A test of the additivity of acute toxicity of binary-metal mixtures of ni with Cd, Cu, and Zn to Daphnia magna, using the inflection point of the concentration-response curves

Abstract

Mixtures of metals are often present in surface waters, leading to toxicity that is difficult to predict. To provide data for development of multimetal toxicity models, Daphnia magna neonates were exposed to individual metals (Cd, Cu, Ni, Zn) and to binary combinations of those metals in standard 48-h lethality tests conducted in US Environmental Protection Agency moderately hard reconstituted water with 3 mg dissolved organic carbon (DOC)/L added as Suwannee River fulvic acid. Toxicity tests were performed with mixtures of Ni and 1) Cd, which is considerably more toxic than Ni; 2) Cu, which is less toxic than Cd but more toxic than Ni; and 3) Zn, which has a toxicity threshold similar to Ni. For each combination of metals in the binary mixtures, the concentration of 1 metal was held constant while the second metal was varied through a series that ranged from nonlethal to lethal concentrations; then the roles of the metals were reversed. Inflection points of the concentration-response curves were compared to test for additivity of toxicity. Sublethal concentrations of Ni caused less-than-additive toxicity with Cd, slightly less-than-additive toxicity with Zn, and greater-than-additive toxicity with Cu. One explanation of these results might be competition among the metals for binding to biological ligands and/or dissolved organic matter. Therefore, models might have to incorporate sometimes competing chemical interactions to accurately predict metal-mixture toxicity. Environ Toxicol Chem 2016;35:1843-1851. © 2015 SETAC.

Keywords: Bioavailability; Independent action; Metal complexation; Metal-metal competition; Response addition.

Figure 1

Hypothetical example of response-additive toxicity of 2 metals in 5 different binary mixtures. In this example, metal 1 was held constant at 5 different background concentrations (represented by the 5 different colors; [Purple] < [Blue] < [Green] < [Orange] < [Red]) while metal 2 was increased along a concentration gradient at each background metal concentration. Higher concentrations of metal 1 cause higher initial mortality, but the inflection point of each curve will occur at the same concentration of metal 2 (at a concentration of ~1.6 in this example) if the toxicity of the metal mixture is response additive (see Supplemental Data, Equation S1). M₁ = metal 1.

Figure 2

Mortality (determined by immobilization of Daphnia magna neonates) in 48-h exposures to Cd–Ni mixtures, in which a constant background concentration of Cd was maintained while Ni was varied along a concentration gradient. Different background concentrations of added Cd were used in different sets of toxicity tests, but with replicate tests conducted at each Cd concentration (k equals the number of replicate tests, indicated in the key). As the concentration of Ni was increased, the Cd-induced mortality decreased until Ni concentrations reached the range at which Ni-induced mortality began to increase, represented by the rising portion of the shaded Ni-only mortality band.

Figure 3

Concentration–response curves of select binary-metal mixtures. (A) Concentration–response relationship when a constant concentration of Cu is present with increasing concentrations of Ni. The inflection points of the curves shift toward lower concentrations as the concentration of Cu is increased. Because less Ni is required to elicit mortality as the background Cu concentration is increased, the toxicity of this mixture is more-than-additive. (B) Mixtures of constant Ni concentrations with increasing concentrations of Cd. The inflection points of the curves shift to a higher concentration of Cd as the concentration of Ni is increased, thus illustrating less-than-additive toxicity.

Figure 4

Toxicity of Cd–Ni, Cu–Ni, and Zn–Ni mixtures to Daphnia magna neonates in 48-h exposures in which the concentration of the metal that remained constant throughout a test is plotted on the horizontal axis versus the concentration at the inflection point of the varied metal’s concentration–response curve (the ECx_infl) plotted on the vertical axis. The solid horizontal line is the average median effect concentration (EC50) of the varied metal, as determined by single-metal toxicity tests run in conjunction with the mixture tests (Supplemental Data, Figure S2), and the dashed lines represent the 84% confidence limits. Open circles indicate that the ECx_infl of the binary mixture was not statistically different (p > 0.05) from the EC50 of the single-metal toxicity, based on overlap of the 84% confidence intervals for the single-metal EC50 and the mixture ECx_infl values (see text). Closed circles indicate trials in which the ECx_infl differed significantly from the single-metal EC50 (p ≤ 0.05). The tests represented in each graph are: (A) a constant background concentration of Cu while Ni was varied along a concentration gradient, (B) a constant background of concentration of Ni while Cu was varied along a concentration gradient, (C) a constant background concentration of Zn while Ni was varied along a concentration gradient, (D) a constant background concentration of Ni while Zn was varied along a concentration gradient, and (E) a constant background concentration of Ni while Cd was varied along a concentration gradient.