The overall objective of the research conducted and described in this thesis was to develop an environmentally relevant bioassay to assess the effects of complex effluents on a sentinel fish species. A short-term fathead minnow (FHM) reproductive bioassay was utilized to assess the effects of industrial effluents on multiple levels of biological organization (sub-organismal to population endpoints). The FHM bioassay was tested in both lab and on-site investigations using an artificial stream system. The incorporation of trophic-transfer into the bioassay was also developed to quantify the importance of contaminated food as a source of exposure. This work was conducted in two key phases. Phase I focused on testing and developing the FHM bioassay, in the lab and on-site with pulp mill effluent (PME), to firstly document response patterns and, secondly, to conduct an investigation of cause study. Phase II focused on developing the trophic-transfer system to document responses to metal mine effluent (MME) in the lab and on-site in an artificial stream system. Development of the trophic-transfer system was also conducted during this phase to compare responses to standard water-only exposures. <p>In Phase I, exposure to PME in both the lab and field studies resulted in disruptions in egg production and spawning events. By focusing on identifying response patterns I was able to determine that the effects observed were indicative of an estrogenic response. I was also able to identify a process stream that was the potential cause of responses observed after exposure to final effluent. Isolation of this process stream will assist the mill in developing approaches for future mitigation. The results from this research will also provide additional data for the environmental effects monitoring (EEM) program for pulp and paper and investigation of cause studies on a national basis. <p> In Phase II, in both the field and laboratory investigations, significant decreases in reproductive output (egg production and spawning events) were observed in the water-only system exposures. Significant decreases in hatching success and increases in deformities were observed in the trophic-transfer system only, suggesting that the combination of both food and water was important in assessing the effects on the F1 generation. Overall, the responses in the trophic-transfer system were not comparable between the lab and field studies. In the lab study, significant decreases in reproductive output occurred, compared to the field study where significant increases in egg production and spawning events occurred. In addition, the effects on the F1 generation in the field study were not as severe as those observed in the lab investigation. It was concluded that the presence of reference water and the environment within the trophic-transfer system were responsible for this reduction in toxicity. <p> Phases I and II of this research have made significant contributions to artificial stream development within Canada for the assessment of industrial effluents and their effects on aquatic biota. The results from these studies have also demonstrated that environmentally relevant testing is essential if we are to accurately assess effects on aquatic biota. Future development and application of this bioassay should be towards developing a standardized approach for not only assessing the effects of industrial effluents in a comparative manner, but also in investigation of cause studies.

Sitosterolemia (STSL) is a sterol storage disorder characterized by very high plasma plant sterol (PS) and 5α-stanol levels, and leads to premature atherosclerosis, xanthomas, macrothrombocytopenia and endocrine disruption. Ezetimibe (EZE), a sterol absorption inhibitor, reduces plasma PS levels in STSL but its effect on tissue pool of sterols has not been investigated yet. The research objectives were to assess if EZE reduces whole body sitosterol and cholesterol pool sizes, improves cholesterol homeostasis, enhance hematologic profile and reduce endocrine disruption in STSL. EZE effects on circulating levels of cholestanol and its precursors (cholesterol and bile acid derivative 7α-hydroxy-4-cholesten-3-one, 7α-H-C4) relative to exogenous stanols (sitostanol) were also studied. Eight STSL patients were taken off EZE for 14 wks. After 4 wks off EZE they received intravenous doses of D7-sitosterol and 18O-cholesterol for sterol pool sizes assessments, and oral doses of 13C-cholesterol and deuterium oxide to measure fractional cholesterol absorption and synthesis rates. EZE (10 mg/d) was resumed and stable isotopes testing repeated. Measurement parameters included isotopic sterol enrichments, blood cell count, plasma and red blood cell (RBC) PS, cholesterol and its precursor (lathosterol), 5α-stanols and plasma 7α-H-C4, and thyroid hormones levels. EZE reduced plasma levels of sitosterol and total cholesterol, whole body sitosterol and cholesterol pool sizes and fractional cholesterol absorption rate while increasing cholesterol synthesis, production and clearance rates. EZE increased platelet count and decreased platelet size without affecting RBC indices of size or mass. A substantial decrease in circulating sitostanol but moderate decrease of cholestanol was noted with EZE. EZE increased lathosterol but not 7α-H-C4, suggesting increases in cholesterol biosynthesis and thus precursor availability for synthesis of cholestanol. In summary, EZE reduces body stores of PS and cholesterol, and increases cholesterol turnover by reducing cholesterol absorption and enhancing its synthesis and clearance. EZE reduces circulating PS and 5α-stanol levels, and improves macrothrombocytopenia and thyroid disruption. Endogenous cholestanol in STSL is mainly derived from cholesterol but not bile acid synthesis pathway. These data suggest that EZE may reduce the risks of developing premature atherosclerosis, bleeding and hormone disruption, thereby reinforcing the rationale for the use of EZE in treatment of STSL. / February 2015

