11 Chapter III

Chapter III
Methodology
The present environmental pollution monitoring study was performed at the Bhavan’s
College campus of Andheri, Mumbai, (Photographs.1-2) to investigate the level of pollution
in water, sediment, fishes and plants.
1.Area of study
Mumbai is a developed and over crowded city of Maharashtra. It is lies between 18° 96’
north latitude and 72° 81’ east longitude. The standard altitude ranges from 10 m to 15 m.
The main portion of the Mumbai city is at the sea level and the maximum height of the city is
at 450 meters.The total area of Mumbai city is 603.4 km2.
2. Water Sampling and preservation
The water samples were collected for four times in a month in morning, afternoon and
evening session from different locations along the Bhavan’s Lake. The samples were
collected and then analysed for an interval of twelve months from June 2011 to May 2012.
Plastic containers were used.
3. Sediment sampling and preservation
The sampling was done with help of hollow plastic pipes having 7cm diameter driving
into the sediment. The sediment cores recovered in a dry place and cut after bringing in the
lab into slices at three intervals for the first 15 cm, then15–25 cm, and finally for the later
sections of the cores. Different layers of sediment samples where mixed very well and
preserved till analysis. Such sampling was done in morning and evening sessions along
different locations of the lake. The grab samples collected in two sessions for a month were
mixed to give gross sample. Such gross samples drawn for a month where air dried,
grounded and stored in polyethylene bags until further analysis.
4. Vegetation and biota (Fish) sampling
The vegetation (mangroves), and fish (C.catla also known as Indian Carp) samples were
collected randomly from different sampling locations.The samples were cleaned by washing
with water twice and then with double distilled water.
5. Requisites
The analytical reagent reagent were taken. The apparatus were washed nitric acid then
with tap water and distilled water Finally were dried in oven and stored in a clean place,
(Radojevic and Bashkin,1999).
6. Sample Preparation
a)Heavy metals
The water samples were evapourated to one tenth, 10 mL of 2% ammonium pyrrolidine
dithiocarbamate, 4 mL of 0.5 M HCl was added and extracted with 10 mL of methyl isobutyl
ketone (MIBK). The MIBK extract containing the desired metals was then diluted to give
final volumes. (Chen and Ma, 2001). The sample solution was then aspirated.
In a glass beaker 2 g of each well- mixed soil samples were taken and digested for 2 hrs
aqua regia on a sand bath and dissolved in 10 mL of 2% nitric acid, and finally diluted with
distilled water (Chen and Ma, 2001).
Vegetation samples were carefully washed to remove all stuck soil particles. Samples dried
at 100 ± 1°C in a hot-air oven for 3 hours. The samples were powdered in warm condition
and passed through sieve. The samples were initially digested with nitric acid and further
with aqua regia on a sand bath (Lark , et al., 2002). After evapouration to a smaller volume
the samples were filtered and diluted upto 50 mL with deionized water.
The fish samples were cut and soft tissues were seperated; then dried in freezer for 3days
and finely grinded to a homogenous powder. The freeze-dried tissue was subjected to nitric
acid digestion in a Teflon polytetrafluroacetate closed digestion vessel (Baldwin, et al,
1994). Digests were stored between 0–5 °C until analysis.
b)Physico-Chemical study
The water samples were directly analysed whereas 1:5 soil suspension was prepared by
adding 100 ml of distilled water to 20 gm of soil.The suspension was stirred mechanically for
about 1 hour at regular intervals and filtered through filter paper using Buchner funnel and
vacuum pump.
7.Heavy metals Analysis
The analysis Copper (Cu), Lead (Pb), Zinc (Zn), Calcium (Ca), Chromium (Cr), Cadmium
(Cd), Nickel (Ni), Iron (Fe), Mercury (Hg) and Arsenic (As) was done by Perkin- Elmer
ASS-280 Flame Atomic Absorption Spectrophotometer (Photograph.15). Details analyses of
individual metals are as follows:
7.1.Copper
1. Instrumentation
Atomic absorption spectrometer
2. Operating parameters
1) Instrument: Atomic absorption spectrometer
i. Make-Perkin Elmer
ii. Model - AAS-280
2) Light source :Hallow Cathode lamp
3) Flame: Acetylene Flame
4) Wave length: 324.8 nm
3. Materials and equipments
Calibrated Class ‘A’ glassware,double distilled water, Reagents.
4. Procedure
Step -1Preparation of standard stock solution:
Copper, 1000µg/ml solution is prepared in 1litre with 1% nitric acid.Prepare standard
solution, having known concentration of the metal to be determined in the same solvent.
Step -2: Preparation of Test solution: Aspirated directly
Calculation:
µg/ml = (sample absorbance) X (Standard concentration in µg/ml)
Standard absorbance
7.2.Lead
1. Instrumentation
ii. Model - AAS-280
4. Procedure

Lead,1000µg/ml solution is prepared in 1litre with 1% nitric acid.Prepare standard solution,
having known concentration of the metal to be determined in the same solvent.
Calculation:
µg/ml = (sample absorbance)X (Standard concentration in µg/ml)
Standard absorbance
7.3.Zinc
1. Instrumentation
ii. Model - AAS-280
4. Procedure
Zinc, 500µg/ml solution is prepared in 1litre with 1% Hydrochloric acid..Prepare standard
Calculation:
Standard absorbance
7.4.Calcium
1. Instrumentation
ii. Model - AAS-280
4. Procedure
Calcium,500µg/ml.To 1.249g of primary standard calcium carbonate in 50ml of deionised
water. Add dropwise a minimum volume of HCl (approximately 10ml) to effect complete
solution of CaCO3. Dilute to 1 litre with deionised water.Prepare standard solution, having
known concentration of the metal to be determined in the same solvent.
Calculation:
Standard absorbance
7.5.Chromium
1. Instrumentation
2. Operaring parameters
ii. Model - AAS-280
4. Procedure
Chromium,1000µg/ml, Potasium chromate solution is prepared in 1 litre deionised
water.Prepare standard solution, having known concentration of the metal to be determined
in the same solvent.
Calculation:
Standard absorbance
7.6.Cadmium
1. Instrumentation
ii. Model - AAS-280
4. Procedure
Cadmium,1000µg/ml, solution is prepared in 1litre with 1% Hydrochloric acid..Prepare
standard solution, having known concentration of the metal to be determined in the same
solvent.
Calculation:
Standard absorbance
7.7.Nickel
1. Instrumentation
ii. Model - AAS-280
4. Procedure
Nickel,1000µg/ml, solution is prepared in 1litre with 1% nitric acid.Prepare standard
Calculation:

