Chapter 3

Material and Methods

3.1 Wan Reservoir

The Wan Reservoir is medium sized reservoir. Government of Maharashtra

construction across Wan River in 20th November 1963 .Wan Reservoir are located at
Nagapur, Parli-Vaijnath, Tehsil , District Beed , Marathwada region.(M.S), India, its
is 14 km, away from Parli –Vaijnath town. This and longitude 760.27 E. the earthen
dam of 1981 meters height and 2188.40 meters in length on wan river. The reservoir
have 2371.40 km. catchments area gross storage capacity 25.181 MCM, more than 51
villages of Beed, Parabhani district have been benefited from this project .this
project used by irrigation, fishery purpose ,it also supply the drinking water to Parli-
Vaijnath town .and Parli-Vaijnath co-operative sugar factory,supply to the Thermal
power station it situated near Parli-Vaijnath town .The reservoir consists of two canals
i,e .left canal 9.65 kames and right canal is 12.87 km. Head discharge capacity is 1.77
m3/s, under irrigation 7,567 hectors .more than 51 villages of Beed ,Parabhani
district has been benefited from this project, and supply water to thermal power
station unit no.3,4,5,6,7,and unit no.8.Agriculture ,and culture of piciculature.
3.2 Selection of sampling station/sites
Sr. no Sampling station /sites Direction
1 Sampling station A South

2 Sampling station B East

3 Sampling station C North

4 Sampling station D North

For convenient monitoring, systematic field study and sampling of water, four
permanent sampling were fixed in the Wan Reservoir. These stations were fixed
according to different in degrees of human interactions within different parts of the
Wan Reservoir systems, and also as zones of special ecological interests. The stations

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were fixed after a detailed survey of the reservoir systems these stations were
designated as Wan Reservoir station (WRS-A),(WRS-B),(WRS-C),(WRS-D).

3.3 Sampling sites3.

3.1 Sampling station A

The sampling station “A” is located on the south side of the reservoir, this side is
a upstream water inflow come from Ambajogai municipal waste water. This sampling
take place time start at the early morning between 9.00 am to 3.00 pm in evening,
every sampling to all sampling ,same time and same day .Sampling were take place
the Second and third week between of every month, water samples were immediately
brought into laboratory for the Estimation of various Physico-chemical parameters.
Was highly polluted side cloth washing and bathing domestic animal and disposal of
domestic sewage.

3.3.2 Sampling station B

The sampling station “B” is located on East side of the reservoir. this side nearby
where the municipal corporation drinking water supply to Parli-Vaijnath city, this site
more visited tourism Person and boat riding, washing cloth, and cattle etc. and due to
create pollution. Which is distance between sites ‘A’ and ‘B’ from ½ Km?

3.3.3 Sampling station C

Sampling station “C” is located on the North side of the reservoir. There were
human activity like that was washing the cloth, this side was showed overflow side of
Reservior, (sandawa),water flow going at downstream river.

3.3.4 Sampling station D

Sampling station ‘D’ is located on the North side .This side show the left the water
through channel for Agriculture purpose, where some human activity like that
washing the cloth and domestic water came up from nearby village .Haswalamba ,
Mandhekhel , Davanapur , & other villages etc.

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3.4 Field visit and sample collection

The sampling was done in first week of every month from each sampling sites A
,B, C,& D. from the Period of June 2013 to May 2015. I was started my Journey From
Parli- Vaijnath to Nagapur village with Taxi , I had been reached Nagapur village of
Wan Reservoir at that time 9.00 am .usually started sampling from station “A” at 9.00
am continued order, WRS-A,WRS-B, WRS-C, and last one sample I usually collected
the from the WRS-D, at 3.00 pm. All the water samples for laboratory analysis were
packed appropriately in well insulated boxes filled with ice cubes. After reached the
laboratory at next day about 10 am, the samples for BOD measurement were
immediately incubated and other was kept in the fridge for next five days analysis.

3.5 Water sampling collection

Taken water sample for surface water less than 1-5cm, at WRS - D, and WRS-A,
were done from the shores but surface and depth sampling at the and collection of
water samples in designated bottles. And these collection and preservation of water
samples for laboratory analysis we always carried with us separate relabeled and
preservatives and all other requirements for this study.

