Ships and marine technology -- Bioassay methods for screening anti-fouling paints

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.

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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 21716-3:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 21716-3:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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 © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 21716-3:2020(E)
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

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 21716-3:2020(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 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).

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, 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
---------------------- Page: 4 ----------------------
ISO 21716-3:2020(E)
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.
© ISO 2020 – All rights reserved v
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ISO 21716-3:2020(E)

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
---------------------- Page: 6 ----------------------
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 and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp;

— I EC E le c t r op e d i a : av a i l able at ht t p:// w w w . ele c t r op e d i a . or g / .

3.1
byssus

attachment organ secreted by a mussel, consisting of stem, byssus threads and adhesive discs

Note 1 to entry: See Figure 1.
© ISO 2020 – All rights reserved 1
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ISO 21716-3:2020(E)
Key
1 foot
2 root (in byssus gland)
3 stem
4 byssus thread
5 adhesive disc (or plaque)
Figure 1 — Attachment organ of a mussel
3.2
shell length

longest linear distance between two points on the outside edge of the shell of a mussel

3.3
purified water

water with an electric conductivity of 2 µS/cm or less, prepared by distillation and/or treatment with

ion exchange resin(s)
4 Principle

The test procedure consists of the following 5 sequential steps, summarized in Figure 2:

— preparation of the test organism and the test seawater;
— preparation of the test panel and control panel;
— operation of the test;
— validation of the test; and
— data treatment and interpretation of the results.
2 © ISO 2020 – All rights reserved
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ISO 21716-3:2020(E)
Figure 2 — Schema of the test procedure

Each bioassay shall consist of three runs at a minimum. Each run shall consist of a test group of three

or more test panels and a control group of three or more control panels. Provided that the validation

criteria are met, then the number of byssus threads for the test and control groups can be compared.

5 Material and apparatus
The items listed in Tables 1 and 2 shall be used for the test.
Table 1 — List of material used
Material Remarks
Adhesive of surgical grade E.g., cyano-acrylate adhesive
Control panels 50 mm square of PVC is recommended.

Cultured stock of live young At least 30 live mussels, Mytilus galloprovincialis sp., with a shell length of

mussels 8 mm to 10 mm, that are attached to natural or artificial substrates.
Other mussel species may be used if Mytilus galloprovincialis sp. mussels are
not available.
Natural seawater Defined in ISO 21716-1:2020, 3.8
Purified water Defined in 3.3
Scissors
Small piece of paper Filter paper may be used as spacer.
Test panels Specified in ISO 21716-1:2020, 4.2. 50 mm square is recommended.
1 µm filters Used to prepare test seawater.
Table 2 — List of apparatus used
Apparatus Remarks
Light White fluorescence or LED
Light intensity meter Accuracy: ±10 lx
© ISO 2020 – All rights reserved 3
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ISO 21716-3:2020(E)
Table 2 (continued)
Apparatus Remarks
pH meter Accuracy: ±0,1
Salinometer Accuracy: ±0,1
Stereo microscope Magnification: 5-30X with fiber light
Thermometer Accuracy: ±0,1 °C

Water flow-through system Specified in ISO 21716-1:2020, 5.2, with a means of maintaining the test sea-

water tank at 20 °C ± 1 °C and alternately illuminating the test seawater tank

with a light intensity of 3 000 lx [Clause 8, d), light conditions] and with a light

intensity of <5 lx [see Clause 8, d), dark conditions].
6 Preparation of the test organism and the test seawater
6.1 General

The cultured stock of live mussels is used to perform the bioassay test in sea water.

6.2 Preparation of the test organism

Live mussels are generally prepared by collecting wild mussels and acclimatizing them in the laboratory

prior to testing. Guidance on this process and on storing mussels can be found in Annex B.

Information on the life cycle of mussels can also be found in Annex B, and information on the

identification of M. galloprovincialis sp. mussels can be found in Annex C.
6.3 Preparation of test seawater

Pass natural seawater through a 1 µm filter unit and adjust to salinity 30,0 ± 0,5 using purified water.

7 Preparation of the test panels and control panels
7.1 General
The same test and control groups shall be used throughout the whole test.
7.2 Preparation of the test panels

Test panels shall be prepared following the specifications described in ISO 21716-1:2020, Clause 4.

