Genotoxic effect of effluents discharged in Ramgarh Lake on Freshwater fish Channa punctatus

 

Kumari Mamta, Ajay Singh*

Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur- 273009 (U.P.) India

 

ABSTRACT:

Pollution of water resources is a serious and growing problem, despite the existence of relevant legislation. Genotoxic studies of effluent pollutants are very important as they tend to accumulate in aquatic animals. Thus use of aquatic organism to detect the genotoxicity is very useful in environment monitoring. Ramgarh Lake is an important water body of Gorakhpur, U.P. India, which received untreated effluents from different sources of Gorakhpur city. It covers an area of 723hectares, with a circumference of 18 kilometers. The aim of the Present study was to evaluate the toxic impact of these effluents on water quality as well as genotoxic effects on fish. Micronuclei test was employed to study genotoxicity inhabiting in lake. A significant (p<0.05) increment in number of micronuclei were found in fishes directly caught from different sites of lake. Seasonal variation in Physico-chemical parameters (i.e. Temperature, pH, DO, BOD, COD, TSS, and TDS) of water body also measured on basis of American Public Health Association (APHA) method. All these parameters were much higher than the tolerance limit recommended by Central Pollution Control Board of India.

 

 

 

INTRODUCTION:

Pollution of the aquatic ecosystem is recognized as potential threat to all living organisms. It is produced by man himself; therefore pollution and its effects are considered man’s greatest crime against himself. Maximum aquatic pollutants come from industrial and agricultural run-off. The importance of the fisheries sector has been highlight as a major food source. Fish makes a vital contribution to the survival and health of a significant portion of the world’s population. Aquatic ecosystems are continuously loaded with anthropogenic pollutants of which heavy metals ions, microbial toxins and polycyclic aromatic hydrocarbons forms an important group of concern.

 

These pollutants are affecting stages of the aquatic food chain which may lead to the disturbance of the whole ecosystem. Interrelationship of physico-chemical and biological conditions has been investigated in various aquatic habitat by a number of workers like [1-8]. Pollutants may be genotoxic and may lead to several human afflictions like cancer, atherosclerosis, cardiovascular diseases and premature ageing. Genotoxicity is a deleterious action, which affects a cells genetic material affecting its integrity [9-10]. Genotoxic pollution of aquatic ecosystem describes the introduction of contaminants with mutagenic, tetragenic and carcinogenic potential into its principal media and genome of the resident organisms [11-12]. These contaminants include certain chemical compound like heavy metals [13-15] Microbial toxins [10] and polycyclic aromatic hydrocarbon (PAHs) [16-19]. These genotoxicants have been reported to cause mutation because they form strong covalent bond with DNA, resulting in the formation of DNA adducts preventing accurate replication [20]. Genotoxins affecting germ cells (sperm and eggs) can pass genetic changes down to descendants and have been implicated to be against sustainable development principles by WHO Portraying them as significant factors in congenital anomalies, which account for 589,000 deaths in human annually. Fishes are most susceptible to genotoxic effects caused by the pollutants usually agricultural waste, chemical, heavy metals etc. The selection of fishes as a model in the ecogenotoxicology studies could be made since fish is a very sensitive bio-indicator of water quality and can highlight the potential danger of new chemicals introduced in the aquatic environment [21] and also respond to toxicants in a manner similar to metabolize xenobiotics and accumulate pollutants [22]. The aim of this study was to evaluate the effects of pollutants released in lake without treatment on water quality along with genotoxic effect on fish inhabiting in this lake.

 

MATERIALS AND METHODS:

Study area:

Ramgarh lake is a large natural lake situated southeast of Gorakhpur in eastern Uttar Pradesh, India, on National Highway-28 lies between Latitude 26˚46’ N and Longitude 83˚22’E, covers the geographical area of 3483.8 Sq km. It lies within the floodplain of River Rapti and outflow into it through a drain called Gurrah Nalla. Lake is cover an area 723hactares and its embankment area is 18 km long. The lake receives storm water runoff and waste water through several drains such as the Kuda Ghat Nalla, Gordhaiya Nalla, Mohaddipur power house Nalla, Bichhiya Nalla, Padley Ganj Nalla etc.

 

Selection and collection of water samples from sites:

Selection of three different sites on the basis of effluent discharged in Ramgarh lake.

Site 1: Padley Ganj Nalla Gorakhpur, U.P.

Site 2: Bichhiya Nalla railway colony Gorakhpur, U.P.

Site 3: R.K.B.K. Maruti servicing centre Kunraghat Gorakhpur, U.P.