Standard absorbance
7.8.Iron
1. Instrumentation
ii. Model - AAS-280
4. Procedure
Iron,1000µg/ml, solution is prepared in 1litre with 1% nitric acid..Prepare standard solution,
having known concentration of the metal to be determined in the same solvent.
Calculation:

Standard absorbance
7.9.Mercury
1. Instrumentation
ii. Model - AAS-280
4. Procedure
Mercury,1000µg/ml, is prepared by with mercury (II) oxide 1litre with 1% Hydrochloric
acid. Prepare standard solution, having known concentration of the metal to be determined in
the same solvent.
Calculation:

Standard absorbance
7.10.Arsenic
1. Instrumentation
ii. Model - AAS-280
4. Procedure
Arsenic,1000µg/ml using primary standard AsO3 solution is prepared in 1litre with 1%
sulphuric acid.Prepare standard solution, having known concentration of the metal to be
determined in the same solvent.
Calculation:
Standard absorbance
8.Physico Chemical study.

The water was analyzed for pH, alkalinity, biological oxygen demand (B.O.D), chemical
oxygen demand (C.O.D), dissolved oxygen (D.O), total hardness, chlorides, sulphates,
conductivity and total bacterial count. The soil sediment samples were collected and
analyzed for pH, chlorides, sulphates and phosphates.The method and procedures followed
for sampling, conservation and elucidation as per ICMR (1975), Trivedy, et
al.,(1984),Hem (1985), APHA (1995) and De (1994).
8.1. pH of water samples
pH of water samples was determined after calibrating digital pH meter with buffer solution of
pH 4 and pH 9.
8.2. Alkanity of water by Titrimetric Method
Reagents
1.Sodium carbonate solution,1N:
Weigh accurately 13.25 g anhydrous sodium carbonate,Na2CO3 (previously dried at 140 oC
for 2 hrs).Dissolve it in little distilled water and make up to 250 ml in volumetric flask.
2.Sulphuric acid,1N:
Place 28 ml conc. Sulphuric acid in a 1000 ml volumetric flask and make up to the mark with
carbon dioxide free distilled water.Standardize it against 1N sodium carbonate solution using
methyl orange indicator.(End point is appearance of faint orange colour).
3.Sulphuric acid,0.02N:
Dilute appropriate volume of 1N H2SO4 (about 20 ml.) to 1000ml. in volumetric flask with
carbon dioxide – free distilled water, to give a 0.02N solution.
1.0 ml. exactly 0.02N H2SO4 = 1.0 mg. CaCO3
4.Phenolphthalein indicator: Dissolve 500 mg phenolphthalein in 50 mL ethyl alcohol
or isopropyl alcohol and add 50 mL distilled water. Then add 0.02 N sodium hydroxide drop-
wise until a faint-pink colour appears.
5.Mixed indicators : Dissolve 20 mg methyl red and 100 mg bromo cresol green in 100
ml. 95% ethyl alcohol or isopropyl alcohol (if the sodium salts of indicator are used
,prepare the solution with distilled water instead alcohol).
Procedure
Phenolphthalein alkanity : Place 25 ml or 50 ml or appropriate volume of the sample
(requiring not more than 25 ml. of the titrant) in a 250 ml. conical flask. Add 5 drops of
Phenolphthalein indicator solution. If no pink colouration occurs, there is no phenolphthalein
alkanity. If pink colour appears then titrate with 0.02N Sulphuric acid ( to pH 8.3 ) until the
solution becomes colourless.
Total alkanity: Add 3 drops of mixed indicator solution to the solution in which
phenolphthalein alkanity has been determined and titrate against 0.02N Sulphuric acid (to pH
4.5) to light pink.(If methyl orange is used as indicator, the end point is from orange to red).
If the sample contains suspended matter remove by filtration or centrifugation and then
determine the alkanity.If the suspended matter contains any alkanity, add a known excess of
standard acid to the sample, boil off carbon dioxide, cool and back- titrate with standard
alkali.
Calculation
If the sulphuric acid used for titration is exactly 0.02N
Phenolphthalein
ml.
alkanity = ml. 0.02 N H2SO4 for phenolphthalein end point x 1x
1000
(asCaCO3) ,mg/l ml of sample taken for titration
Total alkanityml. = ml. 0.02 N H2SO4 for total alkanity end point x 1x
1000
(asCaCO3) ml of sample taken for titration
,mg/l
8.3.Biological Oxygen Demand (B.O.D),

