Original Article

Evaluation of the acute toxicity of the seeds of Anamirta cocculus (Linn.) and its piscicidal effect on three species of freshwater fish

N Jothivel, V Paul

Keywords

acute toxicity, piscicide

Citation

N Jothivel, V Paul. Evaluation of the acute toxicity of the seeds of Anamirta cocculus (Linn.) and its piscicidal effect on three species of freshwater fish. The Internet Journal of Toxicology. 2007 Volume 5 Number 1.

Abstract

Seeds of the Indian fish berry Anamirta cocculus (Linn.) are a potential piscicidal agent used for catching fish from the wild by native people. In the present study, laboratory evaluation of the acute toxicity of the seeds of A. cocculus was done at various time intervals on three species of freshwater predatory fishes viz., Clarias batrachus (Linn.), Channa striatus (Bloch.) and Mystus vitattus (Bloch.). The piscicidal agent was administered as a stomach poison in two different modes viz., after deep-frying and without heating. The LC50(s) and LC99(s) at different durations were calculated in both the modes of administration on all the three species tested. For C. batrachus, the LC50(s) at 1, 2, 24, 48, 72 and 96 hours (h) of piscicidal administration in the heated mode was 203.8463, 170.5797, 137.785, 100.5709, 85.4432 and 62.7660 mg/kg body weight and in the without heated mode 167.2705, 101.2334, 68.4538, 63.9773, 51.6782 and 50.2421 mg/kg body weight respectively. For C. striatus, the LC50(s) at 1, 2, 24, 48, 72 and 96 h in the heated mode was 109.2334, 77.4538, 57.9773, 44.2705, 34.6782 and 24.2421 mg/kg body weight and in the without heated mode 90.7660, 55.4433, 34.5709, 28.7685, 20.5798 and 15.3158 mg/kg body weight respectively. For M. vitattus the LC50(s) at the corresponding durations in the heated mode was 32.2706, 22.4433, 19.7686, 15.2883, 7.2517 and 3.4484 mg/kg body weight and in the without heated mode 26.8463, 17.2705, 10.0193, 7.9773, 4.9850 and 1.9082 mg/kg body weight respectively. LC99(s) in both the modes of administration in the respective exposure periods for all the three species tested were also calculated. The results reveals that of all the species tested, C. batrachus is the most resistant one towards the toxicity of A. cocculus followed by C. striatus and M. vitattus. The present study has also shown that these seeds may be used as a potent aquaculture management tool to eradicate unwanted wild fish from culture ponds before stocking.

 

Introduction

Since prehistoric times, cultures throughout the world have used piscicidal plants for fishing. According to ( 1 ), plants are virtually inexhaustible sources of structurally diverse and biologically active substances. Fossil records dates back the use of plants by human beings for various purposes including medical use at least to the middle Paleolithic age, some 60,000 years ago ( 2 ). Plants with insecticidial, piscicidal and molluscicidal properties have also been used widely by human beings ( 3 ). Fisher folks of various African countries extensively use many plants and plant products for capturing fish ( 4,5 ). Barbascos of ethanobotanical origin and their application in capturing fish have also been reported from other regions of the world such as South America ( 6 ), Nepal ( 7 ), India ( 8,9 ), etc. In addition to their use as traditional piscicidal agents for catching wild fish, plant derived fish toxicants are also used in aquaculture management for controlling the predatory and weed fishes. The eradication of these wild fishes form the culture ponds before the stocking of desired species is an important step in pond management as the former compete and/or prey upon the latter. In this aspect, the air-breathing predatory fish species are of particular importance as they are highly resistant to toxicants ( 10 ) and may survive in moist borrows and mud even when ponds are drained. The use of plant origin ichthyotoxicant as a fisheries management tool has been practiced in at least 30 countries ( 11,12,13 ). As the control and eradication of unwanted fishes in the pond require effective piscicides which are usually not easily accessible, farmers even use synthetic compounds including malachite green, sodium cyanide, antimycin, etc. and even pesticides ( 14,15,16,17 ). However, use of these compounds in culture ponds is seldom appreciated especially due to their long-term persistence in the ecosystem as well as in the cultured species. Therefore alternative piscicides such as botanicals, which are biologically degradable, and having piscicidal activities with shorter residual effects are being appreciated.

