Chemical Forms of Mercury and Cadmium Accumulated in Marine

Marine mammals and seabirds tend to exhibit high accumulations of mercury, cadmium, and selenium in their livers and kidneys. In this study, chemical ...
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Environ. Sci. Technol. 2004, 38, 6468-6474

Chemical Forms of Mercury and Cadmium Accumulated in Marine Mammals and Seabirds as Determined by XAFS Analysis TERUKO ARAI,† TOKUTAKA IKEMOTO,‡ AKIKO HOKURA,† YASUKO TERADA,§ TAKASHI KUNITO,‡ SHINSUKE TANABE,‡ AND I Z U M I N A K A I * ,† Department of Applied Chemistry, Tokyo University of Science, 1-3, Kagurazaka, Shinjuku, Tokyo 162-8601, Japan, Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho, Matsuyama-shi, Ehime 790-8577, Japan, and SPring-8, JASRI, 1-1-1, Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5198, Japan

Marine mammals and seabirds tend to exhibit high accumulations of mercury, cadmium, and selenium in their livers and kidneys. In this study, chemical forms of mercury, cadmium, and selenium accumulated in the livers and kidneys of northern fur seal (Callorhinus ursinus), Risso’s dolphin (Grampus griseus), and black-footed albatross (Diomedea nigripes) were studied by extended X-ray absorption fine structure (EXAFS) spectroscopy to reveal the detoxification mechanisms of these metals. It was found that mercury and selenium exist in the form of HgSe in the liver of northern fur seal. Mercury levels were found to be higher than those of Se, based on their molar ratio, in black-footed albatross. XAFS analysis disclosed an existence of chalcogenide containing both Hg-Se and the Hg-S bonds, suggesting the existence of a solid solution Hg(Se, S) as granules in black-footed albatross. In contrast, Cd concentrations in the kidney were higher than those in the liver for northern fur seal, black-footed albatross, and Risso’s dolphin. It was found that Cd was bound to sulfur, which was probably derived from the metallothionein. The Cd-O bond was observed in the tissues of northern fur seal.

Introduction It is known that marine mammals and seabirds accumulate high amounts of toxic metals such as mercury and cadmium in their tissues in the food web. Alhough most are not directly exposed to industrial pollution sources, they tend to accumulate mercury in the liver at concentrations greater than 100 µg g-1 and cadmium in the kidney without showing apparent symptoms of poisoning. Interestingly, however, high accumulations of mercury have not been observed in the kidneys (1). The mercury accumulated in the liver is mostly found in inorganic form. It is generally accepted that the toxicity of mercury is reduced by being bonded with * Corresponding author phone: +81-3-3260-3662; fax: +81-33235-2214; e-mail: [email protected] † Tokyo University of Science. ‡ Ehime University. § JASRI. 6468