1. Phosphate buffer solution: Dissolve 8.5 g potassium dihydrogen phosphate , 21.75 g
dipotassium hydrogen phosphate , 33.4 g disodium hydrogen phosphate heptahydrate and
1.7 g ammonium chloride in about 500 ml distilled water and dilute to 1 litre. The pH of this
buffer should be 7.2 without further adjustment.
2. Magnesium sulphate solution: Dissolve 22.5 g MgSO4.7H2O in distilled water and dilute to
1 litre.
3. Calcium chloride solution: Dissolve 27.5 g anhydrous CaCl2 in distilled water and dilute to
1 litre.
4. Ferric chloride solution: Dissolve 0.25 g FeCl3.6H2O in distilled water and dilute to 1 litre.
5. Sodium sulphite solution 0.025 N: Dissolve 1.575 g anhydrous Na2SO3 in 1 litre distilled
water. This is to be prepared daily.
Procedure
1. Take the required distilled water .
2. Add buffer, MgSO4 solution, CaCl2 solution and FeCl3 solution 1mL in 1litre distilled
water.
3. Neutralised the samples to pH 7.
4. Add few drops of sodium sulphite to destroy the chlorine in the sample.
5. Prepare suitable dilutions of the samples as per required concentrations.
6. Take the required quantity of sample dilute with water up to the 650 mL mark.
7. Fill solution in one B.O.D. bottle keep incubating and in other to determine the initial DO
in another immediately.
8. Finally fill two B.O.D . bottles with the dilution water alone . One of bottle is incubated
and the other is used for determining the initial dissolved oxygen.
9. The bottles to be incubated are placed in the incubator at 27°C for 3 days .The bottles are
sealed.
10. The initial dissolved oxygen contents in the other set of bottles is determined results are
noted.
11. The dissolved oxygen content in the incubated bottles is determined at the end of 3 days
incubation period.
(Di – Do )
B.O.D.mg/L = P
Where,
P = decimal fraction of sample used.
Di = initial DO .
Do = DO at the end of 3 days incubation.
8.4. Chemical Oxygen Demand (C.O.D)

Apparatus
1. Reflux apparatus
2. Burettes
3. Pipettes
Reagents
1. Standard potassium dichromate solution 0.25 N: Dissolve 12.259 g K2Cr2O7 primary

standard grade previously dried at 103°C for 2 hours and dilute to 1 litre.
2. Sulphuric acid-Silver sulphate reagent: Dissolve 10.1 g Ag2SO4 in 1000 mL conc. H2SO4
and keep it for 2 days. This is the reagent.
3. Standard ferrous ammonium sulphate 0.1 N: Dissolve 39.29g Fe(NH4)2(SO4)2. 6H2O in
distilled water. Add 20 mL conc. H2SO4 , cool and dilute upto 1 litre. Standardise against the
standard dichromate solution. Dilute 10 mL standard K2Cr2O7 solution to about 100 mL. Add
30 mL conc. H2SO4 and cool. Titrate with ferrous ammonium sulphate titrant using 23 drops
of ferroin indicator.
4. Ferroin indicator solution:Dissolve 1.485 g of 1,10- phenonthroline and 0.695g of ferrous
sulphate (FeSO4 .7H2O) in distilled water to make 100 ml of solution.
5. Mercuric sulphate ,Hg SO4,solid.
6. Sulphuric acid crystals
Procedure
1. Place 50.0 mL of sample in a 500 mL flask.
2. Add 1g mercuric sulphate and a few glass beads.
3. Add sulphuric acid to dissolve the mercuric sulphate and cool.
4. Add 25.0 ml (0.25 N) potassium dichromate solution and mix well.
5. Fit the flask to the water condenser .
6. Add (70 mL) acid reagent through the condenser and mix well.
7. Apply heat and reflux for 5 hours.
8. After cooling rinse the condenser thoroughly with distilled water.
9. The mixture is diluted to about two times its volume and then cooled to ambient
temperature.
10. The excess dichromate is titrated against standard ferrous ammonium sulphate adding two
drops of ferroin indicator.
11. The colour change from bluish green to red indicates the end point.
12. Repeat a blank using distilled water in equal volume as that of the sample.
Calculation
(V1 – V2) N x 8000
C.O.D. mg/L =
V
where,
V1 = mL ferrous ammonium sulphate used for blank
V2 = mL ferrous ammonium sulphate used for sample
N = normality of ferrous ammonium sulphate
V = volume of sample used.
8.5.Dissolved Oxygen(D.O) by using modified Winkler’s (Azide modification) method.
1. Manganous sulphate solution: Dissolve 480g MnSO4.4H2O, 400g MnSO4.2H2O or 364 g
MnSO4.H2O in distilled water, filter and dilute to 1 litre.
2. Alkali-iodide-azide reagent: 500 g NaOH or 700 g KOH and 135 g NaI or 150 g KI is
dissolved in distilled water and diluted upto 1 litre in a volumetric flask. 10 g sodium azide
(NaN3) is dissolved in 40 mL distilled water and added. The solution must be colorless with
starch when diluted and acidified.
3. Sulphuric acid concentrated: 1mL is equivalent to about 3 mL alkali-iodide-azide reagent.
4. Standard sodium thiosulphate 0.025 N: Dissolve 6.205 g sodium thiosulphate
(Na2S2O3.5H2O) to boiled and cooled distilled water and dilute to 1 litre. Add 5 mL
chloroform or 0.4 g NaOH/L or 4 g borax and 510 mg HgI2/L for preservation. Standardise
this with 0.025 N potassium dichromate solution which is prepared by dissolving 1.226 g
potassium dichromate in distilled water and diluted to 1 litre.
5. Standard potassium dichromate solution 0.025 N: A solution of potassium dichromate
equivalent to 0.025 N sodium thiosulphate contains 1.226 g/L K2Cr2O7. Dry K2Cr2O7 at
103°C for 2 hrs before making the solution.
6. 0.025 N sodium thiosulphate solution: Dissolve 24.82g of Na2S2O3.5H2O in boiled
distilled water and make up the volume to 1 litre. Add 0.4g of borax or a pallet of NaOH as
stabilizer . This is 0.1 N stock solution. Dilute it to 4 times with boiled distilled water to
prepare 0.025 N sodium thiosulphate solution. Keep in a brown glass stoppered bottle.
7. Starch Indicator: Add cold water suspension of 5 g soluble starch to approximately 800
mL boiling water with stirring. Dilute to 1 litre,heat for a few minutes and kept overnight for
settling. Use supernatant liquor. 1.25 g salicylic acid/1 litre or a few drops of toluene is
added.
Apparatus
1. 300 mL capacity bottle with stopper

