Nas últimas décadas, a ocorrência de atividade estrogênica e interferentes endócrinos (IEs) no ambiente aquático têm se tornado uma crescente preocupação. Dentre as diversas substâncias classificadas como IEs, destacam-se os fármacos, produtos de higiene e cuidados pessoais, hormônios naturais e sintéticos, produtos químicos industriais, praguicidas e muitos outros compostos que atingem o ambiente aquático por meio de descargas de esgoto doméstico, industrial ou de escoamento agrícola. Os objetivos deste estudo foram determinar a atividade estrogênica em amostras de águas superficiais, e avaliar seus efeitos biológicos no desenvolvimento de embriões de Danio rerio, a fim de propor faixas baseadas em valores de desencadeamento de efeitos para categorizar a atividade estrogênica. As amostras ambientais também foram analisadas por cromatografia líquida acoplada com a espectrometria de massas para identificar as substâncias que são suspeitas de causar alteração endócrina. Os compostos analisados foram: praguicidas, hormônios, triclosan, bisfenol A, octilfenol, nonilfenol, e a cafeína como indicador de atividade antrópica. A atividade estrogênica foi medida pelo ensaio Bioluminescent Yeast Estrogen (BLYES), que fornece os resultados em equivalente de 17β-estradiol (EEQ). No entanto, este ensaio não é capaz de prover informações sobre os efeitos adversos em organismos aquáticos. Para observação de possíveis efeitos na biota, os embriões foram expostos a amostras de águas superficiais com resultados acima de 0,1 EEQ no BLYES. Os ensaios foram realizados de acordo com a OECD No. 236 (2013), verificando efeitos agudos como: ausência de batimento cardíaco, não formação de somitos, não desprendimento da cauda, e embrião coagulado. Malformações embrionárias tais como: redução do tamanho do organismo, edema cardíaco e vitelínico, curvatura da coluna vertebral, também foram avaliadas. As informações obtidas pelo ensaio com embriões de Danio rerio foram adequadas para mostrar os efeitos da mistura de contaminantes em organismos não-alvo. A atividade estrogênica medida pelo BLYES ficou abaixo do limite de quantificação (0,1 EEQ) em 44,8% do total de 116 amostras analisadas, e a faixa de atividade estrogênica variou de 0,11 a 14,6 EEQ. Além disso, a presença de contaminantes mesmo que em concentrações baixas ressalta a necessidade de mais estudos para entender os efeitos dessas substâncias nos organismos aquáticos. / Over the last few decades, the occurrence of estrogenic activity and endocrine disrupting chemicals (EDCs) in aquatic environment has become a worldwide issue of increasing environmental concern. The EDCs have the ability to alter the endocrine system of organisms, and includes pharmaceuticals, personal care products, steroid hormones, industrial chemicals, pesticides and many other compounds. Such compounds are present in several industrial and domestic activities and reach the aquatic environment via wastewater discharges or agricultural runoff. The aim of this study was to determine the overall estrogenic activity of surface water, evaluate biological effects on fish embryos development, in order to propose concentrations range based on trigger value to categorize estrogenic activity. Environmental samples were also analyzed by liquid chromatography tandem mass spectrometry to identify substances that are suspected to be an endocrine disruptor. The analyzed compounds were: pesticides, hormones, triclosan, bisphenol A, octylphenol, nonylphenol, and caffeine as an indicative of anthropic activity. The estrogenic activity was measured by Bioluminescent Yeast Estrogen assay (BLYES), with the results expressed in 17β-estradiol equivalent quotient (EEQ). However, this assay is not able to provide information about adverse effects to aquatic organisms. In order to observe effects on aquatic organisms, organic extracts of surface water with results ≥ 0.1 EEQ in BLYES were tested in a bioassay using Danio rerio embryos. The methodology was conducted according OECD No. 236 and verified effects such as: lack of heart beat, lack of somites formation, non-detachment tail and coagulated embryo. Embryonic malformations were also evaluated, such as: reduction of organism size, edema and spine curvature, which are chronic effects. These effects probably are associated with contaminants mixtures. The obtained information by embryonic assay with Danio rerio was suitable to show the effects of contaminants mixture and was used to a categorization proposal of estrogenic activity. Estrogenic activity was below the limit of quantification (0.1 EEQ) in 44.8% of 116 analyzed samples, and range of estrogenic activity was from 0.11 to 14.6 EEQ. The tested samples in FET test were analyzed for acute or chronic toxicity in Danio rerio embryos. Based on the obtained results, even when estrogenic activity is present in surface water, the contaminants mixture can cause toxic effects in non-target organisms. Besides this, the widespread presence of these chemicals highlight the need for further studies in order to understand the harmfulness of these contaminants to aquatic organisms.

