Test organisms

Fathead minnows were from the in-house culture at the US Environmental Protection Agency (USEPA) Great Lakes Toxicology and Ecology Division (Duluth, MN, USA), which is maintained at 25 °C in filtered Lake Superior water (LSW, obtained from an intake located offshore from the laboratory at 46.840°N, 92.004°W). This water typically has a pH of approximately 7.5, a dissolved organic carbon concentration of 1–2 mg C/L, and inorganic constituents as specified in Table 2. For each experiment, a cohort of newly hatched fry (<24 h old) from multiple spawns was isolated, fed brine shrimp nauplii ad libitum for 6 days, and used for testing when 7–8 days old. Preliminary efforts had evaluated the toxicities of NaCl and MgSO₄ to fathead minnows <1, 3–4, 7–8, and 14–15 days old. The selection of organisms that were 7–8 days old was based on them not being significantly less sensitive than other ages, being robust compared with newly hatched fry regarding control survival in the absence of feeding, and being amenable to smaller chambers and less culture effort than older organisms. This work was conducted under an institution-approved animal care and use plan within the project plan (MED-QAPP-DM-2013-07).

Test chemicals

Nine major ion salts were used to evaluate the relative toxicities of and the interactions among the major ions, including the chloride salts of all four cations, the sulfate salts for all cations except Ca (due to limited solubility), and the carbonate salts of Na and Mg. Gluconate salts of Na, Mg, and Ca were also tested to provide an anion expected to have limited interaction with the organisms to further evaluate correlation of toxicity to the cations. Mannitol was tested to provide a comparison of salt toxicity to that of a compound expected to only affect the external osmotic environment.

All chemicals were obtained from the Sigma-Aldrich Chemical Company or Thermo-Fisher Scientific Company. All compounds had a designated hydration and a purity of at least 98%, except for MgCO₃ and Mg gluconate, for which the hydration was not specified. The certificates of analysis for these two salts were used to determine the ratio of the anhydrous salt to total salt weight for computing nominal concentrations. The certificate of analysis for MgCO₃ also specified the Ca content to be 0.44% of the Mg content (on a molar basis), enough to appreciably affect the background Ca for tests on the toxicity of MgCO₃, necessitating additional Ca measurements to document concentrations.

Test waters

The dilution water for most tests was amended Lake Superior water (ALSW), which was developed by Mount et al. (2016) to address concerns that ion ratios in some commonly used dilution waters are atypical of natural waters (e.g., unusually high Mg:Ca ratios in some synthetic waters, low Na in LSW relative to its hardness and alkalinity), which could cause misleading results. Amended Lake Superior water is prepared by amending sand-filtered and UV-treated LSW with major ion salts to provide ion ratios close to median values for United States freshwaters, while maintaining its alkalinity and other natural constituents. The major ion content of ALSW is provided in Table 2.

For some tests, the formula for ALSW was modified to create test waters with different concentrations for some or all ions (Table 2). This included three ways in which Ca was modified. First, Ca was changed in proportion to all other ions by diluting ALSW with deionized water so that all ion concentrations were reduced to a third of that in ALSW (⅓×ALSW) and by adding salts so that ion concentrations were increased to three times that in ALSW (3×ALSW). Second, Ca was changed inversely with Mg while keeping water hardness and other ions approximately constant so that the Ca:Mg molar ratio of 2.2 for ALSW was reduced to 0.2 (LoCa:Mg) and increased to 9 (HiCa:Mg). Third, several test waters were formulated with altered Ca concentrations ranging from 1/8× to 81× that in ALSW, reducing Ca by substituting Na for a portion of the Ca in the water formulation and increasing Ca concentrations by adding CaCl₂, with other ions approximately constant. To test the effects of Na and K concentrations in dilution water, test waters were also formulated with different Na concentrations (1.6 mgNa/L, 10 mgNa/L, 30 mgNa/L), with concomitant changes to Cl, and with the K:Na molar ratio altered from 0.14 for ALSW to 0.015 (LoK:Na) and 3.1 (HiK:Na), with negligible changes to other ions.

Study design

Seventeen experiments, each comprising two to seven simultaneous toxicity tests (92 total tests), were conducted on the acute (96-h) lethality of single compounds to fathead minnows in various dilution waters. Table 3 specifies the experiments addressing the effects of different dilution waters on the lethality of the major ion salts and mannitol. Other experiments compared the toxicities, in ALSW only, of mannitol and of the gluconate salts of Na, Mg, and Ca to the chloride salts of these cations. An additional experiment compared the toxicity of NaHCO₃ in ALSW with pH uncontrolled (9.3) to that with pH reduced to 8.4 by elevating CO₂.

