Environ. Sci. Technol. 2000, 34, 4123-4127
Use of Isotopic Dilution Techniques To Assess the Mobilization of Nonlabile Cd by Chelating Agents in Phytoremediation KATRENA G. STANHOPE, SCOTT D.YOUNG,* JULIAN J. HUTCHINSON, AND ROOPA KAMATH University of Nottingham, School of Biological Sciences, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD U.K.
Metals are unavailable to plants when fixed within relatively inert mineral matrices in soil. The success of phytoremediation may be limited by restricted bioavailability of metals in such nonlabile forms. We conducted a pot trial with EDTA added at rates of 0, 0.1, 1.0, 5.0, and 10 mmol kg-1, to soil historically contaminated with sewage sludge to increase the availability of metals to a test crop, Indian Mustard (Brassica juncea). The soil was found to have large total concentrations (mg kg-1) of Cd (59.3), Cu (664), Pb (605), Ni (489), and Zn (1780); the EDTA-extractable metal contents (mg kg-1) were Cd (36.2), Cu (483), Pb (302), Ni (372), and Zn (1280). Isotopic dilution of 109Cd was used to measure labile Cd both (i) chemically (the E value: 28.3 mg Cd kg-1) and (ii) biologically (the L value). Comparison of E and L values was then used to determine whether nonlabile Cd was mobilized by the chelate treatment. Addition of EDTA increased plant uptake of Cd, Zn, Pb, Ni, and Cu from sludge-amended soil but did not facilitate access to the nonlabile pool of Cd in the soil; E values and L values were similar across the range of EDTA applications. Thus it was apparent that the small EDTA concentrations applied in phytoremediation to increase metal solubility are probably insufficient to mobilize fixed forms of Cd.
Introduction Conventional remediation of contaminated land can be relatively inefficient and costly and may cause local disruption risking greater contaminant exposure (1, 2). Proponents of phytoremediation techniques suggest that “phytoextraction” of soil metals may be a solution to these problems as the technique can be applied in situ and, in some circumstances, may offer cost savings over conventional forms of land remediation (3). Phytoremediation methods are still in the early stages of development, but a search of the World Wide Web suggests a rapidly growing interest within both the scientific and commercial communities. One approach to phytoremediation is to use plants that produce large amounts of biomass and to increase the solubility and translocation of metals by adding chelating agents to the soil. For example, Brassica juncea L. (Indian * Corresponding author phone: 44(0)115 9516256; fax: 44(0)115 9516261; e-mail:
[email protected]. 10.1021/es0010812 CCC: $19.00 Published on Web 08/31/2000
2000 American Chemical Society
Mustard) has been used successfully to remove cadmium, chromium, copper, nickel, lead, and zinc from soil (4). However, restricted bioavailability of metals in contaminated soils limits the efficiency of phytoextraction (5). Metal bioavailability may be limited by occlusion within relatively insoluble mineral matrices in soil (6). Metals in this form are “nonlabile” and are not isotopically exchangeable with metal ions in the soil solution. The proportion of nonlabile metal may be dependent upon the origins of the metal contaminant, its residence time in the soil, and soil conditions such as pH (7). It might be argued that the fraction of soil metal accessed by plants is, by definition, the true bioavailable pool and that removal of this fraction is all that should be required for successful remediation. However, leaving aside the issue of the time required to abstract this portion of soil metal there are also several reasons why this goal may not be sufficient. These include (i) the practical issue of dealing with regulation standards which are often based upon total, rather than bioavailable metal; (ii) the possibility that long-term transfers from the nonlabile to labile pools may occur following removal of the labile metal pool; and (iii) the fact that the “soil f plant f human” pathway may not be the only important route for contaminant transfer and the risk associated with direct inhalation and ingestion of soil metal may still exist following removal of plant-available metal. Thus, promoting the solubility of metals, including the nonlabile forms, and facilitating their transport to the shoots of plants may be important to the wider success of phytoremediation. It is well-known that metal solubility and bioavailability can be increased by the application to soil of synthetic chelating agents such as ethylenediaminetetraacetic acid (EDTA) in the range 0.1-10 mmol kg-1 (4, 8-13). There is also evidence from soil extraction experiments (0.05 M EDTA) to suggest that relatively large concentrations of EDTA (approximately 250 mmol kg-1) are able to solubilize metal ions from pools which are not otherwise isotopically exchangeable, possibly by mineral dissolution (14). However, it is not known whether there is substantial release from the nonlabile fraction of the soil metal at the levels of chelate addition commonly used in phytoremediation studies. The aim of this study was to assess the extent of mobilization of nonlabile Cd caused by EDTA application to a soil contaminated by long-term disposal of sewage sludge. We compared two assays of radio-labile Cd, measured (i) by isotopic