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Final Report Summary - BIOMETA (Biomethylation and Biovolatilisation of Arsenic in Soils: Using Carbon and Hydrogen Isotopes to Unravel the Mechanisms and Pathways Involved)

The project BIOMETA’s aim was primarily to study the biomethylation and biovolatilisation of arsenic (As) by using 13C and 2H labelled As compounds as tracers. Indeed, although two pathways for the formation of methylated and volatile As have been formulated, none could be ascertained. Therefore, the Fellow set out to synthesize labelled As compounds to use in a range of bacterial cultures and to develop the analytical methods needed to extract, separate, collect and measure the labels so that the pathway(s) could finally be known. The Fellow could successfully synthesize monomethylated and dimethylated As compounds and has developed a method that allows for extracting and trapping methylated and volatile As species respectively. The traps and extracts could be measured by High Performance Liquid Chromatography coupled to an Inductively Coupled Plasma Mass Spectrometer (HPLC-ICP-MS). Furthermore, the use of a micro-splitter allowed the fellow to redirect part of the stream to a Fraction Collector, where each species could be collected. However, due to unforeseen instrumental issues with the isotopic ratio mass spectrometers, the project could not be satisfactorily completed. The Project was nonetheless successful in the sense that cultures of microorganisms were incubated and the methylated and volatile As species emitted could be successfully measured. Thus laying the foundation for the use of isotopically labelled As compounds in the future. Although isotopes could not be used and the exact pathway(s) therefore not determined, this work provided nonetheless new information on As biomethylation and biovolatilisation. It was found for example that lichens biovolatilise As at a rate higher than ever measured before. When upscaled, such rate comes close to copper (Cu) smelting which is the highest source of As to the atmosphere. Therefore, not only could lichen represent a very relevant but unknown source of As to the atmosphere but the rate at which they emit volatile As could be very useful to uncover As biomethylation pathway in incubations experiments.
Furthermore, instead of using As labelled compounds due to the above mentioned analytical issues, the Fellow initiated a series of experiments on the biomethylation and biovolatilisation of antimony (Sb), dubbed “As sister element” in the scientific literature, followed by a series of experiments on biomethylation of another highly relevant pollutant: mercury (Hg).
To measure methylated and volatile species of Sb and Hg, the fellow had to develop and validate new methods since they either did not exist or they were simply too complex and too costly. The fellow therefore developed extraction and analysis methods that are user-friendly and cost-efficient using HPLC-ICP-MS. In the case of Sb, a new method to measure volatile Sb species at trace levels using liquid traps and a new quantitative extraction method using oxalic acid to measure methylated Sb species in soils and biota were validated. These methods were used with soil samples originating from Sb-impacted shooting ranges where it was found that trimethylantimony existed in all three studied fields at values as high as 1.35 mg kg-1. In a hydroponic plant experiment, it was also discovered that plants growing on shooting ranges, such as rye grass could take up trimethylantimony and translocate it to the shoots. This is highly relevant since shooting ranges are still used as pastures. Finally, a soil incubation experiment showed that when flooded and amended with 2% organic matter (cow dung), a common agricultural practice, the soil released amounts of Sb in the pore water in the range of several mg.L-1 in the first 2 to 3 days. Furthermore, trimethylantimony represented up to 12% of the total Sb in the porewater after only 6 days.
With regard to Hg, the fellow adapted and validated a new method to quantitatively extract MeHg form soil and biota using 5M HCl followed by a selective extraction with DCM and a back extraction in 1% L-Cysteine for stability. The extract could then be measured by HPLC-ICP-MS and quantified in less than 3 min. The whole process being much faster, cost-effective and more user friendly than the usual certified method derived from the US EPA. This method was used to investigate the presence of MeHg in a contaminated floodplain in Switzerland. It was discovered that although total Hg concentration decreases with depth, the amount of MeHg remained constant at around 2 µg kg-1. Finally, a soil incubation experiment was performed to measure the impact of flooding and agriculture practices (organic matter addition) on Hg polluted soils. Incubated soils showed an important release of Hg within 24 hours of flooding (up to 45 µg L-1) and the incubated soils contained up to 5 times more MeHg after 11 days of flooding.
These new methods, developed to investigate As, Sb and Hg biomethylation and biovolatilisation, will allow the scientific community to study biomethylation and biovolatilisation in depth, thus gaining a better understanding of the biogeochemical cycle of these elements. This in turn will help stake holders to better manage polluted sites or to remediate them by understanding the behaviour of these toxic elements. They will also allow policy makers to create new laws on specific compounds rather than on total concentrations, using new user-friendly and cost-effective techniques.

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