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UF/IFAS Extension Soil Testing Laboratory (ESTL) Analytical Procedures and Training Manual

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University of Florida Institutional Repository IFAS
Permanent Link: http://ufdc.ufl.edu/IR00003469/00001

Material Information

Title: UF/IFAS Extension Soil Testing Laboratory (ESTL) Analytical Procedures and Training Manual
Physical Description: Fact Sheet
Creator: Mylavarapu, Rao S.
Publisher: University of Florida Cooperative Extension Service, Institute of Food and Agriculture Sciences, EDIS
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Notes

Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Melanie Mercer.
Publication Status: Published
General Note: "Original publication date August 2002. Revised February 2009. Reviewed July 2012."
General Note: "Circular 1248"

Record Information

Source Institution: University of Florida Institutional Repository
Holding Location: University of Florida
Rights Management: All rights reserved by the submitter.
System ID: IR00003469:00001

Permanent Link: http://ufdc.ufl.edu/IR00003469/00001

Material Information

Title: UF/IFAS Extension Soil Testing Laboratory (ESTL) Analytical Procedures and Training Manual
Physical Description: Fact Sheet
Creator: Mylavarapu, Rao S.
Publisher: University of Florida Cooperative Extension Service, Institute of Food and Agriculture Sciences, EDIS
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Notes

Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Melanie Mercer.
Publication Status: Published
General Note: "Original publication date August 2002. Revised February 2009. Reviewed July 2012."
General Note: "Circular 1248"

Record Information

Source Institution: University of Florida Institutional Repository
Holding Location: University of Florida
Rights Management: All rights reserved by the submitter.
System ID: IR00003469:00001


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Circular 1248 UF/IFAS Extension Soil Testing Laboratory (ESTL) Analytical Procedures and Training Manual1Rao S. Mylavarapu2 1. This document is Circular 1248, one of a series of the Soil and Water Science Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Original publication date August 2002. Revised February 2009. Reviewed July 2012. Visit the EDIS website at http://edis.ifas.u.edu 2. Rao S. Mylavarapu, assistant professor, Nutrient Management Specialist and Director, UF/IFAS Extension Soil Testing Laboratory, Department of Soil and Water Science, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611.The Institute of Food and Agricultural Sciences (IFAS) is an Equal Opportunity Institution authorized to provide research, educational information and other services only to individuals and institutions that function with non-discrimination with respect to race, creed, color, religion, age, disability, sex, sexual orientation, marital status, national origin, political opinions or aliations. U.S. Department of Agriculture, Cooperative Extension Service, University of Florida, IFAS, Florida A&M University Cooperative Extension Program, and Boards of County Commissioners Cooperating. Millie Ferrer-Chancy, Interim DeanContentsExtension Soil Testing Laboratory: Mission and Purpose Description of Oered Tests Sample Submission How to Submit Samples Sample Submission Forms Supplies Sample Preparation Analytical Procedures for Soil Soil Scooping Soil pH (1:2 V/V) Adams-Evans Buer pH Mehlich-1 Extractable P, K, Ca, Mg, Cu, Mn, and Zn Organic Matter Walkley-Black Method Loss-on-Ignition Electrical Conductivity Analytical Procedures for Container Media Extraction Procedure pH Electrical Conductivity (EC) NO3Water-Extractable P, K, Ca, Mg Analytical Procedures for Calcareous Soils AB-DTPA Extractable P Analytical Procedures for Water pH Electrical Conductivity (EC) Metals Chloride (Cl-) Carbonate Equivalent Suspended Solids Analytical Procedures for Plants Standard Determination of Ca, Mg, P, K, Na, Mn, Cu, Fe, Zn, and B in Plant Tissue Total Kjeldahl Nitrogen (TKN) in Plant Tissue Quality Control Laboratory Safety References AcknowledgementsExtension Soil Testing Laboratory: Mission and Purposee University of Florida (UF), Institute of Food and Agricultural Sciences (IFAS), Extension Soil Testing Laboratory (ESTL) was established to serve the people of

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2 Florida with their soil, plant, and water testing needs for ensuring economically and environmentally sustainable crop production. e ESTL clientele receive accurate agricultural test results, interpretations, and recommendations regarding appropriate rates, use of nutrients and nutrient management techniques developed for Florida.Mission Statemente mission of the UF/IFAS Extension Soil Testing Laboratory is: to serve the citizens of Florida, by providing appropriately selected soil, plant and water testing, interpretation and recommendations as an educational service through the Cooperative Extension Service to guide management decisions aecting lime and fertilizer use eciency. e ESTL provides chemical analyses of inorganic soils, container media, diagnostic plant tissue nutrients, and irrigation and household water samples for all Florida residents. Testing is restricted to samples originating from the state of Florida only. Requests for testing of animal manures should be sent to UF/IFAS Livestock Waste Testing Laboratory at the North Florida Research and Education Center Suwannee in Live Oak, FL. Testing of materials such as drinking water, sewage sludges, wastewater euent, sludges from water-treatment facilities, hazardous chemical or biological tests of water or soil, or limestone are referred to other governmental or private laboratories.Purpose of This Manual and Intended Audienceis Circular consists of revised portions from the IFAS Extension Soil Testing Laboratory Chemical Procedures and Training Manual (CIR 812), authored by Hanlon, Gonzalez and Bartos, which has been withdrawn. e procedures described in this manual reect the current methodologies for agricultural testing oered by the UF/IFAS ESTL. is Circular replaces previous information that is contained in other IFAS publications. e ESTL services are oered as a part of the Nutrient Management Extension Program in fulllment of the public service mandate of the landgrant university mission. Only tests that have been shown through research/experience to assist in crop-management decisions are oered by the ESTL to Florida residents. It is the intention of the Cooperative Extension Service to oer only analytical procedures whose results can be interpreted, and thus render assistance with management decisions involving water, plants, soils, and nutrients. A small number of diagnostic samples submitted by county or state-wide Extension IFAS faculty may be tested free of charge. A limited number of similar services may be extended to IFAS researchers if needed, to assist researchers in making nutrient management decisions when establishing eld research plots. Superscripted numbers throughout the text below denote the corresponding publication in the References section.Description of Tests OeredCommercial Crop Production on Mineral Soils (Agronomic, Vegetable, Ornamental, and Fruit Crops)e ESTL uses Mehlich-1 extraction procedure for extracting soil samples in preparation for further soilfertility analyses. e Mehlich-1 extraction solution, also referred to as the dilute double acid extractant (0.05M HCl + 0.0125M H2SO4), is intended for use in extracting weathered soils that have cation exchange capacities of less than 10 cmol kg-1 and whose soil pH is less than 7.0. e Mehlich-1 extraction method is not suitable for the extraction of alkaline soils.21 For alkaline soils, recommended extractant in Florida is ammonium bicarbonate-DTPA (AB-DTPA). e ESTL oers a standard soil-fertility test for noncalcareous inorganic soils. e standard test includes analyses for soil pH and macronutrients phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) levels in the soil. e ESTL does not test for soil nitrogen (N) as there is no reliable soil test for predicting N availability to the plants. If a crop code is included on the analysis request form, a test for Buer pH may be determined using Adams-Evans Buer solution (pH 8.00) if the soil pH obtained is lower than the Target pH of the crop specied. e Target pH for a crop is that soil pH at which optimal crop performance and yield is achieved and is therefore specic to the crop. Buer pH will not be determined if no crop is specied on the sample submission form or if the dierence between the soil pH and the target pH is less than 0.2 pH units or if the soil pH exceeds the target pH. Subsequent to the Buer pH determination, lime requirement is calculated using the amount of exchangeable (potential) acidity in the soil. Currently, the ESTL does not provide information concerning methods of lowering soil pH. Results from the above soil tests are interpreted for crop response based on Table 1. e interpretation values are determined from several long-term eld calibration

