Water and soil quality - Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) (ISO 10872:2020)

This document specifies a method for determining the toxicity of environmental
samples on growth, fertility and reproduction of Caenorhabditis elegans. The method applies
to contaminated whole fresh water sediment (maximum salinity 5 ‰), soil and waste, as well
as to pore water, elutriates and aqueous extracts that were obtained from contaminated
sediment, soil and waste.

Wasserbeschaffenheit - Bestimmung der toxischen Wirkung von Sediment- und Bodenproben auf Wachstum, Fertilität und Reproduktion von Caenorhabditis elegans (Nematoda) (ISO 10872:2020)

Dieses Dokument legt ein Verfahren zur Bestimmung der Toxizität von Umweltproben auf Wachstum, Fertilität und Reproduktion von Caenorhabditis elegans fest. Das Verfahren kann sowohl für Süßwassersediment (maximale Salinität 5 g/l), Böden, Abfall, als auch für Porenwasser, Eluate und wässrige Extrakte verwendet werden, die aus kontaminierten Sedimenten, Böden und Abfall extrahiert wurden.
WARNUNG - Anwender dieses Dokuments sollten mit der üblichen Laborpraxis vertraut sein. Dieses Dokument gibt nicht vor, alle unter Umständen mit der Anwendung des Verfahrens verbundenen Sicherheitsaspekte anzusprechen. Es liegt in der Verantwortung des Arbeitsgebers, angemessene Sicherheits- und Schutzmaßnahmen zu treffen und sicherzustellen.
WICHTIG - Es ist erforderlich, bei den Untersuchungen nach diesem Dokument Fachleute oder Facheinrichtungen einzuschalten.

Qualité de l'eau et du sol - Détermination de l'effet toxique d'échantillons de sédiment et de sol sur la croissance, la fertilité et la reproduction de Caenorhabditis elegans (Nematodes) (ISO 10872:2020)

Kakovost vode in tal - Določanje učinkov strupenosti vzorcev usedlin in tal na rast, plodnost in razmnoževanje Caenorhabditis elegans (Nematoda) (ISO 10872:2020)

General Information

Status
Published
Public Enquiry End Date
02-May-2021
Publication Date
07-Sep-2021
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
19-Aug-2021
Due Date
24-Oct-2021
Completion Date
08-Sep-2021

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SLOVENSKI STANDARD
SIST EN ISO 10872:2021
01-oktober-2021
Nadomešča:
SIST ISO 10872:2011
Kakovost vode in tal - Določanje učinkov strupenosti vzorcev usedlin in tal na
rast, plodnost in razmnoževanje Caenorhabditis elegans (Nematoda) (ISO
10872:2020)

Water and soil quality - Determination of the toxic effect of sediment and soil samples on

growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) (ISO
10872:2020)
Wasserbeschaffenheit - Bestimmung der toxischen Wirkung von Sediment- und
Bodenproben auf Wachstum, Fertilität und Reproduktion von Caenorhabditis elegans
(Nematoda) (ISO 10872:2020)

Qualité de l'eau et du sol - Détermination de l'effet toxique d'échantillons de sédiment et

de sol sur la croissance, la fertilité et la reproduction de Caenorhabditis elegans

(Nematodes) (ISO 10872:2020)
Ta slovenski standard je istoveten z: EN ISO 10872:2021
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 10872:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN ISO 10872:2021
---------------------- Page: 2 ----------------------
SIST EN ISO 10872:2021
EN ISO 10872
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2021
EUROPÄISCHE NORM
ICS 13.060.70
English Version
Water and soil quality - Determination of the toxic effect of
sediment and soil samples on growth, fertility and
reproduction of Caenorhabditis elegans (Nematoda) (ISO
10872:2020)

Qualité de l'eau et du sol - Détermination de l'effet Wasserbeschaffenheit und Bodenbeschaffenheit -

toxique d'échantillons de sédiment et de sol sur la Bestimmung der toxischen Wirkung von Sediment-

croissance, la fertilité et la reproduction de und Bodenproben auf Wachstum, Fertilität und

Caenorhabditis elegans (Nematodes) (ISO Reproduktion von Caenorhabditis elegans (Nematoda)

10872:2020) (ISO 10872:2020)
This European Standard was approved by CEN on 18 July 2021.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10872:2021 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN ISO 10872:2021
EN ISO 10872:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

---------------------- Page: 4 ----------------------
SIST EN ISO 10872:2021
EN ISO 10872:2021 (E)
European foreword

The text of ISO 10872:2020 has been prepared by Technical Committee ISO/TC 147 "Water quality” of

the International Organization for Standardization (ISO) and has been taken over as EN ISO 10872:2021

by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by February 2022, and conflicting national standards

shall be withdrawn at the latest by February 2022.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

Any feedback and questions on this document should be directed to the users’ national standards body.

A complete listing of these bodies can be found on the CEN website.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 10872:2020 has been approved by CEN as EN ISO 10872:2021 without any modification.

