Regulation of blood haemoglobin and electrolytes in rainbow trout Salmo gairdneri (Richardson) exposed to nitrite

Aquatic Toxicology, 13 (1988) 13-28

13

Elsevier

AQT 00283

Regulation of blood haemoglobin and electrolytes in rainbow trout Salmo gairdneri (Richardson) exposed to nitrite

E.M. Williams and F.B. E d d y Dundee University, Dundee DD1 1HN, Scotland, U.K. (Received 17 July 1987; revision received 2 November 1987; accepted 23 January 1988)

Rainbow trout (30-70 g) were exposed to environmental nitrite for periods varying from 2 to 24 h, and after 12 h blood plasma nitrite concentration was eight times the environmental levels (0.5 mmol 1- 1). The rise was followed by an increase in methaemoglobin levels from around 3070to over 6007o. After 2 h nitrite exposure the concentrations of plasma potassium, sodium and chloride fell, followed 2 h later by an increase in intra-erythrocyte potassium and sodium concentration with increased red cell volume. Twelve h nitrite exposure led to an increase in the red cell population, the new cells being smaller and containing less haemoglobin. After 24 h exposure, fish fell into two groups: nitrite-intolerant fish with high levels of plasma nitrite and methaemoglobin and nitrite-tolerant fish with low plasma levels. All fish surviving 24 h nitrite exposure had lower plasma potassium levels than unexposed fish. Key words: Freshwater fish; Nitrite toxicity; Methaemoglobin; Blood; Electrolytes; Erythrocytes

INTRODUCTION

The tolerance of several freshwater fish species to nitrite has recently been reviewed (EIFAC, 1984; Lewis and Morris, 1986; Eddy and Williams, 1987), and some major points regarding its toxic action are: (1) there is great variety in tolerance to nitrite, species such as salmonids being most susceptible and eels among the least susceptible; (2) the most susceptible species have the highest chloride uptake rates while a low chloride uptake rate is characteristic of tolerant species; (3) chloride strongly ameliorates nitrite toxicity as a competitor at the site of branchial anion exchange. A well-documented effect of nitrite exposure is the production of methaemoglobinaemia. In normal vertebrate blood almost all the iron of Correspondence to: E.M. Williams, Dundee University, Dundee DD1 IHN, Scotland, U.K. 0166-445X/88/$ 03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

14 haemoglobin is in the ferrous state which is essential for oxygen transport while less than 5% is in the ferric state, the oxidised form of haemoglobin known as methaemoglobin, an inactive form incapable of reversible oxygen binding or of combination with carbon monoxide (Gruca and Grigg, 1980; Board et al., 1977; Williams and Eddy, 1988; Jaff6, 1981). Another effect o f nitrite on haemoglobin is to shift the oxygen dissociation curve to the left so that the release of oxygen to the tissues is impaired (Darling and Roughton, 1942). In man, four main pathways are involved in the reduction of methaemoglobin. The system of greatest importance is NADH-methaemoglobin reductase (60-90%), followed by two non-enzymatic pathways, those involving ascorbic acid (16%) or reduced glutathione (GSH) (12%); NADPH-methaemoglobin reductase appears to have a smaller role (5°70) (Scott, 1965). Many species of fish have erythrocytes that exhibit NADH-methaemoglobin reductase activity (Freeman et al., 1983), and in rainbow trout the enzymic activity is similar to that found in human red cells (Scott and Harrington, 1985). Dietary ascorbic acid reduced methaemoglobin levels in rainbow trout exposed to nitrite (Blanco and Meade, 1980) as did intravenous injection (Cameron, 1971). Levels of methaemoglobin around 60-70°/0 can be tolerated by rainbow trout and chinook salmon (Smith and Williams, 1974; Brown and McLeay, 1975), but higher levels prove lethal. Fish exposed to nitrite in the water are able to accumulate the anion in the blood plasma and tissues (Bath and Eddy, 1980; Margiocco et al., 1983), with nitrite freely crossing the red cell membrane oxidizing haemoglobin to methaemoglobin with a reduction of functional haemoglobin. In the short term (24 h) there is little effect on total haemoglobin values (Huey et al., 1980; Scarano et al., 1984), but with longer nitrite exposure total haemoglobin levels decrease even after removal of external nitrite (Scarano and Saroglia, 1984). The energy demand for sustained methaemoglobin reductase activity may shorten red cell life and evidence of increased haemolysis of red cells in the spleen of sea bass exposed to 5.4 mmol 1- 1 nitrite for 96 h was reported by Scarano et al. (1984), whilst Fletcher (1977) observed in nitrite-exposed rainbow trout that, instead of being the normal bright green colour, the gall bladder was swollen with an opaque red-brown fluid, possibly through increased haemolytic breakdown of the red cells. The haematocrit in both rainbow trout and Atlantic salmon fell from around 42% to 30% in 12 h when exposed to 0.7 mmol 1-1 nitrite (Eddy et al., 1983). This study was designed to investigate the haematological consequences of shortterm (less than 12 h) and longer term nitrite exposure (24 h) to trout. Since nitrite ions are concentrated in the blood plasma the concentrations of other plasma electrolytes are likely to be affected and a major objective of this study was to investigate how extracellular electroneutrality is maintained, a point of significant interest in ionic regulation in fish, and one seldom addressed (Evans, 1984; Payan and Girad, 1984). Furthermore, little is known about how methaemoglobin formation and subsequent hypoxia affects the red blood cell as a whole, again an aspect

