Spatiotemporal trends of new psychoactive substances in Slovenia through influent wastewater analysis

• Spatiotemporal trends in new psychoactive substance use are evaluated. • The most common NPS detected were 3-methylmethcathinone (3-MMC) and mitragynine. • The highest population-normalised mass loads were found for mitragynine. • Mass loads of 3-MMC increased during COVID-19. New psychoactive substances (NPS) are a continuously evolving class of psychoactive compounds, marketed initially as legal alternatives to conventional illicit drugs while bypassing legal regulations and, as such, are of international public health concern. Due to their continuous evolution, with new derivatives continually appearing on the drug market, monitoring and surveillance of NPS pose a challenge when employing traditional socio-epidemiological methods. In this study, we applied wastewater-based epidemiology (WBE) to gain additional information on the spatiotemporal consumption patterns of NPS. Samples of influent wastewater were collected from seven wastewater treatment plants (WWTPs) in Slovenia throughout six sampling periods, covering the pre- and during-pandemic periods (summer 2019, winter/spring 2020 and winter/spring 2021), and when most of the restrictions had been eased (spring 2022) due to the COVID-19 pandemic. Following solid-phase extraction, pooled samples were analysed using two separate methods: a targeted LC-MS/MS method for NPS analysis and suspect screening using LC-QTOF-MS in combination with the HighResNPS database of more than 2000 NPS and other compounds of toxicological relevance. Five NPS (3-methylmethcathinone (3-MMC), 4-fluoroamphetamine (4-FA), eutylone, mephedrone, and mitragynine) were semi-quantified across all sites. 3-MMC and mitragynine were the most prevalent NPS detected, with the latter having the highest population-normalised mass loads (up to 40 mg/day/1000 inhabitants). A noticeable increase in 3-MMC mass loads was observed during the pandemic. Following suspect screening, 17 additional compounds were detected at least once across all sites. This study highlights the potential for WBE to provide insight into Slovenia’s NPS market.