 

Sampling was done in summer, rainy and winter season from depth ranging from 25-50 cm at various points. Care was taken to avoid any disturbance by loose sediments. The collected samples transported immediately to the laboratory and the physical and chemical characteristic effluents and water samples estimated. The samples were analyzed for Temperature, pH, DO, BOD, COD, TSS and TDS values. The procedure described in standard methods for the examination of effluents wastes on the basis of APHA [23] has been adopted in the analytical techniques. The values obtained were compared with standards prescribed by WHO and Central Pollution Control Board of India (Table -1).

 

Ramgarh Taal, Gorakhpur, Uttar Pradesh – 273001

 

Genotoxicity experiment:

The common fresh water fish Channa  punctatus were collected from the different polluted site and adjoining place and then were used for micronuclei test next day. Fish had an average weight of 42.03 g and average length 18.22 cm. Blood smear slides were prepared by the method of Das and Nanda [24]. Peripheral blood samples were obtained from caudal vein of fish. At each assessment 2500 cell/fish were analyzed (7500 erythrocytes for each site). Slide just dried in air, fixed in absolute methanol for 10-15 minute, and stained in Geimsa (pH 7.0) for 1 to 2 hours. The average frequencies of abnormalities were determined for each site as presented in table-2. MN frequencies (MN %) was calculated as follows:

 

MN%=             Number of cells containing micronucleus × 1000

                                    Total number of cells counted

 

Then the mean ± standard error for each site was calculated. Student’s t-test was employed for comparison of control group and polluted fishes [25].

 

RESULTS:

Physico-chemical parameters study:

Water is the basic element for the existence of aquatic fauna and its specific properties as a cultural medium are naturally of great significance in the productivity of lake. The fresh water bodies are being greatly affected by excessive influence of human activities. The interaction of physical and chemical parameters of any aquatic system determines the nature organisms inhabiting it. The result of physico-chemical analysis of water samples of different seasons from selected sites are given in Table-1.

 

Temperature of water samples varied with different seasons. The maximum temperature was recorded in summer while minimum in winter season.  The highest temperature observed in summer season at site-1 which is 30-33˚C and lowest temperature 17-20˚C in winter season at site-3.

Table 1: Physico-chemical characteristic of polluted water samples collected from different sites of Ramgarh lake in summer (May to June 2015), rainy (August to September 2015) and winter (November to December 2015)

