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Published online 1 May 2008
Published in Soil Sci Soc Am J 72:641-649 (2008)
DOI: 10.2136/sssaj2007.0099
© 2008 Soil Science Society of America
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SOIL FERTILITY & PLANT NUTRITION

Effect of Zinc Source Applied to Soils on its Availability to Navy Bean

D. Gonzalez, A. Obrador, L. M. Lopez-Valdivia and J. M. Alvarez*

Departamento de Química y Análisis Agrícola, E.T.S.I. Agrónomos, Universidad Politécnica de Madrid (UPM), Ciudad Universitaria s/n, 28040 Madrid, Spain

* Corresponding author (josemanuel.alvarez{at}upm.es).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to compare the mobility, availability, and relative effectiveness of Zn from liquid Zn–diethylenetriamine-pentaacetate–N-2-hydroxyethyl-ethylenediamine-triacetate–ethylenediamine-tetraacetate (Zn-DTPA-HEDTA-EDTA or Zn-D-H-E) and Zn–aminelignosulfonate (Zn-AML) sources applied to weakly acidic (moderate permeability) and calcareous (moderate to rapid permeability) soils at different Zn levels in a navy bean (Phaseolus vulgaris L.) greenhouse experiment during a 60-d period. Zinc behavior in soil was evaluated by single and sequential extractions. The addition of the two Zn complexes produced a considerable increase in Zn concentration in the water-soluble plus exchangeable and organically complexed fractions. The Zn-D-H-E source resulted in greatly increased mobility of Zn through the soils in comparison with the control (no Zn addition) and the Zn-AML source at the highest dosage (10 mg Zn kg–1); 11 and 32% of total applied Zn was lost in weakly acidic and calcareous soils, respectively, through leaching. Zinc application increased the plant dry matter yield compared with the control treatment in the weakly acidic soil; however, there were no significant differences between the Zn rates of 5 and 10 mg kg–1. All sources of Zn increased the amount of Zn uptake in plants. The Zn-D-H-E was more effective than Zn-AML in the calcareous soil but had a similar effect in the weakly acidic soil. Soluble Zn extracted from plant dry matter with reactive 2-(N-morpholino)ethanesulfonic acid (MES) could be used to diagnose the nutritional status of Zn in navy bean.

Abbreviations: AMC, amorphous mineral colloids bound • AML, aminelignosulfonate • AB, ammonium bicarbonate • CAR, carbonate bound • CFeO, crystalline iron oxide bound • DTPA, diethylenetriamine pentaacetate • EDTA, ethylenediamine tetraacetate • HEDTA, N-2-hydroxyethyl-ethylenediamine triacetate • MES, 2-(N-morpholino)ethanesulfonic acid • OC, organically complexed • OM, organically bound • RES, residual • RMO, easily reducible metal oxide bound • TEA, triethanolamine • Zn-D-H-E, Zn-DTPA-HEDTA-EDTA • WSEX, water soluble plus exchangeable


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Micronutrient deficiencies in plants are an old problem occurring in areas of sandy or calcareous soils (Marschner, 1986; Alvarez-Fernandez et al., 2005). Zinc is an essential micronutrient for plants (Römheld and Marschner, 1991) and its deficiency is one of the most widespread disorders in a number of crops (Westfall et al., 1971). The navy bean is a crop particularly sensitive to Zn deficiency (Loué, 1988). Several studies have indicated that organic sources of Zn are more effective than inorganic ones (Hergert et al., 1984; Mortvedt and Gilkes, 1993). A number of fertilizers containing Zn complexes or chelates are therefore being developed to correct this deficiency (Liñán, 2006). Moraghan (1996) found that ZnSO4 and Zn-EDTA produced similar increases in Zn concentrations in navy bean seeds. Band application of the same sources, however, produced lower Zn concentrations in seed than direct incorporation into the soil. Few studies have been conducted, however, on the use of different Zn complexes with navy bean crops in various soil types (Shuman, 1998). It would therefore be advisable to study the effects of these new fertilizers on plants, soils, and the natural environment (Öborn et al., 2003).

