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Effect of nitrite on the respiratory response of grass carp Ctenopharyngodon idella (Val.) with relation to chloride
Camp. Biochem. Physiol.Vol. 106C, No. 3, pp. 161-764, 1993
0742-84I3/93 56.00+ 0.00 Pergamon Press Ltd
Printed in Great Britain
EFFECT OF NITRITE ON THE RESPIRATORY RESPONSE OF GRASS CARP CTENOPHARYNGODON IDELLA (VAL.) WITH RELATION TO CHLORIDE SONIA ESPINA
and GUILLERMINAAXARAZ
Laboratorio de Ecofisiologia, Departamento de Biologia, Facultad de Ciencias, Universidad National Autonoma de Mexico, 04510 Mexico D.F., Mexico [Received 14 June 1993; accepted for publication 23 July 1993) Abstract-l. The oxygen consumption rates of C. idelia decrease when exposed to nitrite but return to normal in the presence of chloride, reaching similar values to those of the control groups. 2. A quadratic polynomial model describes the relationship between metabolic rate, wet weight and chloride concentration in the environment, both with nitrite fixed (~~~-96 hr = 1.71 mg N-NOT/I) as chloride varied (336 mg/l) and chloride fixed (3 mg/l) as nitrite varied from lethal to sublethal concentration (1.71, 1.0, 0.5 and 0.3 mg N-NO;/l). 3. The metabolic rate is a very useful indicator of stress caused by nitrite.
INTRODUCTION increases in cultured fish tanks to critical levels for the survival of fish due to nitrogen excretion and fertilizer addition (Williams and Eddy, 1986). Although the damage caused by nitrite in fish may not be evident, metabolic alterations may result as nitrite induces the oxidation of hemoglobin to ferrihemoglobin, which is unable to transport oxygen (Hilmy et al., 1987; Jensen et al., 1987). The presence of chloride in the environment has been shown to reduce nitrite toxicity, and this is ascribed to the fact that nitrite is a competitive inhibitor of chloride transport through the gill epithelium (Perrone and Meade, 1977; Wedemeyer and Yasutake, 1978; Schwedler and Tucker, 1983; Williams and Eddy, 1986; Tomasso and Carmichael, 1986). In this paper, the effect of nitrite, with respect to the chloride ions, on the respiratory response of juvenile grass carp (Ctenopharyngodon idella, Val.) was evaluated. Distilled water was used as dilution medium to separate the effect of these ions. The herbivorous carp was selected as a model because it is an important species for aquaculture in this country. Nitrite concentration
MATERIALS AND
METHODS
Juveniles Ctenopharyngodon idella (3.04-4.45 cm; 0.17-0.75 g wet weight) were obtained from the Centro de Produccibn Piscicola de Tezontepec de Aldama, Estado de Hidalgo, Mexico. The fish (n = 100) were kept at the laboratory in 60-l aquaria filled with tap water at a constant temperature of 24°C pH 7.0, 0.44 mg HCO;/l of alkalinity, 125 mg Cl-/l (A.P.H.A., 1985) and 6.8mg 0*/l (YSI
54 + 0.1 mg 0,/l). Photoperiod was established at 12 hr of light. The animals were fed every day with commercial balanced food (75%) and lucern (25%). Organisms were allowed to acclimate to this condition for at least 10 days before running any experiment (but fish were fasted 24 hr before and during the experiments). For the experiments, the fish were divided into six groups, two of which were considered controls: one of them was kept in tap water and the other in distilled water containing 3 mg Cl-/l. The remaining four groups were exposed to 1.71 mg N-NOT/I, i.e. the median lethal concentration of nitrite (Davalos, 1991; Alcaraz and Espina, 1993). The first group was kept in distilled water (3 mg Cl-/l) and the others