Endocrine disrupting activity in sewage sludge: Screening method, microbial succession and cost-effective strategy for detoxification.

Sewage sludge (SS) presents a high agronomic potential due to high concentrations of organic matter and nutrients, encouraging its recycling as a soil conditioner. However, the presence of toxic substances can preclude this use. To enable the safe disposal of this waste in agriculture, SS requires additional detoxification to decrease the environmental risks of this practice. Although some alternatives have been proposed in this sense, little attention is provided to eliminating endocrine-disrupting chemicals (EDCs). To fill this gap, this study aimed to develop effective and low-cost technology to eliminate EDCs from SS. For this, a detoxification process combining microorganisms and biostimulating agents (soil, sugarcane bagasse, and coffee grounds) was performed for 2, 4, and 6 months with aerobic and anaerobic SSs. The (anti-)estrogenic, (anti-)androgenic, retinoic-like, and dioxin-like activities of SSs samples were verified using yeast-based reporter-gene assays to prove the effectiveness of the treatments. A fractionation procedure of samples, dividing the target sample extract into several fractions according to their polarity, was conducted to decrease the matrix complexity and facilitate the identification of EDCs. A decrease in the abundance and microbial diversity of the SS samples was noted along the biostimulation with the predominance of filamentous fungal species over yeasts and gram-positive bacteria and non-fermenting rods over enterobacteria. Among the 9 EDCs quantified by LC-ESI-MS/MS, triclosan and alkylphenols presented the highest concentrations in both SS. Before detoxification, the studied SSs induced significant agonistic activity, especially at the human estrogen receptor α (hERα) and the human aryl hydrocarbon receptor (AhR). The raw anaerobic sludge also activated the androgen (hAR), retinoic acid (RARα), and retinoid X (RXRα) receptors. However, no significant endocrine-disrupting activities were observed after the SS detoxification, showing that the technology applied here efficiently eliminates receptor-mediated toxicity.

The wastewater micropollutant carbamazepine in insectivorous birds-an exposure estimate.

Insects with aquatic life stages can transfer sediment and water pollutants to terrestrial ecosystems, which has been described for metals, polyaromatic hydrocarbons, and polychlorinated chemicals. However, knowledge of the transfer of aquatic micropollutants released by wastewater treatment plants is scarce despite some preliminary studies on their occurrence in riparian spiders. In our study, we address a major analytical gap focusing on the transfer of the micropollutant carbamazepine from the larvae to the adult midges of Chironomus riparius using an optimized QuEChERS extraction method and HPLC-MS/MS applicable to both life stages down to the level of about three individuals. We show that the uptake of carbamazepine by larvae is concentration-dependent and reduces the emergence rate. Importantly, the body burden remained constant in adult midges. Using this information, we estimated the daily exposure of insectivorous tree swallows as terrestrial predators to carbamazepine using the energy demand of the predator and the energy content of the prey. Assuming environmentally relevant water concentrations of about 1 µg/L, the daily dose per kilogram of body weight for tree swallows was estimated to be 0.5 µg/kg/day. At places of high water contamination of 10 µg/L, the exposure may reach 5 µg/kg/day for this micropollutant of medium polarity. Considering body burden changes upon metamorphosis, this study fills the missing link between aquatic contamination and exposure in terrestrial habitats showing that wastewater pollutants can impact birds' life. Clearly, further analytical methods for biota analysis in both habitats are urgently required to improve risk assessment.

Plastic Products Leach Chemicals That Induce In Vitro Toxicity under Realistic Use Conditions.

Plastic products contain complex mixtures of extractable chemicals that can be toxic. However, humans and wildlife will only be exposed to plastic chemicals that are released under realistic conditions. Thus, we investigated the toxicological and chemical profiles leaching into water from 24 everyday plastic products covering eight polymer types. We performed migration experiments over 10 days at 40 °C and analyzed the migrates using four in vitro bioassays and nontarget high-resolution mass spectrometry (UPLC-QTOF-MSE). All migrates induced baseline toxicity, 22 an oxidative stress response, 13 antiandrogenicity, and one estrogenicity. Overall, between 17 and 8681 relevant chemical features were present in the migrates. In other words, between 1 and 88% of the plastic chemicals associated with one product were migrating. Further, we tentatively identified ∼8% of all detected features implying that most plastic chemicals remain unknown. While low-density polyethylene, polyvinyl chloride, and polyurethane induced most toxicological endpoints, a generalization for other materials is not possible. Our results demonstrate that plastic products readily leach many more chemicals than previously known, some of which are toxic in vitro. This highlights that humans are exposed to many more plastic chemicals than currently considered in public health science and policies.

