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Active pharmaceutical ingredients (API) represent a group of the most prominent categories of emerging environmental contaminants. However, a vast majority of the currently published studies are focused on their occurrence and fate in abiotic environmental matrices like water and sediments while the data on API concentrations in aquatic organisms are still comparatively rare. Consequently, the existing knowledge on API bioaccumulation and metabolic transformations in the aquatic organisms is still inadequate. The key prerequisite to study bioaccumulation behavior of pharmaceuticals in the aquatic environment is the availability of analytical methods suitable for the reliable determination of trace concentrations of API and their metabolites in complex biological matrices. In this work, a novel multiresidue LC- MS/MS method for quantitative measurement of trace levels of selected APIs and their metabolites in freshwater fish was developed. In order to develop a robust analytical protocol suitable for reliable determination of 44 analytes, characterized by rather wide-range of physicochemical characteristics, in difficult fish matrices, the sample preparation protocol was optimized in a number of model experiments, by examining different combinations of extraction and clean-up techniques. The method development was performed using homogenized muscle tissue of Squalius cephalus (Chub) spiked with target analytes at low ng/g levels. In order to enhance the analytical reliability in complex fish matrices, the method included extensive usage of isotopically labelled surrogate standards. Two different extraction techniques, ultrasonic extraction and pressurized solvent extraction, were compared using different solvent combinations, taking into account two key parameters: absolute recovery (including extract suppression) and reproducibility. At the optimized conditions for LC/MS analysis, a Synergy Polar HPLC column was selected for chromatographic separation, using water and methanol, both containing 0.1% formic acid (v/v), as eluting solvents. The selectivity of the method was assured by highly specific MRM detection, using the two most abundant precursor/product ion transitions for each analyte and surrogate. The developed analytical method was used to assess API concentrations in feral freshwater fish from the Sava River (Croatia).

Opioidni analgetici su psihoaktivne tvari koje nakon primjene dospijevaju u otpadne vode putem ljudskih izlučevina. U okviru ove disertacije razvijene su analitičke metode za multirezidualno određivanje tragova 27 terapijskih opioida i njihovih metabolita u otpadnim i površinskim vodama upotrebom vezanog sustava tekućinske kromatografije i spektrometrije masa. Razvijene metode primijenjene su za istraživanje njihove rasprostranjenosti i biogeokemijskog ponašanja u vodenom okolišu. Ukupna koncentracija opioida u otpadnim vodama Grada Zagreba premašivala je 1 000 ng L-1. Najzastupljeniji predstavnici bili su tramadol i metadon. Budući da se njihovo uklanjanje u uređajima za pročišćavanje otpadnih voda pokazalo neučinkovitim, istraženi su alternativni postupci, uključujući biološku razgradnju adaptiranim mješovitim mikrobnim kulturama i abiotičku razgradnju ozoniranjem. Ozoniranjem je uklonjeno do 90 % roditeljskih spojeva, a biološkom razgradnjom do 82 % uz proporcionalno smanjenje toksičnosti za alge. Međutim, i biotičkom i abiotičkom razgradnjom istraženih spojeva nastala je znatna količina stabilnih transformacijskih produkata što treba uzeti u obzir kod sveoubuhvatne procjene njihova utjecaja na okoliš. Opioid analgesics are a group of psychoactive substances which enter the aquatic environment primarily via human excretion. In this thesis, multiresidue analytical methods for the determination of trace levels of 27 therapeutic opioids in wastewater and surface water matrices were developed, using liquid chromatography-tandem mass spectrometry. These methods were applied to study the occurrence and biogeochemical behaviour of the selected opioids in the aquatic environment. The total concentration of investigated opioids in wastewater of the city of Zagreb exceeded 1 000 ng L-1, with tramadol and methadone being the most prominent ones. Due to their rather inefficient elimination in wastewater treatment plants, two alternative procedures, including biological degradation with enriched mixed microbal cultures and ozonation were investigated. An improved removal of up to 90% and 82% was achieved in the ozonation and biodegradation experiments, respectively, with a proportional decrease of toxicity to algae. However, degradation of parent compounds was associated with the formation of stabile transformation products, which should be taken into account when assessing the overall impact on the environment.

