How tobacco (Nicotiana tabacum) BY-2 cells cope with Eu(III) - a microspectroscopic study.

The extensive use of lanthanides in science, industry and high-technology products is accompanied by an anthropogenic input of rare earth elements into the environment. Knowledge of a metal's environmental fate is essential for reasonable risk assessment and remediation approaches. In the present study, Eu(III) was representatively used as a luminescent probe to study the chemical environment and to elucidate the molecular interactions of lanthanides with a suspension cell culture of Nicotiana tabacum BY-2. Biochemical methods were combined with luminescence spectroscopy, two-dimensional microspectroscopic mappings, and data deconvolution methods to resolve the bioassociation behavior and spatial distribution of Eu(III) in plant cells. BY-2 cells were found to gradually take up the metal after exposure to 100 µM Eu(III) without significant loss of viability. Time-resolved luminescence measurements were used to specify the occurrence of Eu(III) species as a function of time, revealing the transformation of an initial Eu(III) species into another after 24 h exposure. Chemical microscopy and subsequent iterative factor analysis reveal the presence of four distinct Eu(III) species located at different cellular compartments, e.g., the cell nucleus, nucleolus and cell walls, which could be assigned to intracellular binding motifs. In addition, a special type of bioaccumulation occurs through the formation of a Eu(III)-containing oxalate biomineral, which is already formed within the first 24 hours after metal exposure. Oxalate crystals were also obtained in analogous experiments with Gd and Sm. These results indicate that tobacco BY-2 cells induce the precipitation of metal oxalate biominerals for detoxification of lanthanides, although they also bind to other cellular ligands at the same time.

Localization and chemical speciation of europium(III) in Brassica napus plants.

For the reliable safety assessment of repositories of highly radioactive waste, further development of the modelling of radionuclide migration and transfer in the environment is necessary, which requires a deeper process understanding at the molecular level. Eu(III) is a non-radioactive analogue for trivalent actinides, which contribute heavily to radiotoxicity in a repository. For in-depth study of the interaction of plants with trivalent f elements, we investigated the uptake, speciation, and localization of Eu(III) in Brassica napus plants at two concentrations, 30 and 200 µM, as a function of the incubation time up to 72 h. Eu(III) was used as luminescence probe for combined microscopy and chemical speciation analyses of it in Brassica napus plants. The localization of bioassociated Eu(III) in plant parts was explored by spatially resolved chemical microscopy. Three Eu(III) species were identified in the root tissue. Moreover, different luminescence spectroscopic techniques were applied for an improved Eu(III) species determination in solution. In addition, transmission electron microscopy combined with energy-dispersive X-ray spectroscopy was used to localize Eu(III) in the plant tissue, showing Eu-containing aggregates. By using this multi-method setup, a profound knowledge on the behavior of Eu(III) within plants and changes in its speciation could be obtained, showing that different Eu(III) species occur simultaneously within the root tissue and in solution.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life.

Certain f-block elements-the lanthanides-have biological relevance in the context of methylotrophic bacteria. The respective strains incorporate these 4 f elements into the active site of one of their key metabolic enzymes, a lanthanide-dependent methanol dehydrogenase. In this study, we investigated whether actinides, the radioactive 5 f elements, can replace the essential 4 f elements in lanthanide-dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides when presented with a mixture of equal amounts of lanthanides together with americium and curium. Our combined in vivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one-carbon metabolism if they possess the correct size and a +III oxidation state.

Studies of pyrroloquinoline quinone species in solution and in lanthanide-dependent methanol dehydrogenases.

Pyrroloquinoline quinone (PQQ) is a redox cofactor in calcium- and lanthanide-dependent alcohol dehydrogenases that has been known and studied for over 40 years. Despite its long history, many questions regarding its fluorescence properties, speciation in solution and in the active site of alcohol dehydrogenase remain open. Here we investigate the effects of pH and temperature on the distribution of different PQQ species (H3PQQ to PQQ3- in addition to water adducts and in complex with lanthanides) with NMR and UV-Vis spectroscopy as well as time-resolved laser-induced fluorescence spectroscopy (TRLFS). Using a europium derivative from a new, recently-discovered class of lanthanide-dependent methanol dehydrogenase (MDH) enzymes, we utilized two techniques to monitor Ln binding to the active sites of these enzymes. Employing TRLFS, we were able to follow Eu(III) binding directly to the active site of MDH using its luminescence and could quantify three Eu(III) states: Eu(III) in the active site of MDH, but also in solution as PQQ-bound Eu(III) and in the aquo-ion form. Additionally, we used the antenna effect to study PQQ and simultaneously Eu(III) in the active site.

