*2.3. Chemical Analysis and Indices*

Soil samples were collected from each field three times a year, i.e., (i) at the onset of the stem elongation phase of WOSR growth (BBCH 30, STME), (ii) at the onset of flowering (BBCH 60, FL), and (iii) at the crop physiological maturity (BBCH 89). Soil samples at each stage of WOSR growth were taken from three soil depths as follows: 0–0.3, 0.3–0.6, and 0.6–0.9 m.

The soil content of NH4-N and NO3-N was determined in field-fresh (not air dried) soil samples within 24 h after sampling. Twenty-gram soil samples were shaken for 1 h with 100 mL of 0.01 M calcium chloride (CaCl2) solution (soil/solution ratio 1:5; *m*/*v*). Concentrations of NO3-N were determined by the colorimetric method using flow injection analyses (FIAstar5000, FOSS). The method of NO3-N concentration analysis consists of two basic steps: reduction from nitrate to nitrite by using a cadmium column, followed by colorimetric determination of nitrite based on the Griess–Ilosvay reaction with N-(1-Naphtyl) ethylene-diamine dichloride as a diazotizing agent. The measurement was performed at a wavelength of 540 nm. The total content of nitrate nitrogen (NN) was expressed in kg ha−1. The Nmin content for a given soil layer was calculated using indices, which were constructed based on a soil textural class and soil bulk density [42].

Soil pH was measured ina1M potassium chloride (KCl) solution (soil/solution ratio 1:2.5; *m*/*v*). The content of plant available nutrients, including P and K, Mg, and Ca, was determined by the Mehlich 3 method [39]. The P concentration in the extract was determined by the colorimetric method, using the molybdenum blue method. Concentration of K in the collected extracts was determined by flame photometry. Concentration of Mg and Ca were determined by atomic absorption spectrometry (AAS)-flame type.

The partial productivity of NN was calculated based on yield (Y, kg ha−1) and the amount of NN present in a soil layer of 0.0–0.9 m at BBCH 30 (kg ha−1):

$$\text{PPP}\_{\text{N}\%} = \text{Y/N}\_{\text{30}} \text{ kg seeds kg}^{-1} \text{ of N} \tag{1}$$
