Preself-Feeding Medaka Fry Provides a Suitable Screening System for in Vivo Assessment of Thyroid Hormone-Disrupting Potential.

Endocrine-disrupting chemicals are assessed based on their physiological potential and their potential associated adverse effects. However, suitable end points for detection of chemicals that interfere with the thyroid hormone (TH) system have not been established in nonmammals, with the exception of amphibian metamorphosis. The aims of the current study were to develop an in vivo screening system using preself-feeding medaka fry (Oryzias latipes) for the detection of TH-disrupting chemicals and elucidate the underlying molecular mechanism. 17α-Ethinylestradiol (EE2: <100 ng/L) did not induce mRNA expression of estrogen-responsive genes, vitellogenins (vtgs) mRNA. Meanwhile, coexposure with thyroxin (T4) induced an increase of vtg expression. TH-disrupting chemicals (thiourea (TU), perfluorooctanoic acid (PFOA), and tetrabromobisphenol A (TBBPA)) significantly suppressed EE2 (1,000 ng/L)-induced vtg1 expression, while T4 rescued their expression as well as that of thyroid hormone receptor α (tRα) and estrogen receptors (esrs). These results were supported by in silico analysis of the 5'-transcriptional regulatory region of these genes. Furthermore, the esr1 null mutant revealed that EE2-induced vtg1 expression requires mainly esr2a and esr2b in a TH-dependent manner in preself-feeding fry. Application of preself-feeding medaka fry as a screening system might help decipher the in vivo mechanisms of action of TH-disrupting molecules, while providing an alternative to the traditional animal model.

Assessment of the Effects of Double-Stranded RNAs Corresponding to Multiple Vitellogenesis-Inhibiting Hormone Subtype I Peptides in Subadult Female Whiteleg Shrimp, Litopenaeus vannamei.

Vitellogenesis-inhibiting hormone (VIH) negatively regulates reproduction in shrimp and other decapod crustaceans. In order to assess the effects of transcriptional silencing by multiple VIH subtype I sinus gland peptides (SGPs) on ovarian maturation in female whiteleg shrimp, Litopenaeus vannamei, we synthesized five dsRNAs targeting Liv-SGP-A, -B, -C, -F, and -G and injected them into subadults. The following treatments were employed: sgpG-dsRNA (targeting Liv-SGP-G), sgpC-dsRNA (targeting Liv-SGP-C), and mixed-dsRNA (targeting Liv-SGP-A, -B, and -F). The expression of Liv-SGP-G in eyestalks was significantly decreased at 10, 20, and 30 days after the injection of sgpG-dsRNA In addition, it was significantly decreased at 10 and 30 days after the injection of mixed-dsRNA. The expression of vitellogenin (Vg) gene expression in the ovaries, and concentrations of Vg protein in the hemolymph, were not changed by the administration of any dsRNA treatment (the ovaries remained immature in all treated individuals and contained mostly oogonia and previtellogenic oocytes). Although the administration of dsRNAs corresponding to multiple VIHs did not promote ovarian maturation, this is the first report of the co-transcriptional repression of Liv-SGP-G by the injection of dsRNA for homologous genes (Liv-SGP-A, -B, and -F). These results indicate that subadults can respond to the techniques of transcriptional silencing.

Water quality of the Meuse watershed: Assessment using a multi-biomarker approach with caged three-spined stickleback (Gasterosteus aculeatus L.).