Testing the toxicity of mannitol to fathead minnow in dilution waters with different [Ca] was important to the evaluation of the relationship of salt toxicity to osmolarity and to model development in the companion article (Erickson et al., 2022). For C. dubia the effects of Ca on mannitol were not tested by Mount et al. (2016), so an ancillary experiment was conducted to fill this data gap and is described in Supporting Information.

Fifteen binary mixture experiments (78 total tests) were conducted in ALSW. Each experiment consisted of multiple toxicity tests, each test having a fixed ratio of the two chemicals. A typical experiment had five tests with estimated toxic unit ratios of 1:0, 3:1, 1:1, 1:3, and 0:1, but ratios were sometimes changed or added to better establish chemical interactions. These mixture experiments included all possible combinations involving NaCl, Na₂SO₄, MgCl₂, MgSO₄, and CaCl₂. The compounds NaHCO₃ and MgCO₃ were tested only in mixtures with their respective Cl salts. For K salts, the only mixture tests were of KCl with NaCl and MgCl₂ due to the expectation of K-driven toxicity independent of other ions. A final mixture experiment was of NaCl and mannitol to test for concentration additivity on an osmolarity basis.

Toxicity test procedures

Static, 96-h, unfed acute toxicity tests were conducted in accordance with ASTM International Method E729 (ASTM International, 2002). Exposures were in 5 oz (~150 ml) polystyrene cups (Fineline Settings) containing 80 ml of test solution and suspended in a temperature-controlled (25 °C) water bath using perforated floating foam boards, with glass sheets covering all cups. The test compounds were dissolved in the designated test dilution water (Tables 2 and 3) to produce the highest test concentration, with the dissolution of MgCO₃ being CO₂-assisted as described in Mount et al. (2016). This high test concentration was serially diluted with the test dilution water to produce a geometric series of 9–10 concentrations with a dilution factor of approximately 0.80× (i.e., 100%, 80%, 64%, 50% etc.) or 0.707× (i.e., 100%, 70.7%, 50% etc.). Solutions were prepared immediately prior to test initiation, except for two experiments involving NaHCO₃ (NaCl × NaHCO₃ mixture experiment, NaHCO₃ toxicity at different pHs), for which solutions were prepared 3 days in advance to allow CaCO₃ precipitation to occur and Ca concentrations to stabilize. In other, earlier experiments involving NaHCO₃, CaCO₃ precipitation caused Ca concentrations to decline by a factor of up to 20 over the course of the 4-day exposures, making it difficult to associate a specific {Ca} with the LC50 for this salt (Note that braces { } are used for chemical activities, vs. brackets [ ] for concentrations).

Each concentration was tested in duplicate cups, except quadruplicate cups were used for the control for the last two experiments (on NaCl × mannitol mixture interactions and the effects of Ca on mannitol toxicity) to provide better information on control survival. For the experiment on the effect of added CO₂ on NaHCO₃ toxicity, a third cup was added for the control and for three concentrations near the expected LC50 to provide samples for Na and Ca measurements at 48 h, but these destructively sampled cups were not included in effects assessment. Five fathead minnows were added to each cup at test initiation. The number of surviving organisms in each test chamber was recorded at the end of each 24-h period. Death was determined by the change in appearance from translucent to opaque or no reaction to gentle prodding. At the end of the exposures, surviving fish were euthanized with a buffered solution of nonpharmaceutical grade tricaine mesylate (Finquel).

Tests were conducted under ambient laboratory fluorescent lighting with a 16:8-h light:dark photoperiod. At the initiation of each test, alkalinity and hardness were measured in an aliquot of the control water, dissolved oxygen and pH were measured in aliquots of both the control water and the highest test concentration, and conductivity was measured for all test concentrations in one of the replicate test cups. Temperature was measured daily in one chamber of each test. Conductivity, dissolved oxygen, and pH were measured at 24, 48, and 72 h in one replicate of every test concentration reaching 100% mortality during the preceding time interval, and at 96 h in one replicate of all other concentrations. These measurements were only in test chambers being terminated to avoid the probes perturbing test organisms and/or test water chemistry, while still documenting the chemistry at concentrations causing mortality.

