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Evaluation of the effect of water type on the toxicity of nitrate to aquatic organisms
Chemosphere 168 (2017) 435e440
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Evaluation of the effect of water type on the toxicity of nitrate to aquatic organisms Josh A. Baker a, *, Guy Gilron b, Ben A. Chalmers c, James R. Elphick a a
Nautilus Environmental, 8664 Commerce Court, Burnaby, BC, V5A 4N7, Canada Borealis Environmental Consulting, 148 East 25th Street, North Vancouver, BC, V7N 1A1, Canada c The Mining Association of Canada, 350 Sparks Street, Suite 1105, Ottawa, ON, K1R 7S8, Canada b
h i g h l i g h t s Acute and chronic NO3 toxicity to aquatic organisms assessed. Hyalella azteca and Ceriodaphnia dubia most sensitive to effects of nitrate. Strong influence of ionic strength on nitrate toxicity. 2e10-fold reductions in chronic NO3 toxicity as water changed from soft to hard.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 August 2016 Received in revised form 29 September 2016 Accepted 16 October 2016
A suite of acute and chronic toxicity tests were conducted to evaluate the sensitivity of freshwater organisms to nitrate (as sodium nitrate). Acute exposures with rainbow trout (Onchorhynchus mykiss) and amphipods (Hyalella azteca), as well as chronic exposures with H. azteca (14-d survival and growth), midges (Chironomus dilutus; 10-d survival and growth), daphnids (Ceriodaphnia dubia; 7-d survival and reproduction), and fathead minnows (Pimephales promelas; 7-d survival and growth) were used to determine sublethal and lethal effect concentrations. Modification of nitrate toxicity was investigated across a range of ionic strengths, created through the use of very soft water, and standard preparations of synthetic soft, moderately-hard and hard dilution waters. The most sensitive species tested were C. dubia and H. azteca, in soft water, with reproduction and growth IC25 values of 13.8 and 12.2 mg/L NO3-N, respectively. All of the organisms exposed to nitrate demonstrated significantly reduced effects with increasing ionic strength associated with changes in water type. Possible mechanisms responsible for the modifying effect of increasing major ion concentrations on nitrate toxicity are discussed. © 2016 Elsevier Ltd. All rights reserved.
Handling Editor: Jim Lazorchak Keywords: Nitrate Toxicity Hardness Modifying factor Anions
1. Introduction Nitrogen is an essential element for all organisms, being a major component of amino acids, nucleic acids, and other biological materials. In aquatic environments, biologically-available nitrogen occurs primarily in the chemical forms of ammonia, nitrite and nitrate, with the relative concentrations of these materials determined by biological and chemical processes associated with the nitrogen cycle. Nitrate is the most common aqueous form of nitrogen, and is produced primarily from the oxidation of plant and animal debris (Camargo and Alonso, 2006). Anthropogenic sources
* Corresponding author. E-mail address: [email protected] (J.A. Baker). http://dx.doi.org/10.1016/j.chemosphere.2016.10.059 0045-6535/© 2016 Elsevier Ltd. All rights reserved.
of nitrogen have increased concentrations of nitrate in many aquatic environments (Puckett, 1995), exceeding 25 and 100 mg/L NO3-N in contaminated surface and ground waters, respectively (Camargo and Alonso, 2006). Mining activities often discharge nitrate into receiving environments, primarily as a result of use of nitrogen-containing blasting agents, such as ammonium nitrate/ fuel oil or AN/FO (Camargo et al., 2005; Zaitsev et al., 2008). Significant research has been conducted on the toxicity of ammonia and nitrite, however, less information is available regarding the concentrations of nitrate which cause adverse effects to aquatic organisms. Direct nitrate toxicity has previously been considered to be negligible, and concern was primarily associated with the potential for nitrate to stimulate eutrophication (Camargo and Alonso, 2006). However, studies have demonstrated that environmentally-relevant concentrations of nitrate can lead to
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direct toxicity to aquatic organisms (Camargo et al., 2005). Inhibition of the oxygen-carrying capacity of hemoglobin has been implicated as the cause of reduced performance of aquatic organisms in the presence of elevated concentrations of nitrate (Grabda et al., 1974). This toxic action is similar to that of nitrite (Lewis Jr and Morris, 1986), likely due to reduction of nitrate to nitrite in the blood (Guillette and Edwards, 2005) resulting in a similar metabolic pathway involving production of nitric oxide (Hannas et al., 2010). Effects on osmoregulation resulting from nitrate exposures have also been identified (Gulyassy et al., 1962; Hrubec et al., 1997). Research related to the toxicity of ammonia and nitrite has identified key toxicity-modifying factors for these compounds (i.e., pH and chloride, respectively); these factors have been incorporated into water quality benchmarks for these constituents (British Columbia Ministry of Environment, 2009; Canadian Council of Ministers of the Environment, 2010; U.S. Environmental Protection Agency, 2013). Due to a limited focus on evaluating toxicity of nitrate, regulatory agencies have developed guidelines based on limited datasets (British Columbia Ministry of Environment, 2009; Canadian Council of Ministers of the Environment, 2012; Environment Australia, 2000) and the role of toxicity-modifying factors for nitrate has not been comprehensively evaluated, although the effect of chloride on toxicity of nitrate to amphipods has been reported (Soucek and Dickinson, 2016). The aquatic toxicity of sulphate and chloride has been reported to be reduced with increasing water hardness (Davies and Hall, 2007; Elphick et al., 2011a, 2011b; Lasier and Hardin, 2010; Soucek and Kennedy, 2005), although the mechanism associated with this effect has not yet been clearly defined. The effect of water hardness on the toxicity of nitrate has not previously been evaluated, although the potential for this interaction has been hypothesized (Camargo et al., 2005; Scott and Crunkilton, 2000). This study was designed to expand upon the available data on the toxicity of nitrate using additional freshwater species, while
exploring the relationship between nitrate toxicity and the ionic characteristics of water (e.g., hardness). The relationships between nitrate and water quality characteristics are potentially significant in understanding mechanisms of nitrate toxicity and in establishing whether toxicity modifying factors should be incorporated into water quality benchmarks for nitrate. 2. Methods Toxicity tests were conducted at Nautilus Environmental (Burnaby, BC, Canada) in walk-in environmental chambers with temperature (±1 C) and photoperiod (16:8 light:dark) control. Water quality parameters, including dissolved oxygen, pH and temperature, were recorded daily throughout the exposures. Test waters were prepared by addition of reagent-grade salts to achieve the target water hardness types, with the exception of very soft water (i.e., 10e15 mg/L as CaCO3), which was dechlorinated Metro Vancouver municipal tap water. Salt additions followed the ratios of salts specified by USEPA (U.S. Environmental Protection Agency, 2002), with the exception of tests using Hyalella azteca and Chironomus dilutus, which employed a recipe containing a higher concentration of chloride, as described by Environment Canada (Environment Canada, 1997a). Tests using rainbow trout (Onchorhynchus mykiss) and fathead minnows (Pimephales promelas) were conducted at four hardnesses: very soft water (VSW, 10e15 mg/L as CaCO3); soft water (SW, 40e55 mg/L as CaCO3); moderately-hard water (MHW, 80e100 mg/L as CaCO3); and hard water (HW, 160e180 mg/L as CaCO3). The invertebrate species (i.e., Ceriodaphnia dubia, C. dilutus and H. azteca) were tested at the three higher hardnesses (Table 2). These water types are commonly used in evaluations of water hardness as a toxicity modifying factor; however, in addition to ions contributing to hardness (i.e., Ca and Mg) other major ions (i.e., Na, K, HCO3, Cl and SO4) also co-vary in these water types (Table 1). Hardness and alkalinity were measured on waters at test initiation using titration techniques.