Background: Evidence from experimental studies suggests that atrazine and its analytes alter the timing of puberty in laboratory animals. Such associations have not been investigated in humans. Objective: To determine the association between in utero exposure to atrazine analytes and earlier menarche attainment in a nested case-control study of the population-based Avon Longitudinal Study of Parents and Children. Methods: Cases were girls who reported menarche before 11.5 years while controls were girls who reported menarche at or after 11.5 years. Seven atrazine analyte concentrations were measured in maternal gestational urine samples (sample gestation week median (IQR): 12 (8–17)) during the period 1991–1992, for 174 cases and 195 controls using high performance liquid chromatography-tandem mass spectrometry. We evaluated the study association using multivariate logistic regression, adjusting for potential confounders. We used multiple imputation to impute missing confounder data for 29% of the study participants. Results: Diaminochlorotriazine (DACT) was the most frequently detected analyte (58%>limit of detection [LOD]) followed by desethyl atrazine (6%), desethyl atrazine mercapturate (3%), atrazine mercapturate (1%), hydroxyl atrazine (1%), atrazine (1%) and desisopropyl atrazine (0.5%). Because of low detection of other analytes, only DACT was included in the exposure–outcome analyses. The adjusted odds of early menarche for girls with DACT exposures≥median was 1.13 (95% Confidence Interval [95% CI]:0.82, 1.55) and exposure Conclusions: This study is the first to examine the association between timing of menarche and atrazine analytes. We found a weak, non-significant association between in-utero exposure to atrazine metabolite DACT and early menarche, though the association was significant in the subset of girls with complete confounder information. Further exploration of the role of these exposures in female reproduction in other cohorts is needed.

In recent years exposure to endocrine-disrupting chemicals (EDCs) in humans and wildlife has become an increasing concern. These compounds have been found ubiquitously in the environment and are suspected to induce adverse effects on the health of aquatic organisms. The results of health effects due to EDCs are clearly presented in many aquatic organisms, such as the feminization of male fish and a near extinction of some species. No clear effects on human health have been documented at this time. The major sources of these contaminants in the environment are discharges from wastewater treatment plants (WWTP) and diffuse pollution. Conventional wastewater treatment processes are not designed to remove such emerging pollutants and removal efficiency depends on many factors, including treatment technology and pollutant species. Passage through WWTPs and changes due to treatment technologies lead to detection of minute concentrations of EDCs in water downstream from discharge points. In New Orleans, Louisiana, discharge from its East Bank WWTP is being considered for potential reuse for wetland restoration. Therefore, effluents must be treated adequately to prevent adverse effects on the natural biota. Since effluents from wastewater treatment plants using conventional technologies may prove potentially unsafe for the environment due to the presence of EDCs, improved and/or new treatment processes for removal of these contaminants are needed. Ferrate (Fe+6) is known as an alternative oxidant for the treatment of wastewater that can be used as an oxidizing, disinfectant, and/or coagulating agent. Because of its redox potential, ferrate has been used as a disinfection agent and has been reported as a tool for enhanced treatment to remove many micropollutants without producing undesirable disinfection byproducts in contrast to other disinfection processes. Recent research has noted the ability of ferrate to deactivate a wide range of EDCs present in wastewater effluents. The negative effect of effluent's soluble organic matter on ferrate has been reported and higher doses of ferrate may be needed to obtain desired effluent quality. This study found that aerobic biological treatment processes reduce more than half of EDCs in wastewater and that free chlorine disinfection increases estrogenic activity in discharged effluent. Higher organic content in wastewater results in increased