As agressões ao meio ambiente, num contexto de esgotamento de recursos naturais, vêm recebendo crescente importância aos olhos da sociedade e, nesse cenário, o meio ambiente passou a ser protegido pelo Direito Penal. Assim, muitas destas agressões, como a poluição, passaram a ser qualificadas como crimes ambientais tornando se necessária a produção de prova técnica para o seu devido julgamento. Este trabalho apresenta uma nova estratégia para caracterização de crimes ambientais de poluição e correlatos baseado em abordagem multiparamétrica. Para isso foram utilizadas análises de diferentes parâmetros como metais, razões de isótopos estáveis e compostos orgânicos (hidrocarbonetos aromáticos policíclicos), e análise estatística multivariada, com o intuito de obter uma assinatura química robusta dos poluentes da fonte suspeita e assim estabelecer correspondência com os mesmos parâmetros determinados no compartimento ambiental receptor. Ainda, foram incorporados alguns conceitos de metrologia, como o cálculo de incerteza de amostragem, conforme preceituam as novas tendências de desenvolvimento conceitual e metodológico das ciências forenses. Os sedimentos de um curso dágua altamente impactado por descargas diversas foram o objeto das investigações como sendo o compartimento receptor e o percolado de um aterro de resíduos industriais perigosos envolvido em um crime ambiental foi analisado como possível fonte. A abordagem multiparamétrica utilizada neste trabalho proporcionou uma melhor discriminação dos pontos de coleta com base na sua localização com relação às fontes de poluição por meio da Análise de Componentes Principais e as análises de metais realizadas nos sedimentos permitiram caracterizar um crime de poluição ambiental. As estimativas de incerteza de amostragem evidenciaram variações nos resultados principalmente decorrentes da heterogeneidade da distribuição dos contaminantes no meio o que implica que as incertezas devem, preferencialmente, ser estimadas e reportadas nas medições no âmbito forense para um efetivo apoio às tomadas de decisões nelas baseadas. / The aggressions to the environment in a context of depletion of natural resources have been receiving increasing attention in the eyes of society; therefore, the environment became protected by criminal law. For this reason, many assaults, such as pollution, were classified as environmental crimes, generating the need for scientific evidence to be used in the due judgment of these crimes. This paper presents a new method for characterization of environmental pollution and related crimes based on a multiparameter approach. This approach employs the analysis of different parameters such as metals, stable isotope ratio and organic compounds such as polycyclic aromatic hydrocarbons, and statistical techniques of multivariate analysis in order to obtain a robust chemical signature of the suspected source pollutants and then establish correspondence with the same parameters determined in the affected environmental compartment. In addition, some concepts of metrology were incorporated, such as uncertainty of sampling, as demanded by the conceptual and methodological trends in the development of forensic sciences. Watercourse sediments highly impacted by various discharges were investigated as the pollution sink and the leachate from a landfill of hazardous industrial waste involved in an environmental crime was analyzed as a possible source. The multiparameter approach used in this study provided a better discrimination of sampling points based on their location with respect to sources of pollution with the principal components analysis. The metal analysis in sediments allowed the characterization of a pollution crime in the studied stream. The estimates of uncertainty of sampling showed large variations in the results mainly arising from the heterogeneity of the distribution of contaminants in the environment. Hence, the uncertainties should preferably be estimated and reported in the forensic measurements to provide an effective support to decision making.

O aumento na poluição ambiental é na atualidade uma das grandes preocupações em nível mundial. Especificamente, a poluição atmosférica por material particulado tem demostrado ser um fator determinante no desenvolvimento de doenças cardiopulmonares e câncer de pulmão nas populações expostas. O material particulado é constituído por uma mistura complexa de compostos orgânicos e inorgânicos, muitos dos quais possuem potencial mutagênico e genotóxico. As características destes compostos variam em função da suas propriedades físicas, químicas e em razão das condições meteorológicas prevalecentes. A maior parte dos estudos tem se focado em avaliar o potencial genotóxico da fração orgânica do material particulado e poucos estudos têm explorado a fração solúvel em água e a contribuição diferencial das diversas espécies químicas presentes nesta fração para o dano genotóxico. O objetivo deste trabalho foi avaliar os efeitos mutagênicos e genotóxicos in vitro da fração orgânica e da fração solúvel em água de material particulado (MP10) coletado em três locais diferentes do estado de São Paulo e estabelecer a relação entre a composição química e o efeito biológico observado. Para isto, realizou­-se a extração orgânica e solúvel em água de 12 amostras de MP10. A mutagenicidade e genotoxicidade foram avaliadas usando o ensaio de Salmonella/microssoma e o teste de micronúcleos (MN) em células A549 e 16 hidrocarbonetos Policíclicos Aromáticos (HPAs) e 15 metais hidrossolúveis presentes nas amostras foram determinados quimicamente. Adicionalmente, foi determinada a metodologia de extração da fracção solúvel em água e se avaliou a estabilidade química e biológica desta fração. Os resultados indicam que a extração assistida por micro-ondas é um método eficiente para a extração da fração solúvel em agua do MP e que um tempo superior a 60 dias de armazenamento e congelamento deste tipo de extrato tem um efeito significativo sobre os resultados analíticos e a resposta biológica. Foi demonstrado ainda