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NUTRIENT MANAGEMENT & SOIL & PLANT NUTRITION

Modification of the Illinois Soil Nitrogen Test to Improve Measurement Precision and Increase Sample Throughput

Department of Crop and Soil Environ. Sci., Smyth Hall, Virginia Tech, Blacksburg, VA 24061

^* Corresponding author (jspargo{at}vt.edu).

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Quantification of organic N mineralization during the corn (Zea mays L.) growing season should improve the precision of fertilizer N recommendations. The Illinois soil N test (ISNT) has shown promise in some regions as a useful tool for estimating mineralizable soil N; however, the procedure needs to be modified for use in routine soil testing labs that must process hundreds of samples per day. The assay determines alkali hydrolyzable N by treating 1 g of soil with 10 mL of 2 mol L^–1 NaOH in a 473-mL wide-mouth Ball jar, and heating for 5 h at 50°C on a hot plate to liberate (NH₄⁺ + amino sugar)–N as gaseous NH₃, which is collected in H₃BO₃ solution and subsequently determined by acidimetric titration. The objectives of this study were to determine if variance in measurement values could be reduced and sample throughput increased while maintaining accuracy by using an incubator to replace the hot plate as the heat source. Thirty-five soils collected from N-response trials in Virginia were used in this study. Jars were heated in an incubator at 50°C for 5, 6, 7, 8, 9, 10, or 15 h. Soil samples were also analyzed with the unmodified method for comparison. All determinations were conducted in triplicate. Use of an incubator set to 50°C reduced the total recovery of N from the samples, but increasing the diffusion period increased N recovery. The 15-h diffusion period resulted in quantitative recovery of ISNT-N with significantly (P < 0.05) improved measurement precision compared with the unmodified method (CV = 4.3 vs. 7.4, respectively). Modifying the ISNT by using an incubator instead of a hot plate increases measurement precision and allows greater sample throughput.

Abbreviations: EONR, economically optimum nitrogen rate • INC5-, INC6-, INC7-, INC8-, INC9-, INC10-, and INC15-ISNT, Illinois soil nitrogen test incubator method with 5-, 6-, 7-, 8-, 9-, 10-, or 15-h diffusion period, respectively • ISNT, Illinois soil nitrogen test

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Fertilizer N applications for corn are generally based on expected yield. This method fails to account for variations in soil N supplies and often results in over- or underfertilization with N (Mulvaney et al., 2005). Fertilizer N needs of corn often differ widely among and within fields (Scharf, 2001) due to variations in soil N supply. Fertilizer N application recommendations are generally adjusted to account for estimated amounts of N mineralized from organic N sources such as manures, biosolids, and legumes, but there is a great deal of uncertainty in estimates of N availability from soil N supplies due to its dynamic nature. In addition, recommendations do not generally account for the influence of management practices such as tillage, the use of cover crops, and crop residue management on mineralizable soil organic N.

A soil-based approach that attempts to quantify organic N turnover could improve the precision of fertilizer N recommendations and increase N use efficiency. A number of chemical methods have been proposed for estimating soil N availability (Stevenson and Cole, 1999). Chemical methods designed to estimate potentially mineralizable N have been based on an empirical approach, and their use has been limited due to low correlations with mineral N and crop N uptake (Khan et al., 2001; Stevenson and Cole, 1999). Soil NO₃–N testing is currently the best method for identifying soils where yield response to fertilizer N may be limited. Preplant soil profile NO₃–N testing has long been recommended for assessment of N availability in drier areas of the western United States where leaching losses are minimal (Bundy and Meisinger, 1994; Hergert, 1987) and the pre-sidedress soil NO₃–N test (PSNT) has shown potential for modifying fertilizer N recommendations in the humid, eastern United States for corn grown on land receiving manure or where legumes have been grown in the rotation (Andraski and Bundy, 2002; Evanylo and Alley, 1997; Fox et al., 1989; Magdoff et al., 1984; Meisinger et al., 1992; Roth et al., 1992; Sims et al., 1995). Use of the PSNT has been limited by the need to collect samples during the growing season and delay N fertilization until soil samples are analyzed. In addition to the logistical problems, the PSNT is also limited by the variability of soil NO₃–N due to its dependence on a number of factors such as temperature, moisture, and soil texture (Khan et al., 2001).

