Synchrotron-Based X-ray Fluorescence Imaging Elucidates Uranium Toxicokinetics in Daphnia magna.

A combination of synchrotron-based elemental analysis and acute toxicity tests was used to investigate the biodistribution and adverse effects in Daphnia magna exposed to uranium nanoparticle (UNP, 3-5 nm) suspensions or to uranium reference (Uref) solutions. Speciation analysis revealed similar size distributions between exposures, and toxicity tests showed comparable acute effects (UNP LC50: 402 µg L-1 [336-484], Uref LC50: 268 µg L-1 [229-315]). However, the uranium body burden was 3- to 5-fold greater in UNP-exposed daphnids, and analysis of survival as a function of body burden revealed a ∼5-fold higher specific toxicity from the Uref exposure. High-resolution X-ray fluorescence elemental maps of intact, whole daphnids from sublethal, acute exposures of both treatments revealed high uranium accumulation onto the gills (epipodites) as well as within the hepatic ceca and the intestinal lumen. Uranium uptake into the hemolymph circulatory system was inferred from signals observed in organs such as the heart and the maxillary gland. The substantial uptake in the maxillary gland and the associated nephridium suggests that these organs play a role in uranium removal from the hemolymph and subsequent excretion. Uranium was also observed associated with the embryos and the remnants of the chorion, suggesting uptake in the offspring. The identification of target organs and tissues is of major importance to the understanding of uranium and UNP toxicity and exposure characterization that should ultimately contribute to reducing uncertainties in related environmental impact and risk assessments.

Synchrotron XRF and Histological Analyses Identify Damage to Digestive Tract of Uranium NP-Exposed Daphnia magna.

Micro- and nanoscopic X-ray techniques were used to investigate the relationship between uranium (U) tissue distributions and adverse effects to the digestive tract of aquatic model organism Daphnia magna following uranium nanoparticle (UNP) exposure. X-ray absorption computed tomography measurements of intact daphnids exposed to sublethal concentrations of UNPs or a U reference solution (URef) showed adverse morphological changes to the midgut and the hepatic ceca. Histological analyses of exposed organisms revealed a high proportion of abnormal and irregularly shaped intestinal epithelial cells. Disruption of the hepatic ceca and midgut epithelial tissues implied digestive functions and intestinal barriers were compromised. Synchrotron-based micro X-ray fluorescence (XRF) elemental mapping identified U co-localized with morphological changes, with substantial accumulation of U in the lumen as well as in the epithelial tissues. Utilizing high-resolution nano-XRF, 400-1000 nm sized U particulates could be identified throughout the midgut and within hepatic ceca cells, coinciding with tissue damages. The results highlight disruption of intestinal function as an important mode of action of acute U toxicity in D. magna and that midgut epithelial cells as well as the hepatic ceca are key target organs.

Vadose-zone alteration of metaschoepite and ceramic UO2 in Savannah River Site field lysimeters.

Uranium dioxide (UO2) and metaschoepite (UO3•nH2O) particles have been identified as contaminants at nuclear sites. Understanding their behavior and impact is crucial for safe management of radioactively contaminated land and to fully understand U biogeochemistry. The Savannah River Site (SRS) (South Carolina, USA), is one such contaminated site, following historical releases of U-containing wastes to the vadose zone. Here, we present an insight into the behavior of these two particle types under dynamic conditions representative of the SRS, using field lysimeters (15 cm D x 72 cm L). Discrete horizons containing the different particle types were placed at two depths in each lysimeter (25 cm and 50 cm) and exposed to ambient rainfall for 1 year, with an aim of understanding the impact of dynamic, shallow subsurface conditions on U particle behavior and U migration. The dissolution and migration of U from the particle sources and the speciation of U throughout the lysimeters was assessed after 1 year using a combination of sediment digests, sequential extractions, and bulk and µ-focus X-ray spectroscopy. In the UO2 lysimeter, oxidative dissolution of UO2 and subsequent migration of U was observed over 1-2 cm in the direction of waterflow and against it. Sequential extractions of the UO2 sources suggest they were significantly altered over 1 year. The metaschoepite particles also showed significant dissolution with marginally enhanced U migration (several cm) from the sources. However, in both particle systems the released U was quantitively retained in sediment as a range of different U(IV) and U(VI) phases, and no detectable U was measured in the lysimeter effluent. The study provides a useful insight into U particle behavior in representative, real-world conditions relevant to the SRS, and highlights limited U migration from particle sources due to secondary reactions with vadose zone sediments over 1 year.

