There is a growing need for efficient methods to quantify a wide range of psychoactive pharmaceuticals in wastewater and to comprehensively evaluate their in-sample stability for both population-level drug use monitoring and environmental risk assessment. Traditional analytical approaches using solid-phase extraction (SPE) incur high time and cost burdens and generally lack systematic stability assessment, hindering research progress in high-throughput monitoring. In this study, a cost-effective and sensitive method using direct injection LC-MS/MS was developed for the simultaneous quantification of 30 psychoactive pharmaceutical biomarkers, with a comprehensive assessment of their in-sample stability under six storage conditions across multiple freezethaw cycles. The method met all required performance criteria and achieved quantification limits of 0.2-100 ng/ L, comparable to or lower than SPE methods, while saving 5.5 h and 15 Australian dollars per sample processing. This method also reduced solvent and material consumption, contributing to improved analytical sustainability. In our stability assessment, acidification of samples reduced in-sample transformation for most target biomarkers, with <20 % degradation over 28 days at 22 degrees C, even after multiple freeze-thaw cycles. However, certain benzodiazepines exhibited higher stability in untreated wastewater. The method was applied to 68 influent wastewater samples collected from ten wastewater treatment plants from Australia in 2021. Nineteen chemicals from five pharmacological classes were detected at concentration ranging from 1.0 to 7900 ng/L. This highthroughput, cost-effective, and environmentally sustainable method can thus be applied for large-scale wastewater-based epidemiology and long-term monitoring of psychoactive pharmaceutical use.

Melamine is a versatile compound with various industrial and commercial applications, and the long-term use has resulted in its widespread occurrence in the environment. Furthermore, illicit addition of melamine into food products has raised interest in this compound. The aim of this study was to provide the first assessment of exposure to melamine and its derivatives in the Australian population. De-identified urine specimens collected in 2012-2013 and 2022-2023 were obtained from a community-based pathology laboratory and were pooled based on sex and age groups (n = 48 pools of 100, to a total of 4800 individual samples). Melamine, cyanuric acid (CYA), and ammelide were frequently detected in the pooled urine samples, with mean concentrations of 4.3, 94, and 4.2 ng/mL, respectively. A U-shaped trend in urinary concentrations of melamine was identified as age increased. Children and adolescents (< 15 years old) represented key groups for high exposure to CYA, whereas older individuals (≥45 years old) had higher exposure risk to ammelide. No temporal or sex differences was observed in urinary concentrations of melamine and its derivatives. Compared to other countries, high concentrations of CYA were observed in samples collected from the Australian population. From the estimated CYA daily intakes, 21 % of urine pools exceeded reference doses (2.5 μg/kg bw/day). To the best of our knowledge, this is the first study to reveal continued exposure of melamine and its derivatives in the Australian population and underscore the age trends of their urinary concentrations.

Benzotriazoles (BZTs) and benzothiazoles (BTs) are high-production-volume chemicals utilized in many different commercial products and industrial processes, such as metal corrosion inhibitors, vulcanization accelerators, plastic-associated UV stabilizers, and pharmaceutical precursors. This study assessed age, gender, and temporal trends of BZTs and BTs in deidentified surplus pathology urine samples, pooled and stratified by age, gender, and sample collection year from a general Australian population (168 pools representing 16,800 individuals). Tolyltriazole (TTri), 5,6-dimethyl-1H-benzotriazole (DMBZT), 1,3-benzothiazole (BTH), 2-hydroxybenzothiazole (2-OH-BTH), and 2-aminobenzothiazole (2-amino-BTH) were detected in >50% of the pools. TTri was frequently detected in pooled samples representing <= 45-year-olds (both genders). Concentrations of DMBZT, BTH (females), 2-OH-BTH, and 2-amino-BTH (females) increased with age significantly, with adults (>15 years old) showing higher levels than children (<= 15 years old). Gender differences in DMBZT concentrations (females > males) were observed across all sampling years and only in some for TTri (males > females: >45 years old), BTH (females > males), and 2-amino-BTH (males > females). A temporal increase in BTH, 2-OH-BTH, and 2-amino-BTH levels within the studied period (2012-2023) has been observed. Our findings suggest ongoing exposure of the Australian general population to BZTs and BTs, highlighting age, gender, and temporal trends of these compounds as measured via their urinary concentrations.

