Interlaboratory prevalidation of a new in vitro transcriptional activation assay for the screening of (anti-)androgenic activity of chemicals using the UALH-hAR cell line.

We report an interlaboratory evaluation of a recently developed androgen receptor (AR) transactivation assay using the UALH-hAR reporter cell line that stably expresses the luciferase gene under the transcriptional control of androgen receptor elements (AREs) with no glucocorticoid receptor (GR) crosstalk. Herein, a two-step prevalidation study involving three laboratories was conducted to assess performance criteria of the method such as transferability as well as robustness, sensitivity, and specificity. The first step consisted in the validation of the transfer of the cell line to participant laboratories through the testing of three reference chemicals: the AR agonist dihydrotestosterone, the AR antagonist hydroxyflutamide and the glucocorticoid dexamethasone. Secondly, a blinded study was conducted by screening a selection of ten chemicals, including four AR agonists, five AR antagonists, and one non-active chemical. All test compounds yielded the same activity profiles in all laboratories. The logEC50 (agonist assay) or logIC50 (antagonist assay) were in the same range, with intra-laboratory coefficients of variation (CVs) of 0.1-3.4% and interlaboratory CVs of 1-4%, indicating very good within- and between-laboratory reproducibility. Our results were consistent with literature and regulatory data (OECD TG458). Overall, this interlaboratory study demonstrated that the UALH-hAR assay is transferable, produces reliable, accurate and specific (anti)androgenic activity of chemicals, and can be considered for further regulatory validation.

Biological effect and chemical monitoring of Watch List substances in European surface waters: Steroidal estrogens and diclofenac - Effect-based methods for monitoring frameworks.

Three steroidal estrogens, 17α-ethinylestradiol (EE2), 17ß-estradiol (E2), estrone (E1), and the non-steroidal anti-inflammatory drug (NSAID), diclofenac have been included in the first Watch List of the Water Framework Directive (WFD, EU Directive 2000/60/EC, EU Implementing Decision 2015/495). This triggered the need for more EU-wide surface water monitoring data on these micropollutants, before they can be considered for inclusion in the list of priority substances regularly monitored in aquatic ecosystems. The revision of the priority substance list of the WFD offers the opportunity to incorporate more holistic bioanalytical approaches, such as effect-based monitoring, alongside single substance chemical monitoring. Effect-based methods (EBMs) are able to measure total biological activities (e.g., estrogenic activity or cyxlooxygenase [COX]-inhibition) of specific group of substances (such as estrogens and NSAIDs) in the aquatic environment at low concentrations (pg/L). This makes them potential tools for a cost-effective and ecotoxicologically comprehensive water quality assessment. In parallel, the use of such methods could build a bridge from chemical status assessments towards ecological status assessments by adressing mixture effects for relevant modes of action. Our study aimed to assess the suitability of implementing EBMs in the WFD, by conducting a large-scale sampling and analysis campaign of more than 70 surface waters across Europe. This resulted in the generation of high-quality chemical and effect-based monitoring data for the selected Watch List substances. Overall, water samples contained low estrogenicity (0.01-1.3 ng E2-Equivalent/L) and a range of COX-inhibition activity similar to previously reported levels (12-1600 ng Diclofenac-Equivalent/L). Comparison between effect-based and conventional analytical chemical methods showed that the chemical analytical approach for steroidal estrogens resulted in more (76%) non-quantifiable data, i.e., concentrations were below detection limits, compared to the EBMs (28%). These results demonstrate the excellent and sensitive screening capability of EBMs.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Human and Zebrafish Nuclear Progesterone Receptors Are Differently Activated by Manifold Progestins.

The environmental risk of natural and synthetic ligands of the nuclear progesterone receptor (nPR) has been pointed out, however there is still a lack of mechanistic information regarding their ability to interact with nuclear PR in aquatic species. To identify possible interspecies differences, we assessed in vitro the ability of manifold progestins to transactivate zebrafish (zf) and human (h) PRs, using two established reporter cell lines, U2OS-zfPR and HELN-hPR, respectively. Reference ligands highlighted some differences between the two receptors. The reference human agonist ligands promegestone and progesterone induced luciferase activity in both cell lines in a concentration-dependent manner, whereas the natural zebrafish progestin 17α,20ß-dihydroxy-4-pregnen-3-one activated zfPR but not hPR. The potent human PR antagonist mifepristone (RU486) blocked PR-induced luciferase in both cell models but with different potencies. In addition, a set of 22 synthetic progestins were screened on the two cell lines. Interestingly, all of the tested compounds activated hPR in the HELN-hPR cell line, whereas the majority of them acted as zfPR antagonists in U2OS-zfPR. Such zfPR-specific response was further confirmed in zebrafish liver cells. This study provides novel information regarding the activity of a large set of progestins on human and zebrafish PR and highlights major interspecies differences in their activity, which may result in differential effects of progestins between fish and humans.