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3 studies conducted for various crops and soils in Florida and thus form the basis for lime, nutrient and management recommendations detailed on the soil test report sent to the clients. e ESTL Soil Test Report will specify the recommended quantities of macronutrients (N, P, K, Ca and Mg) to be applied to the soil in order to increase the supply of these nutrients to the levels needed for optimum yield and/or quality for the crops requested. Quantities are reported in either pounds per acre, pounds per 100 linear bed feet, pounds per 1000 square feet, or pounds per 100 square feet, depending on the crop.5 e P and K recommendations are both reported as the oxide forms (P2O5 and K2O) in order to comply with current fertilizer-label requirements. Recommended quantities of N, Ca and Mg are reported as the elemental form. e report also will indicate the amount of lime needed, if any, to be added to the soil in order to raise the soil pH to that of the Target pH of the crop requested. It should be noted that recommendations for N are not based on soil testing. e ESTL does not currently test for N in soil due to lack of a meaningful soil test method through which N availability to meet plant needs can be predicted. e recommendations for N shown on the soil test report form are instead based on research studies that measured the response of the indicated crop to various levels of applied N fertilizers. e results of these studies are then used to determine the correct amount of N needed for optimum crop response. If part of the soil N requirements will be met through nutrient release from organic sources such as crop residue or organic soil amendments, the N fertilizer recommendation should be lowered appropriately by estimating the N availability of the amendment material. An integral part of the recommendations are the footnotes. e footnotes included in the report elaborate on many aspects of fertilization and cultural management for the specied crop(s). It is strongly recommended that the producers consider the information contained in these footnotes when making management decisions for ecient fertilizer use. e ESTL also oers a micronutrient test for Mehlich-1 extractable Cu, Mn, and Zn. e primary value of the micronutrient soil test is to determine if adequate levels of micronutrients already exist in the soil. e interpretation of the soil micronutrient test results and proper micronutrient fertilization is included with the report. Micronutrient fertilizers should be used with discretion since it is possible to build up toxic levels of these elements in a soil. Use of the shotgun approach (i.e., addition of micronutrients as insurance) should be avoided. It should also be noted that pesticide formulations frequently contain one or more of these micronutrients. erefore, if pesticides are applied, additional application of micronutrient fertilizer is oen unnecessary.Tests to Choose FromL ANDSCAPE L A WN, AND VEGETABLE GARDEN TEST13e UF/IFAS ESTL oers two soil testing options for the homeowner or home gardener. e rst soil test option is for soil pH and lime requirement determination. No other nutrient analysis or fertilizer recommendation is provided under this option. e measured soil pH is compared to the Target pH for the crop specied by the homeowner or gardener and a lime requirement, if any, is determined using the Adams-Evans Buer pH Index. Both the soil pH and the recommended lime application rate for the specied crop are included in the soil test report. General fertilizer recommendations for landscape, lawns, and vegetable gardens can then be found in a variety of IFAS extension publications by visiting http://edis.ifas.u.edu or the local County Extension Agency. It should be noted that general recommendations do not account for nutrients supplied to the plant from sources already within the soil. Instead, all nutrition is assumed to come only from the fertilizer added to the soil. e second option includes tests for soil pH and lime requirement alongwith macronutrients (Mehlich-1 extractable P, K, Ca and Mg). is information is then used to calculate specic lime and fertilizer recommendations for the crop of interest and is included in the soil test report along with the appropriate footnotes. is allows the homeowner or gardener to develop their fertilization program according to the specic fertilizer needs of the crop they are growing. Recommendations are made for a variety of crops including landscaping plants, ornamentals, vegetable gardens, and lawn grasses (bahia, bermuda, centipede, St Augustine, Table 1. Current interpretation for Mehlich-1 soil test results for agronomic and vegetable crops. Very Low Low Medium High Very High ------------------------------ppm--------------------------------P <10 10-15 16-30 31-60 >60 K <20 20-35 36-60 61-125 >125 Mg -<15 15-30 >30