The seeds of the Indian fish berry Anamirta cocculus (Linn.) are one such piscicidal agent, which is used by the local people in different parts of the country for catching fish for human consumption. However, only few scientific reports regarding the piscicidal plants of India are available ( 8,9 ). Similarly studies regarding A. cocculus are also scanty ( 18 ). In this context, efforts have been made to evaluate the acute toxicity of the seeds of A. cocculus in the stomach poison mode in two ways of administration (viz., heated and without heating) using two freshwater catfishes viz., Clarias batrachus (Linn.) and Mystus vitattus (Bloch.) and the freshwater snakehead fish Channa striatus (Bloch.). While C. batrachus and C. striatus are voracious predatory air-breathing fishes causing considerable loss of yield in the culture pond, M. vitattus preys upon fry and fingerlings. It is also considered as a weed fish in culture ponds. Selection of the stomach poison mode for the administration of the toxicant is because of the fact that the rural folk utilize this route of administration of the piscicidal agent. Their routine method involves deep-frying the seeds and mixing it with minced fish feeds like earthworm, chicken intestine etc., before administration.

Materials and methods

Fishes and their maintenance

Healthy specimens of C. batrachus (130 ± 2 g body weight and 18 ± 1 cm length), C. striatus (78 ± 2 g body weight and 16 ± 1 cm length) and M. vitattus (15 ± 2 g body weight and 10 ± 1 cm length) were collected locally at Chidambaram, Tamilnadu and were reared separately in large plastic aquaria bearing well water. The fishes were acclimated to the laboratory condition for about 30 days (d).

Food and feeding

The fishes were liberally fed with minced earthworm meatballs. While C. batrachus and C. striatus were fed with meatballs weighing 6 ± 0.42 g each, M. vitattus were given meatballs each weighing 3 ± 0.23 g everyday for a period of 3 hours (h), before the renewal of the medium. On average, while an individual C. batrachus consumed 8 ± 1 meatballs, an individual C. striatus had 6 ± 1 meatballs and M. vitattus consumed 4 ± 1 meatballs per feeding. Water was renewed after every 24 h with routine cleaning of the aquaria leaving no fecal mater, dead fish (if any) or unconsumed food.

Preparation of the toxicant

The hard shells of the dried fruits of A. cocculus were broken and the seeds were collected and were processed in two ways for administering it as stomach poison. One part of the seeds was ground well in a grinder without frying. The powdered seeds were administered in the proportion of mg/kg body weight of fish through earthworm meatballs. After preliminary experiments, A. cocculus laced meatballs for C. batrachus and C. striatus were prepared in such a way that the required concentration of the seed per fish was equally divided into 4 parts and were incorporated to the interior of 4 meatballs. For M. vitattus, the required concentration of the seed per fish was equally divided into two parts and was incorporated to the interior of two meatballs. The seeds were totally covered by the meatballs. Another part of the seeds was deep fried (heated) at 100 °C for 15 minutes and ground well. The required quantity of seed incorporated meatballs was prepared in the same way as that of the unheated seeds.

Bioassay

LC50(s) of A. cocculus as stomach poison to all the three species at various exposure periods viz., 1, 2, 24, 48, 72 and 96 h were estimated in two modes of administration (heated and without heating) following Finney's ( 19 ) method. In both the modes of administration, the experiments were done in triplicate for calculating the LC50(s) at each exposure period. For each exposure period, groups of 10 fish each were kept in plastic aquarium bearing 100 liters of well water having an ambient temperature 27 ± 1 °C, pH 7.2 ± 0.2, dissolved oxygen content 5.6 ± 0.2 mg/l, carbon dioxide 3 ± 0.2 mg/l, alkalinity 9.3 ± 0.1 ppm and total hardness 88 ± 2 ppm ( 20 ). Parallel controls were also kept without administering the seeds. The experimental groups were fed with the respective meatballs laced with the A. cocculus seeds 3 h prior to the renewal of the medium. Wherever needed, water in the respective sets was renewed after every 24 h. Dead fish were immediately removed and recorded. The fish was considered dead if it did not respond to prodding by a glass rode. No mortality was recorded in the control groups.

