SIST EN ISO 9439:2000

SLOVENSKI STANDARD
SIST EN ISO 9439:2000
01-december-2000
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Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in
aqueous medium - Carbon dioxide evolution test (ISO 9439:1999)
Wasserbeschaffenheit - Bestimmung der vollständigen aeroben biologischen
Abbaubarkeit organischer Stoffe im wäßrigen Medium - Verfahren mit Kohlenstoffdioxid-
Messung (ISO 9439:1999)
Qualité de l'eau - Evaluation de la biodégradabilité aérobie ultime en milieu aqueux des
composés organiques - Essai de dégagement de dioxyde de carbone (ISO 9439:1999)
Ta slovenski standard je istoveten z: EN ISO 9439:2000
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 9439:2000 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 9439:2000

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SIST EN ISO 9439:2000

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SIST EN ISO 9439:2000

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SIST EN ISO 9439:2000
INTERNATIONAL ISO
STANDARD 9439
Second edition
1999-03-01
Water quality — Evaluation of ultimate
aerobic biodegradability of organic
compounds in aqueous medium — Carbon
dioxide evolution test
Qualité de l'eau — Évaluation de la biodégradabilité aérobie ultime en
milieu aqueux des composés organiques — Essai de dégagement de
dioxyde de carbone
A
Reference number
ISO 9439:1999(E)

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SIST EN ISO 9439:2000
ISO 9439:1999(E)
Contents
1 Scope .1
2 Definitions .1
3 Principle.3
4 Test environment.3
5 Reagents.3
6 Apparatus .4
7 Procedure .5
8 Calculation.7
9 Validity of results.8
10 Test report .9
Annex A (informative) Principle of a test system for measuring carbon dioxide (example) .10
Annex B (informative) Examples of the determination of released carbon dioxide.11
Annex C (informative) Example of a biodegradation curve .13
Annex D (informative) Combined determination of carbon dioxide and DOC .14
Bibliography.17
©  ISO 1999
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic
or mechanical, including photocopying and microfilm, without permission in writing from the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet iso@iso.ch
Printed in Switzerland
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SIST EN ISO 9439:2000
© ISO
ISO 9439:1999(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 3.
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.
International Standard ISO 9439 has been prepared by Technical Committee ISO/TC 147, Water quality,
Subcommittee SC 5, Biological methods.
This second edition cancels and replaces the first edition (ISO 9439:1990), which has been technically revised.
Annexes A to D of this International Standard are for information only.
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SIST EN ISO 9439:2000
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ISO 9439:1999(E)
Introduction
The conditions described in this International Standard do not always correspond to the optimal conditions for
allowing the maximum degree of biodegradation to occur. With this test system, the microbially derived carbon
dioxide (CO ) is measured in the traps through which gas exhausted from the test vessels is passed. Some of the
2
CO remains in the medium in the vessels as dissolved inorganic carbon (DIC), the concentration of which may
2
increase as biodegradation proceeds. As the organic carbon approaches complete removal, the concentration of
DIC gradually falls and tends to reach zero by the end of incubation. It is thus necessary to acidify the medium at
the end of the test to measure the biogenically formed CO completely. The measurement of CO in the external
2 2
traps may differ from the true production of CO and the kinetic rate may also be lower than a rate based on DOC
2
removal measurement. The consequence may be that the biodegradation curves based on the trapped CO may
2
not fully represent the true microbial kinetic rate. For alternative biodegradation methods, see ISO 15462 and in
particular ISO 14593, which is based on CO production as well but does not have this defect.
2
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SIST EN ISO 9439:2000
INTERNATIONAL STANDARD  © ISO ISO 9439:1999(E)
Water quality — Evaluation of ultimate aerobic biodegradability of
organic compounds in aqueous medium — Carbon dioxide
evolution test
WARNING — Activated sludge and sewage may contain potentially pathogenic organisms. Appropriate
precautions should be taken when handling them. Toxic test compounds and those whose properties are
unknown should be handled with care.
