Successful isolation and cultivation of Cylindrospermopsis raciborskii strains isolated from finished drinking water samples

This work presents the successful establishment of Cylindrospermosis raciborskii cultures isolated from water samples collected at the exit point of Water Treatment Plant (WTP). An intense bloom dominated by filamentous cyanobacteria (Aphanizomenon spp., Planktothrix spp., Cylindrospermosis raciborskii, Anabaena spp.) occurred in the summer of 2015 in Roxo reservoir (south Portugal). Several cyanotoxins (microcystins, saxitoxins, cylindrospermopsin) were detected in raw and treated water, although at levels below the corresponding regulatory and/or guideline values. Nevertheless, this bloom caused intense unpleasant odour and taste in the water supplied to the populations and cyanobacterial cells (up to 1000 cells.mL-1) were detected in finished water samples collected at the exit point of WTP. Treated water samples collected at the WTP and at the city water deposit were inoculated in Z8 culture medium and cyanobacterial growth was followed by optical microscopy. After 30 days, cyanobacterial growth was observed showing resistance to the treatment processes and maintenance of reprodution capacity. Interestingly, morphometric and molecular analysis revealed the presence of C. raciborskii. Three isolates of this species were obtained and none were cylindrospermopsin- or microcystins-producers, as confirmed by Enzyme Linked Immunosorbent Assays (ELISA) and by amplification of genes (PS, PKS, mcyA-cd, mcyAB, mcyB) involved in those cyanotoxin synthesis. However, the ELISA for saxitoxins was positive for the 3 isolates and confirmation of this toxin production is in progress. To our knowledge, this is the first report on the establishment of successful cultures of C. raciborskii that survived to conventional water treatment processes.N/

Treatment of water for human consumption: a case of cyanobacterial survival

No verão de 2015 ocorreu um episódio de mau odor/sabor na água de abastecimento proveniente da albufeira do Roxo. Este episódio foi atribuído à presença de um bloom cianobacteriano que incluía espécies produtoras de compostos voláteis (geosmina, MIB). De facto, foi detetada a presença de cianobactérias na água tratada, à saída da ETA, ainda que em densidades relativamente baixas. No entanto, desconhecia-se se estes organismos mantinham a viabilidade celular e a capacidade de se desenvolver na rede de distribuição, a jusante da ETA e, desta forma, contaminar a água para consumo humano. Assim, amostras de água colhidas à saída da ETA e no Reservatório de abastecimento público foram inoculadas em meio de cultura e o crescimento celular foi seguido por microscopia ótica. Após 30 dias, verificou-se o crescimento algal, o que demostra que algumas espécies resistiram ao tratamento na ETA e mantiveram a capacidade de se reproduzir. Curiosamente, uma dessas espécies foi o Cylindrospermopsis raciborskii, considerada até há pouco tempo uma espécie tropical. Irá proceder-se à caracterização molecular e toxigénica desta espécie. Atualmente está a decorrer um protocolo de colaboração entre o Instituto Nacional de Saúde Doutor Ricardo Jorge e a AgdA, com o objetivo de se estudar a distribuição de cianobactérias, cianotoxinas e compostos voláteis na albufeira do Roxo.In the summer of 2015 an unpleasant odour/taste occurred in the water for human consumption supplied from Roxo Reservoir. This episode was attributed to the presence of a cyanobacterial bloom composed by species producing volatile compounds (geosmin, MIB). In fact, cyanobacteria were detected in treated water, at the exit of Water Treatment Plant (WTP), although at relatively low densities. However, it was unknown if these organisms maintained their viability and the growth capacity in the distribution network and, as such, to contaminate the drinking water. Thus, treated water samples collected at the WTP and at the water deposit were inoculated in culture medium and cell growth was followed by optical microscopy. Algal growth was observed 30 days after culturing, which shows that some species resisted the treatment in the WTP and maintained the capacity to reproduce. Interestingly, one of those species was Cylindrospermopsis raciborskii, considered until recently a tropical species. Molecular and toxigenic characterization of this species will be carried out. A collaboration protocol between INSA I.P. and AgdA, is currently being carried out with the aim of studying the distribution of cyanobacteria, cyanotoxins and volatile compounds in the Roxo reservoir.Elsa Dias, bolsa da Fundação para a Ciência e Tecnologia (SFRH/BPD/77981/2011)info:eu-repo/semantics/acceptedVersio

The Kidney Vero-E6 Cell Line: A Suitable Model to Study the Toxicity of Microcystins