The total dry matter content of a micronutrient is most frequently determined by plant analysis, although only a fraction of the total content could be determined; for example, the part that is soluble in water or in diluted acids or chelators sometimes provides a better indication of nutritional status (Marschner, 1986).

In the hope of yielding information for the assessment of metal bioavailability and mobility in soils, single chemical extractions are often performed using selective chemical extractants (Lake et al., 1984; Beckett, 1989; Ure, 1996). The DTPA–triethanolamine (TEA), DTPA–NH4HCO3 (AB) and Mehlich-3 methods are usually used to diagnose Zn bioavailability for plants in different soils (Reed and Martens, 1996) and the easily leachable Zn can be determined by means of the BaCl2 extraction procedure (Schultz et al., 2004). Sequences of different chemical extractants have therefore become increasingly popular for quantifying the amounts of metals present, in their different forms, in soils (Ure et al., 1993). Chemical speciation provides a better understanding of Zn behavior in relation to the transformation of Zn added to soil (Iyengar et al., 1981) and has often been used to determine the relative distribution and chemical forms of Zn in soil (Chao, 1972; Tessier et al., 1979; Shuman, 1985; Krishnamurti and Naidu, 2002). The selectivity of chemical reagents has been criticized, however, and cases of back-adsorption of trace metals during the extraction steps have also been mentioned (Orsini and Bermond, 1993). Despite some of these limitations, sequential extraction is still considered to be a valuable tool for investigating the various forms in which metals are found in soils (D'Amore et al., 2005).

The objectives of this greenhouse study were to determine: (i) the mobility and leaching of Zn applied as different organic complexes (natural vs. synthetic origin) to two soils with different physicochemical characteristics in which a low-growing navy bean crop was grown; (ii) the effectiveness of these two Zn complexes on the navy bean's dry matter yield and total and soluble Zn concentrations; (iii) the Zn fractionation in the soils; (iv) the extractable Zn; and (v) the parameters pH and redox potential in the soils.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Applied Fertilizers
The two Zn sources used in this study were: (i) a liquid solution of Zn chelated with a mixture of three synthetic chelating agents: DTPA, HEDTA, and EDTA at the rate of 90 g water-soluble Zn L–1 solution, and (ii) a liquid solution of Zn chelated by aminelignosulfonic acids at the rate of 59 g water-soluble Zn L–1 solution. These organic Zn sources are made by several commercial companies (Liñán, 2006).

Soil Characterization
The weakly acidic soil came from Navalcarnero (40°21' N, 4°0' W) in the region of Madrid, Spain. It was classified as a Typic Haploxeralf (Soil Survey Staff, 2006). The calcareous soil came from Torrejón del Rey (40°39' N, 3°20' W) in the province of Guadalajara, Spain, and was classified as a Typic Calcixerept. Surface soil was taken from the Ap horizon (0–26 cm in Soil 1 and 0–20 cm in Soil 2); samples were air dried and a fraction of <2 mm was used in the experiment. The soil properties, which are means of three replicates, are reported in Table 1 .


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  		Table 1. Selected soil properties and analytical procedures used in this study.

 
Greenhouse Experiment
Treatments and Experimental Design
A navy bean crop was grown in polypropylene lysimeters (capacity 11 L, internal diameter 24 cm, and height 25 cm). A 1.5-cm-thick layer of washed gravel was placed in the bottom of each lysimeter to facilitate aeration and drainage and a polyester mesh and 10 kg of soil were placed on top of it. The leachate was collected with a silicone tube leading to a polyethylene bottle. Basal fertilization was applied with 50 mg N kg–1 (as urea), 50 mg P kg–1 (as superphosphate), and 50 mg K kg–1 (as K2SO4). Three navy bean seeds (Garrafal Rabona Enana Esmeralda, Fito S.A., Barcelona, Spain) were cultivated in each lysimeter. The soil received 0 (control), 5, or 10 mg Zn kg–1 of soil, which was applied from Zn fertilizers. The control and treatments were replicated three times in a completely randomized design layout. The lysimeters were placed in a greenhouse, where temperatures varied between 16 and 28°C and relative air humidity between 60 and 85%. The soils were irrigated at slightly above field capacity moisture to obtain 10 fractions (each of 200 mL) of leachate. Sixty days after germination, the plants were cut at soil level, washed in deionized water, and then dried in an oven at 65°C to a constant weight. Once weighed, they were ground and kept in sealed containers for later analysis. The soils were also dried at room temperature, homogenized, sieved (<2 mm), and stored for further analysis.

