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Materials and Methods

Page 52

3.1 SAMPLING METHOD

The study was carried out for 13 different lakes of Urban Ahmedabad. Among

all these lake some of the lakes were from eastern Ahmedabad and some were from

western Ahmedabad. Therefore proper sampling method was necessary to get

accurate result. During the study of all the 13 lakes following points were kept in

mind.

3.1.1 SELECTION OF SAMPLING SITES

In the present study the sampling was done during morning hour. The water

samples were collected from five different points of lake. Among these (1) The inlet

point – points where the feeder opens in the lake, (2) Center point - the point which

represent the general water quality of the lake and (3) The outlets point - the place

where the overflows occurs, whereas remaining two point were selected randomly.

3.1.2 COLLECTION OF SAMPLES

The water samples were collected in the polyethylene bottles or in a glass

bottle. Initially the prewashed bottles were rinsed with the sample water. The closed

bottle was dipped in the lake at the depth of 0.5 to 0.7 m, and then a bottle was opened

inside and was closed again to bring it out at the surface. The samples were collected

from five different points and were mixed together to prepare an integrated sample.

3.1.3 SAMPLE HANDLING AND PRESERVATION

From the time of sample collection to the time of actually analyses, many

physical, chemical and biochemical reactions would change the quality of the water

sample, therefore to minimize this change the sample were preserved soon after the

collection. The water samples were preserved by adding chemical preservatives,

lowering the temperature or by the combination of both the method. The water

temperature, pH, dissolved oxygen, electrical conductivity and total dissolve solids

were analyzed immediately at a time of collection, whereas the

parameters were done in the laboratory.

The study was carried for

The collected water samples were brought to the laboratory and relevant analysis was

performed. pH was determined electrometr

conductivity was measured by conductivity meter, d

DO meter, total dissolve solid was measured by

turbidity is measured by Nepthal

magnesium, total hardness, nitrate

suggested by APHA

(1984). Estimation of s

3.2 PHYSICAL AND CHEMICAL

Physico chemical analysis of water means analysis of water for physical and

chemical parameters. The

follows

3.2.1 METHODS FOR PHYSICAL PARAMETERS

3.2.1.1 Temperature Measurement

The temperature of water was measured by using centigrade thermometer

Procedure

Bulb ofthermometer

wasimmersed in

the water


were analyzed immediately at a time of collection, whereas the analyses of remaining

done in the laboratory.

The study was carried for a period of 1 year. (March 2009 to February 2010


performed. pH was determined electrometrically using digital pH meter

easured by conductivity meter, dissolved oxygen is measured by

otal dissolve solid was measured by using TDS meter and similarly

urbidity is measured by Nepthalo turbidity meter. Alkalinity, chloride, TDS, calcium

hardness, nitrate and phosphate were determined by method

APHA (1985); Kumar and Ravindranath (1998);

(1984). Estimation of sodium was done by Flame Photometric method.

AND CHEMICAL METHOD

sico chemical analysis of water means analysis of water for physical and

chemical parameters. The methods used here to check the status of the lakes are as

FOR PHYSICAL PARAMETERS

Temperature Measurement


About 6"below the

surface water

It was keptsteady for a

period ofsufficient time


Page 53

analyses of remaining

a period of 1 year. (March 2009 to February 2010).


ically using digital pH meter, electrical

xygen is measured by

using TDS meter and similarly

o turbidity meter. Alkalinity, chloride, TDS, calcium,

determined by method

Trivedy and Goel

odium was done by Flame Photometric method.

sico chemical analysis of water means analysis of water for physical and

methods used here to check the status of the lakes are as


Temperaturewas noted

3.2.1.2 Electrical Conductivity

EC was determined by using Conductivity meter, Model: EQ

of conductivity measurement is m.mho/cm.

Principle

Water usually

electric current through water. Hence, conductivity may be defined as the measure of

ability of aqueous medium to carry on electric current. Conductivity is totally

dependent upon concentrat

Conductivity is generally measured by conductivity meter which consist

conductance cells having electrode of platinum, these electrode are placed

fixed distance.

As the ionizations

the result is measured at 25

Procedure


3.2.1.2 Electrical Conductivity

EC was determined by using Conductivity meter, Model: EQ

of conductivity measurement is m.mho/cm.

Water usually consists of various types of ions, these ions help in passing the



upon concentration of ions.

Conductivity is generally measured by conductivity meter which consist

conductance cells having electrode of platinum, these electrode are placed

ionizations of solutes are totally dependent on tempe

the result is measured at 250C.

Conductivity cellwas rinsed by

using 0.01 M KCLsolution

Temperature wasadjusted to 25

Then cell constantwas computed.

