Dried cysts were performed as indicated above, and incubated (1 g cyst per liter) in a hatcher at 28–30°C with strong aeration, under a continuous light regime. Approximately 12 h after hatching the phototropic nauplii were collected with a pipette from the lighted side and concentrated in a small vial. Ten brine shrimp were transferred to each well using adequate pipettes. Each test consisted of exposing groups of 10 Artemia aged 12 h to various concentrations of the toxic compound. The toxicity was determined after 12 h (mainly nauplii in instar I/II), 24 h (nauplii in instar II/III) and 48 h (mainly nauplii in instar III/IV) of exposure.
The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation.
The larvae did not receive food. To ensure that the mortality observed in the bioassay could be attributed to bioactive compounds and not to starvation; we compared the dead larvae in each treatment to the dead larvae in the control. In any case, hatched brine shrimp nauplii can survive for up to 48 h without food [15 ] because they still feed on their yolk-sac [10 (link)]. However, in cases where control deaths were detected, the percentage of mortality (% M) was calculated as: % M = percentage of survival in the control - percentage of survival in the treatment.
The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation.
The larvae did not receive food. To ensure that the mortality observed in the bioassay could be attributed to bioactive compounds and not to starvation; we compared the dead larvae in each treatment to the dead larvae in the control. In any case, hatched brine shrimp nauplii can survive for up to 48 h without food [15 ] because they still feed on their yolk-sac [10 (link)]. However, in cases where control deaths were detected, the percentage of mortality (% M) was calculated as: % M = percentage of survival in the control - percentage of survival in the treatment.