Leachate, Plant, and Soil Analyses
The leached liquids were collected, acidified with HCl, and filtered (Whatman no. 41), and their Zn concentrations were determined by atomic absorption spectrophotometry. The electrochemical parameters of the soils and leachates were determined by potentiometry analysis using pH and redox (Pt) electrodes. Temperature was automatically compensated by means of a probe connected to the potentiometer in the case of the pH. To calculate the redox potential, the potential from the reference electrode was added to the measured potential of the cell at the same temperature (ISO, 2002). For soils, the procedure consisted of making a hole 2 mm larger than the diameter of the electrode by inserting a stainless steel bar into the previously humidified soil. The electrode was then inserted to a depth >2 cm. The temperature probe was also inserted into the soil. Measurements were taken between 10 and 15 min when values became stable. In the case of leachates, the electrodes were submerged in the solutions collected from each lysimeter and the measurements stabilized much more quickly than in the soils. The total Zn in the plant was determined by wet digestion in a microwave oven (a two-step process with a maximum pressure of 1.17 MPa) using 0.3 g of dried ground samples, 4 mL of concentrated HNO3 and 2 mL of concentrated HF.

Soluble Zn in the plant dry matter was extracted by the method proposed by Rahimi and Schropp (1984) and Cakmak and Marschner (1987), with slight modifications: 0.25 g of the aerial part of the plant was weighed and its Zn content was extracted with 10 mL of 1 mmol L–1 MES at pH 6 (ratio 1:40 w/v).

The Zn distribution in different soil fractions was determined by the sequential fractionation method proposed by Krishnamurti and Naidu (2002). The fractions were sequentially determined in seven steps with the following extractants: 1 mol L–1 NH4NO3 at pH 7.0 (water soluble plus exchangeable, WSEX); 1 mol L–1 NaOAc at pH 5.0 (carbonate bound, CAR); 0.1 mol L–1 Na4P2O7 at pH 10.0 (organically complexed, OC); 0.1 mol L–1 NH2OH·HCl in 0.01 mol L–1 HNO3 (easily reducible metal oxide bound, RMO); 30% H2O2 at pH 2.0 + 0.02 mol L–1 HNO3 and 2 mol L–1 NH4NO3 in 20% HNO3 (organically bound, OM); 0.2 mol L–1 (NH4)2C2O4 and 0.2 mol L–1 H2C2O4 at pH 3 in the dark (amorphous mineral colloids bound, AMC); and 0.2 mol L–1 (NH4)2C2O4 and 0.2 mol L–1 H2C2O4 at pH 3 in 0.1 mol L–1 ascorbic acid (crystalline Fe oxide bound, CFeO). The residual (RES) fraction was calculated as the difference between total Zn and the sum of the other fractions (the first six steps for Soil 1, all seven steps for Soil 2).

Available Zn in the soil was assessed by extracting it with DTPA-TEA (Lindsay and Norvell, 1978), DTPA-AB (Soltanpour, 1991) and Mehlich-3 (Mehlich, 1984). The easily leachable Zn fraction was extracted with 0.01 mol L–1 BaCl2 (Schultz et al., 2004).

Total Zn in the soil was determined after treating 2 g of dried sample with 14 mL of HNO3 and 6 mL of concentrated HF followed by digestion in Teflon bombs in a microwave oven (three steps, maximum pressure of 689 kPa).

The PerkinElmer pure standard checks were used for the quality assurance system (certified by National Institute of Standards and Technology Standard Reference Material). Standard solutions of Zn were prepared for each extraction in a background solution of the extracting agents. In all cases, Zn concentrations were determined by atomic absorption spectrophotometry (AAnalyst 700; PerkinElmer, 2000).

