Synergistic Mitochondrial Genotoxicity of Carbon Dots and Arsenate in Earthworms Eisenia fetida across Generations: The Critical Role of Binding

The escalating utilization of carbon dots (CDs) in agriculture raises ecological concerns. However, their combined toxicity with arsenic remains poorly understood. Herein, we investigated the combined mitochondrial genotoxicity of CDs and arsenate at environmentally relevant concentrations across successive earthworm generations. Iron-doped CDs (CDs_-Fe) strongly bound to arsenate and arsenite, while nitrogen-doped CDs (CDs_-N) exhibited weaker binding. Both CDs enhanced arsenate bioaccumulation without affecting its biotransformation, with most arsenate being reduced to arsenite. CDs_-Fe generated significantly more reactive oxygen species than did CDs_-N, causing stronger mitochondrial DNA (mtDNA) damage. Arsenate further exacerbated the oxidative mtDNA damage induced by CDs_-N, as evidenced by increased reactive oxygen species, elevated 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG) levels, and a higher correlation between 8-OHdG and mtDNA damage. This was due to arsenic inhibiting the antioxidant enzyme catalase. This exacerbation was negligible with CDs_-Fe because their strong binding with arsenic prevented catalase inhibition. Maternal mitochondrial DNA damage was inherited by filial earthworms, which experienced significant weight loss in coexposure groups coupled with mtDNA toxicity. This study reveals the synergistic genotoxicity of CDs and arsenate, suggesting that CDs could disrupt the arsenic biogeochemical cycle, increase arsenate risk to terrestrial animals, and influence ecosystem stability and health through multigenerational impacts.