A crescente degradação da qualidade dos mananciais que servem os sistemas públicos de abastecimento de água potável faz com que se busquem novas técnicas para remoção de compostos indesejáveis. Os fatores climáticos que predominam no Brasil favorecem o desenvolvimento, em ambientes eutrofizados, de microrganismos conhecidos como cianobactérias que podem ser potencialmente produtores de toxinas. Este trabalho estudou a eficiência de remoção de microcistinas em um sistema que associa coagulação, floculação, flotação por ar dissolvido, adsorção em carvão ativado e filtração, com o intuito de remover a maior quantidade possível de microcistina para minimizar as dosagens de carvão ativado necessárias à remoção de concentrações de microcistinas - hepatotoxinas produzidas por cianobactérias - uma vez que, em uma estação real, uma menor dosagem de carvão ativado representa menores custos. Até o momento, o estado da arte permite afirmar que a aplicação de carvão ativado constitui a etapa final de um sistema de tratamento usualmente utilizado para a eficiente remoção de toxinas. Em escala de laboratório, foram feitos ensaios em equipamentos com alimentação por batelada: jarteste e flotateste. Após a preparação da água de estudo, uma amostra foi submetida à mistura rápida em um equipamento de jarteste, com aplicação de produtos químicos para atingir valores desejados de pH e, em outros ensaios, também foi adicionado o carvão ativado em pó - CAP. Após a coagulação, a amostra ensaiada era transferida para o equipamento de flotateste para a floculação e posterior flotação por ar dissolvido e, em alguns casos, submetido à filtração em papel Whatman 40. Observou-se que o uso dos parâmetros de cor e turbidez não foram suficientes para garantir a também remoção de microcistina. No sistema proposto, a aplicação de CAP antes da mistura rápida apresentou melhor desempenho na remoção de microcistina do que quando aplicado posteriormente a esta etapa; foi necessário dosagem de 60 mg/L de CAP, aplicado a 1,5 hora antes da mistura rápida para que a concentração de microcistina ficasse inferior a 1 &#956g/L. O sistema que associa coagulação, floculação, flotação por ar dissolvido, adsorção em carvão ativado e filtração mostrou ser capaz de remover microcistina a níveis inferiores a 1 &#956g/L, conforme estabelecido na Portaria 518/04 do Ministério da Saúde. / The growing quality degradation of the water sources serving the public distribution of drinking water results in the search of new techniques for the removal of undesirable compounds. The brazilian climate factors are in favor of the development of eutrophic environments, and of microorganisms known as cyanobacteria that are potential sources of toxins. This work studied the microcystin removal efficiency in a system that associates, coagulation, flocculation, dissolved air flotation, adsorption in activated carbon and filtration, with the scope to remote the maximum possible quantity of toxins in order to minimize the activated carbon dosage need to the removal of microcystin concentration - hepatotoxins produced by cyanobacteria - taking into accounts that in a real plant, the less the activated carbon, the less the operation costs. So far, the state of art permits the affirmation that the activated carbon characterizes the final step of a treatment system usually used for the efficient removal of toxins. In the laboratory scale tests were done in batch equipments: jartest and flotatest. After the preparation of the water in study one sample was submitted to a quick mixture in a jartest, with the application of chemical products to reach the desired pH levels, and in other assays powder activated carbon PAC was also added. After the coagulation, the tried sample was transferred to the flotatest equipment to achieve flotation and posterior dissolved air flotation, and in some cases, submitted to filtration in Whatman 40. It could be observed that the use of color and turbidity parameters were not sufficient to guarantee the microcystin removal. In the proposed system, the PAC application before the quick mixture presented better performance in the removal of microcystins than the posterior application to this step; a dosage of 60 mg/L of PAC was necessary when applied 1,5 hour before the quick mixture for the microcystin concentration be as low as 1 &#956g/L. The system which associates coagulation, flocculation, dissolved air flotation, activated carbon adsorption and filtration, exhibits the ability of microcystin removal to levels below 1 &#956g/L as is established in Portaria 518 - 23/03/05 - Ministério da Saúde - Brazil.

