The salinization of freshwaters due to the use of deicing road salts is a serious anthropogenic threat. We investigated the effects of the road deicer, which is mostly composed (ca. 70%) of NaCl, on the life cycle parameters of the cladoceran Moina macrocopa in acute and chronic toxicity tests and on the hatching success of resting eggs exposed to sediments contaminated with the road salt. The negative effects of the road salt on survival and life cycle parameters of animals were observed at concentrations above 5 g L−1. The 6-month exposure of resting eggs to contaminated sediments had a consistent but relatively weak effect on the postexposure hatching of resting eggs. Experiments demonstrated that the concentration of the deicer in the top water layer in the water-sediment systems is more important for the hatching success of resting eggs than the salt content in the sediment. Only 2.2 ± 1.9% of resting eggs hatched when the deicer content in the top water layer was equal to 12 g L−1. Lethal effects on hatchlings were observed starting from the deicer content in the water equal to 1 g L−1, and 97.0 ± 0.8% of hatchlings were dead at the deicer content in the water equal to 5 g L−1. Thus, the resilience of resting eggs to the contaminated sediments can ensure the replenishment of the population from the egg bank after the salinity disturbance is diminished but the negative effects of the elevated salt content in surface waters on active population will be manifested at lower salinities.
Chemical pollution of freshwater ecosystems due to the long-term application of deicing road salts has been identified as a serious water quality problem in northern regions worldwide (Jones et al. 2017; Meter et al. 2011; Schuler et al. 2017; Dugan et al. 2017; Godwin et al. 2003; Iglesias et al. 2020). Numerous studies indicate that the use of deicing agents, primarily ones containing chlorides (e.g., NaCl, CaCl2, MgCl2, and KCl), increases the salinity of aquatic habitats (Schuler et al. 2017; Kotalic et al. 2017). The road salts being transported into surface water or groundwater readily dissociate into respective cations (Na+, Ca2+, Mg2+, and K+) and anion Cl- (Godwin et al. 2003; Velasco et al. 2019; Kotalic et al. 2017; Zhang et al. 2013). The salinity is one of the key factors determining the species composition, structure of the food web and the productivity of aquatic ecosystems (Velasco et al. 2019; Kefford et al. 2007; Rasdi et al. 2019). Thus, the increase in ion concentrations in freshwaters may affect biodiversity and significantly alter ecosystem functioning (Iglesias et al. 2020; Zadereev et al. 2020). Given the rising global concern over salinization of natural waters, it is very important to know critical rates of increase and concentrations of salts that cause the negative effects on biodiversity in freshwater habitats (Kefford et al. 2007). This information is also important for the development of urgently needed ion-specific standards and management guidelines for protecting freshwaters from continued salinization (Schuler et al. 2019).
Cladocera is the dominant group of zooplankton in many freshwater habitats, ranging in size from shallow temporary ponds to deep lakes. Most of the Cladocera species are effective filter feeders, which transfer organic carbon from primary producers to higher food levels and play a key role in water clarity control. Changes in diversity and population density of these species can have a significant effect on biotic interactions in aquatic ecosystems. Many Cladocera species are cyclic parthenogens mostly reproducing by parthenogenetic eggs. It allows them to increase quickly in numbers under favorable environmental conditions. Due to the fast reproduction rate and ease of handling, some species (e.g., of taxa Daphniidae and Moinidae) are well-known bioindicators used in toxicity test (Schuytema et al. 1997; Zadereev et al. 2017).
The acute and chronic tests with cladocerans demonstrated toxicity of water samples during road-salt runoff (Corsi et al. 2010). As sodium chloride (NaCl) is the dominant road salt used for deicing (Godwin et al. 2003; Schuler et al. 2017; Kotalic et al. 2017), studies evaluating the impact of deicers on aquatic habitats are mainly focused on this compound. Laboratory tests demonstrated adverse effects of NaCl on population dynamics (Sarma et al. 2006) and individual survival of cladocerans (Schuytema et al. 1997; Martínez-Jerónimo et al. 2007).
Cladocerans are not always present in active stage in natural habitat (Cáceres 1998). Under adverse environmental conditions, they switch from parthenogenesis to gametogenesis and produce diapausing eggs, which are resistant to harsh external factors (Alekseev et al. 2007; Oskina et al. 2019). Resting eggs accumulating at the bottom of a waterbody form an egg bank. Such banks are a source of genetic diversity and of recruiting of active animals after periods of population decline (Brendonck and De Meester 2003).
