Dilute-and-shoot approach for the high-throughput LC-MS/MS determination of illicit drugs in the field of wastewater-based epidemiology.

The determination of illicit drugs in urban influent wastewater (IWW) enables the monitoring of spatial and temporal drug usage trends and assessment of community lifestyle habits. The increasing number of wastewater surveillance studies has emphasized the necessity for the development of rapid, high-throughput methods that maintain high quality data. This work evaluates the use of a dilute-and-shoot methodology, based on direct injection (DI) of centrifuged samples, as an alternative approach to the widely applied sample pre-treatment based on solid-phase extraction, for the liquid chromatography-tandem mass spectrometry determination of seven widely consumed illicit drugs and their metabolites in IWW (amphetamine; cocaine metabolite, benzoylecgonine; ketamine; 3,4-methylenedioxymethamphetamine (MDMA); methamphetamine; cannabis metabolite, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THCCOOH); heroin metabolite, 6-acetylmorphine (6-MAM)). Comparison of both approaches in terms of matrix effects, sensitivity and accuracy, demonstrates the DI method suitability to correctly quantify these analytes in IWW, with a limit of quantification lower than 30 ng L-1 for most compounds. After validation of the method and participation in an interlaboratory exercise, the DI method was applied to the analysis of 54 IWW samples collected from different Spanish wastewater treatment plants. Additionally, quality controls were incorporated in each analysis batch to support the DI method applicability and robustness. The use of a 10 µL-DI reduces time-consuming sample preparation, analysis time and measurement uncertainty. Moreover, it supports green chemistry by reducing the consumption of organic solvents and it facilitates logistics by collecting, transporting, and storing less sample volume. The methodology is therefore especially appropriate for monitoring illicit drugs in large wastewater-based epidemiology sampling campaigns or when fast near real-time results are needed.

Current state and future perspectives on de facto population markers for normalization in wastewater-based epidemiology: A systematic literature review.

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.

Necroptosis induced by ruthenium (II) complexes as mitochondrial disruptors.

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.

Influent wastewater analysis to investigate emerging trends of new psychoactive substances use in Europe.

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.

Making waves: Wastewater-based surveillance of cannabis use.

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.

Workflow to facilitate the detection of new psychoactive substances and drugs of abuse in influent urban wastewater.

The complexity around the dynamic markets for new psychoactive substances (NPS) forces researchers to develop and apply innovative analytical strategies to detect and identify them in influent urban wastewater. In this work a comprehensive suspect screening workflow following liquid chromatography - high resolution mass spectrometry analysis was established utilising the open-source InSpectra data processing platform and the HighResNPS library. In total, 278 urban influent wastewater samples from 47 sites in 16 countries were collected to investigate the presence of NPS and other drugs of abuse. A total of 50 compounds were detected in samples from at least one site. Most compounds found were prescription drugs such as gabapentin (detection frequency 79%), codeine (40%) and pregabalin (15%). However, cocaine was the most found illicit drug (83%), in all countries where samples were collected apart from the Republic of Korea and China. Eight NPS were also identified with this protocol: 3-methylmethcathinone 11%), eutylone (6%), etizolam (2%), 3-chloromethcathinone (4%), mitragynine (6%), phenibut (2%), 25I-NBOH (2%) and trimethoxyamphetamine (2%). The latter three have not previously been reported in municipal wastewater samples. The workflow employed allowed the prioritisation of features to be further investigated, reducing processing time and gaining in confidence in their identification.

Understanding associations between antimicrobial agents usage and antimicrobial resistance genes prevalence at the community level using wastewater-based epidemiology: A Spanish pilot study.

Understanding the development and spread of antimicrobial resistance (AMR) is important for combating this global threat for public health. Wastewater-based epidemiology (WBE) is a complementary approach to current surveillance programs that minimizes some of the existing limitations. The aim of the present study is to explore WBE for monitoring antibiotics and antibiotic resistance genes (ARGs) in wastewater samples collected during 2021/2022 from the city of Castellon (Spain). Eighteen commonly prescribed antibiotics have been selected and measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), with triple quadrupole mass analysers. Moreover, qPCR for specific ARGs has been performed to obtain information of these genes in co-presence with antibiotics. All selected ARGs, along with a total of 11 antibiotics, were identified. The highest population-normalized daily loads were observed for the macrolide azithromycin, followed by the quinolones ciprofloxacin and levofloxacin. Subsequently, daily consumption estimates based on wastewater data were compared with prescription data of antibiotics. Statistical analyses were conducted to explore if there is correlation between antibiotics and ARGs. While no correlations were found between antibiotics and their corresponding ARGs, certain correlations (p < 0.05) were identified among non-corresponding ARGs. In addition, a strong positive correlation was found between the sum of all antibiotics and the intl1 gene. Moreover, population-normalized ARG loads significantly correlate with the 16S rRNA-normalized ARG loads, serving as an indicator for population size. Results provide a baseline for future work and a proof-of-concept emphasising the need for future work and long-term surveillance, and highlight the need of similar programs at a regional and global levels worldwide.

