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Souza Ts Dr Rcla.Pdf (1013.Kb) UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS - RIO CLARO PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS BIOLOGIA CELULAR E MOLECULAR ANÁLISE DA TOXICIDADE E DA MUTAGENICIDADE DE SOLO DE LANDFARMING, PROVENIENTE DE REFINARIA DE PETRÓLEO, ANTEES E DEPOIS DE PROCESSOS QUE VISAM ESTIMULAR A BIODEGRADAÇÃO DE HIDROCARBONETOS TATIANA DA SILVA SOUZA Tese apresentada ao Instituto de Biociências do Campus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas. Tese apresentada ao Instituto de Biociências do Campus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas. Junho - 2010 ANÁLISE DA TOXICIDADE E DA MUTAGENICIDADE DE UM SOLO DE LANDFARMING, PROVENIENTE DE UMA REFINARIA DE PETRÓLEO, ANTES E DEPOIS DE PROCESSOS QUE VISAM ESTIMULAR A BIODEGRADAÇÃO DE HIDROCARBONETOS TATIANA DA SILVA SOUZA Orientadora: Profa. Dra. CARMEM SILVIA FONTANETTI CHRISTOFOLETTI Tese apresentada ao Instituto de Biociências da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Rio Claro para obtenção do título de Doutor em Ciências Biológicas (Área de Concentração: Biologia Celular e Molecular). Rio Claro Estado de São Paulo-Brasil Maio de 2010 O P # $ H & ' ' H ( ) + , - + ()+,-+ . /+ 01234565789 H O !"P: ; H+ $$ ! H+ %!&&"' ' ' ' H !( )& *&+ ) $ &" :,+#)> )' ) , H .( ' $ ) + +7& @ ; / & )& & $) 1" ) @ A,-+ : ; ' 3(+ + $ , H . ) 4 5 . ) #' ; 4( . 4 , 7 "8 "8 .. /$&"$ . / / 4&" & *" &" "( $) * $( $ $. " ! "' . C D ) 7 "8 . "$ . / ' )" . $ $. (,$4.. ) 7 H : "&&" 4 / * H4'+ ) ! H& '/ 91& SUMÁRIO Página 1. RESUMO............................................................................................................ 1 2. ABSTRACT........................................................................................................ 3 3. INTRODUÇÃO.................................................................................................. 5 4. OBJETIVOS....................................................................................................... 8 5. REVISÃO DE LITERATURA......................................................................... 10 5.1. Biorremediação de hidrocarbonetos de petróleo em landfarming.............................................................................................................. 10 5.2. Casca de arroz e seu emprego na biorremediação de solos contaminados com hidrocarbonetos de petróleo............................................................................. 13 5.3. Vinhaça e seu emprego na biorremediação de solos contaminados com hidrocarbonetos de petróleo..................................................................................... 15 5.4. Teste de anormalidades mitóticas e cromossômicas em Allium cepa.......................................................................................................................... 16 5.5. Morfologia do intestino médio e do corpo gorduroso perivisceral de Rhinocricus padbergi e seu emprego na avaliação de solos contaminados........................................................................................................... 23 6. MATERIAL E MÉTODOS............................................................................... 26 6.1. Área de estudo – Landfarming da Refinaria de Petróleo do Planalto (REPLAN/ PETROBRÁS)...................................................................................... 26 6.2. Solos controle.................................................................................................... 29 6.3. Vinhaça............................................................................................................. 29 6.4. Biorremediação................................................................................................. 30 6.4.1. Adição de casca de arroz à amostras de solo de landfarming.............................................................................................................. 30 6.4.1.2. Ensaio respirométrico em frascos de Bartha & Pramer...................................................................................................................... 30 6.4.2. Adição de casca de arroz, vinhaça e casca de arroz associado com vinhaça à amostras de solo de landfarming........................................................................... 30 6.5. Teste de anormalidades mitóticas e cromossômicas em Allium cepa............... 31 6.6. Bioensaio com Rhinocricus padbergi............................................................... 