Chapter III
Research Methodology
3. A. Hydrobiology
3. A.1: ANALYSIS OF WATER
There are some soluble salts in the irrigation water, irrespective of its
source. The types and amounts of dissolved salts determine the appropriateness
of waters for a precise reason. Some of the constituents and other dissolved
salts may be helpful for crops. The class or appropriateness of waters for
irrigation use is checked in terms of the attendance of objectionable constituents.
Figure No.3.1: - Map showing the sampling stations of Eastern part of Bhima
river
The irrigation waters simply in a restricted situation, is evaluated as a
basis of plant nutrients. a number of of the suspended ions as such nitrates are
helpful for crops. The restrictions of purity of water are known for drinking water.
The waters to be useful for manufacturing intentions and for farming are different.
It may, for that reason, be likely that a water which is not suitable for
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consumption and manufacturing use, may be reasonably suitable for farming.
The most important features that decide the superiority of irrigation waters are:
1. pH
2. Electrical conductivity (EC) to judge total concentration of soluble salts .
3. Sodium Adsorption Ratio (SAR) to test relative quantity of Na to other cations
such as calcium and magnesium .
4. Concentration of elements like B or other that may be poisonous to plants.
5. Residual Sodium Carbonate (RSC) to assess proportion of CO3 and HCO3 as
correlated to the concentration of Ca and Mg .
6. Content of anions such as Cl, NO3and SO4.
The excellence of irrigation water can be decided by using the above
analytical information on parameters.It is also in the form of a directory used to
point up the taking values fixed for each feature.
Many of the definite plant nutrients are available from several waters coming
from manufacturing companies as waste matters and household throw away
water as sewage. The irrigation of pasture crops can be done by these valuable
waters, may be if checked for toxic/pollutant metals and organic and microbial
contents with respect to their suitability or else. Organic contents can be
estimated normally under two categories: i) organic substances which determine
an cumulative amount of organic carbon, and ii) individual or definite organic
substances such as C6H6, DDT, methane, hex abutol, endosulfan, etc. Important
determinations are like the chemical oxygen demand (COD) which indicates the
presence of total organic substances and the bio-chemical oxygen demand
(BOD) which indicates the presence of total biodegradable organic substances in
the water sample.
3. A.2: Methods for Water analysis
Collection of Samples
Collection of samples was carried out as per the methods suggested by
APHA,1985, Trivedi and Goyal, 1884 and Gupta, 2004. The details are given
below.
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Water
Water samples were collected early in the morning from all the selected
sites(Figure No.3.1). 2 L. plastic bottles rinsed with perchloric acid and distilled
water were used for the sample collection. At the time of sampling the bottles
were rinsed with the water to be sampled and then filled by the sample. the
frequency of river water sampling was fixed according to the flow of water in the
river on the basis three seasons during 2011 to 2012.
3.2.1: Analysis of Water
The pH of water samples was noted on spot with digital pH meter and
samples were brought to the laboratory. The analysis of drinkable water samples
was carried out for the parameters Total Dissolved Solids (TDS), Total Hardness
(TH),and Electrical Conductivity (EC), Major Constituents (Calcium (Ca), Sodium
(Na), Potassium (K)and Magnesium (Mg), - Cationic and Chloride (Cl), Total
Alkalinity (TA), Sulphate (SO4)–Anionic), Minor Constituents (Phosphate (PO4)
and Nitrate (NO3)), Indicator Parameter Biological Oxygen Demand (BOD) and
Chemical Oxygen Demand (COD) and (Dissolved Oxygen (DO),) and E Coli
were determined according to the standard methods given by APHA (1985) and
Trivedi and Goyal (1984) in the laboratory.
3.2.1.1: Physical Characteristics
The physical characteristics of Bhima river water analyzed are pH,
Electrical conductivity (EC), Total dissolved solids (TDS) and Total Hardness
(TH).
pH:
pH is considered as the logarithm of the activity of hydrogen ions with
negative sign.
It measures the strength of acidity or alkalinity and the intensity
of hydrogen ions in water. The normal acidity or alkalinity depends upon excess
of H+ or OH- ions over the other. If free H+ ions are more than OH- ions, the water shall
be acidic or alkaline the other way. The pH was calculated with the help of digital
pH meter.
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The Electrical ion Conductivity (EC):
The irrigation waters are assessed for total salt content concentration
using the parameter the electrical conductivity.
The measure of electrical
conductivity is the most important parameter for judgment of the appropriateness
of waters in irrigation The suitable irrigation waters considered are generally,
having conductivity less than 2.25 mS/cm. But in some odd situations like highly
clay soils having meager permeability and very sensitive crops are not suitable
for such waters. The assessment value less than 0.75 mS/cm is Ideal and the
waters generally used have the standards between 0.75-2.25 mS/cm according
to Richards, 1954. The physical values calculated in a laboratory
determination of
for
conductivity is usually of resistance, measured in ohms or
mega ohms. The confrontation of a performer is inversely relative to its cross
sectional area and directly relative to its length. The electrical conductivity was
calculated with the help of digital conductivity meter in dSm-1.
Total Dissolved Solids (TDS):
A well assorted sample was filtered through a Whatman 41 filter paper
and the filtrate was evaporated to dryness in a pre weighted porcelain dish. The
dish was cooled and again weight was taken to see the difference in weight
before and after evaporation of sample. The increase in dish weight was
represented as full amount dissolved solids in water in miligrams per litre.
Total Hardness (TH):
Hardness of water is caused by multivalent metallic cations. Calcium and
magnesium are the most abundant naturally occurring cat ions .In areas where
there is extensive geological formation of limestone hard water is commonly
found at both underground and surface water. The calcium and magnesium
precipitates soap and reduce its cleaning action. These elements cause scale in
[CaCO3 and Mg[OH]2] water distributions main supply and also in the waterheaters.
Ca and Mg form a complex of wine red color with Eriochrome black T
indicator at pH of 10. The Ethylene Diamine Tetra acetic Acid (EDTA) has got a
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stronger attraction towards Ca++ and Mg++ and therefore, by addition of EDTA,
the previous complex is broken down and a new complex of blue color is
produced. The hardness in the water was measured using following formula
ml of EDTA used X 1000
Hardness as mg/l, CaCO3 = _____________________
ml sample
3.2.1.2: Chemical features
The chemical constituents in water are grouped in two broad classes i.e.
Major and Minor constituents.