2. Burette
3. Pipettes, etc.
Procedure
1. Add 2 mL of manganous sulphate solution and 2 mL of alkali-iodide azide reagent to the

bottle containing 300 mL sample, well below the surface of the liquid.(The pipette should be
dipped inside the sample while adding the above two reagents.)
2. Place stopper carefully so that all air bubbles are excluded, invert the bottle minimum
fifteen times for mixing.
3. Allow the bottle for sometime to settle, soon the precipitate settles and a clear supernatant
solution is observed shake well again.
4. After 2 minutes of settling, open the stopper cautiously, add 3 mL concentrated sulphuric
acid immediately allowthe acid to run down along the sides of the bottle.
5. Restopper and gently mix by shaking the bottle until dissolution is complete.
6. Titrate the contents , within 1 hour of dissolution of the precipitate against 0.025 N sodium
thiosulphate solution to a light straw colour. 1–2ml starch solution is added and the titration
is continued the first the blue colour disappears and note down the volume of sodium
thiosulphate solution added (V), which gives directly the D.O. in mg/L.
8.6. Total Hardness of water by EDTA Titration
Reagents
1. Calcium standard solution: Weigh accurately 1.000g of pure calcium carbonate ( dried
previously at 105 oC) and add to a 250 ml. conical flask using 50ml, distilled water.Through
a liquid funnel add 20.5 ml,1N HCl. Warm,cool and adjust to the mark with distill water.
2. Standard EDTA titrant, 0.02 N (0.01 M): Weigh 3.723 g analytical reagent grade EDTA
disodium salt (Na2H2C10H12O8N2.H2O) and dissolve in distilled water and make the upto
1litre.Standardize by titrating against standard calcium solution. Store carefully in polythene
bottles or in a pyrex bottle.
1ml.of exactly 0.02 N EDTA = 1.0 mg of CaCO3
3. Ammonia- Ammonium chloride buffer solution: a) Dissolve 16.9 g ammonium chloride
(NH4Cl) in 143ml concentrated ammonium hydroxide (NH4OH). b)Add 1.179 g of EDTA
disodium salt and 780 mg. MgSO4,7H2O (or 644 mg of MgCl2,6H2O) and dilute to 50ml with
distilled water. Add solution a to solution b and mix well then make up to 250ml. with
distilled water.Do not store more than a months supply. Discard the buffer if 1 – 2 ml added
to the sample does not produce a pH of 10 ±0.1 at the end point of titration. Keep the solution
in a plastic or resistant glass container.
4. Sodium sulphide inhibitor: Dissolve 5.0 g Na2S ,7 H2O and 3.7 g Na2S ,5 H2O in 100ml.
distill water.This inhibitor deteriorates on oxidation and air must be exduded.Keep the bottle
tightly fitted with rubber stopper.
5. Eriochrome black T indicator: Mix 0.5 g Eriochrome black T dye and 100g sodium
chloride to prepare a dry powder mixture.
Procedure
1. Take 25 ml of sample (V) and dilute upto 50 ml with distilled water in an Erlenmeyer
flask.
2.To this add 1 ml of buffer solution per 50 ml volume.The pH of titre solution must be 10.
Add 1 drop Sodium sulphide inhibitor solution or required amount of solid.
3. Put two drops of indicator solution. The solution turns wine red in colour.
4.Titrate against the standard EDTA slowly with constant stirring till reddish shade
disappears. The colour of the solution at the end point is blue under normal conditions.The
whole titration process must be over within 5 mins after the buffer is
added.
5. Note down the volume of EDTA added.
Calculation
ml of EDTA titrant x E x1000
Hardness (as CaCO3)mg/L =
ml of sample taken
where,
E = mg CaCO3 equivalent to 1 ml of EDTA titrant
if the normality of EDTA is exactly 0.02 N,then

Hardness (as CaCO3)mg/L = ml of EDTA titrant x E x1000
ml of sample taken
8.7. Chloride estimation by Silver Nitrate method

Reagents
Use chloride free distilled water for the preparation of all reagents
1. Standard silver nitrate titrant, 0.0282N

Dissolve 4.791g silver nitrate, AgNO3 in distilled water and make upto 1000ml in distilled
water , in a volumetric flask .Standardize it against 0.0282N sodium chloride solution Store it
in an amber bottle.
1 ml of 0.0282N AgNO3= 1mg Cl
2. Standard sodium chloride titrant, 0.0282N
Dissolve 1.648g sodium chloride , NaCl solution and make upto 1000 ml in volumetric
flask
1.0 ml =1.0 mg Cl
3. Potassium chromate indicator solution: Dissolve 25g. of Potassium chromate K2CrO4 in
100 ml distilled water. Add silver nitrate solution dropwise until slight red precipitate is
formed stand . Allow for 12 hrs.Filter and dilute the filtrate to 500ml.
Procedure
1. Before starting the titration rinse the burette with silver nitrate solution .Fill the burette with
0.0282N silver nitrate solution adjust zero and fix the burette to the stand.
2. Take 100 ml sample solution in conical flask (if pH is in range of 7 – 9.5 it can be directly
titrated. If pH is not within this range adjust the pH to be within this range using sulphuric
acid or sodium hydroxide solution).
3. Potassium chromate indicator solution (1 ml) is added to get light yellow solution.
4. Titrate the sample solution against silver nitrate solution until reddish colour persists ie. the
end point.
5. Conduct a blank titration by placing 100ml. chloride-free distilled water instead of sample .A
blank of 0.2ml to 0.3ml is used for the method.
Calculation
Chloride (ml. AgNO3 solution for sample – ml. AgNO3 solution
(asCl-)mg/l = for blank) x Normality of AgNO3 x 35.45 x 1000
ml. of sample taken for estimation
If Normality of AgNO3 is exactly 0.0282N then,
(ml. AgNO3 solution for sample – ml. AgNO3 solution