Exposure monitoring

Samples for analysis of the major ions were collected at the start of each experiment from the dilution water(s) and from the highest concentration of each toxicity test. In addition, samples were collected for each test at 24 h from the lowest concentration with 100% mortality, and at 96 h from the concentration nearest the apparent LC50. Additional samples were taken in some experiments to more fully characterize ion concentrations, especially Ca, in treatments near the LC50. The conductivity measurements made on all treatments confirmed that the intended gradient of exposure existed among treatments not sampled for cation analysis.

Dilution waters were analyzed for all four cations plus Cl and SO₄. Other samples were analyzed for the test salt cation(s), and also for Ca in tests involving NaHCO₃ and MgCO₃ (which caused oversaturation of CaCO₃), for the Na₂SO₄ × CaCl₂ and MgSO₄ × CaCl₂ mixture tests (which caused oversaturation of CaSO₄), and for the toxicity tests with NaCl, MgCl₂, Na₂SO₄, and MgSO₄ over the series of dilution waters with modified Ca.

Samples were filtered through an 0.45 µm nylon syringe filter (Grainger). For cation analyses, samples were acidified to 0.2% (v/v) with concentrated HNO₃ and held at room temperature; for tests with NaHCO₃ and MgCO₃, this acid addition was increased by an amount calculated to neutralize the extra alkalinity. For Cl and SO₄ analyses, samples were refrigerated. Analyses were by flame atomic absorption spectrophotometry for cations and ion chromatography for anions, using the methods and equipment described in Mount et al. (2016). For samples in which SO₄ exceeded 20 mM, there was apparent interference with Ca analysis due to insufficient inhibitor for this interference, resulting in estimated Ca concentrations 10%–30% lower than nominal; such measurements were not used.

For measurements of ions in dilution waters (ALSW and the modifications thereof in Table 2), the ratios of measured to nominal concentrations had means (% relative standard deviation, sample size) of 1.03 (6%, n = 64) for Na, 1.03 (8%, n = 63) for K, 1.01 (6%, n = 63) for Ca, 1.07 (4%, n = 63) for Mg, 1.01 (4%, n = 57) for Cl, and 1.05 (7%, n = 60) for SO₄. For measurements of test salt cations in test solutions near and above the LC50, the ratios of measured to nominal concentrations across all samples were 1.00% (4%, n = 402) for Na, 0.98% (3%, n = 74) for K, 1.02% (5%, n = 353) for Mg, and 0.96% (6%, n = 41) for Ca, excluding Ca analyses for tests in which CaCO₃ or CaSO₄ precipitation was a consideration and for tests with MgCO₃ due to uncertain nominal Ca. For these test salt cation comparisons (n = 870), only 18 measurements (2.1%) deviated by more than 10% from nominal, with a maximum deviation of 19%. Such agreement was considered sufficient to warrant the use of nominal concentrations in the data analyses except as follows.

For the Na₂SO₄ × CaCl₂ and MgSO₄ × CaCl₂ mixture experiments, most mixture ratios had visible precipitates and lower-than-nominal measured Ca concentrations at the highest exposure concentrations. At the LC50, precipitation was less evident, but test solutions were calculated to be oversaturated for CaSO₄ and there were minor (~10%) reductions in measured Ca from nominal. Therefore, for these experiments, the Ca and SO₄ concentrations to associate with the LC50 were based on interpolation of the average Ca measurements in treatments bracketing the LC50.

Measured Ca concentrations were also used in tests with NaHCO₃ and MgCO₃ due to precipitation of CaCO₃ and due to the uncertain Ca content of the MgCO₃ salt. At each measurement time, Ca measurements were used to estimate the Ca concentration at the LC50 by interpolation. If these estimates declined by ≤25% over time, their average was associated with the LC50. If the temporal variation was greater than this, both the nominal and final Ca concentrations are reported to designate a range for the Ca concentration to associate with the LC50. The same procedure was applied to the Na₂SO₄ toxicity test with the highest Ca elevation (81×), where substantial CaSO₄ precipitation at the LC50 was evident.

