SIST EN ISO 16072:2011

SLOVENSKI STANDARD
SIST EN ISO 16072:2011
01-november-2011
.DNRYRVWWDO/DERUDWRULMVNHPHWRGH]DGRORþHYDQMHPLNUREQHJDGLKDQMDYWOHK
,62
Soil quality - Laboratory methods for determination of microbial soil respiration (ISO
16072:2002)
Bodenbeschaffenheit - Laborverfahren zur Bestimmung der mikrobiellen Bodenatmung
(ISO 16072:2002)
Qualité du sol - Méthodes de laboratoire pour la détermination de la respiration
microbienne du sol (ISO 16072:2002)
Ta slovenski standard je istoveten z: EN ISO 16072:2011
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 16072:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16072:2011

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SIST EN ISO 16072:2011


EUROPEAN STANDARD
EN ISO 16072

NORME EUROPÉENNE

EUROPÄISCHE NORM
June 2011
ICS 13.080.30
English Version
Soil quality - Laboratory methods for determination of microbial
soil respiration (ISO 16072:2002)
Qualité du sol - Méthodes de laboratoire pour la Bodenbeschaffenheit - Laborverfahren zur Bestimmung der
détermination de la respiration microbienne du sol (ISO mikrobiellen Bodenatmung (ISO 16072:2002)
16072:2002)
This European Standard was approved by CEN on 10 June 2011.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16072:2011: E
worldwide for CEN national Members.

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SIST EN ISO 16072:2011
EN ISO 16072:2011 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 16072:2011
EN ISO 16072:2011 (E)
Foreword
The text of ISO 16072:2002 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 16072:2011 by
Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by December 2011, and conflicting national standards shall be withdrawn
at the latest by December 2011.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 16072:2002 has been approved by CEN as a EN ISO 16072:2011 without any modification.

3

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SIST EN ISO 16072:2011

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SIST EN ISO 16072:2011

INTERNATIONAL ISO
STANDARD 16072
First edition
2002-12-15

Soil quality — Laboratory methods for
determination of microbial soil
respiration
Qualité du sol — Méthodes de laboratoire pour la détermination de
la respiration microbienne du sol




Reference number
ISO 16072:2002(E)
©
ISO 2002

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SIST EN ISO 16072:2011
ISO 16072:2002(E)
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©  ISO 2002
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
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Published in Switzerland

ii © ISO 2002 — All rights reserved

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SIST EN ISO 16072:2011
ISO 16072:2002(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Procedure. 2
4.1 General conditions. 2
4.2 Choice of the measuring system. 3
5 Measuring systems. 3
5.1 Determination of O consumption by static incubation in a pressure-compensation
2
system. 3
5.2 Determination of CO release by titration in a static system . 4
2
5.3 Coulometric determination of CO release in a static system . 6
2
5.4 Determination of CO release using an infrared gas analyser in a flow-through system. 7
2
5.5 Determination of CO release using gas chromatography in a flow-through system and a
2
static system . 10
5.6 Determination of soil respiration by pressure measurement in a static system . 15
Bibliography . 19

© ISO 2002 — All rights reserved iii

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SIST EN ISO 16072:2011
ISO 16072:2002(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 16072 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
iv © ISO 2002 — All rights reserved

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SIST EN ISO 16072:2011
ISO 16072:2002(E)
Introduction
This International Standard is derived from the German standard DIN 19737 (see [1]). It describes methods
for the determination of microbial soil respiration in the laboratory.
Microbial soil respiration results from the mineralization of organic substances. In this process, organic
substances are oxidized to the end products carbon dioxide and water, with concurrent uptake of O for
2
aerobic microorganisms. The soil respiration is measured by the determination of O consumption and/or by
2
CO release. Respiration is a measure of the overall activity of soil microorganisms.
2

© ISO 2002 — All rights reserved v

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SIST EN ISO 16072:2011

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SIST EN ISO 16072:2011
INTERNATIONAL STANDARD ISO 16072:2002(E)

