New Psychoactive Substances in Wastewater from Rest Areas and Commercial Weigh Stations along Interstate Highways

The use of new psychoactive substances (NPS) poses a persistent threat to public health and safety worldwide. The lack of early and comprehensive detection of NPS has been a significant challenge for harm reduction initiatives. In this study, we demonstrate the use of wastewater-based epidemiology (WBE) for the early detection of NPS, analyzing 547 samples collected over 12 months from five rest areas and two commercial weigh stations along interstate highways in Kentucky. Using a validated ultraperformance liquid chromatography-tandem mass spectrometry method, several target NPS were quantified, including two synthetic opioids (<i>p</i>-fluorofentanyl and metonitazene), a synthetic cannabinoid (MAB-CHMINACA), a phenylpiperazine (mCPP), an alkaloid (mitragynine), and two synthetic cathinones (MDPV and MMMP). Notably, metonitazene was detected for the first time in 30% of wastewater samples─preceding reports in the state's seized and toxicological data. Similarly, <i>p</i>-fluorofentanyl was also quantified for the first time in wastewater collected from these roadway facilities, with concentrations reaching up to 0.48 mg/d/1,000 people in 83% of samples (<i>n</i> = 66) at I-64E. The lower dilution factors of human excreta at these point-of-release facilities, combined with higher-resolution data on wastewater inflow and user populations, highlight the potential of WBE as an effective regional early warning system for NPS.