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.

Integrative biomarker response - Threshold (IBR-T): Refinement of IBRv2 to consider the reference and threshold values of biomarkers.

The Integrated Biomarker Response (IBR) is one of the most used index in biomonitoring, especially the IBRv2 integrating a reference condition. However, some limitations remain for its routine and large-scale use. The IBRv2 is proportional to the total number of biomarkers, is dependent on the nature of biomarkers and considers all biomarkers modulations, even small and biologically non-significant. In addition, IBRv2 relies on reference values but the references are often different between each study, making it difficult to compare results between studies and/or campaigns. To overcome these limitations, the present work proposed a new index called IBR-T ("Integrated Biomarker Response - Threshold") which considers the threshold values of biomarkers by limiting the calculation of the IBR value to biomarkers with significant modulations. The IBRv2 and the IBR-T were calculated and compared on four datasets from active biomonitoring campaigns using Dreissena polymorpha, a bivalve widely used in freshwater biomonitoring studies. The comparison between indices has demonstrated that the IBR-T presents a better correlation (0.907 < r2 < 0.998) with the percentage of biomarkers significantly modulated than the IBRv2 (0.002 < r2 < 0.759). The IBRv2 could not be equal to 0 (0.915 < intercept <1.694) because the value was dependent on the total number of biomarkers, whereas the IBR-T reached 0 when no biomarker was significantly modulated, which appears more biologically relevant. The final ranking of sites was different between the two index and the IBR-T ranking tends to be more ecologically relevant that the IBRv2 ranking. This IBR-T have shown an undeniable interest for biomonitoring and could be used by environmental managers to simplify the interpretation of large datasets, directly interpret the contamination status of the site, use it to decision-making, and finally to easily communicate the results of biomonitoring studies to the general public.

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.

Interest of a multispecies approach in active biomonitoring: Application in the Meuse watershed.

A biomonitoring approach based on a single model species cannot be representative of the contaminations impacts on the ecosystem overall. As part of the Interreg DIADeM program ("Development of an integrated approach for the diagnosis of the water quality of the River Meuse"), a study was conducted to establish the proof of concept that the use of a multispecies active biomonitoring approach improves diagnostic of aquatic systems. The complementarity of the biomarker responses was tested in four model species belonging to various ecological compartments: the bryophyte Fontinalis antipyretica, the bivalve Dreissena polymorpha, the amphipod Gammarus fossarum and the fish Gasterosteus aculeatus. The species have been caged upstream and downstream from five wastewater treatment plants (WWTPs) in the Meuse watershed. After the exposure, a battery of biomarkers was measured and results were compiled in an Integrated Biomarker Response (IBR) for each species. A multispecies IBR value was then proposed to assess the quality of the receiving environment upstream the WWTPs. The effluent toxicity was variable according to the caged species and the WWTP. However, the calculated IBR were high for all species and upstream sites, suggesting that the water quality was already downgraded upstream the WWTP. This contamination of the receiving environment was confirmed by the multispecies IBR which has allowed to rank the rivers from the less to the most contaminated. This study has demonstrated the interest of the IBR in the assessment of biological impacts of a point-source contamination (WWTP effluent) but also of the receiving environment, thanks to the use of independent references. Moreover, this study has highlighted the complementarity between the different species and has emphasized the interest of this multispecies approach to consider the variability of the species exposition pathway and sensibility as well as the mechanism of contaminants toxicity in the final diagnosis.

Application in a biomonitoring context of three-spined stickleback immunomarker reference ranges.

The relevance of a biomarker for biomonitoring programs was influenced both by the knowledge on biomarker natural inter-individual and site variabilities and by the sensitivity of the biomarker towards environmental perturbations. To minimize data misinterpretation, robustness reference values for biomarkers were important in biomonitoring programs. Specific three-spined stickleback, Gasterosteus aculeatus, immune reference ranges for field studies had been determined based on laboratory data and one reference station (Contentieuse river at Houdancourt). In this study, data obtained in one uncontaminated and three contaminated sites were compared to these reference ranges as a validation step before considering them for larger scale biomonitoring programs. When the field reference range were compared to data from the uncontaminated station (Béronelle), only few deviations were shown. In this way, data coming from uncontaminated station (Béronelle) was integrated in the field reference ranges to improve the evaluation of site variability. The new field reference ranges provided better discrimination of sites and spanned a larger range of fish lengths than the initial reference ranges. Furthermore, the results suggest lysosomal presence during several months and phagocytosis capacity in autumn may be the most relevant immunomarkers towards identifying contaminated sites. In the future, combining this reference value approach with active biomonitoring could facilitate the obtention of data in multiple stream conditions.

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.

Effects of chronic exposure to a pharmaceutical mixture on the three-spined stickleback (gasterosteus aculeatus) population dynamics in lotic mesocosms.

