V{tÑàxÜ 3 MATERIALS AND METHODS In the present study, water samples from different stations of Pamba river were collected and analyzed for water quality status during pre-monsoon, monsoon and post- monsoon seasons. Two selected fishes Cyprinus carpio (Linnaeus, 1759) and Puntius sarana (Hamilton-Buchanan, 1822) were maintained in the laboratory in river water collected during different seasons and exposed to different bacterial inocula to study the haematological, biochemical and histopathological changes due to the stress induced by the bacteria. 3.1 Study Area The river Pamba, the third largest river in Kerala, is originating from the Piramedu plateau, second segment of forest in the southern Western Ghats, adjacent to the north of Agasthyamalai range. The main tributaries are Kakki Ar, Azhutha Ar, Kakkad Ar and Kalli Ar which join with the Pamba river in the high lands. The rivers Manimala and Achenkovil join with it in the plains. After meandering through Idukki, Pathanamthitta and Alleppey districts in Kerala over 176 km, the river ultimately flows into the Vembanad lake. The Sabarigiri Hydroelectric Project, the second major Hydel Scheme in Kerala, is situated in this river. The famous forest shrine of Swami Ayyappa (Sabarimala Pilgrim Centre) is in the north western foothills of Pamba plateau and the mount plateau. Over the years it has become one of the most popular pilgrim centres in South India and millions of pilgrims visit the shrine during the months of December and January and also during the first day of every Malayalam month.
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V{tÑàxÜ 3
MATERIALS AND METHODS
In the present study, water samples from different stations of Pamba river were
collected and analyzed for water quality status during pre-monsoon, monsoon and
post- monsoon seasons. Two selected fishes Cyprinus carpio (Linnaeus, 1759) and
Puntius sarana (Hamilton-Buchanan, 1822) were maintained in the laboratory in
river water collected during different seasons and exposed to different bacterial
inocula to study the haematological, biochemical and histopathological changes due
to the stress induced by the bacteria.
3.1 Study Area
The river Pamba, the third largest river in Kerala, is originating from the Piramedu
plateau, second segment of forest in the southern Western Ghats, adjacent to the
north of Agasthyamalai range. The main tributaries are Kakki Ar, Azhutha Ar,
Kakkad Ar and Kalli Ar which join with the Pamba river in the high lands. The rivers
Manimala and Achenkovil join with it in the plains. After meandering through
Idukki, Pathanamthitta and Alleppey districts in Kerala over 176 km, the river
ultimately flows into the Vembanad lake. The Sabarigiri Hydroelectric Project, the
second major Hydel Scheme in Kerala, is situated in this river. The famous forest
shrine of Swami Ayyappa (Sabarimala Pilgrim Centre) is in the north western
foothills of Pamba plateau and the mount plateau. Over the years it has become one
of the most popular pilgrim centres in South India and millions of pilgrims visit the
shrine during the months of December and January and also during the first day of
every Malayalam month.
Chapter 3 Materials and Methods
36
3.1.1 Sampling stations
Ten sampling stations (Site-1 to Site-10) were selected for investigation (Fig.1). The
sampling sites were fixed as per the requirements of the study and extending an area
of about 80 km. from Azhutha Ar in the upstream to Thakazhi area in the down
stream.
3.2 Water quality analysis
The water samples for the present investigation were collected at monthly intervals
from ten sites of Pamba river for a period of two years from January 2005-December
2006. Surface water samples were collected in sterilized bottles from the identified
sites of River Pamba, brought to the laboratory taking necessary precautions and
analyzed for the various physico-chemical and bacteriological parameters following
standard methods (APHA, 1998). The study was carried out during three seasons,
Pre-monsoon, Monsoon and Post-monsoon.
3.2.1 Physico-chemical parameters
Temperature (oC)
The temperature of water was recorded using a digital thermometer at the site itself.
Turbidity
Turbidity of the water sample was analyzed by a Nephelometric turbidity meter
(Systronics). When light is passed through a sample having suspended turbidity,
some of the light is scattered by the particles. The scattering of the light is
proportional to the turbidity. The turbidity of a sample is thus measured from the
amount of light scattered by the sample taking a reference with standard turbidity
suspension. Nephelometer measures the scattered light at the right angle of the path
of the incident light. Turbidity is expressed in NTU.
Chapter 3 Materials and Methods
37
Electrical conductivity (EC)
Electrical conductivity is the measure of the ability of an aqueous solution to carry an
electric current. It was measured with the help of a conductivity meter having a
conductance cell containing electrodes of Platinum coated with Pt Black or Carbon.
The unit of conductivity measurement is µ/m Siemens(S)/cm. The conductivity of
most waters is generally low and hence the unit µ mhos /cm is commonly used.
Total Dissolved Solids (TDS)
Total Dissolved Solids was determined using TDS-conductivity meter (Systronics)
that was calibrated using standard KCl solution and expressed in ppm.
Hydrogen ion concentration (pH)
Hydrogen ion concentration (pH) was measured by a digital pH meter (Eutech).
Total Alkalinity
Total alkalinity is the measure of the capacity of the water to neutralize a strong acid.
