4.9. Larvicidal Bioassay

The WHO procedure was used to evaluate the larvicidal activity [59]. Each test sample (CuO NPs, TCLE, and CS) was individually dissolved in 100 mL of distilled water to prepare the stock solution. Using double-distilled water as a solvent, the appropriate doses were obtained from the stock solution. Twenty mosquito larvae were used in the antilarval experiment, along with different test sample concentrations, in 200 mL of sterilized double-distilled water, which was then placed in an ambient chamber set at 25 °C with a 16:8 h light/dark cycle. For each test, there were five repetitions of each dose of distilled water in a set of control groups. Ae. aegypti and An. stephensi 4th instar larval mortality was tested after 24 h to assess the acute toxicities, and the death of the larvae was confirmed by stoppage of movement when touched by a needle.