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The spread of biogenic matrices for agricultural purposes can lead to plastic input into soils, raising a question on possible consequences for the environment. Nonetheless, the current knowledge concerning the presence of plastics in biogenic matrices is very poor. Therefore, the objective of the present study was a quali-quantitative characterization of plastics in different matrices reused in agriculture as manures, digestate, compost and sewage sludges. Plastics were quantified and characterized using a Fourier Transform Infrared Spectroscopy coupled with an optical microscope (μFT-IR) in Attenuated Total Reflectance mode. Our study showed the presence of plastics in all the investigated samples, albeit with differences in the content among the matrices. We measured a lower presence in animal matrices (0.06–0.08 plastics/g wet weight w.w.), while 3.14–5.07 plastics/g w.w. were measured in sewage sludges. Fibres were the prevalent shape and plastic debris were mostly in the micrometric size. The most abundant polymers were polyester (PEST), polypropylene (PP) and polyethylene (PE). The worst case was observed in the compost sample, where 986 plastics/g w.w. were detected. The majority of these plastics were compostable and biodegradable, with only 8% consisting of fragments of PEST and PE. Our results highlighted the need to thoroughly evaluate the contribution of reused matrices in agriculture to the plastic accumulation in the soil system. [Display omitted] • The spread of biogenic matrices can contribute to plastic accumulation in the agroecosystems. • Manures and digestate are less contaminated with plastics than sewage sludges and compost. • Human wastes can introduce 100 times more plastics in soil with respect to animal ones. • Remarkable amount of compostable and biodegradable plastics found in compost. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • A comprehensive and flexible screening approach based on LC-HRMS was developed. • Almost 200 "priority new psychoactive substances" were investigated in wastewater. • The method allows the inclusion of new NPS based on experimental results. • The screening approach was applied in an extensive European wastewater monitoring. • Thirteen new psychoactive substances were identified at different confidence levels. [ABSTRACT FROM AUTHOR]

Emerging Contaminants (ECs) are ubiquitous in waters, arousing concern because of their potential risks for human health and the environment. This study investigated the presence of multiple classes of ECs in 21 wells over the drinking water network of Milan, in the most inhabited and industrialized area of Italy, and assessed the risks for consumers. Samples were analyzed using liquid chromatography coupled to mass spectrometry. Human risk assessment (HRA) was conducted by comparing the measured concentrations with drinking water thresholds from guidelines or calculated in this study; first considering the exposure to each single EC and then the entire mixture. Thirteen ECs were measured in the low ng/L range, and were generally detected in less than half of the wells. Pharmaceuticals, perfluorinated substances, personal care products, and anthropogenic markers were the most frequently detected. The results of the HRA excluded any risks for consumers in each scenario considered. This is one of the most comprehensive studies assessing the presence of a large number of ECs in the whole drinking water network of a city, and the risks for human health. Results improve the limited information on ECs sources and occurrence in drinking water and help establishing guidelines for regulatory purposes. [ABSTRACT FROM AUTHOR]

The occurrence of several classes of emerging contaminants (ECs) was assessed in the River Lambro basin, one of the most urbanized and industrialized areas of Italy. The study aims were to identify the main sources of ECs, quantify their amounts circulating in the water cycle, and study their fate in the aquatic environment. More than 80 ECs were selected among pharmaceuticals ( PHARM ), personal care products ( PCPs ), disinfectants ( DIS ), illicit drugs ( IDs ), perfluorinated compounds ( PERF ), alkylphenols and bisphenol A ( Alk-BPA ), and anthropogenic markers ( AM ). Specific analytical methods were developed for quantitative analysis based on solid phase extraction and liquid chromatography tandem mass spectrometry. ECs were measured in rivers upstream and downstream of the main city (Milan), and in untreated and treated wastewater from Milan to assess the contribution to river contamination, and in superficial and deep groundwater in the city area to study the relationship between river and groundwater contamination. Samples were collected in a two-year monitoring campaign. Almost all ECs were ubiquitous in untreated wastewater, at concentrations up to the μg/L range, and the most abundant classes were PHARM and AM . Removals during different wastewater treatment processes were studied and the most stable substances were PHARM , PCPs and PERF . The mass loads increased for all the classes of ECs along the River Lambro basin. A mass balance was done in the river basin and allowed to identify the main sources of contamination, which were domestic, from treated or untreated wastewater, for PHARM , PCPs and IDs , mainly industrial for PERF, and both industrial and domestic for Alk-BPA . The study of AM helped to identify direct discharges of untreated wastewater. A substantial contribution of surface water to groundwater contamination was observed. This study improves the knowledge on occurrence, sources and fate of multiple classes of ECs in a highly urbanized area providing useful information to help the establishment of EU regulations for ECs. [ABSTRACT FROM AUTHOR]

