An XAFS Investigation of Mercury and Selenium in Beluga Whale ...

Environmental Bioindicators, 4:291–302, 2009 Copyright © Taylor & Francis Group, LLC ISSN: 1555-5275 print/ 1555-5267 online DOI: 10.1080/15555270903404651

An XAFS Investigation of Mercury and Selenium in Beluga Whale Tissues

1555-5267 1555-5275 UEBI Environmental Bioindicators, Bioindicators Vol. 4, No. 4, November 2009: pp. 0–0

Mercury et Huggins and al.Selenium in Beluga Whales

FRANK E. HUGGINS,1 STEPHEN A. RAVERTY,2 OLE S. NIELSEN,3 NICHOLAS E. SHARP,4 J. DAVID ROBERTSON,4 AND NICHOLAS V.C. RALSTON5 1

Consortium for Fossil Fuel Science and Department of Chemical & Materials Engineering, University of Kentucky, Lexington, KY 2 Animal Health Center, Abbotsford, BC, Canada 3 Central & Arctic Region, Fisheries and Oceans Canada, Winnipeg, Canada 4 Department of Chemistry & Research Reactor, University of Missouri, Columbia, MO 5 Energy & Environmental Research Center, University of North Dakota Grand Forks, ND High dietary methylmercury (MeHg) exposures are often associated with increased accumulation of selenium (Se), particularly in tissues with rapid rates of selenocysteine synthesis. Conversely, increased dietary Se intakes result in increased accumulation of Hg, possibly because of mutual sequestration in insoluble HgSe complexes. In the current study, results from Hg X-ray absorption fine structure (XAFS) spectroscopy of lyophilized liver and pituitary tissues from beluga whales, coupled with instrumental neutron activation analysis determinations of their Hg and Se contents, show that Hg occurs as a mixture of HgS and HgSe. In the liver tissues studied, the proportion of Hg as HgSe varied from 38% to 77%, whereas it was higher in the pituitary tissues (85%–90%). Selenium XAFS spectra showed that Se as HgSe also varied from dominant to minor among Se forms in the same tissues. The distribution of Se between HgSe and a biological form was estimated from analysis of the Hg derivative XANES spectra and from the Se EXAFS spectra. These alternative estimates for the Se distribution were reasonably consistent. These findings confirm that dietary exposure of beluga whales to Se and MeHg results in the formation of HgSe, a potential bioindicator for non-toxic mercury, in their tissues. Keywords Beluga whale, mercury, selenium, XAFS spectroscopy, liver, pituitary, brain

Introduction The molecular forms of mercury (Hg) in biological tissues are of significant interest for understanding its toxicological behavior and the potential ramifications of environmental exposures. In most animals, tissue concentrations of Hg are typically below 1 mg/kg, but it occurs in a variety of forms that are differentially distributed into organs. Birds and marine mammals absorb the bulk of the methylmercury (MeHg) present in their food. Therefore, the levels of Hg in their tissues are proportional to both the amount of MeHg present in the Address correspondence to Dr. Frank E. Huggins, CFFS/CME, University of Kentucky, Lexington, KY 40506, USA. E-mail: [email protected]

