Seven-years of alcohol consumption in Australia by wastewater analysis: Exploring patterns by remoteness and socioeconomic factors.

BACKGROUND: Wastewater analysis provides a complementary measure of alcohol use in whole communities. We assessed absolute differences and temporal trends in alcohol consumption by degree of remoteness and socioeconomics indicators in Australia from 2016 to 2023. METHODS: Alcohol consumption estimates from 50 wastewater treatment plants (WWTP) in the Australian National Wastewater Drug Monitoring Program were used. Trends were analysed based on 1) site remoteness: Major Cities, Inner Regional and a combined remoteness category of Outer Regional and Remote, and 2) using two socioeconomic indexes from the Australian Bureau of Statistics (ABS) relating to advantage and disadvantage for Income, education, occupation, and housing. RESULTS: Consumption estimates were similar for Major Cities and Inner Regional areas (14.3 and 14.4L/day/1000 people), but significantly higher in Outer Regional and Remote sites (18.6L/day/1000 people). Consumption was decreasing in Major cities by 4.5% annually, Inner Regional by 2.4%, and 3.5% in the combined Outer Regional and Remote category. Consumption estimates were higher in socioeconomically advantaged quartiles than those of lower advantage (0%-25% mean = 13.0, 75%-100% mean = 17.4). Consumption in all quartiles decreased significantly over the 7 year period with annual rates of decrease of 0.9%, 3.7%, 3.6%, and 3.0% for the lowest to highest quartile, respectively. CONCLUSIONS: Declines in Australian alcohol consumption have been steeper in large urban areas than regional and remote areas. There were smaller annual decreases in the most socioeconomically disadvantaged areas. If continued, these trends may increase Australian health inequalities. Policy and prevention work should be appropriately targeted to produce more equitable long-term outcomes.

Amphetamine-type drug-related fatalities in the context of 8 years of methamphetamine use measured by wastewater analysis in South Australia.

Methamphetamine is the illicit stimulant of choice in Australia. Countless initiatives have been employed to reduce methamphetamine use and drug-related harm. Wastewater analysis (WWA) has been used effectively as an objective measure of drug use at a population level and can be compared to indicators of harm, such as the number of drug-related fatalities. This paper attempts to describe methamphetamine use in the context of changes in levels measured in wastewater in South Australia whilst recognising considerable interventions over an 8-year period. Validated liquid chromatography-mass spectrometry methods were used to determine methamphetamine (and MDMA) levels in wastewater over an 8-year interval. The number of drug-induced deaths and driver fatalities involving methamphetamine (and MDMA) was presented and described in the context of changes in use measured by WWA. A rise in methamphetamine use according to WWA was evident up to 2017, followed by a gradual decrease to 2020 back to 2015 levels. Both driver fatalities and drug-induced deaths correlated well with use measured by WWA over the 8-year period. Multiple initiatives to curb supply, distribution and harm within the state and nationally have been implemented. The decrease in methamphetamine use after 2017 suggests that timely interventions have successfully reduced overall drug use and has led to fewer fatalities. This study shows that the response to increasing methamphetamine use in South Australia has resulted in a reversal of the upward trend in consumption and fewer drug-related fatalities.

Monitoring the use of novel psychoactive substances in Australia by wastewater-based epidemiology.

