A new analytical framework for multi-residue analysis of chemically diverse endocrine disruptors in complex environmental matrices utilising ultra-performance liquid chromatography coupled with high-resolution tandem quadrupole time-of-flight mass spectrometry.

This manuscript presents a comprehensive analytical framework for identification and quantification of chemically diverse endocrine disrupting chemicals (EDCs) used in personal care and consumer products in diverse solid and liquid environmental matrices with an ultimate goal of evaluating public exposure to EDCs via water fingerprinting. Liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry (UHPLC-ESI-MS/MS) was used for targeted analysis of selected EDCs as well as to identify and quantify a few metabolites using post-acquisition data mining. Solid-phase extraction (SPE) was applied to liquid matrices in order to reduce matrix effects and provide required sample concentration and ultimately, high sensitivity and selectivity of measurements. SPE recoveries in liquid samples ranged from 49 to 140% with method quantification limits not exceeding 1 ng L-1 for the majority of EDCs. Microwave-assisted extraction (MAE) was applied to solid samples and when followed by SPE, it permitted the analysis of EDCs in digested sludge. MAE/SPE recoveries varied from 11 to 186% and MQLs between 0.03 and 8.1 ng g-1 with the majority of compounds showing MQLs below 2 ng g-1. Mass error for quantifier and qualifier ions was below 5 ppm when analysing river water and effluent wastewater and below 10 ppm when analysing influent wastewater and solid samples. The method was successfully applied to environmental samples, with 33 EDCs identified and quantified in wastewater and receiving waters. In addition, several EDCs were found in digested sludge, which confirms that for a more comprehensive understanding of exposure patterns and environmental impact, analysis of solids cannot be neglected. Finally, post-acquisition data mining permitted the identification and quantification of a metabolite of BPA and the identification of a metabolite of 4-Cl-3-methylphenol. Graphical abstract ᅟ.

New Analytical Framework for Verification of Biomarkers of Exposure to Chemicals Combining Human Biomonitoring and Water Fingerprinting.

Molecular epidemiology approaches in human biomonitoring are powerful tools that allow for verification of public exposure to chemical substances. Unfortunately, due to logistical difficulties and high cost, they tend to evaluate small study groups and as a result might not provide comprehensive large scale community-wide exposure data. Urban water fingerprinting provides a timely alternative to traditional approaches. It can revolutionize the human exposure studies as urban water represents collective community-wide exposure. Knowledge of characteristic biomarkers of exposure to specific chemicals is key to the successful application of water fingerprinting. This study aims to introduce a novel conceptual analytical framework for identification of biomarkers of public exposure to chemicals via combined human metabolism and urban water fingerprinting assay. This framework consists of the following steps: (1) in vitro HLM/S9 assay, (2) in vivo pooled urine assay, (3) in vivo wastewater fingerprinting assay, (4) analysis with HR-MSMS, (5) data processing, and (6) selection of biomarkers. The framework was applied and validated for PCMC (4-chloro-m-cresol), household derived antimicrobial agent with no known exposure and human metabolism data. Four new metabolites of PCMC (hydroxylated, sulfated/hydroxylated, sulfated PCMC, and glucuronidated PCMC) were identified using the in vitro HLM/S9 assay. But only one metabolite, sulfated PCMC, was confirmed in wastewater and in urine. Therefore, our study confirms that water fingerprinting is a promising tool for biomarker selection and that in vitro HLM/S9 studies alone, although informative, do not provide high accuracy results. Our work also confirms, for the first time, human internal exposure to PCMC.

Estimation of community-wide multi-chemical exposure via water-based chemical mining: Key research gaps drawn from a comprehensive multi-biomarker multi-city dataset.

This paper explores the strong potential of chemical mining of wastewater for markers of community-wide intake of wide-ranging harmful chemicals belonging to several usage groups: industrial chemicals, personal care products, pesticides, illicit drugs, lifestyle chemicals and prescription pharmaceuticals as a proxy for multi-chemical community-wide exposure. An estimation of chemical intake in five contrasting town/cities based in the Avon River catchment in the South-West UK was undertaken. High-resolution spatiotemporal pharmaceutical prescription databases were used for system calibration, both in terms of biomarker selection and its correction factor, as well as for the overall system performance evaluation, both spatially and temporality. Only metabolism data accounting for phase two metabolism provided correct estimates of pharma intake. Using parent compounds as XCRs (xenobiotic compounds residue) was found to overestimate exposure due to an inclusion of directly disposed (unused) drugs. Spatiotemporal trends in XC intake were observed as a result of occupational exposure (higher bisphenol A (BPA) intake during weekday), and lifestyle choices (higher cocaine and pyrethroid pesticides intake during weekend). WBE is not intended to estimate individual exposure to chemicals. It can however provide estimates at a community level, and as a result, it has the potential to be developed into an early warning system, a powerful tool for large scale screening studies identifying communities at risk and in need of high resolution individual testing at a localised scale.

