Global Journal of Environmental Research 2 (3): 126-132, 2008

ISSN 1990-925X
© IDOSI Publications, 2008

Effect of Temperature on Mortality and Behavioural Responses in

Freshwater Catfish, Heteropneustes fossilis (Bloch) Exposed to Dimethoate

Rakesh Kumar Pandey, Ram Nayan Singh and Vijai Krishna Das

Department of Zoology, Kamla Nehru Institute of Physical and Social Sciences,

Sultanpur, U.P.- 228118, India

Abstract: Temperature has significant influence on physio-chemical responses of all living organisms including
fish. The present study was conducted to evaluate the effect of temperature on dimethoate toxicity (96 h static
bioassay) to freshwater catfish Heteropneustes fossilis. Fish mortality data were recorded at 24, 48, 72 and
96 h and analysed using EPA Probit analysis, Version 1.5, software based on Finney's Probit Analysis method
to calculate LC50 values. The 96 h LC50 values at temperatures, 17.16±0.78°C (January) and 27.50±1.50°C
(August) were found to be 14.39 mg lG1 and 2.98 mg lG1 respectively. Alterations in behavioural responses,
especially the schooling, surfacing, mucus secretion, skin colour, eyelid condition and opercular movement
were also studied. The increase in fish mortality at higher temperature indicated in the present study may be
helpful to determine season specific toxicant concentration for field applications to avoid adverse effect on non
target organisms.

Key words: Organophosphate % Dimethoate % LC50. Behaviour % OCM. Heteropneustes fossilis

INTRODUCTION brain and peripheral nervous system [3, 4]. Dimethoate

Pesticides of various categories viz.,
organophosphates, organochlorines, pyrethroids and
carbamates are used against a number of pests, to
increase the crop production. Besides their use against
agricultural pests, the pesticides are generously used to
control the population of mosquitoes, houseflies, termites,
other household and stored grain pests. The short-
sighted approach to eradicate harmful insects has led to
indiscriminate use of pesticides, unmindful of their
consequences to the environment. The remains of
pesticides either enter into the atmosphere as aerosol
or into the aquatic system through runoff water,
inadvertently exposing the non-target organisms and
finally finding their way to the food chain threatening the
ecological balance and the biodiversity of the nature.
Aquatic contamination of pesticides causes acute and
chronic poisoning of fish and cause severe damage to
vital organs [1, 2].
The toxicity of OP is due to their ability to inhibit
cholinesterase enzyme which results in the accumulation
of acetylcholine in nerve tissues and the effecter
organs with the principal site of action being the
(CAS 60-51-5, O,O-dimethyl s- methyl- carbamoylmethyl
phosphorodithioate), a wide spectrum systemic and
contact insecticide is used against a broad range of
insects and mites. USNTP has classified it as moderately
to highly toxic chemical pesticide and PAN Bad
Actors considered it a chemical of special concern
(http://www.pesticideinfo.org/Docs/ref_toxicity7.html#
BadActors). USEPA has released an Interim Re-
registration Eligibility Decision (IRED) in June 2006 and
concluded that the FQPA (Food Quality Protection
Act-1996) safety factor for dimethoate should be removed
(http://www.epa.gov/oppsrrd1/REDs/dimethoate_ired.p
df). In rats and human, dimethoate is rapidly eliminated
trough urinary route; 76-90% is reported to appear in urine
within 24 h [5]. Dimethoate is hydrolysed by enzymes
esterases and amidases found in mammals. Insects,
deficient in these enzymes, however, can not hydrolyze
dimethoate and become susceptible to it [6].
In present scenario of global warming, the increasing
atmospheric temperature is matter of great concern for
the environmentalists to obtain relevant knowledge on
tolerance of organisms to the temperature. The sensitivity
of fishes exposed to the toxicants depends on biological

