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a Univ. of Florida, Institute of Food and Agricultural Sciences, 700 Experiment Station Rd., Lake Alfred, FL 33850, present address, Center for Marine, Environmental Sci., and Biotechnology Research, Savannah State Univ., P.O. Box 20288, Savannah, GA 31404 USA
b Dep. of Agronomy, Madison Sturgis Hall, Louisiana State Univ., Baton Rouge, LA 70803 USA
siva{at}savstate.edu
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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Abbreviations: RCF, relative centrifugal force • RF, recently fixed
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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60 to 90% of total fixation usually occurs within the first few hours after fertilization (Nommik, 1965; Sippola et al., 1973). Lamm and Nadafy (1973) suggested that fixation of applied NH4 takes place in the interlayer sites of the clay lattices at a medium stage of collapse (in transitional zone) where the fixed and exchangeable ions meet. Long-term application of NH4 fertilizers can increase the amount of nonexchangeable NH4 in soils, and this RF NH4 appears to be more available to plants than native fixed NH4 (Mohammed, 1979; Mengel and Scherer, 1981; Saha and Mukhopadhyay, 1986). Collectively, native and RF NH4 entrapped in 2:1 clay minerals can represent a significant fraction of the total N in soil profiles (Black and Waring, 1972; Mengel and Scherer, 1981; Keerthisinghe et al., 1984; Beathgen and Alley, 1987). The availability of entrapped NH4 to plants or soil microorganisms, however, is less understood than that of other forms of soil N (Allison et al., 1951; Nommik, 1957; Black and Waring, 1972; Mohammed, 1979; Mengel and Scherer, 1981; Saha and Mukhopadhyay, 1986; Drury and Beauchamp, 1991). This uncertainty is due, in part, to the limitations of various methods employed for determination of clay fixed NH4.
Bremner (1965b) suggested the term nonexchangeable NH4 to describe NH4 held by clays and not displaced by 2 M KCl extraction. Although this fraction can be defined, quantitative determination of the amounts of nonexchangeable NH4 in soils poses a challenging analytical problem. Some of the uncertainty is also due to the fact that NH4, like K, is fixed in varying positions between 2:1 clay lattices. Ammonium fixed near an exposed edge will generally be more available than that fixed some distance from an edge.
Bremner et al. (1967) compared many of the numerous methods proposed for the determination of nonexchangeable NH4 in soils and showed that they gave widely divergent results. High values obtained for nonexchangeable NH4 were presumably inflated by the partial recovery of organic N as NH4 during treatment of samples with acid mixtures to release entrapped NH4 or by steam distillation of hydrolysate with strong alkali. Low values resulted when removal of organic N caused partial loss of nonexchangeable NH4 or procedures used to determine clay entrapped NH4 failed to recover this N quantitatively. The KOBr–HF method of Silva and Bremner (1966) gave mid-range values for nonexchangeable NH4 among the various methods evaluated. These studies further revealed defects in all methods evaluated except the KOBr–HF method (1966) proposed by Silva and Bremner. Bremner et al. (1967) concluded that the most reliable estimates of nonexchangeable NH4 in soils could be obtained by the method of Silva and Bremner (1966) involving oxidation of exchangeable NH4 and organically bound N to N2 using alkaline KOBr. However, Bremner et al. (1967) also indicated that the method of Silva and Bremner (1966) underestimated the amount of fixed NH4 in soils by failing to recover NH4 present as metal NH4 phosphates. They further stated that there is currently no evidence to indicate that soils normally contain a significant amount of NH4 that is in the form of tarankites or other complex NH4 phosphates. Therefore, Bremner et al. (1967) cautioned that this method was most dependable for measuring native fixed NH4 that was well protected within the clay lattice.
