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.

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.

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.

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.

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.

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.

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.

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.

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.

Effects of carbamazepine and two of its metabolites on the non-biting midge Chironomus riparius in a sediment full life cycle toxicity test.

The antiepileptic drug carbamazepine (CBZ) and its main metabolites carbamazepine-10,11-epoxide (EP-CBZ) and 10,11-dihydro-10,11-dihydroxy-carbamazepine (DiOH-CBZ) were chosen as test substances to assess chronic toxicity on the non-biting midge Chironomus riparius. All the three substances were tested in a 40-day sediment full life cycle test (according to OECD 233) in which mortality, emergence, fertility, and clutch size were evaluated. In addition, these parameters were considered to calculate the population growth rate which represents an integrated measure to assess population relevant effects. With an LC50 of 0.20 mg/kg (time-weighted mean), the metabolite EP-CBZ was significantly more toxic than the parent substance CBZ (LC50: 1.1 mg/kg). Especially mortality, emergence, and fertility showed to be sensitive parameters under the exposure to CBZ and EP-CBZ. By using classical molecular dynamics (MD) simulations, the binding of CBZ to the ecdysone receptor was investigated as one possible mode of action (MoA) but appeared to be unlikely. The second metabolite DiOH-CBZ did not cause any effects within the tested concentration rage (0.17-1.2 mg/kg). Even though CBZ was less toxic compared to EP-CBZ, CBZ is found in the environment at much higher concentrations and therefore causes a higher potential risk for sediment dwelling organisms compared to its metabolites. Nevertheless, the current study illustrates the importance of including commonly found metabolites into the risk assessment of parent substances.

Field data reveal low critical chemical concentrations for river benthic invertebrates.

River ecosystems are of immense ecological and social importance. Despite the introduction of wastewater treatment plants and advanced chemical authorization procedures in Europe, chemical pollution is still a major threat to freshwater ecosystems. Here, large-scale monitoring data was exploited to identify taxon-specific chemical concentrations beyond which benthic invertebrate taxa are unlikely to occur using Threshold Indicator Taxa Analysis (TITAN). 365 invertebrate taxa and 25 organic chemicals including pesticides, pharmaceuticals, plasticisers, flame retardants, complexing agents, a surfactant and poly- and monocyclic aromatic hydrocarbons from a total of 399 sites were analysed. The number of taxa that responded to each of these chemicals varied between 0% and 21%. These sensitive taxa belonged predominantly to the groups Plecoptera, Coleoptera, Trichoptera, Ephemeroptera, Turbellaria, Megaloptera, Crustacea, and Diptera. Strong effects were observed in response to wastewater-associated compounds, confirming that wastewater is an important cause of biological degradation. The majority of change points identified for each compound were well below predicted no-effect concentrations derived from laboratory toxicity studies. Thus, the results show that chemicals are likely to induce effects in the environment at concentrations much lower than expected based on laboratory experiments. Overall, it is confirmed that chemical pollution is still an important factor shaping the distribution of invertebrate taxa, suggesting the need for continued efforts to reduce chemical loads in rivers.

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.

Toxicity of silver nanoparticles and ionic silver: Comparison of adverse effects and potential toxicity mechanisms in the freshwater clam Sphaerium corneum.

A range of studies has addressed possible environmental impacts of nanosilver, but most focused on acute effects in few species. Moreover, it remains unclear if toxic effects are particle-specific or mediated by released silver ions. We investigated chronic effects of nanosilver and soluble silver (AgNO3) on the freshwater bivalve Sphaerium corneum. Animals were exposed to nanosilver (0-500 µg Ag L(-1)) and AgNO3 (0-318 µg Ag L(-1)) over 28 days, and effects on reproduction and behavioral changes were assessed. To explore toxic mechanisms, we evaluated the effects on intracellular levels of reactive oxygen species (ROS) and the activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase). We further explored the activity of the sodium-potassium adenosine triphosphatase (Na(+)/K(+)-ATPase). Chronic exposure to nanosilver and AgNO3 resulted in negative effects on reproduction at concentrations of 5 and 3.18 µg Ag L(-1) (LOEC), respectively. ROS levels significantly increased after exposure to nanosilver at 10 µg Ag L(-1) and AgNO3 at 63.5 µg Ag L(-1). Both forms of silver altered the activities of antioxidant enzymes. Nanosilver (500 µg Ag L(-1)) and AgNO3 (318 µg Ag L(-1)) inhibited Na(+)/K(+)-ATPase activity by 82.6 and 78.9%, respectively. Nanoparticulate and soluble silver produced similar effects in S. corneum suggesting that toxicity of nanosilver is mainly mediated by dissolution of nanoparticles in the test media or after uptake by the test organisms.

