*2.4. Cobalt Immobilization Analysis in Soil*

After soils were incubation with 5% (w/v) wheat straw biochar, the (Community Bureau of Reference (BCR) sequential extraction procedure was applied to measure the following four fractions of cobalt in the tested soil: exchangeable and bound to carbonates (Fraction 1), reducible or bound to Fe and Mn-oxides (Fraction 2), oxidizable or bound to organic substances (Fraction 3), and residual (Fraction 4). Acetic acid, hydroxyl ammonium chloride, hydrogen peroxide plus ammonium acetate, and aqua regia stages of the sequential extraction procedure were applied to the soil samples, respectively [43–45]. The contents of cobalt, extracted during the BCR procedure, were measured on a microwave plasma-atomic emission spectrometer MP-AES 4200 (Agilent Technologies, Santa Clara, CA, USA). Data are provided as an average result from triplicate with the relative standard deviation (RSD), calculated by MP Expert Software Agilent Technologies. The maximum relative standard deviation (RSD) between replicates was set to 5%. Values that were above 5% were not included in the statistical analyses. To avoid analytical errors, standard solutions (from LGC Standards Ltd., UK) for MP-AES 4200 were used for calibration and certified reference materials as follows: RTH 953 Heavy Clay Soil from LGC Promochem (LGC Standards Ltd., Teddington, UK), total Co content 14.7 mg/kg and CRM055 (Honeywell Fluka, Charlotte, NC, USA) with Co content of 97 mg/kg were analyzed with every sample set. The recovery of Co from Certified Reference Material (CRM) was 89–94% and the maximum values of RSD were 2.6%. Detection limits were 0.01 mg/kg of Co in the soil samples.