Chloride
for blank) x Normality of AgNO3 x 1 x 1000
(asCl-)mg/l =
8.8.Estimation of Sulphate content in water

Reagents
1.Methyl red indicator: Dissolve 100 mg methyl red sodium salt in 100ml distilled water.
2. Hydrochloric acid, 1+1.
3. Barium chloride, 5%: Dissolve 25 mg barium chloride dehydrate, BaCl2.2H2O in 500ml
distilled water. (If any turbidity appears on standing, reject the solution).
4. Silver nitrate – nitric acid reagent (to test chloride): Dissolve 8.5g silver nitrate and 0.5
ml conc. HNO3 in distilled water to prepare 500 ml reagent.
Procedure
1. Measure in to a 400 ml beaker appropriate volume of clear sample containing 8 to 60 mg
sulphate (usually 200ml).Dilute the sample to 200ml with distilled water. (if sulphate content
of the sample is very low take higher volumes of the sample and evapourate on a water bath
to 200ml).
2. pH of the sample is adjusted to 4.5-5.0 with HCl using methyl-red indicator (to orange
color). Then add additional 2 ml HCl . The samples are acidified to eliminate so that BaCO3
does not precipitate which is possible in very alkaline waters near the boiling temperature.
(In case the sample is already digested with conc. HCl to remove suspended impurities, omit
this step).
3. Boil the solution for about one minute and add 10 ml hot barium chloride solution slowly
from pipette with constant stirring.
4. Keep the beaker on a water bath to digest the precipitate at 80-90oC for atleast 2 hrs,
preferably over night.
5. Filter the contents of the beaker through an ashless filter paper (Whatman No.42 or
equivalent).Wash the precipitate with hot water till the washings give no opalescence with
silver nitrate – nitric acid reagent.
6. Place the filter paper with the precipitate in a previously weighed platinum or silica crucible
and heat in low flame with its lid loosely covered. Gradually increase the heat until the paper
chars. Do not allow the filter paper to flame as it may cause losses. After the charring is
complete ignite at 8000C for 1 hr. (with free access of air).Cool in desicator and weigh.
`Note: If silica crucible is used pre-ignite at 8000C for 1 hr, Cool in dessicator and weigh.
Calculation
Sulphate(as SO42-) mg/L = mg BaSO4 X 411.5
ml. of sample taken for determination
8.9.Conductivity
Conductivity was measured with the help of conductometer (EUIP-TRONICS, Model No.
EQ-692) at 25oC.
8.10.Total viable bacteria count Using Multiple tube fermentation method

Apparatus and materials
1. Test tubes
2. Inoculation loop
3. Durham’s vial
4. McConkey’s broth (single strength Lactose 10.0 g

medium):
Bile salts 5.0 g
Sodium chloride 5.0 g
Neutral red 0.075 g
Distilledwater 1L
pH ( at 25°C) 7.4±0.2
5. Brilliant green bile lactose broth (BGLB

Peptone 10.0 g
Broth):
Lactose 10.0 g
Oxgall 2.0 g
Brilliant green 0.0133 g
Distilledwater 1L
pH ( at 25°C) 7.2
Procedure
Presumptive test
Durham’s vials are kept inverted one in each of the test tube and 10 ml double strength
medium is added in 5 tubes, 10 ml single strength in10 tubes .
1. Sterilize the tubes at 121oC for 15 mins.

2. The water samples are thoroughly shaken instantaneously before sampling to inoculate the
test tubes.
3. The samples are put into the test tubes selected for the test with sterilized pipettes and
mixed thoroughly .
4. All these test tubes are placed in an incubator within ½ hour at 35 -37 oC.
5. Each test tube carefully examined after 48 hours. The ones with small amount of gas is
reported as (+) ve.The (+)ve tubes are proceeded for confirmatory test.
Confirmatory test.
Procedure
1. Prepare fermentation tubes with 10 ml BGLB medium and Durham’s vials. The number of
tubes to be prepared is equal to all positive tests in the presumption test.
2. Shake gently, the fermentation tubes with positive results and transfer one loopful of medium
to BGLB broth.
3. The test tubes are then incubated for 48 ± 2 hours at 35-37 oC and record the tubes with gas
formation as positive (+).
Calculation
MPN is calculated by combining of +ve and –ve tubes in the multiple tube test. The main
thing is that combinations of one test can be used.
If the 5 tubes each of 10ml, 1ml and 0.1ml proportions have been used and the results are as
follows
10 ml – 2 tubes +ve, 3 tubes -ve
1ml – 2 tubes +ve, 3 tubes -ve
0.1ml – All tubes -ve
The combination is written as 2-2-0 and MPN index according to the following table is
calculated.
Table 1:MPN/100ml for various results of positive results when 5 tubes each of 10,1,and 0.1ml
sample fractions are used.
Combinations MPN/100ml Combinations MPN/100ml
0-0-0 >2 4-3-0 27
0-0-1 2 4-3-1 33
0-1-0 2 4-4-0 34
0-2-0 4 5-0-0 23
1-0-0 2 5-0-1 31
1-0-1 4 5-0-2 43
1-1-0 4 5-1-0 33
1-1-1 6 5-1-1 46
1-2-0 6 5-1-2 63
2-0-0 5 5-2-0 49
2-0-1 7 5-2-1 70
2-1-0 7 5-2-2 94
2-1-1 9 5-3-0 79
2-2-0 9 5-3-1 110
2-3-0 12 5-3-2 140
3-0-0 8 5-3-3 180
3-0-1 11 5-4-0 130
3-1-0 11 5-4-1 170
3-1-1 14 5-4-2 220
3-2-0 14 5-4-3 280
3-2-1 17 5-4-4 350
4-0-0 13 5-5-0 240
4-0-1 17 5-5-1 350
4-1-0 17 5-5-2 540
4-1-1 21 5-5-3 920
4-1-2 26 5-5-4 1600
4-2-0 22 5-5-5 ≥2400
4-2-1 26
8.11.pH of soil sediments
Prepare 1:5 soil suspension by adding 100 ml of distilled water to 20 gm of soil.Stir the
suspension stirred mechanically for about 1 hour at regular intervals and filter from Whatman
filter paper no.50 through Buchner funnel on vacuum pump. Calibrate the digital pH meter
pH 4 buffer and pH 9 buffer and determine pH of the unfiltered soil suspension.
8.12.Alkanity of soil sediments by Titrimetric Method

