Human urine is a promising circular fertiliser, but concerns remain that excreted pharmaceutical residues may transfer to crops and food products. We investigated the fate of 34 pharmaceuticals in a granulated fertiliser produced using urine collected from public urinals, after application to spring barley through grain harvest, malting, and brewing into beer. Seven compounds (desmethylvenlafaxine, carbamazepine, metoprolol, fexofenadine, lamotrigine, atenolol, and citalopram) were quantified in the fertiliser at 62-642 ng g(-1) dry weight, corresponding to a total load of 0.74 g ha-1 , or 8.7 mg kg-1 N at the applied rate of 85 kg N ha-1 . However, none were quantified above method limits in any downstream matrix, including soil, straw, grain, malt, and beer. Under this realistic recycling scenario, pharmaceutical inputs were too low to produce measurable carryover of parent compounds into beer. These findings extend previous field evidence beyond crop tissues and provide empirical support for evaluating urine-derived fertilisers in circular food systems.

This study measured 39 biomarkers of licit (e.g., alcohol, nicotine, benzodiazepines, antidepressants) and illicit drug consumption (e.g., cocaine, amphetamine, cannabis, new psychoactive substances) in wastewater from three neighbourhoods (NGBs) in Barcelona, each representing different socioeconomic and lifestyle profiles. Four weeklong sampling campaigns were conducted between March 2021 and March 2022. The results revealed significant differences in drug use across neighbourhoods that are not apparent in city-level analyses. These differences were particularly notable for illicit drugs—such as higher morphine and cannabis loads in socioeconomically disadvantaged areas—as well as for specific benzodiazepines and antidepressants, which varied in prevalence between neighbourhoods. Alcohol and nicotine patterns were more strongly associated with the local density of bars and restaurants than with socioeconomic status. A clear weekend peak was observed for substances like alcohol. Temporal analysis over the study period showed increased consumption of certain drugs at the end of 2021, likely linked to the reopening of nightlife venues and elevated anxiety related to the COVID-19 pandemic. This study highlights the value of applying wastewater-based epidemiology (WBE) at the intra-city level. By moving beyond aggregated citywide data, intra-urban WBE can reveal spatial and temporal variations in drug use that may otherwise go unnoticed. This approach also enables the identification of communities or socioeconomic groups at higher risk of substance use or mental health challenges, offering critical insights for public health authorities to develop targeted interventions and preventive strategies.

The reuse of reclaimed wastewater for agricultural irrigation is increasingly adopted as a sustainable response to freshwater scarcity driven by climate change. However, the fate of organic contaminants, particularly persistent, mobile, and toxic (PMT) substances, which are poorly removed by conventional wastewater treatment, remains a concern. In this study, Cichorium endivia (endive) was cultivated under greenhouse conditions and irrigated with water fortified with nine PMT compounds (benzophenone-3, clarithromycin, Dotarem, imazalil, metformin, sulpiride, terbutryn, tiapride and tramadol) at concentrations of 0.5, 5, 50, and 500 & micro;g & centerdot;L-& sup1;. To evaluate their fate in the soil-plant-water system, samples of endive, soil, and drainage water were collected and analysed by mixed-mode liquid chromatography-tandem mass spectrometry, except for Dotarem, determined by LC coupled to inductively coupled plasma mass spectrometry. Results showed distinct partitioning: highly mobile compounds, such as metformin, reached concentrations of 88 ng & centerdot;g(-1) and 53 & micro;g & centerdot;L-1 in endive and drainage water, respectively, whereas moderately mobile ones, such as tramadol, primarily accumulated in soil (6423 ng & centerdot;g(-1)). In contrast, low-mobility substances, such as benzophenone-3, showed minimal translocation (<10 ng & centerdot;g(-1)). These findings reveal compound-specific behaviour of PMTs, emphasizing their differential accumulation and the need for risk-informed management of reclaimed wastewater in agriculture. At low PMTs concentrations, endive plants exhibited a hormetic response, with enhanced photosynthetic and transpiration activity indicating mild, non-damaging stress adaptation. Conversely, elevated PMT levels elicited pronounced physiological damage, characterized by significant growth inhibition, chlorosis, and widespread hormonal dysregulation, reflecting a shift toward defence activation and impaired growth performance.

