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Determining water quality : development of a cell culture cytotoxicity assayHumphries, Petro 19 September 2005 (has links)
Not only in South Africa, but also all over the world, pollution is threatening the quality of water resources. More than 100,000 chemicals are produced worldwide and some of these chemicals enter into the aquatic environment, posing a potential threat to humans, animals as well as other organisms. Therefore, determining the quality of water is of great importance. The testing of the toxicity of water samples are either based on a biological model or chemical model. In the biological model, organisms are utilized as indicators for toxicity of a particular sample, this sample may then be cytotoxic to the cells of the organism. In the chemical model the amount of a specific chemical present in water samples are predicted. Although, within the biological model there are many cytotoxicity screening assays (also called bioassays) that assess quality of water, there is a need for a fast, efficient and cost-effective cell culture system that may act as first screening procedure in a range of tests. Of particular importance in the context of this thesis, are bioassays that measure specific biochemical parameters, e.g. the Uridine uptake bioassay, MTT (3-(4,5¬dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay and the bioluminescent assay. An important advantage of these assays are their sensitivity and the fact that the tests are principally based on cell mortality as a consequence of membrane damage or influence on cell metabolism. Researchers at the Highveld Biological Laboratory in South Africa have the past few years paid particular attention to the MTT assay, originally developed by Mosmann, and have modified this assay. In this thesis the adjusted method is referred to as the Modified Highveld Biological assay. There were, however, still a few adjustments to be made in order to use this modified method to its full potential. The Bioluminescent assay, also were thought to have potential to be modified as water quality screening test. Therefore, the hypotheses investigated in the current thesis were: • The original Mosmann MTT method can be modified successfully to provide a sensitive, reproducible mass screening method for determining chemical cytotoxicity and water quality, by using the K-562 cell line. • Sensitivity of the assay can be increased by utilizing a 3 phase medium cycle (Medium type 1-3) instead of the previously used single culture medium. • Furthermore, this cytotoxicity assay can be successfully utilized to determine whether any chemical solution or water sample is cytotoxic. Thus information was needed to determine whether cytotoxicity correlates with real life toxicity. This requires time-consuming epidemiological surveys unless results can be correlated with previous surveys. The research in this thesis attempted to indicate that the obtained cytotoxicity can be used to predict toxicity of a water sample, and that the cytotoxicity findings of this dissertation may indeed be meaningful. The culturing conditions of the Modified Highveld Biological MTT assay were modified successfully and could thus be utilized as the first assay in a battery of tests to determine overall cytotoxicity by utilising K¬652 cells in culture. Due to successful modification of the assay, this MTT assay now reduces assay duration, thereby saving important resources. An attempt was also made to optimise the Bioluminescent assay. This method is based on the fact that adenosine triphosphate (ATP) is present in all living cells. The method utilizes an enzyme, firefly luciferase, which catalyses the formation of light from ATP and luciferin. The emitted light intensity is linearly dependent on the ATP . concentration and is measured using a luminometer. The following problems were, however, identified that renders this method unsatisfactory as cytotoxicity indicator for water samples. • The luciferase enzyme is too sensitive to too many different substances used in the cell culture medium and in the preparation procedures of the cells (ions, salts etc.) which makes it less suited for usage for fast, effective testing of water toxicity. • This method will probably function better when luciferase can be carried into the cells or form part of the cells' genetic material. • We therefore suggest that, the Bioluminescent assay might possibly be a method to determine cytotoxicity, if the sensitivity of the luciferase enzyme could be further investigated. Thus it could be concluded that luminescence is not suitable for assaying complex mixtures because it is possible for unknown non~toxic agents, present in test samples, to interfere with the process of light emission (quenching). Lastly, known chemical solutions as well as unknown water samples were screened using the adjustments to the Modified Highveld Biological Method proposed in this thesis. The cytotoxicity of unknown water samples and chemical solutions were successfully determined and different cytotoxic effects were obtained, e.g. synergism, antagonism, additive effects and neutral effects. It was also possible to reduce or remove the cytotoxicity of certain water samples by applying pre~treatment with either Na2CO3 (removing possible toxic divalent and polyvalent metals) or SepPak cartridge clean up (removing organic toxicants). Results indicated that the method is very sensitive and can detect even low traces of toxicants. Thus, it can be concluded that the method was successfully adjusted to be useful as a first screening assay for toxicity analysis of a series of environmental water samples. / Dissertation (MSc (Anatomy))--University of Pretoria, 2006. / Anatomy / unrestricted
