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Alkilfenoletoksilati (APEO) so najpogosteje uporabljene neionske površinsko aktivne snovi (PAS) v industriji. V industriji se pretežno uporabljajo v obliki raztopin, ki se po koncu uporabe odplaknejo v komunalne in industrijske odpadne vode, od koder se kasneje transportirajo do čistilnih naprav. Med postopkom čiščenja pride do biorazgradnje, kot rezultat tega procesa pa nastajajo kratkoverižni APEO in alkilfenoli (AP). AP so najpomembnejši razgradni produkti, za katere je znano, da delujejo kot endokrini motilci, in se v okolju akumulirajo. V vodnem okolju so velikokrat v različnih oblikah prisotni halogeni, predvsem klorove in bromove spojine, s katerimi lahko APEO in AP reagirajo in tvorijo halogenirane transformacijske produkte. Ker lahko ti produkti vstopajo v prehranjevalno verigo, je zelo pomembno, da poznamo njihovo usodo v okolju. V nalogi smo se osredotočili na dva AP, dva monoetoksilirana alkilfenola in dva dietoksilirana alkilfenola ter njihove halogenirane produkte, in sicer mono- in diklorirane ter mono- in dibromirane. Uporabili smo dva različna programa za preučevanje spojin in silico, in sicer Endocrine Disruptome in VegaNIC. Z njima smo napovedali njihov okoljski vpliv, akutno toksičnost in endokrino delovanje. S pomočjo literaturnih podatkov in dobljenih rezultatov smo spoznali, da ima pomemben vpliv na usodo teh spojin vrednost logP. Med drugim nas je v nalogi zanimala tudi verjetnost biološko razgradnje, odpornost na biodegradacijo ter faktor bioakumulacije (BCF). Pri napovedovanju obstojnosti smo ugotovili, da so neetoksilirani AP veliko obstojnejši kot APEO, ki se med procesi prečiščevanja in med zadrževanjem v vodnem okolju še vedno razgrajujejo do AP. Poleg tega pa smo opazili tudi, da se obstojnost povečuje tudi s halogeniranjem, še posebej z bromiranjem, kar pomeni, da bodo halogenirani transformacijski produkti po verjetnosti še bolj obstojni kot njihove izhodne spojine. Za najmanj obstojno spojino smo označili oktilfenoldietoksilat, ki ima najnižjo vrednost BCF, najbolj obstojen pa je NP z najvišjo vrednostjo BCF. Kot najbolj akutno toksična spojina se je izkazal NP, kot najmanj pa nonilfenoldietoksilat z najvišjo vrednostjo LC50. Pri primerjavi vrednosti LC50 izhodnih spojin z njihovimi halogeniranimi derivati smo odkrili, da halogeniranje poveča akutno toksičnost. Pri preučevanju endokrinega delovanja smo ugotovili, da imajo AP in APEO največjo verjetnost vezave na androgenski, mineralokortikoidni in tiroidni receptor (AR, MR, TRα in TRβ), pri čemer se verjetnost vezave s halogeniranjem bistveno ne spremeni. Na osnovi teh in ostalih dobljenih rezultatov smo ocenili, da je najvarnejša preučevana spojina NP2EO, najbolj toksična pa NP. Čeprav se AP in APEO v okolju pojavljajo v nizkih koncentracijah, lahko dokazano motijo delovanje človeškega endokrinega sistema. Uporabo so v primerjavi s preteklimi leti omejili, a so še vedno najpogosteje uporabljane neionske PAS v industriji. Alkylphenol ethoxylates (APEO) are the most commonly used non-ionic surfactants in the industry. They are mostly water-insoluble compounds, which are mainly used in form of solution, which, after use, are discharged into municipal and industrial waste water, from which they are subsequently transported to water-treatment plants. Biodegradation occurs during the purification process, resulting in short-chain APEOs and alkylphenols (APs). APs are the most important degradation products known to act as endocrine disruptors and which accumulate in the enviornment. In the aqueous enviornment, halogens are often present in various forms, in particular chlorine and bromine compounds, which can react APEOs and APs to form halogenated transformation products. Since products can enter the food chain, it is very important to know their fate in the enviornment. In the scope of this Master's thesis, we focused on two APs,their mono- and diethoxylated alkylphenols as well as their halogenated transformation products(mono- and dichlorinated and mono- and dibrominated). We used two different programs to study compounds in silico, Endocrine Disruptome and VegaNIC. They allow for the prediction of the environmental impact, acute toxicity and endocrine function of compounds. On the basis of literature survey and our findings, we have corroborated that logP has a significant effect on the fate of these compounds. Among other things, we were interested in the probability of biodegradation, resistance to biodegradation, and bioaccumulation factor (BCF). In predicting persistence, we found that non-ethoxylated APs are much more persistent than APEOs, which still transform to APs during purification processes and during retention in the aqueous environment. In addition, we also observed that the stability also increases with halogenation, especially via bromination, which means that the halogenated transformation products are likely to beeven more stable than their parent compounds. The least persistent compund was designated OP2EO, which has the lowest BCF value, and the most persistent is the NP with the highest BCF value. NP was the most acutely toxic compound, whereas the least toxic was NP2EO with the highest LC50 value. When comparing the LC50 values of the starting compounds with those of their halogenated derivates, we observed that halogenation increased the acute toxicity. When studying endocrine function, we predicted that APs and APEOs have the highest probabilities of binding to the androgen, mineralocorticoid and thyroid receptors (AR, MR, TRα and TRβ), with no significant change in the likelihood of halogen binding. On the basis of all obtained results, we concluded that the safest studied compound was NP2EO and the most toxic was NP. Although APs and APEOs occur in low concentrations in the environment, they have been shown to interfere with the functioning of the human endocrine system. Their usehas been restricted over the years, although they are still the most widely use non-ionic surfactants in the industry.