dilution in an equilibrated soil suspension, the E value, and (ii) as “bioavailable” Cd determined from the specific activity of plants grown in soil spiked with 109Cd, the L value. Techniques for determining E and L values for Cd, [CdE], and [CdL] have been reported previously (14-16). Both [CdE] and [CdL] are measures of the soil labile Cd pool and so might be expected to be equal. However, a ratio of [CdL]: [CdE] greater than unity may suggest that the plant is able to access nonlabile metal. For example, if EDTA dissolves nonlabile Cd, then its addition would cause greater isotopic dilution of added 109Cd and result in a lower specific activity of 109Cd in plant material than expected from the E value. Thus the extent of nonlabile Cd uptake by plants can be quantified by comparing [CdE] and [CdL]. Two assumptions underpin this comparative analysis: (i) that 109Cd added to the soil equilibrates fully with labile Cd in the soil and (ii) that there is no fixation of 109Cd (transfer to nonlabile forms) during the course of the growth trial. We applied five EDTA treatments (0, 0.1, 1, 5, 10 mmol kg-1) to a contaminated soil spiked with 109Cd, and B. juncea VOL. 34, NO. 19, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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was grown as a simulation of phytoremediation practice. Soil and plant samples were analyzed for 109Cd, Cd, Pb, Zn, Cu, and Ni. The E and L values were measured to determine the extent of transfer of fixed Cd to the labile pool as a result of the EDTA treatments.
Experimental Section Topsoil was taken from a designated sewage disposal farm in Nottinghamshire, U.K. which has received sewage sludge for nearly 100 years. Soil was sampled to a depth of 15 cm, air-dried, sieved to < 4 mm, and thoroughly mixed. Soil pH was measured in deionized water using a solid:liquid ratio of 1:2.5 after equilibration for 2 h. The organic matter content was estimated by loss on ignition at 550 °C for 8 h (17) and cation exchange capacity (CEC) by the exchangeable base method (18). Total soil metal contents were measured by flame-atomic absorption spectrometry (FAAS) following digestion of 1 g of finely ground oven-dried soil in 25 mL of concentrated HNO3. EDTA-extractable metal was determined by extraction of 5 g of soil with 25 mL of 0.05 M EDTA for 48 h. Total nitrogen was determined using an NA-2000 N analyzer. Bicarbonate-extractable phosphate was measured by extraction with 0.5 M NaHCO3, and the CaCO3 content was determined from CO2 evolution using a Collin’s Calcimeter. All soil measurements were made in triplicate. Determination of “Plant-Available” Cd in Soil, [CdL]. Sieved soil (field moist) was spiked with carrier-free 109Cd in 0.1 M Ca(NO3)2 (15 mL kg-1) and mixed for five minutes in a food mixer to give a final activity of approximately 250 kBq kg-1. Moist soil, equivalent to a dry weight of 130 g, was then placed in plastic pots (9 cm diameter at top) to give a soil column of 6 cm height and 6 cm diameter, tapering to 5 cm diameter at the base of the pot. The moisture content was increased to 54% (mL g-1 soil) with deionized water. Twentyfive pots were organized in a randomized block design and left to equilibrate in darkness in a controlled environment growth room for one week before sowing plant seeds. Seeds of Brassica juncea were placed just below the soil surface, at a sowing density of five per pot. To reduce evaporative loss, 4 g of “perlite” was added to the surface of each pot. Plants were grown in a controlled environment growth room (15/ 20 °C, 8 h dark/16 h light with an intensity of around 520 ( 30 µmol photons m-2 s-1). Moisture content was maintained by returning the pots to their original weight on a daily basis using deionized water. Two weeks after sowing 20 mL of KNO3 (0.2 M) was added to each pot as fertilizer; with a soil surface area of 28.3 cm2 this was intended to be equivalent to an N application of 200 kg ha-1. Three weeks after sowing the plants were thinned to two per pot. Four weeks after sowing, EDTA (disodium salt) was added to the 25 pots at concentrations of 0 (control), 0.1, 1, 5, and 10 mmol kg-1 to give five replicates of each treatment. The shoots were harvested two weeks after EDTA treatment by cutting them 1 cm above the surface. The entire shoot material from each pot was dried at 80 °C for 72 h, weighed, and digested in concentrated HNO3 (25 mL) prior to analysis for 109Cd (γ-spectrometer) and stable Cd (FAAS). The L value, [CdL], was determined from
[CdL] )
[Asoil][Cdplant] [Aplant]
(1)
where [CdL] is the labile or bioavailable Cd (mg kg-1), [Asoil] and [Aplant] are the gravimetric activities of 109Cd (Bq kg-1) added to the soil and measured in the plant respectively, and [Cdplant] is the Cd concentration in the plant tissue (mg kg-1). In practice, [Asoil] was determined from radio-assay of the stock solution used to spike the soil and was measured at the same time as the plant digests. 4124
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TABLE 1. Total and EDTA-Extractable Metal Content of the Sewage Sludged Soila metal
Cd
Cu
Pb
Ni
Zn
extractable (0.05 M EDTA) total (HNO3 digest)
36.2 (0.40) 59.3 (1.2)
483 (4.8) 664 (13)
302 (3.5) 605 (3.0)
372 (3.1) 489 (3.4)
1280 (27) 1780 (28)
a Standard errors are shown in parentheses, n ) 3. All figures are in mg kg-1.
