Environ. Sci. Technol. 1993, 27, 2466-2471
Luminescence Study of Eu3+ Binding to Immobilized Datura innoxia Huel-Yang David Ke and Gary D. Rayson' Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003
Paul J. Jackson Life Science Division, LS-2, MS M886, Los Alamos National Laboratory, LOS Alamos, New Mexico 87545
The spectrally and temporally resolved Eu3+ excitation signals have been measured for solid samples of Eu3+(silicate polymer), Eu3+-(immobilized Datura), and Eu3+Datura at liquid nitrogen temperature. The binding capacity of Eu3+to Datura innoxia cell material has been found to be significantly enhanced when immobilized in a polysilicate matrix. New binding sites inaccessible to Eu3+ in free cell material have been observed for the binding of Eu3+ after immobilization of the cultured biomaterials. However, chemical alteration of the cell material resulting from this immobilization process has not been observed. The chemical environment of the binding sites responsible for the binding of Eu3+ in immobilized and free D. innoxia cell material has been demonstrated to be significantly different. This difference results from the immobilization of the cell material within a Dolvsilicate matrix.
Introduction The use of biologically derived materials as sources of metal binding for wastewater treatment has been amply studied in recent years (1-9). The majority of these investigations have been focused on algal biomasses. However,our previous studies (10-16)have demonstrated that the same kind of biosorption (adsorption) processes as those observed in algae are likely to occur on the cultured plant cell walls, specifically Datura innoxia. D.innoxia cell wall material has been immobilized within a polysilicate matrix (17) to yield a packing with the mechanical properties of the polymer and the adsorptive properties of this cell material. It was suspected that a fraction of the cell material would be made inaccessible because of the immobilization process. However, a decrease in the Eu3+binding capacity was not observed when the amount of Eu3+ bound was normalized to the mass of cell materials used. Conversely, a significant increase in the normalized binding of Eu3+ was observed when the biomaterial was immobilized. Thus, it is necessary to investigate how the immobilization of the cell material affected its ability to bind Eu3+ ions from aqueous solutions. Eu3+ luminescence has been employed in the present study to better understand the effect of the immobilization on the binding of Eu3+ to D. innoxia cell walls. Two possible explanations of the increased binding capacity were investigated: (1)the availability of more binding sites because of a conformational change or the denaturation of the biopolymer on the cell walls, and (2) added binding sites due to a chemical change of the cell materials. This information will enable D. innoxia cell wall material to be more effectively applied to wastewater treatment. 2466
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The Eu3+ ion is unique in that both the ground state (7Fo)and the excited emissive state (SDo) are nondegenerate. Since neither of these levels can be split by a ligand field, the absorption (and emission) band corresponding to a transition between these levels must consist of a single, unsplit line for a given Eu3+ environment (18, 19). This implies the existence of a one-to-one correspondence between the number of peaks in the excitation spectrum and the number of distinct Eu3+ environments (18, 19). Consequently, useful spectral interpretations are possible without a detailed analysis of the crystal field splitting. This characteristic makes Eu3+luminescence an excellent probe for studying biologicalsystems which usually contain multiple binding sites. Several studies using Eu3+luminescence to probe metal-binding interactions on proteins have been described (10, 11, 16,20-23). The electronic 7Fo 5Do transition of the Eu3+ corresponds to the pairing of two of the six unpaired electrons found in the ground state of Eu3+(i-e.,4f6).Thus, the energy of this transition will be governed by the interelectronic repulsions within Eu3+ion, and any change in energy can be regarded as a nephelauxetic effect (19), which is a measure of the reduction in the interelectronic repulsions between 4f electrons on the Eu3+ion, resulting from a delocalization of the f electrons onto the coordinated ligands. Consequently, any subtle change in energy gap due to the nephelauxetic effect could directly be detected by examining the shift of the excitation peak position of the Eu3+ion. In this paper, the interaction between Eu3+ and silicate polymer, or the functionalities responsible for the binding of Eu3+ on D. innoxia cell wall, will be investigated using Eu3+ luminescence.
