Ostanki zdravilnih učinkovin in metabolitov v okolju so opredeljeni kot novodobna mikroonesnažila. V okolje vstopajo predvsem kot posledica uporabe v humani in veterinarski medicini. Ugotovili so, da so čistilne naprave glavni točkovni vir izpustov, saj njihova tehnologija ni prilagojena za čiščenje tovrstnih mikroonesnažil. Odpadna voda se iz čistilnih naprav zliva v površinske in/ali podtalne vode, ki so vir pitne vode. Koncentracije ostankov zdravilnih učinkovin v okolju so večinoma nizke: do nekaj μg/L v odpadnih vodah, kjer so običajno izmerjene najvišje vrednosti za posamezen analit, in v padajočem trendu v površinskih in pitnih vodah. V slednjih so koncentracije najpogosteje v območju pg/L. Kljub nizkim koncentracijam pa je z vidika toksičnosti nujno treba obravnavati kombinacijo vseh zdravilnih učinkovin, ki imajo lahko negativne učinke na izpostavljene vodne organizme in posredno tudi na človeško zdravje. Nekatere raziskave poročajo o biokopičenju v vodnih organizmih in negativnih učinkih, kot so morfološke, citološke in vedenjske spremembe. Prve raziskave pojavljanja ostankov zdravilnih učinkovin v okolju so bile objavljene že leta 1970, porast pa beležimo v zadnjih letih, kar je posledica razvoja naprednih instrumentalnih metod, ki omogočajo zaznavo in merjenje zdravilnih učinkovin že pri zelo nizkih koncentracijah. Okoljska analitika je običajno sestavljena iz dveh delov: priprave vzorca ter instrumentalne metode. Glede na objavljeno literaturo je metoda prvega izbora za pripravo vzorca ekstrakcija na trdnem nosilcu in instrumentalna analiza s tekočinsko kromatografijo sklopljena z masno spektrometrijo. V doktorski nalogi smo se najprej osredotočili na razvoj ustrezne metodologije za pripravo vzorcev. Ločeno smo razvijali postopke za ekstrakcijo iz odpadnih, površinskih in pitnih voda ter za ekstrakcijo iz ribjih tkiv. Za ekstrakcijo iz odpadnih voda smo razvili dva inovativna pristopa ekstrakcije: prvi je pol-avtomatiziran sistem SPE-DEX, ki je osnovan na ekstrakciji na trdnem nosilcu (ang. solid phase extraction, SPE), drugi pristop pa je ekstrakcija Twister®, ki je osnovana na sorptivnem mehanizmu. Oba pristopa smo ovrednotili, vendar izbrali prvega, ki se je izkazal za bolj primernega za večje število zdravilnih učinkovin (> 100). Razvito metodo smo primerjali z najpogosteje uporabljenim ročnim pristopom SPE ter potrdili primerljive validacijske parametre. Metoda SPE-DEX ima poleg tega številne prednosti: gre za avtomatiziran sistem, ki ne zahteva stalne prisotnosti operaterja, s čimer se izboljša ponovljivost med vzorci ima manjšo možnost mašenja ekstrakcijskih diskov omogoča hkratno filtracijo z ekstrakcijo neposredno na aparaturi, kar znatno skrajša čas priprave vzorca in še pomembnejše prepreči izgube analitov zaradi morebitne adsorpcije na odfiltriran material ali filter. Z metodo SPE-DEX smo preverjali prisotnost ostankov zdravilnih učinkovin in metabolitov v odpadnih vodah. Vzorčili smo na 19 čistilnih napravah, pri čemer smo vzorec 33-krat vzeli na iztoku in 13-krat na vtoku. Ekstrakte smo analizirali z visoko občutljivo metodo LC-MS/MS. Po zahtevnem razvoju in optimizaciji metode je analiza, ki je zajemala ločitev in detekcijo vseh 104 analitov, trajala 8 minut. V odpadnih vodah na iztoku smo potrdili 94 % vseh tarčnih analitov (n = 104) v vsaj enem vzorcu v povprečnih koncentracijah od 1 ng/L do 11 μg/L. Na vtoku smo potrdili prisotnost 87 % vseh tarčnih analitov (n = 104) v vsaj enem vzorcu, povprečne koncentracije pa so bile v območju od 1 ng/L do 116 μg/L. Na vtoku smo najvišjo povprečno koncentracijo ugotovili za metabolit salicilno kislino (116 μg/L), prisotna je bila v 92 % vzorcev (n = 13). V visokih koncentracijah so bili prisotni še paracetamol (18 μg/L) in metformin (17 μg/L), oba v vseh vzorcih, in kofein (16 μg/L) v 92 % vzorcev. Skupno je bilo v koncentracijah nad 1 μg/L prisotnih 12 analitov s pogostostjo