Biogeochemical spatio-temporal transformation of copper in Aspergillus niger colonies grown on malachite with different inorganic nitrogen sources.

This work elucidates spatio-temporal aspects of the biogeochemical transformation of copper mobilized from malachite (Cu2 (CO3 )(OH)2 ) and bioaccumulated within Aspergillus niger colonies when grown on different inorganic nitrogen sources. It was shown that the use of either ammonium or nitrate determined how copper was distributed within the colony and its microenvironment and the copper oxidation state and succession of copper coordinating ligands within the biomass. Nitrate-grown colonies yielded ∼1.7× more biomass, bioaccumulated ∼7× less copper, excreted ∼1.9× more oxalate and produced ∼1.75× less water-soluble copper in the medium in contrast to ammonium-grown colonies. Microfocus X-ray absorption spectroscopy revealed that as the mycelium matured, bioaccumulated copper was transformed from less stable and more toxic Cu(I) into less toxic Cu(II) which was coordinated predominantly by phosphate/malate ligands. With time, a shift to oxalate coordination of bioaccumulated copper occurred in the central older region of ammonium-grown colonies.

Microbial reduction of U(VI) under alkaline conditions: implications for radioactive waste geodisposal.

Although there is consensus that microorganisms significantly influence uranium speciation and mobility in the subsurface under circumneutral conditions, microbiologically mediated U(VI) redox cycling under alkaline conditions relevant to the geological disposal of cementitious intermediate level radioactive waste, remains unexplored. Here, we describe microcosm experiments that investigate the biogeochemical fate of U(VI) at pH 10-10.5, using sediments from a legacy lime working site, stimulated with an added electron donor, and incubated in the presence and absence of added Fe(III) as ferrihydrite. In systems without added Fe(III), partial U(VI) reduction occurred, forming a U(IV)-bearing non-uraninite phase which underwent reoxidation in the presence of air (O2) and to some extent nitrate. By contrast, in the presence of added Fe(III), U(VI) was first removed from solution by sorption to the Fe(III) mineral, followed by bioreduction and (bio)magnetite formation coupled to formation of a complex U(IV)-bearing phase with uraninite present, which also underwent air (O2) and partial nitrate reoxidation. 16S rRNA gene pyrosequencing showed that Gram-positive bacteria affiliated with the Firmicutes and Bacteroidetes dominated in the post-reduction sediments. These data provide the first insights into uranium biogeochemistry at high pH and have significant implications for the long-term fate of uranium in geological disposal in both engineered barrier systems and the alkaline, chemically disturbed geosphere.

Arsenic bioremediation by biogenic iron oxides and sulfides.

Microcosms containing sediment from an aquifer in Cambodia with naturally elevated levels of arsenic in the associated groundwater were used to evaluate the effectiveness of microbially mediated production of iron minerals for in situ As remediation. The microcosms were first incubated without amendments for 28 days, and the release of As and other geogenic chemicals from the sediments into the aqueous phase was monitored. Nitrate or a mixture of sulfate and lactate was then added to stimulate biological Fe(II) oxidation or sulfate reduction, respectively. Without treatment, soluble As concentrations reached 3.9 ± 0.9 µM at the end of the 143-day experiment. However, in the nitrate- and sulfate-plus-lactate-amended microcosms, soluble As levels decreased to 0.01 and 0.41 ± 0.13 µM, respectively, by the end of the experiment. Analyses using a range of biogeochemical and mineralogical tools indicated that sorption onto freshly formed hydrous ferric oxide (HFO) and iron sulfide mineral phases are the likely mechanisms for As removal in the respective treatments. Incorporation of the experimental results into a one-dimensional transport-reaction model suggests that, under conditions representative of the Cambodian aquifer, the in situ precipitation of HFO would be effective in bringing groundwater into compliance with the World Health Organization (WHO) provisional guideline value for As (10 ppb or 0.13 µM), although soluble Mn release accompanying microbial Fe(II) oxidation presents a potential health concern. In contrast, production of biogenic iron sulfide minerals would not remediate the groundwater As concentration below the recommended WHO limit.

Capillarity creates single-crystal calcite nanowires from amorphous calcium carbonate.

Single-crystal calcite nanowires are formed by crystallization of morphologically equivalent amorphous calcium carbonate (ACC) particles within the pores of track etch membranes. The polyaspartic acid stabilized ACC is drawn into the membrane pores by capillary action, and the single-crystal nature of the nanowires is attributed to the limited contact of the intramembrane ACC particle with the bulk solution. The reaction environment then supports transformation to a single-crystal product.

