**3. Materials and methods**

Three herbal extracts and calcium as possible antidotes were tried for their efficacy to counteract Cypermethrin,a synthetic pyrethroid (trade name-Cypermethrin, chemical name-Cyano methyl,2,2-dimethyl,cyclopropane-carboxylate) and Carbofuran, a carbamate pesticide (trade name-Furadan 3G, chemical name- 2,3 dihydro,2-2 dimethyl,7 benzo furymethyl carbamate) toxicosis in fish. Antidotes study was conducted in 2 steps (1) Preparation of Crude extracts and (2) Experimental trials.

Datura (*Datura ripens*), Kalka (*Nerium indicum*) and Neem (*Azadiricta indica*) were the plants selected for antidote study and fruits of datura and kalka, and leaves of neem were the specific materials. These plant materials were minced, then grounded and finally extracted using acetone (50%) solution. The ratio of plant material and acetone solution was maintained at 1:10. The paste was collected in a fine meshed cloth and the filtrate was collected for the study. Everytime fresh extracts were prepared before trials. The doses of datura extract were 2.5 and 5.0 ml/l, for both the pesticides. The doses of kalka extract were 2.5 and 5.0 ml/l, for the Cypermethrin experiment while the same were 0.75 and 1.5 ml/l, for the Carbofuran experiment. The doses of neem extract were 10 and 25 ml/l for the Cypermethrin experiment while the same were 5.0 and 10 ml/l for the Carbofuran study.

For the application of calcium solution in water, calcium chloride was chosen as reagent. Fresh solutions were prepared before experiment and applied with different doses (50, 100, 200, 300 and 400 mg/l for both the pesticides under study) to the experiments.

Rohu fingerlings (2.25±0.16g) were used for the antidote study. The fishes were collected from the institute's pond and acclimatized at laboratory condition for 15 days after treating the fish with 0.1% potassium permanganate solution. Probable lethal doses for both cypermethrin and carbofuran were detected by error and trial method in 40 litre plastic tubs containing 20 litres of water. The antidote study was also performed for both the pesticides in the same type of plastic tubs with 20 L of freshwater. The water used for this study was 7.5 pH and 80 mg/l total alkalinity as CaCO3. Four treatments, viz., control (only fish, no pesticides, no antidote), fishes treated with only pesticide (lethal dose), fishes treated with only antidote and fishes treated with pesticide (lethal dose) and antidote solution, were maintained. In every case, antidotes were applied half an hour after the pesticide treatment. Two different doses of each antidote were tried for each of the pesticides. Three replications were maintained for all the treatments. For each case, sampling was done after 1, 2, 4, 6, 12, 24, 48, 72 and 96 hours, respectively(if any mortality occurs), and observations were made and final data were calculated on the basis of comparison between the different experimental observations. The results were expressed as percentage of fish survivability.

For significance of antidote study, one way ANOVA (Duncan Multiple Range Test) were done (Zar, 1974). Test of significance was examined at 5% level.

#### **4. Results and discussion**

Among the four doses of calcium, both at 100 and 200 mg/l of water calcium levels, 67 percent of fish survived up to 96 hours compared to 100 percent fish mortality at 50,300 and 400 mg/l concentration of water calcium against the lethal concentration of cypermethrin.

To investigate some herbal extracts and water calcium as antidotes to counteract the

Three herbal extracts and calcium as possible antidotes were tried for their efficacy to counteract Cypermethrin,a synthetic pyrethroid (trade name-Cypermethrin, chemical name-Cyano methyl,2,2-dimethyl,cyclopropane-carboxylate) and Carbofuran, a carbamate pesticide (trade name-Furadan 3G, chemical name- 2,3 dihydro,2-2 dimethyl,7 benzo furymethyl carbamate) toxicosis in fish. Antidotes study was conducted in 2 steps (1)

Datura (*Datura ripens*), Kalka (*Nerium indicum*) and Neem (*Azadiricta indica*) were the plants selected for antidote study and fruits of datura and kalka, and leaves of neem were the specific materials. These plant materials were minced, then grounded and finally extracted using acetone (50%) solution. The ratio of plant material and acetone solution was maintained at 1:10. The paste was collected in a fine meshed cloth and the filtrate was collected for the study. Everytime fresh extracts were prepared before trials. The doses of datura extract were 2.5 and 5.0 ml/l, for both the pesticides. The doses of kalka extract were 2.5 and 5.0 ml/l, for the Cypermethrin experiment while the same were 0.75 and 1.5 ml/l, for the Carbofuran experiment. The doses of neem extract were 10 and 25 ml/l for the Cypermethrin experiment while the same were 5.0 and 10 ml/l for the Carbofuran study. For the application of calcium solution in water, calcium chloride was chosen as reagent. Fresh solutions were prepared before experiment and applied with different doses (50, 100,

