The sequential elution technique applied to cryptogams: a literature review

The use of bryophytes and lichens as biomonitors of atmospheric contamination, particularly contamination by heavy metals, is a well-known and widely applied technique. Determination of the total concentrations in organisms is often used in such studies, but has some disadvantages that may be resolved by using the sequential elution technique (SET). This technique involves successive washing steps that enable quantification of the elements in the different cell compartments (intercellular, extracellular, and intracellular), and finally determination of the remaining fraction, i.e. particulate material. The key step in the SET is the correct extraction of the extracellular fraction, for which a suitable extractant must be used for each different element considered. We have found only seven studies that have focused on selecting suitable extractants, which may be metal cations or chelating agents. Ethylenediaminetetraacetic acid is presented as the most appropriate extracellular extractant for Al, Co, Cu, K, Mg, Pb, V, and Zn, for which it has been tested, and for Cd and Fe, for which it has not yet been tested (although it is known to be capable of extracting these elements). The only extractant that is capable of extracting extracellular Hg is dimercaprol. The technique has been used in laboratory studies and less often in field studies carried out in the surroundings of focal points of contamination. The elements analysed include nutrients (e.g. Ca, K, and Mg) and heavy metals (e.g. Cd, Zn, and Cu), usually associated with sources of emission of contaminants. Several problems have arisen in the application of the SET, some of which must be resolved. These include methodological problems (e.g. variability in the extraction process) and problems inherent in the SET itself, which are more difficult to resolve. Other techniques, such as histochemical techniques and electron microscopy with microanalysis, must therefore be used simultaneously with the SET to determine if the uptake of metal is only extracellular. At present, in light of the disadvantages of the quantification of the extracellular fraction, measurement of the intracellular fraction is presented as the best option because it is not affected by the metal burden in the particles, enables evaluation of environmental risks, better represents the average conditions of contamination, and enables better evaluation of phytotoxicity.