15

of red cell physiology which has seldom been addressed (Jaff6, 1980). This study aims to investigate anion homeostasis in fish when perturbed by nitrite. MATERIALS AND METHODS

Rainbow trout (30-70 g) were obtained in a local hatchery and held in running tap water containing (in mmol 1- 1) sodium 0.22, calcium 0.1 and chloride 0.2, pH 7.4, 10°C, under a 12:12 hour photoperiod. All fish were fasted for three days prior to experiments which were all performed during November and December 1986. Rainbow trout were placed in 20-1 tanks containing aerated tapwater (changed daily), for three days, before sodium nitrite addition to a final concentration of 0.5 mmol 1- 1. The nitrite concentration chosen was sufficiently high to have immediate effects, but was lower than the 24-h LCso value of 0.7 mmol 1- ~ (Eddy et al., 1983). After the desired period of nitrite exposure fish were quickly killed by a sharp blow to the head and 0.5 to 1 ml of blood was taken from the caudal vein into heparinized capillary tubes.

Blood electrolytes Plasma nitrite concentration was determined colorimetrically (Schecter et al., 1972), plasma chloride by coulometric titration (Radiometer CMTI0 Chloride titrator) and all cation concentations by atomic absorption spectrometry using a Pye Unicam SP1800 spectrophotometer. Intra-erythrocyte sodium and potassium concentration was determined according to the method of Borne and Cossins (1984).

Blood and haematological indices Total blood haemoglobin and percent methaemoglobin were determined spectrophotometrically using 50/zl whole blood (Evelyn and Malloy, 1938). Red blood cell numbers were determined using a haemocytometer and blood haematocrit by centrifugation of whole blood in heparinized glass capillary tubes. To measure red cell fragility, 50 #1 of freshly drawn heparinized blood was added to ten tubes containing 3 ml of unbuffered saline solutions of 9, 7, 6, 5, 4.5, 4, 3.5, 3, 2, 1 g NaC11-1. After 5 min the tubes were vortexed and centrifuged to sediment the whole red cells, cell fragments and blood clots. The absorbance of the decanted supernatant at 460 nM was determined using a dual beam Pye Unicam SP 1800 spectrophotometer with a 1 cm path length with a 0.9°7o saline blank; both haemoglobin and methaemoglobin absorb strongly at 460 nM. Maximal haemolysis occurred in 0.1 °7o saline, and the absorbance of this sample was compared to the other saline solutions to determine percentage haemolysis. A plot of haemolysis versus saline concentration produced a sigmoid curve and the concentration at which 500 haemolysis occurred describes the median corpuscular fragility in g d l - 1 NaC1.

16

The haematological indices, mean corpuscular volume (MCV) fl cell-1, mean corpuscular haemoglobin (MCH), pg Hb cell-1, mean corpuscular haemoglobin concentration (MCHC), g Hb dl - 1 were calculated as described by Wintrobe 0956). RESULTS

Short-term exposure (0 to 12 hours) Plasma electrolytes Exposure to environmental nitrite caused the blood plasma nitrite concentration to rise from less than 10 #mol l - 1 to around 4 m m o l 1- 1 over 12 h (Fig. 1), rising slowly at first at a rate o f 163 #mol h - 1 over 4 h, and then increasing to around 440/~mol h - 1 from 4 to 12 h with the blood plasma concentration becoming greater

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Time ( h )

Fig. la. Plasma nitrite concentration before and during exposure to 0.5 m m o ] l - ~ environmental nitrite in r a i n b o w trout. A s in all other figures the m e a n _+ SEM is s h o w n and the n u m b e r o f d e t e r m i n a t i o n s indicated. 100

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~ 4o N 3O 20 10

Plasma nitrite(retool 1-1)

Fig. lb. C o r r e l a t i o n b e t w e e n p l a s m a nitrite c o n c e n t r a t i o n a n d b l o o d m e t h a e m o g l o b i n levels. T h e regression e q u a t i o n is Y = 6.18 + 1 2 . 9 X a n d c o r r e l a t i o n coefficient, r = 0.9439, n = 35.

