Study of Action of Sublethal Concentrations of Diazinon on Blood Indices of Male and Female Anabas testudineus

Sunil Chandra Pradhan1* and Ajaya Kumar Patra2

1V.N. Autonmous College, Jajpur road, Jajpur- 302001, India

2Department of Zoology, Utkal University, Vani Vihar, Bhubaneswar-751004, India

*Corresponding Author:
Sunil Chandra Pradhan
V.N. Autonmous College
Jajpur road, Jajpur- 302001, India
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Abstract

Anabas testudineus is a commercially important and nutritionally rich fish. This investigation evaluated haemato-biochemical responses of Anabas testudineus associated with exposure of sublethal concentrations 3.275 ppm (1/2th of LC50) and 1.31 ppm (1/5th of LC50) of diazinon 60 EC under laboratory conditions for one week (7 days), two weeks (14 days) and four weeks (28 days). Increase exposure time and dose lead to decrease of Hemoglobin (Hb), Total Erythrocyte Count (TEC) and Hematocrit (HCT) values, but increase of Total Leucocytes Count (TLC), Mean Corpuscular Hemoglobin Concentration (MCHC) and Mean Corpuscular Hemoglobin (MCH) values in both sexes. Significant (P<0.01) increase in blood glucose (77.44 mg/dl) and cholesterol (232.03 mg/dl) level was more noticed among female whereas total protein contents were significantly decreased. Three factors ANOVA analysis showed significant (p<0.01) variation in TLC (F=5.56), MCV (F=20.445), MCHC (F=12.427), glucose (F=4.029) and cholesterol (F=5.008). Sublethal concentrations of diazinon lead to subtle changes in blood indices of test fish. ANOVA showed that the pesticide concentrations had more influence than the duration of exposure in all cases (P<0.01). Findings of this study showed sex-related and depicted severity of diazinon was both dose and duration dependant.

Keywords

Anabas testudineus, Diazinon, Sublethal concentration, Blood indices

Introduction

Fisheries and aquatic resources are exceptionally valuable natural assets, which provide long-term benefits in return for minimal care and protection. Appreciation of fisheries and aquatic systems has been accompanied by increasing concern about the effects of growing human populations and human activity on aquatic life. Pesticides are one group of toxic compounds linked to human use that have a profound effect on aquatic life and water quality. Pesticides in the aquatic environment can negatively affect the ecosystem [1,2]. Among the various animal groups, fishes have been identified as being very sensitive to pollutants and have been the most popular test organism because they are presumed to be the best understood organism in the aquatic environment [3]. Hematology is used as an index of fish health status in a number of fish species to detect physiological changes following different stress conditions like exposure to pollutants, diseases, metals, hypoxia, etc [4]. Hence, its use is growing and becoming very important for toxicological researches. It is noted that studies on fish blood gives the possibility of knowing physiological conditions within the fish long before outward manifestation of pathological condition. The climbing perch, Anabas testudineus, is one of the potential cultivable air-breathing species attracting attention of the pisciculturists owing to their high production potential from paddy fields and stagnant shallow ponds, high economic value, and preference due to its tolerance to adverse environmental conditions in the derelict water bodies [5,6]. With increased use of pesticides in agriculture, it has become important to know about the toxic effect of pesticides on such non-target organisms as these test fishes.

One such widely used toxicant is diazinon, which is a type of organophosphorous pesticides [7]. Although, the aquatic environment is not the target one for the use of such pesticides the results of a number of monitoring studies have evidenced the presence of diazinon and its metabolite, diazoxon, in surface waters [8,9]. That is why great attention has been paid to the effect of diazinon on fish organism. However, sex-related variations in various hematological values of fishes exposure to pesticide are scanty [10-12]. The aim of the present work was to assess the influence of sublethal concentration of the pesticide on the test fish and observed changes in the haematological parameters of male and female Anabas testudineus in order to show the toxic effects as indices of the pesticide stress.

