Water quality - Adsorption of substances on activated sludge - Batch test using specific analytical methods

This International Standard specifies a screening test method for the determination of the degree of adsorption of substances on to the activated sludge or primary sludge in a waste water treatment plant. The conditions described in this International Standard normally correspond to the optimum conditions for the adsorption to occur at the chosen activated-sludge concentration and water hardness during the test period. The method applies to substances for which an analytical method with sufficient accuracy is available and which, under the conditions of the test and at the test concentration used, a) are water-soluble; b) or, if only slightly water-soluble, allow sufficiently stable suspensions, dispersions or emulsions to be prepared; c) are not significantly removed from the test solution during the test by known abiotic processes such as stripping or foaming; d) do not deflocculate activated sludge; e) are not readily biodegradable (for a discussion of biodegradability, see ISO/TR 15462).

Qualité de l'eau - Adsorption des substances sur la boue activée - Essai de lot utilisant des méthodes analytiques spécifiques

Kakovost vode - Adsorpcija snovi na aktivno blato - Šaržni preskus z uporabo specifičnih analitskih metod

Ta mednarodni standard določa metodo presejalnega preskusa za določevanje stopnje adsorpcije snovi na aktivno blato ali blato v prvotni obliki v čistilni napravi odpadne vode. Pogoji, opisni v tem mednarodnem standardu običajno ustrezajo optimalnim pogojem za adsorbcijo, do katere pride na izbrani koncentraciji aktivnega blata in trdnosti vode med preskusnim obdobjem. Metoda velja za snovi, za katere je na voljo analitska metoda z zadostno točnostjo in ki, pod preskusnimi pogoji in uporabljenimi preskusnimi koncentracijami, a) so topne v vodi; b) ali če so le nekoliko topne v vodi, omogočajo pripravo stabilnih suspenzij, disperzij ali emulzij; c) niso znatno odstranjene iz preskusne raztopine z poznanimi abiotičnimi procesi, kot so odstranjevanje ali  penjenja; d) ne razgradijo aktivnega blata v fine delce; e) niso lahko biorazgradljive (za razpravo o biorazgradljivosti glej ISO/TR 15462).

General Information

Status
Published
Public Enquiry End Date
19-Jul-2009
Publication Date
01-Jul-2010
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
12-May-2010
Due Date
17-Jul-2010
Completion Date
02-Jul-2010

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INTERNATIONAL ISO
STANDARD 18749
First edition
2004-02-15
Water quality — Adsorption of
substances on activated sludge — Batch
test using specific analytical methods
Qualité de l'eau — Adsorption des substances sur la boue activée —
Essai de lot utilisant des méthodes analytiques spécifiques

Reference number
ISO 18749:2004(E)
©
ISO 2004

---------------------- Page: 1 ----------------------
ISO 18749:2004(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall
not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the
unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
© ISO 2004
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56  CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
©
ii ISO 2004 – All rights reserved

---------------------- Page: 2 ----------------------
ISO 18749:2004(E)
Contents Page
1 Scope . 1
2 Terms and definitions . 1
3 Principle . 2
4 Reagents and materials . 2
5 Apparatus . 4
6 Test environment . 5
7 Procedure . 5
8 Calculation . 7
9 Validity of the test . 9
10 Test report . 9
Bibliography . 10
©
ISO 2004 – All rights reserved iii

---------------------- Page: 3 ----------------------
ISO 18749:2004(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 18749 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
©
iv ISO 2004 – All rights reserved

---------------------- Page: 4 ----------------------
ISO 18749:2004(E)
Introduction
This test is used as a screening test to determine the degree of adsorption of substances on activated sludge
or primary sludge in waste water treatment plants. General information on the adsorption and desorption of test
compounds may also be obtained by other tests (see e.g. Reference [5] in the Bibliography).
©
ISO 2004 – All rights reserved v

