Oxidative stress induction of mixtures of commercial pesticide formulations with a copper hydroxide nanopesticide in Daphnia magna is mediated by copper speciation

• Pesticides may affect glutathione S-transferase (GST) or catalase (CAT) activity • Single pesticides and mixtures changed GST and CAT at sublethal concentrations • Trend of GST and CAT induction in high concentrations of prochloraz + Cu(OH) 2 NP • CAT reduction trends in low glyphosate/acetamiprid + Cu(OH)₂ NP concentrations • Mixture effects likely mediated by copper speciation between ions and nanoparticles Current application levels of organic and nanoparticle-based pesticide formulations expose aquatic non-target organisms to complicated mixtures. Acute toxicity tests revealed unpredictable mixture effects of a copper hydroxide particle-based nanopesticide with commercial fungicide, herbicide and insecticide formulations, mediated by speciation of copper nanoparticles. Induction of oxidative stress is one of the suspected mechanisms of action of copper-based nanoparticles. As the first study to assess oxidative stress responses of antioxidant enzymes in daphnids after short-term exposure to mixtures of the copper hydroxide-based nanopesticide Funguran progress (FP) with either acetamiprid (ACE), glyphosate (GLY) or prochloraz (PRO) formulations, this study employed an exploratory design by pooling 20 daphnids from four biological replicates to provide sufficient biomass. Results revealed preliminary trends in mixture interactions. All single formulations showed signs of altered oxidative stress responses. PRO and FP likely induced glutathione S-transferase (GST) and catalase (CAT) activity, GLY additionally indicated GST activity reduction. For ACE, an increase of GST and CAT activity was suggested. In mixtures, expectations differed from single formulation test results. No additive or synergistic effects were observed. Mixture effects were likely mediated by copper speciation. In FP and PRO, stabilization and low dissolution of copper nanoparticles into copper ions may have induced GST and CAT. For FP with GLY and ACE, increased dissolution into copper ions at lower concentrations likely caused GST and CAT inhibition. Alterations of oxidative stress responses caused by the mixtures occurred after short-term exposures at sublethal and environmentally relevant concentrations and may affect D. magna populations in the environment.