Cellular uptake of PET nanoplastics in brittle stars: impairment of arm regeneration and behavioural traits

: Nanoplastics are emerging contaminants of increasing concern in marine ecosystems, yet their in vivo biodistribution and biological effects in benthic organisms remain poorly understood. In this study, we provide an integrated, multi-level assessment of the uptake, translocation, and biological effects of polyethylene terephthalate nanoplastics (PET-NPs) in the brittle star Ophiactis virens, an ecologically relevant benthic invertebrate with high regenerative capacity. Individuals were exposed for 14 days to environmentally relevant (0.05 and 0.5 mg/L) and worst-case (5 mg/L) concentrations of PET-NPs. A multidisciplinary approach combining confocal and electron microscopy, behavioural assays, regeneration analysis, and biochemical biomarkers was applied. Imaging analyses revealed a dose-dependent accumulation of PET-NPs on the body surface and within the vestibulum and digestive systems, demonstrating particle ingestion. Notably, PET-NPs were detected also within internal tissues and intracellular vesicles, providing in vivo evidence of translocation across epithelial barriers and cellular internalization. At the organismal level, the highest tested concentration resulted in increased mortality, impaired righting behaviour and reduced arm regeneration. The latter was significantly reduced also at 0.5 mg/L, dose at which a significant decrease in superoxide dismutase activity was observed as well. This suggests a partial, although dose-independent, modulation of antioxidant homeostasis which however is not accompanied by a clear oxidative damage. Overall, this study provides one of the first integrated in vivo demonstrations of nanoplastic translocation from external exposure to intracellular compartments in a burrowing echinoderm, linking biodistribution to functional impairment. The observed effects on behaviour and arm regeneration highlight the potential ecological consequences of PET-NP exposure for benthic organisms, particularly for species whose fitness depends on locomotor performance and regenerative capacity. These findings underscore the ecological risks associated with PET-NP accumulation in sediment-associated fauna and identify arm regeneration as a sensitive ecotoxicological endpoint for nanoplastic risk assessment.