Despite the obvious importance of egg banks, few studies have estimated the effects of increased salinity on hatching success of resting eggs (e.g., Bailey et al. 2003). The contribution of resting eggs to resilience of zooplankton communities to increased salinity was determined in several studies (Santangelo et al. 2014; Nielsen et al. 2012). It was demonstrated that dormant eggs of zooplankton could persist at salinities and concentrations of toxicants that are deleterious to active animals (Nielsen et al. 2012). However, the reinstating of the native composition of zooplankton community from egg banks depends on the water salinity, which should be low enough. Given the serious investment of the road deicers into salinity increase, the study of the effect of road salts on hatching success of resting eggs and survival of hatchlings is urgently needed.
The aim of this study was to assess the impact of road deicer solutes and contaminated sediments containing up to 70% of sodium chloride on the active and resting stages of freshwater cladoceran Moina macrocopa. First, we exposed animals to the range of deicer solutes in acute and chronic toxicity tests to evaluate sublethal and lethal concentrations of the deicer. Second, to test the effect of road salt on the hatching success of resting eggs and survival of hatchlings, we created experimental systems with eggs exposed to contaminated sediments.
We made tentative assumptions that: (1) the effect of the deicing material is mostly associated with the effect of chlorine salts; (2) the hatching success of resting eggs is inhibited by increased salinity; and (3) the water salinity is more important for hatching success of resting eggs and survival of hatchlings than sediment salt content.
We tested the effects of solutes and contaminated sediments of the «Bionord» road salt (Ural Plant of Deicing Materials, Russia) on the females and resting eggs of freshwater cladoceran Moina macrocopa (Straus 1820) (Cladocera: Moinidae). According to official information, the proportion of the components in the "Bionord" road salt can vary depending on the batch within the following ranges: CaCl2: 10–50%, NaCl: 10–80%, CaCO3: ≤20%, KCl: ≤25%, formates (K and/or Na): ≤35%, CH3COOK: ≤35%) (Technical documentation 2014). Laboratory culture of M. macrocopa had been maintained in the Institute of Biophysics SB RAS (Krasnoyarsk, Russia) for more than ten years. Tap water (pH–7.3; total permanent hardness—62.9 mg·equivalent of CaCO3/L; total content of cations (macro- and trace elements)—26.4 mg L−1) aged for at least 72 h was used as culture medium in all experiments.
Total element contents of road salt solutes, control medium, sediments, and sediment elutriates were quantified using an iCAP 6300 Duo ICP-OES spectrometer («Thermo Scientific», U.K.) according to EPA 200.7. Technical characteristics and operating conditions of the ICP-OES spectrometer were described in detail elsewhere (Oskina et al. 2019). Specific electrical conductivity was measured with a STARTER ST300C conductometer ("Ohaus Corporation," USA), pH–with a PB-11 "Sartorius" pH meter (Germany). Before each series of measurements, the instruments were calibrated.
To perform analysis, sediment samples were dried at 105 °C in a drying oven, ED53 (Binder, Germany), until a constant weight. For ICP-OES analysis, 0.2 g of sediments was microwave digested in PTFE containers in 3 ml HCl and 1 ml HNO3 at 160 °C and 12 atm for 4 min and then at 190 °C and 20 atm for 5 min in an MS-6 oven (Volta, Russia). Then, the sediment was diluted with 18 MΩ water (Anishchenko et al. 2020). To prepare elutriates, we mixed a sediment sample with tap water at a ratio of 1:20 (v/v) and shook it for 10 minutes (OCDE/GD 1993). Separation of elutriate and solid phase was performed by centrifugation for 30 minutes at 8000g.
To determine the proportion of insoluble fraction of the road salt, we centrifuged the solutes for 30 minutes at 8000g and 20 °C. After the primary centrifugation, the resulting pellet was washed three times with distilled water and centrifuged once more; then, it was dried at a temperature of 105 °C and weighed using an analytical balance.
The pH of all solutions (7.7 ± 0.2) did not depend on the concentration of the deicer.
