Assessing the relative toxicity of different road salts and effect of temperature on salinity toxicity: LC x values vs. no effect concentration (NEC) values

Freshwater biota are at risk globally from increasing salinity, including increases from de-icing salts in cold regions. A variety of metrics of toxicity are used when estimating the toxicity of substances and comparing the toxicity between substances. However, the implications of using different metrics is not widely appreciated. Using the mayfly Colobruscoides giganteus (Ephemeroptera: Colobruscoidea) we compare the toxicity of seven different salts where toxicity was estimated using two metrics 1) the no effect concentrations (NEC) and 2) the lethal concentrations for 10, 25 and 50% of the test populations (LC x ). The LC x values were estimated using two different models, the classic log-logistic model and the newer toxicokinetic-toxicodynamic (TKTD) model. We also compare the toxicity of two salts (NaCl and CaCl 2 ) for C. giganteus at water temperatures of 4°C, 7°C and 15°C using the same metrics of toxicity. Our motivation for using a mayfly to assess salinity toxicity was because mayflies are generally salt sensitive, are ecologically important and are common in Australian (sub-)alpine streams. Considering 144-hour LC x values, we found toxicity differed between various salts, i.e., the lowest 144-hour LC 50 (8 mS/cm) for a salt used by a ski resort was half that of the highest 144-hour LC 50 from artificial marine salts and CaCl 2 applied to roads (16mS/cm). 144-hour LC 50


Introduction
Salinity is rising in many freshwaters because of a range of anthropogenic causes including agriculture, mine effluent (Cañedo Argüelles, et al. 2013;Sauer, et al. 2016;Niedrist et al 2021) and in cold regions applications of salts for de-icing i.e., to prevent the build-up of snow and ice (Niedrist, et al. 2020;Shenton, et al. 2021). The application of de-icing salts involves applying salts to roads, typically sodium chloride (NaCl) and/or calcium chloride (CaCl2). It is increasingly been recognised that concentrations and/or ratios of major ions that makeup salinity are as important as the total salinity concentration (Cañedo-Argüelles, et al. 2016) to the toxicological effects on freshwater organisms. Consequently, the ecological and toxicological effects of road de-icing may differ depending on whether NaCl, CaCl2 or both are used. Additionally, those studies which have assessed the toxicity of different salts (Mount et al. 2019) have tended to use high purity, i.e., analytical grade salt (Mount et al. 1997) and it is feasible there may be differences in toxicity between the salts applied in deicing applications and these high purity salts used in toxicity tests, as the former would be expected to have lower purity.
Toxicity tests are a widely used experimental method to measure the toxicity of substances for individual test-species organisms. The results of toxicity tests can be expressed using a range of metrics. When the measured response of a test organism is mortality, the concentration lethal to x% of the test population is the most commonly used metric (LCx, see Table 1). For example, the LC50 is the median concentration of a toxicant in a solution that is lethal to 50% of the test organisms (Burton. 1997). LCx are calculated for a pre-determined exposure period. The duration of acute tests with macroinvertebrates are usually between 24 and 96 hours. For stream macroinvertebrates exposed to salinity, 72-hour LC50 values are commonly used (e.g., Kefford et al. 2012, Castillo et al, 2017. Toxicity tests are typically conducted within ±1-2•C of the desired test temperate, although temperature is acknowledged to alter toxicity (Jackson andFunk,2019, Macaulay et al. 2020). Consequently, the LC50 gives the concentration lethal to 50% of the test population for the indicated period of exposure at the test temperature indicated and makes no assessment of the response of the test organisms at other periods of exposure or test temperatures.
An alternative metric to LCx, is the No Effect Concentration (NEC) estimated with a toxicokinetic-toxicodynamic (TKTD) model (Jager et al 2011, Kon Kam King et al 2015. NEC estimates the maximum concentration that has no observed effect on the test population studied and the response (or endpoint) measured. Such a response may be mortality (as with LCx) but could alternatively measure sub-lethal responses, such as inhibition of a physiological function or the response of a whole organism (e.g., growth or reproductive output). Unlike LCx values, the NEC is time independent (Kon Kam King et al. 2015). LCx values are always associated with some level of mortality as it is not possible to have x=0. Moreover, as x approaches zero, uncertainty in the estimate of the LCx value increases giving imprecise estimates. In contrast, the NEC is an estimate of the maximum concentration that has no effect, e.g., zero mortality from the toxicant, and no mortality beyond which occurs in the control.
The statistical method by which LCx values are classically estimated, e.g., log-logistical models, uses only the data from the exposure period that the LCx is reported. Consequently, when using a log-logistic model to estimate LCx values, not all data that is typically available to the investigators is used. For example, in estimating 96-hour LCx values the investigators would typically have survival data at 24, 48, 72 and 96 hours, but when using a log-logistic model they would only use the data from 96 hours. TKTD model (Jager et al 2011, Kon Kam King et al 2015, that are used to estimate NEC values can also estimate LCx values but they use data from all survival observations, potentially allowing TKTD to produce better estimates of LCx values. We shall refer to LCx values estimated by a log-logistic model as the classic LCx values or estimated by a classic model, and those LCx values estimated by a TKTD model as TKTD LCx values. Another potentially important factor is water temperature during salinity exposure. Recently, Jackson and Funk (2019) observed, that between 5-25º, four mayfly (Ephemeroptera) species tended to have higher 96-hour LC50 values (i.e., are more tolerant) when tested at lower water temperatures. Salinity levels from de-icing salts are typically maximum in months when water temperatures are typically much lower than used in standard toxicity tests. Thus, Jackson and Funk (2019) suggest that standard toxicity tests may overestimate toxicity. However, in aquatic ectotherms, metabolic rates are often highly correlated with water temperatures (Sutcliffe, 1984), and thus, higher LC50 values from a fixed exposure period at colder water may not necessarily reduce toxicity at colder water temperatures. Rather it may indicate that at longer exposure periods there is no effect of water temperature on toxicity.
Here, we have three aims. First is to determine if the toxicity of various salts (artificial marine salts (AMS), analytical grade NaCl and CaCl2 and four salts (Table 1) used in de-icing operations near Perisher, New South Wales (NSW), Australia) differed for the mayfly Colobruscoides giganteus (Ephemeroptera: Colobruscoidea). Second, to determine if water temperatures influenced the toxicity of two salts (analytical grade NaCl and CaCl2) using C. giganteus. Third, we examine the relative merits of using the three separate approaches by assessing toxicity LCx values estimated by a log-logistic model, by a TKTD model, and NEC values estimated by the same TKTD model. We determine if patterns in relative toxicity between the salts and water temperature were consistent across the two methods of estimating LCx and the NEC values.

