SIST EN ISO 10872:2021

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.

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oSIST prEN ISO 10872:2021

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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

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oSIST prEN 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
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below or ISO’s member body in the country of the requester.
ISO copyright office
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Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
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oSIST prEN ISO 10872:2021
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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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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
1
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 .
2
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 .
4
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;
3
— 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).
TM
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;
2
— cation exchange capacity: (10,0 ± 0,4) mmol /100 g;
c
NOTE mmol /100 g is synonymous with meq/100 g.
c
— 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
50
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
50
range of 20 % to 80 %.
Additionally, the EC of the reference substance shall be determined at least every 12 months. The
50
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).
2)
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
...