Comparative toxicity of nitrite to freshwater fishes

Aquatic Toxicology, 8 (1986) 129-137 Elsevier

129

AQT 00193 COMPARATIVE

TOXICITY OF NITRITE TO FRESHWATER

FISHES

J.R. TOMASSO

Aquatic Station, Southwest Texas State University, San Marcos, TX 78666, U.S.A. (Received 1 October 1985; revised version received 19 December 1985; accepted 19 February 1986)

The primary purpose of this study was to determine the toxicity of nitrite to several species of freshwater fishes and to investigate the underlying physiological mechanisms which account for differential toxicity among species. Green sunfish (Lepornis cyanellus) was the most resistant species studied while the channel catfish (Ictaluruspunctatus) was the least resistant. Ninety-six hour median lethal concentrations correlated significantly with both the percentage of hemoglobin in the methemoglobin form and plasma nitrite concentrations among species. Plasma nitrite levels also correlated significantly with percent methemoglobin. Environmental chloride did not increase the tolerance of largemouth bass (Micropterus salmoides) to nitrite toxicity as it did for channel catfish. These results indicate that plasma nitrite concentrations are the primary determinant of the toxicity of nitrite to fishes. Further, plasma nitrite concentrations in various species depend on the discriminatory ability of the active transport system in fish gills which ordinarily transports chloride ions. Key words: nitrite; methemoglobin; plasma nitrite

INTRODUCTION T h e t o x i c i t y o f nitrite to fishes has received m u c h a t t e n t i o n in recent years (Russo a n d T h u r s t o n , 1977; T o m a s s o et al., 1981; P a l a c h e k a n d T o m a s s o , 1984b). Nitrite m a y r e a c h toxic c o n c e n t r a t i o n s in high d e n s i t y a q u a c u l t u r e systems a n d in flowing waters d u e to i n d u s t r i a l c o n t a m i n a t i o n . T h e toxicity o f nitrite is t h o u g h t to be due to its a b i l i t y to o x i d i z e h e m o g l o b i n to m e t h e m o g l o b i n , a f o r m n o t c a p a b l e o f b i n d ing o x y g e n ( T o m a s s o et al., 1979; H u e y et al., 1980). H o w e v e r , a n inconsistent relat i o n s h i p b e t w e e n m e t h e m o g l o b i n e m i a a n d m o r t a l i t y has led o t h e r s to suggest t h a t m e t h e m o g l o b i n e m i a m a y n o t b e the p r i m a r y toxic m e c h a n i s m o f nitrite toxicity. Instead, s o m e as yet u n i d e n t i f i e d m e c h a n i s m m a y be the p r i m a r y cause o f toxicity ( S m i t h a n d W i l l i a m s , 1 9 7 4 ; B r o w n a n d M c L e a y , 1975; C r a w f o r d a n d A l l e n , 1977). T h e t o x i c i t y o f nitrite to f r e s h w a t e r fish species m a y v a r y c o n s i d e r a b l y . R a i n b o w t r o u t (Salmo gairdneri) have been r e p o r t e d to have 96-h m e d i a n lethal c o n c e n t r a tions (LCs0) o f less t h a n 1 m g / l ( R u s s o et al., 1981) while l a r g e m o u t h bass (Micropterus salmoides) e x h i b i t e d a 96-h LCs0 o f 452.7 ___ 24.6 m g / l ( m e a n ___ SE) ( P a l a c h e k a n d T o m a s s o , 1984b). T h e a b i l i t y o f l a r g e m o u t h bass to p r e v e n t nitrite f r o m c r o s s i n g the gill m e m b r a n e a n d e n t e r i n g the b l o o d as c o m p a r e d to the a b i l i t y 0166-445X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

