Avec la hausse mondiale de la fréquence des floraisons de cyanobactéries (CB), dont certaines produisent des cyanotoxines (CT), le développement d’une méthode de détection/quantification rapide d’un maximum de CT s’impose. Cette méthode permettrait de faire un suivi quotidien de la toxicité de plans d’eau contaminés par des CB et ainsi d’émettre rapidement des avis d’alerte appropriés afin de protéger la santé publique. Une nouvelle technologie utilisant la désorption thermique induite par diode laser (LDTD) couplée à l’ionisation chimique sous pression atmosphérique (APCI) et reliée à la spectrométrie de masse en tandem (MS/MS) a déjà fait ses preuves avec des temps d'analyse de l’ordre de quelques secondes. Les analytes sont désorbés par la LDTD, ionisés en phase gazeuse par APCI et détectés par la MS/MS. Il n’y a donc pas de séparation chromatographique, et la préparation de l’échantillon avant l’analyse est minimale selon la complexité de la matrice contenant les analytes. Parmi les quatre CT testées (microcystine-LR, cylindrospermopsine, saxitoxine et anatoxine-a (ANA-a)), seule l’ANA-a a généré une désorption significative nécessaire au développement d’une méthode analytique avec l’interface LDTD-APCI. La forte polarité ou le poids moléculaire élevé des autres CT empêche probablement leur désorption. L’optimisation des paramètres instrumentaux, tout en tenant compte de l’interférence isobarique de l’acide aminé phénylalanine (PHE) lors de la détection de l’ANA-a par MS/MS, a généré une limite de détection d’ANA-a de l’ordre de 1 ug/L. Celle-ci a été évaluée à partir d’une matrice apparentée à une matrice réelle, démontrant qu’il serait possible d’utiliser la LDTD pour effectuer le suivi de l’ANA-a dans les eaux naturelles selon les normes environnementales applicables (1 à 12 ug/L). Il a été possible d’éviter l’interférence isobarique de la PHE en raison de sa très faible désorption avec l’interface LDTD-APCI. En effet, il a été démontré qu’une concentration aussi élevée que 500 ug/L de PHE ne causait aucune interférence sur le signal de l’ANA-a. / Within the context of the worldwide increasing frequency of cyanobacterial (CB) blooms, some containing cyanotoxins (CT), the development of a detection/quantification method for the fast analysis a maximum of CT is necessary. This method would allow daily tracking of the toxicity of CB-contaminated water such that, as warranted, appropriate measures can be taken quickly to protect public health. A new technology using laser diode thermal desorption (LDTD) coupled to atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry (MS/MS) has shown great potential to reduce analysis time to seconds. Analytes are desorbed by the LDTD, ionized in gas-phase by APCI and detected by MS/MS. Therefore, there is no chromatographic separation and sample treatment prior to analysis is minimal, depending on the complexity of the sample matrix. Among the four CT tested (microcystin-LR, cylindrospermopsin, saxitoxin and anatoxin-a (ANA-a)), only ANA-a exhibited sufficient desorption which is necessary to develop an analytical method with the LDTD-APCI interface. The strong polarity or high molecular weight of the other CT probably inhibited their efficient desorption. Optimization of instrumental parameters, while accounting for the isobaric interference caused by the acid amino phenylalanine (PHE) in the detection of ANA-a by MS/MS, generated a detection limit of the order of 1 ug/L ANA-a. This value was obtained in a simulated natural matrix, demonstrating that it would be possible to use LDTD to monitor ANA-a in natural waters within the range of current applicable environmental guidelines (1 to 12 ug/L). Because PHE desorption is limited with the LDTD-APCI interface, this method avoids its interference on ANA-a analysis, even at PHE concentrations as high as 500 ug/L.

