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International Journal of Trend in Scientific Research and Development (IJTSRD)
Volume 4 Issue 4, June 2020 Available Online: www.ijtsrd.com e-ISSN: 2456 – 6470
@ IJTSRD | Unique Paper ID – IJTSRD30908 | Volume – 4 | Issue – 4 | May-June 2020 Page 194
Studies on Seasonal Variations of Total Glycogen, Protein and
Lipids in Estuarine Clam, Meretrix Meretrix (Linnaeus) after
Chronic Exposure to Cadmium Chloride
Sanjay Kumbhar
Department of Zoology, Fergusson College (Autonomous), Pune, Maharashtra, India
ABSTRACT
In the present investigation, estuarineclam,M.meretrixwasemployedtostudy
effect of cadmium chloride stress on biochemical components ofdifferentsoft
tissues. During summer, monsoon and winterclamswere subjectedtochronic
exposure (96 hours) to predetermined sub lethal concentrations of cadmium
chloride. During the experimental period water samples were analyzed for
temperature, pH, salinity, dissolved oxygen and cadmium content. It was
observed that rainfall, salinity, oxygen saturation and temperature of
estuarine water has profound effect on seasonal changes in biochemical
content of M.meretrix. During summer cadmium content in estuarine water
was below detectable level, while in monsoon and winter it was 0.005 and
0.001ug/ml respectively. Clams from control group showed low glycogen
content in gills and high in gonad and foot . Clams from chronic sub lethal
concentration showed increase in glycogen content in gill in monsoon, while
hepatopancreas showed significant increase during winter. Clams from
control and chronic group showed higher protein content in gonad and foot
for three different seasons. In chronicgroup,cadmiuminducedtoxicitycaused
significant depletion in gonad and foot, while gill and hepatopancreasshowed
significant elevation. Chronic exposure of cadmium chloridedepletedlipidsin
all soft tissues, but depletion was significant (p<0.001)in hepatopancreasand
mantle. Hepatopancreas showed 30.85 and 28.55% decrease inmonsoonand
winter respectively. During summer, mantle showed (39.77%) considerable
depletion than hepatopancreas. In the present study, it was found that,
alterations in total glycogen, proteins and lipids from control group were
attributed to highly labile estuarine water parameters and breedingseasonof
clams, while, alterations in clams from chronic group are moreoverattributed
to cadmium chloride stress.
KEYWORDS: Meretrix meretrix, Bhatye estuary, total glycogen, Proteins, Lipids,
Chronic toxicity
How to cite this paper: Sanjay Kumbhar
"Studies on Seasonal Variations of Total
Glycogen, Protein and Lipids in Estuarine
Clam, Meretrix Meretrix (Linnaeus) after
Chronic Exposure to Cadmium Chloride"
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of Trend in Scientific
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INTRODUCTION
Studies of the seasonal alterations in different biochemical
components and enzymes in marine pelecypods have been
studied extensively (Rao, K.V. 19691 Nagbhushnam and
Mane, 19822.; Peter and Albert, 19843; K.K. Appukuttan and
C.M. Arvindan, 19954; Patil, S.S. and U.H. Mane, 19975;
Tendulkar S. P. and B.G.Kulkarni,19986), no study to date,
however, has compared heavy metal induced seasonal
changes in total glycogen, proteins and lipids in gill, mantle,
hepatopancreas, foot and gonads of marine bivalves. These
soft tissues and organs are locationaly, structurally and
functionally different and hence their energy requirement,
biochemical composition and physiological processes are
different. Zandee and Klutman (1971)7 first time recorded
seasonal changes in biochemical composition in Mytilis
eudilis. Of all these studies, impact of cadmium on marine
biota is least studied subject .Geochemistry of cadmium has
been discussed by Eaton (1979)8 it is well established that
although concentration of cadmium in surface water is
negligible it increases manyfoldinsedimentsandstill higher
in marine biota. Recent understanding of biochemical
processes has proved useful in determining the mechanism
of toxicity. Sanjay Kumbhar (2001)9 studiedaccumulation of
cadmium in different soft tissues of M.meretrixandobserved
great varience in concentration of cadmium in different soft
tissues during summer, monsoon and winter. Theremust be
metabolic strategies to utilize or sequester these metals
depending upon the toxic potential and it may be operated
by transport, conversion and utilization of biochemical
components like glycogen, proteins and lipids. The present
work is an attempt to correlate seasonal estuarine water
parameters and biochemical constituents of soft tissues like
gill, mantle, hepatopancreas, foot and gonads under
cadmium chloride stress.
Material and Methods:
Physio-chemical parameters like temperature, salinity, pH
and dissolved oxygen were determined for each season.
Temperature of the aerated estuarinewaterusedforholding
the clams during experimentation was recorded at regular
intervals. Salinity was recorded with the help of hand
salinometer (Hanna instruments, Italy). pH of the estuarine
water was measured by digital pH meter. Dissolved oxygen
was measured by Winkler's titrimetric method. Cadmium in
estuarine water was analyzed on Atomic Adsorption
IJTSRD30908
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@ IJTSRD | Unique Paper ID – IJTSRD30908 | Volume – 4 | Issue – 4 | May-June 2020 Page 195
Spectrophotometer (Perkin Elmer Model 3030.USA). Clams
of medium size measuring 4.0-4.8cms were selected for
experiment. These clams were exposed to predetermined
sub lethal concentrations of cadmiumchloride.Afterchronic
exposure, various tissues (gill, mantle, hepatopancreas, foot
and gonad of live clams were removed from the control and
experimental groups in different seasons. Pooled tissues
were weighed and dried in an oven at 100°C ( + 5°C) until a
constant weight was obtained. Oven dried tissue powder
was used for biochemical analyzed for total glycogen (De
Zwan and Zandee, 1972), proteins (Gornal et al, 1949) and
lipids (Barnes and Blackstock, 1973).