Determination of E Value, [CdE]. Four replicate soil samples of approximately 5 g were shaken with 25 mL of 0.1 M Ca(NO3)2 for 5 days and then centrifuged at 2200g for 30 min. Five milliliter samples of the supernatant were added to an equal volume of 5% HNO3, and the concentration of Cd was determined by graphite furnace-AAS (GFAAS). An aliquot (0.2 mL) of 109Cd in 0.1 M Ca(NO3)2 solution, of approximately 12 kBq mL-1 activity, was added to the soil suspensions, and these were shaken for a further 48 h. After centrifugation, 4 mL was removed and assayed (γ-spectrometer) for 109Cd. It has been shown in previous work (7) that such small differences (48 h) in initial equilibration and radio-isotope contact time do not affect the value of [CdE]. Simultaneous assay of the isotope stock solution enabled the determination of the distribution coefficient, for 109Cd, within the soil suspensions (kd*). The concentration of labile Cd, [CdE], was calculated as
(
[CdE] ) [Cdsol] kd* +
V W
)
(2)
where [CdE] is the concentration of labile soil Cd (mg kg-1), [Cdsol] is the concentration of Cd in solution (mg L-1), kd* is the isotopic distribution coefficient (L kg-1), and V and W are the solution volume (L) and the mass of dry soil (kg). Determination of Cd Extractable by 1 M CaCl2, [CdCl], and EDTA, [CdEDTA]. Four replicate soil samples of approximately 5 g were shaken with 25 mL of 1.0 M CaCl2 for 7 days and then centrifuged at 2200g for 30 min. Samples of the supernatant (5 mL) were added to an equal volume of 5% HNO3, and the concentration of Cd was determined by FAAS or GFAAS. The value of [CdCl], (mg kg-1), was determined as
(WV )
[CdCl] ) [Cdsol]
(3)
A similar approach was used to determine metal extraction with EDTA (e.g. [CdEDTA]) at concentrations of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 mmol EDTA kg-1 soil and also with a standard 0.05 M EDTA extraction, equivalent to 250 mmol EDTA kg-1 at the ratio of soil:solution used.
