Water quality -- Determination of the acute toxicity to the marine rotifer Brachionus plicatilis

ISO 19820:2016 specifies a method for the determination of the lethal effects of toxicants to Brachionus plicatilis after 24 h or 48 h exposure. The method is applicable to the following: a) chemical substances which are soluble or which can be maintained as a stable suspension of dispersions under the conditions of the test; b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration, or centrifugation; c) marine or estuarine waters; d) sediment elutriates/eluates.

Qualité de l'eau -- Détermination de la toxicité aigue envers le rotifère marin Brachionus plicatilis

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Status
Published
Publication Date
07-Mar-2016
Current Stage
9060 - Close of review
Start Date
05-Jun-2021
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INTERNATIONAL ISO
STANDARD 19820
First edition
2016-03-01
Water quality — Determination of the
acute toxicity to the marine rotifer
Brachionus plicatilis
Qualité de l’eau — Détermination de la toxicité aigue envers
le rotifère marin Brachionus plicatilis
Reference number
ISO 19820:2016(E)
ISO 2016
---------------------- Page: 1 ----------------------
ISO 19820:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland

All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 19820:2016(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Test environment ................................................................................................................................................................................................. 2

6 Reagents, test organisms and media ............................................................................................................................................... 2

6.1 Test organisms ........................................................................................................................................................................................ 2

6.2 Culturing and dilution medium ................................................................................................................................................ 2

6.3 Reference substance ........................................................................................................................................................................... 3

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Treatment and preparation of samples ........................................................................................................................................ 3

8.1 Special precautions ............................................................................................................................................................................ 3

8.2 Preparation of the stock solutions of substances to be tested ..................................................................... 4

9 Procedure..................................................................................................................................................................................................................... 4

9.1 Selection of test concentrations .............................................................................................................................................. 4

9.2 Preparation of the test and control solutions ............................................................................................................. 4

9.3 Introduction of the organisms .................................................................................................................................................. 5

9.4 Incubation of the test system .................................................................................................................................................... 6

9.5 Measurements ........................................................................................................................................................................................ 6

10 Estimation of the LC ..................................................................................................................................................................................... 7

11 Reference test .......................................................................................................................................................................................................... 7

12 Validity criteria ...................................................................................................................................................................................................... 7

13 Test report ................................................................................................................................................................................................................... 8

Annex A (informative) Procedure for hatching of Brachionus plicatilis cysts ...........................................................9

Annex B (informative) Preparation of artificial seawater ...........................................................................................................10

Annex C (informative) Performance data .....................................................................................................................................................11

Bibliography .............................................................................................................................................................................................................................13

© ISO 2016 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 19820:2016(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 on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 5,

Biological methods.
iv © ISO 2016 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 19820:2016(E)
Introduction

The evaluation of harmful effects on chemicals and pollutants on the biota in marine and estuarine

environments has, for several years, involved the performance of biological tests.

Rotifers, and especially the species Brachionus plicatilis, are of interest from the ecotoxicological view

because they are often an important component of the zooplankton and serve as prey for small fish and

larger invertebrates.

The test specified in this International Standard involves determination of the lethal effects of toxicants

to the marine rotifer, Brachionus plicatilis, after 24 h or 48 h exposure (depending on the intended use

of the results). Prolongation of the exposure to 48 h is advised since it substantially increases the

sensitivity of the assay.
© ISO 2016 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 19820:2016(E)
Water quality — Determination of the acute toxicity to the
marine rotifer Brachionus plicatilis

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document does not purport to address all of the safety problems, if any, associated with its

use. It is the responsibility of the user to establish appropriate safety and health practices and to

ensure compliance with any national regulatory conditions.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

be carried out by suitably qualified staff.
1 Scope

This International Standard specifies a method for the determination of the lethal effects of toxicants

to Brachionus plicatilis after 24 h or 48 h exposure.
The method is applicable to the following:

a) chemical substances which are soluble or which can be maintained as a stable suspension of

dispersions under the conditions of the test;

b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration, or

centrifugation;
c) marine or estuarine waters;
d) sediment elutriates/eluates.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 5667-16, Water quality — Sampling — Part 16: Guidance on biotesting of samples

ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method

ISO 10523, Water quality — Determination of pH
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
control batch
series of replicates containing control solution
[SOURCE: ISO 6341:2012, 3.1]
3.2

concentration of dilution of the test sample which gives rise to 50 % mortality of the test organisms

© ISO 2016 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 19820:2016(E)
3.3
test batch
series of replicates filled with the same test solution
[SOURCE: ISO 6341:2012, 3.6]
3.4
pure water
deionized or distilled water with a conductivity below 10 µS/cm
4 Principle

The test organisms are exposed to a range of concentrations of the sample under analysis and the

percentage mortality of the test organisms is determined after 24 h exposure and/or 48 h exposure

with subsequent calculation of the 24 h LC and/or the 48 h LC .
50 50

Prolongation of the test to 48 h is recommended since it substantially increases the sensitivity of the assay.