Table 1 Water chemistry for dilution waters used in nitrate exposures. Nominal concentrations based on salt additions (mg/L). Constituent (mg/L)
Na Mg Ca K HCO3 Cl SO4 pH Hardness (as CaCO3)
P. promelas/O. mykiss
C. dubia
H. azteca/C. dilutus
VSW
SW
MHW
HW
SW
MHW
HW
SW
MHW
HW
1.8 0.1 3.9 0.2 11.0 2.0 2.7 6.9e7.3 10e15
15.0 6.2 10.9 1.2 45.9 2.9 43.4 7.3e7.7 40e55
28.1 12.3 17.9 2.3 80.8 3.9 84.0 7.7e8.0 80e100
54.4 24.4 31.8 4.4 150.5 5.8 165.4 7.9e8.3 160e180
13.1 6.1 7.0 1.0 34.9 1.0 40.7 7.6e7.9 40e55
26.3 12.7 14.7 2.1 69.7 1.9 85.4 8.1 80e100
52.5 24.2 27.9 4.2 139.5 3.8 162.7 8.2e8.4 160e180
15.0 3.2 18.7 1.2 45.9 18.8 28.6 7.4e7.7 40e55
28.1 6.2 33.5 2.3 80.8 36.0 54.5 7.7e8.0 80e100
54.4 12.3 63.2 4.4 150.5 69.4 106.4 8.0e8.2 160e180
Very soft water: 10e15 mg/L as CaCO3; soft water: 40e55 mg/L as CaCO3; moderately-hard water: 80e100 mg/L as CaCO3; and, hard water: 160e180 mg/L as CaCO3.
Table 2 Summary of toxicity test endpoints and test concentrations. Test species
Test duration
Test endpoint(s)
Nominal concentrations tested (mg/L NO3-N)
O. mykiss H. azteca
96-hr 96-hr 14-day 10-day 7 ± 1 day 7-day
Survival Survival Survival, Survival, Survival, Survival,
312, 625, 1250, 2500, 5000 103, 206, 412, 824, 1647 10a, 20, 40, 80, 160, 320, 640b 10a, 20, 40, 80, 160, 320, 640b 5c, 10, 20, 40, 80, 160, 320, 640d 50, 100, 200, 400, 800, 1600
C. dilutus C. dubia P. promelas a b c d
Tested Tested Tested Tested
in in in in
soft water only. moderately hard and hard water only. moderately hard water. soft water and hard water.
growth growth reproduction growth
J.A. Baker et al. / Chemosphere 168 (2017) 435e440
Nitrate was added using reagent-grade sodium nitrate; concentrations used in each test are shown in Table 2. Nitrate was measured in all test concentrations at initiation of the tests. In chronic toxicity tests, nitrate was also measured in a subset of concentrations bracketing the effect level at test termination. Nitrate was measured using ion chromatography (U.S. Environmental Protection Agency, 1993) with detection by UV absorbance by ALS Environmental (Burnaby, BC, Canada). 2.1. Rainbow trout Acute toxicity tests using rainbow trout (O. mykiss) were conducted according to the Environment Canada test method for this species (Environment Canada, 2000). Exposures were conducted in 20-L glass aquaria containing 10 L of test solution and ten fry ranging from 0.3 to 0.6 g. Fish were obtained from the Fraser Valley Trout Hatchery (Abbotsford, BC, Canada) and were acclimated in the laboratory for at least two weeks prior to testing. Tests were conducted at 15 ± 1 C and the test solutions were aerated continuously at a rate of 65 ± 10 mL per minute throughout the exposure period. The solutions were not renewed and the test organisms were not fed during the exposure. Survival of the test organisms were recorded daily throughout the tests; control performance of 90% survival was required for the test to be considered acceptable. 2.2. Amphiphods Acute (96-h) and chronic (14-d) toxicity tests using H. azteca were adapted from methods published by Environment Canada (Environment Canada, 1997a); the tests were initiated using 6e8 day old amphipods obtained from Aquatic Biosystems (Fort Collins, CO). The acute exposures were conducted using triplicate 200 mL volumes in glass jars with no solution renewal, and feeding using digested yeast, Cerophyll and trout chow (YCT; 1.5 mL in each replicate) at test initiation and after 48 h of exposure. Chronic toxicity tests were conducted using clean sediment comprised of beach-collected sand, supplemented with peat at a rate of 2% by weight. Test methods were modified from Environment Canada procedures for sediment toxicity tests (Environment Canada, 1997a) by incorporating test solution renewal three times per week throughout exposure with freshly-prepared test solutions, at which time YCT (1.5 mL per replicate) was added as food. These tests were conducted using four replicates per concentration in glass jars containing 100 mL of control sediment and filled to 275 mL with the test solutions. The exposures were conducted at 23 ± 1 C and the overlying water aerated gently throughout exposure. Surviving amphipods were dried at the end of the test on pre-weighed aluminum pans and dry weight was determined. Both acute and chronic exposures were conducted using 10 test organisms per replicate. Control survival of 90 and 80% was required for the acute and chronic exposures, respectively, to meet test acceptability criteria. In addition, the chronic test required that control amphipods achieve at least 0.1 mg dry weight at test termination. 2.3. Chironomids Chronic toxicity tests using C. dilutus were conducted in a similar manner to that described for H. azteca, with the exception that the test duration was 10 rather than 14 days. The tests were initiated with third instar larval midges obtained from Aquatic Biosystems, and the exposure chambers were fed with 6 mg of Tetramin each day. These methods were adapted from Environment Canada test methods for evaluating sediment samples using
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this species (Environment Canada, 1997b). The tests were considered acceptable if the control survival was 70% and dry weight of the surviving larvae was 0.6 mg. 2.4. Daphnids Chronic toxicity tests using C. dubia were conducted according to the Environment Canada test method for this species (Environment Canada, 2007) in 15-mL volumes in 18-mL glass test tubes. Exposures of each concentration comprised 10 replicates, each initiated with a single <24-hr old daphnid obtained from inhouse cultures. The organisms were cultured at the three test water hardnesses for at least two generations prior to test initiation. Exposure waters were renewed with freshly-prepared solutions daily, at which time they were fed a mixture of green algal cells (Pseudokirchneriella subcapitata) and YCT. Exposures were conducted at 25 ± 1 C and survival and reproduction were recorded daily. Exposures were terminated after 7 ± 1 days, when at least 60% of the control organisms had produced their third brood. The tests were considered acceptable if the control organisms had 80% survival and produced an average of 15 neonates in three broods during the test. 2.5. Fathead minnows Fathead minnow tests were conducted according to test methods for this species described by Environment Canada (Environment Canada, 1992), involving a 7-d exposure, initiated with <24-hr post-hatch fish, which were obtained from Aquatic Biosystems. Tests were conducted using three replicates with ten fish in 300 mL glass jars containing 250 mL of solution. The tests were conducted at 25 ± 1 C. Fish were fed with brine shrimp (Artemia salina) and solutions were renewed daily throughout the exposure period. At the end of the test, surviving fish were dried on pre-weighed aluminum pans and weighed. Endpoints from the test were survival and biomass, and the tests were considered acceptable if 80% survival was observed in control exposures. 2.6. Quality assurance/quality control Reference toxicant tests, involving exposure of the organisms to a range of concentrations of a known toxicant, were conducted using all species evaluated in this study, and the results of the tests were compared with a control chart of toxicity test data produced previously in the laboratory. Reference toxicants used were as follows: sodium dodecyl sulphate (SDS) for rainbow trout, potassium chloride for C. dilutus, and sodium chloride for the remaining three species. The duration of the reference toxicant tests were the same as the individual tests, with the exception of the 14 and 10-d tests using H. azteca and C. dilutus, which utilized a 96-hr static exposure for the reference toxicant tests, as recommended by Environment Canada (Environment Canada, 1997a, 1997b). 2.7. Statistical analyses Statistical analyses were conducted using CETIS (Tidepool Software; McKinleyville, CA), according to procedures described by Environment Canada (Environment Canada, 2005) for calculating point estimates. Survival data were analyzed using probit or logit multiple linear estimation, where possible, and quantitative data for reproduction and growth were analyzed using non-linear regression in cases where model assumptions were met. Linear interpolation of log-transformed data was used in cases where the assumptions of the models described above were not met. Simple regression analyses were conducted using Microsoft Excel to
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determine the effect of water chemistry on nitrate endpoints. 3. Results The results of all toxicity tests met requirements specified in the test methods for control performance and water quality parameters remained within the ranges specified in the corresponding test methods. The reference toxicant test results fell within the range of acceptable results (i.e., mean ± 2 standard deviations) from historical tests with these species, indicating that the organisms were of acceptable sensitivity. Measured concentrations of nitrate were in agreement with target concentrations of nitrate; analyzed values were an average of 99 ± 7% of the nominal concentrations across all of the tests. Measured concentrations were used in determining effect concentrations. Test data for each test concentration are provided in the Supplementary data. Acute toxicity testing with juvenile rainbow trout resulted in median lethal concentrations (i.e., 96-hr LC50) that ranged from 808 mg/L NO3-N in very soft water to 1913 mg/L NO3-N in hard water. Testing with 6e8 day old H. azteca produced 96-hr LC50 values that increased from 168 to 485 and 921 mg/L NO3-N in soft, moderately-hard and very hard water, respectively (Table 3). A clear reduction in toxicity with increase in water hardness was apparent in tests using both species (Fig. 1). Lethal (LC50) and sublethal (IC25; concentration resulting in a 25% reduction) effect concentrations from chronic tests conducted with H. azteca, C. dilutus, C. dubia and P. promelas are summarized in Table 4. A similar trend to that observed with the acute tests was observed with each of these species, with lower nitrate toxicity associated with organisms exposed in waters with higher concentrations of major ions.