---------------------- Page: 5 ----------------------
.
vi

---------------------- Page: 6 ----------------------
INTERNATIONAL STANDARD ISO 18749:2004(E)
Water quality — Adsorption of substances on activated
sludge — Batch test using specific analytical methods
WARNING — Activated sludge and sewage contain potentially pathogenic organisms. Take appropriate
precautions when handling them. Handle with care toxic test compounds and those whose properties
are unknown.
1Scope
This International Standard specifies a screening test method for the determination of the degree of adsorption
of substances on to the activated sludge or primary sludge in a waste water treatment plant.
The conditions described in this International Standard normally correspond to the optimum conditions for the
adsorption to occur at the chosen activated-sludge concentration and water hardness during the test period.
The method applies to substances for which an analytical method with sufficient accuracy is available and
which, under the conditions of the test and at the test concentration used,
a) are water-soluble;
b) or, if only slightly water-soluble, allow sufficiently stable suspensions, dispersions or emulsions to be
prepared;
c) are not significantly removed from the test solution during the test by known abiotic processes such as
stripping or foaming;
d) do not deflocculate activated sludge;
e) are not readily biodegradable (for a discussion of biodegradability, see ISO/TR 15462).
An important parameter that can influence the reliability of the test results is the stability of the test compound
during the test. If no information on the stability is available, it is recommended that this be checked before the
test. If any transformation (e.g. due to hydrolysis) is observed, it is recommended that the degree of adsorption
of the transformation products be determined, if possible. Since biodegradability of the test compound may also
lead to an incorrect assessment of the degree of adsorption, it is recommended that the biodegradability be
investigated in advance using standard biodegradation tests which are preferably based on oxygen
consumption or on carbon dioxide production and in which adsorption has no influence on the test result. If
biodegradation cannot be excluded, sterilized sludge may be used (see Clause 7). There is generally no need
to carry out adsorption tests on substances which are readily biodegradable as they are sufficiently removed
biologically in waste water treatment plants. Substances which are easily adsorbed on activated sludge in waste
water treatment plants are preferably removed by adsorbing them in sludge digesters and degrading them
anaerobically. For such substances, high adsorption may be a reason for carrying out anaerobic biodegradation
tests. An overview of standardized biodegradation tests is given in ISO/TR 15462.
The test compound concentrations used in this method are usually very low and therefore no negative effects
are to be expected on the capacity of the activated sludge to adsorb even toxic test compounds. When there is
any doubt, it is recommended that microscopic investigations of the flocs and suitable toxicity tests such as that
specified in ISO 8192 be carried out.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
©
ISO 2004 – All rights reserved 1

---------------------- Page: 7 ----------------------
ISO 18749:2004(E)
2.1
activated sludge
biomass and inert matter produced in the aerobic treatment of waste water by the growth of bacteria and other
microorganisms in the presence of dissolved oxygen
2.2
degree of adsorption on activated sludge
percentage of a test compound eliminated by any process except biodegradation and stripping under the
conditions of a specific aqueous batch test by activated or primary sludge, determined by comparing the
concentration at the beginning of the test with that at the end
2.3
distribution coefficient
the ratio of the concentration of a test compound in the sludge to its concentration in the aqueous phase
2.4
concentration of suspended solids in an activated sludge
amount of solids obtained from a known volume of activated sludge by filtration or centrifugation under specified

conditions and drying at about 105 C to constant mass
3Principle
The method determines the degree of adsorption, and, optionally, the distribution coefficient and mass balance,
of water-soluble organic substances on activated or primary sludge using an aqueous batch-test system. The
test mixture usually comprises an inorganic-salt test medium, activated sludge and the test compound. The
hardness of the test medium, the concentration of suspended solids in the activated sludge and the amount of
test compound added are specified to simulate real conditions of waste water treatment plants. Thus, the initial
concentration of the test compound is usually as low as possible within the sensitivity range of the analytical
methods available.
The concentration of the test compound is measured using substance-specific analytical methods at the
beginning, during and at the end of the test (normally 24 h). The measured values are used as the basis for
calculating the degree of adsorption and, optionally, the distribution coefficient and the mass balance. The
measurement of elimination in the test vessels generally allows no direct differentiation between adsorption and
other elimination mechanisms such as complex formation, flocculation, precipitation, sedimentation or
biodegradation. More information can be obtained by using an abiotic elimination control without activated
sludge, sterilized sludge and/or by determining biodegradation of the test compound with appropriate tests.
4 Reagents and materials
Use only reagents of recognized analytical grade.
4.1 Water
Use distilled or de-ionized water containing less than 1mg of dissolved organic carbon (DOC) per litre.
4.2 Test medium
4.2.1 Preparation of solution A
Anhydrous potassium dihydrogen phosphate (KH PO ) 8,5 g
2 4
Anhydrous dipotassium hydrogen phosphate (K HPO ) 21,75 g
2 4
Disodium hydrogen phosphate dihydrate (Na HPO ·2H O) 33,4 g
2 4 2
Dissolve in water (4.1) and make up to 1 000 ml
©
2 ISO 2004 – All rights reserved

