ISO 16240:2005
(Main)Water quality — Determination of the genotoxicity of water and waste water — Salmonella/microsome test (Ames test)
Water quality — Determination of the genotoxicity of water and waste water — Salmonella/microsome test (Ames test)
ISO 16240:2005 specifies a method for the determination of the genotoxic potential of water and wastewater using the bacterial strains Salmonella typhimurium TA 100 and TA 98. This method includes sterile filtration of water and wastewater prior to the test. ISO 16240:2005 is applicable only to the detection of genotoxic substances which are in the filtered aqueous phase. It is not applicable to the detection of genotoxic substances adsorbed by the retained particles.
Qualité de l'eau — Détermination de la génotoxicité des eaux et des eaux résiduaires — Essai de Salmonella/microsome (essai d'Ames)
Kakovost vode - Določanje genotoksičnosti vode in odpadne vode - Preskus s Salmonella (preskus po Amesu)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2007
.DNRYRVWYRGH'RORþDQMHJHQRWRNVLþQRVWLYRGHLQRGSDGQHYRGH3UHVNXVV
6DOPRQHOOD SUHVNXVSR$PHVX
Water quality -- Determination of the genotoxicity of water and waste water --
Salmonella/microsome test (Ames test)
Qualité de l'eau -- Détermination de la génotoxicité des eaux et des eaux résiduaires --
Essai de Salmonella/microsome (essai d'Ames)
Ta slovenski standard je istoveten z: ISO 16240:2005
ICS:
13.060.30 Odpadna voda Sewage water
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.
INTERNATIONAL ISO
STANDARD 16240
First edition
2005-04-01
Water quality — Determination of the
genotoxicity of water and waste water —
Salmonella/microsome test (Ames test)
Qualité de l'eau — Détermination de la génotoxicité des eaux et des
eaux résiduaires — Essai de Salmonella/microsome (essai d'Ames)
Reference number
©
ISO 2005
PDF disclaimer
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accepts no liability in this area.
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the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
© ISO 2005
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 2005 – All rights reserved
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Interferences. 3
5 Principle . 4
6 Reagents and media . 4
7 Apparatus. 9
8 Procedure. 10
8.1 Pouring and labelling of plates. 10
8.2 Test samples and their preparation . 10
8.3 Number of test samples . 11
8.4 Preparation . 12
8.4.1 On the day prior to the test:. 12
8.4.2 On the day of testing only:. 12
8.5 Study conduct . 12
8.5.1 General. 12
8.5.2 Mutagenicity study. 12
8.6 Titre determination. 13
9 Colony-counting, evaluation and assessment. 13
9.1 Colony-counting. 13
9.2 Evaluation . 13
9.3 Assessment criteria . 13
9.4 Determination of the decisive D value. 13
min
10 Validity criteria . 14
11 Test report. 14
Annex A (normative) Nutrient broth and agar . 15
Annex B (normative) S9 fraction. 16
Annex C (normative) Verification of genotype . 17
Annex D (informative) Examples of results obtained . 18
Bibliography . 20
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 16240 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2005 – All rights reserved
Introduction
It should be decided on a case-by-case basis whether, and to what extent, additional instructions may be
necessary for the application of this International Standard.
INTERNATIONAL STANDARD ISO 16240:2005(E)
Water quality — Determination of the genotoxicity of water and
waste water — Salmonella/microsome test (Ames test)
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 the determination of the genotoxic potential of water and
wastewater using the bacterial strains Salmonella typhimurium TA 100 and TA 98. This method includes
sterile filtration of water and wastewater prior to the test.
This International Standard is applicable only to the detection of genotoxic substances which are in the filtered
aqueous phase. It is not applicable to the detection of genotoxic substances adsorbed by the retained
particles.
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 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes
ISO 5667-2, Water quality — Sampling — Part 2: Guidance on sampling techniques
ISO 5667-3, Water quality — Sampling — Part 3: Guidance on the preservation and handling of water
samples
ISO 5667-14, Water quality — Sampling — Part 14: Guidance on quality assurance of environmental water
sampling and handling
ISO 5667-16, Water quality — Sampling — Part 16: Guidance on biotesting of samples
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
number of revertants
number of mutants
number of visible mutant colonies per plate at the termination of the test
3.2
dilution level
D
denominator of the dilution coefficient (using the numerator 1) of a mixture of water or wastewater with
dilution water (3.16) as integral number
NOTE For undiluted water or wastewater, the dilution coefficient is by definition 1:1. The corresponding and smallest
possible D value is 1.
