Multilevel toxicity assessment of polypropylene microplastics and pyrene on mussels: DNA damage, oxidative stress, and physiological effects

Despite extensive research on microplastic pollution, combined biological effects of microplastics and associated pollutants on marine invertebrates remain unclear. We present an integrative assessment of polypropylene (PP) and pyrene, individually and in co-exposure, in mussel Mytilus galloprovincialis. Mussels were exposed to 1 mg L PP (~40 μm) and 50 μg L of pyrene for 7 and 14 days, representing a scenario relevant to highly polluted coastal areas. DNA damage increased significantly in mussels exposed to pyrene or PP alone, but remained at control levels under combined exposure, suggesting an interaction that may reduce genotoxic potential. Lipid peroxidation remained stable across treatments, despite significant changes in antioxidant enzymes. Catalase activity increased in pyrene and pyrene + PP treatments, with tissue-specific trends, indicating enhanced antioxidant protection. Glutathione S-transferase activity was stable in digestive glands but significantly inhibited in gills after seven days under PP exposure. ETS activity increased in pyrene-containing treatments after 14 days, reflecting elevated metabolic demand after prolonged exposure. Respiration rate declined under PP exposure. Heart rate recovery time after the hyposalinity test was the slowest in the pyrene + PP group, indicating compromised physiological resilience. These findings reveal interactive, tissue- and biomarker-specific effects of PP and pyrene. Their combination suggested attenuation of genotoxicity but enhanced physiological stress responses, highlighting the complexity of pollutant interactions and importance of evaluating multiple biomarkers, tissues and pollutants. Presented data provide the first ever biomarker-based evaluation of PP and pyrene co-exposure, offering novel insights into microplastic-pollutant interactions and potential ecological consequences for marine invertebrates.