Water Research 195 (2021) 116983 Contents lists available at ScienceDirect Water Research journal homepage: www.elsevier.com/locate/watres New psychoactive substances in several European populations assessed by wastewater-based epidemiology Sara Castiglioni a , ∗, Noelia Salgueiro-González a , Lubertus Bijlsma b , Alberto Celma b , Emma Gracia-Lor a , c , Mihail Simion Beldean-Galea d , Tomáš Macku ľ ak e , Erik Emke f , Ester Heath g , Barbara Kasprzyk-Hordern h , Andjelka Petkovic i , Francesco Poretti j , Jeliaz Rangelov k , Miguel M. Santos l , Maja Srema ̌cki m , Katarzyna Styszko n , Felix Hernández b , Ettore Zuccato a a Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Department of Environmental Sciences, Via Mario Negri 2, 20156, Milan, Italy b Environmental and Public Health Analytical Chemistry, Research Institute for Pesticides and Water, University Jaume I, 12071 Castellón, Spain c Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040, Madrid, Spain d Babes-Bolyai University, Faculty of Environmental Science and Engineering, Cluj-Napoca, Romania e Institute of Chemical and Environmental Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 2101/9, 812 37 Bratislava, Slovakia f KWR Water Research Institute, P.O. Box 1072, 3430 BB, Nieuwegein, The Netherlands g Department of Environmental Sciences, Jožef Stefan Institute, Jamova cesta 39, Ljubljana, Slovenia h Department of Chemistry, University of Bath, Bath BA2 7AY, United Kingdom i Jaroslav Cerni Water Institute, 11226 Belgrade, Serbia j Consorzio Depurazione Acque Lugano e Dintorni, Via Molinazzo 1, 6934 Bioggio, Switzerland k Sofiyska Voda AD part of Veolia, Sofia, Bulgaria l CIMAR/CIIMAR - LA, Interdisciplinary Centre of Marine and Environmental Research, Group of Endocrine Disruptors and Emerging Contaminants, FCUP, Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal m University of Novi Sad, Faculty of Technical Sciences, Department of Environmental Engineering and Occupational Safety and Health, Novi Sad, Serbia n AGH University of Science and Technology, Department of Coal Chemistry and Environmental Sciences, Al. Mickiewicza 30, Krakow, Poland a r t i c l e i n f o Article history: Received 29 October 2020 Revised 24 February 2021 Accepted 25 February 2021 Available online 27 February 2021 Keywords: Urban wastewater new psychoactive substances monitoring Europe spatial and temporal trends use profiles a b s t r a c t Wastewater-based epidemiology (WBE) can be a useful tool to face some of the existing challenges in monitoring the use of new psychoactive substances (NPS), as it can provide objective and updated infor- mation. This Europe-wide study aimed to verify the suitability of WBE for investigating the use of NPS. Selected NPS were monitored in urban wastewater by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The main classical illicit drugs were monitored in the same samples to compare their levels with those of NPS. Raw composite wastewater samples were collected in 2016 and 2017 in 14 European countries (22 cities) following best practice sampling protocols. Methcathinone was most frequent ( > 65% of the cities), followed by mephedrone ( > 25% of the cities), and only mephedrone, methcathinone and methylone were found in both years. This study depicts the use of NPS in Europe, confirming that it is much lower than the use of classical drugs. WBE proved able to assess the qualita- tive and quantitative spatial and temporal profiles of NPS use. The results show the changeable nature of the NPS market and the importance of large WBE monitoring campaigns for selected priority NPS. WBE is valuable for complementing epidemiological studies to follow rapidly changing profiles of use of drugs. © 2021 Elsevier Ltd. All rights reserved. 1 r l a f u a h 0 . Introduction Urban wastewater reflects the lifestyle of a population as it rep- esents anonymous urine samples from thousands of people. When ∗ Corresponding Author. Dr. Sara Castiglioni, PhD, Istituto di Ricerche Farmaco- ogiche Mario Negri IRCCS, Via Mario Negri 2, 20156 Milano E-mail address: sara.castiglioni@marionegri.it (S. Castiglioni). a l W t ttps://doi.org/10.1016/j.watres.2021.116983 043-1354/© 2021 Elsevier Ltd. All rights reserved. n individual ingests a substance, it can be excreted in urine or aeces as the parent substance or a metabolite, and enters the rban sewage system. Wastewater-based epidemiology (WBE) is promising approach for estimating the use of a substance in population by chemical analysis of urban wastewater for se- ected urinary metabolites (biomarkers) ( Gracia-Lor et al., 2017 ). BE was developed to estimate the use of illicit drugs more han a decade ago ( Zuccato et al., 2005 , 2008 ; van Nuijs et al., https://doi.org/10.1016/j.watres.2021.116983 http://www.ScienceDirect.com http://www.elsevier.com/locate/watres http://crossmark.crossref.org/dialog/?doi=10.1016/j.watres.2021.116983&domain=pdf