We exposed a native endangered species, bonytail chub (Gila elegans), to graded concentrations of secondarily-treated effluent. At the end of each treatment period, we collected water from all raceways and samples were analyzed for the presence of 83 organic wastewater compounds. We extracted blood plasma from control and treatment fish in each raceway and samples were analyzed for hormones, 17β-estradiol and 11-ketotestosterone, and the egg yolk protein, vitellogenin.17β-estradiol concentrations were consistently lower in treatment females than those detected in control females. The 17β-estradiol and vitellogenin concentrations were always higher in treatment males than those found in control males. Concentrations of 11-ketotestosterone were consistently lower in treatment males than those detected in control males.Endocrine disrupting effects occurred in male and female fish exposed to low concentrations of effluent. Changes in hormone levels suggested a feminizing effect in treatment male fish and an androgenizing effect in treatment female fish.

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.

As part of their reclamation plan, oil sands operators propose to transfer the mature fine tailings, which are a by-product of the oil sands extraction process, to open-pits and cap them with either a layer of surface water or oil sands process-affected waters (OSPW). These oil sands pit lakes are expected to develop habitats with productive capabilities comparable to natural lakes in the region. The studies presented in this thesis evaluate the potential impact of OSPW and its associated constituents [i.e. acid-extractable organics (e.g. naphthenic acids; NAs) and salts] on the reproductive physiology of adult fathead minnow (Pimephales promelas). Through 14-21 day fathead minnow reproduction assays it was demonstrated that aged OSPW can impair spawning, lower plasma sex steroid concentrations, and reduce male secondary sexual characteristics. The acid-extractable organics in OSPW were demonstrated to have an adverse effect on fathead minnow reproductive physiology. Other studies showed that the high salinity which characterizes OSPW also influences toxicity. When fathead minnows were exposed to the OSPW extract and 700 mg/l of NaHCO3, the NaHCO3 reduced the inhibitory effects of the extract on the numbers of reproductive tubercles and plasma testosterone levels by reducing the uptake of NAE to the fish. Embyro and larval bioassays also revealed that NaHCO3 reduces the acute toxic effects of the OSPW extract. An assessment of a wild population of fathead minnows inhabiting an OSPW pond determined that there were differences in the condition factor (CF), gonadosomatic indices (GSIs), liver somatic indices (LSIs), male secondary sexual characteristics, and 11-ketotestosterone concentrations in the fathead minnows from the OSPW pond relative to fish collected at reference sites. The opercula of fathead minnows from the OSPW pond also differed from those of reference fish and an examination of the gills revealed that were a number of proliferative and degenerative alterations relative to reference fish. Collectively, these studies demonstrate that aged OSPW has the potential to negatively affect the reproductive physiology of fathead minnows and suggest that aquatic habitats with high NAs concentrations (>10 mg/l) will have adverse effects on fish. / Canadian Natural Resources Ltd., Canadian Water Network, Canadian Oil Sands Network for Research and Development (CONRAD), Imperial Oil Ltd., NSERC, Shell Canada Energy, Suncor Energy Inc., Syncrude Canada Ltd., and Total E&P Canada