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selenium. The observation of mercuric selenide (HgSe, mineral name: tiemannite) in the livers of marine animals has previously been reported based on the data of transmission electron microscopy (TEM) and X-ray microanalysis (2-5). It seems that HgSe is an inert nontoxic compound in higher trophic marine animals (2, 6). It has also been reported, however, that most of the mercury in the soluble fractions of rat organs is found in high-molecular weight fractions (7), with the mercury and selenium existing in 1:1 molar ratios forming Hg-Se compounds binding to proteins (8). HgSe-S compounds have been identified in the plasma of rabbits by X-ray absorption fine structure (XAFS) analysis (9), and Hg(S,Se) granules have been found in the livers of striped dolphin by X-ray microanalysis and X-ray diffraction analysis (10). The existence of HgS has been hypothesized in seabirds (5). These differences in chemical forms for mercury accumulated in these animals are intriguing, as they could be caused by unique detoxification mechanisms. We have studied the chemical forms of mercury accumulated in liver of northern fur seal in nuclear, lysosomal, mitochondrial, microsomal, and cytosolic fractions. It was found that a large portion of the mercury was distributed in the nuclear, lysosomal, and mitochondrial fractions (11). The fractions containing the microsomes and the cytosol were found to contain the rest of the mercury almost entirely in organic forms. In contrast, in the nuclear, lysosomal, and mitochondrial fractions, the percentage of organic mercury was estimated to be only approximately 1%. The remaining mercury is thought to have been in inorganic form, although the specific chemical form has not yet been determined. In contrast, the cadmium is thought to be detoxified by metallothioneins (MTs). A number of structural studies have provided insight into the metal-binding sites in MT and other metal-binding proteins (12-17). The chemical forms of cadmium in liver of marine animals and seabirds have been also reported to be present in the association form with MTs (18-20). On the other hand, the existence of a Cd-Se complex has also been detected in rat plasma by gel filtration chromatography (21), and some have suggested the formation of a Se-metabolite (22-25). In addition, the existence of cadmium- and calcium-containing granules in kidney tissue has been reported for the Atlantic white-sided dolphin (Lagenorhyncus acutus) (26). It is interesting to consider that the cadmium accumulated in kidney tissue could be detoxified through a biomineralization process and as such be present in an inert nontoxic compound. In the present study, we have used X-ray absorption spectroscopy to determine the chemical forms of mercury, selenium, and cadmium accumulated in the livers and kidneys of northern fur seal, black-footed albatross, Risso’s dolphin, and Japanese common squid and attempted to elucidate their detoxification mechanism. Our attention was especially given to revealing the chemical forms of biominerals containing mercury or cadmium in these animals. X-ray absorption spectroscopy provides a useful tool for investigating the coordination chemistry of a wide range of elements in vitro (9) and in vivo (27). X-ray absorption spectra can be divided into two regions, the near-edge and the extended region. The near-edge region of the spectrum, called the XANES (X-ray absorption near-edge structure) spectrum, is that within 50 eV above and below the absorption edge and contains predominantly dipole-allowed transitions to low-lying unoccupied states. This region thus contains information about the electronic structure (e.g., oxidation state, covalency, etc.) of the absorber atoms in question. Although it is often difficult to assign features within the 10.1021/es040367u CCC: $27.50

 2004 American Chemical Society Published on Web 10/23/2004

TABLE 1. Concentrations of Mercury, Selenium, and Cadmium in Tissue Fractions of Seal, Albatross, Dolphin, and Squid (µg g-1, Dry Weight) and their Molar Ratios concentration no.

species

age

molar ratio

Hg

Se

Cd

liver (N, liver (Mi)c (L) liver (cytosol) (L) liver (whole) kidney (whole) liver (whole) kidney (whole)

2100 110 11 351 6.2 37 3.9

820 49 5.8 140 24 24 34

86.3 33.0 46.0 78.8 255 17.9 174

M)b

Hg

Se

Cd

1 1 1 2.50 0.01 1.16 0.01

0.99 1.13 1.34 2.58 0.13 1.92 0.28

0.07 0.54 7.46 1 1 1 1 0.91 0.23 1.25 1 1

H-1 H-2 H-3 C-1 C-2 C-3 C-4

Northern fur seal

H-4 H-5 H-6 C-5 C-6

black-footed albatross

liver (whole) (L) liver (N, M)b lung (whole) liver (whole) kidney (whole)

230 390 11 220 20

54 130 46 94 62

125 49.4 7.98 124 262

1 1 1 0.99 0.04

0.56 0.85 11.5 1.08 0.34

C-7 C-8

Risso’s dolphin

liver (whole) kidney (whole)

260 28

140 21

119 109

1.22 0.14

1.67 0.27

1 1

C-9

Japanese common squid

liver (whole)

16

336

3 × 10-4

0.07

1

a

18

tissue and MPa

2

0.19

MP: method of preparation. L: lyophilization only. No symbol: digested with papain, etc. b Nucleus, lysosome, and mitochondrion. c Microsome.

near-edge spectrum in a quantitative manner, it can be very useful as a fingerprint in comparison with the spectra of reference materials. In contrast, the higher-energy region of the XANES spectrum, referred to as the EXAFS (extended X-ray absorption fine structure) region, can be analyzed in terms of a local radial structure and necessarily requires comparison to model compounds. Accordingly, this technique is suitable for revealing the chemical form of mercury, selenium, and cadmium in the animals of interest. We have therefore applied this technique.