7.3 Affixing of mussels to the test panels and control panels

Separate the live mussels from their substrate by cutting the byssus threads with scissors, taking care

to avoid damage to other tissue and organs. Affix five mussels to each test with the adhesive, using

filter paper as a spacer between the shell of the mussel and the surface of the panel, providing a 20 mm

diameter circular separation zone around each mussel [see Figure 3 a)].

NOTE A spacer of adhesive-infiltrated filter paper can be used to prevent excessive spreading of the adhesive

and improve adhesion of the mussel to the test and control panels [see Figure 3 b)].

4 © ISO 2020 – All rights reserved
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ISO 21716-3:2020(E)
a) Top view
b) Side view
Key
1 mussel
2 test/ control panel
3 spacer of adhesive-infiltrated filter paper
Figure 3 — The mussels on the test or control panel
8 Operation of the test

The bioassay shall be simultaneously performed on the test group and the control group as follows (see

Figure 4).

The experimental system specified in ISO 21716-1 shall be used for the test. The system is equipped

with the devices that maintain the specified water temperature and light irradiation of the test.

a) Fill the test seawater tank with test seawater and provide a continuous flow of test seawater from

the seawater storage tank. Maintain the temperature of the test seawater at 20 °C ± 1 °C for the

duration of the test. The flow rate should be set to achieve at least about 0,8 turnover per hour of

the water of test seawater tank.

NOTE If the flow rate is too low, the result can be affected by the concentration of biocide in seawater of

the test seawater tank.

b) Place the test and control panels in the test seawater tanks, ensuring all the panels are fully

immersed in the test seawater.

c) Measure and record the temperature, pH and salinity. Measure and record those parameters again

after 24 h from the beginning of the test.

d) Illuminate the test seawater tank for 12 h with a light intensity of 3 000 lx, and then leave the test

seawater tank in darkness for 12 h.
© ISO 2020 – All rights reserved 5
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ISO 21716-3:2020(E)

e) Carefully remove the test and control panels from the test seawater tanks after 24 h from the

beginning of the test.

f) Using a stereo-microscope, count the number of byssus threads for each live and attached mussel

on each test and control panel, and record the results using e.g. Table 3.

Count the number of dead or detached mussels on each test and control panel, and record the results

using e.g. Table 3.
Figure 4 — Flow chart of the procedure for the test
9 Validation of the test

The results of the bioassay are validated by assessing if the results of the test meet the following four

criteria.

The results of the bioassay shall only be considered valid if all four criteria are met.

— The average number of byssus threads formed by living individual in the control panels in each run

is ten or more.
— The mortality of the mussels in the test panels in each run is 10 % or less.

— The mortality of the mussels in the control panels in each run is 10 % or less.

— The percentage of individuals detached from the test surface in each run is 5 or less.

10 Byssus threads formation
10.1 General

The evaluation procedure for screening anti-fouling paint is given in 10.2 and 10.3. The anti-fouling

paint is evaluated by comparing the number of byssus threads between the test and control groups.

The test data should be recorded using e.g. Table 4.
6 © ISO 2020 – All rights reserved
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ISO 21716-3:2020(E)
10.2 Calculation of the average number of byssus threads formed

Calculate the average number of byssus threads formed by the mussels on the control group using

Formula (1) and record the results using e.g. Table 3 to one decimal place.
()NN++N
∑ cn
cc12
j=1
A = (1)
j=1
where
A is the average number of byssus threads in the control groups;
j is the run number;

is the number of byssus threads formed by living individual mussel attaching on the control

panels on the j-th run;

is the number of living mussels attaching on the control panels on the j-th run.

Calculate the average number of byssus threads formed by the mussels on the test group using

Formula (2) and record the results using e.g. Table 3 to one decimal place.
jj j
()NN++N
∑ tt12 tn
j=1
A = (2)
j=1
where
A is the average number of byssus threads in the test groups;
j is the run number;

is the number of byssus threads formed by living individual attaching on the test panels on

the j-th run;
is the number of living mussels attaching on the test panels on the j-th run.
10.3 Data treatment and interpretation of the results