The dynamic new psychoactive substances (NPS) market presents a great challenge for public health officers, law enforcement and analytical and forensic chemists. Wastewater analysis is a complementary tool in the ongoing surveillance of these compounds but the low doses, somewhat unknown metabolism and the different chemical classes have made analytical methods difficult to develop. The current study presents a direct injection method for the quantification of 32 NPS. These include a range of classes: phenethylamines (25C-NBOMe and 4-fluoroamphetamine), synthetic cathinones (3-methylmethcathinone, butylone, dibutylone, eutylone, methcathinone, N-ethylheptedrone, N-ethylhexedrone, N-ethylpentylone and pentylone), synthetic cannabinoid receptor agonists (5F-EMB-PICA, 5F-MDMB-PICA, 5F-MDMB-PINACA, AMB FUBINACA, MDMB-4en-PINACA, cumyl pegaclone and cumyl-5F-pegaclone), opioids (2-methyl AP-237, AP-238, brorphine, isotonitazene, metonitazene and protonitazene), benzodiazepines (clonazolam, etizolam, flualprazolam and flubromazolam), plant-based NPS (7-hydroxymitragynine and mitragynine) and dissociatives (2F-deschloroketamine, 2-oxo-PCE). The method was validated in terms of linearity, range, precision (interday and intraday), limit of detection and limit of quantification, while filtration losses and matrix effects were also examined. The method was applied to wastewater samples collected from New South Wales and Queensland over the 2021-22 New Year period, when recreational drug use was expected to increase. Three NPS were found: eutylone, clonazolam and etizolam, with eutylone having the highest mass loads.

Understanding the impacts of herbicides on soil microbial communities is important, as these organisms mediate a wide range of ecosystem services. Here, we investigated whether the diversity and function of soil microbial communities were significantly influenced by one-off applications of atrazine, diuron, fluazifop-P-butyl, haloxyfop-P-methyl and pendimethalin as pure compounds at their recommended doses over multiple time points (1, 3, 7, 14, 30 and 60 days). Phylogenetic marker gene sequencing revealed that none of the herbicides influenced the numbers of bacterial and archaeal taxa or the evenness of their abundances. Similarly, none of the herbicides influenced the composition of bacterial and archaeal communities, except for diuron, fluazifop-P-methyl and pendimethalin, which were associated with larger relative abundances of a small number of OTUs on day 30 only. Functionally, none of the herbicides significantly influenced fluorescein diacetate hydrolysis (FDA) and beta-glucosidase activities or the induced respiratory responses of soil microbial communities to a range of substrates. These data indicate that the active herbicide ingredients tested may have minimal non-target effects when applied once at their recommended dose. Given their frequent use, it is important to next consider whether these herbicides have more pronounced effects at higher doses and application frequencies.

Abstract The increasing global prevalence of gout and diabetes has led to a rise in the use of their respective medications, allopurinol and metformin. These are excreted via urine as oxypurinol and metformin and are discharged into wastewater and the environment. Current environmental monitoring of those two polar chemicals requires labour intensive and potentially inefficient sample pre‐treatments, such as using solid‐phase extraction or freeze‐drying. This study validated a sensitive and simple method using direct‐injection LC–MS/MS for the simultaneous measurement of oxypurinol and metformin in wastewater. The final method utilised a hydrophilic interaction liquid chromatography together with simple filtration through 0.2 μm regenerated cellulose filter followed by dilution in acetonitrile with a dilution factor of 10. The developed method was validated with the limit of quantifications (LOQ) of 0.11 and 0.34 μg/L for metformin and oxypurinol, respectively. The new method was applied to 42 influent wastewater samples and 6 effluent samples collected from 6 Australian wastewater treatment plants. Both compounds were detected well above the LOQ at concentrations 29–214 μg/L in influent and 2–53 μg/L in effluent for metformin, and 24–248 μg/L in influent and 4–81 μg/L in effluent for oxypurinol, demonstrating its high applicability.