Several monitoring studies have shown that benzoylecgonine (BE) is the main illicit drug residue commonly measured in the aquatic system worldwide. Few studies have investigated the potential toxicity of this molecule towards invertebrate and vertebrate aquatic non-target organisms focusing on effects at low levels of the biological organization, but no one has assessed the consequences at higher ones. Thus, the present study was aimed at investigating the toxicity of a 48-h exposure to two concentrations of BE, similar to those found in aquatic ecosystems (0.5 μg/L and 1.0 μg/L), on the cladoceran Daphnia magna at different levels of the ecological hierarchy. We relied on a multi-level approach focusing on the effects at biochemical/biomolecular (biomarkers), individual (swimming activity) and population (reproduction) levels. We measured the amount of reactive oxygen species and of the activity of antioxidant (SOD, CAT, and GPx) and detoxifying (GST) enzymes to assess if BE exposure can alter the oxidative status of D. magna specimens, while the lipid peroxidation (TBARS) was measured as a marker of oxidative damage. Moreover, we also measured the acetylcholinesterase (AChE) activity because it is strictly related to behavioral changes in aquatic organisms. Changes in swimming behavior were investigated by a video tracking analysis, while the consequences on reproduction were assessed by a chronic toxicity test. Our results showed that BE concentrations similar to those found in aquatic ecosystems induced oxidative stress and inhibited AChE activity, affecting swimming behavior and the reproduction of Daphnia magna individuals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Silver nanoparticles are toxic both in vitro and in vivo . We have investigated the possibility to exploit the cytotoxic potential of silver nanoparticles in T24 bladder carcinoma cells using both bare and PolyVinylPyrrolidone-coated silver nanoparticles. We show that the two types of silver nanoparticles promote morphological changes and cytoskeletal disorganization, are cytotoxic and induce cell death. These effects are due to the increased production of reactive oxygen species which are responsible, at least in part, for the sustained activation of ERK1/2. Indeed, both cytotoxicity and ERK1/2 activation are prevented by exposing the cells to the anti-oxidant N -acetylcysteine. Also blocking the ERK1/2 pathway with the MEK inhibitor PD98059 protects the cells from nanoparticles’ cytotoxicity. Our findings suggest that ERK activation plays a role in silver nanoparticle-mediated cytotoxicity in T24 cells. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Highlights: [•] Mifepristone affects fecundity of zebrafish. [•] Mifepristone alters histology of gonads. [•] Effects occur at low ng/L levels. [Copyright &y& Elsevier]

Abstract: Background: Wastewater analysis can provide estimates of illicit drug (ID) consumption in local communities. Methods: We used repeated raw wastewater analysis in urban wastewater treatment plants to estimate loads of cocaine, heroin, methamphetamine, and cannabis consumed daily by the inhabitants of two cities in Northern Italy, Milan and Como, from 2005 to 2009. Results: Daily cocaine loads did not change in Milan from 2005 to 2008 but fell 45% in 2009 (ANOVA, p <0.001, followed by Tukey–Kramer HSD test [2009 vs. others], p <0.05), and there was a similar drop in Como (41%, p <0.0001, t-test). Heroin also fell from 2008 to 2009 in Milan (66%, ANOVA, p <0.001, followed by Tukey–Kramer [2009 vs. others], p <0.05) and Como (26%, p =0.017, t-test). However, methamphetamine, which had risen in Milan from 2005 to 2008, rose further in 2009 (Kruskal–Wallis test, p <0.001, followed by Steel–Dwass [2009 or 2008 vs. previous], p <0.05), and cannabis, which was falling from 2005 to March 2009, rose again in September 2009 (40%, p =0.027, t-test). Conclusions: Results suggest a trend toward a decrease in consumption of costly ID, such as cocaine and heroin. This might be due to a reduction in the number of consumers and/or to a change in their behaviour since there was also an increase in the consumption of less expensive ID. This itself might reflect a drop in consumers’ money supply, caused by the economic crisis. Wastewater analysis was useful to estimate ID consumption levels in local communities in real time and promptly identify changes in trends. [Copyright &y& Elsevier]