que as duas frações de MP estudadas são responsáveis pela indução do dano ao DNA e que não existe uma relação direta entre a concentração de MP e o efeito genotóxico observado, confirmando a importância do uso de bioensaios na avaliação da genotoxicidade de misturas complexas como o MP. Os HPAs prevalecentes nas amostras de PM10 foram fluorantene e benzo(ghi)perileno. Nos extratos solúveis em água, as maiores concentrações de metais foram determinadas para zinco, ferro e cobre. Confirmou-se que a indução de MN e o ensaio de Salmonella/microssoma representam uma poderosa ferramenta na avaliação da polução atmosférica e que as análises químicas por si só não são suficientes para a proteção e predição dos efeitos biológicos em populações expostas. / The increase of environmental pollution is today one a major concern worldwide. Specifically, air pollution by particulate matter has been shown to be a determining factor in the development of cardiopulmonary disease and lung cancer in exposed populations. The particulate material consists of a complex mixture of organic and inorganic compounds, many of which have mutagenic and genotoxic activity. The characteristics of these compounds vary according to their physical and chemical properties and also to the prevailing weather conditions. Most studies have focused on evaluating the genotoxic potential of the organic fraction of particulate material, but few studies have explored the water­-soluble fraction, and the differential contributions of different chemical species present in this fraction to genotoxic damage. The aim of this study was to evaluate the in vitro mutagenic and genotoxic effects of organic and water-soluble fractions of 12 samples of particulate matter (PM10) collected at three different sites in the state of São Paulo and establish the relationship between the chemical composition and the biological effect observed. The mutagenicity and genotoxicity were evaluated using the Salmonella/microssome test and the micronucleus assay (MN) in A549 cells and 16 Polycyclic Aromatic Hydrocarbons (PAHs) and 15 water-soluble metals present in the samples were chemically determined. Additionally, the extraction method of water- soluble fraction was determined and the chemical and biological stability of this fraction evaluated. The results indicate that the microwave­-assisted extraction is an efficient method for the extraction of the water-soluble compounds of PM and that the freezing and storage of the extract over 60 days has a significant effect on the mutagenic and analytical results of PM samples. It was demonstrated that the two PM fractions studied are responsible for the induction of DNA damage and that there is no direct relationship between the MP concentration and the genotoxic effect observed, confirming the importance of using bioassays in the genotoxicity evaluation of complex mixtures as PM. The PAHs prevailing in our samples were fluoranthene and benzo(ghi)perylene. In the water-soluble extracts, highest concentrations of the elements studied were found for zinc, iron, and copper in the three places of sampling. We confirmed that MN induction and Salmonella/microsome assay represents a powerful tool to evaluate the atmospheric air pollution and that the total concentration of PM and the chemical analyses alone would not be sufficient for the prognosis of biological effects in exposed populations

Para determinar o histórico deposicional e as fontes dos hidrocarbonetos policíclicos aromáticos (HPAs) na região amazônica nos últimos 120 anos, três testemunhos foram coletados na Baía do Guajará - PA. Esses testemunhos também foram datados com 210Pb, e analises da razão isotópica do carbono estável (δ13C) e de n-alcanos foram efetuadas. Ao longo dos três testemunhos, os resultados do δ13C e n-alcanos indicaram a predominância de matéria orgânica terrígena, derivadas de plantas do tipo C3. Entretanto, uma variação nesses valores foi observada em 1965. As concentrações de HPAs também apresentaram uma alteração por volta de 1965. Até 1965, os HPAs alquilados foram predominantes em relação à seu composto parental (metilfenantreno> fenantreno). Esses HPAs estão geralmente associados a fontes petrogênicas, entretanto, nenhuma evidência de petróleo foi observada. A maior proporção de HPAs alquilados pode estar relacionada a processos naturais, diagenéticos ou de biossíntese. Após 1965, as concentrações de HPAs aumentaram. As razões diagnósticas indicaram a predominância de HPAs pirolíticos, provavelmente relacionadas às queimadas efetuadas para criação de pastos. A diminuição do δ13C no período também deve estar relacionada à extração de madeira, que introduz uma grande quantidade de resíduos de vegetação nos sedimentos. / In order to evaluate the historical deposition and sources of PAHs over the last 120 years (1890 to 2011) in the amazon region, three sediment cores were collected from Guajara Bay (state of Pará). The cores were also dated with 210Pb and n-alkanes and carbon isotopic ratios (δ13C) were measured. Throughout the cores, the δ13C and n-alkanes indicated the predominance of organic matter from terrigenous sources (C3 plants). Even though δ13C values and n-alkanes ratios remained representative of terrigenous sources, there was an abrupt variation around the middle 1960s. PAH concentrations also showed a variation around the late 1960s. Before 1960s, alkyl-PAHs concentrations were higher than those of their parental compound (methylphenanthrene > phenanthrene). These PAHs are usually associated with petrogenic inputs; however, no evidence of oil was observed. The higher proportion of alkyl-PAHs could be related to natural diagenetic and biogenic processes. After the 60s, PAH concentrations increased and diagnostic ratios showed a predominance of pyrolytic PAH, probably related to the forest fires used to create pastures. The deforestation can also be associated to the δ13C depletion found in the sediments, due to logging activities, which introduce more organic matter derived from vascular plants into the sediment.

