TOXICITY EVALUATION OF PLASTIC NANOPARTICLES ON AQUATIC ORGANISMS

Plastics are an immense family of unique and versatile materials which play an essential role in modern society and are an indispensable part of our daily lives. With a global production of nearly 440 million tons in 2019, plastic waste has been described to accumulate in virtually any environment, from marine to freshwater ecosystems. The irreversibility and global ubiquity of marine plastic pollution has turned plastics into a potential planetary boundary threat. Nanoplastics (NPs) represent the smallest fraction of plastic litter and can result in the environment as the degradation products or larger plastic items. Nevertheless, only recently such particles have been detected in real environments, albeit not yet accurately quantified. Although today there is still a lack of clarity as regards their toxicological effects, the high exposure potential, together with the physical and chemical heterogeneity of NPs, the likely significant diffusive release of plastic additives and adsorbed substances, combined with their small size and enhanced accessibility to biological tissues, potentially make NPs highly hazardous pollutants. Ecological risk assessment of nanoplastics are urgently needed. In the absence of reliable environmental exposure estimates to date, ecotoxicological research has focused on defining a hazard assessment of NPs. This thesis aimed at investigating the mechanism of toxicity of NPs, taking advantage of high-throughput sequencing technologies. In an initial experiment, described in Chapter 2, adult specimens of the freshwater benthic crayfish Procambarus clarkii were exposed to 100 μg of 100 nm carboxylated polystyrene nanoparticles in a 72h dietary exposure experiment. The cosmopolitan red swamp crayfish Procambarus clarkii, is widely used as bioindicator of environmental pollution and was selected here as a representative non-model decapod of the freshwater ecosystem. An integrated approach was conceived to assess the biological effects of polystyrene NPs, by analyzing both transcriptomic and physiological responses. Total hemocyte counts, basal and total phenoloxidase activities, glycemia and total protein concentration were investigated in crayfish hemolymph at 0h, 24h, 48h and 72h to evaluate general stress response over time. Transcriptomes of hemocytes and hepatopancreas were analyzed after 72h. At the physiological level, crayfish were able to compensate for the induced stress by not exceeding the generic stress thresholds. The RNA-Sequencing analysis revealed the altered expression of few genes involved in immune response, oxidative stress, gene transcription and translation, protein degradation, lipid metabolism, oxygen demand, and reproduction in P. clarkii exposed to NPs. Activation of oxidative stress pathways and inflammatory responses has been widely recognized as primary molecular mechanisms of NPs-induced toxicity, in agreement with our findings. In particular, we note an alteration of several genes related to the ubiquitin-proteasome system, one of the major degradation pathways for maintaining cellular protein homeostasis, which has not been described before. Moreover, a rather clear transcriptomic response to NPs emerged as a strong downregulation of vitellogenin expression in the hepatopancreas of female crayfish, which may indicate a shift in energy allocation induced by plastic exposure from reproduction to organism maintenance, as previously advocated. This finding may provide the basis for a deeper exploration of the potential population-level effects of nanosized polystyrene particles. Overall, this study suggests that a low concentration of PS NPs may induce mild stress in crayfish, and sheds light on molecular pathways possibly involved in nanoplastic toxicity. In Chapter 3 we examined the underlying mechanism of NPs toxicity in a sensitive early developmental stage of a key marine invertebrate species, the bivalve Mytilus galloprovincialis.