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Fish Physiol Biochem (2012) 38:1355–1365 DOI 10.1007/s10695-012-9623-3

Alterations in serum electrolytes, antioxidative enzymes and haematological parameters of Labeo rohita on short-term exposure to sublethal dose of nitrite Alexander Ciji • N. P. Sahu • A. K. Pal S. Dasgupta • M. S. Akhtar



Received: 26 August 2011 / Accepted: 14 February 2012 / Published online: 3 March 2012 Ó Springer Science+Business Media B.V. 2012

Abstract An experiment was conducted to study the effects of short-term exposure to sublethal levels of nitrite on electrolyte regulation, antioxidative enzymes and haematological parameters in Labeo rohita juveniles. The fishes were exposed to graded levels of nitrite (0–15 mg l-1) for different duration (0, 12, 24, 48 and 96 h). The 96-h LC50 value for L. rohita (avg. wt, 66.5 ± 0.5 g) was found to be 11.28 mg l-1. Activities of antioxidative enzymes (catalase and superoxide dismutase), acetylcholine esterase (AChE) and methaemoglobin reductase, serum electrolytes (sodium, potassium and chloride), haematological parameters and blood glucose level significantly varied (P \ 0.05) in a dose-dependent manner. With increasing nitrite concentration and exposure period, a progressive reduction in the total erythrocyte count and haemoglobin were observed. With increase in nitrite concentration, a significant (P \ 0.05) increase

in activities was evidenced in catalase and superoxide dismutase in liver as well as gill, methaemoglobin reductase in blood, while progressive decline in AChE activity in brain was recorded. The serum sodium and chloride content showed a progressive decline, while potassium showed an increasing trend upon increase in nitrite concentration. The serum K? and Cl- after 96-h exposure demonstrated a linear relationship (Y = 0.221x ? 2.542, R2 = 0.938, P \ 0.01 and Y = -5.760x ? 129.5, R2 = 0.952, P \ 0.01, respectively) with nitrite concentrations. This study revealed that nitrite exposure causes alteration in all measured tissue enzymes, serum electrolytes and haematological parameters. Keywords Nitrite  Labeo rohita  Electrolytes  Cortisol  Methaemoglobin reductase

Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10695-012-9623-3) contains supplementary material, which is available to authorized users. A. Ciji (&)  N. P. Sahu  A. K. Pal  S. Dasgupta  M. S. Akhtar Division of Fish Nutrition, Biochemistry and Physiology, Central Institute of Fisheries Education, Fisheries University Road, Versova 400061, Mumbai, India e-mail: [email protected] M. S. Akhtar Fish Nutrition Section, Directorate of Coldwater Fisheries Research, Bhimtal, Nainital 263136, Uttrakhand, India

The global aquaculture production will need to reach 80 million tonnes by 2050 to meet the present level of consumption rate (FAO 2005). Intensification of aquaculture is one of the possible alternatives to meet the demand. But intensification results in excessive use of proteinaceous feed and nitrogenous fertilisers along with higher stocking densities. Moreover, this may also result in increased load of nitrogenous as well as other toxic metabolites in the water bodies (Das et al. 2004b). Imbalance in bacterial nitrification process

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can also elevate nitrite concentrations in water (Cheng and Chen 1998; Deane and Woo 2007). An optimum pH in the range of 7.5–8.6 is required for proper nitrification by Nitrobactor (Huey et al. 1984), and hence increased nitrite toxicity is reported in conditions of lower pH and lower temperature in several teleosts and crustaceans (Huey et al. 1984; Chen and Cheng 2000). Nitrite problems typically are more likely to occur in closed intensive culture systems due to insufficient, inefficient or malfunctioning filtration systems of removing waste ammonia from water by means of nitrification (Kroupova et al. 2005). The accumulation of organic matter also leads to the release of microbial metabolites, such as ammonia, nitrite and hydrogen sulphide, into the water column, causing chronic stress on fish during the culture (Das et al. 2004b). The resultant stress may ultimately leads to growth depression, immunosuppression, disease and mortality in fish (Lewis and Morris 1986). The physiological effects of nitrite have been extensively studied in many fishes (Jensen et al. 1987; Stormer et al. 1996; Haung and Chen 2002; Jensen 2003) and found that toxicity varies widely between species. The blood appears to be the primary target site of nitrite action. From the blood plasma, nitrite diffuses into red blood cells, where it oxidises iron in haemoglobin (Hb) to the 3? oxidation state. The haemoglobin that is oxidised in this way is called methaemoglobin or ferrihaemoglobin, which reduces the total oxygen-carrying capacity of the blood (Cameron 1971). Haemato-immunological parameters are increasingly used as indicators of physiological stress response to endogenous or exogenous changes in fish (Santos and Pacheco 1996). In addition, cortisol and glucose levels can be used as general stress indicators in fish (Santos and Pacheco 1996). The exposure to contaminants in aquatic ecosystems can enhance the intracellular formation of reactive species of oxygen (ROS), such as hydrogen peroxide, superoxide and the hydroxyl radical, which induce oxidative damage to biological systems (Livingstone 2001; Kelly et al. 1998; Livingstone et al. 1990). The ROS can be detoxified by an enzyme defence system, comprising superoxide dismutase (SOD) and catalase (CAT). Changes in the levels of antioxidant enzyme activities can be used as possible stress biomarkers in different aquatic organisms. To the best of our knowledge, ROS formation upon nitrite exposure has not been reported in fish, but the reactive nitrogen

