Mechanistic understanding toward the toxicity of graphene-family materials to freshwater algae

We systematically investigated the toxicity mechanism of three graphene-family materials (GFMs), graphene oxide (GO), reduced graphene oxide (rGO) and multi-layer graphene (MG), to algae (Chlorella pyrenoidosa). GFMs exhibited much higher toxicity than other carbon materials (carbon nanotube and graphite), with the 96 h median effective concentration (EC₅₀) values of 37.3 (GO), 34.0 (rGO), and 62.2 (MG) mg/L. Shading effect contributed approximately 16.4% of growth inhibition by GO due to its higher dispersibility and transformation while the other GFMs did not show any shading effect. Hydrophobic rGO and MG more readily heteroagglomerated with algae than GO, thus likely leading to more direct contacts with algae. Flow cytometry results showed significant decrease of membrane integrity after GFM exposure, and rGO caused the highest membrane damage, which was confirmed by the increased DNA and K⁺ efflux. The observed membrane damage was caused by a combination of oxidative stress and physical penetration/extraction. Moreover, all the three GFMs could adsorb macronutrients (N, P, Mg, and Ca) from the algal medium, thus leading to nutrient depletion-induced indirect toxicity. GO showed the highest nutrient depletion (53% of total toxicity) due to its abundant functional groups. The information provided in this work will be useful for understanding toxicity mechanism and environmental risk of different GFMs in aquatic environments.