Statistical Analysis
Correlation analysis and other statistical studies were performed with Statgraphics Plus software, Version 5.1 (Manugistic, Rockville, MD). Multifactor analysis of variance was performed to determine the main effects and interactions of the different parameters. Multiple comparisons of variables were made using Duncan's separation of means procedure. To establish statistical significance, a probability level of P ≤ 0.05 was chosen.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Zinc Leaching
The amounts of Zn leached in the control (no Zn addition) and in the soils treated with Zn-AML were very small (<1% of applied Zn) in both Soils 1 and 2. In the control soils, the cumulative quantities of collected Zn in 2 L of leachate (at 60 d) were 0.24 and 0.07 mg in Soils 1 and 2, respectively. The total amounts of Zn leached in Soil 1 amended with Zn-AML at rates of 5 and 10 mg kg–1 were 0.27 and 0.28 mg, respectively, while in Soil 2 these quantities were 0.09 and 0.19 mg for the same rates. In Soil 1 amended with Zn-D-H-E, however, the total amounts of Zn leached were 2.51 (5.0% of applied Zn) and 11.18 mg (11.2%), and in Soil 2 they were 13.95 (27.9%) and 32.04 mg (32.0%) for the 5 and 10 mg Zn kg–1 rates, respectively (Fig. 1 ). When this fertilizer was applied, the Zn concentration in the leachate showed a peak in the first leachate fraction (volume of leachate 200 mL) followed by a decline in subsequent leachate fractions collected, with the exception of the 10 mg Zn kg–1 rate applied in Soil 1, which showed maximum concentrations in the first three leachate fractions.


Figure 1
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  		Fig. 1. Amounts and concentrations of Zn in 10 leachate portions vs. volume of leachate from soils amended with 0 (control), 5, and 10 mg Zn kg–1 soil as Zn–diethylenetriamine-pentaacetate–N-2-hydroxyethyl-ethylenediamine-triacetate–ethylenediamine-tetraacetate (Zn–DTPA–HEDTA–EDTA or Zn-D-H-E). The vertical line at each of the data points represents standard error of the mean.

 
Dry Matter Production and Zinc Uptake
The effects of the treatments on dry matter and Zn concentration in 60-d-old navy beans are shown in Table 2 . In Soil 1, Zn-AML applied at rates of 5 and 10 mg Zn kg–1 and Zn-D-H-E at 5 mg Zn kg–1 significantly increased yield in comparison with the control (P < 0.05), but there were no significant differences between the sources or rates of Zn fertilizers. The application of Zn chelates significantly increased the concentration (P < 0.0001) and uptake of Zn in the plant shoot. Both Zn-D-H-E and Zn-AML behaved similarly and a linear relationship was observed between the rate of applied Zn and the concentration of Zn in the plant. Applications at 5 mg Zn kg–1 increased the Zn concentration by factors of 4.5 and 4.2 and at 10 mg Zn kg–1 by factors of 8.5 and 8.0 for Zn-D-H-E and Zn-AML fertilizers, respectively. There were no significant differences between Zn sources.


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  		Table 2. Response of navy bean to rates and forms of Zn complexes in soil (Zn-D-H-E = Zn–diethylenetriamine-pentaacetate–N-2-hydroxyethyl-ethylenediamine-triacetate–ethylenediamine-tetraacetate or Zn-DTPA-HEDTA-EDTA; Zn-AML = Zn–aminelignosulfonate).

 
In Soil 2, the application of Zn fertilizers did not affect the dry matter production of navy bean (P > 0.05). Total Zn concentrations in plants, however, were significantly affected by Zn rate and source (Table 2). The Zn-D-H-E applied to navy bean had a higher Zn concentration.

