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Impact of Distillery Effluent on Carbohydrate Metabolism of Freshwater Fish, Cyprinus carpio
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Abstract.
The impact of distillery effluent on carbohydrate metabolism of Cyprinus carpio was studied at
different days during exposure (7, 14, and 21 days) in the ambient temperature of 28±1 C. Oxygen
consumption in fish decreased with increasing effluent concentrations as well as duration of exposure.
Effluent concentrations and exposure durations (days) had a significant effect on oxygen consumption of
tested fish (p<0.0005). Total carbohydrate, glycogen content and SDH enzyme activity in muscle, liver and
brain tissues of C. carpio exposed to different sublethal concentrations decreased gradually and significantly.
This was also the case with exposure duration. Reduction in glycogen content was greater in liver
tissue i.e., 54.1% in 0.2% effluent concentration on the 21st day of exposure. However, serum glucose and
lactic acid content showed an increasing trend with increase in effluent concentration and time of exposure.
Unlike SDH, LDH enzyme activity of muscle, liver and brain tissues showed an increasing trend and the
enhancement of enzyme activity was more in liver tissue (71.3%). From these results, it could be inferred
that respiratory processes in C. carpio under distillery effluent stress was affected resulting in a shift towards
anaerobiosis at organ level during sublethal intoxication.
Keywords: carbohydrate metabolism; Cyprinus carpio; distillery effluent; oxygen consumption; glucose;
glycogen; LDH; SDH; lactic acid
Introduction
Industrial effluents in developing countries are
indiscriminately discharged into aquatic ecosystems
and even into adjoining fields without any
pretreatment. Industrial effluents contain a variety
of pollutants such as heavy metals, pesticides,
fertilizers, detergents, organic and inorganic salts,
oil, etc. which create serious problems to the nontarget
organisms. They inhabit aquatic water
ecosystems especially fish (Motwani et al., 1956;
Ayyadurai and Krishnamurthy, 1989; Ramakrishnan
et al., 1999). Changes in biochemical
constituents of tissues and enzyme activity are
important in order to determine the nature and
extent of toxicant effects on organisms (Murthy,
1985; Heath, 1987). Reports on the effects of
industrial effluent on carbohydrate metabolism of
fishes are available (McLeay and Brown, 1974,
1979; Stonner and Livingston, 1978; Shaffi, 1980,
1981; Oikari and Nakari, 1985).
*To whom correspondence should be addressed:
Tel./Fax: +91-452-2459080;
E-mail: cmramakritinan[at]yahoo.co.in
Ecotoxicology, 14, 693–707, 2005
2005 Springer Science+Business Media, Inc. Printed in The U.S.A.
DOI: 10.1007/s10646-005-0019-3
Alcohol is one of the raw materials for various
chemical industries. At present, India has more
than 285 distilleries with an annual production of
2.7 billion litres of alcohol (Joshi, 1999). The raw
material of distillery (sugar mill) industry is
molasses, a by-product obtained from sugar mill
industry, which contains 50% of sugar. For each
litre of alcohol produced, a quantity of 20–50 l of
‘spent wash’ is discharged. Approximately 40 billion
litres of effluent is discharged to land and
aquatic systems without any pretreatment. This
can cause considerable stress on the waterways
leading to extensive damage to aquatic life (Joshi,
1999). Wastewater from distillery industry poses a
serious threat to the quality of receiving water
bodies in several regions of the country. The spent
wash is characterized by its colour, high temperature,
low pH, high ash, and high percentage of
dissolved organic and inorganic matter of which
50% may be present in reducing sugars. The spent
wash discharged from Indian distillery industry
contains very high amounts of potassium, calcium,
chloride, sulphate and BOD (around 50,000 mg/l).
The major toxic constituents in distillery effluent
are high volume of dissolved solids, chlorides,
sulphate and low volume of highly toxic sulphides
(Ellis, 1937; Arockiasamy, 1982; Joshi, 1999). In
addition to these, the low pH value, more organic
load and high BOD are some of the other major
pollution problems due to distillery industrial
wastewater. The high BOD causes depletion of
dissolved oxygen and proves harmful to aquatic
life. However, studies dealing with the effects of
distillery (sugar mill) effluent in freshwater fishes
are meager (Saffi, 1980, 1981). The present study
reports the impacts of distillery (sugar mill) effluent
on carbohydrate metabolism of freshwater
fish, Cyprinus carpio.
