ABSTRACT

The relative contribution of phosphorus (P) from agricultural nonpoint sources to surface water quality problems has increased in recent years as point sources of P have been reduced significantly. Phosphorus contributes to eutrophication, a process characterized by increased growth of undesirable algae and aquatic weeds, followed by oxygen shortages as the biomass decomposes. Eutrophication restricts water use for fisheries, recreation, industry, and human consumption. The focus of attention on P has increased the demand for information on methods of analysis of soil, water, and residual materials for environmentally relevant forms of P. The purpose of this publication is to present these methods in a single document. Previously, the methods have appeared across a wide variety of documents or only in the scientific literature. It is not the intent of this publication to define a uniform set of recommended methods for agronomic soils tests or for testing water and residual materials. The methods presented in this manual are intended solely to provide a set of uniform testing methods for environmental scientists working across an enormous range of soil and climatic conditions, with the hope that comparable methods may lead to improved communication and understanding of this complex issue.

FOREWORD

　As scientists focus on the fate of phosphorus applied to agricultural lands, it has become increasingly clear that a uniform set of testing methods is needed to enable comparison of results across county, state, regional, and even national boundaries.
　By contrast, soil testing developed with a high priority on meeting local needs. As a result, many local variations in extractants and laboratory procedures have been developed to achieve timely analysis and improved correlation of soil test results with plant responses within well-defined regions. Over time, enormous amounts of information on individual soils, crops, and extractants have been collected using these localized modifications and laboratory methods. Soil testing labs cannot easily change from one extractant to another. The cost of repeating the calibration experiments for many soils and crops is prohibitively expensive, and the changes would initially preclude users from comparing results across years. Even so, a set of standard reference methods can be useful for laboratories wishing to consider a new analysis for a particular element, and for comparing results across laboratories. In 1992, SERA-IEG-6 selected 15 reference procedures for soil testing laboratories in the southern region. Criteria for selection included the accuracy of the method in predicting crop responses, and general acceptability by workers in the soil testing field.
　This publication in no way attempts to define a uniform set of recommended methods for agronomic soil tests. The methods presented here are intended solely to provide a set of uniform testing methods for environmental scientists working across an enormous range of soil and climatic conditions, with the hope that comparable methods may lead to improved communication and understanding of this complex issue.
　For more information on agronomic soil testing methods, and the source of many of the procedures described here, the reader should refer to recent bulletins compiled by the various regional committees working on nutrient analysis of soils, plants, water, and waste materials (SERA-IEG-6, NCERA-13 and NEC-67). More detailed information on analysis of animal manures can be found in the publication “Recommended Methods for Manure Analysis . A3769” (www.sera17.ext.vt.edu/Documents/Recommended_Methods_Manure_Analysis.pdf).

FOREWORD FOR SECOND EDITION

　During the eight years since the original publication became available, the authors of many of the chapters in the manual learned that some editing, and in some cases clarification, was needed. The development of this 2nd Edition presented that opportunity. This publication now consists of 22 chapters, three of which are new.

TABLE OF CONTENTS

Methods of Phosphorus Analysis for Soils, Sediments, Residuals, and Waters: Introduction....................................................................................1
Gary M. Pierzynski, Kansas State University／Andrew N. Sharpley, University of Arkansas／John L. Kovar, USDA-ARS, Ames, IA

SOIL...............................................................................................................5【表1】
Soil Sample Collection, Handling, Preparation, and Storage..................6
Frank J. Coale, University of Maryland
Soil Test Phosphorus: Principles and Methods........................................9
J. Thomas Sims, University of Delaware
A Phosphorus Sorption Index....................................................................20
J. Thomas Sims, University of Delaware
Water- or Dilute Salt-Extractable Phosphorus in Soil............................22
M.L. Self-Davis, University of Arkansas／P.A. Moore, Jr., USDA-ARS, Fayetteville, AR／B.C. Joern, Purdue University
Phosphorus Extraction with Iron Oxide-Impregnated Filter Paper (Pi test)...............................................................................................................25
W. J. Chardon, Alterra, Wageningen UR. Wageningen, The Netherlands
Determination of the Degree of Phosphate Saturation in Noncalcareous Soils...............................................................................................................29
O.F. Schoumans, Alterra, Wageningen UR. Wageningen, The Netherlands
Phosphorus Sorption Isotherm Determination........................................33
D.A. Graetz, University of Florida／V.D. Nair, University of Florida
Bioavailable Phosphorus in Soil................................................................38
Andrew N. Sharpley, University of Arkansas
Total Phosphorus in Soil............................................................................44
April Leytem, USDA-ARS, Northwest Irrigation and Soils Research Laboratory／Kokoasse Kpomblekou-A, Tuskegee University
Fractionation of Soil Phosphorus..............................................................50【図1】【図2】
Hailin Zhang, Oklahoma State University／John L. Kovar, USDA-ARS National Soil Tilth Laboratory／
Procedures (see flow chart, Fig.2)
Phosphorus Fractionation in Flooded Soils and Sediments...................61
Philip Moore, USDA-ARS, Fayetteville, AR／Frank Coale, University of Maryland