If A is less than A , this can indicate that the byssus settlement on the test group is less than that of the

t c

control group. However, further analysis of the results is required in order to determine if the difference

of the result between test and control groups is statistically significant. There are many possible ways

to perform the statistical analysis. Typical examples are shown in Annex A.
© ISO 2020 – All rights reserved 7
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ISO 21716-3:2020(E)
Table 3 — Example of compilation of the data
Number of byssus threads Number
Number of
of dead de-
Sample name
Mussel Mussel Mussel Mussel Mussel Subto- Aver-
mussel tached
Total
1 2 3 4 5 tal age
(%) mussel
(%)
Control panel 1 25 22 36 20 18 121
Control panel 2 21 31 11 34 19 116 341 22,73
(0,00)
Control panel 3 15 14 25 20 30 104
Run
Test panel 1 10 4 5 10 * 29
(3,3)
Test panel 2 12 5 8 10 5 40 112 8,00
(0,00)
Test panel 3 7 9 3 9 15 43
Control panel 1 25 17 * 20 31 93
Control panel 2 34 22 26 14 13 109 270 19,28
(0,00)
Control panel 3 15 15 8 15 15 68
Run
Test panel 1 8 * 4 8 3 23
(3,3)
Test panel 2 8 4 9 0 3 24 72 5,14
(6,7)
Test panel 3 2 2 8 11 2 25
Control panel 1 16 12 30 22 23 103
Control panel 2 * 8 14 10 22 54 241 18,54
(6,7)
Control panel 3 16 15 41 12 * 84
Run
Test panel 1 3 1 4 6 0 14
(3,3)
Test panel 2 6 * 2 4 2 14 42 3,00
(6,7)
Test panel 3 1 3 2 4 4 14
* Dead or detached mussel (excluded from the table).
Results:
A : 20,2
A : 5,4
11 Test report

Table 4 specifies the minimum required information for the test report. The test results shall be

reported using Table 4.
Table 4 — Minimum required information for the test report
Information Requirement
Materials and size of substrate to be painted ×
General specifications Biocides contained ×
and process for paint
Name of paint ×
Undercoat (×)
Surface treatment (×)
Dry film thickness ×
Methods and time (number of days) for aging test panels ×
x: required; (x): optional.
Information required for each run.
8 © ISO 2020 – All rights reserved
---------------------- Page: 14 ----------------------
ISO 21716-3:2020(E)
Table 4 (continued)
Information Requirement
General information on Identification (species name and identifier) ×
the test organisms
Date of sampling/collection (×)
Place of sampling/collection (×)
Water quality parameters at the time of sampling (temperature, ×
salinity, pH)
Acclimatization condition of the mussel (temperature, salinity, pH, ×
light condition, acclimatization period, survival rate)
Initial test condition Starting date ×
Number of test and control panels ×
Size of the test seawater tank ×
Water quality parameters (temperature, pH, salinity), light ×
condition, rate of water exchange
Other information on the test procedure and test device (×)
Test condition Water quality (temperature, pH, salinity), light condition ×
after 24 h
The mortality of mussels in the test surface, the percentage of ×
individuals detached from the test surface
Accidental/unexpected items observed during the test (×)
Average number of byssus threads ×
Statistical analysis method used (×)

Is there a statistically significant difference between the bioassay results for the test and (×)

control groups, Yes/No?
x: required; (x): optional.
Information required for each run.
© ISO 2020 – All rights reserved 9
---------------------- Page: 15 ----------------------
ISO 21716-3:2020(E)
Annex A
(informative)
Statistical analysis — Examples
A.1 Introduction

It is stated in the Scope that 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. It can be useful to perform statistical analysis to determine if the

difference in mussel settlement is statistically significant. A typical example of statistical analysis was

given in this Annex, based on Reference [1]. Selection of an optimal statistical analysis method from

different approaches should be made depending on the purpose and the data distributions. This Annex

describes some statistical analysis methods as examples.
A.2 Statistical analysis to compare a test group and a control group
A.2.1 Introduction

This clause indicates some statistical analysis methods to calculate the difference between a control

group and a test group as examples. Figure A.1 shows an example for this process. Statistical analysis

of significance difference between the control and test groups is conducted using a one-way ANOVA

(analysis of variance) followed by a multiple comparison test. In this case, Dunnett’s multiple comparison

test is recommended for comparing several treatments with the control groups (Reference [2]).

Probability values (p-value) less than 0,05 are considered significant.
10 © ISO 2020 – All rights reserved
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ISO 21716-3:2020(E)
Figure A.1 — Example of a statistical analysis process
[3]–[7]
A.2.2 One-way analysis of variance

One-way analysis of variance (ANOVA) is a collection of statistical models and their associated

estimation procedures (such as the "variation" among and between groups) used to analyse the

differences among group means in a sample. The ANOVA is based on the law of total variance, where

the observed variance in a particular variable is partitioned into components attributable to different

sources of variation. In its simplest form, ANOVA provides a statistical test of whether two or more

population means are equal, and therefore generalizes the t-test beyond two means.