Materials and methods
Juvenile C. carpio (1.5±0.20 g) were collected
from Aliyar reservoir, Aliyar, Tamilnadu Fisheries
Development Corporation Ltd., Pollachi, Tamilnadu,
India. The collected fish were transported
to the laboratory in an oxygen pack. They were
acclimated for a period of 7 days at 28±1 C in a
FRP aquarium tank (90¢¢ 45¢¢ 45¢¢). During
acclimation, fish fed on ad libitum on chopped goat
liver pieces. The undiluted distillery effluent was
collected from the discharge point of the factory
(Trichy Distilleries, Trichy, Tamilnadu, India) and
was considered to be 100% concentrations. The
physico-chemical nature of raw distillery effluent
was presented in Table 1 (APHA et al., 1975).
Table 1. Physicochemical characteristics of freshwater and undiluted distillery effluent
Environmental variables Units Freshwater Raw distillery effluent
Temperature (C) 28.0±1.00 90–95
PH 7.4±0.20 3.6–5.0
Oxygen (mg l)1) 8.1±0.30 Nil
COD (mg l)1) ND 20,000–65,000
BOD (mg l)1) ND 15,000–30,000
Total solids (mg l)1) 2.4±0.16 35,000–80,000
Suspended solids (mg l)1) ND 2000–3000
Volatile solids (mg l)1) ND 10,000–50,000
Total hardness (mg l)1) 2.2±0.10 ND
Free carbon dioxide (mg l)1) 1.2±0.20 ND
Organic nitrogen (mg l)1) 0.3±0.03 ND
Total nitrogen (%) ND 0.100–1.900
Total phosphorus (%) ND 0.038–1.100
Potassium as K2O (mg l)1) ND 1.200–1.800
Sulphate as SO4 (mg l)1) ND 4500–6000
Ferrous (mg l)1) ND 300–400
Sulphide (mg l)1) ND 200–300
Calcium (mg l)1) 0.2±0.02 (as Ca++) 200–350 (as Ca++)
Chloride (mg l)1) 0.8±0.31 (as Cl)) 600–700 (as Cl))
Salinity (ppt) 0.9±0.04 ND
ND: not determined. Each value is the mean of 10 results ±SE.
694 Ramakritinan et al.
From this, the selected effluent concentrations for
the experiments were obtained by diluting it with
clean non-chlorinated tap water (Table 1).
Acute toxicity test was conducted the method
described by Sprauge (1973). The range finding
test was conducted before the definitive toxicity
test. The definitive toxicity test was carried out for
a period of 96 h. Daily renewal of test solutions
was done through out the study. The mortality
was observed during 24, 48, 72 and 96 h and the
LC50 values calculated using Probit analysis were
1.2%, 1.1%, 1.0% and 0.8%, respectively. The
95% Confidence Interval end points were 1.295–
1.2749%, 1.0489–1.1536%, 0.9538–1.0484% and
0.7126–0.8981% for 24, 48, 72 and 96 h exposure,
respectively (Ramakrishnan, 1991). The 100%
survival of C. carpio when exposed to distillery
effluent has been found as 0.2% during 96 h exposure
(Ramakrishnan et al., 1999). Hence, C. carpio
was exposed to the selected sublethal effluent concentrations
i.e., 0.05%, 0.10%, 0.15% and 0.20%.
For the oxygen consumption study, C. caprio
was exposed at selected sublethal effluent concentrations
i.e., 0.05%, 0.01%, 0.15% and 0.20% and
effluent-free control medium. For each concentration
as well as control medium, five replicates
(five fish in each replicate) were maintained. Fish
was exposed in a glass cylindrical aquarium
(30 30 cm) containing 6 l of test medium for a
total period of 21 days. Fish was offered ad libitum
diet of goat liver pieces once a day for 2 h (10 AM
to 12 Noon). The rate of oxygen consumption was
estimated at 7, 14 and 21 days of exposure. Estimation
of oxygen consumption was made exposing
the fish in a simple respirometer. Fish, in each
concentration, was transferred to the respirometer
(15 10 cm) containing 3 l of test medium and
immediately the upper layer was covered with a
thin film of liquid paraffin which prevent the diffusion
of atmospheric air into the test medium.
The concentration of dissolved oxygen in the
medium was estimated by the Winkler’s volumetric
method (Welch, 1948) and the results were expressed
as ml oxygen consumed per g live fish per
hour (ml O2 g)1 h)1).
