Full life-cycle toxicity assessment using rotifer resting egg production: implications for ecological risk assessment

The majority of standardized toxicity tests incorporate only a fraction of the test organism's life-cycle. However, in natural ecosystems, organisms may be exposed at various times during their life-cycle or throughout their life-cycle. Thus, ecotoxicological data from standardized toxicity tests is of limited ecological relevance. Existing standardized toxicity tests using the freshwater rotifer Brachionus calyciflorus utilize 24-h survival or 48-h asexual reproduction as endpoints, despite evidence that sexual reproduction is more sensitive. A 96-h B. calyciflorus resting egg toxicity test was developed and used to estimate the toxicity of pentachlorophenol (PCP) and copper. Results were compared to a variety of acute and sublethal endpoints for both toxicants. The B. calyciflorus 96-h resting egg production NOEC for PCP of 10 micrograms/l was 20 times lower than the 48-h asexual reproduction no observed effect concentration (NOEC) and 120 times lower than the 24-h acute lethal concentration 50%. The 96-h resting egg production NOEC for copper of 2.8 micrograms/l was 7 times lower than the 48-h asexual reproduction NOEC and nine times lower than the 24-h acute LC50. Resting egg production was a more sensitive indicator of toxicity than several other sublethal endpoints as well. These results indicate that partial life-cycle toxicity tests are not sufficiently sensitive to detect ecologically relevant adverse effects.     

Effects of long-term chlorpyrifos exposure on mortality and reproductive tissues of Banded Gourami (Trichogaster fasciata)

Abstract

This study assessed the long-term toxicity of chlorpyrifos on survival and reproduction of Banded Gourami by using mortality, gonado-somatic index (GSI) and histopathological observations as endpoints. Adult fish were exposed to five different concentrations of chlorpyrifos (0, 15, 50, 150, 500?µg/L) in 15 PVC tanks for 15, 30, 45, 60 and 75?days. Results showed that all male and female fish died after 15?days of 500?µg/L chlorpyrifos exposure. No consistent significant effect was observed for both male and female GSI. Furthermore, results showed dose- and time-dependent histopathological alterations for both ovary and testes. The 60-d No Observed Effect Concentration (NOEC) for most histopathological alterations of Banded Gourami ovary and testes was 50?μg/L, while 60-d NOEC for mortality of both male and female fish was < 15?μg/L. The results show that the long-term exposure to chlorpyrifos not only affect the reproductive tissues of Banded Gourami at exposure concentrations but also cause their mortality. Future studies should evaluate effects at lower concentrations.     

Evaluation of the Daphnia magna reproduction test for detecting endocrine disruptors

The Daphnia 21 d reproduction test is considered as a comprehensive and decisive test in the OECD Conceptual Framework for testing and assessment of endocrine disrupting chemicals (EDCs). However, how to interpret results of the Daphnia 21 d reproduction test for identification, risk assessment and testing strategy of EDCs remains an unsolved issue. This study analysed a total number of 135 published studies encompassing 86 known EDCs and non-EDCs with different modes of action. Our results show that the majority of effects on apical endpoints (survival, molting, growth, time to reproductive maturity, brood size, the number of broods, and the total number of offspring) do not seem to be EDC-specific. In contrast, the endpoint sex ratio is likely specific to juvenile hormones and their mimics. Variability is quantified for three most reported endpoints survival, the total number of offspring and sex ratio. Quantification of the endpoint sensitivity shows that the sensitivity of the sex ratio is lower than that of the total number of offspring. The Daphnia 21 d reproduction test gives insufficient information to conclude if a substance is an EDC or not. EDCs that are potent in assays in vitro may not be potent in the Daphnia 21 d reproduction test. We conclude that the Daphnia 21 d reproduction test is important for deriving No Observed Effect Concentrations for risk assessment but may produce false negatives in identification of EDCs when used on its own. A targeted testing strategy for selection of species, tests, and endpoints is suggested for identifying EDCs.     

