Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas

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(1)RENORBIO Programa de Pós-Graduação em Biotecnologia. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Marcos Vinícius Teles Gomes. São Cristóvão – Sergipe 2013.

(2) RENORBIO Programa de Pós-Graduação em Biotecnologia Universidade Federal de Sergipe. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. MARCOS VINÍCIUS TELES GOMES Tese apresentada como requisito para obtenção do título de Doutor em Biotecnologia com ênfase na área de Biotecnologia Industrial. ORIENTADOR: Prof. Dr. Roberto Rodrigues de Souza – Departamento de Engenharia Química, UFS. Área de Industrial. concentração:. São Cristóvão – Sergipe 2013. Biotecnologia.

(3) FICHA CATALOGRÁFICA ELABORADA PELA BIBLIOTECA CENTRAL UNIVERSIDADE FEDERAL DE SERGIPE. G633f. Gomes, Marcos Vinícius Teles Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas / Marcos Vinícius Teles Gomes ; orientador Roberto Rodrigues de Souza. – São Cristóvão, 2013. 96 f. : il. Tese (doutorado em Biotecnologia) – Rede Nordeste de Biotecnologia – RENORBIO, Universidade Federal de Sergipe, 2013.. 1. Biotecnologia. 2. Fitorremediação. 3. Typha domingensis. 4. Zonas úmidas construídas. 5. Águas residuais – Purificação. 6. Mercúrio. I. Souza, Roberto Rodrigues de, orient. II. Título. CDU 602.4:628.316.12:546.49. iii.

(4) iv.

(5) Dedico à minha família e amigos. v.

(6) AGRADECIMENTOS. Aos meus filhos Vinícius, Victor e Victória, e à minha esposa Cleã, pelo amor incondicional demonstrado em todos os momentos, mesmo quando estive ausente devido ao trabalho. Estou voltando! À minha mãe Terezinha (in memorian) por ter me ensinado a nunca desistir. À minha irmã amiga Zarebe por sempre acreditar em mim. À minha segunda mãe Dora pelo incentivo e confiança. Ao meu orientador Prof. Dr. Roberto Rodrigues de Souza pelo apoio acadêmico, companheirismo e incentivo durante todo o desenvolvimento desta pesquisa. À CODEVASF, pelo apoio financeiro e técnico para realização do doutorado. A um grande amigo, que certa vez me disse: “Reconhecimento e respeito não se exige, se conquista. E muitas vezes temos que conquistá-los externamente, para um dia sermos internamente percebidos.” Aos meus colegas da CODEVASF que direta ou indiretamente contribuíram para realização deste trabalho e desta importante etapa da minha vida. Muito obrigado!!!. vi.

(7) ÍNDICE. LISTA DE ABREVIATURAS ................................................................................... ix RESUMO .................................................................................................................. x ABSTRACT .............................................................................................................. xi RESUMEN ............................................................................................................... xii 1. INTRODUÇÃO ...................................................................................................... 1 2. OBJETIVOS ........................................................................................................... 4 2. 1. Geral ................................................................................................................... 4 2. 2. Específico ........................................................................................................... 4 3. CAPÍTULO 1 – Phytoremediation of mercury water contaminated using Typha domingensis in constructed wetland ........................................................................... 5 4. CAPÍTULO 2 - Phytoremediation of the pisciculture wastewater, using Typha domingensis in constructed wetlands system ............................................................. 28 5. CAPÍTULO 3 – Relação entre o estado trófico e a forma de exploração para piscicultura de lagoas costeiras do pantanal de Pacatuba, Sergipe, Brasil ................... 50 6. CAPÍTULO 4 – Depósito de pedido de patente: PI1103405-0 ............................... 59 7. CONCLUSÕES .................................................................................................... 67 8. REFERÊNCIAS.................................................................................................... 69 ANEXO A – Atividades desenvolvidas no doutorado ............................................... 72 ANEXO B – Barragem de Três Marias (capítulos 1 e 2) ........................................... 73 ANEXO C - Centro Integrado de Recursos Pesqueiros e Aquicultura de Três Marias – 1ª/CIT (capítulos 1 e 2) ............................................................................................. 74 ANEXO D – Viveiro de policultivo da 1ª/CIT (capítulo 2) ....................................... 75 ANEXO E – Sistema de zonas úmidas construídas (capítulos 1 e 2) .......................... 76 vii.

(8) ANEXO F – Equipamento DMA-80 utilizado para análise direta de mercúrio (capítulo 1)............................................................................................................... 77 ANEXO G – Sonda multiparamétrica e espectrofotômetro UV-visível (capítulo 2) ... 78 ANEXO H – Lagoas (Cabaceiras, da Passagem e da Frente) do pantanal de Pacatuba SE (capítulo 3) ........................................................................................... 79 ANEXO I – Índice de estado Trófico (IETMédio) das lagoas Cabaceiras, da Passagem e da Frente (capítulo 3) ................................................................................................ 80 ANEXO J – Utilização da Typha domingensis por artesãos (capítulo 3) .................... 81 ANEXO K – Artesanato produzido pelas comunidades do Tigre e Junça (capítulo 3) ...... 82 ANEXO L – Sistema de bombeamento e lagoa de estabilização da ETE-CW da 1ª/CIT ... 83. ANEXO M – Sistema de zona úmida construída e descarte do efluente tratado da ETE-CW da 1ª/CIT ................................................................................................... 84. viii.

(9) LISTA DE ABREVIATURAS. ABNT - Associação Brasileira de Normas Técnicas CCRMP - Canadian Certified Reference Materials Project CIT - Centro Integrado de Recursos Pesqueiros e Aquicultura de Três Marias CODEVASF - Companhia de Desenvolvimento dos Vales do São Francisco e do Parnaíba CONAMA - Conselho Nacional do Meio Ambiente CW - Constructed Wetlands DBO - Demanda Bioquímica de Oxigênio DBO - Demanda bioquímica de oxigênio DMA - Direct Mercury Analyser DO - Dissolved Oxygen EC - Electric Conductivity ETE – Estação de Tratamento de Efluente IET - Índice de Estado Trófico INPI - Instituto Nacional de Propriedade Intelectual MDL - Method Detection Limit MQL - Method Quantification Limit NRC - National Research Council Canada RENORBIO - Rede Nordeste de Biotecnologia TC - Transference Coefficient TDH - Tempo de Detenção Hidráulica TDS - Total Dissolved Solids US EPA - United States Environmental Protection Agency UTM - Universal Transversa de Mercator. ix.

