to calculate LC values. The 96 h LC values at temperatures, 17.16±0.78°C (January) and 27.50±1.50°C. 50. 50. (August) were found to be 14.39 mg l and 2.98 ...
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 l 1 and 2.98 mg l 1 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
INTRODUCTION
OCM. Heteropneustes fossilis
brain and peripheral nervous system [3, 4]. Dimethoate (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 Reregistration 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
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 shortsighted 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
Corresponding Author: Rakesh Kumar Pandey, Department of Zoology, Kamla Nehru Institute of Physical and Social Sciences, Sultanpur, U.P.- 228118, India
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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.
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].
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 l 1, 70% mortality at 3.25 mg l 1 and 100% mortality at 4.00 mg l 1 in August. The range finding results in January were as follows; no mortality at 12 mg l 1, 70% at 16 mg l 1 and 100% mortality at 19 mg l 1. All determined concentrations were repeated four times. A control was simultaneously run
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 LC 50 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 l 1, 4.8 mg l 1, 4.67 mg l 1 and 4.57 mg l 1 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 l 1 by Begum and Vijayraghvan [15]. In Channa punctatus, Dikshith and Raizada [16] measured 68 mg l 1 LC50 (96 h) for dimethoate which is very high in comparison to 17.9 mg l 1 LC50 value (24 h) in the same
Table 1: Physico-chemical parameters of test water Month
Water temp. (°C)
pH
DO (mg l 1)
Free CO2 (mg l 1)
January
17.16±0.78
7.39±0.23
7.20±0.16
4.05±0.13
115.50±1.29
August
27.50±1.50
7.30±0.17
7.80±0.34
3.25±0.12
112.34±0.25
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Total hardness (mg l 1)
Global J. Environ. Res., 2 (3): 126-132, 2008
Probit
Probit
10+ o 9+ 8+ . 7+ . .. o .. ... .... 6+ ...o .... ..o .... ...o 5+ ... .... ....o o.. .... 4+ o.... .... ... .. .. 3+ . -. 2+ 1+ 0+ -+--------------+--------+---------+---------+--------+--------------+EC01 EC10 EC25 EC50 EC75 EC90
EC99
10+ o o 9+ 8+ . 7+ . .. .. .o. .... 6+ .... .... ...o .... .... 5+ ..o .... .... o... .... 4+ .... .... o... .. .. 3+ . -. 2+ 1+ 0+ -+--------------+--------+---------+---------+--------+--------------+EC01 EC10 EC25 EC50 EC75 EC90
96 h LC50 (Jan)
EC99
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
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Global J. Environ. Res., 2 (3): 126-132, 2008
January (17.16 ± 0.78 oC 70
OCM per Minute
60 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)
August (27.50 ± 1.50 oC) 70
OCM per Minute
60 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 ---------------------------------------------------------------------------LC50 Lower Upper Duration (h) (mg l 1 ) confidence limit confidence limit 24 3.41 3.34 3.49 48 3.13 3.05 3.20 72 3.08 2.99 3.15 96 2.98 2.91 3.05
January ----------------------------------------------------------------------------------LC50 Lower Upper (mg l 1 ) confidence limit confidence limit 16.71 16.44 17.00 15.95 15.63 16.26 15.38 15.06 15.70 14.39 13.99 14.76
Table 3: Comparison between behavioural parameters at two different temperatures January Behaviour (17.16±0.78°C) Schooling Disrupted Excitability and reflex responses Reduced with increase in toxicant conc. And time Swimming pattern Abrupt and Jerky, shows gradual improvement with time Surfacing and gulping No surfacing at low conc. Frequent at higher conc. Opercular movement Mucus secretion Skin colour Eye condition Fin posture Barbels condition
Declines with respect to concentration Huge amount of mucus secretion Moderate secretion at almost all conc. Drastic changes in colour associated with appearance of rashes and patches on the skin Turned white and opaque Drooped at higher conc. Dropped at tips with increase in conc. and time
August (27.50±1.50°C) Disrupted Similar changes similar observations Frequent surfacing at all conc. Within 24 h of exposure Declines with respect to time and concentration
Loss of pigmentation Rashes and patches on skin do not appear Remained bright and transparent Show similar changes 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 Month Parameters of Variance Squares freedom Square F-ratio January Concentration Between Column 7620.14 8 953.00 2.75 Duration Between Rows 486.84 3 162.00 0.47 Residual Error 8330.51 24 347.00 August Concentration Between Column 9977.81 7 1425.41 7.01 Duration Between Rows 2924.31 3 974.80 4.80 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 l 1 and 14.39 mg l 1 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 130
Level of Significance p