Microcystins (MCs) are toxins produced by cyanobacteria from water environments that can induce acute and chronic effects on humans and animals, after ingestion/contact with contaminated water. This group of cyclic heptapeptides comprises approximately 80 variants, being microcystin-LR (MCLR) the most frequent and toxic variant. The studies of MCs effects on cell lines often lead to contradictory results, given the fact that distinct MC toxicity endpoints (mainly cytotoxicity and genotoxicity) have been studied in diverse cell lines (and cell clones) under distinct exposure conditions (different doses-ranges, time of exposure, MCs variants, etc). In our work with Vero-E6 cells we tested MCLR (both pure toxin and from cyanobacterial extracts of M. aeruginosa) within a wide range of concentrations (1 nM- 200 μM), using several endpoints and methodologies (cytotoxicity, morphology, genotoxicity, protein expression). In this chapter we will summarize our results and discuss the utility of Vero-E6 cell line to evaluate the toxicological properties of MCLR

Comparative study of the cytotoxic effects of microcystin-LR in mammalian cell lines: Vero, HepG2, Caco2 and MDCK

Dissertação de Mestrado em Biologia Humana e Ambiente apresentada à Faculdade de Ciências da Universidade de Lisboa, 2009Tese orientada por: Professora Doutora Ana Amorim (Professora Auxiliar da Faculdade de Ciências da Universidade de Lisboa); Doutora Elsa Alverca (Investigadora do Laboratório de Biologia e Ecotoxicologia, Departamento de Saúde Ambiental,Instituto Nacional de Saúde Doutor Ricardo Jorge)Microcystin-LR (MCLR) is a natural occurring freshwater cyanotoxin, recognized as one of the most toxic microcystin variants. It is thought to be responsible for cases of livestock and human intoxication due to consumption of toxic cyanobacteria-contaminated water. Although considered a hepatotoxin, MCLR also targets other organs such as the kidneys and intestines. In spite the cellular mechanisms associated with the toxicity of MCLR are still unclear, a previous work in a monkey kidney cell line suggested that the endoplasmic reticulum was an early target of MCLR toxicity and that autophagy was triggered as a cell defense mechanism at subcytotoxic concentrations of MCLR. In the present work, cytotoxic, morphological and ultrastructural effects of MCLR were compared in HepG2 (human liver), Vero (monkey kidney), MDCK (dog kidney) and Caco2 (human intestine) cell lines. MCLR induced a concentration-dependent decrease in cell viability by the NR assay in all cell lines, with HepG2 and Vero showing the lowest cytotoxic thresholds of 25 and 50 μM MCLR, respectively. In these cells, MCLR exposure induced lysosomal damages previously to mitochondrial disruption, reinforcing the role of lysosomes in MCLR-induced toxicity. Immunolabelling and ultrastructural visualization of autophagosomes, showed that autophagy was a response transversal to both cell lines, triggered at subcytotoxic MCLR concentrations, confirming its importance as a defense mechanism to early damages inflicted by the toxin. The analysis of GRP94, an ER stress protein, did not undoubtedly demonstrate that MCLR targets the ER. However, together with the ultrastructural data, suggested that in both HepG2 and Vero cells, the ER has a role in autophagy induction. Additionally, in HepG2 cells, GRP94 down-regulation with increasing MCLR concentrations supported the ER role in the triggering of apoptosis. At high toxin concentrations, ultrastructural alterations consistent with apoptosis were observed for all four cell lines, proving that this is a general MCLR-induced mechanism

Risk levels of toxic cyanobacteria in portuguese recreational freshwaters

Portuguese surface freshwaters are widely used as a source of drinking water as well as bathing water. Cyanobacterial blooming in these water resources are common and are often associated with cyanotoxin production. The Portuguese legislation for drinking water (Decreto-Lei nº306/2007) establishes the regulatory level of 1 μg/L for total microcystins in treated water. This parameter is determined when eutrophication of water is suspected and when the number of potentially toxic cyanobacteria exceeds 2000 cells/mL. Conversely, the Portuguese legislation concerning the quality of bathing water (Decreto-Lei nº 135/2009), that was transposed form the European Directive nº2006/7/CE, do not include any guideline for cyanobacterial cells nor microcystins concentrations. It only recommends that when the bathing water profile indicates a potential for cyanobacterial proliferation, appropriate monitoring shall be carried out to enable timely identification of health risks. When cyanobacterial proliferation is detected visually, it is the responsibility of the local health delegate to evaluate the associated risk. If any risk has been identified or presumed, health and environmental authorities should implement the adequate management measures to prevent exposure, including information to the public. According to national specificities, some European countries complemented the European Directive nº2006/7/CE and implemented their own guidance or regulations, based on cyanobacterial cell numbers, biovolumes, pigments and/or cyanotoxin concentrations (Ibelings et al., 2015; Chorus, 2012). Prior to establishing regulatory or guideline values, it will be fundamental to characterize Portuguese inland bathing waters concerning the frequency, density, specie composition and toxicity of cyanobacterial blooms. These data are available but not systematized at a national scale. In this work we present the results of the monitoring of cyanobacteria and microcystins in 8 freshwater reservoirs located in the centre of Portugal largely used for bathing and recreational activities. These results will contribute to identify the cyanobacterial blooms profile and to assess the risk level of toxic cyanobacteria in Portuguese recreational freshwaters.N/

