Combined sewer overflow contribution to pharmaceutical pollution: a case study in northern Spain

Combined sewer overflow contribution to pharmaceutical pollution: a case study in northern Spain | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Combined sewer overflow contribution to pharmaceutical pollution: a case study in northern Spain Vladimir Akhrimenko Akhrimenko, Daniel Cerveny, Xiana Gonzalez-Gomez, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6980045/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The use of combined sewer systems is associated with frequent overflow events and discharges of untreated wastewater containing micropollutants, including APIs. The aim of this study was to evaluate the impact of weather-related factors on the presence of pharmaceuticals and illicit substances and assess the related environmental risk. To address this goal, three sites in the surroundings of the city of Vitoria-Gasteiz were sampled in April (dry week) and November (wet week): Betoño Pond in the Salburua Wetlands, and Zadorra River before and after receiving the Crispijana wastewater treatment plant’s (WWTP) effluent. An environmental risk assessment was performed by measuring the hazard quotient. This is the first time when an enantioselective risk assessment has been performed for environmentally relevant concentrations of illicit drugs measured in a wetland of an international importance. A total of 33 out of 102 substances were detected in at least one of the samples and a minimum of 6 APIs in all sample sites. Post-WWTP samples taken in autumn (wet week) presented the highest cumulative concentration (7739 ng L-1). Risk quotients of 12 APIs in the most contaminated site (SP3) in the rainy weather sampling period reached values > 1: diclofenac (3.12) and eprosartan (1.59). Pharmaceutical pollution sewer overflow weather factors wastewater treatment plant national parks drugs of abuse Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Active pharmaceuticals ingredients (APIs) and illicit drugs are organic micropollutants that occur globally in all types of environmental matrices (aus der Beek et al., 2016 ; Mejías et al., 2021 ; UBA, 2020; Wilkinson et al., 2022 ). The main sources of APIs in surface water are effluents from wastewater treatment plants (WWTP), landfill leachates and surface runoff over crop fields irrigated with treated wastewater and sewage sludge (Orive et al., 2022 ). APIs are biologically active molecules capable of interacting with biological receptors in wildlife species and induce toxicity at low concentrations, resulting in changes in reproduction, growth, behaviour and physiology (Brodin et al., 2024 ). Globally, an estimated 52% of all wastewater is treated to some degree although very few WWTPs are capable of removing micropollutants (Jones et al., 2021 ). Surface waters receiving WWTP’s effluent, especially downstream rejection points, tend to be the most contaminated site of the river compared with river sites far from, or upstream of, the WWTPs. (Vaudreuil, M. A. et al. 2024) The European Commission has recently added a list of APIs in the updated Urban Wastewater Treatment Directive, 80% of which must be removed from wastewater by quaternary treatment. (European Commission, 2025) As pharmaceuticals and personal care products have been identified as important sources of micropollutants in urban wastewater. These products are covered by extended producer responsibility, which means that the pharmaceutical and cosmetics industry will have to pay for at least 80% of the quaternary treatment. In addition, by 2027, Member States shall assess the risks posed by urban wastewater to the environment and public health, considering seasonal fluctuations and extreme events. The use of combined sewer systems for wastewater and stormwater, increased domestic flow and surface runoffs attributed to climate change are associated discharges of untreated wastewater containing micropollutants. (Perry, W. B. et al. 2023) Combined sewer systems have overflow channels to alleviate excessive flow (known as “wet weather overflow” when caused by rainfall). In the United Kingdom, 39 million tons of untreated wastewater are released into the Thames river as a result of sewer overflows. (EA, 2023 .) The Urban Wastewater Treatment Directive proposes a non-binding objective for overflows to achieve by 2039. It states that all agglomerations of 100 000 populations equivalent (p.e.) shall limit the annual overflows volume should be less than 2% of the annual collected wastewater load. In the case of the Mediterranean regions, climate change is expected to be accompanied by significant rainfall patterns and an increase of extreme rainfall events. (Gogien, F. et al. 2023 ) One example is the recent catastrophic and extreme rainfall in south-east Spain which likely resulted in a significant number of pharmaceuticals being released directly into the environment. (Plaza Valencia, 2025 ) The updated Urban Wastewater Treatment Directive also requires Member States to present by 2030 a list of areas of concern where there is a high risk of environmental and public health impacts attributed to aquatic pollution. This list may include catchment areas for human consumption, areas of recreational swimming, areas with aquaculture activities, lakes, coastal waters and areas of special interest for conservation forming part of the Natura 2000 ecological network. In most cases, rivers and streams within national parks and special areas of conservation do not receive any kind of water treatment. They are therefore sensitive to micropollutant exposure from animal husbandry, local human population and tourism. The aims of the present study was to monitor pharmaceuticals and illicit substances in various stages of the Zadorra River and Betoño Pond in Salburua Wetlands in the surroundings of the city of Vitoria-Gasteiz (north of Spain). Special focus on those stages near overflow channels and the point of rejection of the Crispijana WWTP’s effluent was applied to evaluate the possible impact of weather-related factors on the presence of pharmaceuticals. MATERIAL AND METHODS • Sampling The Zadorra River belongs to the Ebro drainage basin, and it flows across the province of Álava, Basque Country collecting water from all the rivers and streams of the region. It runs along the northern border of the city of Vitoria-Gasteiz, alongside the outfall pipe that collects all the city’s sewage water. The outfall pipe has 6 overflow channels (Abechuco, Calidad, Yurre, Asteguieta and two Crispijana WWTP’s bypass channels) that drain wastewater directly into the river. (Fig. 1 ) Salburua Wetlands, located within the municipal boundaries of Vitoria-Gasteiz, is a humid area with a series of ponds (Arkaute, Betoño, Duranzarra, Larregana), which are fed by the underlying aquifer. The wetlands interchange water with nearby tributaries of Zadorra River (Alegría, Errekaleor and Santo Tomás Rivers) via groundwater. It has an extension of 70 ha conformed by shallow waters, springs, groundwater emergences and irrigation ditches that house biological communities of interest. The banks of the lush basins are rich in reedbeds and mallow meadows neighboring with a gallery forest, areas left for reforestation and zones of public use. This marsh is included in the RAMSAR convention and constitutes a special protection area for birds, in accordance with the Directive 79/409/EEC (Council Directive 1979). Is also part of the Natura Network 2000. Crispijana WWTP serves as the main plant for the city of Vitoria-Gasteiz and other minor agglomerations nearby. It is a treatment plant based on activated sludge process, with a 480,000-population equivalent of pollution load, prepared to treat 17,500 m 3 h -1 by primary treatment, 5,500 m 3 h -1 by secondary (biologic) treatment and 400 m 3 h -1 by tertiary (phosphorus removal) treatment. It also contains two overflow channels, before and after the primary treatment. In total 3 sites were sampled: Betoño Pond in the Salburua Wetlands (SP1), Zadorra River before receiving the Crispijana WWTP’s effluent (SP2) and Zadorra River after receiving the Crispijana WWTP’s effluent (SP3). (Fig. 1 ) SP2 was 580 m upstream the Crispijana WWTP, while SP3 was 190 m downstream. Four overflow channels are located before SP2, while the other two are installed between SP2 and SP3. Grab samples were collected in duplicate from all three locations on November 4, 2021 during a week with low rainfall intensity and April 26, 2022 during a week with high rainfall intensity. The samples were taken from bridges from the center of the water bodies using a metal bucket. 3 mL samples were stored in rinsed glass vials and fridged at -20ºC until their analysis. • Reagents and methods of active pharmaceutical ingredients analysis Solid phase extraction (SPE) with 6 cc Oasis HLB cartridges containing 200 mg of sorbent (Waters, Milford, US) was used for the extraction of the water samples. The cartridges were conditioned with 5 mL of HPLC grade methanol (Merc, Darmstadt, Germany) and 5 mL of purified water. After that, 200 mL of the water sample was spiked with the mixture of 15 isotopically labelled internal standards. The vacuum manifold VacMaster (Biotage, Uppsala, Sweden) was used to impulse the sample volume through the cartridge at the rate of approximately 2 mL min − 1 . 5 mL of HPLC grade methanol, followed by 5 mL of Ethyl acetate were used to elute the target analytes from the sorbent. The eluent was stored in a 12 mL glass vials and was evaporated to dryness using the TurboVap LV (Biotage, Uppsala, Sweden). After the reconstitution with 150 µL of LC-MS grade methanol (Merc, Darmstadt, Germany), samples were transferred into a 1.5 mL autosampler vials equipped with a 200 µL glass inserts, and finally stored at -20°C. A total of 95 pharmaceuticals were determined based on their bioaccumulation capacity and their potency (effect/concentration ratio). The analysis was performed using a single run liquid chromatography tandem mass spectrometry (LC-MS/MS). A full list of target compounds, basic method performance parameters and internal standards used for quantification are compiled in Supplementary material (Table S1 ). Additional information on the instrumental method including polarities, HESI ionizations, collision energies, precursor/product ions and tube lens values can be found elsewhere. (Grabic et al., 2012 ; Lindberg et al., 2014 ). All internal standards and surrogates added for identification and quantification of the target molecules were categorized as analytical grade (> 98%). For the in-house preparation of purified water, a Mili-Q Advantage system was used, with a UV radiation source (Millipore, Billerica, USA). It was used as a mobile phase in combination with LC-MS grade methanol. The acidification of the mobile chromatographic phases at 0.1% was performed with LC-MS grade formic acid (Sigma-Aldrich, Steinheim, Germany). A triple stage quadrupole mass spectrometer (TSQ Quantiva, Thermo Scientific, San Jose, CA) equipped with a heated-electrospray ionization (HESI) ion source, coupled to an Accela LC pump (Thermo Fisher Scientific, San Jose, CA) and a PAL HTC autosampler (CTC Analytics AG, Zwingen, Switzerland) was used for sample analysis. Target analytes were separated by using a C18 phase Hypersil gold column (50 mm × 2.1 mm ID × 3 µm