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Experimental Section M a t e r i a l s and M e t h o d s . Stock europium(I1I) chloride solutions, prepared from EuzO3 (Molycorp), were standardized with ethylenediaminetetraacetic acid (EDTA) solution (Sigma) at pH 6.0 by methods described elsewhere (10,11, 20-23). A 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) (Sigma) was used as a noncomplexing buffer (24,251. Sodium hydroxide (Mallinckrodt) and nitric acid (Baker) were used to adjust solutions to the desired pH. Distilled deionized water was used throughout the work. The procedures used to grow and wash the D. innoxia biomass material have been described elsewhere (IO). A portion of the cultured cell material was immobilized according to the method described by Darnall et al. (17). Briefly, 5 g of cell materials was added to 75 mL of 5% HzS04 (Mallinckrodt). Then a 6% NazSiO3 (Fisher) solution was added until the mixture reached pH 2.0. After the suspension was stirred for about 1h, the pH was slowly raised to 7 by dropwise addition of 6% NazSiO3. Silicate polymers were formed at this pH. This mixture waa stirred 0013-936X/93/0927-2466$04.00/0
0 1993 American Chemlcal Society
for an additional 30 min. The resulting gel was refrigerated, and the polymer was allowed to settle. The separated aqueous phase was aspirated off and tested with a barium nitrate solution (Aldrich) for the formation of a Bas04 precipitate. When no precipitate was observed, immobilized biomaterial was washed one additional time with distilled deionized water to ensure the complete removal of residual sulfate. The final cell-polymer gel was baked at 87 "C until completely dry. The percentage of D. innoxia cell walls in the immobilized material was calculated by dividing the initial mass of cell material by the total mass of immobilized material. Thematerial used in this study was found to be 68% cell material by weight. Before reacting this immobilized biomaterial with E d + , it was washed and dried by the method described previously (10). Silicate polymers without D. innoxia cell matrial were similarly prepared. In all Eu3+-binding experiments, blank and sample solutions were prepared in parallel. For blank solutions, 250 mg of the immobilized D. innoxia cell material were suspended in 50 mL of a europium(II1)-freebuffer solution at the same pH as that used in the sample solutions. After a contact time of 1h to establish equilibrium, the solution was centrifuged and the supernatant liquid was stored. Then, 0.001 M of the blank and sample Eu3+solutions was prepared by adding the same volume of the stock EuC13 solution to the supernatant liquid obtained above or the buffer solution containing the immobilized cell material (5 mg mL-l). After being agitated for about 1h, the blank and sample solutions were centrifuged, and the supernatant liquids were analyzed using inductively coupled plasma atomic emission spectrometry (ICP-AES). The difference between the sample and blank solutions represents the amount of Eu3+ions bound to immobilized D. innoxia cell materials. The solid Eu3+-(immobilized Datura) samples were air-dried and stored for subsequent Eu3+ luminescence measurements. Solid Eu3+-('immobilized Datura) prepared from different concentrations of Eu3+ at pH 6.0 were similarly dried and stored. Solid Eu3+-Datura and Eu3+-(silicate polymer) samples were also similarly prepared and dried. Europium(II1) Luminescence. Excitation spectra associated with the electronic 7Fo 5Dotransition of the Eu3+ ions were acquired with the laser spectrofluorimeter described previously (10, 11, 20-23). Solid Eu(II1)containing samples (4 mg) were placed in a sample cell (a 3-mm-diameter Pyrex glass tube with one end sealed). All the luminescence experiments were performed at liquid nitrogen temperature to minimize temperature-dependent line-broadening effects. The dye laser was operated during these experiments using a 0.004 M Rhodamine 590 (Exciton Corp.) solution in absolute ethanol. Emission was monitored at 615 nm with an effective bandwidth of 1.67 nm. Luminescence spectra deconvolution was performed using a nonlinear least-squares regression routine. The spectra were fitted to a sum of peaks having a Lorentzian line shape (10,l1). Lifetime measurements were obtained using the instrumental setup described previously (10). The average of 2000 luminescence decay curves was analyzed using either single or multiple exponential functions for the determination of individual lifetimes. Inductively Coupled Plasma Atomic Emission. The ICP-AES system used in this study has been described elsewhere (10, 26). A conventional tangential-flow torch
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60
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0 2
0
4
6
PH Figure 1. Effect of pH on the binding of Eu3+ ions to free Datura (O), immobilizedDatura (B), silicate polymer (A), and the calculated binding to 88% cell material in a polysilicate matrix (0).
