Hormones are endocrine disrupting compounds, which have been shown to alter the sexual development of aquatic organisms. Animal manure applications to agricultural fields for nutrient management can be a source of environmental hormones. This dissertation investigates the occurrence of hormones in fields applied with various manures and their adjacent streams, as well as the effect of manure application technologies on the fate of hormones in soils, sediments, and runoff. A total of 11 hormone compounds were studied. All studied analytes were quantified using liquid chromatography and triple-quadrupole mass spectrometry following various sample extraction and clean-up strategies. The spatial and temporal distribution of manure-associated hormones in a manure surface applied agricultural field and adjacent stream was studied at time points up to 7.5 months after a routine manure application. Hormones were detected mainly in the top 0-5cm soils. Significantly higher levels of hormones were found in the drystack applied area of the field when compared to dairy manure slurry applied portion. New technologies for the subsurface application of poultry litter show promise as a tool to reduce the transportation of environmental hormones in surface runoff. Once adequate sampling protocols were established; it was determined that subsurface injection of both dairy manure and poultry litter reduced the impact of manure surface runoff. Hormones also showed little vertical and lateral movement in the soil. The transformation rates of 1,4-androstadiene-3,17-dione, 4-androstene-3,17-dione and estrone were studied comparing the effects of temperature, soil type, and application type. The calculated half-life of 1,4-androstadiene-3,17-dione in poultry litter surface-applied soils was 1.9 times higher than that in the poultry litter subsurface-injected soils, indicating a faster dissipation rate in the injection slits. Estrone persisted at detectable levels for the duration of the study in all treatments. The continued use of best management practices and innovative manure management techniques for the reduction of nutrients, sediment and other contaminants has the potential to also reduce hormone transport to the natural environment. Monitoring many different types of hormones in all areas of an environmental system will continue to provide better information on the occurrence and fate of hormones sourced from manure amended soils. / Ph. D.

Introduction Epidemiologic data supporting the role of organochlorine pesticides in pubertal development are limited. Methods Using a nested case-control design, serum collected during pregnancy from mothers of 218 girls who reported menarche before 11.5 years of age (cases) and 230 girls who reported menarche at or after 11.5 years of age (controls) was analyzed for 9 organochlorines and metabolites. We analyzed the association between in utero organochlorine concentrations and early menarche using multivariate logistic regression controlling for mother's age at menarche, or mother's prenatal BMI. Results We did not observe an association between in utero exposure to HCB, β-HCH, ϒ-HCH, p,p′-DDT, p,p′-DDE, oxychlordane or trans-nonachlor and early menarche. Conclusions This study is the first to examine the association between in utero exposure to HCB, β-HCH, ϒ-HCH, oxychlordane or trans-nonachlor and early menarche. In utero exposure to organochlorine pesticides does not appear to have a role in the timing of menarche in this study.

Polybrominated diphenyl ethers (PBDEs), flame retardants, have been applied to consumer goods, such as furniture, electrical devices, textiles, and appliances for decades. Due to their physico-chemical properties, PBDEs are semi-volatile and easily leach off the consumer good during aging, stress, or normal wear and tear of the good. Once airborne, they pose an environmental health threat because they can adsorb onto dust particles, soil, or other particulates that can be inhaled, ingested, or come into contact with the dermal layer. Additionally, PBDEs have a molecular structure similar to other persistent organic pollutants, such as polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins and furans. They are a health threat due to their endocrine-disrupting nature by affecting thyroid functioning, fertility, and child development. The purpose of the study is to measure selected PBDEs in a wastewater treatment plant (WWTP) that produces reclaimed water, such that a mass balance can be completed, and to compare this mass balance with theoretically expected concentrations. The mass balance includes the collection of samples from wastewater, sewage sludge, and air at points within the WWTP. The PBDEs examined are BDE-28, 47, 99, 100, 153, 154, and 183. The second part of the study will compare effluent concentrations to reclaimed water concentrations in order to examine the potential exposure (if any) of using reclaimed water. Influent concentration of mean (sum7)PBDE was found to be 49,117 pg/L and effluent concentration was 4,603 pg/L, illustrating a 91% removal rate of PBDEs during the wastewater treatment plant. Sludge samples contained the highest total concentrations of PBDEs with mean (sum7)PBDE ranging from 14.0 to 41.3 ug/kg dry weight. Air samples were highest at the post-aeration (248 pg/m3 mean (sum7)PBDE) step due to the use of highly oxygenated air assisting in the release and volatilization of the PBDEs. Sludge was found to carry the largest mass loading at 14.2 lb/day (sum7)PBDE. Of the total mass loading of PBDEs from the WWTP, sludge is responsible for 86.7%, followed by reclaimed water and effluent (11.7% and 1.6%, respectively). The mass loading from air was negligible with less than 0.01% contribution to the total mass loading. Whereas reclaimed water overall had higher PBDE congener mean concentrations than the effluent, the independent samples t-test found no statistically significant differences between the two groups. The results of this study can be used to improve the wastewater treatment process to reduce the impact of PBDEs being released into the environment by WWTPs, and to educate the public on utilizing reclaimed water in a safe and healthy manner.