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fish they consume and the length of time they have been consuming these fish. Such considerations are especially important for animals that live in the polar regions of the world. For example, after a lifelong exposure, northern fur seals can attain liver Hg concentrations in excess of 2,000 mg/kg. Despite such high accumulations in this key organ, these animals typically show no obvious symptoms of Hg poisoning. It has been suggested that the Hg that accumulates in the liver occurs in a minimally hazardous inorganic form, rather than as a more toxic organomercury species, such as MeHg. It has also been noted that selenium (Se) concentrations are often high in the same tissues in which the Hg contents are high (Koeman et al. 1973, 1975; Becker et al. 1995; Dietz et al. 1996; Endo et al. 2002), leading to the suggestion that formation of relatively inert mercury selenide (HgSe) may be responsible for the apparent lack of Hg toxicity. Such studies imply that the proportion of Hg as HgSe could be developed as a bioindicator and measure of minimally toxic Hg in biological systems and thereby provide more precise determinations and assessments of the actual Hg risks. Recent examination with Hg and Se X-ray absorption fine structure (XAFS) spectroscopy of elevated Hg levels in various organs (liver, kidney, lung) of northern fur seals and a black-footed albatross has confirmed the presence of HgSe (Arai et al. 2004). In this paper, we examine Hg and Se in liver and pituitary tissues of a number of beluga whales with Hg LIII- and Se K-edge XAFS spectroscopy to identify possible Hg and Se species. Here, the Hg levels are mostly significantly lower than the exceptionally high values investigated previously in marine animals (Arai et al. 2004). Although lower levels of Hg and Se in the present study make a complete analysis of the XAFS data more difficult, the findings obtained on the whale tissues generally agree with those obtained for the higher Hg concentrations in the northern fur seals and black-footed albatross. This agreement for three different animal species suggests that formation of HgSe is an important general biological detoxification pathway for relatively low-level ingestion of Hg (see also the review by Yang et al. 2008). It also supports the concepts that high MeHg exposures may be causally associated with the inhibition of Se-dependent enzyme activities that have been noted in Hg-intoxicated animals and that the amount of Hg as HgSe may be a useful bioindicator for minimally toxic Hg in biological systems.

Materials and Methods (a) Description of Samples and Determination of Hg and Se Contents Samples of the liver and pituitary from various beluga whales were obtained from animals harvested by First Nations hunters in 2005. Whole organ samples or representative tissue samples were collected using stainless steel instruments and stored frozen in plastic bags. Tissue samples were freeze-dried prior to analysis. Total Se and Hg contents of these tissues were determined by standard comparator instrumental neutron activation analysis (INAA) using the 74Se(n,g)75Se and the 202Hg(n,g)203Hg reactions. Freeze-dried samples were weighed and sealed into clean quartz vials. The samples were then irradiated for 50 hours at a thermal flux of ca. 5 × 1013 n·cm−2·s−1 and allowed to decay for several weeks. Samples, standards, and quality control (QC) materials were then live-time counted for 4 hours using an automated sample changer. The Hg concentration of each sample was determined by using the average of the 279/136 keV ratio and the 279/264 keV ratio to subtract the 75Se contribution from the total 279 keV peak in the sample spectra. All samples were blank subtracted and corrected for the possible Se interferences from 182Ta, 181W, and 181Hf. The QC samples co-analyzed with the samples were prepared from National

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Table 1 List of beluga whale tissues investigated by Hg and Se (XAFS) spectroscopy with Hg and Se concentrations determined by instrumental neutron activation analysis (INAA). Tissue Sample ID