Rohu fingerlings (2.25±0.16g) were used for the antidote study. The fishes were collected from the institute's pond and acclimatized at laboratory condition for 15 days after treating the fish with 0.1% potassium permanganate solution. Probable lethal doses for both cypermethrin and carbofuran were detected by error and trial method in 40 litre plastic tubs containing 20 litres of water. The antidote study was also performed for both the pesticides in the same type of plastic tubs with 20 L of freshwater. The water used for this study was 7.5 pH and 80 mg/l total alkalinity as CaCO3. Four treatments, viz., control (only fish, no pesticides, no antidote), fishes treated with only pesticide (lethal dose), fishes treated with only antidote and fishes treated with pesticide (lethal dose) and antidote solution, were maintained. In every case, antidotes were applied half an hour after the pesticide treatment. Two different doses of each antidote were tried for each of the pesticides. Three replications were maintained for all the treatments. For each case, sampling was done after 1, 2, 4, 6, 12, 24, 48, 72 and 96 hours, respectively(if any mortality occurs), and observations were made and final data were calculated on the basis of comparison between the different experimental observations. The results were expressed as percentage of fish survivability. For significance of antidote study, one way ANOVA (Duncan Multiple Range Test) were

Among the four doses of calcium, both at 100 and 200 mg/l of water calcium levels, 67 percent of fish survived up to 96 hours compared to 100 percent fish mortality at 50,300 and 400 mg/l concentration of water calcium against the lethal concentration of cypermethrin.

200, 300 and 400 mg/l for both the pesticides under study) to the experiments.

done (Zar, 1974). Test of significance was examined at 5% level.

**4. Results and discussion** 

pesticidal effects on Indian Major Carp, *Labeo rohita* (rohu).

Preparation of Crude extracts and (2) Experimental trials.

**2. Aim of the study** 

**3. Materials and methods** 

There was no significant difference in fish survivability between 100 and 200 mg/l levels of water calcium (Table 1). Fifty percent of fish survivability after 96 hours of exposure with lethal concentration of carbofuran was recorded both at 100 and 200 mg/l levels of water calcium while 100 percent of fish mortality was obtained with 50,300 and 400 mg/l levels of calcium against lethal concentration of carbofuran up to 96 hours (Table 3).


\*Values followed by the same superscript were not significantly different at the 0.05 level.

Table 1. Effect of water calcium on the toxicity of cypermethrin to *Labeo rohita* under laboratory conditions

The effects of neem, datura and kalka extracts as probable antidotes against the cypermethrin toxicity using fish survivability as indicator have shown in Table 2. Datura extract (10%w/v) at the levels of 2.5 and 5.0 ml/l exhibited 37 and 50% fish survivability after 96 hours of exposure with lethal concentration of cypermethrin. The fish survivability between these two levels of datura extract showed a significant (P<0.05) difference. Hundred percent of fish mortality recorded both at neem and kalka extract after 96 hours of exposure with lethal level of cypermethrin. It may be mentioned here that at neem and kalka extracts, the fish died within 12 hours.


\*In control experiment of Kalka (i.e. only kalka), the fish died within 12 hours. \*Values followed by the superscript were not significantly different at the 0.05 level.

Table 2. Efficacy of some antidotes to cypermethrin on survivability (%) of Labeo rohita under laboratory conditions.

Application of Some Herbal Extracts and Calcium as an Antidote to Counteract

Observation at different time intervals (hrs.) Survivability percentage of control Survivability percentage in Carbofuran (15 mg a.i./l) Survivability percentage in carbofuran and different doses of antidote calcium(mg/l)\* 50 100 200 300 400

 100a 100a 50b 50b 25c 0 0 0 100a

 100a 100a 50b 50b 50b 50b 50b 50b

100a 100a 100a 50b 50b 50b 50b 50b 50b

100a 100a 100a 100a 50b 0 0 0 0 100a

 100a 100a 100a 50b

\*Values followed by the same superscript were not significantly different at the 0.05 level.

conditions

level of carbofuran.

