Both released silver ions and particulate Ag contribute to the toxicity of AgNPs to earthworm Eisenia fetida.

To disentangle the contribution of ionic and nanoparticulate Ag to the overall toxicity to the earthworm Eisenia fetida, a semi-permeable membrane strategy was used to separate Ag(+) released from silver nanoparticles (AgNPs) in an aqueous exposure. Internal Ag fractionation, activities of antioxidant enzymes and metabolites in E. fetida were determined after 96 h of exposure to two sizes of polyvinylpyrrolidone-coated AgNPs. The response of the antioxidant system combined with the content of malondialdehyde indicated that the Ag(+) released from AgNPs induced significant oxidative stress to the earthworms. Ag accumulated from AgNPs was predominantly associated with the granules and cell membrane compartments, whereas dissolved Ag was localized in the cytosol-containing fraction. In both Ag(+) exposures, two intermediates in the Krebs cycle, succinate and fumarate, were significantly elevated and depleted, respectively. A similar alteration pattern was seen in groups exposed to both smaller AgNPs (S AgNP, 10 nm) and larger AgNP (L AgNP, 40 nm), indicating that these effects in E. fetida were induced by exposure to released Ag(+). In addition, unique metabolic responses including decreased malate and glucose levels in S AgNP-exposed earthworms could be associated with exposure to nanoparticulate silver. Increased leucine and arginine and decreased ATP and inosine levels were observed in L AgNP exposures only, which clearly demonstrated a size-specific effect of AgNPs. Collectively, this study provided strong evidence that nanosilver acts by a different mechanism than ionic silver to cause acute toxicity to E. fetida, but further verification under different environmental conditions is needed.