Acute Toxicity of Major Geochemical Ions to Fathead Minnows (Pimephales Promelas): Part A-Observed Relationships for Individual Salts and Salt Mixtures

Abstract

The results of a series of experiments on the acute toxicity of major geochemical ions (Na⁺ , K⁺ , Ca²⁺ , Mg²⁺ , Cl^- , SO₄ ^2- , HCO₃ ^- /CO₃ ^2- ) to fathead minnows (Pimephales promelas) are reported. Tests of individual major ion salts in various dilution waters demonstrated that the toxicities of Na, Mg, and K salts decrease as the overall ion content of the dilution water increases. For Na and Mg salts, this is attributable to Ca content as previously reported for Ceriodaphnia dubia. For K salts, the cause is unclear, but it is not due to Na as reported for C. dubia. In an unregulated test at high pH (9.3), NaHCO₃ was also found to be twice as toxic compared to when the pH was reduced to 8.4. Experiments with binary salt mixtures indicated the existence of multiple independent mechanisms of action. These include K-specific toxicity and Ca/Mg-specific toxicity previously reported for C. dubia, but also apparent toxicities related to SO₄ and to high pH/alkalinity in CO₃ /HCO₃ -dominated exposures. Previous work with C. dubia also suggested a general ion toxicity involving all ions that was correlated with osmolarity. For fathead minnow, similar correlations were observed, but multiple mechanisms were indicated. At higher Ca, this general toxicity could be attributable to osmotic effects, but at lower Ca, osmolarity may be more a covariate than a cause, with this toxicity being related to a combined effect of ions other than via osmolarity. Environ Toxicol Chem 2022;41:2078-2094. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Keywords: Aquatic toxicology; Fathead minnow; Major geochemical ions; Mixture toxicity; Pimephales promelas; Toxicity mechanisms.

Figure 1.

Effect of Ca on the toxicity of Mg salts to P. promelas at nontoxic Ca concentrations. Panels A and B provide LC50s for several experiments on the toxicities of MgCl₂, MgSO₄, and MgCO₃ in dilution waters with variable composition, with Panel A expressed as concentrations and Panel B as activities. Error bars are ≥95% confidence limits. Small arrows denote LC50s being slightly less than or greater than the indicated value for tests in which treatments did not bracket the LC50. The dotted blue line indicates the range of Ca concentrations for a test in which significant Ca precipitation occurred. In Panel B, the dashed line is a regression of the MgCl₂ data for {Ca}<0.4 mM, including LC50s for other MgCl₂ toxicity tests in ALSW (black dots). The gray band provides approximate uncertainty limits for predictions from the regression, based on ±2 standard deviations for the inter-experimental variability of all MgCl₂ LC50s in ALSW.

Figure 2.

Effects relationships for binary mixtures of Mg salts. Panel A provides the isobologram for the MgCl₂×MgSO₄ mixture experiment for LC50s (triangles) based on added salt concentrations. Colors indicate the ratio of salts in the mixture, grading from red for MgSO₄ to yellow for MgCl₂, and the error bars are ≥95% confidence limits. The dashed line is a linear regression over all data to illustrate concentration additivity. Panel B provides the total Mg activity at the LC50 versus the mole fraction of MgSO₄ in the mixture. The solid line denotes LC50 predictions based on the MgCl₂ toxicity versus Ca relationship in Figure 1B and the gray band reflects the inter-experimental variability in LC50s. Panels C and D provide comparable information for the MgCl₂×MgCO₃ mixture experiment.

Figure 3.

Effect relationship for the combined MgCl₂×CaCl₂ (unfilled triangles) and MgSO₄×CaCl₂ (dotted triangles) binary mixture experiments. Colors indicate the ratio of salts in the mixtures, grading from red for MgSO₄ to yellow for MgCl₂, and the error bars are ≥95% confidence limits. Additivity of Mg and Ca toxicity is demonstrated by the linearity of the data except at low Ca, where LC50s are reduced by the dependence of Mg-only toxicity on Ca.

Figure 4.