Zinc Fractions, Available Zinc, and Easily Leachable Zinc
The distribution of the Zn fractions and total Zn content in the two soils are shown in Fig. 2 . In Soil 1, total Zn in the control was 9.94 mg kg–1 and the order of Zn distribution between fractions was: CFeO > OC > RES > OM > AMC > WSEX > RMO. Speciation of the original soil indicated that the majority (23.5%) of Zn was present in the CFeO fraction and that the second most abundant form was OC, which accounted for 20.0% of the total. The addition of Zn complexes produced a considerable increase in Zn concentration in the most labile fractions, and particularly in the WSEX and OC fractions, which are potentially bioavailable and therefore important for plant nutrition. In the OC fraction, differences were observed between both fertilizers and rates (P < 0.0001) but in the WSEX fraction there were only differences between rates (P < 0.001). Applying Zn-D-H-E at 10 mg Zn kg–1 increased the Zn concentration in the OC fraction by a factor of 3.6 with respect to the control, while the Zn-AML fertilizer applied at the same rate produced an increase of a factor of 2.9.


Figure 2
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  		Fig. 2. Zinc fractions in soils at the moment of navy bean harvest with 0 (control), 5, and 10 mg Zn kg–1 soil as Zn–diethylenetriamine-pentaacetate–N-2-hydroxyethyl-ethylenediamine-triacetate–ethylenediamine-tetraacetate (Zn–DTPA–HEDTA–EDTA or Zn-D-H-E) and Zn–aminelignosulfonate (Zn-AML). Zinc fractions are residual fraction (RES), crystalline Fe oxide bound (CFeO), amorphous minerals colloids bound (AMC), organically bound (OM), easily reducible metal oxide bound (RMO), organically complexed (OC), and water soluble plus exchangeable (WSEX).

 
In Soil 2, total Zn in the control was 44.11 mg kg–1 and the order of Zn distribution between fractions was: RES > CFeO > OC > OM > AMC > CAR > WSEX > RMO. Speciation of the original soil indicated that the great majority (63.6%) of Zn was present in the RES fraction and the second most abundant form was CFeO, with 20.0% of the total. The fertilizer treatments increased concentrations in all fractions (except RES), but most notably in the WSEX, CAR, and OC fractions, in which there were significant differences between rates and treatments (P < 0.01–0.001). The Zn-D-H-E source increased the Zn concentration in WSEX by factors of 3.6 and 13.9 compared with the untreated soils for the 5 and 10 mg Zn kg–1 rates of application, respectively, and also increased the OC fraction by a factor of 1.8 and 2.3, respectively, for the same rates. The greatest increase associated with Zn-AML was observed in the WSEX fraction, however, with an increase of a factor of 3.1 for the 10 mg Zn kg–1 rate of application.

Electrochemical Parameters
Electrochemical parameters, pH and redox potential (Eh), were determined in soils at two times: after 30 and 60 d after germination for all treatments. Soil pH increased significantly (P < 0.0001) with time, while Eh hardly varied. The differences were significant between the two soils (P < 0.0001) and not significant between fertilizer treatments. At the end of the experiment, the calculated average values for pe (pe = Eh [mV]/59.2 or the negative logarithm of the free electron activity) revealed more oxidizing conditions in weakly acidic soil (pe = 10.7) than in calcareous soil (pe = 9.2), and the parameter pe + pH in Soil 1 had an average value of 16.9, while in Soil 2 it was 16.5.


   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Zinc Leaching
It was found that the applied Zn migrated through the soil with the irrigation water. The amounts of leached Zn depended on the type of soil, the source of Zn used, the rates applied, and the leached volume collected during the experiment. The amounts of Zn leached in the control soils were very small in all cases. In the control soils, the cumulative quantities of Zn recovered in 2000 mL of leachate were 0.24 and 0.07 mg for Soils 1 and 2, respectively. In the soils fertilized with Zn-D-H-E, the total amount of Zn leached was 5 to 32% of the applied Zn and it was significantly greater than in the control and Zn-AML-treated soil. Such values imply a risk of water contamination. These results can be explained by the relative stability of Zn complexes. According to several researchers, synthetic chelating agents (like DTPA, HEDTA, and EDTA) have a higher stability than natural ones (like AML), maintain greater amounts of Zn in the soil solution, migrate through the soil profile, and are leached (Lindsay, 1979; Modaihsh, 1990; Alvarez and Rico, 2003). Alvarez et al. (1996) found almost no micronutrient movement in calcareous soil column leaching studies involving the application of Zn–lignosulfonate, while application of Zn–EDTA showed considerable movement and some leaching from the columns.

