest. High mortality rates of Thamnocephalus platyurus in Thamnotoxkit F^TM test can be caused by high conductivity, high concentration of TOC or the presence of compounds which are not accounted for in the water quality monitoring program.

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Y. Huang, C.C. Aldasoro, G. Persoone and D. Wlodkowic
Automation of Artoxkit M^® Ecotoxicity Biotests using a Microfluidic Lab-on-a-Chip Technology
SETAC Europe 25th Annual Meeting, 3-7 May 2015, Barcelona, Spain
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : test development

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Abstract : Despite its critical importance, the broad deployment of high-throughput whole-organism ecotoxicity tests is profoundly limited by the lack of appropriate bio-compatible automation, integrated optoelectronic sensors, and the associated electronics and analysis algorithms. These major shortcomings have not been yet addressed, and as a result all such biotests are still performed manually, are time consuming, laborious and not available for high-throughput analysis. In this work, we present development of a miniaturized Lab-on-a-Chip (LOC) platform for automation of acute ecotoxicity test based on a marine crustacean Artemia franciscana (Artoxkit M^®). We for the first time demonstrate development of a proof of-concept technology for rapid assessment of sub-lethal, behavioral eco-toxic effects. The system working principles incorporated a time-resolved video data analysis to dynamically assess impact of the reference toxicant on swimming behavior of marine crustacean Artemia franciscana. Our system design combined: (i) innovative LOC flow-through device for on-chip keeping of Artemia sp. ; (ii) automated mechatronic hardware for LOC fluidic system actuation and data acquisition; and (iii) innovative video analysis algorithms for the segmentation, tracking, visualization and movement analysis. The algorithm are written in Matlab and released open-source at the website www.phagosight.org. The chip-based device supported rapid loading and keeping of free swimming crustacean larvae suspended in a continuous microfluidic perfusion as a mean of toxin delivery in both open-loop and closed-loop modalities. This system was also capable of performing fully programmable time-lapse and video-microscopy of multiple samples for rapid ecotoxicity analysis. Custom video algorithms were then developed for scoring dynamic features of video frames, capturing detailed statistical information on test specimen movements. This enabled development of a new test protocol to dynamically detect sub-lethal behavioral end-points based on a swimming behavior of Artemia franciscana. This work provides a foundation for automated high throughput ecotoxicity biotests using innovative Lab-on-a-Chip technologies. A key advantage of this flow-through system as compared to conventional static testing is the miniaturization and automation of analysis and special emphasis on sub-lethal behavioral parameters.

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A.O. Jimenez, S. Novais, F. Beltran, A. Espejo, C. Gravato and M. Lemos
Antioxidant Enzyme Activities and Lipid Peroxidation in Daphnia magna Exposed to an Effluent Spiked with Pharmaceuticals and under Single Ozonation
SETAC Europe 25th Annual Meeting, 3-7 May 2015, Barcelona, Spain
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : effluents; chemicals - drugs

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Abstract : Application of Advanced oxidation processes used to remove pharmaceuticals from wastewater generates reactive oxygen species (ROS) that may reach superficial waters through discharges of effluents from sewage treatment plants. The main aim of this study was to clarify know if increased levels of ROS produced after application of a single ozonation treatment of a real effluent would induce changes in the antioxidant enzymatic activities superoxide dismutase and catalase, that protect tissues against superoxide anion (O2.-) and hydrogen peroxide (H2O2) radicals respectively, and the level of oxidative damage (level of lipid peroxidation) in D. magna.
Firstly, the wastewater was spiked with 9 selected pharmaceuticals (acetaminophen, antipyrine, caffeine, carbamazepine, diclofenac, hydrochlorothiazide, ketorolac, metoprolol and sulfamethoxazole at 200 µgL^-1 each) and then a single ozonation treatment was applied. Oxidative stress biomarkers were studied after an acute exposure period of 48 h to this treated effluent. The most marked change observed was an increase in the activity of SOD in daphnids that could be considered as a response to cope with increased levels of oxyradicals (O2.-) produced during the single ozonation treatment of the spiked effluent.
See corresponding Poster

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L. Marziali, L. Guzzella, G. Tartari, C. Bravi, S. Castelli, A.M. Ribaudo, C. Rondanini and R. Serra
A Protocol for Assessing Sediment Toxicity in Reservoirs before Flushing
SETAC Europe 25th Annual Meeting, 3-7 May 2015, Barcelona, Spain
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments

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Abstract : Reservoirs are often characterized by high siltation rates which impair the storage capacity. Accumulated sediments are frequently removed by flushing, causing physical mechanical impacts on aquatic organisms in the downstream rivers. In some cases chemical compounds (organic and metals) adsorbed on sediments may induce toxic effects, mainly in a long-term perspective.
PrATo is a protocol designed for assessing the toxic potential of sediments released downstream and includes methods for sampling, chemical analysis and ecotoxicological evaluation of sediments, as well as criteria for risk assessment, based on cross-interpretation of results deriving from chemical and ecotoxicological analyses. In particular, different dilution rates of flushing silt are calculated in order to prevent adverse effects. The trial of the PrATo protocol application in some Italian reservoirs will be presented. Preliminary results show that the definition of threshold concentrations may be biased by high natural background values (particularly for trace metals), by different on-site bioavailability and by synergistic effects of pollutants. Therefore, toxicity bioassays are essential to define the toxic potential of sediments.
The protocol is an example of analytical approach useful for supporting decision making.
See corresponding Poster

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Y. Jung, S. Baik, C. Park and S. Kim
Evaluation of Environmental Toxicity for Surface-Modified Silver Nanoparticles on Aquatic Organisms
SETAC Europe 25th Annual Meeting, 3-7 May 2015, Barcelona, Spain
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - nanoparticles; chemicals - metals

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Abstract : With the increased use and application of surface modified silver nanoparticles (AgNPs), many concerns have been raised for their toxicity after releasing and discharging to aquatic ecosystems which may pose possible risks to the environment. Therefore, in this study, ecotoxicological evaluation of surface-modified AgNPs on aquatic organisms was conducted. Aquatic organisms for the ecotoxicological evaluation were luminescent bacteria and Daphnia magna, and four different surface coated AgNPs with the same core diameter of about 20 nm were used. Surface coating materials for AgNPs were citrate (Cit), tannic acid (Tan), polyethleneglycol (PEG), and branched polyethleneimine (BPEI) and ζ potentials of those NPs were -27.1 mV, -10.7 mV, -20.5 mV, and 18.3 mV, respectively.
For an acute (15 min) ecotoxicological screening test, luminous microbial array for risk assessment (LumiMARA), which consists of 2 fresh water and 9 marine bacteria, was applied. The tendency of toxic effects for target AgNPs on the marine bacteria are following order; BPEI-AgNPs > PEG-AgNPs > Tan-AgNPs > Cit-AgNPs, resulting on the positively charged AgNPs with higher salt stability showed more toxic on those marine luminescent bacteria. The other acute (48 h) ecotoxicological test was conducted using Daphnia magna as the aquatic organisms in accordance with OECD guideline 202. Mortality and immobility of Daphnia magna exposed to target AgNPs were investigated. 50% effective concentration (EC50) and 50% lethal concentration (LC50) of BPEI-AgNP on Daphnia magna were calculated as 20.38 µg/L and 64.19 µg/L, respectively, and EC50 for Cit-AgNP was calculated as 110.64 µg/L. However, there is still insufficient understanding of main factors which influence the organisms. The NPs size, surface coating materials or ions released from NPs as well as reactive oxygen species (ROS) can contribute to toxicity of AgNPs to the organisms. Furthermore, regarding the safety of AgNPs with environmentally relevant conditions, it is necessary to deliberate more complicated matrix considering pH, ionic strength, organic matters, etc.
Prior to the critical assessment of the mechanism, we tried to identify the quantification and size distribution of AgNPs taken up by organisms in the test media by inductive coupled plasma mass spectrometry (ICP-MS).

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L. Molano Leno, M. Casado-Martinez, N. Estoppey, F. de Alencastro, I. Werner and B. Ferrari
Bioavailability and Toxicity of Sediment-Bound Contaminants (PCBs and Metals) using an Integrative Approach : a Case Study in the Venoge River (Switzerland)
SETAC Europe 25th Annual Meeting, 3-7 May 2015, Barcelona, Spain
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments

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Abstract : The Venoge is a small river flowing into Lake Geneva, the largest water body of Switzerland. Previous monitoring campaigns have shown increased concentrations of PCBs in fishes, which were reported to exceed maximum levels set by the European Union for human consumption. Water quality of the river is well characterized, both in terms of chemical contamination and benthic macro-invertebrate community structure. However, no information of sediment quality is available so far. To assess the potential impact of sediment bound contaminants on habitat quality, we used an integrated methodology that includes sediment physico-chemical measurements, a battery of sediment toxicity tests, and measurement of contaminants in benthic invertebrates. Sediments were collected at several sampling sites along the Venoge and analyzed for PCBs and metals as well as ancillary parameters important for determining contaminant bioavailability. The battery of tests used for the characterization of sediment ecotoxicity included the growth inhibition test using the aquatic macrophyte Myriophyllum aquaticum (ISO 16191); the growth and mortality test using the ostracod Heterocypris incongruens (ISO 14371); and the emergence test using midge larvae (Chironomus riparius, AFNOR 90-339). Bio-accumulated contaminants were assessed in midge larvae. Four sampling sites were selected according to the identified sources of contamination and previous results of water quality. Results of the toxicity tests showed no effect on ostracod mortality but growth inhibition at the sampling site closest to the mouth of the river. On the contrary, percent emergence of midge larvae was decreased only at the site closest to the effluent outfall of the waste water treatment plant.
Chemical analyses of sediments and midge larvae are on-going. The results will be discussed based on the two objectives of this study : 1) to assess whether sediments are contributing to the impairment of the beneficial uses of this aquatic ecosystem (ecosystem services) and 2) to assess which of the selected tools can provide useful information within a decision-making framework.

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Y. Marneffe, M. Hemart, J. Berger and M. Veschkens
Application of Complementary Methods in Chemical and Ecotoxicological Monitoring of Sediments under WFD in Wallonia (Belgium)
SETAC Europe 25th Annual Meeting, 3-7 May 2015, Barcelona, Spain
Toxkits : Algaltoxkit F^TM; Ostracodtoxkit F^TM; Rotoxkit F^TM short-chronic
Test Species : Brachionus calyciflorus; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : sediments; toxicity monitoring

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Abstract : Sediment remains important matrix for the monitoring of certain substances with significant potential for accumulation to assess long-term impacts of anthropogenic activity and trends. The EQS Directive (2008/105 EC amended by 2013/39/EU) provides that Member States should take measures with the aim of ensuring that existing levels of contamination in sediment will not significantly increase. Analyses of sediment can be a cost-effective approach for initial screening of areas for contamination.
In using sediment as a first level screening for certain chemicals, the monitoring program may be downscaled. Indeed, the initial screening may help to identify areas of concern and areas where additional effort is needed. Chemical analysis of priority pollutants is often not sufficient to explain ecotoxicological effects of these complex environmental samples. Risk assessment based on concentrations, e.g. of priority pollutants in sediments or water, obviously does not reflect the risk of the actual mixture of contaminants, but only the risk of those preselected toxicants. Bioassays are therefore useful tools for the evaluation of sediment in which both known and unknown contaminants are present. Effect-based monitoring is also useful for the development of investigative monitoring. Combining the three assessment methods (chemical, bioassay, ecology) in a Triad approach can give an answer that cannot be given by any of the individual methods by themselves.
From 2010 to 2012, 31 monitoring stations included in the surveillance monitoring program of the WFD in Wallonia (Belgium) were sampled for sediment. Bioassays (Chironomus riparius, Heterocypris incongruens for whole samples and Vibrio fischeri, Pseudokirchneriella subcapitata and Brachionus calyciflorus for pore waters) were carried out to determine the potential impact of contaminated sediment.
According to their level of toxicity for the different tests, the sediments of the surveillance monitoring stations were classified from non-toxic to extremely toxic. Priority substances selected as pertinent for a monitoring in sediments and other pertinent chemicals were analysed in each sample (63 µm fraction). The link between physico-chemical and ecotoxicological parameters was assessed by multivariate statistical analysis.
See corresponding Poster

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C. Park, M. Song, N. Choi and S. Kim
Acute Aquatoxicity Screening of Cellulosic Products Contained Glyoxal, using Multi-Strain Bacteria, Daphnia and Zebrafish Embryos
SETAC Europe 25th Annual Meeting, 3-7 May 2015, Barcelona, Spain
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics :

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Abstract : It is well known that cellulose ethers are water-soluble polymers derived from cellulose which is consisted natural organic compounds. These polymers have been specifically modified in a wide range of application, from construction products, ceramics, paints, personal care products, and pharmaceuticals. From June 2015, the information about the safety of cellulosic products contained an additives such as a cross-linking agent (i.e., glyoxal) on the human- and environment health will need to response in EU CLP (classification, labelling and packaging) legislation.
To screen the aqua-toxic effects of cellulosic products involved glyoxal with different concentration, the acute aqua-toxicity test using multi-strain environmental bacteria (microbial array for toxicity risk assessment, MARA), Daphnia magna (OECD test guideline 202) and zebrafish embryos (OECD test guideline 203) was conducted in this study. The acute aqua-toxicity using 3 different aquatic organisms (multi-strain bacteria, Daphnia manga, and zebrafish embryos) were limit and not significant. To better evaluate the aqua-toxic effects of cellulosic products contained cross-linking agents, the chronic toxicity test will be performed and the further studies regarding the toxic effects of cellulosic products by physical properties (e.g. solubility, viscosity, pH change, etc.) is also required.
See corresponding Poster

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A. Kovacs, N.-A. Abdel-Hameid, A. Acs, A. Ferincz and N. Kovats
A Novel Protocol for Assessing Aquatic Pollution, Based on the Feeding Inhibition of Daphnia magna
Knowledge and Management of Aquatic Ecosystems 2012 404, 07
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : rapid tests; test development; behavioural tests

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Abstract : In this study, sensitivity of a novel acute bioassay based on the feeding activity of Daphnia magna was assessed, using 2 and 4 h of exposure. For calibration purposes, results were compared with those of the standard immobility test as described by the ISO 6341:1996 standard. Using potassium dichromate as the reference chemical, after 4h of exposure the proposed protocol showed similar sensitivity in comparison with the standard, as the EC50 of the immobility test was 1.093 ± 0.098 mg^.L^-1, whereas the EC50 of the feeding inhibition bioassay was 1.742 ± 0.133 mg^.L^-1. In order to test the sensitivity of the bioassay, toxicity of two other contaminants, copper and wastewater, was estimated and the results were compared with those of the standard immobility test. For both cases, the feeding inhibition test showed higher sensitivity, as in the case of copper the EC50s were 0.0952 ± 0.0087 and 0.0753 ± 0.0152 mg^.L^-1, whilst the EC50 recorded for the 24h immobility test was 0.2407 ± 0.0159 mg^.L^-1. In the case of the effluent, EC50 values after 2 and 4 h of exposure were 15.698 ± 2.681 and 12.557 ± 2.358 expressed as % of the wastewater, respectively, whereas the EC50 of the immobility test was calculated to be 36.4688 ± 5.4887. Full article (from open access periodical : Knowledge & Management of Aquatic Ecosystems - KMAE)

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A. Acs, N. Kovats and M. Refaey
Novel Daphnia Test for Detecting Chemical Pollution
Environmental Security 2009 439-444
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : rapid tests; test development; behavioural tests

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Abstract : Daphnia magna (water flea) is perhaps the most commonly used test organism. As they represent primary consumers in freshwater aquatic ecosystems, their relevance in screening bioassays is very high. However, performing the standard Daphnia mortality test requires a 24–48 h exposure time, therefore no rapid response can be acquired on the risk posed by any environmental contamination. A novel bioassay has been elaborated and tested in our laboratory, using the above mentioned Daphnia species, but based on another ecological effect, the inhibition of feeding activity. Applicability and sensitivity of the test was assessed using potassium dichromate. Standard Daphnia test was also carried out as reference. The sensitivity range of the new method was similar to the reference test (EC50 was in the same magnitude in comparison to Daphnia magna mortality test). Its further benefit is that this bioassay is using a sublethal endpoint.

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M. Wieczerzak, G. Kudlak and J. Namiesnik
Environmental Risk Assessment of Exposure to Residues of Pharmaceuticals using a Battery of Bioassays
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - drugs

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Abstract : As a result of anthropopressure the environment becomes a reservoir for a wide range of contaminants. Generated due to human activities hazardous chemicals penetrate into various elements of the environment disrupting the functioning of ecosystems. In each ecosystem there is a delicate balance and a network of relationships between organisms from different trophic levels.
For thousands of years, natural selection has promoted species whose features allow surviving. Evolution has provided living organisms in a number of repair and detoxification mechanisms to avoid intoxication and accumulation of pollutants in the tissues and organs (Sytar et al. 2013, Wieloch et al. 2012).
Sadly the dynamic development of civilization caused that in recent decades, environmental changes occur at an alarming rate, without giving a chance for quick adaptation. Anthropogenic pollutants are a large group of different compounds in terms of physicochemical properties, some of which may adversely affect the biochemical processes going on in the living organism. Examples of biologically active compounds are pharmaceuticals and their residues accumulating in the environment.
Pharmaceuticals are synthetic or natural substances aimed at improving health and saving lives. Chemical with proven therapeutic effect undergoes many phases of clinical trials, to detect whether a given compound will be safe for the patient. Unfortunately, pharmaceuticals’ environmental fate studies are rare.
Interdisciplinary environmental studies require a lot of knowledge, one must take into account many factors such as the solubility of the substances as well as environmental factors (pH, salinity, temperature, etc.) coexistence ions and various other factors, of which the test substances can interact and also biological factors such as response (sensitivity) of the respective organisms from different trophic levels. There are three basic types of interaction between the factors in the environment :
- Additivity - the effect of the factors is the sum effect of various factors present in the mixture;
- Synergy - the effect of factors in total is greater than the sum of the effects of each factor separately;
- Antagonism - the effect of factors in total is lower than the sum of the effects of each factor separately.
The substances in the mixture may interact with each other living organisms, which can reduce or increase their toxicity depending on the sample matrix or observed organism or parameter (US EPA 2000 Wieczerzak et al. 2015).
Research carried out at the Department of Analytical Chemistry, Faculty of Chemistry, Technical University of Gdansk are an attempt to determine the risk of exposure of organisms living on a mixture of selected pharmaceuticals and attempt to determine the possible interactions between them and physico-chemical factors.
Four compounds from the group of biologically active pharmaceuticals are used in various treatments from non-steroidal anti-inflammatory drugs (NSAIDs), antidepressants, antibiotics were chosen for this study.
The model substance selected for the tests are : diclofenac (sodium salt), chloramphenicol, oxytetracycline hydrochloride and fluoxetine hydrochloride (Sigma Aldrich - Germany).
The study was performed using a bioassay battery consisting of Ostracodtoxkit F^TM, XenoScreen YES/YAS and Microtox®.
Like most pollution, residues of pharmaceuticals occur at low concentration levels, in which they do not directly contribute to the death of the organisms in the exposed ecosystem, but may contribute to the long-term and irreversible changes.

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B. Kudlak, M. Wieczerzak and J. Namiesnik
Initial Results on Determination of Toxicity Level of Technological Fluids Utilized During Shale Gas Extraction
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Ostracodtoxkit F^TM;Phytotoxkit
Test Species : Heterocypris incongruens; Sinapis alba
Topics : soils; chemicals-general; surface waters; groundwaters; plant tests

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Abstract : Shale gas, also called blue gas, is a non-conventional one. This terms covers deposits of methane which are more difficult to extract and less efficient from the economic point of view than traditional deposits. Continuously increasing demand for gas, to ensure energy safety of a country and enforce its market position will increase interest in this kind of deposits. On the other hand one should be aware that there are numerous obstacles for the economically efficient utilization of such gas. Technological processes conducted in the non-conventional deposits greatly differ from conventional ones and even between different geological regions. However, the most costs-generating and technologically advanced techniques are drilling processes and hydraulic fracturing.
The “shale gas” term refers to gas captured in strongly diagenised clay-siltstone rocks characterized by low permeability due to low porosity of such rocks. This gas is deposited at the particles easily undergoing fracturing, i.e. shales. Unfortunately until now except for some North American companies (e.g. Total, Schlumberger, BP) none is capable of exploiting these deposits. The main obstacles are high costs and technological advancements of horizontal drilling at high depths, as well as not-fully elaborated technologies of hydraulic fracturing enabling creation of fractures’ cobwebs connecting the rocks with the exploratory drill. There are 3 main stages of fracturing :
* fractures’ opening – pumping the fracturing fluid, most often without proppant, initiation and propagation,
* pumping the proppant - responsible for full opening of fractures and preventing from their close up,
* drill hole flushing - usually with regular water in order to clean it from the proppant fluid.
Thanks to informatics it is possible to virtually determine composition of the technological fluids, including the pressure, volume of pumped fluids, the chemical composition etc.
The planned studies included analyses of the real impact of processes conducted on all environmental compartments in the vicinity of drilling platforms: atmosphere, surface, ground, soil, surface and ground waters in the regions : 1) Lubocino 2) Stare 3) Syczyn 4) Wysin 5) Zawada 6) Lebien 7) Gapowo. The results presented here however cover only toxicological studies in locations where full fracturing cycle took place.
In the samples of technological wastes and fluids the toxicity was determined on the following organisms: Vibrio fischeri (water extracts and fluids), the crustacean Heterocypris incongruens and the higher plant Sinapis alba (solids and aqueous extracts and fluids). These organisms were selected to cover several trophic levels and studies on both fluids and solids in a cost-efficient manner.
At all the locations the water samples did not pose any ecotoxicological threat both from acute and chronic point of view. This was not the case of the proppant samples which were highly toxic even after serial dilution – especially in case of acute toxicity to bacteria. From the chronic toxicity point of view, the samples of sediments and drill cuttings are very dangerous for the environment, especially to crustaceans. For backflow fluids significant decrease of toxicity was noted with time. However, the highest threat is posed by the sediments present in post-technological fluids. Despite extraction, the sediments remained toxic and special safety measures must be taken into account when dealing with this type of waste material.

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I. Josko, E. Skwarek, A. Bogusz and P. Oleszczuk
The Assessment of Selected Nanoparticles Toxicity in the Presence of Surfactants
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Daphtoxkit F^TM magna; Phytotoxkit
Test Species : Daphnia magna; Lepidium sativum
Topics : chemicals - nanoparticles; chemicals - surfactants; plant tests; soils

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Abstract : Rising production and using of different nanoparticles (NP) result in their release in the environment. In the different components of environment, NP get in contact with various compounds (for example surfactants), which may impact the behavior and toxicity of NP. The objective of the study was the estimation of the effect of surfactants on the toxicity of nanoparticles: nano-ZnO, nano-TiO2 and nano-Ni on Daphnia magna and Lepidium sativum. The effect of hexadecyltrimethylammonium bromide (CTAB), Triton X-100 (TX100) and 4-dodecylbenzenesulfonic acid (SDBS) was tested. The Daphtoxkit F^TM microbiotest and the germination/rootgrowth test (OECD 1984) were applied for the toxicity testing. Additionally, the scanning and transmission electron microscopy (SEM and TEM) and the analysis of particle size and zeta potential (photon correlation spectroscopy) were conducted to the characterization and observation of NP in the presence of surfactants. In these studies, a wide range of NP concentrations (0.5-1000 mg/L) was used, whereas the surfactants were added to the solutions at non-toxic doses for D. magna and L. sativum.
The results showed that the influence of surfactants on NP toxicity was dependent/determined of the biotests used. The addition of NPs to a solution of the surfactants caused a significant reduction of their toxicity. The range of decrease of the toxicity of the ENPs depended on the type of ENPs and their concentration in the solution, and also on the kind of surfactant. For nano-ZnO the greatest toxicity decrease was caused by CTAB, while for nano-TiO2 the largest toxicity drop was observed after the addition of TX100. In the case of nano-Ni, the effect of the surfactants depended on its concentration. In the range of the lowest concentrations tested no differences were noted among the surfactants, while at nano-Ni higher concentrations (100 and 1000 mg/L) the toxicity decrease was characteristic of CTAB. In contrast to the Daphtoxkit F^TM, the phytotest showed increase of NP toxicity in the presence of surfactants in most of the cases. However, range of phytotoxicity changes was determined by the kind of NP and surfactants as well as the concentration of NP.

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B. Czech and P. Oleszczuk
Ecotoxicological Evaluation of Selected Pharmaceuticals to Vibrio fischeri (Microtox®) and Daphnia magna (Daphtoxkit F) before and after Photooxidation Process
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : Chemicals - drugs; waste waters

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Abstract : Photocatalysis, as one of the Advanced Oxidation Process, is a widely recognized method that enables the total removal of almost all organic pollutants from air, water or wastewater. The main driving force are *OH radicals that can be generated physically (UV, ultrasounds), chemically (H2O2) or photocatalytically (TiO2/UV). The result of radicals non selective and quick action is decomposition of pollutants into CO2, H2O and simple inorganic acids.
Many groups of pollutants can be decomposed using photocatalysis: pesticides, PAHs, pharmaceuticals. The last group is a great concern as the growing production and consumption will result in their increased release into environment. The main routes of pharmaceuticals introduction into the environment are both hospital ad municipal wastewater. As pharmaceuticals are noted in surface water or even in ground water, this indicates that wastewater treatment methods applied presently are inefficient in their removal. New efficient methods are therefore needed to meet the standards for treated wastewater.
One of the mostly applied photocatalysts is TiO2 which is widely used as a cheap, stable and efficient material. The main two drawbacks: powder form and activity in UV region, can however be bypassed for example by doping (with metals, nonmetals, semiconductors) or application of supports. The effectiveness of the photocatalytic process is often measured as the loss of concentration in time (c/c0, where c is the concentration measured at time t, and c0 the initial concentration). This approach, however does not include the formation of by-products that can be resistant to further decomposition and by which total decomposition cannot be achieved. This may result in increased toxicity of the treated wastewater. A new approach, must hence be undertaken.
The evaluation of the photocatalytic efficiency can be achieved by ecotoxicological tests. The application of bioassays allows to obtain quick and reliable answers on the effect of treatment. For the evaluation of the effectiveness of wastewater treatment tests with aquatic organisms can be successfully applied. Tests such as Daphtoxkit F^TM (Daphnia magna) and Microtox^® (Vibrio fischeri) gained popularity. However, their application in the qualitative analysis of photocatalytically treated waste water containing PPCPs is quite occasional. Generally, many PPCPs are considered to be toxic for water organisms with EC50 39.9 – 69.8 mg/L for Daphnia magna and 11.45 mg/L for Vibrio fischeri for DCF.
The effectiveness of the photocatalytic treatment of wastewater containing various pharmaceuticals was tested. Model pollutants compounds belonging to different therapeutic class drugs: an analgesics and anti-inflammatory drug Diclofenac (DCF), ß-blocker Metoprolol (MT) and antimicrobial agent Chloramphenicol (CPL) were selected.
The toxicity tests were applied to untreated solutions of PPCPs, the model wastewater treated photocatalytically and model wastewater after the treatment, where the samples were left for ca. 2 months as this time span is crucial for DCF biodegradation and the UV irradiation induced chain reactions that ran after the end of irradiation.
The data obtained indicated that, according to the EC50 values obtained with Daphnia magna, untreated pharmaceutical wastewater should be classified as III class toxic compounds using the criteria established according with the Directive 79/831/EEC. The toxicity to Daphnia magna of photocatalytically treated PPCPs solutions was generally decreased. The toxicity of CPL and MT after treatment was decreased at about 60% and 40%, respectively. And the toxicity of DCF oxidation products left for 60 days to Daphnia magna was generally lower in comparison to the model wastewater immediately after treatment. The results obtained for MT, however, indicate that MT oxidation products, although with initial lower toxicity, caused increased immobilization of Daphnia magna at the concentration above 25 mg/L.
The results revealed that photocatalytical treatment of wastewater containing the studied pollutants was efficient, both in mineralization and degradation and in detoxification. The process may be proposed as the polishing or post-treatment method for PPCPs removal from model wastewater. The high sensitivity of Daphnia magna indicates that these organisms should be used for testing the photocatalytic removal of pharmaceutical pollutants.

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M. Cieszynska-Semenowicz, J. Rogowska, W. Ratajczyk, J. Sychowska and L. Wolska
Does the Presence of Elemental Sulfur in the Sediment May Affect Their Toxicity ?
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Sinapis alba
Topics : sediments; chemicals - inorganic compounds; sediment and soil contact tests; plant tests

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Abstract : Sulfur in the form of different compounds, is widely present in all parts of the environment: minerals, soil, water and atmosphere. One of its forms is the elemental sulfur which occurs in nature in the elemental form of S⁰ and forms a rhombus eight-membered S8 cyclocristals. Elemental sulfur as extremely sparingly soluble in water, is considered a form of sulfur with low toxicity and low bioavailability, and thus unable to bioaccumulate in living organisms. However, the effect of sulfur toxicity has not been sufficiently understood. Information on the doses / concentrations of elemental sulfur that cause negative effects and the interaction of sulfur with organic matter and in mixtures with other compounds is not certain and confirmed. Furthermore, there is insufficient information on how the toxicity of elemental sulfur could be modified by the presence of substances with hydrophobic properties, e.g. compounds of oil, grease, or other substances which may be present in sediment samples.

The presentation will show the results of research aimed at increasing knowledge about the impact of elemental sulfur on the observed toxicity of sediments collected from different places in the Gulf of Gdansk. These studies were conducted in several stages :

- Determination whether a relationship between the concentrations of elemental sulfur listed in real samples of sediments and their toxicity exist;

- Comparison of the toxicity of solvent extracts obtained from the sediment samples containing elemental sulfur with the toxicity of the same extracts after elemental sulfur was purified using activated copper.

In the studies, no relationship between the concentration of elemental sulfur and toxicity of sediment or sediment solvent extracts was noted. No difference in toxicity was observed between purified extracts and extracts containing elemental sulphur. This may indicate the presence in the tested samples of sediments compounds responsible for their toxicity.

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M. Wawryniuk, A. Drobniewska, E. Bolimowska, D. Kasprzak, K. Klimiuk and G. Nalecz-Jawecki
Assessment of Direct and Indirect Photodegradation of Chemical Substances in the Aquatic Environment, a Case of Antidepressants
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Ceriodaphtoxkit F^TM; Thamnotoxkit F^TM
Test Species : Ceriodaphnia dubia; Thamnocephalus platyurus
Topics : chemicals - drugs

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Abstract : Every year thousands of tons of pharmacologically active compounds (PhACs) are used in medicine throughout the world. The widespread use of pharmaceuticals has led to significant increase of their detection in environment. Antidepressants are at the forefront and have a very high growth dynamics of consumption. Solar radiation is the main abiotic factor influencing the distribution of chemical compounds present in the environment. Exposure of drugs to irradiation can impact the stability of the formulation, leading to changes in the physicochemical properties of the product. Some pharmaceuticals have a photochemical half life of only a few minutes, while other drugs may be decomposed only a few percent after several weeks’ of exposure. Although many drugs are found to decompose under irradiation the practical consequences may not necessarily be the same for all these compounds. Chemical substances are subject to two types of photoreaction, direct and indirect photodegradation. In the first case, the chemicals absorb sunlight directly and are converted to products, when unstable excited states of the molecule decompose. Chemicals that cannot absorb light above 290 nm are resistant to direct photodegradation. In the second case, photodegradation of the target compound is caused by light-absorbing humic species. In both cases, sunlight may cause conversion of the drugs into more toxic decomposition products. Often photoproducts are characterized by high biological activity and high stability in aqueous solution.
The aim of the studies was a comprehensive assessment of photodegradation of selected antidepressants using the bioassays and physicochemical methods.
Photodegradation experiment was performed in a solar light simulator. The samples were evaluated using the bioassays and high pressure liquid chromatography (HPLC). Toxicity of the samples was tested with standard aquatic organisms, such as: Vibrio fischeri (Microtox^®), Spirostomum ambiguum (Spirotox), Ceriodaphnia dubia (Ceriodaphtoxkit F^TM acute), Thamnocephalus platyurus (Thamnotoxkit F^TM), Lemna minor and Spirodela polyrhiza. At the same time the concentrations of the pharmaceuticals was monitored using HPLC.
Most of the tested antidepressants were toxic for the organisms used in these studies. After exposure to light the presence of photoproducts was confirmed in samples. Toxicity, measured with bioassays, showed that photodegradation of antidepressant drugs caused the formation of more toxic photoproducts that their parent compounds. Moreover, the toxicity of the samples decreased slower than the concentration of the pharmaceuticals. The results indicate that the harmfulness of photoproducts of the antidepressants and other drugs should be taken into consideration in the ecotoxicological studies.

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W. Ratajczyk, M. Cieszynska-Semenowicz, J. Sychowska and L. Wolska
Whether the Discharges of Treated Wastewater into the Gulf of Gdansk May Affect the Quality of its Waters ?
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : waste waters; chemicals - hydrocarbons; chemicals - metals; sediment and soil contact tests

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Abstract : Commonly practiced way of introducing treated sewage to the environment is their point discharges into surface waters. In Poland, according to Regulation of the Minister of Environment of 24 July 2006 (with amendments of 28 January 2009) the quality control of treated wastewater entering to surface waters is based on the physicochemical indicators of pollution. The quality of wastewater discharged into the environment is highly variable in composition and pollution concentrations. Therefore, a reasonable question arises: whether the modern wastewater treatment plants cope with the removal from wastewater a wide range of compounds and what could be the impact of treated wastewater on the receiver, especially when it is the marine ecosystem.
In the study the results of the ecotoxicological and chemical samples of all sewage treatment plants discharge treated wastewater into the Gulf of Gdansk will be discussed. In addition to wastewater research there were also conducted water and sediment toxicity tests around the collectors lead-out sewage treatment plants. For ecotoxicological studies Vibrio fischeri bacteria and Heterocypris incongruens crustaceans were applied. In all samples concentrations of persistent organic pollutants (POPs) such as the compounds from the group of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and the content of heavy metals in sediments was also determined.

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A. Telesinski, W. Michalcewicz, M. Onyszko, M. Platkowski and M. Strek
Assessment of Spinosad Ecotoxicity Effect on Soil Based on Chosen Biotests
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; chemicals - pesticides; plant tests; seidment and soil contact tests

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Abstract : The organic farming system requires the use of specific principles in crop and animal production. This is due to use natural products in plant protection. Spinosad is insecticide, which is formed by the fermentation of soil actinomycetes Saccharopolyspora spinose. Due to the chemical structure spinosad is ranked among the macrocyclic lactones. The spinosad consists of two macrocyclic metabolites: spinosyn A (C41H65NO10) and spinosyn D (C42H67NO10).
The aim of this study was to determine the rate of dissipation and toxicity of spinosad residues in soil.
The experiment was carried out on samples of sandy loam with organic carbon content of 10.91 g.kg^-1. Spinosad, as Spintor 240 SC was added into soil material in dosages: recommended by the manufacturer five-hold, ten-hold and twenty five-hold higher. The soil without insecticide was reference. Moisture samples was adjusted to 60% of the maximum holding water capacity and the samples were stored at 20°C. Spinosad residue content (as a sum spinosynu A and D), soil enzyme activities (dehydrogenase, acid and alkaline phosphatase, and urease), and the content of basic groups of microorganisms (bacteria, fungi and actinomycetes) were determined during the experiment. Spinosad residue content was measured with liquid chromatographic method, and soil enzyme activities was assayed with spectrophotometric method. In addition, on the first and last day of the experiment, tests Phytotoxkit, Ostracodtoxkit F and modified screening test with the bacteria Vibrio fischeri were used.
Based on the obtained results, it was found that after 28 days the content of spinosad in the soil was 95% decomposed – residues of this insecticide have been found in soil with at a dosage of spinosad tenfold and twenty-fivefold higher than recommended . Analysis of soil enzyme activity changes showed little effect of spinosad on the assessed biochemical processes. Among the most sensitive enzymes assayed for the presence of the insecticide proved dehydrogenase. The presence of spinosad residues in the soil resulted in significant changes in the number of soil microorganisms, especially actinomycetes.
Comparing the results of ecotoxicity tests no effect of spinosad residue on germination test was observed. Occurred while the decrease of root growth, bioluminescence in Vibrio fischeri and increased mortality of shellfish Heterocypris incongruens, were shown, particularly immediately after insecticide application.

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M. Koltowski and P. Oleszczuk
Evaluation of the Effectiveness of Soil Remediation using Activated Biochars in Relation to Ecotoxicological Bioassays
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : soils; chemicals - hydrocarbons; plant tests

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Abstract : Polycyclic aromatic hydrocarbons (PAHs) are compounds that may adversely affect the health of humans and other living organisms. Moreover, PAHs could enter to the soil provoking toxic effects also for soil dwelling organisms. One possibility to remediate soils that are contaminated by PAHs is to bind and immobilize them with biochar. The aim of this study was to reduce the ecotoxicity of PAH contaminated soils by adding biochar pyrolysed from willow. To improve the biochar porous structure and binding capacity it was subjected to a range of activations by: 1) microwaves (in a microwave reactor under an atmosphere of superheated steam), 2) carbon dioxide in fluidized bed reactor and 3) superheated steam in fluidized bed reactor. Activated biochars were applied to three types of soils: 1) from landfill, where industrial wastes from coking plant are deposited (KB), 2) from the area of coking battery (KOK), and 3) from the area of bitumen processing plant (POPI).
Soils were mixed with original and activated biochars with 5% w/w dose and conditioned for two months in the dark. A battery of three bioassays was used for ecotoxicological evaluation of samples : Phytotoxkit (Lepidium sativum), Collembolan test (Folsomia candida) and Microtox^® (Vibrio fischeri). Solid phase soil samples were tested in Phytotoxkit and Collembolan test, whereas in the case of Microtox^®, leachates from investigated soils were analysed.\r\nResults showed that biochars can affect on ecotoxicity reduction in soils contaminated by PAHs. The ecotoxicity reduction range depended on type of the soil and type of the biochar activation. Control soils was characterised by high mortality of springtails (53-70%) and low number of juveniles (5-19). Moreover, a high root growth inhibiton (RGI) in contrast to POPI soil was observed in KOK and KB soils (25 and 19%, respectively). Microtox^® test results showed also very high inhibition of bioluminescence of Vibrio fischeri only for KOK soil (89%). The highest toxicity due to all kinds of bioassays and control soils was observed for KOK soil which was characterised by the highest sum of the 16 PAHs (40 mg/kg). After no activated and activated biochar amendment to investigated soils, reduce in ecotoxicity due to range of bioassays was observed. In Collembolan test according to the mortality of Folsomia candida all type of the amended biochars caused significant reduction of mortality in KOK soil and results was ranged from 20 to 40%. The highest number of juveniles (51) was observed after biochars activated by CO2 and microwaves amendment to KOK soil. According to the results obtained from Phytotoxkit test the highest decrease of root growth inhibition (RGI) in KOK soil was observed for biochar activated by microwaves (RGI= -12). A similiar to KOK soil significant reduction of RGI was also noted for POPI soil. In Microtox^® test a significiant decrease of inhibition of bioluminescence in KOK and POPI soil was observed for all kind of amended biochar. The most effective was that modified by water vapour and microwaves.

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A. Zielinska and P. Oleszczuk
Toxicity of Soils Fertilized by Sewage Sludge and Biochar Produced from Sewage Sludge Relative to Lepidium Sativum and Folsomia Candida
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : soils; plant tests; sludges

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Abstract : The presence of contaminants in sewage sludge, such as highly toxic organic compounds, potentially toxic elements, pathogenic microorganisms or warm eggs, restricts its application for soil fertilization and reclamation. Due to this, new solutions are constantly sought. Biochar (BC) can be an answer to the current problems related to sewage sludge reuse. The addition of biochar to soil increases its fertility and affects climate change through carbon sequestration in soil and reduction of N2O and CH4 emission from soil. In addition, biochar increases the water capacity of soil and its pH, prevents leaching of nutrients and immobilises organic and inorganic contaminants in soil.
The aim of the study was to evaluate the effect of the addition of sewage sludges and biochars obtained from them (at temperatures of 500 °C, 600 °C i 700 °C) to soil on its toxicity relative to plants (Lepidium sativum) and arthropod (Folsomia candida). The sewage sludge and biochar were added to soil at a dose of 1 %.
The studies have been shown that a mixture of sewage sludge and soil was toxic to L. sativum. The conversion of the sewage sludge to biochar caused a total reduction of its phytotoxicity. Biochars added to soil, stimulated root growth of L. sativum, both in comparison with the soil itself and a mixture of soil-sewage sludge. Depending on the pyrolysis temperature, stimulation of root growth within the range from 7.8 % (BC700) to 64.2 % (BC500) relative to the soil containing no additives and from 51.3 % (BC700) to 130.6 % (BC500) relation to initial sewage sludge was observed.
In turn, in the case of the test with F. candida the opposite trends were observed. The mixture of soil-sewage sludge did not have any significant effect on the mortality of F. candida relation to soil itself. The impact of the mixture of soil and biochars depended on the pyrolysis temperature. Biochars obtained at 500 °C caused the increase of mortality of F. candida. Biochars produced at 700 °C did not cause any significant effect on survival of F. candida, while biochars obtained at 600 °C caused decrease the mortality of the test organisms. The addition of both biochars and sewage sludges significantly inhibited the reproduction F. candida.

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A. Bogusz and P. Oleszczuk
Phytotoxicity of Biochars after the Adsorption of Heavy Metals
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : plant tests; soils; chemicals - metals

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Abstract : In the view of increasingly stringent regulations on the quality of groundwater and surface water, the aspect of removal of heavy metal ions has become a primary aim of many studies. Given the fact that the heavy metals, in contrast to the organic contaminants, are not biodegradable, and characterized by high stability in the environment, reducing of their bioavailability in the environment is crucial. Hence, in recent years, the particular attention is paid to developing effective methods for removing or immobilization of heavy metal ions. Limiting their mobility in the environment and minimizing the negative effects on living organisms are the main purposes. Considering each available techniques the most commonly used is adsorption. It is associated with the undoubted advantages of the process, such as the availability of a wide variety of adsorbents and the possibility of their recovery, ease of modification, the absence of normally toxic by-products and low cost. Current studies are mainly focused on the search for cheap and readily available adsorbents which will be capable to selective and irreversible removing heavy metal ions. For this purpose, different types of biomass as a feedstock for high-performance carbonaceous materials are tested. Biochar derived from biomass pyrolysis, because of its properties, have become an attractive alternative to activated carbons, whose market price is still growing, while the availability of biomass is virtually unlimited.
During developing of methods of metal ions immobilization on the basis of adsorption, it is necessary to determine whether the process takes place in an irreversible way, without causing the secondary pollution of ecosystems and transfer of these substances into the food chain of animals. For that reason, evaluation of the strength of interactions between biochar and the adsorbate in the form of a metal ion should be done. Stability of these interactions determines the non-toxicity of biochar after the adsorption process by reducing the release of ions from the adsorbents surface.
In the current study, the adsorption ability of biochar obtained by pyrolysis of wheat straw (BCS) in respect to the Cd(II), Cu(II), Cr(VI), Zn(II), Pb(II) and Ni(II) ions was perforfmed. Atomic absorption spectrometry with flame atomization (F AAS) was used for determination the metal ions concentration in the solution. Adsorption process was carried out in a batch mode, and the adsorption capacity was determined from the difference between the concentrations of ions in the starting solution, and after establishing the equilibrium state. In addition, the phytotoxicity of biochar before and after immobilization of the ions was evaluated using the Phytotoxkit test. As a test plant Lepidium sativum was used.

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A. Klimkowicz-Pawlas, B. Smreczak and A. Ukalska-Jaruga
Application of Microbiotests for the Assessment of Areas Subjected to Different Anthropopressure
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Phytotoxkit; Rapidtoxkit
Test Species : Thamnocephalus platyurus; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : plant tests; rapid tests; soils; behavioural tests

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Abstract : Soil protection is one of the most important issues in EU policy, as reflected in the European Parliament resolution of 13 November 2007 on the Thematic Strategy on soil protection. Among the main threats to soil functions decline of organic matter, changes in biodiversity, acidification of soils and soil pollution are mentioned. These threats may be the result of natural processes occurring in nature and anthropogenic activity resulting from agricultural (use of organic and mineral fertilizers, compost and pesticides) or non-agricultural (industrial emissions of pollutants) activities. Soil is the component of the environment in which finally accumulates the majority of pollutants. This may result in reduction of soil biodiversity, thus reducing resistance to stress, deterioration in the quality, fertility and productivity of soils. The hazard assessment associated with soil contamination is often based on the determination of the total content of chemicals. Chemical analysis alone does not provide complete information on a wide range of toxic substances, thus they have to be complemented with the results of ecotoxicological tests at different trophic levels.
The aim of the study was to evaluate the risks associated with contamination of soils subjected to different level of anthropopressure. The study was conducted in two agricultural areas (Czerwionka and Frampol) with similar soil characteristics (organic carbon content, pH, nutrients content), but with different history and intensity of exposure to pollution (metals and 16 PAHs). For detailed characteristic of soil ecotoxicity three microbiotests were applied: the test of bioluminescence inhibition (Vibrio fischeri) using Microtox, the Rapidtoxkit with crustaceans Thamnocephalus platyurus and Phytotestkit for the evaluation the direct impact of the chemicals in soil solution on the plant growth. The level of anthropopressure was determined on the basis of emission indexes.

