MATERIAL AND METHODS

This chapter deals with the details of the methods

followed during investigation carried out from Feb. 2017
to May 2017. The present study was undertaken with a
view to investigate the "PHYSICOCHEMICAL ANALYSIS OF
DRINKING WATER OF MUNICIPAL CORPORATION AREA OF
REWA (M.P.)".

A filed kit, containing standard analytical reagent

corning glass wares, standard Polythene bottles of 1 liters
capacity, graduated pipettes. dropers, measuring tape,
rope, water analyzer kit, secchi disc, dark and light bottle
etc. were carried to the field. A boat was engaged and the
stations were visited in the sequence, which was carefully
followed throughout the investigation period. The
sampling started after a few minutes of arrival at the
sampling station, to minimize the disturbance of water.
All necessary precautions were taken during sampling,
analysis and transportation of samples, prescribed for
limnological study.

Methods for Analysis :-

In the present study, universally accepted standard

Methods in the field of limnology were followed as possible
for all type of analysis, the analysis of water temperature.

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Conductivity, total dissolved solids (TDS), pH, dissolved
oxygen, free carbon dioxide, hardness were done
immediately after collection and completed before
reaching next station. The following methods were used to
analyze different Physico-chemical parameters.

I. PHYSICO- CHEMICAL PARAMETER

1. Ambient Air Temperature and water temperature:

Temperature of the air surrounding the sampling

stations and samples collected from surface water in the
sampler bottle and taking it up the boat, with the help of
fractional centigrade thermometer in degree Celsius.

2. Transparency:

It is the measurement of the depth of light

penetration. It was measured with the help of secchi disc
of about 20 cm. diameter, with four (alternate black and
white) quadrants on the upper surface and a hook in the
centre to tie a graduated rope. The disc was lowered in to
the water till its sight was lost. This depth was noted-
next the disc was drawn up till it become visible. This
depth was also recorded. The average of these two depths
was taken as the depth of light penetration as the degree
of transparency.

3. Hydrogen ion concentration (pH) :

PH of the lake water was also measured with the

help of digital water analyzer kit.

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4. Dissolved oxygen:

Dissolved oxygen was determined by Winklers

modified azide method.
(i) Maganous Sulfate solution : 364 gm of MnSO4. H2O
was dissolved in distilled water, filtered and diluted
to 1000 ml.
(ii) Alkali Iodided Azide solution: 500 gm of NaOH and
150 gm of KI were dissolved in distilled water and
diluted to 1000 ml. 10 gm of sodium azide (NaN 3)
was dissolved in 40 ml of distilled water and added
to the NaOH-KI solution.
(iii) Concentrated Sulphuric Acid: Analytical reagent
grade.
(iv) Starch indicator : 2.0 gm of soluble starch was
added to 100 ml of distilled water and with
constant stirring the solution was heated until
transparent and 0.5 ml of formalin was added as a
preservative.
(v) Sodium thiosulphate solution : 6.3 gm of Na2S2O3.
5H2O was dissolved in freshly boiled and cooled
distilled water and diluted to 1000 ml, 5 drops of
chloroform was added to the solution as
preservative. The solution was restandardized every
month.
(vi) 10% sulphuric acid solution : 5 ml of conc. H2SO4
was added to 45 ml of distilled water.

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(vii) Potassium dichromate solution 0.0250 N : 3 gm of
K2Cr2O7 was dried at 105C and cooled in dessicator.
0.6129 gm of K2Cr2O7 was dissolved in 500 ml
freshly boiled and cooled distilled water.
Procedure:

To start with, 300 ml of BOD bottles were filled

without bubbling and to this were added 2 ml of solution
(i) and (ii) each. The solution was then mixed thoroughly
and after 15 minutes 2 ml of solution (iii) was added and
the precipitate was dissolved by shaking. Some drops of
solution (iv) were added and contents were titrated by
solution (v) until blue colour became colourless. The end
point had been recorded.