Pharmaceutical substances are ubiquitous in the aquatic environment and their concentration levels typically range from ng/L up to several µg/L. Furthermore, as those compounds are designed to be highly biologically active, assessing their impacts on non-target organisms is important. Here, we conducted a mesocosm experiment testing a mixture of five pharmaceuticals (diclofenac, carbamazepine, irbesartan, acetaminophen and naproxen) on fish, three-spined stickleback (Gasterosteus aculeatus). The mixture concentration levels were chosen on the basis of the contamination of the Meuse river in Belgium which had been measured previously during a monitoring campaign undertaken in 2015 and 2016. Three nominal mixture concentration levels were tested: the lowest concentration level mixture was composed by environmentally-relevant concentrations that approximate average realistic values for each pharmaceuticals (Mx1); the two other levels were 10 and 100 times these concentrations. Although no impact on stickleback prey was observed, the mixture significantly impaired the survival of female fish introduced in the mesocosms at the highest treatment level without causing other major differences on fish population structure. Impacts on condition factors of adults and juveniles were also observed at both individual and population levels. Using a modelling approach with an individual-based model coupled to a bioenergetic model (DEB-IBM), we concluded that chronic exposure to environmentally-relevant concentrations of five pharmaceuticals often detected in the rivers did not appear to strongly affect the three-spined stickleback populations. Mechanisms of population regulation may have counteracted the mixture impacts in the mesocosms.

Reliability evaluation of biomarker reference ranges for mesocosm and field conditions: Cellular innate immunomarkers in Gasterosteus aculeatus.

Due to their sensitivity to environmental contamination and their link with fish health status, innate immunomarkers are of great interest for environmental risk assessment studies. Nevertheless, the lack of knowledge about the effect of confounding factors can lead to data misinterpretation and false diagnostics. So, the determination of reference values was of huge interest for the integration of biomarkers in biomonitoring programs. Laboratory immunomarker reference ranges (including cellular mortality, leucocyte distribution, phagocytosis activity, respiratory burst and lysosomal presence) that consider three confounding factors (season, sex and body size) were previously developed in three-spined stickleback, Gasterosteus aculeatus, from our husbandry. Usefulness of these reference ranges in biomonitoring programs depends on how they can be transposed to various experimental levels, such as mesocosm (outdoor artificial pond) and field conditions. Immunomarkers were therefore measured every 2 months over 1 year in one mesocosm and in one site assumed to uncontaminated (Houdancourt, field). Differences between immunomarker seasonal variations in mesocosm and field fish on one side and laboratory fish on the other side were quantified: in some cases, seasonal trends were not significant or did not differ between mesocosm and laboratory conditions, but overall, models developed based on data obtained in laboratory conditions were poorly predictive of data obtained in mesocosm or field conditions. To propose valuable field reference ranges, mesocosm and field data were integrated in innate immunomarker modelling in order to strengthen the knowledge on the effect of confounding factors. As in laboratory conditions, sex was overall a confounding factor only for necrotic cell percentage and granulocyte-macrophage distribution and size was a confounding factor only for cellular mortality, leucocyte distribution and phagocytosis activity. Confounding factors explained a large proportion of immunomarker variability in particular for phagocytosis activity and lysosomal presence. Further research is needed to test the field models in a biomonitoring program to compare the sensitivity of immunomarkers to the confounding factors identified in this study and the sensitivity to various levels of pollution.

Impact of confinement and food access restriction on the three-spined stickleback (Gasterosteus aculeatus, L.) during caging: a multi-biomarker approach.

Caging is an active biomonitoring strategy that employs a sentinel species, sometimes a species naturally absent from the studied site, in the surveillance of water bodies to verify whether biota may be at risk. The main advantage of caging is the possibility to standardize several biotic and abiotic parameters. However, little knowledge is available about the effects of confinement on physiology and metabolism of caged organisms. The aim of this study is to characterize confinement and food access restriction effects, induced via caging experiments using a multi-biomarker approach (biometric data, immunity, antioxidant, metabolic detoxication, and digestive enzymes). The study has been undertaken using the same experiment conducted in ecosystem conditions using three-spined stickleback (Gasterosteus aculeatus) during two different periods: one in April, corresponding to breeding season, and the other in October, outside breeding season. Fifteen fish were maintained for 21 days in different conditions (caged or uncaged and with or without food supply). The main result was that confinement stress had little impact on the biological markers of sticklebacks. However, the stressors seemed to increase the negative effects of food restriction on these biomarkers, when sticklebacks needed more energy, that is, during their breeding period. Outside breeding period, most investigated biomarkers were not impacted by caging. This study showed a way to specify the conditions of application and interpretation of biomarkers during active monitoring to ensure an effective, reliable diagnosis of water body quality.

Impact of multiple stressors on biomarker responses in sympatric dreissenid populations.

Dreissenid mussels, well-known invaders of the northern hemisphere, also constitute good biomonitors for freshwater quality assessment. Whereas the oldest, Dreissena polymorpha, is relatively well-known, the new invasive, Dreissena rostriformis bugensis, has been very little characterized. The aim of this study was to compare subcellular biomarker responses within these species, by taking into account inter- and intra-specific variability in biomarker responses during a multi-stress assessment. Indeed, due to local environmental conditions and genetic background, biomarker responses can be different between populations of a given species, not only between species. To evaluate these differential responses, we sampled mussels on two sites where they are both present in sympatry (plus another D. r. bugensis population), and we exposed these populations in laboratory to different stressors: thermal stress (12 or 17 °C), dietary stress (fed or unfed), contamination (nickel at 0, 20 and 500 µg L-1) and exposure duration (0, 4 or 8 days). Results mainly evidenced strong inter-species and inter-population differences, underlining the need to discriminate correctly between the two species and to know well the populations used in biomonitoring. Results also evidenced thermal and food stress-related effects. The numerous data obtained during this multi-stress experiment also highlight the complexity of working on several stressors and analysing the associated results.