Results and Discussion Soil Characteristics and Metal Extraction. The soil had several characteristics commonly associated with extended applications of sewage sludge. The CEC was large (71.4 cmolc kg-1) with an organic matter content (LOI) of 24.8% and a bicarbonate-extractable phosphate content of 143 mg P kg-1. The P content is approximately 5-10 times the “critical OlsenP” level found for a range of arable soils in different countries (19). The pH, 6.3, was close to the management optimum, and there was a measurable CaCO3 content, 2.3%. Table 1 shows the amounts of metal extracted by HNO3 digestion and equilibration with 0.05 M EDTA. The soil showed considerable enrichment in the five metals determined. In particular, the concentration of Cd was twenty times the U.K. statutory limit for agricultural land receiving sewage
FIGURE 1. Extraction with EDTA of Cd (O), Ni (]), Cu (4), Zn (0), and Pb (b) from soil historically contaminated with sewage sludge. The amount of metal extracted is shown as a proportion (%) of “total” soil metal, determined by HNO3 digest, and as a function of EDTA concentration (mmol kg-1 soil). The solid line is derived from linear regression of the aggregated data for Cd, Ni, Cu, and Zn: y ) 1.76x + 2.28; r ) 0.98. sludge (3.0 mg kg-1). The sampling site is designated specifically for sludge disposal and is therefore subject to various management restrictions but not to the same limits for soil metal content as normal arable land. Figure 1 shows the variation in metal solubility with EDTA concentration following batch equilibration at a soil:solution ratio of 1:5 (w/v). There was a consistent linear relationship between the proportion of soil metal dissolved (% total) and the concentration of EDTA (mmol kg-1) for Cd, Ni, Cu, and Zn; no approach to an asymptote was apparent within the range of EDTA concentrations used. The aggregate slope for these four metals, 1.76, suggests that each mmol kg-1 of added EDTA released just under 2% of the soil metal content. However, this relationship is unlikely to apply universally. Solubility induced by chelation is a product of several metalspecific factors including metal-chelate affinity, competition from other metals and protons, and strength of (metal) adsorption on soil. Thus, the similar trend shown for Cd, Ni, Cu, and Zn may be coincidental. The solubility of Pb followed a markedly different pattern (Figure 1) with considerably lower solubility across the range of EDTA concentrations used. The comparatively low solubility of Pb in the sludged soil may have resulted from low dissolution kinetics of Pbphosphate compounds, considering the exceptionally large phosphate content of the soil. Lead readily forms relatively insoluble Pb-phosphate compounds which can limit availability (20). The radio-labile Cd content, [CdE], of the sludge-amended soil was 28.3 ( 0.50 mg kg-1 which was equivalent to 47.7% of the total soil Cd, [Cdtotal]. Thus almost half of the Cd in the sludged soil was present in the labile pool prior to EDTA application. The accuracy of measurement of [CdE] was confirmed by extraction with 1 M CaCl2 (7), and the results were found to agree closely with E values: [CdCl] was 27.1 ( 1.1 mg kg-1, 96% of [CdE]. Shoot Growth and Development. B. juncea sown in the sludged soil grew rapidly. Plants had flowered by the fourth week and had started to develop seedpods at the time of harvesting. Two or 3 days following EDTA application (week 4 of plant growth), plants exhibited chlorosis and premature moulting of basal leaves, especially at the larger EDTA concentrations of 5 and 10 mmol kg-1. These leaves were collected, dried, and analyzed with the rest of the plant material. At harvest very few older leaves were left on plants treated with 5 and 10 mmol EDTA kg-1, and these plants
FIGURE 2. Increase in metal content of Brassica juncea grown in sewage-sludged soil treated with EDTA over control values as a function of metal extracted by EDTA from soil: Cd (O), Ni (]), Cu (4), Zn (0), and Pb (b). The EDTA extractions were at a suspension ratio of 0.2 g mL-1 and at EDTA loadings equivalent to the treatments used in the pot experiment (0, 0.1, 1, 5, and 10 mmol EDTA kg-1). The value of r2 for correlation of log values was 0.77. exhibited thin, etiolated growth. The mean dry weight yields (g pot-1) for the 5 EDTA treatments (0, 0.1, 1.0, 5.0 and 10 mmol EDTA kg-1) were, respectively, 1.64 (0.14), 2.1 (0.46), 1.43 (0.07), 1.40 (0.09), and 1.28 (0.11); standard errors for the 5 replicates are shown in brackets. Uptake of Metals by B. juncea. Most of the increased uptake of metal with EDTA treatments of 0-10 mmol kg-1 could be explained as a proportional effect of enhanced metal solubility. Figure 2 shows the increase in metal uptake over the control plants as a function of the amount of metal extracted by equilibration with EDTA at concentrations (mmol kg-1) equivalent to the treatments used in the pot trial. The latter were determined by interpolation of the data in Figure 1 for individual metals when there was no direct equivalent value for the concentration of EDTA used in the pot experiment. The increased metal concentrations in the plant were calculated by subtraction of the mean metal concentrations in control plants. Data were not included if they fell within 5% of the control plants: 2 data for Pb and 1 for Cd. If the assimilation of metal from EDTA-treated soils is dominated by passive uptake from the soil solution and root to shoot translocation in the transpiration stream (4, 21), then plants might be expected to extract additional metal in proportion to the amount solubilized by the EDTA treatment. Figure 2 suggests that this may broadly apply, although some differences between individual metals were apparent (r2 ) 0.77). Effect of EDTA on the Bioavailable Soil Cd Pool. Figure 3 shows the effect of EDTA treament on