Discovery of polycyclic aromatic acid metabolites in fish exposed to the petroleum compounds 1-methylphenanthrene and 1,4-dimethylphenanthrene.

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.

Comprehensive study on the potential environmental risk of temporal antibiotic usage through wastewater discharges.

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.

Screening for new psychoactive substances in wastewater from educational institutions.

Drug (ab)use among young people is a serious issue, negatively impacting their well-being and prospects. The emergence of new psychoactive substances (NPS) further complicates the situation as they are easily accessible (e.g., online), but users are at high risk of intoxication as their chemical identity is often unknown and toxicity poorly understood. While surveys and drug testing are traditionally used in educational institutions to comprehend drug use trends and establish effective prevention programs, they are not without their limitations. Accordingly, we investigated the occurrence of NPS in educational institutions through wastewater analysis and critically evaluated the viability of the approach. The study included eight wastewater samples from primary schools (ages 6-15 years), six from secondary schools (ages 15-19 years), three from institutions for both secondary and higher education (ages 15+), and six from higher educational institutions (ages 19+). Samples were obtained mid-week and evaluated in two Slovenian municipalities; the capital Ljubljana and a smaller one (M1). Samples were screened using liquid chromatography-ion mobility-high-resolution mass spectrometry (LC-IMS-HRMS), and NPS identified at three levels of confidence (Level 1: unequivocal, Level 2: probable, Level 3: tentative) from a suspect list containing over 5600 entries. NPS were identified in all types of educational institutions. Most were synthetic stimulants, with 3-MMC, ephedrine, 4-chloro-α-PPP, and ethcathinone being unequivocally identified. Also, NPS were present in wastewater from all educational institution types revealing potential spatial but no inter-institutional trends. Although specific groups cannot be targeted, the study, as a proof-of-concept, demonstrates that a suspect screening of wastewater employing LC-IMS-HRMS can be used as a radar for NPS in educational institutions and potentially replace invasive drug testing.

Three years of wastewater surveillance for new psychoactive substances from 16 countries.

The proliferation of new psychoactive substances (NPS) over recent years has made their surveillance complex. The analysis of raw municipal influent wastewater can allow a broader insight into community consumption patterns of NPS. This study examines data from an international wastewater surveillance program that collected and analysed influent wastewater samples from up to 47 sites in 16 countries between 2019 and 2022. Influent wastewater samples were collected over the New Year period and analysed using validated liquid chromatography - mass spectrometry methods. Over the three years, a total of 18 NPS were found in at least one site. Synthetic cathinones were the most found class followed by phenethylamines and designer benzodiazepines. Furthermore, two ketamine analogues, one plant based NPS (mitragynine) and methiopropamine were also quantified across the three years. This work demonstrates that NPS are used across different continents and countries with the use of some more evident in particular regions. For example, mitragynine has highest mass loads in sites in the United States, while eutylone and 3-methylmethcathinone increased considerably in New Zealand and in several European countries, respectively. Moreover, 2F-deschloroketamine, an analogue of ketamine, has emerged more recently and could be quantified in several sites, including one in China, where it is considered as one of the drugs of most concern. Finally, some NPS were detected in specific regions during the initial sampling campaigns and spread to additional sites by the third campaign. Hence, wastewater surveillance can provide an insight into temporal and spatial trends of NPS use.

Wastewater-based epidemiology for the assessment of population exposure to chemicals: The need for integration with human biomonitoring for global One Health actions.

WBE has now become a complimentary tool in SARS-CoV-2 surveillance. This was preceded by the established application of WBE to assess the consumption of illicit drugs in communities. It is now timely to build on this and take the opportunity to expand WBE to enable comprehensive assessment of community exposure to chemical stressors and their mixtures. The goal of WBE is to quantify community exposure, discover exposure-outcome associations, and trigger policy, technological or societal intervention strategies with the overarching aim of exposure prevention and public health promotion. To achieve WBE's full potential, the following key aspects require further action: (1) Integration of WBE-HBM (human biomonitoring) initiatives that provide comprehensive community-individual multichemical exposure assessment. (2) Global WBE monitoring campaigns to provide much needed data on exposure in low- and middle-income countries (LMICs) and fill in the gaps in knowledge especially in the underrepresented highly urbanised as well as rural settings in LMICs. (3) Combining WBE with One Health actions to enable effective interventions. (4) Advancements in new analytical tools and methodologies for WBE progression to enable biomarker selection for exposure studies, and to provide sensitive and selective multiresidue analysis for trace multi-biomarker quantification in a complex wastewater matrix. Most of all, further developments of WBE needs to be undertaken by co-design with key stakeholder groups: government organisations, health authorities and private sector.