Corresponding Author: Rakesh Kumar Pandey, Department of Zoology, Kamla Nehru Institute of Physical and Social Sciences,
Sultanpur, U.P.- 228118, India
126
 Global J. Environ. Res., 2 (3): 126-132, 2008
factors (age, sex and size) as well as physico-chemical
factors viz., temperature, pH and hardness of test
water [7-9]. This study was conducted to determine
the toxicity level of dimethoate at two different
temperatures, 17.16±0.78°C (January) and 27.50±1.50°C
(August), on the fresh water catfish Heteropneustes
fossilis. Alterations in behavioural activities are important
indicator of stress in fishes exposed to toxicants,
therefore, changes in behaviour was studied and
compared at these temperatures.
MATERIALS AND METHODS
The fish, Heteropneustes fossilis (av. length
17.40±1.14 cm and av. weight 27.14±1.95 g) of both sexes
were collected from local ponds and carefully brought to
the laboratory avoiding any injury to the fish during
transport. The fish were disinfected with 0.05% solution
of KmnO4 for two minutes and then transferred to a
500 l capacity plastic tank to acclimatize for 14 days in
laboratory conditions. They were regularly fed with a
mixture of wheat flour, mustard cake, Soya and dried
prawn powder in ratio of 3:1:1:1 [10]. Fish were given food
in the evening. The leftover was thoroughly cleaned and
water of the tank was changed in the subsequent
morning.
The static bioassay test was conducted under natural
photoperiod for 96 h in the months of January and
August at water temperatures 17.16±0.78°C and
27.50±1.50°C respectively. Water quality characteristics
for both the months are given in Table 1. The experiment
was conducted in glass trough of 15 l capacity. Technical
grade dimethoate (30% EC) popularly known as Rogor,
was procured from Rallis India Ltd., Mumbai (India). The
stock solution was prepared by diluting the dimethoate in
absolute alcohol. Solutions for dose were prepared from
the stock by further dilution with alcohol to get dosing
concentrations. The range finding tests showed no
mortality at concentration 2.50 mg lG1, 70% mortality at
3.25 mg lG1 and 100% mortality at 4.00 mg lG1 in August.
The range finding results in January were as follows;
no mortality at 12 mg lG1, 70% at 16 mg lG1 and 100%
mortality at 19 mg lG1. All determined concentrations were
repeated four times. A control was simultaneously run
Table 1: Physico-chemical parameters of test water
Month Water temp. (°C) pH
January 17.16±0.78 7.39±0.23
August 27.50±1.50 7.30±0.17
with similar number of fish. The experiment was
conducted for 96 h and the fish mortality at each
concentration was recorded at an interval of 24, 48, 72 and
96 h. Fish were not given any food during the test [11].
Dead fish were immediately removed from the trough.
During experiment, temperature, pH and dissolved oxygen
(DO) were routinely measured. Behavioural parameters
such as schooling behaviour, excitability and reflex
responses, swimming pattern, surfacing and gulping air,
mucus secretion, changes in body colour, eyelids
condition, fins and barbels position (Table 3) and also the
opercular movement per minute (OCM) were constantly
monitored and recorded for 96 h (Fig. 2). The change in
OCM with respect to dimethoate concentration and
exposure time was analyzed using two-way ANOVA,
for the statistical significance (Table 4). The bioassay
experiment was carried out following the standard
methods of APHA [12]. The fish mortality data
recorded for 24, 48, 72 and 96 h were subjected to
EPA Probit Analysis, Version 1.5 statistical software
(http://www.epa.gov/nerleerd/stat2.htm#tsk), based on
Finney's method [13].
RESULTS AND DISCUSSION
The physico-chemical properties viz. temperature,
DO, free CO2 and total alkalinity as CaCO3 of test water
were measured during the bioassay in both seasons
(Table 1). The LC50 values for dimethoate for 24, 48, 72 and
96 h of both seasons are summarized in Table 2. At low
temperature fish appear more tolerant towards toxicant in
comparison to high temperature, as it is evident by the
considerable difference in LC50 values of dimethoate
(Table 2). LC50 values have been found to vary in
different fish species and even in the same species at
different conditions. Verma et al. [14] recorded,
5.14 mg lG1, 4.8 mg lG1, 4.67 mg lG1 and 4.57 mg lG1 LC50
value for 24, 48, 72 and 96 h respectively in
Heteropneustes fossilis exposed to dimethoate. A 96 h
LC50 value for dimethoate, on Clarias batrachus was
reported as 65 mg lG1 by Begum and Vijayraghvan [15]. In
Channa punctatus, Dikshith and Raizada [16] measured
68 mg lG1 LC50 (96 h) for dimethoate which is very high
in comparison to 17.9 mg lG1 LC50 value (24 h) in the same
DO (mg lG1) Free CO2 (mg lG1) Total hardness (mg lG1)
7.20±0.16 4.05±0.13 115.50±1.29
7.80±0.34 3.25±0.12 112.34±0.25
127
 Global J. Environ. Res., 2 (3): 126-132, 2008

