10 ch.2 materials and methods -...
TRANSCRIPT
MATERIALS
AND
METHODS
Chapter: 2
���� MATERIALS AND METHODS
In the recent years environmental monitoring through regular assessment of water quality has
become a crucial factor in the exploitation or conservation of aquatic resources.
Generally water quality assessment involves analysis of physical, chemical and biological
parameters, which reflects the status of abiotic and biotic factors of the water reservoir. This in
turn, helps in planning conservation strategies.
Due to the constant exchange of substances between water reservoir and its surrounding
environment, it is necessary to assess and monitor environmental health through water quality
assessment.
Data collection
There are two types of data collection is required. These are referred as Secondary and the
primary data collection methods.
Primary data
It refers to the data which is collected directly during the process of investigation.
Secondary data
It refers to the data sets which already exist. It is important to collect secondary data before
going into field investigations. It may give useful information about the investigation and serve
as the basis. The data may be qualitative, quantitative or spatial.
� SAMPLING METHODS
Sampling is an important tool in understanding the water quality. It is extremely difficult to
analyze the entire water body and it may not be necessary either. A small portion of water is
referred to as a sample which represents the whole water body, is collected for detailed
investigations.
Selection of sampling sites
Sampling points were decided by keeping in mind that the considered sampling points must include
shallow and deep regions of the water body, points of inflow, and outflow of water in the reservoir,
human activities. For deep region centre or mid part of the water body is selected and sample is being
taken from medium depth.
Collection of water sample
Water samples were collected during morning hours (i.e. 7.00 am to 9.00am) on 2nd Sunday of every
month throughout the study period from the selected sites to determine physico-chemical and biological
parameters. Samples for analysis of physico-chemical parameters and biological parameters are
taken separately by applying different method. The method for biological parameters is
discussed elsewhere in this chapter.
Wide mouthed Polyethylene bottle is used to collect water sample for the various physico-
chemical parameters other than DO. For DO, 500 ml BOD glass bottle with glass stopper is used
to collect the water sample. For that BOD bottle is first rinsed with reservoir water and then
dipped into water and then its lid is closed carefully by taking precautions that no bubbles were
formed.
Handling and preservation of water sample
Many physical, chemical and biochemical reactions may change the quality of the water sample
during the period of its collection time and its actual analysis time. To minimize such changes it
is necessary to preserve the samples soon after collection. Analysis of some of the parameters
such as pH and water temp., were performed on the site by using pocket meters and glass
thermometer. To estimate DO, water sample is separately collected in DO bottle as mentioned
earlier and then fixation of the same is performed by adding alkaline KI, MnSO4 and H2SO4 at
the site and later on analysis being carried out in the laboratory along with other parameters by
using standard methods suggested by APHA (1998); Kumar and Ravindranath (1998); Trivedy
and Goel (1984).
Various procedures are employed to preserve the water samples such as addition of chemical
preservative, lowering down the temperature or a combination of both the methods. Few
references are given in the below listed table.
Table: 2.1 Sample Parameters and their preservation methods
Parameter
Contain
-er
Required Volume
(ml)
Method Of
preservation
Holding
time
I. Physical properties 1. Colour
P or G
100
Cool to 40C
48 hrs
2. Odour P or G 100 Cool to 40C 48 hrs
3. Temperature P or G 100 Measure immediately in the field ---
4. EC P or G 100 Cool to 40C 48 hrs
5. Turbidity P or G 100 Cool to 40C (store in dark) 24 hrs
6. TDS (Total dissolved solid)
P or G
300
Cool to 40C
7 days
II. Chemical Properties 7. pH
P or G* 50 Measure immediately in the field
---
8. Total Hardness P or G 100 Adjust pH to < 2 by adding conc.HNO3
6 months
9. Mg Hardness P or G 100 Adjust pH to < 2 by adding conc.HNO3
6 months
10. Ca Hardness P or G 100 Adjust pH to < 2 by adding conc.HNO3
6 months
11. Alkalinity P or G* 100 Cool to 40C 2 weeks
12. Chloride P or G 100 Not required 1 month
13. Phosphate P or G 100 Cool to 40C 48 hours
14. Nitrate P or G 50 Cool to 40C 2 days
15. Dissolved Oxygen
BOD bottle
300
Determine immediately (preferable) or fix DO by adding 2ml of MnSO4 and 2 ml of alkaline KI
6 hours
16. BOD P or G 300 Cool to 40C 24 hours
Source: Water studies Methods for monitoring water quality.
Where, P = Plastic container, G = Glass container, G* = Borosilicate glass container,
HNO3 = Nitric acid, MnSO4 = Manganous sulphate, Alkaline KI= Alkaline Potassium
Iodide
ANALYSIS PROCEDURE FOR PHYSICAL PARAMETER
Seven physical parameters were analyzed. These are Colour, odour, temperature, EC, Turbidity
and TDS.
