Soluble nitrogen forms in sand soil of Pallag: a quantitative report

Introduction

Nitrogen (N) is an essential element for plants, takes various forms in soil¹, and is one of the most limiting nutrients for various crops^2–4. Nitrogen is present in soils as inorganic nitrogen (nitrate, ammonium, and dinitrogen) and organic nitrogen (urea and amino acids)⁵.

Conversions between organic and mineral N forms are primarily affected by soil microorganisms, which enable conversion to forms that plants can uptake⁶. In addition, some pollutant N forms (e.g., nitrite) are present in soil, so nitrogen conversions may indirectly affect human health and load the environment¹. For this reason, quantifying studies aim to better understand nitrogen form ratios in soil and contribute to reaching sustainable agriculture practice. Recently, Jakab published a similar study⁷ quantifying another vital element for plants, phosphorus, in soil from the same region.

Humus is a main fertility component of the soil, 65–75% of its structure being made up of organic matter^8,9. We decided to include humus content in this N-related study as it represents a known indicator of soil quality¹⁰.

Calcium chloride (CaCl₂)-soluble nitrogen forms are the most available N ions for assimilation by plants^11,12. Therefore, we quantified all N forms in CaCl₂ solution. Additionally, we determined nitrate-N via a potassium chloride (KCl)-based method.

The main objectives of our study were: (a) to map out all easily absorbed nitrogen forms for better understanding of available nutrient composition and (b) to determine the ratio of pollutant (nitrite) and absorbable (ammonium ion, nitrate) N forms in humus sand soil in pot experiments.

Methods

Randomized soil sampling, approx. 60 kg; 15 cores from control parcel (no fertilizer was applied, nor crop production, or land-use), was done at the Pallag Experimental Station of Horticulture at the University of Debrecen, Hungary on May 20, 2020, according to Hungarian national standard MSZ 080202¹³ from the -20 cm of topsoil using a vane. On other parcels (different from control parcels), orchards are being cultivated. Sampling point is located on a moderately hot and dry micro-region, the average annual temperature varies between 9.7-10.0 °C. The annual precipitation is of only 520–550 mm¹⁴. Next, samples were transferred for measurements to a greenhouse (where pots had been placed for pot experiments); the greenhouse was located at Department of Agriculture, at the University of Debrecen. To measure chemical parameters, samples were sieved through a 2 mm mesh.

We determined soil moisture content gravimetrically, according to Klimes-Szmik¹⁵, drying the soil samples at 105°C for 24 h and weighing the mass loss. To evaluate texture, Arany-type plasticity index was measured, using the methodology recommended by Hungarian national standard MSZ-08 0206/1-78¹⁶; briefly from a burette distilled water is added to 100g sample until soil reaches the upper limit of its water holding capacity. Soil pH was determined in 1 mol L^-1 KCl solution (soil/water = 1.0/2.5 wt./wt.), according to Buzás¹⁷ using a glass electrode (Model Seven2Go Advanced Single-Channel Portable pH Meter, Mettler, Toledo. We first determined organic-C%. content with potassium dichromate, according to Székely¹⁸, and than we calculated humus% from organic-C% according to: the Hungarian standard MSZ-08 0210-77¹⁹ (E2); briefly, 10 mL K-dichromate was added to 1.0 g of soil sample (solution/soil=10/1) in a 300 mL Erlenmeyer flask, then 0.10 g Ag₂SO₄ aqueous solution was added, boiled for 5 minutes, cooled to room temperature, and titrated with 0.2 N Mohr's salt solution. Ferroin was used was the indicator, given that its color change is reversible, pronounced, and fast. We calculated C% according to Equation (E1):

where a is the volume difference (Mohr’s salt solution loss) between blank and soil sample titration (expressed in mL), multiplied by the Mohr-salt factor (which is 0.0006) and b represents the weight of the soil sample (g), multipled with 100 to convert results to percentage.

Next, humus% was calculated according to Equation (E2):

Where C(%) is organic-Carbon (expressed in percentage), 1.724 is a multiplication factor which was determined by the Hungarian standard MSZ-08 0210-77 based on a number of experimental results; it was concluded that Humus(%) values can be calculated from organic-C% with high reliability.

