Laboratory-to-field extrapolation: Increase in carbamazepine toxicity in a higher tier, multiple-stress experiment.

The toxicity and environmental risk of chemicals, such as the antiepileptic drug carbamazepine (CBZ), is commonly assessed using standardized laboratory tests and laboratory-to-field extrapolation. To investigate the toxicity of CBZ to aquatic key organisms in a more complex and environmentally relevant scenario, we conducted a 32-day multiple-stress experiment in artificial indoor streams. We exposed the non-biting midge Chironomus riparius, the blackworm Lumbriculus variegatus, and the New Zealand mud snail Potamopyrgus antipodarum to 80 and 400 µg CBZ/L in six artificial indoor streams. In addition to hydraulic stress, species' interaction, and low organic content in the sediment, organisms were co-exposed to the herbicide terbutryn (TBY) as a second chemical stressor at a concentration of 6 µg/L. The exposure to CBZ under multiple stress conditions resulted in a 10- to more than 25-fold higher toxicity in C. riparius and P. antipodarum when compared to a previous, standardized laboratory experiment. The co-exposure to TBY enhanced the adverse effects of CBZ on snails (reduced production of embryos). This effect was additive as the single exposure to TBY also reduced the reproduction of snails, most likely through the reduction of biofilm biomass. The emergence of C. riparius declined at a CBZ concentration of 400 µg/L (without the co-exposure to TBY) and at 80 µg/L in combination with TBY. The difference in sensitivity between laboratory and indoor stream experiments is indicative of a potential underestimation of risk when toxicity data are extrapolated to field conditions. The present results suggest the inclusion of non-chemical and chemical stressors in environmental hazard and risk assessments.

A new enzymatic method assessing the impact of wastewater treatment plant effluents on the assimilative capacity of small rivers.

Microorganisms play an important role in maintaining a good water quality in rivers by degrading organic material, including toxic substances. In the present study, we analyzed the potential impact of municipal wastewater treatment plant (WWTP) effluents as a major stress factor on the assimilative capacity of small rivers. It was the aim to develop a new bioassay for assessing such impacts in the receiving rivers by measuring the activity of extracellular enzymes (exoenzymes) in bacteria. Therefore, we established a specific in-vitro assay to detect inhibitory effects of solid phase-enriched water samples on ß-glucosidase (BGL) activity of the actinobacterium Cellulomonas uda as a proxy for the microbial decomposition of organic substances and thus for the assimilative capacity of surface waters. We found significant reductions of BGL activity in the WWTP effluents and in the receiving waters directly downstream as well as a relative quick recovery over the further course of the water bodies. The new bioassay offers a promising tool for the assessment of the assimilative capacity in surface waters and a potential impact of WWTP effluents on this key ecosystem function. Abbreviations WWTP wastewater treatment plant BGL ß-glucosidase EU-WFD European Water Framework Directive FAU Formazin Attenuation Units PE population equivalents REF relative enrichment factor; SPE solid phase extraction MTBE methyl-tert-buthyl-ether DMSO dimethyl-sulfoxide NPG 4-nitrophenol-ß-d-glucopyranoside DOC dissolved organic carbon.

Small but with big impact? Ecotoxicological effects of a municipal wastewater effluent on a small creek.

