SIST EN ISO 8192:2007

SLOVENSKI STANDARD
SIST EN ISO 8192:2007
01-september-2007
1DGRPHãþD
SIST EN ISO 8192:1997
Kakovost vode - Preskus inhibicije porabe kisika z aktivnim blatom za oksidacijo
ogljika in amonija (ISO 8192:2007)
Water quality - Test for inhibition of oxygen consumption by activated sludge for
carbonaceous and ammonium oxidation (ISO 8192:2007)
Wasserbeschaffenheit - Bestimmung der Hemmung des Sauerstoffverbrauchs von
Belebtschlamm nach Kohlenstoff- und Ammonium-Oxidation (ISO 8192:2007)
Qualité de l'eau - Essai d'inhibition de la consommation d'oxygene par des boues
activées pour l'oxydation du carbone et de l'ammonium (ISO 8192:2007)
Ta slovenski standard je istoveten z: EN ISO 8192:2007
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 8192:2007 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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EUROPEAN STANDARD
EN ISO 8192
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2007
ICS 13.060.70 Supersedes EN ISO 8192:1995
English Version
Water quality - Test for inhibition of oxygen consumption by
activated sludge for carbonaceous and ammonium oxidation
(ISO 8192:2007)
Qualité de l'eau - Essai d'inhibition de la consommation Wasserbeschaffenheit - Bestimmung der Hemmung des
d'oxygène par des boues activées pour l'oxydation du Sauerstoffverbrauchs von Belebtschlamm nach
carbone et de l'ammonium (ISO 8192:2007) Kohlenstoff- und Ammonium-Oxidation (ISO 8192:2007)
This European Standard was approved by CEN on 29 December 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 CEN Management Centre 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 CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, 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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2007 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 8192:2007: E
worldwide for CEN national Members.

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EN ISO 8192:2007 (E)





Foreword


This document (EN ISO 8192:2007) 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 August 2007, and conflicting national
standards shall be withdrawn at the latest by August 2007.

This document supersedes EN ISO 8192:1995.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Bulgaria, 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 8192:2007 has been approved by CEN as EN ISO 8192:2007 without any
modifications.

2

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INTERNATIONAL ISO
STANDARD 8192
Second edition
2007-02-01

Water quality — Test for inhibition of
oxygen consumption by activated sludge
for carbonaceous and ammonium
oxidation
Qualité de l'eau — Essai d'inhibition de la consommation d'oxygène par
des boues activées pour l'oxydation du carbone et de l'ammonium




Reference number
ISO 8192:2007(E)
©
ISO 2007

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ISO 8192:2007(E)
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ii © ISO 2007 – All rights reserved

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ISO 8192:2007(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle. 2
5 Reagents, media and inoculum. 3
6 Apparatus . 4
7 Test environment. 5
8 Procedure . 5
9 Calculation and expression of results. 8
10 Validity of the results. 11
11 Test report . 13
Annex A (informative) Examples of measuring units . 14
Annex B (informative) Apparatus for culturing nitrifying activated sludge . 16
Annex C (informative) Overview of the test procedure . 18
Annex D (informative) Mixtures for the preliminary test . 19
Annex E (informative) Example of an inhibition curve . 20
Bibliography . 21

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ISO 8192:2007(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 8192 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 8192:1986), which has been technically revised.
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ISO 8192:2007(E)
Introduction
Information generated by this method for assessing the potential toxicity of substances, mixtures and waste
waters to activated sludge may be helpful in estimating the effect of a test material on mixed bacterial
communities in the aquatic environment, especially in aerobic biological treatment systems. The susceptibility
of oxygen uptake by different sub-populations of the bacterial communities to inhibition by chemicals and
waste waters is not necessarily uniform and selective effects may profoundly influence the outcome of the test.
There are two principal groups of microorganisms contributing to the total oxygen consumption by activated
sludge: heterotrophic organisms mainly responsible for the breakdown of carbon-based substrates
(carbonaceous oxidation) and autotrophic nitrifying organisms causing the oxidation of ammonium to nitrate
(nitrification).
This International Standard may be used to assess the toxicity of substances on total oxygen uptake (i.e.
carbonaceous oxidation and nitrification combined) or, by deliberately adding a specific inhibitor of nitrification,
also to assess toxicity of substances to the carbonaceous and nitrification components separately.
For the determination of the nitrification inhibition with this method, a sufficiently nitrifying activated sludge is
[4]
required. Indications of nitrification may be investigated further by application of ISO 9509 .
The user of this method should be aware that particular problems could require the specification of additional
marginal conditions.
The inhibitory effect of a test material may be exerted on both components or it may be exerted predominantly
on only one of them. Nitrification is the process more commonly prone to selective inhibition.
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INTERNATIONAL STANDARD ISO 8192:2007(E)