---------------------- Page: 8 ----------------------
ISO 18749:2004(E)
It is recommended that this buffer solution be checked by measuring its pH. If it is not about 7,4± 0,5, prepare
a new solution.
4.2.2 Preparation of solution B
Dissolve 12,3 g of magnesium sulfate heptahydrate (MgSO ·7H O) in water (4.1) and make up to 1 000 ml.
4 2
4.2.3 Preparation of solution C
Dissolve 29,4 g of calcium chloride dihydrate (CaCl ·2H O) in water (4.1) and make up to 1 000 ml.
2 2
4.2.4 Preparation of solution D
Dissolve 22,4 g of sodium hydrogen carbonate (NaHCO ) in water (4.1) and make up to 1 000 ml.
3
4.2.5 Preparation of test medium
The test medium is prepared at the beginning of each test by adding the correct amounts of stock solutions A to
D to the test compound as described in Clause 7. It is important to follow the instructions in Clause 7 to avoid
precipitation of the salts. The hardness of the test medium is adjusted to a value which will depend on the usual
water hardness in the region concerned or on the purpose of the test. Mixing 10 ml of each of solutions A to D
2+ 2+
and making up to 1 000 ml with water (4.1) will give a hardness of 2,5 mmol/l (80 mg/l of Ca , 12 mg/l of Mg )

and a hydrogen carbonate (HCO ) concentration of 162 mg/l, which is typical for many waste waters. If
3
required, another hardness may be used. In this case, change the amounts of solutions B and C added, bearing
2+
in mind that an extra 1 ml of solution B corresponds to an increase in Mg concentration of 0,05 mmol/l and an
2+
extra 1 ml of solution C to an increase in Ca concentration of 0,2 mmol/l. Indicate the hardness used and the
Ca/Mg ratio clearly in the test report.
If a test compound influences the pH of the mixture significantly at the chosen concentration (e.g. if the pH is
outside 6,0 to 9,0), an increase in the buffer capacity of the test medium may be required. In such cases, add
more of solution A, e.g. 100 ml instead of 10 ml.
Solutions A to D may be stored for up to 6 months in the dark at room temperature.
4.3 Preparation of stock solutions of test compound and reference material
Dissolve the test compound in water (4.1) or in test medium (4.2) at a suitable concentration. Suitable means a
concentration which simulates real environmental conditions (e.g. of waste water), but which is high enough to
allow a sufficiently accurate quantitative determination of the test compound remaining at the end of the test to
be made, using the intended analytical procedure, even after elimination of about 90 % by adsorption. In the
case of substances which are toxic to activated sludge and might for this reason influence the adsorptive
capacity of the sludge and hence the test result (see Clause 1), the concentration shall be low enough to avoid
this effect. The concentration may also be governed by the intended purpose of the test, for example the
simulation of an exposure scenario at a given environmental concentration. If there is no special request or
other information, a concentration of 1 mg/l to 5 mg/l is appropriate for substance-specific analysis and 40 mg of
DOC/litre in the case of DOC analysis (see Clause 7). Prepare the stock solution freshly before use or store it,

depending on the stability of the test compound, in the dark at about 4 C.
It is generally not necessary to test slightly water-soluble test compounds at levels above their solubility in water,
as they are mechanically removed in waste water treatment plants, e.g. by sedimentation. In such cases,
therefore, the concentration of the solution has to be below the solubility in water under the test conditions, but
high enough to allow a sufficiently accurate determination at the end of the test, even after elimination of about
90 % by adsorption. If substances pass into waste water treatment plants in the form of stable emulsions or
dispersions and hence enter the environment in this form, they can be tested in this form if available.
To decide whether a test compound is sufficiently water-soluble, it is recommended that a sample be taken from
the freshly prepared stock solution and the total organic carbon (TOC) determined directly and, after
©
ISO 2004 – All rights reserved 3

