Influence of Nitrite and Chloride Concentrations on
Survival and Hematological Profiles of Striped Bass
PATRICIA M. MAZ1K
U.S. Fish and Wildlife Service, Southeastern Fish Cultural Laboratory
Route 3, Post Office Box 86, Marion, Alabama 36756, USA
MARK L. HINMAN,1 DOUGLAS A. WINKELMANN,1
STEPHEN J. KLAINE, AND BILL A. SIMCO
Department of Biology, Memphis State University
Memphis, Tennessee 38152, USA
NICK C. PARKER2
U.S. Fish and Wildlife Service, Southeastern Fish Cultural Laboratory
Abstract.The 24-h median lethal concentration of nitrite (NO2-) for striped bass Morone saxatilis was 163 mg L in static toxicity tests. Exogenous chloride ions increased the tolerance of the fish for NO2-; CaCl2 was more than twice as effective as NaCl. Plasma NO2-, cortisol, and methemoglobin were correlated positively with environmental NO2-. Plasma NO2- and methemoglobin were correlated negatively with environmental Cl-, but cortisol was not reduced by the presence of environmental Cl-. Striped bass maintained NO2- in the plasma (0-45 mg NO2-/L) at concentrations below those in the environment (0-250 mg NO2-/L). However, striped bass were sensitive to NO2- that entered the plasma; methemoglobin levels greater than 60% and plasma NO2- levels greater than 70 mg L resulted in significant mortalities.
The toxicity of nitrite ions (NO2-) to fish has received much attention in recent years (Russo and Thurston 1977; Tomasso et al. 1981; Palachek and Tomasso 1984;Lewis and Morris 1986). Nitrite may reach high concentrations in intensive or water-recirculating aquaculture systems where ammonia, the major nitrogenous waste product of fish, is first converted by bacterial action to NO2-, a relatively toxic compound, and then to nitrate, a relatively nontoxic form. Nitrite may also reach toxic levels in streams as a result of contamination by industrial wastes or by the effluents of wastewater treatment plants.
Environmental NO2- enters the circulatory systems of fish through the gills (Perrone and Meade 1977). Nitrite is known to convert hemoglobin to me/hemoglobin, a form incapable of binding and transporting oxygen (Bodansky 1951; Wedemeyer and Yasutake 1978). However, the inconsistency of the relation between methemoglobin and mortality has led to suggestions that the primary cause of death is the toxic reaction to NO2- itself and not methemoglobinemia (Smith and Williams 1974; Brown and McLeay 1975; Crawford and Allen 1977).
' Present address: Exxon Biomedical Sciences, Inc., Mettlers Road CN 2350, East Millstone, New Jersey 08875-2350, USA.
2Present address: Texas Cooperative Fish and Wildlife Research Unit, Texas Tech University, Lubbock Texas 79409-2125, USA.
The toxicity of NO2- varies greatly among fish species. The 96-h median lethal concentration (LC50) of NO2- is 22 mg/L for rainbow trout Oncorhynchus mykiss (Russo and Thurston 1977), but 453 mg/L for largemouth bass Micropterus salmoides (Palachek and Tomasso 1984). This wide difference in toxicity has been attributed to the ability of some species (e.g., largemouth bass) to prevent NO2- from crossing the gill membrane and entering the blood, whereas other fish (e.g., channel catfish Ictalurus punctatus) lack this ability and concentrate NO2- in the blood (Palachek and Tomasso 1984).
The protection afforded by certain environmental ions against NO2- toxicity has also been investigated. The chloride ion (Cl-) has been reported to reduce NO2- toxicity in coho salmon Oncorhynchus kisutch (Perrone and Meade 1977), rainbow trout (Russo and Thurston 1977; Russo et al. 1981), channel catfish (Tomasso et al. 1979), and juvenile steelhead (anadromous rainbow trout) (Wedemeyer and Yasutake 1978). Crawford and Allen (1977) reported that the 48-h LC50 of NO2- for chinook salmon Oncorhynchus tshawytscha is 19 mg/L in fresh water and greater than 100 mg/L in seawater. These authors also strongly implicated environmental calcium as an inhibitor of NO2- toxicity. Wedemeyer and Yasutake (1978) reduced NO2- toxicity to juvenile steelhead by a factor of 3 with 0-200 mg Cl-/L as sodium chloride (NaCl) and by a factor of 50 with 0-200 mg Cl-/L as calcium chloride (CaCl2).