Wastewater-based epidemiology (WBE) has been used to estimate tobacco use in the population. However, the increased use of nicotine replacement therapies and e-cigarettes contributes to the load of nicotine metabolites in wastewater, causing over-estimation of tobacco use if nicotine metabolites were used in WBE back-estimation. This study aims to develop a rapid method for determining the tobacco-specific biomarkers, anabasine and anatabine, in wastewater and to evaluate their in-sewer stability for better estimation of tobacco use by WBE. An enhanced direct injection LC-MS/MS was developed to quantify anabasine and anatabine as well as nicotine biomarkers (nicotine, cotinine and hydroxycotinine). The method was optimal when wastewater was filtered through 0.2 μm RC syringe filters and a pre-conditioned SPE cartridge (Oasis HLB 1 cc, 30 mg) before 50 μL was injected into the LC-MS/MS system. Limits of quantification varied between 2.7 and 54.9 ng/L with recoveries from 76% to 103% for all five compounds. In sewer reactors, anabasine and anatabine were less stable than cotinine and hydroxycotinine. They were more stable in the gravity sewer reactor with <20% loss in 12 h than in the rising main sewer reactor with ~30% loss in the same period. We then applied the new method to 42 daily wastewater influent samples collected from an Australian wastewater treatment plant. The five biomarkers were detected in all samples with concentrations ranging from 9.2 to 7430 ng/L. All five compounds were positively correlated with one another. Our results suggested a high throughput analytical method for feasible application in anabasine and anatabine as biomarkers of tobacco use in routine wastewater monitoring.

Anabolic-androgenic steroids are synthetic compounds prohibited due to their performance-enhancing characteristics. The use of these substances is known to cause health-related issues, which highlights the importance of being able to evaluate the scale of consumption by the general population. However, most available research on the analysis of anabolic steroids is focused on animals and athletes in connection with doping. The potential of wastewater-based epidemiology as an intelligence tool for the assessment of community level use of anabolic steroids is presented herein. A liquid chromatography tandem mass spectrometry method was developed for the analysis of 10 anabolic-androgenic steroids and 14 endogenous hormones in influent wastewater. The validated method was applied to sixteen 24-hour composite wastewater influent samples that were collected over a period of five years from two wastewater treatment plants in Queensland, Australia. Nine investigated compounds were found to be present at concentrations between 14 and 611 ng L-1 which translated into 3-104 mg excreted per 1000 individuals per day. It was concluded that the developed analytical method is suitable for the analysis of AAS in wastewater matrix. Additionally, both the inclusion of metabolites and further investigation into deconjugation by enzymatic hydrolysis would aid in understanding and evaluating community anabolic steroid use. For the first time, this study presents the application of wastewater-based epidemiology on anabolic-androgenic steroids in Australia.

Organophosphate pesticides (OPs) and pyrethroids (PYRs) are widely used for pest control. Exposure to these chemicals has been linked to various negative health outcomes, particularly for young children. Urinary metabolites are commonly used biomarkers of exposure for non-persistent chemicals such as OPs and PYRs. A method is established for quantitatively measuring these biomarkers in human urine samples. Non-specific OP metabolites dialkylphosphates (DAPs; 6 compounds) are analysed using a lyophilisation method coupled with a subsequent derivatisation procedure using pentafluorobenzyl bromide. Samples are analysed using a gas chromatography coupled to triple quadrupole mass spectrometer. Other chemicals (13 compounds) are analysed via solid phase extraction using hydrophilic-lipophilic balance cartridges after hydrolysed by glucuronidase. Target compounds are analysed using liquid chromatography coupled to tandem mass spectrometry. Synthetic urine is prepared and used as blank samples. Method detection limit (MDL) is calculated as average of the levels in blank samples plus three times the standard deviation and ranges from 0.0083 to 0.83 µg L-1 in urine depending on compounds. Quality control (QC) samples are prepared by fortifying the synthetic urine with target compounds. Accuracy of the method is evaluated as the ratio of calculated levels against expected levels and ranges from 0.68 – 1.4. Method precision is expressed as the relative standard deviation of calculated levels from replicated QC samples (n=15) and is generally lower than 20%. The method is then applied to the analysis of 20 pooled urine samples from general Australian population, which are stratified into 5 age strata (0-1, 1-2, 2-3, 3-4, and 4-5 years) and two gender strata (2×males and 2×females). The highest mean concentration is measured for 3,5,6-trichloro-2-pyridinol (TCPy, 12 µg L-1 urine). Significant trend of concentrations by age (P<0.05) is observed for various OP and PYR metabolites.

