## 3 Results and Discussion
### Detection of Water Toxicity  
#### 3.2.1 Fe as a Water Toxicity  
Euglena and Paramecia were initially exposed to varying concentrations of an emerging heavy water pollutant, iron (II) sulphate heptahydrate (FeSO\({}_{4}\cdot\)7H\({}_{2}\)O). The permissible level (PL) for Fe, as determined by the World Health Organization, is 0.1 mg/L. The microorganisms were exposed to concentrations of 2\(\times\) (0.2 mg/L), 10\(\times\) (1 mg/L), and 20\(\times\) (2 mg/L) the PL of Fe. The exposure time was 1.5 h for each study. The experimental results revealed that on increasing the heavy metal concentration, the cross-correlation function was spread out, indicating the slow movement of the Paramecia, as shown in Figure 4a. The correlation time as a function of Fe is shown in Figure 4b, and a concentration dependency can be seen clearly in the case of Paramecia. On increasing the Fe concentration, especially after two times PL, a significant reduction in the swimming ability of Paramecia was observed compared to the control, indicating the toxic water condition. The correlation time increased 1.5 and 2 times compared to the control for concentrations of 10 and 20\(\times\) PL. However, interestingly, at 2\(\times\) PL, the width of the cross-correlation function or the correlation time was reduced by 8% from the control. This indicates that Paramecia exhibited increased activity or improved health conditions compared to the control group at this concentration. It is reported that Fe at low concentration behaves as a growth promotor for Paramecia caudatum, which is well supported by the smaller correlation time [18]. Additionally, at PL, at a concentration of 0.1 mg/L of Fe, no significant change in their swimming ability was observed. This might be because at this concentration, Paramecia developed resistance/tolerance. However, as the Fe concentration increased from 2\(\times\) PL to 10\(\times\) and 20\(\times\) the PL, the correlation time was observed to be significantly higher, indicating the slower movement of the Paramecia under a toxic water environment. This suggests that concentrations above 2\(\times\) the PL of Fe were toxic to Paramecia. Figure 3: (**a**) The cross-correlation function as a function of time for Paramecia under different viscosity media. (**b**) The correlation time as a function of viscosity for Paramecia. The error bars indicate the standard deviation. *, ** indicated the statistical significance of data \(p\) < 0.05 and \(p\) < 0.01. N = 9. On the other hand, when Euglena was exposed to the same set of concentrations, no significant change was observed in their swimming ability. The correlation time curve remained relatively flat compared to Paramecia at this concentration range, as depicted in Figure 4b. More specifically, the correlation times for concentrations of 2\(\times\) PL, 10\(\times\) PL, and 20\(\times\) PL (0.2, 1, and 2 mg/L) were noted to be nearly equal to that of the control, approximately 0.57 s for Euglena. This suggests that Euglena showed tolerance behavior to this concentration range (0.1-2 mg/L). However, as Fe concentrations increased further, specifically at 200\(\times\) PL (20 mg/L), 500\(\times\) PL (50 mg/L), and 1000\(\times\) PL (100 mg/L), a significant change in cross-correlation function and the correlation time was observed as compared to the control for Euglena, shown in Figure 5a,b. This increment indicates the slower movement of Euglena as the environmental toxicity increased. A distinct concentration dependency emerged at elevated Fe concentrations. This behaviour suggested a transition from tolerance to responsiveness at elevated/higher Fe concentrations, as shown in Figure 5b. Specifically, the correlation times were noted to be 1.3-, 7-, and 12-fold larger than that of the control at 200\(\times\) PL, 500\(\times\) PL, and 1000\(\times\) PL, respectively. However, Paramecia retained its sensitivity at elevated concentrations, with the correlation time increasing significantly beyond 2\(\times\) PL.