Our objectives were to determine for striped bass Morone saxatilis (1) the toxicity of NO2-, (2) the effects of NO2- on hematological profiles, and (3) the degree to which NaC1 and CaCl2 inhibit NO2- toxicity.
Striped bass averaging 27 ± 2 cm (SE) in standard length and 250 ± 4.4 g in live weight were maintained at the Southeastern Fish Cultural Laboratory, Marion, Alabama, for at least 4 weeks in 1,200-L fiberglass tanks supplied with aerated well water at a flow rate of two turnovers per hour. Physicochemical characteristics of the water were: temperature 23°C, pH 7.5, hardness 106 mg/L as calcium carbonate, alkalinity 108 mg/L as calcium carbonate, dissolved oxygen >6.0 mg/L, Cl- 38.6 mg/L, NO2- and ammonia < 0.01 mg/L. Fish were fed commercial fish food (40% protein) daily; feeding was discontinued 48 h before the fish were transferred to experimental tanks.
All tests were conducted in 100-L circular tanks supplied with the same water source and with constant aeration to maintain dissolved oxygen near saturation. Ten fish were stocked into each tank 72 h before an experimental treatment began. Water quality characteristics were measured in randomly selected tanks throughout the test and, with the exception of additives, were similar to the above water quality characteristics.
Survival of striped bass during exposure to NO2- (added as sodium nitrite) was determined in triplicate in a series of nine treatments in which the NO2- concentration ranged from 50 to 250 mg/L increments of 25 mg/L. In an experiment to evaluate the protective effects of Cl-, either CaCl2 (62.5-5,000 mg/L as Cl-) or NaCl (500-2,500 ma/ L as Cl- ) was added to tanks containing 250 mg NO2-/L, a concentration normally lethal to striped bass in fresh water. Nitrite (250 mg/L) and freshwater controls were included in triplicate.
Fish were sampled at 24 h, and blood was collected with ammonium-heparinized syringes from vessels in the caudal peduncle. All fish were anesthetized in a 0.02% solution of tricaine (MS-222)3 before they were bled. Sampling was completed within 5 min after initial disturbance and each fish was bled only once. Blood was centrifuged, and plasma was stored at 20°C until analyzed. We used the methods of Hainline (1958) to determine total hemoglobin and of Evelyn and Malloy (1938) to determine methemoglobin in whole blood. The percent methemoglobin was calculated as the percent total hemoglobin in the methemoglobin form. Plasma NO2- concentrations were determined according to USEPA (1974) as modified by Palachek and Tomasso (1984). Plasma concentrations were determined for cortisol, the dominant corticosteroid in fish (Donaldson 1981), by radioimmunoassay with a commercially prepared kit for cortisol (Serono Diagnostics); for glucose and total calcium (Ca) with clinical kits (Sigma Chemical Company); and for C1-, sodium (Na+), and potassium (K+) by selective ion electrodes (Nova Biomedical).
The data are presented as means ± SE unless stated otherwise. The 24-h LC50 was calculated by the trimmed Spearman-Karber method (Hamilton et al. 1977). Analysis of variance (ANOVA), followed by Duncan's multiple-range test, multiple regression, and correlation, were used to test for treatment effects where appropriate. The level of significance established in all tests was P £ 0.05.
Striped bass mortality showed a clear dose-response relation to NO2- concentration (Figure 1). The 24-h LC50 value was 163.0 ± 8.8 mgNO2-/L.
Methemoglobin and plasma NO2- in fish were linearly related to increasing environmental NO2- concentrations (r2 = 0.886 and 0.837, respectively; Figure 2), although plasma NO2- did not differ significantly from that in controls at NO2- concentrations of 100 mg/L or less. As NO2- in the water was increased from 150 mg/L to 175 mg/L, methemoglobin did not change (Figure 2), although mortality increased sharply from 20% to 80% (Figure 1).
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Plasma cortisol concentrations in fish were linearly related to NO2- concentrations in the water (r2 = 0.868; Figure 3), and they increased significantly over controls at environmental NO2- greater than 75 mg/L. Plasma glucose in fish varied but was not linearly related to NO2- concentration (Figure 3).
FIGURE 1.Percent mortality of striped bass exposed for 24 h to various concentrations of NO2- (N = 30).
