We live in a time were plastic and plastic products are all around us, taking part in our everyday life. Several chemical additives can be present in plastic products, such as plastic bottles, and can have a big impact on development, as well as on the endocrine system in adults by the binding to, and disturbance of, the estrogen receptor (ER) in cells. This study has focused on a number of different plastic bottles made from different types of plastic and with variations in size and scope of use. The aim of the study was to see if any estrogenic substances leached from the bottles into water. Non-ionic water was added to the plastic bottles, and the bottles were kept at 70°C for 72 hours. The estrogenic activity in the water was determined with the U2OS-luc assay and expressed as bioassay-derived estradiol equivalents (Bio-EEQ). A difference in Bio-EEQ could be detected between the bottles and the tree plastic types used in the study. The polypropylene (PP) bottle gave the highest Bio-EEQ of 0.5 pg/ml, whereas High Density Polyethylene (HDPE) gave 0.3 pg/ml and Polyethylene Terephthalate (PET) 0.04 pg/ml. These results indicate that there is a small leakage of estrogenic substances from the plastic bottles. Further study is needed to determine whether or not the estrogenic activity in the water could have any significant biological effect in humans. / Vi lever i en tid där plast och plastprodukter finns överallt runt omkring oss och har en stor del i vår vardag. Många kemiska ämnen kan återfinnas i plastprodukter, såsom plastflaskor, och kan ha en stor inverkan inte bara på den växande kroppen hos barn utan även ge endokrina störningar hos vuxna människor. Detta kan till exempel ske genom att ämnen kan binda till och påverka östrogenreceptorn (ER) i cellerna. Den här studien fokuserar på plastflaskor gjorda av olika typer av plast och med varierande storlek och användningsområde. Syftet med studien var att se huruvida några östrogena substanser kunde lakas ut i vatten som förvarades i flaskorna. Flaskorna fylldes med avjoniserat vatten och förvarades i 70°C i 72 timmar. Östrogenaktiviteten i vattnet mättes sedan i en cellbaserad testmetod, kallad U2OS-luc assay, där den samlade biologiska effekten av alla östrogena ämnen kan mätas. Effekten uttrycks som bioassay-derived estradiol equivalents (Bio-EEQ). Vatten från polypropenflaskan (PP) gav det högsta Bio-EEQ på 0,5 pg/ml medan högdensitetspolyeten (HDPE) gav 0,3 pg/ml och polyetylentereftalat (PET) ett Bio-EEQ på 0,04 pg/ml. Dessa resultat indikerar att plastflaskorna läcker ut små mängder östrogenlika ämnen. Vidare studier behöver göras för att avgöra huruvida detta läckage utgör någon risk för människor.

Xenoestrogens are compounds, foreign from the body, that can enter cells and interact with the estrogen receptors (ER) to produce an estrogenic response. Many additives used in plastics are compounds with estrogenic activity. Some of these additives are known to slowly leach from the plastics. When using plastic containers as lunchboxes for reheating or food storage, these additives can leach from the plastics and end up in the food. In this project, food simulates were cooked in six different thermoplastic containers, made of polypropylene, in an oven at 100 °C for 15 minutes. Three of the thermoplastic containers were lunchboxes marketed to be able to withstand cooking in a microwave. The other three were provisional lunchboxes made from various food storing containers originally made for refrigeration purposes. The estrogenic activity in the different samples was measured using an ER-CALUX in vitro assay. The results were measured in 17β-estradiol equivalent (Bio-EEQ) values in pg/ml. The purpose of this project was to investigate whether or not these plastic containers leach xenoestrogens that can be measured with an ER-CALUX assay, and compare the results with the results from other existing toxicological studies, and also to see if there is a difference in Bio-EEQ levels between the plastic containers made for microwave usage and those made for refrigerated purposes. The results from this project indicate that most of these plastic containers do leach estrogenic compounds that can be detected in the ER-CALUX, even the ones made for microwave usage. Fortunately, compared to other toxicological studies, the Bio-EEQ levels in these food samples cooked in plastic containers are low. However the potential adverse effects in prenatally exposed children cannot be ignored as other studies have shown that very low levels of xenoestrogens are enough to potentially cause a disturbance in the reproductive development and fertility.

