EN ISO 10712:1995

Water quality - Pseudomonas putida growth inhibition test (pseudomonas cell multiplication inhibition test)(ISO 10712:1995)

Describes a test method for determining the inhibitory effect of surface, ground and waste water on Pseudomonas putida growth. Not suitable for highly coloured samples or samples containing undissolved or volatile substances which react with the nutrient solution or undergo changes such as biochemical degradation. Also suitable for testing substances soluble in water.

Wasserbeschaffenheit - Pseudomonas putida Wachstumshemmtest (Pseudomonas-Zellvermehrungshemmtest) (ISO 10712:1995)

Diese Internationale Norm legt ein Verfahren zur Bestimmung der Hemmwirkung von Wasser, Abwasser und wasserlöslichen Stoffen gegenüber Pseudomonas putida fest. Das Verfahren ist nicht geeignet für stark gefärbtes Testgut oder Proben, die ungelöste oder flüchtige Stoffe oder solche, die mit der Nährlösung reagieren, enthalten oder die sich während des Tests verändern (z.B. durch Ausfällung, biochemischen oder photochemischen Abbau) und verfälschte Ergebnisse liefern können und/oder die Reproduzierbarkeit beeinträchtigen.

Qualité de l'eau - Essai d'inhibition de la croissance de Pseudomonas putida (essai d'inhibition de la multiplication des cellules de Pseudomonas) (ISO 10712:1995)

La présente Norme internationale prescrit une méthode d'essai pour la détermination de l'effet inhibiteur de l'eau de surface, de l'eau souterraine et des eaux usées vis-à-vis de Pseudomonas putida. La méthode n'est pas applicable aux échantillons de forte coloration, ou contenant des matières non dissoutes ou volatiles ou des substances réagissant avec le milieu nutritif ou qui subissent des changements pendant l'essai (par exemple par précipitation, ou dégradation biochimique ou photochimique), et peuvent donner des résultats faux, et/ou affecter la reproductibilité. La méthode permet également d'expérimenter les substances solubles dans l'eau (voir annexe A).

Kakovost vode - Preskus z zaviranjem rasti bakterije Pseudomonas putida (Preskušanje z zaviranjem razmnoževanja bakterije Pseudomonas) (ISO 10712:1995)

General Information

• Status: Published
• Publication Date: 14-Dec-1995
• Withdrawal Date: 29-Jun-1996
• ICS: 13.060 - Water quality
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 21-May-2008
• Completion Date: 21-May-2008
• Ref Project: SIST EN ISO 10712:1997 - Water quality - Pseudomonas putida growth inhibition test (pseudomonas cell multiplication inhibition test)(ISO 10712:1995)

Overview

EN ISO 10712:1995 is an international standard published by the European Committee for Standardization (CEN) that specifies a reliable test method for assessing the inhibitory effect of water samples on the growth of Pseudomonas putida. This standard focuses on the Pseudomonas putida growth inhibition test, also known as the Pseudomonas cell multiplication inhibition test, used to evaluate biological water quality.

Its scope covers the testing of surface water, groundwater, and waste water samples to determine potential toxic effects on the bacterium Pseudomonas putida, a gram-negative, aerobic rod-shaped microorganism commonly found in freshwater and soil environments. This test is essential for screening water toxicity and evaluating substances soluble in water. However, it is unsuitable for highly coloured samples or samples containing undissolved or volatile substances that may interfere with nutrient solutions or undergo biochemical degradation during testing.

Key Topics

• Purpose and Application
  The standard describes a procedural method for quantifying growth inhibition of Pseudomonas putida after exposure to different concentrations of water samples. Growth is measured by optical density (turbidity), enabling determination of the substance concentrations causing 10% (EC10) and 50% (EC50) inhibition of cell multiplication.

• Test Organism
  Utilizes Pseudomonas putida strains (e.g., MIGULA Berlin 33/Z strain DSM 50026 or NCIB strain 9494) as representative heterotrophic freshwater bacteria sensitive to toxicants.

• Test Procedure

  • Preparation of stock and precultures using defined nutrient solutions.
  • Exposure of test cultures to serial dilutions of the water sample.
  • Incubation at 23 ±1 °C in the dark for 16 ±1 hours.
  • Measurement of bacterial growth by turbidity using spectrometry or turbidimetry at 436 nm.
  • Calculation of percentage inhibition compared to control samples without the test water.