Users of novel psychoactive substances (NPS) are at risk, due to limited information about the toxicity and unpredictable effects of these compounds. Wastewater-based epidemiology (WBE) has been used as a tool to provide insight into NPS use at the population level. To understand the preferences and trends of NPS use in Australia, this study involved liquid chromatography mass spectrometry analysis of wastewater collected from Australian states and territories from February 2022 to February 2023. In total, 59 different NPS were included across two complementary analytical methods and covered up to 57 wastewater catchments over the study. The NPS detected in wastewater were 25-B-NBOMe, buphedrone, 1-benzylpiperazine (BZP), 3-chloromethcathinone, N,N-dimethylpentylone (N,N-DMP), N-ethylheptedrone, N-ethylpentylone, eutylone, 4F-phenibut, 2-fluoro deschloroketamine, hydroxetamine, mephedrone, methoxetamine, methylone, mitragynine, pentylone, phenibut, para-methoxyamphetamine (PMA), alpha-pyrrolidinovalerophenone (α-PVP) and valeryl fentanyl. The detection frequency for these NPS ranged from 3 % to 100 % of the sites analysed. A noticeable decreasing trend in eutylone detection frequency and mass loads was observed whilst simultaneously N,N-DMP and pentylone increased over the study period. The emergence of some NPS in wastewater pre-dates other sources of monitoring and provides further evidence that WBE can be used as an additional early warning system for alerting potential NPS use.

Using wastewater-based epidemiology to evaluate the relative scale of use of opioids.

Wastewater analysis (WWA) has been used as a tool to monitor population drug use, both pharmaceutical and illicit, for over 15 years. Policymakers, law enforcement and treatment services may use WWA-derived data to seek an objective understanding of the extent of drug use in specific areas. Therefore, wastewater data should best be reported in a meaningful form to allow those that are not experts in the field to compare the scale within and between drug classes. Excreted drug loads quantified in wastewater describe the mass of drug present in the sewer. Normalisation for wastewater flow and population is standard practice and critical for comparing drug loads between different catchments and indicates a transition to an epidemiological approach (wastewater-based epidemiology (WBE)). A further consideration is necessary to accurately compare the measured level of one drug to another. The standard dose of a drug taken to elicit a therapeutic effect will vary, with some compounds requiring microgram amounts, while others are administered in the gram range. When WBE data is expressed with units representing excreted or consumed loads without considering dose amounts, the scale of drug use when comparing multiple compounds becomes distorted. To demonstrate the utility and significance of including known excretion rates, potency and typical dose amounts into back-calculations of the measured drug load, this paper compares the levels of 5 prescribed (codeine, morphine, oxycodone, fentanyl and methadone) and 1 illicit (heroin) opioid from South Australian wastewater. The data is presented at each stage of the back-calculation starting with the total mass load measured, to consumed amounts factoring in excretion rates and finally the number of doses the load equates to. This is the first paper to describe the levels of 6 opioids measured in wastewater over a 4-year period in South Australia that demonstrate the relative scale of use.

Three years of wastewater surveillance for new psychoactive substances from 16 countries.

The proliferation of new psychoactive substances (NPS) over recent years has made their surveillance complex. The analysis of raw municipal influent wastewater can allow a broader insight into community consumption patterns of NPS. This study examines data from an international wastewater surveillance program that collected and analysed influent wastewater samples from up to 47 sites in 16 countries between 2019 and 2022. Influent wastewater samples were collected over the New Year period and analysed using validated liquid chromatography - mass spectrometry methods. Over the three years, a total of 18 NPS were found in at least one site. Synthetic cathinones were the most found class followed by phenethylamines and designer benzodiazepines. Furthermore, two ketamine analogues, one plant based NPS (mitragynine) and methiopropamine were also quantified across the three years. This work demonstrates that NPS are used across different continents and countries with the use of some more evident in particular regions. For example, mitragynine has highest mass loads in sites in the United States, while eutylone and 3-methylmethcathinone increased considerably in New Zealand and in several European countries, respectively. Moreover, 2F-deschloroketamine, an analogue of ketamine, has emerged more recently and could be quantified in several sites, including one in China, where it is considered as one of the drugs of most concern. Finally, some NPS were detected in specific regions during the initial sampling campaigns and spread to additional sites by the third campaign. Hence, wastewater surveillance can provide an insight into temporal and spatial trends of NPS use.

Increased Nicotine Consumption in Australia During the First Months of the COVID-19 Pandemic.

