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Environmental Toxicology and Chemistry, Vol. 19, No. 12, pp. 2918–2922, 2000q 2000 SETAC

Printed in the USA0730-7268/00 $9.00 1 .00

ACUTE AND CHRONIC TOXICITY OF NITRATE TO FATHEAD MINNOWS(PIMEPHALES PROMELAS), CERIODAPHNIA DUBIA, AND DAPHNIA MAGNA

GEORGE SCOTT and RONALD L. CRUNKILTON*College of Natural Resources, University of Wisconsin–Stevens Point, Stevens Point, Wisconsin 54481, USA

(Received 7 July 1999; Accepted 15 April 2000)

Abstract—Increasing concentrations of nitrate in surface water and groundwater are becoming a worldwide concern, yet littleinformation has been published on toxicity of nitrate to common organisms used for toxicity testing. The acute and chronic toxicityof nitrate (NO3-N) to Ceriodaphnia dubia, Daphnia magna, and Pimephales promelas was investigated in 48-h to 17-d laboratoryexposures. The 48-h median lethal concentration (LC50) of nitrate to C. dubia and D. magna neonates was 374 mg/L NO3-N and462 mg/L NO3-N. The no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC) for neonateproduction in C. dubia were 21.3 and 42.6 mg/L NO3-N, respectively. The NOEC and LOEC values for neonate production in D.magna were 358 and 717 mg/L NO3-N, respectively. The 96-h LC50 for larval fathead minnows (P. promelas) was 1,341 mg/LNO3-N. The NOEC and LOEC for 7-d larval and 11-d embryo–larval growth tests were 358 and 717 mg/L NO3-N, respectively.Additional exposure of breeding P. promelas and their fertilized eggs to nitrate did not increase susceptibility further. The LC50values for all species tested were above ambient concentrations of nitrate reported for surface water. However, the LOEC for C.dubia was within the range of concentrations that could be found in streams draining areas under extensive agricultural cultivation.

Keywords—Nitrate Acute toxicity Chronic toxicity Ceriodaphnia

INTRODUCTION

Increasing concentrations of nitrate in groundwater and sur-face waters are becoming a worldwide concern. Nitrate nat-urally enters groundwater and surface water via runoff fromdecomposition of vegetation, natural geological deposits, soilnitrogen, and atmospheric deposition [1,2]. Anthropogenic in-puts include septic tank drainage, feedlots, and soil leachingfrom irrigation and fertilizers [3]. During the period from 1950to 1970 fertilizer use in the United States doubled from 20million to 40 million tons per year [4]. The percentage ofnitrogen in fertilizers also increased from 6.1 to 20.4% [2].By the mid-1980s fertilizer consumption reached a high of 41to 53 million tons [5]. Concentrations of NO3-N above 3 mg/L in groundwater are considered elevated because of humanactivity [2]. The United States federal maximum contaminantlevel for drinking water is 10 mg/L NO3-N [6]. Concentrationsin groundwater entering streams draining the Central Sandsregion of Wisconsin, USA, can exceed 10 mg/L [7]. Currently,no safe levels have been established for aquatic life.

The potential toxicity of nitrate to aquatic organisms hasprobably been ignored because other commonly occurringforms of nitrogen such as ammonia and nitrite are more toxic.These nitrogen-containing compounds are interrelated throughthe process of nitrification. Ammonia is converted into nitratethrough a two-step process with nitrite produced as an inter-mediate product [8]. Nitrite is toxic through the conversion ofhemoglobin to methemoglobin, a form incapable of carryingoxygen, resulting in anoxia [9]. Exposure of fish to acutelytoxic concentrations of ammonia causes increased gill venti-lation, hyperexcitability, convulsions, and death [8]. Toxic ef-fects of nitrate on warm-blooded animals are well studied andmay be related to in vivo conversion of nitrate to nitrite [10,11].

* To whom correspondence may be addressed(rcrunkil@uwsp.edu).

Neonatal stages and newborn individuals are especially sus-ceptible because nitrate may be converted to nitrite under an-aerobic conditions in the gut [12].