1 Scope
This International Standard specifies a method, by determination of carbon dioxide (CO ), for the evaluation in an
2
aqueous medium of the ultimate biodegradability of organic compounds at a given concentration by aerobic
microorganisms.
The method applies to organic compounds which are:
a) water-soluble under the conditions of the test, in which case removal of DOC may be determined as additional
information (see annex D);
b) poorly water-soluble under the conditions of the test, in which case special measures may be necessary to
achieve good dispersion of the compound (see, for example, ISO 10634);
c) non-volatile or which have a negligible vapour pressure under the conditions of the test;
NOTE For volatile substances use for example ISO 9408 or ISO 14593.
d) not inhibitory to the test microorganisms at the concentration chosen for the test.
NOTE The presence of inhibitory effects can be determined as specified in 8.3, or by using any other method for
determining the inhibitory effect of a compound on bacteria (see, for example, ISO 8192).
2 Terms and definitions
For the purposes of this International Standard, the following terms and definitions apply.
2.1
ultimate aerobic biodegradation
breakdown of a chemical compound or organic matter by microorganisms in the presence of oxygen to carbon
dioxide, water and mineral salts of any other elements present (mineralization) and the production of new biomass
2.2
primary biodegradation
structural change (transformation) of a chemical compound by microorganisms resulting in the loss of a specific
property
2.3
activated sludge
biomass produced in the aerobic treatment of wastewater by the growth of bacteria and other microorganisms in the
presence of dissolved oxygen
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SIST EN ISO 9439:2000
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ISO 9439:1999(E)
2.4
concentration of suspended solids
 amount of solids obtained by filtration or centrifugation of a known volume of activated sludge
o
and drying at about 105 C to constant mass
2.5
dissolved organic carbon
DOC
that part of the organic carbon in a water sample which cannot be removed by specified phase separation
-2
NOTE For example, by centrifugation at 40 000 m { s for 15 min or by membrane filtration using membranes with pores
of diameter 0,2 μm to 0,45 μm.
2.6
total inorganic carbon
TIC
all that inorganic carbon in the water deriving from carbon dioxide and carbonate
2.7
dissolved inorganic carbon
DIC
that part of the inorganic carbon in water which cannot be removed by specified phase separation
-2
NOTE For example, by centrifugation at 40 000 m { s for 15 min or by membrane filtration using membranes with pores
of diameter 0,2 μm to 0,45 μm.
2.8
theoretical amount of formed carbon dioxide
ThCO
2
theoretical maximum amount of carbon dioxide formed after oxidizing a chemical compound completely
NOTE It is calculated from the molecular formula and expressed in this case as milligrams carbon dioxide per milligram (or
gram) test compound.
2.9
lag phase
time from the start of a test until adaptation and/or selection of the degrading microorganisms are achieved and the
biodegradation degree of a chemical compound or organic matter has increased to about 10 % of the maximum
level of biodegradation
NOTE It is normally recorded in days.
2.10
maximum level of biodegradation
maximum biodegradation degree of a chemical compound or organic matter in a test, above which no further
biodegradation takes place during the test
NOTE It is normally recorded in percent.
2.11
biodegradation phase
time from the end of the lag phase of a test until about 90 % of the maximum level of biodegradation has been
reached
NOTE It is normally recorded in days.
2.12
plateau phase
time from the end of the biodegradation phase until the end of the test
NOTE It is normally recorded in days.
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SIST EN ISO 9439:2000
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ISO 9439:1999(E)
2.13
pre-exposure
pre-incubation of an inoculum in the presence of the test chemical compound or organic matter, with the aim of
enhancing the ability of this inoculum to biodegrade the test material by adaptation and/or selection of the
microorganisms
2.14
preconditioning
pre-incubation of an inoculum under the conditions of the subsequent test in the absence of the test chemical
compound or organic matter, with the aim of improving the performance of the test by acclimatization of the
microorganisms to the test conditions
3 Principle
The biodegradability of organic compounds by aerobic microorganisms is determined using a static aqueous test
system. The test mixture contains an inorganic medium, the organic compound as the nominal sole source of
carbon and energy at a concentration of 10 mg/l to 40 mg/l organic carbon and a mixed inoculum obtained from a
wastewater treatment plant or from another source in the environment. The mixture is agitated in test vessels and
aerated with CO -free air normally up to 28 d (for example see annex A). The CO formed during the microbial
2 2
degradation is trapped in external vessels, determined by an appropriate analytical method (for examples see
annex B), compared with the theoretical amount (ThCO ) and expressed as a percentage.
