Rozprawy doktorskie na temat „Tributyltin”

Tributyltin (TBT) and its degradation products dibutyltin (DBT) and monobutyltin (MBT) have been quantitatively analyzed in environmental water samples using gas chromatography with flame photometric detection and gas chromatography/mass spectrometry (GC/MS). The butyltins were extracted from environmental samples with hexane/0.2% tropolone and derivatized with hexyl magnesium bromide to form hexylbutyltins. Full scanning GC/MS was used for confirmation of peak identifications and quantification was done by selective ion monitoring (methane chemical ionization) at m/z 319 (TBT) and m/z 347 (DBT, MBT and tripentyltin, the internal standard). Calibration curves were linear and detection limits were less than 2 ngL&\sp{lcub}-1{rcub}&. GC/MS and GC with flame photometric detection were compared as quantification methods and were shown to give similar results at the low ngL&\sp{lcub}-1{rcub}& levels. Water samples from Southern Chesapeake Bay were analyzed for TBT in areas of high boating activity. Several sites around a marina were sampled at repeated intervals. High spatial and temporal variability was noted. Reproducible concentration gradients were apparent, with the highest TBT levels near marinas and boatyards. Equilibrium sorption of TBT was measured on selected estuarine and freshwater sediments. Isotherms from twenty-four hour sorption and desorption equilibrations were linear with sorption coefficients between 1.1 &\times& 10&\sp2& and 8.2 &\times& 10&\sp3& Lkg&\sp{lcub}-1{rcub}&. Sorption coefficients decreased with increasing salinity and varied by a factor of two over the salinity range 0-34&\perthous&. Desorption kinetics were measured and indicated an initial fast rate followed by a slower rate. Water and sediment concentrations for TBT at locations in the Chesapeake Bay system were used to calculate apparent sorption coefficients that generally agreed with laboratory measured sorption coefficients. Exceptionally high apparent sorption coefficients were found near areas of high vessel activity and may be the result of TBT paint chips in the sediment.

Tributyltin compounds have been used on a global scale for many decades now, and thus have become global environmental contaminants. These compounds are highly toxic, and deleterious effects on numerous organisms have been demonstrated. Yet, little is known of the molecular mechanisms of tributyltin's extreme toxicity. To obtain a better understanding of such mechanisms, a luxAB gene-fusion library of Escherichia coli was screened for changes in gene expression upon cellular exposure to tributyltin. Two clones, designated TBT1 and TBT3, were thus found, both showing an increased light emission in the presence of added tributyltin. Northern blotting analyses confirmed a marked increase in the transcription of the tributyltin-responsive gene identified from each clone. These genes appear to play a protective role when cells are exposed to tributyltin at concentrations ≧ 10 mug/ml, with minimal-dose responses of 0.1 mug/l when grown on LB media. Speciation studies indicated TBT+ as the active chemical species in eliciting these responses. Mapping and sequencing of these tributyltin-responsive genes revealed that the luxAB reporter element had inserted within the uhpT gene in the TBT1 clone. This gene encodes a sugar-phosphate transporter protein, which has been shown to be up-regulated by external glucose-6-phosphate and 2-deoxyglucose-6-phosphate. On a kinetic level, the increased expression of uhpT by tributyltin closely mirrors that produced by 2-deoxyglucose-6-phosphate. In addition to tributyltin, TBT1 also responds to dibutyltin, monobutyltin, trimethyltin, triethyltin, tripropyltin, trifluoroacetic acid, and vanadium. Similar mapping and sequencing experiments revealed the luxAB reporter genes within the stpA gene in the TBT3 clone, but in an antisense orientation, such that they were not under the regulatory control of stpA. The lack of an appropriate open reading frame for this 140-nucleotide transcript (identified by Northern blo

Tributyltin is arguably the most toxic compound ever to be deliberately introduced into the marine environment as an ingredient of antifouling paints. It has had widespread toxic effects on a range of marine organisms, with some gastropod species being particularly sensitive. Effects of TBT on non-target species have resulted in partial bans on its use in many countries, so that new inputs to the water column have decreased in most areas. One of the physicochemical features of TBT is that it is readily sequestered by suspended particulates due to its low solubility and its hydrophobicity, therefore becoming incorporated into estuarine sediments. The availability of this sediment-bound TBT has been investigated through its potential for re-release back to the water column, and directly from the sediment using the sediment dwelling gastropod Hinia reticulata. The sorption process itself has been investigated using natural components to determine the sediment-water partition coefficient (Kd) together with factors affecting its magnitude. Sorption by sediments has been shown to be rapid (minutes), although the achievement of equilibrium may take longer (hours), and exhibits a Freundlich-like dependence on the TBT concentration due to the variable energies of TBT sorption sites on sediment particles. The major determinant of Kd is sediment type, greater adsorption occurring in fine-grained organic rich sediments compared to low organic sands; although both salinity and pH modify the degree of adsorption. The sorption process has been shown to be reversible, so that previously contaminated sediments may act as reservoirs of TBT, releasing the compound back to the overlying water for many years. Hinia reticulate has been shown to be an effective and quantitative accumulator of both dissolved and sediment-bound TBT, principally acquiring TBT from water across the respiratory surfaces. When additionally exposed to sediments, significantly higher body burdens were accumulated, with up to 80% of the total attributable to the sediment. Uptake of TBT across the surface of the headtfoot appears to be an important pathway for sediment-exposed Hinia reticulata, while the ingestion of contaminated sediment does not appear to occur. Hinia reticulata is capable of metabolising TBT to lesser butylated and presumably less toxic products which are excreted, making its accumulated body burdens responsive to changing environmental TBT levels, and increasing its value as a biomonitor When exposed to a range of TBT contaminated sediments, Hinia reticulata showed there to be greater TBT availability from sediments with a low sorptive capacity (sands), principally through desorption of TBT to the overlying water. Fine-grained organic-rich muds, which have a greater capacity for TBT, produced lower accumulated burdens in Hinia reticulata, but may represent more important long-term sources of TBT to benthic organisms in estuaries.

Doutoramento em Biologia Molecular
O tributilestanho (TBT) é considerado um dos xenobióticos mais tóxicos, produzidos e deliberadamente introduzidos no meio ambiente pelo Homem. Tem sido usado numa variedade de processos industriais e subsequentemente descarregado no meio ambiente. O tempo de meia-vida do TBT em águas marinhas é de várias semanas, mas em condições de anóxia nos sedimentos, pode ser de vários anos, devido à sua degradação mais lenta. Embora o TBT tenha sido descrito como sendo tóxico para eucariotas e procariotas, muitas bactérias podem ser resistentes a este composto. O presente trabalho teve como objetivo principal elucidar o mecanismo de resistência ao TBT em bactérias. Para além disso, pretendeu-se desenvolver um biorepórter para detectar TBT no ambiente. Para atingir estes objetivos foram delineadas várias tarefas cujos principais resultados obtidos se apresentam a seguir. Várias bactérias resistentes ao TBT foram isoladas de sedimento e água do Porto de Pesca Longínqua (PPL) na Ria de Aveiro, Portugal. Entre estas, Aeromonas molluscorum Av27 foi selecionada devido à sua elevada resistência a este composto (concentrações até 3 mM), à sua capacidade de degradar o TBT em compostos menos tóxicos (dibutilestanho, DBT e monobutilestanho, MBT) e também por usar o TBT como fonte de carbono. A. molluscorum Av27 foi caracterizada genotipica e fenotipicamente. Os fatores de virulência estudados mostraram que esta estirpe i) possui atividade lipolítica; ii) não é citotóxica para células de mamíferos, nomeadamente para células Vero; iii) não possui integrões de classe I e II e iv) possui cinco plasmídeos com aproximadamente 4 kb, 7 kb, 10 kb, 100 kb e mais de 100 kb. Estes resultados mostraram que a estirpe Av27 não é tóxica, aumentando assim o interesse nesta bactéria para futuras aplicações, nomeadamente na bioremediação. Os testes de toxicidade ao TBT mostraram que este composto tem um impacto negativo no crescimento desta estirpe, bem como, na densidade, no tamanho e na atividade metabólica das células e é responsável pela formação de agregados celulares. Assim, o TBT mostrou ser bastante tóxico para as bactérias interferindo com a atividade celular geral. O gene Av27-sugE, que codifica a proteína SugE pertencente à família das “small multidrug resistance proteins” (SMR), foi identificado como estando envolvido na resistência ao TBT nesta estirpe. Este gene mostrou ser sobreexpresso quando as células crescem na presença de TBT. O promotor do gene Av27-sugE foi utilizado para construir um bioreporter para detetar TBT, contendo o gene da luciferase do pirilampo como gene repórter. O biorepórter obtido reúne as características mais importantes de um bom biorepórter: sensibilidade (intervalo de limite de detecção de 1-1000 nM), rapidez (3 h são suficientes para a deteção de sinal) e, possivelmente, não é invasivo (pois foi construído numa bactéria ambiental). Usando sedimento recolhido no Porto de Pesca Longínqua da Ria de Aveiro, foi preparada uma experiência de microcosmos com o intuito de avaliar a capacidade de Av27 para bioremediar o TBT, isoladamente ou em associação com a comunidade bacteriana indígena. A análise das amostras de microcosmos por PCR-DGGE e de bibliotecas de 16S rDNA revelaram que a comunidade bacteriana é relativamente estável ao longo do tempo, mesmo quando Av27 é inoculada no sedimento. Para além disso, o sedimento estuarino demonstrou ser dominado por bactérias pertencentes ao filo Proteobacteria (sendo mais abundante as Delta e Gammaproteobacteria) e Bacteroidetes. Ainda, cerca de 13% dos clones bacterianos não revelaram nenhuma semelhança com qualquer dos filos já definidos e quase 100% afiliou com bactérias não cultiváveis do sedimento. No momento da conclusão desta tese, os resultados da análise química de compostos organoestânicos não estavam disponíveis, e por essa razão não foi possível tirar quaisquer conclusões sobre a capacidade desta bactéria remediar o TBT em sedimentos. Esses resultados irão ajudar a esclarecer o papel de A. molluscorum Av27 na remediação de TBT. Recentemente, a capacidade da estirpe Av27 remediar solo contaminado com TBT foi confirmada em bioensaios realizados com plantas, Brassica rapa e Triticum aestivum (Silva 2011a), e também com invertebrados Porcellionides pruinosus (Silva 2011B). Assim, poder-se-á esperar que a bioremediação do sedimento na experiência de microcosmos também tenha ocorrido. No entanto, só a análise química dos compostos organostânicos deverá ser conclusiva. Devido à dificuldade em realizar a análise analítica de organoestânicos, um método de bioensaio fácil, rápido e barato foi adaptado para avaliar a toxicidade do TBT em laboratório, antes de se proceder à análise química das amostras. O método provou a sua utilidade, embora tenha mostrado pouca sensibilidade quando se usam concentrações de TBT baixas. Em geral, os resultados obtidos contribuíram para um melhor entendimento do mecanismo de resistência ao TBT em bactérias e mostraram o potencial biotecnológico de A. molluscorum Av27, nomeadamente, no que refere à sua possível aplicação na descontaminação de TBT no ambiente e também no desenvolvimento de biorepórteres.
Tributyltin (TBT) is considered as one of the most toxic xenobiotics, ever produced and deliberately introduced into the environment by Man. It has been used in a variety of industrial processes and subsequently discharged into the environment. The typical half-life of TBT in marine waters is of several weeks, but in deeper anoxic sediments it may be of several years, due to its slower degradation. Although TBT has been reported to be toxic to both eukaryotes and prokaryotes, bacteria can be resistant to this compound. In the present work a major goal was outlined: to elucidate the TBT resistance mechanism in bacteria. Additionally, it was also an objective developing a bioreporter to detect TBT in the environment. For this purpose a number of tasks were delineated and the main results obtained are bellow described. Several resistant bacteria were isolated from sediment and water at Porto de Pesca Longínqua (PPL) site in Ria de Aveiro, Portugal. Among those, Aeromonas molluscorum Av27 was selected due to its high resistance to TBT (up to 3 mM), its ability to degrade this compound into less toxic compounds (dibutyltin, DBT and monobutyltin, MBT) and also because it uses TBT as carbon source. A. molluscorum Av27 was genotypically and phenotypically characterized. The virulence factors studied showed that Av27 strain: i) has lipolytic activity; ii) is not cytotoxic for mammal cells, namely Vero cells; iii) does not have class I and II integrons and iv) possesses five plasmids with approximately 4 kbp, 7 kbp, 10 kbp, 100 kbp and more than 100 kbp. These results demonstrated that Av27 strain is not toxic, increasing the interest on this bacterium for future applications, namely bioremediation. Different growth parameters were studied revealing that a temperature of 25°C, a salinity range from 1 to 4% and a pH between 6 and 9 were the optimal growth conditions to Av27 strain. Regarding the antibiotic resistance profile, A. molluscorum Av27 is resistant to penicillin (10 μg), amoxicillin/clavulanic acid (30 μg) and cephalothin (30 μg) and also to the vibriostatic agent O/129; moreover, Av27 strain uses various compounds as carbon and energy source. TBT toxicity tests showed that this compound has a negative impact on bacterial growth, cell density, cell size and metabolic activity and promoted the formation of cell aggregates. Thus, TBT was found to be very toxic for this bacterium interfering with general cell activity. A gene, Av27-sugE, encoding the SugE protein belonging to the small multidrug resistance (SMR) proteins family was identified to be involved in the TBT resistance in this strain. This gene showed to be over-expressed when cells grow in the presence of TBT. Based on the Av27-sugE putative promoter, a bioreporter for TBT was constructed, containing the firefly luciferase as the reporter gene. The obtained bioreporter gathers the most important characteristics of a good bioreporter: sensitivity (detection limit range from 1-1000 nM), fastness (3 h for signal detection) and possibly non-invasive (it was constructed in an environmental bacterium). A microcosm experiment using estuarine sediment from PPL, Ria de Aveiro, was established to evaluate the ability of Av27 to bioremediate TBT, alone or in association with the indigenous bacterial community. PCR-DGGE and 16S rDNA libraries analysis of the microcosm samples revealed that the bacterial community is relatively stable over time, even when Av27 is inoculated in the sediment. Additionally, the estuarine sediment demonstrated to be dominated by bacteria belonging to the phyla Proteobacteria (among the most abundant Delta and Gammaproteobacteria) and Bacteroidetes. Moreover, about 13% of the bacterial clones showed no similarity to any of the phyla already defined and almost 100% affiliated with unculturable sediment bacteria. At the time of conclusion of this thesis, the results of organotin compounds chemical analysis were not available; therefore it was not possible to draw any conclusions about the TBT remediation in microcosm. Those results will help clarify the role of A. molluscorum Av27 in the remediation of TBT. Recently the ability of Av27 strain to remediate soil contaminated with TBT was confirmed in bioassays performed with plants, Brassica rapa and Triticum aestivum (Silva 2011a) and also invertebrates Porcellionides pruinosus (Silva 2011b). Thus it could also be expected that bioremediation of the sediment from the microcosm experiment has also occurred. Still only the organotin chemical analysis shall be conclusive. Due to the difficulty in performing analytical analysis, an easy, rapid, and inexpensive bioassay method was adapted to monitor TBT toxicity in the laboratory, prior to chemical analysis. The method proved its usefulness albeit showing low sensitivity when low TBT concentrations are used. Overall, the results obtained contributed to the elucidation of TBT resistance in bacteria and revealed the biotechnological potential of A. molluscorum Av27, namely in its possible application to decontaminating TBT in the natural environment and also in the development of bioreporters.

