Rtuť je v přírodě přirozeně se vyskytujícím toxickým prvkem, jehož globální emise jsou ovlivňovány zejména antropogenními zdroji znečištění. Obrovský globální nárůst v usazování rtuti, zejména ve vodných ekosystémech, byl zaznamenán současně s počátkem průmyslové revoluce. Sedimenty jsou posledním místem úložiště nejrůznějších komplexů rtuti. Rtuť však zde může být přeměněna na toxičtější organickou formu, methylrtuť, pomocí transformačních procesů kontrolovaných různými fyzikálními, chemickými, ale i biologickými faktory. Navíc mohou být specie rtuti remobilizovány ze sedimentů pomocí difuze a resuspenzace a tak se sedimenty mohou stát i potenciálním zdrojem rtuti. Proces bioakumulace a bioobohacování tak pokračuje v potravním řetězci, ve kterém se člověk, i další zvířata, stává konzumentem methylrtuti. Stanovení celkové koncentrace rtuti není dostačující k porozumění osudu rtuti v přírodním prostředí a tak stanovení MeHg poskytuje nezbytnou doplňující informaci. Dostatečně citlivá a přesná analytická metoda pro stanovení specií rtuti je nezbytným nástrojem environmentální chemie. Metody vhodné pro stanovení specií rtuti v sedimentech jsou popsány v části metodologie disertační práce. Metoda stanovení methylrtuti v sedimentech pomocí automatické Headspace vybavené pastí („trap“) a spojené s plynovou chromatografií a fluorescenční detekcí je zde také popsána. Zvláštní pozornost je také věnována potřebám zásad čistého vzorkování, skladování vzorků a přípravě vzorků před samotou analýzou, jakož i samostatné části věnující se terénní studii rtuti a methylrtuti v sedimentech vytipovaných lokalit. Sedimenty jižní Moravy a severní Francie jsou srovnány z hlediska znečištění rtutí. Specie rtuti a další ukazatele (Fe, Mn, S) byly analyzovány v sedimentech, pórové vodě a povrchové vodě řek Dele a Lys (Francie) a Jihlava a Morava (Česká republika). Z hlediska posouzení vodních ekosystémů a jejich znečištění rtutí, je vhodné znát koncentraci rtuti v pórové vodě a posoudit dostupnost rtuti ze sedimentů. Technika difuzního gradientu v tenkém filmu je vhodným způsobem jak stanovit koncentraci rtuti v pórové vodě sedimentů. Do roku 2005 bylo použití této techniky pro měření rtuti značně limitováno. Ale nedávný pokrok především v dostupnosti možných sorpčních gelů vhodných pro stanovení rtuti umožnilo využití této techniky i pro stanovení rtuti. Byly použity různé sorpční gely: Spheron.Thiol, Duolite GT-73 a TiO2. Řeka Dele představuje past enormního množství antropogenní rtuti pocházející z průmyslových zdrojů a je považována za potenciální významný zdroj methylrtuti pro okolní prostředí a živé organismy především. Poslední část dizertační práce se zabývá aplikací dobře zavedeného experimentu využívajícím stabilní isotopy ke studiu metylačních a demethylačních procesů v sedimentech řeky Dele. Obohacené stabilní značkovače rtuti v anorganické formě (199Hg) and methylované formě (201MeHg) byly přidány do sedimentů. Tyto označené specie rtuti tak pomohly sledovat osud specií rtuti a vypočítat rozsah jejich přeměny v průběhu experimentu.

