abstract / Biological Sciences / Doctoral / Doctor of Philosophy

Phytosterols are naturally occurring compounds which regulate membrane fluidity and serve as hormone precursors in plants. They also have the potential to cause endocrine disturbances in aquatic animals at concentrations as low as 10 µg/L. Wastewaters from several industries which process plant matter can contain phytosterols at concentrations in excess of the above-stated level. Despite their endocrine disruption potential, very little is known about phytosterol physical properties and their biotransformation potential in biological treatment systems. Aerated stabilization basins (ASBs) are common biological treatment systems in North American pulp and paper mills. ASBs are large open lagoons which use tapered surface aeration to remove COD and prevent sulfate reduction in the water column. Phytosterols are released from wood during the pulping process and a small fraction enters the wastewater stream during washing of the pulp. Therefore, phytosterols may be exposed to aerobic or anaerobic environments depending on their solubility and solid-liquid partitioning behavior. The overall objective of this research was to systematically and quantitatively assess the biotransformation potential of phytosterols in biological treatment systems and to examine conditions leading to reduction of these compounds in wastewater effluent streams. The results of this research showed that phytosterols are sparingly soluble with aqueous solubility below 1 µg/L when present as a mixture. Phytosterols have a strong affinity to adsorb to solids and dissolved organic matter. The affinity for aerobic biomass was greater than for wastewater solids. The stigmasterol desorption rate and extent from wastewater solids increased with an increase in pH from 5 or 7 to 9. Phytosterols were biotransformed under aerobic conditions but not under sulfate-reducing or methanogenic conditions by stock cultures developed in this study. Biotransformation under nitrate-reducing conditions could not be confirmed conclusively. The continuous-flow system was successful in removing 72 to 96% of phytosterols. Biotransformation accounted for 23, 14 and 41 % of campesterol, stigmasterol and β-sitosterol removal, respectively. Phytosterols accumulated in the reactor sediment and accounted for 97 % of the total phytosterols remaining in the system. Phytosterols can be removed from wastewater streams during biological treatment by a combination of biotransformation and solids partitioning and control of system pH, DO and available carbon and energy sources can increase the degree of phytosterols removal. The results of this research can be used to engineer effective biological treatment systems for the removal of phytosterols from pulp mill wastewaters and other phytosterol-bearing wastewater streams.

Endocrine disruption and high occurrences of intersex have been observed in wild fish associated with wastewater treatment plant (WWTP) effluents in the urbanized reaches of the Grand River watershed located in southern Ontario, Canada. WWTP effluent is a complex matrix with diverse aquatic environmental contaminants and stressors. This study aimed to: (1) characterize the spatio-temporal distribution and fate of antiandrogenic personal care products (triclosan, chlorophene, and dichlorophene), along with selected pharmaceuticals (carbamazepine, ibuprofen, naproxen, and venlafaxine) and the herbicide, atrazine in the Grand River watershed and (2) model the behaviour of these contaminants in the aquatic environment. Water sampling of 29 sites which covered six municipal WWTPs and ~100 km of river length was completed during summer low flows (July 2012). Monthly samples were also collected immediately upstream and downstream of a major WWTP (Kitchener) from August to November 2012. Many of the target pharmaceuticals and triclosan were detected in WWTP effluents in the Grand River watershed, especially those that did not nitrify (minimal treatment with high ammonia). Chlorophene was either undetected or was only found at trace levels in the effluents. Under low flow conditions, triclosan and several other pharmaceuticals exhibited a spatial pattern where concentrations increased directly downstream of the WWTPs, then decreased with distance downstream (dilution and/or degradation). Chlorophene, in contrast, was not found downstream of most of the WWTP outfalls but was first detected at a site 5 km upstream of a WWTP and then continued with relatively constant concentrations for approximately 29 km downstream. It was also only found during the summer sampling period. Atrazine was consistently found in all sampling locations which reflected the agricultural non-point source nature of this compound. The WASP 7.5 model (US Environmental Protection Agency) was adapted and calibrated to a reach of the Grand River associated with the Kitchener WWTP. The simulation of the fate and transport of the target compounds revealed that flow-driven transport processes (advection and dispersion) greatly influence their behaviour in the aquatic environment. However, fate mechanisms such as biodegradation and photolysis also potentially play an important role in the attenuation of most compounds. The exception was carbamazepine where it was shown to act as a conservative tracer compound for wastewater specific contaminants in the water phase. The fate model developed can be applied in the future to predict the fate of a wide variety of contaminants of emerging concern across the watershed to help define the exposure of these biologically active chemicals to sensitive ecosystems.