For the biochemical analysis, fish as a group
(25) was exposed in each concentration in a glass
aquarium (60 60 cm) containing 40 l of test
medium at ambient room temperature (28±1 C).
The medium was not aerated through out the
study. Fish was exposed in freshwater control also
for comparison at the same temperature. The five
replicates were maintained in each test concentration
and also freshwater control. During the
exposure, fish was offered an ad libitum diet of
goat liver pieces once a day for 2 h (10 AM to 12
Noon). The food remains were removed immediately
after the feeding. In all the experiments, the
medium was changed daily with freshly prepared
one to give a constant effect of distillery effluent on
fish. The exposures were conducted for a total
period of 21 days. Effluent treated and control fish
from each concentration were sacrificed at 7, 14,
and 21 days and muscle, liver, and brain tissues
were dissected out and stored at )4 C. Blood was
obtained by terminal puncture of restrained living
fish using 1 ml micro syringe. It was permitted to
clot ()4 C, 45 min), and centrifuged at 5000 g for
10 min. Collected serum was pooled and stored at
)27 C until used.
Total carbohydrate content was estimated by
the method of Roe (1961); glycogen by (Carroll
et al., 1956); LDH and SDH activity by (Nachlas
et al., 1960a, b). Serum glucose (Nelson–Somagy
method) and lactic acid (Barker and Summerson
Method) were estimated as described by Oser
(1965). Data were analyzed using Student’s t-test
(Bailey, 1984) and Two Way Anova (Zar, 2004).
Results
Oxygen consumption
In the present study, when C. carpio was exposed
to different sublethal concentrations of distillery
effluent, it was found that oxygen consumption
decreased from 1.399 ml O2 g)1 h)1 in effluentfree
control medium to 0.825 ml O2 g)1 h)1 when
reared in 0.2% effluent concentration at 7th day of
exposure, from 1.41 to 0.60 ml O2 g)1 h)1 at 14th
day of exposure and 1.39–0.46 ml O2 g)1 h)1
(Fig. 1). The rate of reduction in oxygen consumption
was gradual and significant. Maximal
reduction was observed by the 21st day of exposure
()67.0%), which was highly significant.
Two-way ANOVA indicated that the effluent
concentrations and exposure periods (days) have a
significant effect on oxygen consumption of
C. carpio (Table 2).
Impact of Distillery Effluent on Carbohydrate Metabolism 695
Total carbohydrate and glycogen contents of
muscle, liver and brain tissues of C. carpio decreased
with increasing concentration of distillery
effluent as well as with increasing exposure duration
(days) (Figs. 2 and 3). Total carbohydrate
content of muscle, liver and brain tissues in C.
carpio was significantly reduced to 6.36, 57.15 and
3.36 mg 100 mg)1 at 0.05% effluent concentration
on 7th day of exposure from its respective controls
i.e., from 6.55, 59.1 and 3.6 mg 100 mg)1 (Fig. 2).
This was further reduced to 5.42, 54.43 and
3.20 mg 100 mg)1 at 0.2% effluent concentration
on 7th day of exposure and thereafter showing
declining trend (Fig. 2). After 21 days of exposure,
total carbohydrate content of muscle, liver and
brain tissues were 6.57, 58.6 and 3.71 mg
100 mg)1, respectively, when reared in effluent-free
control medium. In 0.05% effluent concentration,
this level was decreased to 4.7, 46.7 and 3.0 mg
100 mg)1 in respective tissues at 21st day of
exposure. This was gradually and significantly
decreased to 4.0, 36.9 and 2.4 mg 100 mg)1 in the
maximum sublethal effluent concentration at 21st
days of exposure (Fig. 2). This result indicated
that muscle total carbohydrate decreased more
()39.9%) than that of liver ()36.9%) and brain
()34.5%) tissues (p<0.0005). Two-way ANOVA
indicated that the highest effluent concentration as
well as longer exposure period has a significant
effect on total carbohydrate content of tested tissues
(Table 3).
