MATERIALS AND METHODS

Transcription

1 MATERIALS AND METHODS The present investigation was made for the assessment of physicochemical and biological characteristics of river Varuna at Varanasi. The study was conducted during January 2009 to December The entire work has been divided into three sections: SECTION I: PHYSICOCHEMICAL PARAMETERS OF RIVER VARUNA Sample collection: Samples of river water were collected at monthly intervals on the mid of every month from January to December 2009 in the early hours (8.00am am). Water samples were collected in clean 2.0 lt. plastic bottles previously rinsed with river water. Samples of the water were collected from five predetermined sites (Map-1): Site meters upstream to Imaliya Ghat; Site 2- Imaliya Ghat; Site 3- Behind hotel Clarkes; Site 4- Near State Bank of India, Kachahari; Site 5- Chaukaghat. Depending on the magnitude of the pollution load, water samples had been collected from various locations of the river Varuna. So the area under study has the potentiality to investigate the status of water pollution. Site-1 was the entry point from where the river enters into the city, which is, generally, free of sewage. Site-2 is situated in cantonment area and receives domestic sewage of nearby area. Site-3 receives sewage from hotels and adjoining residential area. Site-4 receives sewage from Orderly bazaar area and waste from slaughter house through Orderly bazaar

2 nallah. Site-5 receives heavy sewage from Sampurnanand Sanskrit University and Teliabagh area and waste from battery workshops, automobile servicing stations and dyes from saree printing houses. Analysis of physicochemical Parameters of river water: Samples were brought to the laboratory carefully and were analysed for Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Chloride, Nitrate, and Phosphate. Temperature, Transparency, ph, Dissolved Oxygen (DO), Dissolved CO 2, Alkalinity were analysed at the sampling sites. Standard Methods for Examination of Water and Wastewater, American Public Health Association (APHA), American Water Works Association and Water Pollution Control Federation (2005) were used to analyse different parameters of the river water. Physico-chemical Parameters 1. Temperature - Temperature of river water was recorded by means of Celsius thermometer at the time of sampling and expressed in degree centigrade ( 0 C). 2. Transparency - Secchi disc (20 cm diameter) was used to measure the transparency of river water at the sampling sites using following formula. Transparency = A+B 2 Where, A = the distance (in cm) at which disc disappear from the view in descent. B = the distance (in cm) at which disc reappears in ascent. 3. ph- By ph meter

3 4. Alkalinity - Titration method was used to estimate the dissolved alkalinity of water samples. 100 ml of sample is taken in two conical flasks. 0.5 ml of phenolphthalein indicator is added to one flask, other flask being control. If the sample becomes pink, titrate it with N/50 H 2 SO 4 until the pink colour just disappears. Add two drops of methyl orange indicator in both the conical flasks and titrate one with N/50 H 2 SO 4. The end point is orange (compared with control). Record the ml of acid used both in phenolphthalein and methyl orange titration. Total Alkalinity (mg/l) = No. of ml of N/50 H 2 SO 4 consumed X 1000 ml of sample 5. Dissolved Oxygen (DO) - Titration method was used to estimate the dissolved oxygen. Manganous sulphate (1 ml) and alkaline iodide (1 ml) reagent mixed to the 100 ml sample bottle. A flocculent precipitate formed. 1.0 ml of conc.h 2 SO 4 added to dissolve the precipitate. 50 ml of this solution is transferred to a conical flask and titrated by N Na 2 S 2 O 3 till the colour turns pale yellow. Then added starch solution (1 ml) to give a blue colour and the titration is completed by turning it colourless. Dissolved Oxygen (mg/l) = No. of ml of Na 2 S 2 O 3 solution X 4

4 6. Dissolved Carbon Dioxide - Titration method was used for the estimation of dissolved carbon dioxide. Phenolphthalein indicator (2 drops) mixed to 50 ml sample. If sample remains colourless than titrated with N/44 NaOH, till the colour turns pink. Dissolved CO 2 (mg/l) = No. of ml of N/44 NaOH consumed X Biological Oxygen Demand (BOD) - BOD is the amount of oxygen taken up by microorganisms that decompose organic waste matter in water. It is, therefore, used as a measure of the amount of certain types of organic pollutant in water. Standard BOD determination is done by incubating samples for 5 days at 20 0 C. BOD (mg/l) = DO (initial) DO (5 days) Decimal fraction of dilution 8. Chemical Oxygen Demand (COD) The COD of the water represents the amount of oxygen required to oxidize all the organic matter both biodegradable and non-biodegradable by a strong chemical oxidant (KMnO 4 ). 50 ml of water sample and 50 ml of distilled water (blank) taken in conical flask and 5 ml of KMnO 4 is added to both flasks. Both the flasks were heated on a waterbath at boiling point for one hour. After cooling 5 ml of potassium iodide (10%) and 10 ml of H 2 SO 4 (25% v/v) were added to both flasks. Both flasks titrated with 0.1 N sodium thiosulphate using starch as indicator. COD (mg/l) = (B-S) X N X 8 X 1000 Sample volume in ml B = Volume of titrant used in blank S = Volume of titrant used in sample N = Strength of the titrant

