Oxidative stress related effect of xenobiotics on eukaryotic model organism, Saccharomyces cerevisiae.

Ecotoxicological assays have traditionally focused on the effects of chemicals at the individual level by exploiting mortality and reproduction as endpoints. Although these two parameters are ecologically relevant, they rarely provide information regarding the elemental toxic mechanisms. Obviously, the number of xenobiotics used has been rapidly increased. Thus, any established measurement that shortens the actual outcome and, simultaneously provides information about toxic mechanisms is desirable. This research focused on the study of oxidative stress response as a biomarker in the eukaryotic model organism, Saccharomyces cerevisiae. For this, yeast cells were exposed to a set of selected environmentally relevant chemicals via different approaches, including cellular diagnostics, gene expression analysis and chemo-genetic screening. The results demonstrated that at the cellular level, model organisms reacted to different chemicals in distinct manner. For each xenobiotic, the correlation between toxic response of molecular and cellular levels are presented. Namely, the expression of target genes after chemical exposure affected the cellular alteration as evidenced by an elevated level of superoxide dismutase and a reduced amount of glutathione. Furthermore, the results derived from chemo-genetic screening, in which mutants lacking of gene of interest were employed, exhibited more susceptibility to test chemicals in comparison to the wildtype. The response of oxidative stress upon chemical exposure in budding yeast from this study is potentially useful for an establishment of a proper bio-test system which can eventually be linked to adverse effects at an individual level in higher eukaryotes.

Application of equilibrium passive sampling to assess the influence of anthropogenic activities and bioturbation on the distribution of hydrophobic organic chemicals in North Sea sediment cores.

The pollution state in the German Bight was investigated by determination of pollutant concentrations of sediment samples using equilibrium passive sampling. Polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAH) were determined in the pore water of North Sea sediment. The freely dissolved pore water concentration (Cfree) was measured applying Solid Phase Microextraction (SPME) by using PDMS-coated glass fibers. The obtained results show that the North Sea contamination level with the investigated pollutants is relatively low. However, the stations close to the sediment-dumping site were higher contaminated. A macrofauna analysis showed that bioturbation activities were mostly present in the upper sediment layers, but a direct bioturbation influence on the sediment concentration distribution could not be shown. Overall, the contamination load was below baseline toxicity, but considering that several other priority pollutants will also make a contribution to the baseline toxicity, it can be counted as relatively high.

Spatial and temporal variations in anti-androgenic activity and environmental risk in a small river.

The biological effects of multiple compounds have been widely investigated in aquatic environments. However, investigations of spatial and temporal variations in biological effects are rarely performed because they are time-consuming and labor-intensive. In this study, the variability of the anti-androgen, receptor-mediated activity of surface water samples was observed over 3 years using in vitro bioassays. Large-volume water samples were collected at one site upstream (Wer site) and two sites downstream (Sil and Nien sites) of a wastewater treatment plant (WWTP) outfall in the Holtemme River. Anti-AR activity was persistently present in all surface water samples over the three years. Large spatial variations in anti-androgenic activity were observed, with the lowest activity at the Wer site (mean concentration of 9.5 ± 7.2 µg flutamide equivalents/L) and the highest activity at the Sil site (mean concentration of 31.1 ± 12.0 µg flutamide equivalents/L) directly influenced by WWTP effluents. On the temporal scale, no distinct trend for anti-AR activity was observed among the seasons in all three years. The anti-androgenic activity at the upstream Wer site showed a decreasing trend from 2014 to 2016, indicating improved water quality. A novel bioanalytical-equivalent-based risk assessment method considering the frequency of risk occurrence was developed and then utilized to assess the environmental risk of anti-androgenic activity in the Holtemme River. The results revealed that the highest risk was present at the Sil site, while the risk was considerably reduced at the Nien site. The risk at the upstream Wer site was the lowest.

A plea for the integration of Green Toxicology in sustainable bioeconomy strategies - Biosurfactants and microgel-based pesticide release systems as examples.