Micropollutant fluxes in urban environment - A catchment perspective.

This study provided a holistic understanding of the sources, fate and behaviour of 142 compounds of emerging concern (CECs) throughout a river catchment impacted by 5 major urban areas. Of the incoming 169.3 kg d-1 of CECs entering the WwTWs, 167.9 kg d-1 were present in the liquid phase of influent and 1.4 kg d-1 were present in the solid phase (solid particulate matter, SPM). Analysis of SPM was important to determine accurate loads of incoming antidepressants and antifungal compounds, which are primarily found in the solid phase. Furthermore, these classes and the plasticiser, bisphenol A (BPA) were the highest contributors to CEC load in digested solids. Population normalised loads showed little variation across the catchment at 154 ± 12 mg d-1 inhabitant-1 indicating that population size is the main driver of CECs in the studied catchment. Across the catchment 154.6 kg d-1 were removed from the liquid phase during treatment processes. CECs discharged into surface waters from individual WwTWs contributed between 0.19 kg d-1 at WwTW A to 7.3 kg d-1 at WwTW E, which correlated strongly with the respective contributing populations. Spatial and temporal variations of individual CECs and their respective classes were found in WwTW influent (both solid (influentSPM) and liquid phases (influentAQ)) throughout the catchment, showing that different urban areas impact the catchment in different ways, with key variables being lifestyle, use of over-the-counter pharmaceuticals and industrial activity. Understanding of both spatial and temporal variation of CECs at the catchment level helped to identify possible instances of direct disposal, as in the case of carbamazepine. Analysis of surface waters throughout the catchment showed increasing mass loads of CECs from upstream of WwTW A to downstream at WwTW D, showing clear individual contributions from WwTWs. Many CECs were ubiquitous throughout the river water in the catchment. Daily loads ranged from 0.005 g d-1 (ketamine, WwTW A) up to 1890.3 g d-1 (metformin, WwTW C) for the 84/138 CECs that were detected downstream of the WwTWs. For metformin this represents the equivalent of ∼1,890 tablets (1,000 mg per tablet) dissolved in the river water downstream of WwTW C.

(Fluoro)quinolones and quinolone resistance genes in the aquatic environment: A river catchment perspective.

This study provides an insight into the prevalence of (fluoro)quinolones (FQs) and their specific quinolone qnrS resistance gene in the Avon river catchment area receiving treated wastewater from 5 wastewater treatment plants (WWTPs), serving 1.5 million people and accounting for 75% of inhabitants living in the catchment area in the South West of England.. Ofloxacin, ciprofloxacin, nalidixic acid and norfloxacin were found to be ubiquitous with daily loads reaching a few hundred g/day in wastewater influent and tens of g/day in receiving waters. This was in contrast to other FQs analysed: flumequine, nadifloxacin, lomefloxacin, ulifloxacin, prulifloxacin, besifloxacin and moxifloxacin, which were hardly quantified. Enantiomeric profiling revealed that ofloxacin was enriched with the S-(-)-enantiomer, likely deriving from its prescription as the more potent enantiomerically pure levofloxacin, alongside racemic ofloxacin. While ofloxacin's enantiomeric fraction (EF) remained constant, high stereoselectivity was observed in the case of its metabolite ofloxacin-N-oxide. The removal efficiency of quinolones during wastewater treatment at 5 WWTPs utilising either trickling filters (TF) or activated sludge (AS), was compound and wastewater treatment process dependent, with AS providing better efficiency than TF. The qnrS resistance gene was ubiquitous in wastewater. Its removal was WWTP treatment process dependent with TF performing best and resulting in significant removal of the gene (from 28 to 75%). AS underperformed with only 9% removal in the case of activated sludge and actual increase in the gene copy number within sequencing batch reactors (SBRs). Interestingly, the data suggests that higher removal of antibiotics could be linked with high prevalence of the gene (SBR and WWTP E) and vice versa, low removal of antibiotic is correlated with lower prevalence of the gene in wastewater effluent (TF, WWTP B and D). This is especially prominent in the case of ofloxacin and could indicate that AS might be facilitating antimicrobial resistance (AMR) prevalence to higher extent than TF. Wastewater-based epidemiology (WBE) was also applied to monitor any potential misuse (e.g. direct disposal) of FQs in the catchment. In most cases higher use of antibiotics with respect to official statistics (i.e. ciprofloxacin, ofloxacin) was observed, which suggests that FQs management practice require further attention.

