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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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
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
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
Chapter 4 Results
59
Table 5a Seasonal variation of pH in different sites during 2005
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
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
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
Chapter 4 Results
62
Table 7a Seasonal variation of total hardness (mg/ l) in different sites during 2005
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
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
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
Chapter 4 Results
65
Table 9a Seasonal variation of dissolved oxygen (mgO2/l) in different sites during
2005
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
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
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
Chapter 4 Results
68
Table 11a Seasonal variation of nitrate (mg/ l) in different sites during 2005
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
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
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
Chapter 4 Results
71
Table 13a Seasonal variation of faecal coliforms MPN/100ml (log values) in
different sites during 2005
Sites Pre-monsoon Monsoon Post-monsoon
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
Sites Pre-monsoon Monsoon Post-monsoon
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)
2005 Premonsoon 2005 Monsoon 2005 Postmonsoon2006 Premonsoon 2006 Monsoon 2006 Postmonsoon
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
Pseudomonas sp. * ** ** *
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 ** * *
Pseudomonas sp. * ** ** *
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
30 01.138 00.930 00.000 00.000 Mean of control = 02.596 VR (bacteria) ** CD (bacteria) 0.068 VR (day) ** CD (day) 0.111 VR (interaction) ** CD (interaction) 0.191 ** P<0.01 CD at 5% level
Fig.16 Variation of Total Erythrocyte Count (106/mm3) in Puntius sarana
exposed to different bacteria
00.5
11.5
22.5
33.5
44.5
C 1 3 5 10 15 20 25 30
DAYS
10 6 /m
m3
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
80
Table 19 Total Leucocyte Count (104/mm3) in Cyprinus carpio exposed to different
bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 03.750 03.896 04.210 04.641 Mean of control = 03.362 VR (bacteria) ** CD (bacteria) 0.157 VR (day) ** CD (day) 0.257 VR (interaction) ** CD (interaction) 0.445 ** P<0.01 CD at 5% level
Fig. 17 Variation of Total Leucocyte Count (10 4/ mm3) in Cyprinus carpio
exposed to different bacteria
012345678
C 1 3 5 10 15 20 25 30DAYS
104
/ mm
3
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
81
Table 20 Total Leucocyte Count (10 4 /mm3) in Puntius sarana exposed to different
bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 02.604 02.442 00.000 00.000 Mean of control = 02.720 VR (bacteria) ** CD (bacteria) 0.090 VR (day) ** CD (day) 0.147 VR (interaction) ** CD (interaction) 0.255 ** P<0.01 CD at 5% level
Fig. 18 Variation of Total Leucocyte Count (10 4/ mm3) in Puntius sarana
exposed to different bacteria
012345678
C 1 3 5 10 15 20 25 30DAYS
10
4 /m
m3
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
82
Table 21 Haemoglobin (g /100 ml) content in Cyprinus carpio exposed to different
bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 06.84 05.62 04.50 04.60 Mean of control = 09.260 VR (bacteria) ** CD (bacteria) 0.251 VR (day) ** CD (day) 0.409 VR (interaction) ** CD (interaction) 0.709 ** P<0.01 CD at 5% level
Fig. 19 Variation of Haemoglobin (g /100 ml) content in Cyprinus carpio
exposed to different bacteria
0
2
4
6
8
10
12
C 1 3 5 10 15 20 25 30
DAYS
g /1
00 m
l
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
83
Table 22 Haemoglobin (g /100 ml) content in Puntius sarana exposed to different
bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 05.50 05.00 00.00 00.00 Mean of control = 08.280 VR (bacteria) ** CD (bacteria) 0.220 VR (day) ** CD (day) 0.359 VR (interaction) ** CD (interaction) 0.622 ** P<0.01 CD at 5% level
Fig. 20 Variation of Haemoglobin (g /100 ml) content in Puntius sarana
exposed to different bacteria
