Pressurized Hot Water: an Alternative Method of Nutrient Extraction and Subsequent Analysis for Use in Small-Scale Agriculture

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Theses and Dissertations

2004-07-13

Pressurized Hot Water: An Alternative Method of Nutrient Extraction and Subsequent Analysis for Use in Small-Scale Agriculture

Kristy Susanne Crane Brigham Young University - Provo

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BYU ScholarsArchive Citation Crane, Kristy Susanne, "Pressurized Hot Water: An Alternative Method of Nutrient Extraction and Subsequent Analysis for Use in Small-Scale Agriculture" (2004). Theses and Dissertations. 541. https://scholarsarchive.byu.edu/etd/541

This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. PRESSURIZED HOT WATER: AN ALTERNATIVE METHOD OF

NUTRIENT EXTRACTION AND SUBSEQUENT ANALYSIS

FOR USE IN SMALL-SCALE AGRICULTURE

Kristy S. Crane

A thesis submitted to the faculty of

Brigham Young University

in partial fulfillment of the requirements for the degree of

Master of Science

Department of Plant and Animal Sciences

Brigham Young University

August 2004 BRIGHAM YOUNG UNIVERSITY

GRADUATE COMMITTEE APPROVAL

of a thesis submitted by

Kristy S. Crane

This thesis has been read by each member of the following graduate committee and by majority vote has been found satisfactory.

______Date Phil S. Allen, Chair

______Date Bruce L. Webb

______Date Von D. Jolley

______Date Sheldon D. Nelson

BRIGHAM YOUNG UNIVERSITY

As chair of the candidate’s graduate committee, I have read the thesis of Kristy S. Crane in its final form and have found that (1) its format, citations, and bibliographical style are consistent and acceptable and fulfill university and department style requirements; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the graduate committee and is ready for submission to the university library.

______Date Phil S. Allen Chair, Graduate Committee

Accepted for the Department

______Sheldon D. Nelson Department Chair

Accepted for the College

______Kent D. Crookston Dean, College of Biology and Agriculture

ABSTRACT

PRESSURIZED HOT WATER: AN ALTERNATIVE METHOD OF

ELEMENTAL EXTRACTION AND ANALYSIS FOR USE IN

SMALL-SCALE AGRICULTURE

Kristy S. Crane

Department of Plant and Animal Sciences

Master of Science

Soil analysis for small acreage farms in developing countries is often inconvenient and prohibitively expensive using current procedures, yet the information gained from these soil tests could have economical and environmental benefits. The pressurized hot-water (PHW) extraction coupled with colorimetric or turbidimetric analyses shows promise as an alternative to current procedures. Accepted methods of colorimetric analysis for NO3-N and P exist but an alternative method to atomic absorption spectrometry for K analysis is needed.

Of the many possible methods to quantify K, tests performed in the Brigham

Young University (BYU) laboratory found sodium tetraphenylborate to be unsatisfactory and sodium cobaltinitrite to be simple, inexpensive and consistent.

Test results on 38 arid-zone soils from the Western United States encourage adoption of sodium cobaltinitrite as an acceptable procedure for K quantification in conjunction with PHW extraction [r2 of 0.90 compared to atomic absorption

(AA) analysis and r2 of 0.67 compared to values extracted with ammonium acetate and measured with AA].

Two hundred twenty-eight soils varying widely in soil parameters from different areas of Guatemala and Morocco were analyzed for NO3-N, P and K using standard methods (water-CTA, Olsen-molybdic acid and ammonium acetate- atomic absorption, respectively) and correlated to values obtained from the PHW extraction coupled with colorimetric or turbidimetric analytical procedures at

BYU. The correlations between the values for these tests were good (r2 of 0.96,

0.71 and 0.52 for NO3-N, P, and K; respectively). Soils from each country were simultaneously analyzed for NO3-N and P in laboratories in Guatemala and

Morocco and these results correlated with those from BYU. Correlations between

BYU values and those from other laboratories gave generally positive results with the Guatemala laboratory showing overall closer correlation with BYU values than data from the Morocco laboratory. The results suggest that the PHW extraction and accompanying simplified analyses present a viable, less expensive alternative to current soil nutrient extraction and analysis procedures for the range of soils tested from Morocco and Guatemala. ACKNOWLEDGEMENTS

Grateful acknowledgement is given for the guidance, expertise and encouragement of my faculty advisors Dr. Phil Allen and Bruce Webb. Their love of adventure and enthusiasm for science greatly shaped this project and my life.

Appreciation is also due Dr. Von Jolley and Dr. Sheldon Nelson for their advice and contributions during the revision phase of this project. Particular thanks are due the Benson Food and Agriculture Institute, the Potash and Phosphate Institute and the Department of Plant and Animal Science at Brigham Young University for financial contributions and acknowledgment must be given to Mustapha

Naimi and Hicham at the Institute of Agronomy Hassan II and Gadid Artereaga and Rudolfo Chicas at the University of the East at Cunori. Acknowledgement is in order for Scott Allen, Amanda Silvester, Heather Hunsaker, Darrin Roberts,

Jenny Christopherson and Angela Demeester for many hours of laboratory analysis and to Amanda Shiffler and Necia Bair for camaraderie and understanding. I acknowledge my father, Richard Gibson, who introduced me to the joys of plant science as a child and my mother, Lexia Gibson, for her encouragement. I sincerely appreciate my husband, Peter Crane, for the love and support that was crucial to the completion of this project. Finally, I would like to thank my Heavenly Father by whom all things are possible. TABLE OF CONTENTS

Title Page…………………………………………………………………………..i

Abstract…………………………………………………………………………...iv

Acknowledgements………………………………………………………….……vi

Table of Contents………………………………………………………………...vii

List of Tables…………………………………………………………………...... x

List of Figures………………………………………………………………….....xi

Manuscript 1: A Rapid Turbidimetric Potassium Test Modified for Use with the

Pressurized Hot-Water (PHW) Extraction ……………………………………1

Abstract…………………………………………………………………....1

Introduction………………………………………………………………..1

Method Development……………………………………………………...2

Sodium Tetraphenylborate………………………………………...2

Sodium Cobaltinitrite……………………………………………...3

Test of Method……….……………………………………………………5

Results and Discussion…………………………………………………....6

Conclusions………………………………………………………….…….6

References…………………………………………………………………6

Table 1…………………………………………………………………….9

Table 2…………………………………………………………………...10

Table 3…………………………………………………………………...11

Table 4…………………………………………………………………...12

vii Figure 1…………………………………………………………………..13

Figure 2…………………………………………………………………..14

Figure 3…………………………………………………………………..15

Figure 4…………………………………………………………………..16

Manuscript 2: A Simplified Soil Analysis Procedure for Use in Small-Scale

Agriculture…………………………………………………………………...17

Abstract…………………………………………………………………..17

Introduction………………………………………………………………17

General…………………………………………………………...17

Phosphorus Extraction…………………………………………...19

Experimental Design……………………………………………………..20

Methods and Materials…………………………………………………...21

PHW Extraction and Analysis…………………………………...21

Standard Methods………………………………………………..21

Analyses Completed at BYU…………………………….21

Analyses Completed in Morocco………………………...21

Analyses Completed in Guatemala………………………21

Results and Discussion…………………………………………………..22

Comparison of Methods………………………………………….22

Comparisons among Laboratories……………………………….23

Conclusions………………………………………………………………24

Acknowledgements………………………………………………………25

References………………………………………………………………..25

viii Table 1…………………………………………………………………...27

Table 2…………………………………………………………………...28

Table 3…………………………………………………………………...29

Table 4…………………………………………………………………...30

Table 5…………………………………………………………………...31

Table 6…………………………………………………………………...32

Figure 1…………………………………………………………………..33

Figure 2…………………………………………………………………..34

Figure 3…………………………………………………………………..35

Figure 4…………………………………………………………………..36

Figure 5………………………………………………………………..…37

Figure 6…………………………………………………………………..38

Appendix I: Introduction and Literature Review………………………………...39

Appendix II: A Summary Table of Spectrophotometric Methods of Analysis for

Soil Nutrients………………………………………………………………...44

Appendix III: Methods and Materials…………………………………...…….....70

Appendix IV: Project Data…………………………………………………….....80

ix LIST OF TABLES

Manuscript 1: A Rapid Turbidimetric Potassium Test Modified for Use with the Pressurized Hot-Water Extraction

Table 1. Transmittance (mean of six readings ± standard deviation) for standard solutions (µg g-1) using the sodium tetraphenylborate method. The standard deviations prove that the test is not sensitive enough at agronomically important soil K values. Values were read on a spectrophotometer at 410 nm. ……………………….……………………9

Table 2. Transmittance for 20 and 50 µg K g-1 solution (average of four readings, ± standard deviation) using either solid or solution forms of NaNO3 to promote precipitation. The precipitate for the solid NaNO3 is denser as represented by the lower % transmittance. All values were read on the spectrophotometer at 600 nm. …………………………………………...10

Table 3. Transmittance for 20 and 50 µg K g-1 solution (average of four readings, ± standard deviation) using either 1.25 g weighed or 1.23 cm3 scooped to deliver NaNO3 for precipitation. All values were read on the spectrophotometer at 600 nm. …………………………………………...11

Table 4. Impact of time of reaction (0,5,10,15 and 30 minutes) with sodium cobaltinitrite on % transmittance of five soils with varying amounts of K. Values represent the average of two duplicates read on the spectrophotometer at 600 nm. …………………………….……………..12

Manuscript 2: A Simplified Soil Analysis Procedure for Use in Small-Scale Agriculture

Table 1. A cost analysis of standard methods of extraction and analysis compared to the PHW extraction and subsequent analyses for N, P, and K and pH. All dollar figures are approximations based on current catalog prices. All prices are in American dollars. ………………………………………….27

Table 2. Summary of soil extraction and analysis procedures and related references. ……………………………………………………………….28

Table 3. Standard methods used by the Guatemala1, Morocco2 and BYU3 laboratories for soil extraction and analysis. …………………………….29

Table 4. Coefficients of determination (r2) for regression equations1 defining the relationships between either pressurized hot water, water or 1 M KCl extractions in conjunction with chromotropic acid (CTA) or steam

x distillation-titration analysis for NO3-N. One hundred seventeen soils from Morocco were tested at both Brigham Young University and the Institute of Agronomy Hassan II in Rabat while 111 soils from Guatemala were tested at both Brigham Young University and the University of the East at Cunori in Chiquimula. ………………………………………...…30

Table 5. Coefficients of determination (r2) for regression equations1 defining the relationships between P extracted by either pressurized hot water, Olsen or Mehlich I then analyzed using molybdic acid. One hundred seventeen soils from Morocco were tested at both Brigham Young University and the Institute of Agronomy Hassan II in Rabat while 111 soils from Guatemala were tested at both Brigham Young University and the University of the East at Cunori in Chiquimula. ………………………...31

Table 6. Coefficients of determination (r2) for regression equations1 defining the relationship between P extracted using either Bray I, Mehlich I, Olsen or pressurized hot water then analyzed using molybdic acid. Testing was done at Brigham Young University on 111 soils from Guatemala. ……..32

xi LIST OF FIGURES

Manuscript 1: A Rapid Turbidimetric Potassium Test Modified for Use with the Pressurized Hot-Water (PHW) Extraction

Figure 1: Relationship between K concentration and log transmittance measured at different wavelengths. Data for only three wavelengths is presented. Relationship lines for wavelengths of 725, 700, 650, and 625 nm fall between the regression lines shown in descending order. The regression equation for the 600 nm line is K (µg ml-1) = - 61.0363 * log %T + 126.2748. ………………………………………………………………...13

Figure 2. The relationship between ammonium acetate extracted K analyzed by atomic absorption (AA) and pressurized hot-water (PHW) extracted K analyzed by atomic absorption (AA). Tests were completed at Brigham Young University on 38 soils from the Western United States. ………...14

Figure 3. The relationship between ammonium acetate extracted K analyzed by atomic absorption (AA) and pressurized hot-water (PHW) extracted K analyzed by cobaltinitrite. Tests were completed at Brigham Young University on 38 soils from the Western United States. ………………...15

Figure 4. The relationship between pressurized hot water extracted K analyzed by atomic absorption (AA) and pressurized hot-water (PHW) extracted K analyzed by cobaltinitrite. Tests were completed at Brigham Young University on 38 soils from the Western United States. ………………...16

Manuscript 2: A Simplified Soil Analysis Procedure for Use in Small-Scale Agriculture

Figure 1. Relationship between NO3-N extracted by either water or pressurized hot water, then analyzed by chromotropic acid. Samples included 117 soils from Morocco and 111 soils from Guatemala. Soil tests were completed at Brigham Young University. Regressions were performed separately for each country (A) and for all soils combined (B). ………………………..33

Figure 2. Relationship between P extracted by either Olsen sodium bicarbonate or pressurized hot water, then analyzed by molybdic acid. Samples included 117 soils from Morocco and 111 soils from Guatemala. Soil tests were completed at Brigham Young University. Regressions were performed separately for each country (A) and for all soils combined (B). ………...34

Figure 3. Relationship between K extracted by either ammonium acetate or pressurized hot-water then analyzed using atomic absorption or sodium

xii cobaltinitrite. Samples included 30 soils each from Morocco and Guatemala. Soil tests were completed at Brigham Young University. Regressions were performed separately for each country (A) and for all soils combined (B). ……………………………………………………...35

Figure 4. The relationship between (A) pressurized hot-water extractable K analyzed by atomic absorption (AA) and pressurized hot water extractable K analyzed with the sodium cobaltinitrite procedure (B) ammonium acetate extractable K analyzed by atomic absorption (AA) and pressurized hot water extractable K analyzed by atomic absorption. Tests were completed at Brigham Young University on samples including 25 soils each from Morocco and Guatemala. …………………………………….36

Figure 5. The relationship between NO3-N extracted by pressurized hot water then analyzed by chromotropic acid. (A) The 117 soils from Morocco were tested at Brigham Young University and at the Institute of Agronomy, Hassan II in Rabat. (B) The 111 soils from Guatemala were tested at Brigham Young University and at the University of the East at Cunori in Chiquimula. ……………………………………………………………...37

Figure 6. The relationship between P extracted by pressurized hot water then analyzed by molybdic acid. (A) The 117 soils from Morocco were tested at Brigham Young University and at the Institute of Agronomy, Hassan II in Rabat. (B) The 111 soils from Guatemala were tested at Brigham Young University and at the University of the East at Cunori in Chiquimula. ……………………………………………………………...38

xiii

The following two manuscripts have been prepared for submission to

“Communications in Soil Science and Plant Analysis.” Formatting is in accordance with requirements outlined for this journal. Appendices I-IV include a literature review and data used to make decisions on procedures selected for use, step by step descriptions of each procedure used and summaries of data collected.

xiv A RAPID TURBIDIMETRIC POTASSIUM TEST MODIFIED FOR USE WITH THE

PRESSURIZED HOT-WATER EXTRACTION

Crane, K.S.; Webb, B.L.; Allen, P.S.; Jolley, V.D.

Plant and Animal Sciences Department, Brigham Young University, Provo, UT 84602

ABSTRACT

Colorimetric or turbidimetric quantification of potassium (K) analysis coupled with the pressurized hot-water (PHW) extraction could provide an inexpensive alternative to standard methods for small-scale farmers in developing countries. Two of many methods for K analysis, one using sodium tetraphenylborate and the other using sodium cobaltinitrite, were modified for use with the PHW extraction and evaluated for the following requirements: be readable on the spectrophotometer, have minimal equipment needs, be rapid, simple and comparable in accuracy to proven methods of K analysis. The sodium tetraphenylborate method was unreliable at low K concentrations, did not relate with K extracted using ammonium acetate and analyzed by atomic absorption, required extract filtration and was too expensive or not readily accessible in developing countries. Sodium cobaltinitrite was simple, inexpensive and produced consistent results. Test results from 38 arid soils from the Western United States encourage the use of sodium cobaltinitrite as an acceptable procedure for K quantification (r2 of 0.90) compared to AA analysis and when coupled with PHW related (r2 of 0.67) with ammonium acetate-AA measured K. Use of sodium cobaltinitrite is suited to quantify K to complement PHW extraction on arid soils.

INTRODUCTION

Pressurized hot-water (PHW) soil extraction using an espresso machine holds promise as an alternative soil nutrient extraction procedure (1,2). In previous studies this method proved reliable for the extraction of nitrate-nitrogen (NO3-N), phosphorus (P), potassium (K), boron (B), and sulfur (S) in arid zone soils. Nutrient quantification following PHW extraction has generally been accomplished using combinations of inductively coupled plasma (ICP), atomic absorption (AA) and colorimetric analyses. Eliminating the need for AA or ICP analyses would reduce analytical cost, technical expertise and, coupled with the inexpensive PHW extraction, would enhance soil-testing accessibility to farmers in developing countries. As NO3-N and P already have common colorimetric procedures available (3,4), K presents the greatest challenge for macronutrient analysis adaptation to PHW extraction. Such a K analysis procedure would require minimal specialized equipment, including an espresso machine, spectrophotometer, chemicals and glassware, would be rapid, simple and would be as accurate as proven standard methods.

Soil K extraction and analysis has changed dramatically over the years and many procedures for determining K have been proposed. Sodium cobaltinitrite (5,6,7), sodium tetraphenylborate (8,9,10), dipicrylamine (11,12), platinic chloride and periodate (13) are proposed as methods for simple determination of K. These colorimetric, turbidimetric or volumetric K analysis procedures are based on reagents that selectively react with K, and consequently exhibit little interference from monovalent cations common to soils such as sodium (Na), lithium (Li), and ammonium (NH4). Atomic absorption, which was introduced in the early 1950’s (14), eliminated concerns about interference from other elements and quickly became the accepted standard method for K analysis. However, some procedures in common use before the introduction of AA remain viable alternatives for K analysis and candidates for adaptation for use with the PHW extraction.

Two procedures were identified as possible alternatives to “standard” AA analysis of K. The first is a turbidimetric method using sodium tetraphenylborate. Gloss and Cluley (15,16) reported sodium tetraphenylborate as an exceptional, rapid turbidimetric method for determining K. The second method, sodium cobaltinitrite, is a procedure commonly used before atomic absorption became popular (17) and is currently recommended by Attanandana et al. (18) for rapid analysis of K in Thailand. Both methods were tested in the BYU laboratory and modified as needed to determine their potential for quantifying K in conjunction with the water-based PHW extraction. The first section of this manuscript deals with the challenges of method development of both procedures and the second section reports the relationships established between the sodium cobaltinitrite method and the ammonium acetate “standard” method using 38 arid soil samples from the Western United States.

METHOD DEVELOPMENT

Sodium Tetraphenylborate

In adapting the sodium tetraphenylborate procedure for use with PHW extraction, the procedure outlined by Sunderman and Sunderman (8) was chosen among the many reviewed (8,9,10,15,16,17,19,20,21) primarily because it already involved a water extract and was a turbidimetric procedure read directly on the spectrophotometer. Although the procedure was originally intended to measure blood serum K, the extensively tested protocol had precipitation characteristics similar to soil analysis procedures (9). The proposed protocol uses a 3.0 ml aliquot of a water filtrate placed in a colorimeter cuvet with 3.0 ml of a 2:1 EDTA:formaldehyde mixture, to chelate interfering ions such as NH4, added and then mixed by swirling. To keep the precipitate in suspension, five drops of gum ghatti solution are added and swirled. Finally, 1.0 ml of 5.0% sodium tetraphenylborate solution is added with force to precipitate the K. After precipitate formation the K in solution is quantified by reading the transmittance on a spectrophotometer at 410 nm.

Initial investigation of the necessary reagents and precipitate formation indicated no immediate problems with adapting the procedure to the PHW extract. The purification of

2 the gum ghatti solution previously required is unnecessary because of commercial availability of high quality gum ghatti. A standard curve was developed using stock K solutions between 2 and 100 µg ml-1 K. However, standard solutions gave inconsistent readings, suggesting that the procedure is not sensitive at low K (Table 1). Since most soil tests require readings of solution K in this low concentration range, the sodium tetraphenylborate procedure is not sensitive for soil K analysis. Altering the various reagent concentrations did not overcome the problem. In addition, the PHW extracts varied in color from yellow to dark brown, presumably due to organic matter, which interfered with reading the precipitate that formed using sodium tetraphenylborate. An extract filtration step using activated charcoal overcame the color interference problem but was time-consuming and variable. In dark extracts more activated charcoal was required than in lighter extracts, but in the lighter extracts, excessive amounts of charcoal became difficult to filter. Preliminary soil tests comparing ammonium acetate extracted potassium with the PHW-tetraphenylborate potassium were inconsistent and inaccurate on a set of alkaline soils (data not shown). Finally, in the two developing countries where procedure implementation would be tested, sodium tetraphenylborate was either unavailable or extremely expensive. Thus, the sodium tetraphenylborate procedure was abandoned due to lack of sensitivity at low K concentrations common in agricultural soils, variable precipitate formation, time-consuming filtration and the expense and unavailability of the principal reagent in the procedure.

Sodium Cobaltinitrite

A review of several sodium cobaltinitrite potassium procedures (5,6,7,22,23,24) led to a modification of a protocol by Olson (6) to use in conjunction with the PHW extraction. Olson’s procedure was chosen because it was simple and rapid and did not require specialized equipment.

The original Olson protocol used 7.5 g soil extracted with 15.0 ml of 25.0% sodium nitrate solution. In this procedure Na replaces K on the exchange sites during extraction and the precipitate formed during quantification is a proportional Na/K precipitate that is chemically stable in solutions at approximately 6.0% sodium concentration. A 5.0 ml aliquot of this sodium nitrate/soil extract is transferred to a 5.0 mm glass tube and 3.0 ml of isopropyl alcohol is added to the mixture and swirled. The alcohol aids in the formation of the precipitate and isopropyl alcohol gives a lighter colored solution than other alcohols. Finally, 0.1 g of sodium cobaltinitrite is added and shaken vigorously until dissolution of the sodium cobaltinitrite is complete. The sodium cobaltinitrite is readily dissolved in water and forms a precipitate when K is present in solution. The suspended precipitate is read on the spectrophotometer at 700 nm.

Since PHW was the extractant, four possible procedural adjustments were investigated. The first adjustment was a method for adding NaNO3 to the PHW extract to preserve the 25% NaNO3 concentration that is necessary for predicable precipitate formation. The second adjustment involved adding the sodium cobaltinitrite in solution form for potentially greater ease of analysis and precision in routine laboratory use. The third adjustment was to ascertain the appropriate wavelength at which the samples should be

3 read. Finally, the influence of the amount of time between sodium cobaltinitrite dissolution and spectrophotometeric measurement on the amount of precipitation in suspension (wait time) was investigated.

We replaced the 25% NaNO3 extractant (6) with pressurized hot-water (distilled) (1). The 25% NaNO3 solution used by Olson is important to K precipitate formation. The sodium cobaltinitrite procedure uses cobaltinitrite to form a proportional Na/K precipitate. An excess of Na is necessary to completely precipitate the K in solution but the exact chemical formula of the precipitate varies according to the concentration of Na present. To form a precipitate with a consistent chemical formula, the Na in solution must be adjusted to a concentration that avoids critical points at which the chemical formula of the precipitate changes (7,23). A total Na concentration between approximately 4.5 and 6.6% (6,7) creates an excess of Na leading to stable precipitate formation and negating the impact of any extracted soil Na. Thus, for our purpose it was essential to find an alternative method of adding NaNO3 to PHW extracts to achieve this approximate 4.5- 6.6% Na in solution.

Addition of chemicals by volume in solution form is preferred to weighing for routine laboratory analysis. To add NaNO3 to the PHW sample aliquot to achieve 25% NaNO3 in solution, a concentrated solution (66%) of NaNO3 was prepared. Mixing 5.0 ml of extract and 3.0 ml of 66% NaNO3 solution resulted in an 8.0 ml aliquot with 25% NaNO3. However, the 66% NaNO3 solution proved difficult to prepare and maintain as it was near saturation. Also, a less dense precipitate forms when 5.0 ml aliquots are brought to 25% NaNO3 with 66% NaNO3 solution than with 1.25 g of NaNO3 (equaling 25% NaNO3 in solution), thus making measurement more difficult (Table 2). Adding solid NaNO3 produced more accurate K measurements on low K soils.

An alternative to weighing solids in routine laboratory analysis is volume addition. 3 Addition of NaNO3 to aliquots by weighing (1.25g) or volume addition (1.23 cm ) was compared and found to be equal using a two-sided t-test performed on 20 and 50 µg ml-1 standard solutions (Table 3). There were slightly higher standard deviations when adding by volume as opposed to weighing, but the ease of using the scoop outweighs the slightly less variability of weighing the sample and will promote acceptance of this technique.

