Histopathological analysis of mussels Mytilus galloprovincialis after foodborne exposure to three sizes of polystyrene nanoplastics: Relevance of confounding factors.

Given the widespread occurrence of polystyrene (PS) micro- and nanoplastics in the marine environment, it is important to determine their potential adverse effects on representative sentinel marine species as mussels. Foodborne exposure is more environmentally realistic than waterborne exposure in mussels, but it has received less attention. The aim of this work was to assess the histopathological effects of foodborne exposure to PS nanoplastics (NPs) of different sizes on marine mussels, focusing on inflammatory reactions involving hemocytes, since microplastics (MPs) and NPs have been reported to produce inflammatory responses. Mussels Mytilus galloprovincialis were dietarily exposed through algae Isochrysis galbana to PS NPs of 50, 200 and 1000 nm at nominal low (LD) (10^3 NPs/mL) and high concentrations (HD) (10^8 NPs/mL for 50 and 200 nm NPs and10^6 NPs/mL for 1000 nm NPs). Exposures took place for seven days in duplicate and two independent experiments were carried out per NP (E1 and E4 for 50 nm, E2 and E5 for 200 nm and E3 and E6 for 1000 nm PS NPs). All experiments were developed during three weeks in September-October (E1-E2, E3-E4 and E5-E6) to minimize the influence of confounding factors on studied parameters. High prevalences of hemocytic infiltration, fibrosis and atrophy and necrosis of the digestive tubule epithelium (DE) were detected in the marine mussels' digestive glands. In the gonad, prevalences of the different alterations were generally lower than in the digestive gland. Overall, the GLMM showed that stage of gametogenic development and the presence of parasites were significant confounding factors that influenced atrophy and necrosis of DE, oocyte atresia and hemocytic infiltration in the connective tissue of the gonad. Thus, it does not appear that these responses can be used as biomarkers of NP exposure in mussels, at least at the nominal concentrations tested and in a short-term exposure, since other factors such as reproductive stage and parasitosis affect these responses too. It remains to be determined whether longer term exposures could result in more severe histopathological alterations, independent of the physiological condition of mussels. Further work is required on environmentally realistic NPs that might pose additional risks compared to pristine ones.