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)
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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 16240:2005(E)
©
ISO 2005
---------------------- Page: 1 ----------------------
ISO 16240:2005(E)
PDF disclaimer
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shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
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accepts no liability in this area.
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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
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ISO 16240:2005(E)
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
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ISO 16240:2005(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 16240 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
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ISO 16240:2005(E)
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.
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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
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ISO 16240:2005(E)
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.
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ISO 16240:2005(E)
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 reduced to such an extent (to
several hundred) that the traces of histidine in the minimal softagar are sufficient to allow these bacteria to
grow up to visible colonies mimicking the growth of mutant colonies. This may lead to false positive results.
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ISO 16240:2005(E)
5 Principle
The bacteria are exposed under defined conditions to various doses of the test sample and incubated for 48 h
to 72 h at 37 °C ± 1 °C. Due to this exposure, genotoxic agents contained in the test water or wastewater may
be able to induce mutations in one or both marker genes (hisG46 for TA 100 and hisD3052 for TA 98) in
correlation to the used doses. Such induction of mutations causes a dose-related increase in the numbers of
mutant colonies.
The possible mutagenic activity of the test sample is detected by comparing, for the used bacterial strain and
the respective activation condition [± S9 mix (3.13); Annex B)], the number of mutant colonies on plates
treated with the negative control with those treated with undiluted and diluted test sample.
The lowest dilution (1:N) of the test sample inducing, according to the criteria of this International Standard, no
mutagenic effect under all experimental conditions (if any mutagenic effect is induced by the test sample) is
the parameter relevant for the assessment of the test sample according to this International Standard.
Dilutions above this (1:A, A
Standard in at least one strain under at least one activation condition. The respective D -value is N. If no
min
mutagenic effect is observed under all experimental conditions, this dilution is 1:1 and the respective D -
min
value is 1.
The test facility is qualified for the conduct of this International Standard if the Salmonella/microsome test is
established in this facility according to the following criteria:
several independent experiments are performed;
several known mutagenic and non-mutagenic reagents are tested;
the mutagenic compounds are included in the positive controls of this International Standard (6.18);
the results are reproducible;
the results are in compliance with literature data.
6 Reagents and media
As far as possible, use chemicals of reagent grade. Prepare all aqueous solutions with water of a conductivity
of u 5 µS/cm.
If chemicals with different amounts of crystallisation water are used, recalculate the needed amounts.
Always autoclave for 20 min at 121 °C ± 2 °C. Seal vessels loosely (e.g. with aluminium foil). Sealing should
never be air-tight.
All compositions are given for specific final amounts. Other final amounts (N-fold) may be reached by
multiplying the amounts all single components of the respective composition by N.
Compositions may be subdivided under appropriate conditions into appropriate amounts.
6.1 Hydrochloric acid, c(HCl) = 1 mol/l.
6.2 Sodium hydroxide solution, c(NaOH) = 1 mol/l.
6.3 Dimethyl sulfoxide (DMSO), C H O S.
2 6 4
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ISO 16240:2005(E)
6.4 Nutrient broth
For each 1 l of water, add 3 g of beef extract, 5 g of peptone and 5 g of sodium chloride (or alternatively, 10 g
of beef extract, 10 g of peptone and 5 g of sodium chloride). Warm up and stir to dissolve the compounds.
Adjust the pH to 7,4 ± 0,2 and autoclave in appropriate portions. Store under sterile conditions at 4 °C ± 2 °C
for not longer than one month.
For use of commercial products, see A.1.
6.5 Ampicillin solution
Under sterile conditions at room temperature, dissolve 80 mg of ampicillin in 10 ml of sterile sodium hydroxide
solution (0,02 mol/l). Use immediately.
6.6 Nutrient broth with ampicillin
Under sterile conditions, add 3,15 ml of ampicillin solution (6.5) to 1 l of nutrient broth (6.4). Store under sterile
conditions at 4 °C ± 2 °C for not longer than one week.
6.7 Sodium hydrogen phosphate buffer
The following solutions are needed to prepare the buffer:
solution 1: 13,8 g of sodium dihydrogen phosphate monohydrate (NaH PO · H O) dissolved in 1 l
2 4 2
water;
solution 2: 14,2 g of disodium hydrogen phosphate (Na HPO ) dissolved in 1 l water.
