Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt

Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

भारतीय मानक IS 3025 (Part 31/Sec 1) : 2022

Indian Standard

जल एवं अपशिष्ट जल के नमूने लेने तथा

परीक्षण ( भौतिक एवं रसायन ) की पद्धतियाँ
भाग 31 फॉस्फोरस
अनुभाग 1 वनाडोमोलिब्डो-फॉस्फोरिक एसिड, स्टै नस क्लोराइड,
एस्कॉर्बिक एसिड और पर्सल्फेट विधि द्वारा निर्धारण
(  दसू रा पनु रीक्षण )

Methods of Sampling and Test

( Physical and Chemical )
for Water and Wastewater
Part 31 Phosphorus
Section 1 Determination by vanadomolybdo-phosphoric
acid, stannous chloride, ascorbic acid
and persulphate method

( Second Revision )

ICS 13.060.50

© BIS 2022

भारतीय मानक ब्रयू ो

B U R E A U O F I N D I A N S TA N D A R D S
मानक भवन, 9 बहादरु शाह ज़फर मार्ग, नई िदल्ली – 110002
MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI-110002
         www.bis.gov.in  
www.standardsbis.in

April 2022  Price Group 6

Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

Water Quality Sectional Committee, CHD 36

FOREWORD
‘This Indian Standard (Second Revision) was adopted by the Bureau of Indian Standards, after the draft finalized
by the Water Quality Sectional Committee had been approved by the Chemical Division Council’.
Phosphorus is present in natural water and waste waters mostly in the form of phosphates. These can be classified
into ortho-phosphates, condensed phosphates (pyro, meta and other poly phosphates) and organically bound
phosphates. They are present in solution, in particles or detritus or in the bodies of aquatic organisms.
Variety of sources give rise to various types of phosphates. Orthophosphates in small amounts or certain condensed
phosphates are added to water supplies while treatment. Phosphates in larger quantities may be added during
the process of laundering or other cleaning. Phosphates are extensively used for the treatment of boiler water.
Orthophosphates used as fertilisers in agricultural or residential cultivated land are carried to surface waters with
storm runoff and in relatively smaller amounts with melting snow. Organic phosphates are formed primarily by
biological processes. They are contributed to sewage by body wastes and food residues and also may be formed
from orthophosphates in biological treatment process or by receiving water biota.
This standard was first revised in 1988. In the first revision of the standard, the following two methods for
determination of phosphorus, were given:
a) Vanadomolybdo - phosphoric acid method; and
b) Stannous chloride method.
In this second revision the following modifications have been incorporated:
1) This standard has been renumbered as Part 31/Sec 1 as other sections will be developed for instrument
based methods;
2) Ascorbic acid method has been added; and
3) Persulphate method for simultaneous determination of total nitrogen and total phosphorus has been
added.
This section of Part 31 covers determination of phosphorous by vanadomolybdo-phosphoric acid, stannous
chloride, ascorbic acid and persulphate method. Section 2 of Part 31 specifies flow injection analysis (FIA)
methods for the determination of orthophosphate in the mass concentration range from 0.01 mg/l to 1.0 mg/l (P),
and total phosphorus by manual digestion in the mass concentration range from 0.1 mg/l to 10 mg/l (P). Section 3
of Part 31 specifies continuous flow analysis (CFA) methods for the determination of orthophosphate in the mass
concentration range from 0.01 mg/l to 1.00 mg/l P, and total phosphorus in the mass concentration range from
0.10 mg/l to 10.0 mg/l P.
In the preparation of this standard, considerable assistance has been derived from the method no 4500 P of standard
methods for the examination of water and wastewater‖, published by the American Public Health Association,
Washington, USA, 23nd Edition, 2017.
In reporting the result of a test or analysis made in accordance with this standard, if the final value, observed or
calculated, is to be rounded off, it shall be done in accordance with IS 2 : 2022 ‘Rules for rounding off numerical
values ( second revision )’.
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

Indian Standard
METHODS OF SAMPLING AND TEST
( PHYSICAL AND CHEMICAL )
FOR WATER AND WASTEWATER
PART 31 PHOSPHORUS
Section 1 Determination by vanadomolybdo-phosphoric acid, stannous chloride,
ascorbic acid and persulphate method

( Second Revision )

1 SCOPE 3 PRELIMINARY DIGESTION STEPS

This standard (Part 31/Sec 1) prescribes four methods 3.1 Phosphorus analysis involves the conversion of
for determination of phosphorus, namely: phosphorus present in water in different forms to
a) Vanadomolybdo-phosphoric acid method; dissolved orthophosphate which is then estimated
b) Stannous chloride method; calorimetrically.
c) Ascorbic acid method; and 3.1.1 For Acid Hydrolysable Phosphates
d) Persulphate method for simultaneous determination To a sample of 100 ml volume or to a part of it which
of total nitrogen and total phosphorus. is diluted to 100 ml, phenolphthalein indicator solution
0.05 ml (1 drop) is added. Add strong acid solution
2 SAMPLING AND STORAGE
drop wise, in case the red colour appears more acid
2.1 In order to differentiate between the dissolved is added to discharge the red colour. Again add 1 ml
forms of phosphorus, filter the sample immediately of acid and further boil the solution gently for about
after collecting it. Preserve the sample by freezing it 90 min. Add distilled water in order to keep the volume
at or below – 10 °C. In few cases, 40 mg/l of mercuric between 25 and 50 ml. Alternatively, heat the solution
chloride may be added to the samples, particularly for 30 min in an autoclave or by using pressure cooker
when they are stored for a long period of time before at 98 to137 kPa. Cool the solution and neutralize it to a
the analysis. faint pink colour by using sodium hydroxide solution.
Restore it in 100 ml volumetric flask by making up the
CAUTION — As mercuric chloride is hazardous, care must be
volume using distilled water.
taken for its disposal.