Experimental Procedures Samples. Liver and kidney tissue of northern fur seal (Callorhinus ursinus), Risso’s dolphin (Grampus griseus), and black-footed albatross (Diomedea nigripes) as well as the livers of Japanese common squid (Todarodes pacificus) were used in this study. A mature and an immature northern fur seal (18 and 2 years old) were collected from off Sanriku, Japan in 1997. The seals were caught for commercial and scientific purposes under appropriate permission. A Risso’s dolphin was collected from Ooarai, Ibaraki, Japan in 1999. Data regarding the age of the Risso’s dolphin were not available. Two black-footed albatross (male) were caught in longline fishing activities in the North Pacific in 1998. A Japanese common squid was landed at a fishery in Sakaiminato, Tottori, Japan in 2001. Samples of northern fur seal and black-footed albatross for subcellular fractionation were frozen in liquid nitrogen within 1 h after death of the specimen. These samples were then transported to the laboratory of the Center for Marine Environmental Studies (CMES), Ehime University. The samples for the subcellular fractionation were stored in a deep freezer at -80 °C until analysis. The other samples were stored at -20 °C. Sample Preparation. Liver of northern fur seal was homogenized, after which the nuclear, lysosomal, mitochondrial fration, the microsomal, and cytosol fractions were prepared (28). These sample preparations were performed at 4 °C. To decompose the organic substances, the insoluble material (452 mg, wet weight) of the nucleus, lysosome, and mitochondrion after extraction with 0.25 M 2-mercaptoethanol and 5 M guanidinium thiocyanate was suspended in 8% papain aqueous solution or nonionic surfactant, 1% Triton X-100 aqueous solution. The sample in the papain solution was incubated at 60 °C for 2 days and that in Triton X-100 solution was shaken for 1 day. The digested solution was centrifuged, and the supernatant solution was removed. The