2 4
Stir solution 2 (e.g. with a magnetic stirrer) and add solution 1 until a pH of 7,4 is reached and remains stable.
Subdivide this solution in appropriate amounts and autoclave to sterilize. Store at 4 °C ± 2 °C for not longer
than one month.
6.8 Cofactor solution
Dissolve the following compounds, in the amounts given, in 70 ml of sodium hydrogen phosphate buffer (6.7):
162,6 mg of magnesium chloride hexahydrate (MgCl · 6 H O);
2 2
246,0 mg of potassium chloride (KCl);
179,1 mg of glucose-6-phosphate, disodium salt (G6P);
1)
315,0 mg of NADP , disodium salt.
Filter sterile through appropriate membrane filters. A volume of 70 ml of cofactor solution is needed for the
preparation of 100 ml of S9 mix, sufficient for approximately 200 plates.
6.9 S9 fraction
The preparation of S9 fraction and the treatment for enzyme induction are described in Annex B. If S9 fraction
is purchased commercially, it should also be prepared (including enzyme induction) according to Annex B.
6.10 S9 mix
Prepare the needed amount of S9 fraction (6.9) freshly on the day of test or, if stored frozen, thaw it at room
temperature. Immediately thereafter, prepare S9 mix by mixing the following under sterile conditions:
10 ml of S9 fraction;
1) Nicotinamide adenine dinucleotide phosphate.
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ISO 16240:2005(E)
70 ml of cofactor solution (6.8);
20 ml of potassium chloride solution (6.19).
Cool S9 mix permanently (e.g. in a double-walled separator funnel containing ice water between the walls)
and use it only on the same day. Discard the remaining S9 mix at the end of this day.
6.11 Vogel-Bonner E-medium (50×)
Dissolve by stirring the following amounts of the substances, in the given order, into 670 ml water. Allow each
ingredient to dissolve completely before adding the next.
10 g MgSO · 7 H O
4 2
100 g citric acid · H O
2
500 g K HPO , anhydrous
2 4
175 g NaNH HPO · 4 H O
4 4 2
This results in a final volume of approximately 1 l. Divide in appropriate portions and store at room
temperature.
6.12 Vogel-Bonner plates (see also 8.1)
For the preparation of 1 l of agar:
add 15 g of agar (see A.2) to 880 ml water and dissolve by heating under stirring; seal the container
(e.g. with aluminium foil);
dissolve 20 g of glucose monohydrate in water to a final volume of 100 ml; seal the container (e.g. with
aluminium foil);
transfer 20 ml of Vogel-Bonner E-medium (6.11) into a container and seal container;
autoclave all components;
under sterile conditions, add glucose solution and Vogel-Bonner E-medium to the agar solution.
CAUTION — Delayed boiling leading to splashing can occur.
Mix and pour into Petri dishes (25 ml to 30 ml per dish).
6.13 Nutrient agar plates (see also 8.1)
Mix 23 g of nutrient agar (see A.3) with 1 l of water and dissolve by heating under stirring. Seal container
(e.g. with aluminium foil) and autoclave. Pour into Petri dishes (25 ml per dish).
Alternatively, dissolve 15 g of agar (see A.2) in 1 l of nutrient broth (see A.1) by warming in an appropriate
vessel. Seal vessel loosely (e.g. with aluminium foil) and autoclave the solution. Thereafter pour into Petri
dishes (see 8.1).
6.14 Ampicillin agar plates (see also 8.1)
Dissolve agar (see A.2) in 860 ml water. Prepare 100 ml of glucose-solution and 20 ml of
Vogel-Bonner E-medium as described in 6.11. In addition, the following ingredients are needed:
3,15 ml ampicillin solution (6.5);
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ISO 16240:2005(E)
10,0 ml histidine solution (add 500 mg of L-histidine hydrochloride monohydrate to 100 ml water);
6,0 ml biotin solution [add 12,2 mg of (D+)-biotin to 100 ml of water].
Autoclave all components. Under sterile conditions, add all solutions and Vogel-Bonner E-medium to the
autoclaved agar solution.