Municipal wastewater treatment plants (WWTPs) discharge micropollutants like pharmaceuticals, pesticides, personal care products or endocrine disrupting chemicals but also nutrients. Both can adversely influence the freshwater ecosystem and may finally affect the ecological conditions. Many studies focus on the potential impact of large WWTPs even if smaller ones are more common, often less efficient and discharge into small creeks or the upper reaches of rivers. As a result, the receiving waters are characterized by relatively high shares of treated wastewater. Thus, the primary objective of this study was to investigate the ecotoxicological effects of a small WWTP on freshwater amphipods and mollusks in a small creek using an active and passive monitoring approach, accompanied by laboratory experiments (LE). In vitro assays with recombinant yeasts and the microtox assay with Aliivibrio fischeri were performed in parallel to determine the endocrine potential and the baseline toxicity. The evaluation of the effects of the analysed WWTP was possible due to its shutdown during our study and the application of the same in vivo and in vitro assays before and after the shutdown. During the operation of the WWTP the discharge of treated wastewater caused significantly higher mortalities and lower reproduction of the anaylsed invertebrates in the active and passive montoring as well as in the LEs. Furthermore, the amphipod species assemblage in the creek was affected downstream of the WWTP effluent. Besides, the endocrine activity and baseline toxicity were significantly higher downstream of the effluent. After the shutdown of the WWTP, the in vitro activity levels and adverse in vivo effects in the receiving water recovered quickly with no significant differences downstream of the former WWTP effluent compared to the upstream station. Furthermore, the previously disturbed amphipod species assemblage recovered significantly with a shift in favor of Gammarus fossarum downstream of the effluent. These biological results are consistent with a marked decline by 81.5% for the detected micropollutants in the receiving creek after the shutdown which points to a prominent role of micropollutants for the observed effects.

Effects of metoprolol on aquatic invertebrates in artificial indoor streams.

Aquatic organisms are impacted by various biotic and abiotic stressors such as current, inter- and intraspecific competition for food resources and habitat, neobiota as well as an increasing number of chemicals. The latter also include pharmaceuticals, which are increasingly being detected in surface waters due to their growing use. The aim of our study was to determine effect data for metoprolol as a model compound for beta-blockers under an environmentally realistic exposure scenario on aquatic invertebrates inhabiting lotic environments. To this end we performed a 40-day experiment in artificial indoor streams (AIS) located in a greenhouse. We focussed on three autochthonous invertebrate species with high relevance in stream ecology: the amphipod Gammarus fossarum, the gastropod Potamopyrgus antipodarum, and the oligochaete Lumbriculus variegatus. Effects on reproduction were found with EC10 (40 days) values of 0.092 mg L-1 (G. fossarum), 0.253 mg L-1 (P. antipodarum), and 0.596 mg L-1 (L. variegatus). Considering environmental data, metoprolol seems to pose no hazard for aquatic invertebrates at present exposure levels.

Health effects of metoprolol in epibenthic and endobenthic invertebrates-A basis to validate future in vitro biotests for effect-based biomonitoring.

The aim of this study was to determine the effect data for metoprolol as a model substance for beta-blockers in aquatic invertebrates. The results will be used as a basis for the validation of future mode of action-based in vitro test systems targeting this class of pharmaceuticals. Effects of metoprolol were investigated in two autochthonous species with high relevance in stream ecology: the amphipod Gammarus fossarum and the oligochaete Lumbriculus variegatus. Mortality in G. fossarum was not observed in acute toxicity testing (48 h), and a significant increase of mortality at 45 mg/L was found when amphipods were exposed chronically (40 days). The most sensitive population-relevant endpoints were the juvenile-adult ratio and number of egg-bearing females with NOEC/LOEC-values of 5/15 mg/L. No proteotoxic effects were identified in G. fossarum. The sediment toxicity test with L. variegatus according to the OECD Guideline 225 with an exposure time of 28 days resulted in EC10-values of 92.5 and 126.1 mg/kgdw for the endpoints reproduction and biomass, respectively. In L. variegatus the response kinetics of Hsp70 showed no significant difference between the treatments. A tendency for rising lipid peroxide concentrations was found between 0.03 and 10 mg/kgdw, which were significant between the treatments, but not to the control.

Impact of an estrogenic sewage treatment plant effluent on life-history traits of the freshwater amphipod Gammarus pulex.

