Analytical methodologies for the detection of SARS-CoV-2 in wastewater: Protocols and future perspectives.

In March 2020 the World Health Organization announced a pandemic outbreak. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen for the coronavirus disease-19 (COVID-19) pandemic. The authorities worldwide use clinical science to identify infected people, but this approach is not able to track all symptomatic and asymptomatic cases due to limited sampling capacity of the testing laboratories. This drawback is eliminated by the Wastewater-Based Epidemiology (WBE) approach. In this review, we summarized the peer-reviewed published literature (available as of September 28, 2020), in the field of WBE. The commonly used steps (sampling, storage, concentration, isolation, detection) of the analytical protocols were identified. The potential limitations of each stage of the protocols and good practices were discussed. Finally, new methods for the efficient detection of SARS-CoV-2 were proposed.

Monitoring a large number of pesticides and transformation products in water samples from Spain and Italy.

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.

Wastewater-Based Epidemiology To Monitor Synthetic Cathinones Use in Different European Countries.

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.

Comparison of pharmaceutical, illicit drug, alcohol, nicotine and caffeine levels in wastewater with sale, seizure and consumption data for 8 European cities.

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.

Screening of pharmaceuticals and illicit drugs in wastewater and surface waters of Spain and Italy by high resolution mass spectrometry using UHPLC-QTOF MS and LC-LTQ-Orbitrap MS.

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.

Socioeconomic status and public health in Australia: A wastewater-based study.

Analysis of untreated municipal wastewater is recognized as an innovative approach to assess population exposure to or consumption of various substances. Currently, there are no published wastewater-based studies investigating the relationships between catchment social, demographic, and economic characteristics with chemicals using advanced non-targeted techniques. In this study, fifteen wastewater samples covering 27% of the Australian population were collected during a population Census. The samples were analysed with a workflow employing liquid chromatography high-resolution mass spectrometry and chemometric tools for non-target analysis. Socioeconomic characteristics of catchment areas were generated using Geospatial Information Systems software. Potential correlations were explored between pseudo-mass loads of the identified compounds and socioeconomic and demographic descriptors of the wastewater catchments derived from Census data. Markers of public health (e.g., cardiac arrhythmia, cardiovascular disease, anxiety disorder and type 2 diabetes) were identified in the wastewater samples by the proposed workflow. They were positively correlated with descriptors of disadvantage in education, occupation, marital status and income, and negatively correlated with descriptors of advantage in education and occupation. In addition, markers of polypropylene glycol (PPG) and polyethylene glycol (PEG) related compounds were positively correlated with housing and occupation disadvantage. High positive correlations were found between separated and divorced people and specific drugs used to treat cardiac arrhythmia, cardiovascular disease, and depression. Our robust non-targeted methodology in combination with Census data can identify relationships between biomarkers of public health, human behaviour and lifestyle and socio-demographics of whole populations. Furthermore, it can identify specific areas and socioeconomic groups that may need more assistance than others for public health issues. This approach complements important public health information and enables large-scale national coverage with a relatively small number of samples.

An integrated approach to MS-based identification and risk assessment of pharmaceutical biotransformation in wastewater.

The omnipresence of pharmaceuticals at relatively high concentrations (µg/L) in environmental compartments indicated their inadequate removal by wastewater treatment plants. As such, batch reactors seeded with activated sludge were set up to assess the biotransformation of metformin, ranitidine, lidocaine and atorvastatin. The main objective was to identify transformation products (TPs) through the establishment of an integrated workflow for suspect and non-target screening based on reversed phase liquid chromatography quadrupole-time-of-flight mass spectrometry. To support the identification, hydrophilic interaction liquid chromatography (HILIC) was used as a complementary tool, in order to enhance the completeness of the developed workflow by identifying the more polar TPs. The structure assignment/elucidation of the candidate TPs was mainly based on interpretation of MS/MS spectra. Twenty-two TPs were identified, with fourteen of them reaching high identification confidence levels (level 1: confirmed structure by reference standards and level 2: probable structure by library spectrum match and diagnostic evidence). Finally, retrospective analysis in influent and effluent wastewater was performed for the TPs for four consecutive years in wastewater sampled in Athens, Greece. The potential toxicological threat of the compounds to the aquatic environment was assessed and atorvastatin with two of its TPs showed a potential risk to the aquatic organisms.

Change in the chemical content of untreated wastewater of Athens, Greece under COVID-19 pandemic.

