SIST-TP ISO/TR 11044:2010
Water quality - Scientific and technical aspects of batch algae growth inhibition tests
Water quality - Scientific and technical aspects of batch algae growth inhibition tests
ISO/TR 11044:2008 discusses scientific and technical aspects that have been considered in connection with the development of batch algal growth inhibition test procedures specified in ISO 8692, for freshwater, and ISO 10253, for marine waters.
Previously unpublished results of experiments performed at the Norwegian Institute for Water Research (NIVA) have been included to demonstrate various aspects.
Qualité de l'eau - Aspects scientifiques et techniques des essais d'inhibition de croissance d'un lot d'algues
Kakovost vode - Znanstveni in tehnični vidiki šaržnih preskusov z določevanjem zaviranja rasti alg
To tehnično poročilo obravnava znanstvene in tehnične vidike, ki so bili preučeni v zvezi z razvojem postopkov šaržnih preskusov z določevanjem zaviranja rasti alg, opredeljenih v ISO 8692 za sladko vodo in ISO 10253 za morske vode. Vključeni so še neobjavljeni rezultati eksperimentov, izvedenih na Norveškem inštitutu za vodne raziskave (NIVA), z namenom prikazati različne vidike.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-september-2010
.DNRYRVWYRGH=QDQVWYHQLLQWHKQLþQLYLGLNLãDUåQLKSUHVNXVRY]GRORþHYDQMHP
]DYLUDQMDUDVWLDOJ
Water quality - Scientific and technical aspects of batch algae growth inhibition tests
Qualité de l'eau - Aspects scientifiques et techniques des essais d'inhibition de
croissance d'un lot d'algues
Ta slovenski standard je istoveten z: ISO/TR 11044:2008
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.
TECHNICAL ISO/TR
REPORT 11044
First edition
2008-12-01
Water quality — Scientific and technical
aspects of batch algae growth inhibition
tests
Qualité de l'eau — Aspects scientifiques et techniques des essais
d'inhibition de croissance d'un lot d'algues
Reference number
©
ISO 2008
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 2008
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 2008 – All rights reserved
Contents Page
Foreword. iv
Introduction . v
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 General principles of ISO algal growth inhibition tests.2
5 Test species .4
5.1 General.4
5.2 Pseudokirchneriella subcapitata.6
5.3 Desmodesmus subspicatus .9
5.4 Skeletonema costatum.9
5.5 Phaeodactylum tricornutum.12
6 Test conditions .14
6.1 Growth medium.14
6.2 pH control .16
6.3 Inoculum density .18
6.4 Incubation conditions .21
6.5 Test endpoint .23
Bibliography .25
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.
In exceptional circumstances, when a technical committee has collected data of a different kind from that
which is normally published as an International Standard (“state of the art”, for example), it may decide by a
simple majority vote of its participating members to publish a Technical Report. A Technical Report is entirely
informative in nature and does not have to be reviewed until the data it provides are considered to be no
longer valid or useful.
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/TR 11044 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
iv © ISO 2008 – All rights reserved
Introduction
The growth of microalgae in batch cultures follows a well known pattern, with a lag phase followed by an
exponential growth phase, a phase of declining growth rate, a stationary phase, and ultimately a death phase
(Reference [9]). The characteristics of these phases are dependent on the environmental conditions including
the chemical composition of the growth medium, which provides the basis for using batch cultures of algae as
bioassays to investigate growth stimulating or inhibiting properties of constituents of the growth medium.
The first systematic application of microalgae bioassays for which standard protocols were developed was for
assessment of nutrient status and identification of growth limiting nutrients. Skulberg (Reference [50])
developed a bioassay with the green alga Selenastrum capricornutum Printz, which was used to assess
fertilizing influences of pollution in inland waters. The nutrient bioassay with S. capricornutum was further
developed and standardised in Reference [55]. The strain of S. capricornutum used as test organism in the
nutrient bioassays was originally isolated from the river Nitelva in southeast Norway in 1959. It has become
the most commonly used test algae for bioassays and is available from most major culture collections. Due to
taxonomic revisions, it was first renamed Raphidocelis subcapitata and later Pseudokirchneriella subcapitata
(Korshikov) Hindak (Reference [20]).