Abstract: The most relevant human urinary metabolites of cocaine (nine metabolites) were measured in urban wastewater in Italy and USA. A novel analytical method based on liquid chromatography tandem mass spectrometry allowed the identification of ecgonine, ecgonine methyl ester and the pyrolytic derivatives of cocaine in untreated wastewater. The aim of this study was to verify whether the pattern of cocaine metabolites in wastewater reflected the human excretion profile in urine. The performance of the method was good, with recoveries higher than 60% and limits of quantifications in the low ng/L range. The stability in untreated wastewater was assessed for all metabolites and the best storage condition resulted freezing samples immediately after collection and keep them frozen until analysis. All the selected compounds were measured in wastewater at concentrations up to 1.5 μg/L and their weekly loads were calculated during a five weeks monitoring campaign in Milan (Italy). The profiles of cocaine metabolites in wastewater matched with those in human urine reported in the literature, suggesting that measures in wastewater reflect the real human excretion and that wastewater analysis is suitable for assessing drug consumption. Benzoylecgonine was confirmed as the best target for estimating cocaine use by wastewater analysis, while cocaine itself should not be considered because its amount in wastewater is affected by other environmental sources such as transport, handling and consumption. Results suggested that the measurement of other metabolites in combination with benzoylecgonine might reflect 60% of an administered dose of cocaine providing also information on different patterns of use. [Copyright &y& Elsevier]

Abstract: The consumption of illicit drugs causes indisputable societal and economic damage. Therefore it is necessary to know their usage levels and trends for undertaking targeted actions to reduce their use. Recently, a new approach (namely sewage epidemiology) was developed for the estimation of illicit drug use based on measurements of urinary excreted illicit drugs and their metabolites in untreated wastewater. This review aims at critically evaluating the published literature and identifying research gaps of sewage epidemiology. Firstly, the existing analytical procedures for the determination of the four most used classes of illicit drugs worldwide (cannabis, cocaine, opiates and amphetamine-like stimulants) and their metabolites in wastewater are summarized and discussed. The focus lies on the sample preparation and on the analysis with chromatographic techniques coupled to mass spectrometry. Secondly, back-calculations used to transform measured concentrations in wastewater (in ng/L) into an amount of used illicit drug (in g/day per 1000 inhabitants or doses/day per 1000 inhabitants) are discussed in detail for the four groups of illicit drugs. Sewage epidemiology data from Spain, Belgium, UK, Italy, Switzerland and USA are summarized and compared with data from international organisations, such as the European Monitoring Centre for Drug and Drug Addiction (EMCDDA) and the United Nations Office on Drugs and Crime (UNODC). The results derived from wastewater analysis show in general good agreement with existing prevalence data (percentage of a population that uses illicit drugs at a given time) and demonstrate the potential of sewage epidemiology. However, this review confirms that future work should focus on further optimisation and standardisation of various important parameters (e.g. sample collection and back-calculations). In the future, sewage epidemiology could be used in routine drug monitoring campaigns as a valuable tool in addition to the classical socio-epidemiological studies for the determination of local, national and international illicit drug use. [Copyright &y& Elsevier]

Abstract: Endothelial migration, early step in angiogenesis, is tightly regulated by the coordinated action of tyrosine kinases and tyrosine phosphatases. HD-PTP contributes to endothelial motility, since endothelial cells silencing HD-PTP after transfection with iRNA acquire a scattered and spindle-shaped phenotype and migrate faster than controls. Since (i) the proto-oncogene Src contributes to the regulation of cell motility and (ii) HD-PTP has a potential binding site for Src, we investigated whether an interplay exists between these two proteins. We found that Src binds HD-PTP and this interaction is enhanced after exposure to basic fibroblast growth factor. While HD-PTP does not modulate the levels of Src phosphorylation both in vitro and in vivo, we found that Src phosphorylates HD-PTP on tyrosine residues. Here we show for the first time that (i) HD-PTP has a tyrosine phosphatase activity; (ii) HD-PTP phosphorylation by Src inhibits its enzymatic activity. Interestingly, pharmacological and genetic inhibition of Src abrogates the migratory phenotype of endothelial cells silencing HD-PTP. On these bases, and because we have previously demonstrated that HD-PTP binds and dephosphorylates focal adhesion kinase (FAK), another crucial regulator of cell migration, we hypothesize that HD-PTP participates to the regulation of endothelial motility through its interactions with Src and FAK. [Copyright &y& Elsevier]