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Fate and Transformation of a Conjugated Natural Hormone 17β-Estradiol-3-Glucuronide in Soil-Water SystemsShrestha, Suman Lal January 2011 (has links)
The objectives of the study were to investigate the sorption and degradation of a glucuronide conjugated natural hormone, 17β-estradio1-3-glucuronide (E2-3G), and its estrogenic metabolites in soil-water systems. Radiolabeled E2-3G was first synthesized in the laboratory. Soil-water batch experiments were then conducted using natural and sterilized topsoil (0-6 cm) and subsoil (18-24 cm) with the radiolabeled E2-3G to investigate the effects of soil organic matter content and microbial activity. The aqueous dissipation of 14C in the batch experiments followed a biphasic pattern with an initial rapid dissipation phase followed by a second slower phase. Significant differences in total aqueous 14C dissipation were observed for the different initial concentrations for both soils, with greater persistence of intact E2-3G at higher initial concentrations. / National Science Foundation [Grant No. 0730492]
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Statistical and Machine Learning Methods for Pattern Identification in Environmental MixturesGibson, Elizabeth Atkeson January 2021 (has links)
Background: Statistical and machine learning techniques are now being incorporated into high-dimensional mixture research to overcome issues with traditional methods. Though some methods perform well on specific tasks, no method consistently outperforms all others in complex mixture analyses, largely because different methods were developed to answer different research questions. The research presented here concentrates on answering a single mixtures question: Are there exposure patterns within a mixture corresponding with sources or behaviors that give rise to exposure?
Objective: This dissertation details work to design, adapt, and apply pattern recognition methods to environmental mixtures and introduces two methods adapted to specific challenges of environmental health data, (1) Principal Component Pursuit (PCP) and (2) Bayesian non-parametric non-negative matrix factorization (BN²MF). We build on this work to characterize the relationship between identified patterns of in utero endocrine disrupting chemical (EDC) exposure and child neurodevelopment.
Methods: PCP---a dimensionality reduction technique in computer vision---decomposes the exposure mixture into a low-rank matrix of consistent patterns and a sparse matrix of unique or extreme exposure events. We incorporated two existing PCP extensions that suit environmental data, (1) a non-convex rank penalty, and (2) a formulation that removes the need for parameter tuning. We further adapted PCP to accommodate environmental mixtures by including (1) a non-negativity constraint, (2) a modified algorithm to allow for missing values, and (3) a separate penalty for measurements below the limit of detection (PCP-LOD).
BN²MF decomposes the exposure mixture into three parts, (1) a matrix of chemical loadings on identified patterns, (2) a matrix of individual scores on identified patterns, and (3) and diagonal matrix of pattern weights. It places non-negative continuous priors on pattern loadings, weights, and individual scores and uses a non-parametric sparse prior on the pattern weights to estimate the optimal number. We extended BN²MF to explicitly account for uncertainty in identified patterns by estimating the full distribution of scores and loadings.
To test both methods, we simulated data to represent environmental mixtures with various structures, altering the level of complexity in the patterns, the noise level, the number of patterns, the size of the mixture, and the sample size. We evaluated PCP-LOD's performance against principal component analysis (PCA), and we evaluated BN²MF's performance against PCA, factor analysis, and frequentist nonnegative matrix factorization (NMF). For all methods, we compared their solutions with true simulated values to measure performance. We further assessed BN²MF's coverage of true simulated scores.
We applied PCP-LOD to an exposure mixture of 21 persistent organic pollutants (POPs) measured in 1,000 U.S. adults from the 2001--2002 National Health and Nutrition Examination Survey (NHANES). We applied BN²MF to an exposure mixture of 17 EDCs measured in 343 pregnant women in the Columbia Center for Children’s Environmental Health's Mothers and Newborns Cohort.
Finally, we designed a two-stage Bayesian hierarchical model to estimate health effects of environmental exposure patterns while incorporating the uncertainty of pattern identification. In the first stage, we identified EDC exposure patterns using BN²MF. In the second stage, we included individual pattern scores and their distributions as exposures of interest in a hierarchical regression model, with child IQ as the outcome, adjusting for potential confounders. We present sex-specific results.