During the past 30 yr, the method of Silva and Bremner (1966) has been used extensively to study the amounts and availability of nonexchangeable NH4 in soils. In this method, K is included to prevent the release of fixed NH4 during the removal of organically bound N. While K is effective in protecting fixed NH4 within the clay lattice (Bremner et al., 1967), there is less reason to assume that it is completely effective in preventing the release of RF or other nonexchangeable NH4 retained near the edges of the clay lattices. During treatment of soil with a strongly alkaline (pH 14) oxidizing agent (2 M KOBr) at boiling temperatures, a portion of the less strongly held nonexchangeable NH4 may be released as NH3 due to partial dissolution of clay edges. Ammonium not protected by the clay lattice is oxidized to N2 by this procedure. Concern that the method of Silva and Bremner (1966) does not quantitatively recover RF NH4 was supported by preliminary studies in our laboratory indicating that this method recovered only a portion of NH4 fixed by clay minerals in samples previously treated to remove organic matter.
Our objectives were (i) to develop a method that quantitatively measures the amount of RF as well as native fixed nonexchangeable NH4 in soils, (ii) to assess the accuracy and precision of this method and compare the results obtained with other methods using a range of soils before and after fixation of added NH4, and (iii) to verify that determination of NH4 in 5 M HF:1 M HCl soil digests by a NH3 diffusion technique does not result in values inflated by partial recovery of organic N.
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Materials and methods |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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Ammonium in acid digests and KCl extracts was determined using an automated prototype system employing the technique described by Carlson (1978) where NH4 is determined by measuring the change in electrical conductivity of a 5 g L-1 H3BO3 solution caused by gaseous diffusion of NH3 across a Teflon membrane. A comparable instrument is commercially available from Alltech Associates (Deerfield, IL). During analysis, samples were injected with sufficient 2 M KOH to increase the pH of dilute acid digests to pH 9. This pH is preferred because it optimizes diffusion of NH3 across the membrane and is not sufficiently alkaline to cause detectable hydrolysis of labile organic N in the acid digest during analysis. This alkali solution contains 40 g L-1 of diethylenetriamine pentaacetic acid to prevent the precipitation of metals during analysis. The instrument was calibrated and its performance was monitored by analyzing a series of four standards (0, 5, 1, and 3 mg NH4–N L-1) before determining the NH4 concentration of each set of 20 samples.
Method B
Nonexchangeable NH4 was determined in this procedure by steam distillation of 5 M HF:1 M HCl digests of samples previously treated with 2 M KOBr as described by Silva and Bremner (1966).
Method C
This procedure is similar to Method B except that the determination of NH4 in diluted 5 M HF:1 M HCl acid digests was performed using the automated NH3 diffusion technique as described under proposed Method A.
Method D
This procedure is a modification of that proposed by Rodrigues (1954) where nonexchangeable NH4 was determined as the difference between NH4 in 2 M KCl extracts and soil digests after dissolution of clay minerals with 5 M HF:1 M HCl. The original procedure involved the use of a mixture of hot HF–H2SO4 to release nonexchangeable NH4 prior to distillation with strong alkali. To minimize the hydrolysis of organic N during dissolution of clay minerals, the digestion procedure was altered to use a mixture of 5 M HF:1 M HCl as recommended by Bremner (1965b). This method differs from Method A in that NH4 in 5 M HF:1 M HCl digests was determined by steam distillation after addition of 15 mL of 10 M KOH rather than by the automated NH3 diffusion technique. Steam distillation with strong alkali partially recovers organic N (Bremner, 1959). This method was included primarily to provide an additional estimate of RF NH4 by comparing results obtained before and after exposure of samples to added NH4 on the assumption that the same extent of organic interference would occur with and without addition of NH4.
Effect of 2 M KOBr Treatment on Native Fixed Ammonium
Experiments were performed to determine if significant losses of native fixed NH4 occurred during treatment of soils with 2 M KOBr. In these experiments, samples of Commerce (fine-silty, mixed, nonacid, thermic Aeric Fluvaquent) and Sharkey (very-fine, montmorillonitic, nonacid, thermic Vertic Haplaquept) soils and bentonite clay were treated once or twice with 2 M KOBr to oxidize organic matter and to remove labile organic N and exchangeable NH4. Commerce and Sharkey soils, having contrasting soil properties and representing the agriculturally important alluvial soils of the southern lower Mississippi Delta, were selected along with bentonite for further studies. Samples were treated with 5 M HF:1 M HCl to release entrapped NH4 as described by Silva and Bremner (1966). The amount of NH4 in the acid digest was determined by the automated NH3 diffusion technique described.
