Zebrafish Embryo Exposure to Copper, Anesthetic, and Bioactive Compounds

E3 medium (0.5 mM NaCl, 0.017 mM KCl, 0.033 mM CaCl₂, 0.033 mM MgSO₄, pH 7.0–7.4) was used to prepare all solutions. Copper sulphate pentahydrate (CuSO₄.5H₂O) (Merck, S.A., Algés, Portugal) was used as the source of copper and a stock solution of 4 mM (1000 mg/L) was prepared in ultra-pure water and further diluted in E3 medium to 10 µM (2.5 mg/L) [27 (link)]. Tricaine methanesulfonate (MS-222) (Sigma Aldrich, Lisboa, Portugal) stock solution of 5.74 mM (1500 mg/L) was prepared in buffered water, pH was adjusted to 7.4 and diluted in E3 medium to 574 µM (150 mg/L), according to [28 (link),29 (link)]. Diclofenac (DIC) sodium salt (Alfa Aesar, Kandel, Germany) stock solution was prepared in E3 medium at 2 mM (636 mg/L) and diluted to 1.5 µM (4.75 mg/L) [26 (link)]. THY (5-methyl-2-propan-2-ylphenol) (Sigma Aldrich) stock solution was prepared in ultra-pure water containing 10% ethanol and diluted in E3 medium of 430 µM (64.5 mg/L) and further diluted to 20 µM (3 mg/L). A 20 µM (9.6 mg/L) stock solution of 24-EPI (Sigma Aldrich, Lisboa, Portugal) was prepared in ultra-pure water containing 5% ethanol and diluted to 5 µM (2.4 mg/L) in E3 medium. All other reagents used were purchased from Sigma Aldrich (Lisboa, Portugal) or of the highest commercially available grade.