ferrate demand. The optimum ferrate dosage to deactivate EDCs in lab scale is 6 ppm, and a dosage of 8 ppm is possibly the operational optimum dose. pH neutralization by concentrated sulfuric acid was not found to affect EDCs deactivation efficiency by ferrate when added at the end of designed contact time. Ferrate was observed to have a high oxidation rate in the first10 minutes after application into wastewater and then degraded to other iron states, such as iron III. Higher oxidation rates can be obtained when more organics are present in wastewater as TOC. Higher dosages required longer oxidation reaction times. Ferrate was observed to generate fewer disinfection byproducts as compared to chlorine. Haloacetic acids in ferrate-treated effluent are generated from organics in wastewater and reactions with residual hypochlorite from the incomplete ferrate synthesis process. The reduction of trihalomethanes may be related to EDCs deactivation by ferrate. Because this study was performed on a lab scale, assessment of onsite production and application of ferrate is required to determine the feasibility of the ferrate treatment process at a full-scale treatment plant and to optimize required dosage. / acase@tulane.edu

Les substances perturbatrices du système endocrinien sont des substances qui, de par leur capacité à induire des changements hormonaux chez les organismes vivants, génèrent des préoccupations dans le domaine de la qualité des eaux et, par extension, dans le domaine du traitement des effluents aqueux. Particulièrement, ce projet de recherche s’est attardé sur l’élimination des perturbateurs endocriniens phénoliques nonylphénol (NP), bisphénol A (BPA) et triclosan (TCS) en solution aqueuse à l’aide de la laccase (E.C. 1.10.3.2) sécrétée par la souche fongique Coriolopsis polyzona. Cette oxydase est une métalloprotéine pouvant catalyser l’oxydation d’une vaste gamme de substances phénoliques. En premier lieu, l’impact du pH et de la température sur l’élimination de ces composés à l’aide de la laccase libre en utilisant un design factoriel. L’oxydation de ces composés produit des oligomères (dimère à pentamère) via le couplage des radicaux phénoxy produits par l’action de la laccase. Il s’avère que les substances produites suite à l’oxydation du NP et du BPA par la laccase ont perdu leurs similitudes structurales avec l’estrogène. Ainsi, l’élimination de l’activité estrogénique de ces substances est directement liée à la transformation des composés. Finalement, l’utilisation d’ABTS comme médiateur a permis d’augmenter le taux d’oxydation enzymatique de ces composés chimiques. Puis, de façon à augmenter la possibilité d’utilisation de la laccase dans des biotechnologies environnementales, cette enzyme a été immobilisée sur un support siliceux et via la réticulation d’agrégats. L’impact des conditions d’immobilisation sur l’activité enzymatique, la stabilité du catalyseur et les propriété biocatalytiques apparentes a été déterminé pour différentes stratégies d’immobilisation. Globalement, l’immobilisation génère un biocatalyseur stable vis-à-vis les dénaturations chimique, physique et biologique. Particulièrement, l’immobilisation sur un support solide produit un biocatalyseur facile à utiliser ayant une faible activité massique et des propriétés cinétiques moindres que celle de l’enzyme libre. La formation de CLEAs de laccase a permis d’obtenir une activité massique élevée et des propriétés cinétiques supérieures à celle de l’enzyme soluble. Ces biocatalyseurs solides ont étés utilisés pour éliminer en continu le NP, BPA et TCS dans différents types de bioréacteur. Le biocatalyseur sur silice a été utilisé pour éliminer ces substances dans un réacteur garni, tandis que les CLEAs ont été utilisés dans un réacteur à lit fluidisé et un réacteur à perfusion développé au cours de ce projet. Ces différentes configurations de bioréacteur ont permis d’éliminer efficacement ces différents perturbateurs endocriniens. Globalement, les différents résultats obtenus, à l’échelle de laboratoire, au cours de ce projet de recherche démontrent que la laccase et particulièrement les biocatalyseurs formés via les différentes stratégies d’immobilisation testées représentent des approches extrêmement prometteuses pour le développement de biotechnologies environnementales vouées à l’élimination des perturbateurs endocriniens phénoliques.

