Contaminação de reservatórios de abastecimento público : avaliação de efeitos e depuração de cianotoxinas em Geophagus brasiliensis e utilização da fitoremediação para a remoção de contaminantes emergentes

Orientadora: Profa. Dra. Helena Cristina da Silva de AssisTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ecologia e Conservação. Defesa : Curitiba, 15/02/2019Inclui referênciasResumo: Os reservatórios do Iraí e Alagados, localizados no estado do Paraná, são utilizados principalmente para o abastecimento público de água. Devido às atividades humanas no entorno destes corpos hídricos, têm sido reportado frequentes florações de cianobactérias, além da presença de diferentes contaminantes emergentes. Visto esta problemática, os objetivos do estudo foram i) determinar as concentrações de cianotoxinas e fármacos nos reservatórios do Iraí e Alagados; ii) avaliar como as concentrações de contaminantes podem afetar a saúde de peixes por meio do uso de biomarcadores ambientais; iii) avaliar o potencial de depuração de cianotoxinas nos peixes por 90 dias; iv) estimar o risco de exposição para a saúde humana através da ingestão de peixes presentes nos reservatórios; v) e testar a eficiência da tecnologia de fitoremediação "Fígado Verde" na remoção de contaminantes. Coletas de água e peixes (Geophagus brasiliensis) foram realizadas nos reservatórios no período de 2015 a 2016. Nas amostras de água foram analisados o fitoplâncton, cianotoxinas e fármacos. Os peixes coletados foram divididos em dois grupos, "grupo imediato" e "grupo depuração". No "grupo imediato" os tecidos foram coletados no mesmo dia da coleta para análises químicas e de biomarcadores bioquímicos, osmoregulatórios e genotóxicos; e no "grupo depuração" os peixes permaneceram em aquários com água filtrada e desclorada. Durante os 90 dias os tecidos dos animais do "grupo depuração" também foram coletados para as análises químicas e de biomarcadores. Parte dos peixes coletados no reservatório Alagados e mantidos no laboratório, reproduziram "grupo reprodução" e a geração F1 foi utilizada para análises químicas de cianotoxinas. O experimento de fitoremediação foi realizado com três espécies de macrófitas aquáticas (Egeria densa, Ceratophyllum demersum, Myriophyllum aquaticum), as quais foram expostas às mesmas concentrações de cianotoxinas e fármacos encontradas no reservatório do Iraí. Os resultados do estudo mostraram que os reservatórios do Iraí e Alagados apresentam concentrações de microcistinas e saxitoxinas (PSTs), respectivamente, além de concentrações de diclofenaco e paracetamol. Os peixes coletados apresentaram efeitos tóxicos como estresse oxidativo, alterações na osmorregulação e dano em macromoléculas. Após o tempo de depuração foi observada a recuperação na saúde dos peixes, porém concentrações de cianotoxinas ainda, estavam presentes no tecido muscular. Além disso, as concentrações de PSTs no músculo dos peixes foram estimadas como risco de exposição humana, principalmente para crianças. Entretanto, concentrações de PSTs não foram quantificadas na geração F1. As macrófitas aquáticas utilizadas no experimento "Fígado Verde" foram eficientes na remoção dos contaminantes estudados, sendo boas ferramentas de fitoremediação. Devido à contaminação dos reservatórios de abastecimento público de água do atual estudo é de extrema necessidade a implementação de novas estratégias de monitoramento, gerenciamento e remoção de contaminantes.Abstract: Iraí and Alagados Reservoirs, located in Paraná state, are used mainly as water supply. Due to the several human activities around these water bodies, it has been reported with cyanobacterial blooms, in addition to the presence of different emerging contaminants. In view of this problem, the aims of this study were i) to determinate the concentration of cyanotoxins and pharmaceuticals in Iraí and Alagados Reservoirs; ii) to evaluate as the concentration of contaminants can affect the fish health by using environmental biomarkers; iii) to evaluate the potential of cyanotoxins depuration in fish for 90 days; iv) to estimate the potential risk of the consumption of fish from the reservoirs for human health; v) and to test the phytoremediation technology "Green Liver" to removal of contaminants. Water and fish (Geophagus brasiliensis) samples were carried out during 2015/2016. Water samples were used to phytoplankton and chemical analysis of cyanotoxins and pharmaceuticals. Fish were divided in two groups "immediate group" and "depuration group". On the same day of the sampling, the tissues were collected from "immediate group" in order to chemical analysis and biochemical, osmoregulatories and genotoxic biomarkers; and the "depuration group" were maintained in tanks during 90 days in filtered and dechlorinated water. During the 90 days, the tissues from "depuration group" were also collected to chemical and biomarkers analysis. A part of the fish from Alagados Reservoir was maintained in the laboratory, which reproduced "reproduction group" and the F1 generation was used to cyanotoxin chemical analysis. The phytoremediation experiment was carried out using three aquatic macrophytes species (Egeria densa, Ceratophyllum demersum, Myriophyllum aquaticum), which were exposed to the same cyanotoxins and pharmaceutical concentrations found in Iraí Reservoir. The results of this study showed that the Iraí and Alagados Reservoir present concentrations of microcystins and saxitoxins (PSTs) respectively, in addition diclofenac and paracetamol concentrations. The fish presented toxic effects such as oxidative stress, osmoregulation alterations and molecular damage. After the depuration time the fish health recovery was observed, however cyanotoxins concentrations were still present in the muscular tissue. Furthermore, the saxitoxins concentrations in fish muscle were estimated as potential risk to human health, mainly for children. However, the PSTs concentrations were not quantified in the F1 generation. The aquatic macrophytes used in the "Green Liver" experiment were efficient to the removal of the contaminants, and they are good tools of phytoremediation. Due to the contamination of the water supply reservoirs of the present study, the implementation of new strategies for the monitoring, management and removal of contaminants are required

