Applicability of the OECD Test Guideline 201 to graphene-related materials: Dispersion stability matters

Graphene-related materials (GRMs) are used in many innovative applications for their outstanding physico-chemical properties. Their possible release could have critical consequences for the environment. According to a European Union regulation, one of the test guidelines (TG) that must be applied to check the environmental hazard of new substances is the OECD TG 201 - Algae and Cyanobacteria Growth Inhibition Test. It was developed for water-soluble substances, whereas GRMs are not: dispersed in aqueous media, they tend to aggregate and settle, changing their bioavailability. This work aims to evaluate the applicability of the TG 201 to GRMs by investigating the stability of GRMs dispersions (GDS) in the TG 201 medium, focusing on the stability criterion of TG 201, i.e. maintaining ± 20 % of the nominal initial concentration. Based on flow-cytometry, Turbiscan and Utermöhl sedimentation chamber measurements, the following factors were tested: (i) particle composition and (ii) concentration; (iii) application of turbulence; (iv) addition of dispersants; (v) presence/absence of the target organism. Strong agglomeration/aggregation and sedimentation phenomena were observed for all materials under all tested conditions, thus the stability criterion of TG 201 was not met. Nevertheless, this can be satisfied by allowing an adequate period of time (approx. 6 h) for the GRM dispersion to stabilize after its preparation. Only then, a detailed physico-chemical characterization of the suspended material is required, which must be reiterated at the end of the test.