The test is carried out in one or two of the following stages:

— a “range-finding test” to determine the range of concentrations or dilutions needed for calculation

of the 24 h LC or the 48 h LC ;
50 50

— a “definitive test” conducted when the data of the range-finding test are not sufficient or adequate

for calculation of the 24 h LC or the 48 h LC .
50 50
5 Test environment

The test shall be carried out in the dark, in a temperature-controlled room, or incubator at (25 ± 1) °C in

the test containers.

Maintain the atmosphere free from toxic dusts or vapours. The use of control solutions is a double check

that the test is performed in an atmosphere free from toxic dusts and vapours.
6 Reagents, test organisms and media
Use only reagents of recognized analytical grade, unless otherwise specified.
6.1 Test organisms

The tests organisms are females of the species Brachionus plicatilis obtained from a laboratory culture

(see References [5],[7], and [8]) or hatched from commercially available cysts.

The procedure for hatching of Brachionus plicatilis from cysts is described in Annex A.

6.2 Culturing and dilution medium

A natural or an artificial seawater may be used as the water for stock culturing the rotifers or as dilution

water for the testing. Natural seawater shall be collected from an unpolluted location and should have

salinity between 29 ‰ and 36 ‰. Natural seawater must be filtered (30 µm) and conditioned to test

temperature and oxygen saturation prior to use. Natural seawater can be stored cold (4 ± 1) °C for

several weeks.

An example of artificial seawater suitable for culturing and testing is given in Annex B.

1) MicroBioTests Inc. Mariakerke, Belgium, is an example of a supplier able to provide suitable Brachionus plicatilis

cysts commercially. This information is given for the convenience of the users of this document and does not

constitute an endorsement by ISO of this supplier.
2 © ISO 2016 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 19820:2016(E)
6.3 Reference substance

Potassium dichromate (K Cr O ) or copper sulfate (CuSO ·5H O) are recommended as reference

2 2 7 4 2
chemicals.

NOTE Since K Cr O is a carcinogenic substance toxic via inhalation, the use of a ready-made solution with

2 2 7

a defined concentration of K Cr O for the preparation of the stock solution of the reference substance can

2 2 7
reduce the risk of inhalation of the toxic dust in the laboratory.
7 Apparatus
Usual laboratory equipment and, in particular, the following.
7.1 Temperature-controlled room or chamber.
7.2 Petri dishes.
Small Petri dishes (diameter 5 cm) in glass or in inert plastic material.
7.3 Test containers.
Disposable 48 (6 × 8) microplates made from chemically inert material.

7.4 Pipette for sampling rotifers, with a sufficient diameter for capturing the animals while allowing

sampling of only a small volume of medium.
For example, single use 1 ml capillary mini-pipettes are suitable.

7.5 Stereomicroscope with incident (bottom) illumination, with a magnification of at least eight

times and, if possible, a continuous magnification.

7.6 Light source, providing a range of light intensity in the hatching Petri dish of 3 000 lx to 4 000 lx

-2 -1 -2 -1
corresponding to 40 µmol.m .s to 55 µmol.m .s .
7.7 Sample collecting bottles, as specified in ISO 5667-16.
8 Treatment and preparation of samples
8.1 Special precautions

Special precautions are required for sampling, transportation, storage, and treatment of seawater or

effluent.

Sampling, transportation, and storage of the samples should be performed as specified in ISO 5667-16.

Carry out the toxicity test as soon as possible, ideally within 12 h of collection. If this time interval

cannot be met, cool the sample to 0 °C to 5 °C and test the sample within 24 h. If it is not possible to

perform the test within 72 h, the sample may be frozen and maintained deep-frozen (below –18 °C)

for testing within two months of collection, provided that characteristics are known to be unaffected

by freezing. At the time of testing, homogenize the sample to be analysed by shaking manually, and, if

necessary, allow to settle for 2 h in a container and sample by drawing off (using a pipette) the required

quantity of supernatant maintaining the end of the pipette in the centre of the section of the test tube

and halfway between the surface of the deposited matters and the surface of the liquid.

