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- 4º Congresso Ibérico de Cianotoxinas − Lisboa, 8-10 julho 2015Publication . Churro, Catarina; Pereira, Paulo
- 4º Congresso Ibérico de Cianotoxinas/VI Reunião da Rede Ibérica de Cianotoxinas: livro de resumosPublication . Valério, Elisabete; Dias, Elsa; Menezes, Carina; Churro, Catarina; Vilares, Arminda; Pereira, PauloNeste 4º Congresso Ibérico de Cianotoxinas, foram debatidos os avanços mais recentes no conhecimento deste tipo de compostos e outros compostos bioactivos produzidos por cianobactérias, nos métodos de detecção e na avaliação/gestão do risco de exposição humana a água contaminadas com cianobactérias tóxicas e também nos novos avanços das estratégias de remediação dos blooms de cianobactérias. O Congresso reuniu especialistas principalmente da Península Ibérica, mas também Brasil, Argentina e França, nas áreas da qualidade hídrica, ambiente e saúde, visando a partilha de conhecimento e de experiências no sentido de resolver problemas comuns relacionados com a ocorrência de Cianotoxinas nas águas recreativas e para consumo humano e a sua propagação na cadeia trófica. Esperamos que o 4ºCIC tenha proporcionado a oportunidade de estreitar as colaborações existentes, bem como para estabelecer novas parcerias, designadamente em novas áreas de I&D em Cianobactérias e Cianotoxinas.
- Abordagem multidisciplinar na identificação e monitorização de cianobactérias potencialmente tóxicasPublication . Churro, Catarina; Valério, ElisabeteO risco que as florescências cianobacterianas representam para a saúde humana advêm do facto destes desenvolvimentos excessivos estarem frequentemente associados à produção de cianotoxinas. As principais vias de exposição para o homem são através de água potável contaminada, diálise, consumo de peixe e marisco contaminado e atividades recreativas. A toxicidade destes compostos é elevada, como pode ser constatado no gráfico 1 em que está representada a comparação da toxicidade, com base na dose-letal (LD50%) em murganhos, entre as cianotoxinas e algumas das toxinas mais conhecidas em relação ao cianeto.
- Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural watersPublication . Churro, Catarina; Vasconcelos, Vitor Manuel de OliveiraThe most common cyanotoxins in Portuguese freshwaters are microcystins and their occurrence has been mainly attributed to cyanobacteria from the Microcystis genus. However, most recently, it has been described the production of these toxins by species of the Planktothrix, suggesting that this genus is also a major producer of microcystin in Portuguese surface waters. Nevertheless, and conversely to the Microcystis species, the knowledge on the occurrence, distribution and toxigenesis of Planktothrix is still limited. Planktothrix species exhibits some particularities that difficult their sampling, identification, quantification and toxigenic characterization in natural samples - Chapter 2. Particularly, the morphology of Planktothrix colonies does not allow distinguishing easily the individual cells. This makes their identification and quantification by optical microscopy a very difficult task, although this is the method generally used in cyanobacteria monitoring. Moreover, the most common methods for the detection of microcystins (ELISA, HPLC) do not identify the producer strains. These strains can be identified by conventional PCR, but this method doesnt enable to quantify them. In resume, there is not yet available a method that allow simultaneously identify and quantify microcystin-producing strains. This work aimed to develop a method based on Real-Time PCR applied to the monitoring of toxic species of Planktothrix in surface freshwater reservoirs used as drinking water supply and for recreational activities. The experimental work was developed according to several phases that are listed and explained below. In a first approach, field surveys were conducted to access the occurrence and distribution of Planktothrix – Chapter 3. It was observed that Planktothrix has a wide distribution in Portuguese lakes and that Planktothrix agardhii is the most commonly found specie. Furthermore microcystin production was detected in isolates from this species. In a second stage, it was developed a method based on real-time PCR to detect and quantify Planktothrix agardhii - Chapter 4. The real-time PCR is a promising technique for cyanobacteria research and monitoring. The main advantage of real-time PCR over conventional PCR is the ability to quantify the target gene copy numbers on a sample. Thus, in addition to identifying Planktothrix strains, the real-time PCR also enables to quantify those strains, which constitutes an advantage over the procedures used in the routine monitoring of cyanobacteria. It should be noted that the determination of the cell FCUP Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural waters vii density is critical in the risk assessment of toxic cyanobacteria, as the guideline values for cyanotoxins are based on cyanobacterial concentrations as well as on toxin cell