The use of a multi-biomarker approach with three-spined sticklebacks (Gasterosteus aculeatus) through an active biomonitoring strategy appears to be a promising tool in water quality assessment. The present work proposes to assess the efficiency of these tools in the discrimination of some sites in a large scale on the Meuse basin in Europe. The study was part of an EU program which aims to assess water quality in the Meuse across the French-Belgian border. Sticklebacks were caged 21 days upstream and downstream from the wastewater treatment plants (WWTPs) of Namur (Belgium), Charleville-Mézières (France), Bouillon (Belgium) and Avesnes-sur-Helpe (France). First, the state of a variety of physiological functions was assessed using a battery of biomarkers that represented innate immunity (leucocyte mortality and distribution, phagocytosis activity, respiratory burst), antioxidant system (GPx, CAT, SOD and total GSH content), oxidative damages to the membrane lipids (TBARS), biotransformation enzymes (EROD, GST), synaptic transmission (AChE) and reproduction system (spiggin and vitellogenin concentration). The impacts of the effluents were first analysed for each biomarker using a mixed model ANOVA followed by post-hoc analyses. Secondly, the global river contamination was assessed using a principal component analysis (PCA) followed by a hierarchical agglomerative clustering (HAC). The results highlighted a small number of effects of WWTP effluents on the physiological parameters in caged sticklebacks. Despite a significant effect of the "localisation" factor (upstream/downstream) in the mixed ANOVA for several biomarkers, post-hoc analyses revealed few differences between upstream and downstream of the WWTPs. Only a significant decrease of innate immune responses was observed downstream from the WWTPs of Avesnes-sur-Helpe and Namur. Other biomarker responses were not impacted by WWTP effluents. However, the multivariate analyses (PCA and HAC) of the biomarker responses helped to clearly discriminate the different study sites from the reference but also amongst themselves. Thus, a reduction of general condition (condition index and HSI) was observed in all groups of caged sticklebacks, associated with a weaker AChE activity in comparison with the reference population. A strong oxidative stress was highlighted in fish caged in the Meuse river at Charleville-Mézières whereas sticklebacks caged in the Meuse river at Namur exhibited weaker innate immune responses than others. Conversely, sticklebacks caged in the Helpe-Majeure river at Avesnes-sur-Helpe exhibited higher immune responses. Furthermore, weak defence capacities were recorded in fish caged in the Semois river at Bouillon. This experiment was the first to propose an active biomonitoring approach using three-spined stickleback to assess such varied environments. Low mortality and encouraging results in site discrimination support the use of this tool to assess the quality of a large number of water bodies.

Vitellogenin offsets oxidative costs of reproduction in female painted dragon lizards.

Vitellogenesis ('yolking' of follicles) is a bioenergetically costly stage of reproduction requiring enlargement of the liver to produce vitellogenin (VTG) yolk precursor proteins, which are transported and deposited at the ovary. VTG may, however, serve non-nutritive anti-oxidant functions, a hypothesis supported by empirical work on aging and other life-history transitions in several taxa. We test this hypothesis in female painted dragon lizards (Ctenophorus pictus) by examining covariation in VTG with the ovarian cycle, and relative to reactive oxygen species (ROS) including baseline superoxide (bSO). Plasma VTG decreased prior to ovulation, when VTG is deposited into follicles. VTG, however, remained elevated post-ovulation when no longer necessary for yolk provisioning and was unrelated to reproductive investment. Instead, VTG was strongly and positively predicted by prior bSO. ROS, in turn, was negatively predicted by prior VTG, while simultaneously sampled VTG was a positive predictor. These findings are consistent with the hypothesis that VTG functions as an anti-oxidant to counteract oxidative stress associated with vitellogenesis. The relationship between bSO and VTG was strongest in post-ovulatory females, indicating that its function may be largely anti-oxidant at this time. In conclusion, VTG may be under selection to offset oxidative costs of reproduction in egg-producing species.

Ibuprofen: Fish Short-Term Reproduction Assay with Zebrafish (Danio rerio) Based on an Extended OECD 229 Protocol.