Ideally, a soil N test would estimate a labile organic fraction that supplies plant- available N during the growing season (Khan et al., 2001). This approach depends on fewer N-cycle processes and is therefore less variable. The Illinois soil N test (ISNT) was developed by Khan et al. (2001) as a simple soil assay to identify soils that are unresponsive to N fertilization. Khan et al. (2001) were able to identify soils unresponsive to fertilizer N using the ISNT with a critical range of 225 to 235 mg N kg^–1. They found a wide range of N-test values for both responsive and unresponsive sites and suggested the possibility of using the soil test to quantitatively determine fertilizer N rates in conjunction with expected yield goals.

The ISNT has also been found to provide useful data in other regions of the United States. Klapwyk and Ketterings (2006) were able to identify unresponsive corn silage fields on dairy farms in New York using the ISNT; however, soil organic matter had to be included in the model to accurately identify unresponsive locations. Williams et al.(2007b) successfully predicted the economic optimum N rate (EONR) for corn grown on well (r² = 0.87) or poorly drained soils (r² = 0.78) in North Carolina.

The assay has not proven useful in all regions or with all cropping systems. For example, working in Iowa, Barker et al. (2006) found no relationship between the ISNT and relative corn grain yield, corn response to fertilizer N, or EONR. The soils used in their study had relatively high levels of hydrolyzable NH₄–N relative to amino sugar N and they suggested that this may partially explain the poor performance of the ISNT. Marriott and Wander (2006) used the assay to compare labile soil N in conventional and organic cropping systems and found that the ISNT was not a sensitive index of labile N. The ISNT fraction was not preferentially enriched by organic management and its response was similar to that of total soil C and N. For these reasons, it is not likely that the assay will be adopted as a universal soil N test. Rather, its use will be limited to regions and applications were the assay has proven useful.

The ISNT was developed through work that attempted to find a relationship between different fractions of hydrolyzable soil N and corn yield response to fertilizer N (Mulvaney et al., 2001). Mulvaney et al. (2001) used acid hydrolysis to determine the concentration of hydrolyzable NH₄⁺, amino acid N, amino sugar N, and total hydrolyzable N in soils collected from 18 fertilizer N response studies. Their work showed that hydolyzable amino sugar N was highly correlated (r = 0.79) with check plot yield and fertilizer N response (r = –0.82). The hydrolysis and N fractionation procedures are complicated and time consuming and are therefore unsuitable for routine soil analysis. For these reasons, Khan et al. (2001) developed the simpler ISNT method to estimate amino sugar N.

The ISNT, described in detail in Technical Note 02-01 (University of Illinois, 2004) and Khan et al. (2001), directly diffuses alkali hydrolyzable soil N, eliminating the acid hydrolysis procedures from earlier methods described by Mulvaney and Khan (2001). The test is thought to recover amino sugar N, derived primarily from bacterial and fungal cell walls, plus extractable NH₄–N (Khan et al., 2001). It is probable that some -amino-N is also released (Greenfield, 2001). The ISNT uses hot-plate griddles, Ball jars, Pyrex petri dishes, a microburette or automatic titrator, and commonly available chemical reagents. The test is relatively simple; however, throughput is limited by the number of samples that can be heated on a single hot plate (n = 10–12) and precision of analysis is reduced by uneven heating of the hot plates and environmental fluctuations within the laboratory (i.e., ambient air temperature and drafts) (Klapwyk and Ketterings, 2005).