had 4, 5 and 6mg Cl-/l, respectively. A second experiment was carried out, in which chloride was held constant (3 mg Cl-/l) and nitrite was varied from sublethal concentrations (0.5 and 1.Omg/l) up to the CL,,-96 hr (1.7 mg N-NOT/I). Static 48-hr exposures were performed in 40-l glass aquaria with mild and constant aeration at a temperature of 24 f 1°C. Nitrite and chloride were used in the form of sodium salts (Merk, a.r.). Water was not changed in aquaria during the experimental phase. Oxygen consumption was individually measured in 12-15 fishes in a closed respirometer for 1 hr (Espina et al., 1986). Once the experimental phase was over, the animals were weighed to the nearest 0.01 g. Physiological rates were related to the wet body weight according to the potential model Y = a Xb e, where Y is the physiological variable; X is the wet weight; a and b are constants and ei are the residuals (Schmidt-Nielson, 1984). Logarithmic regression lines (Ln) were fitted by the least square method and the fitness was estimated by residual analysis (Zar, 1974). In order to evaluate the effect of the 761
762
S. ESPINAand G. ALCARAZ Table I. Parameters and estimators of the logarithmic regressions (Ln) between oxygen consumption rate (mgO,/hr) and body weight (g) of C. idella exposed to different environmental concentration of N-nitrite and chloride (me/l) for 48 hr Experimental conditions
Cl_
a
b
R’
P
TW DW
0.0 0.0
125 3
0.025 -0.021
0.790 0.745
99.8 93.8
0.000 0.004
DW DW DW DW
1.7 1.7 1.7 I.7
3 4 5 6
0.598 0.407 0.257 0.145
0.369 0.417 0.974 0.918
99.9 99.8 99.9 99.9
0.000 0.000 0.000 0.000
DW DW DW DW
0.0 0.5 1.0 I.7
3 3 3 3
-0.021 -0.105 -0.262 0.598
0.745 0.805 I.137 0.369
93.8 99.9 99.9 99.9
0.004 0.000 0.000 0.000
N-NO,
TW: tap water; DW: distilled
water.
combination of factors on oxygen consumption, a second degree polynomial model was used and the surfase response was calculated (Montgomery and Peck, 1982). For statistical analysis of the data, STATGRAPHICS programme V. 2.1 (Statist. Graph. Syst. Co., 1985-1986) was employed. RESULTS During the determination of the oxygen consumption rate of juvenile Ctenopharyngodon idella, oxygen concentration did not decrease to unacceptable limits for fish. Mortality was not registered during the acclimation period, nor during the experiments. Parameters and estimators from the logarithmic regressions between oxygen consumption and body weight, are presented in Table 1; all comparisons were significant (P < 0.01). Decodified data of the expected values (mg O,/hr) are presented in Table 2. It can be observed that between control groups, the oxygen consumption rates were similar (P > 0.05). In the group of fish exposed to 1.71 mg N-NO;/1 (CL,,,-96 hr) and 3 mg Cl-/l, the oxygen consumption rate decreased 32% compared to the control group in distilled water (P < 0.05). Then, as chloride increased, the oxygen consumption rate increased until it reached similar values to those of the control group (P > 0.05). Recovery depended on the chloride concentration in the environment. Table 2. Estimated values of oxygen consumption rate (YO,, mg/hr) of C. id& exposed to different combination of nitrite (mg N-NOT/I) and chloride (mg Cl-/l) for 48 hr, in distilled water (DW). Values for controls groups in tap water (TW) and DW are shown. R f SE Cl-
N
V&l
125 3
8 I8
0.71 f 0.05 0.63 + 0.04
1.7 1.7 1.7 1.7
3 4 5 6
13 I1 I8 18
0.43 0.50 0.64 0.67
+ f + f
0.02 0.03 0.05 0.04
DW DW DW DW
0.5 I.0 1.7
3 3 3 3
31 16 16 16
0.64 0.46 0.32 0.57
+ f k +
0.04 0.05 0.05 0.06
N: number
of fish.
