**3. Analytical protocols for sequential extraction**

In recent years a great number of papers have been published on various analytical techniques proposed for the fractionation analysis of trace elements in various environmental samples (soils, sediments, etc.). An approach that has been found to be preferable is the fractionation of heavy metal into operationally defined forms under the sequential action of different extractants [2]. Selective extractants, used in sequential extraction procedures, are aimed at the simulation of natural conditions whereby metals associated with certain soil (sediment) components can be released. For example, changes in the ionic composition affecting adsorp‐ tion–desorption reactions or a decrease in pH may lead to the release of metals, retained on a matrix by weak electrostatic interactions or co-precipitated with carbonates ("exchangeable" and "acid soluble" forms). Decreasing the redox potential can result in dissolution of oxides, unstable under reducing conditions, and liberation of scavenged metals ("reducible" forms). Changes in oxidizing conditions may cause the degradation of organic matter and release of complexed metals ("oxidizable" forms). Finally, the destruction of primary and secondary mineral lattice releases heavy metal retained within the crystal structure, e.g., due to isomor‐ phous substitution ("residual" forms) [2]. The nominal "forms" determined by operational fractionation can help to estimate the amounts of total metals in different reservoirs which could be mobilized under changes in the chemical properties of the soil [34]. Since the 1970s a considerable number of extraction procedures have been proposed for determining the forms of heavy metal [2, 35-39]. Most of these procedures are based on the scheme of Tessier et al. [2]. Although most of the extracting reagents were originally used in the chemical analysis of soils, the procedures proposed have been tested on a wide variety of contaminated environmental samples—sediments, road dust, sewage sludge, etc.

Sequential extraction can be useful to have an operational classification of metals in different geochemical fractions [2] which is the most reliable criteria to quantify the potential effect of soil/sediment contamination by heavy metals. This can provide information about the identification of the main binding sites, the strength of element binding to the particulates and the phase associations of trace elements in soil/sediment. Following this basic scheme, some modified procedures with different sequences of reagents or operational conditions have been developed [40-43]. Considering the diversity of procedures and the lack of uniformity in different protocols, a European Community Bureau of Reference (BCR, now the European Community Standards Measurement and Testing Program) method was proposed [6] and was applied by a large group of researchers [31, 44-47]. In this study, we followed the sequential extraction procedure proposed by the European Union's Standards, Measurements and Testing program [3].
