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.

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.

Evidence of highly dynamic geochemical behaviour of zinc in the Deûle river (northern France).

The monitoring of dissolved zinc in the Deûle river was undertaken at three different periods of the years 2008 and 2009. Electrolabile concentrations of Zn were estimated every 2 hours for several weeks by using an ATMS (Automatic Trace metal Monitoring System) based on voltammetric measurements using a solid Ag-Hg rotating disc working electrode. Complementary measurements were carried out with DGT (Diffusive Gradient in Thin films) pistons deployed directly in the river for 24 hours. Water samples filtrated at 0.45 µm were also analysed by HR-ICP-MS to estimate the total dissolved concentrations of zinc and other trace metals. High frequency monitoring of zinc over several weeks in the Deûle river indicated that the concentration could change significantly over short time periods. Resuspension of polluted sediment and biological activities are two main factors that control the behaviour of zinc in the Deûle river. Furthermore, in May 2009, daily cycles of the electrolabile zinc fraction have been observed at relatively constant total dissolved concentration. It is assumed that this particular behaviour of zinc is based on an exchange between colloids and/or nonelectrolabile forms and free cation and inorganic complexes at a daily time scale.

Diel cycles of reduced manganese and their seasonal variability in the Marque River (northern France).

Electrolabile reduced manganese (II) has been monitored by voltammetry during two periods of one month in summer 2014 and at the end of winter 2015 in a small river (the Marque River) located in northern France and going through a suburban area with agricultural activities. Diel variations, evolution within the one-month periods and seasonal differences have been observed. Taking into consideration the multiple physical, biological and chemical reactions regulating manganese speciation in aquatic systems, it has been demonstrated that manganese speciation is probably controlled by the competition of two antagonist reactions: the photoreduction of manganese oxides (in broad sense and represented thereafter by MnOx) and the biotic oxidation of Mn(II). Depending on the season, the biological activity in the river and the amount of luminosity reaching the MnOx, either the production of reduced labile Mn(II) or the precipitation of MnOx can become the dominant process. Other punctual events such as the drop of oxygen concentration due to large inputs of biodegradable organic matter and eutrophication phenomena, rainy events and high luminosity periods can also affect the behaviour of dissolved Mn(II) in the Marque River and so, of other contaminants.

Comparison of POCIS passive samplers vs. composite water sampling: A case study.

The relevance of Polar Organic Chemical Integrative Samplers (POCIS) was evaluated for the assessment of concentrations of 46 pesticides and 19 pharmaceuticals in a small, peri-urban river with multi-origin inputs. Throughout the period of POCIS deployment, 24h-average water samples were collected automatically, and showed the rapid temporal evolution of concentrations of several micropollutants, as well as permitting the calculation of average concentrations in the water phase for comparison with those estimated from POCIS passive samplers. In the daily water samples, cyproconazol, epoxyconazol and imidacloprid showed high temporal variations with concentrations ranging from under the limit of detection up to several hundreds of ngL-1. Erythromycin, cyprofloxacin and iopromide also increased rapidly up to tens of ngL-1 within a few days. Conversely, atrazine, caffeine, diclofenac, and to a lesser extent carbamazepine and sucralose, were systematically present in the water samples and showed limited variation in concentrations. For most of the substances studied here, the passive samplers gave reliable average concentrations between the minimal and maximal daily concentrations during the time of deployment. For pesticides, a relatively good correlation was clearly established (R2=0.89) between the concentrations obtained by POCIS and those gained from average water samples. A slight underestimation of the concentration by POCIS can be attributed to inappropriate sampling rates extracted from the literature and for our system, and new values are proposed. Considering the all data set, 75% of the results indicate a relatively good agreement between the POCIS and the average water samples concentration (values of the ratio ranging between 0,33 and 3). Note further that this agreement between these concentrations remains valid considering different sampling rates extracted from the literature.

An adaptable automatic trace metal monitoring system for on line measuring in natural waters.

