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Wastewater-based epidemiology (WBE) can provide objective and timely information on the use of new psychoactive substances (NPS), originally designed as legal alternatives of internationally controlled drugs. NPS have rapidly emerged on the global drug market, posing a challenge to drug policy and constituting a risk to public health. In this study, a WBE approach was applied to monitor the use of more than 300 NPS, together with fentanyl and its main metabolite norfentanyl, in influent wastewater collected from 12 European cities during March-June 2021. Quantitative and qualitative analysis of NPS in composite 24 h influent wastewater samples were based on solid phase extraction and liquid chromatography-mass spectrometry. In-sample stability tests demonstrated the suitability of most investigated biomarkers, except for a few synthetic opioids, synthetic cannabinoids and phenetylamines. Fentanyl, norfentanyl and eight NPS were quantified in influent wastewater and at least three substances were found in each city, demonstrating their use in Europe. N,N-dimethyltryptamine and 3-methylmethcathinone (3-MMC) were the most common NPS found, with the latter having the highest mass loads (up to 24.8 mg/day/1000 inhabitants). Seven additional substances, belonging to five categories of NPS, were identified in different cities. Spatial trends of NPS use were observed between cities and countries, and a changing weekly profile of use was observed for 3-MMC. WBE is a useful tool to rapidly evaluate emerging trends of NPS use, complementing common indicators (i.e. population surveys, seizures) and helping to establish measures for public health protection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

New psychoactive substances (NPS) emerged in the mid-2000s as a legal alternative to established illicit drugs. Despite the high individual and public harm associated to NPS, little is known about their real extent of use. New strategies are required to deal with the challenging monitoring of NPS, affected by the high number of substances available in the market, their rapid change and level of innovation, and their easy distribution mainly through the web. In this study, a wastewater-based epidemiology (WBE) approach was applied for a nationwide monitoring of the use of eight categories of NPS in the population, including fentanyl analogues. Sixty-two biomarkers of NPS were selected following an established criterion, that included the most frequently and recently reported. A selective analytical method based on solid-phase extraction and liquid chromatography-tandem mass spectrometry was developed and validated for NPS analysis in wastewater. Composite wastewater samples (24 h) were collected in 33 Italian cities in October-November 2020 and analyzed according the validated method. Results highlighted the use of ten NPS, mainly synthetic cathinones and tryptamines, all over Italy. Methcathinone was found in all the cities and the highest mass loads corresponded to 3-methylmethcathinone with values up to 3.8 mg/day/1000 inhabitants. Low levels of fentanyl (found in 9 cities) and its main metabolite norfentanyl (11) were found whereas no fentanyl analogues were identified. As far as we know, this is the first time that the use of fentanyl and its analogues was investigated in Italy by wastewater analysis. WBE is a useful tool to rapidly evaluate emerging trends of NPS use, complementing common indicators (i.e. population surveys, seizures) and helping to establish measures for public health protection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Wastewater analysis of Δ 9 -tetrahydrocannabinol (THC) biomarkers can provide essential information on trends in cannabis consumption. Although analysis is mostly focused on the aqueous phase, previous studies have illustrated the need of improving the measurements of raw influent wastewater (IWW) considering also suspended solids. This is important for cannabis biomarkers, because a substantial part of them is expected to be found in the suspended solids due to their more lipophilic character compared with other metabolites/drugs included in these types of studies. However, it remains open to which extent trend estimates might be affected by solely analysing the liquid phase. To investigate this aspect, robust analytical methodologies are required to measure both the liquid and solid phases of IWW. In this work, we firstly tested liquid-liquid extraction (LLE) for THC and its major metabolites (THCOH, and THCCOOH). Using LLE, no filtration or centrifugation step was required for raw IWW analysis, and the three analytes were extracted from both the liquid and the solid phase simultaneously. In parallel, the raw IWW was centrifuged and the obtained solid and liquid phases were analyzed separately: the liquid phase by both LLE and solid phase extraction (SPE) for comparison of data, and the suspended solids by solid-liquid extraction (SLE). The separate analysis of both phases in a number of samples revealed that a significant amount of cannabis biomarkers (ranging from 42 to 90%) was found in the suspended solids. In addition, the total amount of cannabis biomarkers obtained by analysing raw IWW on the one hand, and by separate analysis of the liquid and the solid phases, on the other hand, was in good agreement. Data from this study show that the sole analysis of the liquid phase would lead to a notable underestimation of cannabis biomarkers concentrations in IWW., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Wastewater-based viral surveillance was proposed as a promising approach to monitor the circulation of SARS-CoV-2 in the general population. The aim of this study was to develop an analytical method to detect SARS-CoV-2 RNA in urban wastewater, and apply it to follow the trends of epidemic in the framework of a surveillance network in the Lombardy region (Northern Italy). This area was the first hotspot of COVID-19 in Europe and was severely affected. Composite 24 h samples were collected weekly in eight cities from end-March to mid-June 2020 (first peak of the pandemic). The method developed and optimized, involved virus concentration using PEG centrifugation, and one-step real-time RT-PCR for analysis. SARS-CoV-2 RNA was identified in 65 (61%) out of 107 samples, and the viral concentrations (up to 2.1 E + 05 copies/L) were highest in March-April. By mid-June, wastewater samples tested negative in all the cities corresponding to the very low number of cases recorded in the same period. Viral loads were calculated considering the wastewater daily flow rate and the population served by each wastewater treatment plant, and were used for inter- city comparison. The highest viral loads were found in Brembate, Ranica and Lodi corresponding to the hotspots of the first peak of pandemic. The pattern of decrease of SARS-CoV-2 in wastewater was closely comparable to the decline of active COVID-19 cases in the population, reflecting the effect of lock-down. This study tested wastewater surveillance of SARS-CoV-2 to follow the pandemic trends in one of most affected areas worldwide, demonstrating that it can integrate ongoing virological surveillance of COVID-19, providing information from both symptomatic and asymptomatic individuals, and monitoring the effect of health interventions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sub-optimal adherence to pharmacological therapy is one of the main reasons for poor effectiveness, reducing the patient's quality of life and affecting health-care economics. This study investigated the possibility for a wastewater-based epidemiology approach to assess the overall adherence of a population to some pharmacological therapies in a defined area. We selected specific active ingredients and their main urinary metabolites (biomarkers) according to the best practice protocol available and we measured them for the first time in urban wastewater. We conducted this first case study in Italy, considering the whole country and two Italian regions by sampling for five days six cities from north to south. Liquid chromatography tandem mass spectrometry was used to measure the biomarkers in raw wastewater, developing and validating specific analytical methods. We used concentrations measured in influent wastewater, together with a careful review of the excretion profile of the active ingredients selected, to back-calculate consumption (measured consumptions - MC) which were then compared with consumptions calculated from medical prescriptions (expected consumptions - EC) reported annually by the Italian Medicine Agency. In general there was a rough correlation between MC and EC, with ratios sometimes close to unity and always within a 0.3-3.0 range. Interpretation of the results suffers some biases, which are case-by-case discussed in detail, but inclusion of measurements of the urinary metabolites together with the active ingredients gave a fuller understanding of the results., Competing Interests: Declaration of competing interest The authors declare they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Consumption of alcohol and new psychoactive substances (NPS) in a population or during special events (music festivals) is usually monitored through individual questionnaires, forensic and toxicological data, and drug seizures. However, consumption estimates have some biases due mostly to the unknown composition of drug pills for NPS and stockpiling for alcohol. The aim of this study was to evaluate for the first time the real use of alcohol and the occurrence of NPS in Slovakia by wastewater-based epidemiology (WBE). Urban wastewater samples were collected from nine Slovak cities over two years (2017-2018) and during three music festivals. The study included about 20% of the Slovak population and 50 000 festival attendees. The urinary alcohol biomarker ethyl sulfate (EtS) and thirty NPS were analyzed by liquid chromatography tandem mass spectrometry (LC - MS/MS). EtS concentrations were used for estimating the per capita alcohol consumption in each city. The average alcohol consumption in the selected cities and festivals in 2017-2018 ranged between 7 and 126 L/day/1000 inhabitants and increased during the weekends and music festivals. Five NPS belonging to the classes of synthetic cathinones (mephedrone, methcathinone, buphedrone and pentedrone) and phenethylamines (25-iP-NBoMe) were found in the low ng/L range. Methcathinone was the most frequently detected NPS, while the highest normalized mass load corresponded to mephedrone (3.1 mg/day/1000 inhabitants). Wastewater-based epidemiology can provide timely information on alcohol consumption and NPS occurrence at the community level that is complementary to epidemiology-based monitoring techniques (e.g. population surveys, police seizures, sales statistics)., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Wastewater-based epidemiology (WBE) was applied for the first time in seven cities across Europe with the aim of estimating quinolones consumption via the analysis of human urinary metabolites in wastewater. This report is also the first pan-European study focussed on the enantiomeric profiling of chiral quinolones in wastewater. By considering loads of (fluoro)quinolones in wastewater within the context of human stereoselective metabolism, we identified cities in Southern Europe characterised by both high usage and direct disposal of unused ofloxacin. In Northern European cities, S-(-)-ofloxacin loads were predominant with respect to R-(+)-ofloxacin. Much more potent, enantiomerically pure S-(-)-ofloxacin was detected in wastewaters from Southern European cities, reflecting consumption of the enantiomerically pure antibiotic. Nalidixic acid, norfloxacin and lomefloxacin were detected in wastewater even though they were not prescribed according to official prescription data. S,S-(-)-moxifloxacin and S,S-(-)-moxifloxacin-N-sulphate were detected in wastewater due to metabolism of moxifloxacin. For the first time, average population-normalised ulifloxacin loads of 22.3 and 1.5 mg day -1 1000 people -1 were reported for Milan and Castellón as a result of prulifloxacin metabolism. Enrichment of flumequine with first-eluting enantiomer in all the samples indicated animal metabolism rather than its direct disposal. Fluoroquinolone loads were compared with qnrS gene encoding quinolone resistance to correlate usage of fluoroquinolone and prevalence of resistance. The highest daily loads of the qnrS gene in Milan corresponded with the highest total quinolone load in Milan proving the hypothesis that higher usage of quinolones is linked with higher prevalence of quinolone resistance genes. Utrecht, with the lowest quinolones usage (low daily loads) had also one of the lowest daily loads of the qnrS gene. However, a similar trend was not observed in Oslo nor Bristol where higher qnrS gene loads were observed despite low quinolone usage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mycotoxins are among the compounds of most concern for human health because of their common presence in food and their proven toxicity for human health. Human biomonitoring (HBM) studies, foodstuff analysis and dietary surveys are usually used to assess human exposure, but they are costly and time-consuming. Wastewater-based epidemiology (WBE) can complement the established exposure monitoring tools. The aim of this study was to develop a new WBE application for assessing human exposure to mycotoxins by measuring specific biomarkers in raw wastewater. Eleven substances were analysed by liquid chromatography-tandem mass spectrometry in wastewater from four cities in Italy and Spain. Only deoxynivalenol (DON) and fumonisines were found, DON being the most abundant, in accordance with HBM studies. DON intake, back-calculated by WBE, was lower than the established Health-Based Guidance values, indicating moderate exposure in the population, with no risks for human health. WBE intake estimates were very close to those reported in HBM studies, validating WBE for estimating DON intake in the population. This study describes a promising WBE approach to complement HBM studies and assess the human intake of specific classes of mycotoxins, thus helping to identify risks for human health., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study used wastewater-based epidemiology (WBE) to investigate the lifestyle of the inhabitants of Malé, the capital of the Republic of Maldives. Raw wastewater 12-h composite samples were collected from nine pumping stations serving the city area - thus representative of the whole Malé population. Samples were analysed by liquid chromatography coupled to mass spectrometry for estimating the profile of use of a large number of substances including illicit drugs, alcohol, caffeine, tobacco and pharmaceuticals. The illicit drugs most used were cannabis (THC) and heroin (700 and 18 g/day), with lower consumption of cocaine and amphetamines (0.1-1.2 g/day). It is important to note that the consumption of cannabis in Malé was comparable to that measured in other countries, while the consumption of heroin was higher. Among cathinones, mephedrone was detected at the highest levels similar to other countries. Consumption of alcohol, which is not allowed in Maldives, was found (1.3 L/day/1000 inhabitants), but at a low level compared with other countries (6-44 L/day/1000 inhabitants), while the consumption of caffeine and tobacco was generally in line with reports from other countries. Unique information on pharmaceuticals use was also provided, since no official data were available. Human lifestyle was evaluated by applying for the first time the full set of WBE methodologies available in our laboratory. Results provided valuable epidemiological information, which may be useful for national and international agencies to understand population lifestyles better, including illicit drug issues, and for planning and evaluation of drug prevention programs in Malé., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background and Aims: Wastewater-based epidemiology is an additional indicator of drug use that is gaining reliability to complement the current established panel of indicators. The aims of this study were to: (i) assess spatial and temporal trends of population-normalized mass loads of benzoylecgonine, amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) in raw wastewater over 7 years (2011-17); (ii) address overall drug use by estimating the average number of combined doses consumed per day in each city; and (iii) compare these with existing prevalence and seizure data., Design: Analysis of daily raw wastewater composite samples collected over 1 week per year from 2011 to 2017., Setting and Participants: Catchment areas of 143 wastewater treatment plants in 120 cities in 37 countries., Measurements: Parent substances (amphetamine, methamphetamine and MDMA) and the metabolites of cocaine (benzoylecgonine) and of Δ 9 -tetrahydrocannabinol (11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol) were measured in wastewater using liquid chromatography-tandem mass spectrometry. Daily mass loads (mg/day) were normalized to catchment population (mg/1000 people/day) and converted to the number of combined doses consumed per day. Spatial differences were assessed world-wide, and temporal trends were discerned at European level by comparing 2011-13 drug loads versus 2014-17 loads., Findings: Benzoylecgonine was the stimulant metabolite detected at higher loads in southern and western Europe, and amphetamine, MDMA and methamphetamine in East and North-Central Europe. In other continents, methamphetamine showed the highest levels in the United States and Australia and benzoylecgonine in South America. During the reporting period, benzoylecgonine loads increased in general across Europe, amphetamine and methamphetamine levels fluctuated and MDMA underwent an intermittent upsurge., Conclusions: The analysis of wastewater to quantify drug loads provides near real-time drug use estimates that globally correspond to prevalence and seizure data., (© 2019 The Authors. Addiction published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

