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V{tÑàxÜ 4

RESULTS

The present study has been carried out to analyze the physico-chemical and

bacteriological parameters of water samples collected from the selected sites of

Pamba river during different seasons and the variations of the haematological,

biochemical and histopathological changes induced by selected bacterial inocula on

two selected freshwater fishes, Cyprinus carpio and Puntius sarana.

4.1 Water quality analysis

4.1.1. Physico-chemical parameters

The physico-chemical analysis of water samples of Pamba river showed significant

variations between seasons and sampling sites during the study period. Among the

ten sites studied, Site-2, Site-6 and Site-9 indicated more prominent variations. All

the values except BOD were found to be within the permissible limits. Pearson’s

correlation coefficient matrix done for water quality parameters for different seasons

illustrated significant correlation between specific parameters (APPENDIX-III, IV and V).

Temperature (oC)

The seasonal variation of water temperature is presented in Table 1a, b & Fig. 2.

Eventhough the water temperature fluctuated during pre-monsoon, monsoon and

post-monsoon periods, no significant difference was observed between the sites.

Turbidity

In natural waters, turbidity arises due to the presence of suspended matter. Turbidity

was found to be maximum during monsoon and minimum during pre-monsoon

Chapter 4 Results

49

periods. It is significantly correlated with alkalinity during monsoon season. (Table

2a, b & Fig.3). During post-monsoon season, it was highly correlated with chloride.

Maximum value was observed at Site-2 and Site-6 and the minimum value at Site-1.

Electrical conductivity (EC)

The conductivity values indicated a significant variation between seasons and sites.

Seasonal variations of conductivity were given in Table 3a, b & Fig. 4. The highest

conductivity was noted at Site-2 during all seasons. EC is highly correlated with TDS

for all the three seasons. During pre-monsoon period, EC was significantly correlated

with BOD whereas an inverse relation was found with DO.

Total dissolved solids (TDS)

The TDS showed high positive correlation with conductivity and BOD, whereas pH

and DO indicated a negative relationship. A similar trend of variation in EC was

observed for TDS at all sites during different seasons (Table 4a, b& Fig.5).

Hydrogen ion concentration (pH)

Hydrogen ion concentration in water gives a direct picture of its acidic and basic

nature and is considered to be a significant index in water quality assessment. The

seasonal variation of pH of the River Pamba was highlighted in Table 5 a, b and

Fig. 6. During pre-monsoon and post-monsoon season, pH was found to be highly

correlated with dissolved oxygen and showed a negative relation with BOD. A slight

variation of pH was noticed at all stations, but it was found to be within the

permissible limit prescribed for the potability purpose.

Total Alkalinity

The seasonal variations of alkalinity are illustrated in Table 6a, b & Fig.7. Location

wise difference was evident with comparatively higher value of 63 mg /l recorded

during the pre-monsoon at Site-6 and Site-10, but no significant difference between

Chapter 4 Results

50

seasons. Total alkalinity is directly correlated with nitrate during pre-monsoon

whereas a significant relation was found with hardness during monsoon seasons.

Total Hardness

Total hardness is caused by carbonate and bicarbonate ions. Hardness of water

studied at different stations during three seasons was depicted in Table 7a, b& Fig. 8.

Less fluctuation was observed among sites, but it showed a positive relation with

alkalinity during monsoon season.

Chloride

It is one of the important indicators of pollution. The two year study revealed that

there was increasing trend of chloride content in the water samples of Pamba river

(Table 8a, b & Fig.9). The maximum value was recorded at Site-10 during post-

monsoon. Chloride is highly correlated with turbidity during post-monsoon seasons.

It was also found that it had a direct relation with nitrate and faecal coliforms during

pre-monsoon season.

Dissolved Oxygen (DO)

Dissolved oxygen concentration showed significant fluctuation during different

seasons and also between sampling sites (Table 9 a, b & Fig.10). The DO value

ranged between 3.08 mg O2 / l to 7.8 mgO2 / l and the lowest values were found at

Site-2, Site-6 and Site-9 during the pre-monsoon and post-monsoon seasons. It was

significantly correlated with pH, whereas an inverse relationship was noted with EC,

TDS and hardness during all seasons.

Biochemical Oxygen Demand (BOD)

Seasonal variation of BOD at different sites is shown in the Table10 a, b and Fig.11.

Comparatively, higher values were observed during pre-monsoon and post monsoon

seasons for all the sites. It was also observed that the Site-2, Site-6 and Site 9 showed

Chapter 4 Results

51

significantly higher BOD level and was highly correlated with EC, TDS and faecal

coliform count, but a negative correlation was found with DO and pH.

Nitrate

Significant difference in nitrate content was noticed between seasons and sites with

maximum value during monsoon (Table 11 a, b & Fig.12). The nitrate content also

showed an increasing trend from 2005 to 2006 in the water samples and it is

significantly correlated with alkalinity and chloride during pre-monsoon. An inverse

relation was noted with pH during monsoon.

Phosphate

The phosphate variation of water samples at selected sites are shown in (Table12 a, b

& Fig.13). It was found that prominent variations existed between seasons and sites

and the highest value was observed during monsoon and indicating an increasing

trend during the study period.

4.1.2 Bacteriological parameters

The bacteriological characteristics of Pamba river water, analyzed during different

seasons, revealed the presence of significantly high load of faecal coliform count

(MPN) and total heterotrophic bacterial population at selected sites except Site-1.

Faecal coliform

The number of coliforms present is interpreted as an indicator of the extent to which

water has been contaminated by human or non-human faecal discharges. Table13 a,

b and Fig.14 indicated the seasonal variation of faecal coliform load (log values) in

the water samples of River Pamba. The faecal coliform count depicted significant

variations between sites and seasons. The FC value is highly correlated with BOD

and inversely with dissolved oxygen. All the values were found to be above the

permissible limit.

Chapter 4 Results

52

Total heterotrophic bacterial population

Heterotrophic bacteria isolated from the water samples during different seasons were

identified and represented in the Table 14, 15, 16. The distribution of bacteria in

different water samples show the dominance of Gram-negative genus. All the

samples showed considerable levels of coliforms, which is an indication of faecal

pollution of the water bodies. It was also noticed that Site-2, Site-6 and Site-9

indicated the maximum diversity of bacterial population during post-monsoon and

pre-monsoon seasons. A total of sixteen bacterial genera were identified from

different sites of Pamba river. The abundance of Escherichia sp., Enterobacter,

Enterococcus and Klebsiella sp. in the water samples, clearly indicated the bacterial

contamination in the Pamba river. Some of the bacteria identified were pathogens

which are responsible for the various water-borne diseases. The bacterial population

is directly correlated with BOD, turbidity, pH and inversely with DO.

4.2 Fishes of Pamba river

Twenty four species of fishes belonging to thirteen families coming under different

orders were identified from different regions of the river (APPENDIX-VI). Of the families,

Cyprinidae was dominant with six species represented by Puntius sarana,

P. filamentosus, P. amphibius, Gara mullaya, Labeo porcellus and Cyprinus

bacailla. The most abundant genus in this river was found to be Puntius. Labeo

dussumieri, recorded from Pamba river was listed as endangered and Channa

micropeltis and Puntius ophicephalus as critically endangered fishes (Kurup, 2002).

It was also found that Pamba river had lower representatives of fishes compared to

other rivers of Kerala.