Olson (6) added solid sodium cobaltinitrite to form the precipitate, but adding sodium cobaltinitrite solution would be a more practical. However, attempts to use sodium cobaltinitrite solution failed to produce a precipitate and the idea was abandoned. Instead, 0.1 g solid sodium cobaltinitrite was added with a 0.065 cm3 scoop to facilitate analysis. There was no significant difference between a volume scooped and a weight added of sodium cobaltinitrite (data not shown).

In the protocols found in which sodium cobaltinitrite is used to precipitate K for turbidimetric analysis (6,24), a set of standards and 700 nm were used to quantify K. Consequently, once the effective reagent concentrations in the procedure were determined, the proper wavelength to be used to read the precipitate was evaluated. Standard K solutions of 2, 5, 10, 15, 20, 25, 30, 35 and 40 µg ml-1 were analyzed at least

4 in triplicate at wavelengths of 600, 625, 650, 675, 700, 725, and 750 nm. Average percent transmittance for each K concentration averaged was regressed to establish a relationship with K concentration (Figure 1). Below 580 nm, transmittance fluctuated wildly with no apparent pattern, and above 750 nm, the separation of points along the standard curve continually decreased, therefore, no measurements were taken below 600 nm or above 750 nm. The 600 nm wavelength was selected for use because it gave the widest spread in the percent transmittance readings leading to greater separation of points along the line and greater ease of K quantification (Figure 1). The regression equation relating log percent transmittance to concentration K is: K (µg ml-1) = - 61.0363 * log %T + 126.2748.

For some turbidimetric analyses, time allowed for reaction is critical because the precipitate is suspended in solution and can settle out if left for too much time. Impact of time of precipitate formation (or wait time) was determined using five soils known to vary widely in K content. Aliquots of extracts from each of five soils were mixed appropriately and allowed to react for 0, 5, 10, 15 or 30-minute intervals measured from the moment of complete sodium cobaltinitrite dissolution. There were no significant effects of time of reaction on precipitate formation and thus on K measurement in the five soils tested (Table 4; Analysis of variance, p > 0.05). A wait time of five minutes is recommended to insure that the sodium cobaltinitrite is completely dissolved since incomplete dissolution of the sodium cobaltinitrite leads to lower levels of precipitate formation. A characteristic orange coloring associated with the sodium cobaltinitrite test avoided color interference from coloration associated with the PHW extract and filtration was unnecessary.

Based on the series of evaluations completed, we recommend the following procedure for analysis of K following PHW extraction. In a 65 ml plastic test tube in a rack of 24, place a 5.0 ml aliquot of the PHW extract, add 1.25 g sodium nitrate using a 1.23 cm3 plastic spoon and agitate the mixture gently by shaking for approximately one minute until the sodium nitrate is dissolved. Next, add 3.0 ml isopropyl alcohol and swirl several seconds to mix the solution. Add 0.1 g sodium cobaltinitrite using a 0.065 cm3 scoop and shake by hand until the sodium cobaltinitrite is completely dissolved (about 5 min). After allowing the precipitate to stand for at least five minutes, read the transmittance of the solution on the spectrophotometer at 600 nm.

TEST OF METHOD

Thirty-eight soils from the Western United States with a pH range of 6.0 to 8.6 and a texture range of loamy sand to clay (25) were tested for K using the PHW extraction (1,25) and both the sodium cobaltinitrite turbidimetric analysis according to the protocol described above and atomic absorption. The same soils were extracted with ammonium acetate (26) and analyzed by atomic absorption (AA) for comparison. A standard sample was included with every thirty samples analyzed to confirm reproducibility. If the standard varied beyond defined limits for the standard soil, all thirty accompanying samples were reanalyzed. The PHW extract was also analyzed using atomic absorption to provide a comparison between the ammonium acetate extraction and the PHW extraction.

5 The PHW/sodium cobaltinitrite procedure was replicated three times and the results averaged. Results were analyzed using simple linear regression with SAS, Version 8 from SAS Institute (Raleigh, NC).

RESULTS AND DISCUSSION

The relationship between K values obtained using PHW extraction measured using AA and ammonium acetate extraction using AA was highly significant (Figure 2) and is similar to the relationship reported by Hanks et al. (1) for K measured using PHW extraction measured using AA compared to sodium bicarbonate extraction measured using AA (r2 of 0.72). The relationship between K values using PHW extraction and cobaltinitrite analysis compared to ammonium acetate extraction measured using AA was almost as good (Figure 3) indicating that the sodium cobaltinitrite analytical procedure is effective in quantifying K in the extract. The strong relationship between K analyzed by AA and by sodium cobaltinitrite from the same PHW extract further supports this conclusion (Figure 4).

CONCLUSIONS

A simplified soil K extraction and analysis procedure valid over a range of soil types could be advantageous farmers on small farms in developing countries if correlated to yield response. Two methods of K analysis to complement PHW were evaluated. Sodium tetraphenylborate formed a variable precipitate at low K concentrations and the necessary reagents are prohibitively expensive and often unavailable in developing countries where the method would be used. Sodium cobaltinitrite proved to be better suited to complement the PHW extraction. Any variation of color in PHW extracts did not interfere with spectrophotometer readings so filtration was unnecessary, and precipitate formation was stable and sensitive at agronomically important K levels with sodium cobaltinitrite. The data reported indicate that the sodium cobaltinitrite K analysis procedure can be used successfully to complement pressurized hot-water extraction on the arid soils of the Western United States and data reported elsewhere (28) indicate applicability to acid, neutral and calcareous soils.

REFERENCES

1. Hanks, D.; Webb, B.; Jolley, V. A Comparison of Hot Water Extraction to Standard Extraction Methods for Nitrate, Potassium, Phosphorus, and Sulfate in Arid-Zone Soils. Commun. Soil Sci. Plant Anal. 1997, 28, 1393-1402. 2. Fulkey, G.; Czinkota, I. Hot Water Percolation (HWP): A New Rapid Soil Extraction Method. Plant Soil 1993, 157, 131-135. 3. Haby, V.A. Soil NO3-N Analysis in Ca(OH)2 Extracts by the Chromotropic Acid Method. Soil Sci. Soc. Am. J. 1989, 53, 308-310 4. Wantanabe, F.S.; Olsen, S.R. Test of Ascorbic Acid Method for Determining Phosphorus in Water and NaHCO3 Extractants for Soil. Soil Sci. Soc. Amer. Proc. 1965, 29, 677-678.

6 5. Melsted, S.W. A Chemical Study of Quick-Test Technics for Potassium and Calcium. J. Am. Soc. Agron. 1942, 34, 533-543. 6. Olson, R.V. A Turbidimetric Potassium Determination Affected Little by Temperature. Soil Sci. Soc. Amer. Proc. 1953, 17, 20-22. 7. Volk, N.J. The Determination of Small Amounts of Exchangeable Potassium in Soils, Employing the Sodium Cobaltinitrite Procedure. J. Am. Soc. Agron. 1941, 33, 684. 8. Sunderman, F.W.; Sunderman, F.W. Studies in Serum Electrolytes: A Rapid, Reliable Method for Serum Potassium using Tetraphenylboron. Am. J. Clin. Path. 1958, 29, 95- 103. 9. Schall, E.D. Volumetric Determination of Potassium. Anal. Chem. 1957, 29, 1044- 1046. 10. Brabson, J.A. Fertilizers. In Standard Methods of Chemical Analysis 6th Edition Volume 2 pt B. F.J. Welcher, Ed.; Van Nostrand: Princeton, 1966; 1483-1506. 11. Marczenko, Z. Alkali Metals. In Separation and Spectrophotometric Determination of Elements. Halstead Press: New York, 1986; 123-128. 12. Caley, E.R. The Rapid Colorimetric Estimation of Potassium. J. Am. Chem. Soc. 1931, 5, 539-545. 13. Kolthoff, I.M.; Stenger, V.A. Iodometry of Inorganic Substances. In Volumetric Analysis Vol. 3. I.M. Kolthoff; R. Belcher; V.A. Stenger; G. Matsuyama, Eds. Interscience Publishers: New York, 1942; 245-274. 14. Charlot, G. Potassium. In Colorimetric Determination of the Elements, Principles and Methods. Elsevier Publishing Company: Amsterdam, 1964; 350-352. 15. Gloss, G.H. Sodium Tetraphenylboron: A New Analytical Reagent for Potassium, Ammonium, and Some Organic Nitrogen Compounds. Chemist-Analyst 1953, 42, 50-55. 16. Cluley, H.J. The Determination of Potassium by Precipitation as Potassium Tetraphenylboron and its Application to Silicate Analysis. Analyst 1955, 80, 354-364. 17. Kallman, S. Alkali Metals: Determination of Potassium. In Treatise on Analytical Chemistry Part 2 Volume 1. I.M. Kolthoff; P.J. Elving, Eds.; Interscience Encyclopedia, New York, NY, 1959; 369-378. 18. Attanandana, T.; Suwannarat, C.; Vearasilp, T.; Longton, S.; Meesawat, R.; Bunampol, P.; Soitong, K.; Tipanuka, C.; Yost, R.S. Use of Decision-Aids in On-Farm Experiment in Thailand. Regional Workshop on Decision-Aids for Nutrient Management Support System (NuMaSS). PhilRice, January 21-24, 2002. 19. Pflaum, R.T; Howick, L. Spectrophotometric Determination of Potassium with Sodium Tetraphenylborate. Anal. Chem. 1956, 28, 1542-1544. 20. Motomizu, S.; Yoshida, K.; Toei, K. Indirect Spectrophotometric Determination of Potassium Ion in Water Based on the Precipitation with Tetraphenylborate Ion and a Crown Ether Using Flow Injection. Anal. Chim. Acta 1992, 261, 225-231. 21. Dill, A.J.; Popovych, O. Solubility Products of Potassium and Triisoamyl-n- butylammonium Picrates and Tetraphenylborates in Ethanol-water Mixtures at 25 Degrees Celsius. J. Chem. Eng Data 1969, 14, 240-243. 22. Lohse, H.W. Determination of Small Amounts of Potassium by Means of Sodium Cobaltinitrite. Ind. & Eng. Chem., Anal. Ed. 1935, 7, 272-273. 23. Burkhart, L. Potassium Determination by the Cobaltinitrite Method as Affected by Temperature and pH. Plant Physiol. 1941, 16, 411-414. 24. Peech, M.; English, L. Rapid Microchemical Soil Tests. Soil Sci. 1945, 57, 167-195.

7 25. Webb, B.L.; Hanks, D.H.; Jolley, V.D. A Pressurized Hot Water Extraction Method for Boron. Commun. Soil Sci. Plant Anal. 2002, 33, 31-39 26. Council on Soil Testing and Plant Analysis. Determination of Potassium, Magnesium, Calcium, and Sodium by Neutral Ammonium Acetate Extraction. In Handbook on Reference Methods for Soil Testing (Revised Edition); Council on Soil Testing and Plant Analysis, Eds.; The Council on Soil Testing and Plant Analysis: Athens, GA, 1980; 58- 63. 27. Leal, J.E.; Sumner, M.E.; West, L.T. Evaluation of Available P with Different Extractants on Guatemalan Soils. Commun. Soil Sci. Plant Anal. 1994, 25, 1161-1196. 28. Crane, K.S.; Webb, B.L; Allen, P.S; Jolley, V.J. A Simplified Soil Analysis Procedure for Use in Small-Scale Agriculture. In Review.

Concentration Transmittance (µg K g-1 solution) (%, ± standard deviation)

2 95.8 ± 5.3 5 92.4 ± 3.8 10 76.3 ± 9.4 15 60.9 ± 14.4 20 38.8 ± 13.8 25 29.2 ± 7.9 20 24.8 ± 4.9

9 Table 2. Transmittance for 20 and 50 µg K g-1 solution (average of four readings, ± standard deviation) using either solid or solution forms of NaNO3 to promote precipitation. The precipitate for the solid NaNO3 is denser as represented by the lower % transmittance. All values were read on the spectrophotometer at 600 nm.

Concentration NaNO3 added Transmittance ( µg K g-1 solution) as (%, ± standard deviation)

20 Solid 44.5 ± 1.6 Solution 88.6 ± 3.6

50 Solid 17.1 ± 1.8 Solution 26.7 ± 1.0

10 Table 3. Transmittance for 20 and 50 µg K g-1 solution (average of four readings, ± standard deviation) using either 1.25 g weighed or 1.23 cm3 scooped to deliver NaNO3 for precipitation. All values were read on the spectrophotometer at 600 nm.

Concentration NaNO3 added Transmittance ( µg K g-1 solution) (%, ± standard deviation)

20 1.25 g weighed 43.6 ± 1.0 1.23 cm3 scoop 43.1 ± 1.6

50 1.25 g weighed 17.8 ± 1.4 1.23 cm3 scoop 17.1 ± 1.8

11 Table 4. Impact of time of reaction (0,5,10,15 and 30 minutes) with sodium cobaltinitrite on % transmittance of five soils with varying amounts of K. Values represent the average of two duplicates read on the spectrophotometer at 600 nm.

Minutes of Reaction with Sodium Cobaltinitrite Sample # 0 5 10 15 30 Significance†

1 60.5 62.2 65.1 60.6 64.2 NS

2 92.8 93.2 94.0 95.0 95.5 NS

3 85.5 82.9 83.3 83.3 83.5 NS

4 25.0 25.9 25.3 25.3 25.9 NS

5 93.9 95.1 91.7 91.7 93.9 NS

†NS = The variation in transmittance between length of reaction times is not significant at p < 0.05 according to analysis of variance completed using SAS Version 8, SAS Institute

50 Wavelength ● 600 nm 40 ○ 675 nm ▲750 nm

) 30 -1 g µ

K ( 20

0 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 Log % Transmittance Figure 1: Relationship between K concentration and log transmittance measured at different wavelengths. Data for only three wavelengths is presented. Relationship lines for wavelengths of 725, 700, 650, and 625 nm fall between the regression lines shown in descending order. The regression equation for the 600 nm line is K (µg ml-1) = - 61.0363 * log %T + 126.2748.

4000 y = 3.213x – 80.855 r2 = 0.73 A p < 0.0001 e-A

t 3000 eta c A

2000 onium m ), Am -1 1000 g µ K (

0 0 200 400 600 800 1000 1200 K (µg g-1), PHW-AA

4000 y = 4.591x – 245.16 r2 = 0.67 p < 0.0001

e-AA 3000 at

2000 onium Acet m m A

1), -

g 1000 g µ K (

0 0 100 200 300 400 500 600 700 800 K (µg g-1), PHW-Cobaltinitrite

15 1200 y = 1.411x – 47.327 r2 = 0.90 1000 p < 0.0001

800 AA - W PH

, 600 ) -1 g µ 400 K (

200

0 0 100 200 300 400 500 600 700 800 K (µg g-1), PHW-Cobaltinitrite

16 A SIMPLIFIED SOIL ANALYSIS PROCEDURE FOR USE IN SMALL-SCALE AGRICULTURE

Crane, K.S, B.L. Webb, P.S. Allen, and V.D. Jolley

Plant and Animal Sciences Department, Brigham Young University, Provo, UT 84602

ABSTRACT

Soil analysis for small-scale farms in developing countries is often inconvenient and prohibitively expensive using current procedures, yet the information gained from these soil tests could result in economical and environmental benefits. The pressurized hot water (PHW) extraction coupled with colorimetric or turbidimetric analysis shows promise as an alternative to current procedures based on tests done on a limited range of soils. In order to use this extraction and analysis in developing countries, testing is needed across the range of soils found in these countries. At Brigham Young University (BYU), two hundred and twenty-eight soils from different areas of Guatemala and Morocco were analyzed for NO3-N, P and K using standard methods (water-CTA, Olsen- molybdic acid and ammonium acetate-atomic absorption, respectively). Results were correlated to values obtained from the PHW extraction coupled with colorimetric or turbidimetric analytical procedures. The relationships between the values for these tests 2 were good (r of 0.96, 0.71 and 0.52 for NO3-N, P, and K; respectively). Soils from each country were simultaneously analyzed for NO3-N and P in laboratories in Guatemala and Morocco and these results correlated with those from BYU. Correlations between BYU values and those from other laboratories gave generally positive results with the data from the Guatemala laboratory showing overall closer correlation than the Morocco laboratory with BYU values. In an additional study comparing several P extraction methods for Guatemala soils, relationships between PHW P and Olsen, Bray I and Mehlich I (r2 of 0.75, 0.67 and 0.46, respectively) measured at BYU indicate that PHW is a promising alternative P procedure for soils in Guatemala. Overall, the data support PHW extraction and accompanying analyses as a less expensive alternative to current soil nutrient extraction and analysis procedures for the broad range of soils tested from Morocco and Guatemala.

INTRODUCTION

General

Soil testing technology has improved remarkably during the last forty years. Laborious methods of testing have been replaced by simple, more precise ones due to technological and chemical advances (1). These advances have benefited scientists, laboratory technicians and farmers with improved accuracy and increased efficiency but have come at increased cost to the laboratory and consequently the consumer. Higher costs are

17 absorbed into overhead in large-scale agricultural production but are prohibitive for subsistence-level farmers in most underdeveloped countries.

Not only does the expense incurred for soil testing prohibit small-scale farmers from seeking analysis, but the availability of soil testing laboratories is limited (2). Consequently, such farmers generally fail to apply fertilizer or apply it in incorrect amounts. This leads to soil nutrient imbalances, economically inferior yields and environmental pollution (3,4). Fertilizer recommendations leading to a balanced nutrient management system are critical to increasing yields and sustaining crop production; therefore, subsistence farmers in developing countries would benefit from simplified, less expensive soil testing procedures.

The pressurized hot-water (PHW) extraction procedure, originally outlined by Fulkey and Czinkota (5) and adapted by Hanks et al. (6), is a simple and inexpensive method of nutrient extraction that has shown good correlation with standard methods in arid, calcareous soils of the Western United States. The combination of colorimetric or turbidimetric elemental analysis with PHW extraction allows the determination of soil nitrate-nitrogen (NO3-N), phosphorus (P) and potassium (K) with little more than common glassware and chemicals, an espresso machine and a spectrophotometer.

Estimated costs associated with establishing a laboratory for standard soil analysis are approximately $23,300 for equipment compared to $2,250 for PHW (Table 1). Monthly costs of glassware and chemicals are estimated at $600 and $400 per month for standard and PHW, respectively. Since none of the equipment used with PHW requires extensive maintenance or training to operate, training time is minimal (5 days for PHW versus 20 days for standard extraction and analysis). Also, the data generated is not dependent upon the quality of the instrument used. If, as we hypothesize, values from PHW extraction and photometric analyses of soil for the above nutrients correlate strongly with values from standard methods for extraction and analysis of the same elements across a wide range of soils, then further testing to develop an inexpensive laboratory will be warranted. The economic, environmental and social benefits of an inexpensive soil analysis laboratory to small-scale farmers in developing countries could be significant.

Morocco and Guatemala were chosen as countries to participate in this study for several reasons. Soils in Morocco are generally alkaline and low in organic matter while soils in Guatemala are generally acidic and high in organic matter. This range of soils allows a broader comparison of results in an effort to determine the range of utility for the PHW procedure. Contacts in both Morocco and Guatemala associated with the Benson Food and Agriculture Institute, at Brigham Young University, Provo, UT, an organization providing significant funding for this research, facilitated collaboration. Finally, both Morocco and Guatemala have numerous subsistence farmers with little access to regular soil testing.

In the process of developing alternative soil testing methods, several potential colorimetric and turbidimetric elemental analysis procedures were evaluated to determine if they could be used successfully in conjunction with the PHW extraction adapted by

18 Hanks et al. (6). Our objective was to determine the limits of utility for these procedures through testing on soils with highly variable characteristics from Morocco and Guatemala. Testing done in the laboratories of BYU, Morocco and Guatemala not only allowed us to understand the relationship between the PHW extraction and complementary and standard methods of extraction and analysis, but also to explore the limits of reproducibility in the laboratories of interest and to assess the ability of the laboratories to adopt this alternative method of extraction and analyses after minimal training.

Phosphorus Extraction

The standard method for P testing in Guatemala is the Mehlich I extraction followed by molybdic acid analysis. However, laboratories in Guatemala have adopted this extraction procedure as a standard soil testing method with little or no testing or correlation with crop response (7). The variety of P extractions available and Guatemala’s use of a standard method that might not be appropriate for all the soil types of Guatemala prompted us to explore different extraction procedures in the BYU laboratory using the Guatemala soils.

There are several common chemical extractants for P including dilute acid fluoride (Bray I) (8), double-acid (Mehlich I) (9), and sodium bicarbonate (Olsen) (10). These methods were developed to extract a proportion of plant available P from the soil for analysis. These soil tests extract P using dilute acid or bicarbonate ions to chemically react with precipitated soil P compounds causing them to dissolve and the P to be released into solution and then measured. The dilute acids used in Mehlich I and Bray I react with aluminum and iron phosphate precipitates in acid soils and the bicarbonate used in Olsen reacts with calcium and some aluminum phosphate precipitates (11). Because these extractants are specific to the P precipitates that they extract, their uses are generally limited to a narrow range of soils. The Mehlich I extraction for P is considered appropriate for very acid, low CEC soils (12,13), but Tran et al. (12) determined that the Mehlich I test could not be used on soils with varying “chemical and physical properties.” For acid soils with a variety of soil textures, Menon et al. (14) determined that the Mehlich I extraction compared to other common P extractants had the lowest and least significant correlation to plant response. The Bray I procedure is more widely recognized as a better procedure for the acidic clay soils (14) that are characteristic of Guatemala.

Common complaints associated with chemical extractants for P are that they are only viable for a specific range of soil conditions (7,14,15,16,17), and lead to mobilization of non- or slowly- available P (7,12,14,15,18,19,20). A water extraction for P could be ideal because it would not depend upon the changing forms of P during the extraction process (12,14,15) as is common with chemical extractants. Water extraction could also be ideal because it would mimic P movement in soil solutions (14) and field conditions that plants experience (19). The primary concern with the proposed water extractions is that the water-soluble fraction of P is small in comparison to the total P reservoir, thus making

19 interpretation difficult (14,15,17). Therefore, if the PHW procedure extracts measurable amounts of P, it could prove to be an important method of soil P extraction.

To investigate the applicability of the Mehlich I procedure specifically in Guatemala and explore the relationship of the PHW extraction to other standard, potentially applicable extraction procedures, Mehlich I, Bray I, Olsen and PHW extractions were compared. Moroccan soils were not included in this study because the standard method of soil P extraction (Olsen) used in Morocco is considered appropriate for the alkaline soils found there.

EXPERIMENTAL DESIGN

One hundred seventeen soils were collected in Morocco in 2002 and 111 soils were collected in Guatemala in 2003. The Morocco soils were collected from 11 regions in north and central Morocco, from the Atlas Mountains and the coastal plains, with organic matter ranging from 0.9 to 3.5%, pH ranging from 4.7 to 7.7, and texture ranging from clay to loamy sand. The Guatemala soils were collected from fields and forests in six regions across the country, including the volcanic highlands, coastal plains and tropical forests, with organic matter ranging from 1.3 to 17.8%, pH ranging from 4.0 to 7.3, and texture ranging from clay to sandy loam. Soils were dried, ground and mixed in their country of origin before being divided into two parts. Half of the soil was transported to BYU and the rest remained in the country of origin. Both the Morocco and the Guatemala soils were analyzed at BYU in Provo, Utah. In addition, the Moroccan soils were also analyzed at the Institute of Agronomy Hassan II in Rabat, Morocco and the Guatemalan soils at the Central University of the East at Cunori in Chiquimula, Guatemala. In each case, a team from BYU spent approximately four days training personnel to use the techniques employed.

Soils from Morocco and Guatemala were extracted and analyzed for NO3-N, P and K using accepted standard methods (Table 2). They were also extracted using the pressurized hot-water procedure and then quantified using colorimetric or turbidimetric methods. A standard sample was included with every thirty samples analyzed with the standard analysis procedure for each nutrient to confirm reproducibility. If the standard varied beyond defined limits for the standard soil, all thirty accompanying samples were reanalyzed. The PHW extraction and associated analyses were replicated three times on each soil and the results averaged. For the PHW-cobaltinitrite potassium procedure, a subset of fifty soils, twenty-five from Guatemala and twenty-five from Morocco, were used due to insufficient amounts of soil samples remaining. The soils used had the same range of textures that was cited earlier. The Morocco soil subset had organic matter ranging from 1.9 to 3.5% and pH ranging from 6.3 to 7.6. The Guatemala soil subset had organic matter ranging from 1.3 to 8.1% and pH ranging from 4.7 to 7.0. The PHW- cobaltinitrite procedure was replicated three times on each soil and the results were averaged. Data was analyzed with simple linear regression using SAS Version 8 from SAS Institute (Raleigh, NC). Multiple linear regression was also used, but no improvements were noted so data is not presented.