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4 etc) and are reported as either pounds of nutrient per 100 square feet or per 1000 square feet.PINE NURSERY SOIL TEST17Soil samples from a pine nursery should be obtained from the 0to 6-soil depth only, and will be analyzed for soil pH, organic matter, and Mehlich-1 extractable P, K, Ca, and Mg.C ONTAINER MEDIA TEST15e ESTL Container Media Test is used to measure the levels of water-soluble nutrients in soilless media (e.g., mixtures of materials such as perlite, expanded plastics, vermiculite, peat, pine bark, wood shavings, compost, and sand). Analyses include pH, electrical conductivity, nitrate-N, P, K, Ca, and Mg, all of which are measured in a saturated water extract from the soilless media. is test is recommended as a diagnostic tool for fertilizer management in commercial container-plant production as a means of monitoring nutrients in the media throughout the growing season. e test report also provides the fact sheet12 that assists in the interpretation of the results. Test interpretations are meaningful only in commercial nursery situations. Unlike the other soil tests oered by the ESTL, container-media samples should NOT be dried prior to their delivery to the laboratory. Drying these types of media can adversely aect the results of the test by changing the amounts of nutrients extracted from the media. PLANT TISSUE TEST18In addition to soil testing, the ESTL also oers a Plant Tissue Test. is test is oered specically to commercial growers of blueberries and pecans and to IFAS county extension faculty for use as a diagnostic tool. Test results from samples submitted by commercial growers are forwarded to an Extension blueberry or pecan specialist, who evaluates the data and provides a report to the grower. Results from diagnostic samples are provided to the submitting agent or specialist only. e agent/specialist is responsible for interpreting the data for the client.WATER TEST16e ESTL oers testing of both household and other water supplies used for irrigation/microirrigation for mineral determinations only. All health-related and drinking water quality inquiries should be directed to the nearest county Health Department. Additionally, questions concerning municipal water supplies should be referred to the Department of Health and Rehabilitative Services as that agency is responsible for monitoring the quality of municipal water sources. e ESTL Water Test Report includes values for pH, Ca, Mg, Fe, Mn, Na, Cl, hardness, total carbonates, and electrical conductivity. e irrigation water test includes all of the above, as well as a test for suspended solids. e report provides tables assisting with the interpretation of results.3, 4In Florida, many irrigation-water sources originate from limestone aquifers, resulting in high-pH waters. Crops that are pH-sensitive, such as blueberries or pine seedlings, may benet considerably by pretreating such water with acid to destroy carbonates and concurrently lower the pH.10 Results from the total carbonates test can be used to determine the amount of acid required to reduce this high-pH condition. Sample SubmissionHow to Submit Samples to the ESTLA Sample Submission Form and full payment for the requested services should accompany the samples. Sample Submission Forms can be printed directly from the ESTL website (http://soilslab.ifas.u.edu) or can be picked up from the local county Extension oce. Samples may be sent directly to the ESTL via the U.S. Postal Service or express delivery companies. Instructions for collection of a representative sample, proper sample amount, mailing address and other vital information needed for proper sample processing are printed on the forms (described below). Mailing boxes for shipping samples to the ESTL are also available from the county Extension oce. Samples may also be personally delivered directly to the laboratory in order to avoid shipping/mailing delays. Sample analysis generally requires an average of ve working days from the time the sample is received at the ESTL. Results are e-mailed or mailed directly to the address provided on the submission form. Additionally, a copy of these results is sent to the county Extension oce. All county Extension oces have the capacity to receive test results via electronic mail. Clients are encouraged to contact their county Extension oce when seeking further assistance. Clients may also request to receive a copy of their results via fax.Sample SubmissionRelevant sample submission form(s) needs to be lled out completely and accompany all samples submitted for testing. e following forms correspond to the tests and testing options described above and can be downloaded and printed by the clients by from the following links. ese and other information can also be accessed by visiting the

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5 ESTL website (http://soilslab.ifas.u.edu). e forms are also available from the nearest county Extension oce. Producer Soil Test Information Sheet (Fact Sheet SL-135). is form has been designed for use by commercial producers. e information sheet is self-explanatory and provides pertinent information for samples submitted to the ESTL. Landscape and Vegetable Garden Soil Test Information Form (Fact Sheet SL-136). Both private and commercial clients fertilizing plants in the landscape, primarily home horticulture, should use this form. Container Media Test Information Form (Fact Sheet SL134). is form is designed for use by commercial growers using soilless media for container-plant production. Pine Nursery Soil Test Information Sheet (Fact Sheet SL-132). Commercial operators of pine plantations and pine nurseries should use this form. Plant Tissue Analysis Information Sheet (Fact Sheet SL-131). is form is used for submission of plant tissue samples. Only blueberry and pecan leaf samples are tested under this option. All other plant tissue samples must be sent with the consent of an extension agent or state specialist. e agent or specialist assumes the responsibility for interpretation of the plant tissue report. Water Test Information Sheet (Fact Sheet SL-133). is form should be used for analysis of irrigation water or household well water (not municipal or drinking water). Other supplies related to testing and sampling that can also be obtained at the county extension oce include: 1) Soil sample bags. 2) A self-addressed cardboard mailer.Sample PreparationSoil Samples Soil samples should be air-dried before shipment to the ESTL. Drying is best accomplished by spreading a thin layer of soil on clean wrapping paper or newspaper and placing it in a dry shaded area for at least 24 hours. Drying samples in direct sunlight or using a household oven is NOT recommended. Container Media Samples Container media samples should NOT be dried before shipment to the ESTL. Drying media samples will adversely aect the test results decreasing the usefulness of the test. Plant Samples e quality of the tissue samples submitted for analysis is of importance in ensuring proper processing and interpretation of the results. Tissue samples should not be contaminated with soil or sprays. If the tissue is dusty or contaminated, the sample should be gently washed with owing distilled water and allowed to dry overnight prior to shipping. Do not sample diseased or damaged plant materials. Consult the local Extension agent to determine the proper plant part and the proper time to sample. Always place the tissue samples in paper bags only. Plastic bags are NOT recommended. Water Samples e container in which a water sample is sent to the ESTL can inuence results greatly. For example, residual soap from a plastic dish soap container will contaminate the water sample. e container should be clean to avoid contamination of the sample. e sample should be taken several minutes aer the water source has been owing from the spigot or irrigation pump. e container should be ushed thoroughly several times with the owing water. e container should be lled completely with no airspace at the container top. Entrapped air in the container may aect well-water samples due to shis in carbon dioxide, potentially aecting its pH.Analytical Procedures for SoilSoil Scooping Technique8Soil scooping technique is employed to draw an estimated weight of soil sample for testing from the soil sample submitted/prepared. e soil-scooping technique requires practice, despite its unsophisticated appearance. e technique depends upon uniform actions by the technician from sample to sample to produce consistent packing of soil into the scoop. To check scooping consistency, repeatedly scoop soil from one sample and check the weight of each scoop. If the procedure is being carried out properly, the weights should be uniform. e average weights for various scoop volumes are given in Table 2. Scoop weights will vary from soil to soil depending on dierences in soil texture. Procedure 1. Dip the scoop into the center of the soil sample and ll the scoop with a twisting motion so that extra soil is