Chapter 4 Results

53

Table 1a Seasonal variation of water temperature (o

C) in different sites during 2005

Sites Pre-monsoon Monsoon Post-monsoon

1 28.50 25.53 26.03

2 28.50 26.50 27.30

3 27.38 25.60 26.33

4 27.55 24.60 26.17

5 27.68 24.70 26.07

6 29.25 25.70 26.57

7 27.45 25.90 28.20

8 27.53 27.50 26.53

9 29.18 27.40 27.50

10 28.50 27.60 28.50

Table 1b Seasonal variation of water temperature (o

C) in different sites during 2006


1 28.15 24.83 27.50

2 27.50 24.75 27.85

3 26.60 24.55 26.75

4 26.90 24.30 28.50

5 28.63 24.35 27.50

6 28.13 24.45 26.50

7 28.34 24.90 28.50

8 28.85 24.85 27.80

9 30.45 25.65 28.75

10 29.58 25.30 28.75

Chapter 4 Results

54

Table 2a Seasonal variation of turbidity (NTU) in different sites during 2005


1 3.30 4.52 3.67

2 2.53 10.18 5.13

3 3.93 11.58 4.73

4 2.23 12.58 7.67

5 2.57 15.60 9.43

6 3.83 20.43 9.50

7 5.23 13.60 10.30

8 5.73 11.90 10.03

9 4.30 11.68 8.57

10 3.33 9.28 4.33

Table 2b Seasonal variation of turbidity (NTU) in different sites during 2006


1 1.87 9.35 5.30

2 2.37 19.80 16.87

3 1.80 17.80 8.54

4 2.37 15.25 12.30

5 2.57 14.26 9.75

6 1.83 17.30 13.45

7 2.30 15.50 12.5

8 1.73 11.60 8.15

9 2.03 12.45 10.58

10 3.37 14.60 9.96

Chapter 4 Results

55

Fig. 2 Comparison of water temperature (oC) during the study period (2005&2006)

0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10SITES

0 C

2005 Pre-monsoon 2005 Monsoon 2005 Post-monsoon2006 Pre-monsoon 2006 Monsoon 2006 Post-monsoon

Fig. 3 Comparison of turbidity (NTU) during the study period (2005&2006)

0

5

10

15

20

25

1 2 3 4 5 6 7 8 9 10SITES

NTU

2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon

Chapter 4 Results

56

Table 3a Seasonal variation of electrical conductivity (µ mhos/cm) in different

sites during 2005


1 51.10 36.68 46.40

2 67.24 53.66 58.72

3 50.05 36.00 45.85

4 49.13 35.45 43.50

5 39.48 33.48 36.45

6 52.78 39.50 48.33

7 50.38 47.58 47.13

8 47.50 35.68 42.48

9 62.50 51.28 52.58

10 55.08 38.60 47.55

Table 3b Seasonal variation of electrical conductivity (µ mhos/cm) in different

sites during 2006


1 37.35 39.95 48.70

2 72.38 62.82 68.54

3 43.20 54.67 66.98

4 47.95 42.35 46.98

5 53.35 34.50 43.33

6 54.60 44.75 62.65

7 46.77 42.30 46.93

8 57.70 34.02 48.63

9 59.60 43.88 56.70

10 63.25 60.60 61.70

Chapter 4 Results

57

Table 4a Seasonal variation of total dissolved solids (ppm) in different sites during

2005


1 24.150 17.250 19.880

2 46.490 32.830 36.260

3 24.430 16.950 19.680

4 22.200 15.650 20.350

5 18.900 14.550 17.300

6 30.580 15.850 22.180

7 22.900 21.730 21.930

8 21.430 16.100 19.130

9 27.450 24.330 27.780

10 24.680 16.650 21.850

Table 4b Seasonal variation of total dissolved solids (ppm) in different sites during

2006


1 20.430 17.650 25.250

2 51.490 35.400 40.050

3 16.880 16.300 26.430

4 21.430 19.150 22.330

5 29.650 14.650 20.030

6 32.840 24.900 34.900

7 20.350 17.150 16.830

8 19.350 24.400 24.600

9 30.650 24.150 28.850

10 31.870 29.750 30.200

Chapter 4 Results

58

Fig. 4 Comparison of electrical conductivity (µ mhos/cm) during the study

period (2005&2006)

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7 8 9 10SITES

µ m

hos /

cm


Fig. 5 Comparison of total dissolved solids (ppm) during the study period

(2005&2006)

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10SITES

ppm


Chapter 4 Results

59

Table 5a Seasonal variation of pH in different sites during 2005


1 6.34 6.76 6.51

2 6.10 6.21 6.25

3 6.49 6.57 6.73

4 6.50 6.78 6.92

5 6.35 6.80 6.68

6 6.92 6.15 6.43

7 7.11 6.62 6.39

8 6.82 6.27 6.06

9 6.66 6.16 6.19

10 7.04 6.24 6.30

Table 5b Seasonal variation of pH in different sites during 2006


1 6.28 6.80 6.39

2 6.07 6.20 6.06

3 6.29 6.50 6.57

4 6.23 6.90 6.60

5 6.30 7.13 6.60

6 6.25 6.30 5.89

7 6.30 6.60 6.35

8 6.31 6.74 6.63

9 6.27 6.32 6.10

10 6.28 6.87 6.24

Chapter 4 Results

60

Table 6a Seasonal variation of total alkalinity (mg CaCO3 / l) in different sites

during 2005


1 23.00 21.50 20.00

2 40.00 24.50 22.50

3 41.50 24.00 24.00

4 35.50 25.00 23.00

5 42.00 21.00 25.00

6 42.50 26.50 29.50

7 30.50 24.00 26.00

8 42.75 19.00 20.50

9 44.75 22.50 25.50

10 44.00 27.25 32.50

Table 6b Seasonal variation of total alkalinity (mg CaCO3 / l) in different sites

during 2006


1 24.00 13.00 21.00

2 38.00 26.50 36.50

3 47.00 20.00 33.00

4 46.00 23.00 29.50

5 43.00 21.50 36.50

6 63.00 17.00 37.00

7 47.00 17.50 31.00

8 40.00 12.00 29.00

9 45.00 20.00 30.50

10 63.00 21.00 30.00

Chapter 4 Results

61

Fig. 6 Comparison of pH during the study period (2005&2006)

012345678

1 2 3 4 5 6 7 8 9 10SITES

pH


Fig. 7 Comparison of total alkalinity (mg Ca CO3 / l) during the study period

(2005&2006)

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10

SITES

mg

CaC

O3 /

l


Chapter 4 Results

62

Table 7a Seasonal variation of total hardness (mg/ l) in different sites during 2005


1 15.50 16.50 15.50

2 41.20 20.50 15.00

3 40.30 22.00 17.00

4 40.80 21.50 17.00

5 50.50 22.00 21.50

6 48.40 21.00 17.00

7 32.10 17.00 21.00

8 28.40 22.00 14.00

9 32.90 23.50 16.50

10 40.75 23.50 18.00

Table 7b Seasonal variation of total hardness (mg/ l) in different sites during 2006


1 13.40 12.00 11.00

2 44.30 13.50 13.00

3 38.65 11.00 11.00

4 38.80 15.25 11.00

5 52.60 11.00 10.50

6 49.70 10.75 17.00

7 37.40 06.75 09.00

8 38.50 06.50 11.00

9 39.12 10.50 23.00

10 48.70 13.50 27.00

Chapter 4 Results

63

Table 8a Seasonal variation of chloride (mg/ l) in different sites during 2005


1 14.50 10.96 17.47

2 32.00 15.40 19.33

3 30.22 13.14 19.50

4 27.36 15.99 18.03

5 32.55 14.14 20.95

6 26.50 15.83 21.08

7 27.62 10.12 22.82

8 27.67 11.49 20.97

9 34.25 10.51 31.09

10 44.71 10.07 42.59

Table 8b Seasonal variation of chloride (mg / l) in different sites during 2006


1 18.50 20.56 28.40

2 45.70 22.35 30.45

3 43.96 20.02 34.8

4 48.24 25.75 42.46

5 40.89 21.81 38.51

6 48.47 23.21 39.47

7 44.02 22.73 36.70

8 40.47 21.69 34.20

9 64.73 34.13 68.51

10 66.60 40.22 79.80

Chapter 4 Results

64

Fig. 8 Comparison of total hardness (mg/l) during the study period

(2005&2006)

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10SITES

mg

/ l


Fig. 9 Comparison of chloride (mg / l) during the study period (2005&2006)

0

10

20

30

40

50

60

70

80

90

1 2 3 4 5 6 7 8 9 10SITES

mg

/ l


Chapter 4 Results

65

Table 9a Seasonal variation of dissolved oxygen (mgO2/l) in different sites during

2005


1 7.210 6.583 6.520

2 4.980 5.260 4.270

3 5.800 7.200 6.900

4 6.500 7.218 6.875

5 6.938 7.263 6.950

6 4.840 5.797 5.013

7 6.486 6.273 6.388

8 6.691 6.287 6.490

9 5.340 5.817 5.360

10 6.243 7.090 6.030

Table 9b Seasonal variation of dissolved oxygen (mgO2/l) in different sites during