Result and discussion:
Literature survey revealed that, the independent variables
like tides, river discharge, density, differences between
water masses in the estuary caused by differencesinsalinity
and temperature makes estuarine hydronomics very
complex. Quasim,(1980) 12 reported that, magnitude of
variations in the environment depends on a large extent on
the time of the year and the place of the observation In the
present study it was decided to focus on few estuarine
parameters and unveil their probable relationship in
intensifying or decreasing toxicity of cadmium chloride. It
was observed that, average water temperature was
minimum in winter (25.7 0c). The maximum average water
temperature was recorded in summer (26.50c). Maximum
average salinity was recorded in summer (35.1). It was
30.36mg/l in winter. Minimum average salinity was
recorded in monsoon season (5.2mg/l).Inthepresentstudy,
it was observed that, salinity changes according to season.
Differences in seasonal variations in salinity were due
rainfall, heavy influx of fresh water and temperature, pH
fluctuations was observed in monsoon than summer and
winter. The average pH was 8.2, 7.1 and 8.3 in summer,
monsoon and winter respectively. The average dissolved
oxygen was 3.9, 5.6 and 4.7 ml/l in summer, monsoon and
winter respectively. During monsoon estuarine water
showed maximum saturation of oxygen (5.6ml/l). It was
minimum (3.9ml/l) in months of summer. Average pH
during summer, monsoon and winter was 8.2, 7.1 and 8.3
respectively. Variations in pH did not show significant
pattern, however, it was low in monsoon due to heavy influx
of fresh water. Similar observations were recorded by
Chandran and Ramamorthy (1984) 14 while studying
hydronomics of Vellar estuary. The average rainfall during
monsoon was 29.15mm. During winter scanty rainfall was
recorded. Summer received no rainfall. In tropical countries
rainfall is regular cyclic phenomenon. It brings profound
changes in hydronomics of the estuarine environment
(Quasim, 1980) 13. Heavy rainfall in monsoon reduced
salinity, water temperature, pH of estuarine water and
increases turbidity and oxygen saturation.Thehydronomics
of an estuary in general is very complex. It was decided to
analyze preexistence of cadmium before conductingchronic
static bioassay. It was observed that, during summer,
cadmium concentration in water wasbelowdetectablelevel.
In monsoon and winter average concentration of cadmium
was 0.005 and 0.001 µg/l respectively. Maximum
accumulation of cadmium was observed in monsoon.
Unfortunately data is not available stating cadmium
concentration in Bhatye estuary. At Ratnagiri, heavy rainfall
was recorded in monsoon (34.39 mm/day).Rainy freshets
brought heavy load of soil that impregnated with heavy
metals like cadmium. It resulted in increaseinconcentration
of cadmium in monsoon. It also showed rapid loss of
cadmium during summer and winter. Fowler and Oregioni
(1976) 15 suggested that seasonal maximum concentration
appeared in the spring was due to high water run-off which
increased the amount of available metals. Philips (1973) 16
proposed similar conclusion with zinc, cadmium, lead and
copper. In the present investigation concentration of
cadmium in estuarine water was considered while
quantifying sub lethal and lethal concentrations ofcadmium
chloride.
Table1. Cadmium chloride induced alterations in total glycogen content of M.meretrix after chronic exposure
(Results are expressed in mg/100mg. dry wt.basis).
TISSUE SEASON CONTROL CHRONIC GROUP
GILL
SUMMER
MONSOON
WINTER
6.872 + 0.071
4.045 + 0.106
8.121 + 0.046
6.039 + 0.052 (12.12)
5.455 + 0.081 (34.85)* *
6.247 + 0.162 (23.07)* * *
MANTLE
SUMMER
MONSOON
WINTER
14.785 + 0.089
15.410 + 0.025
15.202 + 0.096
19.367 + 0.229 (30.99)* * *
18.950 + 0.158 (22.97)* * *
19.792 + 0.624 (30.19)* * *
FOOT
SUMMER
MONSOON
WINTER
42.063 + 0.012
4.145 + 0.328
52.062 + 0.061
26.239 + 0.123 (37.61)
24.365 + 0.095 (44.80)* *
29.155 + 0.012 (43.99)* * *
GONAD
SUMMER
MONSOON
WINTER
38.802 + 0.920
29.969 + 1.041
32.015 + 1.134
31.091 + 1.024 (19.87)*
26.117 + 0.162 (12.85)
27.980 + 0.524 (12.60)
Values in parenthesis are percent change, ± =S.D. of five animals.
(*=p<0.05; **=p<0.01; ***=p<0.001).
The studies on biochemical changes enable to define the dose response relationship, threshold limit value, revercible and
irrevercible nature of pollutant effect. In the present study significant alterations in total glycogen, proteins and lipids were
observed both in control and chronic group of clams. In summer, the glycogen content in the control group showed ascending
order of gill< hepatopancreas< mantle< gonad< foot, with 6.872 + 0.071, 13.536 + 1.083, 14.785 + 0.089, 29.155 + 0.015,
38.802 + 0.920 mg/100 mg dry tissue respectively. In chronic group, glycogen content was present in increasing orderofgill<
hepatopancreas< mantle< foot< gonad, with 6.036 + 0.052, 12.286 + 0.864, 19.367 + 0.229,and31.091+1.024mg/100mgdry
tissue respectively. As compared to controls, there was 30.99% (p<0.001) increase in mantle, while 9.23 (P<0.05),
37.61(p<0.001) and 19.87% (P<0.05) significant decrease in hepatopancreas, foot and gonad respectively During monsoon,
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control group showed glycogen content in ascending order of gill< hepatopancreas< mantle< gonad< foot, with4.045+0.106,
14.369 + 0.427, 15.410 + 0.025, 29.969 + 1.041, and 44.145 + 0.328 mg/100mg dry tissue respectively. In chronic group,
glycogen content was low in gill, followed by hepatopancreas, mantle, foot and gonad, with 5.455 + 0.081, 12.078 + 0.080,
18.950 + 0.158, 24.365 + 0.095 and 26.117 + 0.162 mg/100mg dry tissue respectively. Based on percent change, there was
34.85 (p<0.001) and 22.97 % (p<0.001) increase in gill and mantle, while 15.94 (P<0.05), 20.47 (p<0.001), 44.80%(p<0.001)
significant decrease in hepatopancreas and foot respectively. During winter, control group exhibited high glycogen contentin
gill, followed by hepatopancreas, mantle, gonad, foot, with 8.121 + 0046, 14.216 + 0.035, 37.485 + 1.050 and 52.062 + 0.061
mg/ 100mg dry tissue respectively. In chronic group, glycogen content was present in ascending order of gill<
hepatopancreas< mantle< gonad<foot, with 6.247 + 0.162, 17.662 + 0.0061,20.616+1.603,27.980+0.521and29.155+0.012
mg in 100mg dry tissue. As compared to control group, there was 24.24 and 30.19 % significant (p< 0.001) increase in
hepatopancreas and mantle while 23.07 and 43.99 % significant (p<0.001) decrease in gill, and foot respectively Clams from
control group showed less glycogen content in gills and high glycogen content in gonad and foot .Glycogen content was at its
peak during gametogenesis and it was low during the spawning period.Similarobservationswerenoted byNagbhushnamand
Talikhedkar (1977)17 in wedge clam, Danus cunaeutus. The result obtained are in good agreement with earlier workers
(Masumoto and Hibino,197318 ;Dhavale and Masurekar,198519 ;George and Mathews,199620) Clams those were subjected to
chronic sub lethal concentration showed increase in glycogen content in gill in monsoon, while hepatopancreas showed
significant increase during winter. Hepatopancreas and gills are more modifiedintheir biochemical compositionthanmuscles
and hence accumulate higher level of cadmium and to reduce toxicity they require more glycogen. Similar observation was
noted by Diaz Mayans et al (1986) 21. In rest of soft tissues there was significant decrease in glycogen content. Decline in
glycogen content was may be due to stress resulting in breakdownoftissueglycogentomeetenergydemandduetotoxicstress
of cadmium chloride.
