Conselho Nacional de Desenvolvimento Científico e Tecnológico / Tintas antifoulings são utilizadas para evitar a incrustação de organismos em estruturas submersas, especialmente casco de embarcações. Os compostos organoestânicos (OTs), utilizados nessas tintas, entre eles o tributilestanho, são desreguladores endócrinos e causaram diversos danos aos ecossistemas marinhos. No caso dos moluscos gastrópodes, esse tipo de poluição faz com que as fêmeas adquiram características masculinas, como vaso deferente e pênis, fenômeno esse conhecido como imposex. A Organização Marítima Internacional (IMO) estabeleceu o banimento de tintas à base de COEs nas embarcações, em 2008. No Brasil, a NORMAM 23, proibiu o uso em 2007, contudo a Marinha já havia suspendido seu uso desde 2003. Entretanto, efeitos deletérios destes compostos ainda são detectados em vários países, inclusive ao longo do litoral brasileiro. Esse trabalho teve como objetivo principal fazer uma avaliação temporal (1997-2012) da poluição por organestânicos na costa do Estado do Rio de Janeiro utilizando como bioindicador a espécie Stramonita haemastoma. A área de estudo abrangeu cinco regiões: Paraty, Ilha Grande, Baia de Ilha Grande, Baia de Guanabara e Arraial do Cabo. Análises químicas de butilestânicos foram feitas em sedimentos superficiais de mangues de Paraty (Mangue do Estaleiro e mangue do Saco do Mamanguá) e Ilha Grande (Mangue do Aventureiro) sendo os dois últimos considerados áreas de referência. Em cada estação de biomonitoramento foram coletados 30 indivíduos sexualmente adultos da espécie S. haemastoma, através de mergulho livre em apnéia e analisados através do método não destrutivo proposto por nosso grupo de pesquisa. Em todas as cinco áreas analisadas ao longo da costa do Estado do Rio de Janeiro foram registradas estações com altos índices de imposex. Apesar de muitas estações apresentarem diminuição nos índices, na maioria ocorreu um aumento ou conservação alta na porcentagem de imposex depois do banimento. As concentrações médias de butilestânicos no mangue (S1), perto de fontes locais, foram 205,7 16,8 ng (Sn) g-1 de TBT, 16,4 1,3 ng (Sn) g-1 de DBT e 10,0 2,9 ng (Sn) g-1 de MBT. Nas áreas de referência: mangue do Saco do Mamanguá (S2) foram 16,0 0,8 ng (Sn) g-1 de TBT, 10,1 1,4 ng (Sn) g-1 de DBT e 10,1 2,2 ng (Sn) g-1 de MBT e mangue do Aventureiro (S3) com 18,1 4,2 ng (Sn) g-1 de TBT, 15,3 0,5 ng (Sn) g-1 de DBT e 10,2 1,5 ng (Sn). g-1 de MBT. As taxas de degradação foram de 01, 1,3 e 1,4 respectivamente indicando inputs recentes desses compostos. Os resultados deste estudo indicam que carbono orgânico dissolvido e particulado, bem como xenoestrógenos podem estar interferindo no desenvolvimento da síndrome, levando a subestimação de avaliação do imposex. As concentrações de butilestânicos, além do aumento ou continuidade alta na incidência de imposex após o banimento na maioria das estações indicam que, apesar da proibição do uso do TBT em tintas antiiincrustantes no Brasil, elas ainda estão sendo utilizadas de forma ilegal, especialmente em pequenos barcos. Esse estudo é fundamental para se propor medidas de mitigação e controle dos compostos organoestânicos, até mesmo dos novos antifoulings TBT- free, que também possuem efeitos prejudiciais ao ambiente. Além disso, as áreas onde se registrou altos índices de imposex serão essenciais para o monitoramento dos efeitos desses novos antifoulings. / Antifoulings paints are used to prevent of fouling organisms on underwater structures, especially hull vessels. Organotin compounds (OTs) used in these paints, including tributyltin, are endocrine disrupters and caused damage to various ecosystems. In the case of the gastropod mollusks, such pollution causes females acquire male characteristics, such as penis and vas deferens, a phenomenon known as imposex. The International Maritime Organization (IMO) established a ban on OTs-based paints on ships, in 2008. In Brazil, the NORMAN 23, prohibited the use in 2007, but the Navy had suspended its use since 2003. However, deleterious effects of these compounds are still detected in several countries, including along the Brazilian coast. This work aimed to make a temporal time series assessment (1997-2012) of organotins pollution on the coast of Rio de Janeiro State using as bioindicator the species Stramonita haemastoma. The study area included five regions: Paraty, Ilha Grande, Ilha Grande Bay, Guanabara Bay and Arraial do Cabo. In each biomonitoring station were collected 30 sexually mature