Comparatively few studies have been conducted on the ef-fects of nitrate to aquatic organisms, particularly to differentlife history stages. No information has been published on theacute and chronic toxicity of nitrate to Pimephales promelasand Ceriodaphnia dubia, species commonly used for aquatictoxicity testing [13]. Toxic effects of nitrate to Daphnia mag-na, another widely used organism for toxicity testing, havebeen reported previously [14–16]. The acute toxicity of nitrateto several species of fish generally falls between 100 and 1,000mg/L NO3-N [17–19]. Limited data are available on longer-term effects of nitrate to fish. Recent studies have raised con-cerns about nitrate toxicity in aquatic insects [13,20] and am-phibians [21–23] because ambient concentrations in surfacewater often exceed effect levels.

The objectives of this study were to determine the acuteand chronic toxicity of nitrate to P. promelas, C. dubia andD. magna, and determine if early life stages of P. promelasare more susceptible to nitrate toxicity than are older individ-uals.

MATERIALS AND METHODS

Acute toxicity

Ceriodaphnia dubia (, 24 h old) and D. magna (, 48 hold) were exposed to a geometric series of five concentrationsof reagent grade NaNO3 (lot 960785, Fisher Scientific, Fair-lawn, NJ, USA) ranging from 150 to 2,500 mg/L NO3-N in48-h static renewal tests. Organisms were cultured in-houseand neonates were selected for testing following proceduresdescribed by Weber and Peltier [24]. Four replicates each con-taining five organisms were assigned to the treatments andcontrol. Polystyrene cups (30 ml) filled to 20 ml served as testchambers. Test solutions were renewed daily with a stock so-

Acute and chronic toxicity of nitrate to fathead minnows Environ. Toxicol. Chem. 19, 2000 2919

lution prepared at the start of the test and stored at 48C. Mor-tality was recorded daily and any dead organisms were re-moved during solution renewal. Organisms were not fed duringthe exposure.

Pimephales promelas larvae from in-house cultures wereexposed to the same concentrations of NO3-N as C. dubia andD. magna in 96-h static renewal tests. Four replicates eachwith 10 larvae (,8 d old) in 300-ml glass beakers filled to250 ml were assigned to the treatments and control. Test so-lutions were renewed daily. Larvae were not fed during thetest. Mortality was recorded daily. Larvae were considereddead if no movement was observed after gentle prodding witha pipette. Test solutions were characterized by total hardness156 to 172 mg/L as CaCO3, total alkalinity 140 to 170 mg/Las CaCO3, pH 7.89 to 8.25, and DO 7.93 to 8.25 at 258C.

Dilution and control water was reconstituted moderatelyhard water prepared according to standard methods [25]. Re-constituted water was from municipal well water that was soft-ened, deionized, and pumped through a high-purity water pro-cessor (Barnstead Nanopure Model D4741, Dubuque, IA,USA) with one carbon, two ion exchange and one organicextraction filter in sequence resulting in type 1 reagent-gradewater. Nitrate concentrations were measured with an ion-spe-cific nitrate electrode (Orion Model 93-07, Orion, Boston, MA,USA) connected to a pH meter (Orion Model 920A). Nitrateconcentrations were measured at the start of the tests and werewithin 95% of nominal concentrations for all acute toxicitytests. All test vessels for acute tests were placed in an envi-ronmental chamber with a temperature of 25 6 18C, a pho-toperiod of 16 h light and 8 h dark, and fluorescent illuminationat 538 to 753 lux.

Chronic toxicity

Ceriodaphnia dubia (,24 h old) and D. magna (6 d old)were exposed in 7-d chronic tests at nominal concentrationsranging from 2.2 to 113 mg/L NO3-N with six treatments andone control. The C. dubia neonates were all released within8 h, and D. magna within 24 h. Ten replicates with one or-ganism per 30-ml cup were assigned to each treatment.

Organisms were fed once daily with yeast cerophyll troutchow and a Selanastrum algal suspension prepared and fed atrecommended rates [24]. Tests were terminated at 7 d. Envi-ronmental exposure conditions were identical to those de-scribed for acute tests.