INTRODUCTION: Mixed findings have been reported about the impact of the COVID-19 pandemic on smoking behavior in different populations. AIMS AND METHODS: In this study, we aimed to quantify changes in smoking prevalence through the proxy of nicotine consumption in the Australian population from 2017 to 2020 inclusive. Estimates of nicotine consumption between 2017 and 2020 were retrieved from a national wastewater monitoring program that covers up to 50% of the Australian population. National sales data for nicotine replacement therapy (NRT) products from 2017 to 2020 were also acquired. Linear regression and pairwise comparison were conducted to identify data trends and to test differences between time periods. RESULTS: The average consumption of nicotine in Australia decreased between 2017 and 2019 but increased in 2020. Estimated consumption in the first half of 2020 was significantly higher (~30%) than the previous period. Sales of NRT products increased gradually from 2017 to 2020 although sales in the first half of the year were consistently lower than in the second half. CONCLUSION: Total nicotine consumption increased in Australia during the early stage of the pandemic in 2020. Increased nicotine consumption may be due to people managing higher stress levels, such as from loneliness due to control measures, and also greater opportunities to smoke/vape while working from home and during lockdowns in the early stage of the pandemic. IMPLICATIONS: Tobacco and nicotine consumption have been decreasing in Australia but the COVID-19 pandemic may have temporarily disrupted this trend. In 2020, the higher impacts of lockdowns and working from home arrangements may have led to a temporary reversal of the previous downward trend in smoking during the early stage of the pandemic.

Neuromodulation with transcutaneous spinal stimulation reveals different groups of motor profiles during robot-guided stepping in humans with incomplete spinal cord injury.

Neuromodulation via spinal stimulation has been investigated for improving motor function and reducing spasticity after spinal cord injury (SCI) in humans. Despite the reported heterogeneity of outcomes, few investigations have attempted to discern commonalities among individual responses to neuromodulation, especially the impact of stimulation frequencies. Here, we examined how exposure to continuous lumbosacral transcutaneous spinal stimulation (TSS) across a range of frequencies affects robotic torques and EMG patterns during stepping in a robotic gait orthosis on a motorized treadmill. We studied nine chronic motor-incomplete SCI individuals (8/1 AIS-C/D, 8 men) during robot-guided stepping with body-weight support without and with TSS applied at random frequencies between 1 and up to 100 Hz at a constant, individually selected stimulation intensity below the common motor threshold for posterior root reflexes. The hip and knee robotic torques needed to maintain the predefined stepping trajectory and EMG in eight bilateral leg muscles were recorded. We calculated the standardized mean difference between the stimulation conditions grouped into frequency bins and the no stimulation condition to determine changes in the normalized torques and the average EMG envelopes. We found heterogeneous changes in robotic torques across individuals. Agglomerative clustering of robotic torques identified four groups wherein the patterns of changes differed in magnitude and direction depending mainly on the stimulation frequency and stance/swing phase. On one end of the spectrum, the changes in robotic torques were greater with increasing stimulation frequencies (four participants), which coincided with a decrease in EMG, mainly due to the reduction of clonogenic motor output in the lower leg muscles. On the other end, we found an inverted u-shape change in torque over the mid-frequency range along with an increase in EMG, reflecting the augmentation of gait-related physiological (two participants) or pathophysiological (one participant) output. We conclude that TSS during robot-guided stepping reveals different frequency-dependent motor profiles among individuals with chronic motor incomplete SCI. This suggests the need for a better understanding and characterization of motor control profiles in SCI when applying TSS as a therapeutic intervention for improving gait.

A method for improved detection of 8-isoprostaglandin F2α/ß and benzodiazepines in wastewater.