A study was conducted to understand the potential for ibuprofen to impact the hypothalamus-pituitary-gonadal endocrine axis resulting in disruption of fish reproduction. The Good Laboratory Practice study was conducted according to the Organisation for Economic Co-operation and Development 229 Protocol, Fish Short-Term Reproduction Assay, and extended an additional 4 d to evaluate hatching success in the F1 generation. Test organisms were exposed to nominal test concentrations of 0.5, 2.4, 11.5, 55.3, and 265.4 µg ibuprofen/L and a negative control (dilution water). To strengthen the statistical power of the study, twice the number of replicates were used in the negative control versus individual treatment levels. A 21-d pre-exposure to identify groups of actively spawning fish was immediately followed by a 36-d exposure. Results for apical endpoints of survival, growth, and reproduction (fecundity and fertility), as well as the biomarker vitellogenin in the F0 generation and time to hatch and hatching success in the F1 generation are presented. Based on mean measured exposure concentrations and effects on fecundity in the F0 generation and hatching success in the F1 generation, overall no-observed-effect concentration and lowest-observed-effect concentration for the present study were 55.2 and 265.9 µg ibuprofen/L, respectively. Results from the present study indicate a lack of endocrine-mediated reproductive effects in zebrafish at environmentally relevant concentrations of ibuprofen. Environ Toxicol Chem 2020;39:1534-1545. © 2020 SETAC.

Adverse Outcome Pathway Network-Based Assessment of the Interactive Effects of an Androgen Receptor Agonist and an Aromatase Inhibitor on Fish Endocrine Function.

Predictive approaches to assessing the toxicity of contaminant mixtures have been largely limited to chemicals that exert effects through the same biological molecular initiating event. However, by understanding specific pathways through which chemicals exert effects, it may be possible to identify shared "downstream" nodes as the basis for forecasting interactive effects of chemicals with different molecular initiating events. Adverse outcome pathway (AOP) networks conceptually support this type of analysis. We assessed the utility of a simple AOP network for predicting the effects of mixtures of an aromatase inhibitor (fadrozole) and an androgen receptor agonist (17ß-trenbolone) on aspects of reproductive endocrine function in female fathead minnows. The fish were exposed to multiple concentrations of fadrozole and 17ß-trenbolone individually or in combination for 48 or 96 h. Effects on 2 shared nodes in the AOP network, plasma 17ß-estradiol (E2) concentration and vitellogenin (VTG) production (measured as hepatic vtg transcripts) responded as anticipated to fadrozole alone but were minimally impacted by 17ß-trenbolone alone. Overall, there were indications that 17ß-trenbolone enhanced decreases in E2 and vtg in fadrozole-exposed fish, as anticipated, but the results often were not statistically significant. Failure to consistently observe hypothesized interactions between fadrozole and 17ß-trenbolone could be due to several factors, including lack of impact of 17ß-trenbolone, inherent biological variability in the endpoints assessed, and/or an incomplete understanding of interactions (including feedback) between different pathways within the hypothalamic-pituitary-gonadal axis. Environ Toxicol Chem 2020;39:913-922. © 2020 SETAC.

Refinement of an OECD test guideline for evaluating the effects of endocrine disrupting chemicals on aromatase gene expression and reproduction using novel transgenic cyp19a1a-eGFP zebrafish.

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 µg/L). In addition to "classical" endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 µg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 µg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.

Medaka (Oryzias latipes) Multigeneration Test with Triclosan.

The medaka extended one-generation reproduction test (MEOGRT) is a tier-2 study in the US Environmental Protection Agency's Endocrine Disruptor Screening Program and a level-5 study in the Organisation for Economic Co-operation and Development's conceptual framework. Integrating nonspecific apical and endocrine-specific mechanistic endpoints, results of a MEOGRT can be used, with other data, in a weight-of-evidence evaluation to establish a dose-response relationship for risk assessment and identify potential causal relationships between an endocrine mode of action and adverse effects. The MEOGRT test design was used to evaluate the multigenerational effects of the antimicrobial agent triclosan. Japanese medaka were exposed to nominal concentrations of 1.4, 2.8, 5.6, 11, and 23 µg/L triclosan and a dilution water control starting with adult medaka (F0) through hatch in the second generation (F2). No consistent or concentration-related responses occurred in the 182-d test that suggested an endocrine-mediated effect. There were no impacts on hepatic vitellogenin, secondary sex characteristics, or sex ratio that were linked to an adverse reproductive outcome. Histopathological responses were consistent with a toxic or stress effect, particularly when considered in context with observed reductions in growth. The overall population-relevant no-observed-effect concentration was 11 µg/L based on effects on growth. The results of the present study support a previously conducted weight-of-evidence evaluation concluding that triclosan does not act as an agonist or antagonist within estrogen, androgen, thyroid, or steroidogenic pathways. Environ Toxicol Chem 2019;38:1770-1783. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Steroidal and phenolic endocrine disrupting chemicals (EDCs) in surface water of Bahe River, China: Distribution, bioaccumulation, risk assessment and estrogenic effect on Hemiculter leucisculus.