A key aspect of the transformation of the economic sector towards a sustainable bioeconomy is the development of environmentally friendly alternatives for hitherto used chemicals, which have negative impacts on environmental health. However, the implementation of an ecotoxicological hazard assessment at early steps of product development to elaborate the most promising candidates of lowest harm is scarce in industry practice. The present article introduces the interdisciplinary proof-of-concept project GreenToxiConomy, which shows the successful application of a Green Toxicology strategy for biosurfactants and a novel microgel-based pesticide release system. Both groups are promising candidates for industrial and agricultural applications and the ecotoxicological characterization is yet missing important information. An iterative substance- and application-oriented bioassay battery for acute and mechanism-specific toxicity within aquatic and terrestrial model species is introduced for both potentially hazardous materials getting into contact with humans and ending up in the environment. By applying in silico QSAR-based models on genotoxicity, endocrine disruption, skin sensitization and acute toxicity to algae, daphnids and fish, individual biosurfactants resulted in deviating toxicity, suggesting a pre-ranking of the compounds. Experimental toxicity assessment will further complement the predicted toxicity to elaborate the most promising candidates in an efficient pre-screening of new substances.

Demonstration of an aggregated biomarker response approach to assess the impact of point and diffuse contaminant sources in feral fish in a small river case study.

The assessment of the exposure of aquatic wildlife to complex environmental mixtures of chemicals originating from both point and diffuse sources and evaluating the potential impact thereof constitutes a significant step towards mitigating toxic pressure and the improvement of ecological status. In the current proof-of-concept study, we demonstrate the potential of a novel Aggregated Biomarker Response (ABR) approach involving a comprehensive set of biomarkers to identify complex exposure and impacts on wild brown trout (Salmo trutta fario). Our scenario used a small lowland river in Germany (Holtemme river in the Elbe river catchment) impacted by two wastewater treatment plants (WWTP) and diffuse agricultural runoff as a case study. The trout were collected along a pollution gradient (characterised in a parallel study) in the river. Compared to fish from the reference site upstream of the first WWTP, the trout collected downstream of the WWTPs showed a significant increase in micronucleus formation, phase I and II enzyme activities, and oxidative stress parameters in agreement with increasing exposure to various chemicals. By integrating single biomarker responses into an aggregated biomarker response, the two WWTPs' contribution to the observed toxicity could be clearly differentiated. The ABR results were supported by chemical analyses and whole transcriptome data, which revealed alterations of steroid biosynthesis and associated pathways, including an anti-androgenic effect, as some of the key drivers of the observed toxicity. Overall, this combined approach of in situ biomarker responses complemented with molecular pathway analysis allowed for a comprehensive ecotoxicological assessment of fish along the river. This study provides evidence for specific hazard potentials caused by mixtures of agricultural and WWTP derived chemicals at sublethal concentrations. Using aggregated biomarker responses combined with chemical analyses enabled an evidence-based ranking of sites with different degrees of pollution according to toxic stress and observed effects.

The Green toxicology approach: Insight towards the eco-toxicologically safe development of benign catalysts.

Green toxicology is a novel approach increasingly applied for the development of materials and chemicals that are more benign to the environment and human health than their conventional counterparts. It includes predictive eco-toxicological assessments of chemicals during the early developmental process to exclude adverse effects. In the present study, two guanidine zinc catalysts for the ring-opening polymerization of lactide were investigated using eco-toxicological tools. Namely, the fish embryo toxicity assay for teratogenic effects, the ER (α) CALUX assay for endocrine activity and the Ames fluctuation assay for mutagenic potential were applied. Both complexes showed no endocrine activity, mutagenicity or acute aquatic toxicity, however a delayed hatch could be observed, therefore suggesting potential effects on a molecular level. This proof-of-concept study aims to assess the toxicity of guanidine zinc catalysts and is a first step towards the incorporation of toxicological assessments into chemical developmental processes to achieve a sustainable and safe production of catalysts.

Evidence of increased estrogenicity upon metabolism of Bisphenol F - Elucidation of the key metabolites.