individuals of the specie S. haemastoma through free diving in apnea and analyzed using the non-destructive method proposed by Fernandez et al, (2007). Butyltin chemical analyzes were made in surface sediments of mangroves of Paraty (Mangrove close to the sources and Saco do Mamanguá mangrove) and Ilha Grande (Aventureiro mangrove) the last two being considered the reference areas. In all five areas studied along the coast of the State of Rio de Janeiro were recorded stations with high levels of imposex. Although many stations present a decrease in rates, in most there was an increase or maintenance in the high percentage of imposex after the ban. The average concentrations of the mangrove butyltin (S1), close to local sources, were 205.7 16.8 ng (Sn) of TBT g-1, 16.4 1.3 ng (Sn) g -1 of DBT and 10.0 2.9 ng (Sn) g-1 MBT. In the areas of reference: the Saco do Mamanguá mangrove (S2) were 16.0 0.8 ng (Sn) g -1 of TBT, 10.1 1.4 ng (Sn) g -1 of DBT and 10, 1 2.2 ng (Sn) g-1 of MBT and Aventureiro mangrove (S3) with 18.1 4.2 ng (Sn) of TBT g-1, 15.3 0.5 ng (Sn)g-1 DBT and 10.2 1.5 ng (Sn). g-1 MBT. The degradation rates were 0,1, 1,3 and 1,4 respectively indicating recent inputs of these compounds. The results of this study indicate that dissolved and particulate organic carbon and xenoestrogens may be interfering with the development of the syndrome, leading to underestimation of assessment of imposex. Butyltins concentrations and increased or high continued incidence of imposex after the ban on most stations show that despite the ban on the use of TBT in antifouling paints in Brazil, they are still being used illegally, especially in small boats. This study is essential to propose measures to mitigate and control of organotin compounds, even the new TBT-free antifoulings, which also have negative environmental effect. This is because the areas where high levels of imposex recorded wil be the more critical for monitoring the effects of these new antifoulings.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Tintas antifoulings são utilizadas para evitar a incrustação de organismos em estruturas submersas, especialmente casco de embarcações. Os compostos organoestânicos (OTs), utilizados nessas tintas, entre eles o tributilestanho, são desreguladores endócrinos e causaram diversos danos aos ecossistemas marinhos. No caso dos moluscos gastrópodes, esse tipo de poluição faz com que as fêmeas adquiram características masculinas, como vaso deferente e pênis, fenômeno esse conhecido como imposex. A Organização Marítima Internacional (IMO) estabeleceu o banimento de tintas à base de COEs nas embarcações, em 2008. No Brasil, a NORMAM 23, proibiu o uso em 2007, contudo a Marinha já havia suspendido seu uso desde 2003. Entretanto, efeitos deletérios destes compostos ainda são detectados em vários países, inclusive ao longo do litoral brasileiro. Esse trabalho teve como objetivo principal fazer uma avaliação temporal (1997-2012) da poluição por organestânicos na costa do Estado do Rio de Janeiro utilizando como bioindicador a espécie Stramonita haemastoma. A área de estudo abrangeu cinco regiões: Paraty, Ilha Grande, Baia de Ilha Grande, Baia de Guanabara e Arraial do Cabo. Análises químicas de butilestânicos foram feitas em sedimentos superficiais de mangues de Paraty (Mangue do Estaleiro e mangue do Saco do Mamanguá) e Ilha Grande (Mangue do Aventureiro) sendo os dois últimos considerados áreas de referência. Em cada estação de biomonitoramento foram coletados 30 indivíduos sexualmente adultos da espécie S. haemastoma, através de mergulho livre em apnéia e analisados através do método não destrutivo proposto por nosso grupo de pesquisa. Em todas as cinco áreas analisadas ao longo da costa do Estado do Rio de Janeiro foram registradas estações com altos índices de imposex. Apesar de muitas estações apresentarem diminuição nos índices, na maioria ocorreu um aumento ou conservação alta na porcentagem de imposex depois do banimento. As concentrações médias de butilestânicos no mangue (S1), perto de fontes locais, foram 205,7 16,8 ng (Sn) g-1 de TBT, 16,4 1,3 ng (Sn) g-1 de DBT e 10,0 2,9 ng (Sn) g-1 de MBT. Nas áreas de referência: mangue do Saco do Mamanguá (S2) foram 16,0 0,8 ng (Sn) g-1 de TBT, 10,1 1,4 ng (Sn) g-1 de DBT e 10,1 2,2 ng (Sn) g-1 de MBT e mangue do Aventureiro (S3) com 18,1 4,2 ng (Sn) g-1 de TBT, 15,3 0,5 ng (Sn) g-1 de DBT e 10,2 1,5 ng (Sn). g-1 de MBT. As taxas de degradação foram de 01, 1,3 e 1,4 respectivamente indicando inputs recentes desses compostos. Os