Data analysis: LC50 estimation

Median lethal concentrations and their confidence limits were estimated similarly to Mount et al. (2016). For tests with at least two partial mortalities, a tolerance distribution analysis was conducted using a three-parameter log-logistic model. Parameters were estimated by maximum likelihood analysis using custom software written with Intel Visual Fortran Compiler XE 2015 (Intel Corporation) and Winteracter 13.0 (Interactive Software Services). Confidence limits (95%) were calculated using the likelihood ratio method (Williams, 1986). For tests with insufficient partial mortalities for this analysis, the same likelihood ratio method was used to calculate confidence limits for the LC50, which can be assigned in the absence of a unique point estimate for the LC50. For these tests, we assigned the geometric mean of these confidence limits to be the point estimate for the LC50. For cases in which there were no partial mortalities, these confidence limits are >95% and are the bracketing concentrations (i.e., the highest concentration with control survival and the lowest concentration with complete mortality), and the LC50 is equivalent to linear interpolation of survival versus log concentration between these two concentrations.

Median lethal concentrations were initially calculated as a percentage of the highest added concentration in the test and then converted to molar concentrations of the added salts. These concentrations were then added to the ion concentrations already present in the dilution water and the resultant total ion concentrations were input to the chemical speciation program Visual MINTEQ (Ver 3.1, https://vminteq.lwr.kth.se/visual-minteq-ver-3-1) to estimate the chemical activity of each chemical species at the LC50. The osmolarity at the LC50 was estimated based on MINTEQ activity estimates using the method described by Robinson & Stokes (1959). The sum of the concentrations of the major inorganic ions, mannitol, and gluconate was also calculated as a potential exposure metric. Confidence limits for LC50s expressed as these various metrics were assigned the same relative values as those for the percentage added concentration LC50s, and do not reflect any additional uncertainty associated with the speciation or other calculations.

Data analysis: Evaluating relationships in binary salt mixture experiments

As discussed in Erickson et al. (2017) and references therein, binary toxicity experiments can be used to infer certain information about toxicity mechanisms, specifically whether the toxicities of the two chemicals show concentration addition (suggesting a shared toxicity mechanism), independent action, synergism, antagonism, or other interactions. Binary mixture toxicity experiments in Erickson et al. (2017) contributed to the inferences about major ion toxicity to C. dubia summarized in Table 1, using isobolograms that compared the observed mixture LC50s to isoboles for concentration addition or independent action fitted to the individual salt LC50s for the experiment. In the present study, isoboles are similarly fitted for some data comparisons, but mixture experiment LC50s are also compared with isoboles predicted based on prior results with individual salts. Furthermore, the prior data was just for Cl salts, so that the predictions address consistency of relationships across different anions. Details on the methods for both fitting and predicting isoboles are provided in Supporting Information.

Effects of dilution water on the toxicity of Mg salts

Effects of dilution water Ca

For all modifications of Ca in dilution water (Tables 2 and 3), Figure 1A provides LC50s for MgCl₂, MgSO₄, and MgCO₃ based on added salt molarity versus Ca concentration. Median lethal concentrations in Figure 1A increase substantially with Ca concentration for all Mg salts and for all methods for modifying Ca concentration. This adds to a consistent pattern of such effects on major ion toxicity, including Ca effects on major ion toxicity to C. dubia (Mount et al., 2016), effects of Ca on Na salt toxicity in the present study, and effects of hardness reported by other authors (e.g., Elphick, Bergh, & Bailey, 2011; Elphick et al., 2011).

The LC50 for the MgCO₃ test in 3×ALSW is uncertain because this is a “greater than” value; however, because there was 30% mortality at the highest concentration (Supporting Information, Table S1), the LC50 should be no more than 20% higher based on typical slopes of the concentration/effect curves. Ca also changed during this test due to CaCO₃ precipitation. The Ca concentration to associate with this LC50 is unlikely to be at either extreme of this Ca range, but could be lower than the indicated midpoint of the range to the extent that mortality reflects increased potencies (due to lower Ca) at the end of the test. Regardless of these uncertainties, this test supports a Ca effect on Mg toxicity.

Implications of single salt tests on exposure metrics for Mg salt toxicity

Interactions in binary mixtures of Mg salts

MgCl₂ × MgSO₄ binary mixture experiment

Figure 2A presents the isobologram for LC50s based on molarity of added salt. The closeness of the data to a straight line indicates a strong degree of additivity, supportive of Mg-driven toxicity. However, [Mg] at the LC50 steadily increases as the proportion of MgSO₄ in the tests increases, reaching nearly 60% greater for the MgSO₄-only test than for the MgCl₂-only test, indicating total concentration is not an appropriate exposure metric for Mg-based toxicity.