particles, Thermo Fisher Scientific, San Jose, CA, USA) with a guard column (2 mm, 2.1 mm, i.d. 5 µm particles). The mobile phase was methanol and water, acidified with formic acid (0.1%). The mobile phase flow started with 100% water and went gradually to 100% methanol in a 10-minute period. After that, it was kept constant for 3 more minutes for each sample run. The positive identification of target compounds was made by two MS/MS transitions. Limit of quantification (LOQ), precision and measurement of blank samples were used to define Quality assurance/Quality control of the analytical method. Recovery has been tested previously (Grabic et al., 2012 ). The quantification of target analytes was performed by using the internal standard approach. Instrumental LOQs were derived from seven-point standard curve prepared in methanol, ranging from 0.1 to 100 ng/ml. The peak area corresponding to the lowest point of the standard curve with a signal/noise ratio of > 10 was then used for calculating the LOQs in individual samples. A relative standard deviation (RSD) of response factors measured for each point of the standard curve from the peak areas of the target analyte and its internal standard was used to express precision. Two processing blanks (Milli-Q purified water and tap water) were prepared in accordance with the extraction protocol used for tissue samples. • Reagents and methods of illicit drug analysis Seven illicit drugs were included in the analysis. Individual solutions of 1 mg mL − 1 of amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), cocaine, benzoylecgonine, 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) and morphine, and 0.1 mg mL − 1 of the deuterated analogues of each substance were used as surrogate internal standards (ISs), were supplied by Cerilliant (Round Rock, TX, USA). A stereoisomeric analysis was performed for amphetamine, metamphetamine and MDMA. Individual solutions of 1 mg mL − 1 of the S-(+) enantiomer of each compound were supplied by Merck (Darmstadt, Germany). LC-MS grade methanol (MeOH), LC-MS grade acetic acid, LC-MS grade formic acid, ammonia (NH 3 ) solution in water (25%) were supplied by Merck. Sodium acetate was obtained from Fluka (Steinheim, Germany). Ultrapure water was obtained with a Genie Water System from RephiLe Bioscience (Boston, MA, USA). The analysis was performed at the University of Santiago de Compostela, after solid-phase extraction with Oasis MCX cartridges (Waters, Milford, MA, USA) and LC-MS/MS analysis (Waters Acquity UPLC® H-class – Xevo TQD system) as described in (González-Mariño et al., 2018 ). This method affords LOQ values in the 0.1 (benzoylecgonine) to 16 (THC-COOH) ng L − 1 range, and satisfactory recoveries in spiked surface water (98–112%, RSD values ≤ 12%). The enantiomeric analysis was conducted as described in Estévez-Danta et al. ( 2021 ). This methodology renders LOQs in the 2.4 to 5.5 ng L − 1 range (referring to pure enantiomers) and recoveries in surface water in the 90–107% range (RSD ≤ 10%). (Table S2) Samples were analyzed in triplicate and each batch was accompanied by one blank sample and two quality control samples spiked at 50 and 500 ng L − 1 , respectively, to verify the correct functioning of the analytical method, following the recommendations of (Hernández et al., 2023 ). • Hydrological data extraction Minimum and maximum river flow rate (m 3 /s) and daily accumulated rainfall (l/m 2 ) was obtained from the Ebro River Hydrological Information System website (Table S3). (Spanish Government, 2025). Information about overflow volumes (m 3 ) discharged from the Crispijana WWTP’s overflow channel (bypass) and the annual WWTP influent flow rate and untreated wastewater discharged annually to the Zadorra River was provided by AMVISA (a private company which provides public services related to integrated water cycle management). (Table S4) This information was used to calculate the percentage of annual untreated wastewater discharge flow using the following formulas: $$\:\%\:of\:discharged\:untreated\:wastewater\:=\frac{{Q}_{WWT{P}^{{\prime\:}}s\:overflow}}{{Q}_{influent}}\:$$ $$\:\%\:of\:discharged\:wastewater\:only\:treated\:with\:1º\:treatment\:=\frac{{Q}_{biologic\:overflow}}{{Q}_{influent}}$$ • Risk assessment An environmental risk assessment was performed for the APIs and illicit drugs detected above LOQ values in at least one sample. For the assessment, risk quotients (RQ) were calculated by dividing the measured environmental concentration (MEC) for each compound at each site by the concentration derived from toxicity data, also known as predicted no-effect concentration (PNEC). A precautionary approach was followed for the assessment; when available, chronic toxicity data was used as the PNEC. For the substances regulated by the Water Framework Directive, environmental quality standards (EQS) were used (European Commission, 2024 ). For the other detected compounds, toxicity data was retrieved from US EPA ECOTOX database (US EPA 2023 ), the Enviro Tox database(Connors et al., 2019 ) or the repository compiled by Giunchi et al. (Giunchi, 2023 ). The species and the endpoint for each PNEC value was also collected. An assessment factor (AF) was to the retrieved toxicity data to mitigate the uncertainty of the data availability and its nature (ECHA, 2011 ). In the case of caffeine, the species sensitivity distribution (SSD) values was used instead of the PNEC. SSD represents the lower limit of the 95% confidence interval LCL of the concentration which would be hazardous to 5% of the species (Rodríguez-Gil et al., 2018 )- We considered that an RQ of < 0.1 indicates that no adverse effects are expected, an RQ between 0.1 and 1 would indicate a low risk, between 1 and 10 would indicate a moderate risk, and above 10 would indicate a high risk. A heatmap of RQ values was generated in R using the ggplot2 libraries. Half-life (DT50) (days), bioaccumulation factor (BCF; L kg − 1 ) and partition factor octanol/water (Log K o/w ) data were also collected from CompTox (Williams et al., 2017). (Table S5) RESULTS Analytical method quality parameters The analytical method performance has shown good reliability parameters. The average LOQs calculated from all water samples ranged between 0.01 and 76 ng L − 1 , and 83 out of 95 target pharmaceuticals expressed average LOQ below 0.5 ng L − 1 . No pharmaceuticals were detected in any of the blank samples. In the case of illicit drugs, LOQs ranged between 0.1 and 5.5 ng L − 1 . More details about the method performance are given as Supplementary material (Table S1 and S2). Presence of pollutants At least six substances were detected at every sampling point both in autumn and spring. Briefly, caffeine, benzoylecgonine, morphine, THC-COOH, R-amphetamine, and cocaine appeared in all the analyzed waters at all seasons. A total of 33 out of 102 substances (27 pharmaceuticals and 6 illicit drugs) were detected in at least one of the samples. Methamphetamine was the only illicit drug that did not appear in any sample. Generally, more pharmaceuticals, and at higher concentrations, were detected in the wet weather season samplings. (Fig. 2 ) Post-WWTP samples taken in autumn presented the highest cumulative concentration (7739 ng L − 1 ), and the highest individual concentrations with paracetamol (2066 ng L − 1 ), telmisartan (1442 ng L − 1 ), caffeine (868 ng L − 1 ) and tramadol (726 ng L − 1 ) at the top spot. (Fig. 3 ) Five illicit drugs and caffeine were detected in Betoño Pond without significant seasonal variations. • Hydrological data The mean flow of Zadorra River was 5.9 m 3 s − 1 (3.4–10.3) on April 26th and 0.87 m 3 s − 1 (0.762–1.03) on November 4th. Days before spring sampling were relatively dry, with daily-accumulated rainfall reaching 1.8 L m − 2 . Rainy weather in the autumn sampling is illustrated by the 10.8 L m − 2 3 days prior to the sampling. There was an overflow outfall on November 4, were 37.400 m 3 of untreated water was released into the Zadorra River. The annual overflows volume discharged from WWTP’s overflow channel was 4.728.400 m 3 in 2021 was and 842.100 m 3 in 2022. The percentage of untreated wastewater or only treated with primary treatment (removal of floatable objects), was 35.07% and 15.76% respectively. Ecotoxicological risk assessment The RQ values of 7 APIs on both season exceeded 0.1 in the most contaminated site (SP3), reaching “medium risk” values (above 1) in the case of diclofenac (31), flecainide (19) and sertraline (1.6). (Fig. 4 ) The RQ values for diclofenac and flecainide at SP2 in the measuring performed in November were 3.2 and 1.4 respectively, which indicates a medium environmental risk. The highest RQ values for illicit drugs were 0.04 (THC-COOH) and 0.01 (cocaine). Five substances (carbamazepine, diclofenac, fluoxetine, propranolol, sulfamethoxazole) regulated by the Water Framework and Urban Wastewater Treatment Directives presented “low-medium risk” levels. (Table S6) DISCUSSION We found that the accumulated concentrations of monitored pharmaceuticals and illicit drugs in surface waters upstream the WWTP were 19 times higher in the rainfall sampling period than in the dry sampling period. This was mainly due to the contribution of 10 APIs that only appeared during the wet weather week. It is also important to note that the combination of low river flow (i.e. reduced dilution) with sewer overflows may result in a difference in the concentration between seasons. Caffeine, an anthropogenic activity marker used in beverages and in pharmaceutical and cosmetic products, and paracetamol, an antipyretic drug, presented the highest concentrations. These two substances have been proposed as tracers of sewer overflow and untreated wastewater discharges (Buerge et al., 2006 ; White et al., 2019 ). During the sampling week in April (with minimal daily accumulated rainfall and consequently no overflow discharges) measured concentrations of caffeine and paracetamol were low and did not vary between sampling sites. The presence of caffeine and paracetamol here could be attributed to rural sources next to Zadorra River and its tributaries. In contrast, in the sampling week in November, samples taken upstream the WWTP in November, from a section of the river receiving untreated wastewater from four overflow channels, showed high concentrations of caffeine. Hence, it is likely that the pharmaceutical burden in the river stage before reaching WWTP can be explained by untreated wastewater overflow discharges. Nevertheless, these assumptions should be treated with care, as other factors, such as seasonal fluctuations in consumption and the river flow rate may vary. Apart from paracetamol and caffeine, angiotensin receptor blockers (ARBs) (telmisartan, irbersartan and eprosartan) and beta-blockers (atenolol, bisoprolol and propranolol), are also present in relatively high concentrations in the Zadorra River. These pharmaceuticals are used worldwide for controlling high blood pressure, the main suspect of cardiovascular disease and premature death (Mills et al., 2020 ). Beta-blockers, ARBs and their active transformation products, which are frequently detected in different aquatic matrices, may cause diverse adverse effects on the reproduction, growth and cardiac development in wildlife (Yi et al., 2020 , Muambo et al., 2024 )). Other substance detected at high concentrations is