N-NO; TW DW
-
DW Dk DW DW
When fish were exposed to the sublethal concentration of nitrite (Table 2) of 0.5 mg N-NO;/1 with 3 mg Cl-/l the rate of their oxygen consumption diminished 28% and at 1.0 mg N-NO;/l, a greater decrease was observed (50%); however, a recovery occurred when ambient N-nitrite concentration augmented to the median lethal concentration as in the former experiment. Juvenile C. idella weights played an important role when both lethal and sublethal nitrite concentrations are considered. Figure 1A shows the surface response generated with data from fish exposed for 48 hr to the nitrite median lethal concentration and chloride concentrations, from 3 to 6mg/l. The quadratic polynomial model that adequately describes this relationship is: P=
-3.16+1.442X,-
1.007X; - 1.556 X; + 0.328 X,X,
where Pis the expected physiological rate (mg O,/hr), and X, and X, are the independent variables body weight (g) and chloride concentration (mg/l), respectively. The estimators of the model were R2 = 0.995, F = 503, and P = 0.001, and there was no autocorrelation since a value of 1.943 was for the DurbinWatson test. Thus, the polynomial model justified 99.9% of the observed variations in the consumption of oxygen by the carps. This rate was greatest between 4.6 and 5.9 mg Cl-/l within a weight range of 0.584.81 g as can be observed in the surface response (Fig. 1). These maximum values corresponded to the group in distilled water, which reflect the protective action of chloride against the effect of nitrite. In the second experiment, where the chloride concentration was constant and nitrite was at sublethal concentrations, the polynomial equation was: E=0.406+0.981
X,-1.075X2+0.513X;
where P is the expected value of fish oxygen consumption rate; X, is fish weight and X2 is nitrite concentration. As before, the model was highly significant, P < 10m4, R2 = 0.996 Durbin-Watson test = 2.05, but there was no significant interaction between the regressor variables. Figure 1A shows
Respiratory response of grass Carp to nitrite
163
Fig. I. Relationship of oxygen consumption rate (mg/l), wet weight (g) and chioride concentration (A, mg/l) or nitrite concentration (8, mg N-NO;;/;) in the media, in juvenile C. idella.
that chloride protected the bigger fish better than the smaller ones. DISCUSSION
Body size often causes a bias in the calculation of physiological variables that do not have a direct relationship with the weight of fish, as in the case of oxygen consumption; thus, regressions between the physiological variable and the body weight were calculated for each experimental condition. The decrease in the rate of oxygen consumption observed in the fish exposed to nitrite in distilled water with lower chloride concentration as compared to the fish in the control groups, may be the result of methemoglobin formation, which reaches a maximum within 24-48 hr of exposure to nitrite (Bowser et al., 1983). But, when chloride was present in the highest concentration, probably the methemoglobin condition, caused by the toxicant, was ameliorated. Similar results were reported for Salmo gairdneri, which did not present measurable levels of nitrite in the blood when exposed to a concentration of 142 mg/l of external chloride; however, when the rainbow trout was kept in water with a low chloride content, internal nitrite was 5-10 times greater than its external concentration (Bath and Eddy, 1980). Smaller chloride concentration than this protected the channel catfish ictalurus purzctatus, since a concentration of 25 mg/l of NaCl (15.17 mg Cl-/I) has
been shown to be effective for this species when exposed to 1 mgji of nitrite at pH 7.0 (Bowser et al., 1983). In this work, it has been established that even smaller concentrations than those previously reported (6mg Cl-[I) protect juveniles of C. idelfa. These discrepancies may be due to many factors among which could be cited the different sizes of the organisms, the physicochemical characteristics of the environment and even interspecific differences. However, when the proportions of environmental ionic concentrations were considered, the results obtained in this work for C. idelfa agree with those reported for lc~alurus punc~atus. Thus, the most effective ratio of nitrite/chloride observed was that within the range 0.22-0.26. Schwedler and Tucker (1983) have stated that at similar ratios, methemoglobinemia is kept at levels considered safe for the channel catfish. The technique of response surfaces was useful to confirm and summarize the complex interactions among the variables; these are: the animals oxygen consumption, their weight and the environmental nitrite and chloride concentrations. It is important to bear in mind, that when the njtrite concentration was the median lethal concentration, the chloride inhibited mortality, although the fish were stressed, which is reflected by a decrease in the metabolic rate at low chloride concentration. This is in agreement with data reported for Resbara daniconius (Jawale, 1985). Also, the protective effect of chloride was more
S. ESPINAand G. ALCARAZ
764
noticeable in the bigger (0.61-0.81 mg w.w.) than in the smaller fish when acutely exposed to nitrite. The fish oxygen uptake was similar to the controls when
the environmental
chloride was 5 mg/l. On the other
hand, when fish were exposed to sublethal nitrite concentrations, almost a specular inverse image from the first experiment was observed (Fig. 1); that is, as N-nitrite increases from 0.5 to 1.1 mg/I, the
oxyaen uvtake decreases. but when nitrite concen*_ . tration increases, up to the CL,,-96 hr, the oxygen consumption rate reached a maximum higher than the control
group, which indicates
stress. As before,
bigger fishes (0.76-0.86 g) seem to be better protected than the smaller ones &hen N-nitrite concentration was I. 1 mgjl. Although extrapolated
the results to culture
of this study management,
could not be they indicate
that in order to establish realistic overational conditions for C. ideffa culture, it is necessary to control the problems caused by nitrite through the maintenance of adequate water quality.