Effect of 2 M KOBr Treatment on Recently Fixed Ammonium
Similar experiments were performed to determine the effects of 2 M KOBr treatment on recovery of recently entrapped nonexchangeable NH4 with the above same two soils and bentonite. Organic matter in these samples was destroyed using 2 M KOBr. The samples were then washed with a dilute salt solution (0.016 M CaCl2, 0.004 M MgCl2, and 0.001 M KCl). This process was repeated two more times to ensure the complete removal of exchangeable and free NH4 in samples after the oxidation of organic matter. Samples were then incubated with 1 mL of an aqueous solution containing 2 mg N kg-1 as (NH4)2 SO4 for 96 h at 27°C. To inhibit microbial immobilization of added NH4, two to three drops of CHCl3 were added prior to capping each centrifuge tube. After incubation for 96 h, 2 M KCl was added at a 1:10 ratio. The sample was shaken on a reciprocal shaker for 2 h and was then centrifuged for 5 min (3000 relative centrifugal force [RCF]). The supernatant was carefully decanted and discarded. Another aliquot of 2 M KCl was added and the tube was shaken manually for 2 to 3 min, and subsequently centrifuged for 5 min. Washing with 2 M KCl was repeated four times to ensure the complete removal of exchangeable and free NH4. Samples were then treated with 5 M HF:1 M HCl to release entrapped NH4 as described by Silva and Bremner (1966). The amount of NH4 in the acid digest was determined by the automated NH3 diffusion technique described.
Enrichment of Soils with Recently Fixed Ammonium
One gram of air-dried soil was placed in a 50-mL screw-capped polypropylene centrifuge tube, and 1 mL of an aqueous solution containing 0.5 mg N as (NH4)2SO4 was added. To inhibit microbial immobilization of added NH4, 2 to 3 drops of CHCl3 were added prior to capping each centrifuge tube. After incubation for 96 h at room temperature (27°C), 10 mL of 2 M KCl was added, the centrifuge tube was placed in a reciprocal shaker for 2 h, and was then centrifuged for 5 min (3000 RCF). The supernatant was carefully transferred to a 100-mL volumetric flask. Another 10 mL aliquot of 2 M KCl was added and the tube was shaken manually for 2 to 3 min, and subsequently centrifuged for 5 min. The supernatant was added to the volumetric flask. This process was repeated two more times to ensure the removal of exchangeable and free NH4 in the soil after the fixation process. The flask containing the supernatant was brought to volume with deionized water, and NH4 was determined by NH3 diffusion through a Teflon membrane as described above. The enrichment of RF NH4 obtained by this procedure was calculated as the difference between the amount of NH4 added and the amount recovered in 2 M KCl washings after incubation for 96 h at 27°C.
Recovery of Recently Fixed Ammonium
The capacity of the various methods to recover RF NH4 was assessed by determining the difference in nonexchangeable NH4 content obtained before and after fixation of added NH4. These values were compared with the amounts calculated as RF in the procedure described above.
Effects of Various Highly Soluble Organic Amendments on Ammonia Diffusion Technique
To confirm that the treatment of soils with 5 M HF:1 M HCl or the determination of NH4 by the NH3 diffusion technique does not give values inflated by organic N, samples of bentonite (W.H. Curtain & Co., New Orleans, LA) were amended with various nitrogenous materials prior to analysis. Because some of the organic N sources used were highly soluble in water, Method A was slightly modified for these experiments. Samples of bentonite were washed with 2 M KCl prior to, rather than after, addition of organic N compounds. Washed bentonite clay samples were amended with 50 g kg-1 alanine, humic acid, rice straw (Oryza sativa L.), clover (Trifolium pratense L.), or microbial cells. The amended bentonite clay samples were incubated for 24 h at 27°C with 5 M HF:1 M HCl in a reciprocal shaker to release NH4–N.
