Wastewater treatment processes are monitored mainly in terms of biogenic substances removal efficiency. Only recently they started to be perceived as a potential sources of pharmaceutical residues to their recipients the and hot-spots for antibiotic resistance dissemination among bacteria. The scale of the problem has not been fully investigated and understood – missing data on the pharmaceuticals consumption, unidentified and dispersed point sources and non-unified sampling strategy of monitoring programs have been identified as the examples of problematic areas. I n scope of project REPHIRA (Reduction of Pharmaceutical Emissions from Dispersed Point Sources in Rural Areas), financed from the Interreg Baltic Sea Region Seed Money, the cooperation has been established between five partners located in the Baltic Sea catchment area: Poland, Germany, Denmark, Sweden and Finland. In order to fulfill the knowledge gap, a preliminary study was conducted by Polish partner on four wastewater treatment plants (WWTPs) localized in the coastal area in northern Poland. Objects differ in terms of load, people equivalent, treatment technology and the recipient of WWTP effluent. The amount of human gut related indicator organism, E. coli has been estimated in raw and treated wastewater, as well as in the receiver, using classical microbiology approach and cultivation method. Additionally, the bacterial resistance to cefotaxime – an antibiotic belonging to third-generation cephalosporin family – has been tested.

Two green chemistry extraction systems, an in-house stainless-steel column Pressurized Hot Water Extraction system (PHWE) and a commercially available Espresso machine were applied for analysing 23 active pharmaceutical ingredients (APIs) in sewage sludge. Final analysis was performed on UPLC-MS/MS using two different chromatographic methods: acid and basic. When analysing all 23 APIs in sewage sludge both extraction methods showed good repeatability. The PHWE method allowed for a more complete extraction of APIs that were more tightly bound to the matrix, as exemplified by much higher concentrations of e.g., ketoconazole, citalopram and ciprofloxacin. In total, 19 out of 23 investigated APIs were quantified in sewage sludge, and with a few exceptions the PHWE method was more exhaustive. Mean absolute recoveries of 7 spiked labelled APIs were lower for the PHWE method than the Espresso method. Under acid chromatographic conditions mean recoveries were 16% and 24%, respectively, but increased to 24% and 37% under basic conditions. The difference between the PHWE method and the Espresso method might be interpreted as the Espresso method giving higher extraction efficiency; however, TIC scans of extracts revealed a much higher matrix co-extraction for the PHWE method. Attempts were made to correlate occurrence of compounds in sewage sludge with chemical properties of the 23 APIs and there are strong indications that both the number of aromatic rings and the presence of a positive charge is important for the sorption processes to sewage sludge.

By using an innovative, positive pressure sample loading technique in combination with an in-line filter of finely ground sand the bottleneck of solid phase extraction (SPE) can be reduced. Recently published work by us has shown the proof of concept of the technique. In this work, emphasis is put on the SPE flow rate and method validation for 26 compounds of emerging environmental concern, mainly from the 1st and 2nd EU Watch List, with various physicochemical properties. The mean absolute recoveries in % and relative standard deviations (RSD) in % for the investigated compounds from spiked pure water samples at the three investigated flow rates of 10, 20, and 40 mL/min were 63.2% (3.2%), 66.9% (3.3%), and 69.0% (4.0%), respectively. All three flow rates produced highly repeatable results, and this allowed a flow rate increase of up to 40 mL/min for a 200 mg, 6 mL, reversed phase SPE cartridge without compromising the recoveries. This figure is more than four times the maximum flow rate recommended by manufacturers. It was indicated that some compounds, especially pronounced for the investigated macrolide molecules, might suffer when long contact times with the sample glass bottle occurs. A reduced contact time somewhat decreases this complication. A very good repeatability also held true for experiments on both spiked matrix-rich pond water (high and low concentrations) and recipient waters (river and wastewater) applying 40 mL/min. This work has shown that, for a large number of compounds of widely differing physicochemical properties, there is a generous flow rate window from 10 to 40 mL/min where sample loading can be conducted. A sample volume of 0.5 L, which at the recommended maximum flow rate speed of 10 mL/min, would previously take 50 min, can now be processed in 12 min using a flow rate of 40 mL/min. This saves 38 min per processed sample. This low-cost technology allows the sample to be transferred to the SPE-column, closer to the sample location and by the person taking the sample. This further means that only the sample cartridge would need to be sent to the laboratory, instead of the whole water sample, like today's procedure.

In environmental trace analysis there is often a need to enrich the compounds of interest from a large sample volume, where the use of solid phase extraction (SPE) is more or less the standard technique. The presence of the sample matrix can cause clogging of the SPE-column, especially at the end of a sample load. Swedish surface waters are often humic rich making the use of traditional sample loading by a vacuum manifold very limited. This obstacle forced the development of a different sample loading technique, based on compressed air and sand as an in-line-filter, designed to load larger sample volumes as needed in trace level analysis of hormones in surface water. The developed technique, combined with a UPLC MS/MS method, showed promising reproducibility and accuracy, and enabled increased sensitivity for the analysis of hormones in humic rich surface water.