---------------------- Page: 9 ----------------------
ISO 18749:2004(E)
2
centrifugation at about 40 000 m/s for 15 min, the dissolved organic carbon (DOC) determined. The test is
applicable to a particular test compound if the DOC is > 90 % of the TOC.
To check the procedure and the adsorptive properties of the sludge, it is recommended that a reference
compound be used which is sufficiently water-soluble, non-volatile and poorly biodegradable, and has a degree
of adsorption > 90 % after 24 h. Use preferably a water-soluble dyestuff at a concentration which gives a
photometric extinction coefficient of 0,4 to 1,0. Basic Violet 4 (light absorption maximum 595 nm) has shown its
suitability in a round-robin test.
Dissolve the reference compound in water (4.1) or in test medium (4.2), at a suitable concentration, and
determine the degree of adsorption.
4.4 Preparation of activated sludge
Take a sample of activated sludge from the aeration tank of a properly operated biological waste water
treatment plant receiving predominantly domestic sewage. The sludge should have distinct flocs, which may be
checked under the microscope, and good settling behaviour, as judged from the sludge volume index (SVI)
which should be < 150 ml/g. To determine the sludge volume index, mix the sample well and place 1 000 ml in
a graduated glass cylinder. Let the sludge sediment for 30 min and read the volume of the settled fraction. Then
wash the activated sludge by repeatedly (e.g. 2 to 3 times) adding tap water or test medium (4.2), centrifuging
or allowing to settle, and decanting off the supernatant liquid. Determine the concentration of suspended solids,
e.g. by ISO 11923, and calculate the sludge volume index by dividing the volume of the settled sludge, in ml, by
the mass of the suspended solids, in g. If required, concentrate the sludge by centrifuging or allowing to settle,
discarding the supernatant liquid and adding less tap water or test medium than was discarded to obtain a
concentration of 5g of suspended solids per litre. Keep the activated sludge aerated at room temperature and
use it in the test at a concentration of 1g± 0,1 g of suspended solids per litre.
If required, other sources and concentrations of activated sludge may be used. If fresh activated sludge is not
available, dried or freeze-dried sludge may be used. Provided it is kept dry, such sludge can be stored with no

change in its adsorptive properties at about 4 C for up to one year. Depending on the purpose of the
investigation, raw waste water or primary sludge derived from untreated waste water may also be used. In this
case, however, the test conditions shall be adapted if necessary. Note that neither the test conditions nor the
test results obtained can be compared with those of investigations using activated sludge.
The sludge used, its origin and its concentration sh
...

SLOVENSKI STANDARD
SIST ISO 18749:2010
01-september-2010
.DNRYRVWYRGH$GVRUSFLMDVQRYLQDDNWLYQREODWRâDUåQLSUHVNXV]XSRUDER
VSHFLILþQLKDQDOLWVNLKPHWRG
Water quality - Adsorption of substances on activated sludge - Batch test using specific
analytical methods
Qualité de l'eau - Adsorption des substances sur la boue activée - Essai de lot utilisant
des méthodes analytiques spécifiques
Ta slovenski standard je istoveten z: ISO 18749:2004
ICS:
13.030.20 7HNRþLRGSDGNL%ODWR Liquid wastes. Sludge
13.060.30 Odpadna voda Sewage water
SIST ISO 18749:2010 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------

SIST ISO 18749:2010

---------------------- Page: 2 ----------------------

SIST ISO 18749:2010
INTERNATIONAL ISO
STANDARD 18749
First edition
2004-02-15
Water quality — Adsorption of
substances on activated sludge — Batch
test using specific analytical methods
Qualité de l'eau — Adsorption des substances sur la boue activée —
Essai de lot utilisant des méthodes analytiques spécifiques