Several modifications have been made to the original ISNT method in an attempt to reduce variability. The method originally published by Khan et al. (2001) was modified by rotating the position of the jars following heating for 1.5 and 3 h to ensure more even heating among samples (University of Illinois, 2004). Klapwyk and Ketterings (2005) enclosed the griddles in a plywood box to eliminate drafts, reduce the effects of ambient laboratory temperature, and eliminate the need to rotate jars. They found that use of the enclosed griddle method slightly reduced the CV (3.8% with open griddles, 2.5% with enclosed griddles; P = 0.013). While these modifications have improved precision of the ISNT, they have done nothing to increase throughput. In order for the method to be adopted for routine use in commercial and institutional soil testing laboratories, where thousands of samples are processed per year, throughput must be increased.

Replacing the griddle with an incubator as the heat source is a way to increase sample throughput, as a single laboratory incubator can easily heat 100 samples simultaneously with uniform heating, eliminating the need to rotate the jars. Khan et al. (1997) found that using an incubator reduced the amount of NH₃ diffused from the soil samples. They attributed the reduced N recovery to a lack of temperature gradient within jars in the incubator.

Williams et al. (2007a,b) used an incubator to replace the griddle as the heat source, but did not report data comparing the incubator procedure with the original method. Williams et al. (2007b) did not find any sites that were unresponsive to fertilizer N, but extrapolation of their EONR vs. ISNT-N regression equation for well-drained sites (EONR, kg ha^–1 = 348 – 3.12 x ISNT, mg N kg^–1) and solving for an economically optimum N rate of 0 gives an ISNT critical value of 112 mg N kg^–1. This is approximately 50% of critical levels identified by Khan et al. (2001) and Klapwyk and Ketterings (2006) and suggests that replacement of the griddle with an incubator may lead to a reduction in recovered ISNT-N. Reduced recovery of N may decrease sensitivity of the assay, since soil test levels would represent a narrower range than the original method.

The objectives of this research were to determine if variation in measurement values could be reduced and sample throughput increased while maintaining accuracy by using an incubator to replace the hot plate as the heat source for the ISNT procedure.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Soils
Sample Collection and Preparation
The 35 soils used in this study (Table 1 ) were collected from locations throughout Virginia used for corn fertilizer N response trials. Locations were selected to represent the wide range of climatic conditions, soil properties, and management practices existing in the region. Soil samples were collected from late March to early April of 2006 or 2007 and consisted of a composite of 10 to 15 soil cores collected to a depth of 15 cm from each location. Soil samples were stored at 4°C immediately following collection, then rapidly air dried after returning to the lab. All soils were ground to pass a 2-mm sieve.

Sample Characterization
Soil samples were analyzed for pH, total C and N, and 2 mol L^–1 KCl extractable NH₄–N and NO₃–N. Soil pH was measured on 1:1 soil/water mixtures after equilibration for 30 min. Total soil C and N were determined using a VarioMax CNS macro elemental analyzer (Elementar, Hanau, Germany). Soil NH₄–N and NO₃–N were extracted with 2 mol L^–1 KCl for 1 h on a reciprocating shaker and determined colorimetrically using a QuickChem automated ion analyzer (Lachat Instruments, Milwaukee, WI).

Illinois Soil Nitrogen Test Procedures
Standard Method
The unmodified ISNT was conducted using the procedures described in detail in Technical Note 02-01 (University of Illinois, 2004) and Khan et al. (1997). Briefly, commercial griddles with the original temperature controls (Model 76220, West Bend Housewares, West Bend, WI) and 473-mL (1-pint) wide-mouth Ball jars (Jarden Home Brands, Daleville, IN) were used. Ball jar lids were fitted with machine screws and cable ties to secure 60-mm-diam. Pyrex petri dishes (Corning Glass Works, Corning, NY) as described by Khan et al. (1997). The forward legs of the griddles were slightly elevated to allow condensation to run down the screw supporting the petri dish and not into the dish (Khan et al., 1997). Before use, temperature controls were set so that a temperature of 50 ± 2°C was achieved when a thermometer was placed in 100 mL of deionized water in an open jar placed in the center of the griddle. Two griddles were used for our research. The griddles were placed side by side, oriented in the same direction, under a laboratory fume hood.