According to Waychunas (1991), the Zn retention in soil is mainly determined by the sheet structure of the surfaces of the Fe oxides, while Ma and Uren (1997) indicated that Zn retention is also determined by the microporosity of the soil components. Furthermore, the movement of water through the soil profile and hence that of the nutrients dissolved in it is clearly related both to the structure and texture of the soil (Duchaufour, 1987; Alvarez et al., 2001). Despite Soil 2 presenting some physical and chemical characteristics that enhance the retention of Zn (alkaline pH, clay percentage, and free CaCO3 content), this soil has a higher permeability than Soil 1 (see Table 1) and this could explain why the leaching of Zn was greater in Soil 2 (see Fig. 1).

Dry Matter Production and Zinc Uptake
An important parameter in the study of the relative efficiency of any fertilizer is the percentage of its use by the crop (Prasad and Sinha, 1981). In this case, we defined the percentage of Zn used (% Zn utilization) by navy bean plants according to the type applied in the following way:

Formula
In Soil 1, Zn-D-H-E and Zn-AML demonstrated similar efficiencies, about 6%. Given that Zn-AML did not produce a loss of Zn by leaching and does not present a potential risk of water contamination, it would be advisable to apply this fertilizer at a rate of 5 mg Zn kg–1 or less, since the Zn concentration in the navy bean dry matter was high (119.37 mg kg–1). The Zn concentration in healthy leaves generally ranges from 15 to 100 mg kg–1 and values of between 30 and 60 mg kg–1 are considered normal in navy bean (Loué, 1988). In Soil 2, however, the most effective treatment was the Zn-D-H-E fertilizer applied at a rate of 5 mg kg–1. This was associated with the highest percentage of utilization (1.7%) and also produced an adequate Zn concentration in the plant. This Zn concentration is important for bean quality since it supports a greater concentration in the seed, which is an important source of nutrition for vegetarians who may be at risk for Zn deficiencies (Wise, 1995).

In both soils, Zn concentrations in navy bean dry matter cultivated in untreated soils (controls) were below the normal range of values, which justifies fertilization with Zn.

On the other hand, in both soils and for all the fertilizers used, the soluble Zn concentration (mg kg–1) extracted from bean tissue with the reactive MES and the total Zn concentration (mg kg–1) behaved similarly and were related according to

Formula
Thus, in our study, the determination of soluble Zn MES in navy bean dry matter could be used to diagnose the nutritional state of Zn in the plant. Rahimi and Schropp (1984) and Cakmak and Marschner (1987) reported that the water-soluble form of Zn, found in plants such as corn (Zea mays L.) and cotton (Gossypium hirsutum L.), reflects their nutritional status much better than total Zn.

Zinc Fractions, Available Zinc, and Easily Leachable Zinc
The Zn concentration in different soil fractions depended on soil type and the Zn fertilizer treatments applied (chelate and rate). Considering that the added Zn could remain in the soil, leach out, or be taken up by the crop, a mass balance calculation was done to determine whether all of the Zn could be accounted for. This calculation produced recoveries between 99 and 105%.

The residual Zn produced by different fertilizer treatments was sufficiently available that later crops could be grown without further micronutrient additions. In Soil 2 (weakly acidic), the DTPA-TEA, DTPA-AB, and Mehlich-3 extractable Zn for the control treatment were greater than for the original soil. This behavior was probably due to the fact that the phosphoric fertilizer contained micronutrient impurities (Table 3 ). The concentrations of available Zn remaining in both soils were much higher than the critical concentrations according to the three methods: 0.5 to 1.0 mg kg–1 by DTPA-TEA extraction (Lindsay and Norvell, 1978), 1.0 to 1.5 mg kg–1 by DTPA-AB extraction (Soltanpour, 1991), and 1.2 to 1.8 mg kg–1 by Mehlich-3 extraction (Tran and Simard, 1993), which was not the case in the control soils. Greater amounts of available Zn were obtained from Soil 1 than from Soil 2. In both cases, the treatment that produced the greatest quantities of available Zn was Zn-D-H-E applied at a rate of 10 mg Zn kg–1 (with values varying between 50.7 and 56.4% of total Zn after the crop in Soil 1 and between 10.1 and 15.0% in Soil 2). The treatment that produced the smallest quantities was Zn-AML applied at a rate of 5 mg Zn kg–1 (with values varying between 15.8 and 21.3% in Soil 1 and between 2.8 and 5.5% in Soil 2).