The aim of this diploma thesis was to detect (anti-)progestagenic activity in wastewater samples from the influent and effluent of six wastewater treatment plants (WWTPs) located in South Bohemia. Subsequently, the efficiency of the treatment process of this WWTPs was assessed from this point of view. The wastewater from WWTPs was transported to the laboratory and extracted oby solid phase extraction. The eluates were washed, evaporated and dissolved in DMSO. Detection of (anti-)progestagenic activity was performed by using the PR-CALUX in vitro bioassay. Transgenic cells were seeded on well plates and were exposed to ORG2058, reference substance for progestagenic activity, or RU-486, reference substance for antiprogestagenic activity, as well as a number of diluted wastewater extracts. After that, luminiscence of the cells was measured and it was was expressed in relative light units which were a measure of (anti-)progestagenic activity.(Anti-)progestagenic activity was reported in equivalent concentrations of the reference substance. Progestagenic activity in WWTPs influent ranged from below LOQ up to 1.8 ng/l ORG2058 eq. In effluent this activity ranged from below LOQ up to 0.5 ng/l ORG2058 eq. The elimination rate of progestagenic activity ranged from -25% to 100%. Antiprogestagenic activity in WWTPs inffluent was below the LOQ up to 1 ng/l RU-486 eq. In effluent this activity was below LOQ up to 9.7 ng/l RU-486 eq. The elimination rate of antiprogestagenic activity ranged from -50% to 100%.(Anti-)progestagenic activity in waste water has been demonstrated by using the PR-CALUX in vitro bioassay. Negative removal efficiency of some WWTPs was also documented. This is probably caused by biotransformation of some substances which don´t have (anti-)progestagenic activity to substances which have this activity or it is caused by deconjugation of metabolits of compounds with (anti-)progestagenic activity. Antiprogestagenic activity may be more hazardous for organisms living in the aquatic environment than progestagenic activity because of concentrations in WWTPs effluent. However this activity should not be underestimated.

Large numbers of samples often need to be prepared and analysed in surveys of organic pollutants in the environment, but while the methods commonly used in such surveys can provide abundant detail they are generally costly, time-consuming and require large amounts of resources, so there is a need for simpler techniques. The work underlying this thesis assessed the potential utility of more convenient sample preparation and bioanalytical techniques for rapidly screening various environmental matrices that could be useful complements to higher resolution methods. Initially, the utility of a simplified extraction technique followed by an enzyme-linked immunosorbent assay (ELISA) for detecting polycyclic aromatic hydrocarbons (PAHs) in authentic (i.e. unspiked) contaminated soils was explored. The results showed that there are relationships between the structure and cross-reactivity among compounds that often co-occur with target PAHs. However, their potential contribution to deviations between estimates of total PAH contents of soils obtained using ELISA and gas chromatography-mass spectrometry (GC-MS) based reference methods were limited. Instead, the cross-reactivity of target PAHs and the failure to extract all of the PAHs prior to the ELISA determinations were the main reasons for these deviations. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were detected in food and feed matrices, as well as in authentic contaminated soils using different bioanalytical techniques – ELISA and two cell-based bioassays: CAFLUX and CALUX (chemically activated fluorescent/luciferase gene expression) assays. In addition, enhanced sample preparation techniques based on accelerated solvent extraction (ASE) were developed. ASE with integrated carbon fractionation (ASE-C) in combination with CAFLUX produced estimates of PCDD and PCDF contents in fish oil and fish meal that agreed well with results obtained using reference methods. Furthermore, results from ELISA and GC-high resolution MS analyses of extracts of PCDD- and PCDF-contaminated soil samples obtained using an adjusted ASE-C technique were strongly correlated. Finally, the thesis reports the first experiments in which the results of CAFLUX, CALUX, and ELISA determinations of PCDDs and PCDFs in extracts of authentic contaminated soil samples were evaluated and compared to those obtained using a reference method. All of the bioanalytical techniques were found to be sufficiently sensitive, selective, and accurate for use in screening in compliance with soil quality assessment criteria. Overall, the improved sample preparation and bioanalytical techniques examined proved to be useful potential complements to conventional methods, enhancing the analytical framework for PAHs, PCDDs, and PCDFs. However, further validation has to be undertaken before they are applied on a large-scale.

