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.

Sub-lethal toxicity of five disinfection by-products on microalgae determined by flow cytometry - Lines of evidence for adverse outcome pathways.

Standardised tests are often used to determine the ecotoxicity of chemicals and focus mainly on one or a few generic endpoints (e.g. mortality, growth), but information on the sub-cellular processes leading to these effects remain usually partial or missing. Flow cytometry (FCM) can be a practical tool to study the physiological responses of individual cells (such as microalgae) exposed to a stress via the use of fluorochromes and their morphology and natural autofluorescence. This work aimed to assess the effects of five chlorine-based disinfection by-products (DBPs) taken individually on growth and sub-cellular endpoints of the green microalgae Raphidocelis subcapitata. These five DBPs, characteristic of a chlorinated effluent, are the following monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), bromochloroacetic acid (BCAA) and 1,1-dichloropropan-2-one (1,1-DCP). Results showed that 1,1-DCP had the strongest effect on growth inhibition (EC50 = 1.8 mg.L-1), followed by MCAA, TCAA, BCAA and DCAA (EC50 of 10.1, 15.7, 27.3 and 64.5 mg.L-1 respectively). Neutral lipid content, reactive oxygen species (ROS) formation, red autofluorescence, green autofluorescence, size and intracellular complexity were significantly affected by the exposure to the five DBPs. Only mitochondrial membrane potential did not show any variation. Important cellular damages (>10%) were observed for only two of the chemicals (BCAA and 1,1-DCP) and were probably due to ROS formation. The most sensitive and informative sub-lethal parameter studied was metabolic activity (esterase activity), for which three types of response were observed. Combining all this information, an adverse outcome pathways framework was proposed to explain the effect of the targeted chemicals on R. subcapitata. Based on these results, both FCM sub-cellular analysis and conventional endpoint of algal toxicity were found to be complementary approaches.

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.

Exposure and hazard of bisphenol A, S and F: a multi-biomarker approach in three-spined stickleback.

Due to the estrogenic behavior of bisphenol (BP) A, industries have developed many substitutes, such as BPS and BPF. However, due to their structural similarities, adverse effects on reproduction are currently observed in various organisms, including fish. Even if new results have shown impacts of these bisphenols on many other physiological functions, their mode of action remains unclear. In this context, we proposed to better understand the impact of BPA, BPS, and BPF on immune responses (leucocyte sub-populations, cell death, respiratory burst, lysosomal presence, and phagocytic activity) and on biomarkers of metabolic detoxification (ethoxyresorufin-O-deethylase, EROD, and glutathione S-transferase, GST) and oxidative stress (glutathione peroxidase, GPx, and lipid peroxidation with thiobarbituric acid reactive substance method, TBARS) in an adult sentinel fish species, the three-spined stickleback. In order to enhance our understanding of how biomarkers change over time, it is essential to determine the internal concentration responsible for the observed responses. Therefore, it is necessary to explore the toxicokinetics of bisphenols. Thus, sticklebacks were exposed either to 100 µg/L of BPA, BPF or BPS for 21 days, or for seven days to 10 and 100 µg/L of BPA or BPS followed by seven days of depuration. Although BPS has very different TK, due to its lower bioaccumulation compared to BPA and BPF, BPS affect oxidative stress and phagocytic activity in the same way. For those reasons, the replacement of BPA by any substitute should be made carefully in terms of risk assessment on aquatic ecosystems.

PBTK-TD model of the phagocytosis activity in three-spined stickleback exposed to BPA.

Due to the high production volume and persistence in the environment of bisphenol A (BPA) and its substitutes, realistic exposure scenarii were proposed in some species to better understand the relationship between external and internal concentrations. For example, a recent PBTK model has been developed and adapted to BPA ADME (Absorption, Distribution, Metabolization, and Excretion) processes in three-spined stickleback. These substances have an impact on organism physiology including reproductive and immune functions. In this context, physiologically-based toxicokinetic models coupled with toxicodynamics (PBTK-TD) have proven to be valuable tools to fill the knowledge gap between external exposure and effect dynamics. The aim of the current work was to explain the impact of BPA on the immune response by determining its temporality. In addition, the relationship between BPA dose and these responses was investigated using a PBTK-TD model. Two experiments were performed on stickleback to characterize their biomarker responses, (i) a short exposure (14 days) at 0, 10 and 100 µg/L, including a depuration phase (7 days), and (ii) a long exposure (21 days) at 100 µg/L to measure the immunomarker dynamic over a long period. The fish spleens were sampled to analyze immune responses of stickleback at various times of exposure and depuration: leucocyte distribution, phagocytic capacity and efficiency, lysosomal presence and leucocyte respiratory burst index. At the same date, blood, muscle, and liver were sampled to quantify BPA and their metabolites (BPA monoglucuronide and BPA monosulfate). All these data enabled the development of the indirect pharmacodynamic models (PBTK-TD) by implementing the responses of biomarkers in the existing BPA PBTK of stickleback. The results shown a high induction of phagocytosis activity by BPA in the two exposure conditions. Furthermore, the immunomarkers exhibit very different temporal dynamics. This study demonstrates the need of a thorough characterization of biomarker response for a further use in Environmental Biomonitoring.

