Agricultural Service Laboratory Soil Analysis Procedures 04/28/00 TABLE OF CONTENTS Sample Preparation Sample Check-In Sample Extraction Soil pH Buffer pH Sulfur (Sulfates) Saturated Extract (SE) Nitrate-Nitrogen Organic Matter Soluble Salts Quality Control for Soil Testing References Sample Preparation 1. Arrange samples by date received, county and grower in sets of 100. 2. Assign a 7 digit lab number to each sample. The 2 left hand digits correspond to the month. The remaining 5 digits are assigned consecutively within the month as the samples are arranged in the sets. The 3 right hand digits designate the position of the sample within the set of 100 samples. 3. Place samples into the set. Sample information on the boxes and record sheets should be compared for agreement. Any special analyses should be noted by lab number on a separate sheet of paper as well as other special requests. 4. Samples received in a damaged condition, missing samples, or duplicated sample numbers should be annotated on the record sheet. Samples received without paperwork should have a record sheet completed from information on the sample box if possible. 5. Within each set, place Check sample boxes at approximately positions 40, 60, and 80 and note the position number shown on a blue Check soil record sheet. 6. After a complete set is arranged, box tops should be removed with the exception of 1 flap of the first sample in the set. The date received and lab number of the first sample should be written on this flap. 7. Place samples in drying racks and allow to dry. 8. After drying, crush and screen samples through a 10 mesh screen. Collect screened samples and return to the original box. Samples are now ready for analysis. 9. Following analysis, place samples into storage racks for 1 month from the date of analysis to allow for recheck if necessary. After that time, samples may be discarded. 10. Soil from Marion, Dillon, and Horry must be treated by heating at 121oC for 2 hr. Stamping Papers with Lab Numbers 1. Collect papers and start stamping with beginning lab number. 2. Write beginning lab number on raw data sheet. Note position of checks and blank on raw data sheet. 3. Be careful to check for any special analyses. Note the special analyses with a check mark on the raw data sheet. 4. Make labels for pH and extracted samples. 5. Turn papers in to data processing. Sample Check-In Sample order must be rechecked prior to any analyses. 1. Retrieve papers for individual sets from data processing with lab numbers already assigned and stamped. 2. Check through each sheet to make sure samples are in the proper order in the trays. 3. Return sheets to data processing. Sample Extraction Procedure After samples are checked for proper order, labeled with Check and Blank locations noted, samples are ready for extraction. 1. Using a 4 mL volumetric scoop (assume 5 g), measure an amount of soil from each sample box using the following method: 1) Dip scoop with sweeping motion and fill to overflowing 2) Hold scoop over box and firmly tap handle to settle 3) Strike off excess soil with leveling rod and transfer 2. Measure samples into extraction racks containing 10 polyethylene cups each. A Check sample is scooped at the appropriate location from a separate Check sample box or no sample is scooped at the blank location. 3. Extract fifty samples at a time. Add twenty milliliters of extracting solution (0.05N HCl + 0.025N H2S04) by automatic pipette to each sample. 4. Shake samples on a mechanical reciprocating shaker, adjusted to 180 oscillations per minute with a 4 cm stroke, for 5 minutes. 5. Place prefolded, high quality filter paper, moistened with deionized water into funnel tubes in racks that correspond with the extraction racks. 