Combined effects of silver nanoparticles and 17α-ethinylestradiol on the freshwater mudsnail Potamopyrgus antipodarum.

Ecotoxicological studies have shown that nanosilver is among the most toxic nanomaterials to aquatic organisms. However, research has so far focused on the determination of acute effects. Combined effects of nanosilver with other substances have not yet been studied in aquatic organisms. The present study aimed to investigate the chronic toxicity of nanosilver as well as the potential of nanosilver to influence the effects of co-occurring substances on the freshwater mudsnail Potamopyrgus antipodarum. In 28-day chronic toxicity experiments, the effects of nanosilver on the reproduction of P. antipodarum were assessed. In order to evaluate the influence of nanosilver on other substances, 17α-ethinylestradiol (EE2) was chosen as model compound due to the well-characterized effects on P. antipodarum. In addition to effects on reproduction, exposure to nanosilver and EE2 was monitored by determining the expression of estrogen-responsive transcripts (estrogen receptor and vitellogenin encoding genes). Exposure to nanosilver decreased the reproduction of P. antipodarum (EC10: 5.57 µg l(-1); EC50: 15.0 µg l(-1)). Exposure to EE2 significantly stimulated the embryo production at 25 ng l(-1). The presence of nanosilver led to increased EE2 effects at EE2 concentrations that had no influence on reproduction when applied in absence of nanosilver. In contrast, combined exposure to nanosilver decreased EE2 effects at concentrations that stimulated reproduction and the expression of estrogen responsive genes when applied in the absence of nanosilver. This is the first study demonstrating an influence of nanosilver on the effects of co-contaminants on aquatic organisms. The study further highlights the need for chronic experiments to properly assess environmental risks of nanosilver and their effects on co-occurring contaminants.

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.

Long-term effects of nanoscaled titanium dioxide on the cladoceran Daphnia magna over six generations.

We investigated the impact of nanoscaled titanium dioxide (nTiO2) on Daphnia magna populations in a multi-generational study over six generations (F0-F5). Each generation was exposed for 21 days to nTiO2 (AEROXIDE(®) TiO2 P25, primary particle size 21 nm) while mortality, individual growth, reproduction and population growth rates (PGR) were assessed as endpoints. The size distribution of nTiO2 in the single test media was analysed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). nTiO2 concentrations were measured using ICP-MS. Mortality and individual growth of D. magna were significantly affected with increasing exposure duration and concentration. Daphnids demonstrated decreasing reproduction over generations in all treatment groups (1.19-6 mg/L) but not in the control. At concentration levels of 1.78 mg/L chronic exposure resulted in a population collapse after five generations. This study indicates that multi-generational studies are suitable for evaluating long-term effects of nanoparticles since they reflect potential effects more accurately than single generation tests.

Comparative toxicity assessment of nanosilver on three Daphnia species in acute, chronic and multi-generation experiments.

The antibacterial properties of nanosilver have led to a versatile application spectrum including medical purposes and personal care products. However, the increasing use of nanosilver has raised concerns about its environmental impacts. Long-term exposure studies with aquatic invertebrates are essential to assess possible adverse effects on aquatic ecosystems. In the present study, acute (48 h), chronic (21 d) and long-term effects of nanosilver (primary size 15 nm) on five successive generations of three Daphnia species (D. magna, D. pulex, and D. galeata) were investigated. Acute EC50 values of nanosilver were 121 µg Ag L(-1) for D. magna being the least sensitive species and 8.95 and 13.9 µg Ag L(-1) for D. pulex and D. galeata, respectively. Chronic exposure provided EC10 values of 0.92 µg Ag L(-1) for D. magna showing the most sensitive chronic reaction and 2.25 and 3.45 µg Ag L(-1) for D. pulex and D. galeata, respectively. Comparative exposure to AgNO3 revealed a generally higher toxicity of the soluble form of silver. The multi-generation experiments resulted in effects on the population level for all tested species. Exposure of D. magna indicated an increased toxicity of nanosilver in the fifth generation of animals exposed to 10 µg Ag L(-1). Neonates from pre-exposed parental daphnids did not completely recover when transferred into clean water. Exposure of D. pulex and D. galeata revealed not only increasing toxicity in some generations, but also greater tolerance to nanosilver. This study contributes to the assessment of the risk potential of nanosilver on aquatic ecosystems. It shows that effects of nanosilver vary within one genus and change with exposure duration. Therefore, long-term studies considering different aquatic species are needed to better understand the possible effects of nanosilver on aquatic 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.