Opioidni analgetici su važna kategorija farmaceutskih spojeva čija raširena upotreba može rezultirati visokim koncentracijama u komunalnim otpadnim vodama. Budući da je konvencionalno pročišćavanje otpadnih voda za uklanjanje opioidnih analgetika neučinkovito, postoji opravdana zabrinutost da bi dugotrajnim ispuštanjem moglo doći do njihovog nagomilavanja u vodenom okolišu sa štetnim posljedicama za akvatičke organizme.1 Kako bi se ovaj problem ublažio, često je potrebno otpadne vode pročistiti dodatnim postupcima, pri čemu napredne oksidacijske metode zauzimaju istaknuto mjesto.2 Cilj ovog rada bio je ispitati uspješnost uklanjanja dvaju istaknutih predstavnika opioidnih analgetika, tramadola i metadona, ozoniranjem. Eksperimenti ozoniranja provedeni su s tri različite matrice, uključujući čistu vodu, fosfatni pufer te sekundarni efluent porijeklom s Centralnog uređaja za pročišćavanje otpadnih voda grada Zagreba. Pri tom je sustavno istražena brzina uklanjanja opioida kao funkcija koncentracije ozona, pH vrijednosti te tipa matrice. Praćenje koncentracija tramadola i metadona, kao i identifikacija njihovih transformacijskih produkata nastalih tijekom eksperimenata, provedeno je tekućinskom kromatografijom visoke djelotvornosti spregnutom sa spektrometrijom masa visoke rezolucije. Eksperimenti su pokazali da se postupkom ozoniranja pri koncentraciji ozona od 0, 05 do 0, 5 mg/L oba opioida u potpunosti uklanjaju u manje od 5 minuta, pod uvjetom da je pH medija za ozoniranje viši od 7. Uklanjanje odabranih opioida znatno je usporeno kod kiselih uvjeta (pH=5), što ukazuje na važnost deprotoniranja amino grupe za učinkovitu reakciju s ozonom. Uklanjanje promatranih spojeva u sekundarnom efluentu odvijalo se dvostruko sporije nego u matricama koje ne sadrže organsku tvar, što se može pripisati smanjenju koncentracije raspoloživog otopljenog ozona zbog interakcije s matricom sekundarnog efluenta. U vremenskom intervalu od 10 minuta koncentracija tramadola u sekundarnom efluentu smanjena je za 91, 1%, a za metadon 99, 1%. Uklanjanje roditeljskih spojeva bilo je povezano sa stvaranjem transformacijskih produkata. Na temelju točne mase i MS/MS eksperimenata utvrđeno je da je najzastupljeniji transformacijski produkt metadona bio EDDP, a tramadola tramadol N-oksid.

The aim of the present study was to investigate the transformation of three prominent representatives of macrolide antibiotics (azithromycin - AZI, clarithromycin - CLA and erythromycin - ERY) in model biodegradation and ozonation experiments. The study included determination of the dissipation kinetics of the parent compounds, identification of transformation products and ecotoxicological evaluation of transformation processes using two different end-points. The biodegradation efficiency was studied using the sludge culture enriched in the presence of AZI (10 mg/L) over a period of 4 months while the ozonation experiments were performed in different matrices by applying selected pH conditions and ozone concentrations. The dissipation kinetics of parent compounds as well as the formation of transformation products (TPs) were followed by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry. Antibiotic activity test was based on the inhibition of bacterial growth (Bacillus subtilis), while toxicity test was performed with the freshwater green algae Desmodesmus subspicatus. At the applied experimental conditions, both biodegradation and ozonation experiments resulted in nearly full elimination of the tested parent compounds. The biotic and abiotic removal of all parent compounds was associated with the formation of different TPs, some of which were rather abundant and persistent to further degradation. The highest number of detected TPs was associated with the elimination of AZI, while the number of CLA and ERY TPs was comparatively much lower either under biotic and abiotic conditions. The environmental relevance of the identified biotransformation products, some of which included previously unknown linearized TPs, was proven by screening the municipal wastewater extracts for their presence. The effect-driven evaluation of the studied transformation processes, based on toxicity to algae and residual antibiotic activity, indicated a significant reduction of harmful effects, however formation of numerous stable transformation products, warrants further ecotoxicological assessment.