Geomicrobiological redox cycling of the transuranic element neptunium.

Microbial processes can affect the environmental behavior of redox sensitive radionuclides, and understanding these reactions is essential for the safe management of radioactive wastes. Neptunium, an alpha-emitting transuranic element, is of particular importance because of its long half-life, high radiotoxicity, and relatively high solubility as Np(V)O(2)(+) under oxic conditions. Here, we describe experiments to explore the biogeochemistry of Np where Np(V) was added to oxic sediment microcosms with indigenous microorganisms and anaerobically incubated. Enhanced Np removal to sediments occurred during microbially mediated metal reduction, and X-ray absorption spectroscopy showed this was due to reduction to poorly soluble Np(IV) on solids. In subsequent reoxidation experiments, sediment-associated Np(IV) was somewhat resistant to oxidative remobilization. These results demonstrate the influence of microbial processes on Np solubility and highlight the critical importance of radionuclide biogeochemistry in nuclear legacy management.

The two Caenorhabditis elegans metallothioneins (CeMT-1 and CeMT-2) discriminate between essential zinc and toxic cadmium.

The nematode Caenorhabditis elegans expresses two metallothioneins (MTs), CeMT-1 and CeMT-2, that are believed to be key players in the protection against metal toxicity. In this study, both isoforms were expressed in vitro in the presence of either Zn(II) or Cd(II). Metal binding stoichiometries and affinities were determined by ESI-MS and NMR, respectively. Both isoforms had equal zinc binding ability, but differed in their cadmium binding behaviour, with higher affinity found for CeMT-2. In addition, wild-type C. elegans, single MT knockouts and a double MT knockout allele were exposed to zinc (340 microm) or cadmium (25 microm) to investigate effects in vivo. Zinc levels were significantly increased in all knockout strains, but were most pronounced in the CeMT-1 knockout, mtl-1 (tm1770), while cadmium accumulation was highest in the CeMT-2 knockout, mtl-2 (gk125) and the double knockout mtl-1;mtl-2 (zs1). In addition, metal speciation was assessed by X-ray absorption fine-structure spectroscopy. This showed that O-donating, probably phosphate-rich, ligands play a dominant role in maintaining the physiological concentration of zinc, independently of metallothionein status. In contrast, cadmium was shown to coordinate with thiol groups, and the cadmium speciation of the wild-type and the CeMT-2 knockout strain was distinctly different to the CeMT-1 and double knockouts. Taken together, and supported by a simple model calculation, these findings show for the first time that the two MT isoforms have differential affinities towards Cd(II) and Zn(II) at a cellular level, and this is reflected at the protein level. This suggests that the two MT isoforms have distinct in vivo roles.

Microbial engineering of nanoheterostructures: biological synthesis of a magnetically recoverable palladium nanocatalyst.

Precious metals supported on ferrimagnetic particles have a diverse range of uses in catalysis. However, fabrication using synthetic methods results in potentially high environmental and economic costs. Here we show a novel biotechnological route for the synthesis of a heterogeneous catalyst consisting of reactive palladium nanoparticles arrayed on a nanoscale biomagnetite support. The magnetic support was synthesized at ambient temperature by the Fe(III)-reducing bacterium, Geobacter sulfurreducens , and facilitated ease of recovery of the catalyst with superior performance due to reduced agglomeration (versus conventional colloidal Pd nanoparticles). Surface arrays of palladium nanoparticles were deposited on the nanomagnetite using a simple one-step method without the need to modify the biomineral surface, most likely due to an organic coating priming the surface for Pd adsorption, which was produced by the bacterial culture during the formation of the nanoparticles. A combination of EXAFS and XPS showed the Pd nanoparticles on the magnetite to be predominantly metallic in nature. The Pd(0)-biomagnetite was tested for catalytic activity in the Heck reaction coupling iodobenzene to ethyl acrylate or styrene. Rates of reaction were equal to or superior to those obtained with an equimolar amount of a commercial colloidal palladium catalyst, and near complete conversion to ethyl cinnamate or stilbene was achieved within 90 and 180 min, respectively.

Vanadium K-edge XAS studies on the native and peroxo-forms of vanadium chloroperoxidase from Curvularia inaequalis.

