Toxic effect of Nerium indicum Latex Powder on Biochemical Profile of Fishes

 

Kamlesh Kumar*, Ajay Singh

Department of Zoology D D U Gorakhpur University, Gorakhpur-273009

 

Abstract:

Presence of predatory and weed fishes in fish cultured pond is also serious problem due to their faster growth rate and better utilization of food and cultured carp habitat. Due to their carnivorous nature they engulf the finger lings of cultured crops and adversely affect the aquaculture production. The WHO and FAO have tested several thousand synthetic compound for the control of weed and predatory fishes through these pesticide so for not provide them self to be entirely satisfactory with a growing awareness of environmental pollution, effort are being made to find out piscicidal product from plant origin. Being product of bio synthesis, these are easily biodegradable in nature. Exposure to 96 hour of fish with sub lethal doses (40% and 80% of LC₅₀, 24h) of latex powder of Nerium indcum caused significant (P<0.05) decrement in the level of total protein, glycogen and nuclic acids and enhancement in level of total free amino acid and activity of enzyme proteases. The reversibility in action and non-toxic effect on mammals are the important positive feature of these botanical piscicides, which reduce the threat of environmental pollution.

 

 

 

INTRODUCTION:

In country like India the intake of meat and milk is low so fish has special importance as a supplement to ill balanced cereal diets. Today protein deficiency is the world’s most serious human multi nutritional problem and perhaps 30 to 40% of the world population is suffering from protein deficiency. It is estimated that about 10 million tons of fish required annually meeting the present day demand of fish protein in the country against an animal production of only 35 million tons (Shukla and Upadhyay, 2008).

 

Fish production in India has increased at a higher rate compared to food grains, milk, eggs and other food items. India ranks second in the world fish production with an annual fish production of about 6.9 million metric tons (Economic Times, 2007). Fisheries sector contributed Rs. 34,758 crores to the GDP during 2005-06, which was 1.2% of the national GDP and to 5.3% of the agricultural GDP. However, the share of fisheries sector in the State Domestic product was estimated to be 1.44% in 2005-06 (Economic Survey, 2007). There has been a gradual shift in the production scenario from marine to inland fisheries in recent years.

 

Indian freshwater aquaculture has evolved itself from the stage of a domestic activity in the Eastern States of West Bengal and Orissa to that of industry and has become an important component of Indian fisheries contributing about one third of total fish production of the country and share about 95% of the total aquaculture production. The mean national fish production levels from tanks and ponds have increased significantly from 600 kg/ha during the 1970’s to 2,200 kg/ha in the 1990s (Anon, 2006). In some areas such as the states of Andhra Pradesh, West Bengal, Punjab and Haryana yield has increased even to 8,000-10,000 kg/ha. Demonstrations on intensive composite carp culture practices had shown a maximum production of 15,000 kg/ha at Central Institute of Freshwater Aquaculture, Bhubaneswar (Anon, 2006). Carps are the backbone of Indian freshwater aquaculture, comprising around 85% of the total freshwater production. Carp culture in India is largely limited to six species; the three Indian ‘major carps’ catla, rohu and mrigal; and three exotic or ‘Chinese carps’, grass carp, silver carp and common carp. Again, Indian major carps contribute the lion’s share of freshwater aquaculture production, around 80% by volume. Among Indian major carp production, the contribution of rohu alone is about 35%. India is regarded as a ‘carp country’ due to its rich diversity of carps in its freshwater ecosystems. About 2,070 species of carps (family Cyprinidae) are available in Indian waters. Though many of the carp species like minor barbs and minnows are not economical from the commercial culture point of view, the country is blessed with at least 15-20 varieties of minor and medium carps that have a high potential for fresh water aquaculture, which has yet to be exploited.

 

A number of predatory fishes belong to Genus Channa (Ophicephalous). Forty two fish species of Genus Channa are available in all over the world (Fishbase, 2002). In India, studies made during the “All India Co-ordinate Research Project on Air-breathing fish Culture” revealed that the ten species of Murrells described by Singh, et al., (2002) only the eight are valid. Out of eight, four species (Channa punctatus; Channa marulius; Channa striatus and Channa gachua) are common in north India. These fishes are predatory in nature and feed upon the fry of cultured carps (Khanna, 1978). Due to the faster growth rate, these fishes share and better utilized the cultured carp habitats as well as food also (Jhingran, 1983). So these predatory fishes adversely affect the aquaculture production and ultimately affect the fish former economy. Most of the predatory and weed fishes breed in ponds, a little earlier than the spawning time of cultured carps and their fry feed vigorously on the available food in the pond. When the spawn of cultured carps released, the young ones of predatory fishes are grown enough to feed upon finger lings of cultured carps (Chakraborty et al., 1972; Jhingran, 1983). Air breathing predatory fish species cause special problem because they may survive in moist burrows, even when ponds are drained (Jhingran, 1983) The aim of present study was to check the picicidal activity of freeze dried Nerium indcum latex powder on the biochemical profile of predatory and weed fish Channa punctatus (bloch) and Mystus mystus.

