SETAC GLB — Gründung und Entwicklung

Goal, Scope, and Background

In the context of the ISO-standardisation of the in vitro micronucleus test for (waste) water testing (ISO/DIS 21427-2), a national collaborative study was organized by the German Federal Institute of Hydrology (BfG) involving ten laboratories of private companies, universities and public authorities. The formation of micronuclei (MN) is a special kind of chromosomal aberration. To meet the standardisation requirements for this method, encoded waste water samples, some of them spiked with known genotoxins, had to be tested in a collaborative study. The study should demonstrate practicability of the in vitro micronucleus test for waste water testing and should provide validity data.

Material and Methods

The micronucleus assay was performed with the permanently growing Chinese hamster lung fibroblast cell line V79. Four encoded samples from one municipal and one industrial wastewater treatment plant were tested with and without metabolic activation by S9-mix. Two of these samples were spiked in advance with defined concentrations of the clastogenic substances cyclophosphamide and mytomycin C. The defined assessment criterion for genotoxicity was the lowest dilution of a sample that does not show any significant induction of micronuclei (LID; lowest ineffective dilution). Cytotoxicity was judged by determining the survival-index, i.e. the percentage growth rate of the cells compared with the respective negative controls. As supplementary qualitative criteria, the mitotic index and the proliferation index were assessed.

Results

Although some of the laboratories had little or even no experience with the protocol of the in vitro micronucleus test described in the ISO-draft, all participants suceeded in establishing the assay within few weeks and in generating viable test results. The two nongenotoxic samples were detected as negative by 90% or 100% of the participants. The mitomycin C-spiked sample (expected to be positive without S9 supplementation) was correctly evaluated as positive by all laboratories. The cyclophosphamide-spiked sample (expected to be positive with S9 supplementation) was evaluated correctly as genotoxic by 80% of the laboratories. A post-test analysis found evidence that the false-negative results were due to technical failure, but not of a methodological nature. The sample LID values varied by no more than one dilution step around the median LID-value for all samples investigated. The survival index was proven to be a robust measure for estimation of cytotoxicity.

Discussion

The measurement of micronuclei is an important parameter for the detection of cytogenetic damage. In contrast to the single-cell gel electrophoresis assay (Comet assay), which is used as an indicator test, the in vitro micronucleus test detects non-repairable and thus manifested genetic damage. Consequently, the in vitro micronucleus test can be regarded as the more significant test system. A more frequent occurrence of micronuclei in treated cells suggests a risk of severe genetic damage for subsequent cell generations. In the interest of a precautionary environmental protection and health protection no municipal or industrial waste-water samples should show any significant induction of micronuclei in the treated cell populations.

Perspectives

The presented collaborative study was the first interlaboratory comparison of the in vitro micronucleus test using wastewater samples. The test system is intended to complement the already DIN- and ISO-standardised bacterial tests, i.e. the umu-test and the Ames plate incorporation assay. The data generated in the course of this project justify the transformation of the draft standard into the final draft international standard (FDIS), the preliminary stage of an international norm, so that a valid, standardised test system for the detection of cukaryotic genotoxicity in water samples might become available.     

über die Notwendigkeit der wirkungsorientierten Analytik in einer umfassenden Wasserforschung

Ohne Zusammenfassung Verantwortliche Herausgeber: Jan Schwarzbauer · Peter Heininger · Evelyn Claus

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A plea for the use of copepods in freshwater ecotoxicology

Standard species used in ecological risk assessment are chosen based on their sensitivity to various toxicants and the ease of rearing them for laboratory experiments. However, this mostly overlooks the fact that species in the field that may employ variable life-history strategies, which may have consequences concerning the vulnerability of such species to exposure with contaminants. We aimed to highlight the importance of copepods in ecology and to underline the need to include freshwater copepods in ecotoxicology. We carried out a literature search on copepods and Daphnia in ecology and ecotoxicology to compare the recognition given to these two taxa in these respective fields. We also conducted a detailed analysis of the literature on copepods and their current role in ecotoxicology to characterize the scale and depth of the studies and the ecotoxicological information therein. The literature on the ecology of copepods outweighed that in ecotoxicology when compared with daphnids. Copepods, like other zooplankton, were found to be sensitive to toxicants and important organisms in aquatic ecosystems. The few studies that were conducted on the ecotoxicology of copepods mainly focused on marine copepods. However, very little is known about the ecotoxicology of freshwater copepods. To enable a more realistic risk higher tier environmental risk assessment, we recommend considering freshwater copepods as part of the hazard assessment process. This could include the establishment of laboratory experiments to analyse the effects of toxicants on copepods and the development of individual-based models to extrapolate effects across species and scenarios.     