O diagnóstico de substâncias poluidoras, com características orgânicas e inorgânicas, encontradas nas águas superficiais e no sedimento de fundo é uma importante ferramenta na avaliação da saúde de um reservatório. Os estudos de corpos dágua e sedimentos contaminados ganharam notoriedade, constituindo objetos importantes de pesquisas ambientais, envolvendo órgãos governamentais e legisladores. A classe dos contaminantes hidrocarbonetos policíclicos aromáticos (HPAs), metais e elementos-traço são as que mais contribuem para o empobrecimento da qualidade das águas superficiais, devido à sua estabilidade química e persistência. Neste trabalho, foram caracterizadas as amostras de água bruta e sedimento de fundo coletadas nos períodos de março e agosto de 2008, agosto de 2010, março e agosto de 2011 e maio de 2014 na Represa Parque Natural do Pedroso em Santo André, SP, sendo em 2008 antes da construção do Rodoanel Mario Covas, 2010 durante a construção, 2011 depois de concluída e 2014 com mais de 3 anos de uso. Foram verificadas as influências sazonais no aporte desses contaminantes durante e após a construção do Rodoanel. As técnicas analíticas empregadas foram a cromatografia líquida em fase reversa com detector UV/vis para determinação dos HPAs e Espectrometria de Emissão Óptica com Plasma de Argônio (ICP-OES) e Espectrometria de Absorção AtômicaAAS para avaliação dos metais. Os teores de vanádio (V), 0,068 a 0,073 &mu;g mL-1, encontrados respectivamente nas duas últimas coletas foram próximos ao limite de enquadramento do corpo dágua de classe II (0,1 &mu;g mL-1). Os teores de cromo (Cr), 0,01 &mu;g mL-1 e de zinco (Zn), em média, 0,06 e 0,04 &mu;g mL-1, respectivamente, nas duas últimas coletas, estiveram bem abaixo dos limites estabelecidos pelo CONAMA 357/05, de 0,05 &mu;g mL-1 para o cromo (Cr) e 0,18 &mu;g mL-1 para o zinco (Zn). Para os elementos alumínio (Al), ferro (Fe), cobre (Cu), manganês (Mn) e chumbo (Pb), foram verificados valores mais elevados em desacordo com Resolução CONAMA 357/05. O comportamento dos HPAs nos cinco pontos de coletas e períodos diferentes é muito semelhante, e esta homogeneidade sugere que a fonte de emissão para esses compostos seja a mesma. Para os HPAs e metais estudados foram encontrados resultados significativos nas coletas de 2011, período em que o anel viário estava funcionando há um ano e meio, em sua plenitude, com aumento expressivo do fluxo de veículos leves, de médio porte e pesado, próximo ao reservatório. Pela somatória dos 13 HPAs para cada ponto se observou que as concentrações obtidas nos sedimentos variaram de 456 a 2723 ng g-1 respectivamente, em agosto de 2010 (ponto 2) e em março de 2011 (ponto 5). Esse aumento na somatória dos HPAs de aproximadamente 6 vezes ultrapassa o TEL 468 ng g-1. Os compostos que percentualmente mais contribuíram com a somatória de HPAs foram: benzo[e]acenaftileno 6,2%; benzo[k]fluoranteno 6,7%; benzo[a]antraceno 7,1%; fenantreno 7,4%; fluoreno 7,8%; antraceno 8,0%; pireno 8,9%; Acenaftileno 9,1%; indeno[1,2,3-c,d]pireno 11,3% e dibenzo[a,h]antraceno 12,6%, com amplitude de concentração de < 20 a 300 ng g-1. / The diagnosis of polluting substances, with both organic and inorganic characteristics, at superficial waters and bottom sediments is an important tool for the assessment of reservoirs pollution. Water bodies and contaminated sediments are important subjects for environmental researches, involving governmental agencies and legislators. Due to its chemical stability and endurance, the polycyclic aromatic hydrocarbons (PAHs) contaminant class and trace elements, are the major contributors for the depletion of superficial waters quality. In this research, gross water and bottom sediments samples were collected in Parque Natural do Pedrosos reservoir in Santo André, SP, in 2008 before the construction of the Mario Covas ring road, in 2010 during the construction and in 2011 after the construction had been concluded and 2014 after over 3 years of use. For analysis, the reversed-phase liquid chromatography with UV/vis for PAHs determination was used as technique. Optical Emission Spectrometry with Argon Plasma (ICP-OES) and Atomic Absorption Spectrometry-AAS were used for evaluation of metals. The PAHs behavior in the five collection points and at different periods is very similar, and this homogeneity suggests that these compounds have the same emission source. For the studied PAHs, significant results were found in 2011s collection, a period in which the beltway had been working for one year and a half, at its full capacity, with an expressive traffic flow increase of smaller, medium-sized and heavy vehicles next to the reservoir. By summing the 13 HPAs for each point, it was observed that concentrations obtained in the sediments ranged from 455 to 2723 ng g-1, in August 2010 (point 2, according to table 1) and in March 2011 (point 5, according to table 1) respectively. This increase in PAHs summing, of approximately 6 times exceeds TEL 468 ng g-1. The compounds that contributed the most to the PAHs summing, in percentage terms, were: benzo[e]acenaphthylene, 6.2%; benzo[k]fluoranthene, 6.7%; benzo[a]anthracene, 7.1%; phenanthrene, 7.4%; fluorene, 7.8%; anthracene, 8.0%; pyrene, 8.9%; Acenaphthylene, 9.1%; indene[1,2,3-c,d]pyrene, 11.3% and dibenzo[a,h]anthracene, 12.6%, with concentration amplitude of <20 to 300 ng g-1. The concentrations of vanadium (V), of 0.068 to 0.073 mg L-1, respectively found in the last two samples collects, are near to the established limit to the water class II (0.1 mg mL-1). The levels of chromium (Cr), 0.01 mg ml-1, and zinc (Zn), on average, 0.06 and 0.04 mg mL-1, respectively, in the last two samples collects, were below of the of 0.05 mg mL-1 for chromium (Cr) and 0.18 mg L-1 to zinc (Zn), which are established limits by CONAMA 357/05. To the aluminum (Al), iron (Fe), copper (Cu), manganese (Mn) and lead (Pb), values at odds with CONAMA Resolution 357/05 were verified. The behavior of PAHs in the five collections points and in different periods is very similar, and this homogeneity suggests that the emission source for these compounds is the same. For the PAHs and metals studied significant results were found in 2011 collected samples, period during which the beltway was in use a year and a half , with significant increase in the flow of light, midsize and heavy vehicles, near the reservoir.

