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Dep. de Química y Análisis Agrícola, E.T.S.I. Agrónomos, Univ. Politécnica de Madrid, Ciudad Universitaria, s/n, 28040 Madrid, Spain
* Corresponding author (jmalvarez{at}qaa.etsia.upm.es)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: AAS, atomic absorption spectrophotometry • DTPA, diethylenetriaminepentaacetate • EDTA, ethylenedinitrilotetraacetate • HEDTA, N-2-hydroxyethyl-ethylenedinitrilotriacetate • Zn-AA, Zn-amino acids • Zn-CH, Zn-DTPA-HEDTA-EDTA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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In calcareous soils, the availability of Zn is largely governed by soil pH, type of soil minerals, kind and amount of anions in the soil solution, and Zn carriers (Sharpless et al., 1969; Shuman, 1975; Lins and Cox, 1988; Maftoun and Karimian, 1989; Thind et al., 1990). Metal-chelates used in soil application must be resistant to microbiological decomposition and not easily precipitated by ions or colloids in soils. Bolton et al. (1993) used soil samples to determine the biodegradation of synthetic chelates and they found that the relative order of chelate persistence was EDTA > DTPA > nitrilotriacetate. Adriano (1986) reported that some cations, such as Zn2+, could enter into the crystal lattice of layer silicates through isomorphous substitution or solid-state diffusion into the crystal structure. This process may be irreversible so that some applied metals may be irreversibly fixed by clay. The chelating agents DTPA, HEDTA, and EDTA are some of the strongest synthetic chelating agents and form much stronger chelates with Zn than naturally occurring organic ligands (Norvell, 1983). According to Dwyer and Miller (1964), the long-chain natural organic compounds, such as Zn-AA, are intermediate in chelating strength. Thus, amino acids can supply Zn effectively when applied to a soil, although at a slightly higher rate of application than is required for chelates (Hsu, 1986). The continuous application of large amounts of Zn chelates to soil has caused concern regarding the possible accumulation of trace elements and potential harm to the environment, as Zn can be transported downward in soil and possibly deteriorate ground water quality (Kiekens, 1995; Li and Shuman, 1997a). The uptake of Zn by maize is markedly influenced by its diffusion rate to the absorbing roots (Prasad and Sinha, 1981). In general, chelating agents, such as DTPA, HEDTA, and EDTA have been shown to contribute largely to Zn movement in soil under conditions of excessive rainfall or irrigation (Adriano, 1986; Modaihsh, 1990; Alvarez et al., 1996b). This movement may produce long-term Zn deficiency in soil and may complicate the study of the behavior of different Zn sources (Mikkelsen and Brandon, 1975; Mikkelsen and Kuo, 1977; Kiekens, 1995). In this way, Goos et al. (2000) showed that Zn-EDTA was not in the long-term a superior source for maize than ZnSO4 and Zn-humate-lignosulfonate in calcareous soil. In addition, chelation of Zn2+ by chelating agents decline in time in calcareous soils, as Zn2+ is slowly replaced on the ligand by Ca2+ (Lindsay, 1979).
The DTPA and Mehlich-3 extractions are methods usually employed to diagnose Zn bioavailability for plant uptake (Reed and Martens, 1996). However, the study of the Zn distribution in various soil fractions provides a better understanding of Zn behavior in relation to the transformation of Zn added to soil (Iyengar et al., 1981). Viets (1962) distinguished the following pools: water soluble—the fraction present in the soil solution; exchangeable—ions bound to soil particles by electrical charges; adsorbed, chelated, or complexed metals—bound to organic ligands; clayey secondary minerals and insoluble metallic oxides; and primary minerals. Although it is relatively easy to conceptually partition an element into different pools in soils, such fractionation is fraught with difficulties, at least with regard to some extractants because of overlapping selectivity. The magnitude of this problem usually increases with the stage in the sequential extraction procedure. For example, the acidification to assess carbonate-bound Zn and oxidation of organic matter with NaOCl can dissolve Mn oxide (Shuman, 1985). Although hydroxylamine hydrochloride appears to be an efficient extractant of manganese oxides in sediments (Chao, 1972), its efficiency with agricultural soils appears to be considerably lower due to nonsignificant correlation between the Mn-oxide-bound Zn fraction and Zn uptake by plants (Shuman, 1986). These topics have been reviewed by Pickering (1986). Despite their severe limitations, sequential extraction procedures have been commonly used to characterize Zn forms. According to McBride (1989), the speciation of metals indicate that organic acids that are good chelating ligands which could explain the higher levels of metal complexation observed in soil solutions, as could N-containing ligands, such as amino acids. The water soluble plus exchangeable metal fraction characterizes the most mobile and immediately bioavailability forms (Li and Shuman, 1997b). They are the most labile metal forms in the soil environment and have greater leaching potential than the other forms (Shuman, 1991).