Acute and chronic toxicity tests with animals
Acute and chronic toxicity tests were performed in a climate chamber at a constant temperature (25 ± 1 °C) and photoperiod (16 h light:8 h dark). To prepare road salt solutes, we used the stock solution of “Bionord” (200 g L−1). Aged tap water was used as the control. To start acute and chronic toxicity test, we placed 1-day-old juvenile females (size 0.5–0.6 mm) hatched in favorable conditions for parthenogenesis individually into beakers with 20 mL of control medium or road salt solutes (Zadereev and Gubanov 1996). For each concentration of the road salt and for the control, we tested 20 individually kept animals in three consecutive acute tests and 15 individually kept animals in chronic test.
The following “Bionord” concentrations were used in three consecutive acute tests: 1.3; 2.5; 4.0; 5.0; 6.0; 8.0; and 10.0 g L−1. The animals were not fed in acute tests. The number of dead individuals after 24 h and 48 h was counted to determine median lethal concentrations (LC50).
In the chronic test, the following “Bionord” concentrations were used: 0.3; 0.6; 1.3; 2.5; 5.0; 6.0; and 8.0 g L−1. The solutes of the road salt were prepared prior the experiment in a volume sufficient for the duration of the chronic test. The animals were fed daily with the Chlorella vulgaris non-axenic green alga (Oskina et al. 2019) at a concentration of 200 thousand cells mL−1 (ca. 2.24 µg C mL−1). The test and the control media in experimental vessels were renewed daily. The chronic test continued until the death of all test animals. For each female, we measured, under 16x magnification, the body length at the first day of the experiment and the day before it produced the first clutch. The body lengths were used to calculate the specific growth rate of juvenile females (Zadereev et al. 2017). For each female, we counted the number of offspring in each clutch and recorded the time of death. The average lifespan (days) and fecundity (hatchlings per female) were calculated for each concentration of the road salt.
Chronic tests with resting eggs
Resting eggs were collected from the batch culture of M. macrocopa cultivated at growth conditions favorable for the mass production of resting eggs (Oskina et al. 2019). We used undamaged ephippia containing two fertilized embryos that were kept in the darkness at 4 °C for 2 months.
The artificial sediment was prepared following the standard protocol (OECD 1984, 2004). Sphagnum peat was air-dried, ground to fine powder (particles ≤ 1 mm) and moistened with deionized water. The suspension was conditioned for 2 days at 20 ± 2 °C, to stabilize pH (6.2 ± 0.5). Then, the peat suspension (5–10%, dry weight) was blended with kaolin clay (20%, dry weight) and quartz sand (70–75%, dry weight). The resulting sediment was mixed thoroughly and moistened with deionized water to obtain a homogeneous substance. The pH of the final mixture was 7.0 ± 0.5.
To enrich the artificial sediments with the road salt, we added the deionized water with dissolved road salt. The amount of water and the amount of the road salt were calculated to adjust sediment moisture content to 35–40% of its dry weight and to achieve road salt concentration in the sediment of 3.0, 6.0 or 12.0 g kg−1. The sediments were thoroughly mixed and kept under experimental conditions for 24 h. Samples were taken from each contaminated sediment to control moisture content, measure the total element content in the sediments, and prepare sediment elutriates. Conductivity, pH, and contents of water-soluble elements of the resulting test sediments were measured in sediment elutriates.
To test the effect of the road salt on the survival of resting eggs, we created two types of experimental systems (Fig. 1): water-sediment systems with contaminated sediments covered with the water layer and dry sediment systems with contaminated sediments (details are described below). Thirty ephippia, each containing two resting eggs, were placed in each test vessel. Each ephippium was buried carefully with a pipette into the sediment to a depth of 5–7 mm. All experimental systems (60 vessels overall) with buried eggs were kept in the darkness at a temperature of 4 °C for 6 months. After exposure, all resting eggs were hatched under a constant temperature (25 ± 1 °C) and photoperiod (16 h light, 8 h dark).
Test with water-sediment systems
To create the experimental system, we used 500-ml vessels. Each vessel contained 100 g of sediments covered with 300 ml of water. We created the following experimental systems, where the first number is the concentration of the road salt in the water (W, g L−1) and the second number is the concentration of the road salt in the sediment (S, g kg−1): contaminated sediments covered with solutes of the road salt (W3S3; W6S6; W12S12); contaminated sediments covered with the tap water (W0S3; W0S6; W0S12); and sediments covered with solutes of road salt (W3S0; W6S0; W12S0). The treatment with sediment covered with the tap water (W0S0) was used as the control. For each combination and for the control, we prepared three vessels.