Enhancing spectral quality in complex environmental matrices: Supporting suspect and non-target screening in zebra mussels with ion mobility.

Identification of bioaccumulating contaminants of emerging concern (CECs) via suspect and non-target screening remains a challenging task. In this study, ion mobility separation with high-resolution mass spectrometry (IM-HRMS) was used to investigate the effects of drift time (DT) alignment on spectrum quality and peak annotation for screening of CECs in complex sample matrices using data independent acquisition (DIA). Data treatment approaches (Binary Sample Comparison) and prioritisation strategies (Halogen Match, co-occurrence of features in biota and the water phase) were explored in a case study on zebra mussel (Dreissena polymorpha) in Lake Mälaren, Sweden's largest drinking water reservoir. DT alignment evidently improved the fragment spectrum quality by increasing the similarity score to reference spectra from on average (±standard deviation) 0.33 ± 0.31 to 0.64 ± 0.30 points, thus positively influencing structure elucidation efforts. Thirty-two features were tentatively identified at confidence level 3 or higher using MetFrag coupled with the new PubChemLite database, which included predicted collision cross-section values from CCSbase. The implementation of predicted mobility data was found to support compound annotation. This study illustrates a quantitative assessment of the benefits of IM-HRMS on spectral quality, which will enhance the performance of future screening studies of CECs in complex environmental matrices.

Prediction of Retention Time and Collision Cross Section (CCSH+, CCSH-, and CCSNa+) of Emerging Contaminants Using Multiple Adaptive Regression Splines.

Ultra-high performance liquid chromatography coupled to ion mobility separation and high-resolution mass spectrometry instruments have proven very valuable for screening of emerging contaminants in the aquatic environment. However, when applying suspect or nontarget approaches (i.e., when no reference standards are available), there is no information on retention time (RT) and collision cross-section (CCS) values to facilitate identification. In silico prediction tools of RT and CCS can therefore be of great utility to decrease the number of candidates to investigate. In this work, Multiple Adaptive Regression Splines (MARS) were evaluated for the prediction of both RT and CCS. MARS prediction models were developed and validated using a database of 477 protonated molecules, 169 deprotonated molecules, and 249 sodium adducts. Multivariate and univariate models were evaluated showing a better fit for univariate models to the experimental data. The RT model (R2 = 0.855) showed a deviation between predicted and experimental data of ±2.32 min (95% confidence intervals). The deviation observed for CCS data of protonated molecules using the CCSH model (R2 = 0.966) was ±4.05% with 95% confidence intervals. The CCSH model was also tested for the prediction of deprotonated molecules, resulting in deviations below ±5.86% for the 95% of the cases. Finally, a third model was developed for sodium adducts (CCSNa, R2 = 0.954) with deviation below ±5.25% for 95% of the cases. The developed models have been incorporated in an open-access and user-friendly online platform which represents a great advantage for third-party research laboratories for predicting both RT and CCS data.

The NORMAN Suspect List Exchange (NORMAN-SLE): facilitating European and worldwide collaboration on suspect screening in high resolution mass spectrometry.