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 &#916;&#916;Ct. 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 &#916;&#916;Ct 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.

The St. Johns River (SJR; Jacksonville, FL, USA) is a large, brackish, estuarine system characterized by considerable anthropogenic pollution, recurrent harmful algal blooms (HABs), and diverse toxin-producing cyanobacteria. The most prevalent toxins in SJR water samples are microcystins/nodularins (MCs/NODs). Additionally, the SJR provides critical habitat for a genetically and behaviorally distinct estuarine community of bottlenose dolphins (Tursiops truncatus) that routinely uses and strands in low mesohaline and oligohaline areas of the river. This population has been subject to two unusual mortality events (UME) since 2010 and has since been described as having substantial declines in population health, characterized by widespread dermatitis and emaciation. Additionally, three dolphins have been recovered from low salinity habitats with epidermal algal mats. Because dolphin illness and strandings overlapped temporally and spatially with confirmed cyanobacterial blooms in the SJR, there is concern that estuarine dolphin health may be declining due to exposure to toxic cyanobacteria and HAB events. Specific to this study, the SJR estuarine community was considered a high-risk group for cyanotoxin exposure in relation to coastal animals. This study analyzed all available hepatic tissues for estuarine dolphins, and used samples from coastal individuals that stranded outside of the known cyanotoxin bloom season as controls. Three analytical methods were used to determine MCs/NODs presence in dolphin liver and epidermal algal mat samples. An Adda ELISA and LC-MS/MS were used to determine free MCs/NODs presence while the MMPB technique was used to determine total (bound and free) concentrations and as confirmatory analyses. ELISA analyses produced high values that were not supported by concurrent LC-MS/MS or MMPB analyses, indicative of false positives. MMPB testing resulted in low-level total MCs/NODs detection in some specimens. Results indicate that both estuarine and coastal dolphins are exposed to MCs/NODs, with potential toxic and immune health impacts.

Made available in DSpace on 2014-12-17T15:55:04Z (GMT). No. of bitstreams: 1 AnaPCS_DISSERT.pdf: 2668661 bytes, checksum: b290eedaaa96f64424423ef0e1573d9c (MD5) Previous issue date: 2013-03-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Water scarcity is a reality for the inhabitants of the Brazilian semiarid region. The problem, in quantitative terms , is caused due to local climatic conditions due mostly to a water deficit. Qualitatively, results of multiple uses and resulting contamination by human activities. Eutrophication is presented as a threat to the sustainable use of water resources, in order to favor the predominance of dense populations of cyanobacteria, which can be potentially harmful to human health. The aim of this study is to understand the population dynamics of phytoplankton and cyanobacteria to assess water quality of reservoirs Santa Cruz do Apodi and Pau dos Ferros belonging to bacia Apodi Mossor?-RN . Water samples were collected monthly between October 2011 and May 2012 in reservoirs Santa Cruz do Apodi and Pau dos Ferros, in this latter , in Jan/12 we performed a diel profile . The abiotic variables obtained in the field and in the laboratory were: water temperature , dissolved oxygen , pH , turbidity , transparency , total nitrogen and total phosphorus . Phytoplankton samples were collected with a plankton net and bottle of Van dorn . Were performed : identification , quantification , calculation of biovolume , classification into functional groups and index calculation phytoplankton assembly ( Q ) , cyanotoxins were quantified by ELISA . Statistical analyzes supported the evaluation of the dynamics