3.2.1.2.1: Major Constituents
The major constituents are grouped in two groups as Cationic and Anionic
for the sake of understanding.
3.2.1.2.1: A: Cationic contents
The cationic contents analyzed for water are Calcium, Magnesium
,Sodium and Potassium .
Calcium (Ca):
When EDTA is added to the waters having both Ca and Mg, it combines
first with the Ca. Calcium was determined directly with EDTA, pH was made
satisfactorily high that the Magnesium was mostly precipitated as the OH and
ammonium purparate was used as an indicator that combines with Ca only.
ml of EDTA used X 400.8
Calcium mg/l= ________________________
ml sample
Magnesium (Mg):
The charge of magnesium was obtained by deducting the value of calcium
from the total Ca++ + Mg++. Magnesium was considered by calculation i.e.
discrimination between solidity and calcium as CaCO3.
Mg, mg/l=
A-B X 400.8
------------------------ml sample X 1.645
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Where,
A= EDTA used in hardness determination
B=EDTA used in Ca determination
Sodium (N) and Potassium (K):
A characteristic light is produced due to excitation of electrons under
controlled condition, when the sample with sodium or potassium is sprayed into
the gas flame. The strength of this characteristic emission is relative to the
concentration of sodium and potassium which can be read using Na and K filters
on flame spectrophotometer. Using the same principal the concentration of Na
and K was measured on flame photometer at wavelength of 589 nm and 766.5
nm respectively. These readings were plotted on the standard curve to find out
the concentration of Na and K.
3.2.1.2.1: B: Anionic Contents
The anionic contents analyzed for water were Chloride (Cl), Total
Alkalinity
(TA) and Sulphate (SO4)
Chloride (Cl):
AgNO3 reacts with Cl, to form very little soluble white precipitate of
AgCl. At the ending point when all chlorides get precipitated, and silver chromate
of radish brown colour are formed by reaction of free silver ions with chromate.
Following formula was used to calculate Cl, concentration
(ml x N) of AgNO3 X 35450
Cl, mg/l= ------------------------------------------ml sample
Total Alkalinity (TA):
Alkalinity in waters generally caused due to CO3, HCO3, phosphates,
NO3, borates etc. together with OH- in free state. The primary need for alkalinity
measurement relates to processing of water .The measurement of alkalinity is
essential for removal of coagulants for turbidity and in softening of water. The
alkalinity measurement is performed in wastewater aeration and in process of
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sludge digestion. Alkalinity is determined by titration analysis. It is like the acidity
estimation by measure of carbon dioxide and other acids in the solution.
Generally it is between the pH range 4.5 and 8.3. For estimation of total alkalinity
the sample is titrated with strong acid like HCL and using indicator
phenolphthalein for pH8.3 and then indicator methyl orange for pH between 4.2
and 5.4.
The values in mg/l were calculated using following Formula
(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
Where,
A = ml of HCl used with only phenolphthalein.
B = ml of HCl used with only phenolphthalein and methyl orange.
PA = Phenolphthalein alkalinity.
TA = Total alkalinity.
Sulphate (SO4):
The precipitation of Sulphate ion can be acheieved by adding barium
chloride in hydrochloric acid medium in the form of barium sulphate. The
determination of concentration can beachieved by
of the sulphate by the
absorbance of light by barium sulphate at 420 nm on Spectrophotometer and
then comparing it with standard curve. Standard curve was used to measure the
concentration of sulphate in the solution.
3.2.1.2.2: Minor Contents
Phosphate (PO4) and Nitrate (NO3) are the minor constituents analyzed
from water in present investigation.
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Phosphate (PO4):
The phosphate in water reacts with the ammonium molybdate and form
composite heteropoly acid (molybdophosphoric acid) which gets reduced to a
compound of blue colour in the presence of SnCl2. The incorporation of light by
this blue colour can be calculated at 690 nm on spectrophotometer to determine
the concentration of phosphate. The concentration of PO4 in the sample was
measured with the help of standard curve.
Nitrate (NO3):
The nitrate in water was measured with phenol disulphonic acid method.
Nitrate in contact with sulphuric acid produces nitric acid which in dry condition
(in presence of excess conc. H2SO4) brings about nitration of phenol disulphonic
acid. This nitrophoenolic product gives intense yellow colour in alkaline medium
which is measured through spectrophotometer at 410 nm. The standard curve
was prepared using standard NO3 solution and the reading of the sample was
plotted on the curve and thus
the concentration was measured.
3.2.1.3: Indicator Parameters
The Indicator
parameters
analyzed in the
water
are Dissolved
Oxygen[DO], Biological Oxygen Demand [BOD] and Chemical Oxygen Demand
[COD].
Dissolved Oxygen:
Dissolved oxygen is one of the significant factor in the water quality, water
pollution cotrol and other treatment processes. Biological decomposition of
organic matter uses dissolved oxygen . Levels
significantly below saturation
values often occur in polluted surface water.
For determine the dissolved oxygen iodine can be titrated against
thiosulphate using starch as an indicator. Dissolved
oxygen was calculated using following formula
(ml x N) of titrant X 8 X 1000
Dissolved Oxygen (mg/l) = ----------------------------------------------------V2 (V1-V / V1)
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Where
N = Normality of Sodiumthiosulphate.
V1= Volume of sample bottle after placing the stopper.
V2 =Volume of part of content titrated.
V = Volume of MnSO4 and KI added.
Biological Oxygen Demand (BOD):
The respiratory demand for oxygen while stabilizing the organic matter
under aerobic conditions is called 'biochemical oxygen demand'. Microorganisms
consume the oxygen produced by degradation of the oxidizable organic material
in the aerobic process.
BOD was evaluated by measuring oxygen concentration in sample
eudiometrically before and after incubation in the dark at 200C for 5 days.
Preliminary dilution and aeration of sample (with the help of dilution water) are
usually necessary to ensure that not all the oxygen is consumed during
incubation. Excess dissolved oxygen must be present during incubation.
Samples absorbing more than 6 mg/l of oxygen should therefore be diluted with
dilution water made from BOD free (distilled) water to which the major
constituents are added in the same concentration as in the sample. Sometimes a
culture of bacteria (seed material) is added so that more of the organic matter will
be used up during incubation. Generally sewage is used as a standard seed
material.