Soil quality — Laboratory methods for determination of
microbial soil respiration
1 Scope
This International Standard describes methods for the determination of soil microbial respiration of aerobic,
unsaturated soils. The methods are suitable for the determination of O uptake or CO release, either after
2 2
addition of a substrate (substrate-induced respiration), or without substrate addition (basal respiration).
This International Standard is applicable to the measurement of soil respiration in order to:
 determine the microbial activity in soil (see [3]);
 establish the effect of additives (nutrients, pollutants, soil improvers, etc.) on the metabolic performance
of microorganisms;
 determine the microbial biomass (see [4]);
 determine the metabolic quotient qCO .
2
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 10381-6:1993, Soil quality — Sampling — Guidance on the collection, handling and storage of soil for the
assessment of aerobic microbial processes in the laboratory
ISO 11274:1998, Soil quality — Determination of the water-retention characteristic — Laboratory methods
ISO 11465:1993, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric
method
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
basal respiration
microbial soil respiration without addition of nutrients
3.2
substrate-induced respiration
SIR
microbial soil respiration after addition of nutrients
NOTE Glucose is an example of an added nutrient.
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SIST EN ISO 16072:2011
ISO 16072:2002(E)
3.3
microbial activity
metabolic performance of microorganisms
NOTE It can be measured, for example, as O uptake or CO release.
2 2
3.4
metabolic quotient
qCO
2
specific metabolic activity of soil microorganisms, which can be calculated as the quotient basal respiration:
microbial biomass
NOTE Metabolic quotient is usually expressed as milligrams of CO carbon released per hour per gram of microbial
2
biomass carbon.
3.5
rate of CO formation [O consumption]
2 2
R [R ]
CO O
2 2
amount of CO released [O consumed] per time unit from a mass unit of soil
2 2
NOTE 1 Soil respiration is usually measured as the rate of CO formation or O consumption.
2 2
–1 –1
NOTE 2 It is usually expressed as milligrams CO [or O ] per gram per hour (mg CO [or O ]·g ·h ).
2 2 2 2
3.6
microbial biomass
mass of intact microbial cells in a given soil
NOTE This is usually estimated from the measurement of carbon or nitrogen content of these cells.
4 Procedure
4.1 General conditions
4.1.1 Soil sampling and storage
Sample, store and pre-incubate soils in accordance with ISO 10381-6, independently of the choice of the
procedure and the respiration parameter to be measured (basal respiration, SIR).
4.1.2 Measuring and incubation conditions
Soil respiration is strongly influenced by water content and temperature. Therefore these parameters should
be recorded in the final report. At suction pressures > 0,03 MPa, the soil respiration will decrease considerably.
The water content of the test soil is optimal when it corresponds with a pore water pressure of – 0,01 MPa to
– 0,03 MPa (measured with an accuracy of 5 %, in accordance with ISO 11274) or 40 % to 60 % of the
maximum water-holding capacity, respectively. A stable temperature should be used. Incubation temperatures
between 20 °C and 30 °C are generally recommended, but other temperatures may be used if required. In the
description of the methods, examples of incubation temperatures are given as well as the accuracy of
temperature maintenance and measurement.
If a method is used for the determination of soil microbial biomass, a temperature of 22 °C is recommended
because biomass calculations have been calibrated to this temperature.
When soil samples are compared with respect to soil respiration, they should have the same moisture status
(pore water pressure or percentage of maximum water-holding capacity).
2 © ISO 2002 — All rights reserved

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SIST EN ISO 16072:2011
ISO 16072:2002(E)
4.2 Choice of the measuring system
Each measurement method has its own advantages and disadvantages. Care is needed, because the results
obtained by O uptake and by CO release are not strictly compatible. It is the responsibility of the investigator
2 2
to decide which of these methods is to be used.
One of the systems described in Clause 5 should be used.
Systems for measuring CO do not distinguish between CO released from microbial activities and CO
2 2 2
resulting from abiotic processes. For alkaline soils and soils with a high organic matter content, which can
release considerable amounts of abiotically released CO , methods using O uptake are recommended.
2 2
NOTE The advantages and disadvantages are described in the individual descriptions of the methods.
5 Measuring systems
5.1 Determination of O consumption by static incubation in a pressure-compensation
2
system
5.1.1 Principle
The determination is based on the measurement of O consumption during incubation of a soil sample in a
2
closed system. The O in the system is replenished electrochemically. The CO released is absorbed by
2 2
calcium hydroxide [Ca(OH) ].
2