the soil Cd indices measured in this study, including L value, E value, 1 M CaCl2extractable Cd, EDTA-extractable Cd (0.05 M and five specific treatments), and total soil Cd (HNO3 digest). The L values measured for B. juncea indicate that the amount of bioavailable soil Cd was very consistent over the five different EDTA treatments (P > 0.05), with an average of 26.1 ( 0.51 mg kg-1 which is equivalent to 44.0 ( 0.86% of total soil Cd. This was very close to the percentage E value (47.7% of Cdtotal) and to the amount of Cd extractable by 1 M CaCl2 (45.7% of Cdtotal). Thus, both the chemical isotopic dilution technique (E value) and extraction with 1 M CaCl2 were found to be reliable estimates of the bioavailable Cd pool in sludge-treated soil (the L value). Neither the “total” soil Cd determined by HNO3 digestion [Cdtotal] nor Cd extracted by 0.05 M EDTA [CdEDTA] correctly determined the bioavailable pool of Cd due to dissolution of nonlabile Cd. It should be noted that VOL. 34, NO. 19, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Effect of EDTA treatment on L value ([CdL], 0) and on Cd solubility (O). Several chemical assays of soil Cd are shown for comparison, including HNO3 digest [Cdtotal], E value [CdE], and extraction with 0.05 M EDTA [CdEDTA] or with 1 M CaCl2 [CdCl]. The EDTA extractions (O) were at a suspension ratio of 0.2 g mL-1 and at EDTA loadings equivalent to the treatments used in a pot experiment (0, 0.1, 1, 5, and 10 mmol EDTA kg-1).
FIGURE 4. Ratio of Cd L value to E value, [CdL]/[CdE], as a function of Cd uptake by Brassica juncea. these findings are not contrary to studies which demonstrate a good correlation between Cd uptake and solubility in 0.05 M EDTA solution across a range of soils and/or contaminant levels (e.g. refs 22 and 23). In this study the objective was to assay the total bioavailable pool and not predict the amount removed by a single crop under prescribed growing conditions. Figure 3 also shows the effect of varying EDTA solution concentration upon the solubility of soil Cd. Even the largest EDTA concentration used in the soil-metal equilibration experiment was unable to solubilize the total bioavailable Cd pool. An extraction concentration of 10 mmol EDTA kg-1 only mobilized approximately 50% of the bioavailable Cd, as indicated by the L value. Furthermore, the consistent L values across the range of EDTA treatments in Figure 3 also show that even the largest EDTA application (10 mmol kg-1) was unable to mobilize any nonlabile Cd. If solubilization of nonlabile and labile Cd had occurred concurrently, even at different rates, the L value would have increased with EDTA application. Similarly, Figure 4 shows that ratios of L:E values for individual pots were (i) close to unity and (ii) independent of the total amount of Cd uptake by B. juncea. The latter point clearly shows that increased uptake of Cd does not involve increased access to the nonlabile Cd pool but simply signifies greater uptake from the labile reservoir of soil Cd. Again, this supports the credibility of the L value as a fundamental characteristic of the soil, independent of the bioassay and growing conditions used in its determination. The latter finding is in accord with similar studies comparing E and L values for Cd uptake by the Zn hyperaccumulator 4126
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Thlaspi caerulescens (24) and Ni uptake by red clover (Trifolium pratense (25)). However, this contrasts with a study of Cd uptake by wheat (Triticum aestivum (26)) where small differences in [CdE] and [CdL] were detected. Hamon et al. (27) evaluated [CdL] and [ZnL] for seven plant species and found a significantly different value of [CdL] in one case. It is possible that L values are a unique function of soil-plant combinations and, for some species of plant, may differ from E values due to processes such as release of rhizo-genic chelates or alteration of rhizosphere pH. More work is needed to resolve these contrasting observations and their underlying causes. Implications for Phytoremediation. The lack of significant difference in L values between treatments shown in Figures 3 and 4 suggests that phytoremediation using B. juncea with soil EDTA application is not an effective way to access nonlabile pools of Cd in sludged soils. Whether this is a serious limitation to phytoremediation depends on the objectives of the remediation program and the relative importance of different pollutant transfer pathways in individual situations. It is important to note that our findings may not apply generally and have been derived for one soil with several characteristics which distinguish it from “normal” agricultural soils (e.g. humus content and phosphate content). However, in previous studies on Cd phytoavailability in sludge-treated soil it has also been suggested that a significant proportion of Cd may be fixed or occluded (28). In the soil used here it is likely that the nonlabile Cd (around 52% of total Cd) is occluded within calcium phosphates given the exceptionally large available P concentration (143 mg kg-1). Although the EDTA treatments used here (0-10 mmol kg-1) were apparently unable to mobilize this nonlabile Cd, extraction with 0.05 M EDTA, equivalent to 250 mmol kg-1, clearly dissolved some fixed Cd ([CdEDTA] > [CdL], Figure 3). However, in practice the concentrations of EDTA needed to mobilize nonlabile Cd in soils might pose problems of excessive phytotoxicity, expense, and leaching of metals to groundwater (13). Galiulina and Galiulin (5) showed that even at concentrations of 1 and 5 mmol kg-1 of soil-applied EDTA, significant increases in water-soluble Zn, Pb, and Cd concentrations were found 2 months after application. EDTA may be degraded over time by rhizobacteria (12), but biodegradation may be quite slow leaving persistent EDTA residues in the environment (29).