filter paper no.50 through Buchner funnel on vacuum pump.
Reagents
1.Sodium carbonate solution,1N:
Weigh accurately 13.25 g anhyd. sodium carbonate,Na2CO3 (dried for 2 hrs at 140
o
C).Dissolve it in little distilled water dilute up to 250 ml in volumetric flask.
2.Sulphuric acid,1N:
Place 28 ml conc. Sulphuric acid in a 1000 ml volumetric flask and dilute to the mark with
carbon dioxide free distilled water.Standardize it against 1N sodium carbonate solution using
methyl orange indicator.(End point is appearance of faint orange colour).
3.Sulphuric acid,0.02N:
Dilute appropriate volume of 1N H2SO4 (about 20 ml.) to 1000ml. in volumetric flask with
carbon dioxide – free distilled water, to give a 0.02N solution.
1.0 ml. exactly 0.02N H2SO4 = 1.0 mg. CaCO3
4.Phenolphthalein indicator: Dissolve 500 mg phenolphthalein in 50 ml ethyl alcohol or
isopropyl alcohol and add 50 ml distilled water. Then add 0.02 N sodium hydroxide drop-
5.Mixed indicators : Dissolve 20 mg methyl red and 100 mg bromo cresol green in 100
ml. 95% ethyl alcohol or isopropyl alcohol (if the sodium salts of indicator are used
,prepare the solution with distilled water instead alcohol).
Procedure
Phenolphthalein alkanity : Place 25 ml or 50 ml or appropriate volume of the sample
(requiring not more than 25 ml. of the titrant) in a 250 ml. conical flask. Add 5 drops of
Phenolphthalein indicator solution. If no pink colouration occurs, there is no phenolphthalein
alkanity. If pink colour appears then titrate with 0.02N Sulphuric acid ( to pH 8.3 ) until the
solution becomes colourless.
Total alkanity: Add 3 drops of mixed indicator solution to the solution in which
phenolphthalein alkanity has been determined and titrate against 0.02N Sulphuric acid (to pH
4.5) to light pink.(If methyl orange is used as indicator, the end point is from orange to red).
If the sample contains suspended matter remove by filtration or centrifugation and then
determine the alkanity.If the suspended matter contains any alkanity, add a known excess of
standard acid to the sample, boil off carbon dioxide, cool and back- titrate with standard
alkali.
Calculation
If the sulphuric acid used for titration is exactly 0.02N
Phenolphthalein
ml.
alkanity = ml. 0.02 N H2SO4 for phenolphthalein end point x 1x
1000
(asCaCO3) ,mg/l ml of sample taken for titration
Total alkanity ml. = ml. 0.02 N H2SO4 for total alkanity end point x 1x
1000
(asCaCO3) ml of sample taken for titration
,mg/l
8.13. Estimation of Chloride by Silver Nitrate method

suspension stirred mechanically for about 1 hour at regular intervals and filtered from
Whatman filter paper no.50 through Buchner funnel over vacuum pump.
Reagents
Use chloride free distilled water for the preparation of all reagents
1. Standard silver nitrate titrant, 0.0282N
Dissolve 4.791g silver nitrate, AgNO3 in distilled water and make upto 1000ml in distilled
water, in a volumetric flask .Standardized it against 0.0282N sodium chloride solution Store
it in an amber bottle.
1ml of 0.0282N AgNO3= 1.0mg Cl
2. Standard sodium chloride titrant, 0.0282N
Dissolve 1.648g sodium chloride , NaCl solution and make upto 1000 ml in volumetric
flask
1.00ml =1.0mg Cl
3. Potassium chromate indicator solution: Dissolve 25g. of Potassium chromate K2CrO4 in
100 ml distilled water. Add silver nitrate solution dropwise until slight red precipitate is
formed stand . Allow for 12 hrs.Filter and dilute the filtrate to 500ml.
Procedure
1. Before starting the titration rinse the burette with silver nitrate solution .Fill the burette with
0.0282N silver nitrate solution adjust zero and fix the burette to the stand.
2. Take 100 ml sample solution in conical flask (if pH is in range of 7 – 9.5 it can be directly
titrated. If pH is not within this range adjust the pH to be within this range using sulphuric
acid or sodium hydroxide solution).
3. Add 1 ml of .Potassium chromate indicator solution to get light yellow solution.
4. Titrate the sample solution against silver nitrate solution until reddish colour persists ie. The
end point.
5. Conduct a blank titration by placing 100ml. chloride-free distilled water instead of sample .A
blank of 0.2ml to 0.3ml is usual for the method.
Calculation
(asCl-)mg/l = for blank) x Normality of AgNO3 x 35.45 x 1000
If Normality of AgNO3 is exactly 0.0282N then,