Reproductive toxicity of bisphenol A and cadmium in Potamopyrgus antipodarum and modulation of bisphenol A effects by different test temperature

An OECD initiative for the development of mollusc-based toxicity tests for endocrine disrupters and other chemicals has recommended three test species with respective test designs for further standardisation. Preparing a subsequent pre-validation study we performed a reproduction test with Potamopyrgus antipodarum, determining the concentration range of the selected test substances, bisphenol A (BPA) and cadmium (Cd). At 16 °C, the recommended test temperature, the number of embryos in the brood pouch was increased by BPA and decreased by Cd (NOEC: 20 μg BPA/L and 1 μg Cd/L). Coinstantaneous BPA tests at 7 °C and 25 °C demonstrated a temperature dependency of the response, resulting in lower NOECs (5 μg/L respectively). As expected, reproduction in control groups significantly varied depending on temperature. Additional observations of the brood stock showed seasonal fluctuations in reproduction under constant laboratory conditions. The recommended temperature range and test conditions have to be further investigated.     

Validating the enchytraeid reproduction test: organisation and results of an international ringtest

In this paper the experiences concerning the organisation and results of the enchytraeid reproduction test (ERT) ringtest are summarised (for details see J. R?mbke, T. Moser, Organisation and Performance of an International Ringtest for the Validation of the Enchytraeid Reproduction Test, vols. I and II. UBA-Texte 4/99, 1999, 150, 223 pp). The performance of this ringtest was in line with requirements published by OECD. It was sponsored by the German Federal Environmental Agency (Umweltbundesamt; UBA, Berlin). The UBA was also actively engaged (together with the European Chemicals Bureau, Ispra) in a scientific task force, which consisted of seven scientists experienced in terrestrial ecology and ecotoxicology. 29 institutions (mainly from universities and contract research laboratories) from 15 countries of Europe and North America participated actively in the ringtest. The co-ordinating laboratory centrally distributed the test chemicals, guidance papers and test organisms to all participants. In addition, several training courses were organised. Most participants performed two tests with the fungicide Carbendazim and two with 4-nitrophenol. For each chemical, one test was designed according to an ECx approach and the other according to an NOEC approach. Several aspects of the test protocol were modified based on the experiences gained during the ringtest. A major aspect of the project was the detailed statistical evaluation of the test results (for details see A. Weyers, J. R?mbke, T. Moser, T. Ratte, Results of and statistical implications from the enchytraeid reproduction ringtest, 2001, submitted), leading to recommendations for an optimised ecotoxicological test design. A total of 92 tests were performed according to the protocol which is among the highest number ever performed in a ringtest. About 72% met the validity criteria (control mortality < 20%, > 25 juveniles per 10 adults). The effects of the two test chemicals on enchytraeid reproduction were in the range expected from data in the literature on oligochaete toxicity. However, statistical evaluation of the data was sometimes difficult as a result of the high variability in the number of juveniles. This variability was caused by several reasons, including hormesis effects or lack of experience of some participants. Comparison of the data from the NOEC and ECx approaches pointed to a clear advantage in favour of the latter. In most cases EC10 values were lower than the NOEC values determined in the same test. For details see A. Weyers et al. (loc. cit.). Reproducibility of the test data and practicability of the ERT ringtest were of the same order of magnitude as other ringtests recently performed. As a result of the ringtest, the ERT draft guideline was significantly improved. The new version is currently being standardised by OECD, ISO and ASTM.     

The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

Background, goals, and scope

In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17??-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.

Methods

A previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and ??negative?? chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.

Results

With a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.

Discussion and conclusions

With one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).

Perspectives

Based on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.     

Effect of chronic exposure to acetaminophen and lincomycin on Japanese medaka (Oryzias latipes) and freshwater cladocerans Daphnia magna and Moina macrocopa, and potential mechanisms of endocrine disruption

Chronic toxicity of acetaminophen and lincomycin were evaluated using freshwater organisms including two crustaceans (Daphnia magna and Moina macrocopa) and a fish (Oryzias latipes). H295R, a human adrenal cell was also used to understand the effects on steroidogenesis. In 21 d D. magna exposure, survival NOEC was found at 5.72 mg L⁻¹ and no reproduction related effects were noted at this level of exposure to acetaminophen, while 21 d survival or growth effects were not observed even at the highest exposure levels (153 mg L⁻¹) for lincomycin. In the chronic fish toxicity test, significant reduction in juvenile survival was observed at 30 d post-hatch (dph) at 95 mg L⁻¹ of acetaminophen, and 0.42 mg L⁻¹ of lincomycin. After the exposure to both pharmaceuticals, vitellogenin levels tended to increase in male fish at 90 dph. In the eggs which were prenatally exposed to 9.5 mg L⁻¹ of acetaminophen, reduced hatchability was observed. The results of H295R cell assay showed that both pharmaceuticals could alter steroidogenic pathway and increase estrogenicity. Endocrine disruption potentials and their ecological implication may deserve further studies. Our observations suggest however that ecological risks of both pharmaceuticals are negligible at the concentrations currently found in the environment.     