After 6 months of exposure, we took 30 ml samples of water from each vessel to measure conductivity, pH, and total element content. Resting eggs were hatched under conditions described above. We recorded daily the number of live and dead hatchlings. Dead hatchlings were removed from the vessels immediately. Live hatchlings were removed from the vessels every fourth day starting from the day when the first hatchling was recorded. We monitored hatching success for 12 days after the start of reactivation.
At the end of the experiment, water was removed from all vessels. To estimate the bioavailable road salt in the sediment, we measured the conductivity of water extracts. The water extract for each vessel was prepared by mixing the entire volume of the sediment in the vessel with the tap water at a ratio of 1:5 (wet weight). Conductivity was measured after 10 minutes of mixing the sediment and tap water with magnetic stirrer.
Hatching success was calculated as the sum of all live and dead hatchlings. Mortality was calculated as the sum of all dead neonates. Also, we calculated 72-h survival of animals as the sum of all live animals observed on every fourth day starting from the day when the first hatchling was recorded.
Test with dry sediment systems
To create the experimental system, we used 500-ml vessels. Each vessel contained 100 g of sediments. We prepared 12 vessels with uncontaminated sediments and six vessels for each concentration of the road salt in contaminated sediment: 3, 6, and 12 g kg−1.
After 6 months of exposure, we divided these 30 experimental systems into two subsets.
Three vessels for each concentration of contaminated sediment (3, 6, and 12 g kg−1) were used to test the effect of exposure to contaminated sediments on survival of resting eggs. We extracted eggs from sediments in each of the nine vessels, washed them in distilled water and placed them into 300 mL of tap water for reactivation under conditions described above.
Twenty-one remaining vessels were used to mimic the effect of spring, when dry sediments are covered with water. We covered sediments with tap water or solutes of the road salt to create the following experimental systems, where the first number is the concentration of the road salt in the tap water (g L−1) and the second number is the concentration of the road salt in contaminated sediment (g kg−1): contaminated sediments covered with tap water (W0S3; W0S6; W0S12); sediments covered with solutes of road salt (W3S0; W6S0; W12S0). The treatment with sediment covered with tap water (W0S0) was used as the control.
The number of hatched neonates for both subsets was recorded daily, and the animals were immediately removed from the vessels. We monitored hatching success for 20 days after the start of reactivation. After 20 days, we measured conductivity of the top water layer. Hatching success was calculated as the ratio of hatched eggs to the total number of eggs.
Data on three consecutive acute tests were combined into one data set based on the absence of statistically significant differences in the survival of animals in the control (test of difference between two proportions, p> 0.32). Data on survival of animals in acute test, life cycle parameters of animals in chronic test, proportions of hatched resting eggs and dead hatchlings did not meet the assumption of normality and heterogeneity of variance. We used nonparametric tests that are usually appropriate for statistical analysis of life cycle parameters and bounded response variables (Zadereev et al. 2017). Differences between treatments and control in the survival of animals in acute test; the proportion of hatched eggs and the proportion of dead hatchlings in water-sediment systems and in dry sediment systems were estimated by nonparametric comparison of two independent samples (Mann–Whitney U test). The effect of the deicer on the specific growth rate of juvenile females, fecundity, and average lifespan of females in chronic test and 72-hour survival of live hatchlings in water-sediment systems were estimated by nonparametric comparison of multiple independent samples (Kruskal–Wallis test), which was followed by multiple comparisons to determine the difference between different concentrations and control. All statistical calculations were performed in STATISTICA 8.0.
The parameters of logistic functions and LC50 and EC50 values in acute and chronic toxicity tests with females of M. macrocopa and water-sediment test with resting eggs were determined in "drc" package for R (Ritz and Streibig 2005).
The road salt chemical analysis
Total element content analysis identified major elements whose concentrations in the solutes linearly depended on the amount of the dissolved deicer (R2 ranged from 0.99 to 0.60). The sum of these elements was equal to 85.0% of the amount of the dissolved deicer (Cl: 48.0 ± 5.8%; Na: 28.7 ± 2.1%; Ca: 7.9 ± 0.5%; S: 0.2 ± 0.1%; K: 0.20 ± 0.01%; Mg: 0.01 ± 0.01%; Sr: 0.006 ± 0.001%; B, Ba, Cr, Cu, Ga, Li, Mn, Mo, V total: 0.003%) (Table 1). The concentrations of Al, As, Bi, Cd, Co, Fe, Ni, P, Pb, Sb, Se, Sn, Ti, Zn were not significantly dependent on the amount of the dissolved deicer (R2 ranged from 0.5 to 0.003). The fraction of the unaccounted elements in the solutes did not exceed 15%. The proportion of the insoluble mineral fraction was equal to 1.18 ± 0.15% (n = 4). The electrical conductivity of the solute (y, mScm−1) linearly depended on the amount of dissolved deicer (x, g L−1) y = 1.22 x+1.57 (R2 = 0.99).