0
2
4
6
8
10
12
C 1 3 5 10 15 20 25 30
DAYS
g / 1
00 m
l
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
84
Table 23 Packed cell volume (%) in Cyprinus carpio exposed to different
bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 25.802 22.400 17.266 15.40 Mean of control = 36.560 VR (bacteria) ** CD (bacteria) 0.713 VR (day) ** CD (day) 1.165 VR (interaction) ** CD (interaction) 2.017 ** P<0.01 CD at 5% level
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
(%)
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
85
Table 24 Packed cell volume (%) in Puntius sarana exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 20.200 19.186 00.000 00.00 Mean of control = 36.960 VR (bacteria) ** CD (bacteria) 0.734 VR (day) ** CD (day) 1.198 VR (interaction) ** CD (interaction) 2.076 ** P<0.01 CD at 5% level
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
(%)
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
86
Table 25 Mean Corpuscular Haemoglobin (pg) in Cyprinus carpio exposed to
different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 50.368 56.940 68.182 86.792 Mean of control = 39.607 VR (bacteria) ** CD (bacteria) 3.140 VR (day) ** CD (day) 5.128 VR (interaction) ** CD (interaction) 8.882 ** P<0.01 CD at 5% level
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
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
87
Table 26 Mean Corpuscular Haemoglobin (pg) in Puntius sarana exposed to
different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 48.33 53.760 00.000 00.000 Mean of control = 31.895 VR (bacteria) ** CD (bacteria) 2.065 VR (day) ** CD (day) 3.372 VR (interaction) ** CD (interaction) 5.840 ** P<0.01 CD at 5% level
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)
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
88
Table 27 Mean Corpuscular Volume (cubic micron) in Cyprinus carpio exposed to
different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
89
Table 28 Mean Corpuscular Volume (cubic micron) in Puntius sarana exposed
to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
90
Table 29 Mean Corpuscular Haemoglobin Concentration (g /100 ml) in Cyprinus
carpio exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
carpio exposed to different bacteria
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
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 27.228 26.061 00.000 00.000 Mean of control = 22.403 VR (bacteria) ** CD (bacteria) 1.136 VR (day) ** CD (day) 1.854 VR (interaction) ** CD (interaction) 3.212 ** P<0.01 CD at 5% level
Fig. 28 Mean Corpuscular Haemoglobin Concentration (g /100 ml) in Puntius
sarana exposed to different bacteria
05
1015202530354045
C 1 3 5 10 15 20 25 30
DAYS
g /
100
ml
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
92
Table 31 Oxygen carrying capacity (g /100 ml) in Cyprinus carpio exposed to
different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 08.550 07.025 05.625 05.750 Mean of control = 11.575 VR (bacteria) ** CD (bacteria) 0.231 VR (day) ** CD (day) 0.512 VR (interaction) ** CD (interaction) 0.822 ** P<0.01 CD at 5% level
Fig. 29 Variation of oxygen carrying capacity (g /100 ml) in Cyprinus carpio
exposed to different bacteria
0
2
4
6
8
10
12
14
C 1 3 5 10 15 20 25 30DAYS
g / 1
00 m
l
E.coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
93
Table 32 Oxygen carrying capacity (g /100 ml) in Puntius sarana exposed to
different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 06.875 06.250 00.000 00.000 Mean of control = 10.35 VR (bacteria) ** CD (bacteria) 0.215 VR (day) ** CD (day) 0.405 VR (interaction) ** CD (interaction) 0.612 ** P<0.01 CD at 5% level
Fig. 30 Variation of oxygen carrying capacity (g /100 ml) in Puntius sarana
exposed to different bacteria
0
2
4
6
8
10
12
14
C 1 3 5 10 15 20 25 30DAYS
g / 1
00 m
l
E.coli A.hydrophilaconsortium-I consortium-II
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…………….
Morphological changes in the blood corpuscles of Cyprinus carpio exposed to
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
Continued…………….
Morphological changes in the blood corpuscles of Cyprinus carpio exposed to
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
Lp -Lobopodial projection
VEr -Vacuolated Erythrocytes,
REr -Ruptured Erythrocytes
VN -Vacuolated Neutrophil
VM -Vacuolated Monocyte
Continued…………….