2
For sufficiently water-soluble compounds, removal of DOC may optionally be measured to obtain additional
information on the ultimate biodegradability. This can be done in the method given, but a convenient procedure is
described in annex D which allows the use of higher concentrations of the test compound and the inoculum, thus
improving the biodegradation potential of the test. If a substance-specific analytical method is available, information
on the primary degradability may also be obtained.
4 Test environment
o o
Incubation shall take place in the dark or in diffused light, at a temperature within the range 20 C to 25 C which
o
shall not vary by more than ± 2 C during the test.
5 Reagents
Use only reagents of recognized analytical grade.
5.1  Water, distilled or deionized, containing less than 1 mg/l DOC.
5.2  Test medium.
5.2.1  Composition
a) Solution a)
Dissolve
anhydrous potassium dihydrogenphosphate (KH PO ) 8,5 g
2 4
anhydrous dipotassium hydrogenphosphate (K HPO ) 21,75 g
2 4
disodium hydrogenphosphate dihydrate (Na HPO ·2H O) 33,4 g
2 4 2
ammonium chloride (NH Cl) 0,5 g
4
in water (5.1), quantity necessary to make up to 1 000 ml
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SIST EN ISO 9439:2000
© ISO
ISO 9439:1999(E)
In order to check this buffer solution it is recommended to measure the pH, which should be about 7,4. If this is
not the case, prepare a new solution.
b) Solution b)
Dissolve 22,5 g magnesium sulfate heptahydrate (MgSO ·7H O) in water (5.1), quantity necessary to make up
4 2
to 1 000 ml.
c) Solution c)
Dissolve 36,4 g calcium chloride dihydrate (CaCl ·2H O) in water (5.1), quantity necessary to make up to
2 2
1 000 ml.
d) Solution d)
Dissolve 0,25 g iron(III) chloride hexahydrate (FeCl ·6H O) in water (5.1), quantity necessary to make up to
3 2
1 000 ml. To avoid precipitation, prepare this solution freshly before use or add a drop of concentrated
hydrochloric acid (HCl).
5.2.2  Preparation of the test medium
For 1 000 ml of test medium add to about 800 ml of water (5.1):
 10 ml of solution a);
 1 ml of each of the solutions b) to d).
Make up to 1 000 ml with water (6.1).
6 Apparatus
Ensure that all glassware is thoroughly cleaned and free from both organic and toxic matter.
6.1  Test vessels. Glass vessels (e.g. Erlenmeyer vessels or bottles) allowing gas purging and shaking or stirring,
including tubing impermeable to CO . Located in a constant-temperature room or in a thermostatically controlled
2
environment (e.g. water bath).
6.2  CO -free air production system, capable of supplying each test vessel at a flowrate between about
2
50 ml/min and 100 ml/min for 3 l of medium, held constant (see example of assembly with the test vessels in
annex A).
6.3  Analytical equipment for determining CO .
2
Any suitable apparatus or technique with sufficient accuracy, e.g. CO - or DIC analyzer or device for titrimetric
2
determination after complete absorption in an alkaline solution (see examples in annex B).
6.4  Analytical equipment for measuring dissolved organic carbon (DOC) (optional).
6.5  Centrifuge or device for filtration, with membrane filters (nominal aperture diameter of 0,2 μm to 0,45 μm
pore size) which adsorb or release organic carbon to a minimum degree.
6.6  pH meter.