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species (RNS) are formed via the formation of nitric oxide from nitrite (Jensen and Hansen 2011). Nitrite exposure also induces ion-regulatory disturbances in freshwater fish (Jensen et al. 1987; Jensen 1990a). As NO2- and Cl- compete for the same uptake pathway (Williams and Eddy 1986; Harris and Coley 1991), part of the active Cl- uptake is converted to NO2- uptake, whereas passive Cl- efflux to the surrounding hypoosmotic environment persists. This leads to net Cl- loss and reduced extracellular Clconcentrations in some species (Jensen et al. 1987; Jensen 1990b; Harris and Coley 1991). Furthermore, accumulation of NO2- disturbs K? homoeostasis leading to loss of potassium from skeletal muscle (Jensen 1990b; Stormer et al. 1996; Knudsen and Jensen 1997) and from red blood cells (Jensen 1990a), resulting in extracellular hyperkalemia in fish (Jensen et al. 1987). Several mechanisms are proposed for detoxification of nitrite. The red blood cells of fish contain methaemoglobin reductase enzyme capable of reconverting methaemoglobin to haemoglobin (Cameron 1971; Huey and Beitinger 1982). This occurs steadily and restores the normal proportion of haemoglobin if a fish is transferred to nitrite-free water (Knudsen and Jensen 1997). Fish is also capable of detoxifying nitrite by oxidising it to low-toxic nitrate (Doblander and Lackner 1996). Dicentrarchus labrax (Scarano and Saroglia 1984) and Onchorhynchus mykiss (Cameron 1971; Stormer et al. 1996; Doblander and Lackner 1997) oxidise nitrite to nitrate by means of oxyhaemoglobin and produce methaemoglobin, which is reduced back to functional haemoglobin by the methaemoglobin reductase system. Indian major carps contribute more than 82% of the total aquaculture production in India (FAO 2003), and high levels of nitrite have been found in carp culture ponds in recent years due to intensification of culture practices (Das et al. 2004b). Under normal conditions, nitrite is quickly converted to non-toxic nitrate in the water by naturally occurring bacteria Nitrobactor spp (Robert et al. 1997). Generally, a 24- to 48-h exposure is required for maximum accumulation of nitrite in fish (Eddy et al. 1983; Aggergaard and Jensen 2001). The lethal concentration (LC50) of nitrite declines after 24 h, and the rate of decline is very low. Therefore, the relevant duration for short-term toxicity testing is probably 24–96 h as is the case for many toxicants (Lewis and Morris 1986). Hence, the present

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investigation was undertaken to elucidate the effects of short-term exposure to sublethal levels of nitrite on serum electrolytes, antioxidative enzymes and haematological parameters of Labeo rohita, the most important candidate species.

