2.5. Brine Shrimp Lethality Bioassay

The cytotoxic activity of the samples (the ethanol and aqueous crude extracts, chloroform, ethyl acetate, n-butanol, and aqueous fractions and the isolated compound) was determined using the Brine shrimp lethality bioassay [29,30]. Briefly, brine shrimp eggs (Artemia salina) were incubated in a vessel containing sterile artificial seawater produced by dissolving 38 g of table salt in 1 L of distilled water at 28–30 °C with good aeration (using air pump), under a continuous light condition (60 W lamp) for 48 h. After hatching, nauplii were collected with a Pasteur pipette, ten brine shrimps were moved into each well containing the seawater.

Sample stock solutions were made by dissolving 5 mg of each sample in 1 mL of DMSO. Test solutions of different concentrations (1000, 800, 400, 200, 100, 50, 25, 12.5, and 6.25 μg/mL) were collected using the serial dilution technique with seawater. The solutions were transferred to individual vials and 5 mL of the seawater including 10 nauplii shrimp were taken into each vial. A control group comprising same volumes of DMSO (as in the sample vials) and 10 nauplii in 5 mL of artificial seawater was used. After 24 h, the vials were examined with a magnifying glass and the amount of nauplii survived in each vial was counted. The percent mortality of the brine shrimp nauplii was determined for control and increasing concentration from the data and the values of the LC₅₀ were determined. Potassium dichromate was used as a reference standard.