Wastewater-based epidemiology is a tool incorporating biomarker analysis that can be used to monitor the health status of a population. Indicators of health include endogenous oxidative stress biomarkers and hormones, or exogenous such as alcohol and nicotine. 8-Iso-prostaglandin F2α/ß is a biomarker of endogenous metabolism that can be used to measure oxidative stress in a community. Benzodiazepines are a harmful subclass of anxiolytics either prescribed or sourced illegally. The analysis of oxidative stress markers and uptake of benzodiazepines in wastewater may provide information about distress in the community. A method has been applied to detect 8-isoPGF2α/ß and the illicit benzodiazepines clonazolam, flubromazolam and flualprazolam in addition to other prescribed benzodiazepines in wastewater. These substances have been sold as counterfeit pharmaceutical products, such as Xanax, which was formulated to include alprazolam. Deconjugation was initially performed on wastewater samples, followed by liquid-liquid extraction for isoprostanes and solid phase extraction for benzodiazepines to determine the total levels of these analytes. Limits of quantification were in the range of 0.5-2 ng/L for all the analytes except 8-isoPGF2α/ß which was 50 ng/L. Stability, recovery and matrix effect studies were also conducted. Finally, this method was applied to influent wastewater from South Australia which showed the prevalence of 8-isoPGF2α/ß and benzodiazepines.

Methcathinone in wastewater: Drug of choice, or artefact?

Methcathinone is a prevalent Novel Psychoactive Substance (NPS) used illicitly in some countries. Routine analysis of wastewater sampled from catchments in South Australia has shown a consistent low-level presence of the compound, inconsistent with NPS use. This raised the question was the occurrence due to regular use as a drug of choice or was it an artefact being produced from other sources in the sewer system? NPS consumption is generally sporadic and would therefore point to the origin of methcathinone in wastewater being due to in-sewer oxidation of its legal precursor, pseudoephedrine. The present study tested this hypothesis by comparing the levels of pseudoephedrine and methcathinone in wastewater samples collected bimonthly from 8 catchment sites in South Australia. Laboratory experiments exposing pseudoephedrine to common household oxidizing agents (hypochlorite and percarbonate) were also performed and the production of methcathinone was demonstrated and monitored. The results of this study showed that the level of pseudoephedrine and methcathinone measured in wastewater followed a similar pattern. However, there were periods when the levels of each compound diverged. Laboratory experiments showed that when exposed to various oxidizing agents, pseudoephedrine is oxidised to non-stoichiometric quantities of methcathinone. Although the use of methcathinone as a drug of choice remains possible, the results of this study indicate that the low and persistent level of methcathinone found in wastewater may arise in part from the oxidation of pseudoephedrine in the sewer system.

Partitioning of phytocannabinoids between faeces and water - Implications for wastewater-based epidemiology.

Evaluating consumption estimates for lipophilic drugs in wastewater has proven to be a challenge. A common feature for these compounds is that they are excreted in faeces and in conjugated form in urine. Limited research with no obvious experimental evidence has been conducted to investigate the degree to which faecal-bound chemical markers contribute towards mass loads in wastewater. Cannabis chemical markers, known as phytocannabinoids, have been suggested in literature to fall into this category. In this study, cannabis users (n = 9) and non-cannabis users (n = 5) were recruited and provided faecal and urine samples after using the substance. The common chemical markers of cannabis consumption, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), were investigated. An extraction method was developed for the cannabis chemical markers in faecal matter and urine and analysis was performed by liquid chromatography-mass spectrometry. Participant samples were used to establish adsorption and desorption dissolution kinetics models and to assess the equilibrium between faeces and water for these compounds. Equilibration between phases were found to be fast (<5 min). THC-COOH, which is the primary metabolite used in wastewater studies, partitioned ~40% in water while the less polar metabolite and CBD remained largely associated with the particulate fraction. Faecal loads of both cannabis users and non-users affected the total measured amounts of cannabinoids in the aqueous phase. The implications for wastewater monitoring of lipophilic substances are discussed.

Adults with a history of recreational cannabis use have altered speech production.

