ISO 9408:1991
(Main)Water quality — Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds — Method by determining the oxygen demand in a closed respirometer
Water quality — Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds — Method by determining the oxygen demand in a closed respirometer
Qualité de l'eau — Évaluation, en milieu aqueux, de la biodégradabilité aérobie "ultime" des composés organiques — Méthode par détermination de la demande en oxygène dans un respiromètre fermé
Kakovost vode - Vrednotenje popolne aerobne biološke razgradljivosti organskih snovi v vodnem okolju - Metoda določanja potrebe po kisiku v zaprtem respirometru
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ISO
INTERNATIONAL
9408
STANDARD
First edition
1991-02-15
- Evaluation in an aqueous
Water quality
medium of the “ultimate” aerobic
biodegradability of organic compounds -
Method by determining the Oxygen demand in a
closed respirometer
Evaluation, en milieu agueux, de la biod&gradabilit&
Qualife de l’eau -
aerobie “ultime” des composes organiques - Methode par
determination de la demande en oxygene dans un respirom6tre ferm6
Reference number
ISO 9408:1991(E)
---------------------- Page: 1 ----------------------
ISO 9408:1991(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. Esch 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, govern-
mental 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.
Draft International Standards adopted by the technical committees are
circulated to the member bodies for voting. Publication as an Inter-
national Standard requires approval by at least 75 % of the member
bodies casting a vote.
International Standard ISO 9408 was prepared by Technical Committee
ISO/TC 147, Water quality.
Annexes A, 6, C and D of this International Standard are for information
only.
0 ISO 1991
All rights reserved. No part of this publication may be reproduced or utilized in any form
or by any means, electronie or mechanical, including photocopying and microfilm, without
Permission in writing from the publisher.
International Organization for Standardization
Case Postale 56 l CH-1211 Geneve 20 l Switzerland
Printed in Switzerland
ii
---------------------- Page: 2 ----------------------
INTERNATIONAL STANDARD
ISO 9408:1991 (E)
Water quality - Evaluation in an aqueous medium of the
“ultimate” aerobic biodegradability of organic compounds -
Method by determining the Oxygen demand in a closed
respirometer
agreements based on this International Standard
1 Scope
are encouraged to investigate the possibility of ap-
plying the most recent editions of the Standards in-
This International Standard specifies a method, by
dicated below. Members of IEC and ISO maintain
determination of the Oxygen demand in a closed
registers of currently valid International Standards.
respirometer, for the evaluation in an aqueous me-
dium of the “ultimate” biodegradability of organic
ISO 60603989, Water quality - Determination of the
compounds at a given concentration by aerobic
Chemical Oxygen demand.
micro-organisms.
The conditions described in this International Stan- ISO 6107-2:1989, Water quality - Vocabulary -
dard do not always correspond to the optimal con- Part 2.
ditions for allowing the maximum degree of
biodegradation to occur. ISO 7827:1984, Water quality - Evaluation in an
aqueous medium of the ” ul tima te”
aerobic
The method applies to organic compounds which
biodegradability of organic compounds - Method by
analysis of dissolved organic carbon (DOC).
a) are soluble in the test conditions;
ISO 8192:1986, Water quality - Test for inhibition of
b) are insoluble in the test conditions, in which case
Oxygen consumption by activated sludge.
special measures may be necessary to achieve
good dispersion of the compound;
3 Definitions
c) do not resch and react with the CO, absorbant;
For the purposes of this International Standard, the
d) are volatile ided that a suitable res-
1 prov
following definitions apply.
pirometer is used;
3.1 ultimate biodegradation: The level of degra-
e) are not inhibitory to the test micro-organisms at
dation achieved when the test compound is totally
the concentration Chosen for the test. The pres-
utilized by micro-organisms resulting in the pro-
ence of inhibitory effects tan be determined as
duction of carbon dioxide, water, mineral salts and
specified in 8.3, or by using any other method for
new microbial cellular constituents (biomass).
determining the inhibitory effect of a compound
on bacteria (See, for example, ISO 8192).
3.2 biochemical Oxygen demand (BOD): The mass
concentration of dissolved Oxygen consumed under
specified conditions by the biological Oxidation of
2 Normative references
organic and/or inorganic matter in water (see
ISO 6107-2;.
The following Standards contain provisions which,
through reference in this text, constitute provisions
of this International Standard. At the time of publi- 3.3 suspended solids (of an activated sludge): Sol-
ids removed by filtration or centrifuging of a known
cation, the editions indicated were valid. All stan-
dards are subject to revision, and Parties to volume of sludge under specified conditions, and,
1
---------------------- Page: 3 ----------------------
ISO 9408:1991(E)
for the purpose of this International Standard, drying
6 Reagents
at about 100 “C.
Use only reagents of recognized analytical grade.
3.4 pre-exposure (or pre-adaptation): The pre-
incubation of an inoculum in the presence of the test
6.1 Distilled or de-ionized water.
compound, with the aim of enhancing the ability of
the inoculum to degrade the test compound.
Containing less than 10 % of the initial DOC content
introduced by the organic compound to be tested.
3.5 pre-conditioning (or pre-acclimatization): The
pre-incubation of an inoculum under the conditions
6.2 Test medium
of the test in the absence of the test compound, to
improve the Performance of the test.
6.2.1 Composition
6.2.1.1 Solution (a).
4 Principle
Anhydrous potassium
Determination of the biodegradation of organic
dihydrogenphosphate (KH,PO,)
875 g
compounds by aerobic micro-organisms, using a
test medium.