mailto:sara.castiglioni@marionegri.it https://doi.org/10.1016/j.watres.2021.116983 S. Castiglioni, N. Salgueiro-González, L. Bijlsma et al. Water Research 195 (2021) 116983 2 o M u W t b n i l d ( o m a B D ( U t o 2 5 d b c s m o i a t m m u p e E v c t c s r S c v r a b i d G h G d i ( a M l m t b i u t b l a p y t 2 2 c t d o 1 n l c D s N d t c s a m p l 2 s a s t p s o U l M n ( g ( 2 3 2 i J i s ( i 011 ), and is now regularly applied worldwide to assess the use f cocaine, amphetamine-like stimulants and cannabis ( González- ariño et al., 2020 ). With its advantage of providing objective and pdated information on a population in a short period of time, BE is considered a new indicator of drug use, complementary to he established drug monitoring tools ( EMCDDA, 2016 ) . WBE can e useful to tackle some of the challenges in monitoring the use of ew psychoactive substances (NPS). NPS are an extremely heterogeneous group of substances orig- nally designed as legal alternatives to the more established il- icit drugs, and they have challenged traditional approaches to rug monitoring, surveillance, control and public health response Peacock et al., 2019 ). These challenges include the large number f substances and the speed with which they enter and exit the arket, the lack of awareness of the exact content of NPS products, nd their often unknown potency and effects ( Peacock et al., 2019 ). y the end of 2019, the European Monitoring Centre for Drugs and rug Addiction (EMCDDA) was recording about 790 NPS in Europe EMCDDA, 2020 ), and the Early Warning Advisory (EWA) of the nited Nations Office on Drugs and Crime (UNODC) reported more han 950 NPS worldwide ( UNODC, 2020 ). In Europe, the number f new substances identified for the first time reached a peak in 014-2015 with 100 newly-reported NPS per year, and dropped to 3 in 2019 ( EMCDDA, 2020 ). The NPS market is highly dynamic epending on the substances availability over time that may vary ecause of the changing laws, i.e. when a substance from licit be- ome protected, illegal producers create rapidly an alternative sub- tance with the same or similar effect. NPS are now increasingly anufactured and marketed as counterfeit prescription medicines r mixed with established illicit substances, but their composition s usually mostly unknown ( EMCDDA, 2015 ). Surveys of self-reported use and seizure data are not suitable lone to assess the prevalence of NPS use, and the current best op- ion is to adopt monitoring systems that triangulate different infor- ation sources, including forensic and toxicology analyses of hu- an biological or drug samples ( Peacock et al., 2019 ). WBE can be sed as a new additional tool to monitor NPS use by the general opulation or in specific sub-populations (e.g. at festivals) as for stablished illicit drugs ( Benaglia et al., 2020 ; Bijlsma et al., 2020 ; MCDDA, 2016 ; González-Mariño et al., 2020 ). The main limitation when assessing NPS use by WBE is the ery low concentration levels expected in urban wastewater be- ause of the lower prevalence of use. This in combination with he often unknown excretion pattern that hinders focusing on spe- ific metabolic products, and the large number of different sub- tances that should ideally be investigated because they were ecorded in the market ( Bade et al., 2019a ; Bijlsma et al., 2019 ; algueiro-González et al., 2019 ) makes monitoring NPS use very hallenging. These limitations can be partially overcome using ad- anced analytical techniques based on mass spectrometry, which eaches very good sensitivity for detecting a substance in wastew- ter even at trace levels, and screening the presence of large num- er of substances ( Hernandez et al., 2016 ). Some recent studies nvestigated urban wastewater for NPS in different countries, ad- ressing a selected set of substances ( Bade et al., 2020 , 2017 ; onzalez-Marino et al., 2016 ; Reid et al., 2014 ) or screening undreds ( Bade et al., 2019b ; Diamanti et al., 2019 ; Salgueiro- onzález et al., 2019 ). Although relatively few substances were etected in wastewater at low levels (few ng/L), literature did dentify temporal trends of synthetic cathinones use in Australia Bade et al., 2019a ; Chen et al., 2013 ; Tscharke et al., 2016 ), nd spatial trends of use in three countries in Europe ( Gonzalez- arino et al., 2016 ). Nevertheless, most of previous studies were imited to a single country and very few cities. As far as we know, the present work is the most extensive onitoring study of NPS use in the general population ever done 2 hrough WBE, and it was part of the NPS Euronet project funded y the European Commission. The aim was to test the suitabil- ty of WBE for investigating the use of NPS in the general pop- lation in an extensive monitoring campaign. This study covered wo years in 14 European countries and 22 cities. A selected num- er of