Amoxicillin is a broad-spectrum β-lactam antibiotic widely used in both human and veterinary medicine. Following administration, approximately 60-80% of the dose is excreted, largely unmetabolized, via urine within a few hours. Consequently, environmental contamination with amoxicillin, especially in aquatic systems, is a significant concern. Due to its potential ecological risks and the current scarcity of data required for a full assessment, it was included in two consecutive European Union Watch Lists (2018, 2020) of substances for comprehensive monitoring to inform water policy. However, data on amoxicillin levels in aquatic environments are contradictory, ranging from high concentrations in some studies to non-detection in others. The presence of intact amoxicillin in aquatic environments is inherently transient, and the risk of substantial analytical errors in its detection, identification, and quantification is considerable unless stringent methodological precautions are applied. Driven by the need for reliable environmental data, inconsistencies in the existing literature and our own extensive analytical experience, we comprehensively investigated amoxicillin degradation in surface water, influent and effluent wastewater under various conditions. Experiments were conducted under both dark and sunlight conditions using water samples spiked with amoxicillin at 5 μg/L and 50 μg/L. The parent compound was quantified by liquid chromatography-tandem mass spectrometry, whereas transformation products were identified by high-resolution mass spectrometry. By critically assessing the factors that influence the analysis and environmental occurrence of amoxicillin, this study resolves discrepancies in prior reports and clarifies the realistic prospects for its detection. We demonstrate that the parent antibiotic should be rarely found in water samples due to its rapid degradation. Therefore, future monitoring efforts should be focused on its primary degradation product, amoxicillin penicilloic acid, as more reliable indicator of contamination in aquatic environments.

Prescription opioids have recently experienced a diversion of use in several countries causing a huge number of overdoses due to their high potency. The aim of this study was to develop a wastewater-based epidemiology (WBE) approach to assess the use of prescription opioids and evaluate the compliance to prescriptions to highlight the potential misuse of these substances in Italy. The most prescribed opioids have been selected and validated as WBE biomarkers and a nation-wide study has been conducted over a three-year period (2020-2022). Wastewater (WW) samples were collected as 24-hour composite samples in eight Italian cities and parent substances or metabolites were quantified using validated analytical methods based on liquid chromatography - tandem mass spectrometry (LC-MS/MS). Daily loads have been normalized to therapeutic doses to obtain a real figure of use that showed higher use of tapentadol and morphine compared to oxycodone and fentanyl. Back-calculation of consumption estimates was done by using the most reliable WBE biomarkers and were compared with prescription data. Spatial and temporal differences were found and highlighted a lower use in the south compared to the north and the center of Italy. Data comparison with prescriptions gave results in the same range indicating that the use of these substances in Italy seems to be predominantly from prescription for therapeutic purposes. This study highlighted the suitability of WBE to investigate the potential non-medical use of prescription pharmaceuticals in a population, providing relevant information for supporting public health emergencies and policies.

Identifying trends in psychoactive substance use is essential for developing effective public health policies. Wastewater-based epidemiology (WBE) offers an objective and rapid approach to monitoring illicit drugs and new psychoactive substances (NPS). Given the continuous evolution of the drug market, it is crucial to establish analytical methods that can simultaneously analyze both conventional illicit drugs and emerging NPS. In this work, we present a novel simultaneous solid-phase extraction procedure coupled with liquid chromatography-tandem mass spectrometry for the qualitative and quantitative determination of 36 psychoactive drugs (30 NPS and 6 illicit drugs) in influent wastewater. The methodology was fully validated, with linearity, accuracy, precision, and robustness meeting all the required standards. Additional assessments were conducted to evaluate filtration loss, matrix effects, and analyte stability. The validated method was applied to 63 influent wastewater samples collected in Türkiye, revealing the presence of several NPS, including 4F-MDMB-BUTICA (detection frequency, 3 %), 4F-MDMB-BINACA-N-4-hydroxybutyl (2 %), ADB-BUTINACA (57 %), mephedrone (5 %), and ketamine (87 %). Biomarkers of illicit drugs (amphetamine, methamphetamine, MDMA, cocaine [i.e., benzoylecgonine], and cannabis [i.e., THCCOOH]) were detected in most samples, with detection frequencies between 65 % and 100 %, although some values were below the limit of quantification. This study represents the first validation of CUMYL-4CN-B7AICA, BZOHEXOXIZID, 5F-BZO-POXIZID, 4F-MDMB-BINACA butanoic acid, and 4F-MDMB-BINACA-N-4-hydroxybutyl in influent wastewater, marking a pioneering effort in Türkiye to monitor NPS and their metabolites through WBE. Considering the lack of historical data on NPS consumption in Türkiye, these findings provide a novel insight into the patterns of NPS use across the country.

