Investigation of pesticides and their transformation products in the Júcar River Hydrographical Basin (Spain) by wide-scope high-resolution mass spectrometry screening.

The Water Framework Directive 2000/60/EC implemented by the European Union established as the main objectives to achieve a "good ecological and chemical status" of the surface water and a "good quantitative and chemical status" of groundwater bodies. One of the major pressures affecting water bodies comes from the use of pesticides and their potential presence in the water ecosystems. For this purpose, the reliable determination of pesticides and their transformation products (TPs) in natural waters (both surface and groundwater) is required. The high number of compounds potentially reaching the aquatic environment makes extraordinary difficult, if not impossible, to investigate all these compounds even using the most powerful analytical techniques. Among these, liquid chromatography coupled to high-resolution mass spectrometry is emphasized due to its strong potential for detection and identification of many organic contaminants thanks to the accurate-mass full spectrum acquisition data. This work focuses on wide-scope screening of many pesticides and their TPs in surface water and groundwater samples, collected between March and May 2017, in the Júcar River Hydrographical Basin, Spain. For this purpose, a home-made database containing more than 500 pesticides and TPs was employed. Analyses performed by liquid chromatography coupled to quadrupole-time of flight mass spectrometry (LC-QTOF MS) allowed the identification of up to 27 pesticides and 6 TPs. The most detected compounds in groundwater were the herbicides atrazine, simazine, terbuthylazine, and their TPs (atrazine-desethyl, terbumeton-desethyl and terbuthylazine-desethyl). Regarding surface water, the fungicides carbendazim, thiabendazole and imazalil, the herbicide terbutryn and the TP terbumeton-desethyl were also detected. These results illustrate the wide use of these compounds (in the present or in the recent past) in the area under study and the vulnerability of the water bodies, and are in accordance with previous findings in other water bodies of the different Spanish Hydrographic systems.

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.

Screening new psychoactive substances in urban wastewater using high resolution mass spectrometry.

Analysis of drug residues in urban wastewater could complement epidemiological studies in detecting the use of new psychoactive substances (NPS), a continuously changing group of drugs hard to monitor by classical methods. We initially selected 52 NPS potentially used in Italy based on seizure data and consumption alerts provided by the Antidrug Police Department and the National Early Warning System. Using a linear ion trap-Orbitrap high resolution mass spectrometer, we designed a suspect screening and a target method approach and compared them for the analysis of 24 h wastewater samples collected at the treatment plant influents of four Italian cities. This highlighted the main limitations of these two approaches, so we could propose requirements for future research. A library of MS/MS spectra of 16 synthetic cathinones and 19 synthetic cannabinoids, for which analytical standards were acquired, was built at different collision energies and is available on request. The stability of synthetic cannabinoids was studied in analytical standards and wastewater, identifying the best analytical conditions for future studies. To the best of our knowledge, these are the first stability data on NPS. Few suspects were identified in Italian wastewater samples, in accordance with recent epidemiological data reporting a very low prevalence of use of NPS in Italy. This study outlines an analytical approach for NPS identification and measurement in urban wastewater and for estimating their use in the population.

Comparison of pharmaceutical, illicit drug, alcohol, nicotine and caffeine levels in wastewater with sale, seizure and consumption data for 8 European cities.

BACKGROUND: Monitoring the scale of pharmaceuticals, illicit and licit drugs consumption is important to assess the needs of law enforcement and public health, and provides more information about the different trends within different countries. Community drug use patterns are usually described by national surveys, sales and seizure data. Wastewater-based epidemiology (WBE) has been shown to be a reliable approach complementing such surveys. METHOD: This study aims to compare and correlate the consumption estimates of pharmaceuticals, illicit drugs, alcohol, nicotine and caffeine from wastewater analysis and other sources of information. Wastewater samples were collected in 2015 from 8 different European cities over a one week period, representing a population of approximately 5 million people. Published pharmaceutical sale, illicit drug seizure and alcohol, tobacco and caffeine use data were used for the comparison. RESULTS: High agreement was found between wastewater and other data sources for pharmaceuticals and cocaine, whereas amphetamines, alcohol and caffeine showed a moderate correlation. methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) and nicotine did not correlate with other sources of data. Most of the poor correlations were explained as part of the uncertainties related with the use estimates and were improved with other complementary sources of data. CONCLUSIONS: This work confirms the promising future of WBE as a complementary approach to obtain a more accurate picture of substance use situation within different communities. Our findings suggest further improvements to reduce the uncertainties associated with both sources of information in order to make the data more comparable.