Calculation of lethal concentrations and statistics

The mortality rates observed during the stipulated exposure periods were recorded and using this data various LC50(s) and LC99(s) were calculated by following Finney's ( 19 ) method along with the slope values. The heterogeneity of the data (if any) in each set was also checked using chi-square (χ 2 ) test.

Results

Behavioural changes

In both the modes of administration of A. cocculus seeds, the acutely intoxicated fishes of all the three species exhibited violent swimming activities. They often came to the water surface and exhibited increased gulping activity. They also expressed highly increased opercular movements indicating respiratory distress and tried many times to jump out of the aquaria. Excessive amounts of mucus were also seen all over the body of the exposed fish. None of the control fish showed any of these behavioral changes. The dying ones in the experimental aquaria exhibited vertical positioning with head above the water surface. They also showed muscular twitching and tetany before death. Finally they lost balance, settled at the bottom of the aquaria and died. No mortality was observed in the control groups.

Mortality rate (Figs. 1 to 6)

The average percentages of mortality of all the three species at various stages of toxicant administration in both the modes are given in Figures 1 to 6. Using these mortality rates, the LC50(s) and LC99(s) were calculated.

Lethal concentrations (Tables 1 to 6)

The respective LC50(s) of A. cocculus at various stages of administration to all the three species are given in Tables 1, 3 and 5. Similarly, the respective LC99(s) are shown in the Tables 2, 4 and 6. The estimated LC50(s) lie within the 95% confidence limits and are given along with the slope and χ 2 values. LC50(s) are found to decrease with increasing exposure periods.

Figure 1
Figure 1: Percentage of mortality of and administered with seeds in different modes of administration at 1 h duration

Figure 2
Figure 2: Percentage of mortality of and administered with seeds in different modes of administration at 2 h duration

Figure 3
Figure 3: Percentage of mortality of and administered with seeds in different modes of administration at 24 h duration

Figure 4
Figure 4: Percentage of mortality of and administered with seeds in different modes of administration at 48 h duration

Figure 5
Figure 5: Percentage of mortality of and administered with seeds in different modes of administration at 72 h duration

Figure 6
Figure 6: Percentage of mortality of and administered with seeds in different modes of administration at 96 h duration

Figure 7
Table 1: LC of to in different modes of administration along with their 95% confidence limits, χ values and slopes at various time intervals

Figure 8
Table 2: LC of to in different modes of administration along with their 95% confidence limits at various times intervals

Figure 9
Table 3: LC of to in different modes of administration along with their 95% confidence limits, χ values and slopes at various time intervals

Figure 10
Table 4: LC of to in different modes of administration along with their 95% confidence limits at various times intervals

Figure 11
Table 5: LC of to in different modes of administration along with their 95% confidence limits, χ values and slopes at various time intervals

Figure 12
Table 6: LC of to in different modes of administration along with their 95% confidence limits at various times intervals

Discussion

From time immemorial, humans explored ways and means to divert poisons of biological origin to their own advantage. The pandemic need to find plants that work well as soap has been pursued by most native cultures and this experience of using various plants for their soap like properties might have lead to the universal discovery that chemicals released from some plants would also stun fish when used in a specific circumstance ( 21 ). A. cocculus (Linn.) is commonly known as fish berry or crow killer. It is a wild woody climber producing poisonous seeds that are being exploited by human beings for several purposes including hunting and fishing ( 22 ). The dried berries of A. cocculus have been used in India to stupefy fish ( 23 ) and are reported to contain picrotoxin ( 24 ), which being a product of biosynthesis are liable to potential degradation. As in many other cases, native fishermen or local people might have identified the piscicidal property of A. cocculus also. However, the traditional fishermen are not aware of the precise quantity of the seeds required to kill fish belonging to any particular species and therefore they usually apply much higher quantities than the optimum such liberal applications seems to be an option, which is ecologically unhealthy.

