Northeastern Naturalist Vol. 25, No. 1 A. Tiwari and J.W. Rachlin 2018 123 2018 NORTHEASTERN NATURALIST 25(1):123–142 A Review of Road Salt Ecological Impacts Athena Tiwari1,* and Joseph W. Rachlin2 Abstract - Road-salt runoff is an increasing problem in areas of North America that receive snow. Its effects include groundwater salinization, loss or reduction in lake turnover, and changes in soil structure. Road-salt runoff can affect biotic communities by causing changes in the composition of fish or aquatic invertebrate assemblages. It also poses threats to birds, mammals, and roadside vegetation. Introduction Paved roads are a major feature of the modern landscape and a ubiquitous element of human presence in the industrialized world. Roads are a source of chemical input to the surrounding landscape and, in areas that receive snow, deliver road salt in the form of runoff to adjacent soil and waterbodies. Research on the persistence of road salt in the environment and its cumulative effects has raised concerns about the long-term implications of this large-scale anthropogenic input. The use of road salt increased as the nation’s highway system grew. In the 1940s, when most winter roadways in the US were kept passable by the addition of sand or cinders, New Hampshire pioneered the use of salt for winter deicing (TRB 1991). By 1955, 1 million tons of road salt were used in the US (TRB 1991). The US currently uses 15–24 million tons of salt annually for road de-icing (Bolen 2016, Godwin et al. 2003, Novotny et al. 2007). Sodium chloride (NaCl) is the least expensive deicer, and is useful at temperatures above -12 °C (10 °F); below that temperature, melting slows down as NaCl approaches its eutectic temperature of -21 °C—the point at which it can no longer lower the freezing point of water. At those temperatures, calcium chloride (CaCl2) is an effective deicer (TRB 1991), but CaCl2 is more than 5 times as expensive as NaCl (rock salt) (Kelly et al. 2010). Magnesium chloride (MgCl2), which is twice as expensive as NaCl, is considered more toxic to aquatic life (Kelly et al. 2010, Kotalik et al. 2 017). After road salt goes into solution as runoff, its 2 major components have different fates. Road salt is approximately 40% sodium and 60% chloride, with up to 5% trace elements or possible contaminants. Most chloride ions move with the water, persisting and accumulating in the aquatic environment (PMRA 2006), but they are not entirely conserved in water. Contrary to prior belief, chloride in runoff is now known to be temporarily retained by soil and gradually released to groundwater (Kincaid and Findlay 2009). Sodium ions tend to bind to soil 1Laboratory for Marine and Estuarine Research (LaMER), City University of New York, 250 Bedford Park West, Bronx, NY 10468. 2The Graduate Center, City University of New York and LaMER, Lehman College, 250 Bedford Park West, Bronx, NY 10468. *Corresponding author - athenatiwari13@gmail.com. Manuscript Editor: David Halliwell Northeastern Naturalist 124 A. Tiwari and J.W. Rachlin 2018 Vol. 25, No. 1 particles (PMRA 2006), and can also be taken up by organisms in freshwater streams. Aquatic insect larvae in acidic environments use Na+/H+ exchangers to substitute sodium for hydrogen ions and thereby maintain acid/base homeostasis (Clark et al. 2004, Harvey 1992, Marshall 2002). In general, aquatic insects have less tolerance to low pH in a sodium-poor environment (Clark et al. 2004). For these reasons, studies of road-salt contamination have often measured Cl- in the environment or its effect on aquatic organisms, rather than measuring the amount or effect of Na+, NaCl, or total salts. Cyanide is a minor component of road salt, which can also contain trace amounts of phosphorus, sulfur, nitrogen, copper, or zinc (Environment Canada 2001). Ferrocyanides such as sodium ferrocyanide Na4Fe(CN)6 .10H20 are used as road salt anti-caking agents. If ferrocyanides are exposed to light while in solution, they dissociate to form cyanide ions, CN-, which then hydrolyze to HCN and volatilize. Ferrocyanides have limited solubility, however, and generally remain stable in the environment (Environment Canada 2001). Tests of the commercial road-salt cyanide components, sodium ferrocyanide and ferric ferrocyanide, showed they had low toxicity to larval and juvenile Villosa iris (Lea) (Rainbow Mussel), a freshwater unionid mussel (Pandolfo et al. 2012). Many mussel species are endangered, and salt is known to be toxic to freshwater mussels, but road salts containing the 2 ferrocyanide forms were not more toxic to the Rainbow Mussel than reference levels of NaCl (Pandolfo et al. 2012). Chloride can come from animal sources, raw sewage, agricultural fertilizers, and water softeners, and it is naturally present in surface and ground water (Jones et al. 1992, Kelly et al. 2008). A mass-balance study of chloride and sodium increases between 1986 and 2005 in the East Branch of Wappinger Creek, Dutchess County, NY, found increases of 1.5 mg/L chloride and 0.9 mg/L sodium in stream water per year, 91% of which were explainable by road-salt use, and less than 10% by sewage and water-softener use (Kelly et al. 2008). Rock salt (halite), may occur as vast deposits on the floor of lakes (Jones et al. 1992). Saline lakes have ≥3000 mg/L total salts (Environment Canada 2001). In contrast, a survey of 417 lakes in non-saline, non-urbanized areas of Canada in Labrador, Newfoundland, Nova Scotia, and New Brunswick found median chloride-ion concentrations between 0.3 mg/L and 4.5 mg/L. Across the US, rain contains 0–2 mg/L of chloride ions (USGS 2007). Road Salt in Streams and Other Surface Waters Chloride concentrations in surface water have been steadily rising in the US for several decades. For example, First Sister Lake, in Ann Arbor, MI, was first sampled in 1965, at which time Cl- concentrations were 33 mg/L. Chloride concentrations rose steadily over a period of decades until, in 2002, First Sister Lake had 295 mg/L Cl- (Benbow and Merritt 2004). Similarly, the change in estimated mean daily yield of chloride ions from the 1950s to the 1990s in the Mohawk River in New York State was 19.93 mg/L, or an increase of 243.02%. Sodium ion concentration also rose sharply during these periods. From the 1950s to the 1990s Na+ in the river increased by 10.10 mg/L, or 129.96% (Godwin et al. 2003). Chloride