M.Sc., Faculty of Science, University of the Witwatersrand, 2011 / Contamination of water bodies by heavy metals and metalloids is an established problem and several studies have been conducted to deal with it. South Africa is amongst those countries whose water systems are most affected as a result of intensive mining activities. This research was dedicated to the development of insoluble chelating polymers for use as adsorbents to abstract heavy metal ions from mining and industrial wastewater. Branched polyethylenimine (PEI), well known for its metal chelating potential, was cross linked by epichlorohydrin in order to convert it into a water-insoluble form. The water-insoluble property gives the advantage of being used in situ and a possibility of regeneration and re-use, making it a more feasible and cost-effective method. Its surface was also modified for selective removal of specifically-targeted heavy metal and metalloid ions. The binding affinity of the synthesized materials to heavy metal and metalloid ions has been determined as well as their ability to be regenerated for reuse. These processes demonstrated that cross-linked polyethylenimine (CPEI) exhibited good complexation ability with high affinity to Cr and some divalent metal ions such as Fe, Zn, and Ni. On the other hand, it showed very poor ability to bind oxo-anions such as SeO32- and AsO2- which has been attributed to the unavailability of suitable functional groups to interact with these ions. The observed order of complexation was: Cr > Zn> Fe >> Ni > Mn > Pb >> As > U > Se. The phosphonated polyethylenimine (PCPEI) showed high selectivity for As, Mn and uranyl ions. The observed order of removal was: U > Mn> Ni > Zn > As >> Cr > Pb > Fe >> Hg > Se; whereas the suffocated polyethylenimine (SCPEI) exhibited high affinity to Se, and Hg. The observed order of adsorption was: Hg > Se >> U > Zn >Pb > Ni >> As > Cr > Fe. v The adsorption behaviour of these polymeric materials involved more than one mechanism such as complexation, normal surface charge exchange, and anion replacement and all these mechanisms are governed by the functional groups. The nitrogen atom on the chelating group (-NH) in the cross-linked polyethylenimine; the phosphorus atom on the chelating group (-PO3H2) in phosphonated cross-linked polyethylenimine; and sulphur atom on the chelating group (-SO3H) in suffocated cross-linked polyethylenimine act as Lewis bases and donate electrons to metal cations which are considered Lewis acids. The existence of the chelating groups in SCPEI and PCPEI facilitate the removal of oxo-anions through anion replacement since they exist as bases in solution and hence cannot be electron acceptors. Thus, the expected mechanism is the normal anion replacement. This mechanism can explain the high removal of Se by SCPEI since Se has similar chemical behaviour as sulphur and are in the same group in the periodic table. As such they can easily replace each other. Sulphur is released from the polymer into the solution by replacing the selenium ions in the polymer. Similar behaviour occurs between phosphorus in PCPEI and arsenic ions as As and P belong to the same group in the periodic table and hence have similarities in their chemical behaviour. The Langmuir and Freundlich isotherm models were used to interpret the adsorption nature of the metal ions onto synthesized polymers. The Freundlich isotherm was found to best fit and describe the experimental data describing the adsorption process of metal and metalloid ions onto the synthesized polymeric materials The kinetic rates were modelled using the pseudo first-order equation and pseudo second-order equation. The pseudo second-order equation was found to explain the adsorption kinetics most effectively implying chemisorption. vi The thermodynamic study of the adsorption of metals and metalloids by the synthesized CPEI, PCPEI and SCPEI resulted in high activation energies > 41 KJ mol-1 which confirm chemisorption as a mechanism of interaction between adsorbate and adsorbent. So far, the developed polymeric materials showed good results and have potential to be applied successfully for remediation of heavy metal-polluted waters, and they have potential for use in filter systems for household use in communities that use borehole water impacted by mining and industrial waste waters. The desorbed metals can be of use to metal processing industries.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2016. / South Africa, as one of the leaders in mining industry, due to the variety and quantity of minerals produced, has been and is still producing a number of mine tailings which are contaminated by heavy metals. Heavy metals are very harmful to plants and especially to human beings and animals due to their non-biodegradable nature. The problem of environmental metal pollution could be combated by the establishment of Arbuscular Mycorrhiza (AM) vegetation on the surface of mine tailings. Besides the toxicity of the substrate, such areas usually lack essential nutrients (mainly N, P, and K) and organic matter. AM fungi contribute to soil structure by forming micro- and macro- soil aggregates within the net of external hyphae. Their presence may reduce stress caused by lack of nutrients or organic matter and increase plant resistance to pathogens, drought and heavy metals. Therefore, mycorrhizal fungi may become the key factor in successful plant revegetation of heavy-metal-polluted areas by promoting the success of plant establishment and increasing soil fertility and quality. The aim of this project was to identify AM fungi from a number of heavy metal sites in South Africa using both morphological and molecular techniques, followed by the evaluation of heavy metal distribution and localisation in mycorrhizal roots. Soil samples were collected from three different provinces, namely: Gauteng, Mpumalanga and North West provinces. The sites were selected based on their historical and current heavy metal contamination. Indigenous AM fungal isolates (which are adapted to local soil conditions) can stimulate plant growth better than non-indigenous isolates. AM fungal spores were isolated from 100g of representative