P503 Acute Toxicities of Diethyl Ether and Ethanol ...

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glycosides — oleandrin and neriine [4]. In fishes, latex of N. indicum(4.42 mg/L – 11.60 mg/L) caused reduction in total protein, nucleic acids, glycogen, AChE in ...
ACUTE TOXICITIES OF DIETHYL ETHER AND ETHANOL EXTRACTED NERIUM INDICUM LEAF TO THE FISH, HETEROPNEUSTES FOSSILIS ManiRam Prasad1, Abhishek Kumar1, Diwakar Mishra1, Sunil K. Srivastav1, Nobuo Suzuki* and Ajai K. Srivastav1 1

Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur 273009, India; *Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa University, Ogi, Noto-cho, Ishikawa 927-0553, Japan Correspondence to: Ajai K. Srivastav (e-mail: [email protected])

Key words: Toxicity, Fish, Nerium, LC 50

ABSTRACT A four-day static renewal acute toxicity test was performed to determine the LC50 value of ethanol and diethyl ether extracted Nerium indicum leaf for the freshwater fish, Heteropneustes fossilis. The LC50 values, their upper and lower confidence limits and slope functions were calculated. The LC50 values for ethanol extracted Nerium indicum leaf at various exposure periods are 155 mg/L for 24 h; 115 mg/L for 48 h; 87.5 mg/L for 72 h; and 60 mg/L for 96 h. The LC50 values for diethyl ether extracted Nerium indicum leaf at various exposure periods are 38 mg/L for 24 h; 27 mg/L for 48 h; 20 mg/L for 72 h; and 14 mg/L for 96 h. The toxicity of Nerium indicum leaf extracts (both ethanol and diethyl ether extracted) exhibits a positive correlation between fish mortality and exposure periods (mortality of fish at a given concentration is increased with increased duration of exposure).

INTRODUCTION The use of the chemical pesticides for the control of insect vectors of diseases and increased yield of many crops has resulted in the pollution of the aquatic environment. The indiscriminate use of these pesticides has produced chronic stress conditions that have negative effect on the aquatic life. Due to high cost of synthetic pesticides and problems caused by their residues, there is a renewed interest in the use of plantderived products (botanical pesticides) which is believed to minimize the undesirable side effects of synthetic pesticides and help preserve the environment for future generations [1,2]. The toxicological and environmental properties of the botanical pesticides must be considered before use. Simply because a compound is a natural product does not ensure that it is safe. Few plant-derived products are photodynamic and they are toxic to all living organisms in the presence of light [3]. Introduction of high levels of toxic natural compounds into the environment could produce more ecological imbalance and cause adverse effects to non-target organisms. Recently, some workers have reported the toxicity of botanical pesticides to some non-target organisms. Nerium indicum is one of the most poisonous plants, the leaves of which contain cardiac glycosides — oleandrin and neriine [4]. In fishes, latex of N. indicum (4.42 mg/L – 11.60 mg/L) caused reduction in total protein, nucleic acids, glycogen, AChE in liver and muscle tissues [5]. Nerium is used as a rat poison, insecticide and piscicide. It is also used for the treatment of inflammation of gums, dysentery, bronchitis, asthma and menorrhagia (although a definite dose/ amount has not been mentioned) [6]. Attempts have been made by few investigators to evaluate the toxicological aspects of various parts of Nerium species on few organisms such as — fish ( 96 h LC50 0.041 g/L [7], 96 h LC50 13.58 mg/L [8], 96 h LC50 44.96 mg/L [9], 96 h LC50 10 mg/L [6]); mammal (50 mg/kg [10], 0.06 g/kg

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[11], 1 g/kg [12], 110 mg/kg [13], 12.5 ml/kg [14], 110 mg/kg [15]); molluscs (13.2 mg/L [16], 24 h LC50 4.9 mg/l [17]) and insect (113.66 ppm [18]). In the present study we aimed to evaluate the comparative toxicity (acute static renewal test) of diethyl ether and ethanol extract of leaf of N. indicum to the fish Heteropneustes fossilis.

