ICAR-CSSRI/Bharuch/Technical Manual/2017/07

Protocols for Soil Sampling, Soil and Water Analysis

Shrvan Kumar, M.V.S. Rajeshwar Rao and Anil R. Chinchmalatpure
ICAR - Central Soil Salinity Research Institute, Regional Research Station, Bharuch, Gujarat

Soil samples collection

Soil sampling includes collection of samples from field and its preparation for the analytical
work. It has to be undertaken with utmost care as all the interpretation and recommendations are
based on the analysis of this representative sample.
Tools required for soil sampling
The tools used for soil sampling includes Auger, Spade, Khurpa, Measuring tape or Scale
Polythene paper Bags ,Cloth bag, marker pen, Information Sheet/Label.
Collection of soil sample
Prepare the farm map, showing the field number/ survey number, boundaries. Where the areas
within field differ distinctly in appearance they may be indicated in map. Depending on the farm size,
topography the sample is to be collected from different spots/field. The sample may be collected after
leaving some gap from the farm periphery/boundary. A composite sample from each field has to be
taken. Scrape the surface litter viz grass, pebbles, and stones preferably to a distance of 30 cm on all
the side. Take a uniform care of the soil from the surface plough (15 cm) with a soil auger or a v
shaped cut up to a depth of 15 cm with a spade or khurpa. Take about 4 to 6 cm thick uniform slice of
soil parallel to the V shaped cut. Depending on the field size about 6-10 samples are to be taken at
random, Collect all these samples in a tray, mix all the soil samples collected from different spots and
mix them well. Divide the mixed sample in four equal parts and make heap. Mix two diagonal heaps
and repeat the process to get a uniform representative sample about two Kilograms. Similarly take the
2nd sample from the depth of 15-30 cm. Now label the polythene bag and the cloth bag with marker
pen. Put the collected soil sample in the polythene bag and close the bag with rubber band or staple
with stapler pin. Now prepare two labels preferably of thin card board indicating the following
information
1. Name and the address of the farmer
2. Field Location and Survey number
3. Crop taken if any or crop to be taken.
4. Date of sampling.
Put one label inside the cloth bag and put the sample collected in polythene bag into the
cloth bag and tightly tie after putting the another label on the outside the mouth of the cloth bag.
The sample collected may be sent to nearest soil laboratory.
Precautions:
1. Sample may not be collected from unusual spots like farm fence, bunds, marshy/wet spots ,
fertilized soils,
2. Samples may not be collected from the place where compost pit or water storage tank or
irrigation source like tube well is located
3. The presence of artifacts (pieces of bricks, pottery etc. or other evidence of man’s activity
occurring below the plough layer should be recorded as evidence of disturbance of very long
period of cultivation
4. In no case the label should be put inside the polythene bag. In case of sampling from salt
affected soils, proper care has to be taken while sampling. Salt crust visible n the surface
should be sampled separately with recording of appropriate depth of sampling. Subsequently,
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the samples should be collected from the surface and the subsurface horizon up to a depth of
about a meter or so, preferably at interval 0-15 cm, 15-30 cm, 30-45 cm, 45-60 cm, 60-90 cm,
90-120 cm.
Preparation of soil samples in the laboratory
Once the soil sample is received in the laboratory, all the information furnished on the sample
bag are recorded in the register thereafter the bag is opened and all the information on inner label and
the polythene bag are tallied. If there is any discrepancy the same is brought to the notice of the
sample collector/ or the agency giving the sample for analysis. The sample is then air dried in shade to
prevent the loss of nutrients. The pebbles, small stones, gravel if any, leaves, grass and such particles
are removed and recorded separately. The weight of stones/ pebbles, gravel etc. may also be recorded
to determine their percentage on the basis of total soil weight. When the sample is completely dried, a
part of the sample (about 500 g) is retained for determination of macro morphological, micro
morphological, microbial and hydrological characteristics. The remaining soil is grinded in mortar
pestle or in the soil grinder and is passed through 2 mm sieve; the sample is mixed thoroughly and is
stored after proper labeling
Preparation of saturation extract
Soil salinity is conventionally defined and measured on the aqueous extract of the saturated
soil paste.
Material required: Plastic Beakers (500 ml capacity), Spatula, Glass Rod, Buchner Funnel, Filter
paper, Vacuum Pump etc
How to extract:
Take about 250 to 300 g of air dried soil in the beaker; add sufficient water to moisten the
soil, stir with glass rod and add water till the soil is saturated. Leave the sample overnight, to get the
soil fully saturated, Next day again stir the sample with glass rod, and add more water if required, at
the saturation the soil paste glistens, flows slightly when the container is tilted, the paste slides
smoothly of spatula/ glass rod and if a depression is made in the surface, no water collects in it and the
depression slowly fills up by the flow of the paste. Allow the sample for about half an hour, remove
the glass rod/ spatula and take the pH of the paste, with pH meter, it is indicated as pH of soil paste
with abbreviation pHs.
Transfer the contents of the beaker (saturated soil paste) to a Buckner funnel having the
moisten filter paper fitted on vacuum flask, apply the vacuum, collect the extract. Once the sufficient
quantity of extract is received the same is transferred to a plastic bottle which is labeled. Immediately
after transferring the extract, take the electrical conductivity of the extracted solution with
conductivity meter which is indicated as electrical conductivity of extract and depicted by the
abbreviation ECe having the unit dS/m. This extract is also used for determination of soluble cations
such as calcium (Ca2+), magnesium (Mg2+), sodium (Na+) and potassium (K+) and anions such as
carbonate (CO32-), bicarbonate (HCO3-), chloride (Cl-) and sulphate (SO42-).
Determination of soil pH and the electrical conductivity (EC) in 1: 2 ratio
Materials required: Glass beakers (100 ml capacity), Distilled water, Conductivity meter, and pH
meter, Standard stock solution of 0.1 N KCL, standard buffer solutions of 4.0, 7.0 and 9.2 pH
Method: Take 25 g of the air dried sample in 100 ml beaker and add 50 ml of distilled water keep for
few minutes and stir the contents with glass rod at an interval of 15-20 minutes after about three to
four stirrings, keep the samples undisturbed for 2-2.5 hours.