The alkalinity in the water is generally imparted by the salts of carbonates,
bicarbonates, phosphates, nitrates, borates, silicates etc. together with the hydroxyl
ions in the free state. Alkalinity is measured as carbonates and bicarbonates by the
titration method with a strong acid (HCl or H2SO4) first to pH 8.3 using
Phenolphthalein as an indicator and then further to pH between 4.2 and 5.4 with
Methyl Orange indicator. In the first case, the value is called as Phenolphthalein
Alkalinity (PA) and in the second case it is Total Alkalinity (TA) and expressed as
mg Ca CO3 / l.
Total Hardness
Hardness of water is defined as the presence of significant concentration of salts of
metallic cations mainly Ca2+ and Mg 2+ions dissolved in water. It is expressed in
terms of CaCO3 concentration in mg/l. The hardness may range from zero to
hundreds of milligram per litre, depending on the source and treatment to which the
Chapter 3 Materials and Methods
38
water has been subjected. It is determined by complexometric titrimetric method
using Ethylene Diamine Tetra Acetic acid (EDTA) as titrant and Eriochrome Black-
T (EBT), a dye used as an indicator.
Chloride
Chloride, in the form of chloride ions is one of the major inorganic anions in water
and wastewater. Chloride is estimated using Argentometric method with standard
silver nitrate as titrant and potassium chromate as the indicator solution. The end
point is indicated by the formation of brick red colour.The chloride content is
expressed in mg / l.
Dissolved Oxygen (DO)
The azide modification of Winkler’s method is used for the determination of DO and
expressed in mg O2 / l.
Biochemical Oxygen Demand (BOD)
BOD is the measure of the degradable organic material present in a water sample,
and can be defined as the amount of oxygen required by the microorganisms in
stabilizing the biologically degradable organic matter under aerobic conditions. A
five day BOD test was conducted to measure the BOD of the sample using BOD
incubator. The reagents used are same as that of DO. The difference of the initial and
the final DO is the BOD. BOD mg O2 / l = Initial DO - Final DO.
Nitrate – nitrogen
Nitrate was determined using the Brucine -sulphanilic acid method. An aliquot of
sample was refluxed with 30% NaCl, 4:1 H2SO
4 and Brucine-sulphanilic acid. The
intensity of the yellow colour formed was measured at 410nm using
spectrophotometer (Genesys-Thermospectronic). The reaction is highly dependent
upon the heat generated during the test and was carried out in controlled heating at
Chapter 3 Materials and Methods
39
constant time. The concentration of NO3N was found out from the standard curve
and expressed as mg / l.
Phosphate- phosphorous
Phosphate was determined by the stannous chloride method. An aliquot of sample
was made into alkaline by adding phenolphthalein followed by NaOH. The solution
decolourised with H2SO4. Then the sample was subjected to persulphate digestion for
converting all the forms of phosphates into one form. After 30 minutes of digestion,
the sample was cooled to room temperature and one drop of phenolphthalein
indicator was added. Then neutralized the solution using NaOH or H2SO4.
Ammonium molybdate and stannous chloride was added to the digested solution to
give intensively coloured molybdenum blue and the colour developed was measured
at 690nm using spectrophotometer. The concentration of phosphate was determined
from the standard curve and expressed as mg / l.
3.2.2 Bacteriological parameters
3.2.2.1 Collection of sample
Water samples for bacteriological analysis were collected in sterilized containers and
kept in icebox and carried to the laboratory as early as possible. The samples were
then analyzed for bacteriological parameters such as faecal coliforms (FC) and total
heterotrophic bacterial (THB) load.
3.2.2.2. Faecal coliform
The most probable number (MPN) of coliform bacteria was determined by the three
tube dilution method using EC broth. A 10ml, 1ml and 0.1ml of appropriate diluted
samples were inoculated into respective dilution tubes containing inverted Durham’s
tubes. 10ml samples were inoculated into 10ml double strength EC broth and 1ml
and 0.1ml sample into single strength medium of 10ml each. Inoculated tubes were
incubated at 44.4°C for 24-48 hours and examined for the growth and gas
Chapter 3 Materials and Methods
40
production. The MPN index was determined by checking the number of positive
tubes in each set and comparing the values with standard MPN table (APPENDIX-II).
3.2.2.3 Isolation of total heterotrophic bacteria (THB)
One ml of the water sample from each site was serially diluted with sterile distilled
water. Appropriately diluted water samples were plated on Nutrient Agar (NA)
medium using spread plate and pour plate technique. The inoculated plates were
incubated at 37oC for 48-96 hours. After incubation plates with countable range (30-
300 colonies) were selected for counting using colony counter and the bacterial load
in the sample was expressed as total colony forming units (cfu) per ml of water
sample.
3.2.2.4 Characterization of total heterotrophic bacteria (THB)
Morphologically different colonies were isolated, restreaked to ensure purity and
maintained on Tryptic Soy Agar (TSA) vials for further characterization. The
cultures were then identified as various genera as per the Bergey’s manual of
determinative bacteriology (Buchanan and Gibbons, 1984).
Gram staining
The Gram staining was done to classify bacteria into two groups, viz., Gram-positive
forms and Gram-negative forms. The Gram-positive forms are those which retain the
primary stain when washed with a decolourizing agent and the Gram-negative forms
are those which do not retain the primary stain when a decolourizing agent is used
but then take up the colour of the counter stain. In this method (Aneja, 1996) the air-
dried and heat fixed bacterial smear is subjected to the following staining reagents in