The increasing concern over bisphenol A (BPA) has directed much attention toward bisphenol F (BPF) and bisphenol S (BPS) as BPA alternatives for the development of "BPA-free" products. Consequently, BPS and BPF were frequently detected in surface water, sediment, sewage effluent, indoor dust, and even in food and biological fluids in humans. Thus, environmental researches start to focus on the potential environmental risks of BPA alternatives. While the estrogenically active metabolites and the specific estrogenically active structure are still unknown. In this study, the MTT assay on acute cytotoxicity and the recombinant transactivation assay were carried out to determine whether BPF and BPS are suitable alternatives to BPA. Our results show that the cytotoxic and estrogenic activities of BPS and BPF are lower than those of BPA. However, after the addition of a rat liver homogenate to simulate mammal metabolism, BPF exhibited higher estrogenic activity than BPA. To identify the chemical structures and estrogen receptor binding affinities of active estrogenic metabolites, LC-MS, MetaPrint2D(-React), and VirtualToxLab were integrated. The observed results indicated that the para-hydroxylated BPF and BPF-OCH3 might have strong ER binding affinities. These results demonstrate that metabolization is important to consider upon investigating endocrine disruption of chemicals getting into contact with humans, such as in dental sealing or food packaging. Alternatives to potentially hazardous substances should be thoroughly tested prior to use.

The EU Green Deal's ambition for a toxic-free environment: Filling the gap for science-based policymaking.

Around the world, many ambitious environmental conventions and regulations have been implemented over recent decades. Despite this, the environment is still deteriorating. An increase in the volume and diversity of chemicals is one of the main drivers of this deterioration, of which biodiversity loss is a telling indicator. In response to this situation, in October 2020, a chemicals strategy for sustainability (CSS) was published in the EU. The CSS is the first regional framework aiming to address chemical pollution in a holistic manner. The CSS covers the complete lifecycle of a chemical, including the design of better substances and remediation options, to remove chemicals from the environment. The strategy contains terms, such as a "toxic-free environment," for which no clear definition exists, potentially hampering the implementation of the CSS. In this paper, a definition for a "toxic-free environment" is proposed on the basis of a survey and a discussion held at the 2020 SETAC Europe Annual Meeting. In addition, key issues that are absent from the CSS but are considered to be key for the realization of a toxic-free environment are identified. To achieve the policy goals, it is recommended to align the definition of risk across the different chemical legislations, to establish a platform for open data and data sharing, and to increase the utility and use of novel scientific findings in policymaking, through the development of a strong science to regulation feedback mechanism and vice versa. The paper concludes that environmental scientists have the tools to address the key challenges presented in the CSS. However, an extra step is needed by both policymakers and scientists to develop methods, processes and tools, to increase the robustness and transparency of deliberation processes, and the utility of science. Integr Environ Assess Manag 2021;17:1105-1113. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Open Science in regulatory environmental risk assessment.

A possible way to alleviate the public skepticism toward regulatory science is to increase transparency by making all data and value judgments used in regulatory decision making accessible for public interpretation, ideally early on in the process, and following the concepts of Open Science. This paper discusses the opportunities and challenges in strengthening Open Science initiatives in regulatory environmental risk assessment (ERA). In this discussion paper, we argue that the benefits associated with Open Science in regulatory ERA far outweigh its perceived risks. All stakeholders involved in regulatory ERA (e.g., governmental regulatory authorities, private sector, academia, and nongovernmental organizations), as well as professional organizations like the Society of Environmental Toxicology and Chemistry, can play a key role in supporting the Open Science initiative, by promoting the use of recommended reporting criteria for reliability and relevance of data and tools used in ERA, and by developing a communication strategy for both professionals and nonprofessionals to transparently explain the socioeconomic value judgments and scientific principles underlying regulatory ERA. Integr Environ Assess Manag 2021;17:1229-1242. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Bioanalytical equivalents and relative potencies for predicting the biological effects of mixtures.