In contrast, LC50s for the MgCl₂ × MgSO₄ mixtures expressed as {Mg} (Figure 2B) are less variable and are consistent with predicted values from the {Mg}-based toxicity model (Equation 1). This prediction includes a small (~10%) reduction of LC50 as the molar fraction of MgSO₄ increases, due to complexation of Ca by SO₄. Due to the unusually low toxicity for the MgCl₂-only exposure in this experiment (which is the lowest black dot in Figure 1B), this Ca effect is not clear in these data. However, a Ca effect consistent with model predictions is evident in mean LC50s in ALSW across the entire study (Supporting Information, Tables S1 and S2), which on the basis of {Mg} drop from 12.1 mM for MgCl₂ tests (n = 9) to 10.9 mM for MgSO₄ tests (n = 6).

Interactions in binary mixtures of Mg and Ca salts

Figure 3 provides a combined isobologram for the MgCl₂ × CaCl₂ and MgSO₄ × CaCl₂ mixture experiments based on Mg and Ca activities, showing good agreement between the Cl-only and combined Cl/SO₄ mixtures, thereby supporting cation-driven toxicity. The linearity of {Mg} versus {Ca} for all but the lowest level of Ca indicates concentration addition, with effects of Ca on Mg toxicity (Figure 1B) causing the LC50 at the lowest Ca to deviate from the line. As for C. dubia (Erickson et al., 2017, 2018), this additivity indicates a common mechanism with {Mg} and {Ca} as the exposure metrics. A prediction for this isobole is not provided in the present study because Equation 1 does not address the complete Ca range; rather, this relationship is further addressed in the companion modeling article (Erickson et al., 2022).

Effects of dilution water chemistry on toxicity of Na salts and mannitol

Effects of dilution water Ca

For all modifications of Ca in dilution water (Tables 2 and 3), Figure 4A provides LC50s for NaCl, Na₂SO₄, NaHCO₃, and mannitol based on added compound concentration versus Ca concentration. Figure 4A also provides the two tests of NaHCO₃ toxicity in 1×ALSW for which test solutions were equilibrated prior to exposure with either ambient air or with 1% CO₂.

Median lethal concentrations for NaCl and Na₂SO₄ increase substantially with Ca concentration for all methods for modifying Ca concentration. Relative to the other salts, the data for NaHCO₃ are more limited, consisting of three nonequilibrated tests in the ⅓×/1×/3×ALSW series and the pre-equilibrated test at high pH in 1×ALSW, and more uncertain due to the large changes in Ca concentration during the nonequilibrated tests in 1× and 3×ALSW. Nevertheless, based on mid-range Ca concentrations, the Ca dependence of NaHCO₃ is similar to that for NaCl and Na₂SO₄. Only if the LC50s for the nonequilibrated 1× and 3×ALSW tests are related to the lowest Ca concentrations (at the end of the tests) would a Ca dependence not be evident. However, the pre-equilibrated test at high pH in 1×ALSW argues against such an interpretation because it was run at a constant Ca concentration similar to those at the end of the nonequilibrated 1× and 3×ALSW tests, and indicates the LC50 to be considerably lower at this Ca concentration.

In contrast to the Ca-dependence of inorganic Na salt toxicity, there is no effect of Ca on mannitol toxicity (Figure 4A). The ancillary experiment for the Ca-dependence of mannitol toxicity C. dubia similarly showed no effects (see Supporting Information, Figure S5). If mannitol toxicity is due to osmotic effects on passive water movement, Ca apparently has little effect on this process, versus its impact on other ion toxicity processes. In any event, these results suggest different mechanisms of toxicity for mannitol and the Na salts at lower Ca concentrations.

Implications of single salt tests on exposure metrics for Na salt toxicity

Interactions in binary mixtures of Na salts

NaCl × Na₂SO₄ binary mixture experiment

Figure 5A presents the isobologram for LC50s based on molarity of added salt. Starting at the NaCl-only LC50 on the abscissa, the data for all the mixtures follow a straight line indicative of additivity, but then abruptly deviate horizontally to the Na₂SO₄-only LC50 on the ordinate. This deviation is consistent with the divergence of NaCl and Na₂SO₄ toxicities at low Ca in Figure 4. Because the only component that increases with this deviation is SO₄, this suggests a SO₄-specific toxicity once SO₄ (or the ratio of SO₄ to another ion) reaches some critical level. Below this critical level, the additive relationship between Na₂SO₄ and NaCl suggests a different mechanism involving only Na or multiple ions.