RESIDUAL MATERIALS AND MANURES.........................................72
Sampling Techniques for Nutrient Analysis of Animal Manures..........73
R.O. Maguire and S.C. Hodges, Virginia Tech University／D.A. Crouse, North Carolina State University
Water-Extractable Phosphorus in Animal Manure and Biosolids........76
Ann M. Wolf, Pennsylvania State University／Philip A. Moore, Jr., USDA-ARS, Fayetteville, AR／Peter J.A. Kleinman, USDA-ARS, University Park, PA／Dan M. Sullivan, Oregon State University
Total Phosphorus in Residual Materials..................................................81
M.R. Bender, St. Cloud State University／C.W. Wood, Auburn University
Bioactive Phosphorus Fractions in Animal Manure, Soil, and Extracts of Soils and Manures..................................................................................87
Phosphorus Speciation in Soils and Manures by Solution 31P NMR Spectroscopy................................................................................................95【表1】
Benjamin L. Turner, Smithsonian Tropical Research Institute, Republic of Panama／April B. Leytem, USDA-ARS, Kimberly, ID

WATER.....................................................................................................101
Water Sample Collection, Handling, Preparation and Storage...........102【図1】
P.M. Haygarth, North Wyke, Okehampton, England／A.C. Edwards, Nether Backhill, Ardallie, By Peterhead, Scotland
Dissolved Phosphorus in Water Samples...............................................110
D.H. Pote, USDA-ARS, Booneville, AR／T.C. Daniel, University of Arkansas
Total Phosphorus and Total Dissolved Phosphorus in Water Samples.....................................................................................................................113
D.H. Pote, USDA-ARS, Booneville, AR／T.C. Daniel and P.B. DeLaune, University of Arkansas
Using the Iron Oxide Method to Estimate Bioavailable Phosphorus in Runoff.........................................................................................................118
R.G. Myers, Kansas State University／G.M. Pierzynski, Kansas State University

図1　無機リンについての連続分画法（元は図）

非石灰質土壌				石灰質土壌
100 mLの遠心分離管に土壌試料1.0 g				100 mLの遠心分離管に土壌試料1.0 g
↓				↓
50 mLの1M NH4Clを加え、30分間振とう、遠心分離	A →	可溶性でゆるく結合したP	A ←	50 mLの0.1M NaOH＋1M NaClを加え、17時間攪拌、遠心分離、飽和NaClで洗浄
残渣 ↓
50 mLの0.5M NH4Fを加え、1時間振とう、遠心分離、飽和NaClで洗浄	B →	Al-P	A ←
残渣 ↓
50 mLの0.1M NaOHを加え、17時間振とう、遠心分離、洗浄	C →	Fe-P	A ←
残渣 ↓				残渣 ↓
40 mLの0.3M Na3C6H5O7・2H2O（クエン酸ナトリウム）と5 mLの1M NaHCO3（炭酸水素ナトリウム）と1.0 gのNa2S2O4（亜ジチオン酸）を加え、.加熱、攪拌、加熱、遠心分離、洗浄	D →	還元剤で可溶なP	B ←	40 mLの0.3M Na3C6H5O7・2H2O（クエン酸ナトリウム）と5 mLの1M NaHCO3（炭酸水素ナトリウム）と1.0 gのNa2S2O4（亜ジチオン酸）を加え、.加熱、攪拌、加熱、遠心分離、洗浄
残渣 ↓				残渣 ↓
50 mLの0.25M H2SO4を加え、1時間振とう、遠心分離、洗浄	E →	Ca-P	C ←	50 mLの0.5M HClを加え、1時間振とう、遠心分離、洗浄