Bowser P. R., Falls W. W., VanZandt J., Collier N. and Phillips J. D. (1983) Methemoglobinemia in channel catfish: Methods of prevention. Prog. Fish-cult 45 (3), I%-158. __-.
Davalos P. (1991) Efecto de1 nitrito sobre la mortalidad y las respuestas respiratorias de Crenopharyngodon idella Val. Tesis de Licenciatura. Facultad de Ciencias. Universidad National Aut~noma de Mexico. 39 pp. Espina S., Diaz F., Rosas C. y Rosas I. (1986) Influencia del
detergente sobre el balance energetic0 de Ctenophurynaodon idella a traves de un bioensavo croniw. Contam. Ambient.
2, 25-37.
Hilmy A. M., El-Domiaty N. A., and Wershana K. (1987) Acute and chronic toxicity of nitrite to Chzrius &era. Cvmp. Biochem. Physiol. 86C, 247-253.
Jawale M. D. (1985) Effects of oesticides on metabolic rate
of freshwater fisn Resbora daniconius. Environ. Ecol. 3, 521-523. Jensen F. B., Andersen N. A. and Heisler N. (1987) Effects of nitrite exposure on blood respiratory properties, acidbase and electrolite regulation in the carp. J. camp. Phvsiol.
1573. 533-541.
Montgomery D: C. and Peck E. A. (1982) Introduction to Linear Regression Analysis. John Wiley and Sons, New York. 504 pp. Perrone S. J. and Meade T. L. (1977) Protective effect of chloride on nitrite toxicity to coho salmon (Oncorhynchus kisutch).
are especially grateful to Centro de Produccibn Piscicola de Tezontepec de Aldama for juveniles grass carp donated for this study. We would like to thank Cecilia Vanegas for technical assistance and Maria Antonieta Arizmendi for helping to translate this manuscript. Acknowledgements-We
REFERENCES Alcaraz G. and Espina S. (1993) The effect of nitrite survival of grass carp, Ctenopharyngodon idella with relation to chloride. Bull. Enoir. Contam. (in press). A.P.H.A. (American Public Health Association)
on the (val.) Toxic.
J. Fish. Res. Board. Can. 34, 486492. K. (1984) ~~~o~ogja Animui Adap?aci~n al Medio Ambiente (Edited by Omega .%A.), Barcelona,
~hmidt-Nielsen
499 pp. Schwedler T. E. and Tucker C. S. (1983) Empirical relationship between percent methemoglobin in channel catfish and dissolved nitrite and chloride in ponds. Trans. Amer. Fish. Sot. 112, 117-119. Tomasso J. R. and Carmichael G. J. (1986) Acute toxicity of ammonia, nitrite, and nitrate to the Guadalupe Bass, Micropterus treculi. Bull. E&r. Coniam. Toxic. 36, 866-870.
Wedemeyer D. E. and Yasutake W. T. (1978) Prevention and treatment of nitrite toxicity in juvenile steelhead trout (Salmo gairdneri).
(1985)
Standard Methods for the Examination of Water Waste Water. A.P.H.A. Washington, 167 pp.
and
Bath R. N. and Eddy F. B. (1980) Transport of nitrite across fish gills. J. exp, Zool. 214, 119-121.
J. Fish. Res. Bd. Can. 35, 822-827.
Williams E. M. and Eddy F. B. (1986) Chloride uptake in freshwater teleosts and its relationship to nitrite uptake and toxicity. J. camp. Physiol. 15 B, 867-872. Zar J. H. (1974) Biosfatistical Analysis. Prentice-Hall. Inc. Englewood Cliffs N.J., 620 pp.