Statistical analysis of regulatory ecotoxicity tests.

ANOVA-type data analysis, i.e.. determination of lowest-observed-effect concentrations (LOECs), and no-observed-effect concentrations (NOECs), has been widely used for statistical analysis of chronic ecotoxicity data. However, it is more and more criticised for several reasons, among which the most important is probably the fact that the NOEC depends on the choice of test concentrations and number of replications and rewards poor experiments, i.e., high variability, with high NOEC values. Thus, a recent OECD workshop concluded that the use of the NOEC should be phased out and that a regression-based estimation procedure should be used. Following this workshop, a working group was established at the French level between government, academia and industry representatives. Twenty-seven sets of chronic data (algae, daphnia, fish) were collected and analysed by ANOVA and regression procedures. Several regression models were compared and relations between NOECs and ECx, for different values of x, were established in order to find an alternative summary parameter to the NOEC. Biological arguments are scarce to help in defining a negligible level of effect x for the ECx. With regard to their use in the risk assessment procedures, a convenient methodology would be to choose x so that ECx are on average similar to the present NOEC. This would lead to no major change in the risk assessment procedure. However, experimental data show that the ECx depend on the regression models and that their accuracy decreases in the low effect zone. This disadvantage could probably be reduced by adapting existing experimental protocols but it could mean more experimental effort and higher cost. ECx (derived with existing test guidelines, e.g., regarding the number of replicates) whose lowest bounds of the confidence interval are on average similar to present NOEC would improve this approach by a priori encouraging more precise experiments. However, narrow confidence intervals are not only linked to good experimental practices, but also depend on the distance between the best model fit and experimental data. At least, these approaches still use the NOEC as a reference although this reference is statistically not correct. On the contrary, EC50 are the most precise values to estimate on a concentration response curve, but they are clearly different from the NOEC and their use would require a modification of existing assessment factors.     

Are individual NOEC levels safe for mixtures? A study on mixture toxicity of brominated flame-retardants in the copepod Nitocra spinipes

In aquatic ecosystems organisms are exposed to mixtures of pollutants. Still, risk assessment focuses almost exclusively on effect characterization of individual substances. The main objective of the current study was therefore to study mixture toxicity of a common group of industrial substances, i.e., brominated flame-retardants (BFRs), in the harpacticoid copepod Nitocra spinipes. Initially, 10 BFRs with high hydrophobicity but otherwise varying chemical characteristics were selected based on multivariate chemical characterization and tested individually for effects on mortality and development using a partial life cycle test (six days) where silica gel is used as a carrier of the hydrophobic substances. Based on these findings, six of the 10 BFRs were mixed in a series of NOEC proportions (which were set to 0.008, 0.04, 0.2, 1, and five times the NOEC concentrations for each individual BFR), loaded on silica gel and tested in a full life cycle test (26 days). Significantly increased mortality was observed in N. spinipes after six and 26 days exposure at a NOEC proportion that equals the NOEC LDR value (x1) for each BFR in the mixture (p=0.0015 and p=0.0105, respectively). At the NOECx5 proportion all animals were dead. None of the other NOEC proportions caused significant negative responses related to development and reproduction. This shows that low concentrations of individual substances can cause toxicity if exposed in mixtures, which highlights the need to consider mixture toxicity to a greater extent in regulatory work.     

Suborganismic and organismic effects of aldicarb and its metabolite aldicarb-sulfoxide to the zebrafish embryo (Danio rerio)