Solution (v) was standardized every month. For this

2 gm of KI was dissolved in 100 ml of distilled water and
to this 10 ml of solution (vi) was added. After adding 10
ml of solution (vii) the flask was placed in complete
darkness. After five minutes the contents of flask were
diluted to 250 ml with distilled water. By using some
drops of solution (iv) the contents were titrated with
solution (v) until the blue colour disappeared. Volume of
solution (v) used was recorded and the normality of the
titrant was calculated by the formula :

NV = NV
Where
N = Product of normality of standard = 0.0250 N
V = ml of standard used in titration = 10 ml.
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N = Product of normality of solution being
standardized
V = Volume of the solution being standardized in ml
= 0.0250 x 10 = N x V
Dissolved oxygen in the sample was calculated as
per the following formula:

(ml x N) of titrant x 8 x 1000

D.O. in mg / lit. =
V2 (V1 V)
V1
N = normality of solution (v) used
V1 = volume of sample in bottle after placing the
stopper
V2 = volume of the parts of the contents titrated
V = Volume of solution (i) and (ii) used.

5. Free carbon dioxide :

Free carbon dioxide was estimated titrimetrically as

per method given by APHA (1985) and Trivedi et al. (1987).
50 ml sample was taken in a conical flask and 3 drops of
Phenolphthalein was titrated with (0.02273N) sodium
Hydroxide solution and Quantity required to turn the
sample pink was noted. The free carbon dioxide in mg/l
was calculated by the formula:

ml of titrant used 1000

Free CO2 mg/l =
ml of Sample

25
 6. Alkalinity :

Alkalinity was determined by titrating the sample

with (0.02N) sulphuric acid in the presence of
phenolphthalein indicators. Different types of alkalinity
were obtained by the method described in APHA (1985).
First phenolphthalein alkalinity was analyzed as CaCO3 in
mg/l by the following formula:

ml of titrant
Phenolphthalein alkalinity = 1000
As CaCO3, mg/l ml of Sample

Then total alkalinity was also estimated by titration

sample with 0.02N H2So4 to 50 ml sample water 2-3 drops
of methyl orange were added. It was titrated with 0.02N
H2So4. The titrant was mixed drop by drop from the
pipette till the end point (faint orange color). The amount
of H2So4 used was noted.

ml of titrant used
Total alkalinity = 1000
ml of Sample

7. Biochemical Oxygen Demand (BOD) :

Regents :

(i) Phosphate Buffer Solution : 8.5 grams of KH2PO4,

21.75 grams of K2HPO4, 33.4 grams of
Na2HPO4.7H2O and 1.7 grams NH4Cl were dissolved
in about 500 ml (distilled) water and diluted to 1
litre. pH was adjusted to 7.2.
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(ii) Magnesium Sulphate solution: 22.5 gram of MgSO4.
7H2O was dissolved in distilled water and diluted to
1 litre.

(iii) Calcium chloride : 27.5 gram CaCl2 was dissolved in

distilled water and diluted to 1 litre.

(iv) Ferric Chloride : 27.5 grams of Ferric Chloride was

dissolved in distilled water and diluted to 1 litre.

(v) Sodium Sulfite solution (0.025 N) : 1.575 grams of

Na2SO3 have been dissolved in 500 ml and diluted to
1000 ml.

Procedure:

The dilution of water was prepared in glass container

by bubbling compressed air in distilled water for about 30
minutes. 1 ml of CaCl2 solution of phosphate buffer,
MgSO4, CaCl2 and FeCl3 solution per litre was added and
mixed thoroughly. Neutralized the sample to pH 7.0 using
NaOH or H2SO4. Since the dissolved oxygen in the sample
could be exhausted, it became usually necessary to
prepare a suitable/dilution of sample. Further dilution in
a bucket be made in a large glass trough and the contents
be mixed thoroughly.

Sets of BOD bottles were filled. One set of the BOD

bottles were kept in BOD incubator at 20C for 5 days
and determined the dissolved content in another set
immediately. Dissolved oxygen content in the sample

27
bottles kept in the incubator have been determined
immediately after the completion of 5 days of incubation.

Similarly for blank, two BOD bottles of diluted water

were taken. In one the dissolved oxygen was determined
and the other was kept incubator for 5 days before D.O.
was determined in the manner described above for water
samples.