Bioanalytical equivalents (BEQs) of mixtures and environmental samples are widely used to reflect the potential threat of pollutants in the environment and can be obtained by bioassays or using chemical analysis combined with relative potencies (REPs). In this study, the relationships between bioassay-detected BEQs (Bio-BEQs) and chemically analyzed BEQs (Chem-BEQs) were studied. BEQs and REPs are correlated with effect level and the concentration-response curves of the reference standard and sample. Thus, effect level (e.g., EC10, EC25 and EC50) should be addressed for the BEQ values obtained from bioassays or chemical analyses. The previous prerequisites for REPs application (i.e., curves that are parallel and have the same maximum response) are redundant, and the use of REPs for the calculation of BEQs or in risk assessment should instead be based on the same effect level. For a complex mixture with many components, all active components can be regarded as dilutions of a standard compound for inducing a specific effect. Relative toxicity estimates based on EC50 ignore the contribution of weak-active components with maximum response below EC50 of the reference standard, especially in complex mixtures or environmental samples. REPs based on an effect level EC10 that can be clearly discriminated from background response are recommended for BEQ calculation. As an example, the aryl hydrocarbon receptor (AhR)-mediated activity of US EPA priority polycyclic aromatic hydrocarbons (PAHs) in RTL-W1 cells was used to assess the reliability of REPs for mixture toxicity prediction based on the effect level EC10.

Comparative toxicity assessment of in situ burn residues to initial and dispersed heavy fuel oil using zebrafish embryos as test organisms.

In situ burning (ISB) is discussed to be one of the most suitable response strategies to combat oil spills in extreme conditions. After burning, a highly viscous and sticky residue is left and may over time pose a risk of exposing aquatic biota to toxic oil compounds. Scientific information about the impact of burn residues on the environment is scarce. In this context, a comprehensive ISB field experiment with approx. 1000L IFO 180 was conducted in a fjord in Greenland. The present study investigated the toxicity of collected ISB residues to early life stages of zebrafish (Danio rerio) as a model for potentially exposed pelagic organisms. The toxicity of ISB residues on zebrafish embryos was compared with the toxicity of the initial (unweathered) IFO 180 and chemically dispersed IFO 180. Morphological malformations, hatching success, swimming behavior, and biomarkers for exposure (CYP1A activity, AChE inhibition) were evaluated in order to cover the toxic response on different biological organization levels. Across all endpoints, ISB residues did not induce greater toxicity in zebrafish embryos compared with the initial oil. The application of a chemical dispersant increased the acute toxicity most likely due to a higher bioavailability of dissolved and particulate oil components. The results provide insight into the adverse effects of ISB residues on sensitive life stages of fish in comparison with chemical dispersant application.

Combining Different In Vitro Bioassays to Evaluate Genotoxicity of Water-Accommodated Fractions from Petroleum Products.

Genotoxicity assessment is of high relevance for crude and refined petroleum products, since oil compounds are known to cause DNA damage with severe consequences for aquatic biota as demonstrated in long-term monitoring studies. This study aimed at the optimization and evaluation of small-scale higher-throughput assays (Ames fluctuation, micronucleus, Nrf2-CALUX®) covering different mechanistic endpoints as first screening tools for genotoxicity assessment of oils. Cells were exposed to native and chemically dispersed water-accommodated fractions (WAFs) of three oil types varying in their processing degree. Independent of an exogenous metabolic activation system, WAF compounds induced neither base exchange nor frame shift mutations in bacterial strains. However, significantly increased chromosomal aberrations in zebrafish liver (ZF-L) cells were observed. Oxidative stress was indicated for some treatments and was not correlated with observed DNA damage. Application of a chemical dispersant increased the genotoxic potential rather by the increased bioavailability of dissolved and particulate oil compounds. Nonetheless, the dispersant induced a clear oxidative stress response, indicating a relevance for general toxic stress. Results showed that the combination of different in vitro assays is important for a reliable genotoxicity assessment. Especially, the ZF-L capable of active metabolism and DNA repair seems to be a promising model for WAF testing.

Microplastics Lead to Hyperactive Swimming Behaviour in Adult Zebrafish.