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6 mounded above the rim of the scoop. Do not press the scoop or force the soil against the side of the container.8 2. Strike the handle near the scoop three times with a plastic rod to settle soil particles. 3. Level the scoop with the plastic rod. Strike o all excess soil above the rim of the scoop in a single stroke so that the soil is not compacted into the scoop.Soil pH (1:2 v/v)is procedure uses a 20-cm3 soil scoop and 40 mL of pure water to obtain a 1:2 soil-to-water ratio. Sample pH may be aected by contaminated water, by microbial activity or by changes in solution chemistry if samples are allowed to sit longer than recommended prior to ananlyisis. Other common errors associated with this method include improper scooping technique and improper electrode use. e pH meter should be calibrated on a daily basis using commercially available buer solutions. Fresh aliquots of buer solution must be used each day. Standard Solutions Obtain commercially available standard buer solutions of pH 4.00, 7.00, and 10.00. Procedure 1. Standardize pH meter according to manufacturers directions. 2. Scoop 20-cm3 of soil and pour into a 90-mL (3-oz) plastic cup. 3. Add 40 mL of pure water to each cup using an automatic pipette. Stir with a glass rod and let the sample stand for 30 min, but not more than 2 hours. Stir sample again just prior to analysis. 4. Continue stirring sample and measure soil pH. 5. Record pH to the nearest 0.1 pH unit (XX.X).Adams-Evans Buer pH1is procedure uses a 15-cm3 soil scoop and 30 mL of Adams-Evans Buer solution for a soil to solution ratio of 1:2. Errors associated with this method include improper standardization of the Adams-Evans buer solution, improper use of the electrode, and delays in analysis beyond the recommended equilibration period. Reagents Reagents used in this procedure are listed in Table 3. Solutions e Adams-Evans Buer solution is prepared as follows: 1. Weigh 180 g of the p-Nitrophenol into a 6-L Erlenmeyer ask containing about 4 L of pure water. Add 135 g of the Boric Acid and dissolve. Use low heat to dissolve, if necessary. 2. Dissolve 95 g of the Potassium Hydroxide in approximately 200 mL of pure water contained in a 500-mL beaker. 3. Using a 20-L carboy calibrated at 18-L volume, add 6 L of pure water. Weigh 666 g of the Potassium Chloride and transfer to the carboy. 4. Combine all solutions by quantitatively transferring the p-Nitrophenol/Boric Acid solution, followed by the Potassium Hydroxide solution, to the carboy containing the Potassium Chloride solution. Bring to 18-L nal volume with pure water. Adjust the solution pH to 8.00 0.02 with small amounts of Potassium Hydroxide (for raising pH) or Hydrochloric Acid (for lowering pH), as needed. Let stand overnight and check pH.Table 2. Applications for scoops used at the Extension Soil Testing Laboratory. Scoop Volume (cm3) Approx. soil weight (g) Application 4 5 Mehlich-1 extraction 15 20 Adams-Evans Buer pH 20 25 Soil pH and electrical conductivity Note: Soil for the AB-DTPA extraction procedure is weighed only.Table 3. List of reagents used in Adams-Evans Buer pH procedure. Name Formula F.W .* p-Nitrophenol NO2C6H4OH 139.11 Boric Acid H3BO361.8 Potassium Hydroxide KOH 56.1 Potassium Chloride KCl 74.6 Formula weight in grams.

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7 Alternately, a commercially prepared Adams-Evans buer solution can be purchased and prepared as per the manufactures instructions. Procedure 1. Standardize the pH meter according to the manufacturers directions. 2. Measure the pH of the Adams-Evans Buer Solution to insure that the solution reads 8.00 0.02. 3. Scoop a 15-cm3 volume of soil into a 50-mL beaker. 4. Add 30 mL of the buer solution using an automatic pipette. 5. Stir for 4 min on a mechanical stirrer. Timing of this test is critical. e reaction starts when the buer solution is added to the sample. 6. Immediately aer stirring, measure the solution pH. Excessive delays will result in low bias in the buer-pH readings. 7. Record pH to the nearest 0.01 pH unit (XX.XX).Mehlich-1 Extractable P, K, Ca, Mg, Cu, Mn, and Zn21is procedure uses a 4-cm3 scoop (approximately 5 g of mineral soil) and 20 mL of the Mehlich-1 extraction solution to provide a soil to solution ratio of 1:4. Once the extraction is complete, the sample is ltered through Whatman 42 lter paper or its equivalent. e ltered solution should be analyzed as soon as possible following the extraction procedure. If refrigeration is not available, the sample must be analyzed the same day as it is extracted. With refrigeration, samples should be analyzed within ve days. Common errors associated with this method include mistakes in sample shake time, delayed ltration, and reagent, lter paper or cup contamination. Reagents A list of reagents is found in Table 4. Solutions Mehlich-1 Extracting Solution (0.0125M H2SO4 and 0.05M HCl) Pour approximately 16 L of pure water into a 20-L plastic carboy. Slowly add 13.9 mL of concentrated Sulfuric acid and 83 mL of concentrated Hydrochloric Acid. Dilute to 20-L nal volume with pure water and mix. Procedure 1. Scoop 4 cm3 of mineral soil and transfer into a 50-mL extracting bottle. 2. Dispense 20 mL of Mehlich-1 extracting solution into each extracting bottle using an automatic pipette. 3. Shake each sample for 5 min on a reciprocating shaker and then lter through lter paper (11 cm Rogers Custom Lab 620, Whatman No. 42 or equivalent) into a plastic cup. 4. Transfer the ltrate to an appropriate vial for analysis. If samples are not to be analyzed immediately, they should be capped or otherwise covered. Sample solutions are stable for 5 days, if refrigerated. 5. e ltrate may be analyzed for nutrients using either ICP (Inductively Coupled Plasma Spectrometer, EPA Method 200.7) or AAS (Atomic Absorption/ame emission Spectrophotometer, EPA Method 200.0) in combination with colorimetric analysis for phosphorus determination (EPA Method 365.2). 6. Instrument readings are recorded in mg L-1 solution concentration. Final results are reported in mg kg-1 -dry weight (ppm) calculated as follows: Soil Organic MatterA. WALKLEYBLACK METHOD19e Walkley Black (WB) method used for determining Soil Organic Matter (SOM) involves a known volume of acidic dichromate solution reacting with an aliquot of soil in order to oxidize the SOM. e oxidation step is then followed by titration of the excess dichromate solution with ferrous sulfate. e SOM is calculated using the dierence between the total volume of dichromate added and the volume titrated aer reaction. Problems associated with this procedure include excessive organic matter in the soil (the limit for this procedure is approximately 6%) and dicult end point determination (dark-colored soil Figure 1. Table 4. List of reagents used in Mehlich-1 Extractable P, K, Ca, Mg, Cu, Mn, and Zn procedure. Name Formula Conc. Sulfuric Acid H2SO418M Hydrochloric Acid HCl 12.1M