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SIST EN ISO 10872:2021
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SIST EN ISO 10872:2021
INTERNATIONAL ISO
STANDARD 10872
Second edition
2020-05
Water and soil quality —
Determination of the toxic effect
of sediment and soil samples on
growth, fertility and reproduction of
Caenorhabditis elegans (Nematoda)
Qualité de l'eau et du sol — Détermination de l'effet toxique
d'échantillons de sédiment et de sol sur la croissance, la fertilité et la
reproduction de Caenorhabditis elegans (Nematodes)
Reference number
ISO 10872:2020(E)
ISO 2020
---------------------- Page: 7 ----------------------
SIST EN ISO 10872:2021
ISO 10872:2020(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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 10872:2021
ISO 10872:2020(E)
Contents Page

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

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

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

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

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

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

5 Reagents and media .......................................................................................................................................................................................... 3

6 Apparatus ..................................................................................................................................................................................................................... 5

7 Reference substance ......................................................................................................................................................................................... 6

8 Organisms .................................................................................................................................................................................................................. 7

8.1 Test organism ........................................................................................................................................................................................... 7

8.2 Food organism ......................................................................................................................................................................................... 7

9 Stock- and pre-cultures .................................................................................................................................................................................. 7

9.1 Stock cultures ........................................................................................................................................................................................... 7

9.1.1 Caenorhabditis elegans .......................................................................................................................................... 7

9.1.2 Escherichia coli................................................................................................................................................................ 7

9.2 Pre-culture .................................................................................................................................................................................................. 8

10 Procedure..................................................................................................................................................................................................................... 8

10.1 Preparation of food medium ....................................................................................................................................................... 8

10.2 Preparation of test material and controls ........................................................................................................................ 8

10.2.1 Soil ................................................................................................................................................................................................ 8

10.2.2 Sediment ................................................................................................................................................................................. 9

10.2.3 Pore water, elutriate, extract.................................................................................................................................. 9

10.2.4 Solution of reference substance .................. ........................................................................................................ 9

10.3 Test .................................................................................................................................................................................................................10

10.4 Nematode separation .....................................................................................................................................................................10

10.5 Measurements and calculations ............................................................................................................................................10

10.5.1 Recovery ...............................................................................................................................................................................10

10.5.2 Males .......................................................................................................................................................................................10

10.5.3 Growth and fertility ....................................................................................................................................................11

10.5.4 Reproduction ...................................................................................................................................................................11

10.6 Timetable of the test .......................................................................................................................................................................12

11 Validity criteria ...................................................................................................................................................................................................13

12 Expression of results .....................................................................................................................................................................................13

13 Test report ................................................................................................................................................................................................................14

Annex A (informative) Determination of maximal water holding capacity (WHC ) ...................................15

max

Annex B (informative) Figures and photos of adult worms C. elegans ...........................................................................16

Annex C (informative) Micropipette method (10.3) ..........................................................................................................................18

Annex D (informative) Performance data ....................................................................................................................................................19

Bibliography .............................................................................................................................................................................................................................22

© ISO 2020 – All rights reserved iii
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SIST EN ISO 10872:2021
ISO 10872:2020(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 147, Water quality, Subcommittee SC 5,

Biological method.

This second edition cancels and replaces the first edition (ISO 10872:2010), which has been technically

revised. The main changes compared to the previous edition are as follows:

— the title has been changed to achieve a better perception in the field soil toxicity testing;

— for soil testing, the method has been modified in terms of a reduced water content of the test

material;
— cited references and standards have been refreshed;

— information on the control soil and restrictions for tested soils has been added;

— the document has been editorially revised.

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
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SIST EN ISO 10872:2021
ISO 10872:2020(E)
Introduction
[1] [2]

Nematodes are one of the most abundant and species-rich metazoans in sediments and soils and

possess key positions in benthic and soil food webs due to the evolution of various feeding types

(bacterial, algal, fungal and plant feeders, omnivores, predators see References [3] and [4]). Moreover,

they are well acknowledged as environmental indicators for assessing the toxicity of chemicals and the

quality of sediments and soils (see References [5], [6], [7], [8] and [9]).

The test organism Caenorhabditis elegans (Maupas, N2 var. Bristol) is a bacterivorous nematode that is

found primarily in microbe-rich, decaying plant material (see Reference [10]) and belongs to the family

of Rhabditidae, frequently found in terrestrial soils and aquatic sediments (see References [11] and

[12]). Moreover, individuals of C. elegans were already found in sediments of polysaprobial fresh-water

[15]

systems (see References [13] and [14]). Due to its easy cultivation and short life cycle , C. elegans has

become a well-studied model organism in biomedical and ecotoxicological research (see References [16],

[17] and [18]).
[19]

The test is designed for measurement of the response to dissolved and particle-bound substances .