The recently developed technique of diffusive gradients in thin films (DGT) for speciation measurement of analytes in the environment was further developed through the development of series of new binding phases including poly(acrylamide-co-acrylic acid) copolymer hydrogel (PAM-PAA), poly(acrylamidoglycolic acid-co-acrylamide) (PAAGA-PAM) hydrogel, the Whatman P81 cellulose phosphate ion exchange membrane (P81) and a liquid binding phase of poly(4-styrenesulfonate) (PSS). A new diffusion layer, cellulose dialysis membrane, was also employed for the liquid binding phase DGT. PAM-PAA copolymer hydrogel was prepared by the controlled hydrolysis of polyacrylamide (PAM) in an alkaline solution of 10% sodium hydroxide. The capacity of the copolymer hydrogel to bind various metal ions was tested under a range of uptake conditions. Ions such as Cu2+ and Cd2+ were bound more strongly to the copolymer hydrogel than the competing ions such as Na+, K+, Ca2+ and Mg2+. Metals bound to the copolymer hydrogel can be efficiently eluted in 2 M HNO3 solution (>94%). Application of this new binding material to DGT technique was validated in a synthetic lake water (Windermere, Lake District, UK) with a recovery of 99.0% for Cu2+. PAAGA-PAM hydrogel was prepared by copolymerising 2-acrylamidoglycolic acid with acrylamide. The metal ion binding properties of the hydrogel were characterised for Cu2+, Cd2+ and competing ions under various experimental conditions. The hydrogel was shown to bind Cu2+ and Cd2+ strongly under non-competitive binding conditions, with binding capacities of 5.3 and 5.1 micromol cm-2, respectively. The binding capacity of each metal decreased, under competitive binding conditions (with a range of metal ions present at 17.8 mN), to 1.3 and 0.17 micromol cm-2, respectively, indicating a strong selective binding towards Cu2+. The metal ions were readily recovered (>94%) by eluting with 2 M HNO3. Finally, the copolymer hydrogel was tested as a binding phase with the DGT technique. A linear mass vs. time relationship was observed for Cu2+ in Windermere water with a recovery of close to 100%. The use of a commercially available solid ion exchange membrane (P81) as the binding phase in DGT analysis was demonstrated. P81 is a strong cation exchange membrane. Its performance characteristics as a new binding phase in DGT measurement of Cu2+ and Cd2+ were systematically investigated. Several advantages over the conventional ion exchange resin-embedded hydrogel based binding phases used in DGT were observed. These include: simple preparation, ease of handling, and reusability. The binding phase preferentially binds to transition metal ions rather than competing ions. Within the optimum pH range (pH 4.0 - 9.0), the maximum non-competitive binding capacities of the membrane for Cu2+ and Cd2+ were 3.22 and 3.07 micromol cm-2, respectively. The suitability of the new membrane-based binding phase for DGT applications was validated experimentally. The results demonstrated excellent agreement with theoretically predicted trends. The reusability of this binding phase was also investigated. Application of a liquid binding phase and a dialysis membrane diffusive layer were proposed for the first time. The binding phase was a 0.020 M solution of poly(4-styrenesulfonate) (PSS) polyelectrolyte using a specially designed DGT device. The binding properties of Cd2+, Cu2+, and a range of alkali and alkaline earth metal ions to the PSS solution were characterised. The PSS behaved like a cation exchanger with preferential binding to Cd2+ (6.0 micromole ml-1, log K = 9.0) and Cu2+ (2.5 micromole ml-1, log K = 8.1) under competitive binding conditions. The DGT devices were successfully validated for Cd2+ and Cu2+ in Windermere water. The speciation performance of the solid and liquid binding phases developed in this study was investigated in solutions containing ethylenediaminetetraacetic acid disodium salt (EDTA), humic acid (HA), glucose (GL), dodecylbenzenesulfonic acid (DBS) and tannic acid (TA) with Cu2+ and Cd2+. The ratios of labile metals over total metals were at good agreement with calculated theoretical values using Stability Constants Database. The results indicated that the DGT-labile concentration measured by DGT with these binding phases is essentially free metal ion concentration in the sample. All newly developed DGT binding phases were successfully applied for environmental speciation. The field deployments were carried out in both freshwater and salt-water test sites.