5 9. Chloride Mohr s method was applied for the determination of the chloride present in the sample water. The water was titrated with silver nitrate using potassium chromate as an indicator. The chloride content was calculated using the following formula Chloride (mg/l) = (a-b) X N X X 100 ml sample a = volume of AgNO 3 used for the sample b = volume of AgNO 3 used for the blank N = normality of AgNO 3 ( N) 10. Phosphate 50 ml of the sample taken and 2 ml of acid- ammonium molybdate reagent added. Then added 2 drop of stannous chloride, and waited for 5 minutes and the blue colour developed is matched with standards prepared in phosphate free distilled water. Phosphate (mg/l) = No of ml of standard phosphate X 0.01 X Nitrate The phenol disulphonic acid method was applied for the analysis of nitrate-nitrogen. The steam dried water samples were dissolved in phenol disulphonic acid (2 ml). The alkaline medium was made by adding ammonium hydroxide (10 ml). The development of yellow colour denoted presence of nitrate-nitrogen. The colour intensity was proportional to the amount of nitrate-nitrogen and was measured with the help of colorimeter at 410 nm in terms of optical density. The final calculations were made with the help of standard graph.

6 SECTION II: HAEMATOLOGICAL PARAMETERS OF FISHES General information on the handling of the experimental fishes: For the experiment, live specimens of Clarias batrachus (both sexes; body wt: , length: cms) and Channa punctatus (both sexes; body wt: gms, length: cms) were locally collected. Only the healthy fishes having no external signs of injury and disease were selected for experiments. The fish were fed daily with formulated fish food, containing 35.2% crude protein, two times per day during acclimatization to laboratory conditions for two weeks in plastic pools. For experiments the fish were kept in four identical glass aquaria each containing 10 litres of the medium. 15 fish were kept in each aquarium. The medium was renewed daily. All care was taken to avoid giving stress to the fish. Fish were not fed 24 hours before and during the experiment. EXPERIMENTAL DESIGN Experiment I: Clarias batrachus were divided into two groups (A and B). They were given the following treatments: Group A: Fish of this group were kept in fresh tap water (Control). Group B: Fish of this group were kept in river Varuna water, procured from site-5 (Experimental). Experiment II: Channa punctatus were divided into two groups (C and D). They were given the following treatments: Group C: Fish of this group were kept in fresh water (Control). Group D: Fish of this group were kept in river Varuna water, procured from site-5 (Experimental).

7 TECHNICAL PROCEDURES Haematological estimation: Blood samples were collected in Ria tubes containing EDTA anticoagulant by sectioning of the caudal peduncle. Five fishes from each group i.e. Group A (Clarias batrachus; Control), Group B (Clarias batrachus; Experimental); Group C (Channa punctatus; Control) and Group D (Channa punctatus; Experimental) were severed for the blood collection and estimation of haematological parameters. Haematological parameters like haematocrit (Ht %), haemoglobin (Hb g/dl), red blood cells (RBC) counts (M/µl), differential leucocyte counts (DLC), mean corpuscular volume (MCV) (fl), mean corpuscular haemoglobin (MCH) (pg) and mean corpuscular haemoglobin concentration (MCHC) (g/dl) were determined by fully automatic digital Haematology Cell Counter machine, Abbott CD 1700 (U.S.A.) whereas differential count was done on blood film stained with May Grumwald- Giemsa stain. SECTION III: CALCIUM METABOLISM IN FISHES Calcium metabolism in fishes from each Group (i.e. A, B, C, and D) was done under following parts: (A) Estimation of plasma calcium and phosphate levels of the fishes. (B) Histological study of calcium regulating endocrine glands of the fishes. (A) Estimation of plasma calcium and phosphate levels of the fishes: Blood of five fishes from each Group (i.e. A, B, C, and D) were collected in five tubes for mineral determination.

8 Blood samples were collected in heparinized tubes by sectioning of the caudal peduncle. The plasma was separated by centrifugation and analysed for calcium level (OCPC method) and inorganic phosphate levels (by Mod. Gomorri s method). (B) Histological study of calcium regulating endocrine glands of the fishes: Preparation of histological slides: After collection of blood samples the tissues for histological studies from each Group (i.e. A, B, C, and D) were fixed as follows- Ultimobranchial gland: The area adjoining the heart along with the oesophagus were removed and fixed in Bouin s fluid. Corpuscles of Stannius: Corpuscles of Stannius along with the adjoining portion of kidney were removed from the fish and fixed in aqueous Bouin s fluid. Pituitary: The pituitary gland along with the brain was fixed in aqueous Bouin s fluid. Tissues were routinely processed in graded series of alcohols, cleared in xylene and embedded in paraffin. Serial sections were cut at 5 μm. The pituitary was stained with Herlant tetrachrome and Heidenhan s azan technique. Ultimobranchial glands were stained with hematoxyline-eosine (H-E). For CS aldehyde fuchsin and HE stains were used. IV Statistical analysis: Values are presented as mean±sd (standard deviation). For statistical analysis, the student s t test was applied to determine the statistical significance.

9 Figure 1: Map of river Varuna showing the study sites (Site-1, 2, 3, 4 and 5).

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