Table 2: Cadmium chloride induced alterations in total protein content of M.meretrix after chronic exposure
(Results are expressed in mg/100mg. dry wt.basis).
TISSUE SEASON CONTROL CHRONIC GROUP
GILL
SUMMER
MONSOON
WINTER
10.620 + 1.013
12.206 + 0.077
10.376 + 0.520
11.427 + 0.907 (7.49)
16.524 + 0.012 (35.37)* *
16.600 + 0.039 (59.93)* * *
MANTLE
SUMMER
MONSOON
WINTER
20.400 + 1.047
21.025 + 0.093
23.552 + 0.034
17.942 + 0.213 (-12.04)* * *
20.102 + 1.054 (-4.39)*
20.763 + 0.615 (-11.84)* *
HEPATOPANCREAS
SUMMER
MONSOON
WINTER
6.526 + 1.022
7.102 + 0.316
7.230 + 1.069
7.902 + 0.091 (21.85)
10.022 + 0.097 (41.11)* *
7.500 + 0.016 (3.73)
FOOT
SUMMER
MONSOON
WINTER
25.059 + 0.085
27.500 + 0.104
27.560 + 0.458
21.709 + 0.007 (13.33)
22.406 + 0.025(18.52)* *
22.970 + 0.044 (16.65)* * *
GONAD
SUMMER
MONSOON
WINTER
27.521 + 0.015
23.746 + 0.705
24.854 + 1.802
21.164 + 0.033 (23.09)***
16.047 + 1.091 (32.42)**
20.803 + 0.196(16.29)
Values in parenthesis are percent change, ± =S.D. of five animals.
(*=p<0.05; **=p<0.01; ***=p<0.001)
Proteins play vital role in the spawning and other metabolic activities. Alteration in protein content after chronic exposure to
cadmium chloride are shown in Table 2. During summer, control group showed protein content in different organs, in
increasing order of hepatopancreas < gill< mantle< foot< gonad with 6.526 + 1.022, 10.620 + 1.013,20.400+1.047.8,25.059+
0.085 and Chronic (0.18 ppm) group, exhibited low protein content in hepatopancreas, followed by gill, mantle, gonads, foot;
with 7.902 + 0.091, 11.427 + 0.907, 17.942 + 0.051, 21.164 + 0.033 and 21.709 + 0.015 mg/ 100mg dry tissue in respective
organs. As compared to control group, there was 12.04, 23.09 and 13.33%significant (p<0.001)decreaseinmantle,gonadand
foot respectively .In monsoon, control group exhibited protein content in increasing order of, hepatopancreas< gill<mantle <
foot < gonad, with 7.102 + 0.316, 12.206 + 0.077, 21.025 + 0.093, 23.746 + 0.705 and 38.640 + 0.070mg proteins in 100mgdry
tissue respectively. Chronic (0.20 ppm) group, showed protein content in the ascending order of hepatopancreas < gill<
mantle< gonad< foot, with 10.022 + 0.097, 16.047 + 1.091, 19.944 + 0.074, 20.102 + 1.054 and 22.406 + 0.025mg/100mgdry
tissue in respective organs. As compared to control, there was 41.11 (p<0.01), 35.37% (p<0.001) increase in hepatopancreas
and gill and 32.42 (p<0.01), 18.52% (p<0.001) increase in gonad and foot respectively. During winter,control group exhibited
protein content in ascending order of hepatopancreas < gill< mantle< female gonad< foot, with 7.230 + 1.069, 10.376 + 0.520,
23.552 + 0.034, 24.854 + 1.802, 27.560 + 0.458mg proteins in 100mg dry tissue respectively. Chronic (0.21 ppm) ground,
showed proteins in ascending order of hepatopancreass< gill< mantle< female gonad< foot, exhibiting7.500+0.016,16.600+
0.039, 003, 20.763 + 0.615, 20.803 + 0.196, 22.970 + 0.044mg proteins in drytissue.Ascomparedtocontrol,there was59.98%
significant (p<0.001) increase in gill, while 11.84 (p<0.01),and16.65%(p<0.01)decreaseinproteincontentinmantleandfoot
respectively. Clams from control group showed marked variations which were attributed to season. During winter protein
content was better in most of the soft tissues. It may be due to abundanceoffoodmaterial,increasedshell valveopeningperiod
and less energy expenditure. Protein content was low in summer and monsoon. It may be due to utilization of proteins for
spawning Mane,U.H et al (1987)22 observed that spawning period of K.opima and M. meretrix from Kalbadevi estuary was in
months of monsoon and summer. Durve and Bal (1961) 23 analyzed Crossostrea gryphoides and reported that low protein
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values coinsides with spawning season. Clams from chronic group showedsignificantdepletionduringthreedifferentseasons.