Mestrado em Toxicologia e Ecotoxicologia
Organotins are an important class man made organometallic compounds and tributyltin (TBT) is one of the most studied chemicals within this class. TBT is a potent endocrine disruptor being also considered as an obesogenic, immunotoxic and neurotoxic compound. Humans were exposed to this and other organotin compounds as a consequence of their widespread commercial applications including plastic stabilizers, catalytic agents and industrial biocides. Their utilization as catalytic agents in the production of silicones leads to the presence of these chemicals in silicone based products including those used in biomedical applications such as breast implants and cardiac valves which may constitute a potential source of exposure. According to the World Health Organization cardiovascular diseases are sharply increasing and constitute the prime cause of death globally. Taking this into account, regulatory agencies, recommend the study of organotin compounds toxicity. Considering the limited number of studies on the cardiovascular effects of organotins, the present thesis aims to elucidate the effects of TBT at the vascular level. The study of TBT effect on the contractility of rat artery (aorta) was performed by the organ bath technique and the L-type calcium channels in A7r5 (cell line derived from the smooth muscle of embryonic rat aorta) was measured by whole-cell configuration of the patch clamp technique. The obtained results demonstrated that TBT seems to relax the rat aorta without endothelium contracted by noradrenaline and potassium chloride but this effect is not significantly different from the respectively ethanol control. The electrophysiological experiments demonstrated that the inhibition of the calcium current by the L-type calcium channels in the A7r5 vascular smooth muscle cells was also not significantly different, which suggests that probably the mode of action of TBT is more complex and involves other pathways.
Os compostos orgânicos de estanho são uma importante classe de compostos organometálicos produzidos pelo homem, e o tributilestanho (TBT) é um dos químicos mais estudados dentro desta classe. O TBT atua como um potente disruptor endócrino, sendo também considerado como um composto obesogénico, imunotóxico e neurotóxico. A exposição humana a este e outros compostos orgânicos de estanho deve-se à sua vasta utilização em aplicações comerciais como estabilizadores de plásticos, agentes catalíticos e biocidas industriais. A sua utilização como agentes catalíticos na produção de silicones faz com que estes compostos estejam presentes em vários produtos à base de silicone, incluindo os usados em aplicações biomédicas como implantes mamários e válvulas cardíacas, o que pode constituir uma fonte potencial de exposição para os humanos. De acordo com a Organização Mundial de Saúde, as doenças cardiovasculares estão a aumentar e são consideradas como a principal causa de morte a nível mundial. Tendo isto em conta, as agências reguladoras recomendam o estudo da toxicidade dos compostos orgânicos de estanho. Considerando o número limitado de estudos sobre os efeitos cardiovasculares dos compostos orgânicos de estanho, o presente trabalho pretende elucidar os efeitos do TBT ao nível vascular. Os efeitos do TBT na contractilidade de artérias de rato (aorta) foram estudados pela técnica do banho de órgãos e a medição dos canais de cálcio tipo L foi realizada em A7r5 (linha celular de músculo liso vascular derivada de aorta embrionária de rato) através da técnica do patch clamp na configuração whole cell. Os resultados obtidos demonstram que o TBT parece induzir relaxamento nas artérias sem endotélio contraídas previamente com noradrenalina e com cloreto de potássio, mas esse efeito não é significativamente diferente do controlo de solvente usado. Nas experiências de eletrofisiologia a inibição das correntes de cálcio através dos canais de cálcio tipo L nas células A7r5 também não mostrou ser significativamente diferente do controlo, o que parece demonstrar que o modo de ação pelo qual o TBT induz efeito nas células vasculares é mais complexo e envolve outras vias.

Tributyltin (TBT) is a pollutant, mainly introduced to the environment as a marine anti-fouling agent. The aim of this work was to assess and develop sustainable and cost-effective remediation technologies for TBT-contaminated dredged materials. For this purpose, analytical methods were developed for sediments and sediment leachates. For the sediments, a triple extraction followed by derivatisation and measurement by gas chromatography with pulsed flame photometric detection was employed, avoiding the lengthy concentration step of the organic layer. The TBT detection limit of ca 0.04 mg Sn/kg in sediment was below the suggested limit of 0.1 mg/kg for sea disposal of TBT-contaminated dredgings (OSPAR Commission). For the leachates, derivatisation and extraction into hexane was used. Also, a new procedure, with the potential for automation, was developed for the simultaneous analysis of multiple water samples, based on in situ extraction and derivatisation on C18 solid phase extraction cartridges. No legislative limits existed for TBT in leachates, therefore the detection limits of ca 6-10 ng Sn/L achieved were regarded satisfactory, as they were below or similar to the EQS for coastal and estuarine waters or freshwaters (2-20 ng/L TBT). A pilot investigation was carried out on a dockyard to evaluate the use of X-Ray fluorescence as a screening method for the presence of TBT in sediments. Due to tin contamination such a technique was not suitable for the site examined. Incineration was found to remove TBT but it would incur very high costs. Ultrasonic destruction was not effective enough, even on TBT-spiked water solutions. Carbon products, pure clays, organically modified clays, zero valent iron, fly ash and cements were screened for their abilities to prevent TBT leaching, using a leaching test. The best performer was a powdered activated carbon product which, even mixed with cement that increases the leaching of TBT, delivered a TBT-free (< 5 ng Sn/L) leaching test result 33 days after the mixing. The result showed that this technique could provide a solution for the immobilisation of TBT in contaminated dredgings by mixing this relatively low-cost, multi-purpose and inert additive, with or without cement according to the site specific requirements.

An improved understanding of the geochemical partitioning of trace metals and tributyltin (TBT) in sediments is of great importance in risk assessment and remedial investigation. The aim of this thesis was to examine the distribution and partitioning behaviour of trace metals and TBT in benthic, estuarine sediments. This was achieved by a series of field- and laboratory-based studies investigating factors controlling the geochemical behaviour of trace metals and TBT in sediments from south-east Queensland, Australia. The distribution and enrichment of selected trace metals in benthic sediments of the Southport Broadwater (a semi-enclosed coastal body of water adjacent to the Gold Coast city, south-eastern Queensland, Australia) was studied. Sediment contamination for Cd, Cr, Cu, Ni, Pb, Sn and Zn was assessed by (1) comparison with Australian sediment quality guidelines, (2) calculation of the index of geoaccumulation based on regional background values, and (3) geochemical noi-malisation against Al (i.e. the abundance of alumino-silicate clay minerals). Based on this approach, several sites were found to be strongly enriched with Cu, Pb, Sn and Zn, arising from sources related to either urban runoff or vessel maintenance activities. The geochemical partitioning of Cu, Pb and Zn was examined in sediments collected from three of these sites of sediment contamination. Total Cu, Pb and Zn concentrations in coarse-textured (65 to 90 % sand sized particles), sub-oxic sediments (Eh + 120 to +260 mV) ranged from 8.3 to 194 mg/kg for Cu, 16.3 to 74.8 mg/kg for Pb and 30.1 to 220 mg/kg for Zn, and were related to vertical trends in sediment texture. The association of Cu, Pb and Zn with amorphous oxides, crystalline oxides and organic matter was linearly dependent on the abundance of each phase. For retention by amorphous oxide minerals, the trace metal retention ranged from 5.2 to 23.7 mgcjgFe oxide as Fe for Cu, 1 2.8 to 21 .5 mgpb/gFe oxide as Fe for Pb, and 23. I to 85.7 mgm/gFe oxide as Fe for Zn. Corresponding values for association with crystalline oxides were an order of magnitude less than those for amorphous oxides, indicating a weaker affinity of trace metals for crystalline oxides. The relationships describing association with organic matter ranged from 17.6 to 54.0 mgcu/gorg c for Cu, 6.1 to 9.6 mgpb/gorg c for Pb and 6.4 to 16.4 mgzn/gorg c for Zn. The in-situ solid/pore-water partitioning of TBT and the degradation products, dibutyltin (DBT) and monobutyltin (MBT), was determined for an estuarine sediment profile with previously identified elevated Sn concentrations. Total butyltin levels were (depending on depth) 220 to 8750 jig/kg for TBT, 150 to 5450 jig/kg for DBT and 130 to 4250 jig/kg for MBT. Pore-water butyltin concentrations ranged from 0.05 to 2.35 jig/L for TBT, 0.07 to 3.25 jiglL for DBT, and 0.05 to 0.53 J.tgIL for MBT. The organic carbon normalised distribution ratios (Doc) were similar for TBT, DBT and MBT, and were io to 106 L/kg. Values for the Butyltin Degradation Index (BDI) were larger than I at depths greater than 10 cm below the sediment/water-column interface, indicating that substantial TBT degradation has occurred in the sediments. This suggests that natural attenuation may be a viable sediment remediation strategy. Factors controlling the partitioning behaviour of Cu, Pb and Zn in nonsulfidic, estuarine sediments were examined in controlled laboratory-based studies with the use of combined sorption curve - sequential extraction analysis. This allowed determination of sorption parameters for Cu, Pb and Zn partitioning to individual geochemical fractions. Partitioning behaviour in sulfidic sediments was also determined by sequentially extracting Cu, Pb and Zn from synthetic sulfide minerals, and from natural sediment and pure quartz sand after spiking with acid-volatile sulfide (AVS). Trace metal sorption to the 'carbonate' fraction (pH 5, NaOAc extraction) increased with metal loading due to saturation of sorption sites associated with the 'Fe-oxide' (NH2OH.HCI extraction) and 'organic' (H202 extraction) fractions in non-sulfidic sediments. Freundlich isotherm parameters describing sorption to the 'Fe-oxide' and 'organic' fractions were dependent on the sediment Fe-oxide and organic carbon content, respectively. Sequential extraction of Cu from pure CuS, AVS-spiked sediment and AVS-spiked quartz sand showed that AVS-bound Cu was quantitatively recovered in association with the 'organic' fraction. However, some AVS-bound Pb and Zn were recovered by the NH2OH.HCI step (which has been previously interpreted as 'Fe-oxide' bound metals) in the sequential extraction procedure used in this study. This indicates that the sequential extraction of Pb and Zn in sulfidic sediments may lead to AVS-bound metals being mistaken as Fe-oxide bound species. Caution should therefore be exercised when interpreting sequential extraction results for Pb and Zn in anoxic sediments. Tributyltin (TBT) sorption to four natural sediment samples in artificial seawater was also examined under a range of modified pH and salinity conditions in controlled laboratory-based studies. Three of the sediment samples were relatively pristine with regard to TBT contamination, but the fourth was a TBT-contaminated sediment from a commercial marina. Sorption of TBT was described well by linear sorption isotherms, with distribution coefficients ranging from 6.1 to 5210 L/kg depending on pH and salinity. Sediment organic C content and particle size distribution were important determinants of sorption behaviour. The presence of resident TBT in the contaminated marina sediment caused a substantial reduction in TBT sorption due to satuaration of high selectivity sites. Desorption of TBT from the marina sediment was described by relatively large observed distribution coefficients ranging from 5100 to 9400 L/kg, suggesting that aging effects may reduce sorption reversibility. Increased artificial seawater salinity generally reduced TBT sorption at pH 4 and pH 6, but enhanced TBT sorption at pH 8. Regardless of salinity, maximum sorption of TBT was observed at pH 6, which is attributed to an optimal balance between abundance of the cationic TBT species and deprotonated surface ligands. Consideration of aqueous TBT speciation along with octanol-water partitioning behaviour suggest that hydrophobic partitioning of TBTCI to non-polar organic matter was important for pH (up to) 6, whilst partitioning of TBTOH was important at higher pH. The effect of aging on the solid/pore-water partitioning and desorption behaviour of TBT in sediments was examined. Three sediment samples with contrasting physical and chemical properties were spiked with 10 mg/kg TBT and aged under sterile conditions for periods of time ranging from I to 84 days. Aging had a negligible effect in a sandy sample with very low organic carbon content (0.2 % w/w). In contrast, for samples with larger amounts of organic carbon (2.6 and 4.8 % w/w), the effect of aging on the solid/pore-water partitioning behaviour was significant. For these samples, the apparent distribution coefficients (Ks) obtained from sequential two hour desorption experiments exhibited a two-fold increase between spiked sediments subjected to aging for 1 day and 84 days. This study demonstrates that aging effects may be an important aspect of TBT fate in contaminated sediments. Overall, the results described in this thesis demonstrate that environmental factors (i.e. pH, salinity, Eh, aging) and sediment composition (i.e. abundance of fine alumino-silicate minerals, organic matter, Fe-oxides, reactive sulfides) exert substantial effects on trace metal and TBT partitioning. The current reliance on measurement of total trace metal and TBT concentrations in contaminated sediment management may consequently lead to inaccurate estimates of environmental risk and inappropriate remediation measures if other factors regulating contaminant distribution and partitioning are ignored.