NPS including mostly synthetic cathinones and phenethy- amines were quantified in urban wastewater to assess their spatial nd temporal profiles of use. The main classical illicit drugs (am- hetamine, methamphetamine, MDMA, and cocaine) were anal- sed in the same samples to compare their levels with those of he NPS. . Material And Methods .1. Compounds selection Thirty NPS were selected according to their frequency of itation in alert reports from the Early Warning Systems of he EMCDDA and UNODC, the availability of analytical stan- ards, their reported presence in urban wastewater in previ- us studies, and stability in wastewater. The selected NPS were 9 synthetic cathinones, 7 phenethylamines, 1 synthetic cannabi- oid, 1 tryptamine, 1 aminorex derivative, and 1 arylcyclohexy- amine/ketamine analog ( Table 1 ). In total, thirteen deuterated ompound analogues of eleven NPS plus amphetamine-d6 (AMPH- 6) and methamphetamine-d9 (METH-D9) were used as internal tandards ( Table 1 ). All the substances are parent drugs as for most PS the metabolism is unknown, and very few analytical stan- ards of metabolites are available. Furthermore, the majority of he NPS considered are synthetic cathinones that are mainly ex- reted as parent compounds ( Uralets et al., 2014 ). The main clas- ical drugs were included in the analysis to compare their use nd were: amphetamine (AMPH), methamphetamine (METH), 3,4- ethylenedioxy-methamphetamine (MDMA), ketamine (KET) as arent substances, and benzoylecgonine (BE) as the main metabo- ite of cocaine. .2. Chemicals and Materials Analytical reference standards of NPS and classical drugs were upplied by LGC (Teddington, UK), Cerilliant (Round Rock, TX, USA) nd Cayman Chemicals (Ann Arbor, MI, USA) as 0.1, 0.4 or 1 mg/mL olutions in acetonitrile (ACN) or methanol (MeOH). Working solu- ions containing either the 30 analytes or the 13 deuterated com- ounds were prepared in MeOH before each analytical run and tored in the dark at -20 °C up to two months. Reference standards f classical drugs were purchased from Cerilliant (Round Rock, TX, SA) as 0.1 or 1 mg/mL solutions in ACN or MeOH. Working so- utions were prepared as described for NPS. HPLC grade ACN and eOH, formic acid (98%), hydrochloric acid (HCl, 37%) and ammo- ium hydroxide solution (NH 4 OH, 25%) were purchased from Fluka Buchs, Switzerland) and Carlo Erba (Italy). Milli-Q water (HPLC rade) was obtained directly from a MILLI-RO PLUS 90 apparatus Millipore, Molsheim, France). .3. Wastewater sampling Composite 24-hour raw wastewater samples were collected for days over the weekend (in 2016) and for-7 consecutive days (in 017) from wastewater treatment plants (WWTP) in different cities n Europe. The sampling period was March-May in 2016, and May- une (plus one city in October) in 2017 (Table S1). The sampling n 2017 was randomly stratified over four weeks taking one/two amples per week to ensure better representability of samples Ort et al., 2014 ). Each sample was collected as a daily compos- te sample and 0.5-1 L aliquots were received by our laboratory. S. Castiglioni, N. Salgueiro-González, L. Bijlsma et al. Water Research 195 (2021) 116983 Table 1 NPS selected for investigation, with their classes and abbreviations. Labelled deuterated analogs listed in the right column were used as internal standards (IS). NPS classes Deuterated compounds (IS) Synthetic cathinones Phenethylamines buphedrone (BUPH) 25-B-NBOMe amphetamine-D6 (AMPH-D6) butylone (BUTL) 25-C-NBOMe butylone-D3 4 ′ -chloro- α-pyrrolidinopropiophenone (4-Cl- α -PPP)] 25-I-NBOMe 25-B-NBOMe-D3 N,N-dimethylcathinone (DCAT) 25-iP- NBOMe 25-C-NBOMe-D3 3,4 dimethylmethcathinone (3,4-DMMC) N-ethyl-1,2-diphenylethylamine (NEDPA) 25-I-NBOMe-D3 ethcathinone (ETHC) para-methoxyamphetamine (PMA) mephedrone-D3 (MEPH-D3) ethylone (ETHL) para-methoxy-N-methylamphetamine (PMMA) methamphetamine-D9 (METH-D9) 4-fluoromethcathinone (4-FMC) Synthetic cannabinoid 3,4-methylenedioxypyrovalerone-D8 (MDPV-D8) mephedrone (MEPH) 5-fluoropentyl-3-pyridinoylindole (5-Fpentyl-3-pyr) methylone -D3) methcathinone (METC) Tryptamine methoxetamine-D3 (MXE-D3) methedrone (METD) 5-methoxy-N-isopropyl-N-methyltryptamine (5-MeO-MiPT) naphyrone-D5 methylenedioxypyrovalerone (MDPV) Aminorex derivative ρ-methoxymethamphetamine-D3 (PMMA-D3) 4-methylethcathinone (4-MEC) 4,4-dimethylaminorex (4,4-DMAR) alpha-pyrrolidinovalerophenone-D8 methylone (METL) Ketamine analog naphyrone (NAPH) methoxetamine (MXE) 1-naphyrone (1-NAPH) pentedrone, (PENTD) pentylone (PENTL) α-pyrrolidinovalerophenone ( α –PVP) P m e d f a N ( 2 p t c t p i M t u p h p ( t ( 2 p s h e t s n d e f t i t m r m d A p t f i u d 2 l ( a i m a b f i w s L ( 2 a t ( w o t l u d 2 ooled weekend and weekday samples were prepared for analysis ixing fixed aliquots from each sample (50-100 mL). Pooled week- nd samples were created by mixing aliquots from Saturday, Sun- ay and