Are Environmental Levels of Nonsteroidal Anti-Inflammatory Drugs a Reason for Concern? Chronic Life-Cycle Effects of Naproxen in Zebrafish.

The nonsteroidal anti-inflammatory drug naproxen (NPX) is among the most consumed pharmaceuticals worldwide, being detected in surface waters within the ng to µg/L range. Considering the limited chronic ecotoxicity data available for NPX in aquatic ecosystems, the present study aimed at evaluating its impact in the model organism Danio rerio, following a full life-cycle exposure to environmentally relevant concentrations (0.1 to 5.0 µg/L). An integration of apical endpoints, i.e., survival, growth, and reproduction, with gonad histopathology and gene transcription (RNA-seq) was performed to provide additional insights into the mode of action (MoA) of NPX. NPX decreased zebrafish growth and reproduction and led to histopathological alterations in gonads at concentrations as low as 0.1 µg/L. At the molecular level, 0.7 µg/L of NPX led to a disruption in gonads transcription of genes involved in several biological processes associated with reproduction, mainly involving steroid hormone biosynthesis and epigenetic/epitranscriptomic machineries. Collectively, these results show that environmentally realistic concentrations of NPX affect zebrafish reproduction and associated signaling pathways, indicating that current hazard and risk assessment data for NPX underestimate the environmental risk of this pharmaceutical.

Determination of the urinary concentrations of six bisphenols in public servants by online solid-phase extraction-liquid chromatography tandem mass spectrometry.

Bisphenols are widely used as monomers and additives in plastic production. Thus, bisphenol A (BPA) and its most prominent substitutes have been detected in many environmental and human samples. This study proposes an online solid-phase extraction analytical methodology coupled to liquid chromatography with tandem mass spectrometry for the determination of six bisphenols (BPA and bisphenols F (BPF), S (BPS), AF (BPAF), B (BPB), and E (BPE)) in urine samples as an efficient and automated methodology. The method was developed and validated for all bisphenols with good recoveries (92-112%) and repeatability (RSD ≤ 10%) despite the variable matrix effects, except BPAF (which would require a dedicated internal standard), achieving method quantification limits in the 0.05-2.2 ng mL-1 range. The methodology was subsequently applied to 435 urine samples from a non-occupational exposure population (civil servants for the regional government) from Santiago de Compostela (Galicia, Spain). Only BPA, BPF, and BPS were positively detected; the last two presented higher detection frequencies than BPA. When the urinary concentrations are extrapolated to human intake and compared to the European Food Safety Agency (EFSA) tolerable daily intake (TDI) of 2 × 10-4 µg kg-1 day-1 (TDI), all BPA positively identified samples would surpass this threshold. Although no TDI exists currently for the other two identified bisphenols, it is evident that human exposure to bisphenols should be limited. Finally, the results stratification by gender revealed higher levels of exposure to BPF in the women group.

Are Fish Populations at Risk? Metformin Disrupts Zebrafish Development and Reproductive Processes at Chronic Environmentally Relevant Concentrations.

The antidiabetic drug Metformin (MET), one of the most prevalent pharmaceuticals in the environment, is currently detected in surface waters in the range of ng/L to low µg/L. As current knowledge regarding the long-term effects of environmentally relevant concentrations of MET in nontarget organisms is limited, the present study aimed at investigating the generational effects of MET, in concentrations ranging from 390 to 14 423 ng/L in the model organism Danio rerio (up to 9 mpf), including the effects on its nonexposed offspring (until 60 dpf). We integrate several apical end points, i.e., embryonic development, survival, growth, and reproduction, with qRT-PCR and RNA-seq analyses to provide additional insights into the mode of action of MET. Reproductive-related parameters in the first generation were particularly sensitive to MET. MET parental exposure impacted critical molecular processes involved in the metabolism of zebrafish males, which in turn affected steroid hormone biosynthesis and upregulated male vtg1 expression by 99.78- to 155.47-fold at 390 and 14 432 MET treatment, respectively, pointing to an estrogenic effect. These findings can potentially explain the significant decrease in the fertilization rate and the increase of unactivated eggs. Nonexposed offspring was also affected by parental MET exposure, impacting its survival and growth. Altogether, these results suggest that MET, at environmentally relevant concentrations, severely affects several biological processes in zebrafish, supporting the urgent need to revise the proposed Predicted No-Effect Concentration (PNEC) and the Environmental Quality Standard (EQS) for MET.