Despite efforts to upgrade sewage treatment plants (STPs) in the last decades, STPs are still a major source for the contamination of surface waters, including emerging pollutants such as pesticides, pharmaceuticals, personal care products and endocrine disrupting chemicals (EDCs). Because many of these substances are not completely removed in conventional STPs they are regularly detected in surface waters where they have the potential to affect local macroinvertebrate communities. The objective of the current work was to investigate the impact of an estrogenic wastewater effluent on the key life-history traits of the freshwater amphipod Gammarus pulex. G. pulex was exposed in artificial indoor flow-channels under constant conditions to different wastewater concentrations (0%, 33%, 66%, 100%). In parallel the estrogenic activity of wastewater samples was determined using the yeast estrogen screen (YES). Estrogenic activities in the STP effluent were up to 38.6 ng/L estradiol equivalents (EEQ). Amphipods exhibited an increasing body length with increasing wastewater concentrations. Furthermore, we observed a shift of the sex ratio in favour of females, a significantly increased fraction of brooding females and increased fecundity indices with increasing wastewater concentrations. The increased body length is likely to be attributed to the additional nutrient supply while the occurrence of EDCs in the wastewater is the probable cause for the altered sex ratio and fecundity in exposed Gammarus cohorts.

Using ICP-qMS to trace the uptake of nanoscale titanium dioxide by microalgae-potential disadvantages of vegetable reference material.

As nanoscale materials have gained in economic importance over recent years, concerns about accumulation in the environment and, consequently, analysis of nanoparticles in biological material have increasingly become the focus of scientific research. A nanomaterial used in a wide range of food, consumer and household products is titanium dioxide (nTiO2). Monitoring of nTiO2 via determination of elemental titanium (Ti) can be very challenging because of a variety of possible interferences. This work describes problems during the development of a quantification method for titanium dioxide (TiO2) using inductively coupled plasma-quadrupole mass spectrometry (ICP-qMS). To evaluate the analytical method, certified vegetable reference material NCS DC 73349 was used. Interestingly, measurements of NCS DC 73349 seemed to result in acceptable recovery values-however, this was without considering interferences or conceivable differences in the natural isotopic abundance of the certified titanium calibration solution and NCS DC 73349. Actually, recoveries were lower than initially assumed. The potential interferences causing augmented recovery could be attributed to the presence of the elements sulfur (S) and phosphorus (P), which were able to form oxide ions and nitrogen-interfering species. The effect of such interfering cluster ions could be prevented by dry ashing as a sample preparation step, to evaporate S and P, before digestion with aqua regia in a high-pressure asher (HPA). Final practicability of the analysis method was proved by monitoring the uptake of nTiO2 by the microalgae Scenedesmus acutus in an environmental exposure study.

Macroinvertebrate communities respond strongly but non-specifically to a toxicity gradient derived by effect-based methods.

Chemical pollution is one of the most important threats to freshwater ecosystems. The plethora of potentially occurring chemicals and their effects in complex mixtures challenge standard monitoring methods. Effect-based methods (EBMs) are proposed as complementary tools for the assessment of chemical pollution and toxic effects. To investigate the effects of chemical pollution, the ecological relevance of EBMs and the potential of macroinvertebrates as toxicity-specific bioindicators, ecological and ecotoxicological data were linked. Baseline toxicity, mutagenicity, dioxin-like and estrogenic activity of water and sediment samples from 30 river sites in central Germany were quantified with four in vitro bioassays. The responses of macroinvertebrate communities at these sites were assessed by calculating 16 taxonomic and functional metrics and by investigating changes in the taxonomic and trait composition. Principal component analysis revealed an increase in toxicity along a joint gradient of chemicals with different modes of action. This toxicity gradient was associated with a decrease in biodiversity and ecological quality, as well as significant changes in taxonomic and functional composition. The strength of the effects suggested a strong impact of chemical pollution and underlined the suitability of EBMs in detecting ecological relevant effects. However, the metrics, taxa, and traits associated with vulnerability or tolerance to toxicity were found to also respond to other stressors in previous studies and thus may have only a low potential as toxicity-specific bioindicators. Because macroinvertebrates respond integratively to all present stressors, linking both ecological and environmental monitoring is necessary to investigate the overall effects but also isolate individual stressors. EBMs have a high potential to separate the toxicity of chemical mixtures from other stressors in a multiple stressor scenario, as well as identifying the presence of chemical groups with specific modes of action.

The biological effects and possible modes of action of nanosilver.

