EN 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)

ISO 11733:2004 specifies a method for the determination of the elimination and the biodegradability of organic compounds by aerobic micro-organisms. The conditions described simulate a wastewater 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 and coupling of the units.
The method applies to organic compounds which, under the conditions of the test, are
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;poorly water-soluble, but which are satisfactorily dispersible in water and allow detection with suitable analytical means (e.g. organic carbon measurements);non-volatile, or which have a negligible vapour pressure under the test conditions;not inhibitory to the test micro-organisms at the concentration chosen for the test. Compounds inhibitory at concentrations used in this test may be tested at concentrations less than their EC20 value, followed by 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).

Wasserbeschaffenheit - Bestimmung der Elimination und der biologischen Abbaubarkeit organischer Verbindungen in einem wässrigen Medium - Belebtschlamm-Simulationtest (ISO 11733:2004)

Diese Internationale Norm legt ein Verfahren zur Bestimmung der Elimination und der biologischen Abbaubarkeit von organischen Substanzen durch aerobe Mikroorganismen bei einer vorgegebenen Konzentration fest. Die beschriebenen Testbedingungen simulieren eine Kläranlage. Zwei Testsysteme können verwendet werden, die Belebtschlammanlage oder der Poröse Topf. Die Prüfungen können optional unter nitrifizierenden/denitrifizierenden Bedingungen (siehe Anhang A) und unter Kopplung der Anlagen (siehe Anhang B) durchgeführt werden.
Das Verfahren ist unter den gegebenen Testbedingungen für organische Stoffe anwendbar, die
a)   bei der gewählten Testkonzentration wasserlöslich sind, wenn davon ausgegangen werden kann, dass keine unlöslichen Metabolite entstehen, sofern zusätzlich zur Elimination die Abbaubarkeit bestimmt wird;
b)   schwer wasserlöslich, aber ausreichend in Wasser dispergierbar sind und eine Bestimmung mit geeigneten analytischen Verfahren (z. B. Messung des organischen Kohlenstoffs) möglich ist;
c)   nicht flüchtig sind oder einen vernachlässigbaren Dampfdruck besitzen;
d)   bei der gewählten Testkonzentration nicht hemmend gegenüber den Mikroorganismen des Inokulums wirken. Eine Hemmung kann mit einem geeigneten Verfahren (siehe z. B. ISO 8192 [15] oder ISO 15522 [27]) ermittelt werden. Stoffe, die bei der üblichen Testkonzentration hemmend wirken, können zunächst in einer Konzentration zudosiert werden, die unter ihrer EC20 liegt und nach Anpassung auf eine höhere, praktikablere Testkonzentration umgestellt werden.
Das Verfahren kann auch verwendet werden, um die biologische Abbaubarkeit und Elimination von gelösten organischen Komponenten in Abwasser zu bestimmen, die hier ebenfalls "Prüfsubstanz" genannt werden.
Wenn weitere oder zusätzliche Informationen erforderlich sind, um das Verhalten von Prüfsubstanzen oder Abwässern in einer Kläranlage vorherzusagen, können auch andere Abbautests durchgeführt werden. Einen Überblick über die Verfahren und ihre Anwen

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)

L'ISO 11733:2004 spécifie une méthode pour la détermination de l'élimination et de la biodégradabilité des composés organiques sous l'action de micro-organismes aérobies. Les conditions décrites simulent une installation de traitement des eaux usées. Deux systèmes d'essai peuvent être utilisés: les unités de traitement utilisant des boues activées ou les dispositifs à cylindres poreux. Les essais peuvent, de façon facultative, être réalisés dans des conditions de nitrification et de dénitrification, et dans des conditions de couplage des unités d'essai.
La méthode s'applique aux composés organiques qui, dans les conditions d'essai, sont
solubles dans l'eau à la concentration d'essai choisie et non susceptibles d'être transformés en métabolites insolubles, s'il faut déterminer la biodégradation en supplément de l'élimination;faiblement solubles, mais aisément dispersables dans l'eau et pouvant être détectés par des moyens d'analyse appropriés (par exemple mesurages du carbone organique);non volatils ou ayant une pression de vapeur négligeable dans les conditions de l'essai;sans effets inhibiteurs vis-à-vis des micro-organismes à expérimenter à la concentration choisie pour l'essai. Des composés à effets inhibiteurs aux concentrations utilisées pour cet essai peuvent être testés à des concentrations inférieures à la valeur de leur EC20, puis à des concentrations pratiques supérieures après une période d'acclimatation.
La méthode peut également être utilisée pour mesurer la biodégradation et l'élimination des composés organiques dissous dans les eaux usées (également appelés «composés à expérimenter» dans la méthode).