BOD mg/l = (Do-D5) X Dilution Factor
Where,
D0 = Initial DO in the Sample
D5 = DO after 5 days
Chemical Oxygen Demand (COD):
Chemical oxygen demand measures the amount of O2 essential for
oxidation of organic compound present in water by means of chemical reactions
involving oxidizing substance such as potassium dichromate. Most of the organic
matter decaying and produces CO2 and H2O when boiled with a mixture of
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potassium dichromate and H2SO4. A sample is refluxed with known amount of
potassium dichromate in H2SO4 medium and the surplus of Cr2O7 is titrated
against ferrous ammonium sulphate(FAS). The quantity of Cr2O7 consumed is
relative to the O2 required to oxidize the organic matter present in the sample.
The COD was calculated using following formula
COD
(b-a) X Normality of Ferrous ammonium sulphate (FAS) X 800
(mg/l) = ------------------------------------------------------------------------------ml of sample
Where,
a= ml of FAS used or blank
b =ml of FAS used for sample
Sodium Carbonate in residual form (RSC)
This key is significant for CO3 and HCO3 loaded irrigation waters. It
shows their inclination to precipitate Ca as CaCO3. The increase in the quantity
of magnesium and calcium disturbs the quality of water. The increased calcium
and magnesium forms carbonate like solids and Mg to form a solid material
(scale) which settles out of the water when the excess carbonate (residual)
concentration becomes too high.
RSC is determined as below.
RSC ( me/litre ) = ( CO3 -- + HCO3 - ) – ( Ca ++ + Mg ++ )
Concentrations of both cations and anions are in miliequivalent per liter. Sodicity
danger in terms of RSC is classified as under
Safe
is less than
Moderate
Unsafe
is
1.25
1.25 – 2.5
> 2.5
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The restrictions can fluctuate depending upon types of soils, climatic
conditions and rainfall. elevated RSC standards can be measured secure for
sandy soils in high rainfall area (greater than 600 mm per annum).
According to the tests and norms of US Salinity Laboratory (USDA, 1954),
Water is harmless for irrigation when RSC value less than (1.25 meq/l), a value
between (1.25 and 2.5 meq/l) is of subsidiary quality and a value more than (2.5
meq/l) is inappropriate for irrigation. If there is no complete precipitation of Ca
and Mg then it is indicated by negative value of RSC. (Tiwari, et.al., 1988).
The Ratio of Sodium Adsorption (SAR)
It is measured to point out the sodicity or alkaline nature dangers of
irrigation water.
Based on the measure of SAR, waters can be rated into different classes of
sodicity as under according to Richards, 1954.
Safe
is less than
Moderately Safe
Moderately unsafe
Unsafe
is
10
between 10-18
is between
is greater than
19-26
26
SAR can be determined by the formula given by (USDA, 1954)
Na
SAR (epm) =-----------------------------(Ca+Mg)1/2/2
The Sodium adsorption ratio is more easily measured property of water .It is
more widely used. The SAR imparts the information about the appearance of Na+ ,
Ca+, and Mg+ in the water . It is calculated by using above formula . Here [Na+],
[Ca+], and [Mg+] are the concentrations of sodium , calcium, and magnesium ions in
the water. An SAR value of 13for the solution extracted from contaminated water is
equivalent to an ESP(exchangeable sodium percentage) value of 15. The SAR is also
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used in characterization of irrigation water applied to crops. By using the Wilcox
diagram and the sodium adsorption index the salinity of the irrigation water can be
determined.
Reduction in crop growth is caused due to presence of trace elements or
heavy metals when their concentration boosts further than a certain point in irrigation
waters and with continuous use of such waters. However, in common irrigation waters,
such elements are normally not a problem. They can be of harmful when
manufacturing runoff water is used for farming.
3. A.2: Water Sample Collection Methods
The container must be thoroughly cleaned before the use and should be rinsed
3 to 4 times with the water from which the sample is to be drawn. Care should be
taken to collect the sample only after continuous discharge of the source for 10 to 20
minutes.
Figure No.3.2: Collecton of water
from sampling Station
of Bhima river
Figure No.3.3 :Conductivitymeter
and pHmeter
If the source of irrigation water is a tank, canal or river, the sample should be
drawn either from a spot, away from the midstream . This can be easily managed
with the help of small bucket tied at long pole. About half a liter of sample is quite
sufficient. The water sample after proper labeling must be sent to the laboratory
immediately for testing in order to avoid any change or deterioration in its quality due
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to the chemical or microbial activity. If delay is inevitable then 2 to 3 drops of pure
toluene may be added to prevent bacterial activity. If sediments are present then filter
the water sample through whatman No. 1.(Figure 3.2)
3. A.3: Analytical Methods
1. Determination of pH
The pH value is the negative normal logarithm of the hydrogen ion activity (mol
per litre.). as a result of the presence of strong bases and weak acids e. g. Na2CO3
increases the pH values, salts of weak bases and strong acids (e. g. CaCL2 ) cause
decreases.
The pH value of neutral water usually lies between 6.5 to 7.5 and lower values
are a result of free CO2 . Biogenic decalcification in surface waters can cause the pH
value to reach 9.5.
Apparatus
pH meter with glass-calomel electrode assembly
Reagents : Buffer solution pH 4, pH 7 and pH 9.2
1. pH 4 Buffer solution
Dissolve 1.012 g anhydrous potassium hydrogen phthalate (KHC8H4O4) in
distilled water and make up to 100mL in a volumetric flask.
2. pH 7 Buffer solution
Dissolve 1.361 g anhydrous potassium dihydrogen phosphate ( KH2PO4)
and 1.420 g anhydrous disodium hydrogen phosphate ( Na2HPO4 )( both dried at
1000 C to 1300 C for two hours.) in distilled water and make up to 1000 mL in a
volumetric flask.
3. pH 9 Buffer solution
Dissolve 3.81 g of sodium borate decahydrate ( borax ) Na2B4O7.10H2O in
distilled water and make up to 1000mL.
4. Readymade commercial buffer solutions, tablets and powders are also
available in the market. these are to be dissolved in water and made up to the
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standard
volume
with
distilled
water
as
per
the
instructions
of
the
manufacturer.(Figure. 3.4)
Procedure
Take50mL of water sample in 100mL beaker, follow the manufacturer's
instructions to operate the pH meter and determine pH of water. Before
measurement of pH of the sample, standardize the pH meter, using standard
buffer solutions of pH near that of the sample to be tested, check the electrode
response occasionally by measuring the pH of another standard buffer solution
with a different pH.