Results: PCP-LOD recovered the true number of patterns through cross-validation for all simulations; based on an a priori specified criterion, PCA recovered the true number of patterns in 32% of simulations. PCP-LOD achieved lower relative predictive error than PCA for all simulated datasets with up to 50% of the data < LOD. When 75% of values were < LOD, PCP-LOD outperformed PCA only when noise was low.
In the POP mixture, PCP-LOD identified a rank three underlying structure. One pattern represented comprehensive exposure to all POPs. The other two patterns grouped chemicals based on known properties such as structure and toxicity. PCP-LOD also separated 6% of values as extreme events. Most participants had no extreme exposures (44%) or only extremely low exposures (18%).
BN²MF estimated the true number of patterns for 99% of simulated datasets. BN²MF's variational confidence intervals achieved 95% coverage across all levels of structural complexity with up to 40% added noise. BN²MF performed comparably with frequentist methods in terms of overall prediction and estimation of underlying loadings and scores.
We identified two patterns of EDC exposure in pregnant women, corresponding with diet and personal care product use as potentially separate sources or behaviors leading to exposure. The diet pattern expressed exposure to phthalates and BPA. One standard deviation increase in this pattern was associated with a decrease of 3.5 IQ points (95% credible interval: -6.7, -0.3), on average, in female children but not in males. The personal care product pattern represented exposure to phenols, including parabens, and diethyl phthalate. We found no associations between this pattern and child cognition.
Conclusion: PCP-LOD and BN^2MF address limitations of existing pattern recognition methods employed in this field such as user-specified pattern number, lack of interpretability of patterns in terms of human understanding, influence of outlying values, and lack of uncertainty quantification.
Both methods identified patterns that grouped chemicals based on known sources (e.g., diet), behaviors (e.g., personal care product use), or properties (e.g., structure and toxicity). Phthalates and BPA found in food packaging and can linings formed a BN²MF-identified pattern of EDC exposure negatively associated with female child intelligence in the Mothers and Newborns cohort. Results may be used to inform interventions designed to target modifiable behavior or regulations to act on dietary exposure sources.
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Toxicological screening of imidazolium based ionic liquidsStenström, Joakim January 2020 (has links)
Ionic liquids are salts that are in liquid form at room temperature. These compounds have been suggested to be environmentally friendly and are proposed to be replacements for commercially available solvents used in laboratory operations today. There is an increasing interest in these compounds, but toxicological data of ionic liquids are still scarce. In this project five imidazolium based ionic liquids, 1-etyl-3-methylimidazolium, 1-butyl-3-methylimidazolium, 1-hexyl-3-methylimidazolium, 1-octyl-3-methylimidazolium and 1-decyl-3-methylimidazolium were evaluated based on responses on aryl hydrocarbon receptor (DR-Ecoscreen), Nrf2 activity (MCF732cARE), androgen receptor (AR-Ecoscreen) and estrogen receptor (VM7Luc4ER). This was done with an effect based in vitro approach using luciferase bioassays. The results show that imidazolium based ionic liquids have the ability to induce androgen and estrogen receptor activity. It is also shown that imidazolium based ionic liquids can act as antagonists on the androgen receptor. Imidazolium based ionic liquids does not seem cause oxidative stress and is shown to not interact with the aryl hydrocarbon receptor. The ability of 1-octyl-3-methylimidazolium and 1-decyl-3-methylimidazolium to pass the gastrointestinal tract was also tested in a modified transwell caco-2 permeability test, which resembled the human GI-tract. It was shown to be difficult to evaluate if 1-octyl-3-methylimidazolium and 1-decyl-3-methylimidazolium have the ability to pass through the GI-tract and antagonize on the androgen receptor. These results are important from both an environmental as well as human health point of view if imidazolium based ionic liquids are to be accidentally or intentionally release into the environment.
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Causal Inference for Health Effects of Time-varying Correlated Environmental MixturesChai, Zilan January 2023 (has links)
Exposure to environmental chemicals has been shown to affect health status throughout the life course. Quantifying the joint effect of environmental mixtures over time is crucial to determine optimal intervention timing. Establishing causal relationships from environmental mixture data can be challenging due to various factors, including multicollinearity, complex functional form of exposure-response relationships, and residual unmeasured confounding. These issues can lead to biased estimates of treatment effects and pose significant obstacles in accurately identifying the true relationship between the pollutants and outcome variables. Causal interpretation of longitudinal environmental mixture studies encounters challenges.