Kakovost vode - Ugotavljanje odstranjevanja in biorazgradljivosti organskih snovi v vodi - Simulacijski preskus z aktivnim blatom (ISO 11733:2004)

General Information

• Status: Published
• Publication Date: 31-Jul-2004
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 14-May-2009
• Completion Date: 14-May-2009
• Ref Project: SIST EN ISO 11733:2005 - Water quality - Determination of the elimination and biodegradability of organic compounds in an aqueous medium - Activated sludge simulation test (ISO 11733:2004)

Relations

• Replaces: EN ISO 11733:1998 - Water quality - Evaluation of the elimination and biodegradability of organic compounds in an aqueous medium - Activated sludge simulation test (ISO 11733:1995)
• Effective Date: 22-Dec-2008

Overview

EN ISO 11733:2004 - "Water quality - Determination of the elimination and biodegradability of organic compounds in an aqueous medium - Activated sludge simulation test" specifies a laboratory method to assess the elimination and biodegradability of organic compounds under conditions that simulate a wastewater treatment plant. The standard uses continuously operated test systems (activated sludge plants or porous pots) run in parallel to measure removal and biodegradation by aerobic micro-organisms. Tests can be extended to include nitrification, denitrification, and coupling of units.

Key topics and technical requirements

• Scope of applicability
  • Applies to water‑soluble compounds, poorly water‑soluble but dispersible substances, and non‑volatile compounds with negligible vapour pressure under test conditions.
  • Compounds must not be inhibitory to test micro‑organisms at chosen concentrations; inhibitory substances can be tested at sub‑EC20 levels with acclimatization.
• Test systems
  • Two parallel units: activated sludge plants or porous pots.
  • Optional modifications for nitrifying–denitrifying treatment (Annex A) and coupling of units (Annex B).
• Operating parameters
  • Typical mean hydraulic retention time (HRT): ~6 h.
  • Typical sludge retention time (SRT): ~6–10 days.
• Measurements and endpoints
  • Determination of degree of elimination from inlet/outlet concentrations.
  • Analytical approaches include DOC/TOC and COD measurements where appropriate.
  • Definitions and terms: lag phase, accelerating removal phase, plateau phase, primary/ultimate biodegradation.
• Validity, reporting and safety
  • Criteria for test validity, calculation methods, and mandatory test report content are specified.
  • Warning: activated sludge can contain pathogenic organisms - follow appropriate biosafety precautions.

Practical applications and users

• Environmental testing laboratories evaluating biodegradability and removal efficiency of industrial or municipal organic compounds.
• Chemical manufacturers and formulators assessing environmental fate for regulatory dossiers and product stewardship.
• Water and wastewater utilities and consultants modeling removal in treatment plants.
• Regulatory agencies establishing discharge limits or assessing environmental risk.
• Researchers studying biodegradation kinetics and effects of SRT/HRT on effluent concentration.

Related standards

• ISO/TR 15462 - selection of biodegradability tests.
• ISO 5667-16 - guidance on biotesting sampling.
• ISO 10634 - preparation/treatment of poorly water‑soluble compounds.
• Test methods for inhibition assessment (e.g., ISO 8192, ISO 15522) are referenced for handling inhibitory compounds.

EN ISO 11733:2004 is essential for consistent, comparable measurement of organic compound removal and biodegradability in simulated activated‑sludge systems, supporting regulatory compliance, environmental risk assessment, and wastewater process design.

Standards Content (Sample)

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

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.