Abstract: Cell migration is fundamental for invasion and metastasis and is modulated by the reversible phosphorylation of tyrosine residues on target proteins. Here we report that the tyrosine phosphatase HD-PTP has a role in modulating the motility of T24 bladder carcinoma cells. Indeed, HD-PTP silencing by RNA interference (RNAi) markedly induced cell migration in a Src dependent fashion. We therefore investigated the interaction and the regulation of Src and HD-PTP. We found that, in Epidermal Growth Factor (EGF) stimulated cells, Src binds to and phosphorylates HD-PTP on tyrosine residues. On the contrary, HD-PTP does not modulate the levels of Src phosphorylation. Interestingly, HD-PTP also binds to FAK, another regulator of cell migration, and this interaction is inhibited after exposure to EGF. FAK phosphorylates HD-PTP and this event reduced the interactions between the two proteins. Interestingly, in cells silencing HD-PTP the phosphorylation of FAK is enhanced and this correlates with its localization in focal complexes both in the presence and in the absence of EGF. We hypothesize that in unstimulated T24 cells HD-PTP does not interact with Src, while it binds to FAK. Following stimulation with EGF, HD-PTP is tyrosine-phosphorylated and releases FAK which will ultimately contribute to the turn-over of focal adhesion and, therefore, to cell motility. [Copyright &y& Elsevier]

Abstract: Antibiotics are commonly detected in the environment as contaminants. Exposure to antibiotics may induce antimicrobial-resistance, as well as the horizontal transfer of resistance genes in bacterial populations. We selected the resistance gene marA, mediating resistance to multiple antibiotics, and explored its distribution in sediment and water samples from surface and sewage treatment waters. Ciprofloxacin and ofloxacin (fluoroquinolones), sulphamethoxazole (sulphonamide), erythromycin, clarythromycin, and spiramycin (macrolides), lincomycin (lincosamide), and oxytetracycline (tetracycline) were measured in the same samples to determine antibiotic contamination. Bacterial populations from environmental samples were challenged with antibiotics to identify resistant isolates. The gene marA was found in almost all environmental samples and was confirmed by PCR amplification in antibiotic-resistant colonies. 16S rDNA sequencing revealed that the majority of resistant isolates belonged to the Gram-positive genus Bacillus, not previously known to possess the regulator marA. We assayed the incidence of marA in environmental bacterial populations of Escherichia coli and Bacillus by quantitative real-time PCR in correlation with the levels of antibiotics. Phylogenetic analysis indicated the possible lateral acquisition of marA by Bacillus from Gram-negative Enterobacteriaceae revealing a novel marA homolog in Bacillus. Quantitative PCR assays indicate that the frequency of this gene in antropised environments seems to be related to bacterial exposure to water-borne antibiotics. [Copyright &y& Elsevier]

It is now well established that residues from therapeutic drugs consumed by humans can end up, through the sewage system, in the surface water of populated areas. Given that the global production of major illicit drugs is comparable to that of widely used pharmaceuticals, we tested for the presence of drugs of abuse (cocaine, opioids, amphetamines and cannabis derivatives), some related opioid pharmaceuticals (codeine and methadone) and/or their metabolites in Italian and British surface waters. Having identified residues of all major drugs of abuse in raw and treated urban wastewater, we now measured their levels in several rivers and lakes by a selective multi-residue assay based on liquid chromatography-tandem mass spectrometry. Recoveries in surface water were generally higher than 80%, with overall variability of the method lower than 10%. LODs were generally lower than 0.2ng/L, and LOQs were lower than 0.6ng/L, with few exceptions. Many of the tested substances were found in both rivers and lakes, at concentrations ranging from high pg/L to high ng/L, with loads in rivers in the range of tenths to hundreds of grams per day. Our data indicate that residues of drugs of abuse have become widespread surface water contaminants in populated areas. Since most of these residues still have potent pharmacological activities, their presence in the aquatic environment may have potential implications for human health and wildlife. [Copyright &y& Elsevier]