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M. Platkowski
Phytotoxicity of Herbicides Avans Premium 360 SL and 300 SL Extra Chwastox in Relation to Certain Crop Species
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - pesticides; plant tests; soils; chemicals - biocides

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Abstract : The use of plant protection products is steadily increasing. In Poland, the most popular of plant protection products include formulations containing glyphosate and 4-chloro-2-methylphenoxyacetic in short MCPA.\r\nAlthough glyphosate and MCPA is known agriculture for many years, researchers conduct further testing to confirm if conducted so far studies fully support this finding toxic effects of these herbicides.
The aim of this study was to evaluate phytotoxicity of glyphosate and MCPA residues in the soil in relation to plant wheat, oats, cress and radishes.
The study was conducted using a modified test of seed germination and early plant growth Phytotoxkit. The modification consisted in changing the test plant species. In the preferred assay dicotyledonous plants selected cress (Lepidium sativum L.) and additionally radish varieties Rowa (Raphanus sativus var. sativus cv. Rowa). To assess the phytotoxicity monocots selected wheat varieties Skagen (Triticum aestivum L. cv. Skagen) and oats variety Bingo (Avena sativa L. cv. Bingo) The study was conducted in laboratory conditions in the loamy sand containing organic carbon 8.7g.kg^-1. For the earthy part of preparations containing glyphosate was introduced Avans Premium 360 SL and MCPA: Chwastox Extra 300 SL. Doses entered aqueous preparation were converted to amount of active substance 1 and 10 mg·kg-1 Reference was an attempt to control soil (without added herbicide). The study was conducted in accordance with the procedure described in the instructions supplied with the test Phytotoxkit.
A consideration that caused the greatest inhibition of root growth in plants studied was the addition of preparation Chwastox Extra 300 SL. All combinations with the preparation of root growth inhibition was over 50% relative to the control samples. The dose of 1 mg·kg-1 of glyphosate caused approximately 40% inhibition of the growth of roots used in monocots. A higher dose of this formulation to a similar extent affected the wheat root growth and radishes.\r\nGiven the inhibition of germination of seeds is also the highest percentage observed in the samples contaminated with both Chwastox 300SL preparation in admixture with the preparation Avans Premium 360 SL. The formulation containing glyphosate significantly affected the germination of oat and cress - a dose of 1 mg·kg^-1 and radish - both applied doses.

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J. Kalka, E. Felis and J. Zackiewicz
Photodegradation and Toxicity of Corrosion Inhibitors in Marine Water
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Artoxkit M^TM; Marine Algaltoxkit
Test Species : Artemia (salina) franciscana; Phaeodactylum tricornutum
Topics : chemicals - general; chemicals - organic compounds; surface waters; waste waters

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Abstract : Benzotriazole (BTA) benzothiazole (BT) have been used mainly as corrosion inhibitors. Introduced in small amounts to the corrosive environment result in a significant reduction of the corrosion rate of the metal, by generating the protective layer on its surface. BT and BTA are also widely used as components of plastics, rubber, pigments, and various coatings to enhance the stability and performance of the product. BT and BTA are used in materials intended for protection against UV radiation. BTA is a component of de-icing and antifreeze liquids, in the road transport and aviation, and as a component of refrigerants. In households, BTA is used as a component of detergents for dishwashers.
Benzothiazoles (BT) and benzotriazole (BTA), are detected in the various environmental matrices. They are both in urban and industrial wastewater as well as in the surface waters e.g. in rivers and lakes, marine waters, as well as in the soil.
During the study the toxicity of BT and BTA was evaluated, in relation to natural organisms inhabiting brackish waters, ie., Vibrio fischeri, Artemia salina and Phaeodactylum tricornutum. A synthetic sea water containing the test compounds at concentrations of 10 mg/L was subjected to irradiation 500 W/m2 in SOLARBOX reactor. Active substances removal was determined by LC-UV, photo-degradation intermediates were identified by LC-MS/MS. Toxicity of postprocessing solutions toxicity was verified with the above-mentioned species.

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L. Manfra, F. Savorelli, B. Di Lorenzo, G. Libralato, S. Comin, D. Conti, B. Floris, M. Francese, M.L. Gallo, I. Gartner, M. Guida, T. Leoni, G. Marino, F. Martelli, D. Palazzi, E. Prato, P. Righini, E. Rossi, A. Volpi Ghirardini and L. Migliore
Intercalibration of Ecotoxicity Testing Protocols with Artemia franciscana
Ecological Indicators 2015 57 47-47
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : test validation; ring testing; test selection; test sensitivity

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Abstract : The brine shrimp, Artemia spp., is widely used in ecotoxicology as a target biological model. Although several protocols were available in the early 1980s, only the 24-h acute mortality toxicity test was evaluated in a European intercalibration exercise during that period. Nevertheless, documentation of standard methods serving to provide specifications, guidelines or detailed characteristics of the 24-h protocol is still unavailable. This paper present the results of an intercalibration study of three toxicity-testing protocols using Artemia franciscana : (a) the 24-h static acute mortality test, (b) the 48-h static hatching test and (c) the 14-d static-renewal long-term mortality test. A first tier of experiments was conducted by a reference laboratory, which investigated the repeatability of the three methods. The feasibility and reproducibility of these protocols were then investigated by an intercomparison exercise involving 11 participants for the acute mortality test, seven for the acute hatching test and nine for the long-term mortality test. Protocols were tested on reference toxicants (copper sulphate pentahydrate and sodium dodecyl sulphate). The coefficients of variation were <20% and <50% for intra- and interlaboratory activities, respectively. These results encourage the standardization of the proposed methods and their use as regulatory procedures.

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G. Persoone, R. Baudo, M. Foudoulakis and G. Arapis
Aquatic Toxicology - a ‘Stock Culture Independent’ Duckweed Microbiotest with Spirodela Polyrhiza : w Practical and Low Cost Alternative to Lemna Bioassays
10th Iberian and 7th Iberoamerican Congress on Environmental Comtamination and Toxicology - Vila Real, Portugal 14-17 July 2015
Toxkits : Spirodela duckweed Toxkit
Test Species : Spirodela polyrhiza
Topics : test development; waste waters; test sensitiviy; test validation

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Abstract :

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M. Isidori, M. Lavorgna, A. Nardelli, A. Parrella, L. Previtera and M. Rubino
Ecotoxicity of Naproxen and its Phototransformation Products
Science of the Total Environment 2005 348 93-101
Toxkits : Algaltoxkit F^TM; Rotoxkit F^TM acute; Rotoxkit F^TM short-chronic; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : chemicals - drugs

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Abstract : The occurrence of pharmaceuticals in the environment is of great concern and only few data are available about the adverse effects of such molecules and their derivatives on non-target aquatic organisms. This study was designed to assess the toxic potential of Naproxen, a nonsteroidal anti-inflammatory, Naproxen Na, its freely water soluble sodium salt and their photoproducts in the aquatic environment. Bioassays were performed on algae, rotifers and microcrustaceans to assess acute and chronic toxicity. Furthermore, possible genotoxic effects of photoderivatives were investigated using SOS chromotest and Ames fluctuation test. The results showed that photoproducts were more toxic than the parent compounds both for acute and chronic values, while genotoxic and mutagenic effects were not found. These findings suggested the opportunity to consider derivatives in ecotoxicology assessment of drugs.

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N. Martins, J.L. Pereira, C.M.G. Duarte, L.C.A. Dias, F.E. Antunes and I. Lopes
Rational Design for Safer Nanomaterials : a Case Study with Nano-Biosur¬factants
10th Iberian and 7th Iberoamerican Congress on Environmental Comtamination and Toxicology - Vila Real, Portugal 14-17 July 2015
Toxkits : Algaltoxkit F^TM; Ostracodtoxkit F^TM; Rotoxkit F^TM acute; Rotoxkit F^TM short-chronic
Test Species : Brachionus calyciflorus; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - nanoparticles; chemicals - surfactants

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Abstract : The increased production and use of nanomaterials (NM) is boosting their release to the environment, which highlights the need to assess their risk to and ensure the protection of recipient ecosystems. With international policies pushing for innovation in the field of green technologies, nanotechnology configures the opportunity to avoid inefficiencies of the classic chemical industry. A major example is the use of rational design principles to reduce the environmental hazardous potential of new NMs. Within this scenario, cooperation between industry and research in early product development stages may be the key to avoid investment in NM variants that will not comply with environmental regulations. As part of such a cooperation project, ecotoxicological trials were conducted for seven variants of a soft organic NM made of biosurfactants; the variants were obtained following the functionalization of the headgroup with an increasing number of ethylene oxide (EO) units. Standard assays with freshwater species belonging to different taxonomic and functional groups were performed: growth rate assay with the algae Raphidocelis subcapitata, mortality and reproduction assay with the rotifer Brachionus calyciflorus, growth rate with the ostracod crustacean Heterocypris incongruens, mortality and embryo development assay with the fish Brachydanio rerio and the amphibian Pelophylax perezi. Even though there was a slight tendency for the reduction of toxicity with the increase of EO units, most species showed an overlap in confidence limits that prevented a clear ranking of the variants. The seven variations of NM biosurfactants were found toxic to freshwater biota. Tolerance to the NMs changed drastically depending on the species, with R. subcapitata being the least sensitive species and B. calyciflorus the most sensitive. Since the EO units functionalization only mildly affects the NMs toxicity, this structural feature can hardly be used alone as a safety variable in their rational design towards meeting a safer biosurfactant variant.

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Y. Huang, C.C. Reyes Aldasoro, G. Persoone and D. Wlodkowic
Integrated Microfluidic Technology for Sub-Lethal and Behavioral Marine Ecotoxicity Biotests
Proceedings of SPIE (International Society for Optics and Photonics, 21-24 September 2015, Toulouse, France
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : test development, rapid tests; behavioural tests

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Abstract : Changes in behavioral traits exhibited by small aquatic invertebrates are increasingly postulated as ethically acceptable and more sensitive endpoints for detection of water-born ecotoxicity than conventional mortality assays. Despite importance of such behavioral biotests, their implementation is profoundly limited by the lack of appropriate biocompatible automation, integrated optoelectronic sensors, and the associated electronics and analysis algorithms. This work outlines development of a proof-of-concept miniaturized Lab-on-a-Chip (LOC) platform for rapid water toxicity tests based on changes in swimming patterns exhibited by Artemia franciscana (Artoxkit M^TM) nauplii. In contrast to conventionally performed end-point analysis based on counting numbers of dead/immobile specimens we performed a time-resolved video data analysis to dynamically assess impact of a reference toxicant on swimming pattern of A. franciscana. Our system design combined: (i) innovative microfluidic device keeping free swimming Artemia sp. nauplii under continuous microperfusion as a mean of toxin delivery; (ii) mechatronic interface for user-friendly fluidic actuation of the chip; and (iii) miniaturized video acquisition for movement analysis of test specimens. The system was capable of performing fully programmable time-lapse and video-microscopy of multiple samples for rapid ecotoxicity analysis. It enabled development of a user-friendly and inexpensive test protocol to dynamically detect sub-lethal behavioral end-points such as changes in speed of movement or distance traveled by each animal.

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C. Blaise, F. Gagné, P. Eullaffroy and F. Férard
Ecotoxicity of Selected Pharmaceuticals of Urban Origin Discharged to the Saint-Lawrence River (Québec, Canada) : a Review
Brazilian Journal of Aquatic Science and Technology 2006 10,2 29-51
Toxkits :
Test Species :
Topics :

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Abstract : Twelve pharmaceuticals comprising anti-inflammatory agents (ibuprofen, naproxen), lipid regulators (bezafibrate, gemfibrozil), an anti-convulsant (carbamazepine), antibiotics (sulfamethoxazole, sulfapyridine, oxytetracycline, novobiocin, trimethoprim), a stimulant (caffeine) and a nicotine metabolite (cotinine) were identified in the final effluent of a major wastewater treatment plant (WWTP) based in the Montreal area (Québec, Canada) discharging to the Saint-Lawrence River. Their measured concentrations ranged from 33 (carbamazepine) to 22187 ng/L (caffeine) in the Montreal WWTP effluent and from 58 (cotinine) to 85000 (ibuprofen) ng/L based on maximum concentrations found in other municipal effluents as reported elsewhere. In general, elimination of these drugs by wastewater treatment is purported to be fairly efficient for ibuprofen, naproxen, bezafibrate, gemfibrozil and caffeine. In contrast, carbamazepine, sulfamethoxazole, sulfapyridine and oxytetracycline appear more resistant to treatment whereas removal efficiency for novobiocin, trimethoprim and cotinine is either unclear or unknown. Despite the degree to which they are eliminated in WWTps, their presence in urban effluents discharging to surface waters is common. To estimate their potential risk to aquatic biota, we undertook acute/chronic toxicity testing with a suite of small-scale bioassays representing four taxonomic groups (bacteria, algae, invertebrates and fish) and complemented our results with those published in the literature. When all acute and chronic toxicity tests were combined, their toxicity responses spanned over five orders of magnitude. Barring caffeine and cotinine, all others are clearly recognized as toxic based on the EU-Directive 93/67/EEC classification, but their concentrations measured in WTTPs are too low to cause acute or chronic effects on an individual basis. Sulfamethoxazole, naproxen and ibuprofen, however, were found to produce toxic effects at concentrations that are < one order of magnitude to those found in municipal effluents. In general, bioassay responses displayed a wide range of sensitivity that justifies continuing use of representative species within test batteries to properly appraise the toxic potential of pharmaceuticals. We also showed that exposure of Hydra attenuate to caffeine for 48 h induced xanthine oxidoreductase activity which may in turn lead to production of oxidative stress at concentrations close to those present in urban effluents. Future studies on the ecotoxicity of pharmaceuticals should therefore also seek to include appropriate biomarkers to assess possible adverse long-term effects to biota resulting from the bioransformation of drugs.

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J.A. Solis-Lemus, Y. Huang, D. Wlodkowic and C.C. Reyes-Aldasoro
Microfluidic Environment and Tracking Analysis for the Observation of Artemia franciscana
Paper presented at the 26th British Machine Vison Conference, 7 - 10 September 2015, Swansea, United Kingdom
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : test development; rapid tests; behavioural tests

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Abstract : The movement of small animals in well-defined environments is increasingly used in ecotoxicology as a sensitive indicator of water contamination with toxicants. This work describes a framework for the analysis of Artemia franciscana within miniaturised chambers. This work combines : (a) a proof-of-concept miniaturised Lab-on-a-Chip (LOC) platform where any changes in swimming patterns exhibited by Artemia franciscana can be related to the toxicity of the environment with (b) a pipeline of image processing steps for the movement analysis of video sequences of test specimens. The analysis pipeline is semi-automatic as it allows a user to define a region of interest and confirm intensity levels that are used for two-level hysteresis segmentation. Tracking is automatic, and a large number of statistical measurements and visualisation tools are provided. In addition, proofreading tools for validation of tracks are provided to link, break or delete tracks. The software consists of Matlab® m-files and is available in the website www.objecttrack.org.uk.

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A. Rotini, L. Manfra, S. Canepa, A. Tornambè and L. Migliore
Can Artemia Hatching Assay Be a (Sensitive) Alternative Tool to Acute Toxicity Test ?
Bulletin of Environmental Contamination and Toxicology 2015
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : test sensitivity; test development

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Abstract : Artemia sp. is extensively used in ecotoxicity testing, despite criticisms inherent to both acute and long-term tests. Alternative endpoints and procedures should be considered to support the use of this biological model. The hatching process comprises several developmental steps and the cyst hatchability seems acceptable as endpoint criterion. In this study, we assessed the reliability of the hatching assay on A. franciscana by comparing with acute and long-term mortality tests, using two chemicals: Diethylene Glycol (DEG), Sodium Dodecyl Sulphate (SDS). Both DEG and SDS tests demonstrated a dose dependent hatching inhibition. The hatching test resulted more sensitive than acute mortality test and less sensitive than the long-term one. Results demonstrate the reliability and high sensitivity of this hatching assay on a short time lag and support its useful application in first-tier risk assessment procedures.

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A. Klimkowicz-Pawlas, B. Maliszewska-Kordybach and B. Smreczak
Chemical and Ecotoxicological Analyses of Soils Historically Contaminated with PAHs
Proceedings of Secotox Conference and International Conference on Environmental Management, Engineering, Planning and Economics, Skiathos, Greece, June 24-28, 2007
Toxkits : Phytotoxkit; Toxi-Screening Kit
Test Species : Lepidium sativum
Topics : soils; chemicals - hydrocarbons

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Abstract : Soil is a sink for a wide range of anthropogenic hazardous contaminants such as polycyclic aromatic hydrocarbons (PAHs) formed in the most of the combustion processes, coke production, petroleum refining, etc. High level of soil contamination with PAHs creates a risk not only for humans, but also for biotic elements of soil ecosystems and may endanger soil habitat function. Chemical determinations, widely applied for evaluation of PAHs content in soils, are not sufficient for comprehensive assessment of ecological risk and have to be complemented with ecotoxicological tests, which reflect reaction of soil organism to biologically available fraction of contaminants.
The aim of the study was to compare two methods - chemical and ecotoxicological - applied for evaluation of the concentrations and effects of PAHs in historically contaminated soils. The study area covered the territory of about 100 km² in the Upper Silesia region in Poland. Samples were collected from the surface layer of agricultural land surrounding coking plant of over 100 years production history. Chemical analyses (GC/MS method) comprised determinations of the total content (CH2Cl2 extraction) of 16 PAHs from US EPA List. The ecotoxicological evaluations covered soils and soil water phases. Soil ecotoxicity tests embraced determinations of the effects on plants (phytotoxicity tests with Sinapis alba) and soil microorganisms (nitrification potential measurement). The acute toxicity of water-extractable pollutants was determined by measuring luminescence inhibition of the bacterium V. fischeri with application of Toxi-Screening kit microbiotest.
Significant negative effects on Sinapis alba growth and activity of nitrifying bacteria were observed in contaminated soils. Toxic effects were depended mainly on the soil properties, such as organic carbon content and soil acidity; relationship with PAHs content (ranged of 0.37–316.0 mg.kg^-1) was rather weak. In historically contaminated soils the sequestration of pollutants (e.g. PAHs) in the soil matrix may lead to the reduction of their concentration in the pore water, and thus their bioavailability. However, in our study the toxicity of soil water extracts was rather high. The observed 40% reduction in bacterial luminescence may suggest the presence of other toxic pollutants than PAHs.
See corresponding Poster

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A. Torokne and K. Toro
Evaluation of the Toxicity of River and Creek Sediments in Hungary with two Different Methods
Environmental Toxicology 2010 25, 5 504-509
Toxkits : Daphtoxkit F^TM magna; Ostracodtoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens
Topics : sediments

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Abstract : The aim of this study is to compare the responses of two different ecotoxicological methods for the determination of the toxic hazard of river or creek sediments in Hungary. Since water quality is intrinsically linked to sediment quality, the ecotoxicological control of sediments is also very important in the water quality policy. The 2000/60 EC Water Framework Directive aims at achieving a good qualitative and quantitative status of all water bodies by 2015. Fifteen sediments and four sludges were collected at different sites in Hungary, which are contaminated by industrial sewage. The first assay is the acute toxicity test with the crustacean Daphnia magna Straus (OECD 202) using larvae less than 1 day old, and an exposure time of 2 days. The second assay is the Ostracodtoxkit F with freshly hatched larvae of the crustacean Heterocypris incongruens and an exposure time of 6 days. The Ostracodtoxkit F test is a direct sediment contact bioassay, in which the test organisms are in continuous contact with the sediments. The Daphnia tests were applied on water extracts of the sediment without any contact of the test organisms with the contaminated sediment. The same 1:4 water/sediment ratio has been applied to both tests. The results showed higher toxic effects of the sediments to H. incongruens than to D. magna confirming the need to complement water only tests with solid phase assays for a meaningful evaluation of the toxic hazard of aquatic environments. The sensitivity of H. incongruens is similar to that of the other test species which are currently used for solid-phase assays. The growth inhibition of H. incongruens is a very sensitive endpoint for sediment toxicity testing. The sediment toxicity tested by ostracods showed strong correlation with concentration of total chromium, lead, and cadmium together.

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P.M. Mwanamoki, N. Devarajan, F. Thevenon, N. Birane, L.F. de Alencastro, D. Grandjean, P.T. Mpiana, K. Prabakar, J.I. Mubedi, C.G. Kabele, W. Wildi and J. Poté
Trace Metals and Persistent Organic Pollutants in Sediments from River-Reservoir Systems un Democratic Republic Of Congo (DRC) : Spatial Distribution and Potential Ecotoxicological Effects
Chemosphere 2014 111 458-492
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals- metals; chemical - organic compounds; sediments

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Abstract : This paper discusses the occurrence and spatial distribution of metals and persistent organic pollutants (POPs : including organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), Polybrominated diphenyl ethers (PBDEs), and polycyclic aromatic hydrocarbons (PAHs) in sediments from a river-reservoir system. Surface sediments were sampled from thirteen sites of the Congo River Basin and Lake Ma Vallée, both situated in the vicinity of the capital city Kinshasa (Congo Democratic Republic). Sediment qualities were evaluated using toxicity test based on exposing Ostracods to the sediment samples. The highest metal concentrations were observed in sediments subjected to anthropogenic influences, urban runoff and domestic and industrial wastewaters, discharge into the Congo River basin. Ostracods exposed to the sediments resulted in 100% mortality rates after 6d of incubation, indicating the ultimate toxicity of these sediments as well as potential environmental risks. The POPs and PAHs levels in all sediment samples were low, with maximum concentration found in the sediments (area of pool Malebo) : OCP value ranged from 0.02 to 2.50 with ΣOCPs : 3.3µgkg(-1); PCB ranged from 0.07 to 0.99 with Total PCBs (Σ7×4.3) : 15.31µgkg(-1); PAH value ranged from 0.12 to 9.39 with ΣPAHs : 63.89µgkg(-1). Our results indicate that the deterioration of urban river-reservoir water quality result mainly from urban stormwater runoff, untreated industrial effluents which discharge into the river-reservoirs, human activities and uncontrolled urbanization. This study represents useful tools incorporated to evaluate sediment quality in river-reservoir systems which can be applied to similar aquatic environments.

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R. Khanal, H. Furumai and F. Nakajima
Characterization of Toxicants in Urban Road Dust by Toxicity Identification Evaluation using Ostracod Heterocypris Incongruens Direct Contact Test
The Science of the Total Environment 2015 15 96-102
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - metals; chemicals - organic compounds; Toxicity Identification Evaluation (T.I.E.)

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Abstract : The current study involves characterization of organic compounds, heavy metals, and ammonia as potential toxicants in one arterial road (St. 3) and two highway (St. 7 and 8) urban road dust (URD) collected in Tokyo, Japan. URD toxicity was evaluated by Toxicity Identification Evaluation using the ostracod Heterocypris incongruens direct contact test. URDs were treated with resins (10% XAD-4, 20% SIR-300 and 20% SIR-600) to determine the reduction in mortality after treatment. The mortality of ostracods exposed to St. 3 URD (baseline 80%) was significantly (p<0.05) reduced to 0% and 47% after XAD-4 and SIR-300 treatment respectively. This reduction led us to suspect hydrophobic organic compounds and heavy metals as potential toxicants. Subsequent elution of the recovered XAD-4 with polar (methanol, acetone) and non-polar (dichloromethane) solvents confirmed the dominance of relatively polar hydrophobic organic toxicants. The dissolved concentration of Cu and Zn after SIR-300 treatment exceeded the 50% lethal concentration (LC50) for ostracods, which led us to suspect Cu and Zn as other probable toxicants. The concentration of Zn in a SIR-300 acidic elutriate, recovered after the termination of toxicity test, confirmed Zn as one of the toxicants in St. 3. The baseline mortality (100%) of St. 7 was significantly reduced (23%) by SIR-300 treatment. This indicated the presence of heavy metals as the probable toxicant. However, the concentration of dissolved heavy metals (Cd, Cr, Cu, Ni, Pb and Zn) in the overlying water was below previously reported LC50 values for each metal in St. 7. Recovery of Zn concentrations exceeding the LC50 in the St. 7 SIR-300 elutriate led us to suspect a dietary exposure route of Zn to the ostracod during the direct contact test. The overall results indicate that the toxicant types can vary widely depending on the road sampled.

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J. Rogowska, M. Cieszynska-Semenowicz, W. Ratajczyk and L. Wolska
The Impact of Anthropogenic Factors on the Ecotoxicity of Sediments of the Gulf of Gdansk
V Polish National Bioindication Conference - Lublin, Poland - 27-29.05.2015
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Sinapis alba
Topics : sediments

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Abstract : Effective protection of marine ecosystems is one of the challenges the international community. The adopted in 2000, within the European Union, The Water Framework Directive and in 2008 The Marine Strategy Framework Directive (RDSM) was the basis for a new, integrated policy to protect aquatic ecosystems. In accordance with the guidelines included in the RDM each Member State is required to develop a marine strategy for its marine waters which will take into account the specificity of these waters and which will also reflect the overall perspective of the marine region or sub region. The programs implemented under maritime strategy will only be effective if developed based on solid knowledge about the state of the marine environment. Therefore, the first step in preparation is to carry out identification and analysis of the characteristics or properties of the pressures and impacts on marine waters in the area.
The classic methodology for evaluation of the quality of the environment, which is required by Polish legislation, is based on the use of chemical analysis techniques. However, information obtained in this way do not fully reflect the risks to ecosystems and, subsequently, human. Accordingly, the complementary chemical information about the results of the ecotoxicological seems inevitable.
The Gulf of Gdansk area is a region which is at risk of pollution and requires special protection due to its specific hydrological and biochemical properties. Industry, and in particular coal-based energy and the dynamically growing road transport, are the main sources of pollution. Equally important is the fact that poor quality fuel is used in homes and in local low emission power stations. This leads to a significant amount of particulate pollutants suspended in the air in the Pomerania region. A large part of the pollution inputs to the marine environment comes from land-based sources and is transmitted primarily from the waters of the Vistula River or direct runoff from the area. What is more through the Gulf of Gdansk sea routes to the harbors of Gdynia and Gdansk are crossed. The waters of the Gulf of Gdansk is also a place for depositing sediments from dredging waterways and reservoirs. It should also be noted that to the waters of the Gulf of Gdansk treated wastewater from sewage treatment plants Gdansk-Wschod, Debogorze and sewage treatment situated in Hel and Jastarnia is discharged. It is also worth mentioning that the area of the southern Baltic Sea was an area of intense battles and much military activity in World War II. The ports of Gdansk and Gdynia were important naval base. Both Gdynia and Gdansk were important naval bases.
In order to determine the levels of toxicity of sediment samples were used indicator organisms representing different trophic levels (bacteria, plants, crustaceans). The search for the relationship between the results of the ecotoxicological studies and chemical analysis was based on a comparison of obtained indicators of toxicity and the results of chemical analyzes.

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R. Baudo, M. Foudoulakis, G. Arapis, K. Perdaen, W. Lanneau, A.-C.M. Paxinou, S. Kouvdou and G. Persoone
History and Sensitivity Comparison of the Spirodela polyrhiza Microbiotest and Lemna Toxicity Tests
Knowledge and Management of Aquatic Ecosystems 2015 416 23
Toxkits : Spirodela duckweed Toxkit
Test Species : Spirodela polyrhiza
Topics : test development; test sensitivity; test validation; ring testing; review

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Abstract : The history of toxicity tests with duckweeds shows that these assays with free-floating aquatic angiosperms are gaining increasing attention in ecotoxicological research and applications. Standard tests have been published by national and international organizations, mainly with the test species Lemna minor and Lemna gibba. Besides the former two test species the great duckweed Spirodela polyrhiza is to date also regularly used in duckweed testing. Under unfavorable environmental conditions, the latter species produces dormant stages (turions) and this has triggered the attention of two research groups from Belgium and Greece to jointly develop a “stock culture independent” microbiotest with S. polyrhiza. A 72 h new test has been worked out which besides its independence of stock culturing and maintenance of live stocks is very simple and practical to perform, and much less demanding in space and time than the conventional duckweed tests. Extensive International Interlaboratory Comparisons on the S. polyrhiza microbiotest showed its robustness and reliability and triggered the decision to propose this new assay to the ISO for endorsement and publication as a standard toxicity test for duckweeds. Sensitivity comparison of the 72 h S. polyrhiza microbiotest with the 7d L. minor assay for 22 compounds belonging to different groups of chemicals revealed that based on growth as the effect criterion both duckweed assays have a similar sensitivity. Taking into account its multiple advantages and assets, the S. polyrhiza microbiotest is a reliable and attractive alternative to the conventional duckweed tests.
Full article (from open access periodical : Knowledge & Management of Aquatic Ecosystems - KMAE)

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Y. Huang, R. Cartlidge, M. Walpitagama, J. Kaslin, O. Campana and D. Wlodkowic
Unsuitable use of DMSO for Assessing Behavioral Endpoints in Aquatic Model Species
Science of the Total Environment 2018 615 107-114
Toxkits : Artoxkit M^TM; Daphtoxkit F^TM magna
Test Species : Artemia (salina) franciscana; Daphnia magna
Topics : chemicals - organic

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Abstract : Dimethyl sulfoxide (DMSO) is a universally used aprotic solvent with the ability to permeate biological membranes and thus is commonly used to achieve appropriate biological availability of hydrophobic toxicants. While DMSO as a carrier medium has a reportedly low toxicity and is routinely employed in ecotoxicology, very little is known about its effect on dynamic behavioral parameters. This study presents a comparative analysis of the lethal and behavioral effects of exposures to DMSO concentrations of 0.1-10% on several test species such as: neonates of the freshwater crustacean Daphnia magna, nauplii of the marine crustacean Artemia franciscana, the marine crustacean Allorchestes compressa, embryos and larvae of the freshwater fish Danio rerio. The results demonstrated that DMSO did not cause statistically significant mortality even at concentrations close to 1% but induced clear and significant behavioral abnormalities in response to sublethal concentrations on all test species. These included hypoactivity syndrome in A. franciscana, A. compressa, D. magna and zebrafish larvae while a slight time-dependent hyperactivity response was observed in zebrafish embryos. For the majority of test species, behavioral changes such as moving distance, acceleration and burst movement were often observed during the first hours of exposure. These results indicate that caution should be exercised when using DMSO as a carrier solvent in experiments assessing behavioral endpoints.

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A. Sieroslawska, A. Rymuszka and T. Skowronski
Application of Biotests in Cyanobacterial Extract Toxicity Assessment
Archives of Environmental Protection 2014 40, 3 115-121
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : cyanobacterial blooms; cyanotoxins

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Abstract : The aim of the study was to determine the toxicity of the extract obtained from the cyanobacterial cells derived from the waters of Zemborzycki dam reservoir with use of a battery of biotests. The taxonomic identification of the bloom-forming cyanobacteria revealed high abundance of Aphanizomenon flos-aquae and Dolichospermum spp. (Anabaena spp.) and in a lower degree of Microcystis aeruginosa and Planktothrix agardhii. In the extract obtained from concentrated cyanobacterial cells, hepatotoxin microcystin-LR at a concentration of 22.89 +/- 3.74 mu g/L and neurotoxin Antx-a at 13.02 +/- 0.01 mu g/L have been detected. Toxicity of the extract was evaluated with the following assays: Daphtoxkit F magna with the crustacean Daphnia magna, Thamnotoxkit F with the crustacean Thamnocephalus platyurus, Rotoxkit F with the rotifer Brachionus calyciflorus and Protoxkit F with ciliate Tetrahymena thermophila. The most sensitive organism among all studied was T. platyurus for which EC50 was estimated to be 1.2% of the initial extract concentration. On the basis of the highest obtained value of the toxicity unit (TU = 83) the studied sample was classified to the IV class, which is of high acute toxicity. Additionally, it was found that reactivity on cyanobacterial products differs greatly among organisms used in bioassays, which indicate the need for using a set of biotests.

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H. Toumi, K.F. Burga-Perez and J.F. Ferard
Acute and Chronic Ecotoxicity of Carbaryl with a Battery of Aquatic Bioassays
Journal of Environmental Science and Health, Part B : Pesticides, Food Contaminants and Agricultural Wastes 2016 51, 1
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - pesticides; chemicals - biocides

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Abstract : The ecotoxic effects of carbaryl (carbamate insecticide) were investigated with a battery of four aquatic bioassays. The nominal effective concentrations immobilizing 50% of Daphnia magna (EC50) after 24 and 48 h were 12.76 and 7.47 µg L(^-1), respectively. After 21 days of exposure of D. magna, LOECs (lowest observed effect concentrations) for cumulative molts and the number of neonates per surviving adult were observed at carbaryl concentration of 0.4 µg L(^-1). An increase of embryo deformities (curved or unextended shell spines) was observed at 1.8 and 3.7 µg L(^-1), revealing that carbaryl could act as an endocrine disruptor in D. magna. Other bioassays of the tested battery were less sensitive: the IC50-72h and IC10-72h of the algae Pseudokirchneriella subcapitata were 5.96 and 2.87 mg L(^-1), respectively. The LC50-6d of the ostracod Heterocypris incongruens was 4.84 mg L(^-1). A growth inhibition of H. incongruens was registered after carbaryl exposure and the IC20-6d was 1.29 mg L(^-1). Our results suggest that the daphnid test sensitivity was better than other used tests. Moreover, carbaryl has harmful and toxic effects on tested species because it acts at low concentrations on diverse life history traits of species and induce embryo deformities in crustaceans.

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V. Tsarpali and S. Dailianis
Toxicity of Two Imidazolium Ionic Liquids, [bmim][BF4] and [omim][BF4], to Standard Aquatic Test Organisms : Role of Acetone in the Induced Toxicity
Ecotoxicology and Environmental Safety 2015 117 62-71
Toxkits : Artoxkit M^TM; Rotoxkit F^TM; Rotoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Brachionus calyciflorus; Brachionus plicatilis; Thamnocephalus platyurus
Topics : chemicals - organic compounds

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Abstract : The main goal of this study was to investigate the toxicity of the imidazolium-based ionic liquids (ILs), [bmim][BF4] (1-butyl-3-methylimidazolium tetrafluoroborate) and [omim][BF4] (1-octyl-3-methylimidazolium tetrafluoroborate), in battery of standard aquatic toxicity test organisms. Specifically, exposure of the algae Scenedesmus rubescens, crustaceans Thamnocephalus platyurus and Artemia franciscana, rotifers Brachionus calyciflorus and Brachionus plicatilis and bivalve Mytilus galloprovincialis to different concentrations of [bmim][BF₄], [omim][BF₄] and/or a binary mixture of [bmim][BF₄]-[omim][BF₄] (1:1) with or without acetone (carrier solvent), revealed that solvent can differentially mediate ILs\' toxic profile. Acetone\'s ability to differentially affect ILs\' cation\'s alkyl chain length, as well as the hydrolysis of [BF₄ (-)] anions was evident. Given that the toxic potency of the tested ILs seemed to be equal or even higher (in some cases) than those of conventional organic solvents, the present study revealed that the characterization of imidazolium-based ILs as green solvents should not be generalized, at least in case of their natural occurrence in mixtures with organic solvents, such as acetone.

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A. Bojanowska-Czajka, G. Kciuk, M. Gumiela, S. Borowieck, G. Nalecz-Jawecki, A. Koc, J.F. Garcia-Reyes, D.S. Ozbay and M. Trojanowicz
Analytical, Toxicological and Kinetic Investigation of Decomposition of the Drug Diclofenac in Waters and Wastes using Gamma Radiation
Environmental Science and Pollution Research International 2015 22, 24
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - drugs; waste waters

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Abstract : The radiolytic decomposition of the drug diclofenac (DCF), and in limited extent, also two other widely used drugs, ibuprofen and carbamazepine, was examined using liquid chromatography (LC) methods. The efficiency of DCF decomposition was examined in function of the absorbed dose of gamma radiation, and also in the presence of selected scavengers of radicals, which are commonly present in natural waters and wastes. Three different tests were employed for the monitoring of toxicity changes in the irradiated DCF solutions. The LC/mass spectrometry (MS) was used for the determination of products of DCF radiolysis. Using pulse-radiolysis method with the spectrophotometric detection, the rate constant values were determined for reactions of DCF with the main products of water radiolysis: hydroxyl radicals (1.24±0.02)x10(10) M^-1 s^-1 and hydrated electrons (3.1±0.2)x10(9) M^-1 s^-1. Their values indicate that both oxidative and reductive processes in radiolytic decomposition of DCF can take place in irradiated diluted aqueous solutions of DCF. The possibility of decomposition of all examined analytes was investigated in samples of river water and hospital waste. Compared to the previous studies, the conducted measurements in real samples were carried out at the concentration levels, which are close to those reported earlier in environmental samples.

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M. Taneez, C. Hurel and N. Marmier
Ex-situ Evaluation of Bauxite Residues as Amendment for Trace Elements Stabilization in Dredged Sediment from Mediterranean Sea : A Case Study
Marine Pollution Bulletin 2015 98, 1-2 229-234
Toxkits : Rotoxkit M^TM
Test Species : Brachionus plicatilis
Topics : sediments

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Abstract : Stabilization of marine dredged sediments contaminated with multi-elements is a challenging task in choosing the appropriate sorbent and application dosage. The present study investigates the possibility of using bauxite residues (Bauxaline® and Bauxsol) as amendment for the treatment of contaminated sediment. A pilot scale experiment was conducted for three months to stabilize trace elements in composted contaminated sediment sample using 5% by-product amendment. The results showed that after 3months of treatment, cationic trace elements were effectively immobilized but increased leaching of anionic pollutants was observed. Increased leaching of anionic pollutants could be limited by addition of higher quantities of amendments. The total content of available pollutants decreased in stabilized sediments but this treatment has no effect on the classification of waste. The leachates were then evaluated for acute toxicity using estuarine rotifers Brachionus plicatilis. Bauxite residues can be inexpensive choices for the stabilization of cationic pollutants in dredged sediments.

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E. Costa, V. Piazza, C. Gambardella, R. Moresco, E. Prato, F. Biandolino, D. Cassin, M. Botter, D. Maurizio, R. D'Adamo, A. Fabbrocini, M. Faimali and F. Garaventa
Ecotoxicological Effects of Sediments from Mar Piccolo, South Italy : Toxicity Testing with Organisms from Different Trophic Levels
Environmental Science and Pollution Research International 2015 October
Toxkits : Artoxkit M^TM; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis
Topics : sediments

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Abstract : The Mar Piccolo of Taranto (Ionian Sea, Southern Italy) is a semi-enclosed and strongly polluted basin. For decades, it has been subjected to different anthropogenic impacts. These stressors caused severe sediments contamination with high concentration of different pollutants (PAHs, PCB, heavy metals). In order to assess the current status of sediments contamination, an ecotoxicological investigation combined with chemical analysis (heavy metals, PAH, and PCB) has been performed. In order to derive ecologically relevant conclusions, a multiorganisms and multiend-points approach has been applied, exposing organisms from different trophic levels to elutriate and whole sediment. The battery of bioassays consists of a microalgal growth inhibition test (Dunaliella tertiolecta), acute and sublethal assays (end-points: mortality, immobilization and swimming speed alteration) on crustaceans larvae and juveniles, and rotifers (Amphibalanus amphitrite, Artemia salina, Corophium insidiosum and Brachionus plicatilis), and embryotoxicity test on echinoderms (Paracentrotus lividus). Considering the high levels of sediment contamination highlighted from chemical analysis, an unexpected very low toxic effect was observed, even considering the sublethal end-point (larval swimming speed alteration). The results of this study suggest a very complex contaminants dynamic in the Mar Piccolo sediments that, despite a strong level of contamination, seems to not affect in a proportional manner the biological compartment.

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I. Blinova, L. Kanarbik, M. Sihtmäe and A. Kahru
Toxicity of Water Accommodated Fractions of Estonian Shale Fuel Oils to Aquatic Organisms
Archives of Environmental Contamination and Toxicology 2016 70 383-391
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : shale oil; chemicals - hydrocarbons

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Abstract : Estonia is the worldwide leading producer of the fuel oils from the oil shale. We evaluated the ecotoxicity of water accommodated fraction (WAF) of two Estonian shale fuel oils (VKG D and VKG sweet) to aquatic species belonging to different trophic levels (marine bacteria, freshwater crustaceans and aquatic plants). Artificial fresh water and natural lake water were used to prepare WAFs. VKG sweet (lower density) proved more toxic to aquatic species than VKG D (higher density). Our data indicate that though shale oils were very toxic to crustaceans, the short-term exposure of Daphnia magna to sub-lethal concentrations of shale fuel oils WAFs may increase the reproductive potential of survived organisms. The weak correlation between measured chemical parameters (C10-C40 hydrocarbons and sum of 16 PAHs) and WAF\'s toxicity to studied species indicates that such integrated chemical parameters are not very informative for prediction of shale fuel oils ecotoxicity.

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H. Hamdi, S. Benzarti, L. Manusadzianas, I. Aoyama and N. Jedidi
Solid-phase Bioassays and Soil Microbial Activities to Evaluate PAH-spiked Soil Ecotoxicity after a Long-term Bioremediation Process Simulating Landfarming
Chemosphere 2007 70, 1 135-143
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - organic compounds; chemicals - hydrocarbons; soils

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Abstract : The residual ecotoxicity of long-term bioremediated soils concomitantly spiked with three PAHs at four levels (15, 75, 150, 300 mg Σ 3 PAHs kg(-1) soil) was evaluated using physico-chemical analyses, solid-phase bioassays and soil microbial activities. The pot-scale bioremediation process consisted of weekly moderate waterings in the presence or absence of sewage sludge compost (SSC) under greenhouse conditions. After 15 months, anthracene and pyrene were almost completely degraded whereas benzo[a]pyrene was still persisting, most apparently in SSC-amended soil treatments. However, no apparent toxic effects of the residual PAHs could be detected. SSC application at 40 t ha(-1) was performed to valorize the biowaste and stimulate PAH biodegradation but caused soil salinization and pH reduction at the end of the bioremediation process. Consequently, SSC-amended soils were characterized by strong phytotoxicity to lettuce and had adverse effects on the ostracod Heterocypris incongruens. Despite the smaller number of culturable bacterial populations in SSC-amended soils, soil enzymatic activities were not affected by the organic amendment and residual PAHs; and the bioremediation efficiency was likely to be more limited by the bioavailability of PAHs rather than by the total number of PAH-degraders.

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H. Zlámalová Gargošová, M. Vávrová, H. Doležalová Weissmannová, L. Mravcová, L. Vydrová and L. Zouhar
The Use of Methods of Environmental Analysis and Ecotoxicological Tests in the Evaluation of Wastewater
In : Waste Water - Evaluation and Management - Ed. : Prof. F.S. García Einschlag 2011 1
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : waste waters

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Abstract : The term “new” environmental pollutant covers all anthropogenic contaminants that have been released in the environment over the last few decades. Research into their fate and impact on the environment has started relatively recently. The group of new environmental pollutants consists of persistent organic pollutants (POPs) including DDT, polychlorinated biphenyls, polycyclic aromatic hydrocarbons and a wide range of organic pesticides. This group has recently been expanded with polybrominated flame retardants, drugs, detergents, and personal hygiene products. Chemicals, used every day in homes, industry and agriculture, can enter the environment in wastewater. According to the United Nations Organization 884 million people do not have access to safe drinking water, and over 2.6 billion people do not have access to proper sanitation systems. On July 28, 2010, the UN General Assembly passed a resolution supporting water as a human right and urging governments and international organizations to address this important human right.
Interest in the presence of pharmaceuticals, personal care products, and endocrine disruptors in the environment has increased significantly over the last decade.
Pharmaceuticals and synthetic musk compounds in personal care products (PPCPs), are groups of organic micro pollutants which are routinely detected in surface water, ground water and even in drinking water (McClellan and Halde, 2010). Hundreds of tons of these compounds are dispensed in communities every year. PPCPs are regarded as being potentially hazardous compounds as some are ubiquitous, persistent and biologically active compounds with recognized endocrine disruption functions (Kasprzyk-Hordern et. al., 2002). Many pharmaceuticals, upon administration, are degraded in the body and may even become inactive, but others, often those applied externally, excreted or not absorbed fully, can leave the body in their active forms. (Roberts &Thomas, 2006). The others only slightly transformed or even unchanged often conjugated to endogenous substrate (e.g. glucuronic acid, sulfuric acid, acetic acid to polar molecules. (Roberts and Thomas, 2006). These conjugates are easily cleaved during sewage treatment and, thus, several pharmaceutically active compounds (PhACs) are discharged almost unchanged from municipal sewage treatment plants (STPs) into the receiving waters (Heberer, 2002). The wide range of trace chemical contaminants persisting in municipal wastewater after conventional treatment includes inorganic compounds, heavy metals, persistent organic pollutants like endocrine disrupting compounds, pharmaceutically active compounds; personal care products (PPCPs), disinfection by-products and many other complex compounds persist through the waste water treatment process. As a result, terrestrial and aquatic organisms can be exposed to these compounds when treatment by-products are subsequently reintroduced into the broader environment. Exposure can be associated with effluents from waste water treatment plants (WWTPs) leach from landfills, and runoff or leach from land that has received applications of reclaimed waters and/or bio solids. Thus, a number of PPCPs are widely detected in water resources throughout the world (Edwards et al., 2009; Lapen et al., 2008; Fatta-Kassinos et al., 2010). From reasons mentioned above is very important to monitor large spectra of chemicals not only in bio solids, which represents inevitable by-products of WWTPs, but in reclaimed water from municipal wastewater treatment. Waste water treatments produces large amount of sewage sludges/bio solids. Part of them is after treatment process used as fertilizer (Jones-Lepp & Stevens, 2006). The Sewage Sludge Directive 86/278/EEC seeks to encourage the use of sewage sludge in agriculture and to regulate its use in such a way as to prevent harmful effects on soil, vegetation, animals and man. On the other hand sludge could be posing a risk for environment they tends to concentrate heavy metals and organic but its, however, rich in nutrients such as nitrogen and phosphorous and contains valuable organic matter. Directive 86/278/EEC sets limit values for seven heavy metals, for some congeners of PCBs and AOX (Absorbable organic halides) other organic compounds and ecotoxicity tests aren’t required The European Commission is currently assessing whether the current Directive should be reviewed – and if so, the extent of this review (Council directive, 1986).
In the 1980’s and 1990’s the focus on the analysis of sewage sludge meanly deal with pathogens, inorganic (metals), polynuclear aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) (Alcock et al.; 1996 Eljarrat et al., 2001). In 21TH century analysis of these matrices is not target only for selected traditional analyses but new xenobiotics as pharmaceuticals and musk compounds in sewage sludge are reported (Herren & Berset 2000; Zorita et al., 2009; McClellan & Halde, 2010). Ecotoxicological evaluation of sewage sludge as integral part of environmental analysis started to appear in this century sparsely (Park et al. 2005, Domene et al., 2008).
Chemicals analysis is integral part of water quality evaluation but to predict effect of this water or used sewage sludge on environment is necessary to use some other possibilities. Useful tool for this could be biotests. Global evaluation of wastewaters should include ecotoxicological tests to complete the chemical characterization. The integrated assessment of biological effects of wastewater discharges in the ecosystems is relevant and ecotoxicity tests are referred as extremely useful tools for the identification of environmental impacts (Mendonca et al., 2009).