It applies to the testing of sediments, soils, waste, pore water, elutriates and aqueous extracts (see

e.g. References [20], [21], [22] and [23]).
© ISO 2020 – All rights reserved v
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SIST EN ISO 10872:2021
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SIST EN ISO 10872:2021
INTERNATIONAL STANDARD ISO 10872:2020(E)
Water and soil quality — Determination of the toxic effect
of sediment and soil samples on growth, fertility and
reproduction of Caenorhabditis elegans (Nematoda)

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.

IMPORTANT — It is absolutely essential that tests conducted according to this document be

carried out by suitably trained staff.
1 Scope

This document specifies a method for determining the toxicity of environmental samples on growth,

fertility and reproduction of Caenorhabditis elegans. The method applies to contaminated whole

freshwater sediment (maximum salinity 5 g/l), soil and waste, as well as to pore water, elutriates and

aqueous extracts that were obtained from contaminated sediment, soil and waste.
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 5667-16, Water quality — Sampling — Part 16: Guidance on biotesting of samples

ISO 7027-2, Water quality — Determination of turbidity — Part 2: Semi-quantitative methods for the

assessment of transparency of waters
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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
agar plate
Petri dish filled with Nematode Growth Medium (NGM) agar
3.2
aqueous control
water that serves as negative control for tests in aqueous samples
3.3
artificial control sediment
defined artificial sediment
3.4
bacterial stock culture
stock culture of food bacteria
© ISO 2020 – All rights reserved 1
---------------------- Page: 13 ----------------------
SIST EN ISO 10872:2021
ISO 10872:2020(E)
3.5
blank replicate

additional replicate that contains no test organism, but is treated in the same way as the other replicates

of a sample
3.6
control

treatment that serves as negative control to which the effect in the respective test material is compared

3.7
control soil
defined standard soil
3.8
dauer larva
developmental stage adopted by C. elegans to endure periods of lack of food
Note 1 to entry: Dauer larvae continue normal development if food is supplied.
3.9
exposed test organisms
individuals of C. elegans that are introduced at the beginning of the test
3.10
food medium
defined aqueous bacterial suspension
3.11
J stage
first of four juvenile stages (J to J ) in the development of C. elegans
1 4
3.12
maximal water holding capacity
WHC
max

maximal amount of water a soil sample is able to take up and keep against gravity

3.13
overnight culture
defined culture of Escherichia coli in Lysogeny Broth (LB)-medium
3.14
starved plate
agar plate (3.1) with dauer larvae
3.15
test material

discrete portion of a contaminated environmental sample or solution of the reference substance

4 Principle

Juvenile organisms of the species C. elegans are exposed to the environmental sample over a period

of 96 h. In the controls, the exposed test organisms are able to complete a whole life cycle within this

period. Toxicity can be quantified by the intensity of the effect as percentage inhibition. A toxic effect

of an environmental sample occurs if the inhibition of growth, fertility or reproduction of C. elegans

in comparison to a control (aqueous control, control sediment or soil) exceeds a certain threshold

value (e.g. as proposed in the following publications: aqueous medium: 10 %, 20 %, 10 % inhibition,

respectively (see Reference [24]); freshwater sediments: 25 %, 20 %, 50 % inhibition, respectively

(see Reference [21]); soil: 10 %, 20 %, 40 % inhibition, respectively (see Reference [20]), and the

performance of toxicity endpoints in the test material is statistically significantly lower compared to

the control performance (p <0,05).
2 © ISO 2020 – All rights reserved
---------------------- Page: 14 ----------------------
SIST EN ISO 10872:2021
ISO 10872:2020(E)
5 Reagents and media
Use only reagents of recognized analytical grade.

5.1 Water, distilled or deionized water or water of equivalent purity, conductivity ≤10 µS/cm.

5.2 LB-medium
Dissolve
— 0,5 g of casein peptone;
— 0,25 g of yeast extract;
— 0,5 g of sodium chloride (NaCl);
in 50 ml water in a 250 ml flask and autoclave for 20 min at 121 °C.
5.3 Cholesterol stock solution

Dissolve 500 mg of powdered cholesterol in 100 ml of absolute ethanol (>99 % purity) by stirring and

gentle heating (<50 °C). Replace ethanol lost through evaporation with ethanol.
5.4 Calcium chloride stock solution, 1 mol/l CaCl .
Dissolve 7,35 g of CaCl ∙2H O in 50 ml water and autoclave for 20 min at 121 °C.
2 2
5.5 Magnesium sulfate stock solution, 1 mol/l MgSO .

Dissolve 12,35 g of MgSO ∙7H O in 50 ml water and autoclave for 20 min at 121 °C.