Materials and methods Experimental animal and experimental set-up Juveniles of L. rohita (avg. wt, 66.5 ± 0.5 g) were procured from Prem Fisheries Consultancy, Gujarat, India, and transported in a circular container (500 L) with sufficient aeration to the experimental facilities at Central Institute of Fisheries Education, Mumbai, India. The fish were acclimatised to the laboratory conditions for 15 days before transferring to the experimental plastic tanks (80 9 57 9 42 cm3) of 150 L capacity. Determination of median lethal concentration (LC50) A static non-renewable acute toxicity bioassay was conducted to determine 96-h LC50 of nitrite for L. rohita (avg. wt, 66.5 ± 0.5 g). Nitrite concentrations (mg NO2-N l-1) were obtained by adding sodium nitrite (Himedia Laboratories, Mumbai, India) in tap water. Initially, a range-finding test was conducted to ascertain the range to be followed in the definitive test. In this trial, the fishes were exposed to a range of concentration, viz. 0, 5, 10, 20, 40, 80 and 160 mg l-1 nitrite for 96 h. The test was carried out with three replicates of each concentration containing eight fishes in each tank. No water exchange was done, and the fishes were not fed during the period of experimental trial in order to minimise nitrogen excretion and to maintain water quality, especially of nitrite concentration. Percentage mortality was recorded at 24, 48, 72 and 96 h. The range of LC50 for L. rohita (avg. wt, 66.5 ± 0.5 g) under given conditions was ascertained to lie below 20 mg l-1. Hence, for the definitive test, 3, 6, 9, 12, 15, 18 and 20 mg l-1 nitrite concentrations were selected, and test was conducted in triplicate (n = 3) for each concentration with 10 fishes in each tank, that is, a total of 30 fish in each concentration were used for the determination of LC50. The fishes

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were not fed during the period of experiment. At the beginning and the end of the trail, nitrite levels were measured spectrophotometrically following the method of Shechter et al. (1972), and in all the treatment groups, nitrite levels remained constant and did not differ from nominal value. The physicochemical parameters of water were within the optimum range (dissolved oxygen, 5.6–7.4 mg l-1; temperature, 26.4–27.3°C; pH, 7.5–8.6; ammonia nitrogen, 0.14–0.27 mg l-1; nitrate nitrogen, 0.02–0.07 mg l-1; and chloride, 10.3 mg l-1) throughout the experimental period. Percentage mortality was recorded at 24, 48, 72 and 96 h. Dead fish were removed from each tank immediately. The data obtained from the experiment were processed by probit analysis using a computer programme, SPSS (version 14; SPSS Inc., Chicago, IL, USA).

Sampling At the end of 96 h, three fishes from each replicate and a total of 9 fishes from each concentration were anesthetised with clove oil at 100 mg l-1 water and sampled for blood and serum. The fish was then dissected under aseptic conditions to remove gill, liver and brain. For enzyme assays, tissues were homogenised with chilled 0.25 M sucrose solution using a mechanical tissue homogeniser (MICCRA D-9, ARTProzess, Labortechnik GmbH KG, Mullheim, Germany). The homogenised samples were centrifuged (7,000 g, 4°C for 10 min), and supernatants were collected and stored at -20°C for subsequent enzyme assays. Blood was collected (as described by Das et al. 2004a, b) from one fish of each replication at 12, 24, 48 and 96 h of exposure by puncturing the caudal vein using a medical syringe (No. 23), which was previously rinsed with 2.7% EDTA solution (as anticoagulant) and shaken gently in order to prevent haemolysis of blood. The sampled fish were immediately returned back and were avoided (by looking at the needle mark in the caudal peduncle) during next blood sampling from the same tank. The blood samples that are not pooled were used for the determination of haemoglobin content, total erythrocyte counts, total leucocyte counts and glucose estimation. Blood samples were also collected in anticoagulant-free syringes only once at 96 h, trans-

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ferred to microcentrifuge tubes and allowed to clot for the estimation of electrolytes. Biochemical assays Haematological parameters Blood glucose Blood glucose level was estimated by the method of Nelson and Somogyi (1945). Haemoglobin and haematocrit The haemoglobin percentage was determined by estimating cyanmethaemoglobin using Drabkin’s fluid provided in the kit (Qualigens, Mumbai, India). The absorbance was measured using a spectrophotometer (MERCK; Merck and Co. Inc., Whitehouse Station, NJ, USA and Nicolet Evolution 100; Thermo Instruments, Canada Inc., ON, Canada) at a wavelength of 540 nm. The final concentration was calculated by comparing with standard cyanmethaemoglobin (Qualigens, Mumbai, India). Haematocrit (Hct %) was determined by the Wintrobe and Westergreen method as described by Blaxhall and Daisley (1973).

