## 2 Materials and Methods
### Total Phenolic Content and Antioxidant Activity Assays  
To evaluate the presence of antioxidant activity in the ciliate exposed to different concentrations of the single and binary mixture of HMs and NPs, three different types of antioxidant assays, namely total phenol content (TPC), \(\alpha\),\(\alpha\)-diphenyl-\(\beta\)-picrylhydrazyl (DPPH), and hydroxyl radical scavenging assay (HRSA), were applied. To analyse the antioxidant activity of these ciliate cells, 2000 cells/mL were taken from an exponentially growing culture flask and transferred to Petri plates in a volume of 10 mL and exposed to different concentrations of single and binary mixtures of HMs and NPs for 1 h and 24 h. For single-metal exposure, concentrations 0.25, 0.5, 0.75, and 1 TUs of Cd, Zn, and ZnO, respectively, were selected and used to analyse the antioxidant activity and enzyme assays. In the binary mixture, minimal cut-off concentrations (less than or equal to 50% mortality) were selected. Based on this minimal cut-off, we selected seven concentrations in the binary mixture of Cd + Zn (0.25 + 0.25, 0.5 + 0.25, 0.25 + 0.5, 0.75 + 0.25, 0.25 + 0.75, 0.75 + 0.5, and 0.75 + 0.75) at 1 h, nine concentrations in the binary mixture of Cd + Zn (0.25 + 0.25, 0.5 + 0.25, 0.25 + 0.5, 0.75 + 0.25, 0.5 + 0.5, 0.25 + 0.75, 1 + 0.75, 0.5 + 1, and 0.75 + 0.75) at 24 h, and six concentrations in the binary mixture of Cd + ZnO (0.25 + 0.25, 0.5 + 0.25, 0.25 + 0.5, 0.75 + 0.25, 0.5 + 0.5, and 1 + 0.25) at 24 h and used them to analyse the antioxidant activity. In the binary mixture of Cd + ZnO at 1 h toxicity test, we could not select any concentrations for the analysis of antioxidant activity assays, because in this test, all the concentrations were above the minimal cut-off concentration. Nanoparticles were not conditioned before antioxidant activity. Antioxidant activity assays were measured in the supernatant of the cell extract (intracellular) according to [PERSON] et al. [48]. The analysis of TPC was based on the Folin assay of [PERSON] [49] with minor modifications. In this method, 100 \(\mu\)Lof cell extract was added to a test tube, mixed with 2 mL of sodium bicarbonate, and incubated for 2 min at 18 \(\pm\) 2 \({}^{\circ}\)C. Then, 100 \(\mu\)Lof the Folin-Ciocalteu reagent was added and incubated in the dark at 18 \(\pm\) 2 \({}^{\circ}\)C for 30 min. After the incubation period, the absorbance was measured at \(\lambda\) = 725 nm using a spectrophotometer. Gallic acid 1 mg/mL was used as standard, and standard curves were obtained using different concentrations of gallic acid. We also used the culture medium (without cells) as a control, as no phenols were observed. We used the DPPH scavenging assay to determine the antioxidant capacity of the _Eupoles_ extracts. Antioxidant activity was determined by its ability to scavenge \(\alpha\),\(\alpha\)-diphenyl-\(\beta\)-picrylhydrazyl (DPPH) radicals. This analysis is based on the procedure of [PERSON] et al. [50] with minor changes. Briefly, 1 mM DPPH stock solution was prepared in 95% ethanol. Then, 800 \(\mu\)Lof 1 mM DPPH solution was added to 200 \(\mu\)Lof sample extract. The samples were mixed well and incubated for 30 min in the dark at room temperature. After incubation, the samples were transferred to centrifuge tubes and centrifuged at 14,000 rpm for 5 min. The supernatant was collected and the absorbance measured at \(\lambda\) = 517 nm using a spectrophotometer. In total, 200 \(\mu\)Lof 95% ethanol was used as a control. Butylated hydroxy anisole (BHA) was used as a reference compound. Antioxidant activity is expressed as a percentage (%). The percentage of DPPH scavenging was calculated using the formula below:  
\[DPPH\ scavenging\ (\%)=\frac{control\ absorbance-extract\ absorbance}{control\ absorbance}\times 100\]  
The HRSA assay is based on the Fenton reaction of [PERSON] and [PERSON] [51] and follows the procedure of [PERSON] et al. [48] with slight modifications.