All test solutions were renewed daily with a stock solutionprepared before the start of the test. The number of neonatesper cup was counted and recorded each day. Young were dis-carded and any excess food was siphoned out with a pipette,leaving a small volume and the adult in the bottom of the cup.Renewal solution was then added to a final volume of 20 ml.

Reconstituted water was prepared as previously described.Test solutions were characterized by alkalinity 120 to 138 mg/L as CaCO3, hardness 150 to 184 mg/L as CaCO3, DO 8.22to 10.13, and pH 7.52 to 8.60 at 258C. Nitrate-N concentrationswere within 95% of nominal concentrations for all replicatesby test end.

To assess the chronic toxicity of nitrate to different devel-opmental stages of P. promelas, tests of three durations wereemployed. One test began with exposure of newly hatchedlarvae, another with fertilized eggs in an embryo–larval test,and the third test exposed spawning adults, their fertilized eggs,and the embryo–larval stage. Newly hatched larvae (,24 hold) were exposed in a 7-d static renewal test following stan-

dard procedure [26]. Larvae were selected from breedingadults and fertilized eggs that were cultured in carbon-filteredmunicipal tap water with background NO3-N concentrationsless than 2.0 mg/L NO3-N.

Test chambers were 300-ml glass beakers filled to 250 ml.Nominal concentrations of NO3-N ranged from 2.2 to 71.7 mg/L NO3-N in five treatments. Four replicates with 10 larvaeeach were assigned to all treatments. Larvae were fed twicedaily at 6-h intervals with 0.15 ml of freshly hatched brineshrimp, but not within the last 2 h of the test. Excess brineshrimp were siphoned from each beaker daily before solutionrenewal. At the end of 7 d, larvae were sacrificed in 70%isopropyl alcohol, rinsed with distilled water, and dried inpreweighed aluminum drying pans for a minimum of 2 h at1058C. After drying, larvae weights were measured on a bal-ance (Mettler AE240, Greifensee, Switzerland) and recorded.

The embryo–larval test was performed with fertilized eggsfrom adults raised in control water. The developing embryoswere exposed to nominal concentrations ranging from 89.6 to1,434 mg/L NO3-N. Ten freshly fertilized eggs (,8 h) withfour replicates per concentration were assigned to the fivetreatment concentrations and a control. Eggs were gently aer-ated until approximately 50% of the eggs had hatched. Afterhatching, the larvae were subjected to the same 7-d staticgrowth test described above. Total exposure was 11 d.

The third test type subjected breeding pairs of P. promelas,their eggs, and hatched larvae in a flow-through exposure sys-tem to nominal concentrations of NO3-N ranging from 2.2 to71 mg/L NO3-N in five treatments and a control. Six 54-Lglass aquaria filled to 41 L were divided into four equal-sized(30 3 14.5 3 29-cm) chambers. One breeding pair was placedin each chamber and allowed to acclimate for at least a one-week period. Spawning substrate consisted of semicircular (103 5-cm) polyvinylchloride tiles.

All fertilized eggs were removed from tiles daily, separated,and counted. Fifty of these fertilized eggs were then transferredinto a glass culture tube (20 3 125 mm) with a 200-mmNytexy screen cover (McMaster-Carr, Chicago, IL, USA) af-fixed to the bottom to permit water exchange. Caps for theculture tubes were drilled to permit insertion of glass pipettesto the bottom that aerated and gently tumbled the eggs. Theglass tubes were suspended inside the same exposure chambersfrom which they were removed and the eggs incubated ap-proximately 4 d until hatching. Dead embryos were removedfrom the tubes daily and recorded. When approximately 50%of the larvae had hatched they were placed in larger polyvi-nylchloride (7.6 3 9-cm) containers with Nytex 200-mm meshscreen bottoms and again returned to the same treatment cham-bers for an additional 7 d for growth assessment. The mesh-covered polyvinylchloride chambers that enclosed the larvaeretained brine shrimp (0.20 ml) supplied twice daily at 6-hintervals, but allowed for water exchange in the surroundingflow-through chamber. Exposure chambers were cleaned dailyto remove excess food from the bottom screen. At the end of7 d, larvae were sacrificed and weighed as described earlier.Total exposure duration including adult acclimation was 18 d.

