ISO 21716-4:2025
(Main)Ships and marine technology — Bioassay methods for screening anti-fouling paints — Part 4: Algae
Ships and marine technology — Bioassay methods for screening anti-fouling paints — Part 4: Algae
This document specifies the laboratory test method for screening anti-fouling paints in a flow-through system using algae as the test organism. It is intended to be used in conjunction with ISO 21716-1, which specifies the general requirements of the test methods described in the other parts of the ISO 21716 series. The purpose of the test specified in this document is to determine the difference in colour of algae on painted test panels compared with algae on inert non-toxic control panels under the test conditions.
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International
Standard
ISO 21716-4
First edition
Ships and marine technology —
2025-08
Bioassay methods for screening
anti-fouling paints —
Part 4:
Algae
Reference number
© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Test procedure . 2
5 Materials and apparatus . 2
6 Preparation of test organism and test seawater . 3
6.1 General .3
6.2 Preparation of test organism . .3
6.3 Preparation of test seawater . .4
7 Preparation of test/control panel . . 4
7.1 General .4
7.2 Preparation of test panels .4
7.3 Affixing algae on the substrates .5
8 Algae viability test . 5
8.1 General .5
8.2 Requirements .5
8.3 Operation of the algae viability test .6
9 Bioassay . 7
9.1 General .7
9.2 Operation .7
10 Colour space . 8
10.1 General .8
10.2 Calculation of the average value of the parameters of colour space .9
10.3 Data treatment and interpretation of the results .10
11 Test report . 10
Annex A (informative) Statistical analysis .12
Annex B (informative) Preparation of test organism.15
Annex C (informative) General information on macroalga, Ectocarpus sp. .16
Bibliography . 19
iii
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.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 8, Ships and marine technology, Subcommittee
SC 2, Marine environment protection.
A list of all parts in the ISO 21716 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
Introduction
Anti-fouling paints that contain biocides are widely used to prevent the fouling of ship hulls by marine
organisms. Effective anti-fouling technologies are critical for maintaining the fuel consumption efficiency of
ships and for minimizing possible translocation of aquatic species through maritime trade. The evaluation
of anti-fouling paints is generally undertaken by adopting a tiered approach whereby paint manufacturers
use a battery of laboratory, raft, patch tests and full vessel trials. Raft, patch tests and full vessel trials are
generally conducted over extended periods of time and are predominantly relied upon for the prediction of
coating performance when used commercially on in-service ships.
The results of raft, patch test and full vessel trials (field testing) can be used as part of the regulatory
process for pesticidal or biocidal products in certain countries in order to demonstrate the efficacy of an
anti-fouling paint. Laboratory testing alone is recognized as being unable to predict in-service performance
[1]
or efficacy. For example, guidance published by the European Chemical Agency (ECHA) on the assessment
and evaluation of the efficacy of anti-fouling products states clearly that laboratory testing of individual
anti-fouling paints is not performed as it is not considered to be a realistic evaluation of the product. Field
testing, which permits anti-fouling products to be tested under similar operating conditions and stresses as
[1]
those encountered when the anti-fouling products are in service, is routinely undertaken instead.
Although laboratory tests are unable to reliably predict in-service coating performance, they have merit in
the screening of experimental coatings for further evaluation during the research and development process.
Reproducible objective data obtained by following standardized screening methods, independent of the
test location or the season, can be a useful tool to support the selection of anti-fouling paints for higher
tier testing, e.g. raft or ship tests. The ISO 21716 series provides a compilation and description of in vitro
bioassay methods intended to aid the process of screening anti-fouling paints prior to higher tier raft or
ship tests. Toxicological screening methods included in each part of the ISO 21716 series can be used for
purposes such as early decision-making in research and product development, rapid feedback on potential
toxicological concerns, or for the preliminary assessment of anti-fouling paints. For instance, the ISO 21716
series provides information on methods that can be used to screen anti-fouling paints in order to determine
whether to continue development of either an experimental paint or a product, or both, that contains a
particular ingredient, or to determine whether to take on the cost of performing the remaining tiers within
a complete tiered-testing strategy.
The ISO 21716 series provides screening bioassays related to certain common genera of fouling organisms,
namely barnacles, mussels and algae. These screening tests are relatively simple and rapid laboratory
tests that can be performed to provide an indication of the toxicity of a painted surface towards selected
test organisms. The screening tests described in each part of the ISO 21716 series can be used as part of
a tiered approach to predict the ability of an anti-fouling paint to prevent fouling on ships. Alternatively,
these screening tests can be used to prevent the translocation of invasive marine species by progressively
involving subsequent semi-field (e.g. raft panels) and field testing (e.g. ship trials). On their own, the
screening tests described in each part of the ISO 21716 series do not reliably predict the ability of an anti-
fouling paint to prevent fouling on ships or the translocation of invasive marine species.
