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Illinois Fertilizer Conference Proceedings
January 25-27, 1999

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Direct Diffusion Methods for Analysis of Soil Nitrogen

S.A. Khan, R.L. Mulvaney, and R.G. Hoeft1
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Introduction
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The supply of N limits the growth and productivity of non-leguminous crops more often than does the supply of any other mineral nutrient. The development, availability, and use of synthetic N fertilizers have played a major role in the dramatic increases in crop yields (especially corn) that have occurred in the United States since World War II. Domestic consumption of N fertilizers has increased tremendously during this period, and nowhere has the increase been greater than in Illinois, which leads the nation in fertilizer use.

The use of N fertilizers is complicated by the dynamic nature of N-cycle processes in soil. Because these processes are greatly affected by weather conditions, N rate recommendations are necessarily subject to considerable uncertainty. Sufficient N must be applied to meet crop demand; however, overfertilization reduces profitability and may have an adverse effect on the environment by promoting loss of N through leaching or denitrification.

In Illinois, N fertilizer rates for corn production are based on the yield goal and the corn:N price ratio, with adjustments to allow for N derived from legumes, manure, and so on. This approach has generally led to profitable fertilizer use, although a recent survey of farmers in Champaign County suggests that the recommended rate is often exceeded, due in part to the use of inadequate N credits.

During the past decade, there has been growing interest in soil testing to estimate N availability, so as to improve the accuracy of N fertilizer recommendations. Numerous tests have been proposed to estimate mineralization of soil organic N (e.g., see Bremner, 1965; Bundy and Meisinger, 1994). Use of these tests has been limited because most are time-consuming, and the results are often poorly correlated with fertilizer responses in the field (Fox and Piekielek, 1984: Thicke et al., 1993).

Soil nitrate (NO3-) testing is an alternative to tests that estimate mineralizable organic N. For several years, a preplant NO3- test (PSNT) has been used with some success west of the Missouri River. More recently, a presidedress NO3- test has been used in Iowa and a few other Corn Belt states. The latter test was originated in Vermont (Magdoff et al., 1984) to improve the accuracy of N rate recommendations by taking into account N derived from manure, but can only be used in cases where N is to be applied as a sidedressing.

In a recent FREC project, the PPNT and PSNT were evaluated for their accuracy in predicting N fertilizer applications to optimize corn yield, relative to those by the conventional approach (Brown et al., 1993). The results showed that neither of the soil tests provided sufficient advantage over the conventional approach to justify their use. No response to N was observed at 33 of the 77 study sites. At 13 of the nonresponding sites, the lack of response could not be attributed to a specific cause (drought, a recent application of manure, or a previous forage legume); however, available P and K tended to be high, indicating the likelihood of manure application in the past. Surprisingly, neither NO3- soil test provided an accurate N recommendation for these 13 nonresponding sites.

When soils are fertilized with N, a substantial proportion of fertilizer N (usually between 10% and 20%) is incorporated into organic forms (i.e., immobilized) during the growing season. In subsequent seasons, some of the residual N becomes plant-available through mineralization. Several studies have demonstrated that recently immobilized N is mineralized much more rapidly than is native soil organic N (e.g., Allen et al., 1973; Shen et al.,1989). There is growing evidence that long-term application of N fertilizers promotes the buildup of rapidly mineralizable (i.e., labile) N in soils, and may reduce crop response to further applications of fertilizer N (Motavalli et al., 1992; Stevens et al., 1996).

As a first step toward developing a soil test to detect the presence of labile organic N in soil, simple Mason-jar diffusion methods recently developed in our laboratory for inorganic N and 15N analysis of soil extracts (Mulvaney et al., 1997a; Khan et al., 1997) were modified to permit these analyses to be carried out directly on the soil sample itself, without extraction. The direct approach has been employed for inorganic N analysis by steam distillation (Keeney and Bremner, 1966); however, interference can arise from hydrolysis of soil organic N and from liberation of CO2 during distillation of calcareous soils (Sahrawat and Ponnamperuma, 1978; Clausen et al., 1981). Besides being less expensive and much more convenient than steam distillation, Masonjar diffusion methods are highly accurate and precise, and because diffusions are performed at a much lower temperature than distillations, there is little risk of interference from hydrolysis of organic N (Mulvaney and Khan, 1999), nor does interference arise from liberation of CO2.