Monday, and pooled weekday samples by mixing aliquots rom Tuesday to Friday. This design was adopted to optimize the nalytical effort to achieve our objectives, i.e. evaluate the use of PS and investigate their weekly pattern of use (week-weekend). The investigation included 20 cities from 14 European countries 21 WWTPs with approximately 6.9 million people connected) in 016, and 7 cities in 5 European countries (7 WWTPs with ap- roximately 3.4 million people connected) in 2017. Unfortunately, he sampling capacity was not the same in 2016 and 2017, but five ities were included in both campaigns in order to follow the time rends (weekend pooled samples from 2016 and 2017 were com- ared). Information on population size and WWTP characteristics ncluding the daily flow rates are reported in the Supplementary aterial (SM) (Table S1). The sampling scheme was designed according to best prac- ice protocols ( Castiglioni et al., 2013 ), in order to limit sampling ncertainty to 5-10%. Samples were collected volume- or time- roportionally, ensuring the collection of at least one aliquot per our. For substances with a low prevalence of use as NPS, sam- ling errors may be higher than for substances used more widely cocaine, alcohol, nicotine), but this can be partially balanced by he large populations investigated, which lowers the uncertainty Ort et al., 2010 ). .4. Chemical analysis The analytical method used to measure NPS was adapted from a revious study ( Gonzalez-Marino et al., 2016 ), including additional ubstances. Samples were solid-phase extracted and analysed by igh-performance liquid chromatography tandem mass spectrom- try (HPLC-MS/MS). The analytical procedure and method valida- ion are described in detail in the SM. Briefly, 50 mL of wastewater amples were filtered and spiked with deuterated labelled inter- al standards to compensate for matrix effects and potential loss uring analysis. Samples were acidified to pH 2, and solid-phase xtracted on Oasis ® MCX (6 mL, 150 mg) cartridges (Waters, Mil- ord, MA, USA) to clean up the sample and enrich the concentra- ions of the target analytes. Cartridges were conditioned by wash- ng with 12 mL MeOH, 6 mL Milli-Q water and 6 mL Milli-Q wa- er acidified to pH 2 and were eluted with 2 mL of MeOH and 2 3 L of 2% NH 4 OH in MeOH. Chromatographic separation was car- ied out at room temperature using an Atlantis® T3 (100 × 2.1 m; 3 μm) column (Waters, Milford, MA, USA), and analyses were one with a triple quadruple mass spectrometer TripleQuad 5500 BSciex (Concord, Ontario, Canada). Analytical parameters are re- orted in Table S2. Quantification was done using the isotopic dilu- ion method with six-point calibration curves prepared freshly be- ore each analytical run. Instrumental and procedural blanks were ncluded in each analytical batch to check for contamination. For classical illicit drugs, samples were prepared and analyzed sing solid phase extraction (SPE) and HPLC-MS/MS, as previously escribed ( Zuccato et al., 2016 ). .5. Method validation and quality control The analytical method was validated for accuracy, precision, inearity and sensitivity according to international guidelines United Nation Industrial Development Organization, 2006 ). Results re reported in Table S3. Recovery and repeatability of the analyt- cal method were tested in raw wastewater (n = 3) by spiking 50 L aliquots with 100 ng/L of each analyte. A non-spiked wastew- ter sample was analysed as well, to correct the recoveries for the ackground levels. Recoveries ranged between 75 and 116%, with ew exceptions, and variability was lower than 14% (Table S3). Lim- ts of detection (LOD) and quantitation (LOQ) of the whole method ere calculated from raw wastewater samples as the values corre- ponding to signal-to-noise ratios (S/N) of 3 and 10, respectively. OQs were all in the low ng/L concentration range (0.06 -15 ng/L) Table S3). .6. Mass loads and statistical analysis All instrumental data were acquired and processed using An- lyst® 1.6.1 and MultiQuantTM 2.1 software (AB Sciex). Concen- rations (ng/L) were multiplied by wastewater daily flow rates m 3 /day) to obtain the mass load of each substance. The loads ere then normalised to the population served by the WWTP to btain the mg/d/10 0 0inh (d = day; inh = inhabitants), which allowed he comparison of results for different cities. Normalised mass oads were used as an indicator of NPS use per day in the pop- lation. The back-calculation normally done considering the illicit rug metabolism to estimate drug consumption ( Castiglioni et al., 013 ; Gracia-Lor et al., 2016 ), was not done in the present study S. Castiglioni, N. Salgueiro-González, L. Bijlsma et al. Water Research 195 (2021) 116983 Table 2 Mean, median and range concentrations of NPS in wastewater, and number of cities and countries where each NPS was found. Sampling 2016 (20 cities, 14 countries) Concentration (ng/L) Number of cities Number of countries Synthetic cathinones Mean Median Range BUTL 2.51 2.51 - 1 1 3,4-DMMC 0.64 0.64 - 1 1 ETHC 3.22 2.97 2.4-4.3 3 1 MDPV 0.65 0.65 - 1 1 MEPH 13.90 16.11 1.6-23 5 5 METC 3.49 2.29 0.9-10.4 13 8 METL 13.89 13.89 4.4-23.3 2 2 PENTL 1.98 1.98 0.8-3.1 2 2 α -PVP 5.26 6.35 1.3-8.1 3 3 Phenethylamines PMA 41.01 28.80 20.4-106 6 5 Sampling 2017 (7 cities, 5 countries) Concentration (ng/L) Number of cities Number of countries Synthetic cathinones Mean Median Range BUPH 3.35 1.30 0.9-7.8 2 2 MEPH 18.31 9.76 2.5-60 4 4 METC 3.30 2.27 1.2-8.3 6 4 METL 4.38 4.38 4.2-4.6 1 1 Phenethylamines 25-iP-NBoMe 3.06 3.06 2.8-3.3 1 1 b r b o c 3 3 o n N 3 w 1 t a ( i s t w T g i n n p s a t ( e N d P w B v f f 2 c M c e a d o v f c t m 3 ( u m B s 5 n l B α L l m ( K ( i d i f r i ( ecause of the lack of information on NPS metabolism. This cur- ently impedes the development of specific correction factors for ack-calculation. The comparison with classical drugs took account f the mass loads too, without back-calculation, for a more realistic omparison. . Results And Discussion .1. NPS occurrence in urban wastewater Table 2 reports the mean, median and concentrations ranges f the NPS quantified in the different cities: only synthetic cathi- ones and phenetylamines were found in wastewater. Twenty-six PS were searched in 2016, and 10 were detected (38%), while 0 NPS were targeted in 2017 and 5 were detected (17%). METC as the substance most frequently detected, as it was found in 3 cities (65% of the cities investigated) and 8 different coun- ries (57% of the countries investigated) in 2016, and 6 cities (86%) nd 4 countries (80%) in 2017. MEPH was also frequently found 5 cities from 5 countries in 2016, and 4 cities from 4 countries n 2017) ( Table 2 ). MEPH, METC and METL were the only sub- tances found in wastewater in both years. METC was also one of he substances found most frequently over the years in Australia, ith a steady profile of use ( Bade et al., 2019a ; Chen et al., 2013 ; scharke et al., 2016 ). In Europe it was found in previous investi- ations in Italy and the UK ( Gonzalez-Marino et al., 2016 ), but not n Croatia ( Senta et al., 2015 ). NPS concentrations were generally in the low ng/L range (1-20 g/L), except for PMA, a phenethylamine, that was found up to 100 g/L in 2016. PMA was not found in 2017, but we detected another henethylamine, 25-iP-NBoMe, which was not identified in 2016, uggesting the variability of the NPS market and the interchange- ble nature of this group of psychoactive stimulants. Concentra- ions above 20 ng/L were also found for MEPH and METL in 2016 up to 23 ng/L), and for MEPH in 2017 (up to 60 ng/L). These lev- ls are generally in agreement with previous investigations where PS were found at trace levels in wastewater, but there were some ifferences among countries for some substances. For instance, α- VP was found in Greece at similar levels to the present study, hile MEPH in Greece was not detected ( Borova et al., 2015 ). In elgium PMA was not detected, but methoxetamine was detected ery often ( Kinyua et al., 2015 ). In Croatia, METL and MEPH were ound at lower levels than in the present study, and MDPV was not 4 ound ( Senta et al., 2015 ), but it was found in China ( Gao et al., 017 ) and in Finland where the levels were quite high in a spe- ific area ( Kankaanpaa et al., 2014 ). In previous European studies, EPH was found at the highest levels in the UK suggesting a de- rease of MEPH use in that country ( Bade et al., 2017 ; Castrignanò t al., 2016 ; Gonzalez-Marino et al., 2016 ), and METL was found lso in Denmark ( Bade et al., 2017 ). Results from the literature give only a snapshot of NPS use in ifferent countries, because different substances were investigated ver the years and studies mainly focused on single countries. In iew of the complex and dynamic market of NPS, it would be use- ul to apply WBE for repeated monitoring campaigns focused on a ore group of relevant substances to identify temporal and spatial rends of use, especially compounds that have an established niche arket and/or are highly potent. .2. Spatial profiles of NPS use Figs. 1 and 2 reports population-normalized mass loads mg/d/10 0 0inh) of the NPS found in this study. Detailed val- es are set out in SM (Tables S4-S5). The highest loads ( > 30 g/d/10 0 0inh) were for PMA in 2016 in Novi Sad (Serbia) and ratislava (Slovakia) ( Fig. 1 ). MEPH was second highest, with con- iderably lower loads: 8.8 mg/d/10 0 0inh in Krakow (Poland), and mg/d/10 0 0inh in Cluj Napoca (Romania) and Ljubljana (Slove- ia). METC was the substance found most frequently, but at ower mass loads, with maximum values of 3 mg/d/10 0 0inh in ucharest (Romania) and Novi Sad (Serbia) ( Fig. 1 ). METL and -PVP were detected less frequently and were found only in jubljana (Slovenia) (5.5 mg/d/10 0 0inh) and Utrecht (The