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The microbial source tracking (MST) methods of antibiotic resistance analysis (ARA) and fluorometry (to detect optical brighteners in detergents) were used in the summers of 2004 and 2005 to determine the origins of fecal pollution at beaches with a past history of, or the potential for, high enterococci counts and posted advisories. At Hilton and Anderson beaches, ARA and fluorometry in the summer of 2004 detected substantial human-origin pollution in locations producing consistently high counts of Enterococcus spp. Investigations by municipal officials led to the fluorometric detection and subsequent repair of sewage infrastructure problems at both beaches. The success of these mitigation efforts was confirmed during the summer of 2005 using ARA and fluorometry, with the results cross-validated by pulsed-field gel electrophoresis (PFGE). Results at other beaches indicated that birds and/or wildlife were largely responsible for elevated enterococci levels during 2004 and 2005. The application of fluorometry proved difficult in opens waters due to high levels of dilution, but showed potential for use in storm drains. An additional study developed and tested a new library-based MST approach based on the pattern of DNA band lengths produced by the amplification of the 16S-23S rDNA intergenic spacer region, and subsequent digestion using the restriction endonuclease MboI. Initial results from small known-source libraries yielded high average rates of correct classification (ARCC). However, an increase in the library size was accompanied by a reduction in the ARCC of the library and the method was deemed unsuccessful, and unsuitable for field application. A final study focused on the potential for classification bias with disproportionate source category sizes using discriminant analysis (DA), logistic regression (LR), and k-nearest neighbor (K-NN) statistical classification algorithms. Findings indicated that DA was the most robust algorithm for use with source category imbalance when measuring both correct and incorrect classification rates. Conversely k-NN was identified as the most sensitive algorithm to imbalances with the greatest levels of distortion obtained from the highest k values. Conclusions of this project include: 1) application of a validation set, as well as a minimum detectable percentage to known-source libraries aids in accurately assessing the classification power of the library and reducing the false positive identification of contributing fecal sources; 2) the validation of MST results using multiple methods is recommended for field applications; 3) fluorometry displayed potential for detecting optical brighteners as indicators of sewage leaks in storm drains; 4) the digestion of the 16S-23S rDNA intergenic spacer region of Enterococcus spp. using MboI does not provided suitable discriminatory power for use as an MST method; and 5) DA was the least, and k-NN the most, sensitive algorithm to imbalances in the size of source categories in a known-source library. / Ph. D.

[pt] O presente trabalho tem como objetivo investigar a contaminação por esgotos domésticos do sistema estuarino formado pelo rio Iguaçu e canal de Sarapuí, localizados na porção noroeste da Baía de Guanabara, através da determinação de esteróis e composição elementar (C e N) da matéria orgânica em amostras de material particulado em suspensão e sedimentos, e da caracterização físico-química e química da água e sedimento. As amostragens foram realizadas em março/2004 e setembro/2004, em 7 estações de água e 10 de sedimento, distribuídas entre 6 km a montante do rio Iguaçu e 4 km dentro da baía. Também foi coletada uma amostra composta de esgoto bruto na Estação de Tratamento da Ilha do Governador (ETIG). Na determinação dos parâmetros foram utilizados métodos analíticos já estabelecidos, tais como cromatografia a gás/espectrometria de massa (esteróis) e oxidação a seco (C e N), após otimização para as condições específicas de campo e de laboratório. As concentrações dos principais esteróis variam em escala temporal e espacial, em função de fatores climáticos e da localização de fontes difusas de esgoto. Há gradientes decrescentes de concentração de esteróis entre rio e a baía, com predomínio de esteróis fecais sobre fitoesteróis. Coprostanona (5b-colestan-3b-ona) atingindo concentrações de até 21 mg gC-1, epicolestanol (5a-colestan-5a-ol) e coprostanol (5b-colestan-3b- ol) foram os mais abundantes. A detecção de epicolestanol (inédita para a baía de Guanabara) em concentrações elevadas no material particulado e sedimento (assim como no esgoto bruto), as diferenças na composição entre esteróis no esgoto bruto em comparação com o material particulado e sedimento, e os resultados das razões entre determinados esteróis, são resultados que refletem a importância dos processos bacterianos em alterar a composição original da matéria orgânica e dos esteróis associados. O