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 barnacle cyprid larvae 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 barnacle settlement on painted test panels compared with barnacle settlement on inert non-toxic control panels under the conditions of the test. Examples of statistical analysis to determine if the difference in barnacle settlement is statistically significant are given in Annex A.

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INTERNATIONAL ISO
STANDARD 21716-2
First edition
2020-11
Ships and marine technology —
Bioassay methods for screening anti-
fouling paints —
Part 2:
Barnacles
Reference number
ISO 21716-2:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 21716-2:2020(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 21716-2:2020(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 the test seawater ............................................................................................................................................. 4

7 Preparation of the triangular prisms.............................................................................................................................................. 4

7.1 General ........................................................................................................................................................................................................... 4

7.2 Preparation of the test panels and control panels ................................................................................................... 4

7.3 Assembly of the triangular prisms ......................................................................................................................................... 4

8 Operation of the test ......................................................................................................................................................................................... 6

8.1 Cyprid viability test ............................................................................................................................................................................. 6

8.2 Bioassay ........................................................................................................................................................................................................ 7

9 Validation of the test ......................................................................................................................................................................................10

9.1 General ........................................................................................................................................................................................................10

9.2 Requirements of the cyprids viability test ...................................................................................................................10

9.3 Requirements of the bioassay .................................................................................................................................................11

10 Settlement rates .................................................................................................................................................................................................12

10.1 General ........................................................................................................................................................................................................12

10.2 Calculation of the settlement rate for the test panel ...........................................................................................12

10.3 Data treatment and interpretation of the results...................................................................................................12

11 Test report ................................................................................................................................................................................................................13

Annex A (informative) Statistical analysis — Examples ................................................................................................................15

Annex B (informative) The barnacle Amphibalanus amphitrite ...........................................................................................18

Annex C (informative) Life cycle of a barnacle ........................................................................................................................................21

Annex D (informative) Materials ...........................................................................................................................................................................23

Annex E (informative) Identification of adult Amphibalanus amphitrite ....................................................................24

Bibliography .............................................................................................................................................................................................................................26

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 21716-2:2020(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 21716-2:2020(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 to maintaining the 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 anti-fouling products are in service is

routinely undertaken instead (see Reference [28]).

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.

Barnacles 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.

This test method utilizes cyprid juveniles to assess settling behaviour in the presence of treated panels.

Cyprid larvae are considered the most relevant life stage for such evaluations as it is at this point that

the barnacle settles on appropriate substrate prior to metamorphosis into the adult. More information

is provided in Annexes B and C.
© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 21716-2:2020(E)
Ships and marine technology — Bioassay methods for
screening anti-fouling paints —
Part 2:
Barnacles
1 Scope

This document specifies a laboratory test method for screening anti-fouling paints in a flow-through

system using barnacle cyprid larvae 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 barnacle settlement on painted test panels compared with barnacle settlement on inert

non-toxic control panels under the conditions of the test. Examples of statistical analysis to determine

if the difference in barnacle 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
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
culturing

growing hatched nauplius larva to cyprid stage under controlled conditions prior to the test

Note 1 to entry: Refer to Figure B.2.
3.2
rearing

growing adult barnacle to enhance larval hatching under controlled conditions prior to the culturing

(3.1) stage
3.3
settlement

stage of the sessile phase involving juvenile (3.4) barnacles and cyprids metamorphosing into juveniles

on the substrates
Note 1 to entry: Refer to Figure B.2.
© ISO 2020 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 21716-2:2020(E)
3.4
juvenile

individual of barnacle after the metamorphosis and molting of cyprid during the test

Note 1 to entry: Refer to Annex B.
3.5
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 1:

— preparation of the test organism and the test seawater;
— preparation of the triangular prisms;
— operation of the test (cyprid viability test and bioassay);
— validation of the test; and
— data treatment and interpretation of the results.
Figure 1 — Schema 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 cyprid viability

and settlement on the control groups are both shown to be acceptable, then the barnacle settlement

rates of the test and control groups can be compared.
2 © ISO 2020 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 21716-2:2020(E)
5 Material and apparatus

The items listed in Tables 1 and 2 shall be used for the test. For recommended items, refer to Annex D.