Calculation:

When dilution water has not been seeded

D1 D2
BOD (Mg/l) = x a
P
When dilution water has been seeded

(D1-D2) (B1 B2)

BOD (Mg/l) = x a
P
Where

D1 = Dissolved oxygen of diluted sample immediately

after preparation in mg/l
D2 = D.O. of diluted sample after 5 days incubation
at 20C (mg./l)
B1 = D.O. of seed control before incubation mg/l
B2 = D.O. of seed control after incubation mg/l
P = Decimal volumetric fraction of sample used.
A = Ratio of seed in sample to be used in control.
= (% seed in D1) (% seed in B1)
(J) = Chemical Oxygen Demand (COD (Closed reflex
titrimetric method)

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8. Chemical Oxygen Demand (COD):

Reagents required

(i) Mercuric sulphate : Mercury(II) sulfate, commonly

called mercuric sulfate is the chemical compound
HgSO4. It is an odorless solid that forms white
granules or crystalline powder. In water, it separates
into an insoluble sulfate with a yellow color and
sulfuric acid

(ii) 0.025 N Potassium dichromate solution : 12.259 gm

of oven dried K2Cr2O7 at 103C was dissolved in
distilled water and diluted to 1000 ml. 120 mg of
sulphuric acid was added to it.

(iii) H2SO4 Ag2SO4 Solution : 2.75 gm of Ag2SO4 was

dissolved in 570 ml of concentrated H2SO4.

(iv) Ferroin Indicator : 1.485 gm 1-10 phenanthroline

mono-hydrate and 0.695 mg of FeSO4.7H2O were
dissolved in 100 ml of distilled water.

(v) 0.1 N Ferrous Ammonium Sulphate: 39 gm of

Fe(NH4)2(SO4)2.6H2O was dissolved in 400 ml of
distilled water. 20 ml of conc. H2SO4 was added and
the solution was made upto one litre.

Procedure:

20 ml of sample was taken in a COD flask and 0.4

gm of reagent (i) was added. 10 ml solution (ii) was added
slowly 30 ml of solution (iii) was added in the flask and

29
reflux condensation was done for 2 hours in a hot water
bath. After 2 hours the mixture was cooled and some
drops of solution (iv) were added and the sample was
titrated with solution (v) until blue green colour changed
to wine red. Similarly a blank experiment was performed
using distilled water and same quantity of reagents at
same temperature. COD was determined by the following
formula:

(a-b)N x 8000
COD (mg./lit.) =
20
where :

a = ml FeSO4(NH4)2SO46H2O for blank

b = ml FeSO4(NH4)2SO46H2O for sample
N = normality of FeSO4(NH4)2SO46H2O

9. Total Dissolved & Suspended Solids :

Dissolved solids are solids that are in dissolved state

in solution. Waters with high dissolved solids generally
are of inferior palatability and may induce an unfavorable
physiological reaction in the transient consumer. The total
dissolved solids are residue upon evaporation of filterable
samples. The term total suspended solids can be referred
to materials which are not dissolved in water and are non
filterable in nature. It is defined as residue upon
evaporation on non filterable sample on a filter paper.

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 Estimation of total dissolved solids is useful to
determine whether the water is suitable for drinking
purpose, agriculture and industrial purpose. High
concentration of dissolved solids about 3000 mg/l may
also produce distress in livestock. In industrial the use of
water with high amount of dissolved solids may lead to
scaling in boilers, corrosion and degraded quality of the
product.
Apparatus required :
An electronic probe, which also measures pH, and
conductivity, is used to measure TDS. The values are
expressed as mg/1 of water.
Procedure :
The Probe is immersed directly in the water collected
in a wide mouthed sampling bottle at the sampling site
immediately after collection for a period of time sufficient
to permit constant reading.

10. Total Hardness:

Reagents required:
(i) Ammonia Buffer Solution : 16.9 gm of ammonium
chloride has been dissolved in 143 ml of conc.
NH4OH and diluted to 250 ml by distilled water.
1.179 gm of disodium salt of EDTA was added to the
solution.
(ii) Eriochrome Black-T indicator : was dissolved in
100 ml of 80% ethyl alcohol.