Isolation and characterization of cylindrospermopsis raciborskii strains from finished drinking water

In the summer of 2015, an intense cyanobacterial bloom producing geosmin/2- methylisoborneol (MIB) occurred in the Roxo freshwater reservoir in Alentejo, Portugal. The drinking water supplied from the Roxo water treatment plant (WTP) exhibited an unpleasant odor/taste and a significant cyanobacteria density was detected in the finished water at the exit of the WTP. Cyanobacteria were not evaluated downstream of the WTP, namely, at the city reservoir. The aim of this work was to isolate and characterize viable cyanobacteria present in finished water (exit of the WTP and city reservoir) that withstand conventional water treatment. Treated water samples collected at both sites were inoculated in Z8 culture medium to provide the conditions for putative cyanobacterial growth. After 30 days, filamentous cyanobacteria were observed in cultures inoculated with samples from the exit point of the WTP. Viable trichomes were isolated and identified as Cylindrospermopsis raciborskii by morphometric and molecular analysis. None of the isolates were cylindrospermopsin/microcystin producers, as confirmed by ELISA and amplification of corresponding genes (PS/PKS and mcyA-cd/mcyAB/mcyB). ELISA results were positive for saxitoxin, but saxitoxin and derivatives were not detected by liquid chromatography with fluorescence detection (LC-FLD), nor were their related genes (sxtA/sxtA4/sxtB/sxtM/sxtPer/sxtI). To our knowledge, this is the first report on the establishment of cultures of C. raciborskii that resisted water treatment processes.Portuguese Foundation for Science and Technology (FCT) for the grant SFRH/BPD/77981/2011 attributed to E.D. This work was partially supported by FCT/MCTES through national funds for the research project “Exploring the Aquatic Resistome” (PTDC/BIA-BMA/31451/2017) and the project UIDB/00211/2020.info:eu-repo/semantics/publishedVersio

Evaluation of phylogenetic markers suitable for Planktothrix spp. discrimination

In Portugal, potentially toxic cyanobacteria from Planktothrix genus have become frequently observed in freshwater reservoirs. Identification of Planktothrix species through optical microscopy is complicated due to limited morphological differences among them. The aim of this work was to determine the most suitable phylogenetic markers that could be used for the molecular identification of Planktothrix species. In order to do so, several genes of interest were selected: rpoB, rpoC1, cpcA, cpcB, rbcX, 16S rRNA genes and 16S rRNA–tRNAIle–tRNAAla-23S rRNA internal transcribed spacer (ITS), and their sequences retrieved from public databases. Phylogenetic analysis showed that 16S rRNA, cpcA, rbcX genes and ITS region trees do not allow a clear discrimination of Planktothrix species, however cpcB and rpoB seemed putative suitable phylogenetic markers for Planktothrix species identification. The applicability of these markers was then evaluated in 20 Planktothrix isolates, isolated over the years from several Portuguese freshwater reservoirs and maintained in the Estela Sousa e Silva Algae Culture Collection (ESSACC). The selected genes, cpcB and rpoB, were amplified by PCR and sequenced and the resulting trees compared with the phylogenetic clustering obtained with our previously characterized rpoC1 phylogenies. The phylogenetic analyses, based on the three gene regions, revealed that Planktothrix isolates analyzed in this study could be phylogenetically resolved into their corresponding species. This work contributes for the discussion of the appropriate genes that can be used in phylogenetic identification of Planktothrix species.The authors would like to thank the Ph.D research grant SFRH/BD65706/2009 to Catarina Churro and the financial support through project PPCDT/AMB/60675/2006 both given by Fundação para a Ciência e Tecnologia (Portugal)