2.2 Use of mercuric chloride is not encouraged. Use 3.1.2 For Total Phosphorus
of acid or chloroform (CHCl3) as a preservative is not 3.1.2.1 Perchloric acid digestion
recommended when different forms of phosphorus are
to be determined. To determine the total phosphorus, This is used for digestion of organic phosphorus and
add sulphuric acid or hydrochloric acid at pH < 2 and samples of sediment and organic bound phosphorus
cool it to 4 °C or freeze it without any additions. compounds. Take the sample that contains the desired
amount of phosphorus in a 125 ml ErlenMeyer flask.
2.3 Samples that contain low concentrations of Acidify the sample using concentrated nitric acid
phosphorus should not be stored in plastic bottles (HNO3) to methyl orange, and then further add 5 ml
unless kept in a frozen state because phosphates can be concentrated HNO3 and evaporate on a steam bath or
absorbed on the walls of the plastic bottles. hot plate to 15 to 20 ml. Add 10 ml of each concentrated
nitric acid and perchloric acid to a conical flask of
2.4 Rinse all the glass containers by using dilute 125 ml capacity by cooling it between the addition of
hydrochloric acid (HCl) and further rinse it several times concentrated nitric acid and perchloric acid. Add some
with reagent water. Never use commercial detergents boiling chips and heat it on a hot plate. Further, gently
that contain phosphate for cleaning glassware that is evaporate till dense white fumes of perchloric acid
to be used in analysis of phosphate. More strenuous appears. In case the solution is not clear, cover the neck
cleaning techniques may be used. of the flask with a watch glass and keep the solution

1
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

boiling till it is clear. If required, 10 ml concentrated 4.2 Interference

nitric acid is added, in order to ease oxidation. Cool If the sample is heated silica and arsenate causes
the digested solution and then add 1 drop of aqueous positive interferences. However arsenate, fluoride,
phenolphthalein solution. Add 6 N sodium hydroxide thorium, bismuth, sulphide, thiosulphate, thiocyanate
solution till the colour of the solution turns pink. As or excess molybdate causes negative interferences.
per the requirement, filter the neutralized solution and Ferrous iron causes blue colour but this cannot be seen if
wash filter using distilled water. Make the volume up to ferrous iron is present in low concentration of 100 mg/l.
100 ml with distilled water. Interference due to sulphide may be removed by using
3.1.2.2 Sulphuric acid –nitric acid digestion bromine water for oxidation. List of ions which do not
interfere if present in concentrations up to 1 000 mg/l
In a micro Kjeldahl flask, sample containing phosphorus are aluminium ion (Al3+), iron ion (Fe3+), magnesium ion
(as determined by colorimetric method) in desired (Mg2+), calcium ion (Ca2+) barium ion (Ba2+) strontium
amount is measured. Add 5 ml of concentrated nitric ion (Sr2+) lithium ion (Li+), sodium (Na+), potassium
acid and 1 ml of concentrated sulphuric acid. Digest the ion (K+), ammonium ion (NH4+) cadmium ion (Cd2+),
solution to a volume of 1 ml and continue till solution manganese ion (Mn2+), lead ion (Pb2+), mercury ion
becomes colourless in order to remove nitric acid. (Hg+,Hg2+), tin ion (Sn2+), copper ion (Cu2+), nickel
Cool the solution and add to it approximately 20 ml ion (Ni2+), silver (Ag+), uranium ion (U4+), arsinite
of distilled water, phenolphthalein indicator 0.05 ml ion (AsO3–), bromide ion (Br –), carbonate (CO32–),
(1 drop), and 1 N sodium hydroxide solution in a perchlorate ion (ClO4–), cyanide (CN–), Iodic acid anion
quantity that is required to produce a faint pink tinge. ( IO3–), silicate (SiO44–), nitrate (NO3–) , nitrite (NO2–),
Into a 100 ml volumetric flask, transfer the neutralized sulphate (SO42–), sulphite (SO32), selenite, molybdate,
solution and filter it if required so as to remove pyrophosphate, teraborate,, citrate, bemzoate, oxalate,
particulate material or any kind of turbidity. Sample lactate, tartrate, formate and salicyate.
volume to 100 ml is adjusted by using distilled water.
4.2.1 Minimum Detectable Concentration
3.1.2.3 Persulphate digestion method
The minimum level of concentration that can be
Take 50 ml or a suitable proportion of sample. Add detected is 200 µg/l in 1 cm spectrometer cells.
phenolphthalein indicator solution (0.05 ml) to it. If
red colour appears, sulphuric acid solution is added 4.3 Apparatus
dropwise in order to discharge the colour. Then 1 ml
of sulphuric acid solution is added along with either 4.3.1 Spectrophotometer
0.5 g of solid potassium persulphate or 0.4 g solid For use at 400 to 470 nm. A wavelength of 470 nm
ammonium persulphate. For about 30 to 40 min, gently usually is used. The concentrations for different
boil on a preheated hot plate or till a final volume of wavelengths are as specified in Table 1.
10 ml is reached. Organic phosphorus compounds, such
as AMP may take 1.5 to 2 h for complete digestion.
Cool the solution and then dilute to 30 ml by using Table 1 Concentrations for Different Wavelengths
distilled water. After that add phenolphthalein indicator ( Clause 4.3.1 )
solution 0.05 ml (1 drop), and neutralize it to a faint
pink colour by using sodium hydroxide. Makeup the Sl No. Concentration of Phosphorous, Wavelength,
mg/l nm
volume up to 100 ml with distilled water. In some
samples a precipitate is formed at this stage, but do not (1) (2) (3)
filter. Shake well for any further subdivision of sample. i) 1.0-5.0 400
In the acidic conditions of the colorimetric reactive ii) 2.0-10.0 420
phosphorus test, the precipitate re-dissolves. iii) 4.0-18.0 470