Microplastic pollution has drawn the attention of both scientists and the public regarding their potential ecotoxicological risks. In the present study, we carried out aqueous exposure experiments to adult zebrafish with polystyrene microplastics (5 µm) at a wide range of concentrations (0.001-20 mg/L, equals to 14.5∼2.9 × 105 particles/mL). Our results showed the gastrointestinal tract (GIT) was the dominant microplastic accumulation site in zebrafish, followed by the gill, whereas no microplastics were detected in the brain or muscle. Microplastic accumulation in GIT did not cause obvious damages to intestinal villi in general. However, the thickness of muscularis layer in the foregut reduced by 32% after 1 mg/L (1.45 × 104 particles/mL) microplastic exposure. As there were no signs of oxidative stress or other histological changes found in the fish, we further investigated the energy-supplying influential factors. We found that the zebrafish became hyperactive after microplastic exposure, whose swimming distance had increased to 1.3-2.4 folds than that of control, and also stayed at manic and active states much longer. The fish behavioural alteration is probably attributed to the particulate matter stimulation and the up-regulation of estrogen contents. Results also showed that the excessive movements of zebrafish also led to decreased glucose and acetaldehyde metabolite contents and increased amino acid amounts, which further proved the shortage of energy-supplying substances. Therefore, the present study suggests that micro-sized microplastics can induce obvious behavioural abnormality at concentrations that some other toxicological endpoints may not warn effects.

Differences in biomarker and behavioral responses to native and chemically dispersed crude and refined fossil oils in zebrafish early life stages.

Petroleum products including crude oils and refined distillates are unique environmental pollutants consisting of thousands of compounds with varying physical-chemical properties and resulting toxicity for aquatic biota. Hence, for a reliable risk assessment individual petroleum product toxicity profiles are needed. Furthermore, the influence of oil spill response strategies like the application of chemical dispersants has to be implemented. The present study addressed the toxicity of water-accommodated fractions (WAFs) of two different oil types on fish early life stages on different biological organization levels in the laboratory model species Danio rerio. Experiments with a 3rd generation dispersant used in loading rated resembling the exposure in experiments with chemically dispersed oils were included, enabling a direct comparability of results. This approach is of high importance as especially the investigation of dispersant toxicity in relevant exposure concentrations is rather scarce. Zebrafish embryos were exposed to different WAFs shortly after and up to 120 hour post fertilization (hpf). Besides phenotypic effects including edema and spine deformations, reduced responses to dark stimuli, increased CYP1A activity and marginal AChE inhibition were observed in sublethal effect concentrations. Both oil types had varying strength of toxicity, which did not correlate with corresponding chemical analysis of target PAHs. Chemically dispersed oils induced stronger acute toxicity in zebrafish embryos compared to native (initial) oil exposure, which was further reflected by very low exposure concentrations for biomarker endpoints. Based on a comparison to the dispersant alone, a higher toxicity of dispersed oils was related to a combination of dispersant toxicity and an elevated crude oil compound bioavailability, due to dispersion-related partitioning kinetics. In contrast to LEWAF and CEWAF neither typical morphological effects nor mechanism-specific toxicity were observed for the dispersant alone, indicating narcosis as the responsible cause of effects.

Receptor-mediated estrogenicity of native and chemically dispersed crude oil determined using adapted microscale reporter gene assays.