The aim of this work was to examine the removal of a difficult-to-degrade opioid analgesic methadone (MTHD) in biodegradation and ozonation studies. The biodegradation study was performed by activated sludge culture adapted to high concentration of methadone (10 mg/L) and included determination of elimination kinetics of the parent compound, taxonomic characterization of microbial culture based on the 16S rRNA gene amplicon sequencing and identification of biotransformation products. The ozonation experiments were performed in three different matrices, including pure water, phosphate buffer and secondary effluent from the Central wastewater treatment plant of the city of Zagreb. The MTHD removal rate was systematically studied as a function of ozone concentration, pH and matrix. The determination of the remaining MTHD concentrations as well as the identification of transformation products formed during the biodegradation and ozonation experiments was performed by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry. Depending on the experimental conditions, the elimination efficiency of methadone (10 mg/L) in biodegradation experiments varied from 9% to 93% with the corresponding half-lives (t1/2) from 11.4 days and 1.5 days. A significantly faster elimination (t1/2 from 1.5 days to 4.1 days) was achieved at cometabolic conditions, using glucose-containing media, as compared to the experiments with MTHD as a single organic carbon source (t1/2 = 11.4 days). Moreover, higher biotransformation rate was associated with the additional supplementation of ammonia, revealing a possible impact of nitrogen availability on the transformation rate. The elimination of the parent compound was associated with the formation of 3 different TPs, characterized by m/z 278, 264 and 324. Two of these TPs were found to be identical to the main human metabolites of MTHD, 2-Ethylidene-1, 5- dimethyl-3, 3-diphenylpyrrolidine (EDDP) and 2- ethyl-5-methyl-3, 3- diphenyl-1- pyrroline (EMDP). EDDP represented over 90% of the total TP concentration at the end of experiment. Ozonation at an ozone dosage of 0.05- 0.5 mg/L completely removed MTHD in less than 5 min in pure water and phosphate buffer solution, providing that pH of the ozonation medium was higher than 7. The elimination of MTHD was significantly slowed down at acidic conditions, which indicated the importance of the amino group deprotonation for an efficient reaction with ozone. MTHD elimination in secondary effluent was much slower (t1/2 = 1.72 min) than in organic- free water matrices (t1/2 = 0.40 - 0.8 min). The removal of MTHD in ozonation experiment was associated with a formation of three transformation products, characterized by m/z 278, m/z 294 and m/z 326, which were identified as EDDP, EDDP N-oxide and MTHD N-oxide, respectively. Both degradation approaches are promising for the development of viable technologies aimed at the removal of methadone from heavily loaded wastewater, however the final assessment of the degradation outcome by both methods warrants a more detailed ecotoxicological evaluation of the TPs formed.

The major sources of opioid compounds to the aquatic environment are human excretion and dumping of unused medications to the sewer. The existing analytical methods include rather limited number of opioid analgesics (OA) representatives and/or their major human metabolites while the methods that comprise a broad spectrum of OAs are still missing. Therefore, the aim of this work was to develop a multiresidue LC-MS/MS method for the simultaneous determination of trace levels of 28 OAs in municipal wastewaters. The method development included: A) optimization of chromatographic and mass spectrometric conditions for the separation and detection of target analytes, respectively ; B) optimization of extraction procedures for the dissolved and particulate OAs ; C) full validation of analytical protocols for untreated wastewater and secondary effluents and D) the method evaluation for real wastewater samples. In order to optimize chromatographic separation different columns (Synergy Polar, Gemini C18 and ACE C18-PFP) and eluents were evaluated. At the final chromatographic conditions, a complete chromatographic separation of all analytes was achieved by using Synergy Polar column with methanol and water, both containing 0, 1% HAc, as eluting solvents. The enrichment of opioid analgesics from aqueous samples and suspended solids were performed employing solid-phase extraction (SPE) based on mixed cation-exchange (Oasis MCX) cartridges and pressurised liquid extraction (PLE), respectively. During extraction optimisation different SPE cartridges were investigated (Oasis MCX and Oasis HLB) in order to optimise the overall recovery and to reduce the matrix effects. The optimal extraction efficiency of dissolved OAs was obtained by using Oasis MCX cartridges (recoveries: 43-81%). For the extraction of particulate phase ultrasonic extraction and PLE as well as different solvents were tested. The best results were achieved by using PLE with 1% ammonia solution in methanol as a solvent. Recoveries were in range from 72-144% for almost all opioids with exception of 6-acetylmorphyne and glucuronides (15-46%). The method was applied for the assessment of OAs in municipal wastewater from Croatia. Most of the OAs were found in relatively low concentrations ranging from 1-50 ng/L. However, some analytes, such as tramadol, morphine, codeine and methadone showed significantly enhanced levels. The total concentration of tramadol and its metabolites exceeded 1 µg/L.