Conductivity cellwas then rinsed

with sample

Temperature wasthen adusted to

250C

Reading wasnoted in mho/cm


Page 54

EC was determined by using Conductivity meter, Model: EQ – 660. The unit

of various types of ions, these ions help in passing the



Conductivity is generally measured by conductivity meter which consists of

conductance cells having electrode of platinum, these electrode are placed parallel at a

of solutes are totally dependent on temperature, therefore all

Temperature wasadjusted to 250C

Conductivity cellwas then rinsed

with sample

Reading wasnoted in mho/cm


Page 55

Method of calibration

1. Approx. method (accuracy 2% to 3%) for cell const. K = 1

only. The following method was proceed

The instrument was allowed to warm up for 15 minutes.

The range switch was thrown in 2mM position (indicated by arrow).

Bring in standard conductance of 1.00mM by throwing the switch

down in EQ-660.

Now with the help of screwdriver turn the standardize shaft was turned

till the Digital display reads 1.000

Throw the standard cond. Switch up

The instrument is now ready for use.

2. Accurate Method (0.5% to 1% accuracy)

KCl Sol was prepared and was allowed to attain the room/ desired

temperature. The solution so prepared is temperature dependant

The cell K = 1 was dipped in the solution

The range switches in appropriate position so that full range of display

is read.

The std. cond. was checked and the switch is in upward position.

The standardize shaft was turned with screwdriver till the display reads

the correct conductance of the solution. The temperature effect has to

be considered.

The instrument thus calibrated also accounts for deviation in cell

constant to the extent of 10% K = 0.5 can also be used in this method.


6

3.2.1.3 Turbidity

Turbidity of water sample was measured by using Digital Nephlo – Turbidity

meter, Model: 132 (Systronics)

Principle

In this method, when light is passed through a turbid sample , the particles

present in sample scatter some of the light and the light scattered is directly

proportional to the turbidity i.e. higher the turbidity, higher will be the light scattered,

the amount of light scattered by the sample is compared with the intensity of light

scattered by a standard suspension.

Turbidity can be determined for any kind of water sample, but it should be

debris and rapidly settable particles.

Apparatus and reagent preparation

(a) Nepthelometer

(b) Sample tubes: Should be free from any scratch

(c) Stock turbidity suspension

I. 1.0 gm of hydrazine sulphate (NH2)2, H2SO4 was dissolve in distilled

water to prepare 100 ml of solution using volumetric flask

II. 10.0 gm hexamethyline tetramine (CH2)6N4, was dissolve in distilled

water to prepare 100 ml of solution in volumetric flask

III. 5 ml of solution (I) and 5 ml of solution (II) were mixed and was allow

to stand for one day at 250C, then it was diluted to 100 ml. Therefore

standard suspension of 400 NTU (Nephelometric turbidity unit) is

prepared. This solution is stable up to one year.

(d) Standard turbidity suspension: 40 NTU solutions were prepared by diluting 10

ml of stock solution to 100 ml.

Procedure

3.2.1.4 Total Dissolved Solid

Total dissolved

is determined as the residue left after evaporation of filtered water at 103

suspended solids are the solids present in a suspended state.

Instrument was setto 100 by using

standardsuspension of 40

Outer surface of thesample tube wascleaned by using

tissue paper .

Reading was notedin NTU.

Calculation

Turbidity (NTU) = Nephelometer reading x 0.4 x dilution factor


Total Dissolved Solid

Total dissolved solid s are the solids present in water in the dissolved state and

is determined as the residue left after evaporation of filtered water at 103

suspended solids are the solids present in a suspended state.

Instrument was setto 100 by using

standardsuspension of 40

NTU.

Sample was addedin Sample tube

Outer surface of thesample tube wascleaned by using

tissue paper .

Sample tube wasthen put inside the

nepthelometer

Reading was notedin NTU.



Page 57

solid s are the solids present in water in the dissolved state and

is determined as the residue left after evaporation of filtered water at 1030C. Total

Sample was added

Sample tube wasthen put inside the



Page 58

Procedure

Calibration Method

Preparation of solution having 1382 ppm TDS:

The 20 ml sachet of HI70032 is dissolved in 1000 ml of distilled water to

prepare solution having TDS value 1382 ppm.

The TDS meter was immersed in the solution.

Then it was allowed to stand until it achieves stable reading.

Then with the help of small screw driver the calibration trimmer was adjusted,

till the display shows the reading of 1382 ppm.

Now the instrument is ready for use.

After calibrating theinstrument.

Water Sample was collectedin the Glass beaker.

TDS meter was thenimmeresed into this water

sample.

It was allowed to stand untilit achieves stable reading.

Reading was noted in ppm.


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3.2.2 METHODS FOR CHEMICAL PARAMETERS

3.2.2.1 pH Measurement

The pH of the sample was determined by digital pH meter Model: 511

(Testronics).

Instrument Calibration

The combination electrode was cleaned with distilled water observing proper

safety precautions

The combination electrode was connected to the meter

The temperature pot was kept on the temperature of the solution under test and

switch (mv/pH) on mv position.

The unit was switched on and was checked weather the led glows.

The electrode was dipped in the reference standard solution of 7 pH (pure

water)

The STAD pot was set and was adjusted so that digital indicator show zero.

Now mv/pH switch position was changed to pH, the digital indicator will read

7 pH.