The objectives of this study were to determine (i) the main chemical forms of Zn applied to a calcareous soil by using a soil incubation experiment with two different types of Zn fertilizers (amino acid complex vs. chelate), (ii) the migration and distribution of Zn in the soil profile and the losses of Zn by leaching via a soil column study, (iii) and the effectiveness of the two selected Zn organic complexes by growing a crop of maize in a greenhouse study.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Extraction Procedures
Fractionation of Zn in the soil was performed by the techniques proposed by Chao (1972), Tessier et al. (1979), Shuman (1985), and Mandal et al. (1992) with slight modifications. The fractions were determined sequentially in seven steps (F1 through F7) with the following extractants: F1, 1 M Mg(NO3)2, pH 7.0, extractable (water soluble plus exchangeable); F2, 0.7 M NaOCl, pH 8.5, extractable (organically complexed); F3, 1 M NaOAc, pH 5.0, extractable (carbonate bound); F4, 0.1 M NH2OH · HCl, pH 2.0, extractable (Mn oxide bound); F5, 0.2 M (NH4)2C2O4 + 0.2 M H2C2O4, pH 3.0, extractable (amorphous Fe oxide bound); F6, solution as in the previous step plus 0.1 M ascorbic acid, extractable (crystalline Fe oxide bound); and F7, determined by using microwave digestion for the sample remaining from Step 6 after air drying and grinding, 2 g soil residue:12 mL acid mixture (2mL HCl, 5 mL HNO3, and 5 mL HF) (residual Zn).
Relative Zn available to the plant was assessed by extracting it with DTPA (5 mM DTPA + 0.01 M CaCl2 + 0.1 M triethanolamine, adjusted to pH 7.3) (Lindsay and Norvell, 1978); and Mehlich-3 (0.2 M HOAc + 0.25 M NH4NO3 + 0.015 M NH4F + 0.013 M HNO3 + 1 mM EDTA) (Mehlich, 1984). The concentration of Zn was determined by atomic absorption spectrophotometry (AAS).
Soil Characterization
The representative soil came from Loeches in the region of Madrid, Spain. Surface soil was taken from the Ap horizon (depth 0–27 cm); samples were air-dried and sieved, and the <2-mm fraction was used for the study. Soil properties were: texture (USDA)—clay loam; clay, 280 g kg-1 (Day, 1965); permeability—moderate (Monturiol and Alcalá, 1990); pH—8.3 (1:2.5 w/v); organic matter—9.1 g kg-1 (Jackson, 1964); total N—0.9 g kg-1 (Bremner, 1996); available P— 34.9 mg kg-1 (Olsen et al., 1954); cation exchange capacity—1.3 cmol+ kg-1 (Bower et al., 1952); base saturation—100%; and total and free CaCO3—205.8 g kg-1 (very calcareous soil) and 24.2 g kg-1, respectively (Nijensohn and Pizarro, 1960; Allison and Moodie, 1965). The soil classification is Typic Xerorthents (Soil Survey Staff, 1998). The predominant clay in the soil was montmorillonite, as determined by electroultrafiltration (Wiklicky and Nemeth, 1981).