Screening of pharmaceuticals and illicit drugs in wastewater and surface waters of Spain and Italy by high resolution mass spectrometry using UHPLC-QTOF MS and LC-LTQ-Orbitrap MS.

The existence of pharmaceuticals and illicit drugs (PIDs) in environmental waters has led many analytical chemists to develop screening methods for monitoring purposes. Water samples can contain a huge number of possible contaminants, commonly at low concentrations, which makes their detection and identification problematic. Liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) has proven itself effective in the screening of environmental contaminants. The present work investigates the use of the most popular HRMS instruments, quadrupole time-of-flight and linear trap quadrupole-Orbitrap, from two different laboratories. A suspect screening for PIDs was carried out on wastewater (influent and effluent) and surface water samples from Castellón, Eastern Spain, and Cremona, Northern Italy, incorporating a database of 107 PIDs (including 220 fragment ions). A comparison between the findings of both instruments and of the samples was made which highlights the advantages and drawbacks of the strategies applied in each case. In total, 28 compounds were detected and/or identified by either/both instruments with irbesartan, valsartan, benzoylecgonine and caffeine being the most commonly found compounds across all samples.

Consumption of illicit drugs and benzodiazepines in six Spanish cities during different periods of the COVID-19 pandemic.

Wastewater-based epidemiology (WBE) can provide objective and real time information about the use of addictive substances. A national study was conducted by measuring the most consumed illicit drugs, other drugs whose consumption is not so widespread but has increased significantly in recent years, and benzodiazepines in untreated wastewater from seven wastewater treatment plants (WWTPs) in six Spanish cities. Raw composite wastewater samples were collected from December 2020 to December 2021, a period in which the Spanish and regional governments adopted different restriction measures to contain the spread of the COVID-19 pandemic. Samples were analyzed using a validated analytical methodology for the simultaneous determination of 18 substances, based on solid-phase extraction and liquid-chromatography tandem mass spectrometry. Except for heroin, fentanyl, 6-acetylmorphine and alprazolam, all the compounds were found in at least one city and 9 out of 18 compounds were found in all the samples. In general, the consumption of illicit drugs was particularly high in one of the cities monitored in December 2020, when the restrictions were more severe, especially for cannabis and cocaine with values up to 46 and 6.9 g/day/1000 inhabitants (g/day/1000 inh), respectively. The consumption of MDMA, methamphetamine and mephedrone was notably higher in June 2021, after the end of the state of alarm, in the biggest population investigated in this study. Regarding the use of benzodiazepines, the highest mass loads corresponded to lorazepam. This study demonstrates that WBE is suitable for complementing epidemiological studies about the prevalence of illicit drugs and benzodiazepines during the COVID-19 pandemic restrictions.

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.

Festivals following the easing of COVID-19 restrictions: Prevalence of new psychoactive substances and illicit drugs.

The market for illicit drugs and new psychoactive substances (NPS) has grown significantly and people attending festivals have been identified as being at high risk (high extent and frequency of substance use). Traditional public health surveillance data sources have limitations (high costs, long implementation times, and ethical issues) and wastewater-based epidemiology (WBE) can cost-effectively support surveillance efforts. Influent wastewater samples were analyzed for NPS and illicit drug consumption collected during New Year period (from 29-Dec-2021 to 4-Jan-2022) and a summer Festival (from 29-June-2022 to 12-July-2022) in a large city in Spain. Samples were analyzed for phenethylamines, cathinones, opioids, benzodiazepines, plant-based NPS, dissociatives, and the illicit drugs methamphetamine, MDA, MDMA, ketamine, heroin, cocaine, and pseudoephedrine by liquid chromatography mass spectrometry. High consumption rates of specific NPS and established illicit drugs were identified at the peak of each event. Furthermore, a dynamic change in NPS use (presence and absence of substances) was detected over a period of six months. Eleven NPS, including synthetic cathinones, benzodiazepines, plant-based NPS and dissociatives, and seven illicit drugs were found across both the New Year and summer Festival. Statistically significant differences (p < 0.05) were seen for 3-MMC (New Year vs summer Festival), eutylone (New Year vs summer Festival), cocaine (summer Festival vs normal week and summer Festival vs New Year), MDMA (New Year vs normal week and summer Festival vs normal week), heroin (summer Festival vs New Year) and pseudoephedrine (summer Festival vs New Year). This WBE study assessed the prevalence of NPS and illicit drugs at festivals following the reduction of the COVID-19 pandemic restrictions highlighting the high use of specific substances at the peak of each event. This approach identified in a cost-effective and timely manner without any ethical issues the most used drugs and changes in use patterns and, thus, can complement public health information.