 

MATERIAL AND METHODS:

The fish Channa punctatus (bloch) of different size (small size 2.5± 0.83 cm, middle size 7.5 ± 0.83cm and large size 15.7±1.35 in total length), and Mystus mystus (small size 1.8± 0.52 cm, middle size 5.0± 0.35 cm and large size 10.2±1.83 in total length) were collected from Mahesara lake of Gorakhpur district. The colleted fishes were stored in Plastic tank containing 100L of de-chlorinated tap water for acclimatization to laboratory condition for seven days. Diseased, injured and dead fish (if any) were removed as soon as possible to prevent the decomposition of the body in the tank. Water was changed every day. Fishes were fed with commercial food. Acclimatized fishes were used for experiment.

 

The yellowish milky latex of Nerium indicum was drained into test tubes by cutting the stem apices, these latices were centrifuged at 1000xg for 20 minutes for removing resin and then this resin free latices was lyophilized at -40°C by freeze drier and lyophilized powder was stored in humidity free chamber for further use.

 

The wet weight of one ml latex of Nerium indicum was 780 mg and dry weight (lyophilized at -40°C) was 200mg.

 

BIOCHEMICAL EXPERIMENT:

Biochemical experiments were performed to determine the biochemical alteration in fishes on exposure to sub- lethal concentrations of latex extracts of the plants. For biochemical studies, the experimental fishes divided into three different groups, and aquarium of each group containing 10 fishes in 6L dechlorinated tap water. The fishes of group one were reared in latex free water and treated as control. Fishes belonging to second and third group were exposed to 40% and 80% of sub lethal concentration of the latex and leaf extracts of all the three plants (Table1). Each experiment was replicated six times; exposures to these extracts were continued up to 96 h exposure periods. Fishes were not fed before 24 h and during the experiment.

 

Table 1: Sub-lethal doses used for biochemical experiments

Name of plant part used	Sub-lethal concentrations (mg/L)
	Fish Mystus Mystus		Fish Channa punctatus
	40% of 24h LC50	80% of 24h LC50	40% 24h LC50	80% 4h LC50
Nerium indicum Latex powder	1.65	3.31	3.84	7.68

 

After 96h fish from control and experimental groups were taken separately and killed by severe blow on head. The fish were opened and muscle tissue from the left side of dorsal fin and liver tissue were quickly dissected out and kept on ice tray and used for biochemical estimation.

 

The following methods were used for the estimation of total protein; Total free amino acids nucleic acids level (DNA and RNA), glycogen, protease in muscle and liver tissues both.

 

Estimation of total protein level:

Total protein was estimated according to the method of Lowry et al., (1951). Tissues were removed and homogenates (5mg/mL, w/v) were prepared in 10% TCA. Tissues were homogenized for 5 minutes using electric homogenizer and centrifuged at 6000g for 20 minutes.

 

Estimation of total free amino acids level:

Free amino acid was measured according to the method of Spices (1957). Homogenates (5mg/mL, w/v) were prepared in 96% ethanol in an electric tissue homogenizer for 5 minutes and centrifuged at 8000g for 20 minutes.

 

Estimation of Glycogen level:

Glycogen was measured according to anthrone method of Van Der Vies (1954) modified by Mahendru and Agarwal (1981). Homogenate (100 mg/mL, w/v) was prepared in 5% TCA by electric homogenizer for five minutes and filtered.

 

Estimation of Nucleic acids (DNA and RNA) level:

Nucleic acids (DNA and RNA) were estimated by the method of Schneider (1957). Homogenates (100 mg/ml, w/v) were prepared in 5% TCA at 90⁰C by electric homogenizer for 5 minutes and centrifuged at 5000g for 20 minutes.

 

DNA:

In 1.0 mL of supernatant 1.0 mL of distilled water and 4.0mL of freshly prepared diphenylamine reagent was added .The reaction mixture was kept in boiling water bath for 10 minutes. The blue colour developed was measured at 600nm. Standard curves were drawn concentrations of calf thymus DNA as standard.