2006


1 7.08 6.72 6.81

2 3.08 6.95 4.86

3 7.05 7.80 6.69

4 6.92 7.44 7.01

5 6.90 7.26 6.75

6 4.25 6.31 4.40

7 6.84 7.16 7.15

8 6.85 7.12 7.08

9 4.51 5.54 3.25

10 5.91 6.56 5.82

Chapter 4 Results

66

Table 10a Seasonal variation of biochemical oxygen demand (mgO2/l) in different

sites during 2005


1 0.357 1.411 1.150

2 3.278 1.248 4.961

3 1.928 1.088 1.265

4 1.513 1.498 1.523

5 1.749 1.333 1.305

6 3.053 3.028 4.526

7 2.083 1.395 2.253

8 0.998 1.048 2.333

9 2.992 2.600 3.180

10 2.255 1.213 2.248

Table 10b Seasonal variation of biochemical oxygen demand (mgO2 /l) in different

sites during 2006


1 0.251 0.523 0.725

2 4.821 1.780 5.160

3 1.238 1.678 1.780

4 1.375 1.820 2.020

5 1.655 1.355 2.080

6 4.750 3.293 4.250

7 1.265 1.088 0.820

8 1.628 1.150 2.050

9 4.140 3.450 4.854

10 1.428 1.980 3.500

Chapter 4 Results

67

Fig. 10 Comparison of dissolved oxygen (mg O2 / l) during the study period

(2005&2006)

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10SITES

mg

O2 /

l


Fig.11 Comparison of biochemical oxygen demand (mgO2 / l) during the study

period (2005&2006)

0

1

2

3

4

5

6

1 2 3 4 5 6 7 8 9 10SITES

mg

O 2

/ l


Chapter 4 Results

68

Table 11a Seasonal variation of nitrate (mg/ l) in different sites during 2005


1 0.047 0.046 0.032

2 0.067 0.089 0.088

3 0.055 0.049 0.052

4 0.043 0.062 0.075

5 0.060 0.074 0.065

6 0.063 0.128 0.072

7 0.045 0.056 0.096

8 0.076 0.089 0.081

9 0.092 0.055 0.061

10 0.103 0.058 0.071

Table 11b Seasonal variation of nitrate (mg/ l) in different sites during 2006


1 0.043 0.067 0.061

2 0.067 0.162 0.088

3 0.055 0.066 0.045

4 0.081 0.111 0.105

5 0.060 0.070 0.083

6 0.111 0.143 0.144

7 0.096 0.126 0.073

8 0.047 0.111 0.079

9 0.092 0.143 0.103

10 0.098 0.088 0.131

Chapter 4 Results

69

Table 12a Seasonal variation of phosphate (mg / l) in different sites during 2005


1 0.041 0.071 0.019

2 0.062 0.181 0.079

3 0.056 0.095 0.035

4 0.052 0.071 0.023

5 0.161 0.081 0.022

6 0.143 0.181 0.137

7 0.094 0.139 0.029

8 0.089 0.091 0.018

9 0.090 0.103 0.026

10 0.105 0.107 0.025

Table 12b Seasonal variation of phosphate (mg / l) in different sites during 2006


1 0.021 0.046 0.072

2 0.046 0.076 0.087

3 0.056 0.059 0.068

4 0.068 0.132 0.061

5 0.088 0.098 0.084

6 0.230 0.164 0.120

7 0.075 0.145 0.080

8 0.052 0.102 0.070

9 0.064 0.162 0.080

10 0.075 0.115 0.086

Chapter 4 Results

70

Fig. 12 Comparison of nitrate (mg / l) during the study period (2005&2006)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

1 2 3 4 5 6 7 8 9 10

SITES

mg

/ l


Fig. 13 Comparison of phosphate (mg / l) during the study period (2005&2006)

0.00

0.05

0.10

0.15

0.20

0.25

1 2 3 4 5 6 7 8 9 10SITES

mg

/ l


Chapter 4 Results

71

Table 13a Seasonal variation of faecal coliforms MPN/100ml (log values) in

different sites during 2005


1 0.67 1.11 1.38

2 3.38 2.63 4.38

3 3.04 2.38 3.32

4 2.63 2.96 2.68

5 3.66 2.36 3.38

6 4.38 3.66 4.38

7 3.66 3.38 4.00

8 1.96 2.87 1.38

9 3.66 3.38 3.66

10 2.63 1.46 2.32

Table 13b Seasonal variation of faecal coliforms MPN/100ml (log values) in

different sites during 2006


1 0.67 0.54 1.02

2 3.38 2.63 4.38

3 3.04 2.38 3.04

4 2.63 2.96 2.63

5 3.66 2.36 3.66

6 4.38 3.66 4.38

7 3.66 3.38 4.00

8 1.96 2.87 3.04

9 3.66 3.38 4.04

10 3.38 1.46 2.98

Chapter 4 Results

72

Fig. 14 Comparison of faecal coliforms MPN / 100 ml (log values) during the

study period (2005&2006)

0

1

2

3

4

5

1 2 3 4 5 6 7 8 9 10SITES

MPN

/100

ml (

log

valu

es)


Chapter 4 Results

73

Table 14 Heterotrophic bacteria identified at different sites during pre-monsoon

season (2005 &2006)

Bacterial genera Control (Site-1) Site-2 Site-6 Site-9

Enterococcus ** *** *** ***

Micrococcus ** ** ** **

Citrobacter * ** ** *

Bacillus nd * * *

Enterobacter * *** ** **

Acinetobacter nd * * *

Aeromonas sp. * ** ** *

Vibrio sp. nd ** * nd

Pseudomonas sp. * ** ** *

Staphylococcus sp. nd * nd nd

Escherichia coli ** *** *** ***

Cornybacterium sp. nd * * *

Proteus sp. nd * nd nd

Klebsiella sp. * *** ** *

Propionobacterium nd * * *

Salmonella sp. nd * nd nd

*** (>70%), ** (40-70%), *(<40%), nd- not detected

Chapter 4 Results

74

Table 15 Heterotrophic bacteria identified at different sites during monsoon season

(2005& 2006)

Bacterial genera Control (Site-1) Site-2 Site-6 Site-9

Enterococcus ** ** *** **

Micrococcus ** ** ** **

Citrobacter * ** ** *

Bacillus nd * * *

Enterobacter * *** ** **

Acinetobacter nd * nd *

Aeromonas sp. * ** * *

Vibrio sp. nd ** * nd


Staphylococcus sp. nd nd * nd

Escherichia coli ** ** *** **

Cornybacterium sp. nd * nd *

Proteus sp. * nd * nd

Klebsiella sp. * *** ** **

Propionobacterium nd * * +

Salmonella sp. nd nd nd *

*** (>70%), ** (40-70%), *(<40%), nd- not detected

Chapter 4 Results

75

Table 16 Heterotrophic bacteria identified at different sites during post-monsoon

season (2005 &2006)

Bacterial genera Control Site-1 Site-2 Site-6 Site-9

Enterococcus * *** *** ***

Micrococcus * *** ** **

Citrobacter * ** * *

Bacillus nd * ** *

Enterobacter * **** ** **

Acinetobacter nd * nd **

Aeromonas sp * ** ** *

Vibrio sp. nd ** * *


Staphylococcus sp. nd * nd nd

Escherichia coli ** **** *** ***

Cornybacterium sp. nd * nd nd

Proteus sp. * * * nd

Klebsiella sp. ** **** *** **

Propionobacterium nd * * *

Salmonella nd * nd nd

**** (> 90%), *** (70-90%), ** (40-70%), *(<40%), nd- not detected

Chapter 4 Results

76

4.3. Haematological parameters

According to Stoskopf (1973) the evaluation of blood parameters may be useful to

monitor the physiological status of fish and for the diagnosis of fish diseases.

Erythrocytes and leucocytes are important indicators of physiological changes.

Haematocrit and haemoglobin concentration have been amply used as

haematological indicators of the secondary stress response in fish. Hence blood

parameters are widely used as ideal tools for toxicological stress studies. The present

study brought out significant results to illustrate the bacterial stress effect on the

freshwater fishes, Cyprinus carpio and Puntius sarana exposed to different bacterial

inocula (E. coli, A. hydrophila, consortium I and consortium II). It was found that

C. carpio could survive throughout the period of exposure (30days) for all the

treatments. But in the case of P. sarana for consortia (I&II) exposures the fish could

not survive beyond 25th day.

4.3.1 Erythrocyte (RBC) count

Erythrocyte (RBC) count showed a prominent reduction in the fishes, C. carpio and

P. sarana irrespective of bacteria and period of exposure. In C. carpio, the RBC

count elevated initially upto 5th day for all the bacterial exposures followed by a

gradual decline and reached a significantly low level by 30th day, whereas in

P. sarana an initial increase was noted upto 3rd day in those fishes exposed to

consortium II followed by a steep decrease and reached the lowest value by 25th day.