Treatment of waste sludge has become an important issue in recent years around the world. However, the trend of waste sludge treatment has shifted from volume minimization and stabilization to reuse of the sludge and recover the energy potential of it. Therefore, anaerobic treatment of sludge is gaining popularity because of byproduct methane production and high percentage of VSS reduction. Pre-treatment of sludge before anaerobic digestion in order to increase methane production, and ozone pre-treatment in this context, is one such option. Domestic sludge also contains the recently recognized, so called, emerging compounds such as Endocrine Disrupting Compounds (EDCs). Therefore treatment of EDCs in sludge is another challenge in waste sludge treatment since direct discharge of such chemicals may harm the environment by causing gender shifts within the fauna. In this context two hormones (estrone and progesterone), three pharmaceuticals (acetaminophen, carbamazepine and diltiazem) and one plasticizer (benzyl-butyl phthalate) were routinely analyzed in sludge samples which were subjected to treatment during this study. Treatment of EDCs during anaerobic digestion and the effect of ozonation both on the performance of digestion and the treatability of EDCs were investigated in this study. Four 2.5L anaerobic jars were used for anaerobic digestion connected to four 1L plastic graduated cylinders immersed in salt-water to collect the off gas. Anaerobic sludge culture of the reactor and the sludge feed to the reactors were obtained from Ankara Tatlar Wastewater Treatment Plant anaerobic digester and return activated sludge (RAS) line, respectively. One of the anaerobic digesters was used as control (no ozonation) and the others were fed with sludge samples ozonated at three different ozone doses 0.65, 1.33 and 2.65 mg ozone/g biomass. Sludge ages of the reactors were initially set to 25 days and the reactors were fed once every 2 days. The TSS, VSS, total gas volume, COD, pH, CH4 percentage and EDCs were analyzed routinely. In the reactors, operated at 25 days, because of the observation of reduction of TSS, SRT was set to infinity / thus, sludge wastage was terminated. Following the startup it was seen that at 2.65 mg ozone/g biomass dose TSS and VSS did not stay constant in the reactor and dropped sharply in the course of operation, indicating that system was not steady at this SRT. However, upon stoppage of sludge wastage from the reactors, thereby setting SRT to infinity, a steady culture could be maintained in the reactors. Both total gas production and CH4 percentage increased with the increasing doses of ozone with respect to control reactor. For 2.65 mg/g ozonated reactor total gas volume doubled the amount produced in the control reactor. All the EDCs within the scope of this study were analyzed in sludge using ultrasound-aided sequential sludge extraction method twice a week and the results showed that ozonation affected treatment of EDCs for up to 96%. The highest removal rate was obtained with natural hormones. Rates of treatment of pharmaceuticals were the second best.

Treatment of waste sludge has become an important issue in recent years around the world. However, the trend of waste sludge treatment has shifted from volume minimization and stabilization to reuse of the sludge and recover the energy potential of it. Therefore, anaerobic treatment of sludge is gaining popularity because of byproduct methane production and high percentage of VSS reduction. Pre-treatment of sludge before anaerobic digestion in order to increase methane production, and ozone pre-treatment in this context, is one such option. Domestic sludge also contains the recently recognized, so called, emerging compounds such as Endocrine Disrupting Compounds (EDCs). Therefore treatment of EDCs in sludge is another challenge in waste sludge treatment since direct discharge of such chemicals may harm the environment by causing gender shifts within the fauna. In this context two hormones (estrone and progesterone), three pharmaceuticals (acetaminophen, carbamazepine and diltiazem) and one plasticizer (benzyl-butyl phthalate) were routinely analyzed in sludge samples which were subjected to treatment during this study. Treatment of EDCs during anaerobic digestion and the