 

RNA:

In 1.0 mL of supernatant 2.0mL of distilled water and 3.0mL of Orcinol reagent was added. The reaction mixture was kept in boiling water bath for 20 minutes. The greenish-blue colour developed was measured at 660 nm. Standard curves were drawn using different known concentrations of yeast RNA as standard.

Nucleic acids (DNA and RNA) contents have been expressed as µg/mg of tissue.

Estimation of protease activity:

Protease activity was measured according to the method of Moore and Stein (1954). Homogenate (50 mg/mL, w/v) was prepared in cold distilled water and centrifuged at 1000g for 15 minutes and supernatant was kept for enzyme assay. The enzyme activity was expressed in μ moles of tyrosine equivalent/mg protein/h.

 

RESULTS:

The observation on biochemical changes in the level of total protein, total free amino acid, glycogen, nucleic acid (DNA and RNA) and activity of enzyme protease in liver and muscle tissues of fish Mystus mystus and Channa puntatus after exposure to sub lethal doses (40% and 80% of LC₅₀; 24h) of ethyl alcohol leaf extracts of extract of Nerium indicum after 96h exposure periods.

 

(A) Freeze dried Latex powder of Nerium indicum (FDLPNI):

Exposure of fish Mystus mystus to sub lethal doses (40% and 80% of LC₅₀; 24h) FDLPNI for 96h exposure periods caused significant (P<0.05) alteration in both nitrogenous and carbohydrate metabolism. Student‘t’ test and analysis of variance showed that these alteration were significantly (p<0.05) time and dose dependent (Table 2)

 

Total protein level was reduced to 56% and 51% of control in liver and 62 % and 57% of control in muscle tissues, total free amino acid level was induced to 152% and 176% of control in liver and 129% and 169% of control in muscle tissue, nucleic acid level such as DNA level was reduced to 65% and 51% of control in liver tissue 58% and 47 % of control in muscle, RNA level was reduced to 60% and 47% of control in liver tissue and 42 % and 40% of control in muscle tissue, glycogen level was reduced to 63% and 48% of control in liver and 58% and 45% in muscle tissue . Activity of enzyme Protease induced to121% and 144% of control in liver and 135% and 164% in muscle tissue of fish Mystus mystus respectively after exposure to 40% and 80% of LC₅₀ 24h of FDLPJG after 96h exposure periods (Table -2) .

 

Withdrawal experiment:

Table- 2 shows the effect of withdrawal of treatment of FDLPNI (80% of 24h LC₅₀) after seven days. There was significant (P<0.05) recovery in the level of total protein (96% and 81%), total free amino acid (112% and 117%), total nucleic acid level such as DNA (91% and 89%) similarly RNA (91% and 92%), glycogen (91% and 84%) and protease (127% and 111%) of controls in liver and muscle tissues of Mystus mystus (Table -2).

Table.2: Changes in total protein , total free amino acids and nucleic acid (DNA and RNA), glycogen level and activity of enzyme protease in liver and muscle tissues of Mystus mystus after exposure to 40% and 80% of LC₅₀ (24h ) of freeze dried latex powder of Nerium indicum after 96h and 7^th day after withdrawal.

Values are mean ±SE of six replicates.

Values in parentheses are % level with control taken as 100%.

Data were analyzed through student’s test.

*, Significant (p>0.05), when treated groups were compared with controls.

+, Significant (p>0.05), when withdrawal groups were compared with treated groups.

Level of total protein, total amino acid, nucleic acid (DNA and RNA) expressed in µg/mg , glycogen in mg/g and activity of enzyme protease expressed in µ moles of tyrosine equivalent/mg protein/h.

 

 

Effect of sub lethal doses of FDLPNI show similar trends of alterations in the level of total protein, total free amino acid, glycogen, nucleic acid (DNA and RNA) and activity of enzyme protease in liver and muscle tissue of fish Channa punctatus after 96h exposure periods (Table- 3). Total protein level was reduced to 38% and 34% of control in liver and 37 % and 34% in muscle tissue, total free amino acid level was induced to 189% and 227% in liver tissue and 147% and 165% in muscle tissue, nucleic acid level such as DNA level was reduced 69% and 53% in liver and 36% and 34 % in muscle, RNA level was reduced to 39% and 35% in liver and 47% and 46% in muscles tissue, Glycogen level was reduced to 71% and 57% of control in liver and 62% and 36% of control in muscle tissue. Activity of enzyme Protease induced 156% and 206% of control in liver and 141% and 175% of control in muscle tissue of fish Channa punctatus respectively after exposure to 40% and 80% of LC₅₀ 24h of FDLPNI after 96h (Table- 3 ). Table 3 represent the results of withdrawal of treatment of FDLPNI (80% LC_50:24h) after seven days. There was significant (P<0.05) recovery in the level of total protein (88% and 99%) in which in Channa punctatus, total free amino acid (150% and 126%), total nucleic acid level such as DNA (88% and 90%), similarly RNA (91% and 94%), glycogen (88% and 81%), and protease (117% and 145%) of control in liver and muscle tissues of Channa punctatus (Table-3).