RESULTS AND DISCUSSION After exposure to various concentrations of leaf extract of Nerium indicum, the fish became restless, tried to come to surface and jump out of water. After about 1 h they settled down with slow movements. Moreover, the fish showed increased opercular beats, fadeness of body colour as compared to control, slimy body, and loss of balance prior to death. The per cent mortality of H. fossilis after exposure to various concentrations of ethanol extracted N. indicum leaf for 24, 48, 72, and 96 h has been depicted in Figures 1a, 1b, 1c and 1d. The LC50 values at various exposure periods are 155 mg/L for 24 h; 115 mg/ L for 48 h; 87.5 mg/L for 72 h; and 60 mg/L for 96 h. The LC50 values, their upper and lower confidence limits and slope functions have been shown in Table 1. The per cent mortality of H. fossilis after exposure to various concentrations of diethyl ether extracted N. indicum leaf for 24, 48, 72, and 96 h has been depicted in Figures 2a, 2b, 2c and 2d. The LC50 values at various exposure periods are 38 mg/L for 24 h; 27 mg/L for 48 h; 20 mg/L for 72 h; and 14 mg/L for 96 h. The LC50 values, their upper and lower confidence limits and slope functions have been shown in Table 1.

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Table 1. LC50 value, slope function and confidence limits for ethanol (EE) and diethyl ether (DEE) extracted Nerium indicum leaf at different intervals of H. fossilis

24 h LC50 (mg/L) Slope Function Upper Co nfidence Limit* Lower Confidence Limit*

48 h EE

72 h DEE

EE

96h

EE

DEE

DEE

hEE

DEE

155 1.259

38 1.141

115 1.440

27 1.257

87.5 1.600

20 1.315

60 2.460

14 1.492

163.28

39.41

124.81

28.76

97.26

21.28

77.02

15.33

147.14

36.63

105.96

25.34

78.75

18.78

46.74

12.79

*The upper and lower confidence limits for LC50 values calculated at 0.05 level

Figure 1a. Per cent mortality of the fish H. fossilis after 24h exposure to different concentrations of ethanol extracted Nerium indicum leaf. Arrow indicates LC50 value (155 mg/L ).

Figure 1c. Per cent mortality of the fish H. fossilis after 72h exposure to different concentrations of ethanol extracted Nerium indicum leaf. Arrow indicates LC50 value (87.5 mg/L ).

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Figure 1b. Per cent mortality of the fish H. fossilis after 48h exposure to different concentrations of ethanol extracted Nerium indicum leaf. Arrow indicates LC50 value (115 mg/L ).

Figure 1d. Per cent mortality of the fish H. fossilis after 96h exposure to different concentrations of ethanol extracted Nerium indicum leaf. Arrow indicates LC50 value (60 mg/L ).

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Figure 2a. Per cent mortality of the fish H. fossilis after 24h exposure to different concentrations of diethyl ether extracted Nerium indicum leaf. Arrow indicates LC50 value (38 mg/L).

Figure 2b. Per cent mortality of the fish H. fossilis after 48h exposure to different concentrations of diethyl ether extracted Nerium indicum leaf. Arrow indicates LC50 value ( 27 mg/L).

Figure 2c. Per cent mortality of the fish H. fossilis after 72h exposure to different concentrations of diethyl ether extracted Nerium indicum leaf. Arrow indicates LC50 value ( 20 mg/L).

Figure 2d. Per cent mortality of the fish H. fossilis after 96h exposure to different concentrations of diethyl ether extracted Nerium indicum leaf. Arrow indicates LC50 value ( 14 mg/L).