>Magister Scientiae - MSc / Tuberculosis still remains one of the world’s killer diseases. Pyrazinamide (PZA) is one of the most commonly prescribed anti-tuberculosis (anti-TB) drugs due to its ability to significantly shorten the TB treatment period. However, excess PZA in the body caused hepatotoxicity and liver damage. This, together with the resistance of the bacteria to treatment drugs, poor medication and inappropriate dosing, contribute significantly to the high incidents of TB deaths and diseases (such as liver damage). This, therefore, calls for new methods for ensuring reliable dosing of the drug, which will differ from person to person due to inter-individual differences in drug metabolism. A novel biosensor system for monitoring the metabolism of PZA was prepared with a nanocomposite of multi-walled carbon nanotubes (MWCNTs), polyaniline (PANI) and cytochrome P450 2E1 (CYP2E1) electrochemically deposited on a glassy carbon electrode (GCE). The nanocomposite biosensor system exhibited enhanced electro-activity that is attributed to the catalytic effect of the incorporated MWCNTs. The biosensor had a sensitivity of 7.80 μA/ μg mL-1 PZA and a dynamic linear range (DLR) of 4.92 – 160 ng/mL PZA. Bisphenol A (BPA) is a hormone-disrupting chemical used in production of epoxy resins and polycarbonates, which produce various products used on a daily basis. However, BPA can leach out of plastic during normal use and cause health effects such as cancer or disrupt the endocrine system. Moreover, BPA has also been proven to degrade from the containers in landfills and accumulate in groundwater and streams, thereby, polluting the environment while destroying aquatic organisms. Therefore, this also calls for new selective and sensitive methods for the monitoring of BPA. A novel biosensor system for monitoring the oxidation of BPA was prepared from a nanocomposite of polyaniline, polymethyl methacrylate and titanium dioxide nanoparticles, also electrochemically deposited on the GCE. Biosensor fabrication was conducted by immobilization of the enzyme manganese peroxidase (MnP) iii onto the nanocomposite film. The nanobiosensor also revealed enhanced electro activity, attributed to the incorporation of TiO2 nanoparticles. The biosensor system had a sensitivity of 0.3 μA/nM and a detection limit of 0.12 nM. This detection limit falls within the range of the allowed daily intake of BPA as recommended by the Food and Drug Administration (FDA, USA) and other regulatory bodies.

>Magister Scientiae - MSc / Current water treatment technologies do not remove many endocrine disruptor compounds (EDCs) such as 17α-ethynylestradiol (EE2) in its entirety, and the amount of these pollutants that continues to enter the aquatic environment through wastewater effluents is still capable of causing harmful health effects. Therefore the development of simpler and more sensitive biosensor system for detection of EE2 must be developed which have high responsiveness, low cost and easy handling. Therefore the aim of this study was to work towards the development of rapid test system of polyaniline-laccase on gold enzyme nanobiosensor (PANI-PSSA/Lac/Glu) for water estrogens. Preliminary studies were first done on the materials used in this study: estrogens, laccase, gold nanoparticles (AuNPs), and electropolymerized PANI-PSSA. Laccase was shown to be active towards EE2 and the enzyme could be stored for over three months. EE2 solution also could be used for over three months. Buffer used in this study was found to be suitable. Phosphoric acid (H3PO4) was a suitable electrolyte than hydrochloric acid (HCl) to be used for the electropolymerization of aniline and was used because it has same ions as the McIlvaine buffer (McIlB) which the post-deposition CVs indicated the formation of electrochemically very stable film. AuNPs were successfully synthesized and its size was identified to be less than 22 nm. McIlB used for testing electrochemical properties of AuNP. CVs of GC/PANI-PSSA and GC/PANIPSSA/ Au showed no difference before and after exposure to aq. EE2 solution, an indication of being re-usable and could also serve as stable immobilising platform in laccase biosensor. When interrogating with electrochemical impedance spectroscopy (EIS), the charge transfer resistance (Rct) of both GC/PANI-PSSA and GC/PANI-PSSA/Lac/Glu showed an average increase by about 2.4% and 21% before and after exposure of EE2, respectively. This shows that the GC/PANI-PSSA/Lac/Glu was a functional EE2 biosensor and showing a positive step towards achieving a re-usable biosensor for EE2 as a model water estrogen. Future work Page | vi will focus on exploring different ways of improving the biosensor’s surface regeneration and its sensitivity to EE2.