Vanadium K-edge X-ray Absorption Spectra have been recorded for the native and peroxo-forms of vanadium chloroperoxidase from Curvularia inaequalis at pH 6.0. The Extended X-ray Absorption Fine Structure (EXAFS) regions provide a refinement of previously reported crystallographic data; one short V=O bond (1.54A) is present in both forms. For the native enzyme, the vanadium is coordinated to two other oxygen atoms at 1.69A, another oxygen atom at 1.93A and the nitrogen of an imidazole group at 2.02A. In the peroxo-form, the vanadium is coordinated to two other oxygen atoms at 1.67A, another oxygen atom at 1.88A and the nitrogen of an imidazole group at 1.93A. When combined with the available crystallographic and kinetic data, a likely interpretation of the EXAFS distances is a side-on bound peroxide involving V-O bonds of 1.67 and 1.88A; thus, the latter oxygen would be 'activated' for transfer. The shorter V-N bond observed in the peroxo-form is in line with the previously reported stronger binding of the cofactor in this form of the enzyme. Reduction of the enzyme with dithionite has a clear influence on the spectrum, showing a change from vanadium(V) to vanadium(IV).

Solid-phase speciation of Pb in urban road dust sediment: a XANES and EXAFS study.

The quality of the urban environment is of growing concern as its human population continues to dramatically increase. X-ray absorption spectroscopy (XAS) and SEM have been used to study the solid-phase speciation of Pb in urban road dust sediments (RDS) in Manchester, UK. XANES analysis and linear combination modeling indicate that PbCrO(4) and Pb-sorbed goethite occur in 1000-500 microm, 250-125 microm, 63-38 microm, and <38 microm size fractions, collectively representing between 51-67% of the contributing Pb-phases. XANES analysis suggests that PbO, PbCl2, and Pb carbonates are also present. EXAFS modeling for all grain size fractions gives best fit models with a first shell of two oxygen atoms at 2.29-2.32 A, which corroborate the possible presence of Pb-sorbed goethite, and also suggest the presence of Pb phosphates and Pb oxides. Second shell Pb-Fe and second and third shell Pb-Pb scattering distances confirm Pb-sorbed to Fe oxide, and PbCl2 and PbCrO4, respectively. Many of the XAS models are corroborated by SEM observations. The Pb-phases may pose a risk to human health if inhaled or ingested, with insoluble phases such as PbCrO4 potentially causing inflammation in the lungs, and soluble phases such as PbO potentially being the most bioaccessible in the digestive tract.

Grain unloading of arsenic species in rice.

Rice (Oryza sativa) is the staple food for over half the world's population yet may represent a significant dietary source of inorganic arsenic (As), a nonthreshold, class 1 human carcinogen. Rice grain As is dominated by the inorganic species, and the organic species dimethylarsinic acid (DMA). To investigate how As species are unloaded into grain rice, panicles were excised during grain filling and hydroponically pulsed with arsenite, arsenate, glutathione-complexed As, or DMA. Total As concentrations in flag leaf, grain, and husk, were quantified by inductively coupled plasma mass spectroscopy and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy. The roles of phloem and xylem transport were investigated by applying a +/- stem-girdling treatment to a second set of panicles, limiting phloem transport to the grain in panicles pulsed with arsenite or DMA. The results demonstrate that DMA is translocated to the rice grain with over an order magnitude greater efficiency than inorganic species and is more mobile than arsenite in both the phloem and the xylem. Phloem transport accounted for 90% of arsenite, and 55% of DMA, transport to the grain. Synchrotron x-ray fluorescence mapping and fluorescence microtomography revealed marked differences in the pattern of As unloading into the grain between DMA and arsenite-challenged grain. Arsenite was retained in the ovular vascular trace and DMA dispersed throughout the external grain parts and into the endosperm. This study also demonstrates that DMA speciation is altered in planta, potentially through complexation with thiols.

Investigating different mechanisms for biogenic selenite transformations: Geobacter sulfurreducens, Shewanella oneidensis and Veillonella atypica.