32 6.6.1. Histologia ...................................................................................................... 33 6.6.1.1. Inclusão em resina (Historesina)................................................................. 33 6.6.2. Histoquímica................................................................................................. 34 6.6.2.1. Técnica do azul de bromofenol para detecção de proteínas totais.............. 34 6.6.2.2. Técnica do PAS para detecção de polissacarídeos neutros......................... 34 6.6.2.3. Método de von Kossa para a detecção de cálcio......................................... 34 6.6.2.4. Técnica do sudan black B para detecção de lipídios totais......................... 35 7. RESULTADOS................................................................................................... 36 7.1. Ecotoxicological assays applied in soils contaminated by petroleum hydrocarbons……………………………………………………………………… 37 7.2. The Allium cepa bioassay to evaluate landfarming soil, before and after the addition of rice hulls to accelerate organic pollutants biodegradation.......................................................................................................... 61 7.3. The effects of two agricultural residues on the bioremediation and genotoxicity of soil contaminated with petroleum refinery sludge……………...... 85 7.4. Clastogenicidade de solo de landfarming associado à vinhaça........................ 107 7.5. O uso de diplópodos como bioindicadores- uma revisão................................. 133 7.6. Morphological biomarkers in the Rhinocricus padbergi midgut exposed to contaminated soil..................................................................................................... 163 7.7. Análise histológica e histoquímica do corpo gorduroso perivisceral de Rhinocricus padbergi, expostos a um solo industrial contaminado......................... 190 8. CONSIDERAÇÕES FINAIS............................................................................ 216 9. CONCLUSÕES.................................................................................................. 220 10. REFERÊNCIAS BIBLIOGRÁFICAS........................................................... 222 Resumo_____________________________________________________________ 1 1. RESUMO O sistema de biorremediação denominado landfarming tem sido utilizado por refinarias de petróleo para dispor e tratar, em grandes áreas de solo, localizadas na própria indústria, o resíduo sólido final gerado, que é composto por uma emulsão formada por água, diversas frações de hidrocarbonetos e metais. Contudo, em landfarmings, a biodegradação de algumas classes de hidrocarbonetos pode levar tempo, meses ou anos. Desse modo, tratamentos complementares podem ser utilizados, visando estimular o metabolismo microbiano, tais como a adição de agentes descompactantes e nutrientes no solo. O progresso da biorremediação pode ser acompanhado por meio de medidas da concentração dos contaminantes de interesse, pela análise do metabolismo microbiano e por meio de ensaios biológicos que mensuram as atividades tóxica, genotóxica e mutagênica das amostras avaliadas. O objetivo do presente trabalho foi investigar os efeitos da adição de dois resíduos agroindustriais, casca de arroz e vinhaça, na biorremediação, na genotoxicidade e na mutagenicidade de um solo de landfarming proveniente de uma refinaria de petróleo, por meio de análises químicas, taxa de CO2 liberado por microrganismos e por meio do teste de anormalidades mitóticas e cromossômicas na espécie vegetal Allium cepa. Também foi objetivo desse trabalho avaliar o potencial bioindicador do invertebrado terrestre Rhinocricus padbergi após exposição a amostras do landfarming desativado. Análises histológicas e histoquímicas no intestino médio e no corpo gorduroso perivisceral do diplópodo foram realizadas. Os resultados do presente trabalho mostraram que a casca de arroz, empregada como agente descompactante do solo, constitui uma alternativa economicamente viável para a biorremediação de resíduos derivados da indústria do petróleo. A adição desse material promoveu um aumento da quantidade de CO2 liberado, diminuiu a concentração de hidrocarbonetos totais de petróleo (TPHs) do solo de landfarming e reduziu sua genotoxicidade. Ao contrário, a vinhaça, quando utilizada in natura, não foi capaz de estimular o metabolismo Resumo_____________________________________________________________ 2 microbiano, visto que a taxa de depleção de TPHs
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