Water quality — Test for inhibition of oxygen consumption by
activated sludge for carbonaceous and ammonium oxidation
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This International 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 International Standard
be carried out by suitably trained staff.
1 Scope
This International Standard specifies a method for assessing the inhibitory effect of a test material on the
oxygen consumption of activated sludge microorganisms.
This method is intended to represent the conditions in biological waste-water treatment plants. It gives
information on inhibitory or stimulatory effects after a short exposure (usually 30 min up to 180 min or even
more) of the test material on activated sludge microorganisms.
This method is applicable for testing waters, waste waters, pure chemicals and mixtures of chemicals.
Concerning the chemicals, the method refers to those which are soluble under the test conditions. Special
care is necessary with materials of low water solubility, high volatility and with materials abiotically consuming
or producing oxygen.
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 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
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 oxygen
[3]
(ISO 6107-1:2004 , definition 2)
3.2
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
[6]
(ISO 9888:1999 , definition 3.4)
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ISO 8192:2007(E)
3.3
oxygen consumption rate
uptake of oxygen by activated sludge microorganisms per unit volume of sludge, in unit time
NOTE This quantity is expressed in milligrams per litre per hour [mg/(l⋅h)].
3.4
specific oxygen consumption rate
uptake of oxygen by activated sludge microorganisms per unit mass of dry sludge (suspended solids), in unit
time
NOTE This quantity is expressed in milligrams per gram per hour [mg/(g⋅h)].
3.5
inhibition of oxygen consumption
decrease of the oxygen consumption rate of an activated sludge plus (a) degradable substance(s) in the
presence of the test material, compared with that of a similar mixture without test material
NOTE 1 This quantity is expressed as a percentage.
NOTE 2 In the absence of a substrate, some chemicals (e.g. uncouplers of phosphorylation) can increase oxygen
uptake.
3.6
toxic range
range of concentration of a test material over which 0 % to 100 % inhibition occurs
3.7
EC
50
effective concentration of the test material giving a calculated or interpolated inhibition of oxygen consumption
of 50 % compared with a blank control
3.8
nitrification
oxidation of ammonium compounds by bacteria
NOTE Usually the intermediate product is nitrite and the end product is nitrate
[3]
[ISO 6107-1:2004 , definition 49].
4 Principle
In the presence of easily biodegradable substances, activated sludge consumes oxygen at a higher rate than
in their absence, depending on, among other factors, the concentration of microorganisms. Addition of a toxic
concentration of a test material results in a decrease in the oxygen consumption rate. The rates are measured
using an oxygen electrode. The percentage inhibition of the oxygen consumption is estimated by comparison
of the rate with that of a control mixture containing no test material.
The sensitivity of the activated sludge may be checked with a suitable reference substance. The inhibition of
the oxygen uptake by all sludge microorganisms, heterotrophic microorganisms and the oxidation of
ammonium salts by nitrifying microorganisms may be separately expressed from measurements of the rate of
uptake in the absence and presence of N-allylthiourea (ATU), a specific inhibitor of the oxidation of ammonium
to nitrite by first-stage nitrifiers. The difference between the two oxygen values is due to nitrification and the
residual value in the presence of allylthiourea is due to the heterotrophs. Any oxygen consumption due to
abiotic processes may be detected by determining the rate in mixtures of the test material, synthetic medium
and water, but omitting activated sludge.
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ISO 8192:2007(E)
Under certain (rare) circumstances, a test substance with strong reducing properties may cause measurable
abiotic oxygen consumption. In such cases, abiotic controls are necessary to discriminate between oxygen
uptake by the test substance and microbial respiration. Abiotic controls may be prepared either by omitting the
inoculum from test mixtures, or by poisoning the inoculum with a solution of mercury(II) chloride.
5 Reagents, media and inoculum
Use only reagents of recognized analytical grade.
5.1 Water, complying with grade 1 as defined in ISO 3696, dissolved respectively distilled or de-ionized
water containing less than 1 mg/l dissolved organic carbon (DOC).
5.2 Specific nitrification inhibitor, N-allylthiourea (ATU).
Dissolve 2,50 g of N-allylthiourea (ATU) in 1 000 ml of water (5.1). The addition of 2,32 ml of this stock
−4
solution to a sample of 500 ml results in a final concentration of 11,6 mg/l (10 mol/l).
5.3 Mercury(II) chloride solution
If required (see Clause 4), prepare a solution of 0,10 g of mercury(II)chloride (HgCl ) in 10 ml of water (5.1).
2
WARNING — Stringent safety precautions and extraordinary waste disposal measures apply to the