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8 solution). e use of a lighted stir plate can be of assistance in the end-point determination. e WB procedure also results in production of chromate, which is categorized as a hazardous chemical. Studies are currently ongoing to develop an alternative method to WB to avoid production any hazardous waste. Reagents Reagents used in this procedure are listed in Table 5. Solutions 0.16M Potassium dichromate Dissolve 98.08 g of oven-dry/desiccated Potassium dichromate in approximately 1500 mL of pure water and dilute to 2 L. Aer preparation of this solution, transfer to a clean glass bottle for use with a repipetter. Do not mix old Potassium dichromate solution with the new solution. 1.0M Ferrous Sulfate Dissolve 556.04 g of Ferrous Sulfate in approximately 1500 mL of pure water. Carefully add 30 mL of concentrated Sulfuric Acid, mix, cool, and dilute to 2 L. Aer preparation, this solution may be transferred to a clean 8-L plastic carboy. Do not mix old Ferrous Sulfate solution with the new solution. e tubing, stopcock, and attachments to the burette should be rinsed three times with new Ferrous Sulfate solution before titrating any blanks or samples. Prepare a new solution every 30 days. Procedure 1. Weigh 1.0 g of mineral soil into a 250-mL wide mouth graduated Erlenmeyer ask. 2. Titrate two blank samples (no soil) before proceeding with any unknown samples in order to standardize the Ferrous Sulfate solution. If the dierence between the two blanks is not within 0.2 mL of Ferrous Sulfate solution, clean the burette and associated tubing. Reanalyze two more blanks to determine if the problem has been eliminated. 3. Pipet 10.0 mL of the Potassium dichromate solution into each ask containing unknown soil and mix by carefully rotating the ask to wet all of the soil. 4. Under a fume hood, carefully add 20 mL of concentrated Sulfuric Acid to each ask and mix gently. 5. Allow asks to stand for 5 min under the fume hood. 6. Add pure water to each ask such that the nal volume is approximately 125-mL. Mix by swirling gently. 7. Add 5 or 6 drops of Phenanthroline complex and immediately titrate with the Ferrous Sulfate solution. As the titration proceeds, the solution will take on a green color that will change abruptly to reddish-brown when the endpoint of the titration is reached. 8. Record each volumetric reading to the nearest X.X mL. 9. e % OM is calculated as follows: (1 S / B) x 10 x 0.68 = organic matter (%) of sample where: S = Volume of Ferrous Sulfate solution required to titrate the sample, in mL. B = Average Volume of Ferrous Sulfate solution required to titrate the two blanks, in mL. 10 = conversion factor for units. 0.68 = a factor derived from the conversion of % organic carbon to % organic matter (1.724), the fraction of Organic Carbon oxidized to CO2 (0.76) and the milliequivalent weight of carbon (0.003 g).B L OSSONIGNITION METHOD11e Loss-on-Ignition (LOI) organic matter determination is used for analyzing soil samples in which the organic matter content is greater than 6%. is procedure involves exposing the soil sample to high temperatures in an oxygen atmosphere in order to convert any organic carbon compounds to carbon dioxide, which is then lost to the atmosphere. e dierence between the soil dry weight and the weight of the sample aer ignition is then used to calculate the amount of organic matter in the sample. is Table 5. List of reagents used in the Walkley-Black Method. Name Formula F.W ./Conc.* Potassium dichromate K2Cr2O7294.19 Ferrous Sulfate FeSO4 7H2O 278.02 Sulfuric Acid H2SO418M 1, 10-Phenanthroline Ferrous Sulfate complex Formula weights in grams or concentration in Molarity.

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9 procedure has been reported to be consistent with even lower SOM levels (<6%) such as sandy soils in Florida. Studies are on-going to determine the suitability and for possible replacement method for WB procedure. Procedure 1. Label and accurately weigh (to 4 decimal places) an oven dried 50 mL Pyrex beaker. 2. Add approximately 5-6 g of soil to the beaker. 3. Place sample in the oven at a constant temperature of 105C and allow sample to dry for a minimum of 2 hrs. 4. Remove sample from the oven at the end of two hours and place immediately into a dessicator to cool. Allow sample to cool to room temperature (approximately 30 minutes) and then accurately weigh sample and beaker 5. Aer weighing, place sample into a mue furnace and heat at 350C for a minimum of 2 hours. Do not exceed this temperature as CaCO3 may be converted to CO2 and cause erroneous results. 6. At the end of the heating period, allow samples to cool slightly and then transfer immediately to a dessicator. Allow samples to cool to room temperature in the dessicator. 7. Aer samples reach room temperature, remove from the dessicator and accurately weigh sample and beaker. 8. e % OM is calculated as follows:Electrical Conductivity (1:2 Soil:W ater)e ESTL oers a test for soil Electrical Conductivity (EC) by which a value for the Soluble Salts in the soil content can be estimated. In this test, 20 cm3 of a mineral soil are mixed with 40 mL of pure water resulting in a soil to water ratio of 1:2. e resultant suspension is allowed to equilibrate for 4 hours in order to allow slowly-soluble constituents to approach solution equilibrium. e suspension is then ltered and the electrical conductivity is immediately determined. Sources of error include improper instrument calibration and incorrect equilibration times. Standards A solution of 0.005M KCl has an electrical conductivity of 720 deciSiemens per meter (dS/m) at 25C. Alternately, a commercially available NIST traceable reference solution of the appropriate concentration and conductivity may be used. Procedure 1. Weigh 20 g of soil and transfer to a plastic 90-mL (3-oz.) cup. 2. Add 40 mL of pure water to each cup. Stir and allow the suspension to stand for 4 hours. 3. At the end of 4 hours, stir the suspension to create slurry. Immediately lter through an 11 cm lter paper (Rogers Custom Lab 620, Whatman No. 42 or equivalent). Collect the ltrate in a 90-mL (3-oz.) plastic cup. 4. Using the reference standard, calibrate the Electrical Conductivity Meter according to manufacturers directions. Measure the EC of the solution and report results to one decimal place in dS/m. While the ESTL reports all electrical conductivity measurements in dS/m, many clients are accustomed to values given in ppm soluble salts. e calculation to convert EC to soluble salts is given below along with the formula for conversion of EC to salt index. ere are many inaccurate assumptions included in these conversions and clients are encouraged to adapt to the more precise and widelyaccepted terminology of EC in dS/m. EC in dS/cm x 700 = soluble salts in ppm Salt index = EC (as direct 2:1 reading) x 8Analytical Procedures for Container MediaW ater-Extractable P, K, Ca, Mg, NO-N, pH, and Electrical Conductivitye entire sample (or that portion of the sample that nearly lls a 600-mL plastic beaker) is used for this diagnostic test.15 De-ionized distilled water is added to the sample to the point of saturation. e sample is then ltered under Figure 2.

