Graphene-based materials do not impair physiology, gene expression and growth dynamics of the aeroterrestrial microalga Trebouxia gelatinosa

Abstract

The effects of two graphene-based materials (GBMs), few-layers graphene (FLG) and graphene oxide (GO), were studied in the aeroterrestrial green microalga Trebouxia gelatinosa. Algae were subjected to short-and long-term exposure to GBMs at 0.01, 1 and 50 lg mL À 1 . GBMs internalization after short-term exposures was investigated with confocal microscopy, Raman spectroscopy and TEM. Potential negative effects of GBMs, compared to the oxidative stress induced by H 2 O 2 , were verified by analyzing chlorophyl a fluorescence (Chl a F), expression of stress-related genes and membrane integrity. Effects of up to 4-week-long exposures were assessed analyzing growth dynamics, Chl a F and photosynthetic pigments. GBMs were not observed in cells but FLG was detected at the interface between the cell wall and plasma membrane, whereas GO was observed adherent to the external wall surface. FLG caused the down-regulation of the HSP70-1 gene, with the protein levels remaining stable, whereas GO had no effect. In comparison, H 2 O 2 produced dose-and time-dependent effects on Chl a F, gene expression and HSP70 protein level. Long-term exposures to GBMs did not affect growth dynamics, Chl a F or photosynthetic pigment contents, indicating that the few observed short-term effects were not dangerous on the long-term. Results suggest that interactions between FLG and plasma membrane were harmless, activating a down-regulation of the HSP70-1 gene similar to that induced by H 2 O 2 . Our work shows that studying GBMs effects on non-model organisms is important since the results of model green microalgae are not representative of the whole taxonomic group. ARTICLE HISTORY

The Interaction of Graphene Oxide with the Pollen−Stigma System: In Vivo Effects on the Sexual Reproduction of Cucurbita pepo L.

“…However, signs of severe damage as cytoplasmic leachates were not detected (Figure 1c,d), as already observed on the stigmatic surface of C. pepo coated with FLG and nanocrystals of the naturally occurring muscovite mica [19]. The absence of severe physical damage is in good agreement with previous works suggesting that the cell wall of plant cells is very difficult to damage and pierce through [18,32], even if the nanomaterials applied are very thin, have sharp edges, and their lateral dimensions are in the order of hundreds of nanometres. In fact, despite our GOs having lateral dimensions bigger than those of the NMs used in the previous study [19], it was as thin as FLG (6 vs. 3−4 layers, respectively) and thinner than muscovite mica (16 ± 13 layers).…”

Applicability of OECD TG 201, 202, 203 for the aquatic toxicity testing and assessment of 2D Graphene material nanoforms to meet regulatory needs

“…Differences in the physiology, cell wall composition and structure (e.g. thickness) among the unicellular algae/cyanobacteria affected the way in which the graphene materials tested interacted with the organisms (Yin et al, 2020, Banchi et al, 2019. The reported EC 50 72h for rGO was 148 mg/L, according to a single study performed (Du et al, 2016).…”

“…Unfortunately, the possible role of impurities in the GO and rGO materials tested was not considered in the majority of studies but independent studies show that a myriad of metallic elements can be introduced following different synthesis processes of graphene (An Wong et al, 2014). In some, a complete elemental analysis of samples was carried out (Banchi et al, 2019;Malina et al, 2020) and distinct levels of particular elements, e.g. manganese, were identified according to the different production processes (Malina et al, 2020).…”

Environmental and Health Impacts of Graphene and Other Two-Dimensional Materials: A Graphene Flagship Perspective

“…Despite the large consensus on these effects, they were not always confirmed even when similar GO concentrations were applied. 138,139 These differences could be due to different exposure modalities or physiological characteristics of the target organisms. Because of the latter factor, effects may vary.…”