Effect of citric acid and malic acid on the uranium uptake into Brassica napus plants in hydroponic culture

The transport and transfer of radionuclides in the environment is an important aspect with regard to the health risk assessment of sites contaminated with naturally occurring radionuclides. In addition, this knowledge is highly relevant for the long-term safety assessment of future nuclear waste repositories. Radionuclides such as uranium are non-essential elements for plants. However, when present in contaminated soil, they can be taken up by plants and thus enter the food chain, posing a health risk to humans. Plants exude organic acids such as citric acid, malic acid, and/or oxalic acid into the rhizosphere. Under deficiency conditions, this can induce the dissolution of previously unavailable insoluble compounds, e.g., iron and phosphate minerals [1]. However, these processes can lead to the acquisition not only of essential nutrients, but also of non-essential ones, such as uranium. Therefore, plants may influence the mobility and bioavailability of radionuclides in the environment.
In the present work, we investigated the effect of citric acid and malic acid, both characteristic plant exudates, on the solubility of uranium in hydroponic plant culture medium as well as on the uranium uptake by Brassica napus (canola) plants in hydroponic culture. Prior to plant exposure, the solubility and speciation of 20 µM uranium(VI) in a phosphate reduced hydroponic solution (HRred, [2]) in the absence and presence of citric acid or malic acid was studied. After 24 h equilibration of uranium(VI) in HRred solution in the absence of citric acid or malic acid, a part of the uranium (~50%) precipitated, most probably in form of a uranyl(VI) phosphate. The control samples without organic acids stayed at the minimum concentration of dissolved uranium for 72 h, whereas those with citric acid or malic acid showed a re-solubilisation to the maximum uranium concentration of 20 µM within the first 24 h after addition of the respective organic acid. Following the uranium speciation in the hydroponic solutions during the exposure period by time-resolved laser-induced fluorescence spectroscopy (TRLFS) indicated the formation of uranium(VI) citrate and malate complexes, whereas in the organic-acid-free solution, the UO2(CO3)34- complex predominated.
B. napus plants were cultivated according to Jessat et al. [2]. To study the influence of the two organic acids on uranium exposure to B. napus, 20 µM U(VI) were added to HRred solution and allowed to equilibrate for 24 h in a plant growth chamber. After pre-equilibration, citric acid or malic acid (100 and 1000 µM) were added to the solutions. Immediately after that, the plants were inserted for exposure to uranium. For comparison, control samples were studied under the same conditions, however, without addition of citric acid or malic acid. The uranium concentration of the solutions was regularly determined by inductively coupled mass spectrometry (ICP-MS) within 72 h. After 72 h, the uranium content in the roots, stems, and leaves was analyzed by ICP-MS after drying and incineration. Furthermore, scanning transmission electron microscopy (STEM) coupled with energy-dispersive X-ray spectroscopy (EDX) was used to qualitatively confirm the presence of uranium in the roots.
The time-dependent bioassociation experiments showed a strong immobilization of uranium by the B. napus plants either in the absence or in the presence of organic acids. However, in the presence of citric acid and malic acid, this process seems to be retarded due to the re-dissolution of the uranium precipitate by the organic acids. Using TRLFS, uranium(VI) citrate and malate complexes were identified in the hydroponic solutions in the presence of the plants at the beginning of exposure. After longer exposure times, however, their contribution decreased and the UO2(CO3)34- complex dominated the speciation. For all conditions, STEM/EDX results verified the uptake of uranium into the whole root tissue. After 72 h of uranium exposure in the presence of citric acid or malic acid, more uranium was found in the leaves of B. napus compared to the control samples, indicating a stronger translocation of uranium in the plants. Despite that, in the presence of 1000 µM malic acid, a significantly lower amount of uranium was observed in the roots. These results demonstrate the ability of plant metabolites to dissolve hardly soluble uranium precipitates and shed more light on the speciation dependent uranium uptake into and translocation in canola plants.
The results of this study may contribute to a more pronounced process understanding on the uranium uptake into plants and their impact on the uranium mobility in the environment. This knowledge is required for the improvement of radioecological models to assess the migration and transfer of radionuclides in the environment, to develop efficient and cost-effective remediation technologies for contaminated sites, and to perform reliable dose predictions for humans and environment.

Acknowledgement
This work was performed within the RadoNorm project. This project has received funding from the Euratom research and training programme 2019-2020 under grant agreement No. 900009.

References
[1] Jones, D. L. (1998). Organic acids in the rhizosphere - a critical review. Plant and Soil 205: 25-44.
[2] Jessat, J., John, W.A., Moll, H., Vogel, M., Steudtner, R., Drobot, B., Hübner, R., Stumpf, T., Sachs, S. (2023), Ecotox. Environ. Saf., under review.