Läkemedelsverket gav i september 2015 ut en rapport med titeln ”Miljöindikatorer inom ramen för nationella läkemedelsstrategin (NLS)”. Rapporten indikerar bland annat stora kvalitetsbrister i tidigare utförda analyser av läkemedel i miljön under åren 2002-2013, men betonar också vikten av att samordna framtida nationella läkemedelsanalyser för att bättre använda våra offentliga resurser. I rapporten skriver man följande: ”Allra högst prioriterad ansåg arbetsgruppen att indikatorn ”mäta halter av läkemedelssubstanser i miljö” vara. Detta beroende på att det utöver att det är av stor vikt att följa utvecklingen av läkemedelsrester i miljön över tid för att utvärdera effekten av genomförda insatser, så ansåg arbetsgruppen att det finns stora möjligheter att optimera användningen av de offentliga resurserna genom en bättre samordning av mätningar i miljön. Många mätningar har gjorts historiskt av olika offentliga aktörer utan någon samordning.” Mot bakgrund av detta, och som svar på ett nationellt behov, åtog sig Högskolan Kristianstad (HKR) genom Ola Svahn och Erland Björklund, båda verksamma vid MoLab, Krinova Incubator and Science Park i Kristianstad, att leda denna interkalibreringsstudie för läkemedelsanalys i miljö-vattenprover av olika typ. Arbetet har skett på uppdrag av Havs- och Vattenmyndigheten som finansierat arbetet via bidrag ur havs- och vattenmiljöanslaget och i samarbete med fyra andra analyslaboratorier vid Umeå Universitet (UU), Sveriges Lantbruksuniversitet Uppsala (SLU), Svenska miljöinstitutet Stockholm (IVL) samt Aarhus Universitet Danmark (AU). Total deltog därmed 5 laboratorier, alla med tidigare erfarenhet av läkemedelsanalys i miljöprover.

I projektet LUSKA (Läkemedelsutsläpp från Skånska Avloppsreningsverk) deltog 6 olika reningsverksorganisationer,    geografiskt fördelade över hela Skåne; Höganäs, Klippan, Höör/Hörby, Svedala, Kristianstad och Simrishamn. Provtagning utfördes i april 2017 på fyra platser vid varje reningsverk. Tre av dessa platser valdes enligt förslag från Länsstyrelsen Skånes tillsynsvägledning; uppströms, nedströms och utgående vatten från reningsverket. Dessutom ingick en fjärde provpunkt bestående av inkommande vatten till reningsverken. I studien analyserades total 21 av 22 läkemedel enligt Läkemedelsverkets föreslagna lista på ämnen från 2015. Analysresultaten från de 8 avloppsreningsverken, samt tillhörande recipient i form av åar och sjöar visar tydligt att stora mängder läkemedel hamnar i våra omgivande skånska vatten varje år. Detta sker som en konsekvens av att reningsverken inte förmår rena läkemedelsutsläpp med befintlig teknik baserad på aktivt slam. Studien visade att de 8 reningsverken släpper ut minst 71 kg läkemedel varje år till skånska vatten enbart av dessa 21 ämnen. Den stora bulken av de uppmätta ämnena utgjordes av blodtryckssänkaren metoprolol och smärtstillaren diklofenak. Men även ämnen som karbamazepin, losartan, naproxen och oxazepam förekom i betydande koncentrationer i avloppsvattnet. Dessa ämnen innefattar flera läkemedelstyper och representerar tre generella och relativt vanliga sjukdomstillstånd som högt blodtryck, inflammation och smärta, samt depression och ångest. Baserat på resultaten i LUSKA-projektet kan man uppskatta att när ett reningsverk behandlar en miljon kubikmeter (1 000 000 m3) avloppsvatten passerar det samtidigt ut ca 4 kg av de 21 läkemedel som Läkemedelsverket tagit upp på sin övervakningslista. Enligt en grov beräkning, innefattande en majoritet av de skånska reningsverken, skulle detta innebära att nästan 600 kg kilo läkemedel läcker ut varje år från skånska reningsverk av Läkemedelsverkets föreslagna ämnen. Samtidigt måste man beakta att dessa 21 ämnen endast utgör en liten del av flera hundra läkemedelssubstanser som används för behandling av sjukdomar. Med stor sannolikhet läcker ett till flera ton läkemedel ut årligen i skånsk recipient. Mätningar i åar och sjöar visar att även om koncentrationerna sjunker nedströms verken som  en konsekvens av företrädesvis utspädning så finns det lokaler där koncentrationerna är an-märkningsvärt höga utifrån ett hållbarhetsperspektiv.