Based on pH values, natural waters can be divided into three distinct
classes:
1. The waters which contain carbonates, with or without bicarbonates, do
not have free carbonic acids. The pH values of these waters are always above 8
.
2. That which contain no carbonates, and carbonic acid .The pH values of
these waters range from 4.5 to 8.0. Most of the natural waters fall under this
category.
3. That which contain free acid in addition to carbonic acid, do not contain
carbonates or bicarbonates. the pH value of these waters is 4.5 or below.
2. Determination of Electrical Conductivity (EC)
The conductivity of electrical ions (EC) is a measure of capacity of water's
to convey electric current. Conductivity of electrical ions of water is directly relative
to its content of dissolved mineral matter . The unit of conductivity is mScm-1. The
result is usually reported at 250C as electrical conductivity varies directly with the
temperature of the sample. Electrical conductivity of water sample is measured
directly by conductivity bridge and values corrected for temperature and cell
constant.
Procedure
Take water sample in a 100 mL beaker for the determination of EC by using the
commercial EC meter. While using these instruments manufacturer's instructions
should be followed carefully. The instrument has to be checked constantly by
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using standard potassium chloride solution, the electrical conductivities of which
are as follows at 250C. The data of Kohlrausch quoted by Metson (1961) are as
below:
Conductivity µScm-1
Classification Class
250and less
Excellent
250-750
Good
750-2000
Permissible
2000-3000
Doubtful
3000 and more
Unsuitable
3. Determination of Ca and Mg
Ca and Mg in water sample are determined by titrating them against standard
EDTA solution using Erichrome Black T ( EBT ) as indicator and NH4Cl + NH4OH
as buffer to give pH of about 10. The color changes from wine red to blue green.
Reagents
1. Buffer solution
Add 67.5 g of pure ammonium chloride in 570mL of concentrated ammonium
hydroxide and make to one litre with distilled water.
2. Erichrome black T, EBT indicator
Weigh 0.5 g of EBT dye and 4.5 g of hydroxylamine hydrochloride and dissolve
both in 100mL of ethyl alcohol (95%).
3. EDTA solution, 0.01 N
Weigh 2 g of disodium salt of EDTA and dissolve in water and volume to 1 litrer
and standardize against 0.01 N calcium solution.
4. Potassium ferrocyanide, K4Fe(CN)6
Dissolve 4 g in 100mL of distilled water.
5. Triethanolamine, TEA
reagent grade.
6. Calcium solution, 0.01 N
Dissolve 0.5 g of pure CaCO3 in 10mL of 2N HCl and dilute to 1000mL volume.
Procedure
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1. Take a known volume (10 mL) water sample in 100mL of clean conical flask
and dilute the content by adding distilled water about 25 mL.
2. Add one mL or enough of NH4Cl + NH4OH buffer to raise pH to 10. Add 10
drops each of NH4OH .HCl, K4Fe( CN )6 and triethnolamine and 4 drops of EBT
indicator. It will give wine red colour to the solution.
3.Titrate against standard EDTA ( 0.01N ) till colour changes from wine red to
blue. No tinge of red colour should remain at the end point , in titrated sample.
4. Note the volume, mL of EDTA used.
5. Run a blank simultaneously using 10mL distilled water in place of sample. Use
all reagents and titrate.
6. Subtract the blank reading from sample reading to get true reading of sample.
Calculation
ml versenate(EDTA)used x normali of EDTA x 1000
Ca+ Mg (me/litre) = -----------------------------------------------------------------ml aliqoot taken
Ca + Mg in me/litre x equivalent wt
Ca+ Mg (me/litre) = ------------------------------------------------1000
Ca + Mg in me/litre x 32.196
= ----------------------------------------------1000
4. Determination of Sodium
The irrigation water generally shows presence of small amount of sodium and
potassium. Sodium constituents are about 50% or more of total cations of saline
and sodic waters. In saline water with EC greater than 1 mScm-1, the content of
sodium may be quite high, and containing relatively a smaller amount of Calcium
and Magnesium. At higher levels, it also exerts a toxic effect on the plant growth.
Therefore, determination of sodium in irrigation waters is very important for
defining its harmful effects on the agricultural land and crops and judging the
appropriateness of water for crops. the concentration of potassium is generally
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low. The measurement of Na and K
is done directly with the help of flame
photometer using the suitable filters and standard curve made by taking known
concentration of Na and K.
Reagents
Standard sodium chloride, NaCl solution
Weigh and dissolve 5.845 g of sodium chloride ( AR) in water and make
upto to 1 litre mark. this gives 100 meqL-1 stock of sodium.
Standard curve for sodium
The concentration of sodium is relatively higher than that of potassium .The
strength of test solution is conveniently expressed in miliequivalents per litre
instead of ppm. For preparing stock solution of 100 meq, weigh and dissolve
5.845 gram of sodium chloride ( AR ) in water and make upto one litre mark.
From this stock solution 0, 1, 2, 3, 4, and 5 mL solution is diluted to
1000mL. . This solution would contain 0, 0.1, 0.2, 0.3, 0.4, and 0.5 meq Na per
litre which means 2.3, 4.6, 6.9, 9.2, and 11.5 µg Na mL-1., respectively . A
Curve is drawn by plotting flame photometer reading on 'Y " axis
concentration of
against
Na on 'X ' axis. .If it is not straight, reduce the range of
concentration of working standards and again draw the above relationship. Now
atomise the unknown samples into the flame and record the readings. Dilute the
samples and bring the sodium concentrations from the graph and multiply by
dilution factor, if any , to get the final value.
5. Determination of Carbonates and Bi-Carboantes
Chiorides , carbonates and bicarbonates, sulphates and nitrates form the
important anions to stdudy the quality of water. Concentration of chloride
generally increases with increase in EC of irrigation waters. Therefore,
magnitude of the total salt may be predicted if chloride concentration is known.
Sum of the carbonate and bicarbonate ions constitutes to be total alkalinity
of water as temporary and raises its pH to more than 7.5 . This alkainity also
causes corrosion in the boilers Aand other metallic pipes, hence their
determination is also important for agricultural as well as industrial purposes.
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Principle
The carbonate and bicarbonate ions in the sample can be determined by
titrating it against standard sulphuric acid ( H2SO4 ) using phenolphthalein and
methyl orange as indicators, respectively. Addition of phenolphthalein ( pH 8.3 )
gives pink red colour in the presence of carbonates and titration with H2SO4
converts these CO3 into HCO3 and decolorizes the pink red colored solution.
