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Testing Methods for Fertilizers(2018)

Incorporated Administrative Agency
Food and Agricultural Materials Inspection Center

Testing Methods for Fertilizers (2018)

Introduction“The Official Methods of Analysis of Fertilizers” stipulated by the Ministry ofAgriculture, Forestry and Fisheries is the only analysis method to assess main
components and harmful components in fertilizers in Japan and contributes tomaintaining quality and securing safety of fertilizers. However, no new revision has
been issued since “The Official Methods of Analysis of Fertilizers 1992” was issued.
Some quarters such as fertilizer manufacturers and inspection instruction agencies
have requested a revised edition of “The Official Methods of Analysis of Fertilizers”
since new kinds of fertilizers and its new components were added into the publicstandard, and analysis instruments and technologies have progressed during the period.
Incorporated Administrative Agency, Food and Agricultural Materials Inspection
Center (FAMIC) has revised the Official Methods of Analysis of Fertilizers byintroducing the analysis conditions and the analysis methods, etc. which meet the latest
situation. Additionally, FAMIC tried to study how to introduce the analysis methods or
the new analysis instruments to cope with new effective components or harmful
components and new fertilizers which are not documented in the Official Methods ofAnalysis of Fertilizers, and established new testing methods. At the same time, FAMIC
conducted a validity test according to the requirements of ISO/IEC 17025 and opened
the results and new testing methods which were discussed and approved by “theTechnical Committee for Fertilizers etc.” including outside experts on FAMIC’s web
site in 2008 as “The Testing Methods for Fertilizers 2008”. Since then, the contents
have been annually added and updated. In this year, High Performance Liquid
Chromatograph, etc. for uric acid within fluid fertilizers which have been studied anew
in FY2017 were merged, and the “Testing Methods for Fertilizes 2018” was opened on
FAMIC’s web site.
The “Testing Methods for Fertilizes” uses reagents and instruments which are
stipulated in JIS standards, etc. and its validity of the testing method is checked byreferencing IUPAC protocols and it is listed as an analysis method whose validity ischecked in “The Management Handbook of Heavy Metal in Sludge Fertilizers”
published by the Ministry of Agriculture, Forestry and Fisheries in August 2010.
Therefore, FAMIC would like people engaged in the quality control and analysis offertilizes to use this as a practical document.
June , 2018
Incorporated Administrative Agency
Food and Agricultural Materials Inspection Center
General Director
Makoto KimuraTesting Methods for Fertilizers (2018)

The members of The Technical Committee
for the Testing Methods for Fertilizers (2017)(Without honorifics, as of March, 2018)
Mayumi AIZAKI

National Federation of Agricultural Cooperative
Associations

Satoshi ISAGAWA

Japan Food Research Laboratories

Toshiaki IMAGAWA

Japan Fertilizer and Feed Inspection Association

Akira KAWASAKI

National Research and Development Agency: National
Agriculture and Food Research Organization

Takayoshi KUROIWA

National Research and Development Agency: National
Metrology Institute of Japan

Shigehiro NAITO

National Research and Development Agency: National
Agriculture and Food Research Organization

Akira NOGUCHI

Incorporated Educational Institution: Nihon
University

Kazuyuki YAZIMACorporate Juridical Person: Niigata Prefectural
Institute of Public Health and Environmental
Sciences

Akemi YASUINational Research and Development Agency: National
Agriculture and Food Research Organization

Testing Methods for Fertilizers (2018)
Contents

1.
General Rule
1.1
Common Items
1.2
Validity check of testing methods
1.3
Adoption of testing methods
1.3.1 Competence evaluation of laboratory
1.3.2 Evaluation of test result

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....
....
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1
1
6
8
8
8

2.
Handling of samples
2.1
Sampling
2.2
Storage of samples
2.3
Preparation of test samples
2.3.1 Pre-drying
2.3.2 Reduction (Separation)
2.3.3 Grinding

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9
9
10
11
12
14
15

3.
General tests
3.1
Moisture or moisture content
3.1.a Loss on drying method with drying apparatus
3.1.b Loss on drying method by moisture analyzers
3.2
Ash content
3.2.a Ignition residue method
3.3pH
3.3.a Glass electrode method
3.4
Electrical conductivity
3.4.a Measurement method with an electrical conductivity meter
3.5
Granularity
3.5.a Dry-type sieving testing method
3.6
Oil content
3.6.a Diethyl ether extraction method

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17
17
17
20
23
23
24
24
26
26
28
28
30
30

4.
Main components, guaranteed components, etc
4.1
Nitrogen4.1.1 Total nitrogen4.1.1.a
Kjeldahl method
4.1.1.b
Combustion method
4.1.1.c
Devarda’s alloy – Kjeldahl method
4.1.1.d
Reduced iron– Kjeldahl method
4.1.1.eCalculation with ammoniac nitrogen and nitrate nitrogen

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32
32
32
32
38
43
50
56

-i-

Testing Methods for Fertilizers (2018)

4.1.2 Ammoniac nitrogen.... 57
4.1.2.aDistillation method.... 57
4.1.2.bFormaldehyde method.... 63
4.1.3 Nitrate nitrogen.... 68
4.1.3.aDevarda’s alloy - distillation method.... 68
4.1.3.b
Reduced iron- distillation method.... 73
4.1.3.cPhenol sulfuric acid method.... 78
4.2
Phosphoric acid.... 82
4.2.1 Total phosphoric acid.... 82
4.2.1.aAmmonium vanadomolybdate absorptiometric analysis.... 82
4.2.1.b
Quinoline gravimetric analysis.... 91
4.2.2 Soluble phosphoric acid.... 95
4.2.2.aAmmonium vanadomolybdate absorptiometric analysis.... 95
4.2.2.b
Quinoline gravimetric analysis.... 101
4.2.3 Citrate-soluble phosphoric acid.... 105
4.2.3.aAmmonium vanadomolybdate absorptiometric analysis.... 105
4.2.3.b
Ammonium vanadomolybdate absorptiometric analysis (Fertilizers containing
phosphorous acid or phosphite).... 1114.2.3.c
Quinoline gravimetric analysis.... 118
4.2.3.d
ICP Optical Emission Spectrometry.... 122
4.2.4 Water-soluble phosphoric acid.... 128
4.2.4.aAmmonium vanadomolybdate absorptiometric analysis.... 128
4.2.4.b
Ammonium vanadomolybdate absorptiometric analysis (Fertilizers containing
phosphorous acid or phosphite).... 136
4.2.4.c
Quinoline gravimetric analysis.... 143
4.2.4.d
ICP Optical Emission Spectrometry.... 146
4.3
Potassium.... 151
4.3.1 Total potassium.... 151
4.3.1.a
Flame atomic absorption spectrometry or flame photometry.... 151
4.3.1.b
Sodium tetraphenylborate gravimetric analysis.... 157
4.3.2 Citrate soluble potassium.... 162
4.3.2.a
Flame atomic absorption spectrometry or flame photometry.... 162
4.3.2.b
Sodium tetraphenylborate gravimetric analysis.... 166
4.3.2.c
Sodium tetraphenylborate volumetric analysis.... 170
4.3.2.d
ICP Optical Emission Spectrometry.... 173
4.3.3 Water-soluble potassium.... 177
4.3.3.a
Flame atomic absorption spectrometry or flame photometry.... 177
4.3.3.b
Sodium tetraphenylborate gravimetric analysis.... 184
4.3.3.c
Sodium tetraphenylborate volumetric analysis.... 188
4.3.3.d
ICP Optical Emission Spectrometry.... 192- ii -

Testing Methods for Fertilizers (2018)

4.4
Silicic acid....
4.4.1 Soluble silicic acid....
4.4.1.aPotassium fluoride method....
4.4.1.b
Potassium fluoride method (Silica gel fertilizers, etc.)....
4.4.1.c
Potassium fluoride method (Fertilizers containing silica gel fertilizers) ....
4.4.1.d
Perchloric acid method....
4.4.2 Water-soluble silicic acid....
4.4.2.aPotassium fluoride method....
4.5
Lime, calcium and alkalinity....
4.5.1 Total lime....
4.5.1.a
Flame atomic absorption spectrometry....
4.5.2 Soluble lime....
4.5.2.a
Flame atomic absorption spectrometry....
4.5.3 Water-soluble calcium....
4.5.3.a
Flame atomic absorption spectrometry....
4.5.3.b
ICP Optical Emission Spectrometry....
4.5.4 Alkalinity....
4.5.4.aEthylenediamine tetraacetate method....
4.5.4.bCalculation with soluble lime and soluble magnesia....
4.6
Magnesia....
4.6.1 Total magnesia....
4.6.1.a
Flame atomic absorption spectrometry....
4.6.2 Soluble magnesia....
4.6.2.a
Flame atomic absorption spectrometry....
4.6.3 Citrate-soluble magnesia....
4.6.3.a
Flame atomic absorption spectrometry....
4.6.3.b
ICP Optical Emission Spectrometry....
4.6.4 Water-soluble magnesia....
4.6.4.a
Flame atomic absorption spectrometry....
4.6.4.b
ICP Optical Emission Spectrometry....
4.7
Manganese....
4.7.1 Soluble manganese....
4.7.1.a
Flame atomic absorption spectrometry....
4.7.2 Citrate-soluble manganese....
4.7.2.a
Flame atomic absorption spectrometry....
4.7.2.b
ICP Optical Emission Spectrometry....
4.7.3 Water-soluble manganese....
4.7.3.a
Flame atomic absorption spectrometry....
4.7.3.b
ICP Optical Emission Spectrometry
....
4.8
Boron
....
- iii -

195
195
195
199
203
209
212
212
216
216
216
222
222
225
225
228
231
231
237
238
238
238
243
243
246
246
251
256
256
259
263
263
263
266
266
271
275
275
281
285

Testing Methods for Fertilizers (2018)

4.8.1 Citrate-soluble boron
4.8.1.aAzomethine-H method
4.8.1.b
ICP Optical Emission Spectrometry
4.8.2 Water-soluble boron
4.8.2.aAzomethine-H method
4.8.2.b
ICP Optical Emission Spectrometry
4.9
Zinc
4.9.1 Total zinc
4.9.1.a
Flame atomic absorption spectrometry
4.9.1.b
ICP Optical Emission Spectrometry
4.9.2 Water-soluble zinc
4.9.2.a
Flame atomic absorption spectrometry
4.9.2.b
ICP Optical Emission Spectrometry
4.10
Copper
4.10.1 Total copper
4.10.1.aFlame atomic absorption spectrometry
4.10.1.b
ICP Optical Emission Spectrometry
4.10.2 Water-soluble copper
4.10.2.aFlame atomic absorption spectrometry
4.10.2.b
ICP Optical Emission Spectrometry
4.11 Organic carbon and carbon-nitrogen ratio
4.11.1 Organic carbon
4.11.1.aDichromate oxidation
4.11.1.bCombustion method
4.11.2 Carbon-nitrogen ratio
4.11.2.aCalculation with organic carbon and total nitrogen4.12 Sulfur
4.12.1 Total sulfur content
4.12.1.aPotassium permanganate analysis
4.12.1.bBarium chloride gravimetric analysis
4.12.1.c
Transmitted light analysis
4.13 Iron
4.13.1 Water-soluble iron4.13.1.aFlame atomic absorption spectrometry
4.13.1.b
ICP Optical Emission Spectrometry
4.14 Molybdenum
4.14.1 Water-soluble molybdenum
4.14.1.aSodium thiocyanate absorptiometric analysis
4.14.1.b
ICP Optical Emission Spectrometry
4.15 Cobalt- iv -

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285
285
291
295
295
299
303
303
303
307
311
311
315
318
318
318
322
326
326
330
333
333
333
337
342
342
343
343
343
345
350
355
355
355
359
362
362
362
366
369

Testing Methods for Fertilizers (2018)

4.15.1 Water-soluble cobalt
4.15.1.aFlame atomic absorption spectrometry
4.15.1.b
ICP Optical Emission Spectrometry.... 369.... 369.... 371

5.
Harmful components.... 374
5.1
Mercury.... 374
5.1.a Cold vapor atomic absorption spectrometry.... 374
5.1.b Cold vapor atomic absorption spectrometry (Fluid sludge fertilizers).... 378
5.2
Arsenic.... 382
5.2.a Hydride generation atomic absorption spectrometry.... 382
5.2.b Silver diethyl dithiocarbamate absorptiometric analysis.... 388
5.2.c ICP Mass Spectrometry (Fluid sludge fertilizers).... 393
5.3
Cadmium.... 398
5.3.a Flame atomic absorption spectrometry.... 398
5.3.b ICP Optical Emission Spectrometry.... 402
5.3.c ICP Mass Spectrometry (Fluid sludge fertilizers).... 406
5.3.d ICP Mass Spectrometry.... 411
5.4
Nickel.... 415
5.4.a Flame atomic absorption spectrometry.... 415
5.4.b ICP Optical Emission Spectrometry.... 419
5.4.c ICP Mass Spectrometry (Fluid sludge fertilizers).... 423
5.5
Chromium.... 428
5.5.a Flame atomic absorption spectrometry (Fertilizers containing organic matters) .... 428
5.5.b Flame atomic absorption spectrometry (Fertilizers mainly containing molten matters, slag,etc.).... 432
5.5.c Flame atomic absorption spectrometry (Fertilizers not containing organic matters). 438
5.5.d ICP Optical Emission Spectrometry.... 444
5.5.e ICP Mass Spectrometry (Fluid sludge fertilizers).... 447
5.6
Lead.... 452
5.6.a Flame atomic absorption spectrometry.... 452
5.6.b ICP Optical Emission Spectrometry.... 456
5.6.c ICP Mass Spectrometry (Fluid sludge fertilizers).... 460
5.7
Sulfamic acid (amidosulfuric acid).... 465
5.7.a Ion Chromatography.... 465
5.7.b High Performance Liquid Chromatograph Mass Spectrometry.... 469
5.8
Ammonium thiocyanate (Sulfurized cyanide).... 475
5.8.a Ion Chromatography.... 475
5.8.b High Performance Liquid Chromatograph Mass Spectrometry.... 479
5.9
Nitrous acid.... 486
5.9.a High Performance Liquid Chromatography.... 486-v-

Testing Methods for Fertilizers (2018)5.10 Biuret nitrogen
5.10.a High Performance Liquid Chromatography
5.11 Titan
5.11.a ICP Optical Emission Spectrometry (1)5.11.b ICP Optical Emission Spectrometry (2)5.12 Sulfurous Acid

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....
....
....
....
....

493
493
499
499
503
507

6.
Testing relating to the other limitations
6.1
Dicyandiamide nitrogen
6.1.a High Performance Liquid Chromatography (1)6.1.b High Performance Liquid Chromatography (2)6.2Chlorine
6.2.a Ion Chromatography
6.2.b Silver nitrate method
6.3
Urea nitrogen
6.3.a Urease method
6.3.b High Performance Liquid Chromatography
6.3.c p-dimethylaminobenzaldehide absorptiometry
6.4
Guanidine nitrogen
6.4.a High Performance Liquid Chromatography
6.5
Cold buffer solution soluble nitrogen (water-soluble nitrogen)6.5.a Cold buffer solution method
6.6
Heat buffer solution soluble nitrogen (hot-water soluble nitrogen)6.6.a Heat buffer solution method
6.7
Activity coefficient of nitrogen
6.7.a Buffer solution method
6.8
Initial elution rate
6.8.a Standing-in-water method
6.9
Humic acid (Acid insoluble - alkali soluble component)
6.9.a Gravimetric analysis
6.10 Sulfate
6.11 Carbon dioxide

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508
508
508
512
518
518
523
526
526
534
541
544
544
550
550
556
556
562
562
570
570
573
573
577
578

7.
7.1
7.1.a
7.2
7.2.a
7.3
7.3.a

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....

579
579
579
583
583
587
587

Nitrification inhibitor
2-amino-4-chloro-6-methylpyrimidine (AM)High Performance Liquid Chromatography
1-amidino-2-thiourea (ASU)
High Performance Liquid Chromatography
4-amino-1,2,4-triazole hydrochloride (ATC)High Performance Liquid Chromatography- vi -

Testing Methods for Fertilizers (2018)

7.4
7.4.a
7.5
7.5.a
7.6
7.6.a

N-2,5-dichlorophenyl succinamic acid (DCS)High Performance Liquid Chromatography
Dicyandiamide (Dd)High Performance Liquid Chromatography
2-sulfanilamide thiazole (ST)High Performance Liquid Chromatography

....
....
....
....
....
....

590
590
594
594
600
600

8.
Others.... 604
8.1
Melamine and its degradation products.... 604
8.1.a Gas Chromatography/Mass Spectrometry.... 604
8.1.b.... 611
8.1.c High Performance Liquid Chromatography (Fertilizers not containing organic matters).... 612
8.1.d High Performance Liquid Chromatography （Fertilizers containing organic matters）.... 618
8.2
Melamine and its degradation products.... 622
8.2.a High Performance Liquid Chromatography/Tandem Mass Spectrometry(1: Simultaneous analysis of three components for clopyralid, etc.).... 622
8.2.b High Performance Liquid Chromatography/Tandem Mass Spectrometry(2: Microanalysis for clopyralid).... 631
8.3
Residue agrichemicals (multicomponent).... 641
8.3.1 Residue agrichemicals multicomponent analysis (1).... 641
8.3.1.a High Performance Liquid Chromatography/Tandem Mass Spectrometry .... 641
8.3.2 Residue agrichemicals multicomponent analysis (2).... 650
8.3.2.a Gas Chromatography.... 650
8.4
Sodium.... 657
8.4.a Flame atomic absorption spectrometry.... 657
8.5
Guanylurea nitrogen.... 661
8.5.a High Performance Liquid Chromatography.... 661
8.6
Uric acid.... 667
8.6.a High Performance Liquid Chromatography.... 667
Appendix. The procedure to validate characteristics of testing methods- vii -.... 672

Testing Methods for Fertilizers (2018)

1. General Rule±
1.1 Common Items(1) Applicable range
The Testing Methods for Fertilizers stipulate the official method of analysis of fertilizers andfertilizer materials. The type of samples in the tests is shown in the summary of respective test
items.
(2) General matters in common, procedures and terms(2.1) Terms related to laws and ordinancesa) Main components or major components: The main components or major components infertilizers in Table 1 are stipulated as components to be calculated by a public notice of the
Ministry of Agriculture, Forestry and Fisheries.
Table 1 Components to be calculated as main component
or major component in fertilizers

Main component or major
component
Phosphate

Component to be calculated
Phosphorus pentoxide (P2O5 )Potassium

Potassium oxide (K2 O)

Silicate
Magnesium
Manganese
Boron

Silicon dioxide (SiO2)
Magnesium oxide (MgO)
Manganese oxide (MnO)
Diboron trioxide (B2 O3)

Sulfur content
Lime

Sulfur trioxide (SO3 )Calcium oxide (CaO)(2.2) General matters and terms that cite Japanese Industrial Standards (JIS)a) General notices: General matters common to analysis are according to JIS K 0050.
b) Definition: The definitions of major terms used in the Testing Methods for Fertilizers areaccording to JIS K 0211, JIS K 0214, JIS K 0215, JIS Z 8101-1, JIS Z 8101-2 or JIS Z
8101-3.
c) Laboratory sample: A sample transferred to a laboratory. Laboratory sample as specified in
JIS K 0211.
d) Test sample: A sample obtained from a laboratory sample after pretreatment such as grinding.
Test sample as specified in JIS K 0211.
e) Analytical sample: A sample measured from a laboratory sample or a test sample and to be
used in one test. Test sample or analytical sample as specified in JIS K 0211.
f) Sample: A sample in the testing methods indicates e) a laboratory sample, f) a test sample org) an analytical sample.
g) Rounding numbers: Methods of rounding the numbers are according to JIS Z 8401.
h) Absorptiometric analysis: General rules for absorptiometric analysis are according to JIS K
0115.
i) Atomic absorption spectrometry: Atomic absorption spectrometry includes flame atomic
absorption spectrometry, electrically heated atomic absorption spectrometry (hereinafter1

Testing Methods for Fertilizers (2018)

j)
k)
l)
m)
n)
o)
p)
q)
r)

referred to as “electrically heated atomic absorption spectrometry”) and other atomic
absorption spectrometry. General matters common to these are according to JIS K 0121.
Gas chromatography: General matters common to gas chromatography are according to JIS
K 0114.
Gas chromatography/Mass spectrometry: General matters common to Gas
chromatography/Mass spectrometry are according to JIS K 0123.
Electrical conductivity measuring method: General matters common to electricalconductivity measuring methods are according to JIS K 0130.
Test sieving: General matters common to test sieving are according to JIS Z 8815.
High Performance Liquid Chromatography: General matters common to High
Performance Liquid Chromatography are according to JIS K 0124.
High Performance Liquid Chromatography/Mass Spectrometry: General matterscommon to High Performance Liquid Chromatography/mass spectrometry are according to
JIS K 0136.
ICP Optical Emission Spectrometry: General matters common to ICP Optical Emission
Spectrometry are according to JIS K 0116.
ICP Mass Spectrometry: General matters common to ICP Mass Spectrometry are according
to JIS K 0133.
Ion Chromatography: General matters common to Ion Chromatography are according to JIS
K 0127.

(2.3) Description methods, procedures and terms in testing methods for fertilizers.
a) Reagent name: Unless otherwise specified, conform to the names by the chemical
nomenclature established by the Chemical Society of Japan [in accordance with theInternational Union of Pure and Applicable Chemistry (IUPAC) nomenclature of inorganic
chemistry and nomenclature of organic chemistry] and the names of JIS reagents.
b) Organic matters: Fertilizers such as Organic fertilizers, sludge fertilizers and compost andfertilizer materials. However, organic compounds such as urea and urea compounds are
excluded.
c) Actual article: A laboratory sample in original state.
d) Drying matter: The matter which remains after drying the actual article.
e) Notes, comments, figures, tables and formulae: Serial numbers for each test item should berecorded in notes, comments, figures, tables and formulae.
f) Dilution of solution: “Transfer accurately a predetermined amount (to a vessel)” means theprocedure to measure any volume of solution with a measuring instrument specified in JIS K
0050 (into a vessel).
Also, “dilute accurately a predetermined amount (with solvent or solution)” means theprocedure to measure any volume of solution with a measuring instrument specified in JIS K
0050 into a volumetric flask of arbitrary volume and fill up to the marked line (with solvent or
solution) (1).
g) Description of mixture solution: Mixture solutions are described as shown in 1) - 4).
1) Reagent + reagent: Describe as reagent name 1−reagent name 2 (V1 + V2). In this case, V1
volume of reagent name 1 is mixed with V2 volume of reagent name 2.
Example: acetonitrile−water (1+1), hexane−ethyl acetate (2+1), methanol−buffer solution(3+1)2) Reagent + water: Describe as reagent name 1 (V1+V2). In the case of reagents described inTable 1 in JIS K 0050, it means V1 volume of reagent name 1 is diluted by mixing with V2volume of water.
Example: hydrochloric acid (1+1), sulfuric acid (1+2), ammonia solution (1+3)
2

Testing Methods for Fertilizers (2018)

3) Solution + reagent: Describe as solution name a (concentration) - reagent name b [V1+V2].
In this case, it means V1 volume of solution name a of a certain concentration is mixed with V2volume of reagent name b.
Example: sodium hydroxide solution (4 g/L)−methanol [1+4]4) Diluted reagent + reagent: Describe as reagents name a (V1+V2) - reagent name b [V3+V4].
In this case, V3 volume of the solution in which V1 volume of reagent name a described inTable 1 in JIS K 0050 diluted by mixing with V2 volume of water, is mixed with V4 volume ofreagent name b.
Example: hydrochloric acid (1+100)−methanol [2+3]h) Preparation of a calibration curve: “Transfer A mL - B mL of the standard solution tovolumetric flasks step-by-step.” means the procedure to transfer a volume of 4 - 6 steps (2) inthe range from A mL to B mL of the standard solution to respective volumetric flasks
step-by-step.
Prepare a calibration curve every time a test is conducted. Also, when the same test item ismeasured under the same conditions for multiple samples continuously, measure the standard
solution at regular intervals to check the indicated value.
i) Washing of apparatus: Wash containers with a detergent and tap water before usage andwash sufficiently with water of A2 specified in JIS K 0577 or water that is confirmed not toaffect a quantification value. In case of sampling a sample to test a metallic element andorganic materials, after previous washing, dip with nitric acid (1+9) or hydrochloric acid(1+9) as appropriate and further wash sufficiently with water of A2, A3 or A4.
j) Handling of reagents and liquid waste, etc.: Handle with care and in compliance with
relevant laws and regulations. When treatment methods are specified in respective test items,
comply with the methods.
k) Referential matters related to the validity of testing methods: Information related to thevalidity of respective testing methods such as quantification range (minimum limits ofquantification, etc.), mean recovery, repeatability, intermediate precision and reproducibility
is described in a Comment, etc. Note that the numerical values such as Minimum Limit of
Quantification, etc. are not standards to be targeted but examples.
Note (1) When the dilution factor is large, accuracy should be secured by procedures such as
repeating the dilution procedure.
(2) Set according to the specification and operation method of the measurement instrumentused. There is no need to include the minimum and the maximum values of thecalibration curb range described in the Testing Methods for Fertilizers.
(3) Watera) Water: Water used in the Testing Methods for Fertilizers herein is water of A2 specified in JIS
K 0557 or water that is confirmed not to affect a quantitation value. However, when otherwise
specified in respective test items, use the specified water.
(4)a)

Reagents
Reagents: When the reagent is JIS-specified, use one of highest quality among those marked
with the JIS symbol; when none of the reagent is marked with the JIS symbol, use one ofquality that will not cause a problem in the test. Use reference materials for volumetricanalysis specified in JIS K 8005 for the standardization of titration solutions.
b) Reference materials: The preparation of standard solutions or standardization of titration
solutions using reference materials 1) - 2) below other than materials specified in respective
testing items is possible.
3

Testing Methods for Fertilizers (2018)

1) Reference materials provided by National Metrology Institute: Reference materials
traceable to International System of Unit (SI) provided by National Metrology Institute (NMI:
National Institute of Advanced Industrial Science and Technology NMIJ, NIST, BAM, etc.)
which signed CIPM MRA (Global Mutual Recognition Arrangement based on the Meter
Convention)2) Reference materials for volumetric analysis: Reference materials for volumetric analysis
specified in JIS K 8005.
c) Standard Solutions: In the cases of specifying in the comment in respective testing items, thepreparation of standard solutions for a calibration curve using the solution which is traceable
to the National Metrology of 1) - 3) below other than specified in respective testing items ispossible. However, use standard solutions which do not cause a problem in the test with thekinds and concentration of chemical compounds or added acid used. In addition, in (2.1) a)
Main components or major components, calculate main components or major components
using conversion factors specified in the comment of respective items.
1) Standard Solutions provided by National Metrology Institute: Standard solutions
traceable to International System of Unit provided by National Metrology Institute (NMI:
National Institute of Advanced Industrial Science and Technology NMIJ, NIST, BAM, etc.)
which signed CIPM MRA.
2) JCSS (Japan Calibration Service System) Standard Solutions: Standard solutions
prepared by JCSS (Japan Calibration Service System) the registered provider for Chemical
Analysis, Atomic Absorption Spectrometry, ICP or Ion Chromatography traceable to specificreference materials based on Measurement Act Article 134. In addition, it is recommended touse standard solutions which indicate the uncertainty of concentration and factors.
3) Standard Solutions traceable to National Metrology: Standard Solutions traceable to
National Metrology (National Institute of Advanced Industrial Science and Technology NMIJ,NIST, BAM reference materials, etc. traceable to International System of Unit) provide by
National Metrology Institutes which signed CIPM MRA, but at the same time they arestandard solutions for Chemical Analysis, Atomic Absorption Spectrometry, ICP or Ion
Chromatography prepared by the providers who obtained the certification of ISO Guide
34(JIS Q 0034： General requirements for the competence of reference material producers).
In addition, it is recommended to use standard solutions which indicate the uncertainty ofconcentration and factors.
d) Titrant: A Titrant described in 1) is usable if it is specified in the comment of a testing item.
In addition, the titrant may be diluted to a predetermined concentration as necessary. In thiscase, however, dilution treatment should be conducted when it is used and factors of thetitrant before dilution should be applied.
1) Titrant conforming to ISO/IEC 17025: A Titrant which is prepared and standardized andwhose factors are calculated by a laboratory which obtained an accreditation (accredited
range: JIS K 8001 JA.5 solutions for titration) based on ISO/IEC 17205. In addition, it isrecommended to use titrant which indicates the uncertainty of concentration and factors.
e) Concentration of reagent solution: Unless otherwise specified, the mass concentration isexpressed as g/L or mg/L, while the molar concentration is expressed as mol/L or mmol/L.
The concentration of the standard solution is expressed as the mass in 1 mL (mg/mL, µg/mL
or ng/mL) except for the ion-selective electrode method.
f) Concentration in parenthesis shown after the name of reagent solution: It indicates thatthe solution is about that concentration except the standard solution. For example, sodiumhydroxide solution (0.1 mol/L) means that it is about 0.1 mol/L sodium hydroxide solution.
Also, the concentration shown in front of the name of solution means that it is the accurate
concentration. However, the concentration is generally expressed as a round figure; calculate
the factor separately.
4

Testing Methods for Fertilizers (2018)(5)a)

Apparatus
Glass apparatus: Unless otherwise specified, use glass apparatus specified in JIS R 3503 andJIS R 3505. Also, when a heating procedure is involved, use borosilicate glass-1 specified in
JIS R 3503.
b) Non-glass apparatus: Unless otherwise specified, use plastic apparatus.
c) Desiccants for desiccators: Unless otherwise specified, use silica gel.
d) Porcelain crucibles and porcelain evaporating dishes: Use ones specified in JIS R 1301
and JIS R 1302.
e) Platinum crucibles and platinum evaporating dishes: Use ones specified in JIS H 6201 andJIS H 6202.
f) Filter paper: Use that specified in JIS P 3801. However, the type of filter paper is specified in
respective test items.
g) Absorbance measurement (absorptiometric analysis) absorbance cells: Unless otherwise
specified, use ones of 10 mm in optical path length.

5

Testing Methods for Fertilizers (2018)

1.2 Validity check of testing methods
The Testing Methods for Fertilizers are methods which have been discussed and approved by theTechnical Committee for Fertilizers etc. or methods in the Official Methods of Analysis of
Fertilizers (1992) whose style was rewritten according to the Testing Methods for Fertilizers. The
Testing Methods for Fertilizers will be revised by adding, modifying, or deleting testing methods
with the approval of the Technical Committee for Fertilizers etc. due to the needs such as progressin analytical techniques and changing social situation.
The procedure for the testing methods validity check is shown in the Appendix of this Testing
Methods for Fertilizers. This procedure was made based on 5.4.5 Validation of methods in JIS Q
17025 “General requirements for the competence of testing and calibration laboratories” or 2.4
Tests for validation requirements in “Guidelines for the design and implementation of surveillance
and monitoring and for the evaluation and publication of the results” which was issued by the
Ministry of Agriculture, Forestry and Fisheries and with reference to the guideline of Codex
Alimentarius Commission (CAC), IUPAC protocol and the guideline of AOAC INTERNATIONAL,
etc. Validated testing methods are methods which are conducted according to this procedure andconfirmed to conform to the standards such as required accuracy (trueness and precision),quantification range (maximum and minimum limits of quantitation) and so on.
In addition, according to the validation levels in Table 1, an individual testing method isclassified from Type A to Type E in Table 2.
Table 1
Symbol of Validation, etc.
Def-M （Defining method）
Def-C （Defining calculation）
Def-E （Defining extraction）
HCV （Harmonized collaborative
validation）
MLV （Multi laboratory validation）
SLV （SingleLaboratory validation）
RNV （Research non validated）

Testing method validation level
Validation method, etc.
Procedures of a testing method define measurement items. No
relation to a validation level.
Only calculation methods in a testing method define measurement
items. The part of the definition has no relation to a validation level.
Only extraction procedures in a testing method define measurement
items. The part of the definition has no relation to a validation level.
Evaluation by a collaboration test using samples of no less than 5concentrations in no less than 8 laboratories with a testing method
validation method regarded as an international standard (Guideline of
AOAC-International, IUPAC protocol, etc.).
Evaluation of validation using multiple laboratories, though it does notsatisfy HCV criteria.
Evaluation in a single laboratory using a testing method validation
method regarded as an international standard (IUPAC/ISO/AOACInternational harmonized guideline, etc.).
A testing method that does not carry out the validation by SLV or
higher level.

6

Testing Methods for Fertilizers (2018)
Table 2

Classification of an individual testing method

Symbol of
Validation level
Classification
Type AA defined testing method
A testing method whose evaluation results by HCV and SLV satisfies the requirements ofType B"Appendix: The procedure to validate characteristics of testing methods".
A testing method whose evaluation results by MLV and SLV satisfies the requirements ofType C"Appendix: The procedure to validate characteristics of testing methods".
A testing method whose evaluation results by SLV satisfies the requirements of "Appendix: The
Type D
procedure to validate characteristics of testing methods".
Type EA testing method that does not carry out the validation by SLV or higher level.

7

Testing Methods for Fertilizers (2018)

1.3 Procedure of testing methods
1.3.1 Competence evaluation of laboratory
When an individual testing method is used, it is recommended that the following competence
evaluation of a laboratory is conducted.
As for the testing methods of Type A, Type B and Type C, conduct a replicate testing with fivesamples, whose measured component concentration is known and to which certified referencematerials or standard solutions are added, to confirm trueness and precision in advance. As for thetesting methods of Type D and Type E, conduct a validity check in a single laboratory anew.
In order to ensure the reliability of a series of testing, conduct internal quality control (internal
quality assurance control, internal precision control) for each testing by a duplicate testing withsamples, whose measured component concentration is known, to confirm trueness and precision.
If it is possible, participate in an external quality assessment (external precision control,
competence test) in order to evaluate the consistency with testing results of other laboratories andconfirm the evaluation by z score.
1.3.2 Evaluation of test result
The results of a testing method which substitutes for the Testing Methods for Fertilizers can beused if it conforms to criteria required in validation of a testing method. However, in case where theresult of the testing method does not agree with the result of the Testing Methods for Fertilizers (1),the testing result of the latter is used to make a final judgement. In addition, if multiple methods aredescribed for a testing component, it is recommended that the result of a testing method for final
judgement is adopted in the following order: Type A, Type B, Type C, Type D and Type E.
Note (1) Refer to the separate sheet: “Target of trueness and criteria of precision in respective
concentration levels” or the reproducibility of respective testing methods” in order todetermine if there is mutual agreement.

8Testing Methods for Fertilizers (2018)2.
Handling of samples
2.1 Sampling
Sampling is according to “2.1 Sampling in the Official Methods of Analysis of Fertilizers (1992)”.
References
1)
National Institute for Agro-Environmental Sciences, the Ministry of Agriculture,
Forestry and Fisheries: The Official Methods of Analysis of Fertilizers 1992, p.4 - 5,
Japan Fertilizers Analysis Association, Tokyo (1992)
2) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p. 12 - 17, Yokendo, Tokyo (1988)

9

Testing Methods for Fertilizers (2018)

2.2 Storage of samples(1) Summary
Put a sample in a container suitable for its characteristics and form and seal it tightly, and then store
the sample at room or cold temperature. Care should be taken not to freeze it when it is stored atcold temperature.
(2) Apparatus and instruments: Apparatus and instruments are shown below.
a) Refrigerator: A refrigerator that can be adjusted to 1 ºC - 8 ºC.
b) Storage container for a sample: A storage container for a sample should be clean, strong andcompletely sealed airtight. In particular, in case it contains sludge for raw materials, thecontainer should be made of non-degradable, non-absorbable material. Additionally, it should
be airtight, water-proof, vapor-proof and non-corrosive.
(3) Procedure: Conduct storage as shown below.
a) Store a relatively stable sample in a tightly sealed container to avoid direct sunlight.
b) Store a sample in a desiccator, etc. by tightly sealing it if test results are affected by moistureabsorption.
c) Store a sample in a tightly sealed container in a dark place at 1 ºC - 8 ºC if it is easily
deteriorated by moisture.

10

Testing Methods for Fertilizers (2018)

2.3
(1)
a)
b)
c)

Preparation of test samples
Summary
Prepare a test sample by pre-drying, reducing, and grinding laboratory samples as necessary.
Conduct pre-drying if a laboratory sample is moist and hard to grind.
A laboratory sample made from such fertilizers as a fluid fertilizer or a particle-fertilizer, etc.
that is sufficiently homogeneous can be used as a test sample.
d) If contamination by apparatus affects a test result, procedures such as pre-drying, reduction
and grinding are prohibitede) Note that part of a test sample should not scatter, nor should surrounding fine particles orother alien substances be mixed with the test sample being prepared
References
1)
JIS M 8100: Particulate materials - General rules for methods of sampling (1992)
2)
JIS K 0060: Sampling method of industrial wastes (1992)

11

Testing Methods for Fertilizers (2018)

2.3.1 Pre-drying(1) Summary
This procedure is applicable to fertilizers whose laboratory sample is moist and hard to grind.
The symbol of the procedure is 2.3.1-2017 or PD.-1.
Conduct pre-drying using a drying apparatus, and measure the loss on drying in this procedure.
In addition, calculate a conversion factor (actual article), if necessary, to convert the component
content obtained in respective tests to the component content in a laboratory sample (actual article).
(2)a)

Apparatus and instruments: Apparatus and instruments are shown below.
Drying apparatus: A drying apparatus that can be adjusted to the pre-drying temperature at± 2 ºC.
b) Sample drying dish: Measure the mass to the order of 0.1g in advance. Additionally, usematerials of a quality that do not affect the measurement of test components.

(3)a)

Procedure: Conduct pre-drying as shown below.
Transfer 250 g - 1 kg of a laboratory sample to a sample drying dish, spread uniformly andmeasure the mass to the order of 0.1g.
b) Place a sample drying dish containing a laboratory sample in a drying apparatus and dry (1).
c) Remove a sample drying dish from a drying apparatus and leave at rest at room temperature
until it is balanced with atmospheric temperature (2).
d) After leaving at rest, measure the mass of c) to the order of 0.1 g.
e) Calculate loss on drying in the pre-drying by the following formula (1). If necessary, calculate
a conversion factor (actual article) by the following formula (2).
Loss on drying (% (mass fraction)) = ((W1 - A)/W1) × 100 ………. (1)Conversion factor (actual article) = A/W1 ………… (2)W1： Mass (g) of the sampled laboratory sample
A： Mass (g) of the sampled laboratory sample
Note (1) Examples of drying temperature and drying time: About 70 hours at 40 ºC, no less than
5 hours at 65 ºC.
(2) An example of time to leave at rest: About 20 minutes
Comment 1 When calculating major components in a laboratory sample (actual article) such ascompost and sludge fertilizers, etc. where the test sample is prepared conducting
pre-drying, convert component contents in the analytical sample obtained in
respective tests by the following formula.
Component content in a laboratory sample (actual article) = B ×C
B： Component content in an analytical sample obtained in each test
C： Conversion factor (actual article)
References
1) Mariko AIZAWA, Yuji SHIRAI, Yasushi SUGIMURA, Yuichi TAKAHASHI, Jun OKI,
Yukio FUKUCHI and Norio HIKICHI: Evaluation of Pre-drying Procedure to Prepare
Test Samples from Sludge Fertilizer, Research Report of Fertilizer Vol.1, p. 122 - 128(2008)
12

Testing Methods for Fertilizers (2018)(4)

Flow sheet for pre-drying
shown below.
250 g – 1 kg ofLaboratory sample
Drying

The flow sheet for the pre-drying of a moist laboratory sample isTransfer to a sample drying dish and spread uniformly.
Measure the mass to the order of 0.1 g
Example: About 70 hours at 40 ºC, no less than 5 hours at 65 ºC

Standing to coolRoom temperature

Measurement

Measure the mass to the order of 0.1 g

Figure Flow sheet for pre-drying

13

Testing Methods for Fertilizers (2018)

2.3.2
Reduction (Separation)(1) Summary
This procedure is applicable to fertilizers. The symbol of the procedure is 2.3.2-2017 or Red.-1.
In order to distinguish a test sample from a sample for granularity test and physical characteristics
test, etc., reduce (separate) a laboratory sample with an increment reduction method, a riffle sampler
method or a conical quartering method.
(2)a)

Apparatus
Scoop for increment reduction: A scoop for increment reduction specified in the attachedchart 1 of JIS M 8100.
b) Riffle sampler: A riffle sampler specified in the attached chart 3 of JIS M 8100.

(3)
a)
b)
c)

Procedure: Conduct reduction (separation) as shown below.
Increment reduction method: Conduct as indicated in 6.5.2 of JIS M 8100.
Riffle sampler method: Conduct as indicated in 6.5.3 of JIS M 8100.
Conical quartering method: Conduct as indicated in 6.5.4 of JIS M 8100.

14

Testing Methods for Fertilizers (2018)

2.3.3
Grinding(1) Summary
This procedure is applicable to fertilizers. The symbol of the procedure is 2.3.3-2017 or GRD.-1.
In order to prepare a homogeneous test sample, grind a laboratory sample with an adequate grinder
until it completely passes through the designated granularity.
(2)a)

Apparatus and instruments: Apparatus and instruments are shown below.
Grinder: Use a grinder (3) of a type and suitability for the granularity and the physicalcharacteristics (1) of a laboratory sample. In addition, the grinder apparatus which come into
contact with a laboratory sample should be made of materials (2) which do not affect theanalytical value.
b) Primary grinder: A grinder (3) that can primarily grind a large lump.
c) Cutter machine: A cutter that can cut long stems, etc.
d) Sieve: A sieve for the test specified in JIS Z 8801-1 or JIS Z 8801-2 or equivalents.
Note (1) The physical characteristics of a laboratory sample are defined by their solidity,
toughness, specific gravity and adhesiveness.
(2) (Ex.) Do not use stainless steel apparatus when preparing a test sample for chromium
or nickel.
(3) A centrifuging type grinder, a cutting mill, a vibration mill type grinder, etc.
(4) A blender with an attachable blade, etc.

(3) Procedure: Conduct grinding as shown below.
(3.1) Fertilizers except ones specified in (3.2): Conduct as specified in 6.4 of JIS M 8100 and as
shown below.
a) Break or cut a laboratory sample with a primary grinder or a cutting machine as necessary.
b) Grind with a grinding machine until it completely passes through a sieve of 500 µm - 1 mm
aperture.
c) Mix ground samples to make the test sample.
Comment 1 If the sampling amount of an analytical sample is less than 1 g, use a test sample
which will completely pass through 500 µm aperture sieve. In addition, in case a test
sample which suits the aforementioned condition cannot be obtained due to adeliquescent laboratory sample, etc., make one by crushing a test sample with amortar and pestle, which completely passes through 1 mm aperture sieve.
(3.2) Fused phosphate fertilizer, slag silicate fertilizer, etc.: Conduct as specified in 6.4 of JIS M
8100 and as shown below.
a) Grind a laboratory sample with a vibration mill type grinder.
b) Transfer the ground laboratory sample to a sieve of 212 µm apertures.
c) Incline the sieve about 20 degrees, supporting it with one hand or a bent arm, and tap thesieve frame with the other hand at the rate of about 120 times per minute. During theprocedure, place the sieve in a horizontal position at the rate of 4 times per minute, rotate it 90
degrees and tap the sieve frame firmly one or two times.
d) When fine powder attaches to the back side of a sieve screen, remove it gently from the backside to make minus sieve.
e) Regarding the plus sieve of a sample, make them pass through by repeating the procedure ina) - d).
f) Combine and mix the sample passed to make the test sample.
15

Testing Methods for Fertilizers (2018)

Comment 2 Conduct the procedures in (3.2) to obtain observed value of citric acid-soluble main
components in a stable manner. For examples of applicable laboratory samples, fusedmatters such as fused phosphate and slag silicate fertilizer, fertilizers made fromfused materials and calcined phosphate, etc. are listed.
Comment 3 The procedures in b) - d) are the procedures in 6.1.3 (1.4) of JIS Z 8815.

16

Testing Methods for Fertilizers (2018)3.
General tests
3.1 Moisture or moisture content
3.1.a Loss on drying method with drying apparatus(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type A (Def-M)
and its symbol is 3.1.a-2017 or Mois.a-1.
Use drying apparatus under conditions suitable for the kind of fertilizers to be measured to heatanalytical samples to measure loss on drying and obtain moisture in an analytical sample or the
moisture content of a quality labeling standard of a special fertilizer (herein after referred to as“moisture”). Additionally, calculate a conversion factor (drying matter) to convert component
content obtained by respective tests to component content in a drying matter as necessary.
This testing method corresponds to loss on heating in the Official Methods of Analysis of Fertilizers(1992).
(2) Apparatus and instruments: Apparatus and instruments are shown below.
a) Drying apparatus: Drying apparatus that can be adjusted to the test temperature ± 2 ºC.
b) Ground-in stoppered weighing bottles (1): Low-form weighing bottles, 50 mm × 30 mm,
specified in JIS R 3503. Dry by heating in advance in a drying apparatus at 75 ºC - 130 ºC,
stand to cool in a desiccator, and measure the mass to the order of 1 mg.
Note (1) Aluminum weighing dishes described in the Handbook of the Feed Analysis Standards-2009- can also be used.
(3)a)

Measurement: Conduct measurement as shown below.
Transfer 2 g-5 g of an analytical sample to a ground-in stoppered weighing bottle, spread sothat the thickness is no more than 10 mm, and measure the mass to the order of 1 mg.
b) Place the ground-in stoppered weighing bottle containing the analytical sample in a drying
apparatus at 100 ºC ± 2 ºC, and heat for 5 hours (2).
c) After heating, fit the stopper into the ground-in stoppered weighing bottle, and immediatelytransfer to a desiccator to let it stand to cool.
d) After standing to cool, remove the ground-in stoppered weighing bottle from the desiccator,and measure the mass to the order of 1 mg.
e) Calculate loss on drying in the analytical sample by the following formula (1) as moisture. If
necessary, calculate a conversion factor (actual article) by the following formula (2).
Loss on drying (% (mass fraction)) = ((W1 - A)/W1) × 100 ………. (1)Conversion factor (drying matter) = W1/A………………………… (2)W1： Mass (g) of the sampled analytical sample
A： Mass (g) of the analytical sample after drying
Note (2) Heat simultaneously the slightly moved or removed stopper of the ground-in stoppered
weighing bottle.
Comment 1 When pre-drying a laboratory sample such as compost and sludge fertilizers to
prepare a test sample, calculate the moisture of the laboratory sample (actual
article) by the following formula:

17

Testing Methods for Fertilizers (2018)

Moisture (% (mass fraction)) in the laboratory sample (actual article) = B + C × ((100 B)/100)
B： Loss on drying (% (mass fraction)) of the laboratory sample (actual article) by the
pre-drying procedure
C： Loss on drying (% (mass fraction)) in the analytical sample by moisturemeasurement
Comment 2 When calculating harmful content in a drying matter of sludge fertilizers, etc.,
convert component content in a test sample obtained from respective tests by thefollowing formula.
Component content in a drying matter = D ×E
D： Component content in an analytical sample obtained in each test
E： Conversion factor (drying matter)
Comment 3 Use drying conditions in Table 1 for fertilizers of the types shown below:
Table 1
Type of fertilizers
Superphosphate of lime, triple
superphosphate of lime, or fertilizers
containing theseAmmonium sulfate, sodium nitrate, andpotassium salts
Urea and urea-containing fertilizers
Silica gel fertilizer and fertilizer
containing silica gel, and silica hydrogel
fertilizer

Drying conditions
Sampling
amount ofDryinganalyticaltemperaturesamples
About 5 g

100 ºC ± 2 ºC

Drying time

3 hours

About 5 g

Until a constant
130 ºC ± 2 ºC
weight isachieved.
75 ºC ± 2 ºC
4 hours

About 5 g

180 °C±5 °C

2 g- 5g

3 hours

Comment 4 For samples containing volatile matters, subtract the volatile matter content by thefollowing a) and b) from loss on drying to obtain moisture.
a) Fertilizers containing guano or diammonium hydrogen phosphate, etc.: Determine
total nitrogen in the test sample, and in the analytical sample after the drying
procedure; convert the difference between the quantitation values into ammonia(NH3) to make the volatile matter content.
b) Potassium hydrogen carbonate: Determine carbon dioxide in the test sample, and in
the analytical sample after the drying procedure; the difference between thequantitation values is the volatile matter content.

18

Testing Methods for Fertilizers (2018)

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p. 20 - 23, Yokendo, Tokyo (1988)
2)
Society for the Study of Feed Analysis Standards: Methods of Analysis of Feeds andFeed Additives-2009-I, p. 37 - 39, Incorporated Administrative Agency Food andAgricultural Materials Inspection Center, Saitama (2009)3)
JIS Z 0701： Silica gel desiccants for packaging (1997)(4)

Flow sheet for moisture: The flow sheet for moisture in fertilizers is shown below:
2 g -5 ganalytical sample
Heating

Transfer to a ground-in stopper weighing bottle, spread so that the
thickness is no more than 10 mm. Measure the mass to the order of 1 mg.
100 ºC ± 2 ºC, 5 hours

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg.

Figure Flow sheet for moisture in fertilizers by loss on drying with drying apparatus.

19

Testing Methods for Fertilizers (2018)

3.1.b Loss on drying method by moisture analyzers(1) Summary
The testing method is applicable to sludge fertilizers, compost, and organic fertilizers, etc. This
testing method is classified as Type B and its symbol is 3.1.b-2017 or Mois.b-1.
Use a moisture analyzer in the heat drying method to measure loss on drying and obtain moisturein an analytical sample or the moisture content of a quality labeling standard of a special fertilizer(herein after referred to as “moisture”). Additionally, calculate a conversion factor (drying matter)to convert component content obtained by respective tests to component content in a drying matteras necessary.
In addition, the performance of this testing method is shown in Comment 3.
(2) Instruments: Instruments are as shown below:a) Moisture analyzer: A moisture analyzer consisted of a heat source to heat an analyticalsample (halogen lamp, infrared heater, ceramic heater, etc.) and a balance (1) with calibration
function.
Note (1) There is a method to calibrate with calibration weights or a method to calibrate
automatically with built-in weights.
(3)

a)
b)
c)
d)

Measurement: Conduct measurement as shown below. However, conduct in advance acomparative test with 3.1.a Loss on drying with drying apparatus using sludge fertilizers,
compost, and organic fertilizers, etc., to confirm that there is no difference in the quantitation
value of moisture.
Transfer 2 g-5 g of an analytical sample to a ground-in stoppered weighing bottle, spread sothat the thickness is no more than 10 mm, and measure the mass to the order of 1 mg.
Heat at 100 ºC (2), until a constant weight is achieved.
After the end of heating (2), measure the mass to the order of 1 mg.
Calculate loss on drying in the analytical sample by the following formula (1) as moisture. If
necessary, calculate a conversion factor (drying matter) by the following formula (2).
Loss on drying (% (mass fraction)) = ((W1 - A)/W1) × 100
Conversion factor (drying matter) = W1/A…. (1)…. (2)

W1： Mass (g) of the sampled analytical sample
A： Mass (g) of the analytical sample after drying
Note (2) The setup of the drying program and the determination parameter for the end of heating(constant weight) is according to the specification and the operation method of the
moisture analyzer used.
Comment 1 When pre-drying is conducted, calculate the moisture of the laboratory sample(actual article) by the following formula:
Moisture (% (mass fraction)) in the laboratory sample (actual article) = B + C × ((100 B)/100)
B： Loss on drying (% (mass fraction)) of the laboratory sample (actual article) by the
pre-drying procedure
C ： Loss on drying (% (mass fraction)) in the analytical sample by moisture20

Testing Methods for Fertilizers (2018)measurement
Comment 2 When calculating harmful content in a drying matter of sludge fertilizers, etc.,
convert component content in a test sample obtained from respective tests by thefollowing formula.
Component content in a drying matter = D × E
D： Component content in an analytical sample obtained in each test
E： Conversion factor (drying matter)
Comment 3 Table 1 shows the results of the comparison of the measurement values by loss on
drying method with drying apparatus and the measurement values by loss on drying
method with a moisture analyzer using organic fertilizers, compost and sludge
fertilizers in order to evaluate trueness.
Table 2 shows results and analysis results from a collaborative study for testing
method validation.

Table 1 Analysis results of the comparison test results of respective methods
Symbol of measurement value
SampleRange ofRegression coefficient
Drying apparatus Moisture
Kind
Number ofyi～yj(y ＝ a ＋bx )1)
2)
3)
samplesabmethodmethod(%)
xi

yi

Sludge fertilizer

4)

26

5)

5.50～90.61

0.188

0.998

Correlation
coefficient
r
0.999

xj
yj
25
2.96～12.33
0.185
0.986
0.994
Organic fertilizer1） 3.1.a Loss on drying method with drying apparatus2） 3.1.b Loss on drying method with a moisture analizer
3） Mass fraction
4） Sewage sludge fertilizer, Human waste sludge fertilizer, Industrial sludge fertilizer, Composted sludge fertilizer5） Fish meal, Byproduct organic fertilizer of vegetable origin, Compost, Steamed leather meal,

Rape seed meal and powdered rape seed meal21

Testing Methods for Fertilizers (2018)

Table 2

Sample nameResults and analysis results from a collaborative study
for the validation of the moisture test method.
Number of1)laboratories

4)

RSD r

3)

(%)

Mean

2)

sr

3)

(%)

(%)

5)

Sewage sludge
9
21.93
0.32
1.4
fertilizer
Human waste
8
13.36
0.14
1.1
sludge fertilizer
Industrial sludge
9
34.28
0.21
0.6
fertilizer
Calcined sludge
9
38.75
0.59
1.5
fertilizer
Composted sludge
9
27.1
0.26
0.9
fertilizer
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

6)

RSD R

(%)

3)

(%)

0.47

2.1

0.37

2.8

0.50

1.5

0.59

1.5

0.60

2.2

sR

7)

References
1)
Takeshi UCHIYAMA and Chiyo SAKASEGAWA: Validation of a Heating Method
Using a Moisture Analyzer for Moisture Content in Sludge Fertilizer. Research Reportof Fertilizer Vol.1, p. 1- 5 (2008)
2)
Takeshi UCHIYAMA and Yuji SHIRAI: Determination of Moisture Content in Sludge
Fertilizer by a Heating Method Using Moisture Analyzer: A Collaborative Study.
Research Report of Fertilizer Vol.1, p. 6 - 11 (2008)
3)
Satono AKIMOTO and Sakiko TAKAHASHI: Validation of a Heating Method Using a
Moisture Analyzer for Moisture Content in Sludge Fertilizer. Research Report of
Fertilizer Vol.2, p. 1 - 5 (2009)(4)

Flow sheet for moisture: The flow sheet for moisture in sludge fertilizers, compost, andorganic fertilizers, etc. is shown below:

About 5 ganalytical sample
Heating

Transfer to a weighing dish, spread so that the thickness is
no more than 10 mm. Measure the mass to the order of 1 mg.
100 ºCEnd of drying

Constant weight

Measurement

Measure the mass to the order of 1 mg

Figure Flow sheet for moisture in sludge fertilizers, compost, and organic fertilizers, etc.
by the loss on drying method with a moisture analyzer.
22

Testing Methods for Fertilizers (2018)

3.2 Ash content
3.2.a Ignition residue method(1) Summary
The method is applicable to organic fertilizers and fertilizers containing organic matters. This
testing method is classified as Type A (Def-M) and its symbol is 3.2.a-2017 or Ash.a-1.
The method ignites an analytical sample with an electric furnace and measures residue on ignition
to obtain ash content in an analytical sample.
(2) Apparatus and instruments: Apparatus and instruments are shown below.
a) Electric furnace: Electric furnace that can be adjusted to 550ºC ± 5 ºC.
b) Crucible: After heating a porcelain crucible for chemical analysis specified in JIS R 1301
with an electric furnace at 550 ºC ± 5 ºC, stand to cool in a desiccator in advance and measure
the mass to the order of 1 mg.
(3) Measurement: Conduct measurement as shown below.
a) Transfer about 2 g of an analytical sample into a crucible, and measure the mass to the orderof 1 mg.
b) Place it into an electric furnace, heat gently until carbonized (1).
c) Heat at 550 ºC ± 5 ºC for no less than 4 hours (1).
d) After heating, move the crucible into a desiccator and let it stand to cool.
e) After standing to cool, remove the crucible from the desiccator and measure the mass to theorder of 1 mg.
f) Calculate the residue on ignition in the analytical sample by the following formula to makeash content.
Residue on ignition (% (mass fraction)) = (A/W) × 100
W： Mass (g) of the sampled analytical sample
A： Mass (g) of the ignited analytical sample
Note

(4)

(1)

Example of carbonizing and incineration procedure: After raising the temperaturefrom room temperature to about 250 °C in 30 minutes to 1 hour, continue heating
for about 1 hour and further raise to 550 °C in 1 to 2 hours.

Flow sheet for ash content: The flow sheet for ash content in fertilizers is shown below:
About 2 g ofanalytical sample

Transfer to a crucible.
Measure the mass to the order of 1 mg.

CharredHeating gently with an electric furnace

Incineration

No less than 4 hours at 550 ºC ± 5 ºC

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1mg

Figure Flow sheet for ash content in fertilizers.

23Testing Methods for Fertilizers (2018)

3.3 pH
3.3.a Glass electrode method(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type A (Def-M)
and its symbol is 3.3.a-2017 or pH.a-1.
Measure the pH of fertilizers with a pH meter using a glass electrode.
(2)a)

Reagent: Reagents are as shown below.
Oxalate pH standard solution: Oxalate pH standard solution class 2 traceable to National
Metrology.
b) Phthalate pH standard solution: Phthalate pH standard solution class 2 traceable to
National Metrology.
c) Neutral phosphate pH standard solution: Neutral phosphate pH standard solution class 2
traceable to National Metrology.
d) Borate pH standard solution: Borate pH standard solution class 2 traceable to National
Metrology.
e) Carbonate pH standard solution: Carbonate pH standard solution class 2 traceable to
National Metrology.
Comment 1 Respective pH standard solutions stored for long time should not be used since thepH value may change during storage period. In particular, note that borate pH
standard solution and carbonate pH standard solution easily absorb carbon dioxide in
the air, so that the pH values deteriorate.
The pH standard solution that was used once or left exposed to the air should not be
used.

(3)
a)

Instruments: Instruments are as shown below:
pH meter: Use type II specified in JIS Z 8802.

Comment 2 Conduct the calibration of a pH meter as indicated in JIS Z 8802. Actual calibration
operation is according to the operation procedure of the pH meter used formeasurement.
When the pH of a sample solution is no more than 7, use neutral phosphate pH
standard solution and oxalate pH standard solution, or phthalate pH standard solution.
When it exceeds 7, use neutral phosphate pH standard solution and borate pH
standard solution, or carbonate pH standard solution.
(4) Test procedures(4.1) Preparation of sample solution: Conduct preparation of a sample solution as shown below.
(4.1.1) Fertilizers except inorganic fertilizersa) Transfer a predetermined amount of an analytical sample (1) into a ground-in stopper flask andadd water 5 - 10 times the volume.
b) Mix with a magnetic stirrer, filter with Type 3 filter paper to make a sample solution.
Note (1) In the case of a moist laboratory sample, it is recommended to use a sample that is not
pre-dried.
(4.1.2) Inorganic fertilizersa) Transfer a predetermined amount of an analytical sample (1) into a ground-in stopper flask and24

Testing Methods for Fertilizers (2018)

add water 100 times the volume.
b) Mix with a magnetic stirrer, filter with Type 3 filter paper to make a sample solution.
Comment 3 The procedure in (4.1.1) is the same as 3.4.a (4.1). Additionally, the sample
solution prepared in 4.2.4.a (4.1) can be used instead of the sample solution
prepared by (4.1.2).
(4.2) Measurement: Conduct measurement as shown below. Actual calibration operation isaccording to the operation procedure of the pH meter used for measurement.
a) Wash the read station of a calibrated pH meter repeatedly no less than 3 times with water andwipe out with clean and soft paper, etc.
b) Transfer a sample solution into a beaker (2), dip the read station in the solution and measure
the pH value.
Note (2) It is necessary to transfer sufficient volume of sample solution to keep a measurement
value stable.
Comment 4 If a pH meter has a temperature correction dial or a digital switching, measure the pH
value after adjusting the graduation of the pH meter with the temperature of asample.
References
1) JIS Z 8802： Methods for determination of pH of aqueous solutions (2011)(5)

Flow sheet for pH value: The flow sheet for pH value in fertilizers is shown below.

Predetermined volume
Ground-in stopper flask.
of analytical sample← Water 5 -10 times or 100 times the volume
Stirring
Filtration
Measurement

Type 3 filter paper
pH meterFigure Flow sheet for pH in fertilizers.

25Testing Methods for Fertilizers (2018)

3.4 Electrical conductivity
3.4.a Measurement method with an electrical conductivity meter(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type A (Def-M)
and its symbol is 3.4.a-2017 or EC.a-1.
Measure the electrical conductivity of organic fertilizers such as compost or sludge fertilizers with
an electrical conductivity meter.
(2)a)

Reagent: Reagents are as shown below.
Potassium chloride: Grind potassium chloride used for measurement of electricalconductivity specified in JIS K 8121 with an agate mortar to powder and heat for 4 hours at
500 ºC ± 5 ºC, and then stand to cool in a desiccator.
b) Potassium chloride standard solution (1): Measure predetermined volume (2) of potassium
chloride of a) on a weighing dish, dissolve in a small amount of water, transfer it into a
1000-mL volumetric flask, and add up to the marked line with water.
Note (1) Store potassium chloride standard solution in a polyethylene or borosilicate glass bottle
and seal the bottle.
(2) The volume that is recommended for an instrument or a cell used.
Comment 1 Potassium chloride standard solution used once or left in the air should not be used.

(3)a)

Instruments: Instruments are as shown below:
Electrical conductivity meter: An electrical conductivity meter specified
0130

in

JIS

K

Comment 2 Check the indicated value as shown in 6.2 in JIS K 0130 as necessary. Actual
procedure to check is according to the operation procedure of the electricalconductivity meter used for measurement.
(4) Test procedures(4.1) Preparation of sample solution: Conduct preparation of a sample solution as shown below.
a) Transfer the predetermined volume of an analytical sample (3) into a ground-in stopper flask
and add water 10 times the equivalent volume of dry matter (4).
b) Mix with a magnetic stirrer, filter with Type 3 filter paper to make a sample solution.
Note (3) In the case of a moist laboratory sample, it is recommended to use a sample that is not
pre-dried.
(4) If the sample solution becomes hard to measure because it is gelled by the influence offlocculants in sludge fertilizer, etc., increase the volume of water added. However, thisfact should be expressed in the test result.
Comment 3 The procedure in (4.1) is the same as 3.3.a (4.1.1).
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0130 and as shown below.
Actual measurement operation is according to the operation procedure of the electricalconductivity meter used for measurement.
a) Wash the read station of an electrical conductivity meter repeatedly no less than 3 times withwater.
b) Transfer a sample solution into a beaker (5), dip the read station and measure electrical26

Testing Methods for Fertilizers (2018)

conductivity.
Note (5) It is necessary to transfer sufficient volume of sample solution to keep a measurement
value stable.
References
1) JIS K 0130：General rules for electrical conductivity measuring method (2008)(5)

Flow sheet for electrical conductivity: The flow sheet for electrical conductivity is showed
below.

Predetermined volume
Ground-in stopper flask.
of analytical sample← Water ,10 times the volume
Stirring
Filtration
Measurement

Type 3 filter paper
Electrical conductivity meter

Figure Flow sheet for electrical conductivity in fertilizers.

27Testing Methods for Fertilizers (2018)

3.5 Granularity
3.5.a Dry-type sieving testing method(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type A (Def-M)
and its symbol is 3.5.a-2017 or P-size.a-1.
Measure the particle diameter distribution of solid fertilizers with a dry-type sieving analysis.
(2) Apparatus: Apparatus are shown below.
a) Sieve: A sieve for testing specified in JIS Z 8815b) Clogging removal brush: A brush which is adequately hard for the apertures and does notdamage the sieving screen.
c) Weighing dish: A container that can contain about 250 g of a sample. Measure the mass tothe order of 0.1g in advance.
(3)

Dry-type sieving analysis procedure: Conduct sieving analysis corresponding to the aperturesize of a sieve used as indicated in JIS Z 8815 and as shown below.
(3.1) More than 1mm and no more than 4mma) Stack a large aperture sieve on an acceptor so that the large sieve is on top.
b) Measure a laboratory sample to the order of 0.1g and transfer it to the sieve at the top section.
c) After putting a stopper on it, hold the stacked sieves with both hands, and vibrate (1) themback and forth along a unidirectional and horizontal plane at about 60 times per minute with
about 70 mm amplitude.
d) Transfer respective plus and minus sieves to a weighing dish (2).
Note (1) Conduct more circular motion at the rate of about 3 revolutions per minute asnecessary.
(2) Turning over the back side of a sieve, remove the clogged particles from the sieve
screen with a clogging removal brush and combine them with the plus sieve.
(3.2) Less than or equal to 1mma) Stack a large aperture sieve on an acceptor so that the large sieve is on top.
b) Measure the laboratory sample or minus sieve of (3.1) c) to the order of 0.1g and transfer it to
the sieve at the top section.
c) After putting a stopper on it, incline the stacked sieves about 20 degrees, supporting with one
hand or a bent arm, and tap the sieve frame with the other hand at the rate of about 120 times
per a minute.
d) During the procedure in c), place the sieve in a horizontal position at the rate of 4 times per
minute, rotate it 90 degrees and tap the sieve frame firmly one or two times.
e) Transfer respective plus and minus sieves (3) to a weighing dish (2).
Note (3) When fine powder attaches to the back side of a sieve screen, remove them gently fromthe back side with a clogging removal brush and combine them with the minus sieve(4)

Measurement of granularity distribution: Calculate the granularity distribution in ananalytical sample as shown below.
a) Measure respective mass of plus and minus sieves to the order of 0.1g.
b) Calculate “plus sieve percentage” and “integrated minus sieve percentage” with the following
formula and round the results to the first decimal place.
c) Confirm that the sum of the mass of the plus sieve and the mass of the minus sieve with thesmallest aperture is in the range of ± 2 % of the mass of sample measured in (3.1) b) or (3.2)
28

Testing Methods for Fertilizers (2018)b).
Mass percentage of plus sieve or minus sieve (%) (R) = (A/T) × 100
A： Mass of plus sieve or minus sieve (g)T： Sum of the mass of plus and minus sieve (g)References
1) JIS Z 8815： Test sieving - General requirements (1994).
2) JIS K 0069： testing methods for sieving of chemical products (1992).
(5)

Flow sheet for granularity: The flow sheet of granularity of solid fertilizers is shown below.

Laboratory sample
or Test sample

Measure the mass to the order of 0.1g

Dry-type sieving
Mass measurement ofrespective plus sieveand minus sieve

Measure the mass to the order of 0.1g

Figure Flow sheet for granularity of solid fertilizers

29Testing Methods for Fertilizers (2018)

3.6 Oil content
3.6.a Diethyl ether extraction method(1) Summary
This testing method is applicable to organic fertilizers. This testing method is classified as Type A(Def-M) and its symbol is 3.6.a-2017 or Oil.a-1.
Extract an analytical sample with diethyl ether using a Soxhlet extractor and measure the extract toobtain oil content in an analytical sample. The oil content contains not only fat but also fat-soluble
pigments (carotenoid, chlorophyll, etc.), wax, and free fatty acids, etc.
(2) Reagent: Reagents are as shown below.
a) Diethyl ether: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Drying apparatus: Drying apparatus that can be adjusted to the test temperature ± 2 ºC.
b) Soxhlet extractor: An inter-changeable Soxhlet extractor, cooling apparatus and weighing
bottles (Example: JIS R 3503, attached figure 71)c) Water bath A water bath that can be adjusted to about 60 ºCd) Weighing bottle: A flat bottle flask connectable to a Soxhlet extractor. After heating with adrying apparatus at 100 ºC - 105 ºC in advance, stand to cool in a desiccator and measure themass to the order of 1mg.
e) Cylindrical filter paper: A cylindrical filter paper made of cellulose. Example: 22 mm
external diameter, 20 mm internal diameter, 90 mm total length (1).
(4)
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
k)

Measurement: Conduct measurement as shown below.
Weigh 2 g - 5 g of an analytical sample to the order of 1mg, and transfer it into a cylindricalfilter paper.
Place absorbent cotton on the upper end of an analytical sample (2), as if gently pushing it, andheat it at 100 ºC - 105 ºC for 2 hours.
As soon as heating is complete, move the cylindrical filter paper to a desiccator and stand to
cool.
After standing to cool, transfer it into a Soxhlet extractor and connect it to a coolingapparatus.
Transfer adequate volume of diethyl ether (3) into a weighing bottle, connect it to the Soxhlet
extractor and heat (4) it for 8 hours to extract.
Recover the diethyl ether (5).
Disconnect the weighing bottle from the Soxhlet extractor and vaporize the diethyl ether (6).
Heat the weighing bottle (7) at 100 ºC - 105 ºC for 3 hours.
As soon as heating is complete, move the weighing bottle to the desiccator and stand to cool.
After standing to cool, remove the weighing bottle from the desiccator and measure the mass
to the order of 1mg.
Calculate oil content with the following formula.
Oil content (% (mass fraction)) = (B/A) × 100
A： Mass (g) of the sampled analytical sample
B： Mass of extract of diethyl ether (g)Note (1) Select a scale according to the volume of a Soxhlet extractor.
30

Testing Methods for Fertilizers (2018)(2) The purpose is to prevent overflow at the upper end of an analytical sample.
(3) The amount of diethyl ether depends on the volume of a weighing bottle.
(4) Adjust the temperature for diethyl ether to circulate 16 - 20 times per hour. (Target
temperature is about 60 ºC)(5) Remove the cylindrical filter paper from the Soxhlet extractor. In the case of a cockattached Soxhlet extractor, open the cock and recover it.
(6) It is dangerous if diethyl ether resides in a weighing bottle when the bottle istransferred to a drying apparatus.
(7) Wipe the outside of a weighing bottle since there is a risk of garbage or stain sticking
to it.
References
1)
Japan Oil Chemist ‘Society: Standard Method for the Analysis of Fats, Oils and RelatedMaterials (2003), 1.5 Oil content p. 1 - 2, Incorporated Foundation Japan Oil Chemist‘Society, Tokyo (2009)
2)
Feed Analysis Standard Task Force: Feed Analysis Method/Handbook -2009 - I, p. 37 39, Incorporated Administrative Agency, Food and Agriculture Materials Inspection
Center, Saitama (2009)(5)

Flow sheet for oil content: The flow sheet for oil content in organic fertilizers is shown
below:
2 g- 5 ganalytical sample
Pre-drying

Weigh to the order of 1mg to a cylindrical filter paper
100 ºC - 105 ºC, for 2 hours

Soxhlet extractor
Extraction

Diethyl ether, heating, for 8 hours

Weighing bottle
Heating

100 ºC - 105 ºC, for 3 hours

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1mg

Figure Flow sheet for oil content in organic fertilizers.

31Testing Methods for Fertilizers (2018)

4.
Main components, guaranteed components, etc
4.1 Nitrogen4.1.1 Total nitrogen4.1.1.a Kjeldahl method(1) Summary
This testing method is applicable to fertilizers containing no nitrate nitrogen. This testing method isclassified as Type C and its symbol is 4.1.1.a-2017 or T-N.a-1.
Add sulfuric acid, potassium sulfate and copper (II) sulfate pentahydrate to an analytical sample,
pretreat by Kjeldahl method to change total nitrogen (T-N) to ammonium ion, and add a sodiumhydroxide solution to subject to steam distillation. Collect isolated ammonia with 0.25 mol/L
sulfuric acid and measure surplus sulfuric acid by (neutralization) titration using a 0.1 mol/L - 0.2
mol/L sodium hydroxide solution to obtain the total nitrogen (T-N) in an analytical sample. Or
collect isolated ammonia with a boric acid solution and measure ammonium ion by (neutralization)
titration using 0.25 mol/L sulfuric acid to obtain the total nitrogen (T-N) in an analytical sample.
This testing method corresponds to the sulfuric acid method in the Official Methods of Analysis of
Fertilizers (1992). In addition, the performance of this testing method is shown in Comment 8.
(2)
a)

Reagent: Reagents are as shown below.
0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C1)
W1： Mass (g) of sulfamic acid sampled
A： Purity (% (mass fraction)) of sulfamic acidV1： Volume (mL) of sulfamic acid solution transferred
V2： Constant volume (250 mL) of sulfamic acid solution
V3： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
c) 0.25 mol/L sulfuric acid (1) (2): Add about 14 mL of sulfuric acid to a beaker containing 100
mL of water in advance, stir well, and add water to make 1000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a 200-mL- 300-mL Erlenmeyer flask, add a few drops of methyl red−methylene blue mixture solution,
32

Testing Methods for Fertilizers (2018)

and titrate with a 0.1 mol/L -0.2 mol/L sodium hydroxide solution until the color of thesolution becomes gray-green (4). Calculate the volume of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the following formula(1). Or, calculate the factor of 0.25 mol/L sulfuric acid by the following formula (2):Volume (B) of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL of 0.25
mol/L sulfuric acid= V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2 × 2)………. (2)V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5： Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 in water tomake 1000 mL.
e) Catalyst (5): Mix potassium sulfate specified in JIS K 8962 and copper (II) sulfate
pentahydrate (6) specified in JIS K 8983 in the ratio of 9 to 1.
f) Sodium hydroxide solution (200 g/L - 500 g/L) (1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water to make 100 mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
i) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in JIS K
8897 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methyl red−methylene blue mixture solution: To 2 volumes of methyl red solution (0.1g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
k) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green specified
in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red−bromocresol green mixture solution: To a methyl red solution (0.1 g/100 mL),
add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL -10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(5) A tablet is commercially available.
(6) Crush into powder as appropriate.
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
Comment 2 0.25 mol/L sulfuric acid in (2) c) can be replaced with 0.25 mol/L sulfuric acid33

Testing Methods for Fertilizers (2018)conforming to ISO/IEC 17025.
(3)
a)
b)
c)

Apparatus and instruments: Apparatus and instruments are shown below.
Steam distillation apparatus
Digestion flask: Kjeldahl flask
Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to a steam
distillation apparatus.

(4) Test procedures(4.1) Kjeldahl method: Conduct digestion as shown below.
a) Weigh 0.5 g - 5 g of an analytical sample to the order of 1 mg, and put it in a 300-mL 500-mL digestion flask.
b) Add 5 g - 10 g of catalyst, and further add 20 mL - 40 mL of sulfuric acid, shake to mix (7) andheat gently.
c) After bubbles cease to form, heat until white smoke of sulfuric acid evolves.
d) Ignite until organic matters are completely digested (8).
e) After standing to cool, add a small amount of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water (9), and further mix by shaking.
f) After cooling is complete, add water up to the marked line to make the digestion solution.
Note (7) Leaving at rest overnight is preferable.
(8) When the solution has finished changing color, heat further for no less than 2 hours.
(9) When the entire sample solution volume is used in measurement, it is not necessary totransfer it to a volumetric flask.
Comment 3 The procedure in (4.1) is the same as that in (4.1.1) a) - f) in 4.2.1.a.
Comment 4 In the case of fish meal containing amino acids that are not easily digested, use 0.5 g-1 g analytical sample, 10 g catalyst and 30 mL - 40 mL sulfuric acid.
Comment 5 In the case of nitrolime, moisten by adding a small amount of water before theprocedure in (4.1) b). Care should be taken because bubbles are produced by theaddition of sulfuric acid.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used in measurement.
a) Transfer a predetermined amount (10) of 0.25 mol/L sulfuric acid to an acceptor (11), add a few
drops of methyl red−methylene blue mixture solution, and connect this acceptor to a steam
distillation apparatus. Or, transfer a predetermined amount (10) of boric acid solution (40 g/L)
to an acceptor (11), add a few drops of methyl red−bromocresol green mixture solution, andconnect this acceptor to a steam distillation apparatus.
b) Transfer a predetermined amount of the digestion solution to a 300-mL distillation flask, add aproper amount of sodium hydroxide solution (200 g/L - 500 g/L) (12), and immediately
connect this distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill at a
distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution in theacceptor with a small amount of water, and pool the washing with the distillate.
Note (10) 5 mL - 20 mL(11) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
34

Testing Methods for Fertilizers (2018)

beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or a boric acid solution (40 g/L).
(12) An amount sufficient to make the solution strong alkalinity. A blue color will appear.
(4.3) Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2):a) Titrate the distillate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes gray-green (4).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= (B × V6 － V7) × C1 × f1 × (V8/V9) × (14.007/W3) × (100/1000)= (B × V6 － V7) × C1 × f1 × (V8/V9) × (1.4007/W3)
B： Volume of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acidV6： Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2) a)
V7： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V8： Predetermined volume (mL) of the digestion solution in (4.1) f)
V9： Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W3： Mass (g) of the analytical sample(4.3.2) When a boric acid solution (40 g/L) is used in (4.2):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomes lightred (13).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= V10 × C2 × 2 × f2 × (V11/V12) × (14.007/W2) × (100/1000)= V10 × C2 × 2 × f2 × (V11/V12) × (2.8014/W2) × (100/1000)
V10：
C2 ：
f2 ：
V11：
V12：

Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acid
Factor of 0.25 mol/L sulfuric acid
Predetermined volume (mL) of the digestion solution in (4.1) f)
Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W2： Mass (g) of the analytical sample
Note (13) The endpoint is reached when the color changes from green to light red.
Comment 6 The titration procedures in (2) a) Standardization, (2) c) Standardization and (4.3)
can be conducted by an automatic titrator. The setup of the titration program, thedetermination parameter for the endpoint and vessels such as acceptors are according
to the specification and the operation method of the automatic titrator used.
Comment 7 The nitrogen content in the analytical sample can be measured by using an automatic
35

Testing Methods for Fertilizers (2018)

nitrogen analyzer (Kjeldahl method) instead of the test procedure in (4). The setup of
the program and the parameter of the analyzer as well as vessels etc. are according to
the specification and the operation method of the automatic nitrogen analyzer used.
However, conduct in advance a comparative test with the test procedure in (4) using
fertilizers containing no nitrate nitrogen, to confirm that there is no difference in thequantitation value of total nitrogen.
Comment 8 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 10 % (mass fraction) 20 % (mass fraction) and 1 % (mass fraction) - 5 % (mass fraction) are 98.5 % 100.6 % and 97.1%-99.2 % as total nitrogen (T-N) respectively.
The results of the collaborative study (limited to reported values with the Kjeldahl
method) to determine a certified reference material fertilizer were analyzed by usinga three-level nesting analysis of variance. Table 1 shows the calculation results ofreproducibility, intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.2 % (mass fraction) for solid fertilizers, and 0.02 % (mass fraction) for fluid
fertilizers.
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatabllity
Intermediate precision
Reproducibility
certified reference laboratory

2)

Average

sr

4)

RSD r
(%)
0.5

1)
3)
3)
material fertilizer
p
(%)
(%)
FAMIC-A-10
11
14.68
0.07
1） The number of laboaratories used
for analysis conducting Kjeldahl method
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

5)

6)

s I(T)

7)

8)

RSD I(T)
sR
RSD R
3)
3)
(%)
(%)
(%)
(%)
0.07
0.5
0.13
0.9
6） Intermediate standard deviation

9)7） Intermediate relative standard deviation
8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p. 27 - 31, Yokendo, Tokyo (1988)
2)
Feed Analysis Standard Task Force: Feed Analysis Method/Handbook -2009 - I, p. 28 33, Incorporated Administrative Agency, Food and Agriculture Materials Inspection
Center, Saitama (2009)3)
Takashi KUBOTA, Tomoko OSHIDA, Kozue YANAI, Yuzuru INOUE, Seiji MATSUI,Takaharu MATSUMOTO, Eiichi ISHIKURO and Akemi YASUI: Improvement of theConditions for the Determination of Total Nitrogen in Fish Meal in Kjeldahl Method
and Its Comparison with Dumas Method, Bunsekikagaku, 60, p. 67 - 74 (2011)
4)
Kimie KATO, Masaki CHIDA and Erina WATANABE: Verification of Performance
Characteristics of Testing Method for Total Nitrogen Content in Fertilizer by Kjeldahl
method, Research Report of Fertilizer, Vol. 5, p. 156 - 166 (2012)36

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total nitrogen: The flow sheet for total nitrogen in fertilizers is shown below:

0.5 g - 5 g
Weigh to the order of 1 mg into a 300-mL digestion flask.
analytical sample← 5 g - 10 g catalyst←20 mL - 40 mL sulfuric acid
Heating
GentlyHeating

After foam no longer evolves, ignite
until organic matters are completely digested.

Standing to cool← small amount of waterTransfer
250-mL - 500-mL volumetric flask, water
Cooling← Water (up to the marked line)
Digestion
solution
Figure 1

The flow sheet for total nitrogen in fertilizers (Kjeldahl method procedure)

Digestion
solution
Aliquot
300-mL distillation flask(predetermined volume)← Sodium hydroxide solution (200 g/L - 500 g/L)
Receiver: 200-mL - 300 - mL Erlenmeyer flask or beakerA predetermined amount of 0.25 mol/L sulfuric acid andSteam distillationa few drops of methyl red-methylene blue mixture solution,
apparatus
or boric acid solution (40 g/L), several drops ofmethyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min -7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L-0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure 2 Flow sheet for total nitrogen in fertilizers (Distillation and mesurement procedure)37

Testing Methods for Fertilizers (2018)

4.1.1.b Combustion method(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 4.1.1.b-2017 or T-N.b-1.
Thermally decompose nitrogen compounds in an analytical sample using a total nitrogen analyzer
by the combustion method to produce nitrogen gas and nitroxide gas. Reduce the nitroxide gas tonitrogen, and measure the nitrogen gas content with a thermal conductivity detector to obtain the
total nitrogen (T-N) in an analytical sample. This testing method is also referred to as a modified
Dumas’ method. In addition, the performance of this testing method is shown in Comment 4.
(2)a)

Instruments: Instruments are as shown below:
Total nitrogen analyzer by the combustion method: A total nitrogen analyzer configuredon the basis of the principle of the combustion method (modified Dumas’ method).
1) Turn on the total nitrogen analyzer by the combustion method (1), and adjust so that stableindicated values can be obtained.
(i) Combustion gas: Oxygen having purity no less than 99.99 % (volume percentage)(ii) Carrier gas: Helium having purity no less than 99.99 % (volume percentage)(3)

Measurement: Conduct measurement as shown below. However, confirm in advance using
an analytical sample that there is no difference from the measured value of total nitrogenobtained according to 4.1.1.a, 4.1.1.c, 4.1.1.d or 4.1.1.e.
a) Measurement conditions for the total nitrogen analyzer by the combustion method: Set
up the measurement conditions for the total nitrogen analyzer considering the following:
Combustion temperature: No less than 870 ºCb) Calibration curve preparation
1) Turn on the total nitrogen analyzer by the combustion method (1), and adjust so that stableindicated values can be obtained.
2) Weigh a predetermined amount of the standard for calibration curves (2) to the order of 0.1 mg
into a combustion vessel.
3) Insert the combustion vessel into the total nitrogen analyzer by the combustion method, andread the indicated value.
4) Conduct the procedure in 3) for another combustion vessel for a blank test, and read theindicated value.
5) Prepare a curve for the relationship between the nitrogen content and the indicated value ofthe standard for calibration curves and the blank test for calibration curves.
c) Sample measurement
1) Weigh a predetermined amount of an analytical sample to the order of 0.1 mg into a
combustion vessel.
2) Insert the combustion vessel containing the analytical sample to the total nitrogen analyzer by
the combustion method, and read the indicated value.
3) Obtain the nitrogen content from the calibration curve, and calculate total nitrogen in theanalytical sample.
Note (1) The setup of the program and the parameter of the analyzer are according to thespecification and the operation method of the total nitrogen analyzer by the
combustion method used.
(2) Standard for calibration curves: DL-Aspartic acid (purity no less than 99 % (massfraction)), EDTA (purity no less than 99 % (mass fraction)), hippuric acid (purity noless than 98 % (mass fraction)) or other reagents having equivalent purity
recommended by the total nitrogen analyzer by the combustion method used.
38

Testing Methods for Fertilizers (2018)

Comment 1 Sample an analytical sample from a test sample prepared in 2.3.3 Grinding (3.1) bygrinding with a mill until it completely passes through a sieve of 500 µm aperture orfrom a test sample prepared in 2.3.3 Grinding Comment 1. Additionally, thesampling amount of an analytical sample is as shown in Table 1. In addition, set the
sampling amounts of analytical samples considering the estimated content of total
nitrogen in the test sample and the measurement range of the total nitrogen analyzer
by the combustion method.
Table 1 Sampling amount of analytical samples
Sampling amount (g)Type of fertilizers
Mixed fertilizers and designated0.02 - 0.5
blended fertilizers
0.05 - 0.5
Organic fertilizers and compost
Sludge fertilizers

0.05 - 0.5

Comment 2 Compound fertilizers, designated blended fertilizers and nitrolime may have highcontents of phosphoric acid (P2O5), alkali metals (Na, K), alkaline earth metals (Ca,Mg), etc., causing contamination of packing or damage in quartz parts, etc. To avoid
their influences, it is recommended to add tungsten oxide (elemental analysis reagent
or heat-treated reagent) to completely cover the analytical sample.
Comment 3 When a sample with a low content of organic compounds, such as mixed fertilizers
and designated blended fertilizers etc., and thus with low combustion efficiency ismeasured, it is recommended to add sucrose to the analytical sample so that the
carbon content will be comparable to the standard for calibration curves. Additionally,
confirm in advance that sucrose to be used has a nitrogen content that does not affectthe measured value of total nitrogen of the analytical sample.
Comment 4 Table 2, in order to evaluate trueness, shows the results of the comparison of
measurement values by the Combustion method and the Kjeldahl method with sludge
fertilizers, organic fertilizers, and inorganic fertilizers, etc.
Table 3 shows results and analysis results from a collaborative study for testing
method validation.
Additionally, the minimum limit of quantification of this testing method is about
0.01 % (mass fraction) for fluid fertilizers for home gardening, and 0.05 % (massfraction) for the other fertilizers.

39Testing Methods for Fertilizers (2018)
Table 2
Symbol of measurement value
Kjaeldahl
Combustion
methodxi

1)methodyi

2)Analysis results of comparison test results between methods
SampleRange ofRegression coefficient Correlation
Kind
Number ofyi - yk(y ＝ a ＋bx )coefficient3)
samples
a
b
r
(%)
4)

81

0.31～8.35-0.006

1.018

0.999

31

1.10～12.90

0.009

1.012

1.000

xk
yk
36
0.60～46.35
0.000
1.004
Inorganic fertilizers, etc.
1） 4.1.1.a Kjeldahl method
2） 4.1.1.b Combustion method
3） Mass fraction
4） Sewage sludge fertilizers, Human waste sludge fertilizers, Industrial sludge fertilizers,
Calcined sludge fertilizers, Composted sludge fertilizers
5） Fish meal, Byproduct organic fertilizer of vegetable origin, Compost, Crustose fertilizer meal,
Rape seed meal and powdered rape seed meal, etc.
6） Nitrogenous fertilizers, Compound fertilizers, Blended fertilizers, Fluid fertilizers

1.000

xj

yj

Sludge fertilizers

Organic fertilizers, etc.

5)

Table 3 Results and analysis results from a collaborative study
for total nitrogen testing method validation
Sample nameCompound fertilizer (containing

nitrate nitrogen)
Compound fertilizer (containing
urea)
Designated blended fertilizer(containing organic fertilizer)
Nitrolime
Fish meal
Steamed wool waste
Rape seed meal and powdered rape
seed meal
Composted sludge fertilizer A
Composted sludge fertilizer B
Human waste sludge fertilizer
Industrial sludge fertilizer
Calcined sludge fertilizer

1)
2)
3)
4)
5)
6)
7)

2)

sr

4)

RSD r

6)

RSD R

3)

(%)

3)

(%)

(%)

3)

(%)

11

9.32

0.07

0.8

0.25

2.7

11

18.34

0.06

0.3

0.45

2.5

12

14.06

0.12

0.9

0.42

3.0

8
10
11

19.96
8.34
13.42

0.07
0.04
0.10

0.4
0.4
0.7

0.17
0.10
0.26

0.8
1.3
2.0

11

6.21

0.07

1.1

0.25

4.0

13
12
11
11
13

6.20
2.36
4.44
8.06
0.80

0.02
0.01
0.02
0.03
0.02

0.3
0.6
0.4
0.4
2.8

0.09
0.04
0.06
0.07
0.03

1.4
1.8
1.3
0.9
4.3

Number oflaboratories

Mean

1)

(%)

Number of laboratories used in analysis
Mean (n = number of laboratories x number of samples (2))
Mass fraction
Repeatability standard deviation
Repeatability relative standard deviation
Reproducibility standard deviation
Reproducibility relative standard deviation

40

5)

sR

7)

Testing Methods for Fertilizers (2018)

References
1)
Mariko AIZAWA, Yasushi SUGIMURA, Yuichi TAKAHASHI, Jun OKI, Yukio
FUKUCHI, Yuji SHIRAI and Norio HIKICHI: Validation of a Combustion Method for
Determination of Total Nitrogen Content in Sludge Fertilizer. Research Report of
Fertilizer Vol.1, p. 12 - 17, (2008)
2)
Mariko AIZAWA and Yuji SHIRAI: Determination of Total Nitrogen content in Sludge
Fertilizer by a Combustion Method: A Collaborative Study. Research Report of
Fertilizer Vol.1, p. 18 - 24, (2008)
3)
Mariko AIZAWA and Yuji SHIRAI: Validation of a Combustion Method for
Determination of Total Nitrogen Content in Organic Fertilizer. Research Report of
Fertilizer Vol.2, p. 6 - 11, (2009)
4)
Mariko AIZAWA and Yuji SHIRAI: Validation of a Combustion Method for
Determination of Total Nitrogen Content in Inorganic Fertilizer. Research Report of
Fertilizer Vol.3, p. 1 - 10, (2010)
5)
Mariko AIZAWA, Yuko SEKINE and Yuji SHIRAI: Determination of Total Nitrogen
Content in Fertilizer by a Combustion Method: A Collaborative Study. Research Reportof Fertilizer Vol.3, p. 11 - 18, (2010)
6)
Kazumi UCHIYAMA and Yoshio MAEBASHI: Effective Analysis of Organic Trace
Element, p. 99, Mimizuku-sha, Tokyo (2008)(4)

Flow sheet for total nitrogen: The flow sheet for total nitrogen in fertilizers is shown
below:
Analytical sample

Weigh to the order of 1 mg into a combustion vessel

Measurement

Total nitrogen analyzer by the combustion method

Figure Flow sheet for total nitrogen in fertilizers by the combustion method.

41

Testing Methods for Fertilizers (2018)

Reference: Chromatograms of the standard for calibration curves and an analytical sample areshown below:

1)Standard for calibration curves (DL-aspartic acid)2)

Analytical sample (sludge fertilizer)

Reference figures

Chromatograms of total nitrogen.

Measurement conditions for total nitrogen analyzer by the combustion method
Combustion gas: Highly pure oxygen, purity no less than 99.9999 % (volume
fraction), flow rate 200 mL/min
Carrier gas: Highly pure helium, purity no less than 99.9999 % (volume fraction),flow rate 80 mL/min
Separation column: A silica gel stainless column (1m)
Detector: Thermal conductivity detector (TCD)
Measurement cycle: Purge time = 60 seconds, circulation combustion time = 200
seconds, measurement time = 100 seconds
Current value of Detector: 160 mA
Temperature conditions: Reaction furnace temperature: 870 ºC
Reaction furnace temperature: 600 °C
Column oven temperature: 70 °C
Detector temperature: 100 °C
42

Testing Methods for Fertilizers (2018)

4.1.1.c Devarda’s alloy – Kjeldahl method(1) Summary
The method is applicable to the fertilizers that contain nitrate nitrogen (N-N) and guarantee total
nitrogen. This testing method is classified as Type E and its symbol is 4.1.1.c-2017 or T-N.c-1.
Add hydrochloric acid (1+1) and tin (II) chloride dihydrate to an analytical sample and further add
devarda’s alloy to reduce nitrate nitrogen (N-N), and then add sulfuric acid (1+1), pretreat by
Kjeldahl method to change total nitrogen (T-N) to ammonium ion and add sodium hydroxide to
subject to steam distillation. Collect isolated ammonia with 0.25 mol/L sulfuric acid and measuresurplus sulfuric acid by (neutralization) titration using a 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution to obtain the total nitrogen (T-N) in an analytical sample. Or collect isolated ammonia witha boric acid solution and measure ammonium ion by (neutralization) titration using 0.25 mol/L
sulfuric acid to obtain the total nitrogen (T-N) in an analytical sample. This testing method
corresponds to the devarda’s alloy - sulfuric acid method in the Official Methods of Analysis of
Fertilizers (1992).
(2)a)

Reagent: Reagents are as shown below.
0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in JIS
K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg. Dissolve
in a small amount of water, transfer to a 250-mL volumetric flask, and add water up to themarked line (1). Transfer a predetermined amount of the solution to a 200-mL - 300-mL
Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as anindicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C1)
W1：
A：
V1：
V2：
V3：

Mass (g) of sulfamic acid sampled
Purity (% (mass fraction)) of sulfamic acid
Volume (mL) of sulfamic acid solution transferred
Constant volume (250 mL) of sulfamic acid solution
Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
c) 0.25 mol/L sulfuric acid (1) (2): Add about 14 mL of sulfuric acid to a beaker containing 100
mL of water in advance, stir well, and add water to make 1000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a 200-mL 300-mL Erlenmeyer flask, add a few drops of methyl red−methylene blue mixture solution,and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of thesolution becomes gray-green (4). Calculate the volume of a 0.1 mol/L - 0.2 mol/L sodium
43Testing Methods for Fertilizers (2018)hydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the following formula (1).
Or, calculate the factor of 0.25 mol/L sulfuric acid by the following formula (2):Volume (B) of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL of 0.25
mol/L sulfuric acid= V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2 × 2)………. (2)V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5： Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 in water tomake 1000 mL.
e) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
f) Tin (II) chloride dihydrate: A JIS Guaranteed Reagent specified in JIS K 8136 or a reagentof mercury analysis grade or equivalent quality.
g) Devarda’s alloy: A reagent of nitrogen analysis grade specified in JIS K 8653 or a reagent ofequivalent quality.
h) Sodium hydroxide solution (200 g/L - 500 g/L) (1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
i) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water to make 100 mL.
j) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
k) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in JIS K
8897 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red−methylene blue mixture solution: To 2 volumes of methyl red solution (0.1g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
m) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green specified
in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
n) Methyl red−bromocresol green mixture solution: To a methyl red solution (0.1 g/100 mL),
add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL -10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Steam distillation apparatusb) Digestion flask: Kjeldahl flask
44Testing Methods for Fertilizers (2018)c)Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to a steam
distillation apparatus.

Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
Comment 2 0.25 mol/L sulfuric acid in (2) c) can be replaced with 0.25 mol/L sulfuric acidconforming to ISO/IEC 17025.
(4) Test procedures(4.1) Reduction and Kjeldahl method: Conduct reduction and digestion as shown below:a) Weigh 0.5 g - 1 g (no more than the equivalents of N-N 50 mg) of an analytical sample to theorder of 1 mg, and put it in a 300-mL- 500-mL digestion flask (5)b) Add 60 mL of hydrochloric acid (1+1) and 2 g of tin (II) chloride dihydrate, and shake to mix
and leave at rest for about 20 minutes.
c) Add 3.5 g of devarda’s alloy and leave at rest for about 40 minutes while sometimes shaking
to mix.
d) Add 70 mL of sulfuric acid (1+1) and one boiling stone as necessary, and heat at low
temperature (6).
e) As soon as white smoke start evolving, strengthen heating gradually and further continue
heating for about 90 minutes.
f) After standing to cool, add 100 mL - 200 mL of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water, and further mix by shaking (7).
g) After cooling is complete, add water up to the marked line to make the digestion solution.
Note (5) In the case of direct distillation, a 500-mL Kjeldahl flask connectable to a steam
distillation apparatus is preferable.
(6) If the bubbles foam strongly and excessively, suspend heating for a little while.
(7) When the entire sample solution volume is used in measurement, it is not necessary to
precisely adjust.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used in measurement.
a) Transfer a predetermined amount (8) of 0.25 mol/L sulfuric acid to an acceptor, (9) add a few
drops of methyl red−methylene blue mixture solution, and connect this acceptor to a steam
distillation apparatus. Or, transfer a predetermined amount (8) of boric acid solution (40 g/L) toan acceptor (9), add a few drops of methyl red−bromocresol green mixture solution, andconnect this acceptor to a steam distillation apparatus.
b) Transfer a predetermined amount of the digestion solution to a 300-mL distillation flask, add aproper amount of sodium hydroxide solution (200 g/L - 500 g/L) (10), and connect this
distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill at a
distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution in theacceptor with a small amount of water, and pool the washing with the distillate.
Note (8) 5 mL - 20 mL(9) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be45

Testing Methods for Fertilizers (2018)

immersed in 0.25 mol/L sulfuric acid or a boric acid solution (40 g/L).
(10) An amount sufficient to make the solution strong alkalinity. A blue color will appear.
(4.3) Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2):a) Titrate the distillate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes gray-green (4).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= (B × V6 － V7) × C1 × f1 × (V8/V9) × (14.007/W2) × (100/1000)= (B × V6 － V7) × C1 × f1 × (V8/V9) × (1.4007/W2)
B： Volume of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acidV6： Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2) a)
V7： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V8： Predetermined volume (mL) of the digestion solution in (4.1) g)
V9： Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W2： Mass (g) of the analytical sample(4.3.2) When a boric acid solution (40 g/L) is used in (4.2):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomes lightred (11).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= V10 × C2 × 2 × f2 × (V11/V12) × (14.007/W3) × (100/1000)= V10 × C2 × f2 × (V11/V12) × (2.8014/W3)
V10： Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidf2： Factor of 0.25 mol/L sulfuric acidV11： Predetermined volume (mL) of the digestion solution in (4.1) g)
V12： Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W3： Mass (g) of the analytical sample
Note (11) The endpoint is reached when the color changes from green to light red.
Comment 3 The titration procedures in (2) a) Standardization, (2) c) Standardization and (4.3)
can be conducted by an automatic titrator. The setup of the titration program, thedetermination parameter for the endpoint and vessels such as acceptors are according
to the specification and the operation method of the automatic titrator used.
References
46Testing Methods for Fertilizers (2018)

1)

Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.31- 33, Yokendo, Tokyo (1988)

47

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total nitrogen: The flow sheet for total nitrogen in fertilizers is shown
below:
0.5 g – 1 g
Weigh to the order of 1 mg into a 300-mL - 500-mL digestion flask.
analytical sample← 60 mL of hydrochloric acid (1+1)← 2 g of tin (II) chloride dihydrate
Leaving at rest
About 20 minutes← 3.5 g devardar's alloy
Leaving at rest
About 40 minutes← 70 mL sulfuric acid (1+1)← 1 boiling stone
Heat at low temperature, and as soon as white smoke evolves,
Heating
strengthen heating gradually and further heat for about 90 minutes.
Standing to cool
← 100 mL - 200 mL Water
Transfer
250-mL - 500-mL volumetric flask, water
Cooling← Water (up to the marked line)
Digedtion
Solution
Figure 1

The flow sheet for total nitrogen in fertilizers (Reduction and Kjeldahl method procedure)48

Testing Methods for Fertilizers (2018)

Digestion
Solution
Aliquot
300-mL distillation flask(predetermined volume)← Sodium hydroxide (200 g/L - 500 g/L)
Receiver: 200-mL - 300-mL Erlenmeyer flask or beaker
Steam distillation
A predetermined amount of 0.25 mol/L sulfuric acid and a few drops ofapparatus
methyl red-methylene blue mixture solution, or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of the
Titration
solution becomes gray-green, or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure 2 Flow sheet for total nitrogen in fertilizers (Distillation and measurement procedure).

49

Testing Methods for Fertilizers (2018)

4.1.1.d Reduced iron - Kjeldahl method(1) Summary
The method is applicable to the fertilizers that contain nitrate nitrogen (N-N) and guarantee total
nitrogen. This testing method is classified as Type E and its symbol is 4.1.1.d-2017 or T-N.d-1.
Add water, reduced iron and sulfuric acid (1+1) to an analytical sample to reduce nitrate nitrogen(N-N) and heat at low temperature, and then add sulfuric acid and pretreat by Kjeldahl method tochange total nitrogen (T-N) to ammonium ion, and add a sodium hydroxide solution to subject tosteam distillation. Collect isolated ammonia with 0.25 mol/L sulfuric acid and measure surplus
sulfuric acid by (neutralization) titration using a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution to
obtain the total nitrogen (T-N) in an analytical sample. Or collect isolated ammonia with a boric
acid solution and measure ammonium ion by (neutralization) titration using 0.25 mol/L sulfuric acidto obtain the total nitrogen (T-N) in an analytical sample. This testing method corresponds to the
reduced iron - sulfuric acid method in the Official Methods of Analysis of Fertilizers (1992).
(2)a)

Reagent: Reagents are as shown below.
0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C1)
W1：
A：
V1：
V2：
V3：

Mass (g) of sulfamic acid sampled
Purity (% (mass fraction)) of sulfamic acid
Volume (mL) of sulfamic acid solution transferred
Constant volume (250 mL) of sulfamic acid solution
Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
c) 0.25 mol/L sulfuric acid (1) (2): Add about 14 mL of sulfuric acid to a beaker containing 100
mL of water in advance, stir well, and add water to make 1000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a 200-mL- 300-mL Erlenmeyer flask, add a few drops of methyl red−methylene blue mixture solution,and titrate with a 0.1 mol/L -0.2 mol/L sodium hydroxide solution until the color of thesolution becomes gray-green (4). Calculate the volume of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the following formula
50

Testing Methods for Fertilizers (2018)(1). Or, calculate the factor of 0.25 mol/L sulfuric acid by the following formula (2):Volume (B) of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL of 0.25
mol/L sulfuric acid= V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2×2)………. (2)V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5： Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 in water tomake 1000 mL.
e) Reduced iron:
Nitrogen content is no more than 0.005 % (mass fraction)f) Catalyst (5): Mix potassium sulfate specified in JIS K 8962 and copper (II) sulfate
pentahydrate (6) specified in JIS K 8983 in the ratio of 9 to 1.
g) Sodium hydroxide solution (200 g/L - 500 g/L) (1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
h) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8842, add water to make 100 mL.
i) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
j) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in JIS K
8897 in 100 mL of ethanol (95) specified in JIS K 8102.
k) Methyl red−methylene blue mixture solution: To 2 volumes of methyl red solution (0.1g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
l) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green specified
in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
m) Methyl red−bromocresol green mixture solution: To a methyl red solution (0.1 g/100mL), add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL -10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(5) A tablet is commercially available.
(6) Crush into powder as appropriate.
(3)
a)
b)
c)

Instruments: Instruments are as shown below:
Steam distillation apparatus
Digestion flask: Kjeldahl flask
Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to a steam
distillation apparatus.
51

Testing Methods for Fertilizers (2018)

Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
Comment 2 0.25 mol/L sulfuric acid in (2) c) can be replaced with 0.25 mol/L sulfuric acidconforming to ISO/IEC 17025.
(4) Test procedures(4.1) Reduction and Kjeldahl method: Conduct reduction and digestion as shown below:a) Weigh 0.5 g - 1 g of an analytical sample to the order of 1 mg, and put it in a 300-mL 500-mL digestion flask.
b) Add 30 mL of water and mix well.
c) As soon as 5 g of reduced iron and 30mL of sulfuric acid (1+1) are added, insert a long stem
funnel to a digestion flask and shake to mix gently while cooling the outside of the container
under flowing water (7).
d) Leave at rest for about 5 minutes (8), boil in a low flame for about 15 minutes.
e) After standing to cool, add 5 g - 10 g of catalyst, 30 mL of sulfuric acid and, if necessary, one
boiling stone, heat gradually until water evaporates and white smoke of sulfuric acid evolves(9).
f) Ignite until it is completely digested (10).
g) After standing to cool, add a small amount of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water, and further mix by shaking.
h) After cooling is complete, add water up to the marked line to make the digestion solution.
Note (7) A sudden reaction generates heat, and unreacted nitric acid vaporizes or digests to makenitrogen oxide etc. through which process losses occur easily. Careful and efficient
operation should be taken(8) Until a sudden reaction is settled.
(9) If the bubbles foam strongly and excessively, suspend heating for a little while.
(10) When the solution has finished changing color, heat further for no less than 2 hours.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used in measurement.
a) Transfer a predetermined amount (11) of 0.25 mol/L sulfuric acid to an acceptor (12), add a few
drops of methyl red−methylene blue mixture solution, and connect this acceptor to a steam
distillation apparatus. Or, transfer a predetermined amount (11) of boric acid solution (40 g/L)
to an acceptor (12), add a few drops of methyl red−bromocresol green mixture solution, andconnect this acceptor to a steam distillation apparatus.
b) Transfer a predetermined amount of the digestion solution to a 300-mL distillation flask, add aproper amount of sodium hydroxide solution (200 g/L - 500 g/L) (13), and connect this
distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill at a
distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution in theacceptor with a small amount of water, and pool the washing with the distillate.
Note (11) 5 mL - 20 mL(12) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
52

Testing Methods for Fertilizers (2018)

beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or a boric acid solution (40 g/L).
(13) An amount sufficient to make the solution strong alkalinity. A blue or reddish-browncolor will appear.
(4.3) Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2):a) Titrate the distillate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes gray-green (4).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= (B × V6 － V7) × C1 × f1 × (V8/V9) × (14.007/W2) × (100/1000)= (B × V6 － V7) × C1 × f1 × (V8/V9) × (1.4007/W2)
B： Volume of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acidV6： Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2) a)
V7： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V8： Predetermined volume (mL) of the digestion solution in (4.1) h)
V9： Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W2： Mass (g) of the analytical sample(4.3.2) When a boric acid solution (40 g/L) is used in (4.2):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomes lightred (14).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= V10 × C2 × 2 × f2 × (V11/V12) × (14.007/W3) × (100/1000)= V10 × C2 × f2 × (V11/V12) × (2.8014/W3)
V10： Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidf2： Factor of 0.25 mol/L sulfuric acidV11： Predetermined volume (mL) of the digestion solution in (4.1) h)
V12： Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W3： Mass (g) of the analytical sample
Note (14) The endpoint is reached when the color changes from green to light red.
Comment 3 The titration procedures in (2) a) Standardization, (2) c) Standardization and (4.3)
can be conducted by an automatic titrator. The setup of the titration program, thedetermination parameter for the endpoint and vessels such as acceptors are according
to the specification and the operation method of the automatic titrator used.
53

Testing Methods for Fertilizers (2018)

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.33- 34, Yokendo, Tokyo (1988)(5)

Flow sheet for total nitrogen: The flow sheet for total nitrogen in fertilizers is shown below:
0.5 g - 1 g
Weigh to the order of 1 mg into a 300-mL digestion flask.
← 30 mL water← 5 g reduced iron
← 30 mL sulfuric acid (1+1)
Immediately insert a long stem funnel to the flask and shake to mix
Mixturewhile cooling outside the container with flowing water.
Leaving at rest
Heating

For about 5 min (Until a sudden reaction is settled.)
Boil in a low flame for about 15 minutes.

Standing to cool
← 5 g - 10 g catalyst← 30 mL sulfuric acidHeat gradually until water evaporates and the white smoke
Heatingof sulfuric acid evolves and ignite to digest completely
Standing to cool
← A small amount of waterTransfer
250-mL - 500-mL volumetric flask, water
Cooling← Water (up to the marked line)
Digestion
Solution
Figure 1

The flow sheet for total nitrogen in fertilizers (Reduction and Kjeldahl method procedure)

54

Testing Methods for Fertilizers (2018)

Digestion
Solution
Aliquot
Distillation flask(predetermined volume)← Sodium hydroxide (200 g/L - 500 g/L)
Receiver: 200-mL - 300-mL Erlenmeyer flask or beaker
Steam distillation
A predetermined amount of 0.25 mol/L sulfuric acid and a few drops ofapparatus
methyl red-methylene blue mixture solution, or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)0.1mol/L - 0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure 2 Flow sheet for total nitrogen in fertilizers (Distillation and measurement procedure).

55

Testing Methods for Fertilizers (2018)

4.1.1.e Calculation with ammoniac nitrogen and nitrate nitrogen(1) Summary
This testing method is applicable to the fertilizers that contain ammoniac nitrogen (A-N) and nitrate
nitrogen (N-N), and that does not contain the fertilizers guaranteeing total nitrogen (T-N). This
testing method is classified as Type A (Def-C) and its symbol is 4.1.1.e-2017 or T-N.e-1.
Calculate total nitrogen (T-N) by adding the ammoniac nitrogen (A-N) obtained in 4.1.2 to thenitrate nitrogen (N-N) obtained in 4.1.3.
(2) The calculation of total nitrogena) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= (A-N) ＋ (N-N)
A-N： Ammoniac nitrogen (% (mass fraction)) in the analytical sample (1) obtainedby 4.1.2
N-N： Nitrate nitrogen (% (mass fraction)) in the analytical sample (1) obtained by
4.1.3
Note (1) A-N and N-N use raw data without rounding numerical value

56

Testing Methods for Fertilizers (2018)

4.1.2 Ammoniac nitrogen
4.1.2.a Distillation method(1) Summary
The testing method is applicable to the fertilizers that contain ammonium salt. However, in somecases, it is not applicable to the fertilizers that contain such compounds as nitrolime that digests byheating. This testing method is classified as Type C and its symbol is 4.1.2.a-2017 or A-N.a-1.
Add water to an analytical sample, further add magnesium oxide or a sodium hydroxide solution to
make the solution alkalinity and subject it to steam distillation. Collect isolated ammonia with 0.25
mol/L sulfuric acid and measure surplus sulfuric acid by (neutralization) titration using a 0.1 mol/L- 0.2 mol/L sodium hydroxide solution to obtain ammoniacal nitrogen (A-N) in an analytical sample.
Or collect isolated ammonia with a boric acid solution and measure ammonium ion by(neutralization) titration using 0.25 mol/L sulfuric acid to obtain ammoniacal nitrogen (A-N) in ananalytical sample. In addition, the performance of this testing method is shown in Comment 7.
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C1)
W1：
A：
V1：
V2：
V3：

Mass (g) of sulfamic acid sampled
Purity (% (mass fraction)) of sulfamic acid
Volume (mL) of sulfamic acid solution transferred
Constant volume (250 mL) of sulfamic acid solution
Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Magnesium oxide: A JIS Guaranteed Reagent specified in JIS K 8432 or a reagent ofequivalent quality.
c) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
d) 0.25 mol/L sulfuric acid (1) (2): Add about 14 mL of sulfuric acid to a beaker containing 100
mL of water in advance, stir well, and add water to make 1000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a 200-mL- 300-mL Erlenmeyer flask, add a few drops of methyl red−methylene blue mixture solution,and titrate with a 0.1 mol/L -0.2 mol/L sodium hydroxide solution until the color of the57

Testing Methods for Fertilizers (2018)

solution becomes gray-green (4). Calculate the volume of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the following formula(1). Or, calculate the factor of 0.25 mol/L sulfuric acid by the following formula (2):Volume (B) of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL of 0.25
mol/L sulfuric acid= V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2×2)………. (2)V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5： Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acide)

Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 in water tomake 1000 mL.
f) Sodium hydroxide solution (200 g/L - 500 g/L) (1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water to make 100 mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
i) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in JIS K
8897 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methyl red−methylene blue mixture solution: To 2 volumes of methyl red solution (0.1g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
k) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green specified
in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red−bromocresol green mixture solution: To a methyl red solution (0.1 g/100mL), add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL -10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
Comment 2 0.25 mol/L sulfuric acid in (2) d) can be replaced with 0.25 mol/L sulfuric acidconforming to ISO/IEC 17025.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Steam distillation apparatusb) Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to a steam58

Testing Methods for Fertilizers (2018)

distillation apparatus.
(4) Test procedures(4.1) Sample solution preparation: Prepare a sample solution as shown below:a) Weigh 0.25 g - 2g (5) (the equivalents of 20 mg - 100 mg as N) of an analytical sample to theorder of 1 mg, and put it in a 300-mL - 500-mL distillation flaskb) Add about 25 mL of water to make a sample solution.
Note (5) The sampling amount of the analytical sample is 5 g when there is less nitrogen content
in the fertilizers such as a home garden-use fertilizer.
Comment 3 When it is a fertilizer which contains uric ammonium acid, humus acid ammonium or
nitrate nitrogen, etc., or when it is not a fertilizer in which phosphate, ammonium andmagnesium coexist, conduct the procedure in (4.1.1) a) - c) in 4.2.4.a or theprocedure in (4.1.2) a) - c) in 3.2.4.a and transfer a predetermined amount ofsuspension (the equivalents of 20 mg - 100 mg as N) to a 300-mL - 500-mL
distillation flask to make a sample solution.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used in measurement.
a) Transfer a predetermined amount (6) of 0.25 mol/L sulfuric acid to an acceptor (7), add a few
drops of methyl red−methylene blue mixture solution, and connect this acceptor to a steam
distillation apparatus.
Or, transfer a predetermined amount (6) of boric acid solution (40(7)g/L) to an acceptor , add a few drops of methyl red−bromocresol green mixture solution,and connect this acceptor to a steam distillation apparatus.
b) Add (9) no less than 2 g of magnesium oxide (8) to the distillation flask, and connect this
distillation flask to a steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill at a
distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution in theacceptor with a small amount of water, and pool the washing with the distillate.
Note (6) 5 mL - 20 mL(7) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or a boric acid solution (40 g/L).
(8) An amount sufficient to make the solution strong alkalinity.
(9) Add a small amount of silicone oil as necessary.
Comment 4 When the sample does not contain organic matters or urea, a proper amount of
sodium hydroxide solution (200 g/L - 500 g/L) (8) can be added instead ofmagnesium oxide.
(4.3) Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2):a) Titrate the distillate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes gray-green (4).
b) Calculate the ammoniacal nitrogen (A-N) in the analytical sample by the following formula:
59

Testing Methods for Fertilizers (2018)

Ammoniacal nitrogen (A-N) (% (mass fraction)) in the analytical sample= (B × V6 － V7) × C1 × f1 × (14.007/W2) × (100/1000)= (B × V6 － V7) × C1 × f1 × (1.4007/W2)
B：

Volume of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1
mL of 0.25 mol/L sulfuric acidV6： Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2)a)
V7： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution
f1： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
W2： Mass (g) of the analytical sample(4.3.2) When a boric acid solution (40 g/L) is used in (4.2):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomes lightred (10).
b) Calculate the ammoniacal nitrogen (A-N) in the analytical sample by the following formula:Ammoniacal nitrogen (A-N) (% (mass fraction)) in the analytical sample= V8 × C2 × 2 × f2 × (14.007/W3) × (100/1000)= V8 × C2 × f2 × (2.8014/W3)
V8：
C2 ：
f2 ：
W3：

Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acid
Factor of 0.25 mol/L sulfuric acid
Mass (g) of the analytical sample

Note (10) The endpoint is reached when the color changes from green to light red.
Comment 5 If it is hard to confirm the endpoint due to the carbon dioxide resulting fromcarbonate in the extract when magnesium oxide is used, it is recommended to boil
the extract for 1-2 minute(s) after distilling and cool, and then titrate.
Comment 6 The titration procedures in (2) a) Standardization, (2) d) Standardization and(4.3) can be conducted by an automatic titrator. The setup of the titration program,the determination parameter for the endpoint and vessels such as acceptors areaccording to the specification and the operation method of the automatic titrator
used.
Comment 7 Recovery testing was conducted to evaluate trueness using a preparation sample. As
a result, the average rate of recovery at the content level of 10 % (mass fraction) 21 % (mass fraction) and 1 % (mass fraction) are 102.2 % - 100.8 % and 102.5 %as ammoniacal nitrogen (A-N) respectively.
The results of the collaborative study (limited to reported values with Distillation
method) to determine a certified reference material fertilizer were analyzed byusing a three-level nesting analysis of variance. Table 1 shows the calculation
results of reproducibility, intermediate precision and repeatability. Additionally, theminimum limit of quantification of this testing method is about 0.1 % (massfraction) for solid fertilizers, and 0.01 % (mass fraction) for fluid fertilizers.
60Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
2)
4)
5)
6)
7)
8)
9)
certified reference laboratory Averagesr
RSD rs I(T)RSD I(T)
sR
RSD R
1)
3)
3)
material fertilizer
(%)
p
(%)
(%)
FAMIC-B-10
11
8.38
0.09
1.0
FAMIC-B-14
11
8.06
0.03
0.4
1） The number of laboaratories used
for analysis conducting Distillation method
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

3)
3)
(%)
(%)
(%)
(%)
0.11
1.3
0.15
1.8
0.05
0.6
0.07
0.9
6） Intermediate standard deviation

7） Intermediate relative standard deviation
8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.36- 37, Yokendo, Tokyo (1988)
2)
Kimie KATO, Masaki CHIDA and Erina WATANABE: Verification of Performance
Characteristics of Testing Method for Ammonia Nitrogen Content in Fertilizer byDistillation Method, Research Report of Fertilizer, Vol. 6, p. 130 - 138 (2013)

61

Testing Methods for Fertilizers (2018)(5)

Flow sheet for ammoniac nitrogen: The flow sheet for ammoniac nitrogen in fertilizers isshown below.
0.25 g - 2 g
Weigh the equivalents of 20 mg – 100 mg as N
analytical sample
to the order of 1 mg, and put it in a 300-mL distillation flask← About 25 mL water← 2 g Magnesium oxide to a distillation flask← A small amount of defoaming agent as necessary
Receiver: 200-mL - 300-mL Erlenmeyer flask or beaker
Steam distillation
A predetermined amount of 0.25 mol/L sulfuric acid and a few drops ofapparatus
methyl red-methylene blue mixture solution, or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure Flow sheet for ammoniac nitrogen in fertilizers.

62Testing Methods for Fertilizers (2018)

4.1.2.b Formaldehyde method(1) Summary
The testing method is applicable to the fertilizers which do not contain a large amount of flora andfauna sample. This testing method is classified as Type C and its symbol is 4.1.2.b-2017 or A-N.b-1.
After adding water or hydrochloric acid (1+20) to an analytical sample to extract ammonium ion,
add an aluminum chloride solution, drop a potassium hydroxide solution and precipitate phosphateand excessive aluminum to make a sample solution. Adjust the sample solution to slight acidity, add
a formaldehyde solution and measure ammonium ion by complexometric titration with 0.1 mol/L 0.2 mol/L sodium hydroxide solution to obtain the ammoniacal nitrogen (A-N) in an analyticalsample. In addition, the performance of this testing method is shown in Comment 8.
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f)= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C)
W1：
A：
V1：
V2：
V3：
C：

Mass (g) of sulfamic acid sampled
Purity (% (mass fraction)) of sulfamic acid
Volume (mL) of sulfamic acid solution transferred
Constant volume (250 mL) of sulfamic acid solution
Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Potassium chloride solution (1 mol/L) (1): Dissolve 75 g of potassium chloride specified in
JIS K 8121 in water to make 1000 mL.
c) Aluminum chloride solution (1 mol/L) (1): Dissolve 240 g of aluminum chloride (III)hexahydrate specified in JIS K 8114 in water to make 1000 mL.
d) Potassium hydroxide solution (170 g/L) (1): Dissolve 170 g of potassium hydroxide in water
to make 1000 mL.
e) Formaldehyde solution: Add one volume of water to one volume of the 36 % (massfraction) - 38 % (mass fraction) formaldehyde specified in JIS K 8872.
f) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, and add water to make 100
63Testing Methods for Fertilizers (2018)mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
i) Bromothymol blue solution (1 g/100 mL): Dissolve 1 g of bromothymol blue specified in
JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, and add water to make 100 mL.
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
Comment 2 Thymol blue can be dissolved if it is sodium salt. The thymol blue specified in JIS K
8643 is slightly hard to dissolve in ethanol and hard to dissolve in water. Therefore,
add about 2.15 mL of sodium hydroxide solution (0.1 mol/L) per 0.1 g of thymol
blue to neutralize, and then prepare the thymol blue solution (1 g/100 mL) throughthe same procedure as (2) i).
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Ammonium saltsa) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL volumetricflask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 3 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.4.a.
(4.1.2) Mixed fertilizersa) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL volumetricflask.
b) Add about 300 mL of hydrochloric acid (1 + 20) and shake to mix at the rate of 30 - 40
revolutions/min for about 30 minutes.
c) Add an aluminum chloride solution (1 mol/L) (2) to the solution and add a few drops of methyl
red solution (1mol/L) as an indicator. After that, add immediately potassium hydroxide (170g/L) while shaking the flask until the color of the solution changes to light yellow (3).
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make a sample solution.
Note (2) Add 3 mL of aluminum chloride per 0.04 g of P or 0.1 g of P2O5 in the sample
solution.
(3) Form precipitate of aluminum hydroxide and aluminum phosphate to separate thephosphate.
Comment 4 In the procedure of (4.1.1) a) and (4.1.2) a), it is also allowed to weigh 2.5g of ananalytical sample to the order of 1 mg, and put it in a 250-mL volumetric flask
64Testing Methods for Fertilizers (2018)

Comment 5 When it is not a fertilizer in which phosphate, ammonium and magnesium coexist,
about 400 mL of potassium chloride solution (1 mol/L) can be used instead of about
300 mL of hydrochloric acid (1+20) in the procedure of (4.1.2) b).
Comment 6 In the case of the mixed fertilizers containing bentonite, after shaking to mix using
about 300 mL of hydrochloric acid (1+20) or about 400 mL of potassium chloride
solution (1 mol/L) according to Comment 5 in (4.1.2) b), add water to the marked
line, filter with Type 3 filter paper and transfer 50 ml - 100 mL to a 250-mL
volumetric flask, and then conduct the procedure in (4.1.2) c) - e).
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined amount of sample solution (the equivalents of 50 mg as A-N) to a
300-mL volumetric flask (4).
b) Add water to the solution to make about 100 mL.
c) Add one or two drop(s) of methyl red solution (0.1 g/100 mL) and add hydrochloric acid
(1+200) until the color of the solution changes to light pink.
d) Add 10 mL of formaldehyde solution.
e) Add one or two drop(s) of thymol blue solution (1 g/100 mL) and titrate with 0.1 mol/L - 0.2
mol/L sodium hydroxide solution until the color of the solution changes to blue (5).
f) As a blank test, transfer 100 mL of water to another 300-mL Erlenmeyer flask and conduct theprocedure in c) - e).
g) Calculate the ammoniacal nitrogen (A-N) in the analytical sample by the following formula:Ammoniacal nitrogen (A-N) (% (mass fraction)) in the analytical sample= (VS － VB) × C × f × (V1/V2) × (14.007/W2) × (100/1000)= (VS － VB) × C × f × (V1/V2) × (1.4007/W2)
VS：

Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration in (4.2) e)
VB： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration of the blank test in (4.2) f)
C： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide
solutionf：
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V1： Predetermined volume (mL) of the sample solution in (4.1.1) c) or (4.1.2) d)
V2： Transferred amount (mL) of the sample solution in (4.2) a)
W2： Mass (g) of the analytical sample
Note (4) The volume to be transferred should be up to 100 mL.
(5) The endpoint is reached when the color changes from green to light red. It is easy toobserve the change of color under fluorescent light.
Comment 7 The titration procedures in (2) a) Standardization and (4.2) e) - f) can be conductedby an automatic titrator. The setup of the titration program, the determination
parameter for the endpoint and vessels such as acceptors are according to thespecification and the operation method of the automatic titrator used.
Comment 8 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 10 % (mass fraction) 21 % (mass fraction) and 1 % (mass fraction) are 100.4 % - 101.0 % and 100.1 % asammoniacal nitrogen (A-N) respectively.
The results of the collaborative study (limited to reported values by Formaldehyde
65Testing Methods for Fertilizers (2018)

method) to determine a certified reference material fertilizer were analyzed by usinga three-level nesting analysis of variance. Table 1 shows the calculation results ofreproducibility, intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.03 % (mass fraction) for solid fertilizers, and 0.02 % (mass fraction) for fluid
fertilizers.
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
4)
5)
6)
7)
8)
9)
certified reference laboratory Average2)
sr
RSD rs I(T)RSD I(T)
sR
RSD R
1)
3)
3)
material fertilizer
(%)
p
(%)
(%)
FAMIC-A-10
10
10.66
0.07
0.7
FAMIC-A-13
9
10.36
0.06
0.5
1） The number of laboaratories used for analysis
conducting Formaldehyde method
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

3)

3)

(%)
(%)
(%)
(%)
0.09
0.8
0.16
1.5
0.08
0.8
0.21
2.0
6） Intermediate standard deviation
7） Intermediate relative standard deviation
8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.39- 42, Yokendo, Tokyo (1988)
2)
Kimie KATO, Masaki CHIDA and Erina WATANABE: Verification of Performance
Characteristics of Testing Method for Ammonia Nitrogen Content in Fertilizer byFormaldehyde Method, Research Report of Fertilizer Vol. 6, p. 139 - 147 (2013)(5)

Flow sheet for ammoniacal nitrogen: The flow sheet for ammoniacal nitrogen in fertilizers
is shown below.
5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask(ammonium salts)← About 400 mL of water
Shaking to mix
Rotary shaker (30 - 40 revolutions/minute), 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 The flow sheet for ammoniac nitrogen in fertilizers (Extraction procedure (4.1.1))

66

Testing Methods for Fertilizers (2018)

5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask(compound fertilizer)← About 300 mL of hydrochloric acid (1 + 20)
Shaking to mix
Rotary shaker (30 - 40 revolutions/minute), 30 minutes← Aluminum chloride solution (1 mol/L)← One or two drop(s) of methyl red← Potassium hydroxide solution (170 g/L)
(Until the solution changes to light yellow)← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 The flow sheet for ammoniac nitrogen in fertilizers (Extraction procedure (4.1.2))
Sample solution
Up to the equivalents of 50 mg as A-N,300-mL Erlenmeyer flask← Add water to make about 100 mL of solution← One or two drop(s) of methyl red← Hydrochloric acid (1+200) [light pink]← 10 mL of formaldehyde solution← One or two drop (s) of thymol blue solution (1 g/100 mL)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution
Titration(until the solution becomes blue)
Aliquot

Figure 2 The flow sheet for ammoniac nitrogen in fertilizers (Measurement solution)

67

Testing Methods for Fertilizers (2018)

4.1.3 Nitrate nitrogen
4.1.3.a Devarda’s alloy - distillation method(1) Summary
The testing method is applicable to the fertilizer containing nitrate. However, it is not applicable tofertilizers containing urea, nitrolime and organic matters that digest by heating and isolate ammonia.
This testing method is classified as Type E and its symbol is 4.1.3.a-2017 or N-N.a-1.
Add water to an analytical sample to dissolve ammoniacal nitrogen (A-N) and nitrate nitrogen(N-N), and further add devarda’s alloy and sodium hydroxide to subject it to steam distillation. In
this process, nitrate nitrogen (N-N) is reduced to ammonia. Collect isolated ammonia with 0.25
mol/L sulfuric acid and measure surplus sulfuric acid by (neutralization) titration using 0.1 mol/L 0.2 mol/L sodium hydroxide solution to obtain nitrogen content (N-N+A-N) in an analytical sample.
Or collect isolated ammonia with a boric acid solution and measure ammonium ion by(neutralization) titration using 0.25 mol/L sulfuric acid to obtain nitrogen content (N-N+A-N) in ananalytical sample. Subtract separately obtained the ammoniacal nitrogen (A-N) by 4.1.2 to calculate
nitrate nitrogen (N-N). This testing method corresponds to the devarda’s alloy method in the
Official Methods of Analysis of Fertilizers (1992).
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C1)
W1：
A：
V1：
V2：
V3：

Mass (g) of sulfamic acid sampled
Purity (% (mass fraction)) of sulfamic acid
Volume (mL) of sulfamic acid solution transferred
Constant volume (250 mL) of sulfamic acid solution
Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
c) 0.25 mol/L sulfuric acid (1) (2): Add about 14 mL of sulfuric acid to a beaker containing 100
mL of water in advance, stir well, and add water to make 1000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a 200-mL- 300-mL Erlenmeyer flask, add a few drops of methyl red−methylene blue mixture solution,
68Testing Methods for Fertilizers (2018)

and titrate with a 0.1 mol/L -0.2 mol/L sodium hydroxide solution until the color of thesolution becomes gray-green (4). Calculate the volume of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the following formula(1). Or, calculate the factor of 0.25 mol/L sulfuric acid by the following formula (2):Volume (B) of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL of 0.25
mol/L sulfuric acid= V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2 × 2)………. (2)V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5： Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 in water tomake 1000 mL.
e) Sodium hydroxide solution (200 g/L - 500 g/L) (1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
f) Devarda’s alloy: A reagent of nitrogen analysis grade specified in JIS K 8653 or a reagent ofequivalent quality.
g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, and add water to make 100mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
i) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in JIS K
8897 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methyl red−methylene blue mixture solution: To 2 volumes of methyl red solution (0.1g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
k) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green specified
in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red−bromocresol green mixture solution: To a methyl red solution (0.1 g/100 mL),
add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL -10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
Comment 2 0.25 mol/L sulfuric acid in (2) c) can be replaced with 0.25 mol/L sulfuric acidconforming to ISO/IEC 17025.
69

Testing Methods for Fertilizers (2018)(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Steam distillation apparatusb) Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to a steam
distillation apparatus.
(4) Test procedures(4.1) Sample solution preparation: Prepare a sample solution as shown below:a) Weigh 0.25 g - 1 g (5) (the equivalents of 20 mg - 100 mg as N) of an analytical sample to theorder of 1 mg, and put it in a 300-mL - 500-mL distillation flaskb) Add about 25 mL of water to make a sample solution.
Note (5) Conduct the procedure in Comment 3 when there is much nitrogen content in thefertilizers such as simple salt fertilizers.
Comment 3 In the case of nitrate fertilizer, etc. containing much nitrogen content, weigh 2 g - 5gof an analytical sample to the order of 1 mg, put it into a 250- mL volumetric flask,
dissolve it in water, and further add water up to the marked line. Put predeterminedvolume of suspension (the equivalents of 20 mg -100 mg as N) into a 300- mL 500-mL distillation flask.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used in measurement.
a) Transfer a predetermined amount (6) of 0.25 mol/L sulfuric acid to an acceptor (7), add a few
drops of methyl red−methylene blue mixture solution, and connect this acceptor to a steam
distillation apparatus. Or, transfer a predetermined amount (6) of boric acid solution (40 g/L) toan acceptor (7), add a few drops of methyl red−bromocresol green mixture solution, andconnect this acceptor to a steam distillation apparatus.
b) Add (10) no less than 3 mg of devarda’s alloy and adequate volume of sodium hydroxide (200g/L - 500 g/L) (8) (9) and connect this distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill at a
distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution in theacceptor with a small amount of water, and pool the washing with the distillate.
Note (6) 5 mL - 20 mL(7) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or a boric acid solution (40 g/L).
(8) Sudden reaction makes bubbles foam drastically and the bubbles overflow from a
distillation flask. Therefore, it is required to add an alkali solution gradually and mix
quietly.
(9) An amount sufficient to make the solution strong alkalinity.
(10) Add a small amount of silicone oil as necessary.
(4.3) Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2):a) Titrate the distillate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes gray-green (4).
70

Testing Methods for Fertilizers (2018)b) Calculate nitrogen content (N-N+A-N) in the analytical sample by the following formula:c) Subtract the ammoniac nitrogen (A-N) separately obtained in 4.1.2 from the obtained nitrogencontent (N-N+A-N) to calculate nitrate nitrogen (N-N) (11) (12).
Nitrogen content (N-N+A-N) (% (mass fraction)) in the analytical sample= (B × V6 － V7) × C1 × f1 × (14.007/W2) × (100/1000)= (B × V6 － V7) × C1 × f1 × (1.4007/W2)
B：

Volume of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1
mL of 0.25 mol/L sulfuric acidV6： Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2)a)
V7： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration in (4.3) a)
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution
f1： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
W2： Mass (g) of the analytical sample
Note (11) The nitrogen content (N-N+A-N) and ammoniacal nitrogen (A-N) use raw data
without rounding the numerical value.
(12) When no ammoniacal nitrogen (A-N) is contained, the nitrogen content (N-N+A-N)
calculated in (4.3) b) is regarded as nitrate nitrogen (N-N).
(4.3.2) When a boric acid solution (40 g/L) is used in (4.2):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomes lightred (13).
b) Calculate nitrogen content (N-N+A-N) in the analytical sample by the following formula:c) Subtract the ammoniac nitrogen (A-N) separately obtained in 4.1.2 from the obtained nitrogencontent (N-N+A-N) to calculate nitrate nitrogen (N-N) (11) (12).
Nitrogen content (N-N+A-N) (% (mass fraction)) in the analytical sample= V10 × C2 × 2 × f2 × (V11/V12) × (14.007/W3) × (100/1000)= V10 × C2 × f2 × (V11/V12) × (2.8014/W3)
V10： Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidf2： Factor of 0.25 mol/L sulfuric acid
W3： Mass (g) of the analytical sample
Note (13) The endpoint is reached when the color changes from green to light red.
Comment 4 The titration procedures in (2) a) Standardization, (2) c) Standardization and (4.3)
can be conducted by an automatic titrator. The setup of the titration program, thedetermination parameter for the endpoint and vessels such as acceptors are according
to the specification and the operation method of the automatic titrator used.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.49- 50, Yokendo, Tokyo (1988)
71

Testing Methods for Fertilizers (2018)(5) Flow sheet for nitrate nitrogen: The flow sheet for nitrate nitrogen in fertilizers is shown
below.
0.25 g - 1 g
Weigh the equivalents of 20 mg - 100 mg as N to the order of 1 mg
analytical sample
into a 300-mL distillation flask.
← 25 mL of water← No less than 3 g of devarda's alloy← Sodium hydroxide solution (200 g/L - 500 g/L)
← A small amount of silicone oil as necessary
Receiver: 200-mL - 300-mL Erlenmeyer flask or beaker
Steam distillation
A predetermined amount of 0.25 mol/L sulfuric acid and a few drops ofapparatus
methyl red-methylene blue mixture solution, or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL - 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure The flow sheet for nitrate nitrogen in fertilizers

72

Testing Methods for Fertilizers (2018)

4.1.3.b Reduced iron- distillation method(1) Summary
The testing method is applicable to the fertilizer containing nitrate. However, it is not applicable tofertilizers containing urea, nitrolime and organic matters that digest by heating and isolate ammonia.
This testing method is classified as Type E and its symbol is 4.1.3.b-2017 or N-N.b-1.
Add water to an analytical sample to dissolve ammoniacal nitrogen (A-N) and nitrate nitrogen(N-N), and add reduced iron and a sulfuric acid solution to boil lightly. In this process, nitrate
nitrogen (N-N) is reduced to ammonia. And further add a sodium hydroxide solution to distillate.
Collect isolated ammonia with 0.25 mol/L sulfuric acid and measure surplus sulfuric acid by(neutralization) titration using 0.1 mol/L - 0.2 mol/L sodium hydroxide solution to obtain nitrogencontent (N-N+A-N) in an analytical sample. Or collect isolated ammonia with a boric acid solution
and measure ammonium ion by (neutralization) titration using 0.25 mol/L sulfuric acid to obtainnitrogen content (N-N+A-N) in an analytical sample. Subtract separately obtained the ammoniacal
nitrogen (A-N) by 4.1.2 to calculate nitrate nitrogen (N-N). This testing method corresponds to the
reduced iron method in the Official Methods of Analysis of Fertilizers (1992).
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C1)
W1：
A：
V1：
V2：
V3：

Mass (g) of sulfamic acid sampled
Purity (% (mass fraction)) of sulfamic acid
Volume (mL) of sulfamic acid solution transferred
Constant volume (250 mL) of sulfamic acid solution
Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
c) 0.25 mol/L sulfuric acid (1) (2): Add about 14 mL of sulfuric acid to a beaker containing 100
mL of water in advance, stir well, and add water to make 1000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a 200-mL- 300-mL Erlenmeyer flask, add a few drops of methyl red−methylene blue mixture solution,and titrate with a 0.1 mol/L -0.2 mol/L sodium hydroxide solution until the color of the73Testing Methods for Fertilizers (2018)

solution becomes gray-green (4). Calculate the volume of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the following formula(1). Or, calculate the factor of 0.25 mol/L sulfuric acid by the following formula (2):Volume (B) of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL of 0.25
mol/L sulfuric acid=V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2 × 2)………. (2)V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5： Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 in water tomake 1000 mL.
e) Sodium hydroxide solution (200 g/L - 500 g/L) (1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
f) Reduced iron: Nitrogen content is no more than 0.005 % (mass fraction)g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, and add water to make 100mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
i) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in JIS K
8897 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methyl red−methylene blue mixture solution: To 2 volumes of methyl red solution (0.1g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
k) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green specified
in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red−bromocresol green mixture solution: To a methyl red solution (0.1 g/100mL), add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL -10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
Comment 2 0.25 mol/L sulfuric acid in (2) c) can be replaced with 0.25 mol/L sulfuric acidconforming to ISO/IEC 17025.
(3)

Instruments: Instruments are as shown below:
74Testing Methods for Fertilizers (2018)a) Steam distillation apparatusb) Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to a steam
distillation apparatus.
(4) Test procedures(4.1) Preparation of sample solution: Conduct preparation of a sample solution as shown below.
a) Weigh 0.5 g - 1 g (5) (the equivalents of 20 mg - 100 mg as N) of an analytical sample to theorder of 1 mg, and put it in a 300-mL - 500-mL distillation flaskb) Add about 30 mL of water and mix well.
c) As soon as adding 5 g of reduced iron and 10 mL of sulfuric acid (1+1), insert a long stem
funnel to a distillation flask and shake to mix gently while cooling the outside of the container
under flowing water (6).
d) After leaving at rest for about 5 minutes (7), heat gradually by low temperature and boil in a
low flame for about 15 minutes, and then stand to cool to make a sample solution.
Note (5) Conduct the procedure in Comment 3 when there is much nitrogen content in thefertilizers such as simple salt fertilizers.
(6) Sudden reaction generates heat, and unreacted nitric acid vaporizes or digests to makenitrogen oxide etc. through which process losses occur easily. Careful and efficient
operation should be taken(7) Until a sudden reaction is settled.
Comment 3 In the case of nitrate fertilizer, etc. containing much nitrogen content, weigh 2 g - 5gof an analytical sample to the order of 1 mg, put it into a 250- mL volumetric flask,
dissolve it in water, and further add water up to the marked line. Put predeterminedvolume of suspension (the equivalents of 20 mg - 100 mg as N) into a 300-mL 500-mL distillation flask.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used in measurement.
a) Transfer a predetermined amount (8) of 0.25 mol/L sulfuric acid to an acceptor (9), add a few
drops of methyl red−methylene blue mixture solution, and connect this acceptor to a steam
distillation apparatus. Or, transfer a predetermined amount (8) of boric acid solution (40 g/L) toan acceptor (9), add a few drops of methyl red−bromocresol green mixture solution, andconnect this acceptor to a steam distillation apparatus.
b) Add adequate volume of sodium hydroxide (200 g/L - 500 g/L) (10) and connect this
distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill at a
distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution in theacceptor with a small amount of water, and pool the washing with the distillate.
Note (8) 5 mL - 20 mL(9) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or a boric acid solution (40 g/L).
(10) An amount sufficient to make the solution strong alkalinity.
(4.3) Measurement: Conduct measurement as shown below.
75Testing Methods for Fertilizers (2018)(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2) a),a) Titrate the distillate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes gray-green (4).
b) Calculate nitrogen content (N-N+A-N) in the analytical sample by the following formula:c) Subtract the ammoniac nitrogen (A-N) separately obtained in 4.1.2 from the obtained nitrogencontent (N-N+A-N) to calculate nitrate nitrogen (N-N) (11) (12).
Nitrogen content (N-N+A-N) (% (mass fraction)) in the analytical sample= (B × V6 － V7) × C1 × f1 × (14.007/W2) × (100/1000)= (B × V6 － V7) × C1 × f1 × (1.4007/W2)
B： Volume of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acidV6： Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2) a)
V7： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
W2： Mass (g) of the analytical sample
Note (11) The nitrogen content (N-N+A-N) and ammoniacal nitrogen (A-N) use raw data
without rounding the numerical value.
(12) When no ammoniacal nitrogen (A-N) is contained, the nitrogen content (N-N+A-N)
calculated in (4.3) b) is regarded as nitrate nitrogen (N-N).
(4.3.2) When a boric acid solution (40 g/L) is used in (4.2) a):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomes lightred (13).
b) Calculate nitrogen content (N-N+A-N) in the analytical sample by the following formula:c) Subtract the ammoniac nitrogen (A-N) separately obtained in 4.1.2 from the obtained nitrogencontent (N-N+A-N) to calculate nitrate nitrogen (N-N) (11) (12).
Nitrogen content (N-N+A-N) (% (mass fraction)) in the analytical sample=V10 × C2 × 2 × f2 × (14.007/W3) × (100/1000)=V10 × C2 × f2 × (2.8014/W3)
V10： Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidf2： Factor of 0.25 mol/L sulfuric acid
W3： Mass (g) of the analytical sample
Note (13) The endpoint is reached when the color changes from green to light red.
Comment 4 The titration procedures in (2) a) Standardization, (2) c) Standardization and (4.3)
can be conducted by an automatic titrator. The setup of the titration program, thedetermination parameter for the endpoint and vessels such as acceptors are according
to the specification and the operation method of the automatic titrator used.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers
76Testing Methods for Fertilizers (2018)(Details), p.48- 49, Yokendo, Tokyo (1988)(5)

Flow sheet for nitrate nitrogen: The flow sheet for nitrate nitrogen in fertilizers is shown
below.
0.5 g – 1 g
Weigh the equivalents of 20 mg - 100 mg of N to the order of 1 mg,
analytical sample
and put it into a 300-mL distillation flask.
30
mL
of water←About←5 g of reduced iron←About 10 mL of sulfuric acid (1+1)
Insert a long stem funnel immediately to a distillation flask and shake
Mixtureto mix gently while cooling the outside of the container under flowing water
Leaving at rest
Heating

For about 5 minutes (until a strong reaction is settled)
Boil in a low flame for about 15 minutes

Standing to cool
←Sodium hydroxide (200 g/L - 500 g/L)
Receiver: 200-mL - 300-mL Erlenmeyer flask or beakerA predetermined amount of 0.25 mol/L sulfuric acid
Steam distillation
and a few drops of methyl red-methylene blue mixture solution,
apparatus
or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL - 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure The flow sheet for nitrate nitrogen in fertilizers

77Testing Methods for Fertilizers (2018)

4.1.3.c Phenol sulfuric acid method(1) Summary
The testing method is applicable to the fertilizer containing nitrate. It is also applicable to thefertilizers containing chemical compounds such as urea, nitrolime and organic matters that digest by
heating and isolate ammonia. This testing method is classified as Type D and its symbol is
4.1.3.c-2017 or N-N.c-1.
Add a copper sulfate - silver sulfate solution, calcium hydroxide and basic magnesium carbonate toan analytical sample, extract nitrate nitrogen (N-N) as well as removing chloride and organic
matters, and measure the absorbance of nitro phenol ammonium sulfate formed by the reaction withphenol sulfuric acid and an ammonia solution to calculate nitrate nitrogen (N-N) in an analyticalsample. In addition, the performance of this testing method is shown in Comment 3.
(2) Reagent: Reagents are as shown below.
a) Nitrate standard solution (N-N 5 mg/mL) (1): Heat potassium nitrate (no less than 99.9 (%)(mass fraction) in purity) at 110 ºC for no less than 1 hour, and after standing to cool in a
desiccator, transfer 36.09 g to a weighing dish. Dissolve in a small amount of water, transfer
to a 1000-mL volumetric flask, and add water up to the marked line.
b) Nitrate standard solution (N-N 0.01 mg/mL): Dilute predetermined volume of nitrate
standard solution (N-N 5 mg/mL) with water to prepare a nitrate standard solution (N-N 0.01
mg/mL).
c) Copper sulfuric - silver sulfuric solution (1): Dissolve 5 g of copper (II) sulfate pentahydrate
specified JIS K 8983 in 900 mL of water, and dissolve while adding 4 g of silver sulfate
specified in JIS K 8965 to make 1000 mL (2).
d) Phenol sulfuric acid: Dissolve 15 g of phenol specified in JIS K 8798 in 100 mL of sulfuric
acid specified in JIS K 8965, heat in a water bath at 80 ºC - 100 ºC for two hours and then let
it stand to cool (2).
e) Ammonia solution: A JIS Guaranteed (NH3 28 % (mass fraction)) reagent specified in JIS
K 8085 or a reagent of equivalent quality.
f) Calcium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8575 or a reagent ofequivalent quality.
g) Basic magnesium carbonate: Basic magnesium carbonate that contains no nitrate nitrogen.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in an amber bottle.
Comment 1 Instead of the nitrate standard solution in (2), a nitrate standard solution for acalibration curve can be prepared using nitrate nitrogen (NO3-N 0.1 mg/mL or 1
mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 250-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Spectrophotometer: A spectrophotometer specified in JIS K 0115c) Water bath: Water bath that can be adjusted to no less than 80 ºC(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add about 200 mL of copper sulfuric - silver sulfuric solution and shake to mix at 30 - 40
78

Testing Methods for Fertilizers (2018)

revolutions /min for about 20 minutes.
c) Add about 1 g of calcium hydroxide and about 1 mg of basic magnesium carbonate and shaketo mix at 30 - 40 revolutions /min for about 10 minutes.
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make a sample solution (3).
Note (3) As soon as the sample solution is prepared, conduct the procedure in (4.2) a).
Comment 2 If the filtrate of (4.1) e) is colored, add no more than 0.5 g of active carbon andfilter with Type 3 filter paper to make a sample solution.
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount of sample solution (the equivalents of 0.01 mg - 0.1 mg asN-N) into a small evaporating dish (4).
b) Evaporate water until dry on a water bath at no less than 80 ºC.
c) After standing to cool, swiftly add 2 mL of phenol sulfuric acid (5) and then rotate theevaporating dish so that the whole residue comes in contact with the acid.
d) After leaving at rest for about 10 minutes, add 20 mL of water (6).
e) After standing to cool, transfer it with water to a 100- mL volumetric flask.
f) Add an ammonia solution (1+2), until the color of a solution changes to light yellow, to allow
it to be weak alkalinity, and further add 3 mL of ammonia solution (1+3) (7).
g) After cooling is complete, add water up to the marked line and leave at rest for 30 minutes.
Note (4) A round-bottom glass or porcelain evaporating dish is preferable.
(5) Add at the center of a small evaporating dish with Komagome pipet.
(6) If residue does not dissolve easily, grind it with a glass rod.
(7) As no color appears from a blank test solution for a calibration curve preparation, add
almost the same volume of ammonia solution (1+2) as the nitrate standard solution.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 410 nmb) Calibration curve preparation
1) Transfer 1 mL - 10 mL of nitrate standard solution (N-N 0.01 mg/mL) to small evaporating
dishes (4) step-by-step.
2) Conduct the same procedure as (4.2) b) - g) to make the nitrate standard solution for thecalibration curve preparation.
3) Transfer 40 mL of water to a 100-mL volumetric flask, and shake to mix while gently adding
2 mL of phenol sulfuric acid. Let it stand to cool and conduct the same procedure as (4.2) f) g) to make the blank test solution for calibration curve.
4) Measure absorbance at wavelength 410 nm of the nitrate standard solution for the calibration
curve preparation using the blank test solution for the calibration curve preparation as thecontrol.
5) Prepare the calibration curve of concentration of the nitrate nitrogen and absorbance of thenitrate standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) g), measure absorbance by the same procedure as b) 4).
79

Testing Methods for Fertilizers (2018)2) Obtain the nitrate nitrogen (N-N) content from the calibration curve and calculate nitrate
nitrogen (N-N) in the analytical sample.
Comment 3 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 16 % (mass fraction) and
1 % (mass fraction) - 3% (mass fraction) are 103.4 % and 101.1 % - 100.9 % asnitrate nitrogen (N-N) respectively.
Additionally, the minimum limit of quantification of this testing method is about
0.01 % (mass fraction) for solid fertilizers, and 0.002 % (mass fraction) for fluid
fertilizers.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.52- 55, Yokendo, Tokyo (1988)
2)
Kimie KATO, Sakiko TAKAHASHI and Yuji SHIRAI: Validation of a Color metric
Method for Determination of Nitrogen, Phosphorus and Boron: Evaluation of
Calibration curve, Research Report of Fertilizer Vol. 2, p. 137 - 144 (2009)3)
Kimie KATO, Masaki CHIDA and Erina WATANABE: Verification of Performance
Characteristics of Testing Method for Nitrate Nitrogen Content in Fertilizer by Phenol
Sulfuric Acid Method, Research Report of Fertilizer Vol. 6, p. 148 - 155 (2013)(5)

Flow sheet for nitrate nitrogen: The flow sheet for nitrate nitrogen in fertilizers is shown
below.
1 mg
Weigh to the order of 1 mg to a 250-mL volumetric flask
analytical sample← About 200 mL of copper sulfate - silver sulfate solution
Shaking to mix
Rotary shaker (30 - 40 revolutions/min) for 20 minutes← About 1 g of calcium hydroxide← About 1 g of basic magnesium carbonate
Shaking to mix
Rotary shaker (30 - 40 revolutions/min) for 10 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1 The flow sheet for nitrate nitrogen in fertilizers (Extraction procedure)

80

Testing Methods for Fertilizers (2018)

Sample solution

Aliquot(predeterminedvolume)Evaporation to dryness

Small evaporating dishNo less than 80 ºC Water bath← 2 mL of phenol sulfuric acid
About 10 minutes after making phenol sulfuric acid
Leaving at rest
contact with residue
20
mL
of
water
←
Standing to coolTransfer
100-mL volumetric flask← Ammonia solution (1+2) ( until the color of a solution changes to light yellow)← Ammonia solution (1+3) 3 mL
← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (410 mm)

Figure Flow sheet for nitrate nitrogen in fertilizers (Coloring and measurement procedure)81

Testing Methods for Fertilizers (2018)

4.2 Phosphoric acid
4.2.1 Total phosphoric acid
4.2.1.a Ammonium vanadomolybdate absorptiometric analysis(1) Summary
This testing method is applicable to fertilizers containing organic matters. This testing method isclassified as Type C and its symbol is 4.2.1.a-2017 or T-P.a-1.
Add sulfuric acid, potassium sulfate and copper (II) sulfate pentahydrate to an analytical sample.
Pretreat by Kjeldahl method or incineration-hydrochloric acid boiling to convert total phosphorus tophosphate ion, and measure the absorbance of phosphovanadomolybdate salt formed by the reaction
with ammonium vanadate (V), hexaammonium heptamolybdate and nitric acid, to obtain total
phosphoric acid (T-P2O5) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 6.
(2) Reagent: Reagents are as shown below.
a) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
b) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
c) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
d) Ammonia solution: A JIS Guaranteed (NH3 28 % (mass fraction)) reagent specified in JIS K
8085 or a reagent of equivalent quality.
e) Catalyst (1): Mix potassium sulfate specified in JIS K 8962 and copper (II) sulfate
pentahydrate (2) specified in JIS K 8983 in the ratio of 9 to 1.
f) Coloring reagent solution (3) (4): Dissolve 1.12 g of ammonium vanadate (V)(5) specified in
JIS K 8747 in water, add 250 mL of nitric acid, then add 27 g of hexaammonium
heptamolybdate tetrahydrate (6) specified in JIS K 8905 dissolved in water, and further add
water to make 1000 mL (7).
g) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in JIS K
8799 in 100 mL of ethanol (95) specified in JIS K 8102.
h) Phosphoric acid standard solution (P2O5 10 mg/mL) (3): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to cool in a
desiccator, and weigh 19.17 g to a weighing dish. Dissolve in a small amount of water,transfer to a 1000-mL volumetric flask, add 2 mL-3 mL of nitric acid, and add water up to themarked line.
i) Phosphoric acid standard solution (P2O5 0.5 mg/mL) (3): Transfer 50 mL of phosphoric acidstandard solution (P2O5 10 mg/mL) to a 1000-mL volumetric flask, add 2 mL - 3 mL of nitricacid, and add water up to the marked line.
Note (1) A tablet is commercially available.
(2) Crush into powder as appropriate.
(3) This is an example of preparation; prepare an amount as appropriate.
(4) This corresponds to reagent “a” reagent solution in the Official Methods of Analysis of
Fertilizers (1992).
(5) This corresponds to ammonium metavanadate in the Official Methods of Analysis of
Fertilizers (1992).
(6) This corresponds to ammonium molybdate in the Official Methods of Analysis of
Fertilizers (1992).
(7) Store in an amber bottle.
82

Testing Methods for Fertilizers (2018)

Comment 1 Instead of the phosphoric acid standard solution in (2), a phosphoric acid standard
solution for a calibration curve can be prepared using a phosphoric acid standard
solution (P 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate the total phosphoric acid (T-P2O5) in an analytical sample bymultiplying the concentration (P) of a phosphoric acid standard solution for acalibration curve or a measured value obtained in (4.3) by a conversion factor(2.2914).
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Spectrophotometer: A spectrophotometer specified in JIS K 0115b) Electric furnace: An electric furnace that can be adjusted to 550 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
d) Digestion flask: Kjeldahl flask(4) Test procedures(4.1) Sample solution preparation: Prepare a sample solution as shown below:(4.1.1) Kjeldahl methoda) Weigh 0.5 g - 5 g of an analytical sample to the order of 1 mg, and put it in a 300-mL
digestion flask.
b) Add 5 g - 10 g of catalyst, and further add 20 mL - 40 mL of sulfuric acid, shake to mix andheat gently.
c) After bubbles cease to form, heat until white smoke of sulfuric acid evolves.
d) Ignite until organic matters are completely digested (8).
e) After standing to cool, add a small amount of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water, and further mix by shaking.
f) After cooling is complete, add water up to the marked lineg) Filter with Type 3 filter paper to make a sample solution.
Note (8) When the solution has finished changing color, heat further for no less than 2 hours.
Comment 2 The procedure in (4.1.1) is the same as the procedure in (4.1) in 4.2.1.b. Also, theprocedures in (4.1.1) a) - f) are the same as in (4.1) in 4.1.1.a.
(4.1.2) Incineration-hydrochloric acid boilinga) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 200-mL - 300-mL tallbeaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (9).
c) Ignite at 550 ºC ± 5 ºC for no less than 4 hours to incinerate (9).
d) After standing to cool, moisten the residue with a small amount of water, gradually add about
10 mL of hydrochloric acid, and further add water to make 20 mL.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to boil forabout 5 minutes.
f) After cooling is complete, transfer to a 250-mL - 500-mL volumetric flask with water.
g) Add water up to the marked line.
h) Filter with Type 3 filter paper to make a sample solution.
Note (9) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about83Testing Methods for Fertilizers (2018)

1 hour and further raise to 550 °C in 1 to 2 hours.
Comment 3 The procedures in (4.1.2) are the same as the procedures in (4.1.1) in 4.3.1.a, (4.1.1)
in 4.5.1.a, (4.1.1) in 4.6.1.a and (4.1) in 8.4.a.
(4.1.3) Incineration-aqua regia digestiona) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 200-mL - 300-mL tallbeaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (10).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (10).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (11), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up (12).
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (13) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL volumetric flask with water, add water up to themarked line, and filter with Type 3 filter paper to make a sample solution.
Note (10) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating forabout 1 hour and further raise to 450 °C in 1 to 2 hours.
(11) The watch glass can be removed.
(12) When drying and hardening it, phosphoric acid may not dissolve completely and theconcentration may become a low value in the procedure in g).
(13) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 4 The procedures in (4.1.3) are the same as in (4.1.2) in 4.3.1.a, (4.1.2) in 4.5.1.a,(4.1.2) in 4.6.1.a, (4.1) in 4.9.1.a, (4.1) in 4.9.1.b, (4.1) in 4.10.1.a and (4.1) in
4.10.1.b. And also, the same as (4.1) a) - h) in 5.3.a, (4.1) a) - h) in 5.3.b, (4.1) a) h) in 5.4.a, (4.1) a) - h) in 5.4.b, (4.1) a) - h) in 5.5.a, (4.1) a) - h) in 5.5.d, (4.1) a) h) in 5.6.a and (4.1) a) - h) in 5.6.b.
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 6 mg as
P2O5) to a 100-mL volumetric flask.
b) Add 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL), and neutralize by adding
ammonia solution (1+1) until the color of the solution becomes light red-purple (14).
c) Add nitric acid (1+10) until the light red-purple color of the solution disappears to make itslightly acidic, and add a proper amount of water (15).
d) Add 20 mL of coloring reagent solution, and further add water up to the marked line, and thenleave at rest for about 30 minutes.
Note (14) It is not necessary to add a phenolphthalein solution (1 g/100 mL) when determination
can be done by the color change of copper ion (light blue → blue-purple).
(15) Without the addition of water, precipitate may be produced when a coloring reagent
84

Testing Methods for Fertilizers (2018)

solution is added.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 420 nmb) Calibration curve preparation
1) Transfer 1 mL - 12 mL of phosphoric acid standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add a proper amount of water (15), and conduct the same procedure as (4.2) d) to make theP2O5 0.5 mg/100 mL - 6 mg/100 mL phosphoric acid standard solutions for the calibration
curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blanktest solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 420 nm of the phosphoric acid standard solutions forthe calibration curve preparation using the blank test solution for the calibration curve
preparation as the control (16).
5) Prepare the calibration curve of the phosphoric acid concentration and absorbance of thephosphoric acid standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) d), measure absorbance by the same procedure as b) 4) (16).
2) Obtain the phosphoric acid (P2O5) content from the calibration curve, and calculate total
phosphoric acid (T-P2O5) in the analytical sample.
Note (16) Measure within 6 hours after adding the coloring reagent solution in the procedure in(4.2) d).
Comment 5 After the procedure in (4.2) a), it is also possible to measure soluble phosphoric acidat the same time by adding 4 mL of nitrate acid (1+1) and 2 mL of Petermans citratesolution and by conducting the procedures from (4.2) d) to (4.3) in 4.2.2.a (using b
reagent solution in the Official Methods of Analysis of Fertilizers (1992)).
Further after the procedure in (4.2) a), it is also possible to measure citrate soluble
phosphoric acid at the same time by adding 4 mL of nitrate acid (1+1) and 17 mL ofcitrate solution and by conducting the procedures from (4.2) d) to (4.3) in 4.2.3.a(using b reagent solution in the Official Methods of Analysis of Fertilizers (1992)).
Comment 6 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 10 % - 20 % (massfraction) and 1 % (mass fraction) - 5 % (mass fraction) are 99.4 % - 100.2 % and101.0 % - 105.7 % as total phosphoric acid (T-P2O5) respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.04 % (mass fraction) for solid fertilizers, and 0.01 % (mass fraction) for fluid
fertilizers.

85Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
4)
5)
6)
7)
8)
9)
certified reference laboratory Average2)
sr
RSD rs I(T)RSD I(T)
sR
RSD R
1)
3)
3)
3)
3)
material fertilizer
(%)
(%)
(%)
p
(%)
(%)
(%)
(%)
FAMIC-C-12
9
8.62
0.03
0.4
0.04
0.4
0.08
0.9
1） The number of laboaratories used for analysis
6） Intermediate standard deviation
conducting Ammonium vanadomolybdate absorptiometric analysis
2） Average （the number of laboratory（p ）×test days（2）7） Intermediate relative standard deviation
×the number of replicate testing（3））3） Mass fraction
8） Reproducibility standard deviation
4） Repeatability standard deviation
9） Reproducibility relative standard deviation
5） Repeatability relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.108- 114, Yokendo, Tokyo (1988)
2)
Kimie KATO, Sakiko TAKAHASHI and Yuji SHIRAI: Validation of a Color metric
Method for Determination of Nitrogen, Phosphorus and Boron: Evaluation of
Calibration curve, Research Report of Fertilizer Vol. 2, p. 137 - 144 (2009)3)
Kimie KATO, Masayuki YOSHIMOTO and Yuji SHIRAI: Systematization of
Determination Methods of Major Components in Sludge Fertilizer, Compost andOrganic Fertilizer, Research Report of Fertilizer, Vol.3 p. 107 - 116 (2010)4)
Yoshiyuki SUNAGA, Yasushi SUGIMURA, Ichiro YOSHIDA and Hidenori KONISHI:
Verification of Performance Characteristics of Testing Methods for Phosphorus Content
in Fertilizer by Ammonium Vanadomolybdate Absorption Photometry, Research Reportof Fertilizer Vol. 5, p. 167 - 179 (2012)86

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total phosphoric acid: The flow sheet for total phosphoric acid in fertilizers isshown below:
0.5 g - 5 g
Weigh to the order of 1 mg into a 300-mL Kjeldahl flask.
analytical sample← About 10 g catalyst← 20 mL - 40 mL sulfuric acid
Heating
GentlyHeating

After bubbles cease to form, ignite
until organic matters are completely digested.
Standing to coolRoom temperature← A small amount of waterTransfer
250-mL - 500-mL volumetric flask, water
Cooling
Room temperature←Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for total phosphate in fertilizers(Kjeldahl method procedure (4.1.1) )87

Testing Methods for Fertilizers (2018)5g
analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL - 300-mL tall beaker.
Heat gently
Ignite at 550 ºC ± 5 ºC for no less than 4 hours

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of hydrochloric acid
← Water (up to about 20 mL)
Heating
Cover with a watch glass, and boil for 5 minutes.
Cooling
Transfer
250-mL - 500-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for total phosphate in fertilizers(Incineration-hydrochloric acid boiling procedure (4.1.2))

88

Testing Methods for Fertilizers (2018)5g
analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC for 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass to digest
Heating

Slightly move the watch glass and remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass to dissolve
Standing to coolTransfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-3 Flow sheet for total phosphate in fertilizers(Incineration-aqua regia digestion orocedure (4.1.3))

89

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot(predeterminedamount)100-mL volumetric flask← 1-2 drop(s) of phenolphthalein solution (1 g/100 mL)← Ammonia solution (1+1) [neutralization]← Nitric acid (1+10) [slightly acidic]← A proper amount of water← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest

About 30 minutes

Measurement

Spectrophotometer (420 nm)

Figure 2 Flow sheet for total phosphate in fertilizers(Coloring and measurement procedure)90

Testing Methods for Fertilizers (2018)

4.2.1.b Quinoline gravimetric analysis(1) Summary
This testing method is applicable to fertilizers containing organic matters. It is suitable for thefertilizers with relatively a high content of phosphoric acid. This testing method is classified as
Type E and its symbol is 4.2.1.b-2017 or T-P.b-1.
Add sulfuric acid, potassium sulfate and copper (II) sulfate pentahydrate to an analytical sample.
Pretreat by the Kjeldahl method to convert the total phosphoric acid (T-P2O5) to phosphate ion, andmeasure the mass of quinolinium phosphomolybdate formed by the reaction with quinoline,
molybdic acid and nitric acid to obtain the total phosphoric acid (T-P2O5) in an analytical sample.
(2) Reagent: Reagents are as shown below.
a) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
b) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
c) Sodium molybdate solution (1): Dissolve 70 g of sodium molybdate dihydrate in 150 mL ofwater.
d) Quinoline solution (1): Add 5 mL of quinolone specified in JIS K 8279 to the mixture solutionof 35 mL of nitric acid and 100 mL of water.
e) Quimosiac solution (1): Add 60 g of citric acid monohydrate specified in JIS K 8283 to the
mixture solution of 85 mL nitric acid and 150 mL of water to dissolve. Add gradually total
volume of the sodium molybdate solution to mix. Add gradually the total volume of thequinoline solution while mixing the solution. After leaving at rest overnight, filter the total
volume with Type 3 filter paper. Add 280 mL of acetone specified in JIS K 8034, and further
add water to make 1000 mL.
f) Catalyst (2): Mix potassium sulfate specified in JIS K 8962 and copper (II) sulfate
pentahydrate (3) specified in JIS K 8983 in the ratio of 9 to 1.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A tablet is commercially available.
(3) Crush into powder as appropriate.
(3)
a)
b)
c)

Apparatus and instruments: Apparatus and instruments are shown below.
Water bath: Water bath that can be adjusted to 60 ºC - 65 ºC.
Drying apparatus: A drying apparatus that can be adjusted to 220 ºC ± 5 ºC.
Crucible type glass filter: A crucible type glass filter 1G4 specified in JIS R 3503. Let it
stand to cool in a desiccator after heating at 220 ºC ± 5 ºC in advance and measure the mass tothe order of 1 mg.
d) Digestion flask: Kjeldahl flask(4) Test procedures(4.1) Kjeldahl method: Conduct digestion as shown below.
a) Weigh 0.5 g - 5 g of an analytical sample to the order of 1 mg, and put it in a 300-mL
digestion flask.
b) Add 5 g - 10 g of catalyst, and further add 20 mL - 40 mL of sulfuric acid, shake to mix andheat gently.
c) After bubbles cease to form, heat until white smoke of sulfuric acid evolves.
d) Ignite until organic matters are completely digested (4).
e) After standing to cool, add a small amount of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water.
91

Testing Methods for Fertilizers (2018)f) After cooling is complete, add water up to the marked lineg) Filter with Type 3 filter paper to make a sample solution.
Note (4) When the solution has finished changing color, heat further for no less than 2 hours.
Comment 1 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.1.a. In
addition, the sample solution prepared in (4.1.2) in 4.2.1.a and (4.1.3) in 4.2.1.a canalso be used.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 10 mg - 30 mg as P2O5 and no more than
5 mL as sulfuric acid) of sample solution to a 300-mL tall beaker.
b) Add 5 mL of nitric acid and add water to make about 80 mL.
c) Cover with a watch glass. After boiling for about 3 minutes, wash the watch glass and theinside the tall beaker with water and add water to make about 100 mL.
d) Immediately, add 50mL of quimosiac solution, heat for about 15 minutes while sometimes
mixing in a water bath at 60 ºC - 65 ºC to produce the precipitate of quinonium
phosphomolybdate.
e) After standing to cool down to room temperature while sometimes mixing, filter under
reduced pressure with a crucible type glass filter, wash the tall beaker 3 times with water andtransfer the whole precipitate into a crucible type glass filter, and further wash 7 - 8 times withwater.
f) Transfer the precipitate together with the crucible type glass filter into a drying apparatus andheat at 220 ºC ± 5 ºC for about 30 minutes.
g) As soon as heating is complete, move it into a desiccator and let it stand to cool.
h) After standing to cool, remove the crucible type glass filter from the desiccator and measure
the mass to the order of 1 mg.
i) Calculate total phosphoric acid (T-P2O5) in the analytical sample by the following formula.
Total phosphoric acid (T-P2O5) (% (mass fraction)) in an analytical sample=A × 0.03207 × (V1/V2) × (1/W) × 100
A：
W：
V1：
V2：

Mass (g) of the precipitate in h)
Mass (g) of the analytical sample
Constant volume (mL) of sample solution
Volume (mL) of the sample solution transferred in a)

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.98- 106, Yokendo, Tokyo (1988)

92

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total phosphoric acid: The flow sheet for total phosphoric acid in fertilizers
is shown below:

0.5 g- 5 g
Weigh to the order of 1 mg into a 300-mL digestion flask.
analytical sample← 5 g - 10 g catalyst← 20 mL - 40 mL sulfuric acid
Heating
GentlyHeating

After bubbles cease to form,
ignite until organic matters are completely digested

Standing to coolRoom temperature← Small mount of waterTransfer
250-mL - 500-mL volumetric flask, water
Cooling← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1 Flow sheet for total phosphate in fertilizers(Kjeldahl method procedure)

93

Testing Methods for Fertilizers (2018)

Sample solution

Aliquot(predetermined
300-mL tall beakervolume)← 5 mL nitrate acid
← Water (to reach about 80 mL)
Cover with a watch glass for 3 minutes
Boiling
Wash a watch glass and inside the tall beaker with water← Water (to be about 100 mL)← Quimosiac solution 50 mL
Produce precipitate
60 ºC - 65 ºC, for 15 minutes, sometimes mixing
Standing to coolRoom temperature

Filtration
under reducedpressure

Crucible type glass filtering apparatus 1G4, 3 times with water

Washing

Wash 7 - 8 times with water

Drying220 ºC ± 5 ºC for about 30 minutes

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure 2 The flow sheet for total phosphate in fertilizers (Measurement procedure)

94

Testing Methods for Fertilizers (2018)

4.2.2 Soluble phosphoric acid
4.2.2.a Ammonium vanadomolybdate absorptiometric analysis(1) Summary
This testing method is applicable to fertilizers that do not contain matter not colored by hydrolysis
with nitrate acids such as phosphonic acid. This testing method is classified as Type C and its
symbol is 4.2.2.a-2017 or S-P.a-1.
Extract by adding water to an analytical sample, then extract by adding an ammonium citric acidsolution, and combine respective pre-determined amounts of extract (equivalent volume). Heat after
adding nitric acid (1+1), hydrolyze nonorthophosphoric acid to orthophosphate ion and measure theabsorbance of phosphovanadomolybdate salt formed by the reaction with ammonium vanadate (V),hexaammonium heptamolybdate and nitric acid to obtain ammonia alkaline ammonium
citrate-soluble phosphoric acid (soluble phosphoric acid (S-P2O5)) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 6.
(2) Reagent: Reagents are as shown below.
a) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
b) Ammonia solution: A JIS Guaranteed (NH3 28 % (mass fraction)) reagent specified in JIS
K 8085 or a reagent of equivalent quality.
c) Petermans citrate solution: Add 173 g of citric acid monohydrate specified in JIS K 8283
in water to dissolve and add gradually an ammonia solution equivalent to 42 g of nitrogen
while cooling. After cooling is complete, add water to make 1000 mL. Additionally, checkthat the specific gravity of the solution is 1.082 - 1.083 (15 ºC) and the nitrogen content per 1
mL is 42 mg.
d) Coloring reagent solution (1) (2): Dissolve 1.12 g of ammonium vanadate (V)(3) specified in
JIS K 8747 in water, add 150 mL of nitric acid, then add 50 g of hexaammonium
heptamolybdate tetrahydrate (4) specified in JIS K 8905 dissolved in water, and further add
water to make 1000 mL (5).
e) Phosphoric acid standard solution (P2O5 10 mg/mL) (1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to cool in a
desiccator, and weigh 19.17 g to a weighing dish. Dissolve in a small amount of water,transfer to a 1000-mL volumetric flask, add 2 mL-3 mL of nitric acid, and add water up to themarked line.
f) Phosphoric acid standard solution (P2O5 0.5 mg/mL) (1): Transfer 50 mL of phosphoric acidstandard solution (P2O5 10 mg/mL) to a 1000-mL volumetric flask, add 2 mL - 3 mL of nitricacid, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to reagent “b” reagent solution in the Official Methods of Analysis of
Fertilizers (1992).
(3) This corresponds to ammonium metavanadate in the Official Methods of Analysis of
Fertilizers (1992).
(4) This corresponds to ammonium molybdate in the Official Methods of Analysis of
Fertilizers (1992).
(5) Store in an amber bottle. However. the reagent solution cannot tolerate long termpreservation.
Comment 1 The coloring reagent solution in d) can also be prepared by the following method.
Dissolve 2.24 g of ammonium vanadate (V) (3) specified in JIS K 8747 in water, add
300 mL of nitric acid, and add water to make 1000 mL. Separately, add 100 g of95

Testing Methods for Fertilizers (2018)hexaammonium heptamolybdate tetrahydrate (4) specified in JIS K 8905 while
dissolving in water, and further add water to make 1000 mL. In the case of usage,
mix equal volumes of the two solutions.
Comment 2 Instead of the phosphoric acid standard solution in (2), a phosphoric acid standard
solution for a calibration curve can be prepared using a phosphoric acid standard
solution (P 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate soluble phosphoric acid (S-P2O5) in an analytical sample bymultiplying the concentration (P) of a phosphoric acid standard solution for acalibration curve or a measured value obtained in (4.3) by a conversion factor(2.2914).
(3) Instruments: Instruments are as shown below:a) Water bath: Water bath that can be adjusted to 65 ºC ± 2 ºC.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Spectrophotometer: A spectrophotometer specified in JIS K 0115
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 2.5 g of an analytical sample to the order of 1 mg, and put it in a small mortar.
b) Add about 20 mL - 25 mL of water, grind well and filter the supernatant with Type 6 filter
paper into a 250-mL volumetric flask.
c) Further, after repeating procedure in b) 3 times, transfer non-dissolved matter in the small
mortar onto a filter paper and wash with water until the filtrate becomes about 200 mL.
d) Add a small amount of nitric acid to the filtrate, and further add water up to the marked line to
make a sample solution (1).
e) Transfer the non-dissolved matter on the filter paper together with the filter paper to another250- mL (6) volumetric flask, and add 100 mL of Petermans citrate solution and stopple. Then
shake to mix until the filter paper breaks down.
f) Heat the volumetric flask in e) in water bath at 65 ºC ± 2 ºC for 1 hour while shaking to mix
every 15 minutes.
g) After immediate cooling is complete, add water up to the marked lineh) Filter with Type 6 filter paper to make a sample solution (2).
Note (6) It is recommended to use a 250-mL short-neck volumetric flask.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.2.2.b.
Comment 4 When the determination is affected by the coloring of the sample solution of d) andh), transfer the predetermined volume (equivalent volume) (7) of the sample solution(1) and the sample solution (2) to a 100-mL volumetric flask, add a few drops ofhydrochloric acid (1+1) to acidify, then add no more than 0.1 g of active carbon.
After leaving at rest for a little while, add water up to the marked line and filter. The
filtrate is to be the mixture solution for the sample solution of (4.2) a). Additionally,as phosphorus contained in active carbon has the possibility to elute and affects thedetermination value, a blank test is required.
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount (the equivalents of 0.5 mg - 6 mg as P2O5 and no more thanthe equivalents of 2 mL of Petermans citrate solution) (7) of the sample solution (1) and thesample solution (2) to a 100-mL volumetric flask.
b) Add the solution to make Petermans citrate solution equivalent to 2 mL.
96

Testing Methods for Fertilizers (2018)c) Add 4 mL of nitric acid (1+1) (8), and heat to boil (9).
d) After cooling is complete, add a proper amount of water (10).
e) Add 20 mL of coloring reagent solution, and further add water up to the marked line, and thenleave at rest for about 30 minutes (8).
Comment 5 The volumetric flask used in the procedure in a) should be distinguished as a flask to
be used for phosphate coloring operation and should not be used for other purposes.
Note (7) The transferred volume of the sample solution (1) and the sample solution (2) should beequivalent.
(8) When the solution is muddled by adding nitric acid (1+1), conduct centrifugation after
the procedure in e).
(9) When it does not contain non-orthophosphate, the boiling operation is not necessary.
(10) Without the addition of water, precipitate may be produced when a coloring reagent
solution is added.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 420 nmb) Calibration curve preparation
1) Transfer 1 mL - 12 mL of phosphoric acid standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add 2 mL of Petermans citrate solution, 4 mL of nitric acid (1+1) and a proper amount ofwater (10), and conduct the same procedure as (4.2) e) to make the P2O5 0.5 mg/100 mL - 6
mg/100 mL phosphoric acid standard solutions for the calibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blanktest solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 420 nm of the phosphoric acid standard solutions forthe calibration curve preparation using the blank test solution for the calibration curve
preparation as the control (11).
5) Prepare the calibration curve of the phosphoric acid concentration and absorbance of thephosphoric acid standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) e), measure absorbance by the same procedure as b) 4) (11).
2) Obtain the phosphoric acid (P2O5) content from the calibration curve, and calculate soluble
phosphoric acid (S-P2O5) in the analytical sample.
Note (11) Measure within 2 hours after adding the coloring reagent solution.
Comment 6 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 10 % (mass fraction) 20 % (mass fraction) and 1 % (mass fraction) - 5 % (mass fraction) are 99.4 % 100.6 % and 98.6 % - 103.0 % as soluble phosphoric acid (S-P2O5) respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
97Testing Methods for Fertilizers (2018)

Additionally, the minimum limit of quantification of this testing method is about
0.08 % (mass fraction).
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
4)
5)
6)
7)
8)
9)
certified reference laboratory Average2)
sr
RSD rs I(T)RSD I(T)sR
RSD R
material fertilizer
FAMIC-B-10

1)

p
10

3)

(%)
8.62

3)

(%)

(%)
0.04

0.4

3)

(%)
0.05

(%)
0.6

3)

(%)
0.06

(%)
0.7

FAMIC-B-14
10
9.18
0.03
0.4
0.04
0.5
0.09
1.0
1） The number of laboaratories used for analysis
6） Intermediate standard deviation
conducting Ammonium vanadomolybdate absorptiometric analysis
2） Average （the number of laboratory（p ）×test days（2）7） Intermediate relative standard deviation
×the number of replicate testing（3））3） Mass fraction

8） Reproducibility standard deviation

4） Repeatability standard deviation
5） Repeatability relative standard deviation

9） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.108- 114, Yokendo, Tokyo (1988)
2)
Kimie KATO, Sakiko TAKAHASHI and Yuji SHIRAI: Validation of a Color metric
Method for Determination of Nitrogen, Phosphorus and Boron: Evaluation of
Calibration curve, Research Report of Fertilizer Vol. 2, p. 137 - 144 (2009)3)
Akira SHIMIZU and Shin ABE: Verification of Performance Characteristics of Testing
Method for Citrate-Soluble Phosphorus Content by Ammonium Vanadomolybdate
Absorption Photometry, Research Report of Fertilizer Vol. 5, p. 180 - 189 (2012)98

Testing Methods for Fertilizers (2018)(5)

Flow sheet for soluble phosphoric acid: The flow sheet for soluble phosphoric acid in
fertilizers is shown below:
2.5 g analytical sample

Repeat 3 times

Weigh to the order of 1 mg to a small mortar← About 20 mL - 25 mL water
Grinding

Supernatant filtration＜Residue＞

Type 6 filter paper, 250-mL volumetric flask

Transfer
Transfer residue to a filter paper
Washingwithwater, until filtrate reaches about 200 mL←＜Residue＞＜Filtrate＞← A small amount of nitric acid
← Water (up to the marked line)
Sample solution (1)Transfer
Together with filter paper, 250-mL volumetric flask← 100 ml of Petermans citrate solution
Shaking to mix
Stopple and shake to mix until a filter paler breaks down
Heating

At 65 ºC ± 2 ºC for 1 hour while shaking to mix every 15 minutes.

Cooling
Immediately← Water (up to the marked line)
Filtration
Type 6 filter paper
Sample solution (2)Figure 1 The flow sheet for soluble phosphoric acid in fertilizers (Extraction procedure)99

Testing Methods for Fertilizers (2018)

Sample solutions (1)Transfer the same volume of sample solutions (1)and (2) Aliquotand (2) to 100-mL volumetric flasks(predeterminedvolume)← Petermans citrate solution, until it reaches the equivalent of 2 mL
← 4 mL of nitric acid (1+1)
Heating
Boiling
Cooling← A proper amount of water← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (420nm)

Figure 2 The flow sheet for soluble phosphoric acid in fertilizers(Coloring and measurement procedure)100

Testing Methods for Fertilizers (2018)

4.2.2.b Quinoline gravimetric analysis(1) Summary
This testing method is applicable to fertilizers containing no Phosphorous acid, etc. It is suitable forthe fertilizers with relatively a high content of phosphoric acid. This testing method is classified as
Type E and its symbol is 4.2.2.b-2017 or S-P.b-1.
Extract by adding water to an analytical sample, then extract by adding an ammonium citric acidsolution, and combine respective pre-determined amounts of extract (equivalent volume). Heat after
adding nitric acid and water, hydrolyze nonorthophosphoric acid to orthophosphate ion and measure
the mass of quinolinium phosphomolybdate formed by the reaction with quinoline, molybdic acidand nitric acid to obtain ammonia alkaline ammonium citrate soluble phosphoric acid (soluble
phosphoric acid (S-P2O5)) in an analytical sample.
(2) Reagent: Reagents are as shown below.
a) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
b) Ammonia solution: A JIS Guaranteed (NH3 28 % (mass fraction)) reagent specified in JIS K
8085 or a reagent of equivalent quality.
c) Petermans citrate solution: Add 173 g of citric acid monohydrate specified in JIS K 8283
in water to dissolve and add gradually an ammonia solution equivalent to 42 g of nitrogen
while cooling. After cooling is complete, add water to make 1000 mL. Additionally, checkthat the specific gravity of the solution is 1.082 - 1.083 (15 ºC) and the nitrogen content per 1
mL is 42 mg.
d) Sodium molybdate solution: Dissolve 70 g of sodium molybdate dihydrate in 150 mL ofwater.
e) Quinoline solution: Add 5 mL of quinolone specified in JIS K 8279 to the mixture solutionof 35 mL of nitric acid and 100 mL of water.
f) Quimosiac solution: Add 60 g of citric acid monohydrate specified in JIS K 8283 to the
mixture solution of 85 mL nitric acid and 150 mL of water to dissolve. Add gradually total
volume of the sodium molybdate solution to mix. Add gradually the total volume of thequinoline solution while mixing the solution. After leaving at rest overnight, filter the total
volume with Type 3 filter paper. Add 280 mL of acetone specified in JIS K 8034, and further
add water to make 1000 mL.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Water bath: Water bath that can be adjusted to 65 °C ± 2 °C and 60 ºC - 65 ºC.
b) Drying apparatus: A drying apparatus that can be adjusted to 220 °C ± 5 ºC.
c) Crucible type glass filter: A crucible type glass filter 1G4 specified in JIS R 3503. Let it
stand to cool in a desiccator after heating at 220 ºC ± 5 ºC in advance and measure the mass tothe order of 1 mg.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 2.5 g of an analytical sample to the order of 1 mg, and put it in a small mortar.
b) Add about 20 mL - 25 mL of water, grind well and filter the supernatant with Type 6 filter
paper into a 250-mL volumetric flask.
c) Further, after repeating procedure in b) 3 times, transfer non-dissolved matter in the small
mortar onto a filter paper and wash with water until the filtrate becomes about 200 mL.
d) Add a small amount of nitric acid to the filtrate, and further add water up to the marked line to
make a sample solution (1).
e) Transfer the non-dissolved matter on the filter paper together with the filter paper to another101Testing Methods for Fertilizers (2018)

250- mL (1) volumetric flask, and add 100 mL of Petermans citrate solution and stopple. Then
shake to mix until the filter paper breaks down completely.
f) Heat the volumetric flask in e) in water bath at 65 ºC ± 2 ºC for 1 hour while shaking to mix
every 15 minutes.
g) After immediate cooling is complete, add water up to the marked lineh) Filter with Type 6 filter paper to make a sample solution (2).
Note (1) It is recommended to use a 250-mL short-neck volumetric flask.
Comment 1 The procedure in (4.1) is the same as the procedure in (4.1) of 4.2.2.a.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined amount (the equivalents of 10 mg - 30 mg as P2O5 and no more thanthe equivalents of 2 mL of Petermans citrate solution) (2) of the sample solution (1) and thesample solution (2) to a 300-mL tall beaker.
b) Add 5 mL of nitric acid and add water to make about 80 mL.
c) Cover with a watch glass. After boiling for about 3 minutes, wash the watch glass and theinside the tall beaker with water and add water to make about 100 mL.
d) Immediately, add 50mL of quimosiac solution, heat for about 15 minutes while sometimes
mixing in a water bath at 60 ºC - 65 ºC to produce the precipitate of quinonium
phosphomolybdate.
e) After standing to cool down to room temperature while sometimes mixing, filter under
reduced pressure with a crucible type glass filter, wash the tall beaker 3 times with water andtransfer the whole precipitate into a crucible type glass filter, and further wash 7 - 8 times withwater.
f) Transfer the precipitate together with the crucible type glass filter into a drying apparatus andheat at 220 ºC ± 5 ºC for about 30 minutes.
g) As soon as heating is complete, move it into a desiccator and let it stand to cool.
h) After standing to cool, remove the crucible type glass filter from the desiccator and measure
the mass to the order of 1 mg.
i) Calculate soluble phosphoric acid (S-P2O5) in the analytical sample by the following formula.
Soluble phosphoric acid (% (mass fraction)) in an analytical sample= A × 0.03207 × (V1/V2) × (1/W) × 100
A：
W：
V1：
V2：

Mass (g) of the precipitate in h)
Mass of an analytical sample (2.5 g)
Predetermined volume (250 mL) of the sample solution
Volume (mL) of the sample solution transferred in a)

Note (2) The transferred volume of the sample solution (1) and the sample solution (2) should beequivalent.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.98- 106, Yokendo, Tokyo (1988)

102

Testing Methods for Fertilizers (2018)(5)

Flow sheet for soluble phosphoric acid: The flow sheet for soluble phosphoric acid in
fertilizers is shown below:
2.5 g analytical sample
Repeat 3 times

Weigh to the order of 1 mg to a small mortar← About 20 mL - 25 mL water
Grinding

Supernatant filtration＜Residue＞

Type 6 filter paper, 250-mL volumetric flask

Transfer
Transfer residue to a filter paper← Wash with water, until filtrate reaches about 200 mL＜Residue＞＜Filtrate＞← A small amount of nitric acid
← Water (up to the marked line)
Sample solution (1)Transfer
Together with filter paper, 250-mL volumetric flask← 100 ml Petermans citrate solution
Shaking to mix
Stopple and shake to mix until a filter paler breaks down
Heating

At 65 ºC ± 2 ºC for 1 hour while shaking to mix every 15 minutes.

Cooling
Immediately← Water (up to the marked line)
Filtration
Type 6 filter paper
Sample solution (2)Figure 1 Flow sheet for soluble phosphoric acid in fertilizers(Extraction procedure)103

Testing Methods for Fertilizers (2018)

Sample solutions (1)and (2) Aliquot
Transfer the same volume of sample solutions (1)(predeterminedand (2) to 300-mL volumetric flasks
volume)← 5 mL nitrate acid
← Water (to reach about 80 mL)
Cover with a watch glass for 3 minutes
Boiling
Wash a watch glass and inside a tall beaker with water← Water (to make about 100 mL)← 50 mL of quimosiac solution
Forming precipitate
60 ºC - 65 ºC, for 15 minutes, sometimes mixing
Standing to coolRoom temperature

Filtration
under reducedpressure

Crucible type glass filtering apparatus 1G4, 3 times with water

Washing

Wash 7 - 8 times with water

Drying220 ºC ± 5 ºC for about 30 minutes

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure 2 Flow sheet for soluble phosphoric acid in fertilizers(Measurement procedure)104

Testing Methods for Fertilizers (2018)

4.2.3 Citrate-soluble phosphoric acid
4.2.3.a Ammonium vanadomolybdate absorptiometric analysis(1) Summary
This testing method is applicable to fertilizers that do not contain matter not colored by hydrolysis
with nitrate acids such as phosphonic acid. This testing method is classified as Type C and its
symbol is 4.2.3.a-2018 or C-P.a-2.
Extract by adding a citric acid solution to an analytical sample. Heat after adding nitric acid (1+1),hydrolyze nonorthophosphoric acid to orthophosphate ion and measure the absorbance ofphosphovanadomolybdate salt formed by the reaction with ammonium vanadate (V),hexaammonium heptamolybdate and nitric acid to obtain citrate soluble phosphoric acid (C-P2O5)
in an analytical sample. In addition, the performance of this testing method is shown in Comment
9.
(2) Reagent: Reagents are as shown below.
a) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
b) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
c) Coloring reagent solution (1) (2): Dissolve 1.12 g of ammonium vanadate (V)(3) specified in
JIS K 8747 in water, add 150 mL of nitric acid, then add 50 g of hexaammonium
heptamolybdate tetrahydrate (4) specified in JIS K 8905 dissolved in water, and further add
water to make 1000 mL (5).
d) Phosphoric acid standard solution (P2O5 10 mg/mL) (1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to cool in a
desiccator, and weigh 19.17 g to a weighing dish. Dissolve in a small amount of water,transfer to a 1000-mL volumetric flask, add 2 mL-3 mL of nitric acid, and add water up to themarked line.
e) Phosphoric acid standard solution (P2O5 0.5 mg/mL) (1): Transfer 50 mL of phosphoric acidstandard solution (P2O5 10 mg/mL) to a 1000-mL volumetric flask, add 2 mL - 3 mL of nitricacid, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to reagent “b” reagent solution in the Official Methods of Analysis of
Fertilizers (1992).
(3) This corresponds to ammonium metavanadate in the Official Methods of Analysis of
Fertilizers (1992).
(4) This corresponds to ammonium molybdate in the Official Methods of Analysis of
Fertilizers (1992).
(5) Store in an amber bottle. However, the reagent solution cannot tolerate long termpreservation.
Comment 1 The coloring reagent solution in c) can also be prepared by the following method.
Dissolve 2.24 g of ammonium vanadate (V) (3) specified in JIS K 8747 in water, add
300 mL of nitric acid, and add water to make 1000 mL. Separately, add 100 g ofhexaammonium heptamolybdate tetrahydrate (4) specified in JIS K 8905 while
dissolving in water, and further add water to make 1000 mL. In the case of usage,
mix equal volumes of the two solutions.
Comment 2 Instead of the phosphoric acid standard solution in (2), a phosphoric acid standard
solution for a calibration curve can be prepared using a phosphoric acid standard
solution (P 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
105

Testing Methods for Fertilizers (2018)

this case, calculate citrate-soluble phosphoric acid (C-P2O5) in an analytical sample
by multiplying the concentration (P) of a phosphoric acid standard solution for acalibration curve or a measured value obtained in (4.3) by a conversion factor(2.2914).
(3) Instruments: Instruments are as shown below:a) Extractor: Constant-temperature rotary shaker or reciprocating water bath shaker as
described below.
aa) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
ab) Reciprocating water bath shaker: A reciprocating water bath shaker: that can be adjusted to
30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocate horizontally at 160 times/ minute with amplitude of 25 mm 40 mm.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Spectrophotometer: A spectrophotometer specified in JIS K 0115
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (6), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (6) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 3 The procedure in (4.1.1) is the same as the procedure in (4.1.1) of 4.2.3.b, (4.1) of
4.2.3.c, (4.1) of 4.2.3.d, (4.1.1) of 4.3.2.a, (4.1) of 4.3.2.b, (4.1) of 4.3.2.c, (4.1.1) of
4.3.2.d, (4.1.1) of 4.6.3.a, (4.1.1) of 4.6.3.b, (4.1.1) of 4.7.2.a, (4.1.1) of 4.7.2.b,(4.1.1) of 4.8.1.a and (4.1.1) of 4.8.1.b.
(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (6).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (6), and shake to mix byreciprocating horizontally at 160 times /min with amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC)
for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (7) Use a 250-mL flat-bottom volumetric flask to stabilize the shaking.
Comment 4 The procedure in (4.1.2) is the same as the procedure in (4.1.2) of 4.2.3.b, (4.1.2) of
4.2.3.d, (4.1.2) of 4.3.2.a, (4.1.2) of 4.3.2.d, (4.1.2) of 4.6.3.a, (4.1.2) of 4.6.3.b,(4.1.2) of 4.7.2.a, (4.1.2) of 4.7.2.b, (4.1.2) of 4.8.1.a and (4.1.2) of 4.8.1.b.
106

Testing Methods for Fertilizers (2018)

Comment 5 For a by-product phosphate fertilizer, if the pH of the sample solution in (4.1.1) d)and (4.1.2) d) is neutral or basic, prepare a sample solution anew by replacing “1 g ofan analytical sample” in the procedures in (4.1.1) a) and (4.1.2) a) with “0.5 g of ananalytical sample”.
Comment 6 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedures of (4.1.1) b) and (4.1.2) b).
Comment 7 When the determination is affected by the coloring of the sample solution of (4.1.1)d) and (4.1.2) d), transfer the predetermined volume of the sample solution to a
100-mL volumetric flask, add a few drops of hydrochloric acid (1+1) to acidify, then
add no more than 0.1 g of active carbon. After leaving at rest for a little while, add
water up to the marked line and filter. The filtrate is prepared as the sample solutionof (4.2) a). Additionally, as phosphorus contained in active carbon has the possibility
to elute and affects the determination value, a blank test is required.
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 6 mg as
P2O5 and no more than the equivalents of 17 mL of citric acid solution) to a 100-mL
volumetric flask.
b) Add the solution to make citric acid solution equivalent to 17 mL.
c) Add 4 mL of nitric acid (1+1) (8), and heat to boil (9).
d) After cooling is complete, add a proper amount of water (10).
e) Add 20 mL of coloring reagent solution, and further add water up to the marked line, and thenleave at rest for about 30 minutes.
Comment 8 The volumetric flask used in the procedure in a) should be distinguished as a flask to
be used for phosphate coloring operation and should not be used for other purposes.
Note (8) When the solution is muddled by adding nitric acid (1+1), conduct centrifugation after
the procedure in e).
(9) When it does not contain non-orthophosphate, the boiling operation is not necessary.
(10) Without the addition of water, precipitate may be produced when a coloring reagent
solution is added.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 420 nmb) Calibration curve preparation
1) Transfer 1 mL - 12 mL of phosphoric acid standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add 17 mL of a citric acid solution, then add 4 mL of nitric acid (1+1), further add a properamount of water (10) and conduct the same procedure as (4.2) e) to make the P2O5 0.5 mg/100
mL - 6 mg/100 mL phosphoric acid standard solution for the calibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blanktest solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 420 nm of the phosphoric acid standard solutions forthe calibration curve preparation using the blank test solution for the calibration curve107Testing Methods for Fertilizers (2018)

preparation as the control (11).
5) Prepare the calibration curve of the phosphoric acid concentration and absorbance of thephosphoric acid standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) e), measure absorbance by the same procedure as b) 4) (11).
2) Obtain the phosphoric acid (P2O5) content from the calibration curve, and calculate citratesoluble phosphoric acid (C-P2O5) in the analytical sample.
Note (11) Measure within 2 hours after adding the coloring reagent solution.
Comment 9 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 10 % (mass fraction) 20 % (mass fraction) and 1 % (mass fraction) - 5 % (mass fraction) are 96.6 % 103.4 % and 102.0 % - 103.8 % as citrate soluble phosphoric acid (C-P2O5)
respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.03 % (mass fraction) for solid fertilizers, and 0.01 % (mass fraction) for fluid
fertilizers.
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
2)
4)
5)
6)
7)
8)
9)
certified reference laboratory Averagesr
RSD rs I(T)RSD I(T)
sR
RSD R
1)
3)
3)
3)
3)
material fertilizer
(%)
(%)
(%)
p
(%)
(%)
(%)
(%)
FAMIC-A-10
11
10.05
0.04
0.4
0.05
0.5
0.13
1.3
FAMIC-A-13
10
10.79
0.06
0.6
0.08
0.8
0.09
0.8
1） The number of laboaratories used for analysis
6） Intermediate standard deviation
conducting Ammonium vanadomolybdate absorptiometric analysis
2） Average （the number of laboratory（p ）×test days（2）7） Intermediate relative standard deviation
×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.108- 114, Yokendo, Tokyo (1988)
2)
Kimie KATO, Sakiko TAKAHASHI and Yuji SHIRAI: Validation of a Color metric
Method for Determination of Nitrogen, Phosphorus and Boron: Evaluation of
Calibration curve, Research Report of Fertilizer Vol. 2, p. 137 - 144 (2009)3)
Yoshiyuki SUNAGA, Yasushi SUGIMURA, Ichiro YOSHIDA and Hidenori KONISHI:
Verification of Performance Characteristics of Testing Methods for Phosphorus Content
in Fertilizer by Ammonium Vanadomolybdate Absorption Photometry, Research Reportof Fertilizer Vol. 5, p. 167 - 179 (2012)108

Testing Methods for Fertilizers (2018)(5)

Flow sheet for citrate soluble phosphoric acid: The flow sheet for citrate soluble
phosphoric acid in fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Shaking to mix
Constant-temperature rotary shaker (30 - 40 revolutions/min)
30 ºC ± 1 ºC, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for citrate soluble phosphoric acid in fertilizers(Extraction procedure (4.1.1))

1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Reciprocating water bath shaker (reciprocation horizontally at
Shaking to mix
160 times /min, with amplitude of 25 mm - 40 mm), at 30 ºC ±1 ºC, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for citrate soluble phosphoric acid in fertilizers(Extraction procedure (4.1.2))

109

Testing Methods for Fertilizers (2018)
Sample solution

Aliquot
100-mL volumetric flask(predetermined volume)← Citric acid solution, until it reaches the equivalents of 17 mL← 4 mL of nitric acid(1+1)
Heating
Boiling
Cooling← A proper amount of water← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (420nm)

Figure 2 Flow sheet for citrate soluble phosphoric acid in fertilizers(Coloring and measurement procedure)110

Testing Methods for Fertilizers (2018)

4.2.3.b Ammonium vanadomolybdate absorptiometric analysis (Fertilizers containing
phosphorous acid or phosphite)(1) Summary
This testing method is applicable to the fertilizers containing phosphorous acid or phosphite. This
testing method is classified as Type B and its symbol is 4.2.3.b-2018 or C-P.b-2.
Extract by adding a citric acid solution to an analytical sample, add hydrochloric acid - sulfuric acidto heat, and oxygenate phosphorous acid ion to orthophosphate ion, and then measure theabsorbance of phosphovanadomolybdate salt formed by the reaction with ammonium vanadate (V),hexaammonium heptamolybdate and nitric acid to obtain citrate-soluble phosphoric acid (C-P2O5)
in an analytical sample. In addition, the performance of this testing method is shown in Comment
7.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
c) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
d) Coloring reagent solution (1) (2): Dissolve 1.12 g of ammonium vanadate (V)(3) specified in
JIS K 8747 in water, add 150 mL of nitric acid, then add 50 g of hexaammonium
heptamolybdate tetrahydrate (4) specified in JIS K 8905 dissolved in water, and further add
water to make 1000 mL (5).
e) Phosphoric acid standard solution (P2O5 10 mg/mL) (1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to cool in a
desiccator, and weigh 19.17 g to a weighing dish. Dissolve in a small amount of water,transfer to a 1000-mL volumetric flask, add 2 mL-3 mL of nitric acid, and add water up to themarked line.
f) Phosphoric acid standard solution (P2O5 0.5 mg/mL) (1): Transfer 50 mL of phosphoric acidstandard solution (P2O5 10 mg/mL) to a 1000-mL volumetric flask, add 2 mL - 3 mL of nitricacid, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to reagent “b” reagent solution in the Official Methods of Analysis of
Fertilizers (1992).
(3) This corresponds to ammonium metavanadate in the Official Methods of Analysis of
Fertilizers (1992).
(4) This corresponds to ammonium molybdate in the Official Methods of Analysis of
Fertilizers (1992).
(5) Store in an amber bottle. However, the reagent solution cannot tolerate long termpreservation.
Comment 1 The coloring reagent solution in d) can also be prepared by the following method.
Dissolve 2.24 g of ammonium vanadate (V) (3) specified in JIS K 8747 in water, add
300 mL of nitric acid, and add water to make 1000 mL. Separately, add 100 g ofhexaammonium heptamolybdate tetrahydrate (4) specified in JIS K 8905 while
dissolving in water, and further add water to make 1000 mL. In the case of usage,
mix equal volumes of the two solutions.
Comment 2 Instead of the phosphoric acid standard solution in (2), a phosphoric acid standard
solution for a calibration curve can be prepared using a phosphoric acid standard
111

Testing Methods for Fertilizers (2018)

solution (P 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate citrate-soluble phosphoric acid (C-P2O5) in an analytical sample
by multiplying the concentration (P) of a phosphoric acid standard solution for acalibration curve or a measured value obtained in (4.3) by a conversion factor(2.2914).
(3) Instruments: Instruments are as shown below:a) Extractor: Constant-temperature rotary shaker or reciprocating water bath shaker as
described below.
aa) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
ab) Reciprocating water bath shaker: A reciprocating water bath shaker: that can be adjusted to
30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocating horizontally at 160 times/ minute with amplitude of 25
mm - 40 mm.
b) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
c) Spectrophotometer: A spectrophotometer specified in JIS K 0115
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (6), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (6) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 3 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.3.a.
(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (7).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (6), and shake to mix byreciprocating horizontally at 160 times/min with amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC)
for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (7) Use a 250-mL flat-bottom volumetric flask to stabilize the shaking.
Comment 4 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.3.a.
Comment 5 For a by-product phosphate fertilizer, if the pH of the sample solution in (4.1.1) d)and (4.1.2) d) is neutral or basic, prepare a sample solution anew by replacing “1 g of112

Testing Methods for Fertilizers (2018)

an analytical sample” in the procedures in (4.1.1) a) and (4.1.2) a) with “0.5 g of ananalytical sample”.
Comment 6 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedures of (4.1.1) b) and (4.1.2) b).
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount (up to 25 mL, the equivalents of 0.5 mg - 6 mg as P2O5) of
the sample solution to a 100-mL - 200-mL tall beaker.
b) Add 3 mL of hydrochloric acid and 1 mL of nitric acid.
c) Cover the tall beaker with a watch glass (8), heat on a hot plate or sand bath at 200 ºC - 250 ºC
and condense until the solution volume becomes about 2 mL (9).
d) After standing to cool, transfer it with water to a 100- mL volumetric flask (10).
e) Add the citric acid solution to make the equivalents of 17 mL of the citric acid and further add
2 mL of nitric acid (1+1).
f) Add 20 mL of coloring reagent solution, and further add water up to the marked line, and thenleave at rest for about 30 minutes.
Note (8) A watch glass should not be uncovered when bubbles form while heating because they
may splash.
(9) It is recommended to transfer 2 mL of water to a 100-mL - 200-mL tall beaker inadvance and confirm the volume.
(10) The volume of the solution after transferring should be up to about 50 mL.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 420 nmb) Calibration curve preparation
1) Transfer 1 mL - 12 mL of phosphoric acid standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add 17 mL of a citric acid solution, then add 4 mL of nitric acid (1+1), further add a properamount of water (11). Conduct the same procedure as (4.2) f) to make the P2O5 0.5 mg/100 mL- 6 mg/100 mL phosphoric acid standard solutions for the calibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blanktest solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 420 nm of the phosphoric acid standard solutions forthe calibration curve preparation using the blank test solution for the calibration curve
preparation as the control (12).
5) Prepare the calibration curve of the phosphoric acid concentration and absorbance of thephosphoric acid standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) f), measure absorbance by the same procedure as b) 4) (12).
2) Obtain the phosphoric acid (P2O5) content from the calibration curve, and calculate citratesoluble phosphoric acid (C-P2O5) in the analytical sample.
Note (11) If water is not added, precipitate may form when adding the coloring reagent solution.
(12) Measure within 2 hours after adding the coloring reagent solution in the procedure in113

Testing Methods for Fertilizers (2018)(4.2) f).
Comment 7 Additive recovery testing was conducted to evaluate trueness using solid fertilizers(10 samples) containing the equivalents of 1.03 % (mass fraction) - 51.04 % (massfraction) as citrate soluble phosphoric acid. As a result, the average recovery is 99 %- 100 %.
The results of the repeatability tests on different days using a solid preparation
sample to evaluate precision were analyzed by one-way analysis of variance. Table 1
shows the calculation results of intermediate precision and repeatability.
Table 2 shows results and analysis results from a collaborative study for testing
method validation.
Additionally, the minimum limit of quantification of this testing method is about
0.05 % (mass fraction).

Table 1 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
4)
5)
samplerepeatability Average2)
sr
RSD r
1)

Preparation sample (solid) 1
Preparation sample (solid) 2

T
5
5

3)

3)

(%)
51.01
2.57

(%)
0.12
0.01

1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

114

(%)
0.2
0.6

Intermediate precision
6)s I(T)

3)

(%)
0.16
0.03

RSD I(T)
(%)

7)

0.3
1.1

6） Intermediate standard deviation
7） Intermediate relative standard deviation

Testing Methods for Fertilizers (2018)

Table 2 Results and analysis results from a collaborative study
for the test method validation of citrate-soluble phosphoric acid4)
5)
6)
Number of Mean2)
sr
RSD r
sR
laboratorie
Sample name
3)
3)
3)
(%)
1)
(%)
(%)
(%)
s
Processed
phosphorus
12
47.21
0.13
0.3
0.69
fertilizers1
Compund fertilizers 1
11
17.71
0.07
0.4
0.19
Compund fertilizers 2
12
5.08
0.08
1.6
Absorptive mixed
11
14.32
0.06
0.4
fertiliser
Regent
11
50.89
0.14
0.3
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

7)

RSD R
(%)
1.5
1.1

0.17

3.3

0.18

1.2

0.57

1.1

References1)
Toshiaki HIROI, Masayuki YAMANISHI Determination Method for Citric
Acid-Soluble Phosphorus in Solid Fertilizer Containing Phosphonate (Phosphite) using
Spectrophotometer, Research Report of Fertilizer, Vol.9, p. 43 - 58 (2016)
2)
Masayuki YAMANISHI, Toshiaki HIROI and Fumika TAKATSU Determination
Method for Citric Acid-Soluble and Water-Soluble Phosphorus in Solid Fertilizer
Containing Phosphonic Acid or Phosphonate (Phosphite) using Spectrophotometer: A
Collaborative Study, Vol.9, p. 59 - 68 (2016)115

Testing Methods for Fertilizers (2018)(5)

Flow sheet for citrate-soluble phosphoric acid containing phosphorus acid, etc.: The flow
sheet for citrate-soluble phosphoric acid in fertilizers containing phosphorus acid, etc.isshown below:

1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask← 150 mL of citric acid solution [about 30ºC]
Constant-temperature rotary shaker (30 - 40 revolutions/min)Shaking to mix
30ºC ± 1ºC, 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1-1 Flow sheet for citrate-soluble phosphoric acidin fertilizers containing phosphorus acid, etc. (Extraction procedure (4.1.1))
1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask← 150 mL of citric acid solution [about 30ºC]
Reciprocating water bath shaker (reciprocation horizontally at 160
Shaking to mix
times /min, with amplitude of 25 mm - 40 mm), at 30 ºC ± 1 ºC,
for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1-2 Flow sheet for citrate-soluble phosphoric acidin fertilizers containing phosphorus acid, etc. (Extraction procedure (4.1.2))

116

Testing Methods for Fertilizers (2018)
Sample solution

Aliquot
100-mL - 200-mL tall beaker(predetermined volume)← 3 mL of hydrochloric acid← 1 mL of nitric acid
MixingHeating

Cover with a watch glass, heat on a hot plate or sand bathat 200 ºC - 250 ºC, condense until the solution volume reaches about 2 mL

Standing to coolTransfer
100-mL volumetric flask, water (solution volume is up to about 50 mL)← Citric acid solution, until the equivalent of 50 mL← 2 mL of Nitric acid (1+1)← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (420 nm)

Figure 2 Flow sheet for citrate-soluble phosphoric acidin fertilizers containing phosphorus acid, etc. (Coloring and measurement procedure)117

Testing Methods for Fertilizers (2018)

4.2.3.c Quinoline gravimetric analysis(1) Summary
This testing method is applicable to fertilizers containing no Phosphorous acid, etc. It is suitable forthe fertilizers with relatively a high content of phosphoric acid. This testing method is classified as
Type E and its symbol is 4.2.3.c-2017 or C-P.c-1.
Extract by adding a citric acid solution to an analytical sample. Heat after adding nitric acid andwater, hydrolyze nonorthophosphoric acid to orthophosphate ion and measure the mass ofquinolinium phosphomolybdate formed by the reaction with quinoline, molybdic acid and nitricacid to obtain citrate soluble phosphoric acid (C-P2O5) in an analytical sample.
(2) Reagent: Reagents are as shown below.
a) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
b) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
c) Sodium molybdate solution: Dissolve 70 g of sodium molybdate dihydrate in 150 mL ofwater.
d) Quinoline solution: Add 5 mL of quinoline specified in JIS K 8279 to the mixture solutionof 35 mL of nitric acid and 100 mL of water.
e) Quimosiac solution: Add 60 g of citric acid monohydrate specified in JIS K 8283 to the
mixture solution of 85 mL nitric acid and 150 mL of water to dissolve. Add gradually total
volume of the sodium molybdate solution to mix. Add gradually the total volume of thequinoline solution while mixing the solution. After leaving at rest overnight, filter the total
volume with Type 3 filter paper. Add 280 mL of acetone specified in JIS K 8034, and further
add water to make 1000 mL.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
b) Water bath: Water bath that can be adjusted to 60 ºC - 65 ºC.
c) Drying apparatus: A drying apparatus that can be adjusted to 220 ºC ± 5 ºC.
d) Crucible type glass filter: A crucible type glass filter 1G4 specified in JIS R 3503. Let it
stand to cool in a desiccator after heating at 220 ºC ± 5 ºC in advance and measure the mass tothe order of 1 mg.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (2) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
118

Testing Methods for Fertilizers (2018)

Comment 1 The procedures in (4.1) are the same as in (4.1.1) in 4.2.3.a.
Comment 2 For a by-product phosphate fertilizer or a fertilizer containing a by-product phosphate,
if the pH of the sample solution of d) is neutral or basic, prepare a sample solution
anew by replacing “1 g of an analytical sample” in the procedure in a) with “0.5 g ofan analytical sample”.
Comment 3 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedure of (4.1) b).
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 10 mg - 30 mg as P2O5) of sample
solution to a 300-mL tall beaker.
b) Add 5 mL of nitric acid and add water to make about 80 mL.
c) Cover with a watch glass. After boiling for about 3 minutes, wash the watch glass and theinside the tall beaker with water and add water to make about 100 mL.
d) Immediately, add 50mL of quimosiac solution, heat for about 15 minutes while sometimes
mixing in a water bath at 60 ºC - 65 ºC to produce the precipitate of quinolinium
phosphomolybdate.
e) After standing to cool down to room temperature while sometimes mixing, filter under
reduced pressure with a crucible type glass filter, wash the tall beaker 3 times with water andtransfer the whole precipitate into a crucible type glass filter, and further wash 7 - 8 times withwater.
f) Transfer the precipitate together with the crucible type glass filter into a drying apparatus andheat at 220 ºC ± 5 ºC for about 30 minutes.
g) As soon as heating is complete, move it into a desiccator and let it stand to cool.
h) After standing to cool, remove the crucible type glass filter from the desiccator and measure
the mass to the order of 1 mg.
i) Calculate citrate soluble phosphoric acid (C-P2O5) in the analytical sample by the following
formula.
Citrate soluble phosphoric acid (C-P2O5) (% (mass fraction)) in an analytical sample= A × 0.03207 × (V1/V2) × (1/W) × 100
A：
W：
V1：
V2：

Mass (g) of the precipitate in h)
Mass of an analytical sample (1 g)
Predetermined volume (250 mL) of the sample solution
Volume (mL) of the sample solution transferred in a)

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.98- 106, Yokendo, Tokyo (1988)

119

Testing Methods for Fertilizers (2018)(5)

Flow sheet for citrate soluble phosphoric acid: The flow sheet for citrate soluble
phosphoric acid in fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Shaking to mix
Constant-temperature rotary shaker (30 - 40 revolutions/min)
30 ºC ± 1 ºC, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1 Flow sheet for citrate soluble phosphoric acid in fertilizers (Extraction procedure)120

Testing Methods for Fertilizers (2018)

Sample solution

Standing to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
300-mL tall beaker(predetermined volume)← 5 mL nitric acid
← Water (to reach about 80 mL)
Cover with a watch glass for 3 minutes
Boiling
Wash a watch glass and the inside the tall beaker with water← Water (to be about 100 mL)← 50mL of quimosiac solution
Forming precipitation 60 ºC - 65 ºC, for 15 minutes, sometimes mixing
Standing to coolRoom temperature

Filtration
under reducedpressure

Crucible type glass filtering apparatus 1G4, 3 times with water

Washing

Wash 7 - 8 times with water

Drying220 ºC ± 5 ºC for about 30 minutes

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure 2 Flow sheet for citrate soluble phosphoric acid in fertilizers (Measurement procedure)

121

Testing Methods for Fertilizers (2018)

4.2.3.d ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fertilizers. It is also suitable for the fertilizers containing
phosphite. This testing method is classified as Type D and its symbol is 4.2.3.d-2018 or C-P.d-1.
Extract by adding a citric acid solution to an analytical sample, introduce it to an ICP OpticalEmission Spectrometer (“ICP-OES”) and measure the phosphorus at a wavelength of 178.287 nm toobtain citric acid-soluble phosphoric acid (citrate-soluble phosphoric acid (C-P2O5)) in an analyticalsample. In addition, the performance of this testing method is shown in Comment 8.
(2) Reagent: Reagents are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
d) Phosphoric acid standard solution (P2O5 10 mg/mL) (1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to cool in a
desiccator, and weigh 19.17 g to a weighing dish. Dissolve in a small amount of water,transfer to a 1000-mL volumetric flask, add 2 mL-3 mL of nitric acid, and add water up to themarked line.
e) Phosphoric acid standard solution (P2O5 1 mg/mL) (1): Transfer 10 mL of phosphoric acidstandard solution (P2O5 10 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid
(1+23) up to the marked line.
f) Phosphoric acid standard solution (P2O5 20 µg/mL - 0.4 mg/mL) for the calibration
curve preparation (1): Transfer 2 mL - 40 mL of phosphoric acid standard solution (P2O5 1
mg/mL) to a 100-mL volumetric flask step by step and add hydrochloric acid (1+23) up to themarked line.
g) Phosphoric acid standard solution ((P2O5 5 µg/mL - 20 µg/mL) for the calibration curve
preparation (1): Transfer 5 mL - 20 mL of phosphoric acid standard solution (P2O5 0.1
mg/mL) to a 100-mL volumetric flask step by step and add hydrochloric acid (1+23) up to themarked line.
h) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in e) - g).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the phosphoric acid standard solution in (2), a phosphoric acid standard
solution for a calibration curve can be prepared using a phosphoric acid standard
solution (P 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate citrate-soluble phosphoric acid (C-P2O5) in an analytical sample
by multiplying the concentration (P) of a phosphoric acid standard solution for acalibration curve or a measured value obtained in (4.2) by a conversion factor(2.2914).
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in f) and g) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, when using the ICP-OES
of an axial observation mode, it is recommended to prepare a phosphoric acid122

Testing Methods for Fertilizers (2018)

standard solution for the calibration curve in the concentration range which issuitable to a device used.
(3)
a)
1)
b)

Instruments: Instruments are as shown below:
ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity
Extractor: Constant-temperature rotary shaker or reciprocating water bath shaker as
described below.
ba) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate a
250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at 30- 40 revolutions/min.
bb) Reciprocating water bath shaker: A reciprocating water bath shaker: that can be adjusted
to 30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocate horizontally at 160 times/ minute with amplitude of 25 mm 40 mm.

(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (2) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 3 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.3.a.
(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (3).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix byreciprocating horizontally at 160 times/min with amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC)
for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (3) Use a 250-mL flat-bottom volumetric flask to stabilize the shaking.
Comment 4 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.3.a.
Comment 5 For a by-product phosphate fertilizer, if the pH of the sample solution in (4.1.1) d)and (4.1.2) d) is neutral or basic, prepare a sample solution anew by replacing “1 g ofan analytical sample” in the procedures in (4.1.1) a) and (4.1.2) a) with “0.5 g of ananalytical sample”.
Comment 6 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved matters
123Testing Methods for Fertilizers (2018)

after the procedures of (4.1.1) b) and (4.1.2) b).
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 178.287 nmb) Calibration curve preparation
1) Spray the phosphoric acid standard solution for the calibration curve preparation and blanktest solution for the calibration curve preparation into inductively coupled plasma, and readthe indicated value at a wavelength of 178.287 nm.
2) Prepare a curve for the relationship between the phosphoric acid concentration and theindicated value of the phosphoric acid standard solution for the calibration curve preparationand the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 40 mg as
P2O5) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the phosphoric acid (P2O5) content from the calibration curve, and calculate citratesoluble phosphoric acid (C-P2O5) in the analytical sample.
Comment 7 The simultaneous measurement of multiple elements by an ICP-OES is available. In
this case, transfer a pre-determined amount of phosphoric acid standard solution (P 1
mg/mL or 10 mg/mL), potassium standard solution (K 1 mg/mL or 10 mg/mL),
magnesium standard solution (Mg 1 mg/mL or 10 mg/mL), manganese standard
solution (Mn 1 mg/mL or 10 mg/mL) and boron standard solution (B 1 mg/mL or 10
mg/mL) traceable to National Metrology to a volumetric flask to mix, addhydrochloric acid (1+5) to make an acid concentration of 0.5 mol/L and further add
water up to the marked line to prepare a primary mixed standard solution. Transfer a
pre-determined volume of primary mixed standard solution to volumetric flasks
step-by-step, add hydrochloric acid (1+23) up to the marked line to prepare mixed
standard solutions for calibration curve preparation within the concentration range inTable 1. In this case, multiply the respective concentrations of mixed standard
solutions for calibration curve preparation or measurement values obtained in (4.2)
by the conversion factors in Table 1 to calculate respective main components in ananalytical sample. The measurement wavelengths of respective elements areaccording to Table 1. In addition, when preserving mixed standard solutions forcalibration curve preparation, use a container, which can be sealed tightly, made ofmaterial such as PTFE that boron hardly elutes.

124

Testing Methods for Fertilizers (2018)

Test item

Table 1 Preparation concentration and mesurement wavelengthof mixed standard solutions for calibration curb preparation
Mixed standard solutions for calibration curb preparation
Conversion
Concentration
Concentration
1of elementof the equivalents to oxide
factor ）（µg/mL）（µg/mL）

citrate solubleP
1 - 200
P2 O5
phosphoric acidcitrate solubleK
1 - 200
K2 O
potassium
citrate soluble
Mg 0.1 - 20
MgO
magnesia
citrate soluble
Mn 0.05 - 10
MnO
manganese
citrate soluble
B 0.05 - 10
B2 O3
boron
1) Factor to convert an element to an oxcide.

Wavelength（nm）

2.291 - 458.2

2.2914178.287

1.205 - 241.0

1.2046766.491

0.1658 - 33.16

1.6583279.5530.06455 - 12.91

1.2912257.6100.1610 - 32.20

3.2199249.773

Comment 8 The comparison of the measurement value (yi：1.74 % (mass fraction) - 49.04 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of Ammonium vanadomolybdate absorptiometric analysis was conducted toevaluate trueness using processed phosphorus fertilizers (2 samples), compound
fertilizers (12 samples), home garden-use mixed fertilizers (1 sample), mixed
compost fertilizers (2 samples), mixed phosphate fertilizers (2 samples), designatedblended fertilizers (4samples), blended fertilizers (5 samples), byproduct mixed
fertilizers (1 sample), byproduct phosphorus fertilizers (2 samples), organic
compound fertilizers (1 sample) and fused phosphate fertilizers (1 sample). As aresult, a regression equation was y = − 0.0027 + 1.001x, and its correlation
coefficient (r) was 1.000. In addition, recovery testing was conducted using apreparation sample. As a result, the average rate of recovery at the content level of
0.260 % (mass fraction) - 49.99 % (mass fraction) was 96.3 % - 100.8 %.
The results of the repeatability tests on different days using compound fertilizers andblended fertilizer to evaluate precision were analyzed by one-way analysis ofvariance. Table 2 shows the calculation results of intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.01 % (mass fraction).

125

Testing Methods for Fertilizers (2018)
Table 2 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
sample2)

repeatability Average
1)

3)

sr

4)
3)

Compound fertilizer

T
7

(%)
20.90

(%)
0.13

Blended fertilizer

7

6.44

0.06

RSD r
(%)

5)

Intermediate precisions I(T)

6)
3)

0.6

(%)
0.18

0.9

0.06

RSD I(T)
(%)

7)

0.9
1.0

1） The number of test days conducting a duplicate test

6） Intermediate standard deviation

2） Average （the number of test days（T ）7） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

References
1) Keisuke AOYAMA: Simultaneous Determination of Water-Soluble Principal Ingredients(W-P2O5, W-K2O, W-MgO, W-MnO and W-B2O3) in Liquid Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), Research
Report of Fertilizer, Vol.8, p. 1 - 9 (2015)(5)

Flow sheet for citrate soluble phosphoric acid: The flow sheet for citrate soluble
phosphoric acid in fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Shaking to mix
Constant-temperature rotary shaker (30 - 40 revolutions/min)
30 ºC ± 1 ºC, for 1 hour
Cooling
Immediately
Water
(up
to
the marked line)←
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for citrate soluble phosphoric acid in fertilizers(Extraction procedure (4.1.1))

126

Testing Methods for Fertilizers (2018)

1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Reciprocating water bath shaker (reciprocation horizontally at
Shaking to mix
160 times /min, with amplitude of 25 mm - 40 mm), at 30 ºC ± 1ºC, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for citrate soluble phosphoric acid in fertilizers(Extraction procedure (4.1.2))

Sample solution

Aliquot
100-mL volumetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
ICP Optical Emission Spectrometry (178.287 nm)
Figure 2 Flow sheet for citrate soluble phosphoric acid in fertilizers(Mmeasurement procedure)

127

Testing Methods for Fertilizers (2018)

4.2.4 Water-soluble phosphoric acid
4.2.4.a Ammonium vanadomolybdate absorptiometric analysis(1) Summary
This testing method is applicable to fertilizers that do not contain matter not colored by hydrolysis
with nitrate acids such as phosphonic acid. This testing method is classified as Type C and its
symbol is 4.2.4.a-2017 or W-P.a-1.
Extract by adding water to an analytical sample, add nitric acid (1+1) to heat, and hydrolyze
nonorthophosphoric acid to orthophosphate ion, then measure the absorbance ofphosphovanadomolybdate salt formed by the reaction with ammonium vanadate (V),hexaammonium heptamolybdate and nitric acid to obtain water-soluble phosphoric acid (W-P2O5)
in an analytical sample. In addition, the performance of this testing method is shown in Comment
9.
(2) Reagent: Reagents are as shown below.
a) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
b) Ammonia solution: A JIS Guaranteed (NH3 28 % (mass fraction)) reagent specified in JIS K
8085 or a reagent of equivalent quality.
c) Coloring reagent solution (1) (2): Dissolve 1.12 g of ammonium vanadate (V)(3) specified in
JIS K 8747 in water, add 250 mL of nitric acid, then add 27 g of hexaammonium
heptamolybdate tetrahydrate (4) specified in JIS K 8905 dissolved in water, and further add
water to make 1000 mL (5).
d) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in JIS K
8799 in 100 mL of ethanol (95) specified in JIS K 8102.
e) Phosphoric acid standard solution (P2O5 10 mg/mL) (1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to cool in a
desiccator, and weigh 19.17 g to a weighing dish. Dissolve in a small amount of water,transfer to a 1000-mL volumetric flask, add 2 mL-3 mL of nitric acid, and add water up to themarked line.
f) Phosphoric acid standard solution (P2O5 0.5 mg/mL) (1): Transfer 50 mL of phosphoric acidstandard solution (P2O5 10 mg/mL) to a 1000-mL volumetric flask, add 2 mL - 3 mL of nitricacid, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to reagent “a” reagent solution in the Official Methods of Analysis of
Fertilizers (1992).
(3) This corresponds to ammonium metavanadate in the Official Methods of Analysis of
Fertilizers (1992).
(4) This corresponds to ammonium molybdate in the Official Methods of Analysis of
Fertilizers (1992).
(5) Store in an amber bottle.
Comment 1 Instead of the phosphoric acid standard solution in (2), a phosphoric acid standard
solution for a calibration curve can be prepared using a phosphoric acid standard
solution (P 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate water-soluble phosphoric acid (W-P2O5) in an analytical sample
by multiplying the concentration (P) of a phosphoric acid standard solution for acalibration curve or a measured value obtained in (4.3) by a conversion factor(2.2914).
128

Testing Methods for Fertilizers (2018)(3) Instruments: Instruments are as shown below:a) Extractor: A rotary shaker or a vertical reciprocating shaker as described below.
aa) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30 40 revolutions/min.
ab) vertical reciprocating shaker: A vertical reciprocating shaker that can shake a 250-mL
volumetric flask using an adapter to reciprocate vertically at 300 times/ minute (amplitude of
40 mm).
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Spectrophotometer: A spectrophotometer specified in JIS K 0115
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test sample(4.1.1.1) Rotary shaker:a) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL volumetricflask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 2 In the procedure of (4.1.1.1) a), it is also allowed to weigh 2.5g of an analyticalsample to the order of 1 mg, and put it in a 250-mL volumetric flask
Comment 3 The procedure in (4.1.1.1) is the same as the procedure in (4.1.1) of 4.1.2.b, (4.1.1.1)
of 4.2.4.b, (4.1) of 4.2.4.c, (4.1) of 4.2.4.d, (4.1.2.1) of 4.3.3.a, (4.1.2) of 4.3.3.b,(4.1.2) of 4.3.3.c, (4.1.1.1) of 4.7.3.a, (4.1.1) of 4.9.2.a, (4.1.1) of 4.10.2.a, (4.1.1) of
4.13.1.a and (4.1.1) of 4.14.1.a.
Comment 4 When the determination is affected by the coloring of the sample solution of (4.1.1.1)d), (4.1.1.2) d) and (4.1.2) d), transfer the predetermined volume of the sample
solution to a 100-mL volumetric flask, add a few drops of hydrochloric acid (1+1) toacidify, then add no more than 0.1 g of active carbon. After leaving at rest for a little
while, add water up to the marked line and filter. The filtrate is prepared as the
sample solution of (4.2) a). Additionally, as phosphorus contained in active carbon
has the possibility to elute and affects the determination value, a blank test isrequired.
(4.1.1.2) Vertical reciprocating shaker:a) Weigh 2.5 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add about 200 mL of water, and shake to mix by reciprocating vertically at 300 times/min(amplitude of 40 mm) for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 5 The procedure in (4.1.1.2) is the same as the procedures in (4.1.1.2) of 4.2.4.b,(4.1.2.2) of 4.3.3.a and (4.1.1.2) of 4.7.3.a.
(4.1.2) Fluid test samplea) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 100-mL volumetricflask.
b) Add about 50 mL of water, and shake to mix.
129

Testing Methods for Fertilizers (2018)c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 6 The procedure in (4.1.2) is the same as the procedure in (4.1.2) of 4.2.4.b, (4.1.3) of
4.3.3.a, (4.1) of 4.3.3.d, (4.1) of 4.5.3.b, (4.1) of 4.6.3.b, (4.1.2) of 4.7.3.a, (4.1) of
4.7.3.b, (4.1) of 4.8.2.b, (4.1.2) of 4.9.2.a, (4.1.2) of 4.9.2.b, (4.1.2) of 4.10.2.a, (4.1)
of 4.10.2.b, (4.1.2) of 4.13.1.a, (4.1) of 4.13.1.b, (4.1.2) of 4.14.1.a, (4.1) of 4.14.1.b,(4.1.2) of 4.14.1.a, (4.1) of 4.14.1.b, (4.1) of 4.15.1.a and (4.1) of 4.15.1.b.
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 6 mg as
P2O5) to a 100-mL volumetric flask.
b) Add 4 mL of nitric acid (1+1) (6), and heat to boil (7).
c) After cooling is complete, add 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL), andneutralize by adding ammonia solution (1+1) until the color of the solution becomes light
red-purple (14).
d) Add nitric acid (1+10) until the light red-purple color of the solution disappears to make itslightly acidic, and add a proper amount of water (8).
e) Add 20 mL of coloring reagent solution, and further add water up to the marked line, and thenleave at rest for about 30 minutes (6).
Comment 7 The volumetric flask used in the procedure in a) should be distinguished as a flask to
be used for phosphate coloring operation and should not be used for other purposes.
Note (6) When the solution is muddled by adding nitric acid (1+1), conduct centrifugation after
the procedure in e).
(7) When it does not contain non-orthophosphate, the procedure in b) is not necessary.
(8) Without the addition of water, precipitate may be produced when a coloring reagent
solution is added.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 420 nmb) Calibration curve preparation
1) Transfer 1 mL - 12 mL of phosphoric acid standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add a proper amount of water (8), and conduct the same procedure as (4.2) e) to make the P2O5
0.5 mg/100 mL - 6 mg/100 mL phosphoric acid standard solutions for the calibration curve
preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blanktest solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 420 nm of the phosphoric acid standard solutions forthe calibration curve preparation using the blank test solution for the calibration curve
preparation as the control (9).
5) Prepare the calibration curve of the phosphoric acid concentration and absorbance of thephosphoric acid standard solutions for the calibration curve preparation.
c) Sample measurement
130Testing Methods for Fertilizers (2018)

1) Regarding the solution in (4.2) e), measure absorbance by the same procedure as b) 4) (9).
2) Obtain the phosphoric acid (P2O5) content from the calibration curve, and calculate
water-soluble phosphoric acid (W-P2O5) in the analytical sample.
Note (9) Measure within 6 hours after adding the coloring reagent solution in the procedure in(4.2) e).
Comment 8 After the procedure in (4.2) a), it is also possible to measure soluble phosphoric acidat the same time by adding 2 mL of Petermans citrate solution and by conducting theprocedures from (4.2) d) to (4.3) in 4.2.2.a (using b reagent solution in the Official
Methods of Analysis of Fertilizers (1992)).
Further after the procedure in (4.2) a), it is also possible to measure citrate soluble
phosphoric acid at the same time by adding 17 mL of citrate solution and by
conducting the procedures from (4.2) c) to (4.3) in 4.2.3.a (using b reagent solution
in the Official Methods of Analysis of Fertilizers (1992)).
Comment 9 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 10 % (mass fraction) 20 % (mass fraction) and 1 % (mass fraction) - 5 % (mass fraction) are 100.5 % 101.2 % and 99.0 % - 101.7 % as water-soluble phosphoric acid (W-P2O5)
respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
The comparison of the measurement value of extraction (yi：0.292 % (mass fraction)- 40.40 % (mass fraction)) with a vertical reciprocating shaker and the measurement
value of extract with a rotary shaker (xi) was conducted to evaluate trueness of theextraction of solid fertilizers using fertilizers (12 samples). As a result, a regression
equation was y = − 0.041 + 0.999x, and its correlation coefficient (r) was 1.000. Also,
the results of the repeatability tests on different days using compound fertilizers anddesignated blended fertilizers to evaluate precision were analyzed by one-way
analysis of variance. Table 2 shows the calculation results of intermediate precisionand repeatability
The comparison of the measurement value of simple extraction (yi：1.92 % (massfraction) - 12.21 % (mass fraction )) and the measurement value of extract (xi) with arotary shaker was conducted to evaluate trueness of the extraction of fluid fertilizers
using fluid fertilizers (12 samples). As a result, a regression equation was y =0.005+1.005x, and its correlation coefficient (r) was 0.999. Also, the results of therepeatability tests on different days using fluid mixed fertilizers to evaluate theprecision of the extraction of fluid fertilizers were analyzed by the one-way analysis
of variance. Table 3 shows the calculation results of intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.03 % (mass fraction) for solid fertilizers, and 0.004 % (mass fraction) for fluid
fertilizers.

131Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the collaborative study to determine certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
4)
5)
6)
7)
8)
9)
certified reference laboratory Average2)
sr
RSD rs I(T)RSD I(T)sR
RSD R
material fertilizer
FAMIC-B-10
FAMIC-B-14

1)

p
9
15

3)

(%)
7.00
6.70

3)

(%)
0.02
0.02

(%)
0.3
0.3

3)

(%)
0.03
0.03

(%)
0.5
0.5

3)

(%)
0.07
0.06

(%)
1.0
0.9

1） The number of laboaratories used for analysis
6） Intermediate standard deviation
conducting Ammonium vanadomolybdate absorptiometric analysis
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））7） Intermediate relative standard deviation

3） Mass fraction
4） Repeatability standard deviation

8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

5） Repeatability relative standard deviation

Table 2 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
Intermediate precision
4)
5)
6)
7)
samplerepeatability Average2)
sr
RSD rs I(T)RSD I(T)
1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)
Designated blended
7
13.77
0.03
0.2
0.06
0.5
fertilizer
Compound fertilizer
7
1.19
0.01
0.5
0.01
0.5
1） The number of test days conducting a duplicate test
6） Intermediate standard deviation
2） Average （the number of test days（T ）7） Intermediate relative standard deviation
×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

Table 3 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
samplerepeatability Averagesr
RSD rs I(T)RSD I(T)
1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)
Fluid mixed fertilizer 1
7
12.19
0.02
0.2
0.05
0.4
Fluid mixed fertilizer 2
7
2.88
0.01
0.2
0.02
0.5
1） The number of test days conducting a duplicate test
6） Intermediate standard deviation
2） Average （the number of test days（T ）7） Intermediate relative standard deviation
×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers
132

Testing Methods for Fertilizers (2018)

2)
3)

4)
5)

(Details), p.108- 114, Yokendo, Tokyo (1988)
Kimie KATO, Sakiko TAKAHASHI and Yuji SHIRAI: Validation of a Color metric
Method for Determination of Nitrogen, Phosphorus and Boron: Evaluation of
Calibration curve, Research Report of Fertilizer Vol. 2, p. 137 - 144 (2009)
Yoshiyuki SUNAGA, Yasushi SUGIMURA, Ichiro YOSHIDA and Hidenori KONISHI:
Verification of Performance Characteristics of Testing Method for Citrate-Soluble
Phosphorus Content by Ammonium Vanadomolybdate Absorption Photometry,
Research Report of Fertilizer Vol. 5, p. 167 - 179 (2012)
Shinji KAWAGUCHI: Simple Extraction Method for Water-Soluble Components in
Liquid Compound Fertilizers, Research Report of Fertilizer, Vol.9, p. 10 - 20 (2016)
Shinji KAWAGUCHI: Extraction Method for the Water-soluble Principal Ingredientsin the Solid Fertilizer using a General-purpose Equipment, Research Report of Fertilizer,
Vol.10, p. 1 - 8 (2017)133

Testing Methods for Fertilizers (2018)(5)

Flow sheet for water-soluble phosphoric acid: The flow sheet for water-soluble
phosphoric acid: in fertilizers is shown below:
5 g analytical sample(powdery)

Weigh to the order of 1 mg into a 500-mL volumetric flask← Water, about 400 mL
Shaking to mix

Rotary shaker (30 - 40 revolutions/min) for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for water-soluble phosphoric acid in fertilizers(Extraction procedure (4.1.1.1))

2.5 g analytical sample(powdery)

Weigh to the order of 1 mg into a 250-mL volumetric flask← Water, about 200 mL
Vertical reciprocating shaker(300 times/min, with amplitude
Shaking to mix
of 40 mm), for 30 minutes
Water
(up
to
the marked line)←
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for water-soluble phosphoric acid in fertilizers(Extraction procedure (4.1.1.2))

1 g analytical sample(fluid)Weigh to the order of 1 mg into a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-3 Flow sheet for water-soluble phosphoric acid in fertilizers(Extraction procedure (4.1.2))

134

Testing Methods for Fertilizers (2018)

Sample solution

Aliquot
100-mL volumetric flask(predetermined volume)← 4 mL of nitric acid(1+1)
Heating
Boiling
Cooling← 1-2 drop(s) of phenolphthalein solution (1 g/100 mL)← Ammonia solution (1+1) [neutralization]← Nitric acid (1+10) [slightly acidic]← A proper amount of water← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (420 nm)

Figure 2 Flow sheet for water-soluble phosphoric acid in fertilizers(Coloring and measurement procedure)135

Testing Methods for Fertilizers (2018)

4.2.4.b Ammonium vanadomolybdate absorptiometric analysis (Fertilizers containing
phosphorous acid or phosphite)(1) Summary
This testing method is applicable to the fertilizers containing phosphorous acid or phosphite. This
testing method is classified as Type B and its symbol is 4.2.4.b-2017 or W-P.b-1.
Extract by adding water to an analytical sample, add hydrochloric acid - sulfuric acid to heat, andoxygenate phosphonic acid ion to orthophosphate ion, and then measure the absorbance ofphosphovanadomolybdate salt formed by the reaction with ammonium vanadate (V),hexaammonium heptamolybdate and nitric acid to obtain water-soluble phosphoric acid (W-P2O5)
in an analytical sample. In addition, the performance of this testing method is shown in Comment
6.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
c) Ammonia solution: A JIS Guaranteed (NH3 28 % (mass fraction)) reagent specified in JIS K
8085 or a reagent of equivalent quality.
d) Coloring reagent solution (1) (2): Dissolve 1.12 g of ammonium vanadate (V)(3) specified in
JIS K 8747 in water, add 250 mL of nitric acid, then add 27 g of hexaammonium
heptamolybdate tetrahydrate(4) specified in JIS K 8905 dissolved in water, and further add
water to make 1000 mL (5).
e) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in JIS K
8799 in 100 mL of ethanol (95) specified in JIS K 8102.
f) Phosphoric acid standard solution (P2O5 10 mg/mL) (1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to cool in a
desiccator, and weigh 19.17 g to a weighing dish. Dissolve in a small amount of water,transfer to a 1000-mL volumetric flask, add 2 mL-3 mL of nitric acid, and add water up to themarked line.
g) Phosphoric acid standard solution (P2O5 0.5 mg/mL) (1): Transfer 50 mL of phosphoricacid standard solution (P2O5 10 mg/mL) to a 1000-mL volumetric flask, add 2 mL - 3 mL ofnitric acid, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to reagent “a” reagent solution in the Official Methods of Analysis of
Fertilizers (1992).
(3) This corresponds to ammonium metavanadate in the Official Methods of Analysis of
Fertilizers (1992).
(4) This corresponds to ammonium molybdate in the Official Methods of Analysis of
Fertilizers (1992).
(5) Store in an amber bottle.
Comment 1 Instead of the phosphoric acid standard solution in (2), a phosphoric acid standard
solution for a calibration curve can be prepared using a phosphoric acid standard
solution (P 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate water-soluble phosphoric acid (W-P2O5) in an analytical sample
by multiplying the concentration (P) of a phosphoric acid standard solution for acalibration curve or a measured value obtained in (4.3) by a conversion factor(2.2914).
136

Testing Methods for Fertilizers (2018)(3) Instruments: Instruments are as shown below:a) Extractor: A rotary shaker or a vertical reciprocating shaker as described below.
aa) Rotary shaker: A rotary shaker that can rotate a 250-mL - 500-mL volumetric flask upside
down at 30 - 40 revolutions/min.
ab) Vertical reciprocating shaker: A vertical reciprocating shaker that can shake a 250-mL
volumetric flask using an adapter to reciprocate vertically at 300 times/ minute (amplitude of
40 mm).
b) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
c) Spectrophotometer: A spectrophotometer specified in JIS K 0115
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test sample(4.1.1.1) Rotary shaker:a) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL volumetricflask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 2 In the procedure of (4.1.1.1) a), it is also allowed to weigh 2.5g of an analyticalsample to the order of 1 mg, and put it in a 250-mL volumetric flask
Comment 3 The procedure in (4.1.1.1) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
(4.1.1.2) Vertical reciprocating shaker:a) Weigh 2.5 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add about 200 mL of water, and shake to mix by reciprocating vertically at 300 times/min(amplitude of 40 mm) for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 4 The procedure in (4.1.1.2) is the same as the procedure in (4.1.1.2) in 4.2.4.a.
(4.1.2) Fluid test samplea) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 100-mL volumetricflask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 5 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.4.a.
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount (the equivalents of 0.5 mg - 6 mg as P2O5) of the sample
solution to a 100-mL - 200-mL tall beaker.
b) Add 3 mL of hydrochloric acid and 1 mL of nitric acid.
137

Testing Methods for Fertilizers (2018)c)

Cover the tall beaker with a watch glass, heat on a hot plate or sand bath at 200 ºC - 250 ºC
and condense (7) until the solution volume becomes about 2 mL (6).
d) After standing to cool, transfer it with water to a 100- mL volumetric flask (8).
e) Add 1 - 2 drop (s) of phenolphthalein solution (1 g/100 mL), and neutralize by adding
ammonia solution (1+1) until the color of the solution becomes light red-purple.
f) Add nitric acid (1+10) until the light red-purple color of the solution disappears to make itslightly acidic.
g) Add 20 mL of coloring reagent solution, and further add water up to the marked line, and thenleave at rest for about 30 minutes.
Note (6) It is recommended to transfer about 2 mL of water to a 100-mL - 200-mL tall beaker inadvance and confirm the volume.
(7) Care should be taken not to dry and harden it. When it dries completely, thedetermined value becomes lower than usual in some cases.
(8) The volume of the solution after transferring should be up to about 50 mL.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 420 nmb) Calibration curve preparation
1) Transfer 1 mL - 12 mL of phosphoric acid standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add a proper amount of water (9), and conduct the same procedure as (4.2) g) to make theP2O5 0.5 mg/100 mL - 6 mg/100 mL phosphoric acid standard solutions for the calibration
curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blanktest solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 420 nm of the phosphoric acid standard solutions forthe calibration curve preparation using the blank test solution for the calibration curve
preparation as the control (10).
5) Prepare the calibration curve of the phosphoric acid concentration and absorbance of thephosphoric acid standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) g), measure absorbance by the same procedure as b) 4) (10).
2) Obtain the phosphoric acid (P2O5) content from the calibration curve, and calculate
water-soluble phosphoric acid (W-P2O5) in the analytical sample.
Note (9) If water is not added, precipitate may form when adding the coloring reagent solution.
(10) Measure within 6 hours after adding the coloring reagent solution in the procedure in(4.2) g).
Comment 6 Recovery testing was conducted to evaluate trueness using a fluid preparation sample.
As a result, the average rate of recovery at the content level of 30 % (mass fraction) 50 % (mass fraction), 10 % (mass fraction) - 20 % (mass fraction), 4 % (massfraction) and 0.2 % (mass fraction) are 101.1 % - 101.8 %, 101.1 % - 101.5 %,100.8 % and 102.5 % as water-soluble phosphoric acid (W-P2O5) respectively. In thecase of using a solid preparation sample, the average rate of recovery at the content
138

Testing Methods for Fertilizers (2018)

level of 30 % (mass fraction) - 59 % (mass fraction), 12 % (mass fraction) - 21 %(mass fraction) and 1 % (mass fraction) - 9 % (mass fraction) are 99.5 % - 100.4 %,99.3 % - 100.3 %, and 96.9 % - 100.4% respectively.
The results of the repeatability tests on different days using a solid preparation
sample to evaluate precision were analyzed by one-way analysis of variance. Table 1
shows the calculation results of intermediate precision and repeatability.
Also, table 2 and table 3 show results and analysis results from a collaborative study
for testing method validation using fluid fertilizers and solid fertilizers.
Additionally, the minimum limit of quantification of this testing method is about
0.04 % (mass fraction) for solid fertilizers, and 0.01 % (mass fraction) for fluid
fertilizers.
Table 1 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
4)
5)
samplerepeatability Average2)
sr
RSD r
1)

Preparation sample (solid) 1

T
7

3)

(%)
59.36

Preparation sample (solid) 2
75.90
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

139

3)

(%)
0.09
0.07

(%)
0.2

Intermediate precision
6)s I(T)

3)

(%)
0.13

7)

RSD I(T)
(%)
0.2

1.2
0.07
1.2
6） Intermediate standard deviation
7） Intermediate relative standard deviation

Testing Methods for Fertilizers (2018)

Table 2 Results and analysis results from a collaborative study
for the test method validation of water-soluble phosphoric acid
Sample name

Number of1)laboratories

4)

RSD r

3)

(%)

Mean

2)

sr

3)

(%)

(%)

5)

Fluid mixed
12
33.56
0.25
0.7
fertilizers 1
Fluid mixed
12
17.93
0.08
0.5
fertilizers 2
Fluid mixed
12
7.99
0.12
1.5
fertilizers 3
Fluid mixed
11
11.93
0.13
1.1
fertilizers 4
Fluid mixed
11
24.1
0.08
0.3
fertilizers 51) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

6)

RSD R

(%)

3)

(%)

0.59

1.8

0.30

1.7

0.31

3.8

0.33

2.8

0.47

2.0

sR

7)

Table 3 Results and analysis results from a collaborative study
for the test method validation of water-soluble phosphoric acid (Solid fertilizer)
Sample name

Number of1)laboratories

4)

RSD r

3)

(%)

Mean

2)

sr

3)

(%)

(%)

Compound
12
58.47
0.13
0.2
fertilizers 1
Compound
12
3.92
0.04
1.0
fertilizers 2
Compound
12
13.37
0.10
0.7
fertilizers 3
Absorptive mixed
12
7.16
0.03
0.4
fertilizers
Blended fertilizers
12
21.80
0.12
0.5
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation
140

5)

6)

RSD R

(%)

3)

(%)

0.42

0.7

0.08

2.1

0.20

1.5

0.16

2.3

0.18

0.8

sR

7)

Testing Methods for Fertilizers (2018)

References1)
Toshiaki HIROI, Masakazu SAIKI and Kimie KATO: Development and Validation ofSpectrophotometry for Determination of Water-Soluble Phosphoric acid in Liquid
Mixed Fertilizer Containing Phosphonic acid, Research Report of Fertilizer, Vol.1 P. 25- 33 (2008)2)
Toshiaki HIROI, Masakazu SAIKI and Kimie KATO: Determination of Water-Soluble
Phosphoric Acid in Liquid Mixed Fertilizer Containing Phosphonic Acid bySpectrophotometry: A Collaborative Study, Research Report of Fertilizer, Vol.1 p. 34 40 (2008)
3)
Fumihiro ABE, Noriyuki SASAKI and Toshio HIRABARA: Determination of
Water-Soluble Phosphorus in Solid Fertilizer Containing Phosphonic Acid bySpectrophotometry, Research Report of Fertilizer, Vol.8, p. 10 - 16 (2015)
4)
Masayuki YAMANISHI, Toshiaki HIROI and Fumika TAKATSU Determination
Method for Citric Acid-Soluble and Water-Soluble Phosphorus in Solid Fertilizer
Containing Phosphonic Acid or Phosphonate (Phosphite) using Spectrophotometer: A
Collaborative Study, Vol.9, p. 59 - 68 (2016)(5)

Flow sheet for water-soluble phosphoric acid containing phosphorus acid, etc.: The flow
sheet for water-soluble phosphoric acid in fertilizers containing phosphorus acid, etc.is shown
below:
5 g analytical sample(powdery)

Weigh to the order of 1 mg into a 500-mL volumetric flask← Water, about 400 mL
Shaking to mix
Rotary shaker (30 - 40 revolutions/min) for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1-1 Flow sheet for water-soluble phosphoric acid in fertilizers
containing phosphorus acid, etc. (Extraction procedure (4.1.1.1))
2.5 g analytical sample(powdery)

Weigh to the order of 1 mg into a 250-mL volumetric flask← Water, about 200 mL
Vertical reciprocating shaker(300 times/min, with amplitude of
Shaking to mix
40 mm), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1-2 Flow sheet for water-soluble phosphoric acid in fertilizers
containing phosphorus acid, etc. (Extraction procedure (4.1.1.2))
141

Testing Methods for Fertilizers (2018)

1 g analytical sample(fluid)Weigh to the order of 1 mg into a 1000-mL volumetric flask← Water, about 200 mL
Shaking to mix← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1-3 Flow sheet for water-soluble phosphoric acid in fertilizers
containing phosphorus acid, etc. (Extraction procedure (4.12))

Sample solution

Aliquot
100-mL - 200-mL tall beaker(predetermined volume)← 3 mL of hydrochloric acid← 1 mL of nitric acid
Cover the tall beaker with a watch glass, heat on a hot plate or sand bath
Heating
at 200 ºC - 250 ºC, condense until the solution volume reaches about 2 mL
Standing to coolTransfer
100-mL volumetric flask, water← 1-2 drop(s) of phenolphthalein solution (1 g/100 mL)← Ammonia solution (1+1) [neutralization]← Nitric acid (1+10) [slightly acidic]← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (420 nm)

Figure 2 Flow sheet for water-soluble phosphoric acid in fertilizers
containing phosphorus acid, etc. (Coloring and measurement procedure)

142

Testing Methods for Fertilizers (2018)

4.2.4.c Quinoline gravimetric analysis(1) Summary
This testing method is applicable to fertilizers containing no phosphorous acid, etc. It is suitable forthe fertilizers with relatively a high content of phosphoric acid. This testing method is classified as
Type E and its symbol is 4.2.4.c-2017 or W-P.c-1.
Extract by adding water to an analytical sample. Heat after adding nitric acid and water, hydrolyze
nonorthophosphoric acid to orthophosphate ion and measure the mass of quinonium
phosphomolybdate formed by the reaction with quinoline, molybdic acid and nitric acid to obtainwater-soluble phosphoric acid (W-P2O5) in an analytical sample.
(2) Reagent: Reagents are as shown below.
a) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
b) Sodium molybdate solution: Dissolve 70 g of sodium molybdate dihydrate in 150 mL ofwater.
c) Quinoline solution: Add 5 mL of quinoline specified in JIS K 8279 to the mixture solutionof 35 mL of nitric acid and 100 mL of water.
d) Quimosiac solution: Add 60 g of citric acid monohydrate specified in JIS K 8283 to the
mixture solution of 85 mL nitric acid and 150 mL of water to dissolve. Add gradually total
volume of the sodium molybdate solution to mix. Add gradually the total volume of thequinoline solution while mixing the solution. After leaving at rest overnight, filter the total
volume with Type 3 filter paper. Add 280 mL of acetone specified in JIS K 8034, and further
add water to make 1000 mL.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Water bath: Water bath that can be adjusted to 60 ºC - 65 ºC.
c) Drying apparatus: A drying apparatus that can be adjusted to 220 ºC ± 5 ºC.
d) Crucible type glass filter: A crucible type glass filter 1G4 specified in JIS R 3503. Let it
stand to cool in a desiccator after heating at 220 ºC ± 5 ºC in advance and measure the mass tothe order of 1 mg.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL volumetricflask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 1 In the procedure in a), it is also allowed to weigh 2.5 g of an analytical sample to theorder of 1 mg, and put it into a 250-mL volumetric flask.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) of 4.2.4.a.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 10 mg - 30 mg as P2O5 and no more than
20 mL as the total solution volume) of sample solution to a 300-mL tall beaker.
b) Add 5 mL of nitric acid and add water to make 80 mL.
c) Cover with a watch glass. After boiling for about 3 minutes, wash the watch glass and the143Testing Methods for Fertilizers (2018)

inside the tall beaker with water and add water to make 100 mL.
d) Immediately, add 50mL of quimosiac solution, heat for about 15 minutes while sometimes
mixing in a water bath at 60 ºC - 65 ºC to produce the precipitate of quinolinium
phosphomolybdate.
e) After standing to cool down to room temperature while sometimes mixing, filter under
reduced pressure with a crucible type glass filter, wash the tall beaker 3 times with water andtransfer the whole precipitate into a crucible type glass filter, and further wash 7 - 8 times withwater.
f) Transfer the precipitate together with the crucible type glass filter into a drying apparatus andheat at 220 ºC ± 5 ºC for about 30 minutes.
g) As soon as heating is complete, move it into a desiccator and let it stand to cool.
h) After standing to cool, remove the crucible type glass filter from the desiccator and measure
the mass to the order of 1 mg.
i) Calculate water-soluble phosphoric acid (W-P2O5) by the following formula.
Soluble phosphoric acid (% (mass fraction)) in an analytical sample= A × 0.03207 × (V1/V2) × (1/W) × 100
A：
W：
V1：
V2：

Mass (g) of the precipitate in h)
Mass of an analytical sample (5 g)Predetermined volume (500 mL) of the sample solution
Volume (mL) of the sample solution transferred in a)

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.98- 114, Yokendo, Tokyo (1988)(5)

Flow sheet for water-soluble phosphoric acid: The flow sheet for water-soluble phosphoricacid: in fertilizers is shown below:
5 g analytical sample
Weigh to the order of 1 mg to a 500-mL volumetric flask← About 400 mL of water
Shaking to mix
Rotary shaker (30 - 40 revolutions/min) for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for water-soluble phosphoric acid in fertilizers(Extraction procedure)

144

Testing Methods for Fertilizers (2018)

Sample solution

Aliquot
300-mL tall beaker(predetermined volume)← 5 mL nitrate acid
← Water (to make about 80 mL)
Cover with a watch glass for 3 minutes
Boiling
Wash a watch glass and the inside the tall beaker with water← Water (to make about 100 mL)← 50 mL quimosiac solution
Forming precipitate
60 ºC - 65 ºC, for 15 minutes, some times mix
Standing to cool
Filtration
under reduced pressure

Room temperature

Crucible type glass filtering apparatus 1G4, 3 times with water

Washing

Wash 7 - 8 times with water

Drying220 ºC ± 5 ºC for about 30 minutes

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure 2 Flow sheet for water-soluble phosphoric acid in fertilizers(Measurement procedure)

145

Testing Methods for Fertilizers (2018)

4.2.4.d ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers and the fluid fertilizers of homegarden-use mixed fertilizers. It is also suitable for the fertilizers containing phosphorus acid(phosphite). This testing method is classified as Type D and its symbol is 4.2.4.d-2017 or W-P.d-1.
Extract by adding water to an analytical sample, introduce it to an ICP Optical Emission
Spectrometer (“ICP-OES”) and measure the phosphorus at a wavelength of 178.287 nm to obtainwater-soluble phosphoric acid W-P2O5) in an analytical sample. In addition, the performance of thistesting method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Phosphoric acid standard solution (P2O5 10 mg/mL) (1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to cool in a
desiccator, and weigh 19.17 g to a weighing dish. Dissolve in a small amount of water,transfer to a 1000-mL volumetric flask, add 2 mL-3 mL of nitric acid, and add water up to themarked line.
d) Phosphoric acid standard solution (P2O5 1 mg/mL) (1): Transfer 10 mL of phosphoric acidstandard solution (P2O5 10 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid
(1+23) up to the marked line.
e) Phosphoric acid standard solution (P2O5 20 µg/mL - 0.4 mg/mL) for the calibration
curve preparation (1): Transfer 2 mL - 40 mL of phosphoric acid standard solution (P2O5 1
mg/mL) to a 100-mL volumetric flask step by step and add hydrochloric acid (1+23) up to themarked line.
f) Phosphoric acid standard solution ((P2O5 5 µg/mL - 20 µg/mL) for the calibration curve
preparation (1): Transfer 5 mL - 20 mL of phosphoric acid standard solution (P2O5 0.1
mg/mL) to a 100-mL volumetric flask step by step and add hydrochloric acid (1+23) up to themarked line.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d), e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the phosphoric acid standard solution in (2), a phosphoric acid standard
solution for a calibration curve can be prepared using a phosphoric acid standard
solution (P 1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case,calculate water-soluble phosphoric acid (W-P2O5) in an analytical sample bymultiplying the concentration (P) of a phosphoric acid standard solution for acalibration curve or a measured value obtained in (4.2) by a conversion factor(2.2914).
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal
observation mode and an axial observation mode. The range of the concentration of
the standard solution for the calibration preparation curve in d) and e) applies to ahorizontal observation mode. While an axial observation mode can observe ameasurement component of low-level concentrations, it cannot gain the linearity ofa calibration curve in the range of high-level concentrations. Therefore, when using
the ICP-OES of an axial observation mode, it is recommended to prepare aphosphoric acid standard solution for the calibration curve in the concentration
146

Testing Methods for Fertilizers (2018)

range which is suitable to a device used.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample (2) to the order of 1 mg, and put it in a 100-mL volumetricflask.
b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (2) The sampling amount of the analytical sample is 10 g when there is less phosphoricacid content in the fertilizers such as a home garden-use fertilizer.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up the measurement
conditions for the ICP Atomic Emission Spectrometer considering the following:
Analytical line wavelength: 178.287 nmb) Calibration curve preparation
1) Spray the phosphoric acid standard solution for the calibration curve preparation and blanktest solution for the calibration curve preparation into inductively coupled plasma, and readthe indicated value at a wavelength of 178.287 nm.
2) Prepare a curve for the relationship between the phosphoric acid concentration and theindicated value of the phosphoric acid standard solution for the calibration curve preparationand the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 40 mg as
P2O5) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the phosphoric acid (P2O5) content from the calibration curve, and calculate
water-soluble phosphoric acid (W-P2O5) in the analytical sample.
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. In
this case, transfer a pre-determined amount of phosphoric acid standard solution (P 1
mg/mL or 10 mg/mL), potassium standard solution (K 1 mg/mL or 10 mg/mL),
magnesium standard solution (Mg 1 mg/mL or 10 mg/mL), manganese standard
solution (Mn 1 mg/mL or 10 mg/mL), boron standard solution (B 1 mg/mL or 10
mg/mL), calcium standard solution (Ca 1 mg/mL), iron standard solution (Fe 1
mg/mL), cobalt standard solution (Co 1 mg/mL), copper standard solution (Cu 1
mg/mL), zinc standard solution (Zn 1 mg/mL) and molybdenum standard solution(Mo 1 mg/mL) traceable to National Metrology to a volumetric flask to mix, addhydrochloric acid (1+5) to make an acid concentration of 0.5 mol/L and further add
water up to the marked line to prepare a primary mixed standard solution. Transfer a147

Testing Methods for Fertilizers (2018)pre-determined volume of primary mixed standard solution to volumetric flasks
step-by-step, add hydrochloric acid (1+23) up to the marked line to prepare mixed
standard solutions for calibration curve preparation within the concentration range inTable 1. In this case, multiply the respective concentrations of mixed standard
solutions for calibration curve preparation or measurement values obtained in (4.2)
by the conversion factors in Table 1 to calculate respective main components in ananalytical sample. The measurement wavelengths of respective elements areaccording to Table 1. In addition, when preserving mixed standard solutions forcalibration curve preparation, use a container, which can be sealed tightly, made ofmaterial such as PTFE that boron hardly elutes.

Table 1Name of test item

Preparation concentration and measurement wavelength of mixed standard solution
for calibration curve preparation
Mixed standard solutions for calibration curve preparation
Concentrationof element(µg/mL)

Water-soluble phosphoric acidP
1~200
Water-soluble potassium
K
1~200
Water-soluble magnesia
Mg 0.1~20
Water-soluble manganese Mn 0.05~10
Water-soluble boron
B 0.05~10
Water-soluble calcium
Ca 0.1 - 20
Water-soluble zinc
Zn 0.1 - 20
Water-soluble copper
Cu 0.1 - 20
Water-soluble ironFe 0.1 - 20
Water-soluble molybdenumMo 0.1 - 20
Water-soluble cobalt
Co 0.1 - 20
1） Factor to convert an element to oxide

Concentration of the equivalents tooxide(µg/mL)
P2O5
K2 O
MgO
MnO
B2O3

2.291~458.2
1.205~241.0
0.1658~33.16
0.06455~12.91
0.1610~32.20-

Conversion
factor1)

2.29141.20461.6583
1.2912
3.2199-Measurement
wavelength(nm)
178.287
766.491279.553257.610249.773
393.366
213.856
327.396259.940202.030228.616

Comment 5 The comparison of the measurement value (yi：0.179 % (mass fraction) - 10.88 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of Ammonium vanadomolybdate absorptiometric analysis was conducted toevaluate trueness using fluid fertilizers (12 samples). As a result, a regression
equation was y = − 0.022 + 1.008x, and its correlation coefficient (r) was 0.999.
Additionally, additive recovery testing was conducted using a fluid mixed fertilizer(1 brand) and a home garden-use mixed fertilizer (1 brand). As a result, the mean
recovery rates at additive level of 10 % (mass fraction) and 1 % (mass fraction) were98.1 % and 101.9 % respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 2 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.02 % (mass fraction).

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Testing Methods for Fertilizers (2018)
Table 2 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
2)4)
5)
samplerepeatability Average
sr
RSD r
1)

T
7

3)

(%)
10.83

3)

(%)
0.10

(%)
0.9

Intermediate precision
6)s I(T)

3)

(%)
0.14

RSD I(T)
(%)
1.3

7)

Fluid mixed fertilizer
Home garder-use
mixed fertilizer
7
0.829
0.008
0.9
0.015
1.8
（fluid)1） The number of test days conducting a duplicate test 6） Intermediate standard deviation
2） Average （the number of test days（T ）7） Intermediate relative standard deviation
×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

References
1)
Keisuke AOYAMA: Simultaneous Determination of Water-Soluble Principal
Ingredients (W-P2O5, W-K2O, W-MgO, W-MnO and W-B2O3) in Liquid Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), Research
Report of Fertilizer, Vol.8, p. 1 - 9 (2015)(5)

Flow sheet: The flow sheet for water-soluble phosphoric acid of the fluid mixed fertilizers isshown below:
1 g analytical sample
Weigh to the order of 1 mg to a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 The flow sheet for water-soluble phosphoric acid in fluid fertilizers(Extraction procedure)

149

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
ICP-OES (178.287nm)
Figure 2 The flow sheet for water-soluble phosphoric acid in fluid fertilizers(Measurement procedure)

150

Testing Methods for Fertilizers (2018)

4.3 Potassium
4.3.1 Total potassium
4.3.1.a Flame atomic absorption spectrometry or flame photometry(1) Summary
This testing method is applicable to fertilizers containing organic matters. This testing method isclassified as Type C and its symbol is 4.3.1.a-2017 or T-K.a-1.
Pretreat an analytical sample with incineration and hydrochloric acid to convert the total potassium
into potassium ion, add an interference suppressor solution, and then spray in an acetylene-air flame,
and measure the atomic absorption with potassium at a wavelength of 766.5 nm or 769.9 nm toquantify the total potassium. Or, determine the intensity of the emission line at a wavelength of
766.5 nm or 769.9 nm produced in flame to obtain the total potassium (T-K2O) in an analyticalsample. In addition, the performance of this testing method is shown in Comment 4.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
c) Interference suppressor solution: Weigh 12.5 g of calcium carbonate specified in JIS K
8617 into a 2000-mL beaker, add a small amount of water, gradually add 105 mL ofhydrochloric acid, and heat for a little while. After cooling is complete, add water to make1000 mL.
d) Potassium standard solution (K2O 1 mg/mL) (1): Heat potassium chloride specified in JIS K
8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, and weigh 1.583 g
into a weighing dish. Dissolve in a small amount of water, transfer to a 1000-mL volumetricflask, and add water up to the marked line. Dissolve in a small amount of water, transfer to a
1000-mL volumetric flask, and add water up to the marked line.
e) Potassium standard solution (K2O 5 µg/mL - 50 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of potassium standard solution (K2O 1 mg/mL) to
500-mL volumetric flasks step-by-step, add about 50 mL of interference suppressor solution(2), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 50 mL ofinterference suppressor solution to a 500-mL volumetric flask (2), and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the potassium standard solution in (2), a potassium standard solution forthe calibration curve preparation can be prepared by using a potassium standard
solution (K 1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case,calculate total potassium (T-K2O) in the analytical sample by multiplying theconcentration (K) of a potassium standard solution for calibration curve preparation
or a measurement value (K) obtained in (4.2) by a conversion factor (1.2046).
(3) Instruments: Instruments are as shown below:a) Analytical instrument: An atomic absorption spectrometer or a flame photometer as shown
below:aa) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in151

Testing Methods for Fertilizers (2018)

JIS K 0121.
1) Light source: A potassium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
ab) Flame photometer:
1) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 550 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Incineration-hydrochloric acid boilinga) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 200-mL - 300-mL tallbeaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (3).
c) Ignite at 550 ºC ± 5 ºC for no less than 4 hours to incinerate (3).
d) After standing to cool, moisten the residue with a small amount of water, gradually add about
10 mL of hydrochloric acid, and further add water to make about 20 mL.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to boil forabout 5 minutes.
f) After cooling is complete, transfer to a 250-mL - 500-mL volumetric flask with water.
g) Add water up to the marked line.
h) Filter with Type 3 filter paper to make a sample solution.
Note (3) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 550 °C in 1 to 2 hours.
Comment 2 The procedure in (4.1.1) is the same as the procedure in (4.1.2) in 4.2.1.a.
(4.1.2) Incineration-aqua regia digestiona) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 200-mL - 300-mL tallbeaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (4).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (6) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL volumetric flask with water, add water up to themarked line, and filter with Type 3 filter paper to make a sample solution.
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Testing Methods for Fertilizers (2018)

Note (4) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 450 °C in 1 to 2 hours.
(5) The watch glass can be removed.
(6) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 3 The procedures in (4.1.2) are the same as in (4.1.3) in 4.2.1.a. and (4.1) a) - h) in
5.3.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer or flame photometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer or flame photometer:
Set up the measurement conditions for the atomic absorption spectrometer or flame
photometer considering the following:
Analytical line wavelength: 766.5 nm or 769.9 nmb) Calibration curve preparation
1) Spray the potassium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 766.5 nm or 769.9 nm.
2) Prepare a curve for the relationship between the potassium concentration and the indicatedvalue of the potassium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 5 mg as
K2O) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (2), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the potassium content from the calibration curve, and calculate total potassium(T-K2O) in the analytical sample.
Comment 4 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 10 % (mass fraction) 20 % (mass fraction) and 1 % (mass fraction) - 5 % (mass fraction) are 97.8 % 100.1 % and 100.9 % - 103.1 % as total potassium acid (T-K2O) respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.08 % (mass fraction) for solid fertilizers, and 0.03 % (mass fraction) for fluid
fertilizers.

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Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
2)
4)
5)
6)
7)
8)
9)
certified reference laboratory Averagesr
RSD rs I(T)RSD I(T)
sR
RSD R
1)
3)
3)
material fertilizer
(%)
p
(%)
(%)
FAMIC-C-12
11
0.584
0.005
0.9
1） The number of laboaratories used for analysis
conducting flame atomic absorbance spectrometry
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

3)

3)

(%)
(%)
(%)
0.011
1.9
0.038
6） Intermediate standard deviation

(%)
6.5

7） Intermediate relative standard deviation
8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.132- 138, Yokendo, Tokyo (1988)
2)
Kimie KATO, Masayuki YOSHIMOTO and Yuji SHIRAI: Systematization of
Determination Methods of Major Components in Sludge Fertilizer, Compost andOrganic Fertilizer, Research Report of Fertilizer, Vol.3 p. 107 - 116 (2010)3)
Yasuharu KIMURA and Hisanori ARAYA Verification of Performance Characteristicsof Testing Methods for Potassium Content in Fertilizer by Atomic Absorption
Spectrometry, Research Report of Fertilizer Vol. 5, p. 190 - 200 (2012)154

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total potassium: The flow sheet for total potassium in fertilizers is shown
below:
5 g analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL - 300-mL tall beaker.
Heat gently
550 ºC ± 5 ºC, no less than 4 hours

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of hydrochloric acid
← Water (up to about 20 mL)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolTransfer

Room temperature
250-mL - 500-mL volumetric flask, water← Water (up to the marked line)
Filtration

Type 3 filter paper

Sample solution
Figure 1-1 Flow sheet for total potassium in fertilizers(Incineration-hydrochloric acid boiling procedure (4.1.1))

155

Testing Methods for Fertilizers (2018)5g
analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC for 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass to digest
Heating

Slightly move the watch glass and remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass to dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for total potassium in fertilizers( Incineration-aqua regia digestion procedure (4.1.2))

Sample solution
Aliquot
100-mL volumetric flask, water← About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer or flame photometer
Figure 2 Flow sheet for total potassium in fertilizers (Measurement procedure)

156

Testing Methods for Fertilizers (2018)

4.3.1.b Sodium tetraphenylborate gravimetric analysis(1) Summary
This testing method is applicable to fertilizers containing organic matters. It is suitable for fertilizers
containing relatively a high content of potassium. This testing method is classified as Type D and its
symbol is 4.3.1.b-2017 or T-K.b-1.
Pretreat an analytical sample by incineration and hydrochloric acid to convert the total potassium
into potassium ion, mask co-existing ammonium and other salts with formaldehyde andethylenediamine tetraacetate, and measure the mass of potassium tetraphenylborate formed by thereaction with tetraphenylborate to obtain the total potassium (T-K2O) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 2.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Formaldehyde solution: A JIS Guaranteed Reagent specified in JIS K 8872 or a reagent ofequivalent quality.
c) Sodium hydroxide solution (200 g/L) (1): Dissolve 200 g of sodium hydroxide specified in
JIS K 8576 in water to make 1000 mL.
d) Aluminum chloride solution (1 mol/L) (1): Dissolve 12 g of aluminum chloride (III)hexahydrate specified in JIS K 8114 in water to make 100 mL.
e) Tetraphenylborate solution (1): Transfer 6.1 g of sodium tetraphenylborate specified in JIS K
9521to a 250-mL volumetric flask, dissolve by adding about 200 mL of water and add 10 mLof aluminum chloride solution. Add a methyl red solution (0.1 g/100 mL) as an indicator, andneutralize with a sodium hydroxide solution (200 g/L) until the color of the solution changes
to yellow, and then add water up to the marked line. Filter with Type 3 filter paper and add 0.5
mL of sodium hydroxide solution (200 g/L) to the total filtrate. Filter with Type 3 filter paperin the case of usage.
f) Tetraphenylborate washing solution (1): Dilute 40 mL of tetraphenylborate solution withwater to make 1000 mL.
g) Ethylenediaminetetraacetate - Sodium hydroxide solution (1): Dissolve 10 g ofethylenediaminetetraacetic acid disodium dihydrogen dihydrate specified in JIS K 8107 and 8
g of sodium hydroxide specified in JIS K 8576 in a proper amount of water. Add 6 mL - 10
mL of tetraphenylborate solution while mixing according to the potassium content coexisting
as impurity after standing to cool, and then add water to make 100 mL. After leaving at rest
for about 30 minutes while sometimes mixing, filter with Type 3 filter paper.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K 8896
in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Electric furnace: An electric furnace that can be adjusted to 550 ºC ± 5 ºC.
b) Drying apparatus: A drying apparatus that can be adjusted to 120 ºC ± 2 ºC.
c) Crucible type glass filter: A crucible type glass filter 1G4 specified in JIS R 3503. Let it
stand to cool in a desiccator after heating at 120 ºC ± 2 ºC in advance and measure the mass tothe order of 1 mg.
d) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
157

Testing Methods for Fertilizers (2018)(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh about 5 g of an analytical sample to the order of 1 mg, and transfer to a 200-mL 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (2).
c) Ignite at 550 ºC ± 5 ºC for no less than 4 hours to incinerate (2).
d) After standing to cool, moisten the residue with a small amount of water, gradually add about
10 mL of hydrochloric acid, and further add water to make 20 mL.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to boil forabout 5 minutes.
f) After cooling, transfer to a 250-mL - 500-mL volumetric flask with water.
g) Add water up to the marked line.
h) Filter with Type 3 filter paper to make a sample solution.
Note (2) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 550 °C in 1 to 2 hours.
Comment 1 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.1.a. In addition,
the sample solution prepared in (4.1) a) - h) in 4.9.1.a can also be used.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined amount (the equivalents of 15 mg - 30 mg as K2O) of the sample
solution to a 100-mL tall beaker.
b) Add water to the solution to reach 50 mL when the procedure in e) is complete.
c) Add hydrochloric acid (1+9), so that the hydrochloric acid becomes equivalent to 0.2 mL.
d) Add 5 mL of formaldehyde solution, and then add 5 mL of ethylenediamine
tetraacetate−sodium hydroxide solution.
e) Add necessary volume (3) of tetraphenylborate solution at the rate of one or two drop(s) per
second while mixing, and further add 4 mL of the same solution in the same manner.
f) Leave at rest for about 30 minutes while sometimes mixing to form the precipitate ofpotassium tetraphenylborate.
g) Filter supernatant under reduced pressure with a crucible type glass filter, wash the tall beaker
5 times with 5 mL of tetraphenylborate washing solution and transfer the whole precipitate to
the crucible type glass filter and further wash 2 times with 2 ml of water.
h) Transfer the precipitate together with the crucible type glass filter into a drying apparatus andheat at 120 ºC ± 2 ºC for about 1hour.
i) As soon as heating is complete, move it into a desiccator and let it stand to cool.
j) After standing to cool, remove the crucible type glass filter from the desiccator and measure
the mass to the order of 1 mg.
k) Calculate total potassium (T-K2O) in the analytical sample by the following formula.
Total potassium (T-K2O) (% (mass fraction)) in an analytical sample= A × 0.1314 × (V1/V2)/W × 100
A： Mass (g) of the precipitate
V1： Predetermined volume (mL) of the sample solution in (4.1) g)
V2： Transferred amount (mL) of the sample solution in (4.2) a)
W： Mass (g) of the analytical sample
158

Testing Methods for Fertilizers (2018)

Note (3) About 3 ml of tetraphenylborate solution per 10 mg of K2O is required to form theprecipitate of potassium tetraphenylborate
Comment 2 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 25 % (mass fraction) 30 % (mass fraction) and 10 % (mass fraction) - 20 % (mass fraction) are 99.5 % 100.8 % and 99.5 % - 100.6 % as total potassium acid (T-K2O) respectively.
Additionally, the minimum limit of quantification of this testing method is about
0.3 % (mass fraction).
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.122- 128, Yokendo, Tokyo (1988)
2)
Keiji YAGI, Aiko YANO and Hideo SOETA: Verification of Performance
Characteristics of Testing Method for Nitrate Nitrogen Content in Fertilizer by Phenol
Sulfuric Acid Method, Research Report of Fertilizer Vol. 5, p. 201 - 211 (2012)159

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total potassium:
below:

5 g analytical sample
Charring
Incineration

The flow sheet for total potassium in fertilizers is shown

Weigh to the order of 1 mg into a 200-mL - 300-mL tall beaker.
Heat gently
Ignite at 550 ºC ± 5 ºC, no less than 4 hours

Standing to coolRoom temperatureA
small
amountof water, moisten the residue←← About 10 mL of hydrochloric acid
← Water (up to about 20 mL)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolRoom temperature

Transfer
250-mL - 500-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1 Flow sheet for total potassium in fertilizers (Extraction procedure)160

Testing Methods for Fertilizers (2018)

Sample solution

Aliquot(predetermined
100-mL tall beakeramount)← Water (add tetraphenylborate solution to reach 50 mL)← Hydrochloric acid (1+9) (the equivalents of 0.2 mL of hydrochloric acid)← 5 mL of formaldehyde solution← 5 mL of ethylenediamine tetraacetate- sodium hydroxide solution← Tetraphenylborate solution (necessary volume + 4 mL)
Forming precipitate
For 30 minutes, sometimes mixing
Filtration
under reducedpressure

Crucible type glass filtering apparatus 1G4,
5 times with 5 mL of tetraphenylborate washing solution

Washing

Wash 2 times with 2 mL of water

Drying120 ºC ± 5 ºC for 1 hour.

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure 2 Flow sheet for total potassium in fertilizers (Measurement procedure)

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Testing Methods for Fertilizers (2018)

4.3.2 Citrate soluble potassium
4.3.2.a Flame atomic absorption spectrometry or flame photometry(1) Summary
This testing method is applicable to fertilizers containing potassium silicate fertilizers, etc. This
testing method is classified as Type D and its symbol is 4.3.2.a-2018 or C-K.a-2.
Extract by adding a citric acid solution to an analytical sample, add an interference suppressor
solution, and then spray in an acetylene−air flame, and measure the atomic absorption withpotassium at a wavelength of 766.5 nm or 769.9 nm to quantify citrate soluble potassium (C-K2O).
Or, measure the intensity of the emission line at a wavelength of 766.5 nm or 769.9 nm produced inflame to quantify citrate soluble potassium (C-K2O) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 5.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
c) Interference suppressor solution (1): Weigh 12.5 g of calcium carbonate specified in JIS K
8617 into a 2000-mL beaker, add a small amount of water, gradually add 105 mL ofhydrochloric acid, and heat for a little while. After standing to cool, add water to make 1000mL.
d) Potassium standard solution (K2O 1 mg/mL) (1): Heat potassium chloride specified in JIS K
8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, and weigh 1.583 g
into a weighing dish. Dissolve in a small amount of water, transfer to a 1000-mL volumetricflask, and add water up to the marked line.
e) Potassium standard solution (K2O 5 µg/mL - 50 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of potassium standard solution (K2O 1 mg/mL) to
500-mL volumetric flasks step-by-step, add about 50 mL of interference suppressor solution(2), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 50 mL ofinterference suppressor solution to a 500-mL volumetric flask (2), and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the potassium standard solution in (2), a potassium standard solution forthe calibration curve preparation can be prepared by using a potassium standard
solution (K 1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case,calculate citrate-soluble potassium (C-K2O) in the analytical sample by multiplying
the concentration (K) of a potassium standard solution for calibration curve
preparation or a measurement value (K) obtained in (4.2) by a conversion factor(1.2046).
(3) Instruments: Instruments are as shown below:a) Extractor: A constant-temperature rotary shaker or a reciprocating water bath shaker as
described below.
aa) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at162

Testing Methods for Fertilizers (2018)30 - 40 revolutions/min.
ab) Reciprocating water bath shaker: A reciprocating water bath shaker: that can be adjusted to
30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocate horizontally at 160 times/ minute with amplitude of 25 mm 40 mmb) Analytical instrument: An atomic absorption spectrometer or a flame photometer as shown
below:ba) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in JIS
K 0121.
1) Light source: A potassium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
bb) Flame photometer:
1) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (3), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (3) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 2 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.3.a.
(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (4).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (3), and shake to mix byreciprocating horizontally at 160 times/min with amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC)
for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (4) Use a 250-mL flat-bottom volumetric flask to stabilize the vibration.
Comment 3 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.3.a.
Comment 4 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedures of (4.1.1) b) and (4.1.2) b).
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
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Testing Methods for Fertilizers (2018)

Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer or flame photometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer or flame photometer:
Set up the measurement conditions for the atomic absorption spectrometer or flame
photometer considering the following:
Analytical line wavelength: 766.5 nm or 769.9 nmb) Calibration curve preparation
1) Spray the potassium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 766.5 nm or 769.9 nm.
2) Prepare a curve for the relationship between the potassium concentration and the indicatedvalue of the potassium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 5 mg as
K2O) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (2), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the potassium content from the calibration curve, and calculate citrate soluble
potassium (C-K2O) in the analytical sample.
Comment 5 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 10 % (mass fraction) 20 % (mass fraction) and 1 % (mass fraction) - 5 % (mass fraction) are 100.2 % 101.7 % and 100.4 % - 101.8 % as citrate soluble potassium (C-K2O) respectively.
Additionally, the minimum limit of quantification of this testing method is about
0.05 % (mass fraction) for solid fertilizers, and 0.06 % (mass fraction) for fluid
fertilizers.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.136- 138, Yokendo, Tokyo (1988)
2)
Yasuharu KIMURA and Hisanori ARAYA Verification of Performance Characteristicsof Testing Methods for Potassium Content in Fertilizer by Atomic Absorption
Spectrometry, Research Report of Fertilizer Vol. 5, p. 190 - 200 (2012)164

Testing Methods for Fertilizers (2018)(5)

Flow sheet for citrate soluble potassium: The flow sheet for citrate soluble potassium in
fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← 150 mL citrate solution [about 30 ºC]
Constant temperature rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1-1 Flow sheet for citrate-soluble potassium in fertilizers (Extract procedure 4.1.1)

1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← 150 mL citrate solution [about 30 ºC]
Shaking to mix

Reciprocating water bath shaker (reciprocation horizontally at
160 times /min, with amplitude of 25 mm - 40 mm), at 30 ºC ± 1 º
C, for 1 hour

Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1-2 Flow sheet for citrate-soluble potassium in fertilizers (Extract procedure 4.1.2)
Sample solution

Aliquot(predetermined
100-mL volumetric flask
amount)← About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer or flame photometer
Figure 2 Flow sheet for citrate-soluble potassium in fertilizers (Measurement procedure)165

Testing Methods for Fertilizers (2018)

4.3.2.b Sodium tetraphenylborate gravimetric analysis(1) Summary
This testing method is applicable to fertilizers containing potassium silicate fertilizers, etc. This
testing method is classified as Type D and its symbol is 4.3.2.b-2017 or C-K.b-1.
Extract by adding a citric acid solution to an analytical sample, mask co-existing ammonium andother salts with formaldehyde and ethylenediamine tetraacetate and measure the mass of citric acidsoluble potassium (citrate-soluble potassium (C-K2O)) and the mass of potassium tetraphenylborate
formed by the reaction with tetraphenylborate to obtain citrate soluble potassium (C-K2O) in ananalytical sample. In addition, the performance of this testing method is shown in Comment 3.
(2) Reagent: Reagents are as shown below.
a) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
b) Formaldehyde solution: A JIS Guaranteed Reagent specified in JIS K 8872 or a reagent ofequivalent quality.
c) Sodium hydroxide solution (200 g/L) (1): Dissolve 200 g of sodium hydroxide specified in
JIS K 8576 in water to make 1000 mL.
d) Aluminum chloride solution (1 mol/L) (1): Dissolve 12 g of aluminum chloride (III)hexahydrate specified in JIS K 8114 in water to make 100 mL.
e) Tetraphenylborate solution (1): Transfer 6.1 g of sodium tetraphenylborate specified in JIS K
9521to a 250-mL volumetric flask, dissolve by adding about 200 mL of water and add 10 mLof aluminum chloride solution. Add a methyl red solution (0.1 g/100 mL) as an indicator, andneutralize with a sodium hydroxide solution (200 g/L) until the color of the solution changes
to yellow, and then add water up to the marked line. Filter with Type 3 filter paper and add 0.5
mL of sodium hydroxide solution (200 g/L) to the total filtrate. Filter with Type 3 filter paperin the case of usage.
f) Tetraphenylborate washing solution (1): Dilute 40 mL of tetraphenylborate solution withwater to make 1000 mL.
g) Ethylenediaminetetraacetate - Sodium hydroxide solution (1): Dissolve 10 g ofethylenediaminetetraacetic acid disodium dihydrogen dihydrate specified in JIS K 8107 and 8
g of sodium hydroxide specified in JIS K 8576 in a proper amount of water. Add 6 mL - 10
mL of tetraphenylborate solution while mixing according to the potassium content coexisting
as impurity after standing to cool, and then add water to make 100 mL. After leaving at rest
for about 30 minutes while sometimes mixing, filter with Type 3 filter paper.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K 8896
in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
b) Drying apparatus: A drying apparatus that can be adjusted to 120 ºC ± 2 ºC.
c) Crucible type glass filter: A crucible type glass filter 1G4 specified in JIS R 3503. Let it
stand to cool in a desiccator after heating at 120 ºC ± 2 ºC in advance and measure the mass tothe order of 1 mg.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
166

Testing Methods for Fertilizers (2018)a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (2) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 1 The procedures in (4.1) are the same as in (4.1.1) in 4.2.3.a.
Comment 2 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedure of (4.1) b).
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer 20 mL of sample solution to a 100-mL tall beaker.
b) Add water to the solution to reach 50 mL when the procedure in d) is complete.
c) Add 5 mL of formaldehyde solution, and then add 5 mL of ethylenediamine tetraacetate sodium hydroxide solution.
d) Add necessary volume (2) of tetraphenylborate solution at the rate of one or two drop(s) per
second while mixing, and further add 4 mL of the same solution in the same manner.
e) Leave at rest for about 30 minutes while sometimes mixing to form the precipitate ofpotassium tetraphenylborate.
f) Filter supernatant under reduced pressure with a crucible type glass filter, wash the vessel 5
times with 5 mL of tetraphenylborate washing solution and transfer the whole precipitate to
the crucible type glass filter and further wash 2 times with 2 ml of water.
g) Transfer the precipitate together with the crucible type glass filter into a drying apparatus andheat at 120 ºC ± 2 ºC for about 1hour.
h) As soon as heating is complete, move it into a desiccator and let it stand to cool.
i) After standing to cool, remove the crucible type glass filter from the desiccator and measure
the mass to the order of 1 mg.
j) Calculate citrate soluble potassium (C-K2O) by the following formula in the analyticalsample.
Citrate soluble potassium (C-K2O) (% (mass fraction)) in an analytical sample=A×0.1314×(V1/V2)/W×100
A：
V1：
V2：
W：

Mass (g) of the precipitate
Predetermined volume (mL) of the sample solution in (4.1) c)
Transferred amount (mL) of the sample solution in (4.2) a)
Mass (g) of the analytical sample

Note (2) About 3 ml of tetraphenylborate solution per 10 mg of K2O is required to form theprecipitate of potassium tetraphenylborate
Comment 3 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 25 % (mass fraction) 30 % (mass fraction) and 10 % (mass fraction) - 20 % (mass fraction) are 98.6 % 100.6 % and 100.6 % - 100.7 % as citrate soluble potassium (C-K2O) respectively.
167

Testing Methods for Fertilizers (2018)

Additionally, the minimum limit of quantification of this testing method is about
0.6 % (mass fraction).
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.122- 128, Yokendo, Tokyo (1988)
2)
Keiji YAGI, Aiko YANO and Hideo SOETA: Verification of Performance
Characteristics of Testing Method for Nitrate Nitrogen Content in Fertilizer by Phenol
Sulfuric Acid Method, Research Report of Fertilizer Vol. 5, p. 201 - 211 (2012)(5)

Flow sheet for the testing method: The flow sheet for citrate soluble potassium in fertilizers
is shown below:
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← 150 mL citrate solution [about 30 ºC]
Constant temperature rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for citrate-soluble potassium in fertilizers (Extraction procedure)

168

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot
100-mL tall beaker(20 mL)← Water (add tetraphenylborate solution to reach 50 mL)← 5 mL of formaldehyde solution← 5 mL of ethylenediamine tetraacetate - sodium hydroxide solution← Tetraphenylborate solution (the equivalents of potassium + 4 mL)
Forming precipitate
For 30 minutes, sometimes mixing
Filtration
under reducedpressure

Crucible type glass filtering apparatus 1G4,
5 times with 5 mL of tetraphenylborate washing solution

Washing

Wash 2 times with 2 mL of water

Drying120 ºC ± 5 ºC for 1 hour.