To illustrate this further, Figure 5D presents the data as {Na} versus {SO₄}, with predictions from the {Na}-based toxicity model (Equation 2). The NaCl-only LC50 for this experiment is the lowest in our study (lowest black dot on Figure 4C) and the mixture data also follow the lower limit of the uncertainty band, declining gradually as {Ca} decreases due to complexation by SO₄. However, for the Na₂SO₄-only test, the LC50 abruptly deviates from predictions as SO₄ apparently reaches a toxic level. It is important to note that the adherence of the data to the model line at lower SO₄ does not provide proof that {Na} is the toxicity driver, but only that it is a good correlate; this toxicity could be due to the combined effect of multiple ions as was the apparent case for C. dubia (Erickson et al., 2017; Mount et al., 2016). Toxicity of SO₄ is also indicated by study-wide mean LC50s in ALSW from Supporting Information, Tables S1 and S2, which on the basis of {Na} drop from 89 mM for NaCl (n = 13) to 55 mM for Na₂SO₄ (n = 6), twice the drop from 89 to 72 mM predicted by the {Na}-based model.

Interactions in binary mixtures of NaCl and mannitol

A mixture experiment involving NaCl and mannitol (Figure 5C) was conducted to further address whether osmolarity is a useful exposure metric for some aspects of ion toxicity. The dashed line connects the molarity of the mannitol-only LC50 to an estimated molarity of NaCl needed to produce the same level of osmolarity, so represents additive toxicity for an exposure metric of osmolarity. Importantly, mixture toxicities are being elicited at combinations of mannitol and NaCl concentrations which individually would not be toxic, demonstrating considerable additivity (i.e., NaCl contributes to mannitol's mechanism of toxicity). There are upward deviations of mixtures from this line, but these are <20%. At higher proportions of NaCl there is a modest trend toward LC50s with lower osmolarities, suggesting a shift away from osmolarity-driven toxicity.

The relationships in Figure 5C are consistent with the data in Figure 4, which suggest that NaCl has a mechanism that is specifically ion-related and more toxic than mannitol at low Ca, but, as this toxicity decreases with increasing Ca, NaCl LC50s converge with that of mannitol and transition to a mechanism correlated with osmolarity. The mixture test in Figure 5C was conducted at Ca levels near but slightly below this transition, which would explain both the substantial degree of additivity on an osmolarity basis and the deviation to lower osmolarities for the NaCl-dominated tests.

Figure 5F plots LC50s on the basis of osmolarity versus {Na} to better illustrate the interaction of these two suggested mechanisms, with predictions based on the Ca-dependent {Na}-based model (Equation 2) and an average study-wide, Ca-independent, osmolarity-based LC50 for mannitol-only tests (278 mOsm/L, n = 7; Supporting Information, Tables S1 and S2). There are undoubtedly complexities to these toxicity relationships that are not fully resolved and data trends are modest relative to uncertainties, but these predictions are reasonably consistent with the data, namely, osmolarities at the LC50 are roughly constant and similar to mannitol for most mixtures and a transition to a specific ion-related mechanism is indicated at the highest proportions of NaCl. If this mechanistic interpretation is correct, the shape of this isobole would be expected to change at different dilution water [Ca], showing more independence at lower Ca and more complete additivity at higher Ca, which would be a useful element of future study.

Given the lack of environmental relevance of exposure to mannitol, the results in Figure 5C,F might seem to be of only academic interest. However, there is a need to address interactions of Na salts with salts of other cations, and interpreting such interactions will be informed by these interactions of NaCl and mannitol.

Interactions in binary mixtures of Na and Ca salts

Figure 6 provides a combined isobologram for LC50s from the NaCl × CaCl₂ and Na₂SO₄ × CaCl₂ mixture experiments, as osmolarity versus {Ca}. It also includes predicted LC50s at {Ca} > 3 mM for independent action of osmolarity-based toxicity (the aforementioned study-wide average of 278 mOsm/L for mannitol toxicity) and Ca-specific toxicity (a study-wide average of 15 mM {Ca} for CaCl₂ toxicity from Supporting Information, Tables S1 and S2). At these higher {Ca}, the observed LC50s are consistent with independent action of these mechanisms, that is, starting with the CaCl₂-only tests at the lower right, Ca activities at the LC50 first remain constant as Na salts are added and then transition to osmolarity-based LC50s similar to those for mannitol. As was the case for C. dubia (Erickson et al., 2017), there is an indication of greater-than-expected toxicity when the Ca-specific and osmolarity mechanisms are at roughly equal toxicity (the bend in the prediction line), but the deviation is only for one test and is <20%.