Reef-building coral populations throughout the world are being threatened by numerous stressors and continue to decline. As potent endocrine-disrupting compounds, exogenous sex steroid contamination has been a largely overlooked stressor to corals. Previous research indicates these compounds are prevalent in marine environments, fluctuate annually along with reproductive cycles, can bioaccumulate, and have had variable effects on growth and reproduction in several cnidarian species. This project had three primary objectives: (1) establish environmental estradiol and progesterone concentrations in Broward County and lower Florida Keys reef environments, (2) conduct 17 β-estradiol and progesterone larval assays on P. astreoides larvae to determine the effects of these compounds on settlement and viability, and (3) conduct 17 β-estradiol and progesterone dosing experiments on adult Acropora cervicornis and Porites astreoides fragments to determine the effects on growth, zooxanthellae, reproduction, and overall tissue health. Estradiol was detected in surface and at-depth water samples from Broward County and lower Keys reef sites at effect level concentrations for marine organisms. Broward County larvae treated with low progesterone (5 ng/L) had decreased survival, while lower Keys larvae in low estradiol treatments (1 ng/L) had increased on-disc settlement. No other treatment effects were observed, however, lower Keys larvae had greater overall survival in comparison to Broward County larvae. There were no significant differences between estradiol and progesterone treatments in the adult-dosing experiment for growth, zooxanthellae density, reproduction, and overall tissue health. This is the first study to detect estradiol at Broward County reefs sites and our results, while inconclusive, indicate these compounds may have the potential to affect coral reef ecosystems.

In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Endocrine disrupting compounds (EDCs) are exogenous compounds/chemicals in the environment that interfere with the synthesis, secretion, distribution and function or elimination of natural hormones in the body. Environmental estrogens are a subclass of EDCs that may mimic or inhibit the effect of endogenous estrogen and can therefore influence developmental and reproductive health in humans and animals. EDCs have been reported to adversely affect the reproductive, immune, endocrine and nervous systems of wildlife and humans. The effects of EDCs include gonadal abnormalities, altered male/female sex ratios, reduced fertility and cancers of the male and female reproductive tract to mention a few. These effects are difficult to detect. Although it is essential to screen for EDCs in aqueous environmental samples, most countries have failed to implement this as part of their routine water quality monitoring programs due to various constraints such as the high cost of assays and the lack of infrastructure and skills required to do the assays. Therefore, there is a clear need for more user-friendly, more economically viable and time saving assays that can be used for routine monitoring of environmental EDCs. The aim of this study was to investigate the comparison between chemical and tissue culture methods to monitor environmental estrogens. 28 environmental water samples were collected from various sites around South Africa and analyzed for EDCs using a battery of rapid in vitro tests. Samples collected for the current study were selected based on various human impacts and also to give approximately 50% high and 50% low estrogen values. The 28 environmental water samples were separated into two groups based on the estradiol ELISA. The estradiol ELISA was chosen because estradiol is the principal estrogen found in all mammalian species during their reproductive years. For this separation, an estradiol level of 5 pg/ml was used as cut-off. Of the 28 samples investigated, 15 had estradiol levels higher than 5 pg/ml and were designated as high estradiol. The remaining 13 samples contained estradiol at 5 pg/ml or less and they were designated as low estradiol The first objective of this study was to compare different rapid ELISAs for EDC monitoring to determine if the data obtained with these assays are similar/identical. The data obtained from the estrogenic ELISAs was related/similar and showed good correlation with each other. This is because the different estrogens are very similar and also due to the fact that the same sub-group in the population (the reproductively active females) is secreting these hormones. Therefore, an estradiol rapid assay was proposed as a first screening system for estrogens in samples. Even though there was a positive correlation between the estradiol rapid assay and