The Amu Darya River, one of the most important water resources for Uzbekistan and Turkmenistan, was declared a World Disaster Zone in 1991. The great increase in irrigation and the use of pesticides has led to both a lack of water and drinking water contamination. The aim of the present study, part of an EU project on water management guidelines, was to evaluate the leachability of 71 organic pesticides commonly employed in the area, and to assess compounds that could potentially contaminate the river and impair drinking water quality. A multivariate approach is proposed for the pesticide screening, condensing information from different environmental partition indexes (GUS, “modified LEACH”, LIN) into a single ranking, the Global Leachability Index (GLI). For a selected data set in water medium this super-index identifies three classes with a risk potential for pesticide leachability, and allows the selection of a small number of chemicals for an analytical survey. [Copyright &y& Elsevier]

• Urban wastewater was used for monitoring new psychoactive substances (NPS) use • This Europe-wide study included 22 cities in 14 countries monitored in 2016 – 2017 • The main classical illicit drugs were also monitored to compare results • The qualitative and quantitative spatio- temporal profiles of drug use were provided • Wastewater-based epidemiology is valuable to follow rapidly changing profiles of use Wastewater-based epidemiology (WBE) can be a useful tool to face some of the existing challenges in monitoring the use of new psychoactive substances (NPS), as it can provide objective and updated information. This Europe-wide study aimed to verify the suitability of WBE for investigating the use of NPS. Selected NPS were monitored in urban wastewater by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The main classical illicit drugs were monitored in the same samples to compare their levels with those of NPS. Raw composite wastewater samples were collected in 2016 and 2017 in 14 European countries (22 cities) following best practice sampling protocols. Methcathinone was most frequent (>65% of the cities), followed by mephedrone (>25% of the cities), and only mephedrone, methcathinone and methylone were found in both years. This study depicts the use of NPS in Europe, confirming that it is much lower than the use of classical drugs. WBE proved able to assess the qualitative and quantitative spatial and temporal profiles of NPS use. The results show the changeable nature of the NPS market and the importance of large WBE monitoring campaigns for selected priority NPS. WBE is valuable for complementing epidemiological studies to follow rapidly changing profiles of use of drugs. [Display omitted] [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Adherence to therapy was assessed through a wastewater-based epidemiology approach. • Urinary metabolites and active ingredients were selected as biomarkers. • All biomarkers were found in measurable concentrations in raw wastewater. • Biomarker concentrations in wastewater may reflect their prescription figures. • Urinary metabolites allowed better assessment of the medical adherence. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • WBE was used to investigate the lifestyle of the inhabitants of Malé, Maldives. • Raw wastewater was collected from nine sites serving the entire city area. • Illicit drugs, alcohol, caffeine, tobacco and pharmaceuticals use were investigated. • Results provided valuable information complementary to epidemiological approaches. • Results may be useful to plan drug use prevention programs and health campaigns. [ABSTRACT FROM AUTHOR]

Illicit drugs and their metabolites have been identified as emerging aquatic pollutants. Cocaine (COC) is one of the most used illicit drug worldwide. After human consumption, COC enters the aquatic ecosystems, where it is commonly detected in ng L−1 concentration range. Although a number of studies have shown that the exposure to environmental concentrations of COC can induce diverse biochemical, molecular and histological effects on aquatic organisms, the information of COC-induced behavioral alterations is scant. Thus, the present study aimed at exploring both biochemical and behavioral effects induced by the exposure to two environmental concentrations (50 ng L−1 and 500 ng L−1) of COC on the freshwater cladoceran Daphnia magna. Specimens were exposed to selected COC concentrations for 21 days and the effects on the oxidative status, including the amount of reactive oxygen species and the activity of antioxidant (SOD, CAT and GPx) and detoxifying (GST) enzymes, and swimming activity were investigated after 7, 14 and 21 days of treatment, while effects on reproductive success was assessed after 21-days only.. Exposure to COC induced an overproduction of reactive oxygen species and a modulation of the activity of defense enzymes. Moreover, COC affected the swimming behavior and altered the reproductive success of treated specimens. Our results highlighted that environmental concentrations of COC can cause adverse effects at different levels of the biological hierarchy in a zooplanktonic species, confirming the potential threat due to this illicit drug for the aquatic community. Unlabelled Image • Cocaine exposure altered the D. magna oxidative status. • Cocaine altered the swimming behavior depending on the tested concentration. • High cocaine concentration reduced the reproductive output of D. magna. • Cocaine might represent a threat for freshwater zooplanktonic species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 1–15 ng/L for the SPE method and 40–800 ng/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 1.1 ng/L to 1.6 μg/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. [ABSTRACT FROM AUTHOR]