Probit Probit

10+ o 10+ o o
- -
- -
- -
- -
9+ 9+
- -
- -
- -
- -
8+ 8+
- -
- -
- . - .
- -
7+ . 7+ .
- .. - ..
- o .. - ..
- ... - .o.
- .... - ....
6+ ...o 6+ ....
- .... - ....
- ..o - ...o
- .... - ....
- ...o - ....
5+ ... 5+ ..o
- .... - ....
- ....o - ....
- o.. - o...
- .... - ....
4+ o.... 4+ ....
- .... - ....
- ... - o...
- .. - ..
- .. - ..
3+ . 3+ .
- -
-. -.
- -
- -
2+ 2+
- -
- -
- -
- -
1+ 1+
- -
- -
- -
- -
0+ -+--------------+--------+---------+---------+--------+--------------+- 0+ -+--------------+--------+---------+---------+--------+--------------+-
EC01 EC10 EC25 EC50 EC75 EC90 EC99 EC01 EC10 EC25 EC50 EC75 EC90 EC99

96 h LC50 (Jan) 96 h LC50 (Aug)

96 h LC50 (January)
Parameter Estimate Std. Err. 95% Confidence Limits
Intercept-12.514366 1.745850 (-15.936233, -9.092500)
Slope 15.123926 1.486897 (12.209608, 18.038244)
Theoretical Spontaneous Response Rate = 0.0000

96 h LC50 (August)
Parameter Estimate Std. Err. 95% Confidence Limits
Intercept -4.838916 1.012849 (-6.824100,-2.853732)
Slope 20.736319 2.085917 (16.647921, 24.824717)
Theoretical Spontaneous Response Rate = 0.0000

Fig. 1: Plot of adjusted Probits and predicted regression line for dimethoate to Heteropneustes fossilis

128
 Global J. Environ. Res., 2 (3): 126-132, 2008

January (17.16 ± 0.78 o C)

70

60
OCM per Minute

50
24 h
40
48 h
30
72 h
20
96 h
10

0
C 12 13 14 15 16 17 18 19
Concentration (mgl-1)

o
August (27.50 ± 1.50 C)
70

60
OCM per Minute

50 24 h
40
48 h
30
72 h
20
96 h
10

0
C 2.5 2.75 3 3.25 3.5 3.75 4
Concentration (mgl-1)

Fig. 2: Opercular Movement (OCM) of Heteropneustes fossilis exposed to Dimethoate in the month of January
and August (C= Control)

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 Global J. Environ. Res., 2 (3): 126-132, 2008

Table 2: LC50 values of dimethoate on Heteropneustes fossilis

August January
---------------------------------------------------------------------------- -----------------------------------------------------------------------------------
LC50 Lower Upper LC50 Lower Upper
Duration (h) (mg lG1 ) confidence limit confidence limit (mg lG1 ) confidence limit confidence limit
24 3.41 3.34 3.49 16.71 16.44 17.00
48 3.13 3.05 3.20 15.95 15.63 16.26
72 3.08 2.99 3.15 15.38 15.06 15.70
96 2.98 2.91 3.05 14.39 13.99 14.76

Table 3: Comparison between behavioural parameters at two different temperatures

January August
Behaviour (17.16±0.78°C) (27.50±1.50°C)
Schooling Disrupted Disrupted
Excitability and reflex responses Reduced with increase in toxicant conc. And time Similar changes
Swimming pattern Abrupt and Jerky, shows gradual improvement with time similar observations
Surfacing and gulping No surfacing at low conc. Frequent at higher conc. Frequent surfacing at all conc. Within 24 h of
exposure
Opercular movement Declines with respect to concentration Declines with respect to time and concentration
Mucus secretion Huge amount of mucus secretion Moderate secretion at almost all conc.

Skin colour Drastic changes in colour associated with appearance of Loss of pigmentation
rashes and patches on the skin Rashes and patches on skin do not appear
Eye condition Turned white and opaque Remained bright and transparent
Fin posture Drooped at higher conc. Show similar changes
Barbels condition Dropped at tips with increase in conc. and time No such effects were observed

Table 4: Result of Two Way ANOVA on OCM with respect to Concentration and Time on Heteropneustes fossilis exposed to Dimethoate
Source Sum of Degree of Mean Level of
Month Parameters of Variance Squares freedom Square F-ratio Significance
January Concentration Between Column 7620.14 8 953.00 2.75 p<0.05
Duration Between Rows 486.84 3 162.00 0.47 NS
Residual Error 8330.51 24 347.00
August Concentration Between Column 9977.81 7 1425.41 7.01 p<0.05)
Duration Between Rows 2924.31 3 974.80 4.80 p<0.05)
Residual Error 4268.22 21 203.25