Determination of electrical conductivity:

Standardize the conductivity meter with 0.1 N KCl solution (7.456 g dry KCl in 1 liter of
distilled water). Once the instrument is standardized, dip the conductivity cell in the supernatant
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solution/layer without disturbing the soil and take the reading. The reading observed is represented
with abbreviation EC2 (dS/ m-1)
Determination of pH:
Once the electrical conductivity is taken the sample is thoroughly stirred for taking the pH.
The pH meter is standardized with the Buffers of 4.0 pH and 7.0 pH standards for two point
calibration units and with 4.0, 7.0 and 9.2 pH buffer standards for three point calibration units. Once
the pH meter is standardized, the electrode of the pH meter is dipped in the soil sample solution and
the reading is recorded. In determining EC and pH care has to be taken that the electrodes do not
touch the walls or the bottom of the beaker (Jakson, 1973). The pH reading observed is represented
with abbreviation pH2.
Analysis of soil saturation extract:
Sodium in the saturation extract is determined by flame photometer
The Standard stock of 1000 ppm sodium chloride is prepared (Dissolve 2.542 g of Sodium
chloride-AR in one liter of double distilled water) which is further diluted to 10 ppm, 20 ppm, 40
ppm, 60 ppm, 80 ppm and 100 ppm. The flame photometer is calibrated with 20 ppm, 40 ppm, 80
ppm and 100 ppm of the stock solution of sodium chloride. Feed the extract and take reading, if the
unit display “out of range” then pipette a known volume of the extract in 25 ml or 50 ml volumetric
flask and make up the volume with double distilled water. Feed this solution to the atomizer of the
flame photometer and take the readings.

Na+ (ppm) = Na+ concentration in ppm × dilution factor

Na+ (me/l) = Na+ concentration in ppm × dilution factor

23
Potassium in the saturation extract is determined by flame photometer
The Standard stock of 1000 ppm potassium chloride is prepared (Dissolve 1.909 g of
potassium chloride-AR in one liter of double distilled water) which is further diluted to 10 ppm, 20
ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm. The flame photometer is calibrated with 20 ppm, 40
ppm, 80 ppm and 100 ppm stock solution of potassium chloride. Feed the extract and take reading, if
the unit display “out of range” then pipette a known volume of the extract in 25 ml or 50 ml
volumetric flask and make up the volume with double distilled water. Feed this solution to the
atomizer of the flame photometer and take the readings.

K (ppm) = K concentration in ppm × dilution factor

K (me/l) = K concentration in ppm × dilution factor

39
Calcium and magnesium in saturation extract:
Reagents required: Standard solution of ethylene diamine tetra acetic acid (EDTA- versanate
solution) 0.01 N: Dissolve 2 g of EDTA disodium salt in about 800 ml of distilled water, transfer the
same to one liter volumetric flask and make up the volume to one liter with distil water, Murexide
indicator: Take 0.2 g ammonium perpurate and 40 g potassium sulphate in mortar and pestle and mix
them to get homogeneous powder, store in plastic bottle, Sodium hydroxide 4N (Dissolve 40 g of
NaOH in 250 ml distilled water), NH4Cl + NH4OH buffer: Dissolve 16.875 g of Ammonium chloride
in 142.5 ml of Ammonium hydroxide and make the volume to 250 ml in volumetric flask, Erichrome
black T indicator (EBT): Take 100 ml ethanol and dissolve 4.5 g of hydroxylamine hydrochloride and
add 0.5 g of EBT indicator powder.
Method
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Determination of calcium plus magnesium: Take 2 ml of the extract and add about 20 ml of
distilled water and add 10-12 drops of NH4Cl+ NH4OH buffer, titrate the same against 0.01 N EDTA
using EBT. The colour changes from pinkish red to Sky blue colour.

Ca2++ Mg2+ (me/l) = Volume of EDTA consumed × Normality of EDTA × 1000

Volume of aliquot taken (ml)
Determination of calcium: Take 2 ml of the extract and add about 20 ml of distilled water and add
0.1g of Muroxide indicator, and 10-12 drops of 4N Sodium Hydroxide buffer titrate the same against
0.01 N EDTA. The colour changes from light pink to violet colour.

Ca2+ (me/l) = Volume of EDTA consumed × Normality of EDTA × 1000

Volume of aliquot taken (ml)

Mg2+ (me/l) = (Ca+Mg) – Ca

Determination of carbonate, bicarbonate, chloride and sulphate in saturation extract

Carbonate (CO32-) and bicarbonate (HCO3-)
Reagents required: Micro-burette 10 ml capacity, Phenolphthalein & Methyl Orange indictor, 0.01N
or 0.1 N sulphuric acid (standardize), 50 ml conical flask or porcelain dish (25-50 ml capacity)
Method for carbonate: Take 2 ml of saturation extract in a 50 ml conical flask or porcelain dish, add
two drops of the phenolphthalein indictor, If the solution gives pink colour, it indicate the presence of
the carbonate, titrate this solution with 0.1 N or 0.01 sulphuric acid adding drop wise till the pink
colour disappear, note the volume of acid consumed (A).
Method for bicarbonate: To the solution in which carbonate has been determined as above, add two
drops of the methyl orange indictor, the solution will turn yellow, or reddish yellow colour titrate this
solution with 0.1 N or 0.01 sulphuric acid adding drop wise till the colour change to red /rose red,
note the volume of acid consumed (B).