Estrogenic activity of surface waters using zebrafish- and human-based in vitro assays: The Danube as a case-study.

Most in vitro reporter gene assays used to assess estrogenic contamination are based on human estrogen receptor α (hERα) activation. However, fish bioassays can have distinct response to estrogenic chemicals and mixtures, questioning the relevance of human-based bioassays for assessing risk to this species. In this study, zebrafish liver cells stably expressing zebrafish ERß2 (ZELHß2) and human breast cancer cells expressing hERα (MELN) were used to quantify the estrogenic activity of 25 surface water samples of the Danube River, for which chemicals have been previously quantified. Most samples had a low estrogenic activity below 0.1 ng/L 17ß-estradiol-equivalents that was more often detected by MELN cells, while ZELHß2 response tend to be lower than predicted based on the chemicals identified. Nevertheless, both bioassays quantified well a higher estrogenic activity at two sites, which was confirmed in vivo using a transgenic zebrafish assay. The results are discussed considering the effect-based trigger values proposed for water quality monitoring.

Chronic simultaneous exposure of common carp (Cyprinus carpio) from embryonic to juvenile stage to drospirenone and gestodene at low ng/L level caused intersex.

Synthetic progestins are emerging contaminants of the aquatic environment with endocrine disrupting potential. The main aim of the present study was to investigate the effects of the synthetic progestins gestodene, and drospirenone on sex differentiation in common carp (Cyprinus carpio) by histological analysis. To gain insights into the mechanisms behind the observations from the in vivo experiment on sex differentiation, we analyzed expression of genes involved in hypothalamus-pituitary-gonad (HPG) and hypothalamus-pituitary-thyroid (HPT) axes, histology of hepatopancreas, and in vitro bioassays. Carp were continuously exposed to concentrations of 2 ng/L of single progestins (gestodene or drospirenone) or to their mixture at concentration 2 ng/L of each. The exposure started 24 h after fertilization of eggs and concluded 160 days post-hatching. Our results showed that exposure of common carp to a binary mixture of drospirenone and gestodene caused increased incidence of intersex (32%) when compared to clean water and solvent control groups (both 3%). Intersex most probably was induced by a combination of multiple modes of action of the studied substances, namely anti-gonadotropic activity, interference with androgen receptor, and potentially also with HPT axis or estrogen receptor.

Differential activity of BPA, BPAF and BPC on zebrafish estrogen receptors in vitro and in vivo.

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 × ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfERα-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfERα-dependent) at lower concentrations, and in the liver (zfERß-dependent) at higher concentrations. BPC induced zfERß-mediated luciferase expression in vitro, and the zfERß agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfERα, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.

Monitoring estrogenic activities of waste and surface waters using a novel in vivo zebrafish embryonic (EASZY) assay: Comparison with in vitro cell-based assays and determination of effect-based trigger values.

This study reports the use of the recently developed EASZY assay that uses transgenic cyp19a1b-GFP zebrafish (Danio rerio) embryos to assess in vivo estrogenic activity of 33 surface (SW) and waste water (WW) samples collected across Europe that were previously well-characterized for estrogen hormones and in vitro estrogenic activity. We showed that 18 out of the 33 SW and WW samples induced estrogenic responses in the EASZY assay leading to a significant and concentration-dependent up-regulation of the ER-regulated cyp19a1b gene expression in the developing brain. The in vivo 17ß-estradiol-equivalents (EEQs) were highly correlated with, both, the chemical analytical risk quotient (RQ) based on steroidal estrogen concentrations and EEQs reported from five different in vitro reporter gene assays. Regression analyses between the vitro and in vivo effect concentrations allowed us to determine an optimal cut-off value for each in vitro assay, above which in vivo responses were observed. These in vitro assay-specific effect-based trigger values (EBTs), ranging from 0.28 to 0.58 ng EEQ/L define the sensitivity and specificity of the individual in vitro assays for predicting a risk associated with substances acting through the same mode of action in water samples. Altogether, this study demonstrates the toxicological relevance of in vitro-based assessment of estrogenic activity and recommends the use of such in vitro/in vivo comparative approach to refine and validate EBTs for mechanism-based bioassays.

