ISO 21716-3:2020

INTERNATIONAL ISO
STANDARD 21716-3
First edition
2020-11
Ships and marine technology —
Bioassay methods for screening anti-
fouling paints —
Part 3:
Mussels
Reference number
©
ISO 2020
© ISO 2020
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ii © ISO 2020 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Material and apparatus . 3
6 Preparation of the test organism and the test seawater . 4
6.1 General . 4
6.2 Preparation of the test organism . 4
6.3 Preparation of test seawater . 4
7 Preparation of the test panels and control panels . 4
7.1 General . 4
7.2 Preparation of the test panels . 4
7.3 Affixing of mussels to the test panels and control panels . 4
8 Operation of the test . 5
9 Validation of the test . 6
10 Byssus threads formation . 6
10.1 General . 6
10.2 Calculation of the average number of byssus threads formed . 7
10.3 Data treatment and interpretation of the results. 7
11 Test report . 8
Annex A (informative) Statistical analysis — Examples .10
Annex B (informative) General information of mussel, Mytilus galloprovincialis .14
Annex C (informative) Identification of Mytilus galloprovincialis .17
Bibliography .19
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 documents 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).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
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expressions related to conformity assessment, as well as information about ISO's adherence to
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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, in collaboration with Technical Committee
ISO/TC 35, Paints and varnishes, Subcommittee SC 9, General test methods for paints and varnishes.
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 © ISO 2020 – All rights reserved

Introduction
Anti-fouling paints that contain biocides are widely used to prevent fouling of ship hulls by marine
organisms. Effective anti-fouling technologies are critical for maintaining fuel consumption efficiency
of ships and also 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 or efficacy. For example, guidance published by the European Chemicals Agency (ECHA)
on the assessment and evaluation of efficacy for anti-fouling products states clearly that laboratory
testing of individual anti-fouling paints is not undertaken 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 those encountered when the antifouling products are in service is
routinely undertaken instead (see Reference [35]).
Whilst 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. ISO 21716 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 ISO 21716 can be used for such
purposes 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, ISO 21716
provides information on methods that can be used to screen anti-fouling paints in order to determine
whether to continue development of an experimental paint and/or a product 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.
ISO 21716 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 ISO 21716 can be used as part of a tiered
approach to predict the ability of an anti-fouling paint to prevent fouling on ships. Alternatively, 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 ISO 21716 do not reliably predict the ability of an anti-fouling paint to prevent fouling on ships
or the translocation of invasive marine species.
ISO 21716 is not intended to provide a list of validated tests for testing the efficacy of anti-fouling
paints; this can be covered in regulations. It is not intended to provide a list of validated tests for this
purpose, nor for predicting the ability of a fouling control paint to prevent fouling on ships or to prevent
the translocation of invasive marine species.
Mussels are typical marine sessile organisms regarded as harmful fouling organisms because of
the impact on fuel consumption and the potential for translocation of non-indigenous species if they
become attached to ship hulls.
This test method utilizes young mussels to assess settling behaviour in the presence of treated panels.
Young mussels are used because they have higher byssus threads production activity as compared to
the adults. More information is provided in Annex B.
vi © ISO 2020 – All rights reserved

INTERNATIONAL STANDARD ISO 21716-3:2020(E)
Ships and marine technology — Bioassay methods for
screening anti-fouling paints —
Part 3:
Mussels
1 Scope
This document specifies a laboratory test method for screening anti-fouling paints in a flow-through
system using mussels as the test organism. It is intended to be used in conjunction with ISO 21716-1,
which specifies the general requirements. The purpose of the test is to determine if there is a difference
in mussel settlement on painted test panels compared with mussel settlement on inert non-toxic control
panels under the conditions of the test. Examples of statistical analysis to determine if the difference in
mussel settlement is statistically significant are given in Annex A.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 21716-1:2020, Ships and marine technology — Bioassay methods for screening anti-fouling paints —
Part 1: General requirements
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 21716-1 and the following apply.
ISO
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