In this study, we applied the wastewater-based epidemiology methodology to investigate the impact of the major music festival and tourist season on the drug, alcohol and tobacco consumption in one of the main coastal tourist areas in Croatia, the city of Split, (about 200, 000 inhabitants). The study was performed in 2016 during three selected one-week periods, covering different situations: a large techno music festival (July), the main tourist season with no special events (August) and the control period outside the tourist season (November). The analyses included the biomarkers of 6 "classical" illicit drugs (cannabis, cocaine, heroin, MDMA, amphetamine and methamphetamine), 20 novel amphetamine-like psychoactive substances (NPS), 27 therapeutic opiods and their metabolites as well as the selected alcohol and nicotine metabolites. All chemical analyses were performed by LC-MS/MS, using the previously validated analytical protocols. Before the instrumental analyses, the drug biomarkers were enriched on Oasis MCX cartridges, while the metabolites of nicotine and alcohol were determined by direct injection of filtered aqueous samples into the instrument, with a previous enzymatic deconjugation in the case of nicotine metabolites. The results indicated a prevalent consumption of "classical" illicit drugs over the investigated NPS. The mostly consumed illicit drug was cannabis (up to 15.7 g/day/1000 inhabitants), but its consumption was not clearly related to any of the investigated seasonal patterns. A rather uniform temporal consumption was observed for nicotine as well. By contrast, both investigated summer periods were characterized by significantly enhanced consumption of different psychostimulant substances, including several illicit drugs and alcohol. The most dramatic change was observed for MDMA, with a 30-fold increase in its consumption during the music festival (average daily consumption of MDMA in July, August and November were 1.8, 0.12 and 0.06 g/day/1000 inhabitants, respectively). The corresponding increase of alcohol (10-18 L/day/1000 inhabitants) and cocaine consumption (0.8-1.6 g/day/1000 inhabitants) was much lower, indicating that MDMA usage strongly predominated among the festival participants.

Modifikacija površinskog sloja metala zbog povećanja njegove korozijske otpornosti primjenom ekološki i ekonomski prihvatljivih metoda, jedan je od izazova s kojima se danas susreću korozijski inženjeri. U tu svrhu, veliki potencijal ima modifikacija metalnih površina tankim, uređenim filmovima dugolančanih karboksilnih kiselina, tzv. samoorganizirajućim monoslojevima. Njihovo formiranje odvija se adsorpcijom kiseline na površinu oksidom prekrivenog metala, prilikom čega dolazi do kiselinsko-bazne reakcije između metalnog oksida i kiseline koju prati samoorganizacija alkilnih lanaca karboksilne kiseline. Na taj način nastaje vrlo tanki, kompaktni zaštitni film na površini metala, koji sprječava penetraciju molekula vode i agresivnih iona na metalnu površinu. Međutim, kako se karboksilne kiseline adsorbiraju na oksidni film, kod čelika, posebice ugljičnog čelika, javlja se potreba za poboljšanjem površinskog sloja oksida. Cilj ovog rada je formirati stabilnije zaštitne slojeve, modifikacijom površine na koju se adsorbiraju, odnosno različitim metodama potpomognuti nastajanje stabilnog i kompaktnog oksidnog sloja na čeliku. Stoga, sloj oksida na čeliku formiran je spontanom oksidacijom pri povišenoj temperaturi, potpomognutom oksidacijom u otopini cerijeva klorida heptahidrata pri sobnoj i povišenoj temperaturi te procesom stvaranja prevlake cerijevog oksida/hidroksida pri sobnoj temperaturi uz prisutstvo oksidansa. Na pojedini sloj oksida, adsorbirani su filmovi stearinske kiseline te je nakon postupka sušenja ispitana djelotvornost pojedine zaštite čelika. Ispitivanja zaštitnih svojstava provedena su u simuliranoj morskoj vodi (3%-tna otopina NaCl) elektrokemijskim metodama: metodom linearne polarizacije, Tafelove ekstrapolacije i elektrokemijske impedancijske spektroskopije. Provedena je i karakterizacija modificirane površina čelika određivanjem kontaktnog kuta vode, kako bi se ispitala hidrofobnost površine, te Fourier transformacijskom infracrvenom spektroskopijom (FTIR-ATR), kojom je utvrđen način vezanja stearinske kiseline na površinu metala. Zahvala: Ovi materijali temelje se na radu koji je financirala Nacionalna zaklada za znanost, visoko školstvo i tehnologijski razvoj Republike Hrvatske pod brojem 09.01/253.