effect of ozonation both on the performance of digestion and the treatability of EDCs were investigated in this study. Four 2.5L anaerobic jars were used for anaerobic digestion connected to four 1L plastic graduated cylinders immersed in salt-water to collect the off gas. Anaerobic sludge culture of the reactor and the sludge feed to the reactors were obtained from Ankara Tatlar Wastewater Treatment Plant anaerobic digester and return activated sludge (RAS) line, respectively. One of the anaerobic digesters was used as control (no ozonation) and the others were fed with sludge samples ozonated at three different ozone doses 0.65, 1.33 and 2.65 mg ozone/g biomass. Sludge ages of the reactors were initially set to 25 days and the reactors were fed once every 2 days. The TSS, VSS, total gas volume, COD, pH, CH4 percentage and EDCs were analyzed routinely. In the reactors, operated at 25 days, because of the observation of reduction of TSS, SRT was set to infinity / thus, sludge wastage was terminated. Following the startup it was seen that at 2.65 mg ozone/g biomass dose TSS and VSS did not stay constant in the reactor and dropped sharply in the course of operation, indicating that system was not steady at this SRT. However, upon stoppage of sludge wastage from the reactors, thereby setting SRT to infinity, a steady culture could be maintained in the reactors. Both total gas production and CH4 percentage increased with the increasing doses of ozone with respect to control reactor. For 2.65 mg/g ozonated reactor total gas volume doubled the amount produced in the control reactor. All the EDCs within the scope of this study were analyzed in sludge using ultrasound-aided sequential sludge extraction method twice a week and the results showed that ozonation affected treatment of EDCs for up to 96%. The highest removal rate was obtained with natural hormones. Rates of treatment of pharmaceuticals were the second best.

Previous research indicates that pharmaceutically active compounds (PhACs) and endocrine-disrupting compounds (EDCs) are poorly removed during conventional drinking water treatment processes including coagulation; however, removal efficiency increases in the presence of natural organic matter (NOM). Therefore, this project investigates the link between various NOM types with EDC/PhAC removal. Bench-scale coagulation tests were conducted on three different source waters spiked with environmentally relevant levels (nominally 1000 ng/L) of EDCs/PhACs. Two different coagulants were used: polyaluminum chloride (PACl) and aluminum sulphate (alum). NOM was characterized using size exclusion liquid chromatography-organic carbon detection (LC-OCD). Results for Lake Ontario, Otonabee and Grand River water indicate that certain EDCs/PhACs are significantly removed during coagulation while others increase in concentration. Concurrently, particular NOM fractions (biopolymers and humic substances) are also being removed. Solvents used for EDC/PhAC spiking (acetone and acetonitrile) did not affect coagulation, but contributed to low molecular weight neutral and hydrophobic NOM fractions.

Previous research indicates that pharmaceutically active compounds (PhACs) and endocrine-disrupting compounds (EDCs) are poorly removed during conventional drinking water treatment processes including coagulation; however, removal efficiency increases in the presence of natural organic matter (NOM). Therefore, this project investigates the link between various NOM types with EDC/PhAC removal. Bench-scale coagulation tests were conducted on three different source waters spiked with environmentally relevant levels (nominally 1000 ng/L) of EDCs/PhACs. Two different coagulants were used: polyaluminum chloride (PACl) and aluminum sulphate (alum). NOM was characterized using size exclusion liquid chromatography-organic carbon detection (LC-OCD). Results for Lake Ontario, Otonabee and Grand River water indicate that certain EDCs/PhACs are significantly removed during coagulation while others increase in concentration. Concurrently, particular NOM fractions (biopolymers and humic substances) are also being removed. Solvents used for EDC/PhAC spiking (acetone and acetonitrile) did not affect coagulation, but contributed to low molecular weight neutral and hydrophobic NOM fractions.