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A. Baran and J. Wieczorek
Assessment of Heavy Metals Bioavability and Toxicity in Soils
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - metals; sediment and contact tests

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Abstract : Nowadays, there is worldwide concern regarding heavy metals pollution in soil, due to their bioaccumulation, their toxicity, and their inability to degrade. The literature bioavailability is described in three steps : availability of metal in the soil (environmental availability); uptake of metals by the organisms (environmental bioavailability) and toxic effect of metals to the organisms (toxicological bioavailability). The aim of this study was an assessment of heavy metals bioavailability and toxicity in soils collected from soils with different degrees of exposure to human pressure. The research was conducted in southern Poland, in the northwestern part of the Malopolska province. The toxicity of soils was determinded with the following tests : Phytotoxkit, Ostracodtoxkit F and Microtox. The general trend observed was an increase in soil toxicity measured by the biotests with increasing bioavailable metal concertation in soils.

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M. Isidori, A. Nardelli, A. Parrella, L. Pascarella, and L. Previtera
A Multispecies Study to Assess the Toxic and Genotoxic Effect of Pharmaceuticals : Furosemide and its Photoproduct
Chemosphere 2006 63 785-793
Toxkits : Ceriodaphtoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Ceriodaphnia dubia; Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - drugs

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Abstract : Pharmaceutical products for humans and animals, as well as their related metabolites end up in the aquatic environment after use. Recent investigations show that concentrations of pharmaceuticals are detectable in the order of ng/l-mug/l in municipal wastewater, groundwater and also drinking water. Little is known about the effects, and the hazard of long-term exposure to low concentrations of pharmaceuticals for non-target aquatic organisms. This study was designed to assess the ecotoxicity of furosemide, a potent diuretic agent, and its photoproduct in the aquatic environment. Bioassays were performed on bacteria, algae, rotifers and microcrustaceans to assess acute and chronic toxicity, while the SOS Chromotest and the Ames test were utilized to detect the genotoxic potential of the investigated compounds. A first approach to risk characterization was to calculate the environmental impact of furosemide by measured environmental concentration and predicted no effect concentration ratio (MEC/PNEC). To do so we used occurrence data reported in the literature and our toxicity results. The results showed that acute toxicity was in the order of mg/l for the crustaceans and absent for bacteria and rotifers. Chronic exposure to these compounds caused inhibition of growth population on the consumers, while the algae did not seem to be affected. A mutagenic potential was found for the photoproduct compared to the parental compound suggesting that byproducts ought to be considered in the environmental assessment of drugs. The risk calculated for furosemide suggested its harmlessness on the aquatic compartment.

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M. DellaGreca, M.R. Iesce, M. Isidori, S. Montanaro, L. Previtera and M. Rubino
Phototransformation of Amlodipine in Aqueous Solution : Toxicity of the Drug and Its Photoproduct on Aquatic Organisms
International Journal of Photoenergy 2007 2007 ID63459
Toxkits : Ceriodaphtoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Ceriodaphnia dubia; Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - drugs

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Abstract : The phototransformation of amlodipine in water was investigated under various conditions. A quantum yield ΦS 2.2 × 10-4 and a half-life time t1/2 0.419 days were calculated when the drug in water (10-4 M) was exposed to sunlight. The only photoproduct found was its pyridine derivative. Formation of this product was explained on the basis of a radical cation intermediate. The acute and chronic toxicity of the drug and its photoproduct were evaluated on different organisms of the freshwater chain (Brachionus calyciflorus, Thamnocephalus platyurus, Daphnia magna, Ceriodaphnia dubia). The photoproduct exhibited a stronger toxic potential than the parent drug on the long time for C. dubia.

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A. Simões, R. Ribeiro, P. Alvarenga, P. Palma and M. Moreira-Santos
Environmental Risk Assessment of using Organic Wastes as Soil Amendments : Effects on Aquatic Organisms Exposed via the Soil-Water Pathways to Aquatic Systems
Paper presented on the Digital Proceedings of the 10th Conference on Sustainable Development of Energy, Water and Environmental Systems – SDEWES (ISSN 1847-7178). September 27 - October 2, Dubrovnik (Croatia) 2015 1036/1-8
Toxkits : Ostracodtoxkit F^TM; Rotoxkit F^TM short-chronic; Spirodela duckweed Toxkit
Test Species : Brachionus calyciflorus; Heterocypris incongruens; Spirodela polyrhiza
Topics : solid wastes; compost; soils; sludges

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Abstract : The interest in the recycling of organic wastes (OW) in agricultural soils is growing. The present study aimed to contribute to the environmental risk assessment of using organic wastes as soil amendments, by assessing effects on aquatic organisms. An experimental field was amended with sewage sludge-SS, mixed municipal solid waste compost-MMSWC and agricultural waste compost-AWC; doses were 6, 12 and 24 ton SS dry matter/ha, while MMSWC and AWC doses were adjusted for the same amount of organic matter/ha. Soil eluates were prepared to perform a battery of seven toxicity tests with key taxonomic and functional species. Unexpectedly, based on chemical/microbiological legislated limits, no toxicity was observed for all OW amendment doses, although up to a 35% D. magna feeding inhibition was observed for all three wastes. In conclusion, most likely due to complex interactions occurring under natural circumstances, it is important to integrate chemical, microbiological and ecotoxicological approaches.

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G. Nalecz-Jawecki, L. Szczesny, D. Solecka and J. Sawicki
Short Ingestion Tests as Alternative Proposal for Conventional Range Finding Assays with Thamnocephalus platyurus and Brachionus calyciflorus
International Journal of Environmental Science & Technology 2011 8, 4 687-694
Toxkits : Rotoxkit F^TM acute; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : chemicals - metals; chemicals - organic compounds; chemicals - general; chemicals - surfactants; chemicals - drugs; test selection; test sensitivity

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Abstract : The goal of this study was to evaluate whether short 1h sublethal assays may predict the results of 24h lethality assays with rotifers Brachionus calyciflorus and anostracan crustaceans Thamnocephalus platyurus. The test bionts were hatched from cysts. Inhibition of ingestion was observed after 15 min of incubation of rotifers and crustaceans with the suspension of carmine and latex beads, respectively. Nine compounds with different modes of action were used as toxicants zinc ions, sodium dodecyl sulphate, p-nitrophenol, 3, 5-dichlorophenol and pharmaceuticals propranolol, fluoxetine, abamectin, doramectin and ivermectin. The toxicity values observed in the ingestion tests were very close to the mortality values over a wide range of toxicity from a low toxic surfactant to very toxic avermectins.
The ratio between the 1h EC50’s in the ingestion test and the 24h LC50’s in the lethality test was below 2 in all cases for rotifers, and 7 in 9 cases for crustaceans. The toxicity of zinc and 3,5-dichlorophenol in the Thamnotoxkit F was 15-fold higher and 10 fold lower than in the ingestion test, respectively. The 24h LC50 values are within the range of 25-400 % of the 1h EC50 values for almost all toxicants tested with the exception of p-nitrophenol for B. calyciflorus and zinc and 3, 5-dichlorophenol for T. platyurus. Short, 1h ingestion assays Rotoxrapid and Rapidtoxkit are good predictors of the mortality over the next 24h and can be used as a range finding tests for representatives of pharmaceuticals and surfactants.

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M.E. Aydin, S. Aydin, S. Tongur, G. Kara, M. Kolb and M. Bahadir
Application of Simple and Low-Cost Toxicity Tests for Ecotoxicological Assessment of Industrial Wastewaters
Environmental Technology 2015 36, 22 2825-2834
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : waste waters

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Abstract : The objective of this study was to identify and to apply appropriate biotests having the advantages of being highly sensitive, easy to run, relatively inexpensive and able to substitute fish toxicity tests due to ethical reasons of animal welfare. To perform an ecotoxicological assessment of industrial wastewaters, different microbiotests were conducted to substitute the fish toxicity test with Lebistes reticulatus through Vibrio fischeri, Thamnocephalus platyurus, Daphnia magna, Lemna minor and Lepidium sativum representing different trophic levels in the aquatic and terrestrial ecosystems. Also, Algaltoxkit F with Pseudokirchneriella subcapitata and Protoxkit F with Tetrahymena thermophila tests were carried out. However, they could not be applied successfully for the wastewater samples. Wastewater samples from seven different industrial zones comprising different industries were subjected to characterization through measuring their physical-chemical parameters and their toxicity versus the above-mentioned organisms. T. platyurus, D. magna and L. reticulatus were the most sensitive test organisms investigated for the wastewaters. Considering toxic unit values, generally wastewater samples were toxic according to Thamnotoxkit F, Daphtoxkit F and fish toxicity tests. As an important outcome, it was concluded that Daphtoxkit F and Thamnotoxkit F could be a good alternative for the fish toxicity test, which is so far the sole toxicity test accepted by the Turkish Water Pollution Control Regulation.

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M. Isidori, A. Nardelli, L. Pascarella, M. Rubino and A. Parrella
Toxic and Genotoxic Impact of Fibrates and Their Photoproducts on Non-Target Organisms
Environment International 2007 33 635-641
Toxkits : Rotoxkit F^TM acute; Rotoxkit F^TM short-chronic; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Thamnocephalus platyurus
Topics : chemicals - organic compounds; chemicals - drugs

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Abstract : Lipid regulators have been detected in effluents from sewage treatment plants and surface waters from humans via excretion. This study was designed to assess the ecotoxicity of fibrates, lipid regulating agents. The following compounds were investigated: Bezafibrate, Fenofibrate and Gemfibrozil and their derivatives obtained by solar simulator irradiation. Bioassays were performed on bacteria, algae, rotifers and microcrustaceans to assess acute and chronic toxicity, while SOS Chromotest and Ames test were utilized to detect the genotoxic potential of the investigated compounds. The photoproducts were identified by their physical features and for the first risk evaluation, the environmental impact of parental compounds was calculated by Measured Environmental Concentrations (MEC) using the available data from the literature regarding drug occurrence in the aquatic environment and the Predicted No Effect Concentrations (PNEC) based on our toxicity data. The results showed that acute toxicity was in the order of dozens of mg/L for all the trophic levels utilized in bioassays (bacteria, rotifers, crustaceans). Chronic exposure to these compounds caused inhibition of growth population on rotifers and crustaceans while the algae seemed to be slightly affected by this class of pharmaceuticals. Genotoxic and mutagenic effects were especially found for the Gemfibrozil photoproduct suggesting that also byproducts have to be considered in the environmental risk of drugs.

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I. Blinova, L. Kanarbik, N. Irha and A. Kahru
Ecotoxicity of Nanosized Magnetite to Crustacean Daphnia magna and Duckweed Lemna minor
Hydrobiologia 2015 10 1-9
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - metals; nanoparticles

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Abstract : Along with the development of nanotechnology, an increase in production and application of nanosized magnetite (Fe3O4) is expected. Though magnetite is considered relatively safe, information concerning potential hazards of synthetic magnetite nanoparticles with unique physico-chemical characteristics to aquatic organisms is still limited. In this study, we evaluated the toxicity of nanosized (27.2 ± 9.8 nm) and bulk (144.2 ± 67.7 nm) magnetite particles to different life stages of the aquatic crustacean Daphnia magna. In addition, phytotoxicity of the magnetite was evaluated using duckweed Lemna minor. The study did not reveal any statistically significant differences between the biological effects of nanosized and bulk magnetite particles. Both forms of magnetite induced very low toxicity (EC50 > 100 ppm) to D. magna and L. minor in the standard acute assays. However, it was demonstrated that at acutely subtoxic magnetite concentrations (10 and 100 ppm), the number of neonates hatched from D. magna ephippia was decreased. Moreover, short-term (48h) exposure of neonate daphnids to these concentrations may significantly affect the long-term survival and reproductive potential of daphnids. These results indicate that substantial contamination of aquatic ecosystems by magnetite may disrupt the stability of cladoceran populations.

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M. Tekere, T. Sibanda and K.W. Maphangwa
An Assessment of the Physicochemical Properties and Toxicity Potential of Carwash Effluents from Professional Carwash Outlets in Gauteng Province, South Africa
Environmental Science and Pollution Research International 2016 (article in print)
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : effluents

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Abstract : The assessment of the quality of carwash effluents has received scant attention as a potential source of public and environmental health hazard in South Africa as demonstrated by the lack of literature in this subject. The physicochemical quality and potential ramifications of carwash effluents on receiving waterbodies were investigated in this study. Grab effluent samples were collected from six carwash outlets in Gauteng Province of South Africa and analyzed for selected physicochemical qualities including biological oxygen demand (BOD), oil and grease, total petroleum hydrocarbons-gasoline range organics (TPH-GRO), pH, dissolved oxygen (DO), total solids (TS) and total dissolved solids (TDS), electrical conductivity (EC), nutrients (nitrates, nitrites and phosphates), anionic surfactants and heavy metals (zinc [Zn], copper [Cu], lead [Pb] and chromium [Cr]). Further, the toxicity potential of the effluent samples was assessed using organisms from four trophic levels ranging from Selenastrum capricornutum (primary producer), Daphnia magna (primary consumer), Poecilia reticulata (secondary-tertiary consumer) and Vibrio fischeri (decomposer). High pollutant levels were observed in all effluents with BOD ranging from 27 + 2.1 to 650 + 4.9 mg/l, TDS from 362 + 8.5 to 686 + 8.5 mg/l, GRO-TPH from 0.01 + 0.0 to 7.6 + 0.2 mg/l, DO from 0.0 to 0.1 mg/l, Zn from 0.79 + 0.08 to 20 + 2.12 mg/l, Cu from 0.77 + 0.03 to 13 + 0.71 mg/l and oil and grease from 12 + 2.8 to 43 + 2.1 mg/l. Ammonium concentrations ranged from 0.4 + 0.1 to 75 + 6.4 mg/l; turbidity from 109 + 0.7 to 4000 + 29.7 mg/l, anionic surfactants from 1.4 + 0.1 to 5.8 + 0.3 mg/l and TPH from <0.01 to 7.6 mg/l. Toxicity assessment assays resulted in 100 % mortality for fish and Daphnia after 96 and 24 h, respectively, and significant bioluminescence and growth reduction in V. fischeri and algae after 15 min and 72 h, respectively. Most of the measured physicochemical parameters were in concentrations above the Environmental Management Agency (EPA) stipulated guidelines. Additionally, the effluents demonstrated acute toxicity against all four test species.

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S. Radic, H. Crnojevic, V. Vujcic, G. Gajski, M. Geric, Z. Cvetkovic, C. Petra, V. Garaj-Vrhovac and V. Orescanin
Toxicological and Chemical Assessment of Arsenic-Contaminated Groundwater after Electrochemical and Advanced Oxidation Treatments
The Science of Total Environment 2016 543-A 147-154
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : groundwaters; chemicals - metals

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Abstract : Owing to its proven toxicity and mutagenicity, arsenic is regarded a principal pollutant in water used for drinking. The objective of this study was the toxicological and chemical evaluation of groundwater samples obtained from arsenic enriched drinking water wells before and after electrochemical and ozone-UV-H2O2-based advanced oxidation processes (EAOP). For this purpose, acute toxicity test with Daphnia magna and chronic toxicity test with Lemna minor L. were employed as well as in vitro bioassays using human peripheral blood lymphocytes (HPBLs). Several oxidative stress parameters were estimated in L. minor. Physicochemical analysis showed that EAOP treatment was highly efficient in arsenic but also in ammonia and organic compound removal from contaminated groundwater. Untreated groundwater caused only slight toxicity to HPBLs and D. magna in acute experiments. However, 7-day exposure of L. minor to raw groundwater elicited genotoxicity, a significant growth inhibition and oxidative stress injury. The observed genotoxicity and toxicity of raw groundwater samples was almost completely eliminated by EAOP treatment. Generally, the results obtained with L. minor were in agreement with those obtained in the chemical analysis suggesting the sensitivity of the model organism in monitoring of arsenic-contaminated groundwater. In parallel to chemical analysis, the implementation of chronic toxicity bioassays in a battery is recommended in the assessment of the toxic and genotoxic potential of such complex mixtures.

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A. Baran and M. Tarnawski
Assessment of Heavy Metals Mobility and Toxicity in Contaminated Sediments by Sequential Extraction and a Battery of Bioassays
Ecotoxicology 2015 24,6 1279-1293
Toxkits : Ostracodtoxkit F^TM; Phytotestkit; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - metals

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Abstract : The aim of this study was to assess heavy metals mobility and toxicity in sediments collected from a dam reservoir in the conditions of intensive human impact by using chemical fractionation and a battery of bioassays. In the studies, the test organisms were exposed to substances dissolved in water (Microtox, Phytotestkit) as well to substances absorbed on the surface of solid particles (Phytotoxkit, Ostracodtoxkit F). The studies showed that sediments from the Rybnik reservoir are toxic, but the tested organisms showed different sensitivity to heavy metals occurring in the bottom sediments. The sediment samples were classified as toxic and very toxic. Moreover, the studies showed a higher toxicity in solid phases and whole sediment than in pore water. The lowest sensitivity was observed in H. incongruens (solid phases) and V. fischeri (pore water, whole sediment). The studies revealed that the toxicity of the sediments is caused mainly by heavy metal forms associated with the solid phase of the sediments. The studies did not confirm the metals occurring in fraction I (exchangeable) to be bioavailable and toxic to living organisms because most correlations between the metal concentration in fraction I and the response of the organisms were negative. The highest mobility from the bottom sediments was found in zinc, average mobility--in copper, cadmium and nickel, and low mobility--in chromium and lead. Organic matter is likely to be the most important factor controlling metal distribution and mobility in the studied sediments.

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P. Alvarenga, C. Mourinha, M. Farto, P. Palma, J. Sengo, M.C. Morais and C. Cunha-Queda
Ecotoxicological Assessment of the Potential Impact on Soil Porewater, Surface and Groundwater from the Use of Organic Wastes as Soil Amendments
Ecotoxicology and Environmental Safety 2016 126 102-110
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : soils; soil leachates; surface waters; groundwaters

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Abstract : This study aimed to assess the potential impact on soil porewater, surface and groundwater from the beneficial application of organic wastes to soil, using their eluates and acute bioassays with aquatic organisms and plants: luminescence inhibition of Vibrio fischeri (15 and 30min), Daphnia magna immobilization (48h), Thamnocephalus platyurus survival (24h), and seed germination of Lolium perenne (7d) and Lactuca sativa (5d). Some organic wastes\' eluates promoted high toxic responses, but that toxicity could not be predicted by their chemical characterization, which is compulsory by regulatory documents. In fact, when organisms were exposed to the water-extractable chemical compounds of the organic wastes, the toxic responses were more connected to the degree of stabilization of the organic wastes, or to the treatment used to achieve that stabilization, than to their contaminant load. That is why the environmental risk assessment of the use of organic wastes as soil amendments should integrate bioassays with eluates, in order to correctly evaluate the effects of the most bioavailable fraction of all the chemical compounds, which can be difficult to predict from the characterization required in regulatory documents. According to our results, some rapid and standardized acute bioassays can be suggested to integrate a Tier 1 ecotoxicological evaluation of organic wastes with potential to be land applied, namely luminescence inhibition of V. fischeri, D. magna immobilization, and the germination of L. perenne and L. sativa.

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P. Oleszczuk, I. Josko, B. Futa, S. Pasieczna-Patkowska, E. Palys and P. Kraska
Effect of Pesticides on Microorganisms, Enzymatic Activity and Plant in Biochar-Amended Soil
Geoderma 2014 214-215 10-18
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : chemicals - pesticides; soils

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Abstract : The objective of the study was the determination of the effect of biochar and pesticides (2,4-D and dicamba) on the enzymatic activity and ecotoxicity of soils. The study was realized within the framework of a field experiment in which biochar was applied to soil at two doses, 30 t/ha and 45 t/ha. Soil samples for analyses were taken 17 months after the application of biochar to the soils. In the soil samples the basic physicochemical properties, enzymatic activity and the ecotoxicological properties were analysed. Biochar stimulated the activity of enzymes in almost all experimental treatments. In addition, it also reduced the negative effect of pesticides on the enzymatic activity and on certain microorganisms in the Microbial Assay for Risk Assessment (MARA). More effective reduction of the negative effect was observed for dicamba than for 2,4-D. Depending on the matrix tested (elutaries or solid phase) varied impacts of the biochar and the pesticides on Lepidium sativum were observed. 2,4-D had an additive effect with the biochar, significantly increasing the phytotoxicity of the soil relative to the soil without any biochar content.

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St. Gheorghe, I. Lucaciu, I. Paun, C. Stoica and E. Stanescu
Environmental Exposure and Effects of Some Micropollutants Found in the Romanian Surface Waters
Journal of Environmental Protection and Ecology 2014 15, 3 878-888
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : surface waters

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Abstract : Ecological risk assessment is required by the European norms to predict or evaluate the effects of chemicals which are discharged into the receiving ‘environment’. Usually the aquatic risk involves two major components: occurrence and hazard effects. In order to estimate the predicted exposure concentrations of chemicals in the water (PEC aquatic) and the predicted no-effect concentration on organisms (PNEC aquatic), literature data collecting and laboratory testing data were necessary. A ratio of PEC/PNEC < 1 indicates no aquatic risk and no further assessment is deemed necessary. In the period of 2009–2013, within different national projects, our aquatic risk studies have been performed on some micropollutants, such as hazardous chemicals (4-chloroaniline, 1-chloro- 4-nitrobenzene and 4-chloro- 2-nitrotoluene), pesticides (azinphos-methyl and bentazone), pharmaceuticals (diclophenac, acetaminophen, ketoprophen, indomethacin, naproxen, ibuprophen, carbamazepine, caffeine, ciprofloxacine and trimethoprim) and surfactants (benzenthonium chloride and cocamidopropyl betaine). Several toxicity bioassays using the sensitivity of living organisms at different trophic levels (fish, planktonic crustacean, green algae and different bacteria species) were performed. The studied chemicals showed a relatively limited acute toxicity and generally the final results have revealed insignificant or low risks on aquatic organisms. Two chemicals (ciprofloxacin and benzenthonium chloride) showed high environmental risk. The ranking of organism sensitivity was crustacean, bacteria and algae. The risk assessment studies were based on environmental concentrations detected in Romanian surface waters (Danube river, Danube delta, Arges river, Mures river, Ciorogarla river and Ghimbasel stream) comparatively with other international rivers.

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St. Gheorghe, I. Lucaciu and R. Grumaz
Microbiotests versus Conventional Toxicity Tests
10th International multidisciplinary Scientific GeoConference- SGEM 2010 - Conference Proceedings 669-676
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - general; regulatory testing

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Abstract : Alternative toxicological tests (microbiotests) are those that reduce the number of animals in a toxicity test, refine procedures to make them less painful or stressful, replace animals with non-animal systems, or replace one animal species with another that is lower on the phylogenetic scale. A number of useful alternative methods have been developed and accepted for the evaluation of the potential toxic effects of chemical products and environment samples. The literature analysis concerning the use of microbiotests for the control of chemicals products and environmental samples, show that these are in full development because of environmental pollution monitoring and control increasingly complexity requirements and in the same time to comply the necessity of new experimental methodology with low cost and reduced complexity, but with relevant, reproducible and qualitative results.

To be conform to the new requirements imposed by the European Regulation REACH concerning chemicals registration, assessment, authorization and restriction, we have analyzed the possibility to reduce / replace the biotests which use fish with microbiotests which use bacteria (Vibrio fischeri sp. ), crustacean (Daphnia magna sp.) and algae (Selenastrum capricornutum sp. ). This paper presents some comparative testing studies performed for environmental samples and chemicals using both conventional toxicity tests with fish and alternative tests - microbiotests. The results obtained have led to the conclusion that the use of microbiotests is very practical and offers a good reproducibility and relevant results, but also it is not recommended to renounce definitively to the conventional toxicity tests with vertebrate organisms (ex. fish) because these represent the principal target of pollution. The toxicity tests with vertebrates have an important role in risk assessment, and can be used in combination with microbiotests. The use of the multi - species microbiotest battery, which can offer relevant answers concerning the chemicals / chemical products / environmental samples risk, may lead to an economical and relevant assessment of acute and chronic toxicity.

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M. Agrawal, S. Yadav, C. Patel, N. Raipuria and M. K. Agrawa
Bioassay Methods to Identify the Presence of Cyanotoxins in Drinking Water Supplies and Their Removal Strategies
European Journal of Experimental Biology 2012 2, 2 321-336
Toxkits : Artoxkit M^TM; Thamnotoxkit F^TM
Test Species : Artemia (salina) franciscana; Thamnocephalus platyurus
Topics : cyanotoxins; cyanobacterial blooms; chemicals - biocides

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Abstract : A diversified group of toxins produced by freshwater cyanobacteria pose threat to human health as they frequently occur in drinking water sources. Though numerous qualitative as well as quantitative chemical analytical methods are now available, relatively simple low cost methods that are able to evaluate the potential health hazard and allow management decisions to be taken, are more useful to agencies that monitor drinking water supplies. Given that there is no single method that can provide adequate monitoring for all freshwater cyanotoxins in the increasing range of sample types, bioassays that can detect the toxic effects and safe levels of cyanobacterial toxins in drinking water supplies are discussed. Methods for removal of cyanobacterial cells as well as dissolved toxins in drinking waters prior to supply are also discussed.

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T. Steliga, D. Kluk and P. Jakubowicz
Analysis of Chemical and Toxicological Properties of Fluids for Shale Hydraulic Fracturing and Flowback Water
Polish Journal of Environmental Studies 2005 24, 5 2185-2196
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - organic compounds; chemicals - general; chemicals - shale oil

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Abstract : An analysis of environmental threats showed that the complete influence of shale gas production on the environment is bigger than in the case of gas production from a traditional oilfield. Hydraulic fracturing of shale, done on a much larger scale, results in huge amounts of liquid waste that must be managed in a rational way. An optimum solution to this problem is reusing flowback water to develop fluids for later fracturing. This article discusses the composition of the fluids used in hydraulic fracturing of non-conventional deposits as well as the flowback water. In the case of the examined liquids, toxicological tests have been carried out using microbiotests of ToxKit types Microtox, MARA, Daphtoxkit F magna, and Thamnotoxkit F. The research was done on a sample of fluids for hydraulic fracturing, and on flowback water obtained in hydraulic fracturing of shale formations in Poland. The tests are essential for correctly managing flowback waters after fracturing.

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S. Peres, M.C.F. Magalhães, M.M.Abreu , S. Leitão, A. Santos and M.J. Cerejeira
Interaction of Contaminated Sediment from a Salt Marsh with Estuarine Water : Evaluation by Leaching and Ecotoxicity Assays and Salts from Leachate Evaporation
Journal of Soils and Sediments 2016 16, 5 1612-1624
Toxkits : Artoxkit M^TM; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis
Topics : sediments; landfill leachates

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Abstract :
Purpose
Wastes from a former Portuguese steel plant were deposited between 1961 and 2001 on the riverbank of a tributary of the Tagus River creating a landfill connected to the river, posing a potential contamination risk to the Tagus estuary ecosystem. This study aims to assess the transfer of chemical elements from contaminated sediments to the estuarine water from cycles of sediment leaching so as to evaluate the ecotoxicity of the leachates, and to analyze the solid phases crystallized from those leachates.
Materials and methods
Landfill sediment and estuarine water samples were collected during low tide. Sediment samples were analyzed for pH, electric conductivity (EC), Corg, NPK, and iron oxides. Leaching assays (four replicates) were done using estuarine water (200 cm³/replicate) and 1.5 kg of sediment per reactor. Each reactor was submitted to four leaching processes (0, 28, 49, and 77 days). The sediment was kept moist between leaching processes. Sediment (total (acid digestion) and available fraction (diluted organic acid extraction-Rhizo)) elemental concentrations were determined by inductively coupled plasma–instrumental neutron activation analysis (ICP/INAA). Leachates, and estuarine and sediment pore waters were analyzed for metals/metalloids by ICP/mass spectrometry (MS) and carbonates/sulfate/chloride by standard methodologies. Ecotoxicity assays were performed in leachates and estuarine and pore waters using Artemia franciscana and Brachionus plicatilis. Aliquots of the leachates were evaporated to complete dryness (23–25 °C) and crystals analyzed by X-ray powder diffraction (XRD).
Results and discussion
Sediment with pH=8 and high EC and Corg was contaminated with As, Cd, Cr, Cu, Pb, and Zn. The element concentrations in the available fraction of the sediment were low compared to the sediment total concentrations (<1 % for Rhizo extraction). The concentrations of potentially hazardous elements in the estuarine water were relatively low, except for Cd. Concentrations of hazardous elements in the leachates were very low. Calcium, K, Mg, Na, and chloride concentrations were high but did not vary significantly among the four leaching experiments. Total concentrations of carbonate were much higher in leachates than in estuarine water. Both estuarine water and leachates showed negligible toxicity. Crystals identified in the solids obtained from the leachates by evaporation were halite, anhydrite, epsomite, dolomite, and polyhalite.
Conclusions
The sediment showed the capacity to retain the majority of the potentially hazardous chemical elements. Remobilization of chemical elements from sediment by leaching was essentially negligible. The variation of total concentrations of Ca, carbonate, and sulfate in leachates indicates that the sediment contained reactive sulfides. Due to its composition, the sediment seems to be a dynamic system of pollution control, which should not be disturbed.

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E.S. Santos, M.M. Abreu, S. Peres, M.C.F. Magalhães, S. Leitão, A. Santos Pereira and M.J. Cerejeira
Potential of Tamarix africana and Other Halophyte Species for Phytostabilisation of Contaminated Salt Marsh Soils
Journal of Soils and Sediments 2015 December 1-15
Toxkits : Artoxkit M^TM; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis
Topics : soils; soil leachates

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Abstract :
Purpose
Salt marsh plants are colonising wastes from a steel plant deposited on the Coina River Banks posing a potential contamination risk to the Tagus estuary ecosystem. The objectives of this study were to assess the uptake, accumulation and translocation of hazardous elements/nutrients in three spontaneous halophytic species, to evaluate the capacity of Tamarix africana to stabilise a contaminated salt marsh soil, and to evaluate the ecotoxicity of the pore water and elutriates from phytostabilised soils.
Materials and methods
The work comprises the following: fieldwork collection of soil samples from Coina River (an affluent of Tagus River) bank landfill, estuarine water and spontaneous plants (Aster tripolium, Halimione portulacoides and Sarcocornia sp.), and greenhouse studies (microcosm assay) with T. Africana growing in one landfill salt marsh soil, for 97 days, and watered with estuarine water. Soils were analysed for pH, EC, Corganic, NPK, iron and manganese oxides. Soils total (acid digestion) elemental concentrations were determined by ICP/INAA. Estuarine waters, plants roots and shoots (acid digestion), soils available fraction (diluted organic acids extraction-RHIZO or pore water), and salts collected from the T. africana leaves surface were analysed for metals/metalloids (ICP-MS). Ecotoxicity assays were performed in T. africana soil elutriates and pore waters using Artemia franciscana and Brachionus plicatilis.
Results and discussion
Soils were contaminated, containing high total concentrations of arsenic, cadmium, chromium, copper, lead and zinc. However, their concentrations in the available fraction were <4 % of the total. The estuarine waters were contaminated with cadmium, but negligible ecotoxicological effect was observed. The spontaneous plants had significant uptake of the above elements, being mostly stored in the roots. Elemental concentrations in the shoots were within the normal range for plants. These species are not hazardous elements accumulators. Tamarix africana was well adapted to the contaminated saline soils, stored the contaminants in the roots, and had small concentrations of hazardous elements in the shoots. Excretion of hazardous elements by the salt glands was also observed. Elutriates from soils with and without plant did not show ecotoxicity.
Conclusions
The salt marsh species play an important role in the stabilisation of the soils in natural conditions. Tamarix africana showed potential for phytostabilisation of saline-contaminated soils. The low translocation of the elements from roots to shoots and/or active excretion of the elements by the salt glands was a tolerance mechanism in T. africana.

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M.C. Casado-Martinez, K.F. Burga-Perez, R. Bebon, J.-F. Ferard, E.L.M. Vermeirssen and I. Werner
The Sediment-Contact Test using the Ostracod Heterocypris incongruens : Effect of Fine Sediments and Determination of Toxicity Thresholds
Chemosphere 2016 151 220-224
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments

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Abstract : The toxicity test using freshwater ostracods of the species Heterocypris incongruens is a sub-chronic static test that exposes individuals to whole sediments over a period of 6 d, the endpoints being mortality and growth. We tested the hypothesis that endpoints of the sediment bioassay using Heterocypris incongruens are affected by the presence of fine sediment particles by testing control sediment supplied with the commercial test kit with increasing proportions of kaolin clay as a proxy for fines. While mortality was not affected, the results showed that increasing the presence of clay reduced ostracod growth. Based on the variability in growth, a sublethal toxicity threshold of 35% is proposed to distinguish effects due to sediment properties from those due to toxicity. The relevance of this threshold was verified using data from toxicity tests of ambient sediment samples with low levels of contamination.

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M.C. Casado-Martínez, T. Benejam, J.L. Loizeau, I. Werner and B.J.D. Ferrari
Toxicity screening of sediments from Lake Geneva using the freshwater ostracod Heterocypris incongruens (ISO 14371)
From : 26th Annual Meeting of Setac-Europe Book of Abstracts 2016
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments

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Abstract : Lake Geneva is the largest water body of freshwater in Western Europe, draining a watershed of about 8000 km2 in Switzerland and France with the Rhone being its major tributary. Lake Geneva is subject to anthropogenic pressures due to a large population, agriculture, and industries, and is periodically monitored for contaminant concentrations in water, sediment and biota [Loizeau et al. 2013]. The last monitoring campaign addressing sediment quality of the whole lake dated back to 1988 and addressed mainly metal and nutrient concentrations in sediments [Arbouille et al. 1989]. More recent studies on the zoobenthos have shown a significant improvement of the quality of sediments over the past twenty years [Lods-Crozet & Reymond 2006]. In 2015, a large sampling campaign supported by CIPEL (International Commission for Lake Geneva Protection) was carried out to provide a comprehensive assessment of micropollutant concentrations in sediments and zoobenthos. We will present the results of testing thirty sediment samples collected from the whole lake, with a sediment-contact test using the freshwater ostracod Heterocypris incongruens [ISO 14371-2012]. This test offers many advantages over other traditional sediment-contact tests such as requiring a small sample volume and the possibility to obtain test organisms from cysts so stock culturing is not needed. Despite the relatively long test duration (6 days) it is a promising method for screening the ecotoxicological quality of freshwater sediments. A preliminary analysis of the results allowed the mapping of the ecotoxicological quality of the sediment, which ranged from no or slightly toxic to highly toxic. We will discuss both the ecotoxicological quality of sediments from Lake Geneva and the performance of this test system.
See corresponding Poster

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K.B. Le Thanh, L.C. Do Hong, T. Combi, P Lara, M.C. Casado-Martínez and I Werner
Sediment quality assessment in Ho Chi Minh City canals
From : 26th Annual Meeting of Setac-Europe Book of Abstracts
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments; chemicals

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Abstract : Ho Chi Minh City metropolitan region is the largest urban and industrial hub of South Vietnam, with more than 6 million inhabitants and a large number of big factories and artisanal plants. Located in the Mekong delta, the area is characterized by a complex system of canals that play a vital role for the region and its inhabitants. They act as reservoirs for water run-off and drainage and they are used for transportation and as a source for natural resources. Due to public health concerns an important sanitation program has been undertaken in the last decade for the reduction of wastewater pollution and flooding. At present, the main canals are recovering after clean-up and clean waters have attracted fish again, with increasing stocks thanks to repopulation initiatives.

In 2015 we carried out a pilot study to assess the quality of recent sediments from Ho Chi Minh City canals. Surface sediments were collected from 6 canals, 4 of them located in the metropolitan area and 2 from suburban areas. Sediments were subject to physico-chemical and ecotoxicological characterisation, the physico-chemical characterisation consisting of the quantification of trace metals and organic micropollutants and the ecotoxicological assessment through the use of existing chemical quality guidelines and standard sediment toxicity tests.

The results show that the sediments from the canals from both the metropolitan and suburban areas accumulate a mixture of contaminants with concentrations of several PAHs and PCBs above the corresponding thresholds for effects to benthic organisms. Several pesticides were quantified in the sediments but the highest concentrations were measured for permethrin in the case of the sediment from the metropolitan area and for bifenthrin in sediments from suburban areas (3692.9 ng/g dw and 477.6 ng/g dw, respectively). Conspicuous concentrations at the µ/g order were found for the fragrances galaxolide and OTNE whereas nonylphenol, triclosan and the UV filters octocrylene, 2-EHDP and EHMC were measured at all canals at concentrations of several tens and even hundreds ng/g. The results of the chemical analyses are corroborated by the different levels of toxicity to crustaceans although no or slight toxicity was observed to rooted macrophytes in laboratory toxicity tests.
See corresponding Poster

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A. Baran, M. Tarnawski and T. Koniarz
Spatial Distribution of Trace Elements and Ecotoxicity of Bottom Sediments in Rybnik reservoir, Silesian-Poland
Environmental Science and Polution Research International 2016 Epub ahead of print
Toxkits : Ostracodtoxkit F^TM;
Test Species : Heterocypris incongruens
Topics : sediments; chemicals - metals

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Abstract : The aim of study was to integrate chemical analyses and toxicity bioassays in order to assess the environmental risk connected with the presence of trace elements in the sediments. This study examined the ecological significance of trace elements in bottom sediments by applying a set of complementary sediment quality assessment methods sediment quality guidelines (SQGs) (mean probable effect concentration quotient (PECQ)), potential ecological risk index (PERI), contamination degree (C d) and two bioassays: the bacterial luminescence inhibition test with Vibrio fischeri on sediment elutriates and the direct contact test with the ostracod crustacean Heterocypris incongruens. The samples were collected from 50 stations of Rybnik reservoir. The reservoir is a region with enormous concentration of industry, mainly hard coal mining, electric power industry, and transportation. Despite the high diversity in metal concentration in the sediments, the spatial distribution of trace elements in the sediments was very similar. Moreover, the strong positive correlations between individual pairs of trace elements indicate that they may derive from a similar source and move together. According to mean PECQs, 68 % of the samples were potentially non-toxic and 32 % of the samples were potentially toxic. PERI values suggested that 70 % of the sediment sampling sites exhibited low ecological risk from metal pollution while 24 % of the samples had severe and serious risk. Based on our combined evaluation, we believe that Cd and Cu in the sediment samples frequently caused adverse biological effects. Higher toxic responses were observed in the Microtox test than in the Ostracodtoxkit test. All the sediment samples were found toxic to V. fischeri, and 96 % of the samples had effect percentages >50 %. For H. incongruens, 12 % of the sediments were not toxic and 44 % had effect percentages >50 %. In order to perform a complex assessment of the environmental impact of metal pollution, both chemical and ecotoxicological analysis should be carried out.

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O. Bemowska-Kalabun and M. Wierzbicka
The Toxicity of the Roundup Herbicide on Railway Tracks
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : chemicals - pesticides; soils

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Abstract : Ecotoxicological studies (biotests) and chemical studies showed that a rail transport is a source of contamination of soil and living organisms among others by oil derivatives, PAHs, PCBs and heavy metals. That is a threat to the environment to a greater extent than previously thought. Moreover, rail areas are regularly sprayed with herbicides, which causes destruction of vegetation. Despite the unfavorable habitat conditions prevailing on the railway tracks, we found mass occurrence of plant species Geranium robertianum L which is a nitrophilous, herbaceous plant with a two-year development cycle, which prefers shaded forest habitats. However, it is puzzling how a species with a two-year life cycle can function on the railway tracks despite spraying of herbicides. Theoretically this is impossible. Roundup is one of the most widely used herbicides for weed control on railway embankments, with glyphosate as its active substance. The dosage recommended by the manufacturer of Roundup for use on railway tracks is 3.5 liters per kilometer of track, twice a year, in the period of intensive plants growth. This consideration was the starting point for studies, on how long glyphosate can remain in the substrate and have a toxic effect on plants. This is a key question, because G. robertianum blooms from May to October and plants seedling of this species occur several times a year. The Phytotoxkit biotest was found to be a good tool to answer this question. The sensitive bioindicator - Lepidium sativum L. was used for the study. A series of biotests was carried out to check the toxicity of the substrate after spraying the herbicide Roundup (track dose) just after spraying and in the following months. The decrease in toxicity of the substrate for testing plants was already noted 3 months after spraying, and its total disappearance after 6 months. Therefore, it is possible that a plant with a biology as G. robertianum was able to survive on the railway tracks despite spraying of herbicides. This can be so, provided that after the disappearance of the toxicity of the substrate new plants will develop which give new seeds before the next spraying. And this indeed seems to be the case. In addition, the conducted experiments show that the seeds retain their germination capacity for several years.

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M. Jedrzejczak
Toxicity of Rainwater Runoff from the Roofs with Varying Coverage
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : rainwater

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Abstract : As a result of the expansion of sewage systems and legal regulations for the disposal of liquid and solid waste, domestic and industrial sewage are becoming less of a threat to the environment. Currently, the urban runoff is considered as a major source of water pollution and it is a huge challenge in terms of environmental protection and the principles of sustainable development of urban areas.
Huge quantities of pollutants are produced in urban areas. These contaminants accumulate on different surfaces, such as roads, car parks or buildings. Rainfalls and thaws cause flushing of impervious surfaces and the transfer of pollutants into the storm water drainage system. Depending on the applied solutions, rain water can get to the treatment plant, or directly to the receiver. Frequent connection of drainage and municipal wastewater transport systems in one combined sewer system causes that during heavy rainfall the capacity of sewers is exceeded and mixed runoff and municipal wastewater overflow over the edge of a channel. This phenomenon is an additional source of water pollution in the receiver.
Water quality is currently defined primarily by the chemical characteristics. Analysis of the chemical composition of water can, however, be insufficient to estimate the potential impact of pollution on the environment. The presence of substances not covered by standard physico-chemical analysis, synergistic effects of chemical compounds which often occur, may cause a greater harmfulness of water pollution to living organisms than it would appear from routine quantitative research. Therefore, in order to determine the total harmfulness of all substances in the water, it is also recommended to perform toxicity tests of rainwater and storm overflows on the indicator organisms.
The main source of toxic substances in the rain wastewater is runoff from multi-line divided highways (more than 100 thousand vehicles per day). Top identified toxic substances in such wastewater are copper, zinc, cadmium, lead, platinum, some PAHs and chlorides. Combined sewer overflows attract less attention due to the lower direct toxicity. Their influence on the water biocenosis of the receiver relies primarily on changing the physical and chemical parameters of water (eutrophication).
An additional source of toxic substances in the rain wastewater is runoff from roofs. The variety of roof coverings used in construction makes that a large amount of chemicals is released into the rainwater flowing down the surface of the roof that can cause environmental degradation. The main toxic compounds in these effluents include: polycyclic aromatic hydrocarbons, sulfur compounds, surfactants, petroleum substances and heavy metals. The concentration and total amount of harmful substances in the runoff from roofs depend primarily on the condition of the roofing and the dynamics of the rainfall.
This study investigated the toxicity of rainwater runoff from four types of roofs : coated with copper, zinc sheet, with tar paper and cement-asbestos sheets (eternit) to the freshwater crustacean Daphnia magna Straus. Samples were taken during four independent rainfalls in March and April 2015. The study used the so-called first wave runoff using a specially designed container placed under the outlet of the launder. Toxicity of rainwater was determined by calculation of 24hEC50 and 48hEC50 by the probit method and by the method of graphical interpolation. In the case where the low toxicity of the sample did not allow to determine the EC50, the EC10 was calculated. Toxicity of runoff from rooftops was compared with the toxicity of rainfall collected directly, without rinsing any surface.
It was found that runoff from the roof covered with copper sheets shows the greatest toxicity to Daphnia magna (the mean 24hEC50 was approximately 0.17%). Runoff from the roof of zinc showed less toxicity (24hEC50 approx. 28%), but because of the prevalence of zinc and the galvanized sheet covering, the importance of this metal in toxicity of urban rainwater can be much greater than copper. The lowest toxicity to Daphnia magna Straus has been found for asbestos covering. Rainwater collected directly showed no toxicity to the test organisms.
Due to the limited availability and increasing consumption of clean fresh water, rainwater and runoff are increasingly being considered as potential sources of water for irrigation and economic purposes. The research shows, however, that runoff from roofs, especially with copper and galvanized coating, are highly toxic and should not be used even for economic purposes without prior cleaning. Moreover, research shows also a need to clean rain wastewater before discharging it into the environment.