The western embayment of Lake Ontario, Hamilton Harbour, is one of the most polluted sites in the Laurentian Great Lakes and in recent years has seen a reoccurrence of cyanobacterial blooms. This study uses a multidisciplinary approach to examine the presences of toxic Cyanobacteria in the harbour in order to gain insight into these recurrent blooms. Microscopic analyses of phytoplankton samples collected during the 2009 summer-fall sampling season from two locations within the harbour showed the spatial and seasonal diversity of the contemporary cyanobacterial community. Microcystis colonies relative abundances in relation to total algal numbers were estimated. The lowest and highest relative abundances of Microcystis in the phytoplankton population were 0.6% and 9.7%, respectively, and showed seasonal variability between stations. Fourteen cyanobacterial genera comprising six families and three orders were identified and for which the most abundant filamentous genera during the summer-fall sampling season were Planktothrix, Aphanizomenon and Limnothrix. Potential microcystin producers Microcystis, Planktothrix, Aphanizomenon and Dolichospermum were also present and during the sampling period Microcystis was recorded at both stations on all dates, however, its relative abundance was below 10 % throughout the study period. The composition and abundance of filamentous cyanobacteria were observed to be positively statistically correlated to water quality environmental parameters dissolved nitrates (NO3/NO2), dissolved inorganic carbon (DIC), and conductivity. Redundancy analysis (RDA) found that 53.35% total variance of Aphanizomenon was correlated to low water column NO3/NO2 and conductivity, and higher water column DIC. 58.13% of the relative abundance of Planktothrix was correlated to high concentrations of dissolved nitrates, while 51.69% of total variance of Limnothrix was correlated to higher DIC and lower water column dissolved nitrate concentrations. Information about past cyanobacterial communities was obtained from the sediment core analysis, using paleolimnological and modern molecular methods. The age of the 100.5 cm long sediment core retrieved from the deepest part of Hamilton Harbour was established to be 140 years (1869-2009), using the Constant Rate of Supply (CRS) 210Pb age model. This age was not sufficient to provide information of harbour’s environmental conditions, presence of the blooms, and triggers for their occurrence before European settlement in the area. Results of the HPLC analysis of fossil pigments indicated that the dominant members of the algal community have not changed over the 140 years and that cyanobacteria were regular members of the phytoplankton community. The composition of the major chlorophyll pigments indicated high presence of Chlorophyta and Bacillariophyta in the harbour at all times. The main algal groups identified on the basis of marker pigments presence, besides the Chlorophyta and Bacillariophyta, were the Dinophyta and the Cryptophyta. The presence of a scytonemin derivative, compound B, indicated that cyanobacterial blooms were occurring in past, before the first officially recorded blooms in the 1960s. Cyanobacterial pigments presence indicated that Cyanobacteria have been a regular but not dominant feature of Hamilton Harbour phytoplankton in the past. To our knowledge, this study is the first one examining fossil pigments from Hamilton Harbour. Results of the PCR-DGGE molecular analysis of 16S rRNA-V3 gene fragments from sedimentary DNA revealed the presence of thirteen cyanobacterial genotypes. The temporal change in the cyanobacterial community composition was indicated by the increasing number of species over time, from the oldest to the most recent sediment layers. The deepest sediment strata showed the lowest number (two bands) and intensity of bands. The most recent sediment layer had the greatest numbers (11) and intensity of bands. This increased diversity indicated changing environmental conditions in the harbour, primarily nutrient pollution and worsening water quality. Results of the PCR-DGGE molecular analysis of mcyE-AMT gene fragments showed that Microcystis aeruginosa and Planktothrix rubescens were two microcystin producers present in Hamilton Harbour over the last 80 years. The persistent presence and resilience of these two genera indicated a more serious and longer-term issue of toxic blooms than previously recognized. Historical records show that noticeable anthropogenic impact on Lake Ontario environment has been measurable since the 1780s, the first dramatic impact on the Lake Ontario watershed was evident from the mid1880s, the earliest evidence of eutrophication in the lake occurred between 1820 and 1850, while human induced environmental changes in Hamilton Harbour date back ca. 350 years. In the 1960s, cyanobacterial blooms were first officially recognized in the harbour and the lower Great Lakes. The present research is the first report of the mcyE module and AMT domain of microcystin genes being amplified from sediment of North American lakes, and showed that toxic Cyanobacterial have been regular members of Hamilton Harbour phytoplankton community for almost a century. This research considerably deepened the knowledge of the past toxic cyanobacterial blooms in Hamilton Harbour and their possible causes. It also showed that in the absence of historical records, both the PCR-DGGE method and the mcyE-AMT gene may be used for reconstruction of the past toxic blooms not only in the Laurentian Great Lakes, but also in other aquatic regions of the world impacted by toxic cyanobacterial blooms. Also, it demonstrated the utility of the combined molecular and paleolimnological analyses, which might become a useful tool in the determination of the bloom causes factors and in the mitigation of the future production of toxic blooms.