4

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SIST EN ISO 9439:2000
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ISO 9439:1999(E)
7 Procedure
7.1 Preparation of the test solutions
7.1.1 Test compound
Prepare a stock solution of a sufficiently water-soluble test compound in water (5.1) or the test medium (5.2) and
add a suitable amount of this to obtain an organic carbon concentration in the final test medium of between 10 mg/l
and 40 mg/l. Depending on the properties of the test compound (e.g. toxicity) and the purpose of the test, other
concentrations may be used. Add compounds of low water solubility directly into the test vessels. Determine the
added amount exactly.
NOTE For more details on handling poorly water-soluble compounds, see ISO 10634.
7.1.2 Reference compound
Use as reference compound an organic compound of known biodegradability, such as aniline or sodium benzoate.
Prepare a stock solution of the reference compound in the test medium (5.2) in the same way as with a water-
soluble test compound (7.1.1), in order to obtain a final organic carbon concentration of 20 mg/l or a concentration
equivalent to that of the test compound.
7.1.3 Solution to check inhibition
If required (when e.g. no information on the toxicity of test compound is available), prepare a solution containing, in
the test medium (5.2), both the test compound (7.1.1) and the reference compound (7.1.2) preferably at
concentrations of organic carbon of 20 mg/l for each.
7.2 Preparation of the inoculum
7.2.1 General
Prepare the inoculum using activated sludge (7.2.2) or the sources described in 7.2.3 and 7.2.4 or a mixture of
these sources to obtain a microbial population that offers sufficient biodegradative activity. Check the activity of the
inoculum by means of the reference compound (7.1.2 and clause 9). The CO production of the blank should fulfil the
2
validity criteria (see clause 9). To reduce the influence of the blank, it may be helpful to precondition the inoculum, e.g.
by washing with medium (5.2.2) and aerating it, from 1 d to 7 d, before use. Use a suitable volume for inoculation (see
note 2 below).
NOTE 1 Normally the inoculum should not be pre-exposed to the test compound to allow a general prediction of the
degradation behaviour in the environment. In certain circumstances, depending on the purpose of the test, pre-exposed inocula
may be used, provided that this is clearly stated in the test report (e. g. percent biodegradation = x %, using pre-exposed
inocula) and the method of pre-exposure is detailed in the test report. Pre-exposed inocula can be obtained from laboratory
biodegradation tests conducted under a variety of conditions (e.g. Zahn-Wellens test ISO 9888 and SCAS test ISO 9887) or
from samples collected from locations where relevant environmental conditions exist (e.g. treatment plants dealing with similar
compounds or contaminated areas).
NOTE 2 Based on experience, suitable volume means:
 sufficient to give a population which offers enough biodegradation activity;
 degrades the reference compound by the stipulated percentage (see clause 9);
3 6
 gives between 10 to 10 colony-forming units per millilitre in the final mixture;
 gives not greater than the equivalent of 30 mg/l suspended solids of activated sludge in the final mixture;
 the quantity of dissolved organic carbon provided by the inoculum should be less than 10 % of the initial concentration of
organic carbon introduced by the test compound;
 generally 1 ml to 10 ml of inoculum are sufficient for 1 000 ml of test solution.
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SIST EN ISO 9439:2000
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ISO 9439:1999(E)
7.2.2 Inoculum from an activated sludge plant
Take a sample of activated sludge collected from the aeration tank of a full-scale or a laboratory wastewater
treatment plant dealing with predominantly domestic sewage. Mix well and determine the concentration of
suspended solids of the activated sludge (use e.g. ISO 11923). If necessary remove coarse particles by filtration
through a sieve and concentrate the sludge by settling, so that the volume of sludge added to the test assay is
minimal. Keep the sample under aerobic conditions and use preferably on the day of collection. Use a suitable
volume to obtain 30 mg/l of suspended solids in the final mixture.
7.2.3 Inoculum from wastewater
Take a sample from the influent or from the effluent of a full-scale or a laboratory wastewater treatment plant
dealing with predominantly domestic sewage. If necessary, remove gross particulate matter by coarse filtration and
concentrate the sample, e.g. by centrifugation. Mix well, keep the sample under aerobic conditions and use
preferably on the day of collection. Before use, let the sample settle for 1 h and take a suitable volume of the
supernatant for inoculation.