Marked depletion was observed in gonad and foot, while gill and hepatopancreas showed considerable elevation in protein
level. It may be due to mobilization of proteins from depositories and conversioninto glycogen.Gillsandhepatopancreaswere
primary target organs for cadmium chloride stress. Thesetissuesandorgansmighthaveutilizedanddemandedmoreglycogen
to reduce cadmium chloride stress.
Table 3: Cadmium chloride induced alterations in total lipid content of M.meretrix after chronic exposure (Results
are expressed in mg/100mg. dry wt. basis).
TISSUE SEASON CONTROL CHRONIC GROUP
GILL
SUMMER
MONSOON
WINTER
5.500 + 1.008
6.102 + 0.065
6.795 + 0.092
4.330 + 0.203 (21.27)
5.702 + 0.041 (6.55)* *
5.967 + 0.396 (12.18)* * *
MANTLE
SUMMER
MONSOON
WINTER
5.187 + 0.086
4.715 + 1.013
4.870 + 0.318
3.124 + 0.008 (39.77)* * *
4.225 + 0.013 (10.39)* * *
4.020 + 0.015 (17.45)* * *
HEPATOPANCREAS
SUMMER
MONSOON
WINTER
5.127 + 0.205
6.080 + 0.516
4.970 + 1.061
4.925 + 0.142 (3.93)*
4.204 + 0.057 (30.85)*
3.551 + 0.016 (28.55)* * *
FOOT
SUMMER
MONSOON
WINTER
42.063 + 0.012
44.145 + 0.328
52.062 + 0.061
26.239 + 0.123 (37.61)
24.365 + 0.095 (44.80)* *
29.155 + 0.012 (43.99)* * *
GONAD
SUMMER
MONSOON
WINTER
38.802 + 0.920
29.969 + 1.041
32.015 + 1.134
31.091 + 1.024 (19.87)*
26.117 + 0.162 (12.85)
27.980 + 0.524 (12.60)
Values in parenthesis are percent change, ± =S.D. of five animals.
(*=p<0.05; **=p<0.01; ***=p<0.001).
Alterations in lipid content after chronic exposuretocadmiumareshownin Table3.In summer,control groupofclamsshowed
variations in lipid content in different organs in ascending order of hepatopancreas< mantle< gill< gonad< foot, with 5.127 +
0.205, 5.187 + 0.086, 5.500 + 1.008 and 9.946 + 0.509 mg/ 100mg dry tissue respectively. Chronic (1.18 ppm)groupexhibited
lipid content with increasing trend of mantle, gill, hepatopancreas,gonad,foot,with3.124+0.008,4.330+0.203,4.925+0.142,
5.210 + 0.081, and 7.630 + 0.091 mg/ 100mg dry tissue respectively.Ascomparedtocontrol group,therewas39.77(p<0.001),
21.27, 3.93, 36.15 (p<0.001), and 23.28% (p<0.01) decrease inrespectiveorgans.Duringmonsoon,control groupshowedlipid
content in ascending order of mantle, followed by hepatopancreas, gill, gonad, foot, with 4.715 + 1.013, 6.080 + 0.516, 6.102 +
0.065, 7.020 + 0.816, and 10.421 + 0.012 mg lipids in 100mgdryweight respectively.Chronicgroup(0.20ppm)exhibitedlipids
in increasing order if hepatopancreas <mantle< gill< gonad< foot, with 4.204 + 0.057, 4.225 + 0.013, 5.702 + 0.041, 6.820 +
0.105, and 9.400 + 1.091 mg/100mg dry tissue respectively. As compared to control group,there wasconsiderable decreasein
lipid content of all organs, but decrease was significant in gill only. During winter, control group, showed lipid content, in
increasing order of mantle< hepatopancreas< gill< gonad < foot. The lipid content was 4.870 + 0.318, 4.970 + 1.061, 6.795 +
0.092, 8.205 + 0.032, and 12.072 + 0.076, mg in 100mg dry tissue respectively. In chronicgroup,(0.21ppm)thelipidcontentin
different organs was present in ascending order of hepatopancreas< mantle< gill< gonad< foot, with 3.551 + 0.016, 4.020 +
0.015, 5.967 + 0.392, 7.010 + 1.030.As compared to control group, there was 12.18 (p<0.001), 16.85 (p<0.01), and 25.22%
(p<0.001) decrease in lipid content in gill, gonad and foot respectively. In general, alterations in lipid content of chronicgroup
showed considerable depletion in all soft tissues, but depletion was significant (p<0.001) in hepatopancreas and mantle.
Hepatopancreas showed 30.85 and 28.55% decrease in monsoon and winter respectively. During summer, mantle showed
(39.77%) considerable depletion than hepatopancreas. Ansari (1983)24 reported copper induced decrease in lipid content of
Crossostrea punctatus .The decrease in lipid content of clams from chronic sub lethal exposure of cadmium chloride is in good
agreement with observations of few workers.( Jana and Bandopadhya,198725; Builter and Wilson, 1998)26.
Conclusion:
The present study gives a detailed account of influence of
cadmium chloride stress and seasonal water parameters on
biochemical alterations in soft tissues like gill, mantle,
hepatopancreas, gonad and foot of M.meretrix.Alterationsin
glycogen, protein and lipids were attributed to various
factors like environmental stress, physiological status of
clams and concentration of cadmium chloride.Alterationsin
glycogen content in clams from control groupwasattributed
to seasonal water parameters and spawning season. In
chronic group of clams, there was significant increase in
glycogen content of gill and hepatopancreas, while in rest of
organs there was marked decrease during summer,
monsoon and winter. The significant increase in glycogen
content in gill and hepatopancreas indicated that these soft
tissues were prominent target organs for cadmium chloride
toxicity. In control group, protein content was better during
winter. It was attributed to favourable environmental
parameters and abundance of food. Clams from chronic
group showed significant elevation in gill and
hepatopancreas. It may be due to mobilization or induction
of new proteins to combat against cadmium stress. Lipid
content was better during winter andgametogenesis period,
Clams from chronic group showed significant decrease in
almost all soft tissues during summer, monsoon and winter.
Acknowledgement:TheauthorgratefullyacknowledgesDr.
D.V Muley, Ret. Professor, Dept. of Zoology andEx.Registrar,
Shivaji University Kolhapur for his consistent and valueable
guidance.