The number of new psychoactive substances (NPS) on the recreational drug market has increased rapidly in the last years, creating serious challenges for public health agencies and law enforcement authorities. Epidemiological surveys and forensic analyses to monitor the consumption of these substances face some limitations for investigating their use on a large scale in a shifting market. The aim of this work was to develop a comprehensive and flexible screening approach for assessing the presence of NPS in urban wastewater by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Almost 200 substances were selected as "priority NPS" among those most frequently and recently reported by the Early Warning Systems (EWS) of different agencies and were included in the screening. Wastewater samples were collected from several cities all over Europe in 2016 and 2017, extracted using different solid-phase cartridges and analysed by LC-HRMS. The screening workflow comprised three successive analytical steps and compounds were identified and confirmed following specific criteria from the current guidelines. Thirteen NPS were identified at different confidence levels by using analytical standards or information from libraries and literature, and about half of them were phenethylamines. As far as we know, this is the first time that four of them (i.e. 3,4-dimethoxy-α-pyrrolidinovalerophenone, para-methoxyamphetamine, 2-phenethylamine and α-methyltryptamine) have been found in urban wastewater. The proposed screening approach was successfully applied in the largest NPS European wastewater monitoring, providing an innovative and easily adapted procedure for investigating NPS. In the light of current challenges and specific future research issues, this approach may complement epidemiological information and help in establishing measures for public health protection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology (WBE) can give valuable light on the extent and actual use of new psychoactive substances (NPS). In this work, a fully validated methodology for the simultaneous determination of illicit drugs and NPS in wastewater by solid-phase extraction followed by UHPLC-MS/MS has been developed. The small sample volume (5 mL) required for analysis is of high interest, especially when performing large sampling campaigns involving many locations of different geographical origin, as it has been performed in the past. The method was applied to wastewater samples from different European locations and permitted the simultaneous monitoring of conventional drugs and NPS. Cocaine, amphetamine, MDMA, methamphetamine and ketamine were found in all wastewater samples, and several NPS (dipentylone, butylone, mephedrone, methedrone and methylone) were observed in some of the samples monitored. It is noteworthy that dipentylone was detected in wastewater for the very first time. Furthermore, a detailed comparison of micro liquid chromatography (μLC) and UHPLC, both coupled to tandem mass spectrometry, in terms of sensitivity and reproducibility has been made for the first time in the application field of WBE. An average increase factor of 14 (mass normalized data) was observed in sensitivity for μLC-MS/MS. The overall method performance was also compared (un-normalized data), and an average increase sensitivity factor of 4.5 was observed for μLC-MS/MS. However, large deviations in retention time (up to 0.4 min) affected the reproducibility and robustness of the μLC-MS/MS method when it was applied to wastewater analysis. Although in this work μLC-MS/MS was strongly influenced by the amount of matrix loaded in the separation device, its enhanced sensitivity and promotion of green chemistry (faster analysis time and less solvent consumption) allow to expect improved future applications, especially when analytes are present at very low concentrations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology is an innovative approach to estimate the consumption of chemicals and their exposure patterns in a population, on the basis of measurements of biomarkers in wastewater. This method can provide objective real-time information on xenobiotics directly or indirectly ingested by a population. This approach was used to examine the exposure of the Martinique population to the three classes of pesticides: triazines, organophosphates and pyrethroids. Martinique island (French West Indies) is a closed market and has been closely monitored since the early 2000's when contamination with chlordecone, an organochlorine insecticide widely applied between 1972 and 1993 in banana plantations, became a critical political issue. The aim of this study was to identify and quantify the patterns of human exposure and compare the results to those from other countries. Wastewater was collected as 24-h composite samples and analysed for selected urinary pesticide metabolites by liquid chromatography-tandem mass spectrometry. Organophosphate and pyrethroid metabolites were found in all the samples up to 330 ng/L, while triazines were found only at trace levels. Mass loads indicated higher exposure to pyrethroids than in some cities in Europe, but lower exposure to triazines and organophosphates. The estimated human intake for pyrethroids was close to the Acceptable Daily Intake, but importation of these pesticides to Martinique was low. This study illustrates the high human exposure with indoor pesticide use in comparison to its use in agriculture., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this work a step forward in investigating the use of prescription drugs, namely erectile dysfunction products, at European level was taken by applying the wastewater-based epidemiology approach. 24-h composite samples of untreated wastewater were collected at the entrance of eight wastewater treatment plants serving the catchment within the cities of Bristol, Brussels, Castellón, Copenhagen, Milan, Oslo, Utrecht and Zurich. A validated analytical procedure with direct injection of filtered aliquots by liquid chromatography-tandem mass spectrometry was applied. The target list included the three active pharmaceutical ingredients (sildenafil, tadalafil and vardenafil) together with (bio)transformation products and other analogues. Only sildenafil and its two human urinary metabolites desmethyl- and desethylsildenafil were detected in the samples with concentrations reaching 60 ng L -1 . The concentrations were transformed into normalized measured loads and the estimated actual consumption of sildenafil was back-calculated from these loads. In addition, national prescription data from five countries was gathered in the form of the number of prescribed daily doses and transformed into predicted loads for comparison. This comparison resulted in the evidence of a different spatial trend across Europe. In Utrecht and Brussels, prescription data could only partly explain the total amount found in wastewater; whereas in Bristol, the comparison was in agreement; and in Milan and Oslo a lower amount was found in wastewater than expected from the prescription data. This study illustrates the potential of wastewater-based epidemiology to investigate the use of counterfeit medication and rogue online pharmacy sales., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The analysis of illicit drugs in urban wastewater is the basis of wastewater-based epidemiology (WBE), and has received much scientific attention because the concentrations measured can be used as a new non-intrusive tool to provide evidence-based and real-time estimates of community-wide drug consumption. Moreover, WBE allows monitoring patterns and spatial and temporal trends of drug use. Although information and expertise from other disciplines is required to refine and effectively apply WBE, analytical chemistry is the fundamental driver in this field. The use of advanced analytical techniques, commonly based on combined chromatography-mass spectrometry, is mandatory because the very low analyte concentration and the complexity of samples (raw wastewater) make quantification and identification/confirmation of illicit drug biomarkers (IDBs) troublesome. We review the most-recent literature available (mostly from the last 5 years) on the determination of IDBs in wastewater with particular emphasis on the different analytical strategies applied. The predominance of liquid chromatography coupled to tandem mass spectrometry to quantify target IDBs and the essence to produce reliable and comparable results is illustrated. Accordingly, the importance to perform inter-laboratory exercises and the need to analyze appropriate quality controls in each sample sequence is highlighted. Other crucial steps in WBE, such as sample collection and sample pre-treatment, are briefly and carefully discussed. The article further focuses on the potential of high-resolution mass spectrometry. Different approaches for target and non-target analysis are discussed, and the interest to perform experiments under laboratory-controlled conditions, as a complementary tool to investigate related compounds (e.g., minor metabolites and/or transformation products in wastewater) is treated. The article ends up with the trends and future perspectives in this field from the authors' point of view. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:258-280, 2018., (© 2016 Wiley Periodicals, Inc.)