Spatially resolved Eu(III) environments by chemical microscopy.

Chemical microscopy combines high-resolution emission spectra with Abbe-limited spatial resolution and is used for studies of inhomogeneous samples at the (sub-)micronscale. The spatial distinction of multiple Eu(III) coordination sites allows for a comprehensive understanding of environmental samples and highlights the applicability of Eu(III) as a molecular probe in medicine and biology.

Dimeric and Trimeric Uranyl(VI)-Citrate Complexes in Aqueous Solution.

This research addresses a subject discussed controversially for almost 70 years. The interactions between the uranyl(VI) ion, U(VI), and citric acid, H3Cit, were examined using a multi-method approach comprising nuclear magnetic resonance (NMR), ultraviolet-visible (UV-vis), attenuated total reflectance Fourier-transform infrared (ATR FT-IR), and extended X-ray absorption fine-structure (EXAFS) spectroscopies as well as density functional theory (DFT) calculations. Combining 17O NMR spectroscopy and DFT calculation provided an unambiguous decision on complex configurations, evidencing for the first time that the dimeric complex, (UO2)2(HCit-H)22-, exists as two diastereomers with the syn-isomer in aqueous solution strongly favored over the anti-isomer. Both isomers interconvert mutually with exchange rates of ∼30 s-1 at -6 °C and ∼249 s-1 at 60 °C in acidic solution corresponding to an activation barrier of about 24 kJ mol-1. Upon increasing the pH value, ternary dimeric mono- and bis-hydroxo as well as trimeric complexes form, that is, (UO2)2(HCit-H)2(OH)3-, (UO2)2(HCit-H)2(OH)24-, (UO2)3(O)(Cit-H)38-, and (UO2)3(O)(OH)(Cit-H)25-, respectively. Stability constants were determined for all dimeric and trimeric species, with log ß° = -(8.6 ± 0.2) for the 3:3 species being unprecedented. Additionally, in the 6:6 sandwich complex, formed from two units of 3:3 species, the 17O NMR resonance of the trinuclear uranyl(VI) core bridging µ3-O is shown for the first time. Species distribution calculations suggest that the characterized polynuclear U(VI)-citrate species do not significantly increase uranium(VI) mobility in the environment. Furthermore, we revise the misconceptions in the aqueous U(VI)-citric acid solution chemistry, that is, structures proposed and repeatedly taken up, and outline generalized isostructural considerations to provide a basis for future U(VI) complexation studies.

Bioassociation of U(VI) and Eu(III) by Plant (Brassica napus) Suspension Cell Cultures-A Spectroscopic Investigation.

In this study, we investigated the interaction of U(VI) and Eu(III) with Brassica napus suspension plant cells as a model system. Concentration-dependent (0-200 µM) bioassociation experiments showed that more than 75% of U(VI) and Eu(III) were immobilized by the cells. In addition to this phenomenon, time-dependent studies for 1 to 72 h of exposure showed a multistage bioassociation process for cells that were exposed to 200 µM U(VI), where, after initial immobilization of U(VI) within 1 h of exposure, it was released back into the culture medium starting within 24 h. A remobilization to this extent has not been previously observed. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to correlate the bioassociation behavior of Eu and U with the cell vitality. Speciation studies by spectroscopy and in silico methods highlighted various U and Eu species over the course of exposure. We were able to observe a new U species, which emerged simultaneously with the remobilization of U back into the solution, which we assume to be a U(VI) phosphate species. Thus, the interaction of U(VI) and Eu(III) with released plant metabolites could be concluded.

Quenching Mechanism of Uranyl(VI) by Chloride and Bromide in Aqueous and Non-Aqueous Solutions.