Organ

Hg mg/kg

Se mg/kg

Se/Hg molar ratio

Max. % Se as HgSe

H2 (wet) H2 (dried) W115 W117 W131 W133 W147 W149 W163 W165

Liver Liver Liver Pituitary Liver Pituitary Liver Pituitary Liver Pituitary

40 151

24 91

1.5 1.5

65 65

74 176 97 32 21 581 213

23 74 60 23 21 284 96

0.8 1.1 1.6 1.8 2.6 1.2 1.1

100 94 64 55 38 81 87

Institute of Standards and Technology (NIST) standard reference material (SRM) 1577 bovine liver and NIST SRM 1633 orchard leaves. Mercury and Se contents of the liver and pituitary tissues of various beluga whales are listed in Table 1. Comparative INAA data on the Hg and Se contents of these and other tissues from a larger set of eleven beluga whales are presented in the associated study by Huggins et al. (2009). (b) Use of Lyophilized Samples Since lyophilization of biological tissues is known to increase the concentration of metals significantly, it was decided to examine lyophilized samples of the whale tissues with XAFS spectroscopy in order to generate better quality XAFS spectra. However, we also considered the possibility that the drying process itself might cause the speciation of the element to change and thereby, generate an altered XAFS spectrum. Hence, selected tissue samples were examined before and after lyophilization with both Hg and Se XAFS spectroscopy. Examples of Hg and Se X-ray absorption near-edge structure (XANES) spectra before (wet) and after (dry) lyophilization are shown in Figure 1. For both sets of spectra, it can be seen that the spectra before and after lyophilization virtually superimpose upon each other. Hence, it can be assumed that lyophilization does not cause significant changes in speciation for these two elements in the whale tissues. All other XAFS spectra shown in the paper were obtained exclusively from lyophilized materials. (c) XAFS Spectroscopy Mercury and selenium XAFS spectroscopy of various tissue samples of whales was conducted at the Stanford Synchrotron Research Lightsource (SSRL), Stanford University, California. Mercury LIII-edge spectra were collected using a multielement solid-state Ge detector tuned to the fluorescent La X-ray lines of Hg. Both Soller slits and a 6-μ gallium

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Figure 1. (a) Se X-ray absorption near-edge structure (XANES) and (b) Hg XANES of liver tissues from beluga whale H2 before (wet) and after (dry) lyophilization. The difference between the two spectra is shown as a dashed line.

filter were employed to enhance the signal/noise ratio of the Hg XAFS spectra. Since the K absorption edge of Se occurs less than 400 eV above the LIII-edge of Hg, the window on the Hg fluorescence was tightly controlled to minimize the observation of the Se K-edge. However, in most cases, a small Se edge was detected that limited the extended X-ray absorption fine structure (EXAFS) region of the Hg LIII-edge to no more than 9.5 Å−1 in kspace. The Hg spectra were recorded from 200 eV below the LIII absorption edges (LIII at 12,284 eV) to as much as 800 eV above the edge. Typically, a scan of about 30 minutes’ duration was set up, and multiple scans (up to as many as ten) were averaged to determine the final spectrum for Hg in the tissue sample. A similar experimental setup and procedures were used to record the Se K-edge XAFS spectra (Se K-edge at 12,658 eV) from the same tissue samples, except that a 6-μ arsenic filter was used. Analysis of the Hg and Se XAFS spectra followed the normal procedures described in detail in the literature (e.g., Lee et al. 1981; Koningsberger and Prins 1988). These procedures were carried out using SIXPack (Webb 2005), one of programs available in the IFEFFIT package for Microsoft Windows-based personal computers (Ravel and Newville 2005). First, the energy scale of the averaged spectrum was calibrated relative to the position of the corresponding absorption edge for elemental Hg or Se, and the absorption spectrum was normalized to the edge-step. It was then divided into separate X-ray absorption near-edge structure (XANES) and EXAFS regions. The EXAFS region was then converted to a k-space representation (chi spectrum), which was weighted by k3 and then subjected to a Fourier transform to generate the corresponding radial structure function (RSF) spectrum. Additional spectra analysis was attempted using least-squares fitting of linear combinations of component spectra to the Hg or Se XANES and EXAFS spectra and also fitting of the coordination shells contributing to the Hg or Se EXAFS regions based on FEFF6 functions generated using the program TKATOMS (Ravel 2001).

Results and Discussion (a) Mercury XAFS Spectra Mercury XANES and derivative XANES spectra for liver tissues from four beluga whales are shown in Figure 2, along with the corresponding spectra for HgS (cinnabar) and HgSe


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Figure 2. Mercury X-ray absorption near-edge structure (XANES) and derivative XANES spectra for HgS, HgSe, and liver tissues from four beluga whales.

Figure 3. Comparison of Hg X-ray absorption near-edge structure (XANES) spectra for liver and pituitary tissues from beluga whales.

