The larvicidal assays were carried out by using the residual film method [7] with slight modifications. Briefly, the effect of different concentrations of tested oils, were checked on 3rd instar larvae (n = 10) to determine the LC50 and LC90 concentrations. To achieve this, 1 mL of the acetone solution containing desired concentration of EO, were spread uniformly on filter paper disc laid inside the glass petri dish of 90-mm-diameter (Fig. 1a). The petri dishes were left open till solvent evaporation followed by inoculation of larvae (n ≥ 10). These tests were carried out under strictly maintained rearing conditions for 24 h. However, in control experiments only solvent, i.e., acetone in place of EO was sprayed following a similar procedure to that in test sets. The larvae were fed on cotton swab soaked in milk solution. The actual dose of EO present in 1 mL mixture was calculated by using the following formula considering the area of petri dish as 63.58 cm2.

The larval mortality was determined after 24 h of exposure with EO to calculate the mean mortality for LC50, and LC90 values using probit analysis [9]. In case of LGEO, the assays were repeated 7 times while the test with TT was repeated 10 times to get the fair results for larval mortality caused by the tested compounds.

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