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure 2 Flow sheet for citrate-soluble potassium in fertilizers (Measurement procedure)

169

Testing Methods for Fertilizers (2018)

4.3.2.c Sodium tetraphenylborate volumetric analysis(1) Summary
This testing method is applicable to fertilizers containing potassium silicate fertilizer, etc. but notorganic matters. This testing method is classified as Type E and its symbol is 4.3.2.c-2017 or
C-K.c-1.
Extract by adding a citric acid solution to an analytical sample, mask co-existing ammonium andother salts with formaldehyde, and make potassium ion and tetraphenylborate react with each other.
Measure unconsumed tetraphenylborate by conducting a precipitate titration to obtaincitrate-soluble potassium (C-K2O) in an analytical sample.
(2) Reagent: Reagents are as shown below.
a) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
b) Formaldehyde solution: A JIS Guaranteed Reagent specified in JIS K 8872 or a reagent ofequivalent quality.
c) Sodium hydroxide solution (120 g/L) (1): Dissolve 30 g of sodium hydroxide specified in JIS
K 8576 in water to make 250 mL.
d) Tetraphenylborate solution (1): Transfer 12.2 g of sodium tetraphenylborate to a 1000-mL
volumetric flask, dissolve by adding about 800 mL of water and add 3 mL of sodiumhydroxide (120 g/L) to the total filtrate, and further add water up to the marked line. Filter
with Type 3 filter paper in the case of usage.
e) Benzalkonium chloride solution (3.3 g/500 mL) (1): Dissolve 3.3 g of benzalkonium chloride
in 500 mL of water.
f) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K 8896
in 100 mL of ethanol (95) specified in JIS K 8102.
g) Titan Yellow solution (0.04 g/100mL): Dissolve 0.04 g of Titan Yellow in 100 mL of waterin the case of usage.
h) Potassium standard solution (K2O 2 mg/mL) (1): Heat potassium chloride specified in JIS K
8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, and weigh 3.166 g
into a weighing dish. Dissolve in a small amount of water, transfer to a 1000-mL volumetricflask, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as shown below:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (2) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
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Testing Methods for Fertilizers (2018)

Comment 1 The procedures in (4.1) are the same as in (4.1.1) in 4.2.3.a.
Comment 2 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedure of (4.1) b).
(4.2) Precipitate formation: Form precipitate as shown below.
a) Transfer 5 mL - 15 mL (no more than the equivalents of 30 mg as K2O) of the extract to a
100-mL volumetric flask.
b) Add water to the solution to make about 30 mL.
c) Add about 5 mL of formaldehyde solution and add 5 mL of sodium hydroxide solution (120g/L).
d) Add 25 mL of tetraphenylborate solution at the rate of one or two drop(s) per second while
shaking to mix.
e) After adding water up to the marked line, leave at rest for 10 minutes.
f) Filter with Type 3 filter paper to make a sample solution.
(4.3) Measurement: Conduct measurement as shown below.
a) Calibration curve preparation
1) Transfer 1 mL - 15 mL of potassium standard solution (K2O 2 mg/mL) to 100-mL volumetricflasks step-by-step.
2) Conduct the same procedures as (4.2) b) - f) to make K2O 2 mg/100 mL - 30 mg/100 mL of
the potassium standard solutions for the calibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blanktest solution for the calibration curve preparation.
4) Transfer 40 mL of the potassium standard solutions for the calibration curve preparation andthe blank test solution for the calibration curve preparation to an Erlenmeyer flask
respectively.
5) Add a few drops of Titan Yellow solution.
6) Titrate with a benzalkonium chloride solution (3.3 g/500 mL) until the color of the solutionchanges to light red (2).
7) Prepare a curve for the relationship between the potassium concentration and the volume of
the benzalkonium chloride solution (3.3 g/500 mL) required for the titration of the potassium
standard solutions for the calibration curve preparation and the blank test solution for thecalibration curve preparation.
b) Sample measurement
1) Transfer 40 mL of the sample solution of (4.2) f) to a 100-mL Erlenmeyer flask.
2) Conduct similarly as in a) 5) - 6) to obtain the volume of the benzalkonium chloride solution(3.3 g/500 mL) required for the titration.
3) Obtain the potassium content from the calibration curve, and calculate citrate soluble
potassium (C-K2O) in the analytical sample.
Note (3) If the solution temperature is no more than 20 ºC, the reaction does not advance insome cases. Therefore, it is recommended to heat the solution up to about 30 ºC.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.128- 132, Yokendo, Tokyo (1988)

171

Testing Methods for Fertilizers (2018)(5)

Flow sheet for citrate soluble potassium: The flow sheet for citrate soluble potassium in
fertilizers is shown below:

1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← 150 mL citrate solution [about 30 ºC]
Constant temperature rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hourStanding to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for citrate-soluble potassium in fertilizers (Extraction procedure)

Sample solution
Aliquot
100-mL volumetric flask(5 mL - 15 mL)← Water (the volume to reach about 30 mL)← About 5 mL of formaldehyde solution← 5 mL of sodium hydroxide solution (120 g/L)
← 25 mL tetraphenylborate solution(one or two drop (s) per second while shaking to mix)
← Water (up to the marked line)
Leaving at rest
10 minutes
Filtration
Type 3 filter paper←A few drops of Titan Yellow solution
Aliquot
100-mL Erlenmeyer flask(40 mL)← A few drops of titan yellow solution
Benzaikonium chloride solution (3.3 g/500 mL)
Titration(until the solution changes to light red)
Figure 2 Flow sheet for citrate-soluble potassium in fertilizers (Measurement procedure)

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Testing Methods for Fertilizers (2018)

4.3.2.d ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type D and its
symbol is 4.3.2.d-2018 or C-K.b-1.
Extract by adding a citric acid solution to an analytical sample, introduce it to an ICP OpticalEmission Spectrometer (“ICP-OES”) and measure the potassium at a wavelength of 766.491 nm toobtain citric acid-soluble potassium (citrate-soluble potassium (C-K2O)) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 6.
(2) Reagent: Reagents are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
d) Potassium standard solution (K2O 1 mg/mL) (1): Heat potassium chloride specified in JIS K
8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, and weigh 1.583 g
into a weighing dish. Dissolve in a small amount of water, transfer to a 1000-mL volumetricflask, and add water up to the marked line.
e) Potassium standard solution (K2O 20 µg/mL - 0.16 mg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 16 mL of potassium standard solution (K2O 1 mg/mL) to
100-mL volumetric flasks step-by-step, add about 25 mL of hydrochloric acid (1+5), and add
water up to the marked line.
f) Potassium standard solution (K2O 2 µg/mL - 20 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 20 mL of potassium standard solution (K2O 0.1 mg/mL) to
100-mL volumetric flasks step-by-step, add about 25 mL of hydrochloric acid (1+23), and add
water up to the marked line.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the potassium standard solution in (2), a potassium standard solution forthe calibration curve preparation can be prepared by using a potassium standard
solution (K 1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case,calculate citrate-soluble potassium (C-K2O) in the analytical sample by multiplying
the concentration (K) of a potassium standard solution for calibration curve
preparation or a measurement value (K) obtained in (4.2) by a conversion factor(1.2046).
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The axial observation mode does not apply topotassium since interference is serious.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Extractor: A constant-temperature rotary shaker or a reciprocating water bath shaker as
described below.
ba) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate a
250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at 30
173

Testing Methods for Fertilizers (2018)- 40 revolutions/min.
bb) Reciprocating water bath shaker: A reciprocating water bath shaker that can be adjusted to
30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocate horizontally at 160 times/ minute with amplitude of 25 mm 40 mm.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (2) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 3 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.3.a.
(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (3).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix byreciprocating horizontally at 160 times/min with amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC)
for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (3) Use a 250-mL flat-bottom volumetric flask to stabilize the vibration.
Comment 3 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.3.a.
Comment 4 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedures of (4.1.1) b) and (4.1.2) b).
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 766.491 nmb) Calibration curve preparation
1) Spray the potassium standard solution for the calibration curve preparation and blank test
solution for the calibration curve preparation into inductively coupled plasma, and read theindicated value at a wavelength of 766.491 nm.
2) Prepare a curve for the relationship between the potassium concentration and the indicatedvalue of the potassium standard solutions for the calibration curve preparation and the174

Testing Methods for Fertilizers (2018)

blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.2 mg - 16 mg
as K2O) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the potassium content from the calibration curve, and calculate citrate soluble
potassium (C-K2O) in the analytical sample.
Comment 5 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.3.d Comment 7.
Comment 6 The comparison of the measurement value (yi：3.57 % (mass fraction) - 34.24 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of Flame atomic absorption spectrometry analysis was conducted to evaluate
trueness using compound fertilizers (9 samples), mixed compost fertilizers (1sample), designated blended fertilizers (1sample), blended fertilizers (4 samples),and byproduct mixed fertilizers (1 sample). As a result, a regression equation was y =− 0.0058 + 1.0027x, and its correlation coefficient (r) was 0.999. In addition,
recovery testing was conducted using a preparation sample. As a result, the averagerate of recovery at the content level of 0.329 % (mass fraction) - 63.18 % (massfraction) was 98.0 % - 100.3 %.
The results of the repeatability tests on different days using compound fertilizers andblended fertilizers to evaluate precision were analyzed by one-way analysis ofvariance. Table 1 shows the calculation results of intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.09 % (mass fraction).
Table 1 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
2)4)
5)
samplerepeatability Average
sr
RSD r
1)

3)

3)

T
(%)
(%)
Compound fertilzier
7
16.17
0.13
Blended fertilizer
7
4.42
0.04
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

175

Intermediate precision
6)

7)

s I(T)RSD I(T)
3)
(%)
(%)
(%)
0.8
0.15
1.0
1.0
0.04
1.6
6） Intermediate standard deviation
7） Intermediate relative standard deviation

Testing Methods for Fertilizers (2018)(5)

Flow sheet for citrate soluble potassium: The flow sheet for citrate soluble potassium in
fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← 150 mL citrate solution [about 30 ºC]
Constant temperature rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1-1 Flow sheet for citrate-soluble potassium in fertilizers (Extract procedure 4.1.1)

1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← 150 mL citrate solution [about 30 ºC]
Reciprocating water bath shaker (reciprocation horizontally at 160
Shaking to mix
times /min, with amplitude of 25 mm - 40 mm), at 30 ºC ± 1 ºC,
for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution

Figure 1-2 Flow sheet for citrate-soluble potassium in fertilizers (Extract procedure 4.1.2)
Sample solution

Aliquot(predetermined
100-mL volumetric flask
amount)← 25 mL hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
ICP-OES (766.491 nm)
Figure 2 Flow sheet for citrate-soluble potassium in fertilizers (Measurement procedure)

176

Testing Methods for Fertilizers (2018)

4.3.3 Water-soluble potassium
4.3.3.a Flame atomic absorption spectrometry or flame photometry(1) Summary
This testing method is applicable to fertilizers containing potassium salts. This testing method isclassified as Type C and its symbol is 4.3.3.a-2017 or W-K.a-1.
Extract by adding water to an analytical sample, add an interference suppressor solution, and thenspray in an acetylene−air flame, and measure the atomic absorption with potassium at a wavelengthof 766.5 nm or 769.9 nm to quantify water-soluble potassium (W-K2O). Or, determine the intensityof the emission line at a wavelength of 766.5 nm or 769.9 nm produced in flame to quantify
water-soluble potassium (W-K2O) in an analytical sample. In addition, the performance of thistesting method is shown in Comment 8.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution: Weigh 12.5 g of calcium carbonate specified in JIS K
8617 into a 2000-mL beaker, add a small amount of water, gradually add 105 mL ofhydrochloric acid, and heat for a little while. After cooling is complete, add water to make1000 mL.
c) Potassium standard solution (K2O 1 mg/mL) (1): Heat potassium chloride specified in JIS K
8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, and weigh 1.583 g
into a weighing dish. Dissolve in a small amount of water, transfer to a 1000-mL volumetricflask, and add water up to the marked line.
d) Potassium standard solution (K2O 5 µg/mL - 50 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of potassium standard solution (K2O 1 mg/mL) to
500-mL volumetric flasks step-by-step, add about 50 mL of interference suppressor solution(2), and add water up to the marked line.
e) Blank test solution for the calibration curve preparation (1): Transfer about 50 mL ofinterference suppressor solution to a 500-mL volumetric flask (2), and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the potassium standard solution in (2), a potassium standard solution forthe calibration curve preparation can be prepared by using a potassium standard
solution (K 1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case,calculate water-soluble potassium (W-K2O) in the analytical sample by multiplying
the concentration (K) of a potassium standard solution for calibration curve
preparation or a measurement value (K) obtained in (4.2) by a conversion factor(1.2046).
(3) Instruments: Instruments are as shown below:a) Extractor: A constant-temperature rotary shaker or a vertical reciprocating shaker as
described below.
aa) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
ab) Vertical reciprocating shaker: A vertical reciprocating shaker that can vibrate a 250-mL
volumetric flask using an adapter to reciprocate vertically at 300 times/ minute (amplitude of177

Testing Methods for Fertilizers (2018)

40 mm).
b) Analytical instrument: An atomic absorption spectrometer or a flame photometer as shown
below:ba) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121.
1) Light source: A potassium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
bb) Flame photometer:
1) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
c) Hot plate : A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Compound fertilizers containing potassium salts and magnesium potassium sulfatea) Weigh 2.5 g of an analytical sample to the order of 1 mg, and transfer to a 300-mL tall beaker.
b) Add about 200 mL of water, and cover with a watch glass and heat on a hot plate to boil forabout 15 minutes.
c) Immediately transfer it with water to a 250- mL volumetric flask.
d) After immediate cooling is complete, add water up to the marked linee) Filter with Type 3 filter paper to make a sample solution.
Comment 2 In the procedure in a), a 250-mL volumetric flask can be used instead of a 300-mLtall beaker. However, the volumetric flask used should be distinguished as an
extraction flask and should not be used for the other purposes. Additionally, “coverwith a watch glass” in b) is replaced by “place a funnel”. Skip “transfer to a 250-mL
volumetric flask with water” in the procedure in c).
Comment 3 The procedure in (4.1.1) is the same as the procedures in (4.1.1) of 4.3.3.b, (4.1.1) of
4.3.3.c and (4.1) of 4.8.2.a.
(4.1.2) Compound fertilizers containing no magnesium potassium sulfate(4.1.2.1) Rotary shaker:a) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL volumetricflask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 4 In the procedure of (4.1.2.1) a), it is also allowed to weigh 2.5g of an analyticalsample to the order of 1 mg, and put it in a 250-mL volumetric flask
Comment 5 The procedure in (4.1.2.1) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
(4.1.2.2) Vertical reciprocating shaker:a) Weigh 2.5 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add about 200 mL of water, and shake to mix by reciprocating vertically at 300 times/min(amplitude of 40 mm) for about 30 minutes.
178

Testing Methods for Fertilizers (2018)c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 6 The procedure in (4.1.2.2) is the same as the procedure in (4.1.1.2) in 4.2.4.a.
(4.1.3) Fluid test samplea) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 100-mL volumetricflask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 7 The procedure in (4.1.3) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer or flame photometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer or flame photometer:
Set up the measurement conditions for the atomic absorption spectrometer or flame
photometer considering the following:
Analytical line wavelength: 766.5 nm or 769.9 nmb) Calibration curve preparation
1) Spray the potassium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 766.5 nm or 769.9 nm.
2) Prepare a curve for the relationship between the potassium concentration and the indicatedvalue of the potassium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 5 mg as
K2O) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (2), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the potassium content from the calibration curve, and calculate water soluble
potassium (W-K2O) in the analytical sample.
Comment 8 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 10 % (mass fraction) 20 % (mass fraction) and 1 % (mass fraction) - 5 % (mass fraction) are 97.9 % 100.2 % and 97.3 % - 100.6 % as water soluble potassium (W-K2O) respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
The comparison of the measurement value of extraction (yi：2.69 % (mass fraction) 26.64 % (mass fraction )) with a vertical reciprocating shaker and the measurement
value of extract with a rotary shaker (xi) was conducted to evaluate trueness of theextraction of solid fertilizers using fertilizers (12 samples). As a result, a regression
equation was y = 0.022 + 1.001x, and its correlation coefficient (r) was 1.000. Also,
the results of the repeatability tests on different days using compound fertilizers and179

Testing Methods for Fertilizers (2018)

designated blended fertilizers to evaluate precision were analyzed by one-way
analysis of variance. Table 2 shows the calculation results of intermediate precisionand repeatability
The comparison of the measurement value of extraction (yi：2.69 % (mass fraction) 26.64 % (mass fraction )) with a vertical reciprocating shaker and the measurement
value of extract with a rotary shaker (xi) was conducted to evaluate trueness of theextraction of fluid fertilizers using fertilizers (12 samples). As a result, a regression
equation was y = 0.022 + 1.001x, and its correlation coefficient (r) was 1.000. Also,
the results of the repeatability tests on different days using fluid mixed fertilizers toevaluate the precision of the extraction of fluid fertilizers were analyzed by the
one-way analysis of variance. Table 3 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.04 % (mass fraction) for solid fertilizers, and 0.007 % (mass fraction) for fluid
fertilizers.
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
4)
5)
6)
7)
8)
9)
certified reference laboratory Average2)
sr
RSD rs I(T)RSD I(T)
sR
RSD R
1)
3)
3)
material fertilizer
(%)
p
(%)
(%)
FAMIC-A-10
11
13.59
0.08
0.6
FAMIC-A-13
10
13.07
0.08
0.6
FAMIC-B-10
9
8.85
0.04
0.4
FAMIC-B-14
14
8.32
0.03
0.4
1） The number of laboaratories used for analysis
conducting frame atomic absorption spectrometry
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

Name ofsample

3)

3)

(%)
(%)
(%)
(%)
0.09
0.6
0.16
1.2
0.11
0.8
0.16
1.2
0.07
0.7
0.12
1.4
0.07
0.8
0.13
1.6
6） Intermediate standard deviation
7） Intermediate relative standard deviation
8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

Table 2 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
7

3)

(%)
19.67

3)

(%)
0.09

Compound fertilzier
Designated
7
6.50
0.07
blended fertilizer
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

180

(%)
0.5

(%)
0.15

3)

(%)
0.7

1.1

0.07

1.1

6） Intermediate standard deviation
7） Intermediate relative standard deviation

Testing Methods for Fertilizers (2018)

Name ofsampleTable 3 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

3)

(%)
9.66

3)

(%)
0.02

(%)
0.2

3)

Fluid mixed fertilizer 1

T
7

(%)
0.07

(%)
0.7

Fluid mixed fertilizer 2

7

2.44

0.01

0.4

0.02

0.8

1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.136- 138, Yokendo, Tokyo (1988)
2)
Yasuharu KIMURA and Hisanori ARAYA Verification of Performance
Characteristics of Testing Methods for Potassium Content in Fertilizer by Atomic
Absorption Spectrometry, Research Report of Fertilizer Vol. 5, p. 190 - 200 (2012)3)
Shinji KAWAGUCHI: Simple Extraction Method for Water-Soluble Components
in Liquid Compound Fertilizers, Research Report of Fertilizer, Vol.9, p. 10 - 20(2016)
4)
Shinji KAWAGUCHI: Extraction Method for the Water-soluble Principal
Ingredients in the Solid Fertilizer using a General-purpose Equipment , Research
Report of Fertilizer, Vol.10, p. 1 - 8 (2017)(5)

Flow sheet for water soluble potassium: The flow sheet for water soluble potassium in
fertilizers is shown below:

2.5 g analytical sample Weigh to the order of 1 mg into a 300-mL tall beaker(potassium salts, etc.)← About 200 mL water
Heating
Cover with a watch dish, boil for 15 minutes
Cooling

Immediately

Transfer
Water, 250-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for water-soluble potassium in fertilizers(Extraction procedure (4.1.1))

181

Testing Methods for Fertilizers (2018)

5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask(compound fertilizer)← About 400 mL water
Rotary shaker (30 - 40 revolutions/min)Shaking to mix
For 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for water-soluble potassium in fertilizers(Extraction procedure (4.1.2.1))

2.5 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask(compound fertilizer)← About 200 mL water
Vertical reciprocating shaker(300 times/min, with amplitude of 40mm), for 30 minutes← Water (up to the marked line)
Type 3 filter paper
Filtration

Shaking to mix

Sample solution
Figure 1-3 Flow sheet for water-soluble potassium in fertilizers(Extraction procedure (4.1.2.2))

Analytical sample
Weigh to the order of 1 mg into a 100-mL volumetric flask(fluid)← About 50 mL water
Shaking to mix← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-4 Flow sheet for water-soluble potassium in fertilizers(Extraction procedure (4.1.3))

182

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot(predetermined
100-mL volumetric flask
volume)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement

Atomic absorption spectrometer or flame photometer

Figure 2 Flow sheet for water-soluble potassium in fertilizers (Measurement procedure)

183

Testing Methods for Fertilizers (2018)

4.3.3.b Sodium tetraphenylborate gravimetric analysis(1) Summary
This testing method is applicable to fertilizers containing potassium salts. This testing method isclassified as Type D and its symbol is 4.3.3.b-2017 or W-K.b-1.
Extract by adding water to an analytical sample, mask co-existing ammonium and other salts withformaldehyde and ethylenediamine tetraacetate and measure the mass of potassium
tetraphenylborate formed by the reaction with tetraphenylborate to obtain water-soluble potassium(W-K2O) in an analytical sample. In addition, the performance of this testing method is shown inComment 5.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Formaldehyde solution: A JIS Guaranteed Reagent specified in JIS K 8872 or a reagent ofequivalent quality.
c) Sodium hydroxide solution (200 g/L) (1): Dissolve 200 g of sodium hydroxide specified in
JIS K 8576 in water to make 1000 mL.
d) Aluminum chloride solution (1 mol/L) (1): Dissolve 12 g of aluminum chloride (III)hexahydrate specified in JIS K 8114 in water to make 100 mL.
e) Tetraphenylborate solution (1): Transfer 6.1 g of sodium tetraphenylborate specified in JIS K
9521to a 250-mL volumetric flask, dissolve by adding about 200 mL of water and add 10 mLof aluminum chloride solution. Add a methyl red solution (0.1 g/100 mL) as an indicator, andneutralize with a sodium hydroxide solution (200 g/L) until the color of the solution changes
to yellow, and then add water up to the marked line. Filter with Type 3 filter paper and add 0.5
mL of sodium hydroxide solution (200 g/L) to the total filtrate. Filter with Type 3 filter paperin the case of usage.
f) Tetraphenylborate washing solution (1): Dilute 40 mL of tetraphenylborate solution withwater to make 1000 mL.
g) Ethylenediaminetetraacetate - Sodium hydroxide solution (1): Dissolve 10 g ofethylenediaminetetraacetic acid disodium dihydrogen dihydrate specified in JIS K 8107 and 8
g of sodium hydroxide specified in JIS K 8576 in a proper amount of water. Add 6 mL - 10
mL of tetraphenylborate solution while mixing according to the potassium content coexisting
as impurity after standing to cool, and then add water to make 100 mL. After leaving at rest
for about 30 minutes while sometimes mixing, filter with Type 3 filter paper.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K 8896
in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Drying apparatus: A drying apparatus that can be adjusted to 120 ºC ± 2 ºC.
c) Crucible type glass filter: A crucible type glass filter 1G4 specified in JIS R 3503. Let it
stand to cool in a desiccator after heating at 120 ºC ± 2 ºC in advance and measure the mass tothe order of 1 mg.
d) Hot plate : A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
184

Testing Methods for Fertilizers (2018)(4.1.1) Compound fertilizers containing potassium salts and magnesium potassium sulfatea) Weigh 2.5 g of an analytical sample to the order of 1 mg, and transfer to a 300-mL tall beaker.
b) Add about 200 mL of water, and cover with a watch glass and heat on a hot plate to boil forabout 15 minutes.
c) Immediately transfer it with water to a 250- mL volumetric flask.
d) After immediate cooling is complete, add water up to the marked linee) Filter with Type 3 filter paper to make a sample solution.
Comment 1 In the procedure in a), a 250-mL volumetric flask can be used instead of a 300-mLtall beaker. However, the volumetric flask used should be distinguished as an
extraction flask and should not be used for the other purposes. Additionally, “coverwith a watch glass” in b) is replaced by “place a funnel”. Skip “transfer to a 250-mL
volumetric flask with water” in the procedure in c).
Comment 2 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.3.3.a.
(4.1.2) Compound fertilizers containing no magnesium potassium sulfatea) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL volumetricflask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 3 In the procedure in a), it is also allowed to weigh 2.5 g of an analytical sample to theorder of 1 mg, and put it into a 250-mL volumetric flask.
Comment 4 The procedure in (4.1.2) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined amount (the equivalents of 15 mg - 30 mg as K2O) of the sample
solution to a 100-mL tall beaker.
b) Add water to the solution to reach 50 mL when the procedure in e) is complete.
c) Add 2 mL of hydrochloric acid (1+9).
d) Add 5 mL of formaldehyde solution, and then add 5 mL of ethylenediamine tetraacetate sodium hydroxide solution.
e) Add necessary volume (2) of tetraphenylborate solution at the rate of one or two drop(s) per
second while mixing, and further add 4 mL of the same solution in the same manner.
f) Leave at rest for about 30 minutes while sometimes mixing to form the precipitate ofpotassium tetraphenylborate.
g) Filter supernatant under reduced pressure with a crucible type glass filter, wash the vessel 5
times with 5 mL of tetraphenylborate washing solution and transfer the whole precipitate to
the crucible type glass filter and further wash 2 times with 2 ml of water.
h) Transfer the precipitate together with the crucible type glass filter into a drying apparatus
adjusted to 120 ºC ± 2 ºC and heat for 1 hour.
i) As soon as heating is complete, move it into a desiccator and let it stand to cool.
j) After standing to cool, remove the ground-in stoppered weighing bottle from the desiccator,and measure the mass to the order of 1 mg.
k) Calculate water soluble potassium (W-K2O) in the analytical sample by the following formula.
Water-soluble potassium (W-K2O) (% (mass fraction)) in an analytical sample=A × 0.1314 × (V1/V2)/W × 100
185

Testing Methods for Fertilizers (2018)

A： Mass (g) of the precipitate
V1： Predetermined volume (mL) of the sample solution in (4.1.1) d) or (4.1.2) c)
V2： Transferred amount (mL) of the sample solution in (4.2) a)
W： Mass (g) of the analytical sample
Note (2) About 3 ml of tetraphenylborate solution per 10 mg of K2O is required to form theprecipitate of potassium tetraphenylborate
Comment 5 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 30 % (mass fraction) 50 % (mass fraction) and 10 % (mass fraction) - 20 % (mass fraction) are 100.2 % 100.8 % and 99.3 % - 102.2 % as water soluble potassium (W-K2O) respectively.
Additionally, the minimum limit of quantification of this testing method is about
0.7 % (mass fraction).
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.122- 128, Yokendo, Tokyo (1988)
2)
Keiji YAGI, Aiko YANO and Hideo SOETA: Verification of Performance
Characteristics of Testing Method for Nitrate Nitrogen Content in Fertilizer by Phenol
Sulfuric Acid Method, Research Report of Fertilizer Vol. 5, p. 201 - 211 (2012)(5)

Flow sheet for water soluble potassium: The flow sheet for water soluble potassium in
fertilizers is shown below:

2.5 g analytical sample
Weigh to the order of 1 mg into a 300-mL tall beaker(potassium salts, etc.)← About 200 mL water
Heating
Cover with a watch glass, boil for 15 minutes
Cooling

Immediately

Transfer
Water, 250-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1

Flow sheet for water-soluble potassium in fertilizers(Extraction procedure (4.1.1))

186

Testing Methods for Fertilizers (2018)

5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask(compound fertilizer)← About 400 mL of water
Rotary shaker (30 - 40 revolutions/min)
Shake to mix
For 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for water-soluble potassium in fertilizers(Extraction procedure (4.1.2))
Sample solution
Aliquot(predetermined
100-mL tall beakeramount)← Water (add tetraphenylborate solution to reach 50 mL)← 2 mL of hydrochloric acid (1+9)← 5 mL of formaldehyde solution← 5 mL of ethylenediamine tetraacetate-sodium hydroxide solution← Tetraphenylborate solution (the equivalents of potassium + 4 mL)
Forming precipitate
For 30 minutes, sometimes mixing
Filtration
under reducedpressure

Crucible type glass filtering apparatus 1G4,
5 times with 5 mL of tetraphenylborate washing solution

Washing

Wash 2 times with 2 mL of water

Drying120 ºC ± 2 ºC for 1 hour.

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure 2

Flow sheet for water-soluble potassium in fertilizers(Coloring and measurement procedure)187

Testing Methods for Fertilizers (2018)

4.3.3.c Sodium tetraphenylborate volumetric analysis(1) Summary
This testing method is applicable to fertilizers containing potassium salt but not organic matters.
This testing method is classified as Type E and its symbol is 4.3.3.c-2017 or W-K.c-1.
Extract by adding water to an analytical sample, mask co-existing ammonium and other salts withformaldehyde, and make potassium ion and tetraphenylborate react with each other. Measureunconsumed tetraphenylborate by conducting a precipitate titration to obtain water-soluble
potassium (W-K2O) in an analytical sample.
(2) Reagent: Reagents are as shown below.
a) Formaldehyde solution: A JIS Guaranteed Reagent specified in JIS K 8872 or a reagent ofequivalent quality.
b) Sodium hydroxide solution (120 g/L) (1): Dissolve 30 g of sodium hydroxide specified in JIS
K 8576 in water to make 250 mL.
c) Tetraphenylborate solution (1): Transfer 12.2 g of sodium tetraphenylborate to a 1000-mL
volumetric flask, dissolve by adding about 800 mL of water and add 3 mL of sodiumhydroxide (120 g/L) to the total filtrate, and further add water up to the marked line. Filter
with Type 3 filter paper in the case of usage.
d) Benzalkonium chloride solution (3.3 g/500 mL) (1): Dissolve 3.3 g of benzalkonium chloride
in 500 mL of water.
e) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K 8896
in 100 mL of ethanol (95) specified in JIS K 8102.
f) Titan Yellow solution (0.04 g/100mL): Dissolve 0.04 g of Titan Yellow in 100 mL of waterin the case of usage.
g) Potassium standard solution (K2O 2 mg/mL) (1): Heat potassium chloride specified in JIS K
8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, and weigh 3.166 g
into a weighing dish. Dissolve in a small amount of water, transfer to a 1000-mL volumetricflask, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Hot plate : A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Compound fertilizers containing potassium salts and magnesium potassium sulfatea) Weigh 2.5 g of an analytical sample to the order of 1 mg, and transfer to a 300-mL tall beaker.
b) Add about 200 mL of water, and cover with a watch glass and heat on a hot plate to boil forabout 15 minutes.
c) Immediately transfer it with water to a 250- mL volumetric flask.
d) After immediate cooling is complete, add water up to the marked linee) Filter with Type 3 filter paper to make a sample solution.
Comment 1 In the procedure in a), a 250-mL volumetric flask can be used instead of a 300-mLtall beaker. However, the volumetric flask used should be distinguished as an
extraction flask and should not be used for the other purposes. Additionally, “coverwith a watch glass” in b) is replaced by “place a funnel”. Skip “transfer to a 250-mL
188

Testing Methods for Fertilizers (2018)

volumetric flask with water” in the procedure in c).
Comment 2 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.3.3.a.
(4.1.2) Compound fertilizers containing no magnesium potassium sulfatea) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL volumetricflask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 3 In the procedure in a), it is also allowed to weigh 2.5 g of an analytical sample to theorder of 1 mg, and put it into a 250-mL volumetric flask.
Comment 4 The procedure in (4.1.2) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
(4.2) Precipitate formation: Form precipitate as shown below.
a) Transfer 5 mL - 15 mL (no more than the equivalents of 30 mg as K2O) of the extract to a
100-mL volumetric flask.
b) Add water to the solution to make about 30 mL.
c) Add about 5 mL of formaldehyde solution and add 5 mL of sodium hydroxide solution (120g/L).
d) Add 25 mL of tetraphenylborate solution at the rate of one or two drop (s) per second while
shaking to mix.
e) After adding water up to the marked line, leave at rest for 10 minutes.
f) Filter with Type 3 filter paper to make a sample solution.
(4.3) Measurement: Conduct measurement as shown below.
a) Calibration curve preparation
1) Transfer 1 mL - 15 mL of potassium standard solution (K2O 2 mg/mL) to 100-mL volumetricflasks step-by-step.
2) Conduct the same procedures as (4.2) b) - f) to make K2O 2 mg/100 mL - 30 mg/100 mL of
the potassium standard solutions for the calibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blanktest solution for the calibration curve preparation.
4) Transfer 40 mL of the potassium standard solutions for the calibration curve preparation andthe blank test solution for the calibration curve preparation to an Erlenmeyer flask
respectively.
5) Add a few drops of Titan Yellow solution.
6) Titrate with a benzalkonium chloride solution (3.3 g/500 mL) until the color of the solutionchanges to light red (2).
7) Prepare a curve for the relationship between the potassium concentration and the volume of
the benzalkonium chloride solution (3.3 g/500 mL) required for the titration of the potassium
standard solutions for the calibration curve preparation and the blank test solution for thecalibration curve preparation.
b) Sample measurement
1) Transfer 40 mL of the sample solution of (4.2) f) to a 100-mL Erlenmeyer flask.
2) Conduct similarly as in a) 5) - 6) to obtain the volume of the benzalkonium chloride solution(3.3 g/500 mL) required for the titration.
3) Obtain the potassium content from the calibration curve, and calculate water soluble
potassium (W-K2O) in the analytical sample.
189

Testing Methods for Fertilizers (2018)

Note (2) If the solution temperature is no more than 20 ºC, the reaction does not advance insome cases. Therefore, it is recommended to heat the solution up to about 30 ºC.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.128- 132, Yokendo, Tokyo (1988)(5)

Flow sheet for water soluble potassium: The flow sheet for water soluble potassium in
fertilizers is shown below:
2.5 g analytical sample
Weigh to the order of 1 mg into a 300-mL tall beaker(potassium salts, etc.)← About 200 mL water
Heating
Cover with a watch glass, boil for 15 minutes
Cooling

Immediately

Transfer
Water, 250-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1

Flow sheet for water-soluble potassium in fertilizers(Extraction procedure (4.1.1))

5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask(compound fertilizer)← About 400 mL of water
Rotary shaker (30 - 40 revolutions/min)
Shake to mix
For 30 minutes
Water
(up
to
the marked line)←
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for water-soluble potassium in fertilizers(Extraction procedure (4.1.2))

190

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot
100-mL volumetric flask(5 mL to 15 mL)← Water (the liquid volume to reach about 30 mL)← About 5mL of formaldehyde solution← 5 mL of sodium hydroxide solution (120 g/L)
← 25 mL tetraphenylborate solution(one or two drop (s) per second while shaking to mix)
← Water (up to the marked line)
Leaving at rest
10 minutes
Filtration

Type 3 filter paper

Aliquot
100-mL Erlenmeyer flask(40 mL)← A few drops of Titan Yellow solution
Benzaikonium chloride solution (3.3 g/500 mL)
Titration(until the solution changes to light red)
Figure 2

Flow sheet for water-soluble potassium in fertilizers(Precipitate formation and measurement procedure)191

Testing Methods for Fertilizers (2018)

4.3.3.d ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers and the fluid fertilizers of homegarden-use mixed fertilizers. This testing method is classified as Type D and its symbol is
4.3.3.d-2017 or W-K.d-1.
Add water to an analytical sample to extract, introduce it to an ICP Optical Emission Spectrometer(“ICP-OES”) and measure the potassium at a wavelength of 766.491 nm to obtain water-soluble
potassium acid (W-K2O). In addition, the performance of this testing method is shown in Comment
5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Potassium standard solution (K2O 1 mg/mL) (1): Heat potassium chloride specified in JIS K
8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, and weigh 1.583 g
into a weighing dish. Dissolve in a small amount of water, transfer to a 1000-mL volumetricflask, and add water up to the marked line.
d) Potassium standard solution (K2O 20 µg/mL - 0.16 mg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 16 mL of potassium standard solution (K2O 1 mg/mL) to
100-mL volumetric flasks step-by-step, add about 25 mL of hydrochloric acid (1+5), and add
water up to the marked line.
e) Potassium standard solution (K2O 2 µg/mL - 20 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 20 mL of potassium standard solution (K2O 0.1 mg/mL) to
100-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in e).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the potassium standard solution in (2), a potassium standard solution forthe calibration curve preparation can be prepared by using a potassium standard
solution (K 1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case,calculate water-soluble potassium (W-K2O) in the analytical sample by multiplying
the concentration (K) of a potassium standard solution for calibration curve
preparation or a measurement value (K) obtained in (4.2) by a conversion factor(1.2046).
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The axial observation mode does not apply topotassium since interference is serious.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 mg of an analytical sample (2) to the order of 1 g, and put it in a 100-mL volumetricflask.
192

Testing Methods for Fertilizers (2018)b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (2) The sampling amount of the analytical sample is 10 g when there is less potassium
content in the fertilizers such as a home garden-use fertilizer.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct measurement according to JIS K 0116 and as shown below. Specificmeasurement procedures are according to the operation method of the ICP Optical Emission
Spectrometry used in measurement.
a) Measurement conditions for the ICP Optical Emission Spectrometer: Set up themeasurement conditions for the ICP Optical Emission Spectrometer considering thefollowing:
Analytical line wavelength: 766.491 nmb) Calibration curve preparation
1) Spray the potassium standard solution for the calibration curve preparation and blank test
solution for the calibration curve preparation into inductively coupled plasma, and read theindicated value at a wavelength of 766.491 nm.
2) Prepare a curve for the relationship between the potassium concentration and the indicatedvalue of the potassium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.2 mg - 16 mg as
K2O) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the potassium content from the calibration curve, and calculate water soluble
potassium (W-K2O) in the analytical sample.
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.4.d Comment 4.
Comment 5 The comparison of the measurement value (yi：0.641 % (mass fraction) - 7.23 %(mass fraction)) of ICP Optical Emission Spectrometry and the measurement value(xi) of Flame atomic absorption spectrometry was conducted to evaluate truenessusing fluid fertilizers (12 samples). As a result, a regression equation was y = − 0.021+ 0.969x, and its correlation coefficient (r) was 0.999. Additionally, additive recovery
testing was conducted using a fluid compound fertilizer (1 brand) and a homegarden-use mixed fertilizer (1 brand). As a result, the mean recovery rates at additive
level of 5 % (mass fraction) and 0.4 % (mass fraction) were 102.3 % and 104.0 %respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.。
Additionally, the minimum limit of quantification of this testing method is about
0.05 % (mass fraction).

193

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
7

3)

3)

(%)
5.69

(%)
0.02

Fluid mixed fertilizer
Home garder-use mixed
7
2.29
0.02
fertilizer（liquid)
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）

(%)
0.4

(%)
0.06

3)

(%)
1.1

0.8

0.04

1.6

6） Intermediate standard deviation
7） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

References
1)
Keisuke AOYAMA: Simultaneous Determination of Water-Soluble Principal
Ingredients (W-P2O5, W-K2O, W-MgO, W-MnO and W-B2O3) in Liquid Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), Research
Report of Fertilizer, Vol.8, p. 1 - 9 (2015)(5)

Flow sheet: The flow sheet for water-soluble potassium of the fluid mixed fertilizers is shown
below:
1 g analytical sample
Weigh to the order of 1 mg to a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 Flow sheet for water-soluble potassium in fluid fertilizers(Extraction procedure)

Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
ICP-OES (766.491nm)
Figure 2 Flow sheet for water-soluble potassium in fluid fertilizers(Measurement procedure)194

Testing Methods for Fertilizers (2018)

4.4 Silicic acid
4.4.1 Soluble silicic acid
4.4.1.a Potassium fluoride method(1) Summary
This testing method is applicable to fertilizers containing no silica gel fertilizers. This testing
method is classified as Type D and its symbol is 4.4.1.a-2017 or S-Si.a-1.
Extract by adding hydrochloric acid (1+23) to an analytical sample, add hydrochloric acid,potassium fluoride solution and potassium chloride and cool in a refrigerator, and then filter after
forming precipitate as potassium silicofluoride (K2SiF6). Put the precipitate in water and heat, andmeasure potassium silicofluoride (K2SiF6) dissolved by precipitation titration to obtain thehydrochloric acid (1+23) soluble silicic acid (soluble silicic acid (S-SiO2)) in an analytical sample.
In addition, the performance of this testing method is shown in Comment 5.
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C)
W1： Mass (g) of sulfamic acid sampled
A： Purity (% (mass fraction)) of sulfamic acidV1： Volume (mL) of sulfamic acid solution transferred
V2： Constant volume (250 mL) of sulfamic acid solution
V3： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
c) Potassium chloride: A JIS Guaranteed Reagent specified in JIS K 8121 or a reagent ofequivalent quality.
d) Potassium chloride solution (1): Add 250 mL of ethanol specified in JIS K 8101 to 750 mL ofwater to mix, and add 150 mL of potassium chloride to dissolve. Add a few drops of methyl
red solution (0.1 g/100 mL) as an indicator and drop hydrochloric acid until the color of thesolution becomes red to make it acidic. After leaving at rest for 1 day, neutralize with the 0.1
mol/L - 0.2 mol/L sodium hydroxide solution.
e) Potassium fluoride solution (1): Dissolve 58 g of potassium fluoride specified in JIS K 8815
195

Testing Methods for Fertilizers (2018)

in 1000 mL of water (2).
f) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K 8896
in 100 mL of ethanol (95) specified in JIS K 8102.
g) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in JIS K
8799 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in a container made of polymer that contains no silicon.
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Beaker made of polymer: A beaker made of polyethylene, etc. of a quality of material
which prevents silicic acid from eluting in the extract procedure in (4.1).
d) Filter made of polymer: A Gooch crucible made of polymer (compatible filter diameter: 25mm) or a funnel for reduced pressure filtering made of polymer (compatible filter diameter:
21 mm) A Gooch crucible made of polyethylene, etc. of a quality of material which prevents
silicic acid from eluting in the extract procedure in (4.1).
Comment 2 A funnel for reduced pressure filtering made of polymer (compatible filter diameter:
21 mm) is sold under the name Polyethylene Kiriyama Funnel SB-21.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add 150 mL of hydrochloric acid (1+23) heated up to about 30 ºC (3), and shake to mix at 30 40 revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (3) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.4.1.d.
Comment 4 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedure of (4.1) b).
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 20 mg - 50 mg as SiO2 and no more than
25 mL of liquid volume) to a 200-mL beaker made of polymer.
b) Add about 10 mL of hydrochloric acid and about 15 mL of potassium fluoride solution, andfurther add about 2 g of potassium chloride to dissolve, and then cool in a refrigerator for196

Testing Methods for Fertilizers (2018)about 30 minutes or more (4) to form the precipitate of potassium fluoride.
c) Filter under reduced pressure with a filter made of polymer (5) topped with Type 6 filter paper,and wash the container 3 times with a potassium chloride solution, then move the whole
precipitate into the filter, and further wash 6 - 7 times with a small amount of potassium
chloride solution (6).
d) Move the precipitate on the filter together with the filter paper into a 300-mL tall beaker withwater, and further add water to make about 200 mL and heat it up to 70 ºC - 80 ºC on a hot
plate.
e) Add a few drops of phenolphthalein solution (1 g/100 mL) to the sample solution as anindicator and titrate with the 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the colorof the solution becomes light red.
f) Calculate soluble silicic acid (S-SiO2) by the following formula.
Soluble silicic acid (S-SiO2) (% (mass fraction)) in an analytical sample= V4 × C × f × (V5/V6) × (15.021/W2) × (100/1000)
V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V5： Predetermined volume (mL) of the extract in (4.1) c)
V6： Volume (mL) of the extract transferred in (4.2) a)
W2： Mass (g) of the analytical sample
Note (4) To be no more than 10 ºC(5) Filter paper pulp can be stuffed to restrain precipitate from outflowing.
(6) Until the filtrate becomes neutral.
Comment 5 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 25 % (mass fraction) 40 % (mass fraction) and 10 % (mass fraction) are 98.4 % - 100.5 % and 101.0 % assoluble silicic acid (S-SiO2) respectively.
Additionally, the minimum limit of quantification of this testing method is about
0.3 % (mass fraction).
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.144- 146, Yokendo, Tokyo (1988)
2)
Yasushi MIYASHITA Verification of Performance Characteristics of Testing Method
for Soluble Silicic Acid in Fertilizer by Potassium Fluoride Method, Research Report of
Fertilizer Vol. 7, p. 123 - 130 (2014)197

Testing Methods for Fertilizers (2018)

5)

Flow sheet for soluble silicic acid: The flow sheet for soluble silicic acid in fertilizers isshown below:
1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask.
← About 150 mL hydrochloric acid (1+23) [about 30 ºC]
Rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hour
Cooling
Immediately
Water
(up
to
the marked line)←
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for soluble silicic acid in fertilizers (Extraction procedure)

Sample solution
Aliquot(predetermined
200-mL beaker made of polymer
amount)← About 10 mL hydrochloric acid
← About 15mL potassium fluoride solution← About 2 g of potassium chloride
Cooling
For 30 minutes or more in a refrigerator
Filtration under
reducedpressure
Washing

Filter made of polymer, Type 6 filter paper

Wash 6 - 7 times with potassium chloride solution

Transfer
300-mL tall beaker, water← Water (until the liquid volume reaches about 200 mL)
Heating
70 ºC - 80 ºC← A few drops of phenolphthalein solution (1 g/100 mL)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution
Titration(Until the solution becomes light red)
Figure 2 Flow sheet for soluble silicic acid in fertilizers (Measurement procedure)198

Testing Methods for Fertilizers (2018)

4.4.1.b Potassium fluoride method (Silica gel fertilizers, etc.)(1) Summary
This testing method is applicable to silica gel fertilizers and silica hydrogel fertilizers. This testing
method is classified as Type B and its symbol is 4.4.1.b-2017 or S-Si.b-1.
Extract by adding hydrochloric acid (1+23) to an analytical sample, add hydrochloric acid,potassium fluoride solution and potassium chloride and cool in a refrigerator, and then filter after
forming precipitate as potassium silicofluoride (K2SiF6). Put the precipitate in water and heat, andmeasure potassium silicofluoride (K2SiF6) dissolved by precipitation titration to obtain the sodiumhydroxide (20 g/L) soluble silicic acid (soluble silicic acid (S-SiO2)) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 3.
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C)
W1： Mass (g) of sulfamic acid sampled
A： Purity (% (mass fraction)) of sulfamic acidV1： Volume (mL) of sulfamic acid solution transferred
V2： Constant volume (250 mL) of sulfamic acid solution
V3： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
c) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
d) Potassium chloride: A JIS Guaranteed Reagent specified in JIS K 8121 or a reagent ofequivalent quality.
e) Potassium chloride solution (1): Add 250 mL of ethanol specified in JIS K 8101 to 750 mL ofwater to mix, and add 150 mL of potassium chloride to dissolve. Add a few drops of methyl
red solution (0.1 g/100 mL) as an indicator and drop hydrochloric acid until the color of thesolution becomes red to make it acidic. After leaving at rest for 1 day, neutralize with the 0.1
mol/L - 0.2 mol/L sodium hydroxide solution.
f) Potassium fluoride solution (1): Dissolve 58 g of potassium fluoride specified in JIS K 8815
199

Testing Methods for Fertilizers (2018)

in 1000 mL of water (2).
g) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K 8896
in 100 mL of ethanol (95) specified in JIS K 8102.
h) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in JIS K
8799 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in a container made of polyethylene, etc. that contains no silicon.
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Water bath: A water bath that can be adjusted to 65 ºC ± 2 ºC.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Volumetric flask and beaker made of polymer: A flask and a beaker that are made ofpolyethylene, etc. of a quality of material which prevents silicic acid from eluting in theextract procedure in (4.1).
d) Gooch crucible made of polymer: A Gooch crucible made of polymer (compatible filter
diameter: 25 mm) or a funnel for reduced pressure filtering made of polymer (compatible
filter diameter: 21 mm) A Gooch crucible made of polyethylene, etc. of a quality of material
which prevents silicic acid from eluting in the extract procedure in (4.1).
Comment 2 A funnel for reduced pressure filtering made of polymer (compatible filter diameter:
21 mm) is sold under the name Polyethylene Kiriyama Funnel SB-21.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask made of polymer.
b) Add about 150 mL of sodium hydroxide solution heated up to about 65 ºC, and heat for 1 hourwhile shaking to mix at every 10 minutes in a water bath at 65 ºC ± 2 ºC.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 20 mg - 50 mg as SiO2 and no more than
25 mL of liquid volume) to a 200-mL beaker made of polymer.
b) Add about 10 mL of hydrochloric acid and about 15 mL of potassium fluoride solution, andfurther add about 2 g of potassium chloride to dissolve, and then cool in a refrigerator for
about 30 minutes (3) to form the precipitate of potassium fluoride.
c) Filter under reduced pressure with a filter made of polymer (4) topped with Type 6 filter paper,and wash the container 3 times with a potassium chloride solution, then move the whole
precipitate into the filter, and further wash 6 - 7 times with a small amount of potassium
chloride solution (5).
d) Move the precipitate on the filter together with the filter paper into a 300-mL tall beaker withwater, and further add water to make about 200 mL and heat it up to 70 ºC - 80 ºC on a hot
plate.
e) Add a few drops of phenolphthalein solution (1 g/100 mL) as an indicator and titrate with the200

Testing Methods for Fertilizers (2018)f)

0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of the solution becomes lightred.
Calculate soluble silicic acid (S-SiO2) by the following formula.
Soluble silicic acid (S-SiO2) (% (mass fraction)) in an analytical sample= V4 × C × f × (V5/V6) × (15.021/W2) × (100/1000)
V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V5： Predetermined volume (mL) of the extract in (4.1) c)
V6： Volume (mL) of the extract transferred in (4.2) a)
W2： Mass (g) of the analytical sample

Note (3) To be no more than 10 ºC(4) Filter paper pulp can be stuffed to restrain precipitate from outflowing.
(5) Until the filtrate becomes neutral.
Comment 3 Table 1 shows results and analysis results from a collaborative study for testing
method validation.
Table 1 Results and analysis results from a collaborative study
for the test method validation of soluble silicic acid in silica gel fertilizers
4)

RSD r

3)

(%)

Silica gel fertilizers 1
8
79.37
0.23
0.3
Silica gel fertilizers 2
8
84.68
0.42
0.5
Silica gel fertilizers 3
8
89.58
0.4
0.4
Silica gel fertilizers 4
8
84.44
0.37
0.4
Silica gel fertilizers 5
8
85.77
0.46
0.5
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories × number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

Sample name

Number of1)laboratories

Mean

2)

sr

3)

(%)

(%)

5)

6)

RSD R

(%)

3)

(%)

0.55
0.85
0.51
0.77
0.59

0.7
1.0
0.6
0.9
0.7

sR

7)

References
1)
Takeshi HASHIMOTO, Akira SHIMIZU and Kaori OKADA: Validation of a Methodof Potassium Fluoride for Determination of Sodium Hydroxide-Soluble Silicic Acid in
Silica Gel Fertilizer, Research Report of Fertilizer, Vol.3, p. 19 - 24 (2010)
2)
Akira SHIMIZU, Shin ABE and Jun ITO: Determination of Solubility Silicic Acid in
Silica gel Fertilizer and Silica gel-including Fertilizer by Potassium Fluoride Method: A201

Testing Methods for Fertilizers (2018)

Collaborative Study, Research Report of Fertilizer, Vol.5, p. 31 - 40 (2012)
5)

Flow sheet for soluble silicic acid: The flow sheet for soluble silicic acid in fertilizers isshown below:
1g
Weigh to the order of 1 mg
analytical sample
into a 250-mL volumetric flask made of polymer.
← About 150 mL sodium hydroxide (20 g/L) [about 65 ºC]
Heating

65 ºC ± 2 ºC, 1 hour, shaking to mix every 10 minutes

Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for soluble silicic acid in silica gel fertilizers (Extraction procedure)

Sample solution
Aliquot(predetermined
200-mL beaker made of polymer
amount)← About 10 mL hydrochloric acid
← About 15mL potassium fluoride solution← About 2 g of potassium chloride
Cooling
For 30 minutes or more in a refrigerator
Filtration under
reducedpressure

Gooch filter made of polymer, Type 6 filter paper

Washing

Wash 6 - 7 times with potassium chloride solution

Transfer
300-mL tall beaker, water
Water(untilthe liquid volume reaches about 200 mL)←
Heating
70 ºC - 80 ºC← A few drops of phenolphthalein solution (1 g/100 mL)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution
Titration(Until the solution becomes light red)
Figure 2 Flow sheet for soluble silicic acid in silica gel fertilizers (Measurement procedure)202

Testing Methods for Fertilizers (2018)

4.4.1.c Potassium fluoride method (Fertilizers containing silica gel fertilizers)(1) Summary
This testing method is applicable to fertilizers containing silica gel fertilizers. This testing method isclassified as Type B and its symbol is 4.4.1.c-2017 or S-Si.c-1.
Mix the equivalent volumes of the extract which is extracted by adding hydrochloric acid (1+23) toan analytical sample and the liquid by extracting non-dissolved matter on a filter paper with sodiumhydroxide (20 g/L), and add hydrochloric acid, a potassium fluoride solution and potassium
chloride. Cool it in a refrigerator, and then filter after forming precipitate as potassium silicofluoride(K2SiF6). Then transfer water in the precipitate, heat and measure potassium silicofluoride (K2SiF6)
dissolved by precipitation titration to obtain soluble silicic acid (S-SiO2) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 3.
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f)= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C)
W1：
A：
V1：
V2：
V3：
C：

Mass (g) of sulfamic acid sampled
Purity (% ) of sulfamic acid
Volume (mL) of sulfamic acid solution transferred
Constant volume (250 mL) of sulfamic acid solution
Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
c) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
d) Potassium chloride: A JIS Guaranteed Reagent specified in JIS K 8121 or a reagent ofequivalent quality.
e) Potassium chloride solution (1): Add 250 mL of ethanol specified in JIS K 8101 to 750 mL ofwater to mix, and add 150 mL of potassium chloride to dissolve. Add a few drops of methyl
red solution (0.1 g/100 mL) as an indicator and drop hydrochloric acid until the color of thesolution becomes red to make it acidic. After leaving at rest for 1 day, neutralize with the 0.1
mol/L - 0.2 mol/L sodium hydroxide solution.
203

Testing Methods for Fertilizers (2018)

Potassium fluoride solution (1): Dissolve 58 g of potassium fluoride specified in JIS K 8815
in 1000 mL of water (2).
g) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K 8896
in 100 mL of ethanol (95) specified in JIS K 8102.
h) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in JIS K
8799 in 100 mL of ethanol (95) specified in JIS K 8102.
f)

Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in a container made of polymer that contains no silicon.
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
(3) Instruments: Instruments are as shown below:a) Water bath: A water bath that can be adjusted to 65 ºC ±2 ºC.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Volumetric flask and beaker made of polymer: A flask and a beaker that are made ofpolyethylene, etc. of a quality of material which prevents silicic acid from eluting in theextract procedure in (4.1).
d) Filter made of polymer: A Gooch crucible made of polymer (compatible filter diameter: 25mm) or a funnel for reduced pressure filtering made of polymer (compatible filter diameter:
21 mm) A Gooch crucible made of polyethylene, etc. of a quality of material which prevents
silicic acid from eluting in the extract procedure in (4.1).
Comment 2 A funnel for reduced pressure filtering made of polymer (compatible filter diameter:
21 mm) is sold under the name Polyethylene Kiriyama Funnel SB-21.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 300-mL tall beaker.
b) Add 150 mL of hydrochloric acid (1+23) warmed up to about 30 ºC, and warm it in a water
bath at 30 ºC ± 2 ºC while stirring every 10minutes with a glass rod for 1 hour.
c) After immediate cooling is complete, filter with Type 6 filter paper to a 250-mL volumetricflask as an acceptor. Wash the tall beaker with water, then move the whole residue on the filter
paper and add water up to the marked line to make a sample solution (1).
d) Transfer the non-dissolved matter on the filter paper together with the filter paper to a 250mL volumetric flask made of polymer.
e) Add 150 mL of sodium hydroxide solution heated up to about 65 ºC, and heat for 1 hour while
shaking to mix at every 10 minutes in a water bath at 65 ºC ± 2 ºC.
f) After immediate cooling is complete, add water up to the marked line and filter with Type 3
filter paper to make a sample solution (2).
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 20 mg - 50 mg as SiO2) (3) of the sample
solution (1) and (2) to a 200-mL beaker made of polymer.
b) Add about 10 mL of hydrochloric acid and about 15 mL of potassium fluoride solution, andfurther add about 2 g of potassium chloride to dissolve, and then cool in a refrigerator for
about 30 minutes or more (4) to form the precipitate of potassium fluoride.
c) Filter under reduced pressure with a filter made of polymer (5) topped with Type 6 filter paper,
204

Testing Methods for Fertilizers (2018)

and wash the container 3 times with a potassium chloride solution, then move the whole
precipitate into the filter, and further wash 6 - 7 times with a small amount of potassium
chloride solution (6).
d) Move the precipitate on the filter together with the filter paper into a 300-mL tall beaker withwater, and further add water to make about 200 mL and heat it up to 70 ºC - 80 ºC on a hot
plate.
e) Add a few drops of phenolphthalein solution (1 g/100 mL) as an indicator and titrate with the
0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of the solution becomes lightred.
f) Calculate soluble silicic acid (S-SiO2) by the following formula.
Soluble silicic acid (S-SiO2) (% (mass fraction)) in an analytical sample= V4 × C × f × (V5/V6) × (15.021/W2) × (100/1000)
V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C： Estimated concentration (mol/L) of sodium hydroxide solution (0.1 mol/L - 0.2
mol/L)
f： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V5： Predetermined volume (mL) of the sample solution in (4.1) c)
V6： Transferred amount (mL) of the sample solution in (4.2) a)
W2： Mass (g) of the analytical sample
Note (3) The transferred volume of the sample solution (1) and the sample solution (2) shouldbe equivalent.
(4) To be no more than 10 ºC(5) Filter paper pulp can be stuffed to restrain precipitate from outflowing.
(6) Until the filtrate becomes neutral.
Comment 3 Table 1 shows results and analysis results from a collaborative study for testing
method validation.
Additionally, the minimum limit of quantification of this testing method is about
0.6 % (mass fraction).

205

Testing Methods for Fertilizers (2018)

Table 1 Results and analysis results from a collaborative study for the test method validationof soluble silicic acid in fertilizers including a silica gel fertilizer
Sample name

Number of1)laboratories

4)

RSD r

3)

(%)

Mean

2)

sr

3)

(%)

(%)

5)

Mixed phosphate
8
24.99
0.16
0.6
fertilizer 1
Mixed phosphate
8
34.50
0.26
0.7
fertilizer 2
Compound
8
30.30
0.13
0.4
fertilizers 1
Compound
8
33.34
0.13
0.4
fertilizers 2
Compound
8
15.76
0.11
0.7
fertilizers 3
1) Number of laboratories used in analysis
2) Total mean (n = number of laboratories × number of replication(2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

6)

RSD R

(%)

3)

(%)

0.33

1.3

0.48

1.4

0.60

2.0

0.47

1.4

0.21

1.3

sR

7)

References
1)
Akira SHIMIZU, Jun ITO and Shin ABE: Method Validation of Potassium Fluoride
Method for Determination of Acid-Soluble and Base-Soluble Silicic Acid in Fertilizer
containing Silica gel, Research Report of Fertilizer, Vol.4, p. 1 - 8 (2011)2)
Akira SHIMIZU: Method Validation of Potassium Fluoride Method for Determinationof Acid-Soluble and Base-Soluble Silicic Acid in Fertilizer containing Silica Gel,
Research Report of Fertilizer, Vol.6, p. 1 - 8 (2013)3)
Shinji KAWAGUCHI and Akira SHIMIZU: Determination of Soluble Silicic Acid in
Fertilizers Containing Silica Gel Fertilizer by Potassium Fluoride Method: A
Collaborative Study, Research Report of Fertilizer Vol. 7, p. 36 - 42 (2014)

206

Testing Methods for Fertilizers (2018)(5)

Flow sheet for soluble silicic acid: The flow sheet for soluble silicic acid in fertilizers
including silica gel fertilizers is shown below:
1g
Weigh to the order of 1 mg
analytical sample
into a 300-mL tall beaker
About
150
mL
hydrochloric acid (1+23) [30 ºC]←
1 hour in water bath at 30 ºC ± 2 ºC
Warming(Stir at every 10 minutes)
Cooling
Filtration(supernatant)<Residue>

Immediately
Type 6 filter paper, a 250-mL volumetric flask

Transfer
Move the whole residue to a filter paper← Wash with water<Filtrate>← Wash (up to the marked line)
Sample solution (1)Put

250-mL volumetric flask made of polyer
together wiyh a filter paper← Add about 150 mL of sodium hydroxide solution (20 g/L) [65ºC] to stopple.
Then, shake to mix until the filter paper completely breaks down
Heating
65 ºC ± 2 ºC, while shaking to mix
at every 10 minutes for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution (2)Figure 1 Flow sheet for soluble silicic acid in fertilizers(Extraction procedure)207

Testing Methods for Fertilizers (2018)

Sample solution (1)and (2) Aliquot(predetermined amount)

Transfer the equal volume of the sample (1) and (2)to a 200-mL beaker made of polymer← About 10 mL of hydrochloric acid
← About 15 mL of potassium fluoride solution← About 2 g of potassium chloride
Cooling
For 30 minutes in a refrigerator
Filtration under
reduced pressure
Washing

Filter made of polymer
Type 6 filter paper
Wash 6 - 7 times with potassium chloride solution

Transfer
300-mL tall beaker, water← Water (until the liquid volume reaches about 200 mL)
Heating
70 ºC - 80 ºC← A few drops of phenolphthalein solution (1 g/100 mL)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution
Titration(Until the color of solution becomes light red)
Figure 2

Flow sheet for soluble silicic acid in fertilizers(Measurement procedure)

208

Testing Methods for Fertilizers (2018)

4.4.1.d Perchloric acid method(1) Summary
This testing method is applicable to fertilizers containing no silica gel fertilizers. This testing
method is classified as Type E and its symbol is 4.4.1.d-2017 or S-Si.d-1.
Extract by adding hydrochloric acid (1+23) to an analytical sample, add perchloric acid and heat,and then measure the formed silicic acid anhydride to obtain hydrochloric acid (1+23) soluble
silicic acid (soluble silicic acid (S-SiO2)) in an analytical sample.
(2) Reagentsa) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Perchloric acid: A JIS Guaranteed Reagent specified in JIS K 8223 or a reagent ofequivalent quality.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Electric furnace: An electric furnace that can be adjusted to 1000 ºC - 1100 ºC.
d) Crucible: After heating a chemical analysis porcelain crucible specified in JIS R 1301 in anelectric funnel at 1000 ºC - 1100 ºC, let it stand to cool in a desiccator and measure the mass
to the order of 1 mg.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add 150 mL of hydrochloric acid (1+23) heated up to about 30 ºC, and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Comment 1 The procedure in (4.1) is the same as the procedure in (4.1) of 4.4.1.a.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume to a 100-mL tall beaker.
b) Add about 10 mL of perchloric acid and heat.
c) When white smoke from the perchloric acid starts evolving, cover with a watch glass, thenheat for 15 - 20 minutes to form precipitate of silicone dioxide.
d) After standing to cool, add about 50 mL of hydrochloric acid (1+4) and cover with a watch
glass and heat at 70 ºC - 80 ºC on a hot plate for several minutes.
e) Immediately after heating, filter with Type 5-C filter paper, wash the container with heatedhydrochloric acid (1+10) and move the whole precipitate to a filter paper.
f) Wash the precipitate and the filter paper 2 times with heated hydrochloric acid (1+10), andfurther wash several times with hot water (1).
g) Put the precipitate together with the filter paper into the crucible.
h) Put the crucible into a drying apparatus and dry at about 120 ºC for 1 hour.
i) After standing to cool, put the crucible into an electric funnel and heat gently to char (2).
j) Ignite at 1000 ºC - 1100 ºC for 1 hour (2).
209

Testing Methods for Fertilizers (2018)k) After ignition, move the crucible to a desiccator and let it stand to cool.
l) After standing to cool, remove the crucible from the desiccator and measure the mass to theorder of 1 mg.
m) Calculate soluble silicic acid (S-SiO2) by the following formula.
Soluble silicic acid (S-SiO2) (% (mass fraction)) in an analytical sample= A × (V1/V2)/W × 100
A：
W：
V1：
V2：

Mass (g) of the precipitate
Mass (g) of the analytical sample
Predetermined volume (mL) of the sample solution in (4.1) c)
Transferred amount (mL) of the sample solution in (4.2) a)

Note (1) Wash until no reaction by chloride appears in the filtrate.
(2) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 1000 °C - 1100 °C in 1 to 2 hours.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.143- 144, Yokendo, Tokyo (1988)
5)

Flow sheet for soluble silicic acid: The flow sheet for soluble silicic acid in fertilizers isshown below:
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← About 150 mL of hydrochloric acid (1+23) [about 30 ºC]
Rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1

Flow sheet for soluble silicic acid in fertilizers (Extraction procedure)

210

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot(predetermined
100-mL tall beakeramount)←About 10 mL of perchloric acid
When white smoke of perchloric acid starts evolving,
Heating
cover with a watch glass, heat for 15 - 20 minutes
Standing to cool
Immediately←About 50 mL of hydrochloric acid (1+4)
Cover with a watch glass,
Heatingheat at 70 ºC - 80 ºC for several minutes
Filtration

Type 5-C filter paper←Wash 2 times with hydrochloric acid (1+10)←Wash several times with hot water
DryingDrying apparatus, about 120 ºC, 1 hourStanding to coolCharring
Incineration

Heat gently in an electric furnace1000 ºC - 1100 ºC for no less than 1 hourStanding to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure 2

Flow sheet for soluble silicic acid in fertilizers (Measurement procedure)211

Testing Methods for Fertilizers (2018)

4.4.2
Water-soluble silicic acid
4.4.2.a Potassium fluoride method(1) Summary
This testing method is applicable to liquid potassium silicate fertilizers. This testing method isclassified as Type D and its symbol is 4.4.2.a-2017 or W-Si.a-1.
Extract by adding water to an analytical sample, add hydrochloric acid, a potassium fluoride
solution and potassium chloride and cool in a refrigerator, and then filter after forming precipitate as
potassium silicofluoride (K2SiF6). Put the precipitate in water and heat, and measure potassium
silicofluoride (K2SiF6) dissolved by precipitation titration to obtain the water-soluble silicic acid
(W-SiO2) in an analytical sample. In addition, the performance of this testing method is shown inComment 5.
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f)= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C)
W1：
A：
V1：
V2：
V3：
C：

Mass (g) of sulfamic acid sampled
Purity (% (mass fraction)) of sulfamic acid
Volume (mL) of sulfamic acid solution transferred
Constant volume (250 mL) of sulfamic acid solution
Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
c) Potassium chloride: A JIS Guaranteed Reagent specified in JIS K 8121 or a reagent ofequivalent quality.
d) Potassium chloride solution (1): Add 250 mL of ethanol specified in JIS K 8101 to 750 mL ofwater to mix, and add 150 mL of potassium chloride to dissolve. Add a few drops of methyl
red solution (0.1 g/100 mL) as an indicator and drop hydrochloric acid until the color of thesolution becomes red to make it acidic. After leaving at rest for 1 day, neutralize with the 0.1
mol/L - 0.2 mol/L sodium hydroxide solution.
e) Potassium fluoride solution (1): Dissolve 58 g of potassium fluoride specified in JIS K 8815
in 1000 mL of water (2).
212Testing Methods for Fertilizers (2018)f)

Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K 8896
in 100 mL of ethanol (95) specified in JIS K 8102.
g) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in JIS K
8799 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in a container made of polyethylene, etc. that contains no silicon.
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Beaker made of polymer: A beaker made of polyethylene, etc. of a quality of material
which prevents silicic acid from eluting in the extract procedure in (4.1).
d) Filter made of polymer: A Gooch crucible made of polymer (compatible filter diameter: 25mm) or a funnel for reduced pressure filtering made of polymer (compatible filter diameter:
21 mm) A Gooch crucible made of polyethylene, etc. of a quality of material which prevents
silicic acid from eluting in the extract procedure in (4.1).
Comment 2 A funnel for reduced pressure filtering made of polymer (compatible filter diameter:
21 mm) is sold under the name Polyethylene Kiriyama Funnel SB-21.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL volumetricflask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 3 In the procedure in a), it is also allowed to weigh 2.5 g of an analytical sample to theorder of 1 mg, and put it into a 250-mL volumetric flask.
Comment 4 The procedure in (4.1) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 20 mg - 50 mg as SiO2 and no more than
25 mL of liquid volume) to a 200-mL beaker made of polymer.
b) Add about 10 mL of hydrochloric acid and about 15 mL of potassium fluoride solution, andfurther add about 2 g of potassium chloride to dissolve, and then cool in a refrigerator for
about 30 minutes or more (3) to form the precipitate of potassium fluoride.
c) Filter under reduced pressure with a filter made of polymer (4) topped with Type 6 filter paper,and wash the container 3 times with a potassium chloride solution, then move the whole
precipitate into the filter, and further wash 6 - 7 times with a small amount of potassium
chloride solution (5).
d) Move the precipitate on the filter together with the filter paper into a 300-mL tall beaker withwater, and further add water to make about 200 mL and heat it up to 70 ºC - 80 ºC on a hot213

Testing Methods for Fertilizers (2018)e)f)plate.
Add a few drops of phenolphthalein solution (1 g/100 mL) to the sample solution as anindicator and titrate with the 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the colorof the solution becomes light red.
Calculate water soluble silicic acid (W-SiO2) by the following formula.
Water soluble silicic acid (W-SiO2) (% (mass fraction)) in an analytical sample= V4 × C × f × (V5/V6) × (15.021/W2) × (100/1000)
V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V5： Predetermined volume (mL) of the extract in (4.1) c)
V6： Volume (mL) of the extract transferred in (4.2) a)
W2： Mass (g) of the analytical sample

Note (3) To be no more than 10 ºC(4) Filter paper pulp can be stuffed to restrain precipitate from outflowing.
(5) Until the filtrate becomes neutral.
Comment 5 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 30 % (mass fraction) and
12 % (mass fraction) - 20% (mass fraction) are 100.7 % and 99.5 % - 100.5 aswater-soluble silicic acid (W-SiO2) respectively.
The results of the repeatability tests on different days using a liquid potassium
silicate fertilizer to evaluate precision were analyzed by the one-way analysis ofvariance. Table 1 shows the calculation results of intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.2 % (mass fraction).
Table 1 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
samplerepeatability Averagesr
RSD rs I(T)RSD I(T)
1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)
Liquid potassium silicate fertilizer 1
7
24.01
0.07
0.3
0.08
0.4
Liquid potassium silicate fertilizer 2
7
16.07
0.03
0.2
0.04
0.3
1） The number of test days conducting a duplicate test
6） Intermediate standard deviation
2） Average （the number of test days （T ）7） Intermediate relative standard deviation
×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.144- 146, Yokendo, Tokyo (1988)
2)
Shinji KAWAGUCHI: Verification of Performance Characteristics of Testing Method214

Testing Methods for Fertilizers (2018)

for Water-Soluble Silicic Acid in Fertilizer by Potassium Fluoride Method, Research
Report of Fertilizer, Vol.8, p. 174 - 181 (2015)

5)

Flow sheet for water-soluble silicic acid: The flow sheet for water-soluble silicic acid in
fertilizers is shown below:
5 g analytical sample
Weigh to the order of 1 mg to a 500-mL volumetric flask.
← About 400 mL of water
Rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for water-soluble silicic acid in fertilizers (Extraction procedure)

Sample solution
Aliquot(predetermined
200-mL beaker made of polymer
amount)← About 10 mL hydrochloric acid
← About 15mL potassium fluoride solution← About 2 g of potassium chloride
Cooling
For 30 minutes or more in a refrigerator
Filtration under
reduced pressure
Washing

Filter made of polymer
Type 6 filter paper
Wash 6 - 7 times with potassium chloride solution

Transfer
300-mL tall beaker, water← Water (until the liquid volume reaches about 200 mL)
Heating
70 ºC - 80 ºC← A few drops of phenolphthalein solution (1 g/100 mL)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution
Titration(Until the solution becomes light red)
Figure 1 Flow sheet for water-soluble silicic acid in fertilizers (Measurement procedure)215

Testing Methods for Fertilizers (2018)

4.5 Lime, calcium and alkalinity
4.5.1 Total lime
4.5.1.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers containing organic matters. This testing method isclassified as Type C and its symbol is 4.5.1.a-2017 or T-Ca.a-1.
Pretreat an analytical sample with incineration and hydrochloric acid, add an interference
suppressor solution, and then spray in an acetylene−air flame, and measure the atomic absorption
with calcium at a wavelength of 422.7 nm to quantify total lime (T-CaO) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 4.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2000-mL beaker, add a small amount of water,
add gradually 420 mL of hydrochloric acid to dissolve, and further add water to make 1000mL.
c) Calcium standard solution (CaO 1 mg/mL) (1): Heat calcium carbonate specified in JIS K
8617 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, and weigh 1.785 g
into a weighing dish. Transfer to a 1000-mL volumetric flask with a small amount of water,
add 20 mL of hydrochloric acid (1+3) to dissolve, and add water up to the marked line.
d) Calcium standard solution (CaO 5 µg/mL - 50 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of a calcium standard solution (CaO 1 mg/mL) to
500-mL volumetric flasks step-by-step, add about 50 mL of interference suppressor solution(3), and add water up to the marked line (4).
e) Blank test solution for the calibration curve preparation (1): Transfer about 50 mL ofinterference suppressor solution to a 500-mL volumetric flask (3), and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can also beused.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
(4) For storage, use a sealable container made of materials which are not likely to dissolve
calcium, such as borosilicate glass-1 specified in JIS R 3503 or Teflon.
Comment 1 Instead of the calcium standard solution in (2), a calcium standard solution for thecalibration curve preparation can be prepared by using a calcium standard solution(Ca 1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case, calculate
total lime (T-CaO) in the analytical sample by multiplying the concentration (Ca) of
the calcium standard solution for calibration curve preparation or a measurement
value (Ca) obtained in (4.2) by a conversion factor (1.3992).
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121.
1) Light source: A calcium hollow cathode lamp
2) Gas: Gas for heating by flame216

Testing Methods for Fertilizers (2018)(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 550 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Incineration-hydrochloric acid boilinga) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 200-mL - 300-mL tallbeaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (5).
c) Ignite at 550 ºC ± 5 ºC for no less than 4 hours to incinerate (5).
d) After standing to cool, moisten the residue with a small amount of water, gradually add about
10 mL of hydrochloric acid, and further add water to make 20 mL.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to boil forabout 5 minutes.
f) After standing to cool, transfer the solution to a 250-mL - 500-mL volumetric flask withwater.
g) Add water up to the marked line.
h) Filter with Type 3 filter paper to make a sample solution.
Note (5) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 550 °C in 1 to 2 hours.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.1.a.
(4.1.2) Incineration-aqua regia digestiona) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 200-mL - 300-mL tallbeaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (6).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (6).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (7), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (8) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL volumetric flask with water, add water up to themarked line, and filter with Type 3 filter paper to make a sample solution.
Note (6) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 550 °C in 1 to 2 hours.
(7) The watch glass can be removed.
(8) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetric217

Testing Methods for Fertilizers (2018)flask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 3 The procedures in (4.1.2) are the same as in (4.1.3) in 4.2.1.a. and (4.1) a) - h) in
5.3.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 422.7 nmb) Calibration curve preparation
1) Spray the calcium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 422.7 nm.
2) Prepare a curve for the relationship between the calcium concentration and the indicated valueof the calcium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 5 mg as
CaO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the calcium content from the calibration curve, and calculate total lime (T-CaO) in theanalytical sample.
Comment 4 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 15 % (mass fraction) and
1 % (mass fraction) are 101.8 % and 97.9% as total lime (T-CaO) respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.05 % (mass fraction).

218

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of

Number of

Repeatability
2)certified reference laboratory Averagematerial fertilizer

p

1)

(%)

3)

FAMIC-C-12

11

5.82

4)

RSD r

3)

0.07

sr

(%)

Intermediate precision

Reproducibilitys I(T)

6)

7)
RSD I(T)

sR

(%)

(%)

3)

(%)

(%)

1.2

0.11

2.0

1） The number of laboaratories used for analysis

5)

8)

RSD R

9)

3)

(%)

0.29

5.0

6） Intermediate standard deviation

conducting flame atomic absorbance spectrometry
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

7） Intermediate relative standard deviation
8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.156- 158, Yokendo, Tokyo (1988)
2)
Kimie KATO, Masayuki YOSHIMOTO and Yuji SHIRAI: 3) Kimie
KATO,
Masayuki YOSHIMOTO and Yuji SHIRAI: Systematization of Determination Methodsof Major Components in Sludge Fertilizer, Compost and Organic Fertilizer, Research
Report of Fertilizer, Vol.3 p. 107 - 116 (2010)3)
Souichi IGARASHI and Yasuharu KIMURA:
Verification of Performance
Characteristics of Testing Methods for Calcium Content in Fertilizer by Atomic
Absorption Spectrometry: Research Report of Fertilizer Vol. 6, p. 183 - 192 (2013)

219

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total lime: The flow sheet for total lime in fertilizers is shown below:
5 g analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL- 300-mL tall beaker.
Heat gently
Ignite at 550 ºC ±5 ºC, no less than 4 hours

Standing to coolRoom temperature← Small amount of water, moisten the residue← About 10 mL of hydrochloric acid
← Water (up to about 20 mL)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolRoom temperature

Transfer
250-mL - 500-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for total lime in fertilizers.
(Incineration-hydrochloric acid boiling prorcedure (4.1.1))

220

Testing Methods for Fertilizers (2018)5g
analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC for 8 - 12 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass to digest
Heating

Slightly move the watch glass and remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass to dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for total lime in fertilizers(Incineration-aqua regia digestion procedure (4.1.2))
Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)←About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer or frame photometer
Figure 2 Flow sheet for total lime in fertilizers (Measurement)

221

Testing Methods for Fertilizers (2018)

4.5.2 Soluble lime
4.5.2.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers that guarantee alkalinity. This testing method isclassified as Type D and its symbol is 4.5.2.a-2017 or S-Ca.a-1.
Add hydrochloric acid (1+23) to an analytical sample, boil to extract, and add an interference
suppressor solution, and then spray in an acetylene−air flame, and measure the atomic absorption
with calcium at a wavelength of 422.7 nm to quantify hydrochloric acid (1+23) soluble lime
(soluble lime (S-CaO)) in an analytical sample. In addition, the performance of this testing method
is shown in Comment 5.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (2): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (1) specified in JIS K 8132 into a 2000-mL beaker, add a small amount of water,
add gradually 420 mL of hydrochloric acid to dissolve, and further add water to make 1000mL.
c) Calcium standard solution (CaO 1 mg/mL) (2): Put calcium carbonate specified in JIS K
8617 in a drying apparatus, heat at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a
desiccator, and weigh 1.785 g into a weighing dish. Transfer to a 1000-mL volumetric flaskwith a small amount of water, add 20 mL of hydrochloric acid (1+3) to dissolve, and add
water up to the marked line.
d) Calcium standard solution (CaO 5 µg/mL - 50 µg/mL) for the calibration curve
preparation (2): Transfer 2.5 mL - 25 mL of a calcium standard solution (CaO 1 mg/mL) to
500-mL volumetric flasks step-by-step, add about 50 mL of interference suppressor solution(3), and add water up to the marked line (4).
e) Blank test solution for the calibration curve preparation (2): Transfer about 50 mL ofinterference suppressor solution to a 500-mL volumetric flask (3), and add water up to themarked line (4).
Note (1) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can also beused.
(2) This is an example of preparation; prepare an amount as appropriate.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
(4) For storage, use a sealable container made of materials which are not likely to dissolve
calcium, such as borosilicate glass-1 specified in JIS R 3503 or Teflon.
Comment 1 Instead of the calcium standard solution in (2), a calcium standard solution for thecalibration curve preparation can be prepared by using a calcium standard solution(Ca 1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case, calculate
soluble lime (S-CaO) in the analytical sample by multiplying the concentration (Ca)of the calcium standard solution for calibration curve preparation or a measurement
value (Ca) obtained in (4.2) by a conversion factor (1.3992).
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121.
1) Light source: A calcium hollow cathode lamp222

Testing Methods for Fertilizers (2018)

2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hot plate : A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 2 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL tall beaker.
b) Add about 20 mL of hydrochloric acid (1+23), cover with a watch glass, and boil on a hot
plate for about 5 minutes (5).
c) After immediate cooling is complete, transfer to a 250-mL - 500-mL volumetric flask withwater.
d) Immediately add water up to the marked linee) Filter with Type 3 filter paper to make a sample solution.
Note (5) Be aware that an analytical sample should not solidify in the bottom of a beaker.
Comment 2 In the case of a by-product magnesia fertilizer or a fertilizer containing a by-productmagnesia, if the pH of the sample solution of d) is neutral or basic, prepare a sample
solution anew by replacing “2 g of an analytical sample” in the procedure in a) with
“1 g - 1.5 g of an analytical sample”.
Comment 3 In the procedure in a), a 500-mL volumetric flask can be used instead of a 500-mLtall beaker. However, the volumetric flask used should be distinguished as an
extraction flask and should not be used for the other purposes. In addition, “coverwith a watch glass” in b) is replaced by “place a funnel”, and “transfer to a 250-mL 500 mL volumetric flask with water” in the procedure in c) is skipped.
Comment 4 The procedure in (4.1) is the same as in (4.1) in 4.6.1.a and 4.7.1.a(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 422.7 nmb) Calibration curve preparation
1) Spray the calcium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 422.7 nm.
2) Prepare a curve for the relationship between the calcium concentration and the indicated valueof the calcium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 5 mg as
CaO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the calcium content from the calibration curve, and calculate soluble lime (S-CaO) in
the analytical sample.
Comment 5 Recovery testing was conducted to evaluate trueness using a preparation sample. As a223

Testing Methods for Fertilizers (2018)

result, the average rate of recovery at the content level of 20 % (mass fraction) and
1 % (mass fraction) are 100.9 % and 101.1% as soluble lime (S-CaO) respectively.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.167- 169, Yokendo, Tokyo (1988)
2)
Souichi IGARASHI and Yasuharu KIMURA: Verification of Performance
Characteristics of Testing Methods for Calcium Content in Fertilizer by Atomic
Absorption Spectrometry: Research Report of Fertilizer Vol. 6, p. 183 - 192 (2013)(5)

Flow sheet for soluble lime: The flow sheet for soluble lime in fertilizers is shown below:

2 g analytical sample
Weigh to the order of 1 mg into a 500-mL tall beaker.
← About 200 mL of hydrochloric acid (1+23)
Heating
Cover with a watch glass, and boil for 5 minutes.
Cooling

Immediately

Transfer
Water, 250-mL - 500-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for soluble lime in fertilizers (Extraction procedure)

Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)←About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer
Figure 2 Flow sheet for soluble lime in fertilizers (Measurement procedure)

224

Testing Methods for Fertilizers (2018)

4.5.3 Water-soluble calcium
4.5.3.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers that indicate calcium content as a response modifier.
This testing method is classified as Type D and its symbol is 4.5.3.a-2017 or W-Ca.a-1.
Extract by adding water to an analytical sample, and add an interference suppressor solution, andthen spray in an acetylene−air flame, and measure the atomic absorption with calcium at awavelength of 422.7 nm to quantify water-soluble calcium (W-Ca) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 3.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2000-mL beaker, add a small amount of water,
add gradually 420 mL of hydrochloric acid to dissolve, and further add water to make 1000mL.
c) Calcium standard solution (Ca 1 mg/mL): Calcium standard solution (Ca 1 mg/mL)
traceable to National Metrology.
d) Calcium standard solution (Ca 5 µg/mL - 50 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of a calcium standard solution (Ca 1 mg/mL) to
500-mL volumetric flasks step-by-step, add about 50 mL of interference suppressor solution(3), and add water up to the marked line (4).
e) Blank test solution for the calibration curve preparation (1): Transfer about 50 mL ofinterference suppressor solution to a 500-mL volumetric flask (3), and add water up to themarked line (4).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can also beused.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
(4) For storage, use a sealable container made of materials which are not likely to dissolve
calcium, such as borosilicate glass-1 specified in JIS R 3503 or Teflon.
Comment 1 Instead of the calcium standard solution in (2), a calcium standard solution for thecalibration curve preparation can be prepared by using a calcium standard solution(Ca 10 mg/mL) traceable to National Metrology.
Comment 2 Instead of the calcium standard solution in (2), a calcium standard solution (CaO 5µg/mL - 50 µg/mL) for the calibration curve preparation prepared in (2) in 4.5.1.acan be used. In this case, calculate water-soluble calcium (W-Ca) in the analyticalsample by multiplying the concentration (CaO) of a calcium standard solution for
calibration curve preparation or a measurement value (CaO) obtained in (4.2) by aconversion factor (0.7147).
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121.
225

Testing Methods for Fertilizers (2018)

1) Light source: A calcium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it in a 500-mL volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 422.7 nmb) Calibration curve preparation
1) Spray the calcium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 422.7 nm.
2) Prepare a curve for the relationship between the calcium concentration and the indicated valueof the calcium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 5 mg asCa) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the calcium content from the calibration curve, and calculate water-soluble calcium(W-Ca) in the analytical sample by the following formula.
Comment 3 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 1 % (mass fraction) - 5 %(mass fraction) is 98.1 % - 101.1% as water-soluble calcium (W-Ca) respectively.
Additionally, the minimum limit of quantification of this testing method is about
0.07 % (mass fraction) for solid fertilizers, and 0.04 % (mass fraction) for fluid
fertilizers.
References
1)
Souichi IGARASHI and Yasuharu KIMURA: Verification of Performance
Characteristics of Testing Methods for Calcium Content in Fertilizer by Atomic
Absorption Spectrometry: Research Report of Fertilizer Vol. 6, p. 183 - 192 (2013)

226

Testing Methods for Fertilizers (2018)(5)

Flow sheet for water soluble calcium: The flow sheet for water soluble calcium in fertilizers
is shown below:
1.00 g500-mL volumetric flask
analytical sample← About 400 mL of water
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for water-soluble calcium in fertilizers (Extraction procedure)

Sample solution
Aliquot(predetermined amount)

100-mL volumetric flask← About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (422.7 nm)
Figure 2 Flow sheet for water-soluble calcium in fertilizers (Measurement procedure)227

Testing Methods for Fertilizers (2018)

4.5.3.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers, liquid microelement mixed fertilizers
and the fluid fertilizers of home garden-use mixed fertilizers. This testing method is classified as
Type D and its symbol is 4.5.3.b-2017 or W-Ca.b-1.
Add water to an analytical sample to extract, introduce it to an ICP Optical Emission Spectrometer(“ICP-OES”) and measure the calcium at a wavelength of 393.366 nm to obtain water-soluble
calcium (W-Ca). In addition, the performance of this testing method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Calcium standard solution (Ca 1 mg/mL): Calcium standard solution (Ca 1 mg/mL)
traceable to National Metrology.
d) Calcium standard solution (Ca 0.1 mg/mL) (1): Transfer 10 mL of calcium standard solution(Ca 1 mg/mL) to a 100-mL flask and add hydrochloric acid (1+23) up to the marked line.
e) Calcium standard solutions (Ca 1 µg/mL - 20µg /mL) for the calibration curve
preparation (1): Transfer 1 mL - 20 mL of calcium standard solution (Ca 0.1 mg/mL) to
100-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) up to the markedline.
f) Calcium standard solutions (Ca 0.1 µg/mL - 1 µg/mL) for the calibration curve
preparation (1): Transfer 1 mL - 10 mL of calcium standard solution (Ca 10 µg/mL) for thecalibration curve preparation to 100-mL volumetric flasks step-by-step, and add hydrochloric
acid (1+23) up to the marked line.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d), e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the calcium standard solution in (2), a calcium standard solution for thecalibration curve preparation can be prepared by using a calcium standard solution(Ca 10 mg/mL) traceable to National Metrology.
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in e) and f) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, when using the ICP-OES
of an axial observation mode, it is recommended to prepare a calcium standard
solution for the calibration curve in the concentration range which is suitable to a
device used.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample (2) to the order of 1 mg, and put it in a 100-mL volumetric
228

Testing Methods for Fertilizers (2018)flask.
b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (2) The sampling amount of the analytical sample is 10 g when the content in theanalytical sample is less than 0.01 % (mass fraction) as water-soluble calcium.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up the measurement
conditions for the ICP Atomic Emission Spectrometer considering the following:
Analytical line wavelength: 393.366 nmb) Calibration curve preparation
1) Spray the calcium standard solution for the calibration curve preparation and blank test
solution for the calibration curve preparation into inductively coupled plasma, and read theindicated value at a wavelength of 393.366 nm.
2) Prepare a curve for the relationship between the calcium concentration and the indicated valueof the calcium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 2 mg asCa) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the calcium content from the calibration curve, and calculate water-soluble calcium(W-Ca) in the analytical sample by the following formula.
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.4.d Comment 4.
Comment 5 The comparison of the measurement value (yi：0.095 % (mass fraction) - 10.93 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of flame atomic absorbance spectrometry was conducted to evaluate truenessusing fluid fertilizers (12 samples). As a result, a regression equation was y = 0.005 +0.978x, and its correlation coefficient (r) was 0.999. Additionally, additive recovery
testing was conducted using a fluid compound fertilizer (1 brand) and a homegarden-use mixed fertilizer (1 brand). As a result, the mean recovery at additive levelof 0.01 % (mass fraction) and 0.1 % (mass fraction) were105.9 % and 106.4 %respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.0005 % (mass fraction).

229

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
7

3)

(%)
2.14

3)

(%)
0.02

Fluid mixed fertilizer
Home garder-use
mixed fertilizer
7
0.103
0.001
（fluid)1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

(%)
0.7

(%)
0.05

3)

(%)
2.1

0.9

0.001

1.0

6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Keisuke AOYAMA: Simultaneous Determination Method for Effect-Development
Promoting Agent (Ca, Fe, Co, Cu, Zn and Mo) in Liquid Compound Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES), Research
Report of Fertilizer, Vol.9, p. 1 - 9 (2016)(5)

Flow sheet: The flow sheet for water-soluble calcium of the fluid mixed fertilizers is shown
below:

1 g analytical sample
Weigh to the order of 1 mg to a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 The flow sheet for water-soluble calcium in fluid fertilizers(Extraction procedure)
Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
ICP-OES (393.366 nm)
Figure 2 The flow sheet for water-soluble calcium in fluid fertilizers(Measurement procedure)230

Testing Methods for Fertilizers (2018)

4.5.4 Alkalinity
4.5.4.a Ethylenediamine tetraacetate method(1) Summary
This testing method is applicable to fertilizers that guarantee alkalinity. This testing method isclassified as Type E and its symbol is 4.5.4.a-2017 or AL.a-1.
Add hydrochloric acid (1+23) to an analytical sample, boil to extract, and mask with
2,2’,2”-nitrilotriethanol and a potassium cyanide solution, add a 0.01 mol/L ethylenediamine
tetraacetate standard solution, and conduct a chelatometric titration with a 0.01 mol/L magnesium
standard solution to obtain alkalinity (AL). Or after masking, conduct a chelatometric titration withan ethylenediamine tetraacetate standard solution to obtain alkalinity (AL) in an analytical sample.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
c) Ascorbic acid: A JIS Guaranteed Reagent specified in JIS K 9502 or a reagent of equivalentquality.
d) 2,2’,2”-nitrilotriethanol (1): A JIS Guaranteed Reagent specified in JIS K 8663 or a reagent ofequivalent quality.
e) Acetone: A JIS Guaranteed Reagent specified in JIS K 8034 or a reagent of equivalentquality.
f) Ammonia solution: A JIS Guaranteed (NH3 28 % (mass fraction)) reagent specified in JIS K
8085 or a reagent of equivalent quality.
g) 0.01 mol/L ethylenediamine tetraacetate standard solution: Dissolve 3.72 g ofethylenediaminetetraacetic acid dihydrogen disodium dihydrate in water to make 1000 mL.
Standardization: Wash zinc reference material for volumetric analysis specified in JIS K
8005 with hydrochloric acid (1+3), water, ethanol (99.5) specified in JIS K 8101, and diethyl
ether specified in JIS K 8103 successively, and immediately leave at rest in a desiccator for
about 12 hours under no more than 2 kPa to dry, and then weigh about 0.65 mg to the orderof 0.1 mg, transfer it to a 1000-mL volumetric flask, add about 10 mL of hydrochloric acidto dissolve and then add water up to the marked line. Transfer 25 mL of the solution to a
200-mL - 300-mL Erlenmeyer flask, add about 15 mL of water and about 5 mL ofammonium chloride buffer solution, and titrate with a 0.01 mol/L ethylenediamine
tetraacetate standard solution, while adding Eriochrome Black T solution as an indicator,
until the color of the solution becomes blue. Calculate the factor of a 0.01 mol/L
ethylenediamine tetraacetate standard solution by the following formula.
Factor of 0.01 mol/L ethylenediamine tetraacetate standard solution (f1)
= W1 × (A/100) × (1/65.409) × (V1/V2) × (1000/V3) × (1/C1)= W1 × A× (1/65.409) × (0.25/V3)
W：
A：
V1：
V2：
V3：

Mass (g) of d zinc sampled
Purity (% (mass fraction)) of zinc
Volume (25 mL) of zinc solution transferred
Constant volume (1000 mL) of zinc solution
Volume (mL) of 0.01 mol/L ethylenediamine tetraacetate standard solution needed
for titration
C1: Set concentration (0.01 mol/L) of 0.01 mol/L ethylenediamine tetraacetatestandard solution
231

Testing Methods for Fertilizers (2018)h) 0.01 mol/L magnesium standard solution: Put 0.24 g of magnesium specified in JIS K
8875 into a 1000-mL beaker, add about 10 mL of hydrochloric acid to dissolve, add a properamount of water, and while adding a methyl red solution (0.1 g/100 mL) as an indicator,
neutralize with an ammonia solution (1+3) until the color of the solution becomes yellow, andthen add water to make 1000 mL.
Standardization: Transfer 25 mL of 0.01 mol/L magnesium standard solution to a 200-mL 300-mL Erlenmeyer flask, add about 15 mL of water and about 5 mL of ammonium chloride
buffer solution, and while adding an Eriochrome Black T solution as an indicator, titratewith 0.01 mol/L ethylenediamine tetraacetate standard solution until the color of the solution
becomes blue. Calculate the factor of a 0.01 mol/L magnesium standard solution by thefollowing formula.
Factor of 0.01 mol/L magnesium standard solution (f2)
= (C1 × f1 × V4) × (1/V5) × (1/C2)= (f1 × V4) × (1/V5)
C1 ：
C2 ：
f1 ：
V4：
V5：

Set concentration (0.01 mol/L) of 0.01 mol/L ethylenediamine tetraacetatestandard solution
Set concentration (0.01 mol/L) of 0.01 mol/L magnesium standard solution
Factor of 0.01 mol/L ethylenediamine tetraacetate standard solution
Volume (mL) of 0.01 mol/L ethylenediamine tetraacetate standard solution
needed for titration
Volume (mL) of 0.01 mol/L magnesium standard solution transferred.

i)Ammonium chloride solution: Dissolve 70 g of ammonium chloride specified in JIS K
8116 and 570 mL of ammonia solution in water to make 1000 mL.
j) 2-aminoethanol solution: Add 400 mL of water to 150 mL of 2-aminoethanol specified in
JIS K 8109, add gradually hydrochloric acid to a pH 10.6.
k) Potassium cyanide solution: Dissolve 100 g of potassium cyanide specified in JIS K 8443
in water to make 1000 mL.
l) Eriochrome Black T solution: Dissolve 0.5 g of Eriochrome Black T specified in JIS K
8736 and 4.5 g of hydroxyl-ammonium chloride in methanol−water (95+5) to make 100 mL.
m) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K 8896
in 100 mL of ethanol (95) specified in JIS K 8102.
n) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
o) Citric acid solution (2): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
Note (1) The reagent corresponds to triethanolamine in the Official Methods of Analysis of
Fertilizers (1992).
(2) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of a 0.01 mol/L ethylenediamine tetraacetate standard solution in (2) g), a 0.1
mol/L ethylenediaminetetraacetic acid dihydrogen disodium solution conforming to
ISO/IEC 17025 can be used.
(3) Instruments: Instruments are as shown below:a) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
232Testing Methods for Fertilizers (2018)(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 2 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL tall beaker.
b) Add about 200mL of hydrochloric acid (1+23), cover with a watch glass, and boil on a hot
plate for about 5 minutes (3).
c) Immediately transfer to a 250-mL - 500-mL volumetric flask with water.
d) Immediately add water up to the marked line (4).
e) Filter with Type 3 filter paper to make a sample solution.
Note (3) Be aware that an analytical sample should not solidify in the bottom of a beaker.
(4) Conduct the procedure in Comment 5 when there is much manganese content in thefertilizers.
Comment 2 In the case of a by-product magnesia fertilizer, if the pH of the sample solution of d)is neutral or basic, prepare a sample solution anew by replacing “2 g of an analyticalsample” in the procedure in a) with “1 g - 1.5 g of an analytical sample”.
Comment 3 In the procedure in a), a 500-mL volumetric flask can be used instead of a 500-mLtall beaker. However, the volumetric flask used should be distinguished as an
extraction flask and should not be used for the other purposes. In addition, “coverwith a watch glass” in b) is replaced by “place a funnel”, and “transfer to a 250-mL 500 mL volumetric flask with water” in the procedure in c) is skipped.
Comment 4 The procedure in (4.1) is the same as the procedure in (4.1) of 4.5.2.a.
Comment 5 Add a predetermined volume of filtrate in (4.1) e), add a drop of methyl red solution
as an indicator and drop ammonia water (28 % (mass fraction)) specified in JIS K
8085 until the color of the solution changes light yellowish red from purplish red.
Add 20 mL of ammonium peroxodisulfate to boil (5). Immediately transfer it to a
100-mL - 200-mL volumetric flask with water. After immediate cooling is complete,
add water up to the marked line. Filter with Type 3 filter paper to make a sample
solution.
Note (5) Precipitation of manganese oxide, etc. is formed.
(4.2) Measurement: Conduct measurement as shown below. Two examples of titration are shownas follows.
(4.2.1) Measurement (A): Titration with a magnesium standard solution (0.01 mol/L)a) Transfer a predetermined volume (the equivalents of 5 mg - 20 mg as CaO + MgO) of sample
solution to a 200-mL - 300-mL Erlenmeyer flask.
b) Add a proper amount of water, add a drop of methyl red solution as an indicator and drop
sodium hydroxide (5 g/100 mL) to neutralize until the color of the solution becomes yellow.
c) Add 0.1 g of ascorbic acid, 1 ml - 10 mL of 2,2’,2”-nitrilotriethanol−water (1+3) and 1 mL 10 mL of potassium cyanide solution (6).
d) Add a predetermined volume of 0.01 mol/L ethylenediamine tetraacetate standard solution (7).
e) Add 20 mL of ammonium chloride solution or 2-aminoethanol solution.
f) Add several drops of Eriochrome Black T solution, and titrate with a 0.01 mol/L magnesium
standard solution until the color of the solution becomes red.
g) Calculate the alkalinity (AL) content in an analytical sample by the following formula.
Alkalinity (AL) (% (mass fraction)) in an analytical sample= ((C1 × f1 × V6/1000) － (C2 × f2 × V7/1000)) × (56.077/W2) × (V8/V9) × 100
233

Testing Methods for Fertilizers (2018)= ((f1 × V6) － (f2 × V7)) × (56.077/W2) × (V3/V4) × (1/1000)
C1： Set concentration (0.01 mol/L) of 0.01 mol/L ethylenediamine tetraacetatestandard solution
C2： Set concentration (0.01 mol/L) of 0.01 mol/L magnesium standard solution
f1： Factor of 0.01 mol/L ethylenediamine tetraacetate standard solution
f2： Factor of 0.01 mol/L magnesium standard solution
V6： Additive volume (mL) of 0.01 mol/L ethylenediamine tetraacetate standard
solution
V7： Volume (mL) of 0.01 mol/L magnesium standard solution needed for titration
V8： Predetermined volume (mL) of the sample solution in (4.1) d)
V9： Transferred volume (mL) of the sample solution subjected to titration in (4.2.1)
a).
W2： Mass (g) of the analytical sample
Note (6) If manganese is present, replace “1 mL - 10 mL of potassium cyanide solution” with
“1g - 5g of potassium cyanide”.
(7) Add excess volume since 1.8 mL of ethylenediamine tetraacetate standard solution(0.01 mol/L) is required for 1 mg of CaO.
(4.2.2) Measurement (B): Titration with an ethylenediamine tetraacetate standard solution (0.01
mol/L)a) Transfer a predetermined volume (the equivalents of 5 mg - 20 mg as CaO+MgO) of sample
solution to a 200-mL - 300-mL Erlenmeyer flask.
b) Add a proper amount of water and 5 mL of citric acid solution (8), add a drop of methyl red
solution as an indicator and drop sodium hydroxide (5 g/100 mL) to neutralize until the colorof the solution becomes yellow.
c) Add 0.1 g of ascorbic acid, 1 ml - 10 mL of 2,2’,2”-nitrilotriethanol−water (1+3) and 1 mL 10 mL of potassium cyanide solution (6).
d) Add 20 mL of ammonium chloride solution or 2-aminoethanol solution.
e) Add several drops of an Eriochrome Black T solution, and immediately titrate with a 0.01
mol/L ethylenediamine tetraacetate standard solution until the color of the solution becomesblue-green.
f) Calculate the alkalinity (AL) content in an analytical sample by the following formula.
Alkalinity (AL) (% (mass fraction)) in an analytical sample= (C1 × f1 × V10/1000) × (56.077/W3) × (V11/V12) × 100= (f1 × V10) × (56.077/W3) × (V11/V12) × (１/1000)
C1： Set concentration (0.01 mol/L) of 0.01 mol/L ethylenediamine tetraacetatestandard solution
f1： Factor of 0.01 mol/L ethylenediamine tetraacetate standard solution
V10： Volume (mL) of 0.01 mol/L ethylenediamine tetraacetate standard solution
needed for titration
V11： Predetermined volume (mL) of the sample solution in (4.1) d)
V12： Transferred volume (mL) of the sample solution subjected to titration in (4.2.2)
a).
W3： Mass (g) of the analytical sample
Note (8) When the sample solution does not contain phosphate, silicate, etc., it is not necessary234Testing Methods for Fertilizers (2018)

to add citric acid.
Comment 6 Care should be fully taken in the case of using potassium cyanide and its solution inaccordance with the Safety Data Sheet (SDS). In addition, observe laws andordinances concerned such as the Poisonous and Deleterious Substance Control Law.
Criteria of the abolition in the Poisonous and Deleterious Substance Control Law (forreference): Add a sodium hydroxide solution to make it alkalinity more than pH 11,
and further add an oxidizer (sodium hypochlorite, bleaching powder) solution to
conduct oxidative degradation processes. After dissolving CN ingredient, neutralize
with sulfuric acid, and discard it after diluting it with a large amount of water. Takeenough time to dissolve the CN ingredient with alkalinity.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.162- 164, Yokendo, Tokyo (1988)(5)

Flow sheet for alkalinity: The flow sheet for alkalinity in fertilizers is shown below:

2 g analytical sample
Weigh to the order of 1 mg to a 500-mL tall beaker← About 200 mL of hydrochloric acid (1+23)
Heating
Cover with a watch glass and boil for 5 minutes
Cooling

Immediately

Transfer
250-mL - 500-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for alkalinity in fertilizers (Extraction procedure)235

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot(predetermined amount)

200-mL - 300-mL Erlenmeyer Flask← Water (proper amount)← A drop of methyl-red solution
Sodium hydroxide (5 g/100 mL)
Neutralization(until the solution becomes yellow )← 0.1 g ascorbic acid← 1 mL - 10 mL of 2,2’,2”-nitrilotriethanol - water (1+3)← 1 mL - 10 mL potassium cyanide solution← Predetermined volume of 0.01 mol/L
ethylenediamine tetraacetate standard solution← 20 mL of ammonium chloride solution or 2-aminoethanol solution← Several drops of Erio-chrome Black T solution
0.01 mol/L magnesium standard solution
Titration(until the solution becomes red)
Figure 2-1 Flow sheet for alkalinity in fertilizers (Measurement procedure (4.2.1) (A))

Sample solution
Aliquot(predetermined amount)

200-mL - 300-mL Erlenmeyer Flask← Water (proper amount)← 5 mL citric acid solution← A drop of methyl-red solution
Sodium hydroxide (5 g/100 mL)
Neutralization(until the solution becomes yellow )← 0.1 g of ascorbic acid← 1 mL - 10 mL of 2,2’,2”-nitrilotriethanol - water (1+3)← 1 mL - 10 mL of potassium cyanide solution← 20 mL of ammonium chloride solution or 2-aminoethanol solution← Several drops of Erio-chrome Black T solution
0.01 mol/L ethylenediamine tetraacetate standard solution
Titration(until the solution becomes blue-green)
Figure 2-2 Flow sheet for alkalinity in fertilizers (Measurement procedure (4.2.2) (B))

236

Testing Methods for Fertilizers (2018)

4.5.4.b Calculation with soluble lime and soluble magnesia(1) Summary
This testing method is applicable to fertilizers that guarantee alkalinity (AL). This testing method isclassified as Type A (Def-C) and its symbol is 4.5.4.b-2017 or AL.b-1.
Multiply the soluble magnesia (S-MgO) obtained in 4.6.2 by the factor (1.3934) and add to thesoluble lime (S-CaO) obtained in 4.5.2 to calculate alkalinity (AL) in an analytical sample.
(2) Calculation of alkalinitya) Calculate the alkalinity (AL) in a teat sample by the following formula.
Alkalinity (AL) (% (mass fraction)) in an analytical sample= (S-CaO)＋1.3914 × (S-MgO)
S-CaO： Soluble lime (% (mass fraction)) (1) obtained in 4.5.2 in an analyticalsampleS-MgO： Soluble magnesia (% (mass fraction)) (1) obtained in 4.6.2 in an analyticalsampleNote (1) S-CaO and S-MgO use raw data without rounding numerical value237

Testing Methods for Fertilizers (2018)

4.6 Magnesia
4.6.1 Total magnesia
4.6.1.a Flame atomic absorption spectrometry(1) Summary
The testing method is applicable to compost, sludge fertilizers (except for calcined sludge fertilizer)
and poultry manure ash. This testing method is classified as Type D and its symbol is 4.6.1.a-2018
or T-Mg.a-1.
Pretreat an analytical sample with incineration and hydrochloric acid or nitric acid−hydrochloric
acid (3+1), add an interference suppressor solution, and then spray in an acetylene−air flame, andmeasure the atomic absorption with magnesium at a wavelength of 285.2 nm to quantify totalmagnesia (T-MgO) in an analytical sample.
In addition, the performance of this testing method is shown in Comment 4.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A JIS Guaranteed Reagent specified in JIS K 8541 or a reagent of equivalentquality.
c) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
d) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2000-mL beaker, add a small amount of water,
add gradually 420 mL of hydrochloric acid to dissolve, and further add water to make 1000mL.
e) Magnesium standard solution (MgO 1 mg/mL) (1): Weigh 0.603 g of magnesium (powder)specified in JIS K 8876 into a weighing dish. Transfer to a 1000-mL volumetric flask with asmall amount of water, add about 10 mL of hydrochloric acid to dissolve, and add water up to
the marked line.
f) Magnesium standard solution (MgO 0.1 mg/mL) (1): Transfer 10 mL of magnesium
standard solution (MgO 1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
g) Magnesium standard solution (MgO 1 µg/mL-10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL-25 mL of magnesium standard solution (MgO 0.1 mg/mL) to
250-mL volumetric flasks step-by-step, add about 25 mL of interference suppressor solution(3), and add water up to the marked line.
h) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution to a 250-mL volumetric flask (3), and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can also beused.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the magnesium standard solution in (2), a magnesium standard solution
for the calibration curve preparation can be prepared by using a magnesium
standard solution (Mg 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National
Metrology. In this case, calculate total magnesia (T-MgO) in the analytical sample
by multiplying the concentration (Mg) of the magnesium standard solution for
calibration curve preparation or a measurement value (Mg) obtained in (4.2) by a238

Testing Methods for Fertilizers (2018)

conversion factor (1.6583).
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121.
1) Light source: A magnesium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 450 °C ± 5 °C or 550 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Incineration-hydrochloric acid boilinga) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 200-mL - 300-mL tallbeaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (4).
c) Ignite at 550 ºC ± 5 ºC for no less than 4 hours to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, gradually add about
10 mL of hydrochloric acid, and further add water to make 20 mL.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to boil forabout 5 minutes.
f) After standing to cool, transfer the solution to a 250-mL - 500-mL volumetric flask withwater.
g) Add water up to the marked line.
h) Filter with Type 3 filter paper to make a sample solution.
Note (4) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 550 °C in 1 to 2 hours.
Comment 2 The procedure in (4.1.1) is the same as the procedure in (4.1.2) in 4.2.1.a.
(4.1.2) Incineration-aqua regia digestiona) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 200-mL - 300-mL tallbeaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (5).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (5).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (6), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (7) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
239

Testing Methods for Fertilizers (2018)

Note (5) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating forabout 1 hour and further raise to 450 °C in 1 to 2 hours.
(6) The watch glass can be removed.
(7) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 3 The procedures in (4.1.2) are the same as in (4.1.3) in 4.2.1.a. and (4.1) a) - h) in
5.3.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 285.2 nmb) Calibration curve preparation
1) Spray the magnesium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read the indicated value ata wavelength of 285.2 nm.
2) Prepare a curve for the relationship between the magnesium concentration and the indicatedvalue of the magnesium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg - 1 mg as
MgO) (8) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the magnesium content from the calibration curve, and calculate the total magnesia(T-MgO) in the analytical sample.
Note (8) If there is a possibility that the concentration of total magnesia in the sample solution
exceeds the maximum limit of the calibration curve, transfer a predetermined amountof the sample solution and dilute by adding water up to the marked line to make 0.1
mg - 1 mg as MgO.
Comment 4 Recovery testing was conducted to evaluate trueness using a preparation sample. As
a result, the average rate of recovery at the content level of 5 % (mass fraction),
1 % (mass fraction) and 0.2 % (mass fraction) are 102.4 %, 101.7 % and 103.0 % astotal magnesia (T-MgO) respectively.
The results of the repeatability tests on different days using swine manure compost,composted sludge fertilizer and poultry manure ash (1 sample for each) to evaluate
precision were analyzed by the one-way analysis of variance. Table 1 shows thecalculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this test method is about 0.2 %(mass fraction).
240

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
4)
5)
6)
7)
repeatability Average2)
sr
RSD rs I(T)RSD I(T)
1)

(%)

5

3.14

T
Swine manure compost

3)

Composted sludge fertilizer
5
0.84
Poultry manure ash
53.971） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

(5)

3)

(%)

(%)

3)

(%)

0.03

1.1

0.05

1.5

(%)

0.01
0.03

1.2
0.01
1.3
0.7
0.04
1.1
6） Intermediate standard deviation
7） Intermediate relative standard deviation

Flow sheet for total magnesia: The flow sheet for total magnesia in fertilizers is shown
below:
5 g analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL- 300-mL tall beaker.
Heat gently
Ignite at 550 ºC ±5 ºC, no less than 4 hours

Standing to coolRoom temperature
Smallamountof water, moisten the residue←← About 10 mL of hydrochloric acid
← Water (up to about 20 mL)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolRoom temperature

Transfer
250-mL - 500-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for total magnesia in fertilizers.
(Incineration-hydrochloric acid boiling prorcedure (4.1.1))

241

Testing Methods for Fertilizers (2018)5g
analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC for 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass to digest
Heating

Slightly move the watch glass and remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass to dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 2 Flow sheet for total magnesia in fertilizers(Incineration-aqua regia digestion procedure (4.1.2))

Sample solution
Aliquot(predetermined amount)

100-mL volumetric flask←About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer
Figure 3 Flow sheet for total magnesia in fertilizers (Measurement procedure)

242

Testing Methods for Fertilizers (2018)

4.6.2 Soluble magnesia
4.6.2.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers containing by-product magnesia fertilizers andfertilizers that guarantee alkalinity. This testing method is classified as Type D and its symbol is
4.6.2.a-2018 or S-Mg.a-1.
Add hydrochloric acid (1+23) to an analytical sample, boil to extract and add an interference
suppressor solution, and then spray in an acetylene−air flame, and measure the atomic absorption
with magnesium at a wavelength of 285.2 nm to obtain hydrochloric acid (1+23) soluble magnesia(soluble magnesia (S-MgO)) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 5.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2000-mL beaker, add a small amount of water,
add gradually 420 mL of hydrochloric acid to dissolve, and further add water to make 1000mL.
c) Magnesium standard solution (MgO 1 mg/mL) (1): Weigh 0.603 g of magnesium (powder)specified in JIS K 8876 into a weighing dish. Transfer to a 1000-mL volumetric flask with asmall amount of water, add about 10 mL of hydrochloric acid to dissolve, and add water up to
the marked line.
d) Magnesium standard solution (MgO 0.1 mg/mL) (1): Transfer 10 mL of magnesium
standard solution (MgO 1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
e) Magnesium standard solution (MgO 1 µg/mL-10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL-25 mL of magnesium standard solution (MgO 0.1 mg/mL) to
250-mL volumetric flasks step-by-step, add about 25 mL of interference suppressor solution(3), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure e) to a 250-mL volumetric flask (3), andadd water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can also beused.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the magnesium standard solution in (2), a magnesium standard solution forthe calibration curve preparation can be prepared by using a magnesium standard
solution (Mg 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate soluble magnesia (S-MgO) in the analytical sample bymultiplying the concentration (Mg) of a magnesium standard solution for calibration
curve preparation or a measurement value (Mg) obtained in (4.2) by a conversion
factor (1.6583).
(3)a)

Instruments: Instruments are as shown below:
Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in243Testing Methods for Fertilizers (2018)

JIS K 0121.
1) Light source: A magnesium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hot plate : A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 2 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL tall beaker.
b) Add about 200mL of hydrochloric acid (1+23), cover with a watch glass, and boil on a hot
plate for about 5 minutes (4).
c) Immediately transfer to a 250-mL - 500-mL volumetric flask with water.
d) Immediately add water up to the marked linee) Filter with Type 3 filter paper to make a sample solution.
Note (4) Be aware that an analytical sample should not solidify in the bottom of a beaker.
Comment 2 In the case of a by-product magnesia fertilizer or a fertilizer containing a by-productmagnesia, if the pH of the sample solution of d) is neutral or basic, prepare a sample
solution anew by replacing “2 g of an analytical sample” in the procedure in a) with
“1 g - 1.5 g of an analytical sample”.
Comment 3 In the procedure in a), a 500-mL volumetric flask can be used instead of a 500-mLtall beaker. However, the volumetric flask used should be distinguished as an
extraction flask and should not be used for the other purposes. In addition, “coverwith a watch glass” in b) is replaced by “place a funnel”, and “transfer to a 250-mL 500 mL volumetric flask with water” in the procedure in c) is skipped.
Comment 4 The procedure in (4.1) is the same as the procedure in (4.1) of 4.5.2.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 285.2 nmb) Calibration curve preparation
1) Spray the magnesium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read the indicated value ata wavelength of 285.2 nm.
2) Prepare a curve for the relationship between the magnesium concentration and the indicatedvalue of the magnesium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg - 1 mg as
MgO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the magnesium content from the calibration curve, and calculate the soluble magnesia(S-MgO) in the analytical sample.
244

Testing Methods for Fertilizers (2018)

Comment 5 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 15 % (mass fraction) and
1 % (mass fraction) are 101.7 % and 99.5% as soluble magnesia (S-MgO)
respectively.
Additionally, the minimum limit of quantification of this testing method is about
0.2 % (mass fraction) for solid fertilizers, and 0.05 % (mass fraction) for fluid
fertilizers.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.167- 169, Yokendo, Tokyo (1988)
2)
Souichi IGARASHI and Yasuharu KIMURA: Verification of Performance
Characteristics of Testing Methods for Magnesia Content in Fertilizer by Atomic
Absorption Spectrometry: Research Report of Fertilizer Vol. 6, p. 193 - 202 (2013)(5)

Flow sheet for soluble magnesia: The flow sheet for soluble magnesia in fertilizers is shown
below:

2 g analytical sample
Weigh to the order of 1 mg into a 500-mL tall beaker.
← About 200 mL of hydrochloric acid (1+23)
Heating
Cover with a watch glass, and boil for 5 minutes.
Cooling

Immediately

Transfer
Water, 250-mL - 500-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for soluble magnesia in fertilizers (Extraction procedure)
Sample solution
Aliquot(predetermined amount)

100-mL volumetric flask← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (285.2 nm)
Figure 2 Flow sheet for soluble magnesia in fertilizers (Measurement procedure)

245

Testing Methods for Fertilizers (2018)

4.6.3 Citrate-soluble magnesia
4.6.3.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing magnesia hydroxide fertilizers, etc. This
testing method is classified as Type C and its symbol is 4.6.3.a-2018 or C-Mg.a-2.
Extract by adding a citric acid solution to an analytical sample and add an interference suppressor
solution, and then spray in an acetylene−air flame, and measure the atomic absorption withmagnesium at a wavelength of 285.2 nm to obtain citrate-soluble magnesia (C-MgO) in ananalytical sample. In addition, the performance of this testing method is shown in Comment 8.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
c) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2000-mL beaker, add a small amount of water,
add gradually 420 mL of hydrochloric acid to dissolve, and further add water to make 1000mL.
d) Magnesium standard solution (MgO 1 mg/mL) (1): Weigh 0.603 g of magnesium (powder)specified in JIS K 8876 into a weighing dish. Transfer to a 1000-mL volumetric flask with asmall amount of water, add about 10 mL of hydrochloric acid to dissolve, and add water up to
the marked line.
e) Magnesium standard solution (MgO 0.1 mg/mL) (1): Transfer 10 mL of magnesium
standard solution (MgO 1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
f) Magnesium standard solution (MgO 1 µg/mL-10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL-25 mL of magnesium standard solution (MgO 0.1 mg/mL) to
250-mL volumetric flasks step-by-step, add about 25 mL of interference suppressor solution(3), and add water up to the marked line.
g) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure f) to a 250-mL volumetric flask (3), andadd water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can also beused.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the magnesium standard solution in (2), a magnesium standard solution forthe calibration curve preparation can be prepared by using a magnesium standard
solution (Mg 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate citrate-soluble magnesia (C-MgO) in the analytical sample bymultiplying the concentration (Mg) of a magnesium standard solution for calibration
curve preparation or a measurement value (Mg) obtained in (4.2) by a conversion
factor (1.6583).
(3) Instruments: Instruments are as shown below:a) Extractor: Constant-temperature rotary shaker or reciprocating water bath shaker as246

Testing Methods for Fertilizers (2018)

described below.
aa) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
ab) Reciprocating water bath shaker: A reciprocating water bath shaker: that can be adjusted
to 30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocate horizontally at 160 times/ minute with amplitude of 25 mm 40 mm.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in JIS
K 0121.
1) Light source: A magnesium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL tall beaker.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (4), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (4) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 2

The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.3.a.

(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (5).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (4), and shake to mix horizontallyat amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC) at 160 times/min for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (5) Use a 250-mL flat-bottom volumetric flask to stabilize the vibration.
Comment 3 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.3.a.
Comment 4 For a by-product magnesia fertilizer, if the pH of the sample solution in (4.1.1) d) and(4.1.2) d) is neutral or basic, prepare a sample solution anew by replacing “1 g of ananalytical sample” in the procedures in (4.1.1) a) and (4.1.2) a) with “0.5 g of ananalytical sample”.
Comment 5 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedures of (4.1.1) b) and (4.1.2) b).
Comment 6 In some slag silicate fertilizers, the variation of measurement value of citratemagnesia (C-MgO) may be observed according to the time variation of heating state
after adding a citric acid solution. Therefore, in the case of slag silicate fertilizers, it
247

Testing Methods for Fertilizers (2018)is necessary to conduct the procedures of (4.1.1) c) - d) and (4.1.2) c) - d) as quickly
as possible after confirming the time of shaking to mix in the procedure in (4.1.1) b)and (4.1.2) b).
Comment 7 In the case of a fertilizer containing Kieserite (magnesium sulfate fertilizers), wash
the non-dissolved matters with water obtained while preparing the sample solution ofwater-soluble magnesia in (4.1) in 4.6.4.a to transfer it to a 250 mL volumetric flask,
and then prepare a sample solution by the procedures in (4.1.1) b) - d) and (4.1.2) b)- d). Mix the magnesia obtained in (4.2) regarding this sample solution with thewater-soluble magnesia in 4.6.4.a regarding the said fertilizers to makecitrate-soluble magnesia.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 285.2 nmb) Calibration curve preparation
1) Spray the magnesium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read the indicated value ata wavelength of 285.2 nm.
2) Prepare a curve for the relationship between the magnesium concentration and the indicatedvalue of the magnesium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg - 1 mg as
MgO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the magnesium content from the calibration curve, and calculate the citrate soluble
magnesia (C-MgO) in the analytical sample.
Comment 8 Recovery testing was conducted to evaluate trueness using a preparation sample. As
a result, the average rate of recovery at the content level of 1 % (mass fraction) - 5 %(mass fraction) are 98.9 % - 100.3 % as citrate-soluble magnesia (C-MgO)
respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this test method is about 0.06 %(mass fraction) for solid fertilizers.

248

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of

Number of

Repeatability
2)certified reference laboratory Average
3)

4)
sr

Intermediate precision

5)
RSD r

6)
s I(T)

3)

(%)

(%)

Reproducibility

7)
RSD I(T)

sR

8)

RSD R

3)

(%)

(%)

3)

(%)

material fertilizer

p

1)

(%)

FAMIC-A-10

11

3.28

0.07

2.0

0.08

2.5

0.11

3.3

FAMIC-A-13

9

3.18

0.03

1.0

0.04

1.4

0.12

3.8

(%)

1） The number of laboaratories used for analysis

9)6） Intermediate standard deviation

conducting flame atomic absorbance spectrometry
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

7） Intermediate relative standard deviation
8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.167- 169, Yokendo, Tokyo (1988)
2)
Souichi IGARASHI and Yasuharu KIMURA: Verification of Performance
Characteristics of Testing Methods for Magnesia Content in Fertilizer by Atomic
Absorption Spectrometry: Research Report of Fertilizer Vol. 6, p. 193 - 202 (2013)3)
Souichi IGARASHI and Yasuharu KIMURA: Effect of Time during Extraction
Operation on Measurements of Citric Acid-Soluble Magnesium in Silicate Slag
Fertilizer Vol. 7, p. 145 - 156 (2014)(5)

Flow sheet for citrate-soluble magnesia: The flow sheet for citrate-soluble magnesia in
fertilizers is shown below:
1 g analytical sample

Weigh to the order of 1 mg into a 250-mL tall beaker.

← About 150 mL of citrate solution [about 30 ºC]
Rotary shaker (30 - 40 revolutions/min),Shaking to mix
30 ºC ± 1 ºC, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for citrate-soluble magnesia in fertilizers(Extraction procedure (4.1.1))

249

Testing Methods for Fertilizers (2018)

1 g analytical sample

Weigh to the order of 1 mg into a 250-mL tall beaker.

← About 150 mL of citrate solution [about 30 ºC]
Reciprocating water bath shaker (reciprocation
Shaking to mix
horizontally at 160 times /min, with amplitude of 25 mm- 40 mm), at 30 ºC ± 1 ºC, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for citrate-soluble magnesia in fertilizers(Extraction procedure (4.1.2))

Sample solution
Aliquot(predetermined amount)

100-mL volumetric flask← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (285.2 nm)
Figure 2 Flow sheet for citrate-soluble magnesia in fertilizers(Measurement procedure)

250

Testing Methods for Fertilizers (2018)

4.6.3.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type D and its
symbol is 4.6.3.b-2018 or C-Mg.b-1.
Extract by adding a citric acid solution to an analytical sample, introduce it to an ICP OpticalEmission Spectrometer (“ICP-OES”) and measure the magnesium at a wavelength of 279.553 nm toobtain citric acid-soluble magnesia (citrate-soluble magnesia (C-MgO)) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 9.
(2) Reagent: Reagents are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
d) Magnesium standard solution (MgO 1 mg/mL) (1): Weigh 0.603 g of magnesium (powder)specified in JIS K 8876 into a weighing dish. Transfer to a 1000-mL volumetric flask with asmall amount of water, add about 10 mL of hydrochloric acid to dissolve, and add water up to
the marked line.
e) Magnesium standard solution (MgO 0.1 mg/mL): Transfer 10 mL of magnesium standard
solution (MgO 1 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid (1+23) up
to the marked line.
f) Magnesium standard solution (MgO 2 µg/mL - 10 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL-16 mL of magnesium standard solution (MgO 0.1 mg/mL) to
100-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) up to the markedline.
g) Magnesium standard solution (MgO 0.2 µg/mL - 2 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 20 mL of magnesium standard solution (MgO 10 µg/mL) forthe calibration curve preparation to 100-mL volumetric flasks step-by-step, and addhydrochloric acid (1+23) up to the marked line.
h) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in e) - g).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the magnesium standard solution in (2), a magnesium standard solution forthe calibration curve preparation can be prepared by using a magnesium standard
solution (Mg 1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate citrate-soluble magnesia (C-MgO) in the analytical sample bymultiplying the concentration (Mg) of a magnesium standard solution for calibration
curve preparation or a measurement value (Mg) obtained in (4.2) by a conversion
factor (1.6583).
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in f) and g) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, when using the ICP-OES
of an axial observation mode, it is recommended to prepare a magnesium standard
solution for the calibration curve in the concentration range which is suitable to a251

Testing Methods for Fertilizers (2018)device used.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Extractor: Constant-temperature rotary shaker or reciprocating water bath shaker as
described below.
ba) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate a
250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at 30- 40 revolutions/min.
bb) Reciprocating water bath shaker: A reciprocating water bath shaker: that can be adjusted
to 30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocate horizontally at 160 times/ minute with amplitude of 25 mm 40 mm.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL tall beaker.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (2) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 2 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.3.a.
(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (3).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix byreciprocating at amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC) at 160 times/min for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (3) Use a 250-mL flat-bottom volumetric flask to stabilize the vibration.
Comment 3 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.3.a.
Comment 4 For a by-product magnesia fertilizer, if the pH of the sample solution in (4.1.1) d)and (4.1.2) d) is neutral or basic, prepare a sample solution anew by replacing “1 g ofan analytical sample” in the procedures in (4.1.1) a) and (4.1.2) a) with “0.5 g of ananalytical sample”.
Comment 5 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedures of (4.1.1) b) and (4.1.2) b).
Comment 6 In some slag silicate fertilizers, the variation of measurement value of citratemagnesia (C-MgO) may be observed according to the time variation of heating state
after adding a citric acid solution. Therefore, in the case of slag silicate fertilizers, it252

Testing Methods for Fertilizers (2018)is necessary to conduct the procedures of (4.1.1) c) - d) and (4.1.2) c) - d) as quickly
as possible after confirming the time of shaking to mix in the procedure in (4.1.1) b)and (4.1.2) b).
Comment 7 In the case of a fertilizer containing Kieserite (magnesium sulfate fertilizers), wash
the non-dissolved matters with water obtained while preparing the sample solution ofwater-soluble magnesia in (4.1) in 4.6.4.a to transfer it to a 250 mL volumetric flask,
and then prepare a sample solution by the procedures in (4.1.1) b) - d) and (4.1.2) b)- d). Mix the magnesia obtained in (4.2) regarding this sample solution with thewater-soluble magnesia in 4.6.4.a regarding the said fertilizers to makecitrate-soluble magnesia.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up the measurement
conditions for the ICP Atomic Emission Spectrometer considering the following:
Analytical line wavelength: 279.553 nmb) Calibration curve preparation
1) Spray the magnesium standard solution for the calibration curve preparation and blank test
solution for the calibration curve preparation into inductively coupled plasma, and read theindicated value at a wavelength of 279.553 nm.
2) Prepare a curve for the relationship between the magnesium concentration and the indicatedvalue of the magnesium standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.02 mg - 1.6 mg
as MgO) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the magnesium content from the calibration curve, and calculate the citrate soluble
magnesia (C-MgO) in the analytical sample.
Comment 8 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.3.d Comment 7.
Comment 9 The comparison of the measurement value (yi：1.59 % (mass farction) - 15.06 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of Flame atomic absorption spectrometry was conducted to evaluate truenessusing processed phosphorus fertilizers (2 samples), compound fertilizers (11samples), slag silicate fertilizer (1 sample), mixed compost fertilizer (1 sample),mixed phosphate fertilizers (2 samples), designated blended fertilizer (1 sample),blended fertilizers (5 samples), byproduct mixed fertilizer (1 sample), organic
compound fertilizer (1 sample), and fused phosphate fertilizer (1 sample). As a result,a regression equation was y = 0.0271+1.0124x, and its correlation coefficient (r) was
0.999. In addition, recovery testing was conducted using a preparation sample. As aresult, the average rate of recovery at the content level of 0.232 % (mass fraction) 18.81 % (mass fraction) was 94.9 % - 102.7 %.
The results of the repeatability tests on different days using compound fertilizers andblended fertilizers to evaluate precision were analyzed by one-way analysis ofvariance. Table 1 shows the calculation results of intermediate precision andrepeatability.
253

Testing Methods for Fertilizers (2018)

Additionally, the minimum limit of quantification of this testing method is about
0.03 % (mass fraction).
Table 1 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
Intermediate precision
4)
5)
6)
7)
samplerepeatability Average2)
sr
RSD rs I(T)RSD I(T)
1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)
Blended fertilizer
7
8.41
0.09
1.1
0.10
1.1
Compound
7
1.66
0.03
1.6
0.03
1.8
fertilizer
1） The number of test days conducting a duplicate test 6） Intermediate standard deviation
2） Average （the number of test days （T ）7） Intermediate relative standard deviation
×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

254

Testing Methods for Fertilizers (2018)(5)

Flow sheet for citrate-soluble magnesia: The flow sheet for citrate-soluble magnesia in
fertilizers is shown below:

1 g analytical sample

Weigh to the order of 1 mg into a 250-mL tall beaker.

← About 150 mL of citrate solution [about 30 ºC]
Rotary shaker (30 - 40 revolutions/min),Shaking to mix
30 ºC ± 1 ºC, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for citrate-soluble magnesia in fertilizers (Extraction procedure (4.1.1))

1 g analytical sample

Weigh to the order of 1 mg into a 250-mL tall beaker.

← About 150 mL of citrate solution [about 30 ºC]
Reciprocating water bath shaker (reciprocation horizontally at
Shaking to mix
160 times /min, with amplitude of 25 mm - 40 mm), at 30 ºC ± 1 º
C, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for citrate-soluble magnesia in fertilizers (Extraction procedure (4.1.2))

Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
ICP-OES (279.553 nm)
Figure 2 Flow sheet for citrate-soluble magnesia in fertilizers (Measurement procedure)255

Testing Methods for Fertilizers (2018)

4.6.4 Water-soluble magnesia
4.6.4.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing magnesia sulfate fertilizers, etc. This testing
method is classified as Type D and its symbol is 4.6.4.a-2018 or W-Mg.a-1.
Add water to an analytical sample and boil to extract. Add an interference suppressor solution, thenspray in an acetylene−air flame and measure the atomic absorption with magnesium at awavelength of 285.2 nm to obtain water-soluble magnesia (W-MgO) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 3.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2000-mL beaker, add a small amount of water,
add gradually 420 mL of hydrochloric acid to dissolve, and further add water to make 1000mL.
c) Magnesium standard solution (MgO 1 mg/mL) (1): Weigh 0.603 g of magnesium (powder)specified in JIS K 8876 into a weighing dish. Transfer to a 1000-mL volumetric flask with asmall amount of water, add about 10 mL of hydrochloric acid to dissolve, and add water up to
the marked line.
d) Magnesium standard solution (MgO 0.1 mg/mL) (1): Transfer 10 mL of magnesium
standard solution (MgO 1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
e) Magnesium standard solution (MgO 1 µg/mL - 10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL-25 mL of magnesium standard solution (MgO 0.1 mg/mL) to
250-mL volumetric flasks step-by-step, add about 25 mL of interference suppressor solution(3), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure d) to a 250-mL volumetric flask (3), andadd water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can also beused.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the magnesium standard solution in (2), a magnesium standard solution forthe calibration curve preparation can be prepared by using a magnesium standard
solution (Mg 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate water-soluble magnesia (W-MgO) in the analytical sample bymultiplying the concentration (Mg) of a magnesium standard solution for calibration
curve preparation or a measurement value (Mg) obtained in (4.2) by a conversion
factor (1.6583).
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121.
1) Light source: A magnesium hollow cathode lamp256

Testing Methods for Fertilizers (2018)

2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Extraction flask (4): A 500-mL volumetric flask made of borosilicate glassc) Hot plate : A hot plate whose surface temperature can be adjusted up to 250 ºC.
Note (4) The volumetric flask used for extraction should be distinguished as an extraction flask
and should not be used for the other purposes.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 400-mL tall beaker.
b) Add 400 mL of water, and cover with a watch glass to boil on a hot plate for about 30 minutes(5).
c) Immediately transfer it to a 500- mL volumetric flask with water.
d) After immediate cooling is complete, add water up to the marked linee) Filter with Type 3 filter paper to make a sample solution.
Note (5) Be aware that an analytical sample should not solidify in the bottom of a beaker.
Comment 2 In the procedure in a), a 500-mL volumetric flask can be used instead of a 500-mLtall beaker. However, the volumetric flask used should be distinguished as an
extraction flask and should not be used for the other purposes. Additionally, “coverwith a watch glass” in b) is replaced by “place a funnel”. Skip “transfer to a 500-mL
volumetric flask with water” in the procedure in c).
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 285.2 nmb) Calibration curve preparation
1) Spray the magnesium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read the indicated value ata wavelength of 285.2 nm.
2) Prepare a curve for the relationship between the magnesium concentration and the indicatedvalue of the magnesium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg - 1 mg as
MgO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the magnesium content from the calibration curve, and calculate the water-soluble
magnesia (W-MgO) in the analytical sample.
Comment 3 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 1 % (mass fraction) - 5 %(mass fraction) are 100.4 % - 100.9 % as water-soluble magnesia (W-MgO)
257

Testing Methods for Fertilizers (2018)

respectively.
Additionally, the minimum limit of quantification of this testing method is about
0.07 % (mass fraction).
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.167- 169, Yokendo, Tokyo (1988)
2)
Souichi IGARASHI and Yasuharu KIMURA: Verification of Performance
Characteristics of Testing Methods for Magnesia Content in Fertilizer by Atomic
Absorption Spectrometry: Research Report of Fertilizer Vol. 6, p. 193 - 202 (2013)(5)

Flow sheet for water-soluble magnesia: The flow sheet for water-soluble magnesia in
fertilizers is shown below:

1 g analytical sample
Weigh to the order of 1 mg into a 500-mL tall beaker.
← About 400 mL of water
Heating
Cover with a watch glass, and boil for about 30 minutes.
Cooling

Immediately

Transfer
Water, 500-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for water-soluble magnesia in fertilizers (Extraction procedure)
Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (285.2 nm)
Figure 2 Flow sheet for water-soluble magnesia in fertilizers (Measurement procedure)

258

Testing Methods for Fertilizers (2018)

4.6.4.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers, liquid microelement mixed fertilizers
and the fluid fertilizers of home garden-use mixed fertilizers. This testing method is classified as
Type D and its symbol is 4.6.4.b-2018 or W-Mg.b-1.
Add water to an analytical sample to extract, introduce it to an ICP Optical Emission Spectrometer(“ICP-OES”) and measure the magnesium at a wavelength of 279.553 nm to obtain water-soluble
magnesia (W-MgO) in an analytical sample. In addition, the performance of this testing method isshown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Magnesium standard solution (MgO 1 mg/mL) (1): Weigh 0.603 g of magnesium (powder)specified in JIS K 8876 into a weighing dish. Transfer to a 1000-mL volumetric flask with asmall amount of water, add about 10 mL of hydrochloric acid to dissolve, and add water up to
the marked line.
d) Magnesium standard solution (MgO 0.1 mg/mL): Transfer 10 mL of magnesium standard
solution (MgO 1 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid (1+23) up
to the marked line.
e) Magnesium standard solution (MgO 2 µg/mL - 16 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL-16 mL of magnesium standard solution (MgO 0.1 mg/mL) to
100-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) up to the markedline.
f) Magnesium standard solution (MgO 0.2 µg/mL - 2 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 20 mL of magnesium standard solution (MgO 10 µg/mL) forthe calibration curve preparation to 100-mL volumetric flasks step-by-step, and addhydrochloric acid (1+23) up to the marked line.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the magnesium standard solution in (2), a magnesium standard solution forthe calibration curve preparation can be prepared by using a magnesium standard
solution (Mg 1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate water-soluble magnesia (W-MgO) in the analytical sample bymultiplying the concentration (Mg) of a magnesium standard solution for calibration
curve preparation or a measurement value (Mg) obtained in (4.2) by a conversion
factor (1.6583).
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in e) and f) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, when using the ICP-OES
of an axial observation mode, it is recommended to prepare a magnesium standard
solution for the calibration curve in the concentration range which is suitable to a
device used.
259

Testing Methods for Fertilizers (2018)(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 mg of an analytical sample (2) to the order of 1 g, and put it in a 100-mL volumetricflask.
b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (2) The sampling amount of the analytical sample is 10 g when there is less magnesia
content in the fertilizers such as a home garden-use fertilizer.
Comment 3

The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.

(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Optical Emission Spectrometer: Set up the measurement
conditions for the ICP Optical Emission Spectrometer considering the following:
Analytical line wavelength: 279.553 nmb) Calibration curve preparation
1) Spray the magnesium standard solution for the calibration curve preparation and blank test
solution for the calibration curve preparation into inductively coupled plasma, and read theindicated value at a wavelength of 279.553 nm.
2) Prepare a curve for the relationship between the magnesium concentration and the indicatedvalue of the magnesium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.02 mg - 1.6 mg
as MgO) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the magnesium content from the calibration curve, and calculate the water-soluble
magnesia (W-MgO) in the analytical sample.
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.4.d Comment 4.
Comment 5 The comparison of the measurement value (yi：0.160 % (mass fraction) - 9.36 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of Flame atomic absorption spectrometry was conducted to evaluate truenessusing fluid fertilizers (12 samples). As a result, a regression equation was y = − 0.006+ 0.985x − 0.006, and its correlation coefficient (r) was 0.999. Additionally, additive
recovery testing was conducted using a fluid mixed fertilizer (1 brand) and a homegarden-use mixed fertilizer (1 brand) and a liquid microelement mixed fertilizer (1sample). As a result, the mean recovery rates at additive level of 1 % (mass fraction)and 0.15 % (mass fraction) were 98.7 % - 102.8 % and 102.3 % respectively.
260

Testing Methods for Fertilizers (2018)

The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.002 % (mass fraction).

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
7

3)

3)

(%)
1.18

(%)
0.004

Fluid mixed fertilizer
Home garder-use
7
0.392
0.002
mixed fertilizer（liquid)
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

(%)
0.3

(%)
0.01

3)

(%)
1.2

0.5

0.008

2.2

6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1) Keisuke AOYAMA: Simultaneous Determination of Water-Soluble Principal
Ingredients (W-P2O5, W-K2O, W-MgO, W-MnO and W-B2O3) in Liquid Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), Research
Report of Fertilizer, Vol.8, p. 1 - 9 (2015)(5)

Flow sheet: The flow sheet for water-soluble magnesia in fluid fertilizers is shown below:

1 g analytical sample
Weigh to the order of 1 mg to a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 Flow sheet for water-soluble magmesia in fluid fertilizers(Extraction procedure)

261

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement

ICP-OES (279.553 nm)

Figure 2 Flow sheet for water-soluble magmesia in fluid fertilizers(Measurement procedure)

262

Testing Methods for Fertilizers (2018)

4.7 Manganese
4.7.1 Soluble manganese
4.7.1.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing manganese carbonate fertilizers. This testing
method is classified as Type D and its symbol is 4.7.1.a-2017 or S-Mn.a-1.
Add hydrochloric acid (1+23) to an analytical sample, boil to extract and add an interference
suppressor solution, and then spray into an acetylene−air flame, and measure the atomic absorption
with manganese at a wavelength of 279.5 nm to obtain the hydrochloric acid (1+23) soluble
manganese (soluble manganese (S-MnO)) in an analytical sample. In addition, the performance of
this testing method is shown in Comment 4.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2000-mL beaker, add a small amount of water,
add gradually 420 mL of hydrochloric acid to dissolve, and further add water to make 1000mL.
c) Manganese standard solution (MnO 1 mg/mL) (1): Weigh 0.775 g of manganese powder(purity no less than 99 % (mass fraction)) into a weighing dish. Transfer to a 1000-mL
volumetric flask with a small amount of water, add about 10 mL of hydrochloric acid todissolve, and add water up to the marked line.
d) Manganese standard solution (MnO 0.1 mg/mL) (1): Transfer 10 mL of manganese standard
solution (MnO 1 mg/mL) to a 100-mL volumetric flask and add water up to the marked line.
e) Manganese standard solution (MnO 1 µg/mL-10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL-25 mL of manganese standard solution (MnO 0.1 mg/mL) to
250-mL volumetric flasks step-by-step, add about 25 mL of interference suppressor solution(3), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure e) to a 250-mL volumetric flask (3), andadd water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can also beused.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the manganese standard solution in (2), a manganese standard solution forthe calibration curve preparation can be prepared by using a manganese standard
solution (Mn 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate soluble manganese (S-MnO) in the analytical sample bymultiplying the concentration (Mn) of a manganese standard solution for calibration
curve preparation or a measurement value (Mn) obtained in (4.2) by a conversion
factor (1.2912).
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in263

Testing Methods for Fertilizers (2018)

JIS K 0121.
1) Light source: A manganese hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hot plate : A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 2 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL tall beaker.
b) Add about 200mL of hydrochloric acid (1+23), cover with a watch glass, and boil on a hot
plate for about 5 minutes (4).
c) Immediately transfer to a 250-mL - 500-mL volumetric flask with water.
d) After immediate cooling is complete, add water up to the marked linee) Filter with Type 3 filter paper to make a sample solution.
Note (4) Be aware that an analytical sample should not solidify in the bottom of a beaker.
Comment 2 In the procedure in a), a 500-mL volumetric flask can be used instead of a 500-mLtall beaker. However, the volumetric flask used should be distinguished as an
extraction flask and should not be used for the other purposes. In addition, “coverwith a watch glass” in b) is replaced by “place a funnel”, and “transfer to a 250-mL 500 mL volumetric flask with water” in the procedure in c) is skipped.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.5.2.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 279.5 nmb) Calibration curve preparation
1) Spray the manganese standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read the indicated value ata wavelength of 279.5 nm.
2) Prepare a curve for the relationship between the manganese concentration and the indicatedvalue of the manganese standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg - 1 mg as
MnO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the manganese content from the calibration curve, and calculate the soluble manganese(S-MnO) in the analytical sample.
Comment 4 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 5 % (mass fraction) and264

Testing Methods for Fertilizers (2018)

0.1 % (mass fraction) are 100.5 % and 101.3 % as soluble manganese (S-MnO)
respectively.
Additionally, the minimum limit of quantification of this testing method is about
0.006 % (mass fraction).
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.176- 177, Yokendo, Tokyo (1988)
2)
Keiji YAGI, Natuki TOYODOME, Tokiya SUZUKI and Hideo SOETA: Verification of
Performance Characteristics of Testing Methods for Manganese Content in Fertilizer by
Atomic Absorption Spectrometry, Research Report of Fertilizer Vol. 6, p. 203 - 212(2013)(5)

Flow sheet for soluble manganese: The flow sheet for soluble manganese in fertilizers isshown below:

2 g analytical sample
Weigh to the order of 1 mg into a 500-mL tall beaker.
← About 200 mL of hydrochloric acid (1+23)
Heating
Cover with a watch glass, and boil for about 5 minutes.
Cooling

Immediately

Transfer
Water, 250-mL - 500-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for soluble manganese in fertilizers (Extraction procedure)
Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (279.5 nm)
Figure 2 Flow sheet for soluble manganese in fertilizers (Mrasurement procedure)265

Testing Methods for Fertilizers (2018)

4.7.2 Citrate-soluble manganese
4.7.2.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing manganese carbonate fertilizers. This testing
method is classified as Type C and its symbol is 4.7.2.a-2018 or C-Mn.a-2.
Extract by adding a citric acid solution to an analytical sample and add an interference suppressor
solution, and then spray into an acetylene−air flame, and measure the atomic absorption withmanganese at a wavelength of 279.5 nm to obtain citric acid soluble manganese (citrate-soluble
manganese(C-MnO)) in an analytical sample. In addition, the performance of this testing method isshown in Comment 5.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
c) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2000-mL beaker, add a small amount of water,
add gradually 420 mL of hydrochloric acid to dissolve, and further add water to make 1000mL.
d) Manganese standard solution (MnO 1 mg/mL) (1): Weigh 0.775 g of manganese powder(purity no less than 99 % (mass fraction)) into a weighing dish. Transfer to a 1000-mL
volumetric flask with a small amount of water, add about 10 mL of hydrochloric acid todissolve, and add water up to the marked line.
e) Manganese standard solution (MnO 0.1 mg/mL): Transfer 10 mL of manganese standard
solution (MnO 1 mg/mL) to a 100-mL volumetric flask and add water up to the marked line.
f) Manganese standard solution (MnO 1 µg/mL - 10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL-25 mL of manganese standard solution (MnO 0.1 mg/mL) to
250-mL volumetric flasks step-by-step, add about 25 mL of interference suppressor solution(3), and add water up to the marked line.
g) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure f) to a 250-mL volumetric flask (3), andadd water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can also beused.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the manganese standard solution in (2), a manganese standard solution forthe calibration curve preparation can be prepared by using a manganese standard
solution (Mn 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate citrate-soluble manganese (C-MnO) in the analytical sample by amultiplying the concentration (Mn) of manganese standard solution for calibration
curve preparation or a measurement value (Mn) obtained in (4.2) by a conversion
factor (1.2912).
(3) Instruments: Instruments are as shown below:a) Extractor: Constant-temperature rotary shaker or reciprocating water bath shaker as266

Testing Methods for Fertilizers (2018)

described below.
aa) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
ab) Reciprocating water bath shaker: A reciprocating water bath shaker: that can be adjusted to
30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocate horizontally at 160 times/ minute with amplitude of 25 mm 40 mm.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121.
1) Light source: A manganese hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL tall beaker.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (4), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (4) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 2

The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.3.a.