The electrode was cleaned with distilled water.

The electrode was kept in 4 pH (reference standard solution) solutions. The

digital indicator read 4 pH. If not the slope and pot was adjusted so that the

digital indicators read 4 pH.

Corresponding mv readout was checked for reference.

Now the digital pH meter is ready to measure any solution

Between 0-14 pH at any temperature between 0 to 100oC


The temperature adjust pot was kept to the temperature of the solution under

test. And no change was made STAD adjust pot and slope adjusts pot.

Procedure

3.2.2.2 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 salt of the

carbonates, bicarbonates, phosphates, nitrates, borates, silicates, etc. together

hydroxyl ions in the Free State. However, most of the ware is rich in carbonates and

bicarbonates with little concentration of other alkalinity imparting ions

alkalinity, carbonates and bicarbonates can be estimated by titrating the sample w

strong acid (HCl or H2

then further to pH between 4.2 and 5.4 with methyl orange or mixed indicator. In first

case, the value is called as phenolphthalein alkalinity (PA) and in second

pH meter was calibratedby following

manufacturer manual.

Half of the electrode wasthen dipped into sample





l Alkalinity



carbonates, bicarbonates, phosphates, nitrates, borates, silicates, etc. together


bicarbonates with little concentration of other alkalinity imparting ions

alkalinity, carbonates and bicarbonates can be estimated by titrating the sample w

2SO4), first to pH 8.3 using phenolphthalein as an indicator and


case, the value is called as phenolphthalein alkalinity (PA) and in second

pH meter was calibratedby following

manufacturer manual.

Electrode was made dryby using tissue paper.

Half of the electrode wasthen dipped into sample

water.pH was noted.


Page 60





carbonates, bicarbonates, phosphates, nitrates, borates, silicates, etc. together with the


bicarbonates with little concentration of other alkalinity imparting ions Total

alkalinity, carbonates and bicarbonates can be estimated by titrating the sample with a

), first to pH 8.3 using phenolphthalein as an indicator and


case, the value is called as phenolphthalein alkalinity (PA) and in second case; it is

Electrode was made dryby using tissue paper.

pH was noted.


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total alkalinity (TA). Values of carbonates, bicarbonates and hydroxyl ion can be

computed from these two types of alkalinities.

Reagents

Hydrochloric acid, 0.1 N: 12N concentrated HCl was diluted to 12 times (8.34

100ml) to prepare 1.0N HCl. It was further diluted to make 0.1N HCl (100 to

1000ml). It was Standardize against sodium carbonate solution.

Methyl orange indicator, 0.05%: 0.5 g of methyl orange was dissolved in

100ml of distilled water.

Phenolphthalein indicator: 0.5g of phenolphthalein was dissolved in 50ml of

95% ethanol and 50ml of distilled water was added. 0.05N CO2 free NaOH

solution was added drop wise, until the solution turns fainty pink.

Sodium carbonate, 0.1N

5.300g of Na2CO3, previously dried at 250 C for about 4 Hours was dissolved

in distilled water to prepare 1Liter of solution.

Calculation for alkalinity

(A X Normality) of HCl X 1000 X 50

PA as CaCO3, mg/l =

ml of sample

(B x Normality) of HCl x 1000 x 50

TA as CaCO3, mg/l =

ml of sample


Page 62

Procedure for Alkalinity

100ml of sample was taken in aconical flask

2 drops of phenolphthalein indicator.was added

The solution remaincolourless

Colour changes topink

PhenophthaleinAlkalinity = 0

Titrated with 0.1NHCl

2-3 drops of methylorange was added

Colour disappears atend point

Titrated with 0.1NHCl

The reading wasnoted as A

Colour changes fromyellow to pink at end

point

The reading wasnoted as B


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Where

A = ml of HCl used with only phenopthalein

B = ml of total HCl used with phenopthalein and methyl orange,

PA = phenopthalein alkalinity

TA = total alkalinity

3.2.2.3 TOTAL HARDNESS

Ethylene diamine tetra acetic acid (EDTA) and its sodium salt form a chelated

soluble complex when added to a solution of certain metal cations. Additional of

small amount of a dye such as Erichrome Black T to an aqueous solution of calcium

and magnesium ions of pH 10.0, turns the solution wine red. When EDTA is added as

titrant, the calcium and magnesium get complexed and the colour of the solution turns

from wine red to blue, marking the end point of the titration. This on calculating using

the formula gives the total hardness.

Metal + Indicator Metal-Indicator (complex).

Metal-Indicator + EDTA Metal-EDTA (Complex) + Indicator.

Reagent Preparation

Buffer Solution: 16.9 gm of ammonium chloride was dissolved in 143ml

ammonium hydroxide, 1.25 gm of magnesium EDTA was added and diluted

to 250 ml by using distill water.

Inhibitor: 4.5 gm of hydroxyl amine hydrochloride was dissolved in 100 ml

of 95% ethanol or isoprophyl alchol.

Eriochrome Black T indicator: 0.5 gm of the dye was mixed with 100 gm of

sodium chloride, to prepare a dry powder.