Though a reduction was noted in those fishes exposed to E. coli and A. hydrophila

inocula, the stress induced by consortia is more severe as it showed the remarkable

variation in the RBC count (Table17, 18 & Fig15, 16).

4.3.2. Leucocyte (WBC) count

Significant changes in the Leucocyte (WBC) count were noted in both fishes

throughout the exposure whether it is single or consortia of bacteria. The WBC count

in C. carpio increased from day 1 and reached highest by 20th day of exposure, but in

P. sarana 15th day showed the maximum level which then reduced sharply in

consortium-I and consortium-II, whereas the fishes exposed to E.coli and Aeromonas

Chapter 4 Results

77

inocula the WBC count showed a slow but uniform increase throughout the period of

exposure (Table 19, 20 and Fig. 17, 18).

4.3.3 Haemoglobin

The variation of haemoglobin content in the experimental fishes is given in Table 21,

22 & Fig. 19, 20. In the case of C. carpio a uniform pattern was observed with an

initial increase upto 5th day followed by a steep decline by 10th day reaching a

significant low level of haemoglobin by 30th day for all exposures. While in

P. sarana an initial increase was noted upto 3rd day in bacterial consortium I and II

followed by a significant reduction and attained maximum reduction by 25thday and

failed to survive, whereas E. coli and A. hydrophila exposed fishes the haemoglobin

content reduced in a more or less a similar pattern and the effect was less severe

when compared to bacterial consortia.

4.3.4 Packed Cell Volume (PCV) or Haematocrit

There observed a slight increase in haematocrit value upto about day 5, beyond

which it significantly decreased in both the fishes (Table 23, 24 & Fig.21, 22). It was

also noted that bacterial consortium exerted more pronounced reduction in PCV.

4.3.5 The haematological indices

The haematological indices such as MCH, MCV and MCHC indicated noticeable

variation in the two fishes, C. carpio and P. sarana (Table 25-30, Fig.23-28). All

these indices exhibited steady increase after an initial almost steady state. The MCH

and MCV exhibited more pronounced increase in P. sarana exposed to consortium II

followed by consortium I. The O2 carrying capacity (Table 31, 32 & Fig. 29, 30) was

directly correlated with haemoglobin concentration and found to be significantly

different in two fishes and the variation was species specific.

4.3.6. Morphological Changes in blood corpuscles

The erythrocytes got enlarged and the oval shaped nucleus becomes rounded. Monocytes showed numerous vacuoles and were comparatively larger. Various lobopodial projections appeared in RBC. Vacuolation of both RBC and WBC cells were more prominent in the infected fishes and the changes were found more noticeable in P. sarana than C. carpio. (PLATE II-IIIa.)

Chapter 4 Results

78

Table 17 Total Erythrocyte Count (10 6/mm3) in Cyprinus carpio exposed to

different bacteria

Duration ( Days)

E. coli A.hydrophila Consortium-I Consortium-II

1 02.408 02.450 02.484 03.250

3 02.488 02.524 03.216 03.960

5 02.578 02.863 03.328 03.150

10 01.752 01.904 01.686 01.860

15 01.687 01.532 01.038 01.274

20 01.624 01.329 00.851 00.730

25 01.523 01.215 00.838 00.620

30 01.358 00.987 00.660 00.530 Mean of control = 02.338 VR (bacteria) ** CD (bacteria) 0.054 VR (day) ** CD (day) 0.089 VR (interaction) ** CD (interaction) 0.154 ** P<0.01 CD at 5% level

Fig.15 Variation of Total Erythrocyte Count (10 6/mm3) in Cyprinus carpio

exposed to different bacteria

00.5

11.5

22.5

33.5

44.5

C 1 3 5 10 15 20 25 30

DAYS

106 /m

m3

E.coli A.hydrophilaconsortium-I consortium-II

Chapter 4 Results

79

Table 18 Total Erythrocyte Count (106/mm3) in Puntius sarana exposed to

different bacteria

Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II

1 02.762 02.654 02.682 02.913

3 02.842 02.848 03.016 03.870

5 02.380 02.168 01.661 01.560

10 02.308 01.974 01.272 01.448

15 02.104 01.671 00.924 00.780

20 01.856 01.290 00.817 00.560

25 01.460 01.038 00.557 00.412


Fig.16 Variation of Total Erythrocyte Count (106/mm3) in Puntius sarana


00.5

11.5

22.5

33.5

44.5

C 1 3 5 10 15 20 25 30

DAYS

10 6 /m

m3


Chapter 4 Results

80

Table 19 Total Leucocyte Count (104/mm3) in Cyprinus carpio exposed to different

bacteria


1 03.412 03.488 03.672 03.842

3 03.878 03.866 04.080 04.620

5 04.098 04.464 04.104 05.020

10 04.360 04.110 04..860 05.850

15 04.642 04.886 05.420 05.970

20 04.516 05.436 05.992 06.790

25 04.056 04.282 06.050 05.060


Fig. 17 Variation of Total Leucocyte Count (10 4/ mm3) in Cyprinus carpio


012345678

C 1 3 5 10 15 20 25 30DAYS

104

/ mm

3


Chapter 4 Results

81

Table 20 Total Leucocyte Count (10 4 /mm3) in Puntius sarana exposed to different

bacteria


1 02.898 03.132 03.228 03.715

3 03.170 03.360 03.690 04.970

5 03.186 03.566 04.423 05.250

10 03.474 03.860 05.012 06.843

15 03.732 04.044 05.460 06..990

20 03.356 03.416 03.352 03.940

25 03.202 03.260 01.664 01.084


Fig. 18 Variation of Total Leucocyte Count (10 4/ mm3) in Puntius sarana


012345678

C 1 3 5 10 15 20 25 30DAYS

10

4 /m

m3


Chapter 4 Results

82

Table 21 Haemoglobin (g /100 ml) content in Cyprinus carpio exposed to different

bacteria


1 09.34 09.44 09.16 09.72

3 09.50 09.48 09.78 09.84

5 09.70 09.88 09.96 10.42

10 08.48 07.96 07.06 08.04

15 08.20 07.42 06.48 06.00

20 07.26 06.86 06.32 06.20

25 07.08 06.72 05.02 05.80


Fig. 19 Variation of Haemoglobin (g /100 ml) content in Cyprinus carpio


0

2

4

6

8

10

12

C 1 3 5 10 15 20 25 30

DAYS

g /1

00 m

l


Chapter 4 Results

83

Table 22 Haemoglobin (g /100 ml) content in Puntius sarana exposed to different

bacteria


1 08.40 08.64 09.92 10.24

3 08.56 08.90 10.06 10.84

5 09.52 09.08 08.22 08.50

10 08.00 07.66 06.62 06.24

15 07.02 06.14 06.36 05.20

20 08.82 05.84 04.54 04.60

25 06.40 05.50 03.76 04.00


Fig. 20 Variation of Haemoglobin (g /100 ml) content in Puntius sarana


0

2

4

6

8

10

12

C 1 3 5 10 15 20 25 30

DAYS

g / 1

00 m

l


Chapter 4 Results

84

Table 23 Packed cell volume (%) in Cyprinus carpio exposed to different

bacteria


1 36.980 37.640 39056 36.80

3 38.420 38.980 39.160 41.56

5 39.120 39.404 40.200 41.40

10 32.100 30.312 25.194 26.80

15 29.400 28.632 23.112 19.80

20 28.880 24.198 20.540 18.50

25 27.472 23.194 18.378 16.60


Fig. 21 Variation of Packed cell volume (%) in Cyprinus carpio exposed to

different bacteria

05

1015

20

25

30

35

40

45

C 1 3 5 10 15 20 25 30DAYS

(%)


Chapter 4 Results

85

Table 24 Packed cell volume (%) in Puntius sarana exposed to different bacteria


1 38.540 37.274 39.368 38.80

3 38.960 38.540 39.680 40.74

5 36.480 35.820 32.730 39.86

10 30.50 23.100 24.700 22.60

15 24.960 21.780 18.080 16.36

20 23.680 22.900 16.400 14.80

25 22.640 20.260 12.50 10.20


Fig. 22 Variation of Packed cell volume (%) in Puntius sarana exposed to

different bacteria

0

10

20

30

40

50

C 1 3 5 10 15 20 25 30DAYS

(%)


Chapter 4 Results

86

Table 25 Mean Corpuscular Haemoglobin (pg) in Cyprinus carpio exposed to

different bacteria


1 38.787 38.531 36.876 29.908

3 38.183 37.559 30.410 24.848

5 37.626 34.509 29.928 33.079

10 48.402 41.807 41.874 43.226

15 48.844 48.433 62.428 47.096

20 44.649 51.618 74.266 84.931

25 46.487 55.309 59.905 93.548


Fig. 23 Mean Corpuscular Haemoglobin (pg) in Cyprinus carpio exposed to

different bacteria.

