2.12. Brine Shrimp Lethality Assay

Synthesized NPs (20 mg/mL stock in water) were tested for probable lethality in the 96-well plate (300 µL) for 24 h against Artemia salina (brine shrimp). Brine shrimp is well-known in study of the toxicological effect of a substance on living organisms [42]. Larvae of Artemia salina was used for this purpose following protocols described by [42]. The eggs of brine shrimp were subjected to incubation for a period of 24–48 h for hatching. During the process, constant oxygen supply in sterile sea water (38 g/L) was ensured. Sterile sea water was supplemented with 6 mg/L dried yeast in a specifically designed plastic tray with two-compartments under proper illumination. Illumination provides the necessary light and temperature (30–32 °C) for hatching. A total of 10 mature nauplii (phototropic) were picked using Pasteur pipette and introduced into the wells. The required volume, as described in developed previous protocols, of the test extracts contain 1% DMSO in sea water. The final concentrations of NPs were adjusted to 25, 50, 100, and 200 μg/mL before transferring it to the wells containing shrimp larvae and sea water. A volume of 300 μL was adjusted in each well. Serial concentrations of doxorubicin (ranging from 1 to 10 µg/mL) were taken as the positive control while that of the DMSO (1%) in sea water served as the negative control. After an incubation period of 24 h, live shrimps were quantified and median lethal concentration (LC50) was determined by using the table curve 2D v5.01 of the test extracts with ≥50% mortality. Doxorubicin was used because it is a renowned chemotherapeutic agent used for a variety of cancer treatments [44].