SIST EN ISO 11733:2005

SLOVENSKI STANDARD
SIST EN ISO 11733:2005
01-februar-2005
1DGRPHãþD
SIST EN ISO 11733:2000
Kakovost vode – Ugotavljanje odstranjevanja in biorazgradljivosti organskih snovi
v vodi – Simulacijski preskus z aktivnim blatom (ISO 11733:2004)
Water quality - Determination of the elimination and biodegradability of organic
compounds in an aqueous medium - Activated sludge simulation test (ISO 11733:2004)
Wasserbeschaffenheit - Bestimmung der Elimination und der biologischen Abbaubarkeit
organischer Verbindungen in einem wässrigen Medium - Belebtschlamm-Simulationtest
(ISO 11733:2004)
Qualité de l'eau - Détermination de l'élimination et de la biodégradabilité des composés
organiques en milieu aqueux - Essai de simulation des boues activées (ISO 11733:2004)
Ta slovenski standard je istoveten z: EN ISO 11733:2004
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 11733:2005 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 11733:2005

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SIST EN ISO 11733:2005
EUROPEAN STANDARD
EN ISO 11733
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2004
ICS 13.060.70 Supersedes EN ISO 11733:1998
English version
Water quality - Determination of the elimination and
biodegradability of organic compounds in an aqueous medium -
Activated sludge simulation test (ISO 11733:2004)
Qualité de l'eau - Détermination de l'élimination et de la
biodégradabilité des composés organiques en milieu
aqueux - Essai de simulation des boues activées (ISO
11733:2004)
This European Standard was approved by CEN on 29 July 2004.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Central Secretariat or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2004 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11733:2004: E
worldwide for CEN national Members.

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SIST EN ISO 11733:2005

EN ISO 11733:2004 (E)





Foreword


This document (EN ISO 11733:2004) has been prepared by Technical Committee ISO/TC 147
"Water quality" in collaboration with Technical Committee CEN/TC 230 "Water analysis", the
secretariat of which is held by DIN.

This European Standard shall be given the status of a national standard, either by publication of
an identical text or by endorsement, at the latest by February 2005, and conflicting national
standards shall be withdrawn at the latest by February 2005.

This document supersedes EN ISO 11733:1998.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.


Endorsement notice

The text of ISO 11733:2004 has been approved by CEN as EN ISO 11733:2004 without any
modifications.

2

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SIST EN ISO 11733:2005


INTERNATIONAL ISO
STANDARD 11733
Second edition
2004-08-01


Water quality — Determination of the
elimination and biodegradability of
organic compounds in an aqueous
medium — Activated sludge simulation
test
Qualité de l'eau — Détermination de l'élimination et de la
biodégradabilité des composés organiques en milieu aqueux — Essai
de simulation des boues activées





Reference number
ISO 11733:2004(E)
©
ISO 2004

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SIST EN ISO 11733:2005
ISO 11733:2004(E)
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ii © ISO 2004 – All rights reserved

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SIST EN ISO 11733:2005
ISO 11733:2004(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 2
4 Principle . 3
5 Test environment . 4
6 Reagents . 4
7 Apparatus. 7
8 Procedure. 8
8.1 General. 8
8.2 Preparation of the inoculum . 8
8.3 Performance of the test. 8
9 Calculation and expression of results . 11
9.1 Calculation of the degree of elimination. 11
9.2 Expression of results. 12
9.3 Indication of biodegradation. 12
9.4 Biodegradation of the organic medium .12
10 Validity of the test . 12
11 Test report. 13
Annex A (informative) Modification of the activated sludge simulation test for nitrifying-
denitrifying sewage treatment plants . 14
Annex B (informative) Coupling of the test units (optional) . 18
Annex C (informative) Test systems. 19
Annex D (informative) Effects of sludge retention time on effluent concentration. 22
Annex E (informative) Example of an elimination/degradation curve. 25
Bibliography . 26

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SIST EN ISO 11733:2005
ISO 11733:2004(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 2.
The main task of technical committees is to prepare International Standards. 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.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 11733 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 11733:1995), which has been technically
revised.