0102030405060708090

100

C 1 3 5 10 15 20 25 30DAYS

pg


Chapter 4 Results

87

Table 26 Mean Corpuscular Haemoglobin (pg) in Puntius sarana exposed to

different bacteria


1 30.13 32.555 36.987 35.153

3 30.12 31.255 33.355 28.010

5 40.00 41.880 49.488 54.487

10 34.662 38.804 52.044 43.094

15 33.365 36.744 68.831 66.667

20 36.907 45.271 55.569 82.143

25 43.836 52.986 67.504 97.087


Fig. 24 Mean Corpuscular Haemoglobin (pg) in Puntius sarana exposed to

different bacteria

0102030405060708090

100

C 1 3 5 10 15 20 25 30

DAYS

(pg)


Chapter 4 Results

88

Table 27 Mean Corpuscular Volume (cubic micron) in Cyprinus carpio exposed to

different bacteria


1 153.571 153.633 159.259 113.231

3 154.421 153.344 121.766 104.950

5 151.746 137.632 122.56 131.429

10 183.219 159.202 149.431 144.086

15 174.274 186.893 222.659 155.416

20 177.833 182.077 241.363 253.425

25 180.381 190.897 219.308 267.742

30 190.000 226.95 261.606 290.566

Mean of control = 156.373 VR (bacteria) ** CD (bacteria) 09.285 VR (day) ** CD (day) 15.162 VR (interaction) ** CD (interaction) 26.261 ** P<0.01 CD at 5% level

Fig. 25 Mean Corpuscular Volume (cubic micron) in Cyprinus carpio exposed to

different bacteria

0

50

100

150

200

250

300

350

C 1 3 5 10 15 20 25 30

DAYS

cubi

c m

icro

n


Chapter 4 Results

89

Table 28 Mean Corpuscular Volume (cubic micron) in Puntius sarana exposed

to different bacteria


1 139.537 1210605 146.786 133.196

3 137.087 135.323 131.565 105.971

5 153.277 165.221 197.051 255.513

10 132.149 117.021 194.182 152.70

15 118.631 130.341 195.671 209.744

20 127.586 177.519 200.734 264.286

25 155.068 195.183 224.417 247.573

30 177.504 206.301 00.000 00.000

Mean of control = 142.373 VR (bacteria) ** CD (bacteria) 8.544 VR (day) ** CD (day) 13.953 VR (interaction) ** CD (interaction) 24.167 ** P<0.01 CD at 5% level

Fig. 26 Mean Corpuscular Volume (µ3) in Puntius sarana exposed to different

bacteria

0

50

100

150

200

250

300

C 1 3 5 10 15 20 25 30

DAYS

cubi

c m

icro

n


Chapter 4 Results

90

Table 29 Mean Corpuscular Haemoglobin Concentration (g /100 ml) in Cyprinus

carpio exposed to different bacteria


1 25.257 25.080 23.155 26.413

3 24.726 24.320 25.090 23.677

5 24.795 25.074 24.776 25.169

10 26.417 26.260 28.023 30.000

15 28.027 25.915 28.037 30.303

20 25.139 28.349 30.769 33.514

25 25.772 28.973 27.315 34.940

30 26. 510 25.089 26.063 29.870 Mean of control = 25.328 VR (bacteria) ** CD (bacteria) 1.331 VR (day) ** CD (day) 2.173 VR (interaction) ** CD (interaction) 3.764 ** P<0.01 CD at 5% level

Fig. 27 Mean Corpuscular Haemoglobin Concentration (g /100 ml) in Cyprinus


0

5

10

15

20

25

30

35

40

C 1 3 5 10 15 20 25 30DAYS

g / 1

00 m

l

E.coli A.hydrophila

consortium-I consortium-II

Chapter 4 Results

91

Table 30 Mean Corpuscular Haemoglobin Concentration (g /100 ml) in Puntius

sarana exposed to different bacteria


1 21.796 23.180 25.198 26.392

3 21.971 23.093 25.353 26.608

5 26.096 25.349 25.115 21.325

10 26.230 33.160 26.802 27.611

15 28.125 28.191 35.177 31.785

20 28.801 25.502 27.683 31.081

25 28.269 27.147 30.080 39.216


Fig. 28 Mean Corpuscular Haemoglobin Concentration (g /100 ml) in Puntius


05

1015202530354045

C 1 3 5 10 15 20 25 30

DAYS

g /

100

ml


Chapter 4 Results

92

Table 31 Oxygen carrying capacity (g /100 ml) in Cyprinus carpio exposed to

different bacteria


1 11.675 11.800 11.450 12.150

3 11.875 11.850 12.225 12.300

5 12.125 12.350 12.450 13.025

10 10.600 09.950 08.825 10.050

15 10.300 09.275 08.100 07.500

20 09.075 08.575 07.900 07.750

25 08.850 08.400 06.275 07.250


Fig. 29 Variation of oxygen carrying capacity (g /100 ml) in Cyprinus carpio


0

2

4

6

8

10

12

14

C 1 3 5 10 15 20 25 30DAYS

g / 1

00 m

l


Chapter 4 Results

93

Table 32 Oxygen carrying capacity (g /100 ml) in Puntius sarana exposed to

different bacteria


1 10.500 10.800 11.150 12.800

3 10.700 11.125 11.425 12.000

5 11.900 12.250 10.275 10.625

10 10.000 09.575 07.025 07.800

15 08.775 07.675 07.950 06.500

20 08.525 07.300 05.675 05.750

25 08.000 06.875 04.700 05.000


Fig. 30 Variation of oxygen carrying capacity (g /100 ml) in Puntius sarana


0

2

4

6

8

10

12

14

C 1 3 5 10 15 20 25 30DAYS

g / 1

00 m

l


Morphological changes in the blood corpuscles of Cyprinus carpio exposed to

different bacterial inocula for one day (x400)

a-control

b- E. coli c- A. hydrophila

d- consortium-I e-consortium-II

Er - Erythrocytes

M - Monocyte,

L -Lymphocyte

N - Neutrophil

Lp - Lobopodial projection

VN- Vacuolated neutrophil

Continued…………….


different bacterial inocula for 25 days (x400)

b(i)-E. coli c(i)-A. hydrophila

d(i)- consortium-I e(i)-consortium-II

Er - Erythrocytes

L -Lymphocyte

Lp - Lobopodial projection

VEr - Vacuolated Erythrocytes

REr - Ruptured Erythrocytes

VN - Vacuolated neutrophil



different bacterial inocula for 30 days (x400)

b (ii)-E. coli c (ii)-A. hydrophila

d (ii) - consortium-I e (ii)-consortium-II

Er - Erythrocytes,

L - Lymphocyte,

Lp -Lobopodial projection

REr -Ruptured Erythrocytes

VM -Vacuolated monocyte

VEr -Vacuolated Erythrocytes

VN -Vacuolated neutrophil

Morphological changes in the blood corpuscles of Puntius sarana exposed to

different bacterial inocula for one day (x400)

a-Control

b(i)-E. coli c(i)-A. hydrophila

d(i)- consortium-I e(i)-consortium-II

Er -Erythrocytes

L -Lymphocyte

M -Monocyte

N -Neutrophil


VEr -Vacuolated Erythrocytes,


VN -Vacuolated Neutrophil

VM -Vacuolated Monocyte


Morphological changes in the blood corpuscles of Puntius sarana exposed to

different bacterial inocula for 25 days(x400)

b (ii)-E. coli c(ii)-A. hydrophila

d (ii)- consortium-I e(ii)-consortium-II

Er -Erythrocytes,

L -Lymphocyte,

N -Neutrophil

M -Monocyte


VEr -Vacuolated Erythrocytes,


VN -Vacuolated Neutrophil

VM -Vacuolated Monocyte

Chapter 4 Results

94

4.4. Biochemical parameters

The pathological state of organisms could also be assessed through biochemical

analysis. The parameters such as lipid peroxidation, protein content, amino acid

concentration and soluble sugar were estimated to evaluate the stress induced by

different bacteria in the selected freshwater fishes under study.