This dissertation explores the use of causal inference in environmental mixture studies, with a particular emphasis on addressing three key challenges. First, there is currently no statistical approach that allows simultaneous consideration of time-varying confounding, flexible modeling, and variable selection when examining the effect of multiple, correlated, and time-varying exposures. Second, the violation of a critical assumption that underpins all causal inference methods - namely, the absence of unmeasured confounding - poses a significant problem, as models that incorporate multiple environmental exposures may exacerbate the degree of bias depending on the nature of unmeasured confounding. Finally, there is a lack of computational resources that facilitate the application of newly developed causal inference methods for analyzing environmental mixtures.
In Chapter 2, we introduce a causal inference method, g-BKMR, which enables to estimate nonlinear, non-additive effects of time-varying exposures and time-varying confounders, while also allowing for variable selection. An extensive simulation study shows that g-BKMR outperforms approaches that rely on correct model specification or do not account for time-dependent confounding, especially when correlation across time-varying exposures is high or the exposure-outcome relationship is nonlinear. We apply g-BKMR to quantify the contribution of metal mixtures to blood glucose in the Strong Heart Study, a prospective cohort study of American Indians.
Chapter 3, we address the issue of time-varying unmeasured confounding when estimating time-varying effects of exposure to environmental chemicals. We review the Bayesian g-formula under the assumption of no unmeasured confounding, and then introduce a Bayesian probabilistic sensitivity analysis approach that can account for multiple, potentially time-varying, unmeasured confounders and continuous exposures. Through a simulation study, we demonstrate that the proposed algorithm outperforms the naive method, which fails to consider the influence of confounding.
Chapter 4, introduces causalbkmr, a novel R package and can be currently be accessed on Github. causalbkmr is designed to support the implementation of g-BKMR, BKMR Causal Mediation Analysis, and Multiple Imputation BKMR, thereby offering a user-friendly and effective platform for executing these state-of-the-art methods in practice in the context of complex mixtures analysis. While the package bkmr is available, the novel package causalbkmr expands upon bkmr by enabling its application specifically to environmental mixture data within a causal inference framework. The implementation of these novel methodologies within causalbkmr allows for the extraction of causal interpretations, thus enhancing the analytical capabilities provided by the package.
Chapter 5 concludes with a discussion and outlines potential future directions for investigation.
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Photochemistry and Toxicity of Triclosan, Triclocarban, and their Photoproducts and Mixtures in Freshwater SystemsAlbanese, Katie 21 December 2016 (has links)
No description available.
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Estudo experimental da exposição ao PCB126 sobre a indução de Diabetes mellitus tipo II / Experimental study of PCB126 exposure on induction of Diabetes mellitus type II.Shimada, Ana Lucia Borges 29 July 2015 (has links)
A exposição ambiental aos poluentes orgânicos persistentes tem recebido amplo destaque na literatura recentemente devido à extensa associação entre o desenvolvimento de doenças metabólicas, obesidade e/ou diabetes mellitus, e a presença destes poluentes, principalmente os organoclorados, como as bifenilas policloradas (PCBs), no organismo. Por outro lado, os mecanismos de ação destes poluentes é controverso devido à elevada quantidade de representantes destas classes, gerando diversidade de protocolos de exposição e escassez de estudos experimentais. Por isto, foi objetivo deste trabalho elucidar os mecanismos de ação tóxica do PCB126, nas doses de 0,1; 1 ou 10 µg/kg de massa corpórea, em ratos Wistar machos, durante quinze dias, expostos por instilação intranasal. O procotolo de exposição empregado foi caracterizado e considerado suficiente para causar toxicidade, uma vez que foram observadas alterações no sistema imune, metabolismo e em parâmetros relacionados à gênese do diabetes mellitus. A caracterização da exposição foi determinada pela quantificação da concentração de PCB126 no fígado e pulmão (CG/MS) e pelo aumento da expressão do receptor aril hidrocarboneto (AhR) no rim, fígado, pulmão e tecido adiposo (Western Blot). O efeito imunossupressor do PCB126 foi evidenciado pelo comprometimento da produção de células na medula óssea e, consequentemente, no número de células totais no sangue circulante. Adicionalmente, foi evidenciada a interferência do poluente na via de ativação mediada por receptores acoplados à proteína G (GPCRs), principalmente em neutrófilos, alterando importantes funções destas células, como a expressão de moléculas de adesão, geração de espécies reativas de oxigênio e