Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium by determination of oxygen demand in a closed respirometer (ISO 9408:1999)

Migrated from Progress Sheet (TC Comment) (2000-07-10): N 315: New TD (980916)

Wasserbeschaffenheit - Bestimmung der vollständigen aerobiologischen Abbaubarkeit organischen Stoffe im wäßrigen Medium über die Bestimmung des Sauerstoffbedarfs in einem geschlossenen Respirometer (ISO 9408:1999)

Diese Internationale Norm legt ein Verfahren zur Bestimmung der vollständigen biologischen Abbaubarkeit einer organischen Substanz bei einer vorgegebenen Konzentration in einem wäßrigen Medium durch aerobe Mikroorganismen über die Bestimmung des Sauerstoffbedarfs in einem geschlossenen Respirometer fest. Das Verfahren ist auf organische Substanzen anwendbar, die folgende Eigenschaften besitzen: a) wasserlöslich unter den Testbedingungen; b) schwer wasserlöslich unter den Testbedingungen. In diesem Fall können besondere Maßnahmen erforderlich sein, um eine gute Dispersion der Prüfsubstanz zu erreichen ; etc.

Qualité de l'eau - Evaluation de la biodégradibilité aérobieultime des composés organiques, en milieux aqueux, par détermination de la demande en oxygène dans un respiromètre ferméomètre fermé (ISO 9408:1999)

La présente Norme internationale spécifie une méthode d'évaluation, en milieu aqueux, de la biodégradabilité ultime de composés organiques et d'eaux résiduaires présents à une concentration donnée sous l'action de microorganismes aérobies, par détermination de la demande en oxygène dans un respiromètre fermé.  La méthode est applicable à des composés organiques :  a) solubles dans l'eau dans les conditions de l'essai;  b) peu solubles dans l'eau dans les conditions de l'essai, auquel cas il peut être nécessaire de prendre des mesures particulières assurant une bonne dispersion du composé (voir, par exemple, l'ISO 10634) ;  c) n'atteignant pas l'absorbant de C02 et ne réagissant pas avec lui ;  d) volatils, à condition d'utiliser un respiromètre adapté ou des conditions appropriées (par exemple un plus petit rapport entre les volumes de l'espace de tête et du milieu liquide) ;  e) n'ayant pas d'effet inhibiteur sur les micro-organismes d'essai à la concentration choisie pour l'essai. La présence d'un effet inhibiteur peut être mis en évidence suivant la méthode spécifiée en 7.3, ou par toute autre méthode de détermination de l'effet inhibiteur d'un composé sur les bactéries (voir, par exemple, l'ISO 8192).  
NOTE Les conditions décrites dans la présente Norme internationale ne correspondent pas toujours aux conditions optimales d'obtention de la biodégradation maximale. D'autres méthodes de biodégradation sont données dans l'ISO 15462.

Kakovost vode – Vrednotenje “končne” aerobne biorazgradljivosti organskih spojin v vodi z določevanjem porabe kisika v zaprtem raspirometru (ISO 9408:1999)

General Information

Status
Published
Publication Date
31-Jul-1999
Current Stage
9093 - Decision to confirm - Review Enquiry
Due Date
21-May-2008
Completion Date
21-May-2008

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SLOVENSKI STANDARD
SIST EN ISO 9408:2000
01-januar-2000
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SIST EN 29408:1998
.DNRYRVWYRGH±9UHGQRWHQMH³NRQþQH´DHUREQHELRUD]JUDGOMLYRVWLRUJDQVNLK
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Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in

aqueous medium by determination of oxygen demand in a closed respirometer (ISO
9408:1999)

Wasserbeschaffenheit - Bestimmung der vollständigen aerobiologischen Abbaubarkeit

organischen Stoffe im wäßrigen Medium über die Bestimmung des Sauerstoffbedarfs in

einem geschlossenen Respirometer (ISO 9408:1999)
Qualité de l'eau - Evaluation de la biodégradibilité aérobieultime des composés

organiques, en milieux aqueux, par détermination de la demande en oxygene dans un

respirometre ferméometre fermé (ISO 9408:1999)
Ta slovenski standard je istoveten z: EN ISO 9408:1999
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 9408:2000 en

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

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SIST EN ISO 9408:2000
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SIST EN ISO 9408:2000
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SIST EN ISO 9408:2000
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SIST EN ISO 9408:2000
INTERNATIONAL ISO
STANDARD 9408
Second edition
1999-08-01
Water quality — Evaluation of ultimate
aerobic biodegradability of organic
compounds in aqueous medium
by determination of oxygen demand
in a closed respirometer
Qualité de l'eau — Évaluation, en milieu aqueux, de la biodégradabilité
aérobie ultime des composés organiques par détermination de la demande
en oxygène dans un respiromètre fermé
Reference number
ISO 9408:1999(E)
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SIST EN ISO 9408:2000
ISO 9408:1999(E)
Contents Page