Combined effects of environmental xeno-estrogens within multi-component mixtures: Comparison of in vitro human- and zebrafish-based estrogenicity bioassays.

Some recent studies showed that in vitro bioassays based on fish or human estrogen receptor (ER) activation may have distinct responses to environmental samples, highlighting the need to better understand bioassay-specific ER response to environmental mixtures. For this purpose, we investigated a 12-compound mixture in two mixture ratios (M1 and M2) on zebrafish (zf) liver cells stably expressing zfERα (ZELHα cells) or zfERß2 (ZELHß2 cells) and on human ER-reporter gene (MELN) cells. The mixture included the well-known ER ligands bisphenol A (BPA) and genistein (GEN), and other compounds representatives of a freshwater background contamination. In this context, the study aimed at assessing the robustness of concentration addition (CA) model and the potential confounding influence of other chemicals by testing subgroups of ER activators, ER inhibitors or ER activators and inhibitors combined. Individual chemical testing showed a higher prevalence of ER inhibitors in zebrafish than human cells (e.g. propiconazole), and some chemicals inhibited zfER but activated hER response (e.g. benzo(a)pyrene, triphenylphosphate). The estrogenic activity of M1 and M2 was well predicted by CA in MELN cells, whereas it was significantly lower than predicted in ZELHß2 cells, contrasting with the additive effects observed for BPA and GEN binary mixtures. When testing the subgroups of ER activators and inhibitors combined, the deviation from additivity in ZELHß2 cells was caused by zebrafish-specific inhibiting chemicals. This study provides novel information on the ability of environmental pollutants to interfere with zfER signalling and shows that non-estrogenic chemicals can influence the response to a mixture of xeno-estrogens in a bioassay-specific manner.

Triclosan Lacks Anti-Estrogenic Effects in Zebrafish Cells but Modulates Estrogen Response in Zebrafish Embryos.

Triclosan (TCS), an antimicrobial agent widely found in the aquatic environment, is suspected to act as an endocrine disrupting compound, however mechanistic information is lacking in regards to aquatic species. This study assessed the ability of TCS to interfere with estrogen receptor (ER) transcriptional activity, in zebrafish-specific in vitro and in vivo reporter gene assays. We report that TCS exhibits a lack of either agonistic or antagonistic effects on a panel of ER-expressing zebrafish (ZELH-zfERα and -zfERß) and human (MELN) cell lines. At the organism level, TCS at concentrations of up to 0.3 µM had no effect on ER-regulated brain aromatase gene expression in transgenic cyp19a1b-GFP zebrafish embryos. At a concentration of 1 µM, TCS interfered with the E2 response in an ambivalent manner by potentializing a low E2 response (0.625 nM), but decreasing a high E2 response (10 nM). Altogether, our study suggests that while modulation of ER-regulated genes by TCS may occur in zebrafish, it does so irrespective of a direct binding and activation of zfERs.

Mixture Concentration-Response Modeling Reveals Antagonistic Effects of Estradiol and Genistein in Combination on Brain Aromatase Gene (cyp19a1b) in Zebrafish.

Comprehension of compound interactions in mixtures is of increasing interest to scientists, especially from a perspective of mixture risk assessment. However, most of conducted studies have been dedicated to the effects on gonads, while only few of them were. interested in the effects on the central nervous system which is a known target for estrogenic compounds. In the present study, the effects of estradiol (E2), a natural estrogen, and genistein (GEN), a phyto-estrogen, on the brain ER-regulated cyp19a1b gene in radial glial cells were investigated alone and in mixtures. For that, zebrafish-specific in vitro and in vivo bioassays were used. In U251-MG transactivation assays, E2 and GEN produced antagonistic effects at low mixture concentrations. In the cyp19a1b-GFP transgenic zebrafish, this antagonism was observed at all ratios and all concentrations of mixtures, confirming the in vitro effects. In the present study, we confirm (i) that our in vitro and in vivo biological models are valuable complementary tools to assess the estrogenic potency of chemicals both alone and in mixtures; (ii) the usefulness of the ray design approach combined with the concentration-addition modeling to highlight interactions between mixture components.