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B. Kudlak
Determining Endocrine and Ecotoxicological Potential of Wastewater Samples with Microtox, Ostracodtoxkit F and Xenoscreen Yes/Yas
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : waste waters

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Abstract : The main aim of the studies undertaken was an attempt to assess the efficiency of EDC removal (thus reduction of endocrine and toxicological potential) of wastewater samples with the biological tests Microtox, Ostracodtoxkit^TM and Xenoscreen YES/YAS. The impact of environmental stressors on samples (collected all over Poland) and the efficiency of WWTP action was assessed with the 3 mentioned biotests.
No serious acute toxicity threat was determined on samples collected in most of the WWTPs. Samples where such threat was found were collected in southern Poland. Based on the results of chronic toxicity studies it can be stated that in most cases increase of toxicity was found in water samples collected after the wastewater release point. This can be due to the presence of chemicals utilized for wastewater treatment and increasing the bioavailability of pollutants present in the water samples.
For numerous locations it was found that an increase of endocrine potential took place for water samples after the WWTP release point when compared to samples prior to the WWTP. Such a situation was particularly occurred at the sampling locations in southern Poland.

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M. Stefaniuk, P. Oleszczuk and A. Zielinska
The Bioavailability of PAHs And their Toxicity in Soils Fertilized with Sewage Sludge and Biochar
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : chemicals - organic compounts; chemicals - hydrocarbons

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Abstract : Sewage sludges are a problematic waste. A limiting factor for their use as fertilizer is their content of heavy metals and organic contaminants, such as polycyclic aromatic hydrocarbons (PAH).
Recent studies showed that addition of biochar or activated carbon to sewage sludge might be considered one of the most effective ways to reduce the bioavailability of PAHs. Depending on the origin of the feedstock and the temperature of biochar production the range of the PAHs immobilization may be different.
The aim of the presented study was determination of the PAHs bioavailability in soil fertilized with sewage sludge (from Przemysl and Biala Podlaska) with addition of biochars obtained from solid residues from biogas plant (at 400°C and 600°C) and from willow (at 600°C). The soil was fertilized with sewage sludge-biochar mixture at the dose of 1% w/w whereas the contribution of biochar in the sewage sludge was at the level of 10%.
The bioavailable fraction of PAHs (Cfree) was determined by using polyoxymethylene (POM). In order to determine the toxicity of the samples ecotoxicological tests were performed with test organisms representing different groups : plants (Lepidium sativum), bacteria (Vibrio fischeri) and arthropods (Folsomia candida). The PAH content and toxicity was determined at the beginning of the experiment and after 90 days of incubation of the sewage sludge with the biochar and soil.
The obtained results show that the addition of biochar to sewage sludges may significantly reduce the risk associated with their environmental use both in terms of PAH content and toxicity of the materials tested. The reduction rate of bioavailable fraction of PAHs and the toxicity depended on the temperature of biochar production and incubation period of biochar – sewage sludge – soil.

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M. Urbaniak, A. Wyrwicka, S. Staniak, P. Kidd and G. Siebielec
The Influence of Urban Sediments and Selected Bacteria Strains and Plants on Soil Phytotoxicity and PCDD/PCDF Concentration
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : sediments; soils; chemicals - metals; chemicals - organic compounds

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Abstract : Urban reservoir sediments contain a broad range of pollutants originating from urban catchments including highly toxic Persistent Organic Pollutants and thus become an important environmental problem due to the risk of mobilization of contaminants under certain conditions and resulting in harmful effects on the environment and as a consequence on human health.
One way of coping with the growing amount of urban reservoir sediments, is to use phytoremediation, i.e. the use of plants to remove pollutants. At the same time bioaugmentation of contaminated sediments with selected bacterial strains may additionally improve the microbial degradation of the pollutants and in this way minimize the risk related to the utilization of sediments.
The aim of this study was to evaluate the influence of urban sediments, selected bacterial strains and plants : Tagetes patula L. and Festuca arundinacea on soil phytotoxicity using the Phytotoxkit test with Lepidum sativum, and concentrations of highly toxic congeners of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs).
Urban sediments were collected from a sequential sedimentation-biofiltration system located on the urban Sokolowka River, Lodz. The sediment was mixed with loamy sand soil in 1:10 proportion. Two bacteria strains: P87, RP92, previously isolated from soils contaminated with heavy metals and organic compounds, respectively, were tested in the study.
The obtained results of soil phytotoxicity demonstrated a positive role of the applied urban sediment showing a 90% increase of the Lepidum sativum root length in comparison to the not fertilized samples. This can be related to the high content of nutrients as well as Ca and Fe which are characterized by immobilizing toxic effects of pollutants. The positive influence of urban sediments also remained after 10 weeks of the experiment duration; however samples inoculated with bacteria strains were characterized by a better growth of Lepidium sativum in comparison to not inoculated samples. The application of plants Tagetes patula L. and Festuca arundinacea additionally improve the soil quality as measured with Lepidium sativum.
The positive role of bioaugmented phytoremediation was also demonstrated in the case of PCDDs/PCDFs removal since aapplication of bacteria and plant led to even 38% and 26% reduction of total and TEQ PCDDs/PCDFs concentration in amended soil.

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A. Baran, M. Taranawski and T. Koniarz
Toxicity of Bottom Sediments of Small Water Retention Reservoirs
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; sediment and soil contact tests

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Abstract : Toxicity assessment of bottom sediments was determined with 3 bioassays. The organisms represented three trophic levels : producers (Sinapis alba, Lepidium sativum, Sorghum saccharatum), consumers (Heterocypris incongruens) and decomposers (Vibrio fischeri). The diversity of test organisms allowed to assess the toxicity of combined substances with different mechanism of action. Most sediment samples (52%) were toxic. The toxicity of sediments was ranked in the following order : Brzoza Krolewska > Ozanna > Bagna Rzeszowskie > Brzoza Stadnicka > Cierpisz > Niedzwiadek > Narozniki > Gluchow. This study confirms the the need to use a battery of bioassays, which combined with chemical analysis gives a comprehensive view of bottom sediment quality.

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R. Biczak, B. Pawlowska and P. Balczewski
Toxicity Of Quaternary Ammonium 2'2-Thiodiacetates
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - organic compounds; sediment and soil contact tests

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Abstract : Quaternary ammonium salts (QAS) are a group of ionic chemicals known for many years for their unusual properties. They are widely used in the industry, biotechnology, medicine, pharmacology. Ionic liquids (ILs) are a special subgroup of QAS and from a definition have melting points below 100 °C, low vapor pressure, they are also non-flammable and feature high thermal and electrochemical stabilities.
One of the representatives of the quaternary ammonium salts are 2,2’-thiodiacetate ammonium salts. Some of them are ILs. In this study, we determined the toxicity of seven 2,2’-thiodiacetates with different ammonium cations (diizopropyloammonium, diisobutyloammonium, triethylammonium, tert-butylammonium, isoquinolinium) to Vibrio fischeri (Microtox) and the crustacean Heterocypris incongruens (Ostracodtoxkit F).
The results allow to conclude that the greatest toxicity of the investigated compounds for Vibrio fischeri and Heterocypris incongruens was found with 2,2-thiodiacetates with aromatic cations, i.e. isoquinolinium 2,2’-thiodiacetate (monosalt) and bis(isoquinolinium) 2,2’-thiodiacetate (disalt).

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J. Chojniak, L. Jalowiecki, I. Biedron, A. Drobniewska and G. Plaza
Evaluation of Toxicity of Silver Nanoparticles (Ag-Nps) Biologically Synthesized using Lemna Test and Algaltoxkit
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - metals; chemicals - nanoparticles

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Abstract : The most common nanomaterial used in many industries is silver, which proved to be a good antibacterial agent and disinfectant. Its easy incorporation into other substances or its use for coating makes silver very helpful for medicine. Nanosilver is used in the production of dressings, surgical instruments or implants. It is also applicable in environmental engineering in, for example, water treatment and purification processes. It finds its application in the production of textiles, underwear and cosmetics. In the context of the wide use of Ag-NPs, human exposure to these materials increases significantly. Current research focuses on the evaluation of the toxicity of silver nanoparticles synthesized by chemical methods, but there are not many articles about nanoparticles synthesized by a biological method.
The aim of the study was to evaluate the toxicity of silver nanoparticles synthesized by a biological method to Selenastrum capricornutum (Algaltoxkit F) and Lemna minor. For the synthesis the supernatant after Bacillus subtilis culture was used. Silver nitrate was added to the supernatant and the final concentration was 1 mM. Solutions were monitored by spectrophotometer UV-Vis to confirm the presence of silver nanoparticles. Toxicity tests were made according to OECD Guideline 201-221 (2006) and other standards (EPA 2012; ISO 2004, 2006). Parameters measured in the test Algaltoxkit F were : inhibition of cell growth of algae and content of chlorophyll a. In the test with Lemna minor : number and area of fronds, biomass and contents of dyes (chlorophyll a and b and carotinoids).

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F. Dawidziak, L. Sikorski, A.I Piotrowicz-Cieslak and B. Adomas
Content of Biogenic Amines in Soybean (Glycine max L. Merr) Seedlings which Grow in Soil Polluted with Herbicide Roundup 360 Sl
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - pesticides; soils

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Abstract : Plants contain aliphatic and aromatic amines that differ in the structure and biological functions. Aliphatic amines include putrescine, spermidine and spermine, which are most often found in the eukaryotes, but recent studies indicate that spermine is found only in angiosperms, whereas thermospermine is found throughout the plant kingdom. Putrescine, a diamine, has been said to be the most important polyamine since it is a precursor for other polyamines. Aliphatic amines are closely correlated with many physiological processes in the plant, such as somatic embryogenesis, cell divisions, seed dormancy breaking, rhisogenesis, maturing and aging of leaves, development of reproductive organs, flowering, fruit ripening, response to stress. Glyphosate (N-(phosphonomethyl)-glycin) the active ingredient in the formulation Roundup 360 SL (41,5 % glyphosate isopropylammonium) has a broad activity spectrum and belongs to the leading herbicides in the world market. It is applied as a salt, most commonly as isopropylamine salt and is an active compound of many herbicides. Glyphosate affects both mono- and dicotyledonous plants and is employed for weed removal in agriculture, orchards, vegetable crops and forestry but also for removing undesirable plants in aquatic ecosystems and urban landscapes. It is a systemic herbicide that is first absorbed by foliage and then translocated throughout the plant via the phloem and further transported to metabolic sinks such as meristems of shoots and roots. Glyphosate is a very polar substance, highly soluble in water and strongly adsorbed to most soils. Glyphosate functions by the means of inhibiting the endogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs, EC 2.5.1.19), which is crucial for the synthesis of aromatic amino acids and all other aromatic compounds in bacteria, algae, fungi and higher plants. Therefore glyphosate prevents the synthesis of chorismate-derived aromatic amino acids and secondary metabolites in plants.
The aim of the study was to investigate the phytotoxicity of low concentration of Roundup 360 SL (0, 1, 3, 7 and 10 µM) of nine-day-old soybean seedlings (Glycine max L. Merr) cv. Mazowia. Morphological (with Phytotoxkit) characteristics and biochemical reactions (using amino acid analyzer AAA400) soybean seedlings were evaluated. The soybean seeds germinated at 100%, regardless of the tested herbicide concentrations. The average length of roots and stems of nine-day-old soybean seedlings growing on control soil was 97.86 and 113.11 mm respectively. Soybean produced shorter roots and stems compared to the control growing on soil contaminated with 1, 3, 7 and 10 µM of glyphosate. But it was found that low concentrations of glyphosate in soil did not change significantly roots and stems length. The fresh weight of the roots and stems of soybean exposed to increasing concentrations of glyphosate decreased and the dry matter slightly increased. The osmotic pressure of soybean seedlings at the highest concentration of the herbicide in soil was increased about 1 PSI compared with controls (6.19 PSI). In control tissues of nine-day-old soybean seedlings, the following biogenic amines were identified: putrescine, cadaverine, and spermidine in the following amounts: 10; 950; and 55 µg.g^-1 FM in roots and 1.9; 446 and 63µg.g^-1 FM in stems, respectively. The content of putrescine and cadaverine in roots and stems of seedlings growing in soil containing glyphosate in the concentration recommended by the manufacturer (7 µM) increased by 35, 50% and 85, 60% respectively. The herbicide in the concentration recommended by the manufacturer decreased the content of spermidine in roots (40.90 µg.g^-1 FM) and increased its content in stems (98.23 µg.g^-1 FM) of soybean seedlings. The content of putrescine and cadaverine was higher in the roots of soybean seedlings than in the stems grown in the same conditions of soil contaminated with the tested concentrations of glyphosate. These studies suggest that low concentration of Roundup 360 SL in soil presents a risk to the of nine-day-old soybean seedlings.

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A. Szulc and A. Parus
The Evaluation of Rhamnolipids Influence on Germination and Initial Growth of Selected Plants
From : 6th National Conference on Bioindicators, Szczecin - Poland, 20-22 April 2016
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - surfactants

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Abstract : The harmful substances in the environment can be identified as : inorganic compounds, such as sulfur compounds, nitrogen, heavy metals, and organic – petroleum substances, harmful detergents, pesticides, etc. These substances should be removed from the environment, to restore it original quality. Besides the traditional physicochemical methods as washing by complexing compounds solutions, phytoextraction is used more and more successfully. To make phytoextraction more effective the substances enhancing the metals mobility in soil are used, such as chelating agents. They increase the bioavailability of metals for plants or stabilize it in the root zone. Both natural and synthetic substances are used, for example biosurfactants. A very popular compound is EDTA, which is a aminopolycarboxylic acid. Also popular are rhamnolipids – the biosurfactant produced by a strain Pseudomonas aeruginosa, short-chain organic acids or aminoacids. Compounds of natural origin are easier biodegradable than synthetic ones, and in the longer term do not constitute a problem for microorganisms which live in a given area. In contrast, synthetic compounds are often hardly biodegradable and may remain in the environment, or penetrate into water. An important aspect of the assessment of the impact of the use of chemical substances on the environment is to determine what effect they would have on plants.
The aim of this study was to investigate the rhamnolipids influence on germination and initial growth of maize (Zea mays) (the representative of the monocotyledon which is used in phytoextraction).
During the research, the impact of rhamnolipids aqueous solution on germination and initial growth of maize in water and soil systems were determined. The assessment of rhamnolipids aqueous solution in hydroponic system was performed using Petri dishes. The soil tests were performed in germination and plants initial growth tests from the Phytotoxkit. In the experiment two types of soil were used, which were characterized by different heavy metals levels (including Pb, Cu, Zn, Fe, Cd). Three independent sets of laboratory experiments in 3 repetitions were performed. On the Petri dish or the soil plate, with aqueous solutions in concentration from 0 to 1000 mg/L, 10 maize seeds were seeded. Controls were made in distilled water. The test plates were placed in an incubator at 21 + 1°C for 6 days. After 6 days of cultivation the number of germinated seeds was counted, and the length of shoot and root was measured. The germination index and the inhibition of shoots and roots growth were calculated.
In the concentration range use the rhamnolipids were not toxic on seed germination; only slightly inhibition of roots and shoots growth was observed in the soil tests cultivation (growth inhibition does not exceed 40%). In the water system a strong influence on seed germination, and roots and shoots development was observed. Germination inhibition (60%) was observed in concentration 25 mg/L. In soil tests the strong influence on maize seed germination was observed using rhamnolipids aqueous solution in concentration from 50 to 250 mg/L. In this case, a maximum of 30% of seed germinated was observed, but also changes in appearance of roots and shoots. A further increase of the rhamnolipids concentration completely stopped the maize seeds development.

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T. Turek, K. Brzozowska-Wojoczek, H. Rzodeczko and P. Fochtman
Sensitivity of Duckweed Species in Different Test Designs
From : 26th Annual Meeting of Setac-Europe Book of Abstracts MO140
Toxkits : Spirodela duckweed Toxkit
Test Species : Spirodela polyrhiza
Topics : test sensitivity; chemicals - organic compounds

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Abstract : The sensitivity of the test organism and the test design are important issues in planning ecotoxicological studies. The aim of the study was to demonstrate that the test design affects the sensitivity of aquatic plants. In the study three species of duckweed : the swollen duckweed Lemna gibba, the common duckweed Lemna minor and the giant duckweed Spirodela polyrhiza were exposed to a reference substance 3,5-dichlorophenol in different test designs. A static and a semi static designs, with or without sediment were used. The tests were performed according to OECD TG 221 (2006). The endpoint values were determined on the basis of the frond number and the dry weight. Generated results were statistically analysed. Lemna minor and Lemna gibba exposed in the static tests were less sensitive, regardless of whether the system was with or without sediment.
However, the presence of sediment in the system affects the sensitivity. Lemna minor and Spirodela polyrhiza seemed to give a similar response, and in the semi-static test without sediment the most toxic impact of the reference substance was observed. Lemna gibba appeared to be the most sensitive in the semi-static test with sediment. When comparing these three species, the generated results indicate that Spirodela polyrhiza is the most sensitive one. Despite that the model organism used is the floating duckweed, the exposure in various test systems may have implications for ecological relevance to generated results.

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C. Hughes, C.V. Eadsforth and S. Harris
Weight of Evidence Approach to Assess the Acute Aquatic Toxicity of GTL Solvents Relative to Other Hydrocarbon Solvents
From : 26th Annual Meeting of Setac-Europe Book of Abstracts TU178
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - hydrocarbons

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Abstract : Hydrocarbon solvents are petrochemicals used in a wide range of industrial and consumer applications globally. These products consist of many different hydrocarbon constituents and are described as UVCB substances (substances of Unknown or Variable composition, Complex reaction products or Biological materials). They are most commonly derived from the processing of crude oil, but other varieties, such as synthetic ‘isoparaffins’ and solvents derived by the FischerTropsch, or ‘Gas-to-Liquid’ (GTL) process, also exist. Although different solvents may share similar performance properties, their composition varies depending on the manufacturing process and they are regarded as different substances under REACH. In order to assess the relative aquatic toxicity of GTL solvents compared with other hydrocarbon solvents a range of screening methods have been used. These included testing of water-accommodated fractions (WAFs) using solid-phase micro-extraction (SPME) combined with gas chromatography (GC) analysis, MICROTOX and DAPHTOXKIT F assays, and toxicity predictions using the PETROTOX model. Results were compared with compositional information for each of the substances and available experimental data from GLP compliant acute aquatic toxicity studies, conducted under OECD guidelines. The results of the screening tests suggested that acute toxicity of GTL Solvents is dependent on hydrocarbon chain length, similar to dearomatised and isoparaffinic hydrocarbon solvents. The white spirits were significantly more toxic based on screening data, which was expected based on the presence of aromatic constituents. On the basis of the experimental screening methods applied, the two most sensitive approaches for detecting toxicity of the various products are SPME-GC and the DAPHTOXKIT F test kit, whereas the MICROTOX assay is the least sensitive. The PETROTOX calculations predicted a similar relationship with chain length, but were generally more conservative than the experimental data. Overall, through the use of a range of screening methods in a weight of evidence approach, the acute aquatic toxicity of GTL solvents was successfully determined relative to other types of hydrocarbon solvents.
See corresponding Poster

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J. Hartmann, C.V. Eadsforth, J. Hermens, T. Halmans, J. Worden and J. Haftka
Toxicity and Bioavailability of Lubricants and Their Additives in the Aquatic Environment
From : 26th Annual Meeting of Setac-Europe Book of Abstracts TU182
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - general; chemicals - organic compounds

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Abstract : For the purpose of communicating environmental hazard via material safety data sheets and product labels, lubricants have to be classified according to their toxicity to the aquatic environment. In line with European Classification, Labeling and Packaging guidelines and GHS classification guidelines, the classification of mixtures should primarily be based upon the available test data on the mixtures as a whole or on similar mixtures. However, in those cases where these data are missing, the classification should be based on calculation (i.e. the GHS summation method) using those ingredients classified for the particular hazard. As experimental data on the aquatic toxicity of lubricants are often not available, hazard assessment of these products is regularly based upon the toxicity of the individual hazardous ingredients, the additives. Fully Formulated Lubricants (FFLs) were tested in screening tests with crustaceans (Daphnia magna), algae (Selenastrum capricornutum) and bacteria (Vibrio fischeri). The test results were then compared with classifications derived from applying the summation method. It is suggested that the additives might not be bioavailable to the aquatic ecosystem due to their incorporation into the lubricant (OECD, 2004). An overestimation of the aquatic toxicity of the final product may be due to a reduction in the bioavailability of the highly toxic additives as a consequence of partitioning into the lubricant base oil. To test this hypothesis, toxicity data were generated (from water accommodated fractions (WAFs)) on the additive packages containing the toxic components on their own and in the presence of base oil as the FFLs.
See corresponding Poster

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B. Opeolu
Chrysene and dibenz(a,h)anthracene contamination of surface waters: toxicity assessment using water flea (Daphnia magna)
From : 26th Annual Meeting of Setac-Europe Book of Abstracts 496
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - hydrocarbons; sediments; surface waters

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Abstract : Water constitutes about 73% of the earth’s surface and only 3% of this is freshwater resources. The surface freshwater resource available as liquid is about 0.3%. Several studies and reports across disciplines had established the universal importance of freshwater for human and ecological health. This limited resource is being exploited and degraded daily especially in the developing world. Climate change and it global impacts continue to exert greater pressure on freshwater resources. The need to develop analytical methods, assess pollution levels, evaluate ecotoxicological effects and remediate wastewaters for possible reuse is now a necessity more than ever. This study aimed at developing an analytical method for the qualitative and quantitative determination of chrysene and dibenz(a,h)anthracene in surface waters and to assess possible toxicity effects of these compounds on water flea, Daphnia magna. A method with good linearity and precision was developed to measure chrysene and dibenz(a,h)anthracene in water and sediment samples. Acute Daphnia toxicity testing was carried out to assess potential harm of these contaminants to ecological health. Results of method development, environmental water monitoring and effective concentrations (ECs) are presented.

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I. Amiano, J. Olabarrieta, J. Vitorica and S. Zorita
Acute toxicity of nanosized TiO2 to Daphnia magna under UVA irradiation
Environmental Toxicology and Chemistry 2012 31, 11 2564-2566
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : nanoparticles; chemicals - nanoparticles

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Abstract : The production and use of nanoparticles (NPs), especially those of titanium dioxide (TiO2) have increased steadily within the last decade. Common knowledge of photoactive nanosized TiO2 (nTiO2) effects on freshwater organisms is largely limited to acute toxicity tests without taking into consideration solar ultraviolet-A (UVA) irradiation. The authors’ approach in the present study, based on a combination of a standardized ecotoxicology method (Organization for Economic Cooperation and Development [OECD] test 202) and the expected solar UVA contribution, will allow a more realistic understanding of the toxicity of TiO2 to Daphnia magna. The results show that the contribution of UVA to TiO2 toxicity cannot be ignored. The half-maximal effective concentration values (median effective concentration [EC50], immobilization as the endpoint) using Organization for Standardization (ISO) test water as well as river water decreased from 29.7 to 33.6 mg/L under dark conditions to 1.2 to 3.4 mg/L TiO2 after exposure to 0.56 mW/cm² UVA radiation. The authors also discuss possible mechanisms of NPs toxicity and the accuracy of results in terms of problems observed, such as solubilization and sedimentation of NPs, in ecotoxicological testing of nTiO2.

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M. Urbaniak, A. Wyrwicka, M. Zielinski and J. Mankiewicz-Boczek
Potential for Phytoremediation of PCDD/PCDF-Contaminated Sludge and Sediments Using Cucurbitaceae Plants : A Pilot Study
Bulletin of Environmental Contamination and Toxicology 2016 97, 3 401-406
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sludges; sediments; wastewaters; soil

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Abstract : The current study evaluates the impact of sewage sludge and urban reservoir sediment on changes in total and Toxic Equivalency (TEQ) PCDD/PCDF concentration in soil and phytotoxicity measured using three test species : Lepidium sativum, Sinapis alba and Sorghum saccharatum, during 5 weeks of Cucurbita pepo L. cv ‘Atena Polka’ (zucchini) cultivation. ‘Atena Polka’ was found to reduce total PCDD/PCDF concentration by 37 % in soil amended with sludge and 32 % in soil treated with sediment from an urban reservoir. The TEQ reduction was almost twofold greater: 68 % in soil amended with sludge and 52 % with urban sediment. Addition of sludge increased root growth inhibition of L. sativum, S. alba and S. saccharatum from 44 % to 90 %. Observed inhibitions were, however, reduced by ‘Atena Polka’ cultivation, and as high as 32 % promotion in root length was noted. Amendment with urban sediment, in turn, resulted in an initial 1 %-36 % promotion of root growth, while ‘Atena Polka’ cultivation reduced this positive effect by inhibition as high as 26 %. Results demonstrated positive influence of ‘Atena Polka’ on the phytotoxicity alleviation and mitigation of total and TEQ PCDD/PCDF concentrations in soil treated with bio-solids from sewage sludge and an urban reservoir.

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P. Oleszczuk
Phytotoxicity of municipal sewage sludge composts related to physico-chemical properties, PAHs and heavy metals
Ecotoxicology and Environmental Safety 2008 69, 3 496-505
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : sludges; waste waters; composts; chemicals - metals; chemicals - hydrocarbons

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Abstract : The aim of the study was to determine the influence of the composting on the phytotoxicity of sewage sludge in relation to their physical–chemical properties, heavy metals and polycyclic aromatic hydrocarbons content. Four municipal sewage sludges were composted for 76 days. A Phytotoxkit test and pot experiment with Lepidium sativum were used for bioassay. The total PAH content in sludges ranged from 3674.1 to 11236.3 µg kg^-1. Heavy metals content was in the range Cd (1.9–76 mg kg^-1), Cr (27.6–120 mg kg^-1), Cu (156–335 mg kg^-1), Pb (37.5–59.5 mg kg^-1), Ni (21.7–155 mg kg^-1) and Zn (1015–1385 mg kg^-1). The results showed a varied toxicity of sewage sludge in relation to the plant tested. In the case of two sludges a 100% inhibition of seed germination were noted. Sludge composting limited their negative influence on most of the phytotoxicity parameters. Only chlorophyll concentration was often lower than in the plant bred on compost obtained from sludge.

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P. Oleszczuk and H. Hollert
Comparison of Sewage Sludge Toxicity to Plants and Invertebrates in three Different Soils
Chemosphere 2011 83, 4 502-509
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sludges; waste waters; soils

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Abstract : Understanding the effect of soil type on the overall toxicity of sewage sludge is one of the most important issues concerning environmental risks associated with the sewage sludge land application. The aim of the study was to determine the influence of different soils (sandy, loamy and OECD soil) on sewage sludges toxicity in relation to plants (Lepidium sativum, Sorghum saccharatum, Sinapis alba) and an invertebrate species (Heterocypris incongruens). The most evident negative influence of sewage sludges on root growth was observed in the case of OECD soil. The EC50 values determined on the basis of the root growth inhibition of all tested plants were in the range 0.1–6.4%, 0.03–9.4% and 6.6–22.1% (% of sewage sludge kg^-1 soil) for OECD, sandy and loamy soil, respectively. Soil type also affects the sewage sludge toxicity in relation to H. incongruens. The LC50 (mortality) values ranged from 0.26% to 11.5% depending on the sludge tested. For EC50 (growth inhibition) values ranged from 10.7% to 36.2%.

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P. Oleszczuk
Testing of Different Plants to Determine Influence of Physico-Chemical Properties and Contaminants Content on Municipal Sewage Sludges Phytotoxicity
Environmental Toxicology 2010 25, 1 38-47
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sludges; waste waters; chemicals - metals; chemicals - hydrocarbons

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Abstract : This study attempts to evaluate the Phytotoxkit as a tool for measuring the toxicity of municipal sewage sludges using 10 common plants: mustard, turnip, cress, red clover, cucumber, tomato, radish, sorrel, and spinach. The results were used to determine a germination index (GI) and a median effective concentration (EC) value for each plant. The trace metal and polycyclic aromatic hydrocarbon (PAH) contaminant content and the physical-chemical properties were examined. Most sewage sludges were characterized by an unfavorable electrical conductivity value of about 5.2 mS/cm(-1). The most abundant trace metals were zinc (Zn) at 871-1680 mg kg(-1), manganese (Mn) at 245-661 mg kg(-1), and copper (Cu) at 88.2-161.0 mg kg(-1). The lowest values were determined for cobalt (Co) at 2.9-3.8 mg kg(-1) and cadmium (Cd) at 0.7-3.7 mg kg(-1). The PAH sum was based on 10 individual compounds (USE EPA), and the PAH content ranged from 4.76 to 27.95 mg kg(-1), most of the sewage sludges showing a predomination by carcinogenic PAHs. The GI values, based on seed germination and root growth inhibition bioassays, showed increasing plant sensitivity to the tested sewage sludges in the following order: cress > turnip > mustard > sorrel > tomato > sorgo > red clover > radish > cucumber > spinach. The EC50 sewage sludge values lay in the range 31-404 g/kg of soil, and significant relationships were found for most of the plants between EC50 and the magnesium content (Mg(2+): alpha = 0.888-0.924, P = 0.05), calcium content (Ca(2+): alpha = 0.813-0.911, P < or = 0.05), and pH (alpha = -0.913-0.948, P = 0.05). In the case of sewage sludge pollutants, significant relationships were found for trace metals such as: strontium (Sr: alpha = 0.851-0.948, P < or = 0.05), chromium (Cr: alpha = 0.858, P < or = 0.05), and nickel (Ni: alpha = 0.955, P = 0.05), as well as pyrene (PAHs).

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A. Malara and P. Oleszczuk
Application of a Battery of Biotests for the Determination of Leachate Toxicity to Bacteria and Invertebrates from Sewage Sludge-Amended Soil
Environmental Science and Pollution Research International 2013 20, 5 3435-3446
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Tetrahymena thermophila
Topics : soils; sludges; waste waters; leachates

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Abstract : The objective of the study was to determine the leachates toxicity from sewage sludge-amended soils (sandy and loamy). Samples originated from a plot experiment realized over a period of 29 months. Two types of soil were fertilized with sewage sludges at the dose of 3 % (90 t/ha). Soil samples were taken after 0, 7, 17, and 29 months from the application of sewage sludges. Leachates were obtained according to the EN 12457-2 protocol. The following commercial tests were applied for the estimation of the toxicity: Microtox (Vibrio fischeri), Microbial assay for toxic risk assessment (ten bacteria and one yeast), Protoxkit F (Tetrahymena thermophila), Rotoxkit F (Brachionus calyciflorus), and Daphtoxkit F (Daphnia magna). The test organisms displayed varied toxicity with relation to the soils amended with sewage sludges. The toxicity of the leachates depended both on the soil type and on the kind of sewage sludge applied. Notable differences were also observed in the sensitivity of the test organisms to the presence of sewage sludge in the soil. The highest sensitivity was a characteristic of B. calyciflorus, while the lowest sensitivity to the presence of the sludges was revealed by the protozoa T. thermophila. Throughout the periods of the study, constant variations of toxicity were observed for most of the test organisms. The intensity as well as the range of those variations depended both on the kind of test organism and on the kind of sludge and soil type. In most cases, an increase of the toxicity of soils amended with the sewage sludges was observed after 29 months of the experiment.

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M. Urbaniak, M. Zielinski and A. Wyrwicka
The Influence of the Cucurbitaceae on Mitigating the Phytotoxicity and PCDD/PCDF Content of Soil Amended with Sewage Sludge
International Journal of Phytoremediation - Epub ahead of print 2016
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sludges; waste waters; soils

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Abstract : The study evaluates the impact of sewage sludge on OECD and vegetable soil phytotoxicity, measured using three test species : Lepidium sativum, Sinapis alba and Sorghum saccharatum, and total and TEQ PCDD/PCDF soil concentration, measured using HRGC/HRMS. It also evaluates the effect of zucchini and cucumber cultivation during 5-weeks period on mitigating these parameters. The application of 3, 9 and 18 t/ha of sewage sludge gradually increases the phytotoxicity of both OECD and vegetable soil. In the case of OECD soil, the highest roots growth inhibitions were observed for S. alba (73%, 86% and 87%, respectively) and the lowest for S. saccharatum (7%, 59% and 70%), while in vegetable soil inhibitions were averagely 25% lower. Sludge application led also to a 38% (3 t/ha), 169% (9 t/ha) and 506% (18 t/ha) increase in PCDD/PCDF concentration, and the TEQs were augmented by 15%, 159% and 251%. Both soil phytotoxicity and total and TEQ PCDD/PCDF concentrations were diminished as a result of zucchini and cucumber cultivation. The maximum reduction of soil phytotoxicity (83%) was observed as an effect of cucumber cultivation, while zucchini was 11% less effective. Zucchini, in turn, was more efficient in PCDD/PCDF removal (37% reduction), followed by cucumber (24%). Such differences were not observed in the case of TEQ reductions (68% and 66% for zucchini and cucumber cultivation, respectively).

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S. Szklarek, M. Stolarska, I. Wagner and J. Mankiewicz-Boczek
The Microbiotest Battery as an Important Component in the Assessment of Snowmelt Toxicity in Urban Watercourses - Preliminary Studies
Environmental Monitoring and Assessment 2015 187, 2 16
Toxkits : Algaltoxkit F^TM; Phytotoxkit; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Phaeodactylum tricornutum; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : stormwaters; surface waters; rainwater

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Abstract : The aim of the study was to use a battery of biotests composed of producers (Selenastrum capricornutum, Sorghum saccharatum, Lepidium sativum and Sinapis alba), consumers (Thamnocephalus platyurus) and decomposers (Tetrahymena thermophila) to evaluate the toxicity of snowmelt and winter storm water samples. The toxicity of the samples collected in the winter period December to February (2010-2011), in one of the largest agglomerations in Poland, the city of Lodz, was compared to that of storm water samples taken under similar conditions in June. The most toxic snowmelt samples were found to be high acute hazard (class IV), while the remaining samples were rated as slight acute hazard (class II). L. sativum (in the Phytotox test) was the most sensitive test organism, giving 27 % of all toxic responses, followed by S. capricornutum with 23 % of all responses. T. thermophila was the least sensitive, with only 2 % of all toxic responses. The greatest range of toxicity was demonstrated by samples from the single family house catchment : no acute hazard (class I) to high acute hazard (class IV).

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M. Salcedo and I. De Vicente Alvarez-Manzaneda
Magnetic Particles' Effects on Survival and Hatching of the Rotifer Brachionus calyciflorus and the Growth of the Chlorella sp. alga
From : 26th Annual Meeting of Setac-Europe Book of Abstracts MO116 2016
Toxkits : Rotoxkit F^TM acute
Test Species : Brachionus calyciflorus
Topics : chemicals - nanoparticles; nanoparticles

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Abstract : The main objective of this research is to study the possible toxicological effects of iron magnetic microparticles on the rotifer Brachionus calyciflorus and the cloroficean alga Chlorella sp. These magnetic microparticles have recently been proposed for the restoration of eutrophic aquatic ecosystem, for remove the phosphorus located in them. With the aim of carrying out the toxicological test on B. calyciflorus, it was followed ASTM protocol. During 24 hours the organisms were in contact with different concentrations of magnetic particles, analyzing the mortality of the organisms. On the other hand, a test about the rotifer’s eggs hatching in contact with dissolved iron from the particles was carried out. In the case of the test with the alga Chlorella sp. , OECD’s standardized protocol was followed. This test last 72 hours and they were used different concentrations of magnetic particles, after these 72 hours the inhibition growth of the alga was calculated. The results obtained were a CE50 of 1.63 g/l of iron for the rotifers and 0.085 g/l for Chlorella sp. , the alga was more sensitive to the particles than planktonic organism being 100% of algal growth inhibition from 0.5 g/l. Hatching of Brachionus calyciflorus eggs in contact with the dissolved iron was total.

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K. Charmpi and P. Makridis
Application of a Battery of Bioassays for Assessing the Effects of WWTP Effluent on the Aquatic Biota
From : 26th Annual Meeting of Setac-Europe Book of Abstracts TU129 2016
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : waste waters; effluents

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Abstract : Since chemical and biological data is considered important for assessing the quality of municipal wastewater treatment plant (WWTP) effluent and its environmental risk, the present study investigates the effects of WWTP influent and effluent on a battery of marine organisms such as the brine shrimp Artemia franciscana (formerly Artemia salina), the sea bream Sparus aurata, and the green algae Dunaliella tertiolecta, while primary culture of hemocytes of the marine bivalve mollusk Mytilus galloprovincialis was also used for investigating the cytotoxic and oxidative effects of samples. Chemical analysis, primarily performed in samples, showed that WWTP processes efficiently reduce the levels of each parameter tested in effluent. On the other hand, the exposure of the green algae Dunaliella tertiolecta to different concentrations of each sample (6.25-50% v/v), showed a significant dose-dependent decrease of WWTP influent-treated algae growth rate, after 24h, compared to that observed in WWTP effluent-treated algae, a fact that attenuated over time (96h). However, a significant depletion of algal growth rate was observed in cells treated with concentrations of WWTP influent higher than 25% v/v. Similarly, a slight attenuation of WWTP effluent toxic effects was observed in the brine shrimp Artemia salina, compared to those occurred in case of WWTP influent (24hLC 50 values of 90.63% and 86.58 v/v respectively). Moreover, Sparus aurata larvae incubated with different amounts of WWTP influent and/or effluent (0.001-10% v/v) for 6 days, showed a time-dependent increase of larvae mortality rates. However, among live larvae, significantly elevated developmental abnormalities/deformities were obtained in WWTP influent-treated larvae, at least in case of larvae treated with 10% v/v of influent. Regarding WWTP effluent-mediated cytotoxic and oxidative potency, a significant increase of cell death as well as increased levels of lipid peroxidation were observed in WWTP influent-treated hemocytes of mussels, compared to those occurring in WWTP effluent-treated cells. In conclusion, the results of the present study showed that even if WWTP processes could efficiently remove organic compounds and pathogenic microbes from municipal influent, WWTP effluent disposal could pose a risk for the aquatic environment. The extent of WWTP risk seems to be species-specific, depending mainly on species’ vulnerability and sensitivity.

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F. Sakaveli, V. Tsiridis, M. Petala and E. Darakas
Ecotoxic Effects of Land Application Of Sewage Sludge
From : 5th International Conference on Industrial & Hazardous Waste Management, Crete - Greece, 27-30 September 2016
Toxkits : Daphtoxkit F^TM magna; Phytotoxkit
Test Species : Daphnia magna; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sludges; waste waters; soils; leachates

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Abstract : Sewage sludge produced from wastewater treatment plants can be used for soil improvement as organic fertilizer or soil conditioner. Application of sludge in agricultural lands reduces the quantities of chemical fertilizer used, improves soil fertility and recycles waste products back to the cycle of soil and plants. However, the release of various harmful constituents during the land application of sewage sludge, such as heavy metals polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs) and other organic pollutants have to be taken into account. These constituents may affect either directly the plants and terrestrial organisms or the aquatic environment through the leachates produced during the land disposal. The evaluation of the performance and the environmental impact of the use of sewage sludge as fertilizer or soil conditioner using chemical analyses is a complicated issue due to a large number of constituents that have to be analyzed. Furthermore, chemical analyses cannot precisely evaluate the fertilization capacity of sewage sludge and the effect of the leachates produced during the disposal on the terrestrial and aquatic environment.

In this study the environmental impact of the application of sewage sludge for soil improvement in agriculture was examined using physicochemical analyses, leaching tests and bioassays. Sewage sludge samples from two different municipal wastewater treatment plants were examined; one sewage sludge with relatively low organic content (21.8% of dry matter determined by loss on ignition) and another one sewage sludge with relatively high organic content (66.4% of dry matter determined by loss on ignition). The effect of the sludge on various plants was evaluated by measuring the rate of both seed germination and root growth of three higher plant species : one monocotyledon Sorghum saccharatum and two dicotyledonous Lepidium sativum and Sinapis alba. A series of mixtures of dried sewage sludge with OECD reference soil at concentrations of 2.5, 5, 10, 15 and 25% (w/w) were examined for their effects on the three plant species tested. The one stage EN 12457-2 leaching test and NEN 7341 availability test were applied for the evaluation of the effect of the sewage sludge to the aquatic environment using two test species, the photobacterium Vibrio fischeri and the crustacean Daphnia magna. The availability test NEN 7341 was applied to dry samples and their residues after heating to 550 °C for the evaluation of the contribution of the organic content to the ecotoxicity of the sewage sludge samples.

Sewage sludge from both wastewater treatment plants exhibited high phytotoxic effect even at low concentrations of 15% (w/w), where a root growth inhibition up to 98% was observed. Toxicity results indicated that the sewage sludge of high organic content was more toxic than that of low organic content when exposed towards both plant and aquatic species. The Lowest Ineffective Dilution value (LID) on D. magna of EN 12457-2 leachates of sludge with high organic content was equal to 5, while the corresponding value for the sludge with low organic content was equal to 1. The leachates obtained by the availability test NEN 7341 for the sludge with low and high organic content caused significant bioluminescence inhibition of V. fischeri up to 66.0% and 72.7%, respectively.

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V. Tsiridis and G. Persoone
Practical and Cost Effective Toxicity Tests for the Evaluation of the Environmental Hazard of Solid Waste
From : 5th International Conference on Industrial & Hazardous Waste Management, Crete - Greece, 27-30 September 2016
Toxkits : All Toxkits
Test Species :
Topics : solid wastes; toxicity classification; solid waste leachates

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Abstract : The application of leaching tests and physicochemical analyses of the resulting leachates is the common practice for the evaluation of the environmental hazard of solid waste. In this context, the results of the physicochemical analyses of leaching tests are compared with the regulatory limit values for the classification of the materials as inert or hazardous waste. Consequently, even though in many cases leaching tests have shown that various heavy metals such as As, Cr, Ni, Pb and Se can be released from the solid matrix of waste, their concentration in the leachates may still be within the leaching limit values for inert or non-hazardous waste. However, it is well recognized that the determination of chemical compounds concentration in the leachates does not dictate the bioavailability of the compounds, and thus their potential effect on the aquatic life cannot be addressed. Many studies have shown that the application of ecotoxicity tests in combination with chemical analyses is a better approach for the evaluation of the environmental hazard of the waste. Constituents detected at concentration lower than the regulatory leaching values may cause harmful effect to aquatic organisms, while interactive effects among constituents may alter the resulting ecotoxicity of a waste. Thus, beyond the consideration of the intrinsic waste properties, the application of ecotoxicity tests is needed for an ecologically meaningful evaluation of the potential environmental hazard resulting from the utilization or disposal of solid waste.
For the classification of waste as hazardous, hazard criteria H1 to H15 are fundamentally defined in Annex III of Council Directive 91/689/EEC on hazardous waste. The criterion H14 refers to “ecotoxic”, while Council Directive 2008/98/EC on wastes extends the criteria for considering a waste as hazardous by appending criterion H15, which imposes the application of H1 to H14 criteria to the leachates of waste. As a result of the need for ecotoxicity testing of waste, the European Standard EN 14735:2005 “Characterization of waste - Preparation of waste samples for ecotoxicity tests” was issued by the Technical Committee CEN/TC 292, in which a collection of bioassays applicable to waste is listed in Annex B.
However, the application of ecotoxicity tests for the evaluation of the environmental hazard of waste (especially on a routine basis) is in practice hampered by the need for the infrastructure and equipment, and the workload and the costs for the culturing and year-round maintenance of live stocks of the test species needed for the assays. This problem has been overcome by the use of “Toxkit microbiotests” which make use of dormant or immobilised stages of selected test species, which can be stored and “de-immobilised” or hatched at the time of performing the assays. A battery of acute and short-chronic “culture/maintenance free” microbiotests with species belonging to several phylogenetic groups (bacteria, micro-algae, protozoans, rotifers, crustaceans and higher plants) is now available and employed worldwide for diverse applications in both aquatic and terrestrial environments. As of to date more than 250,000 Toxkit microbiotests have already been used for research and routine toxicity monitoring in 45 countries, and more than 600 studies are listed on the website www.microbiotests.be.
Most Toxkit microbiotests strictly adhere to the protocols for regulatory testing prescribed by international organizations, e.g. the Algaltoxkit with the microalgae Pseudokirchneriella subcapitata and the Daphtoxkit with the crustaceans Daphnia magna which both follow ISO and OECD test procedures. Ecotoxicity test with higher plants can even be performed directly on the solid waste with the Phytotoxkit microbiotest. This three-day seed germination and early plant growth microbiotest is performed in flat transparent test containers, with measurement of root and shoot growth by image analysis, and employs three plant species, one monocotyledon (Sorghum saccharatum) and two dicotyledons (Lepidium sativum and Sinapis alba).
The stock culture free Toxkit microbiotests are standardized and validated practical, robust and low cost toxicity tests which are excellent tools for the overall evaluation of the environmental hazard of solid waste. Depending on the utilization or disposal scenario of the waste, the most appropriate tests species can be selected for the toxicity tests in combination with the appropriate leaching tests.

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P. Uudeküll, J. Kozlova, H. Mändar, J. Link, M. Sihtmäe, S. Käosaar, I. Blinova, K. Kasemets, A. Kahru, R. Stern, T. Tätte, K. Kuklia and A. Tamm
Atomic Layer Deposition of Titanium Oxide Films on As-Synthesized Magnetic Ni Particles : Magnetic and Safety Properties
Journal of Magnetism and Magnetic Materials 2017 429 299-304
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : nanoparticles; chemicals - metals

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Abstract : Spherical nickel particles with size in the range of 100–400 nm were synthesized by non-aqueous liquid phase benzyl alcohol method. Being developed for magnetically guided biomedical applications, the particles were coated by conformal and antimicrobial thin titanium oxide films by atomic layer deposition. The particles retained their size and crystal structure after the deposition of oxide films. The sensitivity of the coated particles to external magnetic fields was increased compared to that of the uncoated powder. Preliminary toxicological investigations on microbial cells and small aquatic crustaceans revealed non-toxic nature of the synthesized particles.