conjunto de informações levantadas confirma o elevado nível de degradação ambiental da região estudada. Por outro lado, a dinâmica do sistema e processos pré- e pós- deposicionais influenciam na geoquímica dos esteróis e, portanto, seus efeitos devem ser levados em consideração em estudos sobre marcadores moleculares em estuários com características semelhantes ao do presente trabalho. / [en] The contamination by domestic sewage of the Iguaçu River and Sarapuí channel, located in the north-western sector of Guanabara Bay, was investigated by the determination of sterols and elemental composition (C and N) of organic matter in suspended matter and superficial sediments. Physico-chemical and chemical characterization of water and sediments were considered as well. Conventional analytical methods, like gas chromatography/mass spectrometry (sterols) and dry chemical oxidation (C and N), were employed after optimization for specific field and laboratory conditions. In March and September/2004 water/suspended particles (7 sample stations) and sediment (10 stations) were collected in a transect from 6 km up the river until 4 km off the river, in the bay. An additional sample of bulk sewage was also collected at a sewage treatment plant close to the studied area. The faecal sterols predominate over phyto sterols in suspended matter and sediments. Among the 14 sterols quantified, higher concentrations (up to 21 mg gC-1) were measured for coprostanone (5b-cholestan- 3b-one), epicholestanol (5a-cholestan-3a-ol) and coprostanol (5b-cholestan-3b- ol). In general, there is a river-bay decreasing gradient in sterol concentration, associated with the location of diffuse sources of sewage. Seasonal variation in climatic factor also influenced in the sterols distribution. The presence of epicholestanol (reported here for the first time in Guanabara Bay) in relatively high concentrations in suspended matter and sediments (as well as in raw sewage), the differences in sterol composition between raw sewage and suspended matter/sediments, and the values obtained for selected sterols source-diagnostic ratios, suggested the occurrence of significant microbial alteration of organic matter, with implications on the geochemistry of sterols. The elevated degradation

Ambient and recreational surface waters worldwide experience fecal pollution due to a variety of anthropogenic sources. Fecal waste has been proven, for over a century, to harbor pathogenic microorganisms which subsequently cause a variety of disease and illness in human hosts. The benefits of utilizing fecal indicator bacteria (FIB) as a simple, inexpensive means to detect fitful human pathogens within a variety of water matrices are vast. However, no universal agreement exists in regard to which indicator is best suited for detection of fecal contamination and pathogens in environmental waters, and no single standard for bacterial indicators has been federally mandated. This study sought to explore the potential benefits of a multiple-indicator approach to water quality analysis of fresh and brackish surface waters. The distribution and fluctuation of two frequently used, EPA approved groups of FIB – fecal coliform and Enterococcus – were explored, and relationships between the two FIB groups were examined in fresh and brackish surface waters of Central and South Florida. Samples were collected over a period of 12 consecutive months, spanning April 2015 through March 2016, and analyzed using membrane filtration procedures outlined in Standard Methods 9222D and EPA method 1600. Raw and log transformed colony forming unit (CFU) data, per 100 mL, was analyzed annually and seasonally through linear regression, Spearman correlation, and exploratory data analysis techniques performed in R-Studio. The results of this study showed a moderate to strong relationship between fecal coliform and Enterococcus under both fresh and brackish conditions. The presence of a positive, linear relationship between fecal coliform and Enterococcus in both fresh and brackish water was apparent in both seasonal and annual regression analysis; upward and downward fluctuation(s) in one variable was shown to predict similar fluctuation(s) in the other year-round. However, while fecal coliform and Enterococcus showed moderate to strong correlations, causation was not implied. Low R2 values showed that the FIB groups were not dependent upon one another in any case, either annually or seasonally. The results of this study challenge previously accepted views of fecal coliform and Enterococcus effectiveness as ideal fresh and brackish water FIB, their suitability as sole indicators of fecal pollution, and their ideal usage as indicators for waters of varying salinities; results support those previously seen in studies such as Hanes and Fragala 1967, which emphasize the need for a multiple indicator approach to water quality analysis of ambient and recreational waters experiencing brackish conditions.