use of mercury salts in the laboratory. Routine deployment of abiotic controls poisoned with mercuric
chloride is not recommended.
5.4 Antifoam agent, free from silicone.
5.5 Reference substance, stock solution.
Prepare a solution containing 1,00 g of 3,5-dichlorophenol (3,5-DCP) in 1 000 ml of water (5.1). Use warm
water and/or ultrasonication to accelerate the dissolution and make the solution up to volume when it has
cooled to room temperature.
Alternatively N-methylaniline can be used as a reference substance, especially for inhibition of nitrification
processes. When using this substance, prepare a solution containing 1,00 g of N-methylaniline (NMA) in
1 000 ml of water (5.1).
5.6 Test medium, synthetic sewage 1 (100-fold OECD medium).
Peptone 16 g
Meat extract 11 g
Urea [CO(NH )] 3 g
2 2
Sodium chloride (NaCl) 0,7 g
0,4 g
Calcium chloride dihydrate (CaCl ⋅ 2H O)
2 2
0,2 g
Magnesium sulfate heptahydrate (MgSO ⋅ 7 H O)
4 2
Anhydrous potassium monohydrogenphosphate (K HPO) 2,8 g
2 4
Water (5.1) 1 l
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ISO 8192:2007(E)
The pH of this synthetic medium shall be 7,5 ± 0,5.
If the prepared synthetic medium is not used immediately, store it in the dark at 0 °C to 4 °C, for no longer
than 1 week.
Alternatively, sterilize the synthetic medium prior to storage, or add the peptone and meat extract shortly
before carrying out the test. Prior to use, ensure that the medium is mixed thoroughly and adjust the pH as
necessary.
5.7 Test material, stock solution.
The test material may be a pure chemical, a mixture of chemicals, a chemical product or a waste water.
Prepare a stock solution of the test material in water (5.1) at a suitable concentration, for example 1 g/l or
10 g/l. Waste waters may be used without dilution.
For insoluble materials, a suspension or dispersion may be prepared, or the test material may be added
directly to the test vessels. Take care to ensure as much homogeneity as possible. For handling insoluble
[7]
materials, see, for example, ISO 10634 .
5.8 Inoculum
For general use, activated sludge should be taken from the exit of the aeration tank (where substrate
concentrations are lowest) of a waste-water plant, treating predominantly domestic sewage, and working
efficiently. Depending on the purpose of the test, any type of activated sludge, including sludge grown in the
laboratory and sludge grown on industrial waste waters, may also be used at a suitable suspended-solids
concentration of, for example, 2 g/l to 4 g/l. However, activated sludges from different treatment plants are
likely to exhibit different characteristics and sensitivities.
6 Apparatus
General laboratory equipment, and the following (see Annex A).
6.1 Test vessels: 250 ml to 300 ml biochemical oxygen demand (BOD) bottles or Erlenmeyer flasks with
stoppers are recommended (see Figure A.1). Alternatively, larger test vessels may also be used (see
Figure A.2).
When using a BOD bottle for oxygen measurements, a suitable sleeve adapter may be required for sealing
the oxygen electrode against the necks of the test vessels (see Figure A.1). To avoid loss of displaced liquid
on insertion of the oxygen electrode, it is advisable first to insert a funnel or glass tube through the sleeve, or
to use vessels with flared-out rims.
6.2 Device for measuring oxygen concentration: comprising a suitable oxygen electrode, a cell to
contain the sample and a recorder (see Figure A.2).
6.3 Magnetic stirrers, covered with an inert material.
6.4 Aeration device
If necessary, pass compressed air through an appropriate filter to remove dust and oil, and through wash
bottles containing water to humidify the air. Aerate the test vessels with Pasteur pipettes, or other aeration
devices which do not adsorb chemicals.
6.5 pH-meter
2
6.6 Centrifuge, general bench-top centrifuge for sludge, capable of 10 000 m/s .
6.7 Apparatus for culturing nitrifying activated sludge (see Annex B).
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ISO 8192:2007(E)
7 Test environment
Perform the test at a temperature within the range of (22 ± 2) °C and in an atmosphere free from dust and
toxic vapours.
8 Procedure
8.1 General
An overview of the test procedure is shown in Annex C.
The procedures to be applied to nitrifying sludge differ from those applied to non-nitrifying sludge. Therefore, it
is advisable first to check the activated sludge for its nitrification activity (see Annex C).
The use of nitrifying sludge is only necessary when the influence of a test material on nitrification is to be
determined. Nitrifying sludge is not required if only heterotrophic respiration is determined.
In order to check the nitrification activity of the sludge, apply the nitrification test (8.8) and calculate the rate of
nitrification, if any, according to 9.2.
This preliminary test serves as a range-finder for the following definitive test.
See 8.9 for an outline of this preliminary test.
8.2 Elimination of foam
Difficulties can arise if foaming occurs during the incubation, to the extent that the foam, and the sludge solids
carried on it, are expelled from the aeration vessels. Occasionally, foaming may simply result from the
presence of the synthetic sewage, but foaming should be anticipated if the test material is, or contains, a
