ISO 10872:2010
(Main)Water quality — Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda)
Water quality — Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda)
ISO 10872:2010 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.
Qualité de l'eau — Détermination de l'effet toxique d'échantillons de sédiment et de sol sur la croissance, la fertilité et la reproduction de Caenorhabditis elegans (nématodes)
L'ISO 10872:2010 spécifie une méthode de détermination de la toxicité d'échantillons environnementaux sur la croissance, la fertilité et la reproduction de Caenorhabditis elegans. La méthode s'applique aux sédiments d'eau douce (salinité maximale de 5 ‰), sols et déchets contaminés, ainsi qu'à l'eau interstitielle, aux élutriats et aux extraits aqueux obtenus à partir de sédiments, sols et déchets contaminés.
Kakovost vode - Določevanje učinkov strupenosti vzorcev usedlin in tal na rast, plodnost in razmnoževanje Caenorhabditis elegans (Nematoda)
Ta mednarodni standard opredeljuje metodo določevanja strupenosti okoljskih vzorcev rasti, plodnosti in razmoževanja Caenorhabditis elegans. Ta metoda velja za kontaminiran celoten slatkovodni sediment (največja slanost 5 %), tla in odpadke ter tudi za vodo v razpokah, elutriate in vodne ekstrakte, ki so bili pridobljeni iz kontaminiranega sedimenta, tal in odpadkov.
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Standards Content (Sample)
SLOVENSKI STANDARD
SIST ISO 10872:2011
01-junij-2011
.DNRYRVWYRGH'RORþHYDQMHXþLQNRYVWUXSHQRVWLY]RUFHYXVHGOLQLQWDOQDUDVW
SORGQRVWLQUD]PQRåHYDQMH&DHQRUKDEGLWLVHOHJDQV 1HPDWRGD
Water quality - Determination of the toxic effect of sediment and soil samples on growth,
fertility and reproduction of Caenorhabditis elegans (Nematoda)
Qualité de l'eau - Détermination de l'effet toxique d'échantillons de sédiment et de sol sur
la croissance, la fertilité et la reproduction de Caenorhabditis elegans (nématodes)
Ta slovenski standard je istoveten z: ISO 10872:2010
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST ISO 10872:2011 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST ISO 10872:2011
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SIST ISO 10872:2011
INTERNATIONAL ISO
STANDARD 10872
First edition
2010-06-15
Water quality — Determination of the
toxic effect of sediment and soil samples
on growth, fertility and reproduction of
Caenorhabditis elegans (Nematoda)
Qualité de l'eau — 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 10872:2010(E)
©
ISO 2010
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SIST ISO 10872:2011
ISO 10872:2010(E)
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ii © ISO 2010 – All rights reserved
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SIST ISO 10872:2011
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Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Normative references.1
3 Terms and definitions .1
4 Principle.3
5 Reagents.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
9.2 Pre-culture.7
10 Procedure.8
10.1 Preparation of food medium.8
10.2 Preparation of test material and controls .8
10.3 Test .9
10.4 Nematode separation.9
10.5 Measurements and calculations .9
10.6 Timetable of the test.11
11 Validity criteria.12
12 Expression of results.12
13 Test report.13
Annex A (informative) Figures of adult worms C. elegans .14
Annex B (informative) Precision data .15
Bibliography.17
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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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 10872 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2010 – All rights reserved
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Introduction
[1][2]
Nematodes are the most abundant and species-rich group of metazoans in sediments and soils and play
[3][4]
an important role in benthic and soil food webs . Nematodes are endobenthic organisms that are found at
various trophic levels due to the evolution of different feeding types (bacterivorous, algal feeder, omnivorous,
predators).
The test organism Caenorhabditis elegans (Maupas, N2 var. Bristol) is a bacterivorous nematode that is found
[5][6]
primarily in terrestrial soils but it also occurs in aquatic sediments of polysaprobial fresh-water systems .
[7]
C. elegans is a well-studied organism and very easy to cultivate .
[8][9][10]
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.
