EN ISO 9509:2006

SLOVENSKI STANDARD
01-januar-2007
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SIST EN ISO 9509:1998
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QLWULILNDFLMHYDNWLYQHPEODWX,62
Water quality - Toxicity test for assessing the inhibition of nitrification of activated sludge
microorganisms (ISO 9509:2006)
Wasserbeschaffenheit - Toxizitätstest zur Bestimmung der Nitrifikationshemmung in
Belebtschlamm (ISO 9509:2006)
Qualité de l'eau - Essai de toxicité pour l'évaluation de l'inhibition de la nitrification des
micro-organismes des boues activées (ISO 9509:2006)
Ta slovenski standard je istoveten z: EN ISO 9509:2006
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 9509
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2006
ICS 13.060.70 Supersedes EN ISO 9509:1995
English Version
Water quality - Toxicity test for assessing the inhibition of
nitrification of activated sludge microorganisms (ISO 9509:2006)
Qualité de l'eau - Essai de toxicité pour l'évaluation de Wasserbeschaffenheit - Toxizitätstest zur Bestimmung der
l'inhibition de la nitrification des micro-organismes des Nitrifikationshemmung in Belebtschlamm (ISO 9509:2006)
boues activées (ISO 9509:2006)
This European Standard was approved by CEN on 30 June 2006.
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, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
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EUROPÄISCHES KOMITEE FÜR NORMUNG
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© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 9509:2006: E
worldwide for CEN national Members.

Foreword
This document (EN ISO 9509:2006) 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 January 2007, and conflicting national
standards shall be withdrawn at the latest by January 2007.

This document supersedes EN ISO 9509:1995.

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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

Endorsement notice
The text of ISO 9509:2006 has been approved by CEN as EN ISO 9509:2006 without any
modifications.
INTERNATIONAL ISO
STANDARD 9509
Second edition
2006-07-01
Water quality — Toxicity test for
assessing the inhibition of nitrification of
activated sludge microorganisms
Qualité de l'eau — Essai de toxicité pour l'évaluation de l'inhibition de la
nitrification des micro-organismes des boues activées

Reference number
ISO 9509:2006(E)
©
ISO 2006
ISO 9509:2006(E)
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ii © ISO 2006 – All rights reserved

ISO 9509:2006(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 2
4 Principle. 2
5 Reagents and materials . 3
6 Apparatus . 4
7 Procedure . 4
8 Calculation and expression of results. 5
9 Validity of results. 6
10 Precision. 7
11 Test report . 7
Annex A (normative) Determination of the nitrifying activity of an activated sludge. 8
Annex B (normative) Example for preparation of the test . 9
Annex C (informative) Apparatus for culturing nitrifying activated sludge . 10
Bibliography . 12

ISO 9509:2006(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 9509 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 9509:1989), which has been technically revised.
iv © ISO 2006 – All rights reserved

ISO 9509:2006(E)
Introduction
Nitrification is an important process in the treatment of waste waters, since it is necessary to reduce the
polluting effects of ammonium in treated discharges to receiving waters. It is further necessary to convert
ammonium to nitrate in order to allow the subsequent process of denitrification (producing nitrogen gas) in the
anoxic stage of the modified activated sludge process, thus considerably reducing the potential for
eutrophication in the receiving waters. The nitrification process is generally performed by two separate groups
of autotrophic bacterial species This International Standard describes a method for assessing the inhibition of
the production of oxidized nitrogen (nitrite plus nitrate), or of the removal of ammonium, by nitrifying activated
sludge.
INTERNATIONAL STANDARD ISO 9509:2006(E)