Each treatment and the control were replicated by exposingfour different pairs of fish and collecting their eggs. Fiftyfertilized eggs were collected from each of two successiveclutches from each pair, incubated, hatched, and exposed sep-arately as described. Data for the two successive clutches ofeach replicate were combined for statistical analysis.

Water temperature in exposure chambers that housed adults,

2920 Environ. Toxicol. Chem. 19, 2000 G. Scott and R.L. Crunkilton

Table 1. Nominal and measured concentration (range) of nitrate-nitrogen for flow-through tests with Pimephales promelas spawning

adults, eggs, and larvae (n 5 14)

Nominal NO3-N (mg/L) Measured NO3-N (mg/L)

Control4.59.0

17.935.971.7

2.9–4.16.3–9.0

10.1–12.816.6–22.028.2–36.357.1–75.3

Fig. 1. Mean number of neonates produced per adult female Cerio-daphnia dubia in 7-d chronic tests with exposure to nitrate (NO3-N).Error bars are ranges for three separate tests.

Table 3. Effect of nitrate-nitrogen on reproduction in Ceriodaphniadubia and Daphnia magna. Measured end point was the number ofneonates produced per female in 7 d. All values are reported as mg/

L NO3-N

Replicate

NO3-N (mg/L)

NOECa LOECb

C. dubia neonatesTest 1Test 2Test 3Test 4Test 5

7.156.5

7.117.917.9

14.1113

14.135.935.9

D. magna neonatesTest 1Test 2

358358

717717

a NOEC 5 no-observed-effect concentration.b LOEC 5 lowest-observed-effect concentration.

Table 2. Acute toxicity of nitrate-nitrogen to Ceriodaphnia dubia,Daphnia magna, and Pimephales promelas. All values reported as

mg/L NO3-N

Replicate LC50a (95% CI)

C. dubia neonatesTest 1Test 2

374 (300–448)374 (300–449)

D. magna neonatesTest 1Test 2Test 3

323 (198–469)453 (299–659)611 (455–820)

P. promelas larvaeTest 1Test 2Test 3

1,010 (877–1,143)1,607 (1,492–1,723)1,406 (1,236–1,577)

a LC50 5 median lethal concentration.

embryos, and larvae was maintained at 25 6 18C for the du-ration of the test with an external water bath. Lighting wasambient fluorescent (592 lux) with a photoperiod of 16 h lightand 8 h dark.

Because of the large volumes of water required for the flow-through tests, dilution water was carbon-filtered municipal tapwater. Test solutions were characterized by alkalinity 142 to170 mg/L as CaCO3, DO 6.63 to 9.81, pH 7.34 to 8.27, hard-ness 152 to 200 mg/L as CaCO3, and total ammonia as NH3-N at an average of ,1.0 mg/L (range, 0–1.68 mg/L). Waterwas delivered into aquaria at timed 2-L increments resultingin an exchange rate of two volumes per day. Nominal andmeasured concentrations of nitrate-nitrogen are reported in Ta-ble 1.

Statistical analysis

The median lethal concentration (LC50) values for NO3-Nwere calculated with a regression technique following a probittransformation [24] in two replicate tests for C. dubia andthree replicate tests with D. magna. The Spearman–Karberprocedure was used to calculate the LC50 in three replicatetests for P. promelas because the cumulative response curvesdid not conform to a normal probability distribution as mea-sured by a chi-square test for heterogeneity. A one-way anal-ysis of variance model with the Dunnetts method of pairwiseseparation was used to determine the lowest-observed-effectconcentration (LOEC) and the no-observed-effect concentra-tion (NOEC) of NO3-N in 7- to 18-d exposures [27]. Mortalitydata for P. promelas were analyzed with Steel’s many-onerank test because response data could not be transformed to anormal distribution [27].

RESULTS

The LC50 of nitrate to C. dubia and D. magna averaged374 mg/L NO3-N and 462 mg/L NO3-N (Table 2), respectively.The 96-h LC50 values for P. promelas larvae averaged 1,341mg/L NO3-N (Table 2).

