Inflammatory and oxidative responses to PET nanoplastics in the leech Hirudo verbana: a comparative analysis of acute and chronic exposure

Nanoplastics (NPs) are emerging environmental contaminants with the potential to induce cellular stress and immune dysregulation in aquatic organisms. In this study, the freshwater leech Hirudo verbana was used as a non-conventional invertebrate model to investigate the effects of acute (24-72 hours) and chronic (1 week-1 month) exposure to polyethylene terephthalate nanoplastics (PET NPs). A multidisciplinary approach combining microscopy, histology, immunocytochemistry, and qPCR was employed to evaluate PET NP uptake and biological responses. PET NPs were internalised in leech tissues and detected in macrophage-like cells. Both exposure regimes triggered a time- and dose-dependent inflammatory response, characterised by macrophage-like cell recruitment, angiogenic remodelling, and upregulation of the pro-inflammatory marker HmAIF-1. Endothelial activation was confirmed by increased CD31 expression and neovascularisation. Furthermore, oxidative stress was evidenced by altered expression of glutathione S-transferase (GST) and superoxide dismutase (SOD) genes. Overall, PET NPs induced conserved immune and stress responses in H. verbana, supporting its relevance as an alternative model for nanoplastic ecotoxicology. These findings contribute to our understanding of NP-induced pathophysiology and reinforce the need for further investigation into the ecological impact of plastic pollution on freshwater invertebrates.