5.17.1. Hot-Water Method
Boron (B) is a non-metal, in contrast to the other micronutrient elements. Boron is present in small amounts in igneous, sedimentary and metamorphic rocks. It is an essential micronutrient element for plant growth (needed for cell division), hence for the growth of young shoots. Boron in soils is primarily of importance in soil fertility and plant nutrition.The major inorganic sources of B are borate and boron-silicate minerals. Borate minerals occur mostly in arid regions and are formed by the evaporation of water in enclosed salt-water lakes and basins in arid regions.
The hot-water extraction procedure, introduced by Berger and Truog (1939), modified later is still the most popular method for measuring “available” soil B or the fraction of B related to plant growth in alkaline soils. Water soluble B, the available form of B, is extracted from the soil by water suspension.
Boron in soil extracts is measured calorimetrically using reagent Azomethine-H (Bingham, 1982). Also, B can be analyzed by colorimetric methods using reagents such as Carmine, and most recently by Inductively Coupled Plasma (ICP) and Atomic Emission Spectrometry. The colorimetric method, using reagent Azomethine-H, is preferable because the use of AAS poses some limitations as B is not a metal.
Where soil B levels are less than 0.5 ppm, deficiency is likely to occur for most crops. However, where levels are greater than about 5 ppm, toxicity may occur. The normal B concentration in plant tissue is reported to be between 20 and 100 μg/mg dry matter of mature leaves. It is essential in sugar translocation and in the synthesis of hormones and protein in plants.
Apparatus
Dissolve 250 g ammonium acetate (NH4OAc), and 15 g ethylenediamine-tetraacetic acid disodium salt (EDTA disodium) in 400 mL DI water. Slowly add 125 mL glacial acetic acid (CH3COOH), and mix well.
B. Activated Charcoal (Boron - free)
This is prepared by giving repeated washings (8 – 9 times) of DI water (boiling charcoal with water in 1:5 ratio), and subsequent filtering. Boron in the filtrate is checked by Azomethine-H color
development. Continue washing until it is B-free.
C. Azomethine-H Solution (C17H12NNa O8S2)
Dissolve 0.45 g Azomethine-H and 1 g L-ascorbic acid in 100 mL DI water. Fresh reagent should be prepared weekly and stored in a refrigerator.
D. Standard Stock Solution
Dissolve 0.114 g boric acid (H3BO3) in DI water, and bring to 1-L volume. This solution contains 20 ppm B (Stock Solution).
Prepare a series of Standard Solutions from the Stock Solution as follows:
Dilute 2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 mL Stock Solution to 100-mL numbered flasks by adding DI water, and then bring to volume. These solutions contain 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ppm, respectively.
Procedure
A. Extraction
1. Weigh 10 g air-dry soil (2-mm) into a 250-mL Erlenmeyer flask (Pyrex).
2. Add about 0.2 g activated charcoal (B-free).
3. Add 20 mL DI water.
4. Boil on a hot plate for 5 minutes with flask covered by a watch glass.
5. Filter the suspension immediately through Whatman No. 40 filter paper.
B. Measurement
1. Pipette 1-mL aliquot of the extract into a 10-mL polypropylene tube.
2. Add 2 mL buffer solution.
3. Add 2 mL azomethine-H solution, and mix well.
4. Prepare a standard curve as follows:
Pipette 1 mL of each standard (0.5 – 3.0 ppm), and proceed as for the samples.
Also make a blank, pipette 1 mL DI water, and proceed as for the samples.
5. Read the absorbance of blank, standards, and samples after 30 minutes on the Spectrophotometer at 420-nm wavelength.
6. Prepare a calibration curve for standards, plotting absorbance against the respective B concentrations.
7. Read B concentration in the unknown samples from the calibration curve.
Calculation
Where:
V = Total volume of the soil extract (mL) Wt= Weight of air-dry soil (g)
Technical Remarks
1. Use of glassware should be minimal; always use concentrated HCl-treated glassware (for a week) where absolutely essential.
2. The use of Azomethine-H is an improvement over that of carmine and curcumin, since the procedure involving this chemical does not require the use of concentrated acid.
3. The amount of charcoal added may vary with the OM content of the soil and should be just sufficient to produce a colorless extract after 5 minutes of boiling on a hot plate. Excess amounts of charcoal can reduce extractable B values
4. In humid regions soils, borate ions may tend to leach from soils. Therefore, soluble B concentrations are low in highly leached soils. Since highly leached soils usually exhibit low soil pH, acid soils are deficient in B.
5. In arid region soils, borate ions are usually not affected by leaching. Therefore, strongly basic in reaction, they may contain excessive amounts of B for plant growth.
6. Boron deficiency is more likely to occur in Ultisols and Oxisols, whereas B toxicity could be noted in Aridisols. This is in contrast with the other micronutrients.
5.17.2. Dilute Hydrochloric Acid Method
Though the hot water extraction method (HWE) is quite popular for predicting B fertility in alkaline soils, the procedure is tedious and prone to error (because of difficulty in maintaining uniform boiling time). In an effort of having a convenient substitute, researchers (Kausar et al., 1990; Rashid et al., 1994;
Rashid et al., 1997) have found the dilute HCl method of Ponnamperuma et al. (1981), originally designed for acid soils, to be equally effective in diagnosing B deficiency in alkaline and calcareous soils. The HCl method is simple, economical, and more efficient.
Reagents
A. Buffer Solution
Dissolve 250 g ammonium acetate (NH4OAc), and 15 g ethylenediamine-tetraacetic acid disodium salt (EDTA disodium) in 400 mL DI water. Slowly add 125 mL glacial acetic acid (CH3COOH), and mix well.
B. Azomethine-H Solution (C17H12NNa O8S2)
Dissolve 0.45 g Azomethine-H and 1 g L-ascorbic acid in 100 mL DI water. Fresh reagent should be prepared weekly and stored in a refrigerator.
C. Activated Charcoal (Boron - free)
This is prepared by giving repeated washings (8 – 9 times) of DI water (boiling charcoal with water in 1:5 ratio), and subsequent filtering. Check B in the filtrate by Azomethine-H color development;
continue washing until it is B-free.
B. Hydrochloric Acid (HCl), 0.05 N
Dilute 4.14 mL concentrated HCl (37%, sp. gr. 1.19) in DI water, mix well, and bring to 1-L volume.
C. Standard Stock Solution
Dissolve 0.114 g boric acid (H3BO3) in DI water, and bring to 1-L volume. This solution contains 20 ppm B (Stock Solution).
Prepare a series of Standard Solutions from the Stock Solution as follows:
Dilute 2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 mL Stock Solution to 100-mL numbered flask by adding DI water, and then bring to volume. These solutions contain 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ppm,
2. Add about 0.2 g activated charcoal (B-free).
3. Add 20 mL 0.05 N HCl solution.
4. Shake for 5 minutes, and then filter the suspension using a Whatman No. 40 filter paper.
B. Measurement (Azomethine-H method)
1. Pipette 1-mL aliquot of the extract into a 10-mL polypropylene tube.
2. Add 2 mL buffer solution.
3. Add 2 mL azomethine-H solution, and mix well.
4. Prepare a standard curve as follows:
Pipette 1 mL of each standard (0.5 – 3.0 ppm), and proceed as for the samples.
7. Read B concentration in the unknown samples from the calibration curve.
Calculation
Where:
V = Total volume of the soil extract (mL) Wt = Weight of air-dry soil (g)