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
Lp -Lobopodial projection
VEr -Vacuolated Erythrocytes,
REr -Ruptured 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
carpio exposed to different bacteria
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
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98
Table 34 Lipid peroxidation (n moles MDA/g wet wt.) in the gill of Puntius sarana
exposed to different bacteria
Duration
(Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 49.316 53.882 00.000 00.000 Mean of control = 21.958 VR (bacteria) ** CD (bacteria) 0.766 VR (day) ** CD (day) 1.250 VR (interaction) ** CD (interaction) 2.166 ** P<0.01 CD at 5% level
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
carpio exposed to different bacteria
Duration
(Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 33.524 40.078 55.110 58.562 Mean of control = 19.328 VR (bacteria) ** CD (bacteria) 0.617 VR (day) ** CD (day) 1.008 VR (interaction) ** CD (interaction) 1.746 ** P<0.01 CD at 5% level
Fig. 33 Variation of lipid peroxidation (n moles MDA/g wet wt.) in the muscle of
Cyprinus carpio exposed to different bacteria
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
sarana exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 40.110 51.170 00.000 00.000 Mean of control = 19.716 VR (bacteria) ** CD (bacteria) 0.727 VR (day) ** CD (day) 1.187 VR (interaction) ** CD (interaction) 2.056 ** P<0.01 CD at 5% level
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.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
101
Table 37 Lipid peroxidation ((n moles MDA/g wet wt.) in the liver of Cyprinus
carpio exposed to different bacteria
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
30 37.342 42.220 50.348 56.321 Mean of control = 21.626 VR (bacteria) ** CD (bacteria) 0.539 VR (day) ** CD (day) 0.881 VR (interaction) ** CD (interaction) 1.525 ** P<0.01 CD at 5% level
Fig. 35 Variation of lipid peroxidation (n moles MDA/g wet wt.) in the liver of
Cyprinus carpio exposed to different bacteria
0
10
20
30
40
50
60
C* 1 3 5 10 15 20 25 30DAYS
n m
oles
MD
A /g
wet
wt.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
102
Table 38 Lipid peroxidation (n moles MDA/g wet wt.) in the liver of Puntius
sarana exposed to different bacteria
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
30 38.968 40.206 00.000 00.000 Mean of control = 23.818 VR (bacteria) ** CD (bacteria) 0.690 VR (day) ** CD (day) 1.127 VR (interaction) ** CD (interaction) 1.951 ** P<0.01 CD at 5% level
Fig. 36 Variation of lipid peroxidation (n moles MDA/g wet wt.) in the liver of
Puntius sarana exposed to different bacteria
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.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
103
Table 39 Total protein (mg/g wet wt.) content in the gill of Cyprinus carpio
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 20.670 17.476 15.002 12.214 Mean of control = 24.324 VR (bacteria) ** CD (bacteria) 0.546 VR (day) ** CD (day) 0.892 VR (interaction) ** CD (interaction) 1.545 ** P<0.01 CD at 5% level
Fig. 37 Variation of total protein (mg/g wet wt.) content in the gill of Cyprinus
carpio exposed to different bacteria
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.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
104
Table 40 Total protein (mg/g wet wt.) content in the gill of Puntius sarana exposed
to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 18.938 16.262 00.000 00.000 Mean of control = 22.714 VR (bacteria) ** CD (bacteria) 0.478 VR (day) ** CD (day) 0.781 VR (interaction) ** CD (interaction) 1.352 ** P<0.01 CD at 5% level
Fig. 38 Variation of total protein (mg/g wet wt.) content in the gill of Puntius
sarana exposed to different bacteria
0
5
10
15
20
25
30
35
C* 1 3 5 10 15 20 25 30
DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
105
Table 41 Total protein (mg/g wet wt.) content in the muscle of Cyprinus carpio
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
Cyprinus carpio exposed to different bacteria
0
10
20
30
40
50
60
C* 1 3 5 10 15 20 25 30
DAYS
mg
/ g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
106
Table 42 Total protein (mg/g wet wt.) content in the muscle of Puntius sarana
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 26.997 25.870 00.000 00.000 Mean of control = 44.746 VR (bacteria) ** CD (bacteria) 0.730 VR (day) ** CD (day) 1.120 VR (interaction) ** CD (interaction) 2.064 ** P<0.01 CD at 5% level
