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.