Gills are an initial target of zinc oxide nanoparticles in oysters Crassostrea gigas, leading to mitochondrial disruption and oxidative stress

The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnONP) was investigated in Pacific oysters Crassostrea gigas. The nanoscale of ZnONP, in vehicle or ultrapure water, was confirmed, presenting an average size ranging from 28 to 88nm. In seawater, aggregation was detected by TEM and DLS analysis, with an increased average size ranging from 1 to 2μm. Soluble or nanoparticulated zinc presented similar toxicity, displaying a LC₅₀ (96h) around 30mg/L. High zinc dissociation from ZnONP, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONP toxicity. To investigate mechanisms of toxicity, oysters were treated with 4mg/L ZnONP for 6, 24 or 48h. ZnONP accumulated in gills (24 and 48h) and digestive glands (48h). Ultrastructural analysis of gills revealed electron-dense vesicles near the cell membrane and loss of mitochondrial cristae (6h). Swollen mitochondria and a more conspicuous loss of mitochondrial cristae were observed after 24h. Mitochondria with disrupted membranes and an increased number of cytosolic vesicles displaying electron-dense material were observed 48h post exposure. Digestive gland showed similar changes, but these were delayed relative to gills. ZnONP exposure did not greatly affect thiol homeostasis (reduced and oxidized glutathione) or immunological parameters (phagocytosis, hemocyte viability and activation and total hemocyte count). At 24h post exposure, decreased (-29%) glutathione reductase (GR) activity was observed in gills, but other biochemical responses were observed only after 48h of exposure: lower GR activity (-28%) and levels of protein thiols (-21%), increased index of lipid peroxidation (+49%) and GPx activity (+26%). In accordance with ultrastructural changes and zinc load, digestive gland showed delayed biochemical responses. Except for a decreased GR activity (-47%) at 48h post exposure, the biochemical alterations seen in gills were not present in digestive gland. The results indicate that gills are able to incorporate zinc prior (24h) to digestive gland (48h), leading to earlier mitochondrial disruption and oxidative stress. Our data suggest that gills are the initial target of ZnONP and that mitochondria are organelles particularly susceptible to ZnONP in C. gigas.