Plasma electrolyte levels were not significantly altered in striped bass exposed to environmental NO2-. Plasma C1- and Na+ concentrations were significantly correlated (r2 = 0.862) but did not correlate with NO2- in the exposure water (Figure 3). Concentrations of plasma K+ (3.28 ± 0.08 meq/ L) and Ca (4.99 + 0.08 meq/L) did not vary significantly during the experiment.
Calcium Chloride and Sodium Chloride Effects
The addition of chloride salts as either CaCl2 (>250 mg/L as Cl-) or NaCl (³ 1,500 mg/L as Cl-) greatly increased the tolerance of striped bass exposed to a normally lethal concentration of 250 mg NO2- /L (Figure 4).
FIGURE 2.Mean (± SE) plasma NO2- and methemoglobin (as percent of total hemoglobin) in striped bass exposed for 24 h to various concentrations of NO2-. Bold line represents equal concentrations of NO2- in the plasma and environment (N ³ 13 unless otherwise indicated in the figure).
FIGURE 3.Mean (± SE) plasma cortisol, glucose, and Cl levels in striped bass exposed for 24 h to various concentrations of NO2- (N ³ 13 unless otherwise indicated in the figure).
Increasing C1-, as either CaCl2 or NaCl, significantly decreased methemoglobin and plasma NO2- in fish exposed to 250 mg NO2-/L (Figure 5). Methemoglobin and plasma NO2- concentrations in the fish were significantly lower in CaC12 than in NaCl. Methemoglobin was below 50% at 1,000 mg Cl-/L as CaCl2 but remained above 50% in all NaCl treatments.
Increasing levels of Cl as either CaCl2 or NaCl did not significantly decrease plasma cortisol in striped bass exposed to normally lethal concentrations of NO2- (Figure 6). Plasma glucose in fish in CaCl2 was elevated to low Cl- concentrations (£ 500 mg/L) but remained similar to control levels at Cl concentrations of 2,000 mg/L or more. Plasmas glucose in fish in NaCl did not significantly differ from control levels except at 2,500 mg Cl-/L (Figure 6).
FIGURE 4.Percent survival of striped bass exposed for 24 h to NO2- in various concentrations of Cl. Freshwater controls had 100% survival. Each treatment contained a normally lethal concentration (250 mg/L) of NO2- (N= 30).
Except for plasma Cl-, plasma electrolytes in striped bass exposed to NO2- and increasing Cl- concentrations did not significantly differ from control concentrations. Plasma Cl- concentrations were significantly higher in fish exposed to NaCl than in those exposed to CaCl2 at Cl- concentrations less than 2,000 mg/L (Figure 6). Plasma Na+ in fish in CaCl2 (159.6 ± 2.1 meq/L) and NaCl (161.2 ± 2.5 meq/L) did not significantly differ during the experiment. Plasma K+ (3.3 ± 0.09 meq/L) and Ca++ (4.6 ± 0.06 meq/L) concentrations were similar among all treatment groups.
Striped bass tolerated high environmental NO2- without a corresponding increase in plasma NO2-, and thus are among the species most resistant to NO2- toxicity. The 24-h percent methemoglobin and ratio of plasma to environmental NO2- values in striped bass (3.3 ± 1.6 and 0.03 ± 0.01, respectively, means ± SEs) were similar to those reported in largemouth bass (0.6 ± 0.4 and 0.08 ± 0.01) and bluegills Lepomis macrochirus (11.2 ± 1.1 and 0.10 ± 0.02) (Palachek and Tomasso 1984; Tomasso 1986). This is in contrast to channel catfish, where NO2- in the plasma exceeded that in the exposure medium (percent methemoglobin, 85.3 ± 4.8; ration of plasma to environmental NO2-, 3.2 ± 0.2; Tomasso 1986).
The linear relationship between methemoglobin, plasma NO2-, and environmental NO2- has been well documented (Smith and Williams 1974; Brown and McLeay 1975;Smith and Russo 1975; Crawford and Allen 1977; Perrone and Meade 1977; Eddy et al. 1983; Tomasso 1986). Nitrite is transported into freshwater fish by the mechanisms that normally transport chloride ions (Perrone and Meade 1977; Tomasso et al. 1979; Bath and Eddy 1980; Meade and Perrone 1980; Williams and Eddy 1986). The ability of the striped bass to maintain plasma NO2- below environmental concentrations suggests that there is a discrimination between the uptake of nitrite and chloride ions. Williams and Eddy (1988) proposed that fish less sensitive to NO2-, such as common carp Cyprinus carpio have a lower Cl- uptake rate which limits the amount of NO2- absorbed. Striped bass may also have a low Cl- uptake rate, resulting in slower accumulation of NO2- in the fish.