• Sample Requirements and Limitations
  Suitable for samples free from interferences such as strong coloration or volatile/undissolved substances. Samples must be thoroughly mixed and tested preferably soon after collection; preservation by cooling or freezing is only recommended under exceptional conditions.

• Reagents and Media
  Essential nutrient solutions include sodium nitrate, potassium phosphate salts, yeast extract, glucose, magnesium sulfate, and iron(III) citrate. Stock cultures are maintained on slant agar media as per standard specifications.

• Validity and Quality Control
  The method requires parallel replicates for statistical confidence. Inoculum turbidity and culture conditions need precise control to ensure reproducibility. Control and test cultures are compared to assess the growth inhibition.

Applications

The EN ISO 10712:1995 test method is widely used in:

• Environmental Monitoring
  Assessing the toxicity of surface, ground, and wastewater to ensure compliance with environmental regulations protecting aquatic life and ecosystem health.

• Water Treatment Facilities
  Screening incoming raw water or effluent for toxic substances that could impact microbial treatment processes or downstream environments.

• Chemical and Pharmaceutical Industry
  Evaluating the ecotoxicological impact of water-soluble substances and effluents to ensure safe discharge and environmental compatibility.

• Research and Development
  Studying the effects of pollutants, disinfectants, or novel compounds on freshwater bacterial populations.

This bacterial growth inhibition test offers a practical, sensitive bioassay for water quality, complementing physicochemical analyses by detecting biologically relevant toxicity effects.

Related Standards

• ISO 7027:1990 – Water quality - Determination of turbidity, which is used within EN ISO 10712 for measuring bacterial cell suspension optical density.

• EN ISO 5667 series – Water quality sampling standards that guide proper collection and handling of water samples to be tested.

• ISO 19438 – Water quality - Determination of inhibition of bacterial growth by other microbial species, offering complementary bioassays in water toxicity testing.

• OECD Guidelines for Testing of Chemicals – Includes bacterial growth inhibition tests using other bacteria such as Vibrio fischeri, often used alongside Pseudomonas tests for comprehensive ecotoxicity assessment.

Keywords

Water quality testing, Pseudomonas putida, bacterial growth inhibition, EC10, EC50, toxicity bioassay, surface water quality, groundwater toxicity test, wastewater monitoring, biological water analysis, ISO 10712, CEN standards, microbiological water quality.

Standards Content (Sample)

SLOVENSKI STANDARD
01-avgust-1997
Kakovost vode - Preskus z zaviranjem rasti bakterije Pseudomonas putida
(Preskušanje z zaviranjem razmnoževanja bakterije Pseudomonas) (ISO
10712:1995)
Water quality - Pseudomonas putida growth inhibition test (pseudomonas cell
multiplication inhibition test)(ISO 10712:1995)
Wasserbeschaffenheit - Pseudomonas putida Wachstumshemmtest (Pseudomonas-
Zellvermehrungshemmtest) (ISO 10712:1995)
Qualité de l'eau - Essai d'inhibition de la croissance de Pseudomonas putida (essai
d'inhibition de la multiplication des cellules de Pseudomonas) (ISO 10712:1995)
Ta slovenski standard je istoveten z: EN ISO 10712:1995
ICS:
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
IS0
STANDARD
First edition
1995-l 2-15
Water quality - Pseudomonas putida
growth inhibition test (Pseudomonas cell
multiplication inhibition test)
Qualit6 de I’eau - Essai d’inhibition de la croissance de Pseudomonas
putida (essai d’inhibition de la multiplication des cellules de
Pseudomonas)
Reference number
IS0 10712:1995(E)
IS0 10712:1995(E)
Foreword
IS0 (the International Organization for Standardization) is a worldwide
federation of national standards bodies (IS0 member bodies). The work
of preparing International Standards is normally carried out through IS0
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. IS0
collaborates closely with the International Electrotechnical Commission
(I EC) 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 IS0 10712 was prepared by Technical Committee
ISODC 147, INater quality, Subcommittee SC 5, Biological methods.
Annexes A and B of this International Standard are for information only.
0 IS0 1995
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 l CH-1211 Geneve 20 l Switzerland
Printed in Switzerland
ii
0 IS0
IS0 10712:1995(E)
Introduction
The bacterium Pseudomonas putida is used as an organism representative
of heterotrophic microorganisms in fresh water.