On the other hand, predatory and weed fishes pose serious threats to the cultured species and therefore their control is an essential part of the pond management. The problem becomes more aggravated in the case of the predatory fishes like C. batrachus and C. striatus as they possess accessory respiratory organs, which facilitate their survival even under extreme environmental conditions. According to Chiayvareesajja et al. ( 25 ) air breathing species are more tolerant to piscicidal materials and therefore the piscicidal activity should be tested in such species. Critical analysis of the LC50(s) and LC99(s) (Tables 1 to 6) reveals that C. batrachus is the most resistant of all the three species tested, followed by C. striatus and M. vitattus. The higher LC50(s) in case of C. batrachus and C. striatus could be attributed to the presence of accessory respiratory organs in them and this observation is in confirmation with the view of Chiayavareesajja et al. ( 25 ). Therefore, the advantage of selecting C. batrachus as one of the test organisms is that the concentration of the toxicant, which wood kill this species could wipe out almost all the unwanted species of fishes, provided the pond is treated before stocking.

Usually, for harvesting fish from the wild using plant origin piscicides, the plant part to be used as piscicide is thoroughly pounded and the macerated material is thrown into the water body from which the fishes are to be harvested. In such situations, the toxicants act as contact poisons and therefore are more active in standing water bodies. However, in case of A. cocculus, its traditional use as stomach poison facilitates its application in standing as well as running waters to catch fish. It may also be mentioned that the use of the piscicidal agents as contact poisons in natural aquatic ecosystems could probably cause the loss of non-target biodiversity in those ecosystems. In the present study, the highly cohesive mucous content of the earthworm balls holds the seeds well inside the meatballs and thereby prevents the spillage of the seeds directly into medium to a great extend. The mode of administration using the deep fried seeds in the present study is exactly in accordance with the method followed by the native people. However, by also administering the seeds without heating, a comparative assessment of the toxicities of the raw seeds and cooked seeds is made (Tables 1 to 6). The results indicate that the raw seeds are more poisonous than cooked ones as the former is having low LC50(s) at various time intervals. Even though the toxicity of the seeds is noticeably reduced after frying, the seeds become more acceptable to fish. This could be one of the main reasons why the local people fry the seeds before using it as a barbasco. Further, by heating, perhaps some of the active ingredients in the seeds might be getting degraded leading to increase in LC50(s). Such a heat-attenuated toxicity would be a desirable factor as the fish caught is traditionally used for human consumption.

According to Cagauan et al. ( 3 ), concentration causing 100% mortality forms the basis of calculating the piscicidal activity of the test plants. Banerjee ( 26 ) is of the view that determination of LC50 is essential for acute toxicity testing and also for routine bioassay experiments. In view of these reports, the present study provides a range of LC50(s) and LC99(s) of A. cocculus at various durations. As a fishing poison, the knowledge of LC50(s) and LC99(s) at various exposure periods would provide more provisions to the farmers in case they want to kill/eradicate the predatory and weed fishes within a convenient duration from culture ponds before stocking. Further, the mortality rates observed in the present study suggests a clear relationship between dose, mortality and exposure period. The concentration of the toxicant is directly proportional to the mortality rate. On the other hand, as the duration of exposure increases the lethal concentration decreases. As the calculated χ 2 values (representing heterogeneity) are less than the respective table values, the mortality counts are not significantly heterogeneous indicating variables such as individual resistance, etc. do not significantly affect the lethal concentrations as they lie within 95% confidence limits. The steepness of the slope is also an indication of large increase in mortality rate with relatively small increase in the concentration of the piscicidal agent.

Acknowledgement

Authors thank Annamalai University authorities for providing lab facilities and Dr. K.K. Nair, Senior Scientist, KFRI, Kerala, India for plant identification.