6. After shaking, immediately filter and save the collected extract for mineral analysis (P, K, Ca, Mg, Na, Zn, Mn, Cu, B). Transfer to test tubes for ICP. 7. All glassware and cups should be thoroughly rinsed between samples with deionized water. Weekly wash glassware using a minimum of detergent and rinse thoroughly. Mehlich I Extracting Solution (0.05 N HCl + 0.025 N H2S04) To prepare 18 liters: add 77 mL concentrated HCl and 13 mL concentrated H2S04 to approximately 15 liters of deionized water in 20 liter carboy. Bring to 18 liters with deionized water and mix thoroughly. Soil pH Soil pH is determined on all samples using a 1:1 (weight:volume) soil to water ratio. pH 6.86 buffer standard - purchased at pH 7.0 (0.5M Phosphate) Dissolve 3.40 + 0.01 g of dried reagent-grade potassium dihydrogen phosphate (KH2PO4) and 3.55 + 0.01 g of dried reagent-grade anhydrous disodium hydrogen phosphate (Na2HPO4) in sufficient deionized water to make 1000 mL. pH 4.01 buffer standard - purchased at pH 4.0 (0.05M Potassium hydrogen phthalate) Dissolve 10.21 + 0.05 g of dried reagent-grade potassium hydrogen phthalate (KHC8H4O4) in sufficient deionized water to make 1000 mL. Procedure 1. Transfer 16 mL scoop of soil (assume 20 g)to paper cups arranged in numbered trays. After 100 samples (1 set)is transferred, scoop every 20th sample an additional time as rechecks and include at the end of the set. 2. Add 20.0 mL of deionized water by automatic pipette with enough force to mix thoroughly and allow to stand at least 1 hour. 3. A standard pH meter is used to measure pH. Check the filling solution level of the reference electrode daily. 4. Standardize the meter before reading by rinsing the electrodes thoroughly with water and lightly pat dry. Follow instrument procedures for calibrating with buffers; pH 7.0 and pH 4.0. 5. Stir each sample well immediately before reading. Rinse electrodes with deionized water and place electrodes in the sample suspension at the soil-water interface. 6. Allow the meter to stabilize and record pH to the nearest single decimal place (0.1) on the laboratory result sheet. 7. Measure the pH of the check soil. The pH should be read to 1 decimal place (0.1) and should be +0.2 pH units from the standard Check value. If the Check soil is not within tolerance, recalibrate the meter and/or seek assistance. 8. After the set is completed, compare the additional recheck readings with the original readings and record beside the recheck sample number. 9. Any sample with pH less than 4.0 or greater than 8.0 should be scooped again and the pHremeasured for quality control. 10. Store electrodes in storage solution or buffer standard solution with meter in Standby mode. 11. Clean electrodes weekly. Buffer pH (Lime requirement) A routine buffer pH or lime requirement pH is made on all samples. The Adams-Evans buffer method is used to calculate the lime requirement of ground, agricultural limestone required to raise the soil pH of the surface 8 inches to the target pH suggested in EC 476 (Nutrient Management for South Carolina Based on Soil-Test Results). Adams-Evans Buffer - purchased from CCI 1. Dissolve, separately, 360 g of p-nitrophenol and 270 g of H3BO3 in approximately 3 liters each of hot deionized water. 2. Dissolve 225 g KOH in approximately 500 mL of deionized water. 3. Add 1332 g KCl to 6 liters of deionized water in an 18 liter carboy. 4. Add in order, p-nitrophenol, H3BO3 , and KOH solutions mixing thoroughly between additions to the KCl solutions. 5. Bring to 18 liters with deionized water and allow to cool. 6. Adjust pH to 8.00 with either KOH or HCl. (Note: 1 mL concentrated HCl will lower pHapproximately 0.01 unit.) Procedure 1. To each soil-water mixture from the pH determination (20 g soil + 20 mL H20) add 20 mL of the buffer solution by automatic pipette. 2. Stir thoroughly and allow to stand a minimum of 30 minutes. 3. Standardize pH meter with buffer 7.0 and 8.00 (buffer blank = 20 mL buffer + 20 mL deionized water). 4. Stir each sample well immediately before reading. Rinse electrodes with deionized water and place electrodes in the suspension at the soil-solution interface and release the Standby control. 5. Allow the meter to stabilize and record pH to the nearest 0.05 unit on the laboratory sheet. 6. Measure the buffer pH of the Check soil to the nearest 0.05 unit. If the buffer pH is within 0.10 units of the standard check value, read the remainder of the samples in that set. If the Check soil readings are not within tolerance, check standardization and/or seek assistance. 