3.3
dose-response relationship
reduction of the number of visible mutant colonies per plate with increasing D level
3.4
D value
min
smallest value of D at which, under the conditions of this International Standard, no positive increase in the
number of visible mutant colonies per plate is detected
NOTE In the case of more than one D value (a maximum of four are possible), the highest D value is decisive.
min
3.5
stock culture
frozen culture for the preservation of the characteristics (e.g. genotype) of Salmonella typhimurium TA 100
and TA 98
3.6
inoculum
part of a thawed stock culture used to inoculate culture medium
3.7
culture medium
aqueous solution of nutrients which are required for the cultivation of the bacteria
3.8
overnight culture
mixture of inoculum and culture medium, incubated for about 18 h at 37 °C ± 1 °C and gentle agitation
(e.g. shaken at 100 r/min to 150 r/min)
3.9
plate
solidified mixture of water, agar and other possible constituents (e.g. inorganic salts) in Petri dishes
3.10
softagar
mixture of agar, sodium chloride, histidine, biotin and water
NOTE Minimal softagar contains only traces of histidine and is used for the determination of mutants. Maximal
softagar contains histidine in excess and is used for the determination of titres.
3.11
S9 fraction
9 000 g supernatant of a tissue homogenate in 0,15 mol/l KCl, obtained from livers of male rats (200 g to
300 g) pretreated with an appropriate substance or substance combination for enzyme induction
3.12
cofactor solution
aqueous solution of chemicals needed for the activity of the enzymes in the S9 fraction
NOTE Examples of chemicals needed are NADP, glucose-6-phosphate and inorganic salts.
2 © ISO 2005 – All rights reserved
3.13
S9 mix
mixture of S9 fraction and cofactor solution
3.14
titre determination
method for the determination of the number of bacteria (colony-forming units) in an overnight culture and for
the determination of possible bacteriotoxic effects of the test sample
3.15
test sample
sample to be used as test item after all preparative steps (e.g. sterile filtration) have been carried out
3.16
dilution water
sterile water of a conductivity of u 5 µS/cm used for the stepwise dilution of the test sample or used as
negative control
3.17
negative control
dilution water (3.16) without test sample
...
INTERNATIONAL ISO
STANDARD 16240
First edition
2005-04-01
Water quality — Determination of the
genotoxicity of water and waste water —
Salmonella/microsome test (Ames test)
Qualité de l'eau — Détermination de la génotoxicité des eaux et des
eaux résiduaires — Essai de Salmonella/microsome (essai d'Ames)
Reference number
©
ISO 2005
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 2005
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 2005 – All rights reserved
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Interferences. 3
5 Principle . 4
6 Reagents and media . 4
7 Apparatus. 9
8 Procedure. 10
8.1 Pouring and labelling of plates. 10
8.2 Test samples and their preparation . 10
8.3 Number of test samples . 11
8.4 Preparation . 12
8.4.1 On the day prior to the test:. 12
8.4.2 On the day of testing only:. 12
8.5 Study conduct . 12
8.5.1 General. 12
8.5.2 Mutagenicity study. 12
8.6 Titre determination. 13
9 Colony-counting, evaluation and assessment. 13
9.1 Colony-counting. 13
9.2 Evaluation . 13
9.3 Assessment criteria . 13
9.4 Determination of the decisive D value. 13
min
10 Validity criteria . 14
11 Test report. 14
Annex A (normative) Nutrient broth and agar . 15
Annex B (normative) S9 fraction. 16
Annex C (normative) Verification of genotype . 17
Annex D (informative) Examples of results obtained . 18
Bibliography . 20
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 16240 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2005 – All rights reserved
Introduction
It should be decided on a case-by-case basis whether, and to what extent, additional instructions may be
necessary for the application of this International Standard.
INTERNATIONAL STANDARD ISO 16240:2005(E)
Water quality — Determination of the genotoxicity of water and
waste water — Salmonella/microsome test (Ames test)
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 the determination of the genotoxic potential of water and
wastewater using the bacterial strains Salmonella typhimurium TA 100 and TA 98. This method includes
sterile filtration of water and wastewater prior to the test.
