Internal versus External Dose for Describing Ternary Metal Mixture (Ni, Cu, Cd) Chronic Toxicity to Lemna minor

Simultaneous determinations of internal dose ([M]_tiss) and external doses ([M]_tot, {M²⁺} in solution) were conducted to study ternary mixture (Ni, Cu, Cd) chronic toxicity to Lemna minor in alkaline solution (pH 8.3). Also, concentration addition (CA) based on internal dose was evaluated as a tool for risk assessment of metal mixture. Multiple regression analysis of dose versus root growth inhibition, as well as saturation binding kinetics, provided insight into interactions. Multiple regressions were simpler for [M]_tiss than [M]_tot and {M²⁺}, and along with saturation kinetics to the internal biotic ligand(s) in the cytoplasm, they indicated that Ni-Cu-Cd competed for uptake into plant, but once inside, only Cu-Cd shared a binding site. Copper inorganic complexes (hydroxides, carbonates) played a role in metal bioavailability in single metal exposure but not in mixtures. Regardless of interactions, the current regulatory approach of using CA based on [M]_tot can sufficiently predict mixture toxicity (∑TU close to 1), but CA based on [M]_tiss was closest to unity across a range of doses. Internal dose integrates all metal-metal interactions in solution and during uptake into the organism, thereby providing a more direct metric describing toxicity.