Caffeine and salicylic acid: threat for aquatic biota? A case study of mussels under realistic scenarios

The discharge of micropollutants (ng/L-µg/L) like pharmaceutical active compounds (PhACs) in coastal environments constitutes an emerging concern for aquatic biota. Hence, an assessment of the biological impact of PhACs at realistic conditions (environmental concentrations and combined exposures) is essential for defining new guidelines and enhancing more effective recovery strategies. In this work, marine mussels Mytilus galloprovincialis were exposed for 12 days to environmental concentrations of caffeine (CAF) and salicylic acid (SA), two of the most persistent PhACs in the aquatic environment, both individually (CAF: 5 ng/L to 10 µg/L; SA: 0.05 µg/L to 100 µg/L) and in combination (CAF+SA; 5 ng/L+0.05 µg/L to 10 µg/L+100 µg/L). Histological and biochemical analyses were conducted on the gills at different sampling points (T0; T3: 3 days; T5: 5 days; T12: 12 days) to assess tissue organization (H&E staining) and PhACs effect on the antioxidant system (catalase, superoxide dismutase, glutathione S-transferase, lipid peroxidation) and neurotransmission (acetylcholinesterase). A moderate haemocyte infiltration, more marked for the combined CAF+SA exposure, was observed in the gills. A more prominent pro-oxidant effect induced by CAF was observed compared to SA, likely attributable to a mitochondrial damage triggered by this salicylate. However, in the combined exposure counterbalanced patterns occurred between the two PhACs effects on the antioxidant system. The biochemical analysis revealed neurotoxicity at T12 caused by the two single compounds, observed also after their combined exposure at T5. Overall, findings of this work provide new insights into the elucidation of the real health status of non-target organisms exposed to pharmaceutical micropollutants, thus representing a preliminary phase for the implementation of recovery and mitigation approaches towards these emerging pollutants.