pojavljanja nad 85 %, z izjemo lakozamida (7 μg/L), ki je bil prisoten le v 1 vzorcu. Stalno prisotnost v vseh vzorcih smo potrdili za 31 analitov od skupno 104. Na iztoku smo v najvišji povprečni koncentraciji ugotovili tramadol (11 μg/L), v vseh vzorcih (n = 33). V visokih koncentracijah so bili prisotni še: hidroklorotiazid (5 μg/L) v 97 % vzorcev, metformin (4 μg/L) v 76 % vzorcev in salicilna kislina (4 μg/L) v 37 % vzorcev. Skupno je bilo v koncentracijah nad 1 μg/L prisotnih 10 analitov s pogostostjo pojavljanja nad 60 %, z izjemo fluoksetina (1 μg/L), ki je bil prisoten le v treh vzorcih, in salicilne kisline. Stalno prisotnost v vseh vzorcih smo potrdili za 14 analitov od skupno 104. Na osnovi pridobljenih podatkov smo oblikovali ožji izbor analitov za spremljanje njihove prisotnosti v površinskih vodah. Vključili smo le analite, ki smo jih zaznali v več kot polovici vzorcev na iztoku iz čistilne naprave in v višjih koncentracijah skupno smo spremljali 44 analitov. Razvili smo metodo ekstrakcije na ročnem sistemu SPE, ki nam je omogočal zadostno koncentriranje z namenom doseganja čim nižjih mej določitve. Metodo smo ovrednotili in pokazali ustreznost validacijskih parametrov. Uporabnost metode smo preverili z analizo 6 vzorcev površinskih voda, od tega 5 vzorcev rečnih voda in 1 vzorca jezerske vode. Potrdili smo prisotnost 43 tarčnih analitov (od skupno 44), večinoma v koncentracijah pod mejo določitve, merljive koncentracije pa so bile v območju od 0,08 ng/L do 47 ng/L. V vseh vzorcih smo potrdili prisotnost 29 analitov. Najvišja absolutna izmerjena koncentracija je bila za valsartan, ki je znašala 47 ng/L (povprečna koncentracija 11 ng/L, mediana 4 ng/L), in za gabapentin, ki je dosegla 45 ng/L (samo enkrat nad LOQ (LOQ = 40 ng/L)). Pogosto sta bila zaznana še tramadol (v vseh vzorcih, n = 6) v povprečni koncentraciji 5 ng/L (mediana 1,2 ng/L) in sulfametoksazol (v 67 % vzorcev) v povprečni koncentraciji 6 ng/L (mediana 6,3 ng/L). Raziskave smo nadaljevali z razvojem metodologije za ekstrakcijo iz pitnih voda. Uporabili smo razvito metodo SPE za površinske vode in povečali volumen nanešenega nanos vzorca, da bi povišali faktor koncentriranja. Metodo smo ovrednotili in pokazali ustreznost validacijskih parametrov. Preverili smo prisotnost ostankov zdravilnih učinkovin in metabolitov v 18 vzorcih pitnih voda. V vsaj enem vzorcu smo potrdili 24 % izbranih analitov (n = 54). Koncentracije so bile izredno nizke od 0,04 do 26,0 ng/L. V vseh vzorcih je bil prisoten le azitromicin, in sicer v povprečni koncentraciji 2,6 ng/L (mediana 0,7 ng/L). V najvišjih koncentracijah sta bila ugotovljena azitromicin (26 ng/L) in kofein (14 ng/L). Sledil je izračun vseživljenjske izpostavljenosti človeka izbranim zdravilnim učinkovinam zaradi vnosa pitne vode. Pri izračunu smo upoštevali življenjsko dobo 81 let in povprečni vnos pitne vode 2 L/dan. Na osnovi pridobljenih podatkov bi vseživljenjska izpostavljenost znašala 3 mg, iz česar sklepamo, da je tveganje za človeka minimalno. Primerjali smo rezultate glede na posamezne tipe vode in ugotovili, da smo vse zdravilne učinkovine, katerih prisotnost smo potrdili v pitni vodi, zaznali tudi v površinskih in odpadnih vodah. Iz izračunanih koncentracij lahko sklepamo, da je verjetnost pojavljanja v površinskih in pitnih vodah večja, kadar je izmerjena zelo visoka koncentracija v odpadnih vodah (iztok) ter kadar je posamezni analit v odpadnih vodah zaznan zelo pogosto. Raziskave smo dopolnili z ugotavljanjem prisotnosti ostankov zdravilnih učinkovin v ribah. V ta namen smo razvili metodo za ekstrakcijo iz ribjega tkiva, da bi lahko opredelili tveganje za človeka zaradi uporabe rib v prehrani. Za ekstrakcijo smo uporabili metodo QuEChERS, ki smo jo kot prvi prilagodili za ekstrakcijo ostankov zdravilnih učinkovin iz ribjih tkiv. Validirano metodo smo prenesli na 13 vzorcev ribjih mišic, kože in škrg. V vseh treh tipih tkiva