Table 3. Effect of water calcium on the toxicity of carbofuran to *Labeo rohita* under laboratory

The effects of datura, neem and kalka extracts as probable antidotes against carbofuran toxicity using fish survivability as indicator are presented in Table 4. Datura extract (10% w/v) at the concentrations of 2.5 and 5.0 ml/l showed 50 and 37 percent fish survivability after 96 hours of exposure with lethal concentration of carbofuran. The fish survivability at these two levels of datura extract exhibited a significant (P<0.05) difference. Hundred per cent of fish mortality was observed at all the concentrations of neem and kalka extracts with the exposure of lethal level of carbofuran. It is important to note that the fish died within an hour at neem extracts while at kalka extracts, the fish died within 4 hours against the lethal

The toxic effects of both cypermethrin and carbofuran decreased at medium level (100-200 mg/l) of calcium of water. The results indicate that the degradation of these insecticides take place at medium calcium level of water. The decreased toxicity at this level of calcium could be associated with accumulation of insecticides in excess amount which may be metabolized and stored in different tissues. The accumulated insecticides are eliminated through urine of faeces or both with the help of liver, intestine and kidney (Subbiah et al., 1985). Metz and

the Toxic Effects of Cypermethrin and Carbofuran in Indian Major Carp, Labeo Rohita 285

0 0 0 0

\*In control experiment of Kalka (i.e. only kalka), the fish died within 12 hours. \*Values followed by the superscript were not significantly different at the 0.05 level.

different time

intervals (hrs.)

1

100

100

100

100

25

0

0

0

0

0

100

100

100

100

100

100

100

100

2

4

6

12

24

48

72

96

Survivability percentage in cypermethrin and antidotes Observation at

Neem

Neem

Datura@

Datura@

Kalka@

Kalka@

2.5 ml/l

5.0 ml/l

2.5 ml/l

5 ml/l

(10% w/v)@

25 ml/l

100a

100a

100a

100a

100a

100a

100a

100a

100a

100a

100a

100a

100a

0

75b

12b

0

62b

0

0

50c

0

0

50c

0

0

50c

0

0

100a

100a

100a

62b

37c

37c

37c

37c

100a

100a

100a

12b

0

0

0

0

(10%w/v)@

10 ml/l

100a

100a

100a

100a

0

0

0

0

0

Survivability

percentage in

cypermethrin

(37 ml a.i./ha/m

Survivability

percentage of

control

under laboratory conditions.

Table 2. Efficacy of some antidotes to cypermethrin on survivability (%) of Labeo rohita


\*Values followed by the same superscript were not significantly different at the 0.05 level.

Table 3. Effect of water calcium on the toxicity of carbofuran to *Labeo rohita* under laboratory conditions

The effects of datura, neem and kalka extracts as probable antidotes against carbofuran toxicity using fish survivability as indicator are presented in Table 4. Datura extract (10% w/v) at the concentrations of 2.5 and 5.0 ml/l showed 50 and 37 percent fish survivability after 96 hours of exposure with lethal concentration of carbofuran. The fish survivability at these two levels of datura extract exhibited a significant (P<0.05) difference. Hundred per cent of fish mortality was observed at all the concentrations of neem and kalka extracts with the exposure of lethal level of carbofuran. It is important to note that the fish died within an hour at neem extracts while at kalka extracts, the fish died within 4 hours against the lethal level of carbofuran.

The toxic effects of both cypermethrin and carbofuran decreased at medium level (100-200 mg/l) of calcium of water. The results indicate that the degradation of these insecticides take place at medium calcium level of water. The decreased toxicity at this level of calcium could be associated with accumulation of insecticides in excess amount which may be metabolized and stored in different tissues. The accumulated insecticides are eliminated through urine of faeces or both with the help of liver, intestine and kidney (Subbiah et al., 1985). Metz and


\*Values followed by the superscript were not significantly different at the 0.05 level.

Table 4. Efficacy of some antidotes to carbofuran on survivability (%) of Labeo rohita under laboratory conditions.

Branacin (1975) reported that the degradation of endosulfan took place at high pH and hardness of water but in case of triazophos, its toxicity was increased in similar conditions Application of Some Herbal Extracts and Calcium as an Antidote to Counteract

which may have a neutralizing action against pesticide contamination.

toxic in hard water (Mauck et al., 1976).

toxaphene (Mayer et al., 1978).

essential for sustainable growth of aquaculture.

Marathwada University , India. Kirtikar and Basu (1935). Indian Medicinal Plants.

in Channel catfish. Trans. Am. Fish. Soc., 107:326.