The primary purpose of the work reported here was to develop Mason-jar diffusion methods for direct determination of inorganic N in soils. The direct methods were evaluated for analytical accuracy and precision from recovery of N and 15N added as NH4+ or NO3-, and by comparison to inorganic-N analyses by extraction-diffusion.

Materials and Methods
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Soils

The soils used (Table 1) were surface (0-15 cm) samples of 11 soils selected to obtain a wide range in properties. The Fargo soil used was collected in North Dakota. The remaining soils were from Illinois, including a waterlogged organic soil (Houghton), an uncropped sandy soil under coniferous vegetation (Bloomfield), an upland forest soil (Xenia), and seven cultivated soils used for corn (Zea mays L.) and soybean (Glycine max L. Merr.) production. Before use, each sample was air-dried, and unless otherwise specified, the soil was then crushed to pass through a 2-mm screen.

Extraction-Diffusion

Soil extracts were obtained by shaking 20 g of soil with 200 mL of 2 M KCl in a 250-mL polyethylene bottle, and filtering the resulting suspension through Whatman no. 42 filter paper under vacuum (Mulvaney, 1996). Diffusions to determine NH4+-N and (NH4+ + N03-)-N were performed on 10 mL of extract, using the accelerated Mason-jar technique described previously (Khan et al., 1997).

Direct Diffusion

Mason-jar methods for inorganic-N analysis of soil extracts were performed at 25C with or without orbital shaking (Mulvaney et al., 1997a), or at 45-50C with heating on a hot plate (Khan et al., 1997), but instead of soil extract, 1 g of soil was placed in the jar and treated with 10 mL of 2 M KCl.

N-Isotope Analysis

In cases involving diffusion of 15N-treated samples, processing for isotopic analysis was carried out as previously described (Khan et al., 1997; Mulvaney et al., 1997a). Isotopic analyses were performed using a Nuclide Model 3-60-RMS mass spectrometer (Spectrumedix, State College, PA) equipped with an automated Rittenberg system (Mulvaney et al., 1997b).

Results and Discussion
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The determination of inorganic forms of soil N usually involves extraction of the soil with a neutral salt solution such as 2 M KCl. For research applications, the time and effort required for extraction are more than justified by the fact that the extracts can be easily stored for later analysis. This advantage does not apply to soil testing, in which case speed and convenience are at least as important as analytical accuracy and precision. To expedite the processing of samples, determinations of inorganic N should be performed directly on the soil itself.

Recent work in our laboratory has led to simple and inexpensive diffusion methods for determination of inorganic N and 15N in soil extracts and water (Khan et al., 1997; Mulvaney et al., 1997a). These methods are ideally suited for both research and routine testing. In these methods, diffusion is carried out in a 1-pint (473-mL) wide-mouth Mason jar having a lid that supports a petri dish containing boric acid (H3BO3) indicator solution. The H3BO3 solution absorbs NH4+-N liberated as gaseous NH3 by treatment of the sample with MgO, with or without addition of Devarda's alloy to recover NO3--N and/or NO2--N. An especially useful option is to carry out diffusion with gentle heating on a hot plate (Khan et al., 1997), in which case the analysis can easily be completed within a single working day.

Most surface soils contain a large amount of organic N, and this is a potential source of interference in direct determination of inorganic N by diffusion methods, since the alkaline conditions required to effect liberation of NH4+-N as gaseous NH3 promote hydrolysis of organic N, particularly amino sugars. In the Mason-jar methods, such interference is avoided by using MgO as a mild alkali, and by carrying out diffusion at a low temperature. No interference has been detected in analysis of soil extracts when recovery tests were performed using 15N (Khan et al., 1997), and with minor modifications, inorganic-N analyses can even be performed successfully on samples having an exceptionally high content of organic N, such as urine, manure extract, or septic effluent (Mulvaney and Khan, 1999).

To ascertain whether Mason-jar diffusion methods can be used for direct determination of soil inorganic N, a comparison was made of direct analyses by diffusion at 25C with or without orbital shaking or at 45-50C on a hot plate, relative to analyses of soil extracts. The results are summarized in Table 2, which shows that, regardless of the diffusion technique employed, analyses by direct diffusion were in close agreement with those by extraction-diffusion, and analytical precision was comparable. No difference was observed among the 11 soils used, despite the wide range in their physicochemical properties (Table 1). Of particular interest is the fact that the direct approach did not underestimate inorganic N due to liberation of CO2 from a calcareous soil (Harpster), nor did underestimation occur with a soil having a substantial capacity for fixation of NH4+ (Fargo). Moreover, direct analyses were unaffected by the high organicmatter content of a Histosol (Houghton), indicating very little, if any, hydrolysis of labile organic N.