A major hindrance in utilizing uranyl(VI) luminescence as a standard analytical tool, for example, in environmental monitoring or nuclear industries, is quenching by other ions such as halide ions, which are present in many relevant matrices of uranyl(VI) speciation. Here, we demonstrate through a combination of time-resolved laser-induced fluorescence spectroscopy, transient absorption spectroscopy, and quantum chemistry that coordinating solvent molecules play a crucial role in U(VI) halide luminescence quenching. We show that our previously suggested quenching mechanism based on an internal redox reaction of the 1:2-uranyl-halide-complex holds also true for bromide-induced quenching of uranyl(VI). By adopting specific organic solvents, we were able to suppress the separation of the oxidized halide ligand X2·- and the formed uranyl(V) into fully solvated ions, thereby "reigniting" U(VI) luminescence. Time-dependent density functional theory calculations show that quenching occurs through the outer-sphere complex of U(VI) and halide in water, while the ligand-to-metal charge transfer is strongly reduced in acetonitrile.

Uranium(VI) toxicity in tobacco BY-2 cell suspension culture - A physiological study.

For the first time, the physiological and cellular responses of Nicotiana tabacum (BY-2) cells to uranium (U) as an abiotic stressor were studied using a multi-analytic approach that combined biochemical analysis, thermodynamic modeling and spectroscopic studies. The goal of this investigation was to determine the U threshold toxicity in tobacco BY-2 cells, the influence of U on the homeostasis of micro-macro essential nutrients, as well as the effect of Fe starvation on U bioassociation in cultured BY-2 cells. Our findings demonstrated that U interferes with the homeostasis of essential elements. The interaction of U with BY-2 cells confirmed both time- and concentration-dependent kinetics. Under Fe deficiency, a reduced level of U was detected in the cells compared to Fe-sufficient conditions. Interestingly, blocking the Ca channels with gadolinium chloride caused a decrease in U concentration in the BY-2 cells. Spectroscopic studies evidenced changes in the U speciation in the culture media with increasing exposure time under both Fe-sufficient and deficient conditions, leading us to conclude that different stress response reactions are related to Fe metabolism. Moreover, it is suggested that U toxicity in BY-2 cells is highly dependent on the existence of other micro-macro elements as shown by negative synergistic effects of U and Fe on cell viability.

Uranium(VI) Complexes of Glutathione Disulfide Forming in Aqueous Solution.

The interactions between glutathione disulfide, GSSG, the redox partner and dimer of the intracellular detoxification agent glutathione, GSH, and hexavalent uranium, U(VI), were extensively studied by solution NMR (in D2O), complemented by time-resolved laser-induced fluorescence and IR spectroscopies. As expected for the hard Lewis acid U(VI), coordination facilitates by the ligands' O-donor carboxyl groups. However, owing to the adjacent cationic α-amino group, the glutamyl-COO reveal monodentate binding, while the COO of the glycyl residues show bidentate coordination. The log K value for the reaction UO22+ + H3GSSG- → UO2(H3GSSG)+ (pH 3, 0.1 M NaClO4) was determined for the first time, being 4.81 ± 0.08; extrapolation to infinite dilution gave log K⊖ = 5.24 ± 0.08. U(VI) and GSSG form precipitates in the whole pD range studied (2-8), showing least solubility for 4 < pD < 6.5. Thus, particularly GSSG, hereby representing also other peptides and small proteins, affects the mobility of U(VI), strongly depending on the speciation of either component.

How Do Actinyls Interact with Hyperphosphorylated Yolk Protein Phosvitin?

The development of the nuclear industry has raised multiple questions about its impact on the biotope and humans. Proteins are key biomolecules in cell machinery and essential in deciphering toxicological processes. Phosvitin was chosen as a relevant model for phosphorylated proteins because of its important role as an iron, calcium, and magnesium storage protein in egg yolk. A multitechnique spectroscopic investigation was performed to reveal the coordination geometry of two oxocations of the actinide family (actinyl UVI , NpV ) in speciation with phosvitin. IR spectroscopy revealed phosphoryl groups as the main functional groups interacting with UVI . This was confirmed through laser luminescence spectroscopy (U) and UV/Vis absorption spectroscopy (Np). For UVI , X-ray absorption spectroscopy at the LIII edge revealed a small contribution of bidentate binding present, along with predominantly monodentate binding of phosphoryl groups; for NpV , uniquely bidentate binding was revealed. As a perspective to this work, X-ray absorption spectroscopy speciation of UVI and NpV in the extracted yolk of living eggs of the dogfish Scyliorhinus canicula was determined; this corroborated the binding of phosphorous together with a reduction of the actinyl moiety. Such data are essential to pinpoint the mechanisms of heavy metals (actinyls) accumulation and toxicity in oviparous organisms, and therefore, contribute to a shift from descriptive approaches to predictive toxicology.