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I. Blinova
Use of Freshwater Algae and Duckweeds for Phytotoxicity Testing
Environmental Toxicology 2004 19 425-428
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : plant tests; chemicals - mtals

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Abstract : The toxicity of contaminated water of different origins and chemicals [Cr(III), Pb(II), Cu(II), Cd(II), pyrene] were tested using four test species : the alga Selenastrum capricornutum (new name Raphidocelis subcapitata), the duckweed Lemna minor, and the crustaceans Thamnocephalus platyurus and Daphnia magna. On the basis of the results obtained, the sensitivity of plant species and problems concerning the interpretation of the phytotoxicity data are discussed. The data indicated that the sensitivities of crustaceans and plant species both to individual contaminants and to mixtures are unpredictable and that there is no reason to consider plant species less sensitive than animal species.
Lemna minor is more sensitive than Selenastrum capricornutum. With colored samples, duckweed is preferable for toxicity testing. To raise the predictive utility of the phytotoxicity data, it is recommended that natural water be used in the test procedure.

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Y. Marneffe, C. Chalon, D. Leroy and J-F Vaerewyck
Multi-actor Strategy for the Management of an Industrial Effluent Impacting a Sensitive Waterbody : Use of Bioassays to Assess the Efficiency of the Management Measures
27th Annual Meeting SETAC EUROPE, Brussels, Belgium, 7-11 May 2017. Book of Abstracts
Toxkits : Rotoxkit F^TM
Test Species : Brachionus calyciflorus
Topics : effluents; waste waters

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Abstract : Since many years, a highly polluted tannery effluent is impacting a very sensitive waterbody, the Pas-à-Wasmes watercourse. Despite expensive improvements in treatment carried out by the industrial operator, the toxic load as shown by a bioassay battery remained important for the receiving river (waterbody EL13R). The complementary management of the effluent by the urban treatment plant was not possible before and the industrial effluent was directly discharged into the river. In 2013, the main stakeholders (the industrial operator, the regional authority delivering permits, the operator of the urban waste water treatment plant of Pas-à-Wasmes and scientists - ISSeP and Cebedeau) began discussions in order to solve this problem and assess the efficiency of the decided management measures. The improvement of the pretreatment in the tannery and the tannin recycling allowed the urban waste water treatment plant to treat the totality of the industrial effluent without suffering adverse effects on its own treatment capacities. To assess the efficiency of the treatment, complementary to chemical analyses, we used a battery of short term and chronic bioassays with the bacteria Vibrio fischeri, the alga Pseudokirchneriella subcapitata, the rotifer Brachionus calyciflorus and the microcrustacea Daphnia magna. Priority List substances of the WFD and other pollutants discharged in significant quantities were also measured. The analyses were carried out on the effluent of the tannery before and after treatment by the urban waste water treatment plant and in the receiving waterbody, upstream and downstream from the effluent. Results show that the toxicity of the industrial effluent is strongly lowered after the treatment by the urban treatment plant and bioassays show that the receiving waterbody is really significantly less impacted than before. This management resulting from a multi-actor strategy induced an important improvement of the waterbody in a cost-effective way.
See corresponding Poster

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M. Hémart, H. Breulet and Y. Marneffe
TiO2, CNT and Al Nanoparticles Risk Assessment for Sediment-Dwelling Organisms
27th Annual Meeting SETAC EUROPE, Brussels, Belgium, 7-11 May 2017. Book of Abstracts
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : nanoparticles; chemicals - metals; sediments

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Abstract : The increasing use of nanoparticles for the manufacture of numerous and varied materials and products raises questions about the risks they may represent for both the public and industries that use them. As such, nanotechnologies and their products represent a serious challenge to the authorities responsible for environmental policies and human safety. The NANOGRA project proposes a global approach to nanoparticle’s risks using a multidisciplinary assessment based on different fields of study :
- The explosion and inflammation risk of the nano-sized particles by determining different parameters of interest.
- The potential impacts to ecosystems associated with a certain dispersion of nanoparticles in environmental media assessed by laboratory experiments before extrapolating to field experiments.
- The toxicological risks for exposed workers and consumers through the use of some product lines are evaluated using several graded risk management tools (control banding). A comparative study of the various management tools is also conducted on selected nanoparticles for the two previous components.
This presentation focusses on ecotoxicological aspects of NANOGRA. In the case of an accidental release of nanoparticles in the environment in Belgium, sediments will likely be the final sink for the released nanoparticles (leaching of dust for ex). Sediment organisms will probably be the most affected by this new kind of contaminants. A first laboratory approach on artificial sediment spiked with nanoparticles should assess the lethal and sublethal risk for Chironomus riparius (bioaccumulation, growth inhibition, development delay and teratogenicity) and Heterocypris incongruens (growth inhibition). The nanoparticles were selected on the basis of different nanoparticles emission sources identified in Wallonia and/or industrial strategic directions. Three nanoparticles have been chosen: titanium dioxide, multiwall carbon nanotubes and aluminium. The experimental protocols will be presented and the first available results will be discussed.
See corresponding Poster

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D.J.L. Thomas, S.F. Tyrrel, R. Smith and S. Farrow
Bioassays for the Evaluation of Landfill Leachate Toxicity
Journal of Toxicology and Environmental Health, Part B 2009 12 83-105
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : leachates; landfill leachates

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Abstract : This article reviews the application of bioassays for assessing the toxicity hazard posed by landfill leachate discharged to an aquatic environment. Landfill leachate is a complex mixture of chemicals; thus it is difficult to assess the risk posed to aquatic wildlife using standard chemical identification techniques, such as gas chromatography–mass spectroscopy (GC-MS). From this review it is clear that toxicity testing, using species that represent the different trophic levels, is a superior way to predict the risk posed by discharge than chemical analysis. Previous studies assessed leachate toxicity using bacteria, algae, plants, invertebrates, fish, and genotoxicity. Studies showed that leachate exhibits a wide range of toxicities to the species tested. Ammonia, alkalinity, heavy metals, and recalcitrant organics were identified to be the cause of adverse responses from the test organisms. Concentrations of these chemicals were found to depend upon the types of waste landfilled. As part of this review, Slooff analysis was applied to published results to calculate the sensitivity of test species. It was concluded that Lemna minor and Thamnocephalus platyurus were the most sensitive tests and, Vibrio fischeri (Microtox) was the least sensitive test available. Little is known about the sensitivity of each species to the different types of waste that might have been landfilled. A battery of tests needed for a more accurate assessment of landfill leachate is proposed. Some of the more common tests have been replaced by more sensitive tests that produce more relevant results for the industry and regulators.

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S. Le Faucheur, D. Vasiliu, I. Catianis, M. Zazu, P. Dranguet, R. Beauvais-Flück, J.-L. Loizeau, et al.
Environmental Quality Assessment of Reservoirs Impacted by Hg from Chlor-Alkali Technologies : Case Study of a Recovery
Environmental Science and Pollution Research 2016 23 542-553
Toxkits : Daphtoxkit F^TM magna; Ostracodtoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens
Topics : chemicals - metals

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Abstract : Mercury (Hg) pollution legacy of chlor-alkali plants will be an important issue in the next decades with the planned phase out of Hg-based electrodes by 2025 within the Minamata convention. In such a context, the present study aimed to examine the extent of Hg contamination in the reservoirs surrounding the Oltchim plant and to evaluate the possible improvement of the environmental quality since the closure of its chlor-alkali unit. This plant is the largest chlor-alkali plant in Romania, which partly switched to Hg-free technology in 1999 and definitely stopped the use of Hg electrolysis in May 2012. Total Hg (THg) and methylmercury (CH3Hg) concentrations were found to decrease in the surface waters and sediments of the reservoirs receiving the effluents of the chloralkali platform since the closure of Hg units. Hence, calculated risk quotients (RQ) indicated no adverse effect of Hg for aquatic organisms from the ambient water exposure. RQ of Hg in sediments were mostly all higher than 1, showing important risks for benthic organisms. However, ecotoxicity testing of water and sediments suggest possible impact of other contaminants and their mixtures. Hg hotspots were found in soils around the platform with RQ values much higher than 1. Finally, THg and CH3Hg concentrations in fish were below the food safety limit set by the WHO, which contrasts with previous measurements made in 2007 revealing that 92 % of the studied fish were of high risk of consumption.
Discontinuing the use of Hg electrodes greatly improved the surrounding environment of chlor-alkali plants within the following years and led to the decrease environmental exposure to Hg through fish consumption. However, sediment and soil still remained highly contaminated and problematic for the river reservoir management. The results of this ecological risk assessment study have important implications for the evaluation of the benefits as well as limits of the Minamata Convention implementation.

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B.K. Mavakala, S. Le Faucheur, C.K. Mulaji, A. Laffite, N. Devarajan, E.M. Biey, G. Giuliani, J.P. Otamonga, P. Kabatusuila, P.T. Mpiana and J. Poté
Leachates Draining from Controlled Municipal Solid Waste Landfill : Detailed Geochemical Characterization and Toxicity Tests
Waste Management 2016 55 238-248
Toxkits : Ostracodtoxkit F^TM; Thamnotoxkit F^TM
Test Species : Heterocypris incongruens; Thamnocephalus platyurus
Topics : leachates; solid waste leachates; landfill leachates

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Abstract : Management of municipal solid wastes in many countries consists of waste disposal into landfill without treatment or selective collection of solid waste fractions including plastics, paper, glass, metals, electronic waste, and organic fraction leading to the unsolved problem of contamination of numerous ecosystems such as air, soil, surface, and ground water. Knowledge of leachate composition is critical in risk assessment of long-term impact of landfills on human health and the environment as well as for prevention of negative outcomes. The research presented in this paper investigates the seasonal variation of draining leachate composition and resulting toxicity as well as the contamination status of soil/sediment from lagoon basins receiving leachates from landfill in Mpasa, a suburb of Kinshasa in the Democratic Republic of the Congo. Samples were collected during the dry and rainy seasons and analyzed for pH, electrical conductivity, dissolved oxygen, soluble ions, toxic metals, and were then subjected to toxicity tests. Results highlight the significant seasonal difference in leachate physicochemical composition. Affected soil/sediment showed higher values for toxic metals than leachates, indicating the possibility of using lagoon system for the purification of landfill leachates, especially for organic matter and heavy metal sedimentation. However, the ecotoxicity tests demonstrated that leachates are still a significant source of toxicity for terrestrial and benthic organisms. Therefore, landfill leachates should not be discarded into the environment (soil or surface water) without prior treatment. Interest in the use of macrophytes in lagoon system is growing and toxic metal retention in lagoon basin receiving systems needs to be fully investigated in the future. This study presents useful tools for evaluating landfill leachate quality and risk in lagoon systems which can be applied to similar environmental compartments.

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K. De Schamphelaere, J. Teuchies, T. Ysebaert, P. Meire, and R. Blust
Development of a Triad Assessment Method for Brackish Sediments in Flanders
SETAC Europe 27th Annual Meeting, Brussels - Belgium, 7-11 May 2017
Toxkits : Rotoxkit M^TM
Test Species : Brachionus plicatilis
Topics : sediments; toxicity monitoring; toxicity classification

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Abstract : In sediment risk assessment, analysis of the pollutant concentrations is essential in determining the degree and nature of sediment contamination. However, chemical analyses provide no evidence of toxic effects or effects in situ. The Sediment Quality Triad method incorporates measures of various chemical parameters, toxicological effects and benthic community structure in view of conducting an integrated assessment of sediment quality. In framework of developing a triad assessment method for the quality evaluation of brackish sediments in Flanders, in 2015-2016 38 sediment samples were taken along the Scheldt estuary (Sea Scheldt (Flanders) and Western Scheldt (the Netherlands)) and other brackish aquatic systems in Flanders. For these samples, chemical parameters (e.g. metals and organic pollutants), toxicological effects and benthic community structure were assessed. In this study 3 bioassays were carried out to test their suitability for uptake in a quality triad method for brackish sediments as indicator of ecotoxicological effects. Two sediment contact bioassays with the polychaete worm Hediste diversicolor and the amphipod Corophium volutator, and a pore water test with the rotifer Brachionus plicatilis were performed to test the toxicity of the samples. For the sediment contact tests observed mortalities varied between 0%-33% for H. diversicolor, between 1%-22% for Corophium volutator and between 10%-43% for B. plicatilis. Control tests with reference sediment showed for the 3 tests mortalities lower than 10%. For the Scheldt estuary, the 3 tests showed significantly higher mortalities for sediments from the upper part of the estuary in the Sea Scheldt, which was characterized by higher levels of micropollutants, than for the lower part of the estuary (Western Scheldt). Based on a literature inventory of existing sediment quality standards, quality guidelines for chemical parameters for Flemish brackish sediments were formulated. For the biological component of the triad method, comprising an evaluation of the benthic invertebrate community, a separate evaluation method was included for brackish oligohaline more static water systems, and for brackish sediments in the different ecotopes of the Scheldt estuary.
See corresponding Poster

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L. Manusadžianas, K. Sadauskas, R. Vitkus, B. Gylyte, R. Karitonas, A. Krevš, A. Kucinskiene, N. Kazlauskiene and J. Žaltauskaite
Two-level Test-Battery Approach for Routine Effluent Control in Lithuania
From 6th International Conference of Environmental Management, Engineering, Planning & Economics - Book of Abstracts 387 2017
Toxkits : Algaltoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : effluents; test selection; toxicity monitoring

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Abstract : Promoting the methods for Whole Effluent Assessment (WEA) into routine regulation as well as expressing concern on the status of the Baltic Sea, HELCOM has initiated several international programmes such as Cohiba, BaltHaz, etc. In 2011–2014, the Ministry of Environment of Lithuania put forward a national programme with the aim to develop a strategy of implementing toxicity tests for effluent control as well as necessary legal documents. Contrary to the control of individual substances, which is based on knowledge of a single concentration, municipal and industrial effluents can be regulated directly on the basis of harmful effects that occur in testing organisms.
Different countries use laboratory toxicity tests at various levels to monitor effluent discharges into surface waters. For example, acute and chronic toxicity tests are used in the USA and Sweden, whereas exclusively acute tests are used in Canada and several European countries. To date, most countries provide chemical-based effluent controls, however, increasing release of newly developed chemicals and difficulties in measuring them one-by-one make individual-substance approach problematic. In several countries, toxicity data are included for taxation purposes, in parallel to chemical-based taxation, for example, toxicity data from D. magna acute and fish embryos tests are used in France and Germany, respectively.
The main results of the programme include two levels of the test-batteries (acute and chronic toxicity), indices for toxicity evaluation, two criteria for selection of economic subjects to be liable for effluent biotesting (first, criteria of economic activity according to NACE Rev. 2 and, second, ratio of effluent discharge and stream- flow rates), and preparing the drafts of necessary legal environmental documents. In this paper, a system for municipal and industrial effluent control, preliminary data on inventory of effluent monitoring as well as envisaged steps of its implementation into routine will be overviewed.

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T. Pardo, R. Clemente, P. Alvarenga and M. P. Bernal
Efficiency of Soil Organic and Inorganic Amendments on the Remediation of a Contaminated Mine Soil : II. Biological and Ecotoxicological Evaluation
Chemosphere 2014 107 101-108
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : soils; composts

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Abstract : The feasibility of two organic materials (pig slurry and compost) in combination with hydrated lime for the remediation of a highly acidic trace elements (TEs) contaminated mine soil was assessed in a mesocosm experiment. The effects of the amendments on soil biochemical and ecotoxicological properties were evaluated and related with the main physicochemical characteristics of soil and soil solution. The original soil showed impaired basic ecological functions due to the high availability of TEs, its acidic pH and high salinity. The three amendments slightly reduced the direct and indirect soil toxicity to plants, invertebrates and microorganisms as a consequence of the TEs’ mobility decrease in topsoil, reducing therefore the soil associated risks. The organic amendments, especially compost, thanks to the supply of essential nutrients, were able to improve soil health, as they stimulated plant growth and significantly increased enzyme activities related with the key nutrients in soil. Therefore, the use of compost or pig slurry, in combination with hydrated lime, decreased soil ecotoxicity and seems to be a suitable management strategy for the remediation of highly acidic TEs contaminated soils.

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L. Manusadzianas, K. Sadauskas, R. Vitkus, B. Gylyte, R. Karitonas, A. Krevs, A. Kucinskiene, N. Kazlauskiene and J. Zaltauskaite
Two-level Test-Battery Approach for Routine Effluent Control in Lithuania
Book of Abstracts 387 of the Sixth International Conference on Environmental Management, Engineering, Planning & Economics Thessaloniki, Greece, June 25-30, 2017
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : effluents; toxicity classificatino

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Abstract : Promoting the methods for Whole Effluent Assessment (WEA) into routine regulation as well as expressing concern on the status of the Baltic Sea, HELCOM has initiated several international programmes such as Cohiba, BaltHaz, etc. In 2011–2014, the Ministry of Environment of Lithuania put forward a national programme with the aim to develop a strategy of implementing toxicity tests for effluent control as well as necessary legal documents. Contrary to the control of individual substances, which is based on knowledge of a single concentration, municipal and industrial effluents can be regulated directly on the basis of harmful effects that occur in testing organisms.
Different countries use laboratory toxicity tests at various levels to monitor effluent discharges into surface waters. For example, acute and chronic toxicity tests are used in the USA and Sweden, whereas exclusively acute tests are used in Canada and several European countries. To date, most countries provide chemical-based effluent controls, however, increasing release of newly developed chemicals and difficulties in measuring them one-by-one make individual-substance approach problematic. In several countries, toxicity data are included for taxation purposes, in parallel to chemical-based taxation, for example, toxicity data from D. magna acute and fish embryos tests are used in France and Germany, respectively.
The main results of the programme include two levels of the test-batteries (acute and chronic toxicity), indices for toxicity evaluation, two criteria for selection of economic subjects to be liable for effluent biotesting (first, criteria of economic activity according to NACE Rev. 2 and, second, ratio of effluent discharge and stream- flow rates), and preparing the drafts of necessary legal environmental documents. In this paper, a system for municipal and industrial effluent control, preliminary data on inventory of effluent monitoring as well as envisaged steps of its implementation into routine will be overviewed.

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D. Hernandez-Moreno, J. Hernandez-Gonzalo, J. Navas, and M. Fernandez-Cruz
Coating Relevance in the Nanoparticle Toxic Effect to Daphnia magna
From : 26th Annual Meeting of Setac-Europe Book of Abstracts TH051 2016
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : nanoparticles

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Abstract : Coating of nanoparticles (NPs) with a proper surface agent is usually required before their use in any application, to improve its functionality. This coating will determine the NP interaction with the environment as the physicochemical properties of the NPs can be modified due to this cover, leading to different effects in organisms. A major regulatory problem will be the high amount of assays that should be conducted for all coating-NP combinations. In the framework of FP7 Project GUIDEnano we investigated the toxicity produced by different coatings on three metal NPs (synthetized from PlasmaChem GmbH, Germany) to try to establish relationships between coatings and toxicity. The acute effect of NPs, in terms of core composition (TiO2 , CeO2 and Ag), was investigated on neonates of Daphnia magna after 48 h exposure following the OECD guidelines. The two first NPs were functionalized with three different coatings: citrate (TiO2 -CIT/CeO2 -CIT), polyethylene glycol (TiO2 -PEG/CeO2 -PEG), and dodecylphosphonic acid (TiO2 -DDPA/CeO2 -DDPA), moreover those NPs were tested also uncoated (TiO2 -UNC/CeO2 -UNC). AgNPs were coated with CIT, PEG and oleylamine (HPB). Both DDPA and HPB confer hydrophobic characteristics to the metal NPs, whereas CIT and PEG make NPs more hydrophilic. The highest immobilizing effect was found after exposure to AgNPs, showing the lowest IC50 . In those cases, assuming that AgCIT can behave as the uncoated NP, the toxic effect of the AgNPs increased by their functionalization with PEG and HPB. On the other hand, D. magna exposed to TiO2 -UNC and CeO2 -UNC presented higher toxicity than the same core coated with CIT, PEG or DDPA where no toxic effect was observed. Thus the coating conducted to a protective effect over the metallic core. The size of the NPs in the exposure medium could not explain this different behavior between metal-oxide and metal NPs. It is more likely that the coating interaction with the core lead to a different behavior of the NPs. From this study a specific pattern linked to the coating could be established depending on the core composition. The coatings of the metal-oxide nanoparticles eliminated completely the toxicity produced by the uncoated NPs. On the contrary, toxicity of the metal NPs was increased depending on the coating surrounding the AgNP.

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A. Klimkowicz-Pawlas, M. Grela, U. Pasternak and B. Smreczak
Impact Assessment of PAHs Mixture in Soils with Different Properties
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Ostracodtoxkit F^TM; Phytotestkit; Phytotoxkit; Rapidtoxkit
Test Species : Heterocypris incongruens; Thamnocephalus platyurus; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : soils; chemicals - hydrocarbons

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Abstract : Soil protection is one of the most important issues in EU policy; soil protection issues are also included in the Common Agricultural Policy (CAP) and the Rural Development Program (RDP). Among the main threats to soil functions are chemical pollutants, which in the soil environment very rarely occur as individual substances or ions. Soil organisms are exposed to a complex mixture of chemicals with different properties and varying composition, so the toxicity of compounds present in the mixture becomes very important in soil toxicity studies. Previous research on the effects of PAHs were most often focused on evaluating the effects of single compounds, eg. phenanthrene or pyrene (Klimkowicz-Pawlas 2009, Lu et al. 2013, Suszek-Lopatka et al. 2016).
The aim of the study was to evaluate the influence of the PAHs mixture on the soil habitat and retention function. Five compounds (fluorene, anthracene, phenanthrene, pyrene and chrysene) with different properties (water solubility 2 – 1900 µg.dm^-3, log Kow 4,18 – 5,86) were selected for the study.
Soil materials were collected from the humus layer (0-20 cm) of five agricultural soils in which the content of organic pollutants and heavy metals did not exceed the limit values (Reg. Min. Env. 2016). Selected soils were characterized by different biological and physicochemical properties (Corg : 0,68 – 3,41%, pHKCl : 4,7 – 6,8).
Soil samples were artificially contaminated with a mixture of 5 PAHs on five levels in the range of 0-500 mg Σ5WWA kg^-1 (0-100 mg kg^-1 of a single compound). The experiment was conducted under strictly controlled temperature and humidity conditions for a period of 14 days. After incubation the battery of ecotoxicological tests was conducted, and the effects of PAHs mixture on the soil habitat and retention function was assessed. The soil solid-phase tests included: Ostracodtoxkit F, Phytotoxkit test and determinations of dehydrogenases activity, nitrification potential, respiration and microbial biomass; and soil water phase tests: Microtox, Rapidtoxkit, Phytotestkit. The toxic effects caused by PAHs were characterized by toxicity indexes: index of soil resistance and EC20 (EC50) values.
The tested organisms in different way responded to the presence of contaminants; both the negative influence of the 5 PAHs mixture, and stimulation effects were observed. Soil properties were the significant factor influenced the effects caused by the PAH compounds.

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A. Karczewska, K. Lewinska, A. Dradrach, M. Siepak and B. Galka
Arsenic and Antimony in Soils of Historical Metal Ore Mining Sites in the Sudetes - a Solubility and Phytotoxicity
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : chemicals - metals; soils

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Abstract : Arsenic and antimony are the metalloids considerably toxic to biota. Both these elements occur in Polish soils in trace amounts, but locally, in some areas where arsenic and antimony ores, or other polymetallic ores, were mined and processed, high concentrations of one or both of these elements have been confirmed. Their considerable concentrations are present in the material of dumps that remained in former mining sites, and in adjacent soils in Zloty Stok, Srebrna Gora, Radzimowice (The Kaczawskie Mts.), and in the vicinity of the Bardo Pass. Arsenic and antimony are often supposed to behave similarly to each other in terms of their biogeochemistry and toxicological characteristics, however, various authors reported their different environmental behaviour in the soil environment. The factors that govern the ecotoxicity of arsenic have been much better recognized than those related to antimony.
A research presented here focused on the analysis of factors that determine the solubility of both elements in mine dump material and in the soils of former mining sites indicated above, considerably enriched in As and Sb. The effects of soil pH and soil treatment with exogenic organic matter developed from forest litter, as well as of soil amendment with a waste material rich in Fe oxides, on the concentrations of both elements in soil pore water was examined in an incubation experiment. Pore water was acquired using porous MacroRhizon suction samplers. Phytotoxicity of pore water was analysed in a modified assay, based on a Phytotoxkit test, that uses an inhibition of seedlings root and shoot elongation to determine a phytotoxic effects of aqueous media. We used a white mustard Sinapis alba as a testing plant. It was found that soil treatment with Fe oxide-rich material considerably reduced a solubility of As in soils, and a comparable effect was in the case of Sb much poorer. The processes associated with transformation on forest litter, particularly in the conditions of high soil moisture, caused an intensive release of As into soil pore water, and were much less efficient in modification of Sb solubility. The effects of pore water phytotoxicity were strongly correlated with the concentrations of As.

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M. Tankiewicz, P. Pirog and L. Wolska
Acute Toxicity Studies of Selected Surface Active Agents Differing in Chemical Structure and their Mixtures
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - surfactants

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Abstract : Surface active agents, commonly known as surfactants, are the main component of detergents used in households. They are used to reduce or minimize the surface tension occurring at the interface between liquid and the phase of another substance. Thus, the surface nature is changed. Surfactants as additives for various types of products, impart them wetting, emulsifying, foaming, anti-static, softening and biocidal properties. Therefore, they have found such wide application in many areas, including oil, mining, textile, printing, paper, pharmaceutical, cosmetic, metallurgical and rubber industries.
Because of their widespread use in everyday life, they get into the environment in uncontrollably way, mainly through wastewater, causing pollution and consequently the threat to human health. The highest detergent concentrations in surface waters and soils are detected.
Harmfulness of surfactants in the environment result from their physicochemical and toxic properties. These compounds entering into surface water cause it to foam (especially in turbulent sections) and impede diffusion of oxygen from the atmosphere. In addition, they act as emulsifiers of hydrophobic substances and increase the solubility of many dangerous organic pollutants (e.g. PAHs, pesticides) thereby grow the possibility of their penetration into living organisms.
Therefore, it is important to monitor and control emissions of surfactants into the aquatic environment. Knowledge in this area is incomplete. Experimental data is still lacking, especially in the case of mixtures of surfactants that may interact with each other, resulting in multiplication of toxic effects.
The aim of the study was to investigate acute toxicity of selected surfactants differing in chemical structure: anionic - sodium laureth sulfate, cationic - hexadecyltrimethylammonium bromide, and non-ionic - Zonyl FSO^®. Toxicity parameters EC50 and LC50 values were determined using commercially available bioassays: Thamnotoxkit F^TM, Daphtoxkit F^TM and Microtox^®. Subsequently, the toxicity of two-component surfactant mixtures at various volumetric ratios was investigated and the interaction of surfactant components in the mixture was determined based on designated TU toxic units.

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A. Drobniewska, M. Wawryniuk, J. Giebultowicz, A. Maciejczak, P. Stankiewicz and G. Nalecz-Jawecki
Bioaccumulation of antidepressants in Daphnia magna
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - drugs

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Abstract : Antidepressants are frequently prescribed pharmaceuticals. Significant antidepressant usage results in the prevalence of pharmaceuticals in the environment. Antidepressants were detected in surface water at low concentrations (ng L-1) which may not represent isolated threats to non-target organisms when considering the respective contribution. However, since they exert similar effects and are present in the environment as a mixture, it is possible that chronic exposure of aquatic organisms may induce toxicity. Bioaccumulation and chronic toxicity tests are scarce.
In the present study we have analyzed the ability of D. magna to bioaccumulate antidepressants : sertraline, fluoxetine, paroxetine, mianserine. Organisms were incubated at two concentrations of each of the tested pharmaceuticals. The test was conducted in three versions with different periods of drug exposure and drug clearance. The drug concentrations were measured in water in which the organisms were incubated and in D. magna by HPLC MS / MS. The drug concentrations in Daphnia and the bioaccumulative factors for each was calculated.
The results indicate that the greatest potential for bioaccumulation among tested drugs Daphnia magna show for sertraline, and the lowest for fluoxetine.

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J. Ratajczyk J. M. Michalska, M. Bartoszewicz and L. Wolska
Toxicity assessment of post-reaction fluids used in shale fracturing
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Artoxkit M^TM; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis
Topics : effluents; waste waters

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Abstract : Introduction
Ecological hazards arise during the exploitation of all mineral deposits, but in the case of exploitation of shale gas deposits, new not well-known technologies are used. Despite knowing the composition and properties of the fracturing fluid, the composition of the retrograde fluid (flowback water) is unknown and may have negative effects on the environment and humans. The big problem is the necessity of management and disposal of post-reaction fluids. In addition to the components of fracturing fluid, the wastewater may contain toxic substances rinsed from rock layers in the fracturing zone. The shale gas extraction industry is constantly developing and new technologies are being developed to extract this energy resource. Therefore, the analysis of the environmental and human health risks associated with shale fracturing is extremely current and relevant.
Aim of the study
The purpose of this work was to comprehensively assess the toxicity of samples from the water purification plant used for shale fracturing.
Materials and Methods
The samples of technological wastewater from a water purification plant used for shale gas extraction was collected in the northern part of Poland. The toxicity assessment of the specimens was performed on Vibrio fischeri, Artemia salina and Brachionus plicatilis. Chemical analysis included the assessment of physicochemical parameters and estimation of the concentration level of organic compounds using the GC-MS technique.
Results
The acute toxicity of the wastewater was determined in the assay Microtox^®, Rotoxkit M and Artoxkit M. An assessment of the state of activated sludge was made after contact with a sample of process wastewater. The influence of sewage on the morphology of activated sludge and the lytic properties of the Protozoa and Metazoa representatives were determined. GC-MS identification was performed and the level of concentrations of organic compounds in the sample was determined. Physicochemical properties of samples were determined.
Conclusions
Tested samples of process water :
- did not affect the morphology of the activated sludge and did not show lytic properties in relation to the Protozoa and Metazoa representatives present in the settlement;
- cause high toxicity to saltwater microorganisms (Vibrio fischeri, Artemia salina and Brachionus plicatilis);
- consisted of 150 organic compounds with a total content of 85 µg/l.

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S. Szklarek, L. Serwecinska, T. Jurczak and J. Mankiewicz-Boczek
Monitoring of Biological, Physicochemical and Toxicological Parameters in Terms of Ecological Risk Assessment in Mleczna River Catchment – LIFERADOMKLIMA-PL project
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Phytotoxkit; Protoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : surface waters; toxicity classification

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Abstract : Adaptation to climate change in urban areas requires the creation of space and solutions for water retention in order to prevent the effects of floods and droughts. It is also important to provide adequate quality water retention resources, for assure many benefits to society and biodiversity supporting. The implementation of the LIFERADOMKLIMA-PL project addresses these challenges by creating an urban space in Radom city with increased resistance to climate change by demonstration blue-green infrastructure.
For this purpose, in the first year of the project (2016), biological, physicochemical and ecotoxicological parameters assessment of the Mleczna river catchment in the Radom city were carried out. The biological monitoring of catchment included phytobenthic communities, macrophytes, macrozoobenthos, and in the recreation Borki reservoir the bacteria indicator of fecal pollution and toxic cyanobacteria were investigated. Physicochemical monitoring included analysis of nitrogen and phosphorus compounds, chlorides, iron, suspended matter, COD, temperature, pH, dissolved oxygen and conductivity. Ecotoxicity monitoring (not required by law regulations) was carried out in two dates: April and August 2016 in eight sites. Four of them were located on the Radom outskirts (catchment dominated by agricultural areas): R1 - Mazovian River, R4 - Kosowka River, R3 - Mleczna River, above Borki reservoir (connects the water from R1 and R4) and R5 - Cerekwianka River. The remaining points were located in the urbanized area: R7 - Mleczna River below the Borki reservoir, R10 - Mleczna River (connects the water from R7 and R5), R13 – Potok Polnocny and R14 - Mleczna River (connects the water from R10 and R13). Ecotoxicology was based on biotest set representing organisms from all trophic levels : Selenastrum capricornutum (Algaltoxkit); Sorghum saccharatum, Lepidium sativum and Sinapis alba (Phytotoxkit); Daphnia magna (Daphtoxkit) and Tetrahymena thermophila (Protoxkit).
Ecotoxicological studies have shown that three sites are characterized by slight acute (class II) and acute (class III) hazard in both sampling dates, April and August - R1, R3 and R7. The poor quality (noted toxicity) of water in the upper part of the analyzed Mleczna catchment has not been confirmed by other biological and physicochemical analyzes, which show that the quality of water was increasing downstream, along with the increase in the share of the urbanized area.
On the other studied sites, slight acute and acute hazard (class II and III) was recorded only in April, during spring period, characterized by flush off pollutants accumulated in the catchment area during the winter. This was not observed for biological analyzes, which noted lower water quality in autumn. Physicochemical monitoring showed lower average values of conductivity (444 µS/cm) and chloride ions (29 mg/L) in August in comparison to the rest of year (642 µS/cm and 44 mg/L, respectively).
The sensitivity of the organisms was varied. The most sensitive organism was algae Selenastrum capricornutum (Algaltoxkit), then toxic effect was reported in 64% of cases. This may indicate the presence of toxic pollutants for this species or group of organisms, which are not harmful for the other organism tested. On the other hand, in the Phytotoxkit test, water samples stimulated root growth in 60% of cases, which might be a result of high nutrient concentrations in the catchment area, especially high phosphorus levels at many sampling points.
In the monitoring of the recreational reservoir of Borki (conducted between April and November) toxic cyanobacteria were detected, including the genus Microcystis capable to produce cyanitoxins as hepatotoxins - microcystins. The high level of toxigenic cyanobacteria occurred from April to September, which, in accordance with the law regulations, should be monitored at recreational places on the presence and threat of them (Journal of Laws 2016, item 1602). Microbiological studies have shown that since June the number of bacteria, E. coli and Enterococcus increased, to the maximum value in July and August. The highest number of all tested bacterial groups were detected in the Mleczna River below the Borki reservoir, which may indicate contaminated domestic sewage which are introduced into the river below the reservoir with the Cerekwianka River waters and with sewage from rain-drainage sewer system. In addition, at the stations located in the upper and lower part of the Borki reservoir, there was a high number of Enterococcus in the summer, especially in July.

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W. Ratajczyk, N. Chrzczonowska, E. Olkowska, M. Tankiewicz, A. Skwierawska and L. Wolska
The Toxicity of Pharmaceuticals Identified in Treated Wastewater Toward Ostracods Heterocypris incongruens
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - drugs; sediments

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Abstract : The technologies used by wastewater treatment plants do not remove all potentially toxic substances that can eventually get into the environment. The presence of pharmaceutical and personal care products (PPCPs) in treated wastewater is widely reported and discussed in the scientific literature [1-10]. The pharmaceuticals in the treated effluents after discharge to the aquatic environment are mostly present in the water phase, what is related to their hydrophilic properties. Hence, there is little data on the content of pharmaceuticals in sediments. Meanwhile, contaminants are deposited and accumulated in sediments. Many years of ongoing accumulation of pharmaceuticals in bottom sediments can lead to significant pollution by this group of compounds.
The main purpose of the study was to estimate the toxicity of selected pharmaceuticals which presence in treated wastewater was described in the literature. For this purpose, Vibrio fischeri bacteria, which mechanism of toxins absorption is carried out in the water phase were used. Heterocypris incongruens crustaceans, which are feeding in the sediments are exposed to the adsorbed compounds.
Based on the literature review, 8 pharmaceuticals identified in treated effluents were selected: metronidazole, lamotrigine, ciprofloxacin, simvastatin, tramadol, ranitidine, acetylsalicylic acid and caffeine. The toxicity of selected pharmaceuticals toward Vibrio fischeri and Heterocypris incongruens were discussed. The determined toxicity of the selected pharmaceuticals toward Heterocypris incongruens decreased as follows: simvastatin (EC50 = 10 mg/L) >> lamotrigine > tramadol (EC50 = 100 mg/L) > caffeine ≈ acetylsalicylic acid ≈ ciprofloxacin > ranitidine > metronidazole. The tested substances (up to 100 mg/l) showed no toxic effect toward Vibro fischeri. A review of literature on the toxicity of pharmaceuticals shows that simvastatin is the most toxic also toward other bioindicators.

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J. Rogowska, M. Tankiewicz, A. Zielinska and L. Wolska
Assessment of Ecotoxicological Parameters of Bioavailable Bottom Sediments
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments

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Abstract : IntroductionIn the aquatic environment metals can be found in various forms (e.g. exchangeable) associated with carbonate or with Fe/Mn. These forms may have different mobility, bioavailability and toxicity. The bioavailability of the metals occurred in the bottom sediments depends on the content of organic carbon, pH and particle size, or redox potential. Presently, bioavailability is defined as two fractions obtained by sequential analysis: interchangeable and carbonate.
Chemical analysis of metals in sediments allows to obtain information on their origin, routes of spread or concentration levels. However, such research does not allow to identify their bioavailability and effects on living organisms. For identification of possible influence on the organisms living in the ecosystem ecotoxicological assessment should be carried.
The aim of the work
The aim of the study was to investigate that the metals after extraction from sediment samples with HCl aqueous solution (pH = 2) are transformed into a mobile form and thus bioavailable to living organisms.
Materials and methods
The research includes chemical analysis and estimation of toxicity of 3 types of samples: sediment from Turawskie Lake, sediment after extraction with HCl aqueous solution and extracts after extraction. The toxicity assessment was carried out with use of the bacteria Vibrio fischeri and crustaceans Heterocypris incongruens. Chemical analysis includes the determination of metals such as Cr, Zn, Pb, Cu, Ni, Cd, Hg using inductively coupled plasma mass spectrometry (ICP-MS) technique.
Results
The obtained results show that :
- Half of the samples analyzed were characterized by high levels of toxicity (100%) against Heterocypris incongruens. The toxicity of sediments and extract samples after extraction did not change.
- Samples were characterized by different levels of toxicity to Vibrio fischeri bacteria. There is no visible trend in toxicity changes after the extraction process.
- The correlation between Zn, Pb, Cu, Cd content in the sediments before and after extraction was indicated.
- The concentration of metals in the sediments before and after extraction is similar and it indicates that the metals were only slightly extracted.

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W. Podlesinska and H. Dabrowska
Application of Bioassays with Corophium multisetosum (Amphipoda) and Heterocypris incongruens (Ostracoda) in the Toxicity Studies of the Dead Vistula River Bottom Sediments
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments

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Abstract : Evaluation of bottom sediments in terms of their biotoxicity is important for assessing the quality of aquatic ecosystems. The phosphogypsum heap in Wislinka was established at the turn of the sixties and seventies of XXth century and was closed in 2009. Over that time, the chemicals accumulated in the heap could penetrate the Dead Vistula ecosystem, located 50 meters away from the landfill. Currently, the site is secured and meets environmental standards. On the other hand the phosphates and radionuclides contents in the Dead Vistula indicate that harmful substances from the landfill can adversely affect organisms inhabiting the ecosystem.
The study aimed to determine the quality of the Dead Vistula River and Bold Vistula bottom sediments, considering the gradient of distances from the phosphogypsum heap. In July 2016, sediment samples were collected from 5 locations in the central part of the river, in a distance gradient from the phosphogypsum heap. Sediment chronic toxicity bioassays with Corophium multisetosum (Amphipoda) and Heterocypris incongruens (Ostracoda) were conducted. Corophium spp. are amphipods commonly used in biotoxicity studies. They live in sediments, they feed on organic matter, bacteria and diatoms, and are therefore directly exposed to the contaminants contained in sediment and interstitial water. C. multisetosum is an important component of the Bay of Gdansk coastal areas zoobenthos. H. incongruens is a freshwater benthic crustacean of almost global distribution. It reproduces by parthenogenesis, producing cysts. It is used as a bioindication species in the standardized Ostracodtoxkit F bioassay.
C. multisetosum individuals and control sediment were collected from the Bay of Gdansk, from a low pollution area and acclimated to laboratory conditions. Samples of Dead Vistula and Vistula Bold sediments were collected using Van Veen Grab Sampler. Sediment bioassays were carried out in 1L beakers, which contained about 200 g of sediment (2 cm height of the beaker) and were overlaid with saltwater of 7‰ salinity. After 24 hours 15 test individuals of about 2 mm length were placed in each beaker. The tests were performed in 5 replicates for each sample of sediment. After 28 days survival and growth of organisms were determined. In bioassay with Heterocystis incongruens (Ostracodtoxkit F test), sediment samples with enriched water and nutrients were placed in the test wells. Immediately after hatching of the organisms from cysts, 10 individuals of H. incongruens were transferred to the wells. For each sample of sediment, 6 replicates were made. After 6 days, the organisms’ survival and growth were determined and compared to the control wells. The reference test with toxic copper sulphate was performed. The tested sediments were analyzed in terms of granulometric composition and organic matter content in whole sediment and fine fraction (<63 µm). Basic physico-chemical analysis of bottom water (salinity, oxygen content, temperature, pH, phosphates) taken from the same sites as sediments, was performed.
Granulometric analysis showed that the sediments from the C. multisetosum control site were characterized by a low content of grains <63 µm (2.26%), organic matter in whole sediments (0.73%) and fine fraction <63 µm (2.26%) compared to the sediments studied in the Dead Vistula and the Bold Vistula, which were muddy and contained more organic matter. Bottom water were characterized by relatively low salinity, which gradually decreased with the increasing distance from the Bold Vistula mouth and was in the range of 4.52-6.87‰. The content of phosphates in the bottom water was the smallest at the mouth of the Bold Vistula, and the highest in the Dead Vistula at the site near the Mlynowka Canal.
Obtained results showed that in the Ostracodtoxkit F bioassay, the average survival of H. incongruens exposed to the tested sediments exceeded 85%, whereas in C. multisetosum bioassays the average survival of organisms was over 75%. Growth of individuals was higher in the sediment than in the control sediments of both species. These results indicate that the sediments of the Dead Vistula and the Bold Vistula do not have a toxic effect on the survival and growth of the tested species. What is more, the organic matter content of the investigated sediments could have a stimulating effect on the growth of organisms.

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M. Wolf and A. Siedlecka
Ecotoxicity of Wastewater at Various Treatment Stages
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : waste waters

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Abstract : The dynamic development of the human economy and activities may provide the emissions of toxic or potentially toxic substances with unknown properties. According to that new methods of monitoring of the environmental quality are needed, besides the traditional ones.
There is no doubt, that the advantage of the usage of biotests is a possibility to measure the reaction of tested organisms to all sample components, without the necessity of analysing every single one. That is almost impossible in routine tests including environmental samples. A toxicity analysis supplements the chemical analyses, which may determine only the concentration of tested analites. The ecotoxicology monitoring in a different steps of treatment process may play a significant aspect in the protection of not only a receiver, but also microorganisms in bioreactors in wastewater treatment plants.
The advantage of direct ecotoxicity wastewater investigations may allow to take into account the synergistic and antagonistic effects. The influence of toxic compounds on biological units may lead to – in extreme cases – total extinction of activated sludge microorganisms and the toxicology may be an important warning factor, leading to the ptimizing of the technological processes.
The preliminary assessments of the wastewater toxicity may contribute to the consideration of the implementation of toxicology monitoring at different treatment stages. It may improve the functioning of wastewater treatment plants – especially biological units, which are the most vulnerable.
With the purpose of testing raw, mechanically and biologically treated wastewater, the testing organisms of each trophic level were used, included :
- bioluminescent test of acute toxicity Microtox, using bacteria
- commercial biotests of acute / chronic toxicity, using algae and crustaceans biotest using organisms as Daphnia magna, Lemna minor.

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A. Baran, M. Tarnawski and T. Koniarz
Sediment Toxicity Distribution in Vertical Profiles
VII National Bioindication Conference - “Practical use of bioindication systems for evaluation of quality and toxicity of environment and chemical substances” 2017
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments

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Abstract : The aim of the study was to determine the toxicity of bottom sediments of the Roznow reservoir in vertical profiles. The study also investigated the vertical distribution of organic carbon. Rozo reservoir is located in the Malopolska Voivodeship on the Dunajec River. The characteristic feature of the reservoir is its degree of silting, which is about 30%. Bottom sediments were sampled from the most silting part of reservoir - backwater. Sediment was collected using Beeker and Instorf type samplers from 0.5 m to 5.0 m depth. Phytotoxkit (Sinapis albas used to evaluate the bottom sediment toxicity. C-organic content in sediement was determined using an Elementar Vario MAX cube CNS analyzer. The highest sediment toxicity for Sinapis alba was found in sections I, III and 4.0-4.5 m and 4.5-5.0 m depth. The highest C-organic content was found in the sediments collected in section IV and V and in the levels 2.0-2.5 m and 2.5-3.0 m. Significant negative correlation was found between reaction of Sinapis alba and organic carbon content.

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A. Arizzi Novelli, M. Melchiorri, L. Mastrangioli, N. Di Deo, G. Sergiacomo, E. Scamosci, G. Surricchio and C.Spatola Mayo
Assessment of the Effects of Disinfection with Peracetic Acid on Urban Discharge Waters
7° Giornato di studi “l’ecotossicologia come gestione degli ambienti acquatici e terrestri”. Livorno, 22-24 novembre 2016
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : waste waters; chemicals - inorganic compounds

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Abstract : Among the different forms of disinfection in the depuration sector, the use of sodium hypochlorite has been shown to have contraindications due to the formation of chloramines, chlorates, carcinogenic and genotoxic compounds. For this reason, its use has been forbidden, privileging other disinfection methods, including the peracetic acid (PAA), a valid alternative for its high disinfectant power as well as for its low environmental impact. Since January 2015 the Abruzzo Region in the Water Protection Plan has established the replacement of chlorination disinfection systems with alternative systems such as ozone, PAA, UV rays or other equally effective chlorine-free treatments. To assess the effects of the use of PAA, a toxicological test battery (Vibrio fischeri, Daphnia magna; Pseudokirchneriella subcapitata) has been used for monitoring the discharge waters of urban agglomerations with more than 2000 inhabitants equivalent and sampled in 2015/2016 in the provinces of Pescara and Chieti. The results showed a discriminatory response between the organisms used, with greater ability by the algal test to detect the overcoming of the limits on most of the analyzed samples. It was also hypothesized that a residual peroxide concentration of 0.5 mg / L would minimize the risk of a toxic effect induced by the use of the disinfectant.