The new European chemical regulation (REACH) requires a short-term fish test for chemicals where the level of production exceeds 10tons per year. For ethical reasons (3R-concept), an alternative to the acute fish test should be introduced to decrease the number of animal testing with fish. The zebrafish embryo (Danio rerio) test became a valuable tool in ecotoxicology and already replaces the acute fish test for the evaluation of wastewater in Germany. Recent efforts are targeted to use this and other fish embryo tests for the effect assessment of chemicals. The toxic effects of the carbamate insecticide aldicarb and its metabolite aldicarb-sulfoxide to zebrafish embryos were analysed using two approaches with different endpoints. Organismic tests were conducted with zebrafish embryos exposed to the pesticides for 48h. In addition, suborganismic effects were examined analysing the enzyme inhibition of cholinesterases and carboxylesterases. On the organismic level, the only sublethal effect seen was the increase of heart rate at low and decrease at higher concentration with the use of aldicarb-sulfoxide but not with aldicarb (concentration range 0.2-300microM). In contrast, analysis of enzyme inhibitions showed high to very high effects caused by the two carbamates. The enzyme inhibition analysis of whole homogenates of exposed embryos may be advantageous for toxicant screening (biomarker of exposure) and might be used to bridge the gap of sensitivity of the (48h old) zebrafish embryos to adult fish when exposed to anti-cholinesterase substances (biomarker of prospective effect).     

Fluoxetine effects assessment on the life cycle of aquatic invertebrates

Fluoxetine is a serotonin re-uptake inhibitor, generally used as an antidepressant. It is suspected to provoke substantial effects in the aquatic environment. This study reports the effects of fluoxetine on the life cycle of four invertebrate species, Daphnia magna, Hyalella azteca and the snail Potamopyrgus antipodarum exposed to fluoxetine spiked-water and the midge Chironomus riparius exposed to fluoxetine-spiked sediments. For D. magna, a multi-generational study was performed with exposition of newborns from exposed organisms. Effects of fluoxetine could be found at low measured concentrations (around 10mugl(-1)), especially for parthenogenetic reproduction of D. magna and P. antipodarum. For daphnids, newborns length was impacted by fluoxetine and the second generation of exposed individuals showed much more pronounced effects than the first one, with a NOEC of 8.9mugl(-1). For P. antipodarum, significant decrease of reproduction was found for concentrations around 10mugl(-1). In contrast, we found no effect on the reproduction of H. azteca but a significant effect on growth, which resulted in a NOEC of 33mugl(-1), expressed in nominal concentration. No effect on C. riparius could be found for measured concentrations up to 59.5mgkg(-1). General mechanistic energy-based models showed poor relevance for data analysis, which suggests that fluoxetine targets specific mechanisms of reproduction.     

Approach for extrapolating in vitro metabolism data to refine bioconcentration factor estimates

National and international chemical management programs are assessing thousands of chemicals for their persistence, bioaccumulative and environmental toxic properties; however, data for evaluating the bioaccumulation potential for fish are limited. Computer based models that account for the uptake and elimination processes that contribute to bioaccumulation may help to meet the need for reliable estimates. One critical elimination process of chemicals is metabolic transformation. It has been suggested that in vitro metabolic transformation tests using fish liver hepatocytes or S9 fractions can provide rapid and cost-effective measurements of fish metabolic potential, which could be used to refine bioconcentration factor (BCF) computer model estimates. Therefore, recent activity has focused on developing in vitro methods to measure metabolic transformation in cellular and subcellular fish liver fractions. A method to extrapolate in vitro test data to the whole body metabolic transformation rates is presented that could be used to refine BCF computer model estimates. This extrapolation approach is based on concepts used to determine the fate and distribution of drugs within the human body which have successfully supported the development of new pharmaceuticals for years. In addition, this approach has already been applied in physiologically-based toxicokinetic models for fish. The validity of the in vitro to in vivo extrapolation is illustrated using the rate of loss of parent chemical measured in two independent in vitro test systems: (1) subcellular enzymatic test using the trout liver S9 fraction, and (2) primary hepatocytes isolated from the common carp. The test chemicals evaluated have high quality in vivo BCF values and a range of logK(ow) from 3.5 to 6.7. The results show very good agreement between the measured BCF and estimated BCF values when the extrapolated whole body metabolism rates are included, thus suggesting that in vitro biotransformation data could effectively be used to reduce in vivo BCF testing and refine BCF model estimates. However, additional fish physiological data for parameterization and validation for a wider range of chemicals are needed.     

