Determination of trace metals accumulated and internalized by marine phytoplankton; interferences with colloidal organic matter

In the present work, we study the effects that aggregation of humic acids in seawater, and its adsorption to surfaces, can have on the determination of Cu and Pb accumulation by phytoplankton within the context of metal bioavailability studies, using the diatom Thalassiosira weissflogii. Two methods commonly used for collection of algae to analyse metal content, filtration and centrifugation, were compared, and a procedure to differentiate the metal internalized by the alga from that adsorbed to cell walls-washing with 0.01 M ethylenediaminetetraacetic acid (EDTA)- was optimized for its use in seawater and in the presence of humic acids. The present results highlight the possibility of obtaining biased results if filtration is used to isolate the algae, and show that centrifugation is a more suitable procedure minimizing contribution of particulate and colloidal forms of metals to the measured cellular and intracellular metal contents. Thus a combination of centrifugation to isolate the algae from the medium with a 10 minutes washing with a washing agent composed of EDTA 0.01 M at pH 8 and NaCl and KCl to 0.5 M ionic strength is recommended to extract metals adsorbed to the surfaces of marine phytoplankton for a seawater containing humic substances. The importance of discriminating between internalized metal and total cellular metal in bioavailability studies is noted, given the different effects of humic substances on metal accumulation in the different cellular pools.