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A. Arizzi Novelli, M. Melchiorri, E. Scamosci, T. Marcelli, N. Calace and F. Onorati
Application of a Toxicological Test Battery for the Assessment of Fluvial Sediments of Sir Saline Alento
7° Giornato di studi “l’ecotossicologia come gestione degli ambienti acquatici e terrestri”. Livorno, 22-24 novembre 2016
Toxkits : Daphtoxkit F^TM magna; Ostracodtoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens
Topics : sediments; sediment pore waters

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Abstract : According to an agreement between the Region of Abruzzo, ISPRA and ARTA, the Regional Interest Site (SIR) Saline Alento was the subject of a supplemental characterization, planned on the basis of the results obtained from the previous survey of 2009. For this study, sediments of Saline and Alento Rivers were subjected to chemical analyses and ecotoxicological tests. To assess the ecotoxicological hazard, a battery of biological tests has been devised considering the representativeness and ecological value of the organisms, the type of environment, and the matrix to be investigated (such as sediment). The battery foresees the use of a chronic test with Heterocypris incongruens on the whole sediment, and acute tests with Daphnia magna and Pseudokirchneriella subcapitata on the elutriate. The obtained results showed a different sensitivity of the various organisms, confirming the need for a multi-species approach, and a relative absence of ecotoxicological danger.

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R. Croce, F. Cina, F. Albanese, C. Cappelli, M. Vian, A. Lombardo and D. Baderna
Old Molecules, New Challenges : Acute and Short-Term Aquatic Toxicity of Textile Dyes
7° Giornato di studi “l’ecotossicologia come gestione degli ambienti acquatici e terrestri”. Livorno, 22-24 novembre 2016
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Daphnia pulex; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - organic compounds

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Abstract : Dyes are widely used in various sectors such as textile dyeing, paper printing, photography, pharmaceutical, food and cosmetic industries. In addition, it is estimated that more than 10% of total dyes produced is lost to waste streams. Most of these compounds pass conventional wastewater treatment processes and are released into the environment where they persist for a long time because of their high stability to light or temperature and their resistance to water or detergent degradation. These features, coupled with their toxicity, make dyes a possible source of ecological concern, particularly for freshwater aquatic ecosystems. Dyes are often poorly characterized and toxicological/ecotoxicological data are available only for a few. Therefore, new data may be very useful for their risk assessment. In the present study, we investigated the aquatic toxicity of 42 commercial dyes using the application of in silico tools and ecological bioassays. The in silico approach was used to assess the similarities among the dyes. Acute and short-term ecotoxicological assays with daphnids and algae were applied to evaluate the potential impact of these products. The bioassays were able to identify structures with potential ecotoxicity but, in our experimental conditions, algae were more sensitive to dye toxicity, particularly when the effects on biomass were considered.

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G. Genethliou, S. Dailianis and M. Kornaros
Biodegradation of Olive Mill Wastewater Phenolic Compounds in Thermophilic anaerobic digestion and assessment of their toxicity in digested effluent
Book of Abstracts of the Sixth International Conference on Environmental Management, Engineering, Planning & Economics - Thessaloniki, Greece - June 25-30 2017
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : waste waters; chemicals - organic compounds; effluents

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Abstract : The extent of phenolic compounds’ biodegradation was assessed utilizing un-treated Olive Mill Wastewater (OMWW) fed to a high-rate thermophilic (55 °C) anaerobic Upflow Packed Bed Bioreactor (UPBR) and digested effluents collected in different Hydraulic Retention Times (HRTs) under steady-state operating conditions. In parallel the toxicity of each sample was evaluated, using the microbiotest Thamnotoxkit F^TM.
The results indicate complete biodegradation of 5 phenolic compounds and notable removal of hydroxytyrosol and tyrosol. Reaching up to 93.70% and 96.47% respectively. On the contrary, p-coumaric acid, 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid were recognized as the most recalcitrant and persistent compounds in the anaerobic effluents, being capable of modulating the toxic potential of digested effluents.

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I. Blinova, H. Vija, A. Lukjanova, M. Muna, G. Syvertsen-Wiig and A. Kahru
Assessment of the Hazard of Nine (Doped) Lanthanides-Based Ceramic Oxides to Four Aquatic Species
Science of the Total Environment 2018 612 1171-1176
Toxkits : Ostracodtoxkit F^TM; Spirodela duckweed Toxkit; Thamnotoxkit F^TM
Test Species : Heterocypris incongruens; Spirodela polyrhiza; Thamnocephalus platyurus
Topics : chemicals - inorganic compounds; chemicals - metals

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Abstract : The risk of environmental pollution with rare earth oxides rises in line with increasing application of these compounds in different sectors. However, data on potential environmental hazard of lanthanides is scarce and concerns mostly Ce and Gd. In this work, the aquatic toxicity of eight doped lanthanide-based ceramic oxides (Ce0.9Gd0.1O2, LaFeO3, Gd0.97CoO3, LaCoO3, (La0.5Sr0.5)0.99MnO3, Ce0.8Pr0.2O2, (La0.6Sr0.4)0.95CoO3, LaNiO4) and one non-doped oxide (CeO2) with primary size from 23 to 590 nm were evaluated in four short-term laboratory assays with freshwater crustaceans and duckweeds. Results showed no acute toxicity (EC50 > 100 mg/L) or very low acute toxicity for most studied oxides. Observed toxicity was probably due to bioavailable fraction of dopant metals (Ni and Co) but in the case of aquatic plants, decrease of nutrient availability (complexing of phosphorus by lanthanides)was also presumed. Studied oxides/metals accumulated in the aquatic plant tissue and in the gut of crustaceans and thus may be further transferred via the aquatic food chain. Accumulation of metals in the duckweed Lemna minor may be recommended as a cost-effective screening bioassay for assessment of potential hazard of poorly soluble oxides to aquatic ecosystems.

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St. Gheorghe, C. Stoica, I. Paun and S. Cristofor
Ecotoxicological Tests Used as Warning System ror Danube Delta Quality Assessment
ResearchGate 216
Toxkits : Algaltoxkit F^TM; Ostracodtoxkit F^TM; Phytotoxkit; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; surface waters; toxicity classification

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Abstract : The Danube Delta water and sediment toxicity effects have been assessed using a biotests battery based on representative species of primary producers (Selenastrum capricornutum, Sorghum saccharatum, Lepidium sativum, Sinapis alba) and consumers (rotifers – Brachionus calyciflorus, crustaceans – Daphnia magna, Heterocypris incongruens). The ecotoxicological tests results revealed no acute to minimum toxic effect classified as Class I and II of hazard for water samples and Class II and III, in case of sediment samples. In order to control water and sediment-quality, these results indicated the need to integrate within a water and sediment-quality triad different tests such as toxicity tests, chemical analyses as well as field data from water phytoplankton, soft-sediment meio-and macro-invertebrates. Overall, the ecotoxicological results were modulated by organisms sensitivity, sampling locations, season and climate change issues.

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L. Manfra, S. Canepa, V. Piazza and M. Faimali
Lethal and Sublethal Endpoints Observed for Artemia Exposed to Two Reference Toxicants and an Ecotoxicological Concern Organic Compound
Ecotoxicology and Environmental Safety 2016 123 60-64
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : test sensitivity; test selection; chemicals - inorganic compounds; chemicals - metals; chemicals - organic compounds

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Abstract : Swimming speed alteration and mortality assays with the marine crustacean Artemia franciscana were carried out. EC50 and LC50 values after 24-48h exposures were calculated for two reference toxicants, copper sulphate pentahydrate (CuSO4.5H2O) and Sodium Dodecyl Sulphate (SDS) and an ecotoxicological concern organic compound, Diethylene Glycol (DEG).
Different end-points have been evaluated, in order to point out their sensitivity levels. The swimming speed alteration (SSA) was compared to mortality values and also to the hatching rate inhibition (literature data). SSA resulted to be more sensitive than the mortality and with a sensitivity comparable to (or even higher than) the hatching rate endpoint.

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N. Estevez-Calvar, L. Canesi, M. Montagna, M. Faimali, V. Piazza and F. Garaventa
Adverse Effects of the SSRI Antidepressant Sertraline on Early Life Stages of Marine Invertebrates
Marine Environmental Research 2017 128 88-97
Toxkits : Rotoxkit M^TM
Test Species : Brachionus plicatilis
Topics : chemicals - drugs; test sensitivity

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Abstract : Widespread contamination of coastal environments by emerging compounds includes low concentrations of pharmaceuticals. These pollutants are not currently incorporated in monitoring programs despite their effects on non-target organisms are very little documented. Among the selective serotonin reuptake inhibitor (SSRI) antidepressants, sertraline (SRT) is one of the most prescribed globally. In this work, earlier life stages of Amphibalanus Amphitrite, Brachionus plicatilis and Mytilus galloprovincialis were exposed to environmental concentrations of SRT in order to study both sub-lethal and lethal responses in 24/48 h-tests. Low concentrations of SRT altered significantly swimming behavior in A. amphitrite and B. plicatilis giving 48 h-EC50 (µg/L) of 113.88 and 282.23, respectively whereas higher values were observed for mortality and immobilization. C50 embryotoxicity with M. galloprovincialis was 206.80 µg/L. This work add new data about SRT ecotoxicity on marine invertebrates and confirms the applicability of behavioral endpoints to evaluate the environmental impact of antidepressants in marine organisms.

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St. Gheorghe, I. Lucaciu, E. Stanescu and C. Stoica
Romanian Aquatic Toxicity Testing Strategy under Reach
Journal of Environmental Protection and Ecology 2013 14, 2 601-611
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : toxicity classification; test selection

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Abstract : Registration, Evaluation and Authorisation of Chemicals (REACH) is the most important European legislative regulation concerning chemicals and became applicable in Romania since June 2008. In the period 2008–2011, chemicals manufactures/importers and also eco-toxicological laboratories have made great efforts to comply with these requirements. The paper presents our contribution, as laboratory testing, to evaluate the aquatic toxicity of hazard substances and to provide information about environmental risks. Concerning REACH challenges, our laboratory has developed a toxicity testing methodology based on tests involving animals, but at the same time to provide the necessary data for the eco-toxicological characterization of chemicals. As other countries, Romania has chemical pollution problems and in this context we performed a various aquatic toxicity studies for different substances, using an integrated eco-toxicological strategy, in an interdisciplinary collaboration. A reduced number of acute toxicity tests using fish were performed in our laboratory, but to obtain data it was necessary to evaluate the effects of the chemicals on the aquatic organisms, the LC50 bioassays were replaced with algae and crustacean EC50 tests. The fish bioassays were applied for fewer concentrations, the smallest between the EC50 obtained for algae and crustacean and only if the fish were more sensitive than algae and crustacean, acute fish tests were performed for lower concentrations. Our results led to predict the maximum allowable concentrations (MATC) in surface water and non-observed effect concentration (NOEC) and also, to estimate the environmental risk coefficients. Our vision and assessment strategy are in continuous development in order to respond to future demands concerning toxicity on long-term, toxicity pathways, bioaccumulation, recalcitrant metabolites toxicity and inter-species effects extrapolations.

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Y. Foucault, M.J. Durand, K. Tack, E. Schreck, F. Geret, T. Leveque, P. Pradere, S. Goix and C. Dumat
Use of Ecotoxicity Test and Eco-Scores to Improve the Management of Polluted Soils : Case of a Secondary Lead Smelter plant
Journal of Hazardous Materials 2013 246-247 291-299
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : toxicity classification

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Abstract : With the rise of sustainable development, rehabilitation of brownfield sites located in urban areas has become a major concern. Management of contaminated soils in relation with environmental and sanitary risk concerns is therefore a strong aim needing the development of both useful tools for risk assessment and sustainable remediation techniques. For soils polluted by metals and metalloids (MTE), the criteria for landfilling are currently not based on ecotoxicological tests but on total MTE concentrations and leaching tests. In this study, the ecotoxicity of leachates from MTE polluted soils sampled from an industrial site recycling lead-acid batteries were evaluated by using both modified Escherichia coli strains with luminescence modulated by metals and normalized Daphnia magna and Alivibrio fischeri bioassays. The results were clearly related to the type of microorganisms (crustacean, different strains of bacteria) whose sensitivity varied. Ecotoxicity was also different according to sample location on the site, total concentrations and physico-chemical properties of each soil. For comparison, standard leaching tests were also performed. Potentially phytoavailable fraction of MTE in soils and physico-chemical measures were finally performed in order to highlight the mechanisms. The results demonstrated that the use of a panel of microorganisms is suitable for hazard classification of polluted soils. In addition, calculated eco-scores permit to rank the polluted soils according to their potentially of dangerousness. Influence of soil and MTE characteristics on MTE mobility and ecotoxicity was also highlighted.

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K. Van Hoecke, J.T.K. Quik, J. Mankiewicz-Boczek, K.A.C. De Schamphelaere, A. Elsaesser, P. Van der Meeren, C. Barnes, G. McKerr, C. V. Howard, D. Van De Meent, K. Rydzynski, K.A. Dawson, A. Salvati, A. Lesniak, I. Lynch, G. Silversmit, B. De Samber, L. Vincze and C.R. Janssen
Fate and Effects of CeO2 Nanoparticles in Aquatic Ecotoxicity Tests
Environmental Science and Technology 2009 43, 12 4537-4546
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals - nanoparticles

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Abstract : Cerium dioxide nanoparticles (CeO2 NPs) are increasingly being used as a catalyst in the automotive industry. Consequently, increasing amounts of CeO2 NPs are expected to enter the environment where their fate in and potential impacts are unknown. In this paper we describe the fate and effects of CeO2 NPs of three different sizes (14, 20, and 29 nm) in aquatic toxicity tests. In each standard test medium (pH 7.4) the CeO2 nanoparticles aggregated (mean aggregate size approximately 400 nm). Four test organisms covering three different trophic levels were investigated, i.e., the unicellular green alga Pseudokirchneriella subcapitata two crustaceans: Daphnia magna and Thamnocephalus platyurus, and embryos of Danio rerio. No acute toxicity was observed for the two crustaceans and D. rerio embryos, up to test concentrations of 1000, 5000, and 200 mg/L, respectively. In contrast, significant chronic toxicity to P. subcapitata with 10% effect concentrations (EC10s) between 2.6 and 5.4 mg/L was observed. Food shortage resulted in chronic toxicity to D. magna, for wich EC10s of ≥8.8 and ≤20.0 mg/L were established. Chronic toxicity was found to increase with decreasing nominal particle diameter and the difference in toxicity could be explained by the difference in surface area. Using the data set, PNECaquatics ≥ 0.052 and ≤ 0.108 mg/L were derived. Further experiments were performed to explain the observed toxicity to the most sensitive organism, i.e., P. subcapitata. Toxicity could not be related to a direct effect of dissolved Ce or CeO2 NP uptake or adsorption, nor to an indirect effect of nutrient depletion (by sorption to NPs) or physical light restriction (through shading by the NPs). However, observed clustering of NPs around algal cells may locally cause a direct or indirect effect.

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C. Gambardella, T. Mesaric, T. Milivojevic, K. Sepcic, L. Gallus, S. Carbone, S. Ferrando and M Faimali
Effects of Selected Metal Oxide Nanoparticles on Artemia Salina Larvae : Evaluation of Mortality and Behavioural and Biochemical Responses
Environmental Monitoring and Assessment 2014 186-7 4249-4259
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : chemicals - metals; chemicals - nanoparticles

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Abstract : The aim was to investigate the toxicity of selected metal oxide nanoparticles (MO-NPs) on the brine shrimp Artemia salina, by evaluating mortality and behavioural and biochemical responses. Larvae were exposed to tin(IV) oxide (stannic oxide (SnO2)), cerium(IV) oxide (CeO2) and iron(II, III) oxide (Fe3O4) NPs for 48 h in seawater, with MO-NP suspensions from 0.01 to 1.0 mg/mL. Mortality and behavioural responses (swimming speed alteration) and enzymatic activities of cholinesterase, glutathione-S-transferase and catalase were evaluated. Although the MO-NPs did not induce any mortality of the larvae, they caused changes in behavioural and biochemical responses. Swimming speed significantly decreased in larvae exposed to CeO2 NPs.
Cholinesterase and glutathione-S-transferase activities were significantly inhibited in larvae exposed to SnO2 NPs, whereas cholinesterase activity significantly increased after CeO2 NP and Fe3O4 NP exposure. Catalase activity significantly increased in larvae exposed to Fe3O4 NPs. In conclusion, swimming alteration and cholinesterase activity represent valid endpoints for MO-NP exposure, while glutathione-S-transferase and catalase activities appear to be NP-specific.

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I. Kurvet, K. Juganson, H. Vija, M. Sihtmäe, I. Blinova, G. Syvertsen-Wiig and A. Kahru
Toxicity of Nine (Doped) Rare Earth Metal Oxides and Respective Individual Metals to Aquatic Microorganisms Vibrio fischeri and Tetrahymena thermophila
Materials 2017 10-7
Toxkits : Protoxkit F^TM
Test Species : Tetrahymena thermophila
Topics : chemicals - metals

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Abstract : Despite the increasing use of rare earth elements (REEs) and oxides (REOs) in various technologies, the information on their ecotoxicological hazard is scarce. Here, the effects of La³⁺, Ce³⁺, Pr³⁺, Nd³⁺, Gd³⁺, CeO2, and eight doped REOs to marine bacteria Vibrio fischeri and freshwater protozoa Tetrahymena thermophila were studied in parallel with REO dopant metals (Co²⁺, Fe³⁺, Mn²⁺, Ni²⁺, Sr²⁺). The highest concentrations of REOs tested were 100 mg/L with protozoa in deionized water and 500 mg/L with bacteria in 2% NaCl. Although (i) most REOs produced reactive oxygen species; (ii) all studied soluble REEs were toxic to bacteria (half-effective concentration, EC50 3.5-21 mg metal/L; minimal bactericidal concentration, MBC 6.3-63 mg/L) and to protozoa (EC50 28-42 mg/L); and (iii) also some dopant metals (Ni²⁺, Fe³⁺) proved toxic (EC50 </= 3 mg/L), no toxicity of REOs to protozoa (EC50 > 100 mg/L) and bacteria (EC50 > 500 mg/L; MBC > 500 mg/L) was observed except for La2NiO4 (MBC 25 mg/L). According to kinetics of V. fischeri bioluminescence, the toxicity of REEs was triggered by disturbing cellular membrane integrity. Fortunately, as REEs and REOs are currently produced in moderate amounts and form in the environment insoluble salts and/or oxides, they apparently present no harm to aquatic bacteria and protozoa.

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A. Sieroslawska
Evaluation of the Sensitivity of Organisms Used in Commercially Available Toxkits to Selected Cyanotoxins
Polish Journal of Environmental Studies 2013 6 1817-1823
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Protoxkit F^TM; Rotoxkit F^TM; Thamnotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : cyanobacterial blooms

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Abstract : Recently, assays based on living organisms have become a frequently used tool in toxicity assessment of environmental samples containing cyanotoxins. The aim of this study was to determine the sensitivity of different organisms from commercially available toxkits to three commonly detected cyanotoxins: microcystin-LR, cylindrospermopsin, and anatoxin-a. Cyanotoxins were used in purified forms, the highest concentration tested was 4 µg/ml. The most pronounced toxic effects were observed in the presence of cylindrospermopsin, with the lowest LC50 (24h LC50 = 0.27 µg/ml) estimated for the crustacean Thamnocephalus platyurus. On the contrary, anatoxin-a was found to be toxic only to a small degree, with the EC50 only calculated for Daphnia magna (24h EC50 = 2.09 µg/ml; 48h EC50 = 1.70 µg/ml). In that case the test endpoint was manifested as an inability to swim freely rather than lethality, so it was impossible to estimate LC50 values. In turn, the most pronounced toxic effects of microcystin-LR were observed in D. magna after 48-h exposure with EC50 estimated at 0.97 µg/ml. The lowest sensitivity to the cyanotoxins at studied concentrations was demonstrated in acute tests for the rotifer Brachionus calyciflorus, and to some extent the algae Selenastrum capricornutum. It can be concluded, therefore, that organisms used as bioindicators vary considerably in their sensitivity to cyanotoxins and the obtained EC50/LC50 values are much higher than typical toxin concentrations in the environment.

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N.Y. Stepanova, O.V. Nikitin, V.Z. Latypova, I.B. Vybornova, G.S. Galieva and R.V. Okunev
Recovery of ostracod with known ages in differently textured sediments and comparison of toxicity of heavily contaminated sediments with ostracod Heterocypris incongruens and amphipod Hyalella azteca
IOP Conference Series : Earth and Environmental Science 2017 107
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments

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Abstract : The recovery of 1-, 4-, 6,-, and 8-d-old ostracods (Heterocypris incongruens) from sediments with different texture has been evaluated. The recovery of ostracods at all ages has been in agreement with the acceptability criterion of 80% of survival for sediment tests. The recovery of ostracods has turned out to be equal to or more than 80% for sand and silt sediments, respectively. The comparison of survival rates between ostracods and amphipods has shown good convergence in the tests of heavily contaminated sediments (R²=0.75, p<0.05).
The sediment quality criteria (TEC) have been exceeded mostly for total petroleum hydrocarbons (100% samples), Cr (100%), Ni (87%), Cu (87%), Pb (47%), and Cd (53%). The content of acid volatile sulfides (AVS) has been significantly higher than that of simultaneously extracted metals (SEM). The obtained results have indicated that, metals (Cu, Zn, Cd, Ni, and Pb) are non-bioavailable. Only one sample has exceeded TEC for PAHs (dibenz[a,h]anthracene). It was observed that, no significant correlation between the effect of toxicity and the chemical content.

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I. Josko and P. Oleszczuk
Phytotoxicity of Nanoparticles - Problems with Bioassay choosing and Sample Preparation
Environmental Science and Pollution Research 2014 21 10215-10224
Toxkits : Phytotoxkit
Test Species : Lepidium sativum
Topics : chemicals-nanoparticles; soils

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Abstract : For a full estimation of the risk related with the presence of engineered nanoparticles (ENPs) in the environment, the use of the current ecotoxicological methods may prove insufficient. In the study presented herein, various methods of assessment of ecotoxicity were applied to compare the phytotoxicity of three ENPs: nano-ZnO, nano-TiO2 and nano-Ni. The toxicity was assayed both for aqueous solutions of the ENPs (the germination/elongation test and Phytotestkit) and for ENPs added to soil (Phytotoxkit and modified Phytotoxkit). Lepidium sativum was used as a test plant. The scope of the study also included the assessment of the effect of the method of ENP application to the soil (as powder and aqueous suspension) on their phytotoxicity. In the course of the study, no effect of the studied ENPs and their bulk counterparts on the germination of seeds was observed. The root growth inhibition of L. sativum depended on the kind of test applied. The trend between concentration of ENPs and effect depended on the method used and kind of ENPs. For most nanoparticles (despite of the method used), the differences in phytotoxicity between nano and bulk particles were observed. Depending on the kind of ENPs, their phytotoxicity differs between water and soil. ZnO (nano and bulk) and nano-Ni were more toxic in soil than in water. For TiO2 and bulk-Ni, reverse trend was observed. A different method of ENP application to soil differently affects the phytotoxicity.

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M. Mierzwa-Hersztek, K. Gondek, A. Klimkowicz-Pawlas, A. Baran and T. Bajda
Sewage Sludge Biochars Management - Ecotoxicity, Mobility of Heavy Metals and Soil Microbial Biomass
Environmental Toxicology and Chemistry 2017
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sludges; waste waters; soils

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Abstract : Production of biochar from sewage sludge may be a promising solution for sewage sludge management and improvement of soil properties, including carbon dioxide sequestration. The aim of the present study was to compare the effects of biochars derived from different sewage sludges on soil physicochemical and biological properties, ecotoxicity, and plant yield. Three biochars (produced at a temperature of 300 °C) were applied into sandy acid soil in doses of 0.5, 1, and 2% (w/w). Depending on the type and dose, the application of sewage sludge biochars into the soil caused diverse effects on the parameters of soil biological activity (microbial biomass [Cmic], soil respiration, and value of metabolic quotient). No correlation between the applied dose of biochars and inhibition of Vibrio fischeri luminescence was observed. The factor with a stronger impact on the activity of V. fischeri was the type of biochar. The use of the Ostracodtoxkit F test (MicroBioTests) showed that the addition of sewage sludge biochar (regardless of its dose) reduced the soil toxicity to Heterocypris incongruens compared with the control soil. A significant increase of Poa pratensis L. biomass was obtained in soils with 1 and 2% additions of each of the biochars. The addition of biochars in doses of 1 and 2% to the soil had greater effect on the content of mobile forms of Cu, Pb, and Cd than the 0.5% dose compared with the control.

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M. Mierzwa-Hersztek, K. Gondek, A. Klimkowicz-Pawlas and A. Baran
Effect of Wheat and Miscanthus Straw Biochars on Soil Enzymatic Activity, Ecotoxicity and Plant Yield
International Agrophysics 2017 31, 3 367-375
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : soils; composts

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Abstract : The variety of technological conditions and raw materials from which biochar is produced is the reason why its soil application may have different effects on soil properties and plant growth. The aim of this study was to evaluate the effect of the addition of wheat straw and Miscanthus giganteus straw (5 t DM ha-1) and biochar obtained from this materials in doses of 2.25 and 5 t DM ha-1 on soil enzymatic activity, soil ecotoxicity, and plant yield (perennial grass mixture with red clover). The research was carried out under field conditions on soil with the granulometric composition of loamy sand. No significant effect of biochar amendment on soil enzymatic activity was observed. The biochar-amended soil was toxic to Vibrio fischeri and exhibited low toxicity to Heterocypris incongruens. Application of wheat straw biochar and M. giganteus straw biochar in a dose of 5 t DM ha-1 contributed to an increase in plant biomass production by 2 and 14%, respectively, compared to the soil with mineral fertilisation. Biochars had a more adverse effect on soil enzymatic activity and soil ecotoxicity to H. incongruens and V. fischeri than non-converted wheat straw and M. giganteus straw, but significantly increased the grass crop yield.

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A. Zarrelli, M. Dellagreca, M.R. Iesce, M. Lavorgna, F. Temussi, L. Schiavone, E. Criscuolo, A. Parrella, L. Previtera and M. Isidori
Ecotoxicological Evaluation of Caffeine and its Derivatives from a Simulated Chlorination Step
Science of The Total Environment 2013 470-471C 453-458
Toxkits : Algaltoxkit F^TM; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - organic compounds; waste waters

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Abstract : Caffeine is ubiquitous in surface and ground waters and it has been proposed as a marker of the anthropogenic pressure on the environment. Sewage treatment plants based on active sludges seem to be not very efficient in its complete removal from effluents while additional disinfection treatments by chlorination are able to do it. In a simulation of the chlorination step herein we report that caffeine is transformed in six by-products: 8-chlorocaffeine, 1,3-dimethyl-5-azabarbituric acid, N,N’-dimethylparabanic acid, N,N’-dimethyloxalamide, N-methylurea and N,N’-dimethylurea. The ecotoxicity of caffeine and identified compounds was evaluated on the rotifer Brachionus calyciflorus and the alga Pseudokirchneriella subcapitata to assess acute and chronic toxicity, while SOS Chromotest and Ames Test were used to detect the genotoxic potential of the investigated compounds. Moreover, we assessed the possible antigenotoxic effect of the selected compounds using SOS Chromotest after co-incubation with the standard genotoxin, 4-nitroquinoline 1-oxide. Chronic exposure to these compounds caused inhibition of growth population on the rotifer while the algae seemed to be unaffected. Results indicated that caffeine (1), N,N’-dimethyloxamide (4) and N,N’-dimethylparabanic acid (5) reduced ß-galactosidase activity in comparison with positive control, both at 1 and 5mg/L of 4-NQNO with a good dose-response.

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Y. Huang, O. Campana and D. Wlodkowic
A Millifluidic System for Analysis of Daphnia magna Locomotory Responses to Water-born Toxicants
Scientific Reports, Article number 17603 2017 7
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : test development

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Abstract : Aquatic toxicity testing in environmental monitoring and chemical risk assessment is critical to assess water quality for human use as well as predict impact of pollutants on ecosystems. In recent years, studies have increasingly focused on the relevance of sub-lethal effects of environmental contaminants. Sub-lethal toxicity endpoints such as behavioural responses are highly integrative and have distinct benefits for assessing water quality because they occur rapidly and thus can be used to sense the presence of toxicants. Our work describes a Lab-on-a-Chip system for the automated analysis of freshwater cladoceran Daphnia magna locomotory responses to water-born toxicants. The design combines a Lab-on-a-Chip system for Daphnia sp. culture under perfusion with time-resolved videomicroscopy and software tracking locomotory activity of multiple specimens. The application of the system to analyse the swimming behaviour of water fleas exposed to different concentrations of water-born toxicants demonstrated that Lab-on-a-Chip devices can become important research tools for behavioural ecotoxicology and water quality biomonitoring.

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S. Szklarek, E. Kiedrzynska and J. Mankiewicz-Boczek
The Use of A Microbiotest Battery to Assess the Toxicity of treated Wastewater Discharged from Municipal Treatment plants in the Pilica River Catchment
VIII Polish National Bioindication Conference, 18-20 April 2018, Krakow, Poland - Book of Abstracts
Toxkits : Algaltoxkit F^TM; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : waste waters

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Abstract : The quality of surface water resources depends on, among others from, efficiency of pollution removal in municipal wastewater treatment plants. Regulation of the Minister of the Environment of 18 November 2014, about terms that should be meet when introducing sewage into waters or into the ground, and about priority substances harmful to the aquatic environment, defines the highest concentration limits (or levels of pollution reduction), which must be met before the introduction of treated wastewater into the waters only in the scope of five parameters: five-day biochemical oxygen demand (BZT5), chemical oxygen demand (ChZT), total suspension, total nitrogen and total phosphorus.
Required monitoring of the quality of treated wastewater shows that not all sewage treatment plants meet the applicable national standards and the requirements of EU directives, especially Directive 91/271/EWG concerning the treatment of municipal sewage. To improve this situation, a national municipal wastewater treatment program was created (KPOSK) adopted by the Council of Ministers at the 16 December 2003, with updates in subsequent years. The main objective of NPWP is to reduce discharges of insufficiently treated municipal sewage from agglomerations with an equivalent number of inhabitants (PE) over 2000, which should contribute to a significant improvement in the quality of water resources.
However, the above document focuses on improving the quality of treated wastewater only in terms of the five parameters mentioned above. While, literature reports that treated sewage may contain pollutants from the list of priority substances hazardous to the aquatic environment, as well as other contaminants not on this list, which are usually not analyzed, before the introduction of sewage into waters. Therefore, an alternative, complementary solution may be the use of a set of bioassays, which consists of tests with organisms representing three trophic levels (producers, consumers and reducers) for analyzing the quality of treated wastewater before being introduced into waters.
As part of the project carried out in the Pilica river basin, an assessment of the ecotoxicological hazard of treated municipal wastewater was carried out, which is an element of ecohydrological quantification of processes including analysis of a wide range of parameters and factors affecting water quality. Ecotoxicological evaluation was made on samples taken from 17 wastewater treatment plants. Samples were collected in two dates - June 2017 and September 2017. The ecotoxicological assessment was based on a battery of bioassays representing : Selenastrum capricornutum - producers (Algaltoxkit test), Thamnocephalus platyurus - consumers (Thamnotoxkit test) and Tetrahymena thermophila - destructors (Protoxkit test).
The research showed that three treatment plants were characterized by a high acute hazard (IV toxicity class), in both sampling date, and for the next six treatment plants the level of IV class was recorded in the one of the sampling date. Only in one of the seventeen tested treatment plants there was no risk at both dates (class I toxicity), while a low risk (class II toxicity) was observed for three treatment plants (on one or both dates). The remaining four treatment plants were characterized by an acute threat (class III toxicity) in samples of treated wastewater at least one sampling date.
\r\nHigh acute hazard (IV toxicity class) noted in samples of treated wastewater (12 samples out of 34 collected) the mortality of Thamnocephalus platyurus was primarily responsible (10 out of 12 reported cases of IV class toxicity), and next was the inhibition of growth of Selenastrum capricornutum (2 out of 12 reported cases of class IV). However, taking into account all analyzed samples from both sampling dates, the most sensitive organism was the algae Selenastrum capricornutum for which a growth inhibition of more than 20% was noted in 27 out of 34 samples (i.e. in 79% of samples). The second organism in terms of sensitivity was the crustacean Thamnocephalus platyurus for which in 12 of 34 mortality above 20% were noted (i.e., 35% of samples). In 17 sewage treatment plants tested twice - in the spring and summer period, there were three cases of sewage treatment plants in which the toxic effect was observed for all three test organisms.
The two-year monitoring planned under the project will allow to verify the results noted so far, and in combination with physicochemical results will give a full information about the threat to aquatic ecosystems in different seasons. However, it can already be concluded that the standard physicochemical monitoring of wastewater carried out in sewage treatment plants should be supplemented with an assessment of the threat for aquatic ecosystems - e.g. analysis with the use of a microbiotest set.\r\n

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M. Tankiewicz, P. Pirog and L. Wolska
Is Shampoo Necessary for Personal Hygiene of Aquatic Organisms ?
VIII Polish National Bioindication Conference, 18-20 April 2018, Krakow, Poland - Book of Abstracts
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - general

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Abstract : Personal care products are an indispensable part of our everyday life. We use them in various amounts, for multiple purposes. One of the most popular groups among cosmetics are shampoos, due to cleansing, nourishing, moisturizing, anti-static, oiling and glossing, healing and coloring properties. Hair has always been an important element of human appearance. The techniques of their care have changed over the years along with the prevailing fashion and were the result of medicine and cosmetic industry development.
Nowadays, a whole range of products for daily care of the scalp and various hair types is available on the market. Essentially, they are mixtures of many ingredients: surfactants, viscosity agents, thickeners, opacifiers, sequestrants, oiling agents, dyes, fragrances, preservatives and water. Wherein, in the largest quantities consist of water and surfactants. There are also so-called dry shampoos, which are the mixture of adsorbent (sodium carbonate, starch, kaolin, talc) in the form of spray, whose function is merely to refresh hair. Each shampoo is therefore a ‘cocktail’ of many chemical compounds that, after application, are released into the environment along with wastewater, causing water pollution, damage of aquatic ecosystems and pose a health risks. Therefore, it is so important to monitor and control the emission of these products to the environment. An additional problem is the fact that consumers use too much shampoo applying on the hair, causing their increased emissions. Moreover, commercially available personal care products usually consist of mixtures of several surface-active compounds in order to increase their effectiveness. The current state of knowledge regarding the properties of mixtures and interactions of surfactants in this type of products is negligible.
The aim of the research was ecotoxicological evaluation of selected publicly available care products containing surface-active compounds with using bioassays Microtox^®, Thamnotoxkit F^TM and Daphtoxkit F^TM. The results were compared with experimentally determined toxicity parameters for standard mixtures of surfactants and theoretical values for individual compounds. On this basis, their effect on interactions was assessed. As a result, it was possible to investigate the real impact of shampoos on aquatic organisms.

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E. Mierzejewska, A. Baran and M. Urbaniak
The Influence of Selected Plant Secondary Metabolites on the Ecotoxicity and Biodegradation Potential of Soil Contaminated with Herbicide from Phenoxy Acids Group (2,4-D)
VIII Polish National Bioindication Conference, 18-20 April 2018, Krakow, Poland - Book of Abstracts
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sorgum saccharatum
Topics : soils; plant tests; chemicals - drugs

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Abstract : 2,4-D (2,4-dichlorophenoxyacetic acid) is a herbicide from phenoxy acids group, widely used in agriculture to control broadleaf weeds. It is estimated that phenoxy herbicides achieve the 18% of the total herbicide sale in Poland. The intensification of agriculture production may promote contamination of soil matrices, surface and ground water, increased inhibition of plants development and soil toxicity. However, the phenoxy acids herbicides ecotoxicity has not been extensively studied yet.
One of the possible method of 2,4-D removal from the soil matrices, is its degradation by the soil microbiota. Biodegradation is determined by the presence and expression of the functional genes from tfdA cluster, which are involved in the first steps of phenoxy acids degradation pathway. Additionally interactions between soil microbiota and root exudates enriched with plant secondary metabolites (PSMs), structurally related to contaminants, can stimulate and enhance the removal of such xenobiotics from the environment. In order to investigate the soil ecotoxicity and biodegradation potential of soil microbiota, in the presence of 2,4-D and selected PSM (ferulic acid- characteristic for cucurbits), the study was conducted in bacterial cultures (originated from the agricultural soil slurry) in liquid medium enriched with 2,4-D in doses of 0.1 and 0.5 mM and/or ferulic acid 0.25 mM. Bacterial cultures were incubated for 24 days in 25°C. After 24 days of incubation, ecotoxicity tests Phytotoxkit and Microtox^® were performed. In order to analyze the presence of genes from tfdA cluster (tfdA, tfdAα, tfdA CI, tfdA CII and tfdA CIII) and bacterial gene 16S rRNA, samples were collected every 6 days during the incubation period.
The ecotoxicity results showed that 24 days the biodegradation processes in samples contaminated with 2,4- D, have a positive effect on the reduction of root growth inhibition of the tested plants. Especially sensitive to the addition of 2,4-D and ferulic acid were dicotyledonous plants, that after 24-days of incubation still showed 100% inhibition level for L. sativum (both 0.1mM and 0.5 mM 2,4-D), while monocotyledonous S. saccharatum showed an inhibition of root length of 43% (0,1mM 2,4-D) and 14% (0,5mM 2,4-D). This findings indicate that initial concentration of 2,4-D influenced the biodegradation processes, thereby causing the decrease of ecotoxicity level of studied samples. The Microtox^® analysis showed that the decrease of luminescence for V. fischeri reached 100%. The obtained results indicate the diversification of the ecotoxic effect of 2,4-D between the studied species. Metabolites, produced in the course of biodegradation processes of 2,4-D and ferulic acid, might exhibit higher ecotoxicity than primary compounds.
The molecular analysis showed that the addition of PSMs selectively influenced the presence of studied genes from tfdA cluster. In samples amended with 2,4-D and ferulic acid, the presence of tfdAα and tfdA CI genes was observed. tfdA CIII was detected in samples with the addition of 2,4-D only. It was also observed that the concentration of the herbicide influences the detection of genes, because the samples with 0.1mM 2,4-D, were characterized of twofold increase of genes detection from tfdA cluster in comparison to samples with higher concentration of 2,4-D (0.5mM).
\r\nIn general, these results show that the PSMs can have a selective influence on ecotoxicity and the stimulation of biodegradation potential of soil microbiota in terms of the removal of structurally related herbicides form phenoxy acid group.

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A. Baran, M. Mierzwa-Hersztek, K. Gondek, M. Szara and M. Tarnawski
The Content of Organic Carbon and the Toxicity of Bottom Sediments
VIII Polish National Bioindication Conference, 18-20 April 2018, Krakow, Poland - Book of Abstracts
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; sediment and soil contact tests

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Abstract : Organic matter has important influences on fate of environmental pollution in water dam reservoirs. Sorptive abilities of organic matter are important in preventing migration of pollutants in the environment, and also affect the detoxification of pollutants. The aims of studies were to assess content of organic matter in bottom sediment and to determine their impact on toxicity of sediments. Following compounds of carbon were analyzed: total organic carbon (TOC), carbon humic acid (Cha), fulvic acids (Cfa) and carbon hydrolysing (Cnh). The analysis of sediment toxicity was carried out using biotests: Phytotoxkit, Ostracodtoxkit, Microtox. Organic carbon is an important factor controlling the content of contaminants in bottom sediments, and thus their bioavailability and toxicity.

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J. Chojnacka, M. Wawryniuk and G. Nalecz–Jawecki
Assessment of Toxicity of Artificial Sweeteners Using Testbionts
VIII Polish National Bioindication Conference, 18-20 April 2018, Krakow, Poland - Book of Abstracts
Toxkits : Daphtoxkit F^TM magna; Spirodela duckweed Toxkit
Test Species : Daphnia magna; Spirodela polyrhiza
Topics : chemicals - organic chemicals

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Abstract : Artificial sweeteners are the most popular additive used in the industry. Because they have sweet taste, low calories, don`t rise blood sugar and have good physicochemical properties they are often used in the food, cosmetic and pharmaceutical industries.\r\nMost artificial sweeteners are not metabolized and are excreted unchanged. Because of the increased use of artificial sweeteners, their presence in the environment also increases. This may also result in re-consumption of the above compounds without the knowledge of the consumers. Due to their good solubility in water, they may be located in water areas (sewage, surface water).
Our research concerned the most frequently used five artificial sweeteners: aspartam, acesulfame K, sodium saccharinate, sodium cyclamate and sucralose. Toxicity measurements were conducted using the following assays: bacterial (Microtox), plant tests (with using Lemna minor and Spirodela polyrhiza), protozoa (Spirostomum ambiguum), and invertebrates (Daphnia magna). Artificial sweeteners have been tested for safe use in food, but there are no unequivocal results on how they affect the environment.
The research conducted so far in the world has proved that artificial sweeteners may occur in urban wastewater, natural (surface and underground) and drinking water. These substances are not eliminated from sewage in the purification process, as a result, they penetrate into surface waters. Their poor elimination can lead to other ecosystems and trophic structures.
Ask yourself if we can find the presence of these substances in aquatic organisms and what it may have in the environment. Also, it should be noted whether through the natural hydrological cycle, artificial sweeteners do not get into the soil, and thus are not accumulated in edible plants that hit our table.

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L. Sikorski, M. Baciak, A. Bes, K. Warminski and B. Adomas
Indicators of Glyphosate Toxicity (EC) for Common Duckweed (Lemna minor L.) and Water Flea (Daphnia magna L.)
VIII Polish National Bioindication Conference, 18-20 April 2018, Krakow, Poland - Book of Abstracts
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - pesticides; chemicals - biocides

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Abstract : Herbicides containing the active ingredient glyphosate, a chemical compound characterized by high solubility in water, are applied to protect agricultural crops against weeds and water bodies against excessive plant growth. Glyphosate is carried with surface runoffs to surface and ground water, and in water bodies, it affects plants and animals that are not directly targeted by the herbicide. Glyphosate exerts toxic effects at various levels of biological organization in water bodies. Therefore, the aim of this study was to determine glyphosate’s toxicity for common duckweed and water flea by calculating the values of toxicity indicators EC10, EC25, EC50 and EC90.
Glyphosate (Roundup 360 SL) concentrations of 0, 3, 7, 20 and 40 µM were tested, where 7 µM is the field concentration recommended by the manufacturer. The compound’s toxicity was determined in the Lemna chronic toxicity test and the Daphtoxkit acute toxicity test. The percent inhibition of the growth rate and biomass yield of common duckweed was determined after 7 days of the Lemna test, and the immobilization of D. magna was determined 180 and 360 minutes after Daphtoxkit application. Water fleas were regarded as immobilized if they lost their ability to swim 15 seconds after being gently shaken in the test vessel. The effective concentrations of glyphosate which resulted in 10%, 25%, 50% and 90% inhibition in the growth rate and biomass yield of common duckweed and the swimming ability of D. magna were calculated in a regression model.
The 7-day chronic toxicity test revealed that glyphosate was toxic for common weed already at the lowest tested concentration (EC10=1 µM) which inhibited the plant’s growth rate and biomass yield by 25%. The 2.61 µM dose of the herbicide (EC50), which was more than half lower than the recommended dose (7 µM), led to a 50% decrease in the growth rate and biomass yield of common duckweed. The analyzed parameters were inhibited in 90% (EC90) already under exposure to a glyphosate dose of 9.9 µM.
Glyphosate’s toxicity against D. magna was tested at identical concentrations in the Daphtoxkit acute toxicity test. The number of organisms immobilized in multi-well test plates increased gradually with time of exposure to the herbicide. A glyphosate dose of 3.80 µM (EC50) immobilized 50% of the organisms after 180 minutes of the test, and D. magna were completely immobilized when the dose was increased more than 10-fold (40 µM). Prolonged exposure to the herbicide decreased the effective concentrations of the herbicide which were determined at EC10=0.15, EC25=0.36, EC50=1.56 and EC90=16.33 µM after 360 minutes of the Daphtoxkit test.
The calculated values of EC10 indicate that glyphosate adversely affects common duckweed (Lemna minor L.) and water flea (Daphnia magna L.) even at low concentrations. The growth rate and biomass yield of common duckweed and the swimming ability of D. magna were inhibited in 25% and 50% (EC25 and EC25) already under exposure to glyphosate doses lower than 7 µM, the dose which is considered safe by the manufacturer. The above indicates that even the recommended dose of the Roundup 360 SL herbicide is toxic for aquatic ecosystems. The results of this study also confirm that the Lemna test and Daphtoxkit are useful analytical tools for evaluating the toxicity of glyphosate and predicting the consequences of chemical contamination of fresh water based on the calculated values of toxicity indicators.