Fig. 40 Variation of total protein (mg/g wet wt.) content in the muscle of Puntius
sarana exposed to different bacteria
0
10
20
30
40
50
60
C* 1 3 5 10 15 20 25 30
DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
107
Table 43 Total protein (mg/g wet wt.) content in the liver of Cyprinus carpio
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 63.750 59.670 44.208 36.115 Mean of control = 66.84 VR (bacteria) ** CD (bacteria) 1.021 VR (day) ** CD (day) 1.667 VR (interaction) ** CD (interaction) 2.888 ** P<0.01 CD at 5% level
Fig. 41 Variation of total protein (mg /g wet wt.) content in the liver of Cyprinus
carpio exposed to different bacteria
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.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
108
Table 44 Total protein (mg/g wet wt.) content in the liver of Puntius sarana
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 42.860 40.124 00.000 00.000 Mean of control = 65.844 VR (bacteria) ** CD (bacteria) 0.682 VR (day) ** CD (day) 1.113 VR (interaction) ** CD (interaction) 1.928 ** P<0.01 CD at 5% level
Fig. 42 Variation of total protein (mg/g wet wt.) content in the liver of Puntius
sarana exposed to different bacteria
0102030405060708090
C* 1 3 5 10 15 20 25 30
DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
109
Table 45 Total amino acid (mg/g wet wt.) content in the gill of Cyprinus carpio
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
Cyprinus carpio exposed to different bacteria
0102030405060708090
100
C* 1 3 5 10 15 20 25 30
DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
110
Table 46 Total amino acid (mg/g wet wt.) content in the gill of Puntius sarana
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 66.008 70.870 00.000 00.000 Mean of control =19.696 VR (bacteria) ** CD (bacteria) 1.065 VR (day) ** CD (day) 1.739 VR (interaction) ** CD (interaction) 3.013 ** P<0.01 CD at 5% level
Fig. 44 Variation of total amino acid (mg/g wet wt.) content in the gill of Puntius
sarana exposed to different bacteria
0102030405060708090
100
C* 1 3 5 10 15 20 25 30
DAYS
mg
/g w
e w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
111
Table 47 Total amino acid (mg/g wet wt.) content in the muscle of Cyprinus carpio
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 72.970 79.133 84.220 103.415 Mean of control = 32.976 VR (bacteria) ** CD (bacteria) 1.498 VR (day) ** CD (day) 2.446 VR (interaction) ** CD (interaction) 4.237 ** P<0.01 CD at 5% level
Fig. 45 Variation of total amino acid (mg/g wet wt.) content in the muscle of
Cyprinus carpio exposed to different bacteria
0102030405060708090
100110
C* 1 3 5 10 15 20 25 30DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
112
Table 48 Total amino acid (mg/g wet wt.) content in the muscle of Puntius sarana
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 72.656 75.200 00.000 00.000 Mean of control = 27.138 VR (bacteria) ** CD (bacteria) 1.213 VR (day) ** CD (day) 1.980 VR (interaction) ** CD (interaction) 3.430 ** P<0.01 CD at 5% level
Fig. 46 Variation of total amino acid (mg/g wet wt.) content in the muscle of
Puntius sarana exposed to different bacteria
0102030405060708090
100110
C* 1 3 5 10 15 20 25 30DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
113
Table 49 Total amino acid (mg/g wet wt.) content in the liver of Cyprinus carpio
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 72.484 81.454 89.411 93.346 Mean of control = 36.964 VR (bacteria) ** CD (bacteria) 1.782 VR (day) ** CD (day) 2.911 VR (interaction) ** CD (interaction) 5.041 ** P<0.01 CD at 5% level
Fig. 47 Variation of total amino acid (mg/g wet wt.) content in the liver of
Cyprinus carpio exposed to different bacteria
0102030405060708090
100
C* 1 3 5 10 15 20 25 30DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
114
Table 50 Total amino acid (mg/g wet wt.) content in the liver of Puntius sarana
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 80.840 84.846 00.000 00.000 Mean of control = 38.024 VR (bacteria) ** CD (bacteria) 1.772 VR (day) ** CD (day) 2.894 VR (interaction) ** CD (interaction) 5.012 ** P<0.01 CD at 5% level
Fig. 48 Variation of total amino acid (mg/g wet wt.) content in the liver of
Puntius sarana exposed to different bacteria