concentrations in partially Northeastern Naturalist Vol. 25, No. 1 A. Tiwari and J.W. Rachlin 2018 125 urbanized areas often exceed the USEPA level for the chronic water-quality criterion of 230 mg/L as an average over 4 days (Corsi et al. 2015). The trend of rising chloride concentrations can be observed in non-urbanized forested watersheds, which tend to have chloride concentrations below 10 mg/L, along with low amounts of road-salt application (Corsi et al. 2015). Road-salt pollution can be measured in feeder streams that supply lakes, as was documented in 4 headwater streams that descend from Goodnow Mountain and surrounding uplands in the central Adirondacks, in northern New York (Demers and Sage 1990). Those researchers sampled for chloride concentration 1 site above and 2 sites below the state road that crosses all 4 streams and found a significant difference (P < 0.05) in chloride concentration between sites 100 m upstream and downstream at each study stream. Mean upstream concentrations at the 4 streams varied from 0.51 mg/L Cl- to 1.35 mg/L Cl-, while downstream concentrations at 50 m below the road varied from 1.70 mg/L Cl- to 17.05 mg/L Cl-. They observed no significant difference between the 2 downstream sites 50 m and 100 m below the road, indicating that “these elevated chloride levels were not just an immediate roadside phenomenon” (Demers and Sage 1990). This study also demonstrated persistence of chloride runoff over time. Demers and Sage (1990) sampled at least monthly during the periods April–July 1987 and December 1987–September 1988, and elevated chloride concentrations were measured at all downstream stations on all sampling dates. A strong relationship has been established between the concentration of chloride ions in surface water and the percent of local impervious surface (Kaushal et al. 2005). Impervious surfaces are those into which rain cannot percolate, such as buildings, roads, and parking lots (Wang et al. 2001). As impervious surface increased over the span of decades, chloride pollution of streams, rivers, and drinking-water reservoirs closely followed in northeastern and middle Atlantic areas such as Baltimore, MD, and surroundings (R2 = 0.81, linear regression), the Hudson River Valley of New York (R2 = 0.61), and the White Mountains of New Hampshire (R2 = 0.70) (Kaushal et al. 2005). Between 1986 and 2000, 82,000 metric tons of deicing salt were applied in Baltimore, while during the same period, impervious surface in and around Baltimore increased by 39%. Winter chloride-ion spikes can approach 5000 mg/L in streams flowing through urban and suburban Baltimore, MD (Kaushal et al. 2005). Rural streams in the White Mountains sometimes contained more than 100 mg/L Cl- , which Kaushal et al. (2005) point out is “similar to the salt front of the Hudson River Estuary”. Road-salt Effects on Lake Turnover Thermal stratification is the result of the density difference between warmer and cooler water, and turnover of that stratification is an important component of the ecology and productivity of a lake. A top layer of warm water forms in many temperate lakes during summer months. As the weather cools in the fall and winds pick up, the upper layer, the epilimnion, typically gets cooler and denser and then breaks up entirely as wind propels mixing of the entire lake water. This lake turnover occurs because there is less temperature difference between the upper and lower lake Northeastern Naturalist 126 A. Tiwari and J.W. Rachlin 2018 Vol. 25, No. 1 layers in the fall and the force of wind is sufficient for mixing during that time of year. The same turnover (complete vertical mixing), usually occurs again in the spring (Novotny et al. 2007). Lakes that follow this pattern are termed dimictic. A consequence of road salt in lakes may be changes in such lake turnovers. Large inputs of chloride ions to lakes can interfere with normal seasonal mixing of lake waters, causing a chemical form of stratification. A salt addition of 10 mg/L produces as much stratification in a lake as a temperature increase of 1°C (Novotny et al. 2007). The salt runoff from the winter season can prevent the spring turnover. In such cases, as chloride accumulates in the deepest part of the lake, the hypolimnion (lowest layer) becomes denser than normal. For example, in the fall, the salty hypolimnion can be resistant to mixing with the epilimnion and transition zone metalimnion that have sunk lower due to cooling and surface-wind turbulence (Bubeck and Burton 1989). A lake may become monomictic, mixing layers once instead of twice, or meromictic, having no mixing. Changes in mixing have been observed to happen irregularly in the Twin Cities area of Minnesota, with normally dimictic lakes occasionally having a monomictic, heavily chloride-stratified year (Novotny et al. 2007). Low levels of dissolved oxygen in the hypolimnion can stress or kill aquatic life, and cause an increase in the release of heavy metals and phosphorus from the bottom lake sediments into solution (Novotny et al. 2007). In the Twin Cities area of Minnesota (The TCMA), 9 urban lakes showed significant differences in concentrations of Na+ and Cl- between surface and bottom waters, but non-significant differences between surface and bottom concentrations for all other ions tested. Median sodium and chloride concentrations were 73 mg/L and 132 mg/L at the surface, and 105 mg/L and 186 mg/L at the bottom, respectively (Novotny et al. 2007). There are no geological sources of chloride ion in the TCMA; chloride concentrations of 4–10 mg/L are found in the geologically similar Wisconsin North Temperate Lakes Region, (Novotny et al. 2007). Paleontological work on diatom assemblages in sediment cores from TCMA lakes calculated that chloride concentrations during the period 1750–1800 A.D. were ~3 mg/L (Ramstack et al. 2004). Road-salt Effects on Ground Water Groundwater can act as both a source and a sink for chloride ions (Cooper et al. 2014, Kincaid 2006), and chloride levels in groundwater show that it accumulates over time. As with surface water, chloride pollution of groundwater typically reflects the extent to which an area has been urbanized. At sites around Toronto, ON, Canada, measured chloride-ion concentrations have been as high as 1324 mg/L Cl- (with a mean of 1092 mg/L; Williams et al. 1999) and 14,000 mg/L (Howard