resultados deste estudo indicam que carbono orgânico dissolvido e particulado, bem como xenoestrógenos podem estar interferindo no desenvolvimento da síndrome, levando a subestimação de avaliação do imposex. As concentrações de butilestânicos, além do aumento ou continuidade alta na incidência de imposex após o banimento na maioria das estações indicam que, apesar da proibição do uso do TBT em tintas antiiincrustantes no Brasil, elas ainda estão sendo utilizadas de forma ilegal, especialmente em pequenos barcos. Esse estudo é fundamental para se propor medidas de mitigação e controle dos compostos organoestânicos, até mesmo dos novos antifoulings TBT- free, que também possuem efeitos prejudiciais ao ambiente. Além disso, as áreas onde se registrou altos índices de imposex serão essenciais para o monitoramento dos efeitos desses novos antifoulings. / Antifoulings paints are used to prevent of fouling organisms on underwater structures, especially hull vessels. Organotin compounds (OTs) used in these paints, including tributyltin, are endocrine disrupters and caused damage to various ecosystems. In the case of the gastropod mollusks, such pollution causes females acquire male characteristics, such as penis and vas deferens, a phenomenon known as imposex. The International Maritime Organization (IMO) established a ban on OTs-based paints on ships, in 2008. In Brazil, the NORMAN 23, prohibited the use in 2007, but the Navy had suspended its use since 2003. However, deleterious effects of these compounds are still detected in several countries, including along the Brazilian coast. This work aimed to make a temporal time series assessment (1997-2012) of organotins pollution on the coast of Rio de Janeiro State using as bioindicator the species Stramonita haemastoma. The study area included five regions: Paraty, Ilha Grande, Ilha Grande Bay, Guanabara Bay and Arraial do Cabo. In each biomonitoring station were collected 30 sexually mature individuals of the specie S. haemastoma through free diving in apnea and analyzed using the non-destructive method proposed by Fernandez et al, (2007). Butyltin chemical analyzes were made in surface sediments of mangroves of Paraty (Mangrove close to the sources and Saco do Mamanguá mangrove) and Ilha Grande (Aventureiro mangrove) the last two being considered the reference areas. In all five areas studied along the coast of the State of Rio de Janeiro were recorded stations with high levels of imposex. Although many stations present a decrease in rates, in most there was an increase or maintenance in the high percentage of imposex after the ban. The average concentrations of the mangrove butyltin (S1), close to local sources, were 205.7 16.8 ng (Sn) of TBT g-1, 16.4 1.3 ng (Sn) g -1 of DBT and 10.0 2.9 ng (Sn) g-1 MBT. In the areas of reference: the Saco do Mamanguá mangrove (S2) were 16.0 0.8 ng (Sn) g -1 of TBT, 10.1 1.4 ng (Sn) g -1 of DBT and 10, 1 2.2 ng (Sn) g-1 of MBT and Aventureiro mangrove (S3) with 18.1 4.2 ng (Sn) of TBT g-1, 15.3 0.5 ng (Sn)g-1 DBT and 10.2 1.5 ng (Sn). g-1 MBT. The degradation rates were 0,1, 1,3 and 1,4 respectively indicating recent inputs of these compounds. The results of this study indicate that dissolved and particulate organic carbon and xenoestrogens may be interfering with the development of the syndrome, leading to underestimation of assessment of imposex. Butyltins concentrations and increased or high continued incidence of imposex after the ban on most stations show that despite the ban on the use of TBT in antifouling paints in Brazil, they are still being used illegally, especially in small boats. This study is essential to propose measures to mitigate and control of organotin compounds, even the new TBT-free antifoulings, which also have negative environmental effect. This is because the areas where high levels of imposex recorded wil be the more critical for monitoring the effects of these new antifoulings.

Organotin compounds are one of the most produced and most used organometallic compounds. Some of these substances are endocrine disruptors, persistent organic polutants and their high toxic effects are observed. That’s why their presence in the environment caused by human activity could endanger many organisms. The aim of this thesis is summarize their properties and their occurrence in the environment. Then the quick, easy and relatively cheap method for determination of trialkyltin compounds in heavily poluted aquatic sediments using capillary zone electrophoresis is developed.