Liver histopathology and biochemical biomarkers in Gobius niger and Zosterisessor ophiocephalus from polluted and non-polluted Tunisian lagoons (Southern Mediterranean Sea).

The aim of this study was to appraise the response of a multi-marker approach in fish species, Gobius niger and Zosterisessor ophiocephalus, in a polluted lagoon (Bizerte lagoon: MB and ML sites) and in a reference site (Ghar-El-Melh lagoon entrance) by the analysis of physiological indexes, liver histopathology and some biochemical biomarkers. The results showed liver hypertrophy in fish collected from Bizerte lagoon as well as many non-specific lesions, unlike the reference site. All Bizerte lagoon sites had the same prevalence of histopathological lesions, but the mean intensity (MI) of parasites seemed to be more sensible as an indicator of pollution levels. Indeed, parasite MI was more important in MB site that has a higher pollution level. Also, biochemical biomarkers showed an induction in Bizerte lagoon sites with some differences within sites and species. The impact of the continuous release of pollution on the biomarker's response is discussed.

Solid-phase extraction as sample preparation of water samples for cell-based and other in vitro bioassays.

In vitro bioassays are increasingly used for water quality monitoring. Surface water samples often need to be enriched to observe an effect and solid-phase extraction (SPE) is commonly applied for this purpose. The applied methods are typically optimised for the recovery of target chemicals and not for effect recovery for bioassays. A review of the few studies that have evaluated SPE recovery for bioassays showed a lack of experimentally determined recoveries. Therefore, we systematically measured effect recovery of a mixture of 579 organic chemicals covering a wide range of physicochemical properties that were spiked into a pristine water sample and extracted using large volume solid-phase extraction (LVSPE). Assays indicative of activation of xenobiotic metabolism, hormone receptor-mediated effects and adaptive stress responses were applied, with non-specific effects determined through cytotoxicity measurements. Overall, effect recovery was found to be similar to chemical recovery for the majority of bioassays and LVSPE blanks had no effect. Multi-layer SPE exhibited greater recovery of spiked chemicals compared to LVSPE, but the blanks triggered cytotoxicity at high enrichment. Chemical recovery data together with single chemical effect data were used to retrospectively estimate with reverse recovery modelling that there was typically less than 30% effect loss expected due to reduced SPE recovery in published surface water monitoring studies. The combination of targeted experiments and mixture modelling clearly shows the utility of SPE as a sample preparation method for surface water samples, but also emphasizes the need for adequate controls when extraction methods are adapted from chemical analysis workflows.

Mixture effects in samples of multiple contaminants - An inter-laboratory study with manifold bioassays.

Chemicals in the environment occur in mixtures rather than as individual entities. Environmental quality monitoring thus faces the challenge to comprehensively assess a multitude of contaminants and potential adverse effects. Effect-based methods have been suggested as complements to chemical analytical characterisation of complex pollution patterns. The regularly observed discrepancy between chemical and biological assessments of adverse effects due to contaminants in the field may be either due to unidentified contaminants or result from interactions of compounds in mixtures. Here, we present an interlaboratory study where individual compounds and their mixtures were investigated by extensive concentration-effect analysis using 19 different bioassays. The assay panel consisted of 5 whole organism assays measuring apical effects and 14 cell- and organism-based bioassays with more specific effect observations. Twelve organic water pollutants of diverse structure and unique known modes of action were studied individually and as mixtures mirroring exposure scenarios in freshwaters. We compared the observed mixture effects against component-based mixture effect predictions derived from additivity expectations (assumption of non-interaction). Most of the assays detected the mixture response of the active components as predicted even against a background of other inactive contaminants. When none of the mixture components showed any activity by themselves then the mixture also was without effects. The mixture effects observed using apical endpoints fell in the middle of a prediction window defined by the additivity predictions for concentration addition and independent action, reflecting well the diversity of the anticipated modes of action. In one case, an unexpectedly reduced solubility of one of the mixture components led to mixture responses that fell short of the predictions of both additivity mixture models. The majority of the specific cell- and organism-based endpoints produced mixture responses in agreement with the additivity expectation of concentration addition. Exceptionally, expected (additive) mixture response did not occur due to masking effects such as general toxicity from other compounds. Generally, deviations from an additivity expectation could be explained due to experimental factors, specific limitations of the effect endpoint or masking side effects such as cytotoxicity in in vitro assays. The majority of bioassays were able to quantitatively detect the predicted non-interactive, additive combined effect of the specifically bioactive compounds against a background of complex mixture of other chemicals in the sample. This supports the use of a combination of chemical and bioanalytical monitoring tools for the identification of chemicals that drive a specific mixture effect. Furthermore, we demonstrated that a panel of bioassays can provide a diverse profile of effect responses to a complex contaminated sample. This could be extended towards representing mixture adverse outcome pathways. Our findings support the ongoing development of bioanalytical tools for (i) compiling comprehensive effect-based batteries for water quality assessment, (ii) designing tailored surveillance methods to safeguard specific water uses, and (iii) devising strategies for effect-based diagnosis of complex contamination.