Key
A reaction vessel 1 soil sample 4 electrolyte
B oxygen generator 2 CO absorbent 5 electrodes
2
C pressure indicator 3 pressure cell 6 recorder with display
Figure 1 — Determination of O consumption (showing connection of a measuring unit)
2
5.1.2 Apparatus
A detailed description of the apparatus can be found in [4]; the essential features are as follows.
© ISO 2002 — All rights reserved 3

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SIST EN ISO 16072:2011
ISO 16072:2002(E)
The measuring system (see Figure 1) consists of a water bath with temperature control containing measuring
units each comprising a reaction vessel (A) in which a CO absorption device (2) is suspended from the
2
stopper, an O generator (B) and a pressure indicator (C). The vessels (A, B, C) of the measuring unit form
2
together a closed system, connected to each other by tubing. In this way fluctuations in atmospheric pressure
will not influence the results. The CO released is absorbed by the calcium hydroxide (2). The consumption of
2
O due to respiration results in a negative pressure which activates the pressure indicator (C). This drives the
2
electrolytic O formation as well as the display and graphical registration of measuring values on a recorder
2
(6). The O consumption is shown directly, in milligrams of oxygen (mg O ) on a digital display.
2 2
1)
The system can be obtained commercially and detailed instructions should be given in the supplier's manual.
5.1.3 Procedure
Use 50 g to 100 g of field-moist, sieved (2 mm) soil for the measurements. The O consumption should not be
2
measured during the first 2 h, the time needed to reach equilibrium in the system.
5.2 Determination of CO release by titration in a static system
2
5.2.1 Principle
The soil is incubated in a closed vessel and the released CO is absorbed in a solution of sodium hydroxide.
2
After back-titration of the non-consumed sodium hydroxide, the amount of CO released is calculated. The
2
method is suitable for large numbers of samples, and up to 80 respiration measurements per working day are
possible.
5.2.2 Reagents
5.2.2.1 CO -free water
2
Boil distilled water and after cooling store it in flasks closed with stoppers provided with absorption tubes
containing calcium hydroxide.
–1
5.2.2.2 Sodium hydroxide (NaOH) solution, c = 0,05 mol·l .
–1
5.2.2.3 Hydrochloric acid (HCl) solution, c = 0,1 mol·l .
The concentrations of NaOH solution (5.2.2.2) and HCl (5.2.2.3) should be chosen so that less than 20 % of
the NaOH is neutralized by CO . Higher percentages of neutralization will result in less reliable results (see
2
[5]). If other concentrations are used, Equation (1) should be changed accordingly.
–1
5.2.2.4 Barium chloride solution, c = 0,5 mol·l .
Dissolve 10,4 g of BaCl in 100 ml of CO -free distilled water (5.2.2.1).
2 2
5.2.2.5 Indicator.
Dissolve 0,1 g of phenolphthalein in 100 ml of aqueous ethanol (volume fraction ethanol 0,6).
5.2.3 Apparatus
5.2.3.1 Wide-mouth flasks (250 ml content) with screw-caps and pour rim, or preserve flasks (1 l
content) with rubber rings, covers and 2 universal clips.

1) Sapromat is the trade name of a product supplied by H+P Labortechnik AG. This information is given for the
convenience of users of this International Standard and does not constitute an endorsement by ISO of this product.
Equivalent products may be used if they can be shown to lead to the same results.
4 © ISO 2002 — All rights reserved

