A commercial GC-AFS instrument has been developed and optimised for the speciation of organomercury. This instrument couples a GC oven to a modified atomic fluorescence detector via a ceramic pyrolyser. Organomercury compounds in dichloromethane solvent were directly injected through a Programmable Temperature Vaporiser Injector onto a DBl Megabore column. Once separated, the compounds eluted from the column and were atomised in the pyrolyser then detected by AFS. The direct injection technique, ceramic pyrolysis design and argon purged detector have improved previous instrument designs by enhancing and maintaining sensitivity. The instrumental limit of detection was determined to be 0.25 pg Hg absolute. Methods were developed for the extraction of methylmercury from a variety of marine samples. The techniques were validated using mussel homogenate and dogfish liver (IAEA 142, SRM 8044 and DOLT-2) certified reference materials. An interlaboratory comparision exercise was participated in and a method was developed for the detemination of methylmercury in Fucus sea plant (IAEA 140). A concentration of 0.63 ± 0.006 ng g-1 was reported. The material is now certified at 0.626 +0.139 ng g-1. Of all the participating laboratories, this was the closest result to the certified value. The instrument and methods were also applied to soil and sediment samples. Once again validation was performed with a CRM sediment, IAEA 356. Although this material has been reported to give positive artifact formation when using a steam distillation sample preparation procedure, good agreement and no artifects were observed upon analysis. A further contaminated land, an uncontaminated soil and sediment sample were also studied. For all the samples studied by GC-AFS total mercury measurements were also made following an appropriate digestion procedure and CV-AFS. A gas chromatograph was also coupled with ICP-MS and HPLC was coupled to CV-AFS as comparative techniques. Both approaches were optimised and validated with CRM's. The GC-ICP-MS had the advantage of providing additional element information and confirmed the presence of methylmercury bromide in the final mussel homogenate extract. The HPLC approach found to be much less sensitive than the GC techniques and also suffered from vapour generation interferences. The PTV injector was considered for large volume injection and thermal desorption techniques. Injector breakdown problems were overcome by optimising the conditions and solid phase adsorbent for cold splitless injection. A recovery of 70% was achieved for a 50 ul large volume injection of methylmercury chloride in DCM. This technique indicated the possibility that LVI may in the future offer increased method sensitivity.

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Made available in DSpace on 2014-10-09T12:28:00Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:28Z (GMT). No. of bitstreams: 0 / O Reservatório Rio Grande, localizado na Região Metropolitana de São Paulo (RMSP), é um importante manancial de abastecimento de água dessa região. Foram analisadas amostras de água de fundo e de sedimentos coletadas nesse reservatório em 4 pontos, em 4 campanhas, de setembro de 2008 a janeiro de 2010, sendo estas quantificadas em relação à concentração de Hg total pela técnica de CV AAS. Em seguida, adaptou-se uma metodologia da literatura para quantificação de Hg orgânico em sedimentos, que consistiu na extração dos compostos organomercuriais com diclorometano em meio ácido e, posterior destruição dos compostos orgânicos com cloreto de bromo. A validação dessa metodologia, em termos de precisão e exatidão, foi realizada por meio da análise dos materiais de referência BCR CRM 580 e IAEA 405. Para avaliação dos mecanismos de metilação e desmetilação de Hg nesse ambiente, foram determinados os seguintes parâmetros físicos e químicos, in situ: pH, temperatura da água, potencial redox (EH), transparência e profundidade. Nos sedimentos foram determinados também a granulometria, carbono orgânico total, bactérias redutoras de sulfato, N total e P total, além de Co, Cu, Fe e Mn. A seleção destes parâmetros esteve relacionada diretamente a fatores que influenciam o comportamento do MeHg em sedimentos e sua zona de transição. Foram encontradas concentrações para Hg total variando entre 1,0 e 71,0 mg kg- 1 e de Hg orgânico entre <10,0 a 47,2 &mu;g kg-1 e taxas de metilação entre 0,06 e 1,4% em sedimentos, ao longo do reservatório. Discutiram-se também as diferentes condições de metilação e suas influências, ao longo do reservatório. Como estudo suplementar, avaliou-se a concentração de outros metais e elementos traço presentes nos sedimentos, por meio da técnica de ativação neutrônica. Foram quantificados os elementos As, Ba, Br, Co, Cr, Cs, Hf, Fe, Na, Rb, Sb, Sc, Zn e os elementos terras raras Ce, Eu, La, Lu, Nd, Sm, Tb e Yb. Os fatores de enriquecimento calculados em relação aos valores da crosta terrestre utilizando-se Sc como elemento normalizador apresentaram valores acima de 2,0 para os elementos As, Br, Sb, U e Zn. Verificou-se uma forte influência antrópica para alguns dos elementos analisados, principalmente nos pontos localizados na entrada do reservatório, que recebem descarga de efluentes domésticos e industriais dos rios que alimentam o reservatório. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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[pt] Neste estudo, avaliou-se a influência de alguns parâmetros ecológicos e biológicos sobre a acumulação dos elementos- traço e mercúrio orgânico (HgOrg)no fígado e rins de espécies de cetáceos de hábitos costeiros (Pontoporia blainvillei, N = 31; Sotalia guianensis, N = 21) e oceânicos (Stenella coeruleoalba, N = 3; Stenella frontalis, N = 4), acidentalmente capturados em duas áreas distintas da costa brasileira (tropical - litoral do norte do estado do Rio de Janeiro e subtropical - litoral do estado do Rio Grande do Sul). Assim, foram gerados novos dados a respeito das concentrações destes elementos em órgãos de cetáceos marinhos ainda pouco estudados, especialmente em uma região com pouca informação como a porção oeste do Oceano Atlântico Sul. Para tal, foram aplicados métodos analíticos baseados na técnica da espectrometria de absorção atômica (AAS), utilizando a espectrometria de absorção atômica com forno de grafite (GF-AAS) e a espectrometria de absorção atômica com vapor frio (CVAAS), as mais frequentemente aplicadas para a determinação de elementos-traço e mercúrio respectivamente, em amostras biológicas. Estas técnicas apresentaram ótima precisão e exatidão comprovadas através da análise de materiais de referência certificados (CRM) do National Research Council - Canadá (DORM-2, DOLT-2 e TORT-2). Foram observadas diferenças regionais (região tropical e subtropical) e inter-específicas (golfinhos de hábitos costeiro e oceânico) sobre a acumulação dos elementos- traço e HgOrg nos órgãos estudados. As espécies de hábito oceânico apresentaram as maiores concentrações destes elementos em seus órgãos. O sexo dos indivíduos não influiu na bioacumulação dos elementos e HgOrg. Os parâmetros população, comprimento total, e maturidade sexual, influenciaram na acumulação dos elementos-traço e HgOrg em ambos os órgãos, de acordo com a espécie e a área de captura. S. guianensis, co- habita o mesmo ambiente que P. blainvillei, e preferencialmente se alimenta de peixes maiores e predadores, neste estudo, apresentou a razão molar de 1:1 entre o Se e o Hg,mostrando assim, que provavelmente o processo de destoxificação de metilmercúrio hepático esteja ocorrendo. A espécie P. blainvillei se alimenta preferencialmente de cefalópodes e peixes pequenos, e apresentou uma razão molar de 4:1 entre o Se e o Hg no fígado. Os metais de transição Cd e Ag, que também interagem com o Se hepático nos mamíferos, apresentaram correlações significativas no fígado dos cetáceos marinhos estudados. A espécie S. guianensis apresentou correlações significativas entre as concentrações molares de Se e Cd (p = 0,01) e Se e Ag (p < 0,001) no fígado. Para a espécie P. blainvillei, os indivíduos provenientes de ambas as regiões (tropical e subtropical) apresentaram correlações significativas entre as concentrações molares de Se e Hg (p < 0,001) no fígado. Somente os indivíduos de P. blainvillei capturados na região tropical apresentaram correlações significativas entre as concentrações molares de Se e Cd (p < 0,001). Não só as diferentes condições ambientais, mas também, o tempo de vida, o estágio de crescimento e, principalmente a preferência alimentar, influenciaram fortemente sobre a acumulação de elementos- traço e HgOrg nos tecidos dos cetáceos assim como, sobre suas inter-relações. / [en] The present work tested whether ecological and biological parameters have an influence on the accumulation of trace elements and organic mercury (HgOrg) by the liver and kidney of coastal (Pontoporia blainvillei, N = 31; Sotalia guianensis, N = 21) and oceanic (Stenella coeruleoalba, N = 3; Stenella frontalis, N = 4) cetaceans species incidentally caught in fishing nets along two Brazilian coastal areas (tropical - northern Rio de Janeiro State and subtropical -Rio Grande do Sul State). This research added new insight to the existing studies with trace element concentrations in internal organs of marine cetaceans from the southwestern Atlantic Ocean. Few studies have documented trace element concentrations in marine mammals in this area. Analytical methodologies based on atomic absorption spectrometry (AAS), using the graphite furnace (GF-AAS) and cold vapor (CV-AAS) techniques were applied for this purpose. They are the most popular analytical techniques applied for trace element and mercury determination, respectively, in biological samples. Precision and accuracy of the analytical methods were determined and monitored using certified material from National Research Council - Canada (DORM-2, DOLT-2 and TORT- 2). Interspecies (coastal and oceanic cetacean species) and regional (tropical and subtropical regions) differences were observed in the accumulation of trace elements and organic mercury in organs, liver and kidney. The highest concentrations of these elements were observed in the liver of the oceanic cetacean species. However, no