Table 1 — List of material used
Material Remarks

Adhesive tape Used to assemble a prism. Approx. 50 mm long without any harmful effect on

cyprids, e.g., double-sided carbon tape is recommended.
[1]
Abrasive media F20 macrogrit or F20 macrogrit bonded abrasive

Cultured stock of live Amphibalanus amphitrite should be used with a larval density of 2-3 nauplius

barnacle cyprids larvae per ml of seawater.
Other barnacle species may be used if Amphibalanus amphitrite cyprids are
not available.
Natural seawater Defined in ISO 21716-1:2020, 3.8
Pipettes 10 ml capacity, glass or disposable [see 8.2 (i)], used for filling the
microtiter plates.
Plankton net Approx. 11 cm mesh size (NXX13), 100 µm
Plastic legs 2 mm × 2 mm × 30 mm, used to support prism

Polishing agent Used for surface treatment of control panels, sandpaper or other bonded

materials with F-20 macrogrit.
Purified water Defined in 3.5

PVC plates Used as substrates for control panels. Black panels with same size as test/

control panels are recommended.
Test panels Specified in ISO 21716-1:2020, 4.2. 50 mm square is recommended.

White panel White acrylic plates with same size as test/control panels should be used as

they are considered as the material on which cyprids hardly settle, resulting
in increased settlement on the test surface. White plates used to assemble a
prism with control or test panels.
1 µm filters Used to prepare test seawater.
Table 2 — List of apparatus used
Apparatus Remarks
Incubator Thermostatic chamber with a means of maintaining the ambient
temperature at 25 °C
Light White fluorescence or LED
Light intensity meter Accuracy: ±10 lx

6-well (or 12-well) microtiter Made of polystyrene (may be replaced to petri dish)

plates with lids
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 As specified in ISO 21716-1:2020, 5.2, with a means of maintaining the test

seawater tank at 25 °C ± 1 °C and alternately illuminating the test seawater

tank with a light intensity of 3 000 lx (see 8.2 f), light conditions) and with a

light intensity of <50 lx (see 8.2 f) dark conditions).
© ISO 2020 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 21716-2:2020(E)
6 Preparation of the test organism and the test seawater
6.1 General

The cultured stock of live barnacle cyprids is used to perform the bioassay test in seawater.

6.2 Preparation of the test organism

Live barnacle cyprids are generally prepared by collecting and rearing an adult barnacle followed

by preparing and culturing nauplius larvae of the barnacle. Guidance on this process and on storing

cyprids can be found in Annex B. Information on the life cycle of barnacles can be found in Annex C, and

information on the identification of adult Amphibalanus amphitrite barnacles can be found in Annex E.

6.3 Preparation of the test seawater

Pass natural seawater through a 1 µm filter unit and adjust to salinity 28,0 ± 0,5 using purified water.

7 Preparation of the triangular prisms
7.1 General

Each test panel and each control panel shall be used with two white panels to construct a series of

triangular prisms for use in the bioassay (see Figures 2 and 3). The same test and control groups shall

be used throughout the whole test.
7.2 Preparation of the test panels and control panels

Test panels and control panels shall be prepared following the specifications of ISO 21716-1:2020,

Clause 4.

Abrade the surface of the control panels prior to use in the test by gently blasting with F20 macrogrit or

by abrading with F20 macrogrit bonded abrasive (Reference [1]).
7.3 Assembly of the triangular prisms

Construct the required number of prisms for the required number of replicates for each run. Test group

prisms shall use one test panel and two white panels. Control group prisms shall use one control panel

and two white panels. Panels shall be of the same size (see Figure 2). Prisms shall be constructed with

test and control surfaces facing inwards. The bottom of the triangular prism is covered with plankton

net. The panels, the plankton net and a plastic leg at each bottom corner shall be assembled using

adhesive tape. Ensure that all components are tightly fixed together without any gaps between them.