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  		Table 3. Diethylenetriamine pentaacetate–triethanolamine (DTPA-TEA), diethylenetriamine pentaacetate– NH4HCO3 (DTPA-AB) and Mehlich-3 (M-3) extractable Zn and easily leachable Zn (BaCl2) in soil at the moment of navy bean harvest fertilized with 0, 5, and 10 mg Zn kg–1 as Zn–diethylenetriamine-pentaacetate–N-2-hydroxyethyl-ethylenediamine-triacetate–ethylenediamine-tetraacetate (Zn-DTPA-HEDTA-EDTA or Zn-D-H-E) and Zn–aminelignosulfonate (Zn-AML).

 
The concentrations of Zn extracted with BaCl2 were greater in Soil 1 than in Soil 2 (see Table 3), with values reaching 38 and 3% of the respective totals. These concentrations were smaller than those obtained by using the three determination methods of available Zn, and particularly so for Soil 2. The concentrations of easily leachable Zn correlated with the concentrations of available Zn. According to Räisänen et al. (1997) and Schultz et al. (2004), this reagent (BaCl2) only extracts elements that are physically adsorbed on particles. In contrast, DTPA-TEA, DTPA-AB, and Mehlich-3 also extract more strongly retained Zn (Ure, 1995; Reed and Martens, 1996).

The three methods used to estimate available Zn showed highly significant correlations among themselves (P < 0.0001) (Table 4 ). They can therefore be used to predict Zn availability for plants in a similar way. Vocasek and Friedericks (1994) also found a high correlation between Mehlich-3 and DTPA-TEA in soils with a wide range of chemical properties. The order of the average extracted concentrations was as follows: Mehlich-3 > DTPA-AB > DTPA-TEA. These three methods were also correlated with both the easily leachable Zn and the most labile fractions, such as WSEX and OC, and positive correlations were also observed between them. According to Reed and Martens (1996) labile Zn mainly consists of free and complexed Zn in soil solution. This provides the soil intensity required to supply this nutrient to plants, and also the nonspecifically adsorbed Zn, which gives the soil its capacity to replenish the Zn removed from the soil solution. Therefore the Zn associated with the WSEX and OC fractions was more bioavailable to the plants. Novillo et al. (2002) also found a high correlation between WSEX and OC (r = 0.71, P < 0.0001) in three (acidic, neutral, and calcareous soils) soils.


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  		Table 4. Simple correlation coefficients (r) for relationships between diethylenetriamine pentaacetate–triethanolamine (DTPA-TEA), diethylenetriamine pentaacetate–NH4HCO3 (DTPA-AB), Mehlich-3 (M-3) and BaCl2-extractable and sequential extracted Zn fractions in soils (n = 10, except for carbonate-bound fraction where n = 5).

 
Positive and significant relationships were found between the total Zn concentration in the plant dry matter and available Zn in the soil (DTPA-TEA, DTPA-AB, and Mehlich-3) and also with the easily leachable Zn, WSEX, CAR, OC, and RMO fractions (Table 5 ). The correlation coefficients with the RES fraction, however, were negative. Soluble Zn and total Zn showed similar correlations, and dry matter yield only correlated with the OM fraction.


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  		Table 5. Simple correlation coefficients (r) for relationships between parameters of the plants and Zn forms in soils (n = 10, except for carbonate-bound [CAR] fraction n = 5).

 
There was a high degree of correlation between total Zn concentration (mg kg–1) and soluble Zn concentration (mg kg–1) in plant dry matter with soil bioavailable Zn extracted with DTPA-TEA, according to the following:

Formula

Formula
And also, between total and soluble Zn concentration with the easily leachable Zn extracted with BaCl2:

Formula

Formula
A good fit equation would make it possible to forecast Zn uptake by navy bean plants from all fractions sequentially extracted from the soil (R2 = 99%, P < 0.05). In this way, sequential fractionation can be considered useful for the study of the bioavailability of the micronutrient.

