Reactive oxygen species generation is likely a driver of copper based nanomaterial toxicity

Determining the specific nanomaterial features that elicit adverse biological responses is important to inform risk assessments, develop targeted applications, and rationally design future nanomaterials. Embryonic zebrafish are often employed to study nanomaterial-biological interactions, but few studies address the role of the chorion in nanomaterial exposure and toxicity. Here, we used chorion-intact (CI) or dechorionated (DC) embryonic zebrafish to investigate the influence of the chorion on copper-based nanoparticle toxicity. We found that despite higher dissolution and uptake, CuO NPs were less toxic than Cu NPs regardless of chorion status and did not cause 100 % mortality at even the highest exposure concentration. The presence of the chorion inhibited Cu toxicity: DC exposures to Cu NPs had an LC₅₀ of 2.5 ± 0.3 mg/L compared to a CI LC₅₀ of 13.7 ± 0.8 mg/L. This highlights the importance of considering zebrafish chorion status during nanotoxicological investigations, as embryo sensitivity increased by one order of magnitude or more when chorions were removed. Agglomerate size, zeta potential, and dissolved Cu did not sufficiently explain the differences in toxicity between Cu NPs and CuO NPs; however, reactive oxygen species (ROS) generation did. Cu NPs generated ROS in a concentration-dependent manner, while CuO did not and generated less than Cu NPs. We believe that the differences between the toxicities of Cu NPs and CuO NPs are due in part to their ability to generate ROS which could and should be a hazard consideration for risk assessments.