Assessment of bisphenol-A in the urban water cycle.

The plasticizer bisphenol-A (BPA) is common to municipal wastewaters and can exert toxicity to exposed organisms in the environment. Here BPA concentration at 5 sewage treatment works (STW) and distribution throughout a river catchment in South West UK were investigated. Sampling sites included influent and effluent wastewater (n = 5), river water (n = 7) and digested sludge (n = 2) which were monitored for 7 consecutive days. Findings revealed average BPA loads in influent wastewater at two STWs were 10-37 times greater than the other wastewaters monitored. Concentrations up to ~100 µg L-1 were measured considerably higher than previously reported for municipal wastewaters. Temporal variability throughout the week (i.e., highest concentrations during weekdays) suggests these high concentrations are linked with industrial activity. Despite ≥90% removal during wastewater treatment, notable concentrations remained in tested effluent (62-892 ng L-1). However, minimal impact on BPA concentrations in river water was observed for any of the effluents. The maximum BPA concentration found in river water was 117 ng L-1 which is considerably lower than the current predicted no effect concentration of 1.6 µg L-1. Nevertheless, analysis of digested sludge from sites which received these elevated BPA levels revealed average concentrations of 4.6 ±â€¯0.3 and 38.7 ±â€¯5.4 µg g-1. These sludge BPA concentrations are considerably greater than previously reported and are attributed to the high BPA loading in influent wastewater. A typical sludge application regime to agricultural land would result in a predicted BPA concentration of 297 ng g-1 in soil. Further studies are needed on the toxicological thresholds of exposed terrestrial organisms in amended soils to better assess the environmental risk here.

Estimation of community-wide exposure to bisphenol A via water fingerprinting.

Molecular epidemiology in human biomonitoring allows for verification of public exposure to chemical substances. Unfortunately, due to logistical difficulties and high cost, it evaluates only small study groups and as a result does not provide comprehensive large scale community-wide exposure data. Wastewater fingerprinting utilizing metabolic biomarkers of exposure that are excreted collectively by studied populations into urine and ultimately into the community's wastewater, provides a timely alternative to traditional approaches. This study aimed to provide comprehensive spatiotemporal community-wide exposure to bisphenol A (BPA, including BPA intake) using wastewater fingerprinting. Wastewater fingerprinting was undertaken using high resolution mass spectrometry retrospective data mining of characteristic BPA human metabolism marker (bisphenol A sulphate), applied to a large geographical area of 2000 km2 and a population of ~1.5 million served by 5 WWTPs (wastewater treatment plants) accounting for >75% of the overall population in the studied catchment. Community-wide BPA intake was found to be below temporary tolerable daily intake (t-TDI) level of 4 µg kg-1 day-1 set by the European Food Safety Agency (EFSA) suggesting overall low exposure at 3 WWTPs serving residential areas with low industrial/commercial presence. However, at two WWTPs serving communities with higher industrial/commercial presence, higher BPA sulphate loads corresponding to higher (up to 14 times) BPA intakes (exceeding 10 µg kg-1 day-1 at one WWTP and reaching 50 µg kg-1 day-1 at the second WWTP) were observed and they are likely linked with occupational exposure. Characteristic temporal variations of BPA intake were noted in most studied WWTPs with the lowest intake occurring during weekends and the highest during weekdays.

Stereoisomeric profiling of chiral pharmaceutically active compounds in wastewaters and the receiving environment - A catchment-scale and a laboratory study.