surfactant. Loss of sludge solids from the test mixtures will result in artificially lowered respiration rates that
could mistakenly be interpreted as a result of inhibition. In addition, aeration of surfactant solutions
concentrates the surfactant in the foam layer; loss of foam from the test system will lower the exposure
concentrations.
If foaming occurs, add a surfactant-free silicone-emulsion antifoam agent (5.4). If the problem is associated
with the presence of the synthetic sewage, modify the sewage concentrate (5.6) by including an antifoam
agent (5.4) at a rate of 50 µl/l. If foaming is caused by the test material, determine the quantity (generally a
few drops from a Pasteur pipette) needed for abatement at the maximum test concentration, then treat all
individual aeration vessels identically (including those, for example, blank controls and reference vessels,
where foam is absent).
8.3 Preparation of inoculum
Where necessary, remove coarse particles by settling for a short period, for example 15 min, and decanting
the upper layer of finer solids for use. Alternatively, the sludge may be homogenized by using a blender for a
few seconds. Where necessary, remove coarse particles with a suitable sieve.
The sludge may be washed as follows: first centrifuge (6.6) the sludge for about 10 min at approximately
2
10 000 m/s and discard the supernatant liquid. Re-suspend the sludge in chlorine-free tap water, remove this
by re-centrifuging and then repeat, if necessary, the washing and centrifuging process. Determine the dry
mass of a known volume of the sludge. Finally re-suspend the sludge in chlorine-free tap water to obtain the
required activated sludge concentration, of about 3 g/l of suspended solids.
Having adjusted the concentration of suspended solids, continuously aerate the activated sludge and, where
possible, use it within 24 h of collection. If this is not possible, the activated sludge may be fed for up to one
additional day with synthetic medium (see 5.6) at a rate not exceeding 50 ml per litre per day, provided no
significant change in its activity results and that nitrification, if initially present, is not lost. Alternatively,
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ISO 8192:2007(E)
changes in activity may be minimized by refrigerating the activated sludge at 4 °C for up to 4 d without
feeding [13]. In all cases, the origin, the concentration, any pre-treatment and maintenance of the activated
sludge shall be stated in the test report. Knowledge about possible changes occurring to sludges during
storage is insufficient. Therefore, any sludge storage and/or treatment should be the same for all samples in a
study under investigation.
WARNING — Laboratory-grown sludges can be less active, with a narrower spectrum of substrates
than sludges from waste-water treatment plants.
8.4 Test mixtures
Incubate test mixtures under conditions of forced aeration. Start the incubation (aeration) for each preparation
with the initial contact between the activated sludge inoculum and the other mixture constituents, and finalize
after a specified exposure time when the rate of decline in dissolved oxygen concentration is measured.
The capacity of the equipment used to measure oxygen consumption rates determines the manner in which
the incubations begin. For example, if it comprises a single probe, the measurements are made individually. In
this case, prepare the various mixtures required for the test, but withhold the inoculum, adding it to each
vessel of the series and starting each incubation in turn, at timed intervals of, for example, 10 min to 15 min.
Alternatively, the measuring system may comprise multiple probes that facilitate multiple, simultaneous
measurements, in which case inoculum may be added at the same time to appropriate groups of vessels.
The activated sludge concentration in the test mixtures is nominally 1 500 mg/l of suspended solids. Measure
the oxygen consumption after 30 min of incubation. If more information after an extended contact time is found
necessary, carry out additional measurements after 180 min of incubation. Depending on the purpose of the
test, the incubation time may be extended still further, e.g. up to 27 h. For a 27 h test, add the synthetic
medium (5.6) after 24 h of incubation (without synthetic medium) and aerate additionally for 3 h. This shall be
stated in the test report.
NOTE Usually, an incubation time of 30 min is sufficient. Longer incubation may, for example, be required for
substances that are poorly soluble in water. Any extension of the incubation time will result in an increase of work.
Prepare in the test vessels (6.1) mixtures, F , containing dilution water (5.1), synthetic medium (5.6) and test
T
material (5.7), to obtain different known concentrations as required. See Table D.1 of Annex D for examples of
volumes of constituents. Adjust the pH to 7,5 ± 0,5, dilute with water and add the inoculum (5.8) to obtain
equal final volumes. If the inhibitory effect of the pH is to be tested, do not adjust the pH.
8.5 Reference mixtures
Normally, for most cases, prepare mixtures, F , with a suitable reference substance (5.5) in the same way as
R
in 8.4 (see 8.10.2).
8.6 Blank control
Carry out at least one blank control, F , which contains an equal volu
...