3.6 Collection of water samples for Physico-chemicalanalysis

Physico-chemical analysis of water sample of selected four sites was carried out
for different parameter as described in APHA (1989), Trivedy and Goel (1984) and
Khodarkar (1992) water samples were kept in darkness in iceboxes at 40c till the
samples reached the laboratory for analysis.

3.7 Collection of water samples for BOD

The Samples for BOD analysis were collected from surface (1-5cm) two such
bottles were used for each samples. one was fixed on the spot immediately after the
collection following Winkler method (Trivedy and Goel,1986),and second bottles
containing water kept in darkness at 40c(iceboxes) till it reached the laboratory.

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3.8 Collection of water samples for phytoplankton analysis

The sampling was done in first week of every month from each sampling sites, A,
B, C,& D. For period June 2013 to May 2015.The phytoplankton samples was
collected with the help of plankton net made up of blotting silk cloth no.25 with the
mesh size 50µm.konw volume of sample 200 liter was used to collect surface water
.The collected water samples were concentrated plankton was collected from the
plankton bottle fixed at the bottom of net the samples were preserved in 4 % .Lugols
Iodine. The phytoplankton samples were transported in laboratory for analysis.

3.9 Sampling of Zooplankton study

Zooplankton samples was collected monthly from different sampling sites

namely A, B, C, and D, for the period of June 2013 to May. The sample were
collected at 9 am to 3.00 pm .during first week of each month .zooplankton samples
was collected by filtering 200 liters of water through plankton net made up of
standard bolting silk cloth no-25 with the help of zooplankton net. Since majority of
freshwater phytoplankton are smaller than the commonly used mesh size, 50 µm, the
most common is a simple and conical net with bottle attached at its lower end. The
samples collected were concentrated to a 50 ml volume and preserved in 4% formalin,
the samples were brought to laboratory for qualitative and quantitative analysis and
the identification was done with the help of standard text and Identification was made
up to the species level with help of standard texts, keys and monographs. Tonpai and
APHA (1995).the qualitative analysis of zooplankton was carried out using Sedgwick
Rafters plankton counting chamber.

3.9.1 Zooplankton Analysis

Sedgewick Rafter counting cell Method

Sedgwick rafter counting cell is used to count the planktons. Sedgwick rafter cell
is approximately 50mm long, 20mm wide and 1mm deep. A trinocular compound
microscope is used to count the zooplankton with different eyepieces such as 10x and
40x.the microscope is calibrated using micrometer. The following formula is used for
quantities analysis of zooplanktons.
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 Number /ml = C x1000
AxDxF

C = no of organism courted

A = Area of field

D = Depth of field (mm) (S-R) Death 1mm

F = NO. of fields counted

The zooplankton density was expressed on individual /litre. Finally to

zooplankton can be identified by using laboratory with the help of keys provided by
pemak (1978) the zooplankton and phytoplankton taxonomy we identified and
counted under an inverted microscope OLYMPUSK 40 and picture of various
zooplanktons.

3.10.1 Plankton Analysis

Sample drop Method

For phytoplankton analysis was the simple drop method was used. an ordinary 4
ml dropper was taken and samples were sucked in the dropper and 2-3 drops were
taken on the slide for observation. Organisms present in this drop were counted in
high power .numbers of drop forming 1ml for the dropper was taken into
consideration the following formula was used. The phytoplankton density was
expressed on units/ liter.

Phytoplankton (density): N = abc /L

Where

N = No of phytoplankton /lit.

a = average number of individuals in one drop

b=Number of drops forming 1ml

c = Volume of concentrated

L = original water sample (liter)

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 During the year June 2013 to May 2015 phyroplankton analyzed were
Euglenophyceae, chlorophyceae and Bacilillariphyceae and Myxophyceae was
recorded .From Wan Reservoir The samples for phytoplankton study were collected
from all the four station in separate clean plastic bottles .Water samples were
collected separately for the study of all the general phytoplankton and benthos. For
the study of phytoplankton 1 litter samples were collected from the surface water at
all the station in clean plastic Jars and were fixed immediately using Lugholes iodine
solution (1ml: 10) (Tragedy and Goel, 1986).