Endocrine disrupting compounds (EDCs) emerged as a major concern for water quality in the last decade and have been studied extensively since. Besides typical natural and synthetic estrogens also petroleum product compounds such as some PAHs have been identified as potential EDCs, revealing endocrine disruption to be a relevant mode of action for crude oil toxicity. Hence, in the context of a comprehensive retro- or prospective risk assessment of oil spills the implementation of mechanism-specific toxicity such as endocrine disruption is of high importance. To evaluate the exposure risk for the aquatic biota, research focuses on water-soluble fractions underlying an oil slick that could be simulated via water-accommodated fractions (WAF). Against this background human (ERα-CALUX®) and yeast based (A-YES®) reporter gene bioassays were successfully optimized for the application in estrogenicity evaluation of the water-accommodated fraction (WAF) from a crude oil. Combining different approaches, the estrogenicity of the WAFs from a naphthenic North Sea crude oil was tested with and without the addition of a chemical dispersant addressing specific aspects of estrogenicity including the influence of biotransformation capacities and different salinity conditions. Both the WAF free from droplets (LEWAF) as well as the chemically dispersed WAF (CEWAF) gave indications of an ER-mediated estrogenicity with much stronger ERα agonists in the CEWAF treatment. Resulting estradiol equivalents of the WAFs were above the established effect-based trigger values for both bioassays. Results indicate that the dispersant rather increased the fraction of ER-activating crude oil compounds instead of interacting with the receptor itself. Only slight changes in estrogenic responses were observed when cells capable of active metabolism (T47D) were used instead of cells without endogenous metabolism (U2-OS) in the recombinant ER transactivation CALUX assay. With the yeast cells a higher estrogenic activity was observed in the experiments under elevated salinity conditions (6‰), which was in contrast to previous expectations due to typical decrease in dissolved PAH fraction with increasing salinity (salting-out effect) but might be related to increased cell sensitivity.

Toxicity of 10 organic micropollutants and their mixture: Implications for aquatic risk assessment.

Micropollutants, as a serious water pollution issue, raise considerable toxicological concerns, particularly when present as components of complex mixtures. Due to the interactions of environmental pollution components (contaminant), the micropollutant problem is increasingly complex, thus, water quality of organic chemical contamination assessed substance-by-substance might lead to underestimation in aquatic environmental risk assessment. To assess the aquatic environmental risk of micropollutants mixture, a total of 10 organic micropollutants were selected and analysed by an approach of integration of literature data, laboratory experiments and prediction techniques. The experiment results showed that all 10 micropollutants were capable of causing toxicity in zebrafish embryos, aquatic invertebrates and algae with the LC50 (50% lethal concentration) values from 1.14 mg/L to 14.37 mg/L. Triclosan, carbamazepine, diazinon and diuron were the most hazardous compounds in the Danube River and the Rhine River. The artificial mixture presented a strong antagonistic relationship, which demonstrated an independent action (IA) model of the mixture. Based on the observed toxicity data, the risk quotients (RQs) of environmental mixtures of the Danube River and the Rhine River were extrapolated. It can be concluded that the micropollutant mixture may pose a potential risk for aquatic ecosystems with the present environmentally measured concentrations in the Danube River and Rhine River. Mixture risk assessment results suggested that the toxicity of studied chemicals might be induced by dissimilar actions, which is in agreement with the mixture toxicity prediction of the IA model. The observed findings could be useful to establish an overview of the pressures, vision, measures and expectations for hazardous substances pollution, which can help in making to informed decisions to reduce the concentration and bioactive fraction of pollutants.

Optimization of screening-level risk assessment and priority selection of emerging pollutants - The case of pharmaceuticals in European surface waters.

Pharmaceuticals in surface waters have raised significant concern in recent years for their potential environmental effects. This study identified that at present a total of 477 substances (including 66 metabolites and transformation products) have been analyzed in European surface waters. Around 60% (284) of these compounds belonging to 16 different therapeutic groups were positively detected in one or more of 33 European countries. To conveniently and effectively prioritize potential high-risk compounds, an optimized method that considers the frequency of concentrations above predicted no effects levels was developed on the basis of the traditional method, and it was then used to identify and screen candidate priority pollutants in European surface waters. The results proved the feasibility and advantages of the optimized method. Pharmaceuticals detected in European surface waters were classified into 5 categories (high, moderate, endurable, negligible and safe) depending on their potential environmental effects and the distribution of pharmaceuticals. Circa 9% (45 out of 477) analyzed compounds showed a potential environmental risk to aquatic ecosystems. Among these 45 compounds, 12 compounds were indicated to have high environmental risk in aquatic environments, while 17 and 7 compounds showed moderate and small-scale environmental risks, respectively.

Is Hyalella azteca a Suitable Model Leaf-Shredding Benthic Crustacean for Testing the Toxicity of Sediment-Associated Metals in Europe?