(99.999% Alfa AESAR). Figure 3 shows a comparison of the Hg XANES spectra for tissues from the liver and pituitary. As indicated in Figure 4, the XANES and derivative XANES spectra can be simulated by the weighted additions of the separate spectra for HgSe and HgS. However, the XANES spectra for HgS and HgSe are highly correlated (r2 > 0.99, over the range 12,250–12,500 eV), and consequently, the fits may be subject to large systematic errors. The corresponding derivative XANES spectra are less highly correlated (r2 ≈ 0.78, over the range 12,270–12,310 eV), and fitting of the derivative spectra was, therefore, considered more reliable. Even so, the errors of the estimates are likely to be at least ±10%. Results for the mercury speciation in the whale tissue samples based on

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Figure 4. Examples of least-squares fitting of Hg X-ray absorption near-edge structure (XANES) and derivative XANES spectra for liver tissues from a beluga whale.

Table 2 Results of least-squares fitting of mercury d(XANES)/dE spectra for organ tissues from beluga whales. Tissue H2 W115 W117 W131 W133 W147 W149 W163 W165 Est. errors

Organ

% Hg as HgSe

Liver Liver Pituitary Liver Pituitary Liver Pituitary Liver Pituitary

77 38 75 57 85 77 90 ±10

% Hg as HgS

ΔE, eV

23 −0.08 65 0.19 Too weak 25 0.00 Too weak 44 0.17 16 0.02 24 0.10 10 0.26 ±10

Red. c2 × 10−5 0.3 1.5 2.1 1.5 0.8 0.5 0.4

ΔE – energy shift that resulted in the best fit between data and fit. Red. c2 – statistical measure of the adequacy of the least-squares fit.

least-squares fitting of the derivative XANES spectra are summarized in Table 2. More complex fits involving additional mercury standards were not warranted since these two components appeared to account for virtually all of the variation among the spectra. From this analysis, HgSe was found to be the dominant mercury phase in these tissues for four of the six samples, whereas HgS dominated in one sample and one sample contained approximately similar amounts of the two species. It was also observed that the HgSe contents tended to be higher in samples with high Hg contents and that the pituitary tissues were proportionately richer in HgSe than the corresponding liver tissues. For three samples with high concentrations of mercury, the data were of sufficient quality to generate a RSF spectrum from the EXAFS data. These spectra are shown in


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Figure 5. Mercury radial structure function (RSF) spectra for the three whale tissue samples (W165, W163, H2) with high Hg contents. The corresponding spectra for HgSe and HgS are also shown.

Figure 5, along with the corresponding data for HgSe and HgS. It can be seen that the RSF spectra of the three whale tissues are much more similar to that of HgSe than HgS. However, the positions of the peak maxima are displaced slightly from that of HgSe. This displacement was confirmed by performing least-squares fitting analysis of the major Hg-Se coordination shell in these spectra using IFEFFIT procedures (Ravel and Newville 2005; Webb 2005) and parameters derived from crystallographic data for HgSe (Ravel 2001). Examples of fits obtained using this method of analysis are shown in Figure 6 for the RSF spectra of HgSe and a pituitary sample, W165, and derived parameters are listed in Table 3. For the first coordination shell in HgSe, this method of analysis returned values of 3.7 ± 0.8 and 2.63 ± 0.015 Å for the coordination number (CN) and Hg-Se (R) distance, respectively, in agreement with the coordination number (4) and Hg-Se distance (2.635 Å) determined by crystal structure analysis (Wykcoff 1965). The corresponding distances derived

Figure 6. Examples of least-squares fitting of EXAFS/RSF spectra: (a) HgSe and (b) beluga whale pituitary tissue, W165, based on FEFF6 parameters derived for the Hg-Se shell in HgSe. Spectra were fit for the peak in the range 1.5–3.0 Å in R-space obtained by Fourier transform of the 3.0–9.5 Å−1 region in k-space.