20 MATERIALS AND METHODS

PHW Extraction and Analysis

The PHW method consisted of a pressurized hot-water extraction and a colorimetric/turbidimetric elemental analysis. The pressurized hot-water extraction (6) was performed on a Melitta® MEX1B espresso machine. The MEX1B generates an approximate pressure of 2.5 bars and a temperature of 90°C ± 2°C (21). For soil sample extraction, 100.0 ml of distilled water was forced through 5.0 g of soil placed on a 5.0 cm medium filter paper and the extract was collected in a plastic cup.

The colorimetric NO3-N analysis uses the chromotropic acid procedure outlined by Haby (22) with the calcium hydroxide [Ca(OH)2] added and refiltered after the extraction, instead of during extraction, to clarify the extracted sample. Phosphorus is determined with the molybdic acid colorimetric procedure outlined by Wantanabe and Olsen (10). The K analysis uses a sodium cobaltinitrite turbidimetric procedure (23) (Table 2).

Standard Methods

Analyses Completed at BYU

The standard methods used at the BYU laboratory for NO3-N, P and K were water extraction and chromotropic acid (CTA) analysis (22), Olsen bicarbonate extraction and molybdic acid analysis (10) and ammonium acetate extraction and atomic absorption analysis (25), respectively. Despite the range of soil characteristics in the Morocco and Guatemala soils, the Olsen method for P was used as the standard method for both because it is considered to be applicable to the widest variety of soils (10). Using the Olsen method as a standard for soil from both countries was desirable because it allowed joint conclusions to be drawn from the results for both countries. In addition, for the study on P extraction using the Guatemala soils three common P extractants (Mehlich I (9), Bray I (8) and Olsen (10)) were used, followed by molybdic acid analysis to compare with PHW.

Analyses Completed in Morocco

In Morocco the standard NO3-N extraction uses 1M CaCl2 (26) followed by steam distillation and titration analysis. The standard P procedure is the Olsen sodium bicarbonate extraction followed by molybdic acid analysis (10). For K, ammonium acetate extraction followed by atomic absorption analysis is the standard method (25) (Table 3).

Analyses Completed in Guatemala

In Guatemala there is no standard N analysis due to perceptions that N is not deficient in their soils (Rudolfo Chicas, Personal Communication, 2003). The standard P procedure is

21 the Mehlich I (9) extraction followed by molybdic acid analysis (10). For K, Mehlich I (9) extraction is also used followed by atomic absorption analysis (Table 2 and 3).

RESULTS AND DISCUSSION

Comparison of Methods

Even though NO3-N is water soluble, the relationships between the PHW-CTA and water-CTA methods for Morocco and Guatemala soils analyzed at BYU are exceptional (Figure 1). The relationship for Morocco and Guatemala soils between the PHW-CTA and water-CTA methods (Figure 1A) improves when data from both countries are combined (Figure 1B). Thus, a single regression equation is adequate to describe the relationship between the two NO3-N procedures for the soils of both countries. Our results support data of Hanks et al. (6) who reported r2 values of 0.98 for the 38 arid soils tested in the same way and are significantly better than those reported by Fulkey and 2 Czinkota (r of 0.38) (5) who compared NO3-N extracted by CaCl2 and analyzed by steam distillation to PHW-extracted NO3-N followed by steam distillation and titration.

The relationships between P values obtained by the PHW-molybdic acid and the Olsen- molybdic acid methods for the Morocco and Guatemala soils are excellent (Figure 2A) and again suggest that a single equation adequately describes the relationship for both countries (Figure 2B). Using the PHW and Olsen extractions, Hanks et al. (6) reported a significant but less predictable relationship for P with an r2 value of 0.36. The soils used were alkaline and high in calcium, which forms very tight calcium phosphate complexes. The soils from Guatemala and Morocco are acid to slightly alkaline (pH < 8) which helps explain the high r2 value. Fulkey and Czinkota (5) reported similar relationships (r2 of 0.73) on Hungarian soils analogous in soil properties to those found in Guatemala.

The results for the comparison of the PHW-cobaltinitrite method and the ammonium acetate-AA method for the Morocco and Guatemala soils were less predictable for K (Figure 3A) and the relationship defined by combining data from the two countries is not adequate (Figure 3B). This relationship is poorer than that reported by Crane et al. (23) (r2 of 0.67; p < 0.0001) using the same procedures for 38 arid-zone soils from the Western United States. Hanks et al. (6), who used sodium bicarbonate as the standard extractant, also observed better relationships (r2 of 0.72), but their soils had a naturally higher K concentration while the Morocco and Guatemala soils included soils with lower K levels.

Differences in either extraction or quantification of K could contribute to the observed disparity in results when comparing PHW and ammonium acetate methods. Our data suggest this disparity can be attributed to lower extraction of K with PHW than with ammonium acetate (Figure 4B). This is expected since mass action extraction can readily occur with ammonium acetate but not with PHW. Pressurized hot-water extracted K measured using atomic absorption is correlated slightly better to K extracted using ammonium acetate measured on the AA (Figure 4B) than PHW-extracted K measured with the cobaltinitrite method (Figure 3A). The relationship between PHW extracted K

22 measured using atomic absorption and cobaltinitrite is excellent (r2 of 0.88). Similar results were reported by Crane et al. on 38 arid-zone soils (23). Thus, the cobaltinitrite analysis procedure quantifies extracted K in solution and, when coupled with the PHW extraction, could be an inexpensive alternative for measuring K in developing countries.

Comparisons among Laboratories

In addition to comparing a large number of widely divergent soils for NO3-N, P and K from the two countries using PHW extraction and quantified at BYU, we also compared results from PHW extractions and subsequent analyses from laboratories in Morocco and Guatemala to BYU results and to “standard” methods used in each lab. The results presented are limited to NO3-N and P because the procedure to quantify K originally proposed and taught to technicians in the two countries (27) was abandoned due to poor performance (data not shown). Consequently, the cobaltinitrite procedure for quantifying K used in the comparisons was developed and tested independently of the laboratories in Morocco and Guatemala and data further substantiating the cobaltinitrite procedure are presented elsewhere (23).

Relationships between PHW extraction values for NO3-N from Morocco and Guatemala with values generated at BYU for the same soils varied (Figure 5). The good relationship between BYU and Guatemalan values is acceptable (r2 of 0.92), but the poor relationship with Moroccan values is not because identical methods were used (r2 of 0.50). However, this can be explained by a chloride (Cl) contamination that was not able to be corrected before analysis of the soils in Morocco. Analyzing the soils from Morocco in the BYU laboratories revealed that the soils were naturally high in Cl, an element that can interfere with the colorimetric procedure and promote high readings for NO3-N. The scattering of data along the x-axis (Figure 5A), as opposed to the grouping demonstrated in Figure 1A, for the PHW-CTA extraction and analysis from the Moroccan laboratory is indicative of a problem with Cl contamination (Figure 5A). In the procedure outlined by Haby (22) an antimony solution is used to mask Cl. When the solution is cool, antimony sulfate precipitates out. Heating the antimony solution used in the procedure prior to analysis corrects the problem by redissolving the antimony sulfate precipitate, but heating the antimony solution was not part of the protocol used in the Moroccan laboratory and cooperators in Morocco were unavailable to repeat the analyses with the altered protocol. This chloride contamination hypothesis is substantiated by the improvement in the predicted relationship observed when comparing the BYU water-CTA procedure with the Moroccan steam distillation procedure (Table 4) and the poor relationship between Moroccan 1 M KCl-steam distillation and Moroccan PHW-CTA methods.

Pressurized hot water data from the Guatemala laboratory related favorably to both PHW-CTA and water-CTA values obtained from the BYU laboratory (Table 4). This close relationship strengthens the argument that the PHW-CTA procedure is easily adaptable to relatively inexperienced laboratories since the Guatemalan laboratory does not typically do an analysis of NO3-N. The relationship also reflects the fact that the Guatemalan laboratory did not experience the same chloride contamination problem that the Moroccan laboratory did. The range of NO3-N values obtained in Guatemala suggests

23 that a soil test correlated to field response would be helpful for small-scale farmers living in this area of the world.

Like NO3-N, PHW extraction values for P analyzed at BYU compared more favorably to analyses from Guatemala than Morocco (Figure 6). These good relationships suggest relatively easy adaptation across laboratories since neither of these laboratories had previous experience with PHW. In discussions comparing P values from various procedures and laboratories, note that all P was quantified using molybdic acid colorimetric analysis regardless of extraction (Tables 2 and 3).

Moroccan calcareous soils are best compared to PHW using the Olsen bicarbonate extraction (Table 2). The relationship between Olsen and PHW extracted P from BYU laboratory is excellent (Table 5) and better than between Olsen- and PHW-extracted P from Morocco analyses (Table 5). Both relationships are as good as or better than reports of Fulkey and Czinkota (5) on 36 Hungarian soils and Hanks et al. (6) on 38 arid zone soils. The relationship between Olsen P extraction from BYU and Moroccan laboratories is similar to those relationships for the two labs for PHW (Table 4; r2 of 0.75 and 0.74, respectively). Because predictability of relationships between the two laboratories is similar for the two extraction methods, the PHW extraction appears to not sacrifice accuracy as an alternative procedure.

Relating PHW to a “standard” P method is complicated for acidic Guatemala soils due to several choices of extraction chemicals and analytical techniques available (Table 6). Consequently, coefficients of determination for P extracted soils from Guatemala by Bray I, Mehlich I, Olsen, and PHW are reported in Table 6. In general these relationships are better than those reported by Hanks et al. (6) on 38 arid zone soils using PHW and Olsen extractions. Relationships between other methods and PHW are among the best reported with Olsen better than Bray I better than Mehlich I (Table 6). The excellent relationship with Olsen and Bray I extractions (r2 of 0.66) is somewhat surprising since Olsen and Bray I are developed for use on very different soils—one would expect a similarly poor relationship as observed between Mehlich I and Olsen extraction values (r2 of 0.40) and not like the relatively close relationship between values for Mehlich I and Bray I (r2 of 0.69). Thus, the comparison among methods confirms that PHW seems to effectively relate P status in both calcareous and acid soils.

CONCLUSIONS

The PHW extraction and associated analyses for NO3-N, P, and K have strong predictable relationships with standard methods for analysis completed in the BYU laboratory. For NO3-N and P, a single equation derived from the data sets from the two countries (Morocco and Guatemala) described the relationship between PHW and standard methods. For K, separate equations are needed to describe the relationship between PHW-cobaltinitrite and ammonium acetate-atomic absorption for the two countries. Comparison of alternate “standard” P methods on Guatemalan soils indicates that PHW is closely correlated to Olsen and Bray I, suggesting applicability to both acid and alkaline soils. Generally positive regression data obtained from laboratories in Morocco and Guatemala with similar BYU data confirms that the PHW procedure is

24 adapted for application to developing countries where the procedures were successfully implemented to extract and quantify soil NO3-N, P and K. In both countries, these positive relationships existed despite spending no more than five days training personnel. The positive relationships obtained, the minimal training time needed and the generally inexpensive nature of these tests (Table 1) would promote adaptation and use in developing countries.

ACKNOWLEDGEMENTS

Financial support for this research was contributed by the Benson Food and Agriculture Institute, the Potash and Phosphate Institute and the Plant and Animal Sciences Department at BYU.

REFERENCES 1. Greenland, D.J. The Contributions of Soil Science to Society-Past, Present and Future. Soil Sci. 1991, 151, 19-23. 2. Yaalon, D.H. Soil Science in Transition: Soil Awareness and Soil Care Research Strategies. Soil Sci. 1996, 161, 3-8. 3. Dibb, D.W.; Darst, B.C.; Mutert, E.; Beaton, J.D. Nutrient Balance: A Key to Sustainable Agriculture. IFA-FADINAP Regional Fertilizer Conference for Asia and the Pacific, Manila, Philippines. 1993. 4. Lal, R. Soil Management in the Developing Countries. Soil Sci. 2000, 165, 57-72. 5. Fulkey, G.; Czinkota, I. How Water Percolation (HWP): A New Rapid Soil Extraction Method. Plant Soil 1993, 157, 131-135. 6. Hanks, D.; Webb, B.; Jolley, V. A Comparison of Hot Water Extraction to Standard Extraction Methods for Nitrate, Potassium, Phosphorus, and Sulfate in Arid-zone Soils. Commun. Soil Sci. Plant Anal. 1997, 28, 1393-1402. 7. Leal, J.E.; Sumner, M.E.; West, L.T. Evaluation of Available P with Different Extractants on Guatemalan Soils. Commun. Soil Sci. Plant Anal. 1994, 25, 1161-1196. 8. Council on Soil Testing and Plant Analysis. Determination of Bray PI Extraction. In Handbook on reference methods for soil testing (revised edition); Council on Soil Testing and Plant Analysis, Ed; The Council on Soil Testing and Plant Analysis: Athens, GA, 1980; 42-46. 9. Council on Soil Testing and Plant Analysis. Determination of Phosphorus by Mehlich No. 1 (Double acid) Extraction. In Handbook on Reference Methods for Soil Testing (Revised Edition); Council on Soil Testing and Plant Analysis, Ed; The Council on Soil Testing and Plant Analysis: Athens, GA, 1980; 37-41. 10. Wantanabe, F.S.; Olsen, S.R. Test of Ascorbic Acid Method for Determining Phosphorus in Water and NaHCO3 Extractants for Soil. Soil Sci. Soc. Amer. Proc. 1965, 29, 677-678. 11. Kou, S., Phosphorus. In Methods of Soil Analysis, Part 3-Chemical Methods. D.L. Sparks, Ed.; Soil Science Society of America, Inc., Madison, WI, 1996; 869-919. 12. Tran, T.S.; Giroux, M.; Guilbeault, J.; Audesse, P. Evaluation of Mehlich III Extractant to Estimate the Available P in Quebec Soils. Commun. Soil Sci. Plant Anal. 1990, 21, 1-28.

25 13. Mylavarapu, R.S.; Kennelley, E.S. UF/IFAS Extension Soil Testing Laboratory (ESTL) Analytical Procedures and Training Manual. Extension Circular 1248; Florida Cooperative Extension, University of Florida: Gainesville, FL, 2002; 2-4. 14. Menon, R.G.; Chien, S.H.; Hammond, L.L. Development and Evaluation of the Pi Soil Test for Plant-Available Phosphorus. Commun. Soil Sci. Plant Anal. 1990, 21, 1131-1150. 15. Fernandes, M.L.; Indiati, R.; Coutinho, J.; Buondonno, A. Soil Properties Affecting Phosphorus Extraction from Portuguese Soils by Conventional and Innovative Methods. Commun. Soil Sci. Plant Anal. 1999, 30, 921-936. 16. Nesse, P.; Grava, J.; Bloom, P.R. Correlation of Several Tests for Phophorus with Resin Extractable Phosphorus for 30 Alkaline Soils. Commun. Soil Sci. Plant Anal. 1988, 19, 675-689. 17. Fixen, P.E.; Grove, J.H. Testing Soils for Phosphorus. In Soil Testing and Plant Analysis 3rd Ed.; Westerman, R.L., Ed.; American Society of Agronomy: Madison, WI, 1990; 141-180. 18. Ibrikci, H.; Hanlon, E.A.; Rechcigl, J.E. Initial Calibration and Correlation of Inorganic-Phosphorus Soil Test Methods with a Bahiagrass Field Trial. Commun. Soil Sci. Plant Anal. 1992, 23, 2569-2579. 19. Elrashidi, M.A.; Alva, A.K.; Huang,Y.F.; Calvert, D.V.; Obreza, T.A.; He, Z.L. Accumulation and Downward Transport of Phosphorus in Florida Soils and Relationship to Water Quality. Commun. Soil Sci. Plant Anal. 2001, 32, 3099-3119. 20. Sharpley, A.N. Soil Phosphorus Extracted by Iron-Aluminum-Oxide-Impregnated Filter Paper. Soil Sci. Soc. Am. J. 1991, 55, 1038-1041. 21. Webb, B.L.; Hanks, D.H.; Jolley, V.D. A Pressurized Hot Water Extraction Method for Boron. Commun. Soil Sci. Plant Anal. 2002, 33, 31-39. 22. Haby, V.A. Soil NO3-N Analysis in Ca(OH)2 Extracts by the Chromotropic Acid Method. Soil Sci. Soc. Am. J. 1989, 53, 308-310. 23. Crane, K.S.; Webb, B.L; Allen, P.S; Jolley, V.J. A Rapid Turbidimetric Potassium Test Modified for Use with Pressurized Hot Water (PHW) Extraction. (In Review). 24. Sims, J.R.; Jackson, G.D. Rapid Analysis of Soil Nitrate with Chromotropic Acid. Soil Sci. Soc. Amer. Proc. 1971, 35, 603-606. 25. Council on Soil Testing and Plant Analysis. Determination of Potassium, Magnesium, Calcium, and Sodium by Neutral Ammonium Acetate Extraction. In Handbook on Reference Methods for Soil Testing (Revised Edition); Council on Soil Testing and Plant Analysis, Ed; The Council on Soil Testing and Plant Analysis: Athens, GA, 1980; 58-63. 26. Houba, V.J.G.; Novozamsky, I.; Uttenbogaard, J.; Van der Lee, J.J.; Automatic Determination of ‘Total Soluble Nitrogen’ in Soil Extracts. Landw. Forsch. 1987, 40, 295-302. 27. Sunderman, F.W.; Sunderman, F.W. Studies in Serum Electrolytes: A Rapid, Reliable Method for Serum Potassium using Tetraphenylboron. Am. J. Clin. Path. 1958, 29, 95- 103.

26 Table 1. A cost analysis of standard methods of extraction and analysis compared to the PHW extraction and subsequent analyses for N, P, and K and pH. All dollar figures are approximations based on current catalog prices. All prices are in American dollars.

Standard Extraction and Analysis1 $ Value PHW Extraction and Analysis2 $ Value pH meter $300 pH meter $300 Spectrophotometer $1600 Spectrophotometer $1600 Balance $300 Balance $300 Chemicals $200 Chemicals $200 Glassware $400 Glassware $200 Shaker $1100 Espresso machine $50 A.A3 $20,000 - Total: $23,900 Total: $2,650

1Training time estimated to be 20 days 2Training time estimated to be 5 days 3If ICP were used the cost for equipment would increase significantly

27 Table 2. Summary of soil extraction and analysis procedures and related references.

Nutrient Extractant Reference Analysis1 Reference

Used with Pressurized Hot-Water (PHW)

Nitrate- Pressurized Hanks et al. (6) Chromotropic Haby (22) nitrogen Hot-Water Acid

Phosphorus Pressurized Hanks et al. (6) Molybdic Wantanabe and Hot-Water Acid Olsen (10)

Potassium Pressurized Hanks et al. (6) Sodium Crane (23) Hot-Water Cobaltinitrite

Standard Procedures to which PHW Extraction and Accompanying Analyses Were Compared

Nitrate- Distilled Sims and Chromotropic Haby (22) nitrogen Water Jackson (24) Acid

Phosphorus Sodium Wantanabe and Molybdic Wantanabe and Bicarbonate Olsen (10) Acid Olsen (10)

Potassium Ammonium Council on Soil Atomic Council on Soil Acetate Testing and Absorption Testing and Plant Plant Analysis Analysis (25) (25)

1 Following PHW extraction, colorimetric or turbidimetric analytic procedures were selected to promote use in underdeveloped countries.

28 Table 3. Standard methods used by the Guatemala1, Morocco2 and BYU3 laboratories for soil extraction and analysis.

Element Guatemala1 Morocco2 BYU 3

Nitrogen (N) None4 Steam Water-CTA distillation Phosphorus (P) Mehlich I Olsen Olsen Potassium (K) Mehlich I Ammonium Ammonium acetate acetate

1 The Central University of the East at Cunori, Chiquimula, Guatemala 2 The Institute of Agronomy Hassan II, Rabat, Morocco 3 Brigham Young University, Provo, UT 4 Due to a localized perception of adequate N reserves in the soil, no standard method is used. (Rudolfo Chicas, Personal Communication, 2003)

29 Table 4. Coefficients of determination (r2) for regression equations1 defining the relationships between either pressurized hot water, water or 1 M CaCl2 extractions in conjunction with chromotropic acid (CTA) or steam distillation-titration analysis for NO3-N. One hundred seventeen soils from Morocco were tested at both Brigham Young University and the Institute of Agronomy Hassan II in Rabat while 111 soils from Guatemala were tested at both Brigham Young University and the University of the East at Cunori in Chiquimula.

Laboratory Morocco Guatemala BYU location Extractant-analysis PHW-CTA PHW-CTA Water-CTA

Morocco KCl-steam distillation 0.43 - 0.70 BYU Water-CTA - 0.93 - BYU PHW-CTA 0.50 0.92 0.96

1All values were significant at p < 0.001 as determined by simple linear regression.

30 Table 5. Coefficients of determination (r2) for regression equations1 defining the relationships between P extracted by either pressurized hot water, Olsen or Mehlich I then analyzed using molybdic acid. One hundred seventeen soils from Morocco were tested at both Brigham Young University and the Institute of Agronomy Hassan II in Rabat while 111 soils from Guatemala were tested at both Brigham Young University and the University of the East at Cunori in Chiquimula.

Laboratory location Morocco Guatemala BYU Extractant PHW PHW Olsen

Morocco Olsen 0.41 - 0.75 Guatemala Mehlich I - 0.53 0.36 BYU PHW 0.74 0.90 0.71

1All values were significant at p < 0.001 as determined by simple linear regression.

31 Table 6. Coefficients of determination (r2) for regression equations1 defining the relationship between P extracted using either Bray I, Mehlich I, Olsen or pressurized hot water then analyzed using molybdic acid. Testing was done at Brigham Young University on 111 soils from Guatemala.

Extraction Procedure Mehlich I Olsen PHW

Bray I 0.69 0.66 0.67 Mehlich I - 0.40 0.46 Olsen - - 0.75

1All values were significant at p < 0.001 as determined by simple linear regression.

100 y = 0.860x – 4.031 r2 = 0.94 on 80 p < 0.0001

tracti Guatemala Country Ex 60 ● Morocco ter a ○ Guatemala ), W -1 40 g µ

( y = 0.745x – 3.389

-N 2 3 20 r = 0.94

NO p < 0.0001 Morocco A 0 100 y = 0.846x – 4.079 r2 = 0.96 on i 80 p < 0.0001 tract x r E

e 60 t a ), W -1 40 g µ ( -N 3 20 NO B 0

0 20 40 60 80 100 120 140 NO -N (µg g-1), PHW Extraction 3

country (A)andforallsoilscom University. Regressionswere from acid. Sam bicarbonate orpressuri Figure 2.RelationshipbetweenPex P (µg g-1), Olsen Extraction P (µg g-1), Olsen Extraction 12 16 12 16 40 80 40 80 0 0 0 0 0 0 Guatem Morocco p < r y = 0 2 = 1.654 0.0001 0.66 p 2 les included117soilsfrom 04 ala. Soiltestswe x + 5.795 0 P ( µ g zed hotwater,thenanalyzedbym 6 -1 08 ) , PHW Extracti 0 b re com Guatemal p < p < r y = r y = 2 2 ined (B) perform 1 = = on 0 1.349 1.425 0 0.0001 0.0001 0.75 0.71 tracted byeitherOlsensodi Cou ○ ● Guatemala Morocco 1 n p 2 x + x + a tr 0 leted atBrigham Moroccoand111soils e y

7.473 7.070 d separatelyforeach 34 1 A B 4 0

Young o lybdic um

2500 y = 4.183x – 75.718 Country hod r2 = 0.54 ● Morocco Met 2000 p < 0.0001 ○ Guatemala A Guatemala e-A at t 1500 Ace

onium 1000 m y = 1.597x + 15.889 2 ), Am r = 0.28 -1 500

g p < 0.0067 µ Morocco K ( 0 A 2500 y = 4.154x – 164.122 2 hod r = 0.52

Met 2000 p < 0.0001

e-AA at t e

c 1500 A

onium 1000 m Am ),

-1 500 g µ K ( 0 B 0 100 200 300 400 500 K (µg g-1), PHW-Cobaltinitrite Method

Figure 3. Relationship between K extracted by either ammonium acetate or pressurized hot-water then analyzed using atomic absorption or sodium cobaltinitrite. Samples included 30 soils each from Morocco and Guatemala. Soil tests were completed at Brigham Young University. Regressions were performed separately for each country (A) and for all soils combined (B).

500 y = 1.302x – 59.668 r2 = 0.88 400 ption

r p < 0.0001 o Abs

ic 300 Atom -

200 PHW , ) -1 g

µ 100 K ( A 0 0 100 200 300 400 500 K (µg g-1), PHW-Cobaltinitrite 2500 y = 3.103x + 36.403 r2 = 0.56 2000 p < 0.0001

Acetate-AA 1500 monium 1000 1), Am - g

µ 500 K ( B 0 0 100 200 300 400 500 K (µg g-1), PHW-AA

Figure 4. The relationship between (A) pressurized hot-water extractable K analyzed by atomic absorption (AA) and pressurized hot water extractable K analyzed with the sodium cobaltinitrite procedure and (B) ammonium acetate extractable K analyzed by atomic absorption (AA) and pressurized hot water extractable K analyzed by atomic absorption. Tests were completed at Brigham Young University on samples including 25 soils each from Morocco and Guatemala.

