Article pubs.acs.org/est
Tungsten Toxicity, Bioaccumulation, and Compartmentalization into Organisms Representing Two Trophic Levels Alan J. Kennedy,* David R. Johnson, Jennifer M. Seiter, James H. Lindsay, Robert E. Boyd, Anthony J. Bednar, and Paul G. Allison †
U.S. Army Engineer Research and Development Center, Vicksburg, Mississippi 39180, United States S Supporting Information *
ABSTRACT: Metallic tungsten has civil and military applications and was considered a green alternative to lead. Recent reports of contamination in drinking water and soil have raised scrutiny and suspended some applications. This investigation employed the cabbage Brassica oleracae and snail Otala lactea as models to determine the toxicological implications of sodium tungstate and an aged tungsten powder-spiked soil containing monomeric and polymeric tungstates. Aged soil bioassays indicated cabbage growth was impaired at 436 mg of W/kg, while snail survival was not impacted up to 3793 mg of W/kg. In a dermal exposure, sodium tungstate was more toxic to the snail, with a lethal median concentration of 859 mg of W/kg. While the snail significantly bioaccumulated tungsten, predominately in the hepatopancreas, cabbage leaves bioaccumulated much higher concentrations. Synchrotron-based mapping indicated the highest levels of W were in the veins of cabbage leaves. Our results suggest snails consuming contaminated cabbage accumulated higher tungsten concentrations relative to the concentrations directly bioaccumulated from soil, indicating the importance of robust trophic transfer investigations. Finally, synchrotron mapping provided evidence of tungsten in the inner layer of the snail shell, suggesting potential use of snail shells as a biomonitoring tool for metal contamination.
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INTRODUCTION Metallic tungsten has numerous civil and military applications, such as tools, lighting, electrodes, sporting goods, microwaves, televisions, armor plating, kinetic energy penetrators, warheads, and small arms munitions.1,2 Tungsten carbide armor-piercing rounds were employed during World War II3 and later enhanced.4 Tungsten use declined with the advent of depleted uranium (DU) and Pb munitions, although DU and Pb health concerns diverted interest back to W.4 The Green Armament Technology Program (GATP) sought W as an environmentally benign alternative to Pb munitions.2,5−7 The U.S. Fish and Wildlife Service approved W for Pb substitutions, including shotgun ammunition and fishing weights.2 Various W-based munitions consisting of different metal alloys were developed (e.g., Ni, Fe, Sn, Cu, and Co5,8). However, few environmental studies were initially conducted as low solubility and toxicity were projected.2,6 Sources of W contamination include mining operations, industrial activities, military training, and landfills.2,7 Elevated W concentrations in surface soils relative to the background (0.7− 2.7 mg of W/kg) were reported in Fallon, NV (10−67 mg of W/kg), the Kuwait−Iraq border (126.5 mg of W/kg), military firing ranges (5200−5500 mg of W/kg, estimated), and additional international locations such as mining sites in Australia and agricultural soils in the United States, New Zealand, and Europe (0.5−83 mg of W/kg).2,8 Furthermore, W concentrations in fine surface dust deposits within Fallon were This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society
reported to be as high as 934 mg/kg (background body > foot; Figure 3). Micro-XRF data of a hepatopancreas from a snail exposed to 386 mg of W/kg of soil indicated a relatively homogeneous distribution of W in the tissue (Figure 4a). Elemental correlations (r < 0.1) were
and height corresponded to effective median tissue residues of 324 and 636 mg of W/kg, respectively. BAFs were similar between soil concentrations, ranging from 0.53 to 0.72 (mean 0.62 ± 0.08), and fell within a wide range of BAF equivalents (0.1−0.8) for leaf vegetation in the literature.39 Micro-XRF mapping of a B. oleracae leaf exposed to soil (436 mg of W/kg) indicated the highest W levels in the leaf venation, with lower levels of W integrated into the green tissue (Figure 2). Some tissue residue data for W are available in the literature. Cabbage exposed to municipal sludge containing W, among other metals, was previously reported to contain 0.2−1.7 mg of W/kg in tissue.2,23 Butler et al.11 reported tissue residues for Cyperus esculentus exposed to six