Background: The NORMAN Association (https://www.norman-network.com/) initiated the NORMAN Suspect List Exchange (NORMAN-SLE; https://www.norman-network.com/nds/SLE/) in 2015, following the NORMAN collaborative trial on non-target screening of environmental water samples by mass spectrometry. Since then, this exchange of information on chemicals that are expected to occur in the environment, along with the accompanying expert knowledge and references, has become a valuable knowledge base for "suspect screening" lists. The NORMAN-SLE now serves as a FAIR (Findable, Accessible, Interoperable, Reusable) chemical information resource worldwide. Results: The NORMAN-SLE contains 99 separate suspect list collections (as of May 2022) from over 70 contributors around the world, totalling over 100,000 unique substances. The substance classes include per- and polyfluoroalkyl substances (PFAS), pharmaceuticals, pesticides, natural toxins, high production volume substances covered under the European REACH regulation (EC: 1272/2008), priority contaminants of emerging concern (CECs) and regulatory lists from NORMAN partners. Several lists focus on transformation products (TPs) and complex features detected in the environment with various levels of provenance and structural information. Each list is available for separate download. The merged, curated collection is also available as the NORMAN Substance Database (NORMAN SusDat). Both the NORMAN-SLE and NORMAN SusDat are integrated within the NORMAN Database System (NDS). The individual NORMAN-SLE lists receive digital object identifiers (DOIs) and traceable versioning via a Zenodo community (https://zenodo.org/communities/norman-sle), with a total of > 40,000 unique views, > 50,000 unique downloads and 40 citations (May 2022). NORMAN-SLE content is progressively integrated into large open chemical databases such as PubChem (https://pubchem.ncbi.nlm.nih.gov/) and the US EPA's CompTox Chemicals Dashboard (https://comptox.epa.gov/dashboard/), enabling further access to these lists, along with the additional functionality and calculated properties these resources offer. PubChem has also integrated significant annotation content from the NORMAN-SLE, including a classification browser (https://pubchem.ncbi.nlm.nih.gov/classification/#hid=101). Conclusions: The NORMAN-SLE offers a specialized service for hosting suspect screening lists of relevance for the environmental community in an open, FAIR manner that allows integration with other major chemical resources. These efforts foster the exchange of information between scientists and regulators, supporting the paradigm shift to the "one substance, one assessment" approach. New submissions are welcome via the contacts provided on the NORMAN-SLE website (https://www.norman-network.com/nds/SLE/). Supplementary Information: The online version contains supplementary material available at 10.1186/s12302-022-00680-6.

Revealing the differences in collision cross section values of small organic molecules acquired by different instrumental designs and prediction models.

The number of open access databases containing experimental and predicted collision cross section (CCS) values is rising and leads to their increased use for compound identification. However, the reproducibility of reference values with different instrumental designs and the comparison between predicted and experimental CCS values is still under evaluation. This study compared experimental CCS values of 56 small molecules (Contaminants of Emerging Concern) acquired by both drift tube (DT) and travelling wave (TW) ion mobility mass spectrometry (IM-MS). The TWIM-MS included two instrumental designs (Synapt G2 and VION). The experimental TWCCSN2 values obtained by the TWIM-MS systems showed absolute percent errors (APEs) < 2% in comparison to experimental DTIMS data, indicating a good correlation between the datasets. Furthermore, TWCCSN2 values of [M - H]- ions presented the lowest APEs. An influence of the compound class on APEs was observed. The applicability of prediction models based on artificial neural networks (ANN) and multivariate adaptive regression splines (MARS), both built using TWIM-MS data, was investigated for the first time for the prediction of DTCCSN2 values. For [M+H]+ and [M - H]- ions, the 95th percentile confidence intervals of observed APEs were comparable to values reported for both models indicating a good applicability for DTIMS predictions. For the prediction of DTCCSN2 values of [M+Na]+ ions, the MARS based model provided the best results with 73.9% of the ions showing APEs below the threshold reported for [M+Na]+. Finally, recommendations for database transfer and applications of prediction models for future DTIMS studies are made.

Analytical investigation of cannabis biomarkers in raw urban wastewater to refine consumption estimates.

Wastewater analysis of Δ9-tetrahydrocannabinol (THC) biomarkers can provide essential information on trends in cannabis consumption. Although analysis is mostly focused on the aqueous phase, previous studies have illustrated the need of improving the measurements of raw influent wastewater (IWW) considering also suspended solids. This is important for cannabis biomarkers, because a substantial part of them is expected to be found in the suspended solids due to their more lipophilic character compared with other metabolites/drugs included in these types of studies. However, it remains open to which extent trend estimates might be affected by solely analysing the liquid phase. To investigate this aspect, robust analytical methodologies are required to measure both the liquid and solid phases of IWW. In this work, we firstly tested liquid-liquid extraction (LLE) for THC and its major metabolites (THCOH, and THCCOOH). Using LLE, no filtration or centrifugation step was required for raw IWW analysis, and the three analytes were extracted from both the liquid and the solid phase simultaneously. In parallel, the raw IWW was centrifuged and the obtained solid and liquid phases were analyzed separately: the liquid phase by both LLE and solid phase extraction (SPE) for comparison of data, and the suspended solids by solid-liquid extraction (SLE). The separate analysis of both phases in a number of samples revealed that a significant amount of cannabis biomarkers (ranging from 42 to 90%) was found in the suspended solids. In addition, the total amount of cannabis biomarkers obtained by analysing raw IWW on the one hand, and by separate analysis of the liquid and the solid phases, on the other hand, was in good agreement. Data from this study show that the sole analysis of the liquid phase would lead to a notable underestimation of cannabis biomarkers concentrations in IWW.

