Elucidating the mechanisms underlying the cytotoxic effects of nano-/micro-sized graphene oxide on the microalgae by comparing the physiological and morphological changes in different trophic modes

Understanding the cytotoxic mechanisms of environmental contaminants is important to estimate their environmental impacts and prepare guidelines for pollution control. Many studies have assessed the cytotoxic mechanisms of graphene oxide (GO), an emerging aquatic contaminant. However, in many cases, the effect of GO size and putative trophic modes of microalgae on cytotoxicity has been neglected, hindering complete understanding of the cytotoxic mechanisms of GO. In this study, the microalga Euglena gracilis cultivated under light (phototrophic) or dark (heterotrophic) conditions was exposed to two sizes of GO [nano-sized (N) and micro-sized (M)] for assessing the effect of GO on microalgal growth. The cytotoxic effect of GO was higher under phototrophic conditions than under heterotrophic conditions, suggesting that a major cytotoxic mechanism of GO is related to photosynthetic activity inhibition. Moreover, N-GO showed higher toxicity than M-GO. The morphological and physiological changes in N-GO- and M-GO-exposed E. gracilis were assessed to further elucidate the cytotoxic mechanisms. N-GO internalized the cells via endocytic activity/piercing, whereas M-GO partially attached to the cell surface and did not enter the cells. Moreover, N-GO and M-GO negatively affected the cells by inducing oxidative stress; the oxidative stress parameters were higher in N-GO-exposed cells than in M-GO-exposed cells.