residue was further washed with pure water. Then the soluble organic substances were extracted with diethyl ether several times, and the final residue was washed twice with pure water and ethanol in sequential and dried at room temperature. The microsomal fraction of liver from the northern fur seal was also prepared by the same treatment. The liver of blackfooted albatross was also homogenized, and the nuclear, lysosomal, and mitochondrial fractions were prepared by the previous treatment. On the other hand, the whole liver and kidney of the northern fur seal and the whole liver, lung, and kidney of the black-footed albatross were dried by lyophilization. Some lypophilized samples were subjected to the XAFS analysis without any pretreatment, which were listed in Table 1 with the notation of L. All samples were stored in a bag made of Mylar film for the XAFS measurement. Each of the reference materials, including HgIISe-II (mineral name: tiemannite), HgIIS (mineral name: cinnabar and metacinnabar), HgIIO, Na2SeVIO4, Na2SeIVO3, Se0 metal, CdIIO, CdIIS, and CdIISe used for the XAFS measurements, was mixed with boron nitride (BN) to an adequate concentration for the fluorescence XAFS method. The reference materials, tiemanite, cinnabar, and metacinnabar, were natural minerals and were purchased from a mineral dealer. Their crystal structures were examined and confirmed by X-ray powder diffraction analysis prior to XAFS measurements. Measurement. Each fraction was quantitatively analyzed for metals by a previously described method (29). Samples were dried for 12 h at 80 °C and then digested with nitric acid in a Teflon PTFE tube assisted by microwave. The concentrations of mercury and selenium in each fraction before the decomposition of organic substances were determined by using a cold vapor atomic absorption spectrometer (CV-AAS) (Sanso, Model HG-3000) and a hydride generation atomic absorption spectrometer (HG-AAS) (Shimadzu, HVG-1 hydride system), respectively. Analysis of cadmium was performed by an inductively coupled plasma-mass spectrometer (ICP-MS) (Hewlett-Packard, HP-4500) using yttrium as an internal standard element. The accuracy of the determination methods was tested using the standard reference materials of DORM2 (National Research Council, Canada) and SRM1577b (National Institute of Standards and Technology, USA). In the present study, all metal concentrations are expressed as dry weight basis (µg g-1). X-ray absorption spectra of mercury and selenium were measured at BL(Beamline)-12C station at the Photon Factory VOL. 38, NO. 24, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Normalized XANES spectra of liver of northern fur seal and liver and lung of black-footed albatross. (a) Hg LII-edge spectra and (b) Se K-edge spectra. The sample numbers correspond to those given in Table 1. (PF), High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. The incident beam was monochromatized by a Si(111) double-crystal monochromator. A bent cylindrical mirror was used as a focusing optics. Hg LII-edge and Se K-edge XAFS spectra were recorded from 500 eV below to 700 or 1100 eV above the edge threshold energy of 14.210 and 12.655 keV, respectively. Energy calibration was performed by Cu foil, and the energy of the shoulder peak was adjusted to 12.7185˚ (8.9814 keV). X-ray absorption spectra were measured as the fluorescence mode by monitoring the X-ray fluorescence intensities of the Hg-Lβ (11.82 keV) or SeKR (11.21 keV) lines using a 19-element Ge solid-state detector (SSD) or a Lytle detector. All spectra were normalized for the intensities of the incident beam, I0, measured by an ionization chamber filled with N2 gas. The fluorescence intensities were measured for 2-6 s per sampling point. All data were collected at room temperature. On the other hand, the X-ray absorption spectra of Cd were measured at BL01B1 station at SPring-8, Hyogo, Japan. The incident beam was monochromatized by a Si(311) double-crystal monochromator. Cd K-edge XAFS spectra were recorded from 500 eV below to 700 or 1100 eV above the edge energy of 26.715 keV. X-ray absorption spectra were measured as the fluorescence mode by monitoring the X-ray fluorescence intensities of the CdKR (23.110 keV) line using a 19-element Ge SSD. All spectra were normalized for I0, measured by an ionization chamber filled with N2 85% + Ar 15% gas. X-ray absorption spectroscopic data were analyzed using the REX2000 program (Rigaku). Background levels of the fluorescence intensity and µ0 were calculated by the Victoreen method and the cubic spline smoothing method, respectively. Fourier transform (FT) was calculated by using k3-weighted χ(k) data over the R range of approximately 2.5-12 Å-1. Ab initio theoretical amplitude and extended-phase functions were calculated using FEFF ver.8 (30) and were then used for the curve-fitting analysis. The goodness of fit was evaluated by a reliability factor calculated as

R2 )

∑{k χ 3

obs(k)

- k3χcalc(k)}2/Σ{k3χobs(k)}2

The standard deviation of each parameter was estimated from the square root of diagonal element of the covariance matrix (31).

Results and Discussion Total Concentrations of Mercury, Selenium, and Cadmium in Marine Animal Tissues. Table 1 shows the concentration 6470