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SIST EN ISO 11733:2005
INTERNATIONAL STANDARD ISO 11733:2004(E)

Water quality — Determination of the elimination and
biodegradability of organic compounds in an aqueous
medium — Activated sludge simulation test
WARNING AND SAFETY PRECAUTIONS — Activated sludge and sewage contain potentially
pathogenic organisms, therefore 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 for the determination of the elimination and the
biodegradability of organic compounds by aerobic micro-organisms. The conditions described simulate a
waste-water treatment plant. Two test systems can be used: activated sludge plants or porous pots. The tests
can optionally be performed under conditions of nitrification and denitrification (Annex A) and coupling of the
units (Annex B).
The method applies to organic compounds which, under the conditions of the test, are
a) soluble in tap water at the test concentration and not expected to be transformed to insoluble metabolites
if biodegradation, in addition to elimination, is determined;
b) poorly water-soluble, but which are satisfactorily dispersible in water and allow detection with suitable
analytical means (e.g. organic carbon measurements);
c) non-volatile, or which have a negligible vapour pressure under the test conditions;
d) not inhibitory to the test micro-organisms at the concentration chosen for the test. Inhibitory effects can be
[15] [27]
determined by using a suitable test method (e.g. ISO 8192 or ISO 15522 ). Compounds inhibitory at
concentrations used in this test may be tested at concentrations less than their EC value, followed by
20
higher practical concentrations after a period of acclimatization.
The method can also be used to measure the biodegradation and elimination of dissolved organic compounds
in waste water (also called “test compound” in the method).
If more or different information is required to predict the behaviour of test compounds or waste water in a
treatment plant, other degradation tests may be performed. For appropriate use of this method and for
alternative biodegradation methods, see ISO/TR 15462 and for general information on biotesting, see
ISO 5667-16.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 5667-16, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 10634, Water quality — Guidance for the preparation and treatment of poorly water-soluble organic
compounds for the subsequent evaluation of their biodegradability in an aqueous medium
ISO/TR 15462, Water quality — Selection of tests for biodegradability
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SIST EN ISO 11733:2005
ISO 11733:2004(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
accelerating removal phase
〈activated sludge simulation test〉 time from the end of the lag phase until the plateau phase is reached, during
which the biodegradation of a compound or organic matter increases
NOTE Accelerating removal phase is expressed in days.
3.2
activated sludge
biomass and inert matter produced in the aerobic treatment of wastewater by the growth of bacteria and other
micro-organisms in the presence of dissolved oxygen
3.3
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 of oxygen consumed per milligram or per gram of test
compound
3.4
concentration of suspended solids of an activated sludge
amount of solids obtained by filtration or centrifugation at known conditions of a known volume of activated
sludge and drying at about 105 °C to constant weight
3.5
degree of elimination
biodegradation
〈activated sludge simulation test〉 mean eliminated (biodegraded) amount of a chemical compound or organic
matter, calculated from the measured concentrations in the inlet and the outlet of the system
NOTE The degree of elimination (biodegradation) is determined when no further elimination can be measured and is
expressed as a percentage.
3.6
denitrification
reduction of nitrate and nitrite to the end product nitrogen (in the form of the gas) by the action of bacteria