The aim of this paper is to present the first study on spatial and temporal variation in the enantiomeric profile of chiral drugs in eight European cities. Wastewater-based epidemiology (WBE) and enantioselective analysis were combined to evaluate trends in illicit drug use in the context of their consumption vs direct disposal as well as their synthetic production routes. Spatial variations in amphetamine loads were observed with higher use in Northern European cities. Enantioselective analysis showed a general enrichment of amphetamine with the R-(-)-enantiomer in wastewater indicating its abuse. High loads of racemic methamphetamine were detected in Oslo (EF = 0.49 ± 0.02). This is in contrast to other European cities where S-(+)-methamphetamine was the predominant enantiomer. This indicates different methods of methamphetamine synthesis and/or trafficking routes in Oslo, compared with the other cities tested. An enrichment of MDMA with the R-(-)-enantiomer was observed in European wastewaters indicating MDMA consumption rather than disposal of unused drug. MDA's chiral signature indicated its enrichment with the S-(+)-enantiomer, which confirms its origin from MDMA metabolism in humans. HMMA was also detected at quantifiable concentrations in wastewater and was found to be a suitable biomarker for MDMA consumption. Mephedrone was only detected in wastewater from the United Kingdom with population-normalised loads up to 47.7 mg 1000 people -1 day -1 . The enrichment of mephedrone in the R-(+)-enantiomer in wastewater suggests stereoselective metabolism in humans, hence consumption, rather than direct disposal of the drug. The investigation of drug precursors, such as ephedrine, showed that their presence was reasonably ascribed to their medical use., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Wastewater-based epidemiology (WBE) complements existing epidemiology-based estimation techniques and provides objective, evidence-based estimates of illicit drug use. After consumption, biomarkers - drugs and their metabolites - excreted to toilets and flushed into urban sewer networks can be measured in raw wastewater samples. The quantified loads can serve as an estimate for the collective consumption of all people contributing to the wastewater sample. This transdisciplinary approach, further explained in this chapter, has developed, matured and is now established for monitoring substances such as cocaine and amphetamine-type stimulants. Research currently underway is refining WBE to new applications including new psychoactive substances (NPS).

Human biomonitoring, i.e. the determination of chemicals and/or their metabolites in human specimens, is the most common and potent tool for assessing human exposure to pesticides, but it suffers from limitations such as high costs and biases in sampling. Wastewater-based epidemiology (WBE) is an innovative approach based on the chemical analysis of specific human metabolic excretion products (biomarkers) in wastewater, and provides objective and real-time information on xenobiotics directly or indirectly ingested by a population. This study applied the WBE approach for the first time to evaluate human exposure to pesticides in eight cities across Europe. 24 h-composite wastewater samples were collected from the main wastewater treatment plants and analyzed for urinary metabolites of three classes of pesticides, namely triazines, organophosphates and pyrethroids, by liquid chromatography-tandem mass spectrometry. The mass loads (mg/day/1000 inhabitants) were highest for organophosphates and lowest for triazines. Different patterns were observed among the cities and for the various classes of pesticides. Population weighted loads of specific biomarkers indicated higher exposure in Castellon, Milan, Copenhagen and Bristol for pyrethroids, and in Castellon, Bristol and Zurich for organophosphates. The lowest mass loads (mg/day/1000 inhabitants) were found in Utrecht and Oslo. These results were in agreement with several national statistics related to pesticides exposure such as pesticides sales. The daily intake of pyrethroids was estimated in each city and it was found to exceed the acceptable daily intake (ADI) only in one city (Castellon, Spain). This was the first large-scale application of WBE to monitor population exposure to pesticides. The results indicated that WBE can give new information about the "average exposure" of the population to pesticides, and is a useful complementary biomonitoring tool to study population-wide exposure to pesticides., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Analysis of student consumption of illicit drugs (ID) by school population surveys (SPS) provides information useful for prevention, but the results may be influenced by subjective factors. We explored wastewater (WW) analysis to improve the information., Methods: We used WW analysis to measure ID consumption in eight secondary schools in Italy in 2010-13 (students aged 15-19). Samples were collected from the sewage pipes of the schools during lessons for one week each year. Samples were analysed by mass spectrometry to measure ID and consumption by students was compared to that of the general population., Results: We found THCCOOH (human metabolite of THC) concentrations in 2010 indicating significant consumption of cannabis in all the schools and benzoylecgonine (human metabolite of cocaine) suggesting a limited consumption of cocaine in all but one school. Morphine was only found in traces, and amphetamine, methamphetamine, ecstasy, ketamine and mephedrone were not detectable. Repeated analysis showed cannabis stable until 2012 with increases in 2013, low cocaine and morphine levels, and none of the other ID., Discussion: WW analysis suggested that students used amounts of cannabis comparable to the general population, with low, sporadic use of cocaine and opioids, but excluded the use of significant amounts of amphetamine, methamphetamine, ecstasy, ketamine and mephedrone. WW analysis was useful to confirm SPS figures and provides complementary findings for effective prevention strategies. This is the first time WW analysis has been used to investigate consumption of a large number of ID and new psychoactive substances (NPS) in schools., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Assessing the presence of pesticides in environmental waters is particularly challenging because of the huge number of substances used which may end up in the environment. Furthermore, the occurrence of pesticide transformation products (TPs) and/or metabolites makes this task even harder. Most studies dealing with the determination of pesticides in water include only a small number of analytes and in many cases no TPs. The present study applied a screening method for the determination of a large number of pesticides and TPs in wastewater (WW) and surface water (SW) from Spain and Italy. Liquid chromatography coupled to high-resolution mass spectrometry (HRMS) was used to screen a database of 450 pesticides and TPs. Detection and identification were based on specific criteria, i.e. mass accuracy, fragmentation, and comparison of retention times when reference standards were available, or a retention time prediction model when standards were not available. Seventeen pesticides and TPs from different classes (fungicides, herbicides and insecticides) were found in WW in Italy and Spain, and twelve in SW. Generally, in both countries more compounds were detected in effluent WW than in influent WW, and in SW than WW. This might be due to the analytical sensitivity in the different matrices, but also to the presence of multiple sources of pollution. HRMS proved a good screening tool to determine a large number of substances in water and identify some priority compounds for further quantitative analysis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Consumption of 3,4-methylendioxymethamphetamine (MDMA) has been always estimated by measuring the parent substance through chemical analysis of wastewater. However, this may result in an overestimation of the use if the substance is directly disposed in sinks or toilets. Using specific urinary metabolites may overcome this limitation. This study investigated for the first time the suitability of a panel of MDMA metabolites as biomarkers of consumption, considering the specific criteria recently proposed, i.e. being detectable and stable in wastewater, being excreted in a known percentage in urine, and having human excretion as the sole source. A new analytical method was developed and validated for the extraction and analysis of MDMA and three of its main metabolites in wastewater. 24-h composite raw wastewater samples from three European cities were analysed and MDMA use was back-calculated. Results from single MDMA loads, 4-hydroxy-3-methoxymethamphetamine (HMMA) loads and from the sum of MDMA, HMMA and 4-hydroxy-3-methoxyamphetamine (HMA) loads were in line with the well-known recreational use of this drug: consumption was higher during the weekend in all cities. HMMA and HMA turned out to be suitable biomarkers of consumption; however, concentrations measured in wastewater did not resemble the expected pharmacokinetic profiles, quite likely due to the very limited information available on excretion profiles. Different options were tested to back-calculate MDMA use, including the sum of MDMA and its metabolites, to balance the biases associated with each single substance. Nevertheless, additional pharmacokinetic studies are urgently needed in order to get more accurate excretion rates and, therefore, improve the estimates of MDMA use., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pesticides are active substances with potentially adverse effects on human health, and therefore great effort is addressed to study the relation between their widespread use and the effects on humans. To track human exposure to pesticides, novel approaches are needed to give additional information on exposure at population level. In this study, a novel application of Wastewater-Based Epidemiology (WBE) was developed to measure the intake of pyrethroid pesticides in a population. Three human urinary metabolites of pyrethroids were selected and validated as biomarkers of exposure by evaluating their sources and stability in wastewater. They were measured by liquid chromatography-tandem mass spectrometry in raw urban wastewater collected from the wastewater treatment plants of six Italian cities. Their concentrations were used as biomarkers to back-calculate the intake of pyrethroid