The leaf-shredding crustacean Hyalella azteca, which is indigenous to Northern and Central America, is used to assess environmental risks associated with (metal-)contaminated sediments and to propose sediment quality standards also in Europe. Yet, it is unknown if H. azteca is protective for European crustacean shredders. We thus compared the sensitivity of H. azteca with that of the European species Asellus aquaticus and Gammarus fossarum towards copper- and cadmium-contaminated sediments (prepared according to OECD 218) under laboratory conditions employing mortality and leaf consumption as endpoints. H. azteca either reacted approximately fourfold more sensitive than the most tolerant tested species (as for cadmium) or its sensitivity was only 1.6 times lower than the highest sensitivity determined (as for copper), which should be covered by safety factors applied during risk assessments. Therefore, the results for the sediment type and the two heavy metals tested during the present study in combination with the existence of standardized testing protocols, their ease of culture, and short generation time, suggest H. azteca as suitable crustacean model shredder for assessing the toxicity of sediment-associated metals in Europe.

Behavioral profile alterations in zebrafish larvae exposed to environmentally relevant concentrations of eight priority pharmaceuticals.

Although the effects of pharmaceuticals on aquatic organisms have been widely investigated during the last decades, toxic effects, especially delayed toxicity, during the developmental stage at environmental relevant concentrations were rarely known. In this study, a sensitive assay based on behavioral alterations was used for studying the delayed toxicity during the developmental stage on zebrafish embryos. Eight pharmaceuticals that were frequently detected with concentrations ranging from ng/l to µg/l were screened for this study. Behavioral alterations of zebrafish at 118 hpf (hours post fertilization) after exposing to eight single pharmaceuticals with concentrations in the ranges of environmental detected and their mixtures during embryonic development (2-50 h post fertilization, hpf) were observed. Multiple endpoints, including mortality, hatching rate, swimming speed and angular velocity were evaluated. Results showed that behavioral profile alterations in zebrafish larvae are promising for predicting delayed sublethal effects of chemicals. Delayed hatch was observed at 72 hpf following embryonic exposure to triclosan (1 µg/l) and carbamazepine (100 µg/l) up to 50 hpf. The zebrafish larval locomotor behavior following embryonic exposure to 0.1 µg/l triclosan and 1 µg/l caffeine in the early stages of development (2-50 hpf) was altered. Furthermore, the effects of the mixture of 8 pharmaceuticals each with the highest environmental concentration on larval behavior were observed during embryonic development. Generally, this study showed that the effects of pharmaceuticals singly or their mixtures in surface waters cannot be ignored.

Integrated zebrafish-based tests as an investigation strategy for water quality assessment.

Water pollution risks to human health and the environment are emerging as serious concerns in the European Union and worldwide. With the aim to achieve good ecological and chemical status of all European water bodies, the "European Water Framework Directive" (WFD) was enacted. With the framework, bioanalytical techniques have been recognized as an important aspect. However, there are limitations to the application of bioassays directly for water quality assessment. Such approaches often fail to identify pollutants of concern, since the defined priority and monitored pollutants often fail to explain the observed toxicity. In this study, we integrated an effect-based risk assessment with a zebrafish-based investigation strategy to evaluate water sample extracts and fractions collected from the Danube. Four tiered bioassays were implemented, namely RNA-level gene expression assay, protein-level ethoxyresorufin-O-deethylase (EROD) assay, cell-level micronucleus assay and organism-level fish embryo test (FET). The results show that teratogenicity and lethality during embryonic development might be induced by molecular or cellular damages mediated by the aryl hydrocarbon receptor (AhR) -mediated activity, estrogenic activity and genotoxic activity. With the combination of high-throughput fractionation, this effect-based strategy elucidated the major responsible mixtures of each specific toxic response. In particularly, the most toxic mixture in faction F4, covering a log Kow range from 2.83 to 3.42, was composed by 12 chemicals, which were then evaluated as a designed mixture. Our study applied tiered bioassays with zebrafish to avoid interspecies differences and highlights effect-based approaches to address toxic mixtures in water samples. This strategy can be applied for large throughput screenings to support the main toxic compounds identification in water quality assessment.