Field collections
A range of alpine stream macroinvertebrates were collected during the colder months when salts are typically applied (June-September) during 2018 from six sites at 1400-1700 m elevation, near Perisher, Kosciuszko National Park (Supplementary Table S1; Supplementary Figures S1 and S2). Stream water temperature during these collections ranged between 0.4°C-5.2°C. Macroinvertebrates were kept in aerated site water, with some detritus and sediment, during transport. Rapid toxicity tests (RTT) were started in the laboratory within 24-hours of collection and methods are reported in detail in (Moulding. Hnrs. Thesis, 2018) Following the RTTs, Colobruscoides giganteus was chosen to make more precise estimations of the toxicity of various salts and the effect of water temperature on salinity tolerance. This species was chosen because it is a mayfly (Ephemeroptera) which are generally considered salt sensitive (Kefford 2019), are ecologically important and are common in (sub-)alpine and alpine Australian streams. We specifically chose C. giganteus because of its high abundance in field collections and high long-term survival in laboratory under a control conditions, see also (Bray et al. 2021). All C. giganteus came from one site, Rock Creek (S36.41, E148.41, elevation 1700 m). Rock Creek is close to its headwaters, has minimal urban disturbance, no de-icing salt input (Supplementary Table S1; Supplementary Figures S1 and S2) and consistently has low salinity levels (always <0.1 mS/cm but typically <0.01 mS/cm) (Shenton et al. 2021).