soil sample by the wet sieving and decanting method, followed by assessment of spore numbers and infective propagules. The spores of a subset of the pot samples were mounted on microscope slides in polyvinyl lactic acid glycerol and identified by morphological characteristics to the level of genus or species. Most of the spores counted were observed in a 45 μm sieve. These spores were tiny and had different sizes, colours and shapes. The majority of the observed spores were small, brown and oval in shape. For morphological identification, plant roots were stained and hyphae were found to be the most abundant in roots. For molecular identification, two sets of nested PCR primers, namely NS1 & NS4 coupled with AML1 & AML2, were employed in this study due to their ability to amplify all subgroups of arbuscular mycorrhizal fungi (AM fungal, Glomeromycota), while excluding sequences from other organisms. Through both morphological characteristics and molecular identification, the following fungal genera were identified for the first time in the studied sites in South Africa. The study identified a total of 14 AM fungal genera and 55 AM fungal species, which are: Glomus (15), Acaulospora (11), Scutellospora (6), Gigaspora (6), Rhizophagus (3), Funneliformis (3), Archaeospora (2), Claroideoglomus (2), Ambispora (2), Sclerocystis (1), Fuscutata (1), Entrophospora (1), Diversispora (1), Paraglomus (1). Both Glomus and Acualospora have been observed to be the highest occurring genera in the analysed soil samples, followed by Scutellospora and Gigaspora and others mentioned. PIXE technique was successful in localising elemental concentration in both plant roots and AM fungal structures, as well as in indicating the large vesicles in root tissue. AM fungal structures in the outer cortex or outer epidermal layer of the root cross-sections were observable, as shown by the more significantly enriched Si in the vesicles and arbuscules. Distinctive elemental maps can be used to localise sites of colonisation and verification of the symbiotic nature of the tissue. This indicates that a range of metals can be sequestered in AM fungal structures above levels in surrounding host root tissue, and demonstrates the potential of Micro-PIXE to determine metal accumulation and elemental distribution in mycorrhizal plant roots and inter-and intracellular AM fungal structures. This research highlights the potential of AM fungi for inoculation of plants as a prerequisite for successful restoration of heavy metal contaminated soils. It also illustrates the importance of AM fungal diversity in selected high heavy metal (HM) sites in RSA, particularly in the North West and the Gauteng gold mining slime dams. Therefore, phytoremediation of mine tailings by mycorrhizal plants seems to be one of the most promising lines of research on mine tailings contamination by heavy metals. The strategies which evolved during this project have great potential for phytoremediation of toxic mining sites, and thus can help mitigate the environmental problems, especially in the mining waste sites. / LG2017

This PhD thesis follows my master's thesis, which I focused on the problem of uranium determination and content in macrofungal fruit-bodies (the results have been published, Appendix 1). Macrofungi apparently do not accumulate uranium in fruit-bodies but as other studies suggested major roles of fungi in environmental geochemistry of uranium, I hypothesized possible accumulation of uranium and other elements in ectomycorrhizae. I therefore decided to continue the research and focus on investigation of trace elements in ectomycorrhizae. As I had opportunity to use a variety of analytical methods, I also participated in other studies in the field geomycology and the results are included in this thesis. In response to alarmist reports in Czech media, I focused on activity and distribution of radiocaesium in fruit-bodies of Boletus badius. As demonstrated in Appendix 2, the fruit- bodies of this species do not represent a health risk for mushroom consumers. Distribution of mycelium of saprotrophic Agaricus bernardii in a soil profile in Prague was investigated by use of molecular methods (PCR with specific primers). The results have shown that the mycelium reaches the depth of 30 cm. Lead isotopic composition of fruit- bodies suggests lead can be accumulated from soil depth of 13-17 cm (Appendix 3)....

O chumbo (Pb) é um metal tóxico e promove diversos efeitos adversos no organismo que incluem distúrbios neurológicos, hematopoiéticos, esqueléticos, renais e cardiovasculares. A análise proteômica têm se tornado cada vez mais uma ferramenta importante na elucidação de mecanismos de toxicidade, bem como na identificação de potenciais biomarcadores. Nesse sentido, o objetivo deste estudo foi comparar diferenças de abundância de proteínas plasmáticas em amostras de sangue coletadas de trabalhadores de uma fábrica de baterias automotivas, e de um grupo sem histórico de exposição ocupacional ao Pb. Pb no sangue total (Pb-S) foi determinado por espectrometria de massas com plasma acoplado indutivamente (ICP-MS). Para a análise proteômica, o plasma foi separado do sangue total, seguido de depleção de albumina e imunoglobulina G (IgG) através de cromatografia de imunoafinidade. Em seguida, foi realizada digestão das proteínas com tripsina e os peptídeos foram analisados no sistema nanoACQUITY UPLC acoplado ao espectrômetro de massas em tandem SYNAPT® G2-Si HDMS. A identificação das proteínas foi realizada através do banco de dados SwissProt e as proteínas identificadas com pelo menos 3 peptídeos foram classificadas de acordo com o banco de dados do Gene Ontology (GO) e ranqueadas através de algoritmos de classificação utilizando a mineração de dados. A quantificação das proteínas foi realizada por label-free e foram consideradas proteínas com diferença de abundância aquelas com fold-change >=1,5 e p>0,05 através do teste t de Student. A média de Pb-S para os grupos expostos ocupacionalmente e sem histórico de exposição ocupacional foi de 43,7?g/dL e 0,386 ?g/dL, respectivamente. No total, 82 proteínas foram identificadas no plasma e apenas 4 proteínas apresentaram fold change >= 1,5, sendo elas: Apolipoproteína B-100 (APOB) e ?