The doctoral thesis deals with usage of gel techniques for in situ measurement of bioavailable metals in soils and sediments. The DGT technique was applied to soil samples from the localities of Zlín, Tuřany and Chrlice for estimation of bioavailable metals in soils. These soils were amended by sewage sludges in years 1980-1987. The experiment using diffusive gels with different pore size, APA and RG gels, has shown that the DGT can be used for assessment of trace metal soil complexes - small inorganic and larger organic. The results obtained by DGT measurements were approximately up to two orders of magnitude lower than metal concentrations obtained by leaching with sodium nitrate. The leaching with sodium nitrate does not provide true reflection of metal availability to plant root system and soil microorganisms and the DGT technique seems to be a better predictor of metal bioavailability. Only small differences between undisturbed and homogenized soil samples were found out by application of DGT to soil samples from localities of Veverská Bítýška and Ostrovačice. The undisturbed soil is not homogenous and the DGT unit gives information only about the part of the soil which is in direct contact with the unit. The procedure with soil slurry can be recommended for the characterization of large soil areas and estimation of bioavailable metals in soils. Gel techniques were also used for the study of processes occuring in sediments. Mercury species and other metals were analysed in surface water, pore water and sediments collected from two sampling sites in the Dele River in northern France. This river had been polluted during the last century by the activity of Métaleurop factory, which dealt with metal ore treatment. The application of DGT technique using two specific ion-exchange resins, Chelex-100 and Duolite GT-73, provided assessment of mercury in sediment pore water. DGT and DET techniques together with centrifugation provided also high resolution depth profiles of Fe, Mn, Zn, Pb, Cr and Cd in pore water. It is not possible to determine which sampling site was more contaminated. Only small differences in mercury species and metal content were observed between sampling sites. The Dele River can be classified as contaminated river according to total mercury content in sediments and as slightly contaminated river from the point of view of methylmercury content. Correlations between mercury species and sulphide content confirm that total mercury and sulphide content count among factors influencing the mercury release to pore water and controlling processes of methylation and demethylation. Only low toxicity of sediment slurries was found out by the bioluminiscence test with Vibrio fischeri. The results of doctoral thesis demonstrate gel techniques as a very good tool in environmental analysis.

La disponibilitat de metalls per part dels organismes no depèn únicament de la concentració total sinó també de l'especiació d'aquestes subtancias. En general, la disponibilitat de les espècies metàl•liques en medis naturals per a organismes està determinada per un conjunt d'esdeveniments encadenats on el equilibri representa només un cas particular de la situació dinàmica general. Espècies poc definides químicament, com ara àcids húmics i fúlvics, actuen com a lligands de metalls i juguen un paper clau en la circulació i la determinació de les propietats ecotoxicològiques de les espècies metàl•liques en medis naturals. La polifuncionalitat dels àcids húmics i fúlvics és descrita en aquest estudi utilitzant mètodes termodinàmics i mecano-estadístics. La tècnica Diffusive Gradients in Thin films (DGT) s'ha utilitzat per mesurar el flux disponible de metall en un sistema sintètic que conté complexos metàl•lics. La principal conclusió d'aquest estudi és que el disc de resina quelant dels sensors juga un paper clau en la determinació del grau de labilitat d'un complex mesurat amb aquesta tècnica. D'altra banda, a un pH baix, o concentració alta de lligand, es pot aconseguir l'equilibri entre la resina del sensor i el metall en el si de la dissolució, pertorbant l'acumulació lineal a temps de contacte relativament curts. Es presenta una anàlisi d'aquests fenòmens, així com la seva justificació mitjançant models teòrics. / La disponibilidad de metales por parte de los organismos no depende únicamente de la concentración total sino también de la especiación de estas subtancias. En general, la disponibilidad de las especies metálicas en medios naturales hacia organismos está determinada por un conjunto de eventos encadenados cuyo equilibrio representa sólo un caso particular de la situación dinámica general. Especies poco definidas químicamente, tales como ácidos húmicos y fúlvicos, actúan como ligandos de metales y juegan un papel clave en la circulación y la determinación de las propiedades ecotoxicológicas de las especies metálicas en los medios naturales. La polifuncionalidad de los ácidos húmicos y fúlvicos es descrita en este estudio usando métodos termodinámicos y mecano-estadísticos. La técnica Diffusive Gradients in Thin films (DGT) se ha utilizado para medir el flujo disponible de metal en un sistema sintético que contiene complejos metálicos. La principal conclusión de este estudio es que el disco de resina quelatante de los sensores juega un papel clave en la determinación del grado de labilidad de un complejo medido esta técnica. Por otra parte, a un pH bajo, o a concentración alta de ligando, puede ser alcanzado el equilibrio entre la resina del sensor y el metal en el seno de la disolución, perturbando la acumulación lineal a tiempos de contacto relativamente cortos. Se presenta un análisis de estos fenómenos, así como su justificación mediante modelos teóricos. / Availability of metals for organisms does not depend only on the total metal concentration but also on speciation of these species. In general, the availability towards organisms is determined by a set of chained events whose equilibrium approach only represents a simplified limiting case of the general dynamic situation. Poorly defined species such as humic and fulvic acids, act as metal ligands and play a key role in the circulation and determination of the ecotoxicological properties of metal species in the natural media. Thermodynamic and statistical mechanics methods are here applied to describe the polyfunctionality of humic and fulvic acids. The Diffusive Gradients in Thin Films (DGT) has been used to measure the available metal flux in a synthetic system that contains metal complexes. A main conclusion of this study is that the chelating resin disc of the sensor plays a key role in determining the lability degree of a complex measured with DGT. Moreover, at relatively low pH, or in presence of high affinity ligands, the equilibrium with the bulk metal concentration can be approached in the sensor, disturbing linear accumulations at relatively short deployment times. Analysis of these phenomena and theoretical explanations are reported.

