*2.3. Acute Toxicity Test*

Acute toxicity test on nauplii was performed by adding 10 nauplii to each well of 24-well plates, containing 2 mL of solutions at increasing concentrations of NAP (0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.5, 1, 2.5, 5, 10 mg/L), PHE (0, 0.36, 1, 2, 3, 4, 5, 10, 57.5, 115, 230 mg/L), FLT (0, 0.41, 1, 2.5, 5, 12.5, 25, 50, 97.5, 195, 390 mg/L), and BkF (0, 0.025, 0.5, 1, 1.5, 3, 6, 12, 23.5, 47, 94 mg/L) tested in SSW.

Toxicity on adults was evaluated by adding 5 crustaceans to each well of 6-well plates, containing 10 mL with solutions of various concentrations of NAP, PHE, FLT, and BkF mentioned above. The plates were kept at 25 ± 1 ◦C with salinity 35 ppm for 48 h in a light regime of 16:8 h light:dark, without providing food. At 24 and 48 h, the number of nauplii and adults (which were motionless for 10 s) was counted under a stereomicroscope (Leica EZ4 HD) to calculate the mortality. Tests were considered as valid when mortality in control (organisms exposed to 0 mg/L of organic compounds) was <10% after 48 h. All the experiments were performed in triplicates.
