Potentially mineralizable nitrogen is the portion of soil organic nitrogen that can become available to plants through microbial decomposition. Estimating it traditionally can require lengthy biological incubations, which are difficult to incorporate into routine testing because sample processing, storage and controlled laboratory conditions do not fully represent field conditions. Chemical tests offer a faster alternative, but their usefulness depends on whether their measurements distinguish soils that will supply enough nitrogen to reduce fertilizer response from those that will not. The Illinois Soil Nitrogen Test was developed to estimate alkali-hydrolyzable nitrogen as a practical index of soil nitrogen availability, although its original procedure imposed demanding requirements on laboratories.

In ISNT-1, 5 hours of heating at 48–50°C drives nitrogen from a soil–sodium hydroxide mixture into a boric-acid trap. A modified Mason jar lid supports a Petri dish containing the trap, while a commercial griddle provides the heat. Researchers have sought more uniform temperatures using draft shields, repositioning of jars or temperature-controlled incubators, but these additions increase equipment needs. ISNT-2 replaces that arrangement with an unmodified 237-mL Mason jar. A Petri dish rests on a pizza-lid support inside the jar, and the vessel is incubated at 25 ± 1°C for 24 hours. The longer, cooler procedure is designed to improve reproducibility and increase the number of samples a commercial laboratory can process.

For each analysis, investigators weigh 2.00 g of air-dried soil into the Mason jar, add 10 mL of 2 M sodium hydroxide and swirl the mixture for about five seconds. Within 30–45 seconds, they place the support and a Petri dish containing 5 mL of boric-acid indicator solution into the jar and seal it with a screw band. After 24 hours at 25°C, the dish is removed, diluted with 5 mL of deionized water and titrated with standardized 8 mM sulfuric acid. The amount of diffused nitrogen is calculated from the titrant volume and its nitrogen titer, with the calculation adjusted for the 2-g soil sample. A colorimetric alternative, ISNT-2C, was also tested, using absorbance at 520 nanometers in a 96-well microplate.

Optimization experiments showed that temperature and time must be tightly standardized. Recovery generally increased as incubation continued from 20 to 28 hours, but no advantage over 24 hours was found in distinguishing among the soils studied. Altering the duration by two hours produced significant differences in test values. Temperature was similarly important: among one-degree increments from 20 to 30°C, 5% of the differences were significant, compared with 34% when comparisons used two-degree increments. The researchers therefore recommend controlling diffusion temperature to within 1°C. Conditions of 25°C for 24 hours produced the strongest relationship with ISNT-1 among the tested combinations; 30 hours gave an equally strong correlation but offered less practical benefit because it reduced sample throughput.

The simplified method did not recover every form of nitrogen as effectively as heated alkaline extraction. In tests of pure compounds, ISNT-1 recovered 96.5% to 97.0% of the nitrogen supplied as glucosamine, galactosamine and mannosamine, whereas ISNT-2 recovered only 9.6% to 34.9%. Direct steam distillation also produced much higher amino-sugar recoveries. The cooler ISNT-2 conditions and smaller jar, which limited the surface-to-volume ratio of the alkaline mixture, appeared to impede ammonia release. Recovery of ammonium nitrogen was still high at 93.8%, compared with 99.7% for ISNT-1. Most amino acids were poorly recovered by all the alkali-based techniques, while asparagine and glutamine were underestimated by ISNT-2. These differences confirm that the methods are chemically distinct even when their soil-test results are strongly correlated.

Across the 18 Illinois response-trial soils, the mean nitrogen value determined by ISNT-1 was 218 mg N per kilogram of soil, compared with 91 mg per kilogram for ISNT-2. Despite the lower absolute recovery, the ratio of ISNT-2 to ISNT-1 remained tightly grouped between 0.39 and 0.44, with a mean of 0.42. The coefficients of variation were similar: 1.6% for ISNT-1 and 1.7% for ISNT-2. The correspondence was much less consistent for three Brazilian Oxisols, where ratios ranged from 0.23 to 0.52, cautioning against applying a simple conversion factor to soils with contrasting properties. ISNT-2C was less precise, with a coefficient of variation of 7.2% and ratios to ISNT-1 ranging from 0.36 to 0.55.

For predicting fertilizer response, the investigators used data from the 18 Illinois sites and grouped them into three categories: a likely response above 25%, a possible response from 0% to 25%, and an unlikely response of 0%. ISNT-2 followed the same overall pattern as ISNT-1 and direct steam distillation. It identified most responsive sites correctly, although two soils with measured responses of 12% to 13% were exceptions. Their relatively high test values may reflect site-specific influences: one had greater soil organic carbon, which can promote microbial immobilization of mineral nitrogen, while no-till corn production may have intensified that effect at the other. The second site’s crop population was about 20% higher than that of the nonresponsive comparison soils, potentially increasing crop nitrogen demand and limiting the measured response to fertilizer.

The colorimetric version, hot potassium chloride extraction and the Solvita labile amino-N test also placed the two exceptional soils in overlapping regions, but each had other limitations. ISNT-2C underestimated values for two nonresponsive sites, consistent with its greater analytical variability. Hot potassium chloride and Solvita did not show that underestimation, but both overestimated one responsive site, potentially identifying a fertilizer opportunity where the economic response was smaller. The comparison also exposed a practical trade-off: direct steam distillation was highly correlated with ISNT-1 and substantially shortened analytical time, but it is more labor-intensive. ISNT-2 avoids heating hardware and jar modification while retaining predictive performance across the evaluated Illinois sites.

The study supports ISNT-2 as a practical alternative for laboratories that need to estimate soil nitrogen availability and distinguish likely fertilizer-response categories. It does not establish that a soil test alone can determine an economically optimal fertilizer rate. The authors note that mineralization and crop demand are affected by factors including soil acidity, drought, tillage, crop rotation, residue management, plant density, planting date, genotype, fertilizer prices and commodity prices. Weather cannot be controlled and can only be predicted. A further limitation is that most soils used in the project had been stored for at least 18 years, and no biological estimates of potentially mineralizable nitrogen were used in evaluating ISNT-2. Accordingly, the findings demonstrate analytical promise and feasibility in the studied setting rather than a universal rule for reducing fertilizer use or increasing crop yields.

Subject of Research: Development and field-trial evaluation of a simplified chemical soil nitrogen test for estimating fertilizer response

Article Title: Development and evaluation of a simplified Illinois soil nitrogen test

Article References: Nunes, V. L., Zhou, Q., Mulvaney, R. L., & Bassi, L. (2026). Development and evaluation of a simplified Illinois soil nitrogen test. Frontiers in Soil Science, 6, Article 1985825. https://doi.org/10.3389/fsoil.2026.1985825

Image Credits: AI Generated

DOI: 10.3389/fsoil.2026.1985825

Keywords: soil nitrogen, potentially mineralizable nitrogen, Illinois Soil Nitrogen Test, nitrogen fertilizer response, corn, alkali hydrolysis, soil testing, diffusion method, site-specific management, Development, evaluation, simplified