The speciation of the original soil indicated that the great majority (>93%) of Zn was present in the F7 form (Table 1) and the second most abundant form was F5 at 
5% of the total. Diethylenetriaminepentaacetate and Mehlich-3 extractable Zn are shown in Table 2. These soil test levels are commonly known to result in Zn deficiency in maize, especially in alkaline soils (Tran and Simard, 1993).
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Column Experiment
Transparent Plexiglas columns were employed to study the mobility of the two Zn complexes in soil. The soil was packed to a height of 27 cm in 40-cm-long columns with an i.d. of 7.2 cm. Soil columns were built and filled with gravel and two filter papers (Whatman no. 4) at the bottom. These were protected from excessive lateral desiccation by aluminum foil to avoid direct solar radiation. The packed soil with a bulk density of 1.1 Mg m-3 was saturated from below with deionized water. The water was added by means of a capillary tube in the center of the column. Excess water was allowed to drain overnight. The soil from the top to a depth of 1.5 cm in each column was mixed with the respective amendments. The Zn applied in these treatments was 20 mg kg-1 (24 mg of Zn were applied to 1.2 kg of soil in each column). Unamended soil columns were also included in the experiment as control treatments. Three replicate columns were installed at room temperature (18–23°C) for each fertilizer, and experimental time (30 and 60 d) combination. Deionized water was added to the top of each column daily at 30 mL d-1. Leachate was collected in 150-mL fractions (0.235 pore volume) for a total of 1200-mL (1.88 pore volume), which was 60 d. The leachates were acidified with HCl, filtered, and the concentration of Zn was measured in each leachate portion by AAS.
After leaching, the two halves of the columns were separated along the longitudinal axis. The soil columns were air-dried, divided transversally into various portions, and ground to pass a 2-mm sieve. This study was conducted as described by Rico et al. (1995) and Alvarez et al. (1996b). Three layers of the columns were separately analyzed (sequential extraction, DTPA, and Mehlich-3 extractable Zn) and their weights and depths were as follows: Zone a, 312 g (0–7 cm); Zone b, 444 g (7–17 cm); and Zone c, 444 g (17–27 cm).
Greenhouse Pot Experiment
Samples of 8 kg of air-dried soil placed in polyethylene pots with washed gravel at the bottom to facilitate aeration and drainage. The soil received 0, 10, and 20 mg kg-1 of Zn applied as Zn organic sources. In addition to the Zn treatment, a basal dressing of 150 mg N kg-1 in the form of urea (46% N), 75 mg P kg-1 in the form of superphosphate (18% P), and 75 mg K kg-1 in the form of K2SO4 (42% K) were applied uniformly to all pots. Three seeds of a double hybrid maize (A-33, ASGROW Seed Co., Madrid, Spain) were sown in each pot. This variety of maize is used as fodder. The remaining half of the N was added in two equal doses 7 and 30 d after sowing the seeds. Each treatment was replicated thrice. The pots were taken to a greenhouse with average day and night temperatures of 42 and 16°C, respectively, and appropriate amounts of water were added daily to reach and approximately maintain field capacity moisture conditions with limited drainage (35.7% w/w). Forty-five days after seeding, the end of the maximum plant growth period, the part aboveground was cut, washed with tap water, and then rinsed with deionized water for 5 sec. The plant parts were dried at 65°C to a constant weight. Once weighed, they were ground and kept in sealed containers for later analysis. Each of these plant samples was subjected to wet digestion in a microwave oven (programmed in three stages with a maximum pressure of 1173 x 103 Pa) using an acid mixture (HCl + HNO3 + HF) 1:14, plant (g):solution (mL). Zinc was analyzed by AAS in the resulting solutions. Soil Zn was determined after harvesting the maize crop as outlined previously.
