Hot water extraction of soil B (Berger and Truog 1939) is widely used to obtain an index of soil B status for plant growth. Unfortunately, the method presents difficulties for routine analysis because extracts free of colloidal material are difficult to obtain. Cartwright et al. (1983) proposed 0.01 M CaCl2-0.05 M mannitol [C6H8(OH)6] as an alternative to hot water extraction because it was suited to analysis by ICPAES. Subsequent research (Aitken et al. 1987) has shown that hot 0.01 M CaCl2 is a superior predictor of B deficiency, especially in acid and near neutral soils. The method is highly correlated with hot water-soluble B.
The hot CaCl2-extractable soil B method described is based on refluxing air-dry soil for 10 min with 0.01 M CaCl2 at a 1:2 soil/solution ratio. After replacing liquid lost during reflux, extracts are filtered while hot and allowed to cool. In this method, B is determined by an azomethine-H colorimetric procedure based on methods described by Gupta and Stewart (1975), Gupta (1979), Gaines and Mitchell (1979), Parker and Gardner (1981), and Bloesch (1985).
Colorimetric determination of B is an option when ICPAES is not available. The azomethine-H finish is preferred over other colorimetric procedures for soil B as its colour is developed in aqueous medium. Other colorimetric procedures for determining B, including carminic acid (Hatcher and Wilcox 1950) and curcumin (Hayes and Metcalfe 1962) require concentrated acids or organic solvents. The method takes account of evidence (Bloesch 1985) that time affects the absorbance of a B–azomethine-H solution, especially at low B concentrations. Brij 35 Wetting Agent is included to improve the flow of solution to the spectrophotometer.
Comparative data between ICPAES and azomethine-H on CaCl2 extracts of soil (Parker and Gardner 1981; AJ Jeffrey, pers. comm.) suggest that ICPAES will yield slightly lower B values in most cases.
There is confirmation for these observations in results reported in ASPAC interlaboratory proficiency programs. For example, the median values for the azomethine-H method in 2004–05 (Rayment et al. 2007) averaged around 9% higher than results obtained by ICPAES, although there were a few samples where the reverse occurred. Continuous relations between soil B concentrations by azomethine-H and robust % CV are unavailable because of limited numbers of laboratory participants for this test in ASPAC interlaboratory proficiency programs. Method 12C1, however, is consistently less-well performed than its ICPAES alternative (Method 12C2).
Deficiencies of B may occur in soils containing <0.15 mg B/kg by the method described. Bruce and Rayment (1982) have assigned ratings of very low, low, medium, high and very high for hot water-extractable B concentrations of <0.5, 0.5–1.0, 1.0–2.0, 2.0–5.0 and >5.0 mg/B kg.
0.01 M Calcium Chloride Extracting Solution
Dissolve 1.47 g calcium chloride dihydrate (CaCl2 2H2O) in deionised water and make to 1.0 L. Store in B-free glassware or a polyethylene or teflon container.
Activated Charcoal
Add 1.0 L CaCl2 extracting solution to 500 g of activated charcoal and reflux for 10 min. Filter while hot (≈80°C) through Whatman No. 42 filter paper on a buchner funnel. Wash the charcoal four or five times with 200 mL portions of hot (≈80°C) CaCl2 Extracting Solution. Dry the charcoal in an oven at 60°C.
0.025 M EDTA Solutions
Dissolve 4.65 g of disodium EDTA {[CH2.N(CH2.COOH).CH2.COONa]2.2H2O} in 200 mL deionised water and make to 500 mL. Add 1 mL Brij 35 Wetting Agent (see Method 5A2) and mix thoroughly.
Dissolve 250 g ammonium acetate (CH3COONH4) in 500 mL deionised water and adjust solution pH to 5.5 by slowly adding ≈100 mL glacial acetic acid (CH3COOH) with constant stirring. Add 0.5 mL Brij 35 Wetting Agent and remix.
Azomethine-H Solution
Azomethine-H is available commercially or may be prepared as described by Shanina et al. (1967). Dissolve 0.5 g of azomethine-H in ≈50 mL of deionised water. Add 1.0 g l ascorbic acid (C6H8O6) and warm gently (30°C) to obtain a clear solution. Cool and dilute to 100 mL. The solution remains stable for a week if stored at ≈4°C.
Boron Primary Standard
1 L contains 250 mg of B.
Dissolve 1.430 g AR boric acid (H3BO3; dried to constant weight in desiccator over anhydrous CaCl2; Note 1) in 0.01 M HCl and make to 1.0 L in a volumetric flask with 0.01 M HCl. Store in a polyethylene or teflon bottle.
Boron Secondary Standard
1 L contains 20 mg of B.
Pipette 40 mL B Primary Standard into a volumetric flask and make volume to 500 mL with 0.01 M CaCl2 Extracting Solution. Store in a polyethylene bottle; shelf life is about 4 weeks.
Boron Working Standards
Pipette 1.25, 2.5, 5, 10, 15, 20 and 25 mL B Secondary Standard into separate 500 mL volumetric flasks. Dilute to volume with 0.01 M CaCl2 Extracting Solution. These solutions contain 0.05–1.0 mg B/L. For a 1:2 soil/solution ratio and equal volumes of standards and extract solutions, these standards contain concentrations of B equivalent to 0.1, 0.2, 0.4, 0.8, 1.2, 1.6 and 2.0 mg B/kg of soil.
Working standards equivalent to 50 mg B/kg may be required when B toxicity in soil is suspected. The extracting solution serves as a blank.
All glass and plasticware must be washed with dilute (1+4) HCl followed by deionised water before use. Filter papers should be checked for freedom from B contamination. If contaminated, pretreat by washing with hot (≈80°C) CaCl2 Extracting Solution.
Add 10.0 g of air-dry soil (<2 mm) and 20 mL of 0.01 M CaCl2 extracting solution into a B-free flat-bottomed flask or bottle (150 mL). Record weight of each flask plus fresh sample and extracting solution.
Next insert a small plastic funnel in the neck of the flask and quickly bring to the boil, then gently reflux for 10 min. Remove the funnel and immediately bring the flask + sample back to its original weight with hot (>80°C) deionised water. Quickly filter the extract through a Whatman No. 40 filter paper into a polyethylene container.
If the hot (≈80°C) extract is not colourless following soil extraction, add approximately 0.1 g activated charcoal to the filtered extract, shake by hand for about 5 min and filter through Whatman No. 42 paper. Repeat the treatment with activated charcoal if necessary.
Cool then transfer a known suitable aliquot (e.g. 5 mL) into a polyethylene container. Next add 2.0 mL of 0.025 M EDTA solution and 2.0 mL of Buffer Solution. Mix flask contents well then add 2.0 mL Azomethine-H solution and shake well. Set aside for a fixed time of 2 h for colour development, away from sunlight. Treat the same size aliquot of each working standard in a similar manner.
Read absorbance of both standards and assays with a spectrophotometer at 420 nm, using identical times for standards and unknowns. Construct a calibration curve (or a regression equation) and determine concentrations of B in soil extracts. Make allowance for any significant reagent calibration blank.
Report CaCl2-extractable B (mg B/kg) on an air-dry basis.
1. If heated, H3BO3 gradually loses water to initially form metaboric acid (H3BO2) and finally the anhydrous oxide (B2O3). Accordingly, oven heating must not be used to dry H3BO3 (ASTM 1989).