Soluble nitrogen forms in sand soil of Pallag: a quantitative report [version 1; peer review: 3 approved with reservations]

Nitrogen (N) is a crop macronutrient of major importance, which affects both plant growth and yield. In this paper we discuss the humus content (%) and various soluble N forms (NO3, total N, nitrateN, ammonium-N, and organic nitrogen) available in humus sand soil samples originating from the Pallag Experimental Station of Horticulture at the University of Debrecen, Hungary. We found 45.4% nitrate-N and 13.8% nitrite-N of total N content present in the soil. Considering the percentage distribution of soluble N forms present at the Pallag Experimental Station, we recommend using this soil in further pot experiments, given that this has optimal nutrient supply capacity. In addition, we examined possible statistical correlations between humus% and N forms.


Introduction
Nitrogen (N) is an essential element for plants, takes various forms in soil 1 , and is one of the most limiting nutrients for various crops [2][3][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 uptake 6 . In addition, some pollutant N forms are present in soil, so nitrogen conversions may indirectly affect human health and load the environment 1 . 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 study 7 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 quality 10 .
Calcium chloride (CaCl 2 )-soluble nitrogen forms are the most available N ions for assimilation by plants 11,12 . Therefore, we quantified all N forms in CaCl 2 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. 60kg; 15 cores from control parcel) 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 080202 13 from the -20 cm of topsoil using a vane. Next, samples were transferred for measurements to a greenhouse located at Department of Agriculture, at the University of Debrecen. To measure chemical parameters, samples were sieved through a 2mm mesh.
We determined soil moisture content gravimetrically, according to Klimes-Szmik 14 , drying the soil samples at 105°C for 24 hours and weighing the mass loss. To evaluate texture, Arany-type plasticity index was measured, using the methodology recommended by Hungarian national standard MSZ-08 0206/1-78 15 ; 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 Buzás 16 using a glass electrode (Model Seven2Go Advanced Single-Channel Portable pH Meter, Mettler, Toledo. We quantified humus content with potassium dichromate, according to Székely 17 : to calculate humus% we first determined organic-C%. The latter was measured following Hungarian standard MSZ-08 0210-77 18 ; briefly, 10 mL K 2 SO 4 was added to 1.0 g of soil sample in a 300 mL Erlenmeyer flask, then 0.10 g Ag 2 SO 4 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 difference between blank and soil sample titration (mL), multiplied by the Mohr-salt factor and b represents the weight of the soil sample (g). Soil samples were mixed with sodium tetraborate buffer, then oxidized with excess K 2 S 2 O 8 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 0.5 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 CaCl 2 -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 Buzás 16 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 Buzás 16 :

OrganicN TotalN ammoniumN nitrateN nitriteN
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 sand 17 . For these samples, 1.4 % humus content was found, which represents a reasonable value for a sand soil 17 . Nitrate content was determined using two methods: KCl-NO 3 (12.66 mg/kg) and CaCl 2 -NO 3 (13.53 mg/kg), and the average value of nitrate content (12.48 mg/kg) served for the calculation of percentages in Figure 1.
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).
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 capacity 20 .