130 o f fish such as r a i n b o w t r o u t to c o n c e n t r a t e nitrite in their b l o o d has been suggested as the reason for the wide range o f toxicities a m o n g species (Palachek a n d T o m a s s o , 1984b). The p u r p o s e o f this study was to d e t e r m i n e the toxicity o f nitrite to several species o f freshwater fishes, a n d to elucidate the u n d e r l y i n g physiological m e c h a n i s m s which a c c o u n t for differential toxicity a m o n g species. Specifically, 96-h LCs0 values, m e t h e m o g l o b i n levels, a n d p l a s m a nitrite c o n c e n t r a t i o n s were d e t e r m i n e d . T h e effect o f e n v i r o n m e n t a l chloride, a k n o w n i n h i b i t o r o f nitrite toxicity, was also studied in a susceptible a n d a resistant species. I n f o r m a t i o n f r o m previous similar studies is also i n c l u d e d for c o m p a r a t i v e purposes ( P a l a c h e k a n d T o m a s s o , 1984a,b). METHODS C h a n n e l catfish used in this study were o b t a i n e d f r o m the San Marcos, Texas State Fish H a t c h e r y . L a r g e m o u t h bass, bluegill, s m a l l m o u t h bass, a n d green sunfish were provided b y the U.S. Fish a n d Wildlife Service N a t i o n a l Fish H a t c h e r y a n d T e c h n o l o g y C e n t e r (San Marcos). G o l d f i s h were p r o d u c e d in the culture p o n d s at Southwest Texas State University. All fish were acclimated to l a b o r a t o r y c o n d i t i o n s for at least 1 m t h prior to testing ( t e m p e r a t u r e = 23°C; p H = 7.2-7.4; hardness = 300 mg/1 as CaCO3; dissolved oxygen > 6.0 m g / l ; alkalinity = 255 m g / l as CaCO3; chloride --- 22 m g / l ; nitrite < 0.01 m g / l ; sulfate = 31 m g / l ; n i t r a t e - n i t r o g e n = 7.4 mg/1; total p h o s p h o r o u s = < 0.1 mg/1). T a b l e I lists the species discussed a n d their weights. Details o f origin, h o l d i n g a n d testing o f fish TABLE I Species and weights (mean _+ of SE) of organisms used in nitrite toxicity tests. Species

Weight (g) LCso

2.0 12.8 3.0 80.2 Largemouth bass (Micropterus salmoides) 2.8 36.3 Smallmouth bass (Micropterus dolomieui) 3.1 8.3 Bluegill (Lepomis macrochirus) 7.3 Green Sunfish (Lepomis cyanellus) 3.4 Tilapia (Tilapia aurea)

Fathead minnow (Pimephales promelas) Goldfish (Carassius auratus) Channel catfish (Ictalurus punctatus)

_+ 0.4a ___ 0.5b + 0.1 c + 2.8b + 0.0c +__ 1.4b _+ 0.1 b __. 0.7b + 0.3b + 0.2c

Weight (g) sublethal studies 42.7 12.6 10.6 19.6 19.1

_+ 1.9b (MN) + 0.3c (M) _+ 0.2c (N) ___ 1.0c (M) _+ 0.5c (N)

21.9 _+ 1.5b (MN) 31.2 + 2.2c (M) 21.7 + 1.0c (N)

a From Palachek and Tomasso, 1984a. b Present study. From Palachek and Tomasso, 1984b. M, methemoglobin determination only; N, plasma nitrite determination only; MN, both methemoglobin and plasma nitrite determinations.

131

TABLE

II

Water quality in test aquaria during nitrite toxicity tests (range or mean 5: S E ) Time 0

Time 2 4 h

Time 96 h

Nitritea (% nominal) 101 5 : 1

(24) a'f

98 5:2

(12) c

102 5 : 1

102 _+ 1 (23) b

(79) c

9 9 _+ 2 ( 1 0 ) b

102 5 : 1

(20) ~

102 +

1 (19) b

pH 7.7 -

8 . 2 (12) a

7.8 -

8.2 (12) a

7.7 -

8.2 (12) c

7.7 -

8.1 (88) ~

7.7 -

8.2 (20) ¢

7.9 -

8 . 4 (26) b

8.1 -

8 . 4 (26) b

8.0 -

8 . 2 (26) b

Alkalinity e (mg/I as CaC03) 2 5 0 + 4 (12) a

153 _+

4 (12) a

2 3 4 +_ 2 ( 1 2 ) c

223 + 5 (24) c

166 5:

4(27) c

236 _

190 + 8 (18) b

164 _+ 13 (11) b

267 5 : 3 c

255 _+ 4 (37) c

2 0 2 + 4 (27) ¢

2 4 0 _+ 5 (26) b

2 0 9 ___ 5 (18) b

191 _+ 7 (11) b

a From Palachek and T o m a s s o ,

1984a.

