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Microplastics (MPs) are now one of the major environmental problems due to the large amount released in aquatic and terrestrial ecosystems, as well as their diffuse sources and potential impacts on organisms and human health. Still the molecular and cellular targets of microplastics' toxicity have not yet been identified and their mechanism of actions in aquatic organisms are largely unknown. In order to partially fill this gap, we used a mass spectrometry based functional proteomics to evaluate the modulation of protein profiling in zebra mussel (Dreissena polymorpha), one of the most useful freshwater biological model. Mussels were exposed for 6 days in static conditions to two different microplastic mixtures, composed by two types of virgin polystyrene microbeads (size=1 and 10 m) each one. The mixture at the lowest concentration contained 5 × 10 5 MPs/L of 1µm and 5 × 10 5 MPs/L of 10 µm, while the higher one was arranged with 2 × 10 6 MPs/L of 1 µm and 2 × 10 6 MPs/L of 10 µm. Proteomics' analyses of gills showed the complete lack of proteins' modulation after the exposure to the low-concentrated mixture, while even 78 proteins were differentially modulated after the exposure to the high-concentrated one, suggesting the presence of an effect-threshold. The modulated proteins belong to 5 different classes mainly involved in the structure and function of ribosomes, energy metabolism, cellular trafficking, RNA-binding and cytoskeleton, all related to the response against the oxidative stress. Capsule: a mixture of polystyrene microplastics (2 × 10 6 MPs/L of 1 µm and 2 × 10 6 MPs/L of 10 µm) was able to modulate 78 different proteins of the zebra mussels' gills most involved in the oxidative stress response.

Studies related to the evaluation of plastics in freshwaters have been increasing in recent years because approximately 80% of plastic items found in the sea are from inland waters. Despite the ecological relevance of these surveys, no information has been available until now about the hazard related to plastic mixtures in freshwaters. To fill this knowledge gap, we carried out a study aimed to assess the environmental risk associated with the "cocktail" of plastics and environmental pollutants adsorbed on their surface in one of the larger European freshwater basins. Plastic debris was collected by a manta trawl along one transect each in four of the Italian subalpine great lakes (Lake Maggiore, Como, Iseo and Garda) and

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