The triangular prism should be supported by the three legs 10 mm or more from the bottom surface

of the test tank to ensure sufficient flow of test seawater through the prism. The surface of test panels

shall be kept wet with test seawater during the assembly of the prisms and up until immersion in the

test tank. The triangular prisms are assembled according to the process described in Figure 3.

4 © ISO 2020 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 21716-2:2020(E)
Key
1 test panel
2 control panel
3 white panel
Figure 2 — Formation of the triangular prism for the test and control group
© ISO 2020 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO 21716-2:2020(E)
Key
1 adhesive tape
2 white panel
3 test/control panel
4 plankton net
5 plastic leg

Assemble one test/control panel and two white panels to a triangular prism using adhesive tape.

Attach plankton net to the bottom side of the triangular prism using adhesive tape.

Attach plastic leg s to the triangular prisms; length of the legs from the bottom side of the prism ≥10 mm.

Figure 3 — Assembly of triangular prism for the test
8 Operation of the test
8.1 Cyprid viability test

The cyprid viability test is conducted in order to verify the health of cultured cyprids in the bioassay,

and should be performed in parallel with the bioassay. The test shall be performed according to the

following procedure.

a) Place cyprids collected from the cultured stock into a well of a microtiter plate filled with the test

seawater.
6 © ISO 2020 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 21716-2:2020(E)

b) Fill 5 wells of at least three 6-well (or 12-well) microtiter plates with at least 10 cyprids at a

maximum density of 3 individuals/ml of test seawater. Lids should be used to prevent evaporative

loss of test seawater throughout the test period.
c) Record the number of cyprids in each well prior to starting the test.

d) Conduct the test according to 8.2 f) for 48 h. Maintain water temperature at 25 °C ± 1 °C throughout

the test period.

e) After completion of d) above, count the number of juvenile, cyprids and dead individuals using a

stereo microscope and record using e.g. Table 3.
8.2 Bioassay

The bioassay shall be simultaneously performed on the test group and on the control group, using the

triangular prisms as follows (see Figures 4 and 5).

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) Wash each triangular prism thoroughly prior to the test with fresh running test seawater.

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

from the seawater storage tank. Maintain the temperature of the test seawater tank within the

range 25 °C ± 1 °C for the duration of the test. The flow rate should be set to achieve about 1

turnover per hour of the water of test seawater tank.

NOTE If the flow rate is too high, the test seawater can overflow from the prisms and dislodge cyprids

from the test or control surface. If the flow rate is too low, the result can be affected by the concentration of

biocide in seawater of the test seawater tank.

c) Place the prism in the test seawater tank and adjust water level to 1,0 cm ± 0,2 cm below the top

of the prism to ensure the flow rate of the seawater from the bottom of the prisms as shown in

Figure 5.

d) Place cyprids at the density of 2 to 3 individuals/ml inside of the prism ensuring the water does not

overflow from the top of the prism. In case of using 5,0 cm square panels, 60 to 100 cyprids should

be placed.

e) Measure and record the temperature, pH and salinity of the test seawater in the test seawater

tanks at the initial stage of the test. Measure and record these parameters again after 24 h and 48 h

from the beginning of the test.

f) Illuminate the test seawater tank with a light intensity of 3 000 lx for the initial 12 h, maintain a

dark condition for 12 h, and then alternate subsequent 12 h light and dark periods for 48 h in total.

g) After 48 h from the beginning of the test, collect the remaining cyprids and dead individuals inside

the prism around the test seawater surface irradiated with light, using pipettes.

