Hirudo verbana as a freshwater invertebrate model to assess the effects of polypropylene micro and nanoplastics dispersion in freshwater

Plastics are a heterogeneous class of synthetic compounds that, due to their unique characteristics find numerous applications both in industrial and civil fields. However, despite the great advantages that these materials brought in everyday life, the plastic wastes resulting from their massive use represent one of the main envi-ronmental problems at the global level. Once released, plastics persist for a long time and are subjected both to biotic and abiotic processes leading to the formation of small particles, known as micro and to nanoplastics, that interact with organisms, accumulating inside tissues and risking to enter in the trophic chain. Among the different types of plastic, polypropylene (PP) is one of the diffused, widely exploited in food and textile industries for disposable packaging and to produce surgical masks. Owing to the huge distribution and the resultant abundant presence of PP waste products, it results necessary investigate the possible toxicity on living organisms. For these reasons, here we analyzed the effects of PP micro and nanoplastics dispersed in freshwater, using the medicinal leech Hirudo verbana as invertebrate model. To better follow the plastics fate, fluorescent particles, labeled with a fluorophore, have been used. Animals were examined at various timings after plastics exposure and results were analyzed by means of microscopy, immunofluorescent and molecular biology analyses. After assessing the entrance of PP fragments into leech tissues, the activation of the innate immune response was evaluated. The results show that the presence of micro and nanoplastics induces an initial physical protection that consists in the secretion of mucus, followed by an increase of blood vessels and the recruitment of immune cells, in particular macrophages. Moreover, macrophages were directly involved in both phagocytic and encapsulation processes, as demonstrated by acid phosphatase (ACP) histoenzymatic and Thioflavin-T assays, expressing specific pro-inflammatory factors, such as HvRNASET2 and HmAIF-1, as demonstrated by immuno-localization and qPCR experiments. Finally, the expression levels of genes related to oxidative stress-induced enzymes have been investigated, in order to evaluate the possible increase in reactive oxygen species (ROS), due to the entry into the leech tissues of PP micro and nanoplastics. This work allows deepening the current knowledge of the possible harmful effects on human health deriving from micro and nanoplastics dispersion, leading new insight about freshwater ecosystems that often represent the first environments interested in plastic pollution.