4 VANADOMOLYBDO-PHOSPHORIC ACID 4.3.2 Acid Washed Glassware

METHOD
Use glassware that is washed with acid in order to
4.1 Principle determine the low concentrations of phosphorus.
Contamination due to phosphate is common due to
Orthophosphate reacts with ammonium molybdate its absorption on glass surface. Use of commercial
under acid conditions to form a heteropoly acid, detergents that contains phosphate should be avoided.
molybdophosporic acid. In the presence of vanadium, it Clean the entire glassware by using hot dilute
changes into yellow vanadomolybdo-phosphoric acid. hydrochloric acid and after that distilled water should
The intensity of the colour is proportional to phosphate be used to rinse it well. Preferably, reserve the glassware
concentration. for phosphate determination and after use, keep filled

2
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

with water after washing until required. After this, acid 4.5.4 Calibration Curve
treatment is required only occasionally. Calibration curve is prepared by the use of suitable
4.3.3 Filtration Apparatus volumes of standard phosphate solution and by
using the procedure that is given above. At least one
4.3.4 Filter Paper, Whatman No 42 or equivalent. standard with each set of samples should be analysed
and the concentration of the sample should be read
4.4 Reagents from the calibration curve for the given value of
4.4.1 Phenolphthalein Indicator Aqueous Solution absorbance.

4.4.2 Hydrochloric Acid (HCl), 1 : 1 Sulphuric acid, 4.6 Calculation

m
perchloric acid, or nitric acid may be substituted for Phosphorus (P), mg/l = × 1 000
HCl. V
where
4.4.3 Activated Carbon, phosphate free. Remove fine m = amount of phosphorus (in 50 ml of final
particle by rinsing it with distilled water. volume), in mg; and
4.4.4 Vanadate-Molybdate Reagent V = volume of sample, in ml.

4.4.4.1 Solution A, in 300 ml distilled water, dissolve 5 STANNOUS CHLORIDE METHOD

25 g ammonium molybdate.
5.1 Principle
4.4.4.2 Solution B
The molybdo-phosphoric acid is formed and
By heating 300ml distilled water upto boiling, dissolve reduced by stannous chloride and intensely coloured
1.25 g ammonium meta vanadate. After that cool and molybdenum blue is obtained. This method is
add 330 ml concentrated hydrochloric acid. Further, sensitive and makes measurements down to 7 µg P/l
cool solution B up to room temperature. Then solution with minimum amount of interference.
A is poured into B, mixed and further diluted to 1 l.
5.2 Interference
4.4.5 Standard Phosphate Solution
Positive interference is caused by silica and arsenate
In distilled water, dissolve 219.5 mg of anhydrous only when we heat the sample. However, negative
potassium di hydrogen phosphate and dilute the mixture interferences are caused by arsenate, thorium,
to 1 000 ml (1.00 ml = 50.0 µg of orthophosphate P). bismuth, fluoride, thiosulphate, sulphide, thiocyanate
or excess molybdate. Ferrous iron results in blue
4.5 Procedure
colour but this does not affect the results in case
4.5.1 pH Adjustment concentration of ferrous iron is less than 100 mg/l.
Interference due to sulphide may be removed by
If the pH of the sample is more than 10, phenolphthalein oxidation with bromine water.
indicator (0.05 ml) is added to 50.0 ml of sample and
1 + 1 hydrochloric acid is added so that the red colour 5.2.1 Minimum Detectable Concentration
is discharged. Further, the solution is diluted to 100 ml.
The minimum concentration that can be detected is
4.5.2 Colour Removal about 3µg P/l. The sensitivity at 0.301 0 absorbance
is about 10 µg/l for an absorbance change of 0.009.
Remove the excess of colour present in the sample
by shaking 50 ml of sample with 200 mg of activated 5.3 Apparatus
carbon for 5 min using an Erlenmeyer flask. Further,
filter and remove carbon. Check the concentration 5.3.1 Spectrophotometer
of phosphate for each batch of carbon because high For use at 625 nm in the measurement of
reagent blanks are produced by some batches. benene-isobutanol extracts and at 690 nm, use a
wavelength of 650 nm for aqueous solutions, with
4.5.3 Colour Development
somewhat reduced sensitivity and precisions.
Take 35 ml or less of the sample, which contains
0.05 to 1.0 mg of phosphorus (P) in 50 ml flask. Now 5.4 Reagents
add vanadate-molybdate reagent (10 ml) and dilute the
5.4.1 Phenolphthalein Indicator Aqueous Solution
sample upto the mark by using distilled water. After
that substitute the sample with 35 ml of the distilled 5.4.2 Strong Acid Solution
water and after 10 min measure the absorbance at 400 to
Slowly add 300 ml of concentrated sulphuric acid
490 nm of wavelength, depending on sensitivity desired.
to about 600 ml of distilled water. When cool, add
The colour of the sample is stable for days and intensity
4.0 ml of concentrated nitric acid and dilute to 1 l.
is not affected by change in room temperature.