Table 3 Results from 96-hr acute toxicity tests with nitrate (mg/L NO3-N). Test species
Hardness (mg/L CaCO3)
LC50 (95% Confidence limits)
O. mykiss
11 54 90 164 44 100 164
808 (639e1023) 1446 (1192e1754) 1958 (1360e2820) 1913 (1340e2730) 168 (143e197) 485 (435e540) 921 (808e1049)
4. Discussion Data presented in this study add to the current knowledge on the sensitivity of aquatic organisms to nitrate while also highlighting the important role of water chemistry on toxicity. The results from this study show a relationship between the sensitivity to nitrate and the ionic composition of the test solution. Strong correlations were observed between both acute LC50 and chronic IC25 endpoints and water hardness [(i.e., correlation coefficients: R2 ranging from 0.85 to 1.0 (Fig. 1),]. However, other major ions covaried with hardness in the water types used and, therefore, ions other than calcium and magnesium could have contributed to the observed effect on toxicity of nitrate. The sensitivity of rainbow trout observed in this study was similar to a 96-hr LC50 of 1355 mg/L NO3-N for O. mykiss fingerlings reported by Westin (1974), and was within the range of 1010e1975 mg/L NO3-N summarized by Camargo et al. (2005) in a review of nitrate toxicity to freshwater fish. Previous studies have not investigated modifying factors of acute nitrate toxicity to fish, and further work is warranted to identify the mechanism responsible for the effect of water chemistry on toxicity observed during this study. The sensitivity observed in acute exposures to H. azteca in the current study are consistent with those reported by Soucek and Dickinson (2012), Soucek et al. (2015) and the USEPA (2010). Results for this species from each of these studies are shown in Fig. 2 on the basis of chloride concentration in the test waters; chloride has been identified as a modifying factor for acute nitrate toxicity to H. azteca (Soucek and Dickinson, 2016). A significant relationship was observed between LC50 values and chloride concentration (p < 0.01) suggesting that chloride may explain some or all of the effect of water type observed in this study with this species.
10000 IC25 (mg/L NO3-N)
10000 LC50 (mg/L NO3-N)
H. azteca
C. dubia was the most sensitive of the tested species across water types, with reproduction IC25 values ranging from 13.8 to 47.5 mg/L NO3-N, as the water hardness increased. Sensitivities of the chironomid, C. dilutus, and the amphipod, H. azteca, were in a similar range, as growth IC25 values increased from 48.8 to 178 mg/ L NO3-N for C. dilutus and from 12.2 to 181 mg/L NO3-N for H. azteca, across the soft to hard water types. Fathead minnows were the least sensitive of the four species tested for chronic toxicity, with IC25 values ranging from 69.6 to 402 mg/L NO3-N in dilution waters that ranged from very soft to hard conditions.
1000 100 10 1
1000 100 10 1
1
10 100 1000 Water hardness (mg/L, as CaCO3)
Amphipod (96 hr) R² = 1
Rainbow trout (96 hr) R² = 0.95
1
10 100 1000 Water hardness (mg/L, as CaCO3) Amphipod growth (14 d) Chironomid growth (10 d) R² = 0.85 R² = 0.99 Cladoceran repr. (7d) Fathead growth (7 d) R² = 0.98 R² = 0.95
Fig. 1. Acute and chronic effects of nitrate shown as a function of water hardness.
J.A. Baker et al. / Chemosphere 168 (2017) 435e440 Table 4 Results from chronic nitrate toxicity tests using C. dubia, C. dilutus, H. azteca and P. promelas. Test Species
Hardness (as mg/L CaCO3)
Endpoint
H. azteca
46 86 172 46 86 172 46 86 172 46 86 172 44 98 166 44 98 166 12 50 94 168 12 50 94 168
Survival
C. dilutus
C. dubia
P. promelas
mg/L NO3-N (95% Confidence limits)
Growth
Survival
Growth
Survival
Reproduction
Survival
Growth
14-d LC50 14-d LC50 14-d LC50 14-d IC25 14-d IC25 14-d IC25 10-d LC50 10-d LC50 10-d LC50 10-d IC25 10-d IC25 10-d IC25 7-d LC50 7-d LC50 7-d LC50 7-d IC25 7-d IC25 7-d IC25 7-d LC50 7-d LC50 7-d LC50 7-d LC50 7-d IC25 7-d IC25 7-d IC25 7-d IC25
124 (106e145) 275 (243e312) >622 12.2 (0.6e59.0) 116 (47.4e183) 181 (102e274) 114 (96.3e134) 222 (191e257) 342 (301e387) 48.8 (30.8e95.5) 102 (89.4e122) 178 (153e191) 62.0 (37.0e83.0) 120 (110e130) 127 (102e160) 13.8 (4.2e24.5) 23.5 (8.7e31.1) 47.5 (26.5e56.2) 117 (103 - 132) 235 (200e276) 415 (358e481) 465 (398e543) 69.6 (44.4e86.4) 209 (NCe248) 358 (158e567) 402 (193e477)
NC: Not calculable.