36 140 y = 0.714x – 0.360

) 2

U 120 r = 0.50 Y

B p < 0.0001 100

tion ( ac tr

x 80 E

60 PHW

) -1

g 40 µ -N ( 3 20 NO 0 A

0 20406080100120140

-1 NO3-N (µg g ), PHW Extraction (Morocco) 140

) y = 0.726x + 5.232

U 120 2

Y r = 0.92 B

( p < 0.0001

on 100 ti ac tr 80 Ex

60 PHW , ) -1 40 g µ ( -N

3 20 NO 0 B

0 20406080100120140 NO -N (µg g-1), PHW Extraction (Guatemala) 3

140 y = 0.814x – 3.992 2 120

) r = 0.74 U

Y p < 0.0001 B 100

on ( ti c a

r 80 t Ex

W 60 H P , )

-1 40 g µ 20 P (

0 A

0 20406080 P (µg g-1), PHW Extraction (Morocco) 140 y = 1.270x - 0.123 2 120 r = 0.90 )

U p < 0.0001 Y B ( 100 tion

ac 80 tr x E

W 60 PH , )

-1 40 g µ 20 P (

0 B

0 20406080 P (µg g-1), PHW Extraction (Guatemala)

Appendix I

Introduction and Literature Review

39 INTRODUCTION AND LITERATURE REVIEW

Soil testing technology has improved remarkably during the last forty years. Laborious methods of testing have been replaced by simpler and more precise ones due to technological and chemical advances (1). These advances have benefited scientists, laboratory technicians, and farmers with improved accuracy and increased efficiency but have come at increased cost to the laboratory and consequently the consumer. Higher costs are acceptable in large-scale agricultural production, where the cost of soil testing is readily absorbed into overhead, but are prohibitive for subsistence level farmers in most underdeveloped countries.

For these small-scale farmers, not only does the expense of soil testing prohibit them from requesting analysis, but the availability of soil testing laboratories is limited (2). Consequently, such farmers generally fail to apply fertilizer or apply inappropriate fertilizers in incorrect amounts. This leads to soil nutrient imbalances and economically inferior yields (3,4). Fertilizer recommendations leading to a balanced nutrient management system are critical to increasing yield and sustaining crop production; therefore, subsistence farmers in developing countries would benefit from simplified, less expensive soil testing procedures.

The pressurized hot-water (PHW) extraction procedure, originally outlined by Fulkey and Czinkota (5) and adapted by Hanks et al. (6), is performed on a Melitta® MEX1B espresso machine (Melitta USA, Inc., Clearwater, FL). The MEX1B generates an approximate pressure of 2.5 bars and a temperature of 90°C ± 2°C (7). For soil sample extraction, 100.0 ml of distilled water is forced through 5.0 g of soil and the extract collected. This extraction is a simple, inexpensive method of nutrient extraction that has 2 shown good relationships (r of 0.98, 0.85, 0.72, 0.36; NO3-N, SO4-S, K, P, respectively) with standard methods in arid, calcareous soils of the western United States (6). The PHW extraction combined with colorimetric or turbidimetric nutrient analysis would allow elemental determination with little more than common glassware and chemicals, an espresso machine and a spectrophotometer. The need for and cost of atomic absorption or inductively coupled plasma would be eliminated.

The macronutrients (N, P, and K) are the most common fertilizer nutrients and therefore a good focus for the PHW extraction and associated analyses. Both NO3-N and P have accepted colorimetric procedures available. The chromotropic acid (CTA) procedure outlined by Haby (8) is often used to determine NO3-N concentrations and the molybdic acid procedure outlined by Watanabe and Olsen (9) is in common use for P. However, K presents a challenge because it is not commonly analyzed colorimetrically or turbidimetrically. Colorimetric, turbidimetric and volumetric K analyses are based on finding a reagent that will selectively react with K but have little interference from other monovalent cations such as sodium (Na), lithium (Li), and ammonium (NH4). Upon its introduction in the early 1950’s (10), atomic absorption, which eliminated concerns about interference from other elements, quickly became the accepted standard method for K analysis and other methods were discarded. However, many of the procedures in common

40 use before the introduction of atomic absorption remain viable alternatives for K analysis that could be adapted to the PHW extraction.

A review of colorimetric and turbidimetric potassium procedures was conducted and two promising methods of turbidimetric analysis, using sodium cobaltinitrite (11, 12, 13, 14) and sodium tetraphenylborate (15, 16, 17) were identified.

The method of turbidimetric analysis using sodium cobaltinitrite (the sodium cobaltinitrite method) was a common method of turbidimetric K analysis before the introduction of atomic absorption (11). The precipitate formed during the turbidimetric analysis using sodium cobaltinitrite varies in composition according to the sodium concentration in solution (18, 19). Therefore, a sufficiently high concentration of sodium must be present to allow all K in solution to precipitate out.

Burkhart (13) outlined a turbidimetric procedure for K analysis using sodium cobaltinitrite. He emphasized the importance of constant temperature to achieve uniform precipitate particle size and found that the procedure was more sensitive to low concentrations of K when the sodium cobaltinitrite was added as a solid. Olson (12) was also concerned with temperature but found that when solid forms of sodium cobaltinitrite were used for precipitation of K that temperature had a minimal effect and the reaction could be carried out at room temperature. He used a 25 % sodium solution in which to form the precipitate. Peech and English (14) used formaldehyde to complex NH4 during their turbidimetric procedure to eliminate interference and used a solution form of sodium cobaltinitrite. They extracted with sodium acetate in order to have appropriate amounts of sodium in solution for precipitate formation. Both Lohse (19) and Volk (18), though using volumetric analysis procedures, favored a 20 to 25% sodium solution in which to form the K/Na cobaltinitrite precipitate.

Another early method involved using sodium tetraphenylborate to precipitate K. Gloss (17) outlined several advantages to using sodium tetraphenylborate in K analysis. These advantages include a compound formula that is well defined and unchanging (in contrast to sodium cobaltinitrite precipitates) as (Ph)4BK, a low solubility with K and NH4 but a high solubility with other monovalent cations and has few other ion interferences. Pflaum and Howick (16) precipitated K with a sodium tetraphenylborate solution and then redissolved the precipitate in acetonitrile. The resulting colored complex was measured on the spectrophotometer at 266 nm. Sunderman and Sunderman (15) extensively tested a turbidimetric procedure for analyzing blood serum K with reagents similar to those used in soil or fertilizer tests. They used a suspending agent (gum ghatti) to hold the precipitate in suspension and used formaldehyde to complex interfering ions.

To adapt a K analysis procedure for use with the PHW extract, testing must be performed to insure that the procedure is compatible with the water extract, is reasonably simple and correlates with atomic absorption analysis. The methods of analysis using sodium tetraphenylborate and sodium cobaltinitrite showed promise as analytical tools in conjunction with the PHW extract and both were tested in the laboratory at Brigham Young University (BYU). Sodium cobaltinitrite proved superior to sodium

41 tetraphenylborate and has been established as the method of choice for use with the PHW extract.

To use the PHW extraction and subsequent analyses in developing countries for the quantification of soil NO3-N, P, and K concentrations, widely varying soils from two countries, Morocco and Guatemala, were analyzed for NO3-N, P, and K in the BYU laboratory using standard methods of extraction and analysis and the PHW extraction followed by colorimetric or turbidimetric analysis. Soils were simultaneously analyzed at laboratories in Morocco and Guatemala to allow comparison of results from the BYU laboratory to those from countries where the analyses would be taking place.

Standard methods of soil testing in Guatemala include the Mehlich I extraction followed by molybdic acid analysis for P. However, these laboratories have adopted this extraction procedure as a standard soil testing method with little or no testing or correlation with crop response (20). The Mehlich I extraction uses dilute acid which is neutralized in calcareous soils or soils with a high cation exchange capacity and therefore reduces its ability to quantify P in these soils (21). To investigate the applicability of the Mehlich I procedure in Guatemala and explore the relationship of the PHW extraction to other standard, potentially applicable extraction procedures, the Mehlich I, Bray I, Olsen and PHW extractions were performed and compared.

The purpose of this thesis is to develop and test analytical methods for use with the PHW extraction that are relatively inexpensive and simple to perform and then correlate them with standard methods of extraction and analysis for NO3-N, P, and K on widely varying soils from developing countries. If successful, experiments performed to correlate this soil test to plant response will follow. These soil test procedures have the possibility of truly helping subsistence farmers in developing nations to increase crop productivity and better their economic circumstances.

REFERENCES

1. Greenland, D.J. The Contributions of Soil Science to Society-Past, Present and Future. Soil Sci. 1991, 151, 19-23. 2. Yaalon, D.H. Soil Science in Transition: Soil Awareness and Soil Care Research Strategies. Soil Sci. 1996, 161, 3-8. 3. Dibb, D.W.; Darst, B.C.; Mutert, E.; Beaton, J.D. Nutrient Balance: A Key to Sustainable Agriculture. IFA-FADINAP Regional Fertilizer Conference for Asia and the Pacific, Manila, Philippines. 1993. 4. Lal, R. Soil Management in the Developing Countries. Soil Sci. 2000, 165, 57-72. 5. Fulkey, G.; Czinkota, I. How Water Percolation (HWP): A New Rapid Soil Extraction Method. Plant Soil 1993, 157, 131-135. 6. Hanks, D.; Webb, B.; Jolley, V. A Comparison of Hot Water Extraction to Standard Extraction Methods for Nitrate, Potassium, Phosphorus, and Sulfate in Arid-zone Soils. Commun. Soil Sci. Plant Anal. 1997, 28, 1393-1402. 7. Webb, B.L.; Hanks, D.H.; Jolley, V.D. A Pressurized Hot Water Extraction Method for Boron. Commun. Soil Sci. Plant Anal. 2002, 33, 31-39

42 8. Haby, V.A. Soil NO3-N Analysis in Ca(OH)2 Extracts by the Chromotropic Acid Method. Soil Sci. Soc. Am. J. 1989, 53, 308-310 9. Wantanabe, F.S.; Olsen, S.R. Test of Ascorbic Acid Method for Determining Phosphorus in Water and NaHCO3 Extractants for Soil. Soil Sci. Soc. Amer. Proc. 1965, 29, 677-678. 10. Charlot, G. Potassium. In Colorimetric Determination of the Elements, Principles and Methods. Elsevier Publishing Company: Amsterdam, 1964; 350. 11. Melsted, S.W. A Chemical Study of Quick-Test Technics for Potassium and Calcium. J. Am. Soc. Agron. 1942, 34, 533-543. 12. Olson, R.V. A Turbidimetric Potassium Determination Affected Little by Temperature. Soil Sci. Soc. Am. Proc. 1953, 17, 20-22. 13. Burkhart, L. Potassium Determination by the Cobaltinitrite Method as Affected by Temperature and pH. Plant Physiol. 1941, 16, 411-414. 14. Peech, M.; English, L. Rapid Microchemical Soil Tests. Soil Sci. 1945, 57, 167-195. 15. Sunderman, F.W.; Sunderman, F.W. Studies in Serum Electrolytes: A Rapid, Reliable Method for Serum Potassium using Tetraphenylboron. Am. J. Clin. Pathol. 1958, 29, 95- 103. 16. Pflaum, R.T; Howick, L. Spectrophotometric Determination of Potassium with Sodium Tetraphenylborate. Anal. Chem. 1956, 28, 1542-1544. 17. Gloss, G.H. Sodium Tetraphenylboron: A New Analytical Reagent for Potassium, Ammonium, and Some Organic Nitrogen Compounds. Chemist-Analyst 1953, 42, 50-55. 18. Volk, N.J. The Determination of Small Amounts of Exchangeable Potassium in Soils, Employing the Sodium Cobaltinitrite Procedure. J. Am. Soc. Agron. 1941, 33, 684. 19. Lohse, H.W. Determination of Small Amounts of Potassium by Means of Sodium Cobaltinitrite. Ind. & Eng. Chem., Anal. Ed. 1935, 7, 272-273. 20. Leal, J.E.; Sumner, M.E.; West, L.T. Evaluation of Available P with Different Extractants on Guatemalan Soils. Commun. Soil Sci. Plant Anal. 1994, 25, 1161-1196. 21. Kuo, S. Phosphorus. In Methods of Soil Analysis Part 3-Chemical Methods; Sparks, D.L., Ed.; Soil Science Society of America, Inc.: Madison, WI, 1996; 869-919.

Appendix II

A Summary Table of Spectrophotometric Methods of Analysis for Soil Nutrients

44 Nutrient Procedure Name Procedure Interferences Reagents End Point and References Nitrate Chromotropic ⋅ take 2.5 ml aliquot of ⋅ .1% CTA: .184 g CTA into100 ml ⋅ yellow color at

(NO3-N) Acid (1,2,3) sample and place in volumetric flask and dissolve it in 100 ml 410 nm 50 ml Erlenmeyer H2SO4 flask ⋅ sulfite-urea solution: dissolve 5 g ⋅ add one drop sulfite- analytical grade urea and 5 g reagent grade urea solution and two anhydrous sodium sulfite in distilled water ml antimony and dilute to 100 ml solution. Swirl to ⋅ antimony sulfate solution: dissolve .5 g mix. Sb metal in 80 ml of H2SO4, heat in hood ⋅ add 1 ml .1% CTA till clear, cool and add 20 ml of distilled solution and 4.5 ml water conc. H2SO4 ⋅ stock solution ⋅ swirl to mix and cool in water bath ⋅ read color intensity at 410 nm Phosphate Molybdic acid ⋅ to plastic tube, add 5 ⋅ .5 molar NaHCO3: 1 liter of distilled water ⋅ blue color (P) (4,5,6) ml of sample, 10 ml to 42 g NaHCO3-let stand over night develops; run of water, and 5 ml of ⋅ reagent A: dissolve 12 g ammonium transmittance at reagent B; swirl molybdate in 250 ml H2O, dissolve .2908 880 nm vigorously g antimony potassium tartrate in 100 ml ⋅ leave 15 min and H2O, mix 148ml conc. H2SO4 in 1 l H2O; read on mix all three together and bring to volume spectrophotometer at at 2000ml with H2O 880 nm ⋅ reagent B: dissolve .528 g of ascorbic acid in 100 ml reagent A

45 ⋅ stock phosphorus

Potassium Atomic ⋅ make a 1:16 dilution ⋅ standard K solutions ⋅ run on AA (K) Absorption (7) and determine the K concentration on the A.A ⋅ Can be used for any extraction method K Tetraphenylborat ⋅ take 3ml of EDTA- ⋅ ammonium ⋅ sodium tetraphenylborate solution: add 5 ⋅ take optical e- turbidometric formaldehyde ⋅ cesium grams sodium tetraphenylboron solution to density reading at procedure (8) solution and add 1ml ⋅ rubidium 50ml H2O transfer solution to 100ml 420 nm 15-20 of sample volumetric flask and add 10 ml .1 N min after mixing ⋅ add three drops gum NaOH. Fill to volume with H2O ghatti solution and ⋅ alkaline EDTA solution 17: 7.5g of mix ethylenediaminetetraacetic acid disodium ⋅ add 1 ml of salt in 500ml volumetric flask add 44ml 1 tetraphenylborate N NaOH, dilute to mark with H2O solution rapidly with ⋅ folmaldehyde solution: 37% formaldehyde blowout pipet is diluted 1:1 with H2O ⋅ mix contents and ⋅ EDTA-formaldehyde solution: prepare allow to sit for 15 fresh by mixing 2parts EDTA with 1 part min formaldehyde ⋅ gum ghatti solution: 10 grams of gum ghatti are placed in gause bag and suspended in a beaker of 400ml H2O so that upper surface of bag is just covered by H2O. Left over night. Bag discarded, solution mixed, stand for couple of hours

46 Nutrient Procedure Name Procedure Interferences Reagents End Point and References K Tetraphenylborat ⋅ place a 2.5 g sample ⋅ Sodium hydroxide: dissolve 20 g NaOH in ⋅ titrate to blue e -Volumetric of fertilizer in 250 ml 100 ml H2O color with Determination volumetric flsk and ⋅ Reagent grade formaldehyde cetyltrimethyl- (9,10,11,12) add 50 ml 4% ⋅ Sodium tetraphenylborate(TPB): dissolve ammonium ammonium oxalate 23 g TPB in 800 ml H2O, add 20 g bromide solution and 125 ml water Al(OH)3, stir 10 min and filter; add 2 ml ⋅ boil for 30 min and of 20% NaOH to filtrate and dilute to 1 make slightly liter with water, mix well alkaline with NaOH ⋅ cetyltrimethylammonium bromide: ⋅ cool, dilute to dissolve 2.5 g of reagent in H2O and dilute volume with water, to 100 ml mix and pass through ⋅ bromophenol blue indicator: dissolve .040 dry filter or allow to g tetrabromophenolsulfonphthalein in 3 ml stand until clear of .1 N NaOH and dilute to 100 ml with ⋅ transfer a 15 ml H2O aliquot to 50 ml ⋅ K Cl crystals volumetric flask, add ⋅ 4% ammonium oxalate 2 ml 20% NaOH, 5 ml formaldehyde, 1 ml TPB for each 2% potash expected, 2 ml TPB inexcess, ⋅ dilute to volume with water, mix and allow to stand for 5-10 min, pass through

47 dry filter K Tetraphenylborat ⋅ 15 ml aliquot of ⋅ excess NH4 ⋅ 20% NaOH ⋅ titrate with e - titrimetric sample in 100ml ⋅ formaldehyde NH4Cl to pink procedure (13,14) volumetric ⋅ Sodium tetraphenylborate endpoint ⋅ add 2ml of 20% ⋅ Clayton yellow indicator NaOH and 5ml ⋅ Quaternary NH4Cl formaldehyde ⋅ Potassium dihydrogen phosphate ⋅ add 1ml STPB fo each %K expected and then add 8ml more ⋅ dilute to volume, wait 10 min and filter ⋅ to 50ml aliquot of filtrate in 125 ml Erlenmeyer flask add 6-8 drops Clayton yellow ind. ⋅ titrate with NH4Cl

48 Nutrient Procedure Name Procedure Interferences Reagents End Point and References K Tetraphenylborat ⋅ adjust the pH of the ⋅ ammonium ⋅ dilute NaOH ⋅ measure e-colorimetric sample to between 4 ⋅ cesium ⋅ dilute H2SO4 absorbence of

method (15) and 5 ⋅ rubidium ⋅ sodium tetraphenylborate solution: disolve unprecipitated ⋅ in centrifuge tube, 1 gram of reagent and .5 gram aluminum tetraphenyl add 5 ml of sample chloride hexahydrate or aluminum nitrate borate at 266 nm and 5 ml of hexahydrate in 100ml of H2O - filter to tetraphenylborate remove any turbidity solution ⋅ cold saturated potassium salt wash ⋅ centrifuge for three ⋅ 75% acteonitrile, 25% H2O solution minutes and decant the supernant ⋅ wash precipitate twice with 3ml cold saturated solution of potassium salt ⋅ dissolve precipitate by adding 5ml acetonitrile solution ⋅ transfer to 25ml volumetric and fill to volume with H2O ⋅ measure absorbance at 266 K picrate (16,17) ⋅ precipitate K from ⋅ Rb, Cs, ⋅ dipicrylaminate ⋅ measure neutral or slightly NH4, Tl ⋅ ether absorbance at alkaline solution 400 nm

49 with Na, Li, Mg, or ⋅ acetone Ca dipicrylaminate ⋅ red precipitate is washed with ether and dissolved in acetone ⋅ dilute with H20 and adjust pH to 10. ⋅ measure absorbance at 400nm K Cobaltinitrite- ⋅ 10ml aliquot of ⋅ NH4 ⋅ Trisodium cobaltinitrite solution: 10g in ⋅ read at 425 nm colorimetric (18) sample H2O ⋅ compare against ⋅ add 1ml 1 N HNO3 ⋅ Potassium dichromate standard solution blank ⋅ add 5ml trisodium ⋅ Nitric acid (1 N) cobaltinitrite ⋅ Nitric acid (.01 N) ⋅ stand for 2 hours ⋅ centrifuge, decant and wash with 15ml .01 N HNO3 ⋅ centrifuge, decant, add with mixing 10ml K2Cr2O7 and 5ml H2SO4 ⋅ cool and fill with distilled H2O to 100ml

50 Nutrient Procedure Name Procedure Interferences Reagents End Point and References K Cobaltinitrite- ⋅ Extract 7.5 g soil ⋅ 25% NaNO3 solution ⋅ read at 700 nm turbidimetric (19) with 15 ml 25 % ⋅ Reagent grade isopropylalcohol NaNO3 solution ⋅ solid sodium cobaltinitrite ⋅ take 5.0 ml filtrate add 3 ml isopropyl alcohol ⋅ add 0.1 g sodium cobaltinitrite and shake vigorously

K Cobaltinitrite- ⋅ 2.0 ml aliquot of ⋅ NH4 ⋅ Sodium cobaltinitrite: 6.25 g cobaltinitrite ⋅ compare against turbidimetric (20) extract and 75 gm NaNO2 in 175 ml H20. Add standards

51 ⋅ add 6 drops of 5.0 ml 99.5%acetic acid allow to stand formaldehyde, mix overnight and dilute to 250 ml. Store in and allow to stand glass stoppered pyrex bottle for 5 min ⋅ Reagent Grade Isopropyl alcohol ⋅ add 1.0 ml sodium ⋅ 37% Formaldehyde cobaltinitrite solution mix well ⋅ add 2.0 ml isopropylalcohol down side of tube forming a double layer ⋅ mix by rapid swirling for 30 sec. ⋅ let stand 25 min and measure

52 Nutrient Procedure Name Procedure Interferences Reagents End Point and References K Cobaltinitrite- ⋅ extract 30 g soil with ⋅ 20% sodium cobaltinitrite with 4% sodium ⋅ titrate to endpoint volumetric (21) 150 cc ammonium acetate added in. acetate ⋅ permanganate and oxalate (according to ⋅ 10 cc of filtrate Volk and Truog added rapidly to 10 cc 20% soldium cobaltinitrite solution ⋅ Refrigerate for 5 hours at 3 degrees C ⋅ Filter and wash with cold wate ⋅ Titrate with standard permanganate and oxalate K periodate (22) ⋅ dissolve chloride- ⋅ chlorides ⋅ periodic acid solution ⋅ titrate with .1 N free sample in 4-5ml ⋅ solvent - equal volumes of aldehyde-free arsenite solution H2O ethanol and anhydrous ethyl acetate ⋅ add 1 g periodic acid ⋅ borax solution - 5 g. boric acid and 5 g of dissolved in 3ml H20 borax ⋅ add 90ml of solvent ⋅ potassium iodide ⋅ allow to stand in ice ⋅ .1 N arsenite solution bath while stirring ⋅ filter in scintered- glass crucible and wash with anhycrous

53 ethyl acetate cooled to 0 degrees ⋅ place crucible in 250ml beaker and add 125 ml of borax solution ⋅ after precipitate dissolves, add 3g potassium iodide and titrate with .1 N arsenite solution Boron (B) ICP (23,24) ⋅ 5 grams of soil are hot water ⋅ run on ICP extracted on the espresso machine using 50 ml water. ⋅ sample is run on the ICP B Azomethine-H ⋅ to 4 ml sample add 1 ⋅ Buffer-masking reagent: dissolve 280 g ⋅ read at 420 nm (25,26) ml of buffer-masking ammonium acetate, 20 g potassium reagent and 1 ml of acetate, 20 g tetrasodium salt of azomethine-H ethylenedinitrilo tetraacetic acid, 8 g reagent nitrilotriacetic acid in 240 ml H2O, slowly ⋅ mix and allow to add 70 ml acetic acid stand one hour ⋅ Azomethine-H reagent: dissolve 0.9 g ⋅ read at 420 nm azomethine-H, 2 g ascorbic acid in 10 ml H2O with gently heating, fill to 100ml with H2O ⋅ Standard boron solution

54 Nutrient Procedure Name Procedure Interferences Reagents End Point and References B Azomethine-H ⋅ 10 g. soil/ 4 ml ⋅ unclear ⋅ Azomethine-H Reagent: .9g Azomethine- ⋅ Read at 430 nm (w/strong acid) extract filtrate (boil H, 2g ascorbic acid, H2O, ⋅ color? (27,28,29,30) ⋅ 1 ml Buffer/1ml .1% CaCl2- ⋅ Buffer Masking Solution: 250g H2O blank Azomethine-H H2O to help NH4(C2H3O2), 25g tetrasodium salt of solution clear) tetraacetic acid, 10g disodium salt of ⋅ mix immediately, nitrilotriacetic acid, 400 ml H2O cap and stand 1 hour ⋅ Boron Standard B Curcumin ⋅ 1ml of slightly acid ⋅ nitrate, if ⋅ ethanol ⋅ read at 540 nm procedure (31) aqueous solution in present in ⋅ Curcumin-Oxalic Acid reagent: .04 g light within two 250ml beaker large finely ground curcumin, 5 g H2C2O4-2H2O hours ⋅ add 4ml curcumin- quantities in ethanol oxalic acid solution ⋅ Boron standard solution and mix ⋅ evaporate to dryness ⋅ cool and add 25ml ethanol ⋅ triturate and filter B Carminic acid ⋅ 2ml of each sample ⋅ HCl ⋅ will be red, blue, (32,33) in plastic beaker ⋅ H2SO4 or bluish red ⋅ place each beaker on ⋅ Carmine reagent: 460mg carmine40 in 1 L ⋅ measure ice bath H2SO4 absorbance at ⋅ add .1ml HCl and ⋅ Stock boric acid for standards 585 nm in a 1 cm mix cell with a blank ⋅ add 10 ml H2SO4 and for reference mix

55 ⋅ add 10ml carmine reagent and mix ⋅ let stand 2 hours

B Dodd’s Method ⋅ Ash sample in NaOH ⋅ Sodium Hydroxide (NaOH) ⋅ phenothalien/met (34) and obtain boric acid ⋅ .4 N CaOH hyl red

⋅ add 5ml CaCl2-6H2O ⋅ CaCl2-6H2O ⋅ find the ⋅ make alkaline with ⋅ invert sugar - sucrose difference in volume between NaOH until faint ⋅ H2O pink appears and two end points. ⋅ H2SO4 stays ⋅ NaOH ⋅ let stand and filter ⋅ methyl red ⋅ 75 ml of filtrate and ⋅ phenothalien three drops H2SO4 ⋅ titrate with NaOH to neutral ⋅ add sugar ⋅ titrate to phenothalien end point

56 Nutrient Procedure Name Procedure Interferences Reagents End Point and References B Munsell’s ⋅ Ignite in muffle ⋅ H2SO4 ⋅ Blue Quinalizarin Quinalizarin furnace ⋅ Quinalizarin Solution: .01g quinalizarin in color Method (34) ⋅ add 2ml CaOH2 H2O ⋅ run on ⋅ reignite and ⋅ dilute H2SO4 spectrometer evaporate to dry ⋅ CaOH ⋅ take up residue in 5- ⋅ Boric Acid Standard 16 ml dilute H2SO4 ⋅ stir and centrifuge ⋅ transfer 1 ml to test tube ⋅ add 9ml H2SO4 and 0.5ml quinalizarin ⋅ leave overnight Sulfate KCL Extract (35) ⋅ weigh 12.5 g soil in ⋅ 1.0 N KCl: dissolve 74.56 g reagent grade ⋅ run on ICP (S) to 125 ml KCl in water and bring to volume of 1 liter Erlenmeyer flask ⋅ add 25 ml of 1.0 N KCL and shake for 1 hour ⋅ filter through rapid filter paper ⋅ determine on ICP S Turbidimetric ⋅ 20 ml aliquot of ⋅ 5-100 mg of ⋅ BaCl2- gelatin ⋅ 2cm light path (36,37,38,39,40) sample (filtered S per sample ⋅ HCl (Photoelectric through membrane) colorimeter?)

