## 4 Discussion  
Anthropogenic activities contribute significantly to ecosystem pollution, a global challenge, with heavy metals (HMs) and nanoparticles (NPs) emerging as key pollutants [2; 3]. Research on the interaction between HMs/NPs and ciliated protists is limited [29; 31; 36; 41; 57], especially compared to studies on other organisms [37; 58; 59; 60; 61; 62; 63; 64]. Cell biomass plays a pivotal role in bioassay treatments, modifying pollutant bioavailability and enhancing data accu  
Figure 3: Hydroxyl radical scavenging activity measured in _Euplotes 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. The results are \(\pm\) SD of three parallel experiments. racy [65]. The order of toxicity of HMs is Cu \(>\) Cd \(>\) Zn at 1 h and 24 h, a pattern consistent with the findings of [PERSON] and [PERSON] [66] and [PERSON] et al. [29] for HMs. [PERSON] et al. [29] used _Euplotes_ sp. in their study and found concentrations of 0.7, 4.8, and 110.2 mg/1 for Cd, Cu, and Zn, respectively, after a 24 h exposure. Notably, these concentrations are lower than those observed in our study, except for the Cu LC\({}_{50}\) value. The ciliate _Bresslauides_ sp. showed lower LC\({}_{50}\) values for Cd and Zn compared to our study [66]. [PERSON] et al. [67] and [PERSON] et al. [68] observed higher LC\({}_{50}\) values for Cu and Zn in _Colpoda_ genus isolates compared to our study, except for the Cd LC\({}_{50}\) value. Our NP LC\({}_{50}\) values exceed those reported by [PERSON] et al. [69] for the ciliate _Tetrahymena thermophila_. [PERSON] et al. [38] found ZnO values lower and CuO values higher than our results for the ciliated protozoa _T. thermophila_ at 24 h exposure. According to a study by [PERSON] et al. [44], the LC\({}_{50}\) value of CuO on _E. adecitulatus_ was found to be 1.24 mg/L after a 24 h exposure. However, our study showed a higher 24 h LC\({}_{50}\) value of CuO compared to the study by [PERSON] et al. [44]. These different sensitivities may be due to the different origins of the _E. adecitulatus_ strains used in the experiments. Overall, Zn shows fewer toxic effects compared to other HMs, as ciliated protists adapt quickly to excess Zn without adverse effects. In contrast, the _E. adecitulatus_ cells used in this study showed lower resistance to Cu compared to previous studies [29]. In general, the mixtures of Cd + ZnO showed predominantly synergistic effects at both 1 h and 24 h. This particular interaction (Cd + Zn) has been studied and documented in various organisms, with consistent outcomes [37; 67; 70; 71]. On the other hand, the differing responses of _E. adecitulatus_ to heavy metals and nanoparticles can be attributed to the distinct physical and chemical properties of these substances, their differing mechanisms of uptake and bioavailability, and the unique ways in which they interact with biological systems and the environment. Moreover, the toxicity of nanoparticles (NPs) can vary depending on the duration of exposure. Some nanoparticles dissolve slowly, releasing toxic ions over time, which can lead to a delayed onset of toxicity. The level of toxicity may increase or decrease based on the duration of exposure. These differences highlight the complexity of nanoparticle toxicity and the need for further research to fully understand their impacts on soil microorganisms and ecosystems. In our analyses, the independent action (IA) module outperformed the concentration addition (CA) module, as evidenced by the R2 value, which explains 82% of the data variance. Modules taking into account deviations from the norm performed even better.