Development and application of an in-house library and workflow for gas chromatography-electron ionization-accurate-mass/high-resolution mass spectrometry screening of environmental samples.

This work presents an optimized gas chromatography-electron ionization-high-resolution mass spectrometry (GC-EI-HRMS) screening method. Different method parameters affecting data processing with the Agilent Unknowns Analysis SureMass deconvolution software were optimized in order to achieve the best compromise between false positives and false negatives. To this end, an accurate-mass library of 26 model compounds was created. Then, five replicates of mussel extracts were spiked with a mixture of these 26 compounds at two concentration levels (10 and 100 ng/g dry weight in mussel, 50 and 500 ng/mL in extract) and injected in the GC-EI-HRMS system. The results of these experiments showed that accurate mass tolerance and pure weight factor (combination of reverse-forward library search) are the most critical factors. The validation of the developed method afforded screening detection limits in the 2.5-5 ng range for passive sampler extracts and 1-2 ng/g for mussel sample extracts, and limits of quantification in the 0.6-3.2 ng and 0.1-1.8 ng/g range, for the same type of samples, respectively, for 17 model analytes. Once the method was optimized, an accurate-mass HRMS library, containing retention indexes, with ca. 355 spectra of derivatized and non-derivatized compounds was generated. This library (freely available at https://doi.org/10.5281/zenodo.5647960 ), together with a modified Agilent Pesticides Library of over 800 compounds, was applied to the screening of passive samplers, both of polydimethylsiloxane and polar chemical integrative samplers (POCIS), and mussel samples collected in Galicia (NW Spain), where a total of 75 chemicals could be identified.

Screening of Contaminants of Emerging Concern in Surface Water and Wastewater Effluents, Assisted by the Persistency-Mobility-Toxicity Criteria.

Contaminants of emerging concern (CECs) are compounds of diverse origins that have not been deeply studied in the past which are now accruing growing environmental interest. The NOR-Water project aimed to identify the main CECs and their sources in the water environment of Northern Portugal-Galicia (located in northwest Spain) transnational region. To achieve these goals, a suspect screening analytical methodology based on the use of liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) was applied to 29 sampling sites in two campaigns. These sampling sites included river and sea water, as well as treated wastewater. The screening was driven by a library of over 3500 compounds, which included 604 compounds prioritized from different relevant lists on the basis of the persistency, mobility, and toxicity criteria. Thus, a total of 343 chemicals could be tentatively identified in the analyzed samples. This list of 343 identified chemicals was submitted to the classification workflow used for prioritization and resulted in 153 chemicals tentatively classified as persistent, mobile, and toxic (PMT) and 23 as very persistent and very mobile (vMvP), pinpointing the relevance of these types of chemicals in the aqueous environment. Pharmaceuticals, such as the antidepressant venlafaxine or the antipsychotic sulpiride, and industrial chemicals, especially high production volume chemicals (HPVC) such as ε-caprolactam, were the groups of compounds that were detected at the highest frequencies.

Combination of different chromatographic and sampling modes for high-resolution mass spectrometric screening of organic microcontaminants in water.

This study explores the combination of two sampling strategies (polar organic compounds integrative sampler (POCIS) vs. spot sampling) and four chromatographic retention modes (reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), mixed-mode liquid chromatography (MMLC) and supercritical fluid chromatography (SFC)) for high-resolution mass spectrometry (HRMS) screening of organic pollutants in water samples. To this end, a suspect screening approach, using iterative data-dependent tandem mass spectrometry (MS/MS) driven by a library of 3227 chemicals (including pharmaceuticals, pesticides, drugs of abuse, human metabolites, industrial chemicals and other pollutants), was employed. Results show that POCIS can afford a larger number of positive identifications as compared to spot sampling. On the other hand, the best suited retention mechanisms, in terms of identified analytes, are SFC, and followed by RPLC, MMLC and HILIC. However, the best combination (POCIS + SFC) would only allow the identification of 67% of the detected analytes. Thus, the combination of the two sampling strategies, spot and passive sampling, with two orthogonal retention mechanisms, RPLC and SFC, is proposed in order to maximize the number of analytes detected (89%). This strategy was applied to different surface water (river and estuary) samples from Galicia (NW Spain). A total of 155 compounds were detected at a confidence level 2a, from which the major class was pharmaceuticals (61%).

Applicability of mixed-mode chromatography for the simultaneous analysis of C1-C18 perfluoroalkylated substances.