tramadol, which may alter the behaviour as demonstrated with the experimental study performed with European chubs ( Squalius cephalus ). Fish exposed to tramadol showed an anxiolytic-like effect (Santos et al., 2021 ). These alteration may lead to the impairment od key fitness related behaviours of wild animals (Brand et al., 2025 ) In 2020, a micropollutant characterization of the Crispijana WWTP’s effluent and influent was performed, and WWTP’s removal efficiency for each API was also estimated (Lopez-Herguedas et al., 2023 ). The authors showed that the removal efficiency for 62 out of 72 detected APIs (48 presented higher values in the effluent than in the influent) was below 80%. Contrary, Crispijana WWTP showed a removal rate of almost 100% for caffeine and paracetamol. In the current monitorization, paracetamol was absent during April, but reached 883 ng L − 1 in November, likely due to the discharge of untreated water from the 2 overflow channels located in the Crispijana WWTP. The updated Spanish Regulation of Hydraulic Public Domain dictates that flow rates combined sewer overflow must be monitored. (Spanish Government, 2023 ) The Urban Wastewater Treatment Directive takes a step further: “the concentration of the regulated micropollutants and microplastics shall be monitored at relevant points of storm water overflows” (European Commission, 2024 ). The Directive also proposes that the competent authorities (e.g. water management companies) should consider preventive measures to avoid the entry of rain waters into sewers. Some strategies are currently in development to prevent combined sewer overflows using front-of-the-pipe (e.g. soil filters, retention facilities) and end-of-pipe (e.g. chemical coagulation) (Botturi et al., 2020 ). The expansion of green and blue zones in urban areas is also a feasible alternative, although in the case of Vitoria-Gasteiz, a city that has been awarded with the Green Capital title in 2014, is not enough. In the sampling years, the percentage of untreated wastewater reached 35% and 16%, in 2021 and 2022, respectively, which is too far from the non-binding objective proposed by the Directive of 2% of annual discharge. A derivation water channel to decrease the sewer volume that ends in the Crispijana WWTP is currently under construction (URA, 2022). This will likely lead to a decrease of the overflow percentage and the amount of untreated wastewater reaching the river. In addition to the transportation of pollutants, overflow water can compromise the secondary wastewater treatment of WWTP, an essential step for the removal of nutrients, pathogens, APIs and other micropollutants. (Dirckx et al., 2019 ) Consequently, the performance of the WWTP would be impaired until the recovery of the microbial communities affected by the excess stormwater, a process that can take several days. Furthermore, pollutants retained in the sediments can be mobilized during storm events, which can account for up to 65% of the total emissions resulting from wet weather overflows (Del Río et al., 2013 ). There is a strong relationship between log K ow and log K oc (organic carbon to water partition coefficient) values, with the latter representing the tendency of sediment adsorption (EPA, 1996). Del Río et al. ( 2013 ) showed how, following a rainfall event, the sediment fraction of ibuprofen, diclofenac and caffeine are dissolved and mobilised from the sediments. For instance, ARBs are weak acids with high log K ow values and thus have an affinity for the sediment of the combined sewers and are likely to be dissolved and mobilized in a rainfall event. In the present study, ARBs appear in higher concentrations in the rainy sampling period at SP2, with eprosartan reaching low-risk concentrations (RQ = 0.4). The case of diclofenac is similar to ARBs; its physicochemical characteristics could be related to its presence at low-risk concentrations (RQ = 0.32) in the samplings performed in November. Although sampling data in this study is limited, further research on the sewer sediment mobilisation, the relationship with the physicochemical properties of the APIs and the contribution to wet weather overflow discharges are recommended. Betoño Pond consists in stationary waters only connected to groundwater, but it is also exposed to anthropogenic pollution. The presence of APIs and illicit drugs in this wetland could be a consequence of sporadic urination of passerby or due to the exchange between the wetlands and groundwater. (Jurado et al., 2012 ). However, caffeine and some illicit drugs were detected in both seasons at similar concentration, which were higher or close to the concentrations found in the Zadorra River. For example, THC-COOH is a metabolite of THC, an active substance of cannabis which is an illegal drug in Spain. This metabolite has a low percentage of removal in WWTPs, and in some cases the concentration in the effluents augments due to the deconjugation of the glucuronide form. (How et al., 2021) Another example is morphine, it is a prescription drug used for chronic pain with a potential of being abused, and also a metabolite of heroin, and thus, the detection of morphine could be related to the illegal consumption of this drug. As for amphetamine is an illicit drug, but also a metabolite of lisdexamphetamine prescribed used for the treatment of attention deficit hyperactivity disorder. Illicit amphetamine is distinctive by its racemic composition, it is normally composed of even parts of R-amphetamine ang S-amphetamine, while the metabolite of lisdexamphetamine is the S enantiomer. MDMA is also composed of a racemic mixture, whereas methamphetine reaching the illicit Spanish market is normally composed of the most active S enantiomer only (Estévez-Danta et al., 2021 ). The presence of R-amphetamine in the sampled water is indicative of illegal consumption. In a recent study (Estévez-Danta et al., 2021 ), the enantiomeric profiling of wastewater in the Basque Country demonstrated high levels of amphetamine, and only less than 1% of the load was explained by the mentioned prescription drug API lisdexamphetamine. This same study demonstrated that the R-enantiomer of these 3 drugs is enriched during water treatment, while the S-enantiomer is more efficiently removed (Estévez-Danta et al., 2021 ). As far as we know, so far only 4 studies have assessed the presence of illicit drugs in Natural Parks and other natural areas; and the current study represents the first time that an enantiomeric analysis has been performed on samples from natural areas. One study performed in Kruger National Park in South Africa did not find any illicit drugs, while in a monitoring campaign conducted in Antonia Ramos Protected Area in Argentina found benzoylecgonine in the muscle of thararira ( Hoplias Lacerdae ), and in the liver and gills of streaked prochilod ( Prochilodus lineatus ), up to 0.87 µg kg − 1 . (Ondarza et al., 2019 ; Sims et al., 2023 ) In Spain, Pego-Oliva Marsh and L’Albufera Natural Park, both in the province in Valencia, have been monitored for the presence of pharmaceuticals, heavy metals, illicit drugs and other anthropogenic pollutants. (Pascual-Aguilar et al., 2015; Vazquez-Roig., 2012) Both are coastal lagoons that are included in the RAMSAR convention like Salburua Wetlands. Cocainics, amphetamine-like compounds, opiates, cannabinoids and ketamine were analyzed there. Mean concentrations of amphetamine, benzoylecgonine, cocaine and THC-COOH measured in Salburua Wetlands were higher than in two other studies. (Table 1 ) Table 1 Monitoring studies of illicit drugs in National Parks Sampling site Study data Concentrations of selected substances Mean (Maximum) ng L − 1 Main observations AMP BECG COC KET MDMA MET MOR THC-COOH Pego-Oliva Marsh • Study : Vazquez-Roig et al., 2012 • Sampling period : June 2009 • Sampling method : grab sampling at 23 points • Analyzed substances : 14 illicit drugs and metabolites NA 1.92 (15.5) 2.25 ( 11.8 ) 21.33 (414.92 ) 0.062 ( 3.4 ) 0.32 (2.7 ) 3.59 (8.3) 0.07 (1.54) • Near settlements of 600 and 1400 housings. The wetland receives water from a wastewater treatment plant (WWTP) • No rainfall during the sampling period • 8 illicit drugs and metabolites were detected • All samples were contaminated • The most occurring drug was ketamine and MDMA • THC was not detected and its metabolite THC-COOH was found in only one sample • Sampling sites near to the coast presented higher concentrations L’Albufera Natural Park • Study : Pascual-Aguilar et al., 2013 • Sampling period : March 2008 • Sampling method : grab sampling at 21 points • Analyzed substances : 14 illicit drugs and metabolites, and 17 pharmaceuticals 3.38 (3.38) 5.57 ( 78.71 ) 0.48 ( 4.43 ) NA 0.54 (2.48) ND 6 (11.70) ND • The park is surrounded by an urban area of more than 1,200,000 inhabitants. 2 WWTP’s effluents enter the park • No rainfall during the sampling period • 9 illicit drugs and metabolites were detected • 16 samples were contaminated • Highest accumulated concentration was 78 ng L -1 • Cocainics were most frequently detected • Higher concentrations were detected in sites near high population densities. Salburua Wetlands • Study : Current study • Sampling period : November 2021 and April 2022 • Sampling method : grab sampling at 1 pont • Analyzed substances : 7 illicit drugs and metabolites, and 95 pharmaceuticals 9.45 (11) 33.9 (35.1) 2.3 (2.4) NA ND ND 1.6 ( 14.3 ) 17.95 (19) • The park is near a urban zone with more than 250,000 inhabitants. • Is not connected directly with any surface water. • Samples were taken during dry weather period and rainy weather period. • Metamphetamine and MDMA were not detected • An enantiomeric analysis was performed. Only R enantiomer was detected of amphetamine • Highest accumulated concentration was 57 ng L -1 HPLC-MS/MS: High performance liquid chromatography and mass spectroscopy tandem; AMP: amphetamine; BECG: benzoylecgonine; COC: cocaine; KET: ketamine; MDMA: 3,4-methylenedioxymethamphetamine; MOR: morphine; THC-COOH: nor-9-carboxy-Δ9- tetrahydrocannabinol. ND: not detected; NA: not analyzed. Ecotoxicological data regarding illicit drugs are relatively scarce, which was the explanation why a list of illicit drugs was not included in the 4th Watch List. (Gomez Cortes et al., 2022) Amphetamine is known to decrease the bacterial and diatom communities in water stream biofilms, produce reactive oxygen species (ROS) and genetic damage in zebra mussels ( Dreissena polymorpha ). (Lee et al., 2016 ; Parolini et al., 2016 ) Benzoylecgonine decreases the parthenogenic cycle and number of offsprings, and reduces of locomotor activity in Daphnia magna . (Parolini et al., 2017; Parolini et al., 2018 ) Cocaine causes swelling and breakdown of skeletal muscle of European eel ( Anguilla anguilla )) and behavioural changes in Danio rerio . (Darland and Dowling, 2001 ; Capaldo et al., 2018 ) There is differences in the toxicity levels between different enantiomers of several APIs and illicit drugs. (Pérez-Pereira et al., 2024 ) This enantioselectivity is characteristic for amphetamine, MDMA and ketamine. Freshwater algae Chlorella pyrenoidosa showed preferential bioaccumulation and degradation for S-amphetamine, which could explain that only R-amphetamine was detected in the current study. Toxic effects of amphetamine on Daphnia magna seem to be also enantioselective: S enantiomer is related to