(10) RESUMO A presença de mercúrio em ambientes aquáticos tem sido motivo de preocupação por parte da comunidade científica e órgãos ligados à saúde pública de todo o mundo, devido à sua persistência e toxicidade. Atualmente, a demanda mundial por proteína animal tem intensificado a produtividade aquícola, aumentando a geração de águas residuárias com alto teor de compostos nitrogenados e fosforados. A fitorremediação consiste em um grupo de tecnologias baseadas na utilização de plantas de ocorrência natural ou geneticamente modificadas para reduzir, remover, degradar ou imobilizar toxinas, como alternativa aos métodos convencionais de tratamento de efluentes, devido a sua sustentabilidade, baixo custo de manutenção e energia. O presente estudo fornece informações de um experimento realizado em escala piloto, projetado para avaliar o potencial da macrófita aquática Typha domingensis em sistema de zonas úmidas construídas com fluxo subsuperficial, para a fitorremediação de águas residuárias de piscicultura ou contaminadas com mercúrio. A constante de velocidade do sistema foi 7 vezes maior que a linha de controle, demonstrando um maior desempenho e conseguindo reduzir 99.6 ± 0.4% do mercúrio presente na água contaminada. Quando comparadas a outras espécies, os resultados mostraram que a Typha domingensis demonstrou uma maior acumulação de mercúrio (273.3515 ± 0.7234 mg kg-1), quando o coeficiente de transferência foi de 7750.9864 ± 569.5468 L kg-1. Após 120 h de tempo de exposição, o tratamento com 50 brotos por m2 apresentou uma eficiência na remoção do nitrogênio total de 217 % superior à linha de controle (isento de macrófita), mostrando ser a Typha domingensis essencial à fitorremediação do nitrogênio. Para o fósforo total, a eficiência de remoção foi 26% superior a linha de controle, possivelmente por estar presente no material particulado, e este, ser retido predominantemente por filtração e sedimentação. A eficiência de remoção do nitrogênio total e fósforo total foram de aproximadamente 90%, similar ou superior aos obtidos em outros estudos. Os resultados demonstraram o grande potencial da macrófita aquática Typha domingensis, em sistema de zonas úmidas construídas com fluxo subsuperficial, para a fitorremediação de água residuária de piscicultura ou contaminadas com mercúrio. Palavras-chave: bioprocessos, fitorremediação, zonas úmidas construídas, Typha domingensis, tratamento de águas residuais, mercúrio.. x.

(11) ABSTRACT The presence of mercury in aquatic environments has been cause for concern on the part of the scientific community and public health agencies around the world, due to their persistence and toxicity. Currently, the global demand for animal protein has enhanced aquaculture productivity, increasing the generation of wastewater with high content of nitrogen and phosphorous compounds. The phytoremediation consists of a group of technologies based on naturally occurring plants or use genetically modified to reduce, remove, degrade or immobilize toxins, as an alternative to conventional methods of wastewater treatment, due to its sustainability, low maintenance cost and energy. The present study provides information of an experiment conducted on a pilot scale, designed to evaluate the potential of macrophyte Typha domingensis in subsurface flow constructed wetlands for phytoremediation of fish-farming, wastewater or contaminated with mercury. The rate constant of the system was 7 times greater than the control line, demonstrating a higher performance and reduce 99.6 ± 0.4% of mercury present in contaminated water. When compared to other species, the results showed that the Typha domingensis showed greater accumulation of mercury (273.3515 ± 0.7234 mg kg-1), when the transfer coefficient was 7750.9864 ± 569.5468 L kg -1. After 120 h of exposure time, treatment with 50 shoots/m2 showed a total nitrogen removal efficiency of 217% subscript (free from macrophyte), showing the Typha domingensis, essential for phytoremediation of nitrogen. For total phosphorus, the removal efficiency was 26% more than the line of control, possibly by being present in particulate matter, and this, be retained predominantly by filtration and sedimentation. The efficiency of removal of total nitrogen and total phosphorus were approximately 90%, similar or superior to those obtained in other studies. The results demonstrated the great potential of macrophyte Typha domingensis in subsurface flow constructed wetlands for phytoremediation of wastewater from fish farming, or contaminated with mercury. Keywords: bioprocess, phytoremediation, constructed wetlands, Typha domingensis, wastewater treatment, mercury.. xi.

(12) RESUMEN La presencia de mercurio en ambientes acuáticos ha sido motivo de preocupación por parte de los organismos comunitarios y de salud pública científicos del mundo, debido a su persistencia y toxicidad. Actualmente, la demanda mundial de proteína animal ha mejorado la productividad de la acuicultura, aumentar la generación de aguas residuales con alto contenido de nitrógeno y compuestos de fósforo. La fitorremediación consiste en un conjunto de tecnologías basadas en plantas naturalmente o uso modificados genéticamente para reducir, eliminar, degradar o inmovilizar las toxinas, como alternativa a los métodos convencionales de tratamiento de aguas residuales, debido a su sostenibilidad, bajo costo de mantenimiento y energía. El presente estudio proporciona información de un experimento llevado a cabo a escala piloto, diseñado para evaluar el potencial de macrófitos Typha domingensis en flujo subsuperficial humedales de fitorremediación de piscicultura, aguas residuales construidos o contaminados con mercurio. La constante de velocidad del sistema fue 7 veces mayor que la línea de control, demostrando un rendimiento superior y reducir 99,6 ± 0.4% de mercurio presente en agua contaminada. En comparación con otras especies, los resultados mostraron que la Typha domingensis demostró una mayor acumulación de mercurio (273.3515 ± 0.7234 mg kg -1), cuando el coeficiente de transferencia fue 7750.9864 ± 569.5468 L kg-1. Después de 120 horas de tiempo de exposición, el tratamiento con 50 brotes/m2 mostró una eficiencia de remoción de nitrógeno total de subíndice 217% (libre de macrófitos), mostrando la Typha domingensis, esencial para la fitorremediación de nitrógeno. Fósforo total, la eficiencia de remoción fue 26% más de la línea de control, posiblemente por estar presente en partículas y este, conservarse predominante por filtración y sedimentación. La eficiencia de eliminación de nitrógeno total y fósforo total fueron aproximadamente 90%, similar o superior a los obtenidos en otros estudios. Los resultados demostraron el gran potencial de macrófitos Typha domingensis en flujo subsuperficial humedales de fitorremediación de aguas residuales de piscicultura construidos, o estén contaminados con mercurio. Palabras clave: bioprocesos, fitorremediación, humedales construidos, Typha domingensis, tratamiento de aguas residuales, mercurio.. xii.

(13) Programa de Pós-Graduação em Biotecnologia. 1. INTRODUÇÃO A presença de mercúrio em ambientes aquáticos tem sido motivo de preocupação por parte da comunidade científica e órgãos ligados à saúde pública de todo o mundo, devido à sua persistência e toxicidade. Os impactos causados pelo despejo de efluentes contaminados com metais pesados trazem consequências danosas à saúde humana e ao meio ambiente, demandando estudos que viabilizem o seu tratamento. O mercúrio é um elemento presente naturalmente na crosta terrestre, na atmosfera e em ambientes aquáticos (Lacerda et al., 2007). O aporte antrópico pode ocorrer, entre outros, através da queima de combustíveis fósseis, produção eletrolítica de cloro-soda caústica, industrialização de pesticidas, produtos odontológicos e amalgamação durante a extração do ouro (Micaroni et al., 2000). Sendo um dos metais mais tóxicos, o mercúrio não tem função biológica conhecida (Cursino et al., 2003). No meio ambiente, pode ser transformado em formas mais tóxicas como metilmercúrio, acumulando-se na biota aquática e terrestre (Padovani, et al., 1995). Através da biomagnificação e percorrendo a cadeia alimentar, pode alcançar o ser humano, onde a concentração acumulada é proporcional ao nível trófico. A forma de maior distribuição do mercúrio é a elementar (Hg 0), presente na atmosfera na fase de vapor. Quando introduzido na forma inorgânica em um ecossistema aquático, pode transformar-se em uma forma orgânica, sendo convertido a metilmercúrio por bactérias metanogênicas (Bizily et al., 1999). Apesar do íon Hg 2+ ser dominante em ambientes aquáticos, a forma de metilmercúrio (MeHg) é bastante estudada, devido a sua elevada toxicidade a organismos superiores e (Bizily et al., 2000).. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 1 de 96.