FIGURE 5.Mean (± SE) plasma NO2- and methemoglobin (as percent of total hemoglobin) in striped bass exposed for 24 h to NO2- in various concentrations of Cl-. Freshwater control levels of plasma nitrite and methemoglobin in striped bass were 2.02 ± 0.5 and 2.92 ± 0.6 mg/L, respectively. Each treatment contained a normally lethal concentration (250 mg L) of NO2- (N ³ 13 unless otherwise indicated in the figure).
FIGURE 6.Mean (± SE) plasma cortisol, glucose, and Cl- levels in striped bass exposed for 24 h to NO2- in various concentrations of Cl-. Freshwater control levels of plasma cortisol, glucose, and Cl- in striped bass were 95.0 ± 5.5 ng/mL, 91.8 ± 4.4 mg/dL, and 132.8 + 4.1 meq/L, respectively. Each treatment contained a normally lethal concentration (250 mg/L) of NO2- (N ³ 13 unless otherwise indicated in the figure.
Methemoglobin is readily formed in most fish exposed to NO2- in low-chloride waters, but the amount of methemoglobin considered fatal may vary with the species or environmental conditions (Russo 1980). Generally, methemoglobin is considered threatening to fish only at concentrations above 50% (Bowser et al. 1983). Large amounts of methemoglobin were formed in striped bass in response to high NO2concentrations, as seen in largemouth base (Palachek and Tomasso 1984), which may indicate a threshold level in the amount of NO2- excluded by some species.
Our results agree with reports that methemoglobinemia may not be the only toxic mechanism of NO2- in fish. In striped bass exposed to 150-175 mg NO2-/L, methemoglobin remained constant, although mortality increased sharply. Crawford and Allen (1977) observed that chinook salmon exposed to NO2- in seawater had high methemoglobin (74%) but low mortality (10%), whereas in fresh water methemoglobin was lower (44%) but mortality was higher (70%). They also observed that the gills of dying fish were red and not brown (the color characteristic of methemoglobinemia). Observations by Smith and Williams (1974) of mortalities in rainbow trout with methemoglobin lower than that in survivors led them to conclude that the fish died of a toxic reaction to NO2- itself rather than of methemoglobinemia.
The above research suggests that some mechanism other than methemoglobinemia may contribute to NO2- toxicity. Mortalities may be due to the concentration of NO2- in organs (Margiocco et al. 1983), to liver hypoxia followed by liver dysfunction (Arillo et al. 1984; Gaino et al. 1984), to lysosomal damage (Mensi 1982), to biochemical damage in hepatocytes (FAO 1984), and to gill damage (Wedemeyer and Yasutake 1978). However, studies of alternative mechanisms of nitrite-induced mortality have not been conclusive.
Elevations of plasma cortisol and glucose have been well documented as primary and secondary stress responses, respectively, in fish (Mazeaud et al. 1977; Mazeaud and Mazeaud 1981). Tomasso et al. (1981) found that cortisol increased in channel catfish exposed to NO2-; Carmichael et al. (1984a, 1984b) reported an increase in plasma cortisol and glucose in stressed largemouth bass. In striped bass, plasma cortisol increased as environmental NO2- concentration increased. Plasma glucose valued were similar to those normally expected for striped bass (Tisa et al. 1983). The variation in 24-h glucose levels may be due to an earlier secondary stress response in some fish than others. Although plasma glucose remained within the range reported by Tisa et al. (1983) for striped bass, plasma cortisol was elevated, indicating a stress response. This elevation suggests that, although striped bass have the ability to limit NO2- uptake, they still show stress-related symptoms due to the increase in plasma NO2- and methemoglobin.
Changes in plasma electrolyte balance (osmoregulatory dysfunction) caused by NO2- toxicity have not been widely investigated. Williams and Eddy (1986) reported a significant inhibition of Cl influx by environmental NO2- at low external Cl concentrations in rainbow trout and European perch Perca fluviatilis. Furthermore, the authors reported that fish with low Cl- uptake rates such as common carp and tench Tinca tinca were more resistant to NO2- than fish with higher Cl- uptake rates such as rainbow trout, perch, and northern pike Esox lucius. In our experiments, changes in plasma Cl- and Na+ were minor, possibly due to the ability of striped bass to maintain internal NO2- below that of the environment. The constant level of plasma calcium in striped bass was similar to that reported by Jensen et al. (1987) in common carp exposed to NO2-. Fish have been reported to maintain plasma calcium concentrations within a narrow range under a wide variety of environmental conditions (Andreasen 1985; Hunn 1985).