H2SO4 + 2CO3 2- → 2 HCO3- + SO4 2In colorless solution methyl orange is added which gives yellow color to the
solution. Further titration against
H2SO4 neutralises all HCO3 ( original +
converted from CO3 ) into H2O and CO2 and colour of solution changes from
yellow to rosy red. The reaction is as given :
2 HCO3- + H2SO4 → 2H2O + 2CO2 + SO4 2Reagents
• Standard sulphuric acid , (0.01N).
• Phenolphthalein ,0.25% solution in 60% ethyl alcohol( ethanol).
• Methyl orange , 0.5% powder dissolve in in 95% ethyl alcohol
Procedure
1. Take a known volume of ( 10 mL ) of water sample in 100 mL conical flask and
dilute it by 25 mL distilled water.
2. Add 3 drops phenolphthalein . If pink red colour appears , it means CO3 are
present, titrate then against standard H2SO4 till pink color disappears . The
burette reading ( volume used ) is designated as 'y' mL. Here carbonates are
converted into bicarbonates.
3. To this colorless solution add 3 drops of methyl orange or in original sample ( if
pink red color was not observed ) .
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4. again titrate with standard H2SO4till color changes from yellow to orange or
rosy red. Record the volume of H2SO4 as 'z' mL. This volume corresponds to
bicarbonates changed from carbonates plus initial bicarbonates present in
irrigation waters. Here all bicarbonates are destroyed.
Calculation
1000
Carbonates (me/litre) = 2(VolumeH2SO4 ) x Molarity of H2SO4 x--------ml of aliquot
=2Y x 0.01 x1000/5
=2Y x 2 = 4Y
2( Vol. of H2SO4 ) x Molarity x 1000 xEq. Wt. of CO3
Carbonates (g/litre) = -------------------------------------------------------------------ml of sample x 1000
Note:
The Y is the volume of acid used for half-neutralization of carbonate,
hence 2Y has been assumed as for its full neutralization.
Bicarbonates(me/litre) =( Z- 2Y) x molarity of H2SO4
1000
x -------------Ml of aliquot
Z-2Y) x 0.01 x 1000
= ------------------------------5
= ( Z- 2Y ) x 2
Where carbonate is absent: Z x 2
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Figure No.3.4 : Spectrophotometer
Figure No.3.5 :Study of
phytoplankton and
zooplankton using microscope
Sodium Carbonate in residual form (RSC)
This is an important quality for evaluating the appropriateness of water for
irrigation in consideration of likely sodium danger. It is measured from the
investigation data for CO3, HCO3 and calcium plus magnesium in the following
manner:
RSC meq L-1 = meq ( CO3 2- + HCO3 - ) – meq ( Ca 2+ + Mg 2+ )
Note: all expressed in meq
Where concentration of both cations and anions is in. sodicity danger in
terms of RSC is listed as under ( Eaton, 1950).
Sutability class
RSC Rating
Safe
Less than 1.25 meq L-1
Moderate
1.25 - 2.50 meq L-1
Unsafe
More than 2.5 meq L-1
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7. Determination of Chlorides
For determination of chloride the titration method of Mohr’s is most
commonly used. It is dependent upon the formation of precipitate of a scarcely
soluble brick-red silver chromate (AgCrO4) at the end point.
It is formed when
the sample is titrated against standard silver nitrate (AgNO3) solution in the
presence of potassium chromate (K2CrO4) as color indicator.
the reaction involved are as under
AgNO3 + Cl- → AgCl + NO3 White ppt
K2CrO4 + 2AgNo3 → AgCrO4 + 2 KNO3
brick red ppt
The chloride ions are initially precipitated as AgCl and just after the
precipitation of AgCl is over, dark brick-red precipitate of Ag2CrO4 starts
appearing.
Requirements
• Beakers/porcelain dish
•
Burette
Reagents
1. Potassium chromate (K2CrO4) indicator (5%) solution:
The solution can be prepared by dissolving 5 gram of K2CrO4 in about 75
ml distilled water. Then add saturated solution of AgNO3 dropwise until a slight
permanent red precipitate is formed. Filter and dilute to 100 ml.
2. solution of silver nitrate (Standard) 0.02N:
The solution of silver nitrate is made by dissolving 3.40 g of silver nitrate
(AgNO3) in double distilled water and made up to one litre volume .Then its
116 | P a g e
standardization is done against standard NaCl solution. After that it is stored
away from light in amber coloured bottle.
3. Standard sodium chloride , 0.02 N :
Take 1.17 g NaCl ( AR grade, dried as 800 C for 1 hour ), is dissolved in double
distilled water and volume made to one litre.
Procedure
1. Take a known volume of water sample , say 10 mL, and dilute to 25mL or the
same sample used for CO3 and HCO3 analysis may be utilized for chloride
determination.
2. Make it dark yellow by adding 5 drops of K2CrO4 indicator and titrate with the
standard AgNO3( 0.02 N ) solution. Then stir it continuously till the first brick-red
color appears.
Calculation
volume of AgNO3 mL × normality of AgNO3 × 1000mL
Chloride meq L-1 = ------------------------------------------------------water sample, mL
V × 0.02 × 1000mL
Chloride meq L-1 =
--------------------------------------------10 mL
8. Determination of Sulphate
Turbidimetric method
Traces of SO4 occur in all natural waters, its content can be appreciable
in most saline waters showing electrical conductivity values more than 1 mS cm1
at 250C.
Sulphate ions are precipitated as barium sulphate crystals of uniform size
in acid medium. Light absorbed by the precipitate is measured at 420 nm by
using a spectrophotometer.
Reagents
1. Barium chloride
BaCl2 . 2H2O crystals of 20 - 30 mesh .
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2. Standard sulphate solution
Dissolve 0. 1479 g of anhydrous sulphate in distilled water and make up to
1000mL. This gives 100 µg mL-1 solution of sulphate.
3. Conditioning reagent
Dissolve 75 g of sodium chloride in 300mL distilled water and 30 mL of
concentrated HCl and 100 mL 95 % ethyl alcohol ( or isopropyl alcohol ). Add 50
mL of glycerol and mix well and make up volume 500 mL with distilled water.
Procedure
1. Take a known volume ( 100 mL ) of water sample into 250 mL conical flask
with
pipette.
2. Add 5.0 mL of conditioning reagent. Then mix it well using the magnetic stirrer.
Keep speed of stirring same for both sample and standards.