Anhydrous dipotassium
21,75 g
hydrogenphosphate (K,HPO,)
The organic compound is the sole Source of carbon
Disodium hydrogenphosphate dihydrate
and energy in the medium. The concentration of the
(Na,HP0,.2H,O)
3x4 g
test compound is normally 100 mg/l, but its theor-
etical Oxygen demand (ThOD) shall be at least
Ammonium chloride (NH,CI) 095 g
100 mg/l.
Water (6.1) (quantity necessary to make
1000 ml
uP to)
The inoculated medium is stirred in a closed flask
and the consumption of Oxygen is determined either
The pH of this solution should be about 7,4.
by measuring the amount of Oxygen required to
maintain a constant gas volume in the respirometer
6.2.4.2 Solution (b).
flask, or by measuring the Change in volume or
pressure (or a combination of the two) in the appar-
Dissolve 22,5 g of magnesium sulphate hepta-
atus.
hydrate (MgSO,.7H,O) in 1000 ml of the water (6.1).
Evolved carbon dioxide is absorbed in a suitable
6.2.1.3 Solution (c).
substan ce in the test vessel.
Dissolve 27,5 g of anhydrous Calcium chloride
The degradation is followed over a period of
(CaCI,) or 36,4 g of Calcium chloride dihydrate
28 days, or longer if necessary, by determining the
(CaCl,.2H,O) in 1000 ml of the water (6.1).
consumption of Oxygen either automatically or
manually. The amount of Oxygen consumed by the
organic compound (after correction by comparison
6.2.4.4 Solution (d).
with the blank test) is expressed as a percentage of
Dissolve 0,25 g of iron(lll) chloride hexahydrate
the theoretical Oxygen demand (ThOD) calculated
(FeCI,.GH,O) in 1000 ml of the water (6.1). Prepare
from the formula of the compound or the Chemical
this Solution just before use.
Oxygen demand (COD). Additionally, the degree of
biodegradation may also be calculated from sup-
NOTE 1 It is not necessary to prepare this solution just
plemental Chemical analyses, for example, dis-
before use if a drop of concentrated hydrochloric acid
carbon (DOC) for sufficiently
solved organic
(I-ICI) or 0,4 g/l of ethylenediamine-tetraacetic acid (EDTA)
water-soluble compounds or specific analysis (con-
is added.
cerning only primary biodegradation), made at the
beginning and the end of incubation. Evaluation of
6.2.2 Preparation of the test medium.
the biodegradability of the test compound is made
on the basis of these data.
For 1 litre of test medium, just before use add to
800 ml of the water (6.1)
5 Test environment
- 10 ml of Solution (a);
lncubation shall take place in the dark or in diffused
-
and then 1 ml of each Solution (b), (c) and (d), (to
light, in an enclosure which is maintained at a con-
avoid the formation of turbidity in the final me-
stant temperature (within at least + 1 OC) between
dium).
20 OC and 25 “C and which is free from toxic va-
pours.
Make up to 1000 ml with the water (6.1).
2
---------------------- Page: 4 ----------------------
ISO 9408:1991 (E)
NOTE 2 Compounds of low water solubility may be
6.3 Carbon dioxide absorber, Potassium hydroxide
added directly, in solid or liquid form, to the medium in the
Solution (about 10 mol/l), sodium carbonate pellets
appropriate flask; an ISO International Standard will be
or another suitable absorbant.
prepared for guidance.
7 Apparatus
Ensure that all glassware is thoroughly cleaned and,
in particular, free from organic or toxic matter.
8.2 Preparation of the inoculum
Usual laboratory equipment, and
Take a Sample of activated sludge from the aeration
7.1 Closed respirometer.
tank of a biological wastewater treatment plant, or
a laboratory unit, dealing with predominantly dom-
The principle of a closed respirometer is given in
estic sewage. If the sludge is actively respiring on
annex D. When testing volatile compounds, the ap-
external Substrate, bring it to the “endogenous”
paratus used shall be appropriate or adapted to this
Phase (i.e. having no external Substrate remaining)
particular purpose. Care shall be taken that there is
as follows:
no loss of compound due to the apparatus.
-
either aerate for a few hours before use, or
7.2 Water-bath or constant temperature room (to
-
comply with clause 5). centrifuge, wash with medium (6.2), recentrifuge
and resuspend in the medium (this treatment is
recommended if it is suspected that the sludge
7.3 Equipment for measurement of dissolved or-
contains inhibiting matter).
ganic carbon, instrument of sufficient sensitivity for
the measurement of dissolved organic carbon
When the sludge is judged to be in the
(DOC).
“endogenous” Phase, or free from inhibiting matter,
mix Welf, maintain in an aerobic state by stirring or
aeration at the required temperature, and use on the
7.4 Device for determining Chemical Oxygen de-
day of collection or one day Iater. Just before use,
mand (COD).
determine the concentration of suspended solids. If
required, concentrate the sludge by settling, so that
7.5 Device for filtration, with membrane filters of
the volume of sludge added to obtain 30 mg/1 of dry
suitable porosity (nominal aperture diameter be-
matter in the reaction mixture is less than or equal
tween 0,2 pm and 0,45 um) which adsorb organic
to 1 % of the mixture, that is, the sludge should
compounds or release organic carbon to a minimum
contain at least 3 g/l of dry solids.
degree (see note 3 to 8.3).
NOTES
7.6 Centrifuge.
3 A concentration of 30 mg/l of suspended solids in the
final medium has been found suitable for concentrations
of test compound in the range of 50 mg/1 to 150 mg/l. The
7.7 pH=meter.