(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (5).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (4), and shake to mix byreciprocating horizontally at amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC) at 160 times/min for
1 hour.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Note (5) Use a 250-mL flat-bottom volumetric flask to stabilize the vibration.
Comment 3 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.3.a.
Comment 4 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedures of (4.1.1) b) and (4.1.2) b).
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:267

Testing Methods for Fertilizers (2018)

Analytical line wavelength: 279.5 nmb) Calibration curve preparation
1) Spray the manganese standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read the indicated value ata wavelength of 279.5 nm.
2) Prepare a curve for the relationship between the manganese concentration and the indicatedvalue of the manganese standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg - 1 mg as
MnO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the manganese content from the calibration curve, and citrate soluble manganese(C-MnO) in the analytical sample.
Comment 5 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 5 % (mass fraction) and
0.1 % (mass fraction) are 101.9 % and 100.5 % as citrate-soluble manganese(C-MnO) respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.006 % (mass fraction).
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of

Number of

Repeatability
2)certified reference laboratory Average
1)3)material fertilizer
p
(%)

4)
sr
3)

FAMIC-A-10

9

0.403

(%)
0.004

FAMIC-A-13

10

0.356

0.010

Intermediate precision

5)
RSD r

s I(T)

(%)
1.1

(%)
0.01

2.7

0.01

1） The number of laboaratories used for analysis
conducting flame atomic absorbance spectrometry
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

6)
3)

RSD I(T)(%)7)

Reproducibility
8)

sR

3)

1.3

(%)
0.010

3.9

0.018

9)

RSD R
(%)
2.4
4.9

6） Intermediate standard deviation
7） Intermediate relative standard deviation
8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.176- 177, Yokendo, Tokyo (1988)
2)
Keiji YAGI, Natuki TOYODOME, Tokiya SUZUKI and Hideo SOETA: Verification of
Performance Characteristics of Testing Methods for Manganese Content in Fertilizer by
Atomic Absorption Spectrometry, Research Report of Fertilizer Vol. 6, p. 203 - 212
268

Testing Methods for Fertilizers (2018)(2013)(5) Flow sheet for citrate-soluble manganese: The flow sheet for citrate-soluble manganese infertilizers is shown below:

1 g analytical sample

Weigh to the order of 1 mg into a 250-mL tall beaker.

← About 150 mL of citrate-solution [about 30 ºC]
Constant-temperature rotary shaker (30 - 40 revolutions/min),Shaking to mix
30 ºC ± 1 ºC, for 1 hour
Cooling
Immediately
Water
(up
to
the marked line)←
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for citrate-soluble manganese in fertilizers(Extraction procedure (4.1.1))

1 g analytical sample

Weigh to the order of 1 mg into a 250-mL tall beaker.

← About 150 mL of citrate solution [about 30 ºC]
Reciprocating water bath shaker (reciprocation horizontally at
Shaking to mix
160 times /min, with amplitude of 25 mm - 40 mm), at 30 ºC ± 1 º
C, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for citrate-soluble manganese in fertilizers(Extraction procedure (4.1.2))

269

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (279.5 nm)
Figure 2 Flow sheet for citrate-soluble manganese in fertilizers(Measurement procedure)270

Testing Methods for Fertilizers (2018)

4.7.2.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type D and its
symbol is 4.7.2.b-2018 or C-Mn.b-1.
Extract by adding a citric acid solution to an analytical sample, introduce it to an ICP OpticalEmission Spectrometer (“ICP-OES”) and measure the manganese at a wavelength of 257.610 nm toobtain citric acid-soluble manganese (citrate-soluble manganese (C-MnO)) in an analytical sample.
In addition, the performance of this testing method is shown in Comment 6.
(2) Reagent: Reagents are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
d) Manganese standard solution (MnO 1 mg/mL) (1): Weigh 0.775 g of manganese powder(purity no less than 99 % (mass fraction)) into a weighing dish. Transfer to a 1000-mL
volumetric flask with a small amount of water, add about 10 mL of hydrochloric acid todissolve, and further add hydrochloric acid (1+23) up to the marked line.
e) Manganese standard solution (MnO 0.1 mg/mL): Transfer 10 mL of manganese standard
solution (MnO 1 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid (1+23) up
to the marked line.
f) Manganese standard solution (MnO 2 µg/mL - 8 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 8 mL of manganese standard solution (MnO 0.1 mg/mL) to
100-mL volumetric flasks step-by-step and add hydrochloric acid (1+23) up to the markedline.
g) Manganese standard solution (MnO 0.1 µg/mL - 2 µg/mL) for the calibration curve
preparation (1): Transfer 1 mL - 20 mL of manganese standard solution (MnO 10 µg/mL) to
100-mL volumetric flasks step-by-step and add hydrochloric acid (1+23) up to the markedline.
h) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in e) and g).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the manganese standard solution in (2), a manganese standard solution forthe calibration curve preparation can be prepared by using a manganese standard
solution (Mn 1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case,calculate citrate-soluble manganese (C-MnO) in the analytical sample by amultiplying the concentration (Mn) of manganese standard solution for calibration
curve preparation or a measurement value (Mn) obtained in (4.2) by a conversion
factor (1.2912).
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in f) and g) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, when using the ICP-OES
of an axial observation mode, it is recommended to prepare a manganese standard
solution for the calibration curve in the concentration range which is suitable to a271

Testing Methods for Fertilizers (2018)device used.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Extractor: Constant-temperature rotary shaker or reciprocating water bath shaker as
described below.
ba) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate a
250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at 30- 40 revolutions/min.
bb) Reciprocating water bath shaker: A reciprocating water bath shaker: that can be adjusted to
30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocate horizontally at 160 times/ minute with amplitude of 25 mm 40 mm.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL tall beaker.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note(2) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 2 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.3.a.

(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (3).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix byreciprocating horizontally at amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC) at 160 times/min for
1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (3) Use a 250-mL flat-bottom volumetric flask to stabilize the vibration.
Comment 3 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.3.a.
Comment 4 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedures of (4.1.1) b) and (4.1.2) b).
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:272

Testing Methods for Fertilizers (2018)

Analytical line wavelength: 257.610 nmb) Calibration curve preparation
1) Spray the manganese standard solution for the calibration curve preparation and blank test
solution for the calibration curve preparation into inductively coupled plasma, and read theindicated value at a wavelength of 257.610 nm.
2) Prepare a curve for the relationship between the manganese concentration and the indicatedvalue of the manganese standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 0.8 mg
as MnO) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the manganese content from the calibration curve, and calculate the citrate soluble
manganese (C-MnO) in the analytical sample.
Comment 5 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.3.d Comment 7.
Comment 6 The comparison of the measurement value (yi：0.089 % (mass farction) - 1.88 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of Flame atomic absorption spectrometry analysis was conducted to evaluate
trueness using compound fertilizers (7 samples), mixed phosphate fertilizers (2samples), solid fertilizers (2 samples), blended fertilizers (4 samples) and organic
blended fertilizer (1 sample). As a result, a regression equation was y = 0.0015 +0.9988x, and its correlation coefficient (r) was 0.999. In addition, recovery testing
was conducted using a preparation sample. As a result, the average rate of recovery
at the content level of 0.595 % (mass fraction) - 28.94 % (mass fraction) was 98.5 %- 105.5 %.
The results of the repeatability tests on different days using compound fertilizers andblended fertilizers to evaluate precision were analyzed by one-way analysis ofvariance. Table 1 shows the calculation results of intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.01 % (mass fraction).
Table 1 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
samplerepeatability Averagesr
RSD rs I(T)RSD I(T)
1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)
Compound fertilzier
7
0.54
0.01
2.3
0.02
3.1
Blended fertilizer
7
0.089
0.002
1.9
0.002
2.4
1） The number of test days conducting a duplicate test 6） Intermediate standard deviation
2） Average （the number of test days（T ）7） Intermediate relative standard deviation
×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

273

Testing Methods for Fertilizers (2018)(5)

Flow sheet for citrate-soluble manganese: The flow sheet for citrate-soluble manganese infertilizers is shown below:
1 g analytical sample

Weigh to the order of 1 mg into a 250-mL tall beaker.

← About 150 mL of citrate-solution [about 30 ºC]
Constant-temperature rotary shaker (30 - 40 revolutions/min),Shaking to mix
30 ºC ± 1 ºC, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for citrate-soluble manganese in fertilizers(Extraction procedure (4.1.1))

1 g analytical sample

Weigh to the order of 1 mg into a 250-mL tall beaker.

← About 150 mL of citrate solution [about 30 ºC]
Reciprocating water bath shaker (reciprocation horizontally at
Shaking to mix
160 times /min, with amplitude of 25 mm - 40 mm), at 30 ºC ±1 ºC, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for citrate-soluble manganese in fertilizers(Extraction procedure (4.1.2))

Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← About 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
ICP-OES (257.610 nm)
Figure 2 Flow sheet for citrate-soluble manganese in fertilizers(Measurement procedure)274

Testing Methods for Fertilizers (2018)

4.7.3 Water-soluble manganese
4.7.3.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing manganese sulfate fertilizers. This testing
method is classified as Type D and its symbol is 4.7.3.a-2017 or W-Mn.a-1.
Extract by adding water to an analytical sample and add an interference suppressor solution, andthen spray in an acetylene−air flame, and measure the atomic absorption with manganese at awavelength of 279.5 nm to obtain water-soluble manganese (W-MnO) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 6.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2000-mL beaker, add a small amount of water,
add gradually 420 mL of hydrochloric acid to dissolve, and further add water to make 1000mL.
c) Manganese standard solution (MnO 1 mg/mL) (1): Weigh 0.775 g of manganese powder(purity no less than 99 % (mass fraction)) into a weighing dish. Transfer to a 1000-mL
volumetric flask with a small amount of water, add about 10 mL of hydrochloric acid todissolve, and add water up to the marked line.
d) Manganese standard solution (MnO 0.1 mg/mL) (1): Transfer 10 mL of manganese standard
solution (MnO 1 mg/mL) to a 100-mL volumetric flask and add water up to the marked line.
e) Manganese standard solution (MnO 1 µg/mL - 10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL-25 mL of manganese standard solution (MnO 0.1 mg/mL) to
250-mL volumetric flasks step-by-step, add about 25 mL of interference suppressor solution(3), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure d) to a 250-mL volumetric flask (3), andadd water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can also beused.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the manganese standard solution in (2), a manganese standard solution forthe calibration curve preparation can be prepared by using a manganese standard
solution (Mn 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology. In
this case, calculate citrate-soluble manganese (C-MnO) in the analytical sample by amultiplying the concentration (Mn) of manganese standard solution for calibration
curve preparation or a measurement value (Mn) obtained in (4.2) by a conversion
factor (1.2912).
(3) Instruments: Instruments are as shown below.
a) Extractor: Constant-temperature rotary shaker or vertical reciprocating shaker as described
below.
aa) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
275

Testing Methods for Fertilizers (2018)ab) Vertical reciprocating shaker: A vertical reciprocating shaker that can shake a 250-mL
volumetric flask using an adapter for a flack to reciprocate vertically at 300 times/ minute(amplitude of 40 mm).
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121.
1) Light source: A manganese hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test sample(4.1.1.1) Rotary shakera) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL volumetricflask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 2 In the procedure of (4.1.1.1) a), it is also allowed to weigh 2.5g of an analyticalsample to the order of 1 mg, and put it in a 250-mL volumetric flask
Comment 3 The procedure in (4.1.1.1) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
(4.1.1.2) Vertical reciprocating shaker:a) Weigh 2.5 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask.
b) Add about 200 mL of water, and shake to mix by reciprocating vertically at 300 times/min(amplitude of 40 mm) for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 4 The procedure in (4.1.1.2) is the same as the procedure in (4.1.1.2) in 4.2.4.a.
(4.1.2) Fluid test samplea) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 100-mL volumetricflask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 5 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 279.5 nmb) Calibration curve preparation
276

Testing Methods for Fertilizers (2018)

1) Spray the manganese standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read the indicated value ata wavelength of 279.5 nm.
2) Prepare a curve for the relationship between the manganese concentration and the indicatedvalue of the manganese standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg - 1 mg as
MnO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the manganese content from the calibration curve, and water-soluble manganese(W-MnO) in the analytical sample.
Comment 6 Recovery testing was conducted to evaluate trueness using a preparation sample. As
a result, the average rate of recovery at the content level of 6 % (mass fraction) and
0.1 % (mass fraction) are 101.2 % and 101.1 % as water-soluble manganese(W-MnO) respectively.
The comparison of the measurement value of extraction (yi：0.0330 % (mass fraction)- 6.18 % (mass fraction )) by a vertical reciprocating shaker and the measurement
value of extract with a rotary shaker (xi) was conducted to evaluate trueness of theextraction of solid fertilizers using fertilizers (12 samples). As a result, a regression
equation was y = − 0.009 + 1.011x, and its correlation coefficient (r) was 1.000. Also,
the results of the repeatability tests on different days using compound fertilizers andmixed micro element fertilizers to evaluate precision were analyzed by one-way
analysis of variance. Table 1 shows the calculation results of intermediate precisionand repeatability
The comparison of the measurement value of simple extraction (yi：0.0590 % (massfraction) - 1.27 % (mass fraction )) and the measurement value of extract (xi) with arotary shaker was conducted to evaluate trueness of the extraction of fluid fertilizers
using fluid fertilizers (12 samples). As a result, a regression equation was y = − 0.001+ 1.006x, and its correlation coefficient (r) was 1.000. Also, the results of therepeatability tests on different days using fluid compound fertilizers and fluid mixed
micro element fertilizers to evaluate precision were analyzed by one-way analysis ofvariance. Table 2 shows the calculation results of intermediate precision andrepeatability
Additionally, the minimum limit of quantification of this testing method is about
0.004 % (mass fraction).

277

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
Intermediate precision
4)
5)
6)
7)
samplerepeatability Average2)
sr
RSD rs I(T)RSD I(T)
1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)
Mixed micro element
7
3.57
0.03
0.7
0.05
1.5
fertilizers
Blended fertilizer
7
0.226
0.002
1.0
0.004
1.7
1） The number of test days conducting a duplicate test
6） Intermediate standard deviation
2） Average （the number of test days（T ）7） Intermediate relative standard deviation
×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

Name ofsampleTable 2 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
4)
5)
6)
7)
repeatability Average2)
sr
RSD rs I(T)RSD I(T)
1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)

Fluid compound
7
1.28
0.01
fertilizers
Liquid micro element
7
0.230
0.001
compound fertilizers
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

0.4

0.02

1.3

0.5

0.003

1.5

6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.176- 177, Yokendo, Tokyo (1988)
2)
Keiji YAGI, Natuki TOYODOME, Tokiya SUZUKI and Hideo SOETA: Verification of
Performance Characteristics of Testing Methods for Manganese Content in Fertilizer by
Atomic Absorption Spectrometry, Research Report of Fertilizer Vol. 6, p. 203 - 212(2013)
3) Shinji KAWAGUCHI: Simple Extraction Method for Water-Soluble Components in
Liquid Compound Fertilizers, Research Report of Fertilizer, Vol.9, p. 10 - 20 (2016)4) Shinji KAWAGUCHI: Extraction Method for the Water-soluble Principal Ingredientsin the Solid Fertilizer using a Gneral-purpose Equipment , Research Report of Fertilizer,
Vol.10, p. 1 - 8 (2017)

278

Testing Methods for Fertilizers (2018)(5)

Flow sheet for water-soluble manganese: The flow sheet for water-soluble manganese infertilizers is shown below:

5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask.
(Powdery)← About 400 mL of water
Rotary shaker (30 - 40 revolutions/min), 30 minutes
Shaking to mix
Filtration

Type 3 filter paper

Sample solution
Figure 1-1 Flow sheet for water-soluble manganese in fertilizers(Extraction procedure (4.1.1.1))
2.5 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
(Powdery)← About 200 mL of water
Vertical reciprocating shaker(300 times/min, with amplitude
Shaking to mix
of 40 mm), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for water-soluble manganese in fertilizers(Extraction procedure (4.1.1.2))
1 g analytical sample
Weigh to the order of 1 mg into a 100-mL volumetric flask.
(Fluid)← About 50 mL of water
Shaking to mix← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-3 Flow sheet for water-soluble manganese in fertilizers(Extraction procedure (4.1.2))

279

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (279.5 nm)
Figure 2 Flow sheet forwater-soluble manganese in fertilizers(Measurement procedure)280

Testing Methods for Fertilizers (2018)

4.7.3.b ICP Optical Emission Spectrometry(1) Summary
The test method is applicable to fluid mixed fertilizers, liquid microelement mixed fertilizers, liquid
by-product manganese fertilizers and the fluid fertilizers of home garden-use mixed fertilizers. This
testing method is classified as Type D and its symbol is 4.7.3.b-2017 or W-Mn.b-1.
Add water to an analytical sample to extract, introduce it to an ICP Optical Emission Spectrometer(“ICP-OES”) and measure the manganese at a wavelength of 257.610 nm to obtain water-soluble
manganese (W-MnO) in an analytical sample. In addition, the performance of this testing method isshown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Manganese standard solution (MnO 1 mg/mL) (1): Weigh 0.775 g of manganese powder(purity no less than 99 % (mass fraction)) into a weighing dish. Transfer to a 1000-mL
volumetric flask with a small amount of water, add about 10 mL of hydrochloric acid todissolve, and further add hydrochloric acid (1+23) up to the marked line.
d) Manganese standard solution (MnO 0.1 mg/mL) (1): Transfer 10 mL of manganese standard
solution (MnO 1 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid (1+23) up
to the marked line.
e) Manganese standard solution (MnO 2 µg/mL - 8 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 8 mL of manganese standard solution (MnO 0.1 mg/mL) to
100-mL volumetric flasks step-by-step and add hydrochloric acid (1+23) up to the markedline.
f) Manganese standard solution (MnO 0.1 µg/mL - 2 µg/mL) for the calibration curve
preparation (1): Transfer 1 mL - 20 mL of manganese standard solution (MnO 10 µg/mL) to
100-mL volumetric flasks step-by-step and add hydrochloric acid (1+23) up to the markedline.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d), e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the manganese standard solution in (2), a manganese standard solution forthe calibration curve preparation can be prepared by using a manganese standard
solution (Mn 1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case,calculate water-soluble manganese (W-MnO) in the analytical sample by amultiplying the concentration (Mn) of manganese standard solution for calibration
curve preparation or a measurement value (Mn) obtained in (4.2) by a conversion
factor (1.2912).
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in d) and e) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, when using the ICP-OES
of an axial observation mode, it is recommended to prepare a manganese standard
solution for the calibration curve in the concentration range which is suitable to a
device used.
281

Testing Methods for Fertilizers (2018)(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 mg of an analytical sample (2) to the order of 1 mg, and put it in a 100-mL volumetricflask.
b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (2) The sampling amount of the analytical sample is 10 g when there is less manganesecontent in the fertilizers such as a home garden-use fertilizer.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Optical Emission Spectrometer: Set up the measurement
conditions for the ICP Optical Emission Spectrometer considering the following:
Analytical line wavelength: 257.610 nmb) Calibration curve preparation
1) Spray the manganese standard solution for the calibration curve preparation and blank test
solution for the calibration curve preparation into inductively coupled plasma, and read theindicated value at a wavelength of 257.610 nm.
2) Prepare a curve for the relationship between the manganese concentration and the indicatedvalue of the manganese standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 0.8 mg
as MnO) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the manganese content from the calibration curve, and water-soluble manganese(W-MnO) in the analytical sample.
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available.
In that case, see 4.2.4.d Comment 4.
Comment 5 The comparison of the measurement value (yi：0.027 % (mass fraction) - 1.49 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of Flame atomic absorption spectrometry was conducted to evaluate truenessusing fluid fertilizers (12 samples). As a result, a regression equation was y = −0.0013 + 1.025x, and its correlation coefficient (r) was 0.999. Additionally, additive
recovery testing was conducted using a fluid mixed fertilizer (1 brand) and a homegarden-use mixed fertilizer (1 brand) and a liquid micro element mixed fertilizer (1sample). As a result, the mean recovery rates at additive level of 0.15 % (massfraction) - 0.2 % and 0.005 % (mass fraction) were 96.3 % - 96.5 % and 107.0 %282

Testing Methods for Fertilizers (2018)

respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.。
Additionally, the minimum limit of quantification of this testing method is about
0.0002 % (mass fraction).

Table 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
4)
5)
6)
7)
repeatability Average2)
sr
RSD rs I(T)RSD I(T)Name ofsample

1)

(%)

3)

(%)

3)

(%)

(%)

3)

(%)

Fluid compound
fertilzier

7

5.69

0.02

0.4

0.06

1.1

Home garden-use
mixed fertilizers(Liquid)

7

2.29

0.02

0.8

0.04

1.6

T

1） The number of test days conducting a duplicate test2） Average （the number of test days （T ）6） Intermediate standard deviation
7） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
Table 2 Analysis results of the repeatability tests on different days

References
1)
Keisuke AOYAMA: Simultaneous Determination of Water-Soluble Principal
Ingredients (W-P2O5, W-K2O, W-MgO, W-MnO and W-B2O3) in Liquid Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), Research
Report of Fertilizer, Vol.8, p. 1 - 9 (2015)(5)

Flow sheet: The flow sheet for water-soluble manganese in fluid fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg to a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 Flow sheet for water-soluble manganese in liquid fertilizers(Extraction procedure)283

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement

ICP-OES (257.610 nm)

Figure 2 Flow sheet for water-soluble manganese in liquid fertilizers(Measurement procedure)284

Testing Methods for Fertilizers (2018)

4.8 Boron
4.8.1 Citrate-soluble boron
4.8.1.a Azomethine-H method(1) Summary
This testing method is applicable to fertilizers not containing organic matters. This testing method isclassified as Type C and its symbol is 4.8.1.a-2018 or C-B.a-2.
Extract by adding a citric acid solution to an analytical sample, mask co-existing copper, iron andother salts with ethylenediamine tetraacetate and measure the absorbance with azomethine-H borate
formed by the reaction with azomethine-H to obtain citrate soluble boron (C-B2O3) in an analyticalsample. In addition, the performance of this testing method is shown in Comment 6.
(2) Reagent: Reagents are as shown below.
a) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
b) Ethylenediamine tetraacetate solution (1): Dissolve 37.2 g of ethylenediaminetetraacetic
acid dihydrogen disodium dihydrate specified in JIS K 8107 in water to make 1000 mL.
c) Ammonium acetate solution (1): Dissolve 250 g of ammonium acetate specified in JIS K
8359 in water to make 500 mL and adjust pH with sulfuric acid (1+4) to pH 5.2 ± 0.1.
d) Azomethine-H solution: Add water to 0.6 g of azomethine-H and 2 g of L (+) – ascorbic
acid specified in JIS K 9502, and heat up to 35 ºC - 40 ºC to dissolve and add water after
cooling to make 100 mL.
e) Boron standard solution (B2O3 2.5 mg/mL) (1): After leaving boric acid specified in JIS K
8863 at rest in a desiccator for about 24 hours to dry, weigh 4.441 g to a weighing dish.
Dissolve in a small amount of water, transfer to a 1000-mL volumetric flask, and add water up
to the marked line.
f) Boron standard solution (B2O3 0.05 mg/mL): Dilute the predetermined volume of boron
standard solution (B2O3 2.5 mg/mL) with water exactly by a factor of 50.
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the boron standard solution in (2), a boron standard solution for thecalibration curve preparation can be prepared by using a boron standard solution (B 1
mg/mL or 10 mg/mL) traceable to National Metrology. In this case, calculate
citrate-soluble boron (C-B2O3) in the analytical sample by multiplying theconcentration (B) of a boron standard solution for calibration curve preparation or ameasurement value (B) obtained in (4.3) by a conversion factor (3.2199).
(3) Instruments: Instruments are as shown below:a) Extractor: Constant-temperature rotary shaker or reciprocating water bath shaker as
described below.
aa) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
ab) Reciprocating water bath shaker: A reciprocating water bath shaker that can be adjusted to
30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocate horizontally at 160 times/ minute with amplitude of 25 mm 40 mm.
b) Spectrophotometer: A spectrophotometer specified in JIS K 0115
(4) Test procedure285

Testing Methods for Fertilizers (2018)(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL tall beaker.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (2) Shake to mix the volumetric flask gently and disperse an analytical sample into thecitric acid solution.
Comment 2 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.3.a.
(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (3).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (2), and shake to mix byreciprocating horizontally at amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC) at 160 times/min for
1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (3) Use a 250-mL flat-bottom volumetric flask to stabilize the vibration.
Comment 3 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.3.a.
Comment 4 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedures of (4.1.1) b) and (4.1.2) b).
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 mg - 1 mg as
P2O5 and no more than the equivalents of 15 mL of citric acid solution) to a 100-mL
volumetric flask.
b) Add the solution to make citric acid solution equivalent to 15 mL.
c) Add 25 mL of ethylenediamine tetraacetate solution, and then add 10 mL of ammonium
acetate solution.
d) Add 10 mL of azomethine-H solution, and further add water up to the marked line, and thenleave at rest for about 2 hours.
Comment 5 If formaldehyde processed urea, a large quantity of aluminum, copper, iron zinc,organic matters, etc. coexists to affect quantification, transfer a predeterminedamount of the sample solution (the equivalents of 0.05 mg - 1 mg as B2O3, no morethan 10 mL of the solution) to a 100-mL separatory funnel, add 10 mL ofhydrochloric acid (1+3), add water to about 20 mL and add 20 mL of
2-ethyl-1,3-hexanediol – 4-methyl-2-pentanone (1+9) to shake to mix with ashaking apparatus for about 1 minute. After allowing to stand still, remove thelower layer (aqueous phase) and add 20 mL of sodium hydroxide (20 mg/L) to
shake to mix with a shaking apparatus for about 1 minute. After allowing to standstill, transfer the lower layer (aqueous phase) to a 100-mL volumetric flask, add a
few drops of phenolphthalein solution (1 g/100mL) to neutralize with hydrochloric
acid (1+3) until the color of the solution becomes achromatic, and conduct the286

Testing Methods for Fertilizers (2018)

procedure in (4.2) b).
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 415 nmb) Calibration curve preparation
1) Transfer 1 mL - 20 mL of boron standard solution (B2O3 0.05 mg/mL) to 100-mL volumetricflasks step-by-step.
2) Add 15 mL of citric acid solution and conduct the same procedure as (4.2) c) to make theB2O3 0.05 mg/100 mL - 1 mg/100 mL boron standard solution for the calibration curve
preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blanktest solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 415 nm of the boron standard solutions for thecalibration curve preparation using the blank test solution for the calibration curve preparation
as the control.
5) Prepare a curve for the relationship between the boron concentration and absorbance of theboron standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) c), measure absorbance by the same procedure as b) 4).
2) Obtain the boron (B2O3) content from the calibration curve, and calculate citrate solubleboron (C-B2O3) in the analytical sample.
Comment 6 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 10 % (mass fraction) and0.05 % (mass fraction) were 101.5 % and 95.7 % as citrate-soluble boron (C-B2O3)
respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.02 % (mass fraction).

287

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of

Number of

Repeatability
2)certified reference laboratory Averagematerial fertilizer

1)

3)

4)
sr
3)

Intermediate precision

5)
RSD r

s I(T)

6)

RSD I(T)

3)

(%)

(%)
0.209

(%)
0.004

(%)

FAMIC-A-10

p
11

2.0

(%)
0.005

FAMIC-A-13

10

0.203

0.004

1.8

0.005

1） The number of laboaratories used for analysis

7)

Reproducibility
8)

RSD R

3)

(%)

2.2

(%)
0.006

2.5

0.009

4.7

sR

9)

3.1

6） Intermediate standard deviation

conducting Azomethine-H method
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

7） Intermediate relative standard deviation
8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.184- 187, Yokendo, Tokyo (1988)
2)
Kimie KATO, Sakiko TAKAHASHI and Yuji SHIRAI: Validation of a Color metric
Method for Determination of Nitrogen, Phosphorus and Boron: Evaluation of
Calibration curve, Research Report of Fertilizer Vol. 2, p. 137 - 144 (2009)3)
Akira SHIMIZU: Verification of Performance Characteristics of Testing Methods for
Boron Content by Azomethine H Absorption Photometry, Research Report of Fertilizer
Vol. 6, p. 174 -182 (2013)

288

Testing Methods for Fertilizers (2018)(5)

Flow sheet for citrate-soluble boron: The flow sheet for citrate-soluble boron in fertilizers isshown below:
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Shaking to mix

Constant temperature rotary shaker (30 - 40 revolutions/min),
30 ºC ± 1 ºC, for 1 hour

Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for citrate-soluble boron in fertilizers(Extraction procedure (4.1.1))

1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Reciprocating water bath shaker (reciprocation horizontally at 160
Shaking to mix
times /min, with amplitude of 25 mm - 40 mm), at 30 ºC ± 1 ºC,
for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for citrate-soluble boron in fertilizers(Extraction procedure (4.1.2))

289

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← Citric acid solution, until the volume reaches
to the equivalents of 15 mL← 25 mL of ethylenediamine tetraacetate solution← 10 mL of ammonium acetate solution← 10 mL of azomethine-H solution← Water (up to the marked line)
Leaving at rest
For about 2 hours
Measurement

Spectrophotometer (415 nm)

Figure 2 Flow sheet for citrate-soluble boron in fertilizers(Measurement procedure)290

Testing Methods for Fertilizers (2018)

4.8.1.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type D and its
symbol is 4.8.1.b-2018 or C-B.b-1.
Extract by adding a citric acid solution to an analytical sample, introduce it to an ICP OpticalEmission Spectrometer (“ICP-OES”) and measure the boron at a wavelength of 249.773 nm toobtain citric acid-soluble boron (citrate-soluble boron acid (C-B2O3)) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 8.
(2) Reagent: Reagents are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K 8283 inwater to make 1000 mL.
d) Boron standard solution (B2O3 2.5 mg/mL) (1): After leaving boric acid specified in JIS K
8863 at rest in a desiccator for about 24 hours to dry, weigh 4.441 g to a weighing dish.
Dissolve in a small amount of water, transfer to a 1000-mL volumetric flask, and add water up
to the marked line.
e) Boron standard solution (B2O3 0.1 mg/mL) (1): Transfer 4 mL of boron standard solution(B2O3 2.5 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid (1+23) up to themarked line (2).
f) Boron standard solution (B2O3 2 µg/mL - 16 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 16 mL of boron standard solution (B2O3 0.1 mg/mL) to a
100-mL volumetric flask step by step and add hydrochloric acid (1+23) up to the marked line(2).
g) Boron standard solution (B2O3 0.2 µg/mL - 2 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 20 mL of boron standard solution (B2O3 10 µg/mL) to a
100-mL volumetric flask step by step and add hydrochloric acid (1+23) up to the marked line(2).
h) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in e) - g).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) When preserving, use a container, which can be sealed tightly, made of material suchas PTFE that boron hardly elutes
Comment 1 Instead of the boron standard solution in (2), a boron standard solution for thecalibration curve preparation can be prepared by using a boron standard solution (B 1
mg/mL or 10 mg/mL) traceable to National Metrology. In this case, calculate
citrate-soluble boron (C-B2O3) in the analytical sample by multiplying theconcentration (B) of a boron standard solution for calibration curve preparation or ameasurement value (B) obtained in (4.2) by a conversion factor (3.2199).
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in f) and g) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, when using the ICP-OES
of an axial observation mode, it is recommended to prepare a boron standard solution
291

Testing Methods for Fertilizers (2018)

for the calibration curve in the concentration range which is suitable to a device used.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Extractor: Constant-temperature rotary shaker or reciprocating water bath shaker as
described below.
ba) Constant-temperature rotary shaker: A constant-temperature rotary shaker that can rotate
a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC, upside down at
30 - 40 revolutions/min.
bb) Reciprocating water bath shaker: A reciprocating water bath shaker: that can be adjusted
to 30 °C ± 1 °C and shake a 250-mL volumetric flask, set up vertically on the water surface
using a shaking rack, reciprocate horizontally at 160 times/ minute with amplitude of 25 mm 40 mm.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Constant-temperature rotary shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL tall beaker.
b) Add 150 mL of citric acid solution heated up to about 30 ºC (3), and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (3) Use a 250-mL flat-bottom volumetric flask to stabilize the vibration.
Comment 3 The procedure in (4.1.1) is the same as the procedure in (4.1.1) in 4.2.3.a.
(4.1.2) Reciprocating water bath shaker:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL volumetricflask (4).
b) Add 150 mL of citric acid solution heated up to about 30 ºC (3), and shake to mix byreciprocating horizontally at amplitude of 25 mm - 40 mm (30 ºC ± 1 ºC) at 160 times/min for
1 hour.
c) After immediate cooling is complete, add water up to the marked lined) Filter with Type 3 filter paper to make a sample solution.
Note (4) Use a 250-mL flat-bottom volumetric flask to stabilize the vibration.
Comment 4 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.3.a.
Comment 5 The determination may be affected if an analytical sample cakes on the bottom of a
250-mL volumetric flask. Therefore, confirm the status of the non-dissolved mattersafter the procedures of (4.1.1) b) and (4.1.2) b).
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:292

Testing Methods for Fertilizers (2018)

Analytical line wavelength: 249.773 nmb) Calibration curve preparation
1) Spray the boron standard solution for the calibration curve preparation and blank test solution
for the calibration curve preparation into inductively coupled plasma, and read the indicatedvalue at a wavelength of 249.773 nm.
2) Prepare a curve for the relationship between the boron concentration and the indicated valueof the boron standard solution for the calibration curve preparation and the blank test solution
for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.02 mg - 1.6 mg
as B2O3) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the boron (B2O3) content from the calibration curve, and calculate citrate solubleboron (C-B2O3) in the analytical sample.
Comment 6 Wash the sample injector of an ICP-OES sufficiently with water because boron easily
causes the memory effect.
Comment 7 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.3.d Comment 7.
Comment 8 The comparison of the measurement value (yi：0.073 % (mass fraction) - 0.51 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of Flame atomic absorption spectrometry analysis was conducted to evaluate
trueness using compound fertilizers (7 samples), mixed phosphate fertilizers (1sample), solid fertilizers (2 samples), blended fertilizers (3 samples) and organic
compound fertilizer (1 sample). As a result, a regression equation was y = − 0.0408 +1.0456x, and its correlation coefficient (r) was 0.992. In addition, recovery testing
was conducted using a preparation sample. As a result, the average rate of recovery
at the content level of 0.601 % (mass fraction) - 35.51 % (mass fraction) was 97.0 %- 102.0 %.
The results of the repeatability tests on different days using compound fertilizers andblended fertilizers to evaluate precision were analyzed by one-way analysis ofvariance. Table 1 shows the calculation results of intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.01 % (mass fraction).
Table 1 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
samplerepeatability Averagesr
RSD rs I(T)RSD I(T)
1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)
Compound fertilzier
7
0.38
0.01
1.9
0.01
3.1
Blended fertilizer
7
0.076
0.003
4.2
0.006
7.5
1） The number of test days conducting a duplicate test 6） Intermediate standard deviation
2） Average （the number of test days（T ）7） Intermediate relative standard deviation
×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation

293

Testing Methods for Fertilizers (2018)(5)

Flow sheet for citrate-soluble boron: The flow sheet for citrate-soluble boron in fertilizers isshown below:

1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Shaking to mix

Constant temperature rotary shaker (30 - 40 revolutions/min),
30 ºC ± 1 ºC, for 1 hour

Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for citrate-soluble boron in fertilizers(Extraction procedure (4.1.1))
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Reciprocating water bath shaker (reciprocation horizontally
Shaking to mix
at 160 times /min, with amplitude of 25 mm - 40 mm), at 30 º
C ± 1 ºC, for 1 hour
Cooling
Immediately← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for citrate-soluble boron in fertilizers(Extraction procedure (4.1.2))
Sample solution
Aliquot(predeterminedamount)100-mL volumetric flask← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
ICP-OES (249.773 nm)
Figure 2 Flow sheet for citrate-soluble boron in fertilizers(Measurement procedure)294

Testing Methods for Fertilizers (2018)

4.8.2 Water-soluble boron
4.8.2.a Azomethine-H method(1) Summary
This test method is applicable to fertilizers containing borate fertilizers, etc. This testing method isclassified as Type D and its symbol is 4.8.2.a-2017 or W-B.a-1.
Extract by adding water to an analytical sample, boil to extract, and mask co-existing copper, ironand other salts with ethylenediamine tetraacetate and measure the absorbance with azomethine-H
borate formed by the reaction with azomethine-H to obtain water-soluble boron (W-B2O3). In
addition, the performance of this testing method is shown in Comment 7.
(2) Reagent: Reagents are as shown below.
a) Ethylenediamine tetraacetate solution (1): Dissolve 37.2 g of ethylenediaminetetraacetic
acid dihydrogen disodium dihydrate specified in JIS K 8107 in water to make 1000 mL.
b) Ammonium acetate solution (1): Dissolve 250 g of ammonium acetate specified in JIS K
8359 in water to make 500 mL and adjust pH with sulfuric acid (1+4) to pH 5.2 ± 0.1.
c) Azomethine-H solution (1): Add water to 0.6 g of azomethine-H and 2 g of L (+) – ascorbic
acid specified in JIS K 9502, and heat up to 35 ºC - 40 ºC to dissolve and add water after
cooling to make100 mL.
d) Boron standard solution (B2O3 2.5 mg/mL) (1): After leaving boric acid specified in JIS K
8863 at rest in a desiccator for about 24 hours to dry, weigh 4.441 g to a weighing dish.
Dissolve in a small amount of water, transfer to a 1000-mL volumetric flask, and add water up
to the marked line.
e) Boron standard solution (B2O3 0.05 mg/mL): Dilute the predetermined volume of boron
standard solution (B2O3 2.5 mg/mL) with water exactly by a factor of 50.
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the boron standard solution in (2), a boron standard solution for thecalibration curve preparation can be prepared by using a boron standard solution (B
1 mg/mL or 10 mg/mL) traceable to National Metrology. In this case, calculate
water-soluble boron (W-B2O3) in the analytical sample by multiplying theconcentration (B) of a boron standard solution for calibration curve preparation or ameasurement value (B) obtained in (4.3) by a conversion factor (3.2199).
(3) Instruments: Instruments are as shown below:a) Spectrophotometer: A spectrophotometer specified in JIS K 0115b) Hot plate : A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 2.5 g (2) of an analytical sample to the order of 1 mg, and transfer to a 300-mL tallbeaker.
b) Add about 200 mL of water, and cover with a watch glass and heat on a hot plate to boil forabout 15 minutes.
c) Immediately transfer it with water to a 250- mL volumetric flask.
d) After immediate cooling is complete, add water up to the marked linee) Filter with Type 3 filter paper to make a sample solution.
Note (2) The sampling amount of the analytical sample is 1 g when there is a high amount ofboric acid content in the fertilizers such as a borate fertilizer and boric acid fertilizer.
295

Testing Methods for Fertilizers (2018)

Comment 2 In the procedure in (4.1.2) a) and (4.1.2) b), a 250-mL volumetric flask can be usedinstead of a 300-mL tall beaker. However, the volumetric flask used should be
distinguished as an extraction flask and should not be used for the other purposes.
Additionally, “cover with a watch glass” in b) is replaced by “place a funnel”. Skip
“transfer to a 250-mL volumetric flask with water” in the procedure in c).
Comment 3 The procedure in (4.1.2) is the same as the procedure in (4.1.1) in 4.3.3.a.
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 mg - 1 mg as
B2O3) to a 100-mL volumetric flask.
b) Add 25 mL of ethylenediamine tetraacetate solution, and then add 10 mL of ammonium
acetate solution.
c) Add 10 mL of azomethine-H solution, and further add water up to the marked line, and thenleave at rest for about 2 hours.
Comment 4 If formaldehyde processed urea, a large quantity of aluminum, copper, iron zinc,organic matters, etc. coexists to affect quantification, transfer a predeterminedamount of the sample solution (the equivalents of 0.05 mg - 1 mg as B2O3, no morethan 10 mL of the solution) to a 100-mL separatory funnel, add 10 mL ofhydrochloric acid (1+3), add water to about 20 mL and add 20 mL of
2-ethyl-1,3-hexanediol–4-methyl-2-pentanone (1+9) to shake to mix with a shaking
apparatus for about 1 minute. After allowing to stand still, remove the lower layer(aqueous phase) and add 20 mL of sodium hydroxide (20 mg/L) to shake to mix
with a shaking apparatus for about 1 minute. After allowing to stand still, transfer
the lower layer (aqueous phase) to a 100-mL volumetric flask, add a few drops ofphenolphthalein solution (1 g/100mL) to neutralize with hydrochloric acid (1+3)
until the color of the solution becomes achromatic, and conduct the procedure in(4.2) b).
Comment 5 Water-soluble boron can be measured simultaneously with citrate soluble boron byadding 15 mL of citric acid solution before the procedure in (4.2) b).
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 415 nmb) Calibration curve preparation
1) Transfer 1 mL - 20 mL of boron standard solution (B2O3 0.05 mg/mL) to 100-mL volumetricflasks step-by-step.
2) Conduct the same procedures as (4.2) b) to make B2O3 0.05 mg/100 mL - 1 mg/100 mL of theboron standard solutions for the calibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blanktest solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 415 nm of the boron standard solutions for thecalibration curve preparation using the blank test solution for the calibration curve preparation
as the control.
5) Prepare a curve for the relationship between the boron concentration and absorbance of theboron standard solutions for the calibration curve preparation.
296

Testing Methods for Fertilizers (2018)c) Sample measurement
1) Regarding the solution in (4.2) b), measure absorbance by the same procedure as b) 4).
2) Obtain the boron (B2O3) content from the calibration curve, and calculate water-soluble boron(W-B2O3) in the analytical sample.
Comment 6 Water-soluble boron can be measured simultaneously with citrate-soluble boron byadding 15 mL of citric acid solution before the procedure in (4.3) b) 2).
Comment 7 Recovery testing was conducted to evaluate trueness using a preparation sample. As
a result, the average rate of recovery at the content level of 10 % (mass fraction)and 0.05 % (mass fraction) were 101.8 % and 107.1 % as water-soluble boron(W-B2O3) respectively.
Additionally, the minimum limit of quantification of this testing method is about
0.02 % (mass fraction).
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.184- 187, Yokendo, Tokyo (1988)
2)
Kimie KATO, Sakiko TAKAHASHI and Yuji SHIRAI: Validation of a Color metric
Method for Determination of Nitrogen, Phosphorus and Boron: Evaluation of
Calibration curve, Research Report of Fertilizer Vol. 2, p. 137 - 144 (2009)3)
Akira SHIMIZU: Verification of Performance Characteristics of Testing Methods for
Boron Content by Azomethine H Absorption Photometry, Research Report of Fertilizer
Vol. 6, p. 174 -182 (2013)(5)

Flow sheet for water-soluble boron: The flow sheet for water-soluble boron in fertilizers isshown below:
2.5 g analytical sample Weigh to the order of 1 mg into a 300-mL tall beaker.
← About 200 mL of Water
Heating
Cover with a watch glass, boil for 15 minutes
Cooling

Immediately

Transfer
Water, 250-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for water-soluble boron in fertilizers (Extraction procedure)

297

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← 25 mL of ethylenediamine tetraacetate solution← 10 mL of ammonium acetate solution← 10 mL of azomethine-H solution← Water (up to the marked line)
Leaving at rest
For about 2 hours
Measurement

Spectrophotometer (415 nm)

Figure 2 Flow sheet for water-soluble boron in fertilizers (Measurement procedure)

298

Testing Methods for Fertilizers (2018)

4.8.2.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers, liquid microelement mixed fertilizers
and the fluid fertilizers of home garden-use mixed fertilizers. This testing method is classified as
Type D and its symbol is 4.8.2.b-2017 or W-B.b-1.
Extract by adding water to an analytical sample, further dilute the filtered solution, introduce it to an
ICP Optical Emission Spectrometer (“ICP-OES”) and measure the boron at a wavelength of
249.773 nm to obtain water-soluble boron (W-B2O3) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 6.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Boron standard solution (B2O3 2.5 mg/mL) (1): After leaving boric acid specified in JIS K
8863 at rest in a desiccator for about 24 hours to dry, weigh 4.441 g to a weighing dish.
Dissolve in a small amount of water, transfer to a 1000-mL volumetric flask, and add water up
to the marked line.
d) Boron standard solution (B2O3 0.1 mg/mL) (1): Transfer 4 mL of boron standard solution(B2O3 2.5 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid (1+23) up to themarked line (2).
e) Boron standard solution (B2O3 2 µg/mL - 16 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 16 mL of boron standard solution (B2O3 0.1 mg/mL) to a
100-mL volumetric flask step by step and add hydrochloric acid (1+23) up to the marked line(2).
f) Boron standard solution (B2O3 0.2 µg/mL - 2 µg/mL) for the calibration curve
preparation (1): Transfer 2 mL - 20 mL of boron standard solution (B2O3 10 µg/mL) to a
100-mL volumetric flask step by step and add hydrochloric acid (1+23) up to the marked line(2).
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d), e) and f) (2).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) When preserving, use a container, which can be sealed tightly, made of material suchas PTFE that boron hardly elutes
Comment 1 Instead of the boron standard solution in (2), a boron standard solution for thecalibration curve preparation can be prepared by using a boron standard solution (B 1
mg/mL or 10 mg/mL) traceable to National Metrology. In this case, calculate
water-soluble boron (W-B2O3) in the analytical sample by multiplying theconcentration (B) of a boron standard solution for calibration curve preparation or ameasurement value (B) obtained in (4.2) by a conversion factor (3.2199).
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in d) and e) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, when using the ICP-OES
of an axial observation mode, it is recommended to prepare a boron standard solution
for the calibration curve in the concentration range which is suitable to a device used.
299

Testing Methods for Fertilizers (2018)(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5% (volume fraction) in purity(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 mg of an analytical sample (3) to the order of 1 mg, and put it in a 100-mL volumetricflask.
b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (3) The sampling amount of the analytical sample is 10 g when there is less boron content
in the fertilizers such as a home garden-use fertilizer.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Optical Emission Spectrometer: Set up themeasurement conditions for the ICP Optical Emission Spectrometer considering thefollowing:
Analytical line wavelength: 249.773 nmb) Calibration curve preparation
1) Spray the boron standard solution for the calibration curve preparation and blank test solution
for the calibration curve preparation into inductively coupled plasma, and read the indicatedvalue at a wavelength of 249.773 nm.
2) Prepare a curve for the relationship between the boron concentration and the indicated valueof the boron standard solution for the calibration curve preparation and the blank test solution
for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.02 mg - 1.6 mg
as B2O3) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the boron content from the calibration curve, and calculate water-soluble boron(W-B2O3) in the analytical sample.
Comment 4 Wash the sample injector of an ICP-OES sufficiently with water because boron easily
causes the memory effect.
Comment 5 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.4.d Comment 4.
Comment 6 The comparison of the measurement value (yi：0.013 % (mass fraction) - 0.530 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of Azomethine-H method was conducted to evaluate trueness using fluid
fertilizers (12 samples). As a result, a regression equation was y = − 0.0041 + 0.986x,
and its correlation coefficient (r) was 0.999. Additionally, additive recovery testing
was conducted using a fluid mixed fertilizer (1 brand) and a home garden-use mixed
300

Testing Methods for Fertilizers (2018)

fertilizer (1 brand) and a liquid micro element mixed fertilizer (1 sample). As a result,the mean recovery rates at additive level of 0.15 % (mass fraction) - 0.2 % and0.01 % (mass fraction) were 95.5 % - 99.4 % and 96.5 % respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.0005 % (mass fraction).

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
7

3)

(%)
0.166

3)

(%)
0.001

Fluid mixed fertilizer
Home garder-use
mixed fertilizer
70.01340.0001（liquid)1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））

(%)
0.7

(%)
0.002

3)

(%)
1.2

1.0

0.0001

1.0

6） Intermediate standard deviation
7） Intermediate relative standard deviation

3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

References
1)
Keisuke AOYAMA: Simultaneous Determination of Water-Soluble Principal
Ingredients (W-P2O5, W-K2O, W-MgO, W-MnO and W-B2O3) in Liquid Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), Research
Report of Fertilizer, Vol.8, p. 1 - 9 (2015)(5)

Flow sheet: The flow sheet for water-soluble boron in fluid fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg to a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 Flow sheet for water-soluble boron in liquid fertilizers (Extraction procedure)301

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
ICP-OES (249.773 nm)
Figure 2 Flow sheet for water-soluble boron in liquid fertilizers (Measurement procedure)302

Testing Methods for Fertilizers (2018)

4.9 Zinc4.9.1 Total zinc
4.9.1.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type C and its
symbol is 4.9.1.a-2017 or T-Zn.a-1.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3), and thenspray in an acetylene−air flame, and measure the atomic absorption with zinc at a wavelength of
213.9 nm to quantify the total zinc (T-Zn). In addition, the performance of this testing method isshown in Comment 6.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Zinc standard solution (Zn 0.1 mg/mL): A zinc standard solution (Zn 0.1 mg/mL) traceable
to National Metrology.
e) Zinc standard solutions (Zn 0.5 µg - 5 µg/mL) for the calibration curve preparation (1):Transfer 2.5 mL - 25 mL of zinc standard solution (Zn 0.1 mg/mL) to 500-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in e).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the zinc standard solution in (2), a zinc standard solution for thecalibration curve preparation can be prepared by using a zinc standard solution (Zn 1
mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (2) function.
1) Light source: A zinc hollow cathode lamp (when the continuous source method as the
background correction method is used, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 450 °C ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (3).
303

Testing Methods for Fertilizers (2018)c) Ignite at 450 ºC ± 3 ºC for 8 - 16 hours to incinerate (3).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (6) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL volumetric flask with water, add water up to themarked line, and filter with Type 3 filter paper to make a sample solution.
Note (3) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 550 °C in 1 to 2 hours.
(4) The watch glass can be removed.
(5) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers not containing
organic matters.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.b, (4.1) of
4.10.1.a, (4.1) of 4.10.1.b, (4.1) of 5.3.a, (4.1) of 5.3.b, (4.1) of 5.4.a, (4.1) of 5.4.b,(4.1) of 5.5.a, (4.1) of 5.5.d, (4.1) of (5.6.a) and (4.1) of 5.6.b. In addition, thesample solution can be used in 4.2.1.a, 4.2.1.b, 4.3.1.a, 4.3.1.b and 4.5.1.a.
Comment 4 The sample solution prepared in (4.1.3) in 4.2.1.a can also be used.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 213.9 nmb) Calibration curve preparation
1) Spray the zinc standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 213.9 nm.
2) Prepare a curve for the relationship between the zinc concentration and the indicated value ofthe zinc standard solutions for the calibration curve preparation and the blank test solution forthe calibration curve preparation.
c) Sample measurement
1) Subject the sample solution (6) to the same procedure as in b) 1) to read the indicated value.
2) Subject the blank test solution to the same procedure as in b) 1) to read the indicated value,and correct the indicated value obtained for the sample solution.
3) Obtain the zinc content from the calibration curve, and calculate the total zinc (T-Zn) in theanalytical sample.
Note (6) If there is a possibility that the zinc concentration in the sample solution will exceed
the maximum limit of the calibration curve, dilute a predetermined amount with304

Testing Methods for Fertilizers (2018)hydrochloric acid (1+23).
Comment 5 The zinc concentration in the analytical sample can also be corrected by subjecting
the blank test solution to the same procedures as in 1) and 3) to obtain the zinccontent in the blank test solution.
Comment 6 Recovery testing was conducted to evaluate trueness using a preparation sample. As
a result, the average rate of recovery at the content level of 1.2 % (mass fraction) and
90 mg/kg are 99.5 % and 97.8 % as the total zinc (T-Zn) respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this test method is about 5
mg/kg for solid fertilizers.
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
2)
3)
4)
5)
6)
7)
8)
certified reference laboratory Averagesr
RSD rs I(T)RSD I(T)
sR
RSD R
1)
material fertilizer(mg/kg) (mg/kg)(%)(mg/kg)(%)(mg/kg)(%)
p
FAMIC-C-12
12
992
11
1.1
17
1.7
32
3.3
1） The number of laboaratories used for analysis
5） Intermediate standard deviation
conducting Cold vapor atomic absorption spectrometry
2） Average （the number of laboratory（p ）×test days （2）6） Intermediate relative standard deviation
×the number of replicate testing（3））3） Repeatability standard deviation
7） Reproducibility standard deviation
4） Repeatability relative standard deviation
8） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.193- 194, Yokendo, Tokyo (1988)
2)
Kimie KATO, Masayuki YOSHIMOTO and Yuji SHIRAI: 3)
Kimie
KATO,
Masayuki YOSHIMOTO and Yuji SHIRAI: Systematization of Determination Methodsof Major Components in Sludge Fertilizer, Compost and Organic Fertilizer, Research
Report of Fertilizer, Vol.3 p. 107 - 116 (2010)3)
Shin ABE and Yoshiyuki SUNAGA: Verification of Performance Characteristics ofTesting Methods for Zinc in Fertilizer by Atomic Absorption Spectrometry, Research
Report of Fertilizer Vol. 6, p. 156 -164 (2013)305

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total zinc: The flow sheet for total zinc in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Measurement

Atomic absorption spectrometer (213.9 nm)

Figure Flow sheet for total zinc in fertilizers

306

Testing Methods for Fertilizers (2018)

4.9.1.b ICP Optical Emission Spectrometry(1) Summary
The testing method is applicable to sludge fertilizers, etc. This testing method is classified as TypeD and its symbol is 4.9.1.b-2017 or T-Zn.b-1.
Pretreat an analytical sample with incineration, nitric acid−hydrochloric acid (1+3), introduce it toan ICP Optical Emission Spectrometer (“ICP-OES”) and measure the emission with zinc at awavelength of 206.191 nm to obtain the total zinc (T-Zn) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 6.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Zinc standard solution (Zn 0.1 mg/mL): A zinc standard solution (Zn 0.1 mg/mL) traceable
to National Metrology.
e) Zinc standard solution (Zn 25 µg/mL) (1): Dilute a predetermined amount of zinc standard
solution (Zn 0.1 mg/mL) with hydrochloric acid (1+23) to prepare a zinc standard solution(Zn 25 µg/mL).
Note(1) This is an example of preparation; prepare an amount as appropriate.

Comment 1 Instead of the zinc standard solution in (2), a zinc standard solution for thecalibration curve preparation can be prepared by using a zinc standard solution (Zn 1
mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C .
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (2).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (2).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (3), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (4) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare the307Testing Methods for Fertilizers (2018)

blank test solution.
Note (2) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating forabout 1 hour and further raise to 450 °C in 1 to 2 hours.
(3) The watch glass can be removed.
(4) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers not containing
organic matters.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K 0116
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Optical Emission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 206.191 nmb) Calibration curve preparation and sample measurement
1) Put 5mL of sample solution to three 10-mL volumetric flasks respectively.
2) Add 2mL and 4 mL of zinc standard solution (0.25 μg/mL) to volumetric flasks of 1) above,
then add hydrochloric acid (1+23) up to the marked line to make a sample solution ofStandard Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flask of 1)
above to make a sample solution without a standard solution.
4) Spray the sample solution of Standard Addition Method and the sample solution without astandard solution into the induction plasma, and read the indicated value at a wavelength of
206.191 nm.
5) Transfer 5 mL of blank test solution to a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value obtained
from the respective sample solutions.
6) Prepare a curve for the relationship between the added zinc concentration and the correctedindicated value of the sample solution for Standard Addition Method and the sample solution
without a standard solution.
7) Obtain the zinc content from the intercept of the calibration curve to calculate the total zinc
(T-Zn) in the analytical sample.
Comment 4 The total zinc (T-Zn) in the analytical sample can also be corrected by subjecting theblank test solution to the same procedures as in b) 1) - b) 4) and b) 6) - b) 7) toobtain the zinc content in the blank test solution.
Comment 5 The simultaneous measurement of multiple elements by an ICP-OES is available. In
this case, transfer a pre-determined amount of copper standard solution (Cu 0.1
mg/mL, 1 mg/mL or 10 mg/mL), zinc standard solution (Zn 0.1 mg/mL, 1 mg/mL or
10 mg/mL), cadmium standard solution (Cd 0.1 mg/mL, 1 mg/mL or 10 mg/mL),
nickel standard solution (Ni 0.1 mg/mL, 1 mg/mL or 10 mg/mL), chromium standard
solution (Cr 0.1 mg/mL, 1 mg/mL or 10 mg/mL) and lead standard solution (Pb 0.1
308

Testing Methods for Fertilizers (2018)

mg/mL, 1 mg/mL or 10 mg/mL) to a volumetric flask to mix, add hydrochloric acid(1+5) to make acid concentration 0.5 mol/L and further add water up to the marked
line to prepare a primary mixed standard solution. Transfer a pre-determined amountof primary mixed standard solution to a volumetric flask, add hydrochloric acid
(1+23) up to the marked line to prepare a mixed standard solution for addition within
the concentration range in Table 1. The measurement wavelengths of respective
elements are according to Table 1.
In addition, the additive amount of mixed standard solution for addition and the
additive concentrations of respective elements in a sample solution are shown in the
Table below.
Table 1The preparation concentration of mixed standard solutions for addition
The additive concentrations and measurement wavelengthsof respective elements in sample solutions
Additive concentration ofConcentration of
mixed standard
element in sample solution (µg/mL)
1)
1)
1)
Test item
Additive amountAdditive amountAdditive amount Measurement
solution forwavelength
addition (µg/mL)
0 mL
2 mL
4 mL
(nm)
Total zinc
Zn
25
0
5
10
206.191
Total copper
Cu
25
0
5
10
324.754
Cadmium
Cd
0.25
0
0.05
0.1
228.802
Nickel
Ni
2.5
0
0.5
1
231.604
Chromium
Cr
2.5
0
0.5
1
205.552
Lead
Pb
2.5
0
0.5
1
220.351
1) Additive amount of mixed standard solution for addition

Comment 6 The comparison of the measurement value (xi：65.0 mg/kg - 3310 mg/kg) of ICP
Optical Emission Spectrometry and the measurement value (yi) of Flame Atomic
Absorption Spectrometry was conducted to evaluate trueness using sludge fertilizers(49 samples). As a result, a regression equation was y = −47.6 + 1.080x and its
correlation coefficient (r) was 0.995. Triplicates measurement for each one sample ofsewage sludge fertilizer, human waste sludge fertilizer, industrial sludge fertilizer,
mixed sludge fertilizer, calcined sludge fertilizers and composted sludge fertilizer
was conducted. As a result, a repeatability obtained was 0.1 % - 2.3 % as a relative
standard deviation.
Additionally, the minimum limit of quantification of this testing method is about 8
mg/kg.
References
1)
Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI and Tetuya NOMURA:
Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer, Vol.4, p. 30 - 35 (2011)309

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total zinc: The flow sheet for total zinc in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest for 30 minutes
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature
25
mL
50
mL of hydrochloric acid (1+5)←
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for total zinc in fertilizers (Exrtraction procedure)

Sample solution
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0 mL, 2mL and 4 mL of zinc standard solution (25 μg / mL)← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Optical Emission Spectrometer (206.191nm)
Figure 2 Flow sheet for total zinc in fertilizers (Measurement procedure)310

Testing Methods for Fertilizers (2018)

4.9.2 Water-soluble zinc
4.9.2.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers that indicate zinc content as a response modifier. This
testing method is classified as Type D and its symbol is 4.9.2.a-2017 or W-Zn.a-1.
Extract by adding water to an analytical sample, spray in an acetylene−air flame and measure theatomic absorption with zinc at a wavelength of 213.9 nm to obtain water-soluble zinc (W-Zn) in ananalytical sample. In addition, the performance of this testing method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
c) Zinc standard solution (Zn 0.1 mg/mL): A zinc standard solution (Zn 0.1 mg/mL) traceable
to National Metrology.
d) Zinc standard solutions (Zn 0.5 µg - 5 µg/mL) for the calibration curve preparation (1):Transfer 2.5 mL - 25 mL of zinc standard solution (Zn 0.1 mg/mL) to 500-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
e) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the zinc standard solution in (2), a zinc standard solution for the calibration
curve preparation can be prepared by using a zinc standard solution (Zn 1 mg/mL or
10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (2) function.
1) Light source: A zinc hollow cathode lamp (when the continuous source method as the
background correction method is used, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 5.00 g of an analytical sample, and put it in a 500-mL volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 2 In the procedure in (4.1.1) a), it is also allowed to weigh 2.50 g of the analytical311

Testing Methods for Fertilizers (2018)

sample and transfer to a 250-mL volumetric flask.
Comment 3 The procedure in (4.1.1) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 4 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 213.9 nmb) Calibration curve preparation
1) Spray the zinc standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 213.9 nm.
2) Prepare a curve for the relationship between the zinc concentration and the indicated value ofthe zinc standard solutions for the calibration curve preparation and the blank test solution forthe calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 mg - 0.5 mg
as Zn) to a 100-mL volumetric flask.
2) Add about 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the zinc content from the calibration curve, and calculate the water-soluble zinc
(W-Zn) in the analytical sample.
Comment 5 Recovery testing was conducted to evaluate trueness using a preparation sample(solid). As a result, the average rate of recovery at the content level of 10 % (massfraction), 2 % (mass fraction) and 0.01 % (mass fraction) are 101.6 %, 101.9 % and98.9 % as water-soluble zinc (W-Zn) respectively. In addition, recovery testing was
conducted using a preparation sample (fluid). As a result, the average rate ofrecovery at the content level of 1 % (mass fraction), 0.05 % (mass fraction) and 20
mg/kg are 99.6 %, 100.4 % and 100.6 % as water-soluble zinc respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a liquid microelement mixed fertilizer to evaluate the extract precision of fluid
fertilizers were analyzed by the one-way analysis of variance. Table 1 shows thecalculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about 10
mg/kg (solid fertilizers) and 0.9 mg/kg (fluid fertilizers).

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Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
repeatability Average2)
1)

T
7

sr

3)

4)
3)

(%)
1.28

(%)
0.01

Fluid mixed fertilizer
Liquid microelement
7
0.230
0.001
mixed fertilizers
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

RSD r
(%)
0.4
0.5

5)

6)

s I(T)(%)
0.02

RSD I(T)
(%)
1.3

0.003

1.5

3)

7)

6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.192- 194, Yokendo, Tokyo (1988)
2)
Shin ABE and Yoshiyuki SUNAGA: Verification of Performance Characteristics ofTesting Methods for Zinc in Fertilizer by Atomic Absorption Spectrometry, Research
Report of Fertilizer Vol. 6, p. 156 -164 (2013)3) Shinji KAWAGUCHI: Simple Extraction Method for Water-Soluble Components in
Liquid Compound Fertilizers, Research Report of Fertilizer, Vol.9, p. 10 - 20 (2016)(5)

Flow sheet for water-soluble zinc: The flow sheet for water-soluble zinc in fertilizers isshown below:
5.00 g analyticalsample500-mL volumetric flask.
(powdery)← Water, About 400 mL
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), for 30 minutes
Water
(up
to
the marked line)←
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for water-soluble zinc in fertilizers(Extraction procedure(4.1.1))

313

Testing Methods for Fertilizers (2018)

1.00 g analyticalsample100-mL volumetric flask.
(fluid)← Water, About 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for water-soluble zinc in fertilizers(Extraction procedure(4.1.2))
Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (213.9 nm)
Figure 2 Flow sheet for water-soluble zinc in fertilizers(Measurement procedure)314

Testing Methods for Fertilizers (2018)

4.9.2.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers, liquid microelement mixed fertilizers
and the fluid fertilizers of home garden-use mixed fertilizers. This testing method is classified as
Type D and its symbol is 4.9.2.b-2017 or W-Zn.b-1.
Extract by adding water to an analytical sample, introduce it to an ICP Optical Emission
Spectrometer (“ICP-OES”) and measure the zinc at a wavelength of 213.856 nm to obtainwater-soluble zinc (W-Zn) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Zinc standard solution (Zn 1 mg/mL): A zinc standard solution (Zn 1 mg/mL) traceable to
National Metrology.
d) Zinc standard solution (Zn 0.1 mg/mL) (1): Transfer 10 mL of zinc standard solution (Zn 1
mg/mL) to a 100-mL flask and add hydrochloric acid (1+23) up to the marked line.
e) Zinc standard solutions (Zn 1 µg - 20 µg/mL) for the calibration curve preparation (1):Transfer 1 mL - 20 mL of zinc standard solution (Zn 0.1 mg/mL) to 100-mL volumetric flasks
step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Zinc standard solutions (Zn 0.1 µg - 1 µg/mL) for the calibration curve preparation (1):Transfer 1 mL - 10 mL of zinc standard solution (Zn 10 µg/mL) to 100-mL volumetric flasks
step-by-step, and add hydrochloric acid (1+23) to the marked line.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d), e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the zinc standard solution in (2), a zinc standard solution for the calibration
curve preparation can be prepared by using a zinc standard solution (Zn 10 mg/mL)
traceable to National Metrology.
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in e) and f) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, in case of using the
ICP-OES of an axial observation mode, it is recommended to prepare a zinc standard
solution for the calibration curve in the concentration range which is suitable to a
device used.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g (2) of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, shake to mix and add water up to the marked line.
315

Testing Methods for Fertilizers (2018)c)

Filter with Type 3 filter paper to make a sample solution.

Note (2) The sampling amount of the analytical sample is 10 g when the content in the sample
is less than 0.01 % (mass fraction) as water-soluble zinc.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 213.856 nmb) Calibration curve preparation
1) Spray the zinc standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 213.856 nm.
2) Prepare a curve for the relationship between the zinc concentration and the indicated value ofthe zinc standard solutions for the calibration curve preparation and the blank test solution forthe calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 2 mg asZn) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the zinc content from the calibration curve, and calculate the water-soluble zinc
(W-Zn) in the analytical sample.
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.4.d Comment 4.
Comment 5 The comparison of the measurement value (yi：0.0109 % (mass fraction) - 0.0827 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of flame atomic absorbance spectrometry was conducted to evaluate truenessusing fluid fertilizers (12 samples). As a result, a regression equation was y =−0.0007 + 0.984x, and its correlation coefficient (r) was 0.998. Additionally, additive
recovery testing was conducted using a fluid compound fertilizer (1 brand) and ahome garden-use mixed fertilizer (1 brand). As a result, the mean recovery atadditive level of 0.01 % (mass fraction) and 0.1 % (mass fraction) were 91.6 % and95.9 % respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.0005 % (mass fraction).

316

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
7

3)

(%)
0.06773)(%)
0.0004

Fluidd mixed fertilizer
Home garder-use
70.01070.0003
mixed fertilizer（liquid)
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））

(%)
0.6

(%)
0.0005

3)

(%)
0.7

2.3

0.0004

4.2

6） Intermediate standard deviation
7） Intermediate relative standard deviation

3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

References
1)
Keisuke AOYAMA: Simultaneous Determination Method for Effect-Development
Promoting Agent (Ca, Fe, Co, Cu, Zn and Mo) in Liquid Compound Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES), Research
Report of Fertilizer, Vol.9, p. 1 - 9 (2016)(5)

Flow sheet for water-soluble zinc: The flow sheet for water-soluble zinc in fluid fertilizers isshown below:
1.00 g analytical sample
Weigh to the order of 1 mg to a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 Flow sheet for water-soluble zinc in liquid fertilizers (Extraction procedure)

Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement

ICP-OES (213.856 nm)

Figure 2 Flow sheet for water-soluble zinc in liquid fertilizers (Measurement procedure)317

Testing Methods for Fertilizers (2018)4.10 Copper
4.10.1 Total copper
4.10.1.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type C and its
symbol is 4.10.1.a-2017 or T-Cu.a-1.
Pretreat an analytical sample with incineration and nitric acid−hydrochloric acid (1+3), and thenspray in an acetylene−air flame, and measure the atomic absorption with copper at a wavelength of
324.8 nm to obtain the total copper (T-Cu) in an analytical sample. In addition, the performance of
this testing method is shown in Comment 6.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Copper standard solution (Cu 0.1 mg/mL): A copper standard solution (Cu 0.1 mg/mL)
traceable to National Metrology.
e) Copper standard solutions (Cu 0.5 µg - 5 µg/mL) for the calibration curve preparation(1): Transfer 2.5 mL - 25 mL of copper standard solution (Cu 0.1 mg/mL) to 500-mL
volumetric flasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in e).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the copper standard solution in (2), a copper standard solution for thecalibration curve preparation can be prepared by using a copper standard solution(Cu 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (2) function.
1) Light source: A copper hollow cathode lamp (In case of background correction system using
continuous spectrum source, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C .
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (3).
318

Testing Methods for Fertilizers (2018)c) Ignite at 450 ºC ± 3 ºC for 8 - 16 hours to incinerate (3).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (6) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (3) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 450 °C in 1 to 2 hours.
(4) The watch glass can be removed.
(5) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers not containing
organic matters.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
Comment 4 The sample solution prepared in (4.1.2) in 4.2.1.a can also be used.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 324.8 nmb) Calibration curve preparation
1) Spray the copper standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 324.8 nm.
2) Prepare a curve for the relationship between the copper concentration and the indicated valueof the copper standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Subject the sample solution (6) to the same procedure as in b) 1) to read the indicated value.
2) Subject the blank test solution to the same procedure as in b) 1) to read the indicated value,and correct the indicated value obtained for the sample solution.
3) Obtain the copper content from the calibration curve, and calculate the total copper (T-Cu) in
the analytical sample.
Note (6) If there is a possibility that the copper concentration in the sample solution will exceed
the maximum limit of the calibration curve, dilute a predetermined amount withhydrochloric acid (1+23).
319

Testing Methods for Fertilizers (2018)

Comment 5 The copper concentration in the analytical sample can also be corrected by subjecting
the blank test solution to the same procedures as in 1) and 3) to obtain the copper
content in the blank test solution.
Comment 6 Recovery testing was conducted to evaluate trueness using a preparation sample. As
a result, the average rate of recovery at the content level of 0.15 % (mass fraction)and 0.03 % (mass fraction) are 100.4 % and 99.6 % as total copper (T-Cu)
respectively.
The results of the collaborative study to determine a certified reference material
fertilizer were analyzed by using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this test method is about 4
mg/kg.
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
2)
3)
4)
5)
6)
7)
8)
certified reference laboratory Averagesr
RSD rs I(T)RSD I(T)
sR
RSD R
1)
material fertilizer(mg/kg) (mg/kg)(%)(mg/kg)(%)(mg/kg)(%)
p
FAMIC-C-12
11
583
7
1.1
11
1.9
22
3.8
1） The number of laboaratories used for analysis
5） Intermediate standard deviation
conducting Cold vapor atomic absorption spectrometry
2） Average （the number of laboratory（p ）×test days （2）6） Intermediate relative standard deviation
×the number of replicate testing（3））3） Repeatability standard deviation
7） Reproducibility standard deviation
4） Repeatability relative standard deviation
8） Reproducibility relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.254- 255, Yokendo, Tokyo (1988)
2)
Kimie KATO, Masayuki YOSHIMOTO and Yuji SHIRAI: Systematization of
Determination Methods of Major Components in Sludge Fertilizer, Compost andOrganic Fertilizer, Research Report of Fertilizer, Vol.3 p. 107 - 116 (2010)3)
Shin ABE and Yoshiyuki SUNAGA: Verification of Performance Characteristics ofTesting Methods for Copper in Fertilizer by Atomic Absorption Spectrometry, Research
Report of Fertilizer, Vol. 6, p. 165 - 173 (2013)320

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total copper: The flow sheet for total copper in fertilizers is shown below:

5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Measurement

Atomic absorption spectrometer (324.8nm)

Figure Flow sheet for total copper in fertilizers

321

Testing Methods for Fertilizers (2018)

4.10.1.b ICP Optical Emission Spectrometry(1) Summary
The test method is applicable to sludge fertilizers, etc. This testing method is classified as Type D
and its symbol is 4.10.1.b-2017 or T-Cu.b-1.
Pretreat an analytical sample with incineration, nitric acid−hydrochloric acid (1+3), introduce it to
ICP Optical Emission Spectrometry (“ICP-OES”) and measure the emission with copper at awavelength of 324.754 nm to quantify the total copper (T-Cu) in an analytical sample. In addition,
the performance of this testing method is shown in Comment 6.
(2)
a)
b)
c)
d)
e)

Reagent, etc.: Reagents and water are as shown below.
Water: Water of A3 specified in JIS K 0557.
Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Hydrochloric acid:
Copper standard solution (Cu 0.1 mg/mL): A copper standard solution (Cu 0.1 mg/mL)
traceable to National Metrology.
Copper standard solutions (Cu 25 µg/mL) (1): Dilute a predetermined amount of copper
standard solution (Cu 0.1 mg/mL) with hydrochloric acid (1+23) to prepare a copper standard
solution (Cu 25 µg/mL).

Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the copper standard solution in (2), a copper standard solution for thecalibration curve preparation can be prepared by using a copper standard solution(Cu 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C .
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (2).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (2).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (3), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (4) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
322

Testing Methods for Fertilizers (2018)

Note (2) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 450 °C in 1 to 2 hours.
(3) The watch glass can be removed.
(4) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers not containing
organic matters.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 324.754 nmb) Calibration curve preparation and sample measurement
1) Put 5mL of sample solution to three 10-mL volumetric flasks respectively.
2) Add 2mL and 4 mL of copper standard solution (2.5 μg/mL) to volumetric flasks of 1) above,
then add hydrochloric acid (1+23) to the marked line to make a sample solution of Standard
Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flask of 1)
above to make a sample solution without a standard solution.
4) Spray the sample solution of Standard Addition Method and the sample solution without astandard solution into the induction plasma, and read the indicated value at a wavelength of
324.754 nm.
5) Transfer 5 mL of blank test solution to a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value obtained
from the respective sample solutions.
6) Prepare a curve for the relationship between the added copper concentration and the correctedindicated value of the sample solution for Standard Addition Method and the sample solution
without a standard solution.
7) Obtain the copper content from the intercept of the calibration curve to calculate the total
copper (T-Cu) in the analytical sample.
Comment 4 The copper concentration in the analytical sample can also be corrected by subjecting
the blank test solution to the same procedures as in b) 1) - b) 4) and b) 6) - b) 7) toobtain the copper content in the blank test solution.
Comment 5 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.9.1.b Comment 5.
Comment 6 The comparison of the measurement value (xi：12.0 mg/kg - 1400 mg/kg) of ICP
Optical Emission Spectrometry and the measurement value (yi) of Flame Atomic
Absorption Spectrometry was conducted to evaluate trueness using sludge fertilizers(49 samples). As a result, a regression equation was y = − 5.5 + 1.062x and its
correlation coefficient (r) was 0.997. Triplicates measurement for each one sample of323

Testing Methods for Fertilizers (2018)sewage sludge fertilizer, human waste sludge fertilizer, industrial sludge fertilizer,
mixed sludge fertilizer, calcined sludge fertilizers and composted sludge fertilizer
was conducted. As a result, a repeatability obtained was 0.6 % - 1.8 % as a relative
standard deviation.
Additionally, the minimum limit of quantification of this testing method is about 3
mg/kg.
References
1)
Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI and Tetuya NOMURA:
Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer, Vol.4, p. 30 - 35 (2011)(5)

Flow sheet for total copper: The flow sheet for total copper in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for total copper in fertilizers (Extraction procedure)324

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0, 2 and 4 mL of copper standard solution (25 μg / mL)← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Optical Emission Spectrometer (324.754 nm)
Figure 2 Flow sheet for total copper in fertilizers (Measurement procedure)325

Testing Methods for Fertilizers (2018)

4.10.2 Water-soluble copper
4.10.2.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers that indicate copper content as a response modifier.
This testing method is classified as Type D and its symbol is 4.10.2.a-2017 or W-Cu.a-1.
Extract by adding water to an analytical sample, spray in an acetylene−air flame and measure theatomic absorption with copper at a wavelength of 324.8 nm to obtain water-soluble copper (W-Cu)
in an analytical sample. In addition, the performance of this testing method is shown in Comment
5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
c) Copper standard solution (Cu 0.1 mg/mL): A copper standard solution (Cu 0.1 mg/mL)
traceable to National Metrology.
d) Copper standard solutions (Cu 0.5 µg - 5 µg/mL) for the calibration curve preparation(1): Transfer 2.5 mL - 25 mL of copper standard solution (Cu 0.1 mg/mL) to 500-mL
volumetric flasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
e) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the copper standard solution in (2), a copper standard solution for thecalibration curve preparation can be prepared by using a copper standard solution(Cu 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (2) function.
1) Light source: A copper hollow cathode lamp (In case of background correction system using
continuous spectrum source, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 5.00 g of an analytical sample, and put it in a 500-mL volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
326

Testing Methods for Fertilizers (2018)

Comment 2 In the procedure in (4.1.1) a), it is also allowed to weigh 2.50 g of the analyticalsample and transfer to a 250-mL volumetric flask.
Comment 3 The procedure in (4.1.1) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 4 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 324.8 nmb) Calibration curve preparation
1) Spray the copper standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 324.8 nm.
2) Prepare a curve for the relationship between the copper concentration and the indicated valueof the copper standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 mg - 0.5 mg
as Cu) to a 100-mL volumetric flask.
2) Add about 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the copper content from the calibration curve, and calculate the water-soluble copper(W-Cu).
Comment 5 Recovery testing was conducted to evaluate trueness using a preparation sample(solid). As a result, the average rate of recovery at the content level of 10 % (massfraction), 1 % (mass fraction) and 0.03 % (mass fraction) are 100.7 %, 99.4 % and102.6 % as water-soluble copper (W-Cu) respectively. In addition, recovery testing
was conducted using a preparation sample (fluid). As a result, the average rate ofrecovery at the content level of 1 % (mass fraction), 0.05 % (mass fraction) and 20
mg/kg are 98.8 %, 99.3 % and 101.4 % as water-soluble copper respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a liquid microelement mixed fertilizer to evaluate the extract precision of fluid
fertilizers were analyzed by the one-way analysis of variance. Table 1 shows thecalculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about 10
mg/kg (solid fertilizers) and 3 mg/kg (fluid fertilizers).

327

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

3)

(%)

Fluid mixed fertilizer
Liquid microelement
7
0.01720.0001
mixed fertilizers
1） The number of test days conducting a duplicate test

T
7

3)

(%)
0.0540(%)
0.0003

2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

3)

(%)

0.6

(%)
0.00070.7

0.00031.5

1.36） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.254- 255, Yokendo, Tokyo (1988)
2)
Shin ABE and Yoshiyuki SUNAGA: Verification of Performance Characteristics ofTesting Methods for Copper in Fertilizer by Atomic Absorption Spectrometry, Research
Report of Fertilizer, Vol. 6, p. 165 - 173 (2013)3)
Shinji KAWAGUCHI: Simple Extraction Method for Water-Soluble Components in
Liquid Compound Fertilizers, Research Report of Fertilizer, Vol.9, p. 10 - 20 (2016)(5)

Flow sheet for water-soluble copper: The flow sheet for water-soluble copper in fertilizers isshown below:

5.00 g analyticalsample500-mL volumetric flask.
(powdery)← Water, About 400 mL
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for water-soluble copper in fertilizers(Extraction procedure (4.1.1))

328

Testing Methods for Fertilizers (2018)

1.00 g analyticalsample100-mL volumetric flask.
(fluid)← Water, About 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for water-soluble copper in fertilizers(Extraction procedure (4.1.2))
Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (324.8 nm)
Figure 2 Flow sheet for water-soluble copper in fertilizers
Measurement procedure)329

Testing Methods for Fertilizers (2018)

4.10.2.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers, liquid microelement mixed fertilizers
and the fluid fertilizers of home garden-use mixed fertilizers. This testing method is classified as
Type D and its symbol is 4.10.2.b-2017 or W-Cu.b-1.
Extract by adding water to an analytical sample, introduce it to an ICP Optical Emission
Spectrometer (“ICP-OES”) and measure the copper at a wavelength of 327.396 nm to obtainwater-soluble copper (W-Cu) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Copper standard solution (Cu 1 mg/mL): A copper standard solution (Cu 1 mg/mL)
traceable to National Metrology.
d) Copper standard solutions (Cu 0.1 mg/mL) for the calibration curve preparation (1):Transfer 10 mL of copper standard solution (Cu 1 mg/mL) to 100-mL volumetric flask, andadd hydrochloric acid (1+23) to the marked line.
e) Copper standard solutions (Cu 1 µg - 20 µg/mL) for the calibration curve preparation (1):Transfer 1 mL - 20 mL of copper standard solution (Cu 0.1 mg/mL) to 100-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Copper standard solutions (Cu 0.1 µg - 1 µg/mL) for the calibration curve preparation(1): Transfer 1 mL - 10 mL of copper standard solution (Cu 10 µg/mL) to 100-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d), e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the copper standard solution in (2), a copper standard solution for thecalibration curve preparation can be prepared by using a copper standard solution(Cu 10 mg/mL) traceable to National Metrology.
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in e) and f) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, in case of using the
ICP-OES of an axial observation mode, it is recommended to prepare a copper
standard solution for the calibration curve in the concentration range which issuitable to a device used.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample (2) to the order of 1 mg, and put it in a 100-mL volumetric330

Testing Methods for Fertilizers (2018)flask.
b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (2) The sampling amount of the analytical sample is 10 g when the content in the sample
is less than 0.01 % (mass fraction) as water-soluble copper.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 327.396 nmb) Calibration curve preparation
1) Spray the copper standard solution for the calibration curve preparation and blank test
solution for the calibration curve preparation into inductively coupled plasma, and read theindicated value at a wavelength of 327.396 nm.
2) Prepare a curve for the relationship between the copper concentration and the indicated valueof the copper standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 2 mg asCu) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the copper content from the calibration curve, and calculate the water-soluble copper(W-Cu).
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.4.d Comment 4.
Comment 5 The comparison of the measurement value (yi：0.00982 % (mass fraction) - 0.0819 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of flame atomic absorbance spectrometry was conducted to evaluate truenessusing fluid fertilizers (12 samples). As a result, a regression equation was y = −0.0006 + 0.966x, and its correlation coefficient (r) was 0.999. Additionally, additive
recovery testing was conducted using a fluid compound fertilizer (1 brand) and ahome garden-use mixed fertilizer (1 brand). As a result, the mean recovery atadditive level of 0.01 % (mass fraction) and 0.1 % (mass fraction) were 93.5 % and95.3 % respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.0005 % (mass fraction).

331

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)1)

Fluid mixed fertilizer
Home garder-use
mixed fertilizer（liquid)

3)

3)

T
7

(%)
0.0643(%)
0.0006(%)

7

0.009760.00006

3)

(%)

0.9

(%)
0.00110.6

0.00033

3.4

1.7

1） The number of test days conducting a duplicate test

6） Intermediate standard deviation

2） Average （the number of test days（T ）7） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

References
1) Keisuke AOYAMA: Simultaneous Determination Method for Effect-Development
Promoting Agent (Ca, Fe, Co, Cu, Zn and Mo) in Liquid Compound Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES), Research
Report of Fertilizer, Vol.9, p. 1 - 9 (2016)(5)

Flow sheet for water-soluble copper: The flow sheet for water-soluble copper in fluid
fertilizers is shown below:

1.00 g analytical sample 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 Flow sheet for water-soluble copper in fertilizers (Extraction procedure)
Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement

ICP-OES (327.396 nm)

Figure 2 Flow sheet for water-soluble copper in fertilizers (Measurement procedure)

332

Testing Methods for Fertilizers (2018)4.11 Organic carbon and carbon-nitrogen ratio
4.11.1 Organic carbon
4.11.1.a Dichromate oxidation(1) Summary
This test method is applicable to sludge fertilizers and compost, etc. This testing method isclassified as Type C and its symbol is 4.11.1.a-2017 or O-C.a-1.
Add a potassium dichromate-sulfuric acid solution to an analytical sample and heat to oxidizeorganic carbon with potassium dichromate. Quantify unconsumed potassium dichromate byoxidation-reduction titration to obtain organic carbon (O-C). This test method is also referred to as
the Method of Tyulin. In addition, the performance of this testing method is shown in Comment 2.
(2) Reagent: Reagents are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
c) 0.2 mol/L ammonium iron (II) sulfate solution (1): Weigh 80 g of ammonium iron (II)sulfate hexahydrate specified in JIS K 8979 into a 2000-mL beaker, and add 1000 mL of
sulfuric acid (1+50) to dissolve.
Standardization: Grind potassium dichromate reference material for volumetric analysis
specified in JIS K 8005 in an agate mortar to powder, heat at 150 ºC ± 2 ºC for 1 hour, let it
stand to cool in a desiccator, and then transfer about 1 g to a weighing dish, and weigh themass to the order of 0.1 mg. Dissolve in a small amount of water, transfer to a 100-mL
volumetric flask, and add water up to the marked line to make the potassium dichromate
standard solution (1) (2). On each day to use a 0.2 mol/L ammonium iron (II) sulfate solution,transfer 10 mL of the potassium dichromate standard solution to a 100-mL Erlenmeyer flask,
add about 5 mL of sulfuric acid (1+2), and then conduct the procedures in (4.2) b) - c), andcalculate the factor of a 0.2 mol/L ammonium iron (II) sulfate solution by the following
formula:Factor (f) of 0.2 mol/L ammonium iron (II) sulfate solution= W1 × (A/100) × (6/294.18) × (V1/V2) × (1000/V3)/C= (W1×A/V3) × (30/294.18)
W1： Mass (g) of potassium dichromate weighed
A： Purity (% (mass fraction)) of potassium dichromate
V1： Volume (10 mL) of potassium dichromate solution transferred
V2： Constant volume (100 mL) of potassium dichromate solution
V3： Volume (mL) of 0.2 mol/L ammonium iron (II) sulfate solution needed for titration
C： Set concentration (0.2 mol/L) of 0.2 mol/L ammonium iron (II) sulfate solutiond) Potassium dichromate-sulfuric acid solution (1): Weigh 40 g of potassium dichromate
specified in JIS K 8517 to a 3000-mL beaker. Add 1000 mL of water to dissolve, and further
add gradually 1000 mL of sulfuric acid while cooling and mixing.
e) N-Phenylanthranilic acid solution: Dissolve 0.2 g of N-phenylanthranilic acid of no lessthan 98 % (mass fraction) in purity and 0.2 g of sodium carbonate specified in JIS K 8625 in asmall amount of water, and add water to make 100 mL.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard potassium dichromate solution (0.2 M (1/6 K2Cr2O7)
solution) in 7.1 B 1) in the Official Methods of Analysis of Fertilizers (1992).
333

Testing Methods for Fertilizers (2018)(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
b) Sample digestion flask (3): A 100-mL borosilicate glass volumetric flask 100 mL (180 mm
total height, 13 mm mouth diameter)
Note (3) Distinguish the volumetric flask used in digestion as a sample digestion flask and do
not use it for any other purposes.
(4) Test procedure(4.1) Dichromate oxidation: Conduct oxidation as follows:a) Weigh 0.05 g of an analytical sample to the order of 0.1 mg (4), and transfer to a sample
digestion flask.
b) Add 25 mL of potassium dichromate-sulfuric acid solution.
c) Heat on a hot plate at 200 ºC until organic matters are completely digested (5).
d) After immediate cooling is complete, precisely adjust to 100 mL with water to make a sample
solution.
e) As a blank test, conduct the procedures in b) and d) using another sample digestion flask to
prepare the blank test solution.
Note (4) Up to about 28 mg as organic carbon (O-C).
(5) Heat for no less than 1 hour after boiling
Comment 1 Sample an analytical sample from a test sample prepared in 2.3.3 Grinding (3.1) bygrinding with a mill until it completely passes through a sieve of 500 µm aperture orfrom a test sample prepared in 2.3.3 Grinding Comment 1.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
a) Transfer 20 mL of the sample solution to a 100-mL Erlenmeyer flask.
b) Add a 0.2 mol/L ammonium iron (II) sulfate solution drop-by-drop until the brown color ofdichromate ion almost disappears from the sample solution.
c) Add about a 0.25 mL of N-phenylanthranilic acid solution (6), and titrate with a 0.2 mol/Lammonium iron (II) sulfate solution until the color of the solution changes from dark
red-purple to blue-green.
d) Transfer 20 mL of the blank test solution to a 100-mL Erlenmeyer flask, and conduct theprocedures in b) - c) to titrate.
e) Calculate the organic carbon (O-C) in the analytical sample by the following formula:
Organic carbon (% (mass fraction)) in the analytical sample= (V4－V5) × C × f × (12.011/4)/W2× (100/1000) × (V6/ V7)= (V4－V5) × f × (12.011/40)/W2
V4： Volume (mL) of 0.2 mol/L ammonium iron (II) sulfate solution needed for thetitration of the blank test solution
V5： Volume (mL) of 0.2 mol/L ammonium iron (II) sulfate solution needed for thetitration of the sample solution
C： Set concentration (0.2 mol/L) of 0.2 mol/L ammonium iron (II) sulfate solution
f： Factor of 0.2 mol/L ammonium iron (II) sulfate solution
V6： Predetermined volume (100 mL) of the sample solution and the blank test
solution in (4.1) d)334

Testing Methods for Fertilizers (2018)

V7： Transferred volume (20 mL) of the sample solution and the blank test solution
subjected to titration in (4.2) a) and (4.2) d)
W2： Mass (g) of the analytical sample
Note (6) About 5 drops with a 1-mL - 2-mL Komagome pipette. Add the same volume to thesample solution and the blank test solution.
Comment 2 The results of the collaborative study to determine a certified reference material
fertilizer were analyzed using a three-level nesting analysis of variance. Table 1
shows the calculation results of reproducibility, intermediate precision andrepeatability.
Additionally, the minimum limit of quantification of this testing method is about
1.5 % (mass fraction).
Table 1 Analysis results of the collaborative study to determine a certified reference material fertilizer
Name of
Number of
Repeatability
Intermediate precision
Reproducibility
certified reference laboratory Average2)
material fertilizer

1)

3)

4)

sr

3)

RSD r
(%)

p
(%)
(%)
FAMIC-C-12
12
20.2
0.3
1.5
1） The number of laboaratories used for analysis
conducting Dichromate oxidation
2） Average （the number of laboratory（p ）×test days（2）×the number of replicate testing（3））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

5)

6)

s I(T)

3)

RSD I(T)
(%)

7)

sR

8)
3)

9)

RSD R
(%)

(%)
(%)
0.5
2.3
0.6
3.1
6） Intermediate standard deviation

7） Intermediate relative standard deviation
8） Reproducibility standard deviation
9） Reproducibility relative standard deviation

References
1) Yuji SHIRAI, Yuko SEKINE, and Toshiaki HIROI: Validation of Determination Method
for Organic Form Carbon in Sludge Fertilizer and Compost, Research Report of
Fertilizer, Vol.3, p. 117 - 122 (2010)(5)

Flow sheet for organic carbon: The flow sheet for organic carbon in sludge fertilizers andcompost, etc. is shown below:

335

Testing Methods for Fertilizers (2018)0.05 gWeigh to the order of 0.1 mg into a sample digestion flask.

analytical sample(Up to about 28 mg as organic carbon.)← 25 mL potassium dichromate-sulfuric acid solution
Heating
Boil for about 1 hour, 200 ºC
Cooling

Immediately← Water (fill up to100 mL)
20 mL aliquot

100-mL Erlenmeyer flask

Sample solution
Figure 1 Flow sheet for organic carbon in sludge fertilizers and compost, etc.
(Dichromate oxidation procedure)

Sample solution
Addingdrop by drop

0.2 mol/L Ammonium iron (II) sulfate solution(until the brown color of dichromate ion

almost disappears from the solution)← About 0.25 mL N-phenylanthranilic acid solution
Titration

0.2 mol/L ammonium iron (II) sulfate solution(until the solution becomes blue-green)

Figure 2 Flow sheet for organic carbon in sludge fertilizers and compost, etc.
(Measurement procedure)

336

Testing Methods for Fertilizers (2018)

4.11.1.b Combustion method(1) Summary
This testing method is applicable to compost and sludge fertilizers. This testing method is classified
as Type B and its symbol is 4.11.1.b-2017 or O-C.b-1.
Drop hydrochloric acid (1+3) to an analytical sample and evaporate inorganic carbon as carbon
dioxide, then thermally decompose carbon compounds using a total nitrogen-total carbon analyzer
by the combustion method to measure carbon dioxide gas with a thermal conductivity detector andobtain organic carbon (O-C) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 4.
(2) Reagent: Reagents are as shown below.
a) Sea sand: Particle diameter 425 µm - 850 µmb) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
Comment 1 Sea sand (particle diameter 425 µm - 850 µm) is commercially sold by FUJIFILM
Wako Pure Chemical Co., Ltd. and YONEYAMA YAKUHIN KOGYO Co., Ltd.
(3) Instruments: Instruments are as shown below:a) Total nitrogen-total carbon analyzer by the combustion method: A total nitrogen-total
carbon analyzer configured on the basis of the principle of the combustion method (modified
Dumas’ method).
1) Turn on the total nitrogen-total carbon analyzer by the combustion method (1), and adjust sothat stable indicated values can be obtained.
(i) Combustion gas: Oxygen having purity no less than 99.99 % (volume percentage)(ii) Carrier gas: Helium having purity no less than 99.99 % (volume percentage)b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Drying apparatus: A drying apparatus that can be adjusted to 105 ºC ± 2 ºC.
Note (1) The setup of the program and the parameters of the analyzer are according to thespecification and the operation method of the total nitrogen-total carbon analyzer by the
combustion method used.
(4)

Test procedures: Conduct measurement as shown below. However, confirm that there is nodifference from the measured value of organic carbon obtained in advance according to
4.11.1.a by using an analytical sample.

(4.1) Hydrochloric acid treatmenta) Weigh 0.05 g of an analytical sample to the order of 0.1 mg, and transfer to a container for
combustion.
b) Cover the analytical sample with about 0.2 g of sea sand and moisten the analytical sample bydropping a few drops of water.
c) After dropping 0.5 mL - 0.7 mL of hydrochloric acid (1+3) little by little (2), drop about 0.3
mL of water (3) (4).
d) Heat a container for combustion on a hot plate at 100 ºC for 90 minutes to dry and harden it.
e) Put the container for combustion at 105 °C ± 2 °C into a drying apparatus and heat for 30
minutes (5).
f) After heating, let it stand to cool to make a test sample.
Note (2)The additive amount of hydrochloric acid (1+3) is merely a target. It is enough to make337

Testing Methods for Fertilizers (2018)

the whole analytical sample come into contact with hydrochloric acid. Let it stand for ashort time in the case of producing bubbles.
(3) In some cases, it is not necessary to add water due to the capacity of a container.
(4) Shake calmly the container for combustion to make the analytical sample come into
complete contact with hydrochloric acid.
(5) Remove hydrochloric acid completely
Comment 2 Sample an analytical sample from a test sample prepared in 2.3.3 Grinding (3.1) bygrinding with a mill until it completely passes through a sieve of 500 µm aperture orfrom a test sample prepared in 2.3.3 Grinding Comment 1.
Comment 3 When it is confirmed that, for example, the volatilization of hydrogen chloride is notdetected by using a test paper, etc. and hydrochloric is completely removed in theprocedure d), the procedure e) can be skipped.
(4.2) Measurement: Specific measurement procedures are according to the operation method of a
total nitrogen-total carbon analyzer by the combustion method.
a) Measurement conditions for the total nitrogen-total carbon analyzer by the combustion
method: Set up the measurement conditions for the total nitrogen-total carbon analyzer
considering the following:
Combustion temperature: No less than 870 ºCb) Calibration curve preparation
1) Turn on the total nitrogen-total carbon analyzer by the combustion method (1), and adjust sothat stable indicated values can be obtained.
2) Weigh a predetermined amount of the standard for calibration curves (6) to the order of 0.1 mg
into a combustion vessel.
3) Insert the combustion vessel into the total nitrogen-total carbon analyzer by the combustion
method, and read the indicated value.
4) Conduct the procedure in 3) for another combustion vessel for a blank test, and read theindicated value.
5) Prepare a curve for the relationship between the carbon content and the indicated value of thestandard for calibration curves and the blank test for calibration curves.
c) Sample measurement
1) Insert the combustion vessel containing the test sample to the total nitrogen-total carbon
analyzer by the combustion method, and read the indicated value.
2) Obtain the carbon content from the calibration curve, and calculate organic carbon in theanalytical sample.
Note (6) Standard for calibration curves: DL-Aspartic acid (purity no less than 99 % (massfraction)), EDTA (purity no less than 99 % (mass fraction)), hippuric acid (purity no lessthan 98 % (mass fraction)) or other reagents having equivalent purity recommended by
the total nitrogen-total carbon analyzer by the combustion method used.
Comment 4 The comparison of the measurement value (yi：0.21 % (mass fraction) - 45.40 %(mass fraction)) of Combustion method and the measurement value (xi) ofDichromate oxidation was conducted to evaluate trueness using sludge fertilizers andcompost (total 25 samples). As a result, a regression equation was y = 0.004 + 1.009x
and its correlation coefficient (r) were 0.999.
Table 1 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification of this testing method is about
338

Testing Methods for Fertilizers (2018)0.05 % (mass fraction).
Table 1 Results and statistical analysis results from a collaborative study
for the organic carbon method
4)
5)
6)
7)
2)
Number
sr
RSD r
sR
RSD R
Mean
Sample name
of
3)
3)
3)
(%)
(%)
(%)
(%)
laboratori (%)
Human waste sludge
8
34.96
0.07
0.2
0.62
1.8
fertilizer
Industrial sludge
8
15.13
0.20
1.3
0.42
2.8
fertilizer
Calcined sludge
9
9.45
0.17
1.8
0.38
4.0
fertilizer
Composted sludge
9
38.20
0.27
0.7
0.73
1.9
fertilizer
Compost
1)
2)
3)
4)
5)
6)
7)

9

20.50

0.76

3.7

0.94

4.6

Number of laboratories used in analysis
Mean (n = number of laboratories × number of samples (2))
Mass fraction
Repeatability standard deviation
Repeatability relative standard deviation
Reproducibility standard deviation
Reproducibility relative standard deviation

References
1)
Aiko YANO, Satono AKIMOTO, and Yuji SHIRAI: Determination of Organic Carbon
in Sludge Fertilizer and Compost by Hydrochloric Acid-Treated Combustion Method,
Research Report of Fertilizer, Vol.9, p. 9 - 19 (2013)
2)
Aiko YANO and Yuji SHIRAI: Determination of Organic Carbon in Sludge Fertilizer
and Compost by Hydrochloric Acid-Treated Combustion Method: A Collaborative
Study, Research Report of Fertilizer, Vol. 7, p. 22 - 27 (2014)339

Testing Methods for Fertilizers (2018)(5)

Flow sheet for organic carbon: The flow sheet for organic carbon in sludge fertilizers andcompost, etc. is shown below:

0.05 gWeigh to the order of 0.1 mg into a combustion vessel.
analytical sample← Cover the analytical sample with 0.2 g of sea sandand drop a few drops of water.
← Drop 0.5mL - 0.7mL of hydrochloric acid (1+3) little by little← Drop 0.3mL of water
HeatingHeat to make it dry for 90 minutes, at 100 ºC
DryingHeat to make it dry for 30 minutes, at 105 ºC

Test sample
Measurement

Total nitrogen-total carbon analyzer by the combustion method

Figure Flow sheet for organic carbon by the combustion method340

Testing Methods for Fertilizers (2018)

Reference: Chromatograms of the standard for calibration curves and an analytical sample areshown below:

1) Total carbon in a standard calibration curve (DL-Aspartic acid)2) Total carbon in an analytical sample (sludge fertilizer)
Reference figures

Chromatograms of organic carbon.

Measurement conditions for total nitrogen-total carbon analyzer by the combustion
methodCombustion gas: Highly pure oxygen, purity no less than 99.99995 % (volume
fraction), flow rate 200 mL/min
Carrier gas: Highly pure helium, purity no less than 99.9999 % (volume fraction),flow rate 80 mL/min
Separation column: A silica gel stainless column (length 1m)
Detector: Thermal conductivity detector (TCD)
Measurement cycle: Purge time = 60 seconds, circulation combustion time = 300
seconds, measurement time = 270 seconds
Current value of Detector: 160 mA
Temperature conditions: Reaction furnace temperature: 870 ºC
Reaction furnace temperature: 600 °C
Column oven temperature: 70 °C
Detector temperature: 100 °C
341

Testing Methods for Fertilizers (2018)

4.11.2 Carbon-nitrogen ratio
4.11.2.a Calculation with organic carbon and total nitrogen(1) Summary
This test method is applicable to compost and sludge fertilizers. This testing method is classified as
Type A (Def-C) and its symbol is 4.11.2.a-2017 or C/N.a-1.
Calculate carbon-nitrogen ratio (CN ratio) by dividing the organic carbon obtained in 4.11.1 by the
total nitrogen obtained in 4.1.1.
(2) The calculation of carbon-nitrogen ratioa) Calculate the carbon-nitrogen ratio (CN ratio) in an analytical sample by the following
formula:Carbon-nitrogen ratio in an analytical sample= O-C/T-N
O-C： Organic carbon (% (mass fraction)) (1) in the analytical sample obtained in
4.11.1
T-N： Total nitrogen (% (mass fraction)) (1) in the analytical sample obtained in
4.1.1
Note (1) O-C and T-N use raw data without rounding numerical value342

Testing Methods for Fertilizers (2018)4.12 Sulfur
4.12.1 Total sulfur content
4.12.1.a Potassium permanganate analysis(1) Summary
This testing method is applicable to fertilizers mainly containing ferrous sulfate (iron (II) sulfate)(FeSO4)) among sulfur and its compound. This testing method is classified as Type D and its
symbol is 4.12.1.a-2017 or T-S.a-1.
Dissolve an analytical sample in water and diluted sulfuric acid, add phosphoric acid and then titrate
ferrous sulfate (iron (II) sulfate) (FeSO4)) by oxidation-reduction with a potassium permanganate
solution to obtain the total sulfate (T-SO3). In addition, the performance of this testing method isshown in Comment 1.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
c) Phosphoric acid: A JIS Guaranteed Reagent specified in JIS K 9005 or a reagent ofequivalent quality.
d) 0.02 mol/L potassium permanganate solution: Dissolve 3.16 g of potassium permanganate
specified in JIS K 8247 in about 800 mL of water to boil and add water to make 1000 mL andleave at rest 1 - 2 day(s). Further filter with a funnel type glass filter (G4) and store in acolored bottle. Or, a reagent of equivalent quality (volumetric analysis grade) that iscommercially available.
Standardization: Dry sodium oxalate of reference materials for volumetric analysis specified
in JIS K 8005 at 200 ºC for 1 hour and let it stand to cool in a desiccator, and then put about
0.3 g into a weighing dish to measure the mass to the order of 0.1 mg. Add about 250 mL of
sulfuric acid (1+20) cooled down to 25 ºC - 30 ºC after boiling and dissolve. Add about 40
mL of 0.02 mol/L potassium permanganate solution while gently shaking for about 1 minute.
Heat up to 55 ºC - 60 ºC after the red color of a potassium permanganate solution disappears.
Titrate with a 0.02 mol/L potassium permanganate solution while keeping the temperatureand continue titrating until the color of the solution becomes light red (1). Calculate the factor
of a 0.02 potassium permanganate solution by the following formula.
Factor (f) of 0.02 mol/L potassium permanganate solution= W1 × (A/100) × ((2/5)/133.999) × ((1000/V1）/C)= W1 × (A/V1) × 1.4925
W1：
A：
V1：
C：

Mass (g) of sodium oxalate sampled
Purity (% (mass fraction)) of sodium oxalate sampled
Volume (mL) of 0.02 mol/L potassium permanganate needed for titration
0.02 mol/L potassium permanganate solution

Note (1) The endpoint is reached when the color of solution keeps as it is for 30 seconds aftercoloring(3) Instruments: Instruments are as shown below:a) Magnetic stirrer:(4) Test procedure(4.1) Measurement: Conduct measurement as shown below.
343

Testing Methods for Fertilizers (2018)a) Weigh 0.5 g - 1 g of an analytical sample to the order of 0.1 mg, and transfer to a 200-mL tallbeaker.
b) Add about 50 mL of water and about 15 mL of sulfuric acid (1+5) and shake with a magnetic
stirrer to dissolve.
c) Immediately add about 1 mL of phosphate, and then titrate with a 0.02 mol/L potassium
permanganate solution until the color of the solution becomes light red (2).
d) As a blank test, conduct the procedures in b) - c) using another 200-mL tall beaker to titrate(2).
e) Calculate the total sulfur content (T-SO3) in an analytical sample by the following formula.
Total sulfur content (% (mass fraction)) = (5 × 0.02 × f × (V2−V3)/1000×80.064)/W2×100= (f × (V2−V3))/W2 × 0.80064
W2： Mass (g) of the sampled analytical sample
V2： Volume (mL) of 0.02 mol/L potassium permanganate needed for titration
V3： Volume (mL) of 0.02 mol/L potassium permanganate needed for titration
f ： Factor of 0.02 mol/L potassium permanganate solution
Note (2) Titrate using a brown burette.
Comment 1 Recovery testing was conducted using a reagent (ferrous sulfate heptahydrate); as aresult, the average rate of recovery was 29.1 % (mass fraction) as total sulfate
content (T-SO3). The recovery rate to a theoretical value was 101.0 %.
Additionally, the minimum limit of quantification of this testing method is about
0.04 % (mass fraction).
References
1)
Yasushi SUGIMURA and Shinjiro IZUKA: Method validation of Redox Titration for
Determination of Sulfur content (as sulfur trioxide) in Fertilizers of Ferrous sulfate andits mixture materials, Research Report of Fertilizer, Vol.3, 25 - 29 (2010).
2） JIS K 8978： Iron (II) sulfate heptahydrate (Reagent) (2008)(5)

Flow sheet for total sulfur content: The flow sheet for total sulfur content in fertilizers
mainly containing ferrous sulfate is shown below.
0.5 g - 1g
Weigh to the order of 0.1 mg into a 200-mL tall beaker
analytical sample← About 50 mL of water← About 15 mL of sulfuric acid (1+5)
Dissolution
Shake to mix
About
1
mL
of phosphate←
0.02 mol/L potassium permanganate solution
Titration(until the solution becomes light red)
Figure Flow sheet for total sulfur content (raw material: ferrous sulfate)344

Testing Methods for Fertilizers (2018)

4.12.1.b Barium chloride gravimetric analysis(1) Summary
This test method is applicable to fertilizers mainly containing sulfur or sulfuric acid among sulfur
and its compound. This testing method is classified as Type B and its symbol is 4.12.1.b-2017 or
T-S.b-1.
Dissolve an analytical sample in a potassium hydroxide-ethanol solution and add hydrogen peroxide
to oxidize, and measure the mass of barium sulfate (BaSO4) formed by reaction with barium
chloride to obtain the total sulfate content (T-SO3). In addition, the performance of this testing
method is shown in Comment 1.
(2) Reagent: Reagents are as shown below.
a) Potassium hydroxide/ethanol solution: Dissolve 10 g of potassium hydroxide specified in
JIS K 8574 in 50 mL of ethanol (95) specified in JIS K 8102, further add 50 mL of water.
b) Hydrogen peroxide: A JIS Guaranteed Reagent (30 % (mass fraction)) specified in JIS K
8230 or a reagent of equivalent quality.
c) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
d) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K 8541
or a reagent of equivalent quality.
e) Barium chloride solution (1): Dissolve 100 g of barium chloride dihydrate specified in JIS K
8155 in water to make 1000 mL.
f) Silver nitrate solution (2 g/100mL): Dissolve 2 g of silver nitrate specified in JIS K 8550 inwater to make 100 mL.
g) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in JIS K
8799 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
b) Water bath: Water bath that can be adjusted to 80 ºC - 90 ºC.
c) Crucible: After heating porcelain crucible or platinum crucible in an electric furnace at 800ºC in advance, let it stand to cool in a desiccator and measure the mass to the order of 0.1 mg.
d) Drying apparatus: Drying apparatus that can be adjusted to 110 ºC - 120 ºC.
e) Electric furnace: An electric furnace that can be kept at 800 ºC ± 5 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as follows (2):a) Weigh 1 g - 5 g of an analytical sample to the order of 0.1 mg, and transfer to a 200-mL tallbeaker.
b) Add about 50 mL of potassium hydroxide/ethanol solution, cover with a watch glass and heaton a hot plate to boil (3).
c) After standing to cool, transfer to a 250-mL volumetric flask and add water up to the markedline.
d) Filter with Type 3 filter paper (4) to make a sample solution.
Note (2) Omit extraction if fluid fertilizers are made from only sulfuric acid and all materials are
dissolved(3) Until sulfur content is dissolved. About 5 minutes when raw materials, etc. are notdissolved.
345

Testing Methods for Fertilizers (2018)(4) Omit the procedures in d) when all materials are dissolved.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 30 mg - 170 mg as SO3) of sample
solution to a 300-mL tall beaker. (5)b) Add about 50 mL of water and about 5 mL of hydrogen peroxide and heat in a water bath at
80 ºC - 90 ºC for about 1 hour while sometimes shaking (6).
c) After standing cool, add 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL) (7), and addhydrochloric acid (2+1) until the color of the solution disappears (8).
d) Further add hydrochloric acid (2+1), add water to make about 100 mL and boil on a hotplate
for about 5 minutes.
e) Immediately add about 6 mL of thermal barium chloride solution (9) while sometimes shaking
in a water bath at 80 ºC - 90 ºC (10).
f) After leaving at rest for a few minutes (11), add a few drops of thermal barium chloride solution
and check that new precipitate of barium sulfate is no longer formed.
g) Further add about 2 mL of thermal barium chloride solution (100 g/L) while shaking to mix(12).
h) After heating in a water bath at 80 ºC - 90 ºC for about 2 hours, stop the heat source of water
bath and let it stand to cool for no less than 4 hours.(5)i) Filter with filter paper (Type 5 - C), wash a container with water and move the whole
precipitate to a filter paper.
j) Wash the precipitate and the filter paper (Type 5 - C) with water several times (13).
k) Put the precipitate together with the filter paper into the crucible.
l) Put the crucible into a drying apparatus and dry at about 110 ºC - 120 ºC for 1 hour.
m) After standing to cool, put the crucible into an electric funnel and heat gently to char (14).
n) Ignite at about 800 ºC ± 5 ºC for 2 hours (14).
o) After ignition (15), move the crucible to a desiccator and let it stand to cool (16).
p) Measure the mass of crucible to the order of 0.1 mg.
q) Calculate the total sulfur content (T-SO3) in an analytical sample by the following formula.
Total sulfur content (% (mass fraction)) = (A × 0.343)/(W × V2/V1) × 100= 34.3 × A × V1/(W×V2)
A： Mass (g) of the precipitate in p)
W： Mass (g) of the analytical sample
V1： Constant volume (mL) of sample solution
V2： Volume (mL) of sample solution transferred
Note (5) Weigh 1g - 5g of an analytical sample to the order of 0.1 mg if the fluid fertilizers of theanalytical sample are made from only sulfuric acid and all materials are dissolved.
(6) It can be suspended after the procedures end.
(7) A pH meter can be used for neutralization.
(8) Omit the procedures in c) if fluid fertilizers are made from only sulfuric acid and allmaterials are dissolved.
(9) Heated up to 70 ºC - 80 ºC in a water bath in advance.
(10) Add drop-by-drop.
(11) Until precipitate settles.
(12) Add a barium chloride solution slightly excessively to reduce the solubility of barium
sulfate.
(13) Wash the precipitate until a white turbidity is no longer formed when about 5 mL of346

Testing Methods for Fertilizers (2018)

nitric acid (1+2) and about 1 mL of silver nitrate solution (2 g/100 mL) are added toabout 20 mL of washing.
(14) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 800 °C in 1 to 2 hours.
(15) To prevent a crucible from chipping, it is recommended to let it stand to cool gently inan electric furnace until the temperature of an electric furnace falls to no more than
200 ºC.
(16) Time to let it stand to cool in a desiccator should be constant. In the case of a porcelain
crucible, it is about 45 - 60 minutes.
Comment 1 Testing was conducted using sulfur simple substance fertilizers containing nomaterials (2 samples); as a result, the quantitative value of the total sulfur content(T-SO3) was 99.9 % - 100.1 % to a theoretical value.
Table 1 shows results and analysis results from a collaborative study for testing
method validation.
Additionally, the minimum limit of quantification of this testing method is about
0.4 % (mass fraction).
Table 1 Results and analysis results from a collaborative study
for total sulfur content with the barium chroride gravimetric analysis (analized as surfur (S))
Samplename

Number of1)laboratories

4)

RSD r

8)

(%)

Mean

2)

Mean

3)

sr

8)

(%)

8)

(%)

(%)

5)

6)

RSD R

8)

(%)

sR

(%)

7)

Surfur
8
8.32
3.33
0.02
0.7
0.05
1.4
material a
Surfur
10
12.71
5.09
0.03
0.6
0.14
2.8
material b
Surfur
9
247.6
99.17
0.24
0.2
1.39
1.4
material c
Surfur
8
245.6
98.37
0.18
0.2
0.30
0.3
material dSurfur
8
1.41
0.564
0.002
0.4
0.003
0.6
material e
Surfur
9
2.89
1.157
0.001
0.1
0.010
0.9
material f1) Number of laboratories used in analysis
2) Total mean as surfur trioxid (SO3 ) (n =number of laboratories × number of repetition (2))3) Total mean as surfur (S) obtained by dividing the total mean in 2) by 2.4969
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation
8) Mass fraction

347

Testing Methods for Fertilizers (2018)

References
1） JIS K 8088： Sulfur (Regent) (2010)
2） JIS K 8217： Iron ores - Methods for determination of sulfur content (1994)
3)
Edited by KANTO CHEMICAL CO., INC.: Technique on Chemical Analysis of Regent
4)
5)

(5)

- Practical Basic Technique and Knowledge, p. 112 - 120 (2009)
Yasushi SUGIMURA Validation of Gravimetric Analysis for Determination of Sulfur
Content (as Sulfur Trioxide) in Sulfur and its Compounds as Fertilizers, Research
Report of Fertilizer, Vol.4, P. 9 - 15 (2011)
Shin ABE, Chika SUZUKI and Yuji SHIRAI: Testing Method of the Total Amount of
Sulfur Content (as Sulfur Trioxide): A Collaborative Study, Research Report of
Fertilizer, Vol. 7, p. 28 - 35 (2014)

Flow sheet for total sulfur content: The flow sheet for total sulfur content in fertilizers
mainly containing sulfur and sulfuric acid is shown below.
1 g -5 g
Weigh to the order of 0.1 mg into a 200-mL tall beaker
analytical sample← About 50 mL of potassium hydroxide/ethanol solution
Heating
Cover with a watch glass and boil
Standing to coolTransfer
250-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for total sulfur content in fertilizers(Extraction procedure)

348

Testing Methods for Fertilizers (2018)

Sample solution
300-mL tall beaker
Aliquot(predetermined volume)← About 50 mL of water← About 5 mL of hydrogen peroxide
Heating
80 ºC - 90 ºC, 1 hourStanding to cool
← 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL)
Neutralization
Hydrochloric acid (2+1) (until the solution becomes transparent)← About 1mL of hydrochloric acid (2+1)← Water (the liquid volume to reach about 100 mL)
HeatingBoil, for 5 minutes← About 6 mL of thermal barium chloride solution, while shaking
Leaving at rest
For several minutes← A few drops of thermal barium chloride solution(Check that new precipitate is no longer formed)← About 2 mL of thermal barium chloride solution, while shaking
Heating
80 ºC - 90 ºC, 2 hourStanding to cool
Filtration

For no less than 4 hours, in a water bath whose heat source is stopped
Type 5-C filter paper

Transfer
Type 5-C filter paper, water← Wash with water (until reaction of chloric matters disappears in filtrate)
Put
Crucible
DryingDrying apparatus, about 100 ºC - 120 ºC, 1 hourStanding to coolCharring
Incineration

Heat gently in an electric furnace
Ignite at 800 ºC ± 5ºC, 2 hours

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 0.1 mg

Figure 2 Flow sheet for total sulfur content in fertilizers(Measurement procedure)

349

Testing Methods for Fertilizers (2018)

4.12.1.c Transmitted light analysis(1) Summary
This test method is applicable to fertilizers mainly containing sulfur or sulfuric acid among sulfur
and its compound. This testing method is classified as Type D and its symbol is 4.12.1.c-2017 or
T-S.c-1.
Dissolve an analytical sample in a potassium hydroxide-ethanol solution and add hydrogen peroxide
to oxidize, and then measure the intensity of transmitted light of suspension of barium sulfate(BaSO4) formed by the reaction with barium chloride as the absorbance to obtain total sulfur
content (T-SO3) in an analytical sample. In addition, the performance of this testing method isshown in Comment 2.
(2) Reagent, etc.: Reagents are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Potassium hydroxide/ethanol solution: Dissolve 10 g of potassium hydroxide specified in
JIS K 8574 in 50 mL of ethanol (95) specified in JIS K 8102, further add 50 mL of water.
c) Hydrogen peroxide: A JIS Guaranteed Reagent (H2O2 30 % (mass fraction)) specified in JIS
K 8230 or a reagent of equivalent quality.
d) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
e) Glycerin – ethanol solution (1+1): Add 250 mL of ethanol (95) specified in JIS K 8102 to
250 mL of glycerin specified in JIS K 8295.
f) Sodium chloride solution (1): Dilute 240 g of sodium chloride specified on JIS K 8150 inwater containing 20 mL of hydrochloric acid specified in JIS K 8180, further add water tomake 1000 mL.
g) Barium chloride solution: Sieve barium chloride dihydrate specified in JIS K 8155 to makebarium chloride whose particle size is between 710 μm - 500 μm.
h) Sulfate standard solution (SO3 2 mg/mL) (1): Heat potassium sulfate specified in JIS K 8962
until it becomes constant weight value at 800 ºC in advance, stand to cool it in a desiccator,
and then transfer 4.3531 g to a weighing dish. Dissolve in a small amount of water, transfer toa 1000-mL volumetric flask, and add water up to the marked line.
i) Sulfate standard solution (SO3 0.02 mg/mL - 0.1 mg/mL): Transfer 2mL - 10 mL of sulfate
standard solution (SO3 2 mg/mL) to 200-mL volumetric flasks step by step and add water up
to the marked line.
j) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in JIS K
8799 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3)
a)
b)
c)
d)

Apparatus and instruments: Apparatus and instruments are shown below.
Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Water bath: Water bath that can be adjusted to 30 °C ± 2 °C and 80 ºC - 90 ºC.
Magnetic stirrer:
Spectrophotometer: A spectrophotometer specified in JIS K 0115

(4) Test procedure(4.1) Extraction: Conduct extraction as follows (2):a) Weigh 1 g - 2 g of an analytical sample to the order of 0.1 mg, and transfer to a 200-mL tallbeaker.
b) Add about 50 mL of potassium hydroxide/ethanol solution, cover with a watch glass and heaton a hot plate to boil (3).
350

Testing Methods for Fertilizers (2018)c)

After standing to cool, transfer to a 250-mL volumetric flask and add water up to the markedline.
d) Filter with Type 3 filter paper (4) to make the extract.
Note (2) Omit extraction if fluid fertilizers are made from only sulfuric acid and all materials
are dissolved(3) Until sulfur content is dissolved. About 5 minutes when raw materials, etc. are notdissolved.
(4) Omit the procedures in d) when all materials are dissolved.
(4.2) Oxidation: Conduct oxidation as follows:a) Transfer a predetermined volume (the equivalents of 5 mg - 200 mg as SO3) of sample
solution to a 300-mL tall beaker (5).
b) Add about 50 mL of water and about 5 mL of hydrogen peroxide and heat in a water bath at
80 ºC - 90 ºC for about 1 hour while sometimes shaking (6).
c) After standing cool, add 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL) (7), and addhydrochloric acid (2+1) until the color of the solution disappears (8).
d) After standing to cool, transfer to a 200-mL volumetric flask and add water up to the marked
line.
e) Filter with 0.3μm glass filter paper.
Note (5) Weigh 1g - 5g of an analytical sample to the order of 0.1 mg if the fluid fertilizers of
the analytical sample are made from only sulfuric acid and all materials are dissolved.
(6) It can be suspended after the procedures end.
(7) A pH meter can be used for neutralization.
(8) Omit the procedures in c) if fluid fertilizers are made from only sulfuric acid and allmaterials are dissolved.
(4.3)
Precipitate formation: Form precipitate as shown below.
a) Transfer 50 mL of filtrate to a 100-mL Erlenmeyer flask with screw cap.
b) Add about 10 mL of glycerin – ethanol solution (1+1) and about 5 mL of sodium chloride
solution in the Erlenmeyer flask with screw cap.
c) Warm up on a water bath at 30ºC ± 2 ºC.
d) After warming, add 0.30 g of barium chloride and stir with a magnetic stirrer for about 2
minutes.
e) Warm up on a water bath at 30ºC ± 2 ºC for about 4 minutes.
f) After warming, stir with a magnetic stirrer for about 3 minutes to make a sample solution.
g) As a blank test, conduct the procedures in a) - c) and f) using another 100-mL Erlenmeyer
flask with screw cap to prepare the blank test solution.
(4.4) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 450 nmb) Calibration curve preparation
1) Transfer 50 mL of sulfate standard solution (SO3 0.02 mg/mL - 0.1 mg/mL) to a 100-mL
Erlenmeyer flask with screw cap and conduct the procedure (4.3) b) - f) to make the SO3 1
mg/65 mL - 5 mg/65 mL sulfate standard solution for the calibration curve preparation.
351

Testing Methods for Fertilizers (2018)

2) Transfer 50 mL of water to another 100-mL Erlenmeyer flask with screw cap and conduct thesame procedures as 1) to make the blank test solution for the calibration curve preparation.
3) Measure absorbance at a wavelength of 450 nm of the sulfuric acid standard solutions for thecalibration curve preparation using the blank test solution for the calibration curve preparation
as the control (8) (9).
4) Prepare the calibration curve of the sulfuric acid concentration and absorbance of the sulfuric
acid standard solutions for the calibration curve preparation.
c) Sample measurement
1) For the sample solution, conduct procedures similarly as in b) 3) to measure absorbance.
2) For the blank test solution, conduct procedures similarly as in 1) to measure absorbance, andcorrect the absorbance obtained for the sample solution.
3) Obtain the sulfate (SO3) content from the calibration curve, and calculate the total sulfur
content (T-SO3) in the analytical sample.
Note (8) Measure right after stirring because barium sulfate easily precipitates.
(9) A spectrophotometer with automatic sample introducing device is preferable.
Comment 1 The range of calibration curve with linearity is SO3 1 mg/65 mL - 5 mg/65 mL andthe curve does not pass through the origin.
Comment 2 Testing was conducted using sulfur simple substance fertilizers containing nomaterials (2 samples); as a result, the quantitative value of the total sulfur content(T-SO3) was 98.4 % - 99.4 % to a theoretical value.
Additionally, the minimum limit of quantification of this testing method is about 1 %(mass fraction).
References
1） JIS K 8001： General rule for test methods of reagents (2009)
2） JIS K 8088： Sulfur (Regent) (2010)
3) JAPAN Sewage Works Association: Sewage Sludge Analysis -2007-, p. 132 - 134 Tokyo(1964)
4) Edited by KANTO CHEMICAL CO., INC.: Technique on Chemical Analysis of Regent Practical Basic Technique and Knowledge, p. 131 - 135 (2009)5) Yasushi SUGIMURA: Method Validation of Turbidimetry for Determination of Sulfur
Content (as Sulfur Trioxide) in Fertilizers of Sulfur, Sulfuric acid and Its Mixture,
Research Report of Fertilizer Vol.6, p. 20 - 26 (2013)352

Testing Methods for Fertilizers (2018)(5)

Flow sheet for total sulfur content: The flow sheet for total sulfur content in fertilizers
mainly containing sulfur and sulfuric acid is shown below.
1g-2g
Weigh to the order of 0.1 mg into a 200-mL tall beaker
analytical sample← About 50 mL of potassium hydroxide/ethanol solution
Heating
Cover with a watch glass and boil
Standing to coolTransfer
250-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Filtrate

Figure 1 Flow sheet for total sulfur content in fertilizers (Extraction procedure)
Filtrate
Aliquot
300-mL tall beaker(predeterminedvolume)← About 50 mL of water← About 5 mL of hydrogen peroxide
Heating
80 ºC - 90 ºC, 1 hourStanding to cool
← 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL)
Neutralization
Hydrochloric acid (2+1) (until the color of solution becomes transparent)
Transfer

200-mL volumetric flask, water

Filtration

0.3μm glass filter paper

Filtrate

Figure 2 Flow sheet for total sulfur content in fertilizers (Oxidation procedure)

353

Testing Methods for Fertilizers (2018)
Filtrate

Aliquot(50 mL)

Erlenmeyer flaskwith screw cap

Aliquot(50 mL)

Erlenmeyer flaskwith screw cap← About 10 mL of glycerin – ethanol solution (1+1) →← About 5 mL of sodium chloride solution→
Warming
30 ºC← Barium chloride 0.30 g
Stirring
2 minutes
Warming

30 ºC, 4 minutes

Stirring

3 minutes

Sample solution

Warming

30 ºC

Stirring

3 minutes

Blank test solution

Measurement
Spectrophotometer(450 nm)

Measurement
Spectrophotometer(450 nm)

Figure 2 Flow sheet for total sulfur content in fertilizers(precipitate formation and measurement procedure)354

Testing Methods for Fertilizers (2018)4.13 Iron
4.13.1 Water-soluble iron4.13.1.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers that indicate iron content as a response modifier. This
testing method is classified as Type D and its symbol is 4.13.1.a-2017 or W-Fe.a-1.
Extract by adding water to an analytical sample, spray in an acetylene−air flame and measure theatomic absorption with iron at a wavelength of 248.3 nm to obtain water-soluble iron (W-Fe) in ananalytical sample. In addition, the performance of this testing method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Iron standard solution (Fe 0.1 mg/mL): An iron standard solution (Fe 0.1 mg/mL) traceable
to National Metrology.
d) Iron standard solutions (Fe 0.5 µg/ mL - 5 µg/ mL) for the calibration curve preparation(1): Transfer 2.5 mL - 25 mL of iron standard solution (Fe 0.1 mg/mL) to 500-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) up to the marked line.
e) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the iron standard solution in (2), an iron standard solution for thecalibration curve preparation can be prepared by using an iron standard solution (Fe1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (2) function.
1) Light source: An iron hollow cathode lamp (In case of background correction system using
continuous spectrum source, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 5.00 g of an analytical sample, and put it in a 500-mL volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
355

Testing Methods for Fertilizers (2018)

Comment 2 In the procedure in (4.1.1) a), it is also allowed to weigh 2.50 g of the analyticalsample and transfer to a 250-mL volumetric flask.
Comment 3 The procedure in (4.1.1) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 4 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 248.3 nmb) Calibration curve preparation
1) Spray the iron standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 248.3 nm.
2) Prepare a curve for the relationship between the iron concentration and the indicated value ofthe iron standard solutions for the calibration curve preparation and the blank test solution forthe calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 mg - 0.5 mg
as Fe) to a 100-mL volumetric flask.
2) Add about 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the iron content from the calibration curve, and calculate the water-soluble iron (W-Fe)in the analytical sample.
Comment 5 Recovery testing was conducted to evaluate trueness using a preparation sample(solid). As a result, the average rate of recovery at the content level of 10 % (massfraction), 5 % (mass fraction) and 0.05 % (mass fraction) are 101.1 %, 102.8 % and107.0 % as water-soluble iron (W-Fe) respectively. In addition, recovery testing was
conducted using a preparation sample (fluid). As a result, the average rate ofrecovery at the content level of 1 % (mass fraction), 0.1 % (mass fraction) and
0.01 % (mass fraction) are 103.6 %, 105.7 % and 105.1 % as a water-soluble iron
(W-Fe) respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a liquid microelement mixed fertilizer to evaluate the extract precision of fluid
fertilizers were analyzed by the one-way analysis of variance. Table 1 shows thecalculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about 40
mg/kg (solid fertilizers) and 4 mg/kg (fluid fertilizers).

356

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)

Fluid compound fertilzier

7

0.244

0.002

Liquid microelement
7
0.099
0.001
mixed fertilizer
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

0.6

0.003

1.4

0.5

0.003

2.9

6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.252, Yokendo, Tokyo (1988)
2)
Shinei TAKAHASHI, Chika SUZUKI and Noriyuki SASAKI: Verification of
Performance Characteristics of Testing Method for Iron in Fertilizer by Atomic
Absorption Spectrometry, Research Report of Fertilizer, Vol. 7, p. 131 - 137 (2014)3) Shinji KAWAGUCHI: Simple Extraction Method for Water-Soluble Components in
Liquid Compound Fertilizers, Research Report of Fertilizer, Vol.9, p. 10 - 20 (2016)(5)

Flow sheet for water-soluble iron: The flow sheet for water-soluble iron in fertilizers isshown below:
5.00 g analyticalsample500-mL volumetric flask.
(powdery)← Water, About 400 mL
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for water-soluble iron in fertilizers(Extraction procedure (4.1.1))

357

Testing Methods for Fertilizers (2018)

1.00 g analyticalsample100-mL volumetric flask.
(Fluid)← Water, About 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for water-soluble iron in fertilizers(Extraction procedure (4.1.2))

Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (248.3 nm)
Figure 2 Flow sheet for water-soluble iron in fertilizers(Measurement procedure )358

Testing Methods for Fertilizers (2018)

4.13.1.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers, liquid microelement mixed fertilizers
and the fluid fertilizers of home garden-use mixed fertilizers. This testing method is classified as
Type D and its symbol is 4.13.1.b-2017 or W-Fe.b-1.
Extract by adding water to an analytical sample, introduce it to an ICP Optical Emission
Spectrometer (“ICP-OES”) and measure the iron at a wavelength of 259.940 nm to obtainwater-soluble iron(W-Fe) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Iron standard solution (Fe 1 mg/mL): An iron standard solution (Fe 1 mg/mL) traceable to
National Metrology.
d) Iron standard solution (Fe 0.1 mg/mL) (1): Transfer 10 mL of iron standard solution (Fe 1
mg/mL) to a 100-mL volumetric flask and add hydrochloric acid (1+23) up to the marked line.
e) Iron standard solutions (Fe 1 µg - 20 µg/mL) for the calibration curve preparation (1):Transfer 1 mL - 20 mL of iron standard solution (Fe 0.1 mg/mL) to 100-mL volumetric flasks
step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Iron standard solutions (Fe 0.1 µg - 1 µg/mL) for the calibration curve preparation (1):Transfer 1 mL - 10 mL of iron standard solution (Fe 10 µg/mL) to 100-mL volumetric flasks
step-by-step, and add hydrochloric acid (1+23) to the marked line.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d), e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the iron standard solution in (2), an iron standard solution for thecalibration curve preparation can be prepared by using an iron standard solution (Fe10 mg/mL) traceable to National Metrology.
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in e) and f) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, in case of using the
ICP-OES of an axial observation mode, it is recommended to prepare an ironstandard solution for the calibration curve in the concentration range which issuitable to a device used.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample (2) to the order of 1 mg, and put it in a 100-mL volumetricflask.
359

Testing Methods for Fertilizers (2018)b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (2) The sampling amount of the analytical sample is 10 g when the content in the sample
is less than 0.01 % (mass fraction) as water-soluble iron.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 259.940 nmb) Calibration curve preparation
1) Spray the iron standard solution for the calibration curve preparation and blank test solution
for the calibration curve preparation into inductively coupled plasma, and read the indicatedvalue at a wavelength of 259.940 nm.
2) Prepare a curve for the relationship between the iron concentration and the indicated value ofthe iron standard solutions for the calibration curve preparation and the blank test solution forthe calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 2 mg asFe) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the iron content from the calibration curve, and calculate the water-soluble iron (W-Fe)in the analytical sample.
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.4.d Comment 4.
Comment 5 The comparison of the measurement value (yi：0.0191 % (mass fraction) - 0.517 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of flame atomic absorbance spectrometry was conducted to evaluate truenessusing fluid fertilizers (12 samples). As a result, a regression equation was y = 0.001 +0.968x, and its correlation coefficient (r) was 0.999. Additionally, additive recovery
testing was conducted using a fluid compound fertilizer (1 brand) and a homegarden-use mixed fertilizer (1 brand). As a result, the mean recovery at additive levelof 0.01 % (mass fraction) and 0.1 % (mass fraction) were 96.5 % and 93.9 %respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.0005 % (mass fraction).

360

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
7

3)

3)

(%)
0.145

(%)
0.001

Fluid mixed fertilizer
Home garder-use
70.04850.0003
mixed fertilizer（fluid)
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

(%)
0.6

(%)
0.002

3)

(%)
1.1

0.5

0.00050.96） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1) Keisuke AOYAMA: Simultaneous Determination Method for Effect-Development
Promoting Agent (Ca, Fe, Co, Cu, Zn and Mo) in Liquid Compound Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES), Research
Report of Fertilizer, Vol.9, p. 1 - 9 (2016)(5)

Flow sheet: The flow sheet for water-soluble iron in fluid fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1mg into a 100-mL volumetric flask
Water,
about
50 mL
←
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure1

The flow sheet for water-soluble iron in fertilizers (Extraction procedure)

Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement

ICP-OES (259.940 nm)

Figure 2 The flow sheet for water-soluble iron in fertilizers (Measurement procedure)

361

Testing Methods for Fertilizers (2018)4.14 Molybdenum
4.14.1 Water-soluble molybdenum
4.14.1.a Sodium thiocyanate absorptiometric analysis(1) Summary
This test method is applicable to fertilizers that indicate molybdenum content as a response modifier.
This testing method is classified as Type D and its symbol is 4.14.1.a-2017 or W-Mo.a-1.
Extract by adding water to an analytical sample, add sulfuric acid (1+1) and perchloric acid, further
add a sodium thiocyanate solution and a tin (II) chloride solution, and measure the absorbance with
thiocyanate complex formed by the reaction of reduced molybdenum (V) with thiocyanate ion to
obtain water-soluble molybdenum (W-Mo) in an analytical sample. In addition, the performance of
this testing method is shown in Comment 6.
(2) Reagent: Reagents are as shown below.
a) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
b) Perchloric acid: A JIS Guaranteed Reagent specified in JIS K 8223 or a reagent ofequivalent quality.
c) Iron (III) sulfate solution (1): Dissolve 5 g of iron (III) sulfate specified in JIS K 8981 inabout 10 mL of sulfuric acid (1+1) and a proper amount of water, and further add water tomake 100 mL.
d) Sodium thiocyanate solution (1): Dissolve 50 g of sodium thiocyanate specified in JIS K
9002 in water to make 500 mL.
e) Tin (II) chloride solution (1): Dissolve 20 g of tin (II) chloride dihydrate specified in JIS K
8136 in 80 mL of hydrochloric acid (1+1) while heating, then add water to make 200 mL.
f) Molybdenum standard solution (Mo 1 mg/mL) (1): After leaving at rest molybdenum (VI)oxide (2) in a desiccator for about 24 hours to dry, put it to a 1,500 g weighing dish. Dissolve
in a small amount of water, transfer to a 1000 mL volumetric flask and add about 5 g of
sodium hydroxide specified in JIS K 8576 to dissolve and add water up to the marked line.
g) Molybdenum standard solution (Mo 0.01 mg/mL): Dilute a predetermined amount ofmolybdenum standard solution (Mo 1 mg/mL) precisely by a factor of 100 with water.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 99.5 % (mass fraction) in purity is commercially sold as
molybdenum (VI) oxide.
Comment 1 Instead of the molybdenum standard solution in (2), a molybdenum standard solution
for the calibration curve preparation can be prepared by using a molybdenum
standard solution (Mo 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National
Metrology.
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Spectrophotometer: A spectrophotometer specified in JIS K 0115
(4) Test procedure362

Testing Methods for Fertilizers (2018)(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 5.00 g of an analytical sample, and put it in a 500-mL volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 2 In the procedure in (4.1.1) a), it is also allowed to weigh 2.50 g of the analyticalsample and transfer to a 250-mL volumetric flask.
Comment 3 The procedure in (4.1.1) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
Comment 4 When the sample solution in d) contains organic matters that affect the determination,
put a predetermined amount of the sample solution to a 100-mL tall beaker, add asmall amount of sulfuric acid and nitric acid to heat and digest the organic mattersuntil white smoke of sulfuric acid evolves. After standing to cool, transfer thesolution to a 100-mL volumetric flask and add water up to the marked line to filter.
The filtrate is prepared as the sample solution of (4.2) a).
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 5 The procedure in (4.1.2) is the same as the procedure in (4.1.2) in 4.2.4.a.
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 0.3 mg
as Mo) to a 100-mL volumetric flask.
b) Add about 5 mL of sulfuric acid (1+1), about 5 mL of perchloric acid and about 2 mL of iron(III) sulfate solution.
c) Add about 16 mL of sodium thiocyanate solution and about 10 mL of tin (II) chloride solution
successively while shaking to mix and further add water up to the marked line (3).
Note (3) When the solution becomes muddy, centrifuge after the procedure in c). However, if itis presumed that copper (I) thiocyanate caused the muddying, centrifuge after leaving
at rest for 1 hour.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 460 nmb) Calibration curve preparation
1) Transfer 1 mL - 30 mL of molybdenum standard solution (Mo 0.01 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Conduct the same procedure as (4.2) b) - c) to make the 0.01 mg/100 mL - 0.3 mg/100 mL
molybdenum standard solution for the calibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make the blank363

Testing Methods for Fertilizers (2018)

test solution for the calibration curve preparation.
4) Measure absorbance at wavelength 460 nm of the molybdenum standard solution for thecalibration curve preparation using the blank test solution for the calibration curve preparation
as the control.
5) Prepare a curve for the relationship between the molybdenum concentration and theabsorbance of the molybdenum standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) c), measure absorbance by the same procedure as b) 4).
2) Obtain the molybdenum content from the calibration curve, and calculate the water-soluble
molybdenum (W- Mo).
Comment 6 Recovery testing was conducted to evaluate trueness using a preparation sample. As aresult, the average rate of recovery at the content level of 2.5 % (mass fraction) and
0.1 % (mass fraction) are 100.2 % and 100.8 % as water-soluble molybdenum(W-Mo) respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a liquid microelement mixed fertilizer to evaluate the extract precision of fluid
fertilizers were analyzed by the one-way analysis of variance. Table 1 shows thecalculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about 60
mg/kg (solid fertilizers) and 6 mg/kg (fluid fertilizers).
Table 1 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
samplerepeatability Averagesr
RSD rs I(T)RSD I(T)
1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)
Fluid mixed fertilizer
7
0.242
0.001
0.4
0.002
1.0
Liquid microelement
7
0.02280.00010.4
0.00020.8
mixed fertilizers
1） The number of test days conducting a duplicate test
6） Intermediate standard deviation
2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

7） Intermediate relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.281- 283, Yokendo, Tokyo (1988)
2)
Keiji YAGI, Natuki TOYODOME, Tokiya SUZUKI and Hideo SOETA: Verification of
Performance Characteristics of Testing Method for Water-soluble Molybdenum Content
in Fertilizer by Sodium Thiocyante Absorptiometric Analysis, Research Report of
Fertilizer Vol. 7, p. 138 - 144 (2014)3) Shinji KAWAGUCHI: Simple Extraction Method for Water-Soluble Components in
Liquid Compound Fertilizers, Research Report of Fertilizer, Vol.9, p. 10 - 20 (2016)

364

Testing Methods for Fertilizers (2018)(5)

Flow sheet for water-soluble molybdenum: The flow sheet for water-soluble molybdenumin fertilizers is shown below:
5 g analytical sample
500-mL volumetric flask.
(powdery)← About 400 mL of water
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-1 Flow sheet for water-soluble molybdenum in fertilizers(Extraction procedure (4.1.1))

1.00 g analyticalsample100-mL volumetric flask.
(fluid)← About 50 mL of water
Shaking to mix← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1-2 Flow sheet for water-soluble molybdenum in fertilizers(Extraction procedure (4.1.2))

Sample solution
Aliquot(predetermined
100-mL volumetric flask
amount)← About 5 mL of sulfuric acid (1+1)← About 5 mL of perchloric acid
← About 2 mL of iron (III) sulfate solution←16 mL of sodium thiocyanate solution (while shaking to mix)←About 10 mL of tin (II) chloride solution (while shaking to mix)
← Water (up to the marked line)
Leaving at rest
Until the red color of iron disappears
Measurement

Spectrophotometer (460 nm)

Figure 2 Flow sheet for water-soluble molybdenum in fertilizers(Measurement procedure)
365

Testing Methods for Fertilizers (2018)

4.14.1.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers, liquid microelement mixed fertilizers
and the fluid fertilizers of home garden-use mixed fertilizers. This testing method is classified as
Type D and its symbol is 4.14.1.b-2017 or W-Mo.b-1.
Extract by adding water to an analytical sample, introduce it to an ICP Optical Emission
Spectrometer (“ICP-OES”) and measure the molybdenum at a wavelength of 202.030 nm to obtainwater-soluble molybdenum (W-Mo) in an analytical sample. In addition, the performance of thistesting method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Molybdenum standard solution (Mo 1 mg/mL): A molybdenum standard solution (Mo 1
mg/mL) traceable to National Metrology.
d) Molybdenum standard solution (Mo 0.1 mg/mL) (1): Transfer 10 mL of molybdenum
standard solution (Mo 1 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid
(1+23) up to the marked line.
e) Molybdenum standard solutions (Mo 1 µg - 20 µg/mL) for the calibration curve
preparation (1): Transfer 1 mL - 20 mL of molybdenum standard solution (Mo 0.1 mg/mL) to
100-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Molybdenum standard solutions (Mo 0.1 µg - 1 µg/mL) for the calibration curve
preparation (1): Transfer 1 mL - 10 mL of molybdenum standard solution (Mo 0.1 mg/mL) to
100-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d), e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the molybdenum standard solution in (2), a molybdenum standard solution
for the calibration curve preparation can be prepared by using a molybdenum
standard solution (Mo 10 mg/mL) traceable to National Metrology.
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in e) and f) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, when using the ICP-OES
of an axial observation mode, it is recommended to prepare a molybdenum standard
solution for the calibration curve in the concentration range which is suitable to a
device used.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample (2) to the order of 1 mg, and put it in a 100-mL volumetric366

Testing Methods for Fertilizers (2018)flask.
b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (2) The sampling amount of the analytical sample is 10 g when the content in the sample
is less than 0.01 % (mass fraction) as water-soluble molybdenum.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 202.030 nmb) Calibration curve preparation
1) Spray the molybdenum standard solution for the calibration curve preparation and the blanktest solution for the calibration curve preparation into inductively coupled plasma, and readthe indicated value at a wavelength of 202.030 nm.
2) Prepare a curve for the relationship between the molybdenum concentration and the indicatedvalue of the molybdenum standard solution for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 2 mg asMo) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the molybdenum content from the calibration curve, and calculate the water-soluble
molybdenum (W- Mo).
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.4.d Comment 4.
Comment 5 The comparison of the measurement value (yi ： 0.00342 % (mass fraction) 0.20374 % (mass fraction)) of ICP Optical Emission Spectrometry and themeasurement value (xi) of the Sodium thiocyanate absorptiometric analysis was
conducted to evaluate trueness using fluid fertilizers (12 samples). As a result, a
regression equation was y = 0.0004 + 0.982x and its correlation coefficient (r) was
0.999. Additionally, additive recovery testing was conducted using a fluid compound
fertilizer (1 brand) and a home garden-use mixed fertilizer (1 brand). As a result, themean recovery at additive level of 0.01 % (mass fraction) and 0.1 % (mass fraction)
were 95.4 % and 97.6 % respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.0005 % (mass fraction).