3
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

5.4.3 Ammonium Molybdate Reagent I 5.5.3 Colour Measurement

Dissolve 25 gm ammonium molybdate in 175 ml After 10 min but before 12 min, using the same
of distilled water. After that carefully add 280 ml of equal intervals for all determinations, measure colour
concentrated sulphuric acid to 400 ml distilled water. photometrically at 690 nm and compare with a
Cool and add molybdate solution and dilute to 1 l. calibration curve, using a distilled water blank. Light
path lengths suitable for various concentration ranges
5.4.4 Stannous Chloride Reagent I are as specified in Table 2.
Dissolve 2.5 gm fresh stannous chloride in 100 ml Table 2 Light Path Lengths Suitable for Various
glycerol. Heat it on a water bath and stir it using a glass Concentration Ranges
rod to hasten dissolution. This reagent is stable and does
not require any preservatives or special storage. ( Clause 5.5.3 )
Sl No. Approximate P Range, mg/l Light Path, cm
5.4.5 Standard Phosphate Solution
(1) (2) (3)
Take 219.5 mg anhydrous potassium dihydrogen
phosphate and dissolve it in distilled water. Further dilute i) 0.3-2 0.5
it to 1 000 ml. (1.00 ml = 50.0 µg of orthophosphate P). ii) 0.1-1 2
iii) 0.007- 0.2 10
5.4.6 Reagents for Extraction
Always run a blank on distilled water and reagents.
5.4.6.1 Benzene isobutanol solvent Because the colour first develops gradually and
Mix equal volumes of isobutyl alcohol and benzene. later fades, maintain equal intervals for samples and
CAUTION — This solvent system is highly inflammable. standards. Prepare at least one standard for each set
of samples or once each day to conduct the tests. The
5.4.6.2 Ammonium molybdate reagent II calibration curve can deviate from a straight towards
Take 40.1 gm ammonium molybdate and dissolve it in the higher concentrations of the 0.3 -2.0 mg/l.
approximately 500 ml distilled water. After that slowly
add 396 ml ammonium molybdate reagent I. Cool and 5.5.4 Extraction
dilute to 1 l. In order to increase the sensitivity and to overcome
interferences, extraction of phosphate is done as
5.4.6.3 Alcoholic sulphuric acid solution follows:
To 980 ml methyl alcohol with continuous mixing add
20 ml concentrated sulphuric acid cautiously. 5.5.4.1 Pipette out 40 ml sample or one diluted to
that volume, in a 125 ml separating funnel. Further
5.4.6.4 Dilute stannous chloride reagent II add 50 ml benzene-isobutanol solvent and 15 ml
molybdate reagent II. Close the funnel quickly and
Mix 8 ml stannous chloride reagent I and 50 ml glycerol.
shake vigorously for exactly 15 s. In the presence
This reagent is stable and can be kept for about 6 months.
of condensed phosphate, any delay will increase its
5.5 Procedure conversion to orthophosphate. Remove the stopper
and take out 25 ml of separated organic layer, using
5.5.1 Preliminary Sample Treatment a pipet with safety bulb. Transfer it to a 50 ml
To 100 ml sample that does not contains more than volumetric flask, add 15 to 16 ml alcoholic sulphuric
200 µg phosphorous (P) and is free from colour and acid solution. Swirl and add 0.50 ml (10 drops)
turbidity, add phenolphthalein indicator 0.05 ml dilute stannous chloride reagent II. Again swirl and
(1 drop). If sample turns pink, add strong acid solution dilute it with alcoholic sulphuric acid solution. Mix
drop by drop so as to discharge the colour. If more it thoroughly and after 10 min but before 30 min
than 0.25 ml (5 drops) of acid is required, take a small read against the blank at a wavelength of 625 nm.
amount of sample and dilute to 100 ml with distilled Prepare a blank by carrying 40 ml distilled water
water after first discharging the pink colour with acid. by using similar procedure as used for the sample.
Read the phosphate concentration from a calibration
5.5.2 Colour Development curve which is prepared by taking known phosphate
After thorough mixing after every addition, further standards by using similar procedure as used for
add 4.0 ml molybdate reagent I and 0.5 ml (10 drops) samples.
stannous chloride reagent I. Intensity and rate of
5.5.4.2 Direct procedure
colour development depend on temperature of the final
solution which produces about 1 per cent increase in Concentration of phosphorous, mg/l =
colour intensity with each 1oC temperature increase. mg P ( Approximately 104.5 ml finsl volume) * 1000
Hence, hold samples, standards, and reagents within
Volume of sample in ml
2 oC of one another and in the temperature range
between 20 oC and 30 oC.
4
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