V doktorski nalogi smo proučevali vpliv nekaterih spojin okoljskih endokrinih motilcev na plodnost moških z namenom, da bi preverili hipotezo ali vplivajo endokrini motilci na kakovost semenčic. V študijo smo vključili 136 parov, ki so se zdravili zaradi neplodnosti. Za analitiko sledov spojin endokrinih motilcev v urinu smo razvili analizno metodo določevanja s plinsko kromatografijo in masno spektrometrijo. Uporabili smo 13C izotopsko označene standardne spojine. Po dodatku internih standardov smo za dekonjugacijo analitov urinu dodali encim β-glukuronidazo. Vzorec smo nato nakisali s HCl na pH 2. Sledila je ekstrakcija z diklorometanom. Čiščenje ekstraktov smo izvedli na SiO2 koloni ter tako odstranili bazične interference, ki jih predstavljajo aminokisline, sečnina, amini in druge bazične spojine. Očiščeni ekstrakt smo odparili do suhega in dodali derivatizacijski reagent N-metil-N-trimetilsilil trifluoroacetamid (MSTFA). Sililirani ekstrakt smo analizirali s plinsko kromatografijo in masno spektrometrijo. Uporabljena je bila tehnika snemanja izbranih masnih fragmentov (SIM). Sočasno smo določali di-alkil ftalate: di-metil ftalat (DMP), di-etil ftalat (DEP), di-butil ftalat (DBP), benzil-butil ftalat (BzBP) ter di-(2-etil-heksil) ftalat (DEHP), njihove metabolite mono-alkil ftalate: mono-etil ftalat (MEP), mono-izo-butil ftalat (MiBP), mono-n-butil ftalat (MnBP), mono-(2-etilheksil) ftalat (MEHP), mono-benzil ftalat (MBzP), mono-izo-nonil ftalat (MiNP), mono-n-oktil ftalat (MnOP), mono-(2-etil-5-oksoheksil)ftalat (MEOHP) ter mono-(2-etil-5-hidroksiheksil)ftalat (MEHHP). Določali smo alkilfenole: 4-terc-oktilfenol (4-tOP), 4-n-oktilfenol (4nOP) ter 4-n-nonilfenol (4nNP). Prav tako smo določali bisfenol A (BPA). Izvedli smo validacijo metode. Meja določanja je bila za bisfenol A 0,1 µg L-1, za mono-alkil ftalate in alkilfenole 1 µg L-1, in za di-alkil ftalate 5 µg L-1. Linearnost metode smo preverili v koncentracijskem območju od 1 do 200 µg L-1. Uporabljena metoda se je pri kvantitativnem določanju spojin endokrinih motilcev v vzorcih urinov izkazala za uspešno, saj je natančna, točna in selektivna. Omogoča sočasno določanje endokrinih spojin z nizko mejo zaznavnosti v območju µg L-1. Z uporabo tehnike snemanja celotnega masnega spektra smo določili še 60 drugih spojin v urinu (maščobne kisline, steroide, mono-aromatske spojine in druge spojine). Kemometrijsko karakterizacijo smo izvedli s pomočjo podatkov pridobljenih z analizami 136 vzorcev urinov moških, ki imajo težave s plodnostjo. Namen raziskave je bil ugotoviti ali obstajajo pomembne razlike med vzorci, ki smo jih uvrstili v dve skupini, glede na normalno ter zmanjšano kakovost semenčic. Skupini se razlikujeta po vrednostih koncentracije in gibljivosti semenčic. Cilj kemometrijske karakterizacije je bil poiskati korelacije med vsebnostjo spojin endokrinih motilcev v urinu, kot so ftalatni estri in njihovi metaboliti, oktil in nonil alkilfenoli in bisfenol A ter kliničnimi parametri kakovosti semenčic kot sta koncentracija in gibljivost semenčic. Dodatno smo iskali korelacije med kakovostjo semenčic in vsebnostjo hormonov: FSH (folikel-stimulativni hormon), LH (luteinizarajoči hormon), estrogen, testosteron, SHBG (spolne hormone vezoči globulin), inhibinB. Izvedli smo korelacijsko analizo, analizo variance (ANOVA), Mann Whitney test, analizo glavnih osi (PCA), hierarhično razvrščanje (CA), linearno diskriminatno analizo (LDA), metodo gradnje regresijskih dreves (CART) in umetne nevronske mreže (ANN). Študija je pokazala, da obstajajo pomembne razlike med obema skupinama moških, ki imajo težave s plodnostjo, predvsem glede na vsebnost di-(2-etilheksil) ftalata (DEHP), mono-(2-etilheksil) ftalata (MEHP) in obeh sekundarnih metabolitov mono-(2-etil-5-oksoheksil) ftalata (MEOHP) ter mono-(2-etil-5-hidroksiheksil) ftalata (MEHHP). V vzorcih urina moških z zmanjšano kakovostjo semenčic so bile izmerjene vsebnosti endokrinih motilcev v povprečju dvakrat višje od vsebnosti v vzorcih urina moških This