Colour
Water in its pure form is colourless. However, a water body can acquired colour from colloidal
substances and materials in solution. In natural waters, colour may occur due to the presence of
humic acids, metallic ions, suspended matter, Iron, manganese, phytoplankton, weeds, and
industrial effluents.
The method described here is Platinum – cobalt method. It gives quantitative measure of the
hue.
Material
i. Nessler tuabe, 50 ml; pH meter and
ii Colour standards:
Procedure to prepare colour standards: Dissolve 1.245gm of potassium chloroplatinate (K2PtCl6)
and 1.0 gm of crystalline Cobalt chloride (COCl2.6H2O) in a very small amount of distilled
water; add 100 ml of conc. H2SO4 or conc. HCl (Hydrochloric acid) and dilute to 1:1 with
distilled water. This solution bears a colour value of 500 colour units. Prepare standards having
colour value of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 by diluting 0.5, 1.0, 1.5,
2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7 ml of above solution with distilled water to
50ml in standard Nessler tubes. Indicate the color value on each tube, and protect them from
evaporation and contamination.
Method
Centrifuge the sample at high speed to remove suspended matter. Fill standard Nessler tube with
sample to same level as that of standard (50 ml). Label each tube with the colour value and the
volume of sample taken. Cover the tubes with aluminium foil to protect it from evaporation and
contamination. Compare the colour of sample with that of various standard tubes held vertically
above a white surface and find one standard which has same hue as that of sample and read its
colour value.
If the sample shows a colour greater than 70 units, it should be diluted with distilled water. In
such case, original colour is computed as under.
Odour
Water in its pure form does not have any odour. Odour in water can be due to natural or man-
made causes. Natural causes may include the presence of decaying plants, algae and other
organisms in the water body. Decomposition of organic matter liberates gases such as ammonia,
methane and hydrogen sulphide. These gases impart odour to water.
Procedure:
Fill about ¾ th of the beaker with the water sample immediately after collection and smelled.
The same was repeated at least for five times by using different persons and record the result.
Temperature
Materials
100 ml beaker, mercury thermometer.
Fill about ¾ th of the beaker with the water sample immediately after collection. Hold the
thermometer uprightly in it until the mercury become steady. Care should be taken that the
thermometer must completely immersed in water.
You can record the water temperature by this method also. Tide a long thread at the hind end of
the thermometer and leave it (dipped it) into water at approximately 2 to 3 meter depth for about
5 minutes. Lift out the thermometer from the water body, read and note down the reading. It is
in the Celsius.
EC
Pure water is a poor electricity conductor. The EC is measured by conductivity meter. EC is the
capacity of water to carry out electricity.
Material
Conductivity meter, conductivity cell, thermometer, volumetric flask, beakers.
Chemicals/Reagents
Anhydrous Potassium chloride (KCl), distilled water.
Preparation of standard 0.01 M KCl solution: Dissolve 74.56 mg of anhydrous KCl in distilled
water in a volumetric flask. This is considered as the standard reference solution and with
conductivity of 1912 micromohos/cm at 250C.
Method
Study properly the operation manual of the conductivity meter.
Calibrate the instrument by adjusting the calibration knob to the standard value.
Note down the temperature of the water sample.
Dip the electrodes of the conductivity meter into a beaker already filled with sample water. The
instrument used has automatic temperature compensation (i.e. it read out automatically gives
conductivity values at 250C) hence directly record the values.
Turbidity
Turbidity in natural water is due to suspended particles of clay, silt, and organic matter,
phytoplankton, and other microscopic living objects. It can be described as the expression of
optical property (Tyndall effect).
Material
Nephelometer (Turbidimeter), Sample tubes, Standard Turbidity suspension.
Method to prepare standard turbidity suspension: Dissolve 1 gm of hydrazine sulphate in
distilled water to prepare 100 ml of solution, Dissolve 10 g of hexamethylene tetramine in
distilled water to make it 100 ml. Mix 5 ml of each of the solutions in a 100 ml volumetric flask
and allow standing for 24 hours at about 250C. Dilute it by adding distilled water to 100 ml
mark. This is a suspension having 400 NTU (Nephelometric Turbidity Unit), and can be stored
for about a month.
Dilute 10 ml of above stock solution (of 400 NTU) to 100 ml by adding distilled water. This
standard solution has 40 NTU. This can be stored for about a week.
Method
Set the nephelometer at 100 using 40 NTU standard suspension. In doing so, every per cent on
the scale will be equal to 0.4 NTU turbidity. Shake the sample thoroughly and let the air bubbles
subside. Take the sample in nephelometer sample tube and find out the value on scale. If the
sample has turbidity more than 40 NTU, dilute it so that its turbidity can be read on the same
scale.
Turbidity (NTU) = Nephelometer reading x 0.4 x dilution factor
TDS (Total Dissolved Solids)
A large variety of salts are dissolved in natural waters. These are mainly carbonates(CO3-),
bicarbonates(HCO3-), sulphates(SO4
2-), phosphates(PO43-), chlorides(Cl-), and nitrates(NO3
-) of
Calcium, Magnesium, Sodium, Potassium, Iron, and Manganese.