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SIST EN ISO 16072:2011
ISO 16072:2002(E)
5.2.3.2 Centrifuge tubes or reaction tubes with a rim (e.g. polypropylene, external diameter 29 mm,
length 120 mm). Small holes should be drilled in the tubes for gas exchange. Instead of tubes, fine-mesh
nylon bags can also be suspended from the neck in the wide-mouth flasks.
5.2.4 Procedure
Weigh 20 g to 25 g of field-moist soil into the centrifuge tubes (5.2.3.2). Suspend the tubes in the wide-mouth
flasks (5.2.3.1) (see Figure 2), in which 20 ml solution of sodium hydroxide (5.2.2.2) has been previously
pipetted. Close the flasks tightly and incubate for 24 h in a temperature-controlled room at the temperature of
choice, e.g. 22 °C ± 1 °C. Before closing the flasks, they should be flushed with clean air with low CO content
2
(e.g. from outdoors). Then remove the tubes. The CO absorbed will precipitate as barium carbonate upon
2
addition of 2 ml of barium chloride solution (5.2.2.4). Titrate the unused sodium hydroxide with hydrochloric
acid (5.2.2.3) after addition of 3 or 4 drops of indicator solution (5.2.2.5).

Key
1 wide-mouth flask (250 ml) 6 openings for gas exchange
2 screw-cap 7 soil sample
3 pour rim 8 sodium hydroxide solution
4 closing pad 9 plastic thread
5 suspended centrifuge tubes 10 fine-mesh woven plastic bag
Figure 2 — Incubation flasks for the determination of soil respiration
The determination should be carried out at least in triplicate. Controls (triplicate flasks without soil) should be
included.
If the soil respiration is measured in a long-term experiment (> 3 days), then the soil samples should be
incubated in flasks in which the sodium hydroxide solution is renewed every 3 days. Also the water content of
the soil has to be adjusted every 3 days.
Also suitable for incubation are preserve flasks (1 l content) with rubber rings, covers and 2 universal clips.
Weigh the soil samples (up to 200 g) into crystallization disks, which are placed on the bottom of the preserver
flasks. Place sodium hydroxide solution in a beaker. On the bottom of the flasks, place 4 ml of CO -free water
2
(5.2.2.1) to maintain air moisture.
NOTE When determining basal respiration in the laboratory, an increase in CO release is often observed in the first
2
hours. This can be caused by an increased availability of nutrients due to the moving and mixing of soil particles during
sample preparation, but also by the short-term establishment of an equilibrium between gaseous and dissolved CO . The
2
incubation time necessary for reaching a steady basal respiration depends in the first instance on the soil's content of
easily available carbon compounds. This applies to all methods measuring CO release.
2
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SIST EN ISO 16072:2011
ISO 16072:2002(E)
5.2.5 Calculation of results
Calculate the rate of CO evolution using equation (1).
2
2,2VV−
()
bp
R = (1)
CO
2
24⋅⋅mw
sm sd
where
–1 –1
R is the rate of CO evolution a on soil dry mass basis (mg CO ·g ·h );
CO 2 2
2
V is the average volume of HCl consumed in the control, in millilitres;
b
V is the average volume of HCl consumed in the test sample, in millilitres;
p
m is the mass of the moist soil sample, in grams;
sm
–1 –1
2,2 is a factor (1 ml of 0,1 molar HCl corresponds to 2,2 mg of CO per day) (mg·ml ·day );
2
–1
24 is a factor to convert daily evolution to hourly evolution (h⋅day );
w is the dry mass fraction of the moist soil.
sd
NOTE The dry mass fraction (w ) of the moist soil equals the percent dry mass divided by 100.
sd
–1 –1
This calculation is only valid if the specified reagent concentrations (0,05 mol·l NaOH and 0,1 mol·l HCl)
are used.
5.3 Coulometric determination of CO release in a static system
2
5.3.1 Principle
The soil sample is incubated (e.g. in triplicate) in containers at constant temperature, and the CO evolved is
2
absorbed in sodium hydroxide solution. In the reaction:
2 NaOH + CO → Na CO + H O
2 2 3 2
hydroxyl ions are replaced by carbonate ions, which have a different electrical conductivity. The change in
conductivity is registered electronically by the apparatus. A CO content of 0,000 1 % (volume fraction) can be
2
detected with certainty. With the apparatus, basal respiration can be measured as well as substrate-induced
soil respiration after addition of glucose to determine biomass carbon. Depending on the type of apparatus, up
to 90 samples can be measured simultaneously.
The mass of the soil samples used and the temperature setting should be established according to the
manufacturer's instructions.
For more details see [6] and [8].
5.3.2 Test assembly
A simple type of apparatus is depicted schematically in Figure 3.
6 © ISO 2002 — All rights reserved

