2. 20 Furadan effects on fish glycogen metabolism
±2°C) previously washed with potassium permanganate. The fish were fed every
alternate day with boiled eggs and earthworms. A week before the commencement
of experiments, a suitable number of healthy fish were transferred from the stock
and maintained in the laboratory conditions (28 ±1°C) in small cement oisterns.
These fish were fed every day and water in the cisterns was renewed daily.
Feeding was stopped 2 days before the fish were used for experiments in order to
reduce the amount of excreted products in the test tanks.
Technical grade furadan (2, 3-dihydro-2, 2-dimethyl-7-bensofuranyl methyl
carbamate) manufactured by Rallis India Limited, Bangalore, was used. Stock
solution of pesticide was prepared in acetone. Two concentrations, viz., 0.56 mg/
litre (120 h LC0) and 1.56 mg/litre (24 h LC100) were chosen based on the acute
toxicity test (Bakthavathsalam, 1980). Test solutions were prepared from the stock
solution using filtered well-water with the following characteristics: temperature
28 ±1°C, pH 7.4–7.6, dissolved oxygen 7–10 ppm, salinity 0.2-0.6 ppm, alkalinity
240-260 mg/litre as CaCO3 and hardness 360-380 mg/litre as CaCO3.
About 15 fish were exposed to each selected concentration and at each exposure
in 200 litre rectangular fibre glass tanks (100×50× 40 cm). Acetone used in the test
solution never exceeded 0.25 ml/litre of water at any concentration. Parallel
controls were also maintained with a maximum aliquot of acetone (0.25 ml/litre) in
the test chambers. The test solutions were renewed every 24 h.
Six surviving fish from each tank were sacrificed by decapitation after 1, 3, 6, 12,
24, 48, 72, 96 and 120 h in the case of control and 0.56 mg/litre of furadan
treatment. In the case of 1.56 mg/litre of furadan treated lots, the fish were
sacrificed at 1, 3 and 6 h of exposure. Muscle and liver were isolated in the cold
and immediately frozen. Homogenates (5 and 10%) of liver and muscle respectively
were prepared in 10% trichloroacetic acid and centrifuged at 1308 g for 15 min
using Electrical Swing Head Covered Centrifuge and 1 ml of the supernatant was
used for glycogen and lactic acid estimations. The blood samples (0.2 ml) were
collected by caudal puncture using heparinized hypodermic syringe and were
immediately deproteinized with 10 ml of 10% tungstic acid. The protein-free
filtrates were used for glucose and lactic acid estimations.
Glycogen was estimated by the method of Carroll et al. (1956) using a Linear
Readout Grating Spectrophotometer (Cecil model CE 373). Lactic acid was
estimated, using Barker and Summerson's (1941) method. Blood glucose was
determined by the method of Folin and Malmros microprocedure as modified by
Murrel and Nace (1958). To minimise fluctuations due to diel rhythmicity (Datta
Munshi and Patra, 1978) the experiments were carried out between 10.00 a.m. and
12.00 noon each day.
Statistical significance of the difference between the control and experimental
values was calculated by Student’s ‘t’ test. Data were also subjected to one way
analysis of variance (F test) as described by Steel and Torrie (1960).
Results
The mean content of muscle and liver glycogen (table 1), muscle and liver lactic
acid (table 2) and blood lactic acid and blood glucose (table 3) for all the 9 periods
3. Bakthavathsalam and Reddy
of control exposure were not significant (P>0.05) when subjected to analysis of
variance (F test).
Table 1. Glycogen content in muscle and liver of Anabas testudineus treated with furadan.
a
Values expressed as mg/g wet wt of tissue.
Mean ±S.D. of 6 individual observations;
b
=P>0.05; b=P<0.05; Others P<0.01; * PF >0.05; ** PF<0.05. – Not determined.
Table 2. Lactic acid content of muscle and liver in Anabas testudineus treated with furadan.
a
Values expressed as mg/g wet wt. of tissue.
Mean ±S.D. of 6 individual observations;
b
=P 0.05; c
=P<0.05; Others P<0.01; * PF>0.05; ** PF<0.05. – Not determined.
21
4. 22 Furadan effects on fish glycogen metabolism
Table 3. Lactic acid and glucose contents of blood in Anabas testudineus treated with
furadan.
a
mg/ml of blood
Mean ±S.D. of 6 individual observations;
b
=P 0.05; c
=P>0.05; Other P<0.01; * PF>0.05; ** P
F
<0.05. – Not determined
In 0.56 mg/litre furadan exposure, a significant decrease (P<0.01) at 1 h
(followed by a recovery during 3 and 6 h was observed in the muscle glycogen
content of fish (table 1). From 12 h onwards, marked decrease was noted. But a
significant (P<0.01) increase was noted in the muscle glycogen at 120 h of
exposure.
After 1 h exposure, the liver glycogen content showed 50% decrease, which was
further reduced to 85% at 3 h. A sudden increase (P<0.01) was noticed in the
glycogen level at 6 h which followed an acute drop until 72 h of exposure. The
glycogen content attained normal level during 96 h and it was maintained at 120 h
also (table 1).