It was early recognized that bioassays of microalgae could be used to study the growth-inhibiting effects of
toxic chemicals and waste waters, and a modification of the algal assay procedure for toxicity studies was
made in Reference [43]. However, based on compilations of early algae toxicity test data some authors
claimed that the sensitivity of algae generally was low (Reference [26]). The environmental relevance of
results of the tests was also questioned because of the significant interspecies variation in response and lack
of field-validation of results of algal toxicity tests (Reference [28]). On the other hand, microalgae are generally
the most important primary producers in aquatic ecosystems. Excluding the assessment of toxicity to this
group of organisms in risk assessment and environmental management cannot be justified. Development and
standardisation of methods have therefore been undertaken to increase the reproducibility and relevance of
toxicity tests with microalgae. Standardised growth inhibition tests with algae are now a cornerstone in the
environmental management and risk assessment of chemicals. Recent reviews (e.g. Reference [57]) show
that they are often the most sensitive of the “base-set” tests which include also acute toxicity tests with fish
and Daphnia.
In addition to several national organisations, the Organisation for Economic Co-operation and Development
(OECD) and the International Organization for Standardization (ISO) took on the work of developing
guidelines and standards for growth inhibition with microalgae in the late 1970s. The OECD guidelines aim to
test chemical substances, while ISO documents cover tests for composite water samples, such as waste
water and elutriates. However, harmonisation of the procedures was an objective as the two series of
documents were developed in parallel by the two organisations. The development of the freshwater test was
initiated by ISO in 1978. Three ring tests were organised between 1980 and 1982 and included in
ISO 8692:1989, revised as ISO 8692:2004. The first draft of a marine algae inhibition test was produced in
1982, but the first ISO/DIS was not published until 1991, when the method had been ring tested.
ISO 10253:1995 was revised as ISO 10253:2006. In addition to these two standards, ISO 14442:1999,
guidelines for algal growth inhibition tests with poorly soluble matter, volatile compounds, metals and waste
water, was revised as ISO 14442:2006. In this Technical Report, the general principles of the batch culture
growth inhibition tests, and how some critical methological aspects have been addressed in the International
Standards for algal growth inhibition tests, are presented.
TECHNICAL REPORT ISO/TR 11044:2008(E)
Water quality — Scientific and technical aspects of batch algae
growth inhibition tests
1 Scope
This Technical Report discusses scientific and technical aspects that have been considered in connection with
the development of batch algal growth inhibition test procedures specified in ISO 8692, for freshwater, and
ISO 10253, for marine waters.
Previously unpublished results of experiments performed at the Norwegian Institute for Water Research
(NIVA) have been included to demonstrate various aspects.
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 8692:2004, Water quality — Freshwater algal growth inhibition test with unicellular green algae
ISO 10253:2006, Water quality — Marine algal growth inhibition test with Skeletonema costatum and
Phaeodactylum tricornutum
ISO 14442, Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials, volatile
compounds, metals and waste water
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
effective concentration
EC
x
concentration of test sample which results in a reduction of x % in the specific growth rate relative to the
controls
[ISO 8692]
NOTE Unless otherwise stated, the form EC is used in this Technical Report to mean E C where “r” denotes “rate”.
x r x
Effective concentrations based on area under the growth curve can be derived, and these are designated E C , where “b”
b x
denotes “biomass” (see 6.5 for further details).
3.2
specific growth rate
µ
proportional rate of increase in cell density per unit of time:
1dn
µ =
ntd
where
n is the cell density, expressed in cells per millilitre;
t is the time, expressed in days.
