Water hardness reduces the accumulation and toxicity of uranium in a freshwater macrophyte (Ceratophyllum demersum)

Abstract

There is a lack of good quality data and mechanistic understanding on the effects of true water hardness (calcium (Ca) and magnesium (Mg)) on the bioavailability and toxicity of uranium (U) to freshwater biota. This study determined the effect of true water hardness (20, 75, 150, 275 and 400 mg CaCO₃ L^− 1) on the cell surface binding affinity (log K), accumulation and toxicity (growth inhibition) of U in a submerged, rootless, macrophyte (Ceratophyllum demersum) in a synthetic freshwater with constant alkalinity (13 mg CaCO₃ L^− 1) and pH (6.2) over 7 days. A 20-fold increase in water hardness resulted in a 4-fold decrease in U toxicity (median effect concentration (EC50) = 134 μg L^− 1 U at 20 mg CaCO₃ L^− 1 hardness, increasing to 547 μg L^− 1 U at 400 mg CaCO₃ L^− 1 hardness), cell surface binding affinity (log K = 6.25 at 20 mg CaCO₃ L^− 1 hardness, decreasing to log K = 5.64 at 400 mg CaCO₃ L^− 1 hardness) and accumulation (the concentration factor decreased from 63 at 20 mg CaCO₃ L^− 1 hardness to 15 at 400 mg CaCO₃ L^− 1 hardness) of U. Calcium provided a 4-fold greater protective effect against U accumulation and toxicity compared to Mg. Speciation calculations indicated negligible differences in the percentages of key U species (UO₂^2 +, UO₂OH⁺, UO₂(OH)₂) over the range of water hardness tested. The inhibition of U binding at the cell surface, and subsequent uptake, by C. demersum, with increasing Ca and/or Mg concentration, may be explained in terms of (i) competition between Ca^2 +/Mg^2 + and UO₂^2 + (and/or UO₂OH⁺) for physiologically active sites at the cell surface, and/or (ii) reduced negative charge (electrical potential) at the cell surface, resulting in a decrease in the activity of UO₂^2 + (and/or UO₂OH⁺) at the plant/water interface (boundary layer), and consequently, less U bound to physiologically active cell surface sites. In the absence of a biotic ligand model for U, the results of this study (together with previous work) reinforce the need for a more flexible, hardness-dependent, U guideline for the protection of selected freshwater biota.

Introduction

Uranium (U) speciation and toxicity to biota in fresh surface waters may be influenced by a variety of physicochemical variables, particularly water hardness, alkalinity, pH and natural organic matter (Goulet et al., 2012, Markich and Twining, 2012). Many studies that have investigated the effect of water hardness on the uptake and/or toxicity of U in freshwater organisms have confounded the effects of true water hardness (calcium (Ca) and/or magnesium (Mg) concentration) with alkalinity (carbonate concentration) and/or pH (proton concentration) (e.g., Parkhurst et al., 1984, Poston et al., 1984, Barata et al., 1998, Borgmann et al., 2005), since an increase in Ca and/or Mg is often associated with an increase in alkalinity, and hence, pH in fresh surface waters (Meybeck, 2003). It is important to separate the effects of true water hardness, alkalinity and pH, since they have different mechanisms of toxicity. The hardness cations (Ca^2 + and/or Mg^2 +) or protons (H⁺) may competitively inhibit U binding/uptake at the cell surface (Franklin et al., 2000, Riethmuller et al., 2000, Charles et al., 2002, Fortin et al., 2007), whereas alkalinity reduces U binding/uptake at the cell surface via changes in U speciation in solution, where the bioavailability of U (e.g., UO₂^2 +) is reduced through complexation with carbonate (Nakajima et al., 1979, Markich et al., 1996, Tran et al., 2004).

The effect of true water hardness has been successfully uncoupled from the effects of alkalinity and/or pH in studies by Riethmuller et al. (2000), Charles et al. (2002), Vizon SciTec (2004) and Fortin et al. (2007). However, findings on the effects of true water hardness on U toxicity/uptake are mixed. While some studies with fish (Riethmuller et al., 2000, Vizon SciTec, 2004) have shown that U toxicity is independent of water hardness, studies with unicellular green algae (Charles et al., 2002, Fortin et al., 2007), green hydra (Riethmuller et al., 2000) and an amphipod (Vizon SciTec, 2004) have found that U uptake or toxicity decreases with increasing water hardness, where the apparent magnitude of biological response may vary markedly. For example, the toxicity of U decreased 2-fold with a 50-fold increase in water hardness (from 6.6 to 330 mg CaCO₃ L⁻¹) for the green hydra, Hydra viridissima (Riethmuller et al., 2000), while the toxicity of U decreased 20-fold with a 14-fold increase in water hardness (from 17 to 238 mg CaCO₃ L⁻¹) for the amphipod, Hyalella azteca (Vizon SciTec, 2004).

Ceratophyllum demersum is a submerged, free-floating (rootless), freshwater macrophyte with a cosmopolitan distribution in tropical and temperate regions (CABI, 2011), and provides habitat and a food source for a variety of herbivorous fish and macroinvertebrates (Schultz and Dibble, 2012). Uranium is of potential ecotoxicological concern in fresh surface waters at several sites in northern Australia, primarily as a result of past and present mining activities (Markich et al., 2003, Bayliss et al., 2012). Calcium and/or Mg concentrations in mine wastewaters in the region (e.g., Rum Jungle mine) may be up to 500 times higher than receiving waters (Markich et al., 2003). Knowledge of the relationship between true water hardness and U toxicity for C. demersum may provide further data to formulate a hardness-dependent algorithm for future inclusion into site-specific and/or national quality guidelines for protecting selected freshwater biota in Australia, and elsewhere. A dissolved organic carbon (DOC)-dependent algorithm for U has recently been developed (van Dam et al., 2012). Such data will also be useful in the future development of a biotic ligand model (BLM; McGeer et al., 2010) for U.

The aim of this study was to determine whether true water hardness (20, 75, 150, 275 and 400 mg CaCO₃ L^− 1, added as Ca and Mg chloride) affects the toxicity (7 day growth inhibition) of U to C. demersum in a synthetic freshwater, at a constant alkalinity and pH. The accumulation of U and its binding affinity (or conditional stability constant; log K) at the cell surface were also measured to determine whether changes in U toxicity at different levels of water hardness were due to competition between U and Ca/Mg at cell surface binding sites. In addition, U speciation in the test waters was calculated to assess whether speciation changes, if any, could explain changes in U toxicity with varying water hardness.