Exposure medium and particle ageing moderate the toxicological effects of nanomaterials to Daphnia magna over multiple generations: a case for standard test review?

Pristine engineered nanomaterials (NMs) entering the aquatic environment become ‘aged’ during their lifetime via chemical, physical and/or biological process. Therefore, traditional ecotoxicology tests which were designed for soluble chemicals prior to the emergence of NMs, use pristine NMs and salt-only media which are not representative of realistic NM exposure scenarios. Exposure medium and NM ageing moderation of NM toxicity were explored using Daphnia magna multigenerational studies aiming to determine whether the daphnids adapted to continuous exposure and/or if parent-only exposure resulted in epigenetic effects in subsequent generations. Daphnids were continuously, or parent-only, exposed to pristine and aged titanium dioxide (TiO 2 ) and silver (Ag) NMs, in a standard high hardness culture media and synthetic European Class V lowland water. Pristine NMs in the standard culture medium had the most severe toxic consequences, and displayed negative effects in two generations post exposure. NMs aged in the class V water had fewer overall toxic consequences on growth and longevity across all generations in both continuous and parent-only exposure scenarios. Overall, in the absence of environmentally relevant media and aged NMs, standardised Daphnia tests currently overestimate NM toxicity, and fail to consider potential impacts in subsequent generations. The results demonstrate the importance of updating standard testing to reflect scientific advances and increase stakeholder trust in regulation.