Acute and chronic tests with animals
The survival of animals in acute toxicity test was equal to 87–100% at the range of concentrations of the road salt in solutes from 1.2 to 4.0 g L−1 and abruptly decreased to 59% at a concentration of 5.0 g L−1 and to 17% at 6.0 g L−1 (Table 2, Fig. 2). Chronic toxicity test demonstrated similar threshold concentration of the deicer for the adverse effects (Table 2). The effect of the deicer on the lifespan was strongly significant (Kruskal–Wallis test: H (7, N = 119) = 68.0683 p < 0.0001). Lifespan of females in the control was equal to 9.0 ± 3.6 days and sharply decreased to 1.3 ± 0.5 days at a concentration of the deicer of 6.0 g L−1 (Table 2, Fig. 2). The effect of the deicer on the specific growth rate was marginally significant (Kruskal–Wallis test: H (5, N = 84) = 11.149 p = 0.049). The specific growth rate of juvenile females of M. macrocopa was ca. 15% lower at a concentration of 5.0 g L−1 of the deicer than in the control and in all other treatments. We did not observe significant differences between fecundities of females of M. macrocopa at all tested concentrations of the deicing salt (Kruskal–Wallis test: H (5, N = 89) = 4.65 p = 0.46). The average fecundity was equal to 61.2 ± 10.6 neonates/female. The death of all animals in both acute and chronic toxicity tests was observed within 48 hours at the concentration of the deicer equal to 8.0 g L−1 (Table 2). Values of LC50 for survival of animals in acute toxicity tests (LC50 = 5.1 ± 0.1 days) and EC50 for lifespan in chronic toxicity test (EC50 = 5.6 ± 0.2 days) were comparable (Table 3).
Chronic tests with resting eggs
Experiment showed that the effect of the road salt on the hatching success of resting eggs strongly depended on the type of treatment (Fig. 3). Hatching success of resting eggs in the experimental systems with contaminated sediments covered with the control medium was comparable with the control (91.7% in the control (W0S0), from 84.4 to 91.7% in W0S3 W0S6 W0S12 treatments, 87.8% ± 4.5 on average for this subset of treatments). In the experimental systems with the deicer content in the top water layer equal to 3 g L−1 or 6 g L−1 (W3S0, W3S3, W6S0, W6S6 treatments), the hatching success was slightly lower (ca. 80 and 75%, respectively). The hatching success of resting eggs in the W12S0 and W12S12 treatments (the deicer content in the top water layer equal to 12 g L−1) was significantly lower (50 and 90%, respectively) than in the control (Fig. 3).
The proportion of dead hatchlings depended both on the salinity of the top water layer and salt content in the sediments (Fig. 3). The proportion of dead hatchlings in all treatments was higher than in the control. Almost 100 or 100% mortality of hatchlings was observed when the concentration of the deicer in the top water layer was equal to or above 6 g L−1 (W6S0, W6S6, W12S0 and W12S12 treatments).
The survival of live hatchlings was high and independent on the treatment (Kruskal–Wallis test: H (6, N = 21) = 5.27 p = 0.51). The average 72-hour survival for all treatments was equal to 99.7 ± 1.2%.
When we used the equilibrium concentration of the salt in the water as predictor, we found that it strongly determined both the hatching success of resting eggs in our systems (EC50 = 8.5 g L−1) and the mortality of hatchlings (EC50 = 3.8 g L−1) (Table 3, Fig. 2). While hatching success was affected by the water salinity above 9 g L−1, the death of hatchlings was already observed at the water salinity above 1.0 g L−1 (Fig. 3). This value is below the LC50 and EC50 values measured in acute and chronic toxicity tests with females (Table 3).