Reference number
ISO 18749:2004(E)
©
ISO 2004

---------------------- Page: 3 ----------------------

SIST ISO 18749:2010
ISO 18749:2004(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall
not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the
unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
© ISO 2004
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56  CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
©
ii ISO 2004 – All rights reserved

---------------------- Page: 4 ----------------------

SIST ISO 18749:2010
ISO 18749:2004(E)
Contents Page
1 Scope . 1
2 Terms and definitions . 1
3 Principle . 2
4 Reagents and materials . 2
5 Apparatus . 4
6 Test environment . 5
7 Procedure . 5
8 Calculation . 7
9 Validity of the test . 9
10 Test report . 9
Bibliography . 10
©
ISO 2004 – All rights reserved iii

---------------------- Page: 5 ----------------------

SIST ISO 18749:2010
ISO 18749:2004(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 18749 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
©
iv ISO 2004 – All rights reserved

---------------------- Page: 6 ----------------------

SIST ISO 18749:2010
ISO 18749:2004(E)
Introduction
This test is used as a screening test to determine the degree of adsorption of substances on activated sludge
or primary sludge in waste water treatment plants. General information on the adsorption and desorption of test
compounds may also be obtained by other tests (see e.g. Reference [5] in the Bibliography).
©
ISO 2004 – All rights reserved v

---------------------- Page: 7 ----------------------

SIST ISO 18749:2010
.
vi

---------------------- Page: 8 ----------------------

SIST ISO 18749:2010
INTERNATIONAL STANDARD ISO 18749:2004(E)
Water quality — Adsorption of substances on activated
sludge — Batch test using specific analytical methods
WARNING — Activated sludge and sewage contain potentially pathogenic organisms. Take appropriate
precautions when handling them. Handle with care toxic test compounds and those whose properties
are unknown.
1Scope
This International Standard specifies a screening test method for the determination of the degree of adsorption
of substances on to the activated sludge or primary sludge in a waste water treatment plant.
The conditions described in this International Standard normally correspond to the optimum conditions for the
adsorption to occur at the chosen activated-sludge concentration and water hardness during the test period.
The method applies to substances for which an analytical method with sufficient accuracy is available and
which, under the conditions of the test and at the test concentration used,
a) are water-soluble;
b) or, if only slightly water-soluble, allow sufficiently stable suspensions, dispersions or emulsions to be
prepared;
c) are not significantly removed from the test solution during the test by known abiotic processes such as
stripping or foaming;
d) do not deflocculate activated sludge;
e) are not readily biodegradable (for a discussion of biodegradability, see ISO/TR 15462).
An important parameter that can influence the reliability of the test results is the stability of the test compound
during the test. If no information on the stability is available, it is recommended that this be checked before the
test. If any transformation (e.g. due to hydrolysis) is observed, it is recommended that the degree of adsorption
of the transformation products be determined, if possible. Since biodegradability of the test compound may also
lead to an incorrect assessment of the degree of adsorption, it is recommended that the biodegradability be
investigated in advance using standard biodegradation tests which are preferably based on oxygen
consumption or on carbon dioxide production and in which adsorption has no influence on the test result. If
biodegradation cannot be excluded, sterilized sludge may be used (see Clause 7). There is generally no need
to carry out adsorption tests on substances which are readily biodegradable as they are sufficiently removed
biologically in waste water treatment plants. Substances which are easily adsorbed on activated sludge in waste
water treatment plants are preferably removed by adsorbing them in sludge digesters and degrading them
anaerobically. For such substances, high adsorption may be a reason for carrying out anaerobic biodegradation
tests. An overview of standardized biodegradation tests is given in ISO/TR 15462.
The test compound concentrations used in this method are usually very low and therefore no negative effects
are to be expected on the capacity of the activated sludge to adsorb even toxic test compounds. When there is
any doubt, it is recommended that microscopic investigations of the flocs and suitable toxicity tests such as that
specified in ISO 8192 be carried out.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
©
ISO 2004 – All rights reserved 1

