Vario max cn

Undisturbed soil samples for determining aggregate-sized associated C and N fractions were collected in November 2018, right after cowpea harvest, with a 5 cm-diameter auger to a depth of 20 cm. Each soil core was divided into 0–5-cm and 5–20-cm soil depths after extraction. Three random subsamples from each plot and soil depth were pooled together. Soil aggregate classes were determined according to Elliott [48 (link)]. Briefly, moist soil sample was sieved at 8 mm and air-dried. An 80-g subsample was submerged into deionized water for 5 min and wet sieved, obtaining four soil aggregate fractions: large macroaggregates (LM; >2000 µm), small macroaggregates (sM; 250 µm–2000 µm), microaggregates (m; 53 µm–250 µm), and silt and clay fraction (s + c; <53 µm). Fractions within macroaggregates were isolated according to Six et al. [49 (link)], thus obtaining coarse particulate organic matter (cPOM; >250 µm), microaggregates within macroaggregates (mM; 53 µm–250 µm), and silt and clay (s + cM; <53 µm). All fractions were corrected for sand content according to Kemper and Rosenhau [50 ]. One gram of soil per each fraction was then weighed and analyzed for C and N concentrations by the Dumas combustion method with an elemental analyzer (VarioMax C:NS, Elementar, Langenselbold, Germany).

The dried soils were mixed with CO₂-removed water at a soil:water ratio of 1:5 (w/v) prior to measurements of pH and electrical conductivity (EC) using corresponding electrodes. The soil was oxidized with potassium dichromate and measured for TOC using titration with ferrous ammonium sulfate. Soil TN was measured by an elemental analyzer (Vario MAX CNS, Elementar, Germany). AN was measured using the alkali solution diffusion method (Lu, 2000 ). Concentrations of soil ammonium (NH₄⁺) and nitrate (NO₃^–) were analyzed using ion chromatography (ICS-3000, Dionex, United States) following extraction with 2 M KCl. AK and AP were measured using an inductively coupled plasma optical emission spectrometer (Optima 5300DV, PerkinElmer, United States).

The soil pH was measured in a soil/deionized water slurry at a ratio of 1:2.5 using a pH-EC meter (Accumet Excel XL60; Fisher Scientific Inc., Hampton, NH, USA). The available phosphorus in soil samples was extracted using hydrochloric acid in ammonium fluoride and its content determined using molybdenum–antimony anti-colorimetry. Soil nitrate-nitrogen (NO₃⁻-N) and ammonium-nitrogen (NH₄⁺-N) were extracted using 0.01 mol/L anhydrous calcium chloride and quantified using a flow injection autoanalyzer. The soil total carbon content (TC), total nitrogen content (TN), and total sulfur content were measured using an elemental analyzer (Vario MAX CNS; Elementar Analysensysteme GmbH, Berlin, Germany). Heavy metals such as lead (Pb), chromium (Cr), zinc (Zn), nickel (Ni), copper (Cu), Cadmium (Cd) and arsenic (As) were digested by microwave-assisted acid digestion using trace-pure nitric acid (2.5 mL), hydrofluoric acid (1.5 mL), and a closed-vessel high-pressure microwave digester–Multiwave GO (Anton Paar, Graz, Austria), according to Chen et al. [32 (link)]. Finally, the metal concentrations were determined by inductively coupled plasma optical emission spectrometry (Optima 7000DV; PerkinElmer, Waltham, MA, USA). Physiochemical properties of sampled soils are presented in Table 1.