Thesis submitted in fulfilment of the requirements for the degree Doctor of Technology: Chemistry in the Faculty of Applied Sciences at the Cape Peninsula University of Technology 2013 / In this present study, the physicochemical properties, nature and morphology of prepared composite materials involving activated carbon, fly ash, nFe3O4, nSiO2 and nZnO in the 1:1 ratio for two components composite materials and 1:1:1 for three components composite materials were investigated. The nature, morphology and elemental characterizations of these materials were carried out by means of modern analytical methods such as scanning electron and transmission electron microscopy (SEM and TEM), x-ray diffraction (XRD), x-ray fluorescence (XRF), inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma atomic emission spectroscopy (ICP-AES) and Fourier transform infrared spectroscopy (FTIR). Other physicochemical characterizations undertaken were CNH analysis, ash content, pH, point of zero charge and surface area and porosity determination by Brunauer, Emmett and Teller (BET). The precursors and composite materials were then applied to the sorption (remediation) of tributyltin (TBT) and triphenyltin (TPT) from artificial seawater and wastewater and the adsorption efficiencies for the precursors and the composites compared. The adsorption of TBT and TPT onto these materials as a function of adsorbent amount, contact time, pH, stirring speed, initial adsorbate concentration and temperature was investigated. Maximum organotin adsorption was recorded within the pH range of normal saline water (pH 8). Approximately 99.95 %, 95.75 %, 96.78 %, 99.88 %, 96.96 %, 99.98 %, 99.99 %, 99.99 % and 99.99 % TBT were removed from 25 mL of 100 mg/L TBT-contaminated artificial seawater using 0.5 g adsorbents at a contact time of 60 min, pH 8, stirring speed 200 rpm and temperature of 80 oC by activated carbon, fly ash, nFe3O4, nSiO2, nZnO, fly ash/activated carbon, nFe3O4/activated carbon, nSiO2/activated carbon and nZnO/activated carbon composite, respectively and the adsorption of TBT onto these adsorbents was endothermic. Approx. 99.99 %, 96.54 %, 95.50 %, 96.92 %, 97.14 %, 99.99 %, 98.44 %, 98.98 % and 99.66 % TPT were also removed from 25 mL of 100 mg/L TPT-contaminated artificial seawater using 0.5 g adsorbents at a contact time of 60 min, pH 8, stirring speed 200 rpm and a temperature of 20 oC by the activated carbon, fly ash, nFe3O4, nSiO2, nZnO, fly ash/activated carbon, nFe3O4/fly ash, nSiO2/fly ash and nZnO/fly ash composite, respectively. The adsorption of TPT onto activated carbon and fly ash/activated carbon composite from TPT – contaminated artificial seawater was endothermic while TPT adsorption onto fly ash, nFe3O4, nSiO2, nZnO, nFe3O4/fly ash, nSiO2/fly ash and nZnO/fly ash composites from TPT – contaminated artificial seawater was exothermic. The adsorption of TBT and TPT onto nFe3O4/fly ash/activated carbon and nSiO2/fly ash/activated carbon composites from TBT – and TPT – contaminated water, respectively were endothermic and approx. 99.98 % and 99.99 % of TBT and TPT, respectively were removed from the initial concentration of 100 mg/L OTC by the composites at a temperature of 80 oC, 60 min contact time, pH 8 and a stirring speed of 200 rpm. The adsorption kinetics of all the precursors and composite materials fitted well with the pseudo second-order kinetic model while the adsorption isotherm data could be well described by the Freundlich isotherm model except TBT adsorption onto nZnO/activated carbon and nFe3O4/activated carbon composite from TBT contaminated artificial seawater, TPT adsorption onto activated carbon and fly ash/activated carbon from TPT contaminated artificial seawater, and TPT sorption onto nSiO2/fly ash/activated carbon composite from TPT – contaminated water which could be described by both the Freundlich and Dubinin-Radushkevich (D-R) isotherm models. Optimal conditions for the adsorption of TBT and TPT from artificial seawater were further applied to TBT and TPT removal from TBT – and TPT – contaminated natural seawater obtained from Cape Town harbour and the results obtained show that 99.71 %, 79.23 %, 80.11 %, 82.86 %, 80.42 %, 99.75 %, 99.88 %, 99.83 % and 99.88 % TBT were removed from TBT – contaminated natural seawater by activated carbon, fly ash, nFe3O4, nSiO2, nZnO, fly ash/activated carbon, nFe3O4/activated carbon, nSiO2/activated carbon and nZnO/activated carbon composite, respectively while 99.90 %, 96.44 %, 95.37 %, 96.75 %, 97.03 %, 99.92 %, 98.42 %, 98.92 % and 99.58 % TPT were removed from TPT – contaminated natural seawater by activated carbon, fly ash, nFe3O4, nSiO2, nZnO, fly ash/activated carbon, nFe3O4/fly ash, nSiO2/fly ash and nZnO/fly ash composite, respectively. Experimental results therefore show that the composite materials present higher organotin adsorption efficiency than the precursors due to the nature and improved properties of the composite materials and can therefore be utilized for the remediation of organotin contamination from industrial and/or shipyards process wastewater to > 99 % reduction before discharge into the environment.