This study investigated selected steroidal and phenolic endocrine disrupting compounds (EDCs) in the surface water of the Bahe River (China) using gas chromatography mass spectrometry (GC-MS). Their effect on the wild sharpbelly Hemiculter leucisculus was investigated. The concentrations of 4-t-octylphenol, nonylphenol, bisphenol-A, estrone, 17 ß-estradiol, 17 α-Ethinylestradiol, and estriol were up to 126.0, 634.8, 1573.1, 55.9, 23.9, 31.5, and 5.2 ng L-1 in the surface water, and up to 26.4, 103.5, 146.9, 14.2, 9.3, 13.8, and 1.3 ng g-1 in the fish muscle tissue, respectively. High estrogen equivalent levels and hazard quotients were found in the middle and lower reaches of the river, and the pollution in these regions caused enhanced growth conditions, inhibition of gonad growth, and suppression of spermatogenesis in H. leucisculus. The up-regulation of Vitellogenin mRNA expression in male fish, collected from relatively heavily EDCs contaminated areas, indicates a potential estrogenic effect. The differential expression profiles of genes related to steroidogenesis at all sampling sites suggests that these endpoints may play an important role for the pollution monitoring of estrogenic EDCs in the Bahe River.

Assessment of the effects of discontinuous sources of contamination through biomarker analyses on caged mussels.

The present study analysed potential adverse effects of discontinuous sources of contamination, namely the discharge of a combined sewer overflow (CSO) and of runoff in an urban area, the Bay of Santander (North Iberian Peninsula). Water samples and caged mussels were used to analyse concentrations of contaminants and biological responses. Mussels (Mytilus galloprovincialis) were transplanted to a marina receiving runoff from a petrol station and to a CSO discharge site. Samples were collected in synchrony with heavy rains along 62days. Lysosomal membrane stability (LMS) and acyl-CoA oxidase (AOX) activity were measured as core biomarkers and were analysed at all sampling times. Histopathology of digestive gland and gonads, transcription levels of vitellogenin gene, volume density of black silver deposits and micronuclei formation were measured at initial and final stages of the transplant. Chemical analyses of metals, polycyclic aromatic hydrocarbons (PAHs) and endocrine disruptors were performed in water samples and mussel flesh. Mussels accumulated low concentrations of contaminants, which is in accordance with results obtained from exposure biomarkers. AOX activity decreased in all transplanted mussels after the first heavy rain, but this change seems to be related to the seasonal pattern of the enzyme activity. Mussels located close to the CSO discharge site showed a reduction in LMS after the first rain event, when compared to mussels before the transplant and to mussels from the reference location. However, this was attributable to natural environmental changes rather than to pollution. Values of the rest of analysed biomarkers were below threshold values reported for the study area.

Integration of biological effects, fish histopathology and contaminant measurements for the assessment of fish health: A pilot application in Irish marine waters.