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.

Time and dose-dependent impairment of liver metabolism in Gasterosteus aculeatus following exposure to diclofenac (DCF) highlighted by LC-HRMS untargeted metabolomics.

Anthropogenic chemicals as emerging contaminants, such as pharmaceuticals, increased worldwide in the environment. This study aimed to apply metabolomics-based approaches on the fish model species three-spined stickleback (Gasterosteus aculeatus) exposed to diclofenac (DCF) to identify toxicity pathways and potential biomarkers. For this purpose, males and females were exposed to a continuous flow of diclofenac solution in laboratory for 21 days, followed by 3 days of depuration, to nominal concentrations of 1 (low) and 100 µg/L (high) of DCF. A methodology based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) was employed. Uni- and multivariate statistical analyses were combined to evaluate the modulations of the liver metabolome of G. aculeatus after exposure to DCF. The metabolomics data revealed variations both as a function of time and of the DCF concentration. We observed 2487 altered metabolites, with 1460 and 1027 specific to males and females, respectively. Some of them were significantly impaired by the experimental conditions. However, we showed that several metabolites were impacted by other factors as they were already modulated in the control individuals. The results indicated that the energy metabolism was up-modulated in females and down-modulated in males, with the presence of DCF. The antioxidant system was impacted in males, suggesting oxidative stress in the metabolism, while the immunity system was down-regulated in females following exposure. Moreover, our results revealed 1 and 4 metabolites as potential metabolic biomarkers in male and female sticklebacks, respectively. Among them, the glutaryl-carnitine and the adipoyl-carnitine were putatively identified in females, known to be implicated in the energy metabolism. These 5 metabolites showed to be promising biomarkers since they were early modulated during exposure to the stress and showed a notable trend through time.

Bioaccumulation of trace metal elements and biomarker responses in caged juvenile flounder at a polluted site: Effects of fish density and time exposure.

This study investigates the effect of fish density and exposure duration on trace metal elements (TME) bioaccumulation and several biomarkers response. Juvenile flounders were caged at low, medium and high densities and exposed during 15 or 30 days in the Seine estuary. The concentrations of the TME measured in the muscle of the caged fish were all in agreement with their bioavailability percentage in the sediments. Higher concentrations of TME were found in flounders' muscle exposed for 15 days compared with those caged for 30 days. For the same exposure time, the density of fish had no effect on the accumulation of the TME in the flounders' muscle. Biomarkers responses varied according to density and duration of exposure. Special care should be taken in their interpretation. We underline that for an optimal assessment of TME pollution in the field, 15 days with low densities of fish per cage are sufficient.

The toxicokinetics of bisphenol A and its metabolites in fish elucidated by a PBTK model.

Bisphenol A (BPA) is a chemical of major concern due to its endocrine disrupting function, high production volume, and persistence in the aquatic environment. Consequently, organisms such as fish are subject to chronic exposure to BPA. However, physiologically-based toxicokinetic (PBTK) models, which are valuable tools to improve the understanding of a chemical's fate in an organism, have never been specifically adapted to model BPA toxicokinetics (TK) in fish. In our work, an existing PBTK developed for four different fish species was modified to model BPA ADME processes (absorption, distribution, metabolization and excretion). The metabolization of BPA into BPA-monoglucuronide (BPA gluc) and BPA-monosulfate (BPA sulf) and their TK in various organs was taking into account in the model. Experiments were performed to generate BPA TK data in a model species commonly used in ecotoxicology, the stickleback. The model structure had to include two sites of metabolization to simulate BPA TK accurately in stickleback organs. Thus, the fish liver may not be the only site of the metabolization of BPA: plasma or gills could also play a role in BPA metabolization. The PBTK model predictive performance evaluated on literature data in zebrafish and rainbow trout concurs with this conclusion. Finally, a calibration mixing data from the three species was compared to the calibration on stickleback data only.