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of mercury, selenium, and cadmium in each fraction prior to the decomposition of organic substances in the animal samples studied. It was found that mercury and selenium accumulated in a 1:1 molar ratio in the nuclear, lysosomal, mitochondrial (H-1), and the microsomal fractions (H-2) of northern fur seal liver. In contrast, in the liver tissue of blackfooted albatross (H-4 and H-5), the abundance of selenium was found to be lower than that of mercury as expressed by the molar ratio. It was observed that the concentrations of mercury in the lungs of black-footed albatross (H-6) were very low as compared with those in the livers, while the ratio of selenium to mercury in the lungs was much higher. In contrast, the concentrations of cadmium were higher in the kidneys of both northern fur seal and black-footed albatross as compared with those in the livers. This finding is in agreement with previous reports (32). To determine the chemical forms of mercury, selenium, and cadmium in these samples, the samples were subjected to XAFS analysis. Chemical States of Mercury and Selenium as Studied by XANES Analysis. Hg LII-edge XANES spectra of the samples are shown in Figure 1a together with the reference materials. The samples include two fractions of northern fur seal liver (H-1 and H-2), black-footed albatross liver (H-4 and H-5), and the reference samples: HgO, HgSe (tiemannite), and m-HgS (metacinnabar), which have the same crystal structures as HgSe. Unfortunately, the mercury concentrations were not high enough to obtain good spectra for the cytsol fractions of northern fur seal liver (H-3) and black-footed albatross lung (H-6). It was found from the chemical shifts that mercury exists as the Hg(II) state in these samples. Judging from the similarity of the XANES spectra, the local structures around the Hg atoms seems to be similar to those in HgSe and m-HgS rather than that in HgO. The Se K-edge XANES spectra are shown Figure 1b. The samples include the nuclear and mitochondrial fraction of northern fur seal liver (H-1), liver and lung of black-footed albatross (H-4, H-5 and H-6), and the reference samples: Na2SeO4, Na2SeO3, HgSe, and Se metal. Unfortunately, the Se levels in H-2 were too small to obtain a good spectrum. The shape and chemical shifts of the XANES spectra of all the samples indicate that Se exists as the Se(-II) state, and they seem to be more or less similar to those of HgSe. HgSe did not exhibit a clear chemical shift as compared with Se metal (Se(0)) due to the covalent nature of the Hg-Se bonding. Thus, the present XANES analysis suggests that mercury and selenium existed as Hg(II) and Se(-II) states, respectively,

FIGURE 2. EXAFS oscillation k3χ(k) of liver of northern fur seal and liver and lung of black-footed albatross. (a) Hg LII-edge data and (b) Se K-edge data. The sample numbers correspond to those given in Table 1.

FIGURE 3. Fourier transform (FT) of k3-weighted Hg LII-edge (a) and Se K-edge (b) EXAFS spectra of the liver of northern fur seal and black-footed albatross. The sample numbers correspond to those given in Table 1. in all fractions. The chemical forms of mercury and selenium in each fraction were determined by the following EXAFS analysis. Chemical Forms of Mercury in the Northern Fur Seal Studied by EXAFS Analysis. The k3χ(k) oscillations of EXAFS spectra for Hg LII-edge and Se K-edge are shown in Figure 2a,b, respectively. Figure 2a includes the data for the northern fur seal together with those of reference materials, m-HgS and HgSe, which have similar XANES spectra, as seen in Figure 1a. The k3χ(k) oscillations of Hg LII-EXAFS of the nuclear, lysosomal, mitochondrial (H-1), and the microsomal fractions (H-2) of northern fur seal show only one resolved shell of scatters similar to HgSe, as seen in Figure 2a. The k3χ(k) oscillations of Se K-EXAFS of H-1 also exhibited a

structure similar to that of HgSe. Therefore, mercury seems to exist as HgSe in the liver of northern fur seal. The corresponding Fourier transform (FT) data for k3weighted χ(k) values for mercury and selenium are shown in Figure 3 a,b, respectively. The phase shift was not corrected in the FT. In Figure 3a, it can be seen that the FTs of H-1 and H-2 exhibited a strong peak at around 2.2 Å. This peak position was close to the one at 2.2 Å observed in the reference material, HgSe, which was due to the Hg-Se bond in HgSe. Therefore, the peaks of H-1 and H-2 around 2.2 Å were assigned to the Hg-Se interaction. In contrast, the FT of Se K-EXAFS for H-1 has doublet peaks similar to those observed for the reference material of HgSe shown in Figure 3b. These peaks can be interpreted as spurious peaks due to the heavy VOL. 38, NO. 24, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. k3χ(k) of Fourier-filtered (solid) and the fit (dotted) data from curve-fitting analysis. (a) Mercury in the liver of northern fur seal (H-1), (b) selenium in H-1, (c) mercury in the liver of black-footed albatross (H-5), and (d) selenium in H-5.