This study investigates the use of a weight of evidence (WOE) approach to evaluate fish health status and biological effects (BEs) of contaminants for assessment of ecosystem health and discusses its potential application in support of the Marine Strategy Framework Directive (MSFD). External fish disease, liver histopathology and several BEs of contaminant exposure including 7-ethoxy resorufin O-de-ethylase (EROD), acetylcholinesterase (AChE), bile metabolites, vitellogenin (VTG) and alkali labile phosphates (ALP) were measured in two flatfish species from four locations in Ireland. Contaminant levels in fish were generally low with PCBs in fish liver below OSPAR environmental assessment criteria (EAC). There were consistencies with low PCB levels, EROD and PAH bile metabolite levels detected in fish. Dab from Cork, Dublin and Shannon had the highest relative prevalence of liver lesions associated with the carcinogenic pathway. An integrated biomarker response (IBR) showed promise to be useful for evaluation of environmental risk, although more contaminant parameters in liver are required for a full assessment with the present study.

Sharing the Roles: An Assessment of Japanese Medaka Estrogen Receptors in Vitellogenin Induction.

Teleost fish express at least three estrogen receptor (ER) subtypes. To date, however, the individual role of these ER subtypes in regulating expression of estrogen responsive genes remains ambiguous. Here, we investigate putative roles of three ER subtypes in Japanese medaka (Oryzias latipes), using vitellogenin (VTG) I and II as model genes. We identify specific ligand/receptor/promoter dynamics, using transient transactivation assays that incorporate luciferase reporters comprising 3kb promoter/enhancer regions of medaka VTGI and VTGII genes. Four steroidal estrogens (17ß-estradiol, estrone, estriol, and 17α-estradiol) were tested in these assays. Results indicate that all three medaka ERs (mERs) are capable of initiating transactivation of both VTG I and II, with ERß2 exhibiting greatest activity. Promoter deletion analysis suggests that ligand-specific receptor transactivation and utilization of regional-specific estrogen response elements may be associated with differential activities of each medaka ER. Further, cluster analysis of in vivo gene expression and in vitro transactivation suggests that all three ER subtypes putatively play a role in up-regulation of VTG. Results illustrate that preferential ligand/receptor/promoter interactions may have direct implications for VTG gene expression and other ER-mediated regulatory functions that are relevant to the risk assessment of estrogenic compounds.

Assessment of multiple hormone activities of a UV-filter (octocrylene) in zebrafish (Danio rerio).

In this study, zebrafish (Danio rerio) were exposed to a UV-filter-octocrylene (OCT) with elevated concentrations for 28 d. The total body accumulation of OCT in zebrafish was found to reach 2321.01 ("L" level), 31,234.80 ("M" level), and 70,593.38 ng g(-1) ("H" level) when the average OCT exposure concentration was controlled at 28.61, 505.62, and 1248.70 µg L(-1), respectively. Gross and histological observations as well as RT-qPCR analysis were conducted to determine the effects of OCT accumulation on zebrafish. After exposure, the gonad-somatic index and percentage of vitellogenic oocytes were found to increase significantly in the ovaries of female zebrafish at the H accumulation level. Significant up-regulation of esr1 and cyp19b were observed in the gonads, as well as vtg1 in the livers for both female and male zebrafish. At M and H accumulation levels, apparent down-regulation of ar was observed in the ovaries and testis of the female and male zebrafish, respectively. Although the extent of the effects on zebrafish differed at different accumulation levels, the induction of vtg1 and histological changes in the ovaries are indications of estrogenic activity and the inhibition of esr1 and ar showed antiestrogenic and antiandrogenic activity, respectively. Thus, as OCT could easily accumulate in aquatic life such as zebrafish, one of its most of concern hazards would be the disturbance of the histological development and its multiple hormonal activities.

Reproduction impairment and endocrine disruption in female zebrafish after long-term exposure to MC-LR: A life cycle assessment.

