Toxicities of copper oxide nanomaterial and copper sulphate in early life stage zebrafish: Effects of pH and intermittent pulse exposure

Effort has been made to standardise regulatory ecotoxicity tests for engineered nanomaterials (ENMs), but the
environmental realism of altered water quality and/or pulse exposure to these pollutants should be considered.
This study aimed to investigate the relative toxicity to early life-stage zebrafish of CuO ENMs at acid pH and then
under pulse exposure conditions, all compared to CuSO₄. At all pH values, CuSO₄ was more toxic to zebrafish
than CuO ENMs. Additions of H+ were protective of CuSO₄ toxicity, with median lethal concentrations LC₅₀
(with 95% confidence intervals) of: 0.36 (0.33–0.40), 0.22 (0.20–0.24) and 0.27 (0.25–0.29) mg L−1 at pH 5, pH
6 and pH 7, respectively. In contrast, the toxicity of CuO ENMs increased with acidity; LC₅₀ values were: 6.6
(4.5–8.5), 19.4 (11.6–27.2) and>100 mg L−1 at pH 5, pH 6 and pH 7, respectively. The increased toxicity of the
CuO ENMs in acid water corresponded with greater dissolution of dissolved Cu from the particles at low pH,
suggesting free Cu²⁺ ion delivery to the zebrafish was responsible for the pH-effect. In continuous 96 h exposures
to the substances at the LC₁₀ values and at pH 6, both CuSO4 and CuO ENMs caused Cu accumulation,
inhibition of Na+/K+-ATPase and depletion of total glutathione in zebrafish. However, two 24 h pulses of CuSO₄
or CuO ENMs at the same peak concentration caused similar effects to the continuous 96 h exposure, despite the
shorter exposure durations of the former; suggesting that the pulses were more hazardous than the continuous
exposure. In conclusion, the current water quality correction for pH with respect to Cu toxicity to freshwater fish
should not be applied to the nano form. Crucially, CuO ENMs are more toxic in pulse than continuous exposure
and new corrections for both water pH and the Cu exposure profile are needed for environmental risk assessment.