The investigation presented in this thesis examined the microbial and functional diversity of the meltwater ponds Fresh, Orange and Salt Ponds on the McMurdo Ice Shelf, near Bratina Island, Antarctica. These sites were chosen because of the ecological importance and absence of detailed characterisations of their diversity and function as part of Antarctica?s largest wetland. Particular focus was on cyanobacterial diversity, nitrogen fixation and secondary metabolite production. Using 16S rRNA gene and morphological analysis a large diversity of cyanobacteria (more than 22 phylotypes) was identified with high phylogenetic similarities (up to 99% sequence identity) to cyanobacteria from mats in other regions of Antarctica. In addition biogeographical distributions were identified including potentially endemic and cosmopolitan cyanobacteria. High salinities were also connected to the change and reduction of diversity. Lipid marker analyses were performed targeting hydrocarbons, ether-linked hydrocarbons, methylated fatty acid esters (FAME), wax esters, hopanols and sterols. Lipid biomarker profiles were similar to typical cyanobacteria dominated mats with major input from microorganisms including oxygenic and anoxygenic phototrophs, obligate aerobic and anaerobic heterotrophs that conduct the metabolic processes of fermentation, sulphate reduction, sulphate and iron-oxidation, methanogeneses. Signature lipids indicative of Chloroflexus and archaea, as well as branched aliphatic alkanes with quaternary substituted carbon atoms (BAQCs), were identified for the first time in Fresh, Orange and Salt Ponds. Based on nifH gene analysis, the nitrogen fixing diversity characterised in Orange Pond consisted of cyanobacterial Nostoc sp. as well as firmicutes, beta-, gamma- and delta-proteobacteria. Acetylene reduction assays and nifH gene RNA transcript diversity identified Nostoc sp. as a main contributor of nitrogenase activity in these ponds. Furthermore, analytical methods were used to identify the cyanobacterial secondary metabolites microcystins, although the genetic basis for this production and the toxin producer could not been identified. However non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) genes were identified which could be the genetic basis for novel bioactives. The use of a multi-disciplinary approach synthesis and subsequent results significantly increased our understanding of the diversity and function of microbial mat communities in the unique meltwater ponds of the McMurdo Ice shelf, Antarctica.

Planktothricoides raciborskii and Roseofilum reptotaenium are physiologically similar, yet ecologically distinct organisms found in a hot spring outflow and coral black band disease (BBD), respectively. The aim of this study was to elucidate the relationship between R. reptotaenium and sulfide in BBD, to compare microcystin (MC) production in response to environmental factors, and to determine chemotactic responses to MC and sulfide by the two organisms. Results showed that the pathogenicity of R. reptotaenium in BBD is dependent on sulfate-reducing bacteria as secondary pathogens. Roseofilum reptotaenium produced significantly more MC than P. raciborskii, as measured using ELISA. Roseofilum reptotaenium was negatively chemotactic to sulfide, determined using horizontal and vertical gradients in agar, while P. raciborskii was not affected. Neither cyanobacterium was chemotactic to MC in the agar assays. The ecophysiology of P. raciborskii and R. reptotaenium in relation to MC production and response to sulfide reflected their pathogenic versus non-pathogenic status.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-09-15T14:54:18Z No. of bitstreams: 1 TeseNDM.pdf: 2605858 bytes, checksum: bff58bf7890c21ba2d47c2079816f268 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-16T19:26:12Z (GMT) No. of bitstreams: 1 TeseNDM.pdf: 2605858 bytes, checksum: bff58bf7890c21ba2d47c2079816f268 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-16T19:26:23Z (GMT) No. of bitstreams: 1 TeseNDM.pdf: 2605858 bytes, checksum: bff58bf7890c21ba2d47c2079816f268 (MD5) / Made available in DSpace on 2016-09-16T19:26:31Z (GMT). No. of bitstreams: 1 TeseNDM.pdf: 2605858 bytes, checksum: bff58bf7890c21ba2d47c2079816f268 (MD5) Previous issue date: 2015-03-10 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Cyanobacteria, under favorable conditions can form large superficial masses of green color called blooms. Some genera of cyanobacteria are toxins producers, and during the bloom, there may be risks of water bodies contamination and thus of those organisms that depends on this. Among the toxins produced by cyanobacteria, stands the microcystin. This type of toxin has pronounced effects on the liver, although it can exhibit its effects in several organs. Moreover, it is the cyanotoxin which is more widely distributed in various types of ecosystems. In addition to the phosphatases inhibition, microcystins may also affect DNA repair systems and gene expression, and appear to interact with the mitochondria of animal tissues leading to oxidative stress and apoptosis. The aim of this study was to evaluate the cardiorespiratory responses during graded hypoxia and also the biomarkers of oxidative stress in two species of Neotropical teleost, traíra, Hoplias malabaricus, and matrinxã, Brycon amazonicus in control condition and 48 h after of an intraperitoneal injection of lyophilized extract containing microcystin -LR (MC-LR - 100 