the decrease of the number of eggs and body size, while R enantiomer affects the heart rate and size. The decrease of body size in Daphnia magna is also attributed to S-MDMA. No illicit drug had a RQ value higher than 0,1. However, the available ecotoxicological studies only have assessed acute toxicity; the information about how chronic exposure to illicit drugs may affect wild species is scarce. (Maasz et al., 2021 ) In Betoño Pond, concentrations from Autumn and Spring samplings were similar, meaning that there is a chronic exposure to these micropollutants. RAMSAR sites are of high importance for water birds, especially for the globally threatened birds that reside mostly in wetlands. As these birds consume aquatic species, there is a risk for biomagnification. (Herrero-Villar et al., 2024 ) A study conducted in Mai Po RAMSAR site in Hong Kong, China have concluded that birds which seeks food from the sediments (e.g. common sandpiper) and have a small body size are more prone to be exposed to micropollutants. (Man et al., 2021 ) CONCLUSIONS The Zadorra River is exposed to at least 33 APIs and illicit drugs, and their concentrations reaching medium risk levels for the environment. The situation is exacerbated during periods of wet weather, resulting in a 19-fold increase in the accumulated concentrations, attributed mainly to paracetamol, caffeine and telmisartan, in the sections of the river that do not receive WWTP’s effluent. As stipulated by the updated Urban Wastewater Directive, combined sewer overflow and the micropollutants carried by this untreated water shall be monitored. As storm events frequency attributed to climate continue to increase, it is imperative to prioritise the identification of solutions for reducing the volume of wet weather overflow. Betoño Pond located in the protected area of the Salburua Wetlands is also a subjected to organic pollution, including illicit drugs. In contrast to the river, the concentrations across different seasons is stable. The ongoing exposure to these substances could pose a significant threat to local aquatic species and could also potentially impact on the avian communities, foraging in the wetlands, due to biomagnification. Declarations Funding JBQ and XGG would like to acknowledge the financial support from the Spanish Agencia Estatal de Investigación – MCIN/AEI/10.13039/501100011033 (RED2022-134363-T, PID2020-117686RB-C32) and Instituto de Salud Carlos III-EDRF (RD24/0003/0020 – RIAPAd Network) GO acknowledges the Spanish Ministry of Economy, Industry, and Competitiveness (PID2022-139746OB-I00/AEI/10.13039/501100011033) and the University of the Basque Country. VA acknowledges the Basque Government for his Ph.D. scholarship (PRE_2024_1_0142). Competing interests The authors have no relevant financial or non-financial interests to disclose. Author contributions Vladimir Akhrimenko: Investigation, Formal analysis, Writing – original draft. Daniel Cerveny : Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Xiana Gonzalez-Gomez : Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Matías Nicolás Cuenca-Castillo : Formal analysis, Investigation, Writing – original draft. Iker Egaña : Investigation, Writing – original draft. Jose Benito Quintana : Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Tomas Brodin :Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Unax Lertxundi: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. Gorka Orive : Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. DATA AVAILABILITY The authors declare that the results of the analyzed data are available at the online repository ZENODO: 10.5281/zenodo.15342909 References aus der Beek, T., Weber, F. A., Bergmann, A., 2016. 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Alava Campus: Universidad del Pais Vasco - Campus de Alava","correspondingAuthor":false,"prefix":"","firstName":"Unax","middleName":"","lastName":"Lertxundi","suffix":""},{"id":477009819,"identity":"91106a93-8f7c-4dfe-9f6a-23d80b0f0235","order_by":8,"name":"Gorka Orive","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYHACZjDJxsDA+ICxgUQtzAakaQHpkiBKi7x772ODHww2+XwSyccqPu44zMAvkf4ArxbDM8eNE3sY0izbJNLSbs48c5hBckaOAX4tM9KYD/AwHDZg4zljdpu37TCDwY0c/A4DaTn4B6zl/Lfiv0At9jcIOExeIo05GWwLew8bMyPIFokE/A4z4DnGbCxjkAbU0mYs2duWziNx5g1+LfLtbcySbypsDOSbmR9++NlmLcffTsBhBgfAJEKAB69ysC0NBJWMglEwCkbBiAcAmgc+HUZfaVwAAAAASUVORK5CYII=","orcid":"","institution":"University of the Basque Country - Alava Campus: Universidad del Pais Vasco - Campus de Alava","correspondingAuthor":true,"prefix":"","firstName":"Gorka","middleName":"","lastName":"Orive","suffix":""}],"badges":[],"createdAt":"2025-06-26 06:21:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6980045/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6980045/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85836106,"identity":"e1a35714-fdcb-4bf8-8ed8-d4fe5f5c130b","added_by":"auto","created_at":"2025-07-02 08:21:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":195258,"visible":true,"origin":"","legend":"\u003cp\u003eMap of city of Vitoria-Gasteiz and surroundings with the information regarding sampling sites and the sewer overflow channel discharging in the Zadorra River.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980045/v1/17cd148cf37095f19b72e67b.jpg"},{"id":85836100,"identity":"983e4394-70b5-4003-8ce0-4662910684e2","added_by":"auto","created_at":"2025-07-02 08:21:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79831,"visible":true,"origin":"","legend":"\u003cp\u003eCumulative concentration (ng L\u003csup\u003e-1\u003c/sup\u003e) of the detected substances at the Salburua Natural Park (SP1), Zadorra River upstream of the wastewater treatment plant (SP2) and downstream the wastewater treatment plant (SP3) on the wet and dry sampling\u0026nbsp; periods.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980045/v1/b76a2aec63c397576aadd500.jpg"},{"id":85838003,"identity":"4e6d031d-6626-4570-92b1-84d5776983e8","added_by":"auto","created_at":"2025-07-02 08:37:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":714579,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration (ng L\u003csup\u003e-1\u003c/sup\u003e) of each detected substance at the Salburua Natural Park (SP1), Zadorra River upstream of the wastewater treatment plant (SP2) and downstream the wastewater treatment plant (SP3) on the wet and dry sampling\u0026nbsp; periods.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6980045/v1/3ccdf2e0c00f61420191146b.png"},{"id":85838002,"identity":"10b1ef49-8969-4bd1-9d7f-2b7c68a0eb3f","added_by":"auto","created_at":"2025-07-02 08:37:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":143148,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of the hazard quotient (HQ) values of the detected pharmaceuticals and transformations products at the 3 sampling points on the wet and dry sampling periods. No risk is expected with HQ values below 0.1, low risk is expected with HQ values between 0.1 and 1, medium risk with HQ values between 1 and 10, and high risk above 10.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980045/v1/922771793b18b7c5be6f905d.jpg"},{"id":96708204,"identity":"ac623b2a-3f68-4b81-9785-5c6f0eaadb61","added_by":"auto","created_at":"2025-11-25 09:59:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1995878,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6980045/v1/0eef5931-c1dc-424a-95cb-894617cb203c.pdf"},{"id":85836108,"identity":"bf16634b-278c-4814-9f07-6333de3bf31f","added_by":"auto","created_at":"2025-07-02 08:21:28","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":70999,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6980045/v1/cb67c10f5b123550d41325e7.docx"}],"financialInterests":"","formattedTitle":"Combined sewer overflow contribution to pharmaceutical pollution: a case study in northern Spain","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eActive pharmaceuticals ingredients (APIs) and illicit drugs are organic micropollutants that occur globally in all types of environmental matrices (aus der Beek et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mej\u0026iacute;as et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; UBA, 2020; Wilkinson et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The main sources of APIs in surface water are effluents from wastewater treatment plants (WWTP), landfill leachates and surface runoff over crop fields irrigated with treated wastewater and sewage sludge (Orive et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). APIs are biologically active molecules capable of interacting with biological receptors in wildlife species and induce toxicity at low concentrations, resulting in changes in reproduction, growth, behaviour and physiology (Brodin et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGlobally, an estimated 52% of all wastewater is treated to some degree although very few WWTPs are capable of removing micropollutants (Jones et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Surface waters receiving WWTP\u0026rsquo;s effluent, especially downstream rejection points, tend to be the most contaminated site of the river compared with river sites far from, or upstream of, the WWTPs. (Vaudreuil, M. A. et al. 2024) The European Commission has recently added a list of APIs in the updated Urban Wastewater Treatment Directive, 80% of which must be removed from wastewater by quaternary treatment. (European Commission, 2025) As pharmaceuticals and personal care products have been identified as important sources of micropollutants in urban wastewater. These products are covered by extended producer responsibility, which means that the pharmaceutical and cosmetics industry will have to pay for at least 80% of the quaternary treatment. In addition, by 2027, Member States shall assess the risks posed by urban wastewater to the environment and public health, considering seasonal fluctuations and extreme events.\u003c/p\u003e \u003cp\u003eThe use of combined sewer systems for wastewater and stormwater, increased domestic flow and surface runoffs attributed to climate change are associated discharges of untreated wastewater containing micropollutants. (Perry, W. B. et al. 2023) Combined sewer systems have overflow channels to alleviate excessive flow (known as \u0026ldquo;wet weather overflow\u0026rdquo; when caused by rainfall). In the United Kingdom, 39\u0026nbsp;million tons of untreated wastewater are released into the Thames river as a result of sewer overflows. (EA, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e.) The Urban Wastewater Treatment Directive proposes a non-binding objective for overflows to achieve by 2039. It states that all agglomerations of 100 000 populations equivalent (p.e.) shall limit the annual overflows volume should be less than 2% of the annual collected wastewater load. In the case of the Mediterranean regions, climate change is expected to be accompanied by significant rainfall patterns and an increase of extreme rainfall events. (Gogien, F. et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) One example is the recent catastrophic and extreme rainfall in south-east Spain which likely resulted in a significant number of pharmaceuticals being released directly into the environment. (Plaza Valencia, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe updated Urban Wastewater Treatment Directive also requires Member States to present by 2030 a list of areas of concern where there is a high risk of environmental and public health impacts attributed to aquatic pollution. This list may include catchment areas for human consumption, areas of recreational swimming, areas with aquaculture activities, lakes, coastal waters and areas of special interest for conservation forming part of the Natura 2000 ecological network. In most cases, rivers and streams within national parks and special areas of conservation do not receive any kind of water treatment. They are therefore sensitive to micropollutant exposure from animal husbandry, local human population and tourism.