The ISO 21716 series is not intended to provide a list of validated tests for testing the efficacy of anti-fouling
paints; this can be covered by applicable legal regulations in each country or region. It is also not intended
to be used for predicting the ability of a fouling control paint to prevent fouling on ships or to prevent the
translocation of invasive marine species.
Algae are typical marine sessile organisms regarded as harmful fouling organisms because of their impact
on fuel consumption and the potential for translocation of non-indigenous species if they become attached
to ship hulls.
The test method specified in this document utilizes algae to asses
...
International
Standard
ISO 21716-4
First edition
Ships and marine technology —
Bioassay methods for screening
anti-fouling paints —
Part 4:
Algae
PROOF/ÉPREUVE
Reference number
© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
PROOF/ÉPREUVE
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Test procedure . 2
5 Materials and apparatus . 2
6 Preparation of test organism and test seawater . 3
6.1 General .3
6.2 Preparation of test organism . .3
6.3 Preparation of test seawater . .4
7 Preparation of test/control panel . . 4
7.1 General .4
7.2 Preparation of test panels .4
7.3 Affixing algae on the substrates .5
8 Algae viability test . 5
8.1 General .5
8.2 Requirements .5
8.3 Operation of the algae viability test .6
9 Bioassay . 7
9.1 General .7
9.2 Operation .7
10 Colour space . 8
10.1 General .8
10.2 Calculation of the average value of the parameters of colour space .9
10.3 Data treatment and interpretation of the results .10
11 Test report . 10
Annex A (informative) Statistical analysis .12
Annex B (informative) Preparation of test organism.15
Annex C (informative) General information on macroalga, Ectocarpus sp. .16
Bibliography . 19
PROOF/ÉPREUVE
iii
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.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 8, Ships and marine technology, Subcommittee
SC 2, Marine environment protection.
A list of all parts in the ISO 21716 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
PROOF/ÉPREUVE
iv
Introduction
Anti-fouling paints that contain biocides are widely used to prevent the fouling of ship hulls by marine
organisms. Effective anti-fouling technologies are critical for maintaining the fuel consumption efficiency of
ships and for minimizing possible translocation of aquatic species through maritime trade. The evaluation
of anti-fouling paints is generally undertaken by adopting a tiered approach whereby paint manufacturers
use a battery of laboratory, raft, patch tests and full vessel trials. Raft, patch tests and full vessel trials are
generally conducted over extended periods of time and are predominantly relied upon for the prediction of
coating performance when used commercially on in-service ships.
The results of raft, patch test and full vessel trials (field testing) can be used as part of the regulatory
process for pesticidal or biocidal products in certain countries in order to demonstrate the efficacy of an
anti-fouling paint. Laboratory testing alone is recognized as being unable to predict in-service performance
[1]
or efficacy. For example, guidance published by the European Chemical Agency (ECHA) on the assessment
and evaluation of the efficacy of anti-fouling products states clearly that laboratory testing of individual
anti-fouling paints is not performed as it is not considered to be a realistic evaluation of the product. Field
testing, which permits anti-fouling products to be tested under similar operating conditions and stresses as
[1]
those encountered when the anti-fouling products are in service, is routinely undertaken instead.
Although laboratory tests are unable to reliably predict in-service coating performance, they have merit in
the screening of experimental coatings for further evaluation during the research and development process.
Reproducible objective data obtained by following standardized screening methods, independent of the
test location or the season, can be a useful tool to support the selection of anti-fouling paints for higher
tier testing, e.g. raft or ship tests. The ISO 21716 series provides a compilation and description of in vitro
bioassay methods intended to aid the process of screening anti-fouling paints prior to higher tier raft or
ship tests. Toxicological screening methods included in each part of the ISO 21716 series can be used for
purposes such as early decision-making in research and product development, rapid feedback on potential
toxicological concerns, or for the preliminary assessment of anti-fouling paints. For instance, the ISO 21716
series provides information on methods that can be used to screen anti-fouling paints in order to determine
whether to continue development of either an experimental paint or a product, or both, that contains a
particular ingredient, or to determine whether to take on the cost of performing the remaining tiers within
a complete tiered-testing strategy.
The ISO 21716 series provides screening bioassays related to certain common genera of fouling organisms,
namely barnacles, mussels and algae. These screening tests are relatively simple and rapid laboratory
tests that can be performed to provide an indication of the toxicity of a painted surface towards selected
test organisms. The screening tests described in each part of the ISO 21716 series can be used as part of
a tiered approach to predict the ability of an anti-fouling paint to prevent fouling on ships. Alternatively,
these screening tests can be used to prevent the translocation of invasive marine species by progressively
involving subsequent semi-field (e.g. raft panels) and field testing (e.g. ship trials). On their own, the
screening tests described in each part of the ISO 21716 series do not reliably predict the ability of an anti-
fouling paint to prevent fouling on ships or the translocation of invasive marine species.