Phytoremediation: green technology for the removal of mixed contaminants of a water supply reservoir

The Irai Reservoir, a water supply in Brazil, is constantly impacted by anthropogenic activities such as waste inputs from agriculture, hospitals and urbanization, resulting toxic cyanobacterial blooms causing economic, social and environmental problems. This study assessed the concentration of some common contaminants of the Irai Reservoir, namely paracetamol, diclofenac and microcystin-LR and tested whether a laboratory scale Green Liver System (R) would serve as a suitable technology to remove these contaminants. Further, the study investigated whether the pollutants caused adverse effects to the macrophytes using catalase as a biomarker for oxidative stress and investigated whether biotransformation (glutathione S-transferase) was a main route for detoxification. Egeria densa, Ceratophyllum demersum and Myriophyllum aquaticum were exposed to a mixture of the three contaminants for 14 days in a concentration range similar to those detected in the reservoir. The plants removed 93% of diclofenac and 100% of MC-LR after 14 days. Paracetamol could not be detected. Catalase and glutathione S-transferase enzyme activities remained unaltered after the 14-day exposure, indicating that the mixture did not cause oxidative stress. The study showed that the aquatic macrophytes used are suitable tools to apply in a Green Liver System (R) for the remediation of mixed pollutants.Peer reviewe

Genotoxicity detected during cyanobacteria bloom in a water supply reservoir

The aquatic ecosystems are susceptible to cyanobacterial blooms due to the eutrophication of water bodies caused by human activities. In this study, phytoplankton and cyanotoxins analysis, as well as cellular and genetic biomarkers of toxicity (Allium cepa test - higher plant test system), were evaluated in water samples of Alagados Reservoir during a cyanobacterial bloom in South Brazil. The water samples were collected during the wet season at two sites in the Reservoir. Paralytic shellfish toxins (PSTs) were detected in both samples (sites 1 and 2); however, the levels of PSTs were higher in site 1. Gonyautoxin 2 was the major cyanotoxin found in the Reservoir. Both samples were able to induce cytotoxic effects (reduced Mitotic Index) and damage the genetic material (i.e., increased frequencies of chromosome aberration and micronuclei) of meristematic cells of A. cepa. The cellular and genetic damages were higher in the sample site 1, wherein high levels of PSTs were verified. Thus, our findings suggested that cyanotoxins-contaminated waters may damage the genetic material of living organisms, and therefore this group of contaminants should be assessed for their potential genotoxicity

Biochemical and genotoxicity assessment of a polluted urban river using the native fish Astyanax altiparanae Garutti & Britski (Teleostei, Characidae)