CO32- (me/l) = 2 A × Normality of sulfuric acid × 1000

Volume of aliquot taken (ml)

HCO32- (me/l) = (B - 2A) × Normality of sulfuric acid × 1000

Volume of aliquot taken (ml)

Where, A= volume (ml) of the sulphuric acid consumed i.e. disappearance of pink colour
B = volume (ml) of the sulphuric acid consumed i.e. change of colour from yellow to rose red
Chloride (Cl-)
Reagents and material: Potassium Chromate indicator (Dissolve 5 g of Potassium Chromate in 100
ml distilled water), Standard 0.01 Silver Nitrate (Dissolve 1.791 g of AgNO3 in one liter of distilled
water), 50 ml conical flask or porcelain dish.
Method: Take 2 ml of fresh aliquot, add two drops of the potassium chromate indictor, the solution
will turn yellow, or reddish yellow colour, titrate this solution with 0.1N or 0.01N silver nitrate adding
drop wise till the red colored permanent precipitates appears.
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Cl- (me/l) = Volume of AgNO3 consumed × Normality of AgNO3 × 1000

Volume of aliquot taken (ml)

Sulphate (SO42-)
Determination of sulphate in soil extract by tubimetric method. The tubidimetric method of sulphur
determination was given by Chesnin and Yien (1951).
Reagents & material: Sodium acetate Acetic acid buffer (Morgan reagent): Dissolve 100 g Sodium
acetate trihydrate in 800 ml distilled water and add 30 ml of 99.5 % Acetic Acid and make volume to
1 liter, Gum acacia: Dissolve 0.25 g gum acacia in 100 ml distilled water and filter the solution to get
clear solution, Barium chloride: Grind the barium chloride crystal to make fine powder, Standard
sulphur solution 100 ppm S: Dissolve 0.5434 g of Potassium Sulphate in distilled water and dilute to 1
liter.
Standard curve: Pipette 0, 0.2, 0.4, 0.8, 1.0, 2.0, 3.0, 4.0 and 5.0 ml from 100 ppm standard S
solution to get 0, 0.8, 1.6, 3.2, 4.0, 8.0, 12.0, 16.0 and 20.0 ppm S concentration in 25 ml volumetric
flask add 5 ml sodium acetate-Acetic acid buffer, 1 ml gum acacia, add 0.5 g barium chloride and add
distilled water and make up the volume, shake for 1 minute, take turbidity readings at 420 nm within
5 to 20 minutes.
Method: Take 2 ml of saturation extract in 25 ml volumetric flask and add 5 ml sodium acetate-acetic
acid buffer, 1 ml gum acacia. Add 0.5 g barium chloride and add distilled water and make up the
volume, shake for 1 minute. Take turbidity readings at 420 nm within 5 to 20 minutes.
Calculations:
Volume of extract taken = 2 ml
Dilution (2 ml to 25 ml=12.5times) = 12.5 times
% Transmission (Reading of Spectrophotometer) = T
S as obtained from standard curve (ppm) = y
Sulphur in extract (ppm) = y × dilution= y × 12.5= Z
2–
SO4 in extract (ppm) =Z×3
2-
SO4 in extract ( me/l ) = (ppm of SO42-)/ mol. wt. of SO42-
Determination of exchangeable sodium, potassium, calcium and magnesium in neutral soils
Exchangeable sodium and potassium
Reagents: 1 N neutral ammonium acetate of 7.0 pH: Dissolve 77.08 g of ammonium acetate
dissolved in about 800 ml of distilled water and pH adjusted to 7.0 with ammonium hydroxide or
acetic acid, make the final volume to 1liter.
Method: Take 5 g of soil in 100 ml conical flask and add 25 ml of 1N ammonium acetate solution
shake the contents on the mechanical shaker for 20-30 minutes and filter and the make up the volume
to 25 ml.
Determine sodium, potassium on flame photometer as mentioned above and calcium & magnesium by
EDTA titration.

Exch. Na+ (ppm) = Na+ conc. from standard curve (ppm)× Volume of extractant (ml)
Wt. of soil (g)

Exch. Na+ = Exch. Na+ (ppm)

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(me/100g or cmol(p+)/kg (23 × 10)

soil)

Exch. K+ (ppm) = K+ conc. from standard curve (ppm)× Volume of extractant (ml)
Wt. of soil (g)

Exch. K+ = Exch. K+ (ppm)

(me/100g or cmol(p+)/kg (39 × 10)
soil)

Determination of exchangeable calcium and magnesium in calcareous soils

Reagents required: 1 N potassium chloride + tri-ethanol amine (TEA) solution (KCL-TEA solution):
Dissolve 74.6 g of KCl in about 500 ml distilled water and add 25 ml of TEA (Sp-gr.1.12) and stir
well dilute the solution to 850 ml with distilled water and adjust the pH to 8.5 with 1.0 N HCl,
Standard solution of ethylene diamine tetra acetic acid (EDTA- versanate solution) 0.01 N: Dissolve
2 g of EDTA disodium salt in about 800 ml of distilled water, transfer the same to one liter
volumetric flask and make up the volume to one liter with distilled water, Murexide indicator: (0.2 g
ammonium purpurate + 40 g of potassium sulphate, take both the reagents in mortar and pestle and
mix them to get homogeneous powder, store in plastic bottle, Sodium hydroxide 4N (Dissolve 40 g
NaOH in 250 ml distilled water), NH4Cl + NH4OH buffer: Dissolve 16.875 g of Ammonium chloride
in 142.5 ml of Ammonium hydroxide and make the volume to 250 ml in volumetric flask, Erichrome
black T indicator (EBT): Take 100 ml ethanol and dissolve 4.5 g of hydroxylamine hydrochloride and
add 0.5 g of EBT indicator powder.
Method: Weigh 10 g of air dry soil sample in 100 ml beaker and add 40 ml of KCL-TEA solution,
stir the contents thoroughly with glass rod for three to four times at an interval of 20 minutes. Filter
the content through Watman No. 40 filter paper and collect the leachate in 100 ml volumetric flask,
further leach the content with 20 ml KCL-TEA to bring the volume of aliquot to 100 ml.
Determination of calcium plus magnesium: Take 5 ml of the extract and add about 25 ml of
distilled water and add 10-12 drops of ammonium chloride–ammonium hydroxide buffer titrate the
same against 0.01 N EDTA using EBT. The colour changes from pinkish red to sky-blue colour.
Determination of calcium: Take 5 ml of the extract and add about 25 ml of distilled water and add
0.1 g of murexide indicator, and 10-12 drops of 4N sodium hydroxide buffer titrate the same against
0.01 N EDTA. The colour changes from light pink to violet colour.