7. Dump cups then autoclave samples from Marion, Horry, and Dillon. 8. Store electrodes in storage solution or buffer standard solution with meter in the Standby mode. Lime requirement for fish pond samples The same procedure for buffer pH on fish pond and shrimp pond samples is followed. Sulfur (Sulfates) Extractable total sulfur is determined on request and on subsoil samples where the argillic horizon is less than 20 inches from the surface if requested. Sulfate Extracting Solution (0.5N NH4OAC in 0.25N CH3COOH) 1. Dissolve 38.5 g NH4OAC in deionized water in a 1 liter volumetric flask. Add 14.2 mL of glacial acetic acid (CH3COOH) and bring to volume with deionized water. Or, add 66.5 mL of concentrated NH4OH to approximately 1000 mL of deionized water in a 2 liter volumetric flask. Add 86.0 mL of glacial acetic acid (CH3COOH) and bring to volume with deionized water. 2. To prepare 18 liters, add 600 mL of concentrated NH4OH to approximately 10 liters of deionized water in a 20 liter carboy. Add 770 mL of glacial acetic acid (CH3COOH) and dilute to 18 liters with deionized water. Procedure 1. Samples are extracted using sulfate free glassware, with sulfate extracting solution, following the same procedure used in the Mehlich I soil extraction. 2. After shaking, samples are filtered through number 42 Whatman filter paper or equivalent, and the extract saved for S analysis on the ICP. Record results in lb/A with no decimal. Saturated Extract (SE) Samples should be logged in the log book. Procedure 1. Fill 600 mL plastic beakers with sample mix. Bring to field capacity with distilled water, stirring gently to avoid breaking encapsulated fertilizer pellets and allow to equilibrate for 30 minutes. 2. Vacuum filter through # 4 filter paper and collect at least 110 mL of the extract. 3. Pour an aliquot into a test tube to determine extractable P, K, Ca, and Mg routinely in the laboratory, using the ICP. 4. Save 100 mL to use for the rest of the analyses. 5. Determine soluble salt content on the solubridge and convert to mmhos/cm by moving decimal three (3) places to the left. Record value with 2 decimal places. (See Plant and Feed Manual for meter instructions.) 6. Determine soil pH by glass electrode in a separate subsample by routine lab procedure. 7. To determine NO3-N by specific ion electrode see NO3-N procedure for SE. 8. Saturated extract results are reported on the Special Analyses Report sheet. Lab number should be the log book number for that sample as recorded. Method of extraction is SE. Nutrient analyses should be reported in ppm. These values may be calculated by dividing soil test lb/A values by 8 (now done automatically by the computer). NO3-N values do not require calculation. Nitrate-Nitrogen Nitrate-Nitrogen (NO3-N) is determined routinely on saturated extracts and mineral soils when requested. Nitrate Extracting Solution Dissolve 173.2 g AL2(SO4)3.18H20, 12.8 g H3BO3, and 0.7222 g KNO3 in 8 liter of deionized water. Dilute to 9.5 L and check pH. Adjust to pH 3.0 with NaOH. Dilute final volume to 10 liter. Boric Acid Preservation Solution - Dissolve 6.2 g boric acid in 100 mL hot distilled water. Ammonium Sulfate Ionic Strength Adjustor (ISA)- Prepare a 2 M ammonium sulfate solution by placing 26.42 g reagent-grade (NH4)2SO4 in a 100 mL volumetric flask. Dissolve and dilute to the mark with distilled water. Standard Stock Solution (1000 mg/l NO3-N) - Dissolve 0.7218 g KNO3 (dry) in deionized water in a 100 mL volumetric flask. Add 1 mL preservation solution then dilute to 100 mL. Silver sulfate - Reagent grade Ag2SO4. Outer filling solution - Dilute 2 mL ISA to 100 mL with distilled water Inner filling solution - Orion cat. 900002 Procedure (Soils): 1. Measure 20 g soil (16 mL scoop) into a paper cup. 2. Add 40 mL NO3 extracting solution, mix thoroughly, and allow to equilibrate for 1 (one) hour. 3. Filter through # 4 filter paper and collect filtrate in 50 mL beakers. 