---------------------- Page: 9 ----------------------

SIST ISO 18749:2010
ISO 18749:2004(E)
2.1
activated sludge
biomass and inert matter produced in the aerobic treatment of waste water by the growth of bacteria and other
microorganisms in the presence of dissolved oxygen
2.2
degree of adsorption on activated sludge
percentage of a test compound eliminated by any process except biodegradation and stripping under the
conditions of a specific aqueous batch test by activated or primary sludge, determined by comparing the
concentration at the beginning of the test with that at the end
2.3
distribution coefficient
the ratio of the concentration of a test compound in the sludge to its concentration in the aqueous phase
2.4
concentration of suspended solids in an activated sludge
amount of solids obtained from a known volume of activated sludge by filtration or centrifugation under specified

conditions and drying at about 105 C to constant mass
3Principle
The method determines the degree of adsorption, and, optionally, the distribution coefficient and mass balance,
of water-soluble organic substances on activated or primary sludge using an aqueous batch-test system. The
test mixture usually comprises an inorganic-salt test medium, activated sludge and the test compound. The
hardness of the test medium, the concentration of suspended solids in the activated sludge and the amount of
test compound added are specified to simulate real conditions of waste water treatment plants. Thus, the initial
concentration of the test compound is usually as low as possible within the sensitivity range of the analytical
methods available.
The concentration of the test compound is measured using substance-specific analytical methods at the
beginning, during and at the end of the test (normally 24 h). The measured values are used as the basis for
calculating the degree of adsorption and, optionally, the distribution coefficient and the mass balance. The
measurement of elimination in the test vessels generally allows no direct differentiation between adsorption and
other elimination mechanisms such as complex formation, flocculation, precipitation, sedimentation or
biodegradation. More information can be obtained by using an abiotic elimination control without activated
sludge, sterilized sludge and/or by determining biodegradation of the test compound with appropriate tests.
4 Reagents and materials
Use only reagents of recognized analytical grade.
4.1 Water
Use distilled or de-ionized water containing less than 1mg of dissolved organic carbon (DOC) per litre.
4.2 Test medium
4.2.1 Preparation of solution A
Anhydrous potassium dihydrogen phosphate (KH PO ) 8,5 g
2 4
Anhydrous dipotassium hydrogen phosphate (K HPO ) 21,75 g
2 4
Disodium hydrogen phosphate dihydrate (Na HPO ·2H O) 33,4 g
2 4 2
Dissolve in water (4.1) and make up to 1 000 ml
©
2 ISO 2004 – All rights reserved

---------------------- Page: 10 ----------------------

SIST ISO 18749:2010
ISO 18749:2004(E)
It is recommended that this buffer solution be checked by measuring its pH. If it is not about 7,4± 0,5, prepare
a new solution.
4.2.2 Preparation of solution B
Dissolve 12,3 g of magnesium sulfate heptahydrate (MgSO ·7H O) in water (4.1) and make up to 1 000 ml.
4 2
4.2.3 Preparation of solution C
Dissolve 29,4 g of calcium chloride dihydrate (CaCl ·2H O) in water (4.1) and make up to 1 000 ml.
2 2
4.2.4 Preparation of solution D
Dissolve 22,4 g of sodium hydrogen carbonate (NaHCO ) in water (4.1) and make up to 1 000 ml.
3
4.2.5 Preparation of test medium
The test medium is prepared at the beginning of each test by adding the correct amounts of stock solutions A to
D to the test compound as described in Clause 7. It is important to follow the instructions in Clause 7 to avoid
precipitation of the salts. The hardness of the test medium is adjusted to a value which will depend on the usual
water hardness in the region concerned or on the purpose of the test. Mixing 10 ml of each of solutions A to D
2+ 2+
and making up to 1 000 ml with water (4.1) will give a hardness of 2,5 mmol/l (80 mg/l of Ca , 12 mg/l of Mg )

