Metal-based nanoparticles (NPs) can release metal ions that are toxic to aquatic organisms; however, whether the toxicity is from metal ions rather than unique "nano-scale" effects of the NPs is unresolved. The present study aimed to compare the toxicity of Cu(2+)and Cu-NPs in larval zebrafish (Danio rerio) to clarify whether toxic effects are attributable to release of Cu ions and to determine the effect of the chelating agent ethylenediaminetetraacetic acid (EDTA) and calcium hardness (as CaCO3) on the Cu toxicity. First, the acute toxicity (96-h lethality) of Cu-NPs was determined in comparison to aqueous Cu in larvae exposed to CuSO4, and subsequently, sublethal tests with Cu-NPs and CuSO(4)were conducted with additions of EDTA or calcium ions to evaluate alterations in expression of metallothionein-2 (MT2) gene transcripts (quantitative real-time polymerase chain reaction). Acute toxicity of Cu in larvae exposed to CuSO(4)was greater (LC50= 226 mu g Cu/L) than for larvae exposed to Cu-NPs (LC50= 648 mu g Cu/L). The expression ofMT2increased with Cu concentration (p< 0.05), and the slope of the linear regression was significantly greater in fish exposed to CuSO4(slope = 0.090) compared to Cu-NPs (slope = 0.011). Cu(2+)was 2.9-fold more toxic than Cu-NPs. The presence of 5 mg/L EDTA and 220 mg/L CaCO(3)significantly reduced the expression ofMT2(1.8-fold for EDTA, 2.3-fold for CaCO3) in larvae exposed to CuSO4. For larvae exposed to Cu-NPs, the presence of EDTA reduced the expression ofMT2(1.7-fold) relative to Cu-NP concentration. While Cu-NPs inducedMT2expression, the differences in concentration relationships ofMT2expression between Cu-NPs and CuSO(4)indicated that factors other than release of Cu ions from Cu-NPs influenced acute toxicity of Cu-NPs. The conclusion drawn from this ecotoxicological risk assessment was that EDTA and calcium significantly decreased Cu toxicity in freshwater fish.