Nether- ands) (0.7 mg/d/10 0 0inh) for METL, and Lugano (Switzerland) (3.3 g/d/10 0 0inh) and Porto (Portugal) (0.3 mg/d/10 0 0inh) for α-PVP Table S4). In 2017 ( Fig. 2 ), the highest loads were found for MEPH in rakow (Poland) (up to 11 mg/d/10 0 0inh) and Ljubljana (Slovenia) 3 mg/d/10 0 0inh), and for METC and BUPH up to 2 mg/d/10 0 0inh n all the places where they were found. All the other substances etected (BUTL, 3,4-DMMC, ETHC, MDPV, PENTL, BUPH, and 25- P-NBoMe) were at levels lower than 1 mg/d/10 0 0inh and were ound only in one city or country each (Table S5). MEPH was al- eady reported in Krakow few years ago (2012) where it was found n sewage effluents at comparable loads (3.6-7.1 mg/d/10 0 0 inh) Styszko et al., 2016 ). In the same study 4-MEC was also reported S. Castiglioni, N. Salgueiro-González, L. Bijlsma et al. Water Research 195 (2021) 116983 Fig. 1. Mass loads (mg/d/10 0 0 inhabitants) of the NPS detected most frequently in 2016. a d p 2 ( ( d i i t ( k r ( f w a M ( y ( u b ( l r 2 m 2 ( 3 i f e t c 2 o P w M m M p p m w u r T u l t t similar levels (4.8-5.8 mg/d/10 0 0 inh), but it was not anymore etected in this study. Generally, the loads found in this study were similar to those in revious investigations in Australia ( Bade et al., 2019a ; Chen et al., 013 ), Norway ( Baz-Lomba et al., 2016 ), UK, Italy and Spain Gonzalez-Marino et al., 2016 ), China ( Gao et al., 2017 ) and Greece Diamanti et al., 2019 ). Nevertheless, there are some interesting ifferences for certain substances such as PMA that was found n this study at the highest levels ever found before, but only n a few cities. PMA was reported previously only in Greece (up o 11 mg/d/10 0 0inh) ( Diamanti et al., 2019 ), but not in Belgium Kinyua et al., 2015 ), indicating a scattered profile of use. METL was the first synthetic cathinone appearing in the mar- et in Europe in 2005 ( UNODC, 2013 ) and was found in Eu- ope at levels < 10 mg/d/10 0 0inh in a few studies [this study and Baz-Lomba et al., 2016 )], while an increasing trend was seen a ew years ago in Australia ( Chen et al., 2013 ; Thai et al., 2016 ), here it reached loads up to 40 mg/d/10 0 0inh. MDPV was found t levels of 1 mg/d/10 0 0inh in Italy [this study and ( Gonzalez- arino et al., 2016 )], Australia ( Chen et al., 2013 ) and China Gao et al., 2017 ), but increased consumption was recorded a few ears ago in Finland, where it ranged up to 19 mg/d/10 0 0inh Kankaanpaa et al., 2014 ). These results were confirmed by un- sually high rates of MDPV-related crimes and MDPV-positive iological specimens from clinical sources in the same region EMCDDA, 2014 ; Kankaanpaa et al., 2014 ). The ketamine ana- og methoxetamine was not found in the present study, but was ecorded in Belgium (about 1-2 mg/d/10 0 0inh) ( Kinyua et al., 015 ), Norway (0.8 mg/d/10 0 0inh) ( Baz-Lomba et al., 2016 ) and, ore recently, in Australia (1.3 mg/d/10 0 0inh) ( Bade et al., 5 020 ) and the UK (up to more than 100 mg/d/10 0 0inh) Rice et al., 2020 ). .3. Temporal trends of use of NPS Temporal trends were evaluated on the five cities investigated n 2016 and 2017. MEPH, METC and METL were the only substances ound in both years (Figure S1 and Tables S4, S5). No major differ- nces were seen between the loads found in the two years, though here were differences for the single cities (Figure S1). The only ex- eption was PMA that was found only in 2016 and disappeared in 017, indicating a potential change in the market/use of this drug ver the investigated period. MEPH was higher in 2017 than 2016 in Krakow (Poland) and orto (Portugal), and lower in Ljubljana (Slovenia), while METC as generally higher in 2017, except in Bratislava (Slovakia). ETL was found only in Ljubljana and loads decreased from 5.5 g/d/10 0 0inh in 2016 to 1 mg/d/10 0 0inh in 2017 (Table S4, S5). EPH mass loads in Italy and UK resulted in lower levels than in revious studies ( Gonzalez-Marino et al., 2016 ; Rice et al., 2020 ), articularly in the UK where mass loads dropped from about 30 g/d/10 0 0inh in 2015 to 0.5 mg/d/10 0 0inh in 2016. This trend as seen also in other countries such as Australia where MEPH se peaked in 2010-2011 ( Chen et al., 2013 ) then dropped rapidly emaining very low in the next few years ( Bade et al., 2019a ; hai et al., 2016 ). MEPH was one of the first NPS to become pop- lar in the market in the mid-20 0 0s, especially in the UK and Ire- and, and its use increased for a few years (2008-2010), reaching he levels of MDMA in the general population, and cocaine among S. Castiglioni, N. Salgueiro-González, L. Bijlsma et al. Water Research 195 (2021) 116983 Fig. 2. Mass loads (mg/d/10 0 0 inhabitants) of the NPS detected most frequently in 2017. Fig. 3. Weekly profiles of use of MEPH (left) and METC (right) in the 2017 sampling campaign. Weekday = Tuesday to Friday; Weekend = Saturday to Monday. y t r n u i a t o t u d c t c