A intensificaÃÃo da eutrofizaÃÃo nos mananciais à provocada pelas aÃÃes antrÃpicas e tem como predominÃncia as floraÃÃes de cianobactÃrias. Por sua vez, estas floraÃÃes alteram a qualidade da Ãgua para o abastecimento da populaÃÃo, e, ao serem lisadas, podem liberar toxinas (cianotoxinas) causando intoxicaÃÃo. A primeira confirmaÃÃo in loco da morte de seres humanos por intoxicaÃÃo com cianotoxinas no Brasil levou à OMS a publicar rapidamente novas Portarias sobre o monitoramento da qualidade da Ãgua bruta que incorporaram novos indicadores, como a concentraÃÃo das cianobactÃrias e de suas toxinas nos mananciais utilizados para abastecimento de Ãgua potÃvel. Com isso, as tecnologias para o tratamento da Ãgua e para a identificaÃÃo e quantificaÃÃo das cianotoxinas vÃm passando por um processo de aperfeiÃoamento. Neste contexto, o presente trabalho objetivou validar o mÃtodo de cromatografia lÃquida de fase reversa com detector de fluorescÃncia (CLAE-FLD) e derivatizaÃÃo prÃ-coluna para detecÃÃo e quantificaÃÃo das cianotoxinas saxitoxina (STX) e decarbamoil-saxitoxina (dc-STX) proveniente do cultivo da cianobactÃria Cylindrospermopsis raciborskii. Esta validaÃÃo foi realizada para dar credibilidade ao mÃtodo analÃtico e os parÃmetros selecionados foram: seletividade, linearidade, limite de detecÃÃo (LD) e quantificaÃÃo (LQ), exatidÃo, precisÃo e robustez. Os resultados obtidos apresentaram boa seletividade, comprovando que o mÃtodo possuÃa capacidade de medir as toxinas em uma matriz PÃs ExtraÃÃo na presenÃa de outros componentes. As curvas analÃticas foram construÃdas com nove pontos a partir dos padrÃes de STX e dc-STX. O mÃtodo apresentou uma linearidade no intervalo de 4,5 à 150 Âg L-1 para STX e 3,0 à 132 Âg L-1 para dc-STX, e o coeficiente de correlaÃÃo (r) maior que 0,99 para as duas toxinas, mostrando que o mÃtodo tem a capacidade de fornecer resultados diretamente proporcionais à concentraÃÃo dos analitos detectados. A sensibilidade foi medida atravÃs do LD e LQ, obtendo resultados satisfatÃrios para os objetivos do trabalho. O mÃtodo obteve boa precisÃo e exatidÃo, visto que para STX e dc-STX os diferentes nÃveis de concentraÃÃo estavam com valores dentro dos intervalos permitidos pelas normas brasileiras de validaÃÃo, e apresentou-se robusto, pois foi insensÃvel a pequenas variaÃÃes possÃveis de ocorrer durante a anÃlise. Em resumo, pode-se considerar que o mÃtodo utilizado para a detecÃÃo e quantificaÃÃo das cianotoxinas STX e dc-STX apresentou resultados satisfatÃrios, uma vez que os parÃmetros analisados para validÃ-lo estavam em conformidade aos valores aceitos nas normas brasileiras. / The intensification of eutrophication in the watershed is caused by human actions and is the predominant cyanobacteria. In turn, these blooms affecting the quality of the water supply for the population, and, when disrupted, can release toxins (cyanotoxins) causing intoxication. The first in situ confirmation of the death by poisoning of humans with cyanotoxins in Brazil led the WHO to quickly publish new Ordinance on monitoring of raw water quality that incorporate new indicators, as the concentration of the cyanobacteria and their toxins in water sources used for drinking water supply. Thus, technologies for water treatment and for the identification and quantification of cyanotoxins have been going through a process of improvement. In this context, this study aimed to validate the method of reverse phase liquid chromatography with fluorescence detection (HPLC-FLD) and pre-column derivatization for detection and quantification of cyanotoxins saxitoxin (STX) and decarbamoil-saxitoxin (dc-STX) from the cyanobacterium Cylindrospermopsis raciborskii cultivation. This