367

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
7

3)

(%)
0.124

3)

(%)
0.001

Fluid mixed fertilizer
Home garder-use
70.003590.00001
mixed fertilizer（fluid)
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

3)

(%)
0.5

(%)
0.001

(%)
1.2

0.3

0.00014

4.06） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Keisuke AOYAMA: Simultaneous Determination Method for Effect-Development
Promoting Agent (Ca, Fe, Co, Cu, Zn and Mo) in Liquid Compound Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES), Research
Report of Fertilizer, Vol.9, p. 1 - 9 (2016)(5)

Flow sheet: The flow sheet for water-soluble molybdenum in fluid fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1mg into a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 Flow sheet for water-soluble molybdenum in liquid fertilizers(Extraction procedure)
Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
ICP-OES (202.030 nm)
Figure 2 Flow sheet for water-soluble molybdenum in liquid fertilizers(Measurement procedure)
368

Testing Methods for Fertilizers (2018)

4.15
Cobalt
4.15.1 Water-soluble cobalt
4.15.1.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers, liquid microelement mixed fertilizers
and the fluid fertilizers of home garden-use mixed fertilizers. This testing method is classified as
Type E and its symbol is 4.15.1.a-2017 or W-Co.a-1.
Extract by adding water to an analytical sample, spray in an acetylene−air flame and measure theatomic absorption with cobalt at a wavelength of 240.7 nm to obtain water-soluble cobalt (W-Co) inan analytical sample.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Cobalt standard solution (Co 0.1 mg/mL): A cobalt standard solution (Co 0.1 mg/mL)
traceable to National Metrology.
d) Cobalt standard solutions (Co 0.5 µg - 5 µg/mL) for the calibration curve preparation (1):Transfer 1 mL - 25 mL of cobalt standard solution (Co 0.1 mg/mL) to 500-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
e) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the cobalt standard solution in (2), a cobalt standard solution for thecalibration curve preparation can be prepared by using a cobalt standard solution (Co
1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (2) function.
1) Light source: An cobalt hollow cathode lamp (In case of background correction systemusing continuous spectrum source, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample (3) to the order of 1 mg, and put it in a 100-mL volumetricflask.
b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (3) The sampling amount of the analytical sample is 10 g when the content in the sample
is less than 0.01 % (mass fraction) as water-soluble cobalt.
369

Testing Methods for Fertilizers (2018)

Comment 2 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 240.7 nmb) Calibration curve preparation
1) Spray the cobalt standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 240.7 nm.
2) Prepare a curve for the relationship between the cobalt concentration and the indicated valueof the cobalt standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 2 mg asCo) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the cobalt content from the calibration curve, and calculate the water-soluble cobalt(W-Co) in the analytical sample.
(5)

Flow sheet: The flow sheet for water-soluble cobalt in fluid fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1mg into a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 The flow sheet for water-soluble cobalt in fertilizers(Extraction procedure)

Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement

Atomic absorption spectrometer (240.7 nm)

Figure 2 The flow sheet for water-soluble cobalt in fertilizers(Measurement procedure)370

Testing Methods for Fertilizers (2018)

4.15.1.b ICP Optical Emission Spectrometry(1) Summary
This testing method is applicable to fluid mixed fertilizers, liquid microelement mixed fertilizers
and the fluid fertilizers of home garden-use mixed fertilizers. This testing method is classified as
Type D and its symbol is 4.15.1.b-2017 or W-Co.b-1.
Extract by adding water to an analytical sample, introduce it to an ICP Optical Emission
Spectrometer (“ICP-OES”) and measure the cobalt at a wavelength of 228.616 nm to obtainwater-soluble cobalt (W-Co) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Cobalt standard solution (Co 1 mg/mL): A cobalt standard solution (Co 1 mg/mL) traceable
to National Metrology.
d) Cobalt standard solution (Co 0.1 mg/mL) (1): Transfer 10 mL of cobalt standard solution(Co 1 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid (1+23) up to themarked line.
e) Cobalt standard solutions (Co 1 µg - 20 µg/mL) for the calibration curve preparation (1):Transfer 1 mL - 20 mL of cobalt standard solution (Co 0.1 mg/mL) to 100-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Copper standard solutions (Cu 0.1 µg - 1 µg/mL) for the calibration curve preparation(1): Transfer 1 mL - 10 mL of copper standard solution (Cu 10 µg/mL) to 100-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d), e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 Instead of the cobalt standard solution in (2), a cobalt standard solution for thecalibration curve preparation can be prepared by using a cobalt standard solution (Co
10 mg/mL) traceable to National Metrology.
Comment 2 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in e) and f) applies to a horizontal
observation mode. While an axial observation mode can observe a measurement
component of low-level concentrations, it cannot gain the linearity of a calibration
curve in the range of high-level concentrations. Therefore, in case of using the
ICP-OES of an axial observation mode, it is recommended to prepare a cobalt
standard solution for the calibration curve in the concentration range which issuitable to a device used.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0115
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample (2) to the order of 1 mg, and put it in a 100-mL volumetric371

Testing Methods for Fertilizers (2018)flask.
b) Add about 50 mL of water, shake to mix and add water up to the marked line.
c) Filter with Type 3 filter paper to make a sample solution.
Note (2) The sampling amount of the analytical sample is 10 g when the content in the sample
is less than 0.01 % (mass fraction) as water-soluble cobalt.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.2.4.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 228.616 nmb) Calibration curve preparation
1) Spray the cobalt standard solution for the calibration curve preparation and blank test solution
for the calibration curve preparation into inductively coupled plasma, and read the indicatedvalue at a wavelength of 228.616 nm.
2) Prepare a curve for the relationship between the cobalt concentration and the indicated valueof the cobalt standard solution for the calibration curve preparation and the blank test solution
for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 2 mg asCo) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the cobalt content from the calibration curve, and calculate the water-soluble cobalt(W-Co) in the analytical sample.
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.2.4.d Comment 4.
Comment 5 The comparison of the measurement value (yi：0.00105 % (mass fraction) - 0.0213 %(mass fraction )) of ICP Optical Emission Spectrometry and the measurement value(xi) of flame atomic absorbance spectrometry was conducted to evaluate truenessusing fluid fertilizers (12 samples). As a result, a regression equation was y = 0.0001+ 0.927x, and its correlation coefficient (r) was 0.996. Additionally, additive recovery
testing was conducted using a fluid compound fertilizer (1 brand) and a homegarden-use mixed fertilizer (1 brand). As a result, the mean recovery at additive levelof 0.01 % (mass fraction) and 0.1 % (mass fraction) were 94.6 % and 98.4 %respectively.
The results of the repeatability tests on different days using a fluid mixed fertilizer
and a home garden-use mixed fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.0005 % (mass fraction).

372

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
7

3)

(%)
0.05543)(%)
0.0010

Fluid mixed fertilizer
Home garder-use
70.01050.0003
mixed fertilizer（fluid)
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

(%)
1.7

(%)
0.0024

3)

(%)
4.4

3.3

0.0005

4.86） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1) Keisuke AOYAMA: Simultaneous Determination Method for Effect-Development
Promoting Agent (Ca, Fe, Co, Cu, Zn and Mo) in Liquid Compound Fertilizer using
Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES), Research
Report of Fertilizer, Vol.9, p. 1 - 9 (2016)(5)

Flow sheet: The flow sheet for water-soluble cobalt in fluid fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1mg into a 100-mL volumetric flask← Water, about 50 mL
Shaking to mix← Water (up to the marked line)
Filtration
Sample solution
Figure 1 The flow sheet for water-soluble cobalt in liquid fertilizers(Extraction procedure)

Sample solution
Aliquot
100-mL volmetric flask(predetermined volume)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement

ICP-OES (228.616 nm)

Figure 2 The flow sheet for water-soluble cobalt in liquid fertilizers(Measurement procedure)373

Testing Methods for Fertilizers (2018)

5. Harmful components
5.1 Mercury
5.1.a Cold vapor atomic absorption spectrometry(1) Summary
The test method is applicable to fertilizers excluding fluid sludge fertilizers. This testing method isclassified as Type B and its symbol is 5.1.a-2017 or Hg.a-1.
Pretreat an analytical sample with nitric acid- perchloric acid, and then reduce mercury (II) in thesolution with tin (II) chloride. Aerate this solution, and measure the atomic absorption for generated
mercury vapor at a wavelength of 253.7 nm to obtain mercury (Hg) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 3.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Perchloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
e) Tin (II) chloride solution: To 10 g of tin (II) chloride dihydrate (1) specified in JIS K 8136,
add 60 mL of sulfuric acid (1+20), and stir while heating to dissolve. After cooling iscomplete, add water to make 100 mL.
f) L-cystein solution: To 10 mg of L-cystein (HSCH2CH(NH2)COOH) of no less than 98.0 %in purity, add 100 mL of water and 2 mL of nitric acid to dissolve, and further add water tomake 1000 mL. Store in a refrigerator, and do not use after 6 months after preparation.
g) Tri-n-butyl phosphate (2): A reagent of no less than 98 % in purity.
h) Mercury standard solution (Hg 0.1 mg/mL): A mercury standard solution (Hg 0.1 mg/mL)
traceable to National Metrology.
i) Mercury standard solution (Hg 10 µg/mL) (3) (4): Transfer 10 mL of mercury standard
solution (Hg 0.1 mg/mL) to a 100 mL of volumetric flask and add an L-cystein solution up to
the marked line.
j) Mercury standard solution (Hg 0.1 µg/mL) (3) (5): Dilute a predetermined amount of
mercury standard solution (Hg 10 µg/mL) with an L-cystein solution to prepare a mercury
standard solution (Hg 0.1 µg/mL).
Note (1) Use a reagent with low mercury content, such as for mercury analysis or for harmful
metal analysis.
(2) Use as an anti-forming agent.
(3) This is an example of preparation; prepare an amount as appropriate.
(4) Store in a refrigerator, and do not use after 4 months after preparation.
(5) Store in a refrigerator, and do not use after 1 months after preparation.
Comment 1 Instead of the mercury standard solution in (2), a mercury standard solution for thecalibration curve preparation can be prepared by using a mercury standard solution(Hg 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Mercury atomic absorption spectrometer: Mercury atomic absorption spectrometer using
a method for producing the atomic vapor by reduction specified in JIS K 0121.
1) Light source: Low-pressure mercury lampb) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature can374

Testing Methods for Fertilizers (2018)c)

be set to 180 °C - 200 °C.
Sample digestion flask (6): A 100-mL borosilicate glass volumetric flask 100 mL (180 mm
total height, 13 mm mouth diameter)

Note (6) Distinguish the volumetric flask used in digestion as a sample digestion flask and do
not use it for any other purposes.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and transfer to a sample digestion flask.
b) Add about 10 mL of nitric acid, and heat on a hot plate or sand bath for a short time (7).
c) After standing to cool, add about 10 mL of perchloric acid, and digest by heating on a hot
plate or sand bath at 180 ºC - 200 ºC for about 30 minutes - 1 hour (8).
d) After standing to cool, fill up with water to 100 mL to make a sample solution.
e) As a blank test, conduct the procedures in b) - d) using another sample digestion flask to
prepare the blank test solution.
Note (7) If it foams vigorously, leave at rest overnight.
(8) The sample solution and the blank test solution should be stored when they are cooled
after the procedure in (4.1) c). After filling up the sample solution and the blank test
solution with water, immediately conduct the procedure in (4.2).
(4.2) Measurement: Conduct measurement by cold vapor atomic absorption spectrometry specified
in JIS K 0121. Specific measurement procedures are according to the operation method of theatomic absorption spectrometer used. An example of measurement using a mercury atomic
absorption spectrometer is shown below:a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 253.7 nmb) Calibration curve preparation
1) Transfer 1 mL - 20 mL of mercury standard solution (Hg 0.1 µg/mL) to 100-mL volumetricflasks step-by-step, and add water up to the marked line. Transfer 5 mL of these solutions torespective reduction vessels, add 1 drop of tri-n-butyl phosphate (9), to make mercury standard
solutions for the calibration curve preparation.
2) Add 5 mL of water to another reduction vessel, and add 1 drop of tri-n-butyl phosphate (9), tomake the blank test solution for the calibration curve preparation.
3) Connect the reduction vessel to the mercury atomic absorption spectrometer, and introduce
sulfuric acid (1+1) and a tin (II) chloride solution, and circulate air.
4) Read the indicated value at a wavelength of 253.7 nm.
5) Prepare a curve for the relationship between the mercury content (µg) and the indicated valueof the mercury standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer 5 mL of a sample solution to respective reduction vessels, add 1 drop of tri-n-butyl
phosphate (9), and conduct similarly as in b) 3) - 4) to read the indicated value.
2) Transfer 5 mL of the blank test solution to a reduction vessel, add 1 drop of tri-n-butyl
phosphate (9), and conduct similarly as in b) 2) - 4) to read the indicated value, and correct theindicated value obtained for the sample solution.
3) Obtain the mercury content (µg) from the calibration curve, and calculate mercury (Hg) in the375

Testing Methods for Fertilizers (2018)

analytical sample.
Note (9) It is not required to add tri-n-butyl phosphate if not needed.
Comment 2 Instead of the correction method in c) 2), mercury (Hg) can be corrected byobtaining the mercury content in the blank test.
Comment 3 Recovery testing was conducted to evaluate trueness using an industrial sludge
fertilizer (1 sample), composted sludge (3 samples) and a human waste sludge
fertilizer (1 sample). As a result, the average rate of recovery at the content level of
2 mg/kg and 0.2 mg/kg are 98.7 % - 101.6 % and 100.7 % - 105.4 % as mercury(Hg) respectively. In addition, recovery testing was conducted using soybean meal,rape seed meal, compound fertilizers (2 samples) and blended fertilizers. As a result,the average rate of recovery at the content level of 40 mg/kg and 0.5 mg/kg are
98.5 % - 101.5 % and 100.4 % - 103.3 % as mercury (Hg) respectively.
Table 1 shows results and analysis results from a collaborative study for test
method validation.
Additionally, the minimum limit of quantification of this test method is about 0.01
mg/kg.
Table 1 Results and analysis results from a collaborative study
for mercury test method validation
3)
4)
2)
Number of
RSD r
RSD R
Mean
Sample name1)
(mg/kg)(%)(%)
laboratories
Human waste
11
0.651
5.3
11.6
sludge fertilizer A
Human waste
11
1.10
6.3
10.2
sludge fertilizer B
Composted sludge
11
0.489
6.8
10.2
fertilizer A
Composted sludge
11
0.822
8.1
13.1
fertilizer B
Composted sludge
9
0.182
10.6
10.6
fertilizer C
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

References1)
Fumihiro ABE, Takeshi HASHIMOTO and Yasushi SUGIMURA: Determination of
mercury in sludge fertilizer - Improved decomposition method -, Research Report of
Fertilizer Vol.1, p. 60 - 66 (2008)
2)
Fumihiro ABE, Takeshi HASHIMOTO and Norio HIKICHI: Determination of mercury
in sludge fertilizer - Collaborative Test Results -, Research Report of Fertilizer Vol.1, p.
67 - 73 (2008)
3)
Akira SHIMIZU, Kaori OKADA, Takeshi HASHIMOTO, Yasuto IDE and Toshiaki
HIROI: Determination of Mercury in Fertilizer- Expanding the scope of application of
improved method -, Research Report of Fertilizer Vol.2, p. 12 - 17 (2009)376

Testing Methods for Fertilizers (2018)(5)

Flow sheet for mercury: The flow sheet for mercury in fertilizers is shown below:
1.00 gSample digestion flask
analytical sample← About 10 mL nitric acid
Heating
For a short timeStanding to coolRoom temperature← About 10 mL of perchloric acid
Heating on a hot plate or sand bath at 180 ºC - 200 ºC
Decomposition
for 30 minutes -1 hourStanding to cool
← Water (fill up to 100 mL)
Sample solution
Measurement

Atomic absorption spectrometer (253.7 nm) for mercury

Figure Flow sheet for mercury in fertilizers

377

Testing Methods for Fertilizers (2018)

5.1.b Cold vapor atomic absorption spectrometry (Fluid sludge fertilizers)(1) Summary
The test method is applicable to fluid sludge fertilizers. This testing method is classified as Type D
and its symbol is 5.1.b-2017 or Hg.b-1.
Pretreat an analytical sample with nitric acid- perchloric acid, and then reduce mercury (II) in thesolution with tin (II) chloride. Aerate this solution, and measure the atomic absorption for generated
mercury vapor at a wavelength of 253.7 nm to obtain mercury (Hg) in an analytical sample. In
addition, the performance of this testing method is shown in Comment 4.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrogen peroxide: A JIS Guaranteed Reagent specified in JIS K 8230 or a reagent ofequivalent quality.
d) Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
e) Tin (II) chloride solution: To 10 g of tin (II) chloride dihydrate (1) specified in JIS K 8136,
add 60 mL of sulfuric acid (1+20), and stir while heating to dissolve. After cooling iscomplete, add water to make 100 mL.
f) L-cystein solution: To 10 mg of L-cystein (HSCH2CH(NH2)COOH) of no less than 98.0 %in purity, add 100 mL of water and 2 mL of nitric acid to dissolve, and further add water tomake 1000 mL. Store in a refrigerator, and do not use after 6 months after preparation.
g) Mercury standard solution (Hg 0.1 mg/mL): A mercury standard solution (Hg 0.1 mg/mL)
traceable to National Metrology.
h) Mercury standard solution (Hg 10 µg/mL) (2) (3): Transfer 10 mL of mercury standard
solution (Hg 0.1 mg/mL) to a 100 mL of volumetric flask and add an L-cystein solution up to
the marked line.
i) Mercury standard solution (Hg 0.1 µg/mL) (2) (4): Dilute a predetermined amount of
mercury standard solution (Hg 10 µg/mL) with an L-cystein solution to prepare a mercury
standard solution (Hg 0.1 µg/mL).
Note (1) Use a reagent with low mercury content, such as for mercury analysis or for harmful
metal analysis.
(2) This is an example of preparation; prepare an amount as appropriate.
(3) Store in a refrigerator, and do not use after 4 months after preparation.
(4) Store in a refrigerator, and do not use after 1 month after preparation.
Comment 1 Instead of the mercury standard solution in (2), a mercury standard solution for thecalibration curve preparation can be prepared by using a mercury standard solution(Hg 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Mercury atomic absorption spectrometer : Mercury atomic absorption spectrometer using
a method for producing the atomic vapor by reduction specified in JIS K 0121.
1) Light source: Low-pressure mercury lampb) Pressure vessel decomposing device: A device which pressurizes the inside of a vessel by
putting acid, etc.to heat in the airtight vessel, and decomposes a sample by the interaction ofheating, pressurizing and acid. The following requirements should be met.
1) The main part of a decomposing device: In the case of the microwave heating method, adevice should be able to produce a high-frequency wave using a frequency which is permitted
at an industrial frequency facility. It is desirable to be able to monitor pressure and378

Testing Methods for Fertilizers (2018)

temperature, etc. in the airtight vessel with a sensor inside the device. The interior of a device
should be acid-resistant treated and should have a high temperature durability and a high levelof safety.
2) Exhaust system: A system which has an exhaust fan of acid-resistant specification, and has
an air-cool function inside the device to allow constant airflow, keeping operating temperature
below a certain temperature level.
3) Airtight vessel: A vessel which is heat-resistance and pressure-tight and has the durability
required to decompose particles, and has resistance to internal contamination. It should havesafety functions such as causing overheat prevention valve to work and internal pressure todrop by emitting gas, and preventing gas bumping in the case of exceeding a pressure
limitation.
c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 20.0 g (5) of an analytical sample, and put it in a 100-mL volumetric flask.
b) Gradually add 2.5 mL of nitric acid and 2 mL of hydrogen peroxide.
c) Put the airtight vessel into the main part of a decomposing device and heat using a microwave(6).
d) Ignite at 240 ºC ±5 ºC for no less than 10 minutes (6) to decompose (7).
e) After standing to cool, transfer the solution to a 50-mL volumetric flask (8) with water.
f) Add water up to the marked line and transfer it to a 50-mL ground-in stopper centrifugal
precipitate tube (8).
g) Centrifuge it at 1700 × g centrifugal force for about five minutes (9) and use the supernatant as
the sample solution.
h) As a blank test, conduct the procedures in b) - g) using another airtight vessel to prepare theblank test solution.
Note (5) The maximum limit of solid content, which is converted from moisture content, in thesampling volume 20.0 g of an analytical sample is about 0.5 g. If solid content is likely
to exceed the limit, reduce sampling volume as necessary.
(6) Condition examples for a microwave decomposing device: 0 min (room temperature)→ 10min (240 ºC) → 20 min (240 ºC) → 40 min (room temperature), initial output
1400 W(7) When organic matters still remain, for example the digestion solution is colored,
repeat the procedures in (4.1) b) - c).
(8) The vessel should be made of polypropylene, etc. to not affect the measurement.
(9) 16.5-cm of radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) in 5.2.c, 5.3.c, 5.4.c, 5.5.eand 5.6.c.
(4.2) Measurement: Conduct measurement by cold vapor atomic absorption spectrometry specified
in JIS K 0121. Specific measurement procedures are according to the operation method of theatomic absorption spectrometer used. An example of measurement using a mercury atomic
absorption spectrometer is shown below:a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 253.7 nm
379

Testing Methods for Fertilizers (2018)b) Calibration curve preparation
1) Transfer 0.4 mL - 10 mL of mercury standard solution (Hg 0.1 µg/mL) to 100-mL volumetricflasks step-by-step, and add water up to the marked line. Transfer 5 mL of these solutions torespective reduction vessels to make mercury standard solutions for the calibration curve
preparation.
2) Add 5 mL of water to another reduction vessel to make the blank test solution for thecalibration curve preparation.
3) Connect the reduction vessel to the mercury atomic absorption spectrometer, and introduce
sulfuric acid (1+1) and a tin (II) chloride solution, and circulate air.
4) Read the indicated value at a wavelength of 253.7 nm.
5) Prepare a curve for the relationship between the mercury content (µg) and the indicated valueof the mercury standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer 5 mL of a sample solution to respective reduction vessels, and conduct similarly as inb) 3) - 4) to read the indicated value.
2) Transfer 5 mL of the blank test solution to a reduction vessel and conduct similarly as in b) 2)- 4) to read the indicated value, and correct the indicated value obtained for the sample
solution.
3) Obtain the mercury content (µg) from the calibration curve, and calculate mercury (Hg) in theanalytical sample.
Comment 3 Instead of the correction method in c) 2), mercury (Hg) can be corrected by obtaining
the mercury content in the blank test.
Comment 4 Triplicates additive recovery testing was conducted to evaluate trueness using fluid
industrial sludge fertilizers (2 samples), and composted sludge fertilizers (6 samples).
As a result, the mean recovery at the concentration level of 0.2 mg/kg - 0.4 mg/kg,
0.01 mg/kg - 0.09 mg/kg and 0.7µg /kg - 7µg /kg are 100.0 % - 109.1 %, 99.0 % 114.6 % and 100.4 % - 113.4 % as mercury (Hg) in an actual article respectively.
The results of the repeatability tests on different days using two kinds of fluid sludge
fertilizers to evaluate precision were analyzed by the one-way analysis of variance.
Table 1 shows the calculation results of intermediate precision and repeatability.。
Additionally, the minimum limit of quantification of this testing method is about 0.2µg/kg.

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
3)
4)
5)
6)
repeatability Averagesr
RSD rs I(T)RSD I(T)1)

Composted sludge fertilizer 1

T
5(mg/kg)(mg/kg)(%)(mg/kg)(%)

0.05770.00091.50.0014

2.5

Composted sludge fertilizer 2
50.01420.0002
1） The number of test days conducting a duplicate test

1.7
0.00032.2
5） Intermediate standard deviation

2） Average （the number of test days（T ）6） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Repeatability standard deviation
4） Repeatability relative standard deviation380

Testing Methods for Fertilizers (2018)

References1)
Toshiharu YAGI: Determination of Heavy Metals in Fluid Sludge Fertilizers by ICP-MSand CV-AAS, Research Report of Fertilizer Vol. 8, p. 26 - 37 (2015)(5)

Flow sheet for mercury: The flow sheet for mercury in fluid sludge fertilizers is shown
below:
20.0 g analytical sample Airtight vessel← Nitric acid, 2.5 mL← Hydrogen peroxide, 2 mL
Microwave
decomposition
Transfer

Centrifugal separation

50-mL volumetric flask, water
Ground-in stopper centrifugal precipitate tube,
Anout 1700 × g , 5 minutes

Sample solution
Measurement

Atomic absorption spectrometer (253.7nm) for mercury

Figure Flow sheet for mercury in fluid sludge fertilizers

381

Testing Methods for Fertilizers (2018)

5.2 Arsenic
5.2.a Hydride generation atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 5.2.a-2017 or As.a-1.
Pretreat an analytical sample with nitric acid-sulfuric acid-perchloric acid, and then generate arsenic
hydride by the addition of sodium tetrahydroborate in the acidic condition with hydrochloric acid,introduce it with argon gas to a heated absorption cell, and measure the atomic absorption witharsenic at a wavelength of 193.7 nm to obtain arsenic (As) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 7.
(2)
a)
b)
c)
d)

Reagent, etc.: Reagents and water are as shown below.
Water: Water of A3 specified in JIS K 0557.
Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Perchloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
e) Hydrochloric acid: Hydrochloric acid for arsenic analysis specified in JIS K 8180, or of
harmful metal analysis grade, microanalysis grade or equivalents.
f) Potassium iodide solution (1): Dissolve 20 g of potassium iodide specified in JIS K 8913 inwater to make 100 mL.
g) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
h) Sodium tetrahydroborate solution (1): Dissolve 10 g of sodium tetrahydroborate (NaBH4)
for atomic absorption spectrometry in a sodium hydroxide solution (4 g/L) to make 1000 mL.
i) Arsenic standard solution (As 0.1 mg/mL): An arsenic standard solution (As 0.1 mg/mL)
traceable to National Metrology.
j) Arsenic standard solution (As 1 µg/mL) (2) (3): Dilute a predetermined amount of arsenic
standard stock solution (As 0.1 mg/mL) accurately with hydrochloric acid (1+100) to prepare
an arsenic standard solution (As 1 µg/mL).
k) Arsenic standard solution (As 0.1 µg/mL) (2) (4): Dilute a predetermined amount of arsenic
standard solution (As 0.1 µg/mL) with hydrochloric acid (1+100) to prepare an arsenic
standard solution (As 0.1 µg/mL).
Note (1) The concentrations of a potassium iodide solution and a sodium tetrahydroborate
solution vary depending on the instrument used.
(2) This is an example of preparation; prepare an amount as appropriate.
(3) Store in a refrigerator, and do not use after 6 months after preparation.
(4) Store in a refrigerator, and do not use after 1 month after preparation.
Comment 1 Instead of the arsenic standard solution in (2), an arsenic standard solution for thecalibration curve preparation can be prepared by using an arsenic standard solution(As 1 mg/mL or 10 mg/mL) traceable to National Metrology.

(3) Instruments: Instruments are as shown below:a) Atomic absorption spectrometer : To an atomic absorption spectrometer specified in JIS K
0121, connect a hydride generator and parts shown below. Also, an atomic absorption
spectrometer with a built-in hydride generator can be used.
1) Light source: An arsenic hollow cathode lamp or an arsenic high-intensity discharge lamp.
2) Atomizer: Heated absorption cell (5)382Testing Methods for Fertilizers (2018)3) Gas: Gases for heating the heated absorption cell.
(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hydride generator: A batch-type or continuous-type hydride generator specified in JIS K
0121. For continuous hydride generators, there is a method to introduce a potassium iodide
solution on-line in addition to a sample solution, hydrochloric acid, and a sodium
tetrahydroborate solution.
1) Argon: Argon of grade 2 specified in JIS K 1105 or equivalents.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 350 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to no less than 300 °C.
Note (5) For cell heating, there are a method of electric heating and a method of flame heating.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g - 2.00 g of an analytical sample, and transfer to a 200-mL - 300-mL tall beaker.
b) Add about 10 mL of nitric acid and about 5 mL of sulfuric acid, cover the tall beaker with awatch glass, and leave at rest overnight.
c) Heat mildly on a hot plate or sand bath at 170 ºC - 220 ºC for no less than 30 minutes. Afterbubbles cease to form, set the temperature of the hot plate or sand bath to no less than 300 ºC,
and heat until nitroxide (yellow-brown smoke) is no longer generated (6) (7).
d) After standing to cool, add about 5 mL of perchloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or sand bath at no less than
300 ºC for 2 - 3 hours to digest (8).
f) Slightly move the watch glass (9), and keep on heating on the hot plate or sand bath toconcentrate until the liquid volume becomes no more than 2 mL (10).
g) After standing to cool, add about 5 mL of hydrochloric acid (1+10) and about 20 mL of water,
cover the tall beaker with a watch glass and heat mildly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask with water,
add water up to the marked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (6) Carbonization (degradation) of organic matters by sulfuric acid begins by heating when
nitric acid no longer remains. In this state, As5+ may be reduced to As3+ andevaporate; therefore, stop heating immediately after the end of the generation of
nitroxide (yellow-brown smoke).
(7) Oxidation of organic matters by perchloric acid progresses extremely rapidly andexplosively. For that reason, add perchloric acid after fully degrading organic matterswith nitric acid to avoid danger.
(8) When the white smoke of perchloric acid is generated, if the solution is colored suchas black-brown or brown, stop heating immediately, and after standing to cool, add
nitric acid, and heat again to degrade remaining organic matters.
(9) The watch glass can be removed.
(10) The generation of arsenic hydride is inhibited by the presence of nitric acid; therefore,
remove nitric acid by sufficiently generating the white smoke of sulfuric acid.
Comment 2 The procedure in (4.1) is the same as in (4.1) in 5.2.b and 5.5.c. However, thesampling amount of the analytical sample in (4.1) a) in 5.5.c is 1.00 g.
383

Testing Methods for Fertilizers (2018)

Comment 3 When the analytical sample solidifies in the procedure in (4.1) b), moisten theanalytical sample with a small amount of water as necessary in advance.
Comment 4 It is not necessary to conduct the procedure in (4.1) b) “leave at rest overnight” in thecase of fertilizers not containing organic matters.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used. Two examples of measurement procedures with a continuous
hydride generator are shown below.
(4.2.1) Measurement (A): Leaving at rest after adding a potassium iodide solution.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 193.7 nmb) Calibration curve preparation
1) Transfer 2.5 mL - 10 mL of arsenic standard solution (As 0.1 µg/mL) to 50-mL volumetricflasks step-by-step.
2) Add 5 mL of hydrochloric acid and 5 mL of potassium iodide solution, leave at rest for about
15 minutes, and then add water up to the marked line, to make 5 ng/mL -20 ng/mL arsenic
standard solutions for the calibration curve preparation.
3) Conduct the same procedures as 2) for another 50-mL volumetric flask to make the blank test
solution for the calibration curve preparation.
4) While letting argon flow, introduce the arsenic standard solution for the calibration curve
preparation for each step and the blank test solution for the calibration curve preparation
respectively, further introduce hydrochloric acid (1+1) and a sodium tetrahydroborate solution
to the hydride generator to generate arsenic hydride.
5) Separate arsenic hydride and liquid waste, and then introduce the gas containing arsenic
hydride to a heated absorption cell, and read the indicated value at a wavelength of 193.7 nm.
6) Prepare a curve for the relationship between the arsenic concentration and the indicated valueof the arsenic standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution to a 50-mL volumetric flask, andconduct procedures similarly as in b) 2) and b) 4) - 5) to read the indicated value.
2) Transfer a predetermined amount of the blank test solution to a 50-mL volumetric flask, andconduct procedures similarly as in b) 2) and b) 4) - 5) to read the indicated value, and correct
the indicated value obtained for the sample solution.
3) Obtain the arsenic content from the calibration curve, and calculate arsenic (As) in theanalytical sample.
(4.2.2) Measurement (B): On-line introduction of a potassium iodide solution.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 193.7 nmb) Calibration curve preparation
1) Transfer 5 mL - 25 mL of arsenic standard solution (As 0.1 µg/mL) to 50-mL volumetricflasks step-by-step, add water up to the marked line, to make the 10 ng/mL - 50 ng/mL arsenic
standard solution for the calibration curve preparation. Use water as the blank test solution forthe calibration curve preparation.
2) While letting argon flow, introduce arsenic standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation, respectively, and384

Testing Methods for Fertilizers (2018)

further introduce a potassium iodide solution, hydrochloric acid (1+1) and a sodium
tetrahydroborate solution to the hydride generator to generate arsenic hydride.
3) Separate arsenic hydride and liquid waste, and then introduce the gas containing arsenic
hydride to a heated absorption cell, and read the indicated value at a wavelength of 193.7 nm.
4) Prepare a curve for the relationship between the arsenic concentration and the indicated valueof the arsenic standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution to a 50-mL volumetric flask, andconduct procedures similarly as in b) 2) - 3) to read the indicated value.
2) Transfer a predetermined amount of the blank test solution to a 50-mL volumetric flask, add
water up to the marked line, and conduct similarly as in b) 2) - 3) to read the indicated value,and correct the indicated value obtained for the sample solution.
3) Obtain the arsenic content from the calibration curve, and calculate arsenic (As) in theanalytical sample.
Comment 5 The coexistence of iron, nickel, and cobalt at over 5, 10, 80 folds amount of arsenic,
respectively, inhibits the generation of arsenic hydride. However, the inhibition ofarsenic hydride generation even in the coexistence of iron at 1000 folds amount canbe removed by adding or introducing a potassium iodide solution.
Comment 6 Instead of the correction method in c) 2), the arsenic (As) in the analytical sample
can also be corrected by obtaining the arsenic content in the blank test solution.
Comment 7 A recovery testing was conducted using industrial sludge fertilizer, composted sludge
fertilizer (3 samples) and human waste sludge fertilizer; as a result, the recovery at
the concentration level of 50 mg/kg and 5 mg/kg were 94.6 % - 100.6 % and 99.9 %- 103.3 % as arsenic (As), respectively. Also, a recovery testing was conducted using
processed slug phosphate fertilizer, soybean meal, rape seed meal, compound
fertilizer and potassium-magnesium-sulfate fertilizer; as a result, the recovery at theconcentration level of 50 mg/kg and 5 mg/kg were 98.5 % - 109.8 % and 103.5 % 108.6 %, respectively.
Table 1 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification of this test method is about 0.1
mg/kg.

385

Testing Methods for Fertilizers (2018)

Table 1 Results and analysis results from a collaborative study
for arsenic test method validation
3)
4)
2)
Number of
RSD r
RSD R
Mean
Sample name1)
(mg/kg)(%)(%)
laboratories
Sewage sludge
11
6.42
3.5
10.7
fertilizer
Human waste
10
4.62
4.9
7.0
sludge fertilizer
Industrial sludge
12
0.632
5.7
19.7
fertilizer
Calcined sludge
12
5.08
4.1
9.5
fertilizer
Composted sludge
10
1.23
6.1
11.4
fertilizer
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

References
1)
Naoki ASAO, Yukie ISHIDA, Shinjiro IZUKA and Masakazu SAIKI: Determination of
Arsenic in Sludge Fertilizer - Improved Decomposition Method - Research Report of
Fertilizer Vol.1, p. 74 - 81 (2008)
2)
Naoki ASAO, Shinjiro IZUKA and Norio HIKICHI: Determination of Arsenic in
Sludge Fertilizer - Collaborative Test Results - Research Report of Fertilizer Vol.1, p. 82- 89 (2008)
3)
Yasushi SUGIMURA, Naoki ASAO and Shinjiro IZUKA: Determination of Arsenic in
Fertilizer- Expanding the scope of application of improved method -, Research Reportof Fertilizer Vol.2, p. 18 - 24 (2009)386

Testing Methods for Fertilizers (2018)(5)

Flow sheet for arsenic: The flow sheet for arsenic in fertilizers is shown below:
1.00 g - 2.00 g
200-mL - 300-mL tall beaker
analytical sample← A small amount of water to moisten analytical sample(if needed).
← About 10 mL nitric acid
← About 5 mL sulfuric acid
Leaving at rest
Cover with a watch glass
overnight
HeatingHeat mildly on a hot plate or sand bathat 170 ºC - 220 ºC for no less than 30 minutes.

HeatingHeat on a hot plate or sand bath at no less than 300 ºC
until yellow-brown smoke is no longer generated

Standing to coolRoom temperature← About 5 mL of perchloric acid
Cover with a watch glass, and heat on a hot plate or
Heatingsand bath at no less than 300 ºC for 2 - 3 hours
Heating

Slightly move the watch glass, and concentrate
until it becomes no more than 2 mL

Standing to coolRoom temperature← About 5 mL of hydrochloric acid (1+10)← About 20 mL of water
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Measurement

Atomic absorption spectrometerwith a hydride generator (193.7 nm)

Figure Flow sheet for arsenic in fertilizers

387

Testing Methods for Fertilizers (2018)

5.2.b Silver diethyl dithiocarbamate absorptiometric analysis(1) Summary
This test method is applicable to fertilizers other than sulfur and its compound. This testing method
is classified as Type E and its symbol is 5.2.b-2017 or As.b-1.
Pretreat an analytical sample with nitric acid - sulfuric acid - perchloric acid, and then put thepredetermined volume into an arsenic hydride generation bottle, and generate arsenic hydride byadding a potassium iodide solution, a tin chloride solution and zinc successively in the acidic
condition with hydrochloric acid to react with silver diethyl dithiocarbamate in pyridine. Measure
the absorbance with the silver diethyl dithiocarbamate solution, the coloring liquid, at a wavelengthof 510 nm or 519 nm to obtain arsenic (As) in an analytical sample.
(2)
a)
b)
c)
d)

Reagent, etc.: Reagents and water are as shown below.
Water: Water of A3 specified in JIS K 0557.
Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Perchloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
e) Hydrochloric acid: Hydrochloric acid for arsenic analysis specified in JIS K 8180, or of
harmful metal analysis grade, microanalysis grade or equivalents.
f) Potassium iodide solution: Dissolve 20 g of potassium iodide specified in JIS K 8913 inwater to make 100 mL.
g) Tin (II) chloride solution: Dissolve 15 g of tin (II) chloride dihydrate specified in JIS K
8136 in 100 mL of hydrochloric acid (1+1), add a small amount of granular tin specified in
JIS K 8580 then store in a colored bottle.
h) Ascorbic acid: A JIS Guaranteed Reagent specified in JIS K 9502 or a reagent of equivalentquality.
i) Zinc: A reagent of arsenic analysis grade specified in JIS K 8012 or equivalents. (1 mm - 1.5
mm particle diameter)j) Lead acetate glass wool: Glass wool air-dried after moisturizing with 100 mL of the solution,
where 10 g of lead acetate (II) trihydrate specified in JIS K 8374 is dissolved in water.
k) Silver diethyldithiocarbamate solution: Dissolve 0.5 g of silver N, N-diethyl
dithiocarbamate specified in JIS K 9512 in 100 ml of pyridine specified in JIS K 8777, thenstore in cool and dark placel) Arsenic standard solution (As 0.1 mg/mL): An arsenic standard solution (As 0.1 mg/mL)
traceable to National Metrology.
m) Arsenic standard solution (As 1 µg/mL) (1) (2): Dilute a predetermined amount of arsenic
standard stock solution (As 0.1 mg/mL) accurately with hydrochloric acid (1+100) to prepare
an arsenic standard solution (As 1 µg/mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in a refrigerator, and do not use after 6 months after preparation.
Comment 1 Instead of the arsenic standard solution in (2), an arsenic standard solution for thecalibration curve preparation can be prepared by using an arsenic standard solution(As 1 mg/mL or 10 mg/mL) traceable to National Metrology.

(3) Instruments: Instruments are as shown below:a) Arsenic hydride generator: An arsenic hydride generator specified in 61.1 in JIS K 0102 or
equivalents.
b) Spectrophotometer: A spectrophotometer specified in JIS K 0115
388

Testing Methods for Fertilizers (2018)c)

Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 350 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to no less than 300 °C.

(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g - 2.00 g of an analytical sample, and transfer to a 200-mL - 300-mL tall beaker.
b) Add about 10 mL of nitric acid and about 5 mL of sulfuric acid, cover the tall beaker with awatch glass, and leave at rest overnight.
c) Heat mildly on a hot plate or sand bath at 170 ºC - 220 ºC for no less than 30 minutes. Afterbubbles cease to form, set the temperature of the hot plate or sand bath to no less than 300 ºC,
and heat until nitroxide (yellow-brown smoke) is no longer generated (3) (4).
d) After standing to cool, add about 5 mL of perchloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or sand bath at no less than
300 ºC for 2 - 3 hours to digest (5).
f) Slightly move the watch glass (6), and keep on heating on the hot plate or sand bath toconcentrate until the liquid volume becomes no more than 2 mL (7).
g) After standing to cool, add about 5 mL of hydrochloric acid (1+10) and about 20 mL of water,
cover the tall beaker with a watch glass and heat mildly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask with water,
add water up to the marked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (3) Carbonization (degradation) of organic matters by sulfuric acid begins by heatingwhen nitric acid no longer remains. In this state, As+5 may be reduced to As+3 andevaporate; therefore stop heating immediately after the end of the generation of
nitroxide (yellow-brown smoke).
(4) Oxidation of organic matters by perchloric acid progresses extremely rapidly andexplosively. For that reason, add perchloric acid after fully degrading organic matterswith nitric acid to avoid danger.
(5) When the white smoke of perchloric acid is generated, if the solution is colored suchas black-brown or brown, stop heating immediately, and after standing to cool, add
nitric acid, and heat again to degrade remaining organic matters.
(6) The watch glass can be removed.
(7) The generation of arsenic hydride is inhibited by the presence of nitric acid; therefore,
remove nitric acid by sufficiently generating the white smoke of sulfuric acid.
Comment 2
Comment 3
Comment 4

The procedure in (4.1) is the same as the procedure in (4.1) of 5.2.a.
When the analytical sample solidifies in the procedure in (4.1) b), moisten theanalytical sample with a small amount of water as necessary in advance.
It is not necessary to conduct the procedure in (4.1) b) “leave at rest overnight” inthe case of fertilizers not containing organic matters.

(4.2) Reaction: Conduct reaction as shown below.
a) Put a predetermined amount (the equivalents of 1 μg - 20 μg of As and liquid volume is no
more than 40 mL) of the sample solution into an arsenic hydride generation bottle.
b) Add water to the solution to make about 40 mL.
c) Add hydrochloric acid to make it equivalent to 10 mL of hydrochloric acid.
d) Add about 2 mL of potassium iodide solution, shake and leave at rest for a few minutes.
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Testing Methods for Fertilizers (2018)

Add about 1 mL of tin (II) chloride solution, shake and leave at rest for about 10 minutes (8).
Connect arsenic hydride generation bottle, glass tube lightly stuffed with lead acetate glass
wool in advance and 5 mL of silver diethyl dithiocarbamate solution (9), and quickly put 2.5g
of zinc into an arsenic hydride generation bottle.
g) Leave at rest at room temperature (15ºC - 25ºC) for about 45 minutes, and allow generated
arsenic hydride to absorb into the silver diethyl dithiocarbamate solution and color.
h) Transfer a predetermined amount of blank test solution into the arsenic hydride generation
bottle, conduct procedures similarly as in b) - g) to allow generated arsenic hydride to absorb
into the silver diethyl dithiocarbamate solution and color.
e)f)Note (8) When a large amount of iron is contained, add 1 g of ascorbic acid and 2mL of tin (II)chloride solution and shake to mix to leave at rest for about 10 minutes instead of theprocedure in e).
(9) Apply a small amount of silicone grease, etc. to the connecting parts of an arsenic
hydride generation bottle, glass tube and arsenic hydride absorption tube to maintaintheir airtightness.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used.
a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 510 nm or 519 nmb) Calibration curve preparation
1) Transfer 2.5 mL - 20 mL of arsenic standard solution (1 µg/mL) to arsenic hydride generation
bottles step-by-step.
2) Conduct procedures similarly as in (4.2) b) - g) and allow them to react.
3) For another arsenic hydride generation bottle, the silver diethyl dithiocarbamate solution
prepared similarly as in the procedure in 2) is used as the blank test solution for the calibration
curve preparation.
4) Measure the absorbance at wavelength 510 nm of the silver diethyl dithiocarbamate solutionof the arsenic standard solution for the calibration curve preparation using the blank test
solution for the calibration curve preparation as the control.
5) Prepare a curve for the relationship between the arsenic concentration and the indicated valueof the arsenic standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) For the silver diethyl dithiocarbamate solution of (4.2) g), conduct procedures similarly as inb) 4) to measure absorbance.
2) For the silver diethyl dithiocarbamate solution of (4.2) h), conduct procedures similarly as inb) 4) to measure absorbance, and correct the absorbance obtained for the sample solution.
3) Obtain the arsenic content from the calibration curve, and calculate arsenic (As) in theanalytical sample.
Comment 5 Instead of the correction method in c) 2), the arsenic (As) in the analytical sample
can also be corrected by obtaining the arsenic content in the blank test solution.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.270- 273, Yokendo, Tokyo (1988)390

Testing Methods for Fertilizers (2018)(5)

Flow sheet for arsenic: The flow sheet for arsenic in fertilizers is shown below:
1.00 g -2.00 g
200-mL - 300-mL tall beaker
analytical sample← A small amount of water to moisten analytical sample (if needed).
← About 10 mL nitric acid, about 5 mL sulfuric acid
Leaving at rest
Cover with a watch glass
overnight
HeatingHeat mildly on a hot plate or sand bathat 170 ºC - 220 ºC for no less than 30 minutes.

HeatingHeat on a hot plate or sand bath at no less than 300 ºC
until yellow-brown smoke is no longer generated

Standing to coolRoom temperature← About 5 mL of perchloric acid
Cover with a watch glass, and heat on a hot plate or
Heatingsand bath at no less than 300 ºC for 2 - 3 hours
Heating

Slightly move the watch glass, and concentrate
until it becomes no more than 2 mL

Standing to coolRoom temperature← About 5 mL of hydrochloric acid (1+10), about 20 mL of water
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for arsenic in fertilizers(Extraction procedure)

391

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot
Transfer into an arsenic hydride generation bottle(predetermined volume)← Water (until liquid volume becomes about 40 mL)← Hydrochloric acid (until hydrochloric acid becomes equivalent to 10 mL)← About 2 mL of potassium iodide solution
Shaking to mix
Leaving at rest
For a few minutes←About 1 mL of tin (II) chloride solution
Shaking to mix
Leaving at rest
For about 10 minutes←2.5 g of zinc
Generation ofarsenic hydrideººRoom temperature (15 C - 25 C),
about 45 minutes

Absorption

5 mL of silver diethyldithiocarbamate solution

Measurement

Spectrophotometer (wavelength 510 nm or 519 nm)

Figure 2 Flow sheet for arsenic in fertilizers(Reaction and measurement procedure)

392

Testing Methods for Fertilizers (2018)

5.2.c
ICP Mass Spectrometry (Fluid sludge fertilizers)(1) Summary
The testing method is applicable to fluid sludge fertilizers. This testing method is classified as TypeD and its symbol is 5.2.c-2017 or As.c-1.
Add nitric acid – hydrogen peroxide to an analytical sample to heat and extract by microwave
irradiation. After adding an internal standard element, introduce it to an ICP Mass Spectrometer(ICP-MS) and measure the respective indicated values of arsenic and an internal standard elementwith mass/ charge number (m/z) and obtain arsenic (As) in the analytical sample from the ratio of
the indicated value for arsenic and the indicated value for an internal standard element. In addition,
the performance of this testing method is shown in Comment 4.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A4 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Nitric acid: Nitric acid used to dilute a standard solution and a sample solution is a highly
pure reagent specified in JIS K 9901.
d) Hydrogen peroxide: A JIS Guaranteed Reagent specified in JIS K 8230 or a reagent ofequivalent quality.
e) Rhodium standard solution (Rh 1 mg/mL): A rhodium standard solution (Rh 1 mg/mL)
traceable to National Metrology.
f) Rhodium standard solution (Rh 0.1 µg/mL) (1)(2)(3): Dilute a predetermined amount ofrhodium standard solution (Rh 1 mg/mL) with nitric acid (1+19) to prepare a rhodium
standard solution (Rh 0.1 µg/mL).
g) Rhenium standard solution (Re 1 mg/mL) (4): A rhenium standard solution (Re 1 mg/mL)
traceable to National Metrology.
h) Rhenium standard solution (Re 0.1 µg/mL) (1)(2)(3)(4): Dilute a predetermined amount ofrhenium standard solution (Re 1 mg/mL) with nitric acid (1+19) to prepare a rhenium
standard solution (Re 0.1 µg/mL).
i) Arsenic standard solution (As 0.1 mg/mL): An arsenic standard solution (As 0.1 mg/mL)
traceable to National Metrology.
j) Arsenic standard solution (As 5 µg/mL) (1)(2)(3): Transfer 5 mL of arsenic standard solution(As 0.1 mg/mL) to a 100-mL volumetric flask and add nitric acid (1+19) to the marked line.
k) Arsenic standard solutions (As 1 ng/ mL - 100 ng/ mL) for the calibration curve
preparation (1)(2)(3): Transfer 0.02 mL - 2 mL of arsenic standard solution (As 5 µg/mL) to
100-mL volumetric flasks step-by-step, and add 10 mL of rhodium standard solutions (Rh 0.1µg/mL) as internal standard respectively (5) and add nitric acid (1+19) up to the marked line.
l) Blank test solution for the calibration curve preparation (1)(2)(3): transfer 10 mL of rhodium
standard solution (Rh 0.1 µg/mL) to 100-mL volumetric flasks as internal standard (5) and add
nitric acid (1+19) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in a refrigerator, and do not use after 1 months after preparation.
(3) For storage, use a sealable container made of materials such as polypropylene
containing no arsenic.
(4) Use when measuring lead simultaneously.
(5) Add an internal solution that is 1/10 of the volume to be prepared.
Comment 1 Instead of the arsenic standard solution in (2), an arsenic standard solution for thecalibration curve preparation can be prepared by using an arsenic standard solution(As 1 mg/mL or 10 mg/mL) traceable to National Metrology.
393

Testing Methods for Fertilizers (2018)(3) Instruments: Instruments are as shown below:a) ICP Mass Spectrometer: High-frequency plasma mass spectrometer specified in JIS K 0133.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.995 % in purityb) Pressure vessel decomposing device: A device which pressurizes the inside of a vessel by
putting acid, etc.to heat in the airtight vessel, and decomposes a sample by the interaction ofheating, pressurizing and acid. The following requirements should be met.
1) The main part of a decomposing device: In the case of the microwave heating method, adevice should be able to produce a high-frequency wave using a frequency which is permitted
at an industrial frequency facility. It is desirable to be able to monitor pressure andtemperature, etc. in the airtight vessel with a sensor inside the device. The interior of a device
should be acid-resistant treated and should have a high temperature durability and a high levelof safety.
2) Exhaust system: A system which has an exhaust fan of acid-resistant specification, and has
an air-cool function inside the device to allow constant airflow, keeping operating temperature
below a certain temperature level.
3) Airtight vessel: A vessel which is heat-resistance and pressure-tight and has the durability
required to decompose particles, and has resistance to internal contamination. It should havesafety functions such as causing overheat prevention valve to work and internal pressure todrop by emitting gas, and preventing gas bumping in the case of exceeding a pressure
limitation.
c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 20.0 g (6) of an analytical sample, and put it in a 100-mL volumetric flask.
b) Gradually add 2.5 mL of nitric acid and 2 mL of hydrogen peroxide.
c) Put the airtight vessel into the main part of a decomposing device and heat using a microwave(7).
d) Ignite at 240 ºC ± 5 ºC for no less than 10 minutes (7) to decompose (8).
e) After standing to cool, transfer the solution to a 50-mL volumetric flask (9) with water.
f) Add water up to the marked line and transfer it to a 50-mL ground-in stopper centrifugal
precipitate tube (9).
g) Centrifuge it at 1700 × g centrifugal force for about five minutes (10) and use the supernatantas the sample solution.
h) As a blank test, conduct the procedures in b) - g) using another airtight vessel to prepare theblank test solution.
Note (6) The maximum limit of solid content, which is converted from moisture content, in thesampling volume 20.0 g of an analytical sample is about 0.5g. If solid content is likely
to exceed the limit, reduce sampling volume as necessary.
(7) Condition examples for a microwave decomposing device: 0 min (room temperature)→ 10min (240 ºC) → 20 min (240 ºC) → 40 min (room temperature), initial output
1400 W(8) When organic matters still remain, for example the digestion solution is colored,
repeat the procedures in (4.1) b) - d).
(9) The vessel should be made of polypropylene, etc. to not affect the measurement.
(10) 16.5-cm of radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
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Testing Methods for Fertilizers (2018)

Comment 2

The procedure in (4.1) is the same as the procedure in (4.1) of 5.1.b.

(4.2) Measurement: Conduct measurement (Internal Standard Method) according to JIS K 0113
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Mass Spectrometer used in measurement.
a) Measurement conditions for the ICP Mass Spectrometer: Set up the measurement
conditions for the ICP Mass Spectrometer considering the following:
Arsenic: monitor ion (m/z): 75
Rhodium: monitor ion (m/z): 103b) Calibration curve preparation
1) Spray arsenic standard solution for the calibration curve preparation and blank test solution
for the calibration curve preparation into the inductively coupled plasma, and read the ratio of
the respective ion count number for the monitor ion of an element subjected to measurement
and an internal standard element.
2) Prepare a curve for the relationship between the concentration and the ratio of the ion countnumber of an element subjected to measurement.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 µg - 5 µg asAs) to a 50-mL volumetric flask (9).
2) Add 5mL of internal standard solution (5) and add nitric acid (1+19) to the marked line.
3) Conduct procedures similarly as in b) 1) to read the ratio of the ion count numbers.
4) For blank test solution, conduct procedures similarly as in 1) - 3) to correct the ratio of the ion
count numbers obtained for the sample solution.
5) Obtain the arsenic content from the calibration curve, and calculate arsenic (As) in theanalytical sample.
Comment 3 Instead of the correction method in c) 4), the arsenic (As) in the analytical sample
can also be corrected by obtaining the arsenic content in the blank test solution.
Comment 4 Triplicates additive recovery testing was conducted to evaluate trueness using fluid
industrial sludge fertilizers (2 samples) and composted sludge fertilizers (6 samples).
As a result, the mean recovery at the concentration level of 1 mg/kg - 9 mg/kg, 0.1
mg/kg - 0.9 mg/kg and 0.02 mg /kg - 0.04 mg /kg are 85.0 % - 105.9 %, 90.6 % 108.5 % and 95 % as arsenic (As) in an actual article respectively.
The results of the repeatability tests on different days using two kinds of fluid sludge
fertilizers to evaluate precision were analyzed by the one-way analysis of variance.
Table 1 shows the calculation results of intermediate precision and repeatability.。
Additionally, the minimum limit of quantification of this test method is about 3 µg
/kg.

395

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the repeatability tests on different days
Name of

Test days of

Repeatability
2)

samplerepeatability Average
1)

Intermediate precision

3)
sr

4)
RSD r

s I(T)

5)

RSD I(T)6)(mg/kg)(mg/kg)(%)(mg/kg)(%)

Composted sludge fertilizer 1

T
5

1.43

0.07

4.6

0.07

4.8

Composted sludge fertilizer 2

5

0.341

0.017

5.0

0.017

5.0

1） The number of test days conducting a duplicate test

5） Intermediate standard deviation

2） Average （the number of test days （T ）6） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Repeatability standard deviation
4） Repeatability relative standard deviation

Comment 5 Simultaneous measurement of multiple elements by an ICP-MS is available. In thiscase, transfer a pre-determined amount of cadmium standard solution (Cd 0.1 mg/mL,
1 mg/mL or 10 mg/mL), lead standard solution (Pb 0.1 mg/mL, 1 mg/mL or 10
mg/mL), nickel standard solution (Ni 0.1 mg/mL, 1 mg/mL or 10 mg/mL), chromium
standard solution (Cr 0.1 mg/mL, 1 mg/mL or 10 mg/mL) and arsenic standard
solution (As 0.1 mg/mL, 1 mg/mL or 10 mg/mL) traceable to National Metrology toa volumetric flask to mix, dilute with nitric acid (1+19) to prepare a mixed standard
solution (Cd 0.5 µg/mL, Pb 5 µg/mL, Ni 5 µg/mL, Cr 5 µg/mL, As 5 µg/mL) (1)(2)(3).
Transfer 0.02 mL - 2 mL of mixed standard solutions to 100-mL volumetric flasks (9)step-by-step, add 10 mL of rhodium standard solution (Rh 0.1 µg/mL) and rhenium
standard solution (Re 0.1 µg/mL) (5) respectively as internal standard and add nitricacid (1+19) up to the marked line to prepare mixed standard solutions for calibration
curve preparation (1)(2)(3) within the concentration range in Table 2. Conduct
procedures similarly as (4.2) b) - c) under the measurement conditions in Table 2 andcalculate the respective element concentrations in the analytical samples.
Table 2

Preparation concentration and monitor ion of mixed standard solution

for calibration curve preparation
Materials (element)Internal standard

subjected to measurement

materials (element)

Concentration ofelement(ng/mL)

Monitor ion(m /z )

Arsenic

As 1 - 100

Cadmium

Cd 0.1 - 10

Name of test item

NickelNi 1 - 100
Chromium
Cr 1 - 100
Lead
Pb 1 - 100
1） Rh: Rhodium, Re: Rhenium(1)

Monitor ion(m /z )

75

Concentrationof element(ng/mL)Rh 10

111, 114

Rh 10

103

60, 5853, 52, 50
208, 206, 207

Rh 10
Rh 10
Re 10

103
103
187

396

103

Testing Methods for Fertilizers (2018)

Comment 6 The presence and the extent of interference to the measurement mass number of anelement subjected to measurement and an internal standard element can be estimatedby qualitative analysis using an ICP-MS in advance to quantitation. Select ameasurement mass number considering the extent of interference. However, a mass
number cannot be changed in the measurement of arsenic. A magnetic sector
double-focusing mass spectrometer or a collision reaction cell specified in JIS K0133
can be used as a method to reduce spectrum interference.
References1)
Toshiharu YAGI: Determination of Heavy Metals in Fluid Sludge Fertilizers by ICP-MSand CV-AAS, Research Report of Fertilizer Vol. 8, p. 26 - 37 (2015)(5)

Flow sheet for arsenic: The flow sheet for arsenic in fluid sludge fertilizers is shown below:
20.0 g analytical sample Airtight vessel← Nitric acid, 2.5 mL← Hydrogen peroxide, 2 mL
Microwave
decomposition
Transfer

50-mL volumetric flask, water← Water (up to the marked line)
Ground-in stopper centrifugal precipitate tube,
Centrifugal separation
Anout 1700 × g , 5 minutes
Sample solution
Figure 1 Flow sheet for arsenic in fluid sludge fertilizers(Extraction procedure)

Sample solution
50-mL volumetric flask
Aliquot(predetermined volume)← Rhodium standard solution (Rh 0.1µg/mL) 5 mL (internal standard)← Nitric acid (1+19) (up to the marked line)
Measurement
ICP-MS (As: m /z 75, Rh: m /z 103)
Figure 2 Flow sheet for arsenic in fluid sludge fertilizers(Measurement procedure)

397

Testing Methods for Fertilizers (2018)

5.3 Cadmium
5.3.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 5.3.a-2017 or Cd.a-1.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3), spray into anacetylene-air flame, and measure the atomic absorption with cadmium at a wavelength of 228.8 nm
to obtain cadmium (Cd) in an analytical sample. In addition, the performance of this testing method
is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Cadmium standard solution (Cd 0.1 mg/mL): A cadmium standard solution (Cd 0.1
mg/mL) traceable to National Metrology.
e) Cadmium standard solution (Cd 10 µg/mL): Transfer 10 mL of cadmium standard solution(Cd 0.1 mg/mL) to a 100-mL volumetric flask, and add hydrochloric acid (1+23) up to themarked line.
f) Cadmium standard solutions (Cd 0.05 µg - 0.5 µg/mL) for the calibration curve
preparation (1) (2): Transfer 2.5 mL - 25 mL of cadmium standard solution (Cd 0.1 mg/mL) to
500-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
g) Blank test solution for the calibration curve preparation (1) (2): Hydrochloric acid (1+23)
used in the procedures in e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
Comment 1 Instead of the cadmium standard solution in (2), a cadmium standard solution for thecalibration curve preparation can be prepared by using a cadmium standard solution(Cd 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (3) function.
1) Light source: A cadmium hollow cathode lamp (In case of background correction systemusing continuous spectrum source, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C .
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
Note (3) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure398

Testing Methods for Fertilizers (2018)(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (4).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (6) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (4) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 450 °C in 1 to 2 hours.
(5) The watch glass can be removed.
(6) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers not containing
organic matters.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 228.8 nmb) Calibration curve preparation
1) Spray the cadmium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 228.8 nm.
2) Prepare a curve for the relationship between the cadmium concentration and the indicatedvalue of the cadmium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Subject the sample solution (7) to the same procedure as in b) 1) to read the indicated value.
2) Subject the blank test solution to the same procedure as in b) 1) to read the indicated value,and correct the indicated value obtained for the sample solution.
3) Obtain the cadmium content from the calibration curve, and calculate cadmium (Cd) in theanalytical sample.
Note (7) If there is a possibility that the cadmium concentration in the sample solution will399

Testing Methods for Fertilizers (2018)exceed the maximum limit of the calibration curve, dilute a predetermined amountwith hydrochloric acid (1+23).
Comment 4 Instead of the correction method in c) 2), the cadmium (Cd) in the analytical sample
can also be corrected by obtaining the cadmium content in the blank test solution.
Comment 5 Recovery testing was conducted using industrial sludge fertilizer and composted
sludge fertilizer (5 samples); as a result, the recovery at the concentration level of 5
mg/kg and 0.5 mg/kg were 97.5 % - 99.2 % and 96.7 % - 99.7 %, respectively.
Table 1 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification of this test method is about 0.1
mg/kg.
Table 1 Results and analysis results from a collaborative study
for cadmium test method validation
3)
4)
2)
Number of
RSD r
RSD R
Mean
Sample name1)
(mg/kg)(%)(%)
laboratories
Sewage sludge
10
1.50
5.5
6.4
fertilizer a
Sewage sludge
10
3.35
1.2
4.2
fertilizer b
Composted sludge
10
1.96
1.0
4.4
fertilizer a
Composted sludge
11
3.81
1.9
3.2
fertilizer b
Composted sludge
10
1.80
3.5
4.9
fertilizer c1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

References
1)
Yoshinari SAKAKIBARA, Manabu MATSUZAKI and Tadao AMANO: Determinationof Cadmium, Lead, Nickel and Chromium in Sludge Fertilizer - Improved
Decomposition Method - Research Report of Fertilizer Vol.1, p. 41 - 49 (2008)
2)
Yoshinari SAKAKIBARA and Manabu MATSUZAKI: Determination of Cadmium,
Lead, Nickel and Chromium in Sludge Fertilizer - Collaborative Test Results - Research
Report of Fertilizer Vol.1, p. 50 - 59 (2008)
3)
Hisanori ARAYA and Yoshimi TAKEBA: Validation of atomic absorption spectrometryfor determination of cadmium, lead, nickel and chromium in calcined sludge fertilizer Research Report of Fertilizer Vol.3, p. 30 - 42 (2010)

400

Testing Methods for Fertilizers (2018)(5)

Flow sheet for cadmium: The flow sheet for cadmium in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature
About
25
mL
- 50 mL of hydrochloric acid (1+5)←
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Measurement

Atomic absorption spectrometer (228.8 nm)

Figure Flow sheet for cadmium in fertilizers

401

Testing Methods for Fertilizers (2018)

5.3.b ICP Optical Emission Spectrometry(1) Summary
The test method is applicable to sludge fertilizers, etc. This testing method is classified as Type D
and its symbol is 5.3.b-2017 or Cd.b-1.
Pretreat an analytical sample with incineration, nitric acid - hydrochloric acid (1+3), introduce it to
ICP Optical Emission Spectrometry (“ICP-OES”) and measure the emission with cadmium at awavelength of 228.802 nm to quantify the cadmium (Cd) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 6.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Cadmium standard solution (Cd 0.1 mg/mL): A cadmium standard solution (Cd 0.1
mg/mL) traceable to National Metrology.
e) Cadmium standard solution (Cd 0.25µg/mL) (1) (2): Dilute a predetermined amount of
cadmium standard solution (Cd 0.1 mg/mL) with hydrochloric acid (1+23) to prepare a
cadmium standard solution (Cd 0.25 µg/mL)
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
Comment 1 Instead of the cadmium standard solution in (2), a cadmium standard solution for thecalibration curve preparation can be prepared by using a cadmium standard solution(Cd 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purity.
b) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (3).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (3).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (4), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (5) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
402

Testing Methods for Fertilizers (2018)i)

As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.

Note (3) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 450 °C in 1 to 2 hours.
(4) The watch glass can be removed.
(5) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers not containing
organic matters.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K 0116
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Optical Emission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 228.802 nmb) Calibration curve preparation and sample measurement
1) Put 5mL of sample solution to three 10-mL volumetric flasks respectively.
2) Add 2mL and 4 mL of cadmium standard solution (0.25 μg/mL) to volumetric flasks of 1)
above, and further add hydrochloric acid (1+23) to the marked line to make a sample solutionof Standard Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flask of 1)
above to make a sample solution without a standard solution.
4) Spray the sample solution of Standard Addition Method and the sample solution without astandard solution into the induction plasma, and read the indicated value at a wavelength of
228.802 nm.
5) Transfer 5 mL of blank test solution to a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value obtained
from the respective sample solutions.
6) Prepare a curve for the relationship between the added cadmium concentration and thecorrected indicated value of the sample solution for Standard Addition Method and sample
solution without a standard solution.
7) Obtain the cadmium content from the intercept of the calibration curve to calculate cadmium(Cd) in the analytical sample
Comment 4 Instead of the correction method in c) 5), the cadmium (Cd) in the analytical sample
can also be corrected by obtaining the cadmium content in the blank test solution.
Comment 5 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.9.1.b Comment 5.
Comment 6 The comparison of the measurement value (xi：0.003 mg/kg - 3.32 mg/kg) of ICP
Optical Emission Spectrometry and the measurement value (yi) of Flame Atomic
Absorption Spectrometry was conducted to evaluate trueness using sludge fertilizers(49 samples). As a result, a regression equation was y = − 0.03 + 1.009x and its403

Testing Methods for Fertilizers (2018)

correlation coefficient (r) was 0.996. Triplicates measurement for each one sample ofsewage sludge fertilizer, human waste sludge fertilizer, industrial sludge fertilizer,
mixed sludge fertilizer, calcined sludge fertilizers and composted sludge fertilizer
was conducted. As a result, a repeatability obtained was 0.8 % - 4.1 % as a relative
standard deviation.
Additionally, the minimum limit of quantification of this testing method is 0.2
mg/kg.
References
1)
Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI and Tetuya NOMURA:
Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer, Vol.4, p. 30 - 35 (2011)(5)

Flow sheet for cadmium: The flow sheet for cadmium in fertilizers is shown below:

5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for cadmium in sludge fertilizers(Extraction procedure)404

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0, 2 and 4 mL of cadmium standard solution (0.25 μg / mL)← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Optical Emission Spectrometer (228.802 nm)
Figure 2 Flow sheet for cadmium in sludge fertilizers(Measurement procedure)

405

Testing Methods for Fertilizers (2018)

5.3.c ICP Mass Spectrometry (Fluid sludge fertilizers)(1) Summary
The test method is applicable to fluid sludge fertilizers. This testing method is classified as Type D
and its symbol is 5.3.c-2017 or Cd.c-1.
Add nitric acid – hydrogen peroxide to an analytical sample to heat and extract by microwave
irradiation. After adding an internal standard element, introduce it to an ICP Mass Spectrometer(ICP-MS) and measure the respective indicated values of cadmium and an internal standard elementwith mass/ charge number (m/z) and obtain cadmium (Cd) in the analytical sample from the ratio of
the indicated value for cadmium and the indicated value for an internal standard element. In
addition, the performance of this testing method is shown in Comment 4.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A4 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Nitric acid: Nitric acid used to dilute a standard solution and a sample solution is a highly
pure reagent specified in JIS K 9901.
d) Hydrogen peroxide: A JIS Guaranteed Reagent specified in JIS K 8230 or a reagent ofequivalent quality.
e) Rhodium standard solution (Rh 1 mg/mL): A rhodium standard solution (Rh 1 mg/mL)
traceable to National Metrology.
f) Rhodium standard solution (Rh 0.1 µg/mL) (1)(2)(3): Dilute a predetermined amount ofrhodium standard solution (Rh 1 mg/mL) with nitric acid (1+19) to prepare a rhodium
standard solution (Rh 0.1 µg/mL).
g) Rhenium standard solution (Re 1 mg/mL) (4): A rhenium standard solution (Re 1 mg/mL)
traceable to National Metrology.
h) Rhenium standard solution (Re 0.1 µg/mL) (1)(2)(3)(4): Dilute a predetermined amount ofrhenium standard solution (Re 1 mg/mL) with nitric acid (1+19) to prepare a rhenium
standard solution (Re 0.1 µg/mL).
i) Cadmium standard solution (Cd 0.1 mg/mL): A cadmium standard solution (Cd 0.1
mg/mL) traceable to National Metrology.
j) Cadmium standard solution (Cd 0.5 µg/mL) (1)(2)(3): Dilute a predetermined amount of
cadmium standard solution (Cd 1 mg/mL) with nitric acid (1+19) to prepare a cadmium
standard solution (Cd 0.5 µg/mL).
k) Cadmium standard solutions (Cd 0.1 ng/ mL - 100 ng/ mL) for the calibration curve
preparation (1)(2)(3): Transfer 0.02 mL - 2 mL of cadmium standard solution (Cd 0.5 µg/mL) to
100-mL volumetric flasks step-by-step, and add 10 mL of rhodium standard solutions (Rh 0.1µg/mL) as internal standard respectively (5) and add nitric acid (1+19) up to the marked line.
l) Blank test solution for the calibration curve preparation (1)(2)(3): transfer 10 mL of rhodium
standard solution (Rh 0.1 µg/mL) to 100-mL volumetric flasks as internal standard (5) and add
nitric acid (1+19) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in cool and dark place, and do not use after 1 month after preparation.
(3) For storage, use a sealable container made of materials such as polypropylene
containing no cadmium.
(4) Use when measuring lead simultaneously.
(5) Add an internal solution that is 1/10 of the volume to be prepared.
Comment 1 Instead of the cadmium standard solution in (2), a cadmium standard solution for thecalibration curve preparation can be prepared by using a cadmium standard solution
406

Testing Methods for Fertilizers (2018)(Cd 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) ICP Mass Spectrometer: High-frequency plasma mass spectrometer specified in JIS K 0133.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.995 % in purity.
b) Pressure vessel decomposing device: A device which pressurizes the inside of a vessel by
putting acid, etc.to heat in the airtight vessel, and decomposes a sample by the interaction ofheating, pressurizing and acid. The following requirements should be met.
1) The main part of a decomposing device: In the case of the microwave heating method, adevice should be able to produce a high-frequency wave using a frequency which is permitted
at an industrial frequency facility. It is desirable to be able to monitor pressure andtemperature, etc. in the airtight vessel with a sensor inside the device. The interior of a device
should be acid-resistant treated and should have a high temperature durability and a high levelof safety.
2) Exhaust system: A system which has an exhaust fan of acid-resistant specification, and has
an air-cool function inside the device to allow constant airflow, keeping operating temperature
below a certain temperature level.
3) Airtight vessel: A vessel which is heat-resistance and pressure-tight and has the durability
required to decompose particles, and has resistance to internal contamination. It should havesafety functions such as causing overheat prevention valve to work and internal pressure todrop by emitting gas, and preventing gas bumping in the case of exceeding a pressure
limitation.
c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 20.0 g (6) of an analytical sample, and put it in a 100-mL volumetric flask.
b) Gradually add 2.5 mL of nitric acid and 2 mL of hydrogen peroxide.
c) Put the airtight vessel into the main part of a decomposing device and heat using a microwave(7).
d) Ignite at 240 ºC ± 5 ºC for no less than 10 minutes (7) to decompose (8).
e) After standing to cool, transfer the solution to a 50-mL volumetric flask (9) with water.
f) Add water up to the marked line and transfer it to a 50-mL ground-in stopper centrifugal
precipitate tube (9).
g) Centrifuge it at 1700 × g centrifugal force for about five minutes (10) and use the supernatantas the sample solution.
h) As a blank test, conduct the procedures in b) - g) using another airtight vessel to prepare theblank test solution.
Note (6) The maximum limit of solid content, which is converted from moisture content, in thesampling volume 20.0 g of an analytical sample is about 0.5 g. If solid content is likely
to exceed the limit, reduce sampling volume as necessary.
(7) Condition examples for a microwave decomposing device: 0 min (room temperature)→ 10min (240 ºC) → 20 min (240 ºC) → 40 min (room temperature), initial output
1400 W(8) When organic matters still remain, for example the digestion solution is colored,
repeat the procedures in (4.1) b) - d).
(9) The vessel should be made of polypropylene, etc. to not affect the measurement.
(10) 16.5-cm of radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
407

Testing Methods for Fertilizers (2018)

Comment 2

The procedure in (4.1) is the same as the procedure in (4.1) of 5.1.b.

(4.2) Measurement: Conduct measurement (Internal Standard Method) according to JIS K 0113
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Mass Spectrometer used in measurement.
a) Measurement conditions for the ICP Mass Spectrometer: Set up the measurement
conditions for the ICP Mass Spectrometer considering the following:
Cadmium: monitor ion (m/z): 111, 114
Rhodium: monitor ion (m/z): 103b) Calibration curve preparation
1) Spray cadmium standard solution for the calibration curve preparation and blank test solution
for the calibration curve preparation into the inductively coupled plasma, and read the ratio of
the respective ion count numbers for the monitor ion of an element subjected to measurement
and an internal standard element.
2) Prepare a curve for the relationship between the concentration and the ratio of the ion countnumber of an element subjected to measurement.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.005 µg - 0.5 µg
as Cd) to a 50-mL volumetric flask (9).
2) Add 5mL of internal standard solution (5) and add nitric acid (1+19) to the marked line.
3) Conduct procedures similarly as in b) 1) to read the ratio of the ion count numbers.
4) For blank test solution, conduct procedures similarly as in 1) - 3) to correct the ratio of the ion
count number obtained for the sample solution.
5) Obtain the cadmium content from the calibration curve, and calculate cadmium (Cd) in theanalytical sample.
Comment 3 Instead of the correction method in c) 4), the cadmium (Cd) in the analytical sample
can also be corrected by obtaining the cadmium content in the blank test solution.
Comment 4 Triplicates additive recovery testing was conducted to evaluate trueness using fluid
industrial sludge fertilizers (2 samples), and composted sludge fertilizers (6 samples).
As a result, the mean recovery at the concentration level of 0.1 mg/kg - 0.9 mg/kg,
0.01 mg/kg - 0.09 mg/kg and 2 µg /kg - 4 µg /kg are 89.4 % - 108.5 %, 91.0 % 112.0 % and 96.3 % - 108.5 % as cadmium (Cd) in an actual article respectively.
The results of the repeatability tests on different days using two kinds of fluid sludge
fertilizers to evaluate precision were analyzed by the one-way analysis of variance.
Table 1 shows the calculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this test method is about 2 µg
/kg.

408

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the repeatability tests on different days
Name ofsampleTest days of

Repeatability
2)

repeatability Average
1)

sr

3)

RSD r

Intermediate precision
4)s I(T)

5)

RSD I(T)(mg/kg)(mg/kg)(%)(mg/kg)(%)

Composted sludge fertilizer 1

T
5

0.139

0.007

5.1

0.010

7.0

Composted sludge fertilizer 2

50.03600.0011

3.2

0.0020

5.6

6)1） The number of test days conducting a duplicate test

5） Intermediate standard deviation

2） Average （the number of test days （T ）6） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Repeatability standard deviation
4） Repeatability relative standard deviation

Comment 5 Simultaneous measurement of multiple elements by an ICP-MS is available. In thatcase, see 5.2.c Comment 5.
Comment 6 The presence and the extent of interference to the measurement mass number of anelement subjected to measurement and an internal standard element can be estimatedby qualitative analysis using an ICP-MS in advance to quantitation. Select ameasurement mass number considering the extent of interference. However, a mass
number cannot be changed in the measurement of arsenic. A magnetic sector
double-focusing mass spectrometer or a collision reaction cell specified in JIS K0133
can be used as a method to reduce spectrum interference.
References1)
Toshiharu YAGI: Determination of Heavy Metals in Fluid Sludge Fertilizers by ICP-MSand CV-AAS, Research Report of Fertilizer Vol. 8, p. 26 - 37 (2015)(5)

Flow sheet for cadmium: The flow sheet for cadmium in fluid sludge fertilizers is shown
below:
20.0 g analytical sample Airtight vessel← Nitric acid, 2.5 mL← Hydrogen peroxide, 2 mL
Microwave
decomposition
Transfer

50-mL volumetric flask, water← Water (up to the marked line)
Ground-in stopper centrifugal precipitate tube,
Centrifugal separation
Anout 1700 × g , 5 minutes
Sample solution
Figure 1 Flow sheet for cadmium in liquid sludge fertilizers(Extraction procedure)409

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot

50-mL volumetric flask(predetermined volume)← Rhodium standard solution (Rh 0.1µg/mL) 5 mL (internal standard)← Nitric acid (1+19) (up to the marked line)
Measurement

ICP-MS (Cd: m /z 111,114 Rh: m /z 103)

Figure 2 Flow sheet for cadmium in liquid sludge fertilizers(Measurement procedure)410

Testing Methods for Fertilizers (2018)

5.3.d ICP Mass Spectrometry(1) Summary
The testing method is applicable to sludge fertilizers excluding fluid sludge fertilizers. This testing
method is classified as Type D and its symbol is 5.3.d-2017 or Cd.d-1.
Add nitric acid–hydrogen peroxide to an analytical sample to heat and extract by microwave
irradiation. After adding an internal standard element, introduce it to an ICP Mass Spectrometer(ICP-MS) and measure the respective indicated values of cadmium and an internal standard elementwith mass/ charge number (m/z) and obtain cadmium (Cd) in the analytical sample from the ratio of
the indicated value for cadmium and the indicated value for an internal standard element. In
addition, the performance of this testing method is shown in Comment 3.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A4 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Nitric acid: Nitric acid used to dilute a standard solution and a sample solution is a highly
pure reagent specified in JIS K 9901.
d) Hydrogen peroxide: A JIS Guaranteed Reagent specified in JIS K 8230 or a reagent ofequivalent quality.
e) Rhodium standard solution (Rh 1 mg/mL): A rhodium standard solution (Rh 1 mg/mL)
traceable to National Metrology.
f) Rhodium standard solution (Rh 0.1 µg/mL) (1)(2)(3): Dilute a predetermined amount ofrhodium standard solution (Rh 1 mg/mL) with nitric acid (1+19) to prepare a rhodium
standard solution (Rh 0.1 µg/mL).
g) Cadmium standard solution (Cd 0.1 mg/mL): A cadmium standard solution (Cd 0.1
mg/mL) traceable to National Metrology.
h) Cadmium standard solution (Cd 0.5 µg/mL) (1)(2)(3): Dilute a predetermined amount of
cadmium standard solution (Cd 1 mg/mL) with nitric acid (1+19) to prepare a cadmium
standard solution (Cd 0.5 µg/mL).
i) Cadmium standard solutions (Cd 0.1 ng/ mL - 100 ng/ mL) for the calibration curve
preparation (1)(2)(3): Transfer 0.02 mL - 2 mL of cadmium standard solution (Cd 0.5 µg/mL) to
100-mL volumetric flasks step-by-step, and add 10 mL of rhodium standard solutions (Rh 0.1µg/mL) as internal standard respectively (4) and add nitric acid (1+19) up to the marked line.
j) Blank test solution for the calibration curve preparation (1)(2)(3): transfer 10 mL of rhodium
standard solution (Rh 0.1 µg/mL) to 100-mL volumetric flasks as internal standard (4) and add
nitric acid (1+19) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in cool and dark place, and do not use after 1 month after preparation.
(3) For storage, use a sealable container made of materials such as polypropylene
containing no cadmium.
(4) Add an internal solution that is 1/10 of the volume to be prepared.
Comment 1 Instead of the cadmium standard solution in (2), a cadmium standard solution for thecalibration curve preparation can be prepared by using a cadmium standard solution(Cd 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) ICP Mass Spectrometer: High-frequency plasma mass spectrometer specified in JIS K 0133.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.995 % in purityb) Pressure vessel decomposing device: A device which pressurizes the inside of a vessel by411

Testing Methods for Fertilizers (2018)

putting acid, etc.to heat in the airtight vessel, and decomposes a sample by the interaction ofheating, pressurizing and acid. The following requirements should be met.
1) The main part of a decomposing device: In the case of the microwave heating method, adevice should be able to produce a high-frequency wave using a frequency which is permitted
at an industrial frequency facility. It is desirable to be able to monitor pressure andtemperature, etc. in the airtight vessel with a sensor inside the device. The interior of a device
should be acid-resistant treated and should have a high temperature durability and a high levelof safety.
2) Exhaust system: A system which has an exhaust fan of acid-resistant specification, and has
an air-cool function inside the device to allow constant airflow, keeping operating temperature
below a certain temperature level.
3) Airtight vessel: A vessel which is heat-resistance and pressure-tight and has the durability
required to decompose particles, and has resistance to internal contamination. It should havesafety functions such as causing overheat prevention valve to work and internal pressure todrop by emitting gas, and preventing gas bumping in the case of exceeding a pressure
limitation.
c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 0.20 g of an analytical sample, and put it into an airtight vessel.
b) Gradually add 2.5 mL of nitric acid and 2 mL of hydrogen peroxide (5).
c) Put the airtight vessel into the main part of a decomposing device and heat using a microwave(6).
d) Ignite at 240 ºC ± 5 ºC for no less than 10 minutes (6) to decompose (7).
e) After standing to cool, transfer the solution to a 50-mL volumetric flask (8) with water.
f) Add water up to the marked line and transfer it to a 50-mL ground-in stopper centrifugal
precipitate tube (8).
g) Centrifuge it at 1700 × g centrifugal force for about five minutes (9) and use the supernatant as
the sample solution.
h) As a blank test, conduct the procedures in b) - g) using another airtight vessel to prepare theblank test solution.
Note (5) Leaving at rest overnight is preferable.
(6) Condition examples for a microwave decomposing device: 0 min (room temperature)→ 10min (240 ºC) → 20 min (240 ºC) → 40 min (room temperature), initial output
1400 W(7) When organic matters still remain, for example the digestion solution is colored,
repeat the procedures in (4.1) b) - d).
(8) The vessel should be made of polypropylene, etc. to not affect the measurement.
(9) 16.5-cm of radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(4.2) Measurement: Conduct measurement (Internal Standard Method) according to JIS K 0113
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Mass Spectrometer used in measurement.
a) Measurement conditions for the ICP Mass Spectrometer: Set up the measurement
conditions for the ICP Mass Spectrometer considering the following:
Cadmium: monitor ion (m/z): 111
Rhodium: monitor ion (m/z): 103412

Testing Methods for Fertilizers (2018)b) Calibration curve preparation
1) Spray cadmium standard solution for the calibration curve preparation and blank test solution
for the calibration curve preparation into the inductively coupled plasma, and read the ratio of
the respective ion count numbers for the monitor ion of an element subjected to measurement
and an internal standard element.
2) Prepare a curve for the relationship between the concentration and the ratio of the ion countnumber of an element subjected to measurement.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.005 µg - 0.5 µg
as Cd) to a 50-mL volumetric flask (8).
2) Add 5mL of internal standard solution (4) and add nitric acid (1+19) to the marked line.
3) Conduct procedures similarly as in b) 1) to read the ratio of the ion count number.
4) For blank test solution, conduct procedures similarly as in 1) - 3) to correct the ratio of the ion
count number obtained for the sample solution.
5) Obtain the cadmium content from the calibration curve, and calculate cadmium (Cd) in theanalytical sample.
Comment 2 Instead of the correction method in c) 4), the cadmium (Cd) in the analytical sample
can also be corrected by obtaining the cadmium content in the blank test solution.
Comment 3 The comparison of the measurement value (xi：0.18 mg/kg - 3.02 mg/kg) of ICP-MSand the measurement value (yi) of Flame Atomic Absorption Spectrometry was
conducted to evaluate trueness using sludge fertilizers (26 samples). As a result, a
regression equation was y = 0.0402 + 1.01x and its correlation coefficient (r) was
0.997.
The results of the repeatability tests on different days using two kinds of sludge
fertilizers to evaluate precision were analyzed by the one-way analysis of variance.
Table 1 shows the calculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this test method is about 0.1
mg/kg.
Table 1 Analysis results of the repeatability tests on different days
Name ofsampleTest days of

Repeatability
2)

repeatability AveragesrIntermediate precision

3)

4)
RSD r

s I(T)

5)

RSD I(T)1)(mg/kg)(mg/kg)(%)(mg/kg)(%)

Composted sludge fertilizer 1

5

0.263

0.022

8.5

0.031

11.7

Composted sludge fertilizer 2

5

2.39

0.04

1.6

0.10

4.1

T

6)

1） The number of test days conducting a duplicate test

5） Intermediate standard deviation

2） Average （the number of test days（T ）6） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Repeatability standard deviation
4） Repeatability relative standard deviation

Comment 4 The presence and the extent of interference to the measurement mass number of anelement subjected to measurement and an internal standard element can be estimatedby qualitative analysis using an ICP-MS in advance to quantitation. Select ameasurement mass number considering the extent of interference. A magnetic sector
double-focusing mass spectrometer or a collision reaction cell specified in JIS K0133
413

Testing Methods for Fertilizers (2018)

can be used as a method to reduce spectrum interference.
References1)
Toshiharu YAGI, Kenta SAKUMA, Yoshimi HASHIMOTO: Determination of Heavy
Metals in Sludge Fertilizers using Inductively Coupled Plasma-Mass Spectrometer(ICP-MS), Research Report of Fertilizer Vol. 9, p. 21 - 32 (2016)(5)

Flow sheet for cadmium: The flow sheet for cadmium in splid sludge fertilizers is shown
below:
0.20 g analytical sample Airtight vessel← Nitric acid, 2.5 mL← Hydrogen peroxide, 2 mL
Microwave
decomposition
Transfer

50-mL volumetric flask, water← Water (up to the marked line)
Ground-in stopper centrifugal precipitate tube,
Centrifugal separation
Anout 1700 × g , 5 minutes
Sample solution
Figure 1 Flow sheet for cadmium in solid sludge fertilizers(Extraction procedure)

Sample solution
50-mL volumetric flask
Aliquot(predetermined volume)← Rhodium standard solution (Rh 0.1µg/mL) 5 mL (internal standard)← Nitric acid (1+19) (up to the marked line)
Measurement

ICP-MS (Cd: m /z 111 Rh: m /z 103)

Figure 2 Flow sheet for cadmium in solid sludge fertilizers(Measurement procedure)

414

Testing Methods for Fertilizers (2018)

5.4 Nickel
5.4.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 5.4.a-2017 or Ni.a-1.
Pretreat an analytical sample with incineration and nitric acid−hydrochloric acid (1+3), spray into
an acetylene−air flame, and measure the atomic absorption with nickel at a wavelength of 232.0 nm
to obtain nickel (Ni) in an analytical sample. In addition, the performance of this testing method isshown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Nickel standard solution (Ni 0.1 mg/mL): A nickel standard solution (Ni 0.1 mg/mL)
traceable to National Metrology.
e) Nickel standard solutions (Ni 0.5 µg/mL - 5 µg/mL) for the calibration curve preparation(1) (2): Transfer 2.5 mL - 25 mL of nickel standard solution (Ni 0.1 mg/mL) to 500-mL
volumetric flasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Blank test solution for the calibration curve preparation (1) (2): Hydrochloric acid (1+23)
used in the procedures in e).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
Comment 1 Instead of the nickel standard solution in (2), a nickel standard solution for thecalibration curve preparation can be prepared by using a nickel standard solution (Ni1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (3) function.
1) Light source: A nickel hollow cathode lamp (In case of background correction system using
continuous spectrum source, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C .
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
Note (3) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (4).
415

Testing Methods for Fertilizers (2018)c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (6) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (4) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 450 °C in 1 to 2 hours.
(5) The watch glass can be removed.
(6) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers not containing
organic matters.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 232.0 nmb) Calibration curve preparation
1) Spray the nickel standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 232.0 nm.
2) Prepare a curve for the relationship between the nickel concentration and the indicated valueof the nickel standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Subject the sample solution (7) to the same procedure as in b) 1) to read the indicated value.
2) Subject the blank test solution to the same procedure as in b) 1) to read the indicated value,and correct the indicated value obtained for the sample solution.
3) Obtain the nickel content from the calibration curve, and calculate nickel (Ni) in the analyticalsample.
Note (7) If there is a possibility that the nickel concentration in the sample solution will exceed
the maximum limit of the calibration curve, dilute a predetermined amount withhydrochloric acid (1+23).
416

Testing Methods for Fertilizers (2018)

Comment 4 Instead of the correction method in c) 2), the nickel (Ni) in the analytical sample canalso be corrected by obtaining the nickel content in the blank test solution.
Comment 5 Recovery testing was conducted using industrial sludge fertilizer and composted
sludge fertilizer (5 samples); as a result, the recovery at the concentration level of
300 mg/kg and 30 mg/kg were 98.5 % - 100.3 % and 97.1 % - 99.9 %, respectively.
Table 1 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification of this test method is about 1
mg/kg.
Table 1 Results and analysis results from a collaborative study
for nickel test method validation
3)
4)
2)
Number of
RSD r
RSD R
Mean
Sample name1)
(mg/kg)(%)(%)
laboratories
Sewage sludge
11
56.9
1.1
4.6
fertilizer a
Sewage sludge
11
21.8
2.2
3.9
fertilizer b
Composted sludge
11
28.9
1.3
6.4
fertilizer a
Composted sludge
11
28.5
1.8
4.4
fertilizer b
Composted sludge
12
58.3
1.6
4.4
fertilizer c1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

References
1)
Yoshinari SAKAKIBARA, Manabu MATSUZAKI and Tadao AMANO: Determinationof Cadmium, Lead, Nickel and Chromium in Sludge Fertilizer - Improved
Decomposition Method - Research Report of Fertilizer Vol.1, p. 41 - 49 (2008)
2)
Yoshinari SAKAKIBARA and Manabu MATSUZAKI: Determination of Cadmium,
Lead, Nickel and Chromium in Sludge Fertilizer - Collaborative Test Results - Research
Report of Fertilizer Vol.1, p. 50 - 59 (2008)
3)
Hisanori ARAYA and Yoshimi TAKEBA: Validation of atomic absorption spectrometryfor determination of cadmium, lead, nickel and chromium in calcined sludge fertilizer Research Report of Fertilizer Vol.3, p. 30 - 42 (2010)

417

Testing Methods for Fertilizers (2018)(5)

Flow sheet for nickel: The flow sheet for nickel in fertilizers is shown below:

5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← About 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Measurement

Atomic absorption spectrometer (232.0 nm)

Figure Flow sheet for nickel in fertilizers

418

Testing Methods for Fertilizers (2018)

5.4.b ICP Optical Emission Spectrometry(1) Summary
The test method is applicable to sludge fertilizers, etc. This testing method is classified as Type D
and its symbol is 5.4.b-2017 or Ni.b-1.
Pretreat an analytical sample with incineration and nitric acid−hydrochloric acid (1+3), and thenintroduce it to an ICP Optical Emission Spectrometer (“ICP-OES”) and measure the emission withnickel at a wavelength of 231.604 nm to obtain nickel (Ni) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Nickel standard solution (Ni 0.1 mg/mL): A nickel standard solution (Ni 0.1 mg/mL)
traceable to National Metrology.
e) Nickel standard solutions (Ni 2.5 µg/mL) (1) (2): Dilute a predetermined amount of nickel
standard solution (Ni 0.1 mg/mL) with hydrochloric acid (1+23) to prepare a nickel standard
solution (Ni 2.5 µg/mL)
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
Comment 1 Instead of the nickel standard solution in (2), a nickel standard solution for thecalibration curve preparation can be prepared by using a nickel standard solution (Ni1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C .
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (3).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (3).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (4), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (5) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
419

Testing Methods for Fertilizers (2018)i)

As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.

Note (3) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 450 °C in 1 to 2 hours.
(4) The watch glass can be removed.
(5) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 1 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers not containing
organic matters.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K 0116
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Optical Emission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 231.604 nmb) Calibration curve preparation and sample measurement
1) Put 5mL of sample solution to three 10-mL volumetric flasks respectively.
2) Add 2 mL and 4 mL (0.25 μg/mL) of nickel standard solution to volumetric flasks of 1) above,
then add hydrochloric acid (1+23) to the marked line to make a sample solution of Standard
Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flask of 1)
above to make a sample solution without a standard solution.
4) Spray the sample solution of Standard Addition Method and the sample solution without astandard solution into the induction plasma, and read the indicated value at a wavelength of
231.604 nm.
5) Transfer 5 mL of blank test solution to a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value obtained
from the respective sample solutions.
6) Prepare a curve for the relationship between the added nickel concentration and the correctedindicated value of the sample solution for Standard Addition Method and the sample solution
without a standard solution.
7) Obtain the nickel content from the intercept of the calibration curve to calculate nickel (Ni)concentration in the analytical sample.
Comment 3 Instead of the correction method in c) 5), the nickel (Ni) in the analytical sample canalso be corrected by obtaining the nickel content in the blank test solution.
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.9.1.b Comment 5.
Comment 5 The comparison of the measurement value (xi：8.4 mg/kg - 129 mg/kg) of ICP
Optical Emission Spectrometry and the measurement value (yi) of Flame Atomic
Absorption Spectrometry was conducted to evaluate trueness using sludge fertilizers(49 samples). As a result, a regression equation was y = − 0.96 + 1.010x and its
420

Testing Methods for Fertilizers (2018)

correlation coefficient (r) was 0.995. Triplicates measurement for each one sample ofsewage sludge fertilizer, human waste sludge fertilizer, industrial sludge fertilizer,
mixed sludge fertilizer, calcined sludge fertilizers and composted sludge fertilizer
was conducted. As a result, a repeatability obtained was1.0 % - 2.6 % as a relative
standard deviation.
Additionally, the minimum limit of quantification of this testing method is about 8
mg/kg.
References
1)
Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI and Tetuya NOMURA:
Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer, Vol.4, p. 30 - 35 (2011)(5)

Flow sheet for nickel: The flow sheet for nickel in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for nickel in sludge fertilizers (Extraction procedure)

421

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0, 2 and 4 mL of nickel standard solution (0.25 μg / mL)← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Optical Emission Spectrometer (231.604 nm)
Figure 2 Flow sheet for nickel in sludge fertilizers (Measurement procedure)

422

Testing Methods for Fertilizers (2018)

5.4.c ICP Mass Spectrometry (Fluid sludge fertilizers)(1) Summary
The test method is applicable to fluid sludge fertilizers. This testing method is classified as Type D
and its symbol is 5.4.c-2017 or Ni.c-1.
Add nitric acid−hydrogen peroxide to an analytical sample to heat and extract by microwave
irradiation. After adding an internal standard element, introduce it to an ICP Mass Spectrometer(ICP-MS) and measure the respective indicated values of nickel and an internal standard elementwith mass/ charge number (m/z) and obtain nickel (Ni) in the analytical sample from the ratio of theindicated value for nickel and the indicated value for an internal standard element. In addition, theperformance of this testing method is shown in Comment 4.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A4 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Nitric acid: Nitric acid used to dilute a standard solution and a sample solution is a highly
pure reagent specified in JIS K 9901.
d) Hydrogen peroxide: A JIS Guaranteed Reagent specified in JIS K 8230 or a reagent ofequivalent quality.
e) Rhodium standard solution (Rh 1 mg/mL): A rhodium standard solution (Rh 1 mg/mL)
traceable to National Metrology.
f) Rhodium standard solution (Rh 0.1 µg/mL) (1)(2)(3): Dilute a predetermined amount ofrhodium standard solution (Rh 1 mg/mL) with nitric acid (1+19) to prepare a rhodium
standard solution (Rh 0.1 µg/mL).
g) Rhenium standard solution (Re 1 mg/mL) (4): A rhenium standard solution (Re 1 mg/mL)
traceable to National Metrology.
h) Rhenium standard solution (Re 0.1 µg/mL) (1)(2)(3)(4): Dilute a predetermined amount ofrhenium standard solution (Re 1 mg/mL) with nitric acid (1+19) to prepare a rhenium
standard solution (Re 0.1 µg/mL).
i) Nickel standard solution (Ni 0.1 mg/mL): A nickel standard solution (Ni 0.1 mg/mL)
traceable to National Metrology.
j) Nickel standard solution (Ni 5 µg/mL) (1)(2)(3): Transfer 5 mL of nickel standard solution (Ni
0.1 mg/mL) to a 100-mL volumetric flask and add nitric acid (1+19) to the marked line.
k) Nickel standard solutions (Ni 1 ng/ mL - 100 ng/ mL) for the calibration curve
preparation (1)(2)(3): Transfer 0.02 mL - 2 mL of nickel standard solution (Ni 5 µg/mL) to
100-mL volumetric flasks step-by-step, and add 10 mL of rhodium standard solutions (Rh 0.1µg/mL) as internal standard respectively (5) and add nitric acid (1+19) up to the marked line.
l) Blank test solution for the calibration curve preparation (1)(2)(3): transfer 10 mL of rhodium
standard solution (Rh 0.1 µg/mL) to 100-mL volumetric flasks as internal standard (5) and add
nitric acid (1+19) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in cool and dark place, and do not use after 1 month after preparation.
(3) For storage, use a sealable container made of materials such as polypropylene
containing no nickel.
(4) Use when measuring lead simultaneously.
(5) Add an internal solution that is 1/10 of the volume to be prepared.
Comment 1 Instead of the nickel standard solution in (2), a nickel standard solution for thecalibration curve preparation can be prepared by using a nickel standard solution (Ni1 mg/mL or 10 mg/mL) traceable to National Metrology.
423

Testing Methods for Fertilizers (2018)(3) Instruments: Instruments are as shown below:a) ICP Mass Spectrometer: High-frequency plasma mass spectrometer specified in JIS K 0133.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.995 % in purityb) Pressure vessel decomposing device: A device which pressurizes the inside of a vessel by
putting acid, etc.to heat in the airtight vessel, and decomposes a sample by the interaction ofheating, pressurizing and acid. The following requirements should be met.
1) The main part of a decomposing device: In the case of the microwave heating method, adevice should be able to produce a high-frequency wave using a frequency which is permitted
at an industrial frequency facility. It is desirable to be able to monitor pressure andtemperature, etc. in the airtight vessel with a sensor inside the device. The interior of a device
should be acid-resistant treated and should have a high temperature durability and a high levelof safety.
2) Exhaust system: A system which has an exhaust fan of acid-resistant specification, and has
an air-cool function inside the device to allow constant airflow, keeping operating temperature
below a certain temperature level.
3) Airtight vessel: A vessel which is heat-resistance and pressure-tight and has the durability
required to decompose particles, and has resistance to internal contamination. It should havesafety functions such as causing overheat prevention valve to work and internal pressure todrop by emitting gas, and preventing gas bumping in the case of exceeding a pressure
limitation.
c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 20.0 g (6) of an analytical sample, and put it into an airtight vessel.
b) Gradually add 2.5 mL of nitric acid and 2 mL of hydrogen peroxide.
c) Put the airtight vessel into the main part of a decomposing device and heat using a microwave(7).
d) Ignite at 240 ºC ± 5 ºC for no less than 10 minutes (7) to decompose (8).
e) After standing to cool, transfer the solution to a 50-mL volumetric flask (9) with water.
f) Add water up to the marked line and transfer it to a 50-mL ground-in stopper centrifugal
precipitate tube (9).
g) Centrifuge it at 1700 × g centrifugal force for about five minutes (10) and use the supernatantas the sample solution.
h) As a blank test, conduct the procedures in b) - g) using another airtight vessel to prepare theblank test solution.
Note (6) The maximum limit of solid content, which is converted from moisture content, in thesampling volume 20.0 g of an analytical sample is about 0.5 g. If solid content is likely
to exceed the limit, reduce sampling volume as necessary.
(7) Condition examples for a microwave decomposing device: 0 min (room temperature)→ 10min (240 ºC) → 20 min (240 ºC) → 40 min (room temperature), initial output
1400 W(8) When organic matters still remain, for example the digestion solution is colored,
repeat the procedures in (4.1) b) - d).
(9) The vessel should be made of polypropylene, etc. to not affect the measurement.
(10) 16.5-cm of radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
424

Testing Methods for Fertilizers (2018)

Comment 2

The procedure in (4.1) is the same as the procedure in (4.1) of 5.1.b.

(4.2) Measurement: Conduct measurement (Internal Standard Method) according to JIS K 0113
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Mass Spectrometer used in measurement.
a) Measurement conditions for the ICP Mass Spectrometer: Set up the measurement
conditions for the ICP Mass Spectrometer considering the following:
Nickel: monitor ion (m/z): 60, 58
Rhodium: monitor ion (m/z): 103b) Calibration curve preparation
1) Spray nickel standard solution for the calibration curve preparation and blank test solution forthe calibration curve preparation into the inductively coupled plasma, and read the ratio of therespective ion count numbers for the monitor ion of an element subjected to measurement andan internal standard element.
2) Prepare a curve for the relationship between the concentration and the ratio of the ion countnumber of an element subjected to measurement.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 µg - 5 µg asNi) to a 50-mL volumetric flask (9).
2) Add 5mL of internal standard solution (5) and add nitric acid (1+19) to the marked line.
3) Conduct procedures similarly as in b) 1) to read the ratio of the ion count number.
4) For blank test solution, conduct procedures similarly as in 1) - 3) to correct the ratio of the ion
count number obtained for the sample solution.
5) Obtain the nickel content from the calibration curve, and calculate nickel (Ni) in the analyticalsample.
Comment 3 Instead of the correction method in c) 4), the nickel (Ni) in the analytical sample canalso be corrected by obtaining the nickel content in the blank test solution.
Comment 4 Triplicates additive recovery testing was conducted to evaluate trueness using fluid
industrial sludge fertilizers (2 samples) and composted sludge fertilizers (6 samples).
As a result, the mean recovery at the concentration level of 10 mg/kg - 60 mg/kg, 1
mg/kg - 9 mg/kg and 0.1 mg /kg - 0.9 mg /kg are 89.6 % - 99.2 %, 91.5 % - 114.7 %and 96.1 - 103.7 % as nickel (Ni) in an actual article respectively.
The results of the repeatability tests on different days using two kinds of fluid sludge
fertilizers to evaluate precision were analyzed by the one-way analysis of variance.
Table 1 shows the calculation results of intermediate precision and repeatability.。
Additionally, the minimum limit of quantification of this test method is about 6 µg
/kg.

425

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the repeatability tests on different days
Name ofsampleTest days of

Repeatability
2)

repeatability Average
1)(mg/kg)T3)
sr(mg/kg)

RSD r(%)

Intermediate precision
4)

5)

6)

s I(T)(mg/kg)

RSD I(T)(%)

Composted sludge fertilizer 1

5

8.60

0.44

5.1

0.61

7.0

Composted sludge fertilizer 2

5

2.04

0.13

6.1

0.13

6.4

1） The number of test days conducting a duplicate test

5） Intermediate standard deviation

2） Average （the number of test days（T ）6） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Repeatability standard deviation
4） Repeatability relative standard deviation

Comment 5 Simultaneous measurement of multiple elements by an ICP-MS is available. In thatcase, see 5.2.c Comment 5.
Comment 6 The presence and the extent of interference to the measurement mass number of anelement subjected to measurement and an internal standard element can be estimatedby qualitative analysis using an ICP-MS in advance to quantitation. Select ameasurement mass number considering the extent of interference. However, a mass
number cannot be changed in the measurement of arsenic. A magnetic sector
double-focusing mass spectrometer or a collision reaction cell specified in JIS K0133
can be used as a method to reduce spectrum interference.
References1)
Toshiharu YAGI: Determination of Heavy Metals in Fluid Sludge Fertilizers by ICP-MSand CV-AAS, Research Report of Fertilizer Vol. 8, p. 26 - 37 (2015)(5)

Flow sheet for nickel: The flow sheet for nickel in fluid sludge fertilizers is shown below:

20.0 g analytical sample Airtight vessel← Nitric acid, 2.5 mL← Hydrogen peroxide, 2 mL
Microwave
decomposition
Transfer
50-mL volumetric flask, water← Water (up to the marked line)
Ground-in stopper centrifugal precipitate tube,
Centrifugal separation
Anout 1700 × g , 5 minutes
Sample solution
Figure 1 Flow sheet for nickel in fluidsludge fertilizers(Extraction procedure)
426

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot

50-mL volumetric flask(predetermined volume)← Rhodium standard solution (Rh 0.1µg/mL) 5 mL (internal standard)← Nitric acid (1+19) (up to the marked line)
Measurement
ICP-MS (Ni: m /z 60, 58 Rh: m /z 103)
Figure 2 Flow sheet for nickel in fluidsludge fertilizers(Measurement procedure)427

Testing Methods for Fertilizers (2018)

5.5 Chromium
5.5.a Flame atomic absorption spectrometry (Fertilizers containing organic matters)(1) Summary
This testing method is applicable to fertilizers containing organic matters. This testing method isclassified as Type B and its symbol is 5.5.a-2017 or Cr.a-1.
Pretreat an analytical sample with incineration and nitric acid−hydrochloric acid (1+3), spray into
an acetylene−air flame, and measure the atomic absorption with chromium at a wavelength of 357.9
nm or 359.3 nm to obtain chromium (Cr) in an analytical sample. In addition, the performance of
this testing method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Interference suppressor solution (1): Dissolve 100 g of potassium disulfate specified in JIS K
8783 in water to make 1000 mL.
e) Chromium standard solution (Cr 0.1 mg/mL): A chromium standard solution (Cr 0.1
mg/mL) traceable to National Metrologyf) Chromium standard solutions (Cr 0.5 µg/mL - 5 µg/mL) for the calibration curve
preparation (1) (2): Transfer 2.5 mL - 25 mL of chromium standard solution (Cr 0.1 mg/mL) to
500-mL volumetric flasks step-by-step, add about 50 mL of interference suppressor solution(3), and further add hydrochloric acid (1+23) to the marked line.
g) Blank test solution for the calibration curve preparation (1) (2): Transfer about 50 mL ofinterference suppressor solution (3) to a 500-mL volumetric flask, and add hydrochloric acid
(1+23) used in the procedure in f) to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the chromium standard solution in (2), a chromium standard solution forthe calibration curve preparation can be prepared by using a chromium standard
solution (Cr 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (4) function.
1) Light source: A chromium hollow cathode lamp (In case of background correction systemusing continuous spectrum source, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C .
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
Note (4) There are the continuous source method, the Zeeman method, the non-resonance428

Testing Methods for Fertilizers (2018)

spectrum method, and the self-reversal method, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (5).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (5).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (6), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (7) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (5) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 450 °C in 1 to 2 hours.
(6) The watch glass can be removed.
(7) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 357.9 nm or 359.3 nm (8)b) Calibration curve preparation
1) Spray the chromium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame (9), and read the indicated value at awavelength of 357.9 nm or 359.3 nm (8).
2) Prepare a curve for the relationship between the chromium concentration and the indicatedvalue of the chromium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer 25 mL of the sample solution (10) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add hydrochloric acid (1+23) tothe marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Subject the blank test solution to the same procedure as in 1) - 2) and b) 1) to read theindicated value, and correct the indicated value obtained for the sample solution.
429

Testing Methods for Fertilizers (2018)5) Obtain the chromium content from the calibration curve, and calculate chromium (Cr) in theanalytical sample.
Note (8) When background correction is conducted by the Zeeman method, 359.3 nm is
recommended as the analytical line wavelength.
(9) Use low-fuel acetylene−air flame. Acetylene−nitrous oxide flame can also be used.
(10) If there is a possibility that the chromium concentration in the sample solution will
exceed the maximum limit of the calibration curve, decrease the amount to betransferred.
Comment 3 In an acetylene−air flame, sensitivity is enhanced in high-fuel flame, but interferenceby coexisting substances such as iron and nickel will also be enhanced.
In an acetylene−nitrous oxide flame, such interference hardly affects the result.
Comment 4 Instead of the correction method in c) 4), the chromium (Cr) in the analytical sample
can also be corrected by obtaining the chromium content in the blank test solution.
Comment 5 Recovery testing was conducted using industrial sludge fertilizer and composted
sludge fertilizer (5 samples); as a result, the recovery at the concentration level of
500 mg/kg and 50 mg/kg were 97.5 % - 100.0 % and 95.9 % - 101.9 %, respectively.
Table 1 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification of this test method is about 1
mg/kg.
Table 1 Results and analysis results from a collaborative study
for chromium test method validation
3)
4)
2)
Number of
RSD r
RSD R
Mean
Sample name1)
(mg/kg)(%)(%)
laboratories
Sewage sludge
12
33.6
5.3
15.6
fertilizer a
Sewage sludge
12
26.3
4.9
18.7
fertilizer b
Composted sludge
11
41.3
2.1
11.0
fertilizer a
Composted sludge
12
30.2
5.5
13.8
fertilizer b
Composted sludge
12
85.0
6.4
12.5
fertilizer c1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (3))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

References
1)
Yoshinari SAKAKIBARA, Manabu MATSUZAKI and Tadao AMANO: Determinationof Cadmium, Lead, Nickel and Chromium in Sludge Fertilizer - Improved
Decomposition Method - Research Report of Fertilizer Vol.1, p. 41 - 49 (2008)
2)
Yoshinari SAKAKIBARA and Manabu MATSUZAKI: Determination of Cadmium,430

Testing Methods for Fertilizers (2018)

3)

(5)

Lead, Nickel and Chromium in Sludge Fertilizer - Collaborative Test Results - Research
Report of Fertilizer Vol.1, p. 50 - 59 (2008)
Yoshinari SAKAKIBARA, Chie INOUE: Validation of determination methods forchromium in sludge fertilizer - Evaluation of measurement procedure, Research Reportof Fertilizer Vol.2, p. 130 - 136 (2009)

Flow sheet for chromium: The flow sheet for chromium in fertilizers containing organic
matters is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8-16 hours

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot 25 mL
100-mL volumetric flasks← About 10 mL of interference suppressor solution← Hydrochloric acid (1+23) ( up to the marked line)
Sample solution
Measurement

Atomic absorption spectrometer (357.9 nm or 359.3 nm)

Figure Flow sheet for chromium in fertilizers containing organic matters431

Testing Methods for Fertilizers (2018)

5.5.b Flame atomic absorption spectrometry (Fertilizers mainly containing molten matters,slag, etc.)(1) Summary
This testing method is applicable to fertilizers mainly containing molten matters, slag, etc. This
testing method is classified as Type B and its symbol is 5.5.b-2017 or Cr.b-1.
Add ammonium sulfate to prevent the bumping of an analytical sample, pretreat the analyticalsample with nitric acid−sulfuric acid−perchloric acid, and then spray into an acetylene−air flame.
Measure the atomic absorption with chromium at a wavelength of 357.9 nm or 359.3 nm to obtainchromium (Cr) in an analytical sample. The performance of this testing method is shown inComment 4.
In addition, fertilizers containing no organic matters can also be measured by the method of 5.5.c.
However, this method should be used for fertilizers that bump when heating. (Fertilizers made ofmolten matters, slag, etc. may bump in many cases).
(2)
a)
b)
c)
d)
e)

Reagent, etc.: Reagents and water are as shown below.
Water: Water of A3 specified in JIS K 0557.
Ammonium sulfate: A reagent of atomic absorption analysis grade or equivalents
Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Perchloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
f) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
g) Interference suppressor solution (1): Dissolve 100 g of potassium disulfate specified in JIS K
8783 in water to make 1000 mL.
h) Chromium standard solution (Cr 0.1 mg/mL): A chromium standard solution (Cr 0.1
mg/mL) traceable to National Metrologyi) Chromium standard solutions (Cr 0.01 mg/mL) for the calibration curve preparation (1):Transfer 10 mL of chromium standard solution (Cr 0.1 mg/mL) to 100-mL volumetric flask,
and add hydrochloric acid (1+23) to the marked line.
j) Chromium standard solutions (Cr 0.5 µg/mL - 5 µg/mL) for the calibration curve
preparation (1) (2): Transfer 2.5 mL - 25 mL of chromium standard solution (Cr 0.1 mg/mL) or
chromium standard solution (Cr 0.01 mg/mL) to 500-mL volumetric flasks step-by-step, add
about 50 mL of interference suppressor solution (3), and further add hydrochloric acid (1+23)
to the marked line.
k) Blank test solution for the calibration curve preparation (1) (2): Transfer about 50 mL ofinterference suppressor solution (3) to a 500-mL volumetric flask, and add hydrochloric acid
(1+23) used in the procedure in i) and j) to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the chromium standard solution in (2), a chromium standard solution forthe calibration curve preparation can be prepared by using a chromium standard
solution (Cr 1 mg/mL or 10 mg/mL) traceable to National Metrology.

(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in432

Testing Methods for Fertilizers (2018)

JIS K 0121 with the background correction (4) function.
1) Light source: A chromium hollow cathode lamp (In case of background correction systemusing continuous spectrum source, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 350 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to no less than 300 °C.
Note (4) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample and transfer to a 300-mL tall beaker.
b) Add 4 g of ammonium sulfate and moisten the analytical sample with a small amount ofwater.
c) Add about 10 mL of nitric acid, about 5 mL of sulfuric acid and about 5 mL of perchloric acid.
Cover the tall beaker with a watch glass, heat on a hot plate or sand bath gently at 170 °C 220 °C for no less than 1 hour, and then warm to raise temperature gradually to no less than
300 °C taking no less than 30 minutes (5) and further heat at no less than 300 °C for 2-3 hours.
d) Slightly move the watch glass (6), and keep on heating on the hot plate or sand bath toconcentrate until the liquid volume becomes about 3 mL (7).
e) After standing to cool, add about 5 mL of hydrochloric acid (1+10) and about 20 mL of water,
cover the tall beaker with a watch glass and heat mildly for about 10 minutes to dissolve (8).
f) After standing to cool, transfer the solution to a 100-mL volumetric flask with water, add
water up to the marked line, and filter with Type 3 filter paper to make a sample solution.
g) As a blank test, conduct the procedures in b) - f) using another tall beaker to prepare the blanktest solution. However, the heating procedure after adding acid in the procedures in c) should
be conducted as shown below. Cover the tall beaker with a watch glass, heat on a hot plate orsand bath at about 170 °C for a while (8), slightly move the watch glass (6) and then heat gently
for no less than 1 hour to vapor nitric acid. After concentrating to about 15 mL (9), heat andraise the temperature gradually to no less than 300 °C in more than 30 minutes (5). When whitesmoke starts evolving, cover the tall beaker with a watch glass and further heat at no less than
300 °C for 2-3 hours.
Note (5) Sudden temperature rise may cause bumping. Therefore, increase the temperature
gradually.
(6) If there is no possibility of bumping, the watch glass can be removed.
(7) Drying and hardening it up may cause the chromium to become insoluble. Therefore,care should be taken not to concentrate too much.
(8) Heating at high temperature may cause bumping. Therefore, it is recommended toincrease the temperature gradually from about 170 °C to the temperature at which itboils slightly.
(9) If much nitric acid remains, care should be taken because bumping can occur easily
when heating temperature rises.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
433

Testing Methods for Fertilizers (2018)absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 357.9 nm or 359.3 nm (10)b) Calibration curve preparation
1) Spray the chromium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a low-fuel acetylene-air flame (11), and readthe indicated value at a wavelength of 357.9 nm or 359.3 nm (10).
2) Prepare a curve for the relationship between the chromium concentration and the indicatedvalue of the chromium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer 25 mL of the sample solution (12) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add hydrochloric acid (1+17) tothe marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Subject the blank test solution to the same procedure as in 1) - 2) and b) 1) to read theindicated value, and correct the indicated value obtained for the sample solution.
5) Obtain the chromium content from the calibration curve, and calculate chromium (Cr) in theanalytical sample.
Note (10) When background correction is conducted by the Zeeman method, 359.3 nm is
recommended as the analytical line wavelength.
(11) Acetylene−nitrous oxide flame can also be used.
(12) If there is a possibility that the concentration of chromium in the sample solution
exceeds the maximum limit of the calibration curve, transfer no more than 10 mL of
the sample solution to a 100-mL volumetric flask and add about 10 mL of interference
suppressor solution and about 67 mL of hydrochloric acid (1+17), and add water up to
the marked line.
Comment 2 In an acetylene−air flame, sensitivity is enhanced in high-fuel flame, but interferenceby coexisting substances such as iron and nickel will also be enhanced.
In an acetylene−nitrous oxide flame, such interference hardly affects the result.
Comment 3 Instead of the correction method in c) 4), the chromium (Cr) in the analytical sample
can also be corrected by obtaining the chromium content in the blank test solution.
Comment 4 The comparison of the measurement value (yi：54 mg/kg - 4649 mg/kg) of this testing
method and the measurement value (xi) of 5.8 Chromium 5.8.2 Atomic Absorption
Spectrometry in Official Methods of Analysis of Fertilizers (1992) was conducted toevaluate trueness using fertilizers (29 samples). As a result, a regression equation
was y = − 6.842 + 0.998x and its correlation coefficient (r) was 0.999.
The results of the repeatability tests on different days using fused silicic phosphate
fertilizer slag silicic fertilizer and compound fertilizer (1 sample for each) to evaluate
precision were analyzed by the one-way analysis of variance. Table 1 shows thecalculation results of intermediate precision and repeatability. Table 2 shows results
and analysis results from a collaborative study for testing method validation.
Additionally, the minimum limit of quantification of this test method is about 6
mg/kg.

434

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the repeatability tests on different days
Name of
Test days of
Repeatability
Intermediate precision
2)
3)
4)
5)
6)
samplerepeatability Averagesr
RSD rs I(T)RSD I(T)1)(mg/kg)(mg/kg)(%)(mg/kg)

(%)

5

4628

37

0.8

175

3.8

T
Fused silicic
phosphate fertilizer

Compound fertilizer
5
545
5.9
Slag silicic fertilizer
5
319
3.8
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Repeatability standard deviation
4） Repeatability relative standard deviation

1.1
7.3
1.3
1.2
5.7
1.8
5） Intermediate standard deviation
6） Intermediate relative standard deviation

Table 2 Results and analysis results from a collaborative study
for the test method validation of chromium
Mean

sr ）

3

RSD r ）sR ）5

RSD R ）

laboratories（mg/kg）（mg/kg）（%）（mg/kg）（%）

Slag silicic fertilizer
Mixed phosphate fertilizer

12
12

63.75
912.9

2.02
13.0

3.2
1.4

3.87
37.3

6.1
4.1

Slag manganese fertilizer

12

2962

74

2.5

176

5.9

10

4662

135

2.9

166

3.6

10

543.6

10.2

1.9

15.4

2.8

2)

Sample nameFused silicic
phosphate fertilizer
Compond fertilizers

1)
2)
3)
4)
5)
6)

Number of
1)

4

6

Number of laboratories used in analysis
Mean (n = number of laboratories x number of samples (2))
Repeatability standard deviation
Repeatability relative standard deviation
Reproducibility standard deviation
Reproducibility relative standard deviation

References1)
Toshiaki HIROI, Fumika TAKATSU: Evaluation of Digest Method for Determination of
Chromium in Inorganic Fertilizer by Atomic Absorption Spectrometry, Research Reportof Fertilizer Vol.10, p. 9 - 28 (2017)

435

Testing Methods for Fertilizers (2018)(5)

Flow sheet for chromium: The flow sheet for chromium in fertilizers mainly containing
molten matters, slag is shown below:
1.00 g300-mL tall beaker.
analytical sample← 4 g of ammonium sulfate← A small amount of water, moisten the analytical sample← About 10 mL of nitric acid
← About 5 mL of sulfuric acid
← About 5 mL of perchloric acid
Cover with a watch glass, and heat mildly on a hot plate
Heatingor sand bath at 170 ºC - 220 ºC for no less than 1 hour
Heating

Raise temperature gradually to no less than 300 °C in more than
30 minutes

Heating

Digest on a hot plate or sand bath for 2 -3 hours

Heating

Slightly move a watch glass and concentrate
until the liquid volume becomes about 3 mL

Standing to coolRoom temperature← About 5 mL of hydrochloric acid (1+5)← About 20 mL of water
Heating
Cover with a watch glass, and dissolve (heat for about 10 minutes)
Standing to coolRoom temperature

Transfer
100-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for chromium in fertilizers
mainly containing molten matters and slag, etc. (Extraction procedure)

436

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot(no more than 25 mL) 100-mL volumetric flasks← About 10 mL of interference suppressor solution← Hydrochloric acid (1+17) ( up to the marked line)
Measurement
Atomic absorption spectrometer (357.9 nm or 359.3 nm)
Figure 2 Flow sheet for chromium in fertilizers
mainly containing molten matters and slag, etc. (Measurement procedure)

437

Testing Methods for Fertilizers (2018)

5.5.c Flame atomic absorption spectrometry (Fertilizers not containing organic matters)(1) Summary
This testing method is applicable to fertilizers not containing organic matters (including calcinedsludge fertilizer). This testing method is classified as Type B and its symbol is 5.5.c-2017 or Cr.c-1.
In addition, for fertilizers mainly made of molten matters, slag, etc., care should be taken because
they may bump while heating. 5.5.b applies to fertilizers that may bump.
Pretreat an analytical sample with nitric acid−sulfuric acid−perchloric acid, and then spray into anacetylene−air flame, and measure the atomic absorption with chromium at a wavelength of 357.9
nm or 359.3 nm to obtain chromium (Cr) in an analytical sample. In addition, the performance of
this testing method is shown in Comment 8.
(2)
a)
b)
c)
d)

Reagent, etc.: Reagents and water are as shown below.
Water: Water of A3 specified in JIS K 0557.
Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Perchloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
e) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
f) Interference suppressor solution (1): Dissolve 100 g of potassium disulfate specified in JIS K
8783 in water to make 1000 mL.
g) Chromium standard solution (Cr 0.1 mg/mL): A chromium standard solution (Cr 0.1
mg/mL) traceable to National Metrologyh) Chromium standard solutions (Cr 0.01 mg/mL) for the calibration curve preparation (1):Transfer 10 mL of chromium standard solution (Cr 0.1 mg/mL) to 100-mL volumetric flask,
and add hydrochloric acid (1+23) to the marked line.
i) Chromium standard solutions (Cr 0.5 µg/mL - 5 µg/mL) for the calibration curve
preparation (1) (2): Transfer 2.5 mL - 25 mL of chromium standard solution (Cr 0.1 mg/mL) or
chromium standard solution (Cr 0.01 mg/mL) to 500-mL volumetric flasks step-by-step, add
about 50 mL of interference suppressor solution (3), and further add hydrochloric acid (1+23)
to the marked line.
j) Blank test solution for the calibration curve preparation (1) (2): Transfer about 50 mL ofinterference suppressor solution (3) to a 500-mL volumetric flask, and add hydrochloric acid
(1+23) used in the procedure in h) and i) to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of the chromium standard solution in (2), a chromium standard solution forthe calibration curve preparation can be prepared by using a chromium standard
solution (Cr 1 mg/mL or 10 mg/mL) traceable to National Metrology.

(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (4) function.
1) Light source: A chromium hollow cathode lamp (In case of background correction systemusing continuous spectrum source, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame
438

Testing Methods for Fertilizers (2018)(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 350 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to no less than 300 °C.
Note (4) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Add about 10 mL of nitric acid and about 5 mL of sulfuric acid, cover the tall beaker with awatch glass, and leave at rest overnight.
c) Heat mildly on a hot plate or sand bath at 170 ºC - 220 ºC for no less than 30 minutes. Afterbubbles cease to form, set the temperature of the hot plate or sand bath to no less than 300 ºC(5), and heat until nitroxide (yellow-brown smoke) is no longer generated (6).
d) After standing to cool, add about 5 mL of perchloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or sand bath at no less than
300 ºC for 2 - 3 hours to digest (7).
f) Slightly move the watch glass (8), and keep on heating on the hot plate or sand bath toconcentrate until the liquid volume becomes no more than 2 mL (9).
g) After standing to cool, add about 5 mL of hydrochloric acid (1+10) and about 20 mL of water,
cover the tall beaker with a watch glass and heat mildly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask with water,
add water up to the marked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (5) If there is vigorous bumping, increase the temperature gradually.
(6) Oxidation of carbides by perchloric acid progresses extremely rapidly and explosively.
For that reason, add perchloric acid after fully degrading carbides with nitric acid to
avoid danger.
(7) When the white smoke of perchloric acid is generated, if the solution is colored suchas black-brown or brown, stop heating immediately, and after standing to cool, add
nitric acid, and heat again to degrade remaining carbides.
(8) If there is no possibility of bumping, the watch glass can be removed.
(9) When drying and hardening it, chromium may not dissolve completely in g) and theconcentration may become a low value.
Comment 2
Comment 3
Comment 4
Comment 5

The procedure in (4.1) is the same as the procedure in (4.1) of 5.2.a.
When the analytical sample solidifies in the procedure in (4.1) b), moisten theanalytical sample with a small amount of water as necessary in advance.
It is not necessary to conduct the procedures in (4.1) b) “leave at rest overnight”
because the range subjected to analysis does not contain organic matters.
In some cases, about 10 minutes heating in procedure in (4.1) g) is required.

(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
439

Testing Methods for Fertilizers (2018)a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 357.9 nm or 359.3 nm (10)b) Calibration curve preparation
1) Spray the chromium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a low-fuel acetylene−air flame (11), and readthe indicated value at a wavelength of 357.9 nm or 359.3 nm (10).
2) Prepare a curve for the relationship between the chromium concentration and the indicatedvalue of the chromium standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation.
c) Sample measurement
1) Transfer 25 mL of the sample solution (12) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add hydrochloric acid (1+17) tothe marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Subject the blank test solution to the same procedure as in 1) - 2) and b) 1) to read theindicated value, and correct the indicated value obtained for the sample solution.
5) Obtain the chromium content from the calibration curve, and calculate chromium (Cr) in theanalytical sample.
Note (10) When background correction is conducted by the Zeeman method, 359.3 nm is
recommended as the analytical line wavelength.
(11) Acetylene−nitrous oxide flame can also be used.
(12) If there is a possibility that the concentration of chromium in the sample solution
exceeds the maximum limit of the calibration curve, transfer no more than 10 mL of
the sample solution to a 100-mL volumetric flask and add about 10 mL of interference
suppressor solution and about 67 mL of hydrochloric acid (1+17), and add water up to
the marked line.
Comment 6 In an acetylene-air flame, sensitivity is enhanced in high-fuel flame, but interferenceby coexisting substances such as iron and nickel will also be enhanced.
In an acetylene−nitrous oxide flame, such interference hardly affects the result.
Comment 7 Instead of the correction method in c) 4), the chromium (Cr) in the analytical sample
can also be corrected by obtaining the chromium content in the blank test solution.
Comment 8 The comparison of the measurement value (yi：52 mg/kg - 4052 mg/kg) of this testing
method and the measurement value (xi) of 5.8 Chromium 5.8.2 Atomic Absorption
Spectrometry in Official Methods of Analysis of Fertilizers (1992) was conducted toevaluate trueness using fertilizers (27 samples). As a result, a regression equation
was y = − 0.405 + 0.994x and its correlation coefficient (r) was 0.999.
The results of the repeatability tests on different days using mixed
phosphatefertilizer, compound fertilizer and slag silicic fertilizer (1 sample for each) toevaluate precision were analyzed by the one-way analysis of variance. Table 1 shows
the calculation results of intermediate precision and repeatability.
Table 2 shows results and analysis results from a collaborative study for testing
method validation using calcined sludge fertilizers
Additionally, the minimum limit of quantification of this test method is about 6
mg/kg.

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Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
3)
4)
5)
6)
repeatability Averagesr
RSD rs I(T)RSD I(T)T

1)(mg/kg)(mg/kg)

Mixed
5
3966
96
phosphate fertilizer
Compound fertilizer
5
542
6.1
Slag silicic fertilizer
5
288
7.0
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Repeatability standard deviation
4） Repeatability relative standard deviation(%)(mg/kg)

(%)

2.4

107

2.7

1.1
8.6
1.6
2.4
13
4.4
5） Intermediate standard deviation
6） Intermediate relative standard deviation

Table 2 Results and analysis results from a collaborative study
for chromium test method validation
3)
2)
Number of
RSD r
Mean
Sample name1)
(mg/kg)(%)
laboratories

4)

RSD R
(%)

Calcined sludge fertilizer 1

10

107

5.0

9.7

Calcined sludge fertilizer 2

9

136

3.4

3.6

Calcined sludge fertilizer 3

9

182

1.1

2.6

Calcined sludge fertilizer 4

9

213

1.1

3.9

Calcined sludge fertilizer 5

9

117

1.8

4.0

1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

References1)
Hisanori ARAYA, Yoshimi TAKEBA and Toshiaki HIROI: Evaluation of Digest
Method for Determination of Chromium in Calcined Sludge Fertilizer by Atomic
Absorption Spectrometry - Research Report of Fertilizer Vol.4, p. 23 - 29 (2011)
2)
Takeshi UCHIYAMA and Yuji SHIRAI: Evaluation of Digest Method for Determinationof Chromium in Calcined Sludge Fertilizer by Atomic Absorption Spectrometry: A
Collaborative Study. Research Report of Fertilizer Vol.5, p. 41 - 47 (2012)

441

Testing Methods for Fertilizers (2018)(5)

Flow sheet for chromium: The flow sheet for chromium in fertilizers not containing calcinedsludge fertilizer and organic matters is shown below: However, fertilizers that may bump are
excluded from the scope of application.

1.00 g200-mL - 300-mL tall beaker.
analytical sample← A small amount of water, moisten the analytical sample (as necessary)← About10 mL of nitric acid
← About 5 mL of sulfuric acid
Leaving at rest
Cover with a watch glass
overnight
HeatingHeat gently on a hot plate or a sand bath at 170 ºC - 220 ºC
for no less than 30 minutes

HeatingHeat on a hot plate or sand bath at no less than 300 ºC,
until yellow-brown smoke no longer evolves.

Standing to coolRoom temperature← About 5 mL of perchloric acid
Cover with a watch glass,
Heatingdigest on a hot plate or a sand bath at 300 ºC for 2-3 hours
Heating

Slightly move the watch glass,concentrate until it becomes no more than 2mL

Standing to coolRoom temperature← About 5 mL of nitric acid
← About 20 mL of water
Heating
Cover with watch glass, dissolve
Standing to coolRoom temperature

Transfer
100-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for chromium in fertilizers not containing calcined sludge fertilizer
and organic matters (Extraction procedure)(Excluding fertilizers which may bump while heating)442

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot 25 mL
100-mL volumetric flask← About 10 mL of interference suppressor solution← Hydrochloric acid (1+17) ( up to the marked line)
Measurement
Atomic absorption spectrometer (357.9 nm or 359.3 nm)
Figure 2 Flow sheet for chromium in fertilizers not containing calcined sludge fertilizer
and organic matters (Measurement procedure)(Excluding fertilizers which may bump while heating)443

Testing Methods for Fertilizers (2018)

5.5.d ICP Optical Emission Spectrometry(1) Summary
The testing method is applicable to sludge fertilizers, etc. (except for calcined sludge fertilizer) This
testing method is classified as Type D and its symbol is 5.5.d-2017 or Cr.d-1.
Pretreat an analytical sample with incineration, nitric acid−hydrochloric acid (1+3), introduce it to
ICP Optical Emission Spectrometry (“ICP-OES”) and measure the emission with chromium at awavelength of 205.552 nm to quantify the chromium (Cr) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Chromium standard solution (Cr 0.1 mg/mL): A chromium standard solution (Cr 0.1
mg/mL) traceable to National Metrologye) Chromium standard solutions (Cr 2.5 µg/mL) (1) (2): Dilute a predetermined amount ofchromium standard solution (Cr 0.1 mg/mL) with hydrochloric acid (1+23) to prepare achromium standard solution (Cr 2.5 µg/mL)
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
Comment 1 Instead of the chromium standard solution in (2), a chromium standard solution forthe calibration curve preparation can be prepared by using a chromium standard
solution (Cr 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C .
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (3).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (3).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (4), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (5) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
444

Testing Methods for Fertilizers (2018)i)

As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.

Note (3) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 450 °C in 1 to 2 hours.
(4) The watch glass can be removed.
(5) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K 0116
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Optical Emission Spectrometry used in measurement.
a) Measurement conditions for the ICP Optical Emission Spectrometer: Set up themeasurement conditions for the ICP Optical Emission Spectrometer considering thefollowing:
Analytical line wavelength: 205.552 nmb) Calibration curve preparation and sample measurement
1) Put 5mL of sample solution to three 10-mL volumetric flasks respectively.
2) Add 2mL and 4 mL of chromium standard solution (2.5 μg/mL) to volumetric flasks of 1)
above, then add hydrochloric acid (1+23) to the marked line to make a sample solution ofStandard Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flask of 1)
above to make a sample solution without a standard solution.
4) Spray the sample solution of Standard Addition Method and the sample solution without astandard solution into the induction plasma, and read the indicated value at a wavelength of
205.552 nm.
5) Transfer 5 mL of blank test solution to a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value obtained
from the respective sample solutions.
6) Prepare a curve for the relationship between the added chromium concentration and thecorrected indicated value of the sample solution for Standard Addition Method and the sample
solution without a standard solution.
7) Obtain the chromium content from the intercept of the calibration curve to calculate
chromium (Cr) in the analytical sample.
Comment 3 Instead of the correction method in c) 5), the chromium (Cr) in the analytical sample
can also be corrected by obtaining the chromium content in the blank test solution.
Comment 4 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.9.1.b Comment 5.
Comment 5 The comparison of the measurement value (xi：12.9 mg/kg - 193 mg/kg) of ICP
Optical Emission Spectrometry and the measurement value (yi) of Flame Atomic
Absorption Spectrometry was conducted to evaluate trueness using sludge fertilizers(49 samples). As a result, a regression equation was y = 1.74+0.971x and its
correlation coefficient (r) was 0.991. Triplicates measurement for each one sample ofsewage sludge fertilizer, human waste sludge fertilizer, industrial sludge fertilizer,
445

Testing Methods for Fertilizers (2018)

mixed sludge fertilizer and composted sludge fertilizer was conducted. As a result, a
repeatability obtained was 0.9 % - 2.5 % as a relative standard deviation.
Additionally, the minimum limit of quantification of this testing method is 4 mg/kg.
References
1)
Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI and Tetuya NOMURA:
Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer, Vol.4, p. 30 - 35 (2011)(5)

Flow sheet for cadmium: The flow sheet for cadmium in fertilizers is shown below:

5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8 - 16 hours

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest for 30 minutes
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for chromium in sludge fertilizers (Extraction procedure)
Sample solution
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0, 2 and 4 mL of chromium standard solution (2.5 μg / mL) respectively← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Optical Emission Spectrometer (205.552 nm)
Figure 2 Flow sheet for chromium in sludge fertilizers (Measurement procedure)
446

Testing Methods for Fertilizers (2018)

5.5.e ICP Mass Spectrometry (Fluid sludge fertilizers)(1) Summary
The test method is applicable to fluid sludge fertilizers. This testing method is classified as Type D
and its symbol is 5.5.e-2017 or Cr.e-1.
Add nitric acid−hydrogen peroxide to an analytical sample to heat and extract by microwave
irradiation. After adding an internal standard element, introduce it to an ICP Mass Spectrometer(ICP-MS) and measure the respective indicated values of chromium and an internal standard
element with mass/ charge number (m/z) and obtain chromium (Cr) in the analytical sample fromthe ratio of the indicated value for chromium and the indicated value for an internal standard
element. In addition, the performance of this testing method is shown in Comment 4.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A4 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Nitric acid: Nitric acid used to dilute a standard solution and a sample solution is a highly
pure reagent specified in JIS K 9901.
d) Hydrogen peroxide: A JIS Guaranteed Reagent specified in JIS K 8230 or a reagent ofequivalent quality.
e) Rhodium standard solution (Rh 1 mg/mL): A rhodium standard solution (Rh 1 mg/mL)
traceable to National Metrology.
f) Rhodium standard solution (Rh 0.1 µg/mL) (1)(2)(3): Dilute a predetermined amount ofrhodium standard solution (Rh 1 mg/mL) with nitric acid (1+19) to prepare a rhodium
standard solution (Rh 0.1 µg/mL).
g) Rhenium standard solution (Re 1 mg/mL) (4): A rhenium standard solution (Re 1 mg/mL)
traceable to National Metrology.
h) Rhenium standard solution (Re 0.1 µg/mL) (1)(2)(3): Dilute a predetermined amount ofrhenium standard solution (Re 1 mg/mL) with nitric acid (1+19) to prepare a rhenium
standard solution (Re 0.1 µg/mL).
i) Chromium standard solution (Cr 0.1 mg/mL): A chromium standard solution (Cr 0.1
mg/mL) traceable to National Metrologyj) Chromium standard solution (Cr 5 µg/mL) (1)(2)(3): Transfer 5 mL of chromium standard
solution (Cr 0.1 mg/mL) to a 100-mL volumetric flask and add nitric acid (1+19) to themarked line.
k) Chromium standard solutions (Cr 1 ng/ mL - 100 ng/ mL) for the calibration curve
preparation (1)(2)(3): Transfer 0.02 mL - 2 mL of chromium standard solution (Cr 5 µg/mL) to
100-mL volumetric flasks step-by-step, and add 10 mL of rhodium standard solutions (Rh 0.1µg/mL) as an internal standard respectively (5) and add nitric acid (1+19) up to the markedline.
l) Blank test solution for the calibration curve preparation (1)(2)(3): transfer 10 mL of rhodium
standard solution (Rh 0.1 µg/mL) to 100-mL volumetric flasks as internal standard (5) and add
nitric acid (1+19) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in cool and dark place, and do not use after 1 month after preparation.
(3) For storage, use a sealable container made of materials such as polypropylene
containing no chromium.
(4) Use when measuring lead simultaneously.
(5) Add an internal solution that is 1/10 of the volume to be prepared.
Comment 1 Instead of the chromium standard solution in (2), a chromium standard solution for447

Testing Methods for Fertilizers (2018)

the calibration curve preparation can be prepared by using a chromium standard
solution (Cr 1 mg/mL or 10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) ICP Mass Spectrometer: High-frequency plasma mass spectrometer specified in JIS K 0133.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.995 % in purityb) Pressure vessel decomposing device: A device which pressurizes the inside of a vessel by
putting acid, etc.to heat in the airtight vessel, and decomposes a sample by the interaction ofheating, pressurizing and acid. The following requirements should be met.
1) The main part of a decomposing device: In the case of the microwave heating method, adevice should be able to produce a high-frequency wave using a frequency which is permitted
at an industrial frequency facility. It is desirable to be able to monitor pressure andtemperature, etc. in the airtight vessel with a sensor inside the device. The interior of a device
should be acid-resistant treated and should have a high temperature durability and a high levelof safety.
2) Exhaust system: A system which has an exhaust fan of acid-resistant specification, and has
an air-cool function inside the device to allow constant airflow, keeping operating temperature
below a certain temperature level.
3) Airtight vessel: A vessel which is heat-resistance and pressure-tight and has the durability
required to decompose particles, and has resistance to internal contamination. It should havesafety functions such as causing overheat prevention valve to work and internal pressure todrop by emitting gas, and preventing gas bumping in the case of exceeding a pressure
limitation.
c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 20.0 g (6) of an analytical sample, and put it into an airtight vessel.
b) Gradually add 2.5 mL of nitric acid and 2 mL of hydrogen peroxide.
c) Put the airtight vessel into the main part of a decomposing device and heat using a microwave(7).
d) Ignite at 240 ºC ± 5 ºC for no less than 10 minutes (7) to decompose (8).
e) After standing to cool, transfer the solution to a 50-mL volumetric flask (9) with water.
f) Add water up to the marked line and transfer it to a 50-mL ground-in stopper centrifugal
precipitate tube (9).
g) Centrifuge it at 1700 × g centrifugal force for about five minutes (10) and use the supernatantas the sample solution.
h) As a blank test, conduct the procedures in b) - g) using another airtight vessel to prepare theblank test solution.
Note (6) The maximum limit of solid content, which is converted from moisture content, in thesampling volume 20.0 g of an analytical sample is about 0.5 g. If solid content is likely
to exceed the limit, reduce sampling volume as necessary.
(7) Condition examples for a microwave decomposing device: 0 min (room temperature)→ 10min (240 ºC) → 20 min (240 ºC) → 40 min (room temperature), initial output
1400 W(8) When organic matters still remain, for example the digestion solution is colored,
repeat the procedures in (4.1) b) - d).
(9) The vessel should be made of polypropylene, etc. to not affect the measurement.
(10) 16.5-cm of radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
448

Testing Methods for Fertilizers (2018)

force.
Comment 2

The procedure in (4.1) is the same as the procedure in (4.1) of 5.1.b.