smo potrdili prisotnost ostankov zdravilnih učinkovini in metabolitov. V vsaj enem vzorcu smo potrdili 42 vseh tarčnih analitov (n = 56). V vseh je bil prisoten le metoprolol (< LOQ–0,7 ng/g). V več kot polovici vzorcev smo zaznali še kofein (< LOQ–3,2 ng/g), valsartan (0,1–0,4 ng/g) in metabolit kofeina 1,7-dimetilksantin (v vseh vzorcih < LOQ). Najvišje povprečne koncentracije smo izmerili za ciprofloksacin (19,2 ng/g), enalapril (16,2 ng/g) in haloperidol (14 ng/g). Koncentracije zdravilnih učinkovin v mišicah rib, katere so del prehranjevalne verige, so bile zelo nizke, in sicer med 0,07 ng/g in 1,1 ng/g (povprečno 0,3 ng/g). Posledično lahko sklepamo, da je tveganje za človeka zanemarljivo. Najvišje koncentracije smo izmerili v škrgah. Z raziskavami smo potrdili prisotnost zdravilnih učinkovin v odpadnih, površinskih in pitnih vodah ter v ribjih tkivih. Potrdili smo tudi vpliv odpadnih voda na vstopanje zdravilnih učinkovin v okolje, zato smo razvili postopek čiščenja, ki bi učinkovito razgradil ostanke mikroonesnažil. Uporabili smo elektrokemijsko oksidacijsko metodo na osnovi z borom dopirane diamantne elektrode, ki z nastajanjem reaktivnih hidroksilnih radikalov in situ omogoča mineralizacijo organskih snovi. Z razvito elektrolitsko celico smo na laboratorijskem nivoju v sintetični zmesi razgradili več kot 85 % začetne vrednosti za 7 od 10 preiskovanih spojin, za 3 spojine pa smo začetno vrednost razpolovili. Nekoliko slabšo učinkovitost, do 70 % razgradnje, smo dosegli v realnih vzorcih odpadnih voda, ki smo jih izpostavili razvitemu postopku čiščenja na laboratorijskem nivoju. Slabšo učinkovitost pripisujemo krajšemu času postopka, s katerim smo želeli simulirati realne pogoje, in tudi večji količini organskega materiala, za mineralizacijo katerega so se dodatno porabljali nastali hidroksilni radikali. V okviru evropskega projekta LIFE PharmDegrade smo razvito metodologijo prenesli v realno okolje na biološko čistilno napravo kot dodatni postopek čiščenja. Po nadaljnji optimizaciji (večje število vzporedno vezanih celic, večkratni prehod, višja gostota toka in dodatno čiščenje s filtri) smo povečali razgradnjo izbranih zdravilnih učinkovin med procesom čiščenja za povprečno 53 % oziroma smo s celotnim postopkom čiščenja v biološki čistilni napravi in z naprednim oksidacijskem postopkom uspešno razgradili več kot 86 % začetne vrednosti posameznega analita. Rezultati doktorske disertacije so osvetlili problematiko ostankov zdravilnih učinkovin in metabolitov v okolju. Kot novost na področju okoljske analitike smo razvili instrumentalno metodo za sočasno analizo 104 analitov, ki bo znatno olajšala ugotavljanje prisotnosti številnih ostankov zdravilnih učinkovin in metabolitov v okoljskih vzorcih. Z razvito metodologijo smo celovito ovrednotili njihovo prisotnost v odpadnih, površinskih in pitnih vodah, s čimer smo kot prvi predstavili podatke za področje Slovenije in zapolnili vrzel s tega dela Evrope. Na osnovi koncentracij v okoljskih vodah, s poudarkom na pitnih vodah, in ribjem tkivu lahko opredelimo obremenitev okolja in človeka. Glede na izmerjene vrednosti lahko sklepamo o minimalnem tveganju za človeka ter nekoliko večjem za nižje organizme, ki so vse življenje izpostavljeni izpustom iz čistilnih naprav. Pri tem moramo dodati, da se bo s povečevanjem uporabe zdravil tveganje nenehno stopnjevalo. Doktorsko nalogo smo zaključili z razvojem naprednega postopka čiščenja odpadnih voda. Razviti postopek za razgradnjo zdravilnih učinkovin iz okolja predlagamo kot enega od možnih dodatnih postopkov čiščenja na obstoječih čistilnih napravah. Z njim bi znatno zmanjšali izpuste zdravilnih učinkovin v okolje in posledično tudi obremenitev okolja. Predstavljeni podatki so lahko osnova za postopno vpeljavo rutinskega spremljanja izpustov zdravilnih učinkovin v okolje, prav tako pa lahko služijo kot predlog za spremljanje tarčnih snovi oziroma kot