A new analytical method for the determination of 22 perfluoroalkylated (carboxylic and sulfonic) acids in water samples is presented. The method's objective was to achieve the simultaneous quantification of compounds with different chain lengths (from C1 to C18). To this end, 500 mL of water were extracted with Oasis WAX solid-phase extraction cartridges and eluted with 3 mL of 5% ammonia in methanol. After evaporation to dryness, extracts were reconstituted in methanol:ultrapure water (1:1) and analyzed by mixed-mode liquid chromatography-tandem mass spectrometry (MMLC-MS/MS) using a weak anion exchange/reversed-phase column. The method provided good results, with limits of quantification lower than 1 ng/L in river water for most of compounds, except the two perfluorocarboxylic acids with the longest alkyl chain (>C14) and trifluoroacetic acid, for which a blank contamination problem was observed. The method proved good trueness and precision in both ultrapure and river water (R ≥ 81%, RSD ≤ 15%). After validation, the method was applied to the analysis of nine water samples where nine perfluoroalkylated acids were quantified. Seven of them were ultrashort- (C1-C4) and short-chain (C4-C8) perfluoroalkylated acids, pointing out the importance of developing methods capable to target such substances for further monitoring.

Chronic exposure to environmentally relevant levels of simvastatin disrupts zebrafish brain gene signaling involved in energy metabolism.

Simvastatin (SIM), a hypocholesterolaemic drug belonging to the statins group, is a widely prescribed pharmaceutical for prevention of cardiovascular diseases. Several studies showed that lipophilic statins, as SIM, cross the blood-brain barrier and interfere with the energy metabolism of the central nervous system in humans and mammalian models. In fish and other aquatic organisms, the effects of SIM on the brain energy metabolism are unknown, particularly following exposure to low environmentally relevant concentrations. Therefore, the present study aimed at investigating the influence of SIM on gene signaling pathways involved in brain energy metabolism of adult zebrafish (Danio rerio) following chronic exposure (90 days) to environmentally relevant SIM concentrations ranging from 8 ng/L to 1000 ng/L. Real-time PCR was used to determine the transcript levels of several genes involved in different pathways of the brain energy metabolism (glut1b, gapdh, acadm, accα, fasn, idh3a, cox4i1, and cox5aa). The findings here reported integrated well with ecological and biochemical responses obtained in a parallel study. Data demonstrated that SIM modulates transcription of key genes involved in the mitochondrial electron transport chain, in glucose transport and metabolism, in fatty acid synthesis and ß-oxidation. Further, SIM exposure led to a sex-dependent transcription profile for some of the studied genes. Overall, the present study demonstrated, for the first time, that SIM modulates gene regulation of key pathways involved in the energy metabolism in fish brain at environmentally relevant concentrations.

Determination of Persistent and Mobile Organic Contaminants (PMOCs) in Water by Mixed-Mode Liquid Chromatography-Tandem Mass Spectrometry.

The presence of persistent and mobile organic contaminants (PMOC) in aquatic environments has become a matter of concern due to their ability of breaking through natural and anthropogenic barriers, even reaching drinking water. The presence of many of these compounds in surface and drinking water has been reported in screening studies, but there is still a lack of analytical methods capable of quantifying them. Herein, we propose a method combining mixed-mode-solid-phase extraction (MM-SPE) as preconcentration technique and mixed-mode liquid chromatography (MMLC) coupled to tandem mass spectrometry as a determination technique for the quantitative determination of 23 target PMOCs in surface and drinking water samples. When compared to reversed-phase liquid chromatography, the MMLC protocol has proven to be superior in both retentive capabilities and peak shape for ionic compounds, while performing also well for neutrals. The overall method performance was satisfactory with limits of quantification under 50 ng L-1 for most of analytes in both surface and drinking water. The relative standard deviation was lower than 20%, and the average recovery was 78 and 80% in surface and drinking water, respectively. The method was applied to 15 water samples collected in Spain, where 17 out of the 23 target PMOCs were quantified in at least one sample. Among them, 6 chemicals (e.g., benzyltrimethylammonium) are reported and/or quantified here for the first time.

Modeling Dispersal of UV Filters in Estuaries.

