2.5.2. Brine shrimp lethality assay (BSLT)

BSLT was done to assess the preliminary cytotoxicity of plant extracts based on the ability to kill a laboratory cultured larvae (nauplii). About 5 g of brine shrimp eggs were loaded on top of normal saline (1L). A light bulb (60–100 W) was placed a few inches away from the jar. After 20–24 h, the nauplii hatched and they were separated after the next 24 h. A suspension with a concentration of 400 nauplii per ml water was prepared.

For toxicity testing, 24-well plates were used. The test was conducted in different phases; the combinations of extracts and their concentrations to be used for the successive phase were decided on the basis of previous results. Initially, to each well, 100μl of 1% solution of each extract (single/combination) with 2 ml water and 50 μl nauplii suspension was added. The nauplii were exposed to different probable synergistic concentrations of the three plant extracts for 24 h. The number of motile nauplii was calculated for the effectiveness of the extract. The mortality endpoint of these bioassay is defined as the absence of controlled forward motion during 30 s of observation. The percentage lethality of the nauplii/larvae for each concentration and control was calculated.