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SIST EN ISO 16072:2011
ISO 16072:2002(E)
Dimensions in millimetres

Key
1 container with soil sample
2 conductivity cell (in the cover)
3 platinum electrodes
Figure 3 — Apparatus for coulometric measurement of soil respiration
5.4 Determination of CO release using an infrared gas analyser in a flow-through system
2
5.4.1 Principle
Soil samples are flushed with ambient air. The release of CO from the soil samples during the incubation
2
period is determined by an infrared gas analyser in combination with a flow meter. This method is frequently
used for microbial biomass measurements, in which case a temperature of 22 °C is recommended. At a
temperature other than 22 °C, routine evaluation of biomass measurements cannot be carried out, but must
be recalculated using factors.
5.4.2 Reagent
–1 –1
5.4.2.1 CO calibration gas (air with 350 µl CO ·l and 400 µl CO ·l ).
2 2 2
5.4.3 Apparatus
The usual laboratory equipment, as well as an infrared gas analyser (see Figure 4) placed in a temperature-
controlled room at the temperature of choice, e.g. 22 °C ± 1 °C.
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SIST EN ISO 16072:2011
ISO 16072:2002(E)

Key
1 soil sample container 5 valves (3-way) 9 flow adjustment and flow meter
2 air inlet 6 steering 10 infrared CO analyser
2
3 gas pump 7 closure valve 11 control unit
4 moistener 8 reference air 12 control and evaluation equipment
Figure 4 — Example of configuration of an infrared gas analyser system
for the determination of soil respiration
5.4.4 Procedure
Sieve and weigh 10 g to 200 g, depending on the activity expected, of moist soil into the soil sample cylinders
(e.g. in triplicate). To pack the soil sample so that it completely fills the diameter of the cylinder, the use of
porous polyurethane foam stoppers is recommended as shown in Figure 5. To control the baseline of the
apparatus, at least two sample cylinders should remain empty. After connecting the sample cylinders to the
measuring equipment, start the gas pumps and adjust the gas flowrate at each measuring channel to the
required value. Normally, the measured data are registered automatically by the evaluation software.
The infrared gas analyser usually requires recalibration every one to two weeks by means of the calibration
gas.
The continuous flow of surrounding air in the soil sample reduces the disturbance of the soil carbonate
equilibrium (especially in calcareous soils) and therefore reduces the risk of erroneous results due to abiotic
release of CO .
2
A good linearity exists over a wide range between the mass of the soil samples (10 g to 100 g) and the CO
2
release for the measuring equipment described.
If this system is compared with flow-through systems which use CO -free air to measure CO release, no
2 2
inhibition of the citric acid cycle in microorganisms is observed.
8 © ISO 2002 — All rights reserved

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SIST EN ISO 16072:2011
ISO 16072:2002(E)

Key
1 soil sample
2 polyurethane foam stopper
3 rubber stoppers
4 acrylic glass cylinder
Figure 5 — Sample container for determination of soil respiration using an infrared gas analyser
5.4.5 Calculation and results
The infrared gas analyser measures the CO volume fraction in the differential mode, in microlitres per litre.
2
The rate of formation of CO (milligrams CO per gram per hour) is calculated by the evaluation software
2 2
taking into account the actually measured gas flowrate (millitres air per minute) and the mass of the soil
sample (grams dry mass).
()ϕϕ− ⋅ q
se g
R = 0,00183 (2)
CO
2
mw⋅
sm sd
where
–1 –1
R is the rate of CO formation on a soil dry mass basis (mg CO ·g h );
CO 2 2
2
–1
ϕ is the read-out of CO volume fraction in the soil sample cylinder (µl CO ·l );
s 2 2
ϕ is the read-out of CO volume fraction in the empty cylinder which has been analysed most
e 2
–1
recently before or afterwards the soil sample cylinder (µl CO ·l );
2
q is the read-out of the gas flowrate, in litres per hour;
g
–1
0,0
...