Murexide Indicator: 200 mg of murexide and 100 gm of solid sodium

chloride were grinded and mixed.

2 N Sodium hydroxide: 80 gm of sodium hydroxide was dissolved in distilled

water and was diluted to 1000 ml.


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0.01 M Standard EDTA solution: 3.723 gm of EDTA sodium salt was

dissolved and was diluted to 1000 ml in a volumetric flask.

Procedure

50 ml of water sample2 ml of buffer

solution.

1 ml of inhibitor.Pinch of Erichrome

Black T as indicator.

Titrated againstStandard EDTA (0.01

M) solution.

Colour changes fromwine red to blue.

Volume of EDTA Usedwas Noted as A ml.

Calculation

A X 1000Total Hardness mg/l as CaCO3 =

Volume of sample taken (in ml)


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3.2.2.4 Calcium

When the pH is adjusted to 12 or 13 by addition of sodium hydroxide,

magnesium is precipiated as hydroxide. The Murexide indicator gives a colour

change from pink to purple when all the calcium has been complexed.

Reagent preparation

(Same as in Total Hardness)

Procedure

50 ml of watersample.

1 ml of 2 N NaOH.Pinch of

murexideindicator

Titrated againstEDTA.

Colour changesfrom pink to

purple.

Note down thevolume of EDTArequired (A ml).

CalculationA x 1000 ml

Calcium hardness =mg/l as CaCO3 Volume of water sample taken in ml

A x 400.8Calcium mg/l =

Volume of water sample taken in ml


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3.2.2.5 Magnesium

Like calcium magnesium is also found in all natural waters and its sources are

from rocks. Its generally occurs in concentrations lower than those of calcium.

Magnesium is a necessary constituent of chlorophyll. Its high content reduces the

utility of water for domestic use. A concentration above 500 mg/l imparts an

unpleasant taste and renders the water unfit for drinking purposes.

Procedure

Total hardness and calcium hardness of water were determined by following

the above method. From these values the magnesium content is calculated as follows

3.2.2.6 Dissolved Oxygen

Dissolved Oxygen was analyzed by using Dissolved Oxygen meter, Model:

VSI – 14N (VSI – Electronics).

Principle

The measurement of DO is based on Volta metric method. The diffusion of

oxygen through membrane produces current which is proportional to concentration of

dissolved oxygen.

Calibration

The main lead was connected to 230V 10% mains supply.

Calculation

Magnesium mg/l = (T – C) x 0.243

WhereT = Total hardness (mg/l, as CaCO3)

C = Calcium hardness (mg/l, as CaCO3)


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A 200 ma fused was provided at the rear panel for the safety of a

circuit.

The display was adjusted to zero with ‘ZERO’ knob.

The DO probe was connected in the DO sockets provide in the rear

side of the instrument.

The probe was put in 1-2% Sodium Sulphite Na2 S03 solution and

allows about 2 minutes to attain equilibrium.

The instrument read zero or up to 0.2ppm. The probe is good enough.

If it does not become zero, it was adjusted to zero by using ‘ZERO’

knob.

The DO probe was dipped in double distilled water after washing it

thoroughly with distilled water.

Referring to the value of DO in distilled and with the help of ‘CALIB’

knob. The reading was adjusted to the proper value when the sample is

being stirred at continues speed.

Now the instrument was calibrated.

Procedure

After completionof calibration.

Water sample wastaken in a BOD

bottle.

The temperatureof water sample

was noted.

Now DO problewas dipped in the

sample waterstirred by

magnetic stirrer.

The displayshowing reading

was noted.

This was theamount of DO

recorded.


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3.2.2.7 Biochemical Oxygen Demand (BOD)

Principle

Biochemical Oxygen Demand (BOD) is the measure of degradable organic

material present in the water sample, and can be defined as the amount of oxygen

required by the microorganisms in stabilizing the biological degradable organic matter

under aerobic conditions.

The principle of the method involves, measuring the difference of the oxygen

concentration between the sample and after incubating it for 5 days at 20 C

Reagents Preparation

BOD-free water: Double distill water was used

BOD Incubators: Having a temperature control at 20 0C

Phosphate buffer: 8.5 gm KH2PO4, 21.75 gm K2HPO4, 33.4 gm

Na2HPO4.7H2O and 1.7gm NH4Cl was dissolved in distilled water to prepare

1 liter of solution. pH was adjusted to 7.2.

Magnesium Sulphate: 22.5 gm MgSO4.7H2O was dissolved in distilled water

to prepare 1 liter of solution

Calcium Chloride: 27.5 g of anhydrous CaCl2 was dissolved in distilled water

to prepare 1 liter of solution.

Ferric chloride: 0.25 g FeCl3 6H2O distilled water was dissolved to prepare 1

liter of solution.

Allylthiourea solution: 100 mg of allylthiourea was dissolved in distill water

and was diluted to 100 ml.