(14) Programa de Pós-Graduação em Biotecnologia. Atualmente, a demanda mundial por proteína animal tem intensificado a produtividade aquícola, aumentando a geração de resíduos lançados nos corpos hídricos sem um tratamento adequado. Poucas são as pisciculturas que possuem sistemas para tratamento de suas águas residuárias, principalmente para remoção dos constituintes nitrogenados e fosforados. Oriundos da excreção de peixes, ração e fertilizantes, tais constituintes são capazes de provocar a eutrofização das águas, onde estão presentes muitas vezes no material particulado. Nutrientes fosforados são alguns dos principais responsáveis pela eutrofização de ambientes aquáticos, sendo frequentemente o fator limitante para que ocorra tal processo. Como alternativa de um bioprocesso, os sistemas de zonas húmidas construídas cultivadas com macrófitas aquáticas, tem se mostrado eficiente na fitorremediação de águas residuárias com alta carga de matéria orgânica (Vymazal e Kröpfelová, 2009; Bustamante et al., 2011; Abou-Elela & Hellal, 2012), metais (Akinbile et al., 2012; Dotro et al., 2012), nutrientes nitrogenados e fosforados (Ayaz et al., 2012; Białowiec et al., 2012; Chang et al., 2012) e na remoção de macronutrientes em efluentes de tanques de piscicultura (Hussar et al., 2004). Tais sistemas funcionam como filtro biológico, onde ocorre a sedimentação de grande parte do material particulado, nos quais os microorganismos aeróbicos e anaeróbicos são os principais responsáveis pela decomposição da matéria orgânica, com consequente assimilação de parte dos metabólitos pelas plantas. A fitorremediação consiste em um grupo de tecnologias baseadas na utilização de plantas de ocorrência natural ou geneticamente modificadas para reduzir, remover, degradar ou imobilizar poluentes, como alternativa para os métodos convencionais de tratamento de águas residuárias (Lasat, 2002), devido a sua sustentabilidade, e baixo custo de manutenção e energia (Maine et al., 2006). Dentre as principais ações da planta sob os poluentes, destacam-se: a rizofiltração, com remoção de poluentes de um meio aquoso, através da absorção, concentração e/ou precipitação pelas raízes (Dushenkov et al., 1995); a fitoextração, que envolve a remoção pelas raízes, com subsequente transporte à parte aérea da planta (Salt et al., 1998), onde ocorrerá a degradação ou volatilização do poluente; a fitodegradação, Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 2 de 96.

(15) Programa de Pós-Graduação em Biotecnologia. onde a planta, a partir de enzimas internas ou secretadas, via raízes ou microflora associada, degradam os poluentes, convertendo-os em substâncias com reduzida toxicidade (Suresh & Ravishankar 2004); e a fitoestabilização, quando o poluente fica retido ou inativo no tecido vegetal (Sun et al., 2010; Wenzel, 2009). O presente trabalho é o resultado de pesquisa de um experimento realizado em escala piloto, projetado para avaliar o potencial da macrófita aquática Typha domingensis, em sistema de zonas úmidas construídas com fluxo subsuperficial, para a fitorremediação de águas residuárias de piscicultura ou contaminadas com mercúrio.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 3 de 96.

(16) Programa de Pós-Graduação em Biotecnologia. 2. OBJETIVOS 2. 1. Geral. O presente estudo teve como objetivo avaliar o potencial da macrófita aquática Typha domingensis, em sistema de zonas úmidas construídas com fluxo subsuperficial, para a fitorremediação de águas residuárias de piscicultura, ou contaminadas com mercúrio. 2. 2. Específicos . Estudar a variação das concentrações de mercúrio na água e na planta, e do coeficiente de transferência, ambos em função do tempo de exposição.. . Determinar a variação de parâmetros abióticos (pH, condutividade elétrica, oxigênio dissolvido, sólidos totais dissolvido, alcalinidade total, nitrogênio total e fósforo total), e a eficiência de remoção de nutrientes nitrogenados e fosforados, em função do tempo de exposição.. . Caracterizar as lagoas costeiras do Pantanal de Pacatuba-SE, através do Índice de Estado Trófico, mostrando sua relação com a forma de exploração para a piscicultura, e o efeito da presença de macrófitas aquáticas sobre a eficiência de remoção de nutrientes fosforados.. . Desenvolver um protótipo do processo de tratamento de efluentes de piscicultura, utilizando sistema de alagados artificiais de fluxo subsuperficial cultivados com Typha domingensis, no pós tratamento de lagoa de estabilização com pré decantação, para uso em fluxo de efluente de forma contínua ou batelada.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 4 de 96.

(17) Programa de Pós-Graduação em Biotecnologia. 3. CAPÍTULO 1 Phytoremediation of mercury water contaminated using Typha domingensis in constructed wetland. Marcos Vinícius Teles Gomesa,*, Roberto Rodrigues de Souzab, Vinícius Silva Telesb, Érica Araújo Mendesa. a. Centro Integrado de Recursos Pesqueiros e Aquicultura de Três Marias - CODEVASF; Av. Geraldo Rodrigues dos Santos, s/n, Satélite, CEP 39.205-000, CP 11, Três Marias/MG b. Departamento de Engenharia Química, Universidade Federal de Sergipe; Av. Marechal Rondon, s/n, Jardim Rosa Elza, CEP 49.100-000, São Cristovão/SE. * Corresponding author. Tel.: +55-38-3754-1422. E-mail address: marvitego@gmail.com (M. V. T. Gomes).. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 5 de 96.

(18) Programa de Pós-Graduação em Biotecnologia. ABSTRACT The presence of mercury in aquatic environments is a matter of concern by part of the scientific community and public health organizations worldwide due to its persistence and toxicity. The phytoremediation consists in a group of technologies based on the use of natural occurrence or genetically modified plants, in order to reduce, remove, break or immobilize toxins and working as an alternative to replace conventional effluent treatment methods due to its sustainability - low cost of maintenance and energy. The current study provides information about a pilot scale experiment projected to evaluate the potential of the aquatic machrophyte Typha domingensis in a constructed wetland with a subsurface flow for phytoremediation of mercury water contaminated. The efficiency in the reduction of the heavy metal concentration in wetlands, and the relative metal absorption by the Typha domingensis, varied according to the exposure time. The continued rate of the system was 7 times higher than the control line, demonstrating a better performance and reducing 99.6 ± 0.4% of the mercury presents in the water contaminated. When compared to other species, the results showed that the Typha domingensis demonstrated a higher mercury accumulation (273.3515 ± 0.7234 mg kg-1) when the transfer coefficient was 7750.9864 ± 569.5468 L kg -1. The results in this present study shows the great potential of the aquatic machrophyte Typha domingensis in constructed wetlands for phytoremediation of mercury water contaminated. Keywords: Constructed wetlands, Wastewater treatment, Mercury, Typha domingensis. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 6 de 96.

