Zebrafish embryo-larval testing reveals differential toxicity of new psychoactive substances

New psychoactive substances (NPS) have emerged as a significant public health concern, with synthetic cannabinoid receptor agonists (SCRAs) and ketamine derivatives being among the most frequently detected compounds in the forensic context worldwide. The Fish Embryo Acute Toxicity (FET) and Maximum Tolerated Concentration (MTC) tests are used to evaluate the acute toxicity of chemicals. In this study, we used these assays to evaluate the acute toxicity of three NPS in zebrafish embryos and larvae: the SCRA MDMB-4en-PINACA and the ketamine derivatives deschloroketamine (DCK) and 2-fluorodeschloroketamine (2F-DCK). Our findings demonstrated that MDMB-4en-PINACA induced severe developmental abnormalities, including pericardial edema and yolk edema, along with high embryo mortality (10 µM), characterized by endpoints such as coagulation, lack of heartbeat, and lack of somite formation. In contrast, DCK and 2F-DCK exhibited low embryo mortality even at higher concentrations. In larval stages, MDMB-4en-PINACA presented 8 % larvae mortality (10 µM) at eight days post-fertilization (dpf), whereas ketamine derivatives led to 100 % mortality at 2000 µM in the MTC test at eight dpf. The LC₅₀ was calculated for the FET test with MDMB-4en-PINACA, and MTC test for both DCK and 2F-DCK. Additionally<b>,</b> our results support the absence of N-methyl-D-aspartate (NMDA) receptors in the early life stages of zebrafish described in previous studies and highlight the significance of ketamine derivatives intoxications when the NMDA receptor is expressed. Notably, MDMB-4en-PINACA exhibited significantly higher toxicity, with an LC₅₀ of approximately 26 times lower than that of the ketamine derivatives. These results are particularly relevant given the increasing global prevalence of NPS-related intoxications and fatalities. Using zebrafish as an <i>in vivo</i> model for toxicological research provides an efficient approach for screening the acute effects of emerging compounds such as NPS.