Biosolids are the nutrient-rich organic residual materials resulting from the treatment of domestic sewage at a wastewater treatment facility, and are increasingly land-applied for agricultural and land-reclamation purposes as part of the wastewater management process. While the presence and fate of estrogenic endocrine-disruptors (eEDCs) in wastewater has been extensively studied, much less focus has been given to examining the presence and fate of eEDCs during biosolids treatment. In particular, little work has been done to measure the net estrogenic potency of biosolids using in vitro bioassays, such as the Yeast Estrogen Screen (YES) assay. This is despite the fact that widespread land-application of biosolids provides for the direct introduction of eEDCs into terrestrial and aquatic environments. The relative scarcity of bioassay-based net estrogenicity data for sludges and biosolids is in large part due to the analytical challenges involved in working with such a complex sample matrix. Comprehensive sampling at wastewater treatment plants in Guelph and London, ON, demonstrated that the estrogenicity of anaerobically-treated biosolids is considerably lower (12.0-19.7 ng/g estradiol-equivalents) than that reported in earlier published studies. The results of the present study were made possible due to the development of a sample preparation methodology that overcame the toxic effects that sludge and biosolid samples typically exert on yeast cells in the YES assay. An anti-estrogenicity assay was also applied for the first time to sludges/biosolids to measure the extent to which antagonistic compounds ‘block’ the response of the YES assay. The results of these tests suggest that although the net estrogenicity of anaerobically treated solids is indeed low, up to twice the amount of estrogenicity measured by the YES assay may be masked in biosolids by the presence of antagonistic compounds. While aerobic treatment conditions reduced net estrogenicity to at-or-below detectable levels, net estrogenicity remained relatively constant throughout the unit processes of the anaerobic treatment train. Biosolid ageing during storage led to an overall decrease in net estrogenicity of both conventionally-treated “restricted use” and advanced-treated “unrestricted use” anaerobic biosolids. However, levels of net estrogenicity were observed to spike during the early stages of storage, particularly under freeze/thaw conditions. / Natural Science and Engineering Research Council of Canada (NSERC) PGS-D3 scholarship, Water Environment Association of Ontario, Canadian Water Network

There is concern regarding exposure of aquatic organisms to chemicals that interfere with the endocrine system. Disruption of the endocrine system can lead to impacts on sexual development, altered hormone levels, intersex, and ultimately reproductive failure. While effects of endocrine disrupting chemicals (EDCs) on standard laboratory species have been subject of intense study, to this day there is a large gap in knowledge and a high degree of uncertainty regarding the sensitivity of wild fish species to these compounds. One of the main concerns with current toxicity testing approaches is that they require the use of a large number of live animals, particularly when working with native species. Therefore, the aim of this study was to develop in vitro tissue explant assays that would enable the assessment of the sensitivity of different wild fish species native to North America to the exposure with EDCs. Specifically, two in vitro assays were developed: 1) A liver explant assay to assess effects of EDCs that can interact with the estrogen receptor (environmental estrogens), and 2) a gonadal explant assay to assess effects of EDCs on sex-steroid production. The test species selected were northern pike (Esox lucius), walleye (Sander vitreus), and white sucker (Catostomus commersoni) that were sampled from Lake Diefenbaker, Saskatchewan, Canada, and white sturgeon (Acipenser transmontanus) that were randomly selected from an in house stock reared from eggs. Liver tissue was excised from male fishes and exposed for 24 h to a synthetic estrogen, 17α- ethinylestradiol (EE2). Transcript abundance of vitellogenin (VTG), estrogen receptor (ER) α and β in liver tissue were quantified using qPCR. Gonad tissue from both male and female were excised and exposed for 24 h to a model inducer (forskolin) and inhibitor (prochloraz) of steroidogenesis. 11-ketotestosterone (11-KT) and estradiol (E2) were quantified in media by use of ELISA. Exposure to EE2 resulted in a concentration dependent increase in VTG in all species, and an increase in ERα in northern pike. Walleye males showed the greatest sensitivity to EE2. Gonad tissues exposed to forskolin showed a concentration dependent increase in 11-KT and E2. Exposure to prochloraz resulted in a decrease of 11-KTand E2. Male and female white sucker showed greatest sensitivity to forskolin, while male and female walleye showed greatest sensitivity to prochloraz. The seasonal time point during which gonad explants were excised and exposed had an impact on the potency and magnitude of response, resulting in a seasonal effect on sensitivity. Also, gonad explants from these species were found to have greater sensitivity than responses previously reported for in vitro explants of other fish species such as the fathead minnow (Pimephales promelas), and stable cell lines currently used as screening applications to detect chemicals that might disrupt the endocrine system. Therefore, current approaches that use stable cell lines or tissue explants from standardized small bodied laboratory species might not be protective of some wild fish species. These tissue explants represent a promising approach to help understand species sensitivity to EDCs, and if appropriately validated, could be a powerful tool for chemical screening.