3. While stirring add about
0.5 g barium chloride crystals, all at once and
continue to stir exactly 1min.
4. Immediately after one minute dispense some of the solution into absorption ell
of
photometer and compute the optical density at 30 seconds interval for 4
minutes taking the maximum absorbance which will be normally after a period of
2 minutes after finishing point of the stirring at 420 nm.
5. Carry out a blank determination on the reagents used.
6. Pipette 0, 10, 20, 40, and 50 mL standard sulphate solution of 100 µg mL-1
separately
in 250 mL conical flask. Add the balance of distilled water 100, 90,
80, 70, 60 and
50mL to make 100mL proceed as above step '2' step '4' for
each flask and use the
readings for plotting standard curve. From the
standard curve, read sulphate
concentration of a given sample.
Calculation
mg SO4 2- from standard curve X 1000
mg SO4 2- L -1 = ----------------------------------------------------sample mL
9. Determination of Nitrogen , Nitrate (NO3-N)
Apparatus
1. Micro - Kjeldahl glass distillation apparatus.
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2. Micro - burrette, 5 mL or 10 mL graduated.
Reagents
1. Standard H2SO4 (0.02N).
2. Boric acid solution ( 2 % )
2g boric acid + 98 ml distilled water
3. Mixed indicator
Bromocresol green 99 mg + methyl red 66 mg in 100 mL ethanol.
4. Strong NaOH (40 % )
40 g NaOH + 60 mL distilled water.
Procedure
Pipette out 10 mL of boric acid solution into 100mL beaker containing
mixed indicator, place the beaker below the condenser so that the tip of the
condenser dips in the solution. Pipette an aliquot ( usually 10 mL) of digested
acid extract into a distillation apparatus, funnel is washed with 2-3 mL of distilled
water and added 10 mL of 40 % NaOH solution to carry out the distillation.
When all the ammonia is evolved , stop the distillation and titrate the distillation
and titrate the distillate with standard H2SO4 till the color changes from green to
red . Blank should also be run and titration should be carried out to the same end
point as that of sample.
Calculation
X- Y ) x 0.28
NO3 - N ( mg/ litre ) = ------------------------ x 1000 = X – Y x 0.56
50 ( ml of sample )
-
Where,
X = volume (ml) of 0.02M H2SO4 consumed in sample titration.
Y = volume (ml) of 0.02M H2SO4 consumed in blank titration.
0.28 = Factor (1 lit 1M H2SO4) = 14 g N. Therefore,
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B. Method to Study of Phytoplankton and Zooplankton
2. B.1: Field collection methodology
For sampling of water in the larger river three to four samples are taken at
midpoint of equal cross sectional area of such river. The samples are then
combined
together to obtain a composite sample. If only a grab sample is
taken , then the
samples should be collected in the middle of the river stream
at the mid depth. When
a river is mixing with a sewage then sample is taken
down stream sufficiently
away to allow thorough mixing. Generally a distance
of 1 to 3 or 4 km below the tributary is
available.
The sampling was carried out for fixed intervals from the month October
2011 to July 2012 at the aforementioned localities of eastern part of Bhima river.
Sampling was carried out in the month of January (winter), April (summer) and
August (monsoon). Horizontal sampling was carried out by using Plankton Net
(53µm mesh, Wildco), by towing the open waters along the river bank. The
samples were carried to the laboratory in 100 ml plastic containers (Tarsons,
India). The samples were preserved and fixed in 4% formaldehyde. The sample
containers were labeled immediately and stored until further use.
2. B.2: Laboratory methods
The
laboratory
methods
for
identification
of
phytoplankton
and
zooplankton are mainly by the use of microscope. They were identified under
trinocular compound microscope (MLXi).
C. Method to Study of Ichthyofaunal diversity of Eastern part of
Bhima river
METHODS OF FISH PRESERVATION
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Fresh fishes are first fixed in formalin., and then preserved in formalin or
alcohol. Fixation of large specimen is done in the following solution :
Formalin (commercial, i.e. at 40% conc.) 1part
Water
9part
10% formalin
Smaller specimens may be fixed in 5 % formalin. The fixative is used in
neutral state. To obtain this, add one teaspoonful of household borax to one litre
of the above fixative. For best results, belly is cut open by making a slit on the
right side of the midline, and the fixative ( preservative as well) is allowed to enter
it. Large specimen need more care. Besides the slit in the belly, deep cuts are
made into the muscle mass on each side of vertebral column., preferably made
from inside the belly. The fishes are generally fixed for a period of several days.
Then they are transfered to the preservative. The specimens are first washed
well with water and then transferred to either formalin (10%) or ethyl alcohol. In
case of alcohol, specimens are first placed in 50% alcohol, for a few days and
then placed in the final preservative, 70% alcohol, and sealed.(fig.3.6).
Fig. No.3.6: Fish preservation
by labeling and storage
Fig. No.3.7: Visit to ZSI for
guidance and Identification .
for further study.
121 | P a g e
Fig. No.3.8.:Clarias fish
Fig. No.3.9: Collection and preservation
at local market of S4 station of different species at sampling station
There were some large fish species like the catfish Clarius gariepinus(fig2.8)
which were difficult yo preserve , the measurements and identification character
marking were noted down and the photographs of such fish species were taken by
camera.At each sampling station different species were collected and observations
of characters and the avilable number of each fish species were noted and the
species were immediately preserved in 4% formaldehyde solution at the samping
station.(fig. 3.9).
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3.D :Experimental Work
TAXONOMIC IDENTIFICATION OF FISHES
Procedure for identification & verification from the keys
The specimens are first keyed to their proper species designation from
standard available keys or check lists. Then the specimens are compared with
detailed descriptions, photographs, sketches etc. available from published
sources, preferably of the same region, or with museum specimens from a
standard collection, for final verification.
Procedure for identification of new species for a region or for the literature
It is necessary to first ascertain that the fish specimen held to be a species
new for the literature is not identifiable on the basis of known sources of
information either for the region or for the group to which it belongs. Then at least
30 specimens should be collected from the locality by random selection,
comprising both sexes and a wide size range. The new species will require, for
its conformation, support from any of the following :
Skeleton studies, Chromosome studies, protein taxonomy, numerical
taxonomy, quantitative characteristics of the population , Isozyme pattern , DNA
finger-printing etc.