Novel physicochemical and biological properties have led to a versatile spectrum of applications for nanosized silver particles. Silver nanoparticles are applied primarily for their antimicrobial effects, and may variety of commercially available products have emerged. To better predict and prevent possible environmental impacts from silver nanoparticles that are derived from increasing production volumes and environmental release, more data on the biological effects are needed on appropriate model organisms. We examined the literature that addressed the adverse effects of silver nanoparticles on different levels of biological integration, including in vitro and in vivo test systems. Results of in vitro studies indicate a dose-dependent programmed cell death included by oxidative stress as main possible pathway of toxicity. Furthermore, silver nanoparticles may affect cellular enzymes by interference with free thiol groups and mimicry of endogenous ions. Similar mechanisms may apply for antibacterial effects produced by nonasilver. These effects are primary from the interference nanosilver has with bacterial cell membranes. Few in vivo studies have been performed to evaluated the toxic mode of action of nanosilver or to provide evidence for oxidative stress as an important mechanism of nanosilver toxicity. Organisms that are most acutely sensitive to nanosilver toxicity are the freshwater filter-freeding organisms. Both in vitro and in vivo studies have demonstrated tha silver ions released from nanoparticle surface contribute to the toxicity, and, indeed, some findings indicated a unique nanoparticles effect. For an adequate evaluation of the environmental impact of nanosilver, greater emphasis should be placed on combining mechanistic investigations that are performed in vitro, with results obtained in in vivo test systems. Future in vivo test system studies should emphasize long-term exposure scenarios. Moreover, the dietary uptake of silver nanoparticles and the potential to bioaccumulate through the food web should be examined in detail.

Effects of multiple stressors on benthic invertebrates using Water Framework Directive monitoring data.

Multiple stressors affect freshwater systems and cause a deficient ecological status according to the European Water Framework Directive (WFD). To select effective mitigation measures and improve the ecological status, knowledge on the stressor hierarchy and individual and joined effects is necessary. However, compared to common stressors like nutrient enrichment and morphological degradation, the relative importance of micropollutants such as pesticides and pharmaceuticals is largely unaddressed. We used WFD monitoring data from Saxony (Germany) to investigate the importance of 85 environmental variables (including 34 micropollutants) for 18 benthic invertebrate metrics at 108 sites. The environmental variables were assigned to five groups (natural factors, nutrient enrichment, metals, micropollutants and morphological degradation) and were ranked according to their relative importance as group and individually within and across groups using Principal Component Analyses (PCAs) and Boosted Regression Trees (BRTs). Overall, natural factors contributed the most to the total explained deviance of the models. This variable group represented not only typological differences between sampling sites but also a gradient of human impact by strongly anthropogenically influenced variables such as electric conductivity and dissolved oxygen. These large-scale effects can mask the individual importance of the other variable groups, which may act more specifically at a subset of sites. Accordingly, micropollutants were not represented by a few dominant variables but rather a diverse palette of different chemicals with similar contribution. As a group, micropollutants contributed similarly as metals, nutrient enrichment and morphological degradation. However, the importance of micropollutants might be underestimated due to limitations of the current chemical monitoring practices.

Pesticide dynamics in three small agricultural creeks in Hesse, Germany.