Over the past several decades, there has been growing concern among the public and scientific community regarding adverse health effects resulting from exposure to natural and synthetic compounds that act as endocrine disrupters. The structural similarity of many of these compounds to natural hormones and receptors, as well as their ubiquity in the environment, can result in the potential for interference with the endocrine system of wildlife and humans. Much of the research examining the adverse effects of wildlife exposure to endocrine disrupting chemicals (EDCs) has focused on effects on reproduction or short-term changes in hormone physiology. However, organisms exposed to low levels of EDCs at early life stages could also be susceptible to developmental effects, including neurological and other physiological changes affecting later life stages. In birds, migration can be an important component of the annual life cycle and it can be vulnerable to disruption given that it is under endogenous hormonal and neurological control. Previous studies have shown that developmental exposure of birds to thyroid hormone disruptors, such as polychlorinated biphenyls (PCBs), have resulted in reduced hatching success, lower growth rates, teratogenicity, impaired development, and immunotoxicity. In this thesis, I aimed to supplement what is currently known regarding the effects of developmental exposure to low levels of a mixture of endocrine disrupting chemicals in songbirds, as well as further investigate the latent consequences of such an exposure on migratory life stages. I initially investigated the potential physiological and developmental effects of early exposure to Aroclor 1254, a PCB mixture, in two passerine songbird species: European starlings (Sturnus vulgaris) and red-winged blackbirds (Agelaius phoeniceus) during the critical nestling period. In 2011, European starlings and red-winged blackbirds were orally administered Aroclor 1254 over the period of nestling development, which was repeated in 2012 with only European starlings. For both years, morphological parameters (body mass, tarsus, wing-chord and bill-lengths) were measured daily and plasma thyroid hormones were measured. Additional measurements of wing chord and tarsus length fluctuating asymmetry (FA) were taken in the second year, to further assess contaminant-induced alterations in developmental stability. I found that treatment with environmentally-relevant levels of Aroclor 1254 caused increasing liver residues above the controls but did not result in overt effects on morphological growth parameters during the nestling period in either starlings or red-winged blackbirds. However, we did observe significant differences in 2012 starling’s wing chord FA at day 10 and 13, and tarsus length FA between all treatment groups and controls, indicating the potential for PCB-induced stress. Nestling thyroid hormone profiles (T3) sampled throughout the nestling period supported developmental changes but did not reveal any differences among treatment groups. Starlings were subsequently reared in captivity and further tested during a simulated autumn migration. Migratory activity and orientation were tested using Emlen funnel trials over 6 consecutive weeks. Across treatment groups, we found a significant increase in mass, fat, and feather moult, and decreasing plasma thyroid hormones over time. At 12L:12D, control birds showed a peak in activity and a directional preference for 155.95° (South-southeast), while high-dosed birds did not. High-dosed birds showed a delayed directional preference for 197.48° (South-southwest) under 10L:14D, concomitant with apparent delays in moult. These findings link alterations in avian migratory behaviour to contaminant-specific mechanisms. Exposure to a ubiquitous environmental endocrine disruptor exerted only subtle short-term effects during the period of exposure but importantly, latent effects may be far more relevant for individual fitness. We discuss how the impacts of exposure during early stages of development were not significant for short-term nest success, but can still give rise to longer time-scale effects that are potentially relevant for survival and population stability for migratory birds.

Thesis (Ph. D.)--Michigan State University. Dept. of Zoology & Environmental Toxicology, 2008. / Title from PDF t.p. (viewed on Mar. 30, 2009) Includes bibliographical references. Also issued in print.

