2.3.4. The Soil Heavy Metals Fractions Detection

Soil heavy metals from the 2016, 2017, and 2018 samples were sequentially extracted using the modified four-stage procedure recommended by the European Community Bureau of Reference (BCR), as described by Ure et al. and briefly described here. Exchangeable fraction (B1; includes soluble, exchange site- and carbonate-bound) [27]: 40 mL of 0.11 M acetic acid was added to 1 g of soil in a 50 mL centrifuge tube and shaken for 16 h at room temperature, followed by centrifugation and liquid decantation. Iron and manganese oxyhydroxides fraction (B2): 40 mL of a freshly prepared hydroxyl ammonium chloride was added to the residue from the previous step, shaken for 16 h at room temperature, followed by centrifugation and decantation. Organic fraction (B3): the residue from the previous step was treated twice with 10 mL of 8.8 M hydrogen peroxide. The digestion was allowed to stand for 1 h with occasional manual shaking, followed by digestion at 85 ± 2 ◦C until the volume was reduced to 2–3 mL. Samples were allowed to cool, and then 50 mL of 1.0 M ammonium acetate was added to the mixture and shaken for 16 h at room temperature, followed by centrifugation and decantation. Residual fraction (B4; represents metals associated with crystalline mineral phases): residue from the previous step was allowed to air-dry and then digested in a 50 mL digestion tube by first adding 1 mL of deionized water to make a slurry, followed by an aqua regia addition (7 mL HCl and 2 mL HNO3). The mixtures were allowed to predigest overnight at room temperature and then digested at 105 ◦C for 2 h the next day. Then, the mixtures were brought to a 50 mL final volume. The final liquids from all four steps were passed through a 0.45-micron membrane filter prior to Cd and Pb analysis via GFAAS (GFAAS, Zeenit 700 p; Analytik Jena AG, Jena, Germany).