O presente trabalho apresenta resultados de medidas de campo, laboratório e simulações numéricas para o diagnóstico de um sítio de descarte de resíduos de re-refino de óleo lubrificante, localizado no Município de Ribeirão Preto, no Estado de São Paulo. A metodologia laboratorial utilizada mostrou-se viável, sendo descritos também casos nos quais a primeira proposta de trabalho não foi adequada. Adicionalmente, o programa numérico comercial escolhido para simular a situação do sítio mostrou-se suficientemente manuseável e adequado para estudos expeditos. Os dados de contaminação da água subterrânea por metais mostram que os principais contaminantes são o chumbo e o alumínio. Em termos de contaminação do solo por HPAs, verificou-se, de forma geral, baixa concentração (em alguns casos não detectáveis), ficando o fenantreno como o composto com a concentração mais elevada, porém sem padrão de comparação. O naftaleno, que possui esse padrão, mostrou concentração muito inferior àquela considerada como referência. Os resultados da simulação numérica são apresentados em sete camadas ao longo da profundidade, sendo sua resolução, no plano, efetuada com malhas de 50,0 m por 50,0 m. Simulou-se uma pluma poluente de chumbo, com evolução para um tempo de 10 anos, com concentração inicial e de contorno compatível com as medidas efetuadas nos poços de monitoramento (25 &#956g/L). Os resultados apontam para contaminação efetiva da área utilizada para disposição do resíduo, bem como alteração nas circunvizinhanças. O presente trabalho representa uma contribuição para a sistematização da metodologia de estudo de áreas similares. / This research introduces results of field and laboratory applications and numerical simulation to assess a site of oil waste disposal, in Ribeirão Preto, São Paulo, Brazil. The laboratorial methodology applied was useful, but there were also cases where the first try was not adequate. Additionally, the commercial numeric software selected to simulate site conditions was properly handled and applicable to expedited site assessment. The results of metals concentration in ground water highlight lead and aluminum as the main contaminants. In terms of PAHs in soil, in general, low concentrations (in some cases not detectible) were observed, where phenanthrene was the compound with the highest concentration, however without reference level. The naphthalene, has reference level, present concentrations much lower than the reference level. The numerical simulation results are presented in seven subsurface layers, which in a plan view the grid was divided in 50,0 m per 50,0 m. A lead contamination was simulated in the transport model, with 10 years of temporal discretization, and with initial and boundary concentrations compatible with measures in monitoring wells (25 &#956g/L). The results show contamination in the disposal area, and alteration in the neighboring areas. The present research represents a contribution to the methodology for similar site assessment.

La pollution des écosystèmes marins côtiers par les hydrocarbures, en particulier les HAPs, est un problème environnemental majeur. Le même constat est fait pour le littoral Sud de Sfax (Tunisie) dont la pollution presque généralisée menace sérieusement les ressources naturelles existantes dans la région. La caractérisation physico-chimique des échantillons marins prélevés à partir des trois ports (plaisance, commerce et pêche) de la ville de Sfax, prouve une contamination par des micropolluants organiques (hydrocarbures) et inorganiques (métaux) qui sont considérés comme des excellents traceurs de la pollution urbaine et industrielle et ils font partie des composés les plus toxiques étant donné leur faible biodégradation. La méthode d’empreinte moléculaire (PCR-SSCP) montre une dominance du domaine Bacteria suivie des Eucarya et des Archaea au sein des échantillons marins étudiés. Les analyses statistiques par le logiciel R ont montré l’absence de corrélation entre la communauté bactérienne identifiée par PCR-SSCP et les paramètres physico-chimiques étudiés.Dans une autre partie de travail, quatre souches bactériennes marines hydrocarbonoclastes ont été isolées et caractérisées sur les plans phénotypique et phylogénétique, après des enrichissements sur des HAPs et sur le pétrole brut: FLU5 de Bacillus stratosphericus, NAPH6 de Pseudomonas aeruginosa, PYR2 de Bacillus licheniformis isolées sur le fluoranthène, le naphtalène et le pyrène, respectivement, en présence de 30 g/l NaCl; et CO100 de Staphylococcus sp., isolée sur le pétrole brut, en présence de 100 g/l NaCl. Les analyses chromatographiques, GC-MS ou GC-FID, montrent les capacités biodégradatives intéressantes de ces composés récalcitrants par les bactéries isolées. En outre, ces quatre souches bactériennes, sont capables de produire des biosurfactants nommés BS-FLU5, BS-NAPH6, BS-PYR2 et BS-CO100, sur plusieurs sources de carbones, y compris l’huile de friture résiduelle, un substrat bon marché, minimisant ainsi le coût élevé de production de ces tensioactifs. Les analyses MALDI-TOF/MS, des biosurfactants BS-FLU5, BS-PYR2 et BS-CO100 purifiés, montrent qu’il s’agit des lipopeptides, les biosurfactants BS-NAPH6 sont de nature rhamnolipidique, sur la base des analyses FTIR. Ces quatre biosurfactants sont caractérisés par des propriétés tensiactives intéressantes : une faible CMC, une importante réduction de la tension de surface... Ils sont stables