4 2
5.6 Potassium hydroxide, KOH, pellets.
5.7 Potassium phosphate buffer, 1 mol/l KH PO .
2 4

Dissolve 13,6 g of KH PO in 100 ml of water, adjust with KOH (5.6) to pH 6,0 ± 0,2, and autoclave for

2 4
20 min at 121 °C.
5.8 Nematode growth-medium agar (NGM agar)
Dissolve
— 2,5 g of casein peptone;
— 17 g of bacteriological agar;
— 3 g of NaCl;

in 900 ml water in a 1 000 ml flask and autoclave for 20 min at 121 °C. After cooling down to 55 °C, add

the following sterile solutions:
— 1 ml of cholesterol stock solution (see 5.3);
— 1 ml of calcium chloride stock solution (see 5.4);
— 1 ml of magnesium sulfate stock solution (see 5.5);
— 25 ml of potassium phosphate buffer (see 5.7);
and fill up to 1 000 ml with sterile water.
© ISO 2020 – All rights reserved 3
---------------------- Page: 15 ----------------------
SIST EN ISO 10872:2021
ISO 10872:2020(E)
Transfer portions of NGM agar (about 20 ml to 25 ml) to sterile Petri dishes.
5.9 M9-medium
Dissolve
— 6 g of Na HPO ;
2 4
— 3 g of KH PO ;
2 4
— 5 g of NaCl;
— 0,25 g of MgSO ∙7H O;
4 2
in 1 000 ml of water in a 1 000 ml flask.
5.10 Bengal Rose stock solution
Add approximately 30 mg of Bengal Rose to 100 ml of water and stir thoroughly.
5.11 Ludox suspension
1) 3

Dilute Ludox TM 50 (colloidal silica; density: 1,4 g/cm ) with water to a density of (1,13 ± 0,005) g/

3 1)

cm [mix approximately 1 part Ludox TM 50 with 2 parts of water and control the density by weighing

1 ml of the suspension on a balance; 1 ml of the suspension weighs (1,13 ± 0,005) g]. For one 12 or 24-

well multidish, approximately 75 ml or 150 ml of Ludox-suspension are required, respectively.

5.12 Artificial control sediment
Mix the following components thoroughly in the given proportions:
— Al O , 20 % mass fraction;
2 3
— CaCO , 1 % mass fraction;
— dolomite (clay), 0,5 % mass fraction;
— Fe O , 4,5 % mass fraction;
2 3

— silica sand (for example: W4, mean particle size: 0,063 mm), 30 % mass fraction;

— silica sand (0,1 mm to 0,4 mm), 40 % mass fraction;

— peat (decomposed peat from a raised bog, untreated; finely ground and <1 mm sieved), 4 % mass

fraction.
The dry sediment is maintainable without restraint.
This sediment serves as negative control for tests with sediments.

WARNING — Artificial sediments with a kaolin content of >5 % mass fraction (e.g. OECD 218) can

cause deleterious effects on growth, fertility and reproduction of C. elegans. If using a different

artificial control sediment than proposed in this standard, the kaolin content shall be ≤5 %

mass fraction.

WARNING — If available, the use of a site-specific reference sediment is advised, additionally

to the artificial control sediment; the reference sediment shall match following criteria: limit

of contamination; important sediment properties shall be similar to the tested sample (e.g.

particle size distribution, organic content).

1) Ludox 50 is an example of a suitable product available commercially. This information is given for the

convenience of users of this document and does not constitute an endorsement by ISO of this product.

4 © ISO 2020 – All rights reserved
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SIST EN ISO 10872:2021
ISO 10872:2020(E)
5.13 Control soil
Standard soil St. 2.2 from LUFA:
— soil type: loamy sand;
— organic carbon: (2,0 ± 0,5) % mass fraction;
— pH (CaCl ): 5,5 ± 0,5;
— cation exchange capacity: (10,0 ± 0,4) mmol /100 g;
NOTE mmol /100 g is synonymous with meq/100 g.
— water holding capacity: (48,2 ± 5) %;
— clay content: (7,5 ± 2,5) % mass fraction particles <0,002 mm;
— silt content: (12,5 ± 2,5) % mass fraction particles 0,002 mm to 0,063 mm;
— sand content: (80,0 ± 5,0) % mass fraction particles 0,063 mm to 2 mm.
This soil serves as negative control for tests with soil.

NOTE LUFA refers to Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer.

WARNING — If tested soils contain >30 % clay even in uncontaminated soils, inhibiting effects

on C. elegans’ growth and reproduction might exceed the toxicity threshold. In these cases, a

reference soil with a similar clay content shall be tested additionally to the control soil.

WARNING — Artificial soils with a kaolin content of >5 % mass fraction (e.g. OECD 207) can

cause deleterious effects on growth, fertility and reproduction of C. elegans. If using a different

artificial control soil than proposed in this standard, the kaolin content shall be ≤5 % mass

fraction.
5.14 Benzylcetyldimethylammonium chloride monohydrate (BAC-C16) stock solution
Dissolve 30 mg of BAC-C16 (C H CIN∙H O; CAS No.: 122-18-9) in 1 000 ml of water.
25 46 2
5.15 Glycerol (>98 %; Ph.Eu., water free)
5.16 Formazin: Turbidity 4000 NTU calibration standard
6 Apparatus
6.1 Autoclave.