Oxygen consumption of the blank Solution shall not be
greater than 60 mg/l in 28 days and should normally be in
the range of 20 mg/l to 30 mg/l. In Order to reduce the in-
8 Procedure
fluence of the blank, the sludge may be pre-conditioned
(see 3.5) by aeration for up to one week before it is used.
8.1 Preparation of the test solutions
4 Secondary effluent and surface water may also be
inocula may have to be con-
used as inoculum, but these
Prepare the following solutions:
centrifugation to get more
centrated by filtration or
biomass.
a) Solution of the test compound in the test medium
(6.2) to obtain a concentration of 100 mg/l, but at
5 Pre-exposed inocula may be used for certain purposes.
least 100 mg/1 ThOD;
When such inocula are used this should be clearly stated
in the test results (e.g. percentage biodegradation
b) solution of a known organic compound (“refer- = x %, using pre-exposed inocula) and the method of
pre-exposure detailed in the test report.
ence” compound), for example, sodium acetate,
sodium benzoate, aniline, in the test medium
Pre-exposed inocula tan be obtained from laboratory bio-
(6.2) to obtain a concentration of 100 mg/l;
degradation tests run under a variety of conditions (e.g.
Zahn-WeIlens and SCAS tests) or from samples collected
c) Solution containing, in the test medium (6.2), the
from locations where relevant environmental conditions
Same concentrations of the test compound and exist (e.g. treatment plants dealing with identical com-
pounds, contaminated areas, etc.).
the reference compound as used in a) and b).
---------------------- Page: 5 ----------------------
ISO 9408:1991 (E)
When DOC or the concentration of a specific com-
8.3 Test
pound is being monitored, withdraw appropriately
sized samples from the respirometric flasks at the
Set up the closed respirometer (see 7.1 and the ex-
beginning and end of the test period (alternatively,
ample described in annex 0). Assemble a sufficient
the initial value may be calculated). Either filter
number of reaction vessels in Order to have at least
these samples through a membrane filter (see 7.5)
a) two test flasks (Symbol FT), containing the ap- or centrifuge at 4000 g for 15 min (see 7.6). When
measurements of organic carbon are not conducted
propriate volume of test solution [8.1, item a)];
the Same day, keep the samples at 4 OC in the dark
and in tightly stoppered glass flasks. lt should be
b) two flasks for the blank test (Symbol Fg), con-
noted that the maximum allowable storage time is
taining the appropriate volume of the test me-
up to 48 h.
dium (6.2);
If the test compound contains nitrogen, determine
c) if needed, one flask (Symbol FC), for chec
...
SLOVENSKI STANDARD
SIST ISO 9408:1997
01-maj-1997
.DNRYRVWYRGH9UHGQRWHQMHSRSROQHDHUREQHELRORãNHUD]JUDGOMLYRVWLRUJDQVNLK
VQRYLYYRGQHPRNROMX0HWRGDGRORþDQMDSRWUHEHSRNLVLNXY]DSUWHP
UHVSLURPHWUX
Water quality -- Evaluation in an aqueous medium of the "ultimate" aerobic
biodegradability of organic compounds -- Method by determining the oxygen demand in
a closed respirometer
Qualité de l'eau -- Évaluation, en milieu aqueux, de la biodégradabilité aérobie "ultime"
des composés organiques -- Méthode par détermination de la demande en oxygène
dans un respiromètre fermé
Ta slovenski standard je istoveten z: ISO 9408:1991
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST ISO 9408:1997 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
SIST ISO 9408:1997
---------------------- Page: 2 ----------------------
SIST ISO 9408:1997
ISO
INTERNATIONAL
9408
STANDARD
First edition
1991-02-15
- Evaluation in an aqueous
Water quality
medium of the “ultimate” aerobic
biodegradability of organic compounds -
Method by determining the Oxygen demand in a
closed respirometer
Evaluation, en milieu agueux, de la biod&gradabilit&
Qualife de l’eau -
aerobie “ultime” des composes organiques - Methode par
determination de la demande en oxygene dans un respirom6tre ferm6
Reference number
ISO 9408:1991(E)
---------------------- Page: 3 ----------------------
SIST ISO 9408:1997
ISO 9408:1991(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. Esch 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, govern-
mental 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.
Draft International Standards adopted by the technical committees are
circulated to the member bodies for voting. Publication as an Inter-
national Standard requires approval by at least 75 % of the member
bodies casting a vote.
International Standard ISO 9408 was prepared by Technical Committee
ISO/TC 147, Water quality.
Annexes A, 6, C and D of this International Standard are for information
only.
0 ISO 1991
All rights reserved. No part of this publication may be reproduced or utilized in any form
or by any means, electronie or mechanical, including photocopying and microfilm, without
Permission in writing from the publisher.
International Organization for Standardization
Case Postale 56 l CH-1211 Geneve 20 l Switzerland
Printed in Switzerland
ii
---------------------- Page: 4 ----------------------
SIST ISO 9408:1997
INTERNATIONAL STANDARD
ISO 9408:1991 (E)
Water quality - Evaluation in an aqueous medium of the
“ultimate” aerobic biodegradability of organic compounds -
Method by determining the Oxygen demand in a closed
respirometer
agreements based on this International Standard
1 Scope
are encouraged to investigate the possibility of ap-
plying the most recent editions of the Standards in-
This International Standard specifies a method, by
dicated below. Members of IEC and ISO maintain
determination of the Oxygen demand in a closed
registers of currently valid International Standards.
respirometer, for the evaluation in an aqueous me-
dium of the “ultimate” biodegradability of organic
ISO 60603989, Water quality - Determination of the
compounds at a given concentration by aerobic
Chemical Oxygen demand.
micro-organisms.