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Studies on Seasonal Variations of Total Glycogen, Protein and Lipids in Estuarine Clam, Meretrix Meretrix Linnaeus after Chronic Exposure to Cadmium Chloride

  • 1. International Journal of Trend in Scientific Research and Development (IJTSRD) Volume 4 Issue 4, June 2020 Available Online: www.ijtsrd.com e-ISSN: 2456 – 6470 @ IJTSRD | Unique Paper ID – IJTSRD30908 | Volume – 4 | Issue – 4 | May-June 2020 Page 194 Studies on Seasonal Variations of Total Glycogen, Protein and Lipids in Estuarine Clam, Meretrix Meretrix (Linnaeus) after Chronic Exposure to Cadmium Chloride Sanjay Kumbhar Department of Zoology, Fergusson College (Autonomous), Pune, Maharashtra, India ABSTRACT In the present investigation, estuarineclam,M.meretrixwasemployedtostudy effect of cadmium chloride stress on biochemical components ofdifferentsoft tissues. During summer, monsoon and winterclamswere subjectedtochronic exposure (96 hours) to predetermined sub lethal concentrations of cadmium chloride. During the experimental period water samples were analyzed for temperature, pH, salinity, dissolved oxygen and cadmium content. It was observed that rainfall, salinity, oxygen saturation and temperature of estuarine water has profound effect on seasonal changes in biochemical content of M.meretrix. During summer cadmium content in estuarine water was below detectable level, while in monsoon and winter it was 0.005 and 0.001ug/ml respectively. Clams from control group showed low glycogen content in gills and high in gonad and foot . Clams from chronic sub lethal concentration showed increase in glycogen content in gill in monsoon, while hepatopancreas showed significant increase during winter. Clams from control and chronic group showed higher protein content in gonad and foot for three different seasons. In chronicgroup,cadmiuminducedtoxicitycaused significant depletion in gonad and foot, while gill and hepatopancreasshowed significant elevation. Chronic exposure of cadmium chloridedepletedlipidsin all soft tissues, but depletion was significant (p<0.001)in hepatopancreasand mantle. Hepatopancreas showed 30.85 and 28.55% decrease inmonsoonand winter respectively. During summer, mantle showed (39.77%) considerable depletion than hepatopancreas. In the present study, it was found that, alterations in total glycogen, proteins and lipids from control group were attributed to highly labile estuarine water parameters and breedingseasonof clams, while, alterations in clams from chronic group are moreoverattributed to cadmium chloride stress. KEYWORDS: Meretrix meretrix, Bhatye estuary, total glycogen, Proteins, Lipids, Chronic toxicity How to cite this paper: Sanjay Kumbhar "Studies on Seasonal Variations of Total Glycogen, Protein and Lipids in Estuarine Clam, Meretrix Meretrix (Linnaeus) after Chronic Exposure to Cadmium Chloride" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456- 6470, Volume-4 | Issue-4, June 2020, pp.194-198, URL: www.ijtsrd.com/papers/ijtsrd30908.pdf Copyright © 2020 by author(s) and International Journal ofTrendinScientific Research and Development Journal. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (CC BY 4.0) (http://creativecommons.org/licenses/by /4.0) INTRODUCTION Studies of the seasonal alterations in different biochemical components and enzymes in marine pelecypods have been studied extensively (Rao, K.V. 19691 Nagbhushnam and Mane, 19822.; Peter and Albert, 19843; K.K. Appukuttan and C.M. Arvindan, 19954; Patil, S.S. and U.H. Mane, 19975; Tendulkar S. P. and B.G.Kulkarni,19986), no study to date, however, has compared heavy metal induced seasonal changes in total glycogen, proteins and lipids in gill, mantle, hepatopancreas, foot and gonads of marine bivalves. These soft tissues and organs are locationaly, structurally and functionally different and hence their energy requirement, biochemical composition and physiological processes are different. Zandee and Klutman (1971)7 first time recorded seasonal changes in biochemical composition in Mytilis eudilis. Of all these studies, impact of cadmium on marine biota is least studied subject .Geochemistry of cadmium has been discussed by Eaton (1979)8 it is well established that although concentration of cadmium in surface water is negligible it increases manyfoldinsedimentsandstill higher in marine biota. Recent understanding of biochemical processes has proved useful in determining the mechanism of toxicity. Sanjay Kumbhar (2001)9 studiedaccumulation of cadmium in different soft tissues of M.meretrixandobserved great varience in concentration of cadmium in different soft tissues during summer, monsoon and winter. Theremust be metabolic strategies to utilize or sequester these metals depending upon the toxic potential and it may be operated by transport, conversion and utilization of biochemical components like glycogen, proteins and lipids. The present work is an attempt to correlate seasonal estuarine water parameters and biochemical constituents of soft tissues like gill, mantle, hepatopancreas, foot and gonads under cadmium chloride stress. Material and Methods: Physio-chemical parameters like temperature, salinity, pH and dissolved oxygen were determined for each season. Temperature of the aerated estuarinewaterusedforholding the clams during experimentation was recorded at regular intervals. Salinity was recorded with the help of hand salinometer (Hanna instruments, Italy). pH of the estuarine water was measured by digital pH meter. Dissolved oxygen was measured by Winkler's titrimetric method. Cadmium in estuarine water was analyzed on Atomic Adsorption IJTSRD30908