The project LUSKA (a Swedish acronym meaning ‘to figure out’ formed from the name LäkemedelsUtsläpp från SKånska Avloppsreningsverk - Pharmaceutical emissions from Scanian wastewater treatment plants) comprised six different participating wastewater treatment organisations, geographically distributed throughout the whole of the Scania region in Sweden (Skåne): Höganäs, Klippan, Höör/Hörby, Svedala, Kristianstad and Simrishamn. Sampling was done in April 2017 in four locations at each treatment plant. Three of these locations were chosen as suggested by the County Administrative Board of Skåne’s supervisory guide: upstream, downstream and outlet water from the treatment plant. In addition, a fourth sample point was included consisting of inlet water to the treatment plants. In the study, a total of 21 out of 22 pharmaceuticals were analysed according to the Swedish Medical Products Agency’s proposed substance watchlist from 2015. The analysis results from the eight treatment plants and associated recipients in the form of streams and lakes clearly shows that large amounts of pharmaceuticals end up in our surrounding Scanian waters every year. This takes place as a consequence of the wastewater treatment plants not being able to separate pharmaceutical emissions with existing technology based on activated sludge. The study showed that the eight treatment plants release at least 71 kg of pharmaceuticals every year to Scanian waters of these 21 substances alone. The major bulk of the measured substances were comprised of the blood pressure lowering drug metoprolol and the analgesic diclofenac. But even such substances as carbamazepine, losartan, naproxen and oxazepam occurred in significant concentrations in the wastewater. These substances include several pharmaceutical types and represent three general and relatively common illnesses: high blood pressure, inflammation and pain, and depression and anxiety. Based on the results in the LUSKA project, it can be estimated that when a treatment plant treats one million cubic metres (1,000,000 m3) of wastewater, at the same time approximately 4 kg pass of the 21 drugs that the Swedish Medical Products Agency included on its watchlist. According to a rough estimate, including a majority of the Scanian wastewater treatment plants, this would mean that nearly 600 kg of drugs on the Swedish Medical Products Agency’s proposed substances watchlist leak out from Scanian treatment plants every year. At the same time, it must be taken into account that these 21 substances only comprise a small part of the several hundred pharmaceutical substances used for the treatment of diseases. In all likelihood, one or more tonnes of drugs leak out into Scanian recipients annually. Measurements in lakes and streams show that even if the concentrations drop downstream of the plants, probably as a consequence of dilution, there are locales where the concentrations are remarkably high from a sustainability perspective.

In this work the habitual behaviour of low pH in environmental organic trace analysis is challenged by investigating the full potential of building a multi-component UHPLC-ESI-MS/MS method adapted to cover common emerging contaminants of many different polarities, minimizing the elements of compromise in the performance of the final analytical separation and detection. Contributes have been made by taking advantage of common commercially available technology in understanding the impact from solvent components and the ionization of analytes which can facilitate future development of robust, sensitive and precise UHPLC-MS/MS methods. All contaminants were evaluated and optimized without prejudices regarding historical residence in terms of chromatographic conditions and ESI mode; increasing multi-method's flexibility that can be implemented in routine analysis in response to new requests as well as to emerging contaminants yet to be discovered. Our data strongly supports the questioning of the assumption that equilibrium concentrations of ions in solution reflect those produced during the electrospray process. ESI responses of [M+H](+) and limits of detection were comparable, or often better at high pH compared to acidic eluents. Presence of nitrogen basic groups such as tertiary and secondary amines in a compound increased the intensity of the ESI+ signal, and was even further elevated in basic eluent. The proton affinity probably changes for many nitrogen-containing compounds during the ionization process, making the gas-phase processes very important in generation of these ions by ESI+. There were also an unexpected large number of compounds showing their highest response at pH 7 and weak ionic strength. A flow optimized, buffert free, neutral UHPLC-MS/MS method enhanced the sensitivity for the environmental important synthetic hormone ethinyl estradiol significantly.

This study is the first to investigate the pharmaceutical burden from point sources affecting the UNESCO Biosphere Reserve Kristianstads Vattenrike, Sweden. The investigated Biosphere Reserve is a >1000 km(2) wetland system with inflows from lakes, rivers, leachate from landfill, and wastewater-treatment plants (WWTPs). We analysed influent and treated wastewater, leachate water, lake, river, and wetland water alongside sediment for six model pharmaceuticals. The two WWTPs investigated released pharmaceutical residues at levels close to those previously observed in Swedish monitoring exercises. Compound-dependent WWTP removal efficiencies ranging from 12 to 100 % for bendroflumethiazide, oxazepam, atenolol, carbamazepine, and diclofenac were observed. Surface-water concentrations in the most affected lake were ≥100 ng/L for the various pharmaceuticals with atenolol showing the highest levels (>300 ng/L). A small risk assessment showed that adverse single-substance toxicity on aquatic organisms within the UNESCO Biosphere Reserve is unlikely. However, the effects of combinations of a large number of known and unknown pharmaceuticals, metals, and nutrients are still unknown.