Epigenetic effects of (nano)materials in environmental species – Cu case study in Enchytraeus crypticus

Chemical stressors can induce epigenomic changes, i.e., changes that are transferred to the next generation, even when the stressor is removed. Literature on chemical induced epigenetic effects in environmental species is scarce. We here provide the first results on epigenetic effects caused by nanomaterials with an environmental OECD standard soil model species Enchytraeus crypticus species. We assessed the epigenetic potential in terms of global DNA methylation, gene-specific methylation via bisulfite sequencing and MS-HRM (Methylation Sensitive - High Resolution Melting), and gene expression qPCR for genes involved in DNA methylation, histone modifications, non-coding RNA and stress response mechanisms). We have exposed E. crypticus in a multigenerational (MG) test design to Cu (copper oxide nanomaterials (CuO NMs) and copper salt (CuCl₂)). To link possible epigenetic effects to population changes, we used exposure concentrations (ECx) that caused a 10% and 50% reduction in the reproductive output (10% and 50% are the standards for regulatory Risk Assessment), the organisms were exposed for five consecutive generations (F1–F5) plus two generations after transferring to clean media (F5–F7), 7 generations in a total of 224 days. Results showed that MG exposure to Cu increased global DNA methylation and corresponded with phenotypic effects (reproduction). Gene expression analyses showed changes in the epigenetic, stress and detoxification gene targets, depending on the generation and Cu form, also occurring in post-exposure generations, hence indicative of transgenerational effects. There were in general clear differences between organisms exposed to different Cu-forms, hence indicate nanoparticulate-specific effects.