Material
Crucible, glass beakers, funnels, Whatman’s filter paper, oven, chemical balance, desiccators.
Method
� Weigh a glass beaker or crucible and weigh it.
� Filter 250ml of sample water through a filter paper and take the filterate in evaporating
dish or keep it in oven. Put on the switch and evaporate the sample to dryness at 1030C
to 1050C for about 24 hrs or till all the water evaporates and a residue is left behind.
� Cool the residue in desiccators. Weigh and record the reading.
Calculation
Total dissolved solids mg/ l =
Where, A = final weight (g) of evaporating dish or beaker (g), B = initial weight (g) of the
evaporating dish/crucible/beaker, V= volume of sample taken (ml).
���� ANALYSIS PROCEDURE FOR CHEMICAL PARAMETER
pH
There are two methods: Electrometric method and colorimetric methods are being used to
determine the pH of given sample. Now a days pocket pH indicators are also used.
Materials
Portable field pH meter which has a single electrode because it is very convenient to use.,
Thermometer, pH tablets (to make buffer solutions with different pH), were dissolved in 100 ml
of distilled water.
Method
(A-B)
V X 1000
� Read the operation manual of pH meter carefully.
� Put the switched on and make sure that the meter read 7 pH.
� Dip the electrode in the buffer solution of pH 4.0.
� Turn the selector switch to the pH range of 0-7 and adjust the buffer knob to pH 4.0.
Move selector switch to zero.
� Remove the electrode from the buffer solution and wash it with distilled water. Dip the
electrode in a buffer of pH 9.2. Now the meter is calibrated to both the pH ranges i.e., 0-
7 and 7-14.
� Move the selector switch to zero. Remove the electrode from the buffer and wash it with
distilled water. Then dip it in the sample water.
� Record the temperature to the sample.
� Now move the selector knob to pH range of 0-7 and record the pH value of the sample.
If pH exceeds 7, move selector switch to pH range of 7-14 and record the pH value.
� Turn the selector switch to zero before switching off the instrument and remove the
electrode.
� Keep the electrode dipped in distilled water when not in use.
Total hardness
The measure of the capacity of water to react with soap is known as the hardness of water (Sunil
kumar). Magnesium (Mg2+) and Calcium (Ca2+) are the main cations responsible for the
hardness of water, while cations of iron, manganese, strontium and barium are less responsible
for the same.
To determine the Total hardness of sample water the titrimetric method is used.
Materials
Burette, Pipette, flask, measuring cylinder, etc.
Reagents
0.01M EDTA (Ethylene diamine tetra acetate), ammonia buffer solution, eriochrome black T
indicator.
Preparation of reagents
EDTA solution- Prepare by dissolving 3.723 gm. of Di-Na salt of EDTA in distilled water and
dilute to a total volume of 1 litre. It is to be stored in a polythene bottle.
Ammonia buffer solution – Dissolve 13.5 gm of NH4Cl (Ammonium chloride) in 114 ml. of
conc. NH4OH (Ammonium hydroxide) and distilled water is added to make the volume 200 ml.
Eriochrome black T indicator - Dissolve 0.5 gm. of Eriochrome black T dye in 100 ml. of 80
% ethyl alcohol.
Method
Take 50 ml. of water sample in a flask; add 1 ml. of ammonia buffer solution and 3-4 drops of
Erichrome black T indicator. The water sample turns wine red in colour.
Now titrate it against 0.01M EDTA solution until the wine red colour of solution gets changed
into blue. This is the end point of titration. Note down the reading.
Calculation
Total hardness (mg/l as CaCO3) = -----------------------------
Where T = Volume of titrant (ml); and V = Volume of sample (ml)
Calcium hardness
Calcium is found in great amount in all natural waters .
Material
Burette, Pipette, flask, measuring cylinder, etc.
Reagents
T X 1000
V
0.01 M EDTA (Ethylene diamine tetra acetate), NaOH (Sodium hydroxide) solution, Murexide
indicator.
Preparation of reagents
EDTA solution- Prepare by dissolving 3.723 gm. of Di-Na salt of EDTA in distilled water and
dilute to a total volume of 1 litre. It is to be stored in a polythene bottle.
2 N NaOH (Sodium hydroxide solution) - Dissolve 8 gm of NaOH in distilled water to make it
100 ml.
Murexide indicator – It is readily available in the market.
Method
� Now take 50 ml of the sample water in a conical flask
� Add 1 ml of sodium hydroxide (to set the pH between 12 to 13)
� Then add pinch of murexide indicator (solution in a flask turns pink)
� Then titrate against EDTA standard solution until the pink colour turns purple.
� Note down the end point.
Calculation
T X 1000 ml.
Calcium hardness =
mg/l as CaCO3 Sample volume taken in ml.
Calcium hardness (mg/l as Ca2+) = ------------------------
Where T = Volume of titrant (ml); and V = volume of sample water (ml)
T X 400.5
V
Magnesium hardness
Magnesium is also occur in all natural waters and its main source is rocks.