Background: Due to their high biodiversity, small water bodies play an important role for freshwater ecosystems. Nonetheless, systematic pesticide monitoring in small creeks with a catchment <30 km2 is rarely conducted. Methods: In this study, event-driven water samples were taken from May until November 2017 and March until July 2018 after 20 rain events at three sampling sites with catchment areas of <27 km2 in the Wetterau, a region with intensive agriculture in Southern Hesse, Germany. Additionally, enriched extracts of the native water samples from the campaign in 2018 were used for the Microtox assay to determine baseline toxicity to invertebrates over time and sum of toxic units (STU) were calculated to compare the potential toxicity of the samples. Results: Overall, 37 pesticides and 17 transformation products were found, whereby the herbicide metamitron (79 µg/L) showed the highest concentration. Regularly, pesticide concentrations peaked at the time of the highest water level within each sampling event. Within each sampling event maximum pesticide concentration was mostly reached in water samples taken during the first two hours. The sum of the time-weighted mean concentration values of all pesticides was between 2.0 µg/L and 7.2 µg/L, whereby the measured concentrations exceeded their regulatory acceptable concentration (RAC) at 55% of all sampling events for at least one pesticide. The mean EC50 values varied between 28.6 ± 13.1 to 41.3 ± 12.1 REF (relative enrichment factor). The results indicated that several samples caused baseline toxicity, whereby the highest activity was measured at the time of highest water levels and pesticides concentrations, and then steadily decreased in parallel with the water level. Median STUs of invertebrates ranged from -2.10 to -3.91, of algae/aquatic plants from -0.79 to -1.84 and of fish from -2.47 to -4.24. For one of the three sampling sites, a significant linear correlation between baseline toxicity and STUinvertebratewas found (r2 = 0.48). Conclusion: The results of the present study suggest that (1) current pesticide monitoring programs underestimate risks posed by the exposure to pesticides for aquatic organisms and (2) pre-authorization regulatory risk assessment schemes are insufficient to protect aquatic environments.

Comprehensive sediment toxicity assessment of Hessian surface waters using Lumbriculus variegatus and Chironomus riparius.

The objective of this study was a sediment assessment of predominantly small rivers in the German federal state of Hesse. For this purpose, sediment samples were taken at 50 study sites with different contamination levels. The benthic invertebrates Chironomus riparius (Diptera) and Lumbriculus variegatus (Oligochaeta) were used as test species and exposed to whole sediments in chronic laboratory experiments. The bioassays were carried out on the basis of OECD guidelines 218 and 225 for the testing of chemicals. For about 50 % of the study sites chemical analytical data for pollutants from environmentally important substance classes like metals, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and organotin compounds were available. These data were used to analyze correlations between effects in the bioassays and measured chemical contaminations at sampling sites. For 22 % of the sediments ecologically relevant adverse effects were observed. In the majority of these cases effects occurred in only one of the biotests, and only one sediment sample exerted a negative effect on both test organisms. There was no significant correlation between biological responses and chemical data considering substance classes. However, there was a weak positive correlation between arsenic concentration and both worm number and worm biomass as well as a weak positive correlation between single PAHs and worm biomass. In some sediment tests elevated ammonia concentrations occurred in the overlying water so that an influence of these partially toxic concentrations on the test results cannot be ruled out.

Acute and chronic toxicity of four frequently used UV filter substances for Desmodesmus subspicatus and Daphnia magna.

As a consequence of growing public concern about UV radiation effects on human health chemical and physical UV filters are increasingly used in personal care and other products. The release of these lipophilic and often persistent compounds into surface waters may pose a risk for aquatic organisms. The aim of the study was to determine effects of four frequently used UV filters on primary aquatic producers and consumers, the green alga Desmodesmus subspicatus and the crustacean Daphnia magna. Exposure to benzophenone 3 (BP3), ethylhexyl methoxycinnamate (EHMC), 3-benzylidene camphor (3-BC) and 3-(4'-methylbenzylidene)-camphor (4-MBC) resulted in growth inhibition of D. subspicatus with 72 h IC(10) values of 0.56 mg/L (BP 3), 0.24 mg/L (EHMC), 0.27 mg/L (3-BC) and 0.21 mg/L (4-MBC). EC(50) concentrations in the acute test with D. magna were 1.67, 0.57, 3.61 and 0.80 mg/L for BP3, EHMC, 3-BC and 4-MBC, respectively. Chronic exposure of D. magna resulted in NOECs of 0.04 mg/L (EHMC) and 0.1 mg/L (3-BC and 4-MBC). BP 3 showed no effects on neonate production or the length of adults. Rapid dissipation of these substances from the water phase was observed indicating the need for more frequent test medium renewal in chronic tests or the use of flow-through test systems.

The Occurrence of Intersex in Different Populations of the Marine Amphipod Echinogammarus marinus in North-West Brittany - A Longterm-Study.