4. Working Standards (2, 10, 100 mg/l NO3-N) To prepare 2, 10, and 100 ppm working standards from commercial 1000 ppm NO3-N stock solution, pipette 0.2, 1, and 10 mL respectively of the 1000 ppm stock into 100 mL volumetric flasks. Bring to volume with NO3 extracting solution. (Prepare fresh standards every two weeks.) 5. Determine ppm NO3-N by specific ion electrode. 6. Instrument calibration and operation for soils: a) Remove electrodes from storage solution and rinse thoroughly. b) Place electrodes in 2 ppm standard. Press calibration then enter 3-yes for number of standards. Wait for beep then enter 4.0-yes. c) Rinse electrodes then place in 10 ppm standard. Wait for beep then enter 20.0-yes. d) Rinse electrodes then place in 100 ppm standard. Wait for beep then enter 200-yes. Slope should read 54-60. e) To read samples press measure. Red check sample should read 15-19. Brown check sample should read 7-9. 7. Record as ppm NO3-N (no decimals) on lab result sheets for special analyses. Recheck standards periodically and recalibrate as necessary. 8. After analyses are completed, rinse electrodes, return to storage solution, turn off stirrer, and return instrument to Standby. Procedure (Saturated Extract): 1. Working Standards a) To prepare 2, 10, and 100 ppm working standards, add 0.2, 1, and 10 mL respectively standard stock solution (1000 mg/l NO3-N) to 100 mL volumetric flasks and bring to volume with distilled water. b) Prepare fresh standards every two weeks. 2. Add 1 mL preservation solution to each sample and standards. 3. Add 2 mL ISA to the standards and the samples. 4. Check levels of inner and outer filling solutions in reference electrode (see reference electrode manual for specifications). 5. Instrument calibration for SE: (see NO3-N instrument calibration and operation for soils - page 7 except enter exact value for standards, ie., 2, 10 and 100). 6. Rinse electrodes and place into sample. Record reading directly as mg/l NO3-N (no decimal) on Report on Special Soils or Artificial Mixes form. 7. Recheck standards frequently. 8. When finished, place meter in Standby mode and submerge electrodes in Blank. Organic Matter Loss on Ignition for Organic Matter. Apparatus 1. Muffle Furnace 2. "High Form" Porcelain Crucibles Procedure 1. Number crucibles with wax pencil. 2. Weigh crucibles to 4 decimal places. 3. Scoop soil samples into crucibles using 5 g scoop. 4. Put crucibles plus samples on tray in left-hand furnace. 5. Set dial to 100. (This corresponds to a temperature of 105o +/- 5oC.) 6. Ash at this temperature for 2 h. (It takes 30 min for the furnace to reach 105oC, so set the dial at 100 for a total of 2 h and 30 min.) 7. Remove samples from furnace with tongs, place in desiccator, and weigh to 4 decimal places immediately while still warm so moisture won't be absorbed. 8. Replace crucibles plus samples in left furnace. 9. Turn dial up to 360. (This corresponds to a temperature of 360o +/- 5oC.) 10. Ash at this temperature for 2 h. (It takes 1 h for the furnace to reach this temperature, so set the dial at 360 for a total of 3 h.) 11. Turn furnace off and open door. 12. Place samples in desiccator with tongs and allow samples to cool. 13. Weigh samples to 4 decimal places. 14. Calculate OM % and record on data sheet with one decimal. Calculation crucible wt = a crucible wt + sample wt after 105oC heating = b crucible wt + sample wt after 360oC heating = c %OM = ((b-a) - (c-a))(100)/(b-a) Reference E. E. Schulte, Recommended Soil Organic Matter Tests. Walkley-Black procedure. Potassium Dichromate Solution (1.0N K2Cr2O7) Place 49.04 g of potassium dichromate (K2Cr2O7) (dried at 105oC) in a one-liter flask and dilute to volume with demineralized water. Ferrous Sulfate Solution (0.5N FeSO4.7H20) Place 139.01 g of ferrous sulfate (hydrated) FeSO4.7H20 in a one liter flask. Add about 200 mL of demineralized water and 15 mL of concentrated sulfuric acid. Dilute to volume. Indicator (if necessary): Use 0.025 M O-phenanthroline ferrous sulfate (Ferroin) as an indicator (commercially available). 0.5946 g/100 mL phenanthroline = 0.01N 0.2780 g/100 mL FeSO4.7H20 = 0.01N Procedure: 1. Place 1.0 g of soil in a 250-mL Erlenmeyer flask and prepare a Blank by adding no soil. 2. Add exactly 10 mL of 1.00N potassium dichromate and mix by gentle rotation of flask. 