vis-à-vis d’une large gamme de pH, de température et de salinité. De plus, ces agents tensioactifs sont doués d’activité de remobilisation des hydrocarbures contenus dans des sols pollués par. Les biosurfactants BS-FLU5, BS-PYR2 et BS-CO100, présentent des activités anti-adhésives et anti-biofilms intéressantes contre des biofilms de certains microorganismes pathogènes. Par ailleurs, une propriété cicatrisante remarquable sur des plaies d’excision chez un modèle expérimental de rats de race Wistar, a été montrée par les quatre biosurfactants pour des concentrations de l’ordre de 5 et 10 mg/ml, en comparaison avec un cicatrisant de référence (CICAFLORA®). A noter que, l’évaluation de la cytotoxicité des biosurfactants étudiés, a montré qu’ils n’ont pas des effets toxiques sur des cellules rénales humaines HEK-239 à des concentrations jusqu’à 1000 µg/ml pour BS-FLU5 et BS-CO100 et jusqu’à 200 µg/ml pour BS-NAPH6 et BS-PYR2. La production des biosurfactants de la souche FLU5 à l’échelle pilote (deux fermenteurs de 20 et 100 litres, volume total), en présence d’un milieu économique, montre une augmentation des quantités des biosurfactants produits par rapport à l’échelle laboratoire (erlenmeyer, 1 litre). L’ensemble de ces résultats prometteurs, montrent que les souches marines isolées FLU5, NAPH6, PYR2 et CO100, ainsi que leurs biosurfactants demeurent d’intérêts biotechnologiques pour divers types d’applications, tels que la bioremédiation, l’agroalimentaire, la cosmétique... / Pollution of coastal marine ecosystems by hydrocarbons, in particular polycyclic aromatic hydrocarbons (PAHs), is a major environmental problem. The South coast of Sfax (Tunisia) is an example of a polluted ecosystem subject to both urbanization and industrialization including the outfall of untreated domestic sewage and wastewaters, fishery activities, as well as ship traffic and boat pollution. The physico-chemical characterization of the seawater taken from three harbours (pleasure, commercial and fishing) of the city of Sfax, showed a heavy contamination by organic and inorganic micropollutants. These are excellent tracers of urban and industrial pollution, and they are among the most toxic compounds due to their low biodegradation.The molecular fingerprinting technique (PCR-SSCP) showed the dominance of the Bacteria domain followed by Eucarya and Archaea within the studied marine samples. Statistical analysis using the R software, showed that no correlation was identified between the bacterial community identified by PCR-SSCP and the studied physico-chemical parameters.In another part, four marine, aerobic and hydrocarbonoclastic strains: Bacillus stratosphericus FLU5, Pseudomonas aeruginosa NAPH6, Bacillus licheniformis PYR2, isolated after enrichments on fluoranthene, naphthalene and pyrene, respectively, and in the presence of 30 g/l NaCl. Strain Staphylococcus sp. CO100 was isolated after enrichment on crude oil, in the presence of 100 g/l. Chromatographic analysis (GC-MS or GC-FID), showed the interesting biodegradative capacities of these recalcitrant compounds by the isolated bacteria.Besides, these strains showed their capacity to produce efficient surface active agents BS-FLU5, BS-NAPH6, BS-PYR2 and BS-CO100, on several substrates and in particular the residual frying oil, which is a cheap and renewable carbon source alternative, thus minimizing the high cost of producing surfactants. The MALDI-TOF/MS analysis of the purified BS-FLU5, BS-PYR2 and BS-CO100 biosurfactants revealed that they are belonging to lipopeptide family. FTIR analysis showed the glycolipid nature, more precisely the rhamnolipid type, of biosurfactant BS-NAPH6.These four biosurfactants are characterized by interesting tensioactive properties (low CMC, important surface tension reduction...). Furthermore, these surface active agents showed interest stability against a broad range of pH, temperature and salinity. The application of these biosurfactants, in oil recovery, from hydrocarbons-contaminated soil, showed that they were more effective on the hydrocarbon-remobilization than some tested synthetic surfactants. The biosurfactants BS-FLU5, BS-PYR2 and BS-CO100, were found to have notable anti-adhesif and anti-biofilm activities, being able to prevent and eliminate the biofilm formation by pathogenic microorganisms. Moreover, the four tested biosurfactants showed an interesting healing activity, on the wound site in a rat model. They increased significantly the percentage of wound closure when compared to the untreated and CICAFLORA® (a reference pharmaceutical product) treated groups, using two different concentrations (5 and 10 mg/l). Interestingly, the evaluation of the cytotoxicity of the studied biosurfactants, showed that they have no toxic effects on human HEK-239 cells at concentrations up to 1000 μg/ml for BS-FLU5 and BS-CO100 and up to 200 μg/ml for BS-NAPH6 and BS-PYR2. An attempt to produce biosurfactant produce by strain FLU5 on a pilot-scale (fermentors of 20 and 100 liter, as total volume), using a cost-effective medium, was also performed. Preliminary results showed an increase in the quantities of biosurfactantsBS-FLU5 produced on a pilot-scale compared to the lab-scale (Erlenmeyer of 1 liter).These results highlight the interest for potential use of strains FLU5, NAPH6, PYR2 and CO100, as well as their biosurfactants, in a wide variety of industrial, environmental and biotechnological applications.

