Illicit drugs have been recognized as emerging environmental pollutants that could represent a potential risk for aquatic communities. Even if many studies have shown the occurrence of several drugs of abuse and their metabolites in freshwaters in the High ng/L to Low μg/L range worldwide, no information on their potentially harmful effects on non-target organisms is available. The aim of this study was to investigate sub-lethal effects induced by the main metabolite of cocaine, the benzoylecgonine (BE), on the freshwater bivalve Dreissena polymorpha. Mussels were exposed under semi-static conditions for 14 days to two environmentally relevant BE concentrations (0.5 μg/L and 1 μg/L) and induced adverse effects were evaluated through the application of a suite of ten different biomarkers. We applied on bivalve hemocytes the single cell gel electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test) to investigate DNA injuries, while the neutral red retention assay (NRRA) was used to assess BE cytotoxicity. Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities, as well as the lipid peroxidation (LPO) and protein carbonyl content (PCC), were measured as oxidative stress indices in zebra mussel homogenates. Significant decrease in lysosomal membrane stability and imbalances of defense enzyme activities were found at both exposure concentrations, suggesting the involvement of oxidative stress in BE toxicity. Significant increases in LPO and PCC, as well as in primary (DNA strand breaks) and fixed DNA damage (apoptotic and micronucleated cell frequency), were found at the highest BE treatment, confirming that adverse effects to macromolecules were due to the increase of BE-induced oxidative stress.
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