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Table 3 Results from least-squares fitting applied to the Hg-Se coordination shell in EXAFS spectra of three whale tissues based on IFEFFIT back-scattering parameters derived for the Hg-Se shell in HgSe. Sample source

CN*

R, Å

s2, Å2

HgSe (cryst.) HgSe Est. errors (HgSe) H2 (liver) W163 (liver) W165 (pituitary) Est. errors (tissues)

4 3.7 ±0.8 2.4 2.0 2.7 ±1.0

2.635 2.63 ±0.015 2.59 2.60 2.59 ±0.02

– 0.011 ±0.002 0.010 0.007 0.007 ±0.002

Rfactor 0.0118 0.0152 0.0257 0.0106

*CN – coordination number; R – Hg-Se distance; s2 – Debye-Waller factor. Rfactor – statistical measure of the adequacy of the fitting.

from similar fitting of the Hg EXAFS/RSF spectra for the Hg-Se shells in the whale tissues were found to be somewhat shorter, about 2.59 ± 0.02 Å. Moreover, the coordination numbers returned by the IFEFFIT analysis (Table 3) were significantly smaller than four for the whale tissues. The reductions in both coordination number and distance for the Hg compound in the whale tissues compared to those for HgSe are consistent with the substitution of some of the Se by S in the immediate coordination shell around the Hg, as was previously suggested (Arai et al. 2004) and was also indicated by analysis of the Hg XANES spectra (Table 2). However, attempts to model the first coordination shell in the mercury RSF spectra of the whales using separate contributions from Hg-S and Hg-Se shells did not yield meaningful results as the Hg-S distances derived from the two-shell fitting model were inconsistent with Hg-S distances in known HgS structures (Wyckoff 1965). Such inconsistent Hg-S distances were also indicated for the similar two-shell fitting model presented in the paper by Arai et al. (2004). (b) Selenium XAFS Spectra Selenium XANES spectra of selected whale organ tissue samples are shown in Figure 7 along with the corresponding spectrum of HgSe. Qualitatively, it can be seen that there is significant variability among the spectra that can be interpreted as resulting from variation in the fraction of Se as HgSe in the whale organ tissues. The spectrum of the non-HgSe component in the spectrum did not closely resemble that of Se-methionine or of Se-cystine, two model biological Se compounds in our database of XAFS spectra of Se compounds. Hence, in the absence of definitive XAFS data regarding the second Se component, leastsquares fitting of the Se XANES and EXAFS spectra was limited to two components: HgSe and the Se spectra of W147, as this particular sample likely contained the smallest fraction of HgSe since it exhibited the spectra most different from HgSe. Examples of the fitting using this model are shown in Figure 8. Based on the INAA data of Table 1 and the Hg-derivative XANES results of Table 2, it can be estimated that Se as HgSe is about 30% of the total Se in W147. Hence, the estimates shown in Table 4 for %Se in HgSe and a biological Se (“bioSe”) form have been calculated assuming the Se XAFS spectrum of W147


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Figure 7. Se X-ray absorption near-edge structure (XANES) spectra of tissues from (a) liver and (b) pituitary glands from various beluga whales. The corresponding spectrum of HgSe is also displayed in (b).

Figure 8. Examples of least-squares fitting of the Se X-ray absorption near-edge structure (XANES) spectrum (left) and EXAFS (k3chi) (right) spectrum obtained from a liver tissue sample (W163) from a beluga whale. As discussed in the text, the spectra are fit to a combination of the corresponding Se XANES spectra for HgSe and for liver tissue W147.

represents 30% Se as HgSe and 70% Se as “bioSe”. As was noted above for the distribution of Hg, samples with high Se contents tended to have a higher proportion of Se as HgSe. Furthermore, Se as HgSe was generally higher for the pituitary tissues compared to the corresponding liver tissue, although the differences were not as large as those observed for Hg as HgSe. Table 5 summarizes how the Hg and Se are quantitatively distributed between different forms for all tissue samples for which both the INAA and XAFS results were available. The distribution of Se between HgSe and a biological Se-containing phase (“bioSe”) has been estimated by two different methods. One method was based on the amount of Hg as HgSe estimated from analysis of the Hg-derivative XANES spectra (Table 2) from which the Se as HgSe could be calculated using the INAA data (Table 1). The other

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Huggins et al. Table 4 Estimates of %Se in different forms based on Least-Squares Fitting of Se EXAFS spectra of Beluga Whale Liver and Pituitary Tissues. Tissue H2 W115 W117 W131 W133 W147 W149 W163 W165 Est. errors

Organ Liver Liver Pituitary Liver Pituitary Liver Pituitary Liver Pituitary

% Se as HgSe 63 34 68 69 74 30* Too weak 72 76 ±10

% Se as “bioSe” 37 67 32 32 26 70* 28 25 ±10

*assumed values, see discussion in text.