was used with an outer argon gas flow rate of 16 L min-l, an intermediate flow of 1.0 L min-l, and a nebulizer flow rate of 0.8 L min-I. The plasma was operated at 1.50 kW of applied rf power with 10 W reflected. The intensity of the 381.967-nm Eu(I1) line was measured at a viewing position of 12 mm above the load coil. All reported values are the average of three replicates. Results and Discussion pH Dependent Study. The relative binding behaviors of free Eu3+-Datura, Eu3+-(immobilized Datura), and Eu3+-(silicate polymer) occurring at different pH conditions (1-6) are shown in Figure 1. Uncertainties in these measurements were found to be less than the size of the marker and were thus omitted from the figure. The calculated curve represents the binding capacity of Eu3+ calculated from the immobilized D. innoxia cell material containing 68% D.innoria cell wall material and 32% silicate polymer by weight. Thus, it is an indication of the amount of Eu(II1) predicted to be bound. Compared with the calculated curve, it is apparent that the binding capacities of Eu3+ obtained from the immobilized Datura cell material are enhanced by a factor of about 44 % when pH 23. From a practical point of view, this 44 % increment caused by the immobilization process is significant. Carboxylate and sulfate functionalities have been determined to be the two dominant functional groups responsible for the binding of Eu3+ on D. innoxia cell walls (10). The extent of dissociation of these two functionalities is a function of the solution pH, governing the binding capacity of Eu3+ to the cell wall material. Thus, to understand the effect of immobilization on the binding of Eu3+, it is necessary to investigate how the Eu3+ions bind to silicate polymer. Once this binding behavior is understood, it will be possible to ascertain the factors contributing to the binding between Eu3+and immobilized D. innoxia cell material. pH Study of Eu3+Luminescence in Eu3+-(Silicate Polymer). The excitation spectra associated with the electronic 7Fo EDo transition of the Eu3+ ions in a series of solid Eu3+-(silicate polymer) samples (4 mg) prepared from 1mM Eu3+ solution at different pH conditions are shown in Figure 2. The corresponding lifetime data are
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Table I. Calculated Lifetimes of Solid Samples at Different pH Conditions PH first lifetimes (ps) second lifetimes (ws)
Eua+-(Silicate Polymer) 13 f 1 14f 1 14f1 15 f 1
1
2 3 4 5 6
17 f 1
1 2
3 4 5 6 1 2
3 4 5 6
116 f 6 126 f 7 138 f 7 140 f 8 146 f 9 189 f 12
56 f 3 Eu3+-(Immobilized Datura) 16f 1 18f 1 192 f 9 195 f 10 22 f 2 208 f 12 28 f 2 444 f 23 162 f 11 470 f 26 167 k 12 Eu3+-Datura
6
2 0
8
11f 1
12f1 13 f 1 15 f 1 15f 1 15 f 1
4 271 f 7 265 f 5 254 f 5
0
shown in Table I. Because two sets of lifetime data (i.e., 13-56 and 116-189 ks) were obtained (Table I), four typical excitation spectra of Eu3+-(silicate polymer) in Figure 2were each fit with two Lorentzian curves. As shown in Figure 2 (see the dotted lines), the excitation peak positions of the two resolved peaks remained nearly unchanged between pH 1and pH 6. This indicates that the two dominant binding sites responsible for the binding of Eu3+ on silicate polymer are not changed within this pH range. However, the relative peak intensities in both of the two resolved peaks near 578.2 and 579.1nm increase when the solution pH increases from 1to 6. This suggests an ion-exchange mechanism could be involved in the binding process of the Eu3+to the silicate polymer. Also, the binding site associated with the peak near 578.2 nm is more pH dependent than the one near 579.1 nm. This would seem to be consistent with the nephelauxetic effect (19),which predicts the 4f electrons of the Eu3+ in the binding site associated with a shorter wavelength (Le., 578.2 nm) should be more localized on the Eu3+ rather than delocalized onto the coordinated ligands, making the binding between Eu3+and silicate