Perfluoroalkyl substances (PFASs) have been used in large quantities for a variety of applications in Australian industry and household products. Through the course of their everyday use, PFASs enter the wastewater stream however current treatment processes provide only partial removal of these chemicals from wastewater. The release of treated effluent and re-use of biosolids represents an important point source of PFASs into the Australian environment yet the scale of PFAS release from Australian WWTPs is unknown. For the first time, influent, effluent and biosolids samples from 14 WWTPs across Australia were assessed for 9 PFASs and the national loads of these PFASs released from WWTPs estimated. Sigma 9PFASs ranged from 0.98 to 440 ng/L (influent), 21-560 ng/L (effluent) and 5.2-150 ng/g (biosolids). National loads of PFOA and PFOS in effluent were estimated at 65 kg and 26 kg per annum respectively. In biosolids, annual loads were estimated at 2 kg and 8 kg respectively. The continued detection of PFOS over a decade after its phase out, the increasing use of PFOS alternatives together with their resistance to degradation processes suggests that PFASs will be a priority for regulators and waste management to prevent further contamination of Australia's water resources. (C) 2018 Elsevier Ltd. All rights reserved.

The toxicity of herbicide degradation (transformation) products is rarely taken into account, even though these are commonly detected in the marine environment, sometimes at concentrations higher than the parent compounds. Here we assessed the potential contribution of toxicity by transformation products of five photosystem II herbicides to coral symbionts (Symbiodinium sp.), the green algae Dunaliella sp., and prawn (Penaeus monodon) larvae. Concentration-dependent inhibition of photosynthetic efficiency (∆F/F m ′) was observed for all herbicides in both microalgal species. The toxicity of solutions of aged diuron solutions containing transformation products to Symbiodinium sp. and Dunaliella sp. was greater than could be explained by the concentrations of diuron measured, indicating transformation products contributed to the inhibition of ∆F/F m ′. However, the toxicity of aged atrazine, simazine, hexazinone, and ametryn solutions could be explained by the concentration of parent herbicide, indicating no contribution by transformation products. Prawn larval metamorphosis was not sensitive to the herbicides, but preliminary results indicated some toxicity of the transformation products of atrazine and diuron. Risk assessments should take into account the contribution of herbicide transformation products; however, further studies are clearly needed to test the toxicity of a far wider range of transformation products to a representative diversity of relevant taxa.

Since alcohol and tobacco consumption are among the leading causes of population health harm, it is very important to understand the consumption behaviour to develop effective harm reduction strategies. Wastewater-based epidemiology (WBE) is a potential tool for estimating their consumption, but there are several uncertainties that need to be determined, including the stability of biomarkers in the sewer. Utilizing a real rising main sewer, this study investigated the stability of alcohol and tobacco consumption biomarkers. Rhodamine and acesulfame were used as flow tracer and benchmarker to understand the transportation of wastewater in the sewer with a hydraulic retention time between 2.7 and 5.0 h. Ethyl sulphate (EtS) and ethyl glucuronide (EtG), two biomarkers of alcohol consumption, were found to have different in-sewer stability, with EtS much more stable than EtG. The degradation rate of EtS is approximately 8% per hour, while EtG has a half-life of 1.9 h. Formation of nicotine, cotinine and trans-3'-hydroxycotinine, three biomarkers for tobacco consumption, was observed during the experiment, probably due to deconjugation of their glucuronide chemicals. The deconjugation process has prevented the determination of actual stability of the three chemicals However, it is suggested that cotinine is relatively stable, while nicotine and trans-3'-hydroxycotinine degrade to a certain degree in the sewer system. According to our findings, the in-sewer degradation is more important during the interpretation of alcohol consumption estimation than for tobacco consumption estimation. (C) 2018 Elsevier Ltd. All rights reserved.