(4.2) Measurement: Conduct measurement (Internal Standard Method) according to JIS K 0113
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Mass Spectrometer used in measurement.
a) Measurement conditions for the ICP Mass Spectrometer: Set up the measurement
conditions for the ICP Mass Spectrometer considering the following:
Chromium: monitor ion (m/z)：52、53、50
Rhodium: monitor ion (m/z): 103b) Calibration curve preparation
1) Spray chromium standard solution for the calibration curve preparation and blank test solution
for the calibration curve preparation into the inductively coupled plasma, and read the ratio of
the respective ion count numbers for the monitor ion of an element subjected to measurement
and an internal standard element.
2) Prepare a curve for the relationship between the concentration and the ratio of the ion countnumber of an element subjected to measurement.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 µg - 5 µg asCr) to a 50-mL volumetric flask (9).
2) Add 5mL of internal standard solution (5) and add nitric acid (1+19) to the marked line.
3) Conduct procedures similarly as in b) 1) to read the ratio of the ion count number.
4) For blank test solution, conduct procedures similarly as in 1) - 3) to correct the ratio of the ion
count number obtained for the sample solution.
5) Obtain the chromium content from the calibration curve, and calculate chromium (Cr) in theanalytical sample.
Comment 3 Instead of the correction method in c) 4), the chromium (Cr) in the analytical sample
can also be corrected by obtaining the chromium content in the blank test solution.
Comment 4 Triplicates additive recovery testing was conducted to evaluate trueness using fluid
industrial sludge fertilizers (2 samples) and composted sludge fertilizers (6 samples).
As a result, the mean recovery at the concentration level of 10 mg/kg - 90 mg/kg, 1
mg/kg - 9 mg/kg and 0.1 mg /kg - 0.9 mg /kg are 92.4 % - 108.8 %, 94.3 % 115.4 % and 105.8 - 106.8 % as chromium (Cr) in an actual article respectively.
The results of the repeatability tests on different days using two kinds of fluid sludge
fertilizers to evaluate precision were analyzed by the one-way analysis of variance.
Table 1 shows the calculation results of intermediate precision and repeatability.。
Additionally, the minimum limit of quantification of this test method is about 1 µg
/kg.

449

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the repeatability tests on different days
Name ofsampleTest days of

Repeatability
2)

repeatability Average
1)(mg/kg)T3)

sr(mg/kg)

RSD r(%)

Intermediate precision
4)

5)

s I(T)(mg/kg)

RSD I(T)(%)

Composted sludge fertilizer 1

5

14.3

0.6

4.0

0.7

4.7

Composted sludge fertilizer 2

5

3.47

0.20

5.8

0.02

0.5

6)

1） The number of test days conducting a duplicate test

5） Intermediate standard deviation

2） Average （the number of test days（T ）6） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Repeatability standard deviation
4） Repeatability relative standard deviation

Comment 5 Simultaneous measurement of multiple elements by an ICP-MS is available. In thatcase, see 5.2.c Comment 5.
Comment 6 The presence and the extent of interference to the measurement mass number of anelement subjected to measurement and an internal standard element can be estimatedby qualitative analysis using an ICP-MS in advance to quantitation. Select ameasurement mass number considering the extent of interference. However, a mass
number cannot be changed in the measurement of arsenic. A magnetic sector
double-focusing mass spectrometer or a collision reaction cell specified in JIS K0133
can be used as a method to reduce spectrum interference.
References1)
Toshiharu YAGI: Determination of Heavy Metals in Fluid Sludge Fertilizers by ICP-MSand CV-AAS, Research Report of Fertilizer Vol. 8, p. 26 - 37 (2015)450

Testing Methods for Fertilizers (2018)(5)

Flow sheet for chromium: The flow sheet for chromium in fluid sludge fertilizers is shown
below:
20.0 g analytical sample Airtight vessel← Nitric acid, 2.5 mL← Hydrogen peroxide, 2 mL
Microwave
decomposition
Transfer
50-mL volumetric flask, water← Water (up to the marked line)
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
Anout 1700 × g , 5 minutes

Sample solution
Figure 1 Flow sheet for chromium in liquid sludge fertilizers(Extraction procedure)

Sample solution
50-mL volumetric flask
Aliquot(predetermined volume)← Rhodium standard solution (Rh 0.1µg/mL) 5 mL (internal standard)← Nitric acid (1+19) (up to the marked line)
Measurement
ICP-MS (Cr: m /z 53, 52, 50 Rh: m /z 103)
Figure 2 Flow sheet for chromium in liquid sludge fertilizers
Measurement procedure)

451

Testing Methods for Fertilizers (2018)

5.6 Lead
5.6.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 5.6.a-2017 or Pb.a-1.
Pretreat an analytical sample with incineration and nitric acid−hydrochloric acid (1+3), spray into
an acetylene−air flame, and measure the atomic absorption with lead at a wavelength of 217.0 nm
or 283.3 nm to obtain the lead (Pb) in an analytical sample. In addition, the performance of thistesting method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Lead standard solution (Pb 0.1 mg/mL): A lead standard solution (Pb 0.1 mg/mL) traceable
to National Metrology.
e) Lead standard solutions (Pb 0.5 µg - 5 µg/mL) for the calibration curve preparation (1):Transfer 2.5 mL - 25 mL of lead standard solution (Pb 0.1 mg/mL) to 500-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Blank test solution for the calibration curve preparation (1) (2): Hydrochloric acid (1+23)
used in the procedures in e).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
Comment 1 Instead of the lead standard solution in (2), a lead standard solution for the calibration
curve preparation can be prepared by using a lead standard solution (Pb 1 mg/mL or
10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121 with the background correction (3) function.
1) Light source: A lead hollow cathode lamp (In case of background correction system using
continuous spectrum source, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C .
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
Note (3) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedure452

Testing Methods for Fertilizers (2018)(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (4).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (6) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (4) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250°C in 30 minutes to 1 hour, continue heating for about 1
hour and further raise to 450 °C in 1 to 2 hours.
(5) The watch glass can be removed.
(6) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers not containing
organic matters.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 217.0 nm or 283.3 nmb) Calibration curve preparation
1) Spray the lead standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 217.0 nm or 283.3 nm.
2) Prepare a curve for the relationship between the lead concentration and the indicated value ofthe lead standard solutions for the calibration curve preparation and the blank test solution forthe calibration curve preparation.
c) Sample measurement
1) Subject the sample solution (7) to the same procedure as in b) 1) to read the indicated value.
2) Subject the blank test solution to the same procedure as in b) 1) to read the indicated value,and correct the indicated value obtained for the sample solution.
3) Obtain the lead content from the calibration curve, and calculate lead (Pb) in the analyticalsample.

453

Testing Methods for Fertilizers (2018)

Note (7) If there is a possibility that the lead concentration in the sample solution will exceed
the maximum limit of the calibration curve, dilute a predetermined amount withhydrochloric acid (1+23).
Comment 4 Instead of the correction method in c) 2), the lead (Pb) in the analytical sample canalso be corrected by obtaining the lead content in the blank test solution.
Comment 5 Recovery testing was conducted using industrial sludge fertilizer and composted
sludge fertilizer (5 samples); as a result, the recovery at the concentration level of
100 mg/kg and 10 mg/kg were 99.1 % - 100.6 % and 97.5 % - 99.6 %, respectively.
Table 1 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification of this test method is about 1
mg/kg.
Table 1 Results and analysis results from a collaborative study
for lead test method validation
3)
2)
Number of
RSD r
Mean
Sample name1)
(mg/kg)(%)
laboratories
Sewage sludge fertilizer a
10
25.2
4.6
Sewage sludge fertilizer b
11
29.4
3.7
Composted sludge fertilizer a
10
18.6
3.2
Composted sludge fertilizer b
10
22.2
1.8
Composted sludge fertilizer c
11
86.8
1.3
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

4)

RSD R
(%)
3.9
4.3
5.0
7.0
4.0

References
1)
Yoshinari SAKAKIBARA, Manabu MATSUZAKI and Tadao AMANO: Determinationof Cadmium, Lead, Nickel and Chromium in Sludge Fertilizer - Improved
Decomposition Method - Research Report of Fertilizer Vol.1, p. 41 - 49 (2008)
2)
Yoshinari SAKAKIBARA and Manabu MATSUZAKI: Determination of Cadmium,
Lead, Nickel and Chromium in Sludge Fertilizer - Collaborative Test Results - Research
Report of Fertilizer Vol.1, p. 50 - 59 (2008)
3)
Hisanori ARAYA and Yoshimi TAKEBA: Validation of atomic absorption spectrometryfor determination of cadmium, lead, nickel and chromium in calcined sludge fertilizer Research Report of Fertilizer Vol.3, p. 30 - 42 (2010)454

Testing Methods for Fertilizers (2018)(5)

Flow sheet for lead: The flow sheet for lead in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8 - 16 hours

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← About 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water
Water
(up
to
the marked line)←
Filtration
Type 3 filter paper
Sample solution
Measurement

Atomic absorption spectrometer (217.0 nm or 283.3 nm)

Figure Flow sheet for lead in fertilizers

455

Testing Methods for Fertilizers (2018)

5.6.b ICP Optical Emission Spectrometry(1) Summary
The test method is applicable to sludge fertilizers, etc. This testing method is classified as Type D
and its symbol is 5.6.b-2017 or Pb.b-1.
Pretreat an analytical sample with incineration, nitric acid−hydrochloric acid (1+3), introduce it toan ICP Optical Emission Spectrometer (“ICP-OES”) and measure the emission with lead at awavelength of 220.351 nm to obtain the lead (Pb) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 6.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Lead standard solution (Pb 0.1 mg/mL): A lead standard solution (Pb 0.1 mg/mL) traceable
to National Metrology.
e) Lead standard solutions (Pb 2.5 µg/mL) (1) (2): Dilute a predetermined amount of leadstandard solution (Pb 0.1 mg/mL) with hydrochloric acid (1+23) to prepare a lead standard
solution (Pb 2.5 µg/mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
Comment 1 Instead of the lead standard solution in (2), a lead standard solution for the calibration
curve preparation can be prepared by using a lead standard solution (Pb 1 mg/mL or
10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Electric furnace: An electric furnace that can keep the test temperature at 450 °C ± 5 °C .
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (3).
c) Ignite at 450 ºC ± 5 ºC for 8 - 16 hours to incinerate (3).
d) After standing to cool, moisten the residue with a small amount of water, and add about 10
mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (4), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (5) to the digest, cover
the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200 mL volumetric flask with water, add water
up to the marked line, and filter with Type 3 filter paper to make a sample solution.
456

Testing Methods for Fertilizers (2018)i)

As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.

Note (3) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 450 °C in 1 to 2 hours.
(4) The watch glass can be removed.
(5) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of the sample
solution will be hydrochloric acid (1+23). For example, when a 100-mL volumetricflask is used in the procedure in h), about 25 mL of hydrochloric acid (1+5) should be
added.
Comment 2 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers not containing
organic matters.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) of 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K 0116
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Optical Emission Spectrometry used in measurement.
a) Measurement conditions for the ICP Optical Emission Spectrometer: Set up themeasurement conditions for the ICP Optical Emission Spectrometer considering thefollowing:
Analytical line wavelength: 220.351 nmb) Calibration curve preparation and sample measurement
1) Put 5mL of sample solution to three 10-mL volumetric flasks respectively.
2) Add 2 mL and 4 mL of lead standard (0.25 μg/mL) solution to volumetric flasks of 1) above,
then add hydrochloric acid (1+23) to the marked line to make a sample solution of Standard
Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flask of 1)
above to make a sample solution without a standard solution.
4) Spray the sample solution of Standard Addition Method and the sample solution without astandard solution into the induction plasma, and read the indicated value at a wavelength of
220.351 nm.
5) Transfer 5 mL of blank test solution to a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value obtained
from the respective sample solutions.
6) Prepare a curve for the relationship between the added lead concentration and the correctedindicated value of the sample solution for Standard Addition Method and the sample solution
without a standard solution.
7) Obtain the lead content from the intercept of the calibration curve, and calculate lead (Pb) in
the analytical sample.
Comment 4 Instead of the correction method in c) 5), the lead (Pb) in the analytical sample canalso be corrected by obtaining the lead content in the blank test solution.
Comment 5 The simultaneous measurement of multiple elements by an ICP-OES is available. Inthat case, see 4.9.1.b Comment 5.
Comment 6 The comparison of the measurement value (xi：1.1 mg/kg - 69.0 mg/kg) of ICP
Optical Emission Spectrometry and the measurement value (yi) of Flame Atomic
Absorption Spectrometry was conducted to evaluate trueness using sludge fertilizers(49 samples). As a result, a regression equation was y = − 0.31 + 1.045x and its
457

Testing Methods for Fertilizers (2018)

correlation coefficient (r) was 0.993. Triplicates measurement for each one sample ofsewage sludge fertilizer, human waste sludge fertilizer, industrial sludge fertilizer,
mixed sludge fertilizer, calcined sludge fertilizers and composted sludge fertilizer
was conducted. As a result, a repeatability obtained was 0.9 % - 3.3 % as a relative
standard deviation.
Additionally, the minimum limit of quantification of this testing method is about 5
mg/kg.
References
1)
Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI and Tetuya NOMURA:
Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer, Vol.4, p. 30 - 35 (2011)(5)

Flow sheet for lead: The flow sheet for lead in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC, 8 - 16 hours

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for lead in sludge fertilizers (Extraction procedure)

458

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0 mL, 2 mL and 4 mL of lead standard solution (2.5 μg / mL) respectively← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Optical Emission Spectrometer (220.351 nm)
Figure 2 Flow sheet for lead in sludge fertilizers (Measurement procedure)

459

Testing Methods for Fertilizers (2018)

5.6.c ICP Mass Spectrometry (Fluid sludge fertilizers)(1) Summary
The test method is applicable to fluid sludge fertilizers. This testing method is classified as Type D
and its symbol is 5.6.c-2017 or Pb.c-1.
Add nitric acid−hydrogen peroxide to an analytical sample to heat and extract by microwave
irradiation. After adding an internal standard element, introduce it to an ICP Mass Spectrometer(ICP-MS) and measure the respective indicated values of lead and an internal standard element with
mass/ charge number (m/z) and obtain lead (Pb) in the analytical sample from the ratio of theindicated value for lead and the indicated value for an internal standard element. In addition, theperformance of this testing method is shown in Comment 4.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A4 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
c) Nitric acid: Nitric acid used to dilute a standard solution and a sample solution is a highly
pure reagent specified in JIS K 9901.
d) Hydrogen peroxide: A JIS Guaranteed Reagent specified in JIS K 8230 or a reagent ofequivalent quality.
e) Rhenium standard solution (Re 1 mg/mL): A rhenium standard solution (Re 1 mg/mL)
traceable to National Metrology.
f) Rhenium standard solution (Re 0.1 µg/mL) (1)(2)(3): Dilute a predetermined amount ofrhenium standard solution (Re 1 mg/mL) with nitric acid (1+19) to prepare a rhenium
standard solution (Re 0.1 µg/mL).
g) Rhodium standard solution (Rh 1 mg/mL) (4): A rhodium standard solution (Rh 1 mg/mL)
traceable to National Metrology.
h) Rhodium standard solution (Rh 0.1 µg/mL) (1)(2)(3): Dilute a predetermined amount ofrhodium standard solution (Rh 1 mg/mL) with nitric acid (1+19) to prepare a rhodium
standard solution (Rh 0.1 µg/mL).
i) Lead standard solution (Pb 0.1 mg/mL): A lead standard solution (Pb 0.1 mg/mL) traceable
to National Metrology.
j) Lead standard solution (Pb 5 µg/mL) (1)(2)(3): Transfer 5 mL of lead standard solution (Pb
0.1 mg/mL) to a 100-mL volumetric flask and add nitric acid (1+19) to the marked line.
k) Lead standard solutions (Pb 1 ng/ mL - 100 ng/ mL) for the calibration curve
preparation (1)(2)(3): Transfer 0.02 mL - 2 mL of lead standard solution (Pb 5 µg/mL) to
100-mL volumetric flasks step-by-step, and add 10 mL of rhenium standard solutions (Re 0.1µg/mL) as an internal standard respectively (5) and add nitric acid (1+19) up to the markedline.
l) Blank test solution for the calibration curve preparation (1)(2)(3): transfer 10 mL of rhenium
standard solution (Re 0.1 µg/mL) to 10-mL volumetric flasks as internal standard respectively(5)and add nitric acid (1+19) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in cool and dark place, and do not use after 1 month after preparation.
(3) For storage, use a sealable container made of materials such as polypropylene
containing no lead.
(4) Use when measuring arsenic, cadmium, nickel or chromium simultaneously.
(5) Add an internal solution that is 1/10 of the volume to be prepared.
Comment 1 Instead of the lead standard solution in (2), a lead standard solution for the calibration
curve preparation can be prepared by using a lead standard solution (Pb 1 mg/mL or
460

Testing Methods for Fertilizers (2018)10 mg/mL) traceable to National Metrology.
(3) Instruments: Instruments are as shown below:a) ICP Mass Spectrometer: High-frequency plasma mass spectrometer specified in JIS K 0133.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.995 % in purityb) Pressure vessel decomposing device: A device which pressurizes the inside of a vessel by
putting acid, etc.to heat in the airtight vessel, and decomposes a sample by the interaction ofheating, pressurizing and acid. The following requirements should be met.
1) The main part of a decomposing device: In the case of the microwave heating method, adevice should be able to produce a high-frequency wave using a frequency which is permitted
at an industrial frequency facility. It is desirable to be able to monitor pressure andtemperature, etc. in the airtight vessel with a sensor inside the device. The interior of a device
should be acid-resistant treated and should have a high temperature durability and a high levelof safety.
2) Exhaust system: A system which has an exhaust fan of acid-resistant specification, and has
an air-cool function inside the device to allow constant airflow, keeping operating temperature
below a certain temperature level.
3) Airtight vessel: A vessel which is heat-resistance and pressure-tight and has the durability
required to decompose particles, and has resistance to internal contamination. It should havesafety functions such as causing overheat prevention valve to work and internal pressure todrop by emitting gas, and preventing gas bumping in the case of exceeding a pressure
limitation.
c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 20.0 g (6) of an analytical sample, and put it into an airtight vessel.
b) Gradually add 2.5 mL of nitric acid and 2 mL of hydrogen peroxide.
c) Put the airtight vessel into the main part of a decomposing device and heat using a microwave(7).
d) Ignite at 240 ºC ± 5 ºC for no less than 10 minutes (7) to decompose (8).
e) After standing to cool, transfer the solution to a 50-mL volumetric flask (9) with water.
f) Add water up to the marked line and transfer it to a 50-mL ground-in stopper centrifugal
precipitate tube (9).
g) Centrifuge it at 1700 × g centrifugal force for about five minutes (10) and use the supernatantas the sample solution.
h) As a blank test, conduct the procedures in b) - g) using another airtight vessel to prepare theblank test solution.
Note (6) The maximum limit of solid content, which is converted from moisture content, in thesampling volume 20.0 g of an analytical sample is about 0.5 g. If solid content is likely
to exceed the limit, reduce sampling volume as necessary.
(7) Condition examples for a microwave decomposing device: 0 min (room temperature)→ 10min (240 ºC) → 20 min (240 ºC) → 40 min (room temperature), initial output
1400 W(8) When organic matters still remain, for example the digestion solution is colored,
repeat the procedures in (4.1) b) - d).
(9) The vessel should be made of polypropylene, etc. to not affect the measurement.
(10) 16.5-cm of radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
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Testing Methods for Fertilizers (2018)

Comment 2

The procedure in (4.1) is the same as the procedure in (4.1) of 5.1.b.

(4.2) Measurement: Conduct measurement (Internal Standard Method) according to JIS K 0113
and as shown below. Specific measurement procedures are according to the operation methodof the ICP Mass Spectrometer used in measurement.
a) Measurement conditions for the ICP Mass Spectrometer: Set up the measurement
conditions for the ICP Mass Spectrometer considering the following:
Lead: monitor ion (m/z)：208、206、207
Rhenium: monitor ion (m/z): 187b) Calibration curve preparation
1) Spray lead standard solution for the calibration curve preparation and blank test solution forthe calibration curve preparation into the inductively coupled plasma, and read the ratio of therespective ion count numbers for the monitor ion of an element subjected to measurement andan internal standard element.
2) Prepare a curve for the relationship between the concentration and the ratio of the ion countnumber of an element subjected to measurement.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 µg - 5 µg asPb) to a 50-mL volumetric flask (9).
2) Add 5mL of internal standard solution (5) and add nitric acid (1+19) to the marked line.
3) Conduct procedures similarly as in b) 1) to read the ratio of the ion count number.
4) For blank test solution, conduct procedures similarly as in 1) - 3) to correct the ratio of the ion
count number obtained for the sample solution.
5) Obtain the lead content from the calibration curve, and calculate lead (Pb) in the analyticalsample.
Comment 3 Instead of the correction method in c) 4), the lead (Pb) in the analytical sample canalso be corrected by obtaining the lead content in the blank test solution.
Comment 4 Triplicates additive recovery testing was conducted to evaluate trueness using fluid
industrial sludge fertilizers (2 samples) and composted sludge fertilizers (6 samples).
As a result, the mean recovery at the concentration level of 10 mg/kg - 20 mg/kg, 1
mg/kg - 5 mg/kg and 0.1 mg /kg - 0.9 mg /kg are 91.2 % - 103.1 %, 85.0 % 113.9 % and 93.2 - 108.1 % as lead (Pb) in an actual article respectively.
The results of the repeatability tests on different days using two kinds of fluid sludge
fertilizers to evaluate precision were analyzed by the one-way analysis of variance.
Table 1 shows the calculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this test method is about 4 µg
/kg.

462

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the repeatability tests on different days
Name ofsampleTest days of2)

repeatability Average
1)(mg/kg)T

Repeatability

Intermediate precision

3)

RSD I(T)(%)

sr(mg/kg)

4)
RSD r

(%)

s I(T)(mg/kg)5)Composted sludge fertilizer 1

5

2.80

0.09

3.1

0.27

9.7

Composted sludge fertilizer 2

5

0.740

0.014

1.9

0.020

2.7

6)

1） The number of test days conducting a duplicate test
5） Intermediate standard deviation
2） Average （the number of test days（T ）6） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Repeatability standard deviation
4） Repeatability relative standard deviation

Comment 5 Simultaneous measurement of multiple elements by an ICP-MS is available. In thatcase, see 5.2.c Comment 5.
Comment 6 The presence and the extent of interference to the measurement mass number of anelement subjected to measurement and an internal standard element can be estimatedby qualitative analysis using an ICP-MS in advance to quantitation. Select ameasurement mass number considering the extent of interference. However, a mass
number cannot be changed in the measurement of arsenic. A magnetic sector
double-focusing mass spectrometer or a collision reaction cell specified in JIS K0133
can be used as a method to reduce spectrum interference.
References1)
Toshiharu YAGI: Determination of Heavy Metals in Fluid Sludge Fertilizers by ICP-MSand CV-AAS, Research Report of Fertilizer Vol. 8, p. 26 - 37 (2015)(5)

Flow sheet for lead: The flow sheet for lead in fluid sludge fertilizers is shown below:

20.0 g analytical sample
Airtight vessel← Nitric acid, 2.5 mL← Hydrogen peroxide, 2 mL
Microwave
decomposition
Transfer
50-mL volumetric flask, water← Water (up to the marked line)
Ground-in stopper centrifugal precipitate tube,
Centrifugal separation
Anout 1700 × g , 5 minutes
Sample solution
Figure 1 Flow sheet for lead in liquid sludge fertilizers (Extraction procedure)

463

Testing Methods for Fertilizers (2018)

Sample solution
50-mL volumetric flask
Aliquot(predetermined volume)← Rhenium standard solution (Re 0.1µg/mL) 5 mL (internal standard)← Nitric acid (1+19) (up to the marked line)
Measurement
ICP-MS (Pb: m /z 208, 206, 207 Re: m /z 187)
Figure 2 Flow sheet for lead in liquid sludge fertilizers (Measurement procedure)464

Testing Methods for Fertilizers (2018)

5.7 Sulfamic acid (amidosulfuric acid)5.7.a Ion Chromatography(1) Summary
The testing method is applicable to ammonium sulfate. This testing method is classified as Type D
and its symbol is 5.7.a-2017 or AS-acid.a-1.
Add water to an analytical sample to extract sulfamic acid, introduce it to an Ion Chromatograph(IC) or a High-Performance Liquid Chromatograph (HPLC) to isolate it with an ion exchange
column, then measure the sulfamic acid with an electric conductivity detector to obtain sulfamicacid (amidosulfuric acid) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 5.
Sulfamic acid and sulfurized cyanide (ammonium thiocyanate) can be simultaneously quantified byusing this method. (Refer to Comment 4).
(2)
a)
b)
c)
d)
e)
f)

Reagent, etc.: Reagents and water are as shown below.
Water: Water of A3 specified in JIS K 0557.
Phthalic acid: A reagent of no less than 98 % (mass fraction) in purity.
p-hydroxybenzoic acid: A reagent of no less than 95 % (mass fraction) in purity.
1-sodium octane sulfonate: A reagent of no less than 98 % (mass fraction) in purity.
1-sodium hexane sulfonate: A reagent of no less than 98 % (mass fraction) in purity.
Boric acid: A JIS Guaranteed Reagent specified in JIS K 8863 or a reagent of equivalentquality.
g) Eluent (1) (2): Weigh 0.083 g of phthalic acid, 0.552 g of p-hydroxybenzoic acid, 0.195 g of
1-sodium octane sulfonate, 0.376 g of 1-sodium hexane sulfonate and 6.183 g of boric acid toa 1000-mL volumetric flask, add about 500 mL of water to dissolve and add water up to themarked line. Filter with a membrane filter (aperture diameter: no more than 0.5 µm) made ofhydrophilic PTFE.
h) Sulfamic acid standard solution (1000 mg/L) (1): Put 0.1 g of sulfamic acid, referencematerial for volumetric analysis (HOSO2NH2: dried for 48 hours in a silica gel desiccator), ina weighing dish and measure the mass to the order of 0.1 mg. Add a small amount of water todissolve, then transfer to a 100-mL volumetric flask and add water up to the marked line.
i) Sulfamic acid standard solution (10 mg/L) (1): At the time of usage, put 2.5 mL of sulfamicacid standard solution (1000 mg/L) to a 250-mL volumetric flask and add water up to themarked line.
j) Sulfamic acid standard solution for the calibration curve preparation (0.3 mg/L - 3
mg/L): At the time of usage, put 3 mL - 30 mL of sulfamic acid standard solution (10 mg/L)
to 100-mL volumetric flasks step-by-step and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) The concentration of prepared solutions is phthalic acid 0.5 mmol/L,
p-hydroxybenzoic acid 4.0 mmol/L, 1-sodium octane sulfonate 0.9 mmol/L, 1-sodium
hexane sulfonate 2.0 mmol/L and boric acid 100 mmol/L.

(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Ion Chromatograph (IC) or High-Performance Liquid Chromatograph (HPLC): IC
specified in JIS K 0127 or HPLC specified in JIS K 0124 that satisfies following
requirements.
1) Column: A 4-mm inner diameter 100-mm long stainless-steel column tube filled withhydrophilic methacrylate-gel, to which 5-μm particle diameter class 4 ammonium groupchemically bonds (3).
2) Column bath: A column bath whose temperature can be adjusted to 55 ºC - 60 ºC.
465

Testing Methods for Fertilizers (2018)

3) Detection unit: An electric conductivity detectorb) Membrane filters: Pore size is no more than 0.5 μm, made of hydrophilic PTFE
Note (3) A column is commercially sold under the name Shodex IC NI-424, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water and shake to dissolve, and then add water up to the marked line.
c) Transfer a predetermined amount of the solution, and dilute exactly by a factor of 12.5 withwater.
d) Filter with a membrane filter (pore size: no more than 0.5 μm) to make a sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0127 and as shown below.
Specific measurement procedures are according to the operation method of the Ion
Chromatograph (IC) or the High-Performance Liquid Chromatograph (HPLC) used inmeasurement.
a) Measurement conditions for the Ion Chromatograph (IC) or High-Performance Liquid
Chromatograph (HPLC): An example of measurement conditions is shown below. Set up
the measurement conditions considering it:1) Column: A hydrophilic methacrylate-gel column (4-mm inner diameter, 100-mm long, 5-μm
particle diameter) to which quaternary ammonium group chemically bonds.
2) Column bath temperature: 58 °C
3) Eluent: Prepared by the procedures in (2) g)
4) Flow rate: 1 mL/min
5) Injection volume: 20 µL
6) Detection unit: An electric conductivity detectorb) Calibration curve preparation
1) Inject 20 μL of respective standard solutions for the calibration curve preparation to an IC oran HPLC, and record the chromatogram of electric conductivity to obtain peak area.
2) Prepare a curve for the relationship between the concentration and the peak area of electric
conductivity of respective standard solutions for the calibration curve preparation.
Prepare a calibration curve when the sample is measured.
Comment 1 In the measurement of a sample solution, there is a possibility that the recovery rate
becomes lower than actual due to the influence of matrix if the concentration is calculated withpeak height. Therefore, prepare a calibration curve using peak area.
c) Sample measurement
1) Subject 20 μL of sample solution to the same procedure as in b) 1)
2) Obtain the sulfamic acid content from calibration curve by peak area to calculate the sulfamicacid (amidosulfuric acid) in the analytical sample.
Comment 2 Calculate the concentration by the peak area similarly as the calibration curve
preparation to prevent the influence of matrix.
Comment 3 Note that it takes time to stabilize the baseline due to the usage of the ion-pairing
reagent in the elute. It is recommended to take about 120 minutes for stabilization
time before starting measurement.
Comment 4 It is possible for the simultaneous measurement of sulfamic acid and ammonium
466

Testing Methods for Fertilizers (2018)

thiocyanate in this testing method. In that case, mix a predetermined amount of
sulfamic acid standard solution (1000 mg/L) and ammonium thiocyanate standard
solution (1000 mg/L), dilute with water to prepare a mixture standard solution (10mg/L) (1) and use it instead of respective standard solutions (10 mg/L) in (2) i). After
that, conduct the same procedure in (4.2) b) to calculate the respective concentrationsof materials subjected to measurement in the analytical sample.
Comment 5 A recovery testing of ammonium sulfate (3 brands) was conducted. As a result, themean recovery at additive level of 0.25 % (mass fraction) and 0.075 % (massfraction) was 94.4 % - 103.5 % and 94.4 % - 100.8 %.
Additionally, the minimum limit of quantification of this testing method is about
0.04 % (mass fraction).
References1)
Toshiaki HIROI and Yuji SHIRAI: Simultaneous Determination of Sulfamic Acid andAmmonium Thiocyanate in Ammonium Sulfate by Non-suppressed Ion
Chromatography, Research Report of Fertilizer, Vol.5, p. 1 - 23 (2012)(5)

Flow sheet for testing method: The flow sheet for sulfamic acid in ammonium sulfate isshown below:

1.00 gWeigh into a 100-mL volumetric flask← About 50 mL of water
Shaking to mix
Stopple the volumetric flask and dissolve← Water (up to the marked line)
Dilution
Dilute 12.5 times, water
Filtration

Membrane filter (no more than 0.5-µm)

Sample solution
Measurement

Ion Chromatograph

Figure Flow sheet for sulfamic acid in ammonium sulfate

467

Testing Methods for Fertilizers (2018)

Reference: The IC chromatogram of sulfamic acid and thiocyanic acid of the standard solution
for the calibration curve preparation and sample solution (ammonium sulfate) are
shown below.

１2(A) Mixture standard solution (the equivalents of 60 ng as sulfamic acid and ammonium
thiocyanate (3 mg/L, 20 μL), respectively)

Enlarged diagram of peak forsufamic acid １

１2(B) Sample solution (the equivalents of 0.25 % (mass fraction) as sulfamic acid andammonium thiocyanate added in ammonium sulfate (2500 μg/g), respectively)
Reference diagram: IC chromatogram of sulfamic acid and thiocyanic acid.
(Peak: 1. Sulfamic acid, 2. Ammonium thiocyanate)
IC measurement conditions
Column: Shodex IC NI-424 (4.6-mm inner diameter, 100-mm long, 5μm-particle
diameter)Other conditions are according to the example of HPLC measurement conditions in(4.2) a)468

Testing Methods for Fertilizers (2018)

5.7.b High-Performance Liquid Chromatograph Mass Spectrometry(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 5.7.b-2017 or AS-acid.b-1.
Add water to an analytical sample to extract sulfamic acid, introduce it to a High-Performance
Liquid Chromatograph Mass Spectrometer to isolate it with a silica gel column, to whichcrosslink-type diol chemically bonds, and measure with a Selected Ion Monitoring (SIM) method to
obtain sulfamic acid (amidosulfuric acid) in an analytical sample. In addition, the performance of
this testing method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557. Note that water of A4 or equivalent quality
should be used as the eluent which is introduced to LC-MS.
b) Acetonitrile: A reagent of LC-MS analysis grade or equivalents.
c) Formic acid: A reagent of LC-MS analysis grade or equivalents.
d) Ammonium formate buffer solution (pH 3.2): Dilute 3.153 g of ammonium formate (noless than 95 % (mass fraction) in purity) with water to make 500 mL and adjust to pH 3.2 withformic acid.
e) Sulfamic acid standard solution (1 mg/L): Put 0.1 g of sulfamic acid, reference material forvolumetric analysis specified in JIS K 8005, in a weighing dish and measure the mass to theorder of 0.1 mg. Add a small amount of water to dissolve, then transfer to a 100-mL
volumetric flask and add water up to the marked line.
f) Sulfamic acid standard solution (10 μg/L) (1): At the time of usage, put 2.5 mL of standard
solution (1 mg/L) to a 250-mL volumetric flask and add water up to the marked line.
g) Sulfamic acid standard solution (200 ng/L) (1): At the time of usage, put 5 mL of standard
solution (10 µg/L) to a 250-mL volumetric flask and add water up to the marked line.
h) Sulfamic acid standard solution for the calibration curve preparation (10 ng/L - 600ng/L): At the time of usage, put 2.5 mL - 6 mL of sulfamic acid standard solution (10 µg/L) to
100-mL volumetric flasks step-by-step and add water up to the marked line. Similarly put 5
mL - 50 mL of sulfamic acid standard solution (200 ng/L) to 100-mL volumetric flasks
step-by-step and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph/Mass Spectrometer (LC-MS): LC-MS
specified in JIS K 0136 that satisfies the following requirements.
1) High-Performance Liquid Chromatograph(i) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
(ii) Column: A 2-mm - 3-mm inner diameter 100-mm - 150 mm long stainless-steel column
tube filled with silica gel to which 5-μm crosslink-type diol chemically bonds or
polyhydroxymethacrylate. This should comply with the specification of a Mass
Spectrometer.
2) Mass Spectrometer(i) Ionization method: Electro-Spray Ionization (ESI) method(ii) Ion detection method: Selected Ion Monitoring (SIM) methodb) Magnetic stirrer:c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
d) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g - 10000
× g.
469

Testing Methods for Fertilizers (2018)

Comment 1 A high Performance Liquid Chromatograph/Mass Spectrometer (LC-MS/MS) can beused instead of a High-Performance Liquid Chromatograph/Mass Spectrometer(LC-MS). In this case, set up the measurement conditions considering (4.3) a) The
measurement conditions of High-Performance Liquid Chromatograph/Mass
spectrometer and confirm that a calibration curve can be prepared by the procedurein b) in advance.
Comment 2 A column is sold under the name LUNA HILIC or Shodex ODP2 HP-2D.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 1.00 g of an analytical sample, and put it into a 200-mL ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge it at 1700 × g centrifugal force for about five minutes (2) and use the supernatant as
the extract.
Note (2) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
Comment 3 Instead of the procedures in (4.1.1) c) and d), it is allowed to filter with Type 3 filter
paper and the filtrate can be the extract.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water to the marked line to make the extract(4.2) Extraction: Conduct extraction as shown below.
a) Transfer 2 mL of the sample solution to a 200-mL Erlenmeyer flask.
b) Add water up to the marked line and transfer it to a 1.5-mL ground-in stopper centrifugal
precipitate tube (3).
c) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (4) and use thesupernatant as the extract.
Note (3) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(4) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 4 Instead of the procedures in (4.2) b) and c), it is allowed to filter with a membranefilter (aperture diameter: no more than 0.5 µm) made of hydrophilic PTFE and thefiltrate can be the sample solution.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0136 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph Mass Spectrometer used in measurement.
470

Testing Methods for Fertilizers (2018)a) The measurement conditions of High-Performance Liquid Chromatograph/Mass
spectrometer: An example of measurement conditions is shown below. Set up themeasurement conditions considering it:
1) High-Performance Liquid Chromatograph(i) Column: A silica gel column to which crosslink-type diol chemically bonds or
polyhydroxymethacrylate (2-mm - 3-mm inner diameter, 100-mm - 150-mm long, 5-µm
particle diameter).
(ii) Flow rate: 0.2 mL/min(iii) Eluent: Ammonium formate buffer solution - Acetonitrile (1+9）(iv) Temperature of column bath: 40 °C(v) Injection rate: 1 μL(vi) Measurement time: About 20 minutes
2) Mass Spectrometer(i) Ionization method: Electro-Spray Ionization (ESI) method(ii) Mode: Negative(iii) Capillary voltage: − 3.5 kv(iv) Ion source temperature: 300 °C(v) Nebulizer gas rate: 1.5 L/min(vi) Desolvation temperature: 250 °C(vii) Monitor ion: m/z 95.9b) Calibration curve preparation
1) Inject 1 μL of respective standard solutions for the calibration curve preparation to an LC-MS,
and record the chromatogram of monitor ion (m/z) to obtain peak area.
2) Prepare a curve for the relationship between the sulfamic acid concentration and the peak areaof monitor ion of respective standard solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 1 μL of sample solution to the same procedure as in b) 1)
2) Obtain the sulfamic acid content from calibration curve to calculate the sulfamic acid(amidosulfuric acid) in the analytical sample.
Comment 5 Recovery testing was conducted using samples that sulfamic acid equivalent to 1/5 4 times of permissible content are added to ammonium sulfate fertilizer (1 brand),by-product nitrogen fertilizer (1 brand), by-product mixed fertilizer (1 brand),compound fertilizer (1 brand) and fluid mixed fertilizer (1 brand). As a result, themean recovery at the additive level of 0.1 % (mass fraction), 0.025 % (mass fraction)and 0.005 % (mass fraction) are 97.6 % - 104.2 %, 95.2 % - 107.0 % and 96.4 % 111.2 % respectively.
The results of the repeatability tests on different days using ammonium sulfate
fertilizer, by-product nitrogen fertilizer and by-product mixed fertilizer to evaluate
precision were analyzed by the one-way analysis of variance. Table 1 shows thecalculation results of intermediate precision and repeatability.
Table 2 shows results and analysis results from a collaborative study for testing
method validation. In addition, sufficient reproducibility by sulfamic acidconcentration 0.0116 % (mass fraction) was not obtainable. But sufficient
reproducibility was obtained in the scope of sulfamic acid concentration 0.0386 %(mass fraction) - 0.401 % (mass fraction).

471

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the repeatability tests on different days
Name of

Test days of

Repeatability
2)

samplerepeatability Average
T

1)

(%)

3)

sr

4)

(%)

3)

Ammonium sulfate
50.09740.0011
By-product50.06560.0014
nitrogen fertilizer
Compound
5
0.005
0.000
fertilizer
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

RSD r
(%)

Intermediate precision5)s I(T)

6)(%)

RSD I(T)
(%)

3)

1.1

0.00272.7

2.1

0.00172.6

2.4

0.00029

5.8

7)6） Intermediate standard deviation
7） Intermediate relative standard deviation

Table 2 Results and analysis results from a collaborative study
for sulfamic acid testing method validation
Sample name

Number of
1)

Mean

2)

sr

4)

RSD r

5)

6)

RSD R

sR

7)

laboratories

(%)

3)

(%)

3)

(%)

(%)

3)

(%)

Ammonium sulfate
By-product nitrogen
fertilizer
By-product mixed
fertilizer

9

0.203

0.021

10.4

0.024

11.9

9

0.401

0.030

7.5

0.035

8.8

7

0.09570.00434.5

0.0043

4.5

Compounf fertilizer

9990.0166
0.03810.243

0.00280.0022
0.011

16.8
5.8
4.5

0.00480.0049
0.018

29.1
12.8
7.6

Fluid mixed fertilizer 1
Fluid mixed fertilizer 2
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories × number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

References
1)
Kohei ITO, Mariko FUJITA, Yoshimi HASHIMOTO and Yuji SHIRAI: Determinationof Sulfamic Acid in Fertilizer by Liquid Chromatography/Mass Spectrometry (LC-MS),
Research Report of Fertilizer Vol. 8, p. 38 - 49 (2015)
2)
Tomoharu NOZAKI Determination of Sulfamic Acid in Fertilizers using Liquid
Chromatography-Mass Spectrometer (LC-MS): A Collaborative Study, Research Reportof Fertilizer Vol. 9, p. 69 - 76 (2016)
472

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for sulfamic acid in fertilizers is shown
below:

1.00 g analytical sample(powdery)

200-mL ground-in stopper Erlenmeyer flask← 100 mL of water
Extraction
Centrifugal separation

Stir to mix, 10 minites
Ground-in stopper centrifugal precipitate tube,
1700 ×g , 5 minutes

Extract

Figure 1-1 Flow sheet for sulfamic acid in fertilizers(Extraxction procedure (4.1.1))
1.00 g100-mL volumetric flask
analytical sample (fluid)← About 50 mL of water
Extraction
Shake to mix
Water
(up
to
the marked line)←
Extract

Figure 1-2 Flow sheet for sulfamic acid in fertilizers(Extraxction procedure (4.1.2))
Extract

Aliquot (5 mL)
100-mL volumetric flask← Water (up to the marked line)
Centrifugal separation
Ground-in stopper centrifugal precipitate tube,
or filtration
8000 ×g - 10000 ×g , 5 minutes
Sample solution
Measurement

Liquid Chromatograph Mass Spectrometer

Figure 2 Flow sheet for sulfamic acid in fertilizers(Dilution and measurement procedure)473

Testing Methods for Fertilizers (2018)

Reference:

The chromatogram of sulfamic acid for the calibration curve preparation is shown
below.

Sulfamic acidSulfamic acid

Retention time

Retention time(A) Standard solution(The equivalent of 0.6 ng as sulfamic
acid)(The equivalent of 0.1 % of massfraction as sulfamic acid is added to acompound(B) fertilizer)
Sample solution

Reference diagram: Chromatogram of sulfamic acid
LC-MS measurement conditions
Column: LUNA HILIC (2.0-mm inner diameter, 100-mm long, 5-μm particle
diameter)Other conditions are according to the example of LC-MS measurement conditionsin (4.3) a)474

Testing Methods for Fertilizers (2018)

5.8 Ammonium thiocyanate (Sulfurized cyanide)
5.8.a Ion Chromatography(1) Summary
The testing method is applicable to ammonium sulfate. This testing method is classified as Type D
and its symbol is 5.8.a-2017 or SCN.a-1.
Add water to an analytical sample to extract ammonium thiocyanate (sulfurized cyanide), introduce
it to an Ion Chromatograph (IC) or a High-Performance Liquid Chromatograph (HPLC) to isolate itwith an ion exchange column, then measure the thiocyanic acid with an electric conductivity
detector to obtain ammonium thiocyanate (sulfurized cyanide) in an analytical sample. In addition,
the performance of this testing method is shown in Comment 5.
Sulfamic acid and ammonium thiocyanate (sulfurized cyanide) can be simultaneously quantified byusing this method. (Refer to Comment 4).
(2)
a)
b)
c)
d)
e)
f)

Reagent, etc.: Reagents and water are as shown below.
Water: Water of A3 specified in JIS K 0557.
Phthalic acid: A reagent of no less than 98 % (mass fraction) in purity.
p-hydroxybenzoic acid: A reagent of no less than 95 % (mass fraction) in purity.
1-sodium octane sulfonate: A reagent of no less than 98 % (mass fraction) in purity.
1-sodium hexane sulfonate: A reagent of no less than 98 % (mass fraction) in purity.
Boric acid: A JIS Guaranteed Reagent specified in JIS K 8863 or a reagent of equivalentquality.
g) Eluent (1) (2): Weigh 0.083 g of phthalic acid, 0.552 g of p-hydroxybenzoic acid, 0.195 g of
1-sodium octane sulfonate, 0.376 g of 1-sodium hexane sulfonate and 6.183 g of boric acid toa 1000-mL volumetric flask, add about 500 mL of water to dissolve and add water up to themarked line. Filter with a membrane filter (aperture diameter: no more than 0.5 µm) made ofhydrophilic PTFE.
h) Ammonium thiocyanate standard solution (1000 mg/L) (1): Put 0.1 g of ammonium
thiocyanate (3), specified in JIS K 9000 in weighing dish, and measure the mass to the order of
0.1 mg. Add a small amount of water to dissolve, then transfer to a 100-mL volumetric flask
and add water up to the marked line.
i) Ammonium thiocyanate standard solution (10 mg/L) (1): At the time of usage, put 10 mL ofammonium thiocyanate standard solution (1000 mg/L) to a 100-mL volumetric flask and add
water up to the marked line.
j) Ammonium thiocyanate standard solution for the calibration curve preparation (0.3mg/L - 3 mg/L): At the time of usage, put 3 mL - 30 mL of ammonium thiocyanate solutions(10 mg/L) to 100-mLvolumetric flasks step-by-step and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) The concentration of prepared solutions is phthalic acid 0.5 mmol/L,
p-hydroxybenzoic acid 4.0 mmol/L, 1-sodium octane sulfonate 0.9 mmol/L, 1-sodium
hexane sulfonate 2.0 mmol/L and boric acid 100 mmol/L.
(3) It is recommended to store in a desiccator because of deliquescence.

(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Ion Chromatograph (IC) or High-Performance Liquid Chromatograph (HPLC): IC
specified in JIS K 0127 or HPLC specified in JIS K 0124 that satisfies following
requirements.
1) Column: A 4-mm inner diameter 100-mm long stainless steel column tube filled withhydrophilic methacrylate-gel, to which 5-μm particle diameter class 4 ammonium groupchemically bonds (4).
475

Testing Methods for Fertilizers (2018)2) Column bath: A column bath whose temperature can be adjusted to 55 ºC - 60 ºC.
3) Detection unit: An electric conductivity detectorb) Membrane filters: Pore size is no more than 0.5 μm, made of hydrophilic PTFE
Note (4) A column is commercially sold under the name Shodex IC NI-424, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water and shake to dissolve, and then add water up to the marked line.
c) Transfer a predetermined amount of the solution, and dilute exactly by a factor of 12.5 withwater.
d) Filter with a membrane filter (pore size: no more than 0.5 μm) to make a sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0127 and as shown below.
Specific measurement procedures are according to the operation method of the Ion
Chromatograph (IC) or the High-Performance Liquid Chromatograph (HPLC) used inmeasurement.
a) Measurement conditions for the Ion Chromatograph (IC) or High-Performance Liquid
Chromatograph (HPLC):
An example of measurement conditions for an Ion
Chromatograph (IC) is shown below. Set up the measurement conditions considering it:1) Column: A hydrophilic methacrylate-gel column (4-mm inner diameter, 100-mm long, 5-μm
particle diameter) to which quaternary ammonium group chemically bonds.
2) Column bath temperature: 58 °C
3) Eluent: Prepared by the procedures in (2) g)
4) Flow rate: 1 mL/min
5) Injection volume: 20 µL
6) Detection unit: An electric conductivity detectorb) Calibration curve preparation
1) Inject 20 μL of respective standard solutions for the calibration curve preparation to an IC oran HPLC, and record the chromatogram of electric conductivity to obtain peak area.
2) Prepare a curve for the relationship between the concentration and the peak area of electric
conductivity of respective standard solutions for the calibration curve preparation.
Prepare a calibration curve when the sample is measured.
Comment 1 In the measurement of a sample solution, there is a possibility that the recovery rate
becomes lower than actual due to the influence of matrix if the concentration is
calculated with peak height. Therefore, prepare a calibration curve using peak area.
c) Sample measurement
1) Subject 20 μL of sample solution to the same procedure as in b) 1)
2) Obtain the ammonium thiocyanatecontent from the calibration curve by peak area to calculateammonium thiocyanate (sulfurized cyanide) in the analytical sample.
Comment 2 Calculate the concentration by the peak area similarly as the calibration curve
preparation to prevent the influence of matrix.
Comment 3 Note that it takes time to stabilize the baseline due to the usage of the ion-pairing
reagent in the elute. It is recommended to take about 120 minutes for stabilization
time before starting measurement.
476

Testing Methods for Fertilizers (2018)

Comment 4 It is possible for the simultaneous measurement of sulfamic acid and ammonium
thiocyanate (sulfurized cyanide) in this testing method. In that case, mix apredetermined amount of sulfamic acid standard solution (1000 mg/L) andammonium thiocyanate standard solution (1000 mg/L), dilute with water to prepare a
mixture standard solution (10 mg/L) (1) and use it instead of respective standard
solutions (10 mg/L) in (2) i). After that, conduct the same procedure in (4.2) b) to
calculate the respective concentrations of materials subjected to measurement in theanalytical sample.
Comment 5 A recovery testing of ammonium sulfate (3 brands) was conducted. As a result, themean recovery at additive level of 0.25 % (mass fraction) and 0.075 % (massfraction) was 101.8 % - 103.7 % and 93.9 % - 97.4 %.
Additionally, the minimum limit of quantification of this testing method is about
0.04 % (mass fraction).
References1)
Toshiaki HIROI and Yuji SHIRAI: Simultaneous Determination of Sulfamic Acid andAmmonium Thiocyanate in Ammonium Sulfate by Non-suppressed Ion
Chromatography, Research Report of Fertilizer, Vol.5, p. 1 - 23 (2012)(5)

Flow sheet for testing method: The flow sheet for ammonium thiocyanate in ammonium
sulfate is shown below:

1.00 gWeigh into a 100-mL volumetric flask← About 50 mL of water
Shaking to mix
Stopple the volumetric flask and dissolve← Water (up to the marked line)
Dilution
Dilute 12.5 times, water
Filtration

Membrane filter (no more than 0.5-µm)

Sample solution
Measurement

Ion Chromatograph

Figure Flow sheet for ammonium thiocyanate in ammonium sulfate

477

Testing Methods for Fertilizers (2018)

Reference: The IC chromatogram of sulfamic acid and thiocyanic acid of the standard solution
for the calibration curve preparation and sample solution (ammonium sulfate) are shown
below.

１
2(A) Mixture standard solution (the equivalents of 60 ng as sulfamic acid and ammonium
thiocyanate (3 mg/L, 20 μL), respectively)

Enlarged diagram of peak forsufamic acid１１2(B) Sample solution (the equivalents of 0.25 % (mass fraction) as sulfamic acid andammonium thiocyanate added in ammonium sulfate (2500 μg/g), respectively)
Reference diagram: IC chromatogram of sulfamic acid and thiocyanic acid.
(Peak: 1. Sulfamic acid, 2. Thiocyanic acid)IC measurement conditions
Column: Shodex IC NI-424 (4.6-mm inner diameter, 100-mm long, 5μm-particle
diameter)Other conditions are according to the example of HPLC measurement conditions in(4.2) a)478

Testing Methods for Fertilizers (2018)

5.8.b High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 5.8.b-2017 or SCN.b-1.
Add water to an analytical sample, extract ammonium thiocyanate (sulfurized cyanide) and adjust
pH as necessary. Introduce it into a High-Performance Liquid Chromatograph (HPLC), isolate witha silica gel column to which amino group chemically bonds or a vinyl alcohol polymer column towhich amino group chemically bonds, and measure at wavelength 210 nm to obtain ammonium
thiocyanate (sulfurized cyanide) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 5.
Nitrous acid and ammonium thiocyanate (sulfurized cyanide) can be simultaneously quantified byusing this method. (Refer to Comment 4).
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
c) Disodium hydrogen phosphate dodecahydrate: A JIS Guaranteed Reagent specified in JIS
K 9019 or a reagent of equivalent quality.
d) Sodium dihydrogen phosphate dihydrate: A JIS Guaranteed Reagent specified in JIS K
9009 or a reagent of equivalent quality.
e) Sodium perchlorate monohydrate: A JIS Guaranteed Reagent specified in JIS K 8227 or areagent of equivalent quality.
f) Ammonium thiocyanate standard solution (1 mg/L) (1): Put 0.1 g of ammonium thiocyanate
specified in JIS K 9000 in weighing dish, and measure the mass to the order of 0.1 mg. Add asmall amount of water to dissolve, then transfer to a 100-mL volumetric flask and add water
up to the marked line.
g) Ammonium thiocyanate standard solution (10 mg/L) (1): At the time of usage, put 10 mL ofammonium thiocyanate standard solution (1 mg/L) to a 100-mL volumetric flask and add
water up to the marked line.
h) Ammonium thiocyanate standard solution for the calibration curve preparation (1μg/mL - 20 μg/mL): At the time of usage, put 1 mL - 20 mL of ammonium thiocyanate
standard solution (100 μg/mL) to 100-mL volumetric flasks step-by-step and add water up to
the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 4-mm to 6-mm inner diameter 150-mm to 250-mm long stainless steel column
tube filled with poly vinyl alcohol or silica gel (2) , to which 5-μm particle diameter aminogroup chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 210 nm.
b) Magnetic stirrer:c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
d) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g - 10000
× g.
e) pH test paper: A pH test paper infilled with indicator and dried, which can measure the479

Testing Methods for Fertilizers (2018)

value from pH 1 to pH 11 and a color change chart with the pH interval value 1 is attached.
Note (2) Remaining silanol group of silica gel affects the measurement of ion in some cases.
Therefore, use a column which does not affect the measurement of sodium thiocyanateby treating the silanol group. As an example of the treatment, silica gel is to be entirely
covered with the uniform membrane of silicone polymer.
Comment 1 A column is sold under the names CAPCELL PAK NH2 UG80 or Asahipak
NH2P-50 4E.
Comment 2 pH test paper is sold under the name UNIV Test Paper, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 1.00 g of an analytical sample, and put it in a 200-mL volumetric flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge it at 1700 × g centrifugal force for about five minutes (3) and use the supernatant as
the extract.
Note (3) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water to the marked line to make the extract(4.2) pH adjustment: Conduct pH adjustment as shown below.
a) Transfer a small amount of the extract to confirm pH value using a pH-test paper.
b) If the pH value in a) is pH 5 or more, transfer the extract to a 1.5-mL ground-in stopper
centrifugal precipitate tube (4) and conduct the procedure in f) to prepare a sample solution.
c) If the pH value in a) is pH 4 or less, transfer 40 mL of the extract to a 100-mL beaker.
d) Add a sodium hydroxide solution (5 mg/mL), adjust it to pH 5 to pH 7 with a pH meter andtransfer to a 50-mL volumetric flask with water.
e) Add water up to the marked line and transfer it to a 1.5-mL ground-in stopper centrifugal
precipitate tube (4).
f) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (5) and use thesupernatant as the extract.
Note (4) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(5) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 3 Instead of procedures in (4.2) b) and e) - f), it is allowed to filter with a membranefilter (aperture diameter: no more than 0.5-µm) made of hydrophilic PTFE and thefiltrate can be the sample solution.
480

Testing Methods for Fertilizers (2018)(4.3) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High-Performance
Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for an Ion Chromatograph (IC) is shown below. Set up
the measurement conditions considering it:1) Column: A vinyl alcohol polymer column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which amino group chemically bonds or a silica gel column(4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm particle diameter) to whichamino group chemically bonds.
2) Column bath temperature: 30 °C - 40 °C
3) Eluent (1): Dissolve 1.79 g of disodium hydrogen phosphate dodecahydrate, 0.78 g of sodium
dihydrogen phosphate dihydrate and 14.04 g of sodium perchlorate monohydrate in water tomake 1000 mL. Filter with a membrane filter (aperture diameter: no more than 0.5-µm) madeof hydrophilic PTFE.
4) Flow rate: 0.9 mL/min - 1.0 mL/min
5) Injection volume: 10 µL
6) Detection unit: An absorptiometric detector, measurement wavelength: 210 nmb) Calibration curve preparation
1) Inject 10 µL of respective standard solutions for the calibration curve preparation to an HPLC,
record chromatogram at wavelength 210 nm and obtain the peak area.
2) Prepare a curve for the relationship between the concentration and the peak area atwavelength 210 nm of the respective standard solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the ammonium thiocyanate content from the calibration curve by peak area tocalculate ammonium thiocyanate (sulfurized cyanide) in the analytical sample.
Comment 4 This testing method enables the simultaneous measurement of nitrous acid andammonium thiocyanate (sulfurized cyanide). In this case, mix a predeterminedamount of nitrous acid standard solution (1 mg/mL) and ammonium thiocyanate
standard solution (1 mg/mL), dilute with water to prepare a mixture standard solution(100 μg/mL) (1) and use it instead of (2) h) ammonium thiocyanate standard solution(100 μg/mL). After that, conduct the same procedure in (4.3) b) to calculate therespective concentrations of materials subjected to measurement in the analyticalsample.
Comment 5 A recovery testing was conducted using samples that ammonium thiocyanate
equivalent to 1/5 - 5 times of permissible content are added to an ammonium sulfate
fertilizer (1 brand), a coating nitrogen fertilizer (1 brand), a blended fertilizer (2brands), a compound fertilizer (1 brand) and a fluid mixed fertilizer (1 brand). As aresult, the mean recovery at the additive level of 0.025 % (mass fraction), 0.01 %(mass fraction), 0.005 % (mass fraction) and 0.0025 % (mass fraction) are 95.4 % 100.5 %, 94.7 % - 103.8 %, 83.8 % - 109.0 % and 87.2 % - 103.3 % respectively.
Table 1 shows results and analysis results from a collaborative study for test method
validation. Ammonium thiocyanate had sufficient reproducibility in the range from0.00476 % (mass fraction) to 0.204 %. (mass fraction).
Additionally, the minimum limit of quantification of this testing method is about
0.002 % (mass fraction).
481

Testing Methods for Fertilizers (2018)

Table 1 Results and statistical analysis results from a collaborative study
for the ammonium thiocyanate method
Sample name

Number of1)laboratories

4)

RSD r

3)

(%)

Mean

2)

sr

3)

(%)

(%)

Home garden-use mixed
10
0.00476 0.00019
4.1
fertilizer 1
Home garden-use mixed
90.00976 0.00029
2.9
fertilizer 2
Home garden-use mixed
90.0506 0.0019
3.7
fertilizer 3
Compound fertilizer 1
10
0.10
0.002
2.3
Compound fertilizer 2
11
0.204
0.006
2.7
Compound fertilizer 3
100.00989 0.00037
3.81) Number of laboratories used in analysis
2) Mean (n = number of laboratories × number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

5)

6)

RSD R

(%)

3)

(%)

0.00060

12.7

0.00050

4.7

0.0022

4.3

0.003
0.008
0.00060

2.6
3.7
6.5

sR

7)

References
1)
Kohei ITO, Yasuharu KIMURA, Masanori HASEGAWA and Yuji SHIRAI:
Simultaneous Determination of Nitrous Acid and Thiocyanate in Fertilizer by
High-Performance Liquid Chromatography (HPLC), Japanese Journal of Soil Scienceand Plant Nutrition, Vol. 87 (2), p.120 - 124 (2016)
2)
Masanori HASEGAWA and Yasuharu KIMURA: Determination of Nitrous Acid andAmmonium Thiocyanate in Fertilizer by High-Performance Liquid Chromatography(HPLC): A Collaborative Study, Research Report of Fertilizer Vol. 8, p. 70 - 78 (2015)

482

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for ammonium thiocyanate in fertilizers isshown below:
1.00 g analyticalsample200-mL ground-in stopper Erlenmeyer flask(powdery)← 100 mL of water
Extraction
Centrifugal separation

Stir to mix, 10 minites
Ground-in stopper centrifugal precipitate tube,
1700 ×g , 5 minutes

Filtrate

Figure 1-1

Flow sheet for ammonium thiocyanate in fertilizers(Extraction procedure (4.1.1))

1.00 g analyticalsample100-mL volumetric flask(fluid)← 50 mL - 70 mL of water
Extraction
Shake to mix← Water (up to the marked line)
Filtrate

Figure 1-2

Flow sheet for ammonium thiocyanate in fertilizers(Extraction procedure (4.1.2))

483

Testing Methods for Fertilizers (2018)
Filtrate

pH value confirmation pH test paper
pH 5 or more
pH 4 or less
Aliquot (40 mL)
100-mL beaker
pH adjustment(pH 5 - pH 7)

Sodium hydroxide solution (5 mg/mL)Transfer
50-mL volumetric flask, water← Water (up to the marked line)
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

SampleMeasurement

High Performance Liquid Chromatograph

Figure 2 Flow sheet for ammonium thiocyanate in fertilizers(pH adjustment andmeasurement procedure)484

Testing Methods for Fertilizers (2018)

Reference: HPLC chromatogram of nitrous acid and ammonium thiocyanate are shown
below.
←1

2↓

Retention time(A)
Mixture standard solution(The equivalents of 100 ng (10 µg/mL, 10 μL) as nitrous acid and ammonium thiocyanate,respectively)

1→

2
↓

Retention time(B)
Sample solution(The equivalents of 0.1 % (mass fraction) as nitrous acid and ammonium thiocyanate added inblended fertilizer respectively)
Reference diagram: HPLC chromatogram of nitrous acid and ammonium thiocyanate(Peak: 1. Nitrous acid, 2. Thiocyanate)
Measurement conditions for HPLC
Column: CAPCELL PAK NH2 UG80 (4.6-mm inner diameter, 250-mm long,
5-μm particle diameter)
Other conditions are according to the example of HPLC measurement conditionsin (4.3) a)

485

Testing Methods for Fertilizers (2018)

5.9 Nitrous acid
5.9.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 5.9.a-2017 or NO2.a-1.
Add water to an analytical sample, extract nitrous acid and adjust pH as necessary. Introduce it intoa High-Performance Liquid Chromatograph (HPLC), isolate with a silica gel column to whichamino group chemically bonds or a vinyl alcohol polymer column to which amino group chemicallybonds, and measure at wavelength 210 nm to obtain nitrous acid in an analytical sample. In addition,
the performance of this testing method is shown in Comment 5.
Nitrous acid and ammonium thiocyanate (sulfurized cyanide) can be simultaneously quantified byusing this method. (Refer to Comment 4).
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
c) Disodium hydrogen phosphate dodecahydrate: A JIS Guaranteed Reagent specified in JIS
K 9019 or a reagent of equivalent quality.
d) Sodium dihydrogen phosphate dihydrate: A JIS Guaranteed Reagent specified in JIS K
9009 or a reagent of equivalent quality.
e) Sodium perchlorate monohydrate: A JIS Guaranteed Reagent specified in JIS K 8227 or areagent of equivalent quality.
f) Nitrous Acid standard solution (1 mg/mL) (1): Put 0.147 g of sodium nitrite specified in JIS
K 8019 in a weighing dish and measure the mass to the order of 0.1 mg. Add a small amountof water to dissolve, then transfer to a 100-mL volumetric flask and add water up to themarked line.
g) Nitrous acid standard solution (100 μg/mL) (1): At the time of usage, put 10 mL of nitrousacid standard solution (1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
h) Nitrous acid standard solution for the calibration curve preparation (1 μg/mL - 20
μg/mL): At the time of usage, put 1 mL - 20 mL of nitrous acid standard solution (100 μg/mL)
to 100-mL volumetric flasks step-by-step and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel column tube
filled with poly vinyl alcohol or silica gel (2), to which 5-μm particle diameter amino groupchemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 210 nm.
b) Magnetic stirrer:c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
d) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
e) pH test paper: A pH test paper infilled with indicator and dried, which can measure thevalue from pH 1 - pH 11 and a color change chart with the pH interval value 1 is attached.
486

Testing Methods for Fertilizers (2018)

Note (2)Remaining silanol group of silica gel affects the measurement of ion in some cases.
Therefore, use a column which does not affect the measurement of nitrous acid bytreating the silanol group. As an example of the treatment, silica gel is to be entirely
covered with the uniform membrane of silicone polymer.

Comment 1 A column is sold under the names CAPCELL PAK NH2 UG80 or Asahipak
NH2P-50 4E.
Comment 2 pH test paper is sold under the name UNIV Test Paper, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 1.00 g of an analytical sample, and put it into a 200-mL ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge it at 1700 × g centrifugal force for about five minutes (3) and use the supernatant as
the extract.
Note (3) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water to the marked line to make the extract(4.2) pH adjustment: Conduct pH adjustment as shown below.
a) Transfer a small amount of the extract to confirm pH value using a pH-test paper.
b) If the pH value in a) is pH 5 or more, transfer the extract to a 1.5-mL ground-in stopper
centrifugal precipitate tube (4) and conduct the procedure in f) to prepare a sample solution.
c) If the pH value in a) is pH 4 or less, transfer 40 mL of the extract to a 100-mL beaker.
d) Add a sodium hydroxide solution (5 mg/mL), adjust it to pH 5 to pH 7 with a pH meter andtransfer to a 50-mL volumetric flask with water.
e) Add water up to the marked line and transfer it to a 1.5-mL ground-in stopper centrifugal
precipitate tube (4).
f) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (5) and use thesupernatant as the extract.
Note (4) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(5) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 3 Instead of procedures in (4.2) b) and e) - f), it is allowed to filter with a membranefilter (aperture diameter: no more than 0.5-µm) made of hydrophilic PTFE and thefiltrate can be the sample solution.
487

Testing Methods for Fertilizers (2018)(4.3) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High-Performance
Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for an Ion Chromatograph (IC) is shown below. Set up
the measurement conditions considering it:1) Column: A vinyl alcohol polymer column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which amino group chemically bonds or a silica gel column(4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm particle diameter) to whichamino group chemically bonds.
2) Column bath temperature: 30 °C - 40 °C
3) Eluent (1): Dissolve 1.79 g of disodium hydrogen phosphate dodecahydrate, 0.78 g of sodium
dihydrogen phosphate dihydrate and 14.04 g of sodium perchlorate monohydrate in water tomake 1000 mL. Filter with a membrane filter (aperture diameter: no more than 0.5-µm) madeof hydrophilic PTFE.
4) Flow rate: 0.9 mL/min - 1.0 mL/min
5) Injection volume: 10 µL
6) Detection unit: An absorptiometric detector, measurement wavelength: 210 nmb) Calibration curve preparation
1) Inject 10 µL of respective standard solutions for the calibration curve preparation to an HPLC,
record chromatogram at wavelength 210 nm and obtain the peak area.
2) Prepare a curve for the relationship between the concentration and the peak area atwavelength 210 nm of the respective standard solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the nitrous acid content by the peak area using the calibration curve to calculate theconcentration of nitrous acid in the analytical sample.
Comment 4 This testing method enables the simultaneous measurement of nitrous acid andammonium thiocyanate (sulfurized cyanide). In this case, mix a predeterminedamount of nitrous acid standard solution (1 mg/mL) and ammonium thiocyanate
standard solution (1 mg/mL), dilute with water to prepare a mixture standard solution(100 μg/mL) (1) and use it instead of (2) h) nitrous acid standard solution (100μg/mL). After that, conduct the same procedure in (4.3) b) to calculate the respective
concentrations of materials subjected to measurement in the analytical sample.
Comment 5 Recovery testing was conducted using samples that nitrous acid equivalent to 1/5 - 5times of permissible content are added to ammonium sulfate fertilizer (1 brand),coating nitrogen fertilizer (1 brand), blended fertilizer (2brands), compound fertilizer(1 brand) and fluid mixed fertilizer (1 brand). As a result, the mean recovery at theadditive level of 0.1 % (mass fraction), 0.04 % (mass fraction), 0.02 % (massfraction) and 0.01 % (mass fraction) are 99.0 % - 100.8 %, 100.4 % - 102.0 %,103.1 % - 106.6 % and 101.2 % - 105.9 % respectively.
Table 1 shows results and analysis results from a collaborative study for test method
validation. Nitrous acid had sufficient reproducibility in the range from 0.0255 %(mass fraction) to 0.291 %. (mass fraction).
Additionally, the minimum limit of quantification of this testing method is about
0.0003 % (mass fraction).
488

Testing Methods for Fertilizers (2018)

Table 1 Results and statistical analysis results from a collaborative study
for the nitrous acid method
Sample name

Number of1)laboratories

4)

RSD r

3)

(%)

Mean

2)

sr

3)

(%)

(%)

Home garden-use mixed
10
0.0502 0.0005
1.1
fertilizer 1
Home garden-use mixed
11
0.0255 0.00070
2.6
fertilizer 2
Home garden-use mixed
9
0.150
0.004
2.9
fertilizer 3
Compound fertilizer 1
10
0.202
0.004
1.9
Compound fertilizer 2
10
0.291
0.004
1.3
Compound fertilizer 3
100.0498 0.0007
1.41) Number of laboratories used in analysis
2) Mean (n = number of laboratories × number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

5)

6)

RSD R

(%)

3)

(%)

0.00091.7

0.0009

3.5

0.005

3.6

0.004
0.005
0.0010

2.2
1.7
2.0

sR

7)

References
1)
Kohei ITO, Yasuharu KIMURA, Masanori HASEGAWA and Yuji SHIRAI:
Simultaneous Determination of Nitrous Acid and Thiocyanate in Fertilizer by
High-Performance Liquid Chromatography (HPLC), Japanese Journal of Soil Scienceand Plant Nutrition, Vol. 87 (2), p.120 - 124 (2016)
2)
Masanori HASEGAWA and Yasuharu KIMURA: Determination of Nitrous Acid andAmmonium Thiocyanate in Fertilizer by High-Performance Liquid Chromatography(HPLC): A Collaborative Study, Research Report of Fertilizer Vol. 8, p. 70 - 78 (2015)

489

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for nitrous acid in fertilizers is shown below:

1.00 g analyticalsample200-mL ground-in stopper Erlenmeyer flask(powdery)← 100 mL of water
Extraction
Centrifugal separation

Stir to mix, 10 minites
Ground-in stopper centrifugal precipitate tube,
1700 ×g , 5 minutes

Filtrate

Figure 1-1 Flow sheet for niyrous acid in fertilizers(Extraction procedure (4.1.1))

1.00 g analyticalsample100-mL volumetric flask(fluid)← 50 mL - 70 mL of water
Extraction
Shake to mix← Water (up to the marked line)
Filtrate

Figure 1-2

Flow sheet for niyrous acid in fertilizers(Extraction procedure (4.1.2))

490

Testing Methods for Fertilizers (2018)

Filtrate

pH value confirmation pH test paper
pH 5 or more
pH 4 or less
Aliquot (40 mL)
100-mL beaker
pH adjustment(pH 5 - pH 7)

Sodium hydroxide solution (5 mg/mL)Transfer
50-mL volumetric flask, water
Water
(up
to
the marked line)←
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

SampleMeasurement
Figure 2

High Performance Liquid Chromatograph

Flow sheet for nitrous acid in fertilizers

491

Testing Methods for Fertilizers (2018)

Reference: HPLC chromatogram of nitrous acid and ammonium thiocyanate are shown
below.

←1

2↓

Retention time(A)
Mixture standard solution(The equivalents of 100 ng (10 µg/mL, 10 μL) as nitrous acid and ammonium thiocyanate,respectively)

1→

2
↓

Retention time(B)
Sample solution(The equivalents of 0.1 % (mass fraction) as nitrous acid and ammonium thiocyanate added inblended fertilizer respectively)
Reference diagram: HPLC chromatogram of nitrous acid and ammonium thiocyanate
Measurement conditions for HPLC
Column: CAPCELL PAK NH2 UG80 (4.6-mm inner diameter, 250-mm long,
5-μm particle diameter)
Other conditions are according to the example of HPLC measurement conditionsin (4.3) a)492

Testing Methods for Fertilizers (2018)5.10 Biuret nitrogen
5.10.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 5.10.a-2017 or B-N.a-1.
Add water to an analytical sample to extract biuret, introduce it to a High-Performance
Chromatograph (HPLC) to isolate it with a weak acid ion-exchange column, and then measure atwavelength 190 nm to obtain biuret nitrogen (B-N) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 6.
Dicyandiamide nitrogen (Dd-N), urea nitrogen (U-N), guanidine nitrogen (Gd-N) and guanylurea
nitrogen (GU-N) and can be simultaneously quantified by using this method. (Refer to Comment
5).
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Potassium dihydrogen phosphate: A JIS Guaranteed Reagent specified in JIS K 9007 or areagent of equivalent quality.
c) Phosphoric acid: A JIS Guaranteed Reagent specified in JIS K 9005 or a reagent ofequivalent quality.
d) Biuret nitrogen standard solution (B-N 2 mg/mL) (1): Put 0.491 g of biuret [C2H5N3O2] (2)in a weighing dish and measure the mass to the order of 0.1 mg. Add a small amount of water,transfer to a 100-mLvolumetric flask and warm up to 50ºC to dissolve. After standing to cool(3)
, add water up to the marked line (3).
e) Biuret nitrogen standard solution (B-N 200 µg/mL): Put 10 mL of biuret nitrogen standard
solution (B-N 2 mg/mL) to a 100-mL volumetric flask and add water up to the marked line.
f) Biuret nitrogen standard solution (B-N 50 µg/mL - 100 µg/mL): Put 25 mL - 50 mL ofbiuret nitrogen standard solution (B-N 200 µg/mL) to 100-mL volumetric flasks and add
water up to the marked line.
g) Biuret nitrogen standard solution for the calibration curve preparation (B-N 1 μg/mL 50 μg/mL): At the time of usage, put 1 mL - 50 mL of biuret nitrogen standard solution (B-N
100 μg/mL) to 100-mL volumetric flasks step-by-step and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 97 % (mass fraction) in purity is commercially sold as biuret.
(3) If biuret nitrogen standard solution (B-N 2 mg/mL) is stored in a refrigerator,
precipitates may appear. Therefore, it is recommended to store it at room temperature.
In addition, sudden cooling should be avoided.
Comment 1 Biuret is sold by FUJIFILM Wako Pure Chemical Co., Ltd., Kanto Chemical Co., Inc.
and Tokyo Chemical Industry Co., Ltd.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 7.5-mm inner diameter 100-mm long stainless steel column tube filled withweak acid ion-exchange resin.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 190 nm.
b) Magnetic stirrer:c) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g - 10000
493

Testing Methods for Fertilizers (2018)× g.
Comment 2 A column is sold under the production name Asahipak ES-502C 7C.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 1.00 g of an analytical sample, and put it into a 200-mL ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution (4) to a 1.5-mL ground-in stopper
centrifugal precipitate tube (5).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (6) and use thesupernatant as the extract.
Note (4) If there is a possibility that the biuret nitrogen (B-N) concentration in the sample
solution will exceed the maximum limit of the calibration curve, dilute apredetermined amount of supernatant with water.
(5) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(6) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line (7) and transfer to a 1.5-mL ground-in stopper centrifugal
precipitate tube (5).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (6) and use thesupernatant as the extract.
Note (7) If there is a possibility that the biuret nitrogen (B-N) concentration in the sample
solution will exceed the maximum limit of the calibration curve, dilute apredetermined amount of precisely adjusted solution with water.
Comment 3 Instead of procedures in (4.1.1) c) - d) or (4.1.2) c) - d), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5-µm) made of hydrophilic
PTFE and the filtrate can be the sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions is shown below. Set up the measurement conditions
considering it:1) Column: A weak acid ion-exchange resin column (7.5-mm inner diameter, 100-mm long,
5-µm - 10-µm particle diameter)
2) Column bath temperature: 40 °C
3) Eluent (1): Dissolve 3.92 g of potassium dihydrogen phosphate and 0.12 g of phosphoric acidin water to make 1000 mL. Filter with a membrane filter (aperture diameter: no more than494

Testing Methods for Fertilizers (2018)0.5-µm) made of hydrophilic PTFE.
4) Flow rate: 0.6 mL/min
5) Injection volume: 10 µL
6) Detection unit: An absorptiometric detector, measurement wavelength: 190 nm
Comment 4 Eluent can be prepared as follows. Dissolve 19.6 g of potassium dihydrogen
phosphate and 0.584 g of phosphoric acid with water to make 500 mL and store in arefrigerator. At the time of usage, dilute a predetermined volume of the solution by afactor of 10 and filter with a membrane filter (aperture diameter: no more than
0.5-µm) made of hydrophilic PTFE.
b) Calibration curve preparation
1) Inject 10 µL of respective standard solutions for the calibration curve preparation to an HPLC,
record chromatogram at wavelength 190 nm and obtain the peak height.
2) Prepare a curve for the relationship between the biuret nitrogen (B-N) concentration and thepeak height at wavelength 190 nm of the respective standard solutions for the calibration
curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the biuret nitrogen (B-N) content from the peak height using the calibration curve tocalculate the biuret nitrogen (B-N) in the analytical sample.
Comment 5 This testing method enables the simultaneous measurement of biuret nitrogen (B-N),
dicyandiamide nitrogen (Dd-N), urea nitrogen (U-N), guanidine nitrogen (Gd-N) anda guanylurea nitrogen standard solution (GU-N). In this case, mix a predeterminedamount of biuret nitrogen (B-N 2 mg/mL), an urea nitrogen standard solution (U-N 2
mg/mL), a dicyandiamide nitrogen standard solution (Dd-N 2 mg/mL), a guanidine
nitrogen standard solution (Gd-N 2 mg/mL) and a guanylurea nitrogen standard
solution (GU-N 2 mg/mL), dilute with water to prepare a mixture standard solution(200 μg/mL) (1) and use it instead of (2) e) a biuret nitrogen standard solution (B-N
200 μg/mL). After that, conduct the same procedure in (4.2) b) to calculate therespective concentrations of materials subjected to measurement in the analyticalsample.
Comment 6 Additive recovery testing was conducted using one brand of an acetaldehyde
condensed urea fertilizer, a compound fertilizer, a blended fertilizer, a fluid
compound fertilizer and a home garden-use mixed fertilizer respectively. As a result,the mean recovery at additive level of 0.2 % (mass fraction) and 0.1 % (massfraction) and 0.02 % (mass fraction) were 87.0 % - 95.1 %, 90.6 % - 101.1 % and91.2 % - 105 5 % respectively.
The results of the repeatability tests on different days using a blended fertilizer, acompound fertilizer and a home garden-use mixed fertilizer to evaluate precisionwere analyzed by the one-way analysis of variance. Table 1 shows the calculation
results of intermediate precision and repeatability. Table 2 shows results and analysis
results from a collaborative study for testing method validation.
Additionally, the minimum limit of quantification of this testing method is about
0.005 % (mass fraction).

495

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
4)
5)
6)
7)
repeatability Average2)
sr
RSD rs I(T)RSD I(T)
1)

T
5
5

3)

(%)
0.204
0.09693)(%)
0.00060.0006

Blended fertilizer
Compound fertilzierHome garden-use
50.01030.0001
mixed fertilizer
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

(%)
0.3
0.6

(%)
0.00170.0016

3)

(%)
0.9
1.6

0.9

0.0001

0.96） Intermediate standard deviation
7） Intermediate relative standard deviation

Table 2 Results and statistical analysis results from a collaborative study
for the test method validation of biuret nitrogen method
Sample name

Number of1)laboratories

4)

RSD r

3)

(%)

Mean

2)

sr

3)

(%)

(%)

9
0.009630.00030
Compound fertilizer 1
100.02010.0003Compound fertilizer 2
12
0.114
0.013
Compound fertilizer 3
11
0.212
0.017
Compound fertilizer 4
12
0.832
0.050
Nitrogen fertilizer
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories × number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

5)

6)

RSD R

(%)

3)

(%)

3.1
1.6

0.000290.0007

3.1
3.4

11.7
7.8
6.0

0.017
0.026
0.086

15.3
12.4
10.3

sR

7)

References
1) Masahiro ECHI, Yasuharu KIMURA and Yuji SHIRAI: Determination of Urea Nitrogen,
Biuret Nitrogen, Dicyandiamide Nitrogen, Guanidine Nitrogen and Guanyl urea
Nitrogen in Fertilizer by High-Performance Liquid Chromatography: A
Single-Laboratory Validation, Research Report of Fertilizer, Vol.10, p. 72 - 85 (2017)2)
Norio FUNAKI and Yasuharu KIMURA: Determination of Urea Nitrogen, Biuret
Nitrogen, Dicyandiamide Nitrogen, Guanidine Nitrogen and Guanyl urea Nitrogen in
Fertilizer by High-Performance Liquid Chromatography (HPLC): A Collaborative Study,
Research Report of Fertilizer, Vol.10, p. 86 - 100 (2017)496

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for biuret nitrogen in fertilizers is shown
below:
1.00 g200-mL ground-in stopper Erlenmeyer flask
analytical sample (powdery)← 100 mL of water
Extraction

Stir to mix, 10 minites

Stand to coolCentrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

Sample solution

Supernartant

Measurement

Liquid Chromatograph

Figure 1

Flow sheet for biuret nitrogen in fertilizers(Extraction procedure (4.1.1) and measurement)

1.00 g100-mL volumetric flask
analytical sample (fluid)←About 50 mL of water
Extraction
Shake to mix← Water (up to the marked line)
Ground-in stopper centrifugal precipitate tube,
Centrifugal separation
8000 ×g - 10000 ×g , 5 minutes
Sample solution

Supernartant

Measurement

Liquid Chromatograph

Figure 2

Flow sheet for biuret nitrogen in fertilizers(Extraction procedure (4.1.2) and measurement)497

Testing Methods for Fertilizers (2018)

Reference:

The chromatogram of the standard solution of biuret nitrogen for the calibration
curve preparation is shown below.

Reference diagram

HPLC chromatogram of the mixture standard solutions for the calibration
curve preparation (10 mg/L for each)Peak name(1) Urea nitrogen(2) Biuret nitrogen(3) Dicyandiamide nitrogen(4) Guanidine nitrogen(5) Guanylurea nitrogen
Measurement conditions for HPLC
Column: Asahipak ES-502C 7C (7.5-mm inner diameter, 100-mm long, 9-μm
particle diameter)
Other conditions are according to the example of HPLC measurement conditionsin (4.2) a)498

Testing Methods for Fertilizers (2018)5.11 Titanium
5.11.a ICP Optical Emission Spectrometry (1)(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type D and its
symbol is 5.11.a-2017 or Ti.a-1.
Pretreat an analytical sample with nitric acid - sulfuric acid - perchloric acid, introduce it to an ICP
Optical Emission Spectrometer (“ICP-OES”) and measure the emission with titanium at awavelength of 334.941 nm to obtain the titanium (Ti) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 3.
(2)
a)
b)
c)
d)

Reagent, etc.: Reagents and water are as shown below.
Water: Water of A3 specified in JIS K 0557.
Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade or equivalents.
Perchloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
e) Hydrochloric acid: Hydrochloric acid for arsenic analysis specified in JIS K 8180, or of
harmful metal analysis grade, microanalysis grade or equivalents.
f) Titanium standard solution (Ti 1 mg/mL): A titanium standard solution (Ti 1 mg/mL)
traceable to National Metrology.
g) Titanium standard solution (Ti 0.1 mg/mL) (1): Transfer 10 mL of titanium standard
solution (Ti 1 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid (1+23) up to
the marked line.
h) Titanium standard solutions (Ti 0.1 µg - 20 µg/mL) for the calibration curve preparation(1): Transfer 1 mL - 20 mL of titanium standard solution (Ti 0.1 mg/mL) to 100-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
i) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in g) and h).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in h) applies to a horizontal observation
mode. While an axial observation mode can observe a measurement component of
low-level concentrations, it cannot gain the linearity of a calibration curve in therange of high-level concentrations. Therefore, in case of using the ICP-OES of anaxial observation mode, it is recommended to prepare a titanium standard solution
for the calibration curve in the concentration range which is suitable to a device used.

(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: A spectrophotometer specified in JIS K 0116
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 350 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to no less than 300 °C.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
499

Testing Methods for Fertilizers (2018)b) Add about 10 mL of nitric acid and about 5 mL of sulfuric acid, cover the tall beaker with awatch glass, and leave at rest overnight.
c) Heat mildly on a hot plate or sand bath at 170 ºC - 220 ºC for no less than 30 minutes. Afterbubbles cease to form, set the temperature of the hot plate or sand bath to no less than 300 ºC,
and heat until nitroxide (yellow-brown smoke) is no longer generated (2).
d) After standing to cool, add about 5 mL of perchloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or sand bath at no less than
300 ºC for 2 - 3 hours to digest (3).
f) Slightly move the watch glass (4), and keep on heating on the hot plate or sand bath toconcentrate until the liquid volume becomes no more than 2 mL.
g) After standing to cool, add about 5 mL of hydrochloric acid (1+10) and about 20 mL of water,
cover the tall beaker with a watch glass and heat mildly to dissolve.
h) After standing to cool, transfer to a 100-mL volumetric flask with water, add water up to themarked line, and filter with Type 3 filter paper to make a sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (2) Oxidation of organic matters by perchloric acid progresses extremely rapidly andexplosively. For that reason, add perchloric acid after fully degrading organic matterswith nitric acid to avoid danger.
(3) When the white smoke of perchloric acid is generated, if the solution is colored suchas black-brown or brown, stop heating immediately, and after standing to cool, add
nitric acid, and heat again to degrade remaining organic matters.
(4) The watch glass can be removed.
Comment 2 When the analytical sample solidifies in the procedure in (4.1) b), moisten theanalytical sample with a small amount of water as necessary in advance.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 334.941 nmb) Calibration curve preparation
1) Spray the titanium standard solution for the calibration curve preparation and blank test
solution for the calibration curve preparation into inductively coupled plasma, and read theindicated value at a wavelength of 334.941 nm.
2) Prepare a curve for the relationship between the titanium concentration and the indicatedvalue of the titanium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.01 mg - 2 mg as
titanium) to a 100-mL volumetric flask.
2) Add 25 mL of hydrochloric acid (1+23) to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the titanium content from the calibration curve, and calculate titanium (Ti) in theanalytical sample.
500

Testing Methods for Fertilizers (2018)

Comment 3 Additive recovery testing was conducted using an autoclaved lightweight concrete
powdery fertilizer, a mixed phosphate fertilizer, a compound fertilizer, a slag
manganese fertilizer, a solid fertilizer, a fluid compound fertilizer, a mixed compost
compound fertilizer and a blended fertilizer respectively. As a result, the mean
recovery at additive level of 0.01 % (mass fraction) - 0.5 % (mass fraction) were
92.9 % - 99.5 % respectively.
The results of the repeatability tests on different days using a mixed phosphate
fertilizer and a compound fertilizer to evaluate precision were analyzed by the
one-way analysis of variance. Table 1 shows the calculation results of intermediate
precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about0.001 % (mass fraction).

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
3)

(%)

(%)

3)

(%)

Mixed phosphate fertilizer
7
0.950
0.013
Compound fertilizer
7
0.130
0.002
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

1.7
1.4

0.031
0.006

4.3
3.2

T

1)

(%)

3)

(%)

6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Keisuke AOYAMA: Determination of Titanium in Fertilizer using Inductively Coupled
Plasma Optical Emission Spectrometry (ICP-OES) , Research Report of Fertilizer,
Vol.10, p. 29 - 40 (2017)

501

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for titanium in fertilizers is shown below:
1.00 - 2.00g
200-mL - 300-mL tall beaker.
analytical sample← A small amount of water, moisten the analytical sample (as appropriate)← About 10 mL of nitric acid
← About 5 mL of sulfuric acid
Leave at rest overnight Cover with a watch glass
HeatingHeat gently on a hot plate or a sand bath at 170 ºC - 220 ºC
for no less than 30 minutes

HeatingHeat on a hot plate or sand bath at no less than 300 ºC,
until yellow-brown smoke no longer evolves.

Standing to coolRoom temperature← About 5 mL of perchloric acid
Cover with a watch glass,
Heatingdigest on a hot plate or a sand bath at 300 ºC or more for 2-3
hours
Heating

Slightly move the watch glass,concentrate until it becomes no more than 2mL

Standing to coolRoom temperature← About 5 mL of hydrochloric acid (1+10)← About 20 mL of water
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for titanium in fertilizers (Extraction procedure)

Sample solution
Aliquot
100-mL volumetric flask(Predetermined← About 25 mL of hydrochloric acid (1+23)← Water (up to the marked line)
Measurement
ICP Optical Emission Spectrometer (334.941 nm)
Figure 2 Flow sheet for titanium in fertilizers (Measurement procedure)
502

Testing Methods for Fertilizers (2018)

5.11.b ICP Optical Emission Spectrometry (2)(1) Summary
The testing method is applicable to slag silicate fertilizers. This testing method is classified as TypeD and its symbol is 5.11.b-2017 or Ti.b-1.
Melt an analytical sample with ammonium hydrogensulfate, introduce it to an ICP Optical Emission
Spectrometer (“ICP-OES”) and measure the emission with titanium at a wavelength of 334.941 nm
to obtain the titanium (Ti) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 2.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Ammonium hydrogensulfate: A reagent of no less than 98 % (mass fraction) in purity.
c) Hydrochloric acid: Hydrochloric acid for arsenic analysis specified in JIS K 8180, or of
harmful metal analysis grade, microanalysis grade or equivalents.
d) Titanium standard solution (Ti 1 mg/mL): A titanium standard solution (Ti 1 mg/mL)
traceable to National Metrology.
e) Titanium standard solution (Ti 0.1 mg/mL) (1): Transfer 10 mL of titanium standard
solution (Ti 1 mg/mL) to a 100-mL volumetric flask and add hydrochloric acid (1+23) up to
the marked line.
f) Titanium standard solutions (Ti 0.1 µg - 20 µg/mL) for the calibration curve preparation(1): Transfer 1 mL - 20 mL of titanium standard solution (Ti 1 mg/mL) to 100-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
g) Blank test solution for the calibration curve preparation (1): Hydrochloric acid (1+23) usedin the procedures in d), e) and f).
Note (1) This is an example of preparation; prepare an amount as appropriate.
Comment 1 There are two modes to observe emission from an ICP-OES, a horizontal observation
mode and an axial observation mode. The range of the concentration of the standard
solution for the calibration preparation curve in f) applies to a horizontal observation
mode. While an axial observation mode can observe a measurement component of
low-level concentrations, it cannot gain the linearity of a calibration curve in therange of high-level concentrations. Therefore, in case of using the ICP-OES of anaxial observation mode, it is recommended to prepare a titanium standard solution
for the calibration curve in the concentration range which is suitable to a device used.
(3) Instruments: Instruments are as shown below:a) ICP Optical Emission Spectrometry: An Optical Emission Spectrometer specified in JIS K
0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) in purityb) Hot plate : A hot plate whose surface temperature can be adjusted up to 400 ºC.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample and transfer to a 200-mL tall beaker.
b) Add about 10 g of ammonium hydrogensulfate (2).
c) Heat on a hot plate at no less than 350 ºC and make the melted ammonium hydrogensulfate
come into complete contact with the analytical sample (3).
d) Cover with a watch glass, and heat at no less than 350 ºC for 1 hour.
e) After standing to cool, add about 25 mL of hydrochloric acid (1+5), cover the tall beaker with503

Testing Methods for Fertilizers (2018)a watch glass and heat mildly to dissolve.
f) After standing to cool, transfer to a 100-mL volumetric flask with water, add water up to themarked line, and filter with Type 3 filter paper.
g) Transfer 10 mL of the filtrate to another 100-mL volumetric flask and add hydrochloric acid
(1+23) to the marked line to make a sample solution.
h) As a blank test, conduct the procedures in b) - f) using another tall beaker to prepare the blanktest solution.
Note (2) Ammonium hydrogensulfate is corrosive. Therefore, use a dispensing spoon made ofresin.
(3) After the ammonium hydrogensulfate melts, make it come into contact with theanalytical sample by tilting the tall beaker or conducting a similar procedure.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0116 and as shown below.
Specific measurement procedures are according to the operation method of the ICP OpticalEmission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 334.941 nmb) Calibration curve preparation
1) Spray the titanium standard solution for the calibration curve preparation and blank test
solution for the calibration curve preparation into inductively coupled plasma, and read theindicated value at a wavelength of 334.941 nm.
2) Prepare a curve for the relationship between the titanium concentration and the indicatedvalue of the titanium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Subject the sample solution to the same procedure as in b) 1) to read the indicated value.
2) Obtain the titanium content from the calibration curve, and calculate titanium (Ti) in theanalytical sample.
Comment 2 Recovery testing was conducted to evaluate trueness using slag silicate fertilizers (2samples). As a result, the average rate of recovery at additive level of 0.1 % (massfraction) - 0.2 % (mass fraction) are 95.1 % - 98.2 %.
The results of the repeatability tests on different days using slag silicate fertilizers (2samples) to evaluate precision were analyzed by the one-way analysis of variance.
Table 1 shows the calculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.02 % (mass fraction).

504

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

3)

3)

T
(%)
(%)
Slag silicate fertilizer 1
7
0.525
0.005
Slag silicate fertilizer 2
7
0.112
0.002
1） The number of test days conducting a duplicate test2） Average （the number of test days （T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

3)
(%)
(%)
(%)
1.0
0.005
1.0
1.6
0.003
2.4
6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Keisuke AOYAMA: Determination of Titanium in Fertilizer using Inductively Coupled
Plasma Optical Emission Spectrometry (ICP-OES) , Research Report of Fertilizer,
Vol.10, p. 29 - 40 (2017)505

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for titanium in fertilizers is shown below:
1.00 g200-mL tall beaker.
analytical sample← About 10 g of ammonium hydrogensulfate
Heat on a hot plate at no less than 350 ºC
Melting
Make the melted ammonium hydrogensulfate
completely come into contact with the analytical sample
Heating

Cover with a watc glass, heat on a hot plate
at no less than 350 ºC for 1 hourStanding to coolRoom temperature←25 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass to digest
Standing to coolRoom temperature

Transfer
100-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for titanium in fertilizers (Extraction procedure)

Sample solution
Aliquot 10 mL
100-mL volumetric flask← Hydrochloric acid (1+23) (up to the marked line)
Measurement
ICP Optical Emission Spectrometer (334.941 nm)
Figure 2 Flow sheet for titanium in fertilizers (Measurement procedure)

506

Testing Methods for Fertilizers (2018)5.12 Sulfurous Acid
This method is according to 5.3 Sulfurous acid analysis in “The Official Methods of Analysis of
Fertilizers 1992”.
References
1)
National Institute for Agro-Environmental Sciences, the Ministry of Agriculture,
Forestry and Fisheries: The Official Methods of Analysis of Fertilizers 1992, p.78 - 79,
Japan Fertilizers Analysis Association, Tokyo (1992)
2)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p. 196 - 197, Yokendo, Tokyo (1988)

507

Testing Methods for Fertilizers (2018)

6. Testing relating to the other limitations
6.1 Dicyandiamide nitrogen
6.1.a High-Performance Liquid Chromatography (1)(1) Summary
The testing method is applicable to nitrolime and fertilizers containing nitrolime. This testing
method is classified as Type B and its symbol is 6.1.a-2017 or Dd-N.a-1.
Add methanol to an analytical sample to extract dicyandiamide (Dd), introduce it to a
High-Performance Liquid Chromatograph (HPLC), isolate with an amino propyl silica gel column
and measure at wavelength 215 nm to obtain dicyandiamide nitrogen (Dd-N) in an analyticalsample. In addition, the performance of this testing method is shown in Comment 4.
(2) Reagent: Reagents are as shown below.
a) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
b) Methanol: Methanol used in eluent of an HPLC is HPLC analysis grade or a reagent ofequivalent quality.
c) Acetonitrile: A reagent of HPLC grade or equivalents.
d) Dicyandiamide standard solution (1 mg/mL) (1): Put 0.1 g of dicyandiamide [C2H4N4] (2) toa weighing dish and measure the mass to the order of 0.1 mg. Add a small amount ofmethanol to dissolve, transfer to a 100-mL volumetric flask and add the solvent up to themarked line. Store in a refrigerator, and do not use after 6 months after preparation.
e) Dicyandiamide standard solution (0.1 mg/mL): Put 10 mL of dicyandiamide standard
solution (1 mg/ mL) to a 100-mL volumetric flask and add methanol up to the marked line.
f) Dicyandiamide standard solution (10 µg/ mL - 50 µg/ mL) for the calibration curve
preparation: At the time of usage, put 5 mL - 25 mL of dicyandiamide standard solution (0.1
mg/ mL) to 50-mL volumetric flasks step-by-step and add methanol up to the marked line.
g) Dicyandiamide standard solution (1 µg/mL - 10 µg/mL) for the calibration curve
preparation: In the case of usage, transfer 2.5 mL - 25 mL of dicyandiamide standard
solution for the calibration curve preparation (20 µg/mL) to 50-mL volumetric flasks
step-by-step and add methanol up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity as dicyandiamide iscommercially sold.
Comment 1 Dicyandiamide is commercially sold as dicyanodiamide by FUJIFILM Wako Pure
Chemical Co., Ltd. and Kanto Chemical Co., Inc.
(3) Instruments: Instruments are as shown below:a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A column of 4 mm - 6 mm inner diameter and 150 mm - 250 mm long stainlesssteel column tube filled with silica gel, to which amino or amino propyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 215 nm.
b) Shaking apparatusc) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
Comment 2 A column is sold under production names such as Hibar LiChrosorb NH2, Inertsil
508

Testing Methods for Fertilizers (2018)

NH2, Unison UK-Amino, Mightysil NH2, Shim-pack CLC-NH2, Shodex NH-5A,
Unisil Q NH2, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200 mL - 200-mL ground-in stopper
Erlenmeyer flask.
b) Immediately add 100 mL of methanol (3) and shake to mix by using a shaking apparatus forabout 10 minutes.
c) After allowing to stand still, transfer a supernatant solution to a 1.5-mL ground-in stopper
centrifugal precipitate tube (4).
d) Centrifuge at 8000 × g - 10000 × g for about five minutes (5).
e) 1 mL of the supernatant is used as the sample solution.
Note (3) Add methanol immediately as the determined value becomes higher than usual if it isleft in air.
(4) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement(5) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 3 Instead of the procedures in (4.1) c) - e), it is allowed to filter with a membrane filter(aperture diameter: no more than 0.5-µm) made of PTFE and the filtrate can be thesample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for a High-Performance Liquid Chromatograph (HPLC)is shown below. Set up the measurement conditions considering it:1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm
particle diameter) to which amino or amino propyl chemically bonds.
2) Column bath temperature: 30 °C - 40 °C
3) Eluent: Acetonitrile - methanol (6+1)
4) Flow rate: 1 mL/min
5) Detection unit: An absorptiometric detector, measurement wavelength: 215 nmb) Calibration curve preparation
1) Inject 10 µL of respective dicyandiamide standard solutions for the calibration curve
preparation to an HPLC, record a chromatogram at wavelength 215 nm, and obtain the peakarea or the height.
2) Prepare a curve for the relationship between the concentration and the peak area or the heightat wavelength 215 nm of the respective dicyandiamide standard solutions for the calibration
curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain dicyandiamide (Dd) content from the calibration curve to calculate the concentrationof dicyandiamide (Dd) in the analytical sample.
3) Calculate the dicyandiamide nitrogen (Dd-N) by the following formula.
509

Testing Methods for Fertilizers (2018)

Dicyandiamide nitrogen (Dd-N) (% (mass fraction)) in an analytical sample= A × (MW1/MW2)= A × 0.6664
A： Dicyandiamide (Dd) (% (mass fraction)) in an analytical sample
MW1： 4 atomic weight of nitrogen (56.027)
MW2： Molecular weight of dicyandiamide (84.080)
Comment 4 Recovery testing was conducted using nitrolime (3 samples) and a blended fertilizer
containing nitrolime (2 samples), as a result, the recovery of dicyandiamide atconcentration level of 6 % (mass fraction) and 0.6 % (mass fraction) was 94.9 % 105.1 % and 95.6 % - 103.5 % respectively.
Table 1 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification of this testing method is about
0.01 % (mass fraction).
Table 1 Results and analysis results from a collaborative study
for the test method validation of dicyandiamide nitrogen
Sample name

Number of1)laboratories

4)

RSD r

3)

(%)

Mean

2)

sr

3)

(%)

(%)

Nitrolime 1
90.03210.0010
3.2
Nitrolime 2
10
0.159
0.002
1.3
Nitrolime 3
11
0.245
0.002
0.7
Blended fertilizers 1
11
0.124
0.001
0.7
Blebded fertilizers 2
11
0.410
0.007
1.6
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

5)

6)

RSD R

(%)

3)

(%)

0.0012
0.006
0.008
0.002
0.008

3.8
3.8
3.3
2.0
1.9

sR

7)

References
1)
Masakazu SAIKI and Miyuki ASAO: Validation of High-Performance Liquid
Chromatography for Determination of Dicyandiamide in Nitrolime, Research Report of
Fertilizers, Vol.2, p. 25 - 31 (2009)
2)
Masakazu SAIKI and Masayuki YOSHIMOTO: Determination of Dicyandiamide in
Nitrolime by High-Performance Liquid Chromatography: A Collaborative Study,
Research Report of Fertilizers, Vol.2, p. 32 - 37 (2009)(5) Flow sheet for dicyandiamide nitrogen: The flow sheet for dicyandiamide nitrogen innitrolime and fertilizers containing nitrolime is shown below:
510

Testing Methods for Fertilizers (2018)

1.00 g200-mL - 300-mL ground-in stopper Erlenmeyer flask
analytical sample← 100 mL of methanol
Extraction

Shaking, 10 minutes

Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

Sample solution

Supernatant

Measurement

High-Performance Liquid Chromatograph

Figure Flow sheet for dicyandiamide nitrogenin nitrolime and fertilizers containing nitrolime

Reference: HPLC chromatographs of dicyandiamide standard solution and sample solution(nitrolime) for the calibration curve preparation are shown below.

0.09

0.10
0.09
0.08

0.07

0.07

0.06

0.06

AU

0.08

0.05

0.05

0.04

0.04

0.03

0.03

0.02

0.02

0.01

0.01

0.00
0

Dd 8.458

0.10

AU

0.11

Dd 8.541

0.11

0.00
1

2

3

4

5 6
min

7

8

9

10 11

1) Standard solution

0

1

2

3

4

5 6
min

7

8

9

10 11

2) Sample solution

Reference diagram: HPLC chromatogram of dicyandiamide
1) Dicyandiamide standard solution (the equivalents of 100 ng (10 µg/ mL, 10 µL) of
dicyandiamide)2) Sample solution (nitrolime)
Measurement conditions for HPLC
Column: Hibar LiChrosorb NH2 (4.6-mm inner diameter, 25-cm long, 5-µm particle
diameter)Other conditions are according to the example of HPLC measurement conditions in(4.2) a)511

Testing Methods for Fertilizers (2018)

6.1.b High-Performance Liquid Chromatography (2)(1) Summary
This testing method is applicable to fertilizers. In addition, nitrolime is excluded from the scope of
application. This testing method is classified as Type B and its symbol is 6.1.b-2017 or Dd-N.b-1.
Add water to an analytical sample to extract dicyandiamide, introduce it to a High-Performance
Chromatograph (HPLC) to isolate it with a weak acid ion-exchange column, and then measure atwavelength 190 nm to obtain dicyandiamide nitrogen (Dd-N) in an analytical sample. In addition,
the performance of this testing method is shown in Comment 6.
Biuret nitrogen (B-N), urea nitrogen (U-N), guanidine urea (Gd-N) and guanylurea nitrogen(GU-N) can be simultaneously quantified by using this method. (Refer to Comment 5).
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Potassium dihydrogen phosphate: A JIS Guaranteed Reagent specified in JIS K 9007 or areagent of equivalent quality.
c) Phosphoric acid: A JIS Guaranteed Reagent specified in JIS K 9005 or a reagent ofequivalent quality.
d) Dicyandiamide nitrogen standard solution (Dd-N 2 mg/mL) (1): Put 0.300 g ofdicyandiamide [C2H4N4] (2) in a weighing dish and measure the mass to the order of 0.1 mg.
Add a small amount of water to dissolve, then transfer to a 100-mL volumetric flask and add
water up to the marked line.
e) Dicyandiamide nitrogen standard solution for the calibration curve preparation (Dd-N
200 g/mL) (1): Put 10 mL of biuret nitrogen standard solution (B-N 2 mg/mL) to a 100-mL
volumetric flask and add water up to the marked line.
f) Dicyandiamide nitrogen standard solution (Dd-N 50 µg/mL - 100 g/mL) (1): Put 25 mL 50 mL of dicyandiamide nitrogen standard solution (Dd-N 200 µg/mL) to 100-mL volumetricflasks and add water up to the marked line.
g) Dicyandiamide nitrogen standard solution for the calibration curve preparation (Dd-N 1
μg/mL - 50 μg/mL) (1): At the time of usage, put 1 mL - 50 mL of dicyandiamide nitrogenstandard solution (Dd-N 100 μg/mL) to 100-mL volumetric flasks step-by-step and add water
up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity as dicyandiamide iscommercially sold.
Comment 1 Dicyandiamide is sold by Tokyo Chemical Industry Co., Ltd. In addition,dicyandiamide is commercially sold as dicyanodiamide by FUJIFILM Wako Pure
Chemical Co., Ltd. and Kanto Chemical Co., Inc.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 7.5-mm inner diameter 100-mm long stainless steel column tube filled withweak acid ion-exchange resin.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 190 nm.
b) Magnetic stirrer:c) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g - 10000
× g.
512

Testing Methods for Fertilizers (2018)

Comment 2 A column is sold under the production name Asahipak ES-502C 7C.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution (3) to a 1.5-mL ground-in stopper
centrifugal precipitate tube (4).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (5) and use thesupernatant as the extract.
Note (3) If there is a possibility that the dicyandiamide nitrogen (Dd-N) concentration in thesample solution will exceed the maximum limit of the calibration curve, dilute apredetermined amount of supernatant with water.
(4) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(5) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line (6) and transfer to a 1.5-mL ground-in stopper centrifugal
precipitate tube (4).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (5) and use thesupernatant as the extract.
Note (6) If there is a possibility that the dicyandiamide nitrogen (Dd-N) concentration in thesample solution will exceed the maximum limit of the calibration curve, dilute apredetermined amount of solution with water.
Comment 3 Instead of procedures in (4.1.1) c) - d) or (4.1.2) c) - d), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5-µm) made of hydrophilic
PTFE and the filtrate can be the sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph: An example
of measurement conditions is shown below. Set up the measurement conditions considering it:1) Column: A weak acid ion-exchange resin column (7.5-mm inner diameter, 100-mm long,
5-µm - 10-µm particle diameter)
2) Column bath temperature: 40 °C
3) Eluent (1): Dissolve 3.92 g of potassium dihydrogenphosphate and 0.12 g of phosphoric acidin water to make 1000 mL. Filter with a membrane filter (aperture diameter: no more than
0.5-µm) made of hydrophilic PTFE.
4) Flow rate: 0.6 mL/min513

Testing Methods for Fertilizers (2018)

5) Injection volume: 10 µL
6) Detection unit: An absorptiometric detector, measurement wavelength: 190 nm
Comment 4 Eluent can be prepared as follows. Dissolve 19.6 g of potassium
dihydrogenphosphate and 0.584 g of phosphoric acid with water to make 500 mL andstore in a refrigerator. At the time of usage, dilute a predetermined volume of thesolution by a factor of 10 and filter with a membrane filter (aperture diameter: no
more than 0.5-µm) made of hydrophilic PTFE.
b) Calibration curve preparation
1) Inject 10 µL of respective standard solutions for the calibration curve preparation to an HPLC,
record chromatogram at wavelength 190 nm and obtain the peak height.
2) Prepare a curve for the relationship between the dicyandiamide nitrogen (Dd-N) concentrationand the peak height at wavelength 190 nm of the respective standard solutions for thecalibration curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the dicyandiamide nitrogen (Dd-N) content from the peak height using the calibration
curve to calculate the dicyandiamide nitrogen (Dd-N) in the analytical sample.
Comment 5 This testing method enables the simultaneous measurement of biuret nitrogen (B-N),
urea nitrogen (U-N), dicyandiamide nitrogen (Dd-N), guanidine urea (Gd-N) andguanylurea nitrogen (GU-N). In that case, see 5.10.a Comment 5.
Comment 6 Additive recovery testing was conducted using one brand of an acetaldehyde
condensed urea fertilizer, a compound fertilizer, a blended fertilizer, a fluid
compound fertilizer and a home garden-use mixed fertilizer respectively. As a result,the mean recovery at additive level of 3 % (mass fraction) and 1.5 % (mass fraction)and 0.3 % (mass fraction) were 96.3 % - 96.3 %, 94.5 % - 99.7 % and 88.9 % 100.6 % respectively.
The results of the repeatability tests on different days using a blended fertilizer, acompound fertilizer and a home garden-use mixed fertilizer to evaluate precisionwere analyzed by the one-way analysis of variance. Table 1 shows the calculation
results of intermediate precision and repeatability. Table 2 shows results and analysis
results from a collaborative study for testing method validation.
Additionally, the minimum limit of quantification of this testing method is about
0.01 % (mass fraction).

514

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

3)

(%)

Blended fertilizer
Compound fertilizer
Home garden-use
5
0.145
0.001
mixed fertilizer
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）

T
5
5

3)

(%)
3.03
1.45

(%)
0.02
0.01

3)

(%)

0.5
0.9

(%)
0.02
0.02

0.9

0.003

2.3

0.5
1.6

6） Intermediate standard deviation
7） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

Table 2 Results and analysis results from a collaborative study
for the test method validation of dicyandiamide nitrogen
4)5)
6)
Number of Mean2)
sr
RSD r
sR
Sample name
1)
3)
3)
3)
laboratories
(%)
(%)
(%)
(%)
10
0.04640.00235.0
0.0148Compound fertilizer 1
12
0.206
0.011
5.1
0.031
Compound fertilizer 2
12
1.69
0.05
2.8
0.12
Compound fertilizer 3
10
2.76
0.07
2.6
0.12
Compound fertilizer 4
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

7)

RSD R
(%)
31.9
15.2
7.0
4.4

References
1) Masahiro ECHI, Yasuharu KIMURA and Yuji SHIRAI: Determination of Urea Nitrogen,
Biuret Nitrogen, Dicyandiamide Nitrogen, Guanidine Nitrogen and Guanyl urea
Nitrogen in Fertilizer by High-Performance Liquid Chromatography: A
Single-Laboratory Validation, Research Report of Fertilizer, Vol.10, p. 72 - 85 (2017)2)
Norio FUNAKI and Yasuharu KIMURA: Determination of Urea Nitrogen, Biuret
Nitrogen, Dicyandiamide Nitrogen, Guanidine Nitrogen and Guanyl urea Nitrogen in
Fertilizer by High-Performance Liquid Chromatography (HPLC): A Collaborative Study,
Research Report of Fertilizer, Vol.10, p. 86 - 100 (2017)

515

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for dicyandiamide nitrogen in fertilizers isshown below:
1.00 g analytical sample
200-mL ground-in stopper Erlenmeyer flask(powdery)← 100 mL of water
Extraction

Stir to mix, 10 minites

Stand still
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

Sample solution

Supernatant

Measurement

Liquid Chromatograph

Figure 1

Flow sheet for dicyandiamide nitrogen in fertilizers(Extraction procedure (4.1.1) and measurement)

1.00 ganalytical sample (fluid)

100-mL volumetric flask←About 50 mL of water
Extraction
Shake to mix← Water (up to the marked line)
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g, 5 minutes

Sample solution

Supernatant

Measurement

Liquid Chromatograph

Figure 2 Flow sheet for dicyandiamide nitrogen in fertilizers(Extraction procedure (4.1.2) and measurement)

516

Testing Methods for Fertilizers (2018)

Reference: Chromatogram of the standard solution for calibration curve preparation of
dicyandiamide nitrogen is shown below.

Reference diagram

HPLC chromatogram of the mixture standard solutions (10 mg/L foreach) for calibration curve preparation

Peak name(1) Urea nitrogen(2) Biuret nitrogen(3) Dicyandiamide nitrogen(4) Guanidine nitrogen(5) Guanylurea nitrogen
Measurement conditions for HPLC
Column: Asahipak ES-502C 7C (7.5-mm inner diameter, 100-mm long, 9-μm
particle diameter)
Other conditions are according to the example of HPLC measurement conditionsin (4.2) a)517

Testing Methods for Fertilizers (2018)

6.2 Chlorine
6.2.a Ion Chromatography(1) Summary
The testing method is applicable to potassium sulfate, potassium bicarbonate, magnesium potassium
sulfate, fish cakes powder, fish cakes and compost. This testing method is classified as Type D and
its symbol is 6.2.a-2017 or Cl.a-1.
Add water to an analytical sample to extract chloride ions, introduce them to an Ion Chromatograph(IC) to isolate it with an ion exchange column, and then measure with an electric conductivity
detector to obtain chlorine (Cl) in an analytical sample. In addition, the performance of this testing
method is shown in Comment 3.
(2)
a)
b)
c)
d)
e)
f)

Reagent, etc.: Reagents and water are as shown below.
Water: Water of A4 specified in JIS K 0557.
1 mol/L sodium carbonate solution: Solution for ion chromatography.
Phthalic acid: A reagent of no less than 98 % (mass fraction) in purity.
6-aminohexanic acid (1): A reagent of no less than 97 % (mass fraction) in purity.
Phenylboronic acid: A reagent of no less than 97 % (mass fraction) in purity.
Chloride ion standard solution (Cl- 1 mg/mL): A chloride ion standard solution (Cl- 1000
mg/mL) traceable to National Metrology.
g) Chloride ion standard solution (Cl- 100 µg/mL) (2): Put a predetermined amount of chloride
ion standard solution (Cl- 1 mg/mL) to a volumetric flask and add water up to the marked line.
h) Chloride ion standard solution (Cl- 5 µg/ mL - 50 µg/ mL) for the calibration curve
preparation (2): Put 5 mL - 50 mL of Chloride ion standard solution (Cl- 100 µg/mL) to a
100-mL volumetric flask and add water up to the marked line.
i) Chloride ion standard solution (Cl- 1 µg/ mL - 2 µg/ mL) for the calibration curve
preparation (2): Put 5 mL - 10 mL of Chloride ion standard solution (Cl- 20 mg/mL) to a
100-mL volumetric flask and add water up to the marked line.
j) Eluent for suppressor method (2): Put 6.4 mL of 1 mol/L sodium carbonate solution to a
1000-mL volumetric flask and add water up to the marked line. Then filter with membrane
type filter (pore size: no more than 0.5 μm) made of hydrophilic PTFE (2).
k) Eluent for non-suppressor method (2): Put 0.349 g of phthalic acid, 0.380 g of
6-aminohexanic acid and 0.732 g of phenylboronic acid to a 1000-mL volumetric flask andadd about 500 mL of water to dissolve. Add water up to the marked line and filter withmembrane type filter (pore size: no more than 0.5 μm) made of hydrophilic PTFE (3).
Note (1) It is also known as “6-amino-n-caprotic acid”.
(2) This is an example of preparation; prepare an amount as appropriate.
(3) A solution concentrated by a factor of 10 can be prepared in advance and the solution
can be diluted by a factor of 10 at each usage.
Comment 1 Instead of the chloride ion standard solution in (2), a chloride ion standard solution
for the calibration curve preparation can be prepared by using a chloride ion standard
solution (Cl- 0.1 mg/mL) traceable to National Metrology.

(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Magnetic stirrer:b) Centrifugal separator: A centrifugal separator that can work at 1700 × g.
c) Ion Chromatograph (IC): IC specified in JIS K 0127 that satisfies following requirements.
1) Column: In case of a suppressor method, a 4-mm inner diameter 250-mm long 5-μm particle
diameter column tube filled with poly vinyl alcohol porous particles, to which quaternary518

Testing Methods for Fertilizers (2018)

ammonium group chemically bonds (4).
In case of non-suppressor method, a 4.6-mm inner diameter 100-mm long column tube filledwith hydrophilic polymethacrylate-gel, to which quaternary ammonium group chemicallybonds (5).
2) Column bath: A column bath whose temperature can be adjusted to 40 ºC.
3) Suppressor: Cation exchange membrane or resin should be used.
4) Detection unit: An electric conductivity detectord) Membrane filters: Pore size is no more than 0.45 μm, made of hydrophilic PTFE
Note (4) A column is commercially sold under the name Shodex IC SI-52 4E, etc.
(5) A column is commercially sold under the name Shodex IC NI-424, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge it at 1700 × g centrifugal force for about five minutes (6) and use the supernatant as
the extract.
e) Transfer a predetermined amount of the solution, and dilute exactly by a factor of 20 withwater (7).
f) Filter with a membrane filter (pore size: no more than 0.45 μm) to make a sample solution.
Note (6) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(7) In case of exceeding the calibration curve, dilute by a factor of more than 20.
Comment 2 Instead of the procedures in (4.1.1) c) and d), it is allowed to filter with Type 3 filter
paper and the filtrate can be the extract.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0127 and as shown below.
Specific measurement procedures are according to the operation method of an Ion
Chromatograph (IC) used in measurement.
a) Measurement conditions for Ion Chromatograph (IC): An example of measurement
conditions is shown below. Set up the measurement conditions considering it:aa) Suppressor method
1) Column: A polyvinyl alcohol porous particles column (4-mm inner diameter 250-mm long
5-μm particle diameter) to which quaternary ammonium group chemically bonds.
2) Column bath temperature: 40 °C
3) Eluent: Prepared in the procedure in (2) j).
4) Flow rate: 0.8 mL/min
5) Injection volume: 20 µL
6) Detection unit: An electric conductivity detectorab) Non-suppressor method
1) Column: A hydrophilic polymethacrylate-gel column (4.6-mm inner diameter 100 mm long)to which quaternary ammonium group chemically bonds.
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Testing Methods for Fertilizers (2018)2) Column bath temperature: 40 °C
3) Eluent: Prepared in the procedure in (2) k).
4) Flow rate: 1.0 mL/min
5) Injection volume: 20 µL
6) Detection unit: An electric conductivity detectorb) Calibration curve preparation
1) Inject 20 μL of respective standard solutions for the calibration curve preparation to an IC andrecord the chromatogram of electric conductivity to obtain peak area.
2) Prepare a curve for the relationship between the concentration and the peak area of electric
conductivity of respective standard solutions for the calibration curve preparation.
Prepare a calibration curve when the sample is measured.
c) Sample measurement
1) Subject 20 μL of sample solution to the same procedure as in b) 1)
2) Obtain the chloride ion concentration from calibration curve by peak area to calculate thechlorine (Cl) in the analytical sample.
Comment 3 Additive recovery testing was conducted with a suppressor method using samples
that 1.8 % (mass farction) – 33.4 % (mass fraction) of sodium chloride as chlorine is
added to potassium sulfate, magnesium potassium sulfate, potassium bicarbonate,
cow dung compost and fish cakes powder. As a result, the mean recovery at thechlorine additive level of 33.4 % (mass fraction), 10 % (mass farction) - 13.4 %(mass fraction) and 1.8 % (mass farction) - 9.1 % (mass fraction) are 100.8 %,98.6 % - 101.1 % and 96.2 % - 103.2 % respectively. With a non-suppressor method,they are 100.2 %, 96.4 % - 97.2 % and 93.3 % - 101.4 %.
The results of the repeatability tests on different days using potassium sulfate,magnesium potassium sulfate, potassium bicarbonate, cow dung compost and fishcakes powder to evaluate precision were analyzed by one-way analysis of variance.
Table 1 shows the calculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.1 % (mass fraction).

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Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the repeatability tests on different days
Name ofsampleTest days of

Repeatability
2)

repeatability

Average

1)

(%)

T

3)

<Suppressor method>
Pottasium sulfate
5
9.93
Fish cakes powder
5
6.13<Non-supprresor
method>Pottasium sulfate
5
4.86
Magnesium pottasium
5
4.89
sulfatePottasium bicarbonate
5
4.85
Cow dung compost
513.15
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

Intermediate precision

4)
sr
3)

5)
RSD r

s I(T)

6)

(%)

(%)

(%)

0.01
0.03

0.1
0.5

0.03
0.07

0.3
1.1

0.01

0.2

0.08

1.7

0.02

0.4

0.06

1.2

3)

7)

RSD I(T)
(%)

0.02
0.4
0.06
1.3
0.04
0.3
0.16
1.2
6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Satoko SAKAIDA, Mariko FUJITA and Yuji SHIRAI: Determination of Chloride in
Fertilizers by Ion Chromatography (IC), Research Report of Fertilizers, Vol.8, p. 50 - 60
(2015)(5)

Flow sheet for testing method: The flow sheet for chlorine in fertilizers is shown below:
1.00 g200-mL ground-in stopper Erlenmeyer flask
analytical sample← 100 mL of water
Extraction
Magnetic stirrer, 10 minutes
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
2000 ×g , 10 minutes

Dilution

20 times of dilution, water

Filtration

Hydrophilic PTFE membrane filter (0.45 μm)

Sample solution
Measurement

Ion Chromatograph

Figure Flow sheet for chlorine in fertilizers

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Testing Methods for Fertilizers (2018)

Reference:

IC chromatographs of sample solutions (magnesium potassium sulfate and fishcakes powder) are shown below.

Cl-

Cl-

(A) Chromatograph of magnesium
potassium sulfate(Suppressormethod)(B) Chromatograph of magnesium
potassium sulfate(Non-suppressor method)

Cl-

Cl-

(C) Chromatograph of fish cakes powder(Suppressor method)(D) Chromatograph of fish cakes powder(Non-suppressor method)

Reference diagram: IC chromatogram of chloride ion(Peak：1.chloride ion (Cl-))

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Testing Methods for Fertilizers (2018)

6.2.b Silver nitrate method(1) Summary
The test method is applicable to potassium sulfate, potassium bicarbonate and magnesium
potassium sulfate. This testing method is classified as Type E and its symbol is 6.2.b-2017 or
Cl.b-1.
Add water to an analytical sample to extract chloride ions and titrate (precipitate) with a 0.1 mol/Lsilver nitrate standard solution to obtain chlorine (Cl) in an analytical sample.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A JIS Guaranteed (HNO3 60 % (mass fraction)) Reagent specified in JIS K
8541 or a reagent of equivalent quality.
c) 0.1 mol/L silver nitrate solution (1): Weigh 17 g of silver nitrate specified in JIS K 8550 to a
2000-mL beaker, add 1000 mL of water to dissolve and store in a colored bottle.
Standardization: Heat a sodium chloride reference material for volumetric analysis specified
in JIS K 8005 at 600 ºC ± 25 °C for 1 hour, let it stand to cool in a desiccator, and thentransfer about 1.5 g to a weighing dish, and weigh the mass to the order of 0.1 mg. Dissolve
in a small amount of water, transfer to a 250-mL volumetric flask, and add water up to themarked line to make the sodium chloride solution (1). On each day to use a 0.1 mol/L silver
nitrate solution, transfer 10 mL of the sodium chloride solution to a 200-mL Erlenmeyer
flask, add a few drops of potassium chromate solution (5 g/100 mL) as an indicator, andtitrate with a 0.1 mol/L silver nitrate solution until the color of the solution becomes reddish
brown. Calculate the factor of a 0.1 mol/L silver nitrate solution by the following formula:
Factor (f) of 0.1 mol/L silver nitrate solution= W1× (A/100) × (1/58.44) × (V1/V2 × (1000/V3)) × (1/C)= (W1×A/V3) × (4/58.44)
W1： Mass (g) of sodium chloride weighed
A： Purity (% (mass fraction)) of sodium chloride
V1： Volume (10 mL) of sodium chloride solution transferred
V2： Constant volume (250 mL) of sodium chloride solution
V3： Volume (mL) of 0.1 mol/L silver nitrate solution needed for titration
C： Set concentration (0.1 mol/L) of 0.1 mol/L silver nitrate solutiond) Potassium chromate (5 g/ 100 mL) (1): Dissolve 5 g of potassium chromate in 100 mL ofwater.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Magnetic stirrer:b) pH test paper: A pH test paper infiltrated with indicator and dried, which can measure thevalue from pH 1 to pH 11 and a color change chart with the pH interval value 1 is attached.
Comment 1 pH test paper is sold under the name UNIV Test Paper, etc.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
523

Testing Methods for Fertilizers (2018)flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) Filter with Type 3 filter paper to make a sample solution.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer 25 mL of sample solution to a 200-mL tall beaker.
b) Confirm the pH of the solution with a pH-test paper. If the pH is basic, neutralize with nitricacid (1+10).
c) Add a few drops of potassium chromate solution (5 g/100 mL) to the sample solution as anindicator and titrate with a 0.1 mol/L silver nitrate solution until the color of the solution
becomes reddish brown.
d) Calculate chlorine (Cl) by the following formula.
Chlorine (% (mass fraction)) in the analytical sample= V4 × C × f × (35.45)/W2 × (100/1000) × (V5/ V6)= V4 × f × (35.45/25)/W2
V4： Volume (mL) of the 0.1 mol/L silver nitrate solution required for titration
C： Set concentration (0.1 mol/L) of 0.1 mol/L silver nitrate solution
f： Factor of 0.1 mol/L silver nitrate solution
V5： Liquid volume of water (100 mL) subjected to extraction in (4.1) b).
V6： Transferred volume (25 mL) of the sample solution subjected to titration in(4.2) a).
W2： Mass (g) of the analytical sample
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.199- 201, Yokendo, Tokyo (1988)

524

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for chlorine in potassium sulfate is shown
below:
1.00 g analytical sample
200-mL ground-in stopper Erlenmeyer flask← 100 mL of water
Extraction
Magnetic stirrer, 10 minutes
Filtration

Type 3 filter paper

Sample solution
Figure 1 Flow sheet for chroline in potassium sulfate (Extraction procedure)

Sample solution
Aliquot (25 mL)

200-mL tall beaker

pH confirmation
pH-test paper← Nitric acid (1+10) [neutralize in case of basic]← Several drops of potassium chromate (5 g/100 mL)
0.1 mol/L siver nitrate solution
Titration(until the solution becomes reddish brown)
Figure 2 Flow sheet for chroline in potassium sulfate (Measurement procedure)

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Testing Methods for Fertilizers (2018)

6.3 Urea nitrogen
6.3.a Urease method(1) Summary
The test method is applicable to fertilizers containing urea or urea compounds such as acetaldehyde
condensed urea, etc. However, in some cases, it is not applicable to the fertilizers that contain suchcompounds as nitrolime that digests by heating. This testing method is classified as Type D and its
symbol is 6.3.a-2017 or U-N.a-1.
Add water to an analytical sample to extract and add urease to a predetermined amount of theextract to hydrolyze urea into ammonium ion. Add sodium hydroxide to alkalize the solution andsubject it to steam distillation. Collect isolated ammonia with 0.25 mol/L sulfuric acid and titrate(neutralize) surplus sulfuric acid using 0.1 mol/L - 0.2 mol/L sodium hydroxide solution and correct
separately the titers of urease blank test and urease undigested blank test to obtain urea nitrogen(U-N) in the analytical sample. Or collect isolated ammonia with a boric acid solution and titrate(neutralize) the ammonium ion using 0.25 mol/L sulfuric acid and similarly correct to obtainammonia nitrogen (A-N) in the analytical sample. In addition, the performance of this testing
method is shown in Comment 11.
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days. Transfer
5.5 mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000mL of water.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in JIS
K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C1)
W1： Mass (g) of sulfamic acid sampled
A： Purity (% (mass fraction)) of sulfamic acidV1： Volume (mL) of sulfamic acid solution transferred
V2： Constant volume (250 mL) of sulfamic acid solution
V3： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Magnesium oxide: A JIS Guaranteed Reagent specified in JIS K 8432 or a reagent ofequivalent quality.
c) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
d) 0.25 mol/L sulfuric acid (1) (2): Add about 14 mL of sulfuric acid to a beaker containing 100
mL of water in advance, stir well, and add water to make 1000 mL.
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Testing Methods for Fertilizers (2018)

Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a 200-mL- 300-mL Erlenmeyer flask, add a few drops of methyl red−methylene blue mixture solution,and titrate with a 0.1 mol/L -0.2 mol/L sodium hydroxide solution until the color of thesolution becomes gray-green (4). Calculate the volume of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the following formula(1).
Or, calculate the factor of 0.25 mol/L sulfuric acid by the following formula (2):Volume (B) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1 mL of
0.25 mol/L sulfuric acid= V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2 × 2)………. (2)V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5： Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acide)

Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 in water tomake 1000 mL.
f) Urease: A reagent which completely digests 0.25 g of urea by 0.5 g of urease.
g) Sodium hydroxide solution (5 g/L) (1): Dissolve 5 g of sodium hydroxide specified in JIS K
8576 in water to make 1000 mL.
h) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
i) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water to make 100 mL.
j) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
k) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in JIS K
8897 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red−methylene blue mixture solution: To 2 volumes of methyl red solution (0.1g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
m) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green specified
in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
n) Methyl red−bromocresol green mixture solution: To a methyl red solution (0.1 g/100 mL),
add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL -10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
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Testing Methods for Fertilizers (2018)

Comment 2 0.25 mol/L sulfuric acid in (2) d) can be replaced with 0.25 mol/L sulfuric acidconforming to ISO/IEC 17025.
Comment 3 Refined products of urease made from sword beans are commercially sold. The
degree of activation may degrade even if stored in a refrigerator. Therefore, it isrecommended to test similarly and confirm activation by using urea specified in JIS
K 8731 in advance.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Extractor: Rotary shaker or magnetic stirrer as described belowaa) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down at 30 40 revolutions/min.
ab) Magnetic stirrer:b) Steam distillation apparatusc) Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to a steam
distillation apparatus.
d) Water bath: Water bath that can be adjusted to 40 ºC - 45 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Rotary shakera) Weigh 5.00 g of an analytical sample, and put it in a 500-mL volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Comment 4 In the procedure in (4.1.1) a), it is also allowed to weigh 2.50 g of the analyticalsample and transfer to a 250-mL volumetric flask.
Comment 5 The procedure in (4.1.1) is the same as the procedure in (4.1.1.1) in 4.2.4.a.
(4.1.2) Magnetic stirrer:a) Weigh 1.00 g of an analytical sample, and put it into a 200- mL ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) Filter with Type 3 filter paper to make the extract.
Comment 6 The procedure in (4.1.2) is the same as the procedure in (4.1.1) in 6.3.c.
(4.2) Hydrolysis by urease: Conduct hydrolysis as shown below.
a) Transfer a predetermined amount of the extract (the equivalents of 10 mg or more as U- N, theequivalents of 10 mg - 100 mg as N) to a 300-mL distillation flask.
b) Add water to the solution to make about 50 mL.
c) Add a few drops of methyl red solution (0.1 g/100 mL), and add a sodium hydroxide solution(5 g/L) or hydrochloric acid (1+200) until the color of the solution becomes light yellowish
red (6).
d) Add a sufficient amount of urease to digest urea in the extract (7) (8), seal tightly and heat in thewater bath at 40 ºC - 45 ºC.
e) Let it stand to cool to make a sample solution.
f) As an extract blank test, conduct the procedures in a) (9) using another distillation flask to
prepare the undigested test solution.
g) As a urease blank test, conduct the procedures in b), d) and e) (8) (10) using another distillation
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Testing Methods for Fertilizers (2018)flask to prepare the blank test solution.
Note (6) pH 5.6 - pH 5.8(7) An example of the additive volume of urease is shown in Comment 10.
(8) Wash urease off with a small amount of water if it adheres to the surface of a vessel.
(9) Aliquot the same amount of extract as the prepared sample solution.
(10) Add the same amount of urease as the preparation of the sample solution used.
(4.3) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used in measurement.
a) Transfer a predetermined amount (11) of 0.25 mol/L sulfuric acid to an acceptor (12), add a few
drops of methyl red−methylene blue mixture solution, and connect this acceptor to a steam
distillation apparatus. Or, transfer a predetermined amount (11) of boric acid solution (40 g/L)
to an acceptor (12), add a few drops of methyl red−bromocresol green mixture solution, andconnect this acceptor to a steam distillation apparatus.
b) Add 2 g - 3 g of magnesium oxide to a distillation flask containing the sample solution (13),and connect this distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill at a
distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution in theacceptor with a small amount of water, and pool the washing with the distillate.
f) Subject the undigested test solution to the same procedure as in a) - e) to obtain the distillate
from the undigested test solution.
g) Subject the blank test solution to the same procedure as in a) - e) to obtain the distillate fromthe blank test solution.
Note (11) 5 mL - 20 mL(12) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or a boric acid solution (40 g/L).
(13) Add a small amount of silicone oil as necessary.
Comment 7 Immediately conduct the procedure in (4.3) b) not to emit ammonia gas in a vessel.
(4.4) Measurement: Conduct measurement as shown below.
(4.4.1) When 0.25 mol/L sulfuric acid is used in (4.3):a) Titrate the distillate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes gray-green (4).
b) Subject the distillate from the undigested test solution to the same procedure as in a) to titrate.
c) Subject the distillate from the blank test solution to the same procedure as in a) to titrate.
d) Calculate the urea nitrogen (U-N) in an analytical sample by the following formula:
Urea nitrogen (U-N) (% (mass fraction)) in the analytical sample= ((B × V6－V7) －(B × V6－V8)－(B × V6－V9)) × C1 × f1 × (V10/V11) × (14.007/W2) ×(100/1000)= (－(B × V6) － (V7－V8－V9)) × C1 × f1 × (V10/V11) × (1.4007/W2)
B： Volume of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1 mL of
0.25 mol/L sulfuric acid529

Testing Methods for Fertilizers (2018)

V6： Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.3) a)
V7： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration in (4.4.1) a)
V8： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration in (4.4.1) b)
V9： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration in (4.4.1) c)
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V10： Predetermined volume (mL) of the extract in (4.1.1) c) or additive volume ofwater in (4.1.2) b)
V11： Transferred amount (mL) of the extract subjected to hydrolysis in (4.2) a)
W2： Mass (g) of the analytical sample in (4.1.1) a) or (4.1.2) a)(4.4.2) When a boric acid solution (40 g/L) is used in (4.3):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomes lightred (14).
b) Subject the distillate from the undigested test solution to the same procedure as in a) to titrate.
c) Subject the distillate from the blank test solution to the same procedure as in a) to titrate.
d) Calculate the urea nitrogen (U-N) in an analytical sample by the following formula:
Urea nitrogen (U-N) (% (mass fraction)) in the analytical sample= (V12－V13－V14) × C2 × 2 × f2 × (V10/V11) × (14.007/W2) × (100/1000)= (V12－V13－V14) × C2 × f2 × (V10/V11) × (2.8014/W2)
V12： Volume (mL) of 0.25 mol/L sulfuric acid needed for titration in (4.4.2) a)
V13： Volume (mL) of 0.25 mol/L sulfuric acid needed for titration in (4.4.2) b)
V14： Volume (mL) of 0.25 mol/L sulfuric acid needed for titration in (4.4.2) c)
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidf2： Factor of 0.25 mol/L sulfuric acidV10： Predetermined volume (mL) of the extract in (4.1.1) c) or additive volume ofwater (mL) in (4.1.2) b)
V11： Transferred amount (mL) of the extract subjected to hydrolysis in (4.2) a)
W2： Mass (g) of the analytical sample in (4.1.1) a) or (4.1.2) a)
Note (14) The endpoint is reached when the color changes from green to light red.
Comment 8 If it is hard to confirm the endpoint due to the carbon dioxide resulting fromcarbonate in the extract when magnesium oxide is used, it is recommended to boil
the extract for 1-2 minute(s) after distilling and cool, and then titrate.
Comment 9 The titration procedures in (2) a) Standardization, (2) d) Standardization and (4.4)
can be conducted by an automatic titrator. The setup of the titration program, thedetermination parameter for the endpoint and vessels such as acceptors are according
to the specification and the operation method of the automatic titrator used.
Comment 10 An example of the additive volume and titter of urease is shown below.
If the urea content can be estimated, calculate the volume of urea in the transferred
volume of the extract in (4.2) a) by the following formula.
Estimated urea content (mg) in the transferred volume of the extract= (D1/100) × W2 × (V11/ V10)530

Testing Methods for Fertilizers (2018)

D1： Estimated urea content (% ( mass fraction)) in the analytical sample
V10： Predetermined volume (mL) of the extract in (4.1.1) c) or (4.1.2) c)
V11： Transferred amount (mL) of the extract subjected to hydrolysis in (4.2) a)
W2： Mass (g) of the analytical sample in (4.1.1) a) or (4.1.2) a)
If the urea content can not be estimated, subtract ammoniacal nitrogen and nitrate
nitrogen from the total nitrogen of the permissible content or the display component
content of the urea nitrogen in a urea compound to obtain nitrogen content. The obtained
nitrogen content is calculated as around the maximum content of urea nitrogen (U-N). In
this case, calculate the volume of urea in the transferred volume of the extract in (4.1.1) or(4.2) a) after the procedures in (4.1.1) by the following formula.
Estimated urea content (mg) in the transferred volume of the extract= (D2/100) × (60.056/(14.007 × 2)) × W2 × (V11/ V10)
= (D2/100) × 2.1438 × W2 × (V11/ V10)
D2： Estimated urea nitrogen (U-N) content (% (mass fraction)) in the analyticalsampleV10： Predetermined volume (mL) of the extract in (4.1.1) c) or (4.1.2) c)
V11： Transferred amount (mL) of the extract subjected to hydrolysis in (4.2) a)
W2： Mass (g) of the analytical sample in (4.1.1) a) or (4.1.2) a)
The specification of urease shows that “No more than 0.5 g of urease completely digests
0.25 g of urea”. Therefore about 2 mg of urease is required to digest 1 mg of urea.
Assuming that the estimated urea content or the calculated urea content in the transferred
volume of the extract is about 43 mg (about 20 mg as urea nitrogen), about 86 mg ofurease is required. When adding 0.2 g of urease (potency 130 unit - 150 unit), the extractcontaining the equivalents of 10 mg - 100 mg as urea nitrogen (U-N) can be hydrolyzed.
In addition, when about 20 mg is transferred as urea nitrogen, the differences between
the titer of the distillate from the sample solution ((4.4.1) a) or (4.4.2) a)) and the titer ofthe distillate from the undigested test solution ((4.4.1) b) or (4.4.2) b)) are estimated asfollows: About 14 mL, when a 0.1 mol/L sodium hydroxide solution is used as a titrant,
about 7 mL, when 0.2 mol/L sodium hydroxide is used as a titrant and about 3 mL, when
0.25 mol/L sulfuric acid is used as a titrant.
Comment 11 Additive recovery testing was conducted to evaluate trueness using samples
prepared by adding urea to sulfur, a compound fertilizer, isobuttyraldehudecondensed urea, a mixed fertilizer containing formaldehyde processed urea (onesample for each). As a result, the average rate of recovery at additive level of 1.58 %(mass fraction) - 39.96 % (mass fraction) are 98.7 % to 103.7 %The results of the repeatability tests on different days using urea and a compound
fertilizer containing UF (one sample for each), to evaluate precision were analyzed
by one-way analysis of variance. Table 1 shows the calculation results ofintermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.4 % (mass fraction).