and Haynes 1993). In contrast, a spring in the Glen Major Conservation Area had a mean concentration of 2.1 mg/L Cl- (Williams et al. 1999). GIS maps of groundwater chloride values created for the state of Connecticut showed that they varied with proximity to highways, and concentrations increased proportionally with the increase in road-salt application rates in that state (Cassanelli and Robbins 2013). Northeastern Naturalist Vol. 25, No. 1 A. Tiwari and J.W. Rachlin 2018 127 Ions in the enclosed groundwater environment change and migrate according to much slower timeframes than they do in the comparatively chaotic aboveground environment. Chloride ion concentrations in springs (upwellings of groundwater) are more stable seasonally than chloride concentrations in streams (Williams et al. 1999). The speed of groundwater can vary from less than 1 m (a few feet) to upwards of 100 m (several hundred feet) per year (Pollack 1992), and pollutants that enter groundwater may take years to resurface due to baseflow discharge (Environment Canada 2001). Gradual input of road salt to shallow groundwater is one reason why surface-water chloride concentrations have increased more quickly than urbanization in the northern US (Corsi et al. 2015). One way to monitor groundwater and gauge the chloride input to groundwater from road salting is to use the salt-balance method, in which the amount of chloride from road salt used within a watershed or catchment is compared to the amount of chloride in the catchment streams (total salt or conductivity can also be compared); the difference between the 2 amounts is the chloride that is assumed to be in the groundwater. Over time, chloride input to the stream increases to more nearly match the chloride input from road salt to groundwater, until finally a steady state is achieved with chloride inflow to surface water matching chloride outflow from groundwater. For example, the Highland Creek Basin near Toronto, is crossed by a 12-lane highway and a network of arterial and secondary roads, and receives about 10,000 tons of chloride annually in the form of NaCl road salt. Only 45% of the chloride deposited onto the basin by road salting is removed by overland flow into streams and transported with the stream water out of the basin (Howard and Haynes 1993). During 1989–1990, 3427 tons of chloride (31%) left the basin in stream water before the end of April. Another 1609 tons (14%) exited during summer rains before the end of October (Ibid.). If only 45% of chloride applied in a year exits a catchment, 55% is being stored in groundwater. Eventually the amount of chloride entering the basin subsurface waters would match the amount leaving by baseflow. Hydrological calculations indicate that that steady state would be reached in the basin in 20 years (Ibid.). At that time, the average groundwater chloride concentration discharging as baseflow would be 426 ± 50 mg/L, almost twice the maximum acceptable drinking-water chloride concentration (Ibid.). Recently the calculated amount of chloride entering the aquifer and being stored has been amended to 40% (Perera et al. 2013). This revised estimate reflects a better understanding of local groundwater behavior due to rapid subsurface paths of water flow through the “urban karst”—buried pipes and other structures that create a network of flow paths allowing for more rapid summertime groundwater flow than in natural untouched ground (Ibid.). Ultimately, the steady-state chloride concentration in baseflow will be 505 mg/L, well above the current 300 mg/L, itself already above the Canadian drinking-water guideline of 250 mg/L chloride (Perera et al. 2013). No solution to this problem currently presents itself, because, whereas it would be necessary to reduce groundwater recharge of chloride by 50% to get back to the 250 mg/L chloride level, local management practices have already been overhauled, and there seems to be limited Northeastern Naturalist 128 A. Tiwari and J.W. Rachlin 2018 Vol. 25, No. 1 potential for further improvement (Ibid.). If a solution to this long-term problem is possible, it will have to involve creative re-imagining of the issue, as well as longterm thinking and broad community involvement. Chloride Cells: Biological Adaptations for Salt Uptake The main osmoregulatory adaptation for aquatic nymphs of the Ephemeroptera (mayflies), Plecoptera (stoneflies), and Trichoptera (caddisflies) is the chloride cell, or mitochondria-rich cell. Chloride cells show ultrastructural changes when Ephemeropteran nymphs are held in water of different salinities (Wichard et al. 1973). Similarly, after a molt, mayfly nymphs have more or fewer chloride cells depending on the test salinity at which they have been held (Wichard et al. 1973). Although chloride cells in Ephemeroptera and Plecoptera are superficially different, they all have a similar design: an apical porous area of the cuticle over a folded membrane, and abundant mitochondria—apparently adaptations for active transport (Komnick 1977). Chloride has been localized within Ephemeropteran and Plecopteran chloride cells by histochemical precipitation as silver chloride, confirmed by selected-area electron diffraction (Ibid.). The site of salt-ion absorption in caddisfly larvae is either chloride epithelia or anal papillae, depending on which Trichopteran family (Komnick 1977). The Trichopteran families Limnephilidae and Goeridae have oval patches of chloride epithelia on their ventral abdominal surface (sometimes the dorsal surface as well), whereas the Glossosomatidae and Philopotamidae have anal papillae, which also bear chloride cells (Ibid.). When specimens of Limnephilidae or Goeridae are dipped in a dilute solution of silver nitrate, a silver chloride precipitate covers the oval abdominal patches of chloride epithelia. As with chloride cells in Ephemeroptera and Plecoptera, cells that make up the chloride epithelia have abundant mitochondria and highly folded membranes (Ibid.). Experiments using radioactive sodium chloride solution have shown that Trichopteran chloride epithelia absorb both sodium and chloride ions (Komnick 1977). It has also been shown that when the caddisfly Limnephilis stigma Curtis is kept in external media of different salinities, the patches of chloride epithelium on its abdomen enlarge or shrink significantly, in inverse relation to the salinity