(19) Programa de Pós-Graduação em Biotecnologia. 1. Introduction. The presence of mercury in aquatic environments has been a matter of concern by part of the scientific community and public health organizations worldwide due to its persistence and toxicity. The impacts caused by the disposal of effluents contaminated by heavy metals bring harmful consequences to the human health and the environment, demanding a study to enable its treatment. The mercury is an element which is naturally found in the earth crust, in the atmosphere and in aquatic environments (Lacerda et al., 2007). The anthropic contribution may occur, among other things, through the combustion of fossil fuels, the electrolytic clhorine-soda production, the pesticide industrialization, odontological products and amalgamation during gold exctraction (Micaroni et al., 2000). Constituting one of the most toxic metals, the biological function of mercury isn’t known (Cursino et al., 2003). In environment, it may be converted into most toxic forms as the methyl mercury, accumulating itself in the aquatic and terrestrial biota (Padovani, et al., 1995). Through biomagnification and the food chain, it could also reach the human being where the accumulated concentration is proportional to the trophic level. The larger form of mercury distribution is the elementar (Hg 0), found in the atmosphere as vapor. However, when introduced in an aquatic ecosystem as an inorganic form, it shall be transformed into organic, and so being converted into methyl mercury by methanogenic bacteries (Bizily et al., 1999). The ion Hg2+ is predominant in aquatic environments, the methyl mercury form (MeHg) is widely studied, because of its high toxicity to superior organisms (Bizily et al., 2000).. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 7 de 96.

(20) Programa de Pós-Graduação em Biotecnologia. The phytoremediation consists in a group of technologies based on the use of natural occurrence or genetically modified plants, in order to reduce, remove, break or immobilize toxins, working as an alternative to replace conventional effluent treatment methods (Lasat, 2002), due to its sustainability, low cost of maintenance and energy (Maine et al., 2006). Among the main actions of the plant under toxins, stand out: the phytoextraction which involves the removal of toxins, especially metals and metalloids by the roots with subsequent transport to the aerial part of the plant (Salt et al., 1998), when occurs the degradation and volatilization of the contaminants; the phytodegradation, where from interns or secreted enzymes and through roots or associated microflora, the plants degrade the toxins, converting it to substances with reduced toxicity (Suresh and Ravishankar 2004); and the phytoestabilization, which the toxin is retained or inactive in the vegetal tissue (Wenzel, 2009; Sun et al., 2010). In case of phytoremediation of heavy metals contaminated effluents, hyper accumulator plants with a high growth rate are used, so then, being capable of amassing high quantities in the aerial part (Garbisu and Alkorta, 2001; Lorenzen et al., 2001; Weis and Weis, 2004; Bose et al., 2008), being also tolerable to high concentrations (Khan et al., 2000), and being finally able to be disposed in landfills or even recycled for metal recovery. Some Hg phytoremediation studies used genetically modified plants that convert ionic compounds by enzymatic action (Bizily et al., 1999) into elementary mercury, which is less toxic and volatile, being liberated in the atmosphere. Studies indicate the species of the genre Typha spp., as plants that represent high capacity to tolerate and eliminate contaminants present in the water or in the soil (Dordio et al., 2009; Park et al., 2009; Dordio et al., 2010). A great diversity of aquatic macrophytes may be used in the treatment of wastewater in humid zone systems. However, this choice must be associated with the capacity of tolerance to the flooding. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 8 de 96.

(21) Programa de Pós-Graduação em Biotecnologia. conditions and high contaminants concentration. Native plants must be considered once they are adapted to their local weather, insects and diseases. Typha is one of the cosmopolitan types out of about eleven species of flowering plants that belongs to the Thyphaceae family which is distributed in great part of the North hemisphere (Akkol et al., 2011). It rapidly grows and most part shows high evapotranspiration rates (Glenn et al., 1995). The Typha domingensis is an emergent aquatic macrophyte plant that grows in all regions with tropical, hot and temperate climates (Eid et al., 2012), and it is commonly used in constructed wetlands to improve the water quality in treatment systems (Hegazy et al., 2011; Eid et al., 2012a). Native plant and non-endemic from Brazil, this plant is known as “Taboa” (Bove, 2010), and occurs, among other places, in the Caatinga, Cerrado, Rainforest and Swamp (Pott and Pott, 1997), growing naturally in flooded floodplains, marshes, dams and drainage channels. In pilot scale, the present study was planned to evaluate the potential of the aquatic macrophyte Typha domingensis (Typhaceae) in constructed wetlands (CW) with subsurface flow to the phytoremediation of mercury waters contaminated. The specific objectives were to determine the variations of the Hg concentration in water and plant, and the transfer coefficient, both in function of the exposure time.. 2. Material and methods. 2.1 Localization of the constructed wetlands. In Brazil, the use of mercury for gold amalgamation probably began in the XIX century. Nowadays, in the Brazilian state of Minas Gerais, traditionally recognized by Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 9 de 96.

(22) Programa de Pós-Graduação em Biotecnologia. the extraction of ores and the gold-digging, these activities are still made in a predatory manner to the environment and harmful to the health of the workers and the exposed population, due to the use of mercury (Windmöller et al., 2007). A subsurface flow of constructed wetland was built in triplicate in the Três Marias Integrated Center for Fishing Resources and Aquaculture (1ª/CIT), Três Marias city, state of Minas Gerais, Brazil. Aiming to simulate a contaminated environment, was used water of the Três Marias reservoir (23K 0472275, UTM 7985048), and added mercuric chloride salt (HgCl2). The reservoir, located in Alto São Francisco, was formed in 1961, by the impoundment of the São Francisco River, having a water volume of 21 billion m³, and occupying an area of 1040 km².. 2.2 Construction. The construction of the system (Fig. 1) was started in the summer, on January 11th, during the rain period, when the air temperature average oscillated between 18 and 30°C. In a fiberglass recipient with capacity of 3000 liters and containing water of the Três Marias reservoir, was added HgCl2 to obtain an initial mercury total concentration of 9-11 mg L-1. Afterwards, the same volumes of the water contaminated were moved to other two recipients of 1000 liters each. Initially, were filled ¼ with gravel 2 (30mm mesh), granulometry between 19 and 25 mm, and filled ¼ with gravel 0 (12mm mesh), granulometry between 4.8 and 9.5 mm. In one of the smaller recipients (constructed wetland), were cultivated aquatic macrophytes buds of the Typha domingensis species. The drainage, made through the action of the gravity in the inferior part of the system, allowed the direct contact of the water contaminated to the gravel and the roots of the plants. The second recipient, plants free, was used as a control line.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 10 de 96.

(23) Programa de Pós-Graduação em Biotecnologia. Fig. 1 – Schematic presentation of the constructed wetland used in this study.. 2.3 Transplant and acclimation of the plants. In a natural wetland area located nearby to where the experiment was performed, were collected apparently healthy buds and young plants of the Typha domingensis species. Immediately transplanted, they were cultivated with a density of 50 buds per m², for a period of 160 days, enough time to reach a 136 ± 16 cm height. During the acclimation period and plant growth, the system was supplied with reservoir water. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 11 de 96.