The metal-reducing bacteria Geobacter sulfurreducens, Shewanella oneidensis and Veillonella atypica, use different mechanisms to transform toxic, bioavailable sodium selenite to less toxic, non-mobile elemental selenium and then to selenide in anaerobic environments, offering the potential for in situ and ex situ bioremediation of contaminated soils, sediments, industrial effluents, and agricultural drainage waters. The products of these reductive transformations depend on both the organism involved and the reduction conditions employed, in terms of electron donor and exogenous extracellular redox mediator. The intermediary phase involves the precipitation of elemental selenium nanospheres and the potential role of proteins in the formation of these structures is discussed. The bionanomineral phases produced during these transformations, including both elemental selenium nanospheres and metal selenide nanoparticles, have catalytic, semiconducting and light-emitting properties, which may have unique applications in the realm of nanophotonics. This research offers the potential to combine remediation of contaminants with the development of environmentally friendly manufacturing pathways for novel bionanominerals.

In situ spectroscopy and spectroelectrochemistry of uranium in high-temperature alkali chloride molten salts.

Soluble uranium chloride species, in the oxidation states of III+, IV+, V+, and VI+, have been chemically generated in high-temperature alkali chloride melts. These reactions were monitored by in situ electronic absorption spectroscopy. In situ X-ray absorption spectroscopy of uranium(VI) in a molten LiCl-KCl eutectic was used to determine the immediate coordination environment about the uranium. The dominant species in the melt was [UO 2Cl 4] (2-). Further analysis of the extended X-ray absorption fine structure data and Raman spectroscopy of the melts quenched back to room temperature indicated the possibility of ordering beyond the first coordination sphere of [UO 2Cl 4] (2-). The electrolytic generation of uranium(III) in a molten LiCl-KCl eutectic was also investigated. Anodic dissolution of uranium metal was found to be more efficient at producing uranium(III) in high-temperature melts than the cathodic reduction of uranium(IV). These high-temperature electrolytic processes were studied by in situ electronic absorption spectroelectrochemistry, and we have also developed in situ X-ray absorption spectroelectrochemistry techniques to probe both the uranium oxidation state and the uranium coordination environment in these melts.

Arsenic in hair and nails of individuals exposed to arsenic-rich groundwaters in Kandal province, Cambodia.

The health implications of the consumption of high arsenic groundwater in Bangladesh and West Bengal are well-documented, however, little is known about the level of arsenic exposure elsewhere in Southeast Asia, where widespread exploitation of groundwater resources is less well established. We measured the arsenic concentrations of nail and hair samples collected from residents of Kandal province, Cambodia, an area recently identified to host arsenic-rich groundwaters, in order to evaluate the extent of arsenic exposure. Nail and hair arsenic concentrations ranged from 0.20 to 6.50 microg g(-1) (n=70) and 0.10 to 7.95 microg g(-1) (n=40), respectively, in many cases exceeding typical baseline levels. The arsenic content of the groundwater used for drinking water purposes (0.21-943 microg L(-1) (n=31)) was positively correlated with both nail (r=0.74, p<0.0001) and hair (r=0.86, p<0.0001) arsenic concentrations. In addition, the nail and hair samples collected from inhabitants using groundwater that exceeded the Cambodian drinking water legal limit of 50 microg L(-1) arsenic contained significantly more arsenic than those of individuals using groundwater containing <50 microg L(-1) arsenic. X-ray absorption near edge structure (XANES) spectroscopy suggested that sulfur-coordinated arsenic was the dominant species in the bulk of the samples analysed, with additional varying degrees of As(III)-O character. Tentative linear least squares fitting of the XANES data pointed towards differences in the pattern of arsenic speciation between the nail and hair samples analysed, however, mismatches in sample and standard absorption peak intensity prevented us from unambiguously determining the arsenic species distribution. The good correlation with the groundwater arsenic concentration, allied with the relative ease of sampling such tissues, indicate that the arsenic content of hair and nail samples may be used as an effective biomarker of arsenic intake in this relatively recently exposed population.

Can we trust mass spectrometry for determination of arsenic peptides in plants: comparison of LC-ICP-MS and LC-ES-MS/ICP-MS with XANES/EXAFS in analysis of Thunbergia alata.