Effect-based monitoring of the Danube River using mobile passive sampling.

Many aquatic pollutants can be present at low concentrations, but their mixtures can still affect health or behavior of exposed organisms. In this study, toxicological and chemical analyses were combined for spatial contamination profiling using an innovative passive sampling approach. A novel Dynamic Passive Sampler (DPS) was employed as a mobile sampler from a ship cruising along 2130km of the Danube river during the Joint Danube Survey 3 (JDS3). The sampling was performed in eight subsequent river stretches with two types of complementary passive samplers: silicone rubber sheets (SR) used for non-polar chemicals and SDB-RPS Empore™ disks (ED) for more hydrophilic compounds. Besides extensive chemical analyses, the bioactivity of samples was characterized by a battery of reporter gene bioassays. Cross-calibration of the employed passive samplers enabled robust estimation of water concentrations applicable for compounds with a wide range of physicochemical properties. DPS was suitable for sampling of water contaminants even at pgL-1 levels, with 209 of 267 analyzed compounds detected in the samples. Biological effects were detected in both ED and SR extracts across all river stretches by bioassays focused on xenobiotic metabolism mediated by the aryl hydrocarbon and pregnane X receptors, endocrine disruptive potential mediated by estrogen and androgen receptors and the oxidative stress response. The bioassay responses expressed as bioanalytical equivalent concentrations (BEQbio) were comparable with data obtained from large volume active sampling. The extracts of the ED samplers were more biologically active than extracts of SR samplers. Except of estrogenicity, where the analyzed chemicals explained on average 62% of the effects in ED samples, the detected chemicals explained <8% of BEQbio values. The study shows the utility of the combination of the innovative passive sampling approach with effect-based tools for efficient and fast monitoring even in water bodies with relatively low levels of contamination.

Photodegradation of novel oral anticoagulants under sunlight irradiation in aqueous matrices.

Kinetics of photodegradation of novel oral anticoagulants dabigatran, rivaroxaban, and apixaban were studied under simulated solar light irradiation in purified, mineral, and river waters. Dabigatran and rivaroxaban underwent direct photolysis with polychromatic quantum yields of 2.2 × 10-4 and 4.4 × 10-2, respectively. The direct photodegradation of apixaban was not observed after 19 h of irradiation. Kinetics of degradation of rivaroxaban was not impacted by the nature of the aqueous matrix while photosensitization from nitrate ions was observed for dabigatran and apixaban dissolved in a mineral water. The photosensitized reactions were limited in the tested river water (Isle River, Périgueux, France) certainly due to the hydroxyl radical scavenging effect of the dissolved organic matter. The study of photoproduct structures allowed to identify two compounds for dabigatran. One of them is the 4-aminobenzamidine while the second one is a cyclization product. In the case of rivaroxaban, as studied by very high field NMR, only one photoproduct was observed i.e. a photoisomer. Finally, seven photoproducts were clearly identified from the degradation of apixaban under simulated solar light.

Development of a bioanalytical test battery for water quality monitoring: Fingerprinting identified micropollutants and their contribution to effects in surface water.

