2.6. Brine Shrimp Lethality Test (BSLT) Assay

Filtered seawater was prepared in a container separated into two parts and connected with one tunnel, where one part was made dark and the other was lit using 15–20 Watt lamp. Brine shrimp eggs (Artemia salina) were placed in the dark part of the container to hatch (25–29 °C; 48 h) and drawn to the lighted part once hatched. Extract samples (30 mg) were dissolved using a mixture of DMSO 5% (w/v) in 30 mL of filtered seawater and subsequently sonicated until homogenous. The extract concentration ranged from 500 to 1 μg/mL, where seawater containing only DMSO 5% (w/v) was used as a negative control. Each sample was placed into a glass container followed by the addition of 10 brine shrimp and incubated for 24 h at room temperature. Dead brine shrimp were indicated by the absence of movement and counted. The results were expressed in LC₅₀—the minimum required concentration to obtain 50% lethality. This assay was conducted in triplicate.