  • 2. International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 @ IJTSRD | Unique Paper ID – IJTSRD30908 | Volume – 4 | Issue – 4 | May-June 2020 Page 195 Spectrophotometer (Perkin Elmer Model 3030.USA). Clams of medium size measuring 4.0-4.8cms were selected for experiment. These clams were exposed to predetermined sub lethal concentrations of cadmiumchloride.Afterchronic exposure, various tissues (gill, mantle, hepatopancreas, foot and gonad of live clams were removed from the control and experimental groups in different seasons. Pooled tissues were weighed and dried in an oven at 100°C ( + 5°C) until a constant weight was obtained. Oven dried tissue powder was used for biochemical analyzed for total glycogen (De Zwan and Zandee, 1972), proteins (Gornal et al, 1949) and lipids (Barnes and Blackstock, 1973). Result and discussion: Literature survey revealed that, the independent variables like tides, river discharge, density, differences between water masses in the estuary caused by differencesinsalinity and temperature makes estuarine hydronomics very complex. Quasim,(1980) 12 reported that, magnitude of variations in the environment depends on a large extent on the time of the year and the place of the observation In the present study it was decided to focus on few estuarine parameters and unveil their probable relationship in intensifying or decreasing toxicity of cadmium chloride. It was observed that, average water temperature was minimum in winter (25.7 0c). The maximum average water temperature was recorded in summer (26.50c). Maximum average salinity was recorded in summer (35.1). It was 30.36mg/l in winter. Minimum average salinity was recorded in monsoon season (5.2mg/l).Inthepresentstudy, it was observed that, salinity changes according to season. Differences in seasonal variations in salinity were due rainfall, heavy influx of fresh water and temperature, pH fluctuations was observed in monsoon than summer and winter. The average pH was 8.2, 7.1 and 8.3 in summer, monsoon and winter respectively. The average dissolved oxygen was 3.9, 5.6 and 4.7 ml/l in summer, monsoon and winter respectively. During monsoon estuarine water showed maximum saturation of oxygen (5.6ml/l). It was minimum (3.9ml/l) in months of summer. Average pH during summer, monsoon and winter was 8.2, 7.1 and 8.3 respectively. Variations in pH did not show significant pattern, however, it was low in monsoon due to heavy influx of fresh water. Similar observations were recorded by Chandran and Ramamorthy (1984) 14 while studying hydronomics of Vellar estuary. The average rainfall during monsoon was 29.15mm. During winter scanty rainfall was recorded. Summer received no rainfall. In tropical countries rainfall is regular cyclic phenomenon. It brings profound changes in hydronomics of the estuarine environment (Quasim, 1980) 13. Heavy rainfall in monsoon reduced salinity, water temperature, pH of estuarine water and increases turbidity and oxygen saturation.Thehydronomics of an estuary in general is very complex. It was decided to analyze preexistence of cadmium before conductingchronic static bioassay. It was observed that, during summer, cadmium concentration in water wasbelowdetectablelevel. In monsoon and winter average concentration of cadmium was 0.005 and 0.001 µg/l respectively. Maximum accumulation of cadmium was observed in monsoon. Unfortunately data is not available stating cadmium concentration in Bhatye estuary. At Ratnagiri, heavy rainfall was recorded in monsoon (34.39 mm/day).Rainy freshets brought heavy load of soil that impregnated with heavy metals like cadmium. It resulted in increaseinconcentration of cadmium in monsoon. It also showed rapid loss of cadmium during summer and winter. Fowler and Oregioni (1976) 15 suggested that seasonal maximum concentration appeared in the spring was due to high water run-off which increased the amount of available metals. Philips (1973) 16 proposed similar conclusion with zinc, cadmium, lead and copper. In the present investigation concentration of cadmium in estuarine water was considered while quantifying sub lethal and lethal concentrations ofcadmium chloride. Table1. Cadmium chloride induced alterations in total glycogen content of M.meretrix after chronic exposure (Results are expressed in mg/100mg. dry wt.basis). TISSUE SEASON CONTROL CHRONIC GROUP GILL SUMMER MONSOON WINTER 6.872 + 0.071 4.045 + 0.106 8.121 + 0.046 6.039 + 0.052 (12.12) 5.455 + 0.081 (34.85)* * 6.247 + 0.162 (23.07)* * * MANTLE SUMMER MONSOON WINTER 14.785 + 0.089 15.410 + 0.025 15.202 + 0.096 19.367 + 0.229 (30.99)* * * 18.950 + 0.158 (22.97)* * * 19.792 + 0.624 (30.19)* * * FOOT SUMMER MONSOON WINTER 42.063 + 0.012 4.145 + 0.328 52.062 + 0.061 26.239 + 0.123 (37.61) 24.365 + 0.095 (44.80)* * 29.155 + 0.012 (43.99)* * * GONAD SUMMER MONSOON WINTER 38.802 + 0.920 29.969 + 1.041 32.015 + 1.134 31.091 + 1.024 (19.87)* 26.117 + 0.162 (12.85) 27.980 + 0.524 (12.60) Values in parenthesis are percent change, ± =S.D. of five animals. (*=p<0.05; **=p<0.01; ***=p<0.001). The studies on biochemical changes enable to define the dose response relationship, threshold limit value, revercible and irrevercible nature of pollutant effect. In the present study significant alterations in total glycogen, proteins and lipids were observed both in control and chronic group of clams. In summer, the glycogen content in the control group showed ascending order of gill< hepatopancreas< mantle< gonad< foot, with 6.872 + 0.071, 13.536 + 1.083, 14.785 + 0.089, 29.155 + 0.015, 38.802 + 0.920 mg/100 mg dry tissue respectively. In chronic group, glycogen content was present in increasing orderofgill< hepatopancreas< mantle< foot< gonad, with 6.036 + 0.052, 12.286 + 0.864, 19.367 + 0.229,and31.091+1.024mg/100mgdry tissue respectively. As compared to controls, there was 30.99% (p<0.001) increase in mantle, while 9.23 (P<0.05), 37.61(p<0.001) and 19.87% (P<0.05) significant decrease in hepatopancreas, foot and gonad respectively During monsoon,
