Artemia salina acute toxicity test

Commercially avalaible Artemia salina dehydrated cysts were purchased from local aquarium store (ArtemiaCyst Blue Line, Italy). Cysts were first hydrated in ASPM seawater solution (ASPM is a synthetic seawater made of: NaCl = 26.4 g, KCl = 0.84 g, CaCl₂·H₂O = 1.67 g, MgCl·H₂O = 4.6 g, MgSO₄·7H₂O = 5.58 g, NaHCO₃ = 0.17 g and H₃BO₃ = 0.03 g) and then washed to separate the floating cysts (i.e., dead) from those that sink (i.e. alive). The sinking cysts were collected, and approximately 1 g of the precleaned cysts were incubated in 800 mL of ASPM solution seawater in a conical plastic container with graduations. A 1.500 lux daylight was provided continuously by a fluorescent lamp. Aeration was maintained by a small line extending to the bottom of the hatching device from an aquarium air pump. Under conditions of incubation at room temperature (26 ± 1 °C), gentle aeration and continuous illuminations, the nauplii hatched within 24 h. Two stock solutions of as received MoS₂ (5 mg/mL) and ball-milled MoS₂ 40 h (5 mg/mL) after dilution in ASPM solution, were prepared. Then, fresh suspensions with different concentrations of nanopowders (10⁻¹ and 10⁻² mg/mL) were made starting from the stock suspensions. These solutions were vortexed for 30 seconds, and then sonicated in an ultrasonic bath for about 20 minutes. 200 μL of each different concentrations of nanopowders solutions were added to the 96-well microplates. After that, 1 nauplius per well was added and incubated at 26 °C for 24/48 hours. The number of surviving nauplii in each well was counted under a stereomicroscope after 24/48 hours. A control group was also setup with ASPM seawater solution only. Larvae were not fed during the bioassays. The endpoint (immobility, i.e. death) was assessed at the end of the test by a stereomicroscope (a Leica EZ4): a nauplium was considered to be immobile or dead, if it could not move its antennae after slight agitation of the water for 10 seconds. Larvae that were completely motionless were counted as dead, and the percentages of mortality compared to the control were calculated. The death % of the crustacean for each concentration was calculated as follow: (n. dead nauplii/n. total animal treated) • 100. The collected data were analyzed for significance by one-way ANOVA test.