level (Ibid.). When cells of this type were first observed in the 1970s, they were named after fish chloride cells, to which they are functionally similar (Wichard and Komnick 1971). Unlike the chloride cells of saltwater fish however, aquatic nymph chloride cells do not excrete excess chloride. They function in ion uptake only, allowing the absorption of both sodium and chloride (Komnick and Stockem 1973). Experiments using radioactive chloride showed that no chloride ion is excreted from ephemerid chloride cells (Komnick 1977, Wichard et al. 1973). The few Trichoptera species that live in brackish water have no chloride epithelia because they are not useful in a salt-rich environment (Flint and Giberson 2005). Most aquatic insects are optimized for hyperosmotic regulation, creating concentrated haemolymph relative to a hypotonic environment. Numerous aquatic Northeastern Naturalist Vol. 25, No. 1 A. Tiwari and J.W. Rachlin 2018 129 nymph species inhabit low-salinity waters, whereas very few aquatic nymphs are found in brackish conditions (Komnick 1977). Those aquatic invertebrates that survive in very wide salinity ranges, like larval euryhaline species of mosquito (found at up to 20 ppt salinity), may do so by producing amino acids and proteins in their haemolymph, thereby increasing their haemolymph osmotic pressure (Chadwick et al. 2002). A spike in the osmotic concentration of the external medium requires higher haemolymph and cellular osmotic concentration to prevent dehydration and cell shrinkage (Ibid.). Apparently, freshwater aquatic nymphs with relatively narrow salinity tolerances lack the ability to do this (Ibid.). In laboratory tests of Ephemeroptera, Plecoptera, and Trichoptera (EPT) fauna, hypotonic conditions are better tolerated than hypertonic conditions (Kapoor 1979, Wichard et al. 1973). Plecopteran nymphs have been kept in distilled water for 28 d without mortality, despite a significant decrease in their haemolymph osmolality (Kapoor 1979). The Baetidae family of Ephemeroptera can be found in the wild in fresh or brackish water. Baetidae samples held in water more dilute than their normal conditions had significantly higher survival than samples held at higher salt levels (Wichard et al. 1973). It is likely that, in these species’ natural settings, seasonal heavy rains typically create temporary hypotonic conditions, necessitating the ability to withstand this environmental change (Wichard et al. 1973). Ephemeroptera in Appalachian mountain headwater streams normally live in conditions of low ion-concentration. Mayfly abundance was significantly reduced as stream conductivity rose in areas of mining activity, and mayflies were often locally extirpated from mined areas (Pond 2010). Laboratory trials by Johnson et al. (2015) on larval Ephemeropteran Neocloeon triangulifer McDunnough (synonym Centroptilum triangulifer McDunnough) involving mixed NaCl and CaCl2, like those produced as brine wastes associated with mining operations, showed that the organisms had reduced growth rates and elevated mortality in response to higher conductivity. Those researchers tested N. triangulifer individuals for 20 d in mesocosms at varying concentrations of salt mixture. Ninety-five percent of larvae survived at a specific conductance of 1513 μScm-1, and none survived at a level of 8899 μScm-1. No insects emerged at any of the treatment levels, but 65% of N. triangulifer individuals in the control (untreated) mesocosms emerged during the 20-d experiment (Johnson et al. 2015). Most Ephemeropteran nymphs are found at low salinities, although Tricorythus (Tricorythidae) are found in water at up to 3 ppt salinity, Callibaetis floridanus (Banks) (Baetidae) is found at up to 10 ppt salinity, and Hexagenia limbata Serville (Ephemeridae) regularly survives 8-h periods of 25 ppt salinity in tidal portions of the Mobile River, AL (Chadwick et al. 2002). Hexagenia limbata nymphs that had been at 0 ppt salinity in the wild were acclimated in a laboratory over a 7-d period to salinities of 0, 5, 8, and 12 ppt, after which researchers extracted and tested their haemolymph. Chadwick et al. (2001) reported that only the highest of these test salinities resulted in 100% fatality over the 7-d period, although nymphs did tolerate 12 ppt salinity for 8 h, as they would in a tidal pulse. Northeastern Naturalist 130 A. Tiwari and J.W. Rachlin 2018 Vol. 25, No. 1 As with freshwater macroinvertebrates such as the EPT species, the challenge for a freshwater animal is hyperosmoregulation—the process of keeping enough ions in the body. Fish take in necessary ions in fresh water and shed excess ions in salt water to maintain an internal osmolarity around ⅓ that of seawater (Katoh et al. 2001). In fresh water, epithelial chloride cells (currently more often called mitochondria-rich cells) maintain an internal negative charge by actively pumping out hydrogen ions with H+ATPase pumps. This process allows Na+ to passively diffuse into the chloride cell through sodium channels, and, once inside, be actively pumped into the fish bloodstream. HCO3 -/Cl- exchangers allow chloride ions into the cells, and chloride ions exit to fish blood via anion channels (Marshall 2002). Calcium-ion uptake also goes on in chloride cells, in both fresh and salt water (Marshall 2002). Road Salt and Aquatic Macroinvertebrates Nymphs of the EPT taxa, Ephemeroptera (mayflies), Plecoptera (stoneflies), and Trichoptera (caddisflies), are considered to be less tolerant of pollution and are generally associated with clean water (Crawford and Lenat 1989). Ephemeroptera, in particular, may be sensitive to road salt. Observations at Maryland Biological Stream Survey study sites showed that the number of Ephemeropteran genera (but not Trichoptera or Plecoptera) declined with increasing chloride concentration (Stranko et al. 2013). A particularly salt-sensitive Ephemeropteran species, N. triangulifer, has been investigated for possible use as field-indicator species for levels of chloride pollution (Struewing et al. 2014). In a mesocosm study, Cañedo- Argüelles et al. (2012) showed that Ephemeroptera, such as the Baetidae, were most abundant in the control, which had the lowest chloride concentration. In contrast, many Plecoptera and Trichoptera can survive in high levels of chloride (Blasius and Merritt 2002, Williams et al. 1999). Many