Characteristics	Season		Magnitude			General standard by pollution control board
			Site-1	Site-2	Site-3	Inland Surface Water
Temperature	Summer	Upstream	30-33˚C	29-31C	28-30C	Shall not exceed 5˚C above the receiving water temperature
		Entry point	30-32˚C	29-30˚C	28-30˚C
		Downstream	30-33˚C	28-30˚C	28-30˚C
	Rainy	Upstream	26-27˚C	25-26˚C	24-26˚C
		Entry point	26-27˚C	25-27˚C	25-28˚C
		Downstream	26-27˚C	26-27˚C	24-26˚C
	Winter	Upstream	20-22˚C	18-20˚C	17-20˚C
		Entry point	20-22˚C	18-21˚C	17-20˚C
		Downstream	20-22˚C	18-20˚C	17-20˚C
pH	Summer	Upstream	7.2±0.09	6.9±0.03	7.0±0.09	5.5-9.0
		Entry point	7.5±0.06	6.9±0.03	7.7±0.04
		Downstream	7.5±0.06	6.1±0.03	7.0±0.06
	Rainy	Upstream	7.9±0.06	6.1±0.03	7.0±0.06
		Entry point	7.9±0.06	7.0±0.03	7.0±0.03
		Downstream	7.2±0.03	7.5±0.06	7.0±0.03
	Winter	Upstream	8.5±0.03	7.6±0.10	6.6±0.15
		Entry point	8.4±0.07	7.4±0.07	6.4±0.07
		Down stream	8.0±0.06	6.9±0.01	7.2±0.04
DO	Summer	Up stream	7.21±0.043	8.36±0.080	10.04±0.047	4-13mg/L
		Entry point	7.31±0.036	8.55±0.142	10.36±0.019
		Down stream	7.26±0.038	8.13±0.030	10.33±0.069
	Rainy	Up stream	8.22±0.04	10.76±0.038	10.23±0.030
		Entry point	8.06±0.030	10.33±0.069	10.31±0.036
		Down stream	7.25±0.065	10.33±0.030	10.28±0.028
	Winter	Up stream	7.23±0.030	10.46±0.050	10.33±0.030
		Entry point	7.13±0.073	10.5±0.072	10.35±0.061
		Down stream	7.31±0.094	10.33±0.030	10.26±0.038
BOD	Summer	Up stream	468.1±3.87	274.6±0.962	76.36±0.087	30 mg/L
		Entry point	442.8±0.435	270.8±0.366	75.3±0.040
		Down stream	443.5±0.656	271.1±0.366	74.33±0.069
	Rainy	Up stream	481.6±0.384	284.8±1.299	77.38±0.064
		Entry point	480.6±0.384	280.8±0.366	76.58±0.095
		Down stream	481.1±0.030	283±0.849	77.71±0.076
	Winter	Up stream	502.1±0.366	330.8±0.366	72.23±0.019
		Entry point	503.1±0.830	333.6±0.652	70.23±0.038
		Down stream	505.5±1.020	336.3±0.304	73.21±0.036
COD	Summer	Up stream	1126.4±0.069	438.5±0.101	424.3±0.561	250 mg/L
		Entry point	1120.3±0.054	430.25±0.031	421±0.471
		Down stream	1125.1±0.036	435.8±0.208	422±0.471
	Rainy	Up stream	1180±0.030	682.7±0.102	291.5±0.065
		Entry point	1182.4±0.047	680.4±0.038	293.1±0.280
		Down stream	1185.2±0.038	684.3±0.033	293.1±0.280
	Winter	Up stream	1210.2±0.065	454.6±0.652	252.8±0.597
		Entry point	1212.3±0.059	455.3±1.097	251.5±0.874
		Down stream	129.4±0.069	453.8±0.495	252.1±0.497
TSS	Summer	Upstream	1174.8±1.460	532.1±0.471	682.3±0.652	100 mg/L
		Entry point	1181.3±0.384	541.1±0.366	673.3±0.769
		Downstream	1183.3±0.769	521.2±0.344	671.5±0.513
	Rainy	Upstream	1103.5±0.390	281±0.471	312.3±0.451
		Entry point	1112.6±0.561	276.3±0.652	311.3±608
		Downstream	1107.8±0.723	273.6±0.990	316.6±0.962
	Winter	Upstream	1212.1±0.089	423.6±1.065	453.8±0.955
		Entry point	1214.6±0.090	432.8±0.723	450.6±0.304
		Downstream	1215.8±1.422	434±1.247	431.6±0.509
TDS	Summer	Upstream	4668.1±0.280	133.8±0.955	1804.5±1.07	20-1000mg/L
		Entry point	4660.8±0.366	132.6±0.608	1706±0.548
		Downstream	4650.6±0.561	131.6±0.384	1804±1.217
	Rainy	Upstream	6563.8±0.955	311.5±0.565	2316±0.935
		Entry point	6563±1.471	307.5±0.612	2213±0.772
		Downstream	6563±0.641	305±0.471	2315±0.769
	Winter	Upstream	4492.5±0.657	134.8±1.164	2852±1.164
		Entry point	4493±1.3374	133±1.598	2707±0.612
		Downstream	4497.1±0.280	126±0.333	2775±0.435

 

 

 

pH of water is an important environment factors. The pH value measure of the acidity and alkalinity of water and is one of the most stable measurements. Highest value of pH observed in all three season at site-1 i.e. 7.6±0.10 to 8.5±0.06 and minimum in site-2 and 3.

 

During Dissolved oxygen experiment result was found that the highest DO measured in site-3 about 10.04±0.047-10.35±0.061 and minimum in site-1 in summer and winter season which is 7.21±0.043-7.25±0.038 and 7.13±0.073-7.23±0.030 respectively.

 

During all season find that the highest value of Biological Oxygen Demand (BOD) in site-1which is 502.1±0.366 to 505.5±1.020 in winter season observed and lowest value of BOD in summer, rainy and winter season of site-3. Chemical oxygen demand, represents the amount of oxygen required oxidizing all the organic matter both biodegradable and non-biodegradable. Maximum in summer, rainy and winter season at site-1 which is 1126.4±0.069 to 1212.3±0.059 and lowest value of COD summer to winter were found that 424.3±0.0561to 252.1± 0.497 in site-3.

 

The same results of Total suspended solid (TSS) and Total dissolved solid (TDS) were found, the highest TSS in summer 1174.8±1.468 to 1183±0.769, rainy 11.3.5±0.390 to1107±0.723 and winter 1212.8±0.089 to 1215.8±1.422 of site-1.Minimum in site-2 and site-3 in rainy season which is 281±0.471 to 273.6±0.652 and 312.3±0.451 to 316.6±0.962 respectively. The highest value of TDS in rainy of site-1which is 6563.8±0.955 to 6563±0.641 and lowest value of TDS were noted in site-2 and 3.