fish reported by Srivastava and Singh [17]. In this study,
96 h LC50 values in the month of August and January
were recorded as 2.98 mg lG1 and 14.39 mg lG1 respectively
(Fig. 1). This huge difference in LC50 shows that the fish
Heteropneustes fossilis becomes more sensitive to
dimethoate at higher temperature. This is in agreement
with Patra et al. [18] who reported that fish exposed to
chlorpyrifos and endosulfan exhibited increased mortality
with increase in temperature. Variation in LC50 depends on
a number of biological and physico-chemical factors
which have been reported by many earlier workers. The
results of acute toxicity may vary even in same species,
for the same toxicant, depending on the size, age, sex and
condition of the test species together with experimental
factors [7]. The toxicity of cypermethrin to H. fossilis
linearly increases with increase in temperature [19]. The
seasonal variations and pH also has direct effect on fish
mortality in brook trout [20]. Increased mortality at higher
temperature, due to Copper and Cadmium exposure in
Heteropneustes fossilis has been observed by Gupta and
Rajbanshi [21]. The mortality of fishes exposed to mercury
increases with rise in temperature but reduces when the
hardness of test water is increased [8], similarly, pH and
water hardness also affect mortality of Cirrhinus mrigala,
after nickel exposure [9].
A comparative study of behavioural responses
(Table 3) demonstrates that schooling behaviour,
excitability and reflex responses, swimming pattern and
the condition of fins of the exposed fishes were more or
less similar at two different temperatures. However,
parameters such as surfacing and gulping, mucus
secretion, body colour, condition of eyes and barbels and
the opercular movement were remarkably different. At low
temperature the surfacing and gulping exhibit an increase
in comparison to control fish. At higher temperature
very high frequency of it was observed [10]. Increased
frequency of surfacing and gulping may be due to
deficiency in respiratory exchange, as a result of severe
disruption of gill membranes and deposition of mucus
on gills [2]. When toxicant concentration is very high

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 Global J. Environ. Res., 2 (3): 126-132, 2008
(at low temperature), the fish secretes copious mucus from
all over the body in order to neutralize the adverse effect
of large amount of the toxicant, whereas mucus secretion
remains moderate in proportion to the small quantity of
insecticide at higher temperature. The erratic and abrupt
swimming in fish results due to obstruction in acetyl
cholinesterase activity [3, 22]. The skin colour of exposed
fish during January shows drastic changes associated
with appearance of haemorrhagic rashes and loss of
pigmentation at low temperature, this feature was less
apparent in fishes exposed to high temperature (in
August). It could probably be due to the higher toxicant
concentration (in January), to which fishes are exposed.
Severe degenerative changes like necrosis and rupture of
blood vessels leading to hemorrhage have also been
reported [1] at higher concentration. The loss of skin
pigment occurs possibly as a result of impairment of
pituitary functions, causing reduction in number and size
of chromatophores and their pigment content [22, 24]. The
eyes become opaque when fish are exposed to higher
dimethoate concentration at low temperature. Drooping of
fins and loss of extensile nature of the barbels in exposed
fish may be a result of atrophy of muscle proteins [25] and
change in electrolytes balance and decrease in serum
calcium after insecticide exposure [26]. The OCM of
Heteropneustes fossilis falls sharply within 24 h of
dimethoate exposure, thereafter, shows a steady decline
with increase in exposure time and toxicant concentration
(Fig. 2). Two way ANOVA of OCM (Table 4) exhibits a
significant change (P<0.05), when analyzed with respect
to toxicant concentration during January (17.16±0.78°C)
and August (27.50±1.50°C). The decline in OCM with
respect to time was significant during August (P<0.05),
however, it is not significant during January
(P>0.05). The OCM in another teleost Tilapia guineensis,
however, has been reported to declines after an initial
increase on chlorpyrifos exposure [27]. Contrary to it,
the opercular movement in fish has been reported
to increase following the exposure of toxicants
[1, 23, 28, 29]. In present study, decline in the opercular
movement may be due to adaptive response of the
fish to minimize the intake of toxicant by reducing the
frequency of opercular beating and resorting to aerial
respiration with the help of accessory respiratory
organ.
From this study, it may be concluded that the
temperature is an important determinant of toxicity.
Therefore, the application of toxicants should be
synchronized with the seasonal variation in temperature
so that their adverse effects on non-target organisms
131
could be minimized. EPA also considered the effect of
changing application timing of dimethoate to the fall or
spring or modeling application in different areas
(http://www.epa.gov/pesticides/cumulative).
ACKNOWLEDGEMENTS
The financial assistance of University Grant
Commission to one of the authors, R.K.P. (F.5.1.3 (71)/
2004 (MRP / NRCB) to carry out the project is gratefully
acknowledged. We are thankful to Dr. Gunraj Prasad,
Principal, KNIPSS, Sultanpur, for providing required
research facilities.
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