OBJECTIVES:Illicit drug use presents a significant challenge to global health and public safety, requiring innovative and effective monitoring strategies. This study aimed to evaluate the current landscape of wastewater-based epidemiology (WBE) for monitoring illicit drugs in Europe, focusing on collaboration, current practices, and barriers, while identifying opportunities for improvement. STUDY DESIGN:Cross-sectional survey-based study. METHODS:Coordinated by the Sewage Analysis CORe Group Europe (SCORE) and the European Union Drugs Agency (EUDA), two surveys were conducted in 2023 targeting researchers and stakeholders using WBE for illicit drugs. Data were analysed to identify trends, gaps, and opportunities for improving WBE implementation. RESULTS:The findings indicate a robust research infrastructure and diverse analytical methods among European institutions. Two-thirds of the participating countries reported using WBE data to inform policy. However, challenges persist, particularly in securing funding and coordination, as well as generating national estimates from multiple locations and addressing specific local policy needs. CONCLUSIONS:WBE has proven to be a valuable tool for monitoring illicit drug trends and informing drug policies. To unlock its full potential, sustained funding, methodological standardization, and enhanced cooperation are essential. This study provides critical insights into the European WBE landscape, offering a roadmap for strengthening the integration of actionable WBE data into public health and policy frameworks.

New psychoactive substances are a group of synthetic or naturally occurring drugs that mimic the effects of controlled illicit drugs. With limited information around potency, effects and health risks, there is international concern around their use and thus surveillance efforts are needed for public health. This study presents a global assessment of new psychoactive substances in influent wastewater from 52 sites across 20 countries during the 2022/2023 New Year period. Using solid-phase extraction followed by liquid chromatography-tandem mass spectrometry, 21 new psychoactive substances were detected with mitragynine, 3-methylmethcathinone, and eutylone being the most prevalent. Notably, 3,4-methylenedioxy-PV8 was identified for the first time in sites in Australia and the United States. A retrospective analysis of population normalised mass loads for 3-methylmethcathinone at European sites revealed downward trends in 2022/2023 sampling period compared to the previous years, suggesting a possible impact of regional scheduling measures. Additionally, this work demonstrates the stability of new psychoactive substances in loaded cartridges for up to four months when stored at -20 °C. These findings highlight the value of wastewater-based epidemiology for global monitoring of emerging new psychoactive substances threats and policy evaluation.

Reusing wastewater for irrigation is proposed as a strategy to address water scarcity. However, the long-term environmental consequences of this practice are still unknown, especially when reclaimed water contains contaminants of emerging concern, such as persistent, mobile, and toxic (PMT) compounds, due to the inefficiency of wastewater treatment plants in removing certain pollutants. As a result, irrigation with contaminated water could lead to their uptake by crops and enter the food chain. While data on the presence of PMTs in environmental samples is starting to emerge, the analysis of certain compounds in vegetable matrices remains unexplored. In this study, an analytical methodology was developed and validated for the determination of 8 PMTs (i.e., benzophenone-3, clarithromycin, imazalil, metformin, sulpiride, terbutryn, tiapride, and tramadol) in escarole, tomatoes, and tomato leaves. The proposed analytical methodology used a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction method prior to mixed-mode liquid chromatography tandem mass spectrometry analysis. Method validation, performed according to SANTE guidelines, presented satisfactory results at studied concentrations (1, 10 and 100 ng·g-1 for each compound/matrix combination, except for metformin in escarole (50 and 500 ng·g-1). Recoveries ranged from 70 to 120 %, with a precision of ≤ 20 % for most compounds. Benzophenone-3 and tiapride, for which no isotopically labelled internal standard was available, could be adjusted by applying a correction factor. The limit of quantification was 1 ng·g-1 for all compounds in the three matrices, except for benzophenone-3 and metformin in both escarole and tomato leaves. The applicability of the method was tested by analysing samples from an experimental greenhouse plot where crops were irrigated with water spiked with the selected PMTs.

