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Discrimination between intra- and extra-cellular metals

Guidelines are presented for extraction protocols to distinguish between surface-bound and internalised metal, thus facilitating interpretation of bioaccumulation data. The approach is based on the assumption that equilibrium is rapidly attained between the metal in solution and that adsorbed to biological surface sites and that internalisation is the slow rate-determining step. Distinction between adsorption and internalisation can thus be made on the basis of the kinetics of the reactions. A washing agent should rapidly (within a few minutes) desorbs metal from the surface of the organism, whilst not accessing the internalised metal within this time frame.

The utility of several washing agents has been assessed: at pH 6, EDTA was found to be acceptable in most cases. General guidelines for extraction protocols are:

- The washing agent should form metal complexes with high stability and be added in excess over the molar concentration of metal adsorbed on the biological surface.
- The concentration of washing agent must be optimised to limit biological effects within the timeframe of the extraction, e.g. modification of membrane permeability.
- The presence of metal efflux must be verified.

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