Consumidores de produtos orgânicos na economia circular

Purpose: To identify drivers and barriers perceived by consumers of organic products regarding their actions supporting circularity. Theoretical framework: Circular Economy (CE) is seen as solution for food crisis and environmental pollution. It also contributes to growing awareness of social responsibility and sustainability, which increases the interest in organic products. Method/design/approach: Qualitatively, twelve consumers of organic products were interviewed through indepth online interviews. Results and conclusion: Consumers identified several drivers that encourage them to be part of CE, such as less packaging at farmer’s markets, the opportunity to compost at home, and government policies that support waste sorting for recycling. However, the failure of suppliers to provide initiatives to reuse packaging, bags and to return glass containers, the restrictions on home composting, lack of room to separate and dispose of recyclable waste efficiently, not used to reusing vegetable waste, and not used to returning packaging to suppliers, were identified as barriers to CE. Research implications: This study contributes to the development of CE, promotion of sustainability and establishment of CE as an element of organic production. Originality/value: Studies address CE with focus on the industry. Contemplate the importance of the consumer for the development of circularity, this study advances with a look at the consumer, foment to the comprehensive, until then not investigated, of the perception of consumers of organic products regarding their actions supporting circularity.Objetivo: Identificar drivers e barreiras percebidos pelos consumidores de produtos orgânicos em relação às suas ações em prol da circularidade. Referencial teórico: A economia circular (EC) é vista como solução para crise alimentar e poluição ambiental. Também contribui para crescente conscientização sobre responsabilidade social e sustentabilidade, enquanto aumenta o interesse por produtos orgânicos. Método: De forma qualitativa, doze consumidores de produtos orgânicos foram entrevistados por meio de entrevistas em profundidade on-line. Resultados e conclusão: Os consumidores identificaram uma série de drivers que os incentivam a fazer parte da EC, como diminuição do uso de embalagens nas feiras, oportunidade de fazer compostagem em casa e políticas governamentais que apoiam a coleta seletiva de resíduos para reciclagem. No entanto, a falha dos fornecedores em oferecer iniciativas de reutilização de embalagens, sacolas e de devolução de recipientes de vidro, restrições à compostagem domiciliar, falta de espaço para separar e descartar resíduos recicláveis de forma eficiente, não ter como reutilizar resíduos vegetais, e a não devolução de embalagens aos fornecedores, foram identificados como barreiras à EC. Implicações da pesquisa: Este estudo contribui para o desenvolvimento da EC, promoção da sustentabilidade e, estabelecimento da EC como elemento da produção orgânica. Originalidade/valor: Estudos tratam da EC com um foco maior para a indústria. Considerando a significativa importância do consumidor para o desenvolvimento da circularidade, este estudo avança com olhar para o consumidor, contribuindo para o entendimento, até então não investigados, da persepção dos consumidores de produtos orgânicos em relação às suas ações em prol da circularidade

Removal of obturation material in endodontic retreatment: a literature review

Introduction: Removal of filling material from the root canal system is required when a previous endodontic treatment fails, what may result in the permanence of an unfavorable periapical condition. The intent is to completely remove the filling material inside of the root canal to achieve sufficient cleaning and shaping for successful retreatment. Objective: The aims of this article were to provide asystematic review of the different techniques of endodontic filling material associated or not with organic solvents and to analyze them critically in terms of advantages and disadvantages of each technique. Literature review: The descriptors used were “guttapercha”, “obturation,” and “retreatment” in the following databases: PubMed, MEDLINE, Latin American and Caribbean Center on Health Sciences Information (Bireme), Latin-American and CaribbeanHealth Sciences (Lilacs), Brazilian Dentistry Bibliography (BBO), and Scientific Electronic Library Online (SciELO). Publications of in vitro/ ex vivo and in vivo experiments without language restriction between the years 2010 and 2018 were selected. Conclusion: None of the techniques were capable of performing complete root canal cleaning, and the manual method was so effective as the automated method, although it requires longer working time. Furthermore, although this review confirmed that the solvent action did not allow a significantimprovement in the removal of the filling material, ultrasoundactivated irrigation proved to be an efficient adjunctive device as it could significantly reduce the volume of intracanal residuals.Introduction: Removal of filling material from the root canal system is required when a previous endodontic treatment fails, what may result in the permanence of an unfavorable periapical condition. The intent is to completely remove the filling material inside of the root canal to achieve sufficient cleaning and shaping for successful retreatment. Objective: The aims of this article were to provide asystematic review of the different techniques of endodontic filling material associated or not with organic solvents and to analyze them critically in terms of advantages and disadvantages of each technique. Literature review: The descriptors used were “guttapercha”, “obturation,” and “retreatment” in the following databases: PubMed, MEDLINE, Latin American and Caribbean Center on Health Sciences Information (Bireme), Latin-American and CaribbeanHealth Sciences (Lilacs), Brazilian Dentistry Bibliography (BBO), and Scientific Electronic Library Online (SciELO). Publications of in vitro/ ex vivo and in vivo experiments without language restriction between the years 2010 and 2018 were selected. Conclusion: None of the techniques were capable of performing complete root canal cleaning, and the manual method was so effective as the automated method, although it requires longer working time. Furthermore, although this review confirmed that the solvent action did not allow a significantimprovement in the removal of the filling material, ultrasoundactivated irrigation proved to be an efficient adjunctive device as it could significantly reduce the volume of intracanal residuals

Descrição de um surto de intoxicação por cianobactérias (algas verdes-azuis) em bovinos no Alentejo

Suspeita de hepatoxicidade provocada por cianotoxinas, num surto de morte súbita em 25 bovinos numa exploração com 54 vacas de carne em Almodôvar, Alentejo.N/