Effects of cadmium, copper, lead and zinc on growth, reproduction and survival of the earthworm Eisenia fetida (Savigny): Assessing the environmental impact of point-source metal contamination in terrestrial ecosystems

The earthworm Eisenia fetida (Annelida: Oligochaeta) was exposed to a geometric series of concentrations of cadmium, copper, lead and zinc in artificial soil using the OECD recommended protocol. Mortality, growth and cocoon production were measured over 56 days to determine LC50 and EC50 values. No observed effect concentrations (NOECs) were also estimated. Furthermore, the percentage of viable cocoons and number of juveniles emerging per cocoon was recorded. Cocoon production was more sensitive than mortality for all the metals, particularly cadmium and copper for which NOEC reproduction values were an order of magnitude lower than those for NOEC mortality. However, there was no significant effect of metals on the viability of cocoons. The weights of earthworms declined in all treatments (including the controls) during the experiment. This was probably due to the lack of suitable food in the OECD standard soil medium used. It was concluded that future experiments should include animal manure in the test medium. The LC50, EC50 and NOEC values determined in this study were compared with concentrations of metals in soils in the vicinity of a smelting works at Avonmouth, southwest England. The 14-day LC50 for zinc in Eisenia fetida was exceeded in soils covering an area of 75 km2 around the works, compared to 4.2 km2 for copper and 4.7 km2 for lead. Soil values for cadmium did not exceed the LC50 value anywhere in the region. Similar estimates of relative effects on reproduction confirmed that zinc is most likely to be responsible for the absence of earthworms from sites close to the Avonmouth works. However, the OECD standard test overestimated the potential effects of metals on populations, since earthworms can be found as close as 1 km from the smelting works. The discrepancy between test and field observations was probably due to the greater availability of the metals in the artificial soil.     

Toxic Effects of Crude Oil Combined with Oil Cleaner Simple Green on Yolk-sac Larvae and Adult Rainbow Trout Oncorhynchus mykiss (4 pp)

Background, Goal and Scope Cleaner CRYSTAL Simple Green (SG) was used for the cleanup of the oil spill in the Baltic Sea near Lithuania in 2001. No scientific data are available on the effects and consequences of its application for local aquatic life. The aim of this study was to determine and compare sublethal effects of a) solution SG; b) crude oil alone; c) SG in combination with oil on rainbow trout Oncorhynchus mykiss at different stages of its development in laboratory conditions.Methods Laboratory studies were performed on adult rainbow trout (4-day duration) and on yolk-sac larvae (25-day duration) evaluating their biological parameters. Concentrations of water-soluble and thin-dispersed fractions of petroleum hydrocarbons were measured using gas chromatography.Results and Discussion SG solution (0.5 mg/l) did not affect the survival of larvae and adult fish, and no significant changes were determined in respiratory parameters of the exposed larvae and adult fish. The most expressed alterations were found in morphological parameters (a decrease in the average body mass) of larvae and in haematological indices (a decrease in the leukocyte count) of adult fish at the end of the tests. Crude oil (1610 mg/l) did not affect the survival of adult fish during the 4-day exposure. An increase in larvae mortality rate (~ 36%) was recorded at the end of the tests. A significant decrease in the average body mass and heart rate of larvae as well as in gill ventilation frequency of larvae and adult fish were determined. SG combined with oil induced an increase in larval mortality &#61566; 46% of individuals died at the end of the tests. No mortality was recorded in adult fish. The average body mass and heart rate of larvae were significantly decreased. Marked changes were also found in respiratory parameters (gill ventilation frequency of larvae and adult fish significantly decreased, while “coughing” rate increased). A 1-day, 2-day exposure of fish to SG combined with oil induced a significant decrease in the leukocyte count of adult fish, which was also determined at the end of the tests. The augmentation of adverse impact could be explained by the data obtained from our studies. When SG was added into dilution water with crude oil the concentration of petroleum hydrocarbons in the mixture increased 3 ~ 4.5 times after 24 h and 96 h, respectively. Conclusions The comparative study of the effects of crude oil alone, SG and SG combined with oil showed that their toxic effects on fish differed. Oil combined with SG was found to be more toxic to fish (larvae and adults) than SG alone and oil alone. Fish at early stages of development (yolk-sac larvae) were more sensitive to the effects of the compounds studied than adults.Recommendations and Outlook To diminish the negative impact of oil spill cleanup using chemicals on aquatic ecosystems, it is recommended to carry out more comprehensive studies of their effects and after-effects in laboratory conditions using a wide scale of local aquatic organisms. The selected species of the most sensitive aquatic organisms should include those which are unable to escape the impact of combined action of oil and cleaners. Special attention should be directed to the research of effects of these pollutants on studied organisms at their most sensitive stages of life (reproduction, hatching, early stages of development), as after-effects of exposure to pollutants may be observed in future generations.     