6.2 Facilities, with constant temperature for 20 °C and 37 °C, for example incubator or temperature-

controlled chamber.
6.3 Drigalski spatula,
...

SLOVENSKI STANDARD
oSIST prEN ISO 10872:2021
01-april-2021
Kakovost vode in tal - Določanje učinkov strupenosti vzorcev usedlin in tal na
rast, plodnost in razmnoževanje Caenorhabditis elegans (Nematoda) (ISO
10872:2020)

Water and soil quality - Determination of the toxic effect of sediment and soil samples on

growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) (ISO
10872:2020)
Wasserbeschaffenheit - Bestimmung der toxischen Wirkung von Sediment- und
Bodenproben auf Wachstum, Fertilität und Reproduktion von Caenorhabditis elegans
(Nematoda) (ISO 10872:2020)

Qualité de l'eau et du sol - Détermination de l'effet toxique d'échantillons de sédiment et

de sol sur la croissance, la fertilité et la reproduction de Caenorhabditis elegans

(Nematodes) (ISO 10872:2020)
Ta slovenski standard je istoveten z: prEN ISO 10872
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST prEN ISO 10872:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 10872:2021
---------------------- Page: 2 ----------------------
oSIST prEN ISO 10872:2021
INTERNATIONAL ISO
STANDARD 10872
Second edition
2020-05
Water and soil quality —
Determination of the toxic effect
of sediment and soil samples on
growth, fertility and reproduction of
Caenorhabditis elegans (Nematoda)
Qualité de l'eau et du sol — Détermination de l'effet toxique
d'échantillons de sédiment et de sol sur la croissance, la fertilité et la
reproduction de Caenorhabditis elegans (Nematodes)
Reference number
ISO 10872:2020(E)
ISO 2020
---------------------- Page: 3 ----------------------
oSIST prEN ISO 10872:2021
ISO 10872:2020(E)
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oSIST prEN ISO 10872:2021
ISO 10872:2020(E)
Contents Page

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

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

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

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

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

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

5 Reagents and media .......................................................................................................................................................................................... 3

6 Apparatus ..................................................................................................................................................................................................................... 5

7 Reference substance ......................................................................................................................................................................................... 6

8 Organisms .................................................................................................................................................................................................................. 7

8.1 Test organism ........................................................................................................................................................................................... 7

8.2 Food organism ......................................................................................................................................................................................... 7

9 Stock- and pre-cultures .................................................................................................................................................................................. 7

9.1 Stock cultures ........................................................................................................................................................................................... 7

9.1.1 Caenorhabditis elegans .......................................................................................................................................... 7

9.1.2 Escherichia coli................................................................................................................................................................ 7

9.2 Pre-culture .................................................................................................................................................................................................. 8

10 Procedure..................................................................................................................................................................................................................... 8

10.1 Preparation of food medium ....................................................................................................................................................... 8

10.2 Preparation of test material and controls ........................................................................................................................ 8

10.2.1 Soil ................................................................................................................................................................................................ 8

10.2.2 Sediment ................................................................................................................................................................................. 9

10.2.3 Pore water, elutriate, extract.................................................................................................................................. 9

10.2.4 Solution of reference substance .................. ........................................................................................................ 9

10.3 Test .................................................................................................................................................................................................................10

10.4 Nematode separation .....................................................................................................................................................................10

10.5 Measurements and calculations ............................................................................................................................................10

10.5.1 Recovery ...............................................................................................................................................................................10

10.5.2 Males .......................................................................................................................................................................................10

10.5.3 Growth and fertility ....................................................................................................................................................11

10.5.4 Reproduction ...................................................................................................................................................................11

10.6 Timetable of the test .......................................................................................................................................................................12

11 Validity criteria ...................................................................................................................................................................................................13

12 Expression of results .....................................................................................................................................................................................13

13 Test report ................................................................................................................................................................................................................14

Annex A (informative) Determination of maximal water holding capacity (WHC ) ...................................15

max

Annex B (informative) Figures and photos of adult worms C. elegans ...........................................................................16

Annex C (informative) Micropipette method (10.3) ..........................................................................................................................18

Annex D (informative) Performance data ....................................................................................................................................................19

Bibliography .............................................................................................................................................................................................................................22

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oSIST prEN ISO 10872:2021
ISO 10872:2020(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 147, Water quality, Subcommittee SC 5,

Biological method.

This second edition cancels and replaces the first edition (ISO 10872:2010), which has been technically

revised. The main changes compared to the previous edition are as follows:

— the title has been changed to achieve a better perception in the field soil toxicity testing;

— for soil testing, the method has been modified in terms of a reduced water content of the test

material;
— cited references and standards have been refreshed;

— information on the control soil and restrictions for tested soils has been added;

— the document has been editorially revised.

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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Introduction
[1] [2]

Nematodes are one of the most abundant and species-rich metazoans in sediments and soils and

possess key positions in benthic and soil food webs due to the evolution of various feeding types

(bacterial, algal, fungal and plant feeders, omnivores, predators see References [3] and [4]). Moreover,

they are well acknowledged as environmental indicators for assessing the toxicity of chemicals and the

quality of sediments and soils (see References [5], [6], [7], [8] and [9]).