Sodium Sulphite Solution, 0.025 N: 1.575 g Na 2SO4 was dissolved and was

diluted to 1000 ml. Solution was freshly prepared.

Preparation of dilution water: Dilution water was used to dilute water sample,

containing large amount of organic matter and do not contain dissolved

oxygen. Initially the BOD free double distilled water was aerated for about

half an hour using an aerator. Then 1 ml of phosphate buffer, magnesium


Page 69

sulphate, calcium chloride and ferric chloride solution was added to 1000 ml

aerated distilled water This is dilution water.

Preparation of Percentage Dilution of Polluted Water

SamplePercentage

dilution

Dilution mixture

Water sample

ml

Dilution water

ml

Total

ml

Polluted

waters

50 500 ml 500 ml 1000 ml

25 250 ml 750 ml 1000 ml

20 200 ml 800 ml 1000 ml

10 100 ml 900 ml 1000 ml

Procedure

Water sample wasdiluted by dilutionwater if required.

The pH of watersample was

adjusted to 7.0 byusing acid or base.

Two BOD bottlewere filled by this

sample water.

1 ml ofallylthiourea

solution was addedto each bottle.

Dissolved oxygenof one bottle was

analysed.It was noted as D1.

Second bottle waskept in BOD

incubator at 200Cfor 5 days.

Other two BODbottles were filled

with dilutionwater.

Dissolved oxygenof one bottle of

dilution water wasnoted immediately.

Second bootle wasalso incubated for

5 days at 200C.

Dissolved oxygenfor sample waterwas noted as D3.

Dissolved oxygenfor dilution water

was also noted.


Page 70

Calculation

3.2.2.8 Chloride

Principle

Silver nitrate reacts with chloride to form very slightly soluble white

precipitate of AgCl. At the end point when all the chlorides get precipitated, free

silver ion reacts with chromate of reddish brown colour.

Reagent preparation

Potassium chromate indicator: 25 gm of potassium chromate was dissolved in

100 ml distilled water. Then silver nitrate solution was added to it until a

definite red precipitated get formed. Then it was kept for 12 hours. After that it

was diluted to 500 ml by using distill water.

0.0282 N Silver nitrate solution: 4.791 gm of silver nitrate was dissolved in

distilled water and was diluted to 1000 ml.

(D1 – D3 – BC) x 100

BOD (mg/l) =

Percentage dilution of sample

Where

D1 = Initial dissolved oxygen in sample (mg/l).

D3 = Dissolved oxygen left out in the sample after 5 days of

incubation (mg/l).

BC (Blank Correction) = Difference between the DO

content of the blank on initial day and after 5th day of

incubation


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0.0282 n Sodium chloride: 1.648 mg sodium chloride was dissolved in

distilled water and was diluted to 100 ml. 1.0 ml of this solution contain 1.0

mg of chloride.

Procedure

Calculation

100 ml of watersample was taken.

pH was adjustedbetween 7.0 and 8.0.

Add 2 ml K2CrO4 asindicator

Titrated againstsilver nitrate.

Brick red precipitateformed.

Volume of silvernitrate used wasnoted as A (ml).

(A – B) x N x 35.45 x 1000

Chloride mg/l =

Volume of sample (ml)

Where

A= ml of AgNO3 required for titration

N= Normality of AgNO3 used.

35.45= Equivalent weight of chloride and the factor of 1000

is for conversion to one liter


Page 72

3.2.2.9 Sodium

Flame Photometric Method

Principle

Sodium can be determined by using flame photometer at 589nm. When

sample is produced in flame it comes to excitation condition and produce

characteristic light. The light intensity at 589nm is proportional to concentration of

sodium which can be read by using light dispersion devices.

Reagents

Stock sodium solution (100 mg/L Na): 2.542 g NaCl dried at 1400C was

dissolved in distilled water to make one liter of a solution (1 ml = 1.00 mg

Na).

Intermediate sodium solution: 10 ml of stock solution was dissolved in

distilled water and make volume to 100 ml. (1 ml. = 0.10 mg Na)

Nitric acid: HNO3 concentrated.

Hydrochloric acid: HCl concentrated.

Hydrogen peroxide: H2O2, 30%

Ammonium hydroxide: NH4OH concentrated.

Preparation of calibration curve

1. The following procedure was used for pretreatment of the sample The sample

of suitable size in a 250 ml conical flask was taken and was acidify it with

nitric acid. It was evaporated to dryness on a water bath. Again 25ml of

concentrated HNO3 was added to boil until the acid was evaporated to small

volume. The presence of brown fumes indicates the un oxidized organic

matter. Now some more conc. HNO3 and small quantities of H2O2 was added

for complete ashing of organic matter. The final residue is colourless on

drying. The presence of more iron and copper salts may impart a colour to it.

The residue in small amount of HCl and distilled water was warmed. The


Page 73

content was filtered and was neutralize with conc. NH4OH. It was diluted to

suitable volume.

2. For non polluted samples or the sample where only dissolved sodium is to be

estimated, the sample was through a filter paper to remove any suspended

matter which otherwise may clog the capillary of the instrument.