4.4.1. Lipid peroxidation

Analysis of lipid peroxidation in three tissues (gill, muscle and liver) showed a

marked increase in malondialdehyde (MDA) content, a lipid peroxidation product

when exposed to different bacteria. In Cyprinus carpio, lipid peroxidation level in

the gills increased continuously from 3rd day onwards for all exposures but for

consortia (I & II) the increase was more pronounced (Table 33, Fig. 31). In

P. sarana also, the MDA content in gill increased from 1stday reaching the maximum

level by 30th day in E. coli and A. hydrophila exposures, while in consortia (I&II) the

lipid peroxidation activity was more prominent by 25th day (Table 34 and Fig. 32). In

the muscle also significant increase was noted for both the fishes in all the bacterial

exposures. In the case of C. Carpio a steady increase was noted in the muscle tissue,

reaching a maximum level on day 30 in all the treatments. But in P. sarana the lipid

peroxidation activity was greater in consortium I and II indicated by the highest level

of MDA content compared to the other treatments (Table 35, 36 & Fig. 33, 34). In

liver also, remarkable variation was observed irrespective of the bacterial inocula and

period of exposure. The fishes exposed to consortium II showed the maximum MDA

content and least in E. coli treated fishes (Table 37, 38 & Fig. 35, 36).

In C. carpio, the MDA content is significantly high in gill followed by muscle and

liver and reached the highest level by day 30. However, in the case of P. sarana

exposed to E. coli and Aeromonas a remarkable increase was noted throughout the

period whereas in bacterial consortia I & II all the fishes died by 25th day. Of the

three tissues studied the bacterial infection induced oxidative stress (in terms of

MDA level) was more prominent in muscle followed by gill and liver in P. sarana.

Chapter 4 Results

95

4.4.2. Total protein

In both the fishes, there was an overall significant change in total protein content in

gill, muscle and liver exposed to different bacterial inocula. In C. carpio, the protein

content in gill increased initially upto day 5 in all the treated groups (Table 39 and

Fig. 37) followed by significant reduction. In P. sarana also significant depletion in

the total protein content was noted and the percentage of reduction was highest in

bacterial consortia I & II (Table 40 & Fig. 38). In consortium treated fishes highest

elevation of protein was observed by day 3 in gill of P. sarana and C.carpio

respectively. In the muscle, an increase of total protein content was observed upto

day 3 and day 1, followed by pronounced decrease in C. carpio and P. sarana (Table

41, 42 & Fig. 39, 40). In liver, marked reduction in the total protein content was

observed from day 5 in treatments exposed to consortia (I& II) in C. carpio. But in

P. sarana a uniform reduction was observed from day 3 for all treatments. (Table 43,

44 & Fig. 41, 42). For all the three tissues (gill, muscle and liver), the protein content

reduction was least in those fishes exposed to E. coli and A. hydrophila. Among the

three tissues studied for both species of fishes, the maximum percentage of depletion

in the protein content was observed in muscle and gill compared to liver. Between

the two fishes, the percentage of reduction of the total protein content was found to

be more in P. sarana.

4.4.3. Total amino acid

In gill, the total amino acid content of both freshwater fishes, C.carpio and

P. sarana, reduced slightly for E. coli and A. hydrophila treatments. More

pronounced reduction was observed for E. coli treatments initially, followed by

significant increase. But in consortium I and II there was steady increase from the

beginning and reached a maximum at day 30 (Table 45, 46 & Fig.43, 44). In

C. carpio under E. coli treatment, the total amino acid content in muscle showed a

declining trend upto 10th day followed by an increase and its level reached highest by

day 30. In the other treatments, fishes exposed to Aeromonas, consortium I and II the

amino acid content showed a hike from the 1st day onwards. The highest increase

Chapter 4 Results

96

was noted for fishes exposed to consortium II. The variation of total amino acid

content irrespective of treatment was found to be significant (Table 47 & Fig. 45). In

P. sarana the total amino acid content in muscle significantly increased from the 3rd

day reaching a maximum on day 30 for all fishes exposed to E. coli and

A. hydrophila inocula, whereas those treated with bacterial consortia I and II total

amino acid content increased throughout the period of exposure (Table 48& Fig. 46).

In liver, both fishes noted an increase from 5th day of exposure irrespective of the

inocula. Upto the 5th day there observed no significant change (Table 49, 50 & Fig.

47, 48). The percentage of increase in total amino acid content was observed

maximum in the muscle tissue of fishes treated with bacterial consortia than single

species of bacterial inoculum. Among the treatments, the consortia (I&II) exposed

fishes showed the highest increase in the amino acid content.

4.4.4. Soluble sugars

The soluble sugar content in gill, muscle and liver irrespective of bacteria and the

days of exposure significantly reduced after a brief initial elevation throughout the

exposure in C. carpio. In P. sarana soluble sugar level showed an initial increase in

gill and muscle by day 1, whereas in liver the increase was noted upto day 3

followed by a prominent reduction for all treatments (Table 51-56 & Fig. 49-54).

The fishes exposed to E. coli and A. hydrophila, the percentage of reduction was

least in all the tissues compared to those exposed to consortia I and II. Of the three

tissues studied for both species of fishes, the maximum depletion in the soluble sugar

content was observed in liver, whereas the gill showed the minimum reduction.

Among the two fishes, the reduction of the total soluble sugar content was found to

be high in P. sarana.

Chapter 4 Results

97

Table 33 Lipid peroxidation (n moles MDA / g wet wt.) in the gill of Cyprinus


Duration

(Days) E. coli A.hydrophila Consortium-I Consortium-II

1 20.952 21.598 22.786 23.640

3 24.232 23.638 27.844 29.150

5 25.366 25.856 29.832 37.122

10 27.898 29.266 38.322 43.234

15 32.230 35.328 41.910 47.262

20 31.252 36.192 42.310 51.280

25 33.032 38.192 55.200 58.290

30 37.144 45.040 62.392 65.321 Mean of control = 21.908 VR (bacteria) ** CD (bacteria) 0.647 VR (day) ** CD (day) 1.057 VR (interaction) ** CD (interaction) 1.830 **p < 0.01 CD at 5% level

Fig. 31 Variation of lipid peroxidation (n moles MDA/g wet wt.) in the gill of

Cyprinus carpio exposed to different bacteria

0

10

20

30

40

50

60

70

80

C* 1 3 5 10 15 20 25 30

DAYS

n m

oles

MD

A /g

wet

wt.

E. coli A.hydrophilaConsortium-I consortium-II

Chapter 4 Results

98

Table 34 Lipid peroxidation (n moles MDA/g wet wt.) in the gill of Puntius sarana


Duration


1 22.992 28.142 36.484 38.561

3 34.270 33.768 40.884 42.130

5 36.024 38.504 46.852 49.270

10 39.044 42.354 47.974 55.410

15 38.710 41.456 48.902 57.410

20 41.708 43.560 59.780 63.840

25 45.832 46.942 64.032 77.792


Fig. 32 Variation of lipid peroxidation (n moles MDA/g wet wt.) in the gill of

Puntius sarana exposed to different bacteria.

0

10

20

30

40

50

60

70

80

C* 1 3 5 10 15 20 25 30

DAYS

n m

oles

MD

A /g

wet

wt.

E. coli A.hydrophilaConsortium-I consortium-II

Chapter 4 Results

99

Table 35 Lipid peroxidation (n moles MDA/g wet wt.) in the muscle of Cyprinus


Duration


1 20.498 20.388 19.430 18.220

3 21.676 22.542 27.551 31.421

5 24.798 24.360 32.784 37.463

10 26.384 25.008 35.006 38.690

15 27.560 29.034 38.794 49.660

20 26.066 26.376 42.588 43.972

25 29.884 31.934 50.154 44.211


Fig. 33 Variation of lipid peroxidation (n moles MDA/g wet wt.) in the muscle of


0

10

20

30

40

50

60

70

80

90

C* 1 3 5 10 15 20 25 30

DAYS

n m

oles

MD

A /g

wet

wt.

E. coli A.hydrophilaconsortium-I consortium-II

Chapter 4 Results

100

Table 36 Lipid peroxidation (n moles MDA/g wet wt.) in the muscle of Puntius



1 25.134 25.912 28.366 32.650

3 26.196 33.522 36.386 39.890

5 27.354 34.550 46.606 49.354

10 32.390 36.890 49.858 53.547

15 34.610 39.810 53.992 55.241

20 38.760 41.986 55.110 65.083

25 39.830 45.780 72.432 86.705


Fig. 34 Variation of lipid peroxidation (n moles MDA/g wet wt.) in the muscle of

Puntius sarana exposed to different bacteria

0102030405060708090

C* 1 3 5 10 15 20 25 30DAYS

n m

oles

MD

A /g

wet

wt.