An improved understanding of the geochemical partitioning of trace metals and tributyltin (TBT) in sediments is of great importance in risk assessment and remedial investigation. The aim of this thesis was to examine the distribution and partitioning behaviour of trace metals and TBT in benthic, estuarine sediments. This was achieved by a series of field- and laboratory-based studies investigating factors controlling the geochemical behaviour of trace metals and TBT in sediments from south-east Queensland, Australia. The distribution and enrichment of selected trace metals in benthic sediments of the Southport Broadwater (a semi-enclosed coastal body of water adjacent to the Gold Coast city, south-eastern Queensland, Australia) was studied. Sediment contamination for Cd, Cr, Cu, Ni, Pb, Sn and Zn was assessed by (1) comparison with Australian sediment quality guidelines, (2) calculation of the index of geoaccumulation based on regional background values, and (3) geochemical noi-malisation against Al (i.e. the abundance of alumino-silicate clay minerals). Based on this approach, several sites were found to be strongly enriched with Cu, Pb, Sn and Zn, arising from sources related to either urban runoff or vessel maintenance activities. The geochemical partitioning of Cu, Pb and Zn was examined in sediments collected from three of these sites of sediment contamination. Total Cu, Pb and Zn concentrations in coarse-textured (65 to 90 % sand sized particles), sub-oxic sediments (Eh + 120 to +260 mV) ranged from 8.3 to 194 mg/kg for Cu, 16.3 to 74.8 mg/kg for Pb and 30.1 to 220 mg/kg for Zn, and were related to vertical trends in sediment texture. The association of Cu, Pb and Zn with amorphous oxides, crystalline oxides and organic matter was linearly dependent on the abundance of each phase. For retention by amorphous oxide minerals, the trace metal retention ranged from 5.2 to 23.7 mgcjgFe oxide as Fe for Cu, 1 2.8 to 21 .5 mgpb/gFe oxide as Fe for Pb, and 23. I to 85.7 mgm/gFe oxide as Fe for Zn. Corresponding values for association with crystalline oxides were an order of magnitude less than those for amorphous oxides, indicating a weaker affinity of trace metals for crystalline oxides. The relationships describing association with organic matter ranged from 17.6 to 54.0 mgcu/gorg c for Cu, 6.1 to 9.6 mgpb/gorg c for Pb and 6.4 to 16.4 mgzn/gorg c for Zn. The in-situ solid/pore-water partitioning of TBT and the degradation products, dibutyltin (DBT) and monobutyltin (MBT), was determined for an estuarine sediment profile with previously identified elevated Sn concentrations. Total butyltin levels were (depending on depth) 220 to 8750 jig/kg for TBT, 150 to 5450 jig/kg for DBT and 130 to 4250 jig/kg for MBT. Pore-water butyltin concentrations ranged from 0.05 to 2.35 jig/L for TBT, 0.07 to 3.25 jiglL for DBT, and 0.05 to 0.53 J.tgIL for MBT. The organic carbon normalised distribution ratios (Doc) were similar for TBT, DBT and MBT, and were io to 106 L/kg. Values for the Butyltin Degradation Index (BDI) were larger than I at depths greater than 10 cm below the sediment/water-column interface, indicating that substantial TBT degradation has occurred in the sediments. This suggests that natural attenuation may be a viable sediment remediation strategy. Factors controlling the partitioning behaviour of Cu, Pb and Zn in nonsulfidic, estuarine sediments were examined in controlled laboratory-based studies with the use of combined sorption curve - sequential extraction analysis. This allowed determination of sorption parameters for Cu, Pb and Zn partitioning to individual geochemical fractions. Partitioning behaviour in sulfidic sediments was also determined by sequentially extracting Cu, Pb and Zn from synthetic sulfide minerals, and from natural sediment and pure quartz sand after spiking with acid-volatile sulfide (AVS). Trace metal sorption to the 'carbonate' fraction (pH 5, NaOAc extraction) increased with metal loading due to saturation of sorption sites associated with the 'Fe-oxide' (NH2OH.HCI extraction) and 'organic' (H202 extraction) fractions in non-sulfidic sediments. Freundlich isotherm parameters describing sorption to the 'Fe-oxide' and 'organic' fractions were dependent on the sediment Fe-oxide and organic carbon content, respectively. Sequential extraction of Cu from pure CuS, AVS-spiked sediment and AVS-spiked quartz sand showed that AVS-bound Cu was quantitatively recovered in association with the 'organic' fraction. However, some AVS-bound Pb and Zn were recovered by the NH2OH.HCI step (which has been previously interpreted as 'Fe-oxide' bound metals) in the sequential extraction procedure used in this study. This indicates that the sequential extraction of Pb and Zn in sulfidic sediments may lead to AVS-bound metals being mistaken as Fe-oxide bound species. Caution should therefore be exercised when interpreting sequential extraction results for Pb and Zn in anoxic sediments. Tributyltin (TBT) sorption to four natural sediment samples in artificial seawater was also examined under a range of modified pH and salinity conditions in controlled laboratory-based studies. Three of the sediment samples were relatively pristine with regard to TBT contamination, but the fourth was a TBT-contaminated sediment from a commercial marina. Sorption of TBT was described well by linear sorption isotherms, with distribution coefficients ranging from 6.1 to 5210 L/kg depending on pH and salinity. Sediment organic C content and particle size distribution were important determinants of sorption behaviour. The presence of resident TBT in the contaminated marina sediment caused a substantial reduction in TBT sorption due to satuaration of high selectivity sites. Desorption of TBT from the marina sediment was described by relatively large observed distribution coefficients ranging from 5100 to 9400 L/kg, suggesting that aging effects may reduce sorption reversibility. Increased artificial seawater salinity generally reduced TBT sorption at pH 4 and pH 6, but enhanced TBT sorption at pH 8. Regardless of salinity, maximum sorption of TBT was observed at pH 6, which is attributed to an optimal balance between abundance of the cationic TBT species and deprotonated surface ligands. Consideration of aqueous TBT speciation along with octanol-water partitioning behaviour suggest that hydrophobic partitioning of TBTCI to non-polar organic matter was important for pH (up to) 6, whilst partitioning of TBTOH was important at higher pH. The effect of aging on the solid/pore-water partitioning and desorption behaviour of TBT in sediments was examined. Three sediment samples with contrasting physical and chemical properties were spiked with 10 mg/kg TBT and aged under sterile conditions for periods of time ranging from I to 84 days. Aging had a negligible effect in a sandy sample with very low organic carbon content (0.2 % w/w). In contrast, for samples with larger amounts of organic carbon (2.6 and 4.8 % w/w), the effect of aging on the solid/pore-water partitioning behaviour was significant. For these samples, the apparent distribution coefficients (Ks) obtained from sequential two hour desorption experiments exhibited a two-fold increase between spiked sediments subjected to aging for 1 day and 84 days. This study demonstrates that aging effects may be an important aspect of TBT fate in contaminated sediments. Overall, the results described in this thesis demonstrate that environmental factors (i.e. pH, salinity, Eh, aging) and sediment composition (i.e. abundance of fine alumino-silicate minerals, organic matter, Fe-oxides, reactive sulfides) exert substantial effects on trace metal and TBT partitioning. The current reliance on measurement of total trace metal and TBT concentrations in contaminated sediment management may consequently lead to inaccurate estimates of environmental risk and inappropriate remediation measures if other factors regulating contaminant distribution and partitioning are ignored.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environmental Engineering
Full Text

The adsorption of tributyltin onto materials commonly used for the construction of sampling and analytical equipment from aqueous solutions of varying ionic composition has been examined. The adsorption appears to be controlled by non-polar interactions between tributyltin and the surface involved. Since the speciation and hence polarity of tributyltin is affected by the ionic composition of the medium, the extent of adsorption is affected by the salinity and pH of a sample. The adsorption is rapid and, unless strategies are adopted to eliminate its effects, may render analytical results invalid. The electrochemistry of tributyltin, dibutyltin and monobutyltin, individually and in mixtures, has been investigated in aqueous media at mercury electrodes. The basic electrochemistry of each compound is summarised by the reaction BunSn (4-n)+ + (4-n)e- right left harpoons BunSn where n is the number of butyl groups attached to the tin atom. However, the electrochemistry of each compound is largely confined to the surface of the mercury electrode, and the simplicity of the above reaction is disrupted by polymerisation reactions and by butyl exchange processes occurring with the mercury electrode. When mixtures of butyltin compounds are present, the various processes that occur for each individual compound interfere with each other. A direct voltaminetric method for the determination of butyltin compounds in natural waters is therefore probably not possible.

The detrimental effects of tributyltin (TBT) have been recorded on many marine organisms. As a result the UK Government imposed a partial ban on the use of organotin antifouling paints on boats less than 25 m in length, in 1987. In 1988 the Isle of Man Government followed suit introducing a licensing procedure restricting all uses of organotins. At concentrations less than 0.5 ng Sn/I female Nucella lap/Nus develop imposex - the superimposition of male sexual characteristics. To date there have been few studies measuring the recovery of Nucella populations after the introduction of restrictions. This study produces evidence of the extent of recovery in Nucella populations from sites in the south-west of England and on the Isle of Man. The recovery observed was measured by decreasing values of relative penis size, vas deferens sequence and the percentage of sterile adult females in the population. Following the 1987 ban the recovery of Nucella populations in the south-west has shown a linear response allowing predictions to be made for the time scale of complete recovery. In addition concentrations of TBT in the water and tissues of selected indicator organisms also showed decreases. Around the Isle of Man the illegal use of TBT paints was identified and later discouraged by the Marine Administration which was followed by a reduction in TBT concentrations in the water at sites around the Isle of Man. Levels of imposex in dogwhelk populations around the Isle of Man have decreased. Although effects of TBT on Nuce/la have been well documented at the cellular and individual level, the knock on effects on the community have not been investigated. Manipulative field experiments were used to demonstrate the role of Nucella lapd/us in structuring shore communities to allow predictions of the effect of TBT to be made. Rather than using the traditional approach of fences and cages, dogwhelks were removed by hand on regular visits to experimental sites creating treatments with reduced abundances of dogwhelks akin to shores affected by TBT. The role of Nucel/a was examined at different stages of a cycle existing on moderately exposed Manx shores where Fucus vesiculosus and Semibe/anus balanoides fluctuate in abundance. The removal of dogwhelks increased the abundance of Semibalanus ba/anoides on the shore and as a result likelihood of algal escapes from grazing by Patella vulgate also increased. In addition the removal of Nucela increased the size and longevity of newly established Fucus vesiculosus clumps. In a factorial experiment the role of Patella vulgate and Nucella lapillus were examined simultaneously. Nuce/la was found to have an significant effect but less than that of Patella. The presence of Nucella did, however, mediate the effect of Patella. In addition Nucella was found to have a direct effect on the level of Semibalanus balanoides settlement in the field with the number of barnacles settling in cleared areas being reduced on areas which had been previously occupied by Nucella.