3. Calibration curve was prepared in the range of 0 – 1, 0 – 10 or 0 – 100 mg/l,

by using standard sodium solution.

Procedure

Calculation

After calibrating theinstrument.

Suitable amount ofsample was filtered to

remove suspendedmatter.

By using flamephotometer

concentration of sodiumwas recorded.

Calibration curve wasprepared by following

the instruction providedby manufacturer

Sodium was determinedby using calibration

curve.

Sodium mg/l = (mg Na/l in sample) x dilution ratio


Page 74

3.2.2.11 Nitrates

Principal

Nitrate when reacts with brucine produces yellow colour is presence of H2SO4

this can be measured spectrophotometrically at 410nm.

Apparatus

Spectrophotometer, nessler tubes, test tubes, 100 ml beakers, water bath

measuring cylinder, physical balance and pipettes.

Preparation of Reagents

Nitrate Stock solution : 722 mg anhydrous potassium nitrate was dissolved in

100 ml distilled water and final volume was made 100 ml in a volumetric

flask

1 ml of this solution will contain 0.1 mg nitrate nitrogen

Standard nitrate solution: 100 ml. nitrate stock solution was prepared in a

1000ml volumetric flask and the level was made up to 1000 ml.

1 ml of this solution will contain 0.01 mg nitrate nitrogen.

Brucine-sulfanilic acid solution: 1 gm of brucine sulphate and 100 mg.

sulfanilic acid was dissolved in 70 ml. hot distilled water. 3 ml. concentrated

HCl was added to this solution. The solution was cooled and was dilute to 100

ml with distilled water. This solution was stable for several months.

Sulphuric acid solution: 500 ml of conc. sulphuric acid was added to 75 ml

distilled water carefully. Then it is cooled at room temperature. Toxic, avoid

ingestion

Sodium Arsenite solution: 1.83 gm of sodium arsenite (NaAsO2) was

dissolved in 100 ml distilled water.

Sodium Chloride solution: 300 gm NaCl was dissolved in distilled water and

was diluted to 1000 ml.


Page 75

Procedure

Preparation of Standard Curve

Into a series of 50 ml. nessler tubes, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0

ml of nitrate standard solution was pipetted .The volume was made to 5 ml in

each tube by adding the appropriate volume of distilled water. The standards

were labeled.

A beaker containing 5 ml of distilled water was taken as a blank.

2ml of sample wastaken in 50 mlnessler tube.

1 ml of Brucinesulfanilic acid

solution was addedand was mixed nicely.

10 ml of sulphuricacid was added and

was mixed nicely.

Then it was kept indark for 10 minutes

10 ml of distill waterwas added.

Then it was kept indark for 20 to 30

minute

Absorbance wasmeasured at 410 nm.

By using standardcurve concentrationof nitrate nitrogen in

sample wascalculated.


Page 76

1 ml of brucine sulfanilic acid solution was added to the blank and standard,

then they were mixed nicely.

10 ml of sulphuric acid was carefully added to each nessler tube and was

nicely mixed.

.The nessler tubes were kept in the dark for 10 minutes.

10 ml of distilled water was added to each of the standards and blank.

Then all the nessler tubes were kept in the dark for 20 to 30 minutes.

The blank was used to set the spectrophotometer or colorimeter at 100 %

transmittance at a wave length of 410 nm.

The absorbances of the standards were measured.

After that standard curve was prepared to find out the concentration of nitrate

nitrogen in sample.

Calculation

3.2.2.12 TOTAL PHOSPHATE

Principal

Organic phosphorus is converted to orthophosphates by heating or by

persulphate digestion while inorganic phosphates are converted to orthophosphates by

H2SO4 digestion. The phosphates thus released can be determined calorometrically

mg nitrate nitrogen x 1000 mg/l nitrate nitrogen =

Volume of sample taken for estimation (in ml)

Nitrate in mg/l = mg/l nitrate nitrogen x 4.43


Page 77

Chemical

Potassium dihydrogen phosphate (KH2PO4), ammonium molybdate,

concentrated sulphuric acid, stannous chloride (SnCl2.2H2O), glycerol, concentrated

nitric acid and distilled water, phenolphthalein, Sodium hydroxide and

phenolphthalein indicator

Apparatus

Spectrophotometer or colorimeter, volumetric flasks, beakers, test tubes,

nessler tubes, glass rod, measuring cylinder, physical balance, kjeldahl flask, burette,

bunsen burner, tripod stand and wire gauze.

Preparation of reagents

Stock phosphate solution: 439 mg of potassium hydrogen phosphate was

dissolved in distilled water and the volume was made to 1000ml. 1 ml = 100

microgram of phosphate or 0.1 mg of phosphate.

Phosphate working solution: 10 ml stock solution was diluted to 1000 ml

using distilled water. This solution was freshly prepared. 1 ml = 1 microgram

of phosphate or 0.001 mg of phosphate.