Contrary to the results for H. azteca, Soucek and Dickinson (2016) reported that there was no interaction between chloride and toxicity of nitrate in chronic toxicity tests using C. dubia conducted at a hardness of 90 mg/L; EC20 estimates ranged from 80 to 263 mg/L NO3-N demonstrating large variability but no dependence on chloride concentration. Scott and Crunkilton (2000) also reported results from chronic toxicity tests using C. dubia; point estimates were not presented for reproduction, but a MATC value of 30.1 mg/L NO3-N was determined in a hardness range of 150e184 mg/L. The reproduction effect concentrations reported here were similar to those reported by Scott and Crunkilton (2000), but somewhat more sensitive than those reported by Soucek and Dickinson (2016). The water formulation used in Soucek and Dickinson (2016) was lower in sulfate, higher in chloride, and differed in terms of the ratio of calcium to magnesium, in comparison to the waters used in this study (Table 1). None of the major
439
ions were significantly correlated with 7-d C. dubia nitrate IC50 values when the data for the two studies were compared as a whole (p > 0.05). It has been reported that acute toxicity of sulphate is reduced to a greater extent in waters with a higher calcium to magnesium ratio (Davies and Hall, 2007), and it is possible that the lower sensitivity observed by Soucek and Dickinson (2016) could relate to a similar effect. While acute nitrate toxicity data for some benthic organisms are available (Camargo et al., 2005; Pandey et al., 2011; Soucek and Dickinson, 2012), relatively few chronic toxicity data are available. The chronic toxicity endpoints determined here for H. azteca (with EC50s ranging from 50.6 to 181.0 mg/L NO3) were slightly less sensitive than those reported by Soucek and Dickinson (2016) for this species (with EC50s ranging from 32 to 86 mg/L NO3); however, this may be explained by the longer test duration used in their study (42-d). Chronic effects of nitrate on C. dilutus have not previously been reported in the literature. The results presented here indicate that sensitivity of C. dilutus to nitrate is comparable to that of H. azteca; C. dilutus and H. azteca sublethal endpoints were similar in MHW and HW, although H. azteca was four-fold more sensitive to nitrate than C. dilutus in SW. The fathead minnow was the least sensitive species tested using chronic exposures to nitrate; this finding is consistent with previous work by other researchers. Nitrate exposures at a hardness of 156e172 mg/L resulted in a MATC for growth of 506.6 mg/L NO3-N (Scott and Crunkilton, 2000) which is comparable to the IC25 of 402 mg/L NO3-N reported here for the hard water exposure. We have presented compelling evidence that ionic composition influences the toxicity of nitrate. Effects of water type on the toxicity of nitrate could be the result of competitive exclusion at uptake sites by other anions (i.e., chloride, bicarbonate, sulfate), as has been observed by the role of chloride on H. azteca sensitivity to nitrate (Soucek and Dickinson, 2016), or may result from effects of cations such as calcium on membrane permeability. Further investigation into the mechanism(s) behind the effect of water type will help to further refine the role of individual water constituents in modifying nitrate toxicity. In general, with respect to nitrate sensitivity, marine organisms appear to be less sensitive than freshwater organisms (Camargo et al., 2005; Tsai and Chen, 2002) possibly highlighting the importance of physiological aspects of ionoregulation in the toxicity of nitrate. Adverse effects were observed in this study at nitrate concentrations that can be found in polluted surface waters (Camargo et al., 2005). However, sensitivity of all of the organisms tested was reduced with increasing ionic strength of the test waters. Thus,
10000 96-h LC50 (mg/L NO3-N)
US EPA (2010) 1000
Present study R² = 0.91
100
Soucek and Dickinson (2012)
10
Soucek and Dickinson (2016)
1 1
10
100
Chloride in test water (mg/L) Fig. 2. Relationship between the LC50 of nitrate for Hyalella azteca and chloride concentration in the test water.
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water quality guidelines that are based on toxicity tests conducted in very soft water may overestimate the risk of adverse effects in conditions where the ionic strength of the water is higher. Acknowledgements The authors would like to acknowledge member companies of the Mining Association of British Columbia for providing funding for this study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2016.10.059. References British Columbia Ministry of Environment, 2009. Water Quality Guidelines for Nitrogen (Nitrate, Nitrite, and Ammonia). Overview Report Update 29. Camargo, J.A., Alonso, A., 2006. Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: a global assessment. Environ. Int. 32, 831e849. http://dx.doi.org/10.1016/j.envint.2006.05.002. Camargo, J.A., Alonso, A., Salamanca, A., 2005. Nitrate toxicity to aquatic animals: a review with new data for freshwater invertebrates. Chemosphere 58, 1255e1267. http://dx.doi.org/10.1016/j.chemosphere.2004.10.044. Canadian Council of Ministers of the Environment, 2012. Canadian Water Quality Guidelines: Nitrate Ion. Scientific Criteria Document. Canadian Council of Ministers of the Environment, 2010. Canadian Water Quality Guidelines for the Protection of Aquatic Life: Ammonia. Davies, T.D., Hall, K.J., 2007. Importance of calcium in modifying the acute toxicity of sodium sulphate to Hyalella azteca and Daphnia magna. Environ. Toxicol. Chem. 26, 1243e1247. http://dx.doi.org/10.1897/06-510R.1. Elphick, J.R., Bergh, K.D., Bailey, H.C., 2011a. Chronic toxicity of chloride to freshwater species: effects of hardness and implications for water quality guidelines. Environ. Toxicol. Chem. 30, 239e246. http://dx.doi.org/10.1002/etc.365. Elphick, J.R., Davies, M., Gilron, G., Canaria, E.C., Lo, B., Bailey, H.C., 2011b. An aquatic toxicological evaluation of sulfate: the case for considering hardness as a modifying factor in setting water quality guidelines. Environ. Toxicol. Chem. 30, 247e253. http://dx.doi.org/10.1002/etc.363. Environment Australia, 2000. Australian and New Zealand Guidelines for Fresh and Marine Water Quality- Volume 2: Aquatic Ecosystems d Rationale and Background Information, vol. 678. National Water Quality Management Strategy. Australian and New Zealand Environment and Conservation Council. Environment Canada, 2007. Biological Test Method: Test of Reproduction and Survival Using the Cladoceran Ceriodaphnia Dubia. EPS 1/RM/32, Method Development and Application Centre, Environment Canada, Ottawa, ON. Environment Canada, 2005. Guidance Document on Statistical Methods for Environmental Toxicity Tests. EPS 1/RM/46. Method Development and Application Centre, Environment Canada, Ottawa, ON. Environment Canada, 2000. Biological Test Method: Reference Method for Determining Acute Lethality of Effluents to Rainbow Trout. EPS 1/RM/9, Environmental Technology. Method Development and Application Centre, Environment Canada, Ottawa, ON. Environment Canada, 1997a. Biological Test Method: Test for Survival and Growth in Sediment Using the Freshwater Amphipod Hyalella Azteca. EPS 1/RM/33, Method Development and Application Centre, Ottawa, ON. Environment Canada, 1997b. Biological Test Method: Test for Survival and Growth in Sediment Using the Larvae of Freshwater Midges (Chironomus tentans and Chironomus Riparius). EPS 1/RM/32, Method Development and Application Centre, Environment Canada, Ottawa, ON. Environment Canada, 1992. Biological Test Method: Test of Larval Growth and
Survival Using Fathead Minnows. EPS 1/RM/9, Method Development and Application Centre, Environment Canada, Ottawa, ON. Grabda, E., Einszporn-Orecka, T., Felinska, C., Zbanyszek, R., 1974. Experimental methemoglobinemia in rainbow trout. Acta Ichthyol. Piscat. IV, 8e10. Guillette, L.J., Edwards, T.M., 2005. Is nitrate an ecologically relevant endocrine disruptor in vertebrates? Integr. Comp. Biol. 45, 19e27. http://dx.doi.org/ 10.1093/icb/45.1.19. Gulyassy, P.F., Van Ypersele de Strihou, C., Schwartz, W.B., 1962. On the mechanism of nitrate-induced alkalosis. The possible role of selective chloride depletion in acid-base regulation. J. Clin. Investig. 41, 1850e1862. http://dx.doi.org/10.1172/ JCI104642. Hannas, B.R., Das, P.C., Li, H., Leblanc, G.A., 2010. Intracellular conversion of environmental nitrate and nitrite to nitric oxide with resulting developmental toxicity to the crustacean Daphnia magna. PLoS One 5. http://dx.doi.org/ 10.1371/journal.pone.0012453. Hrubec, T.C., Robertson, J.L., Smith, S.A., 1997. Effects of ammonia and nitrate concentration on hematologic and serum biochemical profiles of hybrid striped bass (Morone chrysops x Morone saxatilis). Am. J. Vet. Res. 58, 131e135. Lasier, P.J., Hardin, I.R., 2010. Observed and predicted reproduction of Ceriodaphnia dubia exposed to chloride, sulfate, and bicarbonate. Environ. Toxicol. Chem. 29, 347e358. http://dx.doi.org/10.1002/etc.29. Lewis Jr., W.M., Morris, D.P., 1986. Toxicity of nitrite to fish: a review. Trans. Am. Fish. Soc. 115, 183e195. Pandey, R.B., Adams, G.L., Warren, L.W., 2011. Survival and precopulatory guarding behavior of Hyalella azteca (Amphipoda) exposed to nitrate in the presence of atrazine. Environ. Toxicol. Chem. 30, 1170e1177. http://dx.doi.org/10.1002/ etc.473. Puckett, L.J., 1995. Identifying the major sources of nutrient water pollution. Environ. Sci. Technol. 29, 408Ae414A. http://dx.doi.org/10.1021/es00009a001. Scott, G., Crunkilton, R., 2000. Acute and chronic toxicity of nitrate to fathead minnows (Pimephales promelas), Ceriodaphnia dubia, and Daphnia magna. Environ. Toxicol. Chem. 19, 2918e2922. http://dx.doi.org/10.1002/etc.5620191211. Soucek, D.J., Dickinson, A., 2012. Acute toxicity of nitrate and nitrite to sensitive freshwater insects, mollusks, and a crustacean. Arch. Environ. Contam. Toxicol. 