Agriculture residues and domestic effluents are contaminants that reach aquatic ecosystems leading to toxic effects to environment and human health. These contaminants impacting mainly developing countries, due to precarious treatment of effluents and lack of legislation in the use of pesticides. Perequê River (South of Brazil) is impacted by pesticides (mainly glyphosate) and domestic effluents. The aims of this study were to analyze the concentration of glyphosate in water and sediments of Perequê River; and to evaluate the sublethal effects of the river contamination in Astyanax altiparanae using biomarkers. A sampling of water, sediment and fish were carried out in Perequê River (November/2016). The samples were collected in two sites, site 1 (S1) that is located on agriculture area and water catchment point; and site 2 (S2) located on the area with a domestic effluents influence. Water and sediment samples were used to glyphosate quantification. Fish were collected and tissues (brain, muscle, liver) were used to biochemical and genetic biomarkers. Glyphosate were quantified in S1, which is impacted by agriculture. Alterations in the liver biomarkers were not observed between the sites. However, the decrease in the AChE activity was observed in the S2, showing that the mixture of contaminants (urban and agriculture contaminants) can lead to anticholinesterasic effects. The sublethal effects observed can be a threat of environmental and human health

Monitoramento da qualidade da água do reservatório alagados, Sul do Brasil, com ênfase em cianotoxinas

Orientadora : Profª Drª Helena Cristina da Silva de AssisDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ecologia e Conservação. Defesa: Curitiba, 26/02/2015Inclui bibliografiaResumo: A eutrofizacao, causada pelo enriquecimento de nutrientes, resulta em intensas floracoes de microalgas e cianobacterias. O reservatorio Alagados em Ponta-grossa, PR e utilizado para geracao de energia e para o abastecimento publico de agua, sendo que floracoes persistentes de cianobacterias potencialmente toxicas nesta regiao vem causando problemas ambientais, sociais e economicos. O objetivo deste trabalho foi analisar a qualidade da agua do reservatorio Alagados, Parana, Brasil, atraves da analise de cianotoxinas e de biomarcadores bioquimicos e geneticos. Foram realizadas quatro coletas para amostragem de agua e duas coletas de Geophagus brasiliensis, no periodo de setembro de 2013 a maio de 2014. As amostras de agua foram analisadas quantitativamente e qualitativamente quanto as cianobacterias e cianotoxinas PSTs (Paralytic Shellfish Toxins). Os peixes foram divididos em dois grupos. O primeiro grupo foi eutanasiado no dia da coleta, mensurado o peso e comprimento total, colhido o sangue e coletado o cerebro, o musculo e o figado. O segundo grupo foi submetido ao experimento de depuracao por 40 dias. Apos os 40 dias, foram realizados os mesmos procedimentos que os do primeiro grupo. O sangue foi destinado as analises de biomarcadores geneticos, o musculo, cerebro e o figado para as analises de biomarcadores bioquimicos. Uma amostra do musculo tambem foi retirada para analises quimicas de cianotoxinas. A cianobacteria dominante na agua do reservatorio foi a Cylindrospermopisis raciborskii, apresentando densidade sempre acima do limite estabelecido de 20,000 cels/mL. As concentracoes de PSTs na agua tambem estiveram acima do estabelecido pela legislacao (3ƒÝg/L). No musculo dos peixes houve acumulo destas toxinas, e mesmo apos 40 dias em agua limpa nao foi observado a completa depuracao das toxinas. Os biomarcadores bioquimicos e geneticos apresentaram uma diferenca significativa entre os peixes eutanasiados no dia de coleta em relacao os peixes da depuracao. Essa diferenca sugere a recuperacao do sistema antioxidante assim como a reducao do dano celular apos 40 dias em agua limpa. O fator de condicao e o indice hepatossomatico foram menores nos peixes da segunda coleta, na qual tambem a concentracao de cianotoxinas foi maior. Os resultados sugerem que o reservatorio Alagados apresenta uma persistente contaminacao de cianotoxinas. Alem disso a mistura de contaminantes que pode estar presente neste corpo hidrico pode explicar as respostas sazonais nos peixes nos diferentes pontos amostrados. Palavras-chave: Cylindrospermopsis raciborskii; PSTs; Geophagus brasiliensis; Biomarcadores.Abstract: Eutrophication, caused by high nutrients loads increases microalgae and cyanobacteria blooms. The Alagados reservoir in Ponta Grossa, PR is an important source of water supply and power generation. Blooms of toxic cyanobacteria in this area has caused environmental, social and economic problems. This study aim to monitor the water quality of the Alagados reservoir, Parana, Brazil, through cyanotoxin analysis and biochemical and genetic biomarkers. For this purpose, four water samples and two samples of Geophagus brasiliensis were collected during September 2013 until May 2014. The water samples was analyzed quantitatively and qualitatively in respect to the cyanobacteria and cyanotoxins PSTs (Paralytic Shellfish Toxins). The fish were divided in two main groups. The first one was euthanized after been sampled, then their weight and length were measured and the blood, brain, muscle and liver were collected. The second group was submitted to depuration experiment for 40 days. After that, the same procedures of the first group were carried out. Blood samples were used for the genetic biomarkers; muscle, brain and liver samples for biochemical biomarkers analysis. A muscle sample was also collected for chemical analysis of cyanotoxins. The dominant cyanobacteria in the water of the reservoirs was Cylindrospermopsis raciborskii, with a density above the recommended limit of 20,000 cels/ml. The PSTs concentrations in water also exceed the limits of the established by legislation (3ƒÊg/L). In the muscle samples occurred the accumulation of these toxins and even after 40 days in clean water was not observed a complete elimination. The biochemical and genetic biomarkers showed a significant difference between the fish collected and euthanized and the depuration group, this difference suggests a recovery of the antioxidant system and a reduction of cellular damage after 40 days in clean water. The condition factor and the hepatosomatic index were lower in the fish of the second sampling, which the concentration of cianotoixinas was higher. The results suggested that Alagados reservoir has a persistent contamination of cyanotoxins. Moreover the mixture of contaminants which may be present in the water body can explain the sazonally in fish in the sampled points. Key-words: Cylindrospermopsis raciborskii; PSTs; Geophagus brasiliensis; Biomarkers