In keeping with standard laboratory procedures, the analyses reported in Table 2 were performed on soil that had been crushed to pass through a 2-mm screen. Considering the need for speed and convenience in the use of direct diffusion for inorganic-N analysis, a study was conducted to evaluate the effect of larger particle size on direct analyses, as compared to analyses of extracts obtained from 2-mm soil. The results (Table 3) indicate no need for fine grinding of soil when analyses are to be carried out by direct diffusion, presumably because soil aggregates quickly form a slurry when treated with 2 M KCl.

Table 4 shows the results of recovery tests to evaluate analytical accuracy and precision when direct diffusions were performed with heating on a hot plate. To prevent NH4+ fixation by 2:1 clay minerals that would reduce recovery of NH4+-N, addition of mineral N followed treatment of the soil with 2 M KCl. Recovery of added NH4+-N or NO3--N was quantitative with each soil studied. The coefficient of variation was usually < 1 %.

Although the direct diffusion methods described were developed primarily for routine testing, these methods allow isotope-ratio analysis of the diffused NH3-N and thereby have potential value for use in 15N-tracer investigations. To evaluate the accuracy and precision of 15N analyses by direct diffusion, replicate diffusions were performed from samples of soil that had been treated with a known amount of 15N-labeled (NH4)2SO4 or KNO3 after addition of 2 M KCl. The results are reported in Table 5, which shows that excellent agreement was obtained between measured and expected values. Besides verifying the use of direct diffusion for 15N analysis, the data in Table 5 provide additional evidence against interference by labile organic N.

To avoid the risk of interference by organic N in direct diffusion of inorganic N, care should be taken to ensure that the diffusion periods specified in Table 2 are not exceeded. This is illustrated by Figure 1, which shows that prolonging the period for direct diffusion on a hot plate led to an increase in analyses for (NH4+ + NO3-)-N, owing to mineralization of labile organic N. As expected from their much higher content of organic matter, the increase was much larger with the Drummer, Fargo, and Houghton soils than with Bloomfield soil. Interestingly, the release of mineral N varied among the three former soils and was not directly related to soil organic-matter content. The latter finding may be of considerable significance in regard to developing a simple soil test to detect sites nonresponsive to N fertilization.

Summary
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Mason-jar diffusion methods previously developed for inorganic-N analysis of soil extracts and water were employed for direct soil analysis. Direct diffusions were carried out either at room temperature or with gentle heating on a hot plate, following treatment of 1 g of soil with 10 mL of 2 M KCl in a 1-pint Mason jar. Analyses by direct diffusion were in close agreement with results obtained by diffusion of soil extracts; moreover, recovery of N or 15N added as NH4+ or NO3 was quantitative. Prolonging the period for direct diffusion led to an increase in inorganic-N analyses, presumably because of mineralization of labile organic N.

Tables and Figures
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Table 1. Analysis of soils.

Table 2. Comparison of extraction-diffusion and direct diffusion for inorganic-N analysis of soils.

Table 3. Effect of soil particle size on analyses for exchangable NH4+ by direct diffusion.

Table 4. Recovery by direct diffusion of NH4+-N and NO3--N added to soil.

Table 5. Accuracy and precision of direct diffusion for 15N analysis of soil inorganic N.

Figure 1. Effect of prolonging the diffusion period on direct analysis of inorganic N in soil.

Footnotes and References
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1 S.A. Khan is a Research Associate and R.L. Mulvaney is a Professor, Dept. of Natural Resources and Environmental Sciences, University of Illinois. R.G. Hoeft is a Professor, Dept. of Crop Sciences, Univ. of Illinois.

Allen, A.L., F.J. Stevenson, and L.T. Kurtz. 1973. Chemical distribution of residual fertilizer nitrogen in soil as revealed by nitrogen-15 studies. Journal of Environmental Quality, 2:120-124.

Bremner, J.M. 1965. Nitrogen availability indexes. In: Methods of soil analysis. Part 2 (C. A. Black et al., ed.). Agron. Monogr. 9. American Society of Agronomy, Madison, WI. pp. 1324-1345.