Recognition that the bioavailability of soil As (As) is influenced by its soil dynamics has initiated research into development of more accurate, site-specific soil guideline values, departing from the assumption that the total soil As content is bioavailable. With the aim of deriving predictive models, the relationship between soil properties and As bioavailability (bioaccessibility and phytotoxicity) was examined on a set of naturally contaminated sheep dip soils (n = 30). Sampled soils were extensively characterised, bioaccessibility was estimated through an in vitro procedure, and soil As toxicity and availability to plants were evaluated using an early growth wheat bioassay. The in vitro bioaccessibility was consistently less than the total soil As content. Arsenic bioaccessibility was negatively correlated to soil iron (Fe), manganese (Mn) and aluminium (Al) contents, and it was positively related to the soil As loading. The in vitro extractable soil As concentrations were successfully modelled using linear combinations of soil As content, soil Fe and Mn determinations and soil pH. Differences in As phytotoxicity, expressed in terms of effective toxic concentration (EC50), between soils were directly related to soil Fe, Mn and Al contents. Available soil phosphorous (P) exerted an ameliorating effect on As toxicity, with the available soil As/P ratio representing the single best predictor of plant growth suppression. Plant P nutrition appeared to influence the relative selectivity for As and P by wheat, with greater selectivity for P demonstrated under P deficient conditions. Plant As uptake, its distribution, and also the plant nutrient status were all adversely affected by increasing soil As exposure. Co-contamination by Zn corresponded to a substantial elevation in proportion of the plant As allocated in shoots. Plant As levels exhibited a saturation-dependent relationship with increasing soil As. The best linear predictors of plant As levels in the non-toxic range were RHIZO-extractable and effective soil As concentrations, the latter based on the diffusive gradients in thin films (DGT) technique. Despite the complexity of soil As dynamics, large proportions in the variances exhibited by the two measures of bioavailability were explained using a small set of readily-available soil properties.

