CE and CE-ICP-SFMS in rhizosphere studies

The understanding of fundamental mechanisms of metal uptake in plants is crucial to the progress in phytoremediation research. The proposed project focuses on the specific problem of metal complexation in the plant root environment (rhizosphere). Root exudates, i. e. substances released from intact cells along a concentration gradient have been proposed to be involved in many processes in the rhizosphere, including nutrient acquisition metal mobilization and detoxification because of their capacity to complex metals. Their role in these processes, also with respect to hyperaccumulation, however, can not be fully appreciated unless root exudation pattern and the fate of the exuded compounds in the soil are better known. In the submitted project the problem will be addressed by an interdisciplinary approach: First, adequate analytical techniques for studying of metal species will be developed. Analytical methodological work will be carried out in order to determine potential ligands in real samples i. e. soil solutions by capillary electrophoresis (CE). CE is an attractive technique for analysis of soil solutions because of its very low volume requirements and limited problems with complex matrices. CE hyphenated to inductively coupled plasma sector field mass spectrometry (CE-ICP- SFMS), a novel analytical tool, will be employed for the first time for speciation analysis in rhizosphere solutions. Due to both the advantageous sensitivity of ICP-SFMS and the minor effect of a CE system on the distribution of chemical species, this combination is ideal for element speciation studies. Goal of the research is to perform metal speciation strictly avoiding potential changes of the species during the separation step. CE-ICP-SFMS methods hold great promise for separating potential ligands, free metals in different oxidation states and the metal complexes utilizing both on-line UV detection and ICP-SFMS detection. On the other side the research will focus on development of rhizobox model systems, that simulate soil conditions while allowing for sampling of microbially unchanged root exudates. Moreover the novel rhizobox design will provide experimental data on the spatial distribution of exudation pattern in the root compartment. Information on metal speciation in the rhizosphere will be approached by direct experimental assessment. Three different plants, the hyperaccumulators Thlaspi goesingense (Ni) and Thlaspi caerulsecens (Zn, Cd, Pb) and the non-accumulating Thlaspi arvense will be studied to improve our understanding of rhizosphere processes involved in hyperaccumulation.

The European commission estimated that 1.200.000 contaminated sites were only in Western Europe (estimated remediation costs of Euro 110 millions). The Environmental Agency classified 29.492 sites in Austria as contaminated (2002). Hyperaccumulation of metals by plants represents a promising novel approach for remediation of contaminated soils (phytoremediation). Therefore, fundamental studies on the metal up-take by these plants, in order to understand why hyperaccumulation occurs and how it can be fully exploited for remediation is of vital importance. The rhizosphere is located in the vicinity of the plant root. Interactions at this site are deemed to play a key role in controlling bioavailability of crop plants. Plant roots affect soil solution in their vicinity by ion uptake and root activities such as exudation of organic acids. Processes in the rhizosphere of metal hyperaccumulator species are largely unknown. Quantitative information on gradients of ion concentration across the rhizosphere and organic acid concentration is required to improve our understanding of plant-soil relationships and for the validation of mechanistic rhizosphere models. Methodological studies on experimental set- ups, which allow probing the rhizosphere of plants were carried out. Analytical methods for determination of metal and organic acid traces in soil solution and soil water extracts were established. Rhizoboxes were implemented allowing to grow plants with a defined root monolayer. Novel microsuction cups enabled sampling at defined distances from the root monolayer. In such experiments with the hyperaccumulator Thlaspi goesingense gradients of Ni, Ca, K and Mg could be related to plant uptake and mobilization processes. Moreover, flow-through microdialysis - a technique conventionally used in medical studies- could be implemented as a novel analytical tool for automatic sampling of low molecular weight organic anions, such as oxalate and citrate, in rhizosphere solution. Diffusive gradients in thin films could be applied as tool for evaluation the efficiency of phytoextraction. The arsenic hyperaccumulating brake fern Pteris vittata was studied. In parallel As speciation in soil solution was performed by CE-ICP-MS and HPLC-ICP-MS. In field experiments it could be shown that the oxalic acid content found in the soil water extract of the hyperaccumulator Thlaspi goesingense was significantly higher than the content of the bulk and two other non-hyperaccumulating species Silene vulgaris and Rumex acostella supporting the hypothesis of orgnic acids involved in metal hyperaccumulation mechanism.