3.11 Collection of water samples for Bacteriology

Samples for MPN and fecal coli from analysis were collected from surface water
of each station separately in 100ml pre sterilized dark bottles and kept in darkness at
40c during sampling and were transported to laboratory ,these had been kept in ice
boxes of 40c (Tragedy and Goel,1986).

3.12 Study of primary productivity

Primary productivity studies were conducted for a period of primary productivity

of surface water samples only were measured by light and dark bottle method
(Incubated on the spot for 3 hrs) (Tragedy and Goel,1986)

3.13 Field measurements

Air temperature ,water temperature,(of both the surface ) were measured using a
glass Thermometer, pH, Dissolved oxygen (Do), Conductivity, Total Dissolved solid
.Conductivity and pH were also measured using separate pocket testers, all the data
were reorder in separate field books .The samples for DO were fixed on the spot using
winkers Iodometeric method (Derived and Goel,1986).Total depth and transparency
(Measured with the help of Sochi disc 20cm diameter black and white iron plate were
measured for each station,

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3.4 Laboratory water analysis

3.4.1 Physico –chemical Parameters

DO, Carbon Dioxide (CO2), Total Alkalinity, Total Hardness, Chloride and Sulphates
, were measurement Titrometrically following method

3.4.1.1. Temperature

Temperature of the water was determined on site during sampling by inserting

thermometer (0-1000c) in the water.

3.4.1.2. pH

PH was measured with help of field pH meter Hanno model champ.

3.4.1.3. Conductivity

Electrical conductance is the ability of a substance to conduct the electrical current .in
water, it is the property caused by the presence of various ionic species. It is generally
measured with the help of a conductivity meter having a conductance cell containing
electrodes of platinum coated with carbon .These electrodes are mounted rigidly and
placed parallel at a fixed distance. (Trivedy and Goel, 1986)

3.4.1.4. Transparency

Light penetration in water is greatly influenced by the optical properties which in

turn are mainly influences by the particulates impurities present in it . Light
penetration in water is recorded with the help of a round disc, Secchi , with different
strips of two contrasting colours, white and black .The secchi disc is a circular disc of
metal; generally 20cm. disc Transparency of water to light was measured using by
secchi disc. Calculate light penetration as follows.

Light penetration =Depth at which disc disappears + Depth at which disc reappears 2

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3.4.1.5. Turbidity

The turbidity of water was measured with help of Nephelometer method of range
0.5-40.When light is passes through a sample having suspended turbidity, some of the
light is scattered by the particles .The scattering of the light is generally 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 (Trivedy
and Goel , 1986).

3.4.1.5. Total dissolved solid

Total dissolved solids are determined as the residue left after evaporation of the
filtered sample. (R .K .Trivedy, Goel.P.K , 1984).

Calculation:

TDS, g/l = A-B x 1000

V

(Where, A = Final weight of the dish in gm

B= Initial weight of the dish in gm

V= volume of sample taken in ml.

3.4.1.6 Dissolved oxygen (DO)

The dissolved oxygen was determined by modification of winkers Iodometeric

method (APHA 1989). The samples was collected in 300ml capacity ground stopper
glass B.O.D bottles without any bubbling .Then 2ml of each saturated manganese
sulphates and alkaline iodine Aside solution were added. The precipitate was
dissolved by adding 2ml of con.H2SO4 in sample water and the liberated iodine was
titrated against standard sodium this sulphates (0.025N) solution using starch as an
indicator the calculation was carried out with the help of following formula.

Do (mg/lit) = 0.1x ml.titrant x1000

100

1ml of hypo = 0.1 mg of DO

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3.4.1.7. Dissolved carbon dioxide
The dissolved carbon dioxide was calculator by titrating the water samples against
0.44 NaOH and resultant change in pH from acid to neutrality to alkalinity was
detected by phenolphthalein .The calculation was carried out win the following

formula.CO2 (mg/lit) = ml of titrate x normality of titrate x44x1000

ml of sample

3.4.1.8. Total Alkalinity

It was measured by titrating the sample with 0.1N H2SO4 using methyl orange as
in indicator .the alkalinity was calculated by following formula.

Total alkalinity (as CaCo2) = ml of acid required x normality x 1000

ml of sample
3.4.1.9. Chlorides
Chlorides were determined by Argent metric method using silver nitrate as
indicator. The end point was permanent brick red ppt. The chloride contents in water
sample were calculated by using following formula.