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.

Simultaneous determination of aflatoxins B1, B2, G1 and G2 in commercial rices using immunoaffinity column clean-up and HPLC-MS/MS.

Rice is frequently contaminated with aflatoxins, that are highly toxic fungal substances and strongly involved on hepatic cancer. In this work, different extraction and clean-up methods were evaluated for the simultaneous extraction and clean-up of aflatoxins B1, B2, G1 and G2 from rice. Favourable results were obtained by using methanol - water (80:20, v/v) extraction followed by immunoaffinity columns for clean-up, with recoveries of 86-92%, standard deviations between 5 and 11%, LOD ranged between 0.09 and 0.32 µg/kg, and LOQ between 0.31 and 1.06 µg/kg. Method validation and sample analysis were performed by using HPLC-MS/MS. Nine rice samples from different origin, varieties and specific characteristics, acquired in Spanish supermarkets were analysed. In two basmati samples from the same batch aflatoxin B1 was detected at (1.62 ± 0.08) µg/kg and (0.77 ± 0.03) µg/kg, both lower than the levels established by European Regulation for aflatoxin B1 in cereals.

Analysis of Se and Hg biomolecules distribution and Se speciation in poorly studied protein fractions of muscle tissues of highly consumed fishes by SEC-UV-ICP-MS and HPLC-ESI-MS/MS.

Distribution of Se and Hg in sarcoplasmic, myofibrillar and alkali-soluble protein fractions extracted from muscle tissue of tuna, swordfish and salmon (wild vs. farmed) is investigated for the first time. SEC-UV-ICP-MS analyses revealed that Se and Hg are mostly bound to proteins of 2-12 kDa and up to 574 kDa, respectively. Moreover, Se and Hg appeared associated to proteins of same molecular weight, evidencing that Se-Hg interaction may occur at the level of the fish tissue evaluated. Important differences were found between farmed and wild salmon, suggesting the effect of the type of feed and growing conditions on Se and Hg content and their distribution through protein fractions. Finally, Se speciation studies performed by HPLC-ICP-MS and confirmed by HPLC-ESI-MS/MS showed SeMeSeCys as the only Se specie found in soluble, sarcoplasmic, myofibrillar and alkali-soluble proteins of all fishes analysed, except in soluble proteins extracted from tuna, where SeMet was also identified.

Retrospective LC-QTOF-MS analysis searching for pharmaceutical metabolites in urban wastewater.

The presence of pharmaceuticals in the environment is a matter of major concern because of their wide consumption and their potential negative effect on the water quality and living organisms. After human and/or veterinary consumption, pharmaceuticals can be excreted in unchanged form as the parent compound and/or as free or conjugated metabolites. These compounds seem not to be completely removed during wastewater treatments and might finally arrive to surface and ground waters. Consequently, both parent pharmaceuticals and metabolites are target analytes to be considered in analytical methodologies. The satisfactory sensitivity in full-acquisition mode, high-resolution, exact mass measurements and MS/MS capabilities of hybrid quadrupole time-of-flight (QTOF) mass spectrometry make of this technique a powerful analytical tool for the identification of organic contaminants. In this study, the use of QTOF-MS with the aid of specialised processing-data application managers has allowed the retrospective analysis of pharmaceuticals metabolites in urban wastewater without the need for additional injection of sample extracts. Around 160 metabolites have been investigated in wastewater samples previously analysed only for parent compounds using LC-QTOF under MS(E) mode (simultaneous recording of two acquisition functions, at low and high collision energy). The retrospective analysis was applied to search for pharmaceutical metabolites in parent-positive effluent wastewaters from the Spanish Mediterranean region. Five metabolites, such as clopidogrel carboxylic acid or N-desmethyl clarithromycin, were identified in the samples.

Wastewater-based epidemiology as a novel tool to evaluate human exposure to pesticides: Triazines and organophosphates as case studies.