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SIST ISO 10872:2011
INTERNATIONAL STANDARD ISO 10872:2010(E)
Water quality — Determination of the toxic effect of sediment
and soil samples on growth, fertility and reproduction of
Caenorhabditis elegans (Nematoda)
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 according to this International Standard
be carried out by suitably trained staff.
1 Scope
This International Standard 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.
2 Normative references
The following referenced documents are indispensable for the application 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, Water quality — Determination of turbidity
ISO 10390, Soil quality — Determination of pH
ISO 10523, Water quality — Determination of pH
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric
method
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
agar plate
Petri dish filled with NGM agar (5.8)
3.2
aqueous control
water that serves as negative control for tests in aqueous samples
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3.3
artificial control sediment
defined artificial sediment (5.12)
3.4
bacterial stock culture
stock culture of food bacteria
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 (3.2,
3.3, 3.7)
3.7
control soil
defined standard soil (5.13)
3.8
dauer larva
developmental stage adopted by C. elegans to endure periods of lack of food
NOTE 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 (10.1)
3.11
J stage
1
first of four juvenile stages (J to J ) in the development of C. elegans
1 4
3.12
overnight culture
defined culture of Escherichia coli in LB-medium (9.1.2)
3.13
starved plate
agar plate with dauer larvae
3.14
test material
discrete portion of a contaminated environmental sample (10.2) or solution of the reference substance
(Clause 7)
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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. 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. Toxicity
can by quantified by the intensity of the effect as percentage inhibition.
5 Reagents
Use only reagents of recognized analytical grade.
5.1 Water, distilled or deionized water or water of equivalent purity, conductivity u 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 147 g of CaCl ·2H O in 1 000 ml water and autoclave for 20 min at 121 °C.
2 2
5.5 Magnesium sulfate stock solution, 1 mol/l MgSO .
4
Dissolve 247 g of MgSO ·7H O in 1 000 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 136 g of KH PO in 1 000 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;
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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 (5.3);
⎯ 1 ml of calcium chloride stock solution (5.4);
⎯ 1 ml of magnesium sulfate stock solution (5.5);
⎯ 25 ml of potassium phosphate buffer (5.7);
and fill up to 1 000 ml with sterile water.
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 300 mg of Bengal Rose to 1 000 ml of water and stir thoroughly.
5.11 Ludox suspension.
1) 3 3
Dilute Ludox TM 50 (colloidal silica; density: 1,4 g/cm ) with water to a density of 1,13 ± 0,005 g/cm [mix
1)
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 sample, approximately
50 ml of Ludox-suspension are required.
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 (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.
TM
1) Ludox 50 is an example of a suitable product available commercially. This information is given for the convenience
of users of this International Standard and does not constitute an endorsement by ISO of this product.
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The dry sediment is maintainable without restraint.
This sediment serves as negative control for tests in sediments.
WARNING — If a different artificial control sediment is used (e.g. OECD 218), the kaolin content of the
> 5 % mass fraction can cause deleterious effects on growth, fertility and reproduction of C. elegans.
5.13 Control soil.
2)
Standard soil St. 2.2 from LUFA :
⎯ soil type: loamy sand;
⎯ organic carbon: (2,16 ± 0,4) % mass fraction;
⎯ pH: 5,4 ± 0,1;
⎯ cation exchange capacity: (10 ± 1) mmol /100 g;
c
NOTE mmol /100 g is synonymous with meq/100 g.
c
⎯ water holding capacity: (48,2 ± 5) g/100 g;
⎯ clay content: (6,4 ± 0,9) % mass fraction particles < 0,002 mm;
⎯ silt content: (12,7 ± 2,6) % mass fraction particles 0,002 mm to 0,063 mm;
⎯ sand content: (81,2 ± 5,1) % mass fraction particles 0,063 mm to 2 mm.
This soil serves as negative control for tests in soil.
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 (CAS No.: 56-81-5).
