Exercise 13: Determination of Available Phosphorus in Soils

Phosphorus availability in soils is normally limited by the low solubility of many phosphorus compounds and by the tendency of several soil minerals to adsorb phosphate ions. Estimating the amount of plant-available phosphorus in a soil requires a method that relates to the ability of plant roots to extract phosphorus from the soil system. This ability of chemicals to extract nutrients serves as an index of plant-available phosphorus. Several different extracting solutions have been used for this purpose.

Deionized water, with or without carbon dioxide dissolved in it, is sometimes used as an extractant, but its extracting power is generally considered to be too low for soil testing purposes. Dilute acidic or basic solutions are usually preferred. Sodium bicarbonate solution is used in some of the western states where alkaline soils occur. Plant roots grow in an alkaline environment there, so it seems reasonable that an alkaline extractant would work well on those soils.

Acidic extractants are used more widely than other types for determining available phosphorus. The best known of phosphorus extracting solutions are the Bray-1, Bray-2, and Mehlich-3. The first two were developed by Dr. Roger Bray of Illinois. The Bray-1 extractant has been widely used in the Midwest because it gives values well related to the phosphorus uptake by crops in acid and slightly acid soils. Mehlich-3 is a more recent addition (used at ISU since 1996). Mehlich-3 has the advantage of being a universal extractant that can be used for cations (such as Ca, Mg, K, and Na) and some micronutrients in addition to P. The Mehlich-3 extraction solution was developed by Adolph Mehlich of North Carolina State University and is applicable for soils with a wide range of properties.

Bray-1 contains 0.025 N HCl and 0.03 N NH₄F with pH adjusted to 2.6. Bray-2 is a more acid extractant that contains 0.1 N HCl and 0.03 N NH₄F with pH adjusted to 1.6. Bray-2 is the extractant used in some commercial soil test kits. It is a good extractant for acidic soils but gives poor results on alkaline soils and on soils where rock phosphate has been applied as a fertilizer. Mehlich-3 contains 0.2 N CH₃COOH (acetic acid), 0.25 N NH₄NO₃, 0.015 N NH₄F, 0.013 N HNO₃, and 0.001 M EDTA (ethylenediaminetetraacetic acid). The ammonium and EDTA constituents allow this solution to displace cations and chelate metallic micronutrients, respectively. This solution is acidic (pH 2.5) and contains ammonium fluoride just like Bray-1 and Bray-2. The fluoride component combines with calcium to prevent precipitation of phosphorus as insoluble calcium phosphates, thus maintaining soluble phosphorus in the extracting solution where it can be measured.

The Bray-1 and Mehlich-3 solutions extract part but not all of the plant-available phosphorus from the soil sample. Partial extraction results because H₂PO₄^– ions are adsorbed more strongly to some sites than to others. Most of the extracted ions are thought to have been adsorbed at the edges of silicate clay minerals or on the surfaces of iron and manganese oxides. Stronger extractants like Bray-2 are likely to dissolve too much calcium phosphate. Partial extraction is adequate as long as the amount extracted is a good indicator of how much is available to plants (proportional or otherwise consistently related).

In alkaline soils, a sodium bicarbonate solution adjusted to pH 8.5 (otherwise known as Olsen extraction solution) is often used. Where plant roots grow in an alkaline environment, it seems reasonable that an alkaline extracting solution would work well, just as an acidic extracting solutions works best with acidic soils. Because pH affects the forms of P present in soils, both extracting solutions are attempting to mimic the pH of the rooting environment to estimate available P.

Once extracted from soil, the amount of P in solution is often determined by a colorimetric procedure. Phosphorus as phosphate present in solution will react with ammonium molybdate in acidic solution to form the yellow 12-molybdophosphoric acid ([H₂PMo₁₂O₄₀]^–), which can then later be reduced by ascorbic acid or stannous chloride forming the molybdenum blue complex. The intensity of the blue color of the complex is a measure of the amount of phosphorus in solution.

Any test used for available phosphorus must be calibrated to the results of field trials. The table below is from ISU’s PM 1688 and gives interpretations of soil test P and K values. Note that interpretation of Bray-1 and Mehlich-3 use the same values (Mehlich-3 ICP is determined by inductively coupled plasma (ICP) and results in somewhat higher P test values because it measures some forms of P other than orthophosphate).

 

 

* The recommended amounts of P₂O₅ and K₂O for the optimum soil test category are based on approximate nutrient removal for the harvested yield. The amounts shown in the table for the optimum soil test category are based on 180 bu corn grain per acre. Nutrient removal amounts can be adjusted higher or lower for other yield levels. In the high soil test category, banded NP or NPK starter fertilizer may be advantageous under conditions of limited soil drainage, cool soil, crop residues on the soil surface, or late planting dates with full-season hybrids.

Note: In the table above, remember to convert values obtained in solution to ppm in soil before using the table.

 

Procedure

1. Weigh 1.00 ± 0.05 g (record exact weight) of air-dried soil (< 2.0 mm) into a 50-mL centrifuge tube in duplicate. Add 10.0 ± 0.1 mL of Bray-1 extracting solution to each tube and then cap tightly.

