REPOSITORY > RESULTS

Abstract

The analysis of psychoactive substance residues in wastewater (influent and effluent) and receiving surface waters offers valuable insight into the epidemiological and environmental implications of psychoactive substance use. This thesis describes the use of wastewater-based epidemiology (WBE) to study spatiotemporal trends in the consumption of psychoactive pharmaceuticals and new psychoactive substances (NPS) among the general Slovenian population. It also examines the influence of the COVID-19 pandemic on psychoactive pharmaceuticals and NPS use. Furthermore, this thesis provided an opportunity to assess the removal efficiency of wastewater treatment and the occurrence of psychoactive pharmaceutical residues in the environment. Additionally, the thesis explores the application of novel active-passive sampling strategies in wastewater and compares the results with those obtained from conventional 24-hour composite sampling. Finally, the thesis addresses a significant knowledge gap regarding the impact of psychoactive pharmaceutical residues on aquatic organisms.
First, robust bioanalytical methods were established for the quantification of psychoactive pharmaceutical residues in multiple aquatic matrices. These include the development of target analytical methods based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the simultaneous detection of trace levels of psychoactive pharmaceutical biomarkers, including antidepressants, antipsychotics, and benzodiazepines, in influent wastewater. Additionally, to enhance throughput and reduce sample volume requirements, a novel 96-well solid phase extraction (SPE) protocol was optimised for the quantification of 28 biomarkers of antidepressants, opioids, and drugs of abuse in influent wastewater. This method, which requires only 2 mL of influent wastewater, proved effective for large-scale, long-term monitoring campaigns while maintaining sufficiently low limits of detection (LODs) and adequate analytical sensitivity.
In parallel, analytical protocols based on LC-MS/MS (QTrap-based targeted analysis and QTOF-based “suspect-screening”) were applied to investigate the presence of NPS, highlighting their dynamic emergence and widespread distribution. Targeted LC-MS/MS methods were developed to determine the presence of psychoactive pharmaceutical residues in wastewater (influent and effluent) samples and receiving surface water. These methods assess the removal efficiency of these residues during wastewater treatment processes and determine their occurrence in effluent-receiving surface waters. Furthermore, the acute and chronic toxicity of psychoactive pharmaceutical residues to aquatic organisms was explored using in silico methods (Ecological Structure-Activity Relationships Software – ECOSAR).
Given the uncertainties regarding biomarker stability in wastewater, systematic experiments were conducted to evaluate the stability of 11 antipsychotic biomarkers under various storage and sampling conditions. Results demonstrated compound-specific degradation patterns: several compounds rapidly degraded under acidification or repeated freeze–thaw cycles, while only quetiapine and risperidone remained highly stable across most tested conditions. Storage of extracted samples on SPE cartridges in a freezer (−20 °C) was highly effective, maintaining high stability for up to three months for almost all antipsychotic biomarkers, making them suitable for sample transport and batch analysis. Additionally, the stability of selected NPS on loaded SPE cartridges was evaluated under different temperatures and time intervals to mimic various storage conditions. The findings indicated that keeping extracts at −20 °C maintained recoveries within the acceptable range (80–120%) for most compounds for up to four months.
The major research findings on the use of psychoactive pharmaceuticals and NPS in the general population, based on wastewater (influent) analysis, reveal distinct trends with numerous temporal and spatial variations. Most psychoactive pharmaceuticals showed stable usage throughout the week; however, the few observed differences between weekends and weekdays could not be directly attributed to the pandemic. Moreover, although for most antidepressants the consumption levels increased during the first wave of the pandemic, no clear pandemic-related spatial patterns were found across locations. In contrast, antipsychotics and benzodiazepines consumption decreased during the pandemic, especially during the first wave of COVID-19. Notably, these results align with available annual prescription data for psychoactive pharmaceuticals, supporting the reliability of WBE results. Among the detected NPS, 3-methylmethcathinone (3-MMC) had the highest detection frequency, appearing in almost all sites and sampling periods, followed by mitragynine, which had the highest mass loads. The findings also align with other international wastewater studies, which report that 3-MMC is one of the predominant stimulant NPS in Europe. It is worth noting that mass loads of 3-MMC increased significantly during the pandemic. Suspect screening revealed the presence of additional NPS across at least one site during the sampling campaigns.
Analysis of wastewater treatment plant (WWTP) effluent revealed the incomplete removal of psychoactive pharmaceutical residues during different conventional biological wastewater treatments (activated sludge, sequential batch reactor–SBR, SBR with UV disinfection, membrane bioreactor–MBR, and moving bed biofilm reactor–MBBR), making it possible to detect their presence in effluent-receiving surface waters within the ng/L range. In addition, a good comparison between direct surface water analyses and predicted environmental concentrations indicates that predictive approaches, as described in this thesis, may serve as a practical substitute for conventional water analysis. The ECOSAR model further indicated potential effects on aquatic organisms, especially when exposed to venlafaxine, O-desmethylvenlafaxine, citalopram, and sertraline. These results emphasise the importance of upgrading treatment technologies and the need for further in vivo studies.
Finally, the thesis presents an innovative sampling strategy that utilised active–passive sampling (APS) in real wastewater. APS devices were deployed at the inflow and outflow of a Slovenian wastewater treatment plant to compare the occurrence of psychoactive substances, including antidepressants, illicit drugs, and opioids. The results from APS were compared with those obtained from conventional 24-hour composite sampling followed by SPE. While APS was ineffective for influent samples due to excessive particulate content, a comparison of APS with conventional 24-hour composite sampling in effluent during the same sampling period showed good agreement for most studied compounds. The results highlight the potential of APS for monitoring campaigns.
To summarise, this thesis successfully addressed several gaps related to developing and validating advanced analytical methods for the determination of psychoactive pharmaceuticals and NPS, evaluating their stability, estimating their use through WBE, and understanding the environmental impact of psychoactive pharmaceutical residues. At the same time, it highlighted new research challenges in WBE, such as integrating non-target screening into the early warning system. In addition, investigations are needed into their occurrence in drinking water supplies and the associated risks to human health, as well as further ecotoxicological studies assessing risks to non-target organisms, especially in sensitive environments.