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(iiii) M EDTA Titrant: 3.723 gm of disodium salt of EDTA
was dissolved in water and stored in polyethylene
bottle.

Procedure:

50 ml of sample was taken in a flask and 1.0 ml of

solution (i) and 5 drops of solution (ii) were added. The
contents were titrated with solution (iii) until red colour
changed to clear blue. Total hardness of the sample was
calculated as per the following formula:
ml EDTA used x 1000
Total Hardness as CaCO3 (mg/lit.) =
50
11. Calcium Hardness :

Reagents required

(i) Sodium hydroxide 1N : 40 gm of NaOH was dissolved

in one litre of distilled water.
(ii) Murexide indicator: 0.1 gm of ammonium purpurate
was grinded with 100 gm of NaCl (AR)
(iii) 0.01 M EDTA solution: 3.723 gm of disodium salt of
EDTA was dissolved in one litre of distilled water.
The solution was stored in polyethylene bottle.
Procedure:

Hardness caused by the calcium ions was

determined by employing EDTA titrimetric method. 50 ml
of sample was taken in a flask and 2 ml of solution (i) and
approximately 100 mg of reagent (ii) were added and
mixed. Contents were titrated with solution (iii) until the

32
pink colour changed to dark purple. Calcium hardness
as mg of CaCO3 was calculated by the following formula:

A x B x 1000
Calcium Hardness (mg/ lit.) =
50
Where :
A = ml tiration for sample
B = mg CaCO3 equivalent to 1.0 ml EDTA titrant at
the Calcium indicator end point.

12. Magnesium Hardness :

Magnesium Hardness was estimated by calculation

method magnesium content can be calculated as follows:
Magnesium in mg/l-1 = (Total Hardness-Calcium Hardness) 0.243

BIOLOGICAL ANALYSIS

Coliform bacteria are present in the environment

and feces of all warm-blooded animals and humans.
Coliform bacteria are unlikely to cause illness. However,
their presence in drinking water indicates that disease-
causing organisms (pathogens) could be in the water
system. Most pathogens that can contaminate water
supplies come from the feces of humans or animals.
Testing drinking water for all possible pathogens is
complex, time-consuming, and expensive. It is easy and
inexpensive to test for coliform bacteria. If testing detects

33
coliform bacteria in a water sample, water systems search
for the source of contamination and restore safe drinking
water.

Standardized values for MPN/100 ml in various

combinations of dilutions are available (Table 4.1) for the
direct references. In case of unlikely combinations the
following formula was used to calculate the MPN / 100 ml.

No. of positive tubes x 100

MPN / 100 ml =
Ml sample in x ml sample
Negative tubes in all tubes

The tables of abbreviation and drinking water

standards are as follows -

34
 Table No. 4.1
Drinking water standards
Sl. Characte ICMR INDIA INDIA Inter- Europe USPNS SABS USSR India IS:2296
WHO
No ris-tics/ IS:1050 national an
. Unit 1975 1975 0 1971 1962 1971 1970 1974
1971 1993 1970
a b c d e f g h i j k l m n
1. Arsenic 0.20 - - 0.05 0.05 0.05 0.05 0.05 0.01 0.05 0.05 0.20 - -
A A A A A A A A A A
2. BOD - - - - - - - - - - - 3.00 - 3.00
A A
3. Boran - - - - - 1.00 - - - - - - - 2.00
A A
4. Cadmium - 0.01 - 0.01 0.01 0.01 0.01 0.01 0.01 0.05 0.01 - - -
A A A A A A A A A
5. Calcium 75.00 200.0 200.00 75.00 200.0 75.00 75.00 - - - - - - -
A 0 A A 0 A A I I I I
A A
6. Chloride 200.00 600.0 1000.0 200.0 1000.0 200.00 200.00 200.00 250.00 250.00 - 600.0 - 600.0
A 0 0 0 0 A A A A A I 0 0
A A A A I A
7. Chromiu 0.05 - - 0.05 0.05 0.05 - 0.05 0.05 0.05 0.10 0.01 - -
m A A A A I A A A A A
8. Colour 5.00 50.00 - 5.00 25.00 - - - - - - - - -
C C C D
9. Copper 1.00 1.50 - 0.05 1.50 0.05 0.05 0.05 1.00 1.00 0.10 - - -
A A A A A A A A A A