No description available.

Thesis (M.S.)--Duquesne University, 2006. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p.72) and index.

Abstract Persistent organic pollutants (POPs) are ubiquitous and bioaccumulative man-made chemicals, resistant to chemical, biological and photolytic degradation and widely distributed to sediments, wildlife, and human. Many of these chemicals have adverse effects on a variety of targets, including the endocrine system, organogenesis and reproduction. Due to these effects and wide distribution, many of them are either banned or strictly controlled. However, because of persistency, they continue to interact with organisms globally. Despite the existing knowledge of the adverse effects of POPs, the effects of many chemicals on bone tissue are still poorly known. In the present study, we investigated the adverse effects of three common POPs, including tributyltin (TBT), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and perfluorooctanoic acid (PFOA) on the skeletal system. In vitro models were used to study the effects of PFOA in mouse and in human, and the co-effects of TBT and TCDD on differentiating osteoblasts and osteoclasts of mice. An in vivo model for mice was used to study the developmental effects of maternal PFOA-exposure on pups among with morphometrical and biomechanical property analyses. Mass-spectrometry was used to study the presence of PFOA in bones both in mice and in human, the latter acquired from the bone bank held in the Oulu University Hospital, Finland. The bones were also analyzed with cone beam computer tomography and microcomputer tomography. The results show that PFOA exposure in utero and during lactation leads to the accumulation of PFOA in bone, traceable even 17 months after exposure. PFOA exposure decreased the mineral density of the tibias and increased the medullary area. Nearly all of the human samples contained PFAS, including PFOA. PFOA also disturbed the differentiation of osteoblasts and with lower doses, increased bone resorption of osteoclasts both in mouse and human, the phenomenon being slightly stronger in mice. Co-exposure to TBT and TCDD led to decreased differentiation of osteoblasts and osteoclast, and the co-effect was partially synergistic in osteoblasts. These results show disruption of bone development, bone cell differentiation, and PFAS accumulation in bone. Further studies are recommended to evaluate the co-effects of different POPs and the possible effects of long-term accumulation of POPs in bone and other tissues. / Tiivistelmä Pysyvät orgaaniset ympäristömyrkyt (POP-yhdisteet) ovat kemikaaleja, jotka ovat levinneet ihmisen toiminnan seurauksena laajalle ympäristöön, sen eliöihin ja ihmisiin. Monilla POP-yhdisteillä on haitallisia vaikutuksia esimerkiksi hormonaaliseen toimintaan, elinten muodostukseen ja hedelmällisyyteen. Toksisten vaikutusten ja niiden yleisyyden vuoksi monien POP-yhdisteiden käyttö on joko rajattua tai kielletty kokonaan. Laajan levinneisyytensä ja hitaan puoliintumisaikansa takia POP-yhdisteet ovat kuitenkin edelleen vuorovaikutuksessa ympäristön ja sen eliöiden kanssa. POP-yhdisteiden luustovaikutuksista tiedetään edelleen vähän. Tässä väitöskirjassa tutkittiin kolmen yleisen POP-yhdisteen, tributyylitinan (TBT), 2,3,7,8-tetraklooridibentso-p-dioksiinin (TCDD) ja perfluoro-oktaanihapon (PFOA), vaikutuksia luustoon. PFOA:n vaikutuksia hiiren ja ihmisen luustoon sekä TBT:n ja TCDD:n yhteisvaikutuksia hiiren erilaistuvien osteoblastien ja osteoklastien suhteen selvitettiin in vitro -malleilla. In vivo -mallilla tutkittiin hiiriemon PFOA-altistuksen vaikutusta syntyvien poikasten luuston kehitykseen ja remodelaatioon analysoimalla poikkileikekuvia sekä luiden biomekaanisia ominaisuuksia. Lisäksi luiden PFOA-pitoisuudet mitattiin massaspektrometrilla. Tutkimusta laajennettiin ihmiseen analysoimalla Oulun yliopistollisen sairaalan luupankkinäytteitä. Ihmisnäytteet analysoitiin myös kartiokeila-TT:n ja mikro-TT:n avulla. Tulosten mukaan PFOA kertyy luuhun; hiiriltä voitiin mitata PFOA-pitoisuuksia jopa 17 kuukautta altistumisen jälkeen. Lisäksi PFOA-altistus pienensi luun mineraalitiheyttä ja kasvatti luuydinontelon tilavuutta. Lähes kaikki ihmisluunäytteet sisälsivät PFOA:ta ja muita PFAS-yhdisteitä. Solukokeiden perusteella PFOA-altistus häiritsee osteoblastien erilaistumista ja pienillä pitoisuuksilla lisää osteoklastien luunhajotusta sekä hiirellä että ihmisellä. TBT:n ja TCDD:n yhteisaltistus vaikuttaa puolestaan vähentävän sekä osteoblastien että osteoklastien erilaistumista ja toimintaa; osteoblastien osalta yhteisvaikutus oli osaksi synergistinen. Väitöskirja antaa lisätietoa POP-yhdisteiden vaikutuksista luun kehitykseen ja luusolujen erilaistumiseen sekä PFAS-yhdisteiden kertymisestä luuhun. Väitöksessä myös suositellaan lisätutkimuksia yhdisteiden yhteisvaikutuksista sekä pitkän aikavälin ympäristökemikaalikertymän vaikutuksista luussa ja muissa kudoksissa.