The test organism Caenorhabditis elegans (Maupas, N2 var. Bristol) is a bacterivorous nematode that is

found primarily in microbe-rich, decaying plant material (see Reference [10]) and belongs to the family

of Rhabditidae, frequently found in terrestrial soils and aquatic sediments (see References [11] and

[12]). Moreover, individuals of C. elegans were already found in sediments of polysaprobial fresh-water

[15]

systems (see References [13] and [14]). Due to its easy cultivation and short life cycle , C. elegans has

become a well-studied model organism in biomedical and ecotoxicological research (see References [16],

[17] and [18]).
[19]

The test is designed for measurement of the response to dissolved and particle-bound substances .

It applies to the testing of sediments, soils, waste, pore water, elutriates and aqueous extracts (see

e.g. References [20], [21], [22] and [23]).
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oSIST prEN ISO 10872:2021
INTERNATIONAL STANDARD ISO 10872:2020(E)
Water and soil quality — Determination of the toxic effect
of sediment and soil samples on growth, fertility and
reproduction of Caenorhabditis elegans (Nematoda)

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.

IMPORTANT — It is absolutely essential that tests conducted according to this document be

carried out by suitably trained staff.
1 Scope

This document specifies a method for determining the toxicity of environmental samples on growth,

fertility and reproduction of Caenorhabditis elegans. The method applies to contaminated whole

freshwater sediment (maximum salinity 5 g/l), soil and waste, as well as to pore water, elutriates and

aqueous extracts that were obtained from contaminated sediment, soil and waste.
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 5667-16, Water quality — Sampling — Part 16: Guidance on biotesting of samples

ISO 7027-2, Water quality — Determination of turbidity — Part 2: Semi-quantitative methods for the

assessment of transparency of waters
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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
agar plate
Petri dish filled with Nematode Growth Medium (NGM) agar
3.2
aqueous control
water that serves as negative control for tests in aqueous samples
3.3
artificial control sediment
defined artificial sediment
3.4
bacterial stock culture
stock culture of food bacteria
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3.5
blank replicate

additional replicate that contains no test organism, but is treated in the same way as the other replicates

of a sample
3.6
control

treatment that serves as negative control to which the effect in the respective test material is compared

3.7
control soil
defined standard soil
3.8
dauer larva
developmental stage adopted by C. elegans to endure periods of lack of food
Note 1 to entry: Dauer larvae continue normal development if food is supplied.
3.9
exposed test organisms
individuals of C. elegans that are introduced at the beginning of the test
3.10
food medium
defined aqueous bacterial suspension
3.11
J stage
first of four juvenile stages (J to J ) in the development of C. elegans
1 4
3.12
maximal water holding capacity
WHC
max

maximal amount of water a soil sample is able to take up and keep against gravity

3.13
overnight culture
defined culture of Escherichia coli in Lysogeny Broth (LB)-medium
3.14
starved plate
agar plate (3.1) with dauer larvae
3.15
test material

discrete portion of a contaminated environmental sample or solution of the reference substance

4 Principle

Juvenile organisms of the species C. elegans are exposed to the environmental sample over a period

of 96 h. In the controls, the exposed test organisms are able to complete a whole life cycle within this

period. Toxicity can be quantified by the intensity of the effect as percentage inhibition. A toxic effect

of an environmental sample occurs if the inhibition of growth, fertility or reproduction of C. elegans

in comparison to a control (aqueous control, control sediment or soil) exceeds a certain threshold

value (e.g. as proposed in the following publications: aqueous medium: 10 %, 20 %, 10 % inhibition,

respectively (see Reference [24]); freshwater sediments: 25 %, 20 %, 50 % inhibition, respectively

(see Reference [21]); soil: 10 %, 20 %, 40 % inhibition, respectively (see Reference [20]), and the

performance of toxicity endpoints in the test material is statistically significantly lower compared to

the control performance (p <0,05).
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5 Reagents and media
Use only reagents of recognized analytical grade.

5.1 Water, distilled or deionized water or water of equivalent purity, conductivity ≤10 µS/cm.

5.2 LB-medium
Dissolve
— 0,5 g of casein peptone;
— 0,25 g of yeast extract;
— 0,5 g of sodium chloride (NaCl);
in 50 ml water in a 250 ml flask and autoclave for 20 min at 121 °C.
5.3 Cholesterol stock solution

Dissolve 500 mg of powdered cholesterol in 100 ml of absolute ethanol (>99 % purity) by stirring and

gentle heating (<50 °C). Replace ethanol lost through evaporation with ethanol.
5.4 Calcium chloride stock solution, 1 mol/l CaCl .
Dissolve 7,35 g of CaCl ∙2H O in 50 ml water and autoclave for 20 min at 121 °C.
2 2
5.5 Magnesium sulfate stock solution, 1 mol/l MgSO .