testosterone rapid assay, separation of samples based on estradiol levels wasn’t a good predictor of testosterone levels in the samples. A testosterone rapid assay was therefore recommended as necessary to screen for androgens in samples. The positive correlation between the estradiol rapid assay and progesterone rapid assay was expected because both estradiol and progesterone are secreted and excreted by the same population sub-group (reproductively active females). This study also demonstrated a good predictability of separating samples containing progesterone using the estradiol ELISA. Progesterone is secreted by pregnant women, a sub-group of the reproductively active females. It is advised that a progesterone rapid assay be included to screen samples for progestogens The second objective of this study was to compare estradiol rapid ELISAs with a bioassay for anti-androgenicity using mouse testicular cell cultures. The mouse testicular cell testosterone synthesis bioassay to monitor anti-androgenicity of the samples showed no correlation between the ELISA data for estrogens. This study shows that anti-androgenic effects need to be monitored independently because the data for estrogenic compounds cannot be used as a predictor for anti-androgenic effects. This demonstrated the need for the inclusion of a mouse testicular cell testosterone synthesis bioassay to screen for androgenicity and anti-androgenicity of water samples. In summary, due to the different mechanisms of action of EDCs, this study recommended a battery of assays to monitor for EDCs. The battery of assays suggested is: v progesterone rapid assay was expected because both estradiol and progesterone are secreted and excreted by the same population sub-group (reproductively active females). This study also demonstrated a good predictability of separating samples containing progesterone using the estradiol ELISA. Progesterone is secreted by pregnant women, a sub-group of the reproductively active females. It is advised that a progesterone rapid assay be included to screen samples for progestogens. The second objective of this study was to compare estradiol rapid ELISAs with a bioassay for anti-androgenicity using mouse testicular cell cultures. The mouse testicular cell testosterone synthesis bioassay to monitor anti-androgenicity of the samples showed no correlation between the ELISA data for estrogens. This study shows that anti-androgenic effects need to be monitored independently because the data for estrogenic compounds cannot be used as a predictor for anti-androgenic effects. This demonstrated the need for the inclusion of a mouse testicular cell testosterone synthesis bioassay to screen for androgenicity and anti-androgenicity of water samples. In summary, due to the different mechanisms of action of EDCs, this study recommended a battery of assays to monitor for EDCs. The battery of assays suggested is: Estradiol ELISA as a rapid assay to screen for estrogens. Testosterone ELISA as a rapid assay to screen for androgens. Progesterone ELISA as a rapid assay to screen for progestogens. Mouse testicular cell testosterone synthesis bioassay to screen for androgenicity and anti-androgenicity.

This study was carried out to evaluate the performance of Soil Aquifer Treatment (SAT) under different loading regimes, using wastewater of much higher strength than usually encountered in SAT systems, and also to investigate the removal of the endocrine disruptors triclocarban (TCC), estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). SAT was simulated in the laboratory using a series of soil columns under saturated and unsaturated conditions. Investigation of the removal of Chemical Oxygen Demand (COD), Biochemical Oxygen Demand (BOD), Dissolved Organic Carbon (DOC), nitrogen and phosphate in a 2 meter long saturated soil column under a combination of constant hydraulic loading rates (HLRs) and variable COD concentrations as well as variable HLR under constant COD showed that at fixed HLR, a decrease in the influent concentrations of DOC, BOD, total nitrogen and phosphate improved their removal efficiencies. It was found that COD mass loading applied as low COD wastewater infiltrated over short residence times would provide better effluent quality than the same mass applied as a COD with higher concentration at long residence times. On the other hand relatively high concentrations coupled with long residence time gave better removal efficiency for organic nitrogen. Phosphate removal though poor under all experimental conditions, was better at low HLRs. In 1 meter saturated and unsaturated soil columns, E2 was the most easily removed estrogen, while EE2 was the least removed. Reducing the thickness of the unsaturated zone had a negative impact