SINGLE AND COMBINED EXPOSURE OF POLYSTYRENE NANOPARTICLES AND NORTH SEA MARINE OIL IN TWO IN VITRO FISH MODELS

Abstract: The awareness of micro-and nanoplastic pollution and its impact on the marine ecosystem has increased over the last decade. Nanoplastics may affect aquatic organisms since their small size and high surface-to-volume ratio increases their bioavailability and ability to cross biological membranes. An enhanced understanding of interactions between nanoplastics and other environmental pollutants is needed for risk assessment and management. This study aims to examine possible toxic effects associated with single and combined exposures of polystyrene nanoparticles (PsNPs) and water-accommodated fractions (WAF) of North Sea marine oil, by measuring acute toxicity and exploring sensitive in vitro assays on two in vitro fish models. Rainbow trout gill- and liver cell lines were exposed to 25 nm PsNPs (1-100 μg/mL), WAF (10-90%) or a mixture of them for 24 h. AlamarBlue and Neutral Red assays revealed a dose dependent decrease in cell metabolic activity and lysosomal stability after WAF exposure. 80%- 90% WAF reduced cell membrane integrity significantly in the CFDA-AM viability assay. PsNPs alone did not significantly affect the cytotoxicity. However, higher concentration of PsNPs in the mixtures significantly decreased cell viability, indicating an additive effect. Generation of intracellular reactive oxygen species measured with DCFH-DA assay increased over a 6 h period for all treatments. No significant differences among single exposures of PsNPs or of different PsNP concentrations in the mixtures were found, even if a slightly protective effect can be discussed. A trend of increasing induction of CYP1A dependent EROD-activity was observed in cells exposed to 20-50% WAF, and to mixtures with higher PsNP concentration. Sublethal- and lethal effects in zebrafish embryos tended to increase with higher concentration of WAF alone, and with higher PsNP concentration in the mixtures. To conclude, PsNPs alone were generally not found to be toxic but may increase toxicity of organic pollutants found in crude oil.