Table 5 Estimates of the concentrations of Hg and Se in different forms in beluga whale tissues based on analysis of Hg and Se X-ray absorption fine structure (XAFS) spectra and instrumental neutron activation analysis (INAA) determinations. Hg, mg/kg (Hg XANES) Tissue

Organ

as HgSe

as HgS

H2 W115 W117 W131 W133 W147 W149 W163 W165

Liver Liver Pituitary Liver Pituitary Liver Pituitary Liver Pituitary

116 ± 15

Se, mg/kg (Hg XANES, INAA)

Se, mg/kg (Se EXAFS)

as HgSe

as “bioSe”

as HgSe

as “bioSe”

35 ± 15

46 ± 6

45 ± 6

58 ± 9

33 ± 9

132 ± 18

44 ± 18

52 ± 7

22 ± 7

18 ± 3 18 ± 2 447 ± 58 192 ± 21

14 ± 3 3±2 139 ± 58 21 ± 21

7±1 7±1 176 ± 23 75 ± 8

16 ± 1 14 ± 1 108 ± 23 21 ± 8

16 ± 2 51 ± 7 44 ± 6 7*

7±2 23 ± 7 16 ± 6 16*

204 ± 23 72 ± 10

80 ± 23 24 ± 10

*assumed values, see discussion in text.

method was based on the distribution of Se determined from analysis of the Se EXAFS spectra (Table 4). As can be seen from Table 5, the different estimates of the distribution of Se between HgSe and the “bioSe” form based on analyses of the two types of spectra agree reasonably well.

Conclusions XAFS spectroscopy of Hg and Se in liver and pituitary tissues of beluga whales indicates that formation of HgSe may be a prevalent biochemical pathway that mitigates the toxic


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effects of mercury. Such a pathway has now been confirmed in a number of different marine animals whose diets consist largely of fish that contain MeHg. Consequently, the determination of Hg as HgSe may find application as a bioindicator and measure of minimally toxic Hg in biological tissues. All forms of animal life that possess complex brains require protection against oxidative damage to their brains and endocrine tissues. These tissues employ a series of enzyme families (selenoenzymes) that prevent and reverse oxidative damage in brain and endocrine tissues that require Se in their active sites. However, because MeHg can irreversibly inhibit selenoenzyme activities and selectively sequester Se in insoluble HgSe complexes, high MeHg exposures can result in substantial reductions in selenoenzyme activities and increased oxidative damage. Future risk management decisions will require Hg risk assessments that include consideration of Se when evaluating Hg exposures.

Acknowledgements We thank the Hunters and Trappers Committee of Tuktoyaktuk, NWT, Canada, for providing assistance in obtaining and shipping of the beluga whale tissue samples. The work was funded by a grant from the Fisheries Joint Management Committee (FJMC) of the Inuvialuit Settlement Region and from the Department of Fisheries and Oceans Canada. Mercury and Se XAFS spectroscopy was performed at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy (DOE) Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by DOE Office of Biological and Environmental Research and by the National Institutes of Health National Center for Research Resources Biomedical Technology Program. Nicholas Sharp’s and David Robertson’s participation in this project was supported by the University of Missouri Research Reactor Center. Nicholas Ralston’s participation in this project was supported by the U.S. Environmental Protection Agency grant CR830929-01 and by the U.S. Department of Commerce National Oceanic and Atmospheric Administration grant award NA08NMF4520492 to the Energy & Environmental Research Center at the University of North Dakota.

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