Frederic Been

Population Normalization with Ammonium in Wastewater-Based Epidemiology: Application to Illicit Drug Monitoring

Fluctuations in ammonium (NH4+), measured as NH4-N loads using an ion-selective electrode installed at the inlet of a sewage treatment plant, showed a distinctive pattern which was associated to weekly (i.e., commuters) and seasonal (i.e., holidays) fluctuations of the population. Moreover, population size estimates based on NH4-N loads were lower compared to census data. Diurnal profiles of benzoylecgonine (BE) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) were shown to be strongly correlated to NH4-N. Characteristic patterns, which reflect the prolonged nocturnal activity of people during the weekend, could be observed for BE, cocaine, and a major metabolite of MDMA (i.e., 4-hydroxy-3-methoxymethamphetamine). Additional 24 h composite samples were collected between February and September 2013. Per-capita loads (i.e., grams per day per 1000 inhabitants) were computed using census data and NH4-N measurements. Normalization with NH4-N did not modify the overall pattern, suggesting that the magnitude of fluctuations in the size of the population is negligible compared to those of illicit drug loads. Results show that fluctuations in the size of the population over longer periods of time or during major events can be monitored using NH4-N loads: either using raw NH4-N loads or population size estimates based on NH4-N loads, if information about site-specific NH4-N population equivalents is available.

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 Analysis of Biomarkers of Exposure to Phosphorus Flame Retardants in Wastewater to Monitor Community-Wide Exposure

Phosphorus flame retardants and plasticizers (PFRs) are increasingly used in consumer goods, from which they can leach and pose potential threats to human health. Monitoring human exposure to these compounds is thus highly relevant. Current assessment of exposure through analysis of biological matrices is, however, tedious as well as logistically and financially demanding. Analysis of selected biomarkers of exposure to PFRs in wastewater could be a simple and complementary approach to monitoring, over space and time, exposure at the population level. An analytical procedure, based on solid-phase extraction (SPE) and liquid chromatography coupled to tandem mass spectrometry, was developed and validated to monitor the occurrence in wastewater of human exposure biomarkers of 2-ethylhexyldiphenyl phosphate (EHDPHP), tris(2-butoxyethyl) phosphate (TBOEP), triphenyl phosphate (TPHP), tris(2-chloroisopropyl) phosphate (TCIPP), and tris(2-chloroethyl) phosphate (TCEP). Various SPE sorbents and extraction protocols were evaluated, and for the optimized method, absolute extraction recoveries ranged between 46% and 100%. Accuracy and precision were satisfactory for the selected compounds. Method detection limits ranged from 1.6 to 19 ng L-1. Biomarkers of exposure to PFRs were measured for the first time in influent wastewater. Concentrations in samples collected in Belgium ranged from below the limit of quantitation to 1072 ng L-1, with 2-ethylhexyl phenyl phosphate (EHPHP) and TCEP being the most abundant. Per capita loads of target biomarkers varied greatly, suggesting potential differences in exposure between the investigated communities. The developed method allowed implementation of the concepts of human biomonitoring at the community scale, opening the possibility to assess population-wide exposure to PFRs.

Profiles and changes in stimulant use in Belgium in the period of 2011-2015.

Adapting illicit drug policy strategies requires detailed knowledge on types and amounts of substances consumed by the target population. In this study, we applied wastewater-based epidemiology to detect spatio-temporal changes in the relative amounts of stimulants (amphetamine, methamphetamine, methylenedioxymethamphetamine (MDMA), cocaine) used in seven locations in Belgium over 2011-2015. Clear geographical differences were observed with stimulant users in large cities (Antwerp, Brussels) showing a preference for cocaine, while amphetamine use was most abundant in smaller cities (Geraardsbergen, Koksijde, Lier, Ninove, Ostend). Results obtained across õdifferent years revealed that the investigated substances had a stable share in the total amount of stimulants used, suggesting that habits of stimulant use remained constant, although differences in absolute amounts were observed across years. Investigation of the weekly pattern in stimulant use showed an increase in the use of MDMA on the weekends compared to cocaine and amphetamine.

Keratinous matrices for the assessment of drugs of abuse consumption: A correlation study between hair and nails

Keratinous matrices - hair and nails - accumulate substances over time and allow retrospective investigation of past consumption. Analysis of these matrices can provide information complementary to blood and urine analysis or can be used as standalone. So far, research has primarily focused on the detection of substances in hair, while studies in nails are scarce. In this study, we assessed concentrations of drugs of abuse and their metabolites in hair, fingernails, and toenails collected from the same individuals to evaluate differences and correlations between matrices. A total of 26 hair, 24 fingernail, and 18 toenail samples were collected. Samples were analysed by a validated liquid chromatography-tandem mass spectrometry method able to simultaneously detect the following compounds: amphetamine (AMP), methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine, morphine (MOR), codeine (COD), 6-monoacetylmorphine (6-MAM), methadone (MTD), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine (COC), benzoylecgonine (BE), and ecgonine methyl ester (EME). Strong positive correlations between hair, fingernails, and toenails were present for COC, BE, EME, AMP and MDMA. MOR, COD, 6-MAM, MTD and EDDP showed positive trends. Concentrations were generally higher in nails compared to hair. Ratios between parent compounds and their metabolites were assessed for 6-MAM/MOR, EDDP/MTD, BE/COC and EME/COC. Preliminary cut-off concentrations for COC, BE, EME and AMP in fingernails and toenails were proposed. In light of these results, nails can be considered as a useful alternative to hair for monitoring of long-term drug consumption. However, care should be taken regarding the variability in the accumulation of compounds between the matrices.

Levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in raw wastewater as an innovative perspective for investigating population-wide exposure to third-hand smoke

Tobacco smoking is the major cause of many chronic diseases, especially lung cancer. Knowledge about population-wide tobacco use and exposure is essential to characterise its burden on public health and evaluate policy efficacy. Obtaining such knowledge remains challenging with current methods (e.g., surveys, biomonitoring) but can be achievable with wastewater analysis, a promising tool of retrieving epidemiology information. This study examined population-wide exposure to tobacco toxicants and carcinogens through wastewater analysis and explored relationships among these chemicals. Cotinine, trans-3′-hydroxycotinine, anabasine, anatabine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were analysed in samples from Greece, Switzerland and Belgium, where tobacco control policies are different. Measured per-capita mass loads were ranked as: nicotine biomarkers ≫ tobacco markers > carcinogens. Relationships between nicotine biomarkers and tobacco markers implied substantial use of non-tobacco nicotine items besides tobacco products. Geographic profiles of tobacco markers revealed higher levels in Geneva and Athens than Geraardsbergen and Ninove. Environmental third-hand smoke led to NNK detection, with elevated levels observed in Athens where indoor smoking is widespread, posing potential health risks to the population. Our novel outcomes are relevant for public health authorities as they provide indications about external exposure and can thus be used to plan and evaluate tobacco control policies.

Hair as an alternative matrix to monitor human exposure to plasticizers – Development of a liquid chromatography - tandem mass spectrometry method

The application and production of alternative plasticizers (APs) has been increasing in the last decade in replacement of conventional phthalates due to their toxicity. This calls for the development of non-invasive monitoring approaches to assess human exposure to APs. A method for the simultaneous measurement of exposure biomarkers of di(2‑propylheptyl) phthalate (DPHP), di(isononyl)cyclohexane‑1,2‑dicarboxylate (DINCH), di(2‑ethylhexyl) terephthalate (DEHTP) and di‑2‑ethylhexyl adipate (DEHA) in hair samples was developed and validated in this study. Prior to the analysis, the hair samples were washed in acetone and ultrapure water and pulverized to powder. Further, a solid-liquid and solid-phase extraction, followed by quantification using liquid chromatography - tandem mass spectrometry (LC-MS/MS) was performed. The method was validated in terms of recovery, matrix effects, carry-over, linearity, limits of quantification, within- and between-run precision and trueness. Satisfying results were obtained for all targeted compounds, except for mono(2‑ethylhexyl) adipate (MEHA), which was monitored only qualitatively. The optimized method was implemented in a pilot biomonitoring study with hair samples from 9 healthy volunteers. Detection frequencies of seven metabolites ranged from 11% to 100%. Mono(2‑ethylhexyl) terephthalate (MEHTP) and mono(2‑ethyl‑5‑oxohexyl) adipate (oxo-MEHA) were found in all hair samples. More hydrophobic monoester metabolites were found to be incorporated in hair to a greater extent compared to their oxidized counterparts. Obtained results show that the developed method can detect AP metabolites in hair, supporting the use of this alternative matrix in human biomonitoring.

Mining the Chemical Information on Urban Wastewater: Monitoring Human Exposure to Phosphorus Flame Retardants and Plasticizers

At the individual level, exposure to contaminants is generally assessed through the analysis of specific biomarkers in biological matrices. However, these studies are costly and logistically demanding, limiting their applicability to monitor population-wide exposure over time and space. By focusing on a selection of exposure biomarkers to phosphorus flame retardants and plasticizers (PFRs), this study aims to explore the possibility of using wastewater as a complementary source of information about exposure. Wastewater samples were collected from five cities in Europe and analyzed using a previously established method. Substantial differences in biomarker levels were observed between the investigated catchments, suggesting differences in exposure. Time trends in biomarkers observed between 2013 and 2016 were found to agree with results from human biomonitoring studies and reports about production volumes. Using Monte Carlo simulations, average urinary concentrations were estimated. These were generally higher compared to results from human biomonitoring studies. Various explanations for these differences were formulated (i.e., other excretion routes, external sources and different sampling approaches). Obtained results show that wastewater analysis provides unique information about geographical and temporal differences in exposure, which would be difficult to gather using other monitoring tools.

Analysis of N,N-dimethylamphetamine in wastewater - a pyrolysis marker and synthesis impurity of methamphetamine

The increased availability of high purity crystalline methamphetamine (MA) in Australia raised concerns because of high dosages and its potential consumption through inhalation. The present work investigates the possibility of using wastewater levels of N,N-dimethylamphetamine (DMA), a pyrolysis by-product, as an indirect indicator of MA smoking. A dedicated liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QToF-MS) method was set up to detect and quantify DMA in wastewater samples. Wastewater samples were collected from 8 locations across Australia during the period 2011-2016. Data about the abundance of DMA in MA seizures as well as in residues from drug paraphernalia were obtained from forensic laboratories in Australia. DMA/MA ratios measured in wastewater ranged from 0.0001 to 0.09 (median 0.007). DMA/MA ratios in bulk seizures are generally below 0.0025, with a median value of 0.0004, whilst residues in paraphernalia ranged from 0.031 to 3.37. DMA/MA ratios in wastewater decreased between 2011 and 2016, in parallel to an increase in MA loads. Furthermore, wastewater analyses highlighted a strong positive correlation between DMA/MA ratios and per capita MA use (Pearsons correlation ρ= 0.61, p-value <0.001). Nonetheless, geographical specificities could be highlighted between the investigated locations. The obtained data could help authorities detect hot spots of drug use as well as to plan specific intervention campaigns to tackle the issue. In future, simultaneous analysis of DMA and MA in both wastewater and seizures could improve our understanding about MA use and its consumption patterns.