Ms. Shamrikova Elena, Eco-analytical Laboratory, Department of Soil Science, Institute of Biology of Komi Scientific Center of the Ural Branch, Russian Federation

1. An innovative approach to the
harmonization of GLOSOLAN SOPs
Ms. Shamrikova Elena
Department of Soil Science, Eco-analytical Laboratory,
Institute of Biology of Komi Scientific Center of the Ural Branch
Russian Federation

2. Introduction
• Integration of planetary knowledge in the historical
period about soil carbon is especially relevant in the era
of global warming
• However, the presence of different scientific schools,
which have different, often incomparable results, hinders
the creation of common databases and the development
of knowledge about soils
• A tool for international communication that facilitates
contacts between specialists in the creation of databases
on the world's soils, inventory and monitoring of soil
resources is the harmonization of the method for
measuring soil indicators
2

3. Introduction
• Methods for measuring the content of organic matter in
soils are still not unified*
In Russia, as well as in a number of countries in Europe and Asia, the method of I. Tyurin is
widely used. In the USA, Canada, Australia and other countries, the method of A. Walkley
and I. Black is used in practical soil science
• This makes it difficult to discuss the results of soil studies
in different countries, preventing the generalization of
information into unified data sets
*Orlov D.S. Debatable problems of modern soil chemistry // Eurasian Soil Science, 2001. No 3. P. 336-341.
3

4. Measurement steps
Tyurin method
(Institute of Biology)
- Sieve, d < 2 mm
- 20g K2Cr2O7 + 0,5L H2O, c(K2Cr2O7)= 0,136 M
- “Chromium mixture”: 1V K2Cr2O7+1V H2SO4 conс
c(K2Cr2O7)= 0,068 M
- Soil+10 сm3 “Chromium mixture” (5 сm3 +5 сm3)
added to the sample n(K2Cr2O7)= 0,68 mmol
- Heating in a water bath (t = 100 °С for 60 min)
- +15 см3 H2О
- Centrifugation 6000 rpm for 10 min
- Measure the optical density  = 590 nm
- Сalculation of %Сorg
oxidation correction factor f = 1.15 – our research
oxidation correction factor f = 1.0 – traditionally
Tyurin I.V. New modification of the volumetric method for
determining humus using chromic acid // Pochvovedenie,
1931. No. 6. P. 36-47.
Walkley-Blackmethod
(Colorimetric Method, GLOSOLAN)
- Sieve, d < 0.25 mm
- 50 g K2Cr2O7 +0,5L H2O, c(K2Cr2O7)= 0,34 M
- Soil+ 2 сm3 K2Cr2O7 +5 сm3 H2SO4 conс = 7 сm3
added to the sample n(K2Cr2O7)= 0,68 mmol
- Standing for 30 min
- +20 см3 H2О
- Standingfor 24 hours (without external heating)
- Measure the optical density  = 590 nm
- Сalculation of %Сorg
oxidation correction factor f = 1.3
Walkley A., Black I.A. An examination of the Degtjareff
method for determining soil organic matter, and a
proposed modification of the chromic acid titration
method // Soil Sci., 1934. V. 37. P. 29-38.
4
3 [C]0 + 2 Cr2O7
2- + 16 H+ = 3 CO2 + 4 Cr3+ + 8 H2O