migração. Entre as alterações metabólicas observadas, destacamos o aumento dos níveis de triglicerídeos e colesterol sérico, aumento da liberação de ácidos graxos livres; aumento da atividade da enzima hepática gama glutamil transferase; aumento da resistência à insulina e aumento da geração de óxido nítrico pelas ilhotas de Langerhans, dados estes, possivelmente relacionados ao comprometimento das células beta (β) pancreáticas, confirmados pelo aumento da expressão de GLUT4 no tecido adiposo, aumento da concentração de insulina sérica e aumento do estresse oxidativo nas ilhotas de Langerhans. Em conjunto, os dados obtidos destacam importantes alterações causadas pela exposição intranasal ao PCB126, evidenciando a participação do poluente na gênese do diabetes mellitus do tipo II. / The environmental exposure to persistent organic pollutants has been widely highlighted in recent literature due to the extensive association between the development of metabolic diseases, obesity and/or diabetes mellitus, and presence of these pollutants, especially organochlorines such as polychlorinated biphenyls (PCBs) in organism. Moreover, the mechanisms of action of these pollutants are controversial due to the high number of PCBs congeners, diversity of exposure protocols and lack of experimental studies. Therefore, the aim of this study was to elucidate the mechanisms of PCB126\'s toxic action at doses of 0.1; 1 or 10 µg/kg body weight in male Wistar rats exposed by intranasal instillation for 15 days. The established exposure procotol was characterized and considered sufficient to cause toxicity since changes were observed in the immune system, metabolism and in parameters related to the pathogenesis of diabetes mellitus. Characterization of exposure was determined by quantifying the concentration of PCB126 in liver and lung (GC-MS) and by the increased expression of aryl hydrocarbon receptor (AhR) in kidney, liver, lung, and adipose tissue (Western blot). The immunosuppressive effect of PCB126 was evidenced by impairment of cell production in the bone marrow and thus the total number of cells in the circulation. In addition, the interference of the pollutant in the activation pathway mediated by G-protein coupled receptors (GPCRs), in particular in neutrophils, was observed by changing important functions of these cells such as the expression of adhesion molecules, reactive oxygen species generation, and migration. Among the metabolic changes observed, we highlight the increased levels of triglycerides and serum cholesterol, increased release of free fatty acids; increased gamma glutamyl transferase hepatic enzyme activity; increased insulin resistance and increased generation of nitric oxide by the islets of Langerhans, these data possibly related to the impairment of beta cells (β) pancreatic function, suggested by the increased expression of GLUT4 in adipose tissue, increased serum insulin concentration and increased oxidative stress in the islets of Langerhans. Altogether, these results highlight important changes caused by intranasal exposure to PCB126, suggesting participation of the pollutant in the genesis of diabetes mellitus type II.
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Investigação da biossíntese de toxinas produzidas por cepas de cianobactérias / Investigation on the cyanobacterial strains toxins biossinthesysBortoli, Stella de 05 September 2011 (has links)
A demanda crescente de água doce de boa qualidade são problemas atuais e mundiais, além do descaso com os dejetos lançados nos ambientes aquáticos que comprometem a qualidade dos recursos hídricos. Um dos parâmetros que atesta a potabilidade da água é a presença de cianobactérias e cianotoxinas. Cianobactérias são microrganismos procariontes aeróbicos fotoautróficos que sintetizam as cianotoxinas. Estes compostos podem ser classificados de acordo com seus mecanismos de ação em hepatotóxicos, neurotóxicos e dermatotóxicos. Por sua diversidade, representam diferentes riscos não só ao ecossistema e a outros organismos dos ambientes aquáticos, como também aos seres humanos. Esse projeto visou o isolamento e cultivo de cepas de cianobactérias produtoras de toxinas para a investigação da biossíntese desses compostos. Com este intuito, foram realizadas coletas de água em três reservatórios no estado de São Paulo e um no Paraná. Cepas de cianobactérais foram isoladas, identificadas e analisadas quanto à produção de toxinas. Uma cepa de Microcystis aeruginosa (LTPNA 02) produtora de microcistinas (MC-LR, MC-RR, MC-YR, MC-LF, MC-LW e desm-MC-LR e desm- MC-RR) foi escolhida para ser estudada frente diferentes condições de cultivo e ter o seu crescimento, produção de toxinas e expressão gênica estudados. Foram utilizados os meios de cultura já referidos na literatura: ASM-1 (N:P=1, 10 e 20), MLA (N:P=10), Bold 3N (N:P=16) e BG-11 (N:P=10 e 100). Para acompanhar o crescimento, dois métodos foram utilizados: contagem de células e espectrofotometria. As toxinas foram quantificadas por LC-MS - QTrap. A análise da expressão gênica foi realizada por reação de PCR em tempo real pelo método de quantificação relativa ΔΔCt. Foi observada diferença no