Thesis (M.S.)--University of Alabama at Birmingham, 2010. / Title from PDF t.p. (viewed June 30, 2010). Includes bibliographical references (p. 19-22).

Thesis (MSc. (Human Physiology, Faculty of Health Sciences))--University of Pretoria, 2008. / Includes bibliographical references.

Orientador: Wellerson Rodrigo Scarano / Banca: Patricia Simone Leite Vilamaior / Banca: Luis Fernando Barbisan / Resumo: O TCDD (2,3,7,8-tetraclorodibenzo-p-dioxina), é considerado o contaminante ambiental com maior toxicidade na atualidade e sua ação está associada à interação com o receptor aril-hidrocarboneto (do termo inglês, AhR), responsável pela resposta biológica do TCDD sobre as células. Na próstata, o TCDD age diretamente no UGS inibindo o brotamento prostático através da ativação das vias dependentes de AhR. O Resveratrol destaca-se por suas propriedades anticâncer, antiagregação de plaquetas, antiinflamatória e antialérgica, atuando inclusive como antagonista do AhR. O estudo teve por objetivo avaliar o potencial quimioprotetor do Resveratrol sobre o desenvolvimento da próstata de ratos expostos ao TCDD durante a fase inicial da morfogênese prostática. Para isso, ratas prenhes da linhagem Wistar foram divididas em 4 grupos experimentais: Controle, TCDD, TCDD+Resveratrol e Resveratrol. O TCDD foi administrado em dose única no 15º. dia gestacional (DG15) na dose de 1μg/kg (diluído em óleo de milho) e o Resveratrol foi administrado entre o DG10 e o dia pós-natal 21 (DPN21) na dose de 20mg/kg (diluído em etanol 8%), ambos por via oral. Machos recém-nascidos (DPN1) foram eutanasiados para avaliação dos efeitos imediatos do TCDD (com ou sem a adição do Resveratrol) sobre a morfogênese prostática inicial, através de técnicas morfológicas e imunohistoquímicas, enquanto que, machos adultos (DPN90) foram eutanasiados para avaliação dos efeitos tardios. . No DPN1, os aspectos histológicos e morfométricos apontaram para a redução no número de brotos prostáticos no grupo TCDD quando comparado aos demais grupos Nesse mesmo grupo, as células epiteliais periféricas foram menos imunorreativas para p63 comparado com o Controle e TCDD+Resveratrol. O índice de proliferação celular nos brotos epiteliais e no mesênquima no grupo TCDD foi inferior ao observado nos grupos Controle e TCDD + Resveratrol, bem como o índice... / Abstract: TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is the most toxic environmental contaminant which mechanism of action is through the interaction with the aryl hydrocarbon receptor (AhR). In the prostate, TCDD works inhibiting the prostatic budding and activating the AhR-dependent pathways. Resveratrol is known by your anticancer, antiplatelet, anti-inflammatory and anti-allergic properties, besides acting as AhR antagonist. Therefore, the current research aimed to assess the protective effect of Resveratrol on prostate development in rats exposed to TCDD during early prostate morphogenesis. Pregnant Wistar rars were divided in four experimental groups: Control, TCDD. TCDD+Resveratrol and Resveratrol. TCDD (1μg/kg body weight) diluted in corn oil was administered by gavage as a single dose on 15th gestational day (GD15) while Resveratrol (20mg/kg body weight) was given by gavage (diluted in 8% of ethanol) between GD10 and postnatal day 21 (PND21). Newborn males (PND1) were euthanized to evaluate immediate effects of TCDD and Resveratrol on the initial prostatic morphogenesis, through morphological and immunohistochemical assays, while male rats (PND90) were euthanized for evaluation the late effects. There was decreasing of prostatic buds number in TCDD group. In the same group, basal cells showed weak immunoreactivity for p63 when compared to Control and TCDD+Resveratrol. TCDD reduced the cell proliferation index in the epithelial buds and mesenchyme as well as the number of androgen receptor (AR)-positive mesenchyme cells compared to Control and TCDD+Resveratrol groups. It is worth mentioning that the AR immunostaining was weak in the epithelial buds and intense in the surrounding mesenchyme in all experimental groups. On the other hand, α-actin staining was less intense in the differentiated mesenchymal cell that surrounded the epithelial buds in the TCDD group. Regarding to adult animals (PND90), the histological analysis revealed in ... / Mestre