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.

Evaluation of four different analytical tools to determine the regional origin of Gastrodia elata and Rehmannia glutinosa on the basis of metabolomics study.

Chemical profiles of medicinal plants could be dissimilar depending on the cultivation environments, which may influence their therapeutic efficacy. Accordingly, the regional origin of the medicinal plants should be authenticated for correct evaluation of their medicinal and market values. Metabolomics has been found very useful for discriminating the origin of many plants. Choosing the adequate analytical tool can be an essential procedure because different chemical profiles with different detection ranges will be produced according to the choice. In this study, four analytical tools, Fourier transform near­infrared spectroscopy (FT-NIR), 1H-nuclear magnetic resonance spectroscopy (1H­NMR), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectroscopy (GC-MS) were applied in parallel to the same samples of two popular medicinal plants (Gastrodia elata and Rehmannia glutinosa) cultivated either in Korea or China. The classification abilities of four discriminant models for each plant were evaluated based on the misclassification rate and Q2 obtained from principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS­DA), respectively. 1H-NMR and LC-MS, which were the best techniques for G. elata and R. glutinosa, respectively, were generally preferable for origin discrimination over the others. Reasoned by integrating all the results, 1H-NMR is the most prominent technique for discriminating the origins of two plants. Nonetheless, this study suggests that preliminary screening is essential to determine the most suitable analytical tool and statistical method, which will ensure the dependability of metabolomics-based discrimination.

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.

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.

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.

Wastewater-based epidemiology to assess pan-European pesticide exposure.

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

Estimation of caffeine intake from analysis of caffeine metabolites in wastewater.

Caffeine metabolites in wastewater were investigated as potential biomarkers for assessing caffeine intake in a population. The main human urinary metabolites of caffeine were measured in the urban wastewater of ten European cities and the metabolic profiles in wastewater were compared with the human urinary excretion profile. A good match was found for 1,7-dimethyluric acid, an exclusive caffeine metabolite, suggesting that might be a suitable biomarker in wastewater for assessing population-level caffeine consumption. A correction factor was developed considering the percentage of excretion of this metabolite in humans, according to published pharmacokinetic studies. Daily caffeine intake estimated from wastewater analysis was compared with the average daily intake calculated from the average amount of coffee consumed by country per capita. Good agreement was found in some cities but further information is needed to standardize this approach. Wastewater analysis proved useful to providing additional local information on caffeine use.

Measuring biomarkers in wastewater as a new source of epidemiological information: Current state and future perspectives.

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.

Liquid chromatography-tandem mass spectrometry determination of synthetic cathinones and phenethylamines in influent wastewater of eight European cities.

The popularity of new psychoactive substances (NPS) has grown in recent years, with certain NPS commonly and preferentially consumed even following the introduction of preventative legislation. With the objective to improve the knowledge on the use of NPS, a rapid and very sensitive method was developed for the determination of ten priority NPS (N-ethylcathinone, methylenedioxypyrovalerone (MDPV), methylone, butylone, methedrone, mephedrone, naphyrone, 25-C-NBOMe, 25-I-NBOMe and 25-B-NBOMe) in influent wastewater. Sample clean-up and pre-concentration was made by off-line solid phase extraction (SPE) with Oasis MCX cartridges. Isotopically labelled internal standards were used to correct for matrix effects and potential SPE losses. Following chromatographic separation on a C18 column within 6 min, the compounds were measured by tandem mass spectrometry in positive ionization mode. The method was optimised and validated for all compounds. Limits of quantification were evaluated by spiking influent wastewater samples at 1 or 5 ng/L. An investigation into the stability of these compounds in influent wastewater was also performed, showing that, following acidification at pH 2, all compounds were relatively stable for up to 7 days. The method was then applied to influent wastewater samples from eight European countries, in which mephedrone, methylone and MDPV were detected. This work reveals that although NPS use is not as extensive as for classic illicit drugs, the application of a highly sensitive analytical procedure makes their detection in wastewater possible. The developed analytical methodology forms the basis of a subsequent model-based back-calculation of abuse rate in urban areas (i.e. wastewater-based epidemiology).

Comparative measurement and quantitative risk assessment of alcohol consumption through wastewater-based epidemiology: An international study in 20 cities.

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

Increased levels of the oxidative stress biomarker 8-iso-prostaglandin F2α in wastewater associated with tobacco use.

Wastewater analysis has been demonstrated to be a complementary approach for assessing the overall patterns of drug use by a population while the full potential of wastewater-based epidemiology has yet to be explored. F2-isoprostanes are a prototype wastewater biomarker to study the cumulative oxidative stress at a community level. In this work, 8-iso-prostaglandin F2α (8-iso-PGF2α) was analysed in raw 24 h-composite wastewater samples collected from 4 Norwegian and 7 other European cities in 2014 and 2015. Using the same samples, biomarkers of alcohol (ethyl sulfate) and tobacco (trans-3'-hydroxycotinine) use were also analysed to investigate any possible correlation between 8-iso-PGF2α and the consumption of the two drugs. The estimated per capita daily loads of 8-iso-PGF2α in the 11 cities ranged between 2.5 and 9.9 mg/day/1000 inhabitants with a population-weighted mean of 4.8 mg/day/1000 inhabitants. There were no temporal trends observed in the levels of 8-iso-PGF2α, however, spatial differences were found at the inter-city level correlating to the degree of urbanisation. The 8-iso-PGF2α mass load was found to be strongly associated with that of trans-3'-hydroxycotinine while it showed no correlation with ethyl sulfate. The present study shows the potential for 8-iso-PGF2α as a wastewater biomarker for the assessment of community public health.