 

Table.3 Changes in total protein , total free amino acids, nucleic acid (DNA and RNA), glycogen level and activity of enzyme protease in liver and muscle tissues of Channa puntatus after exposure to 40% and 80% of LC₅₀ (24h ) of freeze dried latex powder of Nerium indicum after 96h and 7^th day after withdrawal.

Values are mean +_ SE of six replicates.

Values in parentheses are % level with control taken as 100%.

Data were analyzed through student’s test.

*, Significant (p>0.05), when treated groups were compared with controls.

 +, Significant (p>0.05), when withdrawal groups were compared with treated groups.

Level of total protein, total amino acid, nucleic acid (DNA and RNA) expressed in µg/mg, glycogen in mg/g and activity of enzyme protease expressed in µ moles of tyrosine equivalent/mg protein/h.

DISCUSSION:

The establishment of piscicides in fish and the subsequent death involves a chemical interaction between the piscicides and the fish, data given in biochemical section of results indicates that extracted compounds affect the several molecular targets at the same time.

 

Data of result, indicates that after exposure to 96h of fish with sub-lethal doses of extracted compounds caused significant (P<0.05) decreasement in the level of total protein, glycogen and nucleic acids and enhancement in level of total free amino acid and activity of enzyme protease.

 

The metabolic potential oriented more towards liver as it forms the set of metabolic regulation and muscle, which forms the mechanical tissues intended for mobility. The proteins mainly constitute enzymes, hormones and free amino acid etc., which play a major role in the metabolism of animal (Sambasiva Rao, 1999). The lesser amount of protein in muscle tissue could be due to the structural organization for mechanical purposes. Toxic compounds enter the animal body through gills and reach the liver, where the liver tries to detoxify the compounds by degrading it. Thus in addition to its usual role in maintaining the dynamic nature of physiological functions liver also perform the function of detoxification and for this need more energy (Arasta, et al., 1996).

 

The depletion of protein fraction in liver and muscle tissues may have been due to their degradation and possible utilization of degraded products for metabolic purposes. Increase in free amino acids level was the result of breakdown of protein for energy requirement and impaired incorporation of amino acids in protein synthesis (Singh, et al., 1996a). It also attributed to lesser use of amino acids (Seshagiri Rao et al., 1987) and their involvement in the maintenance of an acid-base balance (Moorthy et al., 1984). It is likely that the increased free amino acid pool of the respective tissue may be intended to maintaining the osmoregulation of fish whose physiological capabilities are disrupted under toxic stress. Kabeer Ahammad Sahib et al., 1981 and Siva Prasad Rao et al., 1983 reported that exposure of malathion and methyl parathion in fish Tilapia mossambica causes loss of mono as well as bi-valent ions such as Na+, K+, Ca++ etc. from the tissues of pesticides intoxicate fish in preference to the retention of osmotically active organic anions like free amino acid, lactate etc. and water also. Natarajan (1985) suggested that stress conditions induce elevation in the transamination pathway. Inhibition of RNA synthesis may also affect protein and amino acid levels. Inhibitions of DNA synthesis, thus, might affect both protein as well as amino acid level by decreasing the level of RNA in protein synthesis machinery.

 

The incensement in protease activity collaborates with enhancement in free amino acid level in both the tissues may be due to the formation of which might be the result of protein hydrolysis. This is further supported by a remarkable decrease in protein level in liver and muscle tissue of fish Chana punctatus and Mystus mystus. Here the elevation on protease activity is highest in liver, suggesting the highest toxic impact felt on liver tissue. Similar increase in protease activity was reported by several workers in various animals including mammals (Millward, 1970; Sivaiah, 1980; Kabeer Ahammad Sahib, et al., 1984).

 

Active moieties present in the plant extracts are toxic to freshwater fish Channa punctatus and Mystus mystus. Use of these plant products for the control of weed and predatory fishes as well as crustaceans in freshwater bodies will be advantageous due to their easy availability, low cost, easily biodegradability and greater acceptance by users. The reversibility in action and non-toxic effect on mammals are the important positive feature of these botanical piscicides, which reduce the threat of environmental pollution.

 

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