In the past two decades, an increasing body of studies has been published on the intersex phenomenon in separate-sexed crustaceans from marine and freshwater ecosystems. Various causes are being considered that could have an influence on the occurrence of intersex. Besides genetic factors, environmental conditions such as photoperiodicity, temperature, salinity and parasitism, but also environmental pollution with endocrine disrupting chemicals (EDCs) are discussed. As part of a long-term monitoring (2012 - 2020) in north-west Brittany, we recorded the occurrence of intersex in the marine amphipod Echinogammarus marinus. We quantified the intersex incidence at marine and estuarine sites and analyzed the incidence in relation to the endocrine potential of the sediments. Intersex occurred with mean frequencies between 0.87% and 12%. It was striking that the incidence of intersex increased with increasing distance from the sea. Since the highest incidence was observed at the range boundary of this stenohaline species, we assume that intersex is triggered by endocrine potential and increasing stress due to increasing freshwater content - and thus an interplay of different environmental factors.

Effects of cadmium on life-cycle parameters in a multi-generation study with Chironomus riparius following a pre-exposure of populations to two different tributyltin concentrations for several generations.

So far only a few studies have been performed to assess the effects of dynamic pollutant exposure on life-history parameters of invertebrates. In a previous multi-generation approach with the midge Chironomus riparius we tested if a chronic tributyltin pre-exposure alters the ability of a population to cope with subsequent cadmium stress. In the experiment two separate chironomid populations were exposed via sediments to different TBT-concentrations (4.46 and 8.93 µg Sn/kg dw) for several generations, followed by subsequent cadmium exposure (1.2 mg Cd/kg dw) for three generations. While the TBT-exposure to 4.46 µg Sn/kg dw had only small effects on the development and reproduction of C. riparius the higher TBT-concentration of 8.93 µg Sn/kg dw led to negative effects on life-history traits. Therefore, a higher adverse effect of the higher TBT-concentration and thus a higher susceptibility to other stressors could be assumed. Within, this paper only the results of the second stressor experiment were presented; clear effects of Cd on development and reproduction of C. riparius were determined independent of the pre-exposure scenario. While no differences in Cd-sensitivity were found between the population without pre-exposure to TBT and the population pre-exposed to the low TBT-concentration (4.46 µg Sn/kg dw), the pre-exposure of midges to the higher TBT-concentration (8.93 µg Sn/kg dw) resulted in a significantly higher susceptibility to subsequent Cd-stress. These results document that the exposure history may influence the reaction to altered chemical stress. Our findings are relevant to understand and predict the evolutionary fate of populations in rapidly changing, human-impacted environments. However, the fact that chemical-induced reduced genetic diversity, which is not necessarily linked to genetic adaptation, leads to a reduced fitness under altered stress conditions, is to our knowledge a novel finding.

A critical evaluation of the environmental risk assessment for plasticizers in the freshwater environment in Europe, with special emphasis on bisphenol A and endocrine disruption.

Bisphenol A (BPA) and the phthalates di-butyl phthalate (DBP), di-ethylhexyl phthalate (DEHP), di-isodecyl phthalate (DIDP) and di-isononyl phthalate (DINP) are high production volume plasticizers and are regularly detected in aquatic ecosystems due to their continuous release into the environment. These compounds are listed as priority substances in the European Union and therefore subject to an environmental risk assessment (ERA). Final ERA reports are available for DBP, DIDP and DINP, while the process is still ongoing for BPA and DEHP. The current approach critically reviews the exposure and especially the effect analyses in the reports or drafts, considering also potential endocrine effects of the plasticizers.

Endocrine modulation and toxic effects of two commonly used UV screens on the aquatic invertebrates Potamopyrgus antipodarum and Lumbriculus variegatus.