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10 vacuum and the ltrate is analyzed. Underor overestimating the point of sample saturation will introduce some error. If possible, the analysis of the ltrate should be completed on the same day that the extract is prepared. If unable to complete the analysis on the same day, the sample may be refrigerated but analysis must be completed within 48 hours or the sample must be re-extracted. Extraction Procedure 1. Place the entire sample (or a representative sample aliquot) into a 600-mL plastic beaker and conservatively add pure water to the point of complete saturation. At this point, the surface of the mix should glisten, but no water should puddle on the surface. Mix well with a spatula, and let stand for 2 hours. 2. Place a 9-cm, Whatman No.1 lter paper into a large Buchner funnel. Wet the lter paper with approximately 2 mL of pure water and transfer the saturated media onto the lter. 3. Place the funnel under a vacuum and leave until sucient solution is extracted from media to complete the necessary tests. Transfer the ltrate to an appropriate container for analysis. pH Standardize the pH meter according to manufacturers directions and then determine the pH of an aliquot of the ltrate. Results are reported to one decimal place. EC Standardize the EC meter according to manufacturers directions and then determine the electrical conductivity of an aliquot of the ltrate. Report results to one decimal place in dS/m. NO3-N e ESTL uses semi-automated colorimetric analysis (EPA Method 353.2) to determine NO3-N in the media extract. e instrument (Technicon II Auto-Analyzer or equivalent) is set up and calibrated as per manufacturers directions. Instrument results are reported to one decimal place as mg L-1 NO3-N. Water-Extractable P, K, Ca, Mg 1. e ltrate may be analyzed for all other nutrients using either ICP or AAS in combination with colorimetric analysis for phosphorus determination. 2. Results for P, K, Ca, and Mg are reported in mg L-1 (ppm). Analytical Procedures for Calcareous SoilsAmmonium Bicarbonate-DTPA (AB -DTPA) Extractable Pe AB-DTPA extractant works well on soils with high and neutral pH.6, 20 It is being calibrated for the marl and Rockdale soils of southern Florida. Previous studies in Florida have shown that this procedure can be interpreted only for P test results. erefore, results for other nutrients are included in the report. It is not suitable for determination of Ca or Mg. is extraction procedure is used only on soils that have a pH of 7.4 and above. Solutions AB-DTPA Extracting Solution Prepare this solution under a fume hood to avoid possible contact with vapors. Add approximately 700 mL of pure water into a 1-L volumetric ask. Add 0.5 mL (10 drops) of concentrated Ammonium Hydroxide. Dissolve 1.97 g of DTPA in this solution. is dissolution may take several hours. Aer the DTPA has been dissolved, add 79.06 g of Ammonium Bicarbonate, mix, and dilute to 1 L. Adjust to pH 7.6 using concentrated Hydrochloric Acid (for lowering pH) or Ammonium Hydroxide (for raising pH). Prepare this solution daily, as it is pH unstable. Reagents Reagents used in this procedure are listed in Table 6.Table 6. List of Reagents used in Ammonium BircarbonateDTPA (AB-DTPA) Extractable P. Name Formula F.W ./Conc.* Ammonium Hydroxide, concentrated NH4OH 14.8M DPTA (Baker Cat. E 376-.07) C14H23N3O10393.35 Ammonium Bicarbonate NH4HCO379.06 Hydrochloric Acid, concentrated HCl 12.1M Nitric Acid, concentrated HNO315.8M Formula weights in grams or concentration in Molarity.

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11 Analytical Procedures for W ater22e following procedure lists the various subsections that deal with water analyses. To preclude errors introduced by microbial activity, water samples should be analyzed as soon as possible aer sampling. Sample containers should be lled completely with no headspace above the sample surface and should only be opened immediately prior to analysis, since exposure to air can cause changes in the chemical equilibrium of the sample. pH Standardize the pH meter according to manufacturers directions and then determine the pH of an aliquot of the sample. Results are reported to one decimal place. EC Standardize the EC meter according to manufacturers directions and then determine the electrical conductivity of an aliquot of the sample. Report results to one decimal place in dS m-1. Metals Ca, Mg, Fe, Mn, and Na may be analyzed by either ICP (EPA Method 200.7) or AAS (EPA Method 200.0). Cl-e ESTL uses semi-automated colorimetric analysis (EPA Method 325.2) to determine chloride in waters. e instrument (Alpkem Auto-Analyzer) is set up and calibrated as per manufacturers directions. Instrument results are reported to one decimal place as mg L-1 of Clconcentration. Carbonate Equivalent A 50-mL aliquot of water sample is titrated against a standardized hydrochloric acid solution to a pH of 4.0. e volume of acid required is then used to calculate the car bonate and bicarbonate equivalence of the sample. While very low levels of bicarbonates may be present in solution below pH 7.0, these levels are assumed to pose no problems agriculturally. e volume of acid required to titrate the sample to the desired pH is assumed to be entirely due to the neutralization of carbonates and bicarbonates. e most common error associated with this method is degradation of the THAM buer solution. e THAM titrant should be replaced at least once every week. Only newly-opened water samples should be analyzed since changes in carbonate and bicarbonate levels can occur upon exposure to the air. Reagents Reagents used in this procedure are listed in Table 7.Analytical Procedures for PlantsDigestion Procedure for the Determination of Ca, Mg, P, K, Na, Mn, Cu, Fe, Zn, and B in Plant Tissueis digestion procedure has been developed with a suciently large dilution factor to allow accurate determination of macronutrients and secondary nutrients that are oen in relatively high concentrations within the plant. is procedure may not be suitable for certain micronutrient or heavy metal analyses because of the selected dilution factor. If the expected micronutrient concentration in the plant is less than 5 mg kg-1, the element may be diluted below the detection limit of the method. Selection of mue furnace temperature and its control directly aect the analytical results of this process.7 e use of borosilicate glassware can be a source of B and Si contamination. Reagents Reagents used in this procedure are listed in Table 8. Solutions 6.0M Hydrochloric Acid Add approximately 4 L of pure water into a plastic carboy calibrated at 8-L. Under a fume hood, slowly bring to 8-L volume with concentrated Hydrochloric Acid, and mix using a magnetic stir bar with stirrer. Alternately, any repipette container to which equal volumes of pure water Table 7. Reagents used in water analysis procedure. Name Formula F.W/Conc.* THAM C4H11NO3121.14 Hydrochloric Acid HCl 12.1 M Formula weight in grams or concentration in Molarity.Table 8. Reagents used in digestion procedure. Name Formula Conc.* Hydrochloric Acid HCl 12.1 Concentration in Molarity.