The fate and behaviour of estrogens in the environment are of concern due to the compounds’ endocrine disruption potential. Estrogens, namely 17β-estradiol (E2), estrone (E1), and estrogen sulphates, i.e. 17β-estradiol-3-sulphate (E2-3S) and estrone-3-sulphate (E1-3S) excreted by livestock constitute a potential source for estrogen contamination in the environment. A method was developed to separate and quantify the hormones by high-performance-liquid-chromatography (HPLC) and ultraviolet detection (UV). A combination of dichloromethane (DCM) and dicyclohexylamine hydrochloride (DCH·HCl) gave recoveries from 97.3 to 107% for E1-3S extraction from aqueous solutions. The recoveries from soil samples ranged from 80.9 to 95.2% (E2-3S), and from 86.3 to 91.7% (E1-3S), respectively. Results of batch sorption studies showed that Freundlich isotherms were nonlinear (N ≠ 1) with Kf values ranging from 34.2 to 57.2, and from 3.42 to 4.18 mg¹-N LN kg⁻¹ for E1, and E1-3S, respectively, indicating the sorption affinity of E1-3S was about an order of magnitude lower than that of E1. The hydrophilic sulphate group of E1-3S possibly shielded the compound from hydrophobic interactions with the soil organic matter and allophanic clay minerals that were proposed as sorbents for E1. Contraction of clay minerals, “salting out” and competitive sorption of artificial urine constituents were likely to have been responsible for observed changes in Freundlich parameters when artificial urine was used as mediator matrix. Plotting the effective distribution coefficient as a function of hypothetical exposure concentrations facilitated the comparison of the sorption behaviour of both compounds as influenced by the mediator solution. The results emphasized that using the CaCl₂ matrix might result in false inferences for the sorption behaviour of these compounds in a dairying environment. The four hormones rapidly degraded in the agricultural soils under aerobic conditions, and the majority of the compounds degraded > 50% within the first 24 hrs. Soil arylsulphatase activities were directly correlated with degradation rate constants of the estrogen sulphates. Estrone was identified as a metabolite of E2 and E1-3S, and these three compounds were observed as metabolites of E2-3S. Single-first order (SFO) and double first-order in parallel (DFOP) kinetics were used to model the degradation and metabolite formation data. The results showed that the DFOP model was in most cases better able to predict the parent compound degradation than the SFO model, and also enabled to estimate accurate degradation endpoints. ER-CALUX® analysis revealed the formation of estrogenicity during E2-3S degradation, which could partly be explained by the formation of the metabolites E2 and E1. Transport studies with E1-3S and E1 showed that the transport and retention of both compounds were significantly influenced by the mediator matrix. While no breakthrough curves (BTCs) were recorded during hormone application in CaCl₂ (10 mM) both hormones were detected in the leachate when applied in artificial urine. Rate-limited sorption processes were proposed for the delayed arrival of the hormone BTCs compared with a conservative bromide tracer. Intense colouration of the leachate during the artificial urine experiments suggested the hormones were likely to be moved by colloid-facilitated transport. Furthermore, the detection of residue hormone and metabolite concentrations implied that degradation of E1-3S and E1 was hampered by urine constituents such as glycine and urea.