Animal and human wastes introduce pathogens into rivers and streams, creating human health and economic burdens. While direct monitoring for pathogens is possible, it is impractical due to the sporadic distribution of pathogens, cost to identify, and health risks to laboratory workers. To overcome these issues, fecal indicator organisms are used to estimate the presence of pathogens. Although fecal indicators generally protect public health, they fall short in their utility because of difficulties in public health risk characterization, inconsistent correlations with pathogens, weak source identification, and their potential to persist in environments with no point sources of fecal pollution. This research focuses on characterizing the ecology of fecal indicators using both modeling and metabolic indicators to better understand the processes that drive fecal pollution. Fecal indicator impairment was modeled in Sinking Creek, a 303 (d) listed stream in Northeast Tennessee, using the ecological niche model, Maxent, for two different fecal indicators. While the use of Maxent has been well demonstrated at the macroscale, this study introduces its application to ecological niches at the microscale. Stream impairment seasonality was exhibited in two different indicators over multiple years and different resolutions (quarterly versus monthly sampling programs). This stresses the need for multiple year and month sampling to capture heterogeneity in fecal indicator concentrations. Although discharge is strongly associated with dissolved solutes, fecal indicator impairment was governed by other ecological factors such as populations of heterotrophic bacteria, enzyme activity, nutrient conditions, and other metabolic indicators. This research also incorporated metabolic indicators to characterize spatiotemporal variability in microbial community function, making connections to fecal and other pollution gradients. Communities differed in their ability to use a wide variety of substrates, and metabolic inhibition in sediments captured most of the interaction of aquatic and benthic communities. Sediment substrate activity was also indicative of degrees of pollution, suggesting that sediment is a potential reservoir for Escherichia coli in this stream, and there is possibility for resuspension, extended residence times, and increased duration for exposure. This research highlights the benefit of using models and other microbial indicators to better understand how environment shapes the niche of fecal indicators.

O Brasil possui em sua área costeira grandes metrópoles onde diversas atividades humanas são desenvolvidas, podendo trazer prejuízos à qualidade das águas costeiras. O litoral de São Paulo, constituído por 16 municípios incluindo-se Cubatão, apresenta peculiaridades diferenciadas. O tipo de poluição mais encontrado na costa paulista é a poluição fecal causada por despejo de esgotos domésticos, os quais transportam organismos patogênicos cuja presença pode levar a agravos à saúde humana. O objetivo deste estudo é o de avaliar a qualidade sanitária de águas e areias de praias da Baixada Santista. Assim amostras de água e areia (úmida e seca) de três praias, como Gonzaguinha (São Vicente), Boqueirão (Santos) e Pitangueiras (Guarujá) foram coletadas com freqüência mensal pelo período de um ano nas quais foram analisados os seguintes parâmetros: coliformes termotolerantes, Escherichia coli, enterococos, e ainda Staphylococcus coagulase (+), Pseudomonas aeruginosa, Candida albicans e dermatófitos. Para a realização das análises em amostras de água utilizou-se da Técnica da Membrana Filtrante e para as amostras de areia a Técnica de Tubos Múltiplos, de acordo com Normas Técnicas CETESB e Standard Methods. Para a determinação de dermatófitos, as amostras de água foram concentradas pela Técnica de Membrana Filtrante e as amostras de areia (seca e úmida) foram analisadas pela Técnica de inoculação em superfície e, concomitantemente, pela Técnica da Isca de Cabelo. Os resultados obtidos revelaram que 95 por cento das amostras analisadas foram positivas para a presença dos indicadores de contaminação fecal, seguidos por P. aeruginosa, presente em 75 por cento das amostras. C. albicans foi o microrganismo menos freqüente (6,7 por cento) e foi detectada somente nas amostras de água. O dermatófito Microsporum sp foi detectado somente nas amostras de areia e esteve presente nas três praias. De maneira geral, o comportamento das concentrações dos microrganismos foi maior nas amostras de areia seca, independentemente dos eventos de chuva, seguidas pela areia úmida, sendo as menores concentrações observadas nas amostras de água. Concluiu-se que as concentrações dos microrganismos indicadores de poluição fecal e dos patogênicos foram elevadas nas matrizes estudadas e que é necessário dirigir esforços para a melhoria da qualidade das águas e areias, pois as condições observadas trazem preocupação à saúde dos banhistas e freqüentadores das praias / Brazilian seashore comprises many big cities and the diverse activities which are performed in these areas can bring damages to seashore water quality. São Paulo State seashore comprises 16 cities including Cubatão, where each one shows their own particularities. In the São Paulo State seashore is mostly observed fecal pollution due to launching sewage into these waters which transport pathogens with potential to cause diseases. The objective of this study is to assess the sanitary quality of recreational waters and wet and dry sand from three beaches located at Baixada Santista such as Gonzaguinha (São Vicente), Boqueirão (Santos) and Pitangueiras (Guarujá). The samples (water, wet and dry sand) were collected with monthly frequency throughout a year