Effect of Ca and pH on the toxicity of Na salts and mannitol to P. promelas at nontoxic Ca concentrations. Panels A-C provide LC50s for several experiments on the toxicities of NaCl, Na₂SO₄, NaHCO₃, and mannitol in dilution waters with variable Ca, with Panel A expressed as concentrations of added compound versus Ca concentration, Panel B as osmolarity versus Ca activity, and Panel C as Na activity versus Ca activity. Blue dotted lines indicate the range of Ca concentrations for NaHCO₃ tests in which significant Ca precipitation occurred. In Panel B, the black dash-dotted line denotes the average LC50 (no Ca dependence) for mannitol. In Panel C, the dashed line is from a regression analysis of the NaCl data for {Ca}<0.2 mM, including LC50s for other NaCl toxicity tests in ALSW (black dots). The gray band provides approximate uncertainty limits for predictions from the regression, based on ±2 standard deviations for the inter-experimental variability of all NaCl LC50s in ALSW. The light blue circles are for two toxicity tests with NaHCO₃ in which test solutions were equilibrated prior to exposure with either ambient air (open circle), for which the pH was 9.3, or with 1% CO₂ (crossed circle), for which the pH was 8.4.

Figure 5.

Effects relationships for binary mixtures of Na salts and mannitol. Panels A-C provide the isobolograms for the NaCl×Na₂SO₄, NaCl×NaHCO₃, and NaCl×mannitol experiments, for LC50s based on added compound concentrations. Colors indicate the ratio of compounds in the mixture, based on red for Na₂SO₄, yellow for NaCl, blue for NaHCO₃, and black for mannitol. Error bars are ≥95% confidence limits for LC50 estimation. The light blue circles in Panel E are for the separate experiment in which NaHCO3 toxicity is contrasted at the unadjusted high pH (open circle) to a reduced pH (crossed circle). The dashed line in Panel A is a linear regression of data except for the Na₂SO₄-only test. The dashed line in Panel C approximates mixture compositions expected to produce the same level of osmolarity as the mannitol-only test. Panels D-F plot data based on exposure metrics considered relevant to the mixtures. The solid lines denote LC50 predictions based on Ca-dependent NaCl toxicity (regression line in Figure 4C) and Ca-independent mannitol toxicity; for Panel E, the line is limited to not extrapolate beyond the regression limits. The gray band denotes uncertainty based on the inter-experimental variability of LC50s.

Figure 6.

Effect relationship for the combined NaCl×CaCl₂ (unfilled triangles) and Na₂SO₄×CaCl₂ (dotted triangles) binary mixture experiments. Colors indicate the ratio of salts in the mixtures, grading from red for Na₂SO₄ to yellow for NaCl, and the error bars are ≥95% confidence limits. The solid line denotes the predicted relationship at {Ca}>3 mM for independent action of Caspecific toxicity and osmolarity-driven toxicity, with osmolarity-driven toxicity based on the study-wide average LC50s for all mannitol-only tests and the Ca-specific toxicity being based on the study-wide average LC50s for all CaCl₂ tests in ALSW. The gray band denotes uncertainty based on the inter-experimental variability of LC50s. Lower LC50s at the lowest Ca level represent more potent mechanisms than osmolarity-driven that are further addressed in the companion modeling paper.

Figure 7.

Isobolograms for NaCl×MgCl₂ (Panels A-D) and Na₂SO₄×MgSO₄ (Panels E-G) mixture tests. Panel A compares LC50s for the Cl salt mixtures on the basis of added salt concentration to isoboles (dotted lines) for concentration addition and independent action fit to the single-salt LC50s. Panel B compares Cl salt LC50s on the basis of Na and Mg activities to predictions from a two-mechanism model (solid line) for independent action of these two activities, with the dashed lines representing the individual mechanisms and the gray band the model uncertainty. Panel C similarly compares Cl salt LC50s on the basis of Mg activity and osmolarity to a model driven by these two metrics. Panel D compares Cl salt LC50s on the basis of Na and Mg activities to a three-mechanism model that also includes osmolarity-driven toxicity (diagonal dashed line). Panel E compares LC50s for the SO₄ salt mixtures on the basis of added salt concentration to a concentration addition isobole (dashed line) fit by linear regression of the data except for the MgSO₄-only test. Panel F compares SO₄ salt LC50s on the basis of Na and Mg activities to the two-mechanism model for independent action of these activities. Panel G compares SO₄ salt toxicities on the basis of Mg and SO₄ activity to expected LC50s for Mg-driven toxicity (arrow) and SO₄-driven toxicity (dashed line).

Figure 8.

Interaction of KCl toxicity with NaCl and MgCl₂. Panel A shows different effects of Na in the dilution water on KCl toxicity for C. dubia (+’s) from Mount et al. (2016) and fathead minnow from the present study (×’s). For fathead minnow, KCl toxicity is approximately independent of both NaCl (Panel B, NaCl×KCl experiment) and MgCl₂ (Panel C, MgCl₂×KCl experiment) toxicities.