5.5.4.3 Extraction procedure 6.4 Reagents

Concentration of phosphorous, mg/l = 6.4.1 Sulfuric Acid, 5N
mg P ( Approximately 104.5 ml finsl volume) * 1000 Dilute 70 ml of concentrated sulphuric acid to 500 ml
Volume of sample in ml with distilled water.
6.4.2 Antimony Potassium Tartrate Solution
6 ASCORBIC ACID METHOD
In a 500 ml volumetric flask dissolve 1.371 5 gm of
6.1 Principle antimony potassium tartrate in 400 ml of distilled
water. Dilute it to the volume and store it in a
In acidic medium, ammonium molybdate and
glass-stoppered bottle.
antimony-potassium tartrate react with orthophosphate
and form a heteropoly acid-phosphomolybdic 6.4.3 Ammonium Molybdate Solution
acid which can be reduced to intensely coloured
Dissolve 20 gm of ammonium molybdate in 500 ml of
molybdenum blue by ascorbic acid.
distilled water and store it in a glass- stoppered bottle.
6.2 Interference
6.4.4 Ascorbic Acid
Arsenates react with the molybdate reagent and gives
Dissolve 1.76 gm of ascorbic acid in 100 ml of distilled
a blue colour similar to that formed with phosphate. In
water. This solution is stable for about one week at
low concentrations of 0.1 mg As/l arsenic can interfere
4 oC.
with the determination of phosphate. At concentrations
of 1 mg/l nitrite and hexavalent chromium interferes to 6.4.5 Combined Reagent
give 3 percent lower results and 10 to 15 percent lower
results at a concentration of about 10 mg/l. Silicates For 100 ml of the combined reagent, mix all the above
and sodium sulphide do not interfere at concentrations reagents in the given proportions. Take 50 ml of 5 N
of 1.0 and 10 mg/l. sulphuric acid, 5 ml of antimony potassium tartrate
solution, 15 ml of ammonium molybdate solution
6.2.1 Minimum Detectable Concentration, and 30 ml of ascorbic acid solution. Mix it well after
approximately 10 µg P/l. Light path lengths suitable addition of each type of reagent. All the reagents shall
for various concentration ranges are as specified in be mixed in the order given after they reach the room
Table 3. temperature. If turbidity forms with the combined
reagent, shake it and let it stand for a few minutes until
Table 3 Light Path Lengths Suitable for Various
turbidity disappears. The reagent is stable for upto 4 h.
Concentration Ranges
( Clause 6.2.1 ) 6.4.6 Stock Phosphate Solution
Sl No. Approximate P Range, mg/l Light Path, cm Dissolve 219.5mg of anhydrous potassium dihydrogen
phosphate in distilled water and dilute it to 1 000 ml
(1) (2) (3)
(1 ml = 50.0 µg of orthophosphate P).
i) 0.3-2 0.5
ii) 0.15-1.3 1.0 6.4.7 Standard Phosphate Solution
iii) 0.01-0.25 5.0 Dilute 50 ml of stock phosphate solution to 1 000 ml
using distilled water (1 ml = 2.5 µg of P).
6.3 Apparatus
6.5 Procedure
6.3.1 Spectrophotometer, having infrared phototube for
use at 880 nm that can provide a light path of 2.5 cm 6.5.1 Sample Treatment
or longer. In a clean, dry test tube or 125 ml Erlenmeyer flask
pipet out 50 ml of sample. To it add 0.05 ml (1 drop)
6.3.2 Acid Washed Glassware phenolphthalein indicator. In case, a red colour develops
For the determination of lower concentration of add 5 N sulphuric acid solution drop by drop in order
phosphorous glassware washed with acid shall be used. to just discharge the colour. Add 8.0 ml of combined
Contamination with phosphate is common because of reagent and mix it thoroughly. After 10 min but no
its absorption on glass surface. Commercial detergents more than 30 min, measure absorbance of each sample
containing phosphate should not be used for washing. at a wavelength of 880 nm by using reagent blank as
Clean all glassware with hot dilute hydrochloric the reference solution.
acid (HCl) and rinse it well with distilled water.
Preferably, keep aside the glassware only for phosphate 6.5.2 Correction for Turbidity or Interfering Colour
determination and after use, wash it and keep it filled At the higher wavelength, the natural colour of water
with water until required. If this is done, treatment with generally does not interfere. In case of highly coloured
acid is needed occasionally. or turbid waters, prepare a blank sample by adding all
5
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

the reagents other than antimony potassium tartrate and 7.3.2 Glass Culture Tube, 13 mm outer diameter and
ascorbic acid to the sample. Subtract the absorbance of 100 mm long with auto clavable caps.
the blank from the absorbance of the sample.
7.3.3 Autopipettor, should be capable of pipetting a
6.5.3 Preparation of Calibration Curve 6 ml portion.
Prepare the individual calibration curves from a series 7.3.4 Repeating Pipettor, should be capable of
of four up to six standards, including a blank for pipetting a 1.25 ml portion.
calibration, within the phosphate range, as indicated at
6.2.1. The calibration blank solution consists of reagent 7.3.5 Erlenmeyer Flask, 3 000 ml.
water with the combined reagent. Plot the absorbance
verses phosphate concentration and with each set of 7.3.6 Aluminium Foil
samples, test atleast one phosphate standard. 7.3.7 Automated Continuous Flow Instrument System
6.6 Calculation for Nitrate and Phosphate Determination
mg P (in approximately 58 ml final volume) 7.3.7.1 For nitrate determination
×100
mg P / l = An example of a continuous-flow analytical instrument
ml sample is as given in Fig. 1. Tubing volume and flow-rates are
given as example only; manufacturers instruction to be
7 PERSULFATE METHOD FOR followed.
SIMULTANEOUS DETERMINATION
OF TOTAL NITROGEN AND TOTAL 7.3.7.2 For phosphate determination
PHOSPHORUS An example for a kind of continuous flow analytical
instrument consists of the interchangeable components
7.1 Principle are as given in the Fig. 2. A flow cell of 15 or 50 nm
For determining total nitrogen, the oxidation of and a filter of wavelength 650 nm to 660 nm or 880 nm
nitrogenous compounds must take place in an alkaline may be used.
medium while for determining the total phosphorus, 7.4 Reagents
oxidation of phosphorus compounds must occur under
acidic conditions. Methods for the determination of total 7.4.1 Deionised Water, high quality, free of phosphorus
nitrogen have used a persulphate-sodium hydroxide and nitrogen compounds.
system inorder to oxidize nitrogenous compounds to Prepare ammonia free water by ion-exchange or
nitrate. Accordingly, methods for the determination distillation methods.
of total phosphorus have used persulphate in an acidic
7.4.1.2 Ion exchange
medium
Ammonia-free water is prepared by passing distilled
During the initial digestion stage, pH of the sample is
water through an ion exchange column containing a
> 12, that is, alkaline. In the final digestion stage, the
strongly acidic cation exchange resin mixed with a
sodium hydroxide is consumed which causes the pH
strongly basic anion exchange resin. To remove organic
to be < 2, that is, acidic. By using this wide pH range,
compounds which interfere with the ammonium
the procedures allow the oxidation of both nitrogen and
determination resins are selected accordingly. Some
phosphorus compounds. The sample being digested is
anion-exchange resins tend to release ammonia. If
then analysed for nitrate and orthophosphate, yielding
ammonia is released, prepare ammonia-free water with
the amount of total nitrogen and total phosphorus.
a strongly acidic cation-exchange resin. Regenerate the
7.2 Selection of Nitrate/Orthophosphate column according to the manufacturer‘s instructions.
Measurement Methods Check ammonia-free water for the possibility of a high
blank value.
Using auto analyser (dual channel) which performs
nitrate-nitrite by the cadmium reduction method and 7.4.1.3 Distillation
orthophosphate by the ascorbic acid reduction method
The traces of ammonia in distilled water are eliminated
and orthophosphate by the ascorbic acid reduction
by adding 0.1 ml concentrated sulphuric acid to distilled
method, total nitrogen and total phosphorus can be
water and redistilling. Alternatively, treat distilled water
measured simultaneously. Alternatively, other methods
with sufficient bromine or chlorine water to produce a
for orthophosphate and nitrate can be used.
free halogen residual of 2 to 5 mg/l and redistill after
7.3 Apparatus standing at least 1 h. Discard the first 100 ml of the
distillate. Check redistilled water for the possibility of
7.3.1 Autoclave, should be capable of achieving a a high blank.
temperature of 120 °C for minimum of 120 min.