17 than the environmental concentration after 3-4 h exposure. This rise is followed by a similar rise in methaemoglobin levels with an initial slow lag-phase, the formation rate at 2.52°70 h - 1 over the first 4 h, accelerating to 6.64°70 h - 1 over the next 8 h. Blood plasma nitrite concentration and methaemoglobin levels were strongly correlated (r = 0.95, Fig. lb). Blood plasma chloride, sodium and potassium concentrations fell significantly during the first 2 h of nitrite exposure, whereas calcium and magnesium were unaffected (Fig. 2). Plasma potassium concentration showed the greatest fall, decreasing by 48%, plasma chloride by 23% and sodium by 2 2 " . Compensation of plasma chloride and sodium concentration occurred within 4 h and potassium within 6 h. The ratio of the sum of cations (Na + + K + + Ca + ÷ + Mg + +) to the combined

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Fig. 2. Changes in plasma sodium, chloride, potassium, calcium and magnesium prior to and during exposure to nitrite, differences between unexposed and exposed fish were tested in all cases using the Student's t-test, x, P<0.05; × ×, P<0.01.

18

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Fig. 3. The effect of nitrite exposure on intraerythrocyte potassium and sodium levels (/~mol 1- ' 1% Hct-~). ×, significantly different at P < 0 . 0 5 .

anion concentration of chloride and nitrite remained constant around 1.19 throughout the 12 h exposure period. After 12 h nitrite exposure the plasma was brown instead of the normal straw colour. Intraerythrocyte sodium and potassium concentrations sharply increased after 4 h nitrite exposure (Fig. 3), coinciding with the onset of the increased nitrite uptake and methaemoglobin formation rates. Total haemoglobin and red cell volume Whole-blood haemoglobin remained essentially constant during 12 h exposure and methaemoglobin levels steadily increased, while functional haemoglobin concentrations fell significantly after 6 h exposure (Fig. 4). The haematocrit also remained unchanged over 12 h. The red cells swelled slightly over the first 6 h of exposure and then rapidly shrunk by 30070 between 6 and 12 h (Fig. 5), this rapid decrease in size corresponding to an increase in red cell number and a decrease in cellular haemoglobin levels. The ratio of haemoglobin to red cells remained constant over the 12-h period. The nitrite-exposed red cells were less sensitive to osmotic stress than unexposed

19

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Fig. 4. Changes in total haemoglobin (g dl-x), methaemoglobin (%) and functional haemoglobin (g dl-~) on exposure to nitrite. *, significantly different at × P < 0 . 0 5 , × × P < 0 . 0 1 , × × x, P<0.001 (0-12 h, n = 6 ; 24 h, n = 7 ) .

red cells with the median corpuscular fragility being 0.265 and 0.357 g dl- 1 NaCI, respectively (Fig. 6).

Longer-term nitrite exposure (24 hours) After 24 h exposure, surviving trout showed a wide spectrum of tolerance, some showing few external signs of nitrite poisoning while others exhibited external signs of intoxication and were near to death (see Margiocco et al., 1984). The most sensitive of this group (30°70) perished before 24 h nitrite exposure and these were excluded from the study. For convenience, sensitivity to nitrite could be arbitrarily split into two groups, those in the tolerant part of the spectrum termed 'nitritetolerant' while those in the intolerant part of the spectrum were termed 'nitriteintolerant'.

Plasma electrolytes Plasma nitrite concentrations were higher in the nitrite-intolerant fish, while plasma chloride and potassium were lower with plasma sodium, calcium and magnesium concentrations the same in both groups (Table I). Plasma nitrite and chloride values were inversely correlated, as were plasma potassium and nitrite concentrations (Table II). A strong correlation also exists between plasma potassium concentration and methaemoglobin levels, with an unexpectedly poor correlation

20 b e t w e e n p l a s m a chloride a n d m e t h a e m o g l o b i n level. T h e ratio b e t w e e n total c a t i o n c o n c e n t r a t i o n a n d chloride plus nitrite c o n c e n t r a t i o n r e m a i n e d c o n s t a n t at 1.12 in b o t h groups; this ratio was different f r o m 1,19, which was f o u n d as already described in other fish exposed for shorter periods.