Wastewater samples were collected at the influent and effluent of two wastewater treatment plants (WWTPs) in Ho Chi Minh City, Vietnam and then pooled to daily samples over multiple days using 6 hourly grab samples. The aim was to provide a first assessment of the occurrence, consumption, removal and release of a range of organic chemicals including pharmaceuticals and personal care products (PPCPs), illicit drugs, an artificial sweetener, tobacco and its metabolites and alcohol biomarkers (referred to here as DPCPBs). Nineteen DPCPBs were detected via direct measurement of filtered wastewater on LC-MS/MS with a concentration range of 0.05-38μg/L. Caffeine and paracetamol were the most prominent compounds detected in the influent, while acesulfame was found at the highest concentration in the effluent of both WWTPs. Mean concentrations of metabolites of tobacco (nicotine: 7.6μg/L, cotinine: 1.4μg/L and hydroxycotinine: 1.7μg/L) and alcohol (ethyl sulphate: 3.3μg/L) were lower than those of European countries. Consumption rates based on daily mass loads and catchment population data obtained from the WWTPs were <10g/d/1000 pp for the majority of selected PPCPs, except for caffeine (300g/d/1000 pp) and paracetamol (320g/d/1000 pp). Consumption rates for codeine and methamphetamine were 0.05g/d/1000 pp and 0.17g/d/1000 pp, respectively. Consistently across the two WWTPs most of the chemicals (10) showed >80% apparent removal rate from the wastewater, three chemicals showed apparent removal efficiency of approximately 50%; and the removal efficiency could not be assessed for 5 compounds due to their low concentrations in the influent. Based on the fraction of treated and untreated wastewater (10:90) that is released into the receiving environment we estimated a total discharge of approximately 170kg per day of DPCPBs in Ho Chi Minh City.

A key uncertainty of wastewater-based epidemiology is the size of the population which contributed to a given wastewater sample. We previously developed and validated a Bayesian inference model to estimate population size based on 14 population markers which: (1) are easily measured and (2) have mass loads which correlate with population size. However, the potential uncertainty of the model prediction due to in-sewer degradation of these markers was not evaluated. In this study, we addressed this gap by testing their stability under sewer conditions and assessed whether degradation impacts the model estimates. Five markers, which formed the core of our model, were stable in the sewers while the others were not. Our evaluation showed that the presence of unstable population markers in the model did not decrease the precision of the population estimates providing that stable markers such as acesulfame remained in the model. However, to achieve the minimum uncertainty in population estimates, we propose that the core markers to be included in population models for other sites should meet two additional criteria: (3) negligible degradation in wastewater to ensure the stability of chemicals during collection; and (4) < 10% in-sewer degradation could occur during the mean residence time of the sewer network.

To simultaneously quantify and profile the complex mixture of short-, median-, and long-chain CPs (SCCPs, MCCPs, and LCCPs) in Australian sewage sludge, we applied and further validated a recently developed novel instrumental technique, using quadrupole time-of-flight high resolution mass spectrometry running in the negative atmospheric pressure chemical ionization mode (APCI-qTOF-HRMS). Without using an analytical column the cleaned extracts were directly injected into the qTOF-HRMS followed by quantification of the CPs by a mathematical algorithm. The recoveries of the four SCCP, MCCP and LCCP-spiked sewage sludge samples ranged from 86 to 123%. This APCI-qTOF-HRMS method is a fast and promising technique for routinely measuring SCCPs, MCCPs, and LCCPs in sewage sludge. Australian sewage sludge was dominated by MCCPs with concentrations ranging from 542 to 3645 ng/g dry weight (dw). Lower SCCPs concentrations (<57-1421 ng/g dw) were detected in the Australian sewage sludge, which were comparable with the LCCPs concentrations (116-960 ng/g dw). This is the first time that CPs were reported in Australian sewage sludge. The results of this study gives a first impression on the distribution of the SCCPs, MCCPs, and LCCPs in Australia wastewater treatment plants (WWTPs).

Leachate from 27 landfills was analysed for nine perfluoroalkyl substances (PFASs). Five PFASs were detected ubiquitously, with perfluorohexanoate (PFHxA) the predominant PFAS (mean 1700ng/L; range 73-25,000ng/L). Despite the complexity of landfill-specific factors, some general trends in PFAS concentrations were observed. Mean concentrations of eight PFASs were higher in operating landfills (or landfill cells) accepting primarily municipal waste, compared to closed municipal landfills. Landfills accepting primarily construction and demolition wastes produced leachate that had higher mean PFAS concentrations than municipal landfills. Younger landfills appeared to have a higher burden of waste containing PFASs (or their precursors), as significant relationships (p<0.05) were observed between selected PFAS concentrations and landfill age. Increasing pH and total organic carbon (TOC) in leachate were associated with increased concentrations of several PFASs. Eight landfills discharged leachate to wastewater treatment plants (WWTPs). Estimated masses of PFASs discharged reached a maximum of 62g annually (PFHxA), with a national estimate reaching 31kg (PFHxA) annually. The practise of treating leachate at WWTPs allows redistribution of PFASs between the solid and liquid waste streams, although the contribution of leachate to the total load of PFASs entering WWTPs is minor compared to domestic waste water sources.