Whole effluent toxicity assessment at a wastewater treatment plant upgraded with a full-scale post-ozonation using aquatic key species

Ozonation as final wastewater (WW) polishing step, following conventional activated sludge treatment is increasingly implemented in sewage treatment for contaminant degradation to prevent surface water pollution. While the oxidative degradation of chemicals has been extensively investigated, the in vivo toxicological characteristics of ozonated whole effluents are rarely a matter of research.In the present study, whole effluents were toxicologically evaluated with an in vivo test battery before and after full-scale ozonation and subsequent sand filtration on site at a treatment plant. One aquatic plant (duckweed, Lemna minor) and five invertebrate species of different systematic groups (Lumbriculus variegatus, Chironomus riparius, Potamopyrgus antipodarum, Daphnia magna) were exposed to the effluents in a flow-through-designed test system with a test duration of 7-28 d.None of the considered toxicity endpoints correlated with the pollutant elimination. A tendency towards an increased toxicity after ozonation was apparent in three of the test systems showing [statistically] significant adverse effects in the L. variegatus toxicity test (decrease in reproduction and biomass). After sand filtration, adverse effects were reduced to a similar level like after conventional treatment. Solely the Daphnia reproduction test revealed beneficial effects after ozonation in combination with sand filtration.Results of the test battery indicate the formation of adverse oxidation products during WW ozonation. L. variegatus appeared to be the most sensitive of the five test species. Sand filtration effectively removes or detoxifies toxic oxidation products, as toxic effects were subsequently reduced to the level after conventional treatment.     

The weaker points of fish acute toxicity tests and how tests on embryos can solve some issues

Fish acute toxicity tests play an important role in environmental risk assessment and hazard classification because they allow for first estimates of the relative toxicity of various chemicals in various species. However, such tests need to be carefully interpreted. Here we shortly summarize the main issues which are linked to the genetics and the condition of the test animals, the standardized test situations, the uncertainty about whether a given test species can be seen as representative to a given fish fauna, the often missing knowledge about possible interaction effects, especially with micropathogens, and statistical problems like small sample sizes and, in some cases, pseudoreplication. We suggest that multi-factorial embryo tests on ecologically relevant species solve many of these issues, and we shortly explain how such tests could be done to avoid the weaker points of fish acute toxicity tests.     

Ecotoxicological Assessment of Sediment, Suspended Matter and Water Samples in the Upper Danube River. A pilot study in search for the causes for the decline of fish catches (12 pp)

Goals, Scope and Background Fish populations, especially those of the grayling (Thymallus thymallus), have declined over the last two decades in the upper Danube River between Sigmaringen and Ulm, despite intensive and continuous stocking and improvement of water quality since the 1970s. Similar problems have been reported for other rivers, e.g. in Switzerland, Great Britain, the United States and Canada. In order to assess if ecotoxicological effects might be related to the decline in fish catch at the upper Danube River, sediment, suspended matter and waste water samples from sewage treatment plants were collected at selected locations and analyzed in a bioanalytical approach using a battery of bioassays. The results of this pilot study will be used to decide if a comprehensive weight-of-evidence study is needed. Methods Freeze-dried sediments and suspended particulate matters were extracted with acetone in a Soxhlet apparatus. Organic pollutants from sewage water were concentrated using XAD-resins. In order to investigate the ecotoxicological burden, the following bioassays were used: (1) neutral red assay with RTL-W1 cells (cytotoxicity), (2) comet assay with RTLW1 cells (genotoxicity), (3) Arthrobacter globiformis dehydrogenase assay (toxicity to bacteria), (4) yeast estrogen screen assay (endocrine disruption), (5) fish egg assay with the zebrafish (Danio rerio; embryo toxicity) and (6) Ames test with TA98 (mutagenicity). Results and Discussion The results of the in vitro tests elucidated a considerable genotoxic, cytotoxic, mutagenic, bacteriotoxic, embryotoxic and estrogenic burden in the upper Danube River, although with a very inhomogeneous distribution of effects. The samples taken from Riedlingen, for example, induced low embryo toxicity, but the second highest 17β-estradiol equivalent concentration (1.8 ng/L). Using the fish egg assay with native sediments, a broad range of embryotoxic effects could be elucidated, with clear-cut dose-response relationships for the embryotoxic effects of contaminated sediments. With native sediments, embryotoxicity was clearly higher than with corresponding pore waters, thus corroborating the view that – at least for fish eggs – the bioavailability of particle-bound lipophilic substances in native sediments is higher than generally assumed. The effect observed most frequently in the fish egg assay was a developmental delay. A comparison of our own results with locations along the rivers Rhine and Neckar demonstrated similar or even higher ranges of ecotoxicological burdens in the Danube River. Conclusions The complex pattern of ecotoxicological effects caused by environmental samples from the Danube River, when assessed in an in vitro biotest battery using both acute and more specific endpoints, showed that integration of different endpoints is essential for appropriate hazard assessment. Overall, the ecotoxicological hazard potential shown has indeed to be considered as one potential reason for the decline in fish catches at the upper Danube River. However, based on the results of this pilot study, it is not possible to elucidate that chemically induced alterations are responsible for the fish decline. Recommendations and Perspective . In order to confirm the ecological relevance of the in vitro results for the situation in the field and especially for the decline of the grayling and other fishes, further integrated investigations are required. For linking the weight of evidence obtained by in vitro assays and fish population investigations, the application of additional, more specific biomarkers (e.g. vitellogenin induction, EROD and micronucleus assay) has been initiated in fish taken from the field as well as in situ investigations.     

