SIST EN ISO 11348-1:2000

Water quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) - Part 1: Method using freshly prepared bacteria (ISO 11348-1:1998)

Migrated from Progress Sheet (TC Comment) (2000-07-10): N315: New TD (TA/980915)

Wasserbeschaffenheit - Bestimmung der Hemmwirkung von Wasserproben auf die Lichtemission von Vibrio fischeri (Leuchtbakterientest) - Teil 1: Verfahren mit frisch gezüchteten Bakterien (ISO 11348-1:1998)

Qualité de l'eau - Détermination de l'effet inhibiteur des échantillons d'eau sur la luminescence de Vibrio fischeri (Essai de bactéries luminescentes) - Partie 1: Méthode utilisant des bactéries fraîchement préparées (ISO 11348-1:1998)

La CEI 60512-26-100:2008+A1:2011 spécifie les essais et mesures ainsi que le montage de mesure et les dispositifs de référence associés pour les essais d'interopérabilité et de compatibilité amont pour le développement et la qualification des embases et des fiches à 8 voies pour la transmissions des données. Les méthodes d'essai suivantes sont spécifiées:
- perte d'insertion, essai 26a;
- affaiblissement de réflexion, essai 26b;
- paradiaphonie, essai 26c;
- télédiaphonie, essai 26d;
- impédance de transfert, essai 26e;
- perte de conversion transverse, essai 26f;
- perte de transfert de conversion transverse, essai 26g.
Cette publication doit être lue conjointement avec la
CEI 60512-1:2001 et la CEI 60512-1-100:2006.
Cette version consolidée comprend la première édition  (2008) et
son amendement 1 (2011). Il n'est donc pas nécessaire de commander
l'amendement avec cette publication.

Kakovost vode – Določevanje zaviralnega učinka vzorcev vode na oddajanje svetlobe Vibrio Fischeri (preskus luminiscence bakterije) – 1. del: Metoda z uporabo sveže pripravljenih bakterij (ISO 11348-1:1998)

General Information

• Status: Withdrawn
• Publication Date: 31-Dec-1999
• Withdrawal Date: 08-Apr-2009
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: KAV - Water quality
• Current Stage: 9900 - Withdrawal (Adopted Project)
• Start Date: 09-Apr-2009
• Due Date: 02-May-2009
• Completion Date: 09-Apr-2009
• Ref Project: EN ISO 11348-1:1998 - Water quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) - Part 1: Method using freshly prepared bacteria (ISO 11348-1:1998)

Relations

• Replaced By: SIST EN ISO 11348-1:2009 - Water quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) - Part 1: Method using freshly prepared bacteria (ISO 11348-1:2007)
• Effective Date: 01-May-2009

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 11348-1:2000
01-januar-2000
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Water quality - Determination of the inhibitory effect of water samples on the light
emission of Vibrio fischeri (Luminescent bacteria test) - Part 1: Method using freshly
prepared bacteria (ISO 11348-1:1998)
Wasserbeschaffenheit - Bestimmung der Hemmwirkung von Wasserproben auf die
Lichtemission von Vibrio fischeri (Leuchtbakterientest) - Teil 1: Verfahren mit frisch
gezüchteten Bakterien (ISO 11348-1:1998)
Qualité de l'eau - Détermination de l'effet inhibiteur des échantillons d'eau sur la
luminescence de Vibrio fischeri (Essai de bactéries luminescentes) - Partie 1: Méthode
utilisant des bactéries fraîchement préparées (ISO 11348-1:1998)
Ta slovenski standard je istoveten z: EN ISO 11348-1:1998
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 11348-1:2000 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 11348-1:2000

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SIST EN ISO 11348-1:2000

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SIST EN ISO 11348-1:2000

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SIST EN ISO 11348-1:2000

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SIST EN ISO 11348-1:2000

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SIST EN ISO 11348-1:2000
INTERNATIONAL ISO
STANDARD 11348-1
First edition
1998-12-15
Water quality — Determination of the
inhibitory effect of water samples on the
light emission of Vibrio fischeri
(Luminescent bacteria test) —
Part 1:
Method using freshly prepared bacteria
Qualité de l’eau — Détermination de l’effet inhibiteur d’échantillons d’eau
sur la luminescence de Vibrio fischeri (Essai de bactéries luminescentes) —
Partie 1: Méthode utilisant des bactéries fraîchement préparées
A
Reference number
ISO 11348-1:1998(E)

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SIST EN ISO 11348-1:2000
ISO 11348-1:1998(E)
Contents
Page
1 Scope. 1
2 Normative references . 1
3 Principle . 2
4 Interferences. 2
5 Reagents and materials. 2
6 Apparatus . 4
7 Sampling and sample pretreatment. 4
8 Cultivation of luminescent bacteria . 5
9 Procedure . 6
10 Evaluation . 7
11 Expression of results . 9
12 Criteria of validity . 10
13 Precision . 10
14 Test report . 10
Annex A Colour-correction method . 11
Annex B Dilution level D — Preparation of the dilution series. 14
Annex C Precision data . 15
©  ISO 1998
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 the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet [e-mail]
Printed in Switzerland
ii