MORPHOMETRIC AND OTHER ANALYSIS OF FISH BODY
Length of body
measurments of length of body should be taken on a fresh fish which has
yet not been deformed (bending) owing to rigor mortis. A straight line and not a
line following the curvature of the body is used for measurement. A measuring
board is used for the purpose. Measuring board are fabricated according to the
size range of the fishes to be measured. The board has a graduated meter scale
on it . The zero end of the level is matched with a suitable stop against which the
123 | P a g e
anteriormost extremity of the fish is brought to rest when measuring the length.
The board is shaped in V to hold the fish in position. For total length
measurement, mouth is kept closed and caudal fin squeezed (compressed). For
forked fin, tip of the longer lobe is used. For forked A length, measurement is
made from the anterior terminal to the notch of the forked caudal fin i.e.the tip of
the median fin rays. Standard length or A. D. length. measurement is made from
the anterior extremely (mouth closed).Tip of the snout or that of lower jaw, as
may be the case, to the base of caudal fin as determined by the groove or crease
formed when the tail is bent from side to side. This base repredsents the site
where median fin rays meet the median hypural plate. This is the commonest
length used for fishery work.
The different length measurements bof the fish are taken as below :
Total length - It is the distance between the anteriormost extrmity of the
body ( tip of snout or the upper lip and the posteriormost boundary of the body
i,e. the tip of the caudal fin lobe. The angle of the longest lobe if the caudal fin is
forked and has unequal lobes.
Head length : It is the space in a traight line between the anteriormost
part of the snout or the upper lip, whichever is extending farthest forward, and the
posteriormost edge of the opercular bone ( but notits spinous projections ).
Snout length : it is the space in a straight line between the anteriormost
part of the snoutor the upper lip whcihever is extending farthest forward and the
anterior margin of the orbit.
Post orbital length : It is the space in a straight line between the
posterior margin of the orbit and the posteriormost edge of the opercular bone.
Upper jaw length : It is the greastest span of the upper jaw.
Lower jaw length : It is the greastest length of lower jaw.
124 | P a g e
Figure 3.D1 : External Measurments of fish body.
Eye length/ diameter : It is the distance between the front and rear margin of
the eye.
Predorsal length : It is the space between the anteriormost end of the
body and the front end of the dorsal fin base.
Girth length : It is the distance covering the circumference of the body at
its deepest level.
Pre pectoral length : It is the distance between the anteriormost end of
the body and the front position of the pectoral fin base.
Pre anal length : It is the distance between the anteriormost end of the
body and the front point of the anal fin base.
Length of the caudal peduncle : It is the space between the rear point of
the anal fin base and the base of the caudal fin.
125 | P a g e
Figure 3.D2 : Trunk profile in girth Figure 3.D3 : Measurments of fin
Figure 3.D4 : Lateral view of fish head
The length measurements of the following parts may also be required whose
terminology is presented as follows :
Nape : It is the back of the neck.
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Isthmus : It is the interpace which separates the gill openings of the left and right
side. and which lies anterior to the breast.
Breast : It is the region which lies posterior to the isthmus and extends up
to the base of the pectoral fin. Heart lies in this region.
Occiput : It is the point on the mid dorsal line whcih joins the head with
the trunk. Occipital occiput head depth pectoral fin operculum suborbital depth D.
heart
Occipital : It is the distance between the two perpendicularlines touching
the posterior end of the orbit and the occiput.
Head depth : It is the distance between the occiput and the ventral side
of head.
Figure 3.D5 : Front view of fish head Figure3.D6 : Ventral view of fish
head
Weight of body
In weighing the fish, consistency is maintained with respect to the degree
of wetness of the body, the usage of fractional weights and the state of the fish (
fresh or preserved ). A container with enough water to accomodate the fish to
weighed, is first weighed. Then fish is put in it and weight is taken. The difference
gives the weight of the fish.
127 | P a g e
Body ratio H/L of fish
The ratio that height bears to other length of the fish, caudal fin excluded
is body ratio or H/L. This is an index of fleshyness of fish between the individuals
of a given species. The more the fish attains high backs, greater is its importance
as table fish because it would yield relatively more flesh. Wild carps from rivers
have this ratio varying between1/3 to 1/4. A fat raised carp on the other hand
may have this ratio anywhere between 1/2 to 1/3.
Sex determination
It is done by examination of secondary sexual characters only for those
species for which sexual dimorphism has been worked out. The individual has to
be a sexually mature one for this manner of determination of sex. Where sexual
dimorphism is not shown, sexes may be identified by observing the courtship
behaviour : generally, the female is followed by the male. For other cases,
particularly the immature ones, direct examination of the gonads is necessary.
METHODS OF MEASURING CONDITION OF FISH
Condition of fish in general is an expression of relative plumpness of a
raised fish with respect to the same species taken from other water bodies or to
other species of fish taken from the same water body.
To study the ichthyofauna of Eastern part of Bhima river throughout the
observation period from May 2011 to August 2012, fish species were sampled
every week from the fish corridor centers taking the help of
local expert
fishermen by using different fishing equipments like the crafts and gears with
variable mesh size. Sampling locations were distributed throughout the site to
cover its whole area and location was altered for the collection of fish fauna
according to the season. Classification of fishes was done up to species level at
fish landing center with the help of standard literature to get its natural colour,
identification marks like black spot, pattern of scales, fins, bloach on operculum,
mouth pattern, paired and unpaired fins and body parts [ Hamilton Buchanan
128 | P a g e
1822, Day, F.1878, Menon A.G.K. 1999, Jayaram K.C 1981. Datta Munshi J.S
and Shrivastava 1988 Jayaram K.C 1991 Jyoti .M.K and Sharma .A 2006
Yazdani G.M. 1985. ] and etc(fig. No.3.6. fig.3.7)
Figure No.3.D7:Fish collection,
identification of fish by local name
by fishcatcher.
Figure No.3.D8: Collection of fish from
river water through small ‘hodi’.
Fig. No.3.D9: AFish collection method. Fig. No.3.D10: Fish observation at
sampling station
At the sampling stations fish wrere collected from river water using crafts,
from the local fish catchers. (fig. No. 3.B7,Fig.No. 3.B8). The characters were
studied by making notes of measurements and observation of fins , blotches,
mouth , operculum, presence of barbs, colour etc(figure 3.B10),fig.3.B11 and fig.
3.B12)) The local names were also noted from the fishcatchers. The abundance
was counted for different fish species.(fig. 3.B9; andfig.3.B7).
129 | P a g e
Fig. No.3.D11: Study of fish characters
by Measurements.