Lagrangian ocean analysis, where virtual parcels of water are tracked through hydrodynamic fields, provides an increasingly popular framework to predict the dispersal of water parcels carrying particles and chemicals. We conduct the first direct test of Lagrangian predictions for emerging contaminants using (1) the latitude, longitude, depth, sampling date, and concentrations of UV filters in raft cultured mussel ( Mytilus galloprovincialis) of the estuary Ria de Arousa, Spain (42.5°N, 8.9°W); (2) a hydrodynamic numerical model at 300 m spatial resolution; and (3) a Lagrangian dispersion scheme to trace polluted water parcels back to pollution sources. The expected dispersal distances (mean ± SD) are 2 ± 1 km and the expected dispersal times (mean ± SD) are 6 ± 2 h. Remarkably, the probability of dispersal of UV filters from potential sources to rafts decreases 5-fold over 5 km. In addition to predicting dispersal pathways and times, this study also provides a framework for quantitative investigations of concentrations of emerging contaminants and source apportionment using turbulent diffusion. In the coastline, the ranges of predicted concentrations of the UV-filters 4-methylbenzylidene-camphor, octocrylene, and benzophenone-4 are 3.2 × 10-4 to 0.023 ng/mL, 2.3 × 10-5 to 0.009 ng/mL, and 5.6 × 10-4 to 0.013 ng/mL, respectively. At the outfalls of urban wastewater treatment plants these respective ranges increase to 8.9 × 10-4 to 0.07 ng/mL, 6.2 × 10-5 to 0.027 ng/mL, and 1.6 × 10-3 to 0.040 ng/mL.

Linking chemical exposure to lipid homeostasis: A municipal waste water treatment plant influent is obesogenic for zebrafish larvae.

Obesity, a risk factor for the development of type-2 diabetes, hypertension, cardiovascular disease, hepatic steatosis and some cancers, has been ranked in the top 10 health risk in the world by the World Health Organization. Despite the growing body of literature evidencing an association between the obesity epidemic and specific chemical exposure across a wide range of animal taxa, very few studies assessed the effects of chemical mixtures and environmental samples on lipid homeostasis. Additionally, the mode of action of several chemicals reported to alter lipid homeostasis is still poorly understood. Aiming to fill some of these gaps, we combined an in vivo assay with the model species zebrafish (Danio rerio) to screen lipid accumulation and evaluate expression changes of key genes involved in lipid homeostasis, alongside with an in vitro transactivation assay using human and zebrafish nuclear receptors, retinoid X receptor α and peroxisome proliferator-activated receptor γ. Zebrafish larvae were exposed from 4 th day post-fertilization until the end of the experiment (day 18), to six different treatments: experimental control, solvent control, tributyltin at 100 ng/L Sn and 200 ng/L Sn (positive control), and wastewater treatment plant influent at 1.25% and 2.5%. Exposure to tributyltin and to 2.5% influent led to a significant accumulation of lipids, with white adipose tissue deposits concentrating in the perivisceral area. The highest in vitro tested influent concentration (10%) was able to significantly transactivate the human heterodimer PPARγ/RXRα, thus suggesting the presence in the influent of HsPPARγ/RXRα agonists. Our results demonstrate, for the first time, the ability of complex environmental samples from a municipal waste water treatment plant influent to induce lipid accumulation in zebrafish larvae.

Screening for Polar Chemicals in Water by Trifunctional Mixed-Mode Liquid Chromatography-High Resolution Mass Spectrometry.

The presence of persistent and mobile organic contaminants (PMOC) in aquatic environments is a matter of high concern due to their capability of crossing through natural and anthropogenic barriers, even reaching drinking water. Most analytical methods rely on reversed-phase liquid chromatography (RPLC), which is quite limited for the detection of very polar chemicals. Thus, many of these PMOCs may have not been recognized as water pollutants yet, due to the lack of analytical methods capable to detect them. Mixed-mode LC (MMLC), providing the combination of RP and ion-exchange functionalities is explored in this work with a trifunctional column, combining RPLC, anion and cation exchange, which allows the simultaneous determination of analytes with extremely different properties. A nondiscriminant sample concentration step followed by a MMLC-high resolution mass spectrometry method was developed for a group of 37 very polar model chemicals with different acid/base functionalities. The overall method performance was satisfactory with a mean limit of detection of 50 ng/L, relative standard deviation lower than 20% and overall recoveries (including matrix effects) higher than 60% for 54% of model compounds. Then, the method was applied to 15 real water samples, by a suspect screening approach. For those detected PMOC with standard available, a preliminary estimation of concentrations was also performed. Thus, 22 compounds were unequivocally identified in a range of expected concentrations from 6 ng/L to 540 µg/L. Some of them are well-known PMOC, such as acesulfame, perfluorobutanoic acid or metformin, but other novel pollutants were also identified, as for example di-o-tolylguanidine or trifluoromethanesulfonic acid, which had not or were scarcely studied in water so far.