validation was performed to give credibility to the analytical method and the selected parameters were: selectivity, linearity, limit of detection (LOD) and quantification (LOQ), accuracy, precision and robustness. The results showed good selectivity, confirming that the method had the ability to measure the toxins in a Post Extraction matrix in the presence of other components. The analytical curves were constructed with nine points from the patterns of STX and dc-STX. The method showed linearity in the range of 4.5 to 150 mg L-1 for STX and 3.0 to 132 mg L-1 to dc-STX, and the correlation coefficient (r) greater than 0.99 for both toxins, showing that the method has the capacity to deliver results directly proportional to the concentration of analyte detected. The sensitivity was measured by the LD and LQ, obtaining satisfactory for the purposes of work results. The method achieved good precision and accuracy, whereas for STX and dc-STX different concentration levels were with values ​​within the ranges allowed by Brazilian standards for validation, and showed to be robust because it was insensitive to small variations possible to occur during analysis. In summary, one can consider that the method used for the detection and quantification of cyanotoxins STX and dc-STX showed satisfactory results, since the parameters analyzed to validate it were in conformity with the accepted values ​​in Brazilian standards.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-02-11T16:24:22Z No. of bitstreams: 1 rafaelrodriguesdepaiva.pdf: 1188955 bytes, checksum: b04f0f61a4d3103570287581b800ce0b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-02-26T12:03:43Z (GMT) No. of bitstreams: 1 rafaelrodriguesdepaiva.pdf: 1188955 bytes, checksum: b04f0f61a4d3103570287581b800ce0b (MD5) / Made available in DSpace on 2016-02-26T12:03:43Z (GMT). No. of bitstreams: 1 rafaelrodriguesdepaiva.pdf: 1188955 bytes, checksum: b04f0f61a4d3103570287581b800ce0b (MD5) Previous issue date: 2014-03-27 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Florações de cianobactérias podem causar efeitos negativos na qualidade da água e em comunidades biológicas. Dentre estas comunidades, copépodos calanóides possuem uma grande importância, sendo o grupo planctônico dominante em muitos ambientes de água doce. Apesar da importância destes copépodos para a dinâmica planctônica nestes sistemas, estudos sobre a interação entre estes animais e cianobactérias são ainda escassos. No presente estudo, os efeitos da cianobactéria invasiva e tóxica Cylindrospermopsis raciborskii (Woloszyńska) Seenayya et Subba Raju (1972) no copépodo calanóide tropical Notodiaptomus iheringi (Wright 1935) foram avaliados. Alimentação (taxas de remoção e seleção alimentar), sobrevivência (curvas de Kaplan-Meier, probabilidades de sobrevivência e Hazard ratio) e reprodução (% fêmeas com ovos e produção de ovos) deste copépodo foram estudadas usando dietas monoespecíficas e mistas de C. raciborskii e algas nutritivas (Chlamydomonas chlorastera ou Cryptomonas obovata). O aumento na proporção de C. raciborskii (> 50%) esteve relacionado a baixas taxas de alimentação, embora nenhuma diferença entre as taxas de remoção na cianobactéria e na alga nutritiva dentro de cada tratamento tenha sido encontrada. Efeitos negativos (25 ou 75% de C. raciborskii) ou neutros (50% de C. raciborskiii) na sobrevivência foram detectados nos tratamentos. Por outro lado, a reprodução em N. iheringi foi afetada pela presença de C. raciborskii, independente da proporção desta cianobactéria. Características da espécie C. raciborskii tais como toxicidade (e.g. saxitoxinas) e baixos valores nutricionais (e.g. ausência de PUFA) juntamente com não aclimatação