531

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
5

3)

(%)
43.17

Urea
Compound fertilizer
5
2.39
containing UF
1） Gross average (the number of test days (5)× duplicate analysis)
2） Average （the number of test days（T ）× the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

(%)
0.36

3)

(%)
0.8

(%)
0.43

3)

(%)
1.0

0.07

2.9

0.12

5.2

6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.56- 59, Yokendo, Tokyo (1988)
2)
Toshifumi FUJITA, Kimie KATO, Toyokazu NIIMI, Yasuharu KIMURA, Kohei ITO
and Yuji SHIRAI: Determination Method for Urea Nitrogen in Fertilizer by Urease,
Research Report of Fertilizer, Vol.10, p. 195 - 207 (2017)(5)

Flow sheet for urea nitrogen testing method: The flow sheets for urea nitrogen test methodin fertilizers are shown below:
5.00 gWeigh into a 500-mL volumetric flask
Analytical sample← About 400 mL of water
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), 1 hour
← Water (up to the marked line)
Filtration
Type 3 filter paper
Extract
Figure 1-1 Flow sheet for urea nitrogen(Extraction procedure (4.1.1))

532

Testing Methods for Fertilizers (2018)

1.00 gWeigh into a 200-mL ground-in stopper Erlenmeyer flask
Analytical sample← 100 mL of water
Shaking to mix
Magnetic stirrer, 10 minures
Filtration

Type 3 filter paper

Extract
Figure 1-1 Flow sheet for urea nitrogen(Extraction procedure (4.1.2))

Extract
Aliquot(predeterminedvolume)300-mL digestion flask←Water (Until the solution reaches about 50 mL)← A few drops of methyl red solution (0.1 g/100 mL)← Hydrochloric acid (1+200) or sodium hydroxide solution (5 g/L)
(until the color of the solution becomes light yellowish red)
Urease(a sufficient amount of urease to hydrolyze transferred urea)←
Heating
40 ºC - 50 ºC, 1 hourStanding to cool
Sample solution← 2 g - 3 g of magnesium oxide
Receiver: 200-mL - 300-mL Erlenmeyer flask or
200-mL - 300-mL beaker. A predetermined amount of 0.25 mol/L sulfuric acid
Steam distillation
and a few drops of methyl red-methylene blue mixture solution,
apparatus
or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL - 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure 2 Flow sheet for urea nitrogen(Hydrolysis by urease, distillation and measurement procedure)

533

Testing Methods for Fertilizers (2018)

6.3.b High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 6.3.b-2017 or U-N.b-1.
Add water to an analytical sample to extract urea, introduce it to a High-Performance
Chromatograph (HPLC) to isolate it with a weak acid ion-exchange column, and then measure atwavelength 190 nm to obtain urea nitrogen (U-N) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 5.
Biuret nitrogen (B-N), dicyandiamide nitrogen (Dd-N), guanidine urea (Gd-N) and guanylurea
nitrogen (GU-N) can be simultaneously quantified by using this method. (Refer to Comment 4).
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Potassium dihydrogen phosphate: A JIS Guaranteed Reagent specified in JIS K 9007 or areagent of equivalent quality.
c) Phosphoric acid: A JIS Guaranteed Reagent specified in JIS K 9005 or a reagent ofequivalent quality.
d) Urea nitrogen standard solution (U-N 2 mg/mL) (1): Put 0.429 g of urea specified in JIS K
8731 in a weighing dish and measure the mass to the order of 0.1 mg. Add a small amount ofwater to dissolve, then transfer to a 100-mL volumetric flask and add water up to the markedline.
e) Urea nitrogen standard solution for the calibration curve preparation (U-N 200 µg/mL):Put 10 mL of urea nitrogen standard solution (U-N 2 mg/mL) to a 100-mL volumetric flask
and add water up to the marked line.
f) Urea nitrogen standard solution (U-N 50 µg/mL - 100 µg/mL): Put 25 mL - 50 mL of urea
nitrogen standard solution (U-N 200 µg/mL) to 100-mL volumetric flasks and add water up to
the marked line.
g) Urea nitrogen standard solution for the calibration curve preparation (U-N 1 μg/mL - 50
μg/mL): At the time of usage, put 1 mL - 50 mL of urea nitrogen standard solution (U-N 0.1
mg/mL) to 100-mL volumetric flasks step-by-step and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 7.5-mm inner diameter 100-mm long stainless steel column tube filled withweak acid ion-exchange resin.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 190 nm.
b) Magnetic stirrer:c) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g - 10000
× g.
Comment 1 A column is sold under the production name Asahipak ES-502C 7C.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 1.00 g of an analytical sample, and put it into a 200 mL-ground-in stopper Erlenmeyer
534

Testing Methods for Fertilizers (2018)flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution (2) to a 1.5-mL ground-in stopper
centrifugal precipitate tube (3).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (4) and use thesupernatant as the extract.
Note (2) If there is a possibility that the urea nitrogen (U-N) concentration in the sample
solution will exceed the maximum limit of the calibration curve, dilute apredetermined amount of supernatant with water.
(3) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(4) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line (5) and transfer to a 1.5-mL ground-in stopper centrifugal
precipitate tube (3).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (4) and use thesupernatant as the extract.
Note (5) If there is a possibility that the urea nitrogen (U-N) concentration in the sample solution
will exceed the maximum limit of the calibration curve, dilute a predetermined amountof supernatant with water.
Comment 2 Instead of procedures in (4.1.1) c) - d) or (4.1.2) c) - d), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5-µm) made of hydrophilic
PTFE and the filtrate can be the sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions is shown below. Set up the measurement conditions
considering it:1) Column: A weak acid ion-exchange resin column (7.5-mm inner diameter, 100-mm long,
5-µm - 10-µm particle diameter)
2) Column bath temperature: 40 °C
3) Eluent (1): Dissolve 3.92 g of potassium dihydrogenphosphate and 0.12 g of phosphoric acidin water to make 1000 mL. Filter with a membrane filter (aperture diameter: no more than
0.5-µm) made of hydrophilic PTFE.
4) Flow rate: 0.6 mL/min
5) Injection volume: 10 µL
6) Detection unit: An absorptiometric detector, measurement wavelength: 190 nm
Comment 3 Eluent can be prepared as follows. Dissolve 19.6 g of potassium
dihydrogenphosphate and 0.584 g of phosphoric acid with water to make 500 mL andstore in a refrigerator. At the time of usage, dilute a predetermined volume of the535

Testing Methods for Fertilizers (2018)

solution by a factor of 10 and filter with a membrane filter (aperture diameter: no
more than 0.5-µm) made of hydrophilic PTFE.
b) Calibration curve preparation
1) Inject 10 µL of respective standard solutions for the calibration curve preparation to an HPLC,
record chromatogram at wavelength 190 nm and obtain the peak height.
2) Prepare a curve for the relationship between the urea nitrogen (U-N) concentration and thepeak height at wavelength 190 nm of the respective standard solutions for the calibration
curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the urea nitrogen (U-N) content from the peak height using the calibration curve tocalculate the urea nitrogen (U-N) in the analytical sample.
Comment 4 This testing method enables the simultaneous measurement of biuret nitrogen (B-N),
urea nitrogen (U-N), dicyandiamide nitrogen (Dd-N), guanidine urea (Gd-N) andguanylurea nitrogen (GU-N). In that case, see 5.10.a Comment 5.
When Asahipak ES-502C 7C is used as a column for HPLC to analyze urea nitrogen(U-N) in a mixed fertilizer containing formaldehyde processed urea (UF), the peak of
the urea nitrogen (U-N) and the peak of the impurity components originating from
UF are not separated, so that urea nitrogen (U-N) cannot be measured. In this case,the peak of the urea nitrogen (U-N) and the peak of the impurity components
originating from UF are separated by using PRP-X200 as a column for HPLC instead
to be able to analyze the urea nitrogen(U-N) in the compound fertilizer containing
UF. However, when the PRP-X200 column is used as a column for HPLC, thesimultaneous measurement of urea nitrogen (U-N) and biuret nitrogen (B-N), etc. is
impossible.
Comment 5 Additive recovery testing was conducted using one brand of an acetaldehyde
condensed urea fertilizer, a compound fertilizer, a blended fertilizer, a fluid
compound fertilizer and a home garden-use mixed fertilizer respectively. As a result,the mean recovery at additive level of 6 % (mass fraction) and 3 % (mass fraction)and 0.6 % (mass fraction) were 98.3 % - 102.9 %, 98.9 % - 105.2 % and 92.3 % 99.9 % respectively.
The results of the repeatability tests on different days using a blended fertilizer, acompound fertilizer and a home garden-use mixed fertilizer to evaluate precisionwere analyzed by the one-way analysis of variance. Table 1 shows the calculation
results of intermediate precision and repeatability. Table 2 shows results and analysis
results from a collaborative study for test method validation.
Additionally, the minimum limit of quantification of this testing method is about
0.03 % (mass fraction).

536

Testing Methods for Fertilizers (2018)

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
5
5

3)

(%)
6.24
3.01

3)

(%)
0.03
0.03

Blended fertilizer
Compound fertilzierHome garden-use
5
0.315
0.003
mixed fertilizer
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

(%)
0.5
0.7

(%)
0.05
0.04

3)

(%)
0.8
1.4

0.9

0.005

0.9

6） Intermediate standard deviation
7） Intermediate relative standard deviation

Table 2 Results and statistical analysis results from a collaborative study
for the test method validation of urea nitrogen
Sample name

Number of1)laboratories

4)

RSD r

3)

(%)

Mean

2)

sr

3)

(%)

(%)

8
0.296
0.011
Compound fertilizer 1
10
0.589
0.015
Compound fertilizer 2
10
3.08
0.04
Compound fertilizer 3
10
6.03
0.11
Compound fertilizer 4
10
46.5
0.6
Compound fertilizer 5
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories × number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

5)

6)

RSD R

(%)

3)

(%)

3.6
2.6

0.012
0.024

4.1
4.1

1.1
1.7
1.4

0.06
0.20
1.3

2.0
3.4
2.8

sR

7)

References
1) Masahiro ECHI, Yasuharu KIMURA and Yuji SHIRAI: Determination of Urea Nitrogen,
Biuret Nitrogen, Dicyandiamide Nitrogen, Guanidine Nitrogen and Guanyl urea
Nitrogen in Fertilizer by High-Performance Liquid Chromatography: A
Single-Laboratory Validation, Research Report of Fertilizer, Vol.10, p. 72 - 85 (2017)2)
Norio FUNAKI and Yasuharu KIMURA: Determination of Urea Nitrogen, Biuret
Nitrogen, Dicyandiamide Nitrogen, Guanidine Nitrogen and Guanyl urea Nitrogen in
Fertilizer by High-Performance Liquid Chromatography (HPLC): A Collaborative Study,
Research Report of Fertilizer, Vol.10, p. 86 - 100 (2017)

537

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for urea nitrogen in fertilizers is shown
below:

1.00 g analyticalsample200-mL ground-in stopper Erlenmeyer flask(powdery)← 100 mL of water
Extraction

Stir to mix, 10 minites

Stand still
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

Sample solution

Supernatant

Measurement

Liquid Chromatograph

Figure 1 Flow sheet for urea nitrogen in fertilizers(Extraction procedure (4.1.1) and measurement)
1.00 g100-mL volumetric flask
analytical sample (fluid)←About 50 mL of water
Extraction

Shake to mix← Water (up to the marked line)
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

Sample solution

Supernatant

Measurement

Liquid Chromatograph

Figure 2

Flow sheet for urea nitrogen in fertilizers(Extraction procedure (4.1.2) and measurement)

538

Testing Methods for Fertilizers (2018)

Reference: Chromatogram of the standard solution for calibration curve preparation of urea
nitrogen is shown below.

Reference diagram 1 HPLC chromatogram of the mixture standard solutions (10 mg/L foreach) for calibration curve preparation
Peak name(1) Urea nitrogen(2) Biuret nitrogen(3) Dicyandiamide nitrogen(4) Guanidine nitrogen(5) Guanylurea nitrogen
Measurement conditions for HPLC
Column: Asahipak ES-502C 7C (7.5-mm inner diameter, 100-mm long, 9-μm
particle diameter)
Other conditions are according to the example of HPLC measurement conditionsin (4.2) a)539

Testing Methods for Fertilizers (2018)

Reference diagram 2

HPLC chromatogram of the urea nitrogen standard solutions (20 mg/L)
and a formaldehyde processed urea fertilizer for calibration curve
preparation

Peak name(1) Urea nitrogen(2) Biuret nitrogen
Measurement conditions for HPLC
Column: PRP-X200 (4.1-mm inner diameter, 150-mm long, 10-µm particle
diameter)Other conditions are according to the example of HPLC measurement conditionsin (4.2) a)540

Testing Methods for Fertilizers (2018)

6.3.c
p-dimethylaminobenzaldehide absorptiometry(1) Summary
The testing method is applicable to fertilizers containing urea nitrogen. However, isobutyraldehyde
condensed urea fertilizers, formaldehyde processed urea fertilizers, nitrolime, sludge fertilizers, etc.,
and special fertilizers are excluded. This testing method is classified as Type D and its symbol is
6.3.c-2018 or U-N.c-1.
Add water to an analytical sample to extract urea and measure the coloration formed by the reaction
with dimethylaminobenzaldehide by absorbance to obtain urea nitrogen (U-N) in an analyticalsample. In addition, the performance of this testing method is shown in Comment 3.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Coloring reagent solution (1): Dissolve 20 g of p-dimethylaminobenzaldehide specified in JIS
K 8101 in 1000 mL of ethanol (99.5) specified in JIS K 8101 and 100 mL of hydrochloric acidspecified in JIS K 8180 and leave at rest overnight (2).
c) Urea nitrogen standard solution (U-N 2 mg/mL) (1): Put 0.429 g of urea specified in JIS K
8731 in a weighing dish and measure the mass to the order of 0.1 mg. Add a small amount ofwater to dissolve, then transfer to a 100-mL volumetric flask and add water up to the markedline.
d) Urea nitrogen standard solution for the calibration curve preparation (U-N 200 µg/mL)(1): Put 10 mL of urea nitrogen standard solution (U-N 2 mg/mL) to a 100-mL volumetric flask
and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in an amber bottle.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Spectrophotometer: A spectrophotometer specified in JIS K 0115b) Magnetic stirrer:(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200-mL Erlenmeyer flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, filter with Type 3 filter paper to make a sample solution.
Comment 1 The procedure in (4.1) is the same as the procedure in (4.1.2) of 6.3.a.
Comment 2 When the determination is affected by the coloring of the sample solution of (4.1) c),
add about 0.5 g of active carbon and filter with Type 3 filter paper to make a sample
solution from which the coloring disappears.
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 5 mg asU-N) to a 50-mL volumetric flask.
b) Add 20 mL of coloring reagent solution, and further add water up to the marked line, and thenleave at rest for about 30 minutes.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
541

Testing Methods for Fertilizers (2018)a) Measurement conditions of spectrophotometer: Set up the measurement conditions ofspectrophotometer considering the following.
Detection wavelength: 450 nmb) Calibration curve preparation
1) Transfer 2.5 mL - 25 mL of urea nitrogen standard solution (U-N 200 µg/mL) to 50-mL
volumetric flasks step-by-step.
2) Conduct the same procedures as (4.2) b) to make U-N 0.5 mg/50 mL - 5 mg/50 mL of theurea nitrogen standard solutions for the calibration curve preparation.
3) Conduct the same procedures as 2) for another 50-mL volumetric flask to make the blank test
solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 450 nm of the urea nitrogen standard solutions for thecalibration curve preparation using the blank test solution for the calibration curve preparation
as the control.
5) Prepare a curve for the relationship between the urea nitrogen (U-N) concentration and theabsorbance of the urea nitrogen standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) b), measure absorbance by the same procedure as b) 4).
2) Obtain the urea nitrogen (U-N) content from the calibration curve to calculate the urea
nitrogen (U-N) in the analytical sample.
Comment 3 Additive recovery testing was conducted using a compound fertilizer, crustacea
grade fertilizer powdery and a preparation sample. As a result, the mean recovery atadditive level of 20 % (mass fraction), 10 % (mass fraction) and 3 % (mass fraction)
were 100.0 % - 102.4 %, 100.5 % - 102.0 % and 98.0 % - 103.3 % as urea nitrogen(U-N) respectively.
The results of the repeatability tests on different days using urea, a designatedblended fertilizer and a compound fertilizer to evaluate precision were analyzed by
the one-way analysis of variance. Table 1 shows the calculation results ofintermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.2 % (mass fraction).

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
1)

T
7

3)

3)

(%)
45.9

(%)
0.89

Urea
Designated
7
7.45
0.16
blended fertilizer
Compound fertilizer
7
1.12
0.02
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

542

(%)
1.9

(%)
0.91

3)

(%)
2.0

2.1

0.20

2.7

2.2
0.03
2.9
6） Intermediate standard deviation
7） Intermediate relative standard deviation

Testing Methods for Fertilizers (2018)

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.60- 62, Yokendo, Tokyo (1988)(5)

Flow sheet for testing method: The flow sheet for urea nitrogen in fertilizers is shown
below:
1.00 gAnalytical sample
200-mL Erlenmeyer flask(powdery)← 100 mL of water
Extract
Stir for 10 minutes
Stand still
Filtration

Type 3 filter paper

Sample solution
Figure 1 Flow sheet for urea nitrogen in fertilizers (Extraction procedure)

Sample solution
Aliquot(predetermined
50-mL Volumetric flask
volume)← 20 mL of coloring reagent solution← Water (up to the marked line)
Leave at rest
About 30 minutes
Measurement

Spectrophotometer (450 nm)

Figure 2 Flow sheet for urea nitrogen in fertilizers (Measurement procedure)

543

Testing Methods for Fertilizers (2018)

6.4 Guanidine nitrogen
6.4.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 6.4.a-2017 or Gd-N.a-1.
Add water to an analytical sample to extract guanidine, introduce it to a High-Performance
Chromatograph (HPLC) to isolate it with a weak acid ion-exchange column, and then measure atwavelength 190 nm to obtain guanidine nitrogen (Gd-N) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 6.
Biuret nitrogen (B-N), dicyandiamide nitrogen (Dd-N), urea nitrogen(U-N) and guanylurea nitrogen(GU-N) can be simultaneously quantified by using this method. (Refer to Comment 5).
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Potassium dihydrogen phosphate: A JIS Guaranteed Reagent specified in JIS K 9007 or areagent of equivalent quality.
c) Phosphoric acid: A JIS Guaranteed Reagent specified in JIS K 9005 or a reagent ofequivalent quality.
d) Guanidine nitrogen standard solution (Gd-N 2 mg/mL) (1): Put 0.515 g of guanidine sulfate[C2H10N6･H2SO4] (2) in a weighing dish and measure the mass to the order of 0.1 mg. Add asmall amount of water to dissolve, then transfer to a 100-mL volumetric flask and add water
up to the marked line.
e) Guanidine nitrogen standard solution for the calibration curve preparation (Gd-N 200µg/mL) (1): Put 10 mL of guanidine nitrogen standard solution (Gd-N 2 mg/mL) to a 100-mL
volumetric flask and add water up to the marked line.
f) Guanidine nitrogen standard solution (Gd-N 50 µg/mL - 100 µg/mL) (1): Put 25 mL - 50
mL of guanidine nitrogen standard solution (Gd-N 200 µg/mL) to 100-mL volumetric flasks
and add water up to the marked line.
g) Guanidine nitrogen standard solution for the calibration curve preparation (Gd-N 1
μg/mL - 50 μg/mL) (1): At the time of usage, put 1 mL - 50 mL of guanidine nitrogen standard
solution (Gd-N 100 µg/mL) to 100-mL volumetric flasks step-by-step and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity as guanidine sulfate iscommercially sold.
Comment 1 Guanidine sulfate is commercially sold by FUJIFILM Wako Pure Chemical Co., Ltd.,
Kanto Chemical Co., Inc. and Tokyo Chemical Industry Co., Ltd.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 7.5-mm inner diameter 100-mm long stainless steel column tube filled withweak acid ion-exchange resin.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 190 nm.
b) Magnetic stirrer:c) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g - 10000
× g.
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Testing Methods for Fertilizers (2018)

Comment 2 A column is sold under the production name Asahipak ES-502C 7C.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution (3) to a 1.5-mL ground-in stopper
centrifugal precipitate tube (4).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (5) and use thesupernatant as the extract.
Note (3) If there is a possibility that the guanidine nitrogen (Gd-N) concentration in the sample
solution will exceed the maximum limit of the calibration curve, dilute apredetermined amount of supernatant with water.
(4) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(5) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water (6), and shake to mix.
c) Add water up to the marked line and transfer it to a 1.5-mL ground-in stopper centrifugal
precipitate tube (4).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (5) and use thesupernatant as the extract.
Note (6) If there is a possibility that the guanidine nitrogen (Gd-N) concentration in the sample
solution will exceed the maximum limit of the calibration curve, dilute apredetermined amount of supernatant with water.
Comment 3 Instead of procedures in (4.1.1) c) - d) or (4.1.2) c) - d), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5 µm) made of hydrophilic PTFE
and the filtrate can be the sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions is shown below. Set up the measurement conditions
considering it:1) Column: A weak acid ion-exchange resin column (7.5-mm inner diameter, 100-mm long,
5-µm - 10-µm particle diameter)
2) Column bath temperature: 40 °C
3) Eluent (1): Dissolve 3.92 g of potassium dihydrogenphosphate and 0.12 g of phosphoric acidin water to make 1000 mL. Filter with a membrane filter (aperture diameter: no more than 0.5µm) made of hydrophilic PTFE.
545

Testing Methods for Fertilizers (2018)

4) Flow rate: 0.6 mL/min
5) Injection volume: 10 µL
6) Detection unit: An absorptiometric detector, measurement wavelength: 190 nm
Comment 4 Eluent can be prepared as follows. Dissolve 19.6 g of potassium
dihydrogenphosphate and 0.584 g of phosphoric acid with water to make 500 mL andstore in a refrigerator. At the time of usage, dilute a predetermined volume of thesolution by a factor of 10 and filter with a membrane filter (aperture diameter: no
more than 0.5 µm) made of hydrophilic PTFE.
b) Calibration curve preparation
1) Inject 10 µL of respective standard solutions for the calibration curve preparation to an HPLC,
record chromatogram at wavelength 190 nm and obtain the peak height.
2) Prepare a curve for the relationship between the guanidine nitrogen (Gd-N) concentration andthe peak height at wavelength 190 nm of the respective standard solutions for the calibration
curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the guanidine nitrogen (Gd-N) content from the peak height using the calibration curve
to calculate the guanidine nitrogen (Gd-N) in the analytical sample.
Comment 5 This testing method enables the simultaneous measurement of biuret nitrogen (B-N),
urea nitrogen (U-N), dicyandiamide nitrogen (Dd-N), guanidine urea (Gd-N) andguanylurea nitrogen (GU-N). In that case, see 5.10.a Comment 5.
Comment 6 Additive recovery testing was conducted using a preparation sample for a guanylurea
fertilizer (one brand). As a result, the mean recovery at additive level of 3.71 %(mass fraction), 1.85 % (mass fraction) and 0.371 % (mass fraction) were 91.2 %,94.0 % and 100.0 % respectively.
The results of the repeatability tests on different days using a guanylurea fertilizer toevaluate precision were analyzed by one-way analysis of variance. Table 1 shows thecalculation results of intermediate precision and repeatability. Table 2 shows results
and analysis results from a collaborative study for testing method validation.
Additionally, the minimum limit of quantification of this testing method is about
0.02 % (mass fraction).

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
4)
5)
6)
7)
repeatability Average2)
sr
RSD rs I(T)RSD I(T)
1)

3)

3)

T
(%)
(%)
Guanylurea fertilizer
5
1.81
0.01
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

546

3)
(%)
(%)
(%)
0.8
0.04
2.0
6） Intermediate standard deviation
7） Intermediate relative standard deviation

Testing Methods for Fertilizers (2018)

Table 2 Results and analysis results from a collaborative study
for the test method validation of guanidine nitrogen
Sample name

Number of1)laboratories

4)

RSD r

3)

(%)
3.7
4.2
2.0
2.6
4.0

Mean

2)

sr

3)

(%)

(%)

12
4.91
0.18
Compound fertilizer 1
12
3.94
0.16
Compound fertilizer 2
11
3.03
0.06
Compound fertilizer 3
11
2.05
0.05
Compound fertilizer 4
11
5.13
0.21
Guanylurea fertilizer
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

5)

6)

RSD R

(%)

3)

(%)

0.29
0.27
0.12
0.09
0.19

5.8
6.8
4.0
4.2
3.6

sR

7)

References
1) Masahiro ECHI, Yasuharu KIMURA and Yuji SHIRAI: Determination of Urea Nitrogen,
Biuret Nitrogen, Dicyandiamide Nitrogen, Guanidine Nitrogen and Guanyl urea
Nitrogen in Fertilizer by High-Performance Liquid Chromatography: A
Single-Laboratory Validation, Research Report of Fertilizer, Vol.10, p. 72 - 85 (2017)2)
Norio FUNAKI and Yasuharu KIMURA: Determination of Urea Nitrogen, Biuret
Nitrogen, Dicyandiamide Nitrogen, Guanidine Nitrogen and Guanyl urea Nitrogen in
Fertilizer by High-Performance Liquid Chromatography (HPLC): A Collaborative Study,
Research Report of Fertilizer, Vol.10, p. 86 - 100 (2017)

547

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for guanidine nitrogen in fertilizers is shown
below:

1.00 ganalytical sample
200-mL ground-in stopper Erlenmeyer flask(powdery)← 100 mL of water
Extraction

Stir to mix, 10 minites

Stand still
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

Sample solution

Supernatant

Measurement

Liquid Chromatograph

Figure 1 Flow sheet for guanidine nitrogen in fertilizers(Extraction procedure (4.1.1) and measurement)

1.00 ganalytical sample (fluid)

100-mL volumetric flask←About 50 mL of water
Extraction
Shake to mix← Water (up to the marked line)
Ground-in stopper centrifugal precipitate,
Centrifugal separation
8000 ×g - 10000 ×g , 5 minutes
Sample solution

Supernatant

Measurement

Liquid Chromatograph

Figure 2 Flow sheet for guanidine nitrogen in fertilizers(Extraction procedure (4.1.2) and measurement)

548

Testing Methods for Fertilizers (2018)

Reference: Chromatogram of the standard solution for calibration curve preparation of guanidine
nitrogen is shown below.

Reference diagram

HPLC chromatogram of the mixture standard solutions (10 mg/L for
each)

Peak name(1) Urea nitrogen(2) Biuret nitrogen(3) Dicyandiamide nitrogen(4) Guanidine nitrogen(5) Guanylurea nitrogen
Measurement conditions for HPLC
Column: Asahipak ES-502C 7C (7.5-mm inner diameter, 100-mm long, 9-μm
particle diameter)
Other conditions are according to the example of HPLC measurement conditionsin (4.2) a)549

Testing Methods for Fertilizers (2018)

6.5 Cold buffer solution soluble nitrogen (water-soluble nitrogen)6.5.a Cold buffer solution method(1) Summary
The test method is applicable to formaldehyde processed urea fertilizers. This testing method isclassified as Type A (Def-M) and its symbol is 6.5.a-2017 or Buf(C)-N.a-1.
Add a phosphate solution (cold buffer solution) to an analytical sample to extract, add copper (II)sulfate pentahydrate, sulfuric acid and potassium sulfate, pretreat by the Kjeidahl method to change
cold buffer solution soluble nitrogen to ammonium ion and add a sodium hydroxide solution tosubject to steam distillation. Collect isolated ammonia with 0.25 mol/L sulfuric acid and measuresurplus sulfuric acid by (neutralization) titration using a 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution to obtain cold buffer solution soluble nitrogen (water-soluble nitrogen) in the analyticalsample. Or collect isolated ammonia with a boric acid solution and measure ammonium ion by(neutralization) titration using 0.25 mol/L sulfuric acid to obtain cold buffer solution soluble
nitrogen (water-soluble nitrogen) in the analytical sample.
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in JIS
K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C1)
W1： Mass (g) of sulfamic acid sampled
A： Purity (% (mass fraction)) of sulfamic acidV1： Volume (mL) of sulfamic acid solution transferred
V2： Constant volume (250 mL) of sulfamic acid solution
V3： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
c) 0.25 mol/L sulfuric acid (1) (2): Add about 14 mL of sulfuric acid to a beaker containing 100
mL of water in advance, stir well, and add water to make 1000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a 200-mL- 300-mL Erlenmeyer flask, add a few drops of methyl red−methylene blue mixture solution,and titrate with a 0.1 mol/L -0.2 mol/L sodium hydroxide solution until the color of thesolution becomes gray-green (4). Calculate the volume of a 0.1 mol/L - 0.2 mol/L sodium
550

Testing Methods for Fertilizers (2018)hydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the following formula(1). Or, calculate the factor of 0.25 mol/L sulfuric acid by the following formula (2):Volume (B) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1 mL of
0.25 mol/L sulfuric acid=V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2 × 2)………. (2)V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5： Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 in water tomake 1000 mL.
e) Sodium hydroxide solution (200 g/L - 500 g/L) (1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
f) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water to make 100 mL.
g) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
h) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in JIS K
8897 in 100 mL of ethanol (95) specified in JIS K 8102.
i) Methyl red−methylene blue mixture solution: To 2 volumes of methyl red solution (0.1g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
j) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green specified
in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
k) Methyl red−bromocresol green mixture solution: To a methyl red solution (0.1 g/100 mL),
add equal volume of bromocresol green solution (0.5 g/100 mL).
l) Phosphate solution: Dissolve 3.63 g of potassium dihydrogen phosphate specified in JIS K
9007 and 5.68 g of disodium hydrogen phosphate specified in JIS K 9020 in 1000 mL ofwater (5). When it is used, adjust the liquid temperature at about 25 ºC (cold buffer solution).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL -10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(5) pH 7.0 ± pH 0.2
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
Comment 2 0.25 mol/L sulfuric acid in (2) c) can be replaced with 0.25 mol/L sulfuric acidconforming to ISO/IEC 17025.
551

Testing Methods for Fertilizers (2018)(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Rotary shaker: A rotary shaker that can rotate a 250-mL volumetric flask upside down at 30- 40 revolutions/min.
b) Steam distillation apparatusc) Digestion flask: A Kjeldahl flask which can be connected to a steam distillation apparatus.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample (6), and put it into a 250 mL- Erlenmeyer flask.
b) Add about 200 mL of phosphate solution and shake to mix at the rate of 30 - 40
revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make a sample solution.
Note (6) Prepare the test sample according to Comment 3.
Comment 3 Crush a laboratory sample with a mortar or a pestle, etc. until it completely passes
through an 850 µm aperture sieve.
Comment 4 If there is no possibility for an analytical sample to hydrolyze, it is allowed to use
water instead of a phosphoric acid solution.
Comment 5 The temperature of a solution in the procedure in (4.1) b) - d) should be no morethan 26 ºC.
(4.2) Kjeldahl method: Conduct digestion as shown below.
a) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 g or less as
cold buffer solution soluble nitrogen) to a 300-mL distillation flask.
b) Add 0.1 g of copper (II) sulfate pentahydrate (7) specified in JIS K 8962, and further add 5 mLof sulfuric acid, shake to mix and heat gently to evaporate moisture.
c) After standing to cool, add 1 g of potassium sulfate specified in JIS K 8962 and heat to digest.
d) Ignite further for 30 minutes.
e) After standing to cool, add water while shaking to mix until liquid volume reaches about 30
mL and cool it to make the digestion solution.
Note (7) Crush into powder as appropriate.
(4.3) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used in measurement.
a) Transfer a predetermined amount (8) of 0.25 mol/L sulfuric acid to an acceptor, (9) add a few
drops of methyl red−methylene blue mixture solution, and connect this acceptor to a steam
distillation apparatus. Or, transfer a predetermined amount (8) of boric acid solution (40 g/L) toan acceptor, (9) add a few drops of methyl red−bromocresol green mixture solution, andconnect this acceptor to a steam distillation apparatus.
b) Add a proper amount of sodium hydroxide solution (200 g/L - 500 g/L) (10), and immediately
connect this digestion flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill at a
distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution in theacceptor with a small amount of water, and pool the washing with the distillate.
552

Testing Methods for Fertilizers (2018)

Note (8) 5 mL - 20 mL(9) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or a boric acid solution (40 g/L).
(10) An amount sufficient to make the solution strong alkalinity. A blue color will appear.
(4.4) Measurement: Conduct measurement as shown below.
(4.4.1) When 0.25 mol/L sulfuric acid is used in (4.2):a) Titrate the distillate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes gray-green (4).
b) Calculate the cold buffer solution soluble nitrogen in the analytical sample by the following
formula:Cold buffer solution soluble nitrogen (% (mass fraction)) in the analytical sample= (B × V6－V7) × C1 × f1 × (V8/V9) × (14.007/W2) × (100/1000)= (B × V6－V7) × C1 × f1 × (V8/V9) × (1.4007/W2)
B： Volume of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acidV6： Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2) a)
V7： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V8： Predetermined volume (mL) of the extract in (4.1) c)
V9： Transferred amount (mL) of the extract subjected to the Kjeldahl method in(4.2) a)W2： Mass (g) of the analytical sample(4.4.2) When a boric acid solution (40 g/L) is used in (4.2):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomes lightred (11).
b) Calculate the cold buffer solution soluble nitrogen in the analytical sample by the following
formula:Cold buffer solution soluble nitrogen (% (mass fraction)) in the analytical sample= V10 × C2 × 2 × f2 × (V11/V12) × (14.007/W2) × (100/1000)= V10 × C2 × f2 × (V11/V12) × (2.8014/W2)
V10： Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidf2： Factor of 0.25 mol/L sulfuric acidV11： Predetermined volume (mL) of the extract in (4.1) c)
V12： Transferred amount (mL) of the extract subjected to the Kjeldahl method in(4.2) a)W2： Mass (g) of the analytical sample
Note (11) The endpoint is reached when the color changes from green to light red.
Comment 6 The titration procedures in (2) a) Standardization, (2) c) Standardization and (4.4)
553

Testing Methods for Fertilizers (2018)

can be conducted by an automatic titrator. The setup of the titration program, thedetermination parameter for the endpoint and vessels such as acceptors are according
to the specification and the operation method of the automatic titrator used.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.67- 68, Yokendo, Tokyo (1988)(5)

Flow sheet for cold buffer solution soluble nitrogen testing method: The flow sheets forcold buffer solution soluble nitrogen testing method in formaldehyde processed urea
fertilizers are shown below:
1.00 gWeigh into a 250-mL volumetric flask.
analytical sample←150 mL phosphate solution
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), 1 hour← small amount of water
Filtration
Type 3 filter paper
Extract
Figure 1 Flow sheet for cold buffer solution soluble nitrogen testing method
in formaldehyde processed urea fertilizers(Extraction procedure)

Extract
Aliquot
300-mL digestion flask(predeterminedvolume)← 0.1 g copper (II) sulfate pentahydrate← 5 mL sulfuric acid
Heating
GentlyStanding to cool
← 1 g potassium sulfate
Heating
After digestion, ignite further for 30 minutes
Standing to cool
← Water (until liquid volume reaches about 30 mL)
Cooling
Digestion solution
Figure 2 Flow sheet for cold buffer solution soluble nitrogen testing method
in formaldehyde processed urea fertilizers(Kjeldahl method procedure)554

Testing Methods for Fertilizers (2018)

Digestion solution← Sodium hydroxide solution (200 g/L - 500 g/L)
Receiver: 200-mL - 300 - mL Erlenmeyer flask or beakerA predetermined amount of 0.25 mol/L sulfuric acid andSteam distillationa few drops of methyl red-methylene blue mixture solution,
apparatus
or boric acid solution (40 g/L), several drops ofmethyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min -7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L-0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure 3 Flow sheet for cold buffer solution soluble nitrogen testing method
in formaldehyde processed urea fertilizers(Distillation and measurement procedure)

555

Testing Methods for Fertilizers (2018)

6.6 Heat buffer solution soluble nitrogen (hot-water soluble nitrogen)6.6.a Heat buffer solution method(1) Summary
The testing method is applicable to methylolurea polymerization fertilizers. This testing method isclassified as Type A(Def-M) and its symbol is 6.6.a-2017 or Buf(H)-N.a-1.
Add a heat phosphate solution (heat buffer solution) to an analytical sample to elute heat buffer
solution soluble nitrogen, add copper (II) sulfate pentahydrate, sulfuric acid and potassium sulfate,
pretreat non-dissolved matter by Kjeidahl method to change heat buffer solution soluble nitrogen toammonium ion and add a sodium hydroxide solution to subject to steam distillation. Collect isolated
ammonia with 0.25 mol/L sulfuric acid and measure surplus sulfuric acid by (neutralization)
titration using a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution to obtain the nitrogen which is
insoluble in a heat buffer solution in the analytical sample. Or collect isolated ammonia with a boric
acid solution and measure ammonium ion by (neutralization) titration using 0.25 mol/L sulfuric acidto obtain the nitrogen which is insoluble in a heat buffer solution in the analytical sample. Subtract
the nitrogen whichi is insoluble in a heat buffer solution from the separately obtained total nitrogen(T-N) according to the method in 4.1.1 to calculate the nitrogen which is soluble in a heat buffer
solution (hot water-soluble nitrogen).
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days. Transfer
5.5 mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000mL of water.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C1)
W1： Mass (g) of sulfamic acid sampled
A： Purity (% (mass fraction)) of sulfamic acidV1： Volume (mL) of sulfamic acid solution transferred
V2： Constant volume (250 mL) of sulfamic acid solution
V3： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
c) 0.25 mol/L sulfuric acid (1) (2): Add about 14 mL of sulfuric acid to a beaker containing 100
mL of water in advance, stir well, and add water to make 1000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a 200-mL
556

Testing Methods for Fertilizers (2018)- 300-mL Erlenmeyer flask, add a few drops of methyl red−methylene blue mixture solution,and titrate with a 0.1 mol/L -0.2 mol/L sodium hydroxide solution until the color of thesolution becomes gray-green (4). Calculate the volume of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the following formula(1). Or, calculate the factor of 0.25 mol/L sulfuric acid by the following formula (2):Volume (B) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1 mL of
0.25 mol/L sulfuric acid= V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2 × 2)………. (2)V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5： Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 in water tomake 1000 mL.
e) Catalyst (5): Mix potassium sulfate specified in JIS K 8962 and copper (II) sulfate
pentahydrate (6) specified in JIS K 8983 in the ratio of 9 to 1.
f) Sodium hydroxide solution (200 g/L - 500 g/L) (1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water to make 100 mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
i) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in JIS K
8897 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methyl red−methylene blue mixture solution: To 2 volumes of methyl red solution (0.1g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
k) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green specified
in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red−bromocresol green mixture solution: To a methyl red solution (0.1 g/100 mL),
add equal volume of bromocresol green solution (0.5 g/100 mL).
m) Heat phosphate solution: Dissolve 3.63 g of potassium dihydrogen phosphate specified in
JIS K 9007 and 5.68 g of disodium hydrogen phosphate specified in JIS K 9020 in 1000 mLof water (7). When it is used, heat until it reaches boiling point (heat buffer solution).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL - 10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(5) A tablet is commercially available.
(6) Crush into powder as appropriate.
(7) pH 7.0 ± pH 0.2
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Testing Methods for Fertilizers (2018)

Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
Comment 2 0.25 mol/L sulfuric acid in (2) c) can be replaced with 0.25 mol/L sulfuric acidconforming to ISO/IEC 17025.
(3)
a)
b)
c)
d)

Apparatus and instruments: Apparatus and instruments are shown below.
Water bath: A water bath which can boil water.
Steam distillation apparatus
Digestion flask: A Kjeldahl flask which can be connected to a steam distillation apparatus.
Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to a steam
distillation apparatus.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample (8) and transfer to a 300-mL tall beaker.
b) Add 100 mL of heat phosphate solution and stir it gently.
c) Cover the tall beaker with a watch glass, and heat in a boiling water bath while stirring at
ten-minute intervals for about 30 minutes.
d) Immediately filter with Type 3 filter paper, move the whole non-dissolved matter in a vesselon the filter paper with 100 mL of heat phosphate solution and wash the non-dissolved matterwith hot water.
Note (8) Prepare the test sample according to Comment 3.
Comment 3 Crush a laboratory sample with a mortar or a pestle, etc. until it completely passes
through an 850 µm aperture sieve.
(4.2) Kjeldahl method: Conduct digestion as shown below.
a) Put the non-dissolved matter in (4.1) d) together with the filter paper into a 300-mL - 500-mL
digestion flask.
b) Add 5 g - 10 g of catalyst, and further add 20 mL - 40 mL of sulfuric acid, shake to mix andheat gently.
c) After bubbles cease to form, heat until white smoke of sulfuric acid evolves.
d) Ignite until organic matters are completely digested (9).
e) After standing to cool, add a small amount of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water (10), and further mix by shaking.
f) After standing to cool, add water up to the marked line to make the digestion solution.
Note (9) When the solution has finished changing color, heat further for no less than 2 hours.
(10) When the entire sample solution volume is used in measurement, it is not necessary totransfer it to a volumetric flask.
(4.3) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used in measurement.
a) Transfer a predetermined amount (11) of 0.25 mol/L sulfuric acid to an acceptor (12), add a few
drops of methyl red−methylene blue mixture solution, and connect this acceptor to a steam
distillation apparatus. Or, transfer a predetermined amount (11) of boric acid solution (40 g/L)
to an acceptor (12), add a few drops of methyl red−bromocresol green mixture solution, and558

Testing Methods for Fertilizers (2018)

connect this acceptor to a steam distillation apparatus.
b) Transfer a predetermined amount of the digestion solution to a 300-mL distillation flask, add aproper amount of sodium hydroxide solution (200 g/L - 500 g/L) (13), and immediately connect
this distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill at a
distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution in theacceptor with a small amount of water, and pool the washing with the distillate.
Note (11) 5 mL - 20 mL(12) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or a boric acid solution (40 g/L).
(13) An amount sufficient to make the solution strong alkalinity. A blue color will appear.
(4.4) Measurement: Conduct measurement as shown below.
(4.4.1) When 0.25 mol/L sulfuric acid is used in (4.3):a) Titrate the distillate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes gray-green (4).
b) Calculate the heat buffer solution soluble nitrogen in the analytical sample by the following
formula:c) Subtract the nitrogen whichi is insoluble in a heat buffer solution from the separately obtained
total nitrogen (T-N) according to the method in 4.1.1 to calculate the nitrogen which is soluble
in a heat buffer solution (14).
Heat buffer solution soluble nitrogen (% (mass fraction)) in the analytical sample= (B × V6 － V7) × C1 × f1 × (V8/V9) × (14.007/W2) × (100/1000)= (B × V6 － V7) × C1 × f1 × (V8/V9) × (1.4007/W2)
B： Volume of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acidV6： Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2) a)
V7： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V8： Predetermined volume (mL) of the digestion solution in (4.2) f)
V9： Transferred amount (mL) of the digestion solution subjected to distillation in(4.3) b)
W2： Mass (g) of the analytical sample
Note (14) The total nitrogen (T-N) and the nitrogen which is insoluble in a heat buffer solution
use raw data without rounding numerical value.
(4.4.2) When a boric acid solution (40 g/L) is used in (4.3):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomes lightred (15).
b) Calculate the heat buffer solution soluble nitrogen in the analytical sample by the following
formula:559

Testing Methods for Fertilizers (2018)c)

Subtract the nitrogen which is insoluble in a heat buffer solution from the separately obtained
total nitrogen (T-N) according to the method in 4.1.1 to calculate the nitrogen which is soluble
in a heat buffer solution
Heat buffer solution soluble nitrogen (% (mass fraction)) in the analytical sample= V10 × C2 × 2 × f2 × (V11/V12) × (14.007/W2) × (100/1000)= V10 × C2 × f2 × (V11/V12) × (2.8014/W2)
V10： Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidf2： Factor of 0.25 mol/L sulfuric acidV11： Predetermined volume (mL) of the digestion solution in (4.2) f)
V12： Transferred amount (mL) of the digestion solution subjected to distillation in(4.3) b)
W2： Mass (g) of the analytical sample

Note (15) The endpoint is reached when the color changes from green to light red.
Comment 4 The titration procedures in (2) a) Standardization, (2) c) Standardization and (4.4)
can be conducted by an automatic titrator. The setup of the titration program, thedetermination parameter for the endpoint and vessels such as acceptors are according
to the specification and the operation method of the automatic titrator used.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.68- 69, Yokendo, Tokyo (1988)(5)

Flow sheet for heat buffer solution soluble nitrogen testing method: The flow sheets forheat buffer solution soluble nitrogen testing method in methylolurea polymerization fertilizers
are shown below:

1.00 gWeigh into a 300-mL tall beaker
analytical sample←150 mL heat phosphate solution
Stirring
GentlyHeating

Boiling water bathwhile stirring at 10-minute intervals for 30 minutes

Type 3 filter paper
Transfer with 100 mL heat phosphate solution←Wash with hot water
Non-dissolved matter
Filtration

Figure 1 Flow sheet for heat buffer solution soluble nitrogen testing method
in methylolurea polymerization fertilizers(Extraction procedure)

560

Testing Methods for Fertilizers (2018)

Non-dissolved matter
Put
300-mL - 500-mL digestion flask←5 g - 10 g catalyst←20 mL - 40 mL sulfuric acid
Heating
GentlyHeating

After bubbles cease to form,
ignite until organic matters are completely digested

Standing to cool
← A small amount of waterTransfer
250-mL - 500-mLvolumetric flask, water
Cooling← Water (up to the marked line )Digestion solution

Figure 2 Flow sheet for heat buffer solution soluble nitrogen testing method
in methylolurea polymerization fertilizers( Kjeldahl method procedure)

Digestion solution

Aliquot
300-mL distillation flask(predeterminedvolume)← Sodium hydroxide solution (200 g/L - 500 g/L)
Receiver: 200-mL - 300 - mL Erlenmeyer flask or beakerA predetermined amount of 0.25 mol/L sulfuric acid andSteam distillationa few drops of methyl red-methylene blue mixture solution,
apparatus
or boric acid solution (40 g/L), several drops ofmethyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min -7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L-0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure 3 Flow sheet for heat buffer solution soluble nitrogen testing method
in methylolurea polymerization fertilizers( Distillation and measurement procedure)

561

Testing Methods for Fertilizers (2018)

6.7 Activity coefficient of nitrogen
6.7.a Buffer solution method(1) Summary
The testing method is applicable to formaldehyde processed urea fertilizers. This testing method isclassified as Type A(Def-M) and its symbol is 6.7.a-2017 or AI-N.a-1.
Add water to an analytical sample to elute cold water-soluble nitrogen, add potassium sulfate
copper (II) sulfate pentahydrate and sulfuric acid, pretreat non-dissolved matter by Kjeidahl method
to change cold water-soluble nitrogen to ammonium ion and add a sodium hydroxide solution tosubject to steam distillation. Collect isolated ammonia with 0.25 mol/L sulfuric acid and measuresurplus sulfuric acid by (neutralization) titration using a 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution to obtain cold water insoluble nitrogen in the analytical sample. Or collect isolated
ammonia with a boric acid solution and measure ammonium ion by (neutralization) titration using
0.25 mol/L sulfuric acid to obtain cold water-soluble nitrogen in the analytical sample. Separately
add a heat phosphate solution (heat buffer solution) to an analytical sample to elute the nitrogen
which is soluble in a heat buffer solution and conduct the same procedures as above to obtain thenitrogen which is insoluble in a heat buffer solution in the analytical sample. Subtract the nitrogen
which is insoluble in a heat buffer solution from cold water non-dissolved matter and calculate theactivity coefficient of nitrogen by dividing the subtracted value by the cold water non-dissolved
matter.
(2) Reagent: Reagents are as shown below.
a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (1): Transfer about 30 mL of water to apolyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576 byadding in small portions while cooling, seal tightly and leave at rest for 4-5 days. Transfer 5.5
mL -11 mL of the supernatant to a ground-in stoppered storage container, and add 1000 mL ofwater.
Standardization: Dry sulfamic acid reference material for volumetric analysis specified in
JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about 48 hours, thentransfer about 2.5 g to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of water, transfer to a 250-mL volumetric flask, and add water
up to the marked line (1). Transfer a predetermined amount of the solution to a 200-mL 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution (0.1 g/100 mL) as
an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes green. Calculate the factor of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1000/V3) × (1/C1)
W1： Mass (g) of sulfamic acid sampled
A： Purity (% (mass fraction)) of sulfamic acidV1： Volume (mL) of sulfamic acid solution transferred
V2： Constant volume (250 mL) of sulfamic acid solution
V3： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
c) 0.25 mol/L sulfuric acid (1) (2): Add about 14 mL of sulfuric acid to a beaker containing 100
562

Testing Methods for Fertilizers (2018)

mL of water in advance, stir well, and add water to make 1000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a 200-mL- 300-mL Erlenmeyer flask, add a few drops of methyl red−methylene blue mixture solution,and titrate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of thesolution becomes gray-green (4). Calculate the volume of a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the following formula(1). Or, calculate the factor of 0.25 mol/L sulfuric acid by the following formula (2):Volume (B) of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL of 0.25
mol/L sulfuric acid= V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2 × 2)………. (2)V4： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5： Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 in water tomake 1000 mL.
e) Catalyst (5): Mix potassium sulfate specified in JIS K 8962 and copper (II) sulfate
pentahydrate (6) specified in JIS K 8983 in the ratio of 9 to 1.
f) Sodium hydroxide solution (200 g/L - 500 g/L) (1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue specified
in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water to make 100 mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K 8896 in
100 mL of ethanol (95) specified in JIS K 8102.
i) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in JIS K
8897 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methyl red−methylene blue mixture solution: To 2 volumes of methyl red solution (0.1g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
k) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green specified
in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red−bromocresol green mixture solution: To a methyl red solution (0.1 g/100 mL),
add equal volume of bromocresol green solution (0.5 g/100 mL).
m) Heat phosphate solution: Dissolve 1.43 g of potassium dihydrogen phosphate specified in
JIS K 9007 and 9.10 g of disodium hydrogen phosphate specified in JIS K 9020 in 1000 mLof water (7). When it is used, heat until it reaches boiling point (heat buffer solution).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL -10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(5) A tablet is commercially available.
563

Testing Methods for Fertilizers (2018)(6) Crush into powder as appropriate.
(7) pH 7.5 ± pH 0.2
Comment 1 A 0.1 mol/L - 0.2 mol/L sodium hydroxide solution in (2) a) can be replaced with a
0.1 mol/L sodium hydroxide solution or a 0.2 mol/L sodium hydroxide solution
conforming to ISO/IEC 17025.
Comment 2 0.25 mol/L sulfuric acid in (2) c) can be replaced with 0.25 mol/L sulfuric acidconforming to ISO/IEC 17025.
(3)
a)
b)
c)
d)

Apparatus and instruments: Apparatus and instruments are shown below.
Water bath: A water bath which can boil water.
Steam distillation apparatus
Digestion flask: A Kjeldahl flask which can be connected to a steam distillation apparatus.
Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to a steam
distillation apparatus.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below. Subject the non-dissolved matters of (4.1.1)f) and (4.1.2) d) to (4.2) Kjeidahl method respectively.
(4.1.1) Extraction by cold watera) Weigh 1.00 g of an analytical sample (8) and transfer to a 50-mL beaker.
b) Add a small amount of ethanol specified in JIS K 8101 to moisten and add 20 mL of water at
25ºC ± 2ºC to stir.
c) Leave at rest for 15 minutes while stirring at 5-minute intervals.
d) Filter the supernatant with Type 2 filter papere) Wash non-dissolved matter with water at 25ºC ± 2ºC five times and filter the supernatant.
f) Transfer the whole non-dissolved matter on the filter paper using water at 25ºC ± 2ºC andwash the non-dissolved matter using water at the same temperature until filtrate reaches 250
mL.
Note (8) Prepare the test sample according to Comment 3.
Comment 3 Crush a laboratory sample with a mortar or a pestle, etc. until it completely passes
through an 850 µm aperture sieve.
(4.1.2) Extraction by heat phosphate solutiona) Weigh an analytical sample (8) equivalent to 0.12 g of cold non-resolved matter and put it in a
200-mL tall beaker.
b) Add 100 mL of heat phosphate solution and stir it.
c) Cover the tall beaker with a watch glass, and heat in a boiling water bath while stirring at
ten-minute intervals for about 30 minutes.
d) Immediately filter with Type 2 filter paper (9), transfer the whole non-dissolved matter in a
vessel on the filter paper using boiling water and wash the non-dissolved matter using 100 mLof boiling water.
Note (9) If it takes 4 minutes or more for filtrating, conduct extracting anew according toComment 4.
Comment 4 After conducting the procedures in (4.1.2) a) - c), add 1 g of diatomaceous earth tostir and conduct the procedure in (4.1.2) d).
564

Testing Methods for Fertilizers (2018)(4.2) Kjeldahl method: Conduct digestion as shown below.
a) Put the non-dissolved matter in (4.1.1) f) and (4.1.2) d) together with the filter paper into
respective 300-mL - 500-mL digestion flasks.
b) Add 5 g - 10 g of catalyst, and further add 20 mL - 40 mL of sulfuric acid, shake to mix andheat gently.
c) After bubbles cease to form, heat until white smoke of sulfuric acid evolves.
d) Ignite until organic matters are completely digested (10).
e) After standing to cool, add a small amount of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water (11), and further mix by shaking.
f) After cooling is complete, add water up to the marked line to make the digestion solution.
Note (10) When the solution has finished changing color, heat further for no less than 2 hours.
(11) When the entire sample solution volume is used in measurement, it is not necessary totransfer it to a volumetric flask.
(4.3) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used in measurement.
a) Transfer a predetermined amount (12) of 0.25 mol/L sulfuric acid to an acceptor (13), add a few
drops of methyl red−methylene blue mixture solution, and connect this acceptor to a steam
distillation apparatus. Or, transfer a predetermined amount (12) of boric acid solution (40 g/L)
to an acceptor (13), add a few drops of methyl red−bromocresol green mixture solution, andconnect this acceptor to a steam distillation apparatus.
b) Transfer a predetermined amount of the digestion solution to a 300-mL distillation flask, add aproper amount of sodium hydroxide solution (200 g/L - 500 g/L) (14), and immediately connect
this distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill at a
distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution in theacceptor with a small amount of water, and pool the washing with the distillate.
Note (12) 5 mL - 20 mL(13) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or a boric acid solution (40 g/L).
(14) An amount sufficient to make the solution strong alkalinity. A blue color will appear.
(4.4) Measurement: Conduct measurement as shown below.
(4.4.1) When 0.25 mol/L sulfuric acid is used in (4.3):a) Titrate the distillate with a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of
the solution becomes gray-green (4).
b) Calculate the cold-water insoluble nitrogen (N1) and the nitrogen which is insoluble in a heatbuffer solution (N2) in the analytical sample by the following formula (3):c) Obtain the activity coefficient of nitrogen in the analytical sample by the following formula(4) (15).
Cold water insoluble nitrogen (N1) or the nitrogen which is insoluble in a heat buffer
solution (N2) (% (mass fraction)) in the analytical sample= (B × V6 － V7) × C1 × f1 × (V8/V9) × (14.007/W2) × (100/1000)
565

Testing Methods for Fertilizers (2018)= (B × V6 － V7) × C1 × f1 × (V8/V9) × (1.4007/W2)

・・・・・ (3)

B： Volume of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acidV6： Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2) a)
V7： Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1： Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1： Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V8： Predetermined volume (mL) of the digestion solution in (4.2) f)
V9： Transferred amount (mL) of the digestion solution subjected to distillation in(4.3) b)
W2： Mass (g) of the analytical sample
Activity coefficient of nitrogen (%)= ((N1－N2)/N1) × 100

・・・・・ (4)N1： Cold water insoluble nitrogen (% (mass fraction))
N2： Heat buffer solution insoluble nitrogen (% (mass fraction))
Note (15) Cold water insoluble nitrogen (N1) or the nitrogen which is insoluble in a heat buffer
solution (N2) uses raw data without rounding numerical value.
(4.4.2) When a boric acid solution (40 g/L) is used in (4.3):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomes lightred (16).
b) Calculate the cold-water insoluble nitrogen (N1) and the nitrogen which is insoluble in a heatbuffer solution (N2) in the analytical sample by the following formula (5):c) Obtain the activity coefficient of nitrogen in the analytical sample by the formula (4) in (4.4.1)(14).
Cold water insoluble nitrogen (N1) or the nitrogen which is insoluble in a heat buffer
solution (N2) (% (mass fraction)) in the analytical sample= V10 × C2 × 2 × f2 × (V11/V12) × (14.007/W2) × (100/1000)=V10 × C2 × f2 × (V11/V12) × (2.8014/W2)
・・・・・ (3)V10： Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2： Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidf2： Factor of 0.25 mol/L sulfuric acidV11： Predetermined volume (mL) of the digestion solution in (4.2) f)
V12： Transferred amount (mL) of the digestion solution subjected to distillation in(4.3) b)
W2： Mass (g) of the analytical sample
Note (16) The endpoint is reached when the color changes from green to light red.
Comment 5 The titration procedures in (2) a) Standardization, (2) c) Standardization and (4.4)
can be conducted by an automatic titrator. The setup of the titration program, thedetermination parameter for the endpoint and vessels such as acceptors are according
to the specification and the operation method of the automatic titrator used.
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Testing Methods for Fertilizers (2018)

References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.68- 69, Yokendo, Tokyo (1988)(5)

Flow sheet for the activity coefficient of nitrogen testing method: The flow sheets for theactivity coefficient of nitrogen testing method in formaldehyde processed urea fertilizers areshown below:

1.00 gWeigh into a 50-mL beaker
analytical sample←A small amount of ethanol←150 mL water [25ºC±2ºC]
Stirring
Leaving at rest

For 15 minutes
while stirring at 5-minute intervals

Type 2 filter paper
Move with water [25ºC±2ºC]←Wash with 100 mL of water [25ºC±2ºC](until filtrate reaches about 250 mL)
Cold water
non-dissolved mater
Filtration

Figure 1-1 Flow sheet for the activity coefficient of nitrogen testing method
in formaldehyde processed urea fertilizers(Extraction procedure by cold water (4.1.1))
Analytical sample(equivalent to 0.12 g
Weigh into a 200-mL tall beakerof cold water soluble
nitrogen)←150 mL heat phosphate solution
Stirring
Heating

Boiling water bathwhile stirring at 10-minute intervals for 30 minutes

Type 2 filter paper
Move with water (boiling)←Wash with 100 mL of water (boiling)
Heat buffer solution
non-dissolved matter
Filtration

Figure 1-2 Flow sheet for the activity coefficient of nitrogen testing method
in formaldehyde processed urea fertilizers(Extraction procedure by heat phosphate solution (4.1.2))
567

Testing Methods for Fertilizers (2018)

Cold water
non-dissolved mater orheat buffer solution nondissolved matter

Subject Cold water non-dissolved mater orheat buffer solution non-dissolved matter to
Kjeldahl method, disttilation and measurement respectively

Transfer(together with filter
300-mL - 500-mL digestion flask
paper)←5 g - 10 g catalyst←20 mL - 40 mL sulfuric acid
Heating
GentlyHeating

After bubbles cease to form,
ignite until organic matters are completely digested

Standing to cool
← A small amount of waterTransfer
250-mL - 500-mLvolumetric flask, water
Cooling← Water (up to the marked line )Digestion solution
Figure 2 Flow sheet for the activity coefficient of nitrogen testing method
in formaldehyde processed urea fertilizers(Kjeldahl method procedure)

568

Testing Methods for Fertilizers (2018)

Digestion solution
Aliquot (predetermined
300-mL distillation flask
volume)← Sodium hydroxide solution (200 g/L - 500 g/L)
Receiver: 200-mL - 300 - mL Erlenmeyer flask or beakerA predetermined amount of 0.25 mol/L sulfuric acid andSteam distillationa few drops of methyl red-methylene blue mixture solution,
apparatus
or boric acid solution (40 g/L), several drops ofmethyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min -7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L-0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure 3 Flow sheet for the activity coefficient of nitrogen testing method
in formaldehyde processed urea fertilizers(Distillation and measurement procedure)

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Testing Methods for Fertilizers (2018)

6.8 Initial elution rate
6.8.a Standing-in-water method(1) Summary
The testing method is applicable to coated fertilizer. This testing method is classified as TypeA(Def-E) and its symbol is 6.8.a-2017 or SDR.a-1.
An initial elution rate is a quick- acting component of coated fertilizers. Target components include
total nitrogen (T-N), ammoniacal nitrogen (A-N), nitrate nitrogen (N-N), water-soluble phosphoricacid (W-P2O5), water- soluble potassium (W-K2O) and water-soluble magnesia (W-MgO).
Add water to a laboratory sample, leave it at rest while maitaining in water at 30ºC for 24 hours andobtain the initial elution flow of a target component. Separately measure the corresponding
component content by 4.1.1, 4.1.2, 4.1.3, 4.2.4, 4.3.3 or 4.6.3. Calculate an initial elution rate
dividing the initial elution flow of a target component by the corresponding component content.
(2) Reagent: Reagents are as shown below.
a) Reagent solutions for total nitrogen: Reagents corresponding to clauses in 4.1.1 when
determining total nitrogen.
b) Reagent solutions for ammoniacal nitrogen: Reagents corresponding to clauses in 4.1.2
when determining ammoniacal nitrogen.
c) Reagent solutions for nitrate nitrogen: Reagents corresponding to clauses in 4.1.3 when
determining nitrate nitrogen.
d) Reagent solutions for water-soluble phosphoric acid: Reagents corresponding to clauses in
4.2.4 when determining water-soluble phosphoric acid.
e) Reagent solutions for water-soluble potassium: Reagents corresponding to clauses in 4.3.3
when determining water-soluble potassium.
f) Reagent solutions for water-soluble magnesia: Reagents corresponding to clauses in 4.6.3
when determining water-soluble magnesia.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Incubator: An incubator whose temperature is 30 ºC ± 1 ºC.
b) Total nitrogen: Apparatus and instruments corresponding to clauses in 4.1.1 when
determining total nitrogen.
c) Ammonium nitrogen: Apparatus and instruments corresponding to clauses in 4.1.2 when
determining ammoniacal nitrogen.
d) Nitrate nitrogen: Apparatus and instruments corresponding to clauses in 4.1.3 when
determining nitrate nitrogen.
e) Water-soluble phosphoric acid: Apparatus and instruments corresponding to clauses in
4.2.4 when determining water-soluble phosphoric acid.
f) Water-soluble potassium: Apparatus and instruments corresponding to clauses in 4.3.3
when determining water-soluble potassium.
g) Water-soluble magnesia: Apparatus and instruments corresponding to clauses in 4.6.3 when
determining water-soluble magnesia.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 12.5 g of an analytical sample, and put it into a 300 mL- ground-in stopper Erlenmeyer
flask (1).
b) Add 250 mL of water at 30ºC ± 1ºC, put it into an incubator at 30ºC ± 1ºC and leave it at restfor 24 hours (2).
c) Filter with Type 3 filter paper (3) and shake to mix the filtrate to make a sample solution.
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Testing Methods for Fertilizers (2018)

Note (1) Since no grinding is conducted and inhomogeneous laboratory samples are used, it isdesirable to heighten the reliability of determined values by conducting tests using 3 5 laboratory samples.
(2) Since an initial elution flow is estimated to be higher than usual if the laboratory
sample vibrates in water, water should be added gently. Do not shake to mix thesample solution until the filtering in c) is completed.
(3) Filter most of the solution so that non-dissolved matter remains in the Erlenmeyer
flask.
(4.2) Measurement: Conduct the measurements of the initial elution flow of a target component as
specified in respective clauses in a) - f). In addition, specific measurement procedure for eachcomponent is carried out according to a corresponding clause.
a) Total nitrogen: Transfer a predetermined amount of sample solution and quantitate total
nitrogen according to respective clauses in 4.1.1 to make the initial elution flow.
b) Ammonium nitrogen: Transfer a predetermined amount of sample solution and quantitate
ammoniacal nitrogen according to respective clauses in 4.1.2 to make the initial elution flow.
c) Nitrate nitrogen: Transfer a predetermined amount of sample solution and quantitate nitrate
nitrogen according to respective clauses in 4.1.3 to make the initial elution flow.
d) Water-soluble phosphoric acid: Transfer a predetermined amount of sample solution andquantitate water-soluble phosphoric acid according to respective clauses in 4.2.4 to make theinitial elution flow.
e) Water-soluble potassium: Transfer a predetermined amount of sample solution andquantitate water-soluble potassium according to respective clauses in 4.3.3 to make the initial
elution flow.
f) Water-soluble magnesia: Transfer a predetermined amount of sample solution andquantitate water-soluble magnesia according to respective clauses in 4.6.3 to make the initial
elution flow.
(5) Calculation of initial elution ratea) Calculate an initial elution rate (%) by the following formula using the initial elution flow of atarget component obtained in (4.2) and separately determine (4) the corresponding component
content (5).
Initial elution rate (%)= (C1/C2) × 100
C1： Initial elution rate of a target component (% (mass fraction))
C2： Corresponding component content (% (mass fraction))
Note (4) Determine total nitrogen (T-N), ammoniacal nitrogen (A-N), nitrate nitrogen (N-N),
water-soluble phosphoric acid (W-P2O5), water-soluble potassium (W-K2O) andwater-soluble magnesia (W-MgO) in 4.1.1, 4.1.2, 4.1.3, 4.2.4, 4.3.3 or 4.6.3 using test
samples prepared in 2.3 Preparation of test samples.
(5) Initial elution flow and corresponding component content use raw data without
rounding numerical value.
References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.288- 290, Yokendo, Tokyo (1988)

571

Testing Methods for Fertilizers (2018)(6)

Initial elution rate testing method: The flow sheet for initial elution rate testing method in
coated fertilizers is shown below:
12.5 g
300 mL Erlenmeyer flasklaboratory sample←250 mL water [30ºC±1ºC]
Extract
30ºC±1ºC, 24 hours
Filtration

Type 3 filter paper

Determination ofa target component

Initial elution rateof a target component

Figure Flow sheet for initial elution rate testing methodof coated fertilizers

572

Testing Methods for Fertilizers (2018)

6.9 Humic acid (Acid insoluble - alkali soluble component)
6.9.a Gravimetric analysis(1) Summary
The testing method is applicable to humic acid chloride fertilizers. This testing method is classified
as Type A(Def-M) and its symbol is 6.9.a-2017 or H-acid.a-1.
Add hydrochloric acid (1+9) to elute acid dissolved matter, filter non-dissolved matter and measure
the mass of non-dissolved matter to obtain acid non-dissolved matter in the analytical sample.
Separately add hydrochloric acid (1+9) to elute acid dissolved matter, add a sodium hydroxide
solution (10 g/L) to non-dissolved matter to elute alkali dissolved matter, and filter the
non-dissolved matter to obtain acid non-dissolved alkali non-dissolved matter in the analyticalsample. Subtract acid non-dissolved alkali non-dissolved matter from acid dissolved matter tocalculate humic acid (acid insoluble alkali soluble component).
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
(3) Instruments: Instruments are as shown below:a) Shaking apparatusb) Drying apparatus: A drying apparatus that can be adjusted to 105 ºC - 110 ºC.
c) Crucible type glass filter: A crucible type glass filter 1G4 specified in JIS R 3503. Let it
stand to cool in a desiccator after heating at 105 ºC - 110 ºC in advance and measure the mass
to the order of 1 mg.
d) Ground-in stopperweighing bottles (1): Low-form weighing bottles, 50 mm × 30 mm,
specified in JIS R 3503. Let it stand to cool in a desiccator after heating at 105 ºC - 110 ºC in
advance and measure the mass to the order of 1 mg.
Note (1) Aluminum weighing dishes described in the Handbook of the Feed Analysis Standards-2009- can also be used.
(2) A rotary shaker that can rotate a 100-mL ground-in stopper centrifugal precipitate tube
upside down at 30 - 40 revolutions/min may also be used.
(4) Test procedures(4.1) Acid non-dissolved matter(4.1.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg and put it in a 100-mL ground-in
stopper centrifugal precipitate tube.
b) Add 50 mL of hydrochloric acid (1+9) and shake to mix by using a shaking apparatus (2) for
about 1 hour.
c) Centrifuge at about 1700 × g for about five minutes (3) to remove supernatant (4).
d) Add water to stir (5) and centrifuge at about 1700 × g for about five minutes (3) to remove
supernatant (4).
e) Repeat the procedure in d) 3 times.
Note (2) When using a rotary shaker, it should be adjusted to 30 - 40 revolutions/min.
(3) 16.5-cm of radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(4) Remove using Komagome pipet, etc.
573

Testing Methods for Fertilizers (2018)(5) Stir with a glass rod, wash non-dissolved matter which may have adhered to the glassrod with water and add wash to a centrifugal precipitate tube.
(4.1.2) Measurement: Conduct measurement as shown below.
a) Transfer the whole non-dissolved matter in (4.1.1) e) to a crucible type glass filter and filter
under reduced pressure.
b) Put the non-dissolved matter together with the crucible type glass filter into a drying apparatus
and heat at 105 ºC - 110 ºC for 3 hours.
c) As soon as heating is complete, move it into a desiccator and let it stand to cool.
d) After standing to cool, remove the crucible type glass filter from the desiccator and measure
the mass to the order of 1 mg.
(4.2) Acid non-dissolved - alkali non-dissolved matter(4.2.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg and put it in a 100-mL ground-in
stopper centrifugal precipitate tube.
b) Add 50 mL of hydrochloric acid (1+9) and shake to mix by using a shaking apparatus (2) for
about 1 hour.
c) Centrifuge at about 1700 × g for about five minutes (3) to remove supernatant (4).
d) Add water to stir (5) and centrifuge at about 1700 × g for about five minutes (3) to remove
supernatant (4).
e) Repeat the procedure in d) 3 times.
f) Add 50 mL of sodium hydroxide (10 g/L) and shake to mix by using a shaking apparatus (2)for about 1 hour.
g) Centrifuge at about 1700 × g for about five minutes (3) to remove supernatant (4).
h) Add water to stir (5) and centrifuge at about 1700 × g for about five minutes (3) to remove
supernatant (4).
i) Repeat the procedure in h) 3 times.
(4.2.2) Measurement: Conduct measurement as shown below.
a) Put the non-dissolved matter together with a ground -in stopper weighing bottle into a drying
apparatus and heat (6).
b) After standing to cool, transfer the non-dissolved matter into the ground -in stopper weighing
bottle.
c) Put the non-dissolved matter together with a ground -in stopper weighing bottle into a drying
apparatus and heat at 105 ºC - 110 ºC for 3 hours.
d) After heating, fit the stopper into the ground-in stoppered weighing bottle, and immediatelytransfer to a desiccator to let it stand to cool.
e) After standing to cool, remove the ground-in stoppered weighing bottle from the desiccator,and measure the mass to the order of 1 mg.
Note (6) Heat at the temperature to enable the procedure in (4.2.2) b).
(5) Calculation of humic acida) Calculate humic acid by the following formula
Humic acid (% (mass fraction))= (A1/W1) × 100 － (A2/W2) × 100

・・・・・ (1)A1： Mass (g) of the acid non-dissolved matter determined in (4.1.2) d)574

Testing Methods for Fertilizers (2018)

W1： Mass (g) of the analytical sample sampled in (4.1.1)a)A2： Mass (g) of the acid non-dissolved - alkali non-dissolved matter determined in(4.2.2) e)
W2： Mass (g) of the analytical sample sampled in (4.2.1)a)References
1)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.316- 317, Yokendo, Tokyo (1988)(6)

Humic acid testing method: The flow sheet for humic acid testing method is shown below:

1g
Weigh to the order of 1 mg into a 100-mL
analytical sample
ground-in stopper cetrifugal precipitate tubu
←About 50 mL hydrochloric acid (1+9)
Shaking to mix
Shaking, 1 hour
Centrifugal separation

Ground-in stopper centrifugal precipitate tube
About 1700 × g , 5 minutes

Removing supernatant←WaterStirring
Centrifugal separation

Ground-in stopper centrifugal precipitate tube
About 1700 × g , 5 minutes

Removing supernatant
Further repeat 3 times
Transfer

Crucible type glass filter 1G4, water

Heating

105 ºC – 110 ºC, 3 hours

Standing to cool

Desiccator

Measurement

Weigh to the order of 1 mg

Figure 1 Flow sheet for humic acid testing method in humic acid salt fertilizers(Measurement procedure of acid non-dissolved matter (4.1))

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Testing Methods for Fertilizers (2018)

1g
Weigh to the order of 1 mg into a 100-mL
analytical sample ground-in stopper cetrifugal precipitate tubu
← 50 mL hydrochloric acid (1+9)
Shaking to mix
Shaking, 1 hour
Centrifugal separation

Ground-in stopper centrifugal precipitate tube
About 1700 × g , 5 minutes

Removing supernatant←WaterStirring
Centrifugal separation

Ground-in stopper centrifugal precipitate tube
About 1700 × g , 5 minutes

Removing supernatant
Further repeat 3 times← 50 mL sodium hydroxide (10 g/L)
Shaking to mix
Shaking, 1 hour
Centrifugal separation

Ground-in stopper centrifugal precipitate tube
About 1700 × g , 5 minutes

Removing supernatant←WaterStirring
Centrifugal separation

Ground-in stopper centrifugal precipitate tube
About 2000 × g , 5 minutes

Removing supernatant
Further repeat 3 times
HeatingDryingTransfer

Ground-in stopper weighing bottle

Heating

105 ºC – 110 ºC, 3 hours

Standing to cool

Desiccator

Measurement

Weigh to the order of 1 mg

Figure 2 Flow sheet for humic acid testing method in humic acid salt fertilizers(Testing procedure of acid insoluble - alkali non-dissolved matter (4.2))

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Testing Methods for Fertilizers (2018)6.10 Sulfate
This method is according to 5.29.2 Sulfate analysis in “The Official Methods of Analysis of
Fertilizers 1992”.
References
1)
National Institute for Agro-Environmental Sciences, the Ministry of Agriculture,
Forestry and Fisheries: The Official Methods of Analysis of Fertilizers 1992, p.145 147, Japan Fertilizers Analysis Association, Tokyo (1992)
2)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.285- 286, Yokendo, Tokyo (1988)

577

Testing Methods for Fertilizers (2018)6.11 Carbon dioxide
This method is according to 5.20 Carbon dioxide analysis in “The Official Methods of Analysis of
Fertilizers 1992”.
References
1)
National Institute for Agro-Environmental Sciences, the Ministry of Agriculture,
Forestry and Fisheries: The Official Methods of Analysis of Fertilizers 1992, p.121 123, Japan Fertilizers Analysis Association, Tokyo (1992)
2)
Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.259- 261, Yokendo, Tokyo (1988)

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Testing Methods for Fertilizers (2018)

7. Nitrification inhibitor
7.1 2-amino-4-chloro-6-methylpyrimidine (AM)
7.1.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing 2-amino-4-chloro-6-methylpyrimidine(AM). This testing method is classified as Type C and its symbol is 7.1.a-2017 or AM.a-1.
Add methanol − water (1+1) to an analytical sample to extract
2-amino-4-chloro-6-methylpyrimidine, introduce it into a High-Performance Liquid Chromatograph(HPLC), isolate with an octadecyl silylation silica gel column, and measure at wavelength 295 nm
to obtain 2-amino-4-chloro-6-methylpyrimidine (AM) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 6.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
c) Methanol: Methanol used in eluent of an HPLC is HPLC analysis grade or a reagent ofequivalent quality.
d) 2-amino-4-chloro-6-methylpyrimidine standard solution (1 mg/mL) (1): Put 0.1 g of
2-amino-4-chloro-6-methylpyrimidine [C5H6ClN3] (2) in a weighing dish and measure themass to the order of 0.1 mg. Add methanol - water (1+1) to dissolve, transfer to a 100-mL
volumetric flask and add the same solvent up to the marked line. Store in a refrigerator, and do
not use after 6 months after preparation.
e) 2-amino-4-chloro-6-methylpyrimidine standard solution (0.1 mg/mL): In the case ofusage, transfer 10 mL of 2-amino-4-chloro-6-methylpyrimidine standard solution (1 mg/mL)
to a 100-mL volumetric flask and add methanol - water (1+1) up to the marked line.
f) 2-amino-4-chloro-6-methylpyrimidine standard solution (10 µg/mL - 50 µg/mL) for thecalibration curve preparation: In the case of usage, transfer 5 mL - 25 mL of
2-amino-4-chloro-6-methylpyrimidine standard solution (0.1 mg/mL) for the calibration curve
preparation to 50-mL volumetric flasks step-by-step and add methanol - water (1+1) up to themarked line.
g) 2-amino-4-chloro-6-methylpyrimidine standard solution (1 µg/mL - 10 µg/mL) for thecalibration curve preparation: In the case of usage, transfer 2.5 mL - 25 mL of
2-amino-4-chloro-6-methylpyrimidine standard solution (20 µg/mL) for the calibration curve
preparation to 50-mL volumetric flasks step-by-step and add methanol - water (1+1) up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity is commercially sold as
2-amino-4-chloro-6-methylpyrimidine.
Comment 1 2-amino-4-chloro-6-methylpyrimidine is commercially sold by FUJIFILM Wako
Pure Chemical Co., Ltd. and Kanto Chemical Co., Inc.
(3) Instruments: Instruments are as shown below:a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel column tube
filled with silica gel, to which octadecyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
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Testing Methods for Fertilizers (2018)

3) Detection unit: An absorptiometric detector that can measure at wavelength around 295 nm.
b) Magnetic stirrer:c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
d) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
e) Acidic alumina cartridge column: Link a 10-mL cylinder to a column (3) that is filled with
500 mg - 1 g of acidic alumina, put 3 mL of methanol and let it flow down.
Note (3) A cartridge with a 3-mL - 6-mL column filled with 500 mg - 1 g of silica gel can beused.
Comment 2 A column is sold under production names such as Inertsil ODS, Mightysil RP-18,
L-column ODS, Shim-pack VP-ODS, Silica C18M 4D, Puresil C18, COSMOSIL
5C18-MS-II, etc.
Comment 3 An acidic alumina cartridge is commercially sold under production names such as
Bond Elut AL-A, Sep-Pak Alumina-A, Supelclean LC-Alumina-A.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of methanol - water (1+1) and stir it by using a magnetic stirrer for about 30
minutes.
c) After allowing to stand still, transfer a supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge it at about 1700 × g centrifugal force for about five minutes (4) and use thesupernatant (5) as the extract.
Note (4) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(5) If there is a possibility that the 2-amino-4-chloro-6-methylpyrimidine in the sample
solution exceeds the maximum limit of the calibration curve, dilute a predeterminedamount of extract with methanol water (1+1).
(4.2) Cleanup: Conduct cleanup as shown below:a) Transfer the extract to an acidic alumina cartridge column.
b) Dispose of about the first 3 mL of effluent and then transfer about the next 2 mL to a test tube.
c) Transfer the effluent to a 1.5-mL ground-in stopper centrifugal precipitate tube (6).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (7) and use thesupernatant as the extract.
Note (6) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(7) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 4 Instead of the procedures in (4.2) c) - d), it is allowed to filter with a membrane filter(aperture diameter: no more than 0.5-µm) made of PTFE and the filtrate can be thesample solution.
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Testing Methods for Fertilizers (2018)

Comment 5 The test is possible by the following procedures in the case of fertilizers notcontaining organic matters.
The procedures in (4.1) c) - d) and (4.2) a) - b) are omitted and “Transfer effluent” in(4.2) c) is replaced with the “After allowing to stand still, transfer supernatant” tooperate.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for a High-Performance Liquid Chromatograph (HPLC)is shown below. Set up the measurement conditions considering it:1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm
particle diameter) to which octadecyl chemically bonds.
2) Column bath temperature: 30 °C - 40 °C
3) Eluent: Methanol - water (4+6)4) Flow rate: 1 mL/min
5) Detection unit: An absorptiometric detector, measurement wavelength: 295 nmb) Calibration curve preparation
1) Inject 10 µL of respective 2-amino-4-chloro-6-methylpyrimidine standard solutions for thecalibration curve preparation to an HPLC, record a chromatogram at wavelength 295 nm andobtain the peak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area or height atwavelength 295 nm of the respective 2-amino-4-chloro-6-methylpyrimidine standard
solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain 2-amino-4-chloro-6-methylpyrimidine content from the calibration curve to calculate
2-amino-4-chloro-6-methylpyrimidine (AM) in the analytical sample.
Comment 6 A recovery testing was conducted using compound fertilizer (1 sample) and blended
fertilizer (2 samples), as a result, the mean recovery rate of
2-amino-4-chloro-6-methylpyrimidine at concentration level of 1.0 % (mass fraction),
0.4 % (mass fraction) and 0.1 % (mass fraction) were 99.1 % - 100.5 %, 99.3 % 101.6 % and 100.2 % - 100.7 %.
Additionally, the minimum limit of quantification of this testing method is about
0.005 % (mass fraction).
References
1)
Yuji SHIRAI: The volumetric analysis of 2-amino-4-chloro-6-methylpyrimidine infertilizers with High-Performance Liquid Chromatograph, Validation Report of
Fertilizers (in Japanese), 44 (3), p. 26 - 41(1991)

581

Testing Methods for Fertilizers (2018)

(5)

Flow
sheet

sheet for2-amino-4-chloro-6-methylpyrimidine: The
flow
for 2-amino-4-chloro-6-methylpyrimidine in fertilizers is shown below:
1.00 g200-mL of ground-in stopper Erlenmeyer flask
analytical sample← 100 mL of methanol-water (1+1)
Extraction

Centrifugal separation
CleanupCentrifugal separation

Stirring, 30 minutes
Ground-in stopper centrifugal precipitate tube,
1700 × g , 5 minutes
Acidic alumina cartridge column
Ground-in stopper centrifugal precipitate tube,
8000 × g - 10000 × g , 5 minutes

Sample solution
Measurement
FigureHigh Performance Liquid Chromatograph

Flow sheet for 2-amino-4-chloro-6-methylprimidine (AM) in fertilizers.

Reference: HPLC chromatogram of 2-amino-4-chloro-6-methylpyrimidine (AM) standard
solution for the calibration curve preparation is shown below.

Reference diagram: HPLC chromatogram of 2-amino-4-chloro-6-methylpyrimidine(AM) standard solution
Measurement conditions for HPLC
Column: Mightysil RP-18 (4.6-mm inner diameter, 150-mm long, 5-µm particle
diameter)
2-amino-4-chloro-6-methylpyrimidine standard solution (the equivalents of 100 ng)
Other conditions are according to the example of HPLC measurement conditions in(4.3) a)582

Testing Methods for Fertilizers (2018)

7.2 1-amidino-2-thiourea (ASU)
7.2.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing 1-amidino-2-thiourea (ASU). This testing
method is classified as Type B and its symbol is 7.2.a-2017 or ASU.a-1.
Add water to an analytical sample to extract 1-amidino-2-thiourea, introduce it into a
High-Performance Liquid Chromatograph (HPLC), isolate with an octadecyl silylation silica gelcolumn, and measure at wavelength 262 nm to obtain 1-amidino-2-thiourea (ASU) in an analyticalsample. In addition, the performance of this testing method is shown in Comment 4.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Methanol: Methanol used in eluent of an HPLC is HPLC analysis grade or a reagent ofequivalent quality.
c) 1-sodium hexasulfonate: A reagent of ion pair chromatography analysis grade or equivalents.
d) Acetic acid: A reagent of HPLC grade or equivalents.
e) 1-amidino-2-thiourea standard solution (1 mg/mL) (1): Put 0.1 g of 1-amidino-2-thiourea[C2H6N4S] (2) in a weighing dish and measure the mass to the order of 0.1 mg. Add water todissolve, transfer to a 100-mL volumetric flask and add water up to the marked line. Store in arefrigerator, and do not use after 6 months after preparation.
f) 1-amidino-2-thiourea standard solution (0.1 mg/mL): In the case of usage, transfer 10 mLof 1-amidino-2-thiourea standard solution (1 mg/mL) to a 100-mL volumetric flask and add
water up to the marked line.
g) 1-amidino-2-thiourea standard solution (10 µg/mL - 50 µg/mL) for the calibration curve
preparation: In the case of usage, transfer 5 mL - 25 mL of 1-amidino-2-thiourea standard
solution (0.1 mg/mL) to 50-mL volumetric flasks step-by-step and add water up to the markedline.
h) 1-amidino-2-thiourea standard solution (1 µg/mL - 10 µg/mL) for the calibration curve
preparation: In the case of usage, transfer 2.5 mL - 25 mL of 1-amidino-2-thiourea standard
solution (20 µg/mL) for the calibration curve preparation to 50-mL volumetric flasks
step-by-step and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity is commercially sold as
1-amidino-2-thiourea.
Comment 1 1-amidino-2-thiourea is sold under the name guanylthiourea by Tokyo Chemical
Industry Co., Ltd, and under the name amidino thiourea by Kanto Chemical Co., Inc.
(3) Instruments: Instruments are as shown below:a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel column tube
filled with silica gel, to which octadecyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 262 nm.
b) Magnetic stirrer:c) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
583

Testing Methods for Fertilizers (2018)

Comment 2 A column is sold under production names such as Inertsil ODS, Mightysil RP-18,
L-column ODS, Shim-pack VP-ODS, Silica C18M 4D, Puresil C18, COSMOSIL
5C18-MS-II, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution (3) to a 1.5-mL ground-in stopper
centrifugal precipitate tube (4).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (5) and use thesupernatant as the extract.
Note (3) If there is a possibility that the 1-amidino-2-thiourea concentration in the sample
solution exceeds the maximum limit of the calibration curve, dilute a predeterminedamount of supernatant with water.
(4) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement(5) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 3 Instead of the procedures in (4.1) c) - d), it is allowed to filter with a membrane filter(aperture diameter: no more than 0.5-µm) made of PTFE and the filtrate can be thesample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High-Performance
Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for a High-Performance Liquid Chromatograph (HPLC)is shown below. Set up the measurement conditions considering it:1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm
particle diameter) to which octadecyl chemically bonds.
2) Column bath temperature: 30 °C - 45 °C
3) Eluent: Dissolve 0.94 g of sodium 1-hexasulfonic acid in 1000 mL of methanol water (2+8),
adjust to pH 3.15 with acetic acid and filter with a membrane filter (aperture diameter: no
more than 0.5-µm) made of hydrophilic PTFE (1).
4) Flow rate: 1 mL/min
5) Detection unit: An absorptiometric detector, measurement wavelength: 262 nmb) Calibration curve preparation
1) Inject 10 µL of respective 1-amidino-2-thiourea standard solutions for the calibration curve
preparation to an HPLC, record a chromatogram at wavelength 262 nm and obtain the peakarea or height.
2) Prepare a curve for the relationship between the concentration and the peak area or height atwavelength 262 nm of the respective 1-amidino-2-thiourea standard solutions for thecalibration curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
584

Testing Methods for Fertilizers (2018)2) Obtain the 1-amidino-2-thiourea content from the calibration curve to calculate
1-amidino-2-thiourea (ASU) in the analytical sample.
Comment 4 A recovery testing with triplicates measurement was conducted using compound
fertilizer (2 samples), as a result, the mean recovery rate of 1-amidino-2-thiourea atconcentration level of 1.0 % (mass fraction), 0.5 % (mass fraction) and 0.25 % (massfraction) were 99.0 % - 104.3 %, 97.7 % - 100.7 % and 99.7 % - 101.3 %.
Table 1 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification of this testing method is about
0.005 % (mass fraction).
Table 1 Results and analysis results from a collaborative study
for the test method validation of 1-amidino-2-thiourea (ASU)
Mean

2)

sr

3)

4)

laboratories

(%)

(%)

RSD r
(%)

Compound fertilizer 1

10

0.093

0.009

Compound fertilizer 2

10

0.246

Compound fertilizer 3

10

Compound fertilizer 4
Compound fertilizer 5

Sample name

1)
2)
3)
4)
5)
6)
7)

Number of

5)

6)

7)

(%)

RSD R
(%)

9.1

0.010

11.2

0.021

8.6

0.021

8.6

0.511

0.018

3.6

0.025

4.9

10

0.759

0.039

5.1

0.040

5.3

10

1.020

0.039

3.8

0.044

4.3

1)

3)

sR

3)

Number of laboratories used in analysis
Mean (n = number of laboratories × number of samples (2))
Mass fraction
Repeatability standard deviation
Repeatability relative standard deviation
Reproducibility standard deviation
Reproducibility relative standard deviation

References
1)
Kazunori CHIBA： Analysis method of 1-amidino-2-thiourea (ASU) of Nitrification
inhibitor in fertilizers with High-Performance Liquid Chromatograph, Validation Reportof Fertilizers (in Japanese), 43 (4), p. 15 - 22 (1990)
2)
Shigehiro KAI and Erina WATABE: Determination of 1-Amidino-2-thiourea as a
Nitrification Inhibitor in Compound
Fertilizer by High-Performance Liquid
Chromatography: A Collaborative Study, Research Report of Fertilizers, Vol.6, p. 36 42 (2013)585

Testing Methods for Fertilizers (2018)(5)

Flow sheet for 1-amidino-2-thiourea: The flow sheet for 1-amidino-2-thiourea in fertilizers
is shown below:
1.00 g analytical sample

200-mL ground-in stopper Erlenmeyer flask← 100 mL of water
Extraction
Centrifugal separation

Stirring, 10 minutes
Ground-in stopper centrifugal precipitate tube,
8000 × g - 10000 × g , 5 minutes

Sample solution
Measurement
FigureReference:High Performance Liquid Chromatograph

Flow sheet for 1-amidino-2-thiourea (ASU) in fertilizers.

HPLC chromatogram of 1-amidino-2-thiourea (ASU) standard solution for thecalibration curve preparation is shown below.

Reference diagram: HPLC chromatogram of 1-amidino-2-thiourea (ASU) standard solution
Measurement conditions for HPLC
Column: Mightysil RP-18 (4.6-mm inner diameter, 150-mm long, 5-µm particle
diameter)
1-amidino-2-thiourea standard solution (the equivalents of 200 ng)
Other conditions are according to the example of HPLC measurement conditions in(4.2) a)586

Testing Methods for Fertilizers (2018)

7.3 4-amino-1,2,4-triazole hydrochloride (ATC)7.3.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing 4-amino-1,2,4-triazole hydrochloride(ATC) but not containing organic matters. This testing method is classified as Type C and its
symbol is 7.3.a-2017 or ATC.a-1.
Add methanol to an analytical sample to extract 4-amino-1,2,4-triazole hydrochloride, introduce it
into a High-Performance Liquid Chromatograph (HPLC), isolate with an aminopropyl silica gelcolumn, and measure at wavelength 220 nm to obtain 4-amino-1,2,4-triazole hydrochloride (ATC)in an analytical sample. In addition, the performance of this testing method is shown in Comment
4.
(2) Reagent: Reagents are as shown below.
a) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
b) Methanol: Methanol used in eluent of an HPLC is HPLC analysis grade or a reagent ofequivalent quality.
c) Acetonitrile: Acetonitrile used in eluent of an HPLC is a reagent of HPLC analysis grade orequivalents.
d) 4-amino-1,2,4-triazole standard solution (1 mg/mL) (1)(2): Transfer 0.1 g of
4-amino-1,2,4-triazole [C2H4N4] (3) to a weighing dish and measure the mass to the order of
0.1 mg. Add methanol to dissolve, transfer to a 100-mL amber volumetric flask and add
methanol to the marked line. Store in a refrigerator, and do not use after 6 months after
preparation.
e) 4-amino-1,2,4-triazole standard solution (0.1 mg/mL): In the case of usage, transfer 10 mLof 4-amino-1,2,4-triazole standard solution (1 mg/mL) to a 100-mL volumetric flask and add
methanol up to the marked line.
f) 4-amino-1,2,4-triazole standard solution (10 µg/mL - 50 µg/mL) for the calibration curve
preparation: In the case of usage, transfer 5 mL - 25 mL of 4-amino-1,2,4-triazole standard
solution (0.1 mg/mL) to 50-mL volumetric flasks step-by-step and add methanol up to themarked line.
g) 4-amino-1,2,4-triazole standard solution (1 µg/mL - 10 µg/mL) for the calibration curve
preparation: In the case of usage, transfer 2.5 mL - 25 mL of 4-amino-1,2,4-triazole standard
solution for the calibration curve preparation (20 µg/mL) to 50-mL volumetric flasks
step-by-step and add methanol up to the marked line.
Note (1) The solution contains 1.434 mg/mL as 4-amino-1,2,4-triazole hydrochloride.
(2) This is an example of preparation; prepare an amount as appropriate.
(3) A reagent of no less than 98 % (mass fraction) in purity is commercially sold as
4-amino-1,2,4-triazole.
Comment 1 4-amino-1,2,4-triazole is sold under production names such as 4-amino-1,2,4-triazole
by FUJIFILM Wako Pure Chemical Co., Ltd., and Tokyo Chemical Industry Co., Ltd,and 4-amino-4H-1,2,4-triazole by Kanto Chemical Co., Inc.
(3) Instruments: Instruments are as shown below:a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A column of 4 mm - 6 mm inner diameter and 150 mm - 250 mm long stainlesssteel column tube filled with silica gel, to which amino or amino propyl chemically bonds.
587

Testing Methods for Fertilizers (2018)2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 220 nm.
b) Magnetic stirrerc) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
Comment 2 A column is sold under production names such as Hibar LiChrosorb NH2, Inertsil
NH2, Unison UK-Amino, Mightysil NH2, Shim-pack CLC-NH2, Shodex NH-5A,
Unisil Q NH2, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of methanol and stir it with using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution to a 1.5-mL ground-in stopper
centrifugal precipitate tube (4).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (5) and use thesupernatant as the extract.
Note (4) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(5) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 3 Instead of the procedures in (4.1) c) - d), it is allowed to filter with a membrane filter(aperture diameter: no more than 0.5-µm) made of PTFE and the filtrate can be thesample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for a High-Performance Liquid Chromatograph (HPLC)is shown below. Set up the measurement conditions considering it:1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm
particle diameter) to which amino or amino propyl chemically bonds.
2) Column bath temperature: 30 °C - 40 °C
3) Eluent: Acetonitrile - methanol (9+1)4) Flow rate: 1 mL/min
5) Detection unit: An absorptiometric detector, measurement wavelength: 220 nmb) Calibration curve preparation
1) Inject 10 µL of respective 4-amino-1,2,4-triazole standard solutions for the calibration curve
preparation to an HPLC, record a chromatogram at wavelength 220 nm and obtain the peakarea or height.
2) Prepare a curve for the relationship between the concentration and the peak area or height atwavelength 220 nm of the 4-amino-1,2,4-triazole standard solutions for the respective
calibration curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
588

Testing Methods for Fertilizers (2018)2) Obtain the amount of 4-amino-1,2,4-triazole from the calibration curve to calculate
4-amino-1,2,4-triazole in the analytical sample.
3) Calculate the 4-amino-1,2,4-triazole hydrochloride (ATC) by the following formula.
4-amino-1,2,4-triazole hydrochloride in the analytical sample (% (mass fraction))= A × 1.434
A： 4-amino-1,2,4-triazole hydrochloride in the analytical sample (% (mass fraction))
Comment 4 A recovery testing was conducted using compound fertilizer (2 samples), as a result,the mean recovery rate of 4-amino-1,2,4-triazole hydrochloride at concentration levelof 0.5 % (mass fraction), 0.3 % (mass fraction) and 0.2 % (mass fraction) were
100.2 % - 104.9 %, 100.8 % - 103.0 % and 100.7 % - 104.2 %.
Additionally, the minimum limit of quantification of this testing method is about
0.005 % (mass fraction).
References
1)
Koichi SAKAGAMI: Analysis methods of 4-amino-1,2,4-triazole hydrochloride with
High-Performance Liquid Chromatography, Validation Report of Fertilizers (inJapanese), 40 (4), p.9 - 16 (1987)(5)

Flow sheet for 4-amino-1,2,4-triazole hydrochloride (ATC): The flow sheet for
4-amino-1,2,4-triazole hydrochloride in fertilizers is shown below:
1.00 g analytical sample

200-mL ground-in stopper Erlenmeyer flask← 100 mL of Methanol
Extraction
Centrifugal separation

Stirring, 10 minutes
Ground-in stopper centrifugal precipitate tube,
8000 × g - 10000 × g , 5 minutes

Sample solution
Measurement
FigureHigh Performance Liquid Chromatograph

Flow sheet for 4-amino-1,2,4-triazole hydrochloride (ATC) in fertilizers

589

Testing Methods for Fertilizers (2018)

7.4 N-2,5-dichlorophenyl succinamic acid (DCS)
7.4.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing N-2,5-dichlorophenyl succinamic acid
(DCS) but not containing organic matters. This testing method is classified as Type C and its
symbol is 7.4.a-2017 or DCS.a-1.
Add methanol - phosphoric acid (996+4) and water to an analytical sample to extract
N-2,5-dichlorophenyl succinamic acid (DCS), introduce it into a High-Performance Liquid
Chromatograph (HPLC), isolate with octadecyl silylation silica gel column, and measure atwavelength 246 nm to obtain N-2,5-dichlorophenyl succinamic acid (DCS) in an analytical sample.
In addition, the performance of this testing method is shown in Comment 3.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
c) Methanol: Methanol used in eluent of an HPLC is HPLC analysis grade or a reagent ofequivalent quality.
d) Phosphoric acid: A JIS Guaranteed Reagent specified in JIS K 9005 or a reagent ofequivalent quality.
e) N-2,5-dichlorophenyl succinamic acid standard solution (1 mg/mL) (1): Transfer 0.1 g of
N-2,5-dichlorophenyl succinamic acid [C10H9Cl2NO3] to a weighing dish and measure themass to the order of 0.1 mg. Add methanol to dissolve, transfer to a 100-mL volumetric flask
and add methanol to the marked line. Store in a refrigerator, and do not use after 6 months
after preparation.
f) N-2,5-dichlorophenyl succinamic acid standard solution (0.1 mg/mL): In the case of usage,transfer 10 mL of N-2,5-dichlorophenyl succinamic acid standard solution (1 mg/mL) to a
100-mL volumetric flask and add methanol up to the marked line.
g) N-2,5-dichlorophenyl succinamic acid standard solution (10 µg/mL - 50 µg/mL) for thecalibration curve preparation: In the case of usage, transfer 5 mL - 25 mL of
N-2,5-dichlorophenyl succinamic acid standard solution (0.1 mg/mL) to 50-mL volumetricflasks step-by-step and add methanol up to the marked line.
h) N-2,5-dichlorophenyl succinamic acid standard solution (1 µg/mL - 10 µg/mL) for thecalibration curve preparation: In the case of usage, transfer 2.5 mL - 25 mL of
N-2,5-dichlorophenyl succinamic acid standard solution for the calibration curve preparation(20 µg/mL) to 50-mL volumetric flasks step-by-step and add methanol up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as shown below:a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel column tube
filled with silica gel, to which octadecyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 246 nm.
b) Magnetic stirrerc) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
590

Testing Methods for Fertilizers (2018)

Comment 1 A column is sold under production names such as Inertsil ODS, Mightysil RP-18,
L-column ODS, Shim-pack VP-ODS, Silica C18M 4D, Puresil C18, COSMOSIL
5C18-MS-II, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of methanol - phosphate (996+4) and stir it with a magnetic stirrer for about 30
minutes.
c) After allowing to stand still, transfer a supernatant solution (2) to a 1.5-mL ground-in stopper
centrifugal precipitate tube (3).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (4) and use thesupernatant as the extract.
Note (2) If there is a possibility that the N-2,5-dichlorophenyl succinamic acid concentration in
the sample solution exceeds the maximum limit of the calibration curve, dilute apredetermined amount of the outflow solution with methanol.
(3) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement(4) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 2 Instead of the procedures in (4.1) c) - d), it is allowed to filter with a membrane filter(aperture diameter: no more than 0.5-µm) made of PTFE and the filtrate can be thesample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for a High-Performance Liquid Chromatograph (HPLC)is shown below. Set up the measurement conditions considering it:1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm
particle diameter) to which octadecyl chemically bonds.
2) Column bath temperature: 30 °C - 40 °C
3) Eluent: Methanol - water (5) (55 + 45)
4) Flow rate: 0.8 mL/min
5) Detection unit: An absorptiometric detector, measurement wavelength: 246 nm
Note(5) Adjust the water used to pH 3 with phosphpric acid in advance.

b) Calibration curve preparation
1) Inject 10 µL of respective N-2,5-dichlorophenyl succinamic acid standard solutions for thecalibration curve preparation to an HPLC, record a chromatogram at wavelength 246 nm andobtain the peak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area or height atwavelength 246 nm of the respective N-2,5-dichlorophenyl succinamic acid standard solution
for the calibration curve preparation.
c) Sample measurement591

Testing Methods for Fertilizers (2018)

1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the N-2,5-dichlorophenyl succinamic acid content from the calibration curve tocalculate N-2,5-dichlorophenyl succinamic acid (DCS) in the analytical sample.
Comment 3 Recovery testing was conducted using compound fertilizer (2 samples) and blended
fertilizer (1 sample), as a result, the mean recovery rate of N-2,5-dichlorophenyl
succinamic acid at concentration level of 0.4 % (mass fraction), 0.2 % (massfraction) and 0.1 % (mass fraction) were 100.9 % - 101.4 %, 100.8 % - 101.4 % and101.2 % - 103.4 %.
Additionally, the minimum limit of quantification of this testing method is about
0.005 % (mass fraction).
References
1)
Akira KUBO: Analysis methods of N-2,5-dichlorophenyl succinamic acid (DCS) of
Nitrification suppression materials in fertilizers with High-Performance Liquid
Chromatograph, Validation Report of Fertilizers (in Japanese), 44 (4), p.25 - 36 (1991).
(5)

Flow sheet for N-2,5-dichlorophenyl succinamic acid (DCS): The flow sheet for
N-2,5-dichlorophenyl succinamic acid (DCS) in fertilizers is shown below:
1.00 g analytical sample 200-mL ground-in stopper Erlenmeyer flask← 100 mL of methanol phosphoric acid (996+4)
Extraction
Stirring, 30 minutes
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 × g - 10000 × g , 5 minutes

Sample solution
Measurement
FigureHigh Performance Liquid Chromatograph

Flow sheet for N-2,5-dichlorophenyl succinamic acid (DCS) in fertilizers.

592

Testing Methods for Fertilizers (2018)

Reference: HPLC chromatogram of N-2,5-dichlorophenyl succinamic acid (DCS) standard
solution for the calibration curve preparation is shown below.

Reference diagram: HPLC chromatogram of N-2,5-dichlorophenyl succinamic acid
Measurement conditions for HPLC
Column: Mightysil RP-18 (4.6-mm inner diameter, 150-mm long, 5-µm particle
diameter)
N-2,5-dichlorophenyl succinamic acid standard solution (the equivalents of 100 ng)
Other conditions are according to the example of HPLC measurement conditions in(4.2) a)593

Testing Methods for Fertilizers (2018)

7.5 Dicyandiamide (Dd)
7.5.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing dicyandiamide (Dd). This testing method
is classified as Type B and its symbol is 7.5.a-2017 or Dd.a-1.
Add water to an analytical sample, leave at rest for a little while and add methanol to extract
dicyandiamide. After removing interfering substances with a silica gel cartridge column, introduce
it into a High-Performance Liquid Chromatograph (HPLC), isolate with an aminopropyl silica gelcolumn, and measure at wavelength 215 nm to obtain dicyandiamide (Dd) in an analytical sample.
In addition, the performance of this testing method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
c) Methanol: Methanol used in eluent of an HPLC is HPLC analysis grade or a reagent ofequivalent quality.
d) Acetonitrile: A reagent of HPLC grade or equivalents.
e) Dicyandiamide standard solution (1 mg/mL) (1): Put 0.1 g of dicyandiamide [C2H4N4] (2) toa weighing dish and measure the mass to the order of 0.1 mg. Add a small amount ofmethanol to dissolve, transfer to a 100-mL volumetric flask and add the solvent up to themarked line. Store in a refrigerator, and do not use after 6 months after preparation.
f) Dicyandiamide standard solution (0.1 mg/mL): At the time of usage, put 10 mL of
dicyandiamide standard solution (1 mg/ mL) to a 100-mL volumetric flask and add methanol
up to the marked line.
g) Dicyandiamide standard solution (10 µg/ mL - 50 µg/ mL) for the calibration curve
preparation: At the time of usage, put 5 mL - 25 mL of dicyandiamide standard solution (0.1
mg/ mL) to 50-mL volumetric flasks step-by-step and add methanol up to the marked line.
h) Dicyandiamide standard solution (1 µg/mL - 10 µg/mL) for the calibration curve
preparation: In the case of usage, transfer 2.5 mL - 25 mL of dicyandiamide standard
solution for the calibration curve preparation (20 µg/mL) to 50-mL volumetric flasks
step-by-step and add methanol up to the marked line.
i) Sodium sulfate: A JIS Guaranteed Reagent specified in JIS K 8987 or a reagent ofequivalent quality.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % in purity is commercially sold as dicyandiamide.
Comment 1 Dicyandiamide is commercially sold as dicyanodiamide by FUJIFILM Wako Pure
Chemical Co., Ltd. and Kanto Chemical Co., Inc.
(3) Instruments: Instruments are as shown below:a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A column of 4 mm - 6 mm inner diameter and 150 mm - 250 mm long stainlesssteel column tube filled with silica gel, to which amino or amino propyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 40 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 215 nm.
b) Shaking apparatusc) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
594

Testing Methods for Fertilizers (2018)d) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
e) Silica gel cartridge column: Link a 10-mL cylinder to the column (3) filled with 500 mg - 1
g of silica gel, add 3 mL of methanol to let it flow down.
Note (3) A cartridge with a 3-mL - 6-mL column filled with 500 mg – 1 g of silica gel can beused.
Comment 2 A column is sold under production names such as Hibar LiChrosorb NH2, Inertsil
NH2, Unison UK-Amino, Mightysil NH2, Shim-pack CLC-NH2, Shodex NH-5A,
Unisil Q NH2, etc.
Comment 3 A silica gel cartridge column is commercially sold under production names such as
Sep-Pak Plus Silica, InertSep Si.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200 mL ground-in stopper Erlenmeyer
flask.
b) Add 1 mL of water (4) and leave at rest for 5 minutes.
c) Add 100 mL of methanol and shake to mix with a shaking apparatus for about 10 minutes.
d) Add an appropriate amount of sodium sulfate (5).
e) After allowing to stand still, transfer a supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
f) Centrifuge it at about 1700 × g centrifugal force for about five minutes (6) and use thesupernatant (7) as the extract.
Note (4) Mix well until the whole sample comes in contact with water.
(5) About 5 g - 10 g.
(6) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(7) If there is a possibility that the concentration of dicyandiamide in the sample solution
exceeds the maximum limit of the calibration curve, dilute a predetermined amount of
extract with methanol.
(4.2) Cleanup: Conduct cleanup as shown below:a) Transfer extract to a silica gel cartridge column.
b) Dispose of about the first 3 mL of effluent and then transfer about the next 2 mL to a test tube.
c) Transfer the effluent to a 1.5-mL ground-in stopper centrifugal precipitate tube (8).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (9) and use thesupernatant as the extract.
Note (8) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(9) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 4 Instead of the procedures in (4.2) c) - d), it is allowed to filter with a membrane filter(aperture diameter: no more than 0.5-µm) made of PTFE and the filtrate can be thesample solution.
595

Testing Methods for Fertilizers (2018)(4.3) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for a High-Performance Liquid Chromatograph (HPLC)is shown below. Set up the measurement conditions considering it:1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm
particle diameter) to which amino or amino propyl chemically bonds.
2) Column bath temperature: 30 °C - 40 °C
3) Eluent: Acetonitrile - methanol (6+1)
4) Flow rate: 0.5 mL/min - 1 mL/min
5) Detection unit: An absorptiometric detector, measurement wavelength: 215 nmb) Calibration curve preparation
1) Inject 10 µL of respective dicyandiamide standard solutions for the calibration curve
preparation to an HPLC, record a chromatogram at wavelength 215 nm, and obtain the peakarea or the height.
2) Prepare a curve for the relationship between the concentration and the peak area or the heightat wavelength 215 nm of the respective dicyandiamide standard solutions for the calibration
curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain dicyandiamide content from the calibration curve to calculate dicyandiamide (Dd) in
the analytical sample.
Comment 5 Recovery testing was conducted using inorganic compound fertilizer (2 samples) andorganic compound fertilizer (3 samples), as a result, the mean recovery at theconcentration level of 2 % (mass fraction) and 0.2% (mass fraction) were 101.2 % 102.6 % and 98.4 % - 100.6 %.
Table 1 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification of this testing method is about
0.01 % (mass fraction).

596

Testing Methods for Fertilizers (2018)

Table 1 Results and analysis results from a collaborative study
for the test method validation of dicyandiamide
Sample name

Number of
1)

Mean

2)

sr

3)

4)

RSD r

5)

6)

RSD R

sR

7)

laboratories

(%)

(%)

3)

(%)

(%)

3)

(%)

Compound fertilizer 1

11

0.263

0.009

3.2

0.019

7.4

Compound fertilizer 2

11

2.04

0.04

1.7

0.07

3.2

Compound fertilizer 3

13

0.548

0.011

2.0

0.033

6.0

Compound fertilizer 4

12

0.423

0.013

3.2

0.022

5.2

Compound fertilizer 5

12

1.02

0.01

1.4

0.04

4.3

1)
2)
3)
4)
5)
6)
7)

Number of laboratories used in analysis
Mean (n = number of laboratories × number of samples (2))
Mass fraction
Repeatability standard deviation
Repeatability relative standard deviation
Reproducibility standard deviation
Reproducibility relative standard deviation

References
1)
Masakazu SAIKI: Development of High-Performance Liquid Chromatography for
Determination of Dicyandiamide as a Nitrification Inhibitor in Fertilizer. Research
Report of Fertilizer, Vol.3, p. 43 - 50 (2010)
2)
Masakazu SAIKI: Determination of Dicyandiamide as a Nitrification Inhibitor in
Fertilizer by High-Performance Liquid Chromatography: Collaborative Study, Research
Report of Fertilizer, Vol.4, p. 16 - 22 (2011)597

Testing Methods for Fertilizers (2018)(5)

Flow sheet for dicyandiamide: The flow sheet for dicyandiamide in fertilizers is shown
1.00 g200-mL ground-in stopper Erlenmeyer flask
analytical sample← 1 mL of water
Leave at rest
5 minutes← 100 mL of methanol
Shaking
10 minutes← An adequate amount of sodium sulfate
Ground-in stopper centrifugal precipitate tube,
Centrifugal separation
1700 × g , 5 minutes
CleanupCentrifugal separation

Silica gel cartridge column
1.5-mL ground-in stopper centrifugal precipitate tube,
8000 × g - 10000 × g , 5 minutes

Sample solution
Measurement
FigureHigh Performance Liquid Chromatograph

Flow sheet for dicyandiamide (Dd) in fertilizers

598

Testing Methods for Fertilizers (2018)

0.20

0.18

0.18

0.16

0.16

0.14

0.14

8.605 Dd

0.20

0.12
AU

AU

0.12

8.607 Dd

Reference: HPLC chromatogram of dicyandiamide standard solution for the calibration curve
preparation and sample solution (compound fertilizer) are shown below.

0.10

0.10

0.08

0.08

0.06

0.06

0.04

0.04

0.02

0.02

0.00

0.00
0

2

4

6

8
min

10

12

14

0

1) Standard solution
Reference diagram

2

4

6

8
min

10

12

14

2) Sample solution
HPLC chromatogram of dicyandiamide (Dd)1) Dicyandiamide standard solution (the equivalents of 100 ng (10 µg/ mL, 10 µL) of
dicyandiamide)2) Sample solution (compound fertilizer)
Measurement conditions for HPLC
Column: Inertsil NH2 (4.6-mm inner diameter, 250-mm long, 5-µm particle
diameter)
Column oven temperature: 30 °C
Flow rate: 0.5 mL/min
Other conditions are according to the example of HPLC measurement conditions in(4.3) a)599

Testing Methods for Fertilizers (2018)

7.6 2-sulfanilamide thiazole (ST)7.6.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing 2-sulfanilamide thiazole (ST). This testing
method is classified as Type C and its symbol is 7.6.a-2017 or ST.a-1.
Add methanol−water (1+1) to an analytical sample to extract 2-sulfanilamide thiazole, introduce it
into a High-Performance Liquid Chromatograph (HPLC), isolate with an octadecyl silylation silica
gel column, and measure at wavelength 285 nm to obtain 2-sulfanilamide thiazole (ST) in ananalytical sample. In addition, the performance of this testing method is shown in Comment 6.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
c) Methanol: Methanol used in eluent of an HPLC is HPLC analysis grade or a reagent ofequivalent quality.
d) 2-sulfanilamide thiazole standard solution (1 mg/mL) (1): Transfer 0.1 g of 2-sulfanilamide
thiazole [C9H9N3O2S2] (2) to a weighing dish and measure the mass to the order of 0.1 mg.
Add water to dissolve, transfer to a 1000-mL volumetric flask and add methanol−water (1+1)
to the marked line. Store in a refrigerator, and do not use after 6 months after preparation.
e) 2-sulfanilamide thiazole standard solution (0.1 mg/mL): In the case of usage, transfer 10
mL of 2-sulfanilamide thiazole standard solution (100 µg/mL) to a 100-mL volumetric flask
and add methanol−water (1+1) up to the marked line.
f) 2-sulfanilamide thiazole standard solution (10 µg/mL - 50 µg/mL) for the calibration
curve preparation: In the case of usage, transfer 5 mL - 25 mL of 2-sulfanilamide thiazole
standard solution (0.1 mg/mL) to 50-mL volumetric flasks step-by-step and add methanol water (1+1) up to the marked line.
g) 2-sulfanilamide thiazole standard solution (1 µg/mL - 10 µg/mL) for the calibration
curve preparation: In the case of usage, transfer 2.5 mL - 25 mL of 2-sulfanilamide thiazole
standard solution for the calibration curve preparation (20 µg/mL) to 50-mL volumetric flasks
step-by-step and add methanol−water (1+1) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity is commercially sold as
2-sulfanilamide thiazole.
Comment 1 2-sulfanilamide thiazole is sold under the production name sulfathiazole by Tokyo
Chemical Industry Co., Ltd, FUJIFILM Wako Pure Chemical Co., Ltd., and Kanto
Chemical Co., Inc.
(3) Instruments: Instruments are as shown below:a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel column tube
filled with silica gel, to which octadecyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 285 nm.
b) Magnetic stirrer:c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
d) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 600

Testing Methods for Fertilizers (2018)e)

10000 × g.
Acidic alumina cartridge column: Link a 10-mL cylinder to a column (3) that is filled with
500 mg - 1 g of acidic alumina, put 3 mL of methanol and let it flow down.

Note (3) A cartridge with a 3-mL - 6-mL column filled with 500 mg – 1 g of silica gel can beused.
Comment 2 A column is sold under production names such as Inertsil ODS, Mightysil RP-18,
L-column ODS, Shim-pack VP-ODS, Silica C18M 4D, Puresil C18, COSMOSIL
5C18-MS-II, etc.
Comment 3 An acidic alumina cartridge is commercially sold under production names such as
Bond Elut AL-A, Sep-Pak Alumina-A, Supelclean LC-Alumina-A.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of methanol - water (1+1) and stir it by using a magnetic stirrer for about 15
minutes.
c) After allowing to stand still, transfer a supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge it at 1700 × g centrifugal force for about five minutes (4) and use the supernatant (5)as the extract.
Note (4) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(5) If there is a possibility that the 2-sulfanilamide thiazole concentration in the sample
solution exceeds the maximum limit of the calibration curve, dilute a predeterminedamount of the extract with methanol.
(4.2) Cleanup: Conduct cleanup as shown below:a) Transfer the extract to an acidic alumina cartridge column.
b) Dispose of about the first 3 mL of effluent and then transfer about the next 2 mL to a test tube.
c) Transfer the effluent to a 1.5-mL ground-in stopper centrifugal precipitate tube (6).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (7) and use thesupernatant as the extract.
Note (6) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(7) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 4 Instead of the procedures in (4.2) c) - d), it is allowed to filter with a membrane filter(aperture diameter: no more than 0.5-µm) made of PTFE and the filtrate can be thesample solution.
Comment 5 The test is possible by the following procedures in the case of fertilizers notcontaining organic matters.
The procedures in (4.1) c) - d) and (4.2) a) - b) are omitted and “Transfer effluent” in(4.2) c) is replaced with the “After allowing to stand still, transfer supernatant” to601

Testing Methods for Fertilizers (2018)

operate.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for a High-Performance Liquid Chromatograph (HPLC)is shown below. Set up the measurement conditions considering it:1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm
particle diameter) to which octadecyl chemically bonds.
2) Column bath temperature: 30 °C - 40 °C
3) Eluent: Methanol−water (2+8)4) Flow rate: 1 mL/min
5) Detection unit: An absorptiometric detector, measurement wavelength: 285 nmb) Calibration curve preparation
1) Inject 10 µL of respective 2-sulfanilamide thiazole standard solutions for the calibration curve
preparation to an HPLC, record a chromatogram at wavelength 285 nm and obtain the peakarea or height.
2) Prepare a curve for the relationship between the concentration and the peak area or height atwavelength 285 nm of the respective 2-sulfanilamide thiazole standard solutions for thecalibration curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the 2-sulfanilamide thiazole content from the calibration curve to calculate
2-sulfanilamide thiazole (ST) in the analytical sample.
Comment 6 Recovery testing was conducted using compound fertilizer (1 sample) and blended
fertilizer (2 sample), as a result, the mean recovery rate of 2-sulfanilamide thiazole atthe concentration level of 1.0 % (mass fraction), 0.4 % (mass fraction) and 0.1 %(mass fraction) were 101.2 % - 102.1 %, 99.6 % - 101.7 % and 99.4 % - 101.0 %.
Additionally, the minimum limit of quantification of this testing method is about
0.005 % (mass fraction).
References
1)
Yuji SHIRAI: The volumetric analysis of 2-sulfanilamide thiazole in fertilizers with
High-Performance Liquid Chromatograph, Validation Report of Fertilizers (in Japanese),
44 (1), p. 10 - 20 (1991)

602

Testing Methods for Fertilizers (2018)(5)

Flow sheet for 2-sulfanilamide thiazole: The flow sheet for 2-sulfanilamide thiazole infertilizers is shown below:
1.00 g analytical sample

200-mL ground-in stopper Erlenmeyer flask← 100 mL of methanol - water (1+1)
Extraction
Stirring, 15 minutes
Centrifugal separation
CleanupCentrifugal separation

Ground-in stopper centrifugal precipitate tube,
1700 × g , 5 minutes
Acidic alumina cartridge column
Ground-in stopper centrifugal precipitate tube,
8000 × g - 10000 × g , 5 minutes

Sample solution
Measurement
FigureHigh Performance Liquid Chromatograph

Flow sheet for 2-sulfanilamide thiazole (ST) in fertilizers.

Reference: HPLC chromatogram of 2-sulfanilamide thiazole (ST) standard solution for thecalibration curve preparation is shown below.

Reference diagram: HPLC chromatogram of 2-sulfanilamide thiazole (ST)
Measurement conditions for HPLC
Column: Mightysil RP-18 (4.6-mm inner diameter, 150-mm long, 5-µm particle
diameter)2-sulfanilamide thiazole standard solution (the equivalents of 200 ng)
Other conditions are according to the example of HPLC measurement conditions in(4.3) a)603

Testing Methods for Fertilizers (2018)

8. Others
8.1 Melamine and its degradation products
8.1.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type D and its
symbol is 8.1.a-2017 or Mel.a-1.
Extract melamine and its derivative substances (hereinafter referred to as “melamine derivations”)
in organic matters and fertilizers containing organic matters with diethylamine−water−acetonitrile(1+4+5) and derivatize with BSTFA−TMCS (99+1) and then measure with a gas
chromatography/mass spectrometer to obtain melamine deviations in an analytical sample. In
addition, the performance of this testing method is shown in Comment 8.
Comment 1 The structural formula of melamine and its degradation products is shown in the
figure 1. During the production process of melamine, a by-product that replaces“-NH2” of R1 - R3 with “-OH” is formed in some cases.

R1
N
R2

N
N

R3

Melamine
Ammeline
Ammelide
Cyanuric acid

R1
NH2
OH
OH
OH

R2
NH2
NH2
OH
OH

R3
NH2
NH2
NH2
OH

MW
126.12
127.10128.09129.07

Figure 1 Structural formula of melamine and its degradation products(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Acetonitrile: A reagent of agricultural chemicals residue/PCB testing grade (concentration:no less than 300) specified in JIS K 8039 or a reagent of equivalent quality.
c) Diethylamine: A guaranteed reagent or a reagent of equivalent quality.
d) Pyridine (dehydration) (1): A reagent of organic synthesis grade of no less than 99.5 % (massfraction) in purity and no more than 0.05 mg/mL in moisture or a reagent of equivalentquality.
e) Derivatization reagent (2): Bis (trimethylsilyl) trifluoroacetamide−trimethylchlorosilane(99+1).
f) Melamine derivations standard solution (0.5 mg/mL): Put about 0.05 g of melamine[C3H6N6] (3), ammeline [C3H5N5O] (3), ammelide [C3H4N4O2] (3) and cyanuric acid
[C3H3N3O3] (3) into a weighing dish and measure the mass to the order of 0.1 mg. Dissolve in asmall amount of diethylamine−water (1+4), transfer to a 100 mL volumetric flask respectively,
and add the solvent up to the marked line.
g) Mixture standard solution (50 µg/mL) (3): Put 5 mL of respective melamine derivations
standard solutions (0.5 mg/mL) to 50-mL volumetric flasks and add
diethylamine−water−acetonitrile (1+4+5) up to the marked line.
Note (1) After it is opened once, add a proper amount of sodium sulfate (anhydrous) and seal
tightly to store.
(2) A mixed derivatization reagent is commercially sold under the name BSTFA−TMCS(99+1).
(3) The respective standard reagents of melamine, ammeline, ammelide and cyanuric acid604

Testing Methods for Fertilizers (2018)

are commercially sold.
Comment 2 BSTFA−TMCS (99+1) is sold as 1-mL ampoule by SUPELCO. After it is opened
once, use it on the same day.
Comment 3 The standard reagent of melamine, ammeline, ammelide and cyanuric acid are soldby FUJIFILM Wako Pure Chemical Co., Ltd. and Kanto Chemical Co., Inc. andHayashi Pure Chemical Industries., Ltd.
(3) Instruments: Instruments are as shown below:a) Gas Chromatograph/Mass Spectrometer (GC/MS): GC/MS specified in JIS K 0123 thatsatisfies the following requirements.
1) Gas Chromatograph(i) Sample injector: An injector that enables split less system.
(ii) Capillary column: A capillary column (0.25-mm - 0.32-mm inner diameter and 30-mlong) made of fused silica. 5 % phenyl 95 % methyl polysiloxane chemically bonds to theinner surface of a capillary column with 0.25 µm thickness. The column is according to
the specification of mass spectrometer.
(ii) Carrier gas: High purity helium of no less than 99.999 % (volume fraction) in purity.
2) Mass Spectrometer(i) Ionization method: Electron-Impact ionization (EI) method(ii) Ion detection method: Selected Ion Monitoring (SIM) methodb) Ultrasonic generator: An ultrasonic washer can be used.
c) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
d) Concentrator: A Centrifugal evaporator that can be adjusted to 70 ºC ± 2 ºC.
e) Water bath: Water bath that can be adjusted to 70 ºC ± 2 ºC.
Comment 4 A capillary column is commercially sold under the names such as DB-5ms, Rtx-5ms,
HP-5ms, SLB-5ms, BPX-5, CP-Sil 8CB low Bleed/MS and TC -5HT for GC/MS.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 0.50 g of an analytical sample, and put it into a 200 mL - 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 160 mL - 200 mL of diethylamine−water−acetonitrile (1+4+5), and subject toultra-sonication for about 30 minutes using an ultrasonic generator.
c) Transfer about 1.5 mL to a 1.5-mL ground-in stopper centrifugal precipitate tube (4), andcentrifuge at 8000 × g - 10000 × g for about 5 minutes (5).
d) Transfer 1 mL of the supernatant solution to 5-mL - 50-mL volumetric flasks, add
diethylamine−water−acetonitrile (1+4+5) up to the marked line to make the extract.
Note (4) Confirm that the tube is made of polypropylene, etc. to not affect testing results.
(5) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 5 Grind until it completely passes through a sieve of 500 µm aperture to prepare thetest sample.
Comment 6 Weigh 0.5 g of an analytical sample, extract with 200 mL ofdiethylamine−water−acetonitrile (1+4+5). If it is diluted 50 times in the procedure ind), the quantitative range of melamine derivations in the analytical sample is 0.2 %605

Testing Methods for Fertilizers (2018)(mass fraction) - 10 % (mass fraction). In the case of measuring melamine
derivations which do not reach the quantitative range, make the dilution factor in theprocedure in d) decrease. In addition, if the contents of melamine derivations exceed
10 % (mass fraction) respectively, the sampling volume of an analytical sample
should be reduced.
(4.2) Derivatization: Conduct derivatization as shown below.
a) Transfer 0.2 mL of the extract to a 5-mL - 10-mL test tube with a screw stopper.
b) Place a test tube in a concentrator, concentrate under reduced pressure at 70 ºC ± 2 ºC andvaporize the solvent completely (6).
c) Add 0.3 mL of pyridine (dehydration) (1) and 0.2 mL of derivatization reagent (2) to the residue
to mix, and then stopple to seal tightly.
d) After heating in a water bath at 70 ºC ± 2 °C for about 45 minutes (7), let it stand to cool tomake a sample solution (8).
Note (6) A spraying type concentrator can be used.
(7) If moisture remains after the procedure in b) or the reagent used in the procedure in c)contains moisture, the reaction of the derivatization in d) does not advance enough insome cases.
(8) If necessary, transfer the sample solution into a 1.5-mL ground-in stopper centrifugal
precipitate tube (4) to centrifuge at 8000 × g - 10000 × g for about 5 minutes (5).
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0123 and as shown below.
Specific measurement procedures are according to the operation method of the gas
chromatograph/ mass spectrometer used in measurement.
a) Measurement conditions for the gas chromatograph/ mass spectrometer: Set up themeasurement conditions considering it:
1) Gas Chromatograph(i) Sample injection method: split less injection method (1min)(ii) Temperature of sample injector: 280 °C(ii) Capillary column: A capillary column (0.25-mm - 0.32-mm inner diameter, 30-m long,
0.25 µm layer thickness) made of fused silica. 5 % phenyl 95 % methyl polysiloxane
chemically bonds to the inner surface of the capillary column.
(iv) Temperature of column bath: 100 °C (1 min) → (15 °C /min) → 320 °C (3 min)(v) Temperature of GC/MS coupling portion: 250 °C(vi) Carrier gas: helium, flow rate: 1.5 mL/min
2) Mass Spectrometer(i) Ionization method: Electron-Impact ionization (EI) method(ii) Ionization voltage: 70 V(iii) Temperature of ion source: 230 °C(iv) Ion detection method: Selected Ion Monitoring (SIM) method(v) Measurement of ion: Shown in table 1

606

Testing Methods for Fertilizers (2018)

Table 1 Fragment ion of materials to be measured
Materials
Measured fragment ion (m /z)
to be measured Determination Validation Validation Validation Validation
342
Melamine
344
327
285
213
328
Ammeline
345
343
285
214
344
Ammelide
346
329
214
198
345
Cyanuric acid
347
330
215
188
288
DACP(I.S.)
289
290
273
275
b) Calibration curve preparation
1) Transfer 5 mL of mixture standard solution (50 µg/mL) to a 50-mL volumetric flask and add
diethylamine−water−acetonitrile (1+4+5) up to the marked line to make the mixture standard
solution (5 µg/mL).
2) Transfer 1 mL - 20 mL of the mixture standard solution (5 µg/ mL) to 50 mL volumetricflasks step-by-step and add diethylamine−water−acetonitrile (1+4+5) up to the marked line to
make the mixture standard solution (0.1 µg/mL - 2 µg/mL).
3) Conduct the procedures of (4.2) b) - d) for the mixture standard solution (0.1 µg/mL - 2µg/mL) to make the mixture standard solution, the equivalents of 0.04 µg/mL - 0.8 µg/mL, forcalibration curve.
4) Inject 1 µL of respective mixture standard solutions for calibration curve to GC/MS andrecord the chromatogram of ion (m/z) for determination and ion (m/z) for validation ofmaterials subjected to measurement to obtain the peak area or height.
5) Calculate the peak area ratio or height ratio of ion (m/z) for determination and ion (m/z) for
validation of respective materials subjected to measurement.
6) Prepare a curve for the relationship between the concentration of material subjected tomeasurement and the peak area or height of ion (m/z) for determination of respective mixture
solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 1 µL of the sample solution to the same procedure as in b) 4) - 5) (9).
2) Obtain the content of respective materials subjected to measurement from the calibration
curve to calculate respective materials subjected to measurement in the analytical sample.
Note (9) Confirm that the ratio against the peak area ratio or height ratio of the standard
solution is within the range of about ± 30 %. In addition, the peak area ratio or heightratio depends on the concentration.
Comment 7 If the variation of sensitivity of melamine derivations is observed, conduct
measurement by the following method of a) or b).
a) In the procedure in (4.3) c) 1), inject the sample solution into GC/MS
predetermined times, and then correct the calibration curve according to (4.3) b)
4) - 6).
b) Add 2,6-diamino-4-chloropyrimidine (the equivalents of 0.5 µg) as an internalreference material to the standard solution and the sample solution, conduct thesame procedures as (4.2) c) - d), (4.3) b) 4) - 6) and c) 1). However, prepare thecalibration curve from the peak area ratio or height ratio of ion (m/z) for
determination of respective materials subjected to measurement and internalreference material, and calculate the concentration of respective materials
607

Testing Methods for Fertilizers (2018)subjected to measurement in the analytical sample.
Comment 8 Recovery testing of melamine derivations was conducted using soybean meal, fish
meal, fish waste processed fertilizer, mixed organic fertilizer, blended fertilizer andcompound fertilizer, as a result, the mean recovery rate at additive level of 10 %(mass fraction) and 0.2 % (mass fraction) were 92.1 % - 102.9 % and 90.3 % 102.2 %.
Additionally, the minimum limit of quantification of melamine derivations of the test
method is about 0.01 % (mass fraction).
References
1)
Yuji SHIRAI, Jun OKI: Validation of Gas Chromatography/Mass Spectrometry for
Determination of Melamine and Its Degradation Products in Fertilizers, Research
Report of Fertilizer, Vol.1, p. 114 - 121 (2008)

608

Testing Methods for Fertilizers (2018)(5)

Flow sheet for melamine derivations: The flow sheet for melamine derivations in fertilizers
is shown below.

0.50 g analytical sample
200-mL - 300-mL ground-in stopper Erlenmeyer flask← 160 mL - 200 mL of diethylamine - water - acetonitrile（1+4+5）
Extraction
Ultrasonic extraction for 30 minutes
Centrifugal separation

Ground-in stopper centrifugal precipitation tube
8000 × g - 10000 × g, 5 minutes

Extract
Figure 1

Flow sheet for melamine derivations in fertilizers (Extraction procedure)
Extract
Dilution

0.2 mL Aliquot

Transfer 1 mL to 5-mL - 50-mL volumetric flasks,
add diethylamine−water−acetonitrile（1+4+5）up to the marked line
5-mL - 10-mL test tube with a screw stopper

concentration
under reduced pressure
Centrifugal evaporator (70 ºC ± 2 ºC)/ Drying up← 0.3 mL of pyridine (dehydration)← 0.2 mL of derivatization reagent (BSTFA−TMCS (99+1))
Stopple and seal.
Derivatization
70 ºC ± 2 ºC, 45 minutes
Standing to cool
Sample solution
Measurement
Figure 2

Gas Chromatograph/Mass Spectrometer

Flow sheet for melamine derivations in fertilizers(Derivatization and measurement procedure)

609

Testing Methods for Fertilizers (2018)

Reference:

Total Ion Chromatogram (TIC) of GC/MS of mixture standard solution for
calibration curve preparation of melamine derivations is shown below.
↓a

↓b

↓c

↓d

Figure 3 Total Ion Chromatogram (TIC) of GC/MS for melamine derivations
Measurement conditions of GC/MS
Capillary column: Rtx-5ms (0.25-mm inner diameter, 30-m long, 0.25 µm layer
thickness)
Other conditions are according to the example of HPLC measurement conditions in(4.3) a)Peak name of respective total ion chromatogramsa) Cyanuric acidb) Ammelidec) Ammelined) Melamine
Sample and amount injected into GC/MS
Injected sample: Mixture standard solutions (the equivalents of 2 µg/mL) for thecalibration curve preparation of respective melamine derivations.
Injected amount: 1 µL (the equivalents of 2 ng of respective melamine derivations)610

Testing Methods for Fertilizers (2018)

8.1.b

611

Testing Methods for Fertilizers (2018)

8.1.c High-Performance Liquid Chromatography (Fertilizers not containing organic matters)(1) Summary
This testing method is applicable to fertilizers not containing organic matters. This testing method isclassified as Type B and its symbol is 8.1.c-2017 or Mel.c-1.
Add hydrochloric acid (1+15) to an analytical sample, extract melamine and its degradation
products (hereinafter referred to as “melamine derivations”), introduce them into a
High-Performance Liquid Chromatograph (HPLC), isolate with a silica gel column to which
carbamoyl chemically bonds, and measure at wavelength 214 nm to obtain melamine derivations in
an analytical sample. In addition, the performance of this testing method is shown in Comment 4.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Acetonitrile: A JIS Guaranteed Reagent specified in JIS K 8032 or a reagent of equivalentquality. In addition, acetonitrile used in eluent of an HPLC is a reagent of HPLC analysis
grade.
c) Hydrochloric acid: A guaranteed reagent or a reagent of equivalent quality.
d) Phosphate buffer solution (1): Dissolve 0.237 g of disodium hydrogen-phosphate specified in
JIS K 9020 and 0.520 g of sodium dihydrogenphosphate dihydrate specified in JIS K 9009 inwater to make 1000 mL (2). If it is used for eluent of HPLC, filter with a membrane filter (poresize: no more than 0.5-µm) made of hydrophilic PTFE.
e) Melamine derivations standard solution (0.5 mg/mL): Put about 0.05 g of melamine[C3H6N6] (3), ammeline [C3H5N5O] (3), ammelide [C3H4N4O2] (3) and cyanuric acid
[C3H3N3O3] (3) into a weighing dish and measure the mass to the order of 0.1 mg. Dissolve
them with a small amount of hydrochloric acid (1+15), transfer to 100 mL volumetric flasks
respectively, and add the solutions up to the marked line.
f) Mixture standard solution (50 µg/mL) (1): Transfer 5 mL of respective melamine derivations
standard solutions (0.5 mg/mL) to 50-mL volumetric flasks and add acetonitrile - phosphatebuffer solutions (4+1) up to the marked line.
g) Mixture standard solution for calibration curve preparation (1 µg/mL - 5 µg/mL): In thecase of usage, transfer 1 mL - 5 mL of mixture standard solution (50 µg /mL) to 50-mL
volumetric flasks step-by-step and add acetonitrile−phosphate buffer solutions (4+1) up to themarked line.
h) Mixture standard solution for calibration curve preparation (0.05 µg/mL - 0.5 µg/mL):
In the case of usage, transfer 2.5 mL - 25 mL of mixture standard solution (1 µg /mL) to
50-mL volumetric flasks step-by-step and add acetonitrile−phosphate buffer solutions (4+1)
up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) The phosphate buffer solution becomes pH 6.7 ± pH 0.2.
(3) The respective standard reagents of melamine, ammeline, ammelide and cyanuric acidare commercially sold.
Comment 1 The standard reagent of melamine, ammeline, ammelide and cyanuric acid are soldby FUJIFILM Wako Pure Chemical Co., Ltd. and Kanto Chemical Co., Inc. andHayashi Pure Chemical Industries., Ltd.
(3)a)

Instruments: Instruments are as shown below:
High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel column tube
612

Testing Methods for Fertilizers (2018)

filled with silica gel, to which carbamoyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 40 ºC ± 1 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 214 nm.
b) Ultrasonic generator: An ultrasonic washer can be used.
c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
d) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
Comment 2 Column is sold under the production name TSKgel Amide-80, etc. A column which
has actually isolated melamine, ammeline, ammelide and cyanuric acid should be
used.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 0.50 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of hydrochloric acid (1+15) and conduct ultra-sonication for about 30 minutes
using an ultrasonic generator.
c) After allowing to stand still, transfer a supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge it at 1700 × g centrifugal force for about five minutes (4) and use the supernatant as
the extract.
e) Transfer 5 mL of the extract (5) into a 50-mL volumetric flask, and add an
acetonitrile−phosphate buffer solution (4+1) up to the marked line to dilute.
f) Transfer dilution liquid to a 1.5-mL ground-in stopper centrifugal precipitate tube (6).
g) Centrifuge at 8000 × g - 10000 × g for about 5 minutes (7) to make supernatant as the sample
solution
Note (4) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(5) If there is a possibility that the concentration of melamine derivations in the sample
solution exceeds the maximum limit of the calibration curb, the amount of asupernatant solution to be transferred should be1 mL - 2.5 mL.
(6) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement(7) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 3 Instead of the procedures in (4.1) f) - g), it is allowed to filter with a membrane filter(pore size: no more than 0.5-µm) made of hydrophilic PTFE and the filtrate can be
the sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for a High-Performance Liquid Chromatograph (HPLC)is shown below. Set up the measurement conditions considering it:1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm
particle diameter column) to which carbamoyl chemically bonds.
613

Testing Methods for Fertilizers (2018)2) Column bath temperature: 40 °C ± 1 °C
3) Eluent: Acetonitrile - phosphate buffer solution (4+1)
4) Flow rate: 1 mL/min
5) Detection unit: An absorptiometric detector, measurement wavelength: 214 nmb) Calibration curve preparation
1) Inject 10 µL of respective mixture standard solutions for calibration curve preparation into an
HPLC, record a chromatogram at wavelength 214 nm and obtain the peak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area or height atwavelength 214 nm of respective mixture solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the melamine derivations content from the calibration curve to calculate respective
melamine derivations in the analytical sample.
Comment 4 Recovery testing was conducted using 3 brands of nitrolime, 1 brand of compound
fertilizer containing nitrolime, 2 brands of compound fertilizers not containing
nitrolime, 1 brand of ammonium sulfate and 1 brand of urea, as a result, the recovery
rate of melamine derivations at concentration level of 4 % (mass fraction) and 0.1 %(mass fraction) were 90.5 % - 106.3 % and 92.2 % - 107.0 %.
Table 1 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification of the test method is about 0.02 %(mass fraction) for melamine and cyanuric acid and about 0.01 % (mass fraction) for
ammeline and ammelide. In the case of ammelide and cyanuric acid, the sufficient
reproducibility was observed in the range of 0.188 % (mass fraction) - 1.10 % (massfraction) and 0.105 % (mass fraction) - 1.15 % (mass fraction) respectively.

614

Testing Methods for Fertilizers (2018)

Table 1 Results and analysis results from a collaborative study
for the test method validation of melamine deivations
3)
4)
5)
2)
Number of
Agrichemical
sr
RSD r
sR
Mean
Sample name
1)
7)
7)
7)
laboratories
name
(%)
(%)
(%)
(%)
9
2.83
0.04
1.4
0.12
Melamine
Nitrolime 1
10
0.391
0.003
0.8
0.023
Nitrolime 2
Compound fertilizer
9
0.845
0.019
2.2
0.036
containing nitrolime
11
0.198
0.005
2.6
0.012
Compounf ferttilizer
10
0.03430.00154.5
0.0040
Ammonium sulfate
9
1.60
0.02
1.3
0.06
Ammeline
Nitrolime 1
10
0.105
0.001
1.3
0.002
Nitrolime 2
Compound fertilizer
9
0.629
0.027
4.3
0.023
containing nitrolime
11
0.195
0.004
2.1
0.009
Compounf ferttilizer
10
0.03460.00133.7
0.0024
Ammonium sulfate
9
1.10
0.02
2.1
0.08
Ammelide
Nitrolime 1
11
0.361
0.008
2.2
0.023
Nitrolime 2
Compound fertilizer
9
0.188
0.004
2.2
0.014
containing nitrolime
11
0.718
0.028
3.9
0.052
Compounf ferttilizer
11
0.03450.00318.9
0.0056Ammonium sulfate
9
1.15
0.06
4.8
0.09
Cyanuric acid Nitrolime 1
10
0.390
0.018
4.5
0.029
Nitrolime 2
Compound fertilizer
9
0.105
0.003
2.9
0.014
containing nitrolime
9
0.788
0.026
3.2
0.054
Compounf ferttilizer
10
0.03650.00154.2
0.0067Ammonium sulfate
1) Number of laboratories used in analysis
2) Gross mean (n = number of laboratories × number of repeated tests (2))3) Repeatability standard deviation
4) Repeatability relative standard deviation
5) Reproducibility standard deviation
6) Reproducibility relative standard deviation
7) Mass fraction

6)

RSD R
(%)
4.3
5.8
4.2
6.2
11.6
3.8
2.3
3.7
4.5
6.9
7.6
6.5
7.5
7.2
16.1
7.7
7.4
13.2
6.8
18.3

References
1)
Etsuko BANDO and Yuji SHIRAI: Validation of High-Performance Liquid
Chromatography (HPLC) for Determination of Melamine and Its Related Substances in
Fertilizer, Research Report of Fertilizer, Vol.6, p. 27 - 35 (2013)
2)
Etsuko BANDO and Sigehiro KAI: Determination of Melamine and Its Related
Substances in Fertilizer by High-Performance Liquid Chromatography (HPLC): A
Collaborative Study, Research Report of Fertilizer, Vol.7, p. 10 - 21 (2014)

615

Testing Methods for Fertilizers (2018)(5)

Flow sheet for melamine derivations: The flow sheet for melamine derivations in fertilizers
is shown below.
0.50 g analytical sample
200-mL ground-in stopper Erlenmeyer flask←100 mL of hydrochloric acid (1+15）
Extraction
Ultrasonic extraction for 30 minutes
Centrifugal separation

DilutionCentrifugal separation

Ground-in stopper centrifugal precipitation tube
1700 × g, 5 minutes
Transfer 5 mL to a 50-mL volumetric flask,
add acetonitrile−phosphate buffer solution (4+1) up to the marked line
Ground-in stopper centrifugal precipitation tube
8000 × g - 10000 × g, 5 minutes

Sample solution
Measurement

High Performance Liquid Chromatograph

Figure Flow sheet for melamine derivations in fertilizers

616

Testing Methods for Fertilizers (2018)

Reference:

HPLC chromatogram of the mixture standard solution for calibration curve
preparation of melamine derivations is shown below.

Reference diagram HPLC chromatogram of melamine derivations
The names of materials for respective peaks(1) cyanuric acid (2) ammelide(3) melamine(4) ammeline
Measurement conditions for HPLC
Column: TSKgel Amide-80 (4.6-mm inner diameter, 250-mm long, 5-µm particle
diameter)Mixture standard solutions (respective equivalents of 10 ng (1 µg/mL, 10 µL)) for
calibration curve preparation of respective melamine derivations
Other conditions are according to the example of HPLC measurement conditions in(4.2) a)617

Testing Methods for Fertilizers (2018)

8.1.d High-Performance Liquid Chromatography (Fertilizers containing organic matters)(1) Summary
This testing method is applicable to organic fertilizers and fertilizers containing organic matters.
This testing method is classified as Type B and its symbol is 8.1.d-2017 or Mel.d-1.
Add water to an analytical sample to extract melamine, introduce it into a High-Performance
Liquid Chromatograph (HPLC), isolate with a silica gel column to which carbamoyl chemicallybonds, and measure at wavelength 214 nm to obtain melamine in an analytical sample. However,cyanuric acid, ammelide and ammeline which are melamine derivations are excluded fromcomponents subjected to measurement. In addition, the performance of this testing method isshown in Comment 4.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Acetonitrile: A JIS Guaranteed Reagent specified in JIS K 8032 or a reagent of equivalentquality. In addition, acetonitrile used in eluent of an HPLC is a reagent of HPLC analysis
grade.
c) Phosphate buffer solution (1): Dissolve 0.237 g of disodium hydrogen-phosphate
specified in JIS K 9020 and 0.520 g of sodium dihydrogenphosphate dihydrate specified
in JIS K 9009 in water to make 1000 mL (2). If it is used for eluent of HPLC, filter with amembrane filter (pore size: no more than 0.5-µm) made of hydrophilic PTFE.
d) Melamine standard solution (0.5 mg/mL) (1): Put 0.05 g of melamine [C3H6N6] (3) to aweighing dish and measure the mass to the order of 0.1 mg. Dissolve it with a small
amount of water, transfer to a 100 mL volumetric flask and add the solution up to themarked line.
e) Melamine standard solution (50 µg/mL) (1): Transfer 5 mL of melamine solution (0.5mg/mL) to a 50-mL volumetric flask and add acetonitrile - phosphate buffer solutions(82+18) up to the marked line.
f) Melamine standard solution for calibration curve preparation (1 µg/mL - 5 µg/mL):
In the case of usage, transfer 1 mL - 5 mL of melamine standard solution (50 µg /mL) to
50-mL volumetric flasks step-by-step and add acetonitrile−phosphate buffer solutions(82+18) up to the marked line.
g) Melamine standard solution for calibration curve preparation (0.05 µg/mL - 0.5µg/mL): In the case of usage, transfer 2.5 mL - 25 mL of melamine standard solution (1µg/ mL) to 50-mL volumetric flasks step-by-step, add acetonitrile−phosphate buffer
solutions (82+18) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) The phosphate buffer solution becomes pH 6.7 ± 0.2.
(3) A standard reagent as melamine is commercially sold.
Comment 1 A standard reagent of melamine is sold by FUJIFILM Wako Pure Chemical Co., Ltd.,
Kanto Chemical Co., Inc. and Hayashi Pure Chemical Industries., Ltd.
(3) Instruments: Instruments are as shown below:a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel column tube
filled with silica gel, to which carbamoyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 40 ºC ± 1 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 214 nm.
618

Testing Methods for Fertilizers (2018)b) Ultrasonic generator: An ultrasonic washer can be used.
c) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
d) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
Comment 2 Column is sold under the production name TSKgel Amide-80, etc. A column which
has actually isolated melamine, ammeline, ammelide and cyanuric acid should be
used.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 0.50 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of water and conduct ultra-sonication for about 10 minutes using an
ultrasonic generator.
c) After allowing to stand still, transfer a supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge it at 1700 × g centrifugal force for about 10 minutes (4) and use the supernatantas the extract.
e) Transfer 5 mL of the extract (5) into a 50-mL volumetric flask, and add an
acetonitrile−phosphate buffer solution (82+18) up to the marked line to dilute.
f) Transfer dilution liquid to a 1.5-mL ground-in stopper centrifugal precipitate tube (6).
g) Centrifuge at 8000 × g - 10000 × g for about 5 minutes (7) to make supernatant as the
sample solution
Note (4) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(5) If there is a possibility that the concentration of melamine derivations in the sample
solution exceeds the maximum limit of the calibration curb, the amount of asupernatant solution to be transferred should be1 mL - 2.5 mL.
(6) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement(7) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 3 Instead of the procedures in (4.1) f) - g), it is allowed to filter with a membrane filter(pore size: no more than 0.5 µm) made of hydrophilic PTFE and the filtrate can be
the sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High-Performance
Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions for a High-Performance Liquid Chromatograph (HPLC)is shown below. Set up the measurement conditions considering it:1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long, 5-µm
particle diameter column) to which carbamoyl chemically bonds.
2) Column bath temperature: 40 °C ± 1 °C
3) Eluent: Acetonitrile - phosphate buffer solution (82+18)
4) Flow rate: 1 mL/min619

Testing Methods for Fertilizers (2018)

5) Detection unit: An absorptiometric detector, measurement wavelength: 214 nmb) Calibration curve preparation
1) Inject 10 µL of respective melamine standard solutions for calibration curve preparation into
an HPLC, record a chromatogram at wavelength 214 nm and obtain the peak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area or height atwavelength 214 nm of respective melamine solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the melamine content from the calibration curve to calculate melamine in theanalytical sample.
Comment 4 Additive recovery testing was conducted to evaluate trueness using rape seed meal,
soybean meal, compound fertilizer containing lime nitrogen and organic matter,compound fertilizer containing organic matter and blended fertilizer containing
organic matter (1 brand for each). As a result, the mean recovery rate at additive levelof 2 % (mass fraction), 0.4 % (mass fraction) and 0.1 % (mass fraction) were 94.6 %- 99.8 %, 92.4 % - 98.5 % and 93.1 % - 98.4 % respectively.
The results of the repeatability tests on different days using soybean meal andcompound fertilizer containing organic matter, to evaluate precision were analyzed
by one-way analysis of variance. Table 1 shows the calculation results ofintermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.02 % (mass fraction).