A 1-g sample was weighed in to each Ball jar and spread evenly around the bottom before adding 10.0 mL of 2 mol L^–1 NaOH. The NaOH solution was added in such a way as to thoroughly mix with the soil while preventing soil adherence to the walls of the jar. Immediately following the addition of the NaOH solution, the jars were fitted with the lid and petri dish apparatus, containing 5.0 mL of 4% w/v H₃BO_3, and tightly secured with a metal screw band. Twelve sample jars were placed on the griddle and rotated at 1.5 and 3 h after initiation of heating.

In addition to the 12 unknown soil samples, two additional jars containing a quality control soil standard and 1 mL of standard glucosamine solution (1 mg N mL^–1) were placed in the center of each griddle for quality control during every run. The position of the quality control jars was fixed within and between runs to limit the effect of temperature variance across the griddle on recovered N (Klapwyk and Ketterings, 2005).

Following 5 h of heating, jars were removed from the griddles, allowed to cool to room temperature, and opened to release petri dishes from lids. The H₃BO₃ solution was diluted with 5.0 mL of deionized water and titrated with standardized 0.01 mol L^–1 H₂SO₄. Titrations were conducted using a Radiometer TIM 900 Titration Manager and ABU901 autoburette (Radiometer Analytical S.A., Lyon, France). Before titration, the end point was established by measuring the pH of a solution prepared by mixing 5 mL of 4% (w/v) H₃BO₃ with 5 mL of deionized water. Soil test N (mg N kg^–1) was determined as ST, where S is milliliters of titrant and T is the titer (µg N mL^–1) of the standardized H₂SO₄.

Incubator Method
The modified ISNT was conducted using the same procedures described above except that the heat during diffusion was provided by a Precision Model 815 low-temperature incubator (Thermo Fisher Scientific, Waltham, MA) set to 50°C. To determine if N recovery changed with diffusion period in the incubator, a six-sample subset (Samples 1–6, Table 2 ) was placed in jars and heated in the incubator for 5, 6, 7, 8, 9, 10, and 15 h (referred to hereafter as INC5-, INC6-, INC7-, INC8-, INC9-, INC10-, and INC15-ISNT, respectively). All 35 samples were heated using the INC15-ISNT method. Both quality control samples were also included in duplicate with each run in the incubator.

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Analytical Accuracy
Based on our preliminarily work and the reports of others (Khan et al., 1997; Klapwyk and Ketterings, 2005; Williams et al., 2007a,b), we expected that replacing the griddle with an incubator as the heat source during alkaline hydrolysis would result in decreased diffusion rates due to the lack of thermal gradient experienced by the diffusion jars in the incubator. Thus, to recover a similar quantity of N using the incubator, the diffusion period would need to be increased. We also expected that recovery of N using an incubator, regardless of diffusion period, would be well correlated to that recovered using a griddle. To test our initial proposition, we used six samples (Soils 1–6, Table 1) with a range of ISNT-N from 96 to 278 mg N kg^–1 (Table 2) and the glucosamine standard.

Nitrogen recovery was lower when diffusion was performed in the incubator relative to the griddle for the 5-h time period used in the original method. Average recovery of ISNT-N for the six soil samples we examined was only 58% for INC5-ISNT (Table 2). There was a near-perfect relationship between ISNT-N and INC5-ISNT-N (r² = 0.9996) as well as all other incubation periods (Fig. 1 ). As diffusion period in the incubator increased, the percentage of recovered ISNT-N from the six soils examined increased (Table 2) and is described well by the Mitscherlich equation (Fig. 2 ; r² = 0.995). Recovered ISNT-N was 99.9% using INC15-ISNT for the initial six soils we analyzed (Table 2) and the relationship between ISNT-N and INC15-ISNT-N was near unity (slope = 1.01, r² = 0.9998; Fig. 1). During the development of the ISNT method, Khan et al. (1997) found a similar relationship between N determined and diffusion period.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Standard Illinois soil N test method (ISNT) vs. the modified method using an incubator with a diffusion period of 5, 6, 7, 8, 9, 10, and 15 h for a subset of selected sites (Sites 1–6, Table 1).

