PAKISTAN STANDARD FOR BIO ORGANO

C-5 DETERMINATION OF BIOAVAILABLE P IN SOIL SPIKED WITH BOP FERTILIZER
C-5.1 PRINCIPLE: Bioavailability of P after soil spiked with BOP fertilizer over a period ranging from 2
to 13 weeks through sodium bicarbonate extractions and colorimetric analysis. C-5.2 APPARATUS
a. Analytical balance
b. UV visible Spectrophotometer
c. Orbital shaker
d. Centrifuge
e. Extraction bottle, 250 mL with stopper
f. Glassware of standardized laboratory Beakers, volumetric flasks, pipettes, funnels etc.
g. Incubator and plastic beakers for incubation

C-5.3 REAGENTS
C-5.3.1 Sodium Bicarbonate Solution (NaHCO3), 0.5 M. 42 grams of sodium bicarbonate is dissolved in
900 mL of distilled water. pH is adjusted to 8.5 by using 5 N NaOH solution and make the
volume 1L with distilled water. Store the solution in glass or plastic container.
C-5.3.2 Sulfuric Acid Solution (H2SO4), 5 N. 148 mL of concentrated sulfuric acid is diluted with distilled
water in the fume hood. Mix and cool it, then make the volume 1L by using distilled water.
C-5.3.3 Standard Stock Solution of Phosphorus. a. Dry potassium dihydrogen phosphate (KH2PO4) in oven at 105 0C for an hour and is cooled in
desiccator and store it in airtight bottles.
b. Weigh 2.197 g (KH2PO4) and dissolve in to a 1L volumetric flask by using distilled water and
the concentration of this stock solution is 500 ppm
c. Take 50 mL from stock solution into a 250 mL volumetric flask and make the volume by
using distilled water, the concentration of this sub stock is 100 ppm
d. Now prepare working standards of 2, 4, 6, 8, 10, 12, 14, 16 and 18 ppm by dissolving 2, 4, 6,
8, 10, 12, 14, 16 and 18 mL in 100 mL volumetric flask from sub stock solution (100 ppm).
C-5.3.4 Color Developing Reagent (CDR)

a. Reagent A
 Dissolve 12 g of ammonium heptamolybdate (NH4)6 Mo7O24.4H2O in 250 mL of distil water.
 Dissolve 0.2908 g of antimony potassium tartrate (KSbO.C4H4O6) in 100 mL of distil water.
 Add and dissolve both reagents in a 2 L volumetric flask. Also add 5 N Sulfuric acid solution, mix it well and make the volume with distilled water and keep away from light and
heat.

b. Reagent B
 Dissolve 1.056 g of L. ascorbic acid (C6H8O6) in 200 mL of reagent A and mix thoroughly. Reagent B is prepared according to the need as it become ineffective after 24 hours.

C-5.4 PROCEDURE
a. Take six samples of 500 g sieved soil in separate plastic beakers. Label three beakers as
“Fertilizer”, three beakers as “Soil” and make three replications. Spike the soil of beakers
labeled as “Fertilizer” with 100 g of BOP, any other P fertilizer or any other material that
needs to be checked. The beakers labeled as “Soil” should be maintained without addition or
spiking with any fertilizer material. Maintain soil water holding capacity at 50%. Incubation
should be carried out for 13 weeks at 28 ±1°C.
b. Take 5 g well mixed soil sample from each beaker periodically at 7 days interval.
c. Transfer air-dried soil samples in separate flasks of 250 mL each and add 100 mL of sodium bicarbonate (NaHCO3) solution (0.5 M) with pH 8.5 in each flask.
d. Shake each flask at 200 – 300 rpm for 30 minutes.
e. Filter each solution, separately, by using filter paper Whatman No. 42.
f. Transfer 10 mL of each filtrate in separate 100 mL volumetric flasks and add 10 mL of reagent B and make the volume 100 mL with distilled water in each flask.
g. Shake each flask well and take readings against absorbance by feeding; first blank, then
Standards, followed by 6 samples of “Fertilizer” and “Soil”.
h. Make calibration curve for Standards by plotting absorbance against P concentrations.
i. Measure P concentration of “Fertilizer” and “Soil” samples from the calibration curve.