All measurements of soluble N forms in soil were conducted according to Hungarian standard MSZ 20135:1999²⁰; briefly, to determine KCl-NO₃ (mg/kg) content, the sample was prepared with 1 mol L^-1 KCl of soil solution (74.5g KCl were added to 1 L distilled water²¹. Spectra were recorded with a Model PU 8610 UV/VIS kinetics spectrophotometer by Pye Unicam (Cambridge, GB). For assessing total soluble nitrogen (UV degraded) content, soil samples were extracted with 0.01 mol L^-1 CaCl₂ solution. Soil samples were mixed with sodium tetraborate buffer, then oxidized with excess K₂S₂O₈ and passed into an ultraviolet digester. Nitrate was reduced on a cadmium-copper column and then converted to a colored azo compound via the Griess-Ilosvay reaction; dissolving 0.50 g of sulfanilic acid and 0.05 g of 1-naphthylamine in 150 mL of dilute acetic acid. A colored solution was obtained, which was analyzed by visible photometry at 540 nm (SKALAR photometry (San Plus Analyser, S.F.A.S).

We determined CaCl₂-ammonium-N (mg/kg) using a modified Berthelot reaction-based method in which ammonia was initially converted to monochloramine and then to 5-aminosalicylate, according to Buzás¹⁷ using 1% EDTA solution and 1 ml 0.06 N NaOH. After oxidation, a green color complex was obtained, with a maximum light absorption at 660 nm. Nitrate-N was determined as described above for the measurement of N content (after reduction, conversion to azo dye, and photometric measurement at 540 nm). All soluble forms were detected by SKALAR photometry (San Plus Analyser, S.F.A.S) in a segmented continuous flow (SCF) system. Finally, we calculated nitrite-N (mg/kg) using Equation (E3), proposed by Buzás¹⁷:

To better understand the correlation between humus% (Y) and N-forms (X), we carried out two different tests (i) the Pearson test to establish the relation between the variance (Y) and covariance (XY); and (ii) the Kendall test to verify whether the two variables may be regarded as statistically dependent. All statistical analysis was performed with R Statistical Software 3.5.1 (Foundation for Statistical Computing, Vienna, Austria).

Results

Soil pH was slightly acidic: pH (KCl) 5.5. As K_A = 30, according to Arany the soil is considered humus sand¹⁸. For these samples, 1.4 % humus content was found, which represents a reasonable value for a sand soil¹⁸. Nitrate content was determined using two methods: KCl-NO₃ (12.66 mg/kg) and CaCl₂-NO₃ (13.53 mg/kg), and the average value of nitrate content (12.48 mg/kg) served for the calculation of percentages in Figure 1.

Figure 1. Percentage distribution of the soluble forms of nitrogen present in soil.

Average ammonium-N was 6.8 mg/kg, which represents 24.7% of the total-N content. In addition, 3.8 mg/kg of nitrite-N (pollutant nitrogen form) was assessed and found to be one order of magnitude less than the nitrate-N content, as expected for well-ventilated sand soil. Organic-N of 4.4 mg/kg contributed by 16.1% to the total nitrogen content of soil (Table 1).

Considering all forms of N under study, the results of correlation analysis evidenced that only the ammonium-N form was in strong correlation with humus%, with r ≈ 0.97 and T ≈ 0.99 (Figure 2).

Figure 2. Correlation between humus% and ammonium-N (mg/kg).

These promising initial results call for additional experiments to confirm the strong correlation between these two variables. Total-N also showed a good correlation with humus%; however, it was not as strong as that found between humus% and ammonium-N.

Conclusions

Our results reveal that the ratio of nitrate-N and nitrite-N (which is determined mostly by soil oxidation-reduction conditions) is optimal in sand soil samples originating from the Pallag Experimental Station of Horticulture at the University of Debrecen, Hungary. Based on the percentage distribution of soluble N forms present at Pallag Experimental Station, authors recommend using this soil in further pot experiments, given the soil’s optimal nutrient supply capacity²¹.

Data availability