No description available.

Le mercure est présent dans l'environnement à cause de phénomènes naturels (volcans) ou des activités humaines (combustion de combustibles fossiles). Le mercure existe sous forme de mercure élémentaire (Hg0), ionique (HgII) ou organique tel le méthylmercure (MeHg). Ces diverses formes sont en flux constant les uns avec les autres dans le cycle biogéochimique naturel. De par leur grande hydrophobicité et leur capacité à pénétrer les membranes biologiques, les composés organomercuriels contituent la forme la plus toxique de mercure retrouvée dans l’environnement Des niveaux élevés de MeHg ont d’ailleurs été détectés dans la chaire de poissons de nombreuses régions du monde. Conséquemment, une consommation de produits de la mer contaminés représente un grave danger pour la santé humaine. Certaines bactéries isolées à partir d'environnements contaminés par le mercure ont évolué vers un système qui leur permet de convertir efficacement les composés mercuriels présents autant sous forme ionique qu’organique en un mercure élémentaire moins toxique. Cette résistance au mercure s’explique par l'acquisition d'un élément génétique connu sous le nom d’opéron mer. L’opéron mer code entre autre pour deux enzymes importants : la lyase organomercurielle MerB et la réductase mercurielle MerA. MerA catalyse la réduction du HgII conduisant à la formation du mercure élémentaire Hg0 qui est un composé volatile et moins toxique. MerB, quant à elle, catalyse la protonolyse de la liaison carbone-mercure de composés organomercuriels pour produire un composé réduit de carbone et du mercure ionique (HgII). Au vu des effets des organomercuriels et de la réduction de HgII, MerA et MerB sont considérés comme des enzymes clés pouvant servir à la biorestauration des cours d'eau contaminés par les organomercuriels. Une compréhension claire des détails mécanistiques de la façon dont MerA et MerB fonctionnent ensemble au niveau atomique est donc cruciale dans la mise en œuvre de biotechnologies implicant l’opéron mer dans les efforts de bioremédiation. Dans cette étude, nous avons utilisé la résonance magnétique nucléaire (RMN)et la cristallographie aux rayons X pour caractériser la structure et le mécanisme enzymatique de MerB de E. coli. Sur la base d’études structurales précédentes de MerB de E. coli, trois résidus (Cys96, Asp99 et Cys159) ont été identifiés comme constituant la triade catalytique nécessaire au clivage de la liaison carbone-Hg. En guise de suivi aux études antérieures, mon projet consiste d’abord à utiliser la cristallographie aux rayons X afin de définir les rôles de Cys96, Asp99 et Cys159 dans la liaison du substrat et dans le clivage. Deux approches ont été mises en œuvre pour atteindre cet objectif. Tout d'abord, les mutants MerB ont été testés pour définir le rôle des résidus catalytiques. Deuxièmement, les inhibiteurs de MerB et d'autres substrats non organicomercuriels potentiels ont été utilisés pour explorer le site actif de MerB. Une sérine se retrouve à la position de Asp99 dans quatre variants de MerB répertoriés chez les bactéries. Pour mieux comprendre le rôle de Asp99, nous avons comparé la sérine présente dans le variants MerB de Bacillus megaterium (MerB2) et introduit un variant D99S à la protéine MerB du type sauvage d’E. coli (MerB D99S). Nous avons pu constater que la forme purifiée de MerB D99S se caractérisait par une couleur rose après avoir visualisé sa structure cristalline aux rayons X, révélant la présence d'un métal lié au niveau de son site actif. Les analyses par spectrométrie de masse à plasma à couplage inductif (ICP-MS) et par fluorescence des rayons X indiquèrent que MerB D99S se liait au cuivre au niveau du site actif. En outre, les analyses par résonance paramagnétique électronique (EPR) et des études de RMN ont identifié la forme CuII du cuivre. L'addition de substrats organomercuriels a pu déplacer le CuII entrainant ainsi une diminution de l’activité catalytique de MerB D99S. En revanche, MerB2 n'a pu être co-purifié avec le cuivre, bien que la structure aux rayons X du complexe MerB2-Hg soit pratiquement identique à la structure du complexe MerB D99S-Hg. Ceci suggère que le résidu Asp99 est essentiel au clivage des liaisons carbone-Hg de composés organiques du mercure et dirige la spécificité de la liaison au métal. De plus, la liaison cuivre-MerB D99S propose un lien possible entre l'évolution de MerB et son homologue structural, la protéine NosL. Dans la seconde approche, nous nous sommes intéressés au site actif de MerB en testant sa liaison à des composés organostanniques et à des composés organoplombiques avec un inhibiteur de MerB connu sous le nom de triéthylétain (TET) qui se lie au résidu Asp99 sans s’associer aux cystéines du site actif. Une liaison similaire a été observée avec un autre inhibiteur à