between biotic and abiotic factors. A questionnaire was conducted to examine the conceptions of the population, education professionals and students. The reservoir of Pau dos Ferros, shallow , turbid and eutrophic showed dominance of the functional group SN throughout the sample period . The phytoplankton biomass ranged from 20 to 70 mm ? . L - 1, the lowest values coincided with the increase of the mixing zone and transparency, which contributed to the occurrence of a change in species composition phytoplankton . The application of the index Q proved relevant, the reservoir of Pau dos Ferros , depending on the species present , was classified ecologically as bad for almost the entire sample period . The reservoir Santa Cruz do Apodi showed low biomass ( 0.04 and 4.31 mm ? . L - 1 ) and greater diversity in the phytoplankton composition . According to the index assembly (Q ), it showed moderate condition during most of the period influenced by different functional groups of typical meso- eutrophic environment (K S0, H1, C, F , J , E, D and N) . Associations of diatoms and green algae D and X1 succeeded populations of cyanobacteria in periods marked by greater instability in the system , caused by wind or rain. In summary , the occurrence of drought has a direct influence on the hydrological conditions of the reservoirs , in general, these events, reducing the reservoir level is directly related to decreased water quality and increased density of phytoplankton occurring predominance of cyanobacteria , the index Q reflected well to changes in phytoplankton composition , being a good indicator for biomonitoring of reservoirs in this study and survey of previous conceptions showed the need to work on environmental awareness for the preservation of water resources by conducting workshops for Environmental Education / A escassez de ?gua ? uma realidade para os habitantes da regi?o semi?rida brasileira. O problema, em termos quantitativos, ? ocasionado em virtude das condi??es clim?ticas locais refletindo, principalmente, em um d?ficit h?drico. Em termos qualitativos, decorre dos m?ltiplos usos e consequente contamina??o pelas atividades humanas. A eutrofiza??o apresenta-se como uma amea?a para o uso sustent?vel dos recursos h?dricos, tendo em vista favorecer a predomin?ncia de densas popula??es de cianobact?rias, as quais podem ser potencialmente prejudiciais ? sa?de humana. O objetivo deste estudo ? compreender a din?mica das popula??es de cianobact?rias e do fitopl?ncton para avaliar a qualidade de ?gua dos reservat?rios Santa Cruz do Apodi e Pau dos Ferros pertencentes ? bacia hidrogr?fica do rio Apodi-Mossor?. Amostras de ?gua foram coletadas mensalmente entre outubro de 2011 e maio de 2012 nos reservat?rio Santa Cruz e Pau dos Ferros, neste ?ltimo, em jan/12 foi realizado um perfil nictemeral. As vari?veis abi?ticas obtidas em campo e no laborat?rio foram: temperatura da ?gua, oxig?nio dissolvido, pH, turbidez, transpar?ncia, nitrog?nio total e f?sforo total. As amostras do fitopl?ncton foram coletadas com rede de pl?ncton e garrafa de Van. Foram realizados: identifica??o, quantifica??o, c?lculo do biovolume, classifica??o em grupos funcionais e c?lculo do ?ndice de assembleia fitoplanct?nico (Q), cianotoxinas foram quantificadas pelo m?todo ELISA. An?lises estat?sticas deram suporte ? avalia??o da din?mica entre fatores bi?ticos e abi?ticos. Aplica??o de question?rio foi realizada para analisar as concep??es pr?vias da popula??o, profissionais de ensino e alunos. O reservat?rio Pau dos Ferros considerado raso, turvo e eutr?fico apresentou domin?ncia do grupo funcional SN durante todo o per?odo amostral. A biomasa fitoplanct?nica variou entre 20 e 70 mm?.l-1, os menores valores coincidiram com o aumento da zona de mistura e transpar?ncia, que contribu?ram para a ocorr?ncia de altera??o na composi??o de esp?cies da comunidade fitoplanct?nica. A aplica??o do ?ndice Q mostrou-se