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Enzymes of neurotransmission Acetylcholine esterase Acetylcholine esterase (AChE) (EC. 3.1.1.7) activity was measured by the change in OD at 540 nm using the method of Hestrin (1949) modified by Augustinsson (1957). NADH–methaemoglobin reductase activity NADH–methaemoglobin reductase activity in blood was measured by the change in OD at 600 nm using the method described by Avilez et al. (2004). The specific activity is expressed in U (units) per milligram of haemoglobin (U mg total Hb-1). Serum cortisol Cortisol in fish serum was estimated by Enzyme Immuno Assay (EIA KIT DSL-10-2000) kit method. The kit was purchased from Diagnostic Systems Laboratories, Mumbai. Plasma cortisol was expressed as nanogram per millilitre (ng ml-1). Statistical analysis

RBC count, WBC count and mean corpuscular volume (MCV) Total erythrocyte and leucocyte were counted in a haemocytometer using erythrocyte and leucocyte diluting fluids supplied with the kit (Qualigens, Mumbai, India), respectively. The following formula was used to calculate MCV and the number of erythrocytes and leucocytes per millilitre of the blood sample: MCV (fL) = [Haematocrit (%)/No. of RBC] 9 10 No. of cells ml-1 = (No. of cells counted 9 dilution)/(Area counted 9 depth of fluid)

Data were analysed by one-way analysis of variance (ANOVA), and the significant difference between the different nitrite concentrations were determined by Duncan’s multiple range test using SPSS (version 16).

Results Median lethal concentration (LC50)

Serum sodium, potassium and chloride were analysed in an electrolyte analyser (9180 Electrolyte Analyser SN 13513, Roche Diagnostics, GmbH, D-68298 Mannheim, Germany).

The LC50, 95% confidence limits, safe level and slope functions of each response curve for different exposure periods are presented in Online Resource 1. The LC50 values for 24, 48, 72 and 96 h were 29.79, 14.60, 11.63 and 11.28 mg l-1, respectively. The cumulative percentage mortalities in different nitrite concentrations at 24, 48, 72 and 96 h were presented in Online Resource 2.

Antioxidant enzymes

Haematological parameters

Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured by the method of Misra and Fridovich (1972). Catalase (CAT; EC 1.11.1.6) activity was measured by the method of Takahara et al. (1960).

The changes in total leucocyte counts (TLC), total erythrocyte count (TEC), haemoglobin content, haematocrit and MCV values were assessed after 0, 12, 24, 48 and 96 h of exposure to sublethal concentrations

Serum sodium, potassium and chloride

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(0, 3, 6, 9, 12 and 15 mg l-1) of nitrite. The comparisons were made at different time points within each exposure group taking 0 h as the control within each group. L. rohita exposed to different nitrite concentrations showed a reduction in TLC at 12 h followed by an increase at 24 h (Fig. 1). A progressive reduction in the TECs, haemoglobin and haematocrit (Fig. 2) was observed with increasing nitrite concentration and exposure period. TEC count significantly decreased when dose was increased to 9 mg l-1 or above and also at 12-h exposure time. Haemoglobin level in different nitrite-exposed groups showed a progressive decline up to 22% with increasing nitrite exposure dose (0 to 15 mg l-1). Greater reduction in Hb was observed in all the nitrite concentrations at 12 h compared to the reduction in the subsequent exposure. MCV values were significantly lower in nitrite-exposed groups compared to unexposed group (Fig. 2). Two-way ANOVA indicated a significant effect of nitrite concentrations on all studied haematological parameters. The TEC, Hb and Hct were decreased significantly as the nitrite concentration increased, and the lowest values were observed in 15 mg l-1. The highest TLC count was recorded in 9 mg l-1 nitrite-exposed groups, while all the other nitrite-exposed groups showed significantly lower TLC count.