Calculations

Ca2++Mg2+ = Vol. of EDTA used (ml) × Normality of EDTA × Volume of extract (ml) × 100
(me/100g) weight of soil sample taken (g) × aliquot taken (ml)

Ca2+ (me/100g) = Vol. of EDTA used (ml) × Normality of EDTA × Volume of extract (ml)× 100
weight of soil sample taken (g) × aliquot taken (ml)

Mg2+ (me/100g) = [Ca2++Mg2+ (me/100g)] - [Ca2+ (me/100g)]

Determination of cation exchange capacity (CEC)

Materials required: Funnel, beaker, filter paper, flame photometer, volumetric flask
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Reagents: Sodium acetate (1.0 N): Dissolve 136 g of sodium acetate tri hydrate in distilled water and
adjust the pH to 8.5 using sodium hydroxide or acetic acid, Ammonium acetate (1.0 N): Dissolve
77.08 g of Ammonium Acetate dissolved in about 800 ml of distilled water and adjust pH to 7.0 with
Ammonium hydroxide or Acetic acid, make the final volume to 1liter, Ethanol 60%, Standard stock
solution of sodium chloride: Dissolve 2.338 g of dry sodium chloride in 1 liter this will give 40 me/l
Na stock solution, 4 me/l Na stock: Take 25 ml from stock solution from 40 me/l Na solution and
make the volume to 250 ml in 250 ml volumetric flask.
Preparation of working standards and calibration of flame photometer
Pipette out 0, 5, 10, 15, 20, 25, 50 and 75 ml from 4 me/l Na stock in 100 ml volumetric flask and
make the volume to 100 ml by adding 1 N ammonium acetate. This will result 0.0, 0.2, 0.4, 0.6, 0.8,
1, 2, and 3 me/l sodium concentration. Calibrate the flame photometer using above standard stocks.
Method: Weigh 5 g of soil in a 150 ml beaker and add 33 ml of 1N sodium acetate and keep
overnight to saturate the soil with sodium, transfer the contents to funnel fitted with moist Whatman
No. 42 filter paper. Once the leaching of overnight solution is over, add 33 ml of sodium acetate.
Repeat the process for three to four times, discard the leachate. Once the leaching is completed, add
30 ml of 60% ethanol and leach to remove excess of sodium acetate, and check the electrical
conductivity (EC) of the leachate obtained, continue the leaching process with ethanol until the EC of
leachate is between 40-55 micro mhos per centimeter. Discard the leachate of ethanol. Once the
ethanol leaching is over, replace the beaker with 100 ml volumetric flask and add 33 ml of 1 N
ammonium acetate to replace the adsorbed sodium from sample. Continue the process by adding 33
ml of ammonium acetate to get 100 ml of the extract. Determine the sodium concentration by flame
photometer.

CEC{cmol(p+) kg-1} = Na (me/l) from the standard curve × 10

Weight of the soil sample (g)

Determination of exchangeable sodium percent (ESP)

Materials required: Funnel, beaker, filter paper, flame photometer, volumetric flask
Reagents required: Ammonium acetate (1.0 N): Dissolve 77.08 g of ammonium acetate dissolved in
about 800 ml of distilled water and adjust pH to 7.0 with ammonium hydroxide or acetic acid, make
the final volume to 1liter, Ethanol 60%, Standard stock solution of sodium chloride: Dissolve 2.338 g
of dry sodium chloride in 1 liter this will give 40 me/l Na stock solution, 4 me/lit Na stock: Take 25
ml from 40 me/l Na solution and make the volume to 250 ml in 250 ml volumetric flask.
Preparation of working standards and calibration of flame photometer
Pipette out 0, 5, 10, 15, 20, 25, 50 and 75 ml from 4 me/l sodium stock in 100 ml volumetric flask and
make the volume to 100 ml by adding 1 N ammonium acetate. This will result 0.0, 0.2, 0.4, 0.6, 0.8,
1, 2, and 3 me/l Na concentration. Calibrate the flame photometer using above standard stocks.
Method: Take 5 g of soil on 9.5 cm dia. or 11 cm dia. funnel fitted with Whatman filter paper No 42,
moisten the soil with distilled water to remove air bubbles. Add 33 ml of 60 % ethanol and allow to
leach, continue the leaching for another two to three times with 33 ml of 60 % ethanol, check the EC
of the leachate, and add another 33 ml of ethanol portions till the leachate conductivity is between 40-
55 micro mhos per cm. Once the leaching with ethanol is over, add 33 ml of 1N ammonium acetate
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and collect the leachate in 100 ml volumetric flask, continue the process by adding 33 ml of
Ammonium Acetate to get 100 ml of the extract.