Stereotypical depictions of speech in cannabis users often suggest slow, laboured output, yet objective evidence supporting this assumption is extremely limited. We know that depressants or hallucinogenic drugs such as cannabis can cause acute changes in communication and speech rate, but the long-lasting effects of cannabis use on speech are not well described. The aim of this study was to investigate speech in individuals with a history of recreational cannabis use compared to non-drug-using healthy controls. Speech samples were collected from a carefully described cohort of 31 adults with a history of cannabis use (but not use of illicit stimulant drugs) and 40 non-drug-using controls. Subjects completed simple and complex speech tasks including a monologue, a sustained vowel, saying the days of the week, and reading a phonetically balanced passage. Audio samples were analysed objectively using acoustic analysis for measures of timing, vocal control, and quality. Subtle differences in speech timing, vocal effort, and voice quality may exist between cannabis and control groups, however data remain equivocal. After controlling for lifetime alcohol and tobacco use and applying a false discovery rate, only spectral tilt (vocal effort and intensity) differed between groups and appeared to change in line with duration of abstinence from cannabis use. Differences between groups may reflect longer term changes to the underlying neural control of speech. Our digital analysis of speech shows there may be a signal differentiating individuals with a history of recreational cannabis use from healthy controls, in line with similar findings from gait and hand function studies.

A method and its application to determine the amount of cannabinoids in sewage sludge and biosolids.

Xenobiotic cannabinoids (phyto and synthetic) are highly lipophilic compounds and have been shown to accumulate within the particulate fraction of wastewater. Limited research has been conducted to investigate the occurrence of cannabinoids in sewage sludge and/or biosolids. The analysis of excreted cannabinoids from sewage sludge or biosolids can provide information about community health, as well as potentially long-term environmental impacts. In this study, a liquid-liquid extraction method was developed for the extraction and detection method for 50 cannabinoids by liquid chromatography-mass spectrometry, including the cannabis urinary biomarker 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and a variety of different generation synthetic cannabinoids and their respective metabolites. Method validation assessed criteria including linearity, selectivity, recovery, and matrix effects. The method was applied to samples collected from a conventional activated sludge reactor treatment facility from various stages of the treatment process. Three cannabinoids were abundant in primary sludge including THC, THC-COOH, and CBD, where THC was the most ubiquitous with concentrations up to 3200 µg kg-1. Only THC and THC-COOH were detectable in aged biosolids. The detection of some cannabinoids in biosolids demonstrated that these compounds are stable throughout the treatment process.

Pholedrine is a marker of direct disposal of methamphetamine.

Consumption of methamphetamine has primarily been estimated in wastewater-based epidemiology by measuring the parent compound. However, this could lead to overestimation when methamphetamine is directly disposed into the sewer system. In this respect, it would be advantageous to measure a specific metabolite of methamphetamine instead. We identified 4-hydroxymethamphetamine (pholedrine) as a potential marker. Stability experiments were performed in both filtered and unfiltered wastewater. Correlations with relative loads in wastewater were used to establish its potential as a marker of direct disposal of methamphetamine, or even as a wastewater-based epidemiology biomarker of methamphetamine consumption. This study then investigated the use of pholedrine in combination with methamphetamine to better detect direct disposal events and its potential as a marker of methamphetamine consumption. Examples from both South Australia and New Zealand exemplify the use of pholedrine to identify potential instances of direct disposal of methamphetamine.

International snapshot of new psychoactive substance use: Case study of eight countries over the 2019/2020 new year period.

There is considerable concern around the use of new psychoactive substances (NPS), but still little is known about how much they are really consumed. Analysis by forensics laboratories of seized drugs and post-mortem samples as well as hospital emergency rooms are the first line of identifying both 'new' NPS and those that are most dangerous to the community. However, NPS are not necessarily all seized by law enforcement agencies and only substances that contribute to fatalities or serious afflictions are recorded in post-mortem and emergency room samples. To gain a better insight into which NPS are most prevalent within a community, complementary data sources are required. In this work, influent wastewater was analysed from 14 sites in eight countries for a variety of NPS. All samples were collected over the 2019/2020 New Year period, a time which is characterized by celebrations and parties and therefore a time when more NPS may be consumed. Samples were extracted in the country of origin following a validated protocol and shipped to Australia for final analysis using two different mass spectrometric strategies. In total, more than 200 were monitored of which 16 substances were found, with geographical differences seen. This case study is the most comprehensive wastewater analysis study ever carried out for the identification of NPS and provides a starting point for future, ongoing monitoring of these substances.