Treatment of waste sludge has become an important issue in recent years around the world. However, the trend of waste sludge treatment has shifted from volume minimization and stabilization to reuse of the sludge and recover the energy potential of it. Therefore, anaerobic treatment of sludge is gaining popularity because of byproduct methane production and high percentage of VSS reduction. Pre-treatment of sludge before anaerobic digestion in order to increase methane production, and ozone pre-treatment in this context, is one such option. Domestic sludge also contains the recently recognized, so called, emerging compounds such as Endocrine Disrupting Compounds (EDCs). Therefore treatment of EDCs in sludge is another challenge in waste sludge treatment since direct discharge of such chemicals may harm the environment by causing gender shifts within the fauna. In this context two hormones (estrone and progesterone), three pharmaceuticals (acetaminophen, carbamazepine and diltiazem) and one plasticizer (benzyl-butyl phthalate) were routinely analyzed in sludge samples which were subjected to treatment during this study. Treatment of EDCs during anaerobic digestion and the effect of ozonation both on the performance of digestion and the treatability of EDCs were investigated in this study. Four 2.5L anaerobic jars were used for anaerobic digestion connected to four 1L plastic graduated cylinders immersed in salt-water to collect the off gas. Anaerobic sludge culture of the reactor and the sludge feed to the reactors were obtained from Ankara Tatlar Wastewater Treatment Plant anaerobic digester and return activated sludge (RAS) line, respectively. One of the anaerobic digesters was used as control (no ozonation) and the others were fed with sludge samples ozonated at three different ozone doses 0.65, 1.33 and 2.65 mg ozone/g biomass. Sludge ages of the reactors were initially set to 25 days and the reactors were fed once every 2 days. The TSS, VSS, total gas volume, COD, pH, CH4 percentage and EDCs were analyzed routinely. In the reactors, operated at 25 days, because of the observation of reduction of TSS, SRT was set to infinity / thus, sludge wastage was terminated. Following the startup it was seen that at 2.65 mg ozone/g biomass dose TSS and VSS did not stay constant in the reactor and dropped sharply in the course of operation, indicating that system was not steady at this SRT. However, upon stoppage of sludge wastage from the reactors, thereby setting SRT to infinity, a steady culture could be maintained in the reactors. Both total gas production and CH4 percentage increased with the increasing doses of ozone with respect to control reactor. For 2.65 mg/g ozonated reactor total gas volume doubled the amount produced in the control reactor. All the EDCs within the scope of this study were analyzed in sludge using ultrasound-aided sequential sludge extraction method twice a week and the results showed that ozonation affected treatment of EDCs for up to 96%. The highest removal rate was obtained with natural hormones. Rates of treatment of pharmaceuticals were the second best.

Evidence from an abundant number of studies suggests that human female reproductive functions have become impaired over the past half century and that there might be a relationship between endocrine disrupting chemicals (EDCs) and reduced fertility. It is, however, not known by what mechanisms EDCs affect different reproductive functions such as endometrial receptivity, embryo implantation and placentation. The endometrium is continuously changing its morphological and functional properties, responding to cyclic changes of oestrogen and progesterone levels during the menstrual cycle. These changes include monthly preparation for embryo implantation through changed endometrial angiogenic activity and consequent changes in endometrial vasculature. Use of primary human endometrial endothelial cells (HEECs) in this work was evaluated as a possible screening tool for effects caused by EDCs on human endometrial vasculature and subsequently on various endometrial functions. In this study HEEC and endometrial stromal cells were isolated. HEECs were grown in monocultures, and together with stromal cells in co-cultures, and exposed to endocrine active substances. These were cadmium, which has oestrogenic properties, tamoxifen, with anti-oestrogenic effects, mifepristone, which is an anti-progestin, and bisphenol A, with oestrogenic properties. The effects were evaluated by using proliferation and viability assays, migration and tube formation assays, quantitative PCR (qPCR), immunohistochemistry and western blot. Cadmium affected the expression of angiogenesis-related genes, and caused different effects in HEECs cultured alone vs. HEECs co-cultured with stromal cells. Tamoxifen altered the expression of angiogenesis-related genes and reduced HEEC migration, thus having an anti-angiogenic effect. Mifepristone caused reduced formation of tubular structures in tube-formation assays involving HEECs co-cultured with stromal cells. Bisphenol A promoted tube formation in co-cultured HEECs which was related to changes in the expression of several angiogenesis-related genes as well as up-regulated expression of VEGF-D protein. In conclusion, we showed that EDCs have the ability to induce changes in endometrial angiogenic activity in vitro and may thus disturb normal endometrial functions related to fertility and pregnancy. HEECs grown in vitro may provide valuable information on the effects of EDCs on human endometrial functions. However, this model is not suitable as a large-scale screening tool.