3.7
dissolved organic carbon
DOC
part of the organic carbon in a sample of water which cannot be removed by specified phase separation
2
NOTE Phase separation may be obtained, for example, by centrifugation of the water sample at 40 000 m/s for
15 min or by membrane-filtration using membranes with a pore size of 0,45 µm.
3.8
lag phase
〈activated sludge simulation test〉 time from the start of a test until a significant elimination (biodegradation) of
a compound or organic matter can be measured (the beginning of the accelerated removal phase)
NOTE The lag phase is expressed in days.
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SIST EN ISO 11733:2005
ISO 11733:2004(E)
3.9
nitrification
oxidation of ammonium salts by bacteria where usually the intermediate product is nitrite and the end product
nitrate
3.10
plateau phase
〈activated sludge simulation test〉 time from the end of the accelerating removal phase until the end of a test in
which the biodegradation of a compound or organic matter is in a steady state
NOTE The plateau phase is expressed in days.
3.11
pre-exposure
pre-incubation of an inoculum in the presence of the test compound or organic matter, with the aim of
enhancing the ability of this inoculum to biodegrade the test compound by adaptation and/or selection of the
micro-organisms
3.12
pre-conditioning
pre-incubation of an inoculum under the conditions of the subsequent test in the absence of the test
compound and other organic matter, with the aim of improving the performance of the test by acclimatization
of the micro-organisms to the test conditions
3.13
primary biodegradation
structural change (transformation) of a chemical compound by micro-organisms resulting in the loss of a
specific property
3.14
total organic carbon
TOC
all the carbon present in organic matter which is dissolved and suspended in the water
3.15
ultimate aerobic biodegradation
breakdown of a chemical compound or organic matter by micro-organisms in the presence of oxygen to
carbon dioxide, water and mineral salts of any other elements present (mineralization) and the production of
new biomass
4 Principle
This method is designed to determine the elimination and, if possible, the primary or ultimate biodegradation
of water-soluble organic compounds from water by aerobic micro-organisms in a continuously operating test
system simulating the activated-sludge process. An easily biodegradable organic medium and the organic test
compound are the sources of carbon and energy for the micro-organisms.
Two test units (activated sludge plants or porous pots) are run in parallel under identical conditions, normally
with a mean hydraulic retention time, HRT, of 6 h (8.3.1) and a mean sludge retention time, SRT (sludge age),
of 6 d to 10 d (8.3.3).
NOTE 1 HTR is the mean period of retention of waste water in the aeration vessel. It is calculated by dividing the
volume of sludge, expressed in litres, by the rate of flow of waste water, expressed in litres per day.
NOTE 2 SRT is the mean period of retention of activated sludge in the aeration vessel. It is calculated by dividing the
volume or weight of sludge in the aeration vessel by the volume or weight of sludge discarded per day. If a period of
8 days is chosen, remove 1/8 of the volume of the activated sludge of the aeration vessel each working day and discard it.
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SIST EN ISO 11733:2005
ISO 11733:2004(E)
The test compound is added together with the organic medium, usually at a concentration equivalent to a
DOC between 10 mg/l and 20 mg/l, to the influent of only one of the test units. The second unit is used as
control unit to determine the degree of biodegradation of the organic medium when the analysis is based on
DOC or COD.
Samples of the effluents taken at regular intervals are analyzed for DOC or COD. The difference between
values in the effluent of the test and the control unit compared with the influent concentration of the test