Monitoring the evolution of SARS-CoV-2 on a Spanish university campus through wastewater analysis: A pilot project for the reopening strategy.

Wastewater surveillance is a fast and cost-effective tool that enables tracing of both symptomatic and asymptomatic transmission of SARS-CoV-2. In this paper, a pilot program carried out at the University Jaume I for monitoring the trends of the presence of SARS-CoV-2 in wastewater. To the best of our knowledge, this is the first such project conducted on a university campus in Spain. Wastewater samples (n = 838) were collected when students returned to campus, from October 2020 until August 2021, at a confluence sewer point and at the building level including different academic departments and services, the library, administration offices and the university student residence. It has been observed that the probability of SARS-CoV-2 RNA detection in wastewater depended on COVID-19 incidence on campus and visitors/occupants of the buildings i.e., high-, or low-traffic buildings with high or low frequency of potential contacts. Moreover, the third wave in Spain (after Christmas 2020) and an outbreak that occurred at the university student's residence could be carefully followed, allowing confirmation of the end of the outbreak. In addition, viral variants (i.e., mutations and linages) from selected time points were detected by sequencing and gave an indication of the evolution of the virus over time. The results illustrate the potential of wastewater-based epidemiology to provide an early warning for SARS-CoV-2 within the university, especially in buildings with low traffic and more defined populations, like the student residence. The strategy and experience gathered in this study will allow for implementation of improvements for reliable monitoring in the future.

Analytical research of pesticide biomarkers in wastewater with application to study spatial differences in human exposure.

Wastewater-based epidemiology (WBE) relies on the assessment and interpretation of levels of biomarkers in wastewater originating from a well-defined community. It has provided unique information on spatial and temporal trends of licit and illicit drug consumption, and has also the potential to give complementary information on human exposure to chemicals. Here, we focus on the accurate quantification of pesticide biomarkers (i.e., predominantly urinary metabolites) in influent wastewater at the ng L-1 level to be used for WBE. In the present study, an advanced analytical methodology has been developed based on ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), for the simultaneous determination of 11 specific human biomarkers of triazines, urea herbicides, pyrethroids and organophosphates in urban wastewater. The sample treatment consisted of solid-phase extraction using Oasis HLB cartridges. Direct injection of the samples was also tested for all compounds, as a simple and rapid way to determine these compounds without sample manipulation (i.e., minimizing potential analytical errors). However, if extraction recoveries are satisfactory, SPE is the preferred approach that allow reaching lower concertation levels. Six isotopically labelled internal standards were evaluated and used to correct for matrix effects. Due to the difficulties associated with this type of analysis, special emphasis has been placed on the analytical challenges encountered. The satisfactory validated methodology was applied to urban wastewater samples collected from different locations across Europe revealing the presence of 2,6-EA, 3,4-DCA, 3-PBA and 4-HSA i.e, metabolites of metolachlor-s, urea herbicides, pyrethroids and chlorpropham, respectively. Preliminary data reported in this paper illustrate the applicability of this analytical approach for assessing human exposure to pesticides through WBE.

Spatial and temporal assessment of crack cocaine use in 13 European cities through wastewater-based epidemiology.

Already in early 2000s, concerns have been growing in the EU about increasing use of cocaine and it is estimated that below 1 % of the population administer the drug by smoking crack cocaine. New available data suggests an increase in the use of crack cocaine and an increase in the number of crack cocaine users entering treatment has been reported in several European countries. Robust estimations of crack cocaine use are however not available yet. The use of crack cocaine has long been associated with severe adverse socio-economic conditions as well as mental health problems, such as suicide ideation and depression. The aim of this study was to assess spatial trends in population-normalized mass loads of crack cocaine biomarkers (i.e., anhydroecgonine and anhydroecgonine methyl ester) in 13 European cities in six countries (the Netherlands, Belgium, Ireland, Portugal, Spain and Italy). Furthermore, temporal trends over a five-year period were evaluated through the analysis of historic samples collected in the Netherlands. Finally, the stability of the crack cocaine biomarkers in wastewater was investigated through batch experiments. The samples were analyzed with a new developed and validated hydrophilic interaction liquid chromatography coupled to mass spectrometry method. Targeted crack cocaine biomarkers were found in all cities. Also, crack cocaine biomarker was detected in wastewater from 2017 to 2021 in the Netherlands, but no significance between the years were found. With respect to biomarker in-sample stability, AEME was found to be stable in wastewater. This study assessed crack cocaine use for the first time on a broad scale, both temporal and in cities across Europe, with wastewater-based epidemiology and it shows the importance of wastewater analysis to monitor community loads of crack cocaine use.