Dissolve 12,35 g of MgSO ∙7H O in 50 ml water and autoclave for 20 min at 121 °C.

4 2
5.6 Potassium hydroxide, KOH, pellets.
5.7 Potassium phosphate buffer, 1 mol/l KH PO .
2 4

Dissolve 13,6 g of KH PO in 100 ml of water, adjust with KOH (5.6) to pH 6,0 ± 0,2, and autoclave for

2 4
20 min at 121 °C.
5.8 Nematode growth-medium agar (NGM agar)
Dissolve
— 2,5 g of casein peptone;
— 17 g of bacteriological agar;
— 3 g of NaCl;

in 900 ml water in a 1 000 ml flask and autoclave for 20 min at 121 °C. After cooling down to 55 °C, add

the following sterile solutions:
— 1 ml of cholesterol stock solution (see 5.3);
— 1 ml of calcium chloride stock solution (see 5.4);
— 1 ml of magnesium sulfate stock solution (see 5.5);
— 25 ml of potassium phosphate buffer (see 5.7);
and fill up to 1 000 ml with sterile water.
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Transfer portions of NGM agar (about 20 ml to 25 ml) to sterile Petri dishes.
5.9 M9-medium
Dissolve
— 6 g of Na HPO ;
2 4
— 3 g of KH PO ;
2 4
— 5 g of NaCl;
— 0,25 g of MgSO ∙7H O;
4 2
in 1 000 ml of water in a 1 000 ml flask.
5.10 Bengal Rose stock solution
Add approximately 30 mg of Bengal Rose to 100 ml of water and stir thoroughly.
5.11 Ludox suspension
1) 3

Dilute Ludox TM 50 (colloidal silica; density: 1,4 g/cm ) with water to a density of (1,13 ± 0,005) g/

3 1)

cm [mix approximately 1 part Ludox TM 50 with 2 parts of water and control the density by weighing

1 ml of the suspension on a balance; 1 ml of the suspension weighs (1,13 ± 0,005) g]. For one 12 or 24-

well multidish, approximately 75 ml or 150 ml of Ludox-suspension are required, respectively.

5.12 Artificial control sediment
Mix the following components thoroughly in the given proportions:
— Al O , 20 % mass fraction;
2 3
— CaCO , 1 % mass fraction;
— dolomite (clay), 0,5 % mass fraction;
— Fe O , 4,5 % mass fraction;
2 3

— silica sand (for example: W4, mean particle size: 0,063 mm), 30 % mass fraction;

— silica sand (0,1 mm to 0,4 mm), 40 % mass fraction;

— peat (decomposed peat from a raised bog, untreated; finely ground and <1 mm sieved), 4 % mass

fraction.
The dry sediment is maintainable without restraint.
This sediment serves as negative control for tests with sediments.

WARNING — Artificial sediments with a kaolin content of >5 % mass fraction (e.g. OECD 218) can

cause deleterious effects on growth, fertility and reproduction of C. elegans. If using a different

artificial control sediment than proposed in this standard, the kaolin content shall be ≤5 %

mass fraction.

WARNING — If available, the use of a site-specific reference sediment is advised, additionally

to the artificial control sediment; the reference sediment shall match following criteria: limit

of contamination; important sediment properties shall be similar to the tested sample (e.g.

particle size distribution, organic content).

1) Ludox 50 is an example of a suitable product available commercially. This information is given for the

convenience of users of this document and does not constitute an endorsement by ISO of this product.

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5.13 Control soil
Standard soil St. 2.2 from LUFA:
— soil type: loamy sand;
— organic carbon: (2,0 ± 0,5) % mass fraction;
— pH (CaCl ): 5,5 ± 0,5;
— cation exchange capacity: (10,0 ± 0,4) mmol /100 g;
NOTE mmol /100 g is synonymous with meq/100 g.
— water holding capacity: (48,2 ± 5) %;
— clay content: (7,5 ± 2,5) % mass fraction particles <0,002 mm;
— silt content: (12,5 ± 2,5) % mass fraction particles 0,002 mm to 0,063 mm;
— sand content: (80,0 ± 5,0) % mass fraction particles 0,063 mm to 2 mm.
This soil serves as negative control for tests with soil.

NOTE LUFA refers to Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer.

WARNING — If tested soils contain >30 % clay even in uncontaminated soils, inhibiting effects

on C. elegans’ growth and reproduction might exceed the toxicity threshold. In these cases, a

reference soil with a similar clay content shall be tested additionally to the control soil.

WARNING — Artificial soils with a kaolin content of >5 % mass fraction (e.g. OECD 207) can

cause deleterious effects on growth, fertility and reproduction of C. elegans. If using a different

artificial control soil than proposed in this standard, the kaolin content shall be ≤5 % mass

fraction.
5.14 Benzylcetyldimethylammonium chloride monohydrate (BAC-C16) stock solution
Dissolve 30 mg of BAC-C16 (C H CIN∙H O; CAS No.: 122-18-9) in 1 000 ml of water.
25 46 2
5.15 Glycerol (>98 %; Ph.Eu., water free)
5.16 Formazin: Turbidity 4000 NTU calibration standard
6 Apparatus
6.1 Autoclave.