prévia em N. iheringi à C. raciborskii podem explicar os diferentes resultados encontrados. Em geral, copépodos N. iheringi podem se alimentar e sobreviver em dietas com C. raciborskii. Porém, dietas de C. raciborskii não suportam a reprodução destes animais. Este é o primeiro estudo que avalia o efeito de C. raciborskii na sobrevivência e reprodução de copépodos calanóides. Os resultados deste trabalho podem contribuir para o entendimento do comportamento alimentar assim como a dinâmica populacional de copépodos calanóides na presença da cianobactéria C. raciborskii. / Cyanobacterial blooms can cause negative impacts on water quality and biological communities. Among these biological communities, calanoid copepods have a great importance, being the dominant planktonic group in many freshwater systems. Despite the importance of calanoid copepods to plankton dynamics in freshwater systems, studies on the interaction between these animals and cyanobacteria are still scarce. Here the effects of the invasive and toxic cyanobacterium Cylindrospermopsis raciborskii (Woloszyńska) Seenayya et Subba Raju (1972) on the tropical calanoid copepod Notodiaptomus iheringi (Wright 1935) were evaluated. Feeding (clearance rates and selective feeding), survival (Kaplan-Meier curves, survival probabilities and hazard ratios) and reproduction (% females with eggs and egg production) of this copepod were studied using single and mixed diets of C. raciborskii and nutritious algae (Chlamydomonas chlorastera or Cryptomonas obovata). The increase on C. raciborskii proportion (> 50%) was related to lower feeding rates although no differences between the clearance rates on the cyanobacterium and nutritious algae within each treatment were found. Negative (25 or 75% of C. raciborskii) or neutral (50% of C. raciborskiii) effects on survival were detected throughout the treatments. On the other hand, the reproduction in N. iheringi was strongly affected by the presence of C. raciborskii, independently of the proportion of this cyanobacterium. Features of the species C. raciborskii such as toxicity (e.g. saxitoxins) and low nutritional values (e.g. absence of PUFA) along with non-previous acclimation in N. iheringi to C. raciborskii might explain the different outcomes found in the present study. Overall, N. iheringi copepods can feed and survive in diets with C. raciborskii. However, C. raciborskii diets cannot support reproduction of these animals. This is the first study that evaluates the effects of C. raciborskii on survival and reproduction of calanoid copepods. The outcomes found in this work might contribute to the understanding of the feeding interactions as well as the population dynamics of calanoid copepods in the presence of the cyanobacterium C. raciborskii.

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Submitted by (ana.araujo@ufrpe.br) on 2016-08-23T11:22:39Z No. of bitstreams: 1 Hugo Leandro Ferreira Borges.pdf: 956063 bytes, checksum: 64e53a232df271be49887757b5d14d96 (MD5) / Made available in DSpace on 2016-08-23T11:22:40Z (GMT). No. of bitstreams: 1 Hugo Leandro Ferreira Borges.pdf: 956063 bytes, checksum: 64e53a232df271be49887757b5d14d96 (MD5) Previous issue date: 2013-08-23 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Most of the knowledge about cyanobacteria toxin production is traditionally associated with planktonic cynobacterial blooms. However, some studies have been showing that benthic and periphytic cyanobacterias can produce cyanotoxins. According to this, we aimed to evaluate the production of microcystins and saxitoxins and the yield potential of these toxins through the presence of genes involved in their synthesis in benthic and periphytic