The extracting solutions are designed to remove acid-soluble forms of P, largely calcium phosphates, and a portion of the aluminum and iron phosphates. The acid provides sufficient H⁺ activity to dissolve calcium phosphates. Acid solutions also will solubilize some of the aluminum phosphates and iron phosphates. The fluoride ions are very effective in complexing Al ions and, in this manner, releasing P from aluminum phosphates. Calcium is precipitated by F ions and, therefore, the P present in the soil as dicalcium phosphate will be extracted by solutions containing F ions.

2. Set the tubes in a horizontal position on the platform shaker and then shake at high speed for 5 min.
3. Centrifuge for 10 min @ 4000 rpm. Then, pass the supernatant through a 12.5-cm diameter Whatman No. 42 filter paper.

Use dry filter paper (Whatman No. 42 or equivalent) to avoid any change in the phosphorus concentration of the extract. Collect at least 5 mL of clear filtrate. Refilter if the filtrate is turbid; turbidity would absorb light when the reading is taken in step 7. Light absorption by turbidity cannot be distinguished from light absorption indicating the presence of phosphorus.

The extracting solutions are designed to remove acid-soluble forms of P, largely calcium phosphates, and a portion of the aluminum and iron phosphates. The acid provides sufficient H⁺ activity to dissolve calcium phosphates. Acid solutions also will solubilize some of the aluminum phosphates and iron phosphates. The fluoride ions are very effective in complexing Al ions and, in this manner, releasing P from aluminum phosphates. Calcium is precipitated by F ions and, therefore, the P present in the soil as dicalcium phosphate will be extracted by solutions containing F ions.

4. Pipet 1.0 ± 0.1 mL of the sample extracts into separate 20-mL test tubes. To each, add 9.0 ± 0.1 mL of color-developing reagent. Thoroughly Mix.

The color-developing reagent (working solution) is prepared fresh and contains the following added in this order:

1. 1. Acid molybdate stock solution. Dissolve 60 g of ammonium molybdate ((NH₄)6Mo₇O₂₄:4H₂O) in 200 mL of distilled water heated to 60^oC. Cool. Dissolve 1.455 g of antimony potassium tartrate in the molybdate solution. Add 700 mL of concentrated sulfuric acid to the molybdate solution. Dilute to a final volume of 1000 ml. Store in a dark, cool location.
   2. Ascorbic acid stock solution. Dissolve 33 g of ascorbic acid in distilled water and dilute to a final volume of 250 mL. Store in the dark under refrigeration.
   3. Working solution. Prepare fresh each day by adding 25 mL of acid molybdate stock solution to about 800 mL of distilled water, mixing, adding 10 mL of ascorbic acid stock solution and making to a final volume of 1000 mL. Mix well. This solution has a shelf life of about 24 hours.

The reducing agent is ascorbic acid and reduces the molybdenum from +6 to lower valences, +5 and +3. The reduced molybdenum ions react with phosphate ions to form complex phosphomolybdate ions that give the solution a molybdenum-blue color. The antimony potassium tartrate accelerates the reaction and enhances color development. Arsenic compounds interfere by also forming a blue color, but arsenic is not usually found in soils in quantities sufficient to cause much error. Silica may also be a problem in high-pH soils.

12MoO₄^2-(yellow) + H₂PO₄^– + 24H⁺ ó [H₂PMo₁₂O₄₀]^–(blue) + 12H₂O

Allow 10 min for the blue color to develop before taking absorbance measurement.

5. Transfer a portion in a disposable cuvette and then measure the absorbance in a spectrophotometer set at 882 nm. The color is stable for about 2 hr.

Absorbance (sometimes called optical density) refers to the physical process of absorbing light. Mathematically, absorbance equals log₁₀ I_o/I, where I_o is the intensity of the light before it enters the sample and I the intensity after passing through the sample. Absorbance in a specific reading cell is proportional to concentration. The wavelength of light chosen for the measurement is the wavelength of maximum absorbance based on the specific color being measured.

A = log₁₀(I_o/I)

For each extractant (Bray-1 and Mehlich-3), prepare a set of P standards from stock solutions using the P levels as outlined in the table below. Transfer 1.0 mL of each standard to a 20-mL test tube and repeat steps 5 through 7. Prepare the calibration curve for each extractant by plotting standard P concentration on the x-axis and the absorbance reading on the y-axis. Standard solutions should be stored in clean plastic containers to avoid dissolving arsenic from glass. As mentioned earlier, arsenic produces a blue color similar to that produced from phosphorus.

 

6. Calculate P concentration in soil (mg P per kg soil) of each sample and determine its soil test class (relative level) (very low, low, optimum, high, or very high) using Table 1 provided from PM 1688.

(Net sample absorbance – line intercept) ÷ slope = concentration in the extractant

mg P in the 1-mL extractant = concentration in 1 mL × dilution factor

(if the 1-mL aliquot analyzed was diluted)

µg P per g soil (ppm) = 30 (mg P in the 1-mL extractant)/weight of soil extracted

7. After determining the soil test class of P, the next step is to determine the quantities of fertilizers required. Use Table 3 provided from PM 1688 to determine the lbs of P₂O₅ required per acre for corn grain production. For this example, the soil is a fine-textured soil and some coarse soils have very low P. Notice Table 3 also has information for K, which we will use later.

Check out PM 1688 [PDF].^[1] This pamphlet, “A General Guide for Crop Nutrient and Limestone Recommendations in Iowa” serves as the bases for interpretation of soil test reports and lime recommendations for Iowa. Every soil in Iowa has been classified as to its subsoil P and K levels in Table 15 at the end of the pamphlet.

 

 

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