57 in 50ml Erlenmeyer flask ⋅ treat with 2 ml of .5 mol HCl ⋅ swirl in flask and treat with BaCl2- gelatin reagent ⋅ after 30 minutes, swirl again and read S Double ⋅ Excess of Barium ⋅ BaCr (Barium Chromate) ⋅ free chromate precipitation (36) chromate is added to ⋅ NH4OH determined at precipitate barium 436 nm sulfate ⋅ excess Ba is precipitated with dilute NH4OH. ⋅ remove both precipitates, free chromate is left in solution S chloranilate (36) ⋅ react barium ⋅ cation ⋅ analyze on chloranilate in interference spectrometer for alcohol solution chloranilate ion. (Can do 15 per hour)

58 Nutrient Procedure Name Procedure Interferences Reagents End Point and References Zinc (Zn) DTPA (41) ⋅ weigh 12.5 g soil ⋅ DTPA extraction solution: weigh 1.96 g ⋅ run on ICP into 50 ml centrifuge DTPA into a liter volumetric flask. Add tube 14.92 g CaCl2*2H2O and bring to 950 ml ⋅ add 25 ml of DTPA with distilled water, add 26.28 g ⋅ shake for 2 hours on CaCl2*H2O and adjust pH to 7.3 with 6 N reciprocating skaker NaOH or 6 N H Cl; bring to final volume ⋅ centrifuge for 5 min of 1 liter and filter through medium fast filter paper ⋅ determine element concentrations on the ICP Zn Zincon method ⋅ 10ml aliquot of ⋅ Cd in conc. ⋅ zinc free water ⋅ blue colored (18) sample >1 mg/l ⋅ stock zinc solution complex ⋅ add .5g sodium ⋅ Al >5 ⋅ standard zinc solution proportional to ascorbate, mix ⋅ Mn > 5 ⋅ sodium ascorbate the amount of ⋅ 1ml KCN, mix ⋅ Fe (III) > 7 ⋅ potassium cyanide solution zinc ⋅ 5ml buffer solution, ⋅ Fe (II) > 9 ⋅ Buffer solution: dilute NaOH, ptoassium ⋅ measure mix ⋅ Cr > 10 chloride, boric acid absorbance at ⋅ 3ml zincon solution, ⋅ Ni > 20 ⋅ zincon reagent: powdered 2-carboxy-2- 620 nm mix ⋅ Cu > 30 hydroxy-5-sulfoformazyl benzene in ⋅ 3ml chloral hydrate ⋅ Co > 30 methyl alcohol solution, mix ⋅ CrO4 > 50 ⋅ chloral hydrate solution ⋅ measure absorption ⋅ HCl

59 exactly 5 min after ⋅ NaOH adding the chloral hydrate Zn Gravimetric ⋅ render the water ⋅ only zinc in ⋅ NH4OH ⋅ compare to Determination slightly alkaline with solution is ⋅ potassium ferro-cyanide standards (42) ammonium hydrate detected ⋅ boil and filter ⋅ add a few drops of potassium ferro- cyanide

60 Nutrient Procedure Name Procedure Interferences Reagents End Point and References Zn Dithizone ⋅ 10ml aliquot of ⋅ Ferric iron, ⋅ zinc free H2O ⋅ can read red color (18,43,44) solution at pH 2-3 residual ⋅ stock zinc solution of zinc ⋅ add 5ml of acetate chlorine, ⋅ standard zinc solution dithizonate at buffer and 1 ml other ⋅ .02 N HCl 535nm or green sodium thiosulfate oxidizing ⋅ 2 N sodium acetate color of solution and mix agents ⋅ Acetic acid unreacted ⋅ pH should be convert ⋅ Acetate buffer solution: sodium acetate dithizone at 620 between 4-5.5 dithizone to and acetic acid solution nm yellow- ⋅ add 10ml dithizone ⋅ sodium thiosulfate solution brown color solution, stopper and ⋅ Dithizone Solution (I): diphenyl shake for 4 min. thiocarbazone in carbon tetrachloride ⋅ allow layers to ⋅ Dithizone Solution (II): dithizone (I) in separate and dry CCl4 funnel stem with ⋅ Carbon tetrachloride strips of filter paper ⋅ Sodium citrate solution ⋅ run the lower CCl4 layer into a clean dry absorption cell Iron (Fe) DTPA extraction ⋅ weigh 12.5 g soil ⋅ DTPA extraction solution: weigh 1.96 g ⋅ run on ICP (41) into 50 ml centrifuge DTPA into a liter volumetric flask. Add tube 14.92 g CaCl2*2H2O and bring to 950 ml ⋅ add 25 ml of DTPA with distilled water, add 26.28 g ⋅ shake for 2 hours on CaCl2*H2O and adjust pH to 7.3 with 6 N reciprocating skaker NaOH or 6 N H Cl; bring to final volume ⋅ centrifuge for 5 min of 1 liter

61 and filter through medium fast filter paper ⋅ determine element concentrations on the ICP Fe Aqua Regia (42) ⋅ acidify a suitable ⋅ aqua regia: mixture of nitric and ⋅ compare with volume with aqua hydrochloric acid that dissolves gold or known standards regia platinum ⋅ concentrate to 100ml ⋅ ammonium sulfocyanate solution ⋅ place in Nessler jar ⋅ standard iron solution and add 2ml of ammonium sulfocyanate solution

62 Nutrient Procedure Name Procedure Interferences Reagents End Point and References Fe Mercaptoacetic ⋅ 100ml portion of ⋅ aluminum ⋅ Mercaptoic Acid Solution ⋅ Determine Acid (47) well-shaken sample ⋅ chromate ⋅ Ammonium Citrate Solution transmittance at in 250ml beaker ⋅ nitrite ⋅ Ammonium Hydroxide Solution 535 nm ⋅ add 2-3 drops HCl ⋅ cyanide ⋅ Standard Iron Solution ⋅ evaporate to about ⋅ (the first 25ml three are ⋅ cool addressed by ⋅ add 5ml mercaptoic the acid soluiton and mix procedure) ⋅ add 2ml ammonium citrate solution and mix ⋅ add 5ml ammonium hydroxide and mix ⋅ transfer to 50ml volumetric flask and bring to volume Fe Bipyridine (18) ⋅ digest sample ⋅ Iron free distilled H2O ⋅ reddish- purplish ⋅ 10 ml aliquot of ⋅ Bipyridine solution: .1 g bipyridine in HCl ⋅ compare to sample ⋅ Sodium sulfite solution previously made ⋅ add with mixing: 2 ⋅ HCl, 5 N standards or use ml dipyridyl ⋅ Mercuric chloride solution colorimeter solution, 1 ml ⋅ Iron standard solution ⋅ compare with Na2SO4, .1ml of blank HCl

63 ⋅ wait 5 min Fe Gravimetric ⋅ Heat suitable size ⋅ Aluminum, ⋅ Ammonium hydroxide ⋅ Ignite residue to Determination sample to boiling phosphate, ⋅ Ammonium chloride wash solution constant weight (18) ⋅ add 1ml HNO3 silicate ⋅ HNO3 at 1000 degrees C ⋅ add 1:1 NH4OH slowly with constant stirring until in excess (can tell by odor) ⋅ settle precipitate and wash with hot distilled H2O ⋅ filter and wash precipitate with hot NH4Cl wash solution ⋅ Dry

64 Nutrient Procedure Name Procedure Interferences Reagents End Point and References Fe Preliminary ⋅ 100ml of sample in it’s getting rid ⋅ Hydroxylamine Hydrochloride Solution Digestion (46) 250ml Erlenmeyer of ⋅ pH 4 Acetic Acid Buffer Solution flask interferences ⋅ Ammonium Acetate ⋅ add one boiling bead ⋅ Glacial Acetic Acid to flask ⋅ Sulfuric Acid ⋅ add 2ml HCl ⋅ Hydrochloric Acid ⋅ add 2ml hydroxylamine hydrochloride reageant ⋅ heat to near boiling ⋅ evaporate to 20ml ⋅ cool to room temperature and add 50ml distilled H2O ⋅ after residue has dissolved, dilute to 100ml and use 50 mls for color development Fe Phenanthroline ⋅ digest sample in acid metal ions: ⋅ Stock iron solution orange-red (47, 44) ⋅ 10ml aliquot into chromium, ⋅ Standard iron solution precipitate separatory funnel copper, ⋅ Hydroxyamine-hydrochloride solution measure (can add distilled to nickel, cobalt, ⋅ Sodium acetate solution absorbance at bring to volume if zinc, ⋅ Phenanthroline solution 520nm use 5cm cadmium, absorption cell for

65 necessary) mercury, ⋅ HCl amounts of <100 ⋅ add 15ml conc. HCl phosphate, ⋅ Diisopropyl ether mg and 1 cm cell ⋅ extract 3 times with polyphosphat ⋅ H2SO4 for amounts of iron 25ml isopropyl ether, e, fluoride, between 100 mg save the ether layer citrate, and 500 mg in original funnel tartrate, run blank with ⋅ add 25ml H2O to oxalate procedure isopropyl mixture, (eliminated in shake and transfer extraction lower aqueous phase procedure) to 100ml volumetric flask, repeat ⋅ to H2O mixture, add 1ml NH2OH-HCl, 10ml phenanthroline solution, 10 ml sodium acetate solution ⋅ dilute to 100mls and stand for 10 min.

66 REFERENCES 1. Sims, J.R.; Jackson, G.D. Rapid Analysis of Soil Nitrate with Chromotropic acid. Soil Sci. Soc. Amer. Proc. 1971, 35, 603-606. 2. Soltenpour, P.N.; Workman, S. A New Soil Test for Simultaneous Extraction of Macro and Micro Nutrients in Alkaline Soils. Commun. Soil Sci. Plant Anal. 1977, 8, 195-207. 3. Haby, V.A. Soil NO3-N Analysis in Ca(OH)2 Extracts by the Chromotropic Acid Method. Soil Sci. Soc. Am. J. 1989, 53, 308-310. 4. Olsen, S.R.; Cole, C.V.; Watanabe, F.S.; Dean, L.A. Estimation of Available Phosphorus in Soils by Extraction with Sodium Bicarbonate, No 939; USDA: 1954. 5. Thomas, G.W.; Peaslee, D.E. Testing Soils for Phosphorus. In Soil Testing and Plant Analysis; Walsh, L.M., Ed; Soil Science Society of America: Madison, WI, 1973; 115- 131. 6. Wantanabe, F.S.; Olsen, S.R. Test of Ascorbic Acid Method for Determining Phosphorus in Water and NaHCO3 Extractants for Soil. Soil Sci. Soc. Amer. Proc. 1965, 29, 677-678. 7. Council on Soil Testing and Plant Analysis. Determination of Potassium, Magnesium, Calcium, and Sodium by Neutral Ammonium Acetate Extraction. In Handbook on reference methods for soil testing (revised edition); Council on Soil Testing and Plant Analysis, Ed; The Council on Soil Testing and Plant Analysis: Athens, GA, 1980; 58-63. 8. Sunderman, F.W.; Sunderman, F.W. Studies in Serum Electrolytes: A Rapid, Reliable Method for Serum Potassium using Tetraphenylboron. Am. J. Clin. Path. 1958, 29, 95- 103. 9. Cluley, H.J. The Determination of Potassium by Precipitation as Potassium Tetraphenylboron and its Application to Silicate Analysis. Analyst 1955, 80, 354-364. 10. Dill, A.J.; Popovych, O. Solubility Products of Potassium and Triisoamyl-n- butylammonium Picrates and Tetraphenylborates in Ethanol-water Mixtures at 25 Degrees Celsius. J. Chem. Eng Data 1969, 14, 240-243. 11. Gloss, G.H. Sodium Tetraphenylboron: A New Analytical Reagent for Potassium, Ammonium, and Some Organic Nitrogen Compounds. Chemist-Analyst 1953, 42, 50-55. 12. Schall, E.D. Volumetric Determination of Potassium. Anal. Chem. 1957, 29, 1044- 1046. 13. Kallman, S. Alkali Metals: Determination: Potassium. In Treatise on Analytical Chemistry Part 2 Volume 1. I.M. Kolthoff; P.J. Elving, Eds.; Interscience Encyclopedia, New York, NY, 1959; 369-378. 14. Welcher, F.J. Fertilizers. In Standard Methods of Chemical Analysis 6th Edition Volume 2 pt B. N.H. Furman, Ed.; Van Nostrand, Princeton, 1966; 1483-1506. 15. Pflaum, R.T; Howick, L. Spectrophotometric Determination of Potassium with Sodium Tetraphenylborate. Anal. Chem. 1956, 28, 1542-1544. 16. Caley, E.R. The Rapid Colorimetric Estimation of Potassium. J. Am. Chem. Soc. 1931, 5, 539-45. 17. Marczenko, Z. Alkali Metals. In Separation and Spectrophotometric Determination of Elements. Halstead Press: New York, 1986;123-128. 18. American Public Health Association. Physical and Chemical Examination of Natural and Treated Waters in the Absence of Gross Pollution. Standard Methods for the Examination of Water and Wastewater: Including Bottom Sediments and Sludges. American Public Health Association: NY, 1970; 33-366.

67 19. Olson, R.V. A Turbidimetric Potassium Determination Affected Little by Temperature. Soil Sci. Soc. Amer. Proc. 1953, 17, 20-22. 20. Peech, M.; English, L. Rapid Microchemical Soil Tests. Soil Sci. 1945, 57, 167-195. 21. Volk, N.J. The Determination of Small Amounts of Exchangeable Potassium in Soils, Employing the Sodium Cobaltinitrite Procedure. J. Am. Soc. Agron. 1941, 33, 684. 22. Kolthoff, I.M.; Stenger, V.A. Iodometry of Inorganic Substances. In Volumetric Analysis Vol. 3. I.M. Kolthoff; R. Belcher; V.A. Stenger; G. Matsuyama, Eds. Interscience Publishers: New York, 1942; 245-274. 23. Hanks, D.; Webb, B.; Jolley, V. A Comparison of Hot Water Extraction to Standard Extraction Methods for Nitrate, Potassium, Phosphorus, and Sulfate in Arid-Zone Soils. Commun. Soil Sci. Plant Anal. 1997, 28, 1393-1402. 24. Webb, B.L.; Hanks, D.H.; Jolley, V.D. A Pressurized Hot Water Extraction Method for Boron. Commun. Soil Sci. Plant Anal. 2002, 33, 31-39 25. Wolf, B. The Determination of Boron in Soil Extracts, Plant Materials, Composts, Manures, Water and Nutrient Solutions. Commun. Soil Sci. Plant Anal. 1971, 2, 363- 374. 26. Wolf, B. Improvements in the Azomethine-H Method for the Determination of Boron. Commun. Soil Sci. Plant Anal. 1974, 5, 39-44. 27. Lopez, F.J.; Gimenez, E.; Hernandez, F. Analytical Study on the Determination of Boron in Environmental Water Samples. Fresenius’ J. Anal. Chem. 1993, 346, 984-987. 28. Harp, D.L. Modifications to the Azomethine-H Method for Determining Boron in Water. Anal. Chim. Acta. 1997, 346, 373-379. 29. Soil and Plant Analysis Council. Hot Water Boron Extraction. In Soil Analysis Handbook of Reference Methods. CRC Press: Boca Raton, 2000; 118-120 30. Zaijun, L.; Zhu, Z.; Jan, T.; Hsu, C.; Jiomai, P. 4-Methoxy-azomethine-H as a Reagent for the Spectrophotometric Determination of Boron in Plants and Soils. Anal. Chim. Acta. 1999, 402, 253-257. 31. Jackson, M.L. Boron Determinations for Soils and Plant Tissue. In Soil Chemical Analysis. Prentice Hall: New Jersey, 1958; 370-387. 32. Adams, V.D. Methods for the Determination of Inorganics and Nonmetals. In Water and Wastewater Examination Manual. Lewis Publishing: MI, 1990; 73-162. 33. Van Loon, J.C. Dissolved Oxygen and Minor Nutrients. In Chemical Analysis of Inorganic Constituents of Water. CRC Press: Boca Raton, 1982; 201-203. 34. Piper, C.S. The Determination of the Trace Elements. In Soil and Plant Analysis; A Laboratory Manual of Methods for the Examination of Soils and the Determination of the Inorganic Constituents of Plants. Interscience Publishers: NY, 1944; 302-367. 35. Williams, C.H.; Steinbergs, A. The Evaluation of Plant-Available Sulphur in Soils. II. The Availability of Adsorbed and Insoluble Sulphates. Plant Soil 1964, 21, 50-62. 36. Nollet, L. Sulfate, Sulfite, Sulfide. In Handbook of Water Analysis. Marcel Dekker: NY, 2000; 195-199. 37. Chesnin, L.; Yien, C.H.; Turbidimetric Determination of Available Sulfates. Soil Sci. Soc. Amer. Proc. 1950, 15, 149-151. 38. Thomas, J.F.; Cotton, J.E. A Turbidimetric Sulfate Determination. Water and Sewage Works 1954, 101, 462-465. 39. Treon, J.F.; Crutchfield, W.E. Rapid Turbidimetric Method for Determination of Sulfates. Ind. Eng. Chem., Anal. Ed. 1942, 14, 119-121.

68 40. Sheen, R.T.; Kahler, H.L.; Ross, E.M. Turbidimetric Determination of Sulfate in Water. Ind. Eng. Chem., Anal. Ed. 1935, 7, 262-265. 41. Lindsay, W.L.; Norwell, W.A. Development of a DTPA Soil Test for Zinc, Iron, Manganese and Copper. Soil Sci. Soc. Amer. Proc. 1978, 42, 421-428. 42. Mason, W.P. Chemical Examination of Water. In Examination of Water (Chemical and Bacteriological). J. Wiley and Sons: NY, 1899; 7-92. 43. Golterman, H.L. Trace Elements; B, Co, Cu, Fe, Mn, Mo, V, Zn. In Methods for Chemical Analysis of Fresh Waters. International Biological Programme: Oxford, 1969; 79-92. 44. Sandell, E.B. Zinc. In Colorimetric Determination in Traces of Metals. Interscience Publishers: NY, 1959; 941-965. 45. McCoy, J.W. Mineral Content. In Chemical Analysis of Industrial Water. Chemical Publishing Co.: NY, 1969; 47-97. 46. Jackson, G.B. Iron Measurement. In Applied Water and Spentwater Chemistry. Van Nostrand: Reinhold, NY, 1993; 415-425. 47. American Public Health Association. Determination of Inorganic, Nonmetallic Constituents. In Standard Methods for the Examination of Water and Wastewater: Including Bottom Sediments and Sludges. American Public Health Association: NY, 1985; 265-290.

Appendix III

Description of Pressurized Hot-Water Extraction/Standard Extraction and Analytical Methods for N, P, K, pH, Organic Matter, Acid Neutralization Potential and Texture

70 Pressurized Hot-Water Method

Extraction The pressurized hot-water extraction (1) is performed on a Melitta® MEX1B espresso machine. The MEX1B generates an approximate pressure of 2.5 bars and an approximate temperature of 90° Celsius (2). To extract a sample, force 100.0 ml of distilled water through 5.0 g of soil on a 5.0 cm VWR brand medium filter paper and collect the extract in a plastic cup

Procedure Place the filter paper into the filter basket, being sure that all holes are covered and no gaps occur between the paper and the side of the basket. Place the sample into the basket and replace the filter basket holder on the machine, making sure to lock the holder into place by rotating it to the right. Add 100.0 ml distilled water to the water reservoir through the opening on top of the machine. Screw the lid down firmly. Place a labeled plastic sample cup underneath the filter basket and turn the knob on the side of the machine to “brew.” The extraction process takes about four minutes and during this time, pressurized water will be forced through the soil sample in the filter basket and will empty into the plastic cup underneath. Wait until the water has finished dripping from the filter basket (the machine will make a soft “click”) and turn the knob on the left side of the machine completely to the front to release the pressure in the water reservoir. When steam ceases to exit the steam tube, turn the knob to “off.” Remove the sample cup from under the filter basket and place the sample cup to the side. Rotate the filter basket to unlock and release it from the machine. While holding the basket in the holder with a thumb pressed against the black plastic lever, turn the basket and holder upside-down and tap firmly against the inside of a trash can to dislodge and dispose of the filter paper. Rinse the basket with distilled water and place the next filter paper into the basket using the water present to seat the paper. Run the machine once with water but without a soil sample in order to warm up the machine. Warming up the machine gives more consistent results for the first sample. The extract can be stored in a 125.0 ml wide-mouth polypropylene nalgene bottle.

Pressurized Hot-Water (PHW) Procedural Sequence: After obtaining a soil extract from the soil extractor, perform the tests for potassium and phosphorus. These tests do not require clarification of the extract and should be done first. In order to perform the nitrate procedure, filter the remaining sample extract with 0.15 g Ca(OH)2. Add the Ca(OH)2 to the extract and swirl gently in the plastic cup until the mixture is cloudy. Filter the extract/Ca(OH)2 mixture through Whatman® medium filter paper set on the opening of a 125.0 ml wide mouth high density polyethylene nalgene bottle. Collect at least 6.0 ml of filtrate and discard the filter paper. The filtrate should be clear with an opalescent sheen on the water. If the sample is still colored, repeat the filtering process. After the filtrate is clear, follow the procedure for nitrate-nitrogen. The rest of the filtrate can then be discarded.