cyanobacterias isolated from some sample sites of the Mundaú river basin, Pernambuco, and the Environmental Protection Area of Araripe, Ceará. And due to the difficulty of strains morphological classification, phylogenetic analysis were performed using the 16S rDNA and 16S-23S ITS. The material was sampled in rivers and water supply reservoirs, using micromanipulation tec hniques. We isolated 45 clonal strains of cyanobacteria, which were identified morphologically and genetically. In order to evaluate the toxins production, the strains were cultured in controlled laboratory conditions for obtaining biomass for subsequent extraction and analysis of toxins by HPLC. In a complementary way of the toxins analysis, PCR’s were performed screening for genes involved on microcystins and saxitoxins production. Eight species were identified belonging to the Phormidiaceae, Pseudanabaenaceae and Nostocaceae families. This is the first study in Brazil that shows that strains from the Geitlerinema genus correspond to at least three phylogenetic lineages, which possibly correspond to three distinct species to be subsequently reclassified. The strains that showed one of the genes involved in the cyanotoxins production were analyzed by HPLC and Geitlerinema amphibium, G. lemmermannii, Cylindrospermum stagnale and Phormidium uncinatum were identified as producing one or more saxitoxins variants. Thus, this is the first report of saxitoxins producing for those first three species and the first report in Brazil for P. uncinatum. / A maior parte do conhecimento sobre a produção de toxinas por cianobactérias concentra-se no estudo das espécies planctônicas formadoras de florações. Entretanto, alguns estudos vêm mostrando que espécies bentônicas e perifíticas podem produzir várias toxinas. Neste sentido, objetivou-se avaliar a produção de saxitoxinas e microcistinas e a potencialidade de produção dessas toxinas através da presença de genes envolvidos na sua síntese em cianobactérias bentônicas e perifíticas isoladas de alguns pontos da bacia do rio Mundaú, Pernambuco, e da Área de Proteção Ambiental do Araripe, Ceará e, em razão da dificuldade de classificação morfológica das cepas isoladas, foram realizadas análises filogenéticas utilizando os genes 16S rDNA e 16S-23S ITS. O material foi coletado em rios e reservatórios de abastecimento e, usando técnicas de micromanipulação, foram isoladas cepas clonais de cianobactérias, as quais foram identificadas morfologicamente e geneticamente. A fim de avaliar a produção de toxinas, as cepas foram cultivadas em condições controladas de laboratório para obtenção de biomassa para posterior extração de toxinas e análise por cromatografia líquida de alta eficiência (CLAE). De maneira complementar à análise de toxinas, foram realizadas análises genét icas (reação em cadeia da polimerase – PCR) para identificar genes responsáveis pela produção de microcistinas e saxitoxinas. Das 45 cepas isoladas, foram identificadas oito espécies pertencentes às famílias Phormidiaceae, Pseudanabaenaceae e Nostocaceae. Este é o primeiro trabalho feito no Brasil, onde é mostrado que isolados do gênero Geitlerinema correspondem a pelo menos três linhagens filogenéticas, que possivelmente correspondem a três espécies distintas a serem posteriormente reclassificadas. Os isolados que apresentaram um dos genes envolvidos na produção de cianotoxinas foram analisados por CLAE e Geitlerinema amphibium, G. lemmermannii, Cylindrospermum stagnale e Phormidium uncinatum foram identificadas como produtoras de um ou vários análogos de saxitoxinas, sendo o primeiro registro da produção de saxitoxinas para as três primeiras espécies e o primeiro registro no Brasil para P. uncinatum.