O diagnóstico de substâncias poluidoras, com características orgânicas e inorgânicas, encontradas nas águas superficiais e no sedimento de fundo é uma importante ferramenta na avaliação da saúde de um reservatório. Os estudos de corpos dágua e sedimentos contaminados ganharam notoriedade, constituindo objetos importantes de pesquisas ambientais, envolvendo órgãos governamentais e legisladores. A classe dos contaminantes hidrocarbonetos policíclicos aromáticos (HPAs), metais e elementos-traço são as que mais contribuem para o empobrecimento da qualidade das águas superficiais, devido à sua estabilidade química e persistência. Neste trabalho, foram caracterizadas as amostras de água bruta e sedimento de fundo coletadas nos períodos de março e agosto de 2008, agosto de 2010, março e agosto de 2011 e maio de 2014 na Represa Parque Natural do Pedroso em Santo André, SP, sendo em 2008 antes da construção do Rodoanel Mario Covas, 2010 durante a construção, 2011 depois de concluída e 2014 com mais de 3 anos de uso. Foram verificadas as influências sazonais no aporte desses contaminantes durante e após a construção do Rodoanel. As técnicas analíticas empregadas foram a cromatografia líquida em fase reversa com detector UV/vis para determinação dos HPAs e Espectrometria de Emissão Óptica com Plasma de Argônio (ICP-OES) e Espectrometria de Absorção AtômicaAAS para avaliação dos metais. Os teores de vanádio (V), 0,068 a 0,073 &mu;g mL-1, encontrados respectivamente nas duas últimas coletas foram próximos ao limite de enquadramento do corpo dágua de classe II (0,1 &mu;g mL-1). Os teores de cromo (Cr), 0,01 &mu;g mL-1 e de zinco (Zn), em média, 0,06 e 0,04 &mu;g mL-1, respectivamente, nas duas últimas coletas, estiveram bem abaixo dos limites estabelecidos pelo CONAMA 357/05, de 0,05 &mu;g mL-1 para o cromo (Cr) e 0,18 &mu;g mL-1 para o zinco (Zn). Para os elementos alumínio (Al), ferro (Fe), cobre (Cu), manganês (Mn) e chumbo (Pb), foram verificados valores mais elevados em desacordo com Resolução CONAMA 357/05. O comportamento dos HPAs nos cinco pontos de coletas e períodos diferentes é muito semelhante, e esta homogeneidade sugere que a fonte de emissão para esses compostos seja a mesma. Para os HPAs e metais estudados foram encontrados resultados significativos nas coletas de 2011, período em que o anel viário estava funcionando há um ano e meio, em sua plenitude, com aumento expressivo do fluxo de veículos leves, de médio porte e pesado, próximo ao reservatório. Pela somatória dos 13 HPAs para cada ponto se observou que as concentrações obtidas nos sedimentos variaram de 456 a 2723 ng g-1 respectivamente, em agosto de 2010 (ponto 2) e em março de 2011 (ponto 5). Esse aumento na somatória dos HPAs de aproximadamente 6 vezes ultrapassa o TEL 468 ng g-1. Os compostos que percentualmente mais contribuíram com a somatória de HPAs foram: benzo[e]acenaftileno 6,2%; benzo[k]fluoranteno 6,7%; benzo[a]antraceno 7,1%; fenantreno 7,4%; fluoreno 7,8%; antraceno 8,0%; pireno 8,9%; Acenaftileno 9,1%; indeno[1,2,3-c,d]pireno 11,3% e dibenzo[a,h]antraceno 12,6%, com amplitude de concentração de < 20 a 300 ng g-1. / The diagnosis of polluting substances, with both organic and inorganic characteristics, at superficial waters and bottom sediments is an important tool for the assessment of reservoirs pollution. Water bodies and contaminated sediments are important subjects for environmental researches, involving governmental agencies and legislators. Due to its chemical stability and endurance, the polycyclic aromatic hydrocarbons (PAHs) contaminant class and trace elements, are the major contributors for the depletion of superficial waters quality. In this research, gross water and bottom sediments samples were collected in Parque Natural do Pedrosos reservoir in Santo André, SP, in 2008 before the construction of the Mario Covas ring road, in 2010 during the construction and in 2011 after the construction had been concluded and 2014 after over 3 years of use. For analysis, the reversed-phase liquid chromatography with UV/vis for PAHs determination was used as technique. Optical Emission Spectrometry with Argon Plasma (ICP-OES) and Atomic Absorption Spectrometry-AAS were used for evaluation of metals. The PAHs behavior in the five collection points and at different periods is very similar, and this homogeneity suggests that these compounds have the same emission source. For the studied PAHs, significant results were found in 2011s collection, a period in which the beltway had been working for one year and a half, at its full capacity, with an expressive traffic flow increase of smaller, medium-sized and heavy vehicles next to the reservoir. By summing the 13 HPAs for each point, it was observed that concentrations obtained in the sediments ranged from 455 to 2723 ng g-1, in August 2010 (point 2, according to table 1) and in March 2011 (point 5, according to table 1) respectively. This increase in PAHs summing, of approximately 6 times exceeds TEL 468 ng g-1. The compounds that contributed the most to the PAHs summing, in percentage terms, were: benzo[e]acenaphthylene, 6.2%; benzo[k]fluoranthene, 6.7%; benzo[a]anthracene, 7.1%; phenanthrene, 7.4%; fluorene, 7.8%; anthracene, 8.0%; pyrene, 8.9%; Acenaphthylene, 9.1%; indene[1,2,3-c,d]pyrene, 11.3% and dibenzo[a,h]anthracene, 12.6%, with concentration amplitude of <20 to 300 ng g-1. The concentrations of vanadium (V), of 0.068 to 0.073 mg L-1, respectively found in the last two samples collects, are near to the established limit to the water class II (0.1 mg mL-1). The levels of chromium (Cr), 0.01 mg ml-1, and zinc (Zn), on average, 0.06 and 0.04 mg mL-1, respectively, in the last two samples collects, were below of the of 0.05 mg mL-1 for chromium (Cr) and 0.18 mg L-1 to zinc (Zn), which are established limits by CONAMA 357/05. To the aluminum (Al), iron (Fe), copper (Cu), manganese (Mn) and lead (Pb), values at odds with CONAMA Resolution 357/05 were verified. The behavior of PAHs in the five collections points and in different periods is very similar, and this homogeneity suggests that the emission source for these compounds is the same. For the PAHs and metals studied significant results were found in 2011 collected samples, period during which the beltway was in use a year and a half , with significant increase in the flow of light, midsize and heavy vehicles, near the reservoir.

O aumento na poluição ambiental é na atualidade uma das grandes preocupações em nível mundial. Especificamente, a poluição atmosférica por material particulado tem demostrado ser um fator determinante no desenvolvimento de doenças cardiopulmonares e câncer de pulmão nas populações expostas. O material particulado é constituído por uma mistura complexa de compostos orgânicos e inorgânicos, muitos dos quais possuem potencial mutagênico e genotóxico. As características destes compostos variam em função da suas propriedades físicas, químicas e em razão das condições meteorológicas prevalecentes. A maior parte dos estudos tem se focado em avaliar o potencial genotóxico da fração orgânica do material particulado e poucos estudos têm explorado a fração solúvel em água e a contribuição diferencial das diversas espécies químicas presentes nesta fração para o dano genotóxico. O objetivo deste trabalho foi avaliar os efeitos mutagênicos e genotóxicos in vitro da fração orgânica e da fração solúvel em água de material particulado (MP10) coletado em três locais diferentes do estado de São Paulo e estabelecer a relação entre a composição química e o efeito biológico observado. Para isto, realizou­-se a extração orgânica e solúvel em água de 12 amostras de MP10. A mutagenicidade e genotoxicidade foram avaliadas usando o ensaio de Salmonella/microssoma e o teste de micronúcleos (MN) em células A549 e 16 hidrocarbonetos Policíclicos Aromáticos (HPAs) e 15 metais hidrossolúveis presentes nas amostras foram determinados quimicamente. Adicionalmente, foi determinada a metodologia de extração da fracção solúvel em água e se avaliou a estabilidade química e biológica desta fração. Os resultados indicam que a extração assistida por micro-ondas é um método eficiente para a extração da fração solúvel em agua do MP e que um tempo superior a 60 dias de armazenamento e congelamento deste tipo de extrato tem um efeito significativo sobre os resultados analíticos e a resposta biológica. Foi demonstrado ainda que as duas frações de MP estudadas são responsáveis pela indução do dano ao DNA e que não existe uma relação direta entre a concentração de MP e o efeito genotóxico observado, confirmando a importância do uso de bioensaios na avaliação da genotoxicidade de misturas complexas como o MP. Os HPAs prevalecentes nas amostras de PM10 foram fluorantene e benzo(ghi)perileno. Nos extratos solúveis em água, as maiores concentrações de metais foram determinadas para zinco, ferro e cobre. Confirmou-se que a indução de MN e o ensaio de Salmonella/microssoma representam uma poderosa ferramenta na avaliação da polução atmosférica e que as análises químicas por si só não são suficientes para a proteção e predição dos efeitos biológicos em populações expostas. / The increase of environmental pollution is today one a major concern worldwide. Specifically, air pollution by particulate matter has been shown to be a determining factor in the development of cardiopulmonary disease and lung cancer in exposed populations. The particulate material consists of a complex mixture of organic and inorganic compounds, many of which have mutagenic and genotoxic activity. The characteristics of these compounds vary according to their physical and chemical properties and also to the prevailing weather conditions. Most studies have focused on evaluating the genotoxic potential of the organic fraction of particulate material, but few studies have explored the water­-soluble fraction, and the differential contributions of different chemical species present in this fraction to genotoxic damage. The aim of this study was to evaluate the in vitro mutagenic and genotoxic effects of organic and water-soluble fractions of 12 samples of particulate matter (PM10) collected at three different sites in the state of São Paulo and establish the relationship between the chemical composition and the biological effect observed. The mutagenicity and genotoxicity were evaluated using the Salmonella/microssome test and the micronucleus assay (MN) in A549 cells and 16 Polycyclic Aromatic Hydrocarbons (PAHs) and 15 water-soluble metals present in the samples were chemically determined. Additionally, the extraction method of water- soluble fraction was determined and the chemical and biological stability of this fraction evaluated. The results indicate that the microwave­-assisted extraction is an efficient method for the extraction of