Combined effects of heavy metals (Cd, As, and Pb): Comparative study using conceptual models and the antioxidant responses in the brackish water flea.

The combined effect of toxic inducers has emerged as a challenging topic, particularly due to their inconsistent impacts on the environment. Using toxic unit (TU) based on LC50 value, we investigated the 48 h acute toxicities of the following combinations: Cd + As, Cd + Pb, As + Pb, and Cd + As + Pb, and binary and ternary combined effects were interpreted using concentration addition (CA) and independent action (IA) model. The molecular effects of these combinations were further examined on the basis of gene expression (four GST and two SOD isoforms) and antioxidant enzymes activity (SOD and GST). The CA-predicted LC50 was similar to the observed results, indicating that the CA model is more applicable for evaluating the combined effects of the metal mixtures. Synergistic effects (ΣTULC50  < 0.8) were observed for the mixtures As + Pb and Cd + Pb, while additive effects (0.8 < ΣTULC50  < 1.2) were observed for the mixtures Cd + As + Pb and Cd + As. No antagonistic effects were observed in this study. Molecular biomarkers for oxidative stress caused by metals, as well as traditional endpoints such as lethality, have shown a clear response in assessing the toxicity of binary and ternary mixtures. This study opens up a new avenue for the use of biomarkers to assess the combined effects of metals in aquatic environments.