The conditions described in this International Stan- ISO 6107-2:1989, Water quality - Vocabulary -
dard do not always correspond to the optimal con- Part 2.
ditions for allowing the maximum degree of
biodegradation to occur. ISO 7827:1984, Water quality - Evaluation in an
aqueous medium of the ” ul tima te”
aerobic
The method applies to organic compounds which
biodegradability of organic compounds - Method by
analysis of dissolved organic carbon (DOC).
a) are soluble in the test conditions;
ISO 8192:1986, Water quality - Test for inhibition of
b) are insoluble in the test conditions, in which case
Oxygen consumption by activated sludge.
special measures may be necessary to achieve
good dispersion of the compound;
3 Definitions
c) do not resch and react with the CO, absorbant;
For the purposes of this International Standard, the
d) are volatile ided that a suitable res-
1 prov
following definitions apply.
pirometer is used;
3.1 ultimate biodegradation: The level of degra-
e) are not inhibitory to the test micro-organisms at
dation achieved when the test compound is totally
the concentration Chosen for the test. The pres-
utilized by micro-organisms resulting in the pro-
ence of inhibitory effects tan be determined as
duction of carbon dioxide, water, mineral salts and
specified in 8.3, or by using any other method for
new microbial cellular constituents (biomass).
determining the inhibitory effect of a compound
on bacteria (See, for example, ISO 8192).
3.2 biochemical Oxygen demand (BOD): The mass
concentration of dissolved Oxygen consumed under
specified conditions by the biological Oxidation of
2 Normative references
organic and/or inorganic matter in water (see
ISO 6107-2;.
The following Standards contain provisions which,
through reference in this text, constitute provisions
of this International Standard. At the time of publi- 3.3 suspended solids (of an activated sludge): Sol-
ids removed by filtration or centrifuging of a known
cation, the editions indicated were valid. All stan-
dards are subject to revision, and Parties to volume of sludge under specified conditions, and,
1
---------------------- Page: 5 ----------------------
SIST ISO 9408:1997
ISO 9408:1991(E)
for the purpose of this International Standard, drying
6 Reagents
at about 100 “C.
Use only reagents of recognized analytical grade.
3.4 pre-exposure (or pre-adaptation): The pre-
incubation of an inoculum in the presence of the test
6.1 Distilled or de-ionized water.
compound, with the aim of enhancing the ability of
the inoculum to degrade the test compound.
Containing less than 10 % of the initial DOC content
introduced by the organic compound to be tested.
3.5 pre-conditioning (or pre-acclimatization): The
pre-incubation of an inoculum under the conditions
6.2 Test medium
of the test in the absence of the test compound, to
improve the Performance of the test.
6.2.1 Composition
6.2.1.1 Solution (a).
4 Principle
Anhydrous potassium
Determination of the biodegradation of organic
dihydrogenphosphate (KH,PO,)
875 g
compounds by aerobic micro-organisms, using a
test medium.
Anhydrous dipotassium
21,75 g
hydrogenphosphate (K,HPO,)
The organic compound is the sole Source of carbon
Disodium hydrogenphosphate dihydrate
and energy in the medium. The concentration of the
(Na,HP0,.2H,O)
3x4 g
test compound is normally 100 mg/l, but its theor-
etical Oxygen demand (ThOD) shall be at least
Ammonium chloride (NH,CI) 095 g
100 mg/l.
Water (6.1) (quantity necessary to make
1000 ml
uP to)
The inoculated medium is stirred in a closed flask
and the consumption of Oxygen is determined either
The pH of this solution should be about 7,4.
by measuring the amount of Oxygen required to
maintain a constant gas volume in the respirometer
6.2.4.2 Solution (b).
flask, or by measuring the Change in volume or
pressure (or a combination of the two) in the appar-
Dissolve 22,5 g of magnesium sulphate hepta-
atus.
hydrate (MgSO,.7H,O) in 1000 ml of the water (6.1).
Evolved carbon dioxide is absorbed in a suitable
6.2.1.3 Solution (c).
substan ce in the test vessel.
Dissolve 27,5 g of anhydrous Calcium chloride
The degradation is followed over a period of
(CaCI,) or 36,4 g of Calcium chloride dihydrate
28 days, or longer if necessary, by determining the
(CaCl,.2H,O) in 1000 ml of the water (6.1).
consumption of Oxygen either automatically or
manually. The amount of Oxygen consumed by the
organic compound (after correction by comparison
6.2.4.4 Solution (d).
with the blank test) is expressed as a percentage of
Dissolve 0,25 g of iron(lll) chloride hexahydrate
the theoretical Oxygen demand (ThOD) calculated
(FeCI,.GH,O) in 1000 ml of the water (6.1). Prepare
from the formula of the compound or the Chemical
this Solution just before use.
Oxygen demand (COD). Additionally, the degree of
biodegradation may also be calculated from sup-
NOTE 1 It is not necessary to prepare this solution just
plemental Chemical analyses, for example, dis-
before use if a drop of concentrated hydrochloric acid
carbon (DOC) for sufficiently
solved organic
(I-ICI) or 0,4 g/l of ethylenediamine-tetraacetic acid (EDTA)
water-soluble compounds or specific analysis (con-
is added.
cerning only primary biodegradation), made at the
beginning and the end of incubation. Evaluation of
6.2.2 Preparation of the test medium.
the biodegradability of the test compound is made
on the basis of these data.