\u003c/p\u003e \u003cp\u003eThe aims of the present study was to monitor pharmaceuticals and illicit substances in various stages of the Zadorra River and Beto\u0026ntilde;o Pond in Salburua Wetlands in the surroundings of the city of Vitoria-Gasteiz (north of Spain). Special focus on those stages near overflow channels and the point of rejection of the Crispijana WWTP\u0026rsquo;s effluent was applied to evaluate the possible impact of weather-related factors on the presence of pharmaceuticals.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u0026bull; Sampling\u003c/h2\u003e \u003cp\u003eThe Zadorra River belongs to the Ebro drainage basin, and it flows across the province of \u0026Aacute;lava, Basque Country collecting water from all the rivers and streams of the region. It runs along the northern border of the city of Vitoria-Gasteiz, alongside the outfall pipe that collects all the city\u0026rsquo;s sewage water. The outfall pipe has 6 overflow channels (Abechuco, Calidad, Yurre, Asteguieta and two Crispijana WWTP\u0026rsquo;s bypass channels) that drain wastewater directly into the river. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSalburua Wetlands, located within the municipal boundaries of Vitoria-Gasteiz, is a humid area with a series of ponds (Arkaute, Beto\u0026ntilde;o, Duranzarra, Larregana), which are fed by the underlying aquifer. The wetlands interchange water with nearby tributaries of Zadorra River (Alegr\u0026iacute;a, Errekaleor and Santo Tom\u0026aacute;s Rivers) via groundwater. It has an extension of 70 ha conformed by shallow waters, springs, groundwater emergences and irrigation ditches that house biological communities of interest. The banks of the lush basins are rich in reedbeds and mallow meadows neighboring with a gallery forest, areas left for reforestation and zones of public use. This marsh is included in the RAMSAR convention and constitutes a special protection area for birds, in accordance with the Directive 79/409/EEC (Council Directive 1979). Is also part of the Natura Network 2000. Crispijana WWTP serves as the main plant for the city of Vitoria-Gasteiz and other minor agglomerations nearby. It is a treatment plant based on activated sludge process, with a 480,000-population equivalent of pollution load, prepared to treat 17,500 m\u003csup\u003e3\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e by primary treatment, 5,500 m\u003csup\u003e3\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e by secondary (biologic) treatment and 400 m\u003csup\u003e3\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e by tertiary (phosphorus removal) treatment. It also contains two overflow channels, before and after the primary treatment.\u003c/p\u003e \u003cp\u003eIn total 3 sites were sampled: Beto\u0026ntilde;o Pond in the Salburua Wetlands (SP1), Zadorra River before receiving the Crispijana WWTP\u0026rsquo;s effluent (SP2) and Zadorra River after receiving the Crispijana WWTP\u0026rsquo;s effluent (SP3). (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) SP2 was 580 m upstream the Crispijana WWTP, while SP3 was 190 m downstream. Four overflow channels are located before SP2, while the other two are installed between SP2 and SP3. Grab samples were collected in duplicate from all three locations on November 4, 2021 during a week with low rainfall intensity and April 26, 2022 during a week with high rainfall intensity. The samples were taken from bridges from the center of the water bodies using a metal bucket. 3 mL samples were stored in rinsed glass vials and fridged at -20\u0026ordm;C until their analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e• Reagents and methods of active pharmaceutical ingredients analysis\u003c/h3\u003e\n\u003cp\u003eSolid phase extraction (SPE) with 6 cc Oasis HLB cartridges containing 200 mg of sorbent (Waters, Milford, US) was used for the extraction of the water samples. The cartridges were conditioned with 5 mL of HPLC grade methanol (Merc, Darmstadt, Germany) and 5 mL of purified water. After that, 200 mL of the water sample was spiked with the mixture of 15 isotopically labelled internal standards. The vacuum manifold VacMaster (Biotage, Uppsala, Sweden) was used to impulse the sample volume through the cartridge at the rate of approximately 2 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. 5 mL of HPLC grade methanol, followed by 5 mL of Ethyl acetate were used to elute the target analytes from the sorbent. The eluent was stored in a 12 mL glass vials and was evaporated to dryness using the TurboVap LV (Biotage, Uppsala, Sweden). After the reconstitution with 150 \u0026micro;L of LC-MS grade methanol (Merc, Darmstadt, Germany), samples were transferred into a 1.5 mL autosampler vials equipped with a 200 \u0026micro;L glass inserts, and finally stored at -20\u0026deg;C.\u003c/p\u003e \u003cp\u003eA total of 95 pharmaceuticals were determined based on their bioaccumulation capacity and their potency (effect/concentration ratio). The analysis was performed using a single run liquid chromatography tandem mass spectrometry (LC-MS/MS). A full list of target compounds, basic method performance parameters and internal standards used for quantification are compiled in Supplementary material (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Additional information on the instrumental method including polarities, HESI ionizations, collision energies, precursor/product ions and tube lens values can be found elsewhere. (Grabic et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lindberg et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll internal standards and surrogates added for identification and quantification of the target molecules were categorized as analytical grade (\u0026gt;\u0026thinsp;98%). For the in-house preparation of purified water, a Mili-Q Advantage system was used, with a UV radiation source (Millipore, Billerica, USA). It was used as a mobile phase in combination with LC-MS grade methanol. The acidification of the mobile chromatographic phases at 0.1% was performed with LC-MS grade formic acid (Sigma-Aldrich, Steinheim, Germany).\u003c/p\u003e \u003cp\u003eA triple stage quadrupole mass spectrometer (TSQ Quantiva, Thermo Scientific, San Jose, CA) equipped with a heated-electrospray ionization (HESI) ion source, coupled to an Accela LC pump (Thermo Fisher Scientific, San Jose, CA) and a PAL HTC autosampler (CTC Analytics AG, Zwingen, Switzerland) was used for sample analysis. Target analytes were separated by using a C18 phase Hypersil gold column (50 mm \u0026times; 2.1 mm ID \u0026times; 3 \u0026micro;m particles, Thermo Fisher Scientific, San Jose, CA, USA) with a guard column (2 mm, 2.1 mm, i.d. 5 \u0026micro;m particles). The mobile phase was methanol and water, acidified with formic acid (0.1%). The mobile phase flow started with 100% water and went gradually to 100% methanol in a 10-minute period. After that, it was kept constant for 3 more minutes for each sample run.\u003c/p\u003e \u003cp\u003eThe positive identification of target compounds was made by two MS/MS transitions. Limit of quantification (LOQ), precision and measurement of blank samples were used to define Quality assurance/Quality control of the analytical method. Recovery has been tested previously (Grabic et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The quantification of target analytes was performed by using the internal standard approach. Instrumental LOQs were derived from seven-point standard curve prepared in methanol, ranging from 0.1 to 100 ng/ml. The peak area corresponding to the lowest point of the standard curve with a signal/noise ratio of \u0026gt;\u0026thinsp;10 was then used for calculating the LOQs in individual samples. A relative standard deviation (RSD) of response factors measured for each point of the standard curve from the peak areas of the target analyte and its internal standard was used to express precision. Two processing blanks (Milli-Q purified water and tap water) were prepared in accordance with the extraction protocol used for tissue samples.\u003c/p\u003e\n\u003ch3\u003e• Reagents and methods of illicit drug analysis\u003c/h3\u003e\n\u003cp\u003eSeven illicit drugs were included in the analysis. Individual solutions of 1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), cocaine, benzoylecgonine, 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) and morphine, and 0.1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the deuterated analogues of each substance were used as surrogate internal standards (ISs), were supplied by Cerilliant (Round Rock, TX, USA). A stereoisomeric analysis was performed for amphetamine, metamphetamine and MDMA. Individual solutions of 1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the S-(+) enantiomer of each compound were supplied by Merck (Darmstadt, Germany). LC-MS grade methanol (MeOH), LC-MS grade acetic acid, LC-MS grade formic acid, ammonia (NH\u003csub\u003e3\u003c/sub\u003e) solution in water (25%) were supplied by Merck. Sodium acetate was obtained from Fluka (Steinheim, Germany). Ultrapure water was obtained with a Genie Water System from RephiLe Bioscience (Boston, MA, USA).