Microcystin-LR (MC-LR) has been found to cause reproductive and developmental impairments as well as to disrupt sex hormone homeostasis of fish during acute and sub-chronic toxic experiments. However, fish in natural environments are continuously exposed to MC-LR throughout their entire life cycle as opposed to short-term exposure. Here, we tested the hypothesis that the mechanism by which MC-LR harms female fish reproduction and development within natural water bodies is through interference of the reproductive endocrine system. In the present study, zebrafish hatchlings (5 d post-fertilization) were exposed to 0, 0.3, 3 and 30 µg/L MC-LR for 90 d until reaching sexual maturity. Female zebrafish were selected, and the changes in growth and developmental indicators, ovarian ultrastructure as well as the levels of gonadal steroid hormones and vitellogenin (VTG) were examined along with the transcription of related genes in the hypothalamic-pituitary-gonadal-liver axis (HPGL-axis). The results showed for the first time, a life cycle exposure to MC-LR caused growth inhibition, decreased ovary weight and ovarian ultra-pathological lesions. Decreased ovarian testosterone levels indicated that MC-LR disrupted sex steroid hormone balance. Significantly up-regulated transcription of brain FSHß and LHß along with ovarian ERα, FSHR and LHR suggested positive feedback regulation in the HPGL-axis was induced as a compensatory mechanism for MC-LR damage. It was also noted that ovarian VTG content and hepatic ERα and VTG1 expression were all down-regulated, which might be responsible for reduced vitellus storage noted in our histological observations. Our findings indicate that a life cycle exposure to MC-LR impairs the development and reproduction of female zebrafish by disrupting the transcription of related HPGL-axis genes, suggesting that MC-LR has potential adverse effects on fish reproduction and thus population dynamics in MCs-contaminated aquatic environment.

Assessment of biomarkers for contaminants of emerging concern on aquatic organisms downstream of a municipal wastewater discharge.

Contaminants of emerging concern (CECs), including pharmaceuticals, personal care products and estrogens, are detected in wastewater treatment plant (WWTP) discharges. However, analytical monitoring of wastewater and surface water does not indicate whether CECs are affecting the organisms downstream. In this study, fathead minnows (Pimephales promelas) and freshwater mussels Pyganodon grandis Say, 1829 (synonym: Anodonta grandis Say, 1829) were caged for 4 weeks in the North Saskatchewan River, upstream and downstream of the discharge from the WWTP that serves the Edmonton, AB, Canada. Passive samplers deployed indicated that concentrations of pharmaceuticals, personal care products, an estrogen (estrone) and an androgen (androstenedione) were elevated at sites downstream of the WWTP discharge. Several biomarkers of exposure were significantly altered in the tissues of caged fathead minnows and freshwater mussels relative to the upstream reference sites. Biomarkers altered in fish included induction of CYP3A metabolism, an increase in vitellogenin (Vtg) gene expression in male minnows, elevated ratios of oxidized to total glutathione (i.e. GSSG/TGSH), and an increase in the activity of antioxidant enzymes (i.e. glutathione reductase, glutathione-S-transferase). In mussels, there were no significant changes in biomarkers of oxidative stress and the levels of Vtg-like proteins were reduced, not elevated, indicating a generalized stress response. Immune function was altered in mussels, as indicated by elevated lysosomal activity per hemocyte in P. grandis caged closest to the wastewater discharge. This immune response may be due to exposure to bacterial pathogens in the wastewater. Multivariate analysis indicated a response to the CECs Carbamazepine (CBZ) and Trimethoprim (TPM). Overall, these data indicate that there is a 1 km zone of impact for aquatic organisms downstream of WWTP discharge. However, multiple stressors in municipal wastewater make measurement and interpretation of impact of CECs difficult since water temperature, conductivity and bacteria are also inducing biomarker responses in both fish and mussels.

Integrated assessment of wastewater treatment plant effluent estrogenicity in the Upper Murray River, Australia, using the native Murray rainbowfish (Melanotaenia fluviatilis).