The Ca-dependent toxicity at lower Ca is not addressed by the predictions in Figure 6 because it involves only two data points and these have different mechanisms of toxicity. More complete consideration of this part of the relationship is provided in the companion modeling article (Erickson et al., 2022).

Interactions in binary mixtures of Na and Mg salts

NaCl × MgCl₂ binary mixture experiment

Figure 7A compares LC50s based on added concentrations of NaCl and MgCl₂ to fitted isoboles for concentration addition and independent action. The mixture with the highest NaCl fraction does fall on the independent action line, but the data then veer off to be intermediate to the two isoboles (i.e., “partial addition”). Plotting the data as {Mg} versus {Na} and using the independent action isobole predicted from Equations 1 and 2 has no appreciable effect on this pattern (Figure 7B), which was expected due to the strong correlation between added concentrations and cation activities. A primary implication of this is that there is a mechanism of toxicity to which both MgCl₂ and NaCl contribute, instead of or in addition to any mechanisms related just to the individual salts.

For C. dubia, similar plots (Figure 3A–C in Erickson et al., 2017) showed LC50s to also be intermediate between concentration addition and independent action, although closer to the latter. When LC50s were expressed as {Mg} versus osmolarity there was close adherence to independent action (Figure 3D–F in Erickson et al., 2017). This was interpreted as Na salt toxicity involving a mechanism involving multiple ions, to which the Mg salts also contribute. Osmolarity was selected as the metric for this mechanism because of the close concordance of osmolarity-based salt LC50s with mannitol LC50s, at least at the {Ca} for the mannitol tests; these are also in accord with D. magna internal osmolarities indicated in Morris et al. (2021). However, this does not mean that effects are from osmotic stress across all {Ca}.

Because of such relationships for C. dubia, Figure 7C compares LC50s on the basis of {Mg} and osmolarity to a model of independent action for {Mg} (using Equation 1) and osmolarity (278 mOsm/L). The LC50s for the three tests with the highest mole fraction of NaCl have approximately the same osmolarity, consistent with toxicity being related to multiple ions, but are offset from the predictions by 15%–25%, indicating osmolarity is not the best metric. This deviation from the predicted osmolarity is consistent with this mixture experiment having {Ca} low enough that NaCl LC50s are reduced compared with higher {Ca}, where they closely agree wth mannitol LC50s (Figure 4B).

Because this deviation from mannitol LC50s is not great, osmolarity might still contribute to toxicity, jointly with other mechanisms of toxicity. This possibility is considered in Figure 7D, in which LC50s on the basis of Mg and Na activities are compared to a three-mechanism model that includes not only {Mg}-based and {Na}-based toxicity based on Equations 1 and 2, but also osmolarity-driven toxicity using the study-wide average mannitol LC50 of 278 mOsm/L. This moves the isobole closer to the observed LC50s, but falls short enough of the data that this is not a convincing mechanistic explanation.

Interactions of KCl with NaCl and MgCl₂

The LC50s for both KCl and K₂SO₄ were similar on the basis of {K} and showed monotonic increases over the ⅓×/1×/3× ALSW dilution water series (Supporting Information, Table S1, Experiments 13–19 and 13–23). For C. dubia (Mount et al., 2016), similar increases were shown to be related to the Na concentration in the dilution water, based on tests in waters with variable NaCl concentrations (Figure 8A). However, fathead minnows differ from C. dubia in showing no such dependence on Na (Figure 8A). Such interspecies differences might reflect differences in K transporters in fish versus invertebrates (Griffith, 2017). The reason for the effect of different ALSW strengths on K salt toxicity to fathead minnows is unexplained and warrants further testing regarding other attributes of the dilution water, such as Ca concentration.

