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A 12-month active biomonitoring study was performed in 2008-2009 on the Vesle river basin (Champagne-Ardenne, France) using the freshwater mussel Dreissena polymorpha as a sentinel species; allochthonous mussels originating from a reference site (Commercy) were exposed at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle river basin. Selected core biomarkers (acetylcholinesterase (AChE) activity, glutathione-S transferase (GST) activity, metallothionein concentration), along with digestive enzyme activities (amylase, endocellulase) and energy reserve concentrations (glycogen, lipids), were monitored throughout the study in exposed mussels. At the Fismes and Ardre sites (downstream basin), metallic and organic contamination levels were low but still high enough to elicit AChE and GST activity induction in exposed mussels (chemical stress); besides, chemical pollutants had no apparent deleterious effects on mussel condition. At the Bouy and Sept-Saulx sites (upstream basin), mussels obviously suffered from adverse food conditions which seriously impaired individual physiological state and survival (nutritional stress); food scarcity had however no apparent effects on core biomarker responses. Digestive enzyme activities responded to both chemical and nutritional stresses, the increase in energy outputs (general adaptation syndrome-downstream sites) or the decrease in energy inputs (food scarcity-upstream sites) leading to mid- or long-term induction of digestive carbohydrase activities in exposed mussels (energy optimizing strategy). Complex regulation patterns of these activities require nevertheless the use of a multi-marker approach to allow data interpretation. Besides, their sensitivity to natural confounding environmental factors remains to be precised.

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The toxicity of dietary exposure to artificially aged TiO₂ nanomaterial (T-Lite) used in sunscreen cream was studied on Danio rerio. Embryolarval assays were conducted to assess the effects of TiO₂ residues of nanomaterial (RNM) on fish early life stages. Juvenile fishes were exposed by the trophic route in two experiments. During the first experiment, juvenile fishes were exposed to TiO₂ RNM for 14 days by adding RNM to commercial fish food. The second one consisted in producing a trophic food chain. Pseudokirchneriella subcapitata algae, previously contaminated with TiO₂ RNM in growth medium, was used to feed Daphnia magna neonates over a 48-h period. Daphnia were used next to feed juvenile fishes for 7 days. Accumulation of Ti, life traits (survival and growth) and biochemical parameters such as energy reserves, digestive (trypsin, esterase, cellulose and amylase) and antioxidant (superoxide dismutase and catalase) enzyme activity were measured at the end of exposures. As expected in the receiving aquatic system, TiO2 RNM at low concentrations caused a low impact on juvenile zebrafish. A slight impact on the early life stage of zebrafish with premature hatching was observed, and this effect appeared mainly indirect, due to possible embryo hypoxia. When juvenile fish are exposed to contaminated food, digestive enzyme activity indicated a negative effect of TiO₂ RNM. Digestive physiology was altered after 14 days of exposure and seemed to be an indirect target of TiO₂ RNM when provided by food.

Biotransformation enzymatic activities, such as the cytochrome P450 one, have been used as biomarkers for pollution assessment for a long time. Nevertheless, such biochemical tools are destructive processes, because they are performed on fish liver or total larvae homogenates. Moreover, the adaptation of this bioassay to some fish larvae, like Danio rerio ones, is ineffective because it needs a lot of organisms. We thus developed an original, nondestructive method to detect the induction of a biotransformation activity in the prolarva of the fish, Danio rerio, exposed to chemicals. This methodology is based on the assessment of EROD activity, by measurement in the culture medium of the fluorescence increase due to the excretion of resorufin by fish during an ethoxyresorufin exposure. After exposure of fish embryos to known inducers (BaP and beta-naphtoflavone), the prolarvae were exposed to the substrate (ethoxyresorufin), and the kinetic of the fluorescence increase was measured. A dose-effect relationship was observed, with a three to five fold increase of EROD basal activity. This methodology also allowed us to compare between EROD activity induction by different environmental samples. The proposed methodology thus allows to perform a simple, sensitive, and reproducible microbiotest for the detection of sublethal concentrations of AhR chemical inducers in environmental samples.

Wastewater treatment plants (WWTPs) are one of the main sources of freshwater pollution eventually resulting in adverse effects in aquatic organisms. Treated effluents can contain many micropollutants at concentrations often below the limit of chemical quantification. On a regulatory basis, WWTP effluents have to be non-toxic to the aquatic environment, wherefore not only chemical abatement but also ecotoxicological evaluation through relevant bioassays is required. Standardized bioassays currently used are often not sensitive enough to reveal a residual toxicity in treated effluents. Therefore, attention must be paid to the development of better-adapted approaches implementing more sensitive organisms and relevant endpoints. In this study, the toxicity of two differently treated effluents (activated sludge treated effluents with and without ozonation) towards the ecologically relevant species Gammarus fossarum was evaluated. Organism fitness traits such as reproduction and sperm DNA integrity were followed in exposed organisms. In complement, enzymatic biomarkers were measured indicating the presence of neurotoxic compounds (acetylcholinesterase activity), the presence of pathogens likely to increase the toxic effects of chemical compounds (phenol-oxidase activity), and the presence of toxic compounds inducing detoxification mechanisms (glutathione-S-transferase activity). Enzymatic activities were not modified, but significant sub-lethal effects were observed in exposed organisms. In both effluents, females showed a retarded molt cycle, a reduced fecundity and fertility, and >90% of developed embryos exhibited developmental malformations. In addition, a slight but significant genotoxic effect was measured in gammarid sperm. In a whole, no difference in toxicity was found between both effluents. Coupling reproduction impairment and genotoxicity assessment in Gammarus fossarum seems to be a valuable and sensitive tool to reveal residual toxicity in effluents containing a mixture of micropollutants at very low concentrations. Finally, a direct relationship between the observed toxic responses and the quantified micropollutant concentrations could not be evidenced.

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