Likewise, Zn added from different sources remains in the soils in chemical forms that favor its absorption by plants. There are also positive and significant correlations between available and WSEX and OC Zn fractions (in Soil 2, they are also correlated with Zn found with carbonates). This behavior was corroborated by the relationship between Zn uptake by the plant (mg Zn pot–1) and the sum of the most labile fractions (WSEX + OC):

Formula
These results indicate that the degree of Zn uptake by navy bean was controlled by the WSEX and OC fractions in the soils, and consequently, the effectiveness of organic Zn complexes in plant uptake depends on their capacity to maintain the Zn soil content in this labile form.

Electrochemical Parameters
According to Patrick et al. (1996), the values of soil pH and pe correspond to normal (oxic) soils. The pH and pe parameters of the soils also correlated with those determined in the leachates (P < 0.0001). In addition, the negative correlation between pH and pe (P < 0.0001) was very significant, i.e., for a higher pH, the pe is lower.

The pH of the soil showed a significant negative correlation with the RMO fraction (P < 0.05) and a significant positive correlation with the CFeO (P < 0.001). Therefore, in soils with higher pH values, there was less Zn associated with easily reducible metal oxides and more Zn associated with crystalline Fe oxides. According to Reddy et al. (1995), as soil pH decreases, the availability and mobility of metal ions increases. On the other hand, in soils with lower redox potentials (or lower pe) the Zn concentration was lower in the CFeO and RES fractions and higher in the RMO fraction. Ghanem and Mikkelsen (1987) found that Zn retention increased under reduction conditions and attributed this to the transformations of Fe and Mn oxides. In this study, the soil redox potential decreased while the amount of exchangeable and organically complexed Zn decreased, and the amount of Zn bound to amorphous and crystalline sexquioxides increased. Time (not redox), however, was probably the key factor in these changes that occurred during the experiment, that is, an "aging" effect that is well known to occur after adding metal salts to soils (Obrador et al., 2002; Alvarez and Gonzalez, 2006; Ma and Uren, 2006).

In conclusion, the fertilizers applied to the soils migrated through them and leached Zn in amounts that depended on the type of soil involved, the source of the Zn used, the rates applied, and the leached volume collected during the experiment. Zinc applied in the form of synthetic chelates (Zn-D-H-E) leached in higher quantities in Soil 2 than in Soil 1. That was due to the higher permeability of Soil 2, despite other physical and chemical characteristics of this soil, such as its alkaline pH, clay percentage, and free CaCO3 content. In the weakly acidic soil, Zn-D-H-E and Zn-AML demonstrated a similar level of efficiency. Even so, it must be remembered that Zn-D-H-E produced a loss of Zn by leaching, so the use of Zn-AML fertilizer at a rate of 5 mg Zn kg–1 would be advisable. In calcareous soils, however, the highest percentage of Zn used was associated with the fertilizer Zn-D-H-E applied at the rate of 5 mg kg–1. These Zn chelates produced considerable increases in Zn concentrations in the most labile fractions, which are potentially bioavailable and therefore important for plant nutrition. The treatment that produced the greatest quantities of available Zn was Zn-D-H-E applied at a rate of 10 mg Zn kg–1. The methods used to estimate available Zn can be used in a similar way to predict its availability for plants, the quantity of Zn extracted being greater using the Mehlich-3 reactive than that extracted by other methods. Positive and significant relationships were found between total Zn concentrations in plant dry matter and the following micronutrient forms in the soils: available Zn, easily leachable Zn, WSEX, CAR, OC, and RMO fractions. The soluble Zn in plant dry matter could be used as an alternative way of determining total Zn to diagnose the nutritional status of Zn in the plant.


   ACKNOWLEDGMENTS
 
This study was financially supported by the Spanish Ministry of Education and Science (DGI, Project no. AGL2002-02009).


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication March 9, 2007.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





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