Reclaimed wastewater reuse for agricultural irrigation is promoted by policymakers to battle water scarcity. But persistent, mobile and toxic (PMT) substances are often not effectively removed by current wastewater treatments and may end up in soil, drainage water and even receiving aquatic environment. This work forms a part of a broader project on using contaminated water for irrigation of escarole crops. An analytical methodology based on mixed-mode liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been developed for the determination of eight PMTs (benzophenone-3, clarithromycin, imazalil, metformin, sulpiride, terbutryn, tiapride and tramadol) in soil and drainage water samples. Moreover, it has been considered of interest to validate the methodology for environmental water i.e., groundwater and surface water. Soil samples were treated using QuEChERS approach, while water samples were injected directly into the LC-MS/MS system. Isotopically labelled internal standards were used for matrix effect correction and extraction losses. The two methods have been validated satisfactorily (recoveries between 70–120 % and RSD < 20 %). The limits of quantification for all compounds were 5 ng·g−1 and 50 ng·L−1 in soil and water, respectively. Finally, the methodology has been applied to soil and drainage water samples collected from escarole crops irrigated with tap water fortified at 5 μg·L−1. In addition, the methodology was applied to real surface and groundwater samples to demonstrate its applicability. This fully validated methodology is a robust tool for determining the selected PMTs and may provide valuable insights into the environmental fate when performing future irrigation programs.

Wastewater-based surveillance (WBS) is a complementary approach that overcomes some of the limitations of human biomonitoring, such as sampling biases, invasiveness, high costs and ethical issues. This study explores WBS to assess the spatial differences in human exposure in areas with relatively high use of pesticides versus reference areas. Societal concerns have been raised related to reported exposure to pesticides of residents near flower bulb fields. To this aim, several specific human metabolites of triazines, pyrethroids and organophosphates authorized for bulb cultivation have been selected as pesticide exposure biomarkers, investigating their presence in wastewater samples collected near flower bulb fields and areas where those pesticides are less-or not-applied i.e., control areas. In this unique study a total of 71 influent wastewater samples were analyzed during the period of pesticide application on flower cultivation, 31 originated from flower bulb areas and 40 from control areas. Higher population normalized mass loads were found in the flower areas for 2-methyl-6-eth-ylaniline (up to 21 mu g/day/1000 inh.) and hydroxytebuconazole (up to 6 mu g/day/1000 inh.), metabolites corresponding to the pesticides metolachlor-S and tebuconazole, respectively. Our findings illustrate the need for wide WBS monitoring campaigns for assessing exposure of populations living near flower bulb fields. Where WBS provides additional data input for further risk assessment. WBS can be further employed in areas of other crops that are relevant such as potatoes, other vegetables and fruit trees, where pesticides are also widely applied.