Experimental Methods
We conducted two experiments using conventional toxicity tests (Stefania et al. 2010). The first experiment aimed to investigate differences in toxicity between seven salts (Table 1). The second experiment investigated the effect of water temperature on the toxicity of two of these salts: analytical grade of both Sodium Chloride (NaCl) and Calcium Chloride (CaCl2).
All tests were conducted with 144-hours of exposure to salt solutions with C. giganteus (seven individuals for experiment 1; and eight individuals for experiment 2) being placed into beakers with 600 ml of treatment solution and two replicate beakers for each treatment or control. All beakers were aerated using air stones with no further substrate added. All controls and dilution water for solutions was dechlorinated Canberra tap water (≈0.1 mS/cm). No individuals were fed during the tests, illumination was 8-hours per day and water was not refreshed during experiments. All toxicity tests were inspected, and any dead individuals removed daily.
If an individual C. giganteus was suspected of being dead, it was softly prodded with a pipette while the individual was still submerged in treatment solution. If no movement of the individual observed from prodding, it was then collected in a teaspoon (with treatment solution) to witness any small movements e.g., of legs or mandibles. If the individual is confirmed dead it was placed into 80% ethanol, any twitching was noted. In the rare cases where twitching occurs, it is considered alive and become a 'missing' individual from the experiment at subsequent observations. LCx (x= death of 10, 25 and 50% of the test population) values were recorded for time periods (24,48,72,96, 120 and 144 hours)

Experiment 1: What is the difference in toxicity of different salts to Colobruscoides giganteus
A single conventional toxicity test was used to compare relative toxicities of seven different salts to C. giganteus at 7°C ± 1.0°C. The seven salts were: AMS (Ocean Nature), analytical grade CaCl2 and NaCl and four types of salt applied for de-icing in and around Perisher. These salts were CaCl2 (flakes) and NaCl (granulate) used by government authority responsible for de-icing operations, the Roads and Maritime Services of NSW (RMS, hereafter these salts are referred to as RMS CaCl2 and RMS NaCl, respectively) and two types of salts used by the Perisher Environmental Team: evaporated gourmet sea salt (EGSS) and evaporated washed sea salt (EWSS) (both 'mermaid' brand). AMS was included to be able to compare the results to other published toxicity results (Kefford et al. 2012). Analytical grade NaCl and CaCl2 were used as these salts (at lower purity) are widely used as de-icing salts, including near Perisher (Shenton et al. 2021). The salt used by the RMS and the Perisher Environmental Team were used to determine the toxicity of the salt applied to the environment. Treatment solutions in electrical conductivity (EC) were 0.1 (control), 4, 8, 12 & 16 mS/cm.

Experiment 2: Does water temperature effect salinity toxicity to Colobruscoides giganteus.
A conventional toxicity test was also used to compare the sensitivity to analytical grade NaCl and CaCl2 of C. giganteus at a cold temperature (4°C ±1.0°C), a mid-temperature (7°C ±1.0°C) and a high temperature (15°C ±1.0°C). The cold temperature mimics typical winter water temperatures observed at Rock Creek. The mid temperature environment was chosen to mimic observed early spring water temperatures and the high temperature environment was used to mimic observed late spring/early summer water temperatures (Shenton et al. 2021, unpublished data). The EC of treatments for this experiment were 0.1 (control), 5, 6, 7, 8 & 12 mS/cm. Experiment 1 had 0% survival at 16 mS/cm, consequently, this salinity level was removed from experiment 2.

Data Analysis
In both experiments, GUTS-SD (General Unified Threshold Survival) (Jager et al, 2011) was used to determine LCx and NEC results. GUTS-SD is a type of Toxico-Kinetic Toxico-Dynamic model which allows using time-resolved data collected during each experiment (i.e., survival data at 24, 48, 72, 96, 120 and 144 hours). LCx were also estimated using a 2parameter log-logistic model (Ritz et al. 2015). Parameter inference for both models was performed in the Bayesian framework using JAGS (Plummer 2016), with vaguely informative priors adopted by the experimental design (concentration grid, measurement frequency, priors inspired by Delignette-Muller et al. (2017)). Posterior distributions for the LCx could be calculated directly from the posterior distribution on the parameters for the loglogistic model, while a numerical inversion was necessary for the GUTS-SD model. Because of the use of the Bayesian framework uncertainty in estimates of LCx and NEC values are given in 95% credibility intervals (not to be confused with 95% confidence intervals which are commonly used with a frequentist framework).

Results
Survival of C. giganteus in controls (0.1 mS/cm) was 100% over 144-hours across both experiments. At high salinities its survival decreased in all salt and temperature combinations.