Saxitoxins were extracted from a bloom of toxic Anabaena circinalis and used to spike treated water from Hope Valley Reservoir (HVTW) and Milli-Q water. The waters were treated with ozone using the batch method and saxitoxin levels were measured in the samples using HPLC. The results for oxidation of saxitoxins in Milli-Q water versus HVTW show that despite the presence of natural organic matter(NOM) and the production of vastly different ozone residuals, there was a similar removal of all saxitoxins in both waters. The results show that high concentrations of saxitoxins were present in solution after ozonation with doses and contact times typically used in water treatment. Relating the toxin destruction to ozone residual showed that even with a residual ozone concentration of 0.8 mg/L after 10 minutes contact in HVTW, over 60% of the initial saxitoxin content was still present in the samples. The presence of an ozone residual in the water could not be related to saxitoxin destruction and it appeared that saxitoxin removal occurred more rapidly when ozone was consumed rather than stabilised in solution. The results indicate that the mechanism for toxin removal is probably based on the reaction with a hydroxyl radical species as the oxidant rather than molecular ozone. The results obtained during these experiments indicate that ozone is not an effective oxidant for this class of compound. A range of ozone doses were applied to two different treated reservoir waters that had been spiked with microcystins LA (mLA) and LR (mLR). At the ozone dose where a residual was first measured in the sample after 5 minutes exposure time, no microcystins were detected by HPLC in either water. The removal of mLA and mLR was identical in all samples. The absence of mLA and mLR by HPLC was supported by a loss of toxicity using a highly sensitive and specific bioassay (PP2A) and by in vivo studies in mice. In both waters microcystins were removed with an ozone dose typical of that used in drinking water treatment. The results indicate that conventional ozone treatment was effective in removing hepatotoxicity at microcystin levels greater than those likely to be found in drinking water. Two waters were sampled from reservoirs in South Australia. One was collected directly from Happy Valley Reservoir (HVRW) and the other from Myponga Reservoir after treatment but before chlorination (MFCW). They were spiked with mLA and mLR and chlorinated to measure toxin removal and chlorine consumption using the CT concept. In MFCW at pH 7 there was a better removal of both mLA and mLR than in HVRW at pH 8.1. There was also a lesser effect from water temperature upon toxin removal in MFCW. Microcystin LA was less easily removed than mLR at both temperatures in both waters. For HVRW, at the higher pH, this required an initial dose of 7 mg/L of chlorine which corresponded to a CT of around 70 min.mg/L. If the water temperature was reduced to 6??C then under these conditions there would still be 40% of the initial concentration of mLA and mLR present in this water. At this temperature a final chlorine residual of 3.5 mg/L after 30 minutes, requiring a chlorine dose of 8mg/L and corresponding to a CT of about 95 min.mg/L, was required to reduce microcystin levels below the WHO guidelines. This implies that in colder climates the application of chlorine for microcystin removal may require elevated chlorine doses and CT values. Arrhenius activation energies were calculated for mLA and mLR in both waters, revealing different Ea values for both toxins. Due to the complexity of the reactions and the possible effects of pH in solution, this system was considered too complicated to be described by the Arrhenius equation. NOM was collected from Myponga Reservoir in South Australia using magnetic ion exchange (MIEX??) resin. The collected NOM was desorbed and separated into fractions of different molecular weight and character using ultrafiltration and mixed resin ion exchange. At approximately 5 mg/L dissolved organic carbon (DOC) the measured apparent second order rate constant (kapp) for mLA and mLR removal was fairly similar in both the high molecular weight fraction (designated F3), and the intermediate high molecular weight fraction (designated F2). The low molecular weight fraction (designated EN) had slightly higher kapp values as would be expected due to the less reactive nature of the NOM in this fraction. This meant more chlorine was available to react with microcystins in this fraction. Fractions F3 and F2 produced similar kapp values to those from the parent water source following treatment, indicating the similar reactivity of these NOM fractions at comparable DOC levels. Increasing the DOC concentration in the F2 fraction increased kapp for both mLA and mLR due to the additional chlorine needed to react with the additional NOM present. The results showed that pH, temperature and DOC concentration have a higher impact upon chlorination rates, and the efficiency of toxin removal, than NOM character alone. In general it is assumed that chlorine will be more effective at removing toxins in water with a low SUVA and low specific colour as these indicate less 'reactive' NOM in the water. The results of this study show that toxin removal was more effective in the EN fraction as indicated by the higher kapp. This fraction also had the lowest SUVA and lowest specific colour which supports the generally held view in water treatment. Relating the toxin removal to chlorine residual in these reconstituted fractionated NOM samples, indicated that a residual of around 1.5 mg/L after 30 minutes contact was generally adequate to remove all toxins in water with a DOC level of around 5 mg/L. This is consistent with the results obtained in real waters, where at 20??C a chlorine residual of 2 mg/L was found to be sufficient for removal of both mLA and mLR.