The ISO 21716 series is not intended to provide a list of validated tests for testing the efficacy of anti-fouling
paints; this can be covered by applicable legal regulations in each country or region. It is also not intended
to be used for predicting the ability of a fouling control paint to prevent fouling on ships or to prevent the
translocation of invasive marine species.
Algae are typical marine sessile organisms regarded as harmful fouling organisms because of their impact
on fuel consumption and the potential for translocation of non-indigenous species if they become attached
to ship hulls.
The test method specifie
...
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ISO 21716--4:2025(en)
cm, First line: 0 cm, Tab stops: Not at 0.76 cm
ISO/TC 8/SC 2
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Secretariat: ANSI
Date: 2025-04-3006-11 Style Definition: Heading 3: Font: Bold
Ships and marine technology — Bioassay methods for screening anti-fouling paints — Part 4: Style Definition: Heading 4: Font: Bold
Algae
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Style Definition: ANNEX
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Formatted: Font: Not Bold, English (United
Kingdom)
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no
part of this publication may be reproduced or utilized otherwise in any form or by any means,
electronic or mechanical, including photocopying, or posting on the internet or an intranet, without
prior written permission. Permission can be requested from either ISO at the address below or
ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.orgwww.iso.org Formatted: English (United Kingdom)
Formatted: English (United Kingdom)
Published in Switzerland
Formatted: Font: Bold
ii
Formatted: Font: Not Bold, English (United
Kingdom)
Contents
Foreword . v
Introduction. vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Test procedure . 2
5 Materials and apparatus . 2
6 Preparation of test organism and test seawater . 4
6.1 General . 4
6.2 Preparation of test organism . 4
6.3 Preparation of test seawater . 4
7 Preparation of test / control panel . 4
7.1 General . 4
7.2 Preparation of test panels . 4
7.3 Affixing algae on the substrates . 5
8 Algae viability test . 5
8.1 General . 5
8.2 Requirements of the algae viability test . 5
8.3 Operation of the algae viability test. 6
9 Bioassay . 7
9.1 General . 7
9.2 Operation of bioassay . 7
10 Colour space . 9
10.1 General . 9
10.2 Calculation of the average value of the parameters of colour space . 9
10.3 Data treatment and interpretation of the results . 11
11 Test report . 11
Annex A (informative) Statistical analysis . 13
A.1 Introduction . 13
A.2 Statistical analysis between groups. 13
A.2.1 Introduction . 13
A.2.2 One-way analysis of variance among statistical groups . 13
A.2.3 Example of one-way ANOVA . 14
A.2.3.1 Introduction . 14
A.2.3.2 Analytical method . 14
A.2.3.3 Results . 15
Annex B (informative) Preparation of test organism . 16
B.1 Collection of algae . 16
Formatted: Font: Bold
B.2 Culturing algae . 16
iii
Formatted: Font: Not Bold, English (United
Kingdom)
Annex C (informative) General information of macroalga, Ectocarpus sp. . 17
C.1 Introduction . 17
C.2 Life cycle of Ectocarpus sp. . 18
C.3 Identification method . 18
Bibliography . 19
Foreword . v
Introduction. vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Test procedure . 3
5 Materials and apparatus . 4
6 Preparation of test organism and test seawater . 5
6.1 General . 5
6.2 Preparation of test organism . 5
6.3 Preparation of test seawater . 5
7 Preparation of test/control panel . 6
7.1 General . 6
7.2 Preparation of test panels . 6
7.3 Affixing algae on the substrates . 7
8 Algae viability test . 8
8.1 General . 8
8.2 Requirements . 8
8.3 Operation of the algae viability test. 9
9 Bioassay . 10
9.1 General . 10
9.2 Operation . 11
10 Colour space . 12
10.1 General . 12
10.2 Calculation of the average value of the parameters of colour space . 13
10.3 Data treatment and interpretation of the results . 16
11 Test report . 16
Annex A (informative) Statistical analysis . 18
Annex B (informative) Preparation of test organism . 23
Annex C (informative) General information on macroalga, Ectocarpus sp. . 24
Bibliography . 28
Formatted: Font: Bold
iv
Formatted: Font: Not Bold, English (United
Kingdom)
Formatted: Don't adjust space between Latin and
Foreword
Asian text, Don't adjust space between Asian text
and numbers
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
o
...
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