**5. Conclusion** 

**6. References** 

the Toxic Effects of Cypermethrin and Carbofuran in Indian Major Carp, Labeo Rohita 287

while Khalid (1985) reported that water hardness had no significant affect on endosulfan toxicity to four species of fish. Synthetic pyrethroids and natural pyrethrums were more

From the present study, it is clear that both the neem and kalka extracts had no antitoxic effects against cypermethrin and carbofuran poisoning, while acetone extracts of datura served as anti-toxicant against cypermethrin and carbofuran poisoning. The medicinal and therapeutic roles of datura fruit and its seeds have already been established. The young fruits are sedative and slightly intoxicating. The seeds are antispasmodic, narcotic, febrifuge, anthelmintic, works well in inflammation, alexiteric, emetic and useful in leucoderma, ulcers, itching, etc. The seeds contain both hysocyamine and scopolamine (Kirtikar and Basu, 1935). These medicinal qualities have a positive role for the survivability of pesticide intoxicated fish but the specific causes behind it is unknown and its need further investigation. However, it may be due to narcotic and intoxicated properties of datura

It can be mentioned here that vitamins are also having antitoxic effects against insecticide poisoning. For example, vitamins like Macraberin forte (a mixture of vitamins) served as antitoxicant against malathion poisoning to *C. punctatus* (Vaid and Mishra, 1977). When Channel catfish were exposed to various amounts of toxaphene (an organochlorine insecticide) in the diet for 150 days, a dose dependent depression of backbone collagen and spiral deformities were occurred but it recovered effectively when ascorbate (vitamin C) was added to the diet. Vitamin C supplements also reduced the whole body residues of

From the present investigation, it is evident that though, pesticides induced changes of the fish could be improved/protected to a great extent by the application of acetone extract of datura and liming (calcium) up to a certain extent, further investigations are necessary in this regard. Therefore, judicious application of carbofuran in the paddy field to control rice pest and cypermethrin in the pond to control mosquito eggs and Argulus of fish are highly

Ghazaly, K.S. (1994). Efficacy of ascorbic acid (vitamin C) on experimental copper intoxication in Tilapia Zilli. Bull. Natl. Inst. Oceanogr. Fish., Egypt. 20(2):249-257. Khalid, M.S. (1985). Effects of pesticides on freshwater fish, *Rasbora daniconius*. Ph. D. Thesis,

Mayer, F.L., Mehrle, P.M. and Cruteher, P.L. (1978). Interaction of toxaphone and vitamin C

Metz, J. and Branacin, M. (1975). Biochemical and biophysical aspect of salt excretion by

Mauck, W.L., Olson, L.E., and Marking, L.L. (1976). Toxicity of natural pyrethrins and five

chloride cell in teleost. In: Excretion (A.Wessing, Ed.). pp. 127-135.

pyrethroids to fish. Arch. Environ. Contam. Toxicol., 4: 18.

while Khalid (1985) reported that water hardness had no significant affect on endosulfan toxicity to four species of fish. Synthetic pyrethroids and natural pyrethrums were more toxic in hard water (Mauck et al., 1976).

From the present study, it is clear that both the neem and kalka extracts had no antitoxic effects against cypermethrin and carbofuran poisoning, while acetone extracts of datura served as anti-toxicant against cypermethrin and carbofuran poisoning. The medicinal and therapeutic roles of datura fruit and its seeds have already been established. The young fruits are sedative and slightly intoxicating. The seeds are antispasmodic, narcotic, febrifuge, anthelmintic, works well in inflammation, alexiteric, emetic and useful in leucoderma, ulcers, itching, etc. The seeds contain both hysocyamine and scopolamine (Kirtikar and Basu, 1935). These medicinal qualities have a positive role for the survivability of pesticide intoxicated fish but the specific causes behind it is unknown and its need further investigation. However, it may be due to narcotic and intoxicated properties of datura which may have a neutralizing action against pesticide contamination.

It can be mentioned here that vitamins are also having antitoxic effects against insecticide poisoning. For example, vitamins like Macraberin forte (a mixture of vitamins) served as antitoxicant against malathion poisoning to *C. punctatus* (Vaid and Mishra, 1977). When Channel catfish were exposed to various amounts of toxaphene (an organochlorine insecticide) in the diet for 150 days, a dose dependent depression of backbone collagen and spiral deformities were occurred but it recovered effectively when ascorbate (vitamin C) was added to the diet. Vitamin C supplements also reduced the whole body residues of toxaphene (Mayer et al., 1978).
