Enantioseparation and ecotoxicological studies of a cathinone in D. magna

Background: The synthetic cathinone 3-chloromethcathinone (3-CMC) is among the most prevalent new psychoactive substances (NPS), accounting for 46% of NPS seized in Europe in 2023 [1]. Moreover, like most synthetic cathinones that are chiral, 3-CMC enantiomers may exhibit distinct toxicological effects [2,3]. Therefore, enantioselective studies are essential for the comprehensive characterization of their associated environmental risks. Objective: This study aimed to optimize a chromatographic method for the semi-preparative enantioresolution of 3-CMC and assess its ecotoxicity in D. magna. Methods: The enantioresolution of 3-CMC was performed by liquid chromatography (LC) coupled with a UV/Vis detector, using a semi-preparative CHIRALPAK® AD-H column. For the ecotoxicity assessment, neonates aged ≤ 24 h were exposed to 3-CMC at concentrations ranging from 3.13 to 50 mg L⁻¹ in a 48-h acute test, and to 260, 325, and 520 μg L⁻¹ in a 9-day subchronic test. Various key endpoints were evaluated, including behavioural, morphophysiological, reproductive, and biochemical parameters. Results: The optimized method enabled the isolation of the enantiomers of 3-CMC with high purity (> 97.78%). Acute exposure assays to 3-CMC yielded an EC₅₀ (48 h) of 26.14 mg L⁻¹. The racemic mixture of 3-CMC induced significant alterations in behavioural and morphophysiological parameters, while no effects were observed on reproduction. Biochemical analyses revealed a significant increase in thiobarbituric acid reactive substances (TBARS) levels across all exposed groups. However, due to the low stereochemical stability of the isolated enantiomers, enantioselective toxicity assessment was not feasible. Conclusions: These findings demonstrate that 3-CMC can induce toxicity in aquatic organisms even under short-term exposure, emphasizing the need for routine monitoring of this contaminant in surface waters. Moreover, the racemization of the isolated 3-CMC enantiomers in the culture medium prevented the assessment of enantioselective toxicity, highlighting the importance of evaluating stereochemical stability to ensure accurate toxicity risk assessment.