Water quality — Toxicity test for assessing the inhibition of
nitrification of activated sludge microorganisms
WARNING — Sewage and activated sludge contain potentially pathogenic organisms. Appropriate
precautions are necessary when handling them.
Toxic test substances and those with unknown properties are to be handled with care.
Persons using this International Standard should be familiar with normal laboratory practice. This
standard does not purport to address all of the safety problems, if any, associated with its use. It is
the responsibility of the user to establish appropriate safety and health practices and to ensure
compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this standard be carried
out by suitably trained staff.
1 Scope
This International Standard specifies a method for assessing the short-term inhibitory effect of waters, waste
waters or test substances on nitrifying bacteria in activated sludge. The inhibitory effect is estimated over an
exposure period of usually 3 h or up to 24 h with weakly nitrifying sludge.
The method is applicable to nitrifying activated sludge derived from domestic and synthetic sewage and also
to sludges from industrial and mixed domestic and industrial waste waters.
The nitrifying activity of the sludge is verified by testing in the presence and absence of a specific inhibitor (e.g.
N-allylthiourea; see Annex A). If the nitrification rate is within a suitable range for the test, i.e. 2 mg of nitrogen
per gram of suspended solid and hour to 6,5 mg of nitrogen per gram of suspended solids and hour, the
sludge may be used directly. If not, adjustments are necessary (see Clause 9).
The method is applicable to water-soluble, non-volatile chemicals, and to waste waters
Sludges from different sources respond differently to a given concentration of an inhibitor mainly due to
reaction between the inhibitor and components of the sludge. This results in a partial neutralisation of the toxic
effect. Also, since the test lasts only hours, any inhibitory effects may diminish or increase over a longer
period, e.g. in the continuous activated sludge system (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 — Guidance on biotesting of samples
ISO 6777, Water quality — Determination of nitrite — Molecular absorption spectrometric method
ISO 7150-1, Water quality — Determination of ammonium — Part 1: Manual spectrometric method
ISO 9509:2006(E)
ISO 11733, Water quality — Determination of the elimination and biodegradability of organic compounds in an
aqueous medium — Activated sludge simulation test
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
nitrification
oxidation of ammonium compounds by bacteria
NOTE Usually the intermediate product is nitrite and the end product nitrate
[ISO 6107-1:2004, 49]
3.2
test material
pure chemicals, clearly defined mixtures of chemicals, chemical products, waste waters and treated waste
waters
3.3
activated sludge
accumulated biological mass (floc) produced in the treatment of waste water by the growth of bacteria and
other microorganisms in the presence of dissolved oxygen
[ISO 6107-1:2004, 2]
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
[ISO 9888:1999, 3.4]
3.5
toxic range
range of concentration of a test material over which 0 % to 100 % inhibition occurs
1)
[ISO 8192:— ]
3.6
EC
effective concentration of the test material giving a calculated or interpolated inhibition of nitrification of 50 %,
compared with a blank control
4 Principle
The percentage inhibition of nitrification by various concentrations of the test material is calculated by
assessing the difference in concentration of oxidized nitrogen (nitrite plus nitrate) produced, or of ammonium
utilized, under standard conditions by the oxidation of ammonium salts after the parallel aeration of a nitrifying
sludge in the presence and absence of test material.

1) To be published. (Revision of ISO 8192:1986)
2 © ISO 2006 – All rights reserved

ISO 9509:2006(E)
5 Reagents and materials
5.1 Deionized water, for the preparation of defined stock solutions. For washing procedures, tap water is
suitable as well.
2+
Make sure that the water is free from chemicals which may inhibit nitrification processes (e.g. Cu ions).
5.2 Nitrifying activated sludge.
Collect a sufficient volume of a nitrifying activated sludge from a local waste water treatment plant, or from a
laboratory-grown sludge (see Annex C), in which nitrification is known to be occurring. According to the
purpose of the test, sludge may be collected from plants treating predominantly domestic sewage, mixed
domestic industrial waste water or solely industrial waste water; the source of the sludge and the treated
waste water should be reported since the results of the test often depend of the origin of the sludge used (see
Reference [6]). Maintain the sludge in an aerobic condition. Since the toxicity to nitrification may change with
time of storage (see Reference [1]), assessments should be made as soon as possible after collection and
preferably within 24 h (see ISO 5667-16).
Instead of using activated sludge from a waste water plant, nitrifying sludge can be grown in the laboratory
(see Annex C).
Although the sludge may be used as collected, it is preferable to wash the sludge to remove any inhibitors and
nitrate present, before re-suspending in chlorine-free, nitrate-free tap water. This washing procedure may be
─1
carried out by centrifuging or settling and is optional. Centrifuge (e.g. 10 000 min for 5 min) or settle the
sludge and discard the supernatant liquid. Wash the residue with a volume of tap water equal to the original
volume, re-centrifuge or settle and again discard the supernatant liquid. Finally, re-suspend the centrifuged or
settled sludge in an appropriate volume of tap water to give the required concentration of mixed liquor
suspended solids (e.g. 3 g/l) and aerate until use.
5.3 Full medium.
Dissolve 5,04 g of sodium hydrogen carbonate, NaHCO , and 2,65 g of ammonium sulfate, (NH ) SO , in 1 l
3 4 2 4
of water (5.1).
NOTE This medium, when diluted 1:10 (1 + 9) with water (5.1), contains 56 mg of nitrogen per litre and has a pH
value of about 7,6. It allows the production of at least 25 mg/l of oxidized nitrogen without changing the pH value.
5.4 Medium for waste water samples
5.4.1 Medium A.
Dissolve 10,08 g of sodium hydrogen carbonate, NaHCO , in 1 l of water (5.1).
5.4.2 Medium B.
Dissolve 5,3 g of ammonium sulfate, (NH ) SO , in 1 l of water (5.1).
4 2 4
5.5 Reference inhibitor.
Dissolve 1,16 g of N-allylthiourea (ATU) in 1 l of water (5.1).
Other inhibitors may be used as well, e.g. 2-chloro-6-(trichloromethyl)pyridine, but the concentration required
and mode of addition should be investigated in advance.
ISO 9509:2006(E)
5.6 Stock solution of test substance.
Prepare a stock solution or suspension of the test substance in distilled water (5.1) at a suitable concentration,
e.g. 1 g/l or 10 g/l.
If necessary, adjust the pH of the stock solution to 7,6 ± 0,1.
5.7 Waste water samples.
Collect a representative sample of the waste water and store it below 4 °C for as short a period as possible
(see, e.g. ISO 5667-16). The pH of the sample should be adjusted to 7,6 ± 0,1, unless the effect of the whole
sample
...