Statistical Analysis
Correlation analysis and other statistical studies were conducted with Statgraphics Plus software, version 5.0 (Manugistic, Inc., Rockville, MA). Multiple comparisons of variables were made by using Duncan's separation of means procedure. A probability level of P 
0.05 was chosen to establish statistical significance.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Zinc distribution in the control soil (unamended Zn) remained the same during the whole incubation period: residual fraction (F7, 91.9 to 93.2%), amorphous Fe oxide bound fraction (F5, 5.2 to 5.5%), crystalline Fe oxides bound fraction (F6, 1.0 to 2.0%), carbonate bound fraction (F3, 0.4%), organically complexed fraction (F2, 0.1%), and Mn oxide bound fraction (F4, 0.1%). Zinc was not detected in the water soluble plus exchangeable fraction (F1). It is notable that the carbonate bound fraction in a calcareous soil was very low and the water soluble plus exchangeable fraction was below the detection limit of the analytical method.
When the micronutrient was applied to the soil, as Zn complexed by natural amino acids, Zn in the F1 fraction could only be detected 15 d after application. Only 0.2% of the total added was detected at the high Zn rate (20 mg Zn kg-1). The order of Zn distribution between fractions was similar to that in the control soil; with the high Zn rate after 60 d of incubation, this distribution was as follows: 81.6% of the total occurred in F7, followed by F5 (15.8%), and F6 (1.5%). Likewise, 0.5, 0.5, and 0.1%, respectively, were detected in F2, F3, and F4, while no Zn was detected in F1.
When the addition of micronutrient to the soil was Zn chelated by a mixture of the three synthetic chelating agents DTPA-HEDTA-EDTA, the concentration in the most labile forms was larger compared with the other fertilizer. As compared with the same incubation time (60 d) and the same loading level (20 mg kg-1), Zn distribution in the fractions was as following: 76.1% of the total occurred in F7, followed by F5 (11.3%), F1 (9.9%), F6 (1.3%), F2 (0.9%), F3 (0.4%), and F4 (0.1%). Thus, an important amount of total Zn was detected in the water soluble plus exchangeable fraction (F1), which is very important for plant nutrition because it represents the most available Zn (water soluble and clay surface sorbed Zn).
The quantities of Zn extracted with DTPA in the unamended and fertilizer treated soil at the different incubation times are shown in Fig. 1 . In all cases, the quantities of Zn extracted from the samples treated with the fertilizer were much larger than those from the control soil (unamended Zn). During the incubation period, the concentrations of Zn extracted with DTPA decreased in the soil treated with Zn-AA. However, available Zn remained practically constant in the control soil and in the samples treated with Zn-CH during the same time period. For each fertilizer treatment, the concentrations of Zn available were greater for the 20 mg kg-1 Zn rate than for the 10 mg kg-1 Zn rate. However, at the end of the experiment, the concentration of available Zn was greater for the low Zn rate of the Zn-CH fertilizer treatment (6.1 mg kg-1, 5.2% of total Zn) than for the high Zn rate of the Zn-AA fertilizer treatment (5.5 mg kg-1, 6.2% of total Zn).
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A simple linear regression analysis was performed between the different Zn fractions obtained by the sequential extraction procedure. There was a positive correlation between F1-F2, F2-F5, F4-F6, and F5-F7. The correlation coefficients (r) for this statistical analysis varied from 0.71 (P < 0.003) to 0.75 (P < 0.001). A simple regression analysis was also calculated between DTPA extractable Zn and the Zn fractions. The statistically significant correlation coefficients were: DTPA-F1 (r = 0.90, P < 0.0001) and DTPA-F2 (r = 0.95, P < 0.0001). It must be noted that the most important correlation with DTPA values occurred with the first two extracted fractions.
Column Experiment
The amounts of Zn leached in the control soil and the soil treated with Zn-AA were very small in all cases (Fig. 2)
. In the control soil, the cumulative quantity of Zn recovered in 1200 mL of leachate (60 d, 1.88 pore volume) was 0.1 mg. The total amount of Zn leached from the soil columns amended with Zn-AA was only 0.2 mg. In the soil columns amended with Zn-DTPA-HEDTA-EDTA, however, the concentrations of Zn in the leachate fractions increased significantly compared with that from the control soil and Zn-AA treatments. When this fertilizer was applied, the concentration of Zn in the leachate peaked in the sixth leachate fraction (1.41 pore volume), followed by a sharp decline in the subsequent leachate fractions. The total recovery of Zn in the leachates from soil amended with this fertilizer was 11.9 mg (49.4% of the Zn applied).