7.2.4 Inoculum from a surface water
Take a sample of an appropriate surface water. If necessary, concentrate the sample by filtration using a coarse
paper filter or centrifugation. Keep the sample under aerobic conditions and use preferably on the day of collection.
Use a suitable volume as inoculum.
7.3 Test procedure
Provide a sufficient number of vessels (6.1) in order to have
 at least two test vessels (denoted F ) for the test compound (7.1.1);
T
 at least two blank vessels (denoted F ) containing test medium and inoculum;
B
 at least one vessel, for checking the procedure (denoted F ) containing the reference compound (7.1.2);
C
 if needed, one vessel for checking a possible inhibitory effect of the test compound (denoted F ) containing
I
solution 7.1.3;
 if needed, one vessel for checking a possible abiotic elimination (denoted F ) containing the test compound
S
(7.1.1) but no inoculum, sterilized by autoclaving or by addition of a suitable inorganic toxic compound to
prevent microbial activity. Use, for example, 1 ml/l of a solution containing 10 g/l of mercury(II) chloride (HgCl ).
2
Add the same amount of the toxic substance two weeks after the test was begun.
Add appropriate amounts of the test medium (5.2), and the inoculum (7.2) to the vessels as indicated in Table 1 to
obtain a final test volume of e.g. 3 l. Other final test volumes are possible; adapt in such a case all relevant
parameters and the calculation of test results. Connect the vessels to the CO -free air production system (see
2
annex A). Incubate at the desired test temperature (see clause 4) and aerate the vessels for 24 h to purge CO from
2
the system. Agitate throughout the test with a magnetic stirrer. If excessive foaming is observed, replace the air sparge
by headspace aeration while stirring. After the pre-aeration period, connect the air exit of each vessel to the CO
2
trapping or measuring system.
Add the test sample (7.1.1) and the reference compound (7.1.2) at the desired concentrations to the respective
vessels in accordance with Table 1 and start the test by bubbling CO -free air through the vessels with 3 l medium
2
at a rate of about 50 ml/min to 100 ml/min.
Measure the amount of CO released from each vessel at timed intervals, depending on the rate of evolution of
2
CO , using an appropriate and sufficiently accurate method (see annex B). If a nearly constant level of CO
2 2
formation is attained (plateau phase) and no further biodegradation is expected, the test is considered to be
completed. Usually the maximum test period should not exceed 28 d. Extend the test by one to two weeks, if
degradation has obviously started but has not reached a plateau.
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SIST EN ISO 9439:2000
© ISO
ISO 9439:1999(E)
Table 1 — Final distribution of test and reference compounds in the test vessels
Test Test Reference
Vessel Inoculum
medium compound compound
(5.2) (7.1.1) (7.1.2) (7.2)
F Test compound + + 2 +
T
+ + 2 +
F Test compound
T
F Blank + 2 2 +
B
+ 2 2 +
F Blank
B
F Inoculum check + 2 ++
C
F Inhibition control (optional) + + + +
I
F Abiotic elimination check (optional) + + 22
S
On the last day of the test, measure the pH, acidify all the bottles with 1 ml to 10 ml of concentrated hydrochloric
acid in order to decompose the carbonates and bicarbonates and purge the CO . Continue aeration for up to 24 h
2
and measure the amount of CO released from each vessel.
2
NOTE 1 During the handling of samples for the regular measurement of CO in the traps it cannot be excluded that,
2
especially in the case of DIC determinations, small amounts of CO from the air are included and added up during the test. This
2
has normally no effect on the test results as the CO values of the blank vessels, where the same occurs, are subtracted.
2
However, in the case of the abiotic elimination control (vessel F ) this may lead to an apparent and unjustified impression of
S
degradation. Therefore it is recommended to determine the CO evolution from vessel F only at the end of the test.
