Sub-lethal Effects Induced by a Mixture of Three Non-steroidal Anti-inflammatory Drugs (NSAIDs) on the Freshwater Bivalve Dreissena Polymorpha

Non-steroidal anti-inflammatory drugs (NSAIDs) are the sixth top-selling drugs worldwide and are commonly found in freshwater ecosystems in the high ng/l to low μg/l range. Recent studies have investigated both the acute and the chronic toxicity of single NSAIDs on different biological models, but these studies have completely neglected the fact that, in the environment, non-target organisms are exposed to mixtures of drugs that have unforeseeable toxicological behavior. This work investigated the sub-lethal effects induced by a mixture of three common NSAIDs, namely, diclofenac, ibuprofen and paracetamol, on the freshwater bivalve, the zebra mussel (Dreissena polymorpha). The mussels were exposed to three different environmental concentrations of the mixture (Low, Mid and High). A multi-biomarker approach was used to highlight cyto-genotoxic effects and the imbalance of the oxidative status of the treated specimens. The Neutral Red Retention Assay (NRRA) was used as a biomarker of cytotoxicity, whereas the activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase were measured to assess the role played by the oxidative stress enzymes. In addition, the single cell gel electrophoresis assay, the DNA Diffusion assay and the micronucleus test were used to investigate possible genotoxic effects. According to our NRRA results, each treatment was able to induce a significant cellular stress in bivalves, probably due to the raise of oxidative stress, as indicated by the alteration of enzyme activities measured in treated specimens. Moreover, the mixture induced significant enhancements of DNA fragmentation, which preluded fixed genetic damage, as highlighted by the increase of both apoptotic and micronucleated cells.