1 Scope ........................................................................................................................................................................1

2 Terms and definitions .............................................................................................................................................1

3 Principle....................................................................................................................................................................3

4 Test environment.....................................................................................................................................................3

5 Reagents...................................................................................................................................................................4

6 Apparatus .................................................................................................................................................................5

7 Procedure .................................................................................................................................................................5

8 Calculation and expression of results...................................................................................................................8

9 Validity of results...................................................................................................................................................10

10 Test report ............................................................................................................................................................11

Annex A (informative) Example of calculation of theoretical oxygen demand...................................................12

Annex B (informative) Correction of oxygen uptake for interference by nitrification........................................14

(informative)

Annex C Example of a biodegradation curve .................................................................................16

Annex D (informative) Closed respirometer ..........................................................................................................17

Bibliography..............................................................................................................................................................18

© 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 9408:2000
© ISO
ISO 9408:1999(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 9408 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee

SC 5, Biological methods.

This second edition cancels and replaces the first edition (ISO 9408:1991), which has been technically revised.

Annexes A to D of this International Standard are for information only.
iii
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SIST EN ISO 9408:2000
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SIST EN ISO 9408:2000
INTERNATIONAL STANDARD © ISO ISO 9408:1999(E)
Water quality — Evaluation of ultimate aerobic biodegradability
of organic compounds in aqueous medium by determination
of oxygen demand in a closed respirometer

WARNING — Activated sludge and sewage contain potentially pathogenic organisms. Take appropriate

precautions when handling them. Handle with care toxic test compounds and those whose properties are

unknown.
1 Scope

This International Standard specifies a method, by determination of the oxygen demand in a closed respirometer, for

the evaluation in aqueous medium of the ultimate biodegradability of organic compounds and waste waters at a given

concentration by aerobic microorganisms.
The method applies to organic compounds which
a) are water-soluble under the conditions of the test;

b) are 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) do not reach and react with the CO absorbent;

d) are volatile, provided that a suitable respirometer or suitable conditions (e.g. a smaller ratio of volume head

space to volume liquid medium) are used;

e) are not inhibitory to the test microorganisms at the concentration chosen for the test. The presence of inhibitory

effects can be determined as specified in 7.3, or by using any other method for determining the inhibitory effect of

a compound on bacteria (see, for example, ISO 8192).

NOTE The conditions described in this International Standard do not always correspond to the optimal conditions for

allowing the maximum degree of biodegradation to occur. For alternative biodegradation methods, see ISO 15462.

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
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SIST EN ISO 9408:2000
© ISO
ISO 9408:1999(E)
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
2.4
concentration of suspended solids of an activated sludge

amount of solids obtained by filtration or centrifugation of a known volume of activated sludge and drying at about

105 °C to constant mass
2.5
biochemical oxygen demand
BOD

mass concentration of dissolved oxygen consumed under specified conditions by the aerobic biological oxidation of

a chemical compound or organic matter in water

NOTE It is expressed in this case as milligrams oxygen uptake per milligram (or gram) test compound.

2.6
chemical oxygen demand
COD

mass concentration of oxygen equivalent to the amount of a specified oxidant consumed by a chemical compound

or organic matter when a water sample is treated with that oxidant under defined conditions

NOTE It is expressed in this case as milligrams oxygen consumed per milligram (or gram) test compound.

2.7
theoretical oxygen demand
ThOD

theoretical maximum amount of oxygen required to oxidize a chemical compound completely, calculated from the

molecular formula

NOTE It is expressed in this case as milligrams oxygen required per milligram (or gram) test compound.

2.8
dissolved organic carbon
DOC

that part of the organic carbon in the water which cannot be removed by specified phase separation

NOTE Examples of specified phase separation are centrifugation at 40 000 m{s for 15 min or by membrane filtration

using membranes with pores of 0,2 mm to 0,45 mm diameter.
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 expressed 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 expressed in percent.
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SIST EN ISO 9408:2000
© ISO
ISO 9408:1999(E)
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 expressed in days.
2.12
plateau phase
time from the end of the biodegradation phase until the end of the test
NOTE It is expressed in days.
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

Determination of the biodegradation of organic compounds by aerobic microorganisms is carried out using a static

aqueous test system. Organic compounds in the context of this International Standard include waste waters. The

test mixture contains an inorganic medium, the organic compound as the sole source of carbon and energy at a

mass concentration of normally 100 mg/l organic carbon [but its theoretical oxygen demand (ThOD) shall be at least

100 mg/l], and a mixed inoculum obtained from a waste-water treatment plant or from another source in the

environment.

The mixture is agitated in a closed test vessel and the consumption of oxygen is determined either by measuring

the amount of oxygen required to maintain a constant gas volume in the respirometer vessel, or by measuring the

change in volume or pressure (or a combination of the two) in the apparatus. The evolved carbon dioxide is

absorbed in a suitable substance in the test vessel.