Dry sediment systems
When resting eggs exposed to sediments contaminated with the road salt for 6 months were washed to remove the salt and hatched in the tap water, we observed that the overall effect of the salinity in sediments on the hatching success of resting eggs was insignificant (Kruskal–Wallis test: H (3, N = 12) = 6.59 p = 0.09). At the same time, the number of hatchlings from the resting eggs exposed to the sediments contaminated with the road salt at a concentration of 12.0 g kg−1 (Fig. 4) was ca. 20% lower than in the control. However, this effect was weaker than the effect of the solutes of the deicer (compare the hatching success of eggs exposed to sediment contaminated with 12.0 g kg−1 road salt (Fig. 4) and hatching success in W12S0 and W12S12 treatments (Fig. 3)).
The salinity of water that was used to cover sediments in the experimental systems after 20-day exposure (Fig. 5) was comparable with the water salinity in the water-sediment test (Fig. 3). This indicates that the stabilization of salinity between the upper water layer and bottom sediment occurs relatively quickly, within 20 days at the most.
Hatching success of resting eggs in this experimental system was lower than in the water-sediment system. We observed reduced hatching success both in the treatments with contaminated sediments covered with the tap water and sediments covered with solutes of the deicer (Fig. 5). However, the difference with the control was significant only for the treatment with solutes of the deicer at the concentration of 12.0 g L−1.
The road salt chemical analysis
Our analysis demonstrated that the Bionord deicer used in this study is mostly composed of Na and Cl (up to 70% of the total content). Among salts widely used as deicers, such as sodium chloride, magnesium chloride and calcium chloride (Ramakrishna and Viraraghavan 2005; Schuler et al. 2017), sodium chloride is the dominant road salt (Godwin et al. 2003; Kotalic et al. 2017). Usually, the rock salt halite, which is the mineral form of sodium chloride, or multicomponent deicer mixtures with NaCl as the main component are used as deicers. For example, one of the most common deicing agents used across North America is ClearLane™ (Cargill Incorporated, Minneapolis, MN, USA). Similar to the Bionord deicer widely used in Russia, ClearLane mostly consists of NaCl (91–96%) (Schuler et al. 2017). We demonstrate that the electrical conductivity of deicer solutes linearly depends on the amount of the dissolved deicer. Thus, we can use the electrical conductivity and calculated salinity as a proxy of deicer content in our further discussions.
Acute and chronic toxicity tests with animals
As we demonstrated before, the deicer used in our study mostly consists of chloride salts. We observed close correspondence between threshold concentrations of the deicer that induce negative effects on survival and life history parameters of animals in acute and chronic toxicity tests (5–6 g L−1) and the value of critical salinity (5–8%) for freshwater aquatic organisms (Khlebovich 1974). To compare our data with other studies, we used electrical conductivity. Electrical conductivity is an integrated indicator that estimates the amount of cations and anions dissolved in water and indicates the conditions to which the osmotic regulation system of freshwater organisms must adapt.
When we compared the data on the effect of sodium chloride on the survival of Daphnia magna with our data, we observed comparable values of the electrical conductivity of solutions. For example, the LC50 value for NaCl in acute 48-hour tests with D. magna was equal to 5.5–6.6 g L−1 with the corresponding conductivity equal to 9.8–10.0 mScm−1 (Schuytema et al. 1997, Martínez-Jerónimo et al. 2007). In our study, an LC50 value for the deicer was equal to 5.1 g L−1 in an acute 48-hour test, which corresponds to the conductivity of 8.2 mScm−1. Similarly, Sarma et al. (2006) evaluated the effect of sodium chloride concentrations on the population dynamics of several Cladocera species including Moina macrocopa and observed that growth rate was negatively affected when salt content was above the threshold concentrations of 3.0–4.5 g L−1. Thus, it can be assumed that the negative effect of the solutes of the deicer in our experiments is primarily associated with the effect of the chloride salts and can be estimated by the value of the electrical conductivity of the solution and by the recalculated salinity.
Chronic tests with resting eggs
The major results of our tests with resting eggs can be summarized as follows: (1) the hatching success of resting eggs is decreased by the long-term exposure to sediments contaminated with the road salt; (2) the negative effect of contaminated sediments on hatching success of resting eggs is less pronounced than the negative effect of the salinity in the top water layer; and (3) the negative effect of the top saline water layer on the mortality of hatchlings is observed at much lower salinities than the negative effect of the contaminated sediments on the hatching success of resting eggs.