References

1. Istvan U. (2000). Semi-natural products and related substances as alleged botanical pesticides. Pest Manage Sci 56: 703-705.
2. Fabricant D.S. and Farnsworth N.R. (2001). The value of plants used in traditional medicine for drug discovery. Environ Health Perspect 109 suppl 1: 69-75.
3. Cagauan A.G., Galaites M.C. and Fajardo L.J. (2004). Evaluation of botanical piscicides on Nile tilapia Oreochromis niloticus (L.) and mosquito fish Gambusia affinis. Sixth International Symposium on Tilapia in Aquaculture, Manila, Philippines, 179-187.
4. Neuwinger H.D. (2004). Review of plants used for poison fishing in tropical Africa. Toxicon 44(4): 417-430.
5. Fafioye O.O., Adebisi A.A. and Fagade S.O. (2004). Toxicity of Parkio biglobosa and Raphia vinifera extracts on Clarias gariepinus juveniles. Afr J Biotechnol 3: 627-630.
6. Schultes R.E. and Raffauf R.F. (1990). The healing forest, medicinal and toxic plants of the northwest Amazonia, Dioscorides Press, Oregon, 164-166.
7. Kulakkattolickal A.T. (1987). Piscicidal plants of Nepal: Preliminary toxicity screening using grass carp (Ctenopharyngodon idella) finger lings. J Ethnopharmacol 21: 1-9.
8. Singh D. and Singh A. (2002). Piscicidal effect of some common plants of India commonly used in freshwater bodies against target animals. Chemosphere 49: 45-49.
9. Tiwari S. and Singh A. (2005). Possibility of using Latex extracts on Nerium indicum plant for control of predatory fish Channa punctatus. Asian Fish Sci 18: 161-173.
10. Kulakkatolickal A.T. and Kramer D.L. (1988). The role of air-breathing in the resistance of bimodally respiring fish to waterborne toxins. J Fish Biol 32: 119-127.
11. Bettoli P.W. and Maceina M.J. (1996). Sampling with toxicants. In - Eds. B.R. Murphy and D.R. Willis, Fisheries Techniques, 2nd edn. Am Fish Soc, Bethseda, M.D. 303-333.
12. Sanger A.C. and Koehn J.D. (1997). Use of chemicals for carp control. In - Eds. J. Roberts and R. Tiley, Controlling carp; exploring the options for Australia. Proceedings of Workshop 2-24th October 1996, Albury, Canberra, CSIRO, Australia. 37-57.
13. Lintermans M. (2000). Recolonization by the mountain galaxias Galaxias didus of a mountain stream after the eradication of rainbow trout Oncorhynchus mykiss. Mar and Freshwater Res 51: 799-804.
14. Chakraborty D.P., Nandi A.C. and Phillipose M.T. (1972). Barringtonia acutangula (Linn.) as a fish poison. Indian J Exp Biol 10: 78-80.
15. Terazaki M., Tharnbuppa P. and Nakayama Y. (1980). Eradication of predatory fishes in shrimp farms by utilization of Thai tea seed: Aquaculture 19: 235-242.
16. Marking L.L. (1992). Evaluation of toxicants for the control of carp and other nuisance fishes. Fisheries 17: 6-12.
17. Gribgratok S. (1981). The role of cyanide on the fisheries. Thai Fisheries Gazette 34: 499-506.
18. Dumen E., Dorucu M. and Sen D. (2001). Toxic effects of fish-seed (Anamirta cocculus) on carp (Cyprinus carpio). J Biol Sci 1: 1093-1094.
19. Finney D.J. (1971). Probit Analysis, 3rd edn., Cambridge University Press, London 1-333.
20. APHA (American Public Health Association) - AWWA (American Waste Works Association) and WPCF (Water Pollution Control Federation) (1989). Standard methods for the examination of water and wastewater, 17th Ed. Washington, D.C., USA.
21. Kritzon C. (2003). Fishing with Poisons. The Bulletin of Primitive Technology, No. 25. 1-10.
22. Kirtikar K.R., Basu B.D. and I.C.S. (1991). Indian Medicinal Plants. Bishen Singh Mahendra Pal Singh (Publisher), Dehra Dun, India 80-83.
23. Drury H. (1973). The Useful Plants of India. William H. Allen & Co. 1-512.
24. Agarwal S.K., Singh S.S., Verma S. and Kumar S. (1999). Two picrotoxin derivatives from Anamirta cocculus. Phytochemistry 50: 1365-1368.
25. Chiayvareesajja S., Chiayvareesajja J., Rittibhonbhun N. and Wiriyachitra P. (1997). The toxicity of five native Thai plants to aquatic organisms. Asian Fish Sci 9: 261-267.
26. Banerjee T.K. (1993). Estimation of acute toxicity of ammonium sulphate to the freshwater catfish Heteropneustes fossilis. I. Analysis of LC50 values determined by various methods. Biomed Environ Sci 6: 31-36.

Author Information

N. Jothivel
Department of Zoology, Annamalai University

V.I. Paul
Department of Zoology, Annamalai University

Your free access to ISPUB is funded by the following advertisements: