Method 111 Determination of extractable organic and inorganic soil P

Reagents.

• Acetic acid - 8-hydroxyquinoline reagent - dissolve 10 g 8-hydroxy-quinoline in a solution of 2.5 % (v/v) acetic acid and make up to 1 l. (The 8-hydroxyquinoline blocks the readsorption or precipitation of phosphate by active iron and aluminium during acetic acid extraction. Synonyms: hydroxybenzopyridine; oxine; phenopyridine; 8-quinolinol. Not carcinogenic, but may be harmful if swallowed, and causes irritation to eyes, respiratory tract and skin; safety data sheet at http://www. jtbaker.com/msds/q7250.htm.)

• Hydrochloric acid solution, 1.0 M

• Magnesium nitrate, 0.5 M - dissolve 12.82 g Mg(NO3)2.6H2O in water and make up to 100 ml.

• Sodium hydroxide solution, 0.1 M

Procedure (extraction). Transfer 2.5 g air-dry soil, 2 mm mesh size, into a 250 ml polypropylene screw-cap centrifuge bottle/tube and add 100 ml acetic acid - 8-hydroxyquinoline reagent. Cap the tube and shake overnight (17 h) on a reciprocating shaker, at approximately 275 strokes of 25 mm length per minute at a constant temperature (20°C). Centrifuge for 15 min at 2800 rpm and remove an aliquot for the determination of acid extractable inorganic phosphorus (a).

Completely remove the remaining supernatant, dispense 100 ml 0.1 M sodium hydroxide into the tube and shake overnight as previously, being careful to maintain a constant temperature of 20°C, otherwise serious variations in the P extractability will occur. Centrifuge as above, and remove aliquots for determination of both the inorganic (b) and combined inorganic and organic fractions (b + c). If the presence of finely dispersed clay leaves a cloudy extract after centrifugation, dissolution of 0.5 g sodium chloride in the extract before centrifuging should result in a clear supernatant.

Procedure (determination of inorganic phosphate (a) in the acetic acid extract). The 8-hydroxyquinoline forms a precipitate in acidic ammonium molybdate solution, which will interfere unless the aliquot is <5 ml. It should therefore be removed by ignition as follows. Transfer 10 ml acetic acid extract to a 45-ml silica basin, add 0.5 ml 1 M magnesium acetate and evaporate to dryness on a water-bath. (Note: do not use magnesium nitrate, which reacts adversely on heating with 8-hydroxyquinoline.)

Transfer the basin to a cold muffle furnace and raise the temperature to a very dull red heat (545-555°C) and maintain for 20 min to oxidize all organic matter. Dissolve the residue in 15 ml M HCl and evaporate to dryness on a water-bath in a fume cupboard. Add 10 ml hot water and 1 ml 1 M HCl to dissolve the residue, then transfer with washings to a 100-ml beaker until about 40 ml. After cooling, adjust the pH to about 5.0 by dropwise addition of 1 M ammonium hydroxide, then transfer with rinsing to a 50-ml volumetric flask, make up to the mark and mix. Determine acetic acid extractable inorganic phosphate by a suitable procedure (see Methods 5.9a and 5.9b), noting that the results will be in mg P kg-1 soil. Note also that the solution obtained by the Williams procedure (2.5 g soil in 100 ml acetic acid solution, of which 10 ml is made up to 50 ml, i.e. a dilution of x 200 w/v) will be >10 x more dilute than using Olsen's bicarbonate extraction procedure (5 ml soil in 100 ml). Unless sufficient extra P is extracted by the acid compared with bicarbonate, a larger aliquot of the acetic acid extract may be necessary to provide adequate sensitivity for the determination unless the original (Truog and Meyer (1929); Truog (1930)) method is used. The calculation should take these dilutions into account.

Procedure (determination of inorganic phosphate (b) in the alkaline extract).

Pipette 25 ml of the alkaline extract into a 50-ml volumetric flask followed by 3.5 ml 1 M HCl from a dispenser. This will neutralize the sodium hydroxide and leave 1 ml excess of acid, causing precipitation of organic matter. Make up to 50 ml and mix. Filter through a dry Whatman No. 44, 9 cm, filter paper into a dry 100-ml beaker, discarding the first few millilitres. Pipette a suitable aliquot (25-35 ml) into a 50-ml beaker, adjust the pH to approximately 5.0 as previously, make up to 100 ml and mix. Determine the inorganic phosphate (b) in the alkaline extract by a suitable procedure. Note that 2.5 g soil was extracted into 100 ml 0.1 M sodium hydroxide, of which a 25 ml aliquot was made up to 50 ml, of which 25 ml was made up to 100 ml. Thus 2.5 g soil has been extracted into the equivalent of 800 ml solution (x 320 w/v). This should be taken into account in the calculation.

Procedure (determination of inorganic plus organic phosphate (c) in the alkaline extract). Pipette a suitable aliquot (5 or 10 ml) of alkaline extract into a 45-ml silica basin, acidify with a few drops of concentrated nitric acid, add 2 ml 0.5 M magnesium nitrate and evaporate to apparent dryness on a water bath. Complete the evaporation gently on a hotplate in a fume cupboard, then transfer to a cold muffle furnace. Raise the temperature to a very dull red heat (545-555°C) and ignite for 20 min to oxidize all the organic matter and mineralize the organic P. Allow to cool, dissolve the residue in 15 ml of 1 M HCl, cover with a watch glass and heat for 30 min on a boiling water-bath. Remove the watch glass and rinse into the basin, then evaporate to dryness and redissolve the residue in 10 ml hot water plus 1 ml 1 M HCl. Filter through a 7-cm Whatman No. 41 filter paper into a 100-ml beaker. Wash the filter funnel and paper with hot water to bring the contents up to approximately 75 ml. Cool, adjust the pH to approximately 5.0, and transfer to a 100-ml volumetric flask with rinsing; make up to the mark and mix. Determine the inorganic plus organic phosphate (c) in the alkaline extract by a suitable procedure. Note that 2.5 g soil was extracted into 100 ml 0.1 M sodium hydroxide, of which a 5 or 10 ml aliquot was made up to 100 ml. There is therefore a x 400 or 800 w/v dilution factor to be incorporated in the calculation.

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