Nanoparticles (NPs) are engineered in the nanoscale (<100nm) to have unique physico-chemical properties from their bulk counterparts. Nanosilver (nAg) is the most prevalent nanoparticle in consumer products due to its strong antimicrobial action and can be released to the environment during product manufacture, usage and disposal. The predicted environmental concentrations are within the North American guidelines for the protection of aquatic life and in drinking water. While nAg toxicity at high concentrations has been well described, the sublethal effects at environmentally-relevant concentrations are relatively unknown. Initial screening in our lab showed nAg was a potential endocrine disrupting chemical (EDC). Amphibian metamorphosis is mediated by thyroid hormone (TH), and nAg perturbed TH-dependent transcriptional responses in the tailfin of bullfrog (Rana catesbeiana) tadpoles. The primary objective of this thesis was to further investigate and characterize the effects of low, environmentally relevant concentrations of nAg on TH-dependent metamorphosis in R. catesbeiana and Xenopus laevis. Two chronic, 28 day in vivo exposures at 0.06 and 6µg/L nAg were conducted with premetamorphic R. catesbeiana tadpoles using TH to induce precocious metamorphosis. Ionic silver (iAg) was also examined to control for the complete dissolution of Ag. Analysis of metamorphic stage progression demonstrated nAg-induced acceleration of hindlimb growth and development. After 6 days of nAg exposure, analysis with quantitative real-time polymerase chain reaction (QPCR) demonstrated nAg-induced disruption of TH-responsive transcripts in a tissue-specific manner. Furthermore, the nAg effects could not be fully explained by iAg, indicating NP-specific disruption. Two chronic, 28 day exposures to 0.018-1.8 µg/L nAg were conducted on X. laevis premetamorphic and prometamorphic tadpoles. nAg was found to significantly bioaccumulate in tadpole tissue after 28 days. Furthermore, nAg increased the hindlimb length during early premetamorphosis and in post-metamorphic juvenile tadpoles. Using an in-house MAGEX microarray and QPCR transcriptional analysis, 7 biomarkers of nAg exposure were validated. Five of these targets showed disruption to their TH-response. Furthermore, the increased mRNA abundance of two peroxidases indicated that nAg generated reactive oxygen species (ROS) even at low, environmental concentrations. This thesis demonstrates that nAg has consistent EDC actions across two distinct amphibian species, and the data suggest that regulatory guidelines for silver may need revision. A X. laevis derived fibroblast-like TH-responsive cell line, XTC-2, was used in conjunction with the 7 biomarkers of nAg exposure to gain mechanistic insight into the role of ROS in TH signaling disruption. Monocultures were created and validated to increase the specificity of TH-response. While the monocultures were successfully created, the biomarkers were not responsive to nAg in this cell line. Additional investigations were made into the relationship between genetic sex and responsiveness to TH. Genetic sexing methods were used to investigate transcriptional differences between males and females during natural and TH-induced metamorphosis. The sexing protocol was optimized and validated successfully. The genetic sex was determined for premetamorphic and prometamorphic X. laevis tadpoles exposed to TH for 48 h. QPCR and microarray analysis were used to identify three markers that demonstrated transcriptional sex-bias during early gonadal differentiation stages. / Graduate / 0307 / 0383 / 0487 / amanda_carew14@yahoo.ca