Chapter 4 Results

101

Table 37 Lipid peroxidation ((n moles MDA/g wet wt.) in the liver of Cyprinus


Duration ( Days) E. coli A.hydrophila Consortium Consortium-II

1 23.176 23.664 24.206 25.740

3 24.696 25.340 27.846 27.441

5 26.682 27.038 32.980 35.223

10 32.954 34.511 38.218 39.207

15 33.912 36.258 38.582 41.310

20 32.104 35.070 39.351 42.561

25 35.432 38.652 41.006 44.760


Fig. 35 Variation of lipid peroxidation (n moles MDA/g wet wt.) in the liver of


0

10

20

30

40

50

60

C* 1 3 5 10 15 20 25 30DAYS

n m

oles

MD

A /g

wet

wt.


Chapter 4 Results

102

Table 38 Lipid peroxidation (n moles MDA/g wet wt.) in the liver of Puntius


Duration ( Days) E. coli A.hydrophila Consortium Consortium-II

1 25.134 25.912 27.588 29.840

3 26.196 26.864 30.424 34.828

5 27.354 28.080 32.620 40.661

10 31.536 30.942 34.400 39.540

15 33.214 33.756 36.696 42.441

20 35.948 36.078 42.552 45.910

25 37.006 38.168 51.284 58.432


Fig. 36 Variation of lipid peroxidation (n moles MDA/g wet wt.) in the liver of


0

10

20

30

40

50

60

C* 1 3 5 10 15 20 25 30

DAYS

n m

oles

MD

A /g

wet

wt.


Chapter 4 Results

103

Table 39 Total protein (mg/g wet wt.) content in the gill of Cyprinus carpio



1 26.766 26.150 27.878 31.766

3 27.508 27.100 31.612 35.251

5 26.272 28.176 29.346 24.070

10 25.760 24.092 23.540 21.540

15 23.658 21.378 20.910 20.980

20 22.290 24.622 18.348 17.190

25 22.060 22.324 17.356 15.660


Fig. 37 Variation of total protein (mg/g wet wt.) content in the gill of Cyprinus


0

5

10

15

20

25

30

35

40

C* 1 3 5 10 15 20 25 30

DAYS

mg

/ g w

et w

t.


Chapter 4 Results

104

Table 40 Total protein (mg/g wet wt.) content in the gill of Puntius sarana exposed

to different bacteria


1 23.916 25.742 23.602 24.370

3 25.428 27.038 30.213 32.571

5 23.916 26.048 18.712 19.720

10 21.296 22.920 19.166 17.204

15 22.228 21.230 17.154 13.873

20 20.670 19.092 14.858 12.003

25 19.962 17.590 10.856 09.424


Fig. 38 Variation of total protein (mg/g wet wt.) content in the gill of Puntius


0

5

10

15

20

25

30

35

C* 1 3 5 10 15 20 25 30

DAYS

mg

/g w

et w

t.


Chapter 4 Results

105

Table 41 Total protein (mg/g wet wt.) content in the muscle of Cyprinus carpio



1 46.232 44.858 48.340 47.290

3 47.856 49.134 50.860 52.520

5 44.252 42.808 44.750 43.270

10 44.008 38.364 39.954 39.110

15 43.610 34.345 38.730 35.260

20 39.006 32.448 29.541 29.670

25 36.540 30.644 27.646 24.290

30 31.340 28.987 26.540 18.870 Mean of control = 43.764 VR (bacteria) ** CD (bacteria) 0.677 VR (day) ** CD (day) 1.106 VR (interaction) ** CD (interaction) 1.915 ** P <0.01 CD at 5% level

Fig. 39 Variation of total protein (mg/g wet wt.) content in the muscle of


0

10

20

30

40

50

60

C* 1 3 5 10 15 20 25 30

DAYS

mg

/ g w

et w

t.


Chapter 4 Results

106

Table 42 Total protein (mg/g wet wt.) content in the muscle of Puntius sarana



1 46.222 48.432 50.794 55.243

3 48.746 47.980 45.996 41.980

5 38.002 34.982 32.898 30.067

10 37.759 34.924 29.826 26.870

15 36.432 33.221 30.432 24.223

20 39.997 30.420 25.974 18.556

25 29.650 27.038 18.396 14.532


Fig. 40 Variation of total protein (mg/g wet wt.) content in the muscle of Puntius


0

10

20

30

40

50

60

C* 1 3 5 10 15 20 25 30

DAYS

mg

/g w

et w

t.


Chapter 4 Results

107

Table 43 Total protein (mg/g wet wt.) content in the liver of Cyprinus carpio



1 65.400 64.154 71.440 73.560

3 66.632 68.620 69.868 74.920

5 68.510 69.324 72.706 75.810

10 67.860 65.890 56.760 47.121

15 58.632 65.010 49.426 36.765

20 66.770 64.560 47.794 42.671

25 65.420 63.410 45.380 39.982


Fig. 41 Variation of total protein (mg /g wet wt.) content in the liver of Cyprinus


0

10

20

30

40

50

60

70

80

90

C* 1 3 5 10 15 20 25 30

DAYS

mg

/ g w

et w

t.


Chapter 4 Results

108

Table 44 Total protein (mg/g wet wt.) content in the liver of Puntius sarana



1 67.792 68.910 72.013 76.420

3 68.082 69.004 72.336 77.320

5 62.720 61.854 60.740 54.761

10 62.842 59.878 58.186 49.730

15 58.952 56.016 48.742 34.470

20 54.834 48.452 42.430 32.886

25 50.530 43.024 34.231 26.351


Fig. 42 Variation of total protein (mg/g wet wt.) content in the liver of Puntius


0102030405060708090

C* 1 3 5 10 15 20 25 30

DAYS

mg

/g w

et w

t.


Chapter 4 Results

109

Table 45 Total amino acid (mg/g wet wt.) content in the gill of Cyprinus carpio



1 27.348 28.918 37.628 42.231

3 25.518 33.082 48.582 56.650

5 17.456 36.362 50.904 60.120

10 20.352 44.860 51.240 63.760

15 27.138 50.848 58.784 72.860

20 53.082 58.480 65.834 71.124

25 63.434 74.320 81.476 88.410

30 75.288 79.492 90.660 94.656 Mean of control =30.408 VR (bacteria) ** CD (bacteria) 1.527 VR (day) ** CD (day) 2.494 VR (interaction) ** CD (interaction) 4.319 ** P<0.01 CD at 5% level

Fig. 43 Variation of total amino acid (mg/g wet wt.) content in the gill of


0102030405060708090

100

C* 1 3 5 10 15 20 25 30

DAYS

mg

/g w

et w

t.


Chapter 4 Results

110

Table 46 Total amino acid (mg/g wet wt.) content in the gill of Puntius sarana



1 20.866 23.806 28.326 32.430

3 23.030 21.884 26.962 40.680

5 26.192 27.648 38.248 43.689

10 32.924 31.092 62.256 65.926

15 36.548 50.218 65.238 72.873

20 50.992 59.562 78.266 85.080

25 65.592 67.026 82.924 92.650


Fig. 44 Variation of total amino acid (mg/g wet wt.) content in the gill of Puntius


0102030405060708090

100

C* 1 3 5 10 15 20 25 30

DAYS

mg

/g w

e w

t.


Chapter 4 Results

111

Table 47 Total amino acid (mg/g wet wt.) content in the muscle of Cyprinus carpio



1 30.304 31.328 39. 012 37.886

3 24.158 44.228 54.328 65.560

5 27.516 45.560 66.452 73.650

10 25.776 62.220 83.126 85.880

15 53.730 65.540 91.848 93.210

20 70.800 74.930 95.170 97.420

25 73.220 77.240 93.433 99.760


Fig. 45 Variation of total amino acid (mg/g wet wt.) content in the muscle of


0102030405060708090

100110

C* 1 3 5 10 15 20 25 30DAYS

mg

/g w

et w

t.


