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.

Workflow to facilitate the detection of new psychoactive substances and drugs of abuse in influent urban wastewater.

The complexity around the dynamic markets for new psychoactive substances (NPS) forces researchers to develop and apply innovative analytical strategies to detect and identify them in influent urban wastewater. In this work a comprehensive suspect screening workflow following liquid chromatography - high resolution mass spectrometry analysis was established utilising the open-source InSpectra data processing platform and the HighResNPS library. In total, 278 urban influent wastewater samples from 47 sites in 16 countries were collected to investigate the presence of NPS and other drugs of abuse. A total of 50 compounds were detected in samples from at least one site. Most compounds found were prescription drugs such as gabapentin (detection frequency 79%), codeine (40%) and pregabalin (15%). However, cocaine was the most found illicit drug (83%), in all countries where samples were collected apart from the Republic of Korea and China. Eight NPS were also identified with this protocol: 3-methylmethcathinone 11%), eutylone (6%), etizolam (2%), 3-chloromethcathinone (4%), mitragynine (6%), phenibut (2%), 25I-NBOH (2%) and trimethoxyamphetamine (2%). The latter three have not previously been reported in municipal wastewater samples. The workflow employed allowed the prioritisation of features to be further investigated, reducing processing time and gaining in confidence in their identification.

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.

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.

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.

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.

Genotoxicity of advanced glycation end products in vitro is influenced by their preparation temperature, purification and cell exposure time.

Advanced glycation end products (AGEs) are formed via non-enzymatic reactions between amino groups of proteins and the carbonyl groups of reducing sugars. Previous studies have shown that highly glycated albumin prepared using a glucose-bovine serum albumin (Glu-BSA) model system incubated at 60°C for 6 weeks induces genotoxicity in WIL2-NS cells at 9 days of exposure measured by the cytokinesis-block micronucleus cytome (CBMNcyt) assay. However, this AGE model system is not physiologically relevant as normal body temperature is 37°C and the degree of glycation may exceed the extent of albumin modification in vivo. We hypothesised that the incubation temperature and purification method used in these studies may cause changes to the chemical profile of the glycated albumin and may influence the extent of genotoxicity observed at 3, 6 and 9 days of exposure. We prepared AGEs generated using Glu-BSA model systems incubated at 60°C or 37°C purified using trichloroacetic acid (TCA) precipitation or ultrafiltration (UF) and compared their chemical profile (glycation, oxidation, and aggregation) and genotoxicity in WIL2-NS cells using the CBMNcyt assay after 3, 6 and 9 days of exposure. The number of micronuclei (MNi) was significantly higher for cells treated with Glu-BSA incubated at 60°C and purified via TCA (12 ± 1 MNi/1000 binucleated cells) compared to Glu-BSA incubated at 37°C and purified using UF (6 ± 1 MNi/1000 binucleated cells) after 9 days (P < 0.0001). The increase in genotoxicity observed could be explained by a higher level of protein glycation, oxidation, and aggregation of the Glu-BSA model system incubated at 60°C relative to 37°C. This study highlighted that the incubation temperature, purification method and cell exposure time are important variables to consider when generating AGEs in vitro and will enable future studies to better reflect in vivo situations of albumin glycation.

Can a digital slide scanner and viewing technique assist the visual scoring for the cytokinesis-block micronucleus cytome assay?

The cytokinesis-block micronucleus cytome (CBMNcyt) assay is a comprehensive method to measure DNA damage, cytostasis and cytotoxicity caused by nutritional, radiation and chemical factors. A slide imaging technique has been identified as a new method to assist with the visual scoring of cells for the CBMNcyt assay. A NanoZoomer S60 Digital Pathology slide scanner was used to view WIL2-NS cells treated with hydrogen peroxide (H2O2) and measure CBMNcyt assay biomarkers using a high-definition desktop computer screen. The H2O2-treated WIL2-NS cells were also scored visually using a standard light microscope, and the two visual scoring methods were compared. Good agreement was found between the scoring methods for all DNA damage indices (micronuclei, nucleoplasmic bridges and nuclear buds) and nuclear division index with correlation R values ranging from 0.438 to 0.789, P < 0.05. Apoptotic and necrotic cell frequency was lower for the NanoZoomer scoring method, but necrotic frequency correlated well with the direct visual microscope method (R = 0.703, P < 0.0001). Considerable advantages of the NanoZoomer scoring method compared to direct visual microscopy includes reduced scoring time, improved ergonomics and a reduction in scorer fatigue. This study indicates that a digital slide scanning and viewing technique may assist with visual scoring for the CBMNcyt assay and provides similar results to conventional direct visual scoring.