An adaptable automatic trace metal monitoring system (ATMS) was assembled and embedded in a mobile monitoring station belonging to the French Water Agency Artois-Picardie (AEAP) and deployed in the field to measure the concentration of trace metals (electroactive and acid leachable fractions) in natural waters by anodic stripping voltammetry with a hanging mercury drop electrode. Cathodic stripping voltammetry procedures were included to estimate the concentration of dissolved oxygen and reduced sulphur species. The concept of the measuring system enables easy adaptation of methods and procedures to analytes of concern and gives the opportunity to undertake in real-time a routine analysis of the dynamic behaviour of trace metals in river, pond and seawater. The system was tested in two aquatic bodies: in a pond where eutrophication processes occur recurrently and in the Deûle River, where sediments are highly contaminated by several metals such as Pb and Zn and frequently resuspended because of the river traffic. Preliminary field studies demonstrated that trace metal concentrations can evolve quickly as a function of time, depending on the turbidity and luminescence, i.e. day-night cycles. The obtained results were compared with an Environmental Quality Standard (EQS), the Criteria Maximum Concentration (CMC) and the Criterion Continuous Concentration (CCC). The whole system is also prepared for the task of "early warning".

Light modulated toxicity of isoproturon toward natural stream periphyton photosynthesis: a comparison between constant and dynamic light conditions.

This study tested if a variation in light intensity, in comparison to constant light required in well-designed toxicity test, could have measurable consequences on the sensitivity of phototrophic biofilms (periphyton) to isoproturon. Two independent experiments were carried out to investigate the combined effects of light and isoproturon on the photochemical behavior of intact natural biofilms by measurements of chlorophyll fluorescence and pigment composition. Experiment 1 consisted of exposing biofilms to series of isoproturon concentrations (0-2 mg L(-1)) for 7 h under constant light at different irradiance levels (25-300 micromol m(-2) s(-1)). In experiment 2, biofilms were exposed using more environmentally realistic conditions to three selected concentrations of isoproturon (2, 6 and 20 microg L(-1)) during a 7-h-simulated daily light cycle. Our results demonstrated that light, considered here as a direct physical stressor, slightly modulated the acute toxicity of isoproturon on these diatom dominated communities. This was attributed to the fact that these two factors act specifically on the photosynthetic activity. Furthermore, it was shown that a dynamic light regime increased periphyton sensitivity to isoproturon by challenging its photoprotective mechanisms such as the xanthophyll cycle, therefore implying that traditional ecotoxicological bioassays lead to underestimate the effect of isoproturon.

STREAM PERIPHYTON PHOTOACCLIMATION RESPONSE IN FIELD CONDITIONS: EFFECT OF COMMUNITY DEVELOPMENT AND SEASONAL CHANGES(1).

The photochemical behavior of intact stream periphyton communities in France was evaluated in response to the time course of natural light. Intact biofilms grown on glass substrata were collected at three development stages in July and November, and structural parameters of the biofilms were investigated (diatom density and taxonomy). At each season, physiological parameters based on pigment analysis (HPLC) and pulse-amplitude-modulated (PAM) chl fluorescence technique were estimated periodically during a day from dawn to zenith. Regardless of the community studied, the optimal quantum yield of PSII (Fv /Fm ), the effective PSII efficiency (ΦPSII ), the nonphotochemical quenching (NPQ), and the relative electron transport rate (rETR) exhibited clear dynamic patterns over the morning. Moreover, microalgae responded to the light increase by developing the photoprotective xanthophyll cycle. The analysis of P-I parameters and pigment profiles suggests that July communities were adapted to higher light environments in comparison with November ones, which could be partly explained by a shift in the taxonomic composition. Finally, differences between development stages were significant only in July. In particular, photoinhibition was less pronounced in mature assemblages, indicating that self-shading (in relation to algal biomass) could have influenced photosynthesis in older communities.