μg.Kg-1 body weight). The results indicate that exposure to MC-LR induced oxidative stress in liver and gills, and changed the cardiorespiratory responses of both species. In traíra, the exposure to MC-LR increased PCO2 values, caused hyperventilation, increased metabolic rate, increased heart rate values and decreased oxygen extraction, it was also observed oxidative stress in the gill tissue. In matrinxã was observed hyperventilation, increased metabolic rate and PCO2, and reduction in oxygen extraction. This specie was more sensitive to the effects of MC-LR, due to the occurrence of oxidative stress in both tissues (gills and liver), and the death of some animals during the development of graded hypoxia. Therefore, our results indicate that exposure to MC-LR has harmful effects on the two species analyzed, both in cardiorespiratory responses, as in the establishment of oxidative stress, however the magnitude of this effect appears to be species-specific indicating major susceptibility of the specie Brycon amazonicus to the effect of this cyanotoxin. / As cianobactérias, em condições favoráveis podem formar grandes massas superficiais de coloração verde intensa, denominadas florações. Alguns gêneros de cianobactérias são produtoras de toxinas, e durante as florações, pode haver riscos de contaminação da água e consequentemente dos organismos dependentes desta. Dentre as toxinas produzidas pelas cianobactérias, destaca-se a microcistina. Esse tipo de toxina apresenta efeitos pronunciados sobre o fígado, embora possa exibir seus efeitos em diversos órgãos. Ademais, é a cianotoxina que se encontra mais amplamente distribuída nos mais variados tipos de ecossistemas. Além da inibição das fosfatases, as microcistinas podem também afetar os sistemas de reparo de DNA e expressão de genes, e parecem poder interagir com as mitocôndrias de tecidos animais e causar estresse oxidativo e apoptose celular. O objetivo deste estudo foi avaliar as respostas cardiorrespiratórias durante hipóxia gradual e os biomarcadores do estresse oxidativo em duas espécies de teleósteos Neotropicais, a traíra, Hoplias malabaricus e o matrinxã Brycon amazonicus em situação controle e após 48 h de injeção intraperitoneal de extrato liofilizado contendo microcistina-LR (MC-LR - 100 μg.Kg-1 de massa corpórea). Os resultados indicam que a exposição à MC-LR induziu ao estresse oxidativo em fígado e brânquias, bem como alterou as respostas cardiorrespiratórias de ambas as espécies. Na traíra a exposição a MC-LR aumentou os valores de PcO2, causou hiperventilação, aumento da taxa metabólica, aumento dos valores de frequência cardíaca e redução da extração de oxigênio, também foi observado estresse oxidativo no tecido branquial. Em matrinxã foi observada hiperventilação, aumento da taxa metabólica e da PcO2, e redução na extração de oxigênio. Essa espécie se mostrou mais sensível ao efeito da MC-LR, devido a ocorrência de estresse oxidativo em ambos os tecidos analisados (brânquias e fígado) e a morte de alguns animais durante o desenvolvimento da hipóxia gradual. Portanto, nossos resultados indicam que a exposição à MC-LR tem efeitos prejudiciais sobre as duas espécies analisadas, tanto nas respostas cardiorrespiratórias, quanto no estabelecimento de estresse oxidativo, entretanto a magnitude desse efeito parece ser espécie-específica com indicação de maior susceptibilidade da espécie Brycon amazonicus ao efeito dessa toxina.

Made available in DSpace on 2015-09-25T12:20:01Z (GMT). No. of bitstreams: 1 Sativa Barbosa de Brito Lelis Villar.pdf: 821851 bytes, checksum: b1c26304b9ae92f8fe9dbc59a9c0a15c (MD5) Previous issue date: 2012-12-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo deste estudo foi avaliar a remoção de microcistina-LR de água utilizando carvão ativado granular de casca de coco de dendê. A água de estudo utilizada foi preparada a partir da diluição, em água deionizada, de extrato proveniente do cultivo de Microcystis aeruginosa. Realizou-se um planejamento fatoral 23, no qual foram estudados os seguintes fatores e níveis: concentração inicial de microcistina-LR (14,56 e 29,26 µg.L-1), pH (6,4 e 8,3) e tempo de contato (60 e 90 s). Dessa forma, foram monitorados oito tratamentos em triplicata, totalizando 24 experimentos. Cada tratamento consistia na operação de três colunas, alimentadas com mesma água de estudo e com mesmo tempo de contato, sendo que os tratamentos com tempo de contato de 60 s eram compostos por colunas de 20 gramas de carvão ativado granular, enquanto os experimentos que foram realizados com tempo de contato de 90 s, eram compostos por colunas com 30 gramas do carvão. Cada coluna foi monitorada durante 15 horas e as amostras eram coletadas a cada hora. A concentração de microcistina-LR era determinada com o auxílio de kits ELISA. Concentrações efluentes de MC-LR acima do volume máximo permitido (VMP) de 1µg.L-1, determinado pela Portaria 2914/2011, foram detectadas em colunas operadas a partir dos tratamentos 3 e 7. Esses tratamentos apresentavam concentração afluente de MC-LR de 29,26 µg.L-1 e tempo de contato de 60 s. A capacidade adsortiva e a taxa de uso nessas condições foi de 21,2 µg.g-1 e 1,33g.L-1 respectivamente. O tempo de contato foi o fator que mais influenciou no processo de adsorção de microcistina-LR, este comportamento foi observado a partir de 6 horas de monitoramento e se manteve até o final do monitoramento. O pH foi o fator que menos exerceu influência significativa na adsorção de microcistina-LR.