Impact of Haloarchaea on Speciation of Uranium-A Multispectroscopic Approach.

Haloarchaea represent a predominant part of the microbial community in rock salt, which can serve as host rock for the disposal of high level radioactive waste. However, knowledge is missing about how Haloarchaea interact with radionuclides. Here, we used a combination of spectroscopic and microscopic methods to study the interactions of an extremely halophilic archaeon with uranium, one of the major radionuclides in high level radioactive waste, on a molecular level. The obtained results show that Halobacterium noricense DSM 15987T influences uranium speciation as a function of uranium concentration and incubation time. X-ray absorption spectroscopy reveals the formation of U(VI) phosphate minerals, such as meta-autunite, as the major species at a lower uranium concentration of 30 µM, while U(VI) is mostly associated with carboxylate groups of the cell wall and extracellular polymeric substances at a higher uranium concentration of 85 µM. For the first time, we identified uranium biomineralization in the presence of Halobacterium noricense DSM 15987T cells. These findings highlight the potential importance of Archaea in geochemical cycling of uranium and their role in biomineralization in hypersaline environments, offering new insights into the microbe-actinide interactions in highly saline conditions relevant to the disposal of high-level radioactive waste as well as bioremediation.

Ultrafast Transient Absorption Spectroscopy of UO22+ and [UO2Cl].

For the only water coordinated "free" uranyl(VI) aquo ion in perchlorate solution we identified and assigned several different excited states and showed that the 3Δ state is the luminescent triplet state from transient absorption spectroscopy. With additional data from other spectroscopic methods (TRLFS, UV/vis) we generated a detailed Jablonski diagram and determined rate constants for several state transitions, like the inner conversion rate constant from the 3Φ state to the 3Δ state transition to be 0.35 ps-1. In contrast to luminescence measurements, it was possible to observe the highly quenched uranyl(VI) ion in highly concentrated chloride solution by TAS and we were able to propose a dynamic quenching mechanism, where chloride complexation is followed by the charge transfer from the excited state uranyl(VI) to chloride. This proposed quenching route is supported by TD-DFT calculations.

Speciation Studies of Metals in Trace Concentrations: The Mononuclear Uranyl(VI) Hydroxo Complexes.

A direct luminescence spectroscopic experimental setup for the determination of complex stability constants of mononuclear uranyl(VI) hydrolysis species is presented. The occurrence of polynuclear species is prevented by using a low uranyl(VI) concentration of 10­8 M (2.4 ppb). Time-resolved laser-induced fluorescence spectra were recorded in the pH range from 3 to 10.5. Deconvolution with parallel factor analysis (PARAFAC) resulted in three hydrolysis complexes. A tentative assignment was based on thermodynamic calculations: UO22+, UO2(OH)+, UO2(OH)2, UO2(OH)3­. An implementation of a Newton­Raphson algorithm into PARAFAC allowed a direct extraction of complex stability constants during deconvolution yielding log(ß1M,1°C)1:1 = −4.6, log(ß1M,1°C)1:2 = −12.2, log(ß1M,1°C)1:3 = −22.3. Extrapolation to standard conditions gave log(ß0)1:1 = −3.9, log(ß0)1:2 = −10.9, and log(ß0)1:3 = −20.7. Luminescence characteristics (band position, lifetime) of the individual mononuclear hydroxo species were derived to serve as a reference data set for further investigations. A correlation of luminescence spectroscopic features with Raman frequencies was demonstrated for the mononuclear uranyl(VI) hydroxo complexes for the first time. Thereby a signal-to-structure correlation was achieved and the complex assignment validated.

NeptuniumV Retention by Siderite under Anoxic Conditions: Precipitation of NpO2-Like Nanoparticles and of NpIV Pentacarbonate.