relevante, o reservat?rio Pau dos Ferros, em fun??o das esp?cies presentes, foi categorizado, ecologicamente, como ruim durante quase todo o per?odo amostral. O reservat?rio Santa Cruz apresentou baixa biomassa (0,04 e 4,31 mm?. l-1) e maior diversidade na composi??o fitoplanct?nica. De acordo com o ?ndice de assembl?ia (Q), ele apresentou condi??o moderada, durante a maior parte do per?odo, influenciado por diferentes grupos funcionais t?picos de ambientes meso-eutr?ficos (K, S0, H1,C, F, J, P, D e N). Associa??es de diatom?ceas D e clorof?ceas X1 sucederam as popula??es de cianobact?rias em per?odos marcados por maior instabilidade no sistema, causadas pelo vento ou chuva. Em s?ntese, a ocorr?ncia de estiagem tem influ?ncia direta sobre as condi??es hidrol?gicas dos reservat?rios, em geral, nesses eventos, a redu??o do n?vel do reservat?rio est? diretamente associada ? diminui??o da qualidade da ?gua e aumento da densidade fitoplanct?nica ocorrendo predomin?ncia de cianobact?rias, o ?ndice Q refletiu bem ?s mudan?as na composi??o fitoplanct?nica, sendo um bom indicador para o biomonitoramento dos reservat?rios avaliados neste estudo e o levantamento das concep??es pr?vias mostrou a necessidade de se trabalhar a sensibiliza??o ambiental para a preserva??o dos recursos h?dricos atrav?s da realiza??o de oficinas de Educa??o Ambiental

Cyanobacterial blooms and their toxins are a major water quality and potential health risk around the world. This thesis developed an analytical method for microcystin congeners in sediments in order to examine their fate in lakes and establish the history of toxin-producing cyanobacteria in relation to environmental change using lake sediments. A novel method for both intra- and extracellular microcystins in lake sediments was developed, consisting of accelerated solvent extraction, hydrophilic-lipophilic balance solid phase extraction and multiple reaction monitoring-based HPLC-MS/MS quantitation. The method achieved comparable recoveries of intra- and extracellular cyanotoxins based on nine microcystins and nodularin (marine analogue). The analytical method was validated using surficial and deeper sediments from seven lakes of diverse geography and trophic state. To study the fate of microcystins, a multi-year, whole lake study of Microcystis blooms was conducted to obtain both in situ and in vitro half-life estimates of microcystin-LA (MC-LA), an understudied, but increasingly reported microcystin. MC-LA appeared to undergo slower rates of decomposition and persist longer than the more frequently studied MC-LR. Experimentally, high light intensity increased in vitro decomposition of dissolved MC-LA while high temperature enhanced decomposition in the particulate phase. Sediment deposition measurements and estimates of sediment-pore water distribution coefficients, sediment accumulation rates, and diffusive fluxes indicated that microcystin congeners differ in their fate. Notably, MC-LA preferentially distributed into pore water and remobilized (by diffusion) from sediments and into overlying water while MC-RR adsorbed more strongly to sediment particles. Finally, the sediment record of an eutrophic lake of major recreational importance was examined to identify possible drivers of toxigenic cyanobacteria and determine if the perceived increase in toxigenic cyanobacteria could be corroborated. Microcystins were detected to the bottom of the core (early 1800s), indicating that toxigenic cyanobacteria were present prior to the first permanent settlements. Microcystins were significantly correlated with changes in diatom-inferred nutrients (DI-TP and DI-TKN) within the sediment core as well as with specific algal pigments. Sediment microcystins in the upper layers also significantly correlated with a 20-year monitoring record for water column microcystins suggesting that sediment microcystins can be used as a proxy for past surface water conditions.