doctoral dissertation investigated the influence of endocrine compounds’ exposure on male fertility and the hypothesis, if endocrine disruptors affect the quality of sperm was tested. This study included 136 couples being treated for infertility. An analytical method for trace analysis of endocrine disruptors in the urine using gas chromatography and mass spectrometry had been developed. The urine was spiked with 13C isotopically labelled internal standards, β-glucoronidase was added for deconjugation of the analytes. The sample solution was then acidified to pH 2 with HCl. Extraction was performed using dichloromethane. The extracts were purified on the SiO2 column and interference compounds were removed such as amino acids, urea, amines and other compounds. Dichloromethane extracts were then evaporated to dryness and N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was added as a derivatisation reagent. An aliquot of the sililated extract was injected into a GC-MS system and a selected ion monitoring (SIM) technique was used. Di-alkyl phthalates: di-methyl phthalate (DMP), di-ethyl-phthalate (DEP), di-butyl phthalate (DBP), benzyl-butyl phthalate (BzBP) and di-(2-ethyl-hexyl) phthalate (DEHP), their metabolites, mono-alkyl phthalates: mono-ethyl phthalate (MEP), mono-iso-butyl phthalate (MiBP), mono-n-butyl phthalate (MnBP), mono-(2-ethylhexyl) phthalate (MEHP), mono-benzyl phthalate (MBzP), mono-iso-nonyl phthalate (MiNP), mono-n-octyl phthalate (MnOP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), alkylphenols: 4-tert-octylphenol (4-tOP), 4-n-octylphenol (4nOP) and 4-n-nonylphenol (4nNP) and Bisphenol A (BPA) were simultaneously determined. Validation of the method was performed. Limit of quantification (LOQ) was for bisphenol A 0.1 µg L-1, for mono-alkyl phthalates and alkylphenols 1 µg L-1, and for di-alkyl phthalates 5 µg L-1. The linearity of the method was tested in the concentration range from 1 to 200 µg L-1. The method was proved to be successful for the quantitative determination of endocrine disrupting compounds in urine samples. It is precise, accurate and selective, and can be used for simultaneous determination of phthalates, their metabolites, alkylphenols, and bisphenol A in trace amounts in human urine. This method allows the determination of endocrine compounds with low detection limit (LOD) in µg L-1. Sixty other compounds in the urine (fatty acids, steroids, mono-aromatic compounds and other compounds) have also been also determined using the full mass spectra (SCAN) technique. Chemometric characterisation was performed using data obtained from all 136 urine samples from men with fertility problems. The purpose of the study was to investigate whether there were any significant differences between samples, which were classified into two groups according to normal and reduced sperm quality. Groups differ in levels of sperm concentration and motility. The aim of the chemometric characterisation was to find correlations between the content of endocrine disrupting compounds in the urine samples, such as phthalate esters and their metabolites, octyl and nonyl alkylphenols and bisphenol A and clinical parameters of semen quality such as sperm concentration and sperm motility. Additionally, correlations between sperm quality and content of hormones: FSH (follicle-stimulating hormone), LH (luteinising hormone), estrogen, testosterone, SHBG (sex hormone binding globulin), and inhibin B were searched. Correlation analysis, analysis of variance (ANOVA), Mann Whitney test, principal component analysis (PCA), cluster analysis (CA), linear discriminant analysis (LDA), classification and regression tree (CART), and artificial neural networks (ANN) were used for chemometric characterization. The study showed that there are significant differences between the two groups of men with fertility problems, particularly with regard to the content of di-(2-ethylhexyl) phthalate (DEHP), mono-(2-ethylhexyl) phthalate (MEHP) and both secondary metaboli