The one-way ANOVA test is a way to find out if survey or experiment results are significant. In other

words, they help to figure out the need to reject the null hypothesis or to accept the alternate hypothesis.

Bas
...

INTERNATIONAL ISO
STANDARD 21716-3
First edition
Ships and marine technology —
Bioassay methods for screening anti-
fouling paints —
Part 3:
Mussels
PROOF/ÉPREUVE
Reference number
ISO 21716-3:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 21716-3:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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 PROOF/ÉPREUVE © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 21716-3:2020(E)
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

© ISO 2020 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO 21716-3:2020(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 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).

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 environmental 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.
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ISO 21716-3:2020(E)
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 Chemical 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 product 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 coating 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 coating 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; 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.
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ISO 21716-3:2020(E)

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.
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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 and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp;

— I EC E le c t r op e d i a : av a i l able at ht t p:// w w w . ele c t r op e d i a . or g / .

3.1
byssus

attachment organ secreted by a mussel, consisting of stem, byssus threads and adhesive discs

Note 1 to entry: See Figure 1.
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ISO 21716-3:2020(E)
Key
1 foot
2 root (in byssus gland)
3 stem
4 byssus thread
5 adhesive disc (or plaque)
Figure 1 — Attachment organ of a mussel
3.2
shell length

longest linear distance between two points on the outside edge of the shell of a mussel

3.3
purified water

water with an electric conductivity of 2 µS/cm or less, prepared by distillation and/or treatment with

ion exchange resin(s)
4 Principle

The test procedure consists of the following 5 sequential steps, summarized in Figure 2:

— preparation of the test organism and the test seawater;
— preparation of the test panel and control panel;
— operation of the test;
— validation of the test; and
— data treatment and interpretation of the results.
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ISO 21716-3:2020(E)
Figure 2 — Scheme of the test procedure

Each bioassay shall consist of three runs as a minimum. Each run shall consist of a test group of three

or more test panels and a control group of three or more control panels. Provided that the validation

criteria are met, then the number of byssus threads for the test and control groups can be compared.

5 Material and apparatus
The items listed in Tables 1 and 2 shall be used for the test.
Table 1 — List of material used
Material Remarks
Adhesive of surgical grade E.g., cyano-acrylate adhesive.
Control panels 50 mm square of PVC is recommended.

Cultured stock of live young At least 30 live mussels, Mytilus galloprovincialis sp., with a shell length of

mussels 8 mm to 10 mm, that are attached to natural or artificial substrates.
Other mussel species may be used if Mytilus galloprovincialis sp. mussels are
not available.
Natural seawater Defined in ISO 21716-1:2020, 3.8.
Purified water Defined in 3.3.
Scissors
Small piece of paper Filter paper may be used as spacer.
Test panels Specified in ISO 21716-1:2020, 4.2. 50 mm square is recommended.
1 µm filters Used to prepare test seawater.
Table 2 — List of apparatus used
Apparatus Remarks
Light White fluorescence or LED.
Light intensity meter Accuracy: ±10 lx.
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ISO 21716-3:2020(E)
Table 2 (continued)
Apparatus Remarks
pH meter Accuracy: ±0,1.
Salinometer Accuracy: ±0,1.
Stereo microscope Magnification: 5-30X with fiber light.
Thermometer Accuracy: ±0,1 °C.

Water flow-through system Specified in ISO 21716-1:2020, 5.2, with a means of maintaining the test sea-

water tank at 20 °C ± 1 °C and alternately illuminating the test seawater tank

with a light intensity of 3 000 lx [Clause 8, d), light conditions] and with a light

intensity of <5 lx [see Clause 8, d), dark conditions].
6 Preparation of the test organism and the test seawater
6.1 General

The cultured stock of live mussels is used to perform the bioassay test in sea water.

6.2 Preparation of the test organism

Live mussels are generally prepared by collecting wild mussels and acclimatizing them in the laboratory

prior to testing. Guidance on this process and on storing mussels can be found in Annex B.

Information on the life cycle of mussels can also be found in Annex B, and information on the

identification of M. galloprovincialis sp. mussels can be found in Annex C.
6.3 Preparation of test seawater

Pass natural seawater through a 1 µm filter unit and adjust to salinity 30,0 ± 0,5 using purified water.