Experiment 1: What is the difference in toxicity of different salts for Colobruscoides giganteus
Using the classical LCx model, a log-logistic model, LCx values generally decreased with increasing exposure period (Figure 1). However, there were some exceptions. For example, the median estimate of the LC10 for EWSS increased from 120 hours to 144 hours. In the absence of mortality, classical LCx values for short exposures e.g., 24 hours could not be estimated and only a lower bound was available.
Classical LCx values did show that some salts had clearly different toxicity, with these differences being best indicated at prolonged exposure i.e., 120-hours and 144-hours (Table  2). AMS was consistently less toxic than all other salts tested, with a median estimate of 144hour LC50 of about 16 mS/cm. In comparison, EWSS had a value for this parameter at about 10 mS/cm.
Disregarding statistical significance, a ranking of the classical LC50 mean values at 144-hours suggests EGSS is the most toxic salt (8mS/cm), followed by EWSS (10mS/cm), RMS NaCl (11mS/cm), CaCl2 and NaCl (12mS/cm). AMS (16 mS/cm) and RMS CaCl2 appear to be the least toxic salt (16mS/cm) where medians and means were effectively identical ( Table 2). TKTD modelled LCx values always declined with increasing exposure period and the upper 95% credibility interval was always defined i.e., it was never infinity (Figure 2). There were also differences in the toxicity of some salts apparent even after brief exposure periods. For example, at 24 hours of exposure AMS had high a higher LC50 value than EWSS with no over-lapping of their 95% credibility intervals. Nevertheless, credibility intervals did narrow with increasing exposure period across all salts tested ( Table 2).
NEC values for the seven salts at 7ºC showed that EWSS was significantly less toxic compared to NaCl, EGSS and RMS NaCl (Figure 3). However it does appear that NaCl and RMS NaCl have a lower median NEC compared to EGSS, all other salts (AMS, CaCl2 and RMS CaCl2) do have greater confidence intervals (Figure 3). The other salts had fewer precision estimates of their NEC values and it is credible that these salts all had NEC values that encompass estimates for the NEC values of EWSS, NaCl and RMS NaCl. Considering only the median estimate of NEC values, EWSS (~8 mS/cm) was less toxic than all of the other salts, which had similar toxicity's ranging from least toxic to most toxic CaCl2 (~2.5mS/cm), EGSS and RMS CaCl2 (~3mS/cm), AMS(~2.4mS/cm), NaCl(~2mS/cm) then RMS NaCl(~1.75mS/cm). Supplementary Table S3.

Colobruscoides giganteus.
As with the previous experiment with the seven salts, classically estimated LCx values for CaCl2 and NaCl at the three temperatures (4°C, 7°C, 15°C) tended to decrease with increasing exposure period but there were some exceptions ( Figure 4). The LCx values of CaCl2 at 7°C increased between 96 hours and 144 hours. Again, as with the previous experiment, the upper credibility intervals for brief exposures (e.g., 24 and 48 hours) LCx values was at infinity, thus providing very poor estimates of toxicity.
In terms of patterns in toxicity between temperatures, the classical LCx values show that CaCl2 had similar toxicity at 4°C and 7°C but was more toxic at 15°C. In contrast, NaCl toxicity tended to increase with increasing water temperature, see for example the 144-hour LC50 values.
The classic LCx values with long exposure (i.e., 120 and 144 hours) showed NaCl was considerably more toxic than CaCl2 at 7°C, while at 4°C and 15°C the differences in toxicity between these salts were less. At 4°C and 15ºC the credibility intervals overlapped between the salts for the 144-hour LC50 values. The difference in their toxicity was minimal at 4°C and 15ºC compared to 7°C.   24,48,72,96,120 and 144 hours. x= LCx 10,25,50 ( Table S4.See also Supplementary Figure S4.

from left to right). Median indicated by error bars; 95% credibility intervals indicated by boxes. A full listing of all LCx estiamtes shown here is given in Suplemntary
As with the previous experiment, the TKTD modelled LCx values always decreased with increasing exposure period and the upper credibility interval was always defined (i.e., less than infinity) ( Figure   5). For CaCl2 credibility intervals were much narrower at 15°C than at 4°C and 7°C. For NaCl, credibility intervals were slightly wider at 4°C than the 7°C and 15°C.
Similar to the classical LCx values, there was no evidence of difference in the CaCl2 LCx values between 4°C and 7°C. At 15°C CaCl2 was more toxic than at either of the other temperatures tested. NaCl was least toxic at 4°C and this salt had similar toxicity at 7°C and 15°C.