Figure 8B,C shows that, as for C. dubia (Erickson et al., 2017), K appears to involve a specific mechanism of toxicity that is independent of the toxicity of both NaCl and MgCl₂. For D. magna, Morris et al. (2021) reported increased K concentrations in the hemolymph as K exposures approached lethality. This would represent disturbance of the normal K gradients between intracellular and extracellular fluid, consistent with a specific action for K that should be independent of other ions. Such independence of K toxicity means that, in the event that K exposure does get to toxic levels, toxicity data for K salts could be used independently of models for the toxicities of Na, Mg, and Ca salts.

Mg and Ca salt toxicities

An important dilution water characteristic affecting the toxicity of Mg salts is Ca content, consistent with previous results for C. dubia (Erickson et al., 2017; Mount et al., 2016). Toxicities of Mg and Ca salts are best related to their cation activities and are concentration additive with each other, indicating a shared mechanism, as was also the case for C. dubia. However, in contrast to C. dubia, the toxicity of MgCO₃−dominated exposures is greater than that of MgCl₂ and MgSO₄ on the basis of {Mg}, this enhanced toxicity possibly reflecting direct effects of combined high pH and alkalinity, requiring separate consideration in risk assessments.

K salt toxicities

In contrast to findings for C. dubia (Mount et al., 2016), the toxicity of KCl to fathead minnows is not affected by Na, but is reduced modestly by increasing all ions, with the specific cause of this being uncertain. Toxicities of K salts are also best related to cation activity and are independent of the toxicities of other salts, as was the case for C. dubia (Erickson et al., 2017; Mount et al., 2016).

Na salt toxicities

An important dilution water characteristic affecting the toxicity of Na salts is Ca concentration, also consistent with previous results for C. dubia (Erickson et al., 2017; Mount et al., 2016). Another notable effect of dilution water is reduction of NaHCO₃ toxicity when the high unregulated pH (9.3) in tests with this salt is reduced to 8.4 by increasing CO₂ concentration.

The toxicities of Na salts and mixtures are not simply related to Na but involve mechanisms related to multiple ions. For C. dubia, this was addressed across all Ca concentrations using an exposure metric of osmolarity (Erickson et al., 2017, 2018; Mount et al., 2016), although osmolarity per se was not claimed to be causative at all [Ca]. The present study indicates the need to consider multiple mechanisms. Osmolarity is possibly causative at [Ca] above that of ALSW, where osmolarity-based toxicities of Na salts are similar to that of mannitol. At lower Ca, Ca-dependent Na salt toxicities deviate from Ca-independent mannitol toxicity, indicating different mechanisms. Similar considerations are needed for further development of the models for C. dubia toxicity, given the Ca independence of mannitol toxicity for this species reported in Supporting Information.

The toxicities of NaCl, Na₂SO₄, and NaHCO₃ at lower Ca also indicate different mechanisms among these salts. For Na₂SO₄-dominated exposures, toxicity is best attributed to {SO₄}, but becomes additive with NaCl in mixtures that are not SO₄-dominated, suggesting Na₂SO₄ also contributes to a mechanism in common with NaCl. Similar to MgCO₃, the toxicities of NaHCO₃-dominated exposures toxicity are greater than expected based on NaCl toxicity, apparently due to the combined effects of high pH and alkalinity as evidenced by the effect of pH on toxicity. The toxicity of NaHCO₃ also becomes additive with that of NaCl in mixtures that are not HCO₃-dominated, suggesting NaHCO₃ also contributes to a mechanism in common with NaCl.

For Na salt toxicities not attributable to high pH/alkalinity, SO₄, or osmolarity, the appropriate exposure metric is unresolved, despite good correlations with {Na} and osmolarity. Because mixture tests of Na salts with Mg salts indicate some degree of additivity, the exposure metric should not simply be {Na}, but include contributions of other ions. Using osmolarity to address these multi-ion effects would also not be advisable because it suggests that the mechanism involves osmotic effects, which likely is not the case.

Toxicity modeling implications

These results for fathead minnow present a more complicated picture than in Erickson et al. (2018) because C. dubia shows neither enhanced toxicity from high pH/alkalinity nor SO₄-related toxicity, and because different mechanisms at different Ca were not then evident for general ion toxicity to C. dubia. Application of this information for fathead minnow to toxicity models thus presents more challenges, both in how models should be formulated and what range of exposure conditions should be included. Development of these models and certain implications to risk assessment are addressed in the companion article (Erickson et al., 2022).

Supporting Information

The Supporting Information is available on the Wiley Online Library at https://doi.org/10.1002/etc.5390.