0102030405060708090
100
C* 1 3 5 10 15 20 25 30
DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
115
Table 51 Soluble sugar (mg/g wet wt.) content in the gill of Cyprinus carpio
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 166.204 153.452 107.330 90.071 Mean of control = 181.316 VR (bacteria) ** CD (bacteria) 5.481 VR (day) ** CD (day) 8.951 VR (interaction) ** CD (interaction) 15.503 ** P<0.01 CD at 5% level
Fig. 49 Variation of soluble sugar (mg/g wet wt.) content in the gill of Cyprinus
carpio exposed to different bacteria
0
50
100
150
200
250
C* 1 3 5 10 15 20 25 30DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
116
Table 52 Soluble sugar (mg/g wet wt.) content in the gill of Puntius sarana
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 101.336 97.208 00.000 00.000 Mean of control =188.484 VR (bacteria) ** CD (bacteria) 4.015 VR (day) ** CD (day) 6.557 VR (interaction) ** CD (interaction) 11.357 ** P<0.01 CD at 5% level
Fig. 50 Variation of soluble sugar (mg/g wet wt.) content in the gill of Puntius
sarana exposed to different bacteria
0
50
100
150
200
250
C* 1 3 5 10 15 20 25 30
DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
117
Table 53 Soluble sugar (mg/g wet wt.) content in the muscle of Cyprinus carpio
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 204.794 187.892 137.542 121.446 Mean of control = 253.040 VR (bacteria) ** CD (bacteria) 4.091 VR (day) ** CD (day) 6.680 VR (interaction) ** CD (interaction) 11.570 ** P<0.01 CD at 5% level
Fig. 51 Variation of soluble sugar (mg/g wet wt.) content in the muscle of
Cyprinus carpio exposed to different bacteria
0
50
100
150
200
250
300
350
C* 1 3 5 10 15 20 25 30DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
118
Table 54 Total soluble sugar (mg/g wet wt.) content in the muscle of Puntius
sarana exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 144.370 135.318 00.000 00.000 Mean of control =233.586 VR (bacteria) ** CD (bacteria) 4.210 VR (day) ** CD (day) 6.875 VR (interaction) ** CD (interaction) 11.908 ** P<0.01 CD at 5% level
Fig.52 Variation of soluble sugar (mg/g wet wt.) content in the muscle of Puntius
sarana exposed to different bacteria
0
50
100
150
200
250
300
350
C* 1 3 5 10 15 20 25 30DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
119
Table 55 Soluble sugar (mg/g wet wt.) content in the liver of Cyprinus carpio
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 219.242 200.150 163.060 122.536 Mean of control = 266.714 VR (bacteria) ** CD (bacteria) 5.125 VR (day) ** CD (day) 8.369 VR (interaction) ** CD (interaction) 14.496 ** P<0.01 CD at 5% level
Fig. 53 Variation of soluble sugar (mg/g wet wt.) content in the liver of Cyprinus
carpio exposed to different bacteria
0
50
100
150
200
250
300
350
C* 1 3 5 10 15 20 25 30
DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
Chapter 4 Results
120
Table 56 Soluble sugar (mg/g wet wt.) content in the liver of Puntius sarana
exposed to different bacteria
Duration ( Days) E. coli A.hydrophila Consortium-I Consortium-II
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
30 188.540 124.814 00.000 00.000 Mean of control = 269.964 VR (bacteria) ** CD (bacteria) 4.705 VR (day) ** CD (day) 7.684 VR (interaction) ** CD (interaction) 13.309 ** P<0.01 CD at 5% level
Fig. 54 Variation of soluble sugar (mg/g wet wt.) content in the liver of Puntius
sarana exposed to different bacteria
0
50
100
150
200
250
300
350
C* 1 3 5 10 15 20 25 30
DAYS
mg
/g w
et w
t.
E. coli A.hydrophilaconsortium-I consortium-II
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
inocula for 25 days(x400)
a- Control
b -E. coli c -A. hydrophila d - consortium-I e -consortium-II
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
b -E. coli c -A. hydrophila d - consortium-I e -consortium-II
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
inocula for 25 days(x400)
a- Control
b -E. coli c -A. hydrophila d - consortium-I e -consortium-II
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
inocula for 30 days(x400)
a- Control
b -E. coli c -A. hydrophila d - consortium-I e -consortium-II
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
inocula for 25 days(x400)
a- Control
b -E. coli c -A. hydrophila d - consortium-I e -consortium-II
Hc -Hepatocytes
N -Nucleus
Bc -Bile canaliculi
Eh -Enlarged hepatocytes
Lis -Large intercellular space