*3.4. Toxicity Tests*

*Artemia salina* dehydrated cysts were used for the acute toxicity test. Cysts (Hobby, Germany) were hydrated in ASPM seawater solution (ASPM is an artificial 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, NaHCO<sup>3</sup> = 0.17 g, and H3BO<sup>3</sup> = 0.03 g) maintaining standard laboratory conditions (1.500 lux daylight; 26 ± 1 ◦C; continuous aeration), then nauplii hatched within 24 h.

Two stock solutions of CeO<sup>2</sup> (bare CeO<sup>2</sup> and grey CeO2) were prepared after dilution in ASPM solution. Then, fresh suspensions with different concentrations of powders (10−<sup>1</sup> , 10−<sup>2</sup> , 10−<sup>3</sup> mg/mL) were made starting from the stock suspensions (1 mg/10 mL). These solutions were vortexed for 30 s. One nauplius per well in 96-well microplates, was added with 200 µL of each different concentration of powder solutions. They were incubated at 26 ◦C for 24/48 h. The number of surviving nauplii in each well was counted under a stereomicroscope after 24/48 h. A control group was also set up with ASPM seawater solution only. Larvae were not fed during the bioassays.

At the end of the test, the endpoints (immobility, i.e., death) were evaluated with a stereomicroscope (Leica EZ4, Leica Microsystems Srl, Buccinasco (MI), Italy): 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 follows: (n. dead nauplii/n. total animal treated 100). Data were analyzed for differences between the control and treatments using one-way ANOVA followed by Tukey's test, where *p* < 0.05 is considered significant and *p* < 0.01 extremely significant.