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SIST EN ISO 11348-1:2000
©
ISO ISO 11348-1:1998(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 collab-
orates closely with the International Electrotechnical Commission (IEC) on
all matters of electrotechnical standardization.
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.
International Standard ISO 11348-1 was prepared by Technical Committee
ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods.
ISO 11348 consists of the following parts, under the general title Water
quality — Determination of the inhibitory effect of water samples on the
light emission of Vibrio fischeri (Luminescent bacteria test) :
— Part 1: Method using freshly prepared bacteria
— Part 2: Method using liquid-dried bacteria
— Part 3: Method using freeze-dried bacteria
Annexes A, B and C of this part of ISO 11348 are for information only.
iii

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SIST EN ISO 11348-1:2000
©
ISO 11348-1:1998(E) ISO
Introduction
Measurements according to ISO 11348 can be carried out using freshly
prepared bacteria, as well as freeze-dried or liquid-dried bacterial
preparations.
Standardized work carried out by DIN NAW WI and
ISO/TC 147/SC 5 WG 1 has shown that in special cases these different
techniques may give different results, especially where water samples
contain heavy metals.
Such varying sensitivity is caused by differences in media composition
used in the preparation of freeze-dried or liquid-dried bacteria. These
protective media influence the bioavailability of toxicants and/or the light
emission of luminescent bacteria. This means that the origin and type of
preparation need to be taken into account when interpreting the results.
This can be difficult sometimes, as freeze-dried and liquid-dried bacteria
may be obtained from different suppliers. This in turn can mean that the
composition is not known in detail or cannot be revised by the user.
That is why in this International Standard, in addition to toxicity
measurements with liquid-dried bacteria (ISO 11348-2) and freeze-dried
bacteria (ISO 11348-3), a procedure with freshly prepared bacteria is
described (ISO 11348-1), the performance of which can be revised by the
user in every detail.
The laboratories responsible for the results have the opportunity to select
the most suitable technique based on expert judgement and information
about the water sample to be tested.
iv

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SIST EN ISO 11348-1:2000
©
INTERNATIONAL STANDARD  ISO ISO 11348-1:1998(E)
Water quality — Determination of the inhibitory effect of water
Vibrio fischeri
samples on the light emission of (Luminescent
bacteria test) —
Part 1:
Method using freshly prepared bacteria
1  Scope
ISO 11348 describes three methods for determining the inhibition of the luminescence emitted by the marine
bacterium Vibrio fischeri (NRRL B-11177). This part of ISO 11348 specifies a method using freshly prepared
bacteria.
This method is applicable to:
— waste water,
— aqueous extracts and leachates,
— fresh water (surface or ground water) or salt and brackish water, especially the monitoring of changes in
inhibition towards bacteria,
— pore water.
2  Normative references
The following standards contain provisions which, through reference in this text, constitute provisions of this part of
ISO 11348. At the time of publication, the editions indicated were valid. All standards are subject to revision, and
parties to agreements based on this part of ISO 11348 are encouraged to investigate the possibility of applying the
most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid
International Standards.
ISO 5667-16:1998, Water quality — Guidance on biotesting of samples.
1)
ISO 7027:— , Water quality — Determination of turbidity.