2) Titrisol potassium dichromate solution is an example of
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 19820
ISO/TC 147/SC 5
Water quality — Determination of the
Secretariat: DIN
acute toxicity to the marine rotifer
Voting begins on:
2015­10­26 Brachionus plicatilis
Voting terminates on:
Qualité de l’eau — Détermination de la toxicité aigue envers le
2015­12­26
rotifère marin Brachionus plicatilis
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/FDIS 19820:2015(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN­
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. ISO 2015
---------------------- Page: 1 ----------------------
ISO/FDIS 19820:2015(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, Published in Switzerland

All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH­1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2015 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 19820:2015(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Test environment ................................................................................................................................................................................................. 2

6 Reagents, test organisms and media ............................................................................................................................................... 2

6.1 Test organisms ........................................................................................................................................................................................ 2

6.2 Culturing and dilution medium ................................................................................................................................................ 2

6.3 Reference substance ........................................................................................................................................................................... 3

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Treatment and preparation of samples ........................................................................................................................................ 3

8.1 Special precautions ............................................................................................................................................................................ 3

8.2 Preparation of the stock solutions of substances to be tested ..................................................................... 4

9 Procedure..................................................................................................................................................................................................................... 4

9.1 Selection of test concentrations .............................................................................................................................................. 4

9.2 Preparation of the test and control solutions ............................................................................................................. 4

9.3 Introduction of the organisms .................................................................................................................................................. 5

9.4 Incubation of the test system .................................................................................................................................................... 6

9.5 Measurements ........................................................................................................................................................................................ 6

10 Estimation of the LC ..................................................................................................................................................................................... 7

11 Reference test .......................................................................................................................................................................................................... 7

12 Validity criteria ...................................................................................................................................................................................................... 7

13 Test report ................................................................................................................................................................................................................... 8

Annex A (informative) Procedure for hatching of Brachionus plicatilis cysts ...........................................................9

Annex B (informative) Preparation of artificial seawater ...........................................................................................................10

Annex C (informative) Performance data .....................................................................................................................................................11

Bibliography .............................................................................................................................................................................................................................13

© ISO 2015 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/FDIS 19820:2015(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 non­governmental, 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 on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 5,

Biological methods.
iv © ISO 2015 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/FDIS 19820:2015(E)
Introduction

The evaluation of harmful effects on chemicals and pollutants on the biota in marine and estuarine

environments has, for several years, involved the performance of biological tests.

Rotifers, and especially the species Brachionus plicatilis, are of interest from the ecotoxicological view

because they are often an important component of the zooplankton and serve as prey for small fish and

larger invertebrates.

The test specified in this International Standard involves determination of the lethal effects of toxicants

to the marine rotifer, Brachionus plicatilis, after 24 h or 48 h exposure (depending on the intended use

of the results). Prolongation of the exposure to 48 h is advised since it substantially increases the

sensitivity of the assay.
© ISO 2015 – All rights reserved v
---------------------- Page: 5 ----------------------
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 19820:2015(E)
Water quality — Determination of the acute toxicity to the
marine rotifer Brachionus plicatilis

WARNING — Persons using this International Standard should be familiar with normal laboratory

practice. This International Standard does not purport to address all of the safety problems, if

any, associated with its use. It is the responsibility of the user to establish appropriate safety and

health practices and to ensure compliance with any national regulatory conditions.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this

International Standard be carried out by suitably qualified staff.
1 Scope

This International Standard specifies a method for the determination of the lethal effects of toxicants

to Brachionus plicatilis after 24 h or 48 h exposure.
The method is applicable to the following:

a) chemical substances which are soluble or which can be maintained as a stable suspension of

dispersions under the conditions of the test;

b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration, or

centrifugation;
c) marine or estuarine waters;
d) sediment elutriates/eluates.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 5667­16, Water quality — Sampling — Part 16: Guidance on biotesting of samples

ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method

ISO 10523, Water quality — Determination of pH
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
control batch
series of replicates containing control solution
[SOURCE: ISO 6341:2012, 3.1]
3.2

concentration of dilution of the test sample which gives rise to 50 % mortality of the test organisms

© ISO 2015 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO/FDIS 19820:2015(E)
3.3
test batch
series of replicates filled with the same test solution
[SOURCE: ISO 6341:2012, 3.6]
3.4
pure water
deionized or distilled water with a conductivity below 10 µS/cm
4 Principle

The test organisms are exposed to a range of concentrations of the sample under analysis and the

percentage mortality of the test organisms is determined after 24 h exposure and/or 48 h exposure

with subsequent calculation of the 24 h LC and/or the 48 h LC .
50 50

Prolongation of the test to 48 h is recommended since it substantially increases the sensitivity of the assay.