and a hydrogen carbonate (HCO ) concentration of 162 mg/l, which is typical for many waste waters. If
3
required, another hardness may be used. In this case, change the amounts of solutions B and C added, bearing
2+
in mind that an extra 1 ml of solution B corresponds to an increase in Mg concentration of 0,05 mmol/l and an
2+
extra 1 ml of solution C to an increase in Ca concentration of 0,2 mmol/l. Indicate the hardness used and the
Ca/Mg ratio clearly in the test report.
If a test compound influences the pH of the mixture significantly at the chosen concentration (e.g. if the pH is
outside 6,0 to 9,0), an increase in the buffer capacity of the test medium may be required. In such cases, add
more of solution A, e.g. 100 ml instead of 10 ml.
Solutions A to D may be stored for up to 6 months in the dark at room temperature.
4.3 Preparation of stock solutions of test compound and reference material
Dissolve the test compound in water (4.1) or in test medium (4.2) at a suitable concentration. Suitable means a
concentration which simulates real environmental conditions (e.g. of waste water), but which is high enough to
allow a sufficiently accurate quantitative determination of the test compound remaining at the end of the test to
be made, using the intended analytical procedure, even after elimination of about 90 % by adsorption. In the
case of substances which are toxic to activated sludge and might for this reason influence the adsorptive
capacity of the sludge and hence the test result (see Clause 1), the concentration shall be low enough to avoid
this effect. The concentration may also be governed by the intended purpose of the test, for example the
simulation of an exposure scenario at a given environmental concentration. If there is no special request or
other information, a concentration of 1 mg/l to 5 mg/l is appropriate for substance-specific analysis and 40 mg of
DOC/litre in the case of DOC analysis (see Clause 7). Prepare the stock solution freshly before use or store it,

depending on the stability of the test compound, in the dark at about 4 C.
It is generally not necessary to test slightly water-soluble test compounds at levels above their solubility in water,
as they are mechanically removed in waste water treatment plants, e.g. by sedimentation. In such cases,
therefore, the concentration of the solution has to be below the solubility in water under the test conditions, but
high enough to allow a sufficiently accurate determination at the end of the test, even after elimination of about
90 % by adsorption. If substances pass into waste water treatment plants in the form of stable emulsions or
dispersions and hence enter the environment in this form, they can be tested in this form if available.
To decide whether a test compound is sufficiently water-soluble, it is recommended that a sample be taken from
the freshly prepared stock solution and the total organic carbon (TOC) determined directly and, after
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ISO 2004 – All rights reserved 3

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SIST ISO 18749:2010
ISO 18749:2004(E)
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centrifugation at about 40 000 m/s for 15 min, the dissolved organic carbon (DOC) determined. The test is
applicable to a particular test compound if the DOC is > 90 % of the TOC.
To check the procedure and the adsorptive properties of the sludge, it is recommended that a reference
compound be used which is sufficiently water-soluble, non-volatile and poorly biodegradable, and has a degree
of adsorption > 90 % after 24 h. Use preferably a water-soluble dyestuff at a concentration which gives a
photometric extinction coefficient of 0,4 to 1,0. Basic Violet 4 (light absorption maximum 595 nm) has shown its
suitability in a round-robin test.
Dissolve the reference compound in water (4.1) or in test medium (4.2), at a suitable concentration, and
determine the degree of adsorption.
4.4 Preparation of activated sludge
Take a sample of activated sludge from the aeration tank of a properly operated biological waste water
treatment plant receiving predominantly domestic sewage. The sludge should have distinct flocs, which may be
checked under the microscope, and good settling behaviour, as judged from the sludge volume index (SVI)
which should be < 150 ml/g. To determine the sludge volume index, mix the sample well and place 1 000 ml in
a graduated glass cylinder. Let the sludge sediment for 30 min and read the volume of the settled fraction. Then
wash the activated sludge by repeatedly (e.g. 2 to 3 times) adding tap water or test medium (4.2), centrifuging
or allowing to settle, and decanting off the supernatant liquid. Determine the concentration of suspended solids,
e.g. by ISO 11923, and calculate the sludge volume index by dividing the volume of the settled sludge, in ml, by
the mass of the suspended solids, in g. If required, concentrate the sludge by centrifuging or allowing to settle,
discarding the supernatant liquid and adding less tap water or test medium than was discarded to obtain a
concentration of 5g of suspended solids per litre. Keep the activated sludge aerated at room temperature and
use it in the test at a concentration of 1g± 0,1 g of suspended solids per litre.
If required, other sources and concentrations of activated sludge may be used. If fresh activated sludge is not
available, dried or f
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