A filosfera da Mata Atlântica é um importante nicho de colonização por micro-organismos, cuja comunidade ainda é pouco conhecida. Algumas bactérias associadas à superfície das folhas possuem habilidade de fixar nitrogênio, mineralizar substratos orgânicos e também suprir as árvores com dióxido de carbono e fatores de crescimento. Este trabalho teve como objetivo gerar informações sobre a comunidade cianobacteriana que coloniza a filosfera de algumas plantas da Mata Atlântica e investigar a comunidade diazotrófica presente nesse habitat. Um total de 40 linhagens de cianobactérias da filosfera de Merostachys neesii (bambu), Euterpe edulis (palmeira Juçara), Guapira opposita e Garcinia gardneriana foram isoladas e cultivadas. Os isolados foram caracterizados por análises morfológicas e filogenia do gene 16S RNAr. Essa abordagem permitiu a identificação de uma linhagem do gênero Nostoc, sete Desmonostoc, seis Leptolyngbya, uma Oculatella, cinco Brasilonema, uma Pleurocapsa e duas Chroococcidiopsis. Dezessete linhagens (uma Microchaetaceae, dez Nostocaceae e seis Pseudanabaenaceae) não puderam ser identificadas ao nível de gênero. Vinte e seis linhagens (24 pertencentes às ordens Nostocales e duas à Pseudanabaenales) foram caracterizadas como diazotróficas pela amplificação, sequenciamento e filogenia do gene nifH. Além disso, caracterizou-se o perfil de fixação biológica de nitrogênio da linhagem Desmonostoc sp. CENA362. Com relação ao potencial biotecnológico dessas linhagens, treze isolados foram identificados como potenciais produtores de ácido indol acético (IAA) de acordo com o teste Salkowski. Diversas linhagens apresentaram genes associados à via biossintética do inibidor de protease microviridina, sendo que três delas codificam para novas variantes. Além disso, dez linhagens foram identificadas como potenciais produtoras aeruginosina, três de cianopeptolina e três de microcistina. A comunidade bacteriana diazotrófica avaliada por pirosequenciamento do gene nifH apresentou um perfil de variação espécie-específica para Proteobacteria e uma correlação positiva entre a riqueza e a fixação biológica de nitrogênio. Neste estudo, cianobactérias que habitam a filosfera da Mata Atlântica foram isoladas estão sendo mantidas em condições de cultivo. Novos táxons foram descobertos e vários gêneros conhecidos foram descritos pela primeira vez neste hábitat, o que contribuiu para o aprimoramento da sistemática de Cyanobacteria. As linhagens em cultivo e as informações geradas sobre os seus compostos metabólitos representam uma valiosa fonte para estudos posteriores. Além disso, informações sobre a comunidade bacteriana diazotrófica da filosfera pode auxiliar no entendimento da dinâmica do nitrogênio, elemento limitante e pouco disponível na Mata Atlântica / The phyllosphere of the Atlantic Forest is an important niche for colonization by microorganisms, whose community is still little known. Some bacteria associated with leaf surfaces may possess the ability to fix nitrogen, mineralize the organic substrates and also supply the trees with carbon dioxide and growth factors. Therefore, this study aimed to generate information about cyanobacterial community that colonize the phyllosphere of some plants of the Atlantic Forest and investigated the diazotrophic community in this habitat. A total of 40 strains of Cyanobacteria from the phyllosphere of Merostachys neesii (bamboo), Euterpe edulis (Juçara palm), Garcinia gardneriana and Guapira opposita was isolated and cultivated. The isolates were characterized by morphological analyses and phylogeny of the 16S rRNA gene. This approach allowed the identification of one strain of the genus Nostoc, seven Desmonostoc, six Leptolyngbya, one Oculatella, five Brasilonema, one Pleurocapsa and two Chroococcidiopsis. Seventeen strains (one Microchaetaceae, ten Nostocaceae and six Pseudanabaenaceae) could not be identified at the genus level. Twenty-six strains (24 belonging to Nostocales and two belonging to Pseudanabaenales) were characterized as diazotrophic by amplification, sequencing and phylogeny of nifH gene. Also, it was characterized the profile of biological nitrogen fixation for the strain Desmonostoc sp. CENA362. Regarding the biotechnological potential of these strains, thirteen strains were identified as potential producers of indole acetic acid (IAA) according to Salkowski test. Several strains presented genes involved in the biosynthetic pathway of the protease inhibitor microviridin, three of them encoding putative novel variants. Moreover, ten strains were identified as potential producers of aeruginosin, three