pesticides in the population. WBE results were in line with the urinary biomarker levels of biomonitoring studies considering dilution in wastewater. Significant differences in the metabolites levels were observed among different cities. Seasonal variations in human intake of pyrethroids were also seen, as expected, with higher intakes during spring/summer. Intakes in the six cities were compared with the acceptable daily intake (ADI) and it was concluded that some of the populations examined might face significant health risks. Results confirm that this method can provide supplementary information to biomonitoring studies and can be a valuable tool for obtaining objective, direct information on the real levels of exposure to pyrethroids of different populations., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The information obtained from the chemical analysis of specific human excretion products (biomarkers) in urban wastewater can be used to estimate the exposure or consumption of the population under investigation to a defined substance. A proper biomarker can provide relevant information about lifestyle habits, health and wellbeing, but its selection is not an easy task as it should fulfil several specific requirements in order to be successfully employed. This paper aims to summarize the current knowledge related to the most relevant biomarkers used so far. In addition, some potential wastewater biomarkers that could be used for future applications were evaluated. For this purpose, representative chemical classes have been chosen and grouped in four main categories: (i) those that provide estimates of lifestyle factors and substance use, (ii) those used to estimate the exposure to toxicants present in the environment and food, (iii) those that have the potential to provide information about public health and illness and (iv) those used to estimate the population size. To facilitate the evaluation of the eligibility of a compound as a biomarker, information, when available, on stability in urine and wastewater and pharmacokinetic data (i.e. metabolism and urinary excretion profile) has been reviewed. Finally, several needs and recommendations for future research are proposed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The popularity of new psychoactive substances (NPS) has grown in recent years, with certain NPS commonly and preferentially consumed even following the introduction of preventative legislation. With the objective to improve the knowledge on the use of NPS, a rapid and very sensitive method was developed for the determination of ten priority NPS (N-ethylcathinone, methylenedioxypyrovalerone (MDPV), methylone, butylone, methedrone, mephedrone, naphyrone, 25-C-NBOMe, 25-I-NBOMe and 25-B-NBOMe) in influent wastewater. Sample clean-up and pre-concentration was made by off-line solid phase extraction (SPE) with Oasis MCX cartridges. Isotopically labelled internal standards were used to correct for matrix effects and potential SPE losses. Following chromatographic separation on a C 18 column within 6 min, the compounds were measured by tandem mass spectrometry in positive ionization mode. The method was optimised and validated for all compounds. Limits of quantification were evaluated by spiking influent wastewater samples at 1 or 5 ng/L. An investigation into the stability of these compounds in influent wastewater was also performed, showing that, following acidification at pH 2, all compounds were relatively stable for up to 7 days. The method was then applied to influent wastewater samples from eight European countries, in which mephedrone, methylone and MDPV were detected. This work reveals that although NPS use is not as extensive as for classic illicit drugs, the application of a highly sensitive analytical procedure makes their detection in wastewater possible. The developed analytical methodology forms the basis of a subsequent model-based back-calculation of abuse rate in urban areas (i.e. wastewater-based epidemiology)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Wastewater analysis has been demonstrated to be a complementary approach for assessing the overall patterns of drug use by a population while the full potential of wastewater-based epidemiology has yet to be explored. F 2 -isoprostanes are a prototype wastewater biomarker to study the cumulative oxidative stress at a community level. In this work, 8-iso-prostaglandin F 2α (8-iso-PGF 2α ) was analysed in raw 24 h-composite wastewater samples collected from 4 Norwegian and 7 other European cities in 2014 and 2015. Using the same samples, biomarkers of alcohol (ethyl sulfate) and tobacco (trans-3'-hydroxycotinine) use were also analysed to investigate any possible correlation between 8-iso-PGF 2α and the consumption of the two drugs. The estimated per capita daily loads of 8-iso-PGF 2α in the 11 cities ranged between 2.5 and 9.9 mg/day/1000 inhabitants with a population-weighted mean of 4.8 mg/day/1000 inhabitants. There were no temporal trends observed in the levels of 8-iso-PGF 2α , however, spatial differences were found at the inter-city level correlating to the degree of urbanisation. The 8-iso-PGF 2α mass load was found to be strongly associated with that of trans-3'-hydroxycotinine while it showed no correlation with ethyl sulfate. The present study shows the potential for 8-iso-PGF 2α as a wastewater biomarker for the assessment of community public health.

The estimation of illicit drugs use through wastewater analysis has become an important issue in the last few years due to their large worldwide consumption, which results in economic, social and health costs. The amounts of urinary biomarkers of illicit drugs (selected drugs or their metabolites) measured in wastewater are used to back-calculate the consumption of a particular drug by the population and to monitor temporal and spatial trends of illicit drug use in a community. The reliability of back-calculation depends on different factors, one being the accuracy of correction factors. A wide range of correction factors have been used in different studies and some biases must be expected when comparing results. Most of the correction factors were developed several years ago, so they need to be updated to include the latest information on pharmacokinetics. Moreover, new comprehensive methods to treat data should be adopted. The goal of this study is to refine current correction factors for back-calculation of the most widely used illicit drugs: amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and tetrahydrocannabinol (THC). The mean percentages of excretion of the parent drugs and their metabolites were calculated for each route of administration, utilizing all accessible pharmacokinetic studies in the literature. This allowed to select the most suitable drug target residue and a refined correction factor was obtained for each substance considering the most frequent route of administration. The refined correction factors we propose can be used in wastewater based epidemiology to standardize the back-calculation of these drugs. These results can be included in the best practice protocol currently adopted in EU studies in order to reduce uncertainty and improve the comparability of results., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Biomonitoring studies have documented the high exposure of the population to pesticides which are widely used for crop protection, industrial and household purposes. This is the first study which has measured human urinary metabolites of pesticides in urban wastewater as biomarkers of population exposure. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to measure fifteen urinary metabolites selected from the major classes of pesticides. Raw wastewater samples were processed by solid phase extraction (SPE) or direct injection into the LC-MS/MS system. Recoveries ranged from 75 to 115% and the limits of quantification were 1-15ng/L for the SPE method and 40-800ng/L for direct injection. The method was employed for the analysis of 44 composite 24-h wastewater samples collected in seven Italian cities. Most of the target substances were detected at concentrations ranging from 1.1ng/L to 1.6μg/L. The highest concentrations were for some common metabolites of alkyl phosphates and pyrethroids and the specific metabolite of chlorpyrifos and chlorpyrifos-methyl (3,5,6-trichloro-2-pyridinol). The frequency of detection and abundance of most of the measured metabolites were in line with the profiles reported in urine biomonitoring studies. This method is therefore proposed as a novel "biomonitoring approach" for obtaining objective, direct information on the levels of exposure of a specific population to pesticides, and current research is addressed to validate the method identifying the most reliable biomarkers., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Monitoring the scale of pharmaceuticals, illicit and licit drugs consumption is important to assess the needs of law enforcement and public health, and provides more information about the different trends within different countries. Community drug use patterns are usually described by national surveys, sales and seizure data. Wastewater-based epidemiology (WBE) has been shown to be a reliable approach complementing such surveys., Method: This study aims to compare and correlate the consumption estimates of pharmaceuticals, illicit drugs, alcohol, nicotine and caffeine from wastewater analysis and other sources of information. Wastewater samples were collected in 2015 from 8 different European cities over a one week period, representing a population of approximately 5 million people. Published pharmaceutical sale, illicit drug seizure and alcohol, tobacco and caffeine use data were used for the comparison., Results: High agreement was found between wastewater and other data sources for pharmaceuticals and cocaine, whereas amphetamines, alcohol and caffeine showed a moderate correlation. methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) and nicotine did not correlate with other sources of data. Most of the poor correlations were explained as part of the uncertainties related with the use estimates and were improved with other complementary sources of data., Conclusions: This work confirms the promising future of WBE as a complementary approach to obtain a more accurate picture of substance use situation within different communities. Our findings suggest further improvements to reduce the uncertainties associated with both sources of information in order to make the data more comparable.