Exchangeable Na (me/100 g soil) = Na concentration of the extract in me/l × 10

Weight of the soil sample (g)

ESP = Exchangeable Na me/100 soil

CEC
Determination of total nitrogen (Kjeldhal method)
Materials required: Nitrogen analyzer, consisting of Digestion unit attached with exhaust system and
distillation unit, conical flask 250 ml capacity. Micro burette of 10 ml
Chemicals required: Concentrated sulphuric acid, Catalyst/ Digestion mixture: 50 g of Sodium or
potassium sulphate and 5 g of copper sulphate mixed together, 4% Boric acid: 40 g of Boric acid –AR
dissolved in one liter of distilled water, Mixed indicator: 0.3 g of Bromocresol green (BCG) indicator
powder and 0.2 g of Methyl red mixed in 400 ml of 90% ethanol, 40 % Sodium Hydroxide: 400 g of
sodium hydroxide dissolved in 1 liter of distilled water, for titration: 0.1N hydrochloric acid or
sulphuric acid (standardized).
Method: Take 1 g of the soil sample into the digestion tube, add about 5-7 g of digestion mixture and
20 ml of concentrated sulphuric acid, load the insert rack in the digestion block (20 samples at a time)
place the exhaust hood on the tubes, and switch on the exhaust unit then heat the digestion block at a
temperature of 3750 C for about 2 to 3 hour till the digestion is completed, The sample turns colour
less or light green colour at the end of the digestion. Switch off the digester and remove the tubes with
exhaust led intact and place them on the tube support rack and allow them to cool to room
temperature. (Continue the exhaust system to run till tubes are cooled to room temperature and fumes
cease out /stops from the tubes).
Distillation process: Pipette 10 ml of the 4 % boric acid in 250 ml conical flask and add two drops of
mixed indicator place, the conical flask in the distillation chamber and dip the outlet tube/receiver
tube in boric acid. Add 25 ml of 40 % sodium hydroxide in digestion tube and start distillation, collect
distillate of about 150 ml.
Remove the conical flask from the distillation chamber, and allow to cool down to room temperature.
Titrate this distillate with 0.1 N hydrochloric acid or sulphuric acid till the colour changes from green
to red/pinkish red. Run a blank sample without soil sample.

% TN = Vol. of the acid used (sample titer value-blank titer value) × normality of acid × 14 × 100
sample weight (g) × 1000
Determination of available mineralizable nitrogen
Reagents: 0.32% Potassium Permanganate: 3.2 g of potassium permanganate in 1 liter of distilled
water, 2.5 % sodium hydroxide: 25 g of NaOH–AR in one liter distilled water, 4% Boric acid: 40 g of
boric acid –AR dissolved in one liter of distilled water, Mixed indicator: 0.3 g of bromo-cresol green
(BCG) indicator powder and 0.2 g of Methyl red mixed in 400 ml of 90% ethanol, 0.1 N H2SO4:
Standardize with 0.1N Na2CO3 using methyl orange indicator.
Method: Transfer 5 g of soil sample in distillation tube and moisten with distilled water (5 ml) and
add 25 ml of 0.32 % potassium permanganate and load the digestion tube in the distillation unit.
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Distillation process: Pipette 10 ml of the 4 % boric acid in 250 ml conical flask and add two drops of
mixed indicator, place the conical flask in the distillation chamber and dip the outlet tube/receiver
tube in boric acid. Add 25 ml of 2.5 % sodium hydroxide in digestion tube and start distillation,
collect distillate of about 150 ml, remove the conical flask from the distillation chamber, and allow it
to cool to room temperature. Titrate this distillate with 0.1 N hydrochloric acids or sulphuric acid till
the colour changes from green to red/pinkish red. Run a blank sample without soil sample.
1 ml 1N H2SO4 = 0.014 g N

Available = Vol. of acid (Sample titer value-Blank titer value) × N of Acid × 0.014 × 100× 10,000 × 2.24
N (kg/ha) Sample weight (g)

Determination of available phosphorus

Soil available phosphorus is determined by extracting soil with 0.5M NaHCO3 at pH 8.5 for neutral-
alkaline soils and determining P from the filtrate by spectrometric method (Olsen et al., 1954).
Olsen’s method using ascorbic Acid
Reagents: 0.5 N Sodium Bicarbonate: Dissolve 42 g of sodium bicarbonate (AR grade) in 800 ml
distilled water and adjust the pH to 8.5 with dilute sodium hydroxide or hydrochloric acid, Activated
phosphorus free charcoal or Darco G -60 AR (P free; to obtain P-free charcoal, 80 g charcoal material
as a slurry in distilled water leached overnight with 1 liter of 6M HCl in 60 mm diameter column (250
ml capacity) and then with deionized water till the leachate is free of chloride, then material is dried at
1100C) (Sarirullah et al., 1990), Ammonium molybdate P- reagent: Dissolve 20 g of AR ammonium
molybdate in 250 ml of distilled water. In another beaker dissolve 0.291 g antimony potassium
tartarate in about 100 ml distilled water. Both these reagents are added to one liter of 5N sulphuric
acid (140 ml sulphuric acid per liter) mix thoroughly and make the volume to 2 liters with distilled
water, Ascorbic acid: 1.056 g of ascorbic acid is dissolved in 200 ml of ammonium molydate P-
reagent (it has to be prepared a fresh as and when required), P-Nitrophenol: Dissolve 0.5 g of p-
nitrophenol in 100 ml of distilled water, 5N sulphuric Acid: Dilute 140 ml concentrated Sulphuric
acid to one liter, Standard Phosphorus solution: Dissolve 0.4393 g of AR grade potassium dihydrogen
orthophosphate (KH2PO4) in distilled water and make the volume to one liter. This will provide 100
ppm P solution. Dilute 50 ml of this solution to 1000 ml and this will be 5 ppm working standard
stock solution.
Method: Take 2.5 g of soil sample in 150 ml conical flask. Add about 1 g of phosphorus free
activated charcoal and 50 ml of 0.5 N sodium bicarbonate solutions. Shake the contents on
mechanical shaker for 30 minutes and filter them immediately through Whatman filter paper 40.
Pipette out 5 ml aliquot in 25 ml volumetric flask, add 2 drops of p-Nitrophenol indicators, it develops
yellow colour, add 5 N sulphuric acid drop wise till the colour disappear, shake the conical flask to
remove dissolve gases, and keep the contents for two hours to make it free from dissolve gases. Then
add 10 ml of distilled water and 5 ml of ascorbic acid solutions. Make the volume to 25 ml. Blue
colour so developed is measured at 882 nm after calibrating the spectrophotometer with known
standards. Prepare a blank sample without soil.
Standard curve: Take 0, 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of working standard stock solution5 ppm P
in 25 ml flask to prepare 0.0, 0.2, 0.4, 0.6, 0.8 and 1 ppm P solutions, to these add 5 ml of 0.5 N
sodium bicarbonate (8.5 pH), add 2 drops of p-Nitrophenol indicators, it develops yellow colour, add
5 N sulphuric acid drop wise till the colour disappear, shake the conical flask to remove dissolve
gases, and keep the contents for two hours to make it free from dissolve gases. Then add 10 ml of
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distilled water and 5 ml of ascorbic acid solutions. Make the volume to 25 ml, blue colour so
developed is measured at 882 nm.