TABLE 2. Results of Hg LII- and Se K-EXAFS Curve-Fitting Analysis for the First Shell interaction

Na

r (Å)

σ (Å)

R factor (%)

Hg-Se Se-Hg Hg-Se Hg-Se Hg-S Hg-Se Hg-S Se-Hg

3.0 3.7 4.0 2.2 1.5 2.6 2.4 2.8

2.62 ( 0.01 2.57 ( 0.02 2.61 ( 0.02 2.54 ( 0.05 2.25 ( 0.34 2.51 ( 0.04 2.20 ( 0.07 2.50 ( 0.02

0.099 ( 0.016 0.083 ( 0.034 0.103 ( 0.022 0.104 ( 0.090 0.200 ( 0.320 0.098 ( 0.059 0.096 ( 0.057 0.080 ( 0.025

1.6 7.6 1.3 6.9

H-1 H-2 H-4 H-5

7.9 18.0

a

N: coordination number; r: bond length; σ: Debye-Waller factor; R: reliability factor for goodness of fit, which was calculated as Σ{k3χobs(k) - k3χcalc(k)}2/Σ{k3χobs(k)}2.

element, mercury, which was confirmed by simulation using FEFF. As such, the FTs of both Hg LII and Se K EXAFS spectra for the northern fur seal suggest the presence of HgSe in the H-1 and H2 fractions. We then attempted to obtain the structural parameters for mercury-containing compounds in the liver tissue of northern fur seal by curve-fitting analysis. The k3χ(k) of Fourier-filtered and fitted data from the curve-fitting analysis are shown in Figure 4a,b. The bond lengths and the coordination numbers were calculated from the curve-fitting analysis, and the best-fit data are summarized in Table 2. The mean free path of the Hg-Se bond was assumed to be equal to those for the reference materials of HgSe (tiemannite). From the fitting analysis for the Hg LII-edge EXAFS spectra, the Hg-Se bond lengths and the coordination number were calculated to be 2.62 ( 0.01 Å and 3.0 in the nuclear, lysosomal, and mitochondrial fractions (H-1), and 2.61 ( 0.02 Å and 4.0 in the microsomal fraction (H-2). In contrast, the Se-Hg bond length was calculated from the Se K-edge EXAFS of H-1 to be 2.57 ( 0.02 Å. These 6472

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Hg-Se and Se-Hg distances obtained from H-1 correspond to the Hg-Se bond of 2.63 Å in the reference material, HgSe (tiemannite). The coordination number of mercury in HgSe is 4. The coordination number of mercury, 3.0 or 3.7, obtained from H-1 was smaller than 4, likely due to the inherent difficulty of the XAFS analysis in determining an accurate coordination number using X-ray fluorescence data. The Hg-Se interaction of 2.61 ( 0.02 Å was obtained in the microsomal fraction of northern fur seal (H-2). This distance and the obtained coordination number of the HgSe interaction of 4.0 agreed well with these values for HgSe. Unfortunately, it was not possible to obtain the Se K-edge EXAFS data due to the low concentrations of selenium in H-2. However, mercury and selenium were found to exist as a 1: 1.13 molar ratio in this fraction, as seen in Table 1. The existence of HgSe can therefore also be assumed in the microsomal fraction. From these results, it is reasonable to assume the existence of HgSe as an inert nontoxic compound in the nuclear, lysosomal, mitochondrial, and the microsomal fractions of liver of the northern fur seal. Therefore, when mercury and selenium are present at high concentrations in a 1:1 molar ratio, it can be assumed that HgSe has formed and accumulated in the liver, in agreement with previous reports (1-5), primarily in nuclear and lysosomal mitochondrial fractions and partially in the microsomal fractions. Chemical Forms of Mercury in the Black-Footed Albatross as Studied by EXAFS Analysis. The k3χ(k) oscillations of EXAFS spectra for Hg LII-edge and Se K-edge spectra obtained from the black-footed albatross are also shown in Figure 2a,b, respectively. The oscillations of H-4 and H-5 in Figure 2a are similar to but somewhat different from that of HgSe and the samples of northern fur seal (H-1, H-2). On the other hand, the k3χ(k) oscillations of Se K-EXAFS of H-5 are different from those of HgSe and H-1, as shown in Figure 2b.

