4.1. Chemicals and Test Solutions Preparation

Synthetic cathinones (MDPV, mephedrone and methylone) were synthesized in a racemic form as hydrochloride salts as described in [5,57]. Cocaine and MDMA were generously provided by the Spanish National Institute of Toxicology (Madrid, Spain). Drugs were dissolved in pure distilled water to prepare the stock solutions of 250 mM of each drug. Aliquots of the stock solutions were stored at −20 °C until the day of the test. Working solutions were prepared by diluting the stock solutions in Goods Buffer N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonicacid (TAPS 5 mM, CAS 29915-38-6, Merck KGaA, Darmstadt, Germany) at pH 8.6. This buffer was selected according to the speciation profile of the drugs (pKa between 8 and 10), and after previously proving no adverse effects of this pH on survival or developmental parameters, as well as a higher effect of MDMA at pH 8.6 than at pH 7.4 in zebrafish embryos due to a higher bioavailability [58]. Because all the tested drugs are ionizable compounds, the pH influences uptake but not intrinsic toxicity [59].