CAUTION — Delayed boiling leading to splashing can occur.
Mix and pour into Petri dishes (25 ml per dish).
If nutrient agar (6.13) is used as an alternative, add 3,15 ml of ampicillin solution (6.5) per litre of agar solution.
6.15 Amino acid solutions for softagar
The only difference between amino acid solutions for mutant and titre determinations is the amount of histidine
needed.
6.15.1 Minimal medium
Add 105 mg of L-histidine hydrochloride monohydrate and 122 mg of (D+)-biotin to 1 l water, mix, seal and
autoclave. Divide under sterile conditions into appropriate amounts (e.g. 10 ml) in marked vessels, and store
at 4 °C ± 2 °C.
6.15.2 Complete medium
Add 1 050 mg of L-histidine hydrochloride monohydrate and 122 mg of (D+)-biotin to 1 l of water, mix, seal
and autoclave. Divide under sterile conditions into appropriate amounts (e.g. 10 ml) in marked vessels, and
store at 4 °C ± 2 °C.
6.16 Softagar
6.16.1 Softagar for mutant determination
Mix 0,8 g of agar (see A.2) and 0,6 g of sodium chloride with 45 ml water.
Place 10 ml of amino acid solution (minimal medium, 6.15.1) in a separate vessel.
To adjust the volume in experiments without S9 mix, 0,5 ml of sodium hydrogen phosphate buffer (6.7) per
1 ml of softagar may also be needed.
Autoclave all components.
Add minimal medium and, if appropriate, buffer to agar and mix thoroughly. Avoid temperatures below 45 °C.
Place tubes in a water bath (45 °C ± 1 °C) and add 1 ml (for experiments with S9 mix) to each tube or 1,5 ml
(for experiments without S9 mix) of the respective mixture under sterile conditions. Cover tubes again with
aluminium foil. Mark stands in an appropriate form to avoid mistakes. Experience has shown that soft agar
may be stored in this form for up to 24 h.
6.16.2 Softagar for titre determination
Mix 0,8 g of agar (see A.2) and 0,6 g of sodium chloride with 45 ml water.
Place 10 ml of amino acid solution (complete medium, 6.15.2) in a separate vessel.
Autoclave all components.
Add complete medium to agar and mix thoroughly. Avoid temperatures below 45 °C. Place tubes in a water
bath (45 °C ± 1 °C) and add 1 ml of the mixture to each tube under sterile conditions. Cover tubes again with
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ISO 16240:2005(E)
aluminium foil. Mark stands in an appropriate form to avoid mistakes. Experience has shown that soft agar
may be stored in this form for up to 24 h.
6.17 Physiological NaCl solution
Dissolve 9 g of sodium chloride in 1 l water. Separate in appropriate amounts, autoclave and store under
sterile conditions.
6.18 Positive controls
Stock solutions of positive control substances can be stored at – 20 °C as long as validity criteria (Clause 10)
are met.
6.18.1 Nitrofurantoin
Dissolve 20 mg of nitrofurantoin (NF) under sterile conditions in 10 ml of dimethyl sulfoxide, DMSO (6.3)
resulting in a stock solution of 2 mg/ml. Dilute stock solution three times 1:10 with DMSO. Use 0,1 ml of the
−3
final dilution (10 ) (containing 2 µg/ml NF) in the test.
6.18.2 4-Nitro-1,2-phenylene diamine
Dissolve 25 mg of 4-nitro-1,2-phenylene diamine (4-NPDA) under sterile conditions in 5 ml of DMSO (6.3)
resulting in a stock solution of 5 mg/ml. Dilute stock solution three times 1:10 with DMSO. Use 0,1 ml of the
−3
final dilution (10 ) (containing 5 µg/ml 4-NPDA) in the test.
6.18.3 2-Aminoanthracene
Dissolve 30 mg of 2-aminoanthracene (2-AA) under sterile conditions in 10 ml of DMSO (6.3) resulting in a
−2
stock solution of 3 mg/ml. Dilute stock solution twice 1:10 with DMSO. Use 0,1 ml of the final dilution (10 )
(containing 30 µg/ml 2-AA) in the test.