P2O5 (kg/ha) = R (ppm) ×Vol. of extractant (ml) × volume make up (ml) ×2.24 × 2.29
Wt. of soil (g) × Aliquot taken (ml)
R= Conc. of P (ppm) from standard curve
Olsen’s method using stannous chloride
Reagents: 0.5 N Sodium Bicarbonate: Dissolve 42 g of sodium bicarbonate (AR grade) in 800 ml
distilled water and adjust the pH to 8.5 with dilute sodium hydroxide or hydrochloric acid, Activated
phosphorus free charcoal or Darco G -60 AR, 1.5 % ammonium molybedate: Dissolve 15 g
ammonium molybdate in 300 ml warm distilled water. Allow the solution to cool, then add 410 ml of
concentrated hydrochloric acid gradually with mixing and dilute the solution to one liter with distilled
water, Stannous chloride: Dissolve 10 g of stannous chloride in 25 ml concentrated hydrochloric acid
(the solution is highly unstable and has to be prepare a fresh and to be stored in amber bottle, Working
solution of stannous chloride: Take 0.5 ml of the above stock and dilute to 66 ml with distilled water,
Standard P solution (50 ppm): Dissolve 0.2195 g of AR grade potassium dihydrogen orthophosphate
(KH2PO4) in distilled water and make the volume to one liter this will provide 50 ppm P solution.
Dilute 40 ml of this solution to 1000 ml, this will be 2 ppm working standard stock solution,
Method: Take 2.5 g of soil sample in 150 ml conical flask and add about 1 g of phosphorus free
activated charcoal and 50 ml of 0.5N sodium bicarbonate solution. Shake the contents on mechanical
shaker for 30 minutes and filter them immediately through Whatman filter paper 40. Pipette out 10 ml
of aliquot in 50 ml volumetric flask, add 10 ml of 1.5% ammonium molybedate and about 2-3 ml of
distilled water, shake well, add 1 ml of working stannous chloride and make up the volume to 50 ml
with distilled water, the blue colour is developed. Read the OD at 660 nm wavelength on
spectrophotometer within 10-20 minutes.
Standard: Take 0, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml and 10 ml of working standard
stock solution 2 ppm P in 50 ml flask to prepare, 0.0, 0.04, 0.08, 0.12, 0.16, 0.20, 0.24, 0.28, 0.32,
0.36 and 0.40 ppm P. Add 10 ml of 0.5 N sodium bicarbonate (8.5 pH, add 10 ml of 1.5% ammonium
molybdate and about 2-3 ml of distilled water, shake well, add 1 ml of working stannous chloride and
make up the volume to 50 ml with distilled water, blue colour is developed, take the OD at 660 nm
wavelength on spectrophotometer within 15 minute.

P2O5 (kg/ha) = R (ppm) ×Vol. of extractant (ml) × Volume make up (ml) ×2.24 × 2.29
Wt. of soil (g) × Aliquot taken (ml)
R= Conc. of P (ppm) from standard curve

Determination of available Potassium

Soil available potassium is determined by normal neutral 1N ammonium acetate extractant, adjusting
pH 7.0 with using of flame photometer (Jackson, 1973).
Reagents: 1 N neutral ammonium acetate (7.0 pH): Dissolve 77.08 g of ammonium acetate
dissolved in about 800 ml of distilled water and pH is adjusted to 7.0 with ammonium hydroxide or
acetic acid and make the final volume to 1liter.
 ICAR-CSSRI/Bharuch/Technical Manual/2017/07

Method: Take 5 g of soil in 100 ml conical flask and add 25 ml of Ammonium acetate solution.
Shake the contents on the mechanical shaker for 20-30 minutes, filter it and the make up the volume
to 25 ml and determine potassium on flame photometer.

K2O (kg/ha) = Conc. of K (ppm) ×vol. of extractant (ml) × 2.24 × 1.21

Wt. of soil (g)
Soil organic carbon (SOC)
Material required
Conical Flask 500 ml capacity, 10 ml transfer pipette, measuring cylinder or volumetric pipette of 20
ml capacity Burette 25ml capacity
Reagents: Standard 1N potassium dichromate: 49.04 g of potassium dichromate in 1 liter distilled
water, 0.5 N ferrous ammonium sulphate: Dissolve 196.1 g ammonium ferrous sulphate in 800 ml of
double distilled water and add 20 ml of Concentrated Sulphuric acid slowly and cool the contents and
make up the final volume to 1 liter, Concentrated sulphuric acid, Diphenylamine indicator: Dissolve
0.5 g Diphenylamine indicator in 20 ml distilled water and add 100 ml concentrated Sulphuric acid
slowly, cool the reagent and store in amber bottle (To be prepared fresh), 85% ortho-phosphoric acid.
Method: Weigh 1 g or 2 g of soil passed through 0.2 mm sieve in 500 ml conical flask, add 10 ml of
potassium dichromate with pipette, and shake well. Add slowly 20 ml of concentrated sulphuric acid
by swirling the flask during addition. Keep the flask at room temperature for 30 minutes. Similarly
prepare a blank without soil sample. When the content is cooled at room temperature, add 200 ml of
distilled water and 10 ml of 85% orthophosphoric acid and shake the contents, add 10 drops of
diphenylamine indicator which imparts violet colour to the contents. Titrate with 0.5 N ferrous
ammonium sulphate till the colour changes from violet to bright green. At the end note the volume of
the ferrous ammonium sulphate.

