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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. • A novel strategy to monitor aquatic continua using biomarkers is proposed. • A common analysis method was applied in multi-species caged organisms. • Biomarkers provide clear and easily useable assessment of the environmental quality. • Reference values and thresholds are recommended to interpret biomarker responses. • Biomarker-based monitoring on a wide scale complements the WFD evaluation. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • A new Integrative biomarker response index – IBR-T – is proposed to improve IBRv2. • IBR-T integrates reference and threshold values. • IBR-T improves site classification and includes biological variation of biomarkers. • IBR-T appears more ecologically relevant and allows comparing studies and years. [ABSTRACT FROM AUTHOR]

Active biomonitoring of chemical contamination (e.g. , Cd, Hg, Pb, DDT, PCB, PAH) in French Mediterranean coastal waters has been performed for more than two decades. This study aimed at presenting the current contamination in 2021 and the temporal evolution of concentrations from 2000. Based on a relative spatial comparison, low concentrations were measured in 2021 at most sites (>83 %). Also, several stations with moderate to high levels were highlighted in the vicinity of major urban industrial centers (e.g. , Marseille, Toulon) and near river mouths (e.g. , Rhône, Var). Over the last 20 years, no major trend was revealed, mostly, especially for the relative high-level sites. This likely constant contamination over time, plus slight increases of metallic elements at a few sites, still raise questions on the efforts that remain to be made. The decreasing trends of organic compounds, in particular PAH, provide evidence of the efficiency of some management actions. • Contamination in mussels from French waters has been monitored over two decades. • In 2021, most sites showed relative low concentrations. • Most contaminants revealed constant levels over the last 20 years at most stations. • Sites near urban industrial centers or river mouths remain contamination hotspots. • PAH revealed clear decreasing trends for 63 % of stations. [ABSTRACT FROM AUTHOR]

Contaminant bioaccumulation was studied in blue mussels ( Mytilus edulis spp.) using the harbor waters of Kristiansand (Norway) as a case study. A suite of chemical contaminants (trace metals, PAHs and PCBs) was analyzed in caged and native mussels as well as in passive samplers (Diffusive Gradients in Thin films (DGT)-devices and silicone rubbers) placed alongside the mussels for estimation of contaminant concentrations in water and uptake rates and bioaccumulation factors (BAFs) in mussels during a six-months deployment period. Estimated logBAFs were in the ranges 2.3–5.5, 3.8–5.2 and 3.2–4.4 for metals, PCBs and PAHs, respectively. Contaminant levels in caged mussels increased rapidly to stable levels for trace metals, whereas for hydrophobic organic contaminants the increase was steady but slow and for many compounds did not reach the levels observed in native mussels. Some key issues related to mussel caging design, such as mussel deployment time and confounding influence from seasonal fluctuations, are discussed herein. [ABSTRACT FROM AUTHOR]

Within the framework of the biointegrator network (RINBIO), 92 man-made cages containing mussels (Mytilus galloprovincialis), distributed over 1800 km of the French Mediterranean coast, made it possible to assess chemical contamination by heavy metals (Cd, Hg, Zn, Pb, Cu, Ni, Cr, As) and organic compounds (DDT, PCBs, HAP). The caging technique was adopted to compensate for the scarcity of natural shellfish stocks in the Mediterranean and to enable comparison of the sites regardless of their physicochemical and trophic characteristics. Models linking a biometric parameter (the condition index) to pollutant levels make it possible to adjust raw data on contamination for a reference individual, by making a clear distinction between physiological factors (growth) and environmental ones. The results demonstrate significant levels of pollutants in sites heretofore unsampled; the pollution occurs at limited areas, and the sources have been identified. Moreover, average contamination levels are comparable to those measured in wild species and in other areas. Overall, the research demonstrates the reliability of this methodological approach for monitoring marine pollution, especially in the Mediterranean. [Copyright &y& Elsevier]

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. • A multibiomarker approach with caged sticklebacks was used to assess the water quality of the Meuse watershed. • The wastewater treatment plant effluents did not significantly impact the stickleback's biomarker responses. • Fish caged at Bouillon and Avesnes presented a decrease in defence systems and an increase in the immune system respectively. • An immunosuppression and an oxidative stress were respectively shown in fish caged at Namur and Charleville-Mézières. • Based on modulated functions, the tools successfully helped to discriminate the different sites in the Meuse watershed. [ABSTRACT FROM AUTHOR]

The effectiveness of mussel caging for active microplastics (MPs) biomonitoring was investigated for the first time by comparing abundance and characteristics (shape, size, color and type of polymers) of MPs ingested by caged depurated blue mussels with those ingested by native mussels collected at the same sites and with those found in their surrounding environment (surface water and sediments). Mussels were exposed along a pollution gradient originating from a wastewater treatment plant discharge and near an abandoned coastal landfill. After 6 weeks of deployment, the majority (93%) of clean transplanted mussels had ingested MPs with a mean number of items ranging from 0.61 to 1.67 items/g. The occurrence, abundance and properties of MPs ingested by caged mussels were similar to those found in native mussels. Among the debris items detected in caged and native mussels, fragments were the most predominant type, consistent with the MPs found in their surrounding environment. MPs sizes were very similar whether in the water, sediments and both caged and native mussels, with a dominance of items <150 μm. Although some polymers were under-represented or totally absent in the caged mussels compared to overlying seawater or surrounding sediment, there was a good overlap in polymer types proportion being found between caged mussels and sediments (Morisita's index of similarity = 0.93) or seawater (0.86). Polystyrene dominated all samples in all the different matrices. Our study suggests that blue mussels caging may be a promising tool for MPs biomonitoring making monitoring more reliable with an accurate assessment of the biological effects of MPs over a predetermined exposure period. However, further methodological improvements should be considered to define a uniform protocol for blue mussels caging to allow spatial and temporal microplastics active biomonitoring. Unlabelled Image • The effectiveness of using caged blue mussels for MPs biomonitoring is evaluated. • Quantitative and qualitative similarity in MPs ingested by caged and native mussels • Good similarity overlap in MPs size, color and polymers types between all matrices • Highest polymer similarity overlap between caged mussels and sediments • Blue mussels caging is a promising tool for MPs biomonitoring. [ABSTRACT FROM AUTHOR]