Asthma, one of the most common chronic diseases in the world and a leading cause of hospitalization among children, has been associated with outdoor air pollution. We applied the wastewater-based epidemiology (WBE) approach to study the association between the use of salbutamol, a short-acting beta-agonist used to treat acute bronchospasm, and air pollution in the population of Milan, Italy. Composite 24-h samples of untreated wastewater were collected daily and analyzed for human metabolic residues of salbutamol by liquid chromatography tandem mass spectrometry. Corresponding daily outdoor concentrations of particular matter up to 10µm (PM10) and 2.5µm (PM2.5) in aerodynamic diameter, nitrogen dioxide, ozone, sulfur dioxide, and benzene were collected from the public air monitoring network. Associations at different lag times (0-10 days) were assessed by a log-linear Poisson regression model. We found significant direct associations between defined daily doses (DDD) of salbutamol and mean daily concentrations of PM10 and PM2.5 up to nine days of lag time. The highest rate ratio, and 95% confidence interval (CI), of DDD of salbutamol was 1.06 (95% CI: 1.02-1.10) and 1.07 (95% CI: 1.02-1.12) at seven days of lag time and for an increase of 10 μg/m(3) of PM10 and PM2.5, respectively. Reducing the mean daily PM10 concentration in Milan from 50 to 30μg/m(3) means that 852 (95% CI: 483-1504) daily doses of salbutamol per day would not be used. These results confirm the association between asthma and outdoor PM10 and PM2.5 and prove the potential of the WBE approach to quantitatively estimate the relation between environmental exposures and diseases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Synthetic cathinones are among the most consumed new psychoactive substances (NPS), but their increasing number and interchangeable market make it difficult to estimate the real size of their consumption. Wastewater-based epidemiology (WBE) through the analysis of metabolic residues of these substances in urban wastewater can provide this information. This study applied WBE for the first time to investigate the presence of 17 synthetic cathinones in four European countries. A method based on solid-phase extraction and liquid chromatography coupled to tandem mass spectrometry was developed, validated, and used to quantify the target analytes. Seven substances were found, with mephedrone and methcathinone being the most frequently detected and none of the analytes being found in Norway. Population-normalized loads were used to evaluate the pattern of use, which indicated a higher consumption in the U.K., followed by Spain and Italy, in line with the European prevalence data from population surveys. In the U.K., where an entire week was investigated, an increase of the loads was found during the weekend, indicating a preferential use in recreational contexts. This study demonstrated that WBE can be a useful additional tool to monitor the use of NPS in a population.

Quantitative measurement of drug consumption biomarkers in wastewater can provide objective information on community drug use patterns and trends. This study presents the measurement of alcohol consumption in 20 cities across 11 countries through the use of wastewater-based epidemiology (WBE), and reports the application of these data for the risk assessment of alcohol on a population scale using the margin of exposure (MOE) approach. Raw 24-h composite wastewater samples were collected over a one-week period from 20 cities following a common protocol. For each sample a specific and stable alcohol consumption biomarker, ethyl sulfate (EtS) was determined by liquid chromatography coupled to tandem mass spectrometry. The EtS concentrations were used for estimation of per capita alcohol consumption in each city, which was further compared with international reports and applied for risk assessment by MOE. The average per capita consumption in 20 cities ranged between 6.4 and 44.3L/day/1000 inhabitants. An increase in alcohol consumption during the weekend occurred in all cities, however the level of this increase was found to differ. In contrast to conventional data (sales statistics and interviews), WBE revealed geographical differences in the level and pattern of actual alcohol consumption at an inter-city level. All the sampled cities were in the "high risk" category (MOE<10) and the average MOE for the whole population studied was 2.5. These results allowed direct comparisons of alcohol consumption levels, patterns and risks among the cities. This study shows that WBE can provide timely and complementary information on alcohol use and alcohol associated risks in terms of exposure at the community level., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Analytical methods used for pharmaceuticals and drugs of abuse in sewage play a fundamental role in wastewater-based epidemiology (WBE) studies. Here quantitative analysis of drug metabolites in raw wastewaters is used to determine consumption from general population. Its great advantage in public health studies is that it gives objective, real-time data about community use of chemicals, highlighting the relationship between environmental and human health. Within a WBE study on salbutamol use in a large population, we developed a procedure to distinguish human metabolic excretion from external source of contamination, possibly industrial, in wastewaters. Salbutamol is mainly excreted as the sulphate metabolite, which is rapidly hydrolyzed to the parent compound in the environment, so this is currently not detected. When a molecule is either excreted un-metabolized or its metabolites are unstable in the environment, studies can be completed by monitoring the parent compound. In this case it is mandatory to assess whether the drug in wastewater is present because of population use or because of a specific source of contamination, such as industrial manufacturing waste. Because commercial salbutamol mainly occurs as a racemic mixture and is stereoselective in the human metabolism, the enantiomeric relative fraction (EFrel) in wastewater samples should reflect excretion, being unbalanced towards one of two enantiomers, if the drug is of metabolic origin. The procedure described involves chiral analysis of the salbutamol enantiomers by liquid chromatography-tandem mass spectrometry (LC-MS-MS) and calculation of EFrel, to detect samples where external contamination occurs. Samples were collected daily between October and December 2013 from the Milano Nosedo wastewater treatment plant. Carbamazepine and atenolol were measured in the sewage collector, as "control" drugs. Salbutamol EFrel was highly consistent in all samples during this three-month period, but a limited number of samples had unexpectedly high concentrations where the EFrel was close to that observed of the un-metabolized, commercially available drug, supporting the idea of an external source of contamination, besides human metabolic excretion. Results showed that, when present, non-metabolic daily loads could be evaluated indicating an average of 4.12g/day of salbutamol extra load due to non-metabolic sources. The stereoselectivity in metabolism and enantiomeric ratio analysis appears to be a useful approach in WBE studies to identify different sources of drugs in the environment, when no metabolic products are present at useful analytical levels., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Analysis of drug residues in urban wastewater could complement epidemiological studies in detecting the use of new psychoactive substances (NPS), a continuously changing group of drugs hard to monitor by classical methods. We initially selected 52 NPS potentially used in Italy based on seizure data and consumption alerts provided by the Antidrug Police Department and the National Early Warning System. Using a linear ion trap-Orbitrap high resolution mass spectrometer, we designed a suspect screening and a target method approach and compared them for the analysis of 24 h wastewater samples collected at the treatment plant influents of four Italian cities. This highlighted the main limitations of these two approaches, so we could propose requirements for future research. A library of MS/MS spectra of 16 synthetic cathinones and 19 synthetic cannabinoids, for which analytical standards were acquired, was built at different collision energies and is available on request. The stability of synthetic cannabinoids was studied in analytical standards and wastewater, identifying the best analytical conditions for future studies. To the best of our knowledge, these are the first stability data on NPS. Few suspects were identified in Italian wastewater samples, in accordance with recent epidemiological data reporting a very low prevalence of use of NPS in Italy. This study outlines an analytical approach for NPS identification and measurement in urban wastewater and for estimating their use in the population.

