Origin, exposure routes and xenobiotics impart nanoplastics with toxicity on freshwater bivalves

Abstract Various environmental aged plastic wastes were collected in the environment and crushed to the nanometric scale to get a mix of nanoplastics (NPs) of different natures – mostly polyolefins (PE, PP), polyesters (PET) and polyvinylics (PS and PVC) – and undefined shapes (noted NP-L, mean hydrodynamic diameter at 285 nm). We aimed to test the toxicity of NPs of environmentally relevance on freshwater bivalves and compare results to commonly used styrenic NP-PS (206 nm). Corbicula fluminea were exposed to four different conditions with NPs (0.008 to 10 μg/L), for 21 days and kept under depuration conditions for 21 additional days: 1) waterborne exposure to NP-L, 2) diet borne exposure to NP-L, 3) synergic waterborne exposure to NP-L and AlCl 3 salt (1 mg/L), 4) waterborne exposure to NP-PS. Enzyme activities, gene expressions and behavioural tests were assessed. Trophic and synergic exposures with Al triggered more gene modulations than direct exposure to NP-L (namely on 12s, atg12, gal, segpx, p53 and ache ). NP-PS were also more harmful than NP-L, but only at high concentrations (10 μg/L). The effects of each treatment lasted until 7 days of depuration and no more gene inductions were observed after 21 days in clean water. Very few effects were shown on phenol-oxidase (PO), and glutathione S-transferase (GST). However, the inhibition of acetylcholinesterase (AchE) was concomitant with an increase of the filtration activity of bivalves exposed to NP-L (trophic route) and NP-PS, suggesting neurotoxic effects. By disturbing the ventilatory activity, NPs could have direct effects on xenobiotic accumulation and excretion capacities. The results point out how the structure, aging, exposure routes and additional xenobiotics can interact with adverse outcomes of NPs in bivalves. These findings underline the importance to consider naturally aged environmental NPs in ecotoxicological studies rather than synthetic latexes, i . e . crosslinked nanospheres prepared from virgin polymers. This manuscript presents the first data of toxicity on freshwater organisms exposed to nanoplastics coming from natural sources. Whereas the majority of papers are dealing with non-environmentally representative plastics (mainly commercially-available polystyrene latexes) to evaluate nanoplastic effects on organisms, this study develops methods to prepare model nanoplastics from plastic wastes collected in rivers, and to assess their real adverse effects on aquatic organisms. Our results show significant differences between the inflammatory effects triggered by nanoplastics coming from natural sources and polystyrene nanobeads. This work suggests that the data published so far in the literature may underestimate the toxicity of nanoplastics spread into the environment on the aquatic organisms at the bottom of the food chain, which might consequently impart halieutic resources on the long term.