Volume of ferrous ammonium sulphate (FAS) used for titration of blank = A ml

Volume of ferrous ammonium sulphate (FAS) used for titration of sample = B ml
1 ml of potassium dichromate = 0.003 g of organic carbon

Walkey- Black SOC (%) = (A-B) ×0.03 × Normality of FAS × 100

Wt. of soil (g)
By the Walkley-black titration method having 76% recovery of soil organic carbon

Actual SOC (%) = {Walkley Black SOC}× (100/76) = (Walkley-Black SOC × 1.31)

SOC (g/kg) = SOC (%) × 10

Available sulphur
Available sulphur is determined by using 0.15 per cent CaCl2 solution (Williams and Steinbergs,
1959).
Reagents: 0.15% CaCl2.2H2O: 1.5 g of CaCl2.2H2O dissolve in distilled water and make 1 liter
volume, Morgan reagent: Dissolve 100 g sodium acetate in 800 ml distilled water and adjust pH 4.8
by adding 30 ml glacial acetic acid and make volume to 1 liter, Gum acacia: Dissolve 0.5 g gum
acacia in 200 ml distilled water and filter the solution to get clear solution, Barium chloride: grind the
barium chloride crystal to make fine powder; Volumetric flask (50 ml), pipette, burettes.
 ICAR-CSSRI/Bharuch/Technical Manual/2017/07

Method: Transfer 10 g soil into a 100 ml capacity plastic bottle, add 50 ml 0.15% CaCl2 solution,
shake it for 30 minutes on shaker, filter it through Whatman filter No.1. Transfer 20 ml aliquot in 50
ml volumetric flask, add 20 ml Morgan’s reagent, 2 ml Gum acacia and 1 spoon (1.0 g) barium
chloride and make up the volume. After 3-5 minutes, measure the turbidity at 410 nm.
Preparation of standards: Prepare 50 mg/l sulphur by dissolving 0.2717 g potassium sulphate (AR)
in distilled water and make the volume to 1 liter
Take 0ml, 1ml, 2ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml and 10 ml of 50 ppm sulphur in 50 ml
volumetric flask, to get 0 ppm, 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm 6 ppm 7 ppm, 8 ppm 9 ppm and
10 ppm sulphur standards, add 20 ml Morgan’s reagent, 2 ml Gum acacia and 1 spoon (1.0 g) barium
chloride and make up the volume. After 3-5 minutes measure the turbidity at 410 nm.
Calculations

Sulphur in soil = Conc. of S (ppm) from stand. Curve × Volume of extactant (ml)×Volume make up (ml)
(ppm) Wt. of soil taken (g) × volume of aliquot taken (ml)

Determination of DTPA-extractable micronutrients

Reagents: DTPA solution: Take 13.3 ml triethanolamine, 1.967 g DTPA and 1.47 g CaCl2.2H2O,
dissolved in about 500 ml of deionized water and make volume by stirring up to 900 ml. The pH of
the solution is then adjusted to 7.30±0.05 by adding 4N HCl while stirring and finally the contents are
diluted to one liter.
Apparatus and equipment: 1-litre volumetric flasks, 100-ml narrow mouth polyethylene bottles,
pipettes, electric shaker, Whatman No.1 filter paper and atomic absorption spectrophotometer (AAS)
Method: Weigh 10 g of air-dried soil sample and transfer it to a 100-ml narrow mouth polyethylene
bottle. Add 20 ml DTPA solution and put stopper. Shake on an electric shaker for 2 hours at 25 oC.
Filter the contents through Whatman No.1 or Whatman No 42 filter paper. Also, run a blank with only
DTPA solution and no soil.
Standardization: Make 1000 ppm standard stock solution for Zn, Cu, Fe, Mn from their respective
salt i.e. ZnSO4.7H2O, CuSO4.5H2O, FeSO4.7H2O and MnSO4.H2O. From 1000 ppm stock solution of
Zn, Cu, Fe and Mn make 50 ppm working solution (Take 5 ml stock solution in 100-ml volumetric
flask and make up volume with deionized water). From this 50 ppm working solution, prepare
required standards solutions (below mentioned concentration in ppm) of each element in 100-ml
volumetric flask by pipetting out required volume (0.0, 2.0, 4.0, 6.0, 8.0 ml for Fe; 0.0, 1.0, 2.0, 4.0,
6.0 ml for Mn; 0.0, 0.6, 1.2, 1.8, 2.4 ml for Zn; 0.0, 0.8, 1.6, 2.4, 3.2 ml for Cu) of the working
standard and making the final volume with 0.005 M DTPA.
Calibrate the AAS using four standards of each element (Micro or heavy metals).
Fe: 0.0, 1.0, 2.0, 3.0 and 4.0 ppm
Mn: 0.0, 0.5, 1.0, 2.0 and 3.0 ppm
Zn: 0.0, 0.3, 0.6, 0.9 and 1.2 ppm
Cu: 0.0, 0.4, 0.8, 1.2 and 1.6 ppm