crescimento da cepa estudada nos diferentes meios de cultivo empregados. A contagem das células permitiu a identificação das fases logarítmica e total de crescimento. Durante a fase logarítmica, três experimentos demonstraram diferenças estatísticas quando comparadas ao controle (p<0,05). Ao se avaliar o crescimento total, quatro experimentos foram menores (p<0,01). As leituras das absorvâncias e a contagem de células demonstraram alta correlação Para ambas as leituras em 680 nm e 750 nm o coeficiente de correlação (r) esteve entre 0,93 e 0,99. A quantificação das microcistinas (MC) foi realizada por LC-MS - QTrap. Foram quantificadas as variantes MC-LR, MR-RR e MC-YR. Apesar da relação toxina/célula ser distinto para cada experimento, não representou grande variação naqueles realizados com meio ASM-1 (N:P 1; 10 e 20), meio MLA (N:P=10) e BG11(N:P=10). O experimento realizado em Bold3N (N:P=16,6) apresentou menor concentração de toxina/célula e as variantes MC-LR e MC-YR não foram detectadas. Por outro lado, o experimento realizado em BG-11 (N:P=100) apresentou a maior relação toxina por célula. Estes resultados sugerem que o excesso de nitrato seja um fator estressante para o desenvolvimento e crescimento da cepa de M. aeruginosa avaliada e ao mesmo tempo um fator estimulante para a produção das toxinas analisadas. Os experimentos que avaliaram a expressão dos genes 16S e mcyB em relação ao gene da ficocianina (controle endógeno) foram realizados em meio ASM-1 (N:P=10 e 100) e BG 11 (N:P= 10 e 100). Os parâmetros anteriores, como crescimento e produção de toxinas também foram avaliados. Novamente foram encontradas diferenças entre as fases de crescimento e produção de toxina, porém a expressão dos genes avaliados não demonstrou variação significativa entre os experimentos. Porém ambos os genes avaliados demonstraram menor expressão nos experimentos condizidos em (N:P=100). / There is a great concern these days about potable and good quality water due to the increase of the population needs and also to the arising problems with contamination caused by anthropogenic sources. The presence of cyanobacteria and cyanotoxins are some parameters that attest water potability. Cyanobacteria are prokaryotic aerobic photoautotrophic microorganisms that may synthesize cyanotoxins. These compounds can be classified as hepatotoxic, neurotoxic and dermatotoxic according to their action mechanisms. Because of their diversity, they may represent different risks, not only to their ecosystem and other aquatic living organisms, but also to human beings. The aim of this project was the isolation and cultivation of cyanotoxin-producing cyanobacteria for further investigation on the biosynthesis of these compounds. Water samples from three different reservoirs in São Paulo state and one in Paraná state were collected in order to isolate cyanobacteria strains and accomplish their identification and to evaluate the toxin production. The Microcystis aeruginosa (LTPNA 02) microcystin producer strain (MCLR, MC-RR, MC-YR, MC-LF, MC-LW, desm-MC-LR and desm-MC-RR) was chosen to be grown in different cultivation conditions and later analyzed for its growth rate, toxin production and gene expression. All culture media used in this research were chosen according to the literature: ASM-1 (N:P=1, 10 and 20), MLA (N:P=10), Bold 3N (N:P=16) and BG-11 (N:P=10 and 100). To evaluate growth rate, two techniques were used: cell counting and absorbance determination in two different wavelengths (680 nm and 750 nm). Toxins were quantified by LC-MS in a hybrid triple-quadrupole instrument (Qtrap). Gene expression was assessed by real time PCR, using the ΔΔCt relative quantification method. Cell counting allowed total growth and logarithmic phase identification. During the last, three experiments showed statistical difference from control group (p<0,05). Four experiments resulted in a lower total growth rate (p<0,05). A high correlation between cell counting and absorbance levels was found for both wavelengths tested. Correlation coefficients (r) were from 0,93 to 0,99. Three microcystin variants (MC-LR, MR-RR e MC-YR) were quantified by LC-MS. The toxin content per cell was calculated and showed no statistc variation among those experiments performed on ASM-1 (N:P 1; 10 and 20), MLA (N:P=10) and BG-11 (N:P=10). The lowest toxin/cell concentration was found for Bold3N (N:P=16,6) medium, where MC-LR and MC-YR production was not detected. On the other hand, the experiment with BG-11 (N:P=100) medium showed the highest toxin/cell content. These results suggest that high levels of nitrate in the culture medium may be a stressing factor for the development and growth of the M. aeruginosa tested strain, as well as a disturbing factor for microcystin production. Gene expression experiments regarding 16S and mycB genes using the phycocyanin gene as endogen control were performed on ASM-1 (N:P=10 and 100) and BG 11 (N:P= 10 and 100) media, along with the evaluation of growth rate and toxin production. Differences between growth rates and toxin production were once more observed, however gene expression did not show a significant variation among experiments.