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

Orientador: João Lauro Viana de Camargo / Banca: Antonio Francisco Godinho / Banca: Lourival Larini / Acompanha 1 CD-Rom / Resumo: O presente estudo objetivou avaliar o potencial de desregulação endócrina de uma mistura de cinco praguicidas, fornecidos a ratos Lewis macho via ração em nível de seus NOELs. Os praguicidas estudados foram encontrados pela ANVISA em tomates à disposição do consumidor brasileiro. Os animais foram distribuídos em 8 grupos de acordo com a ração experimental, da seguinte maneira: Grupo 1 - ração basal, sem contaminantes; Grupo 2 ("Baixas doses", praguicidas adicionados à ração em nível de seus respectivos NOELs) - diclorvós 0,23 mg/kg, dicofol 0,22 mg/kg, endosulfan 0,6 mg/kg, dieldrin 0,025 mg/kg, permetrina 5,0 mg/kg; Grupo 3 ("Doses efetivas", correspondentes aos respectivos LOEL/LOAEL) - diclorvós 2,3 mg/kg, dicofol 2,5 mg/kg, endosulfan 2,9 mg/kg, dieldrin 0,05 mg/kg, permetrina 25,0 mg/kg e Grupos 4 a 8 (controles positivo) - rações contendo cada praguicida separadamente, em concentrações correspondentes aos seus respectivos LOEL/ LOAEL. Os animais foram sacrificados no final da 8ª. semana de tratamento. O modelo utilizado permitiu verificar que os praguicidas estudados, individualmente ou em misturas em "doses baixas" e em "doses efetivas", provocam alterações variadas na próstata, testículos e fígado. No entanto, a mistura "baixas doses", constituída com os praguicidas em nível de seus NOELs, não diferiu, grosso modo, dos efeitos verificados com a mistura "doses efetivas", em que os praguicidas foram adicionados em níveis de LOEL/LOAELs. Questões de delineamento, como idade dos animais e duração do experimento, podem ter influenciado os resultados, reduzindo a magnitude e significância das alterações encontradas. / Abstract: The present study aimed to evaluate the potential for endocrine disruption of a mixture of five pesticides at their NOELs levels given to Lewis rats through the feed. The pesticides studied were found by ANVISA in tomatoes available at the counter to Brazilian customers. The animals were allocated to 8 groups according to the experimental feed: Group 1 - basal feed, without contaminants; Group 2 ("Low doses"; pesticides were added to the feed at their respective NOEL levels) - dichlorvos 0,23 mg/kg; dicofol 0,22 mg/kg, endosulfan 0,6 mg/kg, dieldrin 0,025 mg/kg, permethrin 5,0 mg/kg; Group 3 ("Effective doses"; corresponding to their respectives LOEL/LOAELs) - dichlorvos 2,3 mg/kg, dicofol 2,5 mg/kg, endosulfan 2,9 mg/kg, dieldrin 0,05 mg/kg, permethrin 25,0 mg/kg and Groups 4 to 8 (positive controls) - feed containing each pesticide separately, at concentrations corresponding to their respective LOEL/LOAELs. The animals were sacrificed at the end of the eighth week of treatment. The model used allowed to verify that the pesticides studied, separated or at "low" or "efective doses" induce a variety of alterations in the prostate, testes and liver. However, the effects verified at the "low doses" did not differ from those registered at the "effective doses". Questions of experiment design, animal's age at the beginning of the experiment and experiment length might have influenced the results, reducing the magnitude and meaning of the alterations found. / Mestre

Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-29Bitstream added on 2014-06-13T20:48:04Z : No. of bitstreams: 1 martinez_mf_me_botfm.pdf: 706611 bytes, checksum: beba68fc5aab4f655aea84c3954a37e1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Toxicam / O presente estudo objetivou avaliar o potencial de desregulação endócrina de uma mistura de cinco praguicidas, fornecidos a ratos Lewis macho via ração em nível de seus NOELs. Os praguicidas estudados foram encontrados pela ANVISA em tomates à disposição do consumidor brasileiro. Os animais foram distribuídos em 8 grupos de acordo com a ração experimental, da seguinte maneira: Grupo 1 - ração basal, sem contaminantes; Grupo 2 (“Baixas doses”, praguicidas adicionados à ração em nível de seus respectivos NOELs) - diclorvós 0,23 mg/kg, dicofol 0,22 mg/kg, endosulfan 0,6 mg/kg, dieldrin 0,025 mg/kg, permetrina 5,0 mg/kg; Grupo 3 (“Doses efetivas”, correspondentes aos respectivos LOEL/LOAEL) - diclorvós 2,3 mg/kg, dicofol 2,5 mg/kg, endosulfan 2,9 mg/kg, dieldrin 0,05 mg/kg, permetrina 25,0 mg/kg e Grupos 4 a 8 (controles positivo) - rações contendo cada praguicida separadamente, em concentrações correspondentes aos seus respectivos LOEL/ LOAEL. Os animais foram sacrificados no final da 8ª. semana de tratamento. O modelo utilizado permitiu verificar que os praguicidas estudados, individualmente ou em misturas em “doses baixas” e em “doses efetivas”, provocam alterações variadas na próstata, testículos e fígado. No entanto, a mistura “baixas doses”, constituída com os praguicidas em nível de seus NOELs, não diferiu, grosso modo, dos efeitos verificados com a mistura “doses efetivas”, em que os praguicidas foram adicionados em níveis de LOEL/LOAELs. Questões de delineamento, como idade dos animais e duração do experimento, podem ter influenciado os resultados, reduzindo a magnitude e significância das alterações encontradas. / The present study aimed to evaluate the potential for endocrine disruption of a mixture of five pesticides at their NOELs levels given to Lewis rats through the feed. The pesticides studied were found by ANVISA in tomatoes available at the counter to Brazilian customers. The animals were allocated to 8 groups according to the experimental feed: Group 1 - basal feed, without contaminants; Group 2 (“Low doses”; pesticides were added to the feed at their respective NOEL levels) – dichlorvos 0,23 mg/kg; dicofol 0,22 mg/kg, endosulfan 0,6 mg/kg, dieldrin 0,025 mg/kg, permethrin 5,0 mg/kg; Group 3 (“Effective doses”; corresponding to their respectives LOEL/LOAELs) – dichlorvos 2,3 mg/kg, dicofol 2,5 mg/kg, endosulfan 2,9 mg/kg, dieldrin 0,05 mg/kg, permethrin 25,0 mg/kg and Groups 4 to 8 (positive controls) – feed containing each pesticide separately, at concentrations corresponding to their respective LOEL/LOAELs. The animals were sacrificed at the end of the eighth week of treatment. The model used allowed to verify that the pesticides studied, separated or at “low” or “efective doses” induce a variety of alterations in the prostate, testes and liver. However, the effects verified at the “low doses” did not differ from those registered at the “effective doses”. Questions of experiment design, animal’s age at the beginning of the experiment and experiment length might have influenced the results, reducing the magnitude and meaning of the alterations found.

