The Freshwater Ciliate <em>Coleps hirtus</em> as a Model Organism for Metal and Nanoparticle Toxicity: Mixture Interactions and Antioxidant Responses

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, TiO₂, SiO₂), and to characterize associated antioxidant responses. Acute toxicity was assessed after 24 h by estimating LC₂₀ and LC₅₀ values, while mixture toxicity for Cd + Zn and Cd + ZnO was analyzed using the Toxic Unit approach and the MixTOX framework. Non-enzymatic (total phenolic content, DPPH, HRSA) and enzymatic (CAT, GST, GPx, SOD) antioxidants were quantified as sub-lethal biomarkers. HMs were markedly more toxic than NPs, with a toxicity ranking of Cu &gt; Cd &gt;&gt; Zn, whereas NPs followed ZnO &gt; CuO &gt;&gt; TiO₂ &gt;&gt; SiO₂. Cd + Zn mixtures showed predominantly antagonistic or non-interactive effects, while Cd + ZnO mixtures exhibited strong, dose-ratio–dependent synergism. Exposure to HMs and NPs induced significant and often coordinated changes in antioxidant biomarkers, with binary mixtures eliciting stronger responses than single contaminants. These results demonstrate that C. hirtus is sensitive to both HMs and metal-oxide NPs and can discriminate among different mixture interaction types. The combination of clear toxicity patterns and robust antioxidant responses supports the use of C. hirtus as a promising bioindicator for freshwater environments impacted by HMs and emerging metal-based nanomaterials.