Chloride mg/lit = (ml x N) of AgNO3x35.5x1000

ml of sample
3.4.1.10. Sulphates

Sulphtes estimation was carried out by Gravimetric method (APHA-1986).

Sulfate is precipitated as barium sulphates in the hydrochloric acid medium by
addition of barium chloride solution.The reaction is carried out near the boiling
temperature .the precipitate is filtered, washed to remove the chlorides ,dried or
ignited and weighed as BaSO4.Many substances interfere in performing this test
.Suspended matter silica, nitrate and sulphate. Alkalinity metal sulphtes cause the low
result .presence of other metals such as iron and chromium also yield low result due to
the formation of metal sulphates. After developing the turbidity by addition of barium
chloride crystals to the acidified water samples.

SO4.ml/lit = mg BaSO4 x 411.5

ml of sample

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3.4.1.11. Hardness

Hardness is generally caused by the calcium and magnesium ions present in

water .Hardness of water determined by EDTA method as described in APHA (1986).
The pH of sample was increased to with the help of ammonium with the buffer
solution. Eriochrome Black T indicator was added in this alkaline water samples,
which forms a wine std .EDTA solution the EDTA breaks the complex and forms
blue colored company .The end point was permanent blue colour .The amount of
EDTA solution required was noted .The hardness of water sampler was calculate by
using following formula.

Hardness (as CaCO3mg/lit) = ml of ETDA used x1000

ml of sample

3.4.1.12. Biological oxygen demand (BOD)

B.O.D of water sample was determined by the method described in (APHA,1986) .the
samples were prepared for each sample, two containing diluted samples and
remaining two containing dilution water (Blank).First day dissolved oxygen was
determined from one bottle and samples of one bottle of blank by Winkler Aside
method (APHA,1989).The remaining bottles were incubated at 200c for five days
.after incubation period; the dissolved oxygen of these water samples was determined
.the BOD was calculated as follows.

B.O.D mg/lit = (S1-S5) – (B1-B5\) x dilution factor

Where = S1 = D.O of sample on first day

S5 = D.O of sample after 5 days is incubation.

B1 = D.O of blank water on first day

B5 = D.O. of blank water after 5 day’s incubation.

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3.5 ANALYSIS OF BIOLOGICAL PARAMETERS-

3.5.1. Phytoplankton

Each of the 1lit samples collected was centrifuged to concentrate of the plankton
organisms. Plankton samples were collected with standard plankton net made of silk
bolting cloth no.25. The amount of water filtered during the plankton net was about
200 lit. the samples collected were concentrated to of 50 ml volume and preserved in
4 % formulain .General phytoplankton were studied for quantitative and qualitative
details counting was made using a spectromicrope with 40x magnification using
Lackeys drop method (Tragedy and Goel,1986), and thus the phytoplankton of the
concentrate was calculated.

The actual count/L were calculated from this by dividing it the with the
concentration factor 10.colonical and filamentous forms were counted a single units.
The Phytoplankton was identified using the standard keys provided by palmer (1980),
and Reyndds (1980).The micro-photo group of each representative sample were taken
using digital camera.

3.5.2. Most probable number of coli forms organism (M.P.N).

It was estimated by multiple tube fermentation technique described analysis kept

in the fridged were carried out immediately in the next morning .The MPN count was
detected with the help of the chart provided by Tragedy and Goel,1986).The
biological studies for E -coil were carried out in the laboratory of microbiology of the
RRI , kottayam , according to the standard procedures of APHA (1995).The samples
were inculcated in Mac Coney’s broth having there .following composition pepton,-
0.5gm,meat-extract,-0.5gm,Lactose,-1.0gm,Na-Taurocholate -0.5gm,Neutral (1%),-
0.7ml.ph,-7.4. set each with five tube was prepared the first set comprised of 5 tubes
each containing 10 ml of double strength medium and third set of 5ml broth in
eachtube.10 ml of water sample was inoculated in each tube of set 2.0 and 0.1ml of
sample was inoculated each tube of set 3.0 all tubes were incubated at 370c for 48
hrs. The numbers of positive tubes (acid and gas) from each tube were noted and
computed on Macconkey’s tube given in APHA (1989) for estimation of most
probable total number of coil from organisms from sampler per 100 ml.

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