(asCl-)mg/l = for blank) x Normality of AgNO3 x 1 x 1000
8.14.Estimation of Sulphate in soil sediments

filter paper no.50 through Buchner funnel over vacuum pump.
Reagents
1.Methyl red indicator: Dissolve 100 mg methyl red sodium salt in 100ml distilled water.
2. Hydrochloric acid, 1+1.
3. Barium chloride, 5%:Dissolve 25 mg barium chloride dehydrate, BaCl2.2H2O in 500ml
distilled water. (If any turbidity appears on standing, reject the solution).
4.Silver nitrate – nitric acid reagent (to test chloride): Dissolve 8.5g silver nitrate and 0.5
ml conc. HNO3 in distilled water to prepare 500 ml reagent.
Procedure
1. Measure in to a 400ml beaker appropriate volume of clear sample containing 8 to 60 mg
sulphate (usually 200ml).Dilute the sample to 200ml with distilled water. (if sulphate content
of the sample is very low take higher volumes of the sample and evapourate on a water bath
to 200ml).
pH of the sample is adjusted to 4.5-5.0 with HCl using methyl-red indicator (to orange color).
Then add additional 2 ml HCl . The samples are acidified to eliminateso that BaCO3 does
not precipitate which is possible in very alkaline waters near the boiling temperature. (In
case the sample is already digested with conc. HCl to remove suspended impurities, this step
can be omitted).
2. Boil the solution for about one minute and add 10 ml hot barium chloride solution slowly
from pipette with constant stirring.
3. Keep the beaker on a water bath to digest the precipitate at 80-90oC for atleast 2 hrs,
preferably over night.
4. Filter the contents of the beaker through an ashless filter paper (Whatman No.42 or
equivalent).Wash the precipitate with hot water till the washings give no opalescence with
silver nitrate – nitric acid reagent.
5. Place the filter paper with the precipitate in a previously weighed platinum or silica crucible
and heat in low flame with its lid loosely covered. Gradually increase the heat until the paper
chars. Do not allow the filter paper to flame as it may cause losses. After the charring is
complete ignite at 8000C for 1 hr. (with free access of air).Cool in desicator and weigh.
Note: If silica crucible is used pre-ignite at 8000C for 1 hr, Cool in dessicator is cooled and
weighed.
Calculation
Sulphate(as SO42-)
mg/L = mg BaSO4 x 411.5
ml. of sample taken for determination
8.15. Estimation of Phosphates in soil sediments by stannous chloride

filter paper no.50 through Buchner funnel over vacuum pump.
Reagent
1. Phenolphthalein indicator: Dissolve 500 mg phenolphthalein in 50 ml ethyl alcohol or
isopropyl alcohol and add 50 ml distilled water. Then add 0.02 N sodium hydroxide drop-
2. Sulphuric acid – nitric acid reagent : 75 ml conc. H2SO4 is added carefully to 150 ml of
distilled water and cooled, conc. HNO3 (1 ml) is then added and the solution is diluted to
250 ml with distilled water.
3.Ammonium Molybdate solution:
Ammonium molybdate (25g) is dissolved in distilled water (150ml) and preserved in
polythene container. Add carefully 280 ml.of . Sulphuric to distilled water (400 ml.) and
cooled. The molybdate solution is added to the diluted acid and diluted to 1000 ml.
4.Stannous chloride solution: Dissolve 2.5g fresh Stannous chloride,SnCl2.2H2O in 100 ml.
glycerol and heat in a water bath. Mix by stirring with glass rod. The reagent being stable
does not need particular storage conditions.
5.Phosphate stock solution: Dissolve 439 mg anhydrous KH2PO4 (fw = 136.09) in
distilled and make up to to 1000 ml. in a volumetric flask. 2 drops toluene is added as
preservative.
1.0 ml. = 100µgP= 306 µg PO4
6.Phosphate Standard solution : Pipet 10.0 ml. phosphate stock solution into 1000 ml.
volumetric flask and make up to to 1000 ml. with distilled water.Prepare freshly.
1.0 ml. = 1µgP= 3.06 µg PO4
Procedure:
1. Place 100 ml. or a suitable aliquot of the sample containing not more than 20µg in a 100ml
Nessler tube . One drop phenolphthalein indicator is added in case of pink colouration,
destroy it withby adding one or two drops of sulphuric-nitric acid solution. If more than 5
drops are required, take a smaller aliquot , add phenolphthalein, discharge the pink colour
and dilute to 100 ml.
2. Into a series of 100 ml Nessler tubes, pipet appropriate volumes of phosphate working
solution covering the range upto 20 µg P.Dilute to 100 ml. Include a Nessler tube containing
distilled water (100 ml) and use as blank.
3. .In the blank, standards and sample add 4.0 ml ammonium molybdate solution and 0.5 ml
stannous chloride solution, mixing after each addition.
4. After 10 mins. but before 12 mins. measure the colour using a spectrophotometer at 690 nm.
5. Prepare a calibration curve and find out the number of micrograms of P equivalent to the
observed optional density of the sample.
6. Express the result as mg phosphate as P per litre of sample.
7. If it has to be expressed in terms of PO4 multiply by 3.06.
9.Air Pollution Tolerance Index (APTI)