savoir le triméthylplomb (TML). Quant au diméthylétain (DMT), il inhibe MerB à l'aide d'un mécanisme alternatif en se liant d'abord à Asp99 puis à Cys96 conduisant à un changement critique dans le site actif perturbant ainsi l’interaction π-cation entre Trp95 et Arg155. D’autres inhibiteurs comme le diéthylétain (DET) et le diéthylplomb (DEL) ont été caractérisés comme étant un substrat de MerB où les deux groupes éthyle ont été clivés pour donner les produits ioniques SnIV PbIV qui se lient au site actif de manière similaire à HgII. DMT, DET et DEL présentent une affinité pour la liaison à MerB supérieure à celle de son substrat initial MeHg. Ces résultats suggèrent que les composés organomercuriels ne sont pas les seuls substrats pour MerB et Asp99 est le premier résidu à se lier aux composés organométalliques suivis de la liaison à Cys96 et Cys159. Ces observations suggèrent un agrandissement de l’éventail d'applications possibles pour MerB dans la bioremédiation de certains sites contaminés par des composés organométalliques tels les organoplombiques et organostanniques. Mot-clé: Organomercuriallyase, Merb, organoplombiques. Organostanniques, protéine de liaison cuivre, carbone liaison métallique clivage, méthylmercure, Organomercuriels, biorestauration, résonance magnétique nucléaire, la cristallographie aux rayons X. / Mercury is introduced into the environment from either natural occurrences (volcanoes) or from human activities (combustion of fossil fuels). Mercury exists as elemental mercury (Hg0), ionic mercury (HgII) or organic mercury like methylmercury (MeHg) and these forms are in constant flux with each other as part of the natural biogeochemical cycle. Organomercurial compounds like MeHg are the most toxic form because of their hydrophobicity and their ability to efficiently permeate membranes and bioaccumulate in organisms. High levels of MeHg have been found in fish in many areas around the world, and therefore human consumption of contaminated seafood represents a serious danger for human health. Bacteria isolated from mercury-contaminated environments have evolved a system that allows them to efficiently convert both ionic and organic mercury compounds to the less toxic elemental mercury. The mercury resistance is due to the acquisition of a transferable genetic element known as the mer operon. The mer operon encodes for several proteins including two enzymes, the organomercurial lyase MerB and the mercuric ion reductase MerA. MerB catalyzes the protonolysis of the carbon-mercury bond of organomercurial compounds to produce a reduced-carbon compound and inorganic ionic mercury HgII. MerA catalyzes the reduction of HgII to elemental mercury Hg0, which is volatile and less toxic. Due to their ability to cleave MeHg and reduce the resulting HgII product, MerB and MerA are considered crucial to bioremediation efforts to clean up MeHg from contaminated waterways. A clear understanding of the mechanistic details of how MerB and MerA function together at the atomic level is crucial for appropriate utilization of the mer system in bioremediation efforts. We have been using nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to structurally and mechanistically characterize E. coli MerB. Based on previous structural studies of E. coli MerB, three residues (Cys96, Asp99 and Cys159) have been identified as a catalytic triad which is required for carbon-Hg bond cleavage. As a follow up to the earlier studies, my project involves using X-ray crystallography to define the roles of Cys96, Asp99 and Cys159 in substrate binding and cleavage. Two different approaches were implemented to fulfill this goal. Firstly, MerB mutants were tested to define the role for the catalytic residues. Secondly, MerB inhibitors and other potential non-organomercurial substrates were used to probe MerB active site. The Cys,-Asp-Cys catalytic triad found in E.coli MerB is conserved in all MerB variants except four variants where aspartic acid is replaced by a serine. To understand the role of Asp99, we compared a serine-containing MerB variant (Bacillus megaterium MerB2) and an E. coli MerB mutant (MerB D99S) to wild type E. coli MerB. Interestingly, the purified MerB D99S protein was found to contain a pink color. X-ray crystal structure indicated the presence of a bound metal in the active site of MerB D99S. Analysis by inductively coupled plasma mass spectrometry (ICP-MS) and X-ray fluorescence indicated that MerB D99S binds copper in the active site. Further, electron paramagnetic resonance (EPR) and NMR studies identified the copper as CuII. Addition of organomercurial substrate displaces bound CuII but MerB D99S shows diminished catalytic activity. In contrast, MerB2 did not co-purify with copper although the X-ray structure of MerB2-Hg complex is virtually identical to the structure