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Fig. 5. Changes in red blood cell count (RBC, x 1 0 6 mm3), haematocrit (Hct, a/0), mean cell volume (MCV, fl cell- ~), mean cell haemoglobin (MCH, pg Hb cell- 1) and mean cell haemoglobin concentration (MCHC, g 100 g- ~ Hb) during nitrite exposure. Differences between unexposed and exposed fish were tested in all cases using the Student's t-test, ×, P<0.05; × ×, P<0.01; × × x, P<0.001. The 24 h haematocrit value is significantly different from values at 2, 4 and 6 h.

21

Haemoglobin and red blood cells The nitrite-intolerant trout had higher levels of methaemoglobin and lower levels of functional haemoglobin than the nitrite-tolerant group (Table I). Total haemoglobin, haematocrit, MCV, MCH, RBC and MCHC were the same in both groups. Fish exposed to nitrite for 24 h, when compared to unexposed fish, had lower plasma potassium concentration with higher plasma nitrite and methaemoglobin levels and a reduced red cell volume (Table I).

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Fig. 6. Effect o f 12 h nitrite exposure o n red b l o o d cell o s m o t i c fragility. N o r m a l red b l o o d cells (circles, n = 8), nitrite exposed b l o o d cells (squares, n = 5). Significantly different at ×, P < 0.05; x ×, p < 0.01; x × ×, P < 0 . 0 0 1 . Inset shows incremental fragility, replotted from the two curves; O - O , exposed red b l o o d cells, x - - x , normal red b l o o d cells.

22 DISCUSSION

Short-term exposure, 0-12 hours Plasma electrolytes N i t r i t e is t r a n s p o r t e d f r o m t h e w a t e r a c r o s s t h e gills a n d is c o n c e n t r a t e d in b l o o d a n d tissues ( B a t h a n d E d d y , 1980; M a r g i o c c o et al., 1983) t o g r e a t e r levels t h a n in t h e w a t e r (Fig. 1). T h e c h l o r i d e t o n i t r i t e r a t i o in t h e w a t e r was 0.2 m M / 0 . 5 -- 0.40, w h i l e t h e b l o o d r a t i o was 128 m M / 0 . 0 1

mM

=

mM

12 800; t h e r e f o r e a net

n i t r i t e u p t a k e p r e d o m i n a t e s , w i t h a m a x i m u m r a t e o f a r o u n d 280 # m o l - ~ k g - l h - 1 in t r o u t ( W i l l i a m s a n d E d d y , 1986). T h e a c c u m u l a t i o n o f p l a s m a n i t r i t e is c l o s e l y f o l l o w e d b y a n i n c r e a s e in m e t h a e m o g l o b i n , more

rapid

rate

(Fig.

4),

a phenomenon

i n i t i a l l y at a s l o w r a t e f o l l o w e d b y a noticed

h a e m o g l o b i n s o l u t i o n s ( R o d k e y , 1976) a n d a l s o h a e m o g l o b i n s o l u t i o n s ( W i l l i a m s a n d E d d y , 1988). The profound

in v i t r o in t r o u t

with whole

mammalian blood

and

d i s t u r b a n c e s in p l a s m a e l e c t r o l y t e levels d u r i n g t h e first 2 h o f

n i t r i t e e x p o s u r e c o u l d b e t h e r e s u l t o f a n initial a d r e n a l i n e - m e d i a t e d stress r e s p o n s e with anion and cation efflux predominating,

and under these conditions entry of

TABLE I Blood haemoglobin, methaemoglobin and plasma electrolytes in normal rainbow trout and trout exposed to 0.5 mmol 1-1 nitrite. Comparison between 'nitrite-intolerant' and 'nitrite-tolerant' fish exposed to nitrite for 24 h is included (see text for details). Parameter/ Units

Hours exposure 0 24

Functional haemoglobin g 100 m1-1 Methaemoglobin °/0 of total Hb Mean cell volume fl cell-1

9.08 0.48 (6) 2.57 0.68 (6) 396 18 (7)

0.004

Plasma electrolytes mmol 1-1 Nitrite

(7) Potassium Chloride

6.98 0.78 (6) 120.25 2.61 (6)