different soils to range from more than 10 to 100 000 mg of W/kg. Johnson et al.15 reported lower tissue residues and BAFs (0.04−0.12) for Helianthus annuus leaves. This suggests B. oleracae leaves more efficiently bioaccumulate W, coinciding with their greater sensitivity, and provide a greater potential to mobilize W into the food chain. Animal Exposures. Higher toxicity (Figure 3) was observed in the Na2WO4 dermal exposure (LC50 = 859 [521−1418] mg of W/kg) relative to the soil exposure (LC50 > 3793 mg of W/kg). This was expected provided the high solubility and bioavailability of the Na2WO412,13,15,24 and the more direct dermal exposure to the cloth relative to the soil, which contained a mixture of tungstates and polytungstates. Inouye et al.14 reported nominal 14 d LC50 values for the earthworm E. fetida of 3960−6250 mg of W/kg in artificial and field soils, respectively, and sublethal 28 and 56 d LOEC end points of ≥704 mg of W/kg. A recent study reported 6 month E. fetida survival (NOEC = 10 000 mg of W/kg, nominal) and 9649
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Figure 3. Survival and measured tungsten concentrations for O. lactea exposed (a) to W-contaminated soil and (b) dermally to a Na2WO4spiked cloth. Values that do not share the same letter designation were statistically significantly different from one another. Note that concentration is plotted on a log scale.
Figure 4. Synchrotron-based X-ray fluorescence map of O. lactea tissues exposed to 386 mg/kg tungsten-contaminated soil. Panel a is a map of the W distribution in the hepatopancreas in counts. Panel b is a map of W, Ca, and P distributions in the shell, with the inside of the shell oriented to the left. Ca counts were 100 times those of W and P. The white bracket in panel b indicates elevated W counts (green and lighter blue) as surface deposits on the inside of the shell, while the white arrow indicates elevated W counts integrated 10−20 μm beneath the shell surface. The scale bar represents 200 μm in panel a and 50 μm in panel b.
insignificant between W and other metals (Ca, Fe, S, P). Previous investigators27,42 reported other metals (e.g., Zn, Fe, Mn, Pb, and Cd) also predominately bioaccumulated in the snail hepatopancreas. While BAF values for the hepatopancreas did not differ significantly between W substrate concentrations, they were significantly higher in the dermal cloth exposure (0.08 ± 0.02 to 0.11 ± 0.02) relative to the soil exposure (0.03 ± 0.01 to 0.04 ± 0.01). BAF values were lower in the foot and body (≤0.02). In the dermal exposure, the LR50 concentration for the hepatopancreas was 80 (52−123) mg of W/kg; however, survival in the soil exposure was slightly higher (73%), where snails bioaccumulated W at levels (84 ± 39 mg of W/kg) similar to the LR50, again suggesting that the diversity of W species21 present in the soil may be less toxic or bioavailable than Na2WO4. While not significant (p ≥ 0.26), lower cabbage consumption was observed in the two highest (53−58% consumed) relative to the two lowest (73−85%) soil concentrations, corresponding to observed snail lethargy and aestivation. Tungsten may affect
sugar homeostasis, glycoprotein production, and mucus secretion and thus mobility, supporting observations of lower activity in higher W exposures. In the dermal exposure, the hepatopancreas concentrations were 6−8 times higher than the foot concentrations (the initial point of exposure). At the two highest soil concentrations, similar hepatopancreas-to-foot ratios (6−7) were found and snails were less active relative to control snails. However, at the two lower soil concentrations, where snail activity and feeding rates were at control levels, hepatopancreas-to-foot ratios were relatively higher (12−13); this greater ratio may suggest the addition of oral exposure (i.e., consumption of soil31,32) at lower W soil concentrations in which soil avoidance was less prevalent; alternatively, the difference may relate to W depuration kinetics. While W is eliminated from soft tissue, mammalian bone is a long-term (8−23 years) storage organ for W.2,19 Similarly, the snail shell may prove useful as a biomonitoring tool for W,30 since once incorporated into the calcium carbonate and collagen matrix,43 it would not be readily eliminated. Micro9650
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investigation of W incorporation into the snail shell for use as a biomonitoring tool will be conducted.