Method & Calculation
The Total hardness and the calcium hardness of water as mg/l CaCO3 are determined as
described above. From these values of magnesium content is calculated as under:
Magnesium(Mg2+mg/l) = (T – C) x 0.244
Where, T = Total hardness (mg/l, as CaCO3); C = Calcium hardness (mg/l, as CaCO3).
Total Alkalinity
Alkalinity of water is its capacity to neutralize a strong acid and is characterized by the presence
of OH- ions capable of combining with H+ ions. However, most of the water is rich in carbonates
and bicarbonates with little concentration of other alkalinity imparting ions. Titration of the
sample water with strong acid (HCl or H2SO4) first to pH 8.3 by adding phenolphthalein indicator
and then it is further titrated to pH between 4.2 and 5.4 by adding few drops of methyl orange or
mixed indicator can give estimated value of Total Alkalinity especially of carbonates and
bicarbonates.
In first case, the value is called as phenolphthalein alkalinity (PA) and in second case; it is
total alkalinity (TA). Values of carbonates, bicarbonates and hydroxyl ion can be computed
from these two types of alkalinities.
Materials
Burettes, pipettes, conical flasks, measuring cylinder, dropper, physical balance, 250ml and
1000ml volumetric flasks.
Reagents/Chemicals
� 0.02 N Standard Sulphuric acid (H2SO4): Dilute 2.8ml of conc. H2SO4 to 1 L by
adding distilled water to form approximately 0.1N Sulphuric acid (H2SO4). Standardize
this against 0.1N sodium carbonate. After standardization, dilute appropriate volume of
this stock solution (0.1N) to 1 L adding distilled water to prepare 0.02N H2SO4.
� 1N Sodium carbonate solution: Weigh 13.25gm of anhydrous sodium carbonate
(Na2CO3). Dissolve it by adding distilled water and make up the volume to 250ml in a
volumetric flask.
� 0.02N Sodium hydroxide (NaOH): Dissolve 0.08 gm of sodium hydroxide in 100ml of
distilled water taken in a volumetric flask.
� Phenolphthalein indicator: Dissolve 0.5gm phenolphthalein in 50ml of 95% methanol.
To it add 50ml of distilled water. Add one drop of 0.02N NaOH (sodium hydroxide) till
a light pink colour appears.
� Methyl orange indicator: Take 50ml of distilled water, add to it 0.1gm of methyl
orange, dissolve and dilute it to 200ml with distilled water.
Method
� First of all determine the phenolphthalein alkalinity in the following manner.
o Take 50ml of the water sample in a conical flask.
o Add 3-4 drops of phenolphthalein indicator. If no pink colour develops, there is
no phenolphthalein alkalinity.
o If pink colour appears, titrate the sample with 0.02N H2SO4 taken in a burette
until the solution becomes colourless.
o Note down the reading of burette and then calculate as (1)
� Now determine and calculate the Total alkalinity as described below.
o To the same beaker in which phenolphthalein alkalinity was determined, add 3-4
drops of indicator methyl orange.
o The soln. turns yellow.
o Titrate till the colour of the solution changes from yellow to orange red against
0.02N H2SO4. Note down the burette reading and calculate as (2).
Calculation
A X Normality of H 2SO4 X 50 X 1000
(1) PA as CaCO3, mg/l =
ml of sample
(2) TA as CaCO3, mg/l =
ml of sample
Where, A = Volume of H2SO4 (ml) used with only phenopthalein
B = Volume of total H2SO4 (ml) used with phenopthalein and
methyl orange
PA = phenopthalein alkalinity
TA = Total alkalinity
Chloride
Generally natural waters, have low chloride concentration, often less than that of bicarbonates
and sulphates.
Material
Burette, 1ml pipettes, measuring cylinder, physical balance, volumetric flask, conical flask,
dropper, etc.
Reagents
Potassium chromate (K2CrO4), Silver nitrate (AgNO3), Sodium chloride (NaCl), Distilled water.
Preparation of reagents
0.02N Silver nitrate (AgNO3) solution: Dissolve 3.397 gm of AgNO3 in distilled water and
then dilute to 1 litre. Store the solution in a dark glass bottle.
Potassium chromate (K2CrO4) indicator: Dissolve 5 gm of Potassium chromate in about 5 ml
of distilled water. Add few drops of AgNO3 soln. to produce red precipitate. Let stand it for
about 12 hours. Filter and dilute the filtrate to 100ml by adding distilled water.
Method
B X Normality of H2SO4 X 50 X 1000
� Take 100 ml of the water sample and adjust the pH to lie between 7.0 and 8.0 by adding
an acid or base accordingly.
� Take 50 ml of this sample water and add 1 ml of K2CrO4 indicator. The sample turns
yellow.
� Titrate against silver nitrate solution until a brick red precipitate or colour is developed.
Note down the reading (i.e. volume A ml).