5 (26) b

Hardness e (mg/l as CaC03) 2 6 8 +__ 3 (12) a

191 _+ 4 (12) a

b Present study. c From Palachek and Tomasso, 1 9 8 4 b . d USEPA,

1974.

e Hach Chemical Company, 1973.

f Pooled data for all time periods. Number of samples are given in parentheses.

used in the previous studies are given in the respective papers. Median lethal concentrations (Thompson, 1947) were determined under static conditions in aerated glass aquaria containing 5 fish each and either 15 or 30 ! of test solution. A series of six aquaria was used to determine each LCs0, and each series was repeated two to eight times for each species. Desired nitrite concentrations were developed by addition of sodium nitrite. Dissolved oxygen concentrations remained near saturation throughout the study. Temperature remained a constant 23°C. Other water quality characteristics are summarized in Table II. The percent of hemoglobin in the methemoglobin form (Evelyn and Malloy, 1938; Hainline, 1958) and plasma nitrite concentrations (USEPA 1974 as modified by Palachek and Tomasso, 1984b) were determined on fish exposed to 40 mg/l nitrite for 24 h. Forty mg/1 nitrite were applied because this is the highest exposure level which the least resistant species (channel catfish) can consistently survive for 24 h. All sublethal tests were conducted as previously described (Palachek and Tomasso, 1984b).

132

The effect of environmental chloride on nitrite toxicity to a susceptible (channel catfish) and a resistant (largemouth bass) species was studied by determining the 24-h LCso of nitrite to channel catfish and largemouth bass in water containing different concentrations o f chloride. The large differences in resistance between the two species precluded direct comparisons (i.e., exposing both species to the same chloride concentrations and then comparing the resulting 24-h LCso). Instead, an initial 24-h LCs0 for each species was determined in water receiving no additional chloride (baseline chloride = 22 mg/l). Subsequent 24-h LCs0 values were determined in water containing a chloride to nitrite ionic ratio of 5 and 10 using the 24-h LCs0 for each species as the basis for the nitrite ion concentration. Desired chloride concentrations were developed by addition of sodium chloride. The 24-h LCso values determined in the chloride augmented water were then divided by the initial 24-h LCs0 value to produce a ratio of chloride augmented to initial 24-h LCs0 values. Analysis of variance (ANOVA) followed by a Tukey range test, regression analysis, correlation, and Student's t-test were applied where appropriate. A probability < 0.05 was considered significant.

RESULTS A N D DISCUSSION

The acute toxicity of nitrite to fishes varied considerably with green sunfish being the most resistant (96-h LCs0 = 526.8 _+ 48.0 mg/l) and channel catfish being the least resistant (96-h LCso = 23.3 +_ 6.5 mg/1) (Fig. 1). As a group the centrarchids were more resistant to nitrite than the other species tested. However, tilapia was one of the most sensitive species tested indicating that the extreme resistance exhibited by the centrarchids is not a characteristic shared by all perciforms. 600

GSF,SMB,LMB 5OO BG,GF,FHM FHM,TLP "~400 TLP,CC ~300

/

•.¢200 IO0

HI 4,

4 H

II

4

3

I-q

FHM GF CC LMBSMBBGGSFTLP Fig. 1. Ninety-six hour median lethal concentrations (LCs0) of nitrite to eight species of freshwater fishes (mean _+ SE). N u m b e r s of replications are given in each column. Species with similar means (ANOVA + Tukey range test) appear on the same line at the top of the figure. F H M , fathead minnow; GF, goldfish; CC, channel catfish; LMB, largemouth bass; SMB, smallmouth bass; BG, bluegill; GSF, green sunfish; T L P , tilapia. CC, LMB and T L P data are from Palachek and T o m a s s o (1984b). F H M data are from Palachek and T o m a s s o (1984a).

133

Nitrite toxicity to fishes is affected by environmental factors such as calcium (Wedemeyer and Yasutake, 1978), pH (Russo et al., 1981), alkalinity (Huey et al., 1980) and chloride (Crawford and Allen, 1977; Perrone and Meade, 1977); thus, it is difficult to compare toxicity values determined in different laboratories. Two generalizations may be made however: first, the acute toxicity values of the centrarchids presented here and elsewhere (Huey et al., 1982) are higher than those reported for any other species. Second, although channel catfish appear very susceptible to nitrite when compared to the other species tested in this study, they are much more resistant to nitrite than salmonids (Russo et al., 1974; Thurston et al., 1978; Russo et al., 1981). The percent of hemoglobin in the methemoglobin form in fish exposed to 40 mg/l nitrite for 24 h varied considerably among species (Fig. 2). The bleugill and largemouth bass generated the lowest percentages of methemoglobin which is consistent with the higher LCs0 values observed. Conversely, the channel catfish demonstrated the highest methemoglobin values and the lowest LCs0. Plasma nitrite concentrations in fish exposed to 40 mg/l nitrite for 24 h varied from a low of 3.5 + 0.3 rag/1 in largemouth bass to a high of 128.7 _+ 8.9 rag/1 in channel catfish. Plasma to environment ratios clearly demonstrated that the centrarchids were maintaining a plasma nitrite concentration below that of the environment, while the other species were concentrating the nitrite in their plasma (Fig. 3). In the non-centrarchids, nitrite was apparently being transported across the gill membranes by the active transport mechanism which ordinarily transports chloride into the fish (Perrone and Meade, 1977; Tomasso et al., 1979; Bath and Eddy, 1980; Meade and Perrone, 1980; Krous et al., 1982). Species with low concentrations of plasma nitrite demonstrated higher LCs0 values than those with higher plasma nitrite levels. Although channel catfish concentrated nitrite in their plasma to levels higher than any other species in this study (3.2 times the environmental concentra-