Arabidopsis thaliana Zn2+-efflux ATPases HMA2 and HMA4 are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina BMM.

Zinc is an essential nutrient at low concentrations, but toxic at slightly higher ones. It has been proposed that hyperaccumulator plants may use the excess zinc to fend off pathogens and herbivores. However, there is little evidence of a similar response in other plants. Here we show that Arabidopsis thaliana leaves inoculated with the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM) accumulate zinc and manganese at the infection site. Zinc accumulation did not occur in a double mutant in the zinc transporters HEAVY METAL ATPASE2 and HEAVY METAL ATPASE4 (HMA2 and HMA4), which has reduced zinc translocation from roots to shoots. Consistent with a role in plant immunity, expression of HMA2 and HMA4 was up-regulated upon PcBMM inoculation, and hma2hma4 mutants were more susceptible to PcBMM infection. This phenotype was rescued upon zinc supplementation. The increased susceptibility to PcBMM infection was not due to the diminished expression of genes involved in the salicylic acid, ethylene, or jasmonate pathways since they were constitutively up-regulated in hma2hma4 plants. Our data indicate a role of zinc in resistance to PcBMM in plants containing ordinary levels of zinc. This layer of immunity runs in parallel to the already characterized defence pathways, and its removal has a direct effect on resistance to pathogens.

Chemical state mapping of simulant Chernobyl lava-like fuel containing material using micro-focused synchrotron X-ray spectroscopy.

Uranium speciation and redox behaviour is of critical importance in the nuclear fuel cycle. X-ray absorption near-edge spectroscopy (XANES) is commonly used to probe the oxidation state and speciation of uranium, and other elements, at the macroscopic and microscopic scale, within nuclear materials. Two-dimensional (2D) speciation maps, derived from microfocus X-ray fluorescence and XANES data, provide essential information on the spatial variation and gradients of the oxidation state of redox active elements such as uranium. In the present work, we elaborate and evaluate approaches to the construction of 2D speciation maps, in an effort to maximize sensitivity to the U oxidation state at the U L3-edge, applied to a suite of synthetic Chernobyl lava specimens. Our analysis shows that calibration of speciation maps can be improved by determination of the normalized X-ray absorption at excitation energies selected to maximize oxidation state contrast. The maps are calibrated to the normalized absorption of U L3 XANES spectra of relevant reference compounds, modelled using a combination of arctangent and pseudo-Voigt functions (to represent the photoelectric absorption and multiple-scattering contributions). We validate this approach by microfocus X-ray diffraction and XANES analysis of points of interest, which afford average U oxidation states in excellent agreement with those estimated from the chemical state maps. This simple and easy-to-implement approach is general and transferrable, and will assist in the future analysis of real lava-like fuel-containing materials to understand their environmental degradation, which is a source of radioactive dust production within the Chernobyl shelter.

Multimodal X-ray microanalysis of a UFeO4: evidence for the environmental stability of ternary U(v) oxides from depleted uranium munitions testing.