Fig. No.3.D12: Study of fish
characters by obseravtions of
fins blotches etc.
Some fish species were collected from the local fishmarkets at the sampling stations
and photographs of some large fish species were taken from the fish sellers at the
markets.
Fig. No.3.D13:Fish observation and
collection of some species
from local fishmarket.
Fig. No.3.D14:Large fish kept in
icebox at the
local fishmarket.
130 | P a g e
Model- Key to the Identification of Fishes
1. Skeleton bony -------
A. Bony fishes (Actinopterygii)
2. No bony skeleton----
B. Cartilaginous fishes (Selachii)
A. Bony Fishes (Actinopterygii)
1. Body elongate or cylindrical ..........
3.
2. Body neither or cylindrical..............
7.
3. A single opening owing to confluence of the
two gill openings .........
Synbranchiformes
4. Two lateral gill openings (non-confluent) ......
5. Dorsal spines.................
5
Mastacembeliformes
(Mastacembelus,Macrognathus)
6. No dorsal spines.........
Anguiliformes
7. Body asymmetrical...........
Pleuronectiformes
8. Body symmetrical........
9. Jaw or jaws projecting into beaks.....
9.
Beloniformes (Xenetodon )
10. No jaw or jaw projecting into beaks.......
11
11. Head snake-like; suprabranchial organ
Present..........
Channiformes(Channa)
12. Head not snake-like : no suprabranchial organ.......
13.
13. Distinct spinous and soft parts in the dorsal fin......
15.
14. No distinct spinous and soft parts in the dorsal fin....
17.
15. Wide interdorsal space......
Mugiliformes (Rhinomugil)
131 | P a g e
16. Narrow interdorsal space......
Perciformes
(Johnius,Colisa )
17. Keeled and serrated abdomen : barbels absent.......
Clupeiformes
18. Not keeled and serrated abdomen ; barbels present........Cypriniformes
Clupeiformes : Key to genera
1. Head scaly; absence of adipose fin; dorsal fin in the caudal
region.......
Notopterus
2. Head not scaly; no adipose fin; dorsal fin in the
trunk region .....
3.
3. Upper jaw prominent ; maxilla much elongate......
5.
4. Upper jaw not prominent, maxilla not elongate.....
7.
5. Caudal fin forked..........
Setipinna
6. Caudal fin pointed........
Coilia
7. Abdomen non-serrated.....
Dussumeria
8. Abdomen serrated ......
9. Toothless.......
9.
Gonialosa,
Nematolosa
10. Teeth present.....
11. Anal fin long ( more than 35 rays ) ......
11.
Pellora
12. Anal fin maderate (less than 23 rays ) .....
13.
13. Lateral line scale count less than 50.....
15.
14. Lateral line scale count more than 75......
Gudusia
15. Upper jaw with a median notch....
Hilsa
16. Upper jaw without a median notch.....
Sardinella
132 | P a g e
Cypriniformes: Key to the genera.
1. Scaly.....
A.(carps, Cyprinoidei )
2. Non- scaly......
B. (catfishes, Siluroidei )
A. Cyprinoidei
1. Barbels 6 to 8 ....
Botic, Noemachellus
2. Barbels 0 to 4 .......
3.
3. Anal spine (last unbranched ray) serrated ....
5.
4. Anal spines non-serrated ......
7.
5. Barbels absent........
Corassius
6. Barbels present (2 parts)....
Cyprinus
7. Abdomen keeled......
Chela, Oxygaster, Hypophthalmichthys
8. Abdomen not keeled ......
9. Barbels absent......
9.
Ctenepharyngodon
10. Barbels present....
11.
11. Anal sheath of large tile-like scales, lateral-line
Scales 94 or less.....
Schizothoras
12. No tile - like scales on anal sheath, lateral-line
Scales 94 or less....
13. Lower lip modified into a sucking disc....
13.
Garra
14. lower lip not modified into a sucking disc ...
15
15. A continuous transverse fold in the lower lip....
16.
16. No continuous transverse fold in the lower lip ....
17.
17. Anal fin having more than 13 rays, body very
133 | P a g e
much compressed ....
Rohtee, Osteobrama
18. Anal fin having less than 11 rays, body not
very much compressed ....
19.
19. Upper lip absent ...
Catla
20. Upper lip present....
21.
21. A symphysal knob in lower jaw......
Cirrhinus
22. No symphysal knob in lower jaw ....
23.
23. Mouth inferior: lower lip fringed ......
Labeo
24. Mouth terminal or sub-terminal, lower lip not fringed...... Puntius
B. Siliuroidei
1. Anal fin long with more than 30 rays .....
3.
2. Anal fin short with less than 20 rays ......
21
3. Caudal fin united with 2nd dorsal fin and anal fin .........
Plotosus
4. Caudal fin not united with 2nd dorsal and anal fin .........
5. First dorsal elongated ......
5
Clarias
6. First dorsal short or absent.......
7
7. Adipose ( dorsal ) fin present .......
9
8. Adipose ( dorsal ) fin absent ....
17
9. Dorsal fin absent. anal fin long with more than 58 rays .... Ailia
10. Dorsal fin present. anal fin short with less than 30 rays ....
11
11. Barbels 8 ( 4 pairs ) ........
13
12. Barbels 2-4 ( 1-2 ) pairs .......
15
13. Gape extends to middle of eye .....
Eutropichthys
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14. Gape extends up to front of eye .....
Clupisoma
15. Barbels 2 ( 1 pair ) and short, anal fin long with
40 rays or more ......
Silonia
16. Barbels 4 ( 2 pairs ) and long, anal fin moderate with
31-34 rays......
Pangasius
17. 4 pairs of barbels : presence of nasal barbels ..... Heteropneustes
18. 2 pairs of barbels : absence of nasal barbels .......
19
19. Gape extends beyond eye ...
Wallago
20. Gape does not extends beyond eye ...
Ompak
21. Nostrils separated by a valve, barbels 6 ( 3 pairs ) ....
Tachysurus
22. Nostril sepsrated by barbel, barbels 3-4 pairs .....
23.Body ( head and trunk ) flat, paired fins horizontal .....
23
Bagarius
24. Body ( head and trunk ) not flat, paired fins not horizontal .....
25
25. Barbels 3 pairs : spines of dorsal fin and pectoral fin
strong and hollow ....
Rita
26. Barbels 4 pairs , spines of dorsal and pectoral fin
neither strong nor hollow......
Mystus.
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