Antibiotic residues can reach aquatic ecosystems through urban wastewater discharges, posing an ecotoxicological risk for aquatic organisms and favoring the development of bacterial resistance. To assess the emission rate and hazardousness of these compounds, it is important to carry out periodic chemical monitoring campaigns that provide information regarding the actual performance of wastewater treatment plants (WWTPs) and the potential impact of the treated wastewater in the aquatic environment. In this study, 18 of the most widely consumed antibiotics in Spain were determined by liquid chromatography-tandem mass spectrometry in both influent (IWW) and effluent wastewater (EWW) samples collected over four seasons along 2021–2022. Eleven antibiotics were detected in EWW with azithromycin, ciprofloxacin and levofloxacin showing the highest concentration levels (around 2 μg L−1 of azithromycin and 0.4 μg L−1 of quinolone compounds). Data showed that only 4 out of the 11 compounds were removed by more than 50 % in the WWTP, with sulfamethoxazole standing out with an average removal efficiency >80 %. The risk that treated water could pose to the aquatic environment was also assessed, with 6 compounds indicating a potential environmental risk by exceeding established ecotoxicological and resistance thresholds. Based on the risk assessment, the WWTP removal efficiency required to reduce such risk for antibiotics was estimated. In addition, pooled wastewater samples were screened by LC coupled to high resolution mass spectrometry with ion mobility separation, searching for metabolites and transformation products of the antibiotics investigated to widen future research. Studies like this are crucial to map the impact of antibiotic pollution and to provide the basis for designing water quality and risk prevention monitoring programs.

Fish exposed to xenobiotics like petroleum-derived polycyclic aromatic hydrocarbons (PAHs) will immediately initiate detoxification systems through effective biotransformation reactions. Yet, there is a discrepancy between recognized metabolic pathways and the actual metabolites detected in fish following PAH exposure like oil pollution. To deepen our understanding of PAH detoxification, we conducted experiments exposing Atlantic haddock (Melanogrammus aeglefinus) to individual PAHs or complex oil mixtures. Bile extracts, analyzed by using an ion mobility quadrupole time-of-flight mass spectrometer, revealed novel metabolites associated with the mercapturic acid pathway. A dominant spectral feature recognized as PAH thiols set the basis for a screening strategy targeting (i) glutathione-, (ii) cysteinylglycine-, (iii) cysteine-, and (iv) mercapturic acid S-conjugates. Based on controlled single-exposure experiments, we constructed an interactive library of 33 metabolites originating from 8 PAHs (anthracene, phenanthrene, 1-methylphenanthrene, 1,4-dimethylphenanthrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene). By incorporation of the library in the analysis of samples from crude oil exposed fish, PAHs conjugated with glutathione and cysteinylglycine were uncovered. This qualitative study offers an exclusive glimpse into the rarely acknowledged mercapturic acid detoxification pathway in fish. Furthermore, this furnishes evidence that this metabolic pathway also succeeds for PAHs in complex pollution sources, a notable discovery not previously reported.

Most of the polycyclic aromatic hydrocarbons (PAHs) in petroleum are alkylated (alkyl PAHs), still the metabolism of these alkyl PAHs to the expected acid products (polycyclic aromatic acids; PAAs) has yet to be demonstrated in oil-exposed fish. Should these compounds be discovered in fish as they have in ragworm, rodents, and humans, they could present an indicative biomarker for assessing oil pollution. In this study, the ability to biotransform alkyl PAHs to PAAs was examined on Atlantic haddock (Melanogrammus aeglefinus). Exposure to phenanthrene, 1-methyphenanthrene or 1,4-dimethylphenanthrene was performed via intraperitoneal injection. An Ion Mobility Quadrupole Time-Of-Flight Mass Spectrometer (IMS-Q-TOF MS) was used in exploratory analysis of extracted bile samples. Acquisition of four-dimensional information by coupling liquid chromatography with the IMS-Q-TOF MS and in-silico prediction for feature prioritization in the data processing workflow allowed several tentative identifications with high degree of confidence. This work presents the first detection of PAAs in fish and suggests the importance of investigating alkyl PAHs in ecotoxicological studies of oil-polluted fish environments.