The study was carried out to understand air pollution impact on different plant species
grown around the Bhavan’s College Campus. Plants of four different species namely
Pollyalthia longifolia,Caesalpinia pulcherrima,Delonix regia and Tamarindus indica
(Photographs. 3-6) were at random selected from the immediate surrounding area of campus
and termed as experimental site (ES). Three sets of wholly developed leaves were plucked
and instantly carried to the laboratory for analysis.Control samples (CS) were selected from
the botanical garden of the Bhavan’s College, Campus, with similar ecological conditions.
The plants from the experimental site were utilised for the study. The leaves taken to
laboratory were instantly weighed for fresh weight. Leaf samples were conserved in a
refrigerator until analysis.
The Air Pollution Tolerance Index (APTI) was determined by calculating the ascorbic acid,
chlorophyll, pH and relative water contents in leaf samples.
9.1.Ascorbic acid (AA)
Ascorbic acid (expressed as mg/g DW ) was estimated by 2, 6 – dichlorophenol dye
method described by Talreja, T.,(2011).
Plant leaves were separately weighed crushed in mortar with 2% Meta Phosphoric Acid(100
mg leaf tissue in 1 ml MPA) This was soaked for one hour. The soaked leaves were
centrifuged separately at low speed (2500 r.p.m.) for 15 min., the above liquid was utilised
to estimate ascorbic acid and the residual tissues were discarded. 1ml of above solution was
taken, to this 5% MPA (2ml) was added and it was allowed to stand for half an hour at
room temperature. After half hour n-amyl alcohol (5ml) and dye (3.2 ml) were added
(prepared by dissolving 5 mg 2, 6-dichlorophenol dye in 100 ml of distilled water at 80°C)
were added. Number of such testtubes were prepared.After bubling of air throughout the
bottom layer the test tubes were closed compactly. The test tubes were shaken briskly and
the above layer was estimated for ascorbic acid content.Analyisis was done on the
Spectronic-20 colorimeter (Baush and lomb) at wavelength 546 nm and 100% transmittance
set up with a mixture extract (1ml), 5% MPA (2ml), n-amyl alcohol (5ml) in distilled
water (3.2 ml), this is the blank solution. This was done before analyzing test samples. The
ascorbic acid content present in 1 ml of extract was measured by using regression formula.
Y = 0.113 – (0.14 × OD),
Where
Y = Concentration of ascorbic acid in mg.
OD = Optical density.
Ascorbic acid content per 100 gm dry weight was calculated as follows:
Free ascorbic acid = [(A × V) / W] × 1000 × 100,
where ,
A = Y = mg AA / 1 ml of original extract,
V = Total volume of original extract (in ml) and
W = Weight of the plant sample (mg) used for analysis.
9.2. Total Chlorophyll (TCH)

Chlorophyll was calculated as per the procedure given by Arnon,1949. 3g of freshly
plucked leaves were mix together and extracted by 80% acetone (10 ml) and allowed to
stand for 15 min. The supernatant liquid was seperated and taken into another testtube and
centrifuged .The top layer was taken and the absorbance was observed at 645 nm and 663
nm on the spectrophotometer. Calculations were as follows:
Chlorophyll a = (12.7DX663 – 2.69DX645 x V) / 1000W mg/g
Chlorophyll b = (22.9DX645 – 4.68DX663 x V) / 1000W mg/g
TCh = Chlorophyll a + Chlorophyll b
Dx = Absorbance of the extract at the wavelength Xnm,
V = total volume of the chlorophyll solution (ml), and
W = weight of the tissue extract (g).
9.3 Leaf extract pH
5 g freshly obtained leaves were crushed to paste in 10 ml distilled water and w filtered .
pH of leaf extracted noted on digital pH meter which was calibrated with pH 4 and pH 9
buffer solutions.
9.4.Relative Water Contents (RWC)
Following the method described by Singh (1977), leaf RWC was determined and calculated
with the formula:
RWC = [(FW –DW)/(TW –DW)] x 100
FW = Fresh weight,
DW = dry weight,
TW = turgid weight.
Freshly obtained leaves were weighed ,this was fresh weight FW. The leaves were soaked
over night in water, wiped till dry and then weighed, this was the turgid weight TW. Finally
the leaves were oven dried overnight at 60o- 70°C and weighed again to give the dry weight.
9.5.APTI Calculation
The air pollution tolerance indices of four common plants (ES & CS) were determined
following the method of Singh and Rao, 1983. The formula of APTI is given as APTI =
[A(T+P) + R]/10
Where A = Ascorbic acid content (mg/g),
T = total chlorophyll (mg/g),
P = pH of leaf extract, and
R = relative water content of leaf (%).
The entire sum was divided by 10 to obtain a small manageable figure.

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Chapter III

Step -1Preparation of standard stock solution:

µg/ml = (sample absorbance)X (Standard concentration in µg/ml)

µg/ml = (sample absorbance)X (Standard concentration in µg/ml)

µg/ml = (sample absorbance)X (Standard concentration in µg/ml)

8.Physico Chemical study.

8.3.Biological Oxygen Demand (B.O.D),

8.4. Chemical Oxygen Demand (C.O.D)

1. Standard potassium dichromate solution 0.25 N: Dissolve 12.259 g K2Cr2O7 primary

1. 300 mL capacity bottle with stopper

1. Add 2 mL of manganous sulphate solution and 2 mL of alkali-iodide azide reagent to the

8.6. Total Hardness of water by EDTA Titration

if the normality of EDTA is exactly 0.02 N,then

8.7. Chloride estimation by Silver Nitrate method

1. Standard silver nitrate titrant, 0.0282N

ml. of sample taken for estimation

If Normality of AgNO3 is exactly 0.0282N then,

(ml. AgNO3 solution for sample – ml. AgNO3 solution

8.8.Estimation of Sulphate content in water

8.10.Total viable bacteria count Using Multiple tube fermentation method

4. McConkey’s broth (single strength Lactose 10.0 g

Sodium chloride 5.0 g

Neutral red 0.075 g

5. Brilliant green bile lactose broth (BGLB

Brilliant green 0.0133 g

1. Sterilize the tubes at 121oC for 15 mins.

8.11.pH of soil sediments

8.12.Alkanity of soil sediments by Titrimetric Method

8.13. Estimation of Chloride by Silver Nitrate method

ml. of sample taken for estimation

If Normality of AgNO3 is exactly 0.0282N then,

ml. of sample taken for estimation

8.14.Estimation of Sulphate in soil sediments

8.15. Estimation of Phosphates in soil sediments by stannous chloride

9.Air Pollution Tolerance Index (APTI)

9.2. Total Chlorophyll (TCH)

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