and the following parameters were analyzed: thermotolerant coliforms, Escherichia coli, enterococos, Staphylococcus coagulase +, Pseudomonas aeruginosa, Candida albicans and dermatophytes. The bacteriological and C. albicans analyzes were performed according to CETESB and Standard Methods 21st edition. Dermatophyte analysis from water samples were carried out by membrane filtering technique and by spread plate technique and hair-baiting technique for sand samples. The results revealed that 95 per cent of the water samples were positive for fecal indicators following by P. aeruginosa (75 per cent). C. albicans was detected only in water samples showing 6.7 per cent of the observed frequency. Microsporum sp specie was detected only in sand samples in all beaches studied. In general, high microorganisms concentrations was found in dry sand samples regardless raining events, following by wet sand and the lowest concentrations were found in water samples. It was possible to conclude that fecal indicators microorganisms and pathogens concentrations were high in all samples analyzed and it highlights to necessity to address efforts to improve the quality of the water and sand because this poor quality condition found poses a significant public health concern

O Brasil possui em sua área costeira grandes metrópoles onde diversas atividades humanas são desenvolvidas, podendo trazer prejuízos à qualidade das águas costeiras. O litoral de São Paulo, constituído por 16 municípios incluindo-se Cubatão, apresenta peculiaridades diferenciadas. O tipo de poluição mais encontrado na costa paulista é a poluição fecal causada por despejo de esgotos domésticos, os quais transportam organismos patogênicos cuja presença pode levar a agravos à saúde humana. O objetivo deste estudo é o de avaliar a qualidade sanitária de águas e areias de praias da Baixada Santista. Assim amostras de água e areia (úmida e seca) de três praias, como Gonzaguinha (São Vicente), Boqueirão (Santos) e Pitangueiras (Guarujá) foram coletadas com freqüência mensal pelo período de um ano nas quais foram analisados os seguintes parâmetros: coliformes termotolerantes, Escherichia coli, enterococos, e ainda Staphylococcus coagulase (+), Pseudomonas aeruginosa, Candida albicans e dermatófitos. Para a realização das análises em amostras de água utilizou-se da Técnica da Membrana Filtrante e para as amostras de areia a Técnica de Tubos Múltiplos, de acordo com Normas Técnicas CETESB e Standard Methods. Para a determinação de dermatófitos, as amostras de água foram concentradas pela Técnica de Membrana Filtrante e as amostras de areia (seca e úmida) foram analisadas pela Técnica de inoculação em superfície e, concomitantemente, pela Técnica da Isca de Cabelo. Os resultados obtidos revelaram que 95 por cento das amostras analisadas foram positivas para a presença dos indicadores de contaminação fecal, seguidos por P. aeruginosa, presente em 75 por cento das amostras. C. albicans foi o microrganismo menos freqüente (6,7 por cento) e foi detectada somente nas amostras de água. O dermatófito Microsporum sp foi detectado somente nas amostras de areia e esteve presente nas três praias. De maneira geral, o comportamento das concentrações dos microrganismos foi maior nas amostras de areia seca, independentemente dos eventos de chuva, seguidas pela areia úmida, sendo as menores concentrações observadas nas amostras de água. Concluiu-se que as concentrações dos microrganismos indicadores de poluição fecal e dos patogênicos foram elevadas nas matrizes estudadas e que é necessário dirigir esforços para a melhoria da qualidade das águas e areias, pois as condições observadas trazem preocupação à saúde dos banhistas e freqüentadores das praias / Brazilian seashore comprises many big cities and the diverse activities which are performed in these areas can bring damages to seashore water quality. São Paulo State seashore comprises 16 cities including Cubatão, where each one shows their own particularities. In the São Paulo State seashore is mostly observed fecal pollution due to launching sewage into these waters which transport pathogens with potential to cause diseases. The objective of this study is to assess the sanitary quality of recreational waters and wet and dry sand from three beaches located at Baixada Santista such as Gonzaguinha (São Vicente), Boqueirão (Santos) and Pitangueiras (Guarujá). The samples (water, wet and dry sand) were collected with monthly frequency throughout a year and the following parameters were analyzed: thermotolerant coliforms, Escherichia coli, enterococos, Staphylococcus coagulase +, Pseudomonas aeruginosa, Candida albicans and dermatophytes. The bacteriological and C. albicans analyzes were performed according to CETESB and Standard Methods 21st edition. Dermatophyte analysis from water samples were carried out by membrane filtering technique and by spread plate technique and hair-baiting technique for sand samples. The results revealed that 95 per cent of the water samples were positive for fecal indicators following by P. aeruginosa (75 per cent). C. albicans was detected only in water samples showing 6.7 per cent of the observed frequency. Microsporum sp specie was detected only in sand samples in all beaches studied. In general, high microorganisms concentrations was found in dry sand samples regardless raining events, following by wet sand and the lowest concentrations were found in water samples. It was possible to conclude that fecal indicators microorganisms and pathogens concentrations were high in all samples analyzed and it highlights to necessity to address efforts to improve the quality of the water and sand because this poor quality condition found poses a significant public health concern