図2　有機リンについての連続分画法（元は図）

100 mLの遠心分離管に湿った土壌試料1.0 ｇ（オーブンで乾燥）				100 mLの遠心分離管に湿った土壌試料1.0 ｇ（オーブンで乾燥）
↓				↓
				2 mLのCHCl3（クロロホルム）（微生物細胞の分解）
50 mLの0.5M NaHCO3（炭酸水素ナトリウム）を加え、16時間振とう、遠心分離、濾過	分液 →	不安定なPi		↓
	分液 →	過硫酸温浸	←	50 mLの0.5M NaHCO3（炭酸水素ナトリウム）を加え、16時間振とう、遠心分離、濾過
残渣 ↓		↓
		不安定なPの総量 不安定なPo＝不安定なPの総量−不安定なPi バイオマスPo＝全PCHCl3−不安定なPの総量
50 mLの1.0M HCｌ（塩酸）を加え、3時間振とう、遠心分離、濾過	分液 →	適度に不安定なPi
	分液 →	過硫酸温浸	→	全P 適度に不安定なPo＝全P−Pi
残渣 ↓
脱イオン水で5分間すすぎ洗い、遠心分離	→	上澄み液廃棄
残渣 ↓
50 mLの0.5M NaOH（水酸化ナトリウム）を加え、6時間振とう、遠心分離、濾過	分液 →	過硫酸温浸	→	全P
	分液 →	pH 1.0-1.5に酸性化、遠心分離	分液 →	過硫酸温浸
				↓
				フルボ酸Po フミン酸Po＝全P−フルボ酸Po
脱イオン水で5分間すすぎ洗い、遠心分離	→	上澄み液廃棄
残渣 ↓
550℃で灰化1時間	→	50 mLの1.0M H2SO4（硫酸）を加えて溶解、振とう24時間	→	不安定でないPo

表1．pH＞13で抽出された土壌と肥やしの溶液³¹P NMR（核磁気共鳴）スペクトルのシグナルの割当

官能基または化合物	シグナル	含まれる化合物
Phosphonates〔ホスホン酸、C-PO(OH)2またはC-PO(OR)2（R=alkyl, aryl）〕	18〜22 ppm	2-aminoethylphosphonic acid（2-AEP、NH2- CH2CH2PO(OH)2）
Phosphate（リン酸塩）	6.1 ppm	無機オルトリン酸塩（orthophosphate）
Phosphate monoesters（リン酸一エステル）	3.0〜6.0 ppm 6.8 ppm	myo-inositol hexakisphosphate（4.6, 4.8, 5.0, 5.9 ppm）（ミオイオノシトール六リン酸、フィターゼ、phytase）、scyllo-inositol hexakisphosphate（4.2 ppm）、その他のイノシトールリン酸、糖リン酸（sygar phosphates）、モノヌクレオチド（mononucleotides）
Phosphate diesters（リン酸ニエステル）	-1.0〜2.5 ppm	DNA（-0.5 ppm）、リン脂質（0.5〜2.0 ppm）（phospholipids）、RNA（0.5 ppm）
Pyrophosphate（ピロリン酸塩、ニリン酸塩、diphosphate、P2O74-）	-4.4 ppm
Polyphosphate（ポリリン酸塩、PnO3n+1(n+2)-）	-4.0 ppm（端グループ） -18〜-23 ppm（中間グループ）	長鎖ポリリン酸塩（long-chain polyphosphate）
Organic polyphosphates	-4.3 ppm（γ-リン酸塩） -9.7 ppm（α-リン酸塩） -19.7 ppm（β-リン酸塩）	アデノシン二リン酸塩（adenosine diphosphate）、アデノシン三リン酸塩（adenosine triphosphate）

図1　水試料中のリンの操作上定義された形態（CNA＝セルロース−ニトレート−アセテート、ICP＝誘導結合プラズマ）（元は図、一部修正）

水試料中のリン
↓	↓	↓	↓
	孔径0.45μmのCNAメンブレンフィルターで濾過
	↓	↓
モリブデン酸−青反応（Murphy and Riley,1962にならった）		温浸の適切な手法（ICPのような手法）
↓	↓	↓	↓
濾過してない反応性P RPunf	濾過した（＜0.45μm）反応性P RP＜0.45μm	濾過した（＜0.45μm）全P TP＜0.45μm	濾過してない全P TPunf
差による計算 反応性P（＞0.45）＝RPunf−RP＜0.45μm 非反応性P（＜0.45）＝TP＜0.45μm−RP＜0.45μm 全P（＞0.45）＝TPunf−TP＜0.45μm 非反応性P（＞0.45）＝全P（＞0.45）−反応性P（＞0.45）