Incubation in Wastewater Reduces the Multigenerational Effects of Microplastics in Daphnia magna.

The aging of microplastics in the environment changes their physicochemical properties. While this may affect their toxicity, comparative data on the effects of aged compared to pristine microplastics are scarce. One of those aging processes is the sorption of chemicals, which has mainly been studied for individual pollutants present in marine ecosystems. To investigate how the sorption of a complex mixture of freshwater pollutants affects the toxicity of microplastics, we incubated irregular polystyrene particles (≤63 µm) in either wastewater or ultrapure water. We exposed Daphnia magna to these aged microplastics and their pristine counterparts (80, 400, 2000, and 10,000 particles mL-1) over four generations using food limitation as an additional, environmentally realistic stressor. Both particle types affect the survival, reproduction, adult and neonate body lengths, and growth. An exposure to pristine microplastics results in the extinction of the third generation of daphnids. In contrast, wastewater-incubated particles induced a lower mortality. The incubation with wastewater does not change the microplastics' size, surface charge, and structure. Consistent with the literature, we assume that the adsorption of dissolved organic matter is a key aging process reducing the toxicity of microplastics. Consequently, toxicity testing using pristine microplastics may overestimate the effects of plastic particles in nature.

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.

Ecotoxicological characterization of the antiepileptic drug carbamazepine using eight aquatic species: baseline study for future higher tier tests.

Ecotoxicological effects of the antiepileptic drug carbamazepine (CBZ) were investigated in one primary producer (Desmodesmus subspicatus) and seven invertebrate species (Daphnia magna, Daphnia pulex, Ceriodaphnia dubia, Gammarus fossarum, Potamopyrgus antipodarum, Lumbriculus variegatus and Chironomus riparius) using OECD and US EPA guidelines for chronic toxicity testing. The present data set was used to conduct a hazard assessment for CBZ including confirmatory data. While most of our results were in accordance with previous studies, published effect data for C. dubia and D. pulex could not be confirmed, even though they have previously been considered to be the most sensitive invertebrate species to CBZ exposure. The non-biting midge, C. riparius, was the most sensitive test organism in the present study. From an EC10 of 406 µg/L and a no observed effect concentration (NOEC) of 400 µg/L, a predicted no effect concentration (PNEC) of 8 µg/L was calculated. With regard to realistic predicted and measured environmental concentrations, the environmental risk can be considered as low for CBZ when the assessment is based on laboratory-based effect data. To conduct a refined and more realistic assessment, this study provides foundational data for two future, higher tier studies: one multiple-stressor experiment and one mesocosm study.

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.

Integrated Evaluation Concept to Assess the Efficacy of Advanced Wastewater Treatment Processes for the Elimination of Micropollutants and Pathogens.

A multidisciplinary concept has been developed to compare advanced wastewater treatment processes for their efficacy of eliminating micropollutants and pathogens. The concept is based on (i) the removal/formation of selected indicator substances and their transformation products (TPs), (ii) the assessment of ecotoxicity via in vitro tests, and (iii) the removal of pathogens and antibiotic resistant bacteria. It includes substances passing biological wastewater treatment plants regulated or proposed to be regulated in the European Water Framework Directive, TPs formed in biological processes or during ozonation, agonistic/antagonistic endocrine activities, mutagenic/genotoxic activities, cytotoxic activities, further activities like neurotoxicity as well as antibiotics resistance genes, and taxonomic gene markers for pathogens. At a pilot plant, ozonation of conventionally treated wastewater resulted in the removal of micropollutants and pathogens and the reduction of estrogenic effects, whereas the in vitro mutagenicity increased. Subsequent post-treatment of the ozonated water by granular activated carbon (GAC) significantly reduced the mutagenic effects as well as the concentrations of remaining micropollutants, whereas this was not the case for biofiltration. The results demonstrate the suitability of the evaluation concept to assess processes of advanced wastewater treatment including ozonation and GAC by considering chemical, ecotoxicological, and microbiological parameters.

Validation of the OECD reproduction test guideline with the New Zealand mudsnail Potamopyrgus antipodarum using trenbolone and prochloraz.

The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals.

Advancing Biological Wastewater Treatment: Extended Anaerobic Conditions Enhance the Removal of Endocrine and Dioxin-like Activities.