NOTE 1 Specific growth rate is expressed in reciprocal days.
NOTE 2 Adapted from ISO 8692.
4 General principles of ISO algal growth inhibition tests
The algae growth inhibition test methods specified in ISO 8692 and ISO 10253 are based on batch cultures
which are inoculated with algae from an exponentially growing inoculum culture and incubated under
continuous illumination. The growth medium, inoculum biomass density, temperature, and illuminance, have
been selected to allow an exponential increase in the algal biomass de
...
TECHNICAL ISO/TR
REPORT 11044
First edition
2008-12-01
Water quality — Scientific and technical
aspects of batch algae growth inhibition
tests
Qualité de l'eau — Aspects scientifiques et techniques des essais
d'inhibition de croissance d'un lot d'algues
Reference number
©
ISO 2008
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 2008
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 2008 – All rights reserved
Contents Page
Foreword. iv
Introduction . v
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 General principles of ISO algal growth inhibition tests.2
5 Test species .4
5.1 General.4
5.2 Pseudokirchneriella subcapitata.6
5.3 Desmodesmus subspicatus .9
5.4 Skeletonema costatum.9
5.5 Phaeodactylum tricornutum.12
6 Test conditions .14
6.1 Growth medium.14
6.2 pH control .16
6.3 Inoculum density .18
6.4 Incubation conditions .21
6.5 Test endpoint .23
Bibliography .25
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.
In exceptional circumstances, when a technical committee has collected data of a different kind from that
which is normally published as an International Standard (“state of the art”, for example), it may decide by a
simple majority vote of its participating members to publish a Technical Report. A Technical Report is entirely
informative in nature and does not have to be reviewed until the data it provides are considered to be no
longer valid or useful.
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/TR 11044 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
iv © ISO 2008 – All rights reserved
Introduction
The growth of microalgae in batch cultures follows a well known pattern, with a lag phase followed by an
exponential growth phase, a phase of declining growth rate, a stationary phase, and ultimately a death phase
(Reference [9]). The characteristics of these phases are dependent on the environmental conditions including
the chemical composition of the growth medium, which provides the basis for using batch cultures of algae as
bioassays to investigate growth stimulating or inhibiting properties of constituents of the growth medium.
The first systematic application of microalgae bioassays for which standard protocols were developed was for
assessment of nutrient status and identification of growth limiting nutrients. Skulberg (Reference [50])
developed a bioassay with the green alga Selenastrum capricornutum Printz, which was used to assess
fertilizing influences of pollution in inland waters. The nutrient bioassay with S. capricornutum was further
developed and standardised in Reference [55]. The strain of S. capricornutum used as test organism in the
nutrient bioassays was originally isolated from the river Nitelva in southeast Norway in 1959. It has become
the most commonly used test algae for bioassays and is available from most major culture collections. Due to
taxonomic revisions, it was first renamed Raphidocelis subcapitata and later Pseudokirchneriella subcapitata
(Korshikov) Hindak (Reference [20]).
It was early recognized that bioassays of microalgae could be used to study the growth-inhibiting effects of
toxic chemicals and waste waters, and a modification of the algal assay procedure for toxicity studies was
made in Reference [43]. However, based on compilations of early algae toxicity test data some authors
claimed that the sensitivity of algae generally was low (Reference [26]). The environmental relevance of
results of the tests was also questioned because of the significant interspecies variation in response and lack
of field-validation of results of algal toxicity tests (Reference [28]). On the other hand, microalgae are generally
the most important primary producers in aquatic ecosystems. Excluding the assessment of toxicity to this
group of organisms in risk assessment and environmental management cannot be justified. Development and
standardisation of methods have therefore been undertaken to increase the reproducibility and relevance of
toxicity tests with microalgae. Standardised growth inhibition tests with algae are now a cornerstone in the
environmental management and risk assessment of chemicals. Recent reviews (e.g. Reference [57]) show
that they are often the most sensitive of the “base-set” tests which include also acute toxicity tests with fish
and Daphnia.