62, 233e242. http://dx.doi.org/10.1007/s00244-011-9705-8. Soucek, D.J., Dickinson, A., 2016. Influence of chloride on the chronic toxicity of sodium nitrate to Ceriodaphnia dubia and Hyalella azteca. Ecotoxicology 25, 1406e1416. http://dx.doi.org/10.1007/s10646-016-1691-1. Soucek, D.J., Kennedy, A.J., 2005. Effects of hardness, chloride, and acclimation on the acute toxicity of sulfate to freshwater invertebrates. Environ. Toxicol. Chem. 24, 1204e1210. http://dx.doi.org/10.1897/04-142.1. Soucek, D.J., Mount, D.R., Dickinson, A., Hockett, J.R., Mcewen, A.R., 2015. Contrasting effects of chloride on growth, reproduction, and toxicant sensitivity in two genetically distinct strains of Hyalella azteca. Environ. Toxicol. Chem. 34, 2354e2362. http://dx.doi.org/10.1002/etc.3070. Tsai, S.-J., Chen, J.-C., 2002. Acute toxicity of nitrate on Penaeus monodon juveniles at different salinity levels. Aquaculture 213, 163e170. http://dx.doi.org/10.1016/ S0044-8486(02)00023-6. U.S. Environmental Protection Agency, 2010. Final Report on Acute and Chronic Toxicity of Nitrate, Nitrite, Boron, Manganese, Fluoride, Chloride and Sulfate on Several Aquatic Animal Species. Illinois, Chicago. U.S. Environmental Protection Agency, 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms Fourth Edition October 2002. U.S. Environmental Protection Agency, 2013. Aquatic Life Ambient Water Quality Criteria for Ammonia e Freshwater, 1-242, EPA-822-R-13-001, Washington, DC. U.S. Environmental Protection Agency, 1993. Method 300.0: Determination of Inorganic Anions by Ion Chromatography. Inorganic Chemistry Branch, Cincinatti, Ohio. Westin, D.T., 1974. Nitrate and nitrite toxicity to salmonoid fishes. Prog. Fish-Culturist 36, 86e89. http://dx.doi.org/10.1577/1548-8659(1974)36[86:NANTTS] 2.0.CO;2. Zaitsev, G., Mett€ anen, T., Langwaldt, J., 2008. Removal of ammonium and nitrate from cold inorganic mine water by fixed-bed biofilm reactors. Min. Eng. 21, 10e15. http://dx.doi.org/10.1016/j.mineng.2007.08.014.
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Evaluation of the effect of water type on the toxicity of nitrate to aquatic organisms Josh A. Baker a, *, Guy Gilron b, Ben A. Chalmers c, James R. Elphick a a
Nautilus Environmental, 8664 Commerce Court, Burnaby, BC, V5A 4N7, Canada Borealis Environmental Consulting, 148 East 25th Street, North Vancouver, BC, V7N 1A1, Canada c The Mining Association of Canada, 350 Sparks Street, Suite 1105, Ottawa, ON, K1R 7S8, Canada b
h i g h l i g h t s Acute and chronic NO3 toxicity to aquatic organisms assessed. Hyalella azteca and Ceriodaphnia dubia most sensitive to effects of nitrate. Strong influence of ionic strength on nitrate toxicity. 2e10-fold reductions in chronic NO3 toxicity as water changed from soft to hard.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 August 2016 Received in revised form 29 September 2016 Accepted 16 October 2016
A suite of acute and chronic toxicity tests were conducted to evaluate the sensitivity of freshwater organisms to nitrate (as sodium nitrate). Acute exposures with rainbow trout (Onchorhynchus mykiss) and amphipods (Hyalella azteca), as well as chronic exposures with H. azteca (14-d survival and growth), midges (Chironomus dilutus; 10-d survival and growth), daphnids (Ceriodaphnia dubia; 7-d survival and reproduction), and fathead minnows (Pimephales promelas; 7-d survival and growth) were used to determine sublethal and lethal effect concentrations. Modification of nitrate toxicity was investigated across a range of ionic strengths, created through the use of very soft water, and standard preparations of synthetic soft, moderately-hard and hard dilution waters. The most sensitive species tested were C. dubia and H. azteca, in soft water, with reproduction and growth IC25 values of 13.8 and 12.2 mg/L NO3-N, respectively. All of the organisms exposed to nitrate demonstrated significantly reduced effects with increasing ionic strength associated with changes in water type. Possible mechanisms responsible for the modifying effect of increasing major ion concentrations on nitrate toxicity are discussed. © 2016 Elsevier Ltd. All rights reserved.
Handling Editor: Jim Lazorchak Keywords: Nitrate Toxicity Hardness Modifying factor Anions
1. Introduction Nitrogen is an essential element for all organisms, being a major component of amino acids, nucleic acids, and other biological materials. In aquatic environments, biologically-available nitrogen occurs primarily in the chemical forms of ammonia, nitrite and nitrate, with the relative concentrations of these materials determined by biological and chemical processes associated with the nitrogen cycle. Nitrate is the most common aqueous form of nitrogen, and is produced primarily from the oxidation of plant and animal debris (Camargo and Alonso, 2006). Anthropogenic sources
* Corresponding author. E-mail address: [email protected] (J.A. Baker). http://dx.doi.org/10.1016/j.chemosphere.2016.10.059 0045-6535/© 2016 Elsevier Ltd. All rights reserved.
of nitrogen have increased concentrations of nitrate in many aquatic environments (Puckett, 1995), exceeding 25 and 100 mg/L NO3-N in contaminated surface and ground waters, respectively (Camargo and Alonso, 2006). Mining activities often discharge nitrate into receiving environments, primarily as a result of use of nitrogen-containing blasting agents, such as ammonium nitrate/ fuel oil or AN/FO (Camargo et al., 2005; Zaitsev et al., 2008). Significant research has been conducted on the toxicity of ammonia and nitrite, however, less information is available regarding the concentrations of nitrate which cause adverse effects to aquatic organisms. Direct nitrate toxicity has previously been considered to be negligible, and concern was primarily associated with the potential for nitrate to stimulate eutrophication (Camargo and Alonso, 2006). However, studies have demonstrated that environmentally-relevant concentrations of nitrate can lead to
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direct toxicity to aquatic organisms (Camargo et al., 2005). Inhibition of the oxygen-carrying capacity of hemoglobin has been implicated as the cause of reduced performance of aquatic organisms in the presence of elevated concentrations of nitrate (Grabda et al., 1974). This toxic action is similar to that of nitrite (Lewis Jr and Morris, 1986), likely due to reduction of nitrate to nitrite in the blood (Guillette and Edwards, 2005) resulting in a similar metabolic pathway involving production of nitric oxide (Hannas et al., 2010). Effects on osmoregulation resulting from nitrate exposures have also been identified (Gulyassy et al., 1962; Hrubec et al., 1997). Research related to the toxicity of ammonia and nitrite has identified key toxicity-modifying factors for these compounds (i.e., pH and chloride, respectively); these factors have been incorporated into water quality benchmarks for these constituents (British Columbia Ministry of Environment, 2009; Canadian Council of Ministers of the Environment, 2010; U.S. Environmental Protection Agency, 2013). Due to a limited focus on evaluating toxicity of nitrate, regulatory agencies have developed guidelines based on limited datasets (British Columbia Ministry of Environment, 2009; Canadian Council of Ministers of the Environment, 2012; Environment Australia, 2000) and the role of toxicity-modifying factors for nitrate has not been comprehensively evaluated, although the effect of chloride on toxicity of nitrate to amphipods has been reported (Soucek and Dickinson, 2016). The aquatic toxicity of sulphate and chloride has been reported to be reduced with increasing water hardness (Davies and Hall, 2007; Elphick et al., 2011a, 2011b; Lasier and Hardin, 2010; Soucek and Kennedy, 2005), although the mechanism associated with this effect has not yet been clearly defined. The effect of water hardness on the toxicity of nitrate has not previously been evaluated, although the potential for this interaction has been hypothesized (Camargo et al., 2005; Scott and Crunkilton, 2000). This study was designed to expand upon the available data on the toxicity of nitrate using additional freshwater species, while
exploring the relationship between nitrate toxicity and the ionic characteristics of water (e.g., hardness). The relationships between nitrate and water quality characteristics are potentially significant in understanding mechanisms of nitrate toxicity and in establishing whether toxicity modifying factors should be incorporated into water quality benchmarks for nitrate. 2. Methods Toxicity tests were conducted at Nautilus Environmental (Burnaby, BC, Canada) in walk-in environmental chambers with temperature (±1 C) and photoperiod (16:8 light:dark) control. Water quality parameters, including dissolved oxygen, pH and temperature, were recorded daily throughout the exposures. Test waters were prepared by addition of reagent-grade salts to achieve the target water hardness types, with the exception of very soft water (i.e., 10e15 mg/L as CaCO3), which was dechlorinated Metro Vancouver municipal tap water. Salt additions followed the ratios of salts specified by USEPA (U.S. Environmental Protection Agency, 2002), with the exception of tests using Hyalella azteca and Chironomus dilutus, which employed a recipe containing a higher concentration of chloride, as described by Environment Canada (Environment Canada, 1997a). Tests using rainbow trout (Onchorhynchus mykiss) and fathead minnows (Pimephales promelas) were conducted at four hardnesses: very soft water (VSW, 10e15 mg/L as CaCO3); soft water (SW, 40e55 mg/L as CaCO3); moderately-hard water (MHW, 80e100 mg/L as CaCO3); and hard water (HW, 160e180 mg/L as CaCO3). The invertebrate species (i.e., Ceriodaphnia dubia, C. dilutus and H. azteca) were tested at the three higher hardnesses (Table 2). These water types are commonly used in evaluations of water hardness as a toxicity modifying factor; however, in addition to ions contributing to hardness (i.e., Ca and Mg) other major ions (i.e., Na, K, HCO3, Cl and SO4) also co-vary in these water types (Table 1). Hardness and alkalinity were measured on waters at test initiation using titration techniques.