6 Apparatus
6.1 Autoclave.
6.2 Facilities, with constant temperature for 20 °C and 37 °C, e.g. incubator or temperature-controlled
chamber.
6.3 Drigalski spatula, glass spatula for distributing bacteria on an agar plate.
6.4 Erlenmeyer flasks, e.g. volume 250 ml.
6.5 Plastic vials, autoclavable and sealed, volume 1,5 ml.
6.6 Filter gauze, 5 µm, 10 µm.
6.7 Freezer, capable of being maintained at −20 °C.
2) Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer.
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6.8 Micropipette.
Draw a Pasteur pipette over a Bunsen burner to a thin capillary. Plug the Pasteur pipette in a section 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 resolution.
6.18 Drying oven, approximately 80 °C.
2
6.19 Multidishes, with 12 wells, 3,5 cm /well.
6.20 Centrifuge with swing-out rotor.
6.21 Piston pipettes, 10 ml to 100 ml, 100 ml to 1 000 ml.
6.22 Sieves, 1 mm and 2 mm.
6.23 Magnetic stirrer and magnetic stirring bar.
6.24 pH-meter.
6.25 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, it is necessary to test a reference substance as
a positive control in parallel with each test, using one concentration near the EC for growth. The test
50
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 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 %.
50
Additionally, the EC of the reference substance shall be determined at least every 12 months. The EC
50 50
(growth) in water shall be in the range of 8 mg to 22 mg BAC/l using an EC design as specified in
x
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 (10.1, 10.2.2, 10.3).
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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 A). 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 days 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
3)
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 (3.13)
serve as stock cultures for C. elegans that should be replenished every two months. If too many males occur
(W 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) with E. coli from
a bacterial lawn on agar [as sent by the Caenorhabditis Genetic Center (CGC)] using an inoculating
loop (6.25) and incubate for 17 h at 37 °C on a shaker (6.11) (overnight culture). Transfer 200 µl glycerol
(5.15) into each 1,5 ml plastic vial (6.5) and sterilize in an autoclave (6.1) at 121 °C for 20 min. Add under
sterile conditions 800 µl of the E. coli overnight culture (3.12), vortex and freeze immediately at −20 °C.
Bacterial stock cultures are thawed only once and discarded after use. Bacterial stock cultures should be
replenished after six months.
9.2 Pre-culture
Inoculate under sterile conditions an agar plate (3.1) with approximately 200 µl of an overnight culture (3.12)
of E. coli, distribute equally using a Drigalski spatula (6.3) and incubate for at least 8 h at (37 ± 2) °C. Cut two
2
or three small pieces (approximately 1 cm ) out of a starved plate (3.13) and transfer them under sterile
3) Supplier for Caenorhabditis elegans and Escherichia coli.
Caenorhabditis Genetic Center
Theresa Stiernagle
University of Minnesota
6-160 Jackson Hall
321 Church Street S.E.
Minneapolis, MN 55455
E-mail: cgc@umn.edu
http://www.cbs.umn.edu/CGC/
This information is given for the convenience of users of this International Standard and does not constitute an
endorsement by ISO of these products.
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conditions on to an agar plate with a fresh lawn of E. coli. After about 3 days at 20 °C, a lot of gravid
hermaphrodites as well as juveniles of stages 1 and 2 are found on the plate. The test starts with worms in the
first juvenile stage. In order to obtain worms synchronized to this life stage, rinse the plate with M9-medium
(5.9). Then filter the suspension containing the nematodes through a cascade of filter gauze (6.6) of 5 µm and
10 µm mesh size to retain larger juveniles and adults. The filtered suspension contains only first-stage
juveniles (J ). Measure the length of 30 J (killed with heat) to obtain the initial length of the introduced
1 1
exposed test organisms (mean value).