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A multifactor variance analysis was performed with the variables DTPA and Mehlich-3 extractable Zn and with the factors fertilizer, time, and depth of the layer. For the two variables, significant differences were obtained between the fertilizers (P < 0.03 for DTPA and P < 0.02 for Mehlich-3) and depths (P < 0.02 for DTPA and P < 0.009 for Mehlich-3), showing a positive interaction between both factors (P < 0.008 for DTPA and P < 0.006 for Mehlich-3). However, the time factor did not have significant influence.
A better understanding of the situation for the remaining Zn in the soil column was obtained by the sequential extraction procedure (Table 3). It must be emphasized that the Zn distribution in the fractions in the a zone of the columns amended with the fertilizers was different if compared with that in the control soil. For example, in the a zone, the Zn distribution in the columns amended with the Zn-AA fertilizer was: in F1, not detected; F2, 21.2%; F3, 0.8%; F4, 0.8%; F5, 9.8%; F6, 2.5%; and F7, 64.9%. When the Zn-CH fertilizer was applied for the same length of the experiment, Zn distribution in the fractions was as follows: in F1, not detected; F2, 5.7%; F3, 0.2%; F4, 0.2%; F5, 5.1%; F6, 2.1%; and F7, 86.7%. Therefore, partitioning Zn among the different soil fractions after the fertilizer treatment also depended on the Zn source used. Zinc distribution in the fractions was affected by the Zn leached from the column, which mostly came from the most mobile fractions. After leaching, Zn was not detected in F1 (the water soluble plus exchangeable fraction) with the Zn-CH treatment. In addition, Zn concentration in F2 (organically complexed-Zn) was 21.2% of total Zn with natural chelate and 5.7% of total Zn with synthetic chelates. In the b and c zones, the extractability of Zn was higher with the Zn-CH than with Zn-AA, and the latter was similar to the control soil.
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In this soil, the behavior of the amino acid formulation of Zn could be due to different causes: (i) the amino acids are such weak or intermediate ligands for Zn that there was no significant chelation and Zn was transferred from the amino acids to soil binding sites, (ii) the complexed Zn was adsorbed as an intact metal-ligand chelate, and/or (iii), the amino acids were rapidly mineralized by soil microbes. This ligand agent does not protect the metal from retention by the soil. In this way, there should be an inverse relation between retention in soil and the stability of this Zn formulation. The different behavior of the Zn-CH formulation is probably due to the greater stability of these chelate molecules (log K ZnDTPA = 19.5, log K ZnHEDTA = 15.3, and log K ZnEDTA = 17.4 with ionic strength 0.01 M) (Lindsay, 1979; McBride, 1989).
Greenhouse Pot Experiment
Growth and Zinc Uptake
The application of Zn fertilizers to this calcareous soil, in a system with minimal leaching, had a significant (P < 0.0001) effect on the dry matter yield (Table 5). In comparison with the control soil, dry matter was increased 601 and 584% by 10 and 20 mg kg-1 of the Zn-CH and 258 and 206% by 10 and 20 mg kg-1 of the Zn-AA. On the other hand, it is clear that the control soil was deficient in Zn available to the plant, being this Zn level is critical for growing maize.
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The Zn source containing synthetic chelates was better than the Zn source containing natural chelates for both Zn uptake and dry matter yield increases. It was observed, however, that maize response to the use of Zn-AA reported in this study is consistent with the results obtained by using other fertilizers, such as ZnSO4 or Zn-humate-lignosulfonate (Maftoun and Karimian, 1989; Thind et al., 1990; Yasrebi et al., 1994).