(24) Programa de Pós-Graduação em Biotecnologia. 2.4 Sampling. The system was evaluated for 27 days during the drought period (July 2011) when the average air temperature oscillated between 12 and 27 °C. Operating in batch, the supply with water contaminated occurred only once. Water samples were collected at the end of both treatments (constructed wetlands and control line) and conserved in polyethylene bottles previously decontaminated. The vegetal material collected, which is constituted by the plants’ aerial part, was chosen randomly at 10 cm higher than the water level and packed in plastic bags. The sampling was placed in a heat at a temperature under 60 °C. Using forced air circulation, the material was dried for 72 hours or until reaching constant mass, being subsequently crushed and homogenized in laboratory.. 2.5 Chemical Analysis. The analysis method used to determine the total mercury concentration in the water samples was based on thermal decomposition detected by atomic absorption spectrometry. The equipment employed was the Milestone DMA-80 (Direct Mercury Analyzer), with direct mercury analysis without the digestion or pre-digestion of the sample, at according to recommendations of the Environmental Protection Agency (U.S. EPA, 1998). The samples were weighed (400 – 600 mg) in nickel nacelles, using an analytical balance with readability of four decimal places. Introduced through the automatic sampler in the catalysis oven, they were initially dried and then, thermally Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 12 de 96.

(25) Programa de Pós-Graduação em Biotecnologia. decomposed in a continuous oxygen flow. The products of the combustion were decomposed in a heated catalyzer, allowing the passage of mercury steams, which were retained in a gold amalgamator. After heating, the Hg was quantitatively released, and after determined through atomic absorption at 253.7 nm. For the certification of the analysis method, were performed mercury determinations in two reference materials (fish protein and lake sediment) certificated from the National Research Council Canada (NRC) (DORM-3) and the Canadian Certified Reference Materials Project (CCRMP) (LKSD-1), and the metal percentage recoveries were of 96.3% and 97.8%, respectively. Considering the average and the standard deviation of ten whites, the Method Detection Limit (MDL) was calculated as the amount of the average with three times the standard deviation. To the Method Quantification Limit (MQL), the average was amounted with ten times the standard deviation. The MDL found was of 0.0518 µg kg-1, and the MQL was of 0.2072 µg kg-1.. 2.6 Statistical Analysis of the data. The experimental results were statistically evaluated using the Origin 6.0 Professional package. Variance comparisons between mercury concentrations of the water in the constructed wetland and in the control line were made with ANOVA parametric test, applying the Tukey test. The differences were considered significant with p < 0.05.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 13 de 96.

(26) Programa de Pós-Graduação em Biotecnologia. 3. Results and discussion. 3.1 Variation of the Hg concentration in the water. The system considered as control line, initiated the study with mercury concentration in the water contaminated, of 10.7322 ± 0.5257 (Table 1), ending with 4.3486 ± 0.7234 mg L-1, after an exposure time of 648 hours (27 days). Without the presence of the Typha domingensis, the Hg remaining was of 40.6 ± 7.1%. The reduction of 59.4 ± 7.1% of the initial mercury concentration in the control line may be associated to the retention by the filtration of the metal connected to the particulate material, as well to the presence of microorganisms responsible for the metal mobilization and immobilization. Depending on the mechanism involved (Gadd, 2004), the balance varies according to the organisms involved, its environment and physicchemical conditions, which the mobilization may result in the volatilization, and the sorption immobilization for biomass, intracellular transport or precipitation as organic and inorganic compounds. Regarding the constructed wetland, the initial concentration was of 9.0176 ± 0.4346, ending with 0.0353 ± 0.0402 mg L -1. With the presence of the plant, the remaining Hg was of 0.4 ± 0.4%, with a 99.6 ± 0.4% reduction. The F obtained (17.9645) was highly significant (P < 0,01), existing difference between the Hg concentrations in the water of the control line and the constructed wetland.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 14 de 96.

(27) Programa de Pós-Graduação em Biotecnologia. Similar results were obtained by Kamal et al. (2004), evaluating the capacity of three aquatic plants, on the heavy metal removal of the water contaminated. The experiment lasted 21 days, beginning with a mercury concentration in the water of 0.501 mg L-1. The systems cultivated with Myriophylhum aquaticum, Ludwigina palustris and Mentha aquatic, obtained a removal efficiency of 99.97, 99.74 and 99.99%, respectively. A study realized by Mant et al. (2005), using Penisetum purpureum, Brancharia decumbens and Phragmites australis, to get the chrome removal in solutions of 10 and 20 mg L-1, reached the removal efficiency of 97.0 and 99.6% in 24 hours. An experiment made in laboratorial scale (Rahman et al., 2011), in a wetland with Juncus effusus, obtained an arsenic removal efficiency in artificial residuary water of 59 to 61% during 491 days. In the plant free control line, the removal was of 44%.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 15 de 96.

(28) Programa de Pós-Graduação em Biotecnologia. Rana et al. (2011) studied the performance of wetlands, composed by anaerobic lakes, facultative and of maturation, to treat cadmium contaminated effluents, obtaining a reduction of 30% with the average retention time of 11.2 days. In a similar system, cultivated with Cyperus haspan and treating landfill leachates, Akinbile et al. (2012) succeeded in 21 days a removal of 75.9 and 89.4% of zinc concentration. The Fig.2 shows the performance of the constructed wetland in the mercury removal, after 648 hours (27 days) of experiment. The constant speed of the removal, first-rate, was determined by the C/Co exponential graphic, corresponding to the relation between the concentration in any exposure time, and the initial concentration. The constant speed k of the control line was of 0.001 hour -1, and 0.007 hour-1 for the constructed wetland.. 1.0. (Control line) (Constructed wetland). 0.9 0.8 0.7. C / C0. 0.6. -0.001 t. y = 0.6585e R² = 0.5602. 0.5 0.4 0.3 0.2. -0.007 t. y = 0.2633e R² = 0.8850. 0.1 0.0 0. 100. 200. 300. 400. 500. 600. 700. Exposure time (hours). Fig. 2 – The performance of the constructed wetland in the mercury removal.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 16 de 96.

(29) Programa de Pós-Graduação em Biotecnologia. The reduction percentage of the heavy metal concentration in wetlands varies, among others, according to the exposure time, to the element and the plant species used. With a constant speed 7 times higher than the control line in the experiment conditions, the system planted with Typha domingensis showed a better performance, being able to reduce 99.6 ± 0.4% of the initial mercury concentration present in the water contaminated. The performance results for the metal removal were similar to the results obtained by other studies that used different plant species, as an example of Kamal et al. (2004) and Mant et al. (2005), for mercury and chrome, respectively. Also, were superior if comparing with the results obtained by Rana et al. (2011) and Akinbile et al. (2012), on the removal of cadmium and zinc.. 3.2 Transfer Coefficient. The water-plant transfer coefficient (TC) was calculated as the relation between the Hg concentration of the aerial part of the plant (dry mass) and the water contaminated, to express the relative metal absorption by the Typha domingensis. The mercury concentration present in the plant began with 0.1785 ± 0.1337 (Table 2), ending with 273.3515 ± 0.7234 mg kg -1, after an exposure time of 648 hours, when the transfer coefficient was of 7750.9864 ± 569.5468 L kg-1.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 17 de 96.