Wastewater-Based Epidemiology as a New Tool for Estimating Population Exposure to Phthalate Plasticizers.

This study proposes the monitoring of phthalate metabolites in wastewater as a nonintrusive and economic alternative to urine analysis for estimating human exposure to phthalates. To this end, a solid-phase extraction-liquid chromatography-tandem mass spectrometry method was developed, allowing for the determination of eight phthalate metabolites in wastewater (limits of quantification between 0.5 and 32 ng L-1). The analysis of samples from the NW region of Spain showed that these substances occur in raw wastewater up to ca. 1.6 µg L-1 and in treated wastewater up to ca. 1 µg L-1. Concentrations in raw wastewater were converted into levels of exposure to six phthalate diesters. For two of them, these levels were always below the daily exposure thresholds recommended by the U.S. Environmental Protection Agency and the European Food Safety Authority. For the other four, however, estimates of exposure surpassed such a threshold (especially the toddler threshold) in some cases, highlighting the significance of the exposure to phthalates in children. Finally, concentrations in wastewater were also used to estimate metabolite concentrations in urine, providing a reasonable concordance between our results and the data obtained in two previous biomonitoring studies.

Optimization of an ultrasound-assisted derivatization for GC/MS analysis of oxygenated organic species in atmospheric aerosol.

A novel ultrasound-assisted derivatization followed by GC/MS analysis was developed for the quantification of oxygenated organic species in ambient aerosol. Derivatization parameters mostly influencing the analytical response were investigated, i.e., solvent type, reagent concentration, and reaction duration. Response surface methodology was used to design experiments and a quadratic model was utilized to predict the variables and establish the optimal conditions. The study was performed on standard solutions of 30 compounds representing the major classes of oxygenated compounds typically found in ambient aerosol, i.e., low molecular weight carboxylic acids, sugars, and phenols. In comparison with conventional methods, the optimized procedure uses mild reaction temperature (room temperature instead of 70 °C), reduces the amount of silyl reagent (24 vs. 40 µL), and shortens derivatization times (45 vs. 70 min), participating in the current trend of analytical chemistry towards clean, green methods that reduce costs and decrease pollution. Once optimized, the ultrasound procedure was validated by assessing for repeatability, linearity, detection limits, and derivative stability. For all oxygenated organic species, the proposed method showed a good reproducibility-as the relative standard deviations (RSDs%, n = 5) of intra-day analysis were ≤7% - a good linearity with the correlation coefficients of calibration curves R 2 ≥ 99.8, and low detection limits, ranging from 0.34 to 6.50 ng µL-1; thus it is suitable for its applicability in air quality monitoring. Finally, this method was successfully applied to determine 30 oxygenated organic species in three ambient PM2.5 samples collected at an urban site in Northern Italy in three different seasons. Graphical abstract Ultrasound-assisted derivatization is a green alternative method for GC/MS analysis of oxygenated organic species in atmospheric aerosol towards reduction of energy and reactive consumption.

Chronic effects of triclocarban in the amphipod Gammarus locusta: Behavioural and biochemical impairment.

Triclocarban (TCC), a common antimicrobial agent widely used in many household and personal care products, has been widely detected in aquatic ecosystems worldwide. Due to its high lipophilicity and persistence in the aquatic ecosystems, TCC is of emerging environmental concern. Despite the frequently reported detection of TCC in the environment and significant uncertainties about its long term effects on aquatic ecosystems, few studies have addressed the chronic effects of TCC in aquatic organisms at ecologically relevant concentrations. Therefore, we aimed at testing a broad range of biological responses in the amphipod Gammarus locusta following a chronic (60 days) exposure to environmentally relevant concentrations of TCC (100, 500 and 2500ng/L). This work integrated biochemical markers of oxidative stress (catalase (CAT), glutathione-s-transferase (GST) and lipid peroxidation (LPO)) and neurotransmission (acetylcholinesterase (AChE)) with several key ecological endpoints, i.e. behaviour, survival, individual growth and reproduction. Significant alterations were observed in all biochemical markers. While AChE showed a dose-response curve (with a significant increased activity at a TCC concentration of 2500ng/L), oxidative stress markers did not follow a dose-response curve, with significant increase at 100 and/or 500ng/L and a decreased activity in the highest concentration (2500ng/L). The same effect was observed in the females' behavioural response, whereas males' behaviour was not affected by TCC exposure. The present study represents a first approach to characterize the hazard of TCC to crustaceans.