quota. Another important aspect in cyanobacteria monitoring is the use of preserved samples. Preservation is used to maintain the morphologic features of cyanobacterial cells to further be used in their identification/quantification and also to avoid sample degradation during transport. In this work, the applicability of the real-time technique in the amplification of DNA from preserved samples was evaluated, by using the method previously developed for cell quantification – Chapter 5. The results indicate that real-time PCR is a robust technique applicable to those types of samples but that the most common preservation methods (Lugol’s solution, formaldehyde, glutaraldehyde) reduce the DNA quantity and quality. Since DNA degrades fast in those samples, the applicability of real-time PCR on preserved samples was tested using other preservation procedure – Chapter 6. The preservation in methanol 100% at -20ºC allowed maintaining the integrity of the samples both for morphologic and molecular analysis up to two years after preservation. The last chapter of this thesis reports the result of two years of monitoring of a reservoir having a persistent bloom of toxic P. agardhii (Appendix A) - Chapter 7. In this reservoir, high cell densities did not always correspond to high amounts of toxin and vice versa. Using the real-time PCR it was demonstrated that both toxic and non-toxic strains are present within the reservoirs and that they can flourish at different times. It was also detected a specie from another genus that also contributes to the production of microcystins. During the monitoring it was observed a chytrid parasite that infected the filaments of Planktothrix (Appendix A). The density of this parasite was also quantified by real-time PCR and the results showed that its development coincides with the increase of toxic Planktotrhix strains and of microcystin levels in the reservoir.
- Acid mine drainage from the Panasqueira mine and its influence on Zêzere River (Central Portugal)Publication . Candeias, C.; Freire Ávila, P.; Silva, E. F; Ferreira, A.; Salgueiro, A. R.; Teixeira, João PauloThe Panasqueira hydrothermal mineralization, located in central Portugal, is the biggest Sn–W deposit of the Western Europe. The main evidences of the mining exploitation and ore treatment operations are testified with huge tailings, mainly, in the Rio and Barroca Grande areas. The mining and benefi- ciation processes, at the site, produces metal rich mine wastes. Oxidation of sulfides tailings and flow from open impoundments are responsible for the mobilization and migration of metals from the mine wastes into the environment. Acid mine drainage (AMD) discharged from Rio tailing has a pH around 3 and high metal concentrations. In Zêzere river, Fe and As are the most rapidly depleted downstream from AMD once As adsorbs, coprecipitate and form compounds with iron oxyhydroxides. The Zêzere river waters are oversaturated with respect to kaolinite and goethite and ferrihydrite can precipitate on stream with a near-neutral pH. At sites having low pH the dissolved Fe species in the water, mainly, occur as sulfate complexes due to a high SO4 concentration. Melanterite (Fe2+(SO4)7(H2O)) and minor amounts of rozenite (Fe2+(SO4)4(H2O)) and szomolnokite (Fe2+(SO4)(H2O)) were observed on Rio tailing basement.
- Activated carbons: efficient materials for the removal of multi- resistant bacteriaPublication . Silva, Sandra; Jordao, Luisa; Duarte, Aida; Carvalho, Ana PaulaThe rapid emergence of multi-resistant bacteria is occurring worldwide being hospital effluent a major source of these microorganisms. To remove these bacteria possible strategies are those involving activated carbon that due to their tunable properties can be suitable adsorbents for different types of microorganisms, for example Gram + or Gram – bacteria. The objective of this study was to test an activated carbon (AC) commercialized for water treatment purpose to reduce the content of bacteria commonly detected in hospital wastewater. The assays were made using the carbon in two different granulometries against multi-resistant strains: Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus. The adsorption tests were performed following a procedure adapted from Naka et al. [1]. In any case in parallel with the adsorption assays, the growth of the bacteria was also assessed.The results displayed at Figure 1 show that carbon AC allows a continue decrease of bacteria amount in the liquid media, despite the continuous increase of bacteria demonstrated by the blank experiment. The images displayed in Figure 2 (b) and (c) show the cells adherent to carbon, after 90 minutes of contact. These preliminary results allow us to conclude that activated carbon can be used to increase the removal efficiency of multi-resistant bacteria present in hospital wastewater. Acknowledgments The financial support of FCT to CQB (PEst-OE/QUI/UI0612/2013) is acknowledged. [1] K. Naka, S.T. Watarai, K. Inoue, Y. Kodama, K. Oguma, T.Yasuda, H.J. Kodama, 2001, Veter. Med. Science., 63, 281-285.