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.
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
ISO 11733:2004(E)
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ii © ISO 2004 – All rights reserved

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

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.
iv © ISO 2004 – All rights reserved

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
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
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
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.
2 © ISO 2004 – All rights reserved

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.
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
4 © ISO 2004 – All rights reserved

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
 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
 sodium chloride (NaCl) 60 mg
.
 iron(III) chloride hexahydrate (FeCl 6HO) 40 mg
 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
ISO 11733:2004(E)
 ammonium chloride (NHCl) 11 mg
.
 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
 trace elements:
 CaCl 5 mg
 NaHCO 25 mg
.
 FeSO 7HO 10 mg
4 2
.
 CuCl 2HO 0,48 mg
2 2
.
 CoCl 6HO 0,05 mg
2 2
 ZnCl 0,18 mg
.
 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.
6 © ISO 2004 – All rights reserved

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 aeration
vessel, either continuously or intermittently. The use of a dosing pump allows the recycling of settled sludge to
the sewage influent and then back to the aeration vessel, so that the settled sludge does not become
anaerobic. The design of the air-lift pump alone does not allow this.
The porous-pot system consists of an inner, porous cylinder with a conical bottom suspended in a slightly
larger vessel of the same shape, but made of impervious material. Separation of the sludge from the treated
organic medium is made by differential passage through the porous wall. The effluent collects in the annular
space from where it overflows into the collecting vessel. No settlement occurs and hence there is neither
sludge return nor formation of anoxic zones. The whole system may be mounted in a thermostatically
controlled room or water-bath. Porous pots can sometimes block and overflow in the initial stages of the test.
In such a case, replace the blocked pot with a clean pot to which the sludge from the blocked pot has been
added. Clean blocked pots by soaking them in dilute sodium hypochlorite solution, then in water, followed by
thorough rinsing with water.
NOTE As material for the cylinder, porous polyethylene with a maximum pore size of 90 µm and a thickness of 2 mm
can be used.
ISO 11733:2004(E)
For aeration of the sludge in the aeration vessels of both systems, suitable techniques, e.g. sintered cubes
(diffuser stones) and compressed air, are required. The air shall be cleaned, if necessary, by passing through
a suitable filter and washed. Sufficient air shall pass through the system to maintain aerobic conditions and
the sludge flocs in suspension at all times during the test.
Neither test system fully mimics full-scale activated sludge plants, but both systems have shown their
suitability for laboratory experiments for many years.
7.2 Analytical equipment, use laboratory carbon analyzer to determine DOC and TOC (see e.g.
[16] [14]
ISO 8245 or equipment for COD determination (see e.g. ISO 6060 and, if necessary, suitable
[2], [17], [18]
equipment for substance-specific analyses or surfactants  . Equipment to determine suspended
[25] [23] [13]
solids , pH , oxygen concentration in water , temperature, if required acidity and alkalinity and, if
[24] [22]
nitrification and denitrification are being investigated, ammonium , nitrite and nitrate .
7.3 Filtration apparatus or centrifuge.
7.3.1 Device for filtration, with membrane filters of suitable porosity (nominal pore size of 0,45 µm) which
adsorb organic compounds and release organic carbon to an insignificant degree.
If filters are used which release organic carbon, wash them carefully with hot water to remove leachable
organic carbon. Be aware that filters treated in this way might be very fragile, therefore centrifugation is
generally recommended.
7.3.2 Centrifuge, suitable for operating at 40 000 m/s .
8 Procedure
8.1 General
The procedure is described for the activated sludge plants (Husmann apparatus). It shall be adapted for the
porous-pot system.
8.2 Preparation of the inoculum
Inoculate the test system at the beginning of the test with either activated sludge or an inoculum containing a
low concentration of micro-organisms. Keep the inoculum aerated at room temperature until it is used and use
it within 24 h.
In the first case, take a sample of activated sludge from the aeration vessel of an efficiently operated biological
waste-water treatment plant (e.g. from the exit end of a plug-flow type) or from a laboratory treatment plant
which receives predominantly domestic sewage.
Determine the concentration of suspended solids. If necessary, concentrate the sludge by settling so that the
volume added to the test system is minimal. Ensure that the starting concentration in the aeration vessel is
about 2,5 g/l dry matter.