 

Genotoxicity Observation:

Control fishes showed almost negligible micronuclei when compared to those fish which were caught from Different sites. In MN test different types of aberrations were determined from three selected site of Ramgarh Lake and these were MN, NN, LN, NB etc. The results of micronucleus and nuclear abnormalities in peripheral erythrocytes of Channa punctatus is as show in Table-2. In all site, which were found that the frequencies of MN and other nuclear abnormalities observed significantly higher p<0.05 when compared with control group. The highest MN frequencies observed in site-1 i.e summer 14.8±1.09, rainy 10.2±0.33 and in winter 12.6±0.87. Lowest MN found in site-3 as compared to site-1 and site-2.

 

Table 2: Frequencies of formation of micronuclei and nuclear anomalies.

Value are mean ± SE of six replicate. Data were analyzed though Student’s t-test. Significant (p<0.05) treated group were compared with control. NN=Notched nucleus, LN=Lobed nucleus, NB=Nuclear bud, MN=Micronucleus. 

 

 

 

DISCUSSION:

Temperature is a very important for its role in chemical and biological activities of organisms in the aquatic media. Temperature of waste water is commonly high because of addition of warm water from industrial activities [26]. Increase in temperature also increases the rate of microbial activity. Temperature increases may become barrier to fish migration and in this way seriously affect on reproduction of species. The fluctuation of pH is linked with chemical changes, species composition and life processes [27]. It is a measure of the acidic and alkalinity of water and is one of the stable measurements. The desirable limit of pH recommended by Central pollution control board of India and WHO. Dissolve oxygen is a very important parameter in water quality assessment. Its presence is highly effective for maintenance of biological life of aquatic ecosystem. Higher DO during winter might also be due to photosynthetic activities of aquatic plants and specific types of algae at upper level of the water body. The increased of DO content with the abundant growth of the phytoplankton bloom [28]. Assessment of dissolved oxygen (DO) is a major characteristic in all pollution related ecological studies according to [29]. Increase in BOD can be due to heavy discharge of industrial waste water effluents, animal and crop waste, house hold materials and domestic waste. The greater the BOD, the more rapidly oxygen is available to higher forms of aquatic life. COD is similar in function to BOD, in that both measure the amount of organic compounds in water. It estimates carbonaceous factor of organic matter [30]. COD gives out the amount of organic pollutants found in surface water, proving COD as useful measure of water quality, which indicates the mass of oxygen consumed [31]. Total dissolved solid are the amount of solid present in dissolved and suspended form [32]. The desirable limit of total solid recommended by Central pollution control board of India WHO, 20-1000 mg/L and 500-1500 mg/L respectively. The suspended solid, the suspended matter consists of the particle of different types colloidal particles of various organic complexes. MN test is one of the best tests for assessing genotoxicity of pollutants in fishes with relative large chromosomes number. Increasing concentration of nitrogenous metabolites in blood affects the series of enzymatic system in fish and induced the formation of reactive oxygen species (ROS). Production of reactive intermediates such as ROS, are highly toxic cause DNA lesion in fish blood [33]. DNA lesion can be different in shape and size such as in bud form (Attaches to the main nucleus), lobed form (envagination of nuclear membrane), or in bridge form, however, each anomalies are the signal of cytogenetic damages in fish that could be resulted with failures in cell division, cell death process with mutagenity [34-36]. Gene amplification via the breakage-fusion-bridge cycle could cause formation of nuclear anomalies during the elimination of amplified DNA from the nucleus [37]. Abnormal cell division due to the blocking of cytokinesis can also cause binucleation that may lead genetic imbalance in the blood [38].

 

CONCLUSION:

It is evident that Ramgarh Lake gets more polluted because effluent directly discharged into water body without any treatment from different sites. Experiment conducted on water quality concluded that, except DO all the physico-chemical parameters i.e. Temperature, pH, BOD, COD, TDS and TSS value was higher as comparison to level recommended by Central Pollution Control Board of India as well as in case of genotoxicity significant damaged the nucleus and form micronuclei and other nuclear abnormalities.

 

ACKNOWLEDGMENTS:

One of the author (Kumari Mamta) is deeply grateful to the University Grant Commission (UGC), New Delhi for awarding Rajiv Gandhi National Fellowship (RGNF), vide sanction letter no. F1-17.2015-16/RGNF-2015-17-Sc-UTT-8159.

 

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