Table 1 Water chemistry for dilution waters used in nitrate exposures. Nominal concentrations based on salt additions (mg/L). Constituent (mg/L)
Na Mg Ca K HCO3 Cl SO4 pH Hardness (as CaCO3)
P. promelas/O. mykiss
C. dubia
H. azteca/C. dilutus
VSW
SW
MHW
HW
SW
MHW
HW
SW
MHW
HW
1.8 0.1 3.9 0.2 11.0 2.0 2.7 6.9e7.3 10e15
15.0 6.2 10.9 1.2 45.9 2.9 43.4 7.3e7.7 40e55
28.1 12.3 17.9 2.3 80.8 3.9 84.0 7.7e8.0 80e100
54.4 24.4 31.8 4.4 150.5 5.8 165.4 7.9e8.3 160e180
13.1 6.1 7.0 1.0 34.9 1.0 40.7 7.6e7.9 40e55
26.3 12.7 14.7 2.1 69.7 1.9 85.4 8.1 80e100
52.5 24.2 27.9 4.2 139.5 3.8 162.7 8.2e8.4 160e180
15.0 3.2 18.7 1.2 45.9 18.8 28.6 7.4e7.7 40e55
28.1 6.2 33.5 2.3 80.8 36.0 54.5 7.7e8.0 80e100
54.4 12.3 63.2 4.4 150.5 69.4 106.4 8.0e8.2 160e180
Very soft water: 10e15 mg/L as CaCO3; soft water: 40e55 mg/L as CaCO3; moderately-hard water: 80e100 mg/L as CaCO3; and, hard water: 160e180 mg/L as CaCO3.
Table 2 Summary of toxicity test endpoints and test concentrations. Test species
Test duration
Test endpoint(s)
Nominal concentrations tested (mg/L NO3-N)
O. mykiss H. azteca
96-hr 96-hr 14-day 10-day 7 ± 1 day 7-day
Survival Survival Survival, Survival, Survival, Survival,
312, 625, 1250, 2500, 5000 103, 206, 412, 824, 1647 10a, 20, 40, 80, 160, 320, 640b 10a, 20, 40, 80, 160, 320, 640b 5c, 10, 20, 40, 80, 160, 320, 640d 50, 100, 200, 400, 800, 1600
C. dilutus C. dubia P. promelas a b c d
Tested Tested Tested Tested
in in in in
soft water only. moderately hard and hard water only. moderately hard water. soft water and hard water.
growth growth reproduction growth
J.A. Baker et al. / Chemosphere 168 (2017) 435e440
Nitrate was added using reagent-grade sodium nitrate; concentrations used in each test are shown in Table 2. Nitrate was measured in all test concentrations at initiation of the tests. In chronic toxicity tests, nitrate was also measured in a subset of concentrations bracketing the effect level at test termination. Nitrate was measured using ion chromatography (U.S. Environmental Protection Agency, 1993) with detection by UV absorbance by ALS Environmental (Burnaby, BC, Canada). 2.1. Rainbow trout Acute toxicity tests using rainbow trout (O. mykiss) were conducted according to the Environment Canada test method for this species (Environment Canada, 2000). Exposures were conducted in 20-L glass aquaria containing 10 L of test solution and ten fry ranging from 0.3 to 0.6 g. Fish were obtained from the Fraser Valley Trout Hatchery (Abbotsford, BC, Canada) and were acclimated in the laboratory for at least two weeks prior to testing. Tests were conducted at 15 ± 1 C and the test solutions were aerated continuously at a rate of 65 ± 10 mL per minute throughout the exposure period. The solutions were not renewed and the test organisms were not fed during the exposure. Survival of the test organisms were recorded daily throughout the tests; control performance of 90% survival was required for the test to be considered acceptable. 2.2. Amphiphods Acute (96-h) and chronic (14-d) toxicity tests using H. azteca were adapted from methods published by Environment Canada (Environment Canada, 1997a); the tests were initiated using 6e8 day old amphipods obtained from Aquatic Biosystems (Fort Collins, CO). The acute exposures were conducted using triplicate 200 mL volumes in glass jars with no solution renewal, and feeding using digested yeast, Cerophyll and trout chow (YCT; 1.5 mL in each replicate) at test initiation and after 48 h of exposure. Chronic toxicity tests were conducted using clean sediment comprised of beach-collected sand, supplemented with peat at a rate of 2% by weight. Test methods were modified from Environment Canada procedures for sediment toxicity tests (Environment Canada, 1997a) by incorporating test solution renewal three times per week throughout exposure with freshly-prepared test solutions, at which time YCT (1.5 mL per replicate) was added as food. These tests were conducted using four replicates per concentration in glass jars containing 100 mL of control sediment and filled to 275 mL with the test solutions. The exposures were conducted at 23 ± 1 C and the overlying water aerated gently throughout exposure. Surviving amphipods were dried at the end of the test on pre-weighed aluminum pans and dry weight was determined. Both acute and chronic exposures were conducted using 10 test organisms per replicate. Control survival of 90 and 80% was required for the acute and chronic exposures, respectively, to meet test acceptability criteria. In addition, the chronic test required that control amphipods achieve at least 0.1 mg dry weight at test termination. 2.3. Chironomids Chronic toxicity tests using C. dilutus were conducted in a similar manner to that described for H. azteca, with the exception that the test duration was 10 rather than 14 days. The tests were initiated with third instar larval midges obtained from Aquatic Biosystems, and the exposure chambers were fed with 6 mg of Tetramin each day. These methods were adapted from Environment Canada test methods for evaluating sediment samples using
437
this species (Environment Canada, 1997b). The tests were considered acceptable if the control survival was 70% and dry weight of the surviving larvae was 0.6 mg. 2.4. Daphnids Chronic toxicity tests using C. dubia were conducted according to the Environment Canada test method for this species (Environment Canada, 2007) in 15-mL volumes in 18-mL glass test tubes. Exposures of each concentration comprised 10 replicates, each initiated with a single <24-hr old daphnid obtained from inhouse cultures. The organisms were cultured at the three test water hardnesses for at least two generations prior to test initiation. Exposure waters were renewed with freshly-prepared solutions daily, at which time they were fed a mixture of green algal cells (Pseudokirchneriella subcapitata) and YCT. Exposures were conducted at 25 ± 1 C and survival and reproduction were recorded daily. Exposures were terminated after 7 ± 1 days, when at least 60% of the control organisms had produced their third brood. The tests were considered acceptable if the control organisms had 80% survival and produced an average of 15 neonates in three broods during the test. 2.5. Fathead minnows Fathead minnow tests were conducted according to test methods for this species described by Environment Canada (Environment Canada, 1992), involving a 7-d exposure, initiated with <24-hr post-hatch fish, which were obtained from Aquatic Biosystems. Tests were conducted using three replicates with ten fish in 300 mL glass jars containing 250 mL of solution. The tests were conducted at 25 ± 1 C. Fish were fed with brine shrimp (Artemia salina) and solutions were renewed daily throughout the exposure period. At the end of the test, surviving fish were dried on pre-weighed aluminum pans and weighed. Endpoints from the test were survival and biomass, and the tests were considered acceptable if 80% survival was observed in control exposures. 