s r i s d E oung people ( EMCDDA-Europol, 2013 ), until it was put under con- rol and its use gradually decreased ( EMCDDA, 2014 ). No particular patterns of use have been identified so far in Eu- ope for other substances, but an interesting trend was recently oted in Australia where different substances were preferentially sed over the few last years (even if not always in the same area), .e. MEPH in 2010-2011, METL in 2012-2013, ETHL in 2014-2016 nd, more recently ( Bade et al., 2019a ), PENTL. It would be useful o start periodic monitoring campaigns also in Europe for a “pri- rity” group, i.e. those substances for which preliminary informa- ion is available in some countries, in order to identify patterns of 6 se better. Selecting this group of “priority” substances is not easy, ue to the high numbers of NPS on the market and the speedy hanges in their profiles of use, but combining different informa- ion such as their presence in wastewater, seizures, epidemiologi- al and forensic data will help to establish a list of selected sub- tances. A prioritization approach was proposed recently by our esearch group for selecting “priority NPS” based on wide screen- ng by HRMS, using a database of substances selected on the ba- is of their reporting frequency by the Early Warning Systems of ifferent agencies and previous detection in wastewater in several uropean countries ( Salgueiro-González et al., 2019 ). Future strate- S. Castiglioni, N. Salgueiro-González, L. Bijlsma et al. Water Research 195 (2021) 116983 Fig. 4. Weekly profiles of amphetamine and MDMA (A) and benzoylecgonine and methamphetamine (B) in the 2017 sampling campaign. Weekday = Tuesday to Friday; Weekend = Saturday to Monday. g c s m 3 w d b t p i p A i T a b h t s M i t i T ( 3 N s Table 3 Mean population-normalized mass loads and ranges (mg/d/10 0 0 inhabitants) of NPS and classical illicit drugs. Sampling 2016 Classical drugs Mean Range NPS Mean Range AMPH 77.3 LOQ-279 BUTL 0.8 0.8 METH 68.6 LOQ-233 3,4-DMMC 0.2 0.2 MDMA 26.8 LOQ-206 ETHC 1.0 LOQ-1.3 BE 114.8 0.4-304 MDPV 0.2 0.2 KET 5.2 LOQ-52 MEPH 4.2 LOQ-9 METC 1.4 LOQ-3.4 METL 3.1 LOQ-5.5 PENTL 1.0 1.0 α -PVP 1.8 LOQ-3.3 PMA 24.0 LOQ-62 Sampling 2017 Classical Drugs Mean Range NPS Mean Range AMPH 35.0 LOQ-79 BUPH 0.7 LOQ-1.8 METH 26.0 0.3-195 MEPH 3.8 LOQ-12.4 MDMA 17.1 3.3-60 METC 0.7 LOQ-1.7 BE 210.4 32-476 METL 0.9 0.9 25-iP-NBoMe 0.7 LOQ-0.8 r s l s c h r t a a a w ies could make use of a first HRMS screening, using broad lists of ompounds, followed by triangulation with information from other ources to build up a panel of “priority NPS” for target quantitative ethods. .4. Weekly profiles of NPS use Pooled samples from weekdays and weekends collected in 2017 ere compared to assess any changing pattern, but no noteworthy ifferences were found ( Fig. 3 ). This may be due to the small num- er of samples investigated that were too limited to identify po- ential changes properly and thus future investigations should ex- and the sample size. However, the weekdays vs weekend compar- son was also done for classical drugs measured in the same sam- les, giving results in line with the known profiles of use ( Fig. 4 and B): increases during the weekends for MDMA and cocaine, .e. BE ( Been et al., 2016 ; Kankaanpaa et al., 2016 ; Lai et al., 2016 ; homas et al., 2012 ; Zuccato et al., 2016 ), but usually not for METH nd AMPH ( Kankaanpaa et al., 2016 ; Zuccato et al., 2016 ). This oosts the reliability of the results also for NPS, which probably ave less marked differences in their weekly profiles of use be- ween weekdays and weekends. For instance, METC shows a con- tant profile of use during the week ( Chen et al., 2013 ; Gonzalez- arino et al., 2016 ; Tscharke et al., 2016 ) and this was confirmed n the present investigation. On the contrary, in previous inves- igations MEPH and METL have shown an increase in use dur- ng the weekend ( Chen et al., 2013 ; Gonzalez-Marino et al., 2016 ; scharke et al., 2016 ); this was also seen here, but only in Porto Portugal) ( Fig. 3 ). .5. Comparison of the use of classical (established) illicit drugs and PS Mass loads of classical illicit drugs were measured in the same amples to compare results and identify differences of use. Table 3 7 eports the means and ranges of the mass loads for each sub- tance in 2016 and 2017. With the exception of PMA, mean mass oads of NPS ranged between 0.2 and 4.2 mg/d/10 0 0inh, thus re- ulting 20 to 50 times lower than classical illicit drugs such as ocaine (expressed as its metabolite BE) or AMPH. PMA instead ad higher loads (up to 62 mg/d/10 0 0inh) in 2016, at a compa- able level with some classical drugs such as AMPH ( Table 3 ). Ke- amine (KET), a dissociative anesthetic used in veterinary medicine nd to a lesser extent in human medicine, thus not classified as n NPS, was also monitored because of its illegal use as a recre- tional drug for