Ammonium molybdate solution: 25 gm of ammonium molybdatewas

dissolved in about 200 ml distilled water. 280 ml of concentrated sulphuric

acid was added to 400 ml distilled water. To the dilute acid, molybdate

solution was added to make final volume 1000 ml.

Strong acid reagent: Carefully add 75 ml concentrated sulphuric acid to 150

ml distilled water. 2 ml of concentrated nitric acid, was added, allowed to cool

and 250 ml was diluted with distilled water.

Stannous chloride: 2.5 gm of stannous chloride was dissolved in 100 ml of

glycerol. This was heated on a water bath to insure complete dissolution. It

was mixed well by stirring with a glass rod.

1 N sodium hydroxide: 40 gm of sodium hydroxide pellets was dissolved in

about 200 ml of distilled water. And total volume was made to 1000 ml in a

volumetric flask.


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Phenolphthalein: It was prepared as it was prepared in the section of the

alkalinity measurement.

Procedure

Preparation of Standard Curve

1. Appropriate amounts of phosphate working solution were pippeted to cover

the range of 0.3 to 1.5 mg/l into the series of 100 ml nessler tubes. These tubes

serve as standards. A nessler tube containing 100 ml distilled water was

included as the blank.

100 ml of thesample was takenin a kjeldahl flask.

1 ml of conc.sulphuric acid and5 ml of conc. nitric

acid was added.

The sample washeated until thesolution become

colourless.

It was Cooled.

20 ml of distilledwater and 2 drops of

phenolphthaleinindicator was added.

Titrated againstsodium hydroxide

until the appearanceof pale pink colour.

The solution wastransfer to the 100ml of volumetric

flask and was diluteup to the mark.

4 ml of ammoniummolybdate solutionwas added and was

mixed well.

0.5 ml of stannouschloride was added.

After 10 to 12minutes colour

developed.

OD was measuredat 690nm using

spectrophotometer.

Comparing the valuewith standard curve.phosphorus content

can be found


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2. To the standards and blank 4 ml of ammonium molybdate solution was added

and was mixed well.

3. 0.5 ml of stannous chloride was added to all the tubes and was mixed well

4. It takes 10 – 12 minutes for the development of colour.

5. The spectrophotometer or colorimeter was calibrated using the blank solution

and distilled water.

6. The intensity of blue coloured complex at 690 nm using a spectrophotometer

was measured.

7. A standard curve was prepared by plotting the phosphate concentration of the

standard solution on the x axis and the optical density on the y axis.

8. The phosphorous content of the sample was found by matching its absorbance

(S) with the standard curve.

9. The result was expressed as mg phosphate as phosphorous. If it has to be

expressed in term of Phosphates multiply by a factor of 3.066.

Calculation

Phosphate mg/l = phosphorus mg/l x 3.066


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3.3 BOTANICAL METHOD

The physical and chemical characteristics of water affect the abundance,

species composition, stability and productivity of the indigenous populations of

aquatic organisms. The botanical methods used for assessing water quality include

collection, counting and identification of phytoplankton. The work involving

phytoplankton analysis would help in:

Explaining the cause of colour and turbidity and the presence of objectionable

odour, tastes and visible particles in waters.

The interpretation of chemical analyses.

Identifying the nature, extent and biological effects of pollution.

3.3.1 Phytoplankton net

The phytoplankton net is a field-equipment used to trap phytoplankton. It has

a polyethylene filter of a defined mesh size and a graduated measuring jar attached to

the other end. A handle holds the net. The mesh size of the net determines the size

range of the plankton trapped

3.3.2 Sampling Procedure

Plankton net number 25 of mesh size 20 μm was used for collecting samples.

100 liters of water was measured in a graduated bucket and filtered through the net

and concentrated in a 100 ml bottle. Samples were collected as close to the water

surface as possible in the morning hours and preserved for further analysis.

3.3.3 Labelling

The samples were labeled with the date, time of sampling, study area-lake

name and the volume measured and pasted on the containers.

3.3.4 Preservation of the sample

Between the time that a sample is collected in the field and until its analysis in

the laboratory, physical, chemical and biochemical changes may take place altering

the intrinsic quality of the sample. It is therefore necessary to preserve the samples


Page 81

before shipping, to prevent or minimize changes. This is done by various procedures

such as keeping the samples in the dark, adding chemical preservatives, lowering the

temperature to retard reactions by freezing or by a combination of these methods. For

a phytoplankton sample to be analyzed for an extended period, commonly two

preservatives are used: Lugol’s iodine using acetic acid which will stain cells

brownish yellow and will maintain cell morphology and of 4% formaldehyde.

Preparation of Lugol Solution.: 100 gm of KI and 5 gm Iodide crystals were

dissolved in 20 ml of distilled water. then 5 gm of anhydrous sodium acetate

was dissolved in 50 ml distilled water now both of this were mixed and Lugol

solution get prepared. 0.5 ml of lugol should be added to preserve 100 ml of

sample

Prparation of 4% Formaline: 4 ml of concentrated formalin was diluted to 100

ml with distill water.