A desregulação endócrina induzida por contaminação ambiental está entre os principais problemas criados pela sociedade moderna de consumo, responsável pela inserção no ambiente de uma série de substâncias interferentes nos sistemas hormonais dos mais diversos organismos, incluindo o próprio homem. A ação destes compostos acarreta, entre outros efeitos, disfunções reprodutivas e estudos apontam que também podem ser indutores de cânceres. A legislação brasileira através do Conselho Nacional do Meio Ambiente (CONAMA) determina os padrões de qualidade das águas, porém muitas substâncias com potencial de desregulação endócrina não tem suas concentrações e emissões especificadas. O objetivo deste trabalho foi executar um levantamento da presença e possíveis conseqüências dos hormônios estrogênicos, uma das classes mais potentes de desreguladores endócrinos (ED), nos compartimentos água e sedimento do rio do Monjolinho. Este rio cruza parte da malha urbana da cidade de São Carlos - SP e recebe lançamentos localizados e difusos de esgotos domésticos e industriais. Portanto, amostras de água e sedimentos foram analisadas através de cromatografia líquida, e exemplares de peixes capturados no rio investigados quanto à presença da proteína vitelogenina (VTG) um biomarcador de exposição. Também ensaios ecotoxicológicos foram desenvolvidos em laboratório com diferentes abordagens para verificação de efeitos diversos. Em uma tentativa de abordar os dados gerados através de uma perspectiva ampla, foi delineada uma avaliação de risco ambiental discutindo as possíveis ameaças a biota e a população humana, já que concentrações de hormônios, principalmente o sintético etinilestradiol (concentração máxima de 30,1 ± 3,41 ng/L), a indução da VTG e efeitos em ensaios ecotoxicológicos foram confirmados. / The environmental endocrine disruption is among the main problems arrived with the modern society way of life. The hormonal systems of several organisms are injured by a number of chemicals disposal on hydric bodies in erroneous way. These compounds causes reproductive disturbs, and studies pointed it be cancer inductors. The Brazilian National Environmental Council (CONAMA) do not regulated standards for discharges and concentrations of these substances. This work aims to investigate the probable presence and effects of sexual estrogens hormones, one of the most powerful groups of endocrine disruptors (EDCs), at the Monjolinho river. This small urban river is placed in São Carlos; a town located in the São Paulo state, southwest Brazil, and receives concentrated and diffuse sewage effluents as industrials as domestics. Samples of water and sediments were analyzed by liquid chromatography, and male fishes captured were investigated to survey the vitellogenin protein (Vtg), a biomarker of exposition. To complement the study, ecotoxicological tests with different approaches were considered. Moreover an environmental risk analyze delineation was made because hormones concentrations, mainly the synthetic ethynilestradiol (EE2), VTG induction, and positive effects in ecotoxicity tests were found.