Materials and Methods

The toxicant diazinon 60 EC had been used in this study. The commercial formulation of diazinon, Basudin 60 EC brand, with the active ingredient diazinon (O, O-Diethyl O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate with purity of 60% and dissolved in 40% acetone, was used to prepare test solutions of diazinon [13]. Toxicity concentrations were defined based on previous studies on diazinon 60 EC toxicity to same fish species. The acute 96 hours LC50 value of diazinon in Anabas testudineus was 96 h 6.55 ppm [14]. Concentrations 3.275 ppm (1/2th of LC50) and 1.31 ppm (1/5th of LC50) of diazinon were selected for the test. Anabas captured from the Tankapani pond, Khurda District, Bhubaneswar (19° 40"N to 20° 25"N Latitude and 24° 55"E to 36°05"E Longitude; area) by using a net and transferred to cement tank (2010). Sexually matured fishes (male: 12.00 ± 0.50 cm, 30 ± 1.00 g; female: 13.50 ± 0.50 cm, 45 ± 1.00 g) were used in this investigation. Fishes were washed with 0.1% KMnO4 solution to avoid dermal infection and APHA et al. [15,16] were followed for maintaining fishes. The collected fishes were acclimated to laboratory conditions in dechlorinated tap water for 30 days. The fishes were fed with commercial fish feed during acclimation period. After acclimation, fishes were separated into different groups of 10 each in similar sized tank. The water in the aerated tank was changed in every 24 hours to maintain the concentration of the diazinon 60 EC during the period of exposure. No mortality occurred in any group during the experimental period. Two hundred seventy test fishes were used for the present study and these were divided into twenty seven groups, each group consisting of ten fishes. The first twelve groups (six male and six female groups) were exposed to 3.27 ppm and the second twelve groups were exposed to 1.31 ppm diazinon for one week (7 days), two weeks (14 days) and four weeks (28 days) while the remaining three groups were maintained as control in diazinon free water.

At the end of the exposure period, fishes were caught very gently using a small dip net. Samples of 2 ml blood were taken from the caudal vein of non-anaesthetized fish by sterilize syringe containing EDTA as an anticoagulant for blood analysis [17].

Hematological analysis

Haemoglobin estimation by cynmethaemoblobin method [12,18]. Estimation of Total Erythrocyte Count (TEC) and Total Leukocyte Count (TLC) as per Pal et al. [19] and Pradhan et al. [12]. The number of cells count was determined as described by Svobodova et al. and Oluyemi et al. [20,21]. The Hct was estimated by using microhemotocrit reader and expressed in percentage. Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin Concentration (MCHC) were calculated as per method Dacie and Lewis [17] and pradhan et al. [12].

Blood biochemical analysis

Estimation of blood plasma protein was carried out as per method of Lowry et al. [22]. Bovine serum albumin was used as standard and different (0.5-5 mg/ml) solutions were prepared by mixing stock BSA solution (1 mg/ml). 0.2 ml protein solution was taken from each dilution, followed by mixing of 2 ml of alkaline copper sulphate solution, which were mixed well and incubated at room temperature for 10 min. Then, Folin Ciocalteau solution (0.2 ml) was added to each tube and incubated for 30 min. The optical density was taken at 660 nm and absorbance was plotted against protein concentration to obtain a standard calibration curve. Blood glucose level was estimated by Glucose Oxidase and Peroxidase (GOD/POD) method using the GOD/POD kit (Coral Clinical Systems), 1 ml of test reagent was added to all the test tubes. Then, 10 ml of distilled water, 10 ml of standard solution and 10 ml of plasma samples were added to the respective marked test tube. These solutions were incubated at 25°C for 30 min and optical density measured the absorbance at 505 nm. Cholesterol amount was determined by the CHOD/PAP method using Kit (Crest Biosystem, India) [23,24].

Statistical analysis

All data were analyzed using ANOVA with the aid of SPSS 17 for windows. The mean values obtained for each variable analyzed at the different experimental concentrations were compared to each other by parametric analysis of variance (ANOVA). The data were statistically analyzed using Duncan's multiple range tests to determine differences in means [25].

Results and Discussion

In the present study, the effect of diazinon on the test fishes in sublethal exposure revealed that pesticide had considerable impact on the different blood parameters (Table 1). Hemoglobin (Hb) concentration varied from 9.16 to 11.54 gm/dl and 7.77 to 11.21 gm/dl in male and females respectively. Hb values at one week (7 days), two weeks (14 days) and four weeks (28 days) followed decrease pattern in both sexes.