This page intentionally left blank

INTERNATIONAL STANDARD 0 ISO IS0 10712:1995(E)
Water quality - Pseudomonas putida growth
inhibition test (Pseudomonas cell multiplication
inhibition test)
3.1 multiplication; growth: Increase in the number
1 Scope
of cells during the test period.
This International Standard specifies a test method for
determining the inhibitory effect of surface, ground 3.2 concentration-effect relationship: Depen-
and waste water on Pseudomonas putida. dence of cell multiplication inhibition on the concen-
tration of the test sample.
This method is not suitable for highly coloured test
samples, or samples containing undissolved or volatile
NOTE 1 The relationship is graphically represented by
materials or substances which react with the nutrient plotting the inhibition values along the ordinate against the
sample concentrations along the abscissa.
solution, or which undergo changes during the test
(for example by precipitation, or biochemical or
3.3 effective concentration (EC): Concentration of
photochemical degradation) and may give false results
the test sample giving a calculated or interpolated
and/or impair the reproducibility.
inhibition of cell multiplication of Pseudomonas putida
The method is also suitable for testing substances
within 16 h + 1 h, compared to that of the control
soluble in water (see annex A).
batch. -
The concentrations of test samples (EC10 and EC50)
2 Normative reference
are determined from the concentration-effect re-
lationship (3.2) at which cell multiplication is inhibited
The following standard contains provisions which,
by 10 % or 50 % respectively, compared to that of the
through reference in this text, constitute provisions
control batch.
of this International Standard. At the time of publi-
cation, the edition indicated was valid. All standards
3.4 stock culture: Bacterial culture obtained from
are subject to revision, and parties to agreements
the collection strain of the laboratory and intended to
based on this International Standard are encouraged
provide an inoculum for the preculture in the test
to investigate the possibility of applying the most re-
procedure.
cent edition of the standard indicated below. Mem-
bers of IEC and IS0 maintain registers of currently
3.5 preculture: Bacterial culture separately used to
valid International Standards.
adapt the test bacteria to the test conditions and to
IS0 7027:1990, Water quality - Determination of produce an adequate number of exponentially multi-
turbidity. plying bacteria as an inoculum for the test culture.
3.6 test culture: Inoculated test medium (3.9).
3 Definitions
For the purposes of this International Standard, the 3.7 inoculum: Suspension of bacteria used to in-
following definitions apply. oculate a nutrient solution.

