Discriminating between intra- and extracellular metals using chemical extractions

Several washing agents ( EDTA, CDTA, NTA, 8 HQS, Ti-EDTA- citrate, Ca2+, and H+) were tested for their ability to extract adsorbed metal from biological surfaces to distinguish between internalized and total cellular metal (Pb, Ni, Cd, Zn, and Cu) for a green alga, Chlorella kesslerii. Although a single extraction protocol cannot be provided that would be acceptable for all biological species and metals, several guidelines are proposed that should facilitate the interpretation of metal bioaccumulation data. It was shown that for the studied metals, a thermodynamic explanation was generally sufficient to explain the metal extraction results. At pH 6.0, a solution of 5 x 10(-3) M ethylenediaminetetraacetic acid ( EDTA) was an acceptable washing agent in most cases, whereas Ca or proton exchange solutions did not appear to quantitatively remove adsorbed metals. Conditions of ligand excess could be attained with relatively small concentrations of added ligand, effectively minimizing potential damage to the organism, including modifications in membrane permeability. Potential deleterious effects of the wash agents were verified. For example, a Ti-EDTA- citrate or an EDTA wash at high concentrations resulted in perturbations of algal membrane permeability. In spite of potential difficulties interpreting extraction data, the usefulness of the extraction protocols was clearly demonstrated for several organisms.