compound is used to determine the degree of elimination of the test compound. Depending on the elimination
characteristics and other available information, e.g. from other tests, ultimate biodegradability can be stated.
If required, the primary biodegradation of the test compound can be determined by substance-specific
analysis. Optionally, the units may be operated under denitrifying conditions (see Annex A) or be coupled (see
Annex B).
5 Test environment
The test shall take place in diffused light or in the dark, in an enclosure which is free from vapours toxic to
micro-organisms and at a controlled temperature in the range of 20 °C to 25 °C. For special purposes, it is
permissible to use a test temperature in another range.
6 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
6.1 Tap water, containing less than 3 mg/l DOC.
6.2 Deionized water, containing less than 1 mg/l DOC.
6.3 Organic media.
6.3.1 General.
Synthetic sewage, domestic sewage or a mixture of both are permissible as an organic medium. Measure the
[16] [14]
DOC (e.g. ISO 8245 ) or COD (e.g. ISO 6060 ) concentration in each new batch of organic medium and
determine the alkalinity, if required and not already known.
[29]
Experience has shown that the so-called OECD medium (6.3.2) might not be suitable in some cases.
Therefore, two more synthetic media which have successfully been tested in laboratories are described in this
International Standard. Domestic sewage (6.3.5) may also be used. Its use is recommended, as a continuous
inoculation takes place and a vastly greater number of nutrients is available to improve the biodegradation
potential of the test.
6.3.2 Synthetic sewage 1 (OECD medium), which gives a mean DOC concentration of about 100 mg/l
and a COD of about 300 mg/l in the influent.
It is composed of the following:
 peptone 160 mg
 meat extract 110 mg
 urea 30 mg
 anhydrous potassium monohydrogenphosphate (K HPO) 28 mg
2 4
 sodium chloride (NaCl) 7 mg
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SIST EN ISO 11733:2005
ISO 11733:2004(E)
.
 calcium chloride dihydrate (CaCl 2HO) 4 mg
2 2
.
 magnesium sulfate heptahydrate (MgSO 7HO) 2 mg
4 2
 tap water (6.1) 1 l
6.3.3 Synthetic sewage 2, which gives a mean DOC concentration of about 150 mg/l and a COD of about
400 mg/l in the influent.
It is composed of the following:
 peptone 192 mg
 meat extract 138 mg
 glucose monohydrate 19 mg
 ammonium chloride (NHCl) 23 mg
4
 anhydrous potassium dihydrogenphosphate (KH PO) 16 mg
2 4
.
 disodium hydrogenphosphate dihydrate (Na HPO 2HO) 32 mg
2 4 2
 sodium hydrogen carbonate (NaHCO) 294 mg
3
 sodium chloride (NaCl) 60 mg
.
 iron(III) chloride hexahydrate (FeCl 6HO) 40 mg
3
2
 tap water (6.1) 1 l
It is strongly recommended to add the iron chloride solution separately and directly to the aeration vessel to
prevent precipitation, especially if a concentrated solution is sterilized (8.3.1). For example, if a stock solution
of 45 g/l iron(III) chloride hexahydrate is prepared, 5 ml should be added daily to the aeration vessel.
6.3.4 Synthetic sewage 3, which gives a mean DOC concentration of about 180 mg/l and a COD of about
470 mg/l in the influent.
The composition is specially balanced for nutrient-removal systems as described in Annex A, but it is equally
usable in the standard test system. It is composed of the following (for more information, see References [4]
and [5]):
 peptone 15 mg
 meat extract 15 mg
 potato starch 50 mg
 milk powder 120 mg
 glycerol 40 mg
 sodium acetate 120 mg
 urea 75 mg
 uric acid 9 mg
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SIST EN ISO 11733:2005
ISO 11733:2004(E)
 ammonium chloride (NHCl) 11 mg
4
.
 magnesium hydrogen phosphate trihydrate (MgHPO 3HO) 25 mg
4 2
.
 tripotassiumphosphate trihydrate (K PO 3HO) 20 mg
3 4 2
 diatomaceous earth 10 mg
 lyophilised, powdered activated sludge 50 mg;
 natural (diet) fibres 80 mg
 linear alkylbenzene sulfonate (LAS) 10 mg