Conventional activated sludge treatment of wastewater does not completely remove micropollutants. Here, extending anaerobic conditions may enhance biodegradation. To explore this, we combined iron-reducing or substrate-limiting and aerobic pilot-scale reactors directly at a wastewater treatment plant. To assess the removal of endocrine disrupting chemicals (EDCs) as group of micropollutants that adversely affects wildlife, we applied a bioanalytical approach. We used in vitro bioassays covering seven receptor-mediated mechanisms of action, including (anti)androgenicity, (anti)estrogenicity, retinoid-like, and dioxin-like activity. Untreated wastewater induced antiandrogenic, estrogenic, antiestrogenic, and retinoid-like activity. Full-scale as well as reactor-scale activated sludge treatment effectively removes the observed effects. Nevertheless, high antiandrogenic and minor dioxin-like and estrogenic effects persisted in the treated effluent that may still be environmentally relevant. The anaerobic post-treatment under substrate-limiting conditions resulted in an additional removal of endocrine activities by 17-40%. The anaerobic pre-treatment under iron-reducing conditions significantly enhanced the removal of the residual effects by 40-75%. In conclusion, this study demonstrates that a further optimization of biological wastewater treatment is possible. Here, implementing iron-reducing anaerobic conditions preceding aerobic treatment appears promising to improve the removal of receptor-mediated toxicity.

Optimizing the design of a reproduction toxicity test with the pond snail Lymnaea stagnalis.

This paper presents the results from two ring-tests addressing the feasibility, robustness and reproducibility of a reproduction toxicity test with the freshwater gastropod Lymnaea stagnalis (RENILYS strain). Sixteen laboratories (from inexperienced to expert laboratories in mollusc testing) from nine countries participated in these ring-tests. Survival and reproduction were evaluated in L. stagnalis exposed to cadmium, tributyltin, prochloraz and trenbolone according to an OECD draft Test Guideline. In total, 49 datasets were analysed to assess the practicability of the proposed experimental protocol, and to estimate the between-laboratory reproducibility of toxicity endpoint values. The statistical analysis of count data (number of clutches or eggs per individual-day) leading to ECx estimation was specifically developed and automated through a free web-interface. Based on a complementary statistical analysis, the optimal test duration was established and the most sensitive and cost-effective reproduction toxicity endpoint was identified, to be used as the core endpoint. This validation process and the resulting optimized protocol were used to consolidate the OECD Test Guideline for the evaluation of reproductive effects of chemicals in L. stagnalis.

The antimicrobial agents triclocarban and triclosan as potent modulators of reproduction in Potamopyrgus antipodarum (Mollusca: Hydrobiidae).

In this study, we assessed the chronic effects of the two antimicrobial substances triclocarban (TCC) and triclosan (TCS) on reproduction of a mollusk species by using the reproduction test with the New Zealand mudsnail Potamopyrgus antipodarum. Snails coming from a laboratory culture were exposed for 28 days to nominal concentrations ranging from 0.1 up to 10 µg/L for both chemicals (measured 0.082-8.85 µg TCC/L; 0.068-6.26 µg TCS/L). At the end of the experiment, snails were dissected and embryos in the brood pouch were counted to assess the individualized reproductive success of adult snails. Exposure to TCC resulted in an inverted u-shaped concentration-response relationship, with a stimulation of reproduction at low concentrations followed by an inhibition at higher concentrations. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were 0.082 and 0.287 µg/L, respectively. TCS caused significantly increased embryo numbers at all tested concentrations, except in the group of 0.170 µg/L. Therefore, the NOEC for TCS was 0.170 µg/L and the LOEC was 0.660 µg/L. These results indicate that TCC and TCS may cause reproductive effects at environmentally relevant concentrations indicating a potential risk for aquatic organisms in the environment.

Comparative sensitivity of juvenile and adult Potamopyrgus antipodarum (Mollusca: Hydrobiidae) under chronic exposure to cadmium and tributyltin.

To investigate a potential extension of a partial life cycle test protocol to a full life cycle test design, a comparative sensitivity analysis with juvenile and adult Potamopyrgus antipodarum was performed. Neonates and adult snails were exposed to the metal cadmium (Cd) and the endocrine disruptor tributyltin (TBT) at nominal concentrations ranging from 1.56 to 50 µg Cd/L and from 25 to 1,000 ng TBT-Sn/L. The experiments were performed over 28 days at 16°C in a semi-static test design. Mortality was assessed for both life stages. Juvenile snails' specific growth rate and reproduction of adults were investigated as main endpoints. We determined effects on snails' survival, juvenile growth and embryo numbers in the brood pouch of adult snails under exposure to both chemicals. Juvenile control mortality was between 25% and 30% and significantly higher than in the control groups with adult snails. A higher sensitivity of juvenile snails compared to adults was observed for the endpoint mortality. Calculated LC50 in Cd exposed snails was 38.2 µg/L for adults and 15.0 µg/L for juvenile snails. Significant effects on mortality in TBT exposed adult snails occurred at the highest test concentration only with a LC50 of 535 ng Sn/L. Juvenile survival was significantly affected at 50.8 ng Sn/L and higher concentrations. Effect concentrations for the main endpoints reproduction and juvenile growth show comparable sensitivities. For Cd exposed groups, EC50 values were 11.3 µg/L for the endpoint reproduction in adult snails and 3.82 µg/L for juvenile growth with overlapping confidence intervals. TBT also significantly affected juvenile snails' growth (EC50: 178 ng Sn /L). EC50 for embryo numbers was 125 ng TBT-Sn/L. Results indicate the manageability of a FLC test starting with newly hatched snails. Precautions have to be taken to guarantee a sufficient number of surviving snails until adulthood so that reproduction can be assessed. For final decision for the practicability of a FLC, further tests are needed.

