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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.

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.