NOTE Cyprids exhibit phototactic behaviour, this can be exploited to facilitate collecting nauplius larvae.

h) Carefully remove the prism from the tank and dismantle immediately after the completion of step

g) above to separate the white panels and test or control panels, noting which surfaces had formed

the inner surface of the prism.

i) Using a pipette, rinse the inner surface of each panel three times with 10 ml of the test seawater to

remove and collect unattached cyprids.
NOTE Unsettled individuals are easily detached by rinsing from the test surface.

j) Using a stereo microscope, count the number of live and dead cyprids and juveniles as shown

in Figure B.1, both on the test surface and on the other surfaces (white panels, edges of the test

© ISO 2020 – All rights reserved 7
---------------------- Page: 12 ----------------------
ISO 21716-2:2020(E)

plates and plankton net), the number of live and dead cyprids and juveniles collected from the

prism [see (g)] and rinsing from the inner surface [see (i)], and record the results using e.g. Table 4.

Metamorphosing cyprids should be counted as cyprids and not as juveniles.
Figure 4 — Flow chart of the procedure for the test
8 © ISO 2020 – All rights reserved
---------------------- Page: 13 ----------------------
ISO 21716-2:2020(E)
a) Triangular prism
b) Top view
c) Side view
Key
1 cyprids of the barnacle
2 charging test water
3 discharging test water
Figure 5 — Setting of the triangular prism in the test seawater tank
© ISO 2020 – All rights reserved 9
---------------------- Page: 14 ----------------------
ISO 21716-2:2020(E)
9 Validation of the test
9.1 General

The results of the bioassay are validated using arithmetic tests to confirm that the viability of the

cyprids used in the bioassay meet a minimum threshold value (see 9.2), and to confirm that the

settlement rate for the control group also meets a minimum threshold value (see 9.3).

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

9.2 Requirements of the cyprids viability test

The degree of settlement is calculated from the results of the cyprid viability test (see 8.1). The degree

of settlement for each well of the microtiter plate for each run is calculated using Formula (1). The

results are recorded using e.g. Table 3 to one decimal place.
R = ×100 (1)
ab++c
where
R is the degree of settlement for verifying the test (%);
a is the number of juveniles on the surface of the wells of microtiter plates;
b is the number of unsettled and live cyprids;
c is the number of dead individuals.

The average number of settlement rate shall be calculated from the values of R for each run. The

viabili
...

INTERNATIONAL ISO
STANDARD 21716-2
First edition
Ships and marine technology —
Bioassay methods for screening anti-
fouling paints —
Part 2:
Barnacles
PROOF/ÉPREUVE
Reference number
ISO 21716-2:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 21716-2:2020(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 21716-2:2020(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 the test seawater ............................................................................................................................................. 4

7 Preparation of the triangular prisms.............................................................................................................................................. 4

7.1 General ........................................................................................................................................................................................................... 4

7.2 Preparation of the test panels and control panels ................................................................................................... 4

7.3 Assembly of the triangular prisms ......................................................................................................................................... 4

8 Operation of the test ......................................................................................................................................................................................... 6

8.1 Cyprid viability test ............................................................................................................................................................................. 6

8.2 Bioassay ........................................................................................................................................................................................................ 7

9 Validation of the test ......................................................................................................................................................................................10

9.1 General ........................................................................................................................................................................................................10

9.2 Requirements of the cyprids viability test ...................................................................................................................10

9.3 Requirements of the bioassay .................................................................................................................................................11

10 Settlement rates .................................................................................................................................................................................................12

10.1 General ........................................................................................................................................................................................................12

10.2 Calculation of the settlement rate for the test panel ...........................................................................................12

10.3 Data treatment and interpretation of the results...................................................................................................12

11 Test report ................................................................................................................................................................................................................13

Annex A (informative) Statistical analysis — Examples ................................................................................................................15

Annex B (informative) The barnacle Amphibalanus amphitrite ...........................................................................................18

Annex C (informative) Life cycle of a barnacle ........................................................................................................................................21

Annex D (informative) Materials ...........................................................................................................................................................................23

Annex E (informative) Identification of adult Amphibalanus amphitrite ....................................................................24

Bibliography .............................................................................................................................................................................................................................26

© ISO 2020 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO 21716-2:2020(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.
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ISO 21716-2:2020(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 to maintaining the 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 anti-fouling products are in service is routinely

undertaken instead (see Reference [28]).