dopolnitev Nadzornega seznama (Izvedbeni sklep Komisije (EU) 2015/495 o določitvi nadzornega seznama snovi za spremljanje na ravni Unije na področju vodne politike v skladu z Direktivo 2008/105/ES Evropskega parlamenta in Sveta Evrope). Pharmaceuticals have been recently classified as emerging pollutants. They enter the environment mainly because of usage in human or veterinary medicine. Wastewater treatment plants are known to be the major point source of releasing considerable amounts of pharmaceuticals into the aquatic environment. Furthermore, they continuously seep into surface water or groundwater, which could be a source of drinking water. The concentrations measured in the environment are relatively low a few μg/L in wastewaters, where the highest concentrations are generally found, while the concentrations measured in surface or drinking waters are notably lower in the range of ng/L or pg/L. Despite these low concentrations of each individual pharmaceutical, the mixture of all present multiple substances may pose a great risk to non-target organisms or even human health. Various studies have already shown the bioaccumulation of pharmaceutical compounds and their negative effects such as morphological, cytological and behavioural malfunctions. Pharmaceutical occurrences in the environment were first published in 1970. However, in recent years, there has been an increase of similar papers, most probably owing to the improvement of analytical methods which enable the analysis of large numbers of substances in extremely low concentrations. Most often, an environmental analysis consist of a combination of appropriate sample preparation and a highly sensitive analytical method. According to the currently available scientific literature, the liquid chromatography coupled with tandem mass spectrometry in combination with a sample pre-concentration step seems to be the method of choice. In this doctoral thesis, we researched the sample preparation and the instrumental analytical methods. Furthermore, we developed a liquid chromatography method coupled with tandem mass spectrometry for the determination of pharmaceutical residues in wastewaters, surface waters, drinking waters, and in fish tissue. Regarding the extraction from wastewaters, two innovative approaches were developed. Firstly, an extraction based on solid phase extraction, namely the semi-automated SPE-DEX method. The second method was the stir-bar sorptive extraction. Both methods were validated however, the first one was better suited for monitoring a large number of active substances (> 100). Afterwards, a comparison between the semi-automated and manual solid phase extraction confirmed the similarity of the validation parameters. Moreover, the semi-automated system has several advantages: automaticity, a high rate of sample processing, simultaneous filtration and extraction, better repeatability, and a lower probability of extraction disc clogging. Therefore, the semi-automated SPE-DEX method was chosen for the analysis of wastewaters from 19 wastewater treatment plants. A total of 46 wastewater samples were collected 33 effluent wastewaters and 13 influent wastewaters in the period from May to August 2016. The presence of 104 pharmaceutical residues was tested using liquid chromatography coupled with tandem mass spectrometry with a single run of 8 minutes per sample. Regarding the influent samples, a total of 90 analytes out of 104 were present in at least one sample at average concentrations between 1 ng/L and 116 μg/L. In the effluent samples, we detected a total of 98 analytes out of 104 in at least one sample. The average concentrations were between 1 ng/L and 11 μg/L. In the influent wastewater samples, salicylic acid had the highest average concentration (116 μg/L) and was present in 92% of the samples. High concentrations were also measured for paracetamol (18 μg/L), metformin (17 μg/L), and caffeine (16 μg/L). Altogether, 12 analytes with a frequency of over 85% were present in concentrations above 1 μg/L, with the exception of lacosamide (7 μg/L), which was present only in one single sample. 