polymer more ionic (i.e., more pH dependent). Concentration Study of Eu3+ Luminescence in Eu3+-(Silicate Polymer). A series of solid Eu3+-(silicate polymer) samples were prepared from different Eu3+ solutions which varied in concentration from 1 to 0.13 mM at pH 6.0. The fitted excitation spectra of four typical samples are shown in Figure 3. As can be seen (from the vertical dotted lines), the excitation peak positions of the two resolved peaks remained unchanged when the initial concentration of Eu3+ was decreased from 1to 0.13 mM. This indicates that the dominant binding sites responsible for the binding of Eu3+ on silicate polymer do not change within this solution concentration range. However, the peak intensity of the peak near 578.2 nm was observed to increase when the Eu3+concentration was decreased from 1 to 0.13 mM. An opposite effect was observed for the peak near 579.1 nm. From the point of view of competition, the binding site associated with the peak near 578.2 nm would be predicted to display a greater binding strength 2468
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0 577.5
578.5
579.5
580.5
Wavelength (nm) Figure 2. Fitted excitation spectra of solid Eu3+ -(silicate polymer) samples prepared from 1 mM Eu3+solution at different pH conditions (+, data points: individual Lorentzlan fitted curves or the sum of the resolved curves) (Aawb = 615 nm).
with a lower availability than the site resultingin the 579.1 nm peak. Characterization of Eu3+Binding to Immobilized Datura innoxia Cell Material. The lifetime data obtained from a series of solid Eu3+-(immobilizedDatura) samples prepared from 1 mM Eu3+ solution at different pH conditions are also shown in Table I. Again, the measurement of two sets of lifetime data at pH 2 2 indicated the presence of two different types of Eu8+ binding sites on the immobilized D. innoxia cell material.
A 20
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Wavelength (nm)
577.5
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Wavelength (nm)
Flgure 3. Fitted excitation spectra of solid Eu3+-(silicate polymer) samples at pH 6.0 with different Eu3+ concentrations of (A) 1.0, (B) 0.66, (C)0.33, and (D) 0.13 mM (+, data points; individual Lorentzlan fitted curves or the sum of the resolved curves) (Aemlsslon = 615 nm).
Figure 4. Fitted excitation spectra of solid Eus+-(immobilized Datura) samples prepared from 1 mM Eus+soiutlon at dlfferent pH conditions (+, data points; Individual Lorentzian fitted curves or the sum of the = 615 nm). resolved curves) (hemlssM
A t pH 1.0, only one lifetime was observed. Accordingly, four typical excitation spectra of Eu3+-(immobilized Datura) were fitted either with one or two Lorentzian curves (Figure 4). Under the same experimental conditions mentioned above, the lifetime data obtained from solid Eu3+-Datura are also shown in Table I. The corresponding excitation spectra of Eu3+-Datura have been previously reported (IO). Interestingly, at pH 1 2 , a red shift of the resolved excitation peaks as a function of solution pH was observed
(the vertical dotted lines in Figure 4). This shift of the measured excitation spectrum was not observed with either of the other materials (i.e., Eu3+-(silicatepolymer) (Figure 2) or Eu3+-Datura (IO)). This spectral shift with solution pH would imply that the chemical environment of the binding sites responsible for the binding of Eu3+ in immobilized Datura cell material is pH dependent. Because two sets of lifetime data were observed in both Eu3+-(immobilized Datura) and Eu3+-Datura, the two dominant functional groups (i.e., carboxylate and sulfate) Environ. Sci. Technol., Vol. 27, No. 12, 1993 2469
( 1 0 , I I ) responsible for the binding of Eu3+on Datura cell walls should remain the same as those present in the immobilized cell material. This could be further reinforced by the similarity in the excitation peak positions observed in Eu3+-(immobilizedDatura) (Figure 4) and Eu3+-Datura
20
(10).