Les suppléments alimentaires à base d’algues sont commercialisés pour leurs bénéfices pour la santé ainsi que pour leur valeur nutritionnelle. Cependant, certains de ces produits peuvent être contaminés par des toxines produites par la cooccurrence d’espèces toxigènes. Afin d’évaluer l’ampleur de la contamination en toxines dans différents suppléments disponibles sur le marché nord-américain, nous avons achetés 37 échantillons de spiruline, d’Aphanizomenon flos-aqua, de Chlorella et de varech et les avons analysés pour 27 toxines par des méthodes de chromatographie liquide et de spectrométrie de masse (LC-MS/MS ou LC-HRMS). Nous avons détecté des microcystines dans les huit échantillons d’Aphanizomenon à des niveaux allant jusqu’à 1000 ng/g et de l’acide 2,4-diaminobutyrique (DAB) dans tous les échantillons de suppléments à des concentrations incluses entre 3 et 1600 ng/g. L’anatoxine A (ANA-a) et le β-amino-N-méthylalanine (BAMA) n’ont pas été détectés tandis que les autres toxines ont été détectées de façon plus aléatoire à des concentrations variables, sans lien clair avec l’espèce de l’algue. En considérant ces résultats, les produits à base d’Aphanizomenon devraient faire l’objet d’un contrôle plus strict pour prévenir leur contamination et restreindre la vente de produits contaminés. Ces découvertes seraient également d’intérêt pour les consommateurs afin qu’ils puissent évaluer les risques associés à la consommation régulière de certains suppléments à base d’algues / Algal dietary supplements are marketed for their health benefits and nutritional value. However, these types of products can be contaminated by toxins produced by co-occurring toxigenic cyanobacteria. In order to evaluate the contamination of different supplements available on the North American market, we purchased 37 samples of spirulina, Aphanizomenon flos-aquae, Chlorella and kelp, and analysed them to identify 27 suspected toxins using liquid chromatography mass spectrometry methods (LC-MS/MS or LC-HRMS). We found microcystins (MC) in the 8 Aphanizomenon samples with levels up to 1000 ng g-1 dw and 2,4-diaminobutyric acid (DAB) in all samples with values ranging from 3 to 1600 ng g-1. Anatoxin-a (ANA) and β-amino-N-methylalanine (BAMA) were not detected, while other toxins were diversely detected with no clear link to the nature of the alga. Considering these results, Aphanizomenon products may require stricter monitoring to prevent further contamination. The findings are also of interest to consumers so that they can properly assess the risks that may be involved in the regular consumption of certain algal dietary supplements.

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Magister Scientiae - MSc / The toxicity of microcystin is associated with the inhibition of serine/threonine protein phosphatases 1 and 2A, which can lead to hepatocyte necrosis and haemorrhage. Analysis of microcystin is most commonly carried out using reversed-phase high performance liquid chromatographic methods (HPLC) combined with ultra-violet (UV) detection .The ability of these techniques to identify unknown microcystin in environmental samples is also restricted by the lack of standard reference materials for the toxins. Highly specific recognition molecules such as antibodies and molecularly imprinted polymers (MIPs) have been employed in the pre-concentration of trace levels of microcystin from water and show great potential for the clean-up of complex samples for subsequent analysis. New biosensor technologies are also becoming available, with sufficient sensitivity and specificity to enable rapid ‗on-site‘ screening without the need for sample processing. In this work we constructed a Protein phosphatase biosensor for detection of microcystin-LR in aqueous medium, onto polyamic acid/graphene oxide (PAA: GO) composite electrochemically synthesised in our laboratory. The composites were synthesised at three different ratios i.e. 50:50, 80:20 and 20:80 to evaluate the effect of each component in the search to produce highly conductive mediator platforms. The electrochemistries of the three different composites were evaluated using CV and SWV to study interfacial kinetics of the materials as thin films at the glassy carbon electrode. The phosphatase biosensor parameters were evaluated using CV, SWV, EIS and Uv-vis spectroscopy. The affinity binding of the microcystin-LR to protein phosphatase 2A was investigated using electrochemical impedance spectroscopy which is a highly sensitive method for measuring interfacial kinetics of biosensor systems.