  • 3. International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 @ IJTSRD | Unique Paper ID – IJTSRD30908 | Volume – 4 | Issue – 4 | May-June 2020 Page 196 control group showed glycogen content in ascending order of gill< hepatopancreas< mantle< gonad< foot, with4.045+0.106, 14.369 + 0.427, 15.410 + 0.025, 29.969 + 1.041, and 44.145 + 0.328 mg/100mg dry tissue respectively. In chronic group, glycogen content was low in gill, followed by hepatopancreas, mantle, foot and gonad, with 5.455 + 0.081, 12.078 + 0.080, 18.950 + 0.158, 24.365 + 0.095 and 26.117 + 0.162 mg/100mg dry tissue respectively. Based on percent change, there was 34.85 (p<0.001) and 22.97 % (p<0.001) increase in gill and mantle, while 15.94 (P<0.05), 20.47 (p<0.001), 44.80%(p<0.001) significant decrease in hepatopancreas and foot respectively. During winter, control group exhibited high glycogen contentin gill, followed by hepatopancreas, mantle, gonad, foot, with 8.121 + 0046, 14.216 + 0.035, 37.485 + 1.050 and 52.062 + 0.061 mg/ 100mg dry tissue respectively. In chronic group, glycogen content was present in ascending order of gill< hepatopancreas< mantle< gonad<foot, with 6.247 + 0.162, 17.662 + 0.0061,20.616+1.603,27.980+0.521and29.155+0.012 mg in 100mg dry tissue. As compared to control group, there was 24.24 and 30.19 % significant (p< 0.001) increase in hepatopancreas and mantle while 23.07 and 43.99 % significant (p<0.001) decrease in gill, and foot respectively Clams from control group showed less glycogen content in gills and high glycogen content in gonad and foot .Glycogen content was at its peak during gametogenesis and it was low during the spawning period.Similarobservationswerenoted byNagbhushnamand Talikhedkar (1977)17 in wedge clam, Danus cunaeutus. The result obtained are in good agreement with earlier workers (Masumoto and Hibino,197318 ;Dhavale and Masurekar,198519 ;George and Mathews,199620) Clams those were subjected to chronic sub lethal concentration showed increase in glycogen content in gill in monsoon, while hepatopancreas showed significant increase during winter. Hepatopancreas and gills are more modifiedintheir biochemical compositionthanmuscles and hence accumulate higher level of cadmium and to reduce toxicity they require more glycogen. Similar observation was noted by Diaz Mayans et al (1986) 21. In rest of soft tissues there was significant decrease in glycogen content. Decline in glycogen content was may be due to stress resulting in breakdownoftissueglycogentomeetenergydemandduetotoxicstress of cadmium chloride. Table 2: Cadmium chloride induced alterations in total protein content of M.meretrix after chronic exposure (Results are expressed in mg/100mg. dry wt.basis). TISSUE SEASON CONTROL CHRONIC GROUP GILL SUMMER MONSOON WINTER 10.620 + 1.013 12.206 + 0.077 10.376 + 0.520 11.427 + 0.907 (7.49) 16.524 + 0.012 (35.37)* * 16.600 + 0.039 (59.93)* * * MANTLE SUMMER MONSOON WINTER 20.400 + 1.047 21.025 + 0.093 23.552 + 0.034 17.942 + 0.213 (-12.04)* * * 20.102 + 1.054 (-4.39)* 20.763 + 0.615 (-11.84)* * HEPATOPANCREAS SUMMER MONSOON WINTER 6.526 + 1.022 7.102 + 0.316 7.230 + 1.069 7.902 + 0.091 (21.85) 10.022 + 0.097 (41.11)* * 7.500 + 0.016 (3.73) FOOT SUMMER MONSOON WINTER 25.059 + 0.085 27.500 + 0.104 27.560 + 0.458 21.709 + 0.007 (13.33) 22.406 + 0.025(18.52)* * 22.970 + 0.044 (16.65)* * * GONAD SUMMER MONSOON WINTER 27.521 + 0.015 23.746 + 0.705 24.854 + 1.802 21.164 + 0.033 (23.09)*** 16.047 + 1.091 (32.42)** 20.803 + 0.196(16.29) Values in parenthesis are percent change, ± =S.D. of five animals. (*=p<0.05; **=p<0.01; ***=p<0.001) Proteins play vital role in the spawning and other metabolic activities. Alteration in protein content after chronic exposure to cadmium chloride are shown in Table 2. During summer, control group showed protein content in different organs, in increasing order of hepatopancreas < gill< mantle< foot< gonad with 6.526 + 1.022, 10.620 + 1.013,20.400+1.047.8,25.059+ 0.085 and Chronic (0.18 ppm) group, exhibited low protein content in hepatopancreas, followed by gill, mantle, gonads, foot; with 7.902 + 0.091, 11.427 + 0.907, 17.942 + 0.051, 21.164 + 0.033 and 21.709 + 0.015 mg/ 100mg dry tissue in respective organs. As compared to control group, there was 12.04, 23.09 and 13.33%significant (p<0.001)decreaseinmantle,gonadand foot respectively .In monsoon, control group exhibited protein content in increasing order of, hepatopancreas< gill<mantle < foot < gonad, with 7.102 + 0.316, 12.206 + 0.077, 21.025 + 0.093, 23.746 + 0.705 and 38.640 + 0.070mg proteins in 100mgdry tissue respectively. Chronic (0.20 ppm) group, showed protein content in the ascending order of hepatopancreas < gill< mantle< gonad< foot, with 10.022 + 0.097, 16.047 + 1.091, 19.944 + 0.074, 20.102 + 1.054 and 22.406 + 0.025mg/100mgdry tissue in respective organs. As compared to control, there was 41.11 (p<0.01), 35.37% (p<0.001) increase in hepatopancreas and gill and 32.42 (p<0.01), 18.52% (p<0.001) increase in gonad and foot respectively. During winter,control group exhibited protein content in ascending order of hepatopancreas < gill< mantle< female gonad< foot, with 7.230 + 1.069, 10.376 + 0.520, 23.552 + 0.034, 24.854 + 1.802, 27.560 + 0.458mg proteins in 100mg dry tissue respectively. Chronic (0.21 ppm) ground, showed proteins in ascending order of hepatopancreass< gill< mantle< female gonad< foot, exhibiting7.500+0.016,16.600+ 0.039, 003, 20.763 + 0.615, 20.803 + 0.196, 22.970 + 0.044mg proteins in drytissue.Ascomparedtocontrol,there was59.98% significant (p<0.001) increase in gill, while 11.84 (p<0.01),and16.65%(p<0.01)decreaseinproteincontentinmantleandfoot respectively. Clams from control group showed marked variations which were attributed to season. During winter protein content was better in most of the soft tissues. It may be due to abundanceoffoodmaterial,increasedshell valveopeningperiod and less energy expenditure. Protein content was low in summer and monsoon. It may be due to utilization of proteins for spawning Mane,U.H et al (1987)22 observed that spawning period of K.opima and M. meretrix from Kalbadevi estuary was in months of monsoon and summer. Durve and Bal (1961) 23 analyzed Crossostrea gryphoides and reported that low protein