Chiral pharmaceutically active compounds (cPACs) are not currently governed by environmental regulation yet are expected to be in the future. As cPACs can exert stereospecific toxicity in the aquatic environment, it is essential to better understand their stereoselective behaviour here. Therefore, this study aims to provide a new perspective towards comprehensive evaluation of cPACs at a river catchment level, including their stereochemistry as a chemical phenomenon driving fate of chiral molecules in the environment. A large spatial and temporal monitoring program was performed in Southwest England. It included 5 sewage treatment works and the receiving waters of the largest river catchment in Southwest England. Simultaneously, lab-scale microcosm studies in simulated activated sludge bioreactors and river water microcosm were performed to evaluate stereoselective degradation of cPACs. A multi-residue enantioselective method allowed the analysis of a total of 18 pairs of enantiomers and 3 single enantiomers in wastewater and river water samples. Our monitoring program revealed: (1) spatial and temporal variations of cPACs in influent wastewaters resulting from different patterns of usage as well as an (2) enantiomeric enrichment of cPACs, likely due to human metabolism, despite their commercialization as racemic mixtures. A similar chiral signature was observed in effluent and receiving waters. Stereoselective degradation was observed in trickling filters (TF) for naproxen, ketoprofen, cetirizine and 10,11-dihydroxy-10-hydroxycarbamazepine, in sequencing batch reactors (SBR) for ifosfamide and in activated sludge (AS) for cetirizine. The extent of enantiomer-specific fate was wastewater treatment dependent in the case of naproxen (TF showed higher stereoselectivity than AS and SBR) and cetirizine (TF and AS showed higher stereoselectivity than SBR) due to differing microbial population. Furthermore, stereoselective degradation of naproxen was highly variable among STWs using similar treatments (TF) and operating in the same region. Microbial stereoselective degradation was also confirmed by both activated and river water simulated microcosm for chloramphenicol, ketoprofen, indoprofen, naproxen and 10,11-dihydroxy-10-hydroxycarbamazepine. Results from our large scale river catchment monitoring study and lab simulated microcosm show wide-ranging implications of enantiomerism of cPACs on environmental risk assessment (ERA). As two enantiomers of the same compound show different biological effects (e.g. toxicity), their non-racemic presence in the environment might lead to inaccurate ERA. This is because current ERA approaches do not require analysis at enantiomeric level.

Verifying community-wide exposure to endocrine disruptors in personal care products - In quest for metabolic biomarkers of exposure via in vitro studies and wastewater-based epidemiology.

This study aimed to identify human specific metabolites of selected known or suspected endocrine disruptors (EDCs), mainly UV filters, used in personal care and consumer products whose metabolism has hardly been explored and to select suitable candidate biomarkers for human exposure studies using wastewater based epidemiology (WBE). The analysis of metabolic biomarkers of target chemicals is crucial in order to distinguish between internal and external exposure, since many sources contribute to chemicals being discharged into wastewater. This was achieved through the employment of a new analytical framework for verification of biomarkers of exposure to chemicals combining human biomonitoring and water fingerprinting. Eight EDCs with unknown metabolic pathways (benzophenone-1 (BP-1); benzophenone-2 (BP-2); 4,4'-dihydroxybenzophenone (4,4'-DHBP); 4-benzylphenol (4-BenzPh); homosalate (HO); octocrylene (OC); 3-benzylidene camphor (3-BC), and two EDCs with known metabolism (bisphenol A (BPA) and benzophenone-3 (BP-3)) were tested. The biotransformation observed consisted mainly of phase I processes such as hydrolysis and hydroxylation together with phase II conjugation reactions such as sulphation and glucuronidation. Only two chemicals (BP-1, BP-3) were identified in urine and three chemicals (BPA, BP-1, BP-3) in wastewater. Five newly discovered metabolites (HO-Met1, OC-Met1, 4-BenzPh-Met4, 4-BenzPh-Met5 and 4-BenzPh-Met6) and one previously known metabolite (BPA-Met3) were detected in tested urine/wastewater samples from five WWTPs serving large communities ranging between 17 and 100 thousand inhabitants. The presence of metabolic biotransformation products of OC, 4-BenzPh, BPA and HO in wastewater provides evidence for internal exposure of studied populations to these chemicals.

Stereochemistry of ephedrine and its environmental significance: Exposure and effects directed approach.

Analysis of drugs and pharmaceuticals in the environment is typically performed with non-chiral chromatographic techniques. The environmental risks posed by chiral compounds analysed in this way must therefore be assumed to be independent of chirality, meaning that each enantiomer is equally potent in toxicity and long-lived in stability. This manuscript examines the degradation of each of the four isomers of ephedrine in river simulating microcosms and links this to toxicity data obtained by exposing three different organisms (D. magna, P. subcapitata and T. thermophila) to each of the isomers individually. Microcosms showed that significant degradation only occurred in biotic conditions and that only two isomers (1R,2S-(-)-ephedrine, 1S,2S-(+)-pseudoephedrine) degraded significantly over a period of fourteen days. This is concerning because at least one of the non-degraded isomers (1S,2R-(+)-ephedrine) has been observed in wastewater effluent, which discharges directly into rivers, meaning these isomers could be persistent in the environment. We also observed formation of 1S,2R-(+)-ephedrine in single isomer 1R,2S-(-)-ephedrine river simulating microcosms. Human liver microsome assays and mass spectrometry based data mining revealed that 1S,2R-(+)-ephedrine is not human derived but it could be formed as a results of microbial metabolic processes. Across all three organisms tested the persistent isomers (1S,2R-(+)-ephedrine and 1R,2R-(-)-pseudoephedrine) were more toxic than those that undergo degradation; meaning that if these isomers are entering or formed in the environment they might represent a potentially hazardous contaminant.

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.