For 1 litre of test medium, just before use add to
800 ml of the water (6.1)
5 Test environment
- 10 ml of Solution (a);
lncubation shall take place in the dark or in diffused
-
and then 1 ml of each Solution (b), (c) and (d), (to
light, in an enclosure which is maintained at a con-
avoid the formation of turbidity in the final me-
stant temperature (within at least + 1 OC) between
dium).
20 OC and 25 “C and which is free from toxic va-
pours.
Make up to 1000 ml with the water (6.1).
2
---------------------- Page: 6 ----------------------
SIST ISO 9408:1997
ISO 9408:1991 (E)
NOTE 2 Compounds of low water solubility may be
6.3 Carbon dioxide absorber, Potassium hydroxide
added directly, in solid or liquid form, to the medium in the
Solution (about 10 mol/l), sodium carbonate pellets
appropriate flask; an ISO International Standard will be
or another suitable absorbant.
prepared for guidance.
7 Apparatus
Ensure that all glassware is thoroughly cleaned and,
in particular, free from organic or toxic matter.
8.2 Preparation of the inoculum
Usual laboratory equipment, and
Take a Sample of activated sludge from the aeration
7.1 Closed respirometer.
tank of a biological wastewater treatment plant, or
a laboratory unit, dealing with predominantly dom-
The principle of a closed respirometer is given in
estic sewage. If the sludge is actively respiring on
annex D. When testing volatile compounds, the ap-
external Substrate, bring it to the “endogenous”
paratus used shall be appropriate or adapted to this
Phase (i.e. having no external Substrate remaining)
particular purpose. Care shall be taken that there is
as follows:
no loss of compound due to the apparatus.
-
either aerate for a few hours before use, or
7.2 Water-bath or constant temperature room (to
-
comply with clause 5). centrifuge, wash with medium (6.2), recentrifuge
and resuspend in the medium (this treatment is
recommended if it is suspected that the sludge
7.3 Equipment for measurement of dissolved or-
contains inhibiting matter).
ganic carbon, instrument of sufficient sensitivity for
the measurement of dissolved organic carbon
When the sludge is judged to be in the
(DOC).
“endogenous” Phase, or free from inhibiting matter,
mix Welf, maintain in an aerobic state by stirring or
aeration at the required temperature, and use on the
7.4 Device for determining Chemical Oxygen de-
day of collection or one day Iater. Just before use,
mand (COD).
determine the concentration of suspended solids. If
required, concentrate the sludge by settling, so that
7.5 Device for filtration, with membrane filters of
the volume of sludge added to obtain 30 mg/1 of dry
suitable porosity (nominal aperture diameter be-
matter in the reaction mixture is less than or equal
tween 0,2 pm and 0,45 um) which adsorb organic
to 1 % of the mixture, that is, the sludge should
compounds or release organic carbon to a minimum
contain at least 3 g/l of dry solids.
degree (see note 3 to 8.3).
NOTES
7.6 Centrifuge.
3 A concentration of 30 mg/l of suspended solids in the
final medium has been found suitable for concentrations
of test compound in the range of 50 mg/1 to 150 mg/l. The
7.7 pH=meter.
Oxygen consumption of the blank Solution shall not be
greater than 60 mg/l in 28 days and should normally be in
the range of 20 mg/l to 30 mg/l. In Order to reduce the in-
8 Procedure
fluence of the blank, the sludge may be pre-conditioned
(see 3.5) by aeration for up to one week before it is used.
8.1 Preparation of the test solutions
4 Secondary effluent and surface water may also be
inocula may have to be con-
used as inoculum, but these
Prepare the following solutions:
centrifugation to get more
centrated by filtration or
biomass.
a) Solution of the test compound in the test medium
(6.2) to obtain a concentration of 100 mg/l, but at
5 Pre-exposed inocula may be used for certain purposes.
least 100 mg/1 ThOD;
When such inocula are used this should be clearly stated
in the test results (e.g. percentage biodegradation
b) solution of a known organic compound (“refer- = x %, using pre-exposed inocula) and the method of
pre-exposure detailed in the test report.
ence” compound), for example, sodium acetate,
sodium benzoate, aniline, in the test medium
Pre-exposed inocula tan be obtained from laboratory bio-
(6.2) to obtain a concentration of 100 mg/l;
degradation tests run under a variety of conditions (e.g.
Zahn-WeIlens and SCAS tests) or from samples collected
c) Solution containing, in the test medium (6.2), the
from locations where relevant environmental conditions
Same concentrations of the test compound and exist (e.g. treatment plants dealing with identical com-
pounds, contaminated areas, etc.).
the reference compound as used in a) and b).
---------------------- Page: 7 ----------------------
SIST ISO 9408:1997
ISO 9408:1991 (E)
When DOC or the concentration of a specific com-
8.3 Test
pound is being monitored, withdraw appropriately
sized samples from the respirometric flasks at the
Set up the closed respirometer (see 7.1 and the ex-
beginning and end of the test period (alternatively,
ample described in annex 0). Assemble a sufficient
the initial value may be calculated). Either filter
number of reaction vessels in Order to have at least
these samples through a membrane filter (see 7.5)
a) two test flasks (Symbol FT), containing the ap- or centrifuge at 4000 g for 1
...