100" Z

~8o. o

LMB,BG GF,TLP ?-,C

._1

~6oW LLI

~20'

,ol 14 12

6

GF CCLMB BG TLP Fig. 2. Percent of hemoglobin in the methemoglobin form (mean _+ SE) in five species of freshwater fishes exposed to 40 m g / l nitrite for 24 h. N u m b e r s o f fish analyzed are given in each column. Species with similar means ( A N O V A + Tukey range test) appear on the same line at the top of the figure. The key to species and references are given in Fig. 1.

134 04"

LMB,BG

I< Q:3'

I-- GF,TLP

,,z, 2.

~1. < _J

H 518 14 GF CC LMB BG TLP

Fig. 3. The ratio (arithmetic mean _+ SE) of plasma to environmental (env.) nitrite in five species of freshwater fishes exposed to 40 mg/1 nitrite for 24 h. Numbers of fish analyzed are given in each column. Species with similar means (ANOVA + Tukey range test) appear on the same line at the top of the figure. The key to species and references are given in Fig. 1. tion), salmonids have been reported to concentrate nitrite in their plasma by a factor greater t h a n 80 (Eddy et al., 1983). Crayfish (Procambarus clarkii) have been observed to concentrate nitrite in their h e m o l y m p h to over 900 rag/1 when exposed to 40 m g / l nitrite for 24 h under conditions similar to the present study (Gutzmer and T o m a s s o , 1985). W h e n c o m p a r i n g the five species for which LCs0, m e t h e m o g l o b i n , and plasma nitrite i n f o r m a t i o n are available, some significant relationships are evident. First, LCs0 values and m e t h e m o g l o b i n e m i a correlate significantly (Fig. 4A). This supports the contention that m e t h e m o g l o b i n e m i a and the ensuing h y p o x i a (Kiese, 1974) is the LMB •.j 500"~ 3 7 5 1 ~ /

A

• "~_

r:-0,929

~ 2501BG 125"1

~ G F

I

.

.

Ty ,

C c

.

20 40 60 80 100 % METHEMOGLOBIN 0 500}LM B B 375|_ 250L ~ r:-0.927 ~o /BG ~: 1251 GF ~ " , ~ L £ ~ C C /

z

o,lool ,,,

'~

.

.

"

=

.

50 100 150 PLASMA NITRITE (mg/liter)

cc.

limb G

C

5"0

1do

156

PLASMA NITRITE (mg/liter)

Fig. 4. Correlation between (A) the 96-h LCso of nitrite and percent methemoglobin in fishes exposed to 40 m g / l nitrite for 24 h; (B) the 96-h LCso and plasma nitrite concentrations in fishes exposed to 40 rag/1 nitrite for 24 h and; (C) percent methemoglobin and plasma nitrite concentrations in fish exposed to 40 m g / l for 24 h. Slopes and y intercepts for each line are (A) 390.70, - 4.25; (B) 369.0, - 2.73; (C) 6.51, 0.62, respectively. The key to species and references are given in Fig. 1.