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M. Szara, A. Baran, A. Klimkowicz–Pawlas, M. Tarnawski and T. Koniarz
Assessment of Bottom Sediment Toxicity Using Phytotoxkit and Rapidtoxkit Biotests
VIII Polish National Bioindication Conference, 18-20 April 2018, Krakow, Poland - Book of Abstracts
Toxkits : Phytotoxkit; Thamnotoxkit F^TM
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum; Thamnocephalus platyurus
Topics : sediments; plant tests; rapid tests; behavioural tests

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Abstract : Bottom sediments are the place where the pollutants are deposited. These pollutants can cause problems with maintaining the quality of drinking water and limit the recreational and tourist values of the reservoirs.
The aims of the study were to assess the toxicity of bottom sediments collected from the Roznow reservoir (Dunajec), located in the Malopolskie Voivodeship. The toxicity of bottom sediments was determined using the Phytotoxkit and Rapidtoxkit tests. The germination inhibition and inhibition of the root growth of the three test plants Sinapis alba, Lepidium sativum and Sorghum saccharatum were tested by the Phytotoxkit test, whereas Rapidtoxkit enabled the detection of the presence of toxic substances with the larvae of the Thamnocephalus platyurus crustacean.
Lepidium sativum was the most sensitive to pollutants in sediments, while Sorghum saccharatum was the least sensitive. An indirect effect was found for Sinapis alba. Larvae of Thamnocephalus platyurus showed inhibition of uptake of food particles (inhibition of particle uptake >/= 30%) in 72% of the tested samples.
In the assessment of bottom sediment toxicity, a good complement to chemical analyzes is the use of biological tests. The use of bioassays allows the assessment of the real threat from contaminants contained in bottom sediments.
See corresponding Poster

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J. Antonkiewicz, A. Baran, R. Pelka and A. Wisla
Ecotoxicity of a Mixture of Sludge from Paper Production and Municipal Sewage Sludge
VIII Polish National Bioindication Conference, 18-20 April 2018, Krakow, Poland - Book of Abstracts
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sludges

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Abstract : Due to chemical contamination in sludge from paper production and sewage sludge, there is a need to treat these wastes prior to their environmental management. Environmental management of the above waste seems to be justified due to the content of organic matter and high fertilizing value. The aim of the study was to assess the ecotoxicity of sludge from paper production and sewage sludge and their mixtures in the aspect of soil environment standards.
The tests were carried out in laboratory conditions. A biological test Phytotoxkit was carried out, which was to indicate the ecotoxicity of waste and mixtures. In addition, the content of macroelements and trace elements in waste was assessed.
The content of heavy metals in waste did not exceed the limits for their agricultural and recultivation use. The sludge from paper production was toxic, while the mixture of carbide lime waste carbide lime waste and sewage sludge was considered to be low-toxic for test plants. Waste mixtures can be used for environmental management. However, before environmental management, biological and chemical tests should be performed.
See corresponding Poster

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B. Wojtasik, A. Baran, M. Tarnawski, M. Szara and T. Koniarz
Toxicity Assessment of Bottom Sediments in Reaction Assays Ostracod
VIII Polish National Bioindication Conference, 18-20 April 2018, Krakow, Poland - Book of Abstracts
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments; sediment and soil contact tests

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Abstract : Toxicity of bottom sediments of the Roznow reservoir (Malopolska province) was tested using two organisms: Heterocypris incongruens (Ostracodtoxkit) and Candona rectangulata Alm, 1914 (CrecTest) [Ostracodtoxkit 2001, Wojtasik 2012]. Candona rectangulata Alm, 1914 is a common, freshwater, parthenogenic Arctic species. This species is very resistant to many environmental factors : temperature, heavy metals, POPs, conductivity, pH (> 5.5). The test used a four-level scale: 1 - normal activity, 2 - low activity, 3 - anabiosis, 4 - lethal effect. In the Ostracodtoxkit test, two parameters of survival and growth of Heterocypris incongruens after 6 days of exposure to bottom sediments were studied. The studies showed a similar reaction of the both organisms, however the time of exposing the ostracod to the bottom sediments was important.
See corresponding Poster

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M. Tarnawski and A. Baran
Use of Chemical Indicators and Bioassays in Bottom Sediment Ecological Risk Assessment
Archives of Environmental Contamination and Toxicology 2018 74, 3 395-407
Toxkits : Ostracodtoxkit F^TM; Phytotestkit; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments; sediment pore waters; sediment and soil contact tests

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Abstract : This study is was designed to assess the ecological risk associated with chemical pollution caused by heavy metals and PAHs on the basis of their ecotoxicological properties in sediments collected from the Rzeszow dam reservoir (Poland). The sediment samples were collected from three sampling stations: S1—inlet, backwater station, S2—middle of reservoir, S3 outlet, near the dam. The sediments’ toxicity was evaluated using a battery of bioassays (Phytotoxkit, Phytotestkit, Ostracodtoxkit F, and Microtox). The highest content of metals (120.5 mg Zn; 22.65 mg Pb; 8.20 mg Cd . kg^-1 dw) and all PAHs (Σ9361 µg . kg^-1 dw) in sediments was found at station S1. The lowest content of metals (86.72 mg Zn; 18.07 mg Cu; 17.20 mg Pb; 3.62 mg Cu; 28.78 mg Ni; 30.52 mg Cr . kg^-1 dw) and PAHs (Σ4390 µg . kg^-1 dw) was found in the sediment from station S2. The ecological risk assessment of the six metals and eight PAHs revealed a high potential toxicity in sediments from stations S1 (PECq = 0.69) and S3 (PECq = 0.56) and a low potential toxicity in sediments from station S2 (PECq = 0.38).
The studies also showed the actual toxicity of sediments for the test organisms. The sediment pore water was least toxic compared to the whole sediment: solid phases > whole sediment > pore water. The most sensitive organism for metals and PAHs in bottom sediments was Lepidium sativum, and in pore water Sorghum saccharatum. The concentration of metals and PAHs in bottom sediments generally did not affect the toxicity for other organisms. Clay content and organic C content are likely to be important factors, which control heavy metal and PAH concentrations in the sediments. Data analysis by PCA found the same origin of metals as well as PAHs—mainly anthropogenic sources. The obtained information demonstrated the need to integrate ecotoxicological and chemical methods for an appropriate ecological risk assessment.

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M. Muna, A. Lukjanova, H. Vija, A. Kahru and I. Blinova
Toxicity of Lanthanides to Freshwater Microcrustaceans
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : chemicals - inorganic compounds

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Abstract : The application of lanthanides (Ln) in different sectors of the world economy has significantly increased during the last two decades. This process has been accompanied by emissions into the environment via different pathways. The anthropogenic anomalies of Ln in soil, surface water, groundwater and even in tap water have already been registered. The disruption of the natural biogeochemical cycle of Ln increases the risk of biota being exposed to elevated concentrations of Ln. However, the ecotoxicological effects of these elements and their fate in the environment are still insufficiently understood. The toxic concentrations reported in the literature, e.g., for Daphnia magna, noticeably vary presumably due to different test conditions. For this study, acute ecotoxicity testing of La, Ce, Pr, Nd and Gd nitrates to freshwater crustaceans Daphnia magna (48 h) and Thamnocephalus platyurus (24 h) were performed in two different test media (synthetic freshwater and natural lake water). Also, long-term (21 days) exposure of D. magna (OECD 211) in lake water was included. It was shown that the Ln fractionation between two main phases (precipitated and settled or remained in the water column) changed during the tests depending on (i) water composition, (ii) nominal concentration, (iii) exposure time, and (iiii) tested chemical element. Therefore, nominal concentrations were used for toxicity calculations. Acute toxicity of investigated Ln to both crustaceans was similarly low. E(L)C50 values obtained in synthetic freshwater varied from 18.2 to 34.6 mg Ln/L for T. platyrus and 18.5–31.1 mg Ln/L for D. magna. Gd was the most toxic to both species, however, difference between E(L)C50 values for Gd and other Ln was statistically significant (p< 0.05) only in T. platyrus. In the lake water, bioavailability of Ln was much lower: mortality of exposed organisms did not exceed 25% at the largest tested concentration (50 mg Ln/L). In contrast to acute assay, the 21 day chronic test performed in the lake water showed high Ln toxicity to D. magna (0.2 to 0.5 mg Ln/L). It was revealed that mortality was a more sensitive endpoint than reproduction. Differences between LC50 of individual Ln were not statistically significant. Thus, our results support the hypothesis that different lanthanides have similar mechanism of toxicity in crustaceans.

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N.B. Martins, A. Pradhan, F. Cassio and C. Pascoal
Impacts of Anti-Cancer Drugs on Freshwater Rotifers at Environmentally Realistic Concentrations
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Rotoxkit F^TM
Test Species : Brachionus calyciflorus
Topics : chemicals - drugs

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Abstract : As human population increases, the presence of emergent chemical contaminants (ECCs) in freshwaters increases. ECCs have shown to be persistent and bio active, reaching the freshwater aquatic systems mostly untreated, where their fate and behavior is little understood. Anti-cancer drugs are among of the ECCs of concern due to their high cytotoxicity and increasing usage. The administration of drugs in cocktails, instead of single drug treatment, make the assessment of the environmental risk of these compounds a difficult task with much information lacking on sub-lethal effects on aquatic species. We used two cytotoxic drugs aiming at linking their effects on the reproduction inhibition of the rotifer Brachionus calyciflorus with processes of oxidative stress. The rotifer was exposed to a range of concentrations of a antimetabolite (5- Fluorouracil; 5FU) and a cytotoxic antibiotic (Doxorubicin; DOX) alone and in mixtures. The results showed that 5-Fluorouracil had a stronger effect (EC50 =0.074 mg L-1) on the population growth rate then Doxorubicin (EC50 =13 mg L-1) and toxicity effects were detected at environmentally relevant concentrations. Two concentrations of each drug were chosen for binary mixtures and two concentrations per drug were used to assess reactive oxygen species (ROS) accumulation and plasma membrane damage with epifluorescence microscopy. In the presence of low concentrations of 5FU, there was a reduction of the toxicity induced by DOX indicating possible antagonistic effects between both drugs. At concentrations, as low as EC05 we found accumulation of ROS in a dose dependent manner showing a clear connection between ROS accumulation and the toxicity of these compounds. Furthermore, this indicates that even in concentrations with no apparent effect on the rotifer reproduction, cellular effects were found with possible consequences for the community at the long term.

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P. Palma, S. Fialho, A. Lima, A. Penha, H. Novais, S. Monllor, N. Guillem-Argiles, M. Lopez de Alda, P. Alvarenga, M. Morais and R. Salgado
Ecotoxicological Tools to Assess the Impact Pollution of Tributaries to the Alqueva Reservoir (Southern Portugal)
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : surface waters

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Abstract : Degradation of surface waters and biodiversity loss at different spatial and temporal scales occurs through multiple stressors whose effects are difficult to separate and identify. Efficient management of water bodies depends on the development and selection of robust, sensitive and easily applicable tools that allow prioritizing the pressures and stressors that act in a basin, and mitigate their effects. The Alqueva reservoir constitutes the most important water supply source in southern Portugal, a semi-arid region with high levels of water scarcity and where agriculture is one of the main activities. The aim of the present study was to assess the use of an ecotoxicological tool-box in tributaries of the Alqueva reservoir for detecting chemical alterations that may influence the water quality of the reservoir. Water samples were collected along 2017 at four tributaries of Alqueva (streams of Zebro, Álamos, Amieira and Lucefécit) and analyzed for: (i) physical chemical support elements (pH, temperature, dissolved oxygen, conductivity, chloride, total phosphorus, Kjeldahl nitrogen, ammonium, nitrite, nitrate, BOD, COD), (ii) hazardous substances (pesticides), and (iii) ecotoxicological endpoints, using bioindicators representing different trophic levels (Vibrio fischeri, Thamnocephalus platyurus, Daphnia magna). In general, Zebro and Lucefécit presented concentrations of BOD (Zebro: 4.0-35.5 mg L-1; Lucefécit: 2.3-7.5 mg L-1) and total phosphorus (Zebro: 0.18-6.23 mg L-1; Lucefécit: 0.02-1.92 mg L-1) that compromise the support of biological life, with regard to nutrient and oxygenation conditions. As regards pesticides, the concentrations detected were low, being bentazone the compound quantified at highest levels. Lucefécit was the tributary that presented higher concentrations of pesticides (with values of bentazone of 1.94 µg L-1), probably due to the intensive agriculture crops around it. Concerning to ecotoxicological analysis, the results highlighted the toxicity in sublethal parameters (reproduction, feed inhibition or growth inhibition) induced, mainly, by samples from Zebro and Lucefécit streams. So, the results from the ecotoxicological tool-box allowed identifying the streams that promote a higher chemical impact to the reservoir, which is essential to delineate management actions to improve the water quality of the reservoir.

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M. Carere, I. Lacchetti, R. Scenati, L. Lucentini, M. Beccaloni, P.M. Gucci, F. Falleni, C. Gavasso, Lega Navale; M. Cicero, G. Marsili, L. Musmeci, V. Frau, L. Zingari and E. Veschetti
Chemical and Ecotoxicological Monitoring of a Marine Coastal Area in Central Italy
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Artoxkit M^TM; Marine Algaltoxkit
Test Species : Artemia (salina) franciscana; Phaeodactylum tricornutum
Topics : seawater; costal waters

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Abstract : A monitoring campaign has been performed in Central Italy with the aim to characterize the chemical quality status of the coastal marine area in order to detect the possible impact of the emissions of a Coal fired power station and other sources of pollution in proximity of the city of Civitavecchia. The sampling has been carried out in two different seasons of the year along the marine coastal area and in a transitional surface waterbody (Saline di Tarquinia). The analysis has been performed in the water column and in the first 20 centimeters of the sediments. The chemical substances analyzed included several priority substances of the WFD (water framework directive) and other chemical substances: Metals, Dioxins, PCB, PAH, Naphtalene. The Ecotoxicological assays have been performed with the use of algae (Phaeodactylum tricornutum) and crustaceans (Artemia franciscana and Tigriopus fulvus). The results have showed a diffuse light exceedance of the sediment environmental quality standards of the Italian legislation for some metals (e.g. arsenic, lead, chromium, mercury) and naphthalene; the data of the water column are in general below the environmental quality standards, but Uranium has been detected in surface water samples at concentrations above the available PNEC (predicted no effect concentration). Ecotoxicological effects have been detected with the algae and could be related to the substances detected (e.g. heavy metals) or other substances released in the area (transitional waterbody). In general the results show a situation in which the quality of the sediments is not in a good status, although the level of concentrations should not cause a high risk for the aquatic ecosystems; the chemical contaminants can derive from different sources of pollution (industrial, urban, agricultural, atmospheric deposition) of the area and management measures should be reinforced in order to achieve the good quality status required by the water framework directive. The presence of Uranium in the marine coastal area should be further investigated to understand the possible role of the Coal fired power station.
See corresponding Poster

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E. Oliva, E. Terrado, J. Navarro, M. Pino, A.M. Mainar and D. Ballestero
Ecotoxicological Evaluation of the Hydrolate Byproduct of Satureja montana on Daphnia magna and Vibrio fischeri
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - plant extracts

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Abstract : The increasing demand of natural bioproducts for cosmetic use, food or phytotherapy is based on the awareness about adverse effects on health and the environment. In particular, this work is focused on the plant Satureja montana (Lamiaceae), which has demonstrated a wide range of applications due to its important antioxidant and antimicrobial activity. Furthermore, Satureja species have a sweet flavor and simple cultivation characteristics. The main components, thymol and carvacrol (oxygenated monoterpens), are supposed to be responsible for these biological activities. Although there are a substantial number of studies where Satureja species are evaluated for their biological and pharmacological activities, as well as its chemical characterization, limited data are available on ecotoxicological characterization. Consequently, the aim of this study is to evaluate the acute ecotoxicity of the hydrolate obtained from Satureja montana (Ejea, Aragón) by the traditional method of steam distillation on two organisms widely used a indicators of ectotoxicity: the freshwater crustacean Daphnia magna and the marine bacterium Vibrio fischeri. Both tests are standardized for the purpose of determining the toxicity expressed as EC50. Our results indicate that the hydrolate of S. montana are likely to cause toxic effects on D. magna and V. fischeri but only at high dilutions (LC50 values in the range of 0,5% in both cases). These studies allow us to know the possible environmental effect that these promising plant extracts can cause as a source of cosmetic and pharmaceutical applications with the aim of ensuring more environment-friendly processes and products.

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G. Amariei, K. Boltes, J. Valimaña-Traverso, M. García, P. Letón, M. Marina and R. Rosal
Effects of Duloxetine and Econazole on Freshwater Species Towards Individual and Combined Conditions
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Spirodela duckweed Toxkit
Test Species : Spirodela polyrhiza
Topics : chemicals - drugs

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Abstract : Thousands of biologically active pharmaceuticals ingredients (APIs) are used in human and veterinary medicine over the world. Nowadays, the occurrence of pharmaceuticals in aquatic environments is a well-established issue, there are still gaps in our knowledge on the fate and effects of these compounds in the environment1 . Evaluating API ecotoxicology is even more challenging due to uncertainties about appropriate dosages, durations of exposure, range of sensitive taxa, sensitivity of developmental stages, and toxicological endpoints2 . More attention should be paid on the nontarget organisms and the chiral nature of contaminants3 . This work assess the toxicity of the antidepressant drug Duloxetine and the antifungal Econazole, individually and combined, on three freshwater species—algae, crustacean and duckweed, using APIs concentration from 0.039 to 100 mg/L. Level and type of drugs interactions were determined using the Combination Index-isobologram method. The enantiomers concentration of the target componds in the culture media were also measured to analyze the relation between degradation profile and the observed toxicity on organisms. Results reveal toxic effects of Duloxetine and Econazole leading to growth reduction and significant changes in the morphology of duckweed fronds. The EC50 values obtained shown Duloxetine as very toxic for algae and toxic for crustacean and plants. Econazole appears as very toxic for all species evaluated. Mixed toxicity profile was different for the organisms studied, showing also dissimilar persistence of R and S enantiomers in culture media.
See corresponding Poster

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S. Caciolli, I. Lacchetti, M. Carere, S. Marcheggiani, C. Puccinelli, A. D'Angelo, S. Marchini, R. Giuseppetti, E. Pierdominici, E. Volpi and L. Mancini
Application of Eco-Genotoxicological and Microbiological Parameters for the Assessment of the Quality of Wastewater Industrial Reuse
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Phytotoxkit
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Sorgum saccharatum
Topics : waste waters

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Abstract : In industrial sectors, water is used in large amount in production cycles. In the field of Medical Devices (MDs) production, for example, water is used, both as an ingredient and as a necessary element for production. The re-use and the recycle of the wastewater represents a priority area in the strategical plan of the European Commission for a sustainable water management and also considering the scarcity of water resources caused by the climate changes. The aim of this study is the assessment of treated wastewater quality discharged from Medical Device and pharmaceutical industries, by identifying a suitable set of tools in order to support the potential reuse of treated wastewater, taking into account both microbiological and eco-/geno-toxicological parameters. The study was carried out on three MDs industries in Italy, which perform the recycle of the wastewater in their own system processes. Samples were collected inlet and outlet of wastewater treatment plants. The microbiological parameters investigated were Bacterial Total Count (BTC), E. coli, Enterococci, Staphylococcus spp., Pseudomonas spp. A set of ecotoxicological bioassays was selected for this study, namely the bacterium Vibrio fischeri, the algae Selenastrum capricornutum, the crustacean Daphnia magna, the fish Danio rerio (Fish Embryo Toxicity Test) and the plant Sorghum saccharatum, in order to represent different trophic levels and thus to assess any potential effects on the aquatic ecosystems. Seeds of Vicia faba were exposed for assessing the possible genotoxic effect of wastewater using the Micronucleus test (MN-test). The study results show a significant decrease in treated water samples of all microbiological parameters and the absence of E. coli. The ecotoxicological assays highlight a significant toxicity of the wastewater before the treatment while an evident decrease has been recorded after it. Sublethal effects for Danio rerio embryos and genotoxic effects for Vicia faba\'s micronucleus frequencies have been reported. Even though not yet conclusive, this approach can be considered a useful and promising tool in the reuse management of industrial wastewater and an initial support to the policy in this field.
See corresponding Poster

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S. Chelinho, C. Venancio, I. Lopes and J. Sousa
Quality Standards for Urban Waste Fertilizers : Putting Ecotoxicology in the Picture
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Ostracodtoxkit F^TM; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Heterocypris incongruens
Topics : solid wastes; decision support system

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Abstract : The recently adopted EU Circular Economy Package intends to boost the production and EU movement of fertilizing products, such as those obtained from urban wastes (UWF) and the harmonization of quality standards (certification) for such products, to avoid market rejection. The Portuguese and EU regulation on UWF production/commercialization relies only on physico-chemical/biochemical analysis, which do not give any insight on the fraction of contaminant/mixture of contaminants bioavailable for organisms, nor the existence of potential antagonistic and/or synergistic effects. The main objective of this study is to develop an environmental quality certification system for the use of UWF in agricultural systems. In the present contribution, it was intended to characterize the ecotoxicological potential of the selected UWF, by evaluating both soil habitat and retention functions using lower-tier laboratory tests. Five UWF, two with origin in source separated organic wastes (group I; theoretically with higher quality, ex. lower metal content) and three originated from the organic fraction of mixed municipal solid waste (group II) were selected and tested using a battery of standardized ecotoxicological assays with plants, soil invertebrates and freshwater species. Five soil-UWF dilutions (0.7; 2.1; 6.3; 18.9; 56.7%) and eluates of pure UWF were used as test-medium. The results show that the highest and lowest toxicity were observed in the two UWF from group I. Among soil organisms the range of sensitivities was: E. Andrei > F. candida > E. crypticus > L. sativa > T. aestivum while for aquatic organisms was: H. viridissima > R. subcapitata > C. vulgaris > H. incongruens > B. calyciflorus. The observed toxicity was probably related with UWF high salinity rather than with metal contents. The obtained data also reinforce the need to include information from biological susceptibility of the receptors potentially at risk on the available regulation to obtain a more realistic view of the potential risks and to adapt the UWF application practices. Ultimately, a sustainable economic growth based on the efficient use of resources/ waste valorization can be promoted.
See corresponding Poster

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A. Romero, E. Joonas, M. Muna, D.A. Vignati and L. Giamberini
Assessment of the Toxic Interaction of Lanthanides on Aquatic Organisms
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - inorganic compounds

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Abstract : The relevance of lanthanides (LNs), in agricultural, industrial and, especially in high-tech applications has increased in the last decades. As consequence, more LNs are expected to enter into the environment and accumulate in the ecosystem. Although no great accumulations have so far been recorded, alterations in the LN natural cycles have already been observed. Most of the available data on LN toxicity focuses on the effect of single elements, however they are commonly found as a group in nature. LNs are expected to have cumulative toxic effects on organisms, owing to their similar chemical properties, but studies as mixtures, more representative of real scenarios, are required to support this hypothesis. In this research, we evaluated the toxic interactions of binary and ternary mixtures of cerium (Ce), gadolinium (Gd), and lutetium (Lu), representative of heavy, middle and light LNs, respectively, on seven aquatic species belonging to different trophic levels. From the seven organism studied (A. fischeri, R. subcapitata, C. vulgaris, B. calyciflorus, H. incongruens, D. magna and D. rerio) potential toxic effects were observed only in five; and the inhibitory LN effects were consistently concentration-dependent only for A. fischeri, R. subcapitata and B. calyciflorus. Bioavailable LN concentrations significantly decreased during all tests and the major decline took place at the beginning of the tests, but the extent of the decrease varied across test media. Thermodynamic speciation calculation highlighted important differences: in distilled water and 1% NaCl, LNs were predicted to occur mainly as free ions; in more complexes media, LNs appeared as free forms and with complexes with groups LNSO 4 +, but these species lowered with the decrease in the ionic radii, and, in detriment of these complexes, species with LNCO 3 + and LN(OH)+2 groups increased. The two multi-toxicity approaches used in this study (concentration addition and toxic unit calculation) showed more than additive effect for the mixtures to the bacterium A. fischeri and the algae R. subcapitata; whereas less than additive toxicity was instead observed for the rotifer B. calyciflorus. Obtained results highlight that it should not be assumed LN toxicity as additive as so far, and predicting the response of aquatic organisms exposed to mixtures should be further research to better understand their toxic interactions in real scenarios.

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S. Goncalves, C. Venâncio, S. Loureiro, R. Vogt, J. Wanzenböck, A.M. Soares and I. Lopes
An Ecotoxicological Assessment of Lake Mondsee, Austria : a Two Year Survey
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : surface waters; waste waters; sediments; sludges

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Abstract : Lake Mondsee is a recreational area in Austria for both bathing and water activities/sports. Nearby exists a wastewater-treatment plant (WWTP) which can represent a point emission for the lake’s contamination. Accordingly, an ecotoxicological assessment of Lake Mondsee was carried out. Water (W) and sediment (S) samples were collected from Lake Mondsee and the reference site, Lake Irrsee, on three different seasons: 1) summer 2015 (preliminary assessment of W and S samples’ toxicity), 2) spring 2016 (possible best-case scenario, since lake was frozen for the winter) and 3) summer 2016 (worst-case scenario, tourist activities peak). The WWTP inflow and outflow, plus pre-thickening (PS) and thickened sludge (TS) were also collected. The toxicity assessment for summer 2015 was made by performing 48-h population growth (r) assays with the rotifer Brachionus calyciflorus (W samples) and the 15-min luminescence inhibition assay with the bacterium Vibrio fischeri (all samples). Regarding the W samples, results showed no luminescence inhibition for V. fischeri and average r inhibition rate (%) of B. calyciflorus was below 26%. The WWTP inflow samples presented high toxicity to B. calyciflorus (EC50 > 60%). Samples of S, PS and TS were extremely/high toxic to V. fischeri. The samples collected during spring 2016 and summer 2016 were analysed through a battery of assays, with species belonging to different trophic levels. In addition to the two above mentioned tests: the 72-h growth inhibition test with Raphidocelis subcapitata and the feeding inhibition test with Daphnia magna with the W samples were performed and the 6- day mortality and growth assessment with Heterocypris incongruens for S, PS and TS samples. Regarding spring 2016, the average r inhibition rate (%) of B. calyciflorus was lower than 30% for most of the W samples and average did not surpass 12%. The Microtox^® tests showed high toxicity only for all W, S, PS and TS samples. Samples collected in the summer 2016, showed similar results for the growth inhibition for R. subcapitata and the Microtox^® test. The r inhibition rate (%) of B. calyciflorus and the 6-day mortality and growth assessment with H. incongruens showed some variation. No evidences of the influence of the WWTP present at lake Mondsee were retrieved, since W and S samples from both Lakes Mondsee and Irrsee showed similar toxicity. Further chemical analysis is necessary to clarify the high toxicity observed in the sediments.
See corresponding Poster

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S. Morgana, V. Piazza, C. Gambardella, E. Costa, F. Garaventa and M. Faimali
Relevance and Suitability of Invertebrates Swimming Behavior as Sub-Lethal Endpoint to be Considered for Ecotoxicological Investigation
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Artoxkit M^TM; Rotoxkit M^TM
Test Species : Artemia (salina) franciscana; Brachionus plicatilis
Topics : rapid tests; test development; behavioural tests

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Abstract : Some pollutants can be resistant to wastewater treatment, hence becoming a risk to aquatic and terrestrial biota even at the very low concentrations (ng L^-1-µg L^-1) they are commonly found at. Tertiary treatments are used for micropollutant removal but little is known about the ecotoxicity of the treated effluent. In this study, a municipal secondary effluent was treated by a solar photo-Fenton reactor at initial neutral pH in a raceway pond reactor, and ecotoxicity was evaluated before and after micropollutant removal. Thirty-nine micropollutants were identified in the secondary effluent, mainly pharmaceuticals, with a total concentration of ~~80 µg L^-1. After treatment, 99 % microcontaminant degradation was reached. As for ecotoxicity reduction, the assayed organisms showed the following sensitivity levels : Tetrahymena thermophila > Daphnia magna > Lactuca sativa > Spirodela polyrhiza ~~ Vibrio fischeri. The initial effluent showed an inhibitory effect of 40 % for T. thermophila and 20 % for D. magna. After 20 min of photo-Fenton treatment, no toxic effect was observed for T. thermophila and toxicity dropped to 5 % for D. magna. Graphical abstract Ecotoxicity removal by solar photo-Fenton at neutral pH.

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S. Morgana, C. Gambardella, M. Faimali and F. Garaventa
Paracentrotus Lividus and Artemia Sp. : Never Too Old Model Organisms to Give New End-Points
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : rapid tests; test development; behavioural tests

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Abstract : In the last few years it has become increasingly important the contribution of ecotoxicological assays to the environmental monitoring, as a fundamental integration of chemical analyses. In environmental risk assessment, in order to fulfill several regulatory requirements, such as the 3R principles (reduction, refinement and replacement), the development of novel approaches to reduce and eventually substitute the use of vertebrate species results to be paramount. Swimming alteration is one of the most frequently used behavioral responses in aquatic ecotoxicology and its evaluation has proved to be a valuable endpoint in ecotoxicological studies with aquatic organisms. Behavioral responses have proven their usefulness in evidencing impacts of chemicals at environmental concentration that do not necessarily cause mortality; therefore, behavioral endpoints are less invasive than traditional acute tests, but still sensitive and more ecological relevant. In this work, we reported a novel research on the use of swimming behavior of two “old” marine model invertebrates in ecotoxicology, the crustacean Artemia sp. and the echinoderm, Paracentrotus lividus, as sub-lethal endpoint. In detail, we optimized and improved an automatic recording system, namely Swimming Behavioral Recorder system (SBR), by developing i) a new swimming speed alteration test using for the first time sea urchin early stages; ii) a new short-term test based on the evaluation of the swimming speed alteration of Artemia nauplii incubated at 39 °C (± 1) for only 6 hours. Thanks to a modern video-based technology, this study provides novel perspectives and future applications applied to two well-known marine model invertebrates, meeting regulatory and market demands, including the reduction in using vertebrate species and the need for early warning technologies.
See corresponding Poster

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B.M. Svensson
Methodology for Evaluation of Hazards from Solid Waste and Landfill Generated Leachate
PhD Thesis 2008
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : landfill leachates; solid waste leachates

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Abstract : A methodology based on an analytical protocol for evaluation of hazards from landfill leachate and solid waste is described. A dynamic analytical protocol, the LAQUA protocol, including measurements of inorganic and water-quality parameters, polar and non-polar organic marker compounds, and toxicity, was constructed. An acute toxicity test, using the brackish water crustacean Artemia salina as test organism, was developed. The methodology was applied to authentic problems such as investigation of different treatment techniques for landfill leachate, evaluation of leaching tests and characterization of solid wastes, and an investigation of a filter material aimed for leachate treatment. Investigated treatment methods comprised in all cases pre-treatment by aeration combined with sedimentation, followed by one of : bioremediation, ozonation, chemical oxidation by Fenton’s reagent, or geo-bed filters. Evaluated filter materials were mixtures of natural or residual waste products. A combination of pre-treatment followed by a geo-bed filter containing a mixture of peat and carbon-containing ash gave an efficient simultaneous removal of metals and organic pollutants.
The performance of two leaching tests for characterization of solid waste, the up-flow percolation test (SIS-CEN/TS 14405:2004) and the batch test (SS-EN 12457 -3), was investigated. Solid waste materials (sludge from street gutters and fragmented metallic waste) were characterized using leaching tests and the hazards of the materials were evaluated in the eluate, obtained at specific liquid-to-solid ratios (L/S). The L/S 2 and L/S 10 values were compared with limit values included in the waste acceptance criteria (WAC). The analyses were extended towards specific organic compounds, such as individual phenolic compounds and polychlorinated biphenyls (PCB). Organic compounds were found in eluates from both types of tests, showing the possibility to use these methods to evaluate the leaching of such compounds from waste materials. The use of authentic leachate as leachant, leads to increased concentrations of heavy metals in the eluate, compared to the prescribed use of demineralised water as leachant. Generally good agreement was found between the results of the two leaching methods. A strategy based on batch tests is described for investigation of a filter material for leachate treatment. Batch tests gave suitable information about the leaching from new and used material, and showed high removal efficiencies of metals and non-polar organic compounds. However, for investigation of removal of polar organic markers (e.g. phenolic compounds) a batch test is not sufficient and should be supplemented by a column test.

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G. Amariei, K. Boltes and P. Letón
Behavior of Cerium Oxide Nanoparticles in Presence of Pharmaceuticals Compounds on Aquatic Specimens
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Algaltoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - nanoparticles; nanoparticles; chemicals - drugs; chemicals - metals

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Abstract : Nanoparticles, in particular metal oxide nanoparticles, have found extensive usage in a wide range of services and industries. Subsequently, they can be released into environment and finally end up in water bodies. That may suppose a potential risk to aquatic environment, exerting toxic effects at the level of cells, tissues or the whole organisms. The present study, evaluate the toxicity behavior of cerium oxide nanoparticles (CeO 2 NPs) on three aquatic specimens- algae Selenastrum capricornutum, bacteria Vibrio fischeri, and activated sludge, by exploring concentration-dependent effect and changes induced due to the presence of Ibuprofen (Ibu) or Levofloxacin (Levo). 72h algae growth-inhibition, marine bacterialuminescence reduction and 24h sludge enzymatic activity and oxidative stress were used as endpoints. Nanoparticles concentration ranged from 0.6 to 160 mg L^-1. The particle size and the ζ-potential of NPs in the culture media were measured to analyze the relation between stability profile and the observed toxicity behavior. The obtained results reveal toxic effects of CeO 2 nanoparticles leading to growth inhibition in algae. The presence of Ibu did not produced significant changes, while Levo showed drastic negative effect in algae growth. Short-term exposure produced significant reduction of luminescence intensity in marine bacteria. The presence of both, Ibu or Levo, reduced the negative effects of single nanoparticles in Vibrio fisheri. Exposure produced significant oxidative stress in bacteria forming activated sludge, with lower damage to enzymatic activity. The presence of pharmaceuticals compounds did not produced significant changes on nanoparticles behavior for activated sludge. Results indicated that algae was more strongly affected than the marine bacteria and activated sludge, respectively. These can be attributed to the culture media and organisms structural characteristics, respectively.
See corresponding Poster

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P. Alvarenga, D. Rodrigues, C. Mourinha, P. Palma, N. Cruz, L.A. Tarelho and S. Rodrigues
Chemical and Ecotoxicological Effects of the Use of Residues from the Pulp and Paper Industry for the Remediation of Soils Degraded by Mining Activities
SETAC Europe 28th Annual Meeting - 13-17 May 2018 - Rome, Italy
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Thamnocephalus platyurus
Topics : pulp and paper; sludges; solid wastes; composts; soils

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Abstract : Biomass ash and biological sludge, both residues from the pulp and paper industry, in different mixture formulations, with and without the application of mixed municipal solid waste compost (MSWC), were used to improve the quality of soils affected by mining activities (Aljustrel mine, Iberian Pyrite Belt). The experiments comprised an incubation experiment, to allow the selection of the application doses, and a pot experiment, with Agrostis tenuis Sibth, to evaluate the possibility of establishing a plant cover in the amended soils. The effects on soil quality were assessed evaluating: (i) soil chemical properties, (ii) plant growth, (iii) immobilization of metals in the soil; (iv) the effects of the amendments on soil eluates toxicity, using organisms from different trophic levels: luminescence inhibition of Vibrio fischeri; 24-h mortality test with Thamnocephalus platyurus; 72-hours population growth of the green microalgae Pseudokirchneriella subcapitata and Daphnia magna acute immobilization test; and (v) soil dehydrogenase activity. Contrary to non-amended control pots, it was possible to establish a plant cover with A. tenuis in pots where correctives were applied, but with some variability between replicates. Phytotoxicity was observed in some of the pots, which compromised plant analysis. The improvement in soil quality, in comparison to non-amended soils (controls) was further evidenced by the increase in the activity of dehydrogenase. The amendments were also able to correct soil acidity, and to increase extractable P and K. However, a significant increase in the organic matter, and N content, was only possible by the simultaneous application of MSWC. Total Cd, Cr, Cu and Zn concentrations in soil decreased in the higher application rates of the correctives, due to a dilution effect. In general, amending the soil with biomass ash and biological sludge decreased the toxicity of soil eluates towards the organisms used. The formulation with 30% of biological sludge (applied in 2.5, 5.0 and 10%, w/w, dry matter), presented a better performance, although inducing a slight toxic effect in the microalgae. Concerning the amendment with MSWC, and despite its beneficial chemical effects, toxicological results did not reflect this improvement, since the presence of MSWC did not promote the decrease of toxicity towards the microalgae. Further research is needed with different plants species, since Agrostis tenuis showed some phytotoxicological response.

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N. Szczerbiñska and M. Galczynska
Biological Methods used to assess Surface Water Quality
Archives of Polish Fisheries 2015 23 185-196
Toxkits : All Toxkits
Test Species :
Topics : review

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Abstract : In accordance with the guidelines of the Water Framework Directive 2000/60 (WFD), both ecological and chemical statuses determine the assessment of surface waters. The profile of ecological status is based on the analysis of various biological components, and physicochemical and hydromorphological indicators complement this assessment. The aim of this article is to present the biological methods used in the assessment of water status with a special focus on bioassay, as well as to provide a review of methods of monitoring water status. Biological test methods include both biomonitoring and bioanalytics. Water biomonitoring is used to assess and forecast the status of water. These studies aim to collect data on water pollution and forecast its impact. Biomonitoring uses organisms which are characterized by particular vulnerability to contaminants. Bioindicator organisms are algae, fungi, bacteria, larval invertebrates, cyanobacteria, macroinvertebrates, and fish. Bioanalytics is based on the receptors of contaminants that can be biologically active substances. In bioanalytics, biosensors such as viruses, bacteria, antibodies, enzymes, and biotests are used to assess degrees of pollution.

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V. Aruoja, S. Pokhrel, M. Sihtmae, M. Mortimer, L. Madler and A. Kahru
Toxicity of 12 Metal-Based Nanoparticles to Algae, Bacteria and Protozoa
Environmental Science : Nano 2015 6 630-644
Toxkits : Algaltoxkit F^TM; Protoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila
Topics : chemicals-nanoparticles; chemicals-metals; nanoparticles

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Abstract : The use of metal based nanoparticles (NPs) is increasing which leads to their release in water bodies via various waste streams and warrants risk assessment. Consistent biological effect data on NPs for environmentally relevant test species that are accompanied by thorough characterization of NPs are scarce but indispensable for understanding possible risks of NPs. We composed and tested a library of 12 metal-based nanoparticles (Al2O3, Co3O4, CuO, Fe3O4, MgO, Mn3O4, Sb2O3, SiO2, ZnO, TiO2, WO3 and Pd) using alga Pseudokirchneriella subcapitata, three bacterial species (Vibrio fischeri, Escherichia coli, Staphylococcus aureus) and protozoan Tetrahymena thermophila. The NPs were characterized for physico-chemical properties, solubility and abiotic reactive oxygen species (ROS) production. Also, respective soluble salts were analyzed for toxic effects. The algal growth inhibition assay proved the most sensitive and yielded EC50 values for 10 NPs ranging from 0.1 to 58 mg/l^-1. Algal toxicity correlated with abiotic ROS production of NPs and the majority of NPs formed agglomerates that entrapped algal cells. Despite of different sensitivity, there was a common trend in the toxicity of NPs across different species and test formats : CuO and ZnO had highest toxicity (EC50 values below 1 mg/l^-1) to all organism groups except protozoa. The high toxicity was mostly due to shedding of toxic concentrations of Zn and Cu ions; for most of the test species Al2O3, SiO2, WO3 and Sb2O3 were not toxic below 100 mg/l^-1 and MgO showed no adverse effect below 100 mg/l^-1 to any test species in any test setting.

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V. Gonzalez, D.A.L. Vignati, M-N. Pons, E. Montarges-Pelletier, C. Bojic and L. Giamberini
Lanthanide ecotoxicity : First Attempt to Measure Environmental Risk for Aquatic Organisms
Environmental Pollution 2015 199 139-147
Toxkits : Ostracodtoxkit F^TM; Rotoxkit F^TM
Test Species : Brachionus calyciflorus; Heterocypris incongruens
Topics : chemicals-inorganic compounds

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Abstract : The geochemical cycles of lanthanides are being disrupted by increasing global production and human use, but their ecotoxicity is not fully characterized. In this study, the sensitivity of Aliivibrio fischeri and Pseudokirchneriella subcapitata to lanthanides increased with atomic number, while Daphnia magna, Heterocypris incongruens, Brachionus calyciflorus and Hydra attenuata were equally sensitive to the tested elements. In some cases, a marked decrease in exposure concentrations was observed over test duration and duly considered in calculating effect concentrations and predicted no effect concentrations (PNEC) for hazard and risk assessment. Comparison of PNEC with measured environmental concentrations indicate that, for the present, environmental risks deriving from lanthanides should be limited to some hotspots (e.g., downstream of wastewater treatment plants). However, considering the increasing environmental concentrations of lanthanides, the associated risks could become higher in the future. Ecotoxicological and risk assessment studies, along with monitoring, are required for properly managing these emerging contaminants.

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K. Burga Pérez
Development and Validation of Solid Phase Microbiotests for the Ecotoxicological Assessment of Sediment
HAL - archives-ouvertes.fr 2018
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments, test validation, test sensitivity, toxicity monitoring

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Abstract : The sediments are a compartment of the aquatic environment that tends to accumulate pollutants through the deposition of suspended solids. Sediment contamination can have potentially negative effects on aquatic organisms, so it is necessary to monitor, assess and protect the sediments to ensure the integrity of the structure and functioning of aquatic ecosystems. The ANR-PRECOD DIESE (diagnostic tools for assessing sediment toxicity) has joined efforts of different partners to develop a strategy for tiered environmental risk assessment of contaminated freshwater sediments. In the framework of this project our research was focused on the development and optimization of solid phase microbiotests to assess the ecotoxicity of freshwater sediments. The microbiotests selected in this study were the tests Microtox^® solid phase (MSPA), the algal solid phase test, the LuminoTox solid phase (Lum-SPA) and Ostracodtoxkit F^TM. These biotests are characterized by different test conditions and assessment criteria. We have obtained different responses for each tested sediment. The algal test was the only ones that have not reported sensitivities with all samples tested. Furthermore, we demonstrated that the response of the test Lum-SPA and MSPA were correlated with some geological characteristics of sediments. Finally, we synthesized the responses of three microbiotests using an index of integration allowing to discriminate between the observed sediment ecotoxicity.
Full article (from HAL – archives-ouvertes.fr)

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I.Blinova, A.Lukjanova, M.Muna, H.Vija and A.Kahru
Evaluation of the Potential Hazard of Lanthanides to Freshwater Microcrustaceans
Science of The Total Environment 2018 642 1100-1107
Toxkits : Daphtoxkit F^TM magna; Ostracodtoxkit F^TM; Thamnotoxkit F^TM
Test Species : Daphnia magna; Heterocypris incongruens; Thamnocephalus platyurus
Topics : chemicals-inorganic compounds

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Abstract : The use of lanthanides in different sectors of industry has significantly increased during the last decades. Although the “anthropogenic” anomalies of lanthanides in the soils, surface and ground waters have already been registered, the ecotoxicological effects of these elements and their fate in the environment are still insufficiently investigated. In this study acute and long-term toxicity of selected lanthanides (La, Ce, Pr, Nd and Gd) nitrates to freshwater crustaceans Daphnia magna, Thamnocephalus platyurus and Heterocypris incongruens were studied and critically evaluated.
The data obtained show that (i) due to the methodical nuances the acute toxicity data of lanthanides are not reliable and have doubtful scientific value even for preliminary toxicity screening and thus should not be used for risk assessment; (ii) toxicity of lanthanides in the 21-day D. magna reproduction test was high whereas the mortality of parent daphnids was more sensitive endpoint than reproduction; (iii) the long-term LC50 values for lanthanides varied from 0.3 to 0.5 mg Ln/L and the differences between individual Ln were not statistically significant.
All in all, the results of this study allow us to conclude that the environmental risk assessment of lanthanides should be performed only using long-term toxicity tests. In the environmental risk assessment, lanthanides may be considered as a uniform group of elements with additive mode of action until future investigations will not reveal differences in the ecotoxicity mechanisms of these elements.

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Y.C. Cloete, B.F. Shaddock and A. Nel
The Use of Two Microbiotests to Evaluate the Toxicity of Sediment from Mpumalanga, South Africa
Africa Journals Online 2017 43, 3 409-412
Toxkits : Ostracodtoxkit F^TM; Phytotoxkit
Test Species : Heterocypris incongruens; Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : sediments

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Abstract : Rapid urbanization throughout the world has resulted in numerous ecological and environmental problems. The release of contaminants into the aquatic environment and the subsequent accumulation in sediment is a specific area of concern due to the potential re-release of the contaminants into solution. The responses of two microbiotests designed to evaluate sediment toxicity (the Ostracodtoxkit F and Phytotoxkit test) were compared once exposed to three samples collected in the vicinity of a power station in Mpumalanga, South Africa. Sediment characterization and chemical analyses were conducted in order to determine possible correlations with the expressed results. Where possible, the concentration of chemicals in the sediment was compared to available sediment guidelines. The study showed that whilst the Phytotoxkit test results did not indicate any acute toxicity (< 50% inhibition), the Ostracodtoxkit F test indicated 100% mortality at the upstream site, with increased growth inhibition at the remaining two sites. The concentration of chrome at all three sites exceeded the interim sediment quality guidelines (37 mg/kg), with the sample collected at the upstream site exceeding the probable effect level (90 mg/kg). The findings from this study indicated that the Phytotoxkit and Ostracodtoxkit F test kits are sensitive enough to evaluate sediment toxicity.