The NpV retention by siderite, an FeII carbonate mineral with relevance for the near-field of high-level radioactive waste repositories, was investigated under anoxic conditions. Batch sorption experiments show that siderite has a high affinity for aqueous NpVO2+ across pH 7 to 13 as expressed by solid-water distribution coefficients, log Rd, > 5, similar to the log Rd determined for the (solely) tetravalent actinide Th on calcite, suggesting reduction of NpV to NpIV by siderite. Np L3-edge X-ray absorption near edge (XANES) spectroscopy conducted in a pH range typical for siderite-containing host rocks (7-8), confirmed the tetravalent Np oxidation state. Extended X-ray absorption fine-structure (EXAFS) spectroscopy revealed a local structure in line with NpO2-like nanoparticles with diameter < 1 nm, a result further corroborated by high-resolution transmission electron microscopy (HRTEM). The low solubility of these NpO2-like nanoparticles (∼10-9 M), along with their negligible surface charge at neutral pH conditions which favors particle aggregation, suggest an efficient retention of Np in the near-field of radioactive waste repositories. When NpV was added to ferrous carbonate solution, the subsequent precipitation of siderite did not lead to a structural incorporation of NpIV by siderite, but caused precipitation of a NpIV pentacarbonate phase.

The Sorption Processes of U(VI) onto SiO2 in the Presence of Phosphate: from Binary Surface Species to Precipitation.

The ternary system containing aqueous U(VI), aqueous phosphate and solid SiO2 was comprehensively investigated using a batch sorption technique, in situ attenuated total reflection Fourier-transform infrared (ATR FT-IR) spectroscopy, time-resolved luminescence spectroscopy (TRLS), and surface complexation modeling (SCM). The batch sorption studies on silica gel (10 g/L) in the pH range 2.5 to 5 showed no significant increase in U(VI) uptake in the presence of phosphate at equimolar concentration of 20 µM, but significant increase in U(VI) uptake was observed for higher phosphate concentrations. In situ infrared and luminescence spectroscopic studies evidence the formation of two binary U(VI) surface species in the absence of phosphate, whereas after prolonged sorption in the presence of phosphate, the formation of a surface precipitate, most likely an autunite-like phase, is strongly suggested. From SCM, excellent fitting results were obtained exclusively considering two binary uranyl surface species and the formation of a solid uranyl phosphate phase. Ternary surface complexes were not needed to explain the data. The results of this study indicate that the sorption of U(VI) on SiO2 in the presence of inorganic phosphate initially involves binary surface-sorption species and evolves toward surface precipitation.

Uranium(VI) chemistry in strong alkaline solution: speciation and oxygen exchange mechanism.

The mechanism by which oxygen bound in UO2(2+) exchanges with that from water under strong alkaline conditions remains a subject of controversy. Two recent NMR studies independently revealed that the key intermediate species is a binuclear uranyl(VI) hydroxide, presumably of the stoichiometry [(UO2(OH)4(2-))(UO2(OH)5(3-))]. The presence of UO2(OH)5(3-) in highly alkaline solution was postulated in earlier experimental studies, yet the species has been little characterized. Quantum-chemical calculations (DFT and MP2) show that hydrolysis of UO2(OH)4(2-) yields UO3(OH)3(3-) preferentially over UO2(OH)5(3-). X-ray absorption spectroscopy was used to study the uranium(VI) speciation in a highly alkaline solution supporting the existence of a species with three U-O bonds, as expected for UO3(OH)3(3-). Therefore, we explored the oxygen exchange pathway through the binuclear adduct [(UO2(OH)4(2-))(UO3(OH)3(3-))] by quantum-chemical calculations. Assuming that the rate-dominating step is proton transfer between the oxygen atoms, the activation Gibbs energy for the intramolecular proton transfer within [(UO2(OH)4(2-))(UO3(OH)3(3-))] at the B3LYP level was estimated to be 64.7 kJ mol(-1). This value is in good agreement with the activation energy for "yl"-oxygen exchange in [(UO2(OH)4(2-))(UO2(OH)5(3-))] obtained from experiment by Szabó and Grenthe (Inorg. Chem. 2010, 49, 4928-4933), which is 60.8 ± 2.4 kJ mol(-1). Both the presence of UO3(OH)3(3-) and the scenario of an "yl"-oxygen exchange through a binuclear species in strong alkaline solution are supported by the present study.

Biostimulation of indigenous microbes for uranium bioremediation in former U mine water: multidisciplinary approach assessment.