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12 and concentrated Hydrochloric Acid have been added is sucient. Procedure 1) Weigh 1.00 g of oven-dry, ground plant tissue into a 50-mL beaker and place in a mue furnance. If boron is requested, use high-form, glazed, porcelain crucibles. a) Duplicate every 20th sample to measure the precision of the test. b) Digest at least one external or internal plant tissue standard sample with each digestion. 2) Place samples in mue furnace. Ensure temperature controls are set to 500C and turn the furnace on. 3) Once the internal temperature of the oven reaches 500C, allow samples to ash for a minimum of 5 hours (ashing time should never exceed 16 hours). Shut oven o and allow oven to cool. 4) Once the furnace temperature is below 200C, carefully open the furnace door to expedite the cooling process. CAUTION: e internal temperature of the mue furnace should be below 200C before opening the furnace door so that the samples are not ignited or disturbed by the rapid inux of air. 5) Once samples reach room temperature, remove them from the oven and moisten the ash by adding approximately 5 drops of pure water using an eyedropper followed by the addition of 5 mL of 6 M Hydrochloric Acid. Let this suspension stand for at least 30 minutes before proceeding. 6) With the aid of a funnel, quantitatively transfer the solution containing the ash to a 50-mL volumetric ask. Rinse beaker with pure water and transfer the rinsate to the ask also. Repeat the rinse steps a second time and then bring to volume with pure water. Mix thoroughly. 7) Transfer an aliquot of the sample to an appropriate container for analysis. If ltration is required, use a Rogers Custom Lab 620 (Whatman No. 42 equivalent) lter paper. 8) e sample solution may be analyzed using either ICP or AAS in combination with colorimetric analysis for P determination. 9) Sample results are reported in mg kg-1 -plant dry weight for B, Cu, Fe, Mn and Zn and in %-plant dry weight for P, K, Ca and Mg.Total Kjeldahl N itrogen (TKN) in Plant Tissuee TKN method is used to analyze for nitrogen in organic materials. Most organically-bound nitrogen (such as that found amines, proteins, etc.) as well as any nitrogen in the form of ammonium ion can be determined using this method. In general, nitrates, nitrites, and some cyclic nitrogenous compounds resistant to digestion are not determined using this method.9 e Kjeldahl digestion process produces a highly acidic solution and is therefore not recommended for nitrate analysis, as it will cause damage to the instrument. Reagents Reagents used in this procedure are listed in Table 9. Digestion Procedure for Plant Samples 1) Weigh 0.200 +/0.005 g of plant tissue onto a nitrogenfree weighing paper. Carefully fold the paper containing the sample and place into a TKN digestion tube (25 mm x 200 mm; Fisher cat. No. 14-960 F). a) Duplicate every 20th sample to measure the precision of the test. b) Digest at least one external or internal plant tissue standard sample with each digestion. 2) Scoop approximately 2.0 g of Kjeldahl digestion mixture (this mixture may be obtained from Pope Inc., Dallas, TX 75221) and transfer to the bottom of the digestion tube with the aid of a long stem funnel. 3) Carefully add 5 mL of concentrated Sulfuric Acid to each tube. 4) Start the digestion by placing samples in a 250C preheated aluminum block digester for 1 hour.Table 9. Reagents used in TKN procedure. Name Formula F.W ./ Conc.* Kjeldahl mixture No. 2 (10 g K2SO4 + 0.30 g CuSO4) Sulfuric Acid H2SO418M Formula weight in grams or concentration in Molarity or percent.