6.19 KCl solution
Dissolve 1,12 g of potassium chloride in 100 ml water and autoclave.
6.20 Tester strains
Use mutants of Salmonella typhimurium LT2, which detect point mutations, to determine the mutagenic
potential of a test sample. Since point mutations can be subdivided into two classes (base-pair substitutions
and frame-shift mutations), two strains are used. These strains are TA 100 and TA 98. TA 100 bears the
base-pair substitution hisG46 as marker, whereas TA 98 contains the frame-shift mutation (+2 type) hisD3052.
In addition, both strains have the following genetic properties.
a) They contain the plasmid pKM101, coding for an ampicillin resistance.
b) They are all deep rough, e.g. partly deficient in lipopolysaccharide side chains, enabling larger molecules
to penetrate the bacterial cell wall and also produce mutations.
c) Due to the mutation uvrB, their capability to repair DNA-damage is limited, as it is induced e.g. by UV-light,
which increases the likelihood that such damage results in mutations.
Immediately upon receipt, spread aliquots of the respective bacterial strain onto the surface of plates with
ampicillin agar (6.14) thus allowing to grow in single colonies. Incubate for 24 h at 37 °C ± 1 °C.
Take samples from individual colonies with a sterile inoculation loop, and transfer them to 20 ml of ampicillin-
containing nutrient broth (6.6).
Incubate again overnight at 37 °C ± 1 °C.
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ISO 16240:2005(E)
Inoculate samples of these cultures onto nutrient agar plates, which have been provided with ampicillin (6.14),
again so as to grow single colonies. Incubate for 24 h at 37 °C ± 1 °C.
Transfer new samples of individual colonies from these plates to flasks containing approximately 20 ml normal
nutrient broth (6.4). Incubate this inoculum overnight at 37 °C ± 1 °C.
Take a small sample to check the genotype (see Annex C).
These investigations are performed to check the presence of the genetic markers “deep rough”, uvrB and
pKM101. In addition, determine the number of spontaneous mutations and the sensitivity to standard
mutagens (positive controls, 6.18).
Stock cultures should fulfil the requirements of Annex C, otherwise they should be discarded.
Treat and mix the remaining parts of the cultures with DMSO (1,8 ml per 20 ml of culture volume) or
glycerol (4 ml per 20 ml of culture volume) to protect against the effects of freezing, and freeze immediately at
u – 70 °C in small portions (0,5 ml to 1 ml).
Whenever new stock cultures need to be produced, inoculate from a frozen stock culture on ampicillin
containing nutrient agar (6.13) so as to grow single colonies. Incubate for 24 h at 37 °C ± 1 °C. Transfer
samples of individual colonies to 20 ml of nutrient broth (6.4), incubate, divide, and check the genotype, then
freeze the cultures a
...
SLOVENSKI STANDARD
SIST ISO 16240:2007
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
SIST ISO 16240:2007 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST ISO 16240:2007
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SIST ISO 16240:2007
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 16240:2005(E)
©
ISO 2005
---------------------- Page: 3 ----------------------
SIST ISO 16240:2007
ISO 16240:2005(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 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
---------------------- Page: 4 ----------------------
SIST ISO 16240:2007
ISO 16240:2005(E)
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
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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.
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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.
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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
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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.
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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 reduced to such an extent (to
several hundred) that the traces of histidine in the minimal softagar are sufficient to allow these bacteria to
grow up to visible colonies mimicking the growth of mutant colonies. This may lead to false positive results.
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5 Principle
The bacteria are exposed under defined conditions to various doses of the test sample and incubated for 48 h
to 72 h at 37 °C ± 1 °C. Due to this exposure, genotoxic agents contained in the test water or wastewater may
be able to induce mutations in one or both marker genes (hisG46 for TA 100 and hisD3052 for TA 98) in
correlation to the used doses. Such induction of mutations causes a dose-related increase in the numbers of
mutant colonies.
The possible mutagenic activity of the test sample is detected by comparing, for the used bacterial strain and
the respective activation condition [± S9 mix (3.13); Annex B)], the number of mutant colonies on plates
treated with the negative control with those treated with undiluted and diluted test sample.
