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

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 (NO 3 -, total N, nitrate-N, 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.

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. 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 080202 13 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 mm 14 . 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-Szmik 15 , 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 0206/1-78 16 ; 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 17 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 Székely 18 , and than we calculated humus% from organic-C% according to: the Hungarian standard MSZ-08 0210-77 19 (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 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 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.

Amendments from Version 1
In the second version of our manuscript we have made the following changes: We clarified the land-use type from where the samples originated, as requested by Yuhua, K. and Juhos, K.; we added the meanings of "control", "greenhouse", and "pollutant nitrogen" (i.e., nitrite) as suggested by Juhos, K.
Regarding conclusions about the test soil suitable for use, there was a misunderstanding, so we tried to clarify in the text, as well; we meant that test soil could be used in pot experiments, according to its characteristics described in the study, which seemed to be confirmed by the total element contribution in test plant (tomato). The latter is the topic of our forthcoming study (we do not present these results here).
We clarified the soil-solution ratio.
We agree with Juhos, K.'s comments that collecting soil samples from various depths is very important to better understand N transformation and mobility in soil. Nevertheless, in the present study we aimed to analyze whether this soil is suitable or not for tomato pot experiments.
In addition, we updated Table 1 according to comments made by Juhos, K. and Yuhua, K.
Regarding Yuhua, K.'s last comment: we found statistical correlation between humus% and ammonium-N (mg/kg) only, as stated at Results and Discussion. This is the reason why we did not display correlations between humus and other 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 (e.g., nitrite) 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 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 17 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 17 :

OrganicN TotalN ammoniumN nitrateN + nitrite
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

Results
Soil pH was slightly acidic: pH (KCl) 5.5. As K A = 30, according to Arany the soil is considered humus sand 18 . For these samples, 1.4 % humus content was found, which represents a reasonable value for a sand soil 18 . 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).

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 capacity 21 .

Open Peer Review
should be added. Why the authors calculated this way the organic and the humus %? The values of the constants should be explained. Maybe they are known for the category of the soil scientists, but for larger dissemination and understanding, additional explanations are needed.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Yes

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate? Partly How many replicates were carried out in the study? In "Soil organic matter (SOM)" is proposed instead of "Humus" and "soil organic carbon (SOC)" is proposed instead of "C%".
In the description of the SOM method, K2SO4 is erroneously used instead of K-dichromate. When giving the averages, the number of items and the standard deviation must also be included (Table 1).

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results? Partly
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com