\u003c/p\u003e \u003cp\u003eThe analysis was performed at the University of Santiago de Compostela, after solid-phase extraction with Oasis MCX cartridges (Waters, Milford, MA, USA) and LC-MS/MS analysis (Waters Acquity UPLC\u0026reg; H-class \u0026ndash; Xevo TQD system) as described in (Gonz\u0026aacute;lez-Mari\u0026ntilde;o et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This method affords LOQ values in the 0.1 (benzoylecgonine) to 16 (THC-COOH) ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range, and satisfactory recoveries in spiked surface water (98\u0026ndash;112%, RSD values\u0026thinsp;\u0026le;\u0026thinsp;12%). The enantiomeric analysis was conducted as described in Est\u0026eacute;vez-Danta et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This methodology renders LOQs in the 2.4 to 5.5 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range (referring to pure enantiomers) and recoveries in surface water in the 90\u0026ndash;107% range (RSD\u0026thinsp;\u0026le;\u0026thinsp;10%). (Table S2)\u003c/p\u003e \u003cp\u003eSamples were analyzed in triplicate and each batch was accompanied by one blank sample and two quality control samples spiked at 50 and 500 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, to verify the correct functioning of the analytical method, following the recommendations of (Hern\u0026aacute;ndez et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e• Hydrological data extraction\u003c/h3\u003e\n\u003cp\u003eMinimum and maximum river flow rate (m\u003csup\u003e3\u003c/sup\u003e/s) and daily accumulated rainfall (l/m\u003csup\u003e2\u003c/sup\u003e) was obtained from the Ebro River Hydrological Information System website (Table S3). (Spanish Government, 2025). Information about overflow volumes (m\u003csup\u003e3\u003c/sup\u003e) discharged from the Crispijana WWTP\u0026rsquo;s overflow channel (bypass) and the annual WWTP influent flow rate and untreated wastewater discharged annually to the Zadorra River was provided by AMVISA (a private company which provides public services related to integrated water cycle management). (Table S4) This information was used to calculate the percentage of annual untreated wastewater discharge flow using the following formulas:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\%\\:of\\:discharged\\:untreated\\:wastewater\\:=\\frac{{Q}_{WWT{P}^{{\\prime\\:}}s\\:overflow}}{{Q}_{influent}}\\:$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\%\\:of\\:discharged\\:wastewater\\:only\\:treated\\:with\\:1\u0026ordm;\\:treatment\\:=\\frac{{Q}_{biologic\\:overflow}}{{Q}_{influent}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003e• Risk assessment\u003c/h3\u003e\n\u003cp\u003eAn environmental risk assessment was performed for the APIs and illicit drugs detected above LOQ values in at least one sample. For the assessment, risk quotients (RQ) were calculated by dividing the measured environmental concentration (MEC) for each compound at each site by the concentration derived from toxicity data, also known as predicted no-effect concentration (PNEC). A precautionary approach was followed for the assessment; when available, chronic toxicity data was used as the PNEC. For the substances regulated by the Water Framework Directive, environmental quality standards (EQS) were used (European Commission, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For the other detected compounds, toxicity data was retrieved from US EPA ECOTOX database (US EPA \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the Enviro Tox database(Connors et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or the repository compiled by Giunchi et al. (Giunchi, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The species and the endpoint for each PNEC value was also collected. An assessment factor (AF) was to the retrieved toxicity data to mitigate the uncertainty of the data availability and its nature (ECHA, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In the case of caffeine, the species sensitivity distribution (SSD) values was used instead of the PNEC. SSD represents the lower limit of the 95% confidence interval LCL of the concentration which would be hazardous to 5% of the species (Rodr\u0026iacute;guez-Gil et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)- We considered that an RQ of \u0026lt;\u0026thinsp;0.1 indicates that no adverse effects are expected, an RQ between 0.1 and 1 would indicate a low risk, between 1 and 10 would indicate a moderate risk, and above 10 would indicate a high risk. A heatmap of RQ values was generated in R using the ggplot2 libraries. Half-life (DT50) (days), bioaccumulation factor (BCF; L kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and partition factor octanol/water (Log K\u003csub\u003eo/w\u003c/sub\u003e) data were also collected from CompTox (Williams et al., 2017). (Table S5)\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eAnalytical method quality parameters\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe analytical method performance has shown good reliability parameters. The average LOQs calculated from all water samples ranged between 0.01 and 76 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 83 out of 95 target pharmaceuticals expressed average LOQ below 0.5 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. No pharmaceuticals were detected in any of the blank samples. In the case of illicit drugs, LOQs ranged between 0.1 and 5.5 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. More details about the method performance are given as Supplementary material (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2).\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003ePresence of pollutants\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAt least six substances were detected at every sampling point both in autumn and spring. Briefly, caffeine, benzoylecgonine, morphine, THC-COOH, R-amphetamine, and cocaine appeared in all the analyzed waters at all seasons. A total of 33 out of 102 substances (27 pharmaceuticals and 6 illicit drugs) were detected in at least one of the samples. Methamphetamine was the only illicit drug that did not appear in any sample. Generally, more pharmaceuticals, and at higher concentrations, were detected in the wet weather season samplings. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) Post-WWTP samples taken in autumn presented the highest cumulative concentration (7739 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and the highest individual concentrations with paracetamol (2066 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), telmisartan (1442 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), caffeine (868 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and tramadol (726 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at the top spot. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) Five illicit drugs and caffeine were detected in Beto\u0026ntilde;o Pond without significant seasonal variations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e• Hydrological data\u003c/h3\u003e\n\u003cp\u003eThe mean flow of Zadorra River was 5.9 m\u003csup\u003e3\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (3.4\u0026ndash;10.3) on April 26th and 0.87 m\u003csup\u003e3\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.762\u0026ndash;1.03) on November 4th. Days before spring sampling were relatively dry, with daily-accumulated rainfall reaching 1.8 L m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. Rainy weather in the autumn sampling is illustrated by the 10.8 L m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e 3 days prior to the sampling. There was an overflow outfall on November 4, were 37.400 m\u003csup\u003e3\u003c/sup\u003e of untreated water was released into the Zadorra River. The annual overflows volume discharged from WWTP\u0026rsquo;s overflow channel was 4.728.400 m\u003csup\u003e3\u003c/sup\u003e in 2021 was and 842.100 m\u003csup\u003e3\u003c/sup\u003e in 2022. The percentage of untreated wastewater or only treated with primary treatment (removal of floatable objects), was 35.07% and 15.76% respectively.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eEcotoxicological risk assessment\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe RQ values of 7 APIs on both season exceeded 0.1 in the most contaminated site (SP3), reaching \u0026ldquo;medium risk\u0026rdquo; values (above 1) in the case of diclofenac (31), flecainide (19) and sertraline (1.6). (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) The RQ values for diclofenac and flecainide at SP2 in the measuring performed in November were 3.2 and 1.4 respectively, which indicates a medium environmental risk. The highest RQ values for illicit drugs were 0.04 (THC-COOH) and 0.01 (cocaine). Five substances (carbamazepine, diclofenac, fluoxetine, propranolol, sulfamethoxazole) regulated by the Water Framework and Urban Wastewater Treatment Directives presented \u0026ldquo;low-medium risk\u0026rdquo; levels. (Table S6)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe found that the accumulated concentrations of monitored pharmaceuticals and illicit drugs in surface waters upstream the WWTP were 19 times higher in the rainfall sampling period than in the dry sampling period. This was mainly due to the contribution of 10 APIs that only appeared during the wet weather week. It is also important to note that the combination of low river flow (i.e. reduced dilution) with sewer overflows may result in a difference in the concentration between seasons. Caffeine, an anthropogenic activity marker used in beverages and in pharmaceutical and cosmetic products, and paracetamol, an antipyretic drug, presented the highest concentrations. These two substances have been proposed as tracers of sewer overflow and untreated wastewater discharges (Buerge et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; White et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). During the sampling week in April (with minimal daily accumulated rainfall and consequently no overflow discharges) measured concentrations of caffeine and paracetamol were low and did not vary between sampling sites. The presence of caffeine and paracetamol here could be attributed to rural sources next to Zadorra River and its tributaries. In contrast, in the sampling week in November, samples taken upstream the WWTP in November, from a section of the river receiving untreated wastewater from four overflow channels, showed high concentrations of caffeine. Hence, it is likely that the pharmaceutical burden in the river stage before reaching WWTP can be explained by untreated wastewater overflow discharges. Nevertheless, these assumptions should be treated with care, as other factors, such as seasonal fluctuations in consumption and the river flow rate may vary.\u003c/p\u003e \u003cp\u003eApart from paracetamol and caffeine, angiotensin receptor blockers (ARBs) (telmisartan, irbersartan and eprosartan) and beta-blockers (atenolol, bisoprolol and propranolol), are also present in relatively high concentrations in the Zadorra River. These pharmaceuticals are used worldwide for controlling high blood pressure, the main suspect of cardiovascular disease and premature death (Mills et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Beta-blockers, ARBs and their active transformation products, which are frequently detected in different aquatic matrices, may cause diverse adverse effects on the reproduction, growth and cardiac development in wildlife (Yi et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Muambo et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)). Other substance detected at high concentrations is tramadol, which may alter the behaviour as demonstrated with the experimental study performed with European chubs (\u003cem\u003eSqualius cephalus\u003c/em\u003e). Fish exposed to tramadol showed an anxiolytic-like effect (Santos et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These alteration may lead to the impairment od key fitness related behaviours of wild animals (Brand et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) In 2020, a micropollutant characterization of the Crispijana WWTP\u0026rsquo;s effluent and influent was performed, and WWTP\u0026rsquo;s removal efficiency for each API was also estimated (Lopez-Herguedas et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The authors showed that the removal efficiency for 62 out of 72 detected APIs (48 presented higher values in the effluent than in the influent) was below 80%. Contrary, Crispijana WWTP showed a removal rate of almost 100% for caffeine and paracetamol. In the current monitorization, paracetamol was absent during April, but reached 883 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in November, likely due to the discharge of untreated water from the 2 overflow channels located in the Crispijana WWTP. The updated Spanish Regulation of Hydraulic Public Domain dictates that flow rates combined sewer overflow must be monitored. (Spanish Government, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) The Urban Wastewater Treatment Directive takes a step further: \u0026ldquo;the concentration of the regulated micropollutants and microplastics shall be monitored at relevant points of storm water overflows\u0026rdquo; (European Commission, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The Directive also proposes that the competent authorities (e.g. water management companies) should consider preventive measures to avoid the entry of rain waters into sewers. Some strategies are currently in development to prevent combined sewer overflows using front-of-the-pipe (e.g. soil filters, retention facilities) and end-of-pipe (e.g. chemical coagulation) (Botturi et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The expansion of green and blue zones in urban areas is also a feasible alternative, although in the case of Vitoria-Gasteiz, a city that has been awarded with the Green Capital title in 2014, is not enough. In the sampling years, the percentage of untreated wastewater reached 35% and 16%, in 2021 and 2022, respectively, which is too far from the non-binding objective proposed by the Directive of 2% of annual discharge. A derivation water channel to decrease the sewer volume that ends in the Crispijana WWTP is currently under construction (URA, 2022). This will likely lead to a decrease of the overflow percentage and the amount of untreated wastewater reaching the river.