1)  To be published. (Revision of ISO 7027:1990)
1

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SIST EN ISO 11348-1:2000
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ISO
ISO 11348-1:1998(E)
3  Principle
The inhibition of light emission by cultures of Vibrio fischeri is determined by means of a batch test. This is
accomplished by combining specified volumes of the test sample or the diluted sample with the luminescent
bacteria suspension in a cuvette.
The test criterion is the decrease of the luminescence, measured after a contact of 15 min and 30 min or optionally
5 min, taking into account a correction factor (f ), which is a measure of intensity changes of control samples during
kt
the exposure time. The inhibitory effect of the water sample can be determined as LID (see annex B) or as EC
20
and/or EC values by means of a dilution series.
50
The dilution level resulting in , 20 % inhibition of light emission is determined. For higher levels of inhibition, the
dilution-effect relationship can be determined graphically or by statistical analysis. The inhibition by a sample is
expressed as the dilutions which result in 20 % and 50 % light reduction compared to the blank (EC and EC ).
20 50
These values are interpolated within the dilution series.
4  Interferences
Insoluble, slightly soluble or volatile substances or substances which react with the dilution water or the test
suspension, or alter their state during the test period, may affect the result or impair the reproducibility of the test
results.
Losses of luminescence caused by light absorption or light scattering may occur in the case of strongly coloured or
turbid waters. This interference sometimes can be compensated, e.g. by using a double-chambered absorption
correction cuvette (see annex A).
Since oxygen at . 0,5 mg/l is required for the bioluminescence, samples with a high oxygen demand (and/or a low
oxygen concentration) may cause a deficiency of oxygen and be inhibitory.
An organic contamination of the sample by readily biodegradable nutrients (e.g. urea, peptone, yeast extract,
usually > 100 mg/l) may cause a pollutant-independent reduction in bioluminescence.
Salt concentrations in the initial sample exceeding 30 g/l NaCl or contents of other compounds giving equal
osmolarity may lead, together with the salt spiking required by the test, to hyperosmotic effects. If the sample
contains between 20 g/l and 50 g/l NaCl-equivalents, no salt shall be added. The resulting concentration in the test
samples shall not exceed the osmolarity of a 35 g/l NaCl solution.
5  Reagents and materials
Use chemicals of recognized analytical grade quality. Water shall be distilled or of equivalent purity.
5.1  Test bacteria
Strain of luminescent bacteria belonging to the species Vibrio fischeri NRRL B-11177. The bacterial suspensions
used for toxicity measurements shall be freshly prepared from cultures.
5.2  Sodium chloride solution, as diluent
Dissolve 20 g of sodium chloride (NaCl) in water and make up to 1 litre with water.
5.3  Sodium hydroxide solution, c(NaOH) = 1 mol/l
5.4  Hydrochloric acid, c(HCl) = 1 mol/l
NOTE  For the adjustment of the pH it may be necessary to use acids or bases of lower or higher concentration.
2

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ISO 11348-1:1998(E)
5.5  Solution for freshly prepared bacteria
— 8,0 g D(1)-Glucose monohydrate (C H O {H O)
6 12 6 2
— 20,0 g Sodium chloride (NaCl)
— 2,035 g Magnesium chloride hexahydrate (MgCl {6H O)
2 2
— 0,30 g Potassium chloride (KCl)
— 11,9 g N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES)
Dissolve in water, stir for about 30 min and adjust the pH to 7,0 – 0,2 with sodium hydroxide solution (5.3) or
hydrochloric acid (5.4). Make up to 1 litre with water.
This solution can be stored in portions at 220 °C.
5.6  Reference substances
— Zinc sulfate heptahydrate (ZnSO ·7H O)
4 2
— 3,5-Dichlorophenol (CHOCl)
6 4 2
— Potassium dichromate (K Cr O )
2 2 7
5.7  Liquid broth for pre- and main cultures
— 30 g Sodium chloride (NaCl)
— 6,10 g Sodium dihydrogenphosphate monohydrate (NaH PO ·H O)
2 4 2
— 2,75 g Dipotassium hydrogenphosphate trihydrate (K HPO ·3H O)
2 4 2
— 0,204 Magnesium sulfate heptahydrate (MgSO ·7H O)
4 2
— 0,500 g Diammonium hydrogenphosphate [(NH ) HPO ]
4 2 4
— 3 ml Glycerol
— 5,00 g Caso-peptone
— 0,50 g Yeast extract
Dissolve in water and adjust the pH to 7,0 – 0,2 with sodium hydroxide (5.3) or hydrochloric acid (5.4). Make up to
1 litre with water. Transfer 50 ml each to Erlenmeyer flasks (approx. vol. 250 ml) and sterilize in an autoclave at
121 °C for 20 min.
NOTE  Caso-peptone and yeast extract offered by different suppliers can be of fluctuating quality. In case of problems (e.g.
growth inhibition), purchase a product from another manufacturer.
5.8  Agar medium for stock cultures
Adjust liquid broth (5.7) to pH 7,0 – 0,2.
Add 12 g of agar per litre and dissolve by gentle warming; sterilize and transfer to sterile Petri dishes.
5.9  Protective medium
— 66 g D(1)-Glucose monohydrate (C H O ·H O)
6 12 6 2
— 4 g Sodium chloride (NaCl)
— 2 g L-Histidine
— 0,5 g Bovine serum albumin, BSA
3