2.6. Quality assurance/quality control Reference toxicant tests, involving exposure of the organisms to a range of concentrations of a known toxicant, were conducted using all species evaluated in this study, and the results of the tests were compared with a control chart of toxicity test data produced previously in the laboratory. Reference toxicants used were as follows: sodium dodecyl sulphate (SDS) for rainbow trout, potassium chloride for C. dilutus, and sodium chloride for the remaining three species. The duration of the reference toxicant tests were the same as the individual tests, with the exception of the 14 and 10-d tests using H. azteca and C. dilutus, which utilized a 96-hr static exposure for the reference toxicant tests, as recommended by Environment Canada (Environment Canada, 1997a, 1997b). 2.7. Statistical analyses Statistical analyses were conducted using CETIS (Tidepool Software; McKinleyville, CA), according to procedures described by Environment Canada (Environment Canada, 2005) for calculating point estimates. Survival data were analyzed using probit or logit multiple linear estimation, where possible, and quantitative data for reproduction and growth were analyzed using non-linear regression in cases where model assumptions were met. Linear interpolation of log-transformed data was used in cases where the assumptions of the models described above were not met. Simple regression analyses were conducted using Microsoft Excel to
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determine the effect of water chemistry on nitrate endpoints. 3. Results The results of all toxicity tests met requirements specified in the test methods for control performance and water quality parameters remained within the ranges specified in the corresponding test methods. The reference toxicant test results fell within the range of acceptable results (i.e., mean ± 2 standard deviations) from historical tests with these species, indicating that the organisms were of acceptable sensitivity. Measured concentrations of nitrate were in agreement with target concentrations of nitrate; analyzed values were an average of 99 ± 7% of the nominal concentrations across all of the tests. Measured concentrations were used in determining effect concentrations. Test data for each test concentration are provided in the Supplementary data. Acute toxicity testing with juvenile rainbow trout resulted in median lethal concentrations (i.e., 96-hr LC50) that ranged from 808 mg/L NO3-N in very soft water to 1913 mg/L NO3-N in hard water. Testing with 6e8 day old H. azteca produced 96-hr LC50 values that increased from 168 to 485 and 921 mg/L NO3-N in soft, moderately-hard and very hard water, respectively (Table 3). A clear reduction in toxicity with increase in water hardness was apparent in tests using both species (Fig. 1). Lethal (LC50) and sublethal (IC25; concentration resulting in a 25% reduction) effect concentrations from chronic tests conducted with H. azteca, C. dilutus, C. dubia and P. promelas are summarized in Table 4. A similar trend to that observed with the acute tests was observed with each of these species, with lower nitrate toxicity associated with organisms exposed in waters with higher concentrations of major ions.
Table 3 Results from 96-hr acute toxicity tests with nitrate (mg/L NO3-N). Test species
Hardness (mg/L CaCO3)
LC50 (95% Confidence limits)
O. mykiss
11 54 90 164 44 100 164
808 (639e1023) 1446 (1192e1754) 1958 (1360e2820) 1913 (1340e2730) 168 (143e197) 485 (435e540) 921 (808e1049)
4. Discussion Data presented in this study add to the current knowledge on the sensitivity of aquatic organisms to nitrate while also highlighting the important role of water chemistry on toxicity. The results from this study show a relationship between the sensitivity to nitrate and the ionic composition of the test solution. Strong correlations were observed between both acute LC50 and chronic IC25 endpoints and water hardness [(i.e., correlation coefficients: R2 ranging from 0.85 to 1.0 (Fig. 1),]. However, other major ions covaried with hardness in the water types used and, therefore, ions other than calcium and magnesium could have contributed to the observed effect on toxicity of nitrate. The sensitivity of rainbow trout observed in this study was similar to a 96-hr LC50 of 1355 mg/L NO3-N for O. mykiss fingerlings reported by Westin (1974), and was within the range of 1010e1975 mg/L NO3-N summarized by Camargo et al. (2005) in a review of nitrate toxicity to freshwater fish. Previous studies have not investigated modifying factors of acute nitrate toxicity to fish, and further work is warranted to identify the mechanism responsible for the effect of water chemistry on toxicity observed during this study. The sensitivity observed in acute exposures to H. azteca in the current study are consistent with those reported by Soucek and Dickinson (2012), Soucek et al. (2015) and the USEPA (2010). Results for this species from each of these studies are shown in Fig. 2 on the basis of chloride concentration in the test waters; chloride has been identified as a modifying factor for acute nitrate toxicity to H. azteca (Soucek and Dickinson, 2016). A significant relationship was observed between LC50 values and chloride concentration (p < 0.01) suggesting that chloride may explain some or all of the effect of water type observed in this study with this species.
10000 IC25 (mg/L NO3-N)
10000 LC50 (mg/L NO3-N)
H. azteca
C. dubia was the most sensitive of the tested species across water types, with reproduction IC25 values ranging from 13.8 to 47.5 mg/L NO3-N, as the water hardness increased. Sensitivities of the chironomid, C. dilutus, and the amphipod, H. azteca, were in a similar range, as growth IC25 values increased from 48.8 to 178 mg/ L NO3-N for C. dilutus and from 12.2 to 181 mg/L NO3-N for H. azteca, across the soft to hard water types. Fathead minnows were the least sensitive of the four species tested for chronic toxicity, with IC25 values ranging from 69.6 to 402 mg/L NO3-N in dilution waters that ranged from very soft to hard conditions.
1000 100 10 1
1000 100 10 1
1
10 100 1000 Water hardness (mg/L, as CaCO3)
Amphipod (96 hr) R² = 1
Rainbow trout (96 hr) R² = 0.95
1
10 100 1000 Water hardness (mg/L, as CaCO3) Amphipod growth (14 d) Chironomid growth (10 d) R² = 0.85 R² = 0.99 Cladoceran repr. (7d) Fathead growth (7 d) R² = 0.98 R² = 0.95
Fig. 1. Acute and chronic effects of nitrate shown as a function of water hardness.
J.A. Baker et al. / Chemosphere 168 (2017) 435e440 Table 4 Results from chronic nitrate toxicity tests using C. dubia, C. dilutus, H. azteca and P. promelas. Test Species
Hardness (as mg/L CaCO3)
Endpoint
H. azteca
46 86 172 46 86 172 46 86 172 46 86 172 44 98 166 44 98 166 12 50 94 168 12 50 94 168
Survival
C. dilutus
C. dubia
P. promelas
mg/L NO3-N (95% Confidence limits)
Growth
Survival
Growth
Survival
Reproduction
Survival
Growth
14-d LC50 14-d LC50 14-d LC50 14-d IC25 14-d IC25 14-d IC25 10-d LC50 10-d LC50 10-d LC50 10-d IC25 10-d IC25 10-d IC25 7-d LC50 7-d LC50 7-d LC50 7-d IC25 7-d IC25 7-d IC25 7-d LC50 7-d LC50 7-d LC50 7-d LC50 7-d IC25 7-d IC25 7-d IC25 7-d IC25
124 (106e145) 275 (243e312) >622 12.2 (0.6e59.0) 116 (47.4e183) 181 (102e274) 114 (96.3e134) 222 (191e257) 342 (301e387) 48.8 (30.8e95.5) 102 (89.4e122) 178 (153e191) 62.0 (37.0e83.0) 120 (110e130) 127 (102e160) 13.8 (4.2e24.5) 23.5 (8.7e31.1) 47.5 (26.5e56.2) 117 (103 - 132) 235 (200e276) 415 (358e481) 465 (398e543) 69.6 (44.4e86.4) 209 (NCe248) 358 (158e567) 402 (193e477)
NC: Not calculable.