macroinvertebrate taxa show tolerance to road salt. Diversity of macroinvertebrate stream communities did not vary significantly with differences in chloride concentration in small Adirondack streams (Demers 1992). High pulses of chloride ion in laboratory and field trials had little effect on the drift of aquatic insect larvae (Crowther and Hynes 1977). In a study of the response of aquatic insects in Michigan to differing concentrations of road salt, Blasius and Merritt (2002) found that insects tolerated high NaCl concentrations and had NaCl LC50 values in excess of field concentrations measured along Michigan streams. Stream sites in this study contained at most 9 mg/L Cl- or16 mg/L Cl-, whereas laboratory acuteexposure experiments used concentrations of 1000–10,000 mg/L NaCl (Blasius and Merritt 2002). Three of the 7 test species—2 Plecopterans (Acroneuria abnormis Newman, Agnetina capitata Pictet), and a crane fly (Tipula abdominalis Say)— were given 96-h LC50 tests and did not exhibit any significant mortality at any of the treatment levels. Black Fly (Diptera: Simuliidae) larvae, accidentally introduced as a contaminant, also showed no significant mortality at any treatment level (Ibid.). Only an amphipod species (Gammarus pseudolimnaeus Bousfield) and 2 caddisflies (Pycnopsyche guttifera Walker and P. lepida Hagen, both family Limnephilidae), Northeastern Naturalist Vol. 25, No. 1 A. Tiwari and J.W. Rachlin 2018 131 exhibited a dose response in these trials, with LC50 values of 7700 for the amphipod and 3526 mg/L NaCl for the 2 caddisflies (Ibid.). Despite evidence of macroinvertebrate resilience to road-salt pollution, it has been shown that road salt can alter macroinvertebrate communities. One study used outdoor mesocosms of different salinities (artificial pools with water, oak leaves, macrozooplankton, and a food source) into which mosquitoes and other flying insects were free to oviposit (Petranka and Doyle 2010). Researchers added road salt to these pools to create a range of salinities that would reflect salinity levels in wetlands near roads that receive de-icing salt. Among the most abundant invertebrates recovered from mesocosms were mosquitoes (Culex restuans Theobald) and cladocerans. Unsalinated and low-salinity pools had relatively few mosquito larvae in them, but they did host abundant cladocerans, including crustaceans such as Daphnia. Cladocerans were rare or absent at concentrations above 1200 mg/L NaCl (664 mg/L Cl), while those mesocosms had abundant mosquitoes. Mesocosm salinity alone, in the absence of cladocerans, did not appear to affect the rates of C. restuans oviposition. The authors note that mosquito larvae and cladocerans may compete for food, and that ovipositing mosquitoes are known to favor pools without high densities of the competitors of mosquito larvae (Petranka and Doyle 2010). Williams et al. (1999) studied chloride contamination and macroinvertebrate fauna in springs in the greater Toronto area. Cluster analysis of species scores derived from canonical correspondence analysis ordination revealed 2 macroinvertebrate groupings—species generally found at higher or lower chloride levels. A chloride-contamination index based on different scores for species in either cluster and intended for use with spring fauna, could assess road-salt contamination of ground water without the need for water sampling (Williams et al. 1999). None of the spring fauna were clearly indicator species because none were exclusively associated with either low- or high-chloride pollution. However, the amphipod Gammarus pseudolimnaeus Bousfield was rare in high-chloride samples, abundant in low-chloride samples, and did not survive well in laboratory salinity trials (Ibid.). No Ephemeropterans were found in the springs. A Plecopteran, Nemoura trispinosa Claassen, was abundant in the more contaminated springs and tolerated high salinity well in trials (Ibid.). In laboratory trials, N. trispinosa and the Trichopteran Lepidostoma sp. survived 96 h of 4500 mg/L Cl- without apparent stress. There is a link between water temperature and salinity tolerance in Trichopterans. Sutcliffe (1961) tested larvae of the freshwater caddisfly species Limnephilus stigma Curtis and Anabolia nervosa Leach (both Limnephilidae) in water of various salinities, from 60 mM NaCl/L to 220 mM NaCl/L. Haemolymph chloride concentration in both species was well regulated, staying hypotonic to all external media, and rising only just before death of the larvae. Haemolymph sodium concentration was less well-controlled, increasing and becoming hypertonic to the external medium in both species. Mortality of both species was high, but was “slightly increased” by lowering the water temperature (Sutcliffe 1961:522). A few species of the Trichopteran family Limnephilidae spend their larval stages in brackish water. One of these taxa, Limnephilus ademus Ross from Eastern Canada, Northeastern Naturalist 132 A. Tiwari and J.W. Rachlin 2018 Vol. 25, No. 1 is found in salinities of 11–25 ppt, or 31–71% of normal seawater (Flint and Giberson 2005). Limnephilus ademus larvae occur very early in the spring in salt-marsh pools on Prince Edward Island, are already pupating at the end of May, and emerge in mid- to late June. This is rapid development compared to freshwater Limnephilids, which typically have an adult summer diapause and don’t oviposit until after midsummer. The early schedule for L. ademus means that these larvae develop when salt-marsh pools are fairly cool, “rarely exceeding 30 °C” (86 °F) (Flint and Giberson 2005:128); this timing may be a strategy for surviving in high salinities. Similarly, in a study on Death Valley caddisflies, Flint and Giberson (2005:128) found that “elevated temperature can reduce tolerance to salinity”. Limnephilis assimilis Banks is found in Death Valley California in waters of widely differing salinity, from fresh to 18 ppt NaCl (Colburn 1983). The larvae are only found in winter, however, never in the warmer months, and never in thermal springs (Ibid.). In laboratory tests at 1–14 ppt salinity, haemolymph chloride levels in L. assimilis were significantly higher at 20 °C than at 8 °C. To control haemolymph chloride at high salinity, the larvae must be able to shed excess chloride ions by active transport using cellular ionic pumps, “a metabolically costly activity” (Ibid.). Dissolved