Calcium Chloride and Sodium Chloride Ejects
The protective effect of Cl- against NO2- toxicity was established by Bath and Eddy (1980) for Atlantic salmon Salmo salar and by Wedemeyer and Yasutake (1978) for steelhead. Tomasso (1986) found that increasing the environmental Cl- concentration increased the 24-h LC50 in channel catfish but not in largemouth bass. Despite the ability of striped bass to maintain plasma NO2- concentrations lower than those in the environment, the presence of higher than normal Cl, as either CaCl2 or NaCl, increased survival of striped bass exposed to normally lethal concentrations of NO2-.
Crawford and Allen (1977) established that NO2- toxicity to chinook salmon depended greatly on the salinity of the water. Tomasso et al. (1980) reported that CaCl2 did not significantly reduce NO2- toxicity in channel catfish compared with NaCl. In the present study, CaCl2 was at least twice as beneficial as NaCl in reducing the toxic effects of NO2- on striped bass. This may indicate a threshold in the amount of NO2- that striped bass are able to exclude. Striped bass exposed to NO2- concentrations above this threshold would then show a protective effect from Cl-. The effect of Cl- on NO2- is now recognized as a major factor in NO2- toxicity. Experiments that do not consider Cl- concentrations are of little use in developing an understanding of NO2- toxicity.
Increasing environmental Cl- significantly decreases methemoglobin and plasma NO2- in various fish (Huey et al. 1980; Meade and Perrone 1980; Tomasso et al. 1980; Hilmy et al. 1987). This relation was also seen in the present study. Perrone and Meade (1977), Russo and Thurston (1977), and Huey et al. (1980) demonstrated that the inhibitory effect of NaCl was due to the chloride ion and not to the sodium ion. Crawford and Allen (1977) found that increasing the calcium concentration (as calcium sulfate) in fresh water and seawater decreased the toxicity of NO2- but did not reduce methemoglobinemia. The calcium ion also seemed to have an inhibitory effect on NO2- toxicity in striped bass. Mortality, methemoglobin, and plasma NO2- were significantly lower in fish in CaCl2 than in fish in NaCl at equivalent Cl- concentrations. The protective effect of the calcium ion may be due to its ability to decrease gill permeability to water (Potts and Fleming 1970; Ogawa 1974; Wendelaar Bonga et al. 1983; Ogasawara and Hirano 1984). High levels of environmental calcium have been reported to reduce the efflux of Na+ and Cl- across the gills (Eddy 1975; McWilliams and Potts 1978). Krous et al. (1982) proposed that reduction in the efflux of Cl- across the gills results in a reduced Cl- uptake and therefore a reduced NO2- uptake.
The elevation of plasma cortisol concentrations above control levels at 24 h indicated that, although increasing environmental Cl- concentrations increased survival of striped bass at lethal (250 mg/L) levels of NO2-, they did not reduce the primary stress response of striped bass to NO2-. Although plasma glucose and electrolyte levels in fish exposed to increasing environmental Cl- were variable, they were still comparable to those found in striped bass exposed to increasing NO2- concentrations in the absence of salt. Striped bass exposed to lethal NO2- concentrations for 24 h in increasing environmental Cl- showed a primary stress response, but they showed no secondary stress response or osmoregulatory dysfunction.
In this study, striped bass tolerated relatively high environmental concentrations of NO2- by limiting NO2- uptake to maintain concentrations in the blood below environmental levels. The presence of high environmental levels of Cl aided in the exclusion of NO2-, permitting the fish to tolerate otherwise lethal NO2- levels. Better protection against NO2- was provided by CaCl2 than by NaCl. However, striped bass were sensitive to NO2- that entered the plasma; methemoglobin levels greater than 60% and plasma NO2- levels greater than 70 mg/L resulted in significant mortalities, as seen in other species.
We than K. Davis, T. Gartner, M. Konikoff, and J. Tomasso for critically reviewing this manuscript and C. Goudie for technical assistance.
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Received December 28, 1989
Accepted July 15, 1990