Contrary to the results for H. azteca, Soucek and Dickinson (2016) reported that there was no interaction between chloride and toxicity of nitrate in chronic toxicity tests using C. dubia conducted at a hardness of 90 mg/L; EC20 estimates ranged from 80 to 263 mg/L NO3-N demonstrating large variability but no dependence on chloride concentration. Scott and Crunkilton (2000) also reported results from chronic toxicity tests using C. dubia; point estimates were not presented for reproduction, but a MATC value of 30.1 mg/L NO3-N was determined in a hardness range of 150e184 mg/L. The reproduction effect concentrations reported here were similar to those reported by Scott and Crunkilton (2000), but somewhat more sensitive than those reported by Soucek and Dickinson (2016). The water formulation used in Soucek and Dickinson (2016) was lower in sulfate, higher in chloride, and differed in terms of the ratio of calcium to magnesium, in comparison to the waters used in this study (Table 1). None of the major
439
ions were significantly correlated with 7-d C. dubia nitrate IC50 values when the data for the two studies were compared as a whole (p > 0.05). It has been reported that acute toxicity of sulphate is reduced to a greater extent in waters with a higher calcium to magnesium ratio (Davies and Hall, 2007), and it is possible that the lower sensitivity observed by Soucek and Dickinson (2016) could relate to a similar effect. While acute nitrate toxicity data for some benthic organisms are available (Camargo et al., 2005; Pandey et al., 2011; Soucek and Dickinson, 2012), relatively few chronic toxicity data are available. The chronic toxicity endpoints determined here for H. azteca (with EC50s ranging from 50.6 to 181.0 mg/L NO3) were slightly less sensitive than those reported by Soucek and Dickinson (2016) for this species (with EC50s ranging from 32 to 86 mg/L NO3); however, this may be explained by the longer test duration used in their study (42-d). Chronic effects of nitrate on C. dilutus have not previously been reported in the literature. The results presented here indicate that sensitivity of C. dilutus to nitrate is comparable to that of H. azteca; C. dilutus and H. azteca sublethal endpoints were similar in MHW and HW, although H. azteca was four-fold more sensitive to nitrate than C. dilutus in SW. The fathead minnow was the least sensitive species tested using chronic exposures to nitrate; this finding is consistent with previous work by other researchers. Nitrate exposures at a hardness of 156e172 mg/L resulted in a MATC for growth of 506.6 mg/L NO3-N (Scott and Crunkilton, 2000) which is comparable to the IC25 of 402 mg/L NO3-N reported here for the hard water exposure. We have presented compelling evidence that ionic composition influences the toxicity of nitrate. Effects of water type on the toxicity of nitrate could be the result of competitive exclusion at uptake sites by other anions (i.e., chloride, bicarbonate, sulfate), as has been observed by the role of chloride on H. azteca sensitivity to nitrate (Soucek and Dickinson, 2016), or may result from effects of cations such as calcium on membrane permeability. Further investigation into the mechanism(s) behind the effect of water type will help to further refine the role of individual water constituents in modifying nitrate toxicity. In general, with respect to nitrate sensitivity, marine organisms appear to be less sensitive than freshwater organisms (Camargo et al., 2005; Tsai and Chen, 2002) possibly highlighting the importance of physiological aspects of ionoregulation in the toxicity of nitrate. Adverse effects were observed in this study at nitrate concentrations that can be found in polluted surface waters (Camargo et al., 2005). However, sensitivity of all of the organisms tested was reduced with increasing ionic strength of the test waters. Thus,
10000 96-h LC50 (mg/L NO3-N)
US EPA (2010) 1000
Present study R² = 0.91
100
Soucek and Dickinson (2012)
10
Soucek and Dickinson (2016)
1 1
10
100
Chloride in test water (mg/L) Fig. 2. Relationship between the LC50 of nitrate for Hyalella azteca and chloride concentration in the test water.
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water quality guidelines that are based on toxicity tests conducted in very soft water may overestimate the risk of adverse effects in conditions where the ionic strength of the water is higher. Acknowledgements The authors would like to acknowledge member companies of the Mining Association of British Columbia for providing funding for this study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2016.10.059. References British Columbia Ministry of Environment, 2009. Water Quality Guidelines for Nitrogen (Nitrate, Nitrite, and Ammonia). Overview Report Update 29. Camargo, J.A., Alonso, A., 2006. Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: a global assessment. Environ. Int. 32, 831e849. http://dx.doi.org/10.1016/j.envint.2006.05.002. Camargo, J.A., Alonso, A., Salamanca, A., 2005. Nitrate toxicity to aquatic animals: a review with new data for freshwater invertebrates. Chemosphere 58, 1255e1267. http://dx.doi.org/10.1016/j.chemosphere.2004.10.044. Canadian Council of Ministers of the Environment, 2012. Canadian Water Quality Guidelines: Nitrate Ion. Scientific Criteria Document. Canadian Council of Ministers of the Environment, 2010. Canadian Water Quality Guidelines for the Protection of Aquatic Life: Ammonia. Davies, T.D., Hall, K.J., 2007. Importance of calcium in modifying the acute toxicity of sodium sulphate to Hyalella azteca and Daphnia magna. Environ. Toxicol. Chem. 26, 1243e1247. http://dx.doi.org/10.1897/06-510R.1. Elphick, J.R., Bergh, K.D., Bailey, H.C., 2011a. Chronic toxicity of chloride to freshwater species: effects of hardness and implications for water quality guidelines. Environ. Toxicol. Chem. 30, 239e246. http://dx.doi.org/10.1002/etc.365. Elphick, J.R., Davies, M., Gilron, G., Canaria, E.C., Lo, B., Bailey, H.C., 2011b. An aquatic toxicological evaluation of sulfate: the case for considering hardness as a modifying factor in setting water quality guidelines. Environ. Toxicol. Chem. 30, 247e253. http://dx.doi.org/10.1002/etc.363. Environment Australia, 2000. Australian and New Zealand Guidelines for Fresh and Marine Water Quality- Volume 2: Aquatic Ecosystems d Rationale and Background Information, vol. 678. National Water Quality Management Strategy. Australian and New Zealand Environment and Conservation Council. Environment Canada, 2007. Biological Test Method: Test of Reproduction and Survival Using the Cladoceran Ceriodaphnia Dubia. EPS 1/RM/32, Method Development and Application Centre, Environment Canada, Ottawa, ON. Environment Canada, 2005. Guidance Document on Statistical Methods for Environmental Toxicity Tests. EPS 1/RM/46. Method Development and Application Centre, Environment Canada, Ottawa, ON. Environment Canada, 2000. Biological Test Method: Reference Method for Determining Acute Lethality of Effluents to Rainbow Trout. EPS 1/RM/9, Environmental Technology. Method Development and Application Centre, Environment Canada, Ottawa, ON. Environment Canada, 1997a. Biological Test Method: Test for Survival and Growth in Sediment Using the Freshwater Amphipod Hyalella Azteca. EPS 1/RM/33, Method Development and Application Centre, Ottawa, ON. Environment Canada, 1997b. Biological Test Method: Test for Survival and Growth in Sediment Using the Larvae of Freshwater Midges (Chironomus tentans and Chironomus Riparius). EPS 1/RM/32, Method Development and Application Centre, Environment Canada, Ottawa, ON. Environment Canada, 1992. Biological Test Method: Test of Larval Growth and
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