In the second case, use 2 ml/l to 10 ml/l of effluent from a domestic biological waste water treatment plant as
an inoculum. The activated sludge develops and grows in the test system. To get as many different species of
bacteria as possible, it might be helpful to combine inocula from various sources. When a smaller inoculum is
used, it usually take longer to achieve sufficient sludge concentrations.
8.3 Performance of the test
8.3.1 Dosage of organic medium
Assemble the test systems (7.1) in a controlled-temperature room (Clause 5) or use water-jacketted test units.
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ISO 11733:2004(E)
Prepare a sufficient amount of the desired organic medium (6.3). Initially fill the aeration vessel and the
separator with organic medium and add the inoculum (8.2). Start aeration such that the sludge is kept in
suspension and aerobic; begin dosing the influent and recycling the settled sludge.
Dose the organic medium out of storage vessels into the aeration vessels of the test and blank units. To get
the normal hydraulic retention time (see note 1, Clause 4) of 6 h in the aeration vessel, pump the organic
medium at 0,5 l/h into the aeration vessel, preferably at appropriate intervals (see note in 8.3.3) to improve the
settleability of the sludge. Measure the amount of organic medium dosed into the units carefully.
If the organic medium is kept for longer than one day, cooling at about 4 °C is necessary to prevent microbial
growth and biodegradation outside of the test units.
If synthetic sewage is used, it is possible to add the concentrated stock solution of synthetic sewage (e.g. 10-
fold strength) and the corresponding amount of tap water separately to get the desired DOC concentration or
COD value in the influent. Store the stock solution at about 4 °C in a refrigerator and use directly or use a
sterilized solution.
If domestic sewage is used, install a pipe, e.g. a ring pipeline, and continuously pump settled (or decanted)
sewage in a closed loop. Discharge waste water from this pipeline into a storage vessel at such an overflow
rate that fresh waste water is always available and that the concentration of dissolved oxygen does not fall
below about 4 mg/l.
8.3.2 Dosage of test compound
Add appropriate amounts of the stock solution of the test compound (6.4) to the storage vessel of the influent
or dose it directly into the aeration vessel continuously or discontinuously with a separate pump. The normal
mean test concentration in the influent should be such that the DOC lies between 10 mg/l and 20 mg/l, with an
upper concentration of no more than 50 mg/l. If the water-solubility of the test compound is low or if toxic
effects are likely to occur, reduce the test concentration, but to not less than 5 mg/l DOC for analytical reasons.
Lower test concentrations may be used if the primary biodegradation is determined using specific analysis.
Check that there is no precipitation when the stock solution is added to the tap water. A dispersed, poorly
water-soluble test compound may be added using special dosing techniques. For more information, see
ISO 10634.
Add the test compound from the beginning of the test or add it only after a stabilization period in which the
DOC has been largely (about 80 %) removed from the organic medium. It is important to check that all units
are working equally efficiently. If they are not, it usually helps to mix the individual sludges and to re-distribute
equal volumes to the individual units. Direct progressive addition of the test compound from the beginning has
the advantage that the activated sludge is better able to adapt to the test compound.
Determine the volume in the storage vessel at regular intervals or measure the flow rate to determine exactly
how much test compound has been dosed to the test system.
8.3.3 Handling of activated sludge
The concentration of solids in activated sludge normally stabilizes during the test, independent of the inoculum
used, in the range of 1 g/l to 3 g/l, depending on the quality and concentration of the organic medium, the
operating conditions, the nature of the micro-organisms present and the influence of the test compound.
[25]
Either determine the suspended solids (e.g. ISO 11923 ) in the aeration vessel at least weekly and discard
the surplus sludge to maintain the concentration at 1 g/l to 3 g/l or, preferably, control the mean sludge
retention time, SRT (sludge age), at a constant value in the range of 6 d to 10 d. The SRT can be controlled
by removing a certain volume each day or by means of an automatic intermittently working pump; see also
Annex D.
NOTE For the volume removal, 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; see also note 2, Clause 4.
ISO 11733:2004(E)
Maintaining a nearly constant concentration of suspended solids does not maintain a constant sludge
retention time, which essentially determines the degree of biodegradation and hence the concentration of the
test compound in the effluent. Because the concentration of suspended solids in a sludge is not an
independent variable, the higher values (e.g. > 3 g/l) are not reached if influents of low concentrations are
treated. Conversely, high removal of DOC is not obtained when low concentrations of suspended solids are