gender differences were found on the bioaccumulation of trace elements and also organic mercury. Biological parameters such as, population, body length and sexual maturity influenced on the trace element and organic mercury accumulation by both organs, according to the cetacean species and caught area. S. guianensis, which co- habits the same coastal marine environment that P. blainvillei species, was the only dolphin species that presented a Se:Hg molar ratio close to 1:1, showing that a probable detoxification process can be occurring in the liver of S. guianensis. This dolphin species feeds mainly on predatory fish, while P. blainvillei feeds mainly on fish and cephalopods. P. blainvillei presented a Se:Hg molar ratio close to 4:1. It`s known that transition metals, such as Hg, Cd and Ag, have an interaction with Se in the liver of marine mammals. The molar concentrations of Cd (p = 0.01) and Ag (p < 0.001) presented significant correlations with the hepatic molar Se in the liver of S. guianensis. P. blainvillei from both sampling areas presented a high significant correlation for molar Se to Hg concentrations (p < 0.001) in liver. Meanwhile, only individuals of P. blainvillei from tropical region showed a significant correlation with hepatic Se and Cd (p < 0.001). Based on the results present here, it is possible to conclude that, not only the different environmental conditions, but also life span, growth stage but mainly the feeding habit influenced the accumulation of trace elements and organic mercury by cetacean tissues. These ecological and biological parameters influenced on the inter-element relationships.

The effects of 1 min 4 hr exposures to mercuric chloride (HgCl2), methyl mercuric chloride (CH3HgCl), p-chloromercuribenzoate (p-CMB) and ethylmercurithiosalicylate (TMS) on cell viability and kinetics of cell death, microtubules, F-actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P), intracellular calcium [Ca2+]i and responses to polarized signals in YAC-1 lymphoma cells were investigated. We hypothesized that immunotoxic effects of HgCl2 (Hg2+) are initiated by global receptor triggering, accompanied by increased protein tyrosine phosphorylation (PTyr-P) and down-regulation of the T-cell receptor (TCR). As a polychloride anion with poor lipid solubility, inorganic Hg2+ may produce effects at the outer cell membrane before significant intracellular accumulation, loss of microtubule integrity (a sensitive target) and activation of cell death through apoptotic pathways. The organomercurial compound p-CMB is likewise thought to penetrate membranes slowly as a result of ionization. In contrast, the highly lipid-soluble organomercurial compounds CH3HgCl and TMS were expected to reduce responses to polarized stimuli only in conjunction with and not prior to loss of microtubule integrity and the onset of necrotic cell death. <p>Two general patterns of effects were observed. In HgCl2-treated YAC-1 cells, inhibition of responses to polarized stimuli preceded loss of microtubules and onset of cell death. Effects on polarized stimuli were preceded by a transient Ca2+ signal; however, this Ca2+ signal appeared abortive, accompanied by a paradoxic decrease in PTyr-P and partial down-regulation of CD3 receptors. Responses to polarised stimuli were inhibited prior to extensive loss of microtubule staining, indicating effects preceded cytosolic Hg2+ accumulation. HgCl2 exposure was followed rapidly by necrotic cell death. <p>Similarly, p-CMB-treated YAC-1 cells failed to respond to polarized stimuli before effects on microtubules or loss of viability, and proceeded rapidly to late apoptosis; however, a transient Ca2+ signal and progressive loss of F-actin preceded effects in all other assays and may account for loss of polarized responses. <p>In CH3HgCl- and TMS-treated YAC-1 cells, CD3 receptor expression, [Ca2+] and PTyr-P were increased immediately, along with loss of microtubules. These reductions preceded inhibition of polarized signaling responses and seemed to indicate a general loss of cellular homeostasis not seen in HgCl2- and p-CMB-treated cells; loss of homeostasis did not necessarily produce simultaneous loss of viability, as TMS-treated cells remained viable for 30 min while CH3HgCl-treated cells became apoptotic within 1 min. Nonetheless, the YAC-1 cells proceeded to cell death more slowly, remaining early apoptotic after 4 hr, when almost all HgCl2- and p-CMB-treated cells were necrotic. These findings indicate the two groups of mercury compounds may alter responses to polarized stimuli and induce cell death by distinct pathways, one involving an apparently abortive signal and the other mediated by much more profound disruption of cellular homeostasis. Within the larger patterns there are further differences between the effects produced by each Hg compound, likely reflecting the combined influence of pharmacokinetic and dynamic factors governing access to and interactions with different cellular targets leading to cell death. These distinct targets may in turn be reflected in the different immune effects produced by these compounds <i>in vivo</i>.

