This study rigorously examined the applicability of two commonly employed models (Free Ion Activity Model, FIAM, and Biotic Ligand Model, BLM) to predict zinc bioaccumulation by phytoplankton (Chlorella kesslerii). Such studies are required to confirm whether the routine analysis recommended to predict metal bioavailability should be based on measurement of the free ion, other bioavailable chemical forms in solution, or metal bound to “biotic ligands” as is currently recommended by the U.S. EPA. In this work, Zn internalization fluxes were related to either the chemical speciation of Zn in solution (FIAM basis) and at the surface of the alga (BLM basis). In order to measure cellular metal content, several cell-washing agents (EDTA, CDTA, NTA, 8 HQS, Ti-EDTA-citrate, Ca^2+and H^+) were tested for ability to extract metal (Pb, Ni, Cd, Zn, Cu) from the algal surface. A thermodynamic explanation can explain the metal extraction results. The most suitable extraction procedure was achieved using a 1-2 min rinsing step with 5 x 10^-3 M EDTA. This resulted in minimal membrane permeability and photosynthetic activity perturbation. Guidelines in order to fully exploit extraction data are presented.
Since Zn is an essential metal, the ability of C. kesslerii to regulate metal accumulation was assessed by growing the algae in defined growth media where only Zn^2+ was varied (10^-11, 10^-9 and 10^-6 M). In all cases, a first order uptake flux as predicted by the FIAM and BLM were not observed. No internalization flux difference was seen for algae grown at 10^-9 (optimal) and 10^-6 (slightly toxic) M Zn^2+, despite a 15-fold difference in initial Zn cellular content. Induction of phytochelatins could not explain this observation. When algae were grown at slightly limiting (10^-11 M) free zinc concentrations, the internalization flux increased and was nearly constant over the range of [Zn^2+] examined; this was attributed to the synthesis of membrane-bound zinc transporters. Several hypotheses were examined to explain the failure of the thermodynamic uptake models. Zn excretion following a first order relationship in regard to cellular Zn content was observed: although it plays a major role in regulating algal cellular content, it was not significant over short-term bioaccumulation experiments using Zn-65. A limitation of Zn bioaccumulation due to diffusion in solution was observed for Zn^2+ concentrations lower than 2 x 10^-10 M. The use of metabolic inhibitors (vanadate and CCCP) demonstrated that Zn transport is an energy-dependent process in C. kesslerii, and it is induced in response to Zn deficiency. Active transport implies that Zn uptake may function independently of the electrochemical Zn gradient and that, in some cases, both uptake and receptor-bound Zn may be independent of solution chemistry. Multiple transport pathways for Zn was suggested because both the internalization rate constant and the equilibrium constant for the binding of Zn to the transport sites varied as a function of [Zn^2+] in the bulk solution. For these reasons, Zn uptake could not be modeled by the steady-state models. Furthermore, competitive effects (Ca, Cd) could not be modeled on Zn internalization flux in the presence of constant [Zn^2+] in solution. Modifications of the algal surface charge due to high [Ca^2+] and the active nature of Ca^2+ transport can partially explain that observation. This work thus highlights the complexity to predict essential trace metal bioavailability based on chemical speciation due to biological regulation.