In addition to several national organisations, the Organisation for Economic Co-operation and Development
(OECD) and the International Organization for Standardization (ISO) took on the work of developing
guidelines and standards for growth inhibition with microalgae in the late 1970s. The OECD guidelines aim to
test chemical substances, while ISO documents cover tests for composite water samples, such as waste
water and elutriates. However, harmonisation of the procedures was an objective as the two series of
documents were developed in parallel by the two organisations. The development of the freshwater test was
initiated by ISO in 1978. Three ring tests were organised between 1980 and 1982 and included in
ISO 8692:1989, revised as ISO 8692:2004. The first draft of a marine algae inhibition test was produced in
1982, but the first ISO/DIS was not published until 1991, when the method had been ring tested.
ISO 10253:1995 was revised as ISO 10253:2006. In addition to these two standards, ISO 14442:1999,
guidelines for algal growth inhibition tests with poorly soluble matter, volatile compounds, metals and waste
water, was revised as ISO 14442:2006. In this Technical Report, the general principles of the batch culture
growth inhibition tests, and how some critical methological aspects have been addressed in the International
Standards for algal growth inhibition tests, are presented.
TECHNICAL REPORT ISO/TR 11044:2008(E)
Water quality — Scientific and technical aspects of batch algae
growth inhibition tests
1 Scope
This Technical Report discusses scientific and technical aspects that have been considered in connection with
the development of batch algal growth inhibition test procedures specified in ISO 8692, for freshwater, and
ISO 10253, for marine waters.
Previously unpublished results of experiments performed at the Norwegian Institute for Water Research
(NIVA) have been included to demonstrate various aspects.
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 8692:2004, Water quality — Freshwater algal growth inhibition test with unicellular green algae
ISO 10253:2006, Water quality — Marine algal growth inhibition test with Skeletonema costatum and
Phaeodactylum tricornutum
ISO 14442, Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials, volatile
compounds, metals and waste water
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
effective concentration
EC
x
concentration of test sample which results in a reduction of x % in the specific growth rate relative to the
controls
[ISO 8692]
NOTE Unless otherwise stated, the form EC is used in this Technical Report to mean E C where “r” denotes “rate”.
x r x
Effective concentrations based on area under the growth curve can be derived, and these are designated E C , where “b”
b x
denotes “biomass” (see 6.5 for further details).
3.2
specific growth rate
µ
proportional rate of increase in cell density per unit of time:
1dn
µ =
ntd
where
n is the cell density, expressed in cells per millilitre;
t is the time, expressed in days.
NOTE 1 Specific growth rate is expressed in reciprocal days.
NOTE 2 Adapted from ISO 8692.
4 General principles of ISO algal growth inhibition tests
The algae growth inhibition test methods specified in ISO 8692 and ISO 10253 are based on batch cultures
which are inoculated with algae from an exponentially growing inoculum culture and incubated under
continuous illumination. The growth medium, inoculum biomass density, temperature, and illuminance, have
been selected to allow an exponential increase in the algal biomass density during the 72 h incubation period
for the recommended test species.
The experimental design of the tests includes a series of five or more concentrations of the test material in
growth medium prepared in triplicate, and six control replicates without test material. After inoculation with test
algae, the solutions are incubated in transparent, inert containers under continuous illumination and constant
temperature. The cultures should be agitated in order to obtain a homogenous suspension of the algae and to
stimulate gas exchange with the atmosphere. The biomass density in the cultures is measured by direct or
indirect methods at 24 h intervals until termination of the test after 72 h.
An example of a growth inhibition test with Pseudokirchneriella subcapitata is shown in Figure 1. The
substance tested was potassium dichromate. The growth curves show close adherence to exponential growth
in the cultures, and decreasing growth rates with increasing concentration of the test substance. Average
specific growth rates may be calculated as the logarithm
...
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