Regulatory testing of ZnO nanoforms - Part 2: Aquatic toxicity testing of selected nanoforms on algae and daphnids

In 2019, the REACH-registration dossier of ZnO was evaluated by the European Chemicals Agency (ECHA), which requested additional data on the fate and effects of all nanoforms present on the EU market. A testing scheme was specified, divided into two steps. First, fate data (OECD GD 29; OECD TG 318) were required for the 28 ZnO nanoforms available on the EU market at that time. In the second step, representative nanoforms were tested for their chronic toxicity to aquatic organisms using the freshwater algal growth inhibition test (OECD TG 201) and the Daphnia magna reproduction test (OECD TG 211), applying nano-specific adaptations available before OECD GD 317 was published. The objective of the testing program was to generate robust, GLP-compliant ecotoxicological data to determine whether Zn ions are the primary toxicity driver for nano ZnO or whether specific nano-related effects occur. Results indicated that in all test systems the main fraction consisted of particles and agglomerates with a hydrodynamic diameter > 200 nm, while only small amounts of dispersed (<200 nm) or dissolved (<200 nm + 3 kDa) fractions were detected of which approx. 1.6% to 2.9% were assumed to be nano. Despite particle-algae hetero-agglomeration, effective concentrations from both test systems showed good comparability between ZnO nanoforms and ionic ZnCl₂, when considering natural variability within the test. Thus, findings point to ionic zinc as the main driver of toxicity, with no clear evidence for an additional nano-specific effect. Furthermore, sequential filtration measurements provided useful insights into the behaviour of ZnO nanomaterials during testing and their interactions with aquatic organisms.