5. Measurement steps
Tyurin method
(Institute of Biology)
- 20g K2Cr2O7 + 0,5L H2O, c(K2Cr2O7)= 0,136 M
- “Chromium mixture”: 1V K2Cr2O7+1V H2SO4 conс
c(K2Cr2O7)= 0,068 M
- Soil+10 сm3 “Chromium mixture” (5 сm3 +5 сm3)
added to the sample n(K2Cr2O7)= 0,68 mmol
- Heating in a water bath (t = 100 °С for 60 min)
- +15 см3 H2О
- Centrifugation 6000 rpm for 10 min
- Measure the optical density  = 590 nm
- Сalculation of %Сorg
correction factor f = 1.15 – our research
correction factor f = 1.0 – traditionally
Tyurin I.V. New modification of the volumetric method for
determining humus using chromic acid // Pochvovedenie,
1931. No. 6. P. 36-47.
Walkley-Blackmethod
(Colorimetric Method, GLOSOLAN)
- 50 g K2Cr2O7 +0,5L H2O, c(K2Cr2O7)= 0,34 M
- Soil+ 2 сm3 K2Cr2O7 +5 сm3 H2SO4 conс = 7 сm3
added to the sample n(K2Cr2O7)= 0,68 mmol
- Standing for 30 min
- +20 см3 H2О
- Standingfor 24 hours (without external heating)
- Measure the optical density  = 590 nm
- Сalculation of %Сorg
correction factor f = 1.3
Walkley A., Black I.A. An examination of the Degtjareff
method for determining soil organic matter, and a
proposed modification of the chromic acid titration
method // Soil Sci., 1934. V. 37. P. 29-38.
! Heating of the reaction mixture occurs
due to the exothermic effect that
occurs when a concentrated solution of
H2SO4 is mixed with distilled water.
! In the Walkley-Black method, the amount of K2Cr2O7 and H2SO4
is equal to the same characteristics as in the Tyurin method, but
the concentration of these components of the mixture is 1.5
times higher.
5

6. At the 3rd GLOSOLAN meeting the proposal to harmonize the
Tyurin colorimetric method was approved.
The matrix to collect information on the procedures implemented
by countries using the method was prepared and sent to
GLOSOLAN members.
BUT there were too many modifications of the method
(to do a standard harmonization)
6

7. Modifications of the Tyurin method
(Russia)
Conditions for the
oxidation
Quantity estimation method
Reference
documents
Cr3+ Cr2O7
2-
Тemperature Time Сolorimetric Тitrimetric
100 °С
(water bath)
1 hour  = 590 nm
(Mohr's salt)
- Turin, 1931;
GOST 26213-91
>140 °С
(electric stove)
5 min - titrant - Mohr's
salt solution Simakov, 1957
>140 °С
(electric stove)
5 min  = 590 nm
(Mohr's salt)
-
Orlov, 1967
20 °С 24 hours  = 590 nm
(Mohr's salt)
- Samoilova,
Rogiznaya, 2013
>140 °С
(water bath)
20 min  = 590 nm
(sucrose)
- Simakov, Tsyplakov,
1969
150 °С
(drying cabinet)
20 min  = 590 nm
(sucrose)
titrant - Mohr's
salt solution Nikitin, 1983
7
WHAT TO DO?

8. The Institute of Biology of Komi Scientific Center of the
Ural Branch has decided to go one step forward
1. The information provided by GLOSOLAN members
was used as a reference
2. The harmonized method was developed to ensure
comparability of the results from the Tyurin,
Walkley-Black and Dry Сombustion methods
8

9. Soil samples
• 3 soil samples from GLOSOLAN;
• 8 quality control samples (Russia);
• Reference materials of soils and bottom
sediments (Elemental Microanalysis Limited, UK);
• More than 110 field soil samples
9

10. Methods / equipment
Methods:
• Tyurin method;
• Walkley-Black method;
• Dry Combustion method (over 120 soil samples)
Equipment:
• Carlo Erba EA-1110 НСNS-O analyzer (Italy);
• Spectrophotometers UNICO 2100 (USA) and KFK-3
(Russia)
• Centrifuge SIGMA 2-16P (Russia)
%Corg by Combustion method is taken as a reference value
10

11. The implementation of large-scale mass
research allowed us to draw
a number of conclusions
11

12. Investigation of calibration functions
The chart shows a graphical calibration
function.
Both reducing agents provide the same
quality of measuring instruments’
calibration, and hence of Corg.
The stability of the absorbance of the
calibration solutions was checked.
After a day, the absorbance of the
solutions decreases by 2-3%, after two
days - by 4-7%.
This happens due to the occurrence of
processes in the system that are not
associated with the oxidation of
organic compounds’ carbon.
12
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0 1 2 3 4 5 6 7
А
Сorg, mg
Mora's Salt
Sucrose