The lowest dilution (1:N) of the test sample inducing, according to the criteria of this International Standard, no
mutagenic effect under all experimental conditions (if any mutagenic effect is induced by the test sample) is
the parameter relevant for the assessment of the test sample according to this International Standard.
Dilutions above this (1:A, A
Standard in at least one strain under at least one activation condition. The respective D -value is N. If no
min
mutagenic effect is observed under all experimental conditions, this dilution is 1:1 and the respective D -
min
value is 1.
The test facility is qualified for the conduct of this International Standard if the Salmonella/microsome test is
established in this facility according to the following criteria:
several independent experiments are performed;
several known mutagenic and non-mutagenic reagents are tested;
the mutagenic compounds are included in the positive controls of this International Standard (6.18);
the results are reproducible;
the results are in compliance with literature data.
6 Reagents and media
As far as possible, use chemicals of reagent grade. Prepare all aqueous solutions with water of a conductivity
of u 5 µS/cm.
If chemicals with different amounts of crystallisation water are used, recalculate the needed amounts.
Always autoclave for 20 min at 121 °C ± 2 °C. Seal vessels loosely (e.g. with aluminium foil). Sealing should
never be air-tight.
All compositions are given for specific final amounts. Other final amounts (N-fold) may be reached by
multiplying the amounts all single components of the respective composition by N.
Compositions may be subdivided under appropriate conditions into appropriate amounts.
6.1 Hydrochloric acid, c(HCl) = 1 mol/l.
6.2 Sodium hydroxide solution, c(NaOH) = 1 mol/l.
6.3 Dimethyl sulfoxide (DMSO), C H O S.
2 6 4
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6.4 Nutrient broth
For each 1 l of water, add 3 g of beef extract, 5 g of peptone and 5 g of sodium chloride (or alternatively, 10 g
of beef extract, 10 g of peptone and 5 g of sodium chloride). Warm up and stir to dissolve the compounds.
Adjust the pH to 7,4 ± 0,2 and autoclave in appropriate portions. Store under sterile conditions at 4 °C ± 2 °C
for not longer than one month.
For use of commercial products, see A.1.
6.5 Ampicillin solution
Under sterile conditions at room temperature, dissolve 80 mg of ampicillin in 10 ml of sterile sodium hydroxide
solution (0,02 mol/l). Use immediately.
6.6 Nutrient broth with ampicillin
Under sterile conditions, add 3,15 ml of ampicillin solution (6.5) to 1 l of nutrient broth (6.4). Store under sterile
conditions at 4 °C ± 2 °C for not longer than one week.
6.7 Sodium hydrogen phosphate buffer
The following solutions are needed to prepare the buffer:
solution 1: 13,8 g of sodium dihydrogen phosphate monohydrate (NaH PO · H O) dissolved in 1 l
2 4 2
water;
solution 2: 14,2 g of disodium hydrogen phosphate (Na HPO ) dissolved in 1 l water.
2 4
Stir solution 2 (e.g. with a magnetic stirrer) and add solution 1 until a pH of 7,4 is reached and remains stable.
Subdivide this solution in appropriate amounts and autoclave to sterilize. Store at 4 °C ± 2 °C for not longer
than one month.
6.8 Cofactor solution
Dissolve the following compounds, in the amounts given, in 70 ml of sodium hydrogen phosphate buffer (6.7):
162,6 mg of magnesium chloride hexahydrate (MgCl · 6 H O);
2 2
246,0 mg of potassium chloride (KCl);
179,1 mg of glucose-6-phosphate, disodium salt (G6P);
1)
315,0 mg of NADP , disodium salt.
Filter sterile through appropriate membrane filters. A volume of 70 ml of cofactor solution is needed for the
preparation of 100 ml of S9 mix, sufficient for approximately 200 plates.
6.9 S9 fraction
The preparation of S9 fraction and the treatment for enzyme induction are described in Annex B. If S9 fraction
is purchased commercially, it should also be prepared (including enzyme induction) according to Annex B.
6.10 S9 mix
Prepare the needed amount of S9 fraction (6.9) freshly on the day of test or, if stored frozen, thaw it at room
temperature. Immediately thereafter, prepare S9 mix by mixing the following under sterile conditions:
10 ml of S9 fraction;
1) Nicotinamide adenine dinucleotide phosphate.