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d c

c

c

c

d d c c b

b

c b

a a a a

Fig. 1 Total leucocyte (9103cells/mm3) count of L. rohita exposed to different concentrations of nitrite (mg NO2-N l-1) for a period of 0, 12, 24, 48 and 96 h. Data expressed as mean ± SE, n = 6. Mean values bearing different superscripts (a, b, c and d) in each series vary significantly (ANOVA, Duncan’s test, P \ 0.05)

A significant (P \ 0.05) difference was observed in the blood glucose level among the control and different exposure groups in a dose-dependent manner after 96 h (Table 1). Blood glucose in different nitrite-exposed groups showed a progressive rise, and a maximum of 25% increase in glucose level was found when nitrite exposure dose increased from 0 to 9 mg l-1. The blood glucose increased up to exposure dose of 9 mg l-1, after which it remained similar. The blood glucose after 96-h exposure showed a second-order polynomial relationship (Y = -0.378x2 ? 4.1646x ? 29.767, R2 = 0.977. P \ 0.01; where ‘Y’ is blood glucose and ‘x’ is nitrite concentration) with nitrite concentrations.

L. rohita exposed to different nitrite levels showed an increasing trend in serum K? concentration, while a declining trend in serum Cl- and Na? concentration in a dose-dependent manner. The serum Na? after 96-h exposure showed a second-order polynomial relationship (Y = 1.536x2 - 14.68x ? 142.3, R2 = 0.968. P \ 0.01; where ‘Y’ is serum Na? and ‘x’ is nitrite concentration) with nitrite concentrations. The serum Cl- concentration decreased by 2.5% upon exposure to 3 mg l-1 nitrite and gradually decreased up to 23% at 15 mg l-1. Serum K? concentration exhibited a gradual and significant increase (P \ 0.05) up to 47% in fish exposed to 15 mg l-1 nitrite than the unexposed group. The serum K? and Cl- after 96-h exposure showed a linear relationship (Y = 0.221x ? 2.542, R2 = 0.938. P \ 0.01 and Y = -5.760x ? 129.5, R2 = 0.952. P \ 0.01, respectively; where ‘Y’ is the serum K? and Cl- and ‘x’ is nitrite concentration) with nitrite concentrations.

Serum sodium, potassium and chloride

Serum cortisol

Statistically significant differences were observed (P \ 0.05) in the serum Cl-, Na? and K? concentrations of fish exposed to different levels of nitrite after 96 h and those in unexposed groups (control) (Table 1).

A significant (P \ 0.05) difference was observed in the serum cortisol level among the control and different exposure groups in a dose-dependent manner after 96 h (Table 1). Serum cortisol in different nitrite-exposed

Blood glucose

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Fig. 2 a Haematocrit; b haemoglobin; c total erythrocyte count; and d MCV of L. rohita exposed to different concentrations of nitrite (mg NO2-N l-1) for a period of 0, 12, 24, 48 and 96 h. Data

expressed as mean ± SE, n = 6. Mean values bearing different superscripts (a, b, c, d and e) in each series under each panel vary significantly (ANOVA, Duncan’s test, P \ 0.05)

groups showed a progressive rise and registered 17% increase at 3 mg l-1 and further rose up to 62% when nitrite exposure dose was increased to 15 mg l-1 nitrite compared to control at 96 h. The regression model revealed a linear relationship (Y = 13.249x ? 100.33, R2 = 0.989, P \ 0.01; where ‘Y’ is the serum cortisol and ‘x’ is nitrite concentration) between cortisol and nitrite concentration.

significant (P \ 0.05) difference in the SOD and CAT activities among the control and different exposure groups in a dose-dependent manner after 96 h. Catalase activity in liver exhibited a gradual and significant increase (P \ 0.05) in the order of 133% in fish exposed to 15 mg l-1 nitrite than the unexposed group. Compared to unexposed (control) group, the activity of SOD in gill increased by 102% and in liver by 50% when nitrite exposure dose increased from 0 to 15 mg l-1.

Antioxidant enzymes Methaemoglobin reductase The activities of CAT and SOD in gill and liver of fishes from unexposed group and different exposure groups are presented in Table 2. In both the organs, there was

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The activity of methaemoglobin reductase in the blood of fishes from unexposed group and different exposure

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Table 1 Serum electrolyte (sodium, potassium and chloride) contents, blood glucose levels and serum cortisol levels of L. rohita exposed to different concentrations of nitrite for a period of 96 h Nitrite concentration (mg NO2-N l-1)

Sodium (mEq l-1)

Potassium (mEq l-1)

Chloride (mEq l-1)

Serum cortisol (ng ml-1)