Application of catecholamine metabolites as endogenous population biomarkers for wastewater-based epidemiology.

Wastewater-based epidemiology studies use catchment populations to normalise chemical marker mass loads in 24-h composite wastewater samples. However, one of the biggest uncertainties within the field is the accuracy of the population used. A population marker in wastewater may significantly reduce the uncertainty. This study evaluated the catecholamine metabolites - homovanillic acid (HVA) and vanillylmandelic acid (VMA) - as potential population biomarkers. Influent wastewater 24-h composite samples were collected from 38 wastewater catchments from around Australia (representing ~33% of Australia's population), extracted and analysed by liquid chromatography tandem mass spectrometry. Measured mass loads were compared to population sizes determined by mapping catchment maps against high-resolution census data. Both biomarkers correlated with coefficient of determinations (r2) of 0.908 and 0.922 for HVA and VMA, respectively. From the regression analysis, a slope (i.e. the daily per-capita excretion) of 1.241 and 1.067 mg.day-1.person-1 was obtained for HVA and VMA, respectively. The mass load ratio between VMA:HVA were very similar to that reported in literature for urinary analysis among all catchments. Overall, this study provided further evidence that catecholamine metabolites are suitable candidates as population biomarkers for future studies.

Impact of COVID-19 Controls on the Use of Illicit Drugs and Alcohol in Australia.

Methamphetamine, MDMA, cocaine, cannabis, and alcohol in samples from 20 wastewater treatment plants servicing the eight state or territory capitals of Australia were analyzed, with equivalent coverage of >45% of the national population. Trends in drug consumption were calculated and assessed from samples collected from 2016 to 2020, with a focus on pre-COVID-19 (August 2016 to December 2019), versus February to June 2020, when Australia observed a nationwide lockdown. Results showed delayed but significant decreases in methamphetamine, >50% in Western Australia. In contrast, significant increases in cannabis in most jurisdictions were observed. This suggests changes in consumption may be somewhat linked to reduced supply of imported substances, with increased use of locally produced drugs. Initial decreases in cocaine and MDMA consumption were evident in many parts of the country, but pre-COVID trends were re-established after April 2020. Interestingly, weekend-weekday differences were narrowed for cocaine, MDMA, and alcohol during lockdown, which might be expected due to bars being closed and social gathering not allowed. With this study providing insight into the first four months of COVID-19 restrictions in Australia, it remains to be seen what the longer-term effect of the pandemic will be.

Changes in alcohol consumption associated with social distancing and self-isolation policies triggered by COVID-19 in South Australia: a wastewater analysis study.

AIM: To assess the effects of social distancing and social isolation policies triggered by COVID-19 on alcohol consumption using wastewater analysis in Adelaide, South Australia. DESIGN: Longitudinal quantitative analysis of influent wastewater data for alcohol concentration. SETTING: Adelaide, South Australia. PARTICIPANTS: Wastewater catchment area representative of 1.1 million inhabitants. MEASUREMENTS: Twenty-four hour composite influent wastewater samples were collected from four wastewater treatment plants in Adelaide, South Australia for 7 consecutive days (Wednesday-Tuesday) every 2 months from April 2016-April 2020. The alcohol metabolite ethyl sulfate was measured in samples using chromatography-tandem mass spectrometry. Data were population-weighted adjusted with consumption expressed as standard drinks/day/1000 people. Weekly consumption and weekend to mid-week consumption ratios were analysed to identify changes in weekday alcohol use pattern. FINDINGS: Estimated weekend alcohol consumption was significantly lower (698 standard drinks/day/1000 people) after self-isolation measures were enforced in April 2020 compared with the preceding sampling period in February 2020 (1047 standard drinks/day/1000 people), P < 0.05. Weekend to midweek consumption ratio was 12% lower than the average ratio compared with all previous sampling periods. April 2020 recorded the lowest alcohol consumption relative to April in previous years, dating back to 2016. CONCLUSIONS: Wastewater analysis suggests that introduction of social distancing and isolation policies triggered by COVID-19 in Adelaide, South Australia, was associated with a decrease in population-level weekend alcohol consumption.