Illicit drugs have been recognized as emerging aquatic pollutants due to their presence in aquatic ecosystems up to µg/L level. Among these, the synthetic psycho-stimulant drug amphetamine (AMPH) is commonly found in both surface and wastewaters worldwide. Even though the environmental occurrence of AMPH is well-known, the information on its toxicity towards non-target freshwater organisms is completely lacking. This study investigated the imbalance of the oxidative status and both oxidative and genetic damage induced by a 14-day exposure to two concentrations (500 ng/L and 5000 ng/L) of AMPH on the freshwater bivalve Dreissena polymorpha by the application of a biomarker suite. We investigated the activity of antioxidant enzymes (SOD, CAT and GPx), the phase II detoxifying enzyme GST, the lipid peroxidation level (LPO) and protein carbonyl content (PCC), as well as primary (Single Cell Gel Electrophoresis assay) and fixed (DNA diffusion assay and Micronucleus test) genetic damage. Our results showed that a current realistic AMPH concentration (500 ng/L) did neither cause notable imbalances in enzymatic activities, nor oxidative and genetic damage to cellular macromolecules. In contrast, the bell-shaped trend of antioxidants showed at the highest tested concentration (5000 ng/L) suggested an overproduction of reactive oxygen species, leading to oxidative damage, as confirmed by the significant increase of protein carbonylation and DNA fragmentation. [ABSTRACT FROM AUTHOR]

Illicit drugs are considered to be emerging aquatic pollutants since they are commonly found in freshwater ecosystems in the high ng L −1 to low μg L −1 range concentrations. Although the environmental occurrence of the most common psychoactive compounds is well known, recently some investigations showed their potential toxicity toward non-target aquatic organisms. However, to date, these studies completely neglected that organisms in the real environment are exposed to a complex mixture, which could lead to dissimilar adverse effects. The present study investigated the oxidative alterations of the freshwater bivalve Dreissena polymorpha induced by a 14-d exposure to an environmentally relevant mixture of the most common illicit drugs found in the aquatic environment, namely cocaine (50 ng L −1 ), benzoylecgonine (300 ng L −1 ), amphetamine (300 ng L −1 ), morphine (100 ng L −1 ) and 3,4-methylenedioxymethamphetamine (50 ng L −1 ). The total oxidant status (TOS) was measured to investigate the increase in the reactive oxygen species’ levels, while the activity of antioxidant enzymes and glutathione S-transferase were measured to note the eventual imbalances between pro-oxidant and antioxidant molecules. In addition, oxidative damage was assessed by measuring the levels of lipid peroxidation and protein carbonylation. Significant time-dependent increases of all the antioxidant activities were induced by the mixture. Moreover, the illicit drug mixture significantly increased the levels of carbonylated proteins and caused a slight variation in lipid peroxidation. Our results showed that a mixture of illicit drugs at realistic environmental concentrations can impair the oxidative status of the zebra mussel, posing a serious hazard to the health status of this bivalve species. [ABSTRACT FROM AUTHOR]

• Target analysis and suspect screening of 311 new psychoactive substances in wastewater. • One of the largest study in Europe (12 cities, 7 countries) performed in the last years. • Synthetic cathinones and arylcyclohexyamines were most stable in wastewater. • Fentanyl, norfentanyl and 15 new psychoactive substances found in wastewater. • The highest mass loads were for 3-methylmethcathinone, especially during weekends. 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. [Display omitted] [ABSTRACT FROM AUTHOR]