  • 4. International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 @ IJTSRD | Unique Paper ID – IJTSRD30908 | Volume – 4 | Issue – 4 | May-June 2020 Page 197 values coinsides with spawning season. Clams from chronic group showedsignificantdepletionduringthreedifferentseasons. Marked depletion was observed in gonad and foot, while gill and hepatopancreas showed considerable elevation in protein level. It may be due to mobilization of proteins from depositories and conversioninto glycogen.Gillsandhepatopancreaswere primary target organs for cadmium chloride stress. Thesetissuesandorgansmighthaveutilizedanddemandedmoreglycogen to reduce cadmium chloride stress. Table 3: Cadmium chloride induced alterations in total lipid content of M.meretrix after chronic exposure (Results are expressed in mg/100mg. dry wt. basis). TISSUE SEASON CONTROL CHRONIC GROUP GILL SUMMER MONSOON WINTER 5.500 + 1.008 6.102 + 0.065 6.795 + 0.092 4.330 + 0.203 (21.27) 5.702 + 0.041 (6.55)* * 5.967 + 0.396 (12.18)* * * MANTLE SUMMER MONSOON WINTER 5.187 + 0.086 4.715 + 1.013 4.870 + 0.318 3.124 + 0.008 (39.77)* * * 4.225 + 0.013 (10.39)* * * 4.020 + 0.015 (17.45)* * * HEPATOPANCREAS SUMMER MONSOON WINTER 5.127 + 0.205 6.080 + 0.516 4.970 + 1.061 4.925 + 0.142 (3.93)* 4.204 + 0.057 (30.85)* 3.551 + 0.016 (28.55)* * * FOOT SUMMER MONSOON WINTER 42.063 + 0.012 44.145 + 0.328 52.062 + 0.061 26.239 + 0.123 (37.61) 24.365 + 0.095 (44.80)* * 29.155 + 0.012 (43.99)* * * GONAD SUMMER MONSOON WINTER 38.802 + 0.920 29.969 + 1.041 32.015 + 1.134 31.091 + 1.024 (19.87)* 26.117 + 0.162 (12.85) 27.980 + 0.524 (12.60) Values in parenthesis are percent change, ± =S.D. of five animals. (*=p<0.05; **=p<0.01; ***=p<0.001). Alterations in lipid content after chronic exposuretocadmiumareshownin Table3.In summer,control groupofclamsshowed variations in lipid content in different organs in ascending order of hepatopancreas< mantle< gill< gonad< foot, with 5.127 + 0.205, 5.187 + 0.086, 5.500 + 1.008 and 9.946 + 0.509 mg/ 100mg dry tissue respectively. Chronic (1.18 ppm)groupexhibited lipid content with increasing trend of mantle, gill, hepatopancreas,gonad,foot,with3.124+0.008,4.330+0.203,4.925+0.142, 5.210 + 0.081, and 7.630 + 0.091 mg/ 100mg dry tissue respectively.Ascomparedtocontrol group,therewas39.77(p<0.001), 21.27, 3.93, 36.15 (p<0.001), and 23.28% (p<0.01) decrease inrespectiveorgans.Duringmonsoon,control groupshowedlipid content in ascending order of mantle, followed by hepatopancreas, gill, gonad, foot, with 4.715 + 1.013, 6.080 + 0.516, 6.102 + 0.065, 7.020 + 0.816, and 10.421 + 0.012 mg lipids in 100mgdryweight respectively.Chronicgroup(0.20ppm)exhibitedlipids in increasing order if hepatopancreas <mantle< gill< gonad< foot, with 4.204 + 0.057, 4.225 + 0.013, 5.702 + 0.041, 6.820 + 0.105, and 9.400 + 1.091 mg/100mg dry tissue respectively. As compared to control group,there wasconsiderable decreasein lipid content of all organs, but decrease was significant in gill only. During winter, control group, showed lipid content, in increasing order of mantle< hepatopancreas< gill< gonad < foot. The lipid content was 4.870 + 0.318, 4.970 + 1.061, 6.795 + 0.092, 8.205 + 0.032, and 12.072 + 0.076, mg in 100mg dry tissue respectively. In chronicgroup,(0.21ppm)thelipidcontentin different organs was present in ascending order of hepatopancreas< mantle< gill< gonad< foot, with 3.551 + 0.016, 4.020 + 0.015, 5.967 + 0.392, 7.010 + 1.030.As compared to control group, there was 12.18 (p<0.001), 16.85 (p<0.01), and 25.22% (p<0.001) decrease in lipid content in gill, gonad and foot respectively. In general, alterations in lipid content of chronicgroup showed considerable depletion in all soft tissues, but depletion was significant (p<0.001) in hepatopancreas and mantle. Hepatopancreas showed 30.85 and 28.55% decrease in monsoon and winter respectively. During summer, mantle showed (39.77%) considerable depletion than hepatopancreas. Ansari (1983)24 reported copper induced decrease in lipid content of Crossostrea punctatus .The decrease in lipid content of clams from chronic sub lethal exposure of cadmium chloride is in good agreement with observations of few workers.( Jana and Bandopadhya,198725; Builter and Wilson, 1998)26. Conclusion: The present study gives a detailed account of influence of cadmium chloride stress and seasonal water parameters on biochemical alterations in soft tissues like gill, mantle, hepatopancreas, gonad and foot of M.meretrix.Alterationsin glycogen, protein and lipids were attributed to various factors like environmental stress, physiological status of clams and concentration of cadmium chloride.Alterationsin glycogen content in clams from control groupwasattributed to seasonal water parameters and spawning season. In chronic group of clams, there was significant increase in glycogen content of gill and hepatopancreas, while in rest of organs there was marked decrease during summer, monsoon and winter. The significant increase in glycogen content in gill and hepatopancreas indicated that these soft tissues were prominent target organs for cadmium chloride toxicity. In control group, protein content was better during winter. It was attributed to favourable environmental parameters and abundance of food. Clams from chronic group showed significant elevation in gill and hepatopancreas. It may be due to mobilization or induction of new proteins to combat against cadmium stress. Lipid content was better during winter andgametogenesis period, Clams from chronic group showed significant decrease in almost all soft tissues during summer, monsoon and winter. Acknowledgement:TheauthorgratefullyacknowledgesDr. D.V Muley, Ret. Professor, Dept. of Zoology andEx.Registrar, Shivaji University Kolhapur for his consistent and valueable guidance.
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