Estuarine environments are threatened by the synergistic effects of anthropogenic disturbance and bioinvasion. The construction of artificial structures (such as pilings and pontoons) provides a habitat resource in close proximity to vessel hulls that may be carrying a wide range of non-indigenous fouling species. In addition, the release and accumulation of toxicants from antifouling (AF) paints on vessel hulls creates a chemical disturbance that may reduce the invasion resistance of native communities. This thesis examines how shipping-related disturbances affect sessile communities, and in particular what role AF paints and artificial structures play in the invasion of estuaries. Using a series of field-based experiments, I found that copper and tributyltin have the potential to influence both the transport of species to a new region, via the application of AF paints on vessel hulls, and their subsequent establishment, via the accumulation of AF biocides in estuaries. Temperature, pH, salinity and turbidity were also related to species distributions. During subtidal surveys of artificial and natural structures I found more non-indigenous species (NIS) on pilings and pontoons than on rocky reef, and shallow floating structures were identified as hotspots for invaders. These findings suggest that artificial structures play an important role in the initial establishment of sessile non-indigenous species in new regions. A subset of NIS were also present on the reefs sampled during the survey and I conducted manipulative experiments to determine factors affecting the invasibility of turf and canopy-forming algal assemblages. The resident assemblage provided a barrier to most invaders, particularly when light and sedimentation levels were also high (i.e. on horizontal substrate). My results suggest that the areas of reef most susceptible to invasion are vertical rock walls and those subjected to disturbances that release space. In summary, this is the first study to relate copper and tributyltin contamination in the field to NIS distributions. My research has also highlighted the potential role of artificial structures in facilitating the establishment of NIS in estuaries and identified invasion threats to rock wall communities and disturbed reefs in estuarine systems.

Tributyltin (TBT) was used as an anti-fouling paint and applied to the hulls of ships in order to prevent buildup of organisms. Major shipping routes are now contaminated with TBT due to the chemical's persistence In the environment. The use of TBT as an anti-fouling paint was banned in the 1980's due to scientific findings that the chemical causes toxic effects in aquatic life. TBT is widely known as a gender-bending chemical due to its endocrine disrupting properties. There are concerns that TBT may also present health problems for humans, and is classed as a Persistent Organic Pollutant (POP). The disposal of sediments contaminated with TBT presents a problem, as disposal to sea will cause unacceptable pollution of the environment. An Integrated pollution control strategy is needed for disposing of dredged sediments in a manner which is cost-effective and sustainable.

The effects of tributyltin (TBT) on phorbol myristate acetate (PMA)- and calcium ionophore (A23187)-stimulated peritoneal macrophages from oyster toadfish (Opsanus tau) were evaluated in a luminol-dependent chemiluminescence (CL) assay. PMA-stimulated CL responses were increased by in vitro exposure to 50 ug/L TBT, but depressed to baseline values by 500 ug/L TBT. A23187-stimulated CL increased with 5 ug/L and 50 ug/L TBT, and depressed by 500 ug/L TBT. Responses stimulated by synergistic doses of PMA and A23187 were increased by 50 ug/L TBT and depressed by 500 ug/L TBT. Enhanced PMA- and A23187-stimulated CL following TBT exposure returned to baseline values after extracellular calcium was removed. In the absence of PMA or A23187, 50 ug/L TBT stimulated a significant CL response that was inhibited by chelation of extracellular calcium. Uptake of calcium-45 was greatly increased with 50 ug/L TBT but depressed by 500 ug/L TBT. Results suggest that low TBT concentrations stimulate calcium influx and enhancement of reactive oxygen intermediate formation, while high TBT concentrations cause membrane dysfunctions which inhibit calcium flux and depress activation sequences. The physiological basis of TBT-stimulated macrophage activation was investigated in vitro. Inhibition of TBT-stimulated CL by sodium benzoate (.OH scavenger) and superoxide dismutase (.O&\sb2& scavenger) was most similar to PMA-stimulated CL, indicating a role of NADPH oxidase activity in TBT-stimulated CL. Inhibition by nordihydroquaiaretic acid (lipoxygenase antagonist) was similar to PMA-, PMA + A23187-, and A23187-stimulated CL, indicating a common role of leukotrienes in activation responses. Inhibition by indomethacin (cyclooxygenase antagonist) was most similar to PMA + A23187-, and A23187-stimulated CL, indicating a common role of prostaglandins in activation responses. Inhibition by isobutylmethylxanthine (phosphodiesterase antagonist) was most similar to PMA- and A23187-stimulated CL, indicating a role of cyclic neucleotides in activation regulation. Inhibition of TBT-stimulated CL by verapamil (calcium channel antagonist) and trifluoperazine (calmodulin antagonist) were most similar to PMA + A23187- and A23187-stimulated CL, indicating the role of calcium in TBT-stimulated macrophage activation. Therefore, TBT-stimulated macrophage activation is initiated by an increased plasma-membrane calcium flux. The influence of in vivo TBT exposure was compared with the effects of in vitro exposure. Fish were treated (i.p. injections) weekly for six weeks with either sham, vehicle, 0.250, 0.750, or 2.5 mg/kg TBT. Macrophages were isolated and PMA + A23187- and TBT (50 ug/L)-stimulated CL was compared between treatment groups. Chemiluminescence was reduced in a dose-dependent manner in response to both PMA + A23187 and TBT (50 ug/L). It is concluded that TBT supresses macrophage function following both in vivo and in vitro exposure. The mode of action appears to be an interference with plasma membrane calcium transport, therefore inhibiting both intracellular and intercellular signal transduction.

Impaired function of the human intestinal epithelial barrier (IEB) might allow for permeability of harmful substances, such as obesogens, which induce obesity and further implications. Tight junction (TJ) proteins are the key component for normal functions of the barrier. In this master thesis, the correlation between increased TJ permeability of the IEB and absorption of obesogens was studied. The effect of obesogens on TJ expression was also investigated. Permeability tests performed on Caco-2 cell monolayers exposed to obesogens showed altered permeability, indicating that obesogens might have an effect on TJ protein expression. Furthermore, impaired monolayers showed increased permeability, which implies that impaired functions of IEB lead to increased absorption of obesogens.

During the 1980s a decline in populations of the bivalve Scrobicularia plana was noted in several U.K. estuaries: tributyltin (TBT) was suspected of being the cause although its toxicity to adults could not be demonstrated except at elevated concentrations; disappearance of clam populations has been also observed in other European countries. Laboratory tests have revealed that: i) D-larvae hatching from embryos after 48 hours in TBT concentrations of 188 ngSn/l amounted to < 50% of control values, and doses of 364 ngSn/l or 20 µgCu/l prevented normal development in = 90% of embryos. ii) planktonic veliger larvae exposed for 10 days to nominal TBT doses ≥ 50 ngSn/l grew at rates which, at maximum, were one third of that exhibited by controls. iii) settling pediveligers subjected for 30 days to levels of TBT ≥70 ngSn/l suffered significant mortalities, and postlarvae kept at 23 ngSn/l displayed some shell growth which was both substantially reduced and grossly abnormal. iv) exposure for 30 days to 2! 300 ngSn/l or ≥20 µgCu/l impaired the burying activity in sand of small spat, and juveniles reared in TBT solutions at ≥ 28 ngSn/l grew significantly less than those in the control treatment. v) while small spat held in heavy metal polluted sediment suffered massive mortalities in 12 days, juveniles exposed for 36 days to butyltin contaminated sediment (0.4 µgSn/g) did not display any limited survival, but both their growth and burying activity were significantly reduced relative to those of juveniles kept in control sediments. It is concluded that in U.K. coastal areas where TBT in water during the summer-autumn months ranged from = 20 ngSn/l to = 200 ngSn/1 (i.e. = 50-500 ngTBT/l) and Scrobicularia plana populations disappeared or declined markedly, a cause-effect relationship is most likely to exist between the former and the latter through the deleterious effects of the chemical on the early life stages of the clam; in addition, the continued presence of sediment-bound TBT may render mudflats unsuitable for the development of larval and juvenile bivalves.

Le bassin d’Arcachon est une lagune semi-fermée qui concentre de forts enjeux économiques grâce à la pratique de l’ostréiculture. Cette activité est affectée depuis une trentaine d’années par des phénomènes de mortalités estivales, et plus récemment par des surmortalités du naissain. Ces travaux se sont intéressés au rôle des polluants majeurs du bassin d’Arcachon dans ce contexte de crise en étudiant leurs effets sur la biologie de Crassostrea gigas. Une approche in situ a d’abord été adoptée afin d’identifier les contaminants les plus présents dans le milieu naturel. Des opérations de transplantation d’huîtres et des prélèvements de sédiments ont permis de quantifier divers contaminants et d’associer leur présence à des réponses biologiques. Les polluants ainsi identifiés ont ensuite été employés en conditions contrôlées au laboratoire. Trois expérimentations ont été réalisées : la première concerne l’étude des voies de contamination par le tributylétain ; la seconde concerne les effets des pesticides et du cuivre ; la troisième concerne l’effet des HAP sur des huîtres diploïdes et triploïdes. Nos résultats indiquent que les organismes transplantés au coeur de la lagune sont plus exposés aux polluants, en lien avec les caractéristiques hydrodynamiques du système. La plupart des paramètres biologiques étudiés sur le terrain ont par ailleurs montré une saisonnalité liée aux processus de gamétogenèse. Au laboratoire, la plupart des contaminants testés ont induit une réponse adaptative chez les huîtres exposées. Notre étude souligne l’importance de coupler approche de terrain et approche expérimentale pour comprendre le fonctionnement des écosystèmes côtiers
The Arcachon Bay is a semi-enclosed lagoon and represents the core of strong economic stakes through the practice of oyster-farming. This activity has been affected for around thirty years by summer mortality events, and more recently by abnormally high death rates of juveniles. This work focused on the role of the Arcachon Bay’s main contaminants in this crisis, by studying their effects on the cupped oyster’s biology. Firstly, an in situ approach was adopted in order to identify the major pollutants of the bay: caged oysters were transplanted and sediments were sampled. The presence of contaminants in the samples was associated to biological responses. Secondly, the contaminants identified in situ were used in controlled conditions at the laboratory. Three experimentations were performed; the first dealt with the contamination pathways of tributyltin; the second focused on the biological effects of pesticides and copper; the third concerned the effects of PAH towards diploid and triploid oysters. Our results indicate that the inner stations present higher accumulation of metals and PAH, in accordance with the hydrodynamic features of the bay. The bioindicators used in situ exhibited seasonal trends related to the oysters’ gametogenesis. In the laboratory, most of the contaminants used at environmental levels induced an adaptive response of the exposed oysters. Our study highlights the importance of coupling in situ and laboratory approaches in order to understand the functioning of coastal ecosystems