Discussion
The different methods of toxicity estimation, i.e., classically modelled (i.e., log-logistic) LCx values, TKTD modelled LCx values and TKTD modelled NEC values, provided different estimates of toxicity. Classical LCx estimates were incalculably high for short exposure periods with only the lower credibility interval estimated and the upper credibility intervals stretching to infinity. This is a function of the salinity concentrations chosen having limited mortality at 24 and often 48 hours. No doubt with the inclusion of high salinity concentrations better estimates of classic 24 and 48-hour LCx values would have been obtained. In contrast, the TKTD modelled LCx values always had upper credibility intervals defined below infinity. Where a toxicity test aims to make estimates of LCx values across a wide range of exposure periods (e.g., 24 to 144 hours, as in the current paper), there are considerable advantages of using a TKTD model.
Classical LCx values tended to, but did not always, decrease with increasing exposure period, while TKTD LCx values always decreased with increasing exposure period. Toxicity depends on both concentration and duration of exposure. With an increase in the duration of exposure toxicity should either be unchanged (if there is no more mortality) or should increase. Consequently, LCx values should not increase with increasing exposure periods, as occasionally occurred with the classical LCx values.
The LCx values, whether estimated classically or by the TKTD model showed differences in toxicity between some salts and the same salt at different water temperatures. The estimated NEC values, however, had overlapping credibility intervals across salts or temperatures, with one exception. This exception was that EWSS had a higher NEC value (~8mS/cm) than all other salts (~1.75 mS/cm for RMS NaCl appearing to be more toxic and CaCl2 being the least toxic of other salts ~3.2 mS/cm). Regardless of this exception, the NEC values were less effective at detecting differences in toxicity between the salts or temperatures than both LCx models. LCx values appear to be better for comparing toxicity between substance or the same substance at different test temperatures. For aiding in the setting of environmental quality guidelines, NEC values are likely to be more useful especially when using one of the more sensitive taxa within a studied environment (C. giganteus), although environmental quality guidelines should also consider sub-lethal toxicity and indirect effects of toxicants (Bray et al. 2018).

Experiment 1: What is the difference in toxicity of different salts to Colobruscoides giganteus
The current study is the first that we are aware of which has compared the toxicity of analytical grade salts to those used by de-icing operations. Our results show considerable variability in the toxicity of NaCl dominated salts for example, in terms of classical mean LC50 values EGSS (8/mS/cm) was considerably more toxic than all other NaCl dominated salts (10-16mS/cm), two-fold RMS CaCl2 (16 mS/cm) at 144 hours ( Table 2). In contradiction to both LCx models, the NEC showed that EWSS was significantly less toxic than all other salts tested. All other salts showed no differences in toxicity between each other when the NEC was applied as indicated by overlapping credibility intervals.

Experiment 2: Does water temperature have an effect on the salinity toxicity to Colobruscoides giganteus
Temperature has fundamental effects on almost all biological processes and it is thus not surprising that it modulates the toxicity of many environmental contaminants including salt toxicity to freshwater ectotherms (Orr andBuchwalter 2020, Verberk et al 2020). Previously, four mayfly species showed lower (classical) 96-hour LC50 values (i.e., more toxic) for NaCl when temperature was increased (Jackson & Funk. 2018). Our results show a similar effect, at least for classical LC50 values for NaCl. For CaCl2, however, we found no apparent difference in toxicity (as indicated by both LCx models) between 4°C and 7°C but higher toxicity at 15°C.
Increased temperature is thought to increase toxicity because of an increase in ion transport rates and a higher energy expense during osmoregulation (Orr andBuchwalter. 2020, Verbeck et al, 2020). However, apparent differences in some LCx values for different salt and/or temperatures do not necessary indicate difference in toxicity long-term as shown by the NEC.
The similarity in the NEC values for NaCl and CaCl2 at the different temperatures means that we cannot exclude the hypothesis that both salts are similarly toxic at all three of the tested temperatures but that it takes longer for the effect to occur at the colder temperatures due to decreased metabolism at colder temperatures. Thus, comparisons between different salt, temperatures, etc. with LCx values need to be treated with caution and may not necessarily indicate real differences in long-term toxicity.