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70 ml of cofactor solution (6.8);
20 ml of potassium chloride solution (6.19).
Cool S9 mix permanently (e.g. in a double-walled separator funnel containing ice water between the walls)
and use it only on the same day. Discard the remaining S9 mix at the end of this day.
6.11 Vogel-Bonner E-medium (50×)
Dissolve by stirring the following amounts of the substances, in the given order, into 670 ml water. Allow each
ingredient to dissolve completely before adding the next.
10 g MgSO · 7 H O
4 2
100 g citric acid · H O
2
500 g K HPO , anhydrous
2 4
175 g NaNH HPO · 4 H O
4 4 2
This results in a final volume of approximately 1 l. Divide in appropriate portions and store at room
temperature.
6.12 Vogel-Bonner plates (see also 8.1)
For the preparation of 1 l of agar:
add 15 g of agar (see A.2) to 880 ml water and dissolve by heating under stirring; seal the container
(e.g. with aluminium foil);
dissolve 20 g of glucose monohydrate in water to a final volume of 100 ml; seal the container (e.g. with
aluminium foil);
transfer 20 ml of Vogel-Bonner E-medium (6.11) into a container and seal container;
autoclave all components;
under sterile conditions, add glucose solution and Vogel-Bonner E-medium to the agar solution.
CAUTION — Delayed boiling leading to splashing can occur.
Mix and pour into Petri dishes (25 ml to 30 ml per dish).
6.13 Nutrient agar plates (see also 8.1)
Mix 23 g of nutrient agar (see A.3) with 1 l of water and dissolve by heating under stirring. Seal container
(e.g. with aluminium foil) and autoclave. Pour into Petri dishes (25 ml per dish).
Alternatively, dissolve 15 g of agar (see A.2) in 1 l of nutrient broth (see A.1) by warming in an appropriate
vessel. Seal vessel loosely (e.g. with aluminium foil) and autoclave the solution. Thereafter pour into Petri
dishes (see 8.1).
6.14 Ampicillin agar plates (see also 8.1)
Dissolve agar (see A.2) in 860 ml water. Prepare 100 ml of glucose-solution and 20 ml of
Vogel-Bonner E-medium as described in 6.11. In addition, the following ingredients are needed:
3,15 ml ampicillin solution (6.5);
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10,0 ml histidine solution (add 500 mg of L-histidine hydrochloride monohydrate to 100 ml water);
6,0 ml biotin solution [add 12,2 mg of (D+)-biotin to 100 ml of water].
Autoclave all components. Under sterile conditions, add all solutions and Vogel-Bonner E-medium to the
autoclaved agar solution.
CAUTION — Delayed boiling leading to splashing can occur.
Mix and pour into Petri dishes (25 ml per dish).
If nutrient agar (6.13) is used as an alternative, add 3,15 ml of ampicillin solution (6.5) per litre of agar solution.
6.15 Amino acid solutions for softagar
The only difference between amino acid solutions for mutant and titre determinations is the amount of histidine
needed.
6.15.1 Minimal medium
Add 105 mg of L-histidine hydrochloride monohydrate and 122 mg of (D+)-biotin to 1 l water, mix, seal and
autoclave. Divide under sterile conditions into appropriate amounts (e.g. 10 ml) in marked vessels, and store
at 4 °C ± 2 °C.
6.15.2 Complete medium
Add 1 050 mg of L-histidine hydrochloride monohydrate and 122 mg of (D+)-biotin to 1 l of water, mix, seal
and autoclave. Divide under sterile conditions into appropriate amounts (e.g. 10 ml) in marked vessels, and
store at 4 °C ± 2 °C.
6.16 Softagar
6.16.1 Softagar for mutant determination
Mix 0,8 g of agar (see A.2) and 0,6 g of sodium chloride with 45 ml water.
Place 10 ml of amino acid solution (minimal medium, 6.15.1) in a separate vessel.
To adjust the volume in experiments without S9 mix, 0,5 ml of sodium hydrogen phosphate buffer (6.7) per
1 ml of softagar may also be needed.