Mathematische Modelle zur Effektabsch?tzung

Zusammenfassung  Die Extrapolation von toxikologischen Daten aus Labortests auf Schwellenwerte für ?kosysteme stellt ein zentrales Problem bei der Effektabsch?tzung im Rahmen der ?kologischen Risikoanalyse dar. Die üblichen statistischen Verfahren werden kurz vorgestellt, bevor Beispiele zur Anwendung von Simulationsmodellen gegeben werden. Ein Verfahren zur Absch?tzung von Schwellenkonzentration für verschiedene Endpunkte mit Hilfe von Monte-Carlo-Simulationen wird vorgestellt und an einem Datensatz für acht Chemikalien mit statistischen Verfahren verglichen. Abschlie?end werden die Vor-und Nachteile gegenübergestellt.      

Phytotoxicity of coloured substances: is Lemna duckweed an alternative to the algal growth inhibition test?

Coloured substances cause problems when interpreting algal tests, because effects due to light absorption can interact with potential toxicity. The Lemna Duckweed growth inhibition test can complement the algal test, on condition that the test is performed on a black, not reflecting surface. On white surfaces, test solution colour can strongly impact Lemna growth. For example, average control sample growth rate of is much higher on white surfaces (0.362 d(-1)) than on black surfaces (0.284 d(-1)). We found that 10 mg l(-1) of the dyestuff "Brilliant Blue R spezial" inhibited average Lemna growth rate about 22% on white surfaces but did not inhibit growth on black surfaces. The reason for this difference stems from the difference in amount of light reflected from below the test beakers. With Brilliant Blue on white surfaces, the test solution colour reduces utilizable light and causes a deterioration of light conditions, whereas on a black surfaces, reflected light is absent a priori, and thus no inhibiting effect was measured. Of particular importance is the choice of test parameter. With Brilliant Blue, a LOEC for average growth rate, based on frond numbers, of 320 mg l(-1) was determined. However, when average growth rate was calculated using dry weights of the plants, the LOEC decreased clearly to 1.0 mg l(-1). In this study, the Lemna test was much more sensitive than the algal test. We recommend Lemna tests be used in addition to algal tests, because doing so may significantly improve the assessment of phytotoxicity of chemicals and sewage.     

A list of fish species that are potentially exposed to pesticides in edge-of-field water bodies in the European Union—a first step towards identifying vulnerable representatives for risk assessment

Surrogate species are used in standard toxicity tests for the environmental risk assessment of chemicals. Test results are then extrapolated to the situation in the field, which is often associated with a large degree of uncertainty. Since a vulnerable species in the field is not only characterised by its intrinsic sensitivity to a stressor but also by its potential for exposure and its population resilience, the identification of focal species based on these three components of vulnerability is needed for a more ecologically relevant risk assessment. This study listed European fish species that are susceptible to pesticide exposure in the field and thus achieved the first step towards identifying focal species for the risk assessment of pesticides for fish in Europe. A step-wise filtering approach was applied to list freshwater fish species that are native to Europe and widespread in the European Union, which inhabit streams, ditches or ponds in agricultural landscapes and therefore, are at an elevated risk of being exposed to pesticides. Out of the 579 fish species occurring in European freshwater, 27 species met the filtering criteria. The resulting list was verified based on monitoring studies that were conducted in agricultural landscapes over the past 20 years. Focal fish species that can be used for a more ecologically relevant environmental risk assessment of pesticides in Europe can be identified from the produced list of species by further assessing their ecological (life history and dispersal characteristics) and intrinsic sensitivities.