� For better accuracy titrate against distilled water (50 ml) in the same way to establish
reagent blank.
� Note down the volume used (B ml).
Calculation
Chlorides (mg/l) =
Where, A = ml of Silver nitrate required for titr ation (burette reading),
B = ml of AgNO3 required for the blank,
N = Normality of AgNO3 used,
35.45 = equivalent weight of chlorine,
1000 = factor added for conversion to one litre.
S = volume of sample water (in ml).
Phosphates
The phosphate in a fresh water body is described to be a total phosphate which includes both
organic and inorganic phosphates (orthophosphates and condensed phosphates).
(A-B) X N X 35.45 X 1000
S
Materials
Volumetric flasks, beakers, test tubes, nessler tubes, glass rod, measuring cylinder, physical
balance and Spectrophotometer or colorimeter.
Reagents
Perchloric acid, Potassium dihydrogen phosphate (KH2PO4), Ammonium molybdate, Con.
Sulphuric acid (H2SO4), Stannous chloride (SnCl2.5H2O), concentrated Nitric acid (HNO3),
Sodium hydroxide solution (NaOH), Phenolphthalein indicator.
Preparation of reagents
Ammonium molybdate: Dissolve 25 gm of ammonium molybdate in 200 ml (approximately)
of distilled water. Add 280 ml of concentrated H2SO4 to 400 ml distilled water. To the diluted
acid, add molybdate solution and make up the volume to 1000 ml.
Strong acid reagent: Add carefully 75 ml concentrated sulphuric acid to 150 ml distilled water
carefully; Add 1 ml of concentrated nitric acid, cool and make it diluted to 250 ml by adding
distilled water.
Stannous chloride: Dissolve 0.5 gm of stannous chloride in 2 ml of concentrated hydrochloric
acid and dilute it to 20 ml with distilled water. Use fresh.
Standard Phosphate solution: Dissolve 4.388 gm of dried anhydrous potassium hydrogen
phosphate in distilled water to make the volume 1 litre. Take 10 ml of the solution and add
distilled water to make 1 litre of stock solution containing 1 mg P/l. Prepare standard
phosphours solution of various strengths (preferably in the range of 0.0 to 1.0 mg P/l at intervals
of 0.1 mg P/l) by diluting the stock solution with distilled water.
Phenolphthalein indicator: Dissolve 1 gm of phenolphthalein in 100 ml of ethyl alcohol and
add 100 ml of distilled water.
Method
• Take 25 ml of sample in a flask and evaporate to dryness.
• Cool and dissolve the residue in 1 ml of perchloric acid. Heat the flask gently so that the
contents become colourless.
• Cool and add 10 ml of distilled water and 2 drops of phenolphthalein indicator.
• Titrate against NaOH solution until the appearance of slight pink colour.
• Make up the volume to 25 ml by adding distilled water.
• Add 1 ml of ammonium molybdate solution and 3 drops of SnCl2 (Stannous chloride)
solution.
• Blue colour will appear.
• Wait for 10 minutes (never more than 15 minutes) and record absorbance (s) on
spectrophotometer at 690 nm. Run simultaneously a distilled water blank in similar
manner.
• Process the standard phosphorus solutions of different strengths in similar manner and
plot a standard curve between absorbances and concentrations of standard phosphorus
solutions.
• Deduce the total phosphorus content of sample by comparing its absorbance (S) with
standard curve and express the result of total phosphorus in mg/l.
Nitrate
When nitrogen gets oxidized, it is converted into nitrate. It is found in natural waters.
Nitrogenous organic matters when undergo biological oxidation it generate nitrates in the water.
Materials
Colorimeter or spectrophotometer, nessler tube, test tubes, 100 ml beakers, water bath,
measuring cylinder, physical balance and pipette.
Reagents
Potassium nitrate, brucine sulphate, sulfanilic acid, concentrated hydrochloric acid, concentrated
sulphuric acid and distilled water.
Preparation of reagents
Nitrate stock solution: 722 mg anhydrous potassium nitrate in 100ml of distilled water and
make up to 1L in a flask.
1 ml of this solution will contain 0.1 mg nitrate nitrogen.
Standard nitrate solution: Pipette out 100 ml nitrate stock solution into a 1000 ml volumetric
flask and make the level up to 1000 ml.
1 ml of this solution will contain 0.01 mg nitrate nitrogen.
Brucine-Sulfanilic acid solution: Take 70 ml of hot distilled water and dissolve 1 gm of
Brucine sulphate and 100 mg Sulfanilic acid in it. Add 3 ml of concentrated Hydrochloric acid
to this solution cool the solution and make it 100 ml with distilled water. This solution is stable
for several months.
Sulphuric acid solution: Add 500 ml of conc.H2SO4 to 75 ml distilled water carefully. Cool to
room temperature.
Method
� Into a series of 50 ml nessler tubes, pipette out 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. Make up the volume to 5 ml in each tube adding the
appropriate volume of distilled water. Label the standards.