Characterizing uranium (U) mine water is necessary to understand and design an effective bioremediation strategy. In this study, water samples from two former U-mines in East Germany were analysed. The U and sulphate (SO42-) concentrations of Schlema-Alberoda mine water (U: 1 mg/L; SO42-: 335 mg/L) were 2 and 3 order of magnitude higher than those of the Pöhla sample (U: 0.01 mg/L; SO42-: 0.5 mg/L). U and SO42- seemed to influence the microbial diversity of the two water samples. Microbial diversity analysis identified U(VI)-reducing bacteria (e.g. Desulfurivibrio) and wood-degrading fungi (e.g. Cadophora) providing as electron donors for the growth of U-reducers. U-bioreduction experiments were performed to screen electron donors (glycerol, vanillic acid, and gluconic acid) for Schlema-Alberoda U-mine water bioremediation purpose. Thermodynamic speciation calculations show that under experimental conditions, U(VI) is not coordinated to the amended electron donors. Glycerol was the best-studied electron donor as it effectively removed 99% of soluble U, 95% of Fe, and 58% of SO42- from the mine water, probably by biostimulation of indigenous microbes. Vanillic acid removed 90% of U, and no U removal occurred using gluconic acid.

Effect of Ba(II), Eu(III), and U(VI) on rat NRK-52E and human HEK-293 kidney cells in vitro.

Heavy metals pose a potential health risk to humans when they enter the organism. Renal excretion is one of the elimination pathways and, therefore, investigations with kidney cells are of particular interest. In the present study, the effects of Ba(II), Eu(III), and U(VI) on rat and human renal cells were investigated in vitro. A combination of microscopic, biochemical, analytical, and spectroscopic methods was used to assess cell viability, cell death mechanisms, and intracellular metal uptake of exposed cells as well as metal speciation in cell culture medium and inside cells. For Eu(III) and U(VI), cytotoxicity and intracellular uptake are positively correlated and depend on concentration and exposure time. An enhanced apoptosis occurs upon Eu(III) exposure whereas U(VI) exposure leads to enhanced apoptosis and (secondary) necrosis. In contrast to that, Ba(II) exhibits no cytotoxic effect at all and its intracellular uptake is time-independently very low. In general, both cell lines give similar results with rat cells being more sensitive than human cells. The dominant binding motifs of Eu(III) in cell culture medium as well as cell suspensions are (organo-) phosphate groups. Additionally, a protein complex is formed in medium at low Eu(III) concentration. In contrast, U(VI) forms a carbonate complex in cell culture medium as well as each one phosphate and carbonate complex in cell suspensions. Using chemical microscopy, Eu(III) was localized in granular, vesicular compartments near the nucleus and the intracellular Eu(III) species equals the one in cell suspensions. Overall, this study contributes to a better understanding of the interactions of Ba(II), Eu(III), and U(VI) on a cellular and molecular level. Since Ba(II) and Eu(III) serve as inactive analogs of the radioactive Ra(II) and Am(III)/Cm(III), the results of this study are also of importance for the health risk assessment of these radionuclides.

The chemical journey of Europium(III) through winter rye (Secale cereale L.) - Understanding through mass spectrometry and chemical microscopy.

A combination of biochemical preparation methods with microscopic, spectroscopic, and mass spectrometric analysis techniques as contemplating state of the art application, was used for direct visualization, localization, and chemical identification of europium in plants. This works illustrates the chemical journey of europium (Eu(III)) through winter rye (Secale cereale L.), providing insight into the possibilities of speciation for Rare Earth Elements (REE) and trivalent f-elements. Kinetic experiments of contaminated plants show a maximum europium concentration in Secale cereale L. after four days. Transport of the element through the vascular bundle was confirmed with Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray analysis (EDS). For chemical speciation, plants were grown in a liquid nutrition medium, whereby Eu(III) species distribution could be measured by mass spectrometry and luminescence measurements. Both techniques confirm the occurrence of Eu malate species in the nutrition medium, and further analysis of the plant was performed. Luminescence results indicate a change in Eu(III) species distribution from root tip to plant leaves. Microscopic analysis show at least three different Eu(III) species with potential binding to organic and inorganic phosphate groups and a Eu(III) protein complex. With plant root extraction, further europium species could be identified by using Electrospray Ionization Mass Spectrometry (ESI MS). Complexation with malate, citrate, a combined malate-citrate ligand, and aspartate was confirmed mostly in a 1:1 stoichiometry (Eu:ligand). The combination of the used analytical techniques opens new possibilities in direct species analysis, especially regarding to the understanding of rare earth elements (REE) uptake in plants. This work provides a contribution in better understanding of plant mechanisms of the f-elements and their species uptake.