135

primary toxic mechanism of nitrite in fishes (Tomasso et al., 1980; Huey et al., 1980; Bowser et al., 1983). However, several investigators (Smith and Williams, 1974; Brown and McLeay, 1975; Crawford and Allen, 1977; Margiocco et al., 1983) have suggested that methemoglobinemia is not the primary toxic mechanism of nitrite. Citing mortalities of largemouth bass with low methemoglobin levels during exposure to high nitrite concentrations, Palachek and Tomasso (1984b) suggested methemoglobinemia is the toxic mechanism in fish which concentrate nitrite in their plasma, while fish such as largemouth bass which maintain plasma nitrite concentrations below environmental levels may die of some other, as yet undescribed, mechanism. The actual contribution of methemoglobinemia to nitrite toxicity, be it a primary or a secondary mechanism, in various fish species remains to be resolved. The second relationship which is evident is the significant correlation between plasma nitrite and LCso values (Fig. 4B). This would suggest that regardless of the mechanism of toxicity, the ability of nitrite to enter the fish is a primary determinant of toxicity. However, the nitrite excluding species tested died apparently with little nitrite in their plasma (present study; Palachek and Tomasso, 1984) indicating that this relationship may be stronger among species which concentrate nitrite than among nitrite excluding species. The third relationship is the significant correlation between plasma nitrite concentrations and methemoglobin percentages (Fig. 4C). This suggests that the primary determinant of methemoglobin levels is the presence of nitrite in the blood. Further, the hemoglobins in the various species seem to be equally susceptible to oxidation by nitrite. Differential methemoglobin reducing activity (Cameron, 197 l) can probably also be discounted as a reason for the differential toxicity among species since

o10' -I 8 ,

c/

'" 6' o

"4' ~2'

LMBj_

0 ._I "-c o

0 ~ ~b CI-:NO~ IONIC RATIO Fig. 5. Relationship between 24-h LCs0 values and environmental chloride ratio for largemouth bass (LMB) and channel catfish (CC). The chloride ratio is a molecular ratio of chloride ions to the nitrite ion concentration that constitutes the 24-h LCs0 in test water with no additional chloride added. The slope o f the LMB line does not differ from 0. The slope of the CC line differs from both 0 and the slope o f the LMB line.

136

variable activity would affect methemoglobin levels at the various plasma nitrite concentrations and decrease the correlation between the two (Fig. 4C). Increasing the environmental chloride concentrations increased the 24-h LCso of channel catfish in a linear fashion (Fig. 5). However, similar treatments did not affect the 24-h LC~0 of nitrite to largemouth bass. These observations support the contention that nitrite is actively transported into fish which concentrate nitrite by the active transport mechanism for chloride. Since channel catfish are nitrite concentrators, environmental chloride competitively excludes nitrite resulting in an increased 24-h LCs0. Largemouth bass, on the other hand, do not concentrate nitrite in their plasma so additional environmental chloride has no effect. This observation also indicates that centrarchids do indeed exclude nitrite from entering the blood and do not maintain low blood concentrations by high excretory activity. A similar failure of chloride to increase the LCs0 in bluegill was reported by Huey et al. (1982). However, the authors attributed the apparent discrepancy to interactions among chloride, nitrite, phosphate buffers and temperature. In summary, the range of toxic concentrations of nitrite to fishes seems to be due mostly to the differential ability of the various species to concentrate or exclude nitrite from the plasma. The basis of this differential ability is the ability of the active transport mechanism on the gill membranes which transports chloride into a fish to discriminate chloride from nitrite ions. ACKNOWLEDGEMENTS

Financial support for this study was provided by a Southwest Texas State University Faculty Research Grant. Fish were provided by the San Marcos State Fish Hatchery and the San Marcos National Fish Hatchery and Technology Center (U.S. Fish and Wildlife Service). P.M. Mazik and C.A. Caldwell provided technical assistance. J.R. Koke and G.J. Carmichael critically reviewed the manuscript.

REFERENCES Bath, R.N. and F.B. Eddy, 1980. Transport of nitrite across fish gills. J. Exp. Zool. 214, 119-121. Bowser, P.R., W.W. Falls, J. Van Zandt, N. Collier and J.D. Phillips, 1983. Methemoglobinemia in channel catfish: methods of prevention. Prog. Fish-Cult. 45, 154-158; 106, 105-109. Brown, D.A. and D.J. McLeay, 1975. Effect of nitrite on methemoglobin and total hemoglobin of juvenile steelhead trout (Salmo gairdneri). J. Fish. Res. Bd. Can. 35, 822-827. Cameron, J.N., 1971. Methemoglobin in erythrocytes of rainbow trout. Comp. Biochem. Physiol. 40A, 743-749. Crawford, R.E. and G.H. Allen, 1977. Seawater inhibition of nitrite toxicity to chinook salmon. Trans. Am. Fish. Soc., 105, 106-109. Eddy, F.B., P.A. Kunzlik and R.N. Bath, 1983. Uptake and loss of nitrite from the blood of rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L., in fresh water and in dilute sea water. J. Fish Biol. 23, 105-116.