Production and application of pesticides have risen remarkably in the last few decades. Even if they provide many benefits, they can be hazardous for humans and ecosystems when they are not used cautiously. Human exposure to pesticides is well documented, but new approaches are needed to boost the available information. This work proposes a new application of wastewater-based epidemiology (WBE) to assess the exposure of the general population to organophosphate and triazine pesticides (pyrethroid pesticides have already been validated). Several human urinary metabolites tested as WBE biomarkers, were suitable. Untreated wastewater samples from different European countries were analyzed by liquid chromatography-tandem mass spectrometry. Biomarker concentrations were converted to mass loads and used to back-calculate the local population's exposure to the parent pesticides, using specific correction factors developed in this study. Exposure to organophosphates and pyrethroids showed spatial and seasonal variations. Finally, pesticide exposure was estimated in twenty cities of ten European countries and compared with the acceptable daily intake, concluding that some populations might face health risks. The study confirms WBE as a suitable approach for assessing the average community exposure to pesticides and is a valuable complementary biomonitoring tool. WBE can provide valuable data for public health.

New psychoactive substances in several European populations assessed by wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) can be a useful tool to face some of the existing challenges in monitoring the use of new psychoactive substances (NPS), as it can provide objective and updated information. This Europe-wide study aimed to verify the suitability of WBE for investigating the use of NPS. Selected NPS were monitored in urban wastewater by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The main classical illicit drugs were monitored in the same samples to compare their levels with those of NPS. Raw composite wastewater samples were collected in 2016 and 2017 in 14 European countries (22 cities) following best practice sampling protocols. Methcathinone was most frequent (>65% of the cities), followed by mephedrone (>25% of the cities), and only mephedrone, methcathinone and methylone were found in both years. This study depicts the use of NPS in Europe, confirming that it is much lower than the use of classical drugs. WBE proved able to assess the qualitative and quantitative spatial and temporal profiles of NPS use. The results show the changeable nature of the NPS market and the importance of large WBE monitoring campaigns for selected priority NPS. WBE is valuable for complementing epidemiological studies to follow rapidly changing profiles of use of drugs.

Monitoring caffeine and nicotine use in a nationwide study in Italy using wastewater-based epidemiology.

Smoking cigarettes and drinking coffee are common habits in today's society. However, it is not easy to get up-to-date information on smoking prevalence and caffeine consumption as it is usually obtained from population surveys. To overcome this limitation and complement epidemiological information, we employed wastewater-based epidemiology (WBE) to gain a picture of the consumption of cigarettes and caffeine per day per person in Italy. A nationwide study was conducted by measuring two urinary metabolites of nicotine (cotinine and trans-3'-hydroxycotinine) and a caffeine metabolite (1,7-dimethyluric acid) in untreated wastewater from 16 cities. The spatial profiles of use depended mostly on the specific population habits in each city, not on the geographical area or the population size. The patterns of consumption were stable over the week, except in Milan where the use of both substances decreased on Sundays, probably because there were no commuters. In Milan, the use of nicotine decreased from 2013 to 2015. There was a significant relationship between smoking and consumption of products containing caffeine (mainly coffee), thus in cities where more cigarettes were smoked, more caffeine was drunk. These results are generally in accordance with findings from epidemiological studies, but provide some additional local profiles of use and closely follow changes over the years. This information could be useful for healthcare professionals and policy-makers to monitor progress towards the reduction of prevalence in tobacco use, and set up new health campaigns.

Wastewater-based epidemiology for tracking human exposure to mycotoxins.

Mycotoxins are among the compounds of most concern for human health because of their common presence in food and their proven toxicity for human health. Human biomonitoring (HBM) studies, foodstuff analysis and dietary surveys are usually used to assess human exposure, but they are costly and time-consuming. Wastewater-based epidemiology (WBE) can complement the established exposure monitoring tools. The aim of this study was to develop a new WBE application for assessing human exposure to mycotoxins by measuring specific biomarkers in raw wastewater. Eleven substances were analysed by liquid chromatography-tandem mass spectrometry in wastewater from four cities in Italy and Spain. Only deoxynivalenol (DON) and fumonisines were found, DON being the most abundant, in accordance with HBM studies. DON intake, back-calculated by WBE, was lower than the established Health-Based Guidance values, indicating moderate exposure in the population, with no risks for human health. WBE intake estimates were very close to those reported in HBM studies, validating WBE for estimating DON intake in the population. This study describes a promising WBE approach to complement HBM studies and assess the human intake of specific classes of mycotoxins, thus helping to identify risks for human health.

Assessment of human exposure to selected pesticides in Norway by wastewater analysis.