Acknowledgments The authors wish to thank Dr. Steve McGrath (IACR Rothamsted, U.K.) for supplying the B. juncea seeds.
Literature Cited (1) Brown, S. K. Bioscience 1995, 15 Oct., 79-582. (2) Baker, A. J. M.; McGrath, S. P.; Sidoli, C. M. D.; Reeves, R. D. Conservation Recycling 1994, 11, 41-49. (3) Moffat, A. S. Science 1995, 269, 302-303. (4) Blaylock, M. J.; Salt, D. E.; Dushenkov, S.; Zakharova, O.; Gussman, C.; Ensley, B. D.; Raskin, I. Environ. Sci. Technol. 1997, 31, 860-865. (5) Galiulina, R. R.; Galiulin, R. V. Proc. 5th Intern. Conf. On The Biogeochem. Of Trace Elements; Vienna, 1999; pp 906-907. (6) McBride, M. B. Adv. Soil Sci. 1989, 10, 1-56. (7) Young, S. D.; Tye, A.; Carstensen, A.; Resende, L.; Crout, N. Eur. J. Soil Sci. 2000, 51, 1-8. (8) Ebbs, S. D.; Kochian, L. V. Environ. Sci. Technol. 1998, 32, 802806. (9) Epstein, A. L.; Gussman, C. D.; Blaylock, C. D.; Yermiyahu, U.; Huang, J. W.; Kapulnik, Y.; Orser, C. S. Plant Soil 1999, 208, 87-94. (10) Huang, J. W.; Cunningham, S. D. New Phytol. 1996, 134, 75-84. (11) Lambrecht, S.; Biester, H.; Haag-Kerwer, A. Proc. 5th Intern. Conf. On The Biogeochem. Of Trace Elements; Vienna, 1999; pp 876-877. (12) Vassil, A. D.; Kapulnik, Y.; Raskin, I.; Salt, D. E. Plant Physiol. 1998, 117, 447-453.
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(22) Pandeya, S. B.; Singh, A. K.; Jha, P. Plant Soil 1998, 203, 1-13. (23) Hooda, P. S.; McNulty, D.; Alloway, D.; Aitken, B. J. J. Sci. Food Agric. 1997, 73, 446-454. (24) Hutchinson, J.; Young, S. D.; McGrath, S. P.; West, H. M.; Black, C. R.; Baker, A. J. M. New Phytologist 2000, 146, 453-460. (25) Echevarria, G.; Klein, S.; Fardeau, J.-C.; Morel, J.-L. Ge´ochime 1997, 324, 221-227. (26) Smolders, E.; Brans, K.; Fo¨ldi, A.; Merkx, R. Soil Sci. Soc. Am. J. 1999, 63, 78-85. (27) Hamon, R.; Wundke, J.; McLaughlin, M.; Naidu, R. Australian J. Soil Res. 1997, 35, 1267-1277. (28) Hyun, H.; Chang, A. C.; Parker, D. R.; Page, A. L. J. Environ. Qual. 1998, 27, 329-334. (29) Li, Z. B.; Shuman, L. M. Soil Sci. 1996, 161, 226-232.
Received for review March 8, 2000. Revised manuscript received June 30, 2000. Accepted July 6, 2000. ES0010812
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