SIST EN ISO 9888:2000

SLOVENSKI STANDARD
SIST EN ISO 9888:2000
01-januar-2000
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Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in
aqueous medium - Static test (Zahn-Wellens method) (ISO 9888:1999)
Wasserbeschaffenheit - Bestimmung der aeroben biologischen Abbaubarkeit
organischer Stoffe im wäßrigen Medium - Statischer Test (Zahn-Wellens-Test) (ISO
9888:1999)
Qualité de l'eau - Evaluation, en milieu aqueux, de la biodégradabilité aérobie des
composés organiques - Essai statique (méthode Zahn-Wellens) (ISO 9888:1999)
Ta slovenski standard je istoveten z: EN ISO 9888:1999
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 9888:2000 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 9888:2000

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SIST EN ISO 9888:2000

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SIST EN ISO 9888:2000

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SIST EN ISO 9888:2000
INTERNATIONAL ISO
STANDARD 9888
Second edition
1999-06-01
Water quality — Evaluation of ultimate
aerobic biodegradability of organic
compounds in aqueous medium — Static
test (Zahn-Wellens method)
Qualité de l'eau — Évaluation, en milieu aqueux, de la biodégradabilité
aérobie ultime des composés organiques — Essai statique
(méthode Zahn-Wellens)
A
Reference number
ISO 9888:1999(E)

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SIST EN ISO 9888:2000
ISO 9888:1999(E)
Contents
1 Scope .1
2 Normative references .1
3 Terms and definitions .2
4 Principle.3
5 Test environment.3
6 Reagents.3
7 Apparatus .5
8 Procedure .5
9 Calculation and expression of results.7
10 Validity of results.8
11 Test report .8
Annex A (informative) Example of a biodegradation curve .10
Bibliography.11
©  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 9888:2000
© ISO
ISO 9888:1999(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 9888 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 9888:1991), which has been technically revised.
Annex A of this International Standard is for information only.
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SIST EN ISO 9888:2000