6
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

It is very difficult to store ammonia-free water in 7.4.4.6 Ammonium chloride-EDTA solution

the laboratory without contamination from gaseous Dissolve 13 gm ammonium chloride and 1.7 gm
ammonia. However, If storage is necessary, store in disodium ethylene di amine tetra-acetate to 900 ml
a tightly stoppered glass container to which is added reagent water. Adjust to pH 8.5 with concentrate
about 10 gm ion exchange resin (preferably strongly ammonium hydroxide and dilute to 1 l with reagent
acidic catio exchange resin)/l ammonia free water. If water. This solution is stable for 1 year.
a high blank value is produced , replace the resin or
prepare fresh ammonia free water. Use low heat EDTA solution to 500 ml with water. Use
this reagent as a cadmium-reduction-column storage
Use ammonia free distilled water for preparing all solution.
reagents, rinsing and sample dilution.
7.4.4.8 Hydrochloric acid, 6 M
7.4.2 Sodium Hydroxide, 3 N
Carefully add 500 ml concentrated hydrochloric acid to
Dissolve about 120 gm low nitrogen sodium hydroxide 400 ml, reagent water. Dilute to1 l with reagent water.
in about 800 ml deionized water taken in a 1 000 ml This solution is stable for 1 year.
volumetric flask. Cool and dilute to volume.
7.4.4.9 Stock nitrate solution A
7.4.3 Oxidizing Reagent
At 103 oC-105 oC dry potassium nitrate in an oven for
In 500 ml deionised water dissolve 64 gm low nitrogen 24 h. Dissolve 0.7218 gm (± 0.000 5 gm) in water and
(< 0.001 percent N ) potassium per sulphate. If necessary dilute to 100 ml (1ml = 100 µg of nitrate). Preserve
use low heat. Add 80 ml of 3 N sodium hydroxide with 2 ml chloroform/l. This solution is stable for at
prepared from low –nitrogen sodium hydroxide and least 6 months.
dilute to 1 000 ml. Store the reagent prepared in a
brown bottle at room temperature. 7.4.4.10 Stock nitrate solution B
7.4.4 Reagents for Determining Nitrate and Nitrite Purchase from a commercial source different from
stock nitrate solution A.
7.4.4.1 Reagent water
7.4.4.11 Intermediate nitrate solution A
Ammonia free distilled water is used for preparing all
reagents, rinsing and sample dilution. Dilute 100 ml stock nitrate solution to 1 000 ml
with water (1ml = 10 µg nitrate N). Preserve with
7.4.4.2 Copper sulphate solution 2 ml chloroform/l. This solution is stable for at least
In 500 ml reagent water, dissolve 20 g copper sulphate 6 months.
and dilute to 1 l with reagent water. This solution is 7.4.5 Reagents for Determining Phosphorus
stable for 1 year.
7.4.5.1 Antimony potassium tartrate solution
7.4.4.3 Wash solution, use reagent water.
Dissolve 0.3 g antimony potassium tartrate in
7.4.4.4 Copper-cadmium (Cd) granules approximately 50 ml distilled water and dilute to
Wash 25 gm new or used 20-100 mesh Cd granules 100 ml with reagent water. Store in 4 oC in a dark, glass
with 6 M hydrochloric acid and rinse with water. For stoppered bottle.
5 min swirl Cd with 100 ml 2 percent copper sulphate 7.4.5.2 Ammonium molybdate solution
solution until blue colour partially fades. Decant
and repeat with fresh copper sulphate until a brown Dissolve 4 g ammonium molybdate in 100 ml distilled
colloidal precipitate begins to develop. Gently flush water. Store in plastic bottle at 4 oC.
with ammonium chloride-ethylene diamine tetraacetic 7.4.5.3 Ascorbic acid
acid (EDTA) solution to remove all precipitated copper.
Store activated Cd covered with dilute ammonium Dissolve 1.76 gm ascorbic acid in 100 ml distilled
chloride-EDTA solution. water. This solution is stable for about 1 week at 4 °C.