35
10. Disolved - - - - - - - - - - - 3.00 3.00 0.00
Oxygen A A A
11. Dissolved - - 1500.0 - - 500.00 500.00 - 500.00 500.00 - - - 2100.
Solids 0 A A I A A 00
A A
12. Elec. - - - - - - - - - - - - 1000x10-6 3000x10-6
Conductan
ce at
25C
13. Fluoride 1.00 1.50 - 1.00 1.50 1.00 0.6-1.7 07-1.7 0.6-1.7 1.0-1.5 1.50 - - -
A A A A A A A A A A
14. Iron 0.30 1.00 - 0.10 1.00 0.30 0.10 0.10 0.30 0.30 0.50 - - -
A A A A A A A A A A
15. Lead 0.10 - - 0.10 0.10 0.05 0.10 0.10 0.05 0.05 0.10 0.10 - -
A A A A A A A A A A
16. Maganese 0.10 0.50 - 0.05 5.00 0.10 0.05 0.05 0.05 0.10 - - - -
A A A A A A A A A I
17. Magnesiu 50.00 150.0 - 30.00 150.00 30.00 30-150 30-125 - 100.00 - - - -
m A 0 A A A A A I I
A
18. Nitrate 45.00 45.00 - 45.00 45.00 45.00 45.00 50.00 45.00 10.00 10.00 50.00 - -
A A A A A A A A A A A
19 pH 7.0- 6.5- 6.5-9.2 7.0- 6.5-9.2 6.5-8.5 7.8-8.5 - - 6.0-9.0 - 6.0- 6.0-9.0 5.5-9.0
8.5 9.2 8.2 I I I 9.0
20. Phenolic 0.001 0.002 - 0.001 0.002 0.001 0.001 0.001 0.001 0.001 0.001 0.005 - -
compound A A A A A A A A AQ A A
21. Selenium 0.05 - - - - 0.01 - - - - - 0.05 - 0.00
A A A
36
22. Sulphate 200.00 400.0 - 200.0 400.00 200.00 200.00 250.00 250.00 250.00 - - - -
A 0 0 A A A A A A I
A A
23. Total - - - - - 200.00 - - - - - - - -
Alkalinity A
24. Total - - 600.00 200.0 600.00 300.00 100.00 100- - 20-200 - - - -
Hardness A 0 A A A 500 I A I
A A
25. Total 500.00 1500. - 500.0 1500.0 - - - - - - - - -
Solid A 00 0 0
A A A
26. Turbidity 5.00 25.00 - 2.50 10.00 5.00 - - - - - - - -
E E E E F
27. Zinc 5.00 15.00 - 5.00 15.00 5.00 5.00 5.00 5.00 5.00 1.00 5000.0 - -
A A A A A A A A A A 0
A
28. Bacterial - - - - - J I H I H I - - -
(Colifora)

37
 Table No. 4.1
ABBREVIATION

Miligram per litre (mg/l) A

Hazen Unit C
Pt Scale D
Jackson Unit E
N.T.U. F
mhos G
Nil Coliform MPN/100 ml H
NS (Not Specified) I
No sample should contain more than 10
Coliform organism/ 100 ml J
WHO (1971) permissible a
WHO (1971) Excessive b
ICMR (1975) c
India (Govt. of India Ministry of
Housing & Work (1975)
- Acceptable d
- Cause of rejection e
India (1993) IS: 10500 f
International (1971) g
European (1970) h
USPNS (1962) i
SABS (1971) j
USSR k
Tolerance limit for inland surface
water subject to pollution
(IS:2296-1974)
- Public water supply & Bathing ghat l
- Fish culture m
- Inland surface water & irrigation n

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