0

129.50c ± 0.76

2.67a ± 0.03

121.33e ± 0.67

109.75a ± 5.05

b

b

Blood glucose (mg dl-1) 33.13a ± 0.31

d

b

128.85 ± 2.95

37.31b ± 0.79

d

c

3

119.73 ± 0.49

6

a

109.50 ± 0.29

3.27 ± 0.09

116.67 ± 0.33

142.90 ± 1.6

38.89bc ± 0.36

9

109.57a ± 0.30

3.30b ± 0.06

105.33c ± 0.88

154.90d ± 0.01

40.19cd ± 0.61

e

12 15

3.13 ± 0.09

a

108.60 ± 0.17

c

b

101.00 ± 0.58

166.00 ± 0.01

40.67d ± 0.72

d

a

f

41.50d ± 0.30

3.60 ± 0.06

a

108.63 ± 0.27

P value

118.33 ± 0.33

b

3.93 ± 0.03

0.001

93.67 ±1.45

0.01

177.80 ± 0.01

0.01

0.01

0.01

Mean values bearing different superscripts (a, b, c, d and e) under each column vary significantly (P \ 0.05). Data expressed as mean ± SE, n = 6

groups is presented in Table 2. Methaemoglobin reductase activity showed a significant increase (P \ 0.05) in fish exposed to nitrite compared to the unexposed group. Its activity increased up to the concentration of 6 mg l-1 after which there were no further changes. The methaemoglobin reductase activity after 96-h exposure showed a second-order polynomial relationship (Y = -0.005x2 ? 0.0479x - 0.02, P \ 0.01, R2 = 0.985) with nitrite concentrations.

presented in Table 2. Compared to unexposed group (control), acetylcholine esterase (AChE) in brain showed a dose-dependent inhibition with significant (P \ 0.05) reduction (54%) in its activity at exposure dose of 15 mg l-1 nitrite after 96 h. AChE activity reduced significantly when exposure dose increased from 0 to 6 mg l-1. After which there was no further decrease even after exposure to higher dose.

Acetylcholine esterase assay

Discussion

The acetylcholine esterase activities in the brain of fishes exposed to different nitrite concentrations are

The present study elucidates the antioxidative and haematological responses of L. rohita juveniles

Table 2 Activities of antioxidative enzymes (catalase and SOD) and serum cortisol of L. rohita exposed to different concentrations of nitrite for a period of 96 h Nitrite concentration (mg NO2-N l-1)

Catalase Gill

0 3 6 9

SOD Liver

2.51a ± 0.15 b

3.36 ± 0.06 c

4.46 ± 0.26 c

4.72 ± 0.10 d

Gill

0.94a ± 0.01 b

1.33 ± 0.01 d

1.59 ± 0.07 1.45

bcd cd

± 0.05

12

5.27 ± 0.17

1.50

15 P value

5.85d ± 0.12 0.01

1.43bc ± 0.07 0.01

± 0.04

Liver

16.00a ± 0.15 b

24.76 ± 0.61 28.85

Acetylcholine esterase Brain

cd

± 0.11

c

27.69 ± 0.65

bc

16.63

± 0.22

c

17.64 ± 0.46 b

15.60 ± 0.23 c

0.28c ± 0.01 bc

0.02a ± 0.01

± 0.01

0.06b ± 0.01

b

0.20 ± 0.04

0.08c ± 0.01

a

0.09c ± 0.01

a

0.25

0.12 ± 0.01

± 0.82

17.10 ± 0.48

0.12 ± 0.01

0.09c ± 0.01

32.30e ± 0.85 0.01

16.89bc ± 0.81 0.01

0.13a ± 0.01 0.01

0.09c ± 0.01 0.01

30.90

de

11.40a ± 0.09

MetHb reductase Blood

Mean values bearing different superscripts (a, b, c, d and e) under each column vary significantly (P \ 0.05). Data expressed as mean ± SE, n = 6 Catalase: mmol H2O2 decomposed/min/mg protein at 37°C SOD (superoxide dismutase): lmol/mg protein/min at 37°C AChE activity: lmol of acetyl choline hydrolysed/min/mg protein at 37°C MetHb reductase (methaemoglobin reductase): activity expressed as U/mg Hb