Toxicological Effects of Thimerosal and Aluminum in the Liver, Kidney, and Brain of Zebrafish (<i>Danio rerio</i>)

Vaccination programs in the first years of a child’s life are effective and extremely important strategies for the successful eradication of diseases. However, as no intervention is without risks, the metal-based components of some vaccines, such as thimerosal (TMS), a preservative composed of ethylmercury, and aluminum (Al), have begun to generate distrust on the part of the population. Therefore, this study evaluated the effects of exposure to thimerosal and aluminum hydroxide (alone or in mixture) on Danio rerio (zebrafish) specimens. The fish were exposed to thimerosal and/or aluminum hydroxide intraperitoneally. The liver, kidney, and brain were removed for a biochemical biomarker analysis, histopathological analysis, and metal quantification. As a result, we observed changes in the activity of the analyzed enzymes (SOD, GST, GPx) in the kidney and brain of the zebrafish, a reduction in GSH levels in all analyzed tissues, and a reduction in MT levels in the kidney and liver as well as in the brain. Changes in AChE enzyme activity were observed. The biochemical results corroborate the changes observed in the lesion index and histomorphology sections. We emphasize the importance of joint research on these compounds to increase the population’s safety against their possible toxic effects

Prognostic Factors for Cancer-Specific Survival and Disease-Free Interval of Dogs with Mammary Carcinomas

Canine mammary tumors (CMTs) are the most diagnosed neoplasms in dogs; however, there are few studies analyzing the influence of epidemiological, clinicopathological, and histopathological data on cancer-specific survival (CSS), disease-free interval (DFI), and overall survival (OS) in a large cohort. To contribute to the understanding of the biological behavior of this neoplasm, 385 cases were analyzed, 89% malignant, 4% benign, and 7% non-neoplastic lesions. Among the dogs diagnosed with malignant neoplasms, 86% had early clinical stages (I–III), while 14% had regional or distant metastasis at the time of diagnosis. Carcinoma in a mixed tumor was the most frequent histological type with 44% of the cases and had the best prognosis. Analyzed factors such as the presence of pseudocyesis, previous history of the disease, advanced clinical stage (IV-V), and presence of ulceration obtained significant results for CSS, DFI, and OS through univariate analysis and had a negative impact on the survival of the patients. Multivariate analysis showed that histological grading and age proved to be the best independent parameters for the prognostic evaluation of CSS and DFI in this study. These factors were also significant in the overall survival analysis. Therefore, these parameters should be considered valuable risk and prognostic factors for CMTs