3. Add 20 mL of concentrated sulfuric acid and mix for one minute by gentle rotation of flask. 4. Allow to stand for 30 minutes. 5. Add 150 mL demineralized water. 6. Add 10 mL of concentrated phosphoric acid and allow to cool to room temperature. 7. Add 4-5 drops of indicator. (If solution turns dark green weigh 1/2 g and multiply answer by 2 or weigh 1/4 g and multiply answer by 4.) 8. Titrate immediately with 0.5N ferrous sulfate solution until end point is reached. 9. Determine % organic matter using given formula: % O.M. = (1 - T/S) (6.8) Where T = sample titration in mL of 0.5N ferrous sulfate Where S = standard blank in mL of 0.5N ferrous sulfate This formula is based on two important assumptions which may not be valid for all soils: (1) only 76% of the organic carbon present is oxidized to CO2, and (2) soil organic matter contains 58% carbon. 10.Record on lab result sheets for special analyses as % O.M. (1 decimal place). Soluble Salts Soluble salts are run on samples as requested in mineral soils and on artificial mixes. Samples should be logged in the log book. Procedure: 1. Scoop 20 mL soil to a 250 erlenmeyer flask. 2. Add 40 mL of distilled water with graduated cylinder, stir, and allow to equilibrate for 1 hour, shaking intermittently. 3. Decant liquid (through screen filter) and collect in small wide mouth bottle. 4. Read mmhos/cm on conductivity meter. (See Plant and Feed Manual for meter instructions). 5. Record 2 decimal places, e.g., 0.200 = 0.20. 6. Report values on Report on lab sheet. Lab number should be either the log book number if only soluble salts are to be reported, or the laboratory number of the mineral soil received by the lab for routine soil analysis. Method of extraction is 1:2. Quality Control for Soil Testing The large number of routine soil samples extracted and analyzed daily in the Soil Testing Laboratory (100-400) requires that a precise system of quality control be used. The system insures that samples remain in their proper order so no mix-up is made in the sample sequence and the quality of the analyses will remain constant from day to day and throughout the year. "Check" samples are established by collecting, drying, homogenizing, and storing several kilograms of a soil that has varying levels of pH, P, K, Mg, and Ca. A "standard" value for pH and extractable nutrients and a standard deviation is established by analyzing this soil ten to twenty times on different days and establishing a mean value. This soil is designated as a Check sample, given a letter, and used daily in maintaining a quality control for soil samples. The laboratory participates in the North America Proficiency Testing soil sample exchange programs. This allows the Soil Testing Laboratory to check its "Check" sample values with those from other laboratories, as well as compare analytical techniques. In a routine, daily soil-test run, two Check samples and one Blank are placed in every group of 100 soil samples as the samples are numbered. An information sheet is included every time a Check sample or Blank is placed in the samples, and the Check or Blank is numbered and analyzed as a routine soil sample. The supervising technician is responsible for comparing the laboratory results of the Check samples and Blanks to make sure they are reading within an accepted range. By careful attention to the values of the Checks and Blanks, errors can be avoided before they leave the laboratory. Samples are held for 1 month after analysis to allow for recheck if necessary. Growers or Extension personnel may request that all or part of their analyses be rechecked for accuracy. This allows for a monitoring of quality control as well as maintaining confidence in the lab. References 1. Isaac, R. A. (ed.). Reference Soil Test Methods for the Southern Region of the United States. Southern Cooperative Series Bulletin 289, September 1983. 2. S. J. Donohue. Reference Soil and Media Diagnostic Procedures for the Southern Region of the United States. Southern Cooperative Series Bulletin No. 374, August 1992.