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Avaliação do praguicida aldicarbe e seus produtos de transformação em matriz ambiental -- desenvolvimento e comparação de técnicas analíticas / Evaluation of aldicarb pesticide and its transformation products in environmental matrix - development and comparison of analytical techniquesSouza, Erica Rodrigues de 02 April 2009 (has links)
Os praguicidas carbamatos surgiram na década de 70. Este grupo de substâncias possui atividade anticolinesterásica, com variado grau de toxicidade. São solúveis em água e termicamente instáveis. O praguicida aldicarbe faz parte do grupo dos carbamatos, sendo um metil carbamato de oxima. Contaminações de águas subterrâneas e superficiais pelo aldicarbe já foram demonstradas, devido a alta solubilidade deste composto em água e sua alta capacidade de lixiviação em solo. Este trabalho visa o desenvolvimento de métodos analíticos para separação e quantificação de aldicarbe, aldicarbe sulfóxido e aldicarbe sulfona, por cromatografia líquida de alta eficiência, cromatografia líquida acoplada à espectrometria de massas e eletroforese capilar. O trabalho objetiva ainda avaliar a toxicidade aguda das três substâncias utilizando a bactéria marinha luminescente Vibrio fischeri. Amostras de água ultrapura, superficial e subterrânea foram submetidas a etapas de extração em diferentes cartuchos de fase sólida para avaliação da recuperação dos analitos e a realização da pré concentração dos mesmos. No método proposto por cromatografia líquida acoplada a espectrometria de massas, obteve-se limite de quantificação de 10 µg.L-1, sendo que o alcançado no método proposto por cromatografia líquida com detecção por arranjo de diodos foi de 2 µg.L-1. Já o método desenvolvido por eletroforese capilar com detecção por arranjo de diodos teve um limite de quantificação de 10 mg.L-1. Os resultados de CE50 obtida para o aldicarbe, aldicarbe sulfona e aldicarbe sulfóxido, no teste de toxicidade com a bactéria luminescente Vibrio fischeri foram respectivamente: 56,0 mg.L-1, 47,0 mg.L-1 e 7,8 mg.L-1. O método desenvolvido por cromatografia líquida se mostrou com sensibilidade satisfatória para análise de aldicarbe e seus produtos de transformação em água com níveis de quantificação dos compostos abaixo do limite determinado pela OMS (10 µg.L-1. O método por eletroforese capilar não se mostrou com sensibilidade ideal para detecção dos analitos em níveis de traços. No teste de toxicidade aguda, observou-se que o aldicarbe sulfóxido é cerca de 7 vezes mais tóxico para a bactéria que o próprio aldicarbe, o que já é descrito na literatura para outras espécies. / Carbamates pesticides first appeared in the 1970s. This group of substances possesses anticholinesterasic activity, with varied toxicity degree. They are soluble in water and thermally unstable. The aldicarb pesticide belongs to the carbamates group, being an oxime methyl carbamate. Contaminations of underground and superficial waters by aldicarb have been demonstrated, due to the high solubility of this compound in water and its high capacity of lixiviation in soil. This dissertation aims to develop analytical methods for separation and quantification of aldicarb, aldicarb sulfoxide, and aldicarb sulfona, by high efficiency liquid chromatography, liquid chromatography-tandem mass spectrometry and capillary electrophoresis. The dissertation also focuses on evaluating the acute toxicity of the three substances using the luminescent marine bacterium Vibrio fischeri. Samples of ultrapure water, superficial and underground, were submitted to extraction stages in different cartridges of solid phase for evaluating the recovery of analytes and conducting their pre-concentration. In the method proposed by liquid chromatography-tandem mass spectrometry, a quantification limit of 10 µg.L-1 was obtained, in comparison with the 2 µg.L-1 achieved in the method proposed by liquid chromatography with diode-array detection. The method developed by capillary electrophoresis with diode-array detection had a quantification limit of 10 mg L-1. The results of CE50 obtained for the aldicarb, aldicarb sulfone and aldicarb sulfoxide, in the toxicity test with the luminescent bacterium Vibrio fischeri were respectively: 56.0 mg.L-1, 47.0 mg.L-1 e 7.8 mg.L-1 The method developed by liquid chromatography showed satisfactory sensitivity for analysis of aldicarb and its transformation products in water with quantification levels of the compounds below the limit determined by OMS (10 µg.L-1</sup) . The method by capillary electrophoresis did not show ideal sensitivity for trace level detection of analytes. In the acute toxicity test, it was observed that the aldicarb sulfoxide is nearly 7 times more toxic for the bacterium than the aldicarb itself, which is already described in the literature for other species.
