An integrated approach to assess the molecular mechanisms and cellular effects of environmentally realistic mixtures of microplastics in Mytilus galloprovincialis.

Microplastics pose great biological concern due to their ubiquity and ability to enter food webs. Despite their environmental heterogeneity, most laboratory studies still rely on pristine, single-polymer microplastics that inadequately reflect complex field conditions. We provide an integrated multilevel toxicological assessment in mussels Mytilus galloprovincialis exposed for 14 days to three 20-50 µm microplastics typologies: EPM, Environmental Plastics Mixture, obtained by micronizing field-collected objects of polyethylene, polyethylene terephthalate, polypropylene, polystyrene, and polyvinyl chloride; VPM, Virgin Plastics Mixture, pristine particles matching EPM polymer composition; RTR, Rapido Trawling Rubber, derived from fisheries-related litter (ethylene-propylene-diene monomer). Additive bioaccumulation, transcriptomic profiles, cellular biomarkers, and microbiota composition were assessed. EPM increased total phthalate concentrations 4.6-fold relative to controls, with DEHP accounting > 90% of the total, and induced immune, oxidative, metabolic, and microbiota alterations. RTR mainly affected redox balance and energy metabolism, whereas VPM produced more limited responses. The integration of results through a Weight of Evidence framework provided synthetic hazard classification, indicating greater integrated toxicological concern for the environmentally derived materials under the tested conditions (EPM and RTR: "MODERATE"; VPM: "SLIGHT"). Overall findings highlight the importance of incorporating environmentally-derived microplastics into toxicological studies to better reflect realistic exposure scenarios and capture their heterogeneous physico-chemical properties.