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ISO 11348-1:1998(E)
Dissolve thoroughly in water at about 37 °C and adjust to pH 7,0 – 0,2 at room temperature with sodium hydroxide
(5.3) or hydrochloric acid (5.4) as necessary. Make up to 100 ml with water.
NOTE  Damage of bacterial cells during the freezing procedure is prevented by the use of the protective medium. BSA offered
by different suppliers can be of fluctuating quality. If problems occur, purchase a product from another manufacturer.
Prepare protective medium freshly before use.
6  Apparatus
6.1  Refrigerator to maintain the stock suspension at a temperature of 3 °C – 3 °C.
6.2  Thermostatically controlled thermoblock to maintain the test samples at a temperature of 15 °C – 1 °C.
Within one test the temperature deviation shall be at most – 0,2 °C.
6.3  Luminometer, measuring cell maintained at 15 °C – 1 °C, equipped with suitable cuvettes.
6.4  Test tubes (vials) made of a chemically inert material, appropriate for the selected luminometer and having a
capacity which facilitates the taking of a reading over the largest possible surface area.
6.5  pH-meter.
6.6  Chronometer.
6.7  Piston pipettes for plastic syringes, nominal capacity 10 μl, 500 μl and 1 000 μl.
6.8  Piston pipettes with variable volume, 10 ml to 200 ml and 200 μl to 5 000 μl.
6.9  Refrigerated centrifuge.
6.10  Magnetic stirrer and magnetic stirring bar.
6.11  Incubator shaker for incubation of Erlenmeyer flasks.
6.12  Autoclave.
6.13  Incubator.
6.14  Spectral- or filterphotometer and cuvettes, optical path 1 cm.
6.15  Inoculating loop (or needle).
6.16  Conductometer.
7  Sampling and sample pretreatment
7.1  Sampling
Sampling shall be conducted in chemically inert, clean containers in accordance with ISO 5667-16. Fill the
containers completely and seal them. Test the samples as soon as possible after collection. Where necessary,
store samples at a temperature of 2 °C to 5 °C in the dark in glass for not longer than 48 h. For periods up to two
weeks, store at 220 °C. Do not use chemicals to preserve the samples. Perform the necessary pH adjustment and
salt addition just before testing.
4

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SIST EN ISO 11348-1:2000
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ISO 11348-1:1998(E)
7.2  Sample preparation
Measure the pH of all samples. If the pH lies between 6 and 8,5 there is generally no adjustment necessary. pH-
adjustment, however, may alter the nature of the sample. On the other hand, the pH of the sample and the pH of the
test batch may differ because of the buffer capacity of the test medium. It may be necessary to carry out tests on
both the pH-adjusted and the non-pH-adjusted samples.

If necessary, adjust the pH of the samples to 7,0 – 0,2 by adding either hydrochloric acid (5.4) or sodium hydroxide
(5.3); choose the concentration of the hydrochloric acid or the sodium hydroxide to restrict the volume added to not
more than 5 % of total volume.
Add 20 g of sodium chloride per litre to the water sample or to the neutralized water sample. For brackish and saline
waters, measure the salinity and calculate the amount of NaCl (if any) required to adjust the osmolarity (clause 4).
Strongly turbid samples should be allowed to sediment for 1 h or centrifuged, for example for 10 min at 5 000 g, or
should be filtered.
8  Cultivation of luminescent bacteria
8.1  Stock culturing
Transfer luminescent bacteria of strain Vibrio fischeri NRRL B-11177 under sterile conditions to Petri dishes
containing the agar for stock cultures (5.8).
Incubate in an incubator for 2 days to 5 days at 20 °C – 1 °C.
Mark luminescent single colonies using visual observations in the dark, and store dishes in the refrigerator
afterwards.
Transfer marked colonies under sterile conditions to fresh dishes after a maximum storage period of 2 weeks.
NOTE 1  Commercially available vials of preserved bacteria are not dispensed under sterile conditions. For cultivation of pure
cultures several single colony passages are recommended. To prevent genetic alterations, a new vial of preserved bacteria
should be opened approximately every 6 months.
NOTE 2  Luminescence of luminescent bacterial colonies can decrease during storage.
8.2  Preparation of precultures
Inoculate 50 ml of preculture broth (5.7) in Erlenmeyer flasks (approx. vol. 250 ml) under sterile conditions with one
luminescent single colony of a stock culture aged 2 days to 5 days.
Shake for 21 h – 1 h at 20 °C – 1 °C at 180 r/min.
Determine the turbidity of a 1:10 dilution in sodium chloride solution (5.2), for example in formazine nephelometric
units (FNU) at 578 nm in accordance with ISO 7027.
8.3  Preparation of main culture
Inoculate 50 ml of the main culture broth (5.7) in 250 ml Erlenmeyer flasks with an appropriate volume of preculture
(8.2) to result in an estimated initially turbidity of 10 FNU.
Shake for 20 h – 1 h at 20 °C – 1 °C at 180 r/min.
Determine turbidity in FNU of a 1:10 dilution in sodium chloride solution (5.2) photometrically at 578 nm.
NOTE  Following the above conditions, the undiluted main culture will normally exhibit a turbidity of 700 FNU to 1800 FNU.
5

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SIST EN ISO 11348-1:2000
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ISO
ISO 11348-1:1998(E)
8.4  Preparation of stock suspension
Precool sodium chloride solution (5.2) and protective medium (5.9) on ice.
Centrifuge bacterial suspension from main culture (8.3) at 4 °C – 2 °C in a precooled refrigerated centrifuge for
...