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.

Barnacles 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 cyprid juveniles to assess settling behaviour in the presence of treated panels.

Cyprid larvae are considered the most relevant life stage for such evaluations as it is at this point that

the barnacle settles on appropriate substrate prior to metamorphosis into the adult. More information

is provided in Annexes B and C.
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INTERNATIONAL STANDARD ISO 21716-2:2020(E)
Ships and marine technology — Bioassay methods for
screening anti-fouling paints —
Part 2:
Barnacles
1 Scope

This document specifies a laboratory test method for screening anti-fouling paints in a flow-through

system using barnacle cyprid larvae 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 barnacle settlement on painted test panels compared with barnacle settlement on inert

non-toxic control panels under the conditions of the test. Examples of statistical analysis to determine

if the difference in barnacle 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
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
culturing

growing hatched nauplius larva to cyprid stage under controlled conditions prior to the test

Note 1 to entry: Refer to Figure B.2.
3.2
rearing

growing adult barnacle to enhance larval hatching under controlled conditions prior to the culturing

(3.1) stage
3.3
settlement

stage of the sessile phase involving juvenile (3.4) barnacles and cyprids metamorphosing into juveniles

on the substrates
Note 1 to entry: Refer to Figure B.2.
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ISO 21716-2:2020(E)
3.4
juvenile

individual of barnacle after the metamorphosis and molting of cyprid during the test

Note 1 to entry: Refer to Annex B.
3.5
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 1:

— preparation of the test organism and the test seawater;
— preparation of the triangular prisms;
— operation of the test (cyprid viability test and bioassay);
— validation of the test; and
— data treatment and interpretation of the results.
Figure 1 — Schema 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 cyprid viability

and settlement on the control groups are both shown to be acceptable, then the barnacle settlement

rates of the test and control groups can be compared.
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ISO 21716-2:2020(E)
5 Material and apparatus

The items listed in Tables 1 and 2 shall be used for the test. For recommended items, refer to Annex D.

Table 1 — List of material used
Material Remarks

Adhesive tape Used to assemble a prism. Approx. 50 mm long without any harmful effect on

cyprids, e.g., double-sided carbon tape is recommended.
[1]
Abrasive media F20 macrogrit or F20 macrogrit bonded abrasive .

Cultured stock of live Amphibalanus amphitrite should be used with a larval density of 2-3 nauplius

barnacle cyprids larvae per ml of seawater.
Other barnacle species may be used if Amphibalanus amphitrite cyprids are
not available.
Natural seawater Defined in ISO 21716-1:2020, 3.8.
Pipettes 10 ml capacity, glass or disposable [see 8.2 (i)], used for filling the
microtiter plates
Plankton net Approx. 11 cm mesh size (NXX13), 100 µm
Plastic legs 2 mm × 2 mm × 30 mm, used to support prism.

Polishing agent Used for surface treatment of control panels, sandpaper or other bonded

materials with F-20 macrogrit.
Purified water Defined in 3.5.

PVC plates Used as substrates for control panels. Black panels with same size as test/

control panels are recommended.
Test panels Specified in ISO 21716-1:2020, 4.2. 50 mm square is recommended.

White panel White acrylic plates with same size as test/control panels should be used as

they are considered as the material on which cyprids hardly settle, resulting
in increased settlement on the test surface. White plates used to assemble a
prism with control or test panels.
1 µm filters Used to prepare test seawater.
Table 2 — List of apparatus used
Apparatus Remarks
Incubator Thermostatic chamber with a means of maintaining the ambient
temperature at 25 °C.
Light White fluorescence or LED.
Light intensity meter Accuracy: ±10 lx.