of cyanopeptolin and three of microcystin. The diazotrophic bacterial community evaluated by pyrosequencing of the nifH gene showed a profile of variation plant species-specific for Proteobacteria, and a positive correlation between richness and biological nitrogen fixation. In this study, cyanobacteria that inhabiting Brazilian Atlantic Forest phyllosphere were isolated and are been maintained in culture conditions. New taxa were discovered and several known genera were described for the first time in this habitat, which contributed to improvement of the cyanobacterial systematic. The culturable strains and the information generated about their metabolites compounds represent a valuable source for further studies. In addition, information about the diazotrophic bacterial community inhabiting the phyllosphere may help in understanding the dynamics of nitrogen, a limiting and low available element in Atlantic Forest

O crescimento desordenado das cidades, a utilização de áreas para plantio próximas aos mananciais e o lançamento de águas residuárias domésticas e industriais sem tratamento são consideradas as principais fontes de poluição dos corpos d´água. Tal poluição contribui para o enriquecimento dos corpos hídricos em relação aos nutrientes. Isto, combinado com fatores climáticos proporciona condições para a ocorrência de florações de algas que podem apresentar espécies potencialmente tóxicas. Desta forma, aumenta a preocupação com desenvolvimento de técnicas de tratamento que forneçam água de qualidade garantindo a saúde da população consumidora. Nesta pesquisa, foram preparadas águas de estudo com diferentes concentrações de microcistina, com as quais foram testados alguns processos de tratamento, em escala de bancada, que consistiram na pré-cloração, adsorção em carvão ativado em pó, coagulação/floculação, flotação por ar dissolvido e centrifugação. O fluxograma de tratamento com aplicação de carvão ativado logo após a mistura rápida foi o que apresentou melhores resultados em termos de cor aparente, turbidez e concentração residual de microcistina. Foi avaliada a influência da concentração de microcistina nos tratamentos simulados. Constatou-se que para 104,92 µg/L de microcistina a aplicação de 3,0 mg\'CL IND.2\'/L (10 segundos antes da mistura rápida) e 30 mg/L de CAP (logo após a mistura rápida) não foram capazes de reduzir a concentração de microcistina para menos de 1 µg/L, conforme o limite estabelecido pela Portaria 518/04 do Ministério da Saúde. Assim, optou-se pela estimativa da dosagem mínima de carvão ativado em pó (CAP) em função da concentração inicial de microcistina das três águas preparadas. O residual de microcistina em cada água preparada, após a aplicação da dosagem estimada de CAP, foi: Água de Estudo 1 = 0,80 µg/L, Água de Estudo 2 = 0,82 µg/L e Água de Estudo 3 = 0,23 µg/L. / The disordered growth of cities, the use of planting areas near the water sources and the launch of domestic and industrial wastewater without treatment are the main sources of water bodies\' pollution. Such pollution contributes to increase the nutrient loading in the water bodies. In association with climatic factors, this enrichment of nutrient provides suitable conditions for the bloom of potentially toxic algae. Thus, increases the concern over to develop treatment techniques that provide water quality able to ensure the health of the consumer population. In this research, waters were prepared with different concentrations of microcystin and some treatments were tested in bench scale. These tests consisted in prechlorination, adsorption on powdered activated carbon, coagulation/flocculation, dissolved air flotation and centrifugation. The diagram of treatment with activated carbon application after the rapid mix showed better results of apparent color, turbidity and residual concentration of microcystin. The influence of the concentration of microcystin in the simulated treatments was evaluated. It was found that for 104.92 µg/L of microcystin the application of 3.0 mg\'CL IND.2\'/L (10 seconds before rapid mix) and 30 mg/L of PAC (after a quick mixture) were not able to reduce the concentration of microcystin to less than 1 µg/L, as the limit established by Order 518/04 of the Health Ministry. Thus, it was decided to estimate the minimum dosage of powdered activated carbon (PAC) according to the initial concentration of microcystin found in the three prepared waters. The residual concentration of microcystin in each water, after the estimated dosage application of PAC was: Water 1 = 0.80 µg/L; Water 2 = 0.82 µg/L; Water 3 = 0, 23 µg/L.

