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It is still not very clear what roles the various Ag species play in the toxicity of silver nanoparticles (AgNPs). In this study, we found that traditional exposure media result in uncontrollable but consistent physicochemical transformation of AgNPs, causing artifacts in determination of median lethal concentration (LC50) and hindering the identification of Ag species responsible for the acute toxicity of AgNPs to Daphnia magna. This obstacle was overcome by using 8 h exposure in 0.1 mmol L−1 NaNO3 medium, in which we measured the 8-h LC50 of seven AgNPs with different sizes and coatings, and determined the concentrations of various Ag species. The LC50 as free Ag+ of the seven AgNPs (0.37-0.44 μg L−1) agreed very well with that of AgNO3 (0.40 μg L−1), and showed the lowest value compared to that as total Ag, total Ag+, and dissolved Ag, demonstrating free Ag+ is exclusively responsible for the acute toxicity of AgNPs to D. magna, while other Ag species in AgNPs have no contribution to the acute toxicity. Our results demonstrated the great importance of developing appropriate exposure media for evaluating risk of nanomaterials. [ABSTRACT FROM AUTHOR]