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13 5) Aer 1 hour at 250C, place glass funnels on all tubes and increase the digestion temperature to 365C. Digest samples for an additional 2.5 to 3.0 hours. 6) Aer digestion is complete, allow block to cool. When tubes are cool enough to handle, remove from the digestion block and place into a wire rack to cool to room temperature. 7) Using a wash bottle, add 5 to 10 mL of pure water washing the sides of each tube. Mix using a Vortex mixer. 8) With the aid of a funnel, transfer the contents of the tube into a 100-mL volumetric ask. Rinse the digestion tube several times with water and add the rinsates to the volumetric ask. Dilute to volume and mix well. 9) Allow ask to cool, dilute to volume, cover with paralm, and mix thoroughly. 10) Transfer an aliquot of the sample to an appropriate container for analysis. If ltration is required to remove particulates, use a Rogers Custom Lab 720 (Whatman No. 2 equivalent) lter paper. 11) e ESTL uses semi-automated colorimetric analysis (EPA Method 351.2) to determine nitrogen in TKN digestates. e instrument (Alpkem autoanalyzer) is set up and calibrated as per manufacturers directions. Instrument calibration standards and quality control samples should be digested in the same manner as the samples. Instrument results are reported to one decimal in mg L-1. Final results are reported as %N-plant dry weight and are converted from mg L-1 using the following equation: Observed value in mg L-1 x (100 mL/0.2 g)/10,000 = % TKNQuality ControlOperations within an analytical laboratory must address quality control in order to maintain both accuracy and precision. is dedication to quality control must begin with detailed procedures and address all steps in which inaccuracies can be introduced. Eorts to control inaccuracies are directed at three levels: quantitative chemical techniques, instrument monitoring, and managerial process inspection. e ESTLs Quality Control Plan addresses each of these areas assuring that the laboratory produces high quality and reliable data. Details concerning the ESTLs Quality control procedures can be obtained by contacting the laboratory director or the manager.Laboratory Safetye following is a general list of safety requirements that should be followed by any person handling laboratory chemicals or working in a chemical laboratory: 1. Always wear an acid/base resistant laboratory coat. 2. Always wear goggles/eyeglasses as minimum eye protection. 3. Always wear appropriate gloves when handling chemicals. 4. Never work alone in a chemical laboratory. 5. Never eat or drink in the laboratory area. 6. Do not store food in chemical refrigerators. 7. If working with an unfamiliar chemical, always read the label and check the MSDS before proceeding. 8. Always transport concentrated acids/bases or other dangerous chemicals in a rubberized safety bucket. 9. Know where the nearest re extinguisher and eye wash station are located. 10. Know the location of the nearest phone and how to reach 911 or the local emergency number. 11. Do not pipet chemicals by mouth. 12. Wear appropriate laboratory clothing including closedtoe shoes and long pants. Tie back long hair. References1. Adams, F., and C.E. Evans. 1962. A rapid method for measuring lime requirement of red-yellow podzolic soils. Soil Sci. Soc. Am. Proc. 26:355-357. 2. Allison, L.E. 1965. Organic carbon. In C.A. Black, D.D. Evans, L.E. Ensminger, J.L. White, and F.E. Clark (ed). Methods of Soil Analysis, Part 2. pp. 1367-1378. 3. Haman, D.Z., and D.B. Bottcher. 1986. Home water quality and safety. Circ 703, UF/IFAS Coop. Extn. Ser., pp. 12. 4. Haman, D.Z., G.A. Clark, and A.G. Smajstrla. 1989. Irrigation of lawns and gardens. Circ. 825, UF/UFAS Coop. Extn. Ser., pp. 28.

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14 5. Hochmuth, G.J. and E.A. Hanlon. 1998. Calculating fertilizer rates for vegetable crops grown in raised-bed cultural systems in Florida. Fact Sheet HS743, UF/ IFAS, Coop. Extn. Ser., pp. 4. 6. Hanlon, E.A., and G.V. Johnson. 1984. Bray/Kurtz, Mehlich III, AB/D and ammonium acetate extractions of P, K and Mg in four Oklahoma Soils. Commun. Soil Sci. Plant Anal. 15(3):277-294. 7. Isaac, R.A., and J.B. Jones, Jr. 1972. Eects of various dry ashing temperatures on the determination of 13 nutrient elements in ve plant tissues. Commun. Soil Sci. Plant Anal. 3:261-269. 8. Jones, Jr., J.B. (ed.). 1992. Handbook on reference methods for soil testing. Council on Soil Testing and Plant Analysis, Athens, GA. 9. Jones, J.B., Jr., and V.W. Case. 1991. Sampling, handling, and analyzing plant tissue samples. pp. 389-415. In R.L. Westerman (ed.) Soil testing and plant analysis. 3rd edition. Book Series No. 3. Soil Sci. Soc. Amer., Madison, WI. 10. Kidder, G., and E.A. Hanlon. 1998. Neutralizing excess bicarbonates from irrigation water. Soil & Water Science Fact Sheet 142, Coop. Ext. Serv., UF/IFAS, pp.6. 11. Magdo, F.R., M.A. Tabatabai, and E.D. Hanlon. 1996. Soil Organic Matter: Analysis and Interpretation. Soil Sci. Spec. Pub. No. 46:21-31. 12. Mylavarapu, R., T. Yeager and J.M. Bartos. 2001. UF/IFAS Nutrient Management Series: Container Media Nutrient Test Interpretations. SL 180, Soil & Water Science, Coop. Extension Service, pp3. 13. Mylavarapu, Rao S., J.M. Bartos. 1999. Landscape and Vegetable Garden Test Information Sheet. SL-136, Soil & Water Science, Cooperative Extension Service, IFAS. pp3. 14. Mylavarapu, Rao S., J.M. Bartos. 1999. Producer Soil Test Information Sheet. SL-135, Soil & Water Science, Cooperative Extension Service, IFAS. pp3. 15. Mylavarapu, Rao S., J.M. Bartos. 1999. Container Media Test Information Sheet. SL-134, Soil & Water Science, Cooperative Extension Service, IFAS. pp3. 16. Mylavarapu, Rao S., J.M. Bartos. 1999. Water Test Information Sheet. SL-133, Soil & Water Science, Cooperative Extension Service, IFAS. pp3. 17. Mylavarapu, Rao S., J.M. Bartos. 1999. Pine Nursery Soil Test Information Sheet. SL-132, Soil & Water Science, Cooperative Extension Service, IFAS. p2. 18. Mylavarapu, Rao S., J.M. Bartos. 1999. Plant Tissue Test Information Sheet. SL-131, Soil & Water Science, Cooperative Extension Service, IFAS. p2. 19. Nelson, D.W., and L.E. Sommers. 1982. Total carbon, organic carbon, and organic matter. pp. 539579. In A.L. Page, R.H. Miller, and D.R. Keeney (ed.). Methods of soil analysis: Part 2. 2nd. Edition. Book Series No. 9. Amer. Soc. Agron. Madison, WI. 20. Soltanpour, P.N. 1991. Determination of nutrient availability and elemental toxicity by AB-DTPA soil and ICPS. In B.A. Stewart (ed.) Advances in Soil Science. 16:165-190. 21. Southern Region Information and Exchange Group on Soil Testing and Plant Analyses (SRIEG 18). 1983. Reference soil test methods for the Southern region of the United States. Southern Cooperative Series Bull. 289. Univ. of Georgia, Athens, GA. 22. U.S. Environmental Protection Agency. 1984. Methods for Chemical Analysis of Water and Wastes, March, EPA600/4-79-020, Environmental Monitoring and Support Laboratory, Oce of Research and Development, Cincinnati, OH 45286.Acknowledgementse authors would like to express their appreciation to the following people for their technical assistance in the preparation of this manual: J. DeVore, L. Moon, P. Reynolds, S. Robinson, C. Simmons, P.Straub, A. Tyer, and B. Welch.