-II-antiplasmina (A2AP2), além de plasminogênio (PLMN) e gelsolina (GELS) cuja diferença de abundância entre os grupos foi significativa (p=0,036 e <0,01, respectivamente). As proteínas com diferença de abundância também foram classificadas entre as mais importantes para a diferenciação dos grupos através da mineração de dados. Assim, o presente estudo foi capaz de demonstrar diferenças de abundância em proteínas plasmáticas entre indivíduos com elevadas concentrações de Pb-S e com baixas concentrações de Pb-S, evidenciando possíveis novos mecanismos de toxicidade do metal. / Lead (Pb) is a toxic metal and may cause several adverse effects including neurological, hematopoietic, skeletal, renal and cardiovascular disorders. Proteomic analyses have become an important tool for the discovery of new mechanism of toxicity as well to identify potential new biomarkers of metal exposure, which may help in early detection of toxic effects. This study aimed to evaluate the differences in the plasma proteome of two groups: i) Pb-exposed workers and ii) a group with no occupational lead exposure history. Blood samples were collected for blood lead levels (BLL) determination by inductively coupled plasma mass spectrometry (ICP-MS) and for proteomic analyses in plasma fraction. Plasma samples were separated from whole blood using centrifugation. Antibody-based affinity columns were used to deplete plasma fractions from albumin and immunoglobulin G (IgG). Thereafter, proteins were digest with trypsin solution and peptide analyses were conducted using a nanoACQUITY UPLC System coupled to a SYNAPT® G2-Si tandem mass spectrometer. Identification of proteins were performed against SwissProt database and proteins identified by at least three peptides were classified according to the Gene Ontology terms (GO). In addition, identified proteins were ranked through classification algorithms by data mining analyses. Proteins quantitation was performed by label-free and only proteins with fold-change >=1.5 were considered differentially expressed. Mean BLL were 43.7?g/dL and 0.386 ?g/dL for occupational and non-occupational lead exposed groups, respectively. Eighty-two proteins were identified in the plasma samples out of which only four - plasminogen (PLMN), gelsolin (GELS), ?-II-anti-plasmin (A2AP2) and apolipoprotein B-100 (APOB) showed >=1.5 folds differential abundance. Differential abundances were confirmed for PLMN and GELS proteins (p=0.036 and <0.01, respectively). In conclusion, the present study indicated that the plasma proteome was a function of BLL in the individuals

A produção de lodo de esgoto vem aumentando exponencialmente com a multiplicação das estações de tratamento de esgoto (ETEs) e a sua disposição final tem se tornado um problema sanitário preocupante. Por ser um resíduo rico em matéria orgânica e nutrientes, muitas pesquisas vêm sendo desenvolvidas na busca de alternativas ecologicamente mais adequadas para o seu descarte. Este trabalho teve como objetivo verificar as diferenças entre lodos de esgoto (biossólidos), produzidos em três diferentes Estações de Tratamento de Esgotos da região metropolitana de São Paulo, quando utilizados como adubo em plantações de Eucalyptus grandis. Procurou-se também avaliar a influência deste resíduo sobre a ciclagem de nutrientes e sobre a dinâmica de metais pesados no ecossistema florestal. O estudo foi realizado na Estação Experimental de Ciências Florestais da ESALQ/USP em Itatinga-SP, cujo solo é predominantemente arenoso e de baixa fertilidade natural. O delineamento experimental utilizado foi em blocos aleatorizados, com 4 repetições e cinco tratamentos, sendo eles: 1)Testemunha Absoluta (TA), sem nenhuma adubação; 2) Fertilização mineral convencional (FM); 3) Aplicação de 15 Mg ha-1 de lodo de esgoto da ETE de Barueri (LB); 4) 15 Mg ha-1 de lodo de esgoto da ETE de São Miguel (LSM); 5) 15 Mg ha-1 de lodo de esgoto da ETE do Parque Novo Mundo (LPNM). Todos os tratamentos com lodo de esgoto foram complementados com potássio mineral (KCl), visto que o lodo é deficiente neste nutriente. O LB e o LSM foram condicionados com polieletrólitos, mas o LPNM foi condicionado com cal hidratada e cloreto férrico. O efeito dos diferentes tratamentos sobre os eucaliptos foi avaliado através de: inventários florestais semestrais, nutrição foliar, estimativa do folhedo produzido mensalmente, estimativa semestral de folhedo acumulado sobre o solo, avaliação da decomposição do folhedo no campo através de bolsas decompositoras, reflexo dos lodos sobre a fertilidade do solo, estimativa da biomassa e do estoque de nutrientes e metais pesados nos componentes do tronco (Lenho+Casca), nas folhas e nas raízes finas dos eucaliptos. Observou-se que a adubação dos eucaliptos com os lodos propiciou, em média, um incremento do volume de madeira da ordem de 65%, superior à testemunha (sem adubação) e similar ao observado no tratamento com fertilização mineral. A concentração dos nutrientes nas folhas dos eucaliptos tratados com os lodos, aos 6 meses de idade, apresentou uma elevação significativa dos nutrientes N, P, Ca, Cu, Fe e Zn em relação à testemunha. Mas, aos 42 meses, observou-se a tendência geral de redução da concentração de todos os nutrientes nas folhas, independente dos tratamentos. A adição dos lodos estimulou em 75% a produção de folhedo pelos eucaliptos, bem como um maior acúmulo de folhedo sobre o piso florestal. Conseqüentemente, houve uma maior transferência de nutrientes, via folhedo, das copas dos eucaliptos para a camada de serapilheira. De maneira geral, a aplicação da FM e dos lodos de esgoto melhorou a fertilidade do solo na linha de plantio (ponto de aplicação), principalmente na camada entre 0 e 5 cm de profundidade. Todavia, poucas mudanças foram observadas na produção de raízes finas em função dos diferentes tratamentos. O lodo de esgoto, quando condicionado na ETE com cal hidratada, tende a elevar o estoque de cálcio em todos os componentes do sistema solo-planta-serapilheira, podendo a longo prazo causar desbalanço nutricional e impactos prejudiciais ao ecossistema. Nos tratamentos com lodo de esgoto, foi observada uma