COVID-19 pandemic spread rapidly worldwide with unanticipated effects on mental health, lifestyle, stability of economies and societies. Although many research groups have already reported SARS-CoV-2 surveillance in untreated wastewater, only few studies evaluated the implications of the pandemic on the use of chemicals by influent wastewater analysis. Wide-scope target and suspect screening were used to monitor the effects of the pandemic on the Greek population through wastewater-based epidemiology. Composite 24 h influent wastewater samples were collected from the wastewater treatment plant of Athens during the first lockdown and analyzed by liquid chromatography mass spectrometry. A wide range of compounds was investigated (11,286), including antipsychotic drugs, illicit drugs, tobacco compounds, food additives, pesticides, biocides, surfactants and industrial chemicals. Mass loads of chemical markers were estimated and compared with the data obtained under non-COVID-19 conditions (campaign 2019). The findings revealed increases in surfactants (+196%), biocides (+152%), cationic quaternary ammonium surfactants (used as surfactants and biocides) (+331%), whereas the most important decreases were estimated for tobacco (-33%) and industrial chemicals (-52%). The introduction of social-restriction measures by the government affected all aspects of life.

Advanced multivariate techniques for the classification and pollution of marine sediments due to aquaculture.

Aquaculture production has globally increased and its environmental impact is not well understood and assessed yet. Therefore, in this work nine metals and metalloids (Cu, Cd, Pb, Hg, Ni, Fe, Mn, Zn and As) and three nutrients (P, N and C) that seem to accumulate in marine sediments, were determined under the fish cages (zero distance) and about 50 and 100 m away from them, in three aquacultures in Greece. The analysis of these data is crucial due to the negative impact of the intensive aquaculture activities on fish population, human health and marine environment. This study investigated the environmental impact associated with aquaculture cages on marine sediments, using Supervised Artificial Neural Networks (ANNs) in parallel with Classification Trees (CTs). Optimised models were constructed in order to detect the significance of each variable, predict the origin of the sediment samples and successfully visualise their results. Three popular ANN architectures, as multi-layer perceptrons (MLPs), radial basis function (RBF) and counter propagation artificial neural networks (CP-ANNs) were used to assess the impact of the intensive aquaculture activities on marine sediments. In addition, more traditional multivariate chemometric techniques like CTs were applied to the same data set for comparison purposes. The modelling study showed that P, N, Cu, Cd were the most critical (and polluting) factors of those metals studied. Moreover, single-element models achieved elevated predictive percentages. The results were justified due to the usual practices used for fish feeding or cages maintenance.

Wastewater-based epidemiology as a novel tool to evaluate human exposure to pesticides: Triazines and organophosphates as case studies.

Production and application of pesticides have risen remarkably in the last few decades. Even if they provide many benefits, they can be hazardous for humans and ecosystems when they are not used cautiously. Human exposure to pesticides is well documented, but new approaches are needed to boost the available information. This work proposes a new application of wastewater-based epidemiology (WBE) to assess the exposure of the general population to organophosphate and triazine pesticides (pyrethroid pesticides have already been validated). Several human urinary metabolites tested as WBE biomarkers, were suitable. Untreated wastewater samples from different European countries were analyzed by liquid chromatography-tandem mass spectrometry. Biomarker concentrations were converted to mass loads and used to back-calculate the local population's exposure to the parent pesticides, using specific correction factors developed in this study. Exposure to organophosphates and pyrethroids showed spatial and seasonal variations. Finally, pesticide exposure was estimated in twenty cities of ten European countries and compared with the acceptable daily intake, concluding that some populations might face health risks. The study confirms WBE as a suitable approach for assessing the average community exposure to pesticides and is a valuable complementary biomonitoring tool. WBE can provide valuable data for public health.

Investigation of Biotransformation Products of p-Methoxymethylamphetamine and Dihydromephedrone in Wastewater by High-Resolution Mass Spectrometry.

There is a paucity of information on biotransformation and stability of new psychoactive substances (NPS) in wastewater. Moreover, the fate of NPS and their transformation products (TPs) in wastewater treatment plants is not well understood. In this study, batch reactors seeded with activated sludge were set up to evaluate biotic, abiotic, and sorption losses of p-methoxymethylamphetamine (PMMA) and dihydromephedrone (DHM) and identify TPs formed during these processes. Detection and identification of all compounds was performed with target and suspect screening approaches using liquid chromatography quadrupole-time-of-flight mass spectrometry. Influent and effluent 24 h composite wastewater samples were collected from Athens from 2014 to 2020. High elimination rates were found for PMMA (80%) and DHM (97%) after a seven-day experiment and degradation appeared to be related to biological activity in the active bioreactor. Ten TPs were identified and the main reactions were O- and N-demethylation, oxidation, and hydroxylation. Some TPs were reported for the first time and some were confirmed by reference standards. Identification of some TPs was enhanced by the use of an in-house retention time prediction model. Mephedrone and some of its previously reported human metabolites were formed from DHM incubation. Retrospective analysis showed that PMMA was the most frequently detected compound.