in Se as compared to the abundance of mercury indicated by the molar ratio. Consequently, it was concluded that mercury in the liver of black-footed albatross forms a chemical compound other than HgSe. The FT data for k3-weighted χ(k)s for Hg and Se are also shown in Figure 3a,b, respectively. The FT of H-4 gave a broad peak at around 1.9 Å and that of H-5 also exhibited a strong somewhat broad peak at around 1.9 Å in Figure 3a. This peak position was close to the one at 1.9 Å in m-HgS (metacinnabar) due to the Hg-S bond. Therefore, the peak at around 1.9 Å found in H-4and H-5 can be assigned to the Hg-S interaction. In contrast, the FT of Se for H-5 shown in Figure 3b shows an asymmetric peak, and the peak positions are not particularly similar to those for HgSe. On the basis of these data, the chemical forms of mercury and selenium in the black-footed albatross were examined by curve-fitting analysis using the model compounds. It was found that twoshell fits (Hg-Se + Hg-S) gave a better fit over one-shell Hg-Se fits in the black-footed albatross (H-4 and H-5). The k3χ(k) of Fourier-filtered and the fit data from the curvefitting analysis are shown in Figure 4c,d. The bond lengths and the coordination number were calculated from the curvefitting analysis, and the best-fit data are summarized in Table 2.

FIGURE 5. Normalized Cd K-edge XANES spectra of tissues of northern fur seal, black-footed albatross, Risso’s dolphin, and squid. The sample numbers correspond to those given in Table 1. Therefore, mercury in black-footed albatross probably has a similar, but not identical, structure to that of HgSe found in the liver of northern fur seal, as described previously. It can be seen in Table 1 that mercury and selenium existed in a 1:0.99 molar ratio in the nuclear and mitochondrial fractions of the liver tissue of northern fur seal (H-1). In contrast, the liver of black-footed albatross (H4) is deficient

From the curve fitting analysis of the Hg LII-edge EXAFS spectrum for H-5, coexistence of the Hg-Se bond of 2.51 ( 0.04 Å and the Hg-S bond of 2.20 ( 0.07 Å can be observed. As has been reported, metal-sulfur clusters exist in metallothionein (12, 14). However, metallothionein is primarily contained in the cytosol fraction. The sulfur of the Hg-S bond in the nuclear, lysosomal, and mitochondrial fractions of black-footed albatross liver (H-5) would be in an inorganic form such as a biomineral. The existence of HgS has been hypothesized for seabirds (5), and Hg(S,Se) granules have been found in the livers of striped dolphin (10). We therefore suggest the existence of a solid solution of Hg(Se, S) in which it is deficient in selenium as compared to the abundance of mercury indicated by the molar ratio in the liver of blackfooted albatross.