Nitrate-Nitrogen: Chromotropic acid (CTA)

71 Reagents: 0.1% solution of chromotropic acid disodium salt (CTA)(4,5-dihydiroxy-2,7- Naphthalenedisulfonic acid): Dissolve 0.184 g of CTA in 100 ml reagent grade H2SO4. (Do not heat the solution to dissolve.) Sulfite-urea solution: Dissolve five grams analytical grade urea and five grams of reagent grade anhydrous sodium sulfite in distilled water and dilute to 100 ml. Antimony sulfate solution: Dissolve 0.5 grams of Antimony (Sb) metal in 80 ml of concentrated H2SO4 at room temperature. Heat the solution until it is clear to dissolve the antimony metal. After the antimony is dissolved, let the solution cool and then bring it to volume with 20.0 ml of distilled water.

Procedure: The pressurized hot-water method utilizes the pressurized hot water extraction (1) and the chromotropic acid colorimetric analysis (3,4). Nitrate (NO3-N) working -1 standards are made from 1000 µg ml stock solution of NO3-N. Working standards of -1 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 µg ml NO3-N in 100 ml distilled water are made for the chromotropic acid acid procedure. In a 50 ml Erlenmeyer flask, place a 2.5 ml aliquot of filtrate, one drop of sulfite- urea solution and two ml of antimony-sulfate solution. Mix the reagents by swirling the flask for a couple of seconds and then add 1.0 ml of CTA solution and 4.5 ml of concentrated H2SO4 to the mixture. Allow the flask and its contents to cool in a water bath and then read the transmittance at 410 nm on the spectrophotometer.

Phosphorus: Molybdic acid

Reagents: Reagent A: Dissolve 12.0 g of ammonium molybdate in 250 ml of distilled water and in a separate volumetric flask, dissolve 0.2908 g of antimony potassium tartrate in 100 ml of distilled water. In a third flask add 148 ml of concentrated H2SO4 to distilled water and bring to volume at one liter to make 5 N H2SO4. Mix these three solutions together in a 2.0 L volumetric flask and bring the solution to a volume of 2000 ml with distilled water. Store in the dark at about 25º Celsius. Reagent B: Dissolve 0.528 grams of ascorbic acid in 100 ml of Reagent A. Reagent B should be made fresh every day.

Procedure: The pressurized hot-water method uses the pressurized hot-water extraction (1) and an ascorbic acid colorimetric analysis procedure (5). Phosphate working standards -1 3- were made from 1000 µg ml stock solution of (P04) . Working standards of 0.25, 0.5, -1 3- 1.0, 2.0, 3.0, 4.0 5.0, 6.0, and 7.0 µg ml (PO4) in 100 ml of distilled water were made for the molybdic acid procedure. In a 95 ml plastic test tube, combine a 5.0 ml aliquot of the soil extract with 10.0 ml of distilled water. Add 5.0 ml of Reagent B to the diluted extract and swirl the test tube vigorously to mix. Allow the solution to stand for 15 minutes and then read the transmittance on the spectrophotometer at 880 nm.

72 Potassium: Tetraphenylborate turbidimetric

Reagents: Ethelynediaminetetra acetic acid (EDTA) solution: Place 7.5 grams of EDTA in a 500 ml volumetric flask and then add 44 ml of 1.0 N NaOH to the flask. Dilute the solution to volume with distilled water. Formaldehyde solution: Dilute reagent grade formaldehyde 1:1 with distilled water. EDTA-Formaldehyde solution (prepared fresh daily): Mix two parts EDTA solution by volume with one part formaldehyde solution. Gum ghatti solution: Mix one gram of gum ghatti powder with 500 ml of distilled water. Solution must be shaken up before each use. Sodium tetraphenylborate solution: Dissolve 5.0 grams of reagent grade sodium tetraphenylborate in 50 ml of distilled water then pour the solution into a 100 ml volumetric flask and add 10.0 ml of 0.1 N NaOH. Using distilled water, bring the solution to volume at 100 ml. The solution should be a clear amber color. If the solution appears “dirty” or brown filter it using Whatman® #2 filter paper.

Procedure: The pressurized hot-water method couples the pressurized hot-water extraction and the sodium tetraphenylborate turbidimetric analysis. This potassium procedure is based on a sodium tetraphenylborate procedure for the quantification of blood serum potassium (6). Potassium working standards were made from 1000 µg ml-1 stock solution of K+. Working standards of 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 80, and 100 µg ml-1 K+ were made for the tetraphenylborate procedure. Filter the extract with 0.15 g activated carbon. To a small plastic sample cup add three ml of EDTA-Formaldehyde solution, 1.0 ml of filtered sample and three drops of gum ghatti solution. Mix ingredients and add 1.0 ml of tetraphenylborate solution with a pipettor. Transfer the contents of the sample cup to a cuvette and allow to stand for 15- 20 minutes. Analyze the transmittance of the suspended precipitate in the treated aliquot on the spectrophotometer at 420 nm.

Potassium: Cobaltinitrite turbidimetric

Procedure: This procedure is based on a turbidimetric procedure for the analysis of potassium suggested by Olson (7). Potassium working standards were made from 1000 µg ml-1 stock solution of K. Working standards of 2, 5, 10, 15, 20, 25, 30, 35, and 40 µg ml-1 K+ were made for the cobaltinitrite procedure. The curve was run at least eight times and due to some degree of variability, the values for each point on the curve were averaged and the standard curve was determined using the averaged point values. The curve was run using wavelengths between 600 and 750 nm. Below 580 nm, the spectrophotometer would not read the samples correctly, and above 750 nm the points on the curve became too close together. In a 95 ml plastic test tube, place a 5.0 ml aliquot of sample extract. Add 1.25 g sodium nitrate using a Fisher Scientific 1.23 cm3 plastic sample spoon and agitate the mixture gently until the sodium nitrate is dissolved. Next, add 3.0 ml isopropyl alcohol

73 and swirl to mix the solution. Add 0.1 g sodium cobalitinitrite using a 0.065 cm3 scoop and shake the tube until the sodium cobaltinitrite is completely dissolved (about 5 min). Read the transmittance of the solution on the spectrophotometer at 600 nm.

Standard Methods

Nitrate-nitrogen: The standard method for nitrate couples the shaking water extraction with a colorimetric chromotropic acid analysis procedure (3).

Reagents: 0.1% solution of chromotropic acid disodium salt (CTA)(4,5-dihydiroxy-2,7- Naphthalenedisulfonic acid): Dissolve 0.184 g of CTA in 100 ml reagent grade H2SO4. (Do not heat to dissolve) Sulfite-urea solution: Dissolve five grams analytical grade urea and five grams of reagent grade anhydrous sodium sulfite in distilled water and dilute to 100 ml. Antimony sulfate solution: Dissolve 0.5 grams of antimony (Sb) metal in 80 ml of concentrated H2SO4 at room temperature. Heat the solution until it is clear to dissolve the antimony metal. After the antimony is dissolved, let the solution cool and then bring it to volume with 20.0 ml of distilled water. NO3-N standard solutions: To develop the standard curve, use 0.5, 1.0, 2.0, 3.0, 4.0, -1 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 µg ml NO3-N in 100 ml distilled water standard solutions and complete the same procedure as found in the chromotropic acid analysis. Plot the log transmittance of each standard sample on the y-axis against the concentration on the x-axis.

Extraction: Place 10.0 g of soil in a 250 ml Erlenmeyer flask and add 0.25 g Ca(OH)2 using a 0.4 cm3 scoop. Then add 50.0 ml of distilled water and shake the solution for 15 minutes on a reciprocating shaker at 180 oscillations per minute. Filter the soil solution into a 15.0 cm Whatman® medium filter paper and collect the extract in a 70 ml plastic cup.

Analysis: In a 50 ml Erlenmeyer flask, place a 2.5 ml aliquot of filtrate, one drop of sulfite- urea solution and two ml of antimony-sulfate solution. Mix the reagents by swirling the flask and then add 1.0 ml of CTA solution and 4.5 ml of concentrated H2SO4 to the mixture. Allow the flask and its contents to cool in a water bath and then read the transmittance at 410 nm on the spectrophotometer.

Phosphorus:

The standard method for phosphate couples extraction with a 0.5 M sodium bicarbonate solution with analysis using an ascorbic acid procedure (5).

Reagents:

74 0.5 M sodium bicarbonate extracting solution: In a 1.0 L volumetric flask, dissolve 42 g of NaHCO3 in distilled water and let the solution stand overnight to equilibrate. The solution should be stored in a plastic container and the pH adjusted to 8.2 before each use with 6.0 N NaOH and 6 N HCl. Reagent A: Dissolve 12.0 g of ammonium molybdate in 250 ml of distilled water and in a separate volumetric flask, dissolve 0.2908 g of antimony potassium tartrate in 100 ml of distilled water. In a third flask add 148 ml of concentrated H2SO4 to distilled water and bring to volume at one liter to make 5 N H2SO4. Mix these three solutions together in a 2.0 L volumetric flask and bring the solution to a volume of 2000 ml with distilled water. Store in the dark at about 25º Celsius. Reagent B: Dissolve 0.528 grams of ascorbic acid in 100 ml of Reagent A. Reagent B should be made fresh every day. 3- (PO4) standard solutions: To make curve, use 0.25, 0.5, 1.0, 2.0, 3.0, 4.0 5.0, 6.0, and -1 3- 7.0 µg ml (PO4) in 100 ml bicarbonate standard solutions and complete the same procedure as found in the ascorbic acid analysis. To find the equation for the standard curve, plot the log transmittance of each standard sample on the y-axis against the concentration on the x-axis.

Extraction: In a 250.0 ml Erlenmeyer flask, mix 5.0 g of soil and 50 ml of sodium bicarbonate (pH 8.2). Shake the solution for 30 minutes on a reciprocating shaker at 180 oscillations per minute and filter the soil solution into 15.0 cm VWR Brand medium fast filter paper. Collect the extract in a 70 ml plastic cup.

Analysis: In a 95 ml plastic test tube, combine a 5.0 ml aliquot of the soil extract with 10.0 ml of distilled water. Add 5.0 ml of Reagent B to the diluted extract and swirl the test tube vigorously to mix. Allow the solution to stand for 15 minutes and then read the transmittance on the spectrophotometer at 880 nm.

Potassium: The standard procedure for potassium couples the ammonium acetate potassium extraction procedure with atomic absorption (A.A.) analysis (8).

Reagents: Ammonium acetate: Add 114.0 ml glacial acetic acid to a 2.0 l volumetric flask and bring to about 1.0 l with distilled water. Add 138.0 ml concentrated ammonium hydroxide and bring to about 1950 ml. Adjust the pH to 7.0 with acetic acid or ammonium acetate and bring to volume at 2.0 l with distilled water. K+ working standards: Make working standards of 0.0, 2.0, 5.0 and 10.0 ppm K+ in 100.0 ml volumetric flasks with 6.25 ml ammonium acetate and filled to volume with distilled water.

Extraction: Weigh 5.0 grams of soil and place them in a 250.0 ml Erlenmeyer flask. Add 25.0 ml ammonium acetate (pH 7.0) and shake for 15.0 minutes on a reciprocating shaker

75 at 180 oscillations per minute. Filter the soil solution into 15.0 cm VWR Brand medium fast filter paper. Collect the extract in a 70 ml plastic cup.

Analysis: In a 95 ml plastic test tube, combine 1.0 ml of the soil extract and 15.0 ml of distilled water (1:16 dilution). Read the absorbance of the dilute solution on the A.A. using 0.0, 2.0, 5.0, 10.0 µg ml-1 solutions of K+ in ammonium acetate to calibrate the machine.

Additional Analysis Procedures

Phosphate: Double-Acid Procedure An alternative phosphate extraction procedure, Melich no.1 (9), is the standard method of phosphate extraction in Guatemala. This phosphate test compares the standard procedures of Guatemala with others that are available and assesses the viability of this procedure, normally confined to acid, sandy soils, as a standard procedure in Guatemala where the soils are generally high in clay content and organic matter. The analysis is done with the ascorbic acid colorimetric procedure.

Reagents: Extracting reagent: In a 1.0 l volumetric flask, add 4.0 ml concentrated HCl and 0.7 ml of concentrated H2SO4. Bring to volume with distilled water. Reagent A: Dissolve 12.0 g of ammonium molybdate in 250 ml of distilled water and in a separate volumetric flask, dissolve 0.2908 g of antimony potassium tartrate in 100 ml of distilled water. In a third flask add 148 ml of concentrated H2SO4 to distilled water and bring to volume at one liter to make 5 N H2SO4. Mix these three solutions together in a 2.0 L volumetric flask and bring the solution to a volume of 2000 ml with distilled water. Store in the dark at about 25º Celsius. Reagent B: Dissolve 0.528 grams of ascorbic acid in 100 ml of Reagent A. Reagent B should be made fresh every day. 3- (PO4) standard solutions: To make curve, use 0.25, 0.5, 1.0, 2.0, 3.0, 4.0 5.0, 6.0, and -1 3- 7.0 µg ml (PO4) in 100 ml of extracting solutions and complete the same procedure as found in the ascorbic acid analysis. To find the equation for the standard curve, plot the log transmittance of each standard sample on the y-axis against the concentration on the x-axis.

Extraction: Weigh 5.0 g of soil and place in a 250 ml Erlenmeyer flask. Add 25.0 ml of the extracting agent and shake the mixture for five minutes on a reciprocating shaker at 180 oscillations per minute. Filter the soil solution and collect the extract in 15 cm VWR medium filter paper set on a 70 ml plastic cup.

Analysis: In a 95 ml plastic test tube, combine a 5.0 ml aliquot of the soil extract with 10.0 ml of distilled water. Add 5.0 ml of Reagent B to the diluted extract and swirl the test

76 tube vigorously to mix. Allow the solution to stand for 15 minutes and then read the transmittance on the spectrophotometer at 880 nm.

Phosphate: Bray P1 Extraction The Bray P1 phosphate extraction procedure (10) is the generally accepted method for extracting plant available phosphate in acid soils. Guatemalan soils can be broadly categorized as acid soils with a high clay and organic matter content. In order to accurately compare the viability of different phosphate extraction procedures for these Guatemalan soils, the Bray procedure must be examined in addition to the sodium bicarbonate and double-acid procedures. The analysis is done with the ascorbic acid colorimetric procedure.

Reagents: 1.0 N NH4F: In a 1.0 l volumetric flask, dissolve 37.0 g of ammonium fluoride in distilled water then fill the flask to volume. The solution should be stored in a polyethylene container to avoid prolonged solution contact with glass. 0.5 N HCl: In a 500 ml volumetric flask, dilute 20.4 ml of 12.0 N HCl to volume with distilled water. Extracting Solution: In a 1.0 l volumetric flask, mix 30 ml of 1.0 N NH4F and 50 ml of 0.5 N HCl. Dilute the mixture to volume and store it in a polyethylene bottle. Reagent A: Dissolve 12.0 g of ammonium molybdate in 250 ml of distilled water and in a separate volumetric flask, dissolve 0.2908 g of antimony potassium tartrate in 100 ml of distilled water. In a third flask add 148 ml of concentrated H2SO4 to distilled water and bring to volume at one liter to make 5 N H2SO4. Mix these three solutions together in a 2.0 l volumetric flask and bring the solution to a volume of 2000 ml with distilled water. Store the Reagent A in a dark cupboard at about 25º Celsius. Reagent B: Dissolve 0.528 grams of ascorbic acid in 100 ml of Reagent A. Reagent B should be made fresh every day. 3- (PO4) standard solutions: To develop the standard curve, use 0.25, 0.5, 1.0, 2.0, 3.0, -1 3- 4.0 5.0, 6.0, and 7.0 µg ml (PO4) in 100 ml of extracting solutions and complete the same procedure as found in the ascorbic acid analysis. To find the equation for the standard curve, plot the log transmittance of each standard sample on the y-axis against the concentration on the x-axis.

Extraction: Weigh 2.5 g of soil and place it in a 250 ml Erlenmeyer flask. Add 25.0 ml of the extracting agent and shake the mixture for five minutes on a reciprocating shaker at 180 oscillations per minute. Filter the soil solution in 15 cm Whatman® #2 filter paper and collect the extract in a 70 ml plastic cup.

77 pH: Saturated Paste In a disposable plastic cup, add distilled water to a small amount of soil, stirring with a spatula, until it meets the conditions of saturation. For a soil sample to be considered saturated, it must 1) glisten as it reflects light 2) flow slightly when the cup is tipped 3) slide cleanly off the spatula (except for soils very high in clay content) and 4) have no standing water on the surface of the sample. After saturation is achieved, let the sample stand for one hour then reexamine the sample to see that it meets the conditions of saturation. If needed, additional soil or distilled water may be added to the soil paste in order that it can meet the conditions of saturation. Allow the sample to stand for at least three more hours in a dessicator with water in the bottom. After the sample sits in the dessicator for an appropriate amount of time, read the pH directly from the saturated paste using a pH meter appropriately calibrated for acid or alkaline soils. (11)

Soil Classification Procedures

Acid Neutralization Potential: Analysis for the acid neutralization potential for the determination of soil carbonates was completed in accordance with the procedure cited by Allison and Moode in Methods of Soil Analysis Part 2 published in 1965 (12).

Soil Texture: The hydrometer method of determining soil texture was carried out according to the procedure cited by Day in Methods of Soil Analysis Part 1 published in 1965 (13). Organic Matter: The Walkley-Black dichromate oxidation was used in the determination of soil organic matter. (14)

REFERENCES

1. Hanks, D.; Webb, B.; Jolley, V. A Comparison of Hot Water Extraction to Standard Extraction Methods for Nitrate, Potassium, Phosphorus, and Sulfate in Arid-Zone Soils. Commun. Soil Sci. Plant Anal. 1997, 28(15-16), 1393-1402. 2. Webb, B.L.; Hanks, D.H.; Jolley, V.D. A Pressurized Hot Water Extraction Method for Boron. Commun. Soil Sci. Plant Anal. 2002, 33, 31-39 3. Haby, V.A. Soil NO3-N Analysis in Ca(OH)2 Extracts by the Chromotropic Acid Method. Soil Sci. Soc. Am. J. 1989, 53, 308-310 4. Sims, J.R.; Jackson, G.D. Rapid Analysis of Soil Nitrate with Chromotropic Acid. Soil Sci. Soc. Amer. Proc. 1971, 35, 603-606. 5. Wantanabe, F.S.; Olsen, S.R. Test of Ascorbic Acid Method for Determining Phosphorus in Water and NaHCO3 Extractants for Soil. Soil Sci. Soc. Amer. Proc. 1965, 29, 677-678. 6. Sunderman, F.W.; Sunderman, F.W. Studies in Serum Electrolytes: A Rapid, Reliable Method for Serum Potassium using Tetraphenylboron. Am. J. Clin. Path. 1958, 29, 95- 103. 7. Olson, R.V. A Turbidimetric Potassium Determination Affected Little by Temperature. Soil Sci. Soc. Amer. Proc. 1953, 17, 20-22.

78 8. Council on Soil Testing and Plant Analysis. Determination of Potassium, Magnesium, Calcium, and Sodium by Neutral Ammonium Acetate Extraction. In Handbook on Reference Methods for Soil Testing (Revised Edition); Council on Soil Testing and Plant Analysis, Eds.; The Council on Soil Testing and Plant Analysis: Athens, GA, 1980; 58- 63. 9. Council on Soil Testing and Plant Analysis. Determination of Phosphorus by Mehlich No. 1 (Double acid) Extraction. In Handbook on Reference Methods for Soil Testing (revised edition); Council on Soil Testing and Plant Analysis, Ed; The Council on Soil Testing and Plant Analysis: Athens, GA, 1980; 37-41. 10. Council on Soil Testing and Plant Analysis. Determination of Bray PI Extraction. In Handbook on Reference Methods for Soil Testing (revised edition); Council on Soil Testing and Plant Analysis, Ed; The Council on Soil Testing and Plant Analysis: Athens, GA, 1980; 42-46. 11. Rhodes, J.D. Soluble Salts. In Methods of Soil Analysis Part 2; Page, A.L.; Miller, R.H.; Keeney, D.R., Eds.; American Society of Agronomy, Inc.: Madison, WI, 1982; 167-179. 12. Allison, L.E.; Moode, C.C. Carbonate. In Methods of soil analysis part 2; Black, C.A.; Evans, D.D.; White, J.L.; Ensminger, L.E.; Clark, F.E, Eds.; American Society of Agronomy, Inc.: Madison, WI, 1965; 1387-1388. 13. Day, P.R. Particle Fractionation and Particle-Size Analysis. In Methods of Soil Analysis Part 1; Black, C.A.; Evans, D.D.; White, J.L.; Ensminger, L.E.; Clark, F.E, Eds.; American Society of Agronomy, Inc.: Madison, WI, 1965; 562-566. 14. Walkley, A.; Black, I.A. An Examination of the Degtjareff Method for Determining Soil Organic Matter and a Proposed Modification of the Chromic Acid Titration Method. Soil Science. 1934, 37, 29-38.

Appendix IV

Project Data

Nitrate done at BYU units in ppm NO3 in soil soil sample Rep 1: PHW Rep 2: PHW Rep 3: PHW water-CTA m1 11.90 6.92 8.88 3.70 m2 8.33 6.06 6.92 3.35 m3 9.66 5.95 7.24 3.38 m4 7.35 5.31 3.10 0.83 m5 5.74 5.20 2.99 0.64 m6 7.03 5.74 3.10 0.88 m7 12.47 6.81 10.44 4.65 m8 10.78 9.32 6.70 2.89 m9 21.31 12.13 14.09 8.83 m10 12.25 12.25 8.00 4.56 m11 9.21 9.44 5.95 2.53 m12 11.79 11.68 9.44 4.71 m13 12.36 6.27 11.56 5.55 m14 14.09 8.88 10.10 6.22 m15 12.13 6.59 12.70 6.44 m16 11.11 6.16 8.88 2.50 m17 12.47 6.59 10.66 2.83 m18 18.84 6.59 12.13 4.86 m19 9.10 5.74 8.22 3.32 m20 12.70 7.46 8.22 4.08 m21 14.32 6.59 11.34 4.65 m22 13.74 5.95 9.88 2.36 m23 10.21 5.74 8.66 1.84 m24 9.55 5.74 9.10 1.84 m25 13.86 5.20 7.35 4.74 m26 12.36 5.52 5.41 3.87 m27 12.70 8.33 7.03 3.58 m28 9.21 8.00 4.04 4.11 m29 8.33 7.57 5.74 3.38 m30 4.25 8.44 4.78 3.43 m31 6.59 8.66 14.09 3.90 m32 5.52 6.70 12.02 3.18 m33 5.31 5.41 13.51 2.39 m34 2.58 4.88 17.75 3.46 m35 2.48 8.22 25.51 6.15 m36 2.89 6.49 10.44 3.58 m37 6.59 4.78 16.20 3.73 m38 6.27 4.67 12.02 2.78

81 m39 7.68 5.31 16.44 6.31 m40 5.20 5.10 26.95 4.92 m41 6.70 5.10 15.96 4.77 m42 4.99 4.36 15.26 5.42 m43 7.24 5.63 16.56 3.61 m44 17.63 12.47 14.20 9.97 m45 6.06 6.16 16.56 5.67 m46 6.38 4.78 13.05 6.09 m47 8.44 8.77 21.68 7.95 m48 6.06 4.78 15.96 6.05 m49 5.31 4.67 13.86 3.32 m50 3.20 3.72 12.36 2.03 m51 4.57 4.36 14.20 1.97 m52 3.52 3.31 13.74 0.54 m53 4.25 3.72 13.16 0.64 m54 2.27 2.58 13.51 1.57 m55 3.20 4.99 21.68 3.32 m56 3.41 4.14 22.18 4.11 m57 3.72 3.10 19.82 2.72 m58 3.93 3.72 22.56 4.23 m59 5.63 4.88 20.07 3.64 m60 8.66 5.31 21.06 3.29 m61 7.79 2.79 12.36 3.55 m62 3.10 2.48 14.67 3.09 m63 5.63 4.25 18.72 3.84 m64 9.77 1.75 19.58 1.84 m65 6.92 1.86 19.09 1.49 m66 6.16 2.48 25.12 1.62 m67 7.46 3.20 20.19 2.06 m68 7.57 2.79 23.96 2.64 m69 6.92 3.31 22.56 2.44 m70 8.77 2.99 19.70 3.73 m71 57.37 52.84 66.72 37.25 m72 98.73 108.92 166.38 89.92 m73 4.99 2.27 24.73 0.52 m74 4.88 1.04 21.93 1.46 m75 6.16 1.34 23.45 0.93 m76 18.72 10.89 30.44 7.61 m77 13.28 11.23 25.12 6.70 m78 15.61 5.31 22.31 3.84 m79 7.68 1.45 23.07 1.06 m80 7.35 1.86 20.31 1.25 m81 14.55 2.79 20.44 1.62