13. Separation of solid and liquid phases
1. Filtration (Tyurin's method)
Some modifications of Tyurin's method
involve filtering soil suspensions.
A significant increase in the
absorbance of the calibration solutions
is observed while using filters.
This indicates the oxidation of organic
matter in the filters by dichromate
ions. The filters are made from
vegetable cellulose and turn green
after filtering.
An assessment of the oxidation
resistance of paper’s organic
compounds is required.
Filtration was not used in our studies.
13
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 2 4 6 8
А
Сorg, mg
with filter
without a filter

14. 2. Separation of solid and liquid phases
QC
sam
ples
pH
Н2О
Oxidation with dichromate ions Dry
Combustion
method
(НСNS-O
analyzer)
f
Tyurin (Т) method
Walkley-Black (W-B)
method
Standing Centrifu-
gation
Standing
24 hours
Centrifu-
gation
T
Сorg3
Сorg6
W-B
Сorg5
Сorg6
24 hours 48 hours
Сorg1 S1() Сorg2 S2() Сorg3 S3() Сorg4 S4() Сorg5 S5() Сorg6 S6()
1 5,10 0,790* 22 0,904 17 0,798 4,5 0,782 2,9 0,773 1,5 0,982 0,7 1,23 1,27
2 4,71 1,002 26 1,072 37 1,070 9,0 1,087 5,0 1,020 0,9 1,323 1,0 1,24 1,30
3 5,78 1,951 12 1,650 21 1,660 3,6 1,551 0,8 1,521 4,7 1,903 2,2 1,15 1,25
4 5,52 2,354 7,3 2,225 19 2,133 0,9 2,100 1,9 2,089 1,2 2,682 2,0 1,26 1,28
5 5,70 3,442 14 3,370 22 3,366 5,9 3,242 1,6 3,230 1,9 4,013 1,5 1,19 1,24
6 5,52** 3,426 10 3,524 9,3 3,330 6,8 3,151 1,7 3,159 1,8 3,926 0,9 1,18 1,24
7 5,54 4,117 17 3,902 34 4,151 2,8 4,052 1,2 3,937 1,8 4,928 1,8 1,19 1,25
8 5,32 0,760 12 - - 0,581 1,1 0,568 1,7 0,549 3,2 0,735 3,6 1,27 1,34
9 5,18 1,227 18 - - 1,230 4,2 1,199 2,5 1,181 2,7 1,736 0,7 1,41 1,47
10 6,55 2,530 14 - - 2,673 1,2 2,547 1,6 2,505 0,7 3,293 1,0 1,23 1,31
Orange unites are related to GLOSOLAN samples
* – each value is the average of 5 parallel measurements, ** – рНKCl,
S() – the relative value of the standard deviation of a single measurement result,%.
14

15. Separation of solid and liquid phases.
Standing suspensions (Tyurin's method)
1. For Tyurin's method, there is a statistical difference between 24 and 48 hours of
standing of suspensions and their centrifugation.
In some soils, sedimentation does not occur even after 48 hours.
0
10
20
30
40
0 1 2 3 4
%Сorg (Tyurin)
S()
Phase separation methods:
Standing, hours
- Centrifugation- 24 - 48
S(), % 26 37 >> 9
±, % 52 74 >> 18
S() – the relative value of the standard deviation of a
single measurement result, %,
± – the boundaries of the interval of the relative error, %
Maximum values for N = 5
15

16. Separation of solid and liquid phases.
Standing suspensions (Tyurin's method)
• The standing of the suspension after Corg oxidation for two days
does not guarantee sedimentation of soil particles.
• Longer standing will lead to poorer results due to discoloration
of the solution.
• Different sedimentation rates of soil particles do not allow
revealing the same holding time for obtaining true solutions of
supernatants and their composition.
• Our recommendation is the centrifugation of soil suspensions.
16