2 S
NOTE 2 If the DOC removal is measured to provide additional information on the biodegradability of a water-soluble test
compound, or if a substance-specific analytical method is used to determine the primary biodegradability, use the information
given in annex D.
8 Calculation
8.1 Amount of theoretical carbon dioxide from the test compound
The theoretical amount, in milligrams, of released carbon dioxide (ThCO ) in the test vessels is given by
2
equation (1):
44
= r ××V (1)
ThCO
2
L
C
12
where
r is the concentration of organic carbon of the test compound in the test vessel, in milligrams per litre,
C
measured or calculated from the stock solution of the test compound (7.1.1);
V is the volume of the test solution in the test vessel, expressed in litres;
L
44 and 12 are the relative molar and atomic masses of CO and carbon, respectively, to calculate the amount
2
of CO from the measured organic carbon.
2
Calculate in the same way the ThCO of the reference compound and the inhibition solution (7.1.3).
2
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SIST EN ISO 9439:2000
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8.2 Percentage biodegradation
Calculate the percentage of biodegradation (%) for each of the test vessels F for each measurement interval
D
m T
using equation (2)
∑∑m - m
TBtt
D =  × 100 (2)
m
ThCO
2
where
(m is the mass, in milligrams, of CO released in vessel F between the start of the test and time t;
Tt 2 T
( is the average mass, in milligrams, of CO released in the blank controls F between the start of the
m
Bt 2 B
test and time t.
Calculate in the same way the biodegradation degree of the reference compound in the inoculum check vessel F ,
C
and, if included, of the mixture of test and reference compound in the inhibition control F , and, without subtracting
I
the blanks, of the test compound in the abiotic elimination control F .
S
NOTE If DOC removal and primary biodegradation by substance-specific analyses were measured, it is recommended that
results be calculated according to annex D.
8.3 Expression of results
Compile a table of CO released ((m and (m ) and the percentages of biodegradation (D ) for each measuring
2 Tt Bt m
interval and each test vessel. Plot a biodegradation curve in percent as a function of time, and indicate lag phase
and degradation phase. Optionally, plot a curve of the net released CO versus time. If comparable results are
2
obtained for the duplicate test vessels F (< 20 % difference), plot a mean curve, otherwise plot curves for each
T
vessel (see example in annex C). Plot in the same way a biodegradation curve of the reference compound F and,
C
if included of the abiotic elimination check F and the inhibition control F .
S I
Determine the mean value of percent biodegradation in the plateau phase or use the highest value, e.g. when the
curve decreases in the plateau phase and indicate this maximum level of biodegradation as "degree of
biodegradation of the test compound" in the test report.
Information on the toxicity of the test compound may be useful in the interpretation of test results showing a low
biodegradation. If in vessel F the degradation percentage is < 25 % and insufficient degradation of the test
I
compound is observed, it can be assumed that the test compound is inhibitory. In this case, the test should be
repeated using a lower test concentration or another inoculum. If in vessel F (abiotic elimination check, if included)
S
a significant amount (> 10 %) of released CO is observed, abiotic degradation processes may have taken place.
2
9 Validity of results
9.1 Validity criteria
The test is considered as valid if
a) the percentage degradation in vessel F (inoculum check) is greater than 60 % on the 14th day;
C
b) the concentration of CO which has evolved from the blank F at the end of the test at a test volume of 3 l is
2 B
about 40 mg/l and does not exceed 70 mg/l;
c) the amount of DIC at the beginning of the test is < 5 % of the organic carbon of the test compound.
If a) and b) are not fulfilled, the test should be repeated using another or a better preconditioned inoculum. If c) is
not fulfilled, verify that the air for aerating the vessels is really free from CO .
2
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SIST EN ISO 9439:2000
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ISO 9439:1999(E)
9.2 Inhibition
If the vessel F (inhibition control) was included, the test compound is assumed to be inhibiting if the degradation
I
percentage of the reference compound in vessel F is lower than 40 % at the end of the test. In this case, it is
I
advisable to repeat the test with a
...