oxygen levels decrease as temperatures increase, which may necessitate a lower rate of ionic pumping (Ibid.). Trichoptera have been shown to have lower salinity tolerance at higher water temperatures (Colburn 1983, Flint and Giberson 2005). In temperate areas, where there are roads, a seasonal salt-pulse occurs at spring melt, when water is cold and oxygen levels are high. In August there may be another salt spike due to evaporation. At this point in the season, the water is warmer, and Trichoptera may possibly be more stressed by salinity, which has the potential to lower local Trichopteran survival, especially under conditions of rising ambient temperatures and increasing concentrations of road salt. Road Salt and Fish Stream fish assemblages have been shown to be structured by conductivity, a result of chloride-ion runoff from impervious surfaces (Morgan et al. 2012). Statewide stream surveys conducted in Maryland since 2000 show that diversity and abundance of stream-fish species in Maryland are strongly related to road density within watersheds (Morgan et al. 2012). Streams with levels of 230–540 μS/cm (33–108 mg/L chloride) supported different fish assemblages than streams at lower conductivities (Morgan et al. 2012). There is a concern that biotic homogenization, the process by which anthropogenic stress re-shapes biotic communities to include only the most tolerant species, may be underway (Morgan et al. 2012). Early life-stages of animals are more vulnerable to pollution, and the alevin and fry stages of salmonids have different responses to the 3 most common deicing salts (Hintz and Relyea 2017). Alevins, the stage after hatching, do not yet have fully functional gills or kidneys, both of which are useful in osmoregulation, and can only swim away from polluted water at the fry stage (Ibid.). In 25-d tests of Oncorhynchus mykiss (Walbaum) (Rainbow Trout) alevins through the fry stage at Northeastern Naturalist Vol. 25, No. 1 A. Tiwari and J.W. Rachlin 2018 133 different concentrations of 3 road salts, Hintz and Relyea (2017) found that salts did not affect the rate at which alevins became fry, but could affect the length and weight they had attained by the end of the experiment. Only the highest concentration of NaCl (3000 mg/L) reduced fish length and mass (P < 0.001). All concentrations of CaCl2 (860, 1500, and 3000 mg/L) reduced fish length and mass (P < 0.05 to P < 0 .001). Magnesium chloride had no effect on fish length or mass (P > 0.05), which is a surprising result because an older study had found MgCl to be the most toxic salt to adult fish (Evans and Frick 2001, Hintz and Relyea 2017). Notemigonus crysoleucas (Mitchill) (Golden Shiner) survived for only 4.6 h in 10,000 mg/L MgCl, whereas this species had survived for 27.6 hours in 10,000 mg/L CaCl2, and 97 hours in 10,000 mg/L NaCl (Evans and Frick 2001). Adult fish typically have an extremely high tolerance of chloride ions. In 96-h LC50 tests (PMRA 2006), Gambusia affinis (Baird and Girard) (Mosquitofish) reached 50% mortality at 10,616 mg/L Cl-, Rainbow Trout at 6743 mg/L Cl-, Pimephales promelas (Rafinesque) (Fathead Minnow) at 4000–6570 mg/L Cl- (different studies reported in PMRA 2006), and Lepomis macrochirus Rafinesque (Bluegill) at 5840 mg/L Cl-. Carassius auratus (L.) (Goldfish) fared worst of adult fish in the 96-h tests, with an LC50 of 4453 mg/L Cl-. Several fish species had no mortality after a 24-h exposure of adults to 10,000 mg/L NaCl (6066 mg/L Cl-), including Rainbow Trout, Fathead Minnow, Bluegill, Salmo trutta L. (Brown Trout), Ictalurus punctatus (Rafinesque) (Channel Catfish), Stizostedion vitreum (Mitchill) (Walleye), and Perca flavescens Mitchill (Yellow Perch) (PMRA 2006). Damage to freshwater fish eggs or embryos generally requires high chloride ion concentrations. Rainbow Trout eggs and embryos have a 7-d EC25 test (effect concentration at which 25% died) of 989 mg/L Cl- (PMRA 2006). Fathead Minnow larvae that were 1-, 4-, and 7-d old had a no-effect concentration (NOEC, normal survival and growth) of 4000 mg/L NaCl (2424 mg/L Cl-) and a subchronic value (SCV, derived from the geometric mean of the NOEC and lowest observed-effect concentration LOEC) of 5700 mg/L (3455 mg/L Cl-) (Pickering et al. 1996). As discussed in the next section, compared to these reports for fish, frogs and other amphibians appear to be more susceptible to salt-induced mortality and deformities. Thus, road-salt runoff could disrupt predator–prey equilibria because fish often prey on amphibians (Sanzo and Hecnar 2006). Road Salt and Amphibians Amphibian vulnerability to water pollution stems from their permeable skin and the dependence on water in their early life-histories. Wood Frog eggs from Canadian wetlands were reared in water containing different concentrations of sodium chloride that reflected salt concentrations measured in the field. Field concentrations of 0.39, 77.5, and 1030 mg/L NaCl, measured in northwestern Ontario, were used to test the chronic effects of salt on Wood Frogs. Chronically salt-exposed Wood Frog tadpoles at all treatment levels had significantly decreased survivorship (P < 0.001). Significantly fewer of the treated tadpoles underwent metamorphosis, with the fewest in the highest-concentration treatment (P = 0.05). Developmental or Northeastern Naturalist 134 A. Tiwari and J.W. Rachlin 2018 Vol. 25, No. 1 behavioral abnormalities, such as bent tails or swimming in circles, were often observed at 77.5 mg/L NaCl and 1030 mg/L NaCl (Sanzo and Hecnar 2006). The 96-h LC50 value for Lithobates sylvaticus LeConte (Wood Frog) tadpoles established by Sanzo and Hecnar (2006) was 2636.5 mg/L NaCl (1598 mg/L Cl-). Unhatched amphibian embryos from same-aged eggs of Taricha granulosa (Skilton) (Roughskinned Newt) were affected by 3 treatment levels of 2 de-icers: NaCl, and MgCl2. The latter has become a commonly used road treatment (Hopkins et al. 2013). All concentrations (1.0, 1.5, and 2.0 g/L Cl-) of either salt produced significantly more deformities than the control (P < 0.0001), or significantly greater numbers and severity of deformities with increasing salt concentration (P < 0.0001), but for the most part, results were not significantly different for type of salt used, P > 0.05 (Hopkins et al. 2013). Not all amphibians are vulnerable to road-salt runoff, whether exposed at egg or larval