7 Preparation of the test panels and control panels
7.1 General
The same test and control groups shall be used throughout the whole test.
7.2 Preparation of the test panels

Test panels shall be prepared following the specifications described in ISO 21716-1:2020, Clause 4.

7.3 Affixing of mussels to the test panels and control panels

Separate the live mussels from their substrate by cutting the byssus threads with scissors, taking care

to avoid damage to other tissue and organs. Affix five mussels to each test with the adhesive, using

filter paper as a spacer between the shell of the mussel and the surface of the panel, providing a 20 mm

diameter circular separation zone around each mussel [see Figure 3 a)].

NOTE A spacer of adhesive-infiltrated filter paper can be used to prevent excessive spreading of the adhesive

and improve adhesion of the mussel to the test and control panels [see Figure 3 b)].

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ISO 21716-3:2020(E)
a) Top view
b) Side view
Key
1 mussel
2 test/ control panel
3 spacer of adhesive-infiltrated filter paper
Figure 3 — The mussels on the test or control panel
8 Operation of the test

The bioassay shall be simultaneously performed on the test group and the control group as follows (see

Figure 4).

The experimental system specified in ISO 21716-1 shall be used for the test. The system is equipped

with the devices that maintain the specified water temperature and light irradiation of the test.

a) Fill the test seawater tank with test seawater and provide a continuous flow of test seawater from

the seawater storage tank. Maintain the temperature of the test seawater at 20 °C ± 1 °C for the

duration of the test. The flow rate should be set to achieve at least about 0,8 turnover per hour of

the water of test seawater tank.

NOTE If the flow rate is too low, the result can be affected by the concentration of biocide in seawater of

the test seawater tank.

b) Place the test and control panels in the test seawater tanks, ensuring all the panels are fully

immersed in the test seawater.

c) Measure and record the temperature, pH and salinity. Measure and record those parameters again

after 24 h from the beginning of the test.

d) Illuminate the test seawater tank for 12 h with a light intensity of 3 000 lx, and then leave the test

seawater tank in darkness for 12 h.
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ISO 21716-3:2020(E)

e) Carefully remove the test and control panels from the test seawater tanks after 24 h from the

beginning of the test.

f) Using a stereo-microscope, count the number of byssus threads for each live and attached mussel

on each test and control panel, and record the results using e.g. Table 3.

Count the number of dead or detached mussels on each test and control panel, and record the results

using e.g. Table 3.
Figure 4 — Flow chart of the procedure for the test
9 Validation of the test

The results of the bioassay are validated by assessing if the results of the test meet the following four

criteria.

The results of the bioassay shall only be considered valid if all four criteria are met.

— The average number of byssus threads formed by living individual in the control panels in each run

is ten or more.
— The mortality of the mussels in the test panels in each run is 10 % or less.

— The mortality of the mussels in the control panels in each run is 10 % or less.

— The percentage of individuals detached from the test surface in each run is 5 or less.

10 Byssus threads formation
10.1 General

The evaluation procedure for screening anti-fouling paint is given in 10.2 and 10.3. The anti-fouling

paint is evaluated by comparing the number of byssus threads between the test and control groups.

The test data should be recorded using e.g. Table 4.
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ISO 21716-3:2020(E)
10.2 Calculation of the average number of byssus threads formed

Calculate the average number of byssus threads formed by the mussels on the control group using

Formula (1) and record the results using e.g. Table 3 to one decimal place.
()NN++N
∑ cn
cc12
j=1
A = (1)
j=1
where
A is the average number of byssus threads in the control groups;
j is the run number;

is the number of byssus threads formed by living individual mussel attaching on the control

panels on the j-th run;

is the number of living mussels attaching on the control panels on the j-th run.

Calculate the average number of byssus threads formed by the mussels on the test group using

Formula (2) and record the results using e.g. Table 3 to one decimal place.
jj j
()NN++N
∑ tt12 tn
j=1
A = (2)
j=1
where
A is the average number of byssus threads in the test groups;
j is the run number;

is the number of byssus threads formed by living individual attaching on the test panels on

the j-th run;
is the number of living mussels attaching on the test panels on the j-th run.
10.3 Data treatment and interpretation of the results