IS0
ATIONALE 9408
Première édition
1991 -02-1 5
Qualité de l’eau - Evaluation, en milieu aqueux,
de la biodégradabilité aérobie (
composés organiques - Méthode par
détermination de la demande en oxygène dan
un respiromètre fermé
Water quality - Evaluation in an aqueous medium of the «ultimate>>
aerobic biodegradability of organic compounds - Method by
determining the oxygen demand in a closed respirometer
Numéro de référence
IS0 9408:1991(F)
---------------------- Page: 1 ----------------------
Avant-propos
L’ISO (Organisation internationale de normalisation) est une fédération
mondiale d’organismes nationaux de normalisation (comités membres
de I’ISO). L’élaboration des Normes internationales est en général
confiée aux comités techniques de I’ISO. Chaque comité membre inté-
ressé par une étude a le droit de faire partie du comité technique créé
à cet effet. Les organisations internationales, gouvernementales et non
gouvernementales, en liaison avec l’lS0 participent également aux tra-
vaux. L’ISO collabore étroitement avec la Commission électrotechnique
internationale (CEI) en ce qui concerne la normalisation électrotech-
nique.
Les projets de Normes internationales adoptés par les comités techni-
ques sont soumis aux comités membres pour vote. Leur publication
comme Normes internationales requiert l’approbation de 75 % au moins
des comités membres votants.
La Norme internationale IS0 9408 a été élaborée par le comité techni-
que ISO/TC 147, Qualité de /’eau.
Les annexes A, B, C et D de la présente Norme internationale sont
données uniquement à titre d’information.
8 IS0 1991
Droits de reproduction reserves. Aucune partie de cette publication ne peut être repro-
duite ni utilisée sous quelque forme que ce soit et par aucun procede, électronique ou
mécanique, y compris la photocopie et les microfilms, sans l’accord écrit de l’éditeur.
Organisation internationale de normalisation
Case Postale 56 CH-I211 Genève 20 Suisse
Imprimé en Suisse
ii
---------------------- Page: 2 ----------------------
NORME INTERNATIONALE IS0 9408:1991(F)
Qualité de l’eau - Évaluation, en milieu aqueux, de la
biodégradabilité aérobie wltime~~ des composés
organiques - Méthode par détermination de la demande en
oxygène dans un respiromètre fermé
1 Domaine d’application 2 References normatives
Les normes suivantes contiennent des dispositions
qui, par suite de la référence qui en est faite,
constituent des dispositions valables pour la pré-
La présente Norme internationale prescrit une mé- sente Norme internationale. Au moment de la pu-
thode pour l’évaluation en milieu aqueux de la bio- blication, les éditions indiquées étaient en vigueur.
Toute norme est sujette à révision et les parties
dégradabilité <(ultime>, de composés organiques
présents à une concentration donnée sous l’action prenantes des accords fondés sur la présente
de micro-organismes aérobies, par détermination Norme internationale sont invitées à rechercher la
de la demande en oxygène dans un respiromètre possibilité d’appliquer les éditions les plus récentes
fermé. des normes indiquées ci-après. Les membres de la
CE1 et de I’lSO possèdent le registre des Normes
Les conditions décrites dans la présente Norme
internationales en vigueur à un moment donné.
internationale ne correspondent pas toujours aux
conditions optimales d‘obtention de la biodégrada-
IS0 6060:1989, Qualité de /’eau - Détermination de
tion maximum.
la demande chimique en oxygène.
La méthode peut s’appliquer à des composés orga-
IS0 6107-2:1989, Qualité de l’eau - Vocabulaire -
niques
Partie 2.
a) solubles dans les conditions de l’essai;
IS0 7827:1984, Qualité de /’eau - Évaluation en mi-
lieu aqueux de la biodégradabilité aérobie «ultime»
b) insolubles dans les conditions de l’essai, auquel
des composés organiques - Méthode par analyse
cas il peut être nécessaire de prendre des me-
du carbone organique dissous (COD).
sures particulières assurant une bonne disper-
sion du composé:
IS0 8192:1986, Qualité de l‘eau - Essai d‘inhibition
de la consommation d‘oxygène par des boues acti-
c) n’atteignant pas l’absorbant de CO, et ne ré-
vées.
agissant pas avec lui;
d) volatils, à condition d’utiliser un respiromètre
3 Definitions
adapté;
Pour les besoins de la présente Norme internatio-
e) n’ayant pas d’action inhibitrice, à la concen-
nale, les définitions suivantes s’appliquent.
tration choisie, sur les micro-organismes utilisés
pour l’essai. L’existence d’une action inhibitrice
peut être mise en évidence suivant la méthode
3.1 dégradation ultime: Niveau de dégradation at-
prescrite en 8.3, ou par toute autre méthode de
teint lorsque le composé soumis à l’essai a été to-
détermination de l’action inhibitrice d’une sub-
talement transformé par les micro-organismes en
stance sur les bactéries (voir par exemple
dioxyde de carbone, eau, sels minéraux et nouveaux
IS0 8192).
con st it u a nts ce I I u lai res microbiens (biomasse).
1
---------------------- Page: 3 ----------------------
IS0 9408:1991 (F)
3.2 demande biochimique en oxygène (DBO): composé organique s’effectue à partir de ces don-
Concentration en masse de l’oxygène dissous nées.
consommé, dans des conditions définies, lors de
I’oxydat ion biologique de matières organ iq ues et/ou
5 Environnement d’essai
inorganiques contenues dans l’eau (voir
IS0 6107-2).
L’incubation doit être menée à l’obscurité ou sous
lumière diffuse dans une enceinte maintenue à
3.3 matières en suspension (dans une boue acti-
température constante (à I 1 OC près) comprise
vée): Matières éliminées par filtration ou centrifu-
entre 20 OC et 25 OC, et ne contenant pas de vapeurs
gation d’un volume connu de boue dans des
toxiques .
conditions définies et, dans le cadre de la présente
Norme internationale. dessiccation à environ 100 OC.