0 IS0
IS0 10712:1995(E)
NOTE 2 The two following strains are suitable for this
38 . nutrient solution: Aque ous solution of nutrients
test:
requi red for bacterial growth.
a) MIGULA, Berlin 33/Z strain (DSM 50026)
3.9 test medium: Mixture of test sample, dilution
This strain is available from the following collection:
water and nutrient solution (without inoculum).
German collection of microorganisms
3.10 sample: The surface, ground, or waste water
Mascheroder Weg 1 b
to be tested.
D-381 24 Braunschweig
Germany
3.11 test sample: The sample, after inclusion of all
b) NCIB strain 9494
preparatory steps such as homogenization, pH ad-
justment, filtration, centrifugation.
This strain is available from the following collection:
3.12 control: Mixture of dilution water, nutrient sol-
Tony Research Station
and inoculum (without test sample). P.O. Box 31
ution
Aberdeen, UK
3.13 formazine nephelometric unit (FNU): Forma-
may be suit-
Any other strain of equivalent sensitivity
zine turbidity units. The optical density of a bacterial
able.
cell suspension at L = 436 nm, measured as
formazine nephelometric units according to IS0 7027.
5.2 Hydrochloric acid, c(HCI) = 1 mol/l.
5.3 Sodium hydroxide solution,
c(NaOH) = 1 mol/l.
4 Principle
More diluted or concent rated acids and alkaline sol-
Determination of the inhibitory effect of the sample
utions are permi ssible to adjust the pH a s necessary.
on Pseudomonas putida by measurement of cell
growth under the influence of varying dilutions of the
test sample, compared to the cell growth of a culture
5.4 Nutrient solution
obtained under the same conditions, but without the
test sample. Prepare the stock solutions I-IV (see 5.4.1 to 5.4.4)
and then sterilize them, for example at 121 “C for
Determination of the cell concentration as optical
IO min.
density after a test period of 16 h + 1 h.
-
The solutions can be stored for several weeks in the
The concentrations of the test sample at which cell
refrigerator at 2 “C to 4 “C.
multiplication is inhibited by IO % and 50 % within
16 h + 1 h. are the basis for assessment.
-
5.4.1 Solution I
Dissolve the following in water and dilute to 500 ml.
- IO,0 g of sodium nitrate (NaNO,)
5 Reagents
- 2,40 g of dipotassium hydrogen phosphate
Use chemicals of analytical grade and deionized water
(K,HPO,)
or water of equivalent purity.
- I,20 g of potassium dihydrogen phosphate
KH2P0,)
5.1 Test organism
- I,0 g of yeast extract
Pseudomonas putida, a gram-negative aerobic bac-
terium of the Pseudomonadaceae family; mobile rods
5.4.2 Solution II
(diameter 0,7 pm to 1 ,I pm, length 2,0 pm to
4‘0 pm) with polar flagellation. It occurs ubiquitously
Dissolve the following in water and dilute to 500 ml.
in soil and surface water. The optimal growth tem-
- IO,0 g of sodium nitrate (NaNO,)
perature is between 25 “C and 30 “C.
0 IS0
IS0 10712:1995(E)
- 2,40 g of dipotassium hydrogen phosphate Dispense 6 ml to IO ml portions of the nutrient me-
dium into culture tubes while still liquid, close the
(K,HPO,)
tubes with plugs and sterilize for IO min at 121 “C.
- I,20 g of potassium dihydrogen phosphate
Allow the nutrient medium to gel at a slant and store
KH,PO,)
at 2 “C to 4 “C.
5.4.3 Solution Ill, glucose solution
5.5.2 Handling of stock culture
Dissolve the following in water and dilute to 500 ml.
- 40,o g of D(+)-glucose monohydrate Store stock cultures of the test strain Pseudomonas
(C6H,206.H20) for biochemical and microbiological putida in slant-agar culture tubes on the solid nutrient
use medium for stock cultures (5.5.1).
Start new stock cultures at intervals of one week, to
5.4.4 Solution IV, magnesium sulfate-iron(lll)
preserve the test strain.
citrate solution
Incubate the inoculated stock cultures for 24 h at
Dissolve the following in water and dilute to
25 “C + 4 “C (and store at 25 “C & 4 “C) for this pur-
1 000 ml.
pose. Long-term recultivation of one strain may cause
changes in the sensitivity of the test organisms. In
- 4,0 g of magnesium sulfate heptahydrate
this case, a new culture of the test strain has to be
(MgSO,.7H,O)
used.
- 0,Ol g of granulated iron(lll) citrate
NOTE 4 A green pigment may be produced after incu-
bation for 24 h. This is normal and does not indicate con-
NOTE 3 In order to reduce the number of steps involved,
solutions I and III can be combined, following sterilization, tamination.
for the procedure in 5.5 and 8.1 and solutions II and Ill for
the procedure in 8.2.
6 Materials and equipment
5.5 Stock culture (see table 1)
Equipment which comes into contact with the test
5.5.1 Nutrient medium for the stock culture
material during preparation of the nutrient medium,
(slant agar)
or during the test period, shall consist of glass or an-
Dissolve 18 g of agar (high purity quality for micro- other chemically inert material.
biology) in water by heating.
All glass equipment and stoppers which come into
Add 50 ml of solution I (5.4.1), 125 ml of solution III contact with the test cultures shall be sterilized before
(5.4.3), 100 ml of solution IV (5.4.4) and dilute to use, if they are not sterilized together with the nutri-
I 000 ml with water.
ent solutions.
Table 1 - Final concentrations in the various media
Stock culture (5.5) Preculture (8.1) Test culture (8.2)
Nutrients
w/l
w/l w/l
I
NaNO, 1 000 500 500
K,HPO, 240 120 120
KH*PO, 120 60 60
Yeast extract 50
10 000
C6H,,06-H20 2 000
MgS0,.7H20 200
Iron(lll) citrate
ItO 0,5
Agar 18 000
0 IS0
IS0 10712:1995(E)
6.1 Spectrometer or turbidimeter. Add 25 m each of solutions I and III (5.4.1 and 5.4.3)
plus 50 m of solution IV (5.4.4).
Alternatively, determine the state of growth of the
The pH of the preculture medium is 7,2 + 0,2.
...