Background: Monitoring consumption by population surveys (PS) is an important way to challenge the spread of illicit drugs (ID). To improve the information, we explored a complementary method, particularly wastewater analysis (WWA)., Methods: We estimated the prevalence of use by PS, and the consumption by WWA, of cocaine, opioids, cannabis, methamphetamine and MDMA (ecstasy) from 2010 to 2014 in Italy and compared the results., Results: According to PS, cannabis and cocaine were the ID most used in Italy (last month prevalence 3.0% and 0.43% respectively in 2010) followed by opioids (0.17%) and amphetamines (0.14%). WWA gave similar findings, with cannabis consumption (4.35 g THC/day/1000 inhabitants) exceeding cocaine (0.78 g), heroin (0.092 g), methamphetamine and MDMA (0.103 g). The time trend investigated by PS showed significant decreases for all ID from 2010 to 2012. WWA also indicated a reduction of consumption for methamphetamine (p<0.0001) and heroin (p<0.01). Both methods showed an increase for cannabis in 2014 (p<0.001) with the other ID unchanged. Spatial investigations by WWA showed that cannabis and cocaine were consumed significantly more in central Italy than in the north and south. PS indicated the same but only for cannabis. WWA was helpful to study weekly patterns of consumption, showing increases in cocaine and MDMA at weekends., Conclusions: PS and WWA were confirmed as complementary methods and when used together improved the information on ID use in Italy. We suggest that the combined use of the two approaches can give better information on ID use in the population., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Wastewater analysis is an innovative approach that allows monitoring illicit drug use at the community level. This study focused on investigating geographical differences in drug consumption by comparing epidemiological, crime and wastewater data., Methods: Wastewater samples were collected in 19 cities across Germany and Switzerland during one week, covering a population of approximately 8.1 million people. Self-report data and consumption offences for the investigated areas were used for comparison and to investigate differences between the indicators., Results: Good agreement between data sources was observed for cannabis and amphetamine-type stimulants, whereas substantial discrepancies were observed for cocaine. In Germany, an important distinction could be made between Berlin, Dortmund and Munich, where cocaine and particularly amphetamine were more prevalent, and Dresden, where methamphetamine consumption was clearly predominant. Cocaine consumption was relatively homogenous in the larger urban areas of Switzerland, although prevalence and offences data suggested a more heterogeneous picture. Conversely, marked regional differences in amphetamine and methamphetamine consumption could be highlighted., Conclusions: Combining the available data allowed for a better understanding of the geographical differences regarding prevalence, typology and amounts of substances consumed. For cannabis and amphetamine-type stimulants, the complementarity of survey, police and wastewater data could be highlighted, although notable differences could be identified when considering more stigmatised drugs (i.e. cocaine and heroin). Understanding illicit drug consumption at the national scale remains a difficult task, yet this research illustrates the added value of combining complementary data sources to obtain a more comprehensive and accurate picture of the situation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Introduction and Aims: Wastewater analysis is a new approach developed to estimate illicit drug (ID) consumption in large communities, such as a city. We tested the ability of this approach to detect differences in consumption in different districts of a city., Design and Methods: Consumption of cocaine, heroin, tetrahydrocannabinol (THC) (cannabis active principle), amphetamine, methamphetamine and ecstasy was estimated by analysis of selected drug excretion residues in composite 24 h samples of untreated urban wastewater by liquid chromatography-tandem mass spectrometry. Samples were collected from the inlet of the three main Milan wastewater treatment plants (WWTP), each serving a district of the city (west, center and east). In each WWTP, samples were taken daily for seven consecutive days in November 2010 and March 2011., Results: We observed significant differences of ID consumption (expressed as mg/day/1000 inhabitants) among districts: consumption of some ID was significantly higher in the eastern district (P < 0.01 for THC, P < 0.001 for cocaine and P < 0.0001 for heroin by one-way analysis of variance), while consumption of methamphetamine and amphetamine was higher in the central area (P < 0.0001). Overall, from 2010 to 2011, ID consumption decreased in all the districts, in line with a recent population survey showing decreases from 25 to 55% in the annual prevalence of ID users in Italy., Discussion and Conclusions: This approach may help to detect ID consumption in different districts of a city and may be useful for planning interventions aimed at specific city areas and substances., (© 2014 Australasian Professional Society on Alcohol and other Drugs.)

This study reports an investigation of omeprazole and venlafaxine parent substances and metabolites in Italian municipal influent wastewaters (IWWs). These pharmaceuticals were selected because they are widely consumed in Italy, but are poorly detected in waste and surface water. The aim of the study was to identify the most relevant pharmaceuticals metabolites in wastewater in order to improve the prioritization step and choose priority pollutants for environmental monitoring campaigns. This was done by investigating omeprazole, venlafaxine and their main metabolites in 30 IWWs from ten Italian cities and by comparing results with information from pharmacokinetic studies. Analysis was performed by solid phase extraction (SPE) and high-performance liquid chromatography (HPLC) coupled to high resolution mass spectrometry (HRMS). We searched for 23 omeprazole and four venlafaxine metabolites using data-dependent and MS/MS methods. Parent omeprazole was never present in the samples. Six omeprazole metabolites were found in IWWs. Venlafaxine and two metabolites were present in all the samples. The metabolic profiles in Italian IWW agreed with results in IWW from Spain and with urinary excretion profiles from pharmacokinetic studies. Comparing results from different sources was useful to improve the identification of pharmaceuticals metabolites in environmental samples and to focus the attention of future studies on the most relevant compounds., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The existence of pharmaceuticals and illicit drugs (PIDs) in environmental waters has led many analytical chemists to develop screening methods for monitoring purposes. Water samples can contain a huge number of possible contaminants, commonly at low concentrations, which makes their detection and identification problematic. Liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) has proven itself effective in the screening of environmental contaminants. The present work investigates the use of the most popular HRMS instruments, quadrupole time-of-flight and linear trap quadrupole-Orbitrap, from two different laboratories. A suspect screening for PIDs was carried out on wastewater (influent and effluent) and surface water samples from Castellón, Eastern Spain, and Cremona, Northern Italy, incorporating a database of 107 PIDs (including 220 fragment ions). A comparison between the findings of both instruments and of the samples was made which highlights the advantages and drawbacks of the strategies applied in each case. In total, 28 compounds were detected and/or identified by either/both instruments with irbesartan, valsartan, benzoylecgonine and caffeine being the most commonly found compounds across all samples.