Cette thèse porte sur le comportement d’un composé prioritaire au titre de la Directive Cadre sur l’Eau au niveau européen : le tributylétain (TBT) et ses métabolites (dibutylétain (DBT) et monobutylétain (MBT)). On s’intéresse plus particulièrement aux eaux usées domestiques lors de leur transit dans deux stations d’épuration par lagunage naturel (STEP), traitement adapté aux effluents provenant des zones faiblement urbanisées. Ce travail de thèse vient combler le manque de résultats scientifiques sur (1) le niveau des concentrations en butylétains dans les effluents domestiques des zones peu urbanisées en entrée des stations par lagunage et en sortie après traitement ; (2) l’efficacité du traitement par lagunage à éliminer les butylétains ; (3)l’identification des processus qui contrôlent le devenir de ces éléments au cours du traitement.Les butylétains sont mesurés dans les matrices particulaires, dissoutes et dans les boues, par couplage GC-ICP-MS, dans toutes les étapes du traitement par lagunage de deux communes rurales du département de l’Hérault (34,France) : Gigean (6000 eq-hab.) et Montbazin (4500 eq-hab.). On montre que les trois espèces butylées sont systématiquement présentes dans tous les échantillons prélevés sur les deux STEP. Les butylétains arrivent principalement sous forme particulaire dans les eaux brutes en entrée des stations. Au cours du traitement, les processus de sorption sur les particules en suspension et la décantation de ces dernières au fond de chaque bassin sont les principaux facteurs permettant l’élimination des butylétains. Les premiers bassins anaérobies, de grande profondeur et de long temps de séjour, permettent l’élimination de plus de 60% à Gigean (93% à Montbazin) des butylétains. Les boues de fond de chaque bassin montrent des concentrations élevées en butylétains. La remise en suspension des butylétains est notée dans les derniers bassins de finition, peu profonds, ce qui peut être due à la re-stratification thermique. En sortie de station, malgré les abattements importants, des concentrations non négligeables en butylétains (12 à 228 et 1.8 à 15 ng(Sn).L-1 respectivement pour Montbazin et Gigean) sont rejetées dans le milieu récepteur. Des expériences de sorption basées sur la mise en contact de butylétains avec des boues de fond de bassin diluées ont été effectuées selon plusieurs approches expérimentales. On a ainsi montré que (1) la distribution des espèces MBT, DBT, TBT, entre les phases solide et liquide, est variable d’un test à l’autre selon les conditions opératoires ; (2) l’affinité entre les butylétains et la boue est importante, quelle que soit sa provenance. 98% des butylétains se retrouvent dans la phase solide, du fait de leur hydrophobicité. Le coefficient de distribution Kd entre le TBT et les boues est plus élevé que celui obtenu pour d’autres type de phases solides (75000 L.kg- 1) ; (3) le processus de sorption est rapide. De plus des processus de sorption, désorption et déalkylation du TBT, expliquant les variations des concentrations en MBT et DBT dans la phase solide ont été identifiés. La désorption du TBT est rapide. Il se dégrade dans la phase liquide en DBT dont une partie se transforme en MBT. La sorption du MBT sur les boues a été systématiquement mesurée. Le phénomène de sorption n’est donc pas complètement réversible au cours du temps. Ces résultats contribuent à expliquer les dysfonctionnements observés dans les stations de traitement par lagunage, par exemple lors d’apports directs d’eau de pluie.Cette thèse démontre la présence systématique des butylétains dans les stations d’épuration par lagunage naturel. Les données acquises in-situ couplées à des essais en laboratoire permettent de comprendre le comportement de ces composés lors de ce traitement : la fraction solide joue un rôle clé dans les processus de sorption-désorption-(bio)dégradation des butylétains
This thesis deals with the behavior of a priority compound on the basis of the framework Directive on water at European level: tributyltin (TBT) ) and its metabolites (dibutyltin (DBT) and monobutyltin (MBT)). The study is focused on two waste stabilization pond treatment plants (WSP), which is an appropriate treatment to domestic effluents from sparsely urbanized areas.This thesis comes to fill the lack of scientific results on (1) the level of butyltin concentrations in domestic effluents from sparsely urbanized areas, in the input of the WSP and the output after treatment; (2) the efficiency of butyltins elimination by the WSP treatment; (3) the identification of the processes that control the fate of these elements during the treatment.Butyltins are measured in particulate and dissolved matrices and in sludge, by GC-ICP-MS, in all stages of the treatment by lagooning of two villages of the Department of Hérault (34, France): Gigean (6000 p.e.) and Montbazin (4500 p.e.).It is shown that the three butyltin species are consistently present in all samples taken from the two WSP. Butyltins arrive primarily in particulate form in the raw water. During treatment, the processes of sorption onto particulate matter and the settling of the latter at the bottom of each basin are the main factors for the elimination of butyltins. The first anaerobic ponds, characterized by deep depth and long residence times, allow the elimination of more than 60% Gigean (93% at Montbazin) of butyltins. Bottom sludge of each basin show high butyltins concentrations. The resuspension of butyltins is noted in the last maturation ponds, characterized by shallow depth, which may be due to re-thermal stratification. At the outlet, despite significant butyltin removal, concentrations in butyltins remain high (12 to 228 and 1.8 to 15 ng (Sn). L-1, respectively for Montbazin and Gigean).Laboratory batch experiments were conducted to study sorption processes following several experimental protocols. It was thus shown that: (1) the distribution of the butyltin species between solid and liquid phases is different from one test to another according to the operating conditions; (2) the affinity between butyltins and bottom sludge is important, regardless of its provenance. 98% of the butyltins are found in the solid phase, due to their hydrophobicity. The TBT sludge-water partition coefficient is higher than that obtained for other type of solid phases (75000 L.kg-1); (3) the process of sorption is fast.Moreover processes of sorption, desorption and dealkylation of TBT, explaining changes in concentrations of MBT and DBT in the solid phase were identified. Desorption of TBT is fast. It degrades in the liquid phase in DBT which part turns into MBT. The sorption of MBT on sludge has been systematically measured. Therefore, the sorption phenomenon is not completely reversible over time. These results help to explain the dysfunctions observed in WSP, for example after direct inputs of rainwater.This thesis shows the systematic presence of butyltin in waste stabilization pond treatment plants. In-situ data coupled with laboratory tests allow to understand the behaviour of these compounds during this treatment: the solid fraction plays a key role in the processes of sorption-desorption-(bio) degradation of butyltins