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In the present study, exposure of Nerium leaf extracts to H. fossilis caused increased surfacing, increased opercular movements, increased mucus secretion and loss of balance. This derives support from the earlier studies which describe similar signs after exposure to latex extract of Nerium indicum [6], permethrin [19, 20], fenvalerate [20-22], cypermethrin [23, 24], deltamethrin [25], dimethyl parathion [26], formothion [27], and propoxure [27]. The above mentioned behavioral responses in fish after exposure to extracts of Nerium leaf may be due to the stress caused by it. Increased mucus secretion in fish seems to be a defense response by which fish try to cope with the exposure of Nerium leaf extract by reducing the entry of it through body surface. Increased mucus secretion forms a thin coat on the gill surface which reduces the gaseous exchange [28] and caused increased opercular beats. In the present study, the data regarding the toxicity of Nerium leaf extract exhibit a positive correlation between fish mortality and exposure periods (mortality of fish at a given concentration is increased with increased duration of exposure which is clearly evident by figures). The LC50 value of ethanol extracted Nerium leaf for 24 h, 48 h, 72 h, and 96 h are 155 mg/L, 115 mg/L, 87.5 mg/ L, and 60 mg/L, respectively. It is important to note that after exposure to diethyl ether extracted Nerium leaf, the LC50 values are quite less (30 mg/L for 24 h, 27 mg/L for 48 h, 20 mg/L for 72 h and 14 mg/L for 96 h) as compared to ethanol extracted Nerium leaf and indicated that diethyl ether extracted Nerium leaf is highly toxic as compared to ethanol extracted Nerium leaf. The differences in toxicity data between diethyl ether and ethanol extracted Nerium leaf may be attributed to the difference in the absorption rate, their translocation to target site, the extent of activation and their excretion. The toxicity observed in fish H. fossilis after exposure to Nerium leaf extract is in conformity with other studies which describe that extract from various parts of Nerium is toxic to fishes. Tiwari and Singh [9] have observed the toxicity of Nerium leaf extract on Channa punctatus. These authors have reported Nerium toxicity in a scaly fish whereas the present report deals with a non-scaly catfish which is directly in contact through skin with the toxicant. Singh and Singh [7] have reported the toxic effects of aqueous extract of stem bark of Nerium indicum and observed negative correlation between LC50 and exposure periods. These authors have used aqueous extract of stem bark whereas we have used in this study the ethanol and diethyl ether extracted Nerium leaf. It is inferred from the present study that Nerium leaf extracts exhibit non-target toxicity to fish. It is suggested that application of plant based pesticides should be encouraged in place of synthetic pesticides as they (synthetic pesticides) are more toxic at low doses than botanical pesticides. Use of plant derived pesticides would eventually help in keeping the environment free from hazardous chemicals. Also care should be taken to use plant based pesticides at moderate levels as these are also harmful to fish species.

MATERIALS AND METHODS Leaves of Nerium indicum were procured from the Botanical Garden of DDU Gorakhpur University campus and dried at room

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temperature. Dried leaves were ground into particles and then extracted either with diethyl ether or ethanol in Soxlet extraction unit. The extract solution thus obtained was evaporated at 37° C to a constant weight. The material was weighed and stock solutions (separately for both solvents i.e. diethyl ether and ethanol) were prepared in 100 % ethanol. Adult freshwater catfish H. fossilis (both sexes; body weight 2531 g) were collected locally (from Ramgarh Lake, Gorakhpur) and acclimatized under laboratory conditions for 15 days in plastic pools and fed daily 2-3 times with wheat flour pellets and ground dried shrimps. To determine the LC50 value of N. indicum leaf (extracted with diethyl ether or ethanol), the four day static renewal acute toxicity test [29] was used. Five replicates (separately) of each containing 10 fish (kept in glass aquaria in 30 L tap water) were exposed to each concentrations of N. indicum leaf extract – 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg/L, for ethanol and 50, 75, 100, 125, 150, 175, and 200 mg/L for diethyl ether. A control group with five replicates each of ten fish kept in 30 L tap water (in glass aquaria) was also run. The media (the control and test solutions) in the aquaria were renewed daily. The fish were not fed 24 h before and during the experiment. Dead fish were removed immediately. The physicochemical conditions of the tap water used in experiment are – temperature – 26.74 ± 2.11 C; pH – 7.26 ± 0.09; hardness – 167.97 ± 5.69 mg/L as CaCO3; dissolved oxygen – 7.85 ± 0.36 mg/L; electrical conductivity – 307.16 ± 65.12 mmho/cm and no free chlorine. LC50 values at different exposure periods were determined by the probit-log analysis [30]. For the calculation of upper and lower confidence limits and slope functions the method of Litchfield and Wilcoxon [31] was used.

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