maior concentração de metais pesados, em comparação à testemunha, nas folhas: Cu, Zn e Ni; no folhedo: Cu e Zn; no solo: Cu e Zn (na linha de plantio) e Zn (na entrelinha de plantio), apenas na camada de 0-5 cm de profundidade e nas raízes finas: Zn e Ni. É imprescindível, portanto, se conhecer previamente a concentração dos diferentes elementos no lodo de esgoto, antes de sua aplicação em plantios florestais. O monitoramento nutricional das árvores e da ciclagem dos nutrientes são práticas de fundamental importância para o bom manejo deste resíduo e do empreendimento florestal. / The production of sewage sludge increased exponentially with the enlargement of sewage treatment plants (ETEs, by its acronym in Portuguese). The final disposal of this product has always been a sanitary problem. Since it is a residue rich in organic matter and nutrients, several researches have been carried out, looking for adequate ecological alternatives for its disposal. This study aims to verify the differences among the sewage sludge (biosolids) from three different sewage treatment plants in the metropolitan region of Sao Paulo when used as fertilizers in Eucalyptus grandis plantations. Moreover it seeks to assess the influence of this residue in the nutrients cycling and in the heavy metals dynamic in forestry ecosystems. This research was carried out in the Experimental Station of Forestry Sciences of ESALQ/USP in Itatinga, Sao Paulo, where the soil is mainly sandy and of low natural fertility. The experimental delineation was of randomized blocks, with 4 replications and 5 treatments: 1) control (TA) without fertilization; 2) mineral conventional fertilization (FM); 3) application of 15 Mg ha-1 of sewage sludge from Barueri ETE (LB); 4) 15 Mg ha-1 of sewage sludge from Sao Miguel ETE (LSM); 5) 15 Mg ha-1 of sewage sludge from Parque Novo Mundo ETE (LPNM). Since the sewage sludge lacks mineral potassium (KCl), it was added to all the treatments. The LB and the LSM were conditioned with polyelectrolyte, but the LPNM was conditioned with CaOH and with FeCl3. The sewage sludge was applied in the planting rows. The effect of the different treatments on the eucalyptus trees was assessed by: six-monthly forestry inventories, leafs nutrition, estimation of monthly production of leaf fall, six-monthly estimation of leaf fall stocked in the soil, assessment of the decomposition of leaf fall in the field with decomposition bags, reflex of the sludge over the soil fertility, estimation of the biomass and the stock of nutrients and heavy metals in the trunk (stem wood + bark), in the leaves and in the fine roots of the eucalyptus trees. It was observed that the fertilization with sludge favored in average the increase of the wood volume around 65%, higher that the control (without fertilization) and similar to what observed in the treatment with mineral fertilization. The concentration of N, P, Ca, Cu, Fe and Zn in the leaves of the eucalyptus treated with the sludge, when six months old, increased significantly compared with the control. When the trees, of all the treatments were 42 months old, there was a decrease in the concentration of all nutrients in the leaves. The addition of sludge stimulated in 75% the production of leaf fall, as well as its high accumulation in the forestry soil. Consequently, the nutrients transfer was high, by the leaf fall, from the top of the trees to the litter. In general, the application of FM and of sewage sludge improved the soil fertility in the inter row (application place), mainly in the depth 0-5 cm. However, few changes were observed in the fine roots production regarding the different treatments. The sewage sludge, when conditioned in the ETE with CaOH, tends to increase the calcium stock in all the components of the system soil-plantlitter, what in long term may cause a nutritional imbalance and harmful impacts to the ecosystem. In the treatments with sewage sludge, a higher concentration of heavy metals in the leaves was observed in comparison with the control: Cu, Zn and Ni; in the leaf fall: Cu and Zn; in the soil: Cu and Zn (in the planting rows) and Zn (between-tree rows), only in the depth 0-5cm and in the fine roots: Zn and Ni. Therefore, before the application of sewage sludge in forestry plantations it is essential to know the concentration of the different elements present in it. The nutritionalmonitoring of the trees and the nutrients cycling are fundamental practices of essential importance for the good management of this residue and for the forestry farmer

A aplicação do lodo de esgoto em sistemas florestais busca o aprimoramento da produção, favorecendo as características físicas, químicas e biológicas do solo e das plantas. Entretanto, o lodo pode conter patógenos e, elementos orgânicos e inorgânicos tóxicos, e o efeito destes contaminantes no solo e na planta ainda é pouco conhecido. Sendo assim, foi estabelecida a hipótese de que o lodo de esgoto, quando aplicado ao solo, com base na norma P 4.230, da CETESB, não causa efeitos tóxicos ao sistema solo-eucalipto. Com o objetivo de testar a hipótese acima, foi proposta a determinação da concentração dos elementos potencialmente tóxicos, a avaliação da toxicidade e genotoxicidade do solo tratado com lodo de esgoto e a verificação da correlação entre essas variáveis. O experimento de campo foi instalado em dezembro de 2004, quando mudas de Eucalyptus grandis foram plantadas e tratadas com lodo de esgoto, nas doses de 0; 7,7; 15,4 e 23,1 tha-1, base seca, em área comercial de cultivo de eucalipto da companhia Suzano Bahia Sul Papel e Celulose S.A., em Itatinga, SP. Amostras de solo foram coletadas aos 18, 21, 24, 27 e 34 meses após a aplicação do lodo. Foram determinadas as concentrações de Cr, Mn, Fe, Ni, Cu, Zn, Cd e Pb no solo, extraídos por DTPA, Mehlich-1 e com HNO3+HCl, e quantificados por ICP-MS. A toxicidade do solo foi avaliada por meio dos testes com Daphnia magna (concentração letal-CL50), com Pseudokcrichirella subcaptata (inibição de crescimento-CI50), com Lactuca sativa (crescimento de raiz) e com Allium cepa (índice mitótico, aberrações cromossômicas e micronúcleos). Os resultados mostraram um aumento nos teores de Cr, Mn, Fe, Ni, Cu, Zn, Cd e Pb no solo de acordo com as doses. Foi observado um aumento da toxicidade para D.magna, P.subcaptata e genotoxicidade e mutagenicidade para A. cepa de acordo com as doses. O lodo de esgoto aplicado ao solo na dose de 15,1 t ha-1, recomendada pela norma da CETESB, não causa efeito tóxico, genotóxico ou mutagênico no sistema solo-planta, de acordo com os testes realizados. Há correlação entre os testes de toxicidade com P. subcaptata, de mutagênicidade com A. cepa (micronúcleos), com os teores dos elementos potencialmente tóxicos no solo. O teste com D. magna constitui uma importante ferramenta na avaliação toxicológica do lodo aplicado ao solo, apesar de não se correlacionar com os teores de elementos potencialmente tóxicos no solo, indicando que a toxicidade do lodo pode estar relacionada também a outros compostos orgânicos tóxicos e/ou agentes patógenos.O teste de toxicidade com L.sativa é inadequado na avaliação toxicológica da aplicação de lodo em relação as doses, por refletir efeitos dos nutrientes e não dos componentes tóxicos do lodo, porém reflete a toxicidade de acordo com o tempo de aplicação do resíduo / The application of sewage sludge in forest systems quests the improvement of the production, favoring the physical, chemistries and biological characteristics of the soil and of the plants. However, the sludge can contain pathogen and, organic and inorganic toxics elements, and the effect of these pollutants in the soil and in the plant is still unknown. It was established the hypothesis that the sewage sludge, when applied to the soil, according to the norm P4.230 of CETESB, doesn\'t cause toxics effects to the soil-eucalyptus system. To test the hypothesis above, it was established as objective, the determination of the concentration of the potentially toxics elements, the evaluation of the toxicity and genotoxicity of the sewage sludge treated soil, and the verification of the correlation among those variables. The field experiment was conducted in December of 2004, when seedlings of Eucalyptus grandis were planted and treated with sewage sludge, in the doses of 0; 7,7; 15,4 and 23,1 tha 1, dry base, in commercial area of eucalyptus cultivation of the Suzano Bahia Sul Papel and Celulose S.A company, in Itatinga, SP. Soil samples were collected to the 18, 21, 24, 27 and 34 months after the application of the sludge. They were established the concentrations of Cr, Mn, Fe, Ni, Cu, Zn, Cd and Pb in the soil, extracted for DTPA, Mehlich-1 and with concentrated HNO3+HCl, and quantified by ICP-MS. The toxicity of the soil was evaluated through the tests with D. magna (lethal concentration -LC50), with Pseudokcrichirella subcaptata (growth inhibition - IC50), with Lactuca sativa (root growth) and with Allium cepa (mitotic index, cromossomic aberration and micronucleus). The results showed an increase in the concentrations of Cr, Mn, Fe, Ni, Cu, Zn, Cd and Pb in the soil in agreement with the doses. An increase of the toxicity was observed for D. magna, P. subcaptata and an increase of the genotoxicity and mutagenicity for A. cepa in agreement with the doses. The applied sewage sludge to the soil in the dose of 15,1t ha-1, recommended by CETESB, doesn\'t cause toxic, genotoxic or mutagenic effect in the system soilplant, in agreement with the accomplished tests. There is correlation among the toxicity tests with P. subcaptata, of mutagenicity with A. cepa (micronucleus) with the concentrations of Cr, Mn, Fe, Ni, Cu, Zn, Cd and Pb in the soil. The test with D. magna is an important tool in the toxicological evaluation of the sludge applied to the soil, however it wasnt correlated with the concentration of potentially toxics elements in the soil, indicating that the toxicity of the sludge can also be related to the other toxic organic compound or pathogen agents. The L.sativa toxicity test is inadequate in the toxicological evaluation of the sludge application, according to the doses, for reflecting effects of the nutrients and it doesnt reflect the toxic components of the sludge, however it reflects the toxicity in agreement with the time of residue application

O feijoeiro comum (Phaseolus vulgaris L.) e caupi [Vigna ungüiculata (L.) Walp] são alimentos básicos em muitos países, principalmente no Brasil, sendo uma importante fonte de proteínas, vitaminas e minerais, incluindo ferro e zinco. O cádmio (Cd) é considerado o metal pesado mais preocupante à saúde humana. Não há estudos sobre a variabilidade genotípica quanto a concentração de Cd em diferentes feijoeiros especialmente quando submetidos próximos a concentração limítrofe para intervenção pela CETESB (3 mg kg-1). O objetivo foi determinar e comparar a concentração de Cd em grãos de diversos cultivares de feijoeiros comuns Phaseolus vulgaris L. e vigna Vigna ungüiculata L. (Walp.) em vasos de 2,5 dm-3 de terra oriunda de um latossolo típico, bem como estudar os efeitos e o acúmulo do elemento em plantas com comportamentos distintos. Os experimentos foram delineados inteiramente ao acaso. No primeiro experimento, sementes de cada espécie (15 variedades de feijoeiro comum e 5 variedades de feijoeiro vigna) foram plantadas em potes sob dois tratamentos: contaminado (3 mg dm-3 Cd) e testemunha, em três repetições. O índice SPAD foi medido nas plantas ao final do estádio vegetativo e a avaliação da produção de matéria seca da parte aérea no final do ciclo. As concentrações de Cd em grãos foram determinadas por ICP-MS. Após a seleção dos cultivares com maior e menor acumulação de Cd no grão, este foram replantados em um experimento com 3 tratamentos: 3 mg dm-3 Cd + 109Cd (solo); 109Cd (solo); 3mg dm-3 Cd (solo) + 109Cd (folha) e cinco repetições. Foram avaliadas a medição da taxa de assimilação de CO2 in vivo e amostras do limbo foliar foram coletadas para visualização através da técnica de microscopia eletrônica de transmissão. A parte aérea de feijoeiros radiomarcados foram expostas a filmes radiográficos e a contagem referente à atividade do 109Cd foi determinada por espectrometria de cintilação líquida. Resultados: O índice SPAD foi diminuído na maioria dos feijoeiros testados, principalmente nas folhas jovens. A menor e maior concentração de Cd foi observada nos cultivares BR3 Tracuateua (0,6 mg kg-1) and BRS Timbó (2,3 mg kg-1) respectivamente. Em ambos a assimilação de CO2 foi alterada pela toxicidade de Cd, mas com a presença de deformidades do limbo, clorose e abscisão da folha no BRS Timbó. Os cloroplastos do feijoeiro comum contaminado mostraram abundância de corpos osmiofílicos quando comparado ao vigna. A auto-radiografia confirmou menor transporte de Cd da raiz para a parte aérea pelo feijoeiro vigna e as concentrações deste metal na parte aérea são maiores no caule e nervuras foliares. Comparado ao BR3 Tracuateua, o feijoeiro comum BRS Timbó absorve e transloca maior quantidade de Cd aplicado na folha para o grão, mas contribui muito pouco na concentração total de Cd no feijão / The common bean (Phaseolus vulgaris L.) and cowpea [Vigna ungüiculata (L.) Walp] are a staple foods in many countries, particularly in Brazil, and it is an important source of protein, vitamins, and minerals including iron and zinc. Cadmium (Cd) is a heavy metal considered one of the most hazardous elements for humans. No data are available on the cadmium (Cd) concentration in grain of these species, especially under conditions of CETESB critical limits in soil (3 mg kg-1). The objective were to determine and compare concentrations of Cd in grain of common beans Phaseolus vulgaris L. and cowpea Vigna ungüiculata L. (Walp.) cultivated in 3 L pots with a typical oxysol, as well as to study the effects and accumulation of Cd in plants with distinct comportment. The trials were arranged as randomized design. In the first experiment, seeds of each species (15 common beans and 5 cowpea cultivars) were sowed in pots (3 mg kg-1 Cd and control), replicated three times. The SPAD índex was measured in plants at the end of vegetative stage and the dry matter yield were evaluated. Cd concentration in grain was determined by ICP-MS. After the selection of major and minor Cd accumulators, these cultivars were sowed in a new trial with 3 treatments 3 mg dm-3 Cd + 109Cd (soil); 109Cd (soil); 3 mg dm-3 Cd (soil) + 109Cd (leaves) with five replications. The CO2 assimilation was measured in vivo. Samples of leaves were collected and the ultrastructure was observed in transmission electronic microscope. The aerial parts of beans plants were exposed to radiographic films and the activity of 109Cd in was determined by liquid scintillation spectrometry. Results: SPAD index were affected in most of plants tested, especially in Young leaves. The minor and major concentration of Cd were observed in BRS Timbó (2,3 mg kg-1) and BR3 Tracuateua (0,6 mg kg-1), respectively. In both cultivars, the CO2 assimilation was affected by Cd phytotoxicity, but there were leaves deformities, chlorosis and leaf abscision in BRS Timbó. Common beans chloroplasts showed more osmiophylics globules compared to cowpea. Autoradiography confirmed low translocation root-to-shoot in caupi BR3 Tracuateua and the concentration of Cd was higher in BR3 Timbó, especially in stem and veins of leaves. In comparison to cowpea BR3 Tracuateua, the common bean BRS Timbó absorb and translocate more Cd applied in leaves to grain, but it is inexpressive in total content of Cd in beans

A disposição de resíduos sólidos e a aplicação de pesticidas e fertilizantes podem levar ao aumento da concentração de metais pesados em solos e águas subterrâneas. A legislação sobre metais pesados quase sempre se refere aos seus teores totais, mas a avaliação do potencial de risco e da toxicidade requer a avaliação da proporção de metal que é móvel e, possivelmente, biodisponível. Alguns parâmetros numéricos têm sido utilizados para tomadas de decisões e para direcionar estratégias de prevenção ou de remediação de áreas contaminadas. O coeficiente de distribuição sólido-solução (Kd), definido como a relação entre as concentrações adsorvida e em solução, permite a comparação do comportamento de elementos em diferentes sistemas. Agências de proteção ambiental utilizam valores genéricos de Kd encontrados em referências bibliográficas que, por terem sido gerados a partir de condições distintas das da região tropical, podem levar a incorretas estimativas de risco. É evidente a necessidade da obtenção de valores de Kd que validem estas estimativas de contaminação ou que norteiem estratégias de intervenção em áreas já contaminadas. Os objetivos deste trabalho foram avaliar a retenção de metais pesados em 30 solos representativos do Estado de São Paulo, a partir da quantificação dos valores do coeficiente de distribuição (Kd) e da relação com atributos dos solos, tais como pH, CTC e teores de argila, matéria orgânica e de diversas formas de óxidos de Fe, Al e de Mn. Os coeficientes de distribuição foram obtidos de isotermas lineares de adsorção, construídas após a adição de 0,1; 0,5; 1,0; 2,5 e 5,0 mg L-1 de Cd, Co, Cr, Cu, Ni, Pb e Zn, em experimentos tipo batch. As menores variações nos valores de Kd foram registradas para o Pb (uma ordem de magnitude - 121 a 7.020 L kg-1) e para o Ni (duas ordens de magnitude - 6 a 998 L kg-1), enquanto variações de quatro ordens de magnitude foram observadas para Cd (7-14.339 L kg-1), Co (2-34.473 L kg-1) e para Cr (1-21.267 L kg-1). Coeficientes de distribuição de zinco estiveram entre 5 a 123.849 L kg-1, variando cinco ordens de magnitude. Os valores foram similares àqueles que vêm sendo utilizados pela CETESB, exceto os de Cr e Ni. Obteve-se a seguinte ordem de afinidade: Pb>>>Cu>>Cd>Zn&#8773;Ni&#8773;Cr>Co. Mais de 55% da variação dos coeficientes de distribuição de cátions metálicos dos grupos IIB (Cd e Zn) e VIIIB (Co e Ni) foram explicados pelo pH. Na análise conjunta, CTC e pH explicaram cerca de 80% da variação dos valores de Kd de Cd, Co e de Ni e evidenciaram que mecanismos de adsorção não-específica estão envolvidos na retenção destes elementos. No caso dos coeficientes de distribuição de Cu e de Pb, o pH e os teores de argila responderam por cerca de 63% da sua variação. Os valores de Kd de Cr apresentaram correlação inversa com o pH, principalmente na análise conjunta com os teores de argila, e 61% da sua variação foi explicada por estas variáveis. / Disposal of solid residues and application of pesticides and fertilizers can lead to an increase in the concentration of heavy metals in soils and groundwater. The legislation on heavy metals nearly always refers to the total contents of these elements; however, an evaluation of their hazard potential and toxicity requires an assessment of the metal fraction that is mobile and possibly bioavailable. A few numerical parameters have been used as a foundation to make decisions and to focus on prevention or remediation strategies in contaminated areas. The solid-solution distribution coefficient (Kd), defined as the relation between metal concentrations that are adsorbed and those present in the solution, allows a comparison between the behavior of elements in different systems. Environmental protection agencies use generic Kd values found in bibliographic references; these values are often obtained under distinct conditions from those found in the tropical region, and may lead to an erroneous hazard estimates. Thus, it becomes clearly necessary to obtain Kd values that will validate these contamination estimates or provide guidance for intervention strategies in already contaminated areas. The objectives of this work were to evaluate heavy metal retention in 30 representative soils of the State of São Paulo, based on a quantification of distribution coefficient values (Kd) and their relation with soil attributes, such as pH, CEC and clay contents, organic matter, and various forms of Fe, Al, and Mn oxides. The distribution coefficients were obtained from linear adsorption isotherms, constructed after the addition of 0.1; 0.5; 1.0; 2.5; and 5.0 mg L-1 Cd, Co, Cr, Cu, Ni, Pb, and Zn, in batch-type experiments. The smallest variations in Kd value were recorded for Pb (one order of magnitude - 121 to 7,020 L kg-1) and Ni (two orders of magnitude - 6 to 998 L kg-1), while variations indicating four orders of magnitude were observed for Cd (7-14,339 L kg-1), Co (2 - 34,473 L kg-1), and Cr (1 - 21,267 L kg-1). Zinc distribution coefficients were between 5 and 123,849 L kg-1, with a variation of five orders of magnitude. The values herein obtained were similar to those that have been used by CETESB, except for Cr and Ni. The following order of affinity was obtained: Pb>>>Cu>>Cd>Zn&#8773;Ni&#8773;Cr>Co. More than 55% of the variation in distribution coefficients for metallic cations of the groups IIB (Cd and Zn) and VIIIB (Co and Ni) were explained by pH. In the joint analysis, CEC and pH explained about 80% of the variation in Kd values for Cd, Co, and Ni, and showed that nonspecific adsorption mechanisms are involved in the retention of these elements. Regarding the distribution coefficients for Cu and Pb, pH and clay content accounted for about 63% of the variation. Kd values for Cr showed an inverse correlation with pH, especially in the joint analysis with clay contents, and 61% of the variation were explained by these variables.

Os xenóbioticos são potenciais contaminantes dos recursos hídricos, o que interfere no equilíbrio ecológico e na saúde humana. Uma potencial solução para tais problemas ambientais é a introdução da técnica de biorremediação. Para tanto, é necessário investigar genes da comunidade microbiana com potencial genético para degradação e resistência desses contaminantes e, assim, desenvolver biotecnologias para a descontaminação de ambientes poluídos. O presente estudo teve como objetivo extrair o DNA da microbiota total, da microbiota cultivável e dos isolados bacterianos provenientes de amostras de água superficial de mananciais e fontes do estado de São Paulo para investigar e padronizar reações de Multiplex PCR para os genes de degradação e resistência aos diferentes xenobióticos alvo do presente projeto de pesquisa. Os genes pesquisados foram amplamente encontrados no DNA da fração total, sendo os genes alkB, alk e merA os mais prevalentes na fração cultivável, indicando que micro-organismos portadores desses genes podem ser isolados para uso em processos de biorremediação. Os genes puhA e puhB foram detectados em menor frequência, tanto na fração total, quanto na cultivável, demonstrando pouca dispersão desses genes nas amostras estudadas. Os gêneros bacterianos portadores de genes de degradação isolados em maior frequência nas amostras de água analisadas foram Bacillus e Aeromonas e os genes mais frequentes nesses isolados foram o atzA, o alk e o merA. Para otimizar a detecção dos genes envolvidos na biodegradação da atrazina, dos hidrocarbonetos aromáticos policíclicos, alcanos e resistência ao cobre, duas reações de Multiplex PCR foram padronizadas / Xenobiotics are potential contaminants of water resources, which interfere in the ecological balance and human health. A potential solution to such environmental problems is the introduction of the bioremediation. Therefore, it is necessary to investigate genes from the microbial community with degrading and resistance genetic potential to these contaminants and, thus, to develop biotechnologies for the decontamination of polluted environments. The present study aimed to extract DNA from the total microbiota, cultivable microbiota and bacterial isolates from 19 surface water samples from sources of the São Paulo State to investigate and standardize Multiplex PCR reactions for degradation and resistance genes to the different xenobiotics target of the present research project. The studied genes were widely found in the DNA from the total fraction, being the alkB, alk and merA genes the most prevalent in the cultivable fraction, indicating that microorganisms carrying these genes can be isolated for use in bioremediation processes. The puhA and puhB genes were detected at lower frequency, both in the total and in the cultivable microbiota, showing little dispersion of these genes in the studied samples. Bacillus and Aeromonas were the most frequent bacterial genera with degradation genes isolated in the analyzed water samples, and the most frequent genes in these isolates were atzA, alk and merA. To optimize the detection of the genes involved in the biodegradation of atrazine, polycyclic aromatic hydrocarbons, alkanes and copper resistance, two reactions of Multiplex PCR were standardized.

