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Abstract

The analysis of drug residues in waste and environmental waters offers valuable insights into the epidemiological and environmental implications of drug use. This study employs a wastewater-based epidemiology (WBE) approach to estimate the use of licit and illicit drugs and new psychoactive substances (NPS) among both general and specific populations. In the latter case, the main focus is on evaluating drug use within educational institutions. Notably, this study comprehensively assessed the presence of illicit drugs in primary schools for the first time. Previously, except for cannabis, such data have not been obtained even through conventional surveys. Additionally, the study evaluates the use of WBE as part of an early warning system capable of detecting the presence of a wide range of psychoactive substances in educational institutions without exposing individuals, as is the case with drug testing. Lastly, this research aims to address a significant knowledge gap regarding the impact of drug use on aquatic ecosystems.
Herein are described the target analytical methods (LC-MS/MS) developed to determine both licit and illicit drug residues in raw wastewater. To study new psychoactive substances (NPS) in educational institutions, suspect screening (LC-IMS-HRMS) was applied. Enantiomeric profiling (chiral derivatization, GC-MS/MS) and determining isotopic composition (GC-C-IRMS) of carbon in biomarkers were explored as complementary approaches to the obtained WBE data. Targeted analytical methods (LC-MS/MS) were also developed to determine drug residues in treated wastewater, surface water, and groundwater. The aim was to assess the removal efficiency of drug residues using conventional biological wastewater treatment processes and determine their occurrence in effluent-receiving rivers and groundwater. In addition, the distribution of drug residues in groundwater was predicted using solute transport modelling.
The toxicity of drug residues towards green algae Chlamydomonas reinhardtii, which serves as a representative primary producer in the aquatic food web, was also explored. This was accomplished through in vivo testing, specifically the algal growth inhibition test. Furthermore, the environmental risks posed by drug residues in effluent-receiving rivers were assessed using in silico methods (ECOSAR).
The research findings on the use of licit, illicit drugs and NPS in the general population reveal distinct patterns. The use of stimulants shows similarities to Western and Southern European countries, with cocaine being the predominant drug detected, as observed through international SCORE monitoring. Among the NPS identified, the use of eutylone, 3-MMC, and mitragynine is confirmed based on international NPS monitoring, with 3-MMC having one of the highest detected mass loads within the study. In educational institutions, drugs are also detected, with nicotine, alcohol, and cannabis being the most prevalent, along with the unequivocal identification of four NPS (stimulants: 3-MMC, ephedrine, 4-chloro-α-PPP, and ethcathinone). It is worth noting that, despite methodological differences, the results obtained through WBE only partially align with data from other sources, regardless of the target population.
In addition to WBE data, a dumping event is confirmed solely through enantiomeric profiling of MDMA, a drug with a straightforward synthesis and excretion profile. Although
the effectiveness of determining the isotopic composition of light elements (C13/C12) in drug residues from raw wastewater for reducing the uncertainty of WBE consumption estimates has not been established, it could be used as a complementary method to drug profiling, potentially providing an early warning system for detecting changes in drug supply. However, further studies are required to substantiate this claim.
Due to the incomplete removal of drug residues during conventional biological wastewater treatment, their presence has been detected in effluent-receiving rivers within the ng/L range. In the algal growth inhibition test conducted on Chlamydomonas reinhardtii, a species of green algae, no adverse effects were observed, even at concentrations higher than those measured (1 mg/L). However, in silico predictions indicate potential effects on aquatic organisms for nicotine, methadone, EDDP, morphine, and MDMA, which warrant further in vivo studies. Furthermore, drug residues have also been found in groundwater within the low ng/L range, which raises concerns considering that groundwater is a primary source of drinking water globally. Contrary to expectations, it was found that the River Sava, which recharges the Ljubljansko polje aquifer and contains drug residues (ng/L range), is not their primary source. Instead, the source has been identified as Ljubljana's leaky sewer infrastructure, emphasizing the need to address raw wastewater as an important source of pollutants when studying urban aquifers.
To summarize, while the thesis successfully addressed several gaps related to estimating drug use through WBE and understanding the environmental impact of drug residues, it has also raised numerous new research questions in epidemiology and environmental and human risk evaluation. For instance, the application of determining the isotopic composition of drug residues in raw wastewater opens up opportunities for further investigation of its applicability as support for forensic intelligence. Additionally, there is a need for ecotoxicological studies to assess the potential harm caused by drug residues (e.g., to subterranean organisms) and studies to examine the occurrence and impact of these residues in drinking water.