The mean number of neonates produced per adult C. dubiagenerally decreased with increasing concentration of nitrate(Fig. 1). No mortality occurred in any of the controls and nosignificant mortality (p , 0.05) occurred in any of the con-centrations tested in the 7-d test. The mean NOEC and LOECvalues for C. dubia reproduction were 21.3 mg/L and 42.6mg/L NO3-N (Table 3), respectively. The mean NOEC andLOEC values for neonate production in D. magna were 358mg/L and 717 mg/L NO3-N (Table 3), respectively.

The NOEC and LOEC values for growth reduction in 7-dP. promelas tests were 358 mg/L and 717 mg/L NO3-N com-pared to 717 mg/L and 1,434 mg/L NO3-N for mortality (Table4), respectively. The NOEC and LOEC for reduced growthand mortality in embryo–larval and larval-only exposures wereidentical. No mortality occurred in control exposures. Larvaewere lethargic and exhibited bent spines before death at 717mg/L NO3-N, with 100% mortality occurring at 1,434 mg/LNO3-N by day 4 of the 7-d test. Fertilized eggs failed to hatchat 1,434 mg/L NO3-N. Hatching was not significantly (p ,

Acute and chronic toxicity of nitrate to fathead minnows Environ. Toxicol. Chem. 19, 2000 2921

Table 4. Chronic toxicity of nitrate-nitrogen to Pimephales promelaslarvae (,24 h old) through 7 d posthatch, embryos and larvae through7 d posthatch, and breeding adults with their offspring through 7 d

posthatch. All values reported as mg/L NO3-N

Life stage

Growth

NOECa LOECb

Mortality

NOEC LOEC

Larvae (,24 h through 7 dposthatch), 7 d exposure 358 717 717 1,434

Embryos and larvae (through 7 dposthatch), 11 d total exposure 358 717 358 717

Breeding adults and offspring(through 7 d posthatch), 181 dtotal exposure — .1,434 — .1,434

a No-observed-effect concentration.b Lowest-observed-effect concentration.

Table 5. Mean dry weight and percent survival of larval and embryo–larval Pimephales promelas exposed to nitrate-nitrogen (n 5 4)

NominalNO3-N(mg/L)

Dry weight (mg)

Larval(7 d)

Embryo–larval(11 d)

Percent survival

Larval(7 d)

Embryo–larval(11 d)

Control4.59.0

18367289

178358717

1,434

0.4180.4180.5000.4880.5020.4980.4210.4700.3870.253

0

0.4740.3710.3950.4040.3710.4020.5610.4730.6190.357

0

10097

1009999

100100

939867

0

100909484849189889544

0

0.05) reduced at any lower concentrations. Average weight persurviving larvae is included in Table 5.

Exposures of breeding pairs and their fertilized eggsthrough 7-d posthatch failed to show statistical reduction inegg survival or growth in the range of concentrations tested(2.24–717 mg/L NO3-N). Thus, the NOEC for spawning suc-cess is greater than 717 mg/L NO3-N.

DISCUSSION

The 96-h LC50 for larval fathead minnows reported hereis similar to previously published data on other fish species.The 96-h LC50 values for Chinook salmon (Oncorhynchustshawytscha), rainbow trout (Oncorhynchus mykiss), channelcatfish (Ictalurus punctatus), and Guadalupe bass (Microp-terus treculi) all fall within the range of 1,250 to1,400 mg/LNO3-N [17–19]. Bluegills (Lepomis macrochirus) exhibit a96-h LC50 range of 420 to 2,000 mg/L NO3-N [28], whereasguppies (Poecilia reticulata) are the most sensitive adult fishreported to date, with 96-h LC50 values ranging from 180 to200 mg/L NO3-N [29].

Adult fish seem to be less sensitive to acute effects of nitrateexposure than some freshwater invertebrates and amphibians.The 96-h LC50 values for two species of net-spinning cad-disflies (Hydropsyche occidentalis and Cheumatopsyche pet-titi) ranged from 97.3 to 165.5 mg/L NO3-N [13]. Hecnar [21]reported 96-h LC50 values for four species of amphibians tobe 13.6 to 39.3 mg/L NO3-N. At 9.0 and 23.0 mg/L NO3-N,decreased rates of growth and differentiation and increased

mortality occurred in the tree frog (Litoria caerrulea) [22].Similar concentrations of nitrate showed adverse effects ongrowth and survival of toad tadpoles (Bufo bufo) [23].