Acute toxicity of the insect larvicide abate® (temephos) on the fish tilapia melanopleura and the dragonfly larvae neurocordelia virginiensis.

Abstract

Acute bioassay tests on the toxic effects of the insect larvicide Abate® (temephos) on the mouth brooder cichlid fish Tilapia melanopleum and the dragonfly larvae (Odonata) Neurocordulia virginiensis were conducted in static non renewal toxicity test set ups. The 96h‐LC50 (95% confidence intervals) was 30.2 (20.5‐ 44.20) mg/L for the fish and 2.0 (1.16–2.0) mg/L for the dragonfly larvae. The dragonfly larvae were 15 times more susceptible to the larvicide than the tilapia. The calculated NOEC (No Observable Effect Concentration) was 14.1 mg/L for the fishand less than 1.0 mg/L for the insect larvae. The estimated ‘safe’ concentration of the pesticide to the fish was 3.0 mg/L and 0.2 mg/L for insect larvae. These figures are far above the concentrations approved for use in the control of mosquito larvae(0.0004–0.01 mg/L). It appears that the application of toxic levels of the insecticide for the elimination of some aquatic invertebrates may be ‘safe’ for normal survival, growth and reproduction offish and some aquatic insect larvae.     

The toxicity of sulfamethazine to Daphnia magna and its additivity to other veterinary sulfonamides and trimethoprim

Sulfonamides (SAs), the oldest chemotherapeutic agents used for antimicrobial therapy, still play an important role in veterinary mass treatments. Consequently, traces of these compounds, alone or in combinations, have been repeatedly detected in the environment. Sulfamethazine (SMZ) deserves particular attention not only because it is the most used veterinary SA, but also due to its proven effects on fertility in mice and on thyroid hormone homeostasis in rats. In this study, after evaluating the acute toxicity to Daphnia magna of six veterinary SAs and trimethoprim (TMP), the additivity of SMZ to each other compound was tested using the isobologram method. Two reproduction tests on the same biological model were also performed in order to derive LOEC and NOEC of SMZ. The acute EC₅₀ was in the range 131–270 mg L⁻¹ for all the compounds tested with the exception of sulfaguanidine (EC₅₀ = 3.86 mg L⁻¹). In acute binary tests SMZ showed a complex interaction with sulfaquinoxaline (superadditivity, additivity or subadditivity) at the three different combination ratios tested, simple additivity to TMP and less than additive interaction when paired to the other SAs. LOEC and NOEC of SMZ obtained from reproduction tests were 3.125 and 1.563 mg L⁻¹, respectively. In conclusion, SMZ should not harm the crustacean population at environmentally realistic concentrations. Its toxicity is comparable to that of other systemic SAs, and their binary interactions are less than additive. The same can not be entirely said for enteric SAs, and considering that these compounds are administered at high doses and mostly excreted in unmetabolised form, further evaluation of their impact to the aquatic environment seems advisable.