Ecotoxicological effects of Graphene-Based Materials

In the recent years, the rapid advancement in the field of nanomaterials has increased their development and consequently their production and commercialization. Among nanomaterials, the carbon-based ones are the most widely researched because of their potential on the most diverse fields, with a predominant role occupied by Graphene-Based Materials (GBMs). Graphene is a two-dimensional, single layer sheet of carbon atoms organized in a honeycombed network with six-membered rings. Since its discovery, the attention of researchers was focused on its unique and exceptional properties, such as mechanical stiffness, strength, elasticity, very high electrical and thermal conductivity, which led to the development of numerous applications. The huge investments brought to an incredible advancement in the industrial field, unfortunately accompanied by a slower progress in the understanding of the impact on human health and the environment. So far, the effects of GBMs have been evaluated mostly on animal and bacterial model organisms, suggesting that GBMs toxicity is dependent on various physiochemical properties such as shape, size, oxidative state and presence of functional groups. The aim of this PhD project was to assess the ecotoxicological effects of two GBMs, few-layers graphene (FLG) and graphene oxide (GO), on aeroterrestrial green microalgae. These GBMs were selected as reference material by the Working Package 4, Health and Environment, in the framework of the European Project Graphene-Flagship. Short-term exposure (30 and 60 minutes) effects were evaluated on the algal species Coccomyxa subellipsoidea and Trebouxia gelatinosa in terms of membrane permeability with the quantification of potassium leakage by inductively coupled plasma-atomic emission spectrometry. Long-term exposure (4 weeks) effects were evaluated on Apatococcus lobatus, Chlorella vulgaris, C. subellipsoidea and T. gelatinosa through chlorophyll a fluorescence measurements (Fv/Fm parameter) and quantifications of the total photosynthetic pigments content. After a short-term exposure of GBMs on the alga T. gelatinosa, internalization was investigated with confocal laser scan microscopy. Potential oxidative effects of GBMs were then studied analyzing the efficiency of the photosystems through measurements of chlorophyll a fluorescence emission (Fv/Fm parameter), changes of gene expression of eight genes of interest through quantitative Real-Time PCR, and quantification of HSP70 protein through western blot. According to the final results obtained, no negative effects were observed for either FLG or GO, in both short- and long-term exposures. Internalization was not clearly observed, even though the FLG exposure after 30 minutes induced the downregulation of the gene coding for HSP70 protein. These results supported the hypothesis that a harmless interaction occurred between GBMs and algae at cell wall – plasma membrane level, involving potentially a yet unknown signaling pathway.