However, as shown in Figure 1, Eu3+ ions have been observed to bind more efficiently to the immobilized cell material than to theDatura cell walls and silicate polymer. One interpretation is that the immobilizationprocess could alter the tertiary structure of macromolecular species within the D.innoxia material. Such a pH-dependent conformation change could result in binding sites which were inaccessible to Eu3+ in free cell material to become available. Those additional sites would then yield the observed increase in binding capacity. The lifetime data for Eu3+-(immobilized Datura) (16-167 and 192-470 p s ) are significantlydifferent from those for Eu3+-Datura (1115 and 271-254 p s ) . This may suggest that the chemical environment of the binding sites in immobilized cell material has been significantly changed. At pH 2 and pH 3, two sets of lifetimes were observed for Eu3+-(immobilized Datura) (18-22 and 192-195 p s ) , but only one lifetime was obtained for the Eu3+-Datura samples (1213 p s ) . This indicates that some new binding sites inaccessible to Eu3+in free cell material become available for the binding of Eu3+after the immobilization process. Concentration Study of Eu3+ Luminescence in Eu3+-(Immobilized Datura). In a manner similar to the study of Eu3+ bound to the polysilicate matrix, four typical excitation spectra of Eu3+-(immobilizedDatura) are shown in Figure 5. These samples were prepared from different Eu3+solution concentrations (1-0.13 mM at pH 6.0). Like the excitation spectra obtained from Eu3+(silicate polymer) (Figure 3) and Eu3+-Datura (II), the excitation peak positions of the two resolved peaks in Figure 5 are independent of the initial Eu3+concentration. This behavior is suggestive that the dominant binding sites responsible for the binding of Eu3+by immobilized D. innoxia cell wall material do not change within this concentration range of Eu3+. However, unlike the preferential binding behavior observed in Eu3+-(silicate polymer) (Figure 3) and Eu3+-Datura (111,the relative peak intensity ratio between the two resolved peaks in Figure 5 remains nearly unchanged as the concentration of Eu3+ was decreased from 1 to 0.13 mM (Le., the same concentration range used for Eu3+-(silicate polymer) and Eu3+Datura studies). This implies that the Eu3+concentrations used in Figure 5 are too high to observe the competitive binding between two dominant binding sites. This is reasonable because more Eu3+ions have been observed to bind to the immobilized cell material (Figure l),lowering the required Eu3+ concentration range for observing a preferential binding phenomenon. Luminescence Spectral Analysis of Eu3+-(Silicate Polymer), Eu3+-(Immobilized Datura), and E u ~ + Datura. For the purpose of comparison, the excitation spectra of Eu3+-(silicate polymer), Eu3+-(immobilized Datura), and Eu3+-Datura, prepared from 1 mM of Eu3+ solution at pH 6.0, are shown in Figure 6A-C, respectively. Interestingly, the resolved excitation peak position near 579.1 nm in Figure 6A is a t almost the same wavelength as one of the resolved excitation peaks shown in Figure 6B (see the dotted lines). This implies that the polysilicate matrix in the immobilized cell material might be involved 2470 Envlron. Scl. Technol., Vol. 27, No. 12, 1993
IO
0
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Wavelength (nm) Figure 5. Fitted excitation spectra of solid Eu3+-(immobilized Datura) at pH 6.0 with different Eu3+ concentrations of (A) 1.0, (B) 0.66, (C) 0.33, and (D) 0.13 mM (+, data points; indivlduai Lorentzian fitted curves or the sum of the resolved curves) (& = 615 nm).
in the binding of Eu3+. It is not apparent why the excitation peak near 578.2 nm in Figure 6A was not observed in Figure 6B. The excitation peak positions of the two resolved peaks shown in Figure 6B,C are only slightly different. This suggests that the dominant functional groups responsible for the binding of Eu3+are similar for both the immobilized and free D. innoxia cell materials. The corresponding lifetime data shown in Table I (167 and 470ps and 15 and 254 p s , respectively), however, are significantly different.