7.4.4.5 Colour reagent 7.4.5.4 Combined reagent

To 800 ml reagent water, add 100 ml 85 per cent Mix the above reagents in the following proportions for
phosphoric acid and 10 gm sulphanilamide. After 100 ml of the combined reagent, 50 ml 5 N sulphuric
dissolving sulphanilamide completely, add 1gm NED. acid, 5 ml antimony potassium tartrate solution, 15 ml
Mix to dissolve, then dilute to 1 litre with water. ammonium molybdate solution and 30 ml ascorbic
Solution is stable for about a month when stored in an acid solution. Mix after addition of each reagent. Let
amber bottle in a refrigerator. Discard if the solution is all reagents reach room temperature before they are
highly coloured or dark or if a precipitate forms. mixed and mix in the order given, If turbidity forms
in the combined reagent, shake and let stand for a few

7
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

minutes until turbidity disappears before proceeding. 7.5.2 Sample Preparation

The reagent is stable for 4 h. If required, dilute the sample with de-ionized water
7.4.5.5 Dilute sulphuric acid solution so that the expected concentrations of nitrogen and
phosphorus fall within the limit of the calibration
Slowly add 140 ml concentrated sulphuric acid to standards. This digestion method cannot be used for
600 ml distilled water. When cool dilute to 1 l. samples that are preserved with acid for determination.
7.4.5.6 Ammonium persulphate, crystalline 7.5.3 Digestion Check Standards
7.4.5.7 Phenolphthalein indicator aqueous solution Analyse the quality control standards that contains
organic nitrogen and phosphorus on each analytical
7.4.5.8 Stock phosphate solution run by using the nicotinic acid p-toluenesulfonate
Dissolve 439.3 mg anhydrous potassium di hydrogen standard (for nitrogen) and adenosine triphosphate
phosphate dried for 1 h at 105 °C, in distilled water and standard (for phosphorus). These standards provide
dilute to 1 000 ml (1.00 ml =100.0 µg of phosphorus). the reference checks on the calibration and test the
ability of the digestion.
7.4.5.9 Intermediate phosphate solution
Dilute 100 ml stock phosphate solution to 1 000 ml 7.5.4 Digestion
with distilled water (1.0 ml = 10.0 µg of phosphorus). Pipet out 6 ml of sample or standard in to the culture
tubes. Further add 1.25 ml oxidizing reagent to each
7.4.5.10 Standard phosphate solution tube using a repeating pipet or and cover the tubes by
Prepare a suitable series of standards by diluting using loose fitting caps. Prepare an auto analyser wash
appropriate volumes of intermediate phosphate water in an Erlenmeyer flask by adding oxidation
solution. reagent to deionized water in the same ratio as it was
added to the samples. Cover the flask by using a foil.
7.4.6 Nicotinic Acid P-toluene Sulfonate Stock and Autoclave the samples and wash water for 55 min at
Working Standards 120 °C. Cool to room temperature. Add about 0.05
For 24 h dry nicotinic acid p-toluene sulfonate in an ml of 3 N NaOH to each tube before proceeding to
oven at a temperature of 150 oC. Dissolve about 2.108 nitrate, nitrite and phosphate analysis. Shake to mix.
4 gm of it in deionized water and dilute to 100 ml Add same proportion of 3 N NaOH to digested wash
(1 ml = 1 mg of nitrogen). Dilute 2 ml of stock solution water.
to 1 000 ml, to prepare a working standard (1ml = 2 µg
7.5.5 Final Nitrate and Nitrite Measurement
of nitrogen).
Use the automated cadmium reduction method to find
7.4.7 Adenosine Triphosphate Stock and Working out the concentration of nitrate-nitrite after digestion.
Standards Other nitrate analysis methods may be applicable for
In deionized water dissolve 0.651 4 gm adenosine this.
triphosphate and dilute it to 100 ml (1ml = 0.1 mg of
7.5.6 Final Phosphate Measurement
phosphorus). To prepare a working standard, dilute
20 ml of stock solution to 1 000 ml (1ml = 2 µg of Use the automated ascorbic acid reduction method
phosphorus). Dilute 1 ml stock solution to 1 000 ml, to to find out the concentration of phosphate after
prepare a low range working standard (1ml = 0.1 µg of digestion. Other phosphate analysis methods may be
phosphorus). applicable.

7.5 Procedure 7.6 Calculation

Prepare standard curves for nitrogen and phosphorus
7.5.1 Calibration Curve
by plotting the instrument response of standards
Prepare at least 5 standards over the desired calibration against the standard concentrations. Compute the
ranges such that a stock calibration standard containing concentrations of nitrogen and phosphorus by
both nitrate and orthophosphate. Treat the standards comparing the response of the sample with the standard
in the same manner just like samples. In calibration curve. Where required, multiply the concentration of
curves include blanks. the sample by the appropriate dilution factor to find
out the final concentration.

8
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

Fig. 1 Nitrate-Nitrite Manifold

Fig 2. Phosphate Manifold for Utomated Analytical System

9
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

ANNEX A
( Foreword )
COMMITTEE COMPOSITION
Water Quality Sectional Committee, CHD 36

Organization Representative(s)

Chief Scientist, EPTRI, Hyderabad Shri N. Raveendhar (Chairman)