17. Separation of solid and liquid phases.
Standing suspensions (Walkley-Black method)
2. For the Walkley-Black method, the error in measuring %Corg both with 24-hour
standing of suspension and with centrifugation is significantly lower compared to
Tyurin's method.
Both methods of phase separation in the Wilkeley-Black method have close errors.
%Сorg (Walkley-Black)
S()
Phase separation methods:
S() – the relative value of the standard deviation of a
single measurement result, %,
± – the boundaries of the interval of the relative error, %
0
10
20
30
40
0 1 2 3 4
- Standing 24 hours - Centrifugation
S(), % 4,7 ~ 5
±, % 10 ~ 9,4
Maximum values for N = 5
17

18. Separation of solid and liquid phases.
Standing suspensions (Walkley-Black
method)
For Walkley-Black technique, heating the system is short-term
and mixing is one-time. Solid particles do not have time to
collapse. Therefore, keeping from the soil with a sulfuric acid
solution of potassium dichromate for a day leads to soil
sedimentation (the reproducibility index of measurement results
does not exceed 5%).
Additional centrifugation of the supernatant system is not
required, since it does not lead to a significant improvement in the
quality of measurement results.
18

19. Separation of solid and liquid phases.
Standing suspensions
3. The error in measuring of %Corg after centrifugation of soil suspensions in the
Tyurin method in all samples is slightly higher than in the Walkley-Black method
%Сorg (T, W-B)
S()
Phase separation methods:
S() – the relative value of the standard deviation of a single
measurement result, %,
± – the boundaries of the interval of the relative error, %
0
10
20
30
40
0 1 2 3 4
Centrifugation
- Tyurin method - Walkley-Black method
S(), % 9 > 4,7
±, % 18 > 9,4
Maximum values for N = 5
19

20. An oxidation correction factor f
f = %Сorg (Tyurin) / %Corg (Dry Combustion method)
0.9
1.0
1.1
1.2
1.3
1.4
1.5
0 1 2 3 4 5 6 7 8 9
1
2
3
4
5
%Сorg (Tyurin)
Average value of f = 1.15
1 – reference materials of soils (Elemental Microanalysis Limited, UK)
2, 3 – standard soil samples (Russia)
4 – soil samples from GLOSOLAN
5 – soil samples of various types
more than 120 soil samples in total
20

21. An oxidation correction factor f
f = %Сorg (Tyurin) / %Corg (Dry Combustion method)
0.9
1.0
1.1
1.2
1.3
1.4
1.5
0 1 2 3 4 5 6 7 8 9
1
2
3
4
5
%Сorg (Tyurin)
Average value of f = 1.15
It should be noted that Ivan Tyurin recommended using f = 1.17 to take into
account the incompleteness of oxidation (Arinushkina, 1962).
However, in practice, this recommendation was not used.
21

22. without taking into account the f = 1,15 taking into account the f = 1,15
-30
-20
-10
0
10
20
30
0 1 2 3 4 5 6 7 8 9
1 2 3 4 5
-30
-20
-10
0
10
20
30
0 1 2 3 4 5 6 7 8 9
1 2 3 4 5
%Сorg (Tyurin) %Сorg (Tyurin)
K K
%Сorg (Tyurin) K, % (Р = 0,95)
0,170 - 8,7 20
[%Сorg (Tyurin) - %C (Dry Combusion)] * 100%
%C (Dry Combusion)
K =
22
Quality control of measurements
%Соrg (Tyurin)

23. These conclusions formed the basis for our
development of a new modification of the Tyurin
method.
We have certified this new modification of the
Tyurin method and the Walkley-Black method at
the Center for Metrology and Certification
"Sertimet" (No. 88-17641-001-2020).
The methods are included in the Federal
Information Fund for ensuring the uniformity of
measurements.
23