\u003c/p\u003e \u003cp\u003eIn addition to the transportation of pollutants, overflow water can compromise the secondary wastewater treatment of WWTP, an essential step for the removal of nutrients, pathogens, APIs and other micropollutants. (Dirckx et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) Consequently, the performance of the WWTP would be impaired until the recovery of the microbial communities affected by the excess stormwater, a process that can take several days. Furthermore, pollutants retained in the sediments can be mobilized during storm events, which can account for up to 65% of the total emissions resulting from wet weather overflows (Del R\u0026iacute;o et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). There is a strong relationship between log K\u003csub\u003eow\u003c/sub\u003e and log K \u003csub\u003eoc\u003c/sub\u003e (organic carbon to water partition coefficient) values, with the latter representing the tendency of sediment adsorption (EPA, 1996). Del R\u0026iacute;o et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) showed how, following a rainfall event, the sediment fraction of ibuprofen, diclofenac and caffeine are dissolved and mobilised from the sediments. For instance, ARBs are weak acids with high log K\u003csub\u003eow\u003c/sub\u003e values and thus have an affinity for the sediment of the combined sewers and are likely to be dissolved and mobilized in a rainfall event. In the present study, ARBs appear in higher concentrations in the rainy sampling period at SP2, with eprosartan reaching low-risk concentrations (RQ\u0026thinsp;=\u0026thinsp;0.4). The case of diclofenac is similar to ARBs; its physicochemical characteristics could be related to its presence at low-risk concentrations (RQ\u0026thinsp;=\u0026thinsp;0.32) in the samplings performed in November. Although sampling data in this study is limited, further research on the sewer sediment mobilisation, the relationship with the physicochemical properties of the APIs and the contribution to wet weather overflow discharges are recommended.\u003c/p\u003e \u003cp\u003eBeto\u0026ntilde;o Pond consists in stationary waters only connected to groundwater, but it is also exposed to anthropogenic pollution. The presence of APIs and illicit drugs in this wetland could be a consequence of sporadic urination of passerby or due to the exchange between the wetlands and groundwater. (Jurado et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, caffeine and some illicit drugs were detected in both seasons at similar concentration, which were higher or close to the concentrations found in the Zadorra River. For example, THC-COOH is a metabolite of THC, an active substance of cannabis which is an illegal drug in Spain. This metabolite has a low percentage of removal in WWTPs, and in some cases the concentration in the effluents augments due to the deconjugation of the glucuronide form. (How et al., 2021) Another example is morphine, it is a prescription drug used for chronic pain with a potential of being abused, and also a metabolite of heroin, and thus, the detection of morphine could be related to the illegal consumption of this drug. As for amphetamine is an illicit drug, but also a metabolite of lisdexamphetamine prescribed used for the treatment of attention deficit hyperactivity disorder. Illicit amphetamine is distinctive by its racemic composition, it is normally composed of even parts of R-amphetamine ang S-amphetamine, while the metabolite of lisdexamphetamine is the S enantiomer. MDMA is also composed of a racemic mixture, whereas methamphetine reaching the illicit Spanish market is normally composed of the most active S enantiomer only (Est\u0026eacute;vez-Danta et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The presence of R-amphetamine in the sampled water is indicative of illegal consumption. In a recent study (Est\u0026eacute;vez-Danta et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the enantiomeric profiling of wastewater in the Basque Country demonstrated high levels of amphetamine, and only less than 1% of the load was explained by the mentioned prescription drug API lisdexamphetamine. This same study demonstrated that the R-enantiomer of these 3 drugs is enriched during water treatment, while the S-enantiomer is more efficiently removed (Est\u0026eacute;vez-Danta et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs far as we know, so far only 4 studies have assessed the presence of illicit drugs in Natural Parks and other natural areas; and the current study represents the first time that an enantiomeric analysis has been performed on samples from natural areas. One study performed in Kruger National Park in South Africa did not find any illicit drugs, while in a monitoring campaign conducted in Antonia Ramos Protected Area in Argentina found benzoylecgonine in the muscle of thararira (\u003cem\u003eHoplias Lacerdae\u003c/em\u003e), and in the liver and gills of streaked prochilod (\u003cem\u003eProchilodus lineatus\u003c/em\u003e), up to 0.87 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. (Ondarza et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sims et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) In Spain, Pego-Oliva Marsh and L\u0026rsquo;Albufera Natural Park, both in the province in Valencia, have been monitored for the presence of pharmaceuticals, heavy metals, illicit drugs and other anthropogenic pollutants. (Pascual-Aguilar et al., 2015; Vazquez-Roig., 2012) Both are coastal lagoons that are included in the RAMSAR convention like Salburua Wetlands. Cocainics, amphetamine-like compounds, opiates, cannabinoids and ketamine were analyzed there. Mean concentrations of amphetamine, benzoylecgonine, cocaine and THC-COOH measured in Salburua Wetlands were higher than in two other studies. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMonitoring studies of illicit drugs in National Parks\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSampling site\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStudy data\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c10\" namest=\"c3\"\u003e \u003cp\u003eConcentrations of selected substances\u003c/p\u003e \u003cp\u003eMean (Maximum) ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMain observations\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAMP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBECG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCOC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKET\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMDMA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMET\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMOR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eTHC-COOH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePego-Oliva Marsh\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026bull; \u003cb\u003eStudy\u003c/b\u003e: Vazquez-Roig et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2012\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u0026bull; \u003cb\u003eSampling period\u003c/b\u003e: June 2009\u003c/p\u003e \u003cp\u003e\u0026bull; \u003cb\u003eSampling method\u003c/b\u003e: grab sampling at 23 points\u003c/p\u003e \u003cp\u003e\u0026bull; \u003cb\u003eAnalyzed substances\u003c/b\u003e: 14 illicit drugs and metabolites\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.92 (15.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.25 (\u003cb\u003e11.8\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e21.33 (414.92\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.062 (\u003cb\u003e3.4\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.32 (2.7\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3.59 (8.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.07 (1.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026bull; Near settlements of 600 and 1400 housings. The wetland receives water from a wastewater treatment plant (WWTP)\u003c/p\u003e \u003cp\u003e\u0026bull; No rainfall during the sampling period\u003c/p\u003e \u003cp\u003e\u0026bull; 8 illicit drugs and metabolites were detected\u003c/p\u003e \u003cp\u003e\u0026bull; All samples were contaminated\u003c/p\u003e \u003cp\u003e\u0026bull; The most occurring drug was ketamine and MDMA\u003c/p\u003e \u003cp\u003e\u0026bull; THC was not detected and its metabolite THC-COOH was found in only one sample\u003c/p\u003e \u003cp\u003e\u0026bull; Sampling sites near to the coast presented higher concentrations\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eL\u0026rsquo;Albufera Natural Park\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026bull; \u003cb\u003eStudy\u003c/b\u003e: Pascual-Aguilar et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u0026bull; \u003cb\u003eSampling period\u003c/b\u003e: March 2008\u003c/p\u003e \u003cp\u003e\u0026bull; \u003cb\u003eSampling method\u003c/b\u003e: grab sampling at 21 points\u003c/p\u003e \u003cp\u003e\u0026bull; \u003cb\u003eAnalyzed substances\u003c/b\u003e: 14 illicit drugs and metabolites, and 17 pharmaceuticals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.38 (3.38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.57 (\u003cb\u003e78.71\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.48 (\u003cb\u003e4.43\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.54\u003c/b\u003e (2.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e (11.70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026bull; The park is surrounded by an urban area of more than 1,200,000 inhabitants. 