3.3.5 Concentration technique

The phytoplankton nets were used to collect samples for the quantitative

estimation, by filtering a known volume of water (100 litres) through the net. The

sample was allowed to settle for 24-48 hours and was further concentrated to

approximately 30 ml by decanting. The concentration factor is used during the

calculations.

3.3.6 Qualitative and quantitative analysis of phytoplankton

Detailed analyses of phytoplanktonic populations are done by estimating the

numbers in each species. The phytoplankton consisting of individual cells, filaments

and colonies are counted as individual cells. When colonies of species are counted,

the average number of cells per colony is counted, and in filamentous algae, the

average length of the filament has to be determined.

Device used for this analysis is Sedgwick Rafter counting cell. Sedgwick Rafter cell is

approximately 50 mm long, 20 mm wide and 1 mm deep. The total volume of the cell

is 1 ml.


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3.3.7 Mounting the slides

Concentrated samples in a bottle are mixed uniformly by gentle inversion.

Then by using bore pipette 1 ml of sample was transfer on Sedgwick Rafter count

cell. Now it was covered by using cover slip, avoiding any kind of air bubble. Then it

was kept for 10-15 minutes so that all plankton may settle down. Now the Sedgwick

rafter counting cell is placed under microscope and then plankton was counted by

moving the cell horizontally and vertically. The process was repeated twice.

3.3.8 Microscope

A binocular compound microscope is used in the counting of plankton with

different eyepieces such as 10X and 40X. The microscope is calibrated using an

ocular micrometer.

3.3.9 Procedure for Plankton Measurement

100 liter of waterwas filtered through

plankton net.

Suitable amount ofpreservative was

added in thecollected samples.

The sample wasallowed to settle for

24-48 hours.

Furtherconcentrated to

approximately 30 mlby decanting.

1 ml ofconcentrated

sample was addedto sedgwick Rafter

counting cell.

The cell wascounted by moving

the cellhorizontally and

vertically.

The observednumber of planktonwas then applied to

the formula.


Page 83

3.3.10 Phytoplankton Identification

From the concentrated sample, the slides for the plankton were prepared. Then

these slides were placed under microscope, and the phytoplanktons were observed in

100 X in binocular microscope. The images of the phytoplankton were captured by

using digital camera. Later on the phytoplankton were identified by using the book “

The Fresh Water Algae” by Prescott (1970).

The status of the water can be evaluated by performing all the above methods-

physical, chemical and botanical. The result are then compared with the WHO and

BIS standard and on the basis of this the quality of water can be judged. And presence

of some algal blooms also shows the richness of the nutrient in the water.

Formula =

where,n = Number of plankton / liter of water.a = Average no of plankton in one small counting chamber of S-R cell.c = ml of plankton concentrate.l = Volume of original water filtered in litre.

(a x 1000) cn =

l


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3.4 Correlation

It is essential to understand the relationship between different water quality

parameter when the study is completed.

3.3.1 Relationship between the parameters (Correlation)

Any relationship between the two variable is known as correlation. If one

variable increases or decreases with a corresponding increase or decrease of the other

variable, a direct positive correlation exists between the two variables. If one variable

decrease with an increase in the other variable, then there is a negative or inverse

correlation. There are two different methods to study correlation

3.3.1.1 Graphic method

It is the simplest method of showing the relationship between two variable . in

this one variable is represented on X-axis and other variable on Y-axis on graph

paper. Data corresponding to X and Y axis were plotted in form of dots. And then

estimated lines joining first and last points was drawn on the graph paper to find out

correlation.

3.3.1.2 Correlation coefficient

The graphic method indicates the existence of a correlation. But it is not

possible to calculate the extent or degree of relationship using these graph. This was

calculated by using following formula

∑ (dx . dy) r =

√∑(dx)2 . ∑(dy)2

Where,

r is the correlation coefficient,

x and y are the two variable

dx is the deviation from the x-mean of the x variable,

dy is the deviation from the y mean of the y variable,

∑ (dx . dy) is the sum of the products of the deviations,

∑ (dx)2 is the sum of the squares of the deviations of the x variable,

∑ (dy)2 is the sum of the squares of the deviations of the y variable,


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The variable in table were written in short form, its full form were as follows

TEMP = TEMPERATURE

EC = ELCTRICAL CONDUCTIVITY

TUR = TURBIDITY

TDS = TOTAL DISSOVED SOLIDS

ALK = ALKALINITY

TH = TOTAL HARDNESS

CAL = CALCIUM

MAG = MAGNESIUM

DO = DISSOLVED OXYGEN

BOD = BIOCHEMICAL OXYGEN DEMAND

Cl = CHLORIDE

Na = SODIUM

NO3 = NITRATE

PO4 = PHOSPHATE

CYN = CYANOPHYCEAE

CHL = CHLOROPHYCEAE

BAC = BACCILARIOPHYCEAE

EUG =EUGLENOPHYCEAE

The correlations were done seasonally for 13 different lakes. All the 19

parameters were correlated with each other to check there relationship.

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