Inducing necroptosis in cancer cells has emerged as an effective strategy to overcome drug resistance. However, while organic small molecules have been extensively studied for this purpose, metal-based compounds have received relatively little attention as triggers of necroptosis. The development of ruthenium (II) hybrid compounds, particularly those containing triazene (Ru-TRZ), highlights a novel avenue for modulating necroptotic cell death. Here we show that incorporating a methyltriazene moiety, a known alkylating warhead, confers superior mitochondrial-targeting properties and enhances cell death compared to amide-containing counterparts. Ru-hybrid TRZ2 exhibits also antitumor efficacy against in vivo drug-resistant cancer cells. Mechanistically, we demonstrate that Ru-TRZ hybrids induce apoptosis. In addition, by activating downstream RIPK3-driven cell death, TRZ2 proficiently restrains normal mitochondrial function and activity, leading to cancer cell necroptosis. Finally, TRZ2 synergizes anti-proliferative activity and cell death effects induced by conventional drugs. In conclusion, Ru-TRZ2 stands as a promising ruthenium-based chemotherapeutic agent inducing necroptosis in drug resistant cancer cells.

Monitoring cannabis consumption holds great interest due to the increasing trend towards its legalization for both medicinal and recreational purposes, despite the potential risks and harms involved. Wastewater-based surveillance (WBS) offers a valuable tool for assessing shifts and patterns in drug consumption and to evaluate law enforcement strategies and harm reduction programs. However, WBS-derived cannabis use estimates have been linked to greater uncertainties compared to other drugs, in part due to the many different routes of administration and a substantial excretion of metabolites in faecal matter. Therefore, the usual approach for estimating consumed amounts and scaling consumption compared to other problem drugs requires a rethink. This viewpoint highlights the progress made in this area and describes the current existing barriers related to in-sewer and in-sample behaviour (e.g., adsorption/desorption mechanisms), analytical procedures used (e.g., sample preparation), and pharmacokinetic aspects (e.g., administration route) linked to cannabis biomarkers in influent wastewater. These need to be addressed to improve the estimation of cannabis use and reflect spatial and temporal trends in the same way as for other drugs. Until then, we recommend being cautious when interpreting wastewater-based cannabis consumption estimates.

Wastewater-based epidemiology (WBE) can provide objective and timely information on the use of new psychoactive substances (NPS), originally designed as legal alternatives of internationally controlled drugs. NPS have rapidly emerged on the global drug market, posing a challenge to drug policy and constituting a risk to public health. In this study, a WBE approach was applied to monitor the use of more than 300 NPS, together with fentanyl and its main metabolite norfentanyl, in influent wastewater collected from 12 European cities during March-June 2021. Quantitative and qualitative analysis of NPS in composite 24 h influent wastewater samples were based on solid phase extraction and liquid chromatography-mass spectrometry. In-sample stability tests demonstrated the suitability of most investigated biomarkers, except for a few synthetic opioids, synthetic cannabinoids and phenetylamines. Fentanyl, norfentanyl and eight NPS were quantified in influent wastewater and at least three substances were found in each city, demonstrating their use in Europe. N,N-dimethyltryptamine and 3-methylmethcathinone (3-MMC) were the most common NPS found, with the latter having the highest mass loads (up to 24.8 mg/day/1000 inhabitants). Seven additional substances, belonging to five categories of NPS, were identified in different cities. Spatial trends of NPS use were observed between cities and countries, and a changing weekly profile of use was observed for 3-MMC. WBE is a useful tool to rapidly evaluate emerging trends of NPS use, complementing common indicators (i.e. population surveys, seizures) and helping to establish measures for public health protection.

Nontargeted screening (NTS) utilizing liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI/HRMS) is increasingly used to identify environmental contaminants. Major differences in the ionization efficiency of compounds in ESI/HRMS result in widely varying responses and complicate quantitative analysis. Despite an increasing number of methods for quantification without authentic standards in NTS, the approaches are evaluated on limited and diverse data sets with varying chemical coverage collected on different instruments, complicating an unbiased comparison. In this interlaboratory comparison, organized by the NORMAN Network, we evaluated the accuracy and performance variability of five quantification approaches across 41 NTS methods from 37 laboratories. Three approaches are based on surrogate standard quantification (parent-transformation product, structurally similar or close eluting) and two on predicted ionization efficiencies (RandFor-IE and MLR-IE). Shortly, HPLC grade water, tap water, and surface water spiked with 45 compounds at 2 concentration levels were analyzed together with 41 calibrants at 6 known concentrations by the laboratories using in-house NTS workflows. The accuracy of the approaches was evaluated by comparing the estimated and spiked concentrations across quantification approaches, instrumentation, and laboratories. The RandFor-IE approach performed best with a reported mean prediction error of 15x and over 83% of compounds quantified within 10x error. Despite different instrumentation and workflows, the performance was stable across laboratories and did not depend on the complexity of water matrices.