� Take a beaker containing 5 ml of distilled water, this will serve as the blank.
� Take 2 ml of the sample in another 50 ml nessler tube.
� Add 1 ml of Brucine sulfanilic acid solution to the blank, the standards and the sample
and mix well.
� Add 10 ml sulphuric acid carefully to each nessler tube and mix well.
� Keep the nessler tubes in the dark for about ten minutes.
� Add 10 ml of distilled water to each of the standards, blank and the sample.
� Keep all the nessler tubes in the dark for 20 to 30 minutes.
� Use the blank to set the spectrophotometer or colorimeter at 100% transmittance at a
wave length of 410 nm.
� Determine the absorbance of the standards and the sample.
� Prepare a standard curve as for phosphorous and with the help of this curve find out the
concentration of nitrate nitrogen in the sample.
Calculation
mg/l nitrate nitrogen =
Nitrate in mg/l = mg/l nitrate nitrogen X 4.43
Dissolved Oxygen (DO)
Oxygen, dissolved in water, also termed as DO, is one of the important parameter of water
quality. Modified Wrinkler’s method can be used to measure DO.
mg nitrate nitrogen X 1000
Volume of sample taken for estimation (in ml)
To avoid the obstruction due to organic matter and chlorides present in sample, Sodium azide is
used along with alkaline Potassium iodide solution.
Material
BOD bottle (500ml), Burette, Burette stands, pipettes, conical flask, beakers, etc.
Reagents
0.025N Na2S2O3 (Sodium thiosulphate) solution, Manganous sulphate (MnSO4), Alkaline
Potassium iodide-azide reagent, Sulphuric acid (H2SO4), Starch solution as indicator.
Preparation of reagents
O.O25 N Sodium thiosulphate: Dissolve 6.205 gm. of Sodium thiosulphate in previously boil-
distilled water and make up the volume to 1000 ml. Add a pellet of NaOH (Sodium hydroxide)
as a preservative. Keep the solution in coloured bottle.
Manganous sulphate: Take 480 gm. of MnSO4.4H2O or 400 gm. of MnSO4.2H2O or 365gm of
MnSO4.H2O and dissolve it in 750 ml. of distilled water; boil, if necessary, cool, filter and make
the volume to 1000 ml.
Alkaline Potassium iodide azide solution: Dissolve 500 gm. of NaOH + 13.5 gm of NaI or 70
gm. of KOH + 150 gm. of KI in 700 ml. of distilled water. Take40 ml of distilled water,
dissolve 10 gm. Sodium azide (NaN3) in it and then add to the previous solution. Make the final
volume to 1 litre.
Sulphuric acid: Con. H2SO4 - Sp. Gr. 1.84.
Starch indicator: Take 100 ml of warm distilled water, dissolve 1 gm of starch in it stir well
and then add few drops of formaldehyde or toluene as preservative if necessary.
Method
� Take a 500 ml glass stoppered BOD bottle and fill it with sample avoiding any bubbling.
� Make sure that no air should be trapped in bottle after the stopper is placed.
� Now open the bottle and add 2 ml each of MnSO4 and alkaline Potassium iodide-azide
solution to it with the help of separate pipettes. Make sure that the pipettes must dip
inside the solution within the bottle. Precipitate (brownish coloured) will appear.
� Close the stopper and shake the bottle thoroughly.
� Add 2 ml. of conc. H2SO4 and invert the stoppered bottle few times to make sure that the precipitate get dissolved.
� Fill the burette with 0.025 N Sodium thiosulphate.
� Take 50 ml. of a solution to a 250 ml. conical flask and titrate against O.O25 N Sodium thiosulphate (freshly prepared) till the colour of the solution turn pale yellow.
� Add O.5 ml. of freshly prepared starch solution as an indicator. Blue colour appears, then carry on the titration carefully. At the end point, the blue colour will suddenly change to colourless.
� Note down the reading.
Calculation
DO (mg/l) =
Where, R = Volume of titrant (ml), 0.025 = Normality of the titrant, V= volume of sample
used for titration, The factor of 1000 = it is for converting to 1 litre, 8 = equivalent wt. of
oxygen.
Biochemical Oxygen Demand (BOD)
The amount of oxygen used by microorganisms in the aerobic oxidation of organic matter is
termed as BOD.
Materials
BOD bottles, Burette, Burette stands, pipettes, conical flask, beakers, BOD incubator, aerator.
R X 0.025 X 1000
V X 8
Reagents
0.025N Sodium thiosulphate (Na2S2O3) solution, Manganous sulphate (MnSO4), Alkaline
Potassium iodide-azide reagent, Sulphuric acid (H2SO4), Starch solution as indicator, BOD-free
water, Phosphate buffer solution, Magnesium sulphate (MgSO4), Calcium chloride(CaCl2),
Ferric chloride, Allythiourea solution.
Preparation of reagents
O.O25 N Sodium Thiosulphate: Take 6.205 gm. of Sodium thiosulphate, dissolve it in
previously boil distilled water and make it up to the 1 litre. Add a pellet of NaOH (Sodium
hydroxide) as a preservative. Keep the reagent in coloured bottle.
Manganous sulphate: Take 480 gm. of MnSO4.4H2o or 400 gm. of MnSO4.2H2O or 365 gm.
of MnSO4.H2O and dissolve it in 750 ml. of distilled water; boil, if necessary, cool, filter and
make the volume to 1000 ml.
Alkaline Potassium iodide azide solution: Take 500 gm. of NaOH + 13.5 gm of NaI or 70 gm.
of KOH + 150 gm. of KI and dissolve in 700 ml. of distilled water. Now dissolve 10 gm. of
sodium azide (NaN3) in 40 ml of distilled water and add to the previous solution. Make the final
volume to 1 litre.
Sulphuric acid: Con. H2SO4 - Sp. Gr. 1.84.
Starch indicator: Take 100 ml of warm distilled water, dissolve 1 gm of starch in it stir well
and then add few drops of formaldehyde or toluene as preservative if necessary.
BOD- free water: Pass distilled water through a column of activated carbon and redistill it. If
this is not possible, use double distilled water.
Phosphate buffer solution: Dissolve 33.4 gm of disodium hydrogen phosphate, 8.5 gm of
potassium di hydrogen phosphate, 21.75 gm of di potassium hydrogen phosphate and 1.7 gm of
NH4Cl in 500 ml distilled water and dilute to 1000 ml in volumetric flask. Adjust pH to 7.2 by
adding acid or base.
Magnesium sulphate: Dissolve 22.5 gm of magnesium sulphate and make to 1000 ml.
Calcium chloride: Dissolve 27.5 gm of anhydrous CaCl2 in distilled water and make up to 1
litre.
Allylthiourea solution: Take 100 mg of allylthiourea and dissolve in distilled water and make
up the volume to 100 ml.
Preparation of dilution water: Dilution water is used to dilute water sample which contain a
large amount of organic matter and do not contain dissolved oxygen. First aerate BOD free
distilled water in a glass container for about half an hour using an aerator. Add 1 ml each of
phosphate buffer, MgSO4 (Magnesium sulphate), CaCl2 (Calcium chloride) and FeCl3 (Ferric
chloride) solution to 1000 ml of aerated distilled water. This is the dilution water.
Method
� Dilute water sample with the dilution water if sample contain large amount of organic
matter and do not contain dissolved oxygen.
� Adjust the pH of the sample to 7.0 (neutralize) using 1N NaOH or 1N H2SO4.
� Fill two BOD bottles with this sample water and add 1 ml of allylthiourea solution to
each bottle.
� Analyse one bottle for dissolved oxygen (D1) (as per 3.4.9) and keep the other for
incubation in a BOD incubator at 270C for 3 days or 200C for 5 days, whichever is
suitable.
� The bottle should not be exposed to light during incubation. Exposure of the bottles to
light induces photosynthetic activity which adds oxygen to the sample.
� Determine the dissolve oxygen from one bottle which is not incubated.
� Take out the sample bottles from BOD incubator after 5 or 3 days of incubation and
determine the dissolved oxygen content D(3) or D(5).
Calculation
BOD (mg/l) =
OR
BOD (mg/l) =
Where, D1 = Initial value of DO in mg/l,
D3 = Final value of DO after 3 or 5 day (whatever applicable) in mg/l,
BC (Blank correlation) = difference between the DO contents of the
blank on initial day and after 3 or 5 days of
incubation,
100 = Dilution factor.
BIOLOGICAL PARAMETERS
Plankton are one of the organisms who can tell us about the water quality. They can also
indicate about what kind of changes are going to taking place regarding water quality of a
particular water reservoir in nearby future. That is why they are considered as biological
parameter to analyze water quality. Hence quantitative and qualitative analysis is of great
importance.
For the quantitative and qualitative study of plankton various steps such as collection of water
sample, its filtration, dilution, counting, and identification are involved.
(D1 – D3) X 100
(D1 – D3 - BC) X 100
Percentage dilution of sample
Collection of sample
Plankton are heterogynous group of organisms which include both phytoplankton and
zooplankton. The sample is collected in the morning hours between 7.00 to 9.00 am. to collect
phytoplankton and zooplankton. For that approximately 50 litre of water is collected by wide
mouthed bucket or measuring container and is filtered by using specific plankton net no.25
(made up of bolting silk, mesh size 55 µm). To the other end of the net a graduated glass bottle
is fitted. To collect zooplankton net is dipped in the water and then slowly (@10 cm/sec) pulled
out. Plankton collected in the plankton bottle fitted at the end of the net are taken out. The value
of water filtered through plankton net is calculated employing specific formula.
Fig.2.1 Plankton net
Preservation
Sample containing zooplankton is preserved in 4% formalin solution. While those having
phytoplankton, are preserved with Lugol’s iodine solution.