6 Réactifs
3.4 préexposition (ou préadaptation): Préincu bation
Utiliser exclusivement des réactifs de qualité analy-
d’un inoculum en présence du composé à expéri-
tique reconnue.
menter, destinée à accroître l‘aptitude de I‘inoculum
à dégrader le composé.
6.1 Eau distillée ou déminéralisée.
3.5 préconditionnement: Préincubation d’un
Elle doit contenir moins de 10 % de la concentration
inoculum dans les conditions d’essai, mais en I’ab-
0
initiale du COD apporté par le composé organique.
sence du composé à expérimenter, destinée à
améliorer l’efficacité de l’essai.
6.2 Milieu d’essai
4 Principe
6.2.1 Composition
Évaluation de la biodégradation de composés orga-
6.2.1.1 Solution (a).
niques par des micro-organismes aérobies, dans un
milieu d’essai.
Dihydrogénophosphate de potassium
anhydre (KH,PO,) 8,5 g
Le composé organique à expérimenter constitue la
seule source de carbone et d’énergie dans le milieu.
Monohydrogénophosphate de potas-
Sa concentration est normalement de 100 mg/l,
sium anhydre (K,HPO,) 21,75 g
mais sa demande théorique en oxygène (DThO) doit
Monohydrogénophosphate de sodium
être au moins égale à 100 mg/l.
dihydraté (Na2HP0,,2H,O) 33,4 g
Le milieu ensemencé est agité dans une fiole fer-
Chlorure d’ammonium (“,CI) 0,5 g
mée et la consommation d’oxygène déterminée par
Eau (6.1): compléter à 1 O00 ml
mesurage de la quantité d’oxygène nécessaire au
maintien d’un volume de gaz constant dans le
Le pH de la solution doit normalement être d’envi-
respiromètre, ou des variations de volume et/ou de
0
ron 7.4.
pression dans l’appareil.
6.2.1.2 Solution (b).
Le dioxyde de carbone dégagé est absorbé par une
substance appropriée, à l’intérieur de la fiole d’es-
Dissoudre dans 1 O00 ml d’eau (6.1), 22,5 g de sul-
sai.
fate de magnésium heptahydraté (MgS0,,7H20).
On suit la dégradation sur une période de 28 jours,
6.2.1.3 Solution (c).
ou davantage si nécessaire, en déterminant la
consommation d’oxygène de facon automatique ou
Dissoudre dans 1 O00 ml d’eau (6.1), 27,5 g de chlo-
manuelle. La quantité d‘oxygène consommé par le
rure de calcium anhydre (CaCi,) ou 36,4 g de chlo-
composé organique (après correction par compa-
rure de calcium dihydraté (CaC1,,2H20).
raison avec l’essai à blanc) est exprimée en pour-
centage de la demande théorique en oxygène
6.2.1.4 Solution (d).
(DThO) calculée à partir de la formule chimique du
composé ou de la demande chimique en oxygène
Dissoudre dans 1 O00 ml d’eau (6.1), 0,25 g de chlo-
(DCO). Par ailleurs, le taux de biodégradation peut
rure de fer (Ill) hexahydraté (FeCI,,GH,O). Préparer
également être calculé à partir de résultats d’ana-
la solution juste avant emploi.
lyses complémentaires, par exemple le dosage du
carbone organique dissous (COD), ou d’analyses
NOTE 1 Cette précaution n’est pas nécessaire si l’on
spécifiques (concernant seulement la biodégrada-
ajoute à la solution une goutte d’acide chlorhydrique
tion primaire), réalisées en début ou fin
concentré (HCI) ou 0,4 g/l d‘acide
d’incubation. L’évaluation de la biodégrabilité du éthylènediaminetétraacétique (EDTA).
2
---------------------- Page: 4 ----------------------
IS0 9408:1991 (F)
6.2.2 Préparation du milieu d’essai.
8 Mode opératoire
Pour 1 I de milieu d’essai, ajouter juste avant em-
ploi, à 800 ml d’eau (6.1)
8.1 Préparation des solutions d’essai
- 10 ml de solution (a);
Préparer les solutions suivantes:
- puis (pour éviter l’apparition de turbidité dans le
a) solution à 100 mg/l, mais de DThO au moins
milieu final) 1 ml de chacune des solutions (b),
égale à 100 mg/l, du composé organique à ex-
(cl et (d).
périmenter dans le milieu d’essai (6.2);
Compléter à 1 O00 ml avec de l’eau (6.1).
b) solution à 100 mg/l d’un composé organique
connu (dit composé de référence) dans le milieu
6.3 Absorbant du dioxyde de carbone, solution
d’essai (6.2). Le composé de référence peut être,
d‘hydroxyde de potassium (à environ 10 mol/l),
par exemple, de l’acétate de sodium, du
pastilles de carbonate de sodium ou tout autre ab-
benzoate de sodium ou de l’aniline;
sorbant approprié.
c) solution contenant, dans le milieu d’essai (6.2),
le composé à expérimenter et le composé de
0 7 Appareillage
référence aux mêmes concentrations que dans
les solutions a) et b).
S’assurer que la verrerie de laboratoire a été soi-
gneusement nettoyée et, notamment, qu’elle est
NOTE 2 Les composés faiblement solubles dans l’eau
exempte de toute trace de substances organiques peuvent être ajoutés directement au milieu, sous forme
liquide ou solide, dans la fiole appropriée. Une Norme
ou toxiques.
internationale sera ultérieurement élaborée comme di-
rective à ce sujet.