Wastewater-based epidemiology (WBE) and wastewater surveillance have become a valuable complementary data source to collect information on community-wide exposure through the measurement of human biomarkers in influent wastewater (IWW). In WBE, normalization of data with the de facto population that corresponds to a wastewater sample is crucial for a correct interpretation of spatio-temporal trends in exposure and consumption patterns. However, knowledge gaps remain in identifying and validating suitable de facto population biomarkers (PBs) for refinement of WBE back-estimations. WBE studies that apply de facto PBs (including hydrochemical parameters, utility consumption data sources, endo- and exogenous chemicals, biological biomarkers and signalling records) for relative trend analysis and absolute population size estimation were systematically reviewed from three databases (PubMed, Web of Science, SCOPUS) according to the PRISMA guidelines. We included in this review 81 publications that accounted for daily variations in population sizes by applying de facto population normalization. To date, a wide range of PBs have been proposed for de facto population normalization, complicating the comparability of normalized measurements across WBE studies. Additionally, the validation of potential PBs is complicated by the absence of an ideal external validator, magnifying the overall uncertainty for population normalization in WBE. Therefore, this review proposes a conceptual tier-based cross-validation approach for identifying and validating de facto PBs to guide their integration for i) relative trend analysis, and ii) absolute population size estimation. Furthermore, this review also provides a detailed evaluation of the uncertainty observed when comparing different de jure and de facto population estimation approaches. This study shows that their percentual differences can range up to ±200 %, with some exceptions showing even larger variations. This review underscores the need for collaboration among WBE researchers to further streamline the application of de facto population normalization and to evaluate the robustness of different PBs in different socio-demographic communities.

Current wastewater-based epidemiology (WBE) studies are predominantly focused on the analysis of urinary biomarkers present in the liquid phase of influent wastewater (IWW). This approach systematically underestimates less polar metabolites, such as cannabis biomarkers. These biomarkers can potentially sorb to and desorb from suspended particulate matter (SPM) present in IWW. This study investigates the bidirectional partitioning of THCCOOH between the liquid phase and SPM of IWW by performing multiple sorption experiments using THCCOOH-D9 as a surrogate due to the unavailability of blank SPM and blank IWW. In addition, this study involves the analysis of IWW collected from eight wastewater treatment plants (WWTP) (n = 56) across four European countries, where raw IWW, the liquid phase and SPM were separately analysed to identify potential trends in the fraction of THCCOOH in the SPM between and withing the examined locations. Based on the performed sorption experiments, bidirectional partitioning of THCCOOH between the liquid phase and SPM was noted, showing partition between both phases when only one of the two phases was spiked. It was illustrated that the concentration of SPM had a notable influence on the THCCOOH partitioning between both phases. In addition to the inter location variability (average THCCOOH present in the SPM ranged 31-59 %), a substantial intra location variation was also observed, e.g., in one location ranged 17-58 %. While the determination of a correction factor for the amount of THCCOOH present in SPM would be ideal, this is challenging, since the amount of SPM is not fixed in all IWW samples. Although SPM has influence on the THCCOOH partition, no correlation (p value Spearman correlation = 0.3160) was observed between the SPM concentration and the fraction of THCCOOH in the solid phase. Moreover, the collection of homogenized samples is difficult, and the time required to reach an equilibration in partitioning of THCCOOH between both IWW phases remains unclear. Due to i) the large inter-and intra-location variation of THCCOOH present in the SPM, ii) the variability in SPM concentration in IWW samples, and iii) the time required to reach a partitioning equilibration, an analytical procedure based on liquid-liquid extraction (LLE) that considers both the liquid phase and SPM of IWW is recommended to reduce the overall uncertainty for THCCOOH measurement in IWW. It was illustrated that this extraction method is capable to recover the total concentration of THCCOOH in both phases.