## 4 Discussion
The chlorination process negatively affects the behavior and mortality of chironomid larvae. As the chlorine dose increases, there is a decrease in the behavior of chironomid larvae, which becomes more significant at higher doses. The impact on larval mortality varies depending on the dose of chlorine used. Low doses of chlorine have minimal impact on larval mortality, while medium and high doses result in much higher mortality rates, especially after five days of exposure. The mortality rate is highest in instar 1 and gradually decreases in instars 2 to 4, suggesting that larvae at earlier stages of development are more susceptible to the toxic effects of chlorine. This is due to the fact that the surface of Chironomid larvae is comprised of several layers of cell tissue, which offers them significant resistance against oxidative agents. To effectively inactivate chironomid larvae using chlorine, it is necessary for the oxidizing agent to penetrate through the surface structure and access the inner portion of the organism. However, at lower doses and longer exposure times, chlorine may not be able to fully penetrate the chironomid larvae surface, leading to decreased efficacy of the treatment. These findings are consistent with a previous study conducted by [PERSON] et al. [3], which compared the effectiveness of chlorine dioxide and chlorine in inactivating chironomid larvae in raw water. The color change of larvae is also a useful indicator for detecting dead body larvae, as they often exhibit a green to dark green coloration (Figure 5a). This color change occurs as a result of the decomposition of the larval tissue, which produces pigments that cause the color change. Additionally, the addition of high doses of chlorine to water can accelerate the physical or chemical breakdown of the larvae tissues and structure, causing larvae to die and \"melt\" within a few days [20]. This is because chlorine is a strong oxidizing agent that can be harmful to the environment and cause severe damage or death to larvae or other organisms. In our study, we observed that the first melting larvae was instar 1, and this occurred when a chlorine dose of 4 mg/L was used. As the chlorine dose increased, the number of melting larvae in instars 1 and 2 also increased. After 5 days of observation, a total of 7 melting instar 1 and 6 melting instar 2 larvae. However, only 2 melting larvae for each instar 3 and instar 4 after 5 days of exposure to chlorine (Figure S4). The melting of larvae after exposure to a high dose of chlorine is likely due to the chlorine causing a change in the structure or physical properties of the larvae that leads to their melting (Figure 5b). This change can occur because chlorine reacts with components in the larval body, such as protein, lipid, or carbohydrate, causing a change in the structure or chemical composition of the larval body. Similar to the coagulation process, larvae can continue to develop during exposure to chlorine, particularly at low doses. This finding suggests that chlorine may not effectively eliminate all chironomid larvae during the water treatment process. During chlorination, especially in stages 3 and 4, the larvae have the potential to develop into adult mosquitoes after only two days (Figure S5). Chlorination is one of the most widely used methods for disinfecting water, but it is not without its drawbacks. This study has highlighted the potential negative impacts of chlorination on aquatic organisms, particularly on chironomid larvae. While high doses of chlorine can effectively inactive chironomid larvae, they can also cause taste and odor problems in drinking water. In addition, excessive amounts of chlorine in water can combine with naturally occurring organic material to form disinfection byproducts (DBPs) during water treatment, which can have potential long-term health impacts. The World Health Organization Drinking Water Guidelines recognize the importance of disinfection but also emphasize the need to balance the benefits of disinfection with potential negative impacts [21].