137 Evelyn, H.A. and H.T. Malloy, 1938. Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood. J. Biol. Chem. 126, 655-662. Gutzmer, M.P. and J.R. Tomasso, 1985. Nitrite toxicity to the crayfish Procambarus clarkii. Bull. Environ. Contam. Toxicol. 34, 369-376. Hach Chemical Company, 1973. Hach water analysis handbook. Hach Chemical Co., Ames, IA. 140 p. Hainline, A., 1958. Hemoglobin. In: Standard methods of clinical chemistry, edited by D. Seligson, W.B. Saunders Co., Philadelphia, PA., pp. 268-269. Huey, D.W., B.A. Simco and D.W. Criswell, 1980. Nitrite-induced methemoglobin formation in channel catfish. Trans. Am. Fish. Soc. 109, 558-562. Huey, D.W., M.C. Wooten, L.A. Freeman and T.L. Beitinger, 1982. Effect of pH and chloride on nitrite-induced lethality in bluegill (Lepomis rnacrochirus). Bull. Environ. Contain. Toxicol. 28, 3-6. Kiese, M., 1974. Methemoglobinemia: a comprehensive treatise. CRC Press, Inc., Cleveland, OH, 259 p. Krous, S.R., V.S. Blazer and T.L. Meade, 1982. Effect of acclimation time on nitrite movement across the gill epithelia of rainbow trout: the role of 'chloride-cells'. Prog. Fish-Cult. 44, 126-130. Margiocco, C., A. Arillo, P. Mensi and G. Schenone, 1983. Nitrite bioaccumulation in Salmo gairdneri Rich. and hematological consequences. Aquat. Toxicol. 3, 261-270. Meade, T.L. and S.J. Perrone, 1980. Effect of chloride ion concentration and pH on the transport of nitrite across the gill epithelia of coho salmon (Oncorhynchus kisutch). Prog. Fish-Cult. 42, 71-72. Palachek, R.M. and J.R. Tomasso, 1984a. Nitrite toxicity to fathead minnows: effect of fish weight. Bull. Environ. Contain. Toxicol. 32, 238-242. Palachek, R.M. and J.R. Tomasso, 1984b. Toxicity of nitrite to channel catfish (lctalurus punctatus), tilapia (Tilapia aurea), and largemouth bass (Micropterus salmoides): evidence for a nitrite exclusion mechanism. Can. J. Fish. Aquat. Sci. 41, 1739-1744. Perrone, S.J. and T.L. Meade, 1977. Protective effect of chloride on nitrite toxicity to coho salmon (Oneorhynchus kisutch). J. Fish. Res. Bd. Can. 34, 486-492. Russo, R.C. and R.V. Thurston, 1977. The acute toxicity of nitrite to fishes. In: Recent advances in fish toxicology, edited by R.A. Tubb, EPA Ecol. Res. Ser., EPA-600/3-77-085, pp. 118-131. Russo, R.C., C.E. Smith and R.V. Thurston, 1974. Acute toxicity of nitrite to rainbow trout (Salmo gairdnert). J. Fish. Res. Bd. Can. 31, 1653-1655. Russo, R.C., R.V. Tburston and K. Emerson, 1981. Acute toxicity of nitrite to rainbow trout (Salmo gairdnerl3: effects of pH, nitrite species, and anion species. Can. J. Fish. Aquat. Sci. 38, 387-393. Smith, C.E. and W.G. Williams, 1974. Experimental nitrite toxicity in rainbow trout and chinook salmon. Trans. Am. Fish. Soc. 103, 389-390. Thurston, R.V., R.C. Russo and C.E. Smith, 1978. Acute toxicity of ammonia and nitrite to cutthroat trout fry. Trans. Am. Fish. Soc. 107, 361-367. Tomasso, J.R., B,A. Simco and K.B. Davis, 1979. Chloride inhibition of nitrite-induced methemoglobinemia in channel catfish (Ictalurus punctatus). J. Fish. Res. Bd. Can. 36, 1141-1144. Tomasso, J.R., K.B. Davis and B.A. Simco, 1981. Plasma corticosteroid dynamics in channel catfish (lctalurus punctatus) exposed to ammonia and nitrite. Can. J. Fish. Aquat. Sci. 38, 1106-1112. U.S. Environmental Protection Agency (USEPA), 1974. Methods for chemical analysis of water and wastes. EPA-625/6-74-003. Wedemeyer, G.A. and W.T. Yasutake, 1978. Prevention and treatment of nitrite toxicity in juvenile steelhead trout (Salmo gairdnerl). J. Fish. Res. Bd. Can. 35, 822-827.