31 analytes were detected in all samples. Regarding the highest concentrations found in effluent samples, the following analytes were present: tramadol (11 μg/L), hydrochlorothiazide (5 μg/L), metformin (4 μg/L) and salicylic acid (4 μg/L). Altogether 10 analytes with a frequency of over 60% were present in concentrations above 1 μg/L, with the exception of fluoxetine (1 μg/L), which was present in three samples and salicylic acid in one third of the samples. 14 analytes were detected in all samples. The analysis was followed by the development of an extraction method for surface waters. A manual solid phase extraction was used because it enables a higher concentration step. Based on the previous analysis, the most frequent effluent wastewater concentrations of pharmaceuticals were used as the basis for pre-selection of target compounds in surface waters. Altogether 44 analytes were included. The optimised solid phase extraction was evaluated and it showed the suitable validation parameters. Finally, six samples from different Slovene regions (five river samples and one lake sample) were analysed. 42 of all target pharmaceuticals (n = 43, one analyte was excluded due to the presence of a high background signal) were detected in at least two samples 29 of them were detected in all samples. The concentration range of the measured pharmaceuticals was between 0.08 and 47 ng/L, the highest measured concentration was for valsartan (average 11 ng/L, median 4 ng/L). Furthermore, high concentrations were measured for tramadol, which had an average concentration of 5 ng/L (median 1.2 ng/L), and sulfamethoxazole with an average concentration of 6 ng/L (median 6.3 ng/L). The developed extraction method was adjusted for the extraction from drinking water samples. The sample volume was higher, thus increasing the concentration factor. The optimised method was evaluated and was shown to provide suitable validation parameters, especially the higher sensitivity. The method was applied to 18 drinking water samples. Among the 54 investigated substances, we confirmed the presence of 13 pharmaceuticals in at least one sample. Azithromycin was present in all samples. In general, the concentrations were very low, typically below 1 ng/L the highest concentration was determined for azithromycin (26 ng/L) and caffeine (14 ng/L). The obtained data was used to speculate the amount of the analysed pharmaceuticals that humans take in with drinking water throughout their lifespan. We postulated the life span of 81 years and the consumption of 2 L of water daily. The sum of all monitored contaminants based on their highest measured values would be 3 mg in 81 years. Therefore it can be concluded that the risks the monitored pharmaceuticals present to human health are negligible. Regarding all three types of tested samples, it can be observed that all the pharmaceuticals present in drinking waters were also detected in surface and wastewaters. From the measured concentrations, the following conclusion can be drawn: The probability of pharmaceutical occurrence in surface and drinking water is greater when a very high concentration was measured in wastewater effluents, and/or when the individual analyte in the wastewaters was detected very frequently. In the last part of the sample preparation development, we optimized a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction protocol for extraction of drug residues from fish tissue. The optimised method was validated for 56 analytes. The method was used for monitoring target analytes in fish tissue. 