Evaluation of the efficacy of the sequential elution technique, by use of electron microscopy methods

In the present study, scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM–EDS) was used in conjunction with the sequential elution technique (SET) to evaluate the efficacy of extraction of Zn from the moss Pseudoscleropodium purum (Hedw.) M. Fleisch. Moss samples were collected from two areas, a contaminated area (with high concentrations of Zn) in the surroundings of a steel works, and an uncontaminated area. Some samples from the uncontaminated area were used as controls, and others were incubated in a solution of ZnCl2 for subsequent analysis of uptake and location of Zn. The concentrations of Zn in the different cell compartments of the moss were determined by use of the SET, and different points on the surface of the moss and in deposited particles were analysed by SEM–EDS. The results showed that the extracellular fraction obtained by SET includes the Zn bound to cation exchange sites in the moss and Zn bound to particles on the moss surface, which leads to overestimation of extracellular Zn. After extracellular extraction, Zn was not detected on the surface of the moss or in the particles. To avoid problems associated with the presence of particles on the moss surface, a washing procedure should be used to remove such particles before application of the SET. Another possibility is to use the intracellular concentration, as this is not affected by the metal load in the particles and better reflects those contaminants that affect the moss metabolism.