Matériel courant de laboratoire. et
7.1 Respiromètre fermé.
8.2 Préparation de I’inoculum
Le principe de fonctionnement d’un respiromètre
Prélever un échantillon de boue activée dans le
fermé est décrit dans l’annexe D. Pour les essais
bassin d’aération d’une usine de traitement biologi-
portant sur des composés volatils, il faut utiliser un
que des eaux résiduaires, ou d’un laboratoire, trai-
appareil spécifique ou adapté à cet usage particu-
tant essentiellement des déchets domestiques. Si la
lier. Aucune perte de composant ne doit se produire
boue respire activement sur substrat externe, la
du fait de l’appareil.
faire passer en phase <
supprimer tout substrat externe) suivant l’une des
7.2 Bain-marie ou enceinte isotherme, (voir pres-
deux méthodes suivantes:
0 criptions de l‘article 5).
- aérer pendant quelques heures avant l’emploi,
ou
7.3 Matériel de mesurage du carbone organique
dissous, instrument de mesure du carbone organi-
- centrifuger, laver avec le milieu (6.2),
que dissous (COD) de sensibilité suffisante.
recentrifuger et remettre en suspension dans le
milieu (ce traitement est recommandé lorsqu’on
soupqonne la présence dans la boue de sub-
7.4 Dispositif de détermination de la demande chi-
stances inhibitrices).
mique en oxygène (DCO).
Lorsqu’on estime qu’elle est en phase <,
ou exempte de substances inhibitrices, bien mélan-
7.5 Dispositif de filtration, comportant des mem-
ger la boue, la maintenir en conditions aérobies en
branes de porosité adéquate (diamètre nominal des
l’agitant ou en l’aérant à la température requise, et
pores de 0,2 pm à 0,45 pm), dans lequel l’adsorption
l’utiliser le jour du prélèvement ou au plus tard le
des composés organiques ou le relargage du car-
lendemain. Déterminer, juste avant l’emploi, la
bone organique sont réduits au minimum (voir note
concentration des matières en suspension. Si né-
3 en 8.3 ).
cessaire, concentrer la boue par décantation, afin
que le volume de boue à utiliser pour obtenir
30 mg/l de matière sèche dans le mélange
7.6 Centrifugeuse.
réactionnel soit inférieur ou égal à 1 % du mélange,
c’est-à-dire que la boue doit normalement contenir
7.7 pH-mètre. au moins 3 g/l de matière sèche.
3
---------------------- Page: 5 ----------------------
IS0 94081991(F)
NOTES
e) pour la mise en évidence d’une éventuelle action
inhibitrice du composé à expérimenter sur I’ac-
3 On a pu constater qu’une concentration de matières
tivité microbienne, une fiole (symbole FI) (voir
en suspension de 30 mg/l convient pour des concen-
note 7).
trations du composé à experimenter comprises entre
50 mgll et 150 mg/l. La consommation d’oxygène dans la
Mesurer le pH et l’ajuster à 7,4 si nécessaire.
solution à blanc ne doit pas dépasser 60 mg/l en 28 jours
et doit normalement être comprise entre 20 mg/l et
Introduire l’absorbant (6.3) dans les compartiments
30 mg/l. Pour réduire l’influence du blanc, on peut
d’absorption de CO, du respiromètre.
préconditionner la boue (voir 34, par aération pendant
au plus une semaine avant utilisation.
Placer les fioles du respiromètre dans le bain-marie
ou l’enceinte isotherme (7.2) et attendre qu’elles
4 L‘utilisation d’inocula provenant d’effluents secondai-
aient toutes atteint la température désirée (article
res ou d’eau de surface est également admise, mais il
5). Introduire dans les fioles FT, FB, Fc et éven-
peut être nécessaire de concentrer ces inocula, par fil-
tration ou centrifugation, pour obtenir une biomasse plus tuellement FI, le volume d’inoculum nécessaire pour
importante.
obtenir, dans chacune, une concentration de matiè-
res en suspension de 30mg/l. Fermer herméti-
5 Dans certaines circonstances, il est admis d’utiliser
quement les fioles, effectuer les branchements
des inocula préexposés, à condition que cela soit clai-
nécessaires pour les respiromètres automatiques,
rement mentionné dans les résultats d’essai (par exem-
et mettre l’agitateur en marche.
ple: pourcentage de biodégradation = x %, avec inocula
préexposés) et que la méthode de préexposition soit dé-
Effectuer les lectures nécessaires sur les manomè-
taillée dans le rapport d’essai.
tres (essai manuel) ou vérifier que l’enregistreur de
Des inocula préexposés peuvent être obtenus à partir
consommation d’oxygène fonctionne correctement
d’essais de biodégradation en laboratoire effectués dans
(res piromèt re automatique).
différentes conditions (par exemple, essais SCAS ou de
Zahn-Wellens) ou à partir d’échantillons prélevés à des
Arrêter l’essai au bout de 28 jours, ou plus tôt si le
emplacements où sont réunies les conditions d’environ-
plateau de la courbe de consommation d’oxygène
nement appropriées (par exemple, usines assurant le
est atteint. Prolonger l’essai de 1 semaine à 2 se-
traitement de composés identiques, zones contaminées,
maines si, au 28ème jour, la dégradation a visi-
etc.).
blement commencé, mais que le palier n‘a pas été
atteint.
Vérifier le pH des mélanges réactionnels finaux à la
fin de l’essai.
8.3
...
Questions, Comments and Discussion
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