on removal efficiencies of the estrogens whereas increased DOC improved the removal in the saturated columns. Better removal efficiencies were also obtained at lower HLRs and in the presence of silt and clay. Sorption and biodegradation were found to be responsible for TCC removal in a 300 mm long saturated soil column, the latter mechanism however being unsustainable. TCC removal efficiency was dependent on the applied concentration and decreased over time and increased with column depth. Within the duration of the experimental run, TCC negatively impacted on treatment performance, possibly due to its antibacterial property, as evidenced by a reduction in COD removals in the column. COD in the 2 meter column under saturated conditions was modelled successfully with the advection dispersion equation with coupled Monod kinetics. Empirical models were also developed for the removal of TCC and EE2 under saturated and unsaturated conditions respectively. The empirical models predicted the TCC and EE2 removal profiles well. There is however the need for validation of the models developed

Desreguladores endócrinos são compostos que podem alterar o sistema endócrino dos indivíduos, bem como sua fertilidade e por em risco a saúde dos mesmos. Muitas questões relacionadas a esses compostos ainda não foram elucidadas, apesar de serem muito estudados recentemente. Diversas pesquisas têm sido feitas investigando-se o emprego de processos oxidativos avançados na degradação dos desreguladores endócrinos em águas naturais e residuárias. O presente trabalho teve como objetivo estudar a remoção do hormônio 17\'beta\'-estradiol em soluções aquosas por oxidação química. A pesquisa foi dividida em duas etapas, sendo que a primeira etapa consistiu na investigação do uso de brometo de prata/dióxido de titânio (\'AG\'BR\'/\'TI\'O IND.2\') como catalisador no processo de fotocatálise com luz visível. Para isso, foram avaliadas a influência da concentração de \'AG\'BR\'/\'TI\'O IND.2\' (0,020; 0,025; 0,050; 0,070 e 0,100 g/L) e a intensidade de luz visível incidente (6,6 x \'10 POT.5\' e 1,3 x \'10 POT.6\' Ix) na eficiência de degradação do 17\'beta\'-estradiol. Na segunda etapa avaliou-se o emprego de ozônio na degradação de 17\'beta\'-estradiol em soluções aquosas, variando-se as dosagens de ozônio (1,7; 3,3, 5,0 e 7,5 mg \'0 IND.3\'/L) aplicadas e o pH (3; 5; 7; 9 e 11) das amostras. Na primeira etapa, os resultados demonstram que E2 foi bastante sensível aos processos oxidativos empregados. A fotocatálise foi eficiente para oxidar 17\'beta\'-estradiol, demandando tempo de exposição pequeno, em torno de 4 minutos para obtenção de remoção da ordem de 98%, quando aplicada concentração de \'AG\'BR\'/\'TI\'O IND.2\' de 0,100 g/L e a intensidade de luz visível incidente de 1,3 x \'10 POT.6\' Ix. Na segunda etapa, observou-se que o aumento do pH promoveu maior rapidez na degradação do E2 e o aumento da massa de ozônio aplicada promoveu maior eficiência de degradação do 17\'beta\'-estradiol. / Endocrine disrupting chemicals are compounds that can alter the endocrine system of individuals and their fertility and threaten their health. Many issues related to these compounds have not been elucidated, although much studied recently. Many researchers have investigated the use of advanced oxidative processes in the degradation of endocrine disrupting compounds in water and wastewater. This work is a study of the removal of 17\'beta\'-estradiol in aqueous solutions by chemical oxidation. This work was divided into two stages. In the first one, the use of bromide of silver/titanium dioxide (\'AG\'BR\'/\'TI\'O IND.2\') was investigated as catalyst in the photocatalysis activity under visible light irradiation. It was evaluated the influence of the concentration of \'AG\'BR\'/\'TI\'O IND.2\' (0.020; 0.025; 0.050; 0.070 and 0.100 g/L) and the incident light intensity (6.6 x \'10 POT.5\' and 1.3 x \'10 POT.6\' Ix) in the efficiency of degradation of 17\'beta\'-estradiol. In the second step, it was evaluated the use of ozone in the degradation of 17\'beta\'-estradiol in aqueous solutions, ranging up the masses of applied ozone (1.7; 3.3; 5.0 and 7.5 mg \'0 IND.3\'/L) and the pH (3, 5, 7, 9 and 11) of the samples. In the first stage, the results show that E2 was very sensitive to oxidative processes applyed. The photocatalysis was efficient to oxidize the 17\'beta\'-estradiol, requiring short exposure time, around 4 minutes, to obtain the removal of 98% (0.100 g/L of \'AG\'BR\'/\'TI\'O IND.2\' and incident visible light intensity of 1.3 x \'10 POT.6\'. In the second step, it was observed that the raise in pH promoted faster degradation of E2 and the increase of the mass of applied ozone promoted high efficiency of degradation of 17\'beta\'-estradiol.