The weakest step in the analytical procedure for speciation analysis is extraction from a biological material into an aqueous solution which undergoes HPLC separation and then simultaneous online detection by elemental and molecular mass spectrometry (ICP-MS/ES-MS). This paper describes a study to determine the speciation of arsenic and, in particular, the arsenite phytochelatin complexes in the root from an ornamental garden plant Thunbergia alata exposed to 1 mg As L(-1) as arsenate. The approach of formic acid extraction followed by HPLC-ES-MS/ICP-MS identified different As(III)-PC complexes in the extract of this plant and made their quantification via sulfur (m/z 32) and arsenic (m/z 75) possible. Although sulfur sensitivity could be significantly increased when xenon was used as collision gas in ICP-qMS, or when HR-ICP-MS was used in medium resolution, the As:S ratio gave misleading results in the identification of As(III)-PC complexes due to the relatively low resolution of the chromatography system in relation to the variety of As-peptides in plants. Hence only the parallel use of ES-MS/ICP-MS was able to prove the occurrence of such arsenite phytochelatin complexes. Between 55 and 64% of the arsenic was bound to the sulfur of peptides mainly as As(III)(PC(2))(2), As(III)(PC(3)) and As(III)(PC(4)). XANES (X-ray absorption near-edge spectroscopy) measurement, using the freshly exposed plant root directly, confirmed that most of the arsenic is trivalent and binds to S of peptides (53% As-S) while 38% occurred as arsenite and only 9% unchanged as arsenate. EXAFS data confirmed that As-S and As-O bonds occur in the plants. This study confirms, for the first time, that As-peptides can be extracted by formic acid and chromatographically separated on a reversed-phase column without significant decomposition or de-novo synthesis during the extraction step.

Microbial manufacture of chalcogenide-based nanoparticles via the reduction of selenite using Veillonella atypica: an in situ EXAFS study.

The ability of metal-reducing bacteria to produce nanoparticles, and their precursors, can be harnessed for the biological manufacture of fluorescent, semiconducting nanomaterials. The anaerobic bacterium Veillonella atypica can reduce selenium oxyanions to form nanospheres of elemental selenium. These selenium nanospheres are then further reduced by the bacterium to form reactive selenide which could be precipitated with a suitable metal cation to produce nanoscale chalcogenide precipitates, such as zinc selenide, with optical and semiconducting properties. The whole cells used hydrogen as the electron donor for selenite reduction and an enhancement of the reduction rate was observed with the addition of a redox mediator (anthraquinone disulfonic acid). A novel synchrotron-based in situ time-resolved x-ray absorption spectroscopy technique was used, in conjunction with ion chromatography and inductively coupled plasma-atomic emission spectroscopy, to study the mechanisms and kinetics of the microbial reduction of selenite to selenide. The products of this biotransformation were also assessed using electron microscopy, energy-dispersive spectroscopy, x-ray diffraction and fluorescence spectroscopy. This process offers the potential to prepare chalcogenide-based nanocrystals, for application in optoelectronic devices and biological labelling, from more environmentally benign precursors than those used in conventional organometallic synthesis.

Arsenate detoxification in a Pseudomonad hypertolerant to arsenic.

Pseudomonas sp. strain As-1, obtained from an electroplating industrial effluent, was capable of growing aerobically in growth medium supplemented with up to 65 mM arsenate (As (V)), significantly higher concentrations than those tolerated by other reference arsenic resistant bacteria. The majority of the arsenic was detected in culture supernatants as arsenite (As (III)) and X-ray absorbance spectroscopy suggested that 30% of this cell-bound arsenic was As (V), 65% As (III) and 5% of arsenic was associated with sulphur. PCR analysis using primers designed against arsenic resistance genes of other Gram-negative bacteria confirmed the presence of an arsenic resistance operon comprising of three genes, arsR, arsB and arsC in order of predicted transcription, and consistent with a role in intracellular reduction of As (V) and efflux of As (III). In addition to this classical arsenic resistance mechanism, other biochemical responses to arsenic were implicated. Novel arsenic-binding proteins were purified from cellular fractions, while proteomic analysis of arsenic-induced cultures identified the upregulation of additional proteins not normally associated with the metabolism of arsenic. Cross-talk with a network of proteins involved in phosphate metabolism was suggested by these studies, consistent with the similarity between the phosphate and arsenate anions.

Reoxidation behavior of technetium, iron, and sulfur in estuarine sediments.