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exposed to different nitrite concentrations and its consequences on ionic balance. In the present study, the 96-h LC50 of nitrite for L. rohita was found to be 11.28 mg l-1 (at chloride level of 10.3 mg l-1), a value similar to that reported for mrigal (Das et al. 2004b) and grass carp (Alcaraz and Espina 1997) as 10.4 and 10.6 mg l-1, respectively, but lower than that reported in other fishes, viz. 16.2 mg l-1 in Tilapia (Palachek and Tomasso 1984) and 40 mg l-1 in common carp (Solbe’ 1981) in similar freshwater environment. However, no literature is available regarding the LC50 of nitrite for L. rohita to compare our result. Leucocytes (WBC) play an important role in nonspecific or innate immunity, and their count can be considered as an indicator of the health status of fish (Roberts 1978). In the present study, all the nitriteexposed groups showed a reduction in WBC followed by an increase at 12 and 24 h, respectively (Fig. 1). The initial decline in TLCs when exposed to sublethal levels of nitrite may be due to the reaction of fish to the stressor resulting from increased pituitary–interrenal activity. The stress induced by nitrite might have immediately blocked and suppressed leucopoietic tissue (Nussey et al. 2002) and activation of leukopoiesis took longer time resulting in a later increase in TLC at 24 h as suggested by Das et al. (2004b) who found that on nitrite exposure, TLC reduced at 6 h and thereafter increased. Prolonged exposure (48 h) to nitrite probably led to the failure or exhaustion of leucopoiesis resulting in reduction in TLC at 48 h. Reduction in haemoglobin content could be attributed due to methaemoglobinemia (Knudsen and Jensen 1997; Jensen 2003) caused by nitrite and depression and exhaustion of the haemopoiesis under the hypoxic condition as suggested by Gill et al. (1991). The observed decrease in RBCs, haemoglobin and haematocrit could be due to the lysis of blood cells and an increased removal of RBCs from the circulation as suggested in carp (Jensen 1990b; Knudsen and Jensen 1997). The formation of methaemoglobin along with the reduced erythrocyte count and haemoglobin concentration might have resulted in hypoxic condition within the fish. The increased energetic demand of RBCs by the increased activity of NADH-methaemoglobin reductase to reconvert the methaemoglobin to haemoglobin might have resulted in a faster destruction of RBCs by the spleen and kidney as suggested by Scarano and Saroglia (1984). The observed higher

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haematocrit values in the nitrite-unexposed groups (Fig. 2) may be attributed to release of catecholamines, causing beta-adrenergic swelling of the erythrocytes and release of RBCs from the spleen due to the handling/sampling procedure. The decrease in haematocrit with nitrite exposure may relate both to decreased number of RBCs and inhibition of adrenergic swelling by methaemoglobin formation (Nikinmaa and Jensen 1992). In the present study, K? efflux from RBCs, which is evident from the corresponding rise in serum K? on nitrite-exposed groups, probably resulted in the shrinkage of RBCs and thereby led to the observed reduction in haematocrit, which is also manifested by reduced MCV values. There was also a defined negative correlation (r2 = 0.92) found between serum K? with haematocrit value. Nitrite is assumed to enter fish body by competing with chloride for the active branchial chloride uptake (Williams and Eddy 1986) and leads to alterations in extracellular electrolyte composition in carp, with a large decline in plasma chloride as a major effect (Jensen et al. 1987). Aggergaard and Jensen (2001) have reported that rainbow trout exposed to nitrite showed a rise in plasma K? and a decrease in plasma Cl-. This rise in plasma K? is probably due to the release of K? from intracellular compartments (Knudsen and Jensen 1997). In the present study, serum K? increased and Na? and Cl- decreased significantly as a result of nitrite exposure (Table 1). A similar extracellular hyperkalemia has been observed in carp (Jensen et al. 1987) and rainbow trout (Stormer et al. 1996) during nitrite exposure and is related to loss of intracellular potassium from RBCs (Jensen 1990b, 1992) and skeletal muscle (Knudsen and Jensen 1997). The decline in plasma chloride in nitrite-exposed fish in the present study can be correlated to the shift in the part of the active Cl- uptake across the gill to NO2uptake and the persistence of the passive Cl- efflux to the surrounding hypoosmotic environment. This leads to net Cl- loss and reduced extracellular Cl- concentrations as reported in some species (Jensen et al. 1987; Jensen 1990b; Harris and Coley 1991). The possible explanation for the observed rise in serum K? and decline in Na? in the present study could be the inhibition of Na?–K?-ATPase even though we have not estimated the activity of this particular enzyme. Jensen et al. (1987) explained that the significant reduction in plasma Na? in nitrite-exposed carp might be due to the inhibition of Na?–K?-ATPase, resulting