0742-84I3/93 56.00+ 0.00 Pergamon Press Ltd
Printed in Great Britain
EFFECT OF NITRITE ON THE RESPIRATORY RESPONSE OF GRASS CARP CTENOPHARYNGODON IDELLA (VAL.) WITH RELATION TO CHLORIDE SONIA ESPINA
and GUILLERMINAAXARAZ
Laboratorio de Ecofisiologia, Departamento de Biologia, Facultad de Ciencias, Universidad National Autonoma de Mexico, 04510 Mexico D.F., Mexico [Received 14 June 1993; accepted for publication 23 July 1993) Abstract-l. The oxygen consumption rates of C. idelia decrease when exposed to nitrite but return to normal in the presence of chloride, reaching similar values to those of the control groups. 2. A quadratic polynomial model describes the relationship between metabolic rate, wet weight and chloride concentration in the environment, both with nitrite fixed (~~~-96 hr = 1.71 mg N-NOT/I) as chloride varied (336 mg/l) and chloride fixed (3 mg/l) as nitrite varied from lethal to sublethal concentration (1.71, 1.0, 0.5 and 0.3 mg N-NO;/l). 3. The metabolic rate is a very useful indicator of stress caused by nitrite.
INTRODUCTION increases in cultured fish tanks to critical levels for the survival of fish due to nitrogen excretion and fertilizer addition (Williams and Eddy, 1986). Although the damage caused by nitrite in fish may not be evident, metabolic alterations may result as nitrite induces the oxidation of hemoglobin to ferrihemoglobin, which is unable to transport oxygen (Hilmy et al., 1987; Jensen et al., 1987). The presence of chloride in the environment has been shown to reduce nitrite toxicity, and this is ascribed to the fact that nitrite is a competitive inhibitor of chloride transport through the gill epithelium (Perrone and Meade, 1977; Wedemeyer and Yasutake, 1978; Schwedler and Tucker, 1983; Williams and Eddy, 1986; Tomasso and Carmichael, 1986). In this paper, the effect of nitrite, with respect to the chloride ions, on the respiratory response of juvenile grass carp (Ctenopharyngodon idella, Val.) was evaluated. Distilled water was used as dilution medium to separate the effect of these ions. The herbivorous carp was selected as a model because it is an important species for aquaculture in this country. Nitrite concentration
MATERIALS AND
METHODS
Juveniles Ctenopharyngodon idella (3.04-4.45 cm; 0.17-0.75 g wet weight) were obtained from the Centro de Produccibn Piscicola de Tezontepec de Aldama, Estado de Hidalgo, Mexico. The fish (n = 100) were kept at the laboratory in 60-l aquaria filled with tap water at a constant temperature of 24°C pH 7.0, 0.44 mg HCO;/l of alkalinity, 125 mg Cl-/l (A.P.H.A., 1985) and 6.8mg 0*/l (YSI
54 + 0.1 mg 0,/l). Photoperiod was established at 12 hr of light. The animals were fed every day with commercial balanced food (75%) and lucern (25%). Organisms were allowed to acclimate to this condition for at least 10 days before running any experiment (but fish were fasted 24 hr before and during the experiments). For the experiments, the fish were divided into six groups, two of which were considered controls: one of them was kept in tap water and the other in distilled water containing 3 mg Cl-/l. The remaining four groups were exposed to 1.71 mg N-NOT/I, i.e. the median lethal concentration of nitrite (Davalos, 1991; Alcaraz and Espina, 1993). The first group was kept in distilled water (3 mg Cl-/l) and the others had 4, 5 and 6mg Cl-/l, respectively. A second experiment was carried out, in which chloride was held constant (3 mg Cl-/l) and nitrite was varied from sublethal concentrations (0.5 and 1.Omg/l) up to the CL,,-96 hr (1.7 mg N-NOT/I). Static 48-hr exposures were performed in 40-l glass aquaria with mild and constant aeration at a temperature of 24 f 1°C. Nitrite and chloride were used in the form of sodium salts (Merk, a.r.). Water was not changed in aquaria during the experimental phase. Oxygen consumption was individually measured in 12-15 fishes in a closed respirometer for 1 hr (Espina et al., 1986). Once the experimental phase was over, the animals were weighed to the nearest 0.01 g. Physiological rates were related to the wet body weight according to the potential model Y = a Xb e, where Y is the physiological variable; X is the wet weight; a and b are constants and ei are the residuals (Schmidt-Nielson, 1984). Logarithmic regression lines (Ln) were fitted by the least square method and the fitness was estimated by residual analysis (Zar, 1974). In order to evaluate the effect of the 761
762
S. ESPINAand G. ALCARAZ Table I. Parameters and estimators of the logarithmic regressions (Ln) between oxygen consumption rate (mgO,/hr) and body weight (g) of C. idella exposed to different environmental concentration of N-nitrite and chloride (me/l) for 48 hr Experimental conditions
Cl_
a
b
R’
P
TW DW
0.0 0.0
125 3
0.025 -0.021
0.790 0.745
99.8 93.8
0.000 0.004
DW DW DW DW
1.7 1.7 1.7 I.7
3 4 5 6
0.598 0.407 0.257 0.145
0.369 0.417 0.974 0.918
99.9 99.8 99.9 99.9
0.000 0.000 0.000 0.000
DW DW DW DW
0.0 0.5 1.0 I.7
3 3 3 3
-0.021 -0.105 -0.262 0.598
0.745 0.805 I.137 0.369
93.8 99.9 99.9 99.9
0.004 0.000 0.000 0.000
N-NO,
TW: tap water; DW: distilled
water.