Gender Duration Dose Hb TEC TLC HCT MCV MCH MCHC
Male 1st week Control Mean 11.35a 2.93a 13.74c 34.18a 153.21b 39.56b 31.78d
SE 0.16 0.08 0.60 0.15 0.29 0.15 0.16
1.31 ppm Mean 11.15a 2.96a 14.36c 29.40a 156.25a 39.53b 33.47bc
SE 0.15 0.06 0.49 0.43 0.12 0.28 0.40
3.227 ppm Mean 10.78a 2.68a 16.14c 28.79a 156.87a 39.70b 36.24a
SE 0.17 0.13 0.33 0.23 0.09 0.58 0.15
2nd week Control Mean 11.54a 2.96a 14.11c 33.76a 153.96b 40.03b 34.37b
SE 0.19 0.09 0.29 0.13 0.07 0.07 0.44
1.31 ppm Mean 11.48a 2.93a 19.65b 27.87a 157.14a 38.62bc 32.30c
SE 0.07 0.19 0.86 0.43 0.12 0.20 0.10
3.227 ppm Mean 10.29a 2.48a 18.75ab 24.66a 159.77a 39.62b 34.86b
SE 0.04 0.17 0.38 0.67 0.34 1.28 0.12
4th week Control Mean 10.54a 3.05a 14.23c 31.48a 156.30a 40.80b 35.23ab
SE 0.13 0.12 0.33 0.60 0.27 0.16 0.17
1.31 ppm Mean 10.45a 2.94a 21.92a 29.49a 158.55a 44.94a 35.97ab
SE 0.17 0.13 0.13 1.79 0.25 0.68 0.32
3.227 ppm Mean 9.16a 2.36a 20.63a 22.70a 170.02a 41.52ab 38.25a
SE 0.47 0.20 0.28 0.24 0.62 0.87 0.34
Female 1stweek Control Mean 11.13a 2.58a 13.40c 30.03a 144.49c 41.47ab 30.70d
SE 0.19 0.04 0.58 0.21 0.20 0.18 0.20
1.31 ppm Mean 11.08a 2.80a 14.24c 26.75a 146.83c 41.19ab 32.07c
SE 0.38 0.13 0.20 0.33 0.73 0.31 0.23
3.227 ppm Mean 9.05a 2.25a 14.15d 25.31a 147.02c 45.89a 38.10a
SE 0.53 0.09 0.17 0.25 0.11 1.23 0.12
2nd week Control Mean 11.16a 2.46a 12.28d 30.23a 144.34c 44.66a 33.24bc
SE 0.17 0.05 0.18 0.33 0.31 0.22 0.11
1.31 ppm Mean 10.34a 2.40a 18.24b 24.66a 147.74c 39.69b 31.59d
SE 0.45 0.07 0.55 0.08 0.63 0.48 0.28
3.227 ppm Mean 8.91a 2.14a 18.82b 25.75a 150.95b 37.48c 35.10ab
SE 0.23 0.05 0.42 0.84 0.51 0.39 0.21
4th week Control Mean 11.21a 2.43a 14.17c 29.30a 147.29c 45.98a 33.36bc
SE 0.14 0.08 0.39 0.47 0.21 0.11 0.26
1.31 ppm Mean 9.84a 2.26a 18.50bc 25.89a 149.52b 48.16a 34.83b
SE 0.19 0.04 0.23 0.71 0.31 0.37 0.13
3.227 ppm Mean 7.77a 2.05a 18.57b 21.91a 152.65b 40.30b 35.63b
SE 0.18 0.12 0.45 0.61 1.11 0.30 0.36

Table 1: Comparative evaluation of haematological parameters of A. testudineus under different treatments of diazinon 60 EC

The total number of erythrocyte ranged from 2.36 × 106 to 3.05 × 106 per cubic mm of blood and 2.05 to 2.8 × 106/ mm3 blood in male and female fish respectively. Total erythrocyte values followed decrease pattern in both the sexes when compared with the control specimens and most significant reduction was observed in sublethal (3.227 ppm) concentration on 28th day. Total Leukocyte Count (TLC) value was higher in 4th week (28 days) and TLC varied between 13.74 × 103 per cubic mm of blood and 21.92 × 103 per cubic mm of blood and 12.28 and 18.82 × 103 per cubic mm of blood in males and females respectively. The Hct value ranged from 22.70 to 34.18% in males and varied between 21.91 and 30.23% in females indicating that this was higher for males than that of female. MCV value ranged between 153.21 and 170.02 μ3 for male and 144.49 and 152.65 μ3 for female fishes. MCH value was ranged from 38.62 to 44.94 pg in male whereas in female variation range was 37.48 and 45.98 pg.