At least 24 hours are requiring for sedimentation of phytoplankton. After 24 hours supernatant is
removed with the help of dropper or pipette. After that distilled water is added to the sediment
and the sample is now ready for the subsequent study. Lugol is ideal for that phytoplankton
which have cilia or flagella.
Lugol’s iodine solution: To prepare Lugol’s iodine solution dissolve 10gm of neutral
Potassium iodide in 20 ml of distilled water and add 5 gm of iodine crystals, + 50 ml of
additional distilled water + 5 gm of sodium acetate to it.
4% formalin: Take 4 ml of concentrated formalin and add 100 ml of distilled water to it.
Quantitative analysis
The quantitative analysis of plankton is being performed by estimating the numbers in each
species. Many phytoplankton are multi celled filamentous, others are colonized while some
are solitary cell. Hence they are more conveniently expressed as units/l in counting.
For the estimation, two methods are widely used. These are SR cell method (Sedgwick-Rafter
cell method) and Lackey’s drop method. Generally Sedgwick-Rafter cell method is used when
the density of plankton and filamentous micro algae are less abundant in sample. While
Lackey’s drop method is being used when high density of plankton population is observed in the
sample.
Sedgwick-Rafter cell method:
Material
Sedgwick-Rafter cell, graduated dropper/pipette, compound microscope more preferable inverted
microscope.
Method
Sedgwick-Rafter cell is a slide with a rectangular cavity (50mm x 20mm x 1mm) of volume 1
ml.
Fig. 2.2 Sedgwick-Rafter chamber
� Shake gentle by inverting twice or thrice the concentrated sample bottle and quickly
transfer 1 ml of sample in the cavity of Sedgwick-Rafter cell with the help of dropper or
graduated pipette.
� Place the cover slip or cover glass of appropriate size diagonally and makes sure that no
air bubble develops as well as no sample runs out from the cell.
� Allow the plankton to settle and count them under the microscope by moving the cell
horizontally and vertically.
� Note down the reading and calculate the average count per ml by applying following
formula.
Calculation
Plankton / ml =
N = number of plankton counted in 1 ml of concentrate,
C = total volume of (plankton) concentrate (ml),
V = total volume of sample in l
Lackey’s drop method
Material
Glass slide, Cover slip or cover glass, graduated medicinal dropper, compound microscope,
preferably inverted plankton microscope.
Method
Shake gently the concentrated sample of plankton by inverting the sample bottle twice or thrice
and quickly transfer 0.1 ml volume of it on a glass slide with the help of a graduated dropper.
Cover the sample carefully by putting coverglass or cover slip diagonally. Make sure that no air
bubble develops as well as no fraction of sample runs out. Study and count the plankton present
under cover slip. Study a good number of replicates and calculate the average count per 0.1 ml.
Calculation
Plankton (units/l) = X 10
N X C
V
N X C
V
Where, N = number of plankton counted in 0.1 ml of concentrate,
C = total volume of sample in l (this represents total volume of water
filtered through net when collection is made by filtration; or total
volume of water taken in bottle for sedimentation when collection is
made by sedimentation method).
Identification and documentation of plankton
Plankton are observed carefully under 4x, 10X or 45 X objective lens wherever required. Then
with the help of digital camera attached with microscope is being used to capture images of
plankton. Then identification is being carried out by using various reference books:
“Freshwater Biology by W. T. Edmondson (1959)”, “The Fresh Water Algae” by Prescott
(1970), Indian freshwater microalgae” by Dr. N. Anand (1998), APHA plates, “Freshwater
Biology” by Ward & Whipple, Freshwater zooplankton of India by Battish S. K. (1992).
To find out correlation between two variables, following formula of Karl Pearson is used.
Where,
r = correlation coefficient,
x and y two variable,
dx = deviation from the x-mean of the x variable,
dy = deviation from the y mean of the y variable,
Σ(dx.dy) = sum of the products of the deviations,
Σ (dx)2 = sum of the squares of the deviations of the x variable,
Σ (dy)2 = sum of the squares of the deviations of the y variable
The degree or the intensity of relationship between two variables was ascertained by computing
the value of coefficient of correlation. Karl Pearson’s has given formula for measuring
correlation. The result of this formula varies between +1 and -1. The following chart shows
degrees of correlation according to Karl Pearson’s formula.
Table shows degree of correlation (Karl Pearson’s)
Table: 2.2 Degree of correlation
Degree of correlation Positive Negative
Perfect correlation +1 -1
Very high degree of Correlation +o. 9 or more -o. 9 or more
Fairly high degree of Correlation From +0.75 to +0.9 From -0.75 to -0.9
Moderate degree of Correlation From +0.50 to +0.75 From -0.50 to -0.75
Low degree of Correlation From +0.25 to +0.50 From -0.25 to -0.50
Very low degree of Correlation Less than +0.25 Less than -0.25
Absence of correlation 0 0
(Source: D.C. Sancheti and V.K. Kapoor, (1998): Statistic (Theory, methods and
Applications), Sultan Chand & Sons.