Liquid chromatography-high resolution mass spectrometry with immunoaffinity clean-up for the determination of the oxidative stress biomarker 8-iso-prostaglandin F2alpha in wastewater.

A reliable oxidative stress biomarker, 8-iso-prostaglandin F2α (8-iso-PGF2α), was for the first time quantitatively analysed in wastewater using an analytical method consisting of liquid chromatography-high resolution mass spectrometry coupled to immunoaffinity clean-up (IAC-LC-HRMS). Factors influencing the method's robustness were investigated, including analyte stability in sewage and enzymatic deconjugation with ß-glucuronidase. The IAC-LC-HRMS method was linear over the range of 0.1-100ng/mL with correlation coefficient (R(2)) of 0.999. The quantification limits were sufficiently low to detect 8-iso-PGF2α in sewage (method quantification limit of 0.3ng/L) and precision, expressed as relative standard deviation was less than 7% and the accuracy expressed as relative recovery was in the 103-113% range. As a result, the application of the method to 24-h composite wastewater samples from Oslo showed 8-iso-PGF2α concentrations of 18.9-23.3ng/L for 8 days in March 2015. This study demonstrates a standard method to analyse 8-iso-PGF2α in sewage that will contribute to the further investigation of the potential use of 8-iso-PGF2α as a sewage biomarker for assessing the status of community health.

Using biomarkers in wastewater to monitor community drug use: a conceptual approach for dealing with new psychoactive substances.

Data obtained from the analysis of wastewater from large-scale sewage treatment plants has been successfully applied to study trends in the use of classical illicit drugs such as cocaine, but the dynamic nature of the new psychoactive substances (NPS) market presents a unique set of challenges to epidemiologists. In an attempt to overcome some of the challenges, this paper presents a framework whereby a collection of tools and alternative data-sources can be used to support the design and implementation of wastewater-based studies on NPS use. Within this framework the most likely and most suitable biomarkers for a given NPS are predicted via in-silico metabolism, biotransformation and sorption models. Subsequent detection and confirmation of the biomarkers in samples of wastewater are addressed via high-resolution mass spectrometry (HRMS). The proposed framework is applied to a set of test substances including synthetic cannabinoids and cathinones. In general, the in-silico models predict that transformation via N-dealkylation and hydroxylation is likely for these compounds, and that adsorption is expected to be significant for cannabinoids in wastewater. Screening via HRMS is discussed with examples from the literature, and common-fragment searching and mass-defect filtering are successfully performed on test samples such that spectral noise is removed to leave only the information that is most likely to be related to the NPS biomarkers. HRMS screening is also applied to a set of pissoir-sourced wastewater samples and a total of 48 pharmaceuticals and drugs including 1-(2-methoxyphenyl)piperazine (oMeOPP) are identified. The framework outlined in this paper can provide an excellent means of maximizing the chances of success when identifying and detecting biomarkers of NPS in wastewater.

An in situ benzoylation-dispersive liquid-liquid microextraction method based on solidification of floating organic droplets for determination of biogenic amines by liquid chromatography-ultraviolet analysis.

A novel analytical method consisting of in situ derivatization combined with liquid phase microextraction followed by liquid chromatography-ultraviolet detection (LC-UV) was developed to determine the biogenic amines (BAs) of alcoholic beverages. Nine BAs (putrescine, cadaverine, 1,3-diaminopropane, tryptamine, phenylethylamine, spermidine, spermine, histamine, and tyramine) were derivatized in situ with benzoyl chloride, extracted by dispersive liquid-liquid microextraction based on solidification of floating organic droplets (DLLME-SFO), and then chromatographed by LC-UV. Factors influencing the derivatization and extraction efficiency were optimized, including the reaction buffer pH and concentration, amount of derivatization reagent, reaction time, types and volumes of extraction and dispersive solvents, and extraction time. Under the optimized conditions, the method was linear over 0.05-8.0µgmL(-1) with an r(2)≥0.992 and exhibited intra- and inter-day precision less than 8.8% and 11.5%, respectively. The limit of detection ranged between 0.005 and 0.01µgmL(-1). The developed method using a basic LC-UV system is sensitive, rapid, convenient, green, and cost-effective. Moreover, it is versatile and practical for the analysis of BAs, as demonstrated by the successful application in four different types of popular alcoholic beverages (white wine, red wine, rice wine, and beer).

Combined application of dispersive liquid-liquid microextraction based on the solidification of floating organic droplets and charged aerosol detection for the simple and sensitive quantification of macrolide antibiotics in human urine.

A novel analytical method combining dispersive liquid-liquid microextraction based on the solidification of floating organic droplets (DLLME-SFO) and liquid chromatography with charged aerosol detection (LC-CAD) was established. For the first time, CAD was applied for the detection of macrolide antibiotics lacking chromophores. Parameters influencing the microextraction efficiency were systematically investigated, and the optimized microextraction conditions yielded high enrichment factors in the range of 60-106. The combined application of DLLME-SFO and LC-CAD provided the sensitivity of the method, expressed as the limit of detection (LOD), as low as 10 to 40ngmL(-1) and intra-day and inter-day precisions below 8.7% and 12.6%, respectively. The measured absolute recovery values were approximately 100%, indicating that the extraction efficiency was very high. Direct comparisons of the liquid-liquid extraction and organic solvent precipitation methods demonstrated that the proposed method was more sensitive, specific, rapid, and environmentally friendly for the determination of five macrolide antibiotics in human urine. The results suggest that the combined use of DLLME-SFO and LC-CAD may be applicable to the analysis of various compounds with poor to no chromophores in complex matrices.