The quantitative determination of urinary biomarkers in raw wastewater has emerged in recent years as a promising tool for estimating the consumption of illicit drugs, tobacco and alcohol in a population and for comparing local and temporal trends. In this study, a three-year monitoring campaign (2012-2014) was conducted to compare alcohol and cocaine use in two European cities (Santiago de Compostela, Spain, and Milan, Italy) by wastewater analysis. Ethyl sulphate and benzoylecgonine were used, respectively, as biomarkers of ethanol and cocaine consumption and cocaethylene as an indicator of co-consumption of both substances. Biomarkers were measured using liquid chromatography-tandem mass spectrometry and concentrations were converted to rates of consumption using specific correction factors. Results were statistically compared in terms of geographic and temporal tendencies. Alcohol intake was significantly higher in Santiago than in Milan (13.6L versus 5.1L ethanol/1000 people day, averages). Cocaine use was higher in Milan than in Santiago de Compostela (800 versus 632 mg/1000 people day, averages). A significant higher consumption of both alcohol and cocaine was observed during the weekends (~23-75% more than on weekdays) in both cities. In terms of years, slight changes were observed, but no clear trends as representative of the whole year could be identified because of the limited number of days sampled. Co-consumption was evaluated using the cocaethylene/benzoylecgonine ratio, which was higher during the weekend in both cities (58% in Santiago and 47% in Milan over the non-weekend day means), indicating a greater co-consumption when cocaine is used as a recreational drug. Wastewater-based epidemiology gave estimates of alcohol and cocaine use in agreement with previous wastewater studies and with recent European surveillance and prevalence data, and weekly profiles of use and preferential patterns of consumption could be plot., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Wastewater analysis was applied in a four-year monitoring study to assess temporal and spatial patterns of ketamine and mephedrone use in the general population in Italy. Composite raw wastewater samples were collected from sewage treatment plants (STPs) in 17 cities. Target analytes were measured using a validated method based on solid phase extraction and liquid chromatography coupled to tandem mass spectrometry analysis. Mass loads were use to assess ketamine and mephedrone use and were normalized to the population served by the plants. Ketamine was detected in wastewater in all except one (Palermo) of the cities investigated, while mephedrone was detected only in Bologna and Florence. Ketamine mass loads progressively increased from 2010 to 2013, and in Milan rose from 1 to 1.5 g/day in 2008-2010 to 3.4-3.6 g/day in 2013-2014. Mass loads were higher in north and central Italy than in the south, and in larger rather than small cities. Wastewater analysis was suitable to provide objective and up-to-date information on the use of ketamine in Italy, to identify ketamine spatial and temporal changes, and to confirm the low use of mephedrone. These results can complement information from population surveys which often provide only scant and incomplete figures for these substances.

The use of caffeine, nicotine and some major metabolites was investigated by wastewater analysis in 13 sewage treatment plants (STPs) across Italy, and their suitability was tested as qualitative and quantitative biomarkers for assessing population size and dynamics. A specific analytical method based on mass spectrometry was developed and validated in raw urban wastewater, and included two caffeine metabolites, 1-methylxanthine and 7-methylxanthine, never reported in wastewater before. All these compounds were found widely at the μg/L level. Mass loads, calculated by multiplying concentrations by the wastewater daily flow rate and normalized to the population served by each plant, were used to compare the profiles from different cities. Some regional differences were observed in the mass loads, especially for nicotine metabolites, which were significantly higher in the south than in the center and north of Italy, reflecting smoking prevalences from population surveys. There were no significant weekly trends, although the mean mass loads of caffeine and its metabolites were slightly lower during the weekend. Most caffeine and nicotine metabolites fulfilled the requirements for an ideal biomarker for the assessment of population size, i.e. being easily detectable in wastewater, stable in sewage and during sampling, and reflecting human metabolism. Nicotine metabolites were tested as quantitative biomarkers to estimate population size and the results agreed well with census data. Caffeine and its metabolites were confirmed as good qualitative biomarkers, but additional information is needed on the caffeine metabolism in relation to the multiple sources of its main metabolites. This exploratory study opens the way to the routine use of nicotine metabolites for estimating population size and dynamics., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: We propose a novel approach for measuring tobacco use in a community through the chemical analysis of nicotine metabolites in urban wastewater. It offers frequent monitoring and 'real-time', 'evidence-based' estimates of tobacco consumption which may complement epidemiological surveillance systems normally repeated only every few years., Methods: Two urinary metabolites of nicotine, namely cotinine and trans-3'-hydroxycotinine, were selected as biomarkers of tobacco consumption in urban wastewater. During smoking, a known amount of nicotine is absorbed and after metabolism excreted as metabolites in urine, ending up in the wastewater; quantitative analysis of the metabolites in the wastewater allows back-calculation of the nicotine collectively absorbed by the population producing the sewage and, indirectly, their tobacco use. Representative samples of wastewater were collected from sewage treatment plants in eight Italian cities and analysed by mass spectrometry. Mass loads of the metabolites were used to estimate nicotine consumption., Results: Wastewater analysis in the cities under study was used to estimate the number of cigarettes smoked, in order to compare the results of this study with those obtained from population surveys. The number of cigarettes calculated with the two methods were closely comparable and wastewater analysis was sufficiently sensitive to confirm the differences in tobacco consumption between northern and southern Italy, previously described in population surveys., Conclusions: The described approach can serve as a supplementary indicator of tobacco consumption in local communities. This approach can provide objective and updated information, which are useful to assess the efficacy of tobacco-control interventions, with the aim of designing and implementing effective tobacco control plans., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Illicit drug use is a global phenomenon involving millions of individuals, which results in serious health and social costs. The chemical analysis of urban wastewater for the excretion products of illicit drugs is a potent approach for monitoring patterns and trends of illicit drug use in a community. The first international and multidisciplinary conference on this topic was recently organized to present the epidemiological knowledge of patterns in drug use and the information obtained from wastewater analysis. This paper gives an overview of the main issues that emerged during the conference, focusing on the identified research gaps and requirements and on the future challenges and opportunities from bringing together wastewater analysis and drug epidemiology. The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) uses an established multi-indicator system to monitor illicit drug use and to identify the emergence of new psychoactive substances. The methodological challenges of monitoring a hidden and stigmatized behavior like drug use include the limitations of self-report data and reporting delays. An increasing evidence base suggests that wastewater analysis can address some of these problems. Specifically this technique can: monitor temporal and spatial trends in drug use at different scales, provide updated estimates of drug use, and identify changing habits and the use of new substances. A best practice protocol developed by a Europe-wide network of experts is available to produce homogeneous and comparable data at different sites. The systematic evaluation of uncertainties related to wastewater analysis has highlighted which areas require careful control and those that need further investigation to generally improve the approach. Wastewater analysis has considerable potential to complement existing approaches for monitoring drug use due to its ability to produce objective, real-time estimates of drug use and to give timely information of any change in the patterns of use.