Made available in DSpace on 2014-12-17T14:33:08Z (GMT). No. of bitstreams: 1 LeideAPS_DISSERT.pdf: 986741 bytes, checksum: e18d4b42b9411720a51bd3f7786fa126 (MD5) Previous issue date: 2012-09-25 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Cyanobacterial blooms are common in eutrophic reservoirs in Brazilian northeastern semi-arid. Given this reality, the present study aimed to analyze the effect of potentially toxic cyanobacterial blooms in Gargalheiras reservoir (semi-arid) on the cladocerans Ceriodaphnia dubia and Daphnia gessneri. In vitro chronic bioassays were performed with reservoir water dilutions from August/2011 to May/2012 and the following effects were evaluated on: intrinsic rate of population growth (r), reproductive parameters (age of first reproduction and fecundity per capita) and cladocerans movements. Phytoplankton was dominated by Cylindrospermopsis raciborskii and Planktothrix agardhii and saxitoxin and microcystin were detected in reservoir water. In most months C. dubia showed differences in r between control (absence of cyanobacteria) and treatments, and has shown negative effects on reproductive parameters. In all months paralysis of swimming movements was observed in C. dubia when both C. raciborskii and saxitoxin (cyanotoxin neurotoxic) were present in water. While C. dubia was sensitive to the reservoir water containing cyanobacteria, D. gessneri showed less intense negative effects in r and reproductive parameters. Furthermore, D. gessneri showed no paralysis of swimming movements. These results support the hypothesis in the literature that D. gessneri is resistant to the Cylindrospermopsis effects. The clone&#8223;s life history may be a key to understand the results. The C. dubia clone, isolated from eutrophicated environment, is in the lab for ten years and it is an exotic species in Brazil. D. gessneri is a common species in the country and this clone was isolated from the Gargalheiras reservoir (where there are constant blooms of potentially toxic cyanobacteria) a year ago. Perhaps the recent contact with cyanobacteria explain the higher resistance presented by this D. gessneri clone. In conclusion, the cladocerans studied have different levels of sensitivity to cyanobacteria, characterizing species-specific responses / Flora??es de cianobact?ras s?o frequentes em reservat?rios eutr?ficos do semi-?rido do nordeste brasileiro. Diante dessa realidade o presente trabalho objetivou analisar o efeito de flora??es de cianobact?rias potencialmente t?xicas do reservat?rio Gargalheiras (semi-?rido brasileiro) sobre os clad?ceros Ceriodaphnia dubia e Daphnia gessneri. Foram realizados ensaios cr?nicos in vitro com dilui??es de ?gua do reservat?rio de agosto/2011 a maio/2012 e foram avaliados efeitos sobre: taxa intr?nseca de crescimento populacional (r), par?metros reprodutivos (idade da primeira reprodu??o e fecundidade per capita) e movimenta??o dos clad?ceros. O fitopl?ncton foi dominado por Cylindrospermopsis raciborskii e Planktothrix agardhii e houve detec??o de saxitoxina e microcistina na ?gua do reservat?rio. Na maioria dos meses C. dubia apresentou diferen?a em r entre controle (aus?ncia de cianobact?rias) e tratamentos, al?m de ter apresentado efeitos negativos nos par?metros reprodutivos. Observou-se ainda um efeito de paralisia dos movimentos natat?rios de C. dubia em todos os meses, concomitante ? presen?a de C. raciborskii e saxitoxina (cianotoxina neurot?xica) na ?gua. Enquanto C. dubia apresentou-se sens?vel ? ?gua do reservat?rio contendo cianobact?rias, D. gessneri apresentou efeitos negativos menos intensos em r e nos par?metros reprodutivos. Al?m disso, esta esp?cie n?o teve os movimentos paralisados. Esses resultados apoiam a hip?tese levantada na literatura de que D. gessneri apresenta resist?ncia aos efeitos da Cylindrospermopsis. A hist?ria de vida dos clones pode ser uma chave para compreens?o dos resultados. O clone de C. dubia, isolado de ambiente eutrofizado, est? em laborat?rio h? dez anos e essa ? uma esp?cie ex?tica no Brasil. D. gessneri ? uma esp?cie comum no pa?s e este clone foi isolado h? um ano do reservat?rio Gargalheiras, onde h? constantes flora??es de cianobact?rias potencialmente t?xicas. A hip?tese mais prov?vel ? que o contato recente deste clone de D. gessneri com cianobact?rias tenha lhe conferido maior resist?ncia. Conclui-se que os clad?ceros estudados apresentam diferentes n?veis de sensibilidade ?s cianobact?rias, caracterizando respostas esp?cie-espec?ficas