Daphnia magna in this study were shown to be slightlymore sensitive than those in previous studies. Dowden andBennett [14] reported a 96-h LC50 of 693 mg/L NO3-N, where-as Andersen [15] reported a 96-h LC50 of 835 mg/L NO3-N.The highest concentration that failed to immobilize the animalsunder prolonged exposure was 1,400 mg/L NO3-N [16].

Of the three organisms exposed in this experiment, C. dubiawas most sensitive to nitrate. No reports have been publishedon nitrate toxicity in C. dubia [13] even though this speciesis widely used for aquatic toxicology testing.

The LOEC for reduced reproduction in C. dubia was withinthe range reported for surface waters draining areas of exten-sive cultivation. Concentrations of nitrate in surface watersdraining agricultural land range from 10 to 60 mg/L NO3-N)[30]. Ceriodaphnia dubia are an important link in aquatic foodchains [31] and effect levels calculated here suggest the po-tential for adverse effects in aquatic ecosystems. Reproductionin D. magna was less affected by nitrate exposure. The LOECfor reproduction of D. magna was greater than the concentra-tion reported in surface waters.

Exposure of spawning adults and their fertilized eggs tonitrate concentrations used in this study did not result in in-creased susceptibility based on the LOEC for growth. TheLOEC for mortality in this longer exposure was equal to thatof the larvae-only exposure. This contrasts with findings ofKincheloe et al. [32], who reported a statistically significantincrease in mortality in embryos of rainbow trout at 2.3 mg/L NO3-N, steelhead trout (O. mykiss) at 1.1 mg/L NO3-N, andcutthroat trout (Salmo clarki) at 4.5 mg/L NO3-N. Mortalityin these tests ranged from 29 to 31% [32]. The toxic actionof nitrate occurs when hemoglobin in red blood cells is oxi-dized to methemoglobin, a form incapable of carrying oxygen[33,34]. Different tolerances may be due to physiologic dif-ferences in embryonic development of salmonids and fatheadminnows, water hardness (soft vs moderately hard), species-specific effects (cold-water vs warm-water physiology), or amore lengthy incubation period (.30 d) for salmonids com-pared to 4 d for P. promelas. The greater sensitivity of sal-monid embryos may be related to increased permeability ofthe chorion of fertilized eggs before hatching [35].

Limited information is available on effects of long-termexposure of nitrate to fish. Three species of salmonids, Chi-nook salmon, rainbow trout, and cutthroat trout larvae, showedsignificant mortality at NO3-N concentrations from 2.3 to 7.6mg/L in a 30-d exposure [32]. Channel catfish and largemouthbass (Micropterus salmoides) tolerated a nitrate concentrationof 96 mg/L NO3-N without affecting their growth and feedingactivity in a 164-d period [36]. Reduced growth was observedafter 84 d in 100 mg/L NO3-N in the anemone fish (Amphiprionocellaris), but survival was not affected. Sticklebacks (Gas-terosteus aculeatus) survived concentrations of 82 mg/L NO3-N for 10 d [37].

Adult fish may be less sensitive to nitrates because uptakeat the gill surface is limited. Stormer et al. [38] reported thatnitrate was taken up passively in rainbow trout, with plasmaconcentrations remaining below ambient [NO3] after 8 d ofexposure. This contrasts with [NO2], which was concentratedin the plasma of rainbow trout [38], where it competes withthe active chloride uptake mechanism in the gill [39].

Apparently, a wide range exists in susceptibility of aquatic

2922 Environ. Toxicol. Chem. 19, 2000 G. Scott and R.L. Crunkilton

organisms to nitrate. Additional studies should test other spe-cies as well as explore the joint effects of water hardness, pH,temperature, and DO on the toxicity of nitrate in aquatic or-ganisms.

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