If A is less than A , this can indicate that the byssus settlement on the test group is less than that of the

t c

control group. However, further analysis of the results is required in order to determine if the difference

of the result between test and control groups is statistically significant. There are many possible ways

to perform the statistical analysis. Typical examples are shown in Annex A.
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ISO 21716-3:2020(E)
Table 3 — Example of compilation of the data
Number of byssus threads Number
Number of
of dead de-
Sample name
Mussel Mussel Mussel Mussel Mussel Subto- Aver-
mussel tached
Total
1 2 3 4 5 tal age
(%) mussel
(%)
Control panel 1 25 22 36 20 18 121
Control panel 2 21 31 11 34 19 116 341 22,73
(0,00)
Control panel 3 15 14 25 20 30 104
Run
Test panel 1 10 4 5 10 * 29
(3,3)
Test panel 2 12 5 8 10 5 40 112 8,00
(0,00)
Test panel 3 7 9 3 9 15 43
Control panel 1 25 17 * 20 31 93
Control panel 2 34 22 26 14 13 109 270 19,28
(0,00)
Control panel 3 15 15 8 15 15 68
Run
Test panel 1 8 * 4 8 3 23
(3,3)
Test panel 2 8 4 9 0 3 24 72 5,14
(6,7)
Test panel 3 2 2 8 11 2 25
Control panel 1 16 12 30 22 23 103
Control panel 2 * 8 14 10 22 54 241 18,54
(6,7)
Control panel 3 16 15 41 12 * 84
Run
Test panel 1 3 1 4 6 0 14
(3,3)
Test panel 2 6 * 2 4 2 14 42 3,00
(6,7)
Test panel 3 1 3 2 4 4 14
* Dead or detached mussel (excluded from the table).
Results:
A : 20,2
A : 5,4
11 Test report

Table 4 specifies the minimum required information for the test report. The test results shall be

reported using Table 4.
Table 4 — Minimum required information for the test report
Information Requirement
Materials and size of substrate to be painted ×
General specifications Biocides contained ×
and process for paint
Name of paint ×
Undercoat (×)
Surface treatment (×)
Dry film thickness ×
Methods and time (number of days) for aging test panels ×
x: required; (x): optional.
Information required for each run.
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ISO 21716-3:2020(E)
Table 4 (continued)
Information Requirement
General information on Identification (species name and identifier) ×
the test organisms
Date of sampling/collection (×)
Place of sampling/collection (×)
Water quality parameters at the time of sampling (temperature, ×
salinity, pH)
Acclimatization condition of the mussel (temperature, salinity, pH, ×
light condition, acclimatization period, survival rate)
Initial test condition Starting date ×
Number of test and control panels ×
Size of the test seawater tank ×
Water quality parameters (temperature, pH, salinity), light ×
condition, rate of water exchange
Other information on the test procedure and test device (×)
Test condition Water quality (temperature, pH, salinity), light condition ×
after 24 h
The mortality of mussels in the test surface, the percentage of ×
individuals detached from the test surface
Accidental/unexpected items observed during the test (×)
Average number of byssus threads ×
Statistical analysis method used (×)

Is there a statistically significant difference between the bioassay results for the test and (×)

control groups, Yes/No?
x: required; (x): optional.
Information required for each run.
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ISO 21716-3:2020(E)
Annex A
(informative)
Statistical analysis — Examples
A.1 Introduction

It is stated in the Scope that 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. It can be useful to perform statistical analysis to determine if the

difference in mussel settlement is statistically significant. A typical example of statistical analysis was

given in this Annex, based on Reference [1]. Selection of an optimal statistical analysis method from

different approaches should be made depending on the purpose and the data distributions. This annex

describes some statistical analysis methods as examples.
A.2 Statistical analysis to compare a test group and a control group
A.2.1 Introduction

This clause indicates some statistical analysis methods to calculate the difference between a control

group and a test group as examples. Figure A.1 shows an example for this process. Statistical analysis

of significance difference between the control and test groups is conducted using a one-way ANOVA

(analysis of variance) followed by multiple comparison test. In this case, Dunnett’s multiple comparison

test is recommended for comparing several treatments with the control groups (Reference [2]).

Probability values (p-value) less than 0,05 are considered significant.
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ISO 21716-3:2020(E)
Figure A.1 — Example of a statistical analysis process
[3]–[7]
A.2.2 One-way analysis of variance

One-way analysis of variance (ANOVA) is a collection of statistical models and their associated

estimation procedures (such as the "variation" among and between groups) used to analyse the

differences among group means in a sample. The ANOVA is based on the law of total variance, where

the observed variance in a particular variable is partitioned into components attributable to different

sources of variation. In its simplest form, ANOVA provides a statistical test of whether two or more

population means are equal, and therefore generalizes the t-test beyond two means.

The one-way
...

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