A modified BCR sequential extraction procedure was used
in this study (Nemati et al., 2010). The procedures of BCR
sequential extraction were:
Exchangeable fraction: 40 mL of 0.11 mol/L acetic acid
was mixed with 1 g sample and shaken for 16 hr. Reducible
fraction: 40 mL of 0.5 mol/L hydroxylammonium chloride,
pH 1.5, were mixed with the remaining solid from step
1 and shaken for 16 hr. Oxidizable fraction: 10 mL of
8.8 mol/L hydrogen peroxide was mixed twice with the
remaining solid from step 2 and heated until the volume
was reduced to 2–3 mL. Then 50 mL of 1.0 mol/L
ammonium acetate was added to the cooled mixture and
shaken for 16 hr. Residual fraction: the residue from step 3
was digested using aqua regia.
All extractions were carried out at room temperature.
The supernatant was separated from the solid by centrifugation
(4000 r/min) for 20 min and transferred into another
centrifuge tube. The residue was washed by adding 20
mL of deionized water, shaking for 15 min on the shaker,
and then centrifuging for the next extraction. All heavy
metal concentrations of each fraction were also measured
by ICP-OES (OPTIAM2000, PerkinElmer, USA).