This International Standard is applicable only to the detection of genotoxic substances which are in the filtered
aqueous phase. It is not applicable to the detection of genotoxic substances adsorbed by the retained
particles.
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 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes
ISO 5667-2, Water quality — Sampling — Part 2: Guidance on sampling techniques
ISO 5667-3, Water quality — Sampling — Part 3: Guidance on the preservation and handling of water
samples
ISO 5667-14, Water quality — Sampling — Part 14: Guidance on quality assurance of environmental water
sampling and handling
ISO 5667-16, Water quality — Sampling — Part 16: Guidance on biotesting of samples
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
number of revertants
number of mutants
number of visible mutant colonies per plate at the termination of the test
3.2
dilution level
D
denominator of the dilution coefficient (using the numerator 1) of a mixture of water or wastewater with
dilution water (3.16) as integral number
NOTE For undiluted water or wastewater, the dilution coefficient is by definition 1:1. The corresponding and smallest
possible D value is 1.
3.3
dose-response relationship
reduction of the number of visible mutant colonies per plate with increasing D level
3.4
D value
min
smallest value of D at which, under the conditions of this International Standard, no positive increase in the
number of visible mutant colonies per plate is detected
NOTE In the case of more than one D value (a maximum of four are possible), the highest D value is decisive.
min
3.5
stock culture
frozen culture for the preservation of the characteristics (e.g. genotype) of Salmonella typhimurium TA 100
and TA 98
3.6
inoculum
part of a thawed stock culture used to inoculate culture medium
3.7
culture medium
aqueous solution of nutrients which are required for the cultivation of the bacteria
3.8
overnight culture
mixture of inoculum and culture medium, incubated for about 18 h at 37 °C ± 1 °C and gentle agitation
(e.g. shaken at 100 r/min to 150 r/min)
3.9
plate
solidified mixture of water, agar and other possible constituents (e.g. inorganic salts) in Petri dishes
3.10
softagar
mixture of agar, sodium chloride, histidine, biotin and water
NOTE Minimal softagar contains only traces of histidine and is used for the determination of mutants. Maximal
softagar contains histidine in excess and is used for the determination of titres.
3.11
S9 fraction
9 000 g supernatant of a tissue homogenate in 0,15 mol/l KCl, obtained from livers of male rats (200 g to
300 g) pretreated with an appropriate substance or substance combination for enzyme induction
3.12
cofactor solution
aqueous solution of chemicals needed for the activity of the enzymes in the S9 fraction
NOTE Examples of chemicals needed are NADP, glucose-6-phosphate and inorganic salts.
2 © ISO 2005 – All rights reserved
3.13
S9 mix
mixture of S9 fraction and cofactor solution
3.14
titre determination
method for the determination of the number of bacteria (colony-forming units) in an overnight culture and for
the determination of possible bacteriotoxic effects of the test sample
3.15
test sample
sample to be used as test item after all preparative steps (e.g. sterile filtration) have been carried out
3.16
dilution water
sterile water of a conductivity of u 5 µS/cm used for the stepwise dilution of the test sample or used as
negative control
3.17
negative control
dilution water (3.16) without test sample
3.18
positive control
known mutagen used to verify the sensitivity of the method or the activity of the S9 mix
NOTE The positive controls are dissolved in DMSO prior to use.
3.19
test mixture
mixture of test sample [pure or diluted with dilution water (3.16)], negative or positive control, bacterial
suspension, softagar and S9 mix or buffer
3.20
induction rate
I
difference between the mean value of mutant colonies counted on the plates treated with a dose of the test
sample or with a positive control and the mean value of the corresponding plates treated with the negative
control using the same strain under identical activation conditions
3.21
background growth
bacterial lawn formed by microcolonies of non-mutated bacteria on a plate with minimal softagar due to the
traces of histidine contained in this softagar
4 Interferences
A strong bacteriotoxic effect of the test sample can lead to a reduction of viable bacteria and to a reduction of
mutant colonies compared to the corresponding negative control counts.
In an extreme case of bacteriotoxicity, the number of surviving bacteria may be r
...
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