maintained with influents of high DOC concentrations.
At least daily throughout the test, remove any sludge adhering to the walls of the aeration vessel and the
separator so that it is re-suspended. Check and clean regularly all tubes to prevent the growth of biofilm. If a
distinct sludge age is required, remove sludge from the aeration vessel at least once a day. Recycle the
settled sludge from the separator to the aeration vessel, preferably by intermittent pumping.
NOTE In the (Husmann) activated-sludge plants (7.1), poor settlement and loss of sludge might occur. This may be
rectified by a number of actions which can be performed in parallel in test and control units:
 Add appropriate amounts of fresh sludge at regular intervals (e.g. weekly).
 Dose the organic medium at intervals (e.g. for 3 min to 10 min every hour) into the aeration vessel.
 Pump sludge intermittently (e.g. for 5 min every 2,5 h to recycle 1 l/h to 1,5 l/h) from the separator to the aeration
vessel.
 Replace the air lift by a peristaltic pump and use a sludge re-circulation flow which approximately equals the influent
flow.
 Pass air in short bursts (e.g. for 10 s every hour) through the settled sludge in the separator.
 Use a non-toxic anti-foaming agent, e.g. silicone oil, at a minimal concentration to prevent loss by foaming.
 Add, in an appropriate way, a suitable flocculant, e.g. about 2 ml of an iron(III) chloride solution (50 g/l FeCl ), per
unit; ensure in advance that no reaction or precipitation of the test compound occurs.
8.3.4 Sampling and analyses
[13]
At regular intervals, measure the dissolved oxygen concentration (e.g. ISO 5814 ), the temperature and the
[23]
pH (e.g. ISO 10523 ) of the activated sludge in the aeration vessels. Ensure that sufficient oxygen (> 2 mg/l)
is always available and that the temperature is kept in the desired range (normally 20 °C to 25 °C). Keep the
pH in a range of (7,5 ± 0,5) by dosing small amounts of inorganic base or acid into the aeration vessel or into
the influent, or by increasing the buffer capacity of the organic medium (6.3). The frequency of measuring
depends on the parameter to be measured and the stability of the system and can vary from daily to weekly
measurements.
[16] [14]
For ultimate biodegradation, measure the DOC (e.g. ISO 8245 ) or COD (e.g. ISO 6060 ) in the influent to
and the effluent from the test and the control units. For primary biodegradation, measure the test compound
concentration by substance-specific analyses in the influent to and the effluent from the test unit. The
relatively small difference between two relatively large DOC concentrations or COD in the organic medium
and that spiked with test compound can lead to variable data. Therefore, alternatively, estimate these
parameters from the concentration of the stock solution of the test compound (6.4), the organic medium (6.3)
and the volumes dosed into the test and control units.
To reduce the number of samples and the variability of the data of the influent, it is recommended to measure
the COD and the concentrations of DOC or the test compound in each new batch of the stock solution and
organic medium and to calculate the concentration in the influent from these values instead of measuring it
directly in the influent.
For measurements in the effluent, take suitable samples (e.g. 24-h composites) from the collected effluent and
filter (7.3.1) them or centrifuge (7.3.2) them at about 40 000 m/s for about 15 min. Centrifuging is preferred,
especially if filtering is difficult. Determine DOC or COD at least in duplicate to measure the ultimate
biodegradation and, if desired, the primary biodegradability by an analysis specific for the test compound.
The use of COD as summary parameter might give rise to analytical problems at low values. It is therefore
recommended only if a sufficiently high (about 30 mg/l COD) test concentration is used.
10 © ISO 2004 – All rights reserved

ISO 11733:2004(E)
In the case of adsorbing test compounds, it is recommended to measure the amount of adsorbed substance
on the sludge using an analytical technique specific for the test substance. The adsorption potential of
[28]
compounds on sludge can also be determined by a special adsorption test (e.g. ISO 18749 ).
The frequency of sampling depends on the expected duration of the test. A recommended frequency is three
samples per week. Once the units are operating efficiently, allow from one week to a maximum of six weeks
after the test compound has been introduced for adaptation to reach a steady state. Then, obtain at least
15 valid values in the plateau phase for the evaluation of the test result. The test may be finished when a
sufficient reduction (e.g. > 90 %) has been reached and 15 values are available. The normal test duration is
not more than 12 weeks after addition of the test compound.
All analyses should be performed as soon as possible. If analyses are postponed, store the samples at about
4 °C in the dark in full, tightly stoppered bottles. If samples are stored for more than 48 h, preserve them by
deep-freezing, acidification (e.g. the addition of 10 ml/l of a 400-g/l sulfuric acid solution) or by the additio
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