The contamination of major continental river systems by endocrine-active chemicals (EACs) derived from the discharge of wastewater treatment plant (WWTP) effluents can affect human and ecosystem health. As part of a long-term effort to develop a native fish model organism for assessment of endocrine disruption in Australia's largest watershed, the Murray-Darling River Basin, the present study evaluated endocrine disruption in adult males of the native Australian Murray rainbowfish (Melanotaenia fluviatilis) exposed to effluent from an activated sludge WWTP and water from the Murray River during a 28-d, continuous-flow, on-site experiment. Analysis of the WWTP effluent and river water detected estrone and 17ß-estradiol at concentrations up to approximately 25 ng L(-1) . Anti-estrogenicity of effluent samples was detected in vitro using yeast-based bioassays (yeast estrogen screen) throughout the experiment, but estrogenicity was limited to the first week of the experiment. Histological evaluation of the testes indicated significant suppression of spermatogenesis by WWTP effluent after 28 d of exposure. Plasma vitellogenin concentrations and expression of vitellogenin messenger RNA in liver were not significantly affected by exposure to WWTP effluent. The combination of low contaminant concentrations in the WWTP effluent, limited endocrine disrupting effects in the Murray rainbowfish, and high in-stream dilution factors (>99%) suggest minimal endocrine disruption impacts on native Australian fish in the Murray River downstream from the WWTP outfall.

Assessment of the health status of wild fish inhabiting a cotton basin heavily impacted by pesticides in Benin (West Africa).

To determine the impact of agricultural pesticides used in cotton cultivation on the health status of fish living in a Beninese cotton basin, we compared the reproductive and hepatic systems of fish sampled from rivers located in both contaminated and pristine conditions. Different types of biomarkers, including biometric indices (a condition factor K, a gonadosomatic index GSI, and a hepatosomatic index HSI), plasma levels of sex steroids (11-ketotestosterone 11-KT, testosterone T and estradiol-17ß E2) and the histopathology of the gonads and liver, were investigated for two different trophic levels of the following two fish species: the Guinean tilapia Tilapia guineensis and the African catfish Clarias gariepinus. The fish were captured during both the rainy season (when there is heavy use of pesticides on cotton fields) and the dry season from one site, in Pendjari River (reference site), which is located outside the cotton-producing basin, and from three other sites on the Alibori River within the cotton-producing basin. Comparing fish that were sampled from contaminated (high levels of endosulfan, heptachlor and DDT and metabolites) and reference sites, the results clearly indicated that agricultural pesticides significantly decreased K and GSI while they increased HSI, regardless of the season, species and sex of the fish. These pesticides also induced a decrease in the plasma levels of 11-KT and T and increased those of E2. The histopathology of the testes revealed, in both species, a high rate of testicular oocytes, up to 50% in the African catfish, downstream of the Alibori River, which indicated estrogenic effects from the pesticides. The disruption of male spermatogenesis primarily included necrosis, fibrosis and the presence of foam cells in the lobular lumen. The histopathology of the ovaries revealed high levels of pre-ovulatory follicular atresia, impaired oogenesis, a decrease in the oocyte vitellogenic diameter and other lesions, such as fibrosis, vacuolation and melano-macrophagic centers. The histopathology of the liver revealed the presence of necrosis, hypertrophic hepatocytes, foci of vacuolation, glycogen depletion and hemosiderin. An assessment of the general health of the fish indicated that all of the sampled fish from the polluted sites were in poorer health compared with those from the reference site but that the African catfish appeared much more affected than the Guinean tilapia, regardless of the sex and season. In conclusion, the overall results indicated that agricultural pesticides significantly impair the endocrine regulation of fish living in the Beninese cotton basin and that this would most likely be one of the causes of the severe damage observed in the liver and gonads and the reduced health condition.

Caged mudsnail Potamopyrgus antipodarum (Gray) as an integrated field biomonitoring tool: exposure assessment and reprotoxic effects of water column contamination.