the water-soluble compounds of PM and that the freezing and storage of the extract over 60 days has a significant effect on the mutagenic and analytical results of PM samples. It was demonstrated that the two PM fractions studied are responsible for the induction of DNA damage and that there is no direct relationship between the MP concentration and the genotoxic effect observed, confirming the importance of using bioassays in the genotoxicity evaluation of complex mixtures as PM. The PAHs prevailing in our samples were fluoranthene and benzo(ghi)perylene. In the water-soluble extracts, highest concentrations of the elements studied were found for zinc, iron, and copper in the three places of sampling. We confirmed that MN induction and Salmonella/microsome assay represents a powerful tool to evaluate the atmospheric air pollution and that the total concentration of PM and the chemical analyses alone would not be sufficient for the prognosis of biological effects in exposed populations

Thesis (M.Sc. (Chemistry)) --University of Limpopo, 2017 / The presence of polycyclic aromatic hydrocarbons (PAHs) in the environment is of major concern since these compounds are highly persistent, toxic and wide spread pollutants. The aim of this study was to evaluate the levels of PAHs in water and sediment samples collected from Blood and Mokolo Rivers in Limpopo Province, South Africa. Liquid-liquid extraction (LLE) was used for the extraction of PAHs from water, whereas PAHs in sediments were extracted using optimised microwaveassisted extraction (MAE). Furthermore, ultrasonication and a combination of ultrasonication and mechanical agitation were used for the extraction of PAHs from sediments samples. The quantification of sixteen (16) PAHs in water and sediment was carried out using gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode and by GC-flame ionisation detector (GC-FID). Concentrations of PAHs in sediments were higher than in water. The highest concentrations of PAHs were obtained in Mokolo River sediments, with the concentration ranging between 0.044 and 51.9 mg/kg. The levels of PAHs recorded in Blood River sediments were lower than those obtained in Mokolo River with concentrations ranging between 0.014 and 3.10 mg/kg. In water samples, higher levels of PAHs were observed in Mokolo River (between 0.0219 and 1.53 µg/L) while lower concentrations were recorded in Blood River (between 0.0121 and 0.433 µg/L). In water and sediment samples from both Rivers, higher molecular weight (HMW) PAH compounds (4-6 rings) were found at greater concentration levels than lower molecular weight (LMW) PAHs (2-3 rings), and this can be attributed to pyrogenic activities in the study areas. The efficiencies and accuracy of the methods for the extraction of PAHs were determined by assessing the recoveries of samples spiked with known amount of standards (for water samples), while a certified reference material (CRM) was used for sediments. Percentage recoveries ranged from 67.6 to 115% for LLE and 83.8 to 125% for MAE for both sample types. Diagnostic ratio was used for the source identification of PAHs in sediment samples. Several PAHs ratios indicated that both pyrogenic and petrogenic could be the sources of these compounds in both rivers. Toxic equivalency factors (TEFs) and benzo(a)pyrene equivalent (BaPE) were used to quantitatively estimate the PAHs potential human health risk. The assessment of ecotoxicological risk indicated that the v sediment samples collected from Mokolo River are at high toxicity risk while sediments from Blood River are at low sediment toxicity risk. / Water Research Commission (WRC), National Research Foundation (NRF) and Sasol Inzalo Foundation

The Deepwater Horizon (DWH) oil spill in the Gulf of Mexico (GoM) released an estimated 4.9 million barrels of oil between April 20, 2010 and July 15, 2010. An estimated 36% of the oil formed a neutrally buoyant intrusion, containing both dissolved compounds and oil microdroplets, between 1000 and 1300 m depth. This study used geographic information systems software, and data from water samples that were collected as part of the National Resource Damage Assessment (NRDA), to determine that an area of at least 1,600 km2 was exposed to DWH oil. Toxic BTEX (benzene, toluene, ethylbenzene, and xylenes) compounds and polycyclic aromatic hydrocarbons (PAHs) reached concentrations 950 and 50 times higher than maximum background concentrations, respectively. BTEX and n-alkane concentrations above pre-2010 values were present through late August, more than a month after the wellhead was capped. This study is the first to examine the DWH intrusion over such a large temporal and spatial extent. We further estimated that an area between 500 and 1000 km2 may have been exposed to harmful PAH concentrations, based on studies of PAH toxicity and U.S. Environmental Protection Agency (EPA) guidelines. We also found evidence of aggregation and deposition of oil near the DWH wellhead, as well as an area of 400 km2 where the intrusion may have impinged on the seafloor. While relative rates of dilution, degradation, and deposition in the intrusion are unknown, we have shown evidence that supports two previously proposed processes that may have deposited DWH oil from this deep intrusion onto sediments, where toxic compounds could be resuspended and continue to be bioavailable to benthic organisms.