13 samples of muscle, skin and gill tissues were analysed. The study showed the detection of 75% of all target analytes in at least one sample. Metoprolol was detected in all samples (< LOQ–0.7 ng/g). Furthermore, caffeine (< LOQ–3.2 ng/g), its metabolite 1,7-dimethylxanthine (< LOQ) and valsartan (0.1–0.4 ng/g) were detected in more than half of the samples. The highest average concentrations were measured for ciprofloxacin (19.2 ng/g), enalapril (16.2 ng/g) and haloperidol (14.0 ng/g) in the skin and gills. Interestingly, the concentrations measured in the muscles were lower or even below the limits of quantification. Regarding these data, human exposure via the ingestion of fish muscles is most probably low or negligible. The research of this doctoral thesis confirmed the presence of pharmaceuticals in wastewaters, surface waters, drinking waters, and fish tissue. Furthermore, the impact of wastewater on the continuous release of pharmaceuticals into the environment was confirmed. Therefore, we developed a treatment process that would effectively degrade pharmaceuticals in wastewaters. Firstly, the electrochemical degradation efficiency of the investigated compounds by boron-doped diamond electrode material was tested under laboratory conditions. The material in situ produced free radicals which effectively reacted with organic matter and provoked degradation. The treatment in an electrolytic cell with a boron-doped diamond electrode degraded more than 85% of the initial concentration for 7 out of 10 target compounds however, slightly lower degradation efficiency in real wastewaters was observed. The latter fact is tightly correlated with a shorter treatment time in order to demonstrate the sewage treatment process under real conditions and with higher amount of organic material, which also react with free radicals. Furthermore, the developed electrolytic cell was afterwards used as an additional treatment step in a hospital wastewater treatment plant within European project LIFE. Some extra optimisation was needed, such as a higher number of electrolytic cells, a higher current density, and the use of extra cleaning filter. Finally, the comparison between biological and electrochemical treatment revealed a superiority of the latter approach based on 53% overall improvement in terms of pharmaceuticals degradation. The entire treatment process of the biological treatment plant with an advanced oxidation process successfully degraded more than 86% of the initial concentration of each analyte. To summarize, the doctoral thesis highlighted the presence of pharmaceutical residue in the environment. To the best of our knowledge, this was the first demonstration of a simultaneous analysis of over 100 analytes using a QqQ mass analyser. The developed methods will greatly facilitate the detection of numerous residues of pharmaceuticals in environmental samples. Our observation of wastewaters, surface waters, drinking waters, and fish tissue represents the first research with comprehensive data for the Slovene region. Based on the found concentrations in environmental waters, with the emphasis on drinking water and fish tissue, we can estimate the environmental burden and risk for humans. We can conclude that the risk for humans is minimal and that it is somewhat higher for organisms that are exposed to wastewater discharges throughout their live cycles. Finally, the research resulted in the development of a highly efficient process for the degradation of pharmaceuticals. We can say that this new technology could be a viable and feasible option for upgrading existing wastewater treatment plants in order to achieve a significantly greater cleaning efficiency, and for lessening the ecological burden. The presented data can be the basis for further introduction of routine monitoring and might represent the preposition for an extension to the Watch List from Water Framework Directive (established in Directive 2008/105/EC of the European Parliament and of the Council of Europe).