The two UV screens 3-benzylidene-camphor (3-BC) and 3-(4'-methylbenzylidene)-camphor (4-MBC) were tested regarding their toxicity and estrogenic activity. The Yeast Estrogen Screen (YES) and two sediment assays with the freshwater invertebrates Lumbriculus variegatus and Potamopyrgus antipodarum were performed. In the YES, both substances activated the human estrogen receptor alpha with EC50 values of 44.2 microM for 3-BC and 44.3 microM for 4-MBC, whereby 4-MBC attained only 8% of the maximal response of 17beta-estradiol. For P. antipodarum embryo production increased after exposure to both substances (EC50 of 4.60 microM 4-MBC=1.17 mg kg(-1)dw) while mortality increased at high concentrations. The reproduction of L. variegatus was decreased by 3-BC with an EC50 of 5.95 microM (=1.43 mg kg(-1)dw) and also by 4-MBC, where no EC50 could be calculated. While reproduction decreased, the worms' weight increased after exposure to 3-BC with an EC50 of 26.9 microM (=6.46 mg kg(-1) dw), hence the total biomass remained unaffected.

Variation in sensitivity to cadmium among genetically characterized laboratory strains of the midge Chironomus riparius.

Estimating the extent of intraspecific variation in sensitivity to contaminant exposure is important in order to explain variation in the outcome of toxicity tests and to predict the effects of chemical stress on natural populations of plants and animals. However, only few studies provide evident data concerning intraspecific variation in life-history traits caused by a differential response to chemical stress. In this study, we compared the life-history response of six laboratory strains of the midge Chironomus riparius to cadmium exposure in a full life-cycle assay. In addition, the level of genetic variation in all strains was measured at five variable microsatellite loci. Several significant differences in life-history traits among the strains were observed in controls and cadmium treatments. The extent of variation among strains was largest at moderate cadmium concentration (0.42 mg Cd/kg dw). At increased Cd concentrations all strains showed similar levels of high mortality and reduced reproductive success. All strains exhibited considerable levels of genetic impoverishment compared to field populations. Strains with low genetic variation showed reduced fitness in the controls and were more susceptible to Cd exposure. For instance, no reproductive success in the lowest Cd treatment was observed for the strain with the lowest level of genetic diversity. In contrast, this Cd concentration had no negative effects on life-history traits of more variable strains. These results confirm recent findings, that inbreeding and reduced genetic variation influence the reaction of populations towards environmental stress. In addition, we show that the level of genetic variation and inbreeding directly influences the outcome of toxicity tests and contributes to the lack of reproducibility of test results among laboratories.

Effectivity of advanced wastewater treatment: reduction of in vitro endocrine activity and mutagenicity but not of in vivo reproductive toxicity.

Conventional wastewater treatment plants (WWTPs) have a limited capacity to eliminate micropollutants. One option to improve this is tertiary treatment. Accordingly, the WWTP Eriskirch at the German river Schussen has been upgraded with different combinations of ozonation, sand, and granulated activated carbon filtration. In this study, the removal of endocrine and genotoxic effects in vitro and reproductive toxicity in vivo was assessed in a 2-year long-term monitoring. All experiments were performed with aqueous and solid-phase extracted water samples. Untreated wastewater affected several endocrine endpoints in reporter gene assays. The conventional treatment removed the estrogenic and androgenic activity by 77 and 95 %, respectively. Nevertheless, high anti-estrogenic activities and reproductive toxicity persisted. All advanced treatment technologies further reduced the estrogenic activities by additional 69-86 % compared to conventional treatment, resulting in a complete removal of up to 97 %. In the Ames assay, we detected an ozone-induced mutagenicity, which was removed by subsequent filtration. This demonstrates that a post treatment to ozonation is needed to minimize toxic oxidative transformation products. In the reproduction test with the mudsnail Potamopyrgus antipodarum, a decreased number of embryos was observed for all wastewater samples. This indicates that reproductive toxicants were eliminated by neither the conventional nor the advanced treatment. Furthermore, aqueous samples showed higher anti-estrogenic and reproductive toxicity than extracted samples, indicating that the causative compounds are not extractable or were lost during extraction. This underlines the importance of the adequate handling of wastewater samples. Taken together, this study demonstrates that combinations of multiple advanced technologies reduce endocrine effects in vitro. However, they did not remove in vitro anti-estrogenicity and in vivo reproductive toxicity. This implies that a further optimization of advanced wastewater treatment is needed that goes beyond combining available technologies.