Advancing environmental monitoring across the water continuum combining biomarker analysis in multiple sentinel species: A case study in the Seine-Normandie Basin (France).

Nowadays, biomarkers are recognized as valuable tools to complement chemical and ecological assessments in biomonitoring programs. They provide insights into the effects of contaminant exposures on individuals and establish connections between environmental pressure and biological response at higher levels. In the last decade, strong improvements in the design of experimental protocols and the result interpretation facilitated the use of biomarker across wide geographical areas, including aquatic continua. Notably, the statistical establishment of reference values and thresholds enabled the discrimination of contamination effects in environmental conditions, allowed interspecies comparisons, and eliminated the need of a reference site. The aim of this work was to study freshwater-estuarine-coastal water continua by applying biomarker measurements in multi-species caged organisms. During two campaigns, eight sentinel species, encompassing fish, mollusks, and crustaceans, were deployed to cover 25 sites from rivers to the sea. As much as possible, a common methodology was employed for biomarker measurements (DNA damage and phagocytosis efficiency) and data interpretation based on guidelines established using reference values and induction/inhibition thresholds (establishment of three effect levels). The methodology was successfully implemented and allowed us to assess the environmental quality. Employing multiple species per site enhances confidence in observed trends. The results highlight the feasibility of integrating biomarker-based environmental monitoring programs across a continuum scale. Biomarker results align with Water Framework Directive indicators in cases of poor site quality. Additionally, when discrepancies arise between chemical and ecological statuses, biomarker findings offer a comprehensive perspective to elucidate the disparities. Presented as a pilot project, this work contributes to gain insights into current biomonitoring needs, providing new questions and perspectives.

Application of the Fpg-modified comet assay on three-spined stickleback in freshwater biomonitoring: toward a multi-biomarker approach of genotoxicity.

Aquatic species are exposed to a wide spectrum of substances, which can compromise their genomic integrity by inducing DNA damage or oxidative stress. Genotoxicity biomarkers as DNA strand breaks and chromosomal damages developed on sentinel species have already proved to be relevant in aquatic biomonitoring. However, these biomarkers do not reflect DNA oxidative lesions, i.e., the 8-oxodG, recognized as pre-mutagenic lesion if not or mis-repaired in human biomonitoring. The relevance to include the measure of these lesions by using the Fpg-modified comet assay on erythrocytes of the three-spined stickleback was investigated. An optimization step of the Fpg-modified comet assay considering enzyme buffer impact, Fpg concentration, and incubation time has been performed. Then, this measure was integrated in a battery of genotoxicity and cytotoxicity biomarkers (considering DNA strand breaks, DNA content variation, and cell apoptosis/necrosis and density) and applied in a freshwater monitoring program on six stations of the Artois Picardie watershed (3-week caging of control fish). These biomarkers allowed to discriminate the stations regarding the genotoxic potential of water bodies and specifically by the measure of oxidative DNA lesions, which seem to be a promising tool in environmental genotoxicity risk assessment.

Chemical occurrence of pesticides and transformation products in two small lentic waterbodies at the head of agricultural watersheds and biological responses in caged Gasterosteus aculeatus.

Recent monitoring campaigns have revealed the presence of mixtures of pesticides and their transformation products (TP) in headwater streams situated within agricultural catchments. These observations were attributed to the use of various agrochemicals in surrounding regions. The aim of this work was to compare the application of chemical and ecotoxicological tools for assessing environmental quality in relation to pesticide and TP contamination. It was achieved by deploying these methodologies in two small lentic water bodies located at the top of two agricultural catchments, each characterized by distinct agricultural practices (ALT: organic, CHA: conventional). Additionally, the results make it possible to assess the impact of contamination on fish caged in situ. Pesticides and TP were measured in water using active and passive samplers and suspended solid particles. Eighteen biomarkers (innate immune responses, oxidative stress, biotransformation, neurotoxicity, genotoxicity, and endocrine disruption) were measured in Gasterosteus aculeatus encaged in situ. More contaminants were detected in CHA, totaling 25 compared to 14 in ALT. Despite the absence of pesticide application in the ALT watershed for the past 14 years, 7 contaminants were quantified in 100 % of the water samples. Among these contaminants, 6 were TPs (notably atrazine-2-hydroxy, present at a concentration exceeding 300 ng·L-1), and 1 was a current pesticide, prosulfocarb, whose mobility should prompt more caution and new regulations to protect adjacent ecosystems and crops. Regarding the integrated biomarker response (IBRv2), caged fish was similarly impacted in ALT and CHA. Variations in biomarker responses were highlighted depending on the site, but the results did not reveal whether one site is of better quality than the other. This outcome was likely attributed to the occurrence of contaminant mixtures in both sites. The main conclusions revealed that chemical and biological tools complement each other to better assess the environmental quality of wetlands such as ponds.

Integration of Genotoxic Biomarkers in Environmental Biomonitoring Analysis Using a Multi-Biomarker Approach in Three-Spined Stickleback (Gasterosteus aculeatus Linnaeus, 1758).

Water is impacted by a variety of increasing pressures, such as contaminants, including genotoxic pollutants. The proposed multi-biomarker approach at a sub-individual level gives a complementary indicator to the chemical and ecological parameters of the Water Framework Directive (WFD, 2000/60/EC). By integrating biomarkers of genotoxicity and erythrocyte necrosis in the sentinel fish species the three-spined stickleback (Gasterosteus aculeatus) through active biomonitoring of six stations of the Artois-Picardie watershed, north France, our work aimed to improve the already existing biomarker approach. Even if fish in all stations had high levels of DNA strand breaks, the multivariate analysis (PCA), followed by hierarchical agglomerative clustering (HAC), improved discrimination among stations by detecting an increase of nuclear DNA content variation (Etaing, St Rémy du Nord, Artres and Biache-St-Vaast) and erythrocyte necrosis (Etaing, St Rémy du Nord). The present work highlighted that the integration of these biomarkers of genotoxicity in a multi-biomarker approach is appropriate to expand physiological parameters which allow the targeting of new potential effects of contaminants.