Brown, H.M., R.G. Hoeft, and E.D. Nafziger. 1993. Evaluation of three N recommendation systems for corn yield and residual soil nitrate. In: 1993 Illinois Fertilizer Conference Proceedings (R. G. Hoeft, ed.). pp. 43-49.

Bundy, L.G., and J.J. Meisinger. 1994. Nitrogen availability indices. In: Methods of soil analysis. Part 2. Microbiological and biochemical properties (R. W. Weaver et al., ed.). SSSA Book Set. 5. Soil Science Society of America, Madison, WI. pp. 951-984.

Clausen, C.R., M.P. Russelle, A.D. Flowerday, and R.A. Olson. Problems in direct steam distillation of soil for mineral nitrogen determination due to carbonates. Soil Science Society of America Journal, 45:1238-1240.

Fox, R.H., and W.P. Piekielek. 1984. Relationships among anaerobically mineralized nitrogen, chemical indexes, and nitrogen availability to corn. Soil Science Society of America Journal, 48:1087-1090.

Keeney, D.R., and J.M. Bremner. 1966. Determination and isotope-ratio analysis of different forms of nitrogen in soils: 4. Exchangeable ammonium, nitrate, and nitrite by directdistillation methods. Soil Science Society of America Proceedings, 30:583-587.

Khan, S.A., R.L. Mulvaney, and C.S. Mulvaney. 1997. Accelerated diffusion methods for inorganic-nitrogen analysis of soil extracts and water. Soil Science Society of America Journal, 61:936-942.

Magdoff, F.R., D. Ross, and J. Amadon. 1984. A soil test for nitrogen availability to corn. Soil Science Society of America Journal, 48:1301-1304.

Motavalli, P.P., L.G. Bundy, W.W. Andrasaki, and A.E. Peterson. 1992. Residual effects of long-term nitrogen fertilization on nitrogen availability to corn. Journal of Production Agriculture, 5:363-368.

Mulvaney, R.L. 1996. Nitrogen-Inorganic forms. In: Methods of soil analysis. Part 3. Chemical methods (D. L. Sparks et al., ed.). SSSA Book Ser. 5. Soil Science Society of America, Madison, WI. pp. 1123-1184.

Mulvaney, R.L., and S.A. Khan. 1999. Use of diffusion to determine inorganic nitrogen in a complex organic matrix. Soil Science Society of America Journal (in press).

Mulvaney, R.L., S.A. Khan, W.B. Stevens, and C.S. Mulvaney. 1997a. Improved diffusion methods for determination of inorganic nitrogen in soil extracts and water. Biology and Fertility of Soils, 24:413-420.

Mulvaney, R.L., S.A. Khan, G.K. Sims, and W.B. Stevens. 1997b. Use of nitrous oxide as a purge gas for automated nitrogen isotope analysis by the Rittenberg technique. Journal of Automatic Chemistry, 19:165-168.

Mulvaney, R.L., and L.T. Kurtz. 1982. A new method for determination of 15N-labeled nitrous oxide. Soil Science Society of America Journal, 46:1178-1184.

Sahrawat, K.L., and F.N. Ponnamperuma. 1978. Measurement of exchangeable NH4+ in tropical rice soils. Soil Science Society of America Journal, 42:282-283.

Shen, S.M., P.B.S. Hart, D. S. Powlson, and D. S. Jenkinson. 1989. The nitrogen cycle in the Broadbalk wheat experiment: 15N-labelled fertilizer residues in the soil and in the soil microbial biomass. Soil Biology & Biochemistry, 21:529-533.

Stevens, W.B., R.G. Hoeft, and R.L. Mulvaney. 1996. Effect of N fertilization on accumulation and release of readily-mineralizable organic N. In: 1996 Illinois Fertilizer Conference Proceedings (R. G. Hoeft, ed.). pp. 91-104.

Sumner, M.E., and W.P. Miller. 1996. Cation exchange capacity and exchange coefficients. In: Methods of soil analysis. Part 3. Chemical methods (D. L. Sparks et al., ed.). SSSA Book Ser. 5. Soil Science Society of America, Madison, WI. pp. 1201-1229.

Thicke, F.E., M.P. Russelle, O.B. Hesterman, and C.C. Sheaffer. 1993. Soil nitrogen mineralization indexes and corn response in crop rotations. Soil Science, 156:322-335.

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