Microbial cells were produced by inoculating Bacto tryptic soy broth (Difco, Detroit, MI) with a few drops of a suspension of freshly collected soil. After incubation for 1 wk at 27°C, cells were harvested by centrifugation and washed three times in deionized water before addition to washed bentonite clay. To confirm that the NH3 diffusion technique quantitatively recovered NH4 in acid digests, 500 mg N kg-1 as (NH4)2SO4 was added to purified bentonite samples prior to digestion, and NH4 in acid digests was determined.
Effects of Leaching Samples Prior to 5 M HF:1 M HCl Treatment
Experiments were performed to determine whether leaching prior to acid digestion could minimize partial recovery of organic N in samples containing appreciable amounts of plant residues. Nitrogen-enriched bentonite samples containing 100 mg of recently fixed NH4–N kg-1 were amended with 50 or 100 g kg-1 of finely ground fresh rice straw, wheat straw (Triticum aestivum L.), clover, or vetch (Vicia sativa L.) and then subsequently leached with 0.01, 0.1, or 1.0 M HCl or with 2 M KCl prior to 5 M HF:1 M HCl digestion. Ammonium in acid digests was determined by the NH3 diffusion technique described.
Statistical Analyses
All the experiments were conducted with four replicates, and the data were analyzed using SAS statistical package (SAS Institute, 1988). Differences among means were tested using Duncan multiple range test (DMRT) for significant difference with a significance level of P < 0.05.
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Results and discussion |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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Effect of 2 M KOBr Treatment on Fixed Ammonium
Experiments performed to determine the effect of 2 M KOBr treatment on the recovery of fixed NH4 indicated a significant loss of fixed NH4. Samples that received a second treatment of 2 M KOBr contained 6 to 33% less NH4 than those receiving a single treatment (Table 2)
. These observations demonstrate the potential of the 2 M KOBr pretreatment to cause significant inadvertent losses of native nonexchangeable NH4 in soils. The decrease from a second KOBr treatment was much larger for the Commerce soil than for the Sharkey soil, which had a much greater content of organic C (Table 1) and less nonexchangeable NH4 (Table 2). The larger decrease of native fixed nonexchangeable NH4 for the Commerce soil was possibly due to substantial amount of nonexchangeable NH4 present in hydroxy-interlayered vermiculite and low-charge vermiculite components of the clay minerals. These two 2:1 clay minerals are typically characterized by lower charge density compared with other 2:1 type expandable clay minerals (Table 1). These minerals tend to hold NH4 very loosely compared with other 2:1 type expandable clay minerals. The effectiveness of KOBr treatment to remove labile organic N was thoroughly demonstrated by Bremner et al. (1967) with soils having a wide range of organic C.
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Regardless of the mechanisms and sample types involved, the results reported in Table 2 clearly demonstrate that treating soils with hot 2 M KOBr appears to result in partial loss of nonexchangeable NH4 that is not well protected within the clay lattice. Because RF and other NH4 entrapped near the edges of the clay lattice are most likely to contribute plant-available N, a method was sought that quantitatively recovers these fractions.
Rodrigues (1954) proposed a method of determining nonexchangeable NH4 released upon steam distillation of HF–H2SO4 soil digest with a strong alkali (Method D). Bremner and Harada (1959) showed that values obtained by this method were inflated by partial recovery of organically bound N during digestion and distillation. Bremner (1959) proposed an alternative method employing digestion of soil with 1 M HF:1 M HCl and distillation of neutralized digests with phosphate buffer (pH 8.8). Bremner (1965b) subsequently concluded that this distillation procedure resulted in partial recovery of organic N and recommended the use of the method of Silva and Bremner (1966) employing 2 M KOBr (Method B).