Technetium is a redox active radionuclide, which is present as a contaminant at a number of sites where nuclear fuel cycle operations have been carried out. Recent studies suggest that Tc(VII), which is soluble under oxic conditions, will be retained in sediments as Fe(III)-reducing conditions develop, due to reductive scavenging as hydrous TcO2. However, the behavior of technetium during subsequent reoxidation of sediments remains poorly characterized. Here, we describe a microcosm-based approach to investigate the reoxidation behavior of reduced, technetium-contaminated sediments. In reoxidation experiments, the behavior of Tc was strongly dependent on the nature of the oxidant. With air, reoxidation of Fe(II) and, in sulfate-reducing sediments, sulfide occurred accompanied by approximately 50% remobilization of Tc to solution as TcO4-. With nitrate, reoxidation of Fe(II) and, in sulfate-reducing sediments, sulfide only occurred in microbially active experiments where Fe(II) and sulfide oxidation coupled to nitrate reduction was occurring. Here, Tc was recalcitrant to remobilization with <10% Tc remobilized to solution even when extensive Fe(II) and sulfide reoxidation had occurred. X-ray absorption spectroscopy on reoxidized sediments suggested that 15-50% of Tc bound to sediments was present as Tc(VII). Overall, these results suggest that Tc reoxidation behavior is not directly coupled to Fe or S oxidation and that the extent of Tc remobilization is dependent on the nature of the oxidant.

Zinc phosphate transformations by the Paxillus involutus/pine ectomycorrhizal association.

In this research, we investigate zinc phosphate transformations by Paxillus involutus/pine ectomycorrhizas using zinc-resistant and zinc-sensitive strains of the ectomycorrhizal fungus under high- and low-phosphorus conditions to further understand fungal roles in the transformation of toxic metal minerals in the mycorrhizosphere. Mesocosm experiments with ectomycorrhizas were performed under sterile conditions with zinc phosphate localized in cellophane bags: zinc and phosphorus mobilization and uptake by the ectomycorrhizal biomass were analyzed. In the presence of a phosphorus source, an ectomycorrhizal association with a zinc-resistant strain accumulated the least zinc compared to a zinc-sensitive ectomycorrhizal association and non-mycorrhizal plants. Under low-phosphorus conditions, mycorrhizal seedlings infected with the zinc-resistant strain increased the dissolution of zinc phosphate and zinc accumulation by the plant. Extended X-ray absorption fine structure analysis of both mycorrhizal and nonmycorrhizal roots showed octahedral coordination of zinc by oxygen-containing ligands such as carboxylates or phosphate. We conclude that zinc phosphate solubilization and zinc and phosphorus uptake by the association depend on ectomycorrhizal infection, strain of the mycobiont, and the phosphorus status of the matrix.

Metal compartmentation and speciation in a soil sentinel: the earthworm, Dendrodrilus rubidus.

Earthworms are well-studied organisms in ecotoxicology because of their keystone ecological status and metal-accumulating capacity. However, the direct estimation of the bioreactive fractions of accumulated metal burdens remains technically elusive. In this study we exploited two physical techniques, electron probe X-ray microanalysis (EPXMA) and X-ray absorption spectroscopy (XAS), to improve understanding of the subcellular spatial distributions, ligand affinities, and coordination chemistries of Cd, Pb and Zn in a field population of the epigeic earthworm, Dendrodrilus rubidus. EPXMA and XAS analyses were performed on cryopreparations to maintain compositional fidelity; EPXMA data were analyzed by multivariate statistics. XAS provided whole-worm insights; EPXMA provided in situ, subcellular data from the major metal-sequestering tissue, the chloragog. Both techniques showed that Cd is coordinated with S; the measured Cd-S bond distance in XAS suggests a metallothionein-type ligand. The mean Cd:S molar ratio (EPXMA) of 0.36 is higher than the ratio of 0.29 estimated from published biochemical data. EPXMA and XAS data also found that Ca, Pb, and Zn are predominantly bound to one or more O-donating, probably phosphate-rich, ligands. X-ray distribution maps (EPXMA) of the hepatocyte-resembling chloragocytes revealed that the O-seeking (Ca, Pb, Zn) metals and S-seeking Cd bioaccumulate in distinct organelles. Extended X-ray absorption fine structure showed that the Pb complex is not biogenic pyromorphite, although X-ray absorption near edge structure did not eliminate the possibility. XAS provided no evidence of Pb spillage from the "sequestration compartment" within D. rubidus. However, the correspondence of Pb with Ca and P in EPXMA is not as strong as that of Zn. This is indicative either of spillover or of a second, hitherto unidentified, sequestered-Pb pool. By exploiting the complimentary techniques of EPXMA and XAS,we are closer to describing the mechanistic link between equilibrated body burdens and biomarker responses in earthworms.