Cellular effects of microplastics are influenced by their dimension: Mechanistic relationships and integrated criteria for particles definition.*

The definition of microplastics (MPs) is nowadays too generic from a biological perspective, since different characteristics of these particles might influence their effects. To provide experimental evidence that size is an important factor to be considered, Mediterranean mussels Mytilus galloprovincialis were exposed to five size classes of polyethylene fragments (PE-MPs, 20-50 mu m, 50-100 mu m, 100-250 mu m, 250-500 mu m, 500-1000 mu m). After 10 days of exposure, MPs ingestion and mechanistic relationships between particles size and cellular effects were analysed through a wide panel of biological alterations, including immune system responses, cholinergic function, antioxidant system, lipid metabolism and peroxidation. Results were further elaborated through a Weight of Evidence approach, summarizing the overall biological significance of obtained results in a hazard index based on the number and magnitude of variations and their toxicological relevance. PE-MPs 500-1000 mu m were identified as the less biologically reactive size class due to the limited ingestion of particles coupled with the lack of biological effects, followed by PE-MPs 250-500 mu m, which slightly altered the cholinergic function and lysosomal membranes. Conversely, PE-MPs smaller than 250 mu m provoked a more consistent onset of biological alterations in terms of immune system composition and functioning, redox homeostasis, and lipid metabolism. The overall findings of this study highlight the importance of considering the size of particles for monitoring and risk assessment of MPs, introducing a more integrated evaluation of plastic pollution that, beside particles concentration, should adequately weigh those characteristics triggering the onset of biological effects.