How the recreational stimulant market has changed: Case study in Adelaide, Australia 2016-2019.

The human consumption of stimulant drugs is known to increase over festive periods. In this work, four illicit stimulants: 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), cocaine and methamphetamine and three new psychoactive substances (NPS): ethylone, mephedrone and N-ethylpentylone were monitored in influent wastewater over the Christmas-New Year period in South Australia from 2016 to 2019 using liquid chromatography-mass spectrometry. The differences in Christmas - New Year consumption between years were evaluated and daily mass loads were compared to the average for that year to determine drug levels over the festive period. Although MDMA, MDA and cocaine showed year-on-year increases, the use over the New Year period was far higher than over the rest of the year, consistent with recreational drug use. These were also the drugs that were used predominantly on weekends during the year. Methamphetamine, which does not have a pattern of predominant weekend use, and the NPS showed variable trends. These results suggest that during holiday periods there are increases in the use of a limited set of drugs only and these can be predicted from patterns of use during the non-holiday periods.

What is the drug of choice of young festivalgoers?

BACKGROUND: Drug and alcohol consumption are commonplace at festivals including those aimed at younger attendees. However, there is little quantitative information about the extent of this consumption. This work investigates drug use at a school-leaver festival and how it compares to non-festival weeks. METHODS: Influent wastewater was collected over three consecutive weeks from a location where a school-leaver festival occurs. Multiple liquid chromatography-mass spectrometry methods were used to analyse the use of illicit drugs, pharmaceuticals with abuse potential, new psychoactive substances (NPS), alcohol and cannabis. A method for human neurotransmitter metabolites was also utilised to show the population change and allow the drugs found to be normalised to a population. RESULTS: A total of 12 compounds were quantifiable: methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), alcohol, cannabis, cocaine, morphine, codeine, fentanyl, buprenorphine, oxycodone and nicotine. The NPS methylone was found solely over the festival weekend but at levels below the limit of quantification of the analytical method. The catecholamine metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA) were found over the entire three weeks, with identical trends - an increase over the festival weekend - indicating a population increase. HVA was used to normalise the drug mass loads to derive a population normalised mass load. Statistical differences using Hedges' g showed large changes in the use of MDMA and MDA over the festival week. Smaller increases were also seen for alcohol and cocaine. CONCLUSIONS: The drugs of choice for the attendees of this school-leaver festival were MDMA and MDA.

The complexities associated with new psychoactive substances in influent wastewater: The case of 4-ethylmethcathinone.

Consumption of new psychoactive substances (NPS) is an international problem for health, policing, forensic, and analytical laboratories. The transience of these substances in the community, combined with continual slight structural changes to evade legislation makes the elucidation of NPS an analytical challenge. This is amplified in a matrix as complex as wastewater. For that reason, suspect and non-target methodologies, employing high resolution mass spectrometry are the most appropriate current tool to facilitate the identification of new and existing compounds. In the current work, a qualitative screening method of influent wastewater using liquid chromatography-high resolution mass spectrometry showed a strong signal at m/z 192.1382 - identical to that of two NPS standards that were in our method (pentedrone and 4-methylethcathinone), and with identical fragment ions, but the retention times did not match. This work shows the methodology followed to identify this compound, highlighting the challenges of the identifying "new" compounds in influent wastewater.