(30) Programa de Pós-Graduação em Biotecnologia. Samecka-Cymerman and Kempers (1996) used Scapania undulata to remove mercury from wastewaters (0.005 mg L-1) from pesticide factories. The initial concentration present in the dry mass of the plant was of 0.05 mg kg -1, and after 14 days it turned into 2.40 mg kg-1. At a hydroponics system, Qian et al. (1999) was cultivated 12 plant species in an incubator for the treatment of water contaminated by mercury (1 mg L -1). After 10 days of experiment, the metal concentration in the aerial part of the plants oscillated between 5 mg kg-1 (Pistia stratiotes L and Wedelia trilobata Hitchc) and 90 mg kg-1 (Sesbania drummondii). During a study regarding the metal accumulation in aquatic machrophytes from six sampling sites on the Southeast of Queensland, Australia, Cardwell et al. (2002), found in the aerial part of the Typha domingensis the concentrations of cadmium (0.00 to 0.40 mg kg-1), copper (3.13 to 16.40 mg kg-1), lead (1.35 to 5.39 mg kg -1) and zinc (19.8 to 90.2 mg kg-1).. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 18 de 96.

(31) Programa de Pós-Graduação em Biotecnologia. In systems cultivated with Myriophylhum aquaticum, Ludwigina palustris and Mentha aquatic for 21 days, Kamal et al. (2004) obtained a final concentration in the plants of 208, 335 and 179 mg kg-1, respectively. A study made (Greger et al., 2005) with six plants, in solution of 0.200 mg L -1 of Hg, for 2 or 3 days, were able to accumulate about 0.16 to 1.40 mg kg -1 on the aerial part of the plant (2.2–16.7 times higher than in the control). Even with reduced Hg translocation to the aerial part (0.17–2.50%), the study concluded that the absorbed metal that hit the leaves are stuck, not having its liberation to the air. Hadad et al. (2006) evaluated the growth of the machrophytes in a constructed wetland, in pilot scale, for the treatment of industrial residual water contaminated by metal. At the end of the experiment, comparing to other species, the Typha domingensis showed a competitive hierarchical tolerance, until it becomes the only species that covered almost the entire surface, reaching a bigger biomass than the verified in the undisturbed environments. The concentrations of chrome, nickel and zinc in the aerial part of the plant were of: 36.0, 32.0 and 39.0 mg kg-1, respectively. The work results showed that the Typha domingensis was capable of accumulating more mercury than the species used by Samecka-Cymerman and Kempers (1996), Qian et al. (1999), Greger et al. (2005) and two of the three species studied by Kamal et al. (2004). When compared to studies that also used the Typha domingensis, the mercury removal, the present study had superior results when comparing with the one found for cadmium, copper, lead and zinc (Cardwell et al., 2002), and chrome, nickel and zinc (Hadad et al., 2006). The low mercury concentration, non-essential to the plant, in residual water samples and their high water-plant transfer coefficient, may be a reasonable explanation to the high efficiency of the removal of this metal. The relative lack of selectivity on the transmembrane transport of the metals would be a reason for the input of non-essential heavy metals in the cells (Soda et al., 2012).. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 19 de 96.

(32) Programa de Pós-Graduação em Biotecnologia. Through the simple exponential regression chart (Fig. 3), having the transfer coefficient in function of the time of exposure, was obtained a R 2 determination coefficient of 0.7888.. -1. Transfer coefficient ( L kg ). 10000. TC = 1.2255e R² = 0.7888. 8000. 0.0152 t. 6000. 4000. 2000. 0 0. 100. 200. 300. 400. 500. 600. 700. Exposure time (hours). Fig. 3 - Variation of the water-plant transfer coefficient as a function of exposure time.. The relative metal absorption by the Typha domingensis, represented by the crescent transfer coefficient in function of the exposure time, agrees with the observation made by Ding et al. (1994) and Lai et al. (2010), wherein the time increasing promotes the heavy metal accumulation.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 20 de 96.

(33) Programa de Pós-Graduação em Biotecnologia. 4. Conclusions. The efficiency on the reduction of heavy metal concentration in constructed wetlands, and the relative metal absorption by the Typha domingensis, varied according to the exposure time. The system’s constant speed was 7 times higher than the control line, demonstrating a better performance and managing to reduce 99.6 ± 0.4% of the mercury present in water contaminated. When compared to other species, the results showed that the Typha domingensis demonstrated a higher mercury accumulation (273.3515 ± 0.7234 mg kg -1), when the transfer coefficient was of 7750.9864 ± 569.5468 L kg-1. Results of the present study showed high potential of the aquatic macrophyte Typha domingensis in constructed wetlands with subsurface flow, for the phytoremediation of mercury waters contaminated.. Acknowledgements. The authors would like to thank the Três Marias Integrated Center for Fishing Resources and Aquaculture – 1ª CIT / CODEVASF and the Northeast Network of Biotechnology – RENORBIO.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 21 de 96.

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(38) Programa de Pós-Graduação em Biotecnologia. Rana, S., Jana, J., Bag, S.K., Mukherjee, S., Biswas, J.K., Ganguly, S., Sarkar, D., Jana, B.B., 2011. Performance of constructed wetlands in the reduction of cadmium in a sewage treatment cum fish farm at Kalyani, West Bengal, India. Ecological Engineering 37, 2096-2100. Rugh, C.L., Wilde, H.D., Stack, N.M., Thompson, D.M., Summers, A.O., Meagher, R.B., 1996. Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene. Proceedings of the National Academy of Sciences 93, 3182-3187. Salt, D.E., Smith, R.D., Raskin, I., 1998. Phytoremediation. Annual Review of Plant Physiology and Plant Molecular Biology 49, 643-668. Samecka-Cymerman, R.B., Kempers, A.J., 1996. Bioaccumulation of heavy metals by aquatic macrophytes around Wroclaw, Poland. Ecotoxicology and Environmental Safety 35, 242-247. Soda, S., Hamada, T., Yamaoka, Y., Ike, M., Nakazato, H., Saeki, Y., Kasamatsu, T., Sakurai, Y., 2012. Constructed wetlands for advanced treatment of wastewater with a complex matrix from a metal-processing plant: Bioconcentration and translocation factors of various metals in Acorus gramineus and Cyperus alternifolius. Ecological Engineering 39, 63-70. Sun, T.R.; Cang, L; Wang, Q.Y.; Zhou, D.M.; Cheng, J.M.; Xu, H., 2010. Roles of abiotic losses, microbes, plant roots, and root exudates on phytoremediation of PAHs in a barren soil. Journal of Hazardous Materials 176, 919-925. Suresh, B., Ravishankar, G.A., 2004. Phytoremediation - a novel and promising approach for environmental clean-up. Critical Reviews Biotechnology 24, 97-124. U.S EPA, 1998. Mercury in Solids and Solutions by Thermal Decomposition, Amalgamation and Atomic Absorption Spectrophotometry (Method 7473).. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 26 de 96.