stage. Lithobates catesbeianus Shaw (American Bullfrog) hatched and reared for 60 d at 3 concentrations of road-salt solution (100, 500, and 1000 mg/L Cl-) showed no significant survival or malformation differences from controls (Matlaga et al. 2014). American Bullfrog tadpoles reared at these concentrations were neither more nor less likely to be preyed upon by dragonfly nymphs (Odonata) compared to controls (Matlaga et al. 2014). Embryonic and larval Rana clamitans (Latreille) (Green Frog) also showed relative insensitivity to road salt, with 93% surviving more than 2 months in chloride concentrations of 33, 145, and 465 mg/L in a laboratory study (Karraker 2007). Survivorship declined to 80% at the 945-mg/L chloride level. These survival rates of 80–93% may not be meaningfully different from survival rates under natural conditions, as other frog species show a wide range of embryonic survival rates in the wild and in laboratory settings (Karraker 2007). Why would one amphibian species tolerate road salt and another not? Amphibian embryos develop inside the innermost membranous chamber of the egg, and are dependent upon water passage into and out of this space. Salinated water flows in and out of the egg’s inner chamber at a reduced rate, which may account for developmental abnormalities (Karraker and Ruthig 2009). Ambystoma maculatum (Shaw) (Spotted Salamander) spend 5–6 weeks in the egg stage, whereas Green Frogs remain in the egg for only a week or less before hatching (Karraker 2007, Karraker and Ruthig 2009). The relatively brief egg developmental period of the Green Frog may make it less vulnerable to altered conditions during the egg stage (Karraker and Ruthig 2009). Conceivably, as pools in the vicinity of roads become increasingly salt-polluted, some amphibians may exhibit developmental plasticity allowing different incubation periods within a species. Developmental plasticity is seen in the Litoria ewingii (Duméril & Bibron) (Australian Brown Tree Frog), in which immature stages occupy different naturally occurring salinities (Kearny et al. 2015). Brown Tree Frog tadpoles inhabiting brackish sites reach metamorphosis sooner and at a smaller size (Kearny et al. 2015). Chloride-ion concentrations appear to structure amphibian assemblages in roadside ponds. In a study of 26 ponds within 60 m of roads in Nova Scotia, measured Northeastern Naturalist Vol. 25, No. 1 A. Tiwari and J.W. Rachlin 2018 135 values averaged 118.5 mg/L chloride ion in spring, 82.3 mg/L in early summer, and 97.1 mg/L in late summer. These concentrations represent a salt pulse at spring snow-melt, dilution of ponds by spring and early summer rains, and then concentration of pond water by evaporation in summer heat. Surveyors documented 8 amphibian species in these ponds. Amphibian species richness declined significantly with increasing chloride concentration (P < 0.05). No Spotted Salamanders or Wood Frogs were found in the ponds with the higher Cl- concentrations, whereas no pattern was seen regarding study-pond Cl- concentration and presence/absence of Pseudacris crucifer (Wied-Neuwied) (Spring Peeper), Green Frogs, or Bufo americanus (Holbrook) (American Toad) (Collins and Russell 2009). Road-salt runoff can also potentially structure sex ratios in amphibians. Same-age Wood Frog tadpoles reared through metamorphosis in mesocosms with leaf litter and road-salt solution showed 10% fewer females in the presence of road-salt solution (800 mg/L chloride; Lambert et al. 2016). Apparently, road salt caused sex reversal rather than higher mortality of males, because survival did not vary with treatment (Lambert et al. 2016). Road Salt and Terrestrial Vertebrates De-icing salt attracts some mammals to roadsides, and contributes to car accidents. Mammals that seek salt at roadsides and are often struck by cars include Alces alces L. (Moose), Odocoileus virginianus Zimmermann (White-tailed Deer), Odocoileus hemionus (Rafinesque) (Mule Deer), Ovis canadensis Shaw (Bighorn Sheep), Marmota monax L. (Woodchuck), Erethizon dorsatum (L.) (Porcupine), Lepis americanus Erxleben (Snowshoe Hare), and Lepis sylvaticus Bachman (Cottontail Rabbit) (Environment Canada 2001, Kelting and Laxson 2010). Radiocollared Moose in New Hampshire “extended their range to include pools heavily contaminated by road salt … there were twice as many Moose–vehicle collisions per km where roadside pools were present than where there were no pools” (Kelting and Laxson 2010:42). Male Moose grow antlers and females lactate in the spring; thus, their sodium hunger is greatest then, as is the frequency of Moose–vehicle collisions, although it is not the season with the heaviest car traffic (Kelting and Laxson 2010). Birds are also attracted to road salt, probably both as a source of sodium and for its resemblance to the pebbles that birds use to aid in digestion. Bird–vehicle strikes are unlikely to be reported; however, Mineau and Brownlee (2005) found 12 published reports of bird strikes by vehicles. Species included Colinus virginianus (L.) (Bobwhite Quail), Phasianus colchicus (L.) (Ring-necked Pheasant), Loxia leucoptera Gmelin (White-winged Crossbill), L. curvirostra L. (Common Crossbill), Hesperiphona vespertina (Cooper) (Evening Grossbeak), and Spinus pinus Wilson (Pine Siskin). Several of these bird kills were obviously reported because of the large numbers involved; for example, ~1000 Evening Grossbeaks were killed in British Columbia over a period of 2 weeks in 1980 after feeding on road salt (Ibid.). Grossbeaks, crossbills, and siskins are collectively called winter finches because they often move south out of boreal forests in winter when Northeastern Naturalist 136 A. Tiwari and J.W. Rachlin 2018 Vol. 25, No. 1 conifer seeds are less available. In some areas, the winter finches are called “grill birds” because they are so often hit by cars (Ibid.). Birds do not have the physiological defenses against salt that some other animal groups have. While the kidneys of mammals have the ability for precise regulation of sodium and chloride retention or excretion, avian kidneys are not as well equipped for this task (Mineau and Brownlee 2005). And while marine birds have nasal glands that excrete excess salt, non-marine birds do not have this mechanism (Ibid.). Birds may ingest considerable quantities of salt. In one case from the 1950s, the consumed road