3.5. Artemia salina Leach Lethality Bioassay

To establish the acute toxicity of the hydroalcoholic extracts of M. fruticulosa subsp. fruticulosa and subsp. coronopifolia, the brine shrimp (Artemia salina Leach) lethality bioassay was carried out according to the method of Meyer et al. [32]. Brine shrimp eggs were placed in a brine shrimp hatchery dish filled with sterile artificial seawater for 48 h. After hatching, active nauplii free from eggshells were collected from the brighter portion of the hatchery dish and used for the assay. Ten brine shrimp larvae were incubated for 24 h at 25–28 °C in 5 mL of artificial seawater mixed with different amounts of the extracts (10–1000 µg/mL). Then, the surviving larvae were counted using a magnifying glass, and median lethal concentration (LC₅₀) values were determined by Litchfield and Wilcoxon’s method. The assay was carried out in triplicate. The toxicity level of the extracts was assessed according to the toxicity scale reported by Clarkson et al. [26]; extracts giving LC₅₀ values greater than 1000 μg/mL were considered nontoxic.