## 1 Introduction  
In recent years, the proliferation of heavy metals in the environment has emerged as a significant environmental concern, with profound implications for ecosystem health and human wellbeing [1]. Among these metals, iron (Fe) and zinc (Zn) play pivotal roles in various industrial processes, agricultural practices, and household applications, leading to their widespread presence in natural ecosystems [2]. While essential in trace amounts for biological functions, elevated concentrations of these metals can harm aquatic organisms, terrestrial flora, and, ultimately, human populations through biomagnification and bioaccumulation processes [3, 4]. The emerging heavy metal pollution requires innovative approaches that quantify metal concentrations and assess their biological impacts with precision and sensitivity. Direct toxicity assessment (DTA) methods or bioassays have recently garnered significant attention for their ability to comprehensively evaluate toxicity without specifying individual chemical substances. These bioassays measure the response of organisms to contaminants compared to controls [5, 6]. By using microorganisms that are sensitive to environmental change, the combined biological activities of a mixture of substances extracted from a water sample or any other polluted environment can be assessed. It enables unknown compounds to be detected based on their activity and provides toxicological relevance to the broad chemical characterization of this mixture [7].  
Bioassays using microorganisms or bacteria are called microbiassays. Microbioassays for toxicity assessments have many advantages over conventional methods; they are inexpensive, have short life cycles, less time-consuming, and are sensitive to different toxic chemicals, such as heavy metals, phenolic compounds, organic and inorganic pollutants, and other effluents from industrial activities [8]. However, in this technique, microscopic observation is necessary to derive the critical features of the targeted microorganisms, such as their state (alive/dead) and swimming ability. As the microorganism's size becomes smaller, observation becomes more difficult due to the narrower focal depth of the imaging system [9].  
In our study, to overcome these difficulties, we have proposed a novel technique for the microbiotaassay utilizing biospeckles in the diffraction field generated from the microorganisms under coherent light illumination [10]. Based on the dynamic properties of the biospeckle generated by the microorganisms under different toxicity conditions, it is easy to determine the organisms' activities or healthiness. Here, we employed two microorganisms, Paramecia and Euglena. When a living biological organism is illuminated by a coherence light, a biospeckle pattern is generated [11]. If a sample that is being observed is a biological object, such as a microorganism that can move, then the intensity of the speckle ceases to be constant, and a dynamic biospeckle pattern is observed [12]. The inhibition of the swimming ability of microorganisms due to toxicity can be evaluated by analyzing the dynamic properties of biospeckles generated from their movement. The biospeckle pattern reflects Paramecia and Euglena's movement and the organelles' internal activity within microorganisms.  
The proposed method offers several advantages, including the ability to process many microorganisms simultaneously without the need for imaging systems. Owing to a non-imaging system, an enormous number of microorganisms, such as Paramecia and Euglena, can be processed at a time, and a statistically significant estimation of the swimming ability of microorganisms under different water toxicity concentrations can be derived immediately and efficiently.