EN ISO 9439:2000

SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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)
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
ii
© 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.
iii
© ISO
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
CO remains in the medium in the vessels as dissolved inorganic carbon (DIC), the concentration of which may
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
removal measurement. The consequence may be that the biodegradation curves based on the trapped CO may
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.
iv
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
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
© ISO
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
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.
© ISO
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.
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
in water (5.1), quantity necessary to make up to 1 000 ml
© 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),
...