Advanced glycation end-products accelerate telomere attrition and increase pro-inflammatory mediators in human WIL2-NS cells.

This study investigated the effect of dietary sugars and advanced glycation end-products (AGE) on telomere dynamics in WIL2-NS cells. Dietary sugars [glucose (Glu) and fructose (Fru); 0.1 M each] were incubated with bovine serum albumin (BSA) (10 mg/ml) at 60 ± 1°C for 6 weeks to generate AGE-BSA. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed total AGE levels as 87.74 ± 4.46 nmol/mg and 84.94 ± 4.28 nmol/mg respectively in Glu-BSA and Fru-BSA model. Cell treatment studies using WIL2-NS cells were based on either glucose, fructose (each 2.5-40 mM) or AGE-BSA (200-600 µg/ml) in a dose-dependent manner for 9 days. Telomere length (TL) was measured using qPCR. Nitric oxide (NO) production and tumour necrosis factor-α (TNF-α) levels were measured in WIL2-NS culture medium. An increasing trend for TNF-α and NO production was observed with higher concentration of glucose (R2 = 0.358; P = 0.019; R2 = 0.307; P = 0.027) and fructose (R2 = 0.669; P = 0.001; R2 = 0.339; P = 0.006). A decreasing trend for TL (R2 = 0.828; P = 0.000), and an increasing trend for NO production (R2 = 0.352; P = 0.031) were observed with increasing Glu-BSA concentrations. Fru-BSA treatment did not show significant trend on TL (R2 = 0.135; P = 0.352) with increasing concentration, however, a significant reduction was observed at 600 µg/ml (P < 0.01) when compared to BSA treatment. No trends for TNF-α levels and a decreasing trend on NO production (R2 = 0.5201; P = 0.019) was observed with increasing Fru-BSA treatment. In conclusion, this study demonstrates a potential relationship between dietary sugars, AGEs and telomere attrition. AGEs may also exert telomere shortening through the production of pro-inflammatory metabolites, which ultimately increase the risk of diabetes complications and age-related disease throughout lifespan.

Dietary sugars and related endogenous advanced glycation end-products increase chromosomal DNA damage in WIL2-NS cells, measured using cytokinesis-block micronucleus cytome assay.

This study investigated the effect of glucose and fructose, and advanced glycation end-products (AGEs) on genome damage in WIL2-NS cells, measured using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The effect of AGEs was investigated using the bovine serum albumin (AGE-BSA) model system induced either with glucose (Glu-BSA) or with fructose (Fru-BSA). Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed higher Nε-carboxymethyllysine (CML; 26.76 ± 1.09 nmol/mg BSA) levels in the Glu-BSA model. Nε-Carboxyethyllysine (CEL; 7.87 ± 0.19 nmol/mg BSA) and methylglyoxal-derived hydroimidazolone-1 (MG-H1; 69.77 ± 3.74 nmol/mg BSA) levels were higher in the Fru-BSA model. Genotoxic effects were measured using CBMN-Cyt assay biomarkers [binucleated(BN) cells with micronuclei (MNi), BN with nucleoplasmic bridges (NPBs) and BN with nuclear buds (NBuds)] following 9 days of treatment with either glucose, fructose, Glu-BSA or Fru-BSA. Fructose treatment exerted a significant genotoxic dose-response effect including increases of BN with MNi (R2 = 0.7704; P = 0.0031), BN with NPBs (R2 = 0.9311; P < 0.0001) and BN with NBuds (R2 = 0.7118; P = 0.0091) on cells, whereas the DNA damaging effects of glucose were less evident. High concentrations of AGEs (400-600 µg/ml) induced DNA damage; however, there was no effect on cytotoxicity indices (necrosis and apoptosis). In conclusion, this study demonstrates a potential link between physiologically high concentrations of reducing sugars or AGEs with increased chromosomal damage which is an important emerging aspect of the pathology that may be induced by diabetes. Ultimately, loss of genome integrity could accelerate the rate of ageing and increase the risk of age-related diseases over the long term. These findings indicate the need for further research on the effects of glycation on chromosomal instability and to establish whether this effect is replicated in humans in vivo.