Autoclave all components.
Add minimal medium and, if appropriate, buffer to agar and mix thoroughly. Avoid temperatures below 45 °C.
Place tubes in a water bath (45 °C ± 1 °C) and add 1 ml (for experiments with S9 mix) to each tube or 1,5 ml
(for experiments without S9 mix) of the respective mixture under sterile conditions. Cover tubes again with
aluminium foil. Mark stands in an appropriate form to avoid mistakes. Experience has shown that soft agar
may be stored in this form for up to 24 h.
6.16.2 Softagar for titre determination
Mix 0,8 g of agar (see A.2) and 0,6 g of sodium chloride with 45 ml water.
Place 10 ml of amino acid solution (complete medium, 6.15.2) in a separate vessel.
Autoclave all components.
Add complete medium to agar and mix thoroughly. Avoid temperatures below 45 °C. Place tubes in a water
bath (45 °C ± 1 °C) and add 1 ml of the mixture to each tube under sterile conditions. Cover tubes again with
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aluminium foil. Mark stands in an appropriate form to avoid mistakes. Experience has shown that soft agar
may be stored in this form for up to 24 h.
6.17 Physiological NaCl solution
Dissolve 9 g of sodium chloride in 1 l water. Separate in appropriate amounts, autoclave and store under
sterile conditions.
6.18 Positive controls
Stock solutions of positive control substances can be stored at – 20 °C as long as validity criteria (Clause 10)
are met.
6.18.1 Nitrofurantoin
Dissolve 20 mg of nitrofurantoin (NF) under sterile conditions in 10 ml of dimethyl sulfoxide, DMSO (6.3)
resulting in a stock solution of 2 mg/ml. Dilute stock solution three times 1:10 with DMSO. Use 0,1 ml of the
−3
final dilution (10 ) (containing 2 µg/ml NF) in the test.
6.18.2 4-Nitro-1,2-phenylene diamine
Dissolve 25 mg of 4-nitro-1,2-phenylene diamine (4-NPDA) under sterile conditions in 5 ml of DMSO (6.3)
resulting in a stock solution of 5 mg/ml. Dilute stock solution three times 1:10 with DMSO. Use 0,1 ml of the
−3
final dilution (10 ) (containing 5 µg/ml 4-NPDA) in the test.
6.18.3 2-Aminoanthracene
Dissolve 30 mg of 2-aminoanthracene (2-AA) under sterile conditions in 10 ml of DMSO (6.3) resulting in a
−2
stock solution of 3 mg/ml. Dilute stock solution twice 1:10 with DMSO. Use 0,1 ml of the final dilution (10 )
(containing 30 µg/ml 2-AA) in the test.
6.19 KCl solution
Dissolve 1,12 g of potassium chloride in 100 ml water and autoclave.
6.20 Tester strains
Use mutants of Salmonella typhimurium LT2, which detect point mutations, to determine the mutagenic
potential of a test sample. Since point mutations can be subdivided into two classes (base-pair substitutions
and frame-shift mutations), two strains are used. These strains are TA 100 and TA 98. TA 100 bears the
base-pair substitution hisG46 as marker, whereas TA 98 contains the frame-shift mutation (+2 type) hisD3052.
In addition, both strains have the following genetic properties.
a) They contain the plasmid pKM101, coding for an ampicillin resistance.
b) They are all deep rough, e.g. partly deficient in lipopolysaccharide side chains, enabling larger molecules
to penetrate the bacterial cell wall and also produce mutations.
c) Due to the mutation uvrB, their capability to repair DNA-damage is limited, as it is induced e.g. by UV-light,
which increases the likelihood that such damage results in mutations.
Immediately upon receipt, spread aliquots of the respective bacterial strain onto the surface of plates with
ampicillin agar (6.14) thus allowing to grow in single colonies. Incubate for 24 h at 37 °C ± 1 °C.
Take samples from individual colonies with a sterile inoculation loop, and transfer them to 20 ml of ampicillin-
containing nutrient broth (6.6).
Incubate again overnight at 37 °C ± 1 °C.
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Inoculate samples of these cultures onto nutrient agar plates, which have been provided with ampicillin (6.1
...
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