of the MerB D99S-Hg. This suggests that the aspartic acid residue is crucial for the cleavage of carbon-Hg bonds of organomercurials as well as metal-binding specificity. Furthermore, the binding of copper to the MerB D99S protein suggests a possible evolutionary link between MerB and its structural homolog, the copper-binding protein NosL. In the second approach, we probed the active site of MerB through testing its binding to organotin and organolead compounds. The known MerB inhibitor triethyltin (TET) binds to Asp99 without binding to any of the active site cysteines. A similar binding has been observed with trimethylead (TML). Dimethyltin (DMT) inhibits MerB using an alternative mechanism. It first binds to Asp99 then Cys96, which induces a dramatic change in the active site by disrupting a cation-π interaction between Try95 and Arg155. In contrast, diethyltin (DET) and diethylead (DEL) were found to be substrates for MerB, where both ethyl groups were cleaved and the SnIV and PbIV products bound to the active site in a similar manner to HgII. DMT, DET and DEL show higher binding affinity to MerB than its initial substrate MeHg. These results suggest that organomercurials may not be the only substrates for MerB and Asp99 is the first residue to bind to organometals followed by subsequent binding to Cys96 and Cys159. In addition, these observations suggest that there are other possible applications for employing MerB in bioremediation of organolead and organotin contaminated sites while other organometals may have implications when using MerB in bioremediation systems. Keyword: Organomercuriallyase, MerB, Organolead. Organotin, Copper binding protein, Carbon metal bond cleavage, Methylmercury, Organomercuriels, Bioremédiation, Nuclear magnetic resonance, X ray crystallography.

[pt] A liberação de compostos butílicos de estanho, como o tributilestanho (TBT), presentes em tintas anti-incrustantes de embarcações marítimas, tem provocado efeitos nocivos sobre a reprodução e o crescimento das várias formas de vida marinha. Em resposta a estes riscos ambientais, vários países regulamentaram ou baniram o uso destes produtos anti-incrustantes. No Brasil, não existe ainda qualquer controle sobre o uso destes compostos em embarcações trafegando em território nacional. O presente trabalho visou estudar a presença de tributilestanho (TBT) e de seus produtos de degradação, dibutilestanho (DBT) e monobutilestanho (MBT), em sedimentos superficiais provenientes de 17 estações de coleta na Baía de Todos os Santos. A seleção das estações para o estudo proposto foi baseada no histórico do fluxo de navios cargueiros, presença de marinas e pólos industriais localizados próximos a cada estação. A especiação dos compostos orgânicos de estanho foi realizada por cromatografia em fase gasosa com detecção por fotometria de chama pulsante, após extração por solvente, derivação, limpeza dos extratos e eliminação de enxofre. As maiores concentrações detectadas (em ng g-1 sedimento, como Sn) para os diferentes compostos organoestânicos foram: TBT (15,9), DBT (28,8) e MBT (4). Os sedimentos superficiais analisados encontram-se levemente contaminados, segundo a classificação reportada por Waite e colaboradores (1991), mas mostram valores bem inferiores aos encontrados na Baía de Guanabara (Almeida et al., 2004), possivelmente devido a propriedades como transparência da água, que favorece a fotodegradação, e ambiente sedimentar oxidante. / [en] The organotin compounds such as tributyltin (TBT) present in anti-fouling paints applied to ships, pleasure boats and vessels are proved to have harmful effects on the reproduction and the growth of several forms of marine life. In response to this environmental threat many countries introduced restriction to their use or even banishment. In Brazil there are no such rules on the use of these compounds. The present work aimed at the speciation of organotin compounds (tributyltin (TBT) and its of degradation products, dibutyltin (DBT) and monobutyltin (MBT)) in surface layer of sediments collected from 15 stations in the Todos os Santos Bay. The selection of stations was based on information on ship traffic, presence of marinas and industries installation. Organotin speciation was carried out by gas chromatography with pulsed flame photometric detection after solvent extraction, derivatization, clean-up and sulfur elimination. The highest organotin concentrations (ng g -1 as tin) found were: TBT (15,9), DBT (28,8) e MBT (4). Sediments in the studied locations are slightly contaminated, according to the classification reported by Waite and collaborators (1991). Values are lower than those found in Guanabara Bay (Almeida et al., 2004) possibly due to the higher water transparence that favors photo degradation, and to the oxidizing sedimentary environment.