XRF data (Figure 4b) indicated W on the inner layers of a shell cross section from a snail exposed to soil (386 mg of W/kg). While the presence of some W on the inside of the shell may relate to surface deposits (shells were washed), the presence of detectable W on the inside of the shell beneath a 10−20 μm thick Ca layer suggests W incorporation into the shell matrix (Figure 4b). Metals incorporate into the shell through the channels that deliver Ca to grow and maintain the shell; ultimately, the snail mantle delivers Ca, other elements, and proteins to the shell.28,32 Snail shells were previously reported to accumulate and store metals (Pb, Cu, Cd, Zn). An effort to assess the relative bioavailability of W to snails through soil (541 mg of W/kg) versus dietary exposures (cabbage 134 mg of W/kg) suggested through higher BAFs that trophic transfer (hepatopancreas BAF = 0.64, body BAF = 0.13) was more important than direct soil contact (hepatopancreas BAF = 0.04, body BAF = 0.01). All snail compartments (hepatopancreas, remaining body, feces) contained higher residues in the dietary (86.3, 17.1, and 74.7 mg of W/kg) relative to the soil (19.2, 5.4, and 12.2 mg of W/kg) exposure. The assimilation efficiency in the feeding experiment was 44− 58%, calculated by the ratio and mass balance methods, respectively. Assimilation of W may be more efficient via trophic transfer due to greater exposure to higher concentrations or if digestion facilitates the oxidation of zerovalent tungsten to tungstate species with subsequent dissemination to organs and tissues. Rapid elimination of W via excretion is possible (80−95% within 24 h), as previously reported for rodents.19 This investigation provides toxicity reference values and bioaccumulation factors for regulatory consideration. The data also provide evidence that while Na2WO4 was generally more toxic and bioavailable than the W powder in soil, the more environmentally realistic, aged soil-laden W still readily bioaccumulated into cabbage leaves and the snail hepatopancreas, as confirmed by liquid and solid (μXRF) phase analytical techniques. Since multiple species of W were detected in the study soil (this study and refs 12 and 13), these results suggest that environmental regulations based solely on total measurable W may be an oversimplification; consideration of soil geochemistry will more accurately estimate W toxicity and bioavailability and better inform management decisions. It is noteworthy that cabbage bioaccumulated W from the soil much more efficiently relative to the snails. Synchrotron-based (μXRF) mapping indicated that W was distributed throughout the cabbage leaves but was most concentrated in the leaf veins. Although an adverse effect on cabbage growth was observed at a lower W soil concentration compared to that on snail survival (likely due to a greater assimilation efficiency), cabbage showed a relatively greater tolerance to higher accumulated W concentrations in tissue without adverse effects relative to snails. That is, a median effect on cabbage growth was not observed until 324 mg of W/kg in leaf tissue, while only 80 mg of W/kg in snail tissue resulted in 50% mortality. The ability of cabbage, or plants in general, to grow despite bioaccumulation of high levels of W highlights the importance of trophic transfer to mobilize W in the food web. Preliminary dietary exposures (trophic transfer) suggested greater assimilation when snails consumed W-contaminated vegetation relative to being directly exposed to the contaminated soil. Future work will involve robust experimentation to determine bioaccumulation kinetics and develop definitive trophic transfer factors and elimination rates for total W and W species. Furthermore, a more detailed
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ASSOCIATED CONTENT
S Supporting Information *
Measured tungsten concentrations used in bioassays (Table S1), cabbage growth curves (Figure S1), and photographic documentation of cabbage size (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: (601) 634-3344; fax: (601) 634-2263; e-mail: Alan.J.
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded by the Army Environmental Quality Technology Basic Research Program (U.S. Army Engineer Research and Development Center, Mr. Martin Savoie and Dr. Elizabeth Ferguson, Technical Directors). Synchrotron analysis was conducted in collaboration with Dr. Sam Webb at the Stanford Synchrotron Radiation Lightsource, a Directorate of the SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy Office of Science. We appreciate Ashley Harmon for sectioning tissue and constructive comments from Drs. Afrachanna Butler, Chris McGrath, and two anonymous reviewers. Permission was granted by the Chief of Engineers to publish this information.
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