As very few studies are available on the effect of road salts on resting eggs of cladocerans, we compared our data with similar studies on the effect of salinity on hatching success of resting eggs. Bailey et al. (2004) demonstrated that hatching rate of diapausing eggs of Bosmina liederi, Daphnia longiremis and Brachionus calyciflorus isolated from ballast sediments from transoceanic vessels was suppressed under the effect of 16.0 and 32.0‰ salinities. However, the eggs were hatched after they were transferred to 0‰ medium. Similarly, in experiments with resting eggs of 13 rotifers and three cladocerans, lower hatching rates were observed in the 16.0 and 32.0 g L−1 treatments when compared to the 0.0, 4.0, 8.0 treatments (Santangelo et al. 2014). Some of the salinity-exposed unhatched resting eggs were able to hatch when transferred to freshwater, but the reinstitution of freshwater allowed the hatching of a small proportion of eggs (~9%).
Similar to these observations, our results demonstrated that resting eggs exposed to saline sediments are able to hatch when transferred to freshwater. The proportion of hatched eggs was reduced only after exposure to sediments with the high salinity (12 g kg−1). At the same time, in experimental systems with the top freshwater layer and contaminated sediment, we observed hatching success comparable to the control. The hatching success was significantly reduced only when the salinity of the top water layer was above threshold concentration for survival of animals. Moreover, we observed elevated mortality of hatchlings at the concentrations of the deicer in the top water layer above 1 g L−1. The hatching success was still high when salinity of the top water layer and salt content in the sediment were above the concentration of the deicer critical for the survival of animals, which resulted in 100% mortality of hatchlings. Thus, the effect of water salinity on hatching success and mortality of hatchlings is more important for the recovery of zooplankton from the egg bank than the effect of the salt content in the contaminated sediment on the hatchability of resting eggs.
We created our experimental systems to mimic different scenarios occurring in nature. With these systems, we simulated scenarios where contaminated sediment is flooded with freshwater, polluted water floods the pristine sediment or where both water and sediment are contaminated with the road salt. For systems with contaminated sediment covered with freshwater or with pristine sediment covered with the solute of the deicer, the salt content in the sediment and water should reach equilibrium after some time. The comparison of water-sediment systems and dry sediment systems demonstrated that this equilibrium was reached relatively fast, during ca. 20 days. With this experimental setup, we established the gradient of salinity concentrations in both the top water layer and bottom sediment. Despite similarity in salinities of the top water layer in the test with water-sediment systems and test with dry sediment systems, the hatching success in dry sediment systems was consistently lower in all treatments and the control.
In natural habitats, the fraction of resting eggs hatched from the egg bank can vary from 5 to 60% (Caceres and Tessier 2003). In general, the number of hatchlings from resting eggs is negligible compared to the size of the egg banks in a lake. A number of external factors can cause low hatching success of resting eggs in natural habitats, including deposition of eggs deep into the sediments (Caceres 1998, Hairston et al. 2000). When we added water to dry sediment systems after 6-month exposure, resting eggs were probably buried deep into the sediments. This could be the reason for the lower hatching success compared with the water-sediment systems. However, at high salinity, the negative effect of salt was still manifested. In our case, even for the systems where we observed the negative effect of sediment disturbance on the hatching success, the salinity was the master factor when it reached the high level, as almost none of resting eggs recovered. Obviously, to hatch under favorable light and temperature, the resting egg should be near the surface, but saline water would be an additional negative factor reducing the re-establishment of the population from the egg bank.
Tests with water-sediment systems and dry sediment systems demonstrated that the effect of saline water on the hatching success of resting eggs and survival of hatchlings could be critical. Adverse effects on the survival of hatchlings were observed at salinity that corresponded to LC50 for survival and lifespan of active animals in acute and chronic toxicity tests. We can predict that temporary water bodies or small water bodies can be contaminated with road salts relatively quickly. Field observations demonstrate that during the spring runoff, the concentrations of salt ions, mostly Na and Cl (Zhang et al. 2013), in samples taken from streams near the city, where the road salt was used, can reach 10 g L−1 (Corsi et al. 2010), which is above critical salinity (Khlebovich 1974).