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R. Cartlidge, O. Campana, D. Nugegoda and D. Wlodkowic
Optofluidic Technology for Monitoring Rotifer Brachionus calyciflorus Responses to Regular Light Pulses
Conference Paper - SPIE BioPhotonics Australasia 2016 10013
Toxkits : Rotoxkit F^TM
Test Species : Brachionus calyciflorus
Topics : behavioural tests; test development; rapid tests

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Abstract : Behavioural alterations can occur as a result of a toxicant exposure at concentrations significantly lower than lethal effects that are commonly measured in acute toxicity testing. The use of alternating light and dark photoperiods to test phototactic responses of aquatic invertebrates in the presence of environmental contaminants provides an attractive analytical avenue. Quantification of phototactic responses represents a sublethal endpoint that can be employed as an early warning signal. Despite the benefits associated with the assessment of these endpoints, there is currently a lack of automated and miniaturized bioanalytical technologies to implement the development of toxicity testing with small aquatic species. In this study we present a proof-of-concept microfluidic Lab-on-a-Chip (LOC) platform for the assessment of rotifer swimming behavior in the presence of the toxicant copper sulfate. The device was designed to assess impact of toxicants at sub-lethal concentrations on freshwater crustacean Brachionus calyciflorus, testing behavioral endpoints such as animal swimming distance, speed and acceleration. The LOC device presented in this work enabled straightforward caging of microscopic crustaceans as well as non-invasive analysis of rapidly swimming animals in a focal plane of a video-microscopy system. The chip-based technology was fabricated using a new photolithography method that enabled formation of thick photoresist layers with minimal distortion. Photoresist molds were then employed for replica molding of LOC devices with poly(dimethylsiloxane) (PDMS) elastomer. The complete bioanalytical system consisted of: (i) microfluidic PDMS chip-based device; (ii) peristaltic microperfusion pumping manifold; (iii) miniaturized CMOS camera for video data acquisition; and (iv) video analysis software algorithms for quantification of changes in swimming behaviour of B. calyciflorus in response to reference toxicants.

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R. Cartlidge and D. Wlodkowic
Caging of Planktonic Rotifers in Microfluidic Environment for Sub-Lethal Aquatic Toxicity Tests
Biomicrofluidics 2018 3, 12 (4)
Toxkits : Rotoxkit F^TM
Test Species : Brachionus calyciflorus
Topics : behavioural tests; test development; rapid tests; test sensitivity

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Abstract : Quantification of neuro-behavioural responses of intact small model organisms has been proposed as a sensitive, sub-lethal alternative to conventional toxicity testing. Such bioassays are characterized by a high physiological and ecological relevance, short response times, increased sensitivity, and non-invasive nature. Despite a significant potential for predictive aquatic toxicology analysis of behavioural traits of micro-invertebrates in microfluidic environment has received little attention. In this work, we demonstrate a new Lab-on-a-Chip technology capable of effectively caging freshwater rotifers Brachionus calyciflorus for real-time video-microscopy analysis. We demonstrate that behavioural bioassays performed under microfluidic perfusion can significantly enhance the sensitivity of conventional ecotoxicology test protocols.

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J. Zaltauskaite and Vaisiunaite
Evaluation of Municipal Effluent Toxicity Using Higher Plants and Invertebrates
Environmental Research, Engineering and Management 2010 3, 53 17-23
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : effluents, waste waters

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Abstract : Toxicity of Kaunas municipal effluent was evaluated using bioassays with aquatic invertebrates and terrestrial higher plants. Toxicity tests were performed on samples of both untreated and mechanically and biologically treated wastewater. Wastewater toxicity was assessed using seed germination and short-term early seedling growth tests of lettuce (Lactuca sativa L.) and acute microcrustaceans Thamnocephalus platyurus (24 h) assays. Undiluted untreated wastewater was severely toxic to T. platyurus and led to death of all exposed organisms. Twofold decrease in wastewater concentration in the solution (from 100 % to 50 %) led to sharp transition between extremely toxic to medium toxic to tested organisms. In four and more times diluted wastewater only 10-20 % of the total exposed T. platyurus died. Measured endpoints in higher plants tests were : seed germination, root length, shoot height and total biomass. Raw sewage was slightly toxic (0.6 TU) to the root growth of lettuce, but mechanically and biologically treated effluents exhibited no adverse effects or even started to stimulate the growth. The growth of shoots and total biomass were stimulated due to exposure to effluents. Stimulation effect was determined by sufficiently high concentrations of nutrients.

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A.V. Terekhova, K. Wadhia, E.V. Fedoseeva and P.V. Uchanov
Bioassay Standardization Issues in Freshwater Ecosystem Assessment : Test Cultures and Test Conditions
Knowledge and Management of Aquatic Ecosystem 2018 419 32
Toxkits : All Toxkits
Test Species :
Topics : review, surface waters, test selection, test sensitivity

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Abstract : The use of bioassays for environment contamination assessment, in particular for aquatic ecosystems, has gained topicality and found extensive applications over the past decades. The methodologies are well established, but the standardization of bioassay conditions needs to be better defined in order to assure applicability as an efficient analytic tool and that results are ecologically relevant. This paper addresses the analysis of the current international situation and the specifics of Russian practice in culturing and use of test organisms for freshwater bioassays. Standardization of bioassays calls for the utilization of pertinent sources of test cultures and the provision of appropriate cultivation conditions. The paper discusses and reviews recommendations for the selection of test species and their culturing in the context of established ecotoxicological targets and the assessment of sensitivity to various reference toxicants. The significance of the quality of water utilized for test cultures and sample dilution is highlighted. Strict water quality requirements and synthetic media options are analyzed and due consideration is given to temperature and illumination conditions. Toxkit microbiotests are discussed as an alternative to toxicity bioassays alleviating the need for continuous stock test culturing and maintenance.
Full article (from open access periodical : Knowledge & Management of Aquatic Ecosystems - KMAE)

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E.P.C. Lai
Bioassay Kits for Ecotoxicological Testing of Wastewaters with Nanoparticles
Biochemistry & Analytical Biochemistry 2013 2, 3 1-2
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : waste waters, rapid tests; behavioural tests

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Abstract : Evaluation of complex wastewaters should include acute/chronic ecotoxicological tests and the potential for endocrine disruption to complement the physicochemical characterization. The advantages of bioassay (with a bacterium, alga or crustacean) are simplicity, speed and low cost for monitoring toxicity over time. Its strongest attribute is usefulness as a primary screening test for a broad spectrum of toxicants. Several bioassay kits are commercially available, including the Lux-fluoro, Polytox, Microtox and Thamnotoxkit that require no specialized equipment. A combined approach using instrumental methods for chemical analysis and bioassays for ecotoxicological testing would be extremely important to hazard/risk assessment of wastewater treatment plant discharges.
Full article (from open access periodical : Biochemistry & Analytical Biochemistry)

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J. Antonkiewicz, A. Baran, R. Pelka, A. Wisla-Swider, E. Nowak and P. Konieczka
A Mixture of Cellulose Production Waste with Municipal Sewage as New Material for an Ecological Management of Wastes
Ecotoxicology and Environmental Safety 2019 169 607-614
Toxkits : Phytotoxkit
Test Species : Lepidium sativum; Sinapis alba; Sorgum saccharatum
Topics : waste waters, solid wastes

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Abstract : Chemical contaminants present in cellulose production waste and municipal sewage sludge condition the necessity to treat these wastes before they are introduced into the environment. Environmental use of the these wastes appears to be justified owing to the content of organic substances as well as the considerable fertilizing value. The aim of the study was to assess chemical composition and ecotoxicity of cellulose production waste and municipal sewage sludge in terms of their eco-friendly application. The assessment was done based on the established soil environment according to regulations in Poland. The research on the chemical composition of the wastes was conducted under laboratory conditions, determining the content of macroelements, microelements and heavy metals with respect to using these wastes in the environment. Phytotoxkit and Microtox tests were performed in order to determine the ecotoxicity of the wastes in the aspect of their environmental use. It was established that, compared to the cellulose production waste, the municipal sewage sludge used in the incubation experiment had a higher content of macroelements and microelements. The content of heavy metals in the waste did not exceed the permissible content that conditions their use in agriculture and reclamation.
Based on the results of bioassays it was established that the cellulose production waste was toxic, whereas the combination of cellulose production waste and sewage sludge was found to be of low toxicity to test organisms.
The applied wastes and their mixtures were ranked according to phytotoxicity in the following order: cellulose production waste> cellulose production waste +soil> cellulose production waste +sewage sludge. The research results indicate that waste mixtures qualify for environmental use. With respect to the variable chemical composition of cellulose production waste and municipal sewage sludge as well as their ecotoxicity, it was established that each batch intended for environmental use must be subjected to chemical analysis and ecotoxicological tests.

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J.W. Kim, H. Ishibashi, R. Yamauchi, N. Ichikawa, Y. Takao, M. Hirano, M. Koga and K. Arizono
Acute toxicity of Pharmaceutical and Personal Care Products on Freshwater Crustacean (Thamnocephalus platyurus) and Fish (Oryzias latipes)
The Journal of Toxicological Sciences 2009 34, 2 227-232
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : chemicals–drugs

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Abstract : Pharmaceutical and personal care products (PPCPs) enter aquatic environments via sewage treatment facilities and their potentially toxic effects on biota, particularly aquatic organisms, are of considerable concern. In this study, we investigated the acute toxicity of selected PPCPs on a freshwater crustacean (Thamnocephalus platyurus) and a fish species (Oryzias latipes). The 24-hr median lethal concentration (LC50) values of ibuprofen, mefenamic acid, indometacin, carbamazepine, propranolol, ifenprodil, clarithromycin and triclosan for T. platyurus were estimated to be 19.59, 3.95, 16.14, > 100, 10.31, 4.43, 94.23 and 0.47 mg/l respectively. Conversely, the 96-hr LC50 values for these PPCPs were estimated at > 100, 8.04, 81.92, 45.87, 11.40, 8.71, > 100 and 0.60 mg/l for O. latipes, respectively. The toxic sensitivity of T. platyurus to these PPCPs, except for carbamazepine, was therefore higher than for O. latipes. No acute toxicity effects were associated with PPCPs, such as atenolol, disopyramide, famotidine, fluconazole, erythromycin and levofloxacin, in the two aquatic organisms at the concentrations tested in this study (> 100 mg/l). These findings may help us to understand the potential biological effects and risks associated with PPCP exposure in aquatic organisms. Further long-term studies are required to fully assess the growth and reproduction of these compounds on aquatic biota.

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M. Renzi, P. Fastelli, E. Grazioli and A. Blaskovic
Ecotoxicological Answers in D. magna due to the Exposure to Microplastics and Surfactants
8th Biannual ECOtoxicology MEeting (BECOME 2018), 26-29 November 2018, Livorno, Italy - Book of Abstracts
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - surfactants; chemicals - plastics, chemicals - microplastics

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Abstract : This study uses as a model species D. magna (Cladocera) to evaluate the ecotoxicological effects recorded daily in the period 24-96 h and induced by exposure to irregular microplastics (0.05 g/L; PP, PE, PVC), surfactants (0.001%, Triton X-100) and their mixtures (microplastic + surfactant). The experimental exposure doses were defined on the basis of the results of a preliminary screening. The tests were conducted both in conditions of fasting (standard exposure according to the OECD method), and feeding the animals with Spirulina every 24 hours for the duration of the exposure.
The results show that the animals actively ingest microplastics in both experimental conditions (fasting, feeding), and remain easily trapped in the microplastic aggregates forming clusters. Furthermore, they show that: i) all tested substances and mixtures have an increasing toxicological effect over time; ii) immobilization is a more sensitive endpoint than mortality; iii) the highest toxicity is observed for the PVC + Triton X-100 mixture; iv) tests carried out by feeding the exposed animals showed survival rates of controls at 96 h higher than 90% allowing greater discrimination of the toxic effect of substances and mixtures both after 24 and after 96 hours of exposure.

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M. Carere, I. Lacchetti, W. Cristiano, S. Caciolli, F. Marcon, R. Crebelli, C. Andreoli, L. Conti, A. Antoccia, G. Frenzilli, P. Guidi, A. Buschini, G. Gorbi, A. Suppa, O. Serra, P. Venier and L. Mancini
Application of an Eco-Genotoxicology Project for the Assessment of the Quality of the Urban Stretch of the River Tiber
8th Biannual ECOtoxicology MEeting (BECOME 2018), 26-29 November 2018, Livorno, Italy - Book of Abstracts
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : surface water

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Abstract : The application of ecotoxicological tests for the evaluation of the quality of aquatic ecosystems is increasingly necessary considering the complexity of environmental chemical pollution due to the presence of thousands of chemicals released, discharged and emitted from sources of different nature (agricultural, urban, industrial). This need is also considered at present as part of the implementation strategy of the Water Framework Directive in which ecotoxicological tests (biomarkers, in vitro and in vivo bioassays) are recommended to support the chemical analysis for monitoring surface water bodies. The ecotoxicological tests are usefully implemented by the eco-genotoxicological assays, based on the assessment of genotoxicity biomarkers able to detect the presence in the environment of agents with genotoxic activity that may indirectly represent a risk to human health.\r\nIn this context, through the collaboration between the Istituto Superiore di Sanità and several Italian Universities, a project was developed for the application of eco-genossicological tests in order to evaluate the quality of the Roman urban stretch of the Tiber River characterized mainly by the presence of pollution sources typical of a large city (e.g. small business discharges, urban wastewater, use of pesticides in urban areas, illegal landfills of waste). Moreover, due to climate change, floods and drought are increasingly frequent in this area, therefore raising the need to use innovative monitoring tools. Sampling sites included a stretch upstream (Farfa) potentially little impacted, a site at the entrance to the city (Castel Giubileo), and a station downstream of the urban sewage treatment plant (Mezzo Cammino). The tests used include genotoxicity tests (e.g. Comet assays and Micronucleus test), as well as tests to detect other effects, such as FET (Fish Embryo Toxicity test) with Zebrafish (embryotoxicity, cardiotoxicity, neurotoxicity), and also identification of teratogenic effects by using Hydra, and acute effects on Daphnia magna and algae.\r\nThe results of this study will contribute to the knowledge of the quality of river Tiber urban stretch, which represents a precious resource considering its different uses (e.g. irrigation) but could potentially pose a risk to human health as well. Moreover, the goal of this study is also the development of a battery of broad-spectrum eco-genotoxic tests that can be used in similar contexts and recommended for the implementation of national and European legislations.

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A. Cabrera-Reina, S. Miralles-Cuevas, G. Rivas and J.A. Sánchez Pérez
Comparison of Different Detoxification Pilot Plants for the Treatment of Industrial Wastewater by Solar photo-Fenton : Are Raceway Pond Reactors a Feasible Option ?
Science of The Total Environment 2019 648 601-608
Toxkits : Protoxkit F^TM
Test Species : Tetrahymena thermophila
Topics : waste waters; effluents

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Abstract : This paper represents a first approach in the study of photoreactor selection to treat industrial wastewater using solar photo-Fenton. In this context, simulated textile industry effluent containing a mixture of four dyes at different initial dissolved organic carbon (DOC) concentrations (45, 90, 180 and 270 mg/L) was treated by using three different solar reactor geometries : (i) tubular (5 cm diameter) provided with compound parabolic collector (ii) tubular (5 cm diameter) provided with flat collector and (iii) open channels forming raceway ponds with two liquid depths (5 and 15 cm). For comparison purposes, mineralization percentages over 75% and chronic toxicity reduction were set as treatment goals. Regardless of the initial DOC concentration, negligible differences in terms of treatment time and hydrogen peroxide consumptions were found between the flat collector and compound parabolic collector photoreactors. Conversely, the treatment in the raceway pond reactors always resulted in higher values. In spite of this, when the photoreactors were compared in terms of treatment capacity (mg of DOC removed/m² min) the raceway pond reactor at 15 cm of liquid depth presented the best results, with values as much as two or three times higher than those of the tubular reactors, except for the wastewater with 270 mg of DOC/L for which the raceway pond reactor at 5 cm liquid depth became the best option. When the treatment capacity is modified to include the photoreactor investment (mg of DOC removed/€ m²) the differences between the raceway pond reactor at both liquid depths and the photoreactors with solar collectors increased by as much as two orders of magnitude, which demonstrates the potential application of the former for the treatment of industrial wastewater by solar photo-Fenton.

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I. Blinova, J. Niskanen, P. Kajankari, L. Kanarbik, A. Käkinen, H. Tenhu, O.P. Penttinen and A. Kahru
Toxicity of Two Types of Silver Nanoparticles to Aquatic Crustaceans Daphnia magna and Thamnocephalus platyurus
Environmental Science Pollution Research International 2013 20, 5 3456-3463
Toxkits : Daphtoxkit F^TM magna; Thamnotoxkit F^TM
Test Species : Daphnia magna; Thamnocephalus platyurus
Topics : nanoparticles

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Abstract : Although silver nanoparticles (NPs) are increasingly used in various consumer products and produced in industrial scale, information on harmful effects of nanosilver to environmentally relevant organisms is still scarce. This paper studies the adverse effects of silver NPs to two aquatic crustaceans, Daphnia magna and Thamnocephalus platyurus. For that, silver NPs were synthesized where Ag is covalently attached to poly(vinylpyrrolidone) (PVP). In parallel, the toxicity of collargol (protein-coated nanosilver) and AgNO3 was analyzed. Both types of silver NPs were highly toxic to both crustaceans : the EC50 values in artificial freshwater were 15-17 ppb for D. magna and 20-27 ppb for T. platyurus. The natural water (five different waters with dissolved organic carbon from 5 to 35 mg C/L were studied) mitigated the toxic effect of studied silver compounds up to 8-fold compared with artificial freshwater. The toxicity of silver NPs in all test media was up to 10-fold lower than that of soluble silver salt, AgNO3. The pattern of the toxic response of both crustacean species to the silver compounds was almost similar in artificial freshwater and in natural waters. The chronic 21-day toxicity of silver NPs to D. magna in natural water was at the part-per-billion level, and adult mortality was more sensitive toxicity test endpoint than the reproduction (the number of offspring per adult).

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N.Y. Stepanova, O.V. Nikitin, V.Z. Latypova, I.B. Vybornova, G.S. Galieva and R.V. Okunev
Recovery of Ostracod with Known Ages in Differently Textured Sediments and Comparison of Toxicity of Heavily Contaminated Sediments with Ostracod Heterocypris incongruens and Amphipod Hyalella azteca
IOP Conference Series : Earth and Environmental Science - Conference 1 - Book of Abstracts 2018 107
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : sediments; test sensitivity

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Abstract : The recovery of 1-, 4-, 6,-, and 8-d-old ostracods (Heterocypris incongruens) from sediments with different texture has been evaluated. The recovery of ostracods at all ages has been in agreement with the acceptability criterion of 80% of survival for sediment tests. The recovery of ostracods has turned out to be equal to or more than 80% for sand and silt sediments, respectively. The comparison of survival rates between ostracods and amphipods has shown good convergence in the tests of heavily contaminated sediments (R²=0.75, p<0.05). The sediment quality criteria (TEC) have been exceeded mostly for total petroleum hydrocarbons (100% samples), Cr (100%), Ni (87%), Cu (87%), Pb (47%), and Cd (53%). The content of acid volatile sulfides (AVS) has been significantly higher than that of simultaneously extracted metals (SEM). The obtained results have indicated that, metals (Cu, Zn, Cd, Ni, and Pb) are non-bioavailable. Only one sample has exceeded TEC for PAHs (dibenz[a,h]anthracene). It was observed that, no significant correlation between the effect of toxicity and the chemical content.

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A. Jemec, A. Kahru, A. Potthoff, D. Drobne, M. Heinlaan, S. Böhme, M. Geppert, S. Novak, K. Schirmer, R. Rekulapally, S. Singh, V. Aruoja, M. Sihtmäe, K. Juganson, A. Käkinen and D. Kühnel
An Interlaboratory Comparison of Nanosilver Characterisation and Hazard Identification : Harmonising Techniques for High Quality Data
Environment International 2016 87 20-32
Toxkits : Algaltoxkit F^TM; Artoxkit M^TM; Daphtoxkit F^TM magna; Protoxkit F^TM
Test Species : Artemia (salina) franciscana; Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila
Topics : chemicals - nanoparticles; interlaboratory testing

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Abstract : Within the FP7 EU project NanoValid a consortium of six partners jointly investigated the hazard of silver nanoparticles (AgNPs) paying special attention to methodical aspects that are important for providing high-quality ecotoxicity data. Laboratories were supplied with the same original stock dispersion of AgNPs. All partners applied a harmonised procedure for storage and preparation of toxicity test suspensions. Altogether ten different toxicity assays with a range of environmentally relevant test species from different trophic levels were conducted in parallel to AgNP characterisation in the respective test media. The paper presents a comprehensive dataset of toxicity values and AgNP characteristics like hydrodynamic sizes of AgNP agglomerates and the share (%) of Ag(+)-species (the concentration of Ag(+)-species in relation to the total measured concentration of Ag). The studied AgNP preparation (20.4+6.8 nm primary size, mean total Ag concentration 41.14 mg/L, 46-68% of soluble Ag(+)-species in stock, 123.8+12.2 nm mean z-average value in dH2O) showed extreme toxicity to crustaceans Daphnia magna, algae Pseudokirchneriella subcapitata and zebrafish Danio rerio embryos (EC50<0.01 mg total Ag/L), was very toxic in the in vitro assay with rainbow trout Oncorhynchus mykiss gut cells (EC50 : 0.01-1 mg total Ag/L); toxic to bacteria Vibrio fischeri, protozoa Tetrahymena thermophila (EC50 : 1-10 mg total Ag/L) and harmful to marine crustaceans Artemia franciscana (EC50 : 10-100 mg total Ag/L). Along with AgNPs, also the toxicity of AgNO3 was analyzed. The toxicity data revealed the same hazard ranking for AgNPs and AgNO3 (i.e. the EC50 values were in the same order of magnitude) proving the importance of soluble Ag(+)-species analysis for predicting the hazard of AgNPs. The study clearly points to the need for harmonised procedures for the characterisation of NMs. Harmonised procedures should consider : (i) measuring the AgNP properties like hydrodynamic size and metal ions species in each toxicity test medium at a range of concentrations, and (ii) including soluble metal salt control both in toxicity testing as well as in Ag(+)-species measurements. The present study is among the first nanomaterial interlaboratory comparison studies with the aim to improve the hazard identification testing protocols.

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O.M. Bondarenko, M. Heinlaan, M. Sihtmäe, A. Ivask, I. Kurvet, E. Joonas, A. Jemec, M. Mannerström, T. Heinonen, R. Rekulapelly, S. Singh, J. Zou, I. Pyykkö, D. Drobne and A. Kahru
Multilaboratory Evaluation of 15 Bioassays for (Eco)Toxicity Screening and Hazard Ranking of Engineered Nanomaterials : FP7 Project NANOVALID
Nanotoxicology 2016 10, 9 1229-1242
Toxkits : Algaltoxkit F^TM; Daphtoxkit F^TM magna
Test Species : Daphnia magna; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : chemicals - nanoparticles; interlaboratory testing

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Abstract : Within EU FP7 project NANOVALID, the (eco)toxicity of 7 well-characterized engineered nanomaterials (NMs) was evaluated by 15 bioassays in 4 laboratories. The highest tested nominal concentration of NMs was 100 mg/l. The panel of the bioassays yielded the following toxicity order : Ag > ZnO > CuO > TiO > MWCNTs > SiO2 > Au. Ag, ZnO and CuO proved very toxic in the majority of assays, assumingly due to dissolution. The latter was supported by the parallel analysis of the toxicity of respective soluble metal salts. The most sensitive tests/species were Daphnia magna (towards Ag NMs, 24-h EC50 = 0.003 mg Ag/l), algae Raphidocelis subcapitata (ZnO and CuO, 72-h EC50 = 0.14 mg Zn/l and 0.7 mg Cu/l, respectively) and murine fibroblasts BALB/3T3 (CuO, 48-h EC50 = 0.7 mg Cu/l). MWCNTs showed toxicity only towards rat alveolar macrophages (EC50 = 15.3 mg/l) assumingly due to high aspect ratio and TiO2 towards R. subcapitata (EC50 = 6.8 mg Ti/l) due to agglomeration of TiO2 and entrapment of algal cells. Finally, we constructed a decision tree to select the bioassays for hazard ranking of NMs. For NM testing, we recommend a multitrophic suite of 4 in vitro (eco)toxicity assays: 48-h D. magna immobilization (OECD202), 72-h R. subcapitata growth inhibition (OECD201), 30-min Vibrio fischeri bioluminescence inhibition (ISO2010) and 48-h murine fibroblast BALB/3T3 neutral red uptake in vitro (OECD129) representing crustaceans, algae, bacteria and mammalian cells, respectively. Notably, our results showed that these assays, standardized for toxicity evaluation of regular chemicals, proved efficient also for shortlisting of hazardous NMs. Additional assays are recommended for immunotoxicity evaluation of high aspect ratio NMs (such as MWCNTs).

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L.K. Pandey, I. Lavoie, S. Morin, S. Depuydt, J. Lyu, H. Lee, J. Jung, D.-H. Yeom, T. Han and J. Park
Towards a Multi-Bioassay-Based Index for Toxicity Assessment of Fluvial Waters
Environmental Monitoring and Assessment 2019 191, 112
Toxkits : Algaltoxkit F^TM; Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens; Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum
Topics : toxicity classification; toxicity index; surface waters

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Abstract : Despite their proven reliability for revealing ‘acceptable’ degrees of toxicity in waste- and reclaimed waters, bioassays are rarely used to assess the toxicity of hazardous contaminants present in natural waters. In this study, we used organisms from different trophic levels to assess the toxicity of water samples collected from four different South Korean rivers. The main objective was to develop a multi-descriptor index of toxicity for undiluted river water. The responses of six test organisms (Aliivibrio fischeri, Pseudokirchneriella subcapitata, Heterocypris incongruens, Moina macrocopa, Danio rerio and Lemna minor) after laboratory exposure to water samples were considered for this index, as well as the frequency of teratologies in diatom assemblages. Each individual test was attributed a toxicity class and score (three levels : no toxicity=0, low toxicity=1, confirmed toxicity=2) based on the organism’s response after exposure and a total score was calculated. The proposed index also considers the number of test organisms that received the highest toxicity score (value=2). An overall toxicity category was then attributed to the water sample based on those two metrics : A=no toxicity, B=slight toxicity, C=moderate toxicity; D=toxicity and E=high toxicity. The susceptibility of the test organisms varied greatly and the sensitivity of their response also differed among bioassays. The combined responses of organisms from different trophic levels and with different life strategies provided multi-level diagnostic information about the intensity and the nature of contamination.

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M. Muna, I. Blinova, A. Kahru, I. Vinkovic Vrcek, B. Pem, K. Orupold and M. Heinlaan
Combined Effects of Test Media and Dietary Algae on the Toxicity of CuO and ZnO Nanoparticles to Freshwater Microcrustaceans Daphnia magna and Heterocypris incongruens : Food for Thought
Nanomaterials 2018 9, 1
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : nanoparticles ; chemicals-nanoparticles ; chemicals-metals

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Abstract : The chemical composition of the test medium as well as the presence of algae (microcrustaceans\' food) affects the bioavailability and thus the toxicity of metal nanoparticles (NP) to freshwater microcrustaceans. This study evaluated the effect of the addition of algae (Rapidocelis subcapitata at 7.5 × 10⁶ cells/mL) on the toxicity of CuO (primary size 22-25 nm) and ZnO NP (10-15 nm) to planktic Daphnia magna and benthic Heterocypris incongruens in artificial (mineral) and natural freshwater (lake water). The toxicity of ionic controls, CuSO4 and ZnSO4, was evaluated in parallel. When algae were added and the toxicity was tested in mineral medium, 48 h EC50 of CuO and ZnO NP to D. magna was ~2 mg metal/L and 6-day LC50 of H. incongruens was 1.1 mg metal/L for CuO and 0.36 mg metal/L for ZnO. The addition of algae to D. magna test medium mitigated the toxicity of CuO and ZnO NP 4-11-fold when the test was conducted in natural water but not in the artificial freshwater. The addition of algae mitigated the toxicity of CuSO4 (but not ZnSO4) to D. magna at least 3-fold, whatever the test medium. In the 6-day H. incongruens tests (all exposures included algae), only up to 2-fold differences in metal NP and salt toxicity between mineral and natural test media were observed. To add environmental relevance to NP hazard assessment for the freshwater ecosystem, toxicity tests could be conducted in natural water and organisms could be fed during the exposure.

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A.M. Freitas, G. Rivas, M.C. Campos-Manas, J.L. Casas Lopez, A. Aguera and J.A. Sanchez Perez
Ecotoxicity Evaluation of a WWTP Effluent Treated by Solar Photo-Fenton at Neutral pH in a Raceway Pond Reactor
Environmental Science and Pollution Research International 2017 24, 2 1093-1104
Toxkits : Daphtoxkit F^TM magna; Protoxkit F^TM; Spirodela duckweed Toxkit
Test Species : Daphnia magna; Spirodela polyrhiza; Tetrahymena thermophila
Topics : effluents; waste waters

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Abstract : Some pollutants can be resistant to wastewater treatment, hence becoming a risk to aquatic and terrestrial biota even at the very low concentrations (ng L^-1-µg L^-1) they are commonly found at. Tertiary treatments are used for micropollutant removal but little is known about the ecotoxicity of the treated effluent. In this study, a municipal secondary effluent was treated by a solar photo-Fenton reactor at initial neutral pH in a raceway pond reactor, and ecotoxicity was evaluated before and after micropollutant removal. Thirty-nine micropollutants were identified in the secondary effluent, mainly pharmaceuticals, with a total concentration of ?80 µg L^-1. After treatment, 99 % microcontaminant degradation was reached. As for ecotoxicity reduction, the assayed organisms showed the following sensitivity levels : Tetrahymena thermophila > Daphnia magna > Lactuca sativa > Spirodela polyrhiza ? Vibrio fischeri. The initial effluent showed an inhibitory effect of 40 % for T. thermophila and 20 % for D. magna. After 20 min of photo-Fenton treatment, no toxic effect was observed for T. thermophila and toxicity dropped to 5 % for D. magna. Graphical abstract Ecotoxicity removal by solar photo-Fenton at neutral pH.

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C. Genethliou
Biodegradation of Olive Mill Wastewater Phenolic Compounds in Thermophilic Anaerobic Digestion and Their Toxicity in Digested Effluent
Master's Thesis (University of Patras - School of Natural Sciences - Department of Chemistry) - 2017
Toxkits : Thamnotoxkit F^TM
Test Species : Thamnocephalus platyurus
Topics : waste waters; chemicals - organic compounds

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Abstract : The olive mill wastewater (OMWW) generated from the 3-phase olive oil production process constitutes a crucial environmental issue, especially in Mediterranean areas. It is ranked among the most polluting by-products of the agro-industrial sector, mainly because of its extremely high phenolic content. Phenolic compounds are characterized by intense phytotoxic and antimicrobial properties, thus resulting in poor biodegradation of OMWW. As large amounts of OMWW end-up to rivers or ponds, massive problems may emerge to the entire ecosystem and specifically to the aquatic life. One of the most promising processing methods of this by-product is the biological process of anaerobic digestion, according to which the organics contained in OMWW (phenolic compounds included) are removed via biodegradation. Simultaneously, the produced biogas during the process can be further deployed generating electricity and/or thermal energy.
The aim of this work was to assess the extent of phenolic compounds’ biodegradation during the operation of a high-rate thermophilic (55 °C) anaerobic Upflow Packed Bed Bioreactor (UPBR) fed with twice-centrifuged OMWW, and furthermore, the investigation of raw and anaerobic effluent samples toxicity to freshwater aquatic organisms. The identification of individual phenolics took place both in the un-treated OMWW samples fed to the UPBR and the digested effluents which were collected in different HRTs (Hydraulic Retention Times) under steady state operating conditions. Following the recovery of phenolics from each sample, identification and quantification was performed using High-Performance Liquid Chromatography (HPLC) connected with DAD (Diode-Array Detector) and FLD (Fluorescence Detector). In parallel, the acute toxicity of raw and digested samples, was assessed by performing acute toxicity tests in the fairy shrimp Thamnocephalus platyurus (Thamnotoxkit F^TM screening toxicity test), which is a worldwide recognized freshwater biomarker.
The obtained results indicate that as the HRT was decreased during the anaerobic digestion process, the concentration of the individual phenolic compounds and therefore the concentration of the total phenolic content in the digested effluents was reduced. Subsequently, the toxicity occurred against the fairy shrimp Thamnocephalus platyurus was lower (higher EC50 values) in the digested effluents compared to un-treated samples. The entire biodegradation of 5 biophenols-caffeic acid, syringic acid, o-coumaric acid, oleuropein and 4-ethylphenol- was achieved. Extremely high removals were also obtained by hydroxytyrosol and tyrosol which reached up to 93.70% and 96.47%, respectively.
See full Master’s Thesis

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P. Dvorak, K. Benova and J. Vitek
Alternative Biotest on Artemia franciscana
IntechOpen (open access) 2012
Toxkits : Artoxkit M^TM
Test Species : Artemia (salina) franciscana
Topics : review

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Abstract : Recent toxicology, in accordance with recommendations from the European Council, has demanded decrease in the number of vertebrates used in toxicology testing and their partial replacement with invertebrate animals, plants or even organ, tissue, or cell cultures. During the last 50 years various invertebrate species have been tested for their sensitivity to many chemical or physical agents to prove their possible use for pre-screening tests. To the most valuable organisms available for ecotoxicity testing belong crustaceans of the Artemia genus, commonly known as brine shrimps.

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M.E. Aydin, S. Ozcan and F. Beduk
Acute Toxicity of Organophosphorus Pesticides and Their Degradation By-products to Daphnia magna, Lepidium sativum and Vibrio fischeri
Pesticides - The Impacts of Pesticides Exposure 2011 261-276
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics : chemicals - pesticides

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Abstract : Full article

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R. Hanczaruk
Bioindication of Surface Water Contamination using Daphtoxkit Microbiotest
Research Papers of Wroclaw University of Economics 2017 494 43-49
Toxkits : Daphtoxkit F^TM magna
Test Species : Daphnia magna
Topics :

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Abstract : Acute toxicity bioassays are widely used in the biomonitoring of aquatic ecosys¬tems. An example of bioassay, conform to OECD Guideline 202 and ISO 6341 is the Daphtox¬kit F magna. Crustaceans Daphnia magna are common in freshwater biocenoses, character¬ized by sensitivity to a wide range of environmental contamination and play an important role in food chain. The aim of the study was to evaluate the usefulness of Daphnia magna in the bioindication of cadmium compounds in the surface water. The results show high sensitivity of Daphnia magna to cadmium chloride (48h-LC50 = 0.77 mg/l). Values of lethal concentra¬tion indicate also the very high acute toxicity of cadmium chloride (48h-TU = 130.30). The results of the study pointed to the necessity of regular monitoring of cadmium compounds in aquatic ecosystems and the usefulness of Daphnia magna in their bioindication.

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N.Y. Stepanova, O.V. Nikitin, V.Z. Latypova, I.B. Vybornova, G.S. Galieva and R.V. Okunev
Recovery of Ostracod with Known Ages in Differently Textured Sediments and Comparison of Toxicity of Heavily Contaminated Sediments with Ostracod Heterocypris Incongruens and Amphipod Hyalella Azteca
IOP Conference Series Earth and Environmental Science 2018 107
Toxkits : Ostracodtoxkit F^TM
Test Species : Heterocypris incongruens
Topics : chemicals - metals; chemicals - hydrocarbons; sediments

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Abstract : The recovery of 1-, 4-, 6,-, and 8-d-old ostracods (Heterocypris incongruens) from sediments with different texture has been evaluated. The recovery of ostracods at all ages has been in agreement with the acceptability criterion of 80% of survival for sediment tests. The recovery of ostracods has turned out to be equal to or more than 80% for sand and silt sediments, respectively. The comparison of survival rates between ostracods and amphipods has shown good convergence in the tests of heavily contaminated sediments (R²=0.75, p<0.05). The sediment quality criteria (TEC) have been exceeded mostly for total petroleum hydrocarbons (100% samples), Cr (100%), Ni (87%), Cu (87%), Pb (47%), and Cd (53%). The content of acid volatile sulfides (AVS) has been significantly higher than that of simultaneously extracted metals (SEM). The obtained results have indicated that, metals (Cu, Zn, Cd, Ni, and Pb) are non-bioavailable. Only one sample has exceeded TEC for PAHs (dibenz[a,h]anthracene). It was observed that, no significant correlation between the effect of toxicity and the chemical content.

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P. Uchanov
Toxicity of the Lead Anions to Plant Growth in Model Soil Conditions
6th YOUNG ENVIRONMENTAL SCIENTISTS MEETING - 16 - 20 February 2017 - Stockholm University, Sweden 2017
Toxkits : Phytotoxkit
Test Species :
Topics : chemicals - metals

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Abstract : A literature review on the role of anions of heavy metal salts in modifying the deleterious effects of pollutants reveals imprecise conclusions. Some authors indicate a clear relationship between toxicity and lead salts composed of different anions, the indication from others is one of inaccurate distinctions, for example, reference to cytotoxicity and plant growth characteristics. The toxicity of metals is dependent on variety of environmental factors. To minimize the influence of external factors for the assessment of effects of heavy metals (particularly concerning plants) can be controlled in the laboratory experiments using standard artificial soil or soil extract. The purpose of this work was to compare the effect of different lead salts on the growth characteristics of plants using Phytotoxkit. The aim was try and quantify the median effective concentration of lead cations (Pb2+) in reference to specific salts tested - nitrate, chloride or acetate. Toxicity of lead salts was investigated on germination and development of plant seeds of monocots Avena sativa and dicots Raphanus sativus. Toxicity was assessed using substrate and eluate methods employing Microbiotest Phytotoxkit disposable plates. The substrate method entailed direct contact of seeds with the soil, whereas in the eluate method seeds were exposed to water extract of soil samples treated to varying degrees of lead salts. The soil used as artificial soil was prepared in accordance with international standard ISO 11268. Lead salts tested were in the range 50-2500 mg/kg. Plant responses observed showed that: at lower concentrations up to 300 mg / kg growth of the vegetative part of the plant was enhanced whereas the root system was suppressed at these levels. Seedlings of Avena sativa exhibited significantly less inhibition to the toxicant concentration range tested than the seedlings of Raphanus sativus. The greatest inhibition of root growth was evident in the treatment using lead acetate (this was attributed to decrease in pH to 3-4) - at 1300 mg / kg. At this maximum inhibition with lead nitrate, in the case of soil extract (eluate method) inhibition was only 60%; and lead chloride - 30%. Thus, in the study of the toxicity of lead salts both with the eluate and substrate methods it was shown that the degree of growth inhibition of plant seedlings grown from seed depends on the nature of the anion. In particular, it was evident that lead nitrate was less toxic than the lead acetate.

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R. Rico-Martinez, M.A. Arzate-Cardenas, D. Robles-Vargas, I.A. Perez-Legaspi, A.-F. Jesus and G.E. Santos-Medrano
Rotifers as Models in Toxicity Screening of Chemicals and Environmental Samples
IntechOpen 2016 4 57-99
Toxkits :
Test Species : Brachionus calyciflorus; Brachionus plicatilis
Topics : review

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Abstract : An important objective of aquatic ecotoxicology is to determine the effects of toxic compounds in organisms that play a central role in aquatic communities where rotifers have a large impact on several important ecological processes. The contribution of the rotifers to secondary production in many aquatic communities is substantial as they are often the larger fraction of zooplankton biomass at certain times of the year. In addition to the importance of their ecological roles in aquatic communities, the rotifers are attractive organisms for ecotoxicological studies by its short life cycles and rapid reproduction, their small size, and little volumes needed for culture and toxicity assays. The main end points used in ecotoxicological studies are mortality, reproduction, behavior, and biomarkers. Such parameters are included in international regulations from all over the world, where different species are used to evaluate the effect of environmental samples or chemical compounds. The high diversity of rotifers is an important issue because it can modify their relative susceptibility to toxicants. Thus, more studies are needed to know the relations and mechanisms involved in clonal variation, sensitivity, and development, which can be all assessed by state-of-the-art procedures.

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M. Kondras, D. Czepinska-Kaminska, J. Karczewska and K. Wojewoda
The Influence of two Pesticides in Soils on Selected Plants and Earthworms
Roczniki Gleboznawcze (Natural Science Journal) 2011 2 219-225
Toxkits : Phytotoxkit
Test Species :
Topics : chemicals - pesticides

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Abstract : The paper presents the influence of two pesticides - Dual Gold 960 EC and Pervicur Energy 840 SL on three plants species (white mustard, maize and rape) and one of them - pesticide Dual Gold 960 EC - on the earthworm species Eisenia foetida. The studies indicate that physical and chemical properties of soils significantly influence the toxicity of the analyzed agent on organisms (earthworms and plants). Largely, the percentage content of the particle of < 0,02 mm in diameter may have crucial influence on the activity of a particular pesticide and its toxicity. Thus, procedures should be introduced when permitting the trade of particular biocides on various soil types with different physical and chemical properties, because the analysis of pesticides on artificial substrates with regard to their toxicity is not always reflected in the real influence on soil organisms when applied in natural conditions.

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S. Boteva, M. Lyubenova, Y. Petkov and A. Ch?kalanov
Biomarkers and Kits for Early and Rapid Testing - Current Knowledge and Gaps
15th International Conference on Environmental Science and Technology Rhodes, Greece, 31 August - 2 September 2017
Toxkits : All Toxkits
Test Species :
Topics : toxicity monitoring

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Abstract : The use and development of biomarkers is related to the establishment of hidden toxic effect on molecular, sub-cellular, cellular and tissue level. The development of new biomarkers is very important for successful early diagnosis of toxic effects on the macrobiological systems. The effects on these levels often come from complex impact and are hard to spot until the onset of extinction of the population or degradation of the community. Developing of a novel biomarker is time consuming and in most cases very expensive, but very perspective issue. The other contemporary tool for rapid assessment of toxicity is the biomarker assay kits. They are very often used due to their ease of operation and representativeness of obtained results. The discussed information about early and rapid ecotoxicological testing was obtained by Review Publication screener software using published developments for recent five years. The frequency and characteristics of biomarkers and kits used in the recent studies are examined and statistically processed. The main advantages, disadvantages and trends in developments are outlined.

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J. Mankiewicz-Boczek, S. Szklarek, E. Kiedrzynska, A. Bednarek, L. Serwecinska, A. Font Najera, M. Kiedrzynski and M. Zalewski
Biomonitoring of Threats and Biotechnological Solutions to Improve the Quality of Surface Waters
IX National Bioindication Conference - “Practical use of bioindication systems to assess the quality and toxicity of the environment and substances chemical” 2019
Toxkits : Algaltoxkit F^TM; Protoxkit F^TM; Thamnotoxkit F^TM
Test Species : Pseudokirchneriella subcapitata; Raphidocelis subcapitata; Selenastrum capricornutum; Tetrahymena thermophila; Thamnocephalus platyurus
Topics : surface waters

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Abstract : Classical biomoniotoring of surface waters in the framework of existing legislation (Water Framework Directive 2000/60 / EC and D20161187) is conducted on the basis of an assessment of the composition and abundance of selected organisms, using the developed indexes and indicators, among others, multi- and multimetric. However, in order to assess possible changes in the state of water quality, it is necessary to compare the obtained results from at least two consecutive monitoring periods. This approach does not allow an immediate risk assessment, with an acute or chronic effect on organisms. Therefore, more and more often to biomonitoring are also introduced bioassays (biotests) involving organisms representing the three main trophic groups, through which it is possible to determine the toxicity of the total (whole) sample of water or sludge, in order to immediately assess the threat to the state of the examined ecosystem.
The aim of the present study was to assess water quality in terms of threat for selected organisms : Selenastrum\r\nCapricornutum - producers (Algaltoxkit test, chronic toxicity), Thamnocephalus platyurus - consumers (Thamnotoxkit test, acute toxicity) and Tetrahymena thermophila - destruents (Protoxkit test, chronic toxicity).
The analyzed samples came from two sources, i.e. 1) municipal wastewater treatment plant - treated wastewater was taken directly to the Pilica river and 2) Pilica river water samples. Samples were collected on six dates in 2017 and 2018. The sensitivity of the above-mentioned organisms was compared and hazard classification in\r\nthe I-V scale was prepared.
The most sensitive organisms in both types of samples (treated wastewater and river water) proved to be the algae represented by S. capricornutum, also protozoan T. thermophila, showed sensitivity in some samples of treated wastewater and river water. In contrast, mortality of the T. platyurus crustacean was recorded only for samples of treated wastewater. In the case of river water, the threat for organisms was estimated in the majority of monitored samples at the level of class III - acute hazard. However, biomonitoring of treated wastewater showed a greater diversity of hazard, class I-IV was noted, which could be related to the variable composition of wastewater as well as the various effectiveness of their treatment.
Taking into account the results obtained in river water biomonitoring and the class determined on the basis of selected chemical parameters (for uniform surface water bodies), it was observed that an important threat could be an excess of phosphate and nitrogen compounds in water. Therefore, in order to limit the inflow of above mentioned nutrients to surface waters, it is proposed to use solutions based on ecohydological biotechnology methods (biotechnology for ecology) with the participation of vegetation ecotonic zones, which can be strengthened by the construction of denitrification barriers (acceleration of nitrogen reduction) ) or geochemical barriers based on, among others dolomite or calcite (binding of soluble phosphorus compounds). The system solution for limiting pollution from point sources are sequential sedimentation and biofiltration systems that allow the elimination of both nitrogen, phosphorus and other organic and inorganic impurities, among others through the use of the ability of microorganisms involved in the mineralization, nitrification and denitrification process as well as plants capable to biofiltration and assimilation of pollutants.\r\n

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