Les objectifs de cette étude comparative de deux bassins versants en France et en Ukraine sont de trois ordres. i) déterminer l'occurrence des micropolluants émergents (métaux traces et molécules pharmaceutiques) dans les eaux de surface, ii) permettre leur évaluation qualitative et quantitative et enfin iii) estimer leur distribution en fonction du contexte socio-économique. Cette recherche porte sur l'analyse de l'origine, sur les propriétés physico-chimiques et sur le comportement des micropolluants (métaux traces et molécules pharmaceutiques) dans les eaux des rivières Jalle (Bordeaux, France) et Udy & Lopan (Kharkiv, Ukraine). Un suivi des métaux traces et des produits pharmaceutiques dans les eaux naturelles de surface dans différents contextes et conditions climatiques a été réalisé. La description et l'analyse des facteurs environnementaux et socio-économiques influençant l'hydrochimie locale en vue de conclure sur l'opportunité et l'intérêt des micropolluants émergents comme indicateurs anthropogéniques du contexte socio-économique et environnemental d'un bassin versant ont été mis en oeuvre. L'étude a combiné des techniques de prélèvements d'échantillons d'eau classiques et par capteurs passifs in situ. L'analyse semi quantitative des micropolluants émergents a porté sur les métaux traces (sondes DGT) et les produits pharmaceutiques (POCIS). L'usage de ces capteurs a permis d'évaluer les différentes formes d'éléments, les sources, les variations saisonnières et spatiales, l'accumulation et les risques environnementaux. A partir des données environnementales collectées, acquises, actualisées et validées il a été possible de modéliser la consommation régionale de médicaments dans le cas de (1) la région de Kharkiv, en Ukraine, sur les rivières Lopan et Udy du bassin Seversky Donets et (2) la région de Bordeaux, en France, sur la rivière Jalle, du bassin de la Garonne. Les sites ont été sélectionnés pour représenter une diversité tant du point de vue de l'état de pollution des eaux (nature et flux) que de celui du paysage socio-économique (contexte urbain, social et indicateurs économiques). Les résultats majeurs de cette étude sont : (i) méthodologique pour le développement et la validation de protocoles d'échantillonnage classique et passif (intérêt, limites, recommandations), ii) analytique quant à la mesure des éléments traces des eaux de surface naturelles dans des conditions extrêmes, iii) scientifique par l'obtention d'un jeu de données sur l'hydrochimie des éléments traces ( présence, accumulation, origine et variabilité spatiale et temporelle et iv) prospectif quant à l'usage des métaux traces et de produits pharmaceutiques comme traceurs anthropiques de l'état des eaux naturelles de surfaces et reflet du contexte socio économique. La modélisation socio-économique (statistique) et environnementale (balance de masse) aide à comprendre l'évolution de la qualité des eaux de surface dans leur contexte régional et permet d'identifier certains contaminants comme des indicateurs des activités anthropiques d'un bassin versant et permet de définir une typologie. L'ensemble des résultats de la thèse ont présentés sous la forme d'articles publiés ou soumis dans des revues scientifiques internationales.

<p dir="ltr">Bacterial Wilt, caused by <i>Ralstonia solanacearum</i>, is among the most devastating plant diseases in the world. This pathogen causes significant loss in crops such as tobacco, potato, and tomato. <i>R. solanacearum</i> root infection and xylem colonization determine disease outcome. To date, little is known about the defense mechanism mediated by roots to prevent <i>R. solanacearum</i> vascular colonization during the initial infection stages. Plant early responses are important since they may impact disease outcomes<i>.</i><i> </i>Here, we report the formation of root hairs and primary root growth inhibition in tomato seedlings as <i>Ralstonia</i>-induced phenotypes that depend on tomato genotype and <i>Ralstonia</i> species. The <i>Ralstonia</i>-induced root phenotypes are independent of a functional type III secretion system and exopolysaccharide production (EPS). We also found that <i>R. solanacearum</i><i> </i>K60 infection increased auxin levels throughout the root meristem in wilt-susceptible tomato roots. Our data suggest proper auxin signaling and transport are important for susceptibility to <i>R. solanacearum</i> K60. Blocking auxin transport pharmacologically or genetically led to fewer wilting symptoms, suggesting that auxin is important during early infection stages and disease outcomes in tomato. We previously found that a tomato mutant defective in auxin transport and signaling, known as <i>diageotropica</i> (<i>dgt</i>), has enhanced resistance to <i>R. solanacearum</i> K60. We characterized the resistant response in the <i>dgt</i> mutant, and we found that the resistant response in the <i>dgt</i> mutant may be due to increased lignin content preventing pathogen vasculature colonization. <i>DGT</i> encodes a cyclophilin protein that regulates auxin transport and signaling. Mutations in the cyclophilin DGT promote resistance to <i>R. solanacearum</i> K60. DGT has been reported to regulate auxin transport and signaling. However, the molecular mechanism regarding how DGT mediates these processes is still unknown. We used Yeast Two-Hybrid to identify candidate protein interactors, and we found that SlbZIP1/SlbZIP29, Sl14-3-3, and SlMYB110 may interact with DGT to regulate both development and defense responses. Understanding the role of DGT, auxin, and lignin in defense responses to <i>R. solanacearum</i> K60 in tomato is necessary for Solanaceae crop improvement.</p>