An environmentally aged radioactive particle of UFeO4 recovered from soil contaminated with munitions depleted uranium (DU) was characterised by microbeam synchrotron X-ray analysis. Imaging of uranium speciation by spatially resolved X-ray diffraction (µ-XRD) and X-ray absorption spectroscopy (µ-XAS) was used to localise UFeO4 in the particle, which was coincident with a distribution of U(v). The U oxidation state was confirmed using X-ray Absorption Near Edge Structure (µ-XANES) spectroscopy as +4.9 ± 0.15. Le-Bail fitting of the particle powder XRD pattern confirmed the presence of UFeO4 and a minor alteration product identified as chernikovite (H3O)(UO2)(PO4)·3H2O. Refined unit cell parameters for UFeO4 were in good agreement with previously published values. Uranium-oxygen interatomic distances in the first co-ordination sphere were determined by fitting of Extended X-ray Absorption Fine Structure (µ-EXAFS) spectroscopy. The average first shell U-O distance was 2.148 ± 0.012 Å, corresponding to a U valence of +4.96 ± 0.13 using bond valence sum analysis. Using bond distances from the published structure of UFeO4, U and Fe bond valence sums were calculated as +5.00 and +2.83 respectively, supporting the spectroscopic analysis and confirming the presence of a U(v)/Fe(iii) pair. Overall this investigation provides important evidence for the stability of U(v) ternary oxides, in oxic, variably moist surface environment conditions for at least 25 years.

Metaschoepite Dissolution in Sediment Column Systems-Implications for Uranium Speciation and Transport.

Metaschoepite is commonly found in U-contaminated environments and metaschoepite-bearing wastes may be managed via shallow or deep disposal. Understanding metaschoepite dissolution and tracking the fate of any liberated U is thus important. Here, discrete horizons of metaschoepite (UO3·nH2O) particles were emplaced in flowing sediment/groundwater columns representative of the UK Sellafield Ltd. site. The column systems either remained oxic or became anoxic due to electron donor additions, and the columns were sacrificed after 6- and 12-months for analysis. Solution chemistry, extractions, and bulk and micro/nano-focus X-ray spectroscopies were used to track changes in U distribution and behavior. In the oxic columns, U migration was extensive, with UO22+ identified in effluents after 6-months of reaction using fluorescence spectroscopy. Unusually, in the electron-donor amended columns, during microbially mediated sulfate reduction, significant amounts of UO2-like colloids (>60% of the added U) were found in the effluents using TEM. XAS analysis of the U remaining associated with the reduced sediments confirmed the presence of trace U(VI), noncrystalline U(IV), and biogenic UO2, with UO2 becoming more dominant with time. This study highlights the potential for U(IV) colloid production from U(VI) solids under reducing conditions and the complexity of U biogeochemistry in dynamic systems.

Correlated X-Ray 3D Ptychography and Diffraction Microscopy Visualize Links between Morphology and Crystal Structure of Lithium-Rich Cathode Materials.

The search for higher performance, improved safety, and lifetime of lithium-ion batteries relies on the understanding of degradation mechanisms. Complementary to methods and studies on primary particles or crystalline structure on bulk materials, here we use spatially correlated ptychographic X-ray computed nanotomography with a 35 nm resolution and scanning X-ray diffraction microscopy with 1 µm resolution to visualize in 3D the hidden morphological and structural degradation processes in individual secondary particles of lithium-rich nickel, cobalt, and manganese oxides. From comparative examination of pristine and cycled particles, we suggest that morphological degradation could have radial dependency and secondary particle size dependency. The same particles were examined to correlate the degradation to crystallinity, which shows surprising core-shell structures. This study reveals the inner 3D structure of the secondary particles while opening up questions on the unexpected crystalline structural distributions, which could offer clues for future studies on this promising cathode material for lithium-ion batteries.

Etiolated Seedling Development Requires Repression of Photomorphogenesis by a Small Cell-Wall-Derived Dark Signal.

Etiolated growth in darkness or the irreversible transition to photomorphogenesis in the light engages alternative developmental programs operating across all organs of a plant seedling. Dark-grown Arabidopsis de-etiolated by zinc (dez) mutants exhibit morphological, cellular, metabolic, and transcriptional characteristics of light-grown seedlings. We identify the causal mutation in TRICHOME BIREFRINGENCE encoding a putative acyl transferase. Pectin acetylation is decreased in dez, as previously found in the reduced wall acetylation2-3 mutant, shown here to phenocopy dez. Moreover, pectin of dez is excessively methylesterified. The addition of very short fragments of homogalacturonan, tri-galacturonate, and tetra-galacturonate, restores skotomorphogenesis in dark-grown dez and similar mutants, suggesting that the mutants are unable to generate these de-methylesterified pectin fragments. In combination with genetic data, we propose a model of spatiotemporally separated photoreceptive and signal-responsive cell types, which contain overlapping subsets of the regulatory network of light-dependent seedling development and communicate via a pectin-derived dark signal.

