Integrative multi-biomarker approach on caged rainbow trout: A biomonitoring tool for wastewater treatment plant effluents toxicity assessment.

The complex mixtures of contaminants released in wastewater treatment plant (WWTP) effluents are a major source of pollution for aquatic ecosystems. The present work aimed to assess the environmental risk posed by WWTP effluents by applying a multi-biomarker approach on caged rainbow trout (Oncorhynchus mykiss) juveniles. Fish were caged upstream and downstream of a WWTP for 21 days. To evaluate fish health, biomarkers representing immune, reproductive, nervous, detoxification, and antioxidant functions were assayed. Biomarker responses were then synthesized using an Integrated Biomarker Response (IBR) index. The IBR highlighted similar response patterns for the upstream and downstream sites. Caged juvenile females showed increased activities of innate immune parameters (lysozyme and complement), histological lesions and reduced glycogen content in the hepatic tissue, and higher muscle cholinergic metabolism. However, the intensity of the observed effects was more severe downstream of the WWTP. The present results suggest that the constitutive pollution level of the Meuse River measured upstream from the studied WWTP can have deleterious effects on fish health condition, which are exacerbated by the exposure to WWTP effluents. Our results infer that the application of IBR index is a promising tool to apply with active biomonitoring approaches as it provides comprehensive information about the biological effects caused by point source pollution such as WWTP, but also by the constitutive pollutions levels encountered in the receiving environment.

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.

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.

Applications in environmental risk assessment of leucocyte apoptosis, necrosis and respiratory burst analysis on the European bullhead, Cottus sp.

The use of a biochemical multi-biomarker approach proved insufficient to obtain clear information about ecosystem health. The fish immune system is considered as an attractive non-specific marker for environmental biomonitoring which has direct implications in individual fitness and population growth. Thus, the present work proposes the use of fish immunomarkers together with more common biochemical biomarkers in sampling conditions optimized to reduce biomarker variability and increase parameter robustness. European bullheads (Cottus sp.) from 11 stations in the Artois-Picardie watershed (France) were sampled. In the multiple discriminant analysis, the sites were highly correlated with apoptosis, respiratory burst, GST and EROD activities. Moreover, the use together of biochemical and immune markers increased the percentage of fish correctly classed at each site and enhanced site separation. This study argues in favor of the utilization of apoptosis, necrosis and respiratory burst for the determination of environmental risk assessment in addition to the set of biochemical biomarkers commonly used in fish.

Adverse effects in wild fish living downstream from pharmaceutical manufacture discharges.

A set of biochemical and histological responses was measured in wild gudgeon collected upstream and downstream of urban and pharmaceutical manufacture effluents. These individual end-points were associated to fish assemblage characterisation. Responses of biotransformation enzymes, neurotoxicity and endocrine disruption biomarkers revealed contamination of investigated stream by a mixture of pollutants. Fish from sampled sites downstream of the industrial effluent exhibited also strong signs of endocrine disruption including vitellogenin induction, intersex and male-biased sex-ratio. These individual effects were associated to a decrease of density and a lack of sensitive fish species. This evidence supports the hypothesis that pharmaceutical compounds discharged in stream are involved in recorded endocrine disruption effects and fish population disturbances and threaten disappearance of resident fish species. Overall, this study gives argument for the utilisation of an effect-based monitoring approach to assess impacts of pharmaceutical manufacture discharges on wild fish populations.