Why are there differences in toxicity indicated by LCx and NEC?
We showed apparent differences in toxicity between salts or between the same salt at different exposure temperatures, indicated by one or both LCx models; however, this was not reflected with NEC values. This appears to be because there was similar overall toxicity of the different salt or exposure temperatures, but that for some salt or at some exposure temperatures, it took longer exposures for the similar toxicity to be apparent.
NEC values are time independent, that is, they estimate a threshold of toxicity that has no effect on the test population for any length of exposure for the relevant endpoint (Kon Kam King et al. 2015), in our case mortality. This is because any LCx values are an estimate of the concentration which cause the indicated proportion of mortality (i.e., x%) for the relevant exposure period. In the case of LC50 values, 50% is a substantial amount of mortality, which if this were to occur to natural populations could have profound consequences for their persistence. While it is possible to estimate LCx values that are more protective, e.g., LC5 values, they still represent a concentration where an ecologically relevant effect (mortality) is occurring. Moreover, any LCx value is representative of the toxicity for a given exposure period, e.g., 72 hours, and make no estimation of what effect would occur if exposure were longer, other than longer exposure would have on less of an effect. NEC values are therefore more meaningful for determining environmental effects than LCx values and is widely suggested as an improvement to the LCx (Proctor, 2019) at least when the exposure period is lengthy, and especially when studying biota from cold regions as toxicity test duration should have a lengthy exposure period, up to 42 days (Proctor, 2019).
NEC vs. LCx comparisons are quite limited in current scientific literature but see (Proctor, 2019). Proctor (2019), similarly with our results, found increased temperature lowered the LCx value of subantarctic isopod, Limnoria stephenseni when exposed to copper. Like our results Proctor (2019) also found no/little differences in NEC values (for copper) between temperatures. She also observed at long exposure periods (~40 days) where LCx values between temperatures become similar. She hypothesises that this is a result of polar taxa requiring extended exposure to illicit an acute response as they respond slower to toxicants than warmer temperature taxa.
When applying the NEC for example a few deaths late in the exposure period (as was the case with EWSS) it can make a big difference to the precision of the NEC estimate. When the aim of a toxicity test is to estimate NEC values, exposure periods should be long enough that there is little or no more mortality at the later time periods (as in the case of EWSS).

Short term and long-term salinity inputs and outputs
In the case of short-term spikes in salinity, as can happen with de-icing salting operations (Shenton et al. 2021), if the spike is short enough, they will not have time for long term effects and thus LCx values might be more relevant than NEC values, although this remains to be demonstrated, as short-term exposure may result in latent effects which only become apparent after exposure ceases. However, de-icing salts (as was the focus of these experiments) store in the land and leach out into streams throughout the entire year, including in our study region (Shenton et al. 2021, Moulding 2018, so that chronic exposure may well be relevant. The highest salinity spikes in our study region (and other alpine areas) occurred in winter or early spring due to de-icing activities (Shenton et al. 2021, Moulding 2018, where water temperatures are generally low. To determine the long-term effect of salinity at such low water temperatures requires lengthy experiments. The NEC may be a powerful tool for testing long term survival of organisms from relatively short-term tests and would provide a more environmentally sound estimate that can take other variables such as landscape salinity output into consideration to understand any long-term effects from outputs in summer and autumn.

Conclusion
Our results suggest that when toxicological studies aim to compare the toxicity of different substances (e.g., salts) or the same substances at different water temperatures, then generally LCx values are better suited to detect differences in toxicity between the substances than NEC values. In contrast, for studies that aim to estimate the concentration of a substance which, with long term exposure, will not produce toxicity, then NEC values will generally be better because it is independent of time and generally has a lower value than LCx results, producing more conservative protection for freshwater biodiversity. While NEC values can be estimated from short duration experiments, more precise estimates of NEC values will be produced with experiments which last long enough that latter observations are recording little or no mortality. Where studies aim to estimate LCx values across a range of exposure periods, then better estimates will generally be obtained using a TKTD model.
We also recommend that de-icing operations consider the toxicity of the salts that they are using and not analytical grade salts, as we observed considerable variability between the toxicity of NaCl dominated salts. Careful selection of salts with relatively less toxicity, has the potential to reduce the environmental harm of de-icing salts. We propose that the least toxic salts were RMS CaCl2 (least toxic by 144-hour LC50 results) and EWSS (least toxic by NEC). Further investigation is required, however, to consider toxicity to other species, sublethal toxicity and effects of de-icing salts in natural or semi-natural (e.g., mesocosms) system where multiple species are interacting (Clements and Kotalik 2016;Bray et al. 2018). It is highly plausible that such studies could find the effect of de-icing salts at lower concentrations than the concentrations we observed mortality in one species, under single species laboratory test conditions.