The test is carried out in one or two of the following stages:

— a “range-finding test” to determine the range of concentrations or dilutions needed for calculation

of the 24 h LC or the 48 h LC ;
50 50

— a “definitive test” conducted when the data of the range-finding test are not sufficient or adequate

for calculation of the 24 h LC or the 48 h LC .
50 50
5 Test environment

The test shall be carried out in the dark, in a temperature­controlled room, or incubator at (25 ± 1) °C in

the test containers.

Maintain the atmosphere free from toxic dusts or vapours. The use of control solutions is a double check

that the test is performed in an atmosphere free from toxic dusts and vapours.
6 Reagents, test organisms and media
Use only reagents of recognized analytical grade, unless otherwise specified.
6.1 Test organisms

The tests organisms are females of the species Brachionus plicatilis obtained from a laboratory culture

(see References [5],[7], and [8]) or hatched from commercially available cysts.

The procedure for hatching of Brachionus plicatilis from cysts is described in Annex A.

6.2 Culturing and dilution medium

A natural or an artificial seawater may be used as the water for stock culturing the rotifers or as dilution

water for the testing. Natural seawater shall be collected from an unpolluted location and should have

salinity between 29 ‰ and 36 ‰. Natural seawater must be filtered (30 µm) and conditioned to test

temperature and oxygen saturation prior to use. Natural seawater can be stored cold (4 ± 1) °C for

several weeks.

An example of artificial seawater suitable for culturing and testing is given in Annex B.

1) MicroBioTests Inc. Mariakerke, Belgium, is an example of a supplier able to provide suitable Brachionus plicatilis

cysts commercially. This information is given for the convenience of the users of this document and does not

constitute an endorsement by ISO of this supplier.
2 © ISO 2015 – All rights reserved
---------------------- Page: 7 ----------------------
ISO/FDIS 19820:2015(E)
6.3 Reference substance

Potassium dichromate (K Cr O ) or copper sulfate (CuSO ·5H O) are recommended as reference

2 2 7 4 2
chemicals.

NOTE Since K Cr O is a carcinogenic substance toxic via inhalation, the use of a ready-made solution with

2 2 7

a defined concentration of K Cr O for the preparation of the stock solution of the reference substance can

2 2 7
reduce the risk of inhalation of the toxic dust in the laboratory.
7 Apparatus
Usual laboratory equipment and, in particular, the following.
7.1 Temperature-controlled room or chamber.
7.2 Petri dishes.
Small Petri dishes (diameter 5 cm) in glass or in inert plastic material.
7.3 Test containers.
Disposable 48 (6 × 8) microplates made from chemically inert material.

7.4 Pipette for sampling rotifers, with a sufficient diameter for capturing the animals while allowing

sampling of only a small volume of medium.
For example, single use 1 ml capillary mini-pipettes are suitable.

7.5 Stereomicroscope with incident (bottom) illumination, with a magnification of at least eight

times and, if possible, a continuous magnification.

7.6 Light source, providing a range of light intensity in the hatching Petri dish of 3 000 lx to 4 000 lx

­2 ­1 ­2 ­1
corresponding to 40 µmol.m .s to 55 µmol.m .s .
7.7 Sample collecting bottles, as specified in ISO 5667-16.
8 Treatment and preparation of samples
8.1 Special precautions

Special precautions are required for sampling, transportation, storage, and treatment of seawater or

effluent.

Sampling, transportation, and storage of the samples should be performed as specified in ISO 5667-16.

Carry out the toxicity test as soon as possible, ideally within 12 h of collection. If this time interval

cannot be met, cool the sample to 0 °C to 5 °C and test the sample within 24 h. If it is not possible to

perform the test within 72 h, the sample may be frozen and maintained deep-frozen (below –18 °C)

for testing within two months of collection, provided that characteristics are known to be unaffected

by freezing. At the time of testing, homogenize the sample to be analysed by shaking manually, and, if

necessary, allow to settle for 2 h in a container
...

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