Tolerant trout

Intolerant P < trout

4.49 *** 0.54 (7) 43.50 *** 7.10 (7) 329 * 26 (5)

5.82 0.63 (3) 24.28 3.82 (2) 355 68.5 (2)

3.50 * 0.20 (4) 57.92 *** 3.29 (4) 312 N.S. 16.6 (3)

2.54 0.84 (6) 4.38 N.S. 0.74 (7) 122.57 N.S. 1.91 (7)

0.89 0.38 (3) 6.09 0.69 (3) 125.50 2.02 (3)

4.19 * 0.81 (3) 3.10 * 0.65 (4) 120.38 N.S. 2.64 (4)

P<

The mean _+ the SEM are shown with parentheses indicating the number of fish sampled. Student's t-test was used to determine significance between the normal and treated animals and between 'nitrite-tolerant' and 'intolerant fish' (see text), N.S., not significant at P<0.1, *P<0.05, **P<0.01, and ***P<0.001. All other parameters tested were not significantly affected by 24 h nitrite exposure.

23 TABLE II Relationship between plasma chloride, nitrite, potassium and methaemoglobin levels in rainbow trout exposed to 0.5 mmol l-~ nitrite for 24 h. Parameters

Plasma chloride vs. plasma nitrite Plasma chloride vs. MetHb Plasma potassium vs. plasma nitrite Plasma potassium vs. MetHb

Regression equation

P<

Y = A (+ SE) - B(_+ SE)Xn=7

Correlation coefficient r

Y = 44.17 (11.26) - 0.3419 (0.0918)X

-0.8576

*

Y = 130.01 (4.29) - 0.1710 (0.0914)X

-0.6415

N.S.

Y=

6.24 (0.69) - 0.8220 (0.2353)X

-0.8422

*

Y=

8.55 (0.84) - 0.0957 (0.0178)X

-0.9231

**

*Significant at P<0.05, **, P<0.01 and N.S., not significant at P<0.1.

nitrite into the plasma would be retarded, as would subsequent methaemoglobin formation. Such reductions in plasma nitrite concentration and methaemoglobin formation have been observed during simultaneous nitrite and adrenaline exposure in rainbow trout (Williams and Eddy, 1987). Alternatively, since nitrite is a vasodilator, a general systemic vaso-dilation could cause plasma electrolyte concentrations to fall as a result of an increased blood volume. Although plasma electrolyte concentrations fell, the ratio of cations to anions remained constant, thus maintaining electroneutrality and plasma osmolarity. Blood potassium content was most sensitive to changes in plasma anion concentration (Fig. 3). Complete compensation of plasma ion concentration occurs within 2 h. During the following period (4-6 h), plasma nitrite levels rose rapidly, suggesting that compensation of plasma ion concentrations is achieved by increasing net ionic uptake from the water. Net chloride flux in resting unexposed rainbow trout was found to be near equilibrium, with influx balancing efflux but on exposure to environmental nitrite (0.5 mmol 1- 1) for 2 to 3 h, chloride efflux became predominant, followed 1 h later by a net chloride influx (unpublished results), thus balancing net chloride flux.

R e d blood cell volume The initial stress response at the gill-water interface was followed by secondary effects in red cells and in the red cell population so that after 6 h nitrite exposure when the plasma nitrite concentration exceeded the environmental level the red cells had swollen, with mean cell volume increased by 8°7o. As the erythrocytes swell, intracellular sodium and potassium increase, the sodium drawing potassium and osmotically-obliged kvater into the erythrocyte (Borgese et al., 1987). Swelling may be in response to methaemoglobin-induced hypoxia or as a result of adrenaline

24 stimulation of the membrane bound Na + / K + ATPase (Nikinmaa, 1983; Nikinmaa and Heustis, 1984). After 12 h exposure the red cell population increased, probably by recruitment from the spleen, but the new cells were smaller and contained less haemoglobin, and thus the cell volume to haemoglobin ratio remained unchanged, with no alteration in total blood haemoglobin or haematocrit. A depression in mean cell haemoglobin following red cell recruitment was observed in rainbow trout suffering from experimentally induced anaemia (Lane, 1979). The smaller cells in this study were less fragile to osmotic stress than unexposed red blood cells and could withstand greater osmotic swelling before rupture (Wintrobe, 1956). The incremental fragility plot indicated that at 12 h of nitrite exposure a heterogeneous population of red blood cells existed, as denoted by a wide haemolysis peak, unlike control cells, which had a sharp well-defined peak suggestive o f a homogeneous cell population (Ezell et al., 1969). The recruitment of new cells may be a direct hypoxic response by the spleen or as a result of stress-induced adrenaline stimulation of this organ. The new cells must have been exposed to nitrite while in the spleen, since no perturbations in methaemoglobin levels were seen which might be expected if new methaemoglobinfree cells were released into the circulation. In fact, release of splenic cells caused a fall in functional haemoglobin during this period (Fig. 4). Evidence of red cell haemolysis is provided by the brown colouration of the plasma, probably as a result of methaemalbumin formation, which is produced when red cells undergo rapid haemolysis and the released methaemaglobin binds to plasma albumin (Wintrobe, 1956).