salt had been stained blue, and the bird gut contents were also blue (Ibid.). Winter finches often appeared sick or unnaturally fearless after feeding on road salt. Symptoms of salt toxicosis include partial paralysis, tremors, inability to fly or perch, and retropulsion (involuntary backwards movemen ts; Ibid.). In toxicity testing using Passer domesticus (L.) (House Sparrow) as a model, water was withheld for 6 h after salt ingestion to replicate winter conditions. Overt signs of toxicity and first mortality were recorded at 1500 mg/kg road salt, or 2.5 road-salt particles of 2.4-mm diameter. The median lethal dose was 3000 mg/kg road salt, or 5.2 road-salt particles of 2.4-mm diameter (Mineau and Brownlee 2005). However, the House Sparrow originated in the Middle East, and, in that arid environment, they may have become more salt tolerant than winter finches (Mineau and Brownlee 2005). Thus, it is likely that these findings underestimate the toxicity of road-salt particles to native North American birds. Road Salt and Plants Challenges to roadside vegetation from road salt include aerial deposition, changes in soil ion content, changes in soil nitrogen cycling, and changes in physical soil structure. Aerial deposition of salt from road de-icing has been measured as a plume of salt up to 15 m in height (Kelsey and Hootman 1992). Earthen berms designed to protect roadside forests from road salt runoff may result in higher aerial-salt plumes because berms can guide air currents and airborne particles upwards (Ibid.). Aerial deposition of road salt onto forests has been reported to occur from as far away as 500 m (Ibid.). A more conservative estimate of typical aerial road salt exposure is 40–100 m from the paved salted surface. Within that distance, road salt can be measured in plant tissues, and salt injury is visible on plant surfaces (reviewed in Cain et al. 2000). Aerial-salt deposition can cause necrosis and death of conifer needles, which normally last for 3 years (Cain et al. 2000, Kelsey and Hootman 1992). In deciduous trees, aerial-salt deposition can kill shoots, which stimulates the growth of adventitious shoots in a so-called “witch’s broom” formation (Cain et al. 2000, Kelsey and Hootman 1992). Road salt also affects trees and other plants by altering the chemical composition of soils and waters that receive runoff. When chloride concentrations are high in downgradient groundwater, that water also contains significantly more sodium, calcium, magnesium, potassium, barium, strontium, copper, iron, and zinc than upgradient water (Granato et al. 1995). This ion increase is apparently an effect of sodium ions being exchanged for calcium and other cations in the Northeastern Naturalist Vol. 25, No. 1 A. Tiwari and J.W. Rachlin 2018 137 soil, increasing the mobility of these cations and causing them to be detectable in downgradient groundwater (Ibid.). Clay and organic matter in soil have negative surface charges which attract groundwater cations. Normally Na+ cannot outcompete ions such as Ca2+ or Mg2+, which are more positively charged, or K+, a smaller cation than sodium (smaller atoms outcompete larger ones for exchange sites). However, sodium becomes a much better competitor when its concentration relative to other ions is increased, as when an area receives road salt runoff (Kelting and Laxson 2010). Roadside vegetation and surface waterbodies are both vulnerable to changes in soil nutrient particles and structure that result from road-salt inputs. The plant cation-macronutrients calcium, magnesium, and potassium, and micronutrients copper, zinc, manganese, and molybdenum are available to plants when electrostatically bound to clay and organic soil particles. When cations are unbound because they have been out-competed for binding sites by sodium, they may leach out of the soil, depriving plants of these nutrients (Kelting and Laxson 2010). Sodium also out-competes hydrogen ions, freeing the H+ and making the soil less acidic. Roadsalt runoff would have one positive effect on very acidic soil, in that a pH lower than 5 decreases the action of soil nitrifying bacteria, and raising pH may increase nitrate (NO3 -) levels in the soil. However, soil nitrifying bacteria are sensitive to salinity, and their activity is significantly reduced at NaCl concentrations ≥ 0.25 mg/L (Green et al. 2008). Ammonium ions (NH4 +) are displaced from their soil binding sites by sodium, and can leach out of soil (Green et al. 2008), which decreases soil fertility. Calcium ions structure soil by bridging negatively-charged clay particles and holding them together. Cation bridging prevents clay particles from being leached into surface waters, and allows the soil to absorb more water. The sodium cation, with its large size and single positive charge, does not bridge clay particles. Loading soil with sodium can therefore increase runoff of rain into local waterbodies along with increased sediment load due to greater erosion (Kelting and Laxson 2010). Summary Chloride ions from the runoff of road-deicing salt have accumulated in surface waters such as streams and in groundwater. The increase in chloride concentration in streams and other receiving waters reflects local increases in impervious surface. Road-salt runoff into lakes can increase the density of the lowest stratified layer, and make it less likely that the layers will mix. When a lake remains stratified, the lowest layer does not get oxygenated, which degrades the lake as fish habitat. Excess salt can damage animal health, including that of aquatic invertebrates, fish, amphibians, birds, and mammals. A special concern with regard to fish is that salt runoff into streams potentially leads to biotic homogenization until only tolerant species remain. Both fish and aquatic invertebrates such as Ephemeroptera, Plecoptera, and Trichoptera have chloride cells that compensate for a hypotonic environment. Chloride cells on the EPT taxa can only serve to take in chloride, however. They cannot release excess ions, unlike such cells in fish. Road salt can have deleterious effects Northeastern Naturalist 138 A. Tiwari and J.W. Rachlin 2018 Vol. 25, No. 1 on plant health by the direct effects of salt deposition on leaves, and because excess sodium ions change soil structure and nutrient cation composition. 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