combination of factors on oxygen consumption, a second degree polynomial model was used and the surfase response was calculated (Montgomery and Peck, 1982). For statistical analysis of the data, STATGRAPHICS programme V. 2.1 (Statist. Graph. Syst. Co., 1985-1986) was employed. RESULTS During the determination of the oxygen consumption rate of juvenile Ctenopharyngodon idella, oxygen concentration did not decrease to unacceptable limits for fish. Mortality was not registered during the acclimation period, nor during the experiments. Parameters and estimators from the logarithmic regressions between oxygen consumption and body weight, are presented in Table 1; all comparisons were significant (P < 0.01). Decodified data of the expected values (mg O,/hr) are presented in Table 2. It can be observed that between control groups, the oxygen consumption rates were similar (P > 0.05). In the group of fish exposed to 1.71 mg N-NO;/1 (CL,,,-96 hr) and 3 mg Cl-/l, the oxygen consumption rate decreased 32% compared to the control group in distilled water (P < 0.05). Then, as chloride increased, the oxygen consumption rate increased until it reached similar values to those of the control group (P > 0.05). Recovery depended on the chloride concentration in the environment. Table 2. Estimated values of oxygen consumption rate (YO,, mg/hr) of C. id& exposed to different combination of nitrite (mg N-NOT/I) and chloride (mg Cl-/l) for 48 hr, in distilled water (DW). Values for controls groups in tap water (TW) and DW are shown. R f SE Cl-
N
V&l
125 3
8 I8
0.71 f 0.05 0.63 + 0.04
1.7 1.7 1.7 1.7
3 4 5 6
13 I1 I8 18
0.43 0.50 0.64 0.67
+ f + f
0.02 0.03 0.05 0.04
DW DW DW DW
0.5 I.0 1.7
3 3 3 3
31 16 16 16
0.64 0.46 0.32 0.57
+ f k +
0.04 0.05 0.05 0.06
N: number
of fish.
N-NO; TW DW
-
DW Dk DW DW
When fish were exposed to the sublethal concentration of nitrite (Table 2) of 0.5 mg N-NO;/1 with 3 mg Cl-/l the rate of their oxygen consumption diminished 28% and at 1.0 mg N-NO;/l, a greater decrease was observed (50%); however, a recovery occurred when ambient N-nitrite concentration augmented to the median lethal concentration as in the former experiment. Juvenile C. idella weights played an important role when both lethal and sublethal nitrite concentrations are considered. Figure 1A shows the surface response generated with data from fish exposed for 48 hr to the nitrite median lethal concentration and chloride concentrations, from 3 to 6mg/l. The quadratic polynomial model that adequately describes this relationship is: P=
-3.16+1.442X,-
1.007X; - 1.556 X; + 0.328 X,X,
where Pis the expected physiological rate (mg O,/hr), and X, and X, are the independent variables body weight (g) and chloride concentration (mg/l), respectively. The estimators of the model were R2 = 0.995, F = 503, and P = 0.001, and there was no autocorrelation since a value of 1.943 was for the DurbinWatson test. Thus, the polynomial model justified 99.9% of the observed variations in the consumption of oxygen by the carps. This rate was greatest between 4.6 and 5.9 mg Cl-/l within a weight range of 0.584.81 g as can be observed in the surface response (Fig. 1). These maximum values corresponded to the group in distilled water, which reflect the protective action of chloride against the effect of nitrite. In the second experiment, where the chloride concentration was constant and nitrite was at sublethal concentrations, the polynomial equation was: E=0.406+0.981
X,-1.075X2+0.513X;
where P is the expected value of fish oxygen consumption rate; X, is fish weight and X2 is nitrite concentration. As before, the model was highly significant, P < 10m4, R2 = 0.996 Durbin-Watson test = 2.05, but there was no significant interaction between the regressor variables. Figure 1A shows
Respiratory response of grass Carp to nitrite
163
Fig. I. Relationship of oxygen consumption rate (mg/l), wet weight (g) and chioride concentration (A, mg/l) or nitrite concentration (8, mg N-NO;;/;) in the media, in juvenile C. idella.