Twenty-four hour nitrite exposure Plasma electrolytes The nitrite-intolerant group of fish had high plasma nitrite levels, and as a consequence of maintenance o f electroneutrality, plasma chloride concentrations were correspondingly lower, although not significantly (Table I). Margiocco et al. (1983) found no change in plasma chloride levels in rainbow trout after several days exposure to 6.5 #mol 1- 1 nitrite as did Bath (1980) studying the same species after 24 h exposure to 0.7 mmol 1- 1 nitrite; these results may be explained by failure to detect a relatively small reduction in plasma chloride levels. The ratio of cations to anions was the same in both nitrite-intolerant and -tolerant fish, but differed from that in briefly exposed fish. This difference can be accounted for by the decreased potassium levels. Concentrations of other anions such as bicarbonate and lactate may also change during nitrite exposure, with plasma bicarbonate levels decreasing as a result of nitrite inhibiting carbonic anhydrase (Gaino et al., 1984) and plasma lactate levels increasing due to increased anaerobic metabolism. In both cases extracellular acidosis would result, but Bath (1980) found

25 no increase in arterial blood pH in cannulated rainbow trout exposed to nitrite for 24 h. When nitrite is oxidised during methaemoglobin formation, plasma nitrate levels may increase; in rats, Imaizumi et al. (1980) found plasma nitrate levels rose in parallel with plasma nitrite levels. In the nitrite-intolerant group the significant fall in plasma potassium led to a profound uncompensated hypokalemia, while other plasma cation concentrations remained unchanged. This suggests that potassium is the main counter-cation to nitrite (Table I). The major effects of hypokalemia are cardiac failure, muscle weakness and paralysis (Black, 1960); therefore, low plasma potassium levels could be an important cause of death in nitrite-intoxicated fish. Methaemoglobinaemia per se may not be the cause of death, for some fish can survive with low haematocrits ( < 10o70)or no haemoglobin whatsoever (Holeton, 1970, 1971) and rely instead on dissolved oxygen in the blood plasma. Furthermore, the vasodilatory action of nitrite may increase gill perfusion and thus maintain adequate dissolved oxygen levels.

Blood haemoglobin The major difference between the two groups was the methaemoglobin level with intolerant fish having more than double the levels found in tolerant fish (Table I), while any fish with very high levels probably died during the 24-h period. Rainbow trout exposed to identical external chloride levels may vary widely in their chloride uptake rates (Williams and Eddy, 1986). Fish with high anion uptake rates would tend to accumulate nitrite in the plasma at a greater rate compared to those with lower uptake rates; thus, fish from the nitrite-intolerant group may have higher uptake rates than fish from the tolerant group. Alternatively, the nitriteintolerant group may exhibit a low initial stress response and lack the ability to ameliorate nitrite accumulation (Williams and Eddy, 1987). Other species of fish, such as carp, which have less variable chloride uptake rates (Williams and Eddy, 1988), have not been shown to exhibit two distinct groups of tolerant and intolerant fish when exposed to nitrite. In conclusion, the response by rainbow trout to environmental nitrite can be divided into two phases: first, over 2-3 h there is a net anion efflux, probably mediated by catecholamines representing an attempt to remove nitrite from the body; in the second phase, methaemoglobin formation is advanced with changes in red cell electrolyte content and volume together with the release of splenic erythrocytes. In both phases, circulatory effects through the vasodilatory action of nitrite may be important. There is a wide spectrum of nitrite tolerance in trout populations as exemplified by nitrite-tolerant and nitrite-intolerant individuals, which may be of considerable consequence in trout populations where nitrite is a significant environmental factor.

26 ACKNOWLEDGEMENTS The authors are indebted to Karen Williams and Shona McClean for preparation of the figures. This work was supported by NERC

grant no. Gr/3/5422.

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