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SIST EN ISO 9888:2000
INTERNATIONAL STANDARD  © ISO ISO 9888:1999(E)
Water quality — Evaluation of ultimate aerobic biodegradability
of organic compounds in aqueous medium — Static test
(Zahn-Wellens method)
WARNING — Activated sludge and sewage may contain potentially pathogenic organisms. Take
appropriate precautions when handling them. Handle with care toxic test compounds and those with
unknown properties.
1 Scope
This International Standard specifies a method for the evaluation in aqueous medium of the ultimate
biodegradability and, as additional information, the primary biodegradability and the total elimination from water, of
organic compounds at a given concentration by aerobic microorganisms.
The conditions described in this International Standard normally correspond to optimal conditions for allowing the
maximum value of biodegradation to occur with the chosen inoculum in the test time. These conditions may even be
more favourable than in full-scale wastewater treatment plants, especially if their hydraulic retention time, sludge
age or the adaptation of the activated sludge is not optimal.
The method applies to organic compounds which are
a) water-soluble at the concentration used under the test conditions and not expected to be transformed to
insoluble metabolites if biodegradation and not elimination only shall be determined;
b) nonvolatile, or which have a negligible vapour pressure under the test conditions;
c) not lost by foaming from the test solution;
d) not inhibitory to the test microorganisms at the concentration chosen for the test. Inhibitory effects can be
determined using a suitable test method (e.g. see ISO 8192). If the test compound is toxic, the test
concentration must be lowered, or a pre-exposed inoculum can be used.
This International Standard is also applicable to the measurement of biodegradation and elimination of dissolved
organic compounds in wastewater (also called "test compound" in the method).
NOTE If more information is required to predict the behaviour of test compounds or wastewater in a treatment plant, a
simulation test (e.g. the activated sludge simulation test ISO 11733) should be performed. For appropriate use of this method
and for alternative biodegradation methods, see ISO 15462.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
ISO 6060:1989, Water quality — Determination of the chemical oxygen demand.
ISO 8245, Water quality — Guidelines for the determination of total organic carbon (TOC) and dissolved organic
carbon (DOC).
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SIST EN ISO 9888:2000
© ISO
ISO 9888:1999(E)
3 Terms and definitions
For the purposes of this International Standard, the following terms and definitions apply.
3.1
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
3.2
primary biodegradation
structural change (transformation) of a chemical compound by microorganisms resulting in the loss of a specific
property
3.3
activated sludge
biomass produced in the aerobic treatment of wastewater by the growth of bacteria and other microorganisms in the
presence of dissolved oxygen
3.4
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
3.5
total organic carbon
TOC
all that carbon present in organic matter which is dissolved and suspended in the water
3.6
dissolved organic carbon
DOC
that part of the organic carbon in the water which cannot be removed by specified phase separation
–2
NOTE Phase separation may be specified for example by 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.
3.7
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 COD is expressed in this case as milligrams oxygen consumed per milligram or gram of test compound.
3.8
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 Lag phase is recorded in days.
3.9
maximum level of biodegradation
maximum degree of biodegradation of a chemical compound or organic matter in a test, above which no further
biodegradation takes place during the test
NOTE Maximum level of biodegradation is recorded in percent.
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SIST EN ISO 9888:2000
© ISO
ISO 9888:1999(E)
3.10
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 Biodegradation phase is recorded in days.
3.11
plateau phase
time from the end of the biodegradation phase until the end of the test
NOTE Plateau phase is recorded in days.
3.12
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
3.13
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
4 Principle
The biodegradation or elimination of water-soluble organic compounds or wastewater ingredients by aerobic
microorganisms is determined using a static aqueous test system. The test mixture contains an inorganic medium,
activated sludge as a mixed inoculum and an organic test compound as the sole source of carbon and energy other
than the sludge. The amount of test compound added is chosen to result in an initial concentration of dissolved
organic carbon (DOC) between 50 mg/l and 400 mg/l, or of chemical oxygen demand (COD) between 100 mg/l and
1000 mg/l, depending on its water solubility and on its toxicity to the bacteria in the inoculum.
Measurement of the concentration of DOC (or COD) is made at the beginning and end of the test (normally 28 d)
and at intermediate time intervals, as required. To allow for any significant adsorption of the test compound onto the
sludge, samples are also taken 3 h after the beginning of the test. Values obtained at this time are used as the basis
for calculating the percentage of ultimate biodegradability at each sampling time. As additional information, the total
elimination of DOC or COD from the aqueous phase may be obtained by calculating the removal based on a
measured value before addition of the salts of the test medium and the inoculum. Possible other abiotic elimination
processes, such as stripping into the air, may be determined by an abiotic elimination control without inoculum.
Use of specific analysis may give additional information on primary biodegradability of test compounds.
5 Test environment
o o
Incubation shall take place in the dark or in diffuse light, at a temperature within the range 20 C to 25 C which shall
o
not vary by more than 2 C during the test and in an enclosure which shall be free from gases and vapours which
are toxic to microorganisms.
6 Reagents
Use only reagents of recognized analytical grade, including the following.
6.1  Water, distilled or deionized, containing less than 1 mg/l DOC.
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SIST EN ISO 9888:2000
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ISO 9888:1999(E)
6.2  Test medium
6.2.1  Composition
6.2.1.1  Solution a)
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
Dissolve in water (6.1) to make up to 1000 ml
NOTE In order to check this buffer solution, it is recommended that the pH is measured. If it is not at about 7,4 a fresh
solution should be prepared.
6.2.1.2  Solution b)
Dissolve 22,5 g magnesium sulfate heptahydrate (MgSO ×7H O) in water (6.1) to make up to 1000 ml.
4 2
6.2.1.3  Solution c)
Dissolve 36,4 g calcium chloride dihydrate (CaCl ×2H O) in water (6.1) to make up to 1000 ml.
2 2
6.2.1.4  Solution d)
Dissolve 0,25 g iron(III) chloride hexahydrate (FeCl ×6H O) in water (6.1) to make up to 1000 ml. Prepare this
3 2
solution freshly before use or add a drop of concentrated hydrochloric acid (HCl) to avoid precipitation.
6.2.2  Preparation of the test medium
1000 ml test medium shall contain 10 ml of solution a) and 1 ml of each of solutions b) to d). Add the correct
amounts of the stock solutions as described in 8.3. 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.
NOTE If a test compound influences the pH value of the mixture at the chosen test concentration, an increase in the buffer
capacity of the test medium may be required. At high test concentrations, a lack of nitrogen may also occur. In these cases it is
recommended that the nutrient composition of the test medium be improved by adding, for example, 100 ml of solution a)
instead of 10 ml or just the phosphate amount of solution a). The C:N:P ratio for a test concentration of 400 mg/l DOC is then
changed from 100:0,3:30 to 100:3:300. As at higher nitrogen concentrations nitrification may occur, higher phosphate buffer
concentrations are required to keep the pH stable.
6.3  Sodium hydroxide solution
Dissolve sodium hydroxide (NaOH) in the water (6.1) to obtain a solution of concentration 0,1 mol/l to 0,5 mol/l.
6.4  Hydrochloric acid solution
Dissolve hydrochloric acid (HCl) in the water (6.1) to obtain a solution of concentration 0,1 mol/l to 0,5 mol/l.
6.5  Mercury chloride solution
Dissolve 1 g of mercury(II) chloride (HgCl ) in 100 ml of the water (6.1).
2
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