Biochemical parameters, glucose and protein were noted as slightly higher in female than males (Table 2). Glucose concentration varied from 64.62 to 83.24 mg/dl and 63.63 to 86.78 mg/dl in male and females respectively. Blood glucose values at 1, 2 and 4 weeks followed increase pattern in both sexes and most significantly increased value was observed in 1.31 ppm in female (86.78 mg/dl) compared with the control groups. Protein values among the test fishes varied from 2.30 to 3.05 gm/dl in male and 1.96 and 2.76 gm/dl in female. Cholesterol content of fish showed variations ranging from 191.89 to 220.07 mg/dl in male and 200.78 mg/dl and 232.03 mg/dl. Blood cholesterol level (P<0.01) increased significantly throughout study period in both sublethal concentrations.

Gender Duration Dose Glucose Protein Cholesterol
Male 1st Week Control Mean 64.95b 3.05a 195.27b
SE 0.45 0.04 1.47
1.31 ppm Mean 71.18a 2.59a 210.94ab
SE 0.37 0.13 4.48
3.227 ppm Mean 72.91a 2.43a 222.07a
SE 0.51 0.06 5.34
2nd Week Control Mean 67.62a 2.92a 193.47b
SE 0.33 0.03 2.23
1.31 ppm Mean 75.24a 2.36a 205.43ab
SE 0.54 0.12 4.26
3.227 ppm Mean 83.24a 2.36a 215.77a
SE 1.57 0.03 7.13
4th Week Control Mean 65.25a 2.92a 191.89b
SE 0.33 0.02 0.73
1.31 ppm Mean 73.85a 2.53a 206.43ab
SE 0.57 0.04 5.98
3.227 ppm Mean 73.24a 2.30a 214.30ab
SE 0.49 0.05 6.54
Female 1st Week Control Mean 62.98b 2.48a 210.87ab
SE 0.17 0.02 1.74
1.31 ppm Mean 69.60a 2.18a 226.48a
SE 0.30 0.04 3.13
3.227 ppm Mean 71.94a 1.96a 231.63a
SE 0.19 0.05 3.26
2nd Week Control Mean 64.18b 2.44a 202.27ab
SE 0.25 0.05 1.90
1.31 ppm Mean 73.78a 2.26a 216.60a
SE 0.52 0.03 6.73
3.227 ppm Mean 76.84a 2.19a 221.20a
SE 0.14 0.12 9.17
4th Week Control Mean 63.63b 2.76a 200.78a
SE 0.27 0.04 0.76
1.31 ppm Mean 72.28a 2.56a 226.47a
SE 0.46 0.10 11.73
3.227 ppm Mean 77.44a 2.21a 232.03a
SE 6.84 0.08 9.58

Table 2: Comparative evaluation of blood biochemical parameters of A. testudineus under different treatments of diazinon 60 EC

Correlations among blood indices showed significant negative correlation between Hb-Cholesterol (r=-0.49, p<0.01), TEC-Cholesterol (r=-0.495, p<0.01) and TLC-Hct (r=-0.547, p<0.01) (Table 3). A positive correlation found between Hb-Hct (r=0.678, p<0.01). TLC-glucose (r=0.655, p<0.01), MCV-MCH (r=0.523, p<0.01). Significant (p<0.01) influence of pesticide on different blood parameters was seen the basis of gender, duration and dose (Table 4). There was significant (p<0.01) interaction between gender and duration observed in most parameters except Hb (ANOVA, F=0.126 Sig.=0.882), TEC, glucose and cholesterol (ANOVA, F=0.611, Sig.=0.542). Gender-Dose interaction also significant (p<0.01) except glucose (ANOVA, F=0.220 Sig.=0.803) and cholesterol (ANOVA, F=0.320 Sig.=0.728). Interactions were also significant (p<0.01) between duration and dose in most parameters except protein, cholesterol and Duration-Dose had also significant (p<0.01) influence in most parameters except (ANOVA, F=0.230 Sig.=0.920) (Table 5). Randomized Block Design, Three-way ANOVA showed significant (p<0.01) gender-dose-duration interaction for TLC, MCV, MCH, MCHC, glucose and cholesterol except Hb, TEC and protein (ANOVA, F=0.618, Sig.=0.652) (Table 6). ANOVA showed that the pesticide concentrations have more influence (P<0.01) than the duration of exposure and gender in all cases.