Les cyanobactéries ont une place très importante dans les écosystèmes aquatiques et un nombre important d’espèces considéré comme nuisible de par leur production de métabolites toxiques. Ces cyanotoxines possèdent des propriétés très variées et ont souvent été associées à des épisodes d’empoisonnement. L’augmentation des épisodes d’efflorescence d’origine cyanobactériennes et le potentiel qu’ils augmentent avec les changements climatiques a renchéri l’intérêt de l’étude des cyanobactéries et de leurs toxines. Considérant la complexité chimique des cyanotoxines, le développement de méthodes de détection simples, sensibles et rapides est toujours considéré comme étant un défi analytique. Considérant ces défis, le développement de nouvelles approches analytiques pour la détection de cyanotoxines dans l’eau et les poissons ayant été contaminés par des efflorescences cyanobactériennes nuisibles a été proposé. Une première approche consiste en l’utilisation d’une extraction sur phase solide en ligne couplée à une chromatographie liquide et à une détection en spectrométrie de masse en tandem (SPE-LC-MS/MS) permettant l’analyse de six analogues de microcystines (MC), de l’anatoxine (ANA-a) et de la cylindrospermopsine (CYN). La méthode permet une analyse simple et rapide et ainsi que la séparation chromatographique d’ANA-a et de son interférence isobare, la phénylalanine. Les limites de détection obtenues se trouvaient entre 0,01 et 0,02 μg L-1 et des concentrations retrouvées dans des eaux de lacs du Québec se trouvaient entre 0,024 et 36 μg L-1. Une deuxième méthode a permis l’analyse du b-N-méthylamino-L-alanine (BMAA), d’ANA-a, de CYN et de la saxitoxine (STX) dans les eaux de lac contaminés. L’analyse de deux isomères de conformation du BMAA a été effectuée afin d’améliorer la sélectivité de la détection. L’utilisation d’une SPE manuelle permet la purification et préconcentration des échantillons et une dérivatisation à base de chlorure de dansyle permet une chromatographie simplifiée. L’analyse effectuée par LC couplée à la spectrométrie de masse à haute résolution (HRMS) et des limites de détections ont été obtenues entre 0,007 et 0,01 µg L-1. Des échantillons réels ont été analysés avec des concentrations entre 0,01 et 0,3 µg L-1 permettant ainsi la confirmation de la présence du BMAA dans les efflorescences de cyanobactéries au Québec. Un deuxième volet du projet consiste en l’utilisation d’une technologie d’introduction d’échantillon permettant des analyses ultra-rapides (< 15 secondes/échantillons) sans étape chromatographique, la désorption thermique à diode laser (LDTD) couplée à l’ionisation chimique à pression atmosphérique (APCI) et à la spectrométrie de masse (MS). Un premier projet consiste en l’analyse des MC totales par l’intermédiaire d’une oxydation de Lemieux permettant un bris de la molécule et obtenant une fraction commune aux multiples congénères existants des MC. Cette fraction, le MMPB, est analysée, après une extraction liquide-liquide, par LDTD-APCI-MS/MS. Une limite de détection de 0,2 µg L-1 a été obtenue et des concentrations entre 1 et 425 µg L-1 ont été trouvées dans des échantillons d’eau de lac contaminés du Québec. De plus, une analyse en parallèle avec des étalons pour divers congénères des MC a permis de suggérer la possible présence de congénères ou d’isomères non détectés. Un deuxième projet consiste en l’analyse directe d’ANA-a par LDTD-APCI-HRMS pour résoudre son interférence isobare, la phénylalanine, grâce à la détection à haute résolution. La LDTD n’offre pas de séparation chromatographique et l’utilisation de la HRMS permet de distinguer les signaux d’ANA-a de ceux de la phénylalanine. Une limite de détection de 0,2 µg L-1 a été obtenue et la méthode a été appliquée sur des échantillons réels d’eau avec un échantillon positif en ANA-a avec une concentration de 0,21 µg L-1. Finalement, à l’aide de la LDTD-APCI-HRMS, l’analyse des MC totales a été adaptée pour la chair de poisson afin de déterminer la fraction libre et liée des MC et comparer les résultats avec des analyses conventionnelles. L’utilisation d’une digestion par hydroxyde de sodium précédant l’oxydation de Lemieux suivi d’une purification