Doutoramento em Biologia
A Directiva Quadro da Água (DQA) foi introduzida pela União Europeia (UE) em 2000 com o objetivo de proteger as águas de superfície (interiores, de transição e costeiras) e subterrâneas. Esta ambiciosa directiva lançou novos desafios, entre os quais a necessidade de desenvolver novas ferramentas ecotoxicológicas para a avaliação do estado ecológico das massas de água. O tributilestanho (TBT), um biocida amplamente utilizado em tintas antiincrustantes e identificado como agente causador do fenómeno de imposex / intersex em gastrópodes, está incluído na lista de substâncias prioritárias da DQA. No entanto, esta directiva não considera qualquer ferramenta de biomonitorização específica para avaliar os efeitos nocivos do TBT nos ecossistemas. O único objetivo concreto para esta substância é referido nas Normas de Qualidade Ambiental (NQA) e atingido a 0,2 ng TBT / L (NQA - Média Anual). Mais tarde, em 2008, a Directiva Quadro da Estratégia Marinha (DQEM) foi introduzida na UE com objectivos semelhantes à DQA, mas direcionada para águas marinhas. No entanto, uma vez mais, não foi proposta nenhuma ferramenta ecotoxicológica para a monitorização dos efeitos biológicos provocados por este poluente. Assim, de forma a integrar a biomonitorização da poluição por TBT ao abrigo destas directivas, esta tese tem como objetivo desenvolver e testar, sob diferentes cenários, ferramentas baseadas nos biomarcadores imposex/intersex para avaliar o estado ecológico de massas de água. Assim, é proposto um sistema de classificação baseado nos níveis de imposex/intersex de populações de gastrópodes para avaliar o estado ecológico de águas costeiras e de transição, relativamente à poluição por TBT. Utilizando a Ria de Aveiro (NW Portugal) como estudo de caso, três bioindicadores - os gastrópodes Nucella lapillus, Nassarius reticulatus e Littorina littorea - foram utilizados tendo em conta o elemento de qualidade biológica para invertebrados bentónicos da DQA, de forma a classificar o estado das massas de água desta região. O índice de sequência do vaso deferente (VDSI) e o índice de intersex (ISI) foram os parâmetros de imposex/intersex selecionados para avaliar o estado ecológico, relativamente à poluição por TBT. Os limites definidos pelos Rácios de Qualidade Ecológica (EQR) foram obtidos para cada espécie, a fim de definir as cinco classes de estado ecológico (Excelente, Bom, Razoável, Medíocre e Mau). A espécie N. lapillus é proposta como bioindicador chave na monitorização da poluição por TBT, no entanto, o uso combinado das outras espécies revela-se muito útil porque permite monitorizar uma área maior e uma maior diversidade de habitats. Esta ferramenta de monitorização multiespecífica demonstrou ser bastante útil para avaliar a evolução temporal do estado ecológico das massas de água da Ria de Aveiro entre 1998 e 2013, que demonstrou uma clara melhoria, alcançando um bom estado ecológico em 2013. Este trabalho propõe também uma outra ferramenta destinada a avaliar a qualidade dos sedimentos. Esta ferramenta consiste num bioensaio em que se expõem fêmeas de N. reticulatus a sedimentos recolhidos no ambiente para determinar se ocorre um aumento de imposex. Este bioensaio provou ser uma ferramenta complementar e com relevância ecológica na monitorização da poluição por TBT na medida em que fornece informação sobre a fracção biodisponível de TBT nos sedimentos e permite avaliar locais onde não existem bioindicadores disponíveis. Para testar o uso de ambas ferramentas em programas de monitorização da DQA, estas foram aplicadas em dois sistemas estuarinos - Minho e Lima - localizados no Norte de Portugal. A monitorização de imposex evidenciou um bom estado ecológico no Minho mas o estuário do Lima não conseguiu alcançar o objetivo de bom estado ecológico, apresentando locais com um estado "moderado" ou de qualidade inferior. Em conformidade, o bioensaio com sedimentos confirmou a presença de TBT biodisponível apenas nos sedimentos do estuário do Lima, já que se observou o aumento de alguns parâmetros de imposex com estes sedimentos. O uso combinado destas ferramentas permitiu aumentar a área de monitorização nestes sistemas estuarinos, uma vez que não foi possível encontrar bioindicadores em todas as estações de amostragem, sendo nesse caso obtidas apenas amostras de sedimento. O critério proposto para a monitorização de imposex na DQA foi também testado ao longo da costa portuguesa de forma a avaliar a evolução do estado ecológico de 2000 a 2014. Em 2014, as fêmeas de N. lapillus colhidas em praias ao longo da costa exibiram baixos níveis de imposex, exceto numa estação situada na Zambujeira do Mar (SW Portugal) onde foram encontradas fêmeas estéreis. Já as fêmeas de N. reticulatus mostraram baixos níveis de imposex na maioria das estações amostradas, porém elevados níveis foram observados em áreas sujeitas a um intenso tráfego naval. Comparando estes valores com os observados em anos anteriores, é possível perceber que ambas as espécies recuperaram de forma significativa ao longo dos últimos 15 anos. N. lapillus apresentou uma redução dos níveis de imposex mais pronunciada do que N. reticulatus, porém esta diminuição parece ter abrandado nos últimos anos (2011-2014), prevendo-se uma estabilização por algum tempo em níveis muito baixos. N. reticulatus também mostrou uma diminuição gradual, mas os valores de imposex indicam uma recuperação mais lenta em portos de pesca e marinas. Isto representa uma mudança das principais fontes de poluição por TBT, já que os níveis de imposex em grandes portos comerciais, outrora locais altamente poluídos, caíram rapidamente. Assim, na costa Portuguesa, o bom estado ecológico foi alcançado na maior parte das estações localizadas em águas costeiras, no entanto uma qualidade ecológica inferior foi observada em massas de água de transição, especialmente onde estão localizados portos de pesca e marinas. A classificação da qualidade ecológica da costa portuguesa segundo a DQA (proposta apresentada nesta tese) e a OSPAR, relativamente à poluição por TBT, é analisada no presente trabalho. Esta comparação é da maior importância, pois tanto as premissas da DQA, como as ferramentas de monitorização utilizadas pelas convenções regionais marinhas (por exemplo a OSPAR) devem ser integradas na aplicação da DQEM. As classificações obtidas segundo a DQA e a OSPAR apresentam um resultado global muito semelhante para 2014, e ambas denotam uma apreciável recuperação do estado ecológico ao longo dos últimos anos. A integração da monitorização de imposex/intersex na DQEM é aqui recomendada e discutida, tendo em conta possíveis divergências nos objetivos definidos por cada legislação e os meios para alcançá-los. Mesmo que a utilidade das ferramentas empregadas para monitorizar a poluição por TBT tenham sido comprovadas, a monitorização de imposex/intersex a ser aplicada na DQA e DQEM só será eficaz se: (i) houver uma boa escolha de bioindicadores; (ii) o imposex/intersex constituírem uma resposta específica para a poluição por TBT porque se outros contaminantes tiverem a capacidade de induzir imposex ou intersex, estes biomarcadores perdem grande parte do seu valor operacional. Por estas razões, esta tese apresenta ainda alguns resultados que devem ser considerados para a validação da utilização do imposex/intersex na monitorização da poluição por TBT. A Ria de Aveiro é uma área que tem sido intensamente estudada em relação à poluição por TBT e, conforme descrito anteriormente, apresentou uma importante melhoria do estado ecológico em relação a este poluente prioritário. No entanto, uma tendência contrária foi observada na evolução dos níveis de imposex no gastrópode Peringia ulvae. Esta espécie - considerada por vários autores como um bom bioindicador da poluição por TBT - não demonstrou uma recuperação dos níveis de imposex, apesar dos níveis de TBT e de imposex/intersex noutras espécies terem vindo consistentemente a diminuir nesta área. Em vez disso, houve um aumento global na percentagem de fêmeas afetadas por imposex e nos níveis de VDSI até 2012, sendo que os níveis de imposex em 2015 são ainda semelhantes aos encontrados em 1998. As possíveis razões para o padrão contracorrente observado nesta espécie podem estar relacionados com a interferência de outros factores que influenciam a expressão do imposex; por exemplo, a indução de imposex por parasitas - que são difíceis de controlar quando a monitorização é realizada com esta espécie - pode enviesar a interpretação dos resultados. É, portanto, necessária uma escolha criteriosa dos bioindicadores a utilizar no âmbito das directivas DQA e DQEM. O imposex não é apenas um efeito exclusivo do TBT, uma vez que já foi descrito que o trifenilestanho (TPT) pode também causar este fenómeno em algumas espécies de gastrópodes. Aqui, é descrita, pela primeira vez, a indução de imposex por TPT em N. lapillus, a espécie que foi proposta como bioindicador chave na DQA. Curiosamente, o desenvolvimento do imposex em fêmeas de N. lapillus injetadas com TPT é diferente das injectadas com TBT, uma vez que as primeiras desenvolvem uma via de imposex principalmente afálica (sem desenvolvimento do pénis). Estes resultados sugerem que o TPT e o TBT podem agir de forma diferente no processo de masculinização de fêmeas de gastrópodes, lançando novas perspectivas sobre as hipotéticas vias subjacentes ao desenvolvimento de imposex. É importante saber se existem outros contaminantes, para além dos organoestânicos, capazes de induzir (ou interferir com) o desenvolvimento de imposex ou intersex, com vista à validação das ferramentas de monitorização propostas nesta tese. Para tal, um ensaio com um gene repórter foi realizado em células transfectadas com recetores humanos, cujos ortólogos foram anteriormente identificados em gastrópodes (RXR, PPAR, ER e RAR). Sabendo que o imposex é causado pela ativação dos recetores nucleares RXR e PPARy por parte do TBT e TPT, é importante perceber se outros contaminantes são capazes de ativar estes recetores. Testaram-se vários contaminantes ambientais e mesmo que algumas substâncias tenham tido a capacidade de ativar ligeiramente o RXR e PPARy, nenhuma foi capaz de o fazer com uma potência semelhante ao TBT e TPT, sugerindo que estas são as únicas substâncias testadas capazes de induzir imposex para concentrações ambientalmente relevantes. Não obstante, todas as substâncias que foram capazes de ativar os recetores foram identificadas e o seu potencial para causar disrupção endócrina nos gastrópodes é discutido. Em conclusão, as ferramentas propostas nesta tese podem ser extremamente úteis na monitorização do ambiente aquático ao abrigo das recentes directivas da União Europeia. Em Portugal existem três peças legislativas que se sobrepõem espacialmente nas águas costeiras (DQA, DQEM e OSPAR) e águas de transição (DQA e OSPAR), e podem diferir nos seus objetivos específicos relativamente à poluição por TBT. Logo, existe uma necessidade urgente de encontrar objetivos comuns na UE, tanto químicos e biológicos, para este poluente. Para além disto, e num momento em que a busca de modelos biológicos não vertebrados está a aumentar em ecotoxicologia, esta tese realça o uso de gastrópodes como uma possível opção. O trabalho desenvolvido nesta tese mostra que a poluição por TBT tem vindo a diminuir em Portugal mas ainda existem alguns locais onde os níveis são altos e não atingem os objectivos definidos pela OSPAR e pela DQA (segundo a proposta aqui apresentada). As ferramentas propostas nesta tese podem ser úteis para acompanhar a evolução da situação em Portugal e na UE nos próximos anos e identificar os locais onde será necessário melhorar a qualidade ecológica das massas de água relativamente a esta substância prioritária, de acordo com as metas estabelecidas pela legislação vigente.
The Water Framework Directive (WFD) was introduced in the European Union (EU) in 2000 with the objective to protect the EU surface waters (inland, transitional and coastal) and ground waters. This ambitious directive raised new challenges, as the need to develop quick and low-cost effect-based monitoring tools, which are increasingly being recommended to perform a proper environmental assessment under this directive. Tributyltin (TBT), a biocide largely used in antifouling paints and identified as a causative agent of imposex/intersex in gastropods, is listed as a priority substance in WFD. However, this directive does not consider any particular biomonitoring tool to assess TBT deleterious effects on the ecosystems. The only explicit objective for TBT regards the Environmental Quality Standard (EQS – Annual Average = 0.2 ng TBT/L). Later on, in 2008, the Marine Strategy Framework Directive (MSFD) was introduced in the EU with similar objectives but specifically related to marine waters that again failed to propose an effect based tool to monitor this pollutant. Therefore, to integrate TBT pollution monitoring within these legislative frameworks, it is the aim of this thesis to develop effect based tools centred on the imposex/intersex biomarkers in order to assess the ecological quality status of EU water bodies regarding TBT pollution. This work proposes a scoring system based on imposex/intersex levels to assess the WFD ecological quality status regarding TBT pollution of transitional and coastal waters. Taking Ria de Aveiro (NW Portugal) as a case study, three bioindicators – the gastropods Nucella lapillus (dog-whelk), Nassarius reticulatus (netted-whelk) and Littorina littorea (periwinkle) - were used under the general WFD benthic invertebrate quality element to classify the ecological status of this area. The vas deferens sequence index (VDSI) and the intersex index (ISI) were selected as biomarkers to assess the condition of this quality element regarding the impact of TBT pollution. EQR boundaries were set for each species in order to define the five ecological status classes (High, Good, Moderate, Poor and Bad). N. lapillus is proposed as a key bioindicator species, however the combined use of further species is very useful to cover a wider monitoring area and a higher diversity of habitats. This multi-species monitoring tool was useful to assess the temporal evolution of the ecological status of Ria de Aveiro water bodies between 1998 and 2013, showing that all the surveyed area improved and reached a good ecological status in 2013. This work also proposes other imposex based tool that allows to evaluate the sediment quality, regarding TBT pollution. The use of this bioassay has proven to be a practical and ecological relevant tool as (i) it can give information for sites with no native populations of snails, (ii) it provides early identification of polluted sites anticipating future imposex levels or early identification of recovering, and (iii) it yields information on the bioavailable fraction of the TBT in the sediment. Therefore, this tool can also be of extreme usefulness under the scope of recent European legislative frameworks. To confirm the suitability of the above mentioned tools in WFD monitoring programs, they were both applied in two estuarine systems - Minho and Lima - located in NW Portugal. The imposex field monitoring evidenced a good ecological status in Minho while the Lima estuary fail to reach the WFD good ecological status objective by presenting sites with “Moderate” and “At Best Poor” ecological status. Accordingly, the sediment bioassay confirmed the presence of bioavailable TBT only in the Lima sediments. The combined use of these tools allowed to increase the monitoring area in these estuarine systems and enhanced the robustness of the assessment, as the bioindicators fail to exist at many sites of the study area. The proposed criteria for imposex monitoring under the WFD was tested along the Portuguese coast to assess the evolution of the ecological status from 2000 to 2014, using N. lapillus and N. reticulatus as bioindicators. Lately, the dogwhelks collected in coastal shores exhibited low imposex levels, except at one station in Zambujeira do Mar (SW Portugal) where sterile females were still found. Accordingly, the netted-whelk showed low levels of imposex at the majority of the sampled stations but high levels were still observed at areas subjected to intense naval traffic. When comparing these data regarding the most recent years with past imposex levels, it is perceived that both species recovered significantly over the last 15 years. N. lapillus presented a more pronounced decrease in imposex levels than N. reticulatus, however this declining trend decelerated from 2011 to 2014. N. reticulatus also showed a gradual decrease but the imposex values indicate a slower recovery at fishing harbours and marinas, which represents an apparent shift in the TBT hotspots as imposex levels in large commercial ports fell rapidly. Consequently, in the Portuguese coast a good ecological status was achieved in most of the stations located in coastal waters while a worse ecological quality was observed in transitional water bodies where fishing ports and marinas are located. A comparison between the proposed WFD imposex tool and the OSPAR Assessment Criteria for imposex shows that both classifications schemes present a general similar result for 2014 and point to a clear recovery of the quality status over the years. This comparison between two legislations is of foremost importance since both WFD premises and Regional Sea Conventions (e.g OSPAR) knowledge and monitoring tools should be integrated by member states on the application of the MSFD. Therefore the imposex/intersex monitoring integration within MSFD is discussed taking into account possible divergences between the objectives defined by each piece of legislation and the means to achieve it. Even if the usefulness of the employed tools to monitor TBT pollution has been proved, the WFD’s monitoring proposal will only be effective if: i) there is a good choice of the bioindicators species; and ii) imposex/intersex are specific responses to TBT pollution, i.e., if other contaminants induce imposex or intersex, then these biomarkers lose much of their operational value. For that reason this thesis presents novel information regarding these aspects in order to validate this methodology and better interpret the obtained results. Ria de Aveiro is an area that has been intensively studied regarding TBT pollution and, as previously reported, presented a major improvement of the ecological status regarding this priority pollutant. An opposite trend has been however observed in the imposex evolution in the gastropod Peringia ulvae. This species - regarded by some authors as a good bioindicator of TBT pollution - did not show an imposex recovery despite the fact that TBT levels have been consistently decreasing in this area. Instead, there was a global increase in the percentage of females affected by imposex and VDSI levels until 2012, and the imposex levels in 2015 were similar to those found in 1998. Possible reasons for this counter current pattern could be related with other factors that can influence imposex expression, such as parasitism, that probably cannot be totally controlled when monitoring is performed with this species. Hence, there is the need to carefully choose the right bioindicators to implement TBT monitoring assessments under WFD or MSFD, and P. ulvae is not recommended for this purpose. Imposex is not an exclusive response to TBT since triphenyltin (TPT), a related organotin, is already known to cause the same phenomenon in some gastropod species. This thesis describes, for the first time, that TPT also induces the development of imposex in N. lapillus, the species considered the key bioindicator in the WFD monitoring proposal. However, imposex development in TPT-injected females followed mostly an aphallic route (no penis development). These results suggest that TPT and TBT may act differently in the sequential process of female masculinization casting new insights about the hypothetical pathways underlying imposex development. Besides TBT and TPT, it is important to know if other environmental contaminants can induce or interfere with the imposex/intersex development to ensure that the proposed tools are specific to organotin pollution. With this purpose, a reporter gene assay was performed using the GAL4/UAS system in cells transfected with human receptors, which were already identified in gastropods: Retinoid X Receptor (RXR), Peroxisome Proliferator-Activated Receptor gamma (PPARγ), Retinoic Acid receptor (RAR) and Estrogenic Receptor (ER). It has been reported in the literature that imposex is caused by the activation of the nuclear receptors RXR and/or PPARγ by TBT or TPT, and therefore if other contaminants can bind these receptors they could have the potential to induce imposex/intersex as well. Even if there were other substances that slightly activated RXR and PPARγ, none was able to do it with similar potency than the organotins (TBT and TPT), suggesting so far that these are the only tested substances capable to induce imposex at realistic environmental concentrations. Nevertheless, all substances that were able to activate the reporter gene were identified and their potential to cause endocrine disruption in gastropods is discussed. Concluding, the tools proposed in this thesis can be of extreme utility to monitor the aquatic environment under the frame of the recent EU directives. Moreover, in Portugal there are three legislative pieces that are spatially overlapped regarding coastal waters (WFD, MSFD and OSPAR) and transitional waters (WFD and OSPAR), and they may differ in their specific objectives regarding TBT pollution. Therefore there is a need to find common EU objectives, both chemical and biological, for this pollutant. Furthermore, in a time where the search of non-vertebrate biological models in ecotoxicology is increasing, this thesis enhances the use of gastropods as an option. Also the work here developed shows that TBT pollution has been decreasing in Portugal but there are some sites where levels are still high. In this scenario, the tools here proposed can be useful to track the evolution of TBT pollution during the next years and to identify waters bodies that will require an action to improve their ecological status.