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in tissue K? efflux and Na? uptake. Nitrite also inhibits carbonic anhydrase in trout gills (Gaino et al. 1984), and this inhibition reduces the production of H?, the counterion for Na? uptake, reducing the Na? influx rate (Harris and Coley 1991). An increase in plasma sodium levels has been reported in Lates calcarifer exposed to nitrite (Woo and Chiu 1997). Generally, cortisol and blood glucose are used as markers of various stress levels. Stress increases the glucose content in blood because of intensive glycogenolysis and the synthesis of glucose from extrahepatic tissue proteins and amino acids (Almeida et al. 2001; Akhtar et al. 2010) in order to provide energy for the fight-or-flight reaction. Nakno and Tomlinson (1967) pointed out that all types of stress elevated the secretion of catecholamine, which in turn increases the breakdown of glycogen and elevates blood glucose level. Elevated cortisol levels upon different environmental stressors were reported in Cirrhinus mrigala (Tejpal et al. 2009) and Labeo rohita (Akhtar et al. 2010). In the present study, the elevation of serum cortisol observed in fish exposed to nitrite (Table 1) suggests that nitrite induces the activation of the hypothalamic–pituitary–interrenal (HPI) axis with a subsequent elevation of serum cortisol (Pickering and Pottinger 1989). Acetylcholine esterase catalyses the hydrolysis of acetylcholine, the chemical responsible for the transmission of nerve impulses. Under normal conditions, the pseudocholinesterase produced in liver hydrolyses cholinesters capable of inhibiting AChE and releases AChE from their blocking effect. In the present study, 96-h nitrite exposure caused a progressive decline in the AChE activity (Table 2), which indicates nitriteinduced stress in L. rohita. Our results are in agreement with Das et al. (2004a) who reported a reduction in brain and liver AChE activity in Indian major carps on nitrite exposure. Reduction in AChE activity in fish exposed to different stressors was reported by many workers (Sancho et al. 1997; Bretaud et al. 2000; Akhtar et al. 2010). Such inhibition of enzyme activity might be due to an increased accumulation of cholinesters at the synaptic region due to reduced release of pseudocholinesterase, the one responsible for the hydrolysis of cholinesters. Several studies demonstrated that changes in antioxidant enzyme activities could be used as stress indicators (Akhtar et al. 2010; Alexander et al. 2010). The exposure to various contaminants in aquatic ecosystems can enhance the intracellular formation of

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reactive oxygen species (ROS) capable of inducing oxidative damage (Livingstone et al. 1990). The ROS can be detoxified by an enzyme defence system, comprising superoxide dismutase (SOD) and catalase (CAT). During nitrite exposure, reactive nitrogen species (RNS) are formed via the formation of nitric oxide from nitrite which may cause oxidative damage (Jensen and Hansen 2011). In the present study, the elevated activities of catalase and superoxide dismutase in both liver and gill following 96-h exposure to nitrite were observed (Table 2), apparently to provide protection against oxidative damage. When a nitrite-exposed fish transferred to nitritefree water, the haemoglobin level is restored to normal level (Knudsen and Jensen 1997). In channel catfish, this recovery of normal haemoglobin has been attributed to the NADH-methaemoglobin reductase system (Schoore et al. 1995). In the present study, enhanced activity of methaemoglobin reductase has been observed with nitrite exposure (Table 2) as a protective mechanism by reducing haemoglobin Fe3? to Fe2?. A similar result has been observed in neotropical teleost Brycon cephalus (Avilez et al. 2004). In conclusion, the overall results of the present study revealed that short-term nitrite exposure causes alterations in all measured tissue enzymes, serum electrolytes, blood glucose, serum cortisol and haematological parameters. Haemoglobin appears to be more sensitive, and the methaemoglobin reductase system seems to be induced in L. rohita by environmental nitrite and thereby helps in the nitrite detoxification process. It is very important to monitor the water nitrite level regularly, ensuring proper water quality management in intensive carp culture systems to enhance aquaculture production. Acknowledgments The authors are grateful to Dr. W. S. Lakra, director and vice chancellor, Central Institute of Fisheries Education (CIFE), Mumbai, for providing facilities and financial assistance for carrying out the work. The first author is grateful to CIFE for awarding the Institutional Fellowship.

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