The degradation is followed over a period of 28 d, or longer if necessary, by determining the consumption of oxygen

either automatically or manually. The amount of oxygen consumed by the organic compound (after correction by

comparison with blank control) is expressed as a percentage of either the theoretical oxygen demand (ThOD),

calculated from the formula of the compound, or the chemical oxygen demand (COD).

For sufficiently water-soluble compounds, removal of dissolved organic carbon (DOC) may be determined

(optionally) by measuring the concentration of DOC at the beginning and the end of incubation to obtain additional

information on the ultimate biodegradability. If a substance-specific analytical method is available, information on the

primary degradability may 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

shall not vary by more than ± 1 C during the test.
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SIST EN ISO 9408:2000
© ISO
ISO 9408:1999(E)
5 Reagents
Use only reagents of recognized analytical grade.
5.1 Water
Distilled or deionized water containing less than 1 mg/l DOC.
5.2 Test medium
5.2.1 Composition
5.2.1.1 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
water (5.1), in quantity necessary to make up to 1000 ml

In order to check this buffer solution, it is recommended to measure the pH, which should be at about 7,4. If this is

not the case prepare a new solution.
5.2.1.2 Solution b)

Dissolve 22,5 g magnesium sulfate heptahydrate (MgSO {H O) in water (5.1), quantity necessary to make up to

4 2
1000 ml.
5.2.1.3 Solution c)

Dissolve 36,4 g calcium chloride dihydrate (CaCl {2H O) in water (5.1), quantity necessary to make up to 1000 ml.

2 2
5.2.1.4 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

1000 ml. Prepare this solution freshly before use, or add a drop of concentrated hydrochloric acid (HCl) to avoid

precipitation
5.2.2 Preparation of the test medium
For 1000 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 1000 ml with the water (5.1). Prepare the test medium freshly before use. The solutions a) to c) may be

stored up to 6 months in the dark at room temperature.
5.3 Carbon dioxide absorber

Potassium hydroxide solution (about 10 mol/l), soda lime pellets or other suitable absorbent.

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SIST EN ISO 9408:2000
© ISO
ISO 9408:1999(E)
5.4 Mercury chloride solution
Dissolve 1 g of mercury(II) chloride (HgCl ) in 100 ml of the water (5.1).
5.5 Sodium hydroxide solution

Dissolve sodium hydroxide (NaOH) in the water (5.1) to obtain a solution of concentration 0,1 mol/l to 0,5 mol/l.

5.6 Hydrochloric acid solution

Dilute concentrated hydrochloric acid (HCl) in the water (5.1) to obtain a solution of concentration 0,1 mol/l to

0,5 mol/l.
6 Apparatus

Ensure that all glassware is thoroughly cleaned and free from organic or toxic matter.

6.1 Closed respirometer

The principle of a closed respirometer is given in annex D. The respirometer contains test vessels allowing oxygen

supply and stirring, including tubing nonpermeable to oxygen and carbon dioxide. The respirometer vessels are

located in a constant temperature room or in a thermostatically controlled water-bath. When testing volatile

compounds, the apparatus used shall be appropriate or adapted to this particular purpose. Care shall be taken that

there is no loss of compound due to the apparatus.
6.2 Water-bath or constant temperature room (to comply with clause 4)
6.3 Equipment for measurement of dissolved organic carbon

Instrument of sufficient sensitivity for the measurement of dissolved organic carbon (DOC) (optional).

6.4 Device for determining chemical oxygen demand (COD) (optional
6.5 Centrifuge or device for filtration
The centrifuge shall be capable of producing an acceleration of 4 000 g.

The filtration apparatus shall be equipped with membrane filters (nominal aperture diameter of 0,2 mm to 0,45 mm

pore size) which do not adsorb or release organic carbon.
6.6 pH meter (usual laboratory equipment)
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 the test medium (5.2) and add a suitable

amount of this solution to the test vessels to obtain a final mass concentration of 100 mg/l test compound, but

equivalent to at least 100 mg/l ThOD. 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. Determine, if required, the COD of the test compound using e.g.

ISO 6060.

NOTE For more details on handling poorly water-soluble compounds, see ISO 10634.

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SIST EN ISO 9408:2000
© ISO
ISO 9408:1999(E)
7.1.2 Solution of the reference compound

Use as reference compound an organic compound of known biodegradability, such as aniline or sodium benzoate

which have degradation degrees >60 %. 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 mass concentration of

100 mg reference compound per litre test medium.
7.1.3 Solution to check inhibition

If required (e.g. when 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 mass

concentrations of 100 mg/l for each.
7.2 Preparation of the inoculum
7.2.1 General

Prepare the inoculum, using preferably activated sludge or the following sources (7.2.2 to 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 BOD of the blank shall fulfil the validity

criteria (see clause 9). To reduce the influence of the blank, it may be helpful to precondition the inoculum, e.g. by

aerating it, up to one week before use. Use a suitable volume for inoculation.

NOTE 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).
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 inocu
...

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