Mestrado em Toxicologia e Ecotoxicologia
Organotins (OTs) are organometallic compounds. Despite of the multiple applications of the OTs, the notoriety of these compounds is due to tributyltin (TBT), a potent biocide used in antifouling paints since the 60s to prevent biofouling on submerged surfaces, including the hulls of ships. Despite of the extreme effectiveness of these paints in combating biofouling, they are also extremely toxic to non-target organisms, therefore several legislative measures restricting their use were implemented in several countries worldwide. TBT causes a huge variety of adverse effects on non-target species (sublethal / lethal) of different taxonomic groups (from bacteria to mammals). The first evidence of adverse effects induced by TBT on non-target species emerged in the 70s in oysters of the species Crassostrea gigas in the Bay of Arcachon, France. Their shells have suffered thickening with consequent decrease in the volume of edible portion, reducing its commercial value. In the same decade was verified the occurrence of male sexual characteristics in female prosobranch gastropods (formation of a penis and / or development of a vas deferens), by exposure to TBT. This phenomenon was coined as "imposex" by Smith in 1971. The imposex is the best known example of endocrine disruption caused by a pollutant being used as a biomarker to monitoring the environmental pollution by TBT. The gastropods are used globally for monitoring environmental pollution by TBT. In the present work we studied how gastropods have been used to track the course of TBT pollution worldwide during the last decade (2003-2013), and the main results obtained. We searched for the number of published articles by year / continent and country regarding the bioindicator species most used in biomonitoring TBT pollution in function of their geographical location; we evaluated the efficacy of the AFS Convention in reducing TBT pollution worldwide; we evaluated the temporal evolution (2003-2013) of the levels of imposex by bioindicator species and geographical area, as well as the concentration of OTs in the biota, water and sediments. There was noticed an increase of the number of publications for monitoring environmental pollution by TBT in 2008, coincident with the time of entry into force of the AFS Convention and a gradual decrease of the number of publications from 2008 to 2013, after the entry into force of this Convention; most of the monitoring studies developed in the period 2003-2013 were conducted in Europe, followed by the continents: America, Asia, Africa e Oceania; in this period were used 96 bioindicator species of imposex, being that the 5 more used were: Nucella lapillus, Hexaplex trunculus, Nassarius reticulatus, Stramonita haemastoma and Thais clavigera. A greater diversity of bioindicators was used in America and Asia compared to Europe, whist Africa and Oceania were the continents with smaller number of bioindicator species used. Regardless of the geographic region, and despite the wide diversity of species used, the bioindicators were all caenogastropods (Subclass Caenogastropoda) and most frequently of Order Neogastropoda and Littorinimorpha, being the majority from family Muricidae. There was a decrease of the levels of imposex in the different bioindicators used by continent and country, as well as a decline of the concentrations of OTs in biota, water and sediments, by continent and country, since the entry into force of the AFS Convention in September 2008.
Os organoestânicos (OTs) são compostos organometálicos. Apesar das múltiplas aplicações dos OTs, a notoriedade destes compostos, no que diz respeito a fenómenos de poluição, é devida ao tributilestanho (TBT), um potente biocida usado em tintas antivegetativas desde a década de 60 para prevenir a bioincrustação nas superfícies imersas, nomeadamente nos cascos de embarcações. Apesar da extrema eficácia destas tintas no combate à bioincrustação são também extremamente tóxicas para organismos não-alvo pelo que várias medidas legislativas restringindo o seu uso foram implementadas em vários países. O TBT provoca uma enorme variedade de efeitos nefastos em espécies não- alvo (sub-letais / letais) de diferentes grupos taxonómicos (de bactérias a mamíferos). A primeira evidência de efeitos nefastos induzidos pelo TBT em espécies não alvo surgiu na década de 70 em ostras da espécie Crassostrea gigas na Baía de Arcachon, França. As suas conchas sofreram um espessamento com a consequente diminuição do volume da parte comestível, diminuindo o seu valor comercial. Na mesma década verificou-se a ocorrência de características sexuais masculinas em fêmeas de gastrópodes prosobrânquios (formação de um pénis e/ou desenvolvimento de um vaso deferente), por exposição ao TBT. Esse fenómeno foi designado de "imposex" por Smith em 1971. O imposexo é o melhor exemplo conhecido de disrupção endócrina provocada por um poluente sendo utilizado como biomarcador para monitorizar a poluição ambiental por TBT. resumo (cont.) Nesta tese fez-se um levantamento da metodologia utilizada na monitorização da poluição por TBT a nível mundial durante a última década (2003-2013), nomeadamente, o número de artigos publicados por ano/continente e país sobre as espécies bioindicadoras do imposexo mais utilizadas na biomonitorização da poluição por TBT nesta década e em função da sua localização geográfica. Neste trabalho avaliou-se, também, a eficácia da Convenção AFS na redução da poluição por TBT a nível mundial, através da evolução temporal (2003-2013) dos níveis de imposexo por espécie bioindicadora e zona geográfica, e avaliou-se a evolução temporal (2003-2013) da concentração de OTs no biota, água e sedimentos por zona geográfica. Verificou-se um aumento do número de publicações de monitorização da poluição ambiental por TBT em 2008, coincidente com o momento de entrada em vigor da Convenção AFS; um decréscimo gradual do número de publicações de 2008 a 2013, após a entrada em vigor da Convenção AFS; a maioria dos estudos de monitorização no período 2003-2013 foi realizada na Europa, seguindo-se os continentes: America, Ásia, África e Oceania; neste período foram usadas 96 espécies bioindicadoras de gastrópodes (imposex), sendo que as 5 mais utilizadas foram: Nucella lapillus, Hexaplex trunculus, Nassarius reticulatus, Stramonita haemastoma e Thais clavigera. Neste trabalho é possível verificar a utilização de uma maior diversidade de espécies bioindicadoras na América e na Ásia em relação à Europa, África e Oceania são os continentes que apresentam menor número de espécies bioindicadoras. Independentemente da região geográfica onde foram realizados os estudos, e apesar da grande diversidade de espécies utilizadas, os bioindicadores foram todos caenogastropodes (subclasse Caenogastropoda) e mais frequentemente das ordens Neogastropoda e Littorinimorpha, sendo a maioria da família Muricidae. Neste trabalho também é possível verificar descida dos níveis de imposexo nas diferentes espécies bioindicadoras, utilizadas por continente e país, e descida das concentrações de OTs no biota, água e sedimentos, por continente e país, desde a entrada em vigor da convenção AFS em Setembro de 2008.

Un régime alimentaire déséquilibré et/ou la présence de composés contaminantsexogènes peuvent modifier la signalisation endocrine et l’homéostasie des lipides et induirel’obésité. Les travaux réalisés dans le cadre de cette thèse ont permis, dans un premier temps,de développer une méthode simple et rapide, dénommée "zebrafish obesogenic (ZO) test",pour identifier in vivo, par utilisation de la larve de poisson zèbre, des facteurs qui peuventaugmenter ou diminuer la taille de l’adipocyte blanc et ainsi moduler le niveau de l’adiposité(Tingaud-Sequeira, Ouadah, Babin, J. Lipid Res. 52, 1765-1772, 2011). Ce test permetd’identifier des composés et des mélanges de molécules obésogènes et anti-obésogènes etfournit des informations pertinentes pour l'évaluation des risques liés leur présence maiségalement pour élucider les mécanismes impliqués. Les travaux ont, dans un second temps,permis d’apporter des réponses quant aux modalités d’action d’un obésogène puissant, lechlorure de tributylétain, contaminant retrouvé très largement dans notre environnement.Cette molécule agit sur l’adipocyte blanc à une concentration de l’ordre du nano molaire viales récepteurs nucléaires RXR et LXR, et non pas via les isoformes PPARgamma/delta(Ouadah et Babin, manuscrit en préparation)
An unbalanced diet and / or the presence of exogenous compounds contaminants mayalter endocrine signaling and lipid homeostasis and induce obesity. The work done in thisthesis have, at first, developed a simple and rapid method, called "zebrafish obesogenic (ZO)test" to identify in vivo by using the zebrafish larva, the factors that may increase or decreasethe size of the white adipocyte and therefore modulate the level of adiposity (Tingaud-Sequeira, Ouadah, Babin, J. Lipid Res. 52, 1765-1772, 2011). This test helps to identifycompounds and mixtures of obesogenic and anti-obesogenic molecules and providesinformation relevant to the risk assessment of their presence but also to elucidate themechanisms involved. Work in a second time allowed to answer as to how the action oftributyltin chloride, a powerful obesogenic contaminant found widely in the environment.This molecule acts in vivo on white adipocytes in a concentration of the order of nano molarvia nuclear receptors LXR and RXR, and not via the PPARgamma isoforms / delta (Ouadahand Babin, manuscript in preparation)

La cyclisation radicalaire d'alpha-halogénoacétals et d'alcools beta-halogènes, issus de sucres insaturés conduit, stéréospécifiquement, à des systèmes furanno-pyranniques CIS avec de bons rendements. Ce type de réaction, 5 exo trig, permet également d'aboutir à des vinylstannanes bicycliques CIS, par hydrostannylation d'alcynes issus de sucres insaturés. Les substrats obtenus se prêtent bien à des transformations chimiques telles que l'ouverture sélective des systèmes furanniques, la substitution du groupement tributylstannyle ou encore la coupure des vinylstannanes

L'emploi du tributylétain (TBT) comme agent biocide dans les peintures antisalissures pour bateaux a été réglementé en France en 1982. Il était intéressant, pour évaluer l'efficacité de cette réglementation et les risques subsistant, d'étudier sa répartition géographique dans des sites pollués et son évolution dans la phase sédimentaire. Nous avons tout d'abord présenté la méthode de spéciation des organoétains par génération d'hydrures-chromatographie gazeuse-spectrométrie d'absorption atomique four en quartz. Les limites de détection obtenues (ng g#-#1 pour des sédiments secs, ng l#-#1 pour des eaux) permettent d'atteindre des concentrations significatives dans l'environnement marin. L'étude des sédiments du bassin d'Arcachon a mis en évidence l'omniprésence du TBT et de ses produits de dégradation avec des concentrations faibles dans les zones de mariculture (contamination diffuse apportée par les courants et marées) et des concentrations élevées dans les ports et les zones de mouillage, laissant présumer de l'existence d'apports continus malgré la législation existante. La spéciation des butylétains a enfin été étudiée dans une carotte sédimentaire profonde prélevée dans le port de Boyardville caractérisé par une sédimentation rapide. Les profils de concentration obtenus indiquent la prépondérance du TBT en surface, du MBT en profondeur et présentent des maxima saisonniers. L'historique du site a permis d'estimer la datation de la carotte et de modéliser la dégradation du TBT en DBT, MBT puis SN(IV) (cinétique d'ordre 1). Les temps de demi-vie ont été estimes à 2,09 ans pour le TBT et 1,9 et 1,1 ans pour le DBT et le MBT respectivement. Le TBT introduit dans le milieu aquatique se trouve stabilisé par la sédimentation et ne se dégrade alors que très lentement. La présence d'un stock important du biocide constitue une menace permanente pour les organismes marins en raison de l'éventualité d'un relargage massif dans la colonne d'eau lors de perturbations causées par les tempêtes, marées ou dragages.

Les contaminants chimiques ont des origines très diverses résultant d'activités anthropiques présentes ou passées pouvant avoir des conséquences dramatiques sur l'environnement et sur les équilibres des milieux naturels. C'est le cas du TBT, composé toxique très largement utilisés dès le début des années 70 en tant qu'agent biocide dans les peintures anti-salissures. Cette utilisation massive est à l'origine de l'ubiquité de ce composé dans l'environnement marin. Les premiers effets néfastes ont été mis en évidence dans le Bassin d'Arcachon à la fin des années 70, par une forte perturbation de la production ostréicole qui s'est d'abord traduite par une baisse importante de la reproduction et une baisse de captage du naissain. Bien que les apports aient cessé du fait d'une réglementation restrictive sur l'usage du TBT en tant que biocide, le TBT persiste dans le milieu, principalement au niveau des sédiments portuaires. Ces travaux de thèse ont été mis en place pour évaluer la contamination des sédiments du Bassin d'Arcachon par les composés organostanniques. L'analyse de carottes sédimentaires a permis d'obtenir un enregistrement biogéochimique de la contamination ainsi que les profils de concentrations des organoétains dans la colonne sédimentaire. De plus, la mesure du 210Pb a permis d'évaluer le taux de sédimentation dans certaines zones portuaires et un enregistrement sédimentaire de la contamination. Enfin, compte tenu des enjeux actuels concernant le devenir des matériaux de dragages contaminés, de trouver une solution pour décontaminer les sédiments. Nous avons donc réalisé une étude préliminaire sur la décontamination des sédiments portuaires par la technique d'électromigration
The majority of environmental contaminants arise from various anthropic activities. Most of them could have dramatic consequences on the ecosystems and on the natural equilibrium. TBT is one of the most toxic contaminants introduced in the marine environment on a large scale since the early 70'. The damaging effects caused by TBT to aquatic organisms have initially been observed in the Bay of Arcachon at the end of the 70's. TBT contamination was found to be responsible for failure in reproduction and for anomalies occurring in the shell calcification leading to stunted growth. Although TBT inputs have decreased due to restrictive regulations, important contamination levels are still reported by numerous and various environmental studies, particularly in port and harbour areas. The aim of the present work was to assess contamination of the sediment by organotin compounds in the Bay of Arcahon. Sediment core analysis permitted a recording of the contamination and organotin depth profil of the sediment. Additionally sedimentation rates in different port areas and a chronology of the contamination have been estimated by 210Pb analysis. Finally, due to the problems concerning dredging material management, we have performed a preliminary study on the sediment decontamination by electrokinetic process

La reaction de l'hydrure de tributyletain avec des halogenures allyliques et acetyleniques, des dienes conjugues et des dithioesters a ete etudiee. L'influence des conditions experimentales est determinante en terme de selectivite. Ainsi la regio-stereo ou chemoselectivite de ces reactions est grandement amelioree par l'utilisation de la technique des hautes pressions (800-1300 mpa)

L'etude de l'imposex chez nucella lapillus en bretagne occidentale permet de mettre en evidence une forte source de contamination par le tributyletain (tbt) au niveau de la zone portuaire brestoise. Cette pollution est exportee non seulement vers l'ensemble de la rade, mais egalement a l'exterieur de celle-ci, vers le nord. Certaines populations de n. Lapillus ont disparu de la rade de brest, consecutivement aux effets sterilisants du tbt. Cependant, un maintien est observe en de nombreuses localites, qui s'expliquerait par la presence du syndrome de dumpton: une defectuosite de l'expression du sexe male, d'origine genetique. Des analyses biochimiques portent sur les taux de tbt couples a ceux de lipides dans les tissus, sur le cytochrome p-450 et des activites enzymatiques associees, et sur les concentrations en steroides. Elles ne fournissent aucun renseignement sur les possibles etapes du mecanisme biologique de l'imposex

La partition du tributylétain entre l'eau et diverses matieres en suspension (MES) dans des eaux de salinité variable a été étudiée au laboratoire selon un plan d'expérience impliquant 6 facteurs indépendants (temps, température, nature (4 types) et concentration des MES, salinité de l'eau et concentration initiale de tributylétain dans l'eau). Les capacités d'adsorption maximum des diverses MES spécifiques et les vitesses d'adsorption ont été étudiées. L'utilisation des isothermes de Langmuir et de la loi d'Arrhenius ont permis de calculer que les énergies d'activation mises en jeu sont d'environ 40 kj. Mol##1 et amènent à penser que l phénomène d'adsorption est une physisorption par liaison de faible énergie. Ensuite, la reconstitution en laboratoire d'écosystèmes représentatifs des milieux pollués (marin côtier, estuarien et eau douce) a permis de suivre l'évolution, la dégradation et la mobilité du tributylétain (tbt) dans un système eau/sédiment. Pour mener à bien cette étude, nous avons établi un plan d'expérience intégrant six facteurs et l'avons réalisé en trois périodes de six mois : température (6#c et 25#c), lumière (absence ou présence), phase dopée (eau ou sédiment), taux de dopage (1 et 10 mg/l dans l'eau; 0,5 et 5 ppm dans les sédiments), site reconstitué (estuaire, eau douce et marin côtier) et aération (absence ou présence). Les résultats expérimentaux sont interprétables en faisant intervenir l'action des micro-organismes dans ces milieux reconstitués où l'on observe une biodégradation par débutylétain progressive qui semble être dans certaines conditions la voie principale d'élimination du tbt. On observe en plus de la biodégradation de nouvelles espèces méthylées et hydrurées. Mais les mécanismes biochimiques régissant les intéractions tbt-microorganismes n'ont pas pu être parfaitement compris.