par SPE a permis d’obtenir une limite de détection de 2,7 µg kg-1. Des échantillons de poissons contaminés ont été analysés, on a retrouvé des concentrations en MC totales de 2,9 et 13,2 µg kg-1 comparativement aux analyses usuelles qui avaient démontré un seul échantillon positif à 2 µg kg-1, indiquant la possible présence de MC non détectés en utilisant les méthodes conventionnelles. / Cyanobacteria have a very important place in aquatic ecosystems and a significant number of species are considered harmful given their production of toxic metabolites. These cyanotoxins have various chemical proprieties and have often been associated with poisoning episodes. The frequency of cyanobacterial blooms is increasing and the study of cyanobacteria and their toxins is of increasing interest, especially considering the potential increase associated with climate changes. Given the chemical complexity of the cyanotoxins, the development of simple, sensitive and fast detection methods is an analytical challenge. Considering these issues, the development of new analytical approaches for the detection of cyanotoxins in water and fish samples contaminated with harmful cyanobacterial blooms have been proposed. A first approach consists of the use of an on-line solid phase extraction coupled to liquid chromatography and tandem mass spectrometry (SPE-LC-MS/MS) for the analysis of six microcystins (MCs), anatoxin-a (ANA-a) and cylindrospermopsin (CYN). This method allows a simple and rapid analysis and enables the chromatographic separation of ANA-a and its isobaric interference, phenylalanine. The detection limits ranged from 0.01 to 0.02 µg L-1 and concentrations in lake waters were found between 0.024 and 36 µg L-1. A second method consists of using manual solid phase extraction (SPE) coupled to high resolution mass spectrometry (HRMS) for the determination of b-N-methylamino-L-alanine (BMAA), ANA-a, CYN and saxitoxin (STX) in contaminated lake water. The analysis of two conformational isomers of BMAA was done to improve the selectivity. Dansyl chloride-based derivatization allows simplified chromatography. The detection limits were obtained between 0.007 and 0.01 µg L-1. The analysis of bloom water samples detected concentrations of cyanotoxins between 0.01 and 0.3 µg L-1 allowing the confirmation of the presence of BMAA in algal blooms in Québec. A second part of the project consists in the use of an alternative sample introduction technology for MS analysis. It enables ultra-fast analysis (< 15 seconds/sample) without the use of a chromatographic step, and is called laser diode thermal desorption (LDTD) coupled with atmospheric pressure chemical ionization (APCI). The first LDTD project consists of the analysis of total MCs via Lemieux oxidation in order to obtain a common moiety of all MCs existing congeners. This fraction, the MMPB, is analyzed after a liquid-liquid extraction step, with the LDTD-APCI-MS/MS. A value of 0.2 µg L-1 was obtained for detection limit and concentrations between 1 and 425 µg L-1 have been found in contaminated water samples. In addition, a comparison with a parallel analysis using MCs congeners’ standards suggested the possible presence of undetected MCs or isomers. A second project involves the direct analysis of ANA-a using LDTD-APCI-HRMS in order to solve the isobaric interference, phenylalanine, which is possible due to the high resolution detection. The LDTD offers no chromatographic separation and by using HRMS, we can distinguish ANA-a signals from those of phenylalanine. A value of 0.2 µg L-1 was obtained as detection limit and the method has been applied on water bloom samples with a positive concentration of 0.21 µg L-1. Finally, using the LDTD-APCI-HRMS combination, analysis of total MCs has been adapted to fish tissues to determine the unbound and bound MCs and compare the results with standard analysis. The use of digestion with sodium hydroxide prior to Lemieux oxidation followed by SPE purification yielded a detection limit of 2.7 µg kg-1. Total MCs concentrations were found between 2.9 and 13.2 µg kg-1 in real field-collected contaminated fish samples and comparison was made with standard analysis which yield a single positive sample with a concentration of 2 µg kg-1. This indicates the possible presence of undetected MCs using conventional analytical methods.