its psychoactive effects ( EMCDDA, 2002 ). KET use as lower than for the other classical illicit drugs, and the mean S. Castiglioni, N. Salgueiro-González, L. Bijlsma et al. Water Research 195 (2021) 116983 m i v l t d v w n b e fi b a f v U 4 v a s r t i l t a r l t i c r i t t y “ s s s r n s t a t c l h m D c i A t g b C H w P s K o v E U t F H w o S f R B B B B B B B B B B ass load (5.2 mg/d/10 0 0inh) was comparable to those of the NPS n this study and to those already found in Italy in a previous in- estigation ( Castiglioni et al., 2015 ). These results showed that, with the exception of the phenethy- amine PMA, which might be used at a similar level to AMPH, he consumption of NPS is normally much lower than classical rugs in the general population, confirming observations from pre- ious studies. Moreover, among the 30 NPS selected only a few ere found in wastewater, suggesting that many of them were ot used anymore or were used at very low extent not detectable y wastewater analysis. This, in addition to the fact that sev- ral substances identified in 2016 were not found in 2017, con- rms the changing patterns of these substances. Data provided y wastewater analysis are referred to the general population nd complement the epidemiological information that is scanty or NPS and mainly focused on specific populations (e.g. festi- al attendees, students and young people) ( EMCDDA, 2020 , 2015 ; NODC, 2020 ). . Conclusions The present study demonstrates the suitability of WBE for in- estigating NPS use in the general population as this approach was ble to identify spatial profiles and temporal trends for selected ubstances in 14 European countries. Despite the limitations still elated to monitoring NPS in wastewater, WBE can provide quan- itative and qualitative information on the use of these substances n a population. This is particularly difficult for other epidemio- ogical tools such as population surveys, because consumers of- en do not know exactly which drug or mixture of drugs they re taking. This study gives information on the use of NPS in Eu- ope, confirming that it is lower than classical drugs, but also high- ights the changeable nature of the market and consequently of he profiles of use. This is a useful contribution to epidemiolog- cal studies, in view of the difficulties of following these rapid hanges. This study also highlighted the scattered nature of the cur- ent WBE information for NPS, at least in Europe, where stud- es so far have addressed different substances and few loca- ions. It would therefore be very useful in the next few years o implement large international NPS monitoring campaigns (e.g. early), employing appropriate strategies for choosing a set of priority” substances and monitoring them over time. The best trategy for selecting NPS will be the triangulation of different ources of information, including epidemiological surveys, foren- ic and toxicology analyses, analysis of seized drugs, and WBE esults from previous studies. Despite its focus on a limited umber of substances, target analysis of selected priority sub- tances has the advantage of providing qualitative and quantita- ive information on use, facilitating comparisons among different reas. For WBE studies on NPS, the main current limitation is related o the scant information on human metabolism that prevents back- alculation of drug consumption, as is done for the classical il- icit drugs. Further studies in this field are required in order to elp identify the best biomarkers of use (parent drugs or urinary etabolites), and refine WBE back-calculation. eclaration of Competing Interest The authors declare that they have no known competing finan- ial interests or personal relationships that could have appeared to nfluence the work reported in this paper. 8 cknowledgements Lubertus Bijlsma wishes to thank Ettore Zuccato, Sara Cas- iglioni and the Istituto di Ricerche Farmacologiche Mario Ne- ri (Milan, Italy) for hosting him as a post-doc researcher. Al- erto Celma acknowledges the Spanish Ministry of Economy and ompetitiveness for his predoctoral grant (BES-2016-076914). Félix ernández acknowledges MINECO (Project CTQ2015-65603-P), as ell as Generalitat Valenciana (Research Group of Excellence, rometeo 2019/040). The collecting of samples in Krakow was upported by ‘‘Municipal Waterworks and Sewer Enterprise in rakow’’. We greatly acknowledge Igor Bodík, Slovak University f Technology, Bratislava, Slovakia; Fiona Regan Dublin City Uni- ersity, Dublin, Ireland; Jaroslav Slobodnik and Natalia Glowacka, nvironmental Institute of Kos, Slovak Republic; Mariya Skobley, kraine, for the help provided for samples collection. Wessex Wa- er is acknowledged for samples provision in the UK. unding This work was supported by the European Commission [grant OME/2014/JDRUG/AG/DRUG/7086 - NPS-Euronet ]. 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