Membrane Diffusion Technique to Minimize Interference of Labile Organic Nitrogen
Method A is similar to that described by Bremner (1959) but employs an alternative technique for measuring NH4 in an effort to avoid interferences from organic N associated with distillation. The automated NH3 membrane diffusion technique used in Method A to determine the amount of NH4 released by 5 M HF:1 M HCl digestion of soils minimizes the recovery of labile organic N. Results indicated that addition of 50 g kg-1 of alanine or humic acid or 150 g kg-1 of a washed microbial consortium did not result in a detectable increase in the values obtained for nonexchangeable NH4 (Table 3)
. However, addition of 50 g kg-1 of finely ground rice straw or clover residues caused slight increases in the values obtained. Although these increases were detectable (10.5–24.2 mg NH4 –N kg-1), they represented <1% of the N contained in these materials (Table 3). These findings are similar to those reported by Bremner and Harada (1959) who conducted an exhaustive study on the release of NH4 by treatment of organic nitrogenous materials with HF–HCl. They found that generally <1% of the N was converted to NH4 and concluded that acid-labile organic nitrogenous compounds occur only in trace amounts in most soils and therefore such interferences are typically negligible. Results also indicated that Method A quantitatively recovered NH4 added as a solution [500 mg (NH4)2 SO4– N kg-1] to bentonite clay 2 h prior to analysis (Table 3). This result further confirmed the ability of the membrane diffusion technique employed in the proposed method to recover the added NH4 that existed as exchangeable and nonexchangeable NH4 in 5 M HF:1 M HCl soil digests.
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100 mg RF NH4–N kg-1) containing 50 or 100 g kg-1 of finely ground fresh rice straw, wheat straw, clover, or vetch prior to 5 M HF:1 M HCl digestion indicated very little difference in recovery of fixed NH4 (Table 4)
. The observed slight increase (<0.4%) in NH4–N for N-enriched bentonite clay samples amended with plant materials was possibly due to the conversion of plant N to NH4 or the presence of free NH4 in plant materials. Among the various preleaching reagents used, the data obtained with 2 M KCl tended to be lower than those with no extractant (Table 4). These findings suggest that interferences in samples containing plant residues were due largely to the presence of readily extractable NH4 rather than to labile organic N released during acid digestion or NH4 determination. Extraction of samples with 2 M KCl prior to acid digestion as described for Method A minimizes these interferences.
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The standard deviations of four replicate analyses were similar for the four methods, and averaged 1.1, 1.1, 1.6, and 1.2 mg NH4 kg-1 for Methods A, B, C, and D, respectively (Table 5). However, the NH3 membrane diffusion technique may be less subject to systematic errors during routine use than in steam distillation because the instrument is continually recalibrated by periodic analysis of a series of standards and because it does not require the subtraction of a "blank" value.
The samples used in the experiments reported in Table 5 had been stored in an air-dried state for several months or more prior to their use. Therefore, they cannot be considered to contain appreciable amounts of RF NH4. To compare the ability of the various methods to recover RF NH4, samples of each horizon were equilibrated with 500 mg (NH4)2 SO4– N kg-1 of soil for 96 h in sealed containers after addition of a few drops of CHCl3 to inhibit microbial immobilization. A similar NH4 enrichment procedure described above was adapted to obtain samples enriched with RF NH4. After thoroughly washing samples with 2 M KCl, the amounts of nonexchangeable NH4 present were determined by each method. Table 6 shows the difference in nonexchangeable NH4 recovered by each method before and after fixation of added NH4. Values obtained by Methods A and D were similar to the amounts of RF NH4 calculated as the difference between the amounts of NH4 added and the amounts recovered in 2 M KCl washings after equilibration (RF). Values obtained by methods employing 2 M KOBr pretreatment (Methods B and C) were an average of 35% less than those obtained by methods not employing 2 M KOBr. Regression analyses showed no clear relationships between the losses caused by 2 M KOBr pretreatment and the soil properties given in Table 1 for 17 additional chemical, physical, and mineralogical properties that have been determined for this set of soils (Paramasivam and Breitenbeck, 1994).
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Conclusions |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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Received for publication January 18, 1999.
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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This article has been cited by other articles:
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  G. D. Eudoxie and G. A. Gouveia Microdiffusion of Strongly Fixed Ammonium in Soil-Acid Digests Prior to Automated Nitrogen-15 Analysis Soil Sci. Soc. Am. J., November 1, 2001; 65(6): 1846 - 1852. [Abstract] [Full Text] [PDF]
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