Glycogen content of tested fish in distillery
effluent medium also followed the same trend as
seen for total carbohydrate. For all the three tissues
tested, reduction in glycogen content of
muscle was 5.7%, 24.3% and 36.9% in the lowest
sublethal effluent concentration at 7, 14 and 21st
day of exposure, respectively. This was significantly
reduced to 23.2%, 39.3% and 47.1% on 7,
14 and 21st day of exposure, respectively when
exposed in the maximum sublethal effluent concentration
(Fig. 3). Reduction in liver content was
27.1%, 41.5% and 54.1% and reduction in brain
content was 17.6%, 32.5% and 43.1% at 21st day
of exposure (Fig. 3). The reduction in content was
highly significant in all the three tested tissues
(p<0.0005). It revealed that the highest inhibition
in glycogen content was found in liver tissues
of C. carpio reared at 0.2% effluent
concentration on 21st day of exposure, followed
Figure 1. Sublethal effects of distillery effluent on oxygen consumption (ml O2 g)1 h)1) in C. carpio to different sublethal concentrations.
Table 2. Summary of two-way analysis of variance of data on
oxygen consumption of C. carpio through Fig. 1
Source of variation S.S. D.F. M.S.
F-value
calculated
Between effluent
concentration
1.0391 4 0.2598 433.000*
Between days 0.0918 2 0.0464 77.333*
Residual (error) 0.2514 8 0.0006 –
*p<0.0005 significant.
696 Ramakritinan et al.
by muscle (47.1%) and brain (34.5%). Distillery
effluent has significant effects on glycogen content
of tested tissues (p<0.001). However, in fish
exposed to a minimum sublethal concentration
through the 7th day of exposure, the reduction
in total carbohydrate content of brain tissue and
Figure 2. Levels of total carbohydrate content in organs/tissues of C. carpio exposed to different sublethal concentrations of distillery
effluent.
Impact of Distillery Effluent on Carbohydrate Metabolism 697
glycogen content of liver was insignificant. The
present results indicated that the lowest effluent
concentration has less impact on tested tissues
and marked effect was found to be observed at
higher concentration as well as increasing exposure
duration. Two-way ANOVA indicated
that the distillery effluent concentrations and
exposure periods have significant effects on glycogen
content in muscle, liver and brain tissues
of C. carpio (Table 3).
However, serum glucose and lactic acid were
elevated with increasing sublethal concentration as
well as exposure duration (Fig. 4a, b). In control
samples, serum glucose content of C. carpio was
63.5, 62.9 and 63.4 mg 100 ml)1 on the 7th, 14th
and 21st day of exposure respectively, and it increased
to 72.5, 81.5 and 93.7 mg 100 ml)1 at
0.05% effluent concentration on the 7th, 14th and
21st day of exposure (p<0.0005)(Fig. 4a). This
contents were significantly elevated to 103.5, 114.3
and 138.3 mg 100 ml)1 in the highest sublethal
concentration of distillery effluent (0.2%) at
corresponding exposure periods (p<0.0005). In
the maximum sublethal effluent concentration,
glucose content was raised to 63.0%, 81.7% and
118.5% on – 7th, 14th and 21st day of exposure.
The above result revealed that fish reared on
chronic exposure to distillery effluent medium, the
rate of increase in serum glucose content was
gradual and significant with increasing concentration
of distillery effluent concentrations as
well as periods of exposure. The analysis of twoway
ANOVA indicated that glucose content was
significantly influenced by the effluent concentration
and longer exposure period (Table 4).
On rearing C. carpio in effluent-free medium, it
was found that serum lactic acid content was significantly
increased to 64.1, 80.3 and 95.4 mg
100 ml)1 (p<0.0005) to a maximum sublethal
concentration of distillery effluent (0.2%) during
the same exposure periods, respectively (Fig. 4b).
Increased serum lactic acid content was manifold
Table 3. Summary of two-way analysis of data on total carbohydrate and glycogen contents of different tissues of C. carpio
through Figs. 2 and 3
Total carbohydrate Glycogen
Source of variation S.S. D.F. M.S. F-value S.S. D.F. M.S. F-value
Muscle
Between effluent concentration 13.881 4 3.470 20.533* 1.069 4 0.267 12.136*
Between days 5.022 2 2.511 14.857* 0.635 2 0.318 14.455*
Residual (error) 1.352 8 0.169 – 0.176 8 0.022 –
Liver
Between effluent concentration 373.847 4 93.462 6.704* 570.011 4 142.503 17.724*
Between days 887.625 2 193.813 13.902* 286.467 2 143.234 17.815*
Residual (error) 111.527 8 13.941 – 64.319 8 8.040 –
Brain
Between effluent concentration 1.290 4 0.323 8.958* 0.663 4 0.166 9.765*
Between days 0.753 2 0.377 10.458* 0.557 2 0.279 16.412*
Residual (error) 0.289 8 0.036 – 0.138 8 0.017 –
*p<0.05 significant; *p<0.005 significant; *p <0.0005 significant.