that chloride protected the bigger fish better than the smaller ones. DISCUSSION
Body size often causes a bias in the calculation of physiological variables that do not have a direct relationship with the weight of fish, as in the case of oxygen consumption; thus, regressions between the physiological variable and the body weight were calculated for each experimental condition. The decrease in the rate of oxygen consumption observed in the fish exposed to nitrite in distilled water with lower chloride concentration as compared to the fish in the control groups, may be the result of methemoglobin formation, which reaches a maximum within 24-48 hr of exposure to nitrite (Bowser et al., 1983). But, when chloride was present in the highest concentration, probably the methemoglobin condition, caused by the toxicant, was ameliorated. Similar results were reported for Salmo gairdneri, which did not present measurable levels of nitrite in the blood when exposed to a concentration of 142 mg/l of external chloride; however, when the rainbow trout was kept in water with a low chloride content, internal nitrite was 5-10 times greater than its external concentration (Bath and Eddy, 1980). Smaller chloride concentration than this protected the channel catfish ictalurus purzctatus, since a concentration of 25 mg/l of NaCl (15.17 mg Cl-/I) has
been shown to be effective for this species when exposed to 1 mgji of nitrite at pH 7.0 (Bowser et al., 1983). In this work, it has been established that even smaller concentrations than those previously reported (6mg Cl-[I) protect juveniles of C. idelfa. These discrepancies may be due to many factors among which could be cited the different sizes of the organisms, the physicochemical characteristics of the environment and even interspecific differences. However, when the proportions of environmental ionic concentrations were considered, the results obtained in this work for C. idelfa agree with those reported for lc~alurus punc~atus. Thus, the most effective ratio of nitrite/chloride observed was that within the range 0.22-0.26. Schwedler and Tucker (1983) have stated that at similar ratios, methemoglobinemia is kept at levels considered safe for the channel catfish. The technique of response surfaces was useful to confirm and summarize the complex interactions among the variables; these are: the animals oxygen consumption, their weight and the environmental nitrite and chloride concentrations. It is important to bear in mind, that when the njtrite concentration was the median lethal concentration, the chloride inhibited mortality, although the fish were stressed, which is reflected by a decrease in the metabolic rate at low chloride concentration. This is in agreement with data reported for Resbara daniconius (Jawale, 1985). Also, the protective effect of chloride was more
S. ESPINAand G. ALCARAZ
764
noticeable in the bigger (0.61-0.81 mg w.w.) than in the smaller fish when acutely exposed to nitrite. The fish oxygen uptake was similar to the controls when
the environmental
chloride was 5 mg/l. On the other
hand, when fish were exposed to sublethal nitrite concentrations, almost a specular inverse image from the first experiment was observed (Fig. 1); that is, as N-nitrite increases from 0.5 to 1.1 mg/I, the
oxyaen uvtake decreases. but when nitrite concen*_ . tration increases, up to the CL,,-96 hr, the oxygen consumption rate reached a maximum higher than the control
group, which indicates
stress. As before,
bigger fishes (0.76-0.86 g) seem to be better protected than the smaller ones &hen N-nitrite concentration was I. 1 mgjl. Although extrapolated
the results to culture
of this study management,
could not be they indicate
that in order to establish realistic overational conditions for C. ideffa culture, it is necessary to control the problems caused by nitrite through the maintenance of adequate water quality.
Bowser P. R., Falls W. W., VanZandt J., Collier N. and Phillips J. D. (1983) Methemoglobinemia in channel catfish: Methods of prevention. Prog. Fish-cult 45 (3), I%-158. __-.
Davalos P. (1991) Efecto de1 nitrito sobre la mortalidad y las respuestas respiratorias de Crenopharyngodon idella Val. Tesis de Licenciatura. Facultad de Ciencias. Universidad National Aut~noma de Mexico. 39 pp. Espina S., Diaz F., Rosas C. y Rosas I. (1986) Influencia del
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