6.2 Facilities, with constant temperature for 20 °C and 37 °C, for example incubator or temperature-

controlled chamber.
6.3 Drigalski spatula, glass spatula for distributing bacteria on an agar plate.
6.4 Erlenmeyer flasks, for example volume 250 ml.
6.5 Plastic vials, autoclavable and sealed, volume 1,5 ml.
6.6 Filter gauze, 5 µm, 10 µm.
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6.7 Freezer, capable of maintaining temperature at −20 °C.
6.8 Micropipette (see Annex C).

Draw a Pasteur pipette over a Bunsen burner to a thin capillary. Plug the Pasteur pipette in a suction

cup at the thicker end.
6.9 Microscope, 100-fold magnification, with measurement scale.
6.10 Thermometer, minimum-maximum.
6.11 Shaker, for 250 ml Erlenmeyer flasks.
6.12 Stereo microscope, 4-fold to 20-fold magnification, with transmitted light.
6.13 Clean bench.
6.14 Sterile Petri dishes, of diameters 3 cm, 6 cm or 10 cm.
6.15 Spectrophotometer, capable of operating at wavelength 600 nm.
6.16 Test tube mixer.
6.17 Balance, 0,001 g readability.
6.18 Drying oven, approximately 80 °C.
2 2

6.19 Multidishes, with 12 wells, 3,5 cm /well (sediment, water); with 24 wells, 1,9 cm /well (soil).

6.20 Centrifuge tubes, 10 ml to 15 ml.
6.21 Centrifuge with swing-out rotor.
6.22 Piston pipettes, 10 ml to 100 ml, 100 ml to 1 000 ml.
6.23 Sieves, 1 mm and 2 mm.
6.24 Magnetic stirrer and magnetic stirring bar.
6.25 pH-meter.
6.26 Inoculating loop.
7 Reference substance

To ensure that the laboratory test conditions (including the condition and sensitivity of the exposed

test organisms) are adequate and have not changed significantly, the laboratory shall test a reference

substance as a positive control in parallel with each test, using one concentration near the EC for

growth. The test parameters “fertility” and “reproduction” are not analysed when testing the reference

substance. Use benzylcetyldimethylammonium chloride monohydrate (BAC-C16; 5.14), which has been

shown to affect growth of C. elegans, as reference substance. The positive control is tested in water

according to the instructions for testing aqueous substrates (10.1, 10.2.2, 10.3). The inhibition of

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growth at a concentration of 15 mg/l (EC for BAC-C16, 5.14) compared to the control should be in the

range of 20 % to 80 %.

Additionally, the EC of the reference substance shall be determined at least every 12 months. The

EC (growth) in water shall be in the range of 8 mg to 22 mg BAC/l using an EC design as specified

50 x

in ISO 5667-16. Stock solutions of BAC in the concentrations of 7,1 mg/l, 10,6 mg/l, 16 mg/l, 24 mg/l,

36 mg/l, 54 mg/l and 81 mg/l are prepared in water and tested according to the instructions for testing

aqueous substrates (see 10.1, 10.2.2, 10.3).
8 Organisms
8.1 Test organism

Caenorhabditis elegans (Maupas, 1899) is a widespread, free-living soil nematode that feeds primarily

on bacteria. Adult worms are about 1,0 mm to 1,5 mm in length and can be distinguished into

hermaphrodites and rarely occurring males (see Annex B). Hermaphrodites usually reproduce by

self-fertilization although they can also be fertilized by males. After hatching, C. elegans develops to

the adult stage through four juvenile stages separated by moults. Under starvation conditions, a

developmentally arrested stage, the dauer larva, can be formed as an alternative third larval stage. The

life cycle for worms grown on E. coli is about 3 d at 20 °C. The biology of C. elegans has been extensively

studied and, in many respects, it is the most thoroughly characterized animal. The “wild type” strain

N2 is used as test organism.
8.2 Food organism

As food organism for C. elegans, the bacterium E. coli (OP50; uracil-deficient strain) is used.

9 Stock- and pre-cultures
9.1 Stock cultures
9.1.1 Caenorhabditis elegans

C. elegans is maintained on agar plates (3.1) with a bacterial lawn (E. coli OP 50; 8.2) at (20 ± 2) °C.

When bacteria are used up, C. elegans forms dauer larvae (3.8) due to lack of food. These starved plates

(3.14) serve as stock cultures for C. elegans that should be replenished every two months. If too many

males occur (≥10 % in tests), new stock cultures should be ordered.
9.1.2 Escherichia coli

Inoculate under sterile conditions 50 ml of LB-medium (5.2) in a 250 ml Erlenmeyer flask (6.4

...

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