View larger version (7K): [in this window] [in a new window]   Fig. 2. Average percentage of Illinois soil N test (ISNT) N recovered using the incubator method with a diffusion period of 5, 6, 7, 8, 9, 10, or 15 h on a subset of selected sites (Sites 1–6, Table 1).

View larger version (11K): [in this window] [in a new window]   Fig. 3. Percentage of recovered N from the glucosamine standard (1 mg N mL–1) using the incubator method with a diffusion period of 5, 6, 7, 8, 9, 10, or 15 h.

Recovery of glucosamine N following 15 h of diffusion in the incubator was equivalent to that recovered using the unmodified method; however, there was a significant difference between the two methods for our quality control soil standard (Table 3 ). The ISNT-N of the quality control soil standard was about 8% higher than INC15. This may have resulted from the fixed position of the quality control soil standard near the center of the griddle. Klapwyk and Ketterings (2005) found that the elevated temperature of jars placed in the center position resulted in higher measured ISNT-N when jars were not rotated. The reason this difference was not observed for the glucosamine standard could be because all N in the standard was recovered by both methods. Quantitative recovery of N from a standard glucosamine solution confirms that ISNT-N has not been underestimated, but it does not address overestimation. For this reason, a standard soil may be a better quality control sample for routine use.

View this table: [in this window] [in a new window]   Table 3. Average (n = 6) and coefficient of variance (n = 3) of recovered N from two quality control samples using the standard Illinois soil N test method with fixed griddle position (ISNT) or the modified method using an incubator with 15 h of diffusion (INC15).

View larger version (7K): [in this window] [in a new window]   Fig. 4. The relationship between measured N using the standard Illinois soil N test method (ISNT) vs. the modified method using an incubator with a 15-h diffusion period (INC15).

	CONCLUSIONS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
The purpose of this research was to determine if measurement precision could be improved and sample throughput increased while maintaining accuracy by using an incubator to replace a hot plate as the heat source for the ISNT.

The use of an incubator set to 50°C reduced both the quantity of recovered N and the sensitivity of the assay when compared with the standard ISNT. Increasing the diffusion period increased N recovery and a 15-h diffusion period resulted in quantitative recovery of ISNT-N with significantly improved measurement precision. Since there was no significant difference between ISNT-N and INC15-ISNT-N for a set of 35 soil samples with a wide range of ISNT-N values, no correction factor is needed to compare values obtained by either of the two methods.

In addition to improved measurement precision, there are several ancillary benefits to using the INC15-ISNT method. Rotation of jars is not necessary for the incubation method, thus reducing labor demand. On an average day, one person can titrate approximately 100 samples using an autotitrator similar to the one used in our research. A standard upright laboratory incubator can easily accommodate 100 diffusion jars. To run the same number of samples using the standard ISNT method would require 9 or 10 griddles with a technician to rotate the jars throughout the incubation period. Thus, greater sample throughput may be achieved with a smaller laboratory footprint using the incubator method. Logistically, the 15-h incubation period works well with an average 8-h work day. Samples may be placed in the incubator at the end of the standard work day, incubated for 15 h, and titrated the following morning. One of the most attractive characteristics of the ISNT is its simplicity and convenience, and our modification of the assay does not change this.

It is clear that the assay will probably not be adopted as a universal soil N test (Barker et al., 2006; Marriott and Wander, 2006); however, the assay has shown promise for prediction of corn N needs, particularly in the eastern United States (Khan et al., 2001; Klapwyk and Ketterings, 2006; Williams et al., 2007b). Our modification of the ISNT improves measurement precision and throughput, making the assay more appropriate for routine use in commercial and institutional soil testing laboratories.

	ACKNOWLEDGMENTS

 
Financial support for this research was provided by the USDA-NRCS through a Conservation Innovation Grant, the Virginia Agricultural Council, and Phillip Morris USA.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication July 16, 2007.

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