6-well (or 12-well) microtiter Made of polystyrene (may be replaced to petri dish)

plates with lids
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 As specified in ISO 21716-1:2020, 5.2, with a means of maintaining the test

seawater tank at 25 °C ± 1 °C and alternately illuminating the test seawater

tank with a light intensity of 3 000 lx (see 8.2 f), light conditions) and with a

light intensity of <50 lx (see 8.2 f) dark conditions).
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ISO 21716-2:2020(E)
6 Preparation of the test organism and the test seawater
6.1 General

The cultured stock of live barnacle cyprids is used to perform the bioassay test in seawater.

6.2 Preparation of the test organism

Live barnacle cyprids are generally prepared by collecting and rearing an adult barnacle followed

by preparing and culturing nauplius larvae of the barnacle. Guidance on this process and on storing

cyprids can be found in Annex B. Information on the life cycle of barnacles can be found in Annex C, and

information on the identification of adult Amphibalanus amphitrite barnacles can be found in Annex E.

6.3 Preparation of the test seawater

Pass natural seawater through a 1 µm filter unit and adjust to salinity 28,0 ± 0,5 using purified water.

7 Preparation of the triangular prisms
7.1 General

Each test panel and each control panel shall be used with two white panels to construct a series of

triangular prisms for use in the bioassay (see Figures 2 and 3). The same test and control groups shall

be used throughout the whole test.
7.2 Preparation of the test panels and control panels

Test panels and control panels shall be prepared following the specifications of ISO 21716-1:2020,

Clause 4.

Abrade the surface of the control panels prior to use in the test by gently blasting with F20 macrogrit or

by abrading with F20 macrogrit bonded abrasive (Reference [1]).
7.3 Assembly of the triangular prisms

Construct the required number of prisms for the required number of replicates for each run. Test group

prisms shall use one test panel and two white panels. Control group prisms shall use one control panel

and two white panels. Panels shall be of the same size (see Figure 2). Prisms shall be constructed with

test and control surfaces facing inwards. The bottom of the triangular prism is covered with plankton

net. The panels, the plankton net and a plastic leg at each bottom corner shall be assembled using

adhesive tape. Ensure that all components are tightly fixed together without any gaps between them.

The triangular prism should be supported by the three legs 10 mm or more from the bottom surface

of the test tank to ensure sufficient flow of test seawater through the prism. The surface of test panels

shall be kept wet with test seawater during the assembly of the prisms and up until immersion in the

test tank. The triangular prisms are assembled according to the process described in Figure 3.

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ISO 21716-2:2020(E)
Key
1 test panel
2 control panel
3 white panel
Figure 2 — Formation of the triangular prism for the test and control group
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ISO 21716-2:2020(E)
Key
1 adhesive tape
2 white panel
3 test/control panel
4 plankton net
5 plastic leg

Assemble one test/control panel and two white panels to a triangular prism using adhesive tape.

Attach plankton net to the bottom side of the triangular prism using adhesive tape.

Attach plastic leg s to the triangular prisms; length of the legs from the bottom side of the prism ≥10 mm.

Figure 3 — Assembly of triangular prism for the test
8 Operation of the test
8.1 Cyprid viability test

The cyprid viability test is conducted in order to verify the health of cultured cyprids in the bioassay,

and should be performed in parallel with the bioassay. The test shall be performed according to the

following procedure.

a) Place cyprids collected from the cultured stock into a well of a microtiter plate filled with the test

seawater.
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ISO 21716-2:2020(E)

b) Fill 5 wells of at least three 6-well (or 12-well) microtiter plates with at least 10 cyprids at a

maximum density of 3 individuals/ml of test seawater. Lids should be used to prevent evaporative

loss of test seawater throughout the test period.
c) Record the number of cyprids in each well prior to starting the test.

d) Conduct the test according to 8.2 f) for 48 h. Maintain water temperature at 25 °C ± 1 °C throughout

the test period.

e) After completion of d) above, count the number of juvenile, cyprids and dead individuals using a

stereo microscope and record using e.g. Table 3.
8.2 Bioassay

The bioassay shall be simultaneously performed on the test group and on the control group, using the

triangular prisms as follows (see Figures 4 and 5).