Calculation

DTPA extractable micro or heavy = Reading (ppm) of AAS × vol. of DTPA solution taken
 ICAR-CSSRI/Bharuch/Technical Manual/2017/07

metals (mg/kg) Weight of soil (g)

DTPA extractable micro or heavy = DTPA extractable micro or heavy metals (mg/kg) ×
metals (kg/ha) 2.24

Determination of Calcium Carbonate from soils

Material required: 25 ml burette and conical flask of 100 ml and 250 ml
Reagents: Hydrochloric acid standard (1 M): Add 81 ml of hydrochloric acid in about 500 ml
distilled water, cool and make the volume to 1 liter, Sodium hydroxide (0.5M): Dissolve 20g of
sodium hydroxide in 800 ml distilled water, cool and make the volume to 1 liter, Phenolphthalein
indicator solution, Method: Weigh 5 g of soil sample in 250 ml conical flask and add100 ml 1 M
hydrochloric acid, boil the flask for 5 minutes and cool down to room temperature or cover the flask
with aluminum foil and keep overnight. Filter the contents, take 10 ml of the filtrate in 100 ml conical
flask and add 2 to 3 drops of phenolphthalein indicator and titrate with 0.5 M sodium hydroxide.

% Calcium carbonate =

[(ml HCL × 1M)-(ml of NaOH ×0.5 M)]× [vol. of HCL/vol. of filtrate aliquot] × [100/(1000×2)]× [100g/wt. of
soil]

Analysis of irrigation water:

Irrigation water should be collected with utmost care. The sample should not be collected
from standing water, field irrigation channel, pond with stagnated water, or any contaminations like
growth of algae, water used for bathing of animals etc. In order to get reliable results the time gap
between collection of sample and its deposition in laboratory should be as short as possible, as the
delay in giving to laboratory may result in some changes in biochemical, and physico-chemical
properties.
Sample should be collected in a clean glass bottle or plastic bottle which should be rinsed
atleast two to three times with sample water. In case of collection of tube well water, the tube well
should run for at least 30-45 minutes before collecting the water. The sample is to be tightly corked
and label giving the following information should be tied securely on the bottle.
Tag for water sampling
Collector/ farmers name, date of collection, field from where it is collected along with survey
number, name of the village along with the address of the farmer. Any other water resources like
canal, pond etc. adjacent to the source from where the water is collected. If the sample is collected
from tube well, then depth of well, installation year, approximate area under irrigation may also be
recorded. The collectors may record general problems such as ingress of water from nearby source of
irrigation, or any effluent etc., failure of crop after irrigation, water stagnation etc.
Analysis of water
Determination of pH and the electrical conductivity
Materials Required: Glass beakers of 100 ml capacity, Distilled water, Conductivity meter, and pH
meter
 ICAR-CSSRI/Bharuch/Technical Manual/2017/07

Standard stock solution of 0.1N KCL,

Standard buffer solutions of 4.0, 7.0 and 9.2 pH
Method: Take about 100 ml water in a glass beaker to determine EC and pH
Determination of electrical conductivity:
Standardize the conductivity meter with 0.1 N KCl solution (7.456g dry KCl in 1 liter of distilled
water). Once the instrument is standardized, dip the conductivity cell in the glass beaker and take the
reading. The reading observed is represented with abbreviation ECw/dSm
Determination of pH
The pH meter is standardized with the buffers of 4.0 pH and 7.0 pH standards for two point
calibration units and with 4.0, 7.0 and 9.2 pH buffer standards for three point calibration units. Once
the pH meter is standardized the electrode of the pH meter is dipped in the water sample solution in
the glass beaker and the reading is recorded which is represented as pHw. In determining EC and pH
care has to be taken that the electrodes do not touch the walls or the bottom of the beaker
Determination of sodium and potassium: Procedure for estimation of Sodium and Potassium in
water is same as followed in estimation of Sodium and Potassium in saturation extract of soils.
Estimation of Calcium and Magnesium in water samples: Procedure for estimation of Calcium
and Magnesium in water is same as followed in estimation of Calcium and Magnesium in saturation
extract of soils.
Estimation of carbonate, bicarbonate, chloride and sulphate in water samples: The same
procedure will be followed as used in determination of carbonate, bicarbonate, chloride and sulphate
in saturation extract of soils.
References
Chesnin, L. and Yien, C.H. (1951). Turbidimetric determination of available sulphates. Soil Sci. Soc.
Amer. Proc., 15: 149-151.
Jackson, M.L. (1973). Soil Chemical Analysis. Prentice Hall of India Private Limited, New Delhi.
Lindsay, W.L. and Norvell, W.A. (1978). Development of DTPA. Soil test for zinc, iron, manganese
and copper. Soil Sci. Soc. Am. J., 42: 421-428.
Olsen, S.R., Cole, C.V., Watanabe, F.S. and Dean, L.A. (1954). Estimation of available phosphorus in
soils by extraction with sodium bicarbonate. U.S. Dep. Agric.., Washington, D.C. Circ. 939.
Richards, L.A. (1954). Diagnosis and Improvement of Saline and Alkali Soils, USDA Agriculture
Handbook No. 66, US Government Printing Office, Washington, DC.
Sarirullah, M., T.H. Flowers, and I.D. Pulford. (1990). Removal of phosphorus contamination from
charcoal prior to decolorisation of soil extracts. Commun. Soil Sci. Plant Anal., 21: 2227-2231.
Subbaih, B.V. and Asija, G.L. (1956). Rapid procedure for the estimation of available nitrogen in soil.
Current Sci., 125: 259-260.
Walkley, A. and Black, I.A. (1934). An examination of the Kjeldahl method for determining soil
organic matter. Soil Sci., 37: 29-38.
Williams, C.H. and Steinbergs, A. (1959). Soil sulphur fractions as chemical indices of available
sulphur in some Australian soils. Australian J. Agric. Res., 10: 340-352.
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