(39) Programa de Pós-Graduação em Biotecnologia. Weis, S.J., Weis, P., 2004. Metal uptake, transport and release by wetland plants: implications for phytoremediation and restoration. Environment International. 30, 685700. Wenzel, W.W., 2009. Rhizosphere processes and management in bioremediation (phytoremediation) of soils. Plant Soil 321,385-408. Windmöller, C.C., Santos, R.C., Athayde, M., Palmieri, H.E.L., 2007. Distribuição e especiação de mercúrio em sedimentos de áreas de garimpo de ouro do Quadrilátero Ferrífero (MG). Química Nova 30 (5), 1088-1094.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 27 de 96.

(40) Programa de Pós-Graduação em Biotecnologia. 4. CAPÍTULO 2 Phytoremediation of the pisciculture wastewater, using Typha domingensis in constructed wetlands system. Marcos Vinícius Teles Gomesa,*, Roberto Rodrigues de Souzab, Vinícius Silva Telesb, Érica Araújo Mendesa. a. Centro Integrado de Recursos Pesqueiros e Aquicultura de Três Marias - CODEVASF;. Av. Geraldo Rodrigues dos Santos, s/n, Satélite, CEP 39.205-000, CP 11, Três Marias/MG b. Departamento de Engenharia Química, Universidade Federal de Sergipe; Av. Marechal. Rondon, s/n, Jardim Rosa Elza, CEP 49.100-000, São Cristovão/SE. * Corresponding author. Tel.: +55-38-3754-1422. E-mail address: marvitego@gmail.com (M. V. T. Gomes).. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 28 de 96.

(41) Programa de Pós-Graduação em Biotecnologia. Abstract This actual study offers information of an experiment accomplished in pilot scale and projected to evaluate the aquatic macrophytes Typha domingensis in constructed wetland systems with subsurface flow in order to have the phytoremediation of wastewater of piscicultures. It is considerable important to understand the dynamics of the abiotic parameters variation, showing that the increasing of the pH, EC, TDS and total alkalinity, in addition of the reduction of DO, total nitrogen and total phosphorus oscillated in function of the exposure time and the density of the macrophyte, suggesting being those the preponderant factors in the constructed wetlands cultivated with Typha domingensis. After 120 h of exposure time, the treatment with 50 shoots per m² showed a total nitrogen removal efficiency 217% higher than the control line (machrophyte free), showing the Typha domingensis is, essential to the nitrogen phytoremediation. For the total phosphorus, the removal efficiency was 26% higher than the control line, possibly for being present in the particulate matter, and this, has been retained predominantly by filtration and sedimentation. The total nitrogen and phosphorus removal efficiency were about 90%. The results showed the great potential of the aquatic macrophyte Typha domingensis for the phytoremediation of the wastewater of pisciculture. Keywords: pisciculture, phytoremediation, constructed wetlands, Typha domingensis, nutrients.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 29 de 96.

(42) Programa de Pós-Graduação em Biotecnologia. INTRODUCTION Nowadays, the world demand for animal protein has enhanced the aquaculture productivity, increasing the generation of waste, usually released in the water bodies without proper treatment which could use efficient biotechnological processes. There are a small amount of piscicultures that have wastewater treatment systems, especially for the removal of the nitrogen and phosphorus constituents. The fish excretion, ration and fertilizer, they are capable of provoking the eutrophication of the water, where are present many times in the particulate matter. Studies show that the phosphorous nutrients are some of the main responsible for the eutrophication of aquatic environments, and are often the limiting factor for such a process (Salas et al., 1991; Esteves, 1998). As alternative to a bioprocess, the constructed wetland systems cultivated with aquatic macrophytes, has showed efficiency in the phytoremediation of the wastewaters with high load of organic matter (Vymazal and Kröpfelová, 2009), and in the removal of the macronutrients of effluents from pisciculture tanks (Hussar et al., 2004). Such systems work as biological filter, and occur the sedimentation of great part of the particulate matter, in which aerobic and anaerobic microorganisms are the main responsible for the decomposition of organic matter, with consequent assimilation of part of the metabolic by plant. The phytoremediation consists in a group of technologies based on the use of natural occurrence or genetically modified plants to reduce, remove, degrade or immobilize pollutants, as alternative to conventional wastewater treatment methods (Lasat, 2002), due to its sustainability, low maintenance and energy costs (Maine et al., 2006). Among the main actions of the plant under the pollutants, stand out: the rhizofiltration with removal of pollutants of an aqueous environment through the. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 30 de 96.

(43) Programa de Pós-Graduação em Biotecnologia. absorption, concentration and/or precipitation through roots (Dushenkov et al., 1995); the phytoextraction, that involves the removal by roots, with subsequent transport to the aerial part of the plant (Salt et al., 1998), when occurs the degradation or volatilization of the pollutant; the phytodegradation, where the plant, from intern or secreted enzymes, via roots or associated microflora, degrade the pollutants, converting them in substances with low toxicity (Suresh and Ravishankar, 2004); and the phytoestabilization, when the pollutant stays retained or inactive in the vegetal tissue (Wenzel, 2009; Sun et al., 2010). The aquatic macrophytes perform important function in the maintenance and balance of the aquatic environments (Rodella et al., 2006), contributing into physical, chemical transformations and in the microbiological process of nutrients removal (Sipaúba-Tavares et al., 2003). The process partially reduces the metabolic load from the cultivation of aquatic organisms, substantially improving the quality of the water (Sipaúba-Tavares et al., 2002). Studies indicate the species of the Typha spp. type, as plants that show high capacity to tolerate and eliminate contaminants present in water or soil (Dordio et al., 2009; Park et al., 2009; Dordio et al., 2010). A great variety of aquatic macrophytes may be used in the treatment of wastewaters in constructed wetlands systems. But its choice must be associated to the capacity of tolerate the flooding conditions and the high pollutant concentrations. Native plants must be considered, once they are adapted to the local weather, bugs and diseases. Typha is a cosmopolitan type out of eleven species of floriferous plants from Typhaceae family (Akkol et al., 2011). It has fast growth, and the majority shows high evapotranspiration rates (Glenn et al., 1995). The Typha domingensis is an emergent aquatic macrophyte plant that grows in all regions with tropical, hot and temperate climates (Eid et al., 2012), and it is commonly used in constructed wetlands to improve the water quality in treatment systems (Hegazy et al., 2011; Eid et al., 2012a). Native plant and non-endemic from Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 31 de 96.