- Actividade antibacteriana de extractos de Planktothrix agardhii contra Staphylococcus aureusPublication . Menezes, Carina; Dias, ElsaA resistência de bactérias patogénicas à antibioterapia é um dos principais problemas de saúde pública, face à crescente ineficácia dos antibióticos no tratamento de doenças infeciosas. Assim, a pesquisa de novos antibióticos é um importante desafio da investigação em saúde. O potencial farmacológico de cianobactérias tem sido avaliado e descrito, mas, no entanto, ainda não há nenhum composto cianobacteriano que tenha sido aprovado pelas autoridades do medicamento como agente antibacteriano. Neste trabalho avaliámos a atividade de extratos de 40 estirpes de Planktothrix agardhii contra duas bactérias patogénicas: Klebsiella pneumonia e Staphylococus aureus. Para cada espécie bacteriana alvo usou-se uma estirpe de referência (ATCC) e uma estirpe clínica, isolada a partir de amostras de pacientes e estudadas no Laboratório de Resistência aos Antibióticos e Infeções Associadas aos Cuidados de Saúde. As estirpes de P. agardhii foram isoladas de albufeiras portuguesas e têm sido mantidas na coleção “Estela Sousa e Silva Algae Culture Collection” do Laboratório de Biologia e Ecotoxicologia. A biomassa de P. agardhii (200mg) foi extraída com metanol (70%, 10mL/100mg) overnight e sujeita a ciclos de sonicação. O metanol dos extratos foi evaporado e os extratos aquosos resultantes foram purificados em cartuchos C18. A atividade dos extratos contra as bactérias K. pneumonia e S.aureus foi avaliada através do método da Difusão em Disco (EUCAST), usando discos impregnados com 2, 4, 6, 8 e 10 mg de cada extrato. De acordo com os resultados preliminares, nenhum dos extratos apresenta atividade contra K. pneumonia, mas o extrato de P. agardhii LMECYA 256 apresenta um ligeiro halo de inibição relativamente às estirpes de S. aureus, de uma forma aparentemente dependente da dose de extrato aplicada. O S. aureus é uma bacteria Gram-positiva, frequentemente associada a uma vasta gama de patologias, desde simples infeções na pele, até infeções graves como pneumonia e meningite. Assim, revela-se do maior interesse explorar as propriedades antibacterianas de cianobactérias, designadamente do género Planktothrix, contribuindo, desta forma, para um dos desafios atuais da ciência: a identificação de novos antibióticos de origem natural.
- Acute and chronic toxicity assessment of haloacetic acids using Daphnia magnaPublication . Melo, Armindo; Ferreira, Cláudia; Ferreira, Isabel M.P.L.V.O.; Mansilha, CatarinaHaloacetic acids (HAAs) are undesirable disinfection by-products (DBPs), released into aquatic ecosystems from various anthropogenic and natural sources. The aim of this study was to examine the ecological risk of exposure to three HAAs commonly detected in water, such as monobromoacetic acid (MBA), monochloroacetic acid (MCA), and trichloroacetic acid (TCA), in in vivo acute and chronic toxicity tests using Daphnia magna as a model. Acute tests showed that MBA was the most toxic of these compounds followed by MCA and TCA as evidenced by immobilization. Aquatic organisms in natural conditions might be exposed simultaneously to numerous compounds; thus, binary mixtures of selected HAAs and a ternary mixture of these were tested. Concentration addition (CA) and independent action (IA) models were used for a predictive assessment of mixture toxicity. Data demonstrated that CA appeared to be the most reliable indicator for HAAs binary and ternary mixtures suggestive of an additive behavior. Median effective concentration (EC50) values from the mixed exposure tests were significantly lower than results obtained from single tests for all three HAAs where an increase of toxicity greater than 50%. Multigenerational chronic tests were also performed exposing daphnids to the ternary mixture of HAAs. A markedly decreased sexual maturity and number of offspring and broods per daphnid especially in the second generation were noted.