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Avaliação ambiental de exposição a fumos metálicos em fundições / Environmental assessment of exposure to metallic fumes in foundriesFelix, Fabiana da Silva 17 June 2004 (has links)
A Universidade de São Paulo (Instituto de Química e Faculdade de Ciências Farmacêuticas) e FUNDACENTRO têm desenvolvido um projeto nos pólos de fabricação de peças de torneiras e registros de Loanda/PR, visando a avaliação do ambiente de trabalho. Considerando as etapas do processo de fundição de latão para a confecção dos produtos, há a presença de fumos metálicos, provenientes da fundição e vazamento, que podem acarretar na intoxicação dos trabalhadores e, conseqüentemente, faz-se necessária a avaliação do risco ocupacional. Esta avaliação consiste da caracterização das partículas através da determinação de seus constituintes em massa por volume de ardo ambiente de trabalho. A concentração para particulados é expressa em µg m-3. Para isso, foi aplicada metodologia específica e reconhecida do National Institute of Organization Safety (NIOSH), a qual recomenda a utilização de espectrometria de emissão óptica com plasma acoplado indutivamente (ICP OES) para a determinação de As, Cd, Mn, Ni e Pb em amostras de ar. Foi proposta uma alternativa utilizando a voltametria de acumulação com redissolução anódica com eletrodo de gota pendente de mercúrio para a determinação de Cd e Pb, além da voltametria adsortiva com redissolução catódica com o mesmo eletrodo para Ni e voltametria de redissolução catódica com eletrodo de diamante dopado com boro para Mn, sendo que utilizou-se onda quadrada para a obtenção de todos os voltamogramas. De acordo com os resultados obtidos, foram propostas alterações tecnológicas no processo e adequação dos equipamentos de proteção no intuito de proteger a saúde e o bem estar dos trabalhadores, bem como evitar danos ao meio ambiente. / The Universidade de São Paulo, Instituto de Química e Faculdade de Ciências Farmacêuticas (University of São Paulo, Institute of Chemistry and Pharmaceutical Sciences College) and FUNDACENTRO have developed a project at the manufacturing facilities of stopcocks and faucet parts in Loanda/PR, aiming the evaluation of workplaces. Considering the stages of the brass foundry process in manufacturing those products, there are metallic fumes, originating from metal melting and pouring, which can result in human intoxication and, therefore, it is necessary to make an assessment of the occupational exposure. This assessment consists of collecting the particulate, measuring the mass of the contaminant, and then calculating the concentration by dividing the mass by the volume of sampled air. For particulates, concentration is expressed as µg m-3. For this assessment, it was applied a specific and recognized method of the National Institute of Organization Safety Health (NIOSH) which recommends the use of the technique of inductively coupled plasma optical emission spectrometry (ICP OES) to determine As, Cd, Mn, Ni and Pb in air samples. It was proposed an alternative method using the anodic stripping voltammetry with hanging mercury drop electrode (HMDE) to determine Cd and Pb, in addition to cathodic adsorptive stripping voltammetry with HMDE to determine Ni and cathodic stripping voltammetry with boron-doped diamond electrode to determine Mn. For these procedures were used square wave voltammetry to record all the voltammograms. According to the results of this work, we proposed technological changes in the production process and the adequacy of protection equipment in order to protect the health and well-being of workers, as well as to avoid damages to the environment.
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