Abstract
Heavy metals (HMs) and metal-oxide nanoparticles (NPs) frequently co-occur in freshwater systems, yet their combined effects on microbial predators remain poorly understood. Here, the freshwater ciliate Coleps hirtus was used to evaluate the cytotoxicity of single and binary mixtures of HMs (Cd, Cu, Zn) and NPs (ZnO, CuO, TiO(2), SiO(2)), and to characterize associated antioxidant responses. Acute toxicity was assessed after 24 h by estimating LC(20) and LC(50) values, while mixture toxicity for Cd + Zn and Cd + ZnO was analyzed using the Toxic Unit approach and the MixTOX framework. Non-enzymatic (TPC, DPPH, HRSA) and enzymatic (CAT, GST, GPx, SOD) antioxidants were quantified as sublethal biomarkers at concentrations below lethal thresholds. HMs were markedly more toxic than NPs, with a toxicity ranking of Cu > Cd >> Zn, whereas NPs followed ZnO > CuO >> TiO(2) >> SiO(2). Cd + Zn mixtures showed predominantly antagonistic or non-interactive effects, while Cd + ZnO mixtures exhibited strong synergistic toxicity with a non-linear dependence on mixture composition, as supported by MixTox modeling. Exposure to HMs and NPs induced significant and often coordinated changes in antioxidant biomarkers, with binary mixtures eliciting stronger responses than single contaminants. Together, these findings indicate that mixture composition strongly influences both toxicity outcomes and oxidative stress responses in C. hirtus. The combination of clear, mixture-dependent toxicity patterns and robust oxidative stress responses makes C. hirtus a promising bioindicator for freshwater environments impacted by HMs and NPs.