Grandes extensões de áreas vêm sendo afetadas por metais pesados em concentrações que podem representar perigo ambiental. Em decorrência do aumento da atividade industrial e à agricultura altamente tecnificada, torna-se fundamental o monitoramento dos níveis de metais pesados nos solos, pois as plantas se comportam como mecanismo de transferência de contaminantes do solo para níveis mais altos da cadeia trófica. A ingestão de vegetais contendo elevadas concentrações de metais pesados é uma das principais vias de acesso desses elementos ao organismo. No corpo humano, os metais pesados depositam-se no tecido ósseo e gorduroso, causando possíveis efeitos nocivos à saúde humana. Lentamente liberados no organismo, podem provocar uma série de doenças. O projeto foi dividido em duas etapas, cada qual com um objetivo específico: 1ª determinar as concentrações de zinco, cobre, ferro e manganês em vegetais frequentemente consumidos no Estado de São Paulo e compará-las com os limites estabelecidos na legislação brasileira; calcular a ingestão diária de metais pesados essenciais para estimar o risco à saúde humana; 2ª avaliar os teores de zinco em hortaliças e nos solos dos campos de produção; identificar a cultura que apresentou maior potencial de translocação de zinco para a cadeia alimentar. As amostras vegetais foram coletadas na Companhia de Entrepostos e Armazéns Gerais de São Paulo e a concentração de metais pesados foi determinada por espectrometria de absorção atômica. Todos os vegetais analisados apresentaram concentrações de zinco e cobre muito abaixo do limite tolerável estabelecido pela legislação brasileira. Até o momento, não há nenhuma recomendação estabelecida de limite máximo de tolerância para ferro e manganês no Brasil. Quanto ao hábito de consumo da população do Estado de São Paulo, a ingestão diária de metais pesados está abaixo da máxima permitida, consequentemente o consumo desses alimentos pode ser considerado seguro, pois não estão em excesso. Porém, os valores reportados nesse estudo não atingiram a ingestão dietética recomendada (RDA) para homens e mulheres, o que poderia demonstrar um caso de deficiência nutricional nos indivíduos que fazem uso desse tipo de dieta. Algumas áreas agrícolas do Estado de São Paulo apresentaram teores totais zinco nos solos acima do valor de referência de qualidade, porém nenhuma área estudada atingiu os valores de prevenção e intervenção, estabelecidos pela CETESB. As hortaliças cultivadas nesses locais apresentaram teores de zinco inferiores ao limite máximo de tolerância, estabelecido pela ANVISA. A rúcula foi a hortaliça que mais acumulou zinco na parte comestível / Large areas have been affected by heavy metals in concentrations which may pose environmental hazard. Due to increased industrial activity and highly technical agriculture, it becomes critical to monitor the levels of heavy metals in soil, because plants behave as a mechanism for transfer of contaminants from soil to higher levels of the food chain. Ingestion of vegetables containing high concentrations of heavy metals is one of the main ways in which these elements enter the human body. Once in human body, heavy metals are deposited in the bone and fat tissues, causing potential harmful effects to human health. Slowly released into the body, heavy metals may cause an array of diseases. The project was divided in two stages, each one with a specific objective: 1st) to determine the concentrations of zinc, copper, iron and manganese in the most frequently consumed foodstuff in the São Paulo State, Brazil and to compare the heavy metal contents with the permissible limits established by the Brazilian legislation; to calculate the daily intake of heavy metals essential to estimate the risk to human health; 2nd) to evaluate the zinc content in leafy vegetables and soils at crop fields; to identify crop with the greatest potential for translocation of zinc into food chain. The vegetable samples were collected at São Paulo General Warehousing and Centers Company, and heavy metal content was determined by atomic absorption spectrophotometry. All vegetables sampled presented average concentrations of zinc and copper lower than the permissible limits established by the Brazilian legislation. So far, the Brazilian legislation has not established a permissible limit for iron and manganese contents. Regarding the consumption habit of the population in the São Paulo State, the daily ingestion of heavy metals is below the oral dose of reference, therefore, the consumption of these vegetables can be considered safe, because they are not in excess. However, the values reported in this study did not reach the Recommended Dietary Allowances (RDA) for men and women, which could prove a case of nutritional deficiency in individuals who use this type of diet. Some agricultural areas of the São Paulo State showed soil total Zn concentrations above the benchmark of quality, but none of studied area reached prevention and intervention values, established by CETESB. The vegetables grown in these locations had Zn levels below the tolerance limits established by ANVISA. The rocket plant was the vegetables that more Zn accumulated in the edible part