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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-Δ 9 -tetrahydrocannabinol (THC-COOH), 11-hydroxy-Δ 9 -tetrahydrocannabinol (THC-OH), Δ 9 -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., Competing Interests: Declaration of competing interest For the submission our manuscript entitled ‘Partitioning of phytocannabinoids between faeces and water – implications for wastewater-based epidemiology’ to the Journal of Science of the Total Environment. We here by acknowledge the following., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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-Δ 9 -tetrahydrocannabinol (THC-COOH), Δ 9 -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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Wastewater-based epidemiology is currently being utilized to monitor the dissemination of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), on a population scale. The detection of SARS-CoV-2 in wastewater is highly influenced by methodologies used for its isolation, concentration and RNA extraction. Although various viral concentration methods are currently employed, including polyethylene glycol (PEG) precipitation, adsorption-extraction, ultracentrifugation and ultrafiltration, to our knowledge, none of these methods have been standardized for use with a variety of wastewater matrices and/or different kits for RNA extraction and quantification. To address this, wastewater with different physical characteristics was seeded with gamma-irradiated SARS-CoV-2 and used to test the efficiency of PEG precipitation and adsorption-extraction to concentrate the virus from three physiochemically different wastewater samples, sourced from three distinct wastewater plants. Efficiency of viral concentration and RNA extraction was assessed by reverse-transcriptase polymerase chain reaction and the recovery yields calculated. As co-purification of inhibitors can be problematic for subsequent detection, two commonly used commercial master mixes were assessed for their sensitivity and efficiency to detect two SARS-CoV-2 target nucleocapsid (N) gene sequences. Recovery rates varied greatly between wastewater matrices and concentration methods, with the highest and most reproducible recovery rates (46.6-56.7%) observed when SARS-CoV-2 was precipitated with PEG and detected by the Luna® Universal master mix. The adsorption-extraction method was less effective (0-21.7%). This study demonstrates that PEG precipitation is the more robust method, which translates well to varying wastewater matrices, producing consistent and reproducible recovery rates. Furthermore, it is compatible with different kits for RNA extraction and quantitation., Competing Interests: Declaration of competing interest The authors declare not conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 (r 2 ) 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., Competing Interests: Declaration of competing interest The authorshave no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript, (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Owing to the risks and dangers of xenobiotic cannabinoids (phyto and synthetic), studies are required to evaluate community consumption. The analysis of excreted cannabinoids in wastewater can provide information about community consumption for a defined catchment area. The recovery of cannabinoids from complex wastewater matrices is difficult due to the hydrophobic properties of these compounds. In this study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was optimised for the recovery of 30 cannabinoids from wastewater, including the cannabis urinary biomarker 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol (THC-COOH), cannabidiol (CBD), and a variety of different generational synthetic cannabinoids and metabolites. Method validation assessed criteria including linearity, selectivity, recovery, ion-suppression, filtration losses and matrix effect. Two sample preparation approaches-liquid-liquid extraction (LLE) and solid phase extraction (SPE)-were compared, with comparable limits of quantification between 0.001 and 0.5 µg L -1 in wastewater. Filtration was found to reduce the recovery for many of the investigated cannabinoids, where up to 97% of analyte was lost. The method was applied to 15 different catchment areas across Australia to gauge the community use of the cannabinoids in this study. The cannabis biomarker THC-COOH was quantified at all locations, and cannabidiol was measured at eight catchment areas. Three synthetic cannabinoids were detected at the limit of detection: 5-fluoro APINACA, JWH-073 (4-hydroxypentyl), and MDMB-CHMICA., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology is a growing research field which provides valuable information on community drug use and chemical exposure. One parameter critical to estimations of drug use is the catchment area population. A population biomarker could be used to provide this information. This study evaluated the analytical suitability of three endogenous biomarkers of human activity: the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) which has previously been proposed and two further candidates, the catecholamine metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA). An analytical method involving derivatization was developed and validated for two candidates, 5-HIAA and HVA by liquid chromatography - mass spectrometry. The best performance was obtained for VMA as the underivatized analyte. The derivatized extracts produced a 100 times better sensitivity. The three neurotransmitter metabolites were evaluated as population biomarkers in wastewater samples. All were stable in sample, not lost upon filtration and showed stable inter-day mass loads over seven days for a metropolitan wastewater treatment plant. When applied to a small community during a festival period, mass loads of both HVA and VMA reflected the increase in the catchment population, whilst 5-HIAA proved to be more variable., Competing Interests: Declaration of Competing Interest All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript, (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human consumption of illicit novel psychoactive substances (NPS) is a continuing problem. New derivatives are constantly appearing, circumventing national and international laws. The use of these compounds tend to be sporadic and many are consumed as mixtures, meaning very low amounts of each are detectable at any one time. The analysis of excreted NPS in wastewater provides information on community prevalence. A wastewater-based epidemiology approach has been applied in the current study for the quantification of 21 NPS. These include three phenethylamines (25B-NBOMe, 25C-NBOMe, 25I-NBOMe), ten synthetic cathinones (3-ethylmethcathinone (3-EMC), 3-methylbuphedrone, 3-methylmethcathinone (3-MMC), 4-fluoromethcathinone (4-FMC), 4-methylbuphedrone, 4-methylethcathinone (4-MEC), buphedrone, butylone, N-ethylpentylone and pentylone), five synthetic opioid analgesics (AH-7921, butyryl fentanyl, furanyl fentanyl, U-47700 and valeryl fentanyl) as well as the synthetic amphetamine 4-fluoroamphemtaine (4-FA), ketamine analogue methoxetamine and methiopropamine. Limits of detection were between 0.01 and 0.5 ng/L and limits of quantification were between 0.05 and 1 ng/L. The method was applied to wastewater samples from South Australia collected over the Christmas-New Year period when recreational drug use tends to be high. Seven NPS (butylone, butyryl fentanyl, furanyl fentanyl, methoxetamine, N-ethylpentylone, pentylone and valeryl fentanyl) were found, with N-ethylpentylone showing the highest mass loads at 36 mg/day/1000 inhabitants., (Copyright © 2019 Elsevier B.V. All rights reserved.)