2 WWTP\u0026rsquo;s effluents enter the park\u003c/p\u003e \u003cp\u003e\u0026bull; No rainfall during the sampling period\u003c/p\u003e \u003cp\u003e\u0026bull; 9 illicit drugs and metabolites were detected\u003c/p\u003e \u003cp\u003e\u0026bull; 16 samples were contaminated\u003c/p\u003e \u003cp\u003e\u0026bull; Highest accumulated concentration was 78 ng L\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u0026bull; Cocainics were most frequently detected\u003c/p\u003e \u003cp\u003e\u0026bull; Higher concentrations were detected in sites near high population densities.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSalburua Wetlands\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026bull; \u003cb\u003eStudy\u003c/b\u003e: \u003cem\u003eCurrent study\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026bull; \u003cb\u003eSampling period\u003c/b\u003e: November 2021 and April 2022\u003c/p\u003e \u003cp\u003e\u0026bull; \u003cb\u003eSampling method\u003c/b\u003e: grab sampling at 1 pont\u003c/p\u003e \u003cp\u003e\u0026bull; \u003cb\u003eAnalyzed substances\u003c/b\u003e: 7 illicit drugs and metabolites, and 95 pharmaceuticals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e9.45 (11)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e33.9\u003c/b\u003e (35.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.3 (2.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.6 (\u003cb\u003e14.3\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e17.95 (19)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026bull; The park is near a urban zone with more than 250,000 inhabitants.\u003c/p\u003e \u003cp\u003e\u0026bull; Is not connected directly with any surface water.\u003c/p\u003e \u003cp\u003e\u0026bull; Samples were taken during dry weather period and rainy weather period.\u003c/p\u003e \u003cp\u003e\u0026bull; Metamphetamine and MDMA were not detected\u003c/p\u003e \u003cp\u003e\u0026bull; An enantiomeric analysis was performed. Only R enantiomer was detected of amphetamine\u003c/p\u003e \u003cp\u003e\u0026bull; Highest accumulated concentration was 57 ng L\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eHPLC-MS/MS: High performance liquid chromatography and mass spectroscopy tandem; AMP: amphetamine; BECG: benzoylecgonine; COC: cocaine; KET: ketamine; MDMA: 3,4-methylenedioxymethamphetamine; MOR: morphine; THC-COOH: nor-9-carboxy-Δ9- tetrahydrocannabinol. ND: not detected; NA: not analyzed.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eEcotoxicological data regarding illicit drugs are relatively scarce, which was the explanation why a list of illicit drugs was not included in the 4th Watch List. (Gomez Cortes et al., 2022) Amphetamine is known to decrease the bacterial and diatom communities in water stream biofilms, produce reactive oxygen species (ROS) and genetic damage in zebra mussels (\u003cem\u003eDreissena polymorpha\u003c/em\u003e). (Lee et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Parolini et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) Benzoylecgonine decreases the parthenogenic cycle and number of offsprings, and reduces of locomotor activity in \u003cem\u003eDaphnia magna\u003c/em\u003e. (Parolini et al., 2017; Parolini et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) Cocaine causes swelling and breakdown of skeletal muscle of European eel (\u003cem\u003eAnguilla anguilla\u003c/em\u003e)) and behavioural changes in \u003cem\u003eDanio rerio\u003c/em\u003e. (Darland and Dowling, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Capaldo et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) There is differences in the toxicity levels between different enantiomers of several APIs and illicit drugs. (P\u0026eacute;rez-Pereira et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) This enantioselectivity is characteristic for amphetamine, MDMA and ketamine. Freshwater algae \u003cem\u003eChlorella pyrenoidosa\u003c/em\u003e showed preferential bioaccumulation and degradation for S-amphetamine, which could explain that only R-amphetamine was detected in the current study. Toxic effects of amphetamine on \u003cem\u003eDaphnia magna\u003c/em\u003e seem to be also enantioselective: S enantiomer is related to the decrease of the number of eggs and body size, while R enantiomer affects the heart rate and size. The decrease of body size in \u003cem\u003eDaphnia magna\u003c/em\u003e is also attributed to S-MDMA.\u003c/p\u003e \u003cp\u003eNo illicit drug had a RQ value higher than 0,1. However, the available ecotoxicological studies only have assessed acute toxicity; the information about how chronic exposure to illicit drugs may affect wild species is scarce. (Maasz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) In Beto\u0026ntilde;o Pond, concentrations from Autumn and Spring samplings were similar, meaning that there is a chronic exposure to these micropollutants. RAMSAR sites are of high importance for water birds, especially for the globally threatened birds that reside mostly in wetlands. As these birds consume aquatic species, there is a risk for biomagnification. (Herrero-Villar et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) A study conducted in Mai Po RAMSAR site in Hong Kong, China have concluded that birds which seeks food from the sediments (e.g. common sandpiper) and have a small body size are more prone to be exposed to micropollutants. (Man et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe Zadorra River is exposed to at least 33 APIs and illicit drugs, and their concentrations reaching medium risk levels for the environment. The situation is exacerbated during periods of wet weather, resulting in a 19-fold increase in the accumulated concentrations, attributed mainly to paracetamol, caffeine and telmisartan, in the sections of the river that do not receive WWTP\u0026rsquo;s effluent. As stipulated by the updated Urban Wastewater Directive, combined sewer overflow and the micropollutants carried by this untreated water shall be monitored. As storm events frequency attributed to climate continue to increase, it is imperative to prioritise the identification of solutions for reducing the volume of wet weather overflow. Beto\u0026ntilde;o Pond located in the protected area of the Salburua Wetlands is also a subjected to organic pollution, including illicit drugs. In contrast to the river, the concentrations across different seasons is stable. The ongoing exposure to these substances could pose a significant threat to local aquatic species and could also potentially impact on the avian communities, foraging in the wetlands, due to biomagnification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJBQ and XGG would like to acknowledge the financial support from the Spanish Agencia Estatal de Investigaci\u0026oacute;n \u0026ndash; MCIN/AEI/10.13039/501100011033 (RED2022-134363-T, PID2020-117686RB-C32) and\u0026nbsp;Instituto de Salud Carlos III-EDRF (RD24/0003/0020 \u0026ndash; RIAPAd Network)\u003c/p\u003e\n\u003cp\u003eGO acknowledges the Spanish Ministry of Economy, Industry, and Competitiveness (PID2022-139746OB-I00/AEI/10.13039/501100011033) and the University of the Basque Country. VA acknowledges the Basque Government for his Ph.D. scholarship (PRE_2024_1_0142).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVladimir Akhrimenko: Investigation, Formal analysis, Writing \u0026ndash; original draft.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDaniel Cerveny\u003c/strong\u003e: Formal analysis, Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eXiana Gonzalez-Gomez\u003c/strong\u003e: Formal analysis, Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eMat\u0026iacute;as Nicol\u0026aacute;s Cuenca-Castillo\u003c/strong\u003e: Formal analysis, Investigation, Writing \u0026ndash; original draft. \u0026nbsp;\u003cstrong\u003eIker Ega\u0026ntilde;a\u003c/strong\u003e: \u003cstrong\u003eInvestigation, Writing \u0026ndash; original draft.\u003c/strong\u003e \u003cstrong\u003eJose Benito Quintana\u003c/strong\u003e: Formal analysis, Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eTomas Brodin\u003c/strong\u003e:Formal analysis, Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eUnax Lertxundi: \u003c/strong\u003eConceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eGorka Orive\u003c/strong\u003e: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the results of the analyzed data are available at the online repository ZENODO: 10.5281/zenodo.15342909\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eaus der Beek, T., Weber, F. 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Tracking changes in the occurrence and source of pharmaceuticals within the River Thames, UK; from source to sea. \u003cem\u003eEnvironmental pollution (Barking, Essex : 1987)\u003c/em\u003e, \u003cem\u003e249\u003c/em\u003e, 257\u0026ndash;266. https://doi.org/10.1016/j.envpol.2019.03.015\u003c/li\u003e\n\u003cli\u003eYi, M., Sheng, Q., Sui, Q., \u0026amp; Lu, H. (2020). \u0026beta;-blockers in the environment: Distribution, transformation, and ecotoxicity. \u003cem\u003eEnvironmental pollution (Barking, Essex : 1987)\u003c/em\u003e, \u003cem\u003e266\u003c/em\u003e(Pt 2), 115269. https://doi.org/10.1016/j.envpol.2020.115269\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pharmaceutical pollution, sewer overflow, weather factors, wastewater treatment plant, national parks, drugs of abuse","lastPublishedDoi":"10.21203/rs.3.rs-6980045/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6980045/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe use of combined sewer systems is associated with frequent overflow events and discharges of untreated wastewater containing micropollutants, including APIs. The aim of this study was to evaluate the impact of weather-related factors on the presence of pharmaceuticals and illicit substances and assess the related environmental risk. To address this goal, three sites in the surroundings of the city of Vitoria-Gasteiz were sampled in April (dry week) and November (wet week): Beto\u0026ntilde;o Pond in the Salburua Wetlands, and Zadorra River before and after receiving the Crispijana wastewater treatment plant\u0026rsquo;s (WWTP) effluent. An environmental risk assessment was performed by measuring the hazard quotient. This is the first time when an enantioselective risk assessment has been performed for environmentally relevant concentrations of illicit drugs measured in a wetland of an international importance. A total of 33 out of 102 substances were detected in at least one of the samples and a minimum of 6 APIs in all sample sites. Post-WWTP samples taken in autumn (wet week) presented the highest cumulative concentration (7739 ng L-1). Risk quotients of 12 APIs in the most contaminated site (SP3) in the rainy weather sampling period reached values\u0026thinsp;\u0026gt;\u0026thinsp;1: diclofenac (3.12) and eprosartan (1.59).\u003c/p\u003e","manuscriptTitle":"Combined sewer overflow contribution to pharmaceutical pollution: a case study in northern Spain","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-02 08:21:23","doi":"10.21203/rs.3.rs-6980045/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7a72a62a-d82a-44cb-98d9-d3b0523eaad5","owner":[],"postedDate":"July 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-21T17:58:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-02 08:21:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6980045","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6980045","identity":"rs-6980045","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}