Mind the Gap: Persistent and Mobile Organic Compounds-Water Contaminants That Slip Through.

The discharge of persistent and mobile organic chemicals (PMOCs) into the aquatic environment is a threat to the quality of our water resources. PMOCs are highly polar (mobile in water) and can pass through wastewater treatment plants, subsurface environments and potentially also drinking water treatment processes. While a few such compounds are known, we infer that their number is actually much larger. This Feature highlights the issue of PMOCs from an environmental perspective and assesses the gaps that appear to exist in terms of analysis, monitoring, water treatment and regulation. On this basis we elaborate strategies on how to narrow these gaps with the intention to better protect our water resources.

Wastewater-Based Epidemiology To Monitor Synthetic Cathinones Use in Different European Countries.

Synthetic cathinones are among the most consumed new psychoactive substances (NPS), but their increasing number and interchangeable market make it difficult to estimate the real size of their consumption. Wastewater-based epidemiology (WBE) through the analysis of metabolic residues of these substances in urban wastewater can provide this information. This study applied WBE for the first time to investigate the presence of 17 synthetic cathinones in four European countries. A method based on solid-phase extraction and liquid chromatography coupled to tandem mass spectrometry was developed, validated, and used to quantify the target analytes. Seven substances were found, with mephedrone and methcathinone being the most frequently detected and none of the analytes being found in Norway. Population-normalized loads were used to evaluate the pattern of use, which indicated a higher consumption in the U.K., followed by Spain and Italy, in line with the European prevalence data from population surveys. In the U.K., where an entire week was investigated, an increase of the loads was found during the weekend, indicating a preferential use in recreational contexts. This study demonstrated that WBE can be a useful additional tool to monitor the use of NPS in a population.

Solid-phase extraction of perfluoroalkylated compounds from sea water.

This study describes an in-depth investigation of the parameters involved in the solid-phase extraction performance of perfluoroalkylated compounds (seven carboxylates and one sulfonate), particularly with sea water samples. The two most popular sorbents, Oasis WAX and Oasis HLB, were considered and it was observed that the high ionic strength of sea water may impair solid-phase extraction recoveries. In the final protocol, Oasis HLB cartridges were selected, incorporating a 10% methanol clean-up step before elution with methanol, since less matrix effects were obtained. The proposed method allows successful recoveries, higher than 71%, and relative standard deviations lower than 20%. It also provides excellent limits of detection values between 0.01 and 0.21 ng/L. Finally, the method was applied to fresh and sea water samples, where several perfluoroalkylated compounds were found at concentrations ranging between 0.16 and 64 ng/L. In the case of perfluorooctane sulfonate, recently included in the Water Frame Directive, its concentration reached the highest values among the perfluoroalkylated compounds measured (64 ng/L in river samples).

Assessment of local tobacco consumption by liquid chromatography-tandem mass spectrometry sewage analysis of nicotine and its metabolites, cotinine and trans-3'-hydroxycotinine, after enzymatic deconjugation.

Cotinine (COT), trans-3'-hydroxycotinine (OH-COT), cotinine-N-ß-glucuronide (COT-GLUC), and trans-3'-hydroxycotinine-O-ß-glucuronide (OH-COT-GLUC) are excreted in urine following the intake of nicotine (NIC), and, as such, they have been detected in sewage. Thus, they also constitute convenient biomarkers for NIC tracing through the sewage epidemiology approach at the local scale. Such estimation requires granting a good stability of the target biomarkers in sewage. However, it was found that glucuronides are not stable, particularly in the case of OH-COT-GLUC, which could render variable concentrations of COT, OH-COT, and their glucuronides, depending on sampling and storage time or temperature. Thus, an enzymatic deconjugation with ß-glucuronidase was optimized. With the optimized method, after enzymatic deglucuronization, the limits of quantification obtained were in the range of 0.2-1 µg L(-1), relative standard deviations were <10%, and the trueness in terms of recovery was in the 95%-112% range. The application of the method to composite sewage samples collected during 1 week in three different years in Santiago de Compostela (Galicia, Spain) showed COT and OH-COT concentrations of 0.3-1.9 µg L(-1) and 1.0-3.3 µg L(-1), respectively. Thereby, the average NIC consumption derived was in the 1.7-1.9 mg per day and person range, being comparable to those derived from tobacco sales statistics.