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Alcohol use has increased in the last years and is a priority public health issue worldwide. Wastewater-based epidemiology (WBE) is a reliable approach for estimating substance use at the population level and can complement population surveys and sales statistics to obtain objective and updated information on alcohol use in a community. This work reports the first nationwide study of alcohol consumption conducted in Italy by WBE, covering 11.4% of the Italian population. A total of 264 raw wastewater samples (24-h composite) were collected from 17 Italian cities in 2013–2014 and one large city (Milan) over a 7-year period (2013–2019). Besides of normal days, special periods/events (i.e. Halloween, Milan Fashion Week and summer holidays) were studied in Milan. The urinary biomarker of ethanol (namely, ethyl sulphate) was measured by liquid chromatography-tandem mass spectrometry and was used to estimate alcohol consumption. Results were statistically evaluated to assess spatial and temporal differences and were compared with data from other epidemiological indicators. In 2013, alcohol consumption was significantly higher in small cities than in large ones but no significant differences were observed in relation to the geographical position. An increase in alcohol use was observed in seven large cities from 2013 to 2014. The 7-year monitoring in Milan showed no marked changes, except for remarkable increases in 2014 and 2019. Alcohol consumption significantly increased (ca. 25%) during weekends in Italy and in Milan, and during special periods/events, for instance summer holidays and Halloween. WBE estimates were in agreement with national prevalence data, demonstrating that WBE is suitable for objectively evaluating geographic, annual and weekly patterns of alcohol consumption at local and national scales. WBE information would be helpful to implement and evaluate measures for reducing alcohol consumption and preventing the related health risks.

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.

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.

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 (WWTPs), 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

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

Aldehyde-oxidase-4 (AOX4) is one of the mouse aldehyde oxidase isoenzymes and its physiological function is unknown. The major source of AOX4 is the Harderian-gland, where the enzyme is characterized by daily rhythmic fluctuations. Deletion of the Aox4 gene causes perturbations in the expression of the circadian-rhythms gene pathway, as indicated by transcriptomic analysis. AOX4 inactivation alters the diurnal oscillations in the expression of master clock-genes. Similar effects are observed in other organs devoid of AOX4, such as white adipose tissue, liver and hypothalamus indicating a systemic action. While perturbations of clock-genes is sex-independent in the Harderian-gland and hypothalamus, sex influences this trait in liver and white-adipose-tissue which are characterized by the presence of AOX isoforms other than AOX4. In knock-out animals, perturbations in clock-gene expression are accompanied by reduced locomotor activity, resistance to diet induced obesity and to hepatic steatosis. All these effects are observed in female and male animals. Resistance to obesity is due to diminished fat accumulation resulting from increased energy dissipation, as white-adipocytes undergo trans-differentiation towards brown-adipocytes. Metabolomics and enzymatic data indicate that 5-hydroxyindolacetic acid and tryptophan are novel endogenous AOX4 substrates, potentially involved in AOX4 systemic actions.

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.

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.

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.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.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.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.