Identifying the major intermediate species by combining time-resolved X-ray solution scattering and X-ray absorption spectroscopy.

Identifying the intermediate species along a reaction pathway is a first step towards a complete understanding of the reaction mechanism, but often this task is not trivial. There has been a strong on-going debate: which of the three intermediates, the CHI2 radical, the CHI2-I isomer, and the CHI2(+) ion, is the dominant intermediate species formed in the photolysis of iodoform (CHI3)? Herein, by combining time-resolved X-ray liquidography (TRXL) and time-resolved X-ray absorption spectroscopy (TR-XAS), we present strong evidence that the CHI2 radical is dominantly formed from the photolysis of CHI3 in methanol at 267 nm within the available time resolution of the techniques (∼20 ps for TRXL and ∼100 ps for TR-XAS). The TRXL measurement, conducted using the time-slicing scheme, detected no CHI2-I isomer within our signal-to-noise ratio, indicating that, if formed, the CHI2-I isomer must be a minor intermediate. The TR-XAS transient spectra measured at the iodine L1 and L3 edges support the same conclusion. The present work demonstrates that the application of these two complementary time-resolved X-ray methods to the same system can provide a detailed understanding of the reaction mechanism.

Microanalytical X-ray imaging of depleted uranium speciation in environmentally aged munitions residues.

Use of depleted uranium (DU) munitions has resulted in contamination of the near-surface environment with penetrator residues. Uncertainty in the long-term environmental fate of particles produced by impact of DU penetrators with hard targets is a specific concern. In this study DU particles produced in this way and exposed to the surface terrestrial environment for longer than 30 years at a U.K. firing range were characterized using synchrotron X-ray chemical imaging. Two sites were sampled: a surface soil and a disposal area for DU-contaminated wood, and the U speciation was different between the two areas. Surface soil particles showed little extent of alteration, with U speciated as oxides U3O7 and U3O8. Uranium oxidation state and crystalline phase mapping revealed these oxides occur as separate particles, reflecting heterogeneous formation conditions. Particles recovered from the disposal area were substantially weathered, and U(VI) phosphate phases such as meta-ankoleite (K(UO2)(PO4) · 3H2O) were dominant. Chemical imaging revealed domains of contrasting U oxidation state linked to the presence of both U3O7 and meta-ankoleite, indicating growth of a particle alteration layer. This study demonstrates that substantial alteration of DU residues can occur, which directly influences the health and environmental hazards posed by this contamination.

Remediation of soils contaminated with particulate depleted uranium by multi stage chemical extraction.

Contamination of soils with depleted uranium (DU) from munitions firing occurs in conflict zones and at test firing sites. This study reports the development of a chemical extraction methodology for remediation of soils contaminated with particulate DU. Uranium phases in soils from two sites at a UK firing range, MOD Eskmeals, were characterised by electron microscopy and sequential extraction. Uranium rich particles with characteristic spherical morphologies were observed in soils, consistent with other instances of DU munitions contamination. Batch extraction efficiencies for aqueous ammonium bicarbonate (42-50% total DU extracted), citric acid (30-42% total DU) and sulphuric acid (13-19% total DU) were evaluated. Characterisation of residues from bicarbonate-treated soils by synchrotron microfocus X-ray diffraction and X-ray absorption spectroscopy revealed partially leached U(IV)-oxide particles and some secondary uranyl-carbonate phases. Based on these data, a multi-stage extraction scheme was developed utilising leaching in ammonium bicarbonate followed by citric acid to dissolve secondary carbonate species. Site specific U extraction was improved to 68-87% total U by the application of this methodology, potentially providing a route to efficient DU decontamination using low cost, environmentally compatible reagents.

Beam-induced oxidation of monomeric U(IV) species.

