## 3 Results
### 1 h and 24 h Binary Mixture of Cd + ZnO--MixTOX Analysis
In the binary mixture, the TPC ranged from 122.58 to 218.54 \(\upmu\)g/L. The highest TPC was observed in the binary mixtures of 1 + 0.25 TU (Cd + Zn) and 0.25 + 0.25 TU (Cd + ZnO) ciliate extracts after a 24 h exposure period, respectively (_p_ < 0.001) (Figure 1). Importantly, the TPC of the ciliate extract, an indicator of antioxidant capacity, was significantly increased in cells exposed to different single heavy metal conditions and bimetallic mixtures compared to untreated cells (_p_ < 0.001). The free radical scavenging activity of the ciliate extracts were evaluated using the DPPH assay, with BHT employed as a standard. Extracts obtained from _E. aediculatus_ showed a maximum scavenging activity of 34.90% at the concentration of 0.75 TU Cd (_p_ < 0.001) and a minimum activity of 26.97% at the concentration of 0.25 TU ZnO (_p_ < 0.001) after 1 h. After 24 h, the maximum activity was found at 0.25 TU Cd, while the minimum activity was found at 0.5 TU Cd, with values of 48.97% and 33.04% (_p_ < 0.001), respectively (Figure 2). Figure 1: Total phenolic content measured in the _Euplotes aediculatus_ cell extracts at single-metal (Cd, Zn, and ZnO) and bimetallic mixture concentrations. (**a**) Single metal, (**b**) bimetallic mixture of Cd + Zn at 1 h exposure period, (**c**) bimetallic mixture of Cd + Zn at 24 h exposure period, and (**d**) bimetallic mixture of Cd + ZnO at 24 h exposure time. The results are presented as means of three replicate experiments. The DPPH scavenging activity ranged from 25.69% to 48.97%. In the single-metal treatments, concentrations of 0.75 TU and 0.25 TU Cd showed higher DPPH scavenging activity, with significant values of \(p<\) 0.001 at 1 h and 24 h, respectively (Figure 2). In the bimetallic mixtures, Cd + Zn showed higher DPPH activity during chronic exposure compared to other bimetallic mixture exposure periods. The maximum activity was observed with 0.5 + 0.25 TU (Cd + Zn) ciliate extracts at 24 h (\(p<\) 0.001), while the minimum value was found at the concentration of 0.5 + 0.25 TU (Cd + ZnO) at 24 h (\(p<\) 0.001) (Figure 2). Among the concentrations of the bimetallic mixture, the values ranged from 28.87% to 57.56%. To establish correlations between the results obtained by different methods, a regression analysis was conducted, and the correlation coefficients (\(R\)) are presented in Table 4.  
Significant correlations were observed between different methods used to determine the antioxidant potential, with notable associations between total phenolic content (TPC) and DPPH assays showing positive correlations of \(R\) = 0.90 and \(R\) = 0.80 at 1 h and 24 h, respectively. For the bimetallic mixture of Cd + Zn at acute exposure, a robust correlation of \(R\) = 0.93 was observed between the TPC and DPPH assays at 24 h. Similarly, Cd + ZnO showed very strong correlations in TPC and DPPH assays, with \(R\) = 0.95 at 24 h (Table 4). A significant positive correlation was observed between DPPH and TPC, as well as DPPH and MRSA (\(R\) = 0.90; \(R\) = 0.86; \(p<\) 0.001), reflecting the antioxidant capacity of the single-metal treatments at both 1 h and 24 h (Table 4). In the acute exposure of the bimetallic mixture \begin{table}
**DPPH** & 1 & & & **DPPH** & 1 & & & & \\
**DPPH** & 1 & & & **DPPH** & 1 & & & **DPPH** & 1 & & \\
**HRSA** & 0.93 & 1 & & **HRSA** & 0.7 & 1 & & **HRSA** & 0.91 & 1 & \\
**TPC** & 0.93 & 0.74 & 1 & **TPC** & 0.77 & 0.89 & 1 & **TPC** & 0.95 & 0.81 & 1 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Correlation coefficient (\(R\)) between single and binary mixture metal exposure of different antioxidant assays on _Eupoles acidiculatus_. Figure 2: DPPH scavenging activity measured in _Eupoles acidiculatus_ cell extracts at single-metal (Cd, Zn, and ZnO) and bimetallic mixture concentrations. (**a**) Single metal, (**b**) bimetallic mixture of Cd + Zn at 1 h exposure time, (**c**) bimetallic mixture of Cd + Zn at 24 h exposure time, and (**d**) bimetallic mixture of Cd + ZnO at 24 h exposure time.