FIGURE 6. Fourier transform (FT) of k3-weighted Cd K-edge EXAFS spectra of the liver and kidney of the animals. (a) Northern fur seal (C-1 to C-4) and (b) black-footed albatross and Risso’s dolphin and the liver of squid (C-5 to C-9). The sample numbers correspond to those given in Table 1. VOL. 38, NO. 24, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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In the present study, the chemical form of mercury accumulated in the liver of black-footed albatross, as estimated from the EXAFS analysis, was found to be different from the form found in the northern fur seal. This interesting difference could be attributable to the lack of selenium. Further works are expected to shed light on the workings of detoxification for mercury accumulated in the body. Chemical Form of Cd in the Northern Fur Seal and the Black-Footed Albatross Studied by XAFS Analysis. Cd K-edge XANES spectra of the samples are shown together with the reference materials in Figure 5. The samples include the liver and the kidney of northern fur seal of 18 years (C-1 and C-2) and 2 years (C-3 and C-4), those of black-footed albatross (C-5 and C-6), those of Risso’s dolphin (C-7 and C-8), the liver of Japanese common squid (C-9), and the reference materials: CdO, CdS, CdSe, and Cd metal. It was found from the chemical shifts of XANES spectra that Cd existed in the divalent state in all samples. A comparison of edge features of the XANES spectra and EXAFS oscillation above 26.75 keV suggests that the local structures around the Cd atoms in C-1 to C-3 are similar to those in CdO rather than those in CdS and CdSe, and those in C-4 to C-9 are similar to those in CdS and CdSe. The corresponding Fourier transform (FT) of k3-weighted χ(k) are shown in Figure 6. The phase shift was not corrected in the FT. The FTs of C-1, C-2, and C-3 of northern fur seal exhibited a strong peak at around 1.8 Å, as shown in Figure 6a. This peak position was close to that at 1.8 Å observed in CdO, which is due to the nearest Cd-O bond in CdO. This peak could therefore be assigned to the Cd-O interaction. On the other hand, C-4 for the kidney of northern fur seal (age 2) gave a strong peak at around 2.0 Å, and it is between the one in CdO and the one at 2.1 Å observed in CdS. The latter peak was due to the Cd-S bond in CdS. Therefore, the peak for C-4 includes a significant contribution from the Cd-S interaction, but this phase is not CdS because this peak in C-4 is shorter than that in CdS. It has been reported that the Cd-S bond length in CdS is 2.47 Å and that in MT is slightly shorter (2.43 Å) (12). XANES spectra in Figure 5 also showed that C-4 was distinct from CdS. It therefore appears more likely that the peak for C-4 is due to the Cd-S interaction in MT rather than that in CdS. On the other hand, the FTs of the liver and kidney (C-5 and C-6, respectively) of black-footed albatross and those of Risso’s dolphin (C-7 and C-8, respectively), and the liver of squid (C-9) exhibited a strong peak at around 2.0 Å in Figure 6b. Considering that XANES spectra of C-5, C-6, C-7, and C-8 are similar to that of C-4, these peaks could also be assigned to the Cd-S interaction in MT. A further study on the chemical forms of Cd in the liver of Japanese squid by using gel filtration and XAFS techniques was reported in the literature (33). The Cd-Se bond was not observed in any of the samples, suggesting that cadmium has a different detoxification mechanism from mercury. In contrast, the Cd-Se complex has been detected in rat plasma (18), and granules containing cadmium and calcium have been found in kidney tissue of the Atlantic white-sided dolphin (Lagenorhyncus acutus) (26). In the present study, an inorganic form of cadmium such as CdSe or CdS was expected for the detoxification of Cd accumulated in the tissues through biomineralization, like HgSe for mercury. However, the XAFS analysis has suggested that the Cd-S bond exists in almost all samples, excluding the sample of northern fur seal (C-1 to C-3), and that the sulfur would be derived from the metallothionein. In contrast, the Cd-O bond was observed in northern fur seal tissues (C-1 to C-3).

KEK-PF. The EXAFS experiments were carried out under approval of the SPring-8 and PF program Advisory Committees (Proposal 2002A0505-NX-np and 2000G336, respectively). We are grateful to Drs. T. K. Yamada, N. Baba, and H. Tanaka for providing samples.

Acknowledgments

Received for review February 24, 2004. Revised manuscript received August 18, 2004. Accepted August 23, 2004.

The authors thank Dr. Chiya Numako and Mr. Katsutoshi Fukuda for their kind help for the XAFS measurement at 6474

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