Table 4. Summary of two-way analysis of variance of data on serum glucose, and lactic acid levels of C. carpio through Fig. 4a, b
Source of variation S.S. D.F. M.S. F-value calculated S.S. D.F. M.S. F-value calculated
Serum glucose Serum lactic acid
Between effluent concentration 5299.727 4 1324.932 2.114* 8670.524 4 2167.631 6.526*
Between days 1253.360 2 626.680 12.689* 664.321 2 332.161 10.704*
Residual (error) 395.109 8 49.389 – 248.252 8 31.032 –
*p>0.05 insignificant; *p <0.05 significant.
698 Ramakritinan et al.
i.e. at 0.2% effluent concentration; it was increased
to 722.4% above the control. Maximum sublethal
concentration(s) of distillery effluent as well as
longer exposure duration had a significant effect
on serum lactic acid content of tested fish. Twoway
ANOVA indicated that serum lactic acid was
significantly influenced by the effluent concentration
and longer exposure period (Table 4).
Figure 3. Levels of glycogen content in organs/tissues of C. carpio exposed to different sublethal concentrations of distillery effluent.
Impact of Distillery Effluent on Carbohydrate Metabolism 699
Like serum glucose and lactic acid, LDH enzyme
activity in all three tested tissues, under distillery
effluent stress, was significantly increased
(p<0.0005). For the highest sublethal concentration
of distillery effluent i.e., 0.2%, LDH activity
of muscle was elevated to 0.266, 0.297, and 0.350 l
mole formozan formed mg protein)1 h)1 at the
end of 7, 14 and 21st day of exposure, respectively.
In liver, it increased to 0.649, 0.745, and 0.856 l
mole formozan formed mg protein)1 h)1 at the
respective exposure periods (Fig. 5). In brain tissue,
it increased to 0.948, 1.063, and 1.164 l mole
formozan formed mg protein)1 h)1, respectively
(Fig. 5). The percent increase in LDH activity was
higher in liver (71.3%) than that of muscle
(66.7%) and brain (47.5%) tissues. The summary
of two-way ANOVA indicated that the maximum
sublethal concentration of distillery effluent as well
as longer exposure period have a significant effect
on LDH activity in muscle, liver, and brain tissues
of C. carpio (Table 5).
Figure 4. (a) Sublethal effects of distillery effluent on serum glucose content (mg 100 mg wet tissue)1) in C. carpio to different sublethal
concentrations. (b) Sublethal effects of distillery effluent on serum lactic acid content (mg 100 mg wet tissue)1) in C. carpio
to different sublethal concentrations.
700 Ramakritinan et al.
SDH activity of tested tissues was inhibited as a
function of effluent concentrations as well as
exposure duration (days) (Fig. 6). SDH activity in
muscle was reduced by 15.7%, 36.0%, and 52.2%
when reared in 0.2% distillery effluent concentration
at the end of 7, 14 and 21st day of exposure.
In liver, it was reduced by 27.4%, 51.2%, and
62.4% at the corresponding exposure periods and
in brain was by 19.5%, 37.5%, and 50.6% at the
end of same exposure period, respectively (Fig. 6).
This reduction was more significant (p<0.0005). It
indicated that in C. carpio exposed to 0.2% distillery
effluent, SDH activity in liver was highly
inhibited by the 21st day of exposure. The summary
of two-way ANOVA indicated that there
was a significant effect on SDH activity of muscle,
liver and brain tissues of C. carpio exposed in the
sublethal concentration of distillery effluent and
also exposure periods (Table 5).
In the present study, except serum glucose,
serum lactic acid and LDH enzyme activity, all
other biochemical constituents and SDH enzyme
activity of C. carpio decreased with increasing
concentration of distillery effluent as well as with
increasing exposure duration (days). The analysis
of variance (two-way) applied indicated that except
serum glucose, there was a significant effect
on oxygen consumption, total carbohydrate,
glycogen, SDH and LDH enzyme activity of tested
tissues, and serum lactic acid of C. carpio exposed
to sublethal distillery effluent as well as exposure
duration (days).