10 Procedure
10.1 Preparation of food medium
After thawing, vortex (6.20) a vial with the E. coli stock vigorously. Inoculate under sterile conditions x × 50 ml
of LB-medium (5.2) (depending on the demand) in 250 ml Erlenmeyer flasks (6.4) with approximately 20 µl of
the E. coli stock each and incubate for 17 h at 37 °C on a shaker (6.11). To control the bacterial density after
incubation, dilute an aliquot of the bacterial suspension 1→10 with LB-medium and measure the optical
density at 600 nm against the LB-medium. The turbidity of the bacterial suspension is given as specified in
ISO 7027 in FAU (formazine absorption units). OD600 is usually > 200 FAU. After centrifugation of the
bacterial suspension (20 min, 2 000g), remove the supernatant and resuspend the pellet in M9-medium (5.9).
After repeated centrifugation and removing of the supernatant, resuspend the pellet in approximately
⎯ x × 8 ml of M9-medium and adjust the bacterial density to (12 000 ± 600) FAU for testing sediment, soil
and waste;
⎯ x × 100 ml of M9-medium and adjust the bacterial density to (1 000 ± 50) FAU for testing pore water,
elutriates, extracts or solutions of reference substance.
Finally, the accurate volume of cholesterol stock solution is added (0,2 % of volume of bacterial suspension;
e.g. 100 µl of cholesterol stock solution in 50 ml bacterial suspension).
NOTE Densities of bacteria differ between tests with solid and liquid test material due to different exposure conditions.
10.2 Preparation of test material and controls
10.2.1 Sediment, soil and waste
Pass the test material (3.14) through a 2 mm sieve (6.22). Determine the dry mass of the test material and
control sediment or control soil in accordance with ISO 11465 by drying a small portion of the test sample.
Determine the pH of the test material and control sediment or control soil in accordance with ISO 10523
(aqueous test material, sediments) and ISO 10390 (soils).
Prepare at least four replicates for each test material and the control [artificial control sediment (5.12) or
control soil (5.13)]. Prepare one additional blank replicate (without test organisms) to estimate the number of
indigenous nematodes in the samples. For artificial substrates, such as the artificial control sediment, it is not
necessary to set up a blank replicate. For test material with W 40 % water content (based on total mass),
transfer (0,500 ± 0,010) g (wet mass) of test material into each test well (6.19). For test material with < 40 %
water content (based on total mass), artificial control sediment and control soil, transfer m ± 0,010 g into each
test well, add (0,500 − m) ml of M9-medium (5.9), and stir with a spatula to achieve a homogenous suspension.
Calculate m, expressed in grams, as given in Equation (1):
0,5 × 0,60
m = (1)
m
o,t
where m is the measured dry mass of the test material.
o,t
Store in a refrigerator at (8 ± 2) °C to avoid loss of moisture.
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Stir the food medium (3.10, 10.1; 12 000 FAU) to ensure homogeneity and add, immediately before the start
of the test, 0,5 ml of homogenized food medium (3.10) to each test well. Mix artificial control sediment or
control soil and test material with the added food medium thoroughly with a spatula.
10.2.2 Pore water, elutriate, extract
Prepare at least four replicates for each test material (3.14) and the control (aqueous control; 3.2). Transfer
0,5 ml of test material and aqueous control into each test well (6.19). Stir the food medium (3.10, 10.1;
1 000 FAU) to ensure homogeneity and add, immediately before the start of the test, 0,5 ml of homogenized
food medium to each test well.
10.2.3 Solution of reference substance
Prepare at least four replicates for each solution of the reference substance (5.14, Clause 7) and the control
(aqueous control; 3.2). Transfer 0,5 ml of the solution of reference substance and aqueous control into each
test well (6.19). Stir the food medium (3.10, 10.1; 1 000 FAU) to ensure homogeneity and add, immediately
before the start of the test, 0,5 ml of homogenized food medium to each test well.
10.3 Test
At the start of the test, transfer ten first-stage juveniles (J ; exposed test organisms) from the filtrate by
1
micropipette (6.8) to each of the test wells containing test material (3.14) and food medium (3.10) (after
allowing temperature to equilibrate to room tempe
...