Uranium L(III)-edge X-ray absorption spectroscopy is often used to probe the oxidation state and coordination of uranium in environmental samples, and micrometre-sized beams can be used to spatially map the distribution of uranium relative to other elements. Here a variety of uranium-containing environmental samples are analyzed at both microbeam and larger beam sizes to determine whether reoxidation of U(IV) occurred. Monomeric U(IV), a recently discovered product of U(VI) reduction by microbes and certain iron-bearing minerals at uranium-contaminated field sites, was found to be reoxidized during microbeam (3 µm × 2 µm) analysis of biomass and sediments containing the species but not at larger beam sizes. Thus, care must be taken when using X-ray microprobes to analyze samples containing monomeric U(IV).

High spatial resolution quantitative imaging by cross-calibration using Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Synchrotron micro-X-ray Fluorescence technique.

High spatial resolution, quantitative chemical imaging is of importance to various scientific communities, however high spatial resolution and robust quantification are not trivial to attain at the same time. In order to achieve microscopic chemical imaging with enhanced quantification capabilities, the current study links the independent and complementary advantages of two micro-analytical techniques - Synchrotron Radiation-based micro X-ray Fluorescence (SR-microXRF) and Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICPMS). A cross-calibration approach is established between these two techniques and validated by one experimental demonstration. In the presented test case, the diffusion pattern of trace level Cs migrating into a heterogeneous geological medium is imaged quantitatively with high spatial resolution. The one-dimensional line scans and the two-dimensional chemical images reveal two distinct types of geochemical domains: calcium carbonate rich domains and clay rich domains. During the diffusion, Cs shows a much higher interfacial reactivity within the clay rich domain, and turns out to be nearly non-reactive in the calcium carbonate domains. Such information obtained on the micrometer scale improves our chemical knowledge concerning reactive solute transport mechanism in heterogeneous media. Related to the chosen demonstration study, the outcome of the quantitative, microscopic chemical imaging contributes to a refined safety assessment of potential host rock materials for deep-geological nuclear waste storage repositories.

Selenium speciation assessed by X-ray absorption spectroscopy of sequentially extracted anaerobic biofilms.

Wet chemical methods such as sequential extraction procedures are commonly used to assess selenium fractionation in anoxic environments, allowing an estimation of the mobility and bioavailability of selenium. However, the interpretation can be biased by unselective extraction of targeted species and artifacts introduced during the extraction. Here, the selectivity of the single extraction steps to gain reliable selenium speciation information are scrutinized for the first time by direct, nondestructive X-ray absorption near edge structure (XANES) spectroscopy at the selenium K-edge. The sequential extraction procedures seriously overestimated the elemental selenium fraction, as major parts (58%) of the total selenium were present as metal selenides and organic selenium compounds, although extracted in the elemental fraction. Spectral fitting of the XANES spectra by the least-squares linear combinations utilizing a large set of model compounds, including previously neglected Se(-I) selenides, showed a novel degree of complexity in the speciation of selenium treating anaerobic biofilms, with up to 4 modeled selenium species contributing to the speciation, i.e., different elemental, organic, and metal-bound selenium species. Furthermore, a short exposure (10 min) to ambient air during the sequential extraction procedure induced the oxidation of organic selenium compounds, revealing the fragility of selenium speciation in anaerobic biofilms.

Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry.

Brown algae of the Laminariales (kelps) are the strongest accumulators of iodine among living organisms. They represent a major pump in the global biogeochemical cycle of iodine and, in particular, the major source of iodocarbons in the coastal atmosphere. Nevertheless, the chemical state and biological significance of accumulated iodine have remained unknown to this date. Using x-ray absorption spectroscopy, we show that the accumulated form is iodide, which readily scavenges a variety of reactive oxygen species (ROS). We propose here that its biological role is that of an inorganic antioxidant, the first to be described in a living system. Upon oxidative stress, iodide is effluxed. On the thallus surface and in the apoplast, iodide detoxifies both aqueous oxidants and ozone, the latter resulting in the release of high levels of molecular iodine and the consequent formation of hygroscopic iodine oxides leading to particles, which are precursors to cloud condensation nuclei. In a complementary set of experiments using a heterologous system, iodide was found to effectively scavenge ROS in human blood cells.