A
Literature Cited
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(1) Crist, H. R.; Oberholser, K.; McGarrlty, J.; Crist, D. R.; Johnson, J. K.; Brittsan, J. M. Environ. Sci. Technol. 1992, 26, 496-502. (2) Crist, R. H.; Oberholser, K.; Wong, B.; Crist, D. R. Environ. Sci. Technol. 1992,26, 1523-1526. (3) Garnham, G. W.; Codd, G. A.; Gadd, G. M. Enuiron. Sci. Technol. 1992, 26, 1764-1770. (4) Wright, P. J.; Weber, J. H. Enuiron. Sci. Technol. 1991,25, 287-294. (5) Gardea-Torresdey, J. L.; Becker-Hapak, M. K.; Hosea, J. M.: Darnall, D. W. Environ. Sci. Technol. 1990.24. 13721378. (6) Majidi, V.; Laude, D. A., Jr.; Holcombe, J. A. Environ. Sci. Technol. 1990,24, 1309-1312. (7) Harris, P. 0.;Ramelow, G. J. Enuiron. Sci. Technol. 1990, 24, 220-228. (8) Gardea-Torresdey, J.; Darnall, D.; Wang, J. Anal. Chem. 1988,60, 72-76. (9) Kubiak, W .W.; Wang, J.; Darnall, D. Anal. Chem. 1989,61, 468-471. (10) Ke, H. Y.; Birnbaum, E. R.; Darnall, D. W.; Rayson, G. D.; Jackson, P. J. Appl. Spectrosc. 1992, 46, 479-488. (11) Ke, H. Y.; Birnbaum, E. R.; Darnall, D. W.; Rayson, G. D.; Jackson, P. J. Environ. Sci. Technol. 1992, 26, 782-788. (12) Ke, H. Y.; Rayson, G. D. Environ. Sci. Technol. 1992, 26, 1202-1205. (13) Ke, H.Y.; Rayson, G. D. Appl. Spectrosc. 1992,46, 11681175. (14) Ke, H. Y.; Rayson, G. D. Appl. Spectrosc. 1992,46, 13761381. (15) Ke, H. Y.; Rayson, G. D. Appl. Spectrosc. 1993,47,44-51. (16) Ke, H. Y.; Rayson, G. D. Enuiron. Sci. Technol., in press. (17) Darnall, D. W.; Alexander, M. K.; Henzl, M.; Green, D.; Hosea, M.; McPherson, R. W. U.S.Patent No. 4,992,207, Feb 12, 1991. (18) Horrocks, W. D., Jr.; Sudnick, D. R. Acc. Chem. Res. 1981, 14, 384-392. (19) Albin, M.; Horrocks, W. D., Jr. Znorg. Chem. 1985,24,895900. (20) Henzl, M. T.; Birnbaum, E. R. J. Biol. Chem. 1988, 263, 10674-10680. (21) Hapak, R. C.; Lammers, P. J.; Palmisano, W. A.; Birnbaum, E. R.; Henzl, M. R. J . Biol.Chem. 1989,264,18751-18760. (22) Treviiio, C. L.; Palmisano, W. A.; Birnbaum, E. R.; Henzl, M. T. J . Biol. Chem. 1990,265,9694-9700. (23) Palmisano, W. A.; Treviiio, C. L.; Henzl, M. R. J.Biol. Chem. 1990,265, 14450-14456. (24) Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa, S.; Singh, R. M. M. Biochemistry 1966,5, 467-477. (25) Good, N. E.;Izawa, D. Methods Enzymol. 1968,24B, 53-68. (26) Rayson, G. D.; Shen, D. Y. Appl. Spectrosc. 1992,46,12451250.
578.5
579.5
580'5
Wavelength (nm) Figure 6. Fitted excitation spectra of solid (A) Eu3+-(silicate polymer), (B) Eu3+-(lmmobilized Datura),and (C) Eu3+-Datura, preparedfrom 1 mM Eus+solution at pH 6.0 (+, data polnts; individual Lorentzian fitted = 615 nm). curves or the sum of the resolved curves) ,A,(,
These differences could be indicative of an alteration of the chemical environments associated with the two dominant binding sites in immobilized and free cell materials during the immobilization process. Acknowledgments
Financial support by the U.S.Department of Energy through the New Mexico Waste-Management Education and Research Consortium is gratefully acknowledged.
,
Received for review February 11, 1993. Revised manuscript received July 26, 1993. Accepted August 3, 1993.' ~~~~~
~
* Abstract published in Advance ACSAbstracts, October 1,1993.
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