(44) Programa de Pós-Graduação em Biotecnologia. Brazil, it is known as “Taboa” (Bove, 2010), and occurs in the Caatinga, Cerrado, Rainforest and Swamp (Pott and Pott, 1997), spontaneously growing in flooded lowlands, marsh, dams and draining channels. In pilot scale, the present study was projected to evaluate the potential of the aquatic macrophyte Typha domingensis (Typhaceae), in a constructed wetland system (Constructed Wetland – CW) with subsurface flow, for the phytoremediation of wastewater of pisciculture. The specific objectives were to determine the variation of abiotic parameters (pH, electrical conductivity, dissolved oxygen, total dissolute solids, total alkalinity, total nitrogen and total phosphorus), and the efficiency on the removal of phosphorus and nitrogen nutrients, according to exposure time.. MATERIAL AND METHODS Construction and conception of constructed wetlands A constructed wetland system with subsurface flow was built in triplicate in the Três Marias Integrated Center for Fishing Resources and Aquaculture (1ª/CIT), Três Marias city, state of Minas Gerais, Brazil. The system operated by treating wastewaters from a polyculture pond (23K 0473273, UTM 7987393), which was provided with water from the Três Marias reservoir (23K 0473273, UTM 7987393). The reservoir situated at Alto São Francisco was formed in 1961 by the impoundment of the São Francisco River, having an water volume of 21 billion per m³, and occupying an area of 1040 km². Among the fishes present in the polyculture pond, the most abundant species were the following: curimatã pacu (Prochilodus argenteus), curimatã pioa (Prochilodus costatus), matrinxã (Brycon orthotaenia), piau verdadeiro (Leporinus obtusidens), pacamã (Lophiosilurus alexandri) and surubim (Pseudoplatystoma corruscans). The construction of the system (Fig. 1) was started in the summer, on January th. 11 , during the rain period, when the air temperature average oscillated between 18 and 30°C. In a region of natural wetlands located close to where the experiment was made, Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 32 de 96.

(45) Programa de Pós-Graduação em Biotecnologia. shoots were collected from young and apparently healthy plants of the Typha domingensis species. Immediately transplanted, they were cultivated with densities of 10, 20, 30, 40 and 50 shoots per m² (E10, E20, E30, E40 e E50 respectively), over a period of 160 days, time enough to reach 136 ± 16 cm high. During the plants acclimation and growth, the system was loaded with water from the Três Marias reservoir, exclusively. The treatment system (Figure 1) was formed by a recipient of 3000 liters that was loaded by pumping the wastewater from the polyculture pond, and then, distributing it by gravity to six other recipients of 1000 liters each. Initially, they were filled ¼ with gravel 2 (30 mm mesh), granulometry varying between 19 and 25 mm, and ¼ with gravel 0 (12 mm mesh), granulometry between 4.8 and 9.5 mm. In five of the smaller recipients (constructed wetland), were cultivated shoots of the Typha domingensis. The exposure time varied between zero to five days. The draining, made through the gravity action in the lower part of the system, allowed the direct contact of the wastewater with the gravel and with the plants’ roots. The sixth recipient - plants free, was used as control line.. Figure 1 Schematic presentation of the constructed wetland used for the study. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 33 de 96.

(46) Programa de Pós-Graduação em Biotecnologia. Sampling and Chemical analysis The system was evaluated for 5 (five) days during drought period (August 11th), when the average of air temperature varied between 12 and 27°C. Operating in batch, the supply with wastewater from the polyculture pond occurred only once. Water samples were collected in the treatments output (constructed wetlands and control line). every 24h,. and preserved. in polyethylene. flasks previously. decontaminated. In order to determine the pH, electrical conductivity (EC), dissolved oxygen (DO), and total dissolved solids (TDS), was used a Horiba mulitparameter probe, model W22XD. The calibration of the equipment was made according to the manufacturer’s recommendations (Horiba, 2007), and after performing three readings per sample. To determine the total alkalinity, the potentiometric method was used (ABNT NBR 13736, 1996). The total nitrogen and phosphorus concentrations were determined according to the method written by Valderrama (1981).. RESULTS AND DISCUSSION On the beginning of the experiment, the wastewater that supplied the system showed pH of 7.13 ± 0.11, EC of 116 ± 3 µS cm-1, DO of 6.91 ± 0.04 mg L-1, TDS of 76 ± 2 mg L-1, total alkalinity of de 29.37 ± 0.26 mg L -1, total nitrogen of 826.05 ± 17.43 µg L-1, and total phosphorus of 101.80 ± 3.71 µg L-1. The CONAMA 357/05 Resolution states the classification of water bodies and environmental guidelines for their framework as well as establishes the conditions and standards for effluent release. For class 2 water, the pH must be in between 6 and 9, DO greater than 5 mg L-1, TDS less than 500 mg L-1, total nitrogen less than 2180 µg L-1, and total phosphorus less than 100 µg L-1. At the beginning of the experiment, only the. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 34 de 96.

(47) Programa de Pós-Graduação em Biotecnologia. total phosphorus values presented was greater than those established by CONAMA 357/05. In all treatments occurred a pH variation (Figure 2) already in the first 24 h. In the end of the experiment, the control line presented the highest value (7.46 ± 0.09), and the E40 treatment the lowest (6.97± 0.06). The presence of Typha domingensis kept the pH close to neutral, fact that has not occurred with the line control, where there has been in basicity increase that may be associated to the degradation of the organic matter and consequent formation of metabolic, in addition to denitrification.. Shoots per square meter: 0 10 20 30 40 50. 8.0. 7.8. 7.6. pH. 7.4. 7.2. 7.0. 6.8. 6.6 0. 20. 40. 60. 80. 100. 120. Exposure time (hours). Figure 2 PH variation depending on exposure time and density of the macrophyte.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 35 de 96.

(48) Programa de Pós-Graduação em Biotecnologia. After 120 h of exposure time, the minimum EC was of 179 ± 3 µS cm-1 has been found in the control line (Figure 3), and maximum of 488 ± 5 µS cm-1, in E40 treatment. In the presence of the macrophyte occurred a strong increase of the EC in all treatments, suggesting its effective participation in the particulate matter degradation and consequent cycling of the nutrients, releasing them in the dissolved form. After 48 h of exposure time, occurs a reduction of electrical conductivity in the E50 treatment, possibly due to the great absorption of dissolved form arising from the higher density of macrophytes.. Shoots per square meter: 0 10 20 30 40 50. 500 450 400. -1. EC (µS cm ). 350 300 250 200 150 100 50 0. 20. 40. 60. 80. 100. 120. Exposure time (hours). Figure 3 Variation of electric conductivity (EC) depending on exposure time and density of the macrophyte.. Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 36 de 96.

(49) Programa de Pós-Graduação em Biotecnologia. In all treatments, including the control line, occurred a strong DO reduction within the exposure time of 48 h (Figure 4). In the end of the experiment, the lowest concentration was found in the E50 treatment (2.41 ± 0.03 mg L -1), probably influenced by the increase of the microbial activity over the degradation of the organic matter.. Shoots per square meter: 0 10 20 30 40 50. 9. 8. 7. -1. DO (mg L ). 6. 5. 4. 3. 2 0. 20. 40. 60. 80. 100. 120. Exposure time (hours). Figure 4 Variation of dissolved oxygen (DO) depending on exposure time and density of the macrophyte.. After 120 h of exposure time, the minimal TDS value (Figure 5) was of 117 ± 1 mg L-1, found in the control line (Figure 5), and maximum of 313 ± 2 mg L -1, in the E40 treatment. In the presence of the macrophyte occurred a strong increase of the TDS values in all treatments. Similarly, the highest concentration of total dissolved solids Fitorremediação utilizando Typha domingensis em sistema de zonas úmidas construídas. Página 37 de 96.