This study highlights the usefulness of gastropods for water quality monitoring. Gastropods were caged upstream and downstream of an effluent discharge. Exposure was assessed by measurement of organic contaminants in water. Contamination of the Potamopyrgus antipodarum mudsnail was also measured using innovative techniques at the end of the 42 days of exposure. Biological effects were measured at the individual level (growth, reproduction) and subindividual level (energy reserves, vitellin-like proteins, steroid levels, expression of genes involved in estrogen signaling pathways), thus providing a better understanding of reprotoxic effects. The effluent was mainly contaminated by pharmaceutical compounds, as was the mudsnail. The highest concentrations were measured for oxazepam and were higher than 2 mg/kg downstream of the effluent discharge. Alkylphenols, bisphenol A, and vertebrate-like sex-steroid hormones were also bioaccumulated by the mudsnail downstream of the effluent. The combined use of water and snail contamination provided a complete exposure assessment. Exposure was further linked to biological effects. The mudsnail was shown to be a better adapted species for in situ exposures than Valvata piscinalis. Reproduction was sharply decreased after 6 weeks of exposure in the mudsnail. Feeding issues were excluded, confirming the toxic origin. These effects were related to estrogen signaling pathways using genomic analysis. Genes coding for proteins involved in nongenomic signaling pathways were inhibited, and those of genomic pathway repressors were induced. These results suggest that the chemical contamination due to the effluent discharge altered steroid control of reproduction and blocked the transition between oocyte and unshelled embryo, resulting in a drastic decrease of embryo production, while survival was not affected.

Assessment of wastewater and recycled water quality: a comparison of lines of evidence from in vitro, in vivo and chemical analyses.

We investigated water quality at an advanced water reclamation plant and three conventional wastewater treatment plants using an "ecotoxicity toolbox" consisting of three complementary analyses (chemical analysis, in vitro bioanalysis and in situ biological monitoring), with a focus on endocrine disruption. The in vitro bioassays were chosen to provide an appropriately wide coverage of biological effects relevant to managed aquifer recharge and environmental discharge of treated wastewater, and included bioassays for bacterial toxicity (Microtox), genotoxicity (umuC), photosynthesis inhibition (Max-I-PAM) and endocrine effects (E-SCREEN and AR-CALUX). Chemical analysis of hormones and pesticides using LCMSMS was performed in parallel to correlate standard analytical methods with the in vitro assessment. For two plants with surface water discharge into open drains, further field work was carried out to examine in situ effects using mosquitofish (Gambusia holbrooki) as a bioindicator species for possible endocrine effects. The results show considerable cytotoxicity, phytotoxicity, estrogenicity and androgenicity in raw sewage, all of which were significantly reduced by conventional wastewater treatment. No biological response was detected to RO water, suggesting that reverse osmosis is a significant barrier to biologically active compounds. Chemical analysis and in situ monitoring revealed trends consistent with the in vitro results: chemical analysis confirmed the removal trends observed by the bioanalytical tools, and in situ sampling did not reveal any evidence of endocrine disruption specifically due to discharge of treated wastewater (although other sources may be present). Biomarkers of exposure (in vitro) and effect (in vivo or in situ) are complementary and together provide information with a high level of ecological relevance. This study illustrates the utility of combining multiple lines of evidence in the assessment of water quality.

Assessment of the effects of a marine urban outfall discharge on caged mussels using chemical and biomarker analysis.

To assess the presence of endocrine disruptors in treated marine outfall discharges and their possible effects, mussels (Mytilus galloprovincialis) were caged in the environmental mixing zone of the outfall of the Santander sanitation system and in one control area. After 30, 60 and 90 days, samples were collected to perform chemical analyses (metals, anionic surfactants, alkylphenols, bisphenol A, phthalates and estrogenic hormones), biomarkers of general stress (lysosomal membrane stability-LMS, histopathology) and biomarkers of endocrine disruption (vitellogenin-like proteins and gonad index). There were no significant differences between outfall and control sites on contaminant levels, except for 4-tert-octylphenol which was higher in the outfall site. Bacteriological counts were higher in the outfall area. No relevant differences in biomarkers were detected between treated and control mussels. A significant reduction in LMS occurred in both groups after 90 days caging, indicating a stress situation possibly related to caging or to post-spawning reproductive state.