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) Wash each triangular prism thoroughly prior to the test with fresh running test seawater.

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

from the seawater storage tank. Maintain the temperature of the test seawater tank within the

range 25 °C ± 1 °C for the duration of the test. The flow rate should be set to achieve about 1

turnover per hour of the water of test seawater tank.

NOTE If the flow rate is too high, the test seawater can overflow from the prisms and dislodge the

cyprids barnacle from the test or control surface. If the flow rate is too low, the result can be affected by the

concentration of biocide in seawater of the test seawater tank.

c) Place the prism in the test seawater tank and adjust water level to 1,0 cm ± 0,2 cm below the top

of the prism to ensure the flow rate of the seawater from the bottom of the prisms as shown in

Figure 5.

d) Place cyprids at the density of 2 to 3 individuals/ml inside of the prism ensuring the water does not

overflow from the top of the prism. In case of using 5,0 cm square panels, 60 to 100 cyprids should

be placed.

e) Measure and record the temperature, pH and salinity of the test seawater in the test seawater

tanks at the initial stage of the test. Measure and record these parameters again after 24 h and 48 h

from the beginning of the test.

f) Illuminate the test seawater tank with a light intensity of 3 000 lx for the initial 12 h, maintain a

dark condition for 12 h, and then alternate subsequent 12 h light and dark periods for 48 h in total.

g) After 48 h from the beginning of the test, collect the remaining cyprids and dead individuals inside

the prism around the test seawater surface irradiated with light, using pipettes.

NOTE Cyprids exhibit phototactic behaviour, this can be exploited to facilitate collecting nauplius larvae.

h) Carefully remove the prism from the tank and dismantle immediately after the completion of step

g) above to separate the white panels and test or control panels, noting which surfaces had formed

the inner surface of the prism.

i) Using a pipette, rinse the inner surface of each panel three times with 10 ml of the test seawater to

remove and collect unattached cyprids.
NOTE Unsettled individuals are easily detached by rinsing from the test surface.

j) Using a stereo microscope, count the number of live and dead cyprids and juveniles as shown

in Figure B.1, both on the test surface and on the other surfaces (white panels, edges of the test

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ISO 21716-2:2020(E)

plates and plankton net), the number of live and dead cyprids and juveniles collected from the

prism [see (g)] and rinsing from the inner surface [see (i)], and record the results using e.g. Table 4.

Metamorphosing cyprids should be counted as cyprids and not as juveniles.
Figure 4 — Flow chart of the procedure for the test
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ISO 21716-2:2020(E)
a) Triangular prism
b) Top view
c) Side view
Key
1 cyprids of the barnacle
2 charging test water
3 discharging test water
Figure 5 — Setting of the triangular prism in the test seawater tank
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ISO 21716-2:2020(E)
9 Validation of the test
9.1 General

The results of the bioassay are validated using arithmetic tests to confirm that the viability of the

cyprids used in the bioassay meet a minimum threshold value (see 9.2), and to confirm that the

settlement rate for the control group also meets a minimum threshold value (see 9.3).

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

9.2 Requirements of the cyprids viability test

The degree of settlement is calculated from the results of the cyprid viability test (see 8.1). The degree

of settlement for each well of the microtitre plate for each run is calculated using Formula (1). The

results are recorded using e.g. Table 3 to one decimal place.
R = ×100 (1)
ab++c
where
R is the degree of settlement for verifying the test (%);
a is the number of juveniles on the surface of the wells of microtiter plates;
...

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