In large lakes, the road salt that enters the waterbody with runoff will be dissolved in a large volume of water, which may reduce its threat to zooplankton community. However, the long-term consequences of salinization of surface water and groundwater should be taken into consideration. The tendency of the gradual accumulation of chlorides in aquatic ecosystems located near the territories where salt-containing deicers have been used for a long time has been confirmed by numerous studies (Godwin et al. 2003; Meter et al. 2011; Schuler et al. 2017; Ramakrishna and Viraraghavan 2005; Howard and Maier 2007; Thunqvist 2004). Also, several studies demonstrated the negative effect of road salts on the structure of food webs (Meter et al. 2011; Schuler et al. 2017), predator–prey relationships (Hintz and Relyea, 2017), and mortality and decreased fertility of Daphnia (Arnott et al. 2020), even at a relatively low level of salt pollution. Even though several recent studies demonstrated that freshwater Daphnia could rapidly adapt to elevated salinity (Coldsnow et al., 2017; Hintz et al., 2019), up to 2 g L−1 of chlorine, this salinity is still below the values of critical salinity.
In the case of dry salt-contaminated sediments with the egg bank, the re-establishment of the zooplankton can be expected following the freshwater runoff. Our test demonstrated that the inhibition of hatching by contaminated sediment was reduced when we added freshwater to the system. On the other hand, the success of the hatchlings will depend on the resulting salinity of the water. Thus, to predict consequences of salinization for a specific ecosystem, the total amount of salt in water and in sediments should be estimated to calculate resulting equilibrium water salinity.
Salinity fluctuations can strongly influence the population density of planktonic organisms in freshwater lakes. Our results and published data (Neilson et al. 2012; Santangello et al. 2014) show the potential of the resting egg bank for the resilience of zooplankton populations to increased salinity, assuming a subsequent decrease in salinity. This suggestion is supported by the field data. Gimatullina et al. (2017) examined the effect of drought-induced salinity changes on aquatic communities and demonstrated that the species richness of Rotifera, Cladocera, and Copepoda was inversely correlated with salinity. Authors observed post-drought recovery of species richness due to replenishment of active zooplankton populations from the egg bank. Nevertheless, the resilience of the community to the drought-induced salinity increase was observed for a relatively short time (1–2 years). Longer salinity anomalies can affect the survival of resting eggs in the bottom sediments and reduce the abundance of some species even when the salinity decreases (Gimatullina et al. 2017). Santangelo et al. (2014) also argued that the resistance of resting eggs to the prolonged exposure to saline sediments was still poorly understood.
Increased salinity can not only affect the viability of resting eggs in an egg bank, but also decrease the amount of resting eggs produced by active animals (Hintz and Relyea, 2017). Seasonal timing of the effect of high salinity may be an important factor. Spring, summer or autumn disturbances due to high salinity can cause mortality of active population, which results in lower investment into the egg bank (Gimatullina et al. 2017). Ineffective re-establishment of the egg bank can reduce its size and diversity, which in turn will affect the re-establishment of the community.
Both the timing of the production of resting eggs with unfavorable environmental conditions and the timing of hatching of resting eggs with favorable conditions are critically important for the ecological success of animals with the diapause in the life cycle (Galimov and Yampolsky 1998). Thus, to be effective, resting eggs must be able to delay hatching and to survive being exposed to salt-contaminated sediments (Santangelo et al. 2014). Given the growing threat of salinization of surface water and groundwater due to application of deicing agents (Schuler et al. 2017), salinity tolerance of freshwater zooplankton needs to be closely studied. Many studies have been focused on salinity tolerance of active zooplankters (Sarma et al. 2006), but the tolerance of resting eggs should be also taken into account (Bailey et al. 2004) in order to predict long-term consequences of freshwater salinization due to the widespread use of road salt and other salt-containing contaminants.
Data, associated metadata, and calculation tools are available from the corresponding author (firstname.lastname@example.org).
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The reported study was funded by the Russian Foundation for Basic Research, the Krasnoyarsk Krai Government, and the Krasnoyarsk Regional Fund for supporting scientific and technical activities, project number 19-44-240014. We are grateful to two anonymous reviewers for the valuable comments and suggestions and to Elena Krasova for linguistic check and improvements.
The reported study was funded by Russian Foundation for Basic Research, the Krasnoyarsk Krai Government, and the Krasnoyarsk Regional Fund for supporting scientific and technical activities, project number 19-44-240014.
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Lopatina, T., Anishchenko, O., Oskina, N. et al. Threshold concentrations of the road salt for adverse effects on females and resting eggs of cladoceran Moina macrocopa. Aquat Ecol 55, 283–297 (2021). https://doi.org/10.1007/s10452-021-09830-z
- Resting eggs
- Road salt
- Aquatic ecosystems