82 m82 13.05 2.68 23.45 1.81 m83 15.49 3.31 21.56 2.44 m84 15.14 6.59 20.81 2.72 m85 14.67 4.46 20.44 3.00 m86 11.11 4.25 22.94 2.61 m87 12.25 1.34 17.03 2.17 m88 11.90 3.10 18.72 3.43 m89 14.79 5.41 17.03 6.44 m90 13.97 5.63 20.44 6.38 m91 11.00 4.99 8.22 6.02 m92 16.67 6.27 5.20 8.27 m93 13.05 4.36 8.33 5.23 m94 17.03 4.78 5.52 4.77 m95 19.94 14.67 22.81 11.72 m96 23.32 13.51 24.47 13.08 m97 11.56 4.57 9.88 3.78 m98 14.09 4.14 8.55 3.84 m99 12.82 3.20 4.67 2.19 m100 15.49 7.57 7.24 5.42 m101 11.34 3.72 7.13 2.83 m102 13.86 5.20 2.58 3.73 m103 15.14 8.55 8.55 7.88 m104 48.99 41.25 32.37 31.88 m105 13.62 8.33 4.67 7.00 m106 15.37 2.48 0.63 5.05 m107 13.05 1.86 3.62 6.77 m108 12.13 7.24 6.27 4.05 m109 9.32 4.88 4.14 2.61 m110 14.55 8.33 5.95 4.62 m111 8.66 3.31 15.49 1.78 m112 14.44 6.81 16.44 3.90 m113 14.55 12.13 15.49 2.61 m114 17.03 13.28 22.81 7.07 m115 19.82 14.32 27.61 8.30 m116 24.09 14.79 26.95 8.44 m117 11.90 8.11 18.24 4.56 g001 17.03 21.31 15.84 16.78 g002 25.51 28.01 23.96 20.53 g003 54.21 43.71 41.09 34.82 g004 44.80 44.96 42.16 35.53 g005 30.85 28.41 22.94 20.76 g006 29.22 37.37 31.26 28.84 g007 42.16 40.19 35.20 29.81

83 g008 27.22 1.04 32.09 29.00 g009 31.95 35.91 28.82 25.09 g010 19.82 23.71 21.56 16.00 g011 26.56 24.99 19.58 16.29 g012 31.26 40.19 31.54 29.65 g013 70.10 58.81 55.25 50.89 g014 31.95 37.37 30.99 30.31 g015 12.02 15.73 10.55 10.89 g016 38.84 37.07 43.09 28.69 g017 38.84 35.77 40.34 31.26 g018 17.87 25.64 29.22 15.77 g019 28.01 27.35 32.65 16.39 g020 26.16 27.88 35.48 19.80 g021 22.06 19.58 19.21 14.52 g022 21.18 17.75 16.79 10.50 g023 58.45 62.69 59.90 51.47 g024 109.81 93.48 91.96 76.35 g025 17.27 19.33 14.67 9.49 g026 78.33 71.74 67.51 54.58 g027 19.94 22.94 17.03 12.33 g028 15.49 15.14 14.67 9.97 g029 24.73 24.47 24.09 17.99 g030 18.48 16.79 14.67 11.76 g031 50.48 40.34 34.62 30.40 g032 79.66 85.86 84.68 69.51 g033 33.63 26.82 25.51 19.42 g034 45.60 44.33 42.62 34.22 g035 19.21 13.05 11.90 7.54 g036 10.21 9.21 9.88 4.05 g037 46.87 36.35 35.63 24.75 g038 39.59 39.29 36.35 26.21 g039 42.93 36.64 33.92 21.46 g040 23.07 23.32 22.18 12.37 g041 49.48 32.79 29.49 23.89 g042 22.56 15.84 13.86 10.12 g043 24.99 12.93 11.45 8.98 g044 41.86 34.20 30.99 28.53 g045 29.62 17.27 15.37 11.56 g046 18.60 11.45 7.13 4.77 g047 53.35 31.40 33.07 24.95 g048 22.06 9.77 9.55 5.70 g049 9.55 10.21 11.11 4.74 g050 7.57 9.55 5.20 4.71

84 g051 19.70 24.09 16.20 15.48 g052 9.32 8.44 7.03 7.17 g053 28.55 27.35 25.77 18.30 g054 28.28 26.42 23.83 18.93 g055 35.91 35.63 32.37 26.14 g056 37.51 34.20 33.77 25.23 g057 22.69 21.31 17.03 12.74 g058 22.06 20.44 15.73 13.00 g059 24.73 25.64 18.36 13.42 g060 56.84 61.75 48.50 36.06 g061 17.75 15.73 15.14 11.08 g062 34.62 38.54 35.63 26.71 g063 44.96 47.52 44.33 37.92 g064 48.50 50.81 46.87 38.15 g065 40.49 40.94 36.78 33.07 g066 15.14 16.08 8.99 7.41 g067 38.25 37.95 41.25 29.32 g068 28.95 27.22 26.56 17.43 g069 19.94 19.33 17.39 8.80 g070 28.95 27.61 25.38 15.67 g071 56.66 54.04 47.04 36.70 g072 24.86 17.87 13.97 10.77 g073 28.55 23.58 17.15 10.73 g074 24.35 17.87 11.00 6.51 g075 26.82 19.58 11.11 7.17 g076 47.84 36.35 32.93 22.80 g077 12.70 18.36 13.62 8.87 g078 14.32 25.25 16.20 9.97 g079 16.44 24.73 19.33 10.69 g080 11.79 19.21 14.09 7.14 g081 31.82 40.79 33.21 25.64 g082 46.71 55.43 51.14 33.74 g083 29.35 36.06 25.12 16.05 g084 41.09 46.55 38.54 28.61 g085 65.16 68.90 61.01 46.37 g086 25.38 28.82 21.56 15.02 g087 18.60 21.06 11.45 7.04 g088 28.68 32.09 22.18 13.00 g089 20.44 13.74 12.59 5.89 g090 24.09 22.06 13.51 8.59 g091 14.90 14.79 16.44 12.95 g092 41.86 40.19 42.16 36.38 g093 31.68 32.93 33.92 29.90

85 g094 35.20 33.21 35.20 28.92 g095 28.95 26.69 28.41 23.69 g096 18.96 14.09 17.75 11.88 g097 38.25 29.35 32.37 26.35 g098 47.52 37.51 40.34 33.07 g099 64.97 50.14 55.60 51.86 g100 45.12 38.25 35.48 30.48 g101 38.40 35.48 25.12 21.05 g102 62.50 60.82 48.82 46.05 g103 31.40 18.60 17.15 13.90 g104 44.49 45.12 47.20 41.07 g105 35.05 35.77 36.78 36.59 g106 40.19 38.25 45.60 40.31 g107 102.01 113.80 108.62 86.56 g108 49.15 51.82 54.56 45.89 g109 61.19 58.63 62.50 56.18 g110 40.19 38.69 44.49 35.64 g111 54.21 49.81 56.48 45.12

Phosphorus done at BYU all units in ppm P in soil soil sample Rep 1: PHW Rep 2: PHW Rep 3: PHW Olsen and molybdic acid m1 4.21 3.57 2.71 4.79 m2 2.94 2.77 4.44 5.38 m3 3.74 3.57 2.60 5.20 m4 0.82 3.28 0.71 4.67 m5 0.77 1.48 0.71 2.77 m6 0.77 0.77 0.66 4.50 m7 11.76 9.27 11.09 16.01 m8 15.86 10.43 10.69 12.77 m9 11.62 6.89 8.19 12.77 m10 1.48 1.54 2.04 3.51 m11 1.37 1.98 1.76 3.63 m12 1.10 1.48 2.21 3.11 m13 1.15 1.37 1.54 4.73 m14 1.15 1.48 1.65 3.86 m15 1.37 1.70 1.54 4.38 m16 4.32 3.80 4.79 8.44 m17 1.70 3.40 4.79 7.32 m18 5.20 3.51 4.32 7.88 m19 1.70 0.77 1.54 13.18 m20 1.10 0.55 1.43 11.22 m21 1.76 1.26 2.94 12.16 m22 1.54 0.55 0.99 6.52 m23 1.15 0.77 0.88 4.97 m24 0.82 0.71 0.93 5.98 m25 1.87 2.94 2.49 16.15 m26 2.09 2.43 2.94 17.85 m27 4.15 5.62 3.57 24.39 m28 1.04 0.99 1.48 5.44 m29 0.71 0.88 0.88 5.09 m30 0.60 0.71 0.93 4.56 m31 6.46 7.44 7.94 18.00 m32 2.37 5.50 4.26 14.01 m33 1.10 1.70 1.76 4.61 m34 0.93 2.32 1.81 9.72 m35 1.59 1.43 1.32 18.30 m36 0.66 1.76 1.48 9.52 m37 15.93 22.39 27.71 86.11 m38 37.68 48.73 37.25 90.57

87 m39 27.35 29.26 36.08 82.24 m40 2.37 2.21 2.71 9.85 m41 1.76 2.32 2.21 6.04 m42 0.93 1.48 1.48 9.40 m43 0.77 1.98 2.09 9.65 m44 2.21 1.37 1.43 9.01 m45 1.32 1.26 1.10 7.26 m46 0.99 0.99 1.04 4.15 m47 0.77 0.99 0.82 3.74 m48 0.99 1.37 0.88 4.61 m49 12.84 16.52 13.32 54.94 m50 14.36 17.93 11.62 40.93 m51 15.64 17.18 13.73 54.65 m52 3.00 4.32 3.63 7.51 m53 3.34 4.73 3.91 5.92 m54 2.21 4.21 3.11 4.61 m55 1.21 1.37 1.43 3.97 m56 0.93 1.32 1.10 4.61 m57 1.10 1.59 1.43 4.73 m58 0.77 1.81 1.21 8.19 m59 0.66 1.43 0.99 7.44 m60 0.33 1.59 1.76 6.46 m61 1.26 2.43 1.76 9.40 m62 0.66 0.93 1.26 7.57 m63 1.48 3.86 2.32 11.22 m64 3.11 3.05 4.73 6.10 m65 2.21 4.38 4.15 4.67 m66 4.56 5.26 5.26 6.46 m67 34.62 39.23 28.34 29.08 m68 38.89 26.11 38.45 29.45 m69 34.83 66.04 43.53 38.67 m70 31.83 33.41 17.78 25.59 m71 20.70 28.34 36.29 34.11 m72 21.74 23.63 24.47 35.04 m73 7.94 8.32 9.85 19.06 m74 7.51 9.91 6.10 16.88 m75 11.96 9.33 8.51 14.15 m76 1.32 1.04 2.09 9.14 m77 1.54 1.98 1.54 9.78 m78 1.81 0.99 1.54 10.30 m79 2.15 1.59 1.98 8.76 m80 5.62 4.03 6.89 9.85 m81 3.68 3.57 3.74 9.08

88 m82 19.14 23.21 19.14 40.24 m83 20.70 23.38 21.18 37.68 m84 26.81 35.14 19.68 49.27 m85 1.26 1.32 1.21 12.09 m86 0.82 1.15 1.04 11.36 m87 0.82 1.10 0.77 9.01 m88 0.49 0.88 0.66 5.56 m89 0.66 0.99 0.39 6.71 m90 0.77 0.66 0.66 9.33 m91 0.88 0.88 0.66 11.82 m92 1.04 1.04 1.48 10.43 m93 0.82 1.21 0.88 8.89 m94 40.93 46.00 34.52 100.07 m95 33.71 21.42 25.07 98.70 m96 24.98 36.72 36.82 90.28 m97 11.36 8.57 9.78 34.62 m98 8.82 11.16 13.11 42.92 m99 6.46 5.03 4.79 26.37 m100 2.04 2.66 3.05 12.50 m101 2.54 2.77 2.26 9.72 m102 3.22 3.00 3.86 17.48 m103 0.55 1.15 0.71 7.88 m104 1.04 3.00 1.21 13.04 m105 0.60 0.93 0.66 7.57 m106 1.48 1.32 1.15 16.15 m107 1.32 1.70 1.21 13.32 m108 1.59 1.48 1.98 18.23 m109 16.81 16.30 15.79 17.55 m110 16.88 16.59 16.08 20.86 m111 16.08 15.14 18.15 15.57 m112 2.77 1.87 2.77 7.57 m113 1.54 2.04 2.21 4.91 m114 1.15 1.26 1.54 6.28 m115 1.21 1.37 1.65 9.85 m116 0.93 0.88 1.04 4.03 m117 0.93 0.99 1.81 9.14 g001 49.81 1.76 0.44 3.68 g002 1.15 1.98 0.33 2.43 g003 1.54 1.04 0.39 2.49 g004 1.37 0.60 0.49 3.00 g005 1.48 2.66 0.55 4.73 g006 0.99 0.77 0.28 4.73 g007 1.04 0.77 0.39 2.60

89 g008 1.48 5.74 1.59 11.76 g009 1.26 3.00 0.49 8.19 g010 0.88 1.21 0.44 2.60 g011 1.10 1.15 0.66 2.15 g012 1.65 4.03 2.88 19.68 g013 2.15 2.49 1.15 7.44 g014 1.04 2.49 0.66 4.67 g015 1.04 2.43 0.99 7.94 g016 4.56 3.51 2.71 23.71 g017 0.55 1.65 0.49 2.04 g018 2.94 6.10 3.17 7.38 g019 8.51 8.76 10.30 15.07 g020 53.03 43.89 40.01 82.49 g021 13.94 12.77 9.20 18.45 g022 17.93 18.00 18.23 23.38 g023 2.49 3.05 1.54 8.70 g024 3.17 10.04 8.95 22.14 g025 57.24 55.39 44.50 66.60 g026 9.65 13.18 12.09 123.62 g027 0.77 1.76 0.60 7.88 g028 0.60 0.88 0.71 6.95 g029 0.88 1.37 0.77 11.42 g030 9.85 7.94 9.59 32.51 g031 42.92 31.44 30.20 109.94 g032 1.32 0.60 1.48 30.48 g033 3.22 2.49 3.57 9.40 g034 2.88 0.93 2.66 10.24 g035 4.91 1.32 2.77 4.97 g036 3.51 1.48 2.83 7.69 g037 5.86 3.68 3.86 7.69 g038 3.11 1.32 2.32 10.96 g039 10.63 1.65 8.76 9.27 g040 2.49 2.77 2.54 29.73 g041 4.73 2.26 2.94 7.14 g042 5.09 5.44 6.22 9.65 g043 1.93 1.04 1.54 14.72 g044 3.34 1.21 2.43 5.03 g045 1.76 1.21 1.81 9.46 g046 8.07 6.59 5.03 6.04 g047 3.11 1.21 2.88 10.76 g048 2.32 0.60 1.70 10.04 g049 0.71 1.32 1.93 5.56 g050 2.21 1.10 1.65 5.32

90 g051 0.93 0.55 1.04 6.95 g052 1.15 0.33 1.15 6.04 g053 8.25 5.38 5.03 10.17 g054 1.65 1.26 1.65 6.77 g055 7.26 7.26 6.22 14.22 g056 5.74 4.03 4.61 13.94 g057 1.04 1.10 1.32 6.46 g058 1.54 1.70 1.48 6.34 g059 12.91 10.11 11.49 15.50 g060 2.32 1.93 1.59 7.20 g061 6.95 6.59 6.34 20.39 g062 0.77 0.66 0.44 7.88 g063 0.77 0.44 0.55 10.43 g064 1.21 1.10 1.21 8.82 g065 8.89 7.51 6.89 26.90 g066 2.09 2.43 2.09 5.38 g067 0.88 0.60 0.44 9.72 g068 2.66 2.32 2.66 10.43 g069 34.42 47.54 44.87 34.01 g070 4.85 2.88 1.15 4.85 g071 16.15 15.93 12.63 39.56 g072 1.59 1.76 0.99 15.00 g073 24.13 19.76 26.20 49.95 g074 26.11 28.43 26.55 53.76 g075 24.13 22.23 11.55 30.01 g076 43.89 69.66 47.94 103.66 g077 24.22 38.01 26.99 60.62 g078 56.15 55.70 45.37 67.72 g079 26.20 26.55 28.07 47.41 g080 33.21 25.93 20.94 45.50 g081 22.55 19.30 16.96 40.70 g082 139.80 118.01 128.10 148.45 g083 35.24 51.19 47.81 61.29 g084 21.02 20.70 18.38 33.11 g085 19.68 22.88 20.94 35.35 g086 15.00 15.14 14.08 21.74 g087 4.50 4.91 4.79 13.18 g088 31.34 30.77 30.86 31.05 g089 1.93 3.22 1.98 12.70 g090 7.57 5.98 5.44 18.91 g091 1.10 0.39 0.55 3.51 g092 2.04 0.12 0.44 3.80 g093 1.43 0.39 0.39 5.26

91 g094 0.71 0.49 0.49 4.97 g095 0.71 0.22 0.71 3.57 g096 1.04 0.60 0.71 2.94 g097 0.99 0.22 0.55 4.15 g098 1.43 0.44 0.60 9.72 g099 0.49 0.22 0.55 5.20 g100 0.60 0.33 0.55 5.03 g101 0.44 0.44 0.49 4.09 g102 0.44 0.17 0.49 4.97 g103 1.15 0.60 0.77 4.21 g104 1.10 0.28 0.66 8.07 g105 0.66 0.22 0.39 5.32 g106 0.71 0.39 0.49 6.89 g107 1.48 0.93 0.44 8.89 g108 1.04 0.44 0.33 2.88 g109 0.71 0.55 0.49 8.95 g110 2.37 0.93 0.60 7.44 g111 57.39 79.10 76.13 125.81

Potassium done at BYU all units in cobaltinitrite ppm K in soil soil sample Rep 1: Rep 2: Rep 3: Ammonium acetate-AA m2 95.30 96.38 136.96 246.4 m6 75.63 84.57 89.91 110.4 m11 94.75 107.35 129.98 320 m16 111.24 115.72 96.92 345.6 m21 127.10 105.13 106.24 173.6 m23 114.04 114.60 96.38 412.8 m24 109.57 113.47 133.46 412.8 m30 114.04 116.28 125.38 124.8 m36 166.48 135.21 135.79 500 m39 124.23 110.68 101.29 127.2 m46 124.80 125.95 127.67 342.4 m49 84.57 151.21 156.05 106.4 m54 93.67 98.01 114.60 20.8 m55 105.13 93.13 120.81 170.4 m62 84.04 93.67 82.98 261.6 m67 104.03 119.11 123.66 217.6 m72 239.40 337.48 169.58 526.4 m76 179.01 205.00 297.94 425.6 m84 110.68 115.72 127.67 256 m86 81.40 104.58 136.96 259.2 m88 113.47 90.44 59.69 149.6 m99 125.38 92.06 103.48 426.4 m102 112.35 120.81 150.01 143.2 m105 143.45 94.75 154.84 184.8 m117 121.95 127.10 162.16 156.8 g2 120.81 73.55 49.66 101.6 g9 121.38 120.81 71.47 282.4 g12 98.01 130.56 64.27 400 g17 111.80 93.13 46.20 196 g23 77.72 138.72 46.69 186.4 g27 177.11 277.70 182.83 400 g30 98.01 144.63 120.81 376.8 g33 95.84 103.48 109.01 212.8 g39 92.06 96.92 97.47 65.6 g41 139.90 141.67 133.46 301.6 g48 74.59 92.06 106.79 105.6 g52 74.59 70.95 86.17 68.8 g59 148.21 177.74 135.21 1955

93 g61 104.03 119.11 89.37 497.6 g66 73.03 87.77 114.60 607.2 g70 97.47 94.21 45.21 71.2 g76 321.47 479.83 387.66 2320 g77 87.77 129.40 53.65 595.2 g78 161.54 243.68 151.21 1245 g82 405.83 583.24 380.20 1260 g88 85.64 96.92 87.77 476.8 g96 95.30 115.72 81.40 136 g106 100.19 121.95 97.47 70.4 g107 156.66 350.46 166.48 420 g111 400.99 188.61 . 1355

Morocco done in Morocco units are in ppm in soil Sample Morocco steam Morocco Morocco Morocco distillation NO3-N PHW NO3-N Olsen P PHW P m1 22.68 8.26 7.38 10.45 m2 13.16 7.38 8.23 10.15 m3 13.72 8.09 4.28 10.33 m4 6.44 0.94 8.79 7.70 m5 4.20 0.91 4.70 7.23 m6 11.76 0.91 5.55 7.05 m7 11.48 10.27 17.10 13.22 m8 8.40 10.34 9.76 13.16 m9 13.16 10.27 12.17 13.53 m10 15.12 3.29 6.54 9.37 m11 9.24 1.10 3.72 7.94 m12 10.36 4.68 3.30 8.18 m13 10.92 18.17 4.85 17.08 m14 8.96 30.71 11.18 5.70 m15 12.04 13.48 5.83 4.99 m16 7.28 10.23 7.66 14.68 m17 10.92 10.06 4.91 11.53 m18 12.18 10.23 7.10 13.10 m19 11.20 8.40 14.28 8.18 m20 14.56 6.19 15.27 8.00 m21 7.00 6.06 7.33 7.41 m22 8.40 4.75 4.70 4.12 m23 13.16 6.53 4.15 4.00 m24 8.40 4.78 2.79 4.41 m25 9.52 4.58 14.00 5.46 m26 13.16 3.99 12.17 4.99 m27 9.52 4.68 38.79 5.81 m28 10.36 15.53 3.86 5.40 m29 10.08 16.59 4.15 4.58 m30 9.80 15.82 4.70 6.05 m31 10.92 12.69 15.83 9.61 m32 12.88 12.62 33.44 7.23 m33 17.36 12.41 4.70 5.87 m34 8.96 12.84 9.35 6.88 m35 5.60 12.76 16.88 6.93 m36 12.32 12.73 7.94 7.05 m37 2.24 12.73 90.21 45.00 m38 2.24 12.94 85.82 46.39

95 m39 3.64 13.05 73.55 44.09 m40 12.04 22.16 5.52 17.70 m41 19.04 22.05 3.88 17.08 m42 11.20 20.42 10.06 17.70 m43 10.08 12.98 9.91 13.59 m44 14.56 16.59 9.00 13.22 m45 22.40 13.01 6.12 13.53 m46 10.92 19.25 3.39 13.16 m47 12.04 19.81 4.30 13.10 m48 8.96 18.99 6.93 13.77 m49 7.56 15.49 51.13 24.99 m50 10.08 14.91 30.90 23.84 m51 6.72 14.91 39.77 23.78 m52 7.28 16.48 11.04 17.02 m53 8.68 17.62 4.70 16.96 m54 46.90 15.67 10.90 16.46 m55 9.80 17.47 4.70 13.16 m56 12.04 17.47 1.58 13.40 m57 12.32 19.63 4.70 13.89 m58 10.36 6.63 10.76 12.98 m59 15.12 5.82 6.73 13.71 m60 4.76 6.66 21.75 13.77 m61 6.44 12.45 7.52 13.71 m62 7.84 14.05 5.69 13.04 m63 12.04 12.09 11.18 13.65 m64 6.44 25.50 9.61 21.00 m65 8.96 27.00 5.27 21.50 m66 11.20 25.46 6.39 22.07 m67 4.76 24.17 27.52 44.86 m68 4.20 28.04 37.66 45.90 m69 6.72 27.64 38.23 43.12 m70 27.16 36.03 44.61 40.72 m71 42.56 36.03 35.91 45.21 m72 97.16 100.93 34.90 39.01 m73 2.80 18.28 9.83 7.94 m74 6.16 20.15 8.38 8.65 m75 11.48 15.82 9.83 9.31 m76 11.20 24.29 6.64 6.82 m77 8.96 24.25 6.35 6.46 m78 7.00 24.21 11.13 6.17 m79 9.24 24.64 5.62 7.29 m80 5.88 21.25 7.36 6.40 m81 8.12 22.51 3.88 5.46

96 m82 6.44 15.31 37.07 19.19 m83 7.28 16.08 34.00 20.56 m84 11.76 18.51 48.09 22.96 m85 7.56 19.03 12.79 3.30 m86 6.72 17.76 9.91 3.07 m87 4.76 18.99 8.39 3.94 m88 7.00 17.17 9.30 3.36 m89 34.44 34.50 4.56 3.13 m90 20.72 17.54 12.94 2.95 m91 10.64 21.02 18.09 3.36 m92 9.52 24.09 9.91 2.95 m93 16.80 10.58 6.42 4.06 m94 6.72 18.36 93.09 29.25 m95 28.56 34.08 90.97 27.75 m96 12.32 18.62 17.33 30.03 m97 7.56 18.51 89.45 12.62 m98 10.92 8.30 25.82 9.55 m99 9.80 5.35 37.66 8.48 m100 17.08 18.65 33.55 7.94 m101 7.84 10.69 19.76 8.00 m102 8.96 1.27 9.49 8.83 m103 11.76 31.78 16.88 4.82 m104 31.64 39.05 5.97 5.99 m105 13.16 15.28 11.12 5.17 m106 10.64 25.15 4.61 8.95 m107 8.68 20.79 11.42 9.07 m108 7.28 20.57 14.17 8.71 m109 10.36 25.77 17.52 14.01 m110 9.52 18.02 14.00 16.22 m111 5.32 6.43 17.80 15.79 m112 6.16 0.62 15.27 5.70 m113 5.88 0.23 9.07 6.05 m114 17.08 4.72 8.23 5.64 m115 15.96 3.65 4.46 6.05 m116 8.40 0.23 16.27 6.52 m117 6.44 0.72 3.30 6.93