Pesticides offer many benefits for humanity and agriculture, but at the same time pose a potential risk to human health because of their widespread use and high biological activity. Human biomonitoring (HBM) studies are the main tool to investigate human exposure to pesticides and other chemicals, but face limitations such as sampling biases, long time to complete and high costs. Wastewater-based epidemiology (WBE) is an alternative approach that is centered on the chemical analysis of biomarkers of (pesticide) exposure in urban wastewater. The present study used WBE to assess human exposure to selected classes of pesticides, triazines, pyrethroids and organophosphates, in Norway. Untreated wastewater samples were collected from four cities, covering approximately 20% of the Norwegian population. The highest population weighted mass loads (mg/day/1000 inhabitants) were for alkyl phosphates and the lowest for triazines. Some differences were observed for the two metabolites, 2-isopropyl-6-methyl-4-pyrimidinol (IMPY) and 3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (DCCA), which were higher in the rural city of Hamar. WBE figures were comparable with HBM findings for the specific metabolite of chlorpyrifos and chlorpyrifos methyl (3,5,6-trichloro-2-pyridinol; TCPY) but were different for the alkyl phosphates. Pyrethroid intake was calculated and was lower than the acceptable daily intake in all the cities, indicating low risk for human health. This is the most extensive WBE study performed to date to assess national human exposure to pesticides. This study demonstrated that WBE has the potential to be a useful complementary biomonitoring tool for assessing population-wide exposure to pesticides, overcoming some of the limitations of HBM.

Use of legal and illegal substances in Malé (Republic of Maldives) assessed by wastewater analysis.

This study used wastewater-based epidemiology (WBE) to investigate the lifestyle of the inhabitants of Malé, the capital of the Republic of Maldives. Raw wastewater 12-h composite samples were collected from nine pumping stations serving the city area - thus representative of the whole Malé population. Samples were analysed by liquid chromatography coupled to mass spectrometry for estimating the profile of use of a large number of substances including illicit drugs, alcohol, caffeine, tobacco and pharmaceuticals. The illicit drugs most used were cannabis (THC) and heroin (700 and 18 g/day), with lower consumption of cocaine and amphetamines (0.1-1.2 g/day). It is important to note that the consumption of cannabis in Malé was comparable to that measured in other countries, while the consumption of heroin was higher. Among cathinones, mephedrone was detected at the highest levels similar to other countries. Consumption of alcohol, which is not allowed in Maldives, was found (1.3 L/day/1000 inhabitants), but at a low level compared with other countries (6-44 L/day/1000 inhabitants), while the consumption of caffeine and tobacco was generally in line with reports from other countries. Unique information on pharmaceuticals use was also provided, since no official data were available. Human lifestyle was evaluated by applying for the first time the full set of WBE methodologies available in our laboratory. Results provided valuable epidemiological information, which may be useful for national and international agencies to understand population lifestyles better, including illicit drug issues, and for planning and evaluation of drug prevention programs in Malé.

Enantiomeric profiling of quinolones and quinolones resistance gene qnrS in European wastewaters.

Wastewater-based epidemiology (WBE) was applied for the first time in seven cities across Europe with the aim of estimating quinolones consumption via the analysis of human urinary metabolites in wastewater. This report is also the first pan-European study focussed on the enantiomeric profiling of chiral quinolones in wastewater. By considering loads of (fluoro)quinolones in wastewater within the context of human stereoselective metabolism, we identified cities in Southern Europe characterised by both high usage and direct disposal of unused ofloxacin. In Northern European cities, S-(-)-ofloxacin loads were predominant with respect to R-(+)-ofloxacin. Much more potent, enantiomerically pure S-(-)-ofloxacin was detected in wastewaters from Southern European cities, reflecting consumption of the enantiomerically pure antibiotic. Nalidixic acid, norfloxacin and lomefloxacin were detected in wastewater even though they were not prescribed according to official prescription data. S,S-(-)-moxifloxacin and S,S-(-)-moxifloxacin-N-sulphate were detected in wastewater due to metabolism of moxifloxacin. For the first time, average population-normalised ulifloxacin loads of 22.3 and 1.5 mg day-1 1000 people-1 were reported for Milan and Castellón as a result of prulifloxacin metabolism. Enrichment of flumequine with first-eluting enantiomer in all the samples indicated animal metabolism rather than its direct disposal. Fluoroquinolone loads were compared with qnrS gene encoding quinolone resistance to correlate usage of fluoroquinolone and prevalence of resistance. The highest daily loads of the qnrS gene in Milan corresponded with the highest total quinolone load in Milan proving the hypothesis that higher usage of quinolones is linked with higher prevalence of quinolone resistance genes. Utrecht, with the lowest quinolones usage (low daily loads) had also one of the lowest daily loads of the qnrS gene. However, a similar trend was not observed in Oslo nor Bristol where higher qnrS gene loads were observed despite low quinolone usage.