Name of

Table 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precisionsamplerepeatability

Average

1)

(%)
1.91

T
5

2)

3)

Soybean meal
Compoundfertilizer containing
50.100
organic matter
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

4)

RSD r

s I(T)

(%)
0.03

3)

(%)
1.7

0.001

1.4

sr

5)

6)

RSD I(T)

(%)
0.04

3)

(%)
2.2

0.002

2.5

6） Intermediate standard deviation
7） Intermediate relative standard deviation

References
1)
Etsuko FUNAMIZU: Determination Method for Melamine in Organic Fertilizer andFertilizer Containing Organic Matter using High-Performance Liquid Chromatograph(HPLC), Research Report of Fertilizers, Vol.9, p. 33 - 42 (2016)

620

7)

Testing Methods for Fertilizers (2018)(5)

Flow sheet for melamine derivations: The flow sheet for melamine derivations in fertilizers
is shown below.
0.50 g analytical sample
200-mL ground-in stopper Erlenmeyer flask←100 mL of water
Extraction
Ultrasonic extraction for 10 minutes
Centrifugal separation

Ground-in stopper centrifugal precipitation tube
1700 × g, 10 minutes
Transfer 5 mL to a 50-mL volumetric flask,
add acetonitrile−phosphate buffer solution (82+18) up to the marked line

DilutionCentrifugal separation

Ground-in stopper centrifugal precipitation tube
8000 × g - 10000 × g, 5 minutes

Sample solution
Measurement

High Performance Liquid Chromatograph

Figure Flow sheet for melamine in fertilizers containing organic matter

Reference: HPLC chromatogram of the standard solution for calibration curve preparation ofmelamine is shown below.

←melamin

Reference diagram HPLC chromatogram of melamine
Measurement conditions for HPLC
Column: TSKgel Amide-80 (4.6-mm inner diameter, 250-mm long, 5-µm particle
diameter)Melamine standard solution (the equivalents of 10 ng (1 µg/mL, 10 µL) for each)
Other conditions are according to the example of HPLC measurement conditionsin (4.2) a)621

Testing Methods for Fertilizers (2018)

8.2 Clopyralid and its degradation products
8.2.a High-Performance Liquid Chromatography/Tandem Mass Spectrometry(1: Simultaneous analysis of three components for clopyralid, etc.)(1) Summary
This testing method is applicable to compost and composted sludge fertilizers. This testing method
is classified as Type B and its symbol is 8.2.a-2017 or CLP.a-1.
Extract clopyralid, aminopyralid and picloram with methanol under alkaline condition, refine with acleanup cartridge by taking advantage of characteristics that the behavior of elution varies between
acidity and alkaline, and then measure with a High-Performance Liquid Chromatograph Tandem
Mass Spectrometer to obtain clopyralid, aminopyralid and picloram in an analytical sample. In
addition, the performance of this testing method is shown in Comment 6.
Comment 1 Structural formulas of clopyralid, aminopyralid and picloram are as shown in Figure
1.

Figure 1 Structural formula of clopyralid, aminopyralid and picloram(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557. Note that water of A4 should be used as theeluent which is introduced to LC-MS/MS.
b) Acetonitrile: A reagent of agricultural chemicals residue/PCB testing grade (concentration:no less than 300) specified in JIS K 8039 or a reagent of equivalent quality.
c) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
d) Methanol: Methanol used in eluent of LC-MS/MS is a reagent of LC-MS analysis grade orequivalents.
e) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
f) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
g) Ammonia solution: A JIS Guaranteed Reagent of 28 % (mass fraction) specified in JIS K
8085 or a reagent of equivalent quality.
h) formic acid: A JIS Guaranteed Reagent specified in JIS K 8264 or a reagent of equivalentquality.
i) Ammonia solution (0.0028 % (mass fraction)) (1): Add 0.1 mL of ammonia solution to 1000mL of water.
j) Respective agrichemical standard solutions (0.1 mg/mL) (1): Put about 0.01 g of clopyralid[C6H3Cl2NO2] (2), aminopyralid [C6H4Cl2N2O2] (2), picloram [C6H3Cl3N2O2] (2) to weighing
dishes and measure the mass to the order of 0.1 mg. Dissolve with a small amount of622

Testing Methods for Fertilizers (2018)acetonitrile, transfer to 100-mL volumetric flasks and add the solvent up to the marked line.
k) Mixture standard solution (100 ng/mL) (1): Dilute a predetermined amount of respective
agrichemical standard solutions (0.1 mg/mL) with formic acid (1+1000) to prepare mixture
standard solution (100 ng/mL).
l) Mixture standard solution for calibration curve preparation (5 ng/mL - 50 ng/mL) (1): In
the case of usage, transfer 2.5 mL - 25 mL of mixture standard solution (100 ng/mL) to 50 mL
volumetric flasks step-by-step, and add formic acid (1+1000) up to the marked line.
m) Mixture standard solution for calibration curve preparation (0.5 ng/mL - 5 ng/mL) (1): In
the case of usage, transfer 2.5 mL - 25 mL of mixture standard solution (10 ng/mL) to 50 mL
volumetric flasks step-by-step, and add formic acid (1+1000) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A standard reagent is commercially sold.
Comment 2 Standard reagents of clopyralid, aminopyralid and picloram are sold by FUJIFILM
Wako Pure Chemical Co., Ltd., Kanto Chemical Co., Inc. and Hayashi Pure
Chemical Industries., Ltd.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph/Mass Spectrometer (LC-MS/MS)：
LC-MS/MS specified in JIS K 0136 that satisfies the following requirements.
1) High-Performance Liquid Chromatograph(i) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
(ii) Column: A 2-mm - 3-mm inner diameter 50-mm - 150-mm long 1.6-µm - 2.2-µm
particle diameter stainless steel column tube filled with silica gel to which octadecyl
chemically bonds. The specification is according to the mass spectrometer specification.
2) Mass Spectrometer(i) Ionization method: Electro-Spray Ionization (ESI) method(ii) Ion detection method: Selected Reaction Monitoringb) Shaking apparatusc) Manifoldd) Centrifugal separator: A centrifugal separator that can work at about 1700 × g.
e) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
f) Concentrator: An evaporator that can adjust to 40 ºC ± 2 ºC.
g) Copolymer cartridge column: A divinylbenzene-N-vinylpyrrolidone copolymer minicolumn (200 mg)
Comment 3 Column is sold under the production name ACQUITY UPLC HSS C18, etc.
Comment 4 A copolymer cartridge is sold under the production names such as Oasis HLB 6cc(200 mg), Oasis PRiME HLB Plus Short Cartridge (225 mg).
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample, and put it into a 200-mL - 300-mL ground-in stopper
Erlenmeyer flask.
b) Add 1 mL of sodium hydroxide solution (40 g/L) and 99 mL of methanol (3), shake to mix
with a shaking apparatus for about 30 minutes.
c) After allowing to stand still, transfer a supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
623

Testing Methods for Fertilizers (2018)d)Centrifuge it at about 1700 × g centrifugal force for about five minutes (4) and use thesupernatant as the extract.

Note (3) It is also allowed to add 100 mL of sodium hydroxide solution (40 g/L)−methanol[1+99](4) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
Comment 5 Grind until it completely passes through a sieve of 500 µm aperture to prepare thetest sample.
(4.2) Cleanup (1) (5): Conduct cleanup (1) as shown below.
a) Wash a cartridge column with about 5 mL of methanol and 5 mL of water in advance.
b) Place a 100-mL round-bottom flask (6) under the cartridge column, transfer 5 mL or 7 mL (7) of
the extract to the cartridge column and allow the extract to overflow until the surface of theliquid reaches the top of the packing materials.
c) Add about 5 mL of sodium hydroxide solution (0.4 g/L)−methanol (1+1) to the cartridge
column 2 times and allow the liquid to overflow in the same manner in b).
d) Add 5 mL of methanol.
Note (5) Use a pressure reducing device in the procedure in (4.2) and (4.3) as appropriate.
(6) When pretreating many analytical samples, a free-standing type vessel that can containa solution with a liquid volume of 20 mL may be used. In this case, instead ofprocedure d), put an effluent into a round-bottle flask, wash the vessel 2 times with 2.5
mL of methanol and add washing to the previous effluent.
(7) When using Oasis HLB 6cc (200 mg), add 5 mL of the extract 2 times.
(4.3) Cleanup (2) (5): Conduct cleanup (2) as shown below.
a) Wash a new cartridge column with about 5 mL of acetonitrile and 5 mL of hydrochloric acid
(1+120) in advance.
b) After conducting vacuum concentration of the effluent in (4.2) c) until no more than 5 mL on
a water bath at no more than 40 °C, add 3 mL of hydrochloric acid (1 +11).
c) Put the concentrated effluent into the cartridge column and allow the effluent to overflow until
the surface of liquid reaches the top of packing materials.
d) Wash a round-bottom flask with about 5 mL of hydrochloric acid (1+120) 2 times and add
washing into the cartridge successively.
e) Then, add about 5 mL of hydrochloric acid (1+120)−acetonitrile (9+1) and about 5 mL ofwater into the cartridge successively and allow the liquid to overflow.
f) Place a 5-mL volumetric flask under the cartridge column, add 4 mL of ammonia solution(0.0028 % (mass fraction))−acetonitrile (9+1) to the cartridge column and allow clopyralid,aminopyralid and picloram to elute quickly.
g) Add formic acid (1+1000) up to the marked line (8) and transfer it to 1.5-mL ground-in stopper
centrifugal precipitate tube (9).
h) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (10) and use thesupernatant as the sample solution
Note (8) If there is a possibility that the clopyralid, aminopyralid and picloram concentration in
the sample solution exceed the maximum limit of the calibration curve, dilute apredetermined amount of effluent with formic acid (1 +1000).
(9) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
624

Testing Methods for Fertilizers (2018)etc. to not affect the measurement(10) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
(4.4) Measurement: Conduct the measurement as indicated in JIS K 0136 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph Mass Spectrometer used in measurement.
a) The measurement conditions of High-Performance Liquid Chromatograph/Mass
spectrometer: An example of measurement conditions for a High-Performance Liquid
Chromatograph/Mass Spectrometer is shown below. Set up the measurement conditions
considering it:
1) High-Performance Liquid Chromatograph(i) Column: A silica gel column (2-mm - 3-mm inner diameter, 50-mm - 150-mm long,
1.6-µm - 2.2-µm particle diameter column) to which octadecyl chemically bonds.
(ii) Flow rate: 0.2 mL/min - 0.5 mL/min(iii) Eluent: A: Formic acid (1+1000) B: Methanol:(iv) Gradient: 0 min (5 %B) → 5 min (60 %B) → 6 min (95 %B) → 7 min (5 %B)(v) Temperature of column bath: 40 °C(vi) Injection volume: 5 µL
2) Mass Spectrometer(i) Ionization method: Electro-Spray Ionization (ESI) method(ii) Mode: Positive(iii) Capillary voltage: 1.0 kV(iv) Ion source temperature: 120 °C(v) Desolvation temperature: 400 °C(vi) Cone voltage: Shown in table 1(vii) Collision energy: Shown in table 1(viii) Monitor ion: Shown in table 1

Substance
nameClopyralid
Aminipyralid
Oicrloram

Table 1 Monitor ion, etc. of respective agrichemicals
Precussor
Product ion
Product ion Cone voltage
Collision
Collisionion(Determination) (Validation)(v)
energyenergy
(m/z)
(m/z)
(m/z)
(Determination) (Validation)
(eV)
(eV)
192
207
241

146
161
195

110
189
223

20
22
28

20
22
22

30
16
16

b) Calibration curve preparation
1) Inject 5 μL of respective mixture standard solutions for calibration curve into the LC-MS/MS,
record the chromatogram of ion (m/z) for determination and ion (m/z) for validation of
clopyralid, aminopyralid and picloram and obtain respective peak areas.
2) Calculate the peak area ratio or height ratio of ion (m/z) for determination and ion (m/z) for
validation of clopyralid, aminopyralid and picloram.
3) Prepare a curve for the relationship between the concentration of respective agrichemicals andthe peak area of ion (m/z) for determination of respective mixture standard solutions for the625

Testing Methods for Fertilizers (2018)calibration curve preparation.
c) Sample measurement
1) Subject 5 µL of the sample solution to the same procedure as in b) 2) - 3) (11).
2) Obtain the content of material subjected to measurement from the calibration curve tocalculate the material subjected to measurement in the analytical sample.
Note (11) Confirm that the ratio against the peak area ratio or height ratio of the standard
solution is within the range of about ± 30 %. In addition, the peak area ratio or heightratio depends on the concentration.
Comment 6 Additive recovery testing of clopyralid, aminopyralid and picloram was conductedusing cow dung compost (2 kinds), composted sludge fertilizers containing cow
manure (2 kinds) and composted sludge fertilizers containing pig manure (1 kind), asa result, the mean recovery rates at additive level of 1000 µg/kg, 400 µg/kg and 40µg/kg were 78.1 % - 90.0 %, 81.0 % - 117.6 % and 71.2 % - 101.3 % respectively.
Table 2 shows results and analysis results from a collaborative study for testing
method validation.
Additionally, the minimum limits of quantification of clopyralid, aminopyralid andpicloram of the test method are about 10 µg/kg respectively.

626

Testing Methods for Fertilizers (2018)

Table 2 Results and analysis results from a collaborative study
for the test method validation of clopyralid and its degradation products
Agrichemical
nameSample name

Mean

sr

3)

RSD r

(µg/kg)(µg/kg)(%)2)

Number of1)laboratories

Clopyralid

4)

sR

5)

RSD R

6)

(µg/kg)

(%)

10
128
6
4.5
21
16.4
Compost 1
10
835
41
4.9
100
11.9
Compost 2
Composted sludge
9
16.2
1.7
10.6
5.2
31.8
fertilizer 1
Composted sludge
10
89.6
11.3
12.6
11.3
12.6
fertilizer 2
Composted sludge
10
339
28
8.3
28
8.3
fertilizer 3
8
324
15
4.5
39
12.0
Aminopyralid Compost 1
8
21.2
5.2
24.7
6.4
30.3
Compost 2
Composted sludge
7
5.39
1.41
26.2
2.22
41.2
fertilizer 1
Composted sludge
10
701
146
20.8
263
37.6
fertilizer 2
Composted sludge
9
59.5
8.9
15.0
16.6
28.0
fertilizer 3
10
840
50
5.9
175
20.8
PicloramCompost 1
9
37.7
3.5
9.4
10.3
27.3
Compost 2
Composted sludge
9
90.2
11.1
12.3
30.3
33.5
fertilizer 1
Composted sludge
8
341
19
5.6
67
19.8
fertilizer 2
Composted sludge
8
182
16
8.6
56
31.0
fertilizer 3
1) Number of laboratories used in analysis
2) Grossl mean (n = number of laboratories × number of repeated tests (2))3) Repeatability standard deviation
4) Repeatability relative standard deviation
5) Reproducibility standard deviation
6) Reproducibility relative standard deviation
References1)
Toshiharu YAGI, Yuko SEKINE, Yuji SHIRAI: Determination of Clopyralid in
Fertilizer by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS),
Research Report of Fertilizer Vol. 3, p. 51 - 59 (2010)
2)
Hisanori ARAYA, Toshiharu YAGI, Yoshimi HASHIMOTO and Yuji SHIRAI:
Determination of Clopyralid, Aminopyralid and Picloram in Compost and Composted
Sludge Fertilizer by Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS),
Research Report of Fertilizer Vol. 7, p. 1 - 9 (2014)3)
Kenji KOZUKA, Mayu OSHIMA, Yoshimi HASHIMOTO, Naoko TAMARU, and Yuji
SHIRAI: Determination of Clopyralid, Aminopyralid and Picloram in Compost and627

Testing Methods for Fertilizers (2018)

Composted Sludge Fertilizer by Liquid Chromatography-Tandem Mass Spectrometry(LC-MS/MS): A Collaborative Study, Research Report of Fertilizer Vol. 10, p. 61 - 71
(2017)

628

Testing Methods for Fertilizers (2018)(5)

Flow sheet for clopyralid and its derivative substances: The flow sheet for clopyralid andits derivative substances in compost and composted sludge fertilizer is shown below:

5.00 g analytical sample
200-mL - 300-mL ground-in stopper Erlenmeyer flask← 1 mL of sodium hydroxide solution (4 g/L)
← 99 mL of methanol
shaking
30 minutes
Centrifugal separation

50 mL centrifugal precipitate tube, 1700 × g, 5 minutes

Extract
Figure 1 Flow sheet for clopyralid and its derivative substances in compostand composted sludge fertilizers (Extraction procedure)
Extract← 10 mL of extract
copolymer cartridge column (1)Cleanup (1)(Wash with about 5 mL of methanol, then 5 mL of water in advance）[Collection, 100-mL eggplant-shaped flask]← About 5 mL of sodium hydroxide solution (0.4 g/L)−methanol [1+1] × 2 times[Collection, same as above vessel]← About 5 mL of methanol
Concentration
under reduced
40 ºC, until the volume reaches about 5 mLpressure← 3 mL of hydrochloric acid (1+11)
copolymer cartridge column (2)Cleanup (2)(Wash with about 5 mL of acetonitrile,then 5 mL of hydrochloric acid (1+120) in advance)← Wash the vessel 2 times with about 5mL of hydrochloric acid (1+120)← Wash with about 5mL of hydrochloric acid (1+120)−acetonitrile [9+1]← Wash with about 5mL of water← 4 mL of ammonia solution (0.0028 w/v %)−acetonitrile [9+1][Elute, 10-mL centrifugal precipitate tube]
Elute
Figure 2 Flow sheet for clopyralid in compost and composted sludge fertilizers(Cleanup (1) and cleanup (2) and measurement procedure)

629

Testing Methods for Fertilizers (2018)

References: Selected Reaction Monitoring chromatograms of mixture standard solution for
calibration curve preparation and sample solution (cow dung compost) are shown
below
Peak No.

2

A

Intensity / arb.units

10

Retention Time / min

5

Peak No.1: Picloram
No.2: Aminipyralid
No.3: Clopyralid
Reference diagram: SRM chromatograms of respective agrichemicals
Mixture standard solution (the equivalents of 200 pg as respective
agrichemicals)
LC-MS/MS measurement conditions
Column: ACQUITY UPLC HSS C18 (2.1-mm inner diameter, 100-mm long, 1.8-µm
particle diameter)
Other conditions are according to the examples of the measurement conditions in (4.4)a) LC-MS/MS.

630

Testing Methods for Fertilizers (2018)

8.2.b High-Performance Liquid Chromatography/Tandem Mass Spectrometry(2: microanalysis for clopyralid)(1) Summary
This testing method is applicable to compost and composted sludge fertilizers. This testing method
is classified as Type B and its symbol is 8.2.b-2018 or CLP.b-1.
Extract clopyralid in compost and composted sludge fertilizer with methanol under alkalinecondition, refine with a cleanup cartridge and dichloromethane by taking advantage ofcharacteristics that the behavior of elution varies between acidity and and then measure with a
High-Performance Liquid Chromatograph Mass Spectrometer to obtain clopyralid in an analyticalsample. In addition, the performance of this testing method is shown in Comment 8.
Comment 1 Structural formula of clopyralid is as shown in Figure 1.

Figure 1 Structural formula of clopyralid(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557. Note that water of A4 should be used as theeluent which is introduced to LC-MS/MS.
b) Acetonitrile: A reagent of agricultural chemicals residue/PCB testing grade (concentration:no less than 300) specified in JIS K 8039 or a reagent of equivalent quality.
c) Methanol: A reagent of agricultural chemicals residue/PCB testing grade (concentration: noless than 300) or a reagent of equivalent quality.
d) Methanol: Methanol used in eluent of LC-MS/MS is a reagent of LC-MS analysis grade orequivalents.
e) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
f) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
g) Ammonia solution: A JIS Guaranteed Reagent of 28 % (mass fraction) specified in JIS K
8085 or a reagent of equivalent quality.
h) Formic acid: A JIS Guaranteed Reagent specified in JIS K 8264 or a reagent of equivalentquality.
i) Formic acid: Formic acid used in eluent of LC-MS/MS is a reagent of LC-MS analysis
grade or equivalents.
j) Dichloromethane: A reagent of agricultural chemicals residue/PCB testing grade(concentration: no less than 300) specified in JIS K 8117 or a reagent of equivalent quality.
k) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent of equivalentquality.
l) Acetone: A reagent of agricultural chemicals residue/PCB testing grade (concentration: noless than 300) specified in JIS K 8040 or a reagent of equivalent quality.
m) Ammonia solution (0.0028 % (mass fraction)) (1): Add 0.1 mL of ammonia solution to 1000mL of water.
n) Clopyralid standard solution (0.1 mg/mL) (1): Put about 0.01 g of clopyralid [C6H3C12NO2]
631

Testing Methods for Fertilizers (2018)
(2)

o)
p)
q)

in a weighing dish and measure the mass to the order of 0.1 mg. Dissolve with a small
amount of acetonitrile, transfer to 100-mL volumetric flasks and add the solvent up to themarked line.
Clopyralid standard solution (250 ng/mL) (1): Dilute a predetermined amount of clopyralidl
standard solutions (0.1 mg/mL) with formic acid (1+1000) to prepare clopyralid standard
solution (250 ng/mL).
Clopyralid standard solution for calibration curve preparation (5 ng/mL - 50 ng/mL) (1):In the case of usage, transfer 2.5 mL - 25 mL of clopyralid standard solution (100 ng/mL) to
50 mL volumetric flasks step-by-step, and add formic acid (1+1000) up to the marked line.
Clopyralid standard solution for calibration curve preparation (0.5 ng/mL - 5 ng/mL) (1):In the case of usage, transfer 2.5 mL - 25 mL of clopyralid standard solution (10 ng/mL) to 50
mL volumetric flasks step-by-step, and add formic acid (1+1000) up to the marked line.

Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A standard reagent is commercially sold.
Comment 2 A standard reagent of clopyralid is sold by FUJIFILM Wako Pure Chemical Co., Ltd.,
Kanto Chemical Co., Inc. and Hayashi Pure Chemical Industries., Ltd.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph/Mass Spectrometer (LC-MS/MS)：
LC-MS/MS specified in JIS K 0136 that satisfies the following requirements.
1) High-Performance Liquid Chromatograph(i) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
(ii) Column: A 2-mm - 3-mm inner diameter 50-mm - 150-mm long 1.6-µm - 2.2-µm
particle diameter stainless steel column tube filled with silica gel to which octadecyl
chemically bonds. The specification is according to the mass spectrometer specification.
2) Mass Spectrometer(i) Ionization method: Electro-Spray Ionization (ESI) method(ii) Ion detection method: Selected Reaction Monitoringb) Shaking apparatusc) Manifoldd) Centrifugal separator: A centrifugal separator that can work at 700 × g - 2000 × g.
e) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g 10000 × g.
f) Concentrator: An evaporator that can adjust to 40 ºC ± 2 ºC.
g) Copolymer cartridge column: A divinylbenzene-N-vinylpyrrolidone copolymer mini column(200 mg or 335 mg)h) Filter: A funnel for filtering under reduced pressure (compatible filter diameter: 60 mm)i) Glass fiber filter paper: A filter paper made of glass fiber (filter diameter: 60 mm) that cankeep particle diameter 0.8 µm.
j) Test tube mixer: Vortex mixer
Comment 3 Column is sold under the production name ACQUITY UPLC HSS C18, etc.
Comment 4 A copolymer cartridge is sold under the production names such as Oasis HLB 6cc(225 mg), Oasis PRiME HLB Plus Short Cartridge (225 mg).
Comment 5 A funnel for filtering under reduced pressure is sold under the production nameKIRIYAMA Funnel SB-60, KIRIYAMA Funnel SU-60, etc.
Comment 6 A glass fiber filter is sold under the production name Glass filter paper GFP-60, etc.
632

Testing Methods for Fertilizers (2018)(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample to the order of 1 mg and put it in a 100-mL centrifugal
precipitate tube with a screw cap (3) (4).
b) Add 50 mL of sodium hydroxide solution (40 g/L)−methanol [1+99] and shake to mix with ashaking apparatus for about 30 minutes.
c) Centrifuge at about 1700 × g for about 3 minutes (5) and transfer the supernatant to a 100-mL
Erlenmeyer flask.
d) Add 40 mL of sodium hydroxide solution (40 g/L)−methanol [1+99] to residue and shake tomix with a shaking apparatus for about 30 minutes.
e) Centrifuge at about 1700 × g for about five minutes (5).
f) Filter supernatant in c) and e) under reduced pressure with a filter that places a glass fiber
filter paper to a 100-mL short-neck volumetric flask as an acceptor (6).
g) Wash the vessel and residue with a small amount of sodium hydroxide (40 g/L)−methanol[1+99] several times and transfer the washing to the previous filter to filter under pressure.
h) Add sodium hydroxide solution (40 g/L) - methanol [1+99] to the marked line to make theextract.
Note (3) A vessel used for an extract procedure should be made of glass or polypropylene and it
must enable vibration and centrifugation procedure.
(4) A 100-mL - 200-mL ground-in stopper or screw cap Erlenmeyer flask can also be
used. In this case, however, suspension should be transferred to a ground-in stopper orscrew cap centrifugal precipitate tube before the procedure c) and e).
(5) 16.5-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g centrifugal
force.
(6) An Erlenmeyer flask can also be used. In this case, however, filtrate should betransferred to a 100-mL volumetric flask before the procedure h).
Comment 5 Grind until it completely passes through a sieve of 500 µm aperture to prepare thetest sample.
(4.2) Cleanup (1) (7): Conduct cleanup (1) as shown below.
a) Wash a cartridge column with about 5 mL of methanol and 5 mL of water in advance.
b) Place a 100-mL round-bottom flask (8) under the cartridge column, transfer 9 mL (9) of theextract to the cartridge column and allow the extract to overflow until the surface of the liquidreaches the top of the packing materials.
c) Add about 5 mL of sodium hydroxide solution (0.4 g/L)−methanol [1+1] to the cartridge
column 2 times and allow the liquid to overflow in the same manner in b).
d) Add 5 mL of methanol.
Note (7) Use a pressure reducing device in the procedure in (4.2) and (4.3) as appropriate.
(8) When making a pretreatment of many analytical samples, a free-standing type vesselthat can contain a solution with a liquid volume of 20 mL may be used. In this case,instead of procedure d), put an effluent into a round-bottle flask, wash the vessel 2
times with 2.5 mL of methanol and add washing to the previous effluent.
(9) When using Oasis HLB 6cc (200 mg), add 5 mL of the extract 2 times.
(4.3) Cleanup (2) (7): Conduct cleanup (2) as shown below.
a) Wash a new cartridge column with about 5 mL of acetonitrile and 5 mL of hydrochloric acid
(1+120) in advance.
633

Testing Methods for Fertilizers (2018)

b)
c)
d)
e)
f)

After conducting vacuum concentration of the effluent in (4.2) c) until no more than 5 mL on
a water bath at no more than 40 °C, add 3 mL of hydrochloric acid (1 +11).
Put the concentrated effluent into the cartridge column and allow the effluent to overflow until
the surface of liquid reaches the top of packing materials.
Wash a round-bottom flask with about 5 mL of hydrochloric acid (1+120) 2 times and add
washing into the cartridge successively.
Then, add about 5 mL of hydrochloric acid (1+120)−acetonitrile (9+1) and about 5 mL ofwater into the cartridge successively and allow the liquid to overflow.
Place a 10-mL cone shaped centrifugal precipitate tube with a screw cap (10) under the
cartridge column, add 4 mL of ammonia solution (0.0028 % (mass fraction))−acetonitrile[9+1] to the cartridge column and allow clopyralid to elute.

Note(10) The part under 2 mL from the bottom forms a cone shape.

(4.4) Cleanup (3): Conduct cleanup (3) as shown below.
a) Add 0.1 mL of sodium hydroxide (40 g/L) to the elute in (4.3) f) and shake to mix by using atest tube mixer.
b) Add about 2 mL of dichloromethane, and shake to mix using a test tube mixer for about 30
minutes.
c) Centrifuge at about 740 × g for about five minutes (11) and remove a low layer by using a
Pasteur pipet (12) or a syringe.
d) Repeat the procedure in b) - c) 1 time.
e) Add about 0.15 mL of sulfuric acid (1+2), and shake to mix using a test tube mixer.
f) Add about 2 mL of dichloromethane, and shake to mix using a test tube mixer for about 30
minutes (13).
g) Centrifuge at about 740 × g for about five minutes (11) and transfer a low layer to a 50mL
round-bottle flask by using a Pasteur pipet (14) or a syringe.
h) Repeat the procedure in f) - g) 2 times. Note that lower layers are added to the sameround-bottle flask.
i) Add 5 mL of acetone.
j) Concentrate under reduced pressure in a water bath of no more than 40 ºC until most of theelute dries up and send a nitrogen gas to dry up.
k) Add 1 mL of formic acid (1+1000) and transfer it to a 1.5-mL ground-in stopper centrifugal
precipitate tube (15).
l) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (16) and use thesupernatant as the sample solution
Note (11) 16.5-cm of rotor radius and 2000 rpm of revolutions makes about 740 × g centrifugal
force. Confirm the permissible range of centrifugal force of a 10-mL cone shaped
bottle centrifugal precipitate tube with a screw cap used.
(12) When using a Pasteur pipet, use the same Pasteur pipet through a series of proceduresin c) - d).
(13) Disperse dichloromethane sufficiently. If vibration occurs while dichloromethane layer
remains caked, the extraction efficiency of clopyralid deteriorates and mesurements
are affected.
(14) When using a Pasteur pipet, use the same Pasteur pipet through a series of proceduresin g) - h). Do not use the Pasteur pipet used in Note (9).
(15) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement(16) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 634

Testing Methods for Fertilizers (2018)

10000 × g centrifugal force.
(17) If there is a possibility that the clopyralid concentration in the sample solution exceed
the maximum limit of the calibration curve, dilute a predetermined amount of effluent
with formic acid (1 +1000).
Comment 6 Aa an alternative to the procedures in (4.4) k) - l), filtration is allowed with amembrane filter (pore size: no more than 0.5-µm) made of hydrophilic PTFE or
conduct centrifugal filtration with a centrifugal type filter unit (Ultrafree-MC PVDF
membrane (0.22 µm), etc) and the filtrate can be the sample solution.
Comment 7 When further concentration is required to ensure minimum quantitation, dissolve
concentrated matters in the procedure in j) by adding acetone, transfer to a nitrogenconcentration tube with the same solvent and send a nitrogen gas to dry up. Then add
0.2 mL of formic acid (1+1000) and conduct centrifugal filtration with a centrifugal
type filter unit (Ultrafree-MC PVDF membrane (0.22 µm), etc) and the filtrate canbe the sample solution. In this case, don not conduct the procedure in i).
(4.5) Measurement: Conduct the measurement as indicated in JIS K 0136 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph Mass Spectrometer used in measurement.
a) The measurement conditions of High-Performance Liquid Chromatograph/Mass
spectrometer: An example of measurement conditions for a High-Performance Liquid
Chromatograph/Mass Spectrometer is shown below. Set up the measurement conditions
considering it:
1) High-Performance Liquid Chromatograph(i) Column: A silica gel column (2-mm - 3-mm inner diameter, 50-mm - 150-mm long,
1.6-µm - 2.2-µm particle diameter column) to which octadecyl chemically bonds.
(ii) Flow rate: 0.2 mL/min - 0.5 mL/min(iii) Eluent: A: Formic acid (1+1000) B: Methanol:(iv) Gradient: 0 min (5 %B) → 5 min (60 %B) → 6 min (95 %B) → 7 min (5 %B)(v) Temperature of column bath: 40 °C(vi) Injection volume: 5 µL
2) Mass Spectrometer(i) Ionization method: Electro-Spray Ionization (ESI) method(ii) Mode: Positive(iii) Capillary voltage: 1.0 kV(iv) Ion source temperature: 120 °C(v) Desolvation temperature: 400 °C(vi) Cone voltage: 20 V(vii) Collision energy: 20 eV for determination, 30 eV for validation(viii)Monitor ion: Precursor ion m/z 192
Product ion m/z 146 for determination, m/z 110 for validationb) Calibration curve preparation
1) Inject 5 μL of respective clopyralid standard solutions for calibration curve into the
LC-MS/MS, record the chromatogram of ion (m/z) for determination and ion (m/z) for
validation of clopyralid and obtain respective peak areas.
2) Calculate the peak area ratio or height ratio of ion (m/z) for determination and ion (m/z) for
validation of clopyralid.
3) Prepare a curve for the relationship between the concentration of respective agrichemicals andthe peak area of ion (m/z) for determination of respective mixture standard solutions for the635

Testing Methods for Fertilizers (2018)calibration curve preparation.
c) Sample measurement
1) Subject 5 µL of the sample solution to the same procedure as in b) 2) - 3) (18).
2) Obtain the clopyralid content from the calibration curve to calculate clopyralid in theanalytical sample.
Note (18) Confirm that the ratio against the peak area ratio or height ratio of the standard
solution is within the range of about ± 30 %. In addition, the peak area ratio or heightratio may depend on the concentration.
Comment 8 Additive recovery testing of clopyralid was conducted using cow dung compost (1kinds), as a result, the mean recovery rates at additive level of 50 µg/kg, 10 µg/kg
and 2 µg/kg were 78.9 %, 78.3 % and 71.5 % respectively. In addition, additive
recovery testing of clopyralid was conducted using swine manure compost, poultry
manure compost and composted sludge fertilizer and (1 sample for each), as a result,the mean recovery rates at additive level of 200µg/kg, 2 µg/kg and 80µg/kg were
88.6 %, 81.2 % and 94.2 % respectively.
Table 1 shows results and analysis results from a collaborative study for testing
method validation.
Additionally, the minimum limit of quantification of clopyralid of this test method isabout 2 µg/kg.

Table 1 Results and analysis results from a collaborative study
for the test method validation of clopyralid
Mean

sr

3)

RSD r

laboratories(µg/kg)(µg/kg)Cow dung compost 1

10

128

Cow dung compost 2

10

Swine manure compost
Poultry manure compost

Sample name

sR

5)

RSD R

(%)

(µg/kg)

(%)

10

7.9

15

11.4

2.28

0.35

15.3

0.40

17.6

9

22.5

2.3

10.3

3.4

15.3

9

1.20

0.06

5.0

0.14

12.0

5.6

11.6

Number of
1)

2)

4)

Composted sludge
9
48.1
1.2
2.5
fertilizer
1) Number of laboratories used in analysis
2) Grossl mean (n = number of laboratories × number of repeated tests (2))3) Repeatability standard deviation
4) Repeatability relative standard deviation
5) Reproducibility standard deviation
6) Reproducibility relative standard deviation

6)

References
1)
National Research and Development Agency: Institute for Agro-Environmental
Sciences, National Agriculture and Food Research Organization: High-sensitive analysis
636

Testing Methods for Fertilizers (2018)

for clopyralid in cow dung compost (Reference method)< http://www.naro.affrc.go.jp/publicity_report/pub2016_or_later/pamphlet/tech-pamph/078229.html>(5)

Flow sheet for clopyralid: The flow sheet for clopyralid in compost is shown below:
5.00 ganalytical sample

100-mL centrifugal precipitate tube with a screw cap← 50 mL of sodium hydroxide solution (40 g/L)−methanol [1+99]
Shaking
About 30 minutes
Centrifugal separation

1700× g , 5 minutes

Transfer supernatant［Acceptor, 100-mL Erlenmeyer flask］（Supernatant）（Residue）← 40 mL of sodium hydroxide solution (40 g/L)−methanol [1+99]
Shaking
30 minutes
Centrifugal separation

1700× g , 5 minutes

Filter under
Glass fiber filter paper
reduced pressure［Acceptor, 100-mL short-neck volumetric flask］←Wash the vessel and residue with a small amount of
sodium hydroxide (40 g/L)−methanol [1+98]←Sodium hydroxide solution (40 g/L)−methanol [1+99](up to the marked line)
Extract

Figure 1

Flow sheet for clopyralid in compostand composted sludge fertilizers (Extraction procedure)

637

Testing Methods for Fertilizers (2018)

Extract← 10 mL of extract
copolymer cartridge column (1)Cleanup (1)(Wash with about 5 mL of methanol, then 5 mL of water in advance）[Collection, 100-mL eggplant-shaped flask]← About 5 mL of sodium hydroxide solution (0.4 g/L)−methanol [1+1] × 2 times[Collection, same as above vessel]← About 5 mL of methanol
Concentration
under reduced
40 ºC, until the volume reaches about 5 mLpressure← 3 mL of hydrochloric acid (1+11)
copolymer cartridge column (2)Cleanup (2)(Wash with about 5 mL of acetonitrile,then 5 mL of hydrochloric acid (1+120) in advance)← Wash the vessel 2 times with about 5mL of hydrochloric acid (1+120)← Wash with about 5mL of hydrochloric acid (1+120)−acetonitrile [9+1]← Wash with about 5mL of water← 4 mL of ammonia solution (0.0028 w/v %)−acetonitrile [9+1][Elute, 10-mL centrifugal precipitate tube]
Elute
Figure 2 Flow sheet for clopyralid in compost and composted sludge fertilizers(Cleanup (1) and cleanup (2) and measurement procedure)

638

Testing Methods for Fertilizers (2018)

EluteCleanup (3)← 0.1 mL of sodium hydroxide (40 g/L)
Vortex
Shake to mix← 2 mL of dichloromethane
Vortex, 30 seconds

Shake to mix

Centrifugal separation

740 × g (2000 rpm, 165 mm), 3 minutes

Remove a low layer

Pasteur pipet or micro syringe

Repeat 1 time
← 0.15 mL of sulfuric acid (1+2)
Vortex

Shake to mix← 2 mL of dichloromethane
Vortex, 30 seconds

Shake to mix

Centrifugal separation

740 × g (2000 rpm, 165 mm), 3 minutes

Transfer a low layer

Pasteur pipet or micro syringe［Acceptor, 50-mL round-bottle flask］

Repeat 2 times←5 mL of acetone
Concentration
under reduced pressure

40 °C

Exsiccation

Nitrogen← 1 mL of formic acid (1+1000)
Transfer
1.5 mL centrifugal precipitate tube made of polymer

Centrifugal separation

8100 × g (10000 rpm, 72.5 mm), 5 minutes

Supernatant
Measurement

Figure 2

LC-MS/MS

Flow sheet for clopyralid in compost and composted sludge fertilizers(Cleanup (3) and measurement procedure)

639

Testing Methods for Fertilizers (2018)

Intensity /References: Selected Reaction Monitoring chromatogram of clopyralid standard solution for
calibration curve preparation is shown below

Retention

Reference diagram: SRM chromatograms of clopyralid
Clopyralid standard solution (the equivalents of 100 pg as clopyralid)
LC-MS/MS measurement conditions
1) Column: ACQUITY UPLC HSS C18 (2.1-mm inner diameter, 100-mm long,
1.8-µm particle diameter)
Other conditions are according to the examples of the measurement conditions in (4.4)a) LC-MS/MS.

640

Testing Methods for Fertilizers (2018)

8.3 Residue agrichemicals (multicomponent)
8.3.1 Residue agrichemicals multicomponent analysis (1)8.3.1.aHigh-Performance Liquid Chromatography/Tandem Mass Spectrometry(1) Compounds subjected to analysis Abamectin: abamectin B1a, ivermectin: 22, 23-dihydroavermectin B1a (Another name: ivermectin B1a), eprinomectin: eprinomectin B1a, rotenone:
rotenone, piperonylbutoxide: piperonylbutoxide, pyrethrin: pyrethrin I and pyrethrin II(2) Summary
This testing method is applicable to fluid home garden-use mixed fertilizer and fluid mixed fertilizer.
This testing method is classified as Type B and its symbol is 8.3.1.a-2017 or AG-C-1.a-1.
Dissolve respective agricultural chemicals in fertilizers with acetonitrile and water, and extract.
Refine by using 2 kinds of cleanup cartridge, and then measure with a High-Performance Liquid
Chromatograph/Mass Spectrometer to obtain compounds subjected to analysis in an analyticalsample. In addition, the performance of this testing method is shown in Comment 3.
(3) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Acetonitrile: A reagent of agricultural chemicals residue/PCB testing grade (concentration:no less than 300) specified in JIS K 8039 or a reagent of equivalent quality.
c) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
d) Methanol: Methanol used in eluent of an HPLC is a reagent of LC-MS analysis grade orequivalents.
e) Ethyl acetate: A JIS Guaranteed Reagent specified in JIS K 8361 or a reagent of equivalentquality.
f) Toluene: A JIS Guaranteed Reagent specified in JIS K 8680 or a reagent of equivalentquality.
g) Ammonium formate: A JIS Guaranteed Reagent (no less than 95 % (mass fraction) inpurity) or a reagent of equivalent quality.
h) Ammonium formate solution (0.1 mol/L) (1): Add 6.306 g of ammonium formate into 1000mL of water.
i) Ammonium formate solution (0.1 mmol/L) (1): Add 1mL of ammonium formate solution(0.1 mol/L) into 1000 mL water.
j) Formic acid: A JIS Guaranteed Reagent specified in JIS K 8264 or a reagent of equivalentquality.
k) Formic acid solution (0.1 v/ v %) (1): Add 1 mL of formic acid to 1,000 mL of water.
l) Acetonitrile formate solution (0.1 v/v %) (1): Add 1 mL of formic acid to 1,000 mL ofacetonitrile.
m) Respective agricultural chemicals standard solutions (0.1 mg/mL) (1): Put about 0.01 g ofabamectin [C48H72O14] (2), ivermectin [C48H74O14] (2), eprinomectin [C50H75NO14] (2), rotenone[C23H22O6] (2), piperonylbutoxide [C19H30O5] (2) and pyrethrin [pyrethrin I: C21H28O3 andpyrethrin II: C22H28O5] (2) to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve with a small amount of methanol, transfer to a 100-mL volumetric flask and add thesolvent up to the marked line. (However, 0.1 mg/mL of pyrethrin contains total of pyrethrin
I/II)n) Mixture standard solution (10 µg/mL): Transfer 10 mL of respective agricultural chemicals
standard solutions to a 100-mL volumetric flask and add methanol up to the marked line.
o) Mixture standard solution (1000 ng/mL): Transfer 10 mL of mixture standard solution (10µg/mL) to a 100-mL volumetric flask and add methanol up to the marked line.
p) Mixture standard solution for calibration curve preparation (50 ng/mL - 500 ng/mL): In
641

Testing Methods for Fertilizers (2018)q)

the case of usage, transfer 2.5 mL - 25 mL of mixture standard solution (1000 ng/ mL) to
50-mL volumetric flasks step-by-step, and add methanol up to the marked line.
Mixture standard solution for calibration curve preparation (5 ng/mL - 50 ng/mL): In
the case of usage, transfer 2.5 mL - 25 mL of mixture standard solution (100 ng/ mL) to
50-mL volumetric flasks step-by-step, and add methanol up to the marked line.

Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A standard reagent is commercially sold.
Comment 1 A standard reagent of respective agricultural chemicals is sold by FUJIFILM Wako
Pure Chemical Co., Ltd., Kanto Chemical Co., Inc. and Hayashi Pure Chemical
Industries., Ltd.
(4) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph/Mass Spectrometer (LC-MS/MS)：
LC-MS/MS specified in JIS K 0136 that satisfies the following requirements.
1) High-Performance Liquid Chromatograph(i) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
(ii) Column: A 2-mm - 3-mm inner diameter 50-mm - 150-mm long 1.6-µm - 3.0-µm
particle diameter stainless steel column tube filled with silica gel to which octadecyl
chemically bonds. The specification is according to the mass spectrometer specification(3).
2) Mass Spectrometer(i) Ionization method: Electro-Spray Ionization (ESI) method(ii) Ion detection method: Selected Reaction Monitoringb) Ultrasonic generator: An ultrasonic washer can be used.
c) Concentrator: An evaporator whose temperature can be adjusted up to 40 ºC.
d) Porous diatomaceous earth cartridge column: A column that is filled with the porous
diatomaceous earth (capacity: 5 mL) (4)e) Graphite carbon-NH2 laminate cartridge column: A 6-mL cylinder on which 500 mg ofgraphite carbon and 500 mg of aminopropyl silylation silica gel is laminated (5).
Note (3) The column is sold under the names ACQUITY UPLC HSS C18, etc.
(4) The column is sold under the names Chem Elut (5 mL), etc.
(5) The column is sold under the names Envi-carb/LC-NH2 (500 mg/500 mg, 6 mL), etc.
(5) Test procedures(5.1) Extraction: Conduct extraction as shown below.
a) Put about 5.00 mL (6) of an analytical sample into a 10-mL volumetric flask.
b) Add 3 mL of acetonitrile to the same volumetric flask, and add water up to the marked line toshake to mix well.
c) Conduct ultra-sonication for about 30 minutes using an ultrasonic generator (7) to make theextract.
Note (6) After measuring the specific gravity of sample, calculate the concentration of materials
subjected to measurement in the analytical sample.
(7) Note that the volume of the solution may expand as a result of ultra-sonication. It is
recommended to leave it at room temperature for a while when it expands.
Comment 2 The specific gravity (density) can be calculated by placing a 10-mL volumetric flask
642

Testing Methods for Fertilizers (2018)

on an electric balance, aligning the scale to zero, transferring 5.00 mL of theanalytical sample to the volumetric flask and reading the weighing value.
(5.2) Cleanup (1): Conduct cleanup (1) as shown below.
a) Put 5 mL of extract into a porous diatomaceous earth cartridge column and keep it in thecolumn for about 5 minutes.
b) Place a 100-mL round-bottom flask under the same cartridge column, add about 5 mL of ethyl
acetate into the same cartridge column 4 times successively and allow the solution to elute
until the surface of the solution reaches the top of packing materials (8).
c) After conducting vacuum concentration of elute in a water bath of no more than 40 ºC until
most of the elute dries up, send a nitrogen gas to dry up the elute (9), and add 2 mL ofacetonitrile−toluene (3+1) to dissolve the residue.
Note (8) Confirm the solution to elute before conducting the testing.
(9) There is a possibility for agricultural chemicals to vaporize if it is dried up excessively.
(5.3) Cleanup (2): Conduct cleanup (2) as shown below.
a) Wash the graphite carbon-NH2 laminate cartridge column with about 10 mL ofacetonitrile−toluene (3+1) in advanceb) Place a 100-mL round-bottom flask under the same cartridge column, put the solution in (5.2)c) to the same cartridge column, and allow the solution to overflow until the surface of thesolution reaches the top of packing materials.
c) Wash the vessel with about 5 mL of acetonitrile−toluene (3+1) 5 times and add washing to thesame cartridge successively to allow it to overflow.
d) After conducting vacuum concentration of elute in a water bath of no more than 40 ºC until
most of the elute dries up, send a nitrogen gas to dry up the elute (10), and add 2 mL of
methanol (11) to dissolve the residue. Transfer a predetermined amount of the solution
precisely and dilute with methanol exactly by a factor of 5 to make the solution as the sample
solution.
Note (10) There is a possibility for agricultural chemicals to vaporize if it is dried up excessively.
(11) If there is a possibility that the concentration of agricultural chemicals in the sample
solution exceeds the maximum limit of the calibration curve, dilute a predeterminedamount of the sample solution with methanol.
(5.4) Measurement: Conduct the measurement as indicated in JIS K 0136 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph Mass Spectrometer used in measurement.
a) The measurement conditions of High-Performance Liquid Chromatograph/Mass
spectrometer: An example of measurement conditions for a High-Performance Liquid
Chromatograph/Mass Spectrometer is shown below. Set up the measurement conditions
considering it:
1) High-Performance Liquid Chromatograph(i) Column: A silica gel column (2-mm - 3-mm inner diameter, 50-mm - 150-mm long,
1.6-µm - 3.0-µm particle diameter column) to which octadecyl chemically bonds.
(ii) Flow rate: 0.2 mL/min - 0.5 mL/min(iii) Eluent: A: Ammonium formate solution (0.1 mmol/L)−formic acid solution (0.1 v/v%)[1+1]
B: Acetonitrile formate solution (0.1 v/v%)(iv) Gradient: 0 min (50 %B) → 15 min (95 %B) → 20 min (98 %B) → 30 min (50 %B)
643

Testing Methods for Fertilizers (2018)(v) Temperature of column bath: 40 °C(vi) Injection volume: 5 µL
2) Mass Spectrometer(i) Ionization method: Electro-Spray Ionization (ESI) method(ii) Mode: Positive(iii) Capillary voltage: 3.0 kV(iv) Ion source temperature: 120 °C(v) Desolvation temperature: 400 °C(vi) Cone voltage: Shown in table 1(vii) Collision energy: Shown in table 1(viii)Monitor ion: Shown in table 1

Table 1 Monitoring ion conditions, etc. of respective agrichemicals
Precursor
Product ion
Product ion
Cone voltage
Agrichemicals
ion(determination)(validation)(V)(m/z )(m/z )(m/z )
Abamectin B1a
891
305
567
20
Ivermectin B1a
893
307
551
25
Eprinomectin B1a
915
186
298
20
Rotenone
395
213
192
35
Piperonylbutoxide
356
177
147
20
PyrethrinⅠ
329
161
133
20
PyrethrinⅡ
373
161
133
20

Collisionenergy
(eV)
25
25
20
25
15
10
10

b) Calibration curve preparation
1) Inject 5 μL of respective mixture standard solutions for calibration curve preparation into the
LC-MS/MS, record the chromatogram of ion (m/z) for determination and ion (m/z) for
validation of materials subjected to measurement.
2) Calculate the peak area ratio or height ratio of ion (m/z) for determination and ion (m/z) for
validation of respective materials subjected to measurement.
3) Prepare a curve for the relationship between the concentration of material subjected tomeasurement and the peak area or height of ion (m/z) for determination of respective mixture
solutions for the calibration curve preparation. Prepare a calibration curve when the sample ismeasured.
c) Sample measurement
1) Subject 5 µL of the sample solution to the same procedure as in b) 2) - 3) (12).
2) Obtain the content of materials subjected to measurement from the calibration curve of thepeak area or height to calculate materials subjected to measurement in the analytical sample.
Note (12) Confirm that the ratio against the peak area ratio or height ratio of the standard
solution is within the range of about ± 30 %. In addition, the peak area ratio or heightratio depends on the concentration.
(5.5)
Calculation
Calculate the respective concentration of agricultural chemicals in the analytical sample by thefollowing formula. in the analytical sample by the following formula:644

Testing Methods for Fertilizers (2018)

Respective concentration of agricultural chemicals in the analytical sample (µg/kg)= (A × B × 10) /C
A： Concentration (ng/mL) of respective materials subjected to measurement in the final
sample solution obtained from the calibration curve
B： Dilution factor in the case that the final sample solution is further diluted because it
exceeds the upper limit of the calibration curve.
C： Specific gravity of the analytical sample (density) (g/mL)
Comment 3 A recovery testing was conducted using fluid home garden-use mixed fertilizer (3kinds) and fluid mixed fertilizer (2 kinds), as a result, the mean recovery at additive
level of 4000 µg/kg and 400 µg/kg (However, 4000 µg/kg and 400 µg/kg ofpyrethrin contain total of pyrethrin I/II) were 77.0 % - 104.5 % and 85.6 % - 107.9 %respectively.
Table 2 shows results and analysis results from a collaborative study for test method
validation.
Additionally, the minimum limit of quantification for respective agrichemicals of thetest method is about 10 µg/kg.

645

Testing Methods for Fertilizers (2018)
Table 2 Analysis results of a collaborative study for the testing method validation of multicomponent analysis ofAdditive
3)
4)
2)
Recovery RSD r
RSD R
Mean
Number ofamountAgrichemicals
Sample name1)（µg/kg） （µg/kg）
（%）
（%）
（%）
laboratories
8
286.8
333.3
86.1
13.3
14.4
Abamectin Home garden-use mixed fertilizer1
B1aHome garden-use mixed fertilizer2
8
358.9
416.7
86.1
13.4
14.8
Home garden-use mixed fertilizer3
8
425.8
500.0
85.2
8.6
11.6
Fluid mixed fertilizer1
8
288.6
333.3
86.6
7.1
8.5
Fluid mixed fertilizer2
8
405.5
500.0
81.1
7.1
7.2
8
298.9
333.3
89.7
14.9
15.0
Ivermectin Home garden-use mixed fertilizer1
B1aHome garden-use mixed fertilizer2
8
382.5
416.7
91.8
14.1
19.3
Home garden-use mixed fertilizer3
8
431.1
500.0
86.2
9.8
10.9
Fluid mixed fertilizer1
8
298.8
333.3
89.6
10.1
12.8
Fluid mixed fertilizer2
8
405.2
500.0
81.0
3.8
5.8
8
293.5
333.3
88.1
7.0
10.4
Eprinomectin Home garden-use mixed fertilizer1
B1aHome garden-use mixed fertilizer2
8
361.9
416.7
86.9
9.2
14.3
Home garden-use mixed fertilizer3
8
425.3
500.0
85.1
7.0
10.0
Fluid mixed fertilizer1
8
277.3
333.3
83.2
9.0
12.0
Fluid mixed fertilizer2
8
398.2
500.0
79.6
7.5
11.6
Home
garden-use
mixed
fertilizer1
8
276.8
333.3
83.1
5.7
7.8
Rotenone
Home garden-use mixed fertilizer2
8
353.5
416.7
84.8
9.8
12.5
Home garden-use mixed fertilizer3
8
426.6
500.0
85.3
6.6
8.5
Fluid mixed fertilizer1
8
263.5
333.3
79.1
11.0
12.3
Fluid mixed fertilizer2
8
385.2
500.0
77.0
5.7
12.1
8
318.2
333.3
95.5
8.1
13.2
Piperonyl Home garden-use mixed fertilizer1
butoxideHome garden-use mixed fertilizer2
8
395.6
416.7
94.9
8.4
13.6
Home garden-use mixed fertilizer3
8
450.3
500.0
90.1
4.6
9.3
Fluid mixed fertilizer1
8
299.7
333.3
89.9
7.4
11.0
Fluid mixed fertilizer2
8
435.8
500.0
87.2
5.8
7.4

1) Number of laboratories used in analysis

2) Gross mean (n = number of laboratories × number of repeated tests (2))3) Repeatability (relative standard deviation)4) Reproducibility (relative standard deviation)

Table 2 （Continued）
2)

Agrichemicals

Pyrethrin I

Pyrethrin II

Sample name

Mean

Number oflaboratories
Home garden-use mixed fertilizer1
8Home garden-use mixed fertilizer2
8Home garden-use mixed fertilizer3
8
Fluid mixed fertilizer1
8
Fluid mixed fertilizer2
8Home garden-use mixed fertilizer1
8Home garden-use mixed fertilizer2
8Home garden-use mixed fertilizer3
8
Fluid mixed fertilizer1
8
Fluid mixed fertilizer2
8

646

1)

（µg/kg）
160.7
202.2
228.6
158.2
223.1
131.1
163.2
182.0
126.2
180.2

Additive
Recoveryamount（µg/kg）
（%）
186.0
86.4
232.5
87.0
279.0
81.9
186.0
85.1
279.0
80.0
147.3
89.0
184.2
88.6
221.0
82.4
147.3
85.7
221.0
81.5

3)

4)

RSD r

RSD R

（%）
9.3
12.6
5.4
6.8
8.5
6.5
10.8
5.4
7.8
6.3

（%）
11.9
12.8
8.8
10.4
9.1
9.7
13.6
8.9
11.4
8.3

Testing Methods for Fertilizers (2018)

References1)
Toshiharu YAGI, Masayuki YAMANISHI, Yuji SHIRAI: Simultaneous Determinationof Agricultural chemicals in Fluid Fertilizer by Liquid Chromatography/Tandem Mass
Spectrometry, Research Report of Fertilizer, Vol.4, p. 36 - 48 (2011)2)
Toshiharu YAGI, Masayuki YAMANISHI, Yuji SHIRAI and Masato SHIBATA:
Simultaneous Determination of Six Kind of Agricultural Chemicals in Fluid Fertilizer
by Liquid Chromatograph-Tandem Mass Spectrometer (LC-MS/MS): A Collaborative
Study, Research Report of Fertilizer, Vol.5, p. 48 - 59 (2012)(6)

Flow sheet for simultaneous analysis of 6 kinds of agrichemicals: The flow sheet for
simultaneous analysis of 6 kinds of agrichemicals in fertilizer is shown below.

5.00 mL analytical sample

10-mL volumetric flask← About 3 mL of acetonitrile← Water (up to the marked line)
Ultra sonication
5 minutes
Extract
Figure 1 Flow sheet for residue agrichemicals multicomponent analysis((1) : simultaneous analysis of 6 kinds of agrichemicals) in fertilizers(Extraction procedure)

647

Testing Methods for Fertilizers (2018)

Extract
Aliquot (5 mL)
Keep the extract for 5 minutes
in the cartridge column (1)← About 5 mL of ethyl acetate ×4 times[elute, 100-mL round-bottom flask ]Concentration
40 °C
under reduced pressure← Dissolve with about 2 mL of acetonitrile−toluene(3+1)
Cartridge column (2)Cleanup (2)(wash with about 5 mL of acetonitrile−toluene (3+1)
× 2 times in advance)[100-mL round-bottom flask]← Wash the vessel 5 times with about 5 mL of acetonitrile−toluene (3+1)[overflow, the same vessel]
Concentration
40 °C
under reduced pressure← 5 mL of methanol
Dilution
Dilute the solution 5 times
Cleanup (1)Sample solution
Measurement

LC-MS/MS

Figure 2 Flow sheet for residue agrichemicals multicomponent analysis((1) : simultaneous analysis of 6 kinds of agrichemicals) in fertilizers(Cleanup (1) and cleanup (2) and measurement procedure)

648

Testing Methods for Fertilizers (2018)

Reference: Selected Reaction Monitoring chromatograms of mixture standard solution for
calibration curve preparation and sample solution (fluid home garden-use mixed
fertilizer) are shown below.

Peak No.
１
2
3

5
6
7

Intensity / arb.units

40Retention Time / min

20 0 0Retention Time / min

20

Peak No.1: AbamectinB1a
No.2: IvermectinB1a
No.3: Eprinomectin B1a
No.4: Rotenone
No.5: Piperonylbutoxide
No.6: Pyrethrin I
No.7: Pyrethrin II
1) Mixed standard solution
Reference diagram

2) Sample solution

Selected Reaction Monitoring chromatograms of respective agriculturalchemicals1) Mixture standard solution (the equivalents of 2,500 pg as respective agrichemicals)(For pyrethrin, the equivalents of 2,500 pg of the total of pyrethrin I/II)
2) Sample solution (fluid home garden-use mixed fertilizer, additive of the equivalents of 400µg/ kg in the sample)(For pyrethrin, the equivalents of 400 µg/kg of the total of pyrethrin I/II)
LC-MS/MS measurement conditions
Column: ACQUITY UPLC HSS C18 (2.1-mm inner diameter, 100-mm long, 1.8-µm
particle diameter)
Flow rate: 0.2 mL/min
Other conditions are according to the examples of the measurement conditions in (5.4)a) LC-MS/MS.
649

Testing Methods for Fertilizers (2018)

8.3.2 Residue agrichemicals multicomponent analysis (2)8.3.2.a Gas Chromatography(1) Compounds subjected to analysis β-HCH (β-BHC), γ-HCH (γ-BHC), o,p'-DDD,
p,p'-DDD, o,p'-DDE, p,p'-DDE, o,p'-DDT, p,p'-DDT,aldrin,endrin, dieldrin, trans-chlordane,
cis-chlordane, trans-nonachlor, cis-nonachlor,heptachlor, heptachlor epoxide andhexachlorobenzene(2) Summary
This testing method is applicable to compost and straw, raw materials of compost. This testing
method is classified as Type D and its symbol is 8.3.2.a-2017 or AG-C-2.a-1.
Extract respective agricultural chemicals in fertilizers or raw materials with acetonitrile and water,refine by using a porous diatomaceous earth column, a gel permeation chromatograph and asynthetic magnesium silicate cartridge column, and then measure with an electron capture detector
equipped gas chromatograph to obtain compounds subjected to analysis in an analytical sample. In
addition, the performance of this testing method is shown in Comment 7.
(3) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Acetonitrile: A reagent of agricultural chemicals residue/PCB testing grade (concentration:no less than 300) specified in JIS K 8039 or a reagent of equivalent quality.
c) Hexane: A reagent of agricultural chemicals residue/PCB testing grade (concentration: noless than 300) specified in JIS K 8825 or a reagent of equivalent quality.
d) Sodium chloride: A reagent of agricultural chemicals residue/PCB testing grade or a reagentof equivalent quality.
e) Cyclohexane: A reagent of agricultural chemicals residue/PCB testing grade (concentration:no less than 300) or a reagent of equivalent quality.
f) Acetone: A reagent of agricultural chemicals residue/PCB testing grade (concentration: noless than 300) specified in JIS K 8040 or a reagent of equivalent quality.
g) Diethyl ether: A reagent of agricultural chemicals residue/PCB testing grade (concentration:no less than 300) specified in JIS K 8357 or a reagent of equivalent quality.
h) 2,2,4-trimethylpentane: A reagent of HPLC grade or a reagent of equivalent quality.
i) Respective agricultural chemicals standard solutions (0.2 mg/mL) (1): Put about 0.02 g of
β-HCH (β-BHC) ［ C6H6Cl6 ］ (2), γ-HCH (γ-BHC) ［ C6H6Cl6 ］ (2), o,p'-DDD ［ C14H10Cl4 ］ (2),p,p'-DDD［C14H10Cl4］(2), o,p'-DDE［C14H8Cl4］(2), p,p'-DDE［C14H8Cl4］, o,p'-DDT［C14H9Cl5］(2), p,p'-DDT ［ C14H9Cl5 ］ ,aldrin ［ C12H8Cl6 ］ ,endrin ［ C12H8Cl6O ］ , dieldrin ［ C12H8Cl6O ］ (2),trans-chlordane ［ C10H8Cl6 ］ (2), cis-chlordane ［ C10H8Cl6 ］ (2), trans-nonachlor ［ C10H5Cl9 ］ (2),cis-nonachlor［C10H5Cl9］(2), heptachlor［C10H5Cl7］(2), heptachlor epoxide［C10H5Cl7O］(2) andhexachlorobenzene［C6Cl6］(2) to a weighing dish, and measure the mass to the order of 0.1 mg.
Dissolve them with 20 mL of acetone, transfer to 100 mL volumetric flasks respectively, andadd 2,2,4-trimethylpentane up to the marked line.
j) Mixture standard solution (1 µg/mL) (1): Transfer 1 mL of respective agricultural chemicals
standard solutions to a 100-mL volumetric flask and add 2,2,4-trimethylpentane-acetone (4+1)
up to the marked line.
k) Mixture standard solution for calibration curve preparation (0.02 µg/mL - 0.2 µg/mL)(1): In the case of usage, transfer 1 mL - 10 mL of mixture standard solution (1 µg/mL) to 50
mL volumetric flasks step-by-step, and add 2,2,4-trimethylpentane−acetone (4+1) up to themarked line.
l) Mixture standard solution for calibration curve preparation (0.005 µg/mL - 0.02 µg/mL)(1): In the case of usage, transfer 2.5 mL - 10 mL of mixture standard solution (0.1 µg/mL) to
50 mL volumetric flasks step-by-step, and add 2,2,4-trimethylpentane−acetone (4+1) up to the650Testing Methods for Fertilizers (2018)

marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A standard reagent is commercially sold.
Comment 1 A standard reagent of respective agricultural chemicals is sold by FUJIFILM Wako
Pure Chemical Co., Ltd., Kanto Chemical Co., Inc. and Hayashi Pure Chemical
Industries., Ltd.
(4) Apparatus and instruments: Apparatus and instruments are shown below.
a) Gas Chromatograph (GC): GC specified in JIS K 0114 that satisfies the following
requirements.
1) Sample injector: An injector that enables split less system.
2) Capillary column: A capillary column (0.25-mm inner diameter and 30-m long) made of
fused silica. 14 % cyanopropylphenyl -86 % dimethyl polysiloxane chemically bonds to theinner surface of a capillary column with 0.25 µm thickness.
3) Detection unit: Electron capture detector (ECD)b) Gel permeation chromatograph (GPC): Preparative liquid chromatograph specified in JIS
K 0135 that satisfies the following requirements. No detector is required.
1) Sample injector: A sample injector that can inject 5 mL of sample solution.
2) Column: A 20-mm inner diameter 300-mm long stainless-steel column tube filled withstyrendivynylbenzene copolymer system hard gel3) Guard column: A 20-mm inner diameter 100-mm long stainless steel column tube filledwith styrendivynylbenzene copolymer system hard gel
4) Fraction collector: A fraction collector that can set up a fraction to which agrichemicalcomponents elute.
c) Shaking apparatusd) Concentrator: An evaporator whose temperature can be adjusted up to 40 ºC.
e) Filter: A funnel for filtering under reduced pressure (compatible filter diameter: 60 mm)f) Porous diatomaceous earth cartridge column: A column that is filled with the porous
diatomaceous earth (capacity: 20 mL).
g) Synthetic magnesium silicate cartridge column: A cartridge column that is filled with 910
mg of synthetic magnesium silicate.
h) Membrane filters: Made of PTFE (pore size is no more than 0.5 μm)
Comment 2 Column for GC is sold under the production name DB-1701, Rtx-1701, SPB-1701,
etc. A column which has actually isolated compounds subjected to analysis should be
used.
Comment 3 Gel permeation chromatograph (GPC) is a preparative liquid chromatograph thatcollects a fraction of a material subjected to measurement sieved and isolated bypacking materials of the column for GPC according to the size of the molecule of thematerial. A column for GPC is sold under the production name Shodex CLNpak
EV-2000 AC, etc. In addition, a gurd column for GPC is sold under the production
name Shodex CLNpak EV-G AC, etc.
Comment 4 A funnel for filtering under educed pressure is sold under the production nameKIRIYAMA Funnel SB-60, KIRIYAMA Funnel SU-60, etc.
Comment 5 A porous diatomaceous earth cartridge is commercially sold under production nameChem Elut (20 mL), etc.
Comment 6 Synthetic magnesium silicate is commercially sold under the production names suchas Sep-Pak Florisil Plus Long Cartridge (910 mg).
651

Testing Methods for Fertilizers (2018)

Comment 7 A membrane filter is sold under the production name HLC-DISK 25 Solvent system(pore size: 0.45 µm), DISMIC 25JP050, Millex FH (diameter: 25 mm, pore size:
0.45 µm), etc.
(5) Test procedures(5.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample, and put it into a 200-mL ground-in stopper Erlenmeyer
flask.
b) Add 20 mL of acetonitrile−water (3 +1) to moisten.
c) After leaving at rest for ten minutes, add 100 mL of acetonitrile and shake to mix for about 30
minutes.
d) Place a 300-mL round-bottom flask under the filter and filtrate the extract under reducedpressure with a filter paper (type 5 B)e) Conduct cleanup of the previous Erlenmeyer flask and residue with 50 mL of acetonitrile
successively, similarly filtrate under reduced pressure and pool with the filtrate in d) to makethe extract.
(5.2) Cleanup (1): Conduct cleanup (1) as shown below.
a) Concentrate under reduced pressure in a water bath of no more than 40 ºC until most of theextract dries up.
b) Add 20 mL of sodium chloride saturated solution, put into a porous diatomaceous earth
cartridge column and leave it at rest for about 5 minutes.
c) Place a 300-mL round-bottom flask under the same cartridge column, wash the vessel 3 times
with about 20 mL of hexane to add the washing to the same cartridge column successively
and allow the solution to elute until the surface of the solution reaches the top of packing
materials.
d) Further add about 60 mL of hexane into the same cartridge and allow the solution to elute
until the surface of the solution reaches the top of packing materials.
e) After conducting vacuum concentration of elute in a water bath of no more than 40 ºC until
most of the elute dries up, send a nitrogen gas to dry up the elute (3), and add 10 mL of
cyclohexane−acetone (4+1) to dissolve the residue.
f) Filter with a membrane filter (pore size: no more than 0.5 μm).
Note (3) There is a possibility for agricultural chemicals to vaporize if it is dried up excessively.
(5.3) Cleanup (2): Conduct cleanup (2) as shown below.
a) Inject 5 mL of the filtrate in (5.2) e) into a gel permeation chromatograph and transfer afraction eluted from respective agrichemicals which are determined according to theprocedure condition in b) into a 100-mL round-bottom flask.
b) Procedure conditions for a gel permeation chromatograph (GPC): An example ofprocedure conditions for a gel permeation chromatograph (GPC) is shown below. Set up themeasurement conditions considering it:1) Column: A styrendivynylbenzene copolymer column (20-mm inner diameter, 300-mm long,
15-µm particle diameter)
2) Guard column: A styrendivynylbenzene copolymer column (20-mm inner diameter,
100-mm long, 15-µm particle diameter)
3) Eluent: Cyclohexane-acetone (4 +1)
4) Flow rate: 5 mL/min
5) Preparative fraction: 70 mL -120 mLc) After conducting vacuum concentration of elute in a water bath of no more than 40 ºC until
652

Testing Methods for Fertilizers (2018)

most of the elute dries up, send a nitrogen gas to dry up the elute (3), and add 2 mL of hexane
to dissolve the residue.
(5.4) Cleanup (3): Conduct cleanup (3) as shown below.
a) Conduct cleanup of a synthetic magnesium silicate cartridge column (910 mg) with about 5
mL of hexane.
b) Place a 50-mL round-bottom flask under the same cartridge column, put the solution in (5.3)c) to the same cartridge column, and allow the solution to overflow until the surface of thesolution reaches the top of packing materials.
c) Wash the vessel with about 2 mL of hexane 2 times and add washing to the same cartridge
successively to allow it to overflow.
d) Further add about 15 mL of hexane−diethyl ethel (9+1) into the same cartridge and allow
respective materials subjected to measurement to elute.
e) After conducting vacuum concentration of the elute in a water bath of no more than 40 ºC
until most of the elute dries up, send a nitrogen gas to dry up the elute (3). And add 1 mL of
2,2,4-trimethylpentane−acetone (4+1) (4) to dissolve the residue, making the sample solution.
Note (4) If there is a possibility that the concentration of respective agricultural chemicals inthe sample solution exceed the maximum limit of the calibration curve, dilute apredetermined amount of the sample solution with 2,2,4-trimethylpentane−acetone(4+1).
(5.4) Measurement: Conduct the measurement as indicated in JIS K 0114 and as shown below.
Specific measurement procedures are according to the operation method of a Gas
Chromatograph used in measurement.
a) The measurement conditions of Gas Chromatograph: An example of measurement
conditions for a Gas Chromatograph is shown below. Set up the measurement conditions
considering it:1) Sample injection method: split less injection method (1min)
2) Temperature of sample injector: 250 °C
3) Capillary column: A capillary column (0.25-mm inner diameter, 30-m long, 0.25 µm layer
thickness) made of fused silica. 14 % cyanopropylphenyl−86 % dimethyl polysiloxane
chemically bonds to the inner surface of the capillary column with 0.25 µm thickness.
4) Column bath temperature: 60 °C (1 min) → (20 °C/min) → 180 °C→ (2 °C/min) →260 °C→ (5 °C/min) → 275 °C (1 min)5) Carrier gas: helium, Flow rate: 1.5 mL/min
6) Addition gas: Nitrogen, Flow rate: 60 mL/min
7) Detection unit: Electron capture detector (ECD)
8) Detector temperature: 280 °Cb) Calibration curve preparation
1) Inject 1 µL of respective mixture standard solutions for calibration curve preparation into a
GC, record a chromatogram and obtain the peak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area or height ofrespective mixture solutions for the calibration curve preparation. Prepare a calibration curvewhen the sample is measured.
c) Sample measurement
1) Subject 1 μL of sample solution to the same procedure as in b) 1)
2) Obtain the content of materials subjected to measurement from the calibration curve of the653

Testing Methods for Fertilizers (2018)

peak area or height to calculate materials subjected to measurement in the analytical sample.
Comment 8 Additive recovery testing of compounds subjected to analysis in compost was
conducted, as a result, the mean recovery rates at additive level of 20 µg/kg and 50µg/kg were 82.1 % - 118.1 % and 62.5 % - 120.2 % respectively. The results of therepeatability tests on different days using compost to evaluate precision wereanalyzed by one-way analysis of variance. Table 1 shows the calculation results ofintermediate precision and repeatability. Note that α-HCH (α-BHC), δ-HCH (δ-BHC),
and oxychlordane, which were subjected to simultaneous analysis, were excluded
from the compounds subjected to analysis because sufficient recovery rates were notobtained.
Additionally, the minimum limit of quantification for respective agrichemicals of thetest method is no more than 20 µg/kg.

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
3)
4)
5)
6)
repeatability Averagesr
RSD rs I(T)RSD I(T)1)(μg/kg)(μg/kg)T
β -BHC5
15.7
1.3
γ -BHC
5
14.7
1.3
δ-BHC
5
50
11.8
hexachlorobenzene
5
15.3
1.4
heptachlor
5
16.9
1.1
aldrin512.8
1.0
oxychlordane
5
32.4
2.8
heptachlor epoxide (1)
5
17.8
2.0
heptachlor epoxide (2)
5
17.9
1.8
trans-chlordane
5
17.6
1.7
cis-chlordane
5
17.8
1.3
trans-nonachlor
5
15.9
1.3
cis-nonachlor
5
16.7
1.5
dieldrin
5
16.6
1.4
endrin5
17.8
1.4
o ,p '-DDE
5
18.7
2.7
p ,p '-DDE
5
16.8
1.6
o ,p '-DDD
5
16.9
1.2
p ,p '-DDD
5
16.3
1.7
o ,p '-DDT
5
17.9
1.4
p ,p '-DDT
5
16.6
1.4
1） The number of test days conducting a duplicate test2） Average （the number of test days （T ）×the number of duplicate testing（2））3） Repeatability standard deviation
4） Repeatability relative standard deviation(%)(μg/kg)
(%)
8.3
2.0
12.8
8.6
1.6
10.9
23.6
14.5
29.1
9.3
2.5
16
6.7
2.4
14.3
7.8
3.4
26.4
8.7
2.8
8.6
11.1
1.6
9
10.1
1.8
10
9.9
1.9
10.8
7.2
2.0
11.3
8.3
1.5
9.6
8.8
2.1
12.4
8.5
2.0
11.9
7.9
1.7
9.5
14.4
2.7
14.6
9.8
1.8
10.9
7.3
1.3
7.8
10.7
1.8
10.9
7.8
2.2
12
8.1
2.2
13
5） Intermediate standard deviation
6） Intermediate relative standard deviation

References
1)
Tomoharu NOZAKI: Determination of Organic Chloride Pesticides in Composts using
Gas Chromatography-Electron Capture Detector (GC-ECD), Research Report of
Fertilizers, Vol.10, p. 41 - 60 (2017)654

Testing Methods for Fertilizers (2018)(6)

Flow sheet for simultaneous analysis of chloride pesticides: The flow sheet for
simultaneous analysis of chloride pesticides in fertilizer is shown below.
5.00 mL analytical sample

200 mL- ground-in stopper Erlenmeyer flask← About 20 mL of acetonitrile - water (3 +1)
Leave at rest
10 minutes← About 100 mL of acetonitrile
30 minutes
Shake to mix

FilterFilter paper (type 5 B), 300-mL round-bottom flask (1)under reduced presuure←Cleanup of the vessel and residue with 50 mL of acetonitrile
Pool washing with the filtrate
Extract

Figure 1 Flow sheet for residue agrichemicals multicomponent analysis((2) : simultaneous analysis of chloride agrichemicals) in fertilizers(Extraction procedure)

Extract
Concentration
40 °C
under reduced presuure← 20 mL of sodium chloride saturated solution,
Porous diatomaceous earth cartridge column
Cleanup (1)Leave at rest for 5 minutes［300-mL round-bottom flask (2)］
←Wash the 300-mL round-bottom flask (1) 3 times with about 20 mLof hexane, add to the same cartridge column successively←60 mL of hexane
Concentration
40 °C
under reduced presuure
Exsiccation
Nitrogen gas←10 mL of cyclohexane−acetone (4+1)
Membrane filter (pore size: no more than 0.45 µm)
Filter

Filtrate
Figure 2 Flow sheet for residue agrichemicals multicomponent analysis((2) : simultaneous analysis of chloride agrichemicals) in fertilizers(Cleanup (1) procedure)

655

Testing Methods for Fertilizers (2018)
Gel permeation chromatographInject 5 mL of the filtrate［Transfer 70 mL - 120 mL of eluted fraction into a 100-mL round-bottom flask］
Concentration
40 °C
under reduced presuure
Exsiccation
Nitrogen gas←About 2 mL of hexane
Cleanup (3)Synthetic magnesium silicate cartridge column

Cleanup (2)［50-mL round-bottom flask］←Wash the 100-mL round-bottom flask 3 times with about 2 mLof hexane, add to the same cartridge column successively←About 15 mL of hexane-diethyl ethel (9+1)
Concentration
under reduced presuure

40 °C

Exsiccation

Nitrogen gas←1 mL of 2,2,4-trimethylpentane−acetone (4+1)

Sample solution
Measurement

GC（ECD）

Figure 3 Flow sheet for residue agrichemicals multicomponent analysis((2) : simultaneous analysis of chloride agrichemicals) in fertilizers(Cleanup (2) and cleanup (3) and measurementprocedure)

656

Testing Methods for Fertilizers (2018)

8.4 Sodium
8.4.a Flame atomic absorption spectrometry(1) Summary
This testing method is applicable to fertilizers containing organic matters. This testing method isclassified as Type D and its symbol is 8.4.a-2017 or Na.a-1.
Pretreat an analytical sample with incineration and hydrochloric acid, spray into an acetylene−air
flame, and measure the atomic absorption with sodium at a wavelength of 589.0 nm or 589.6 nm toobtain sodium (Na) in an analytical sample. In addition, the performance of this testing method isshown in Comment 3.
(2) Reagent: Reagents are as shown below.
a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Sodium standard solution (Na 1 mg/mL) (1): Heat sodium chloride specified in JIS K 8150
at 600 ºC ± 10 ºC for about 1 hour, let it stand to cool in a desiccator, and weigh 2.542 g to aweighing dish. Dissolve in a small amount of water, transfer to a 1000-mL volumetric flask,
and add water up to the marked line.
c) Sodium standard solution (Na 0.1 mg/mL) (1): Transfer 20 mL of sodium standard solution(Na 1 mg/mL) to a 200-mL volumetric flask, and add hydrochloric acid (1+23) up to themarked line.
d) Sodium standard solutions (Na 1 µg/mL - 10 µg/mL) for the calibration curve
preparation (2): Transfer 2.5 mL - 25 mL of sodium standard solution (Na 0.1 mg/mL) to
250-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) up to the marked
line (2).
e) Blank test solution for the calibration curve preparation: Hydrochloric acid (1+23) usedin the procedures in d) (3).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) When using a device model which cannot degrade the sensitivity of the device bytilting the burner head, conduct the model appropriate dilution. (For example, 0.1µg/mL - 4 µg/mL)(3) When preserving, use a container, which can be sealed tightly, made of material suchas polypropylene, PTFE, etc. that sodium hardly elutes.
Comment 1 Instead of the sodium standard solution in (2) b), a sodium standard solution (Na 0.1
mg/mL 1 mg/mL or 10 mg/mL for Atomic Absorption Spectrometry traceable to
National Metrology can also be used.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified in
JIS K 0121.
1) Light source: A sodium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 550 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Adjust the amounts of gas and silica sand of a sand bath so that the sand bath temperature canbe set to 250 ºC.
657

Testing Methods for Fertilizers (2018)(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (4).
c) Ignite at 550 ºC ± 5 ºC for no less than 4 hours to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, gradually add about
10 mL of hydrochloric acid, and further add water to make 20 mL.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to boil forabout 5 minutes.
f) After standing to cool, transfer the solution to a 250-mL - 500-mL volumetric flask with waterg) Add water up to the marked line.
h) Filter with Type 3 filter paper to make a sample solution.
Note (4) Example of charring and incineration procedure: After raising the temperature fromroom temperature to about 250 °C in 30 minutes to 1 hour, continue heating for about
1 hour and further raise to 550 °C in 1 to 2 hours.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1.2) in 4.2.1.a.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up the measurement
conditions for the atomic absorption spectrometer considering the following:
Analytical line wavelength: 589.0 nm or 589.6 nmb) Calibration curve preparation
1) Spray the sodium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation into a flame, and read the indicated value at awavelength of 589.0 nm or 589.6 nm.
2) Prepare a curve for the relationship between the sodium concentration and the indicated valueof the sodium standard solutions for the calibration curve preparation and the blank test
solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg - 1 mg asNa) (5) to a 100-mL volumetric flask.
2) Add hydrochloric acid (1+23) to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Obtain the sodium content from the calibration curve, and calculate the sodium (Na) in theanalytical sample.
Note (5) Sample a predetermined amount of sample solution according to the device model in
Note (2).
Comment 3 Additive recovery testing with triplicates measurement was conducted using fish
caked powder, fish waste processed fertilizers, rape seed meal and its powder,composted sludge fertilizers and compost, as a result, the mean recovery rate at theadditive concentration of sodium in the range of 1 % (mass fraction) - 10 % (massfraction) was 97.0 % - 103 %.
The results of the repeatability tests on different days using fish caked powder(sample to which sodium chloride is added) and compost to evaluate precision were658

Testing Methods for Fertilizers (2018)

analyzed by one-way analysis of variance. Table 1 shows the calculation results ofintermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.02 % (mass fraction).

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)

1)
3)
3)
3)
(%)
(%)
T
(%)
(%)
(%)
Fish cakes powder
5
9.08
0.06
0.6
0.09
1.0
Compost5
0.09730.00192.0
0.00373.8
1） The number of test days conducting a duplicate test 6） Intermediate standard deviation
2） Average （the number of test days（T ）7） Intermediate relative standard deviation

×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

References
1) Kimie KATO, Masaki CHIDA and Toshifumi FUJITA:
Method Validation for
Determination of Sodium in Fertilizer by Atomic Absorption Spectrometry , Research
Report of Fertilizers, Vol.8, p. 61 - 69 (2015)(5)

Flow sheet for sodium testing method: The flow sheet for sodium testing method in
fertilizers is shown below:
5.00 g analytical sample
Charring
Incineration

Weigh into a 200-mL - 300-mL tall beaker.
Heat gently
550 ºC ± 5 ºC, no less than 4 hours

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of hydrochloric acid
← Water (up to about 20 mL)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolRoom temperature

Transfer
250-mL - 500-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for sodium in fertilizers (Extraction procedure)
659

Testing Methods for Fertilizers (2018)

Sample solution
Aliquot(predetermined amount)

100-mL volumetric flask, water←Hydrochloric acid (1+23) (up to the marked line)
Measurement
Atomic absorption spectrometer(589.0 nm or 589.6 nm)
Figure 2 Flow sheet for sodium in fertilizers (Measurement procedure)

660

Testing Methods for Fertilizers (2018)

8.5 Guanylurea nitrogen
8.5.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type B and its
symbol is 8.5.a-2017 or GU-N.a-1.
Add water to an analytical sample to extract guanylurea, introduce it to a High-Performance
Chromatograph (HPLC) to isolate it with a weak acid ion-exchange column, and then measure atwavelength 190 nm to obtain guanylurea nitrogen (GU-N) in an analytical sample. In addition, theperformance of this testing method is shown in Comment 6.
Biuret nitrogen (B-N), dicyandiamide nitrogen (Dd-N), urea nitrogen(U-N) and guanidine nitrogen(Gd-N) can be simultaneously quantified by using this method. (Refer to Comment 5).
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Potassium dihydrogen phosphate: A JIS Guaranteed Reagent specified in JIS K 9007 or areagent of equivalent quality.
c) Phosphoric acid: A JIS Guaranteed Reagent specified in JIS K 9005 or a reagent ofequivalent quality.
d) Guanylurea nitrogen standard solution (GU-N 2 mg/mL) (1): Put 0.540 g of guanylurea
sulfate [C4H12N8O2･H2SO4] (2) in a weighing dish and measure the mass to the order of 0.1
mg. Add a small amount of water to dissolve, then transfer to a 100-mL volumetric flask andadd water up to the marked line.
e) Guanylurea nitrogen standard solution (GU-N 200 g/mL): Put 10 mL of guanylurea
nitrogen standard solution (GU-N 2 mg/mL) to a 100-mL volumetric flask and add water up
to the marked line.
f) Guanylurea nitrogen standard solution (GU-N 50 µg/mL - 100 µg/mL): Put 25 mL - 50
mL of guanylurea nitrogen standard solution (GU-N 200 µg/mL) to 100-mL volumetric flasks
and add water up to the marked line.
g) Guanylurea nitrogen standard solution for the calibration curve preparation (GU-N 1
μg/mL - 50 μg/mL): At the time of usage, put 1 mL - 50 mL of guanylurea nitrogen standard
solution (GU-N 100 µg/mL) to 100-mL volumetric flasks step-by-step and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity as guanylurea sulfate iscommercially sold.
Comment 1 Guanylurea sulfate is commercially sold by Kanto Chemical Co., Inc. and Tokyo
Chemical Industry Co., Ltd.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 7.5-mm inner diameter 100-mm long stainless steel column tube filled withweak acid ion-exchange resin.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 190 nm.
b) Magnetic stirrer:c) High speed centrifugal separator: A centrifugal separator that can work at 8000 × g - 10000
× g.
661

Testing Methods for Fertilizers (2018)

Comment 2 A column is sold under the production name Asahipak ES-502C 7C.
(4) Test procedure(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery test samplea) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of water and stir it by using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer a supernatant solution (3) to a 1.5-mL ground-in stopper
centrifugal precipitate tube (4).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (5) and use thesupernatant as the extract.
Note (3) If there is a possibility that the guanyourea nitrogen (GU-N) concentration in thesample solution will exceed the maximum limit of the calibration curve, dilute apredetermined amount of supernatant with water.
(4) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement.
(5) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
(4.1.2) Fluid test samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, and shake to mix.
c) Add water up to the marked line (6) and transfer to a 1.5-mL ground-in stopper centrifugal
precipitate tube (4).
d) Centrifuge it at 8000 × g - 10000 × g centrifugal force for about five minutes (5) and use thesupernatant as the extract.
Note (6) If there is a possibility that the guanyourea nitrogen (GU-N) concentration in thesample solution will exceed the maximum limit of the calibration curve, dilute apredetermined amount of solution with water.
Comment 3 Instead of procedures in (4.1.1) c) - d) or (4.1.2) c) - d), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5-µm) made of hydrophilic
PTFE and the filtrate can be the sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions is shown below. Set up the measurement conditions
considering it:1) Column: A weak acid ion-exchange resin column (7.5-mm inner diameter, 100-mm long,
5-µm - 10-µm particle diameter)
2) Column bath temperature: 40 °C
3) Eluent (1): Dissolve 3.92 g of potassium dihydrogenphosphate and 0.12 g of phosphoric acidin water to make 1000 mL. Filter with a membrane filter (aperture diameter: no more than
0.5-µm) made of hydrophilic PTFE.
662

Testing Methods for Fertilizers (2018)

4) Flow rate: 0.6 mL/min
5) Injection volume: 10 µL
6) Detection unit: An absorptiometric detector, measurement wavelength: 190 nm
Comment 4 Eluent can be prepared as follows. Dissolve 19.6 g of potassium
dihydrogenphosphate and 0.584 g of phosphoric acid with water to make 500 mL andstore in a refrigerator. At the time of usage, dilute a predetermined volume of thesolution by a factor of 10 and filter with a membrane filter (aperture diameter: no
more than 0.5-µm) made of hydrophilic PTFE.
b) Calibration curve preparation
1) Inject 10 µL of respective standard solutions for the calibration curve preparation to an HPLC,
record chromatogram at wavelength 190 nm and obtain the peak height.
2) Prepare a curve for the relationship between the guanylurea nitrogen (GU-N) concentrationand the peak height at wavelength 190 nm of the respective standard solutions for thecalibration curve preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the guanylurea nitrogen (GU-N) content from the peak height using the calibration
curve to calculate guanylurea nitrogen (GU-N) in the analytical sample.
Comment 5 This testing method enables the simultaneous measurement of biuret nitrogen (B-N),
urea nitrogen (U-N), dicyandiamide nitrogen (Dd-N), guanidine urea (Gd-N) andguanylurea nitrogen (GU-N). In that case, see 5.10.a Comment 5.
Comment 6 Additive recovery testing was conducted using a preparation sample for a guanylurea
fertilizer (one brand). As a result, the mean recovery at additive level of 36.7 %(mass fraction), 35.2 % (mass fraction) and 33.4 % (mass fraction) were 103.8 %,104.6 % and 105.6 % respectively.
The results of the repeatability tests on different days using a guanylurea fertilizer toevaluate precision were analyzed by one-way analysis of variance. Table 1 shows thecalculation results of intermediate precision and repeatability. Table 2 shows results
and analysis results from a collaborative study for testing method validation.
Additionally, the minimum limit of quantification of this testing method is about
0.006 % (mass fraction).

Name ofsampleTable 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)
T

1)

(%)

3)

(%)

3)

Guanylurea nitrogen
5
37.0
0.3
fertilzier
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

663

(%)

(%)

3)

(%)

0.7

0.3

0.8

6） Intermediate standard deviation
7） Intermediate relative standard deviation

Testing Methods for Fertilizers (2018)

Table 2 Results and analysis results from a collaborative study
for the test method validation of guanylurea nitrogen
4)

RSD r

3)

(%)

12
2.20
0.09
Compound fertilizer 1
11
4.38
0.07
Compound fertilizer 2
11
5.83
0.08
Compound fertilizer 3
12
7.43
0.43
Compound fertilizer 4
Guanylurea nitrogen
12
30.3
0.4
fertilizers
1) Number of laboratories used in analysis
2) Mean (n = number of laboratories x number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

Sample name

Number of1)laboratories

Mean

2)

sr

3)

(%)

(%)

5)

6)

RSD R

(%)

3)

(%)

4.2
1.5
1.4

0.17
0.19
0.52

7.7
4.3
8.9

5.7

0.78

10.5

1.5

1.1

3.6

sR

7)

References
1) Masahiro ECHI, Yasuharu KIMURA and Yuji SHIRAI: Determination of Urea Nitrogen,
Biuret Nitrogen, Dicyandiamide Nitrogen, Guanidine Nitrogen and Guanyl urea
Nitrogen in Fertilizer by High-Performance Liquid Chromatography: A
Single-Laboratory Validation, Research Report of Fertilizer, Vol.10, p. 72 - 85 (2017)2)
Norio FUNAKI and Yasuharu KIMURA: Determination of Urea Nitrogen, Biuret
Nitrogen, Dicyandiamide Nitrogen, Guanidine Nitrogen and Guanyl urea Nitrogen in
Fertilizer by High-Performance Liquid Chromatography (HPLC): A Collaborative Study,
Research Report of Fertilizer, Vol.10, p. 86 - 100 (2017)664

Testing Methods for Fertilizers (2018)(5)

Flow sheet for testing method: The flow sheet for guanylurea nitrogen in fertilizers is shown
below:
1.00 g200-mL ground-in stopper Erlenmeyer flask
analytical sample (powdery)← 100 mL of water
Extraction

Stir to mix, 10 minites

Stand still
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

Sample solution

Supernatant

Measurement

Liquid Chromatograph

Figure 1

Flow sheet for guanylurea nitrogen in fertilizers(Extraction procedure (4.1.1) and mesurement)

1.00 ganalytical sample (fluid)

100-mL volumetric flask←About 50 mL of water
Extraction

Shake to mix← Water (up to the marked line)
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

Sample solution

Supernatant

Measurement

Liquid Chromatograph

Figure 2 Flow sheet for guanylurea nitrogen in fertilizers(Extraction procedure (4.1.2) and mesurement)665

Testing Methods for Fertilizers (2018)

Reference:

Chromatogram of the standard solution for calibration curve preparation ofguanylurea nitrogen is shown below.

Reference diagram

HPLC chromatogram of the mixture standard solutions (10mg/L for each)Peak name(1) Urea nitrogen(2) Biuret nitrogen(3) Dicyandiamide nitrogen(4) Guanidine nitrogen(5) Guanylurea nitrogen
Measurement conditions for HPLC
Column: Asahipak ES-502C 7C (7.5-mm inner diameter, 100-mm long, 9-μm
particle diameter)
Other conditions are according to the example of HPLC measurement conditionsin (4.2) a)666

Testing Methods for Fertilizers (2018)

8.6 Uric acid8.6.a High-Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers. This testing method is classified as Type D and its
symbol is 8.6.a-2018 or U-acid.a-1.
Add phosphate solution (pH 8) to an analytical sample to extract uric acid, introduce it to a
High-Performance Chromatograph (HPLC) to isolate it with a multi mode ODS (reverse
phase+strong anion-exchange+strong cation-exchange+normal phase) column, and then measure atwavelength 290 nm to obtain uric acid (U-acid) in an analytical sample. The performance of thistesting method is shown in Comment 5.
(2) Reagent, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557. Note that water of A4 should be used as theeluent which is introduced to an HPLC.
b) Potassium dihydrogenphosphate: A reagent specified in JIS K 9007 or a reagent ofequivalent quality.
c) Disodium hydrogenphosphate: A reagent specified in JIS K 9020 or a reagent of equivalentquality.
d) Phosphate solution: Dissolve 9.073 g of potassium dihydrogenphosphate in water to make1000 mL and dissolve 9.464 g of disodium hydrogenphosphate in water to make 1000 mL.
Mix these solutions so that pH 8.0 ± 0.1 is reached.
e) Lithium carbonate solution: Dissolve 0.739 g of lithium carbonate (Li2CO3) of no less than
99 % (mass fraction) in purity in water to make 1000 mL.
f) Uric acid standard solution (U-acid 1 mg/mL) (1): Put 0.100 g of uric acid in a weighing
dish and measure the mass to the order of 0.1 mg. Dissolve it with a small amount of lithium
carbonate solution, transfer to a 100 mL volumetric flask and add the solution up to themarked line.
g) Uric acid standard solution for the calibration curve preparation (U-acid 100 µg/mL):Put 10 mL of uric acid standard solution (U-acid 1 mg/mL) to a 100-mL volumetric flask andadd phosphate solution up to the marked line.
h) Uric acid standard solution for the calibration curve preparation (U-acid 10 µg/mL - 50
µg/mL): Put 10 mL - 50 mL of uric acid standard solution (U-acid 100 µg/mL) to a 100-mL
volumetric flask and add phosphate solution up to the marked line.
i) Uric acid standard solution for the calibration curve preparation (U-acid 0.1 μg/mL - 5
μg/mL): At the time of usage, put 1 mL - 50 mL of uric acid standard solution (U-acid 10
μg/mL) to 100-mL volumetric flasks step-by-step and add phosphate solution up to themarked line.
j) Ammonium acetate: A reagent specified in JIS K 8359 or a reagent of equivalent quality.
k) Methanol: A reagent of HPLC grade or a reagent of equivalent quality.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) High-Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124 thatsatisfies following requirements.
1) Column: A 4.6-mm inner diameter 250-mm long stainless steel column tube filled withsilica gel, to which octadecyl, ion-exchange group for strong acidity and strong basic
anion-exchange group, chemically bond.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detection unit: An absorptiometric detector that can measure at wavelength around 290 nm.
667

Testing Methods for Fertilizers (2018)

b)
c)
d)
e)

Water bath: Water bath that can be adjusted to 60 ºC ± 2 ºC.
Magnetic stirrer
Centrifugal separator: A centrifugal separator that can work at 1700 × g.
High speed centrifugal separator: A centrifugal separator that can work at 8000 × g - 10000
× g.

Comment 1 A column is sold under the production name Scherzo SS-C18.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200 mL- ground-in stopper Erlenmeyer
flask.
b) Add 100 mL of phosphate solution (2) and heat for 30 minutes while shaking to mix (3) at every
10 minutes in a water bath at 60 ºC ± 2 ºC.
c) Immediately stir it by using a magnetic stirrer for about 10 minutes.
d) After allowing to stand still, transfer supernatant to a 15-mL or 50 mLground-in stopper
centrifugal precipitate tube (4), and centrifuge at 1700 × g for about 5 minutes (5).
e) Transfer supernatant (6) to a 1.5-mL ground-in stopper centrifugal precipitate tube (4), andcentrifuge at 8000 × g - 10000 × g for about 5 minutes (7) to make supernatant as a sample
solution.
Note (2) Use a silicone stopper instead of a glass stopper as the solution is heated.
(3) Steam easily expels silicon stoppers, so while lightly holding down the stopper fromthe top with your finger, shake it so that the water drops on the inside of the flask
come down as much as possible. In addition, conduct this procedure before and after
the heating procedure.
(4) The ground-in stopper centrifugal precipitate tube should be made of polypropylene,
etc. to not affect the measurement(5) 7.2 cm - 18.9-cm of rotor radius and 3000 rpm of revolutions makes about 1700 × g
centrifugal force.
(6) If there is a possibility that the uric acid (U-acid) concentration in the sample solution
will exceed the maximum limit of the calibration curve, dilute a predetermined amountof supernatant with phosphate solution.
(7) 7.2-cm - 8.9-cm of rotor radius and 10000 rpm of revolutions makes about 8100 × g 10000 × g centrifugal force.
Comment 2 Instead of the procedures in (4.1) e), it is allowed to filter with a membrane filter(pore size: no more than 0.5-µm) made of hydrophilic PTFE and the filtrate can be
the sample solution.
(4.2) Measurement: Conduct the measurement as indicated in JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of aHigh-Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for a High-Performance Liquid Chromatograph (HPLC): An
example of measurement conditions is shown below. Set up the measurement conditions
considering it:1) Column: A silica gel column, to which octadecyl, ion-exchange group for strong acidity andstrong basic anion-exchange group chemically bond (4.6-mm inner diameter 250-mm long 3μm particle diameter )2) Column bath temperature: 40 °C
668

Testing Methods for Fertilizers (2018)

3) Eluent (1): Dissolve 1.54 g of ammonium acetate with water to make 1000 mL. Transfer 900
mL of the solution to mix with 100 mL of methanol. Filter with a membrane filter (aperture
diameter: no more than 0.5-µm) made of hydrophilic PTFE.
4) Flow rate: 0.4 mL/min
5) Injection volume: 10 µL
6) Detection unit: An absorptiometric detector, measurement wavelength: 290 nm
Comment 3 Eluent can be prepared as follows. Dissolve 15.4 g of ammonium acetate with water
to make 1000 mL and store in a refrigerator. At the time of usage, dilute apredetermined volume of the solution by a factor of 10 to mix with methanol ofvolume ratio 1/9 and filter with a membrane filter (aperture diameter: no more than
0.5-µm) made of hydrophilic PTFE.
b) Calibration curve preparation
1) Inject 10 µL of respective standard solutions for calibration curve preparation into an HPLC,
record a chromatogram at wavelength 290 nm and obtain the peak area or height.
2) Prepare a curve for the relationship between the uric acid (U-acid) concentration and the peakarea or height at wavelength 290 nm of respective standard solutions for the calibration curve
preparation.
c) Sample measurement
1) Subject 10 μL of sample solution to the same procedure as in b) 1)
2) Obtain the uric acid (U-A) content from the peak area or height using the calibration curve tocalculate the uric acid (U-A) in the analytical sample.
Comment 4 In addition to uric acid, allantoin and allantoic acid can be measured simultaneously
by this measurement method (when using a Scherzo SS-C18 column). Note that thedetection wavelength of allantoin and allantoic acid is 210 nm.
Comment 5 Additive recovery testing was conducted using one brand of a compound fertilizer, acomposted sludge fertilizer, a mixed compost fertilizer and compost. As a result, themean recovery at additive level of 0.1 % (mass fraction), 0.01 % (mass fraction) and
0.005 % (mass fraction) were 92.4 % - 101.8 %, 85.3 % - 105.0 % and 92.5 % 114.1 % respectively.
The results of the repeatability tests on different days using one brand of a compound
fertilizer, a composted sludge fertilizer, a mixed compost fertilizer and compost toevaluate precision were analyzed by one-way analysis of variance. Table 1 shows thecalculation results of intermediate precision and repeatability.
Additionally, the minimum limit of quantification of this testing method is about
0.0008 % (mass fraction).

669

Testing Methods for Fertilizers (2018)
Table 1 Analysis results of the repeatability tests on different days
Test days of
Repeatability
Intermediate precision
2)
4)
5)
6)
7)
repeatability Averagesr
RSD rs I(T)RSD I(T)Name ofsample1)

3)(%)
0.09890.01023)(%)
0.00060.0001

(%)
0.6
0.7

3)

Compound fertilzier

T
7
7

(%)
0.00150.00042

(%)
1.6
4.2

Composted sludge
fertilizer

7

0.93200.00040.5

0.00161.7

70.009380.000090.9

0.000313.3

70.09240.00040.40.0015

1.7

Mixed compost
fertilizer

70.009210.00005
Compost7
0.10100.001
70.00966
0.00018
1） The number of test days conducting a duplicate test2） Average （the number of test days（T ）×the number of duplicate testing（2））3） Mass fraction
4） Repeatability standard deviation
5） Repeatability relative standard deviation

(5)

0.6
0.000323.5
1.3
0.00292.8
1.8
0.00049
5.0
6） Intermediate standard deviation
7） Intermediate relative standard deviation

Flow sheet for testing method: The flow sheet for irea acid in fertilizers is shown below:
1.00 g200-mL ground-in stopper Erlenmeyer flask
analytical sample (powdery)← 100 mL of phosphate solution
Heat at 60 ºC ± 2 ºC for 30 minutes
Heating
while shaking to mix at every 10 minutes
Extraction

Stir to mix, 10 minites

Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
1700 ×g , 10 minutes

Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
8000 ×g - 10000 ×g , 5 minutes

Sample solution

Supernatant

Measurement

Liquid Chromatograph

Figure Flow sheet for uric acid in fertilizers

670

Testing Methods for Fertilizers (2018)

Reference: Chromatogram of the standard solution for calibration curve preparation of uric acid isshown below.

Reference diagram

HPLC chromatogram of the uric acid standard solution (50µg/mL) for calibration curve preparation

Peak name (↓) Uric acid
Measurement conditions for HPLC
Column: Scherzo SS-C18 (4.6-mm inner diameter, 250-mm long, 3-µm particle
diameter)Other conditions are according to the example of HPLC measurement conditionsin (4.2) a)

671

Testing Methods for Fertilizers (2018)

Appendix. The procedure to validate characteristics of testing methods(1) Purposes
This article explains the procedure to validate characteristics of testing methods which will be listed
in the Testing Methods of Fertilizers. In addition, when testing institutes conduct a test which is
not included in the Testing Methods of Fertilizers, a procedure to evaluate the validity of the test
method should conform to a method stipulated in this article.
Additionally, this article targets chemical testing methods. However, this article is not applicable tothe extraction method of the content of effective figures (acid-, alkaline-, citrate- and water-soluble)
in a powdery sample or a solidified fertilizer.
Comment 1 The contents of effective figures (acid-, alkaline-, citrate- and water-soluble) are
stipulated in a notification of the Ministry of Agriculture, Forestry and Fisheries. In
addition, the change of measurement conditions such as an extraction temperature
may affect an observed value in some cases. Therefore, no changes will be
implemented in the extraction method of the contents of effective figures in a
powdery fertilizer and a solidified fertilizer for the present and the application of thisarticle is limited to the change of a measurement method (including refining ofextract, etc.).
(2) Definition of terminology
The definition of terminology in this article is as shown below.
a) Selectivity: Capability to accurately measure components subjected to analysis under the
existence of materials which seem to exist in a sample.
b) Trueness: The degree of agreement between the mean obtained from multiple measurement
results and the true value (1).
c) Precision: The degree of agreement among the independent measurement results which arerepeatedly measured under the determined conditions.
d) Repeatability: The precision of the measurement results of analytical samples, which are
regarded to be all identical, obtained under condition (repeatability conditions) thatindependent measurement results are measured in a short time, using the same method, in thesame laboratory, by the same operator and with the same instrument.
e) Intermediate precision: The precision of a measurement result of analytical samples, which
are regarded to be all identical, obtained under condition (intermediate conditions) thatindependent measurement results are measured, using the same method, in the samelaboratory and in different factors (such as different time and a different operator).
f) Reproducibility: The precision of a measurement result of analytical samples, which are
regarded to be all identical, obtained under condition (reproducibility conditions) thatindependent measurement results are measured, using the same method, in different
laboratories, by different operators and with different instruments.
g) Minimum Limit of Quantification (LOQ): The quantifiable lowest volume or minimum
concentration of a component subjected to analysis which is contained in an analyticalsample.
h) Minimum Limit of Detection (LOD): The detectable lowest volume or minimum
concentration of a component subjected to analysis which is contained in an analyticalsample.
i) Reference material: A material which is uniform and stable enough for one or moreprescribed properties, and is made suitable for the purpose of use in a measurement process.
j) Certified reference material: A reference material, whose values of one or more prescribed
properties are characterized by a reasonable metrological procedure, having a certificate of672

Testing Methods for Fertilizers (2018)attestation on which the characteristics of prescribed properties and their uncertainty andmetrological traceability are stated.
k) Blank sample: An analytical sample not containing components subjected to analysis (2).
l) Addition sample: An analytical sample the content of whose components subjected toanalysis is known, or an analytical sample to which reference materials are added (3) (4) orcompounded (3).
m) Natural contamination sample: A test sample prepared from fertilizers which naturally
contain the components subjected to analysis such as harmful components.
n) Distribution sample: An analytical sample prepared from fertilizers (5) which are
manufactured in a fertilizer production factory, etc.
o) Surrogate: A material which is added to an analytical sample in order to conduct a
pre-process operation, correct yields in respective steps of measurement procedure andconfirm recovery, whose chemical structure is identical or similar to a target component.
p) SN ratio: Intensity ratio of a signal (response value) S originating from the analysis target anda signal (usually noise) N based on the other factors.
Note (1) In reality, the certified value of a certified reference material, the chemical compositionof a compound, the added content of a reference material, etc. and others.
(2) Reagents, etc. containing a target matrix can be used in the case that there is no
distribution fertilizer used as a blank sample for a recovery test and the confirmationof the minimum limit of quantification, etc.
(3) Mix a component subjected to analysis with a mortar, etc. to sufficient uniformity(4) In the case of adding a standard solution, vaporize the solvent sufficiently conducting
measures such as letting it stand for one night.
(5) A fertilizer containing components subjected to analysis whose formation or formchanged due to a chemical or physical process (a granulation process, etc.).
References
1) JIS K 0211： Technical terms for analytical chemistry (General part) (2013)
2) JIS K 0214： Technical terms for analytical chemistry (Chromatography part) (2013)
3) JIS Q 0035： Reference materials−General and statistical principles for certification (2008)
4) JIS Z 8101-2： Statistics−Vocabulary and symbols−Part 2: Statistical quality control terms(1999)
5) JIS Z 8402-1： Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions (1999)
6) ALINORM 09/32/23 Joint FAO/WHO Food Standards Prorgamme: Repot of the Thirtieth
Session of the Codex Committee on Methods of Analysis and Sampling, Codex
Alimentarius Comission Thirty-second Session (2009)
7) ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text andMethodology Q2(R1), International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) (2005)(3) Validation method
Test necessary items of (3.1) to (3.8) in a planned manner and estimate performance parametersfrom the obtained results.
Confirm whether the estimated values of performance parameters are suitable to target values(performance norm) respectively, and evaluate that the test method is validated if they are all
suitable.
673

Testing Methods for Fertilizers (2018)(3.1) Scope of application
As a result of a validation test in a single laboratory and a collaborative study, if the result issuitable up to reproducibility, the test method is evaluated as a validated test method as far as thekind of a fertilizer used in the test and the range of concentration are concerned. Therefore, alaboratory where the said test is conducted can use the performance (reproducibility, etc.) as avalidated method through implementing internal quality control, etc.
As a result of a validation test in a single laboratory, if the result is suitable to trueness,
repeatability and intermediate precision, etc., the test method is evaluated as a validated test method
as far as the laboratory where the test was conducted and as far as the kind of a fertilizer used in thetest and the range of concentration are concerned. Therefore, another laboratory which wants tointroduce the test method is required to carry out the validation anew in an individual laboratory
with the above test method.
(3.2) Selectivity(3.2.1) Case of Chromatography
Conduct a procedure for a blank sample and confirm that there is no peak (interference peak)which affects the measurement of components subjected to analysis (6). In addition, in the case of
the simultaneous measurement of multi components, confirm that adjacent peaks are sufficiently
separated (6).
Note (6) Resolution (R) should be 1.0 or more at minimum though 1.5 or more is preferable.
Comment 2 Resolution (R) is used as a separation indicator of peaks. If Resolution (R) is 1.5 or
more, the adjacent two peaks are sufficiently separated and they do not affect ameasurement, whether a peak height or a peak area is used. If Resolution (R) is 1.0 or
more, the adjacent two peaks are sufficiently separated and they do not affect ameasurement, whether a peak height or a peak area is used.
Resolution (R) can be obtained using a peak width by the formula (1a). In addition, if
the peak is a normal distribution, it can be obtained using a peak width at half heightby the formula (1b). With the data processing device of a chromatograph, the formula(1b) is often used to obtain Resolution (R).

Resolution ( ) =Resolution ( ) =

−

1
(
2×

1.18 × (,･･･(1a)
+

+

−

)
,

： Retention time of Peak 1： Peak widh of Peak 1,,)･･･(1b)： Retention time of Peak 2： Peak width of Peak 1： Peak width at half height of Peak 1： Peak width at half height of Peak 2674

Testing Methods for Fertilizers (2018)(3.2.2) Case of a method other than Chromatography (7)Conduct a procedure for a blank sample and confirm that there is no response which originatesfrom other components than a component subjected to analysis and can be a factor of positive errorof a quantification value (8).
Note (7) A test method such as Molecular absorption spectrometry, Atomic absorption
spectrometry or Titration analysis which does not isolate with a measurement
instrument.
(8) Absorbance, titer, etc.
References
1) AOAC Official Methods of Analysis Appendix K: Guidelines for Dietary Supplements
and Botanicals, AOAC INTERNATIONAL (2012)
2) JIS K 0114： General rules for gas chromatography (2012)
3) JIS K 0124： General rules for high performance liquid chromatography (2011)
4) The notification by the Director of Evaluation and Licensing Division, Pharmaceuticaland Food Safety Bureau, the Ministry of Health, Labour and Welfare: “Guideline on
Bioanalytical Method Validation in Pharmaceutical Development”, July 11, 2013,
Yaku-shoku-Sinsa-Hatsu-0711 No.1 (2013)(3.3) Calibration curve
Measure respective standard solutions for the calibration curve preparation of the concentrationor the content (9) of level 6 to 8 a few times (10) to make a figure plotting the obtained signals (11) as afunction of the concentration or the content of a component subjected to analysis and evaluate its
linearity visually using the figure.
If linearity is recognized, calculate the inclination (b) and the intercept (a) of a calibration curve,
its confidence interval and the coefficient of determination (r2) using a statistical method such as thecalculation of a regression equation by the least square method. Moreover make the plot of residuals(12)
in respective levels.
Note (9) The blank test solution for the calibration curve preparation can be included.
(10) In order to avoid nonlinear confusion due to the variation of sensitivity, etc., conduct
measurements randomly for each replicate determination.
(11) Absorbance, fluorescence intensity, peak height, peak area, etc.
(12) The difference between a signal obtained by measurement and a signal estimated usinga regression equation.
Comment 3 It is recommended that the 95% confidence interval of an intercept (a) includes theorigin (0).
Comment 4 Though it is usable if the coefficient of determination (r2) is 0.99 or more, it isrecommended that the coefficient of determination (r2) is 0.999 or more for a precise
analysis. If it is less than 0.99, use the equation of a higher order or study theconversion of a numerical value.
Comment 5 The mean of residuals is 0 and the residuals indicate a random pattern.
References
1) AOAC Official Methods of Analysis Appendix K, Guidelines for Dietary Supplements
and Botanicals, AOAC INTERNATIONAL (2012)
2) Thompson, M., Ellison, S.L.R, Wood, R., Harmonized guidelines for
single-laboratoryvalidation of methods of analysis, Pure & Appl. Chem. 74 (5), 835-855
675

Testing Methods for Fertilizers (2018)(2002)
3) CLSI EP9 A2 Ed. 2, Method Comparison and Bias Estimation Using Patient Samples,
Clinical and Laboratory Standards Institute (2002)(3.4)
Trueness
As the estimation method of trueness, the methods are recommended in the following order. (1)Use of a certified reference material (3.4.1), (2) Comparison with an observed value by a validated
method (3.4.2) and (3) Recovery test (3.4.3).
In addition, if a surrogate is used, it is recommended that a recovery is about 40% or more.
(3.4.1) Use of a certified reference materialWith regard to a component which has matrixes similar to a fertilizer subjected to test and can use
a certified reference material containing components subjected to measurement of the concentration
in a measurement level, conduct repeatability testing using 3 or more analytical samples (n)according to the test method of the certified reference material. As a result, the mean of theobserved values should be within the warning level to the certified value (characteristic value) or
the absolute value of the difference between the mean of the observed values and the certified value(characteristic value) should not exceed 2 times of the standard uncertainty composed of respective
uncertainties of the mean of the observed values and the certified value (13).
Comment 6 A warning limit is given using the formula (2) which is obtained from a collaborative
study for the characterization of a certified reference material.
A warning limit for a certified value ( )
=

±2× (

−

)+

=

±2×

+

･･･(2)µ： Certified value： Reproducibility standard deviation in a collaborative study： Repeatability standard deviation in a collaborative study (14)n： The number of analytical samples to repeatability test
： Pure between-laboratory standard deviation in a collaborative study
Note (13) The evaluation procedure of the difference between a measurement result and acertified value (characteristic value) is shown in Reference 1 Procedure to compare
an observed value and a certified value.
(14) It may be expressed as within-laboratory standard deviation ( ) in some cases.
(3.4.2) Use of another validated test method
For a component for which a certified reference material is not usable but another validated test
method (hereinafter referred to as “a standard test method”) is applicable, confirm that the conditiona) or b) is satisfied.
a) In case 12 or more samples are available: Conduct respective tests of 12 or more test
samples composed of addition samples, natural contamination samples or distribution samples
according to a new test method and a standard test method, create the correlation chart of676

Testing Methods for Fertilizers (2018)

observed values with two methods for each sample and calculate the inclination (b) and theintercept (a) of a regression line, and a correlation coefficient (r). Further confirm a prediction
interval.
However, in case that the width between the minimum and the maximum observed value issmall, conduct the paired samples t-test to confirm that a significant difference is notobserved.
Comment 7 It is recommended that the 95% confidence interval of an inclination (b) includes 1,
the 95% confidence interval of an intercept (a) includes the origin (0) and thecorrelation coefficient (r) is no less than 0.99.
b) In case fewer samples are available: With regard to 3 or more test samples of different
concentration, conduct respective repeatability addition tests using 4 analytical samples
according to the new test method and a standard test method, confirm the homoscedasticity of
the results of 2 groups and conduct a t-test for each concentration to confirm that significant
difference is not observed under the two-sided significant level of 5%.
(3.4.3) In case neither certified reference material nor other validated test methods are usable
For 3 or more test samples of different concentration, conduct respective repeatability tests using
3 analytical samples and evaluate by obtaining the recovery using the mean of the observed values.
The criteria of the trueness are shown in Separate sheet: The target of trueness and the criteria
of precision in respective concentration levels.
References
1)
AOAC Official Methods of Analysis Appendix K: Guidelines for Dietary Supplements
and Botanicals, AOAC INTERNATIONAL (2012)2)
Thompson, M., Ellison, S.L.R, Wood, R.,: Harmonized guidelines for
single-laboratoryvalidation of methods of analysis, Pure & Appl. Chem. 74 (5), 835-855(2002)
3)
Linsinger, T.,: Comparison of a measurement result with the certified value, European
Reference Materials' application note 1, European Commission - Joint Research Centre
Institute for Reference Materials and Measurements (IRMM) (2010)
4)
Joint FAO/WHO Food Standards Programme: Procedural manual Twenty-second
edition, Codex Almentarius Comission (2013)5)
ICH Harmonised Tripartite Guideline: Validation of Analytical Procedures: Text andMethodology Q2(R1), International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) (2005)(3.5) Precision
Evaluate reproducibility and repeatability by a collaborative study (3.5.1). Or evaluate anintermediate precision and repeatability by a repeatability test (3.5.2).
(3.5.1) Reproducibility and repeatability by a collaborative study
The number of laboratories to obtain effective data should be 8 or more (15). Conduct undisclosed
duplicate collaborative studies for 5 or more kinds of samples with different concentration. Obtainreproducibility and repeatability from the observed values (16) to evaluate.
Criteria to evaluate these precisions are shown in Separate sheet: The target of trueness andthe criteria of precision in respective concentration levels.
Note (15) In case the number of laboratories which have required facility/instruments is limited,677

Testing Methods for Fertilizers (2018)

this should be 5 or more.
(16) The calculation method is shown in Reference 2: Calculation of reproducibility orintermediate precision and repeatability(3.5.2) Intermediate precision and repeatability by a repeatability test in a single laboratory
on different days
Conduct a duplicate test (17) per test day for 5 to 7 days using two analytical samples of different
concentration which is included in a normal range (18). Obtain intermediate precision andrepeatability from the observed values (19) to evaluate.
Criteria to evaluate these precisions are shown in Separate sheet: The target of trueness andthe criteria of precision in respective concentration levels.
Note (17) The data of internal quality control can be used.
(18) It is not necessary for the same tester to conduct a test through 5 to 7 days.
(19) The calculation method is shown in Reference 2: Calculation of reproducibility orintermediate precision and repeatability
References
1) AOAC Official Methods of Analysis Appendix K: Guidelines for Dietary Supplements
and Botanicals, AOAC INTERNATIONAL (2012)
2) Thompson, M., Ellison, S.L.R, Wood, R.,: Harmonized guidelines for
single-laboratoryvalidation of methods of analysis, Pure & Appl. Chem. 74 (5), 835-855(2002)
3) AOAC Official Methods of Analysis Appendix D: Guidelines for Collaborative Study
Procedures To Validate Characteristics of a Method of Analysis, AOAC
INTERNATIONAL (2005)4) Horwitz ， W.: Protocol for the Design ， Conduct and Interpretation of
Method-Performance Studies，Pure & Appl. Chem.，67 (2), 331 - 343 (1995)(3.6) Minimum Limit of Quantification (LOQ)
Estimate Minimum Limit of Quantification according to (3.6.1) to (3.6.3). Prepare test samples
which include the concentration estimated to be near the Minimum Limit of Quantification step bystep as necessary. And conduct respective repeatability tests using 3 analytical samples and define
the concentration of a prepared test sample as the Minimum Limit of Quantification, where themean of the obtained values using the prepared test sample is suitable to the target value of trueness.
Comment 8 In case permissible content and equivalent level is 1.0 mg/kg or more, the Minimum
Limit of Quantification (LOQ) of harmful components and restricted components,
etc. should be no more than 1/5 of the permissible content and equivalent level. In
case permissible content and equivalent level is no more than 1.0 mg/kg, theMinimum Limit of Quantification (LOQ) should be no more than 2/5 of thepermissible content. Moreover, it is recommended that the Minimum Limit of
Quantification of main components/major components and material components
should be no more than 1/5 of minimum volume to be contained and the minimum
content of a distribution fertilizer. In addition, in case the Minimum Limit of
Quantification exceeds 1/5 of these minimum volumes, conduct the above-mentioned
repeatability test, confirm the Minimum Limit of Quantification and state clearly thefact in the applicable range of a test method.
Comment 9 There are some methods to estimate Minimum Limit of Quantification. The methods
differ depending on whether they are based on an instrument analysis or not and678

Testing Methods for Fertilizers (2018)

depending on instruments used. A method different from the methods shown in(3.6.1) to (3.6.3) is allowed. However, the definition of a method and Minimum
Limit of Quantification by the method should be clearly stated.
(3.6.1) Estimation method by a repeatability test
With regard to a test sample with concentration near Minimum Limit of Quantification, conduct a
repeatability test using 7 to 10 analytical samples, obtain repeatability standard deviation andestimate Minimum Limit of Quantification (LOQ) in an analytical sample by the formula (3).
Estimated value of Minimum Limit of Quantification (LOQ) = 10 ×・・・(3)： Repeatability standard deviation(3.6.2) Estimation method using a calibration curve
In case a calibration curve is linear, estimate Minimum Limit of Quantification (LOQ) in ananalytical sample by the formula (4) using the standard deviation of the residuals of a calibration
curve or estimated signals in concentration 0 and the inclination of a calibration curve.
Estimated value of Minimum Limit of Quantification (LOQ) =10 ×・・・(4)： The standard deviation of residuals. Or the standard deviation of signasls inconcentration 0, which are estimated from a regression line
： The inclination of a calibration curve(3.6.3) Estimation method using an SN ratio
In a test method such as Chromatography, etc. which has a baseline noise, calculate from a
concentration in an analytical solution whose SN ratio is 10 to 1 at the peak and estimate Minimum
Limit of Quantification (LOQ) in an analytical sample.
References
1)
ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text andMethodology Q2(R1), International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) (2005)
2)
The notification by the Director of Evaluation and Licensing Division, Pharmaceuticaland Food Safety Bureau, the Ministry of Health, Labour and Welfare: The text
(operation method) on Bioanalytical Method Validation”, October 28, 1997, Iyaku-Shin
No. 338 (1997)(3.7) Minimum Limit of Detection (LOD)
Estimate Minimum Limit of Detection according to (3.7.1) to (3.7.3).
Comment 10 There are some methods to estimate Minimum Limit of Detection. The methods
differ depending on whether they are based on an instrument analysis or not anddepending on instruments used. A method different from the methods shown in(3.7.1) to (3.7.3) is allowed. However, the definition of a method and Minimum
679

Testing Methods for Fertilizers (2018)

Limit of Detection by the method should be clearly stated.
(3.7.1) Estimation method by a repeatability test
With regard to a test sample or a blank sample with concentration near Minimum Limit of
Quantification, conduct repeatability tests using 7 to 10 analytical samples, obtain repeatability
standard deviation and estimate Minimum Limit of Detection (LOD) in an analytical sample by theformula (5).
Estimated value of Minimum Limit of of Detection (LOD)in an analytical sample= 2 × ( − 1, 0.05) ×・・・(5)： Repeatability standard deviation( − 1, 0.05): The Student value of Significance Level 5% (one side) (20)： The number of analytical samples in a repeatability test

Note (20) In case of a repeatability test using 7 analytical samples, the value is 1.94. In case ofusing 10 analytical samples, the value is 1.83.
(3.7.2) Estimation method using a calibration curve
In case a calibration curve is linear, estimate Minimum Limit of Detection (LOD) in an analyticalsample by the formula (6) using the standard deviation of the residuals of a calibration curve or
estimated signals in concentration 0 and the inclination (b) of a calibration curve.
Estimated value of Minimum Limit of of Detection (LOD)=

2 × ( − 2, 0.05) ×・・・(6)： The standard deviation of residuals. Or the standard deviation of signasls inconcentration 0, which are estimated from a regression line
： The inclination of a calibration curve( − 2, 0.05)： The Student value of Significance Level 5% (one side)： The number of a measurement point on a calibration curve(3.7.3) Estimation method using an SN ratio
In a test method such as Chromatography, etc. which has a baseline noise, calculate from a
concentration in an analytical solution whose SN ratio is 3 to 1 at the peak and estimate Minimum
Limit of Detection (LOD) in an analytical sample.
References
1)
ICH Harmonised Tripartite Guideline: Validation of Analytical Procedures: Text andMethodology Q2(R1), International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) (2005)
2)
The notification by the Director of Evaluation and Licensing Division, Pharmaceuticaland Food Safety Bureau, the Ministry of Health, Labour and Welfare: The text
(operation method) on Bioanalytical Method Validation”, October 28, 1997, Iyaku-Shin
680

Testing Methods for Fertilizers (2018)

No. 338 (1997)(3.8) Robustness
Robustness should be studied when an analysis method is developed, and the estimation method
depends on the type of analysis method to be developed. Robustness expresses the reliability of ananalysis method when its analysis conditions are intentionally changed. If an observed value tends
to be easily affected by the variation of an analysis condition, it is necessary to consider a method tocontrol an analysis condition appropriately or to state the fact as a precaution in a testing method.
The evaluation of robustness enables the establishment of a series of parameters such as Resolution
related to system conformance. Similarly, the confirmation of these parameters ensures that thevalidation of an analysis method is maintained in a daily analysis.
Typical variation factors are as follows.
(3.8.1) Common variation factors: Typical variation factors common to various kinds of test
methods are as follows.
a) Extraction time, extraction temperatureb) Stability of a test solution in respective stepsc) Reagent’s grade(3.8.2) Variation factors in Chromatography, etc.: Typical variation factors of measurements by
Chromatography or refining by solid phase extraction are as follows.
a) Change of a column or a cartridge (A different lot or a different brand)b) Influence by the variation of pH and composition of an eluent or a washc) Temperatured) Flow ratee) Influence of a matrix and effect of dilution
References
1)
ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text andMethodology Q2(R1), International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) (2005)
2)
The notification by the Director of Evaluation and Licensing Division, Pharmaceuticaland Food Safety Bureau, the Ministry of Health, Labour and Welfare: The text
(operation method) on Bioanalytical Method Validation”, October 28, 1997, Iyaku-Shin
No. 338 (1997)
3)
Thompson, M., Ellison, S.L.R, Wood, R., : Harmonized guidelines for
single-laboratoryvalidation of methods of analysis, Pure & Appl. Chem. 74 (5), 835-855(2002)

681

Testing Methods for Fertilizers (2018)

Reference 1:

Procedure to compare an observed value and a certified value

Obtain the total mean (m) of the replication test results and the certified value (μ), and theabsolute value ( ) of the difference of the two values by the formula (R1.1). Next, obtain thestandard uncertainty () of the certified value of a certified reference material by the formula(R1.2), and obtain the standard uncertainty ( ) of the total mean by the formula (R1.3). Calculate
the combined standard uncertainty ( (∆ ) ) of
by the formula (R1.4) using the obtainedand. Further, calculate an expanded uncertainty ( ∆ ) by the formula (R1.5) using thecoverage factor ( = 2).
Compareand ∆ to confirm that the criterion (the formula (R1.6)) is satisfied, that is,is no more than ∆ .
The absolute value ()

of the difference of the total mean of repeatability test
results and a certified value= | − |・・・(R1.1)
The standard uncertainty () of the certified value =%The standard uncertainty of the measurement of a total mean (The combined standard uncertainty
The expanded uncertainty
Criterion

≦

∆

∆

of

・・・(R1.6)

(∆ )

=

of

(∆ ) ×=+(∆ ) = 2 ×・・・(R1.2)

)=

√

(∆ )

・・・(R1.3)
・・・(R1.4)
・・・(R1.5)： The total mean of observed valuesµ： A certified value%：

The expanded uncertainty of a certified value： The coverage factor of an expanded uncertainty of a standard reference material： Repeatability standard deviation： The number of repeatability test samples( (∆ ) = 2)(∆ ) ： The coverage factor of an expanded uncertainty of682

Testing Methods for Fertilizers (2018)

Reference 2:

Calculation of reproducibility or intermediate precision and repeatability(1) Structure of an observed value
An observed value (xij) in Table 1, as is shown in the formula (R2.1), consists of a true value (μ), a
variation (β) by a factor and a variation (e) by an accidental error under repeatability conditions(hereinafter referred to as “an accidental error”). When p laboratories conduct a collaborative study
in which respective laboratories conduct repeatability tests using n samples, the formula (R2.2) isintroduced on the assumption that the distribution of β is equivalent to N(0,) which depends ona pure between-laboratory variation and the distribution of e is equivalent to N(0,) whichdepends on an accidental error. In addition, when the same laboratory conducts replicate tests for p
days using n samples on respective test days, the formula (R2.3) is introduced on the assumptionthat the distribution of β is equivalent to N(0, ( ) )which depends on test days variation (factor T)
and the distribution of e is equivalent to (0,Ovserved value= ＋ ＋

Ovserved value= ＋

Ovserved value

= ＋

0,

： Variation of a factor
0,＋・・・ (R2.1)

0, ( ) ＋： True value0,) which depends on an accidental error.

： Normal distribution of： Normal distrubution of0,0,( ) ： Test days variance・・・ (R2.3)： Accidental error

with the mean 0 and standard deviation

with the mean 0 and standard deviation： Pure between − laboratory variance0, ( ) ： Normal distribution of・・・ (R2.2)： Repeatability variancewith the mean 0 and standard deviation

683

( )

Testing Methods for Fertilizers (2018)
Table 1 Test results of collaborative studies or repeatability tests on different days
Laboratory or test day
Analytical sample number
123j(Factor)
･･･
･･･
1
2
3
･･･
i
･･･
p

x 11
x 21
x 31
･･･
xi1
･･･
xp1

x 12
X 22
X 32
･･･
X i2
･･･
X p2

x 13
x 23
x 33
･･･
xi3
･･･
xp3

･･･
･･･
･･･
･･･
･･･
･･･
･･･

x 1j
x 2j
x 3j
･･･
x ij
･･･
x pj

･･･
･･･
･･･
･･･
･･･
･･･
･･･

n
x 1n
x 2n
x 3n
･･･
x in
･･･
x pn

(2) Calculation procedure of reproducibility and repeatability of the results of acollaborative study(2.1) Estimation of true value and variance
In an actual statistical analysis, a true value (μ), a true and pure between-laboratory variance( ) and a true repeatability variance ( ) are unknown. Therefore, they are replaced withestimated values obtained from the results of a collaborative study and are expressed as a mean (m),a pure between-laboratory variance ( ) and a repeatability variance ( ) respectively.
(2.2) One-way analysis of varianceExclude ineffective observed values which have clearly objective reasons such as deviation froma protocol and malfunction of instruments from the report values by laboratories which participatedin a collaborative study. Further exclude outliers by conducting Cochran’s test and Grubb’s test.
And conduct one-way analysis of variance for the remaining results to obtain the unbiased variance(V) of respective variation factors in Table 2.

Variation factor
Between-laboratory(L)Accidental error (e)Table 2 Table of one-way analysis of variance
Unbiased variance
Sum ofDegree of( )squares
freedom−1× ( − 1)

Expectation of variance

+

( )

×

Comment 1 It is possible to conduct one-way analysis of variance easily using a statisticalprogram or a tool of a spreadsheet program. In this case, it should be noted thatdifferent terminologies may be used (Between-laboratory (L)→ Between-group,
Accidental error (e) → Within-group, Unbiased variance→ Mean square, etc.).
Comment 2 Unbiased variance (V) is calculated by (Sum of squares)/ (Degree of freedom).
(2.3) The calculation of reproducibility and repeatability
The relation of the expectation of variance E (V) of respective factors in Table 2 holds true.
Therefore, calculate repeatability variance (sr2) and pure between-laboratory variance (sL2) by theformula (R2.4) and (R2.5), and further calculate reproducibility variance (sR2) by the formula (R2.6)(1) (2).
684

Testing Methods for Fertilizers (2018)

Repeatability variance () =・・・ (R2.4)−
Pure between − laboratory variance ( ) =Reproducibility variance () =・・・ (R2.5)

・・・ (R2.6)＋： The unbiased variance of a variation factor (accidental error (e))
in the table of one − way analysis of variance (Table 2)： The unbiased variance of a variation factor (between − laboratories (L))
in the table of one − way analysis of variance (Table 2)

Calculate a repeatability standard deviation ( ) and a reproducibility standard deviation ( ) by
the formula (R2.7) and (R2.8) using the obtained repeatability variance and reproducibility variance,
and further calculate a repeatability relative standard deviation (RSDr)and a reproducibility relative
standard deviation (RSDR) by the formula (R2.9) and (R2.10) (2) (3).
Repeatability standard deviation ( ) =Reproducibility standard deviation ( ) =Repeatability relative standard deviation (Reproducibility relative standard deviation (・・・ (R2.7)

・・・ (R2.8), %) =, %) =

× 100

× 100

・・・ (R2.9)

・・・ (R2.10)： the total mean ( )of the effective data of collaborative study results

In case<, assume=(that is, the pure between-laboratory variance( )= 0 in the formula (R2.5)) and let the formula (R2.6) form=.
(2) The rounding of a numerical value is not executed in the middle of the calculation.
(3) The mean and the standard deviation are expressed rounding to the digit of theobserved value. The relative standard deviation is expressed rounding to the firstdecimal place.

Note (1)(3) Calculation procedure of intermediate precision and repeatability by the replicate test
results on different days
(3.1) Estimation of a true value and a variance
In an actual statistical analysis, a true value (μ), a true test day variance (σ(T)2) and a true
repeatability variance (σr2) are unknown. Therefore, they are replaced with estimated values
obtained from the eplicate test results on different days and are expressed as a mean (m), test days
variance (s(T)2) and a repeatability variance (sr2) respectively.
(3.2) One-way analysis of variance
Conduct one-way analysis of variance for the replicate test results on different days to obtain the685

Testing Methods for Fertilizers (2018)

unbiased variance (V) of respective variation factors in Table 3.

Variation factor
Test days (T)Accidental
error(e)Table 3 Table of one-way analysis of variance
Unbiased variance
Expectation of variance
Sum ofDegree of( )( )squares
freedom+ × ( )
−1× ( − 1)

Comment 3 It is possible to conduct one-way analysis of variance easily using a statisticalprogram or a tool of a spreadsheet program. In this case, it should be noted thatdifferent terminologies may be used (Test days (T) → between-group, Accidental
error (e) → within-group, Unbiased variance → Mean square, etc.).
Comment 4 Unbiased variance (V) is calculated by (Sum of squares)/ (Degree of freedom).
(3.3) The calculation of intermediate precision and repeatability
The relation of the expectation of variance E (V) of respective factors in Table 3 holds true.
Therefore, calculate repeatability variance ( ) and test days variance ( ( ) 2) by the formula(R2.11) and (R2.12), and further calculate intermediate variance ( ( ) ) by the formula (R2.13) (2)(4).
Repeatability variance (Test days variance( )Intermediate Variance

) =
=

( )

・・・ (R2.11)−=

・・・ (R2.12)

・・・ (R2.13)( ) ＋： The unbiased variance of a variation factor (accidental error (e))
in the table of one − way analysis of variance (Table 3)： The unbiased variance of a variation factor (test days (T))
in the table of one − way analysis of variance (Table 3)
Calculate a repeatability standard deviation ( ) and an intermediate standard deviation ( ( ) ) by
the formula (R2.14) and (R2.15) using the obtained estimated values of repeatability variance andintermediate variance, and further calculate a repeatability relative standard deviation (RSDr) and anintermediate relative standard deviation (RSDI(T)) by the formula (R2.16) and (R2.17) (2) (3).
Repeatability standard deviation ( ) =Internediate standard deviation

( )

=

686

( )

・・・ (R2.14)

・・・ (R2.15)

Testing Methods for Fertilizers (2018)

Repeatability relative standard deviation (Internediate relative standard deviation, %) =( ), %

=

× 100

× 100

・・・ (R2.16)

・・・ (R2.17)： Total mean of the replicate test results on different days

Note (4) In case<assume=(that is, the test days variance ( ( ) ) in theformula (R2.12) = 0) and let the formula (R2.13) form ( ) =.

(4) Examples of the calculation of intermediate precision and repeatability by the replicate
test results on different days.
An example of repeatability test results on different days of citric acid-soluble phosphoric acidusing sample 1 and sample 2 containing phosphite is shown in Table 4. Conduct one-way analysis
of variance for the test results of respective samples to obtain the unbiased variance (V) ofrespective variation factors (Table 5).
Examples of the calculation of intermediate precision and repeatability for the sample 1 andsample 2 using the formula (R2.11) to the formula (R2.17) are shown in Table 6-1 and 6-2. In
addition, the results of respective standard deviation are expressed rounding to the digit of theobserved value and the results of the respective relative standard deviations are expressed rounding
to the first decimal place.
Table 4 Example of repeatability test results on different days

(mass fraction (%))

Test day (factor)
Sample

1

2

3

4

Total
5

6

7)

mean
( )1)

No.
Sample 1
Sample 2

51.20

52.15

51.00

51.35

51.35

51.38

51.28

51.45

51.85

51.09

51.28

51.10

51.38

51.43

5.18

4.90

5.01

5.15

5.14

5.13

5.21

5.00

5.12

5.06

5.14

5.07

5.11

5.18

1) The mean is expressed rounding to the digit of the observed value.

687

51.38
5.10

Testing Methods for Fertilizers (2018)
Table 5

Table of one-way analysis of varianceSampleVariation factor

No.
Teat days (T)Sample 1

Accidental
error (e)Test days (T)Sample 2

Accidental
error (e)Unbiased variance

Sum ofDegree ofsquares

freedom

1.05706

0.176160.125370.017890.04786

0.00797

0.044870.00640( )Expectation of variance
( )

+2×

( )

+2×

( )

Table 6-1 Calculation of intermediate precision and repeatability using the sample 1 replicate test results on
different days1)
Variation factor

Unit

Repeatability variance()

Repeatability standard

Mass

2)

Deviation ( )fraction (%)

Repeatability relative
3)

standard deviation(
Test days variance( )Intermediate varianceIntermediate standard
Deviation

( )

2)

)

( )

%

Mass

Intermediate relative
deviation

%
( )

3)

Calculation== 0.01789== √0.01789==

fraction (%)

standard

Formula

× 100
−

=

( ) ＋

=

( )

=

( )

× 100

=
=

0.1338× 100
51.38

0.17616 − 0.01789
2= 0.07914＋0.01789= √0.09703=

0.3115× 100
51.381) The rounding of a numerical value is not executed in the middle of the calculation.
2) The standard deviation is expressed rounding to the digit of the observed value.
3) The relative standard deviation is expressed rounding to the first decimal place.

688

Result0.01789
0.13
0.3

0.079140.09703
0.31
0.6

Testing Methods for Fertilizers (2018)
Table 6-2 Calculation of intermediate precision and repeatability using the sample 2 replicate test results on
different days1)
Variation factor

Unit

Repeatability variance()

Repeatability standard
2)

Deviation ( )Mass fraction(%)

Repeatability relative
standard deviation

%

3)

(

)

Test days varianceIntermediate varianceIntermediate standard
Deviation( )2)

Mass fraction(%)

Intermediate relative
standard
deviation

%
( )

3)

Footnote: Refer to Table 6-1

Calculation== 0.00640== √0.00640

=
=

( )

( )

Formula

=
=
=

× 100
−

=

( ) ＋
( )
( )

=

× 100

689

0.0800× 100
5.10

0.00797 − 0.00640
2= 0.00078＋0.00640

Result0.006400.08
1.6
0.00078
0.00718= √0.00718

0.08

=

1.7

0.0848× 100
5.10

Testing Methods for Fertilizers (2018)

Separate sheet: The target of trueness and the criteria of precision in respective concentrationlevels
The target of trueness (recovery) and the criteria of precision in respective concentration levels toevaluate Chromatography (1) and test methods other than Chromatography are shown in Table 1 andTable 2. The target of trueness is generally within the recovery of Table 1. As for precision, thepermissible level may exceed them by a factor of 2.0.
Note (1) Gas chromatography, Gas Chromatography/Mass Spectrometry, High-Performance
Liquid Chromatography, High-Performance Liquid Chromatography/Tandem Mass
Spectrometry, Ion Chromatography, etc.
Table 1

The target of trueness in respective concentration levels
Chromatography

Test methods other than
Chromatography

Concentration level

Recovery (%)

Recovery (%)≧25% (mass fraction)

90 - 10898 - 102≧10% (mass fraction)

90 - 10897 - 103≧1% (mass fraction)

85 - 110

96 - 104≧0.1% (mass fraction)

85 - 110

94 - 106≧100 mg/kg

80 - 115

92 - 108≧10 mg/kg

70 - 120

90 - 110≧1 mg/kg

70 - 120

85 - 115≧100 μg/kg

70 - 120

85 - 115≧10 μg/kg

70 - 120

80 - 120<10 μg/kg

60 - 125

75 - 125

690

Testing Methods for Fertilizers (2018)

Table 2 Criteria of precision 1) in respective concentration levels
Chromatography

Test methods other than Chromatography

Reproducibilit

Intermediate

Reproducibilit

Reproducibilit

Intermediate

Reproducibil

y relative

relativey relative

y relative

relative

ity relative

standard

standard

standard

standard

standard

standard

deviation

deviation

deviation (%)

deviation

deviation

deviation

(%)

(%)

(%)

(%)

(%)

8

6.5

4

2.5

2

1

8

6.5

4

3

2.5

1.5

8

6.5

4

4

3.5

2

8

6.5

4

6

4.5

3

≧100 mg/kg

8

6.5

4

8

6.5

4

≧10 mg/kg

11

9

6

11

9

6

≧1 mg/kg

16

13

8

16

13

8

≧100 μg/kg

22

18

11

22

18

11

≧10 μg/kg

22

18

11

22

18

11

<10 μg/kg

22

18

11

22

18

11

Concentration
level

≧25 %

(mass
fraction)≧10 %(mass fraction)≧1 %(mass fraction)≧0.1 %(mass fraction)

1) As for precision, the permissible level may exceed them by a factor of 2.0.

691