Andhra Pradesh Pollution Control Board, Vijaywada Shrimati M. Sreeranjan
Shrimati A. Sri Samyuktha (Alternate)
Bhabha Atomic Research Centre, Mumbai Dr S. K. Sahu
Shri I. V. Saradhi (Alternate)
Central Institute of Mining and Fuel Research, Dr (Mrs) Bably Prasad
Dhanbad Dr Abhay Kumar Singh (Alternate)
Central Pollution Control Board, New Delhi Shri N. C. Durgapal
Dr J. C. Babu (Alternate)
Confederation of Indian Industry, New Delhi Shri Shikhar Jain
Ms Kamal Sharma (Alternate)
Delhi Jal Board Shri Ashutosh Kaushik
Shri Sanjeev Kumar (YP) (Alternate)
Department of Civil Engineering, IIT Madras Dr Ligy Philip
Dr S. Mathava Kumar (Alternate)
Envirocare Laboratories Pvt Ltd, Thane Dr Priti Amritkar
Shri Nilesh Amritkar (Alternate)
Environment Protection and Training Research Dr J. S. Srinivas
Institute, Hyderabad
FAD 14, Member Secretary
Gujarat Pollution Control Board, Gandhinagar Dr D. N. Vansadia
Shri K. B. Vaghela (Alternate)\
In personal capacity (1221, Mahatma Gandhi Road, Shri Sanjib Kumar Goswami
P. O., Haridevpur, Kolkata, 700 082)
In Personal Capacity Shri. Rakesh Malhotra
Indian Agricultural Research Institute – Water Dr Khajanchi Lal
Technology Centre, New Delhi Dr Ravinder Kaur (Alternate)
Indian Chemical Council, Mumbai Shri J. I. Sevak
Dr Mritunjay Chaubey (Alternate I)
Dr N. D. Gangal (Alternate II)
Indian Institute of Toxicology Research, Lucknow Dr S. C. Barman
Dr Satyakam Patnaik (Alternate)
Indian Institute Of Chemical Technology, Hyderabad Dr Sundergopal Sridhar
Dr Nivedita Sahu (Alternate)
Indian Water Works Association Shri Vijay Charhate
Karnataka State Pollution Control Board, Bengaluru Dr H. Rupadevi
Maharashtra State Pollution Control Board, Mumbai Dr V. R. Thakur
Shri S. C. Kollur (Alternate)
National Environmental Engineering Research Dr S. K. Goyal
Institute, Nagpur Dr P. K. Labhasetwar (Alternate I)
Dr Noor A. Khan (Alternate II)

10
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

IS 3025 (Part 31/Sec 1) : 2022

Organization Representative(s)

National Institute of Oceanography, Vishakhapatnam Dr V. V. S. S. Sarma

NTPC Ltd, New Delhi Shri V. Ravi Babu
Shri Sudhir Dahiya (Alternate)
Sriram Institute for Industrial Research, New Delhi Ms Shobha Kumar
Shri Sumant Shekhar (Alternate)
In Personal Capacity, Hyderabad Shri N. Murali Mohan
BIS Directorate General Shri Ajay Kumar Lal, Scientist ‘E’ and Head (CHD)
[ Representing Director General ( Ex-officio ) ]

Member Secretary
Ms Shubhanjali Umrao
Scientist ‘B’ (CHD), BIS

11
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].
Free Standard provided by BIS via BSB Edge Private Limited to Kailtech Test & Research Centre Pvt
Ltd - INDORE([email protected]) 122.168.190.124 [for non-commercial use only].

Bureau of Indian Standards

BIS is a statutory institution established under the Bureau of Indian Standards Act, 2016 to promote harmonious
development of the activities of standardization, marking and quality certification of goods and attending to
connected matters in the country.

Copyright

BIS has the copyright of all its publications. No part of these publications may be reproduced in any form without
the prior permission in writing of BIS. This does not preclude the free use, in the course of implementing the
standard, of necessary details, such as symbols and sizes, type or grade designations. Enquiries relating to
copyright be addressed to the Head (Publication & Sales), BIS.

Review of Indian Standards

Amendments are issued to standards as the need arises on the basis of comments. Standards are also reviewed
periodically; a standard along with amendments is reaffirmed when such review indicates that no changes are
needed; if the review indicates that changes are needed, it is taken up for revision. Users of Indian Standards
should ascertain that they are in possession of the latest amendments or edition by referring to the website-
www.bis.gov.in or www.standardsbis.in
This Indian Standard has been developed from Doc No.: CHD 36 (16994).

Amendments Issued Since Publication

Amend No. Date of Issue Text Affected

BUREAU OF INDIAN STANDARDS

Headquarters:
Manak Bhavan, 9 Bahadur Shah Zafar Marg, New Delhi 110002
Telephones: 2323 0131, 2323 3375, 2323 9402 Website: www.bis.gov.in
Regional Offices: Telephones
Central : 601/A, Konnectus Tower-1, 6th Floor, DMRC Building,
Bhavbhuti Marg, New Delhi 110002 { 2323 7617
Eastern : 8th Floor, Plot No 7/7 & 7/8, CP Block, Sector V,
Salt Lake, Kolkata, West Bengal 700091 { 2367 0012
2320 9474
Northern : Plot No. 4-A, Sector 27-B, Madhya Marg
Chandigarh 160019 { 265 9930
Southern : C.I.T. Campus, IV Cross Road, Taramani, Chennai 600113
{ 2254 1442
2254 1216
Western : Plot No. E-9, Road No.-8, MIDC, Andheri (East),
Mumbai 400093 { 2821 8093
Branches : AHMEDABAD. BENGALURU. BHOPAL. BHUBANESHWAR. CHANDIGARH. CHENNAI.
COIMBATORE. DEHRADUN. DELHI. FARIDABAD. GHAZIABAD. GUWAHATI.
HIMACHAL PRADESH. HUBLI. HYDERABAD. JAIPUR. JAMMU & KASHMIR.
JAMSHEDPUR. KOCHI. KOLKATA. LUCKNOW. MADURAI. MUMBAI. NAGPUR.
NOIDA. PANIPAT. PATNA. PUNE. RAIPUR. RAJKOT. SURAT. VISAKHAPATNAM.
Published by BIS, New Delhi