Distribution of Zinc Forms in Soil after Maize Harvest
Total Zn content (sum of the fractions) in soil and their distributions in various soil forms are shown in Table 6. Total Zn determined in the soil as a whole with no application of Zn (control) was 85.7 mg kg-1. The relative Zn recovered in the different fractions, DTPA, and Mehlich-3 extractable Zn did not vary much compared with the original soil. After the fertilizer treatments, the order of Zn distribution between fractions was: F7 >> F5 >> F6 > F2 > F3 > F4 > F1, for the low Zn rate of both organic Zn chelates. With the high Zn rate of both fertilizers, this order was similar except F2 > F6. Thus, the Zn concentration in F2 (organically complexed Zn) increased when the high Zn rate was applied. These results differ of the incubation study. In this way, Ahumada et al. (1999) reported differences between cultivated and noncultivated soil in sequential extraction of different metals. These differences would be due to physicochemical changes produced in the soil as a consequence of the plant. It must be emphasized that the only treatment that presented Zn content in F1 was the high Zn rate of the Zn-CH fertilizer. This fraction is very important for plant nutrition because it potentially represents the most bioavailable Zn. Thus, the speciation technique accomplished can be employed to predict the degree of Zn complexation in soil, depending on the particular ligand type. Aboulroos (1981) suggested that ineffectiveness of chelating compounds in soil depends on the fixation of the entire metal chelate molecule on clay colloids and the displacement by other cations from soil with the subsequent precipitation of the metal. This author indicated that a high percentage of synthetic Zn chelates (Zn-EDTA and Zn-DTPA) remained in the soil solution and that adsorption of Zn-chelate molecules by soil accounted only for a small part of the Zn loss. McBride (1989) indicated that some metal-complexing ligands suppress metal adsorption and others enhance adsorption by forming stable surface metal-ligand complexes.
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Finally, the results obtained in the incubation experiment provide an explanation of the differences observed in the Zn uptake by maize. In the incubation experiment, soil Zn fractionation showed significant differences between Zn sources. The differences observed in water soluble plus exchangeable Zn fraction and DTPA extractable Zn correlated with the Zn uptake by maize obtained in the greenhouse experiment. On the other hand, the mobility of Zn-CH in the soil columns caused changes in the most bioavailable forms of Zn in soil. Thus, the movement of Zn in the soil can produce a loss of Zn by leaching if excessive rainfall or irrigation occurs. In accordance with Prasad and Sinha (1981), the diffusion of Zn is the main mechanism that contributes to Zn nutrition of maize in calcareous soil, which quite frequently suffers from micronutrient deficiency. According to Maftoun and Karimian (1989), the more enhancing influence of synthetic chelate over other forms in terms of growth and Zn utilization by maize in calcareous soils might be due to less retention, and greater transport and movement of chelated Zn to plant roots. Thus, the applied Zn-CH can increase the concentration gradient of Zn toward the maize roots and Zn could be retained as a bioavailable Zn form in the soil solution, which contributes considerably to the uptake of Zn by the plant.
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ACKNOWLEDGMENTS |
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Received for publication August 3, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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intensification fourragère. Ann. Agron. 21:635–669.This article has been cited by other articles:
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  N. A. Slaton, E. E. Gbur Jr., C. E. Wilson Jr., and R. J. Norman Rice Response to Granular Zinc Sources Varying in Water-Soluble Zinc Soil Sci. Soc. Am. J., March 1, 2005; 69(2): 443 - 452. [Abstract] [Full Text] [PDF]
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in soil with 0 (control), and 20 mg Zn kg-1 soil as Zn-amino acid complex (Zn-AA) or Zn-chelate (Zn-CH) at 0, 30, and 60 d of a column experiment (depth a, 0–7 cm; depth b, 7–17 cm; and depth c, 17–27 cm). Zinc fractions are: organically complexed (F2), carbonate bound (F3), Mn oxide bound (F4), amorphous Fe oxide bound (F5), crystalline Fe oxide bound (F6), and residual (F7).