## 4 Discussion
Remarkably, the bimetallic mixture at 24 h showed robust DPPH scavenging activity, surpassing other bimetallic mixtures such as Cd + Zn at 1 h and Cd + ZnO at 24 h. Additionally, while the DPPH scavenging activity of single metals showed lower activity at both 1 h and 24 h, the combination of single metals with other metals resulted in higher activity. This phenomenon can be attributed to the joint toxicity of the chemical mixture (_p_ < 0.001). In this study, the bimetallic mixtures showed potent antioxidant capacity. Consistent with our findings, a bimetallic mixture of Cd + Zn at chronic exposure has previously demonstrated effective antioxidant activity using the DPPH radical scavenging method [48]. Although no data are available for the analysis of DPPH scavenging activity in ciliated protists, DPPH has been extensively used as a radical to evaluate reducing substances in food materials [79]. [PERSON] et al. [80] analysed the antioxidant activity in alligator pipefish and showed lower DPPH scavenging activity compared to our values. It is worth noting that DPPH is a stable free radical that becomes a diamagnetic molecule when it accepts an electron or hydrogen radical. As previously stated, there are no available data on hydroxyl radical scavenging activity (HRSA) in ciliated protists, but numerous results are available for other organisms. For instance, [PERSON] et al. [80] demonstrated HRSA ranging from 84.89% to 91.02%, and these levels of activity are relatively higher than those observed in our results. The robust correlation observed between TPC and HRSA strongly suggests that phenolic compounds play a crucial role in donating electrons to hydroxyl radicals, effectively neutralizing them into water [81]. The efficiency of _E. aeticulatus_ cell extract is demonstrated by the significant positive correlation (_p_ < 0.001) between HRSA and other kinds of antioxidant activity. This positive correlation suggests that the ciliate cell extract stabilizes lipid peroxidation through its hydrogen-donating potential, contributing to its overall antioxidant capacity. This study has some limitations. First, the findings are based on a single ciliate species, which may not be representative of other aquatic organisms. Additionally, the experiments were conducted in controlled environments, which may not fully replicate natural conditions, potentially limiting the applicability of results to real-world scenarios. Furthermore, the study tested only specific combinations of heavy metals and nanoparticles, leaving out numerous possible mixtures and interactions that could occur in natural settings. The study primarily focused on acute toxicity (1 h) and chronic toxicity (24 h), as well as immediate antioxidant responses, potentially overlooking long-term ecological impacts such as effects over 2 days, 4 days, 6 days, and one week for long-term chronic exposure. Moreover, the study may have used concentrations that do not accurately reflect environmental levels, either being too high or too low compared to what is typically found in polluted habitats. Addressing these limitations in future research could provide a more comprehensive understanding of the ecological risks posed by heavy metals and nanoparticles.