 alcohol ethoxylate C to C EO5 or any other easily biodegradable surfactant 10 mg
12 14
 ethylene diamine tetraacetic acid tetra sodium salt (Na-EDTA) 0,29 mg
4
 trace elements:
 CaCl 5 mg

2
 NaHCO 25 mg

3
.
 FeSO 7HO 10 mg
4 2
.
 CuCl 2HO 0,48 mg
2 2
.
 CoCl 6HO 0,05 mg
2 2
 ZnCl 0,18 mg
2
.
 MnSO HO 0,1 mg
4 2
 K MoO 0,020 mg
2 4
.
 Cr(NO ) 9HO 0,68 mg

3 3 2
.
 NiSO 6HO 0,3 mg
4 2
 Tap water 1 l
NOTE This medium contains surfactants and therefore might not be suitable for the determination of the
biodegradability of surface-active agents.
6.3.5 Domestic sewage, fresh, settled, largely free from coarse particles and, if necessary, neutralized to
(pH 7 ± 0,5).
Preferably use sewage (8.2) from the same plant as the sludge inoculum. Sewage can be stored for several
days at about 4 °C if it has been proven that the DOC or COD does not significantly decrease during storage
(for example, by less than about 20 % compared to the initial concentration). In order to limit disturbances to
the activated sludge system, adjust each new batch to an appropriate constant value of, for example, 100 mg/l
DOC or 300 mg/l COD by dilution with tap water.
6.3.6 Modified organic medium, a dilution of an organic medium (6.3.2 to 6.3.5) with tap water.
EXAMPLE If synthetic sewage 1 (6.3.2) is diluted 1:1, a DOC concentration in the influent of about 50 mg/l is
obtained.
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SIST EN ISO 11733:2005
ISO 11733:2004(E)
Domestic sewage of low acidity or alkalinity or synthetic sewage prepared from tap water of low acidity or
alkalinity can require the addition of a suitable buffer to improve the biological processes, especially
nitrification. A pH of about 7,5 ± 0,5 in the aeration vessel during the test may be achieved, for example, by
adding a buffer solution of 1 500 mg/l potassium dihydrogen phosphate (KH PO ) to the synthetic sewage 1
2 4
(6.3.2). When and how much buffer is added shall usually be decided on an individual basis, depending on the
acidity or alkalinity of the organic medium and the pH values measured in the aeration vessel.
6.4 Test compound stock solution, a solution of a suitable concentration, e.g. 5 g/l, of the test compound
in tap water (6.1) or deionized water (6.2).
Check that diluting this solution with tap water to give the required test concentration does not produce a
precipitate.
Determine the DOC and TOC of the stock solution. If the difference between the DOC and TOC is < 20 %,
DOC can be used as analytical parameter. If the difference between the DOC and TOC is > 20 %, check that
the test compound is completely water-soluble at the desired test concentration (8.3.2). It is recommended to
repeat at least the DOC measurement for each new batch of the stock solution to ensure its correct
preparation. Compare the DOC of the stock solution with the theoretical value to ascertain whether the
analytical recovery is good enough (normally > 90 % can be expected). Ensure, especially for dispersions,
whether or not the DOC can be used as an analytical parameter. For dispersions, centrifugation of the
samples is required. If primary biodegradation shall be determined, check the test-compound concentration of
the stock solution measured by specific analysis with the theoretical value.
[23]
Determine the pH of the stock solution . Extreme pH values indicate that the test compound may have an
influence on the pH of the activated sludge in the test system. In this case, neutralize the stock solution to get
a pH value of, preferably, (7 ± 0,5) with small amounts of inorganic acid or base, but avoid precipitation of the
test compound. In the event of precipitate formation, use another pH range which yields no precipitate.
7 Apparatus
7.1 Test system, consisting, for one test compound, of a test unit and a control unit.
The test unit shall be either an activated-sludge plant (a so-called Husmann apparatus) or a porous pot (see
Annex C). In both cases, storage vessels sufficiently large for the influent and the effluent are needed, as well
as pumps to dose the influent. One control unit can be used for several test units. In the case of coupling (see
Annex B), use one control unit for each test unit.
Each activated-sludge plant consists of an aeration vessel with a capacity of about 3 l for activated sludge and
a separator (secondary clarifier) which holds about 1,5 l. Vessels of different size are permissible if they are
operated with comparable hydraulic loads. If it is not possible to keep the test temperature in the test room in
the desired range, use, for example, water-jacketed vessels with water at a controlled temperature. Use a
dosing pump or a suitable air-lift pump to recycle the activated sludge from the separator to the
...