Les efflorescences de cyanobactéries sont susceptibles d’avoir des effets néfastes sur les organismes des écosystèmes aquatiques, ainsi que sur les populations environnantes, notamment à travers la production de nombreuses molécules potentiellement toxiques (appelées cyanotoxines). Jusqu'à présent, une des cyanotoxines les plus étudiées est la microcystine (MC). Cette thèse a pour objectif d’évaluer la toxicité potentielle sur la reproduction de la MC-LR et de l'extrait d'une souche de Microcystis productrice de MCs en étudiant leurs effets toxiques sur le foie et les gonades de poissons medaka adultes exposés de manière aiguë ou chronique.Une étude complète du foie des poissons médaka deux sexes a été menés par ailleurs, attestant d'un fort dimorphisme sexuel aussi bien au niveau cellulaire que moléculaire et souligne les importantes spécificités métaboliques du foie entre les deux sexes, notamment pour le maintien de la compétence de reproduction chez les poissons medaka adultes femelles.Dans l'étude des effets induits par une exposition aiguë, les poissons medaka adultes ont été exposés par gavage à 10 μg.g-1 bw de MC-LR pure pendant 1 heure. L'examen histologique et l'immunolocalisation des MCs du foie de poisson traité par la MC-LR ont révélé des lésions hépatiques sévères ainsi qu'une distribution intense de la MC-LR dans le foie, localisée particulièrement dans le cytoplasme et dans le noyau des hépatocytes. Dans la gonade des poissons traités, la MC-LR a été détectée dans les tissus conjonctifs de l'ovaire et des testicules. De plus, l’observation par microscopie électronique couplé à la technique d’immunogold a révélé, pour la première fois, que la MC-LR était également détectable dans le chorion, le cytoplasme et le vitellus des ovocytes matures.Au cours des études des effets induits par l’exposition chronique, les poissons medaka adultes ont été exposés durant 28 jours par balnéation à 1 et 5 μg.L-1 de MC-LR et à un extrait de la souche de Microcystis aeruginosa (PCC 7820) productrice de microcystines (5 μg.L-1 équivalent MC-LR). Ces résultats ont révélé que la MC-LR et l'extrait de Microcystis induisent des effets délétères sur différents paramètres de reproduction, tels la fécondité et le taux d’éclosion des embryons. La cause principale de ces perturbations de la reproduction semblent principalement résulté d’un dysfonction hépatique globale induite par les traitements aux MCs (hépatotoxiques, notoires), plutôt qu’à des effets directs sur les gonades. Dans l'ensemble, les résultats de cette thèse démontrent que même si les microcystines pourraient avoir un impact direct, mais modéré, sur la fonction gonadique en induisant une cytotoxicité dans les cellules somatiques gonadiques et les cellules reproductrices, elle semble avoir principalement avoir un impact indirect sur la fonction reproductrice en perturbant la fonction hépatique générale. Ces données améliorent notre compréhension des processus liés à la toxicité potentielle des cyanotoxines pour la reproduction chez un poisson modèle, et fait d’une manière générale progresser questionnement quant à la protection des populations exposées à ces cyanotoxines. / Cyanobacterial blooms threaten human health as well as other living organisms of the aquatic environment, particularly due to the production of natural toxic components (called cyanotoxins). So far, one of the most studied cyanotoxins is the microcystin (MC). This thesis evaluated the potential reproductive toxicity of MC-LR and the extract of one Microcystis strain (MC-producing) by investigating their toxic effects on the liver and gonad of adult medaka fish with one acute and one chronic study.An investigation of the metabolic specificities of the liver in two genders of medaka fish was performed prior to the MC-containing exposure, which attests to a strong sexual dimorphism of medaka liver, and highlights the importance of metabolic adjustments of the liver for maintaining the reproductive competency in adult medaka fish.In the acute study, adult medaka fish were administered with 10 μg.g-1 bw of pure MC-LR for 1 hour by gavage. The histological examination and immunolocalization of the MC-treated fish liver revealed a severe liver lesion along with an intense distribution of MC-LR in the liver, being particularly localized in the cytoplasm and nucleus of hepatocytes. In the gonad of MC-treated fish, MC-LR was shown to be present in the connective tissue of ovary and testis. Additionally, immunogold electron microscopy, for the first time, revealed that MC-LR was also localized in the chorion, cytoplasm and yolk vesicles of oocytes.Overall, the results of this thesis demonstrates that MC might directly impact gonadal function by inducing cytotoxicity in gonadal somatic cells and reproductive cells, and it could also impact the reproductive function indirectly by disturbing the general liver function. This improves our understanding of the potential reproductive toxicity of cyanotoxins in model fish, and advances our current knowledge on the protection of aquatic organism populations as well as human health from cyanotoxin issues.