Većina industrijskih postrojenja pozicionirana uz obalu koriste morsku vodu, koja je korozivan medij i agresivno djeluje na izložene površine brojnih metalnih konstrukcija. Kako je nužno da postrojenja rade sigurno i isplativo, vrlo se često za njihovu izvedbu koriste visokootporne legure poput nehrđajućeg čelika 1.4404. Međutim, zbog nepostojanosti zaštitnog pasivnog filma u kontaktu s kloridnim ionima dolazi do propadanja čeličnih konstrukcija te implatanata, stoga se nameće potreba za adekvatnom zaštitom koja bi omogućila njihovo sigurnije i dugotrajnije korištenje. Cilj ovih istraživanja je pronalaženje djelotvorne, a istovremeno i ekonomične metode zaštite čelika od korozije u kloridnom mediju primjenom spojeva koji nemaju negativan učinak na okoliš. Jedna od mogućnosti je primjena tankih uređenih nano filmova, odnosno samoorganizirajućih monoslojeva koji se formiraju adsorpcijom iz otopina dugolančanih organskih kiselina na površinu oksidom prekrivenog metala te sprječavaju prodiranje vode i agresivnih iona do površine metala. Velika prednost ove metode je mali utrošak kemikalija, što znači i jeftiniju zaštitu. Stearinska i oktadecil fosfonska kiselina izabrane su za istraživanja jer su netoksični spojevi te pokazuju sposobnost stvaranja samoorganizirajućih monoslojeva na površini metalnih oksida. Navedene kiseline karakterizira i završna metilna skupina koja modificiranu površinu čini hidrofobnom što doprinosi povećanju korozijske otpornosti materijala. Kako bi se postigla što bolja zaštita ispitan je utjecaj podloge, metalnog oksida, na adsorpciju organske kiseline. Oksidni sloj formiran je spontanom oksidacijom čelika pri različitim temperaturama kao i kemijskim postupkom pomoću cerijeva klorida heptahidrata koji predstavlja „zelenu“ alternativu do sada korištenom toksičnom Cr6+. Zaštitno djelovanje dobivenih tankih filmova utvrđeno je elektrokemijskim ispitivanjima, metodama Tafelove ekstrapolacije, linearne polarizacije te elektrokemijske impedancijske spektroskopije. Osim elektrokemijskih ispitivanja, uzorci su podvrgnuti analizama pretražnom elektronskom mikroskopijom, goniometrijom i Fourier transformacijskom infracrvenom spektroskopijom kako bi se dobile kvalitativne i kvantitativne informacije o dobivenom zaštitnom sloju. Analizom dobivenih rezultata može se zaključiti da je moguće značajno poboljšati korozijsku otpornost nehrđajućeg čelika u kloridnom mediju relativno jednostavnim, ekonomičnim i ekološki prihvatljivim postupkom koji uključuje kemijsku oksidaciju čelika te formiranje samoorganizirajućih monoslojeva dugolančanih organskih kiselina. Zahvala: Ovi materijali temelje se na radu koji je financirala Nacionalna zaklada za znanost, visoko školstvo i tehnologijski razvoj Republike Hrvatske pod brojem 09.01/253.