Potassium units are in ppm K in soil soil sample BYU ammonium BYU Morocco Morocco acetate tetraphenylborate ammonium tetraphenylborate acetate m1 248.8 169.2 246.55 77.43 m2 246.4 169.67 266.94 76.35 m3 233.6 163.75 299.57 77.74 m4 97.6 165.3 124.20 79.28 m5 103.2 165.12 124.20 80.13 m6 110.4 169.04 127.09 78.74 m7 214.4 186.93 249.07 77.35 m8 132.8 175.35 160.01 78.43 m9 129.6 173.29 156.83 78.43 m10 309.6 173.67 334.26 79.82 m11 320 170.93 340.07 79.82 m12 430.4 177.26 428.04 80.37 m13 368 170.46 392.34 81.61 m14 374.4 172.5 369.11 78.51 m15 383.2 174.72 392.34 80.60 m16 345.6 183.36 356.67 84.20 m17 319.2 179.5 345.87 82.39 m18 376.8 182.07 359.43 82.39 m19 194.4 167.64 206.47 87.60 m20 168.8 169.67 198.73 87.44 m21 173.6 171.71 201.69 87.84 m22 452.8 172.5 411.70 82.71 m23 412.8 175.67 392.34 84.43 m24 429.6 174.4 411.70 85.93 m25 233.6 172.98 273.06 78.05 m26 234.4 171.56 266.94 80.44 m27 224 169.99 258.79 79.51 m28 140 168.42 169.06 80.83 m29 150.4 171.08 199.65 81.85 m30 124.8 169.51 150.97 80.52 m31 143.2 187.25 179.37 79.05 m32 129.6 171.08 171.62 79.90 m33 60 163.13 79.52 79.59 m34 519.2 199.3 434.70 86.88 m35 404.8 171.08 340.80 87.04 m36 500 202.99 424.49 86.80

98 m37 113.6 167.48 114.23 95.99 m38 120 167.48 116.27 100.36 m39 127.2 167.33 124.43 99.61 m40 329.6 176.78 302.02 87.68 m41 292.8 168.89 273.44 93.54 m42 325.6 176.62 316.31 88.87 m43 501.6 177.58 440.82 106.56 m44 372.8 182.55 334.68 110.66 m45 384 171.4 357.13 110.57 m46 342.4 166.23 314.27 97.88 m47 330.4 163.91 314.27 97.88 m48 336.8 165.15 328.55 97.96 m49 106.4 164.99 116.27 98.21 m50 59.2 165.92 59.11 98.37 m51 75.2 170.61 77.48 100.77 m52 20.8 163.75 26.45 105.21 m53 19.2 165.46 16.25 101.27 m54 20.8 163.13 52.83 101.27 m55 170.4 166.08 179.26 95.83 m56 228.8 170.14 262.87 100.94 m57 253.6 172.34 261.19 97.88 m58 309.6 168.89 375.02 102.86 m59 267.2 166.23 279.18 107.58 m60 279.2 165.77 281.22 105.38 m61 272.8 184.49 315.88 110.40 m62 261.6 173.92 299.57 114.38 m63 286.4 173.29 279.18 115.77 m64 111.2 173.45 132.36 118.94 m65 96.8 169.2 118.09 118.23 m66 112.8 176.3 136.44 118.05 m67 217.6 201.31 244.51 116.03 m68 211.2 226.34 244.51 122.40 m69 216.8 212.71 291.41 111.95 m70 427.2 217.38 417.84 114.46 m71 409.6 256.4 419.88 119.20 m72 526.4 306.8 36.52 112.30 m73 28.8 172.66 40.60 72.62 m74 29.6 172.82 40.60 73.00 m75 28 168.11 40.60 73.30 m76 425.6 277.12 392.34 90.24 m77 393.6 317.49 402.02 93.79 m78 428 340.79 407.64 90.40 m79 23.2 176.78 28.35 73.68

99 m80 50.4 174.4 63.03 73.23 m81 36.8 167.64 51.59 73.15 m82 204 175.99 241.32 82.47 m83 231.2 182.55 252.94 83.10 m84 256 190.69 277.14 82.78 m85 276 183.03 309.77 73.08 m86 259.2 172.19 303.65 77.81 m87 262.4 170.77 280.05 73.08 m88 149.6 170.61 177.22 72.77 m89 201.6 169.51 244.51 73.08 m90 180.8 173.77 222.08 73.30 m91 200.8 167.64 246.55 73.68 m92 225.6 167.79 275.10 74.22 m93 159.2 167.79 187.42 92.41 m94 206.4 169.51 256.81 87.20 m95 183.2 170.61 218.09 87.28 m96 230.4 180.03 262.62 87.28 m97 404 175.99 431.06 87.28 m98 439.2 170.14 469.79 89.83 m99 426.4 177.42 431.06 97.71 m100 162.4 172.98 198.73 98.87 m101 154.4 174.4 198.73 99.53 m102 143.2 170.77 179.37 96.89 m103 133.6 180.3 160.01 97.38 m104 206.4 210.99 237.45 98.04 m105 184.8 203.33 218.09 97.71 m106 137.6 173.13 167.75 84.43 m107 166.4 175.19 198.73 87.60 m108 172 175.51 198.73 87.44 m109 32 178.38 43.84 91.36 m110 48.8 172.66 67.07 89.99 m111 22.4 175.67 43.84 91.04 m112 189.6 177.9 223.90 82.47 m113 181.6 175.35 218.09 88.63 m114 146.4 170.61 179.37 83.96 m115 129.6 199.97 154.20 100.19 m116 95.2 190.69 109.67 100.27 m117 156.8 206.55 190.99 97.38

100

Guatemala Done in Nitrate Guatemala units are in ppm NO3-N in soil soil sample Rep 1: PHW Rep 2: PHW Rep 3: PHW g001 23.76 20.41 18.76 g002 36.16 25.47 38.02 g003 38.02 51.77 37.09 g005 25.47 21.24 19.58 g006 47.71 47.71 49.73 g007 51.77 45.72 46.71 g008 42.78 47.71 31.61 g009 38.96 29.83 29.83 g010 26.33 17.13 19.58 g011 29.83 17.94 32.51 g012 44.73 48.71 28.07 g013 78.90 66.97 65.83 g014 49.73 38.96 36.16 g015 18.76 12.35 17.13 g016 42.78 45.72 35.24 g017 45.72 41.82 34.32 g018 15.52 18.76 25.47 g019 36.16 27.20 36.16 g020 25.47 26.33 25.47 g021 32.51 22.07 36.16 g022 13.92 22.92 12.35 g023 72.81 71.63 85.26 g024 96.05 115.51 94.65 g025 10.01 16.32 16.32 g026 80.15 96.05 76.43 g027 12.35 17.94 13.92 g028 12.35 19.58 13.92 g029 40.86 26.33 36.16 g030 10.01 30.72 15.52 g031 57.02 51.77 51.77

101 g032 128.94 130.71 125.47 g033 34.32 28.95 27.20 g034 65.83 50.74 60.26 g035 17.94 18.76 12.35 g036 17.94 3.97 12.35 g037 45.72 41.82 39.91 g038 60.26 38.02 49.73 g039 43.75 39.91 62.46 g040 28.95 25.47 19.58 g041 44.73 38.96 42.78 g042 22.07 13.92 15.52 g043 22.92 13.92 13.92 g044 49.73 43.75 47.71 g045 27.20 12.35 9.24 g046 18.76 13.13 14.72 g047 43.75 34.32 41.82 g048 16.32 3.97 6.21 g049 15.52 3.97 9.24 g050 13.92 9.24 15.52 g051 26.33 19.58 23.76 g052 16.32 17.13 10.79 g053 45.72 38.96 35.24 g054 42.78 29.83 27.20 g055 53.85 41.82 47.71 g056 47.71 38.02 45.72 g057 53.85 24.61 22.07 g058 21.24 23.76 19.58 g059 33.41 24.61 27.20 g060 66.97 60.26 55.95 g061 30.72 17.94 15.52 g062 54.90 49.73 39.91 g063 69.28 58.09 53.85 g064 66.97 64.70 64.70 g065 57.02 45.72 45.72 g066 15.52 17.94 15.52 g067 53.85 45.72 41.82

102 g068 38.02 28.95 28.07 g069 28.07 15.52 15.52 g070 36.16 23.76 19.58 g071 69.28 60.26 59.17 g072 9.24 21.24 10.79 g073 15.52 24.61 16.32 g074 5.46 18.76 9.24 g075 7.72 18.76 10.79 g076 41.82 39.91 38.96 g077 17.13 15.52 12.35 g078 10.79 22.92 13.13 g079 11.56 22.07 16.32 g080 5.46 17.94 10.79 g081 34.32 44.73 43.75 g082 55.95 64.70 62.46 g083 22.07 39.91 27.20 g084 51.77 49.73 45.72 g085 71.63 87.88 74.01 g086 16.32 25.47 17.94 g087 3.23 15.52 7.72 g088 27.20 25.47 19.58 g089 4.71 17.13 5.46 g090 17.13 14.72 13.13 g091 20.41 22.07 21.24 g092 60.26 55.95 50.74 g093 41.82 36.16 46.71 g094 44.73 38.96 43.75 g095 33.41 33.41 31.61 g096 20.41 16.32 15.52 g097 37.09 35.24 34.32 g098 44.73 49.73 53.85 g099 65.83 72.81 76.43 g100 39.91 43.75 44.73 g101 27.20 34.32 28.07 g102 58.09 58.09 58.09 g103 11.56 18.76 5.46

103 g104 55.95 59.17 55.95 g105 44.73 45.72 44.73 g106 50.74 58.09 54.90 g107 130.71 128.94 125.47 g108 62.46 74.01 68.12 g109 81.41 75.22 75.22 g110 51.77 55.95 48.71 g111 60.26 76.43 71.63

104

Phosphorus done in all units are in Guatemala ppm P in soil soil sample Rep 1: PHW Rep 2: PHW Rep 3: PHW Mehlich I done in Guatemala g001 0.09 1.14 1.62 1.62 g002 3.66 1.14 0.49 0.44 g003 0.00 0.58 0.49 1.05 g005 0.00 1.90 1.14 3.02 g006 1.62 1.33 0.39 0.20 g007 1.71 0.67 0.39 0.25 g008 0.10 3.06 3.06 6.76 g009 0.58 2.28 1.62 2.05 g010 1.90 0.67 0.39 0.58 g011 0.00 0.77 1.14 1.43 g012 0.39 3.46 1.90 11.13 g013 3.26 2.86 1.62 4.98 g014 3.26 2.28 0.67 2.34 g015 3.46 3.06 1.90 9.78 g016 0.77 3.26 3.46 33.97 g017 0.00 0.58 0.49 1.24 g018 4.27 5.30 3.66 35.12 g019 10.25 8.85 8.60 51.71 g020 40.92 45.83 43.61 84.03 g021 11.99 10.99 10.01 32.13 g022 16.88 18.08 16.01 48.57 g023 2.57 2.57 1.90 28.49 g024 9.07 8.14 8.14 36.06 g025 55.61 56.48 40.35 86.55 g026 10.25 10.25 8.85 4.48 g027 0.95 0.77 0.39 1.33 g028 1.14 1.62 0.39 0.53 g029 1.90 1.62 0.58 0.77 g030 10.74 6.27 8.60 5.94 g031 33.69 30.99 29.71 63.14 g032 2.47 1.14 0.58 1.81 g033 2.09 1.33 1.14 52.88 g034 0.95 1.14 0.77 51.15 g035 0.77 2.28 1.71 11.50 g036 1.62 2.09 0.58 51.83 g037 0.00 2.57 1.33 56.86

105 g038 1.62 1.71 1.33 6.27 g039 7.24 8.14 4.68 18.22 g040 1.62 1.14 0.58 1.95 g041 0.49 3.86 2.47 8.74 g042 2.47 2.47 5.94 13.45 g043 0.77 0.77 0.39 1.05 g044 0.00 1.90 0.67 4.38 g045 0.95 1.71 0.39 2.67 g046 1.90 3.86 3.46 25.39 g047 1.33 1.90 1.90 1.48 g048 0.29 1.33 1.14 2.92 g049 0.77 1.71 0.95 25.78 g050 0.10 1.62 0.58 1.05 g051 0.09 0.77 0.58 2.87 g052 0.09 0.67 0.39 1.24 g053 5.94 6.50 4.06 17.77 g054 2.28 1.33 0.49 3.67 g055 6.27 5.94 6.27 15.48 g056 3.46 4.06 2.57 22.56 g057 0.77 0.77 0.39 0.39 g058 2.28 1.62 1.62 4.78 g059 10.74 9.54 7.47 26.38 g060 1.90 1.90 0.39 15.19 g061 6.27 5.94 3.86 39.24 g062 1.62 0.67 0.09 0.54 g063 1.62 1.90 0.39 0.86 g064 1.62 1.71 0.67 1.95 g065 5.94 6.15 4.88 30.15 g066 3.06 2.57 2.47 8.37 g067 2.28 1.71 0.29 1.14 g068 3.46 3.26 1.62 81.95 g069 44.53 26.90 42.08 78.60 g070 1.33 1.14 0.39 6.98 g071 10.99 13.54 10.99 48.82 g072 2.28 1.14 1.14 1.15 g073 25.39 23.24 26.12 44.53 g074 30.13 23.94 26.12 63.56 g075 15.73 12.76 15.17 49.35 g076 68.15 53.92 70.66 77.35 g077 31.86 18.08 15.17 42.69 g078 35.18 32.26 40.35 56.02

106 g079 21.21 20.55 23.24 19.76 g080 18.67 22.89 18.08 61.47 g081 15.73 17.46 15.17 41.82 g082 59.80 64.81 75.68 70.24 g083 20.55 20.23 39.79 18.84 g084 16.30 14.35 17.17 58.11 g085 18.08 10.99 14.08 58.96 g086 8.14 7.69 10.50 55.62 g087 3.26 4.06 3.86 23.94 g088 21.21 15.73 18.08 4.37 g089 2.28 2.28 3.46 24.66 g090 7.47 11.23 4.06 11.85 g091 1.14 0.49 1.14 0.15 g092 0.29 0.58 2.86 0.19 g093 0.95 0.77 0.58 0.39 g094 0.58 0.95 0.95 0.05 g095 0.67 0.95 0.67 0.25 g096 0.49 1.90 1.14 0.39 g097 0.95 0.95 0.49 0.05 g098 0.58 0.95 0.49 0.10 g099 0.67 0.67 2.47 0.09 g100 0.67 0.58 0.39 0.20 g101 0.29 0.29 0.29 0.43 g102 0.39 0.77 0.67 0.05 g103 0.39 3.06 0.67 0.20 g104 0.77 0.49 0.77 0.15 g105 0.49 0.58 1.14 0.34 g106 0.77 0.77 0.77 0.39 g107 0.67 0.29 0.67 0.00 g108 0.39 0.39 0.49 0.10 g109 0.77 0.95 0.77 0.10 g110 1.33 1.62 0.95 0.77 g111 46.62 59.80 51.52 56.45

107

Phosphorus Tests done at BYU on Guatemala soils soil sample Mehlich I Bray I g001 2.61 2.56 g002 0.52 1.19 g003 0.39 0.56 g004 0.33 0.59 g005 4.56 9.57 g006 0.01 2.36 g007 0.19 0.33 g008 10.92 6.70 g009 5.99 6.80 g010 1.01 1.32 g011 0.74 1.30 g012 20.88 16.36 g013 9.73 8.66 g014 2.97 4.97 g015 15.58 9.79 g016 35.36 26.07 g017 1.71 2.72 g018 32.49 19.52 g019 39.39 27.17 g020 61.76 69.47 g021 31.51 26.21 g022 41.32 45.08 g023 20.88 28.96 g024 32.66 54.08 g025 55.03 84.06 g026 8.35 17.66 g027 0.32 0.54 g028 0.27 0.41 g029 1.26 1.35 g030 10.98 26.87 g031 51.44 93.81

108 g032 2.16 9.68 g033 29.34 6.11 g034 33.02 5.79 g035 9.01 5.53 g036 30.02 5.00 g037 33.94 7.43 g038 5.83 13.07 g039 18.06 22.15 g040 2.83 4.15 g041 7.65 10.44 g042 9.68 14.44 g043 1.68 1.92 g044 4.05 9.42 g045 5.59 4.00 g046 29.07 7.33 g047 8.13 9.98 g048 12.33 5.34 g049 16.93 3.88 g050 1.63 3.21 g051 4.26 7.43 g052 1.78 3.27 g053 10.77 15.45 g054 4.31 8.27 g055 11.61 20.60 g056 19.79 23.02 g057 0.19 0.54 g058 5.34 4.54 g059 19.91 21.60 g060 16.83 3.94 g061 33.02 28.33 g062 0.45 -0.28 g063 1.01 0.02 g064 1.55 7.20 g065 27.04 40.33 g066 5.87 4.42 g067 0.61 0.02

109 g068 72.33 14.53 g069 61.76 70.30 g070 6.37 2.70 g071 36.03 48.20 g072 1.29 5.06 g073 38.53 49.27 g074 47.46 61.78 g075 44.01 31.25 g076 61.76 70.73 g077 37.99 51.76 g078 49.29 58.41 g079 25.10 42.45 g080 48.65 48.37 g081 39.10 37.33 g082 60.09 86.36 g083 27.04 51.76 g084 44.91 29.45 g085 45.38 38.13 g086 44.91 19.25 g087 24.01 10.13 g088 4.49 14.12 g089 17.18 39.58 g090 12.61 10.79 g091 0.04 0.23 g092 0.00 0.10 g093 0.11 - g094 0.01 0.46 g095 0.19 0.80 g096 0.44 0.90 g097 0.08 0.38 g098 0.41 1.51 g099 0.29 - g100 0.33 1.30 g101 0.35 0.46 g102 0.31 1.06 g103 0.33 0.64

110 g104 0.66 2.78 g105 0.50 0.90 g106 0.85 2.22 g107 -0.04 0.13 g108 - - g109 0.08 0.64 g110 1.43 3.33 g111 46.91 -

111

Morocco Soil Descriptions soil sample REGION SOIL TYPE m1 MA PE m2 MA PE m3 MA PE m4 MA CA m5 MA CA m6 MA CA m7 MA PE m8 MA PE m9 MA PE m10 MA FE m11 MA FE m12 MA FE m13 MA VE m14 MA VE m15 MA VE m16 MA IS m17 MA IS m18 MA IS m19 SA IS m20 SA IS m21 SA IS m22 SE IS m23 SE IS m24 SE IS m25 SE VE m26 SE VE m27 SE VE m28 SE VE m29 SE VE m30 SE VE m31 SE FE m32 SE FE m33 SE FE

112 m34 SE CA m35 SE CA m36 SE CA m37 SE PE m38 SE PE m39 SE PE m40 ST PE m41 ST PE m42 ST PE m43 SL CA m44 SL CA m45 SL CA m46 SS IS m47 SS IS m48 SS IS m49 MG FE m50 MG FE m51 MG FE m52 SY FE m53 SY FE m54 SY FE m55 MG PE m56 MG PE m57 MG PE m58 SL VE m59 SL VE m60 SL VE m61 BS VE m62 BS VE m63 BS VE m64 BS HY m65 BS HY m66 BS HY m67 BS FE m68 BS FE m69 BS FE

113 m70 BS PE m71 BS PE m72 BS PE m73 ZE HY m74 ZE HY m75 ZE HY m76 ZE PE m77 ZE PE m78 ZE PE m79 ZE FE m80 ZE FE m81 ZE FE m82 SA FE m83 SA FE m84 SA FE m85 SA CA m86 SA CA m87 SA CA m88 SA CA m89 SA CA m90 SA CA m91 SA VE m92 SA VE m93 SA VE m94 SA FE m95 SA FE m96 SA FE m97 SA VE m98 SA VE m99 SA VE m100 SA FE m101 SA FE m102 SA FE m103 SA CA m104 SA CA m105 SA CA

114 m106 SA CA m107 SA CA m108 SA CA m109 SA FE m110 SA FE m111 SA FE m112 SA IS m113 SA IS m114 SA IS m115 AH SA m116 AH SA m117 AH SA

115

Guatemala Soil Description soil sample Region Number g001 PET (Peten) 1 g002 PET (Peten) 2 g003 PET (Peten) 3A g004 PET (Peten) 3B g005 PET (Peten) 4 g006 PET (Peten) 5 g007 PET (Peten) 6 g008 PET (Peten) 7 g009 PET (Peten) 8 g010 PET (Peten) 9 g011 PET (Peten) 10 g012 PET (Peten) 11 g013 PET (Peten) 12 g014 PET (Peten) 13 g015 PET (Peten) 14 g016 PET (Peten) 15 g017 PET (Peten) 16 VOL (Volcanic, western g018 highlands) 1 VOL (Volcanic, western g019 highlands) 2 VOL (Volcanic, western g020 highlands) 3 VOL (Volcanic, western g021 highlands) 4 VOL (Volcanic, western g022 highlands) 6 VOL (Volcanic, western g023 highlands) 7 VOL (Volcanic, western g024 highlands) 8 VOL (Volcanic, western g025 highlands) 10 VOL (Volcanic, western g026 highlands) 11 VOL (Volcanic, western g027 highlands) 12

116 VOL (Volcanic, western g028 highlands) 13 VOL (Volcanic, western g029 highlands) 14 VOL (Volcanic, western g030 highlands) 15 VOL (Volcanic, western g031 highlands) 16 VOL (Volcanic, western g032 highlands) 17 g033 LAG (Lago Izabel) 1 g034 LAG (Lago Izabel) 2 g035 LAG (Lago Izabel) 3 g036 LAG (Lago Izabel) 4 g037 LAG (Lago Izabel) 5 g038 LAG (Lago Izabel) 6 g039 LAG (Lago Izabel) 7 g040 LAG (Lago Izabel) 8 g041 LAG (Lago Izabel) 9 g042 LAG (Lago Izabel) 10 g043 LAG (Lago Izabel) 11 g044 LAG (Lago Izabel) 12 g045 LAG (Lago Izabel) 13 g046 LAG (Lago Izabel) 14 g047 LAG (Lago Izabel) 15 g048 LAG (Lago Izabel) 16 g049 LAG (Lago Izabel) 17 g050 LAG (Lago Izabel) 18 g051 LAG (Lago Izabel) 19 g052 LAG (Lago Izabel) 20 g053 MI (Chiquimula area) 100601 g054 MI (Chiquimula area) 120615 g055 MI (Chiquimula area) 120612 g056 MI (Chiquimula area) 200652 g057 MI (Chiquimula area) 180631 g058 MI (Chiquimula area) 200650 g059 MI (Chiquimula area) 200653 g060 MI (Chiquimula area) 180635

117 g061 MI (Chiquimula area) 130619 g062 MI (Chiquimula area) 180630 g063 MI (Chiquimula area) 180626 g064 MI (Chiquimula area) 180629 g065 MI (Chiquimula area) 120614 g066 MI (Chiquimula area) 130622W g067 MI (Chiquimula area) 190637 g068 MI (Chiquimula area) 200651 g069 MI (Chiquimula area) 200656 g070 MI (Chiquimula area) 190639 g071 MI (Chiquimula area) 200660 g072 COA (Coastal) 1 g073 COA (Coastal) 2 g074 COA (Coastal) 4 g075 COA (Coastal) 5 g076 COA (Coastal) 6 g077 COA (Coastal) 7 g078 COA (Coastal) 8 g079 COA (Coastal) 9 g080 COA (Coastal) 10 g081 COA (Coastal) 11 g082 COA (Coastal) 12 g083 COA (Coastal) 13 g084 COA (Coastal) 14 g085 COA (Coastal) 15 g086 COA (Coastal) 16 g087 COA (Coastal) 17 g088 COA (Coastal) 18 g089 COA (Coastal) 19 g090 COA (Coastal) 20 g091 COB (Coban) 1 g092 COB (Coban) 2 g093 COB (Coban) 3 g094 COB (Coban) 4 g095 COB (Coban) 5 g096 COB (Coban) 6

118 g097 COB (Coban) 7 g098 COB (Coban) 8 g099 COB (Coban) 9 g100 COB (Coban) 10 g101 COB (Coban) 11 g102 COB (Coban) 12 g103 COB (Coban) 13 g104 COB (Coban) 14 g105 COB (Coban) 15 g106 COB (Coban) 16 g107 COB (Coban) 17 g108 COB (Coban) 18 g109 COB (Coban) 19 g110 COB (Coban) 20 g111 COB (Coban) 20B

119