Surface waters can contain a diverse range of organic pollutants, including pesticides, pharmaceuticals and industrial compounds. While bioassays have been used for water quality monitoring, there is limited knowledge regarding the effects of individual micropollutants and their relationship to the overall mixture effect in water samples. In this study, a battery of in vitro bioassays based on human and fish cell lines and whole organism assays using bacteria, algae, daphnids and fish embryos was assembled for use in water quality monitoring. The selection of bioassays was guided by the principles of adverse outcome pathways in order to cover relevant steps in toxicity pathways known to be triggered by environmental water samples. The effects of 34 water pollutants, which were selected based on hazard quotients, available environmental quality standards and mode of action information, were fingerprinted in the bioassay test battery. There was a relatively good agreement between the experimental results and available literature effect data. The majority of the chemicals were active in the assays indicative of apical effects, while fewer chemicals had a response in the specific reporter gene assays, but these effects were typically triggered at lower concentrations. The single chemical effect data were used to improve published mixture toxicity modeling of water samples from the Danube River. While there was a slight increase in the fraction of the bioanalytical equivalents explained for the Danube River samples, for some endpoints less than 1% of the observed effect could be explained by the studied chemicals. The new mixture models essentially confirmed previous findings from many studies monitoring water quality using both chemical analysis and bioanalytical tools. In short, our results indicate that many more chemicals contribute to the biological effect than those that are typically quantified by chemical monitoring programs or those regulated by environmental quality standards. This study not only demonstrates the utility of fingerprinting single chemicals for an improved understanding of the biological effect of pollutants, but also highlights the need to apply bioassays for water quality monitoring in order to prevent underestimation of the overall biological effect.

European demonstration program on the effect-based and chemical identification and monitoring of organic pollutants in European surface waters.

Growing concern about the adverse environmental and human health effects of a wide range of micropollutants requires the development of novel tools and approaches to enable holistic monitoring of their occurrence, fate and effects in the aquatic environment. A European-wide demonstration program (EDP) for effect-based monitoring of micropollutants in surface waters was carried out within the Marie Curie Initial Training Network EDA-EMERGE. The main objectives of the EDP were to apply a simplified protocol for effect-directed analysis, to link biological effects to target compounds and to estimate their risk to aquatic biota. Onsite large volume solid phase extraction of 50 L of surface water was performed at 18 sampling sites in four European river basins. Extracts were subjected to effect-based analysis (toxicity to algae, fish embryo toxicity, neurotoxicity, (anti-)estrogenicity, (anti-)androgenicity, glucocorticoid activity and thyroid activity), to target analysis (151 organic micropollutants) and to nontarget screening. The most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity. In most bioassays, major portions of the observed effects could not be explained by target compounds, especially in case of androgenicity, glucocorticoid activity and fish embryo toxicity. Estrone and nonylphenoxyacetic acid were identified as the strongest contributors to estrogenicity, while herbicides, with a minor contribution from other micropollutants, were linked to the observed toxicity to algae. Fipronil and nonylphenol were partially responsible for the fish embryo toxicity. Within the EDP, 21 target compounds were prioritized on the basis of their frequency and extent of exceedance of predicted no effect concentrations. The EDP priority list included 6 compounds, which are already addressed by European legislation, and 15 micropollutants that may be important for future monitoring of surface waters. The study presents a novel simplified protocol for effect-based monitoring and draws a comprehensive picture of the surface water status across Europe.

In vitro and in vivo estrogenic activity of BPA, BPF and BPS in zebrafish-specific assays.

Bisphenol A (BPA) is a widely used chemical that has been extensively studied as an endocrine-disrupting chemical (EDC). Other bisphenols sharing close structural features with BPA, are increasingly being used as alternatives, increasing the need to assess associated hazards to the endocrine system. In the present study, the estrogenic activity of BPA, bisphenol S (BPS) and bisphenol F (BPF) was assessed by using a combination of zebrafish-specific mechanism-based in vitro and in vivo assays. The three bisphenols were found to efficiently transactivate all zebrafish estrogen receptor (zfER) subtypes in zebrafish hepatic reporter cell lines (ZELH-zfERs). BPA was selective for zfERα while BPS and BPF were slightly more potent on zfERß subtypes. We further documented the estrogenic effect in vivo by quantifying the expression of brain aromatase using a transgenic cyp19a1b-GFP zebrafish embryo assay. All three bisphenols induced GFP in a concentration-dependent manner. BPS only partially induced brain aromatase at the highest tested concentrations (>30µM) while BPA and BPF strongly induced GFP, in an ER-dependent manner, at 1-10µM. Furthermore, we show that BPF strongly induced vitellogenin synthesis in adult male zebrafish. Overall, this study demonstrates the estrogenic activity of BPA, BPF and BPS in different cell- and tissue-contexts and at different stages of development. Differences between in vitro and in vivo responses are discussed in light of selective ER activation and the fate of the compounds in the models. This study confirms the relevance of combining cellular and whole-organism bioassays in a unique model species for the hazard assessment of candidate EDCs.