24. Conclusion
• A comparative study of quality control samples and field soil samples by
the Tyurin, Walkley-Black and Dry Combustion methods confirmed the
possibility of taking into account incomplete organic carbon oxidation.
For this, it is necessary to enter a coefficient of 1.15 for the Tyurin
method and 1.3 for the Walkley-Black method.
• The accumulated large array of Corg measurements by the Tyurin and
Walkley-Black methods can be included in the global soil quality
monitoring network with the introduction of these coefficients.
• In our opinion, the Walkie-Black method is less laborious than the Tyurin
method. The rejection of the procedure for heating soil suspensions with
a chromium mixture less pollutes the working areas, therefore, it is more
environmentally friendly and less harmful to the performers.
W-B · 1.3 = Tyurin · 1.15 = Dry Combustion (P = 0.95)
24

25. Ultimately…
• In some cases, harmonization does not mean only finding a
scientifically sound compromise between the procedures in use
• GLOSOLAN should not work only on harmonizing procedures and
methods but on standardising data obtained using different
methods. In this regard, GLOSOLAN SOPs can be harmonized to
ensure that results that are obtained by using different methods
are comparable by default
• Many other methods of soil research "await" us for decades to be
unified. All laboratories that have the capacity to study “new”
procedures for a known method are welcomed to do so
Please note that we are now publishing a scientific article about this work.
The procedure we developed will be published as GLOSOLAN SOP.
25

26. Acknowledgments
We would like to express our gratitude to GLOSOLAN for supporting our
initiative to carry out this work, as well as for providing soil samples for
research.
The authors are also grateful to the administration of the Institute of Biology,
represented by its director, Doctor of Biological Sciences. S.V. Dyogteva for the
opportunity to implement this project.
This work was supported by the Russian Fund for Basic Research under grant
№ 20-04-00445а.
I personally thank my GLOSOLAN colleagues for their assistance in the
preparation of this report.
Special thanks to Nopmanee Suvannang and Lucrezia Сaon.
26

27. Main authors:
1. Davydova A. – lead chemical engineer
2. Dubrovckiy Y. – translator
3. Kondratenok B. – Deputy Director in Science, Candidate of Chemistry
4. Kostrova S. – lead chemical engineer
5. Lapteva E. – HeadoftheDepartmentofSoilScience,CandidateofBiology
6. Lu-Lyan-Min K. – chemical engineer
7. Shamrikova E. – Doctor of Science (Biology)
8. Shamrikova Ev. – translator
9. Tumanova E. – lead chemical engineer
10. Vanchikova E. – Candidate of Chemistry
11. Zonova T. – lead chemical engineer
27

28. Thanks for your
attention!
28

29. Сompounds Oxidation reactions
М(1/ZC) М(1/4С)
М(1/Z С)g/mol
Paraffinic hydrocarbons
С8Н18 + 25/2 О2 = 8СО2 + 9 Н2О 1,92 1,56
С9Н20 + 14 О2 = 9 СО2 + 10 Н2О 1,93 1,56
С16Н32 + 49/2 О2 = 16 СО2 + 17 Н2О 1,96 1,53
Olefinic hydrocarbons С9Н18 + 27/2 О2 = 9 СО2 + 9 Н2О 2,00 1,50
Aromatic hydrocarbons
С6Н6 + 15/2 О2 = 6 СО2 + 3 Н2О 2,40 1,25
С6Н5–С3Н7 + 12 О2 = 9 СО2 + 6 Н2О 2,25 1,33
Naphthenic hydrocarbons С10Н8 + 12 О2 = 10 СО2 + 4 Н2О 2,50 1,20
Fragment of fulvic and humic
acids
С47Н33О7N3 + 207/4 О2 =
47 СО2 + 33/2 Н2О + 3/2 N2
2,67 1,13
The average oxidation state of carbon SOM is close to zero, M(1/4C) = 3 g/mol.
However, arable soils, soils near highways, oil wells, etc. contaminated with organic
compounds that make up gasoline, fuel oil, oil.
In these compounds, M(1/ZC) significantly differs from the conventionally accepted one.
29