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.

Modeling acetylcholine esterase inhibition resulting from exposure to a mixture of atrazine and chlorpyrifos using a physiologically-based kinetic model in fish.

Aquatic organisms are exposed to mixtures of chemicals that may interact. Mixtures of atrazine (ATR) and chlorpyrifos (CPF) may elicit synergic effects on the permanent inhibition of acetylcholinesterase (AChE) in certain aquatic organisms, causing severe damage. Mechanistic mathematical models of toxicokinetics and toxicodynamics (TD) may be used to better characterize and understand the interactions of these two chemicals. In this study, a previously published generic physiologically-based toxicokinetic (PBTK) model for fish was adapted to ATR and CPF. A sub-model of the kinetics of one of the main metabolites of CPF, chlorpyrifos-oxon (CPF-oxon), was included, as well as a TD model. Inhibition of two esterases, AChE and carboxylesterase, by ATR, CPF and CPF-oxon, was modeled using TD modeling of quantities of total and inactive esterases. Specific attention was given to the parameterization and calibration of the model to accurately predict the concentration and effects observed in the fish using Bayesian inference and published data from fathead minnow (Pimephales promelas), zebrafish (Danio rerio) and common carp (Cyprinus carpio L.). A PBTK-TD for mixtures was used to predict dose-response relationships for comparison with available adult fish data. Synergistic effects of a joint exposure to ATR and CPF could not be demonstrated in adult fish.

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.

Enhancing the Antioxidant Activity of Technical Lignins by Combining Solvent Fractionation and Ionic-Liquid Treatment.

A grass soda technical lignin (PB1000) underwent a process combining solvent fractionation and treatment with an ionic liquid (IL), and a comprehensive investigation of the structural modifications was performed by using high-performance size-exclusion chromatography, 31 P NMR spectroscopy, thioacidolysis, and GC-MS. Three fractions with distinct reactivity were recovered from successive ethyl acetate (EA), butanone, and methanol extractions. In parallel, a fraction deprived of EA extractives was obtained. The samples were treated with methyl imidazolium bromide ([HMIM]Br) by using either conventional heating or microwave irradiation. The treatment allowed us to solubilize 28 % of the EA-insoluble fraction and yielded additional free phenols in all the fractions, as a consequence of depolymerization and demethylation. The gain of the combined process in terms of antioxidant properties was demonstrated through 2,2-diphenyl-1-picrylhydrazyl (DPPH. ) radical-scavenging tests. Integrating further IL safety-related data and environmental considerations, this study paves the way for the sustainable production of phenolic oligomers competing with commercial antioxidants.

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.

Effects of a chronic exposure to different water temperatures and/or to an environmental cadmium concentration on the reproduction of the threespine stickleback (Gasterosteus aculeatus).

Knowledge about combined effects of chemicals and temperature on reproductive capacity of fish are rare in literature, especially when it comes to the effects of chronic low-dose chemical exposure combined to the thermal stress. The aim of the study was to evaluate the single and combined effects of temperature (16, 18, 21 °C) and an environmentally relevant concentration of waterborne cadmium (1 µg L-1, nominal concentration) on the reproductive outputs of threespine stickleback (Gasterosteus aculeatus), and their consequences on offspring survival parameters. The high temperature (21 °C) was the only factor that affected parental parameters (gonadosomatic index "GSI", and vitellogenin "VTG" particularly). On females, 21 °C had a stimulating effect on gonadal development evaluated by an early increase, followed by a sharp decrease of GSI, probably indicating gonadal atresia. Promoting effect of temperature was corroborated by an early production of VTG. In vitro fertilization assays showed interesting results, particularly cadmium effects. As it was supposed, high temperature had a negative impact on offspring parameters (significant decrease in survival and an increase of unhatched embryos). Parental exposure to the very low concentration of cadmium had also negative consequences on mortality rate (significant increase) and hatching rate (significant decrease). Our results indicate that in a global warming context, high temperature and its combination with contaminant may impact reproductive capacity of G. aculeatus, by decreasing parental investment (low eggs and/or sperm quality).