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.

Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda).

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis. In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol. Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys® exposed to nominal concentrations of cadmium chloride (from 53 to 608µgCdL(-1)). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20±1°C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56days were reproducible between laboratories (68

Rapid development of increased neonicotinoid tolerance in non-target freshwater amphipods.

The comprehensive assessment of the long-term impacts of constant exposure to pollutants on wildlife populations remains a relatively unexplored area of ecological risk assessment. Empirical evidence to suggest that multigenerational exposure affects the susceptibility of organisms is scarce, and the underlying mechanisms in the natural environment have yet to be fully understood. In this study, we first examined the arthropod candidate species, Gammarus roeselii that - unlike closely related species - commonly occurs in many contaminated river systems of Central Europe. This makes it a suitable study organism to investigate the development of tolerances and phenotypic adaptations along pollution gradients. In a 96-h acute toxicity assay with the neonicotinoid thiacloprid, we indeed observed a successive increase in tolerance in populations coming from contaminated regions. This was accompanied by a certain phenotypic change, with increased investment into reproduction. To address the question of whether these changes are plastic or emerged from longer lasting evolutionary processes, we conducted a multigeneration experiment in the second part of our study. Here, we used closely-related Hyalella azteca and pre-exposed them for multiple generations to sublethal concentrations of thiacloprid in a semi-static design (one week renewal of media containing 0.1 or 1.0 µg/L thiacloprid). The pre-exposed individuals were then used in acute toxicity assays to see how quickly such adaptive responses can develop. Over only two generations, the tolerance to the neonicotinoid almost doubled, suggesting developmental plasticity as a plausible mechanism for the rapid adaptive response to strong selection factors such as neonicotinoid insecticides. It remains to be discovered whether the plasticity of rapidly developed tolerance is species-specific and explains why closely related species - which may not have comparable adaptive response capabilities - disappear in polluted habitats. Overall, our findings highlight the neglected role of developmental plasticity during short- and long-term exposure of natural populations to pollution. Moreover, our results show that even pollutant levels seven times lower than concentrations found in the study region have a clear impact on the developmental trajectories of non-target species.

One like all? Behavioral response range of native and invasive amphipods to neonicotinoid exposure.

Native and invasive species often occupy similar ecological niches and environments where they face comparable risks from chemical exposure. Sometimes, invasive species are phylogenetically related to native species, e.g. they may come from the same family and have potentially similar sensitivities to environmental stressors due to phylogenetic conservatism and ecological similarity. However, empirical studies that aim to understand the nuanced impacts of chemicals on the full range of closely related species are rare, yet they would help to comprehend patterns of current biodiversity loss and species turnover. Behavioral sublethal endpoints are of increasing ecotoxicological interest. Therefore, we investigated behavioral responses (i.e., change in movement behavior) of the four dominant amphipod species in the Rhine-Main area (central Germany) when exposed to the neonicotinoid thiacloprid. Moreover, beyond species-specific behavioral responses, ecological interactions (e.g. parasitation with Acanthocephala) play a crucial role in shaping behavior, and we have considered these infections in our analysis. Our findings revealed distinct baseline behaviors and species-specific responses to thiacloprid exposure. Notably, Gammarus fossarum exhibited biphasic behavioral changes with hyperactivity at low concentrations that decreased at higher concentrations. Whereas Gammarus pulex, Gammarus roeselii and the invasive species Dikerogammarus villosus, showed no or weaker behavioral responses. This may partly explain why G. fossarum disappears in chemically polluted regions while the other species persist there to a certain degree. But it also shows that potential pre-exposure in the habitat may influence behavioral responses of the other amphipod species, because habituation occurs, and potential hyperactivity would be harmful to individuals in the habitat. The observed responses were further influenced by acanthocephalan parasites, which altered baseline behavior in G. roeselii and enhanced the behavioral response to thiacloprid exposure. Our results underscore the intricate and diverse nature of responses among closely related amphipod species, highlighting their unique vulnerabilities in anthropogenically impacted freshwater ecosystems.

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.