Blood glucose showed a marked increase at 6 h and 120 h (table 3) which
coincided with periods of highest glycogen content in liver and muscle. Lactic
acid content of muscle (table 2) showed a greater depletion at later periods of
exposure from 24 to 120 h when the liver glycogen content was at low levels. Liver
lactic acid (table 2) content followed the changes in the liver glycogen content in
the initial periods of exposure. For example during 1 h, the increase was 31.3% but
in 3 h a decrease of 25.1 % was noticed, when liver glycogen also showed a remark-
able decrease. At 6 and 12 h of exposure, lactic acid content of liver showed an
increase in its levels at all periods of exposure but the increase observed at 12, 96,
72 and 16 h are noteworthy (table 3).
Anabas testudineus exposed to 1.56 mg/litre furadan showed a marked decrease
in muscle glycogen and lactic acid contents at all periods. Liver glycogen and
lactic acid and blood glucose and lactic acid, on the other hand, showed an upward
shift in their levels at all the three periods of exposure (tables 1, 2 and 3).
5. 23 Bakthavathsalam and Ready
Further, the changes observed in the mean content of various metabolites at both
the concentrations were statistically significant (P<0.05) except muscle glycogen
contents of fish exposed to 1.56 mg/litre furadan, when analysis of variance was
applied on them.
Discussion
Glycogen content of muscle and liver decreased to a greater extent viz., 69.0 and
50.1% respectively (table 1) at 1 h with 0.56 mg/litre of furadan exposure compared
to similar exposure to disyton, (O, O-diethyl S-[2-(ethylthio)-ethyl] phosphoro
dithioate) (Bakthavathsalam, 1980). Concomitant with this active glycolytic
metabolism, the muscle, liver, and blood lactic acid levels were high in furadan-
treated fish. Liver glycogen levels were further decreased at 3 h whereas muscle
glycogen levels were slightly improved. The fall in muscle and liver lactic acid
levels suggested that glycogenesis should have prevented the otherwise signi-
ficant reduction of liver glycogen. But at 6 h, the presumable depletion of liver
glycogen was averted, the glycogen levels being considerably improved showing an
increase of 158.4% over control. Blood glucose levels were highest at 6 h
emphasizing an adverse effect on glycogen metabolism. Liver lactic acid levels
were also high (table 2) and since muscle and liver lactic acid levels at 3 h could not
quantitatively account for this apparent rise, it could be safely assumed that either
lactic acid formed in other tissues was transported to liver or it was a product of
liver metabolism itself or a combination of both was responsible for high lactic acid
levels in the liver at 6 h.
Both muscle and liver glycogen levels were low at 12 h with an apparent increase
in lactic acid content of blood and muscle. Blood glucose and liver lactic acid also
decreased to 100.3 and 60.1% respectively. All these changes evidently suggest
extensive utilisation of glycogen leading to a serious reduction in the glycogen
content of liver and muscle, lactic acid content of muscle, liver and blood, and
blood glucose at 24 h when compared to their levels in 12 h of exposure. The
insignificant change (P>0.05) in the muscle and liver glycogen levels at 48 h over 24
h values may not mean that the glycogen metabolism may have reached a steady
state. The rate of utilization probably equalled the rate of supplementation.
Increase in blood glucose and blood lactic acid and a fall in muscle and liver lactic
acid all contribute to an active flux of metabolites. In the subsequent periods of
exposure, the glycogen stores were slowly repleted, the improvement first starting
in the muscle as reflected by the change in the mean glycogen content from 0.41 at
48 h to 0.72 mg/g at 72 h.
But at 72 h, though muscle lactic acid did not changes much, high blood lactic
acid content indicated transport of large amounts of lactic acid formed in muscle
and/or other tissues to liver. This event probably led to a decrease in muscle
glycogen and rise in liver glycogen at 96 h. Blood lactic acid continued to be high
at 96 h which perhaps helped to maintain liver glycogen and increase in muscle
glycogen at 120 h in the face of sustained high transport of glucose at 96 h.
Increase in blood glucose at 96 h and 120 h probably was the reason for the upward
shift in the muscle glycogen levels observed at 120 h. The elevation observed in
glycogen content of fish after 120 h of exposure may be an indication for successful
6. 24 Furadan effects on fish glycogen metabolism
adaptation to furadan toxicity or may be a response to an abnormal increase in
succinate dehydrogenase activity levels observed in various tissues of fish at this
period (Bakthavathsalam, 1980).
In contrast to 0.56 mg/litre furadan exposure, 1.56 mg/litre exposure was
characterized by significant changes in muscle and liver glycogen levels in singular
but opposite directions. The decrease in muscle glycogen might be due to the rate
of utilization gaining upperhand over the rate of supplementation. If the blood
lactic acid and blood glucose were taken to reflect the former and the latter,
respectively, the difference in the per cent increases observed in the two, makes
this point clear (table 3). The increase observed in liver glycogen and blood
glucose may be due to the inhibition of glycogenosis or due to the initiation of
gluconeogenesis and/or glycogenesis. These results are in agreement with the
findings of Grant and Mehrle (1973) that glycogenosis was inhibited by the high
endrin concentration, or that gluconeogenesis and glycogenesis were greatly
increased, resulting in high liver glycogen content. Further high blood glucose
and liver glycogen in 1.56 mg/litre treated group suggests an impairment in the
carbohydrate metabolism.
Acknowledgements
The authors thank Profs. P. Govindan and Mrs. P. Shankarmurthy for providing
facilities. One of the authors (R.B.) thanks the University and the University
Grants Commission for providing financial assistance.
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