Florescimentos de cianobactérias têm sido cada vez mais frequentemente detectados em reservatórios do Estado de São Paulo, especialmente naqueles da bacia do Médio rio Tietê. A presença de cianobactérias pode acarretar prejuízos à qualidade da água nestes ambientes, em razão de seu potencial tóxico à biota aquática, incluindo riscos à saúde humana. O presente estudo teve por objetivo analisar a estrutura da comunidade zooplanctônica de três reservatórios do Médio rio Tietê, SP (Barra Bonita, Bariri e Ibitinga) e sua relação com as cianobactérias presentes nas florações, com ênfase nos efeitos da toxicidade destas sobre o dafinídeo Ceriodaphnia silvestrii, uma espécie nativa. Para tanto, amostragens foram realizadas nos períodos de junho e setembro de 2008, e janeiro e maio de 2009. Foram efetuadas medições de diversos parâmetros físicos, químicos e biológicos em cada reservatório. As florações de cianobactérias foram coletadas com redes de plâncton, e armazenadas para análise e quantificação de cianotoxinas e também para a realização de ensaios ecotoxicológicos. Verificou-se que a comunidade zooplanctônica dos reservatórios é dominada por copépodos e rotíferos na maior parte das camadas de água dos reservatórios. Os copépodes Cyclopoida foram mais abundantes que os Calanoida, com grande representatividade de formas jovens (náuplios e copepoditos). A espécie de Cladocera Bosminam longirostris e grande parte das espécies de rotifera, como Brachionus calyciflorus foram frequentes em todas as represas, indicando que podem ter estratégias para coexistir com as toxinas dos blooms de cianobactérias. As concentrações de microcistinas LR foram detectadas em todas as amostras de florações dos reservatórios do médio rio Tietê, com concentrações variando e 18,2 a 100 \'mü\'g/L. Os testes de toxicidade aguda revelaram que os extratos brutos das florações de cianobactérias foram tóxicos ao dafínideo Ceriodaphnia silvestrii, provocando inibição dos movimentos natatórios mesmo em baixas concentrações de microcistina-LR, podendo portanto influenciar negativamente a biota aquática. / Cyanobacteria blooms are increasingly being detected in São Paulo state reservoirs, particularly in those of middle Tietê river basin. The presence of cyanobacteria can cause deterioration of water quality in these systems due to its potential toxicity to the aquatic biota, and also risks to human health. The aim of the present srtudy was to analyse the structure of the zooplankton community in three reservoirs placed in middle Tietê river basin, SP (Barra Bonita, Bariri and Ibitinga) and its relationships with blooms of cyanobacteria, with emphasis on its toxic effects upon the daphnid Ceriodaphnia silvestrii, a native species. Sampling was carried out in June and September 2008 and January and May 2009. Measurements of several physical, chemical and biological parameters were carried out in each reservoir. Samples of cyanobacteria blooms were taken with phytoplankton nets and properly kept for posterior analysis of species composition, cyanotoxin quantification and also for acute toxicity testing. It was found that zooplankton communities in the reservoirs are dominated by rotifers and copepods in most of the water layers sampled. The cyclopoid copepods were more abundant than calanoid ones with great representativiness of the young forms (nauplii and copepodids). The Cladocera species Bosmina longirostris and some species of Rotifera as Brachionus calyciflorus were frequent in all reservoirs, indicating that they might have strategies to cope with the cyanobacteria bloom toxicity. Microcystin LR concentrations were detected in all samples of blooms from Middle Tietê River reservoirs with concentrations varying from 18,2 to 100 \'mü\'g/ L. Acute toxicity tests revealed that raw extracts from cyanobacteria scums were toxic to the daphnid Ceriodaphnia silvestrii, causing immobilization of natatory movements even at low LR microcystin concentrations, thus negatively affecting the aquatic biota.