Chapter 4 Results

112

Table 48 Total amino acid (mg/g wet wt.) content in the muscle of Puntius sarana



1 25.948 25.970 28.994 33.971

3 29.126 27.294 32.286 37.680

5 31.976 33.874 44.586 49.510

10 34.164 43.770 70.848 67.110

15 38.646 59.916 73.278 78.260

20 50.750 64.918 75.110 83.490

25 67.886 67.066 91.860 104.493


Fig. 46 Variation of total amino acid (mg/g wet wt.) content in the muscle of


0102030405060708090

100110

C* 1 3 5 10 15 20 25 30DAYS

mg

/g w

et w

t.


Chapter 4 Results

113

Table 49 Total amino acid (mg/g wet wt.) content in the liver of Cyprinus carpio



1 33.476 32.392 32.620 31.750

3 30.742 31.026 36.548 39.433

5 29.254 30.378 44.982 47.670

10 32.400 37.424 44.690 46.980

15 42.638 47.056 57.282 62.640

20 54.280 60.244 65.574 72.081

25 61.908 71.148 77.218 80.710


Fig. 47 Variation of total amino acid (mg/g wet wt.) content in the liver of


0102030405060708090

100

C* 1 3 5 10 15 20 25 30DAYS

mg

/g w

et w

t.


Chapter 4 Results

114

Table 50 Total amino acid (mg/g wet wt.) content in the liver of Puntius sarana



1 35.636 35.002 34.214 31.756

3 32.506 31.046 37.470 39.656

5 34.798 40.282 42.454 49.670

10 42.092 51.854 55.120 58.321

15 50.526 61.852 66.986 75.630

20 62.908 73.738 80.878 88.120

25 73.076 78.580 83.958 98.780


Fig. 48 Variation of total amino acid (mg/g wet wt.) content in the liver of


0102030405060708090

100

C* 1 3 5 10 15 20 25 30

DAYS

mg

/g w

et w

t.


Chapter 4 Results

115

Table 51 Soluble sugar (mg/g wet wt.) content in the gill of Cyprinus carpio



1 183.638 196.022 200.604 202.904

3 186.348 190.088 205.638 225.490

5 198.086 176.414 207.518 184.498

10 179.768 186.580 176.158 153.050

15 178.478 171.770 161.708 128.410

20 176.672 167.642 145.226 125.180

25 169.448 160.934 134.102 108.270


Fig. 49 Variation of soluble sugar (mg/g wet wt.) content in the gill of Cyprinus


0

50

100

150

200

250

C* 1 3 5 10 15 20 25 30DAYS

mg

/g w

et w

t.


Chapter 4 Results

116

Table 52 Soluble sugar (mg/g wet wt.) content in the gill of Puntius sarana



1 191.522 195.422 210.872 219.169

3 188.294 198.648 200.552 208.574

5 182.234 183.008 184.298 199.880

10 181.452 162.812 155.588 142.910

15 144.814 133.306 117.590 112.021

20 118.364 119.396 108.560 95.440

25 112.386 115.526 88.864 79.059


Fig. 50 Variation of soluble sugar (mg/g wet wt.) content in the gill of Puntius


0

50

100

150

200

250

C* 1 3 5 10 15 20 25 30

DAYS

mg

/g w

et w

t.


Chapter 4 Results

117

Table 53 Soluble sugar (mg/g wet wt.) content in the muscle of Cyprinus carpio



1 259.232 260.522 277.704 289.673

3 266.198 285.420 265.940 297.658

5 273.938 274.454 267.488 235.761

10 252.524 244.526 234.464 205.369

15 245.042 235.238 215.888 189.095

20 231.110 223.886 198.148 165.908

25 214.856 210.986 171.378 154.643


Fig. 51 Variation of soluble sugar (mg/g wet wt.) content in the muscle of


0

50

100

150

200

250

300

350

C* 1 3 5 10 15 20 25 30DAYS

mg

/g w

et w

t.


Chapter 4 Results

118

Table 54 Total soluble sugar (mg/g wet wt.) content in the muscle of Puntius



1 240.552 248.808 254.904 296.321

3 248.168 256.806 267.322 284.610

5 219.602 251.130 248.176 239.633

10 216.542 234.876 236.682 217.367

15 210.100 230.232 183.524 171.181

20 227.910 185.260 131.216 113.375

25 204.174 171.100 87.682 74.873


Fig.52 Variation of soluble sugar (mg/g wet wt.) content in the muscle of Puntius


0

50

100

150

200

250

300

350

C* 1 3 5 10 15 20 25 30DAYS

mg

/g w

et w

t.


Chapter 4 Results

119

Table 55 Soluble sugar (mg/g wet wt.) content in the liver of Cyprinus carpio



1 262.328 259.748 247.364 266.480

3 270.108 265.386 268.004 282.833

5 276.260 268.262 273.670 281.731

10 250.976 230.078 226.466 210.191

15 232.142 222.080 217.436 201.345

20 245.042 213.050 204.536 172.412

25 238.076 223.628 184.412 143.060


Fig. 53 Variation of soluble sugar (mg/g wet wt.) content in the liver of Cyprinus


0

50

100

150

200

250

300

350

C* 1 3 5 10 15 20 25 30

DAYS

mg

/g w

et w

t.


Chapter 4 Results

120

Table 56 Soluble sugar (mg/g wet wt.) content in the liver of Puntius sarana



1 286.992 297.570 306.858 313.189

3 303.998 313.566 332.240 342.530

5 318.714 321.822 318.984 287.310

10 232.658 238.592 248.138 212.250

15 223.370 218.468 168.674 159.796

20 196.022 180.026 150.872 143.504

25 192.340 139.778 95.798 78.354


Fig. 54 Variation of soluble sugar (mg/g wet wt.) content in the liver of Puntius


0

50

100

150

200

250

300

350

C* 1 3 5 10 15 20 25 30

DAYS

mg

/g w

et w

t.


Chapter 4 Results

121

4.4. Histopathological studies

In experimentally infected fishes, C. carpio and P. sarana, clear histological changes

were observed. By 25th day more prominent damage was noted in the gills, liver and

intestine of P. sarana compared to C.carpio. It was found that maximum damage

was noticed in those fishes treated with consortia I and II.

In gill, changes were confined mainly to the lamellar epithelial cells of gill filaments

in the beginning. In later stages the gills showed marked changes like fusion of

secondary lamellae, proliferation of the gill epithelium, and reduction of the

interlamellar space, disruption of the primary lamellae and hypertrophy of the

epithelial cells. PLATE IV-V

The intestine was severely affected with degeneration of intestinal muscularis and

mucosa. The inner epithelial lining was found eroded. More prominent changes were

brought about by the consortia. PLATE VI-VII

In liver, bacterial stress induced changes like enlargement of hepatocytes and nuclei,

disruption of the tissue, necrosis, vacuolation and increase in the intercellular space

were observed. More prominent changes were found in P. sarana. PLATE VIII-IX

Histological changes in the gill of Cyprinus carpio exposed to different bacterial

inocula for 30 days(x400)

a- Control

b -E. coli c -A. hydrophila d - consortium-I e -consortium-II

Pl - Primary lamellae

Sl -Secondary lamellae

Ht - Hypertrophy

Dsl - Disrupted secondary lamellae

De - Disintegrated epithelium

I - Interlamellar space

Slf - Secondary lamellar fusion

Dpl - Disrupted primary lamellae

Histological changes in the gill of Puntius sarana exposed to different bacterial


a- Control


Pl -Primary lamellae

Sl -Secondary lamellae

Ht -Hypertrophy

I -Interlamellar space

De -Disintegrated epithelium

Slf -Secondary lamellar fusion

Dpl -Disrupted primary lamellae

Histological changes in the intestine of Cyprinus carpio exposed to different

bacterial inocula for 30 days (x400)

a- Control


S- Serosa

M-Musularis

E- Epithelium

Ee- Eroded epithelium

Rml- Rupture of muscular layer

Histological changes in the intestine of Puntius sarana exposed to different bacterial


a- Control


S -Serosa

M -Musularis

E -Epithelium

Ee -Eroded epithelium

Rml -Rupture of muscular layer

Histological changes in the liver of Cyprinus carpio exposed to different bacterial


a- Control


Hc -Hepatocytes

N -Nucleus

Bc -Bile canaliculi

Eh -Enlarged hepatocytes

Lis -Large intercellular space

Dt -Distorted tissue

Histological changes in the liver of Puntius sarana exposed to different bacterial


a- Control


Hc -Hepatocytes

N -Nucleus

Bc -Bile canaliculi

Eh -Enlarged hepatocytes

Lis -Large intercellular space

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