The effects of 1 min 4 hr exposures to mercuric chloride (HgCl2), methyl mercuric chloride (CH3HgCl), p-chloromercuribenzoate (p-CMB) and ethylmercurithiosalicylate (TMS) on cell viability and kinetics of cell death, microtubules, F-actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P), intracellular calcium [Ca2+]i and responses to polarized signals in YAC-1 lymphoma cells were investigated. We hypothesized that immunotoxic effects of HgCl2 (Hg2+) are initiated by global receptor triggering, accompanied by increased protein tyrosine phosphorylation (PTyr-P) and down-regulation of the T-cell receptor (TCR). As a polychloride anion with poor lipid solubility, inorganic Hg2+ may produce effects at the outer cell membrane before significant intracellular accumulation, loss of microtubule integrity (a sensitive target) and activation of cell death through apoptotic pathways. The organomercurial compound p-CMB is likewise thought to penetrate membranes slowly as a result of ionization. In contrast, the highly lipid-soluble organomercurial compounds CH3HgCl and TMS were expected to reduce responses to polarized stimuli only in conjunction with and not prior to loss of microtubule integrity and the onset of necrotic cell death. <p>Two general patterns of effects were observed. In HgCl2-treated YAC-1 cells, inhibition of responses to polarized stimuli preceded loss of microtubules and onset of cell death. Effects on polarized stimuli were preceded by a transient Ca2+ signal; however, this Ca2+ signal appeared abortive, accompanied by a paradoxic decrease in PTyr-P and partial down-regulation of CD3 receptors. Responses to polarised stimuli were inhibited prior to extensive loss of microtubule staining, indicating effects preceded cytosolic Hg2+ accumulation. HgCl2 exposure was followed rapidly by necrotic cell death. <p>Similarly, p-CMB-treated YAC-1 cells failed to respond to polarized stimuli before effects on microtubules or loss of viability, and proceeded rapidly to late apoptosis; however, a transient Ca2+ signal and progressive loss of F-actin preceded effects in all other assays and may account for loss of polarized responses. <p>In CH3HgCl- and TMS-treated YAC-1 cells, CD3 receptor expression, [Ca2+] and PTyr-P were increased immediately, along with loss of microtubules. These reductions preceded inhibition of polarized signaling responses and seemed to indicate a general loss of cellular homeostasis not seen in HgCl2- and p-CMB-treated cells; loss of homeostasis did not necessarily produce simultaneous loss of viability, as TMS-treated cells remained viable for 30 min while CH3HgCl-treated cells became apoptotic within 1 min. Nonetheless, the YAC-1 cells proceeded to cell death more slowly, remaining early apoptotic after 4 hr, when almost all HgCl2- and p-CMB-treated cells were necrotic. These findings indicate the two groups of mercury compounds may alter responses to polarized stimuli and induce cell death by distinct pathways, one involving an apparently abortive signal and the other mediated by much more profound disruption of cellular homeostasis. Within the larger patterns there are further differences between the effects produced by each Hg compound, likely reflecting the combined influence of pharmacokinetic and dynamic factors governing access to and interactions with different cellular targets leading to cell death. These distinct targets may in turn be reflected in the different immune effects produced by these compounds <i>in vivo</i>.

This study examined mercury and methylmercury concentrations in shark species in order to determine differences between shark species and between locations. Stable isotope ratios were determined to see if interaction existed between approximate trophic level and mercury concentrations. Sharks were sampled from Florida Bay where muscle tissue biopsies and blood samples were extracted upon capture. Stable isotope ratios for carbon-13 (δ13C) and nitrogen-15 (δ15N) from freezedried blood samples were compared with levels of organic and inorganic mercury species from tissue samples in seven different shark species, focusing on blacknose, blacktip, bull, and lemon sharks. Pre-caudal length (PCL) was strongly correlated to mercury concentration and δ13C but not with δ15N. Ratios of δ15N between shark species were in agreement with observed trophic-level behavior of blacktip sharks feeding on blacknose sharks, indicated by greater values for δ15N ratios in blacktip sharks. Calculated length-normalized stable isotope ratio values correlated strongly to values for mercury concentrations in blacktip sharks, indicating a potential method for forecasting inorganic or organic mercury concentrations without the use of more expensive mercury tests. This calculation also provided validity for a new metric involving the use of stable carbon isotope ratio divided by length (PCL) to be compared with other metrics. Correlations between δ15N values and mercury values indicate some level of interaction between trophic level and quantity of mercury contaminants in shark muscle tissue.