INTERNATIONAL ISO
STANDARD 10872
First edition
2010-06-15
Water quality — Determination of the
toxic effect of sediment and soil samples
on growth, fertility and reproduction of
Caenorhabditis elegans (Nematoda)
Qualité de l'eau — 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 10872:2010(E)
©
ISO 2010
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ISO 10872:2010(E)
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ii © ISO 2010 – All rights reserved
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ISO 10872:2010(E)
Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Normative references.1
3 Terms and definitions .1
4 Principle.3
5 Reagents.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
9.2 Pre-culture.7
10 Procedure.8
10.1 Preparation of food medium.8
10.2 Preparation of test material and controls .8
10.3 Test .9
10.4 Nematode separation.9
10.5 Measurements and calculations .9
10.6 Timetable of the test.11
11 Validity criteria.12
12 Expression of results.12
13 Test report.13
Annex A (informative) Figures of adult worms C. elegans .14
Annex B (informative) Precision data .15
Bibliography.17
© ISO 2010 – All rights reserved iii
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ISO 10872:2010(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 10872 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2010 – All rights reserved
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ISO 10872:2010(E)
Introduction
[1][2]
Nematodes are the most abundant and species-rich group of metazoans in sediments and soils and play
[3][4]
an important role in benthic and soil food webs . Nematodes are endobenthic organisms that are found at
various trophic levels due to the evolution of different feeding types (bacterivorous, algal feeder, omnivorous,
predators).
The test organism Caenorhabditis elegans (Maupas, N2 var. Bristol) is a bacterivorous nematode that is found
[5][6]
primarily in terrestrial soils but it also occurs in aquatic sediments of polysaprobial fresh-water systems .
[7]
C. elegans is a well-studied organism and very easy to cultivate .
[8][9][10]
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.
© ISO 2010 – All rights reserved v
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INTERNATIONAL STANDARD ISO 10872:2010(E)
Water quality — Determination of the toxic effect of sediment
and soil samples on growth, fertility and reproduction of
Caenorhabditis elegans (Nematoda)
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 according to this International Standard
be carried out by suitably trained staff.
1 Scope
This International Standard 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.
2 Normative references
The following referenced documents are indispensable for the application 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, Water quality — Determination of turbidity
ISO 10390, Soil quality — Determination of pH
ISO 10523, Water quality — Determination of pH
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric
method
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
agar plate
Petri dish filled with NGM agar (5.8)
3.2
aqueous control
water that serves as negative control for tests in aqueous samples
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ISO 10872:2010(E)
3.3
artificial control sediment
defined artificial sediment (5.12)
3.4
bacterial stock culture
stock culture of food bacteria
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 (3.2,
3.3, 3.7)
3.7
control soil
defined standard soil (5.13)
3.8
dauer larva
developmental stage adopted by C. elegans to endure periods of lack of food
NOTE 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 (10.1)
3.11
J stage
1
first of four juvenile stages (J to J ) in the development of C. elegans
1 4
3.12
overnight culture
defined culture of Escherichia coli in LB-medium (9.1.2)
3.13
starved plate
agar plate with dauer larvae
3.14
test material
discrete portion of a contaminated environmental sample (10.2) or solution of the reference substance
(Clause 7)
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ISO 10872:2010(E)
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. 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. Toxicity
can by quantified by the intensity of the effect as percentage inhibition.
5 Reagents
Use only reagents of recognized analytical grade.
5.1 Water, distilled or deionized water or water of equivalent purity, conductivity u 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 147 g of CaCl ·2H O in 1 000 ml water and autoclave for 20 min at 121 °C.
2 2
5.5 Magnesium sulfate stock solution, 1 mol/l MgSO .
4
Dissolve 247 g of MgSO ·7H O in 1 000 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 136 g of KH PO in 1 000 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;
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ISO 10872:2010(E)
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 (5.3);
⎯ 1 ml of calcium chloride stock solution (5.4);
⎯ 1 ml of magnesium sulfate stock solution (5.5);
