Influence of calcium and EDTA on copper ion bioavailability in copper nanoparticle toxicity tests improves understanding of nano-specific effects

Abstract

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 Cu²⁺ 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 CuSO₄ 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 CuSO₄ was greater (LC₅₀ = 226 µg Cu/L) than for larvae exposed to Cu-NPs (LC₅₀ = 648 µg Cu/L). The expression of MT2 increased 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). Cu²⁺ was 2.9-fold more toxic than Cu-NPs. The presence of 5 mg/L EDTA and 220 mg/L CaCO₃ significantly reduced the expression of MT2 (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 of MT2 (1.7-fold) relative to Cu-NP concentration. While Cu-NPs induced MT2 expression, the differences in concentration relationships of MT2 expression between Cu-NPs and CuSO₄ 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.