Les principaux objectifs de cette étude sont de mettre en évidence l'influence de la progestérone sur la gamétogenèse et d'étudier l'effet du tributylétain (TBT) sur le niveau des hormones sexuelles (progestérone, testostérone et 17 -estradiol) chez Mya arenaria. Après caractérisation de la progestérone dans la gonade chez Mya arenaria, un pic de cette hormone en relation avec la période de maturation sexuelle a été mesuré. Le délai de la maturation sexuelle constaté chez les myes de Rimouski semblerait être lié aux faibles niveaux de la progestérone mesurés chez ces organismes. La présence des perturbateurs endocriniens tels que le TBT et DBT dans la gonade de ces myes pourrait être la cause des teneurs faibles en progestérone. Les organismes exposés à une contamination nominale de 100 ng TBTCl/L renferment dans leur gonade 491 ng Sn (TBT)/g (poids sec) après deux mois d'exposition et 1031 ng Sn/g après trois mois d'exposition. Les niveaux de testostérone augmentent de 50% chez les femelles exposées à 10 et 100 ng TBTCl/L après un mois d'exposition suivi par une augmentation du niveau en -oestradiol après trois mois d'exposition comparativement aux contrôles
The goals of this study were to examine the link between progesterone and gametogenesis and Tributyltin (TBT) effects on steroid hormones (progesterone, testosterone and 17 -estradiol) in Mya arenaria. Progesterone has been characterized in the gonad of Mya arenaria and for the first time the link has been established between the gametogenesis cycle and the level of progesterone in this species. The level of progesterone in the gonad of Mya arenaria from Rimouski was 12% of those of levels found in Anse à l'Orignal. Inhibition of synthesis of progesterone by the presence of endocrine disruptors as TBT and DBT in the Rimouski gonad clams may cause the delay in the sexual maturation. When Mya arenaria was exposed to 100 ng TBTCl/L we obtained an accumulation of 491 ng of TBT as Sn/g gonad dry weight for a period of two months and of 1031 ng TBT as Sn/g gonad dry weight after three months of exposure. Mesocosms experiments have shown an increase of testosterone by 50% in August followed by an increase of 17 -estradiol between September and October in female gonads exposed to 10 and 100 ng TBTCl/L in comparison to the controls

L’objectif de ce travail est de comprendre les interactions du tributylétain avec un sable de quartz d'origine naturelle. Le comportement du système complexe a été modélisé en effectuant le même type d'étude sur les principales phases minéralogiques le composant : ce même sable sans les argiles (sable traite), un quartz et une kaolinite. Les analyses couplent à la fois l'analyse de la solution et la caractérisation des surfaces. L’analyse du sable par différentes techniques complémentaires (MEB, AFM, XPS) a permis de mettre en évidence la grande hétérogénéité de surface du solide. En ce qui concerne l'adsorbat, des expériences de titrage du TBT par de la soude ainsi que des mesures de conductivité indiquent que seuls les monomères TBT+ et TBTOH sont présents en solution dans nos conditions expérimentales : concentration en TBT comprise entre 1. 10-5 et 5. 10-4 m et présence de méthanol (entre 0,1 et 5%). Le comportement tensioactif du TBT et ses conséquences sur la sorption ont été pris en compte pour l'interprétation des résultats. Les isothermes présentent une allure qui est indicative de la formation d'une première monocouche suivie par un phénomène de précipitation de surface. La première monocouche résulte de la fixation de TBT+ ou de TBTOH avec formation de complexes tel (SO-), (SOH)(TBT+) et (SOH)(TBTOH), la liaison entre les groupements hydroxyles de surface et le TBT étant probablement de nature électrostatique. Par contre, la précipitation de surface est associée a des interactions hydrophobes et fait intervenir des espèces électriquement neutres vraisemblablement TBTOH. Nos résultats indiquent de plus que les sites d'adsorption préférentiels du TBT sont situés sur le quartz et non sur les argiles. Ainsi, par unité de surface, la capacité de sorption du TBT par les différents adsorbants suit l'ordre suivant : kaolinite << sable naturel < sable traite < quartz

La réaction de substitution nucléophile allylique de Trost et Tsuji, catalysée par un complexe soluble du palladium, est ici considérée du point de vue du groupe partant elle apparait donc comme une réaction de déprotection de diverses fonctions de la chimie organique initialement protégées sous forme de dérivés allyliques. L'hydrure de tributylétain, qui acquiert en présence d'une quantité catalytique de palladium, une grande réactivité de donneur d'hydrure, est le nucléophile entrant. La coupure réductrice des dérivés allyliques par le système hydrure de tributylétain-palladium est instantanée à température ambiante et hautement sélective ; elle fait l'objet de deux applications en synthèse peptidique sur support solide : -la déprotection de la fonction amine des acides aminés, protégée par le groupement allyloxycarbonyle. Cette étude s'achève par la synthèse d'un peptide biologique, la substance P, sur support classique (résine méthylben­ zhydrylamine), -la coupure, au terme d'une synthèse peptidique classique [stratégie Boc] d'un lien ester allylique reliant le résidu C-terminal et le polymère. Le peptide ainsi obtenu est un segment protégé utilisable en synthèse convergente
The palladium catalyzed nucleophilic substitution of allyl derivatives- the Trost-Tsuji reaction- is viewed here for its leaving group entity. It appears, there-fore, as a deprotection procedure for various functional groups initially protected as their allyl derivatives. Tributyltin hydride, whose hydride donor properties are considerably enhanced in the presence of catalytic amounts of palladium complexes, is the entering nucleophilic species. The hydrostannolytic cleavage of allyl derivatives by palladium catalyst/tributyltin hydride system is highly selective and instantaneous at room temperature. Two applications of this reaction to solid phase peptide synthesis are described: -the deprotection of the amino group of amino-acids and peptides, initially protected by the allyloxycarbonyl group. The reaction has been repeatedly used in the synthesis of the biologically active undecapeptide substance P which was carried out on a classical p-methylbenzhydrylamine resin support, -the cleavage of an allylic resin-peptide link after the assembly of the peptide chain had been carried out under standard conditions (Boc strategy). Such method allows the obtention of free protected peptide fragments which con be later used in convergent synthesis

Les écosystèmes aquatiques se révèlent particulièrement exposés à des situations de contamination chronique par le tributylétain (TBT) et le mercure (Hg). Ces micropolluants métalliques, ubiquistes et persistants, sont présents dans l'environnement à l'état de traces, néanmoins suffisantes pour induire des implications écotoxicologiques graves. Les divers mécanismes de transformation et de transfert du TBT et du Hg, conditionnant leur dynamique biogéochimique, ont ainsi été étudiés au moyen d'outils analytiques et expérimentaux innovants et permettant une meilleure évaluation des risques. La première partie de ce travail porte sur l'étude mécanistique de la réactivité et du transfert du TBT et du Hg dans des écosystèmes aquatiques reconstitués. Ces expériences associent des techniques analytiques de spéciation performantes, basées en partie sur la quantification par dilution isotopique, à des outils expérimentaux simulant les conditions environnementales. Les cinétiques de distribution du TBT et du Hg (adsorption, bioaccumulation, biodégradation, dépuration) ont ainsi été caractérisées simultanément dans les différents compartiments des microcosmes présentant une organisation simple. Dans une seconde étape, les cinétiques de volatilisation du TBT aux interfaces naturelles ont été étudiées afin d'estimer les voies potentielles de remobilisation et d'élimination des butylétains. Enfin dans une troisième partie, des traceurs isotopiques stables du Hg ont été utilisés pour discriminer et quantifier les cinétiques couplées de méthylation et déméthylation dans des sédiments estuariens, en fonction de différents forçages environnementaux (oxygénation, activité microbienne)
Aquatic ecosystems are particularly affected by tributyltin (TBT) and mercury (Hg) chronic contamination. These micropollutants are ubiquitous and persistent and occurred at trace level, likely to drastically impair aquatic environments. TBT and Hg biogeochemical cycles are driven by transformation and transfer mechanisms between the different environmental compartments. These natural processes have been studied in details by using novel analytical methods and environmental design to improve the risk assessment. The first part of this work focus on the mechanistic study of TBT and Hg reactivity and transfer in reconstituted aquatic ecosystems. These experiments involve both state-of-the-art analytical speciation techniques, partly based on quantification by isotopic dilution and experimental tools simulating the environmental conditions. Kinetics of TBT and Hg distribution (adsorption, bioaccumulation, biodegradation, clearance) have been simultaneously characterized in al compartments of the microcosms presenting a simple biological organization. In a second step, volatilization kinetics of TBT at real interfaces have been studied to assess the potential remobilization and elimination pathways of butyltin compounds. Finally, in a third part, stable isotopic tracers of Hg have been employed to discriminate and quantify the coupled methylation and demethylation kinetics in estuarine sediments, by forcing different environmental factors (oxygenation, microbial activity)

Avec une législation devenant de plus en plus stricte concernant les sédiments de dragages, la décontamination des polluants et plus particulièrement, à cause de leur forte toxicité, des composés organostanniques, notamment le tributylétain (TBT) représente un enjeu très important. Différents procédés de décontamination des sédiments pollués existent. Certains reposent sur les phénomènes d'atténuation naturelle souvent longs et limités à certains polluants, tandis que d'autres consistent à stabiliser et confiner la pollution par voie chimique ou thermique. L'électrodécontamination est une technique innovante qui n'a encore reçu que peu d'applications au stade pilote ou sur le terrain au plan international. Le principe de base de ce procédé consiste à exploiter le déplacement d'espèces ioniques polluantes sous l'effet d'un champ électrique entre une ou plusieurs cathodes et une ou plusieurs anodes implantées dans un milieu poreux. D'une manière simplifiée, la dépollution qui s'effectue essentiellement par électromigration pour les espèces ioniques (métaux libres...) vers l'électrode correspondante (cathode pour les cations et anode pour les anions) et par électroosmose pour les espèces neutres (composés organiques ou organométalliques neutres...) le plus souvent vers la cathode. Son efficacité est contrôlée par le flux de polluants dans la solution du sol donc par leur vitesse de transport et de leur concentration dans la phase liquide. L'optimisation du procédé dépend donc simultanément de facteurs électrochimiques qui contrôlent le transport des polluants et de facteurs physico-chimiques qui influencent le transfert de matière. Plusieurs expériences ont été menées sur un modèle de matrice solide (kaolinite dopée en tributylétain (TBT)) et sous diverses conditions de pH et de gradient de potentiel. Des essais ont été également réalisés sur un sédiment naturel avec diverses conditions opératoires concernant le pH, le gradient de potentiel et la température. Les résultats montrent que nous obtenons des pourcentages d'abattements en TBT supérieurs à 90 % pour un modèle de matrice solide, la kaolinite dopée en TBT, et peuvent atteindre jusqu'à 70 % d'abattement pour un sédiment naturel. Ils montrent également que les composés migrent préférentiellement vers la cathode. Les résultats obtenus ont permis de mettre en évidence la compétition entre deux mécanismes : le transfert / transport des composés butylés de l'étain vers les électrodes et le transport du réactif facilitant la dégradation in-situ de ces espèces. Les travaux ont montré des résultats très satisfaisants et extrêmement prometteurs quant à l'application du procédé au stade pilote. Ils ont ouverts une voie particulièrement intéressante sur la décontamination des sédiments et leur gestion future.

Les restrictions d'utilisation des peintures antisalissures pour bâteaux à base de tributylétain (ou TBT) ont permis d'écarter les risques majeurs de pollution de l'environnement marin dûs à ce composé. Néanmoins, la surveillance de certains sites demeure nécessaire en raison de la rémanence du toxique adsorbé sur les sédiments. Il faut donc disposer de techniques analytiques performantes permettant le dosage du TBT dans ces matrices complexes. La calibration de ces méthodes n'est pas simple et requiert un matériau de référence international dont l'élaboration nous a été confie par la C. E. E. Cette étude a nécessité de s'assurer de la qualité de chacune des étapes de préparation de l'échantillon. La méthode de spéciation retenue (Gh/Cg/Saa), a donc été optimisée et la limite de détection absolue obtenue pour le TBT atteint 0,09 Ng. Plusieurs procédés d'extraction du TBT dans les sédiments ont été testés et c'est avec l'acide acétique pur que nous observons le meilleur recouvrement. Les conditions de transport et stockage du matériau avant l'analyse ont été définies ainsi qu'un traitement de stabilisation à 120°c permettant de limiter la biodégradation du TBT sans causer sa décomposition thermique. L'objectif de ces mises au point préliminaires était de se placer dans des conditions optimales de quantitativité. Les travaux que nous avons effectués sur deux échantillons différents ont conduit à des résultats satisfaisants en ce qui concerne la stabilité du matériau au cours du temps et son homogénéité. La C. E. E a donc autorisé la mise en oeuvre d'une campagne de certification, qui constitue l'aboutissement de nos recherches.