Assessment of Environmental Pollution and Human Exposure to Pesticides by Wastewater Analysis in a Seven-Year Study in Athens, Greece.

Pesticides have been used in large amounts around the world for decades and are responsible for environmental pollution and various adverse effects on human health. Analysis of untreated wastewater can deliver useful information on pesticides' use in a particular area and allow the assessment of human exposure to certain substances. A wide-scope screening method, based on liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry, was applied, using both target and suspect screening methodologies. Daily composite influent wastewater samples were collected for seven or eight consecutive days in Athens between 2014 and 2020 and analyzed for 756 pesticides, their environmental transformation products and their human metabolites. Forty pesticides were quantified at mean concentrations up to 4.9 µg/L (tralkoxydim). The most abundant class was fungicides followed by herbicides, insect repellents, insecticides and plant growth regulators. In addition, pesticide transformation products and/or metabolites were detected with high frequency, indicating that research should be focused on them. Human exposure was evaluated using the wastewater-based epidemiology (WBE) approach and 3-ethyl-carbamoyl benzoic acid and cis-1,2,3,6-tetrahydrophthalimide were proposed as potential WBE biomarkers. Wastewater analysis revealed the presence of unapproved pesticides and indicated that there is an urgent need to include more transformation products in target databases.

Monitoring caffeine and nicotine use in a nationwide study in Italy using wastewater-based epidemiology.

Smoking cigarettes and drinking coffee are common habits in today's society. However, it is not easy to get up-to-date information on smoking prevalence and caffeine consumption as it is usually obtained from population surveys. To overcome this limitation and complement epidemiological information, we employed wastewater-based epidemiology (WBE) to gain a picture of the consumption of cigarettes and caffeine per day per person in Italy. A nationwide study was conducted by measuring two urinary metabolites of nicotine (cotinine and trans-3'-hydroxycotinine) and a caffeine metabolite (1,7-dimethyluric acid) in untreated wastewater from 16 cities. The spatial profiles of use depended mostly on the specific population habits in each city, not on the geographical area or the population size. The patterns of consumption were stable over the week, except in Milan where the use of both substances decreased on Sundays, probably because there were no commuters. In Milan, the use of nicotine decreased from 2013 to 2015. There was a significant relationship between smoking and consumption of products containing caffeine (mainly coffee), thus in cities where more cigarettes were smoked, more caffeine was drunk. These results are generally in accordance with findings from epidemiological studies, but provide some additional local profiles of use and closely follow changes over the years. This information could be useful for healthcare professionals and policy-makers to monitor progress towards the reduction of prevalence in tobacco use, and set up new health campaigns.

Assessment of human exposure to selected pesticides in Norway by wastewater analysis.

Pesticides offer many benefits for humanity and agriculture, but at the same time pose a potential risk to human health because of their widespread use and high biological activity. Human biomonitoring (HBM) studies are the main tool to investigate human exposure to pesticides and other chemicals, but face limitations such as sampling biases, long time to complete and high costs. Wastewater-based epidemiology (WBE) is an alternative approach that is centered on the chemical analysis of biomarkers of (pesticide) exposure in urban wastewater. The present study used WBE to assess human exposure to selected classes of pesticides, triazines, pyrethroids and organophosphates, in Norway. Untreated wastewater samples were collected from four cities, covering approximately 20% of the Norwegian population. The highest population weighted mass loads (mg/day/1000 inhabitants) were for alkyl phosphates and the lowest for triazines. Some differences were observed for the two metabolites, 2-isopropyl-6-methyl-4-pyrimidinol (IMPY) and 3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (DCCA), which were higher in the rural city of Hamar. WBE figures were comparable with HBM findings for the specific metabolite of chlorpyrifos and chlorpyrifos methyl (3,5,6-trichloro-2-pyridinol; TCPY) but were different for the alkyl phosphates. Pyrethroid intake was calculated and was lower than the acceptable daily intake in all the cities, indicating low risk for human health. This is the most extensive WBE study performed to date to assess national human exposure to pesticides. This study demonstrated that WBE has the potential to be a useful complementary biomonitoring tool for assessing population-wide exposure to pesticides, overcoming some of the limitations of HBM.

Use of legal and illegal substances in Malé (Republic of Maldives) assessed by wastewater analysis.

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.

Enantiomeric profiling of quinolones and quinolones resistance gene qnrS in European wastewaters.

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.

Exposure of an urban population to pesticides assessed by wastewater-based epidemiology in a Caribbean island.

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.

Comparison of phosphodiesterase type V inhibitors use in eight European cities through analysis of urban wastewater.

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.

Enantiomeric profiling of chiral illicit drugs in a pan-European study.

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.