Parameters Hb TEC TLC HCT MCV MCH MCHC Glucose Protein Cholesterol
Hb 1                  
TEC .689** 1                
TLC -.307* -.106 1              
HCT .687** .578** -.547** 1            
MCV -.027 .251 .671** -.164 1          
MCH -.158 -.180 -.152 -.017 -.302* 1        
MCHC -.494** -.217 .307* -.387** .523** .235 1      
Glucose -.326* -.250 .655** -.600** .433** -.251 .382** 1    
Protein .454** .517** -.258 .743** .100 .007 -.256 -.498** 1  
Cholesterol -.490** -.495** .167 -.648** -.121 .090 .269* .426** -.747** 1

Table 3: Correlation analysis of blood parameters of A. testudineus

Source Dependent Variable Sum of Squares df Mean Square F Sig.
Gender Hb 12.098 1 12.098 59.645 .000**
TEC 2.973 1 2.973 76.286 .000**
TLC 56.222 1 56.222 105.166 .000**
HCT 84.275 1 84.275 75.778 .000**
MCV 1387.456 1 1387.456 2.394E3 .000**
MCH 69.996 1 69.996 74.051 .000**
MCHC 10.288 1 10.288 54.496 .000**
Glucose 36.457 1 36.457 4.225 .047
Protein .963 1 .963 71.861 .000**
Cholesterol 2119.387 1 2119.387 21.547 .000**
Duration Hb 11.717 2 5.859 28.883 .000**
TEC .179 2 .090 2.299 .115
TLC 215.152 2 107.576 201.225 .000**
HCT 47.052 2 23.526 21.154 .000**
MCV 230.438 2 115.219 198.780 .000**
MCH 120.872 2 60.436 63.937 .000**
MCHC 43.253 2 21.627 114.559 .000**
Glucose 187.808 2 93.904 10.882 .000**
Protein .163 2 .082 6.087 .005**
Cholesterol 457.573 2 228.786 2.326 .112
Dose Hb 24.405 2 12.203 60.159 .000**
TEC 1.691 2 .845 21.692 .000**
TLC 251.652 2 125.826 235.362 .000**
HCT 405.289 2 202.645 182.212 .000**
MCV 357.182 2 178.591 308.112 .000**
MCH 20.278 2 10.139 10.726 .000**
MCHC 117.452 2 58.726 311.080 .000**
Glucose 1185.498 2 592.749 68.689 .000**
Protein 2.517 2 1.258 93.938 .000**
Cholesterol 5309.805 2 2654.902 26.991 .000**

Table 4: Analysis of Variance of blood parameters of A. testiudineus on exposure to diazinon 60 EC toxicity

Source Dependent Variable Sum of Squares df Mean Square F Sig.
Gender * Duration Hb .051 2 .025 .126 .882
TEC .036 2 .018 .467 .631
TLC 6.006 2 3.003 5.617 .003**
HCT 6.002 2 3.001 2.698 .081
MCV 18.730 2 9.365 16.157 .000**
MCH 9.710 2 4.855 5.136 .01**
MCHC 7.077 2 3.538 18.744 .000**
Glucose 37.996 2 18.998 2.202 .125
Protein .384 2 .192 14.334 .000**
Cholesterol 120.248 2 60.124 .611 .548
Gender * Dose Hb 2.108 2 1.054 5.196 .010
TEC .120 2 .060 1.543 .227
TLC 11.382 2 5.691 10.645 .000**
HCT 14.032 2 7.016 6.308 .002**
MCV 23.804 2 11.902 20.534 .000**
MCH 20.321 2 10.161 10.749 .000**
MCHC 3.470 2 1.735 9.190 .001**
Glucose 3.802 2 1.901 .220 .803
Protein .141 2 .071 5.279 .010
Cholesterol 62.975 2 31.488 .320 .728
Duration* Dose Hb 2.096 4 .524 2.584 .053
TEC .347 4 .087 2.229 .085
TLC 111.733 4 27.933 52.250 .000**
HCT 43.612 4 10.903 9.804 .000**
MCV 91.283 4 22.821 39.371 .000**
MCH 147.846 4 36.961 39.103 .000**
MCHC 45.627 4 11.407 60.423 .000**
Glucose 54.014 4 13.503 1.565 .205
Protein .112 4 .028 2.092 .102
Cholesterol 90.521 4 22.630 .230 .920

Table 5: Evaluation of the variables (ANOVA) to determine whether blood parameters of A. testiudineus is dependent on gender, duration or dose

Source Dependent Variable Sum of Squares df Mean Square F Sig.
Gender * Duration * Dose Hb 1.510 4 .377 1.861NS .139
TEC .217 4 .054 1.393NS .256
TLC 11.889 4 2.972 5.560** .001**
HCT 13.465 4 3.366 3.027NS .030
MCV 47.402 4 11.851 20.445** .000**
MCH 65.780 4 16.445 17.398** .000**
MCHC 9.384 4 2.346 12.427** .000**
Glucose 139.070 4 74.767 4.029** .003**
Protein .033 4 .008 .618NS .652
Cholesterol 2001.911 4 500.478 5.088** .000**

Table 6: Three-way ANOVA (Randomized Block Design) evaluating the influence of gender, duration and dose on various blood indices of A. testiudineus

Study based on the effect induced by sevin on haematological indices of Clarias batrachus showed reduction in the number of Hb, red blood cells, packed cell volume indicating anemia [26]. Significant reduction in erythrocytes and haemoglobin has been observed at sublethal concentrations as observed in this experiment is in agreement with [27,28] who reported a decrease in Hb, RBC count and Hct content in malathion exposed freshwater carp, Cyprinus carpio. The significant decrease in the Hb concentration may be due to either an increase in the rate at which the Hb is destroyed or to a decrease in the rate of Hb synthesis. Ambient-toxicants might have caused disintegration of RBC, which in turn has caused reduction of hemoglobin and hematocrit count. It is observed that erythrocyte and haemoglobin concentrations follow a direct and physiological interrelationship as remarked by Chatterjee and Ganguly, and Reddy and Bashamohideen [29,30]. Increase TLC perhaps a typical immune response of the fish against a toxic invasion and may be due to leukemia during which the number of WBC increase. This is in agreement with the findings of Sampat et al. [31] when they exposed the O. niloticus to a toxic environment. The toxicant induced severe physiological stress that possibly disturbed the neurohormonal axis leading to alteration in the leukocyte count. Similar findings in fishes exposed to environmental pollutants were also reported [32]. The decrease in Hct in fish exposed to the pesticide was maximum in higher sublethal exposures may be due to decreased erythrocyte numbers. Increase MCH and MCHC also noted [33] following a short-term exposure of tench (Tinca tinca) to lead. In addition, increase in MCV, MCH and decreased MCHC values indicate that the anemia was of a macrocytic type. Similar result was observed in acute effect of diazinon on carp [13].

Exposure to different concentrations of pesticides caused an increase in the blood glucose levels, which could be attributed to differences in respiration, physiological activities and an imbalance between the hepatic output of glucose and the peripheral uptake of the sugar. It is also noted by previous workers [34] that stressful stimuli elicit rapid secretion of both glucocorticoid and catecholamines hormones from the adrenal tissue. The adrenergic effects may result in the increase of blood sugar within minutes after the onset of stress due to pesticides. This investigation showed decrease protein level when test fish exposed to sublethal concentrations in different exposure periods. Similar findings also noted by Das and Mukherjee [35]. The experiments conducted [36] C. punctatus exposed to sublethal concentrations of carbamate lead to decrease in blood proteins. Fall in protein level may be due to impaired function of kidney or due to reduced protein synthesis owing to liver cirrhosis [37]. The present work also supports the observations of Sastry and others [38] in this regard who opined that such an interference results in the depletion of total protein in the plasma of fish when exposed to quinalphos. Pesticide has also been induces blood cholesterol level, which might be utilized in the conversion of bile salt and in the synthesis of steroid hormone. Hypercholesterolemia may enhances denovo cholesterogenesis in liver, which is responsible for increased blood cholesterol [39,40].

Conclusions

Finally, it is concluded that the fish hematological parameters such as HB, RBC and MCHC are decreased showing anemia conditions. The plausible explanation of all these altered forms of blood cells could be attributed to the severity of "toxic stress" induced by diazinon, which can be due to abnormal erythropoiesis, inadequate haemoglobin formation, damage to the red cells after they leave bone marrow or increased erythropoiesis by bone marrow to compensate for anaemic conditions. Toxicants caused hypoproteinaemia, hypoglycemia and hypocholesterolemia. Further investigations for the quantitative and qualitative parameters are required to mark the erythrocytes as direct pollution indicators.

Acknowledgement

Authors are grateful to HOD, Department of Zoology, Utkal University, Vanivihar, Bhubaneswar, India.

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