## 4 Discussion  
The main objective of this study was to demonstrate the fast, reliable, and simple biospeckle technique for microbioassays using two different types of microorganisms and two heavy metal pollutants, i.e., Zn and Fe. In addition, a rheological study of their swimming speed was conducted using different viscosity media. The result of the proposed bioassay method revealed that significant reductions in the swimming ability of Paramecia and Euglena under different water toxicities were observed just after 1.5 h at higher concentrations of Fe and Zn. This demonstrates the potential of the observation speed of this method. Several studies suggested that Fe and Zn are essential micronutrients for various organisms, including microorganisms such as Euglena and Paramecium [18,19]. Fe and Zn play critical roles in various cellular processes, such as growth, metabolism, and survival. At low concentrations, Fe and Zn act as cofactors for enzymes involved in various biochemical reactions, including photosynthesis, respiration, and DNA replication [20,21]. However, at elevated concentrations, they can become toxic and adversely affect cellular functions. The obtained results show that Euglena's tolerance to low Fe levels can be attributed to its ability to regulate iron uptake and utilization through sophisticated physiological and molecular mechanisms. At low Fe concentrations, Euglena employs high-affinity Fe transporters and Fe scavenging systems to acquire iron from the environment efficiently [22]. Additionally,  
Figure 8: (**a**) The correlation time for Paramecia and (**b**) Euglena as a function of Fe concentrations for 1.5, 24, and 48 h, (**c**) the correlation time for Paramecia and (**d**) Euglena as a function of Zn concentration for 1.5, 24, and 48 h.  
cells, including Euglena, possess iron storage proteins, such as ferritin, which sequester excess iron to prevent oxidative damage and maintain cellular homeostasis [23]. Furthermore, microorganisms can adjust their metabolic pathways and cellular processes to optimize iron utilization under low iron conditions. For example, Euglena may upregulate the expression of iron-dependent enzymes involved in photosynthesis and respiration to maximize energy production. This adaptive response allows for Euglena to thrive in environments with limited iron availability, exhibiting enhanced growth and metabolic activity compared to other microorganisms. It is also confirmed by [PERSON] et al. that iron at optimal levels enhances cellular functions and promotes growth in microorganisms [24]. Euglena employs intracellular sequestration to mitigate the toxic effects of heavy metals such as Fe. Metals are compartmentalized within chloroplasts and vacuoles as a protective mechanism against the damaging effects of this metal and also reduces their interaction with vital cellular components [25]. Additionally, Euglena gracilis, a facultative photosynthetic alga, utilizes a cytoplasmic detoxification mechanism involving the synthesis of metal-binding peptides such as phytochemicals and glutathione to inactivate heavy metal ions. This process enables Euglena gracilis to survive in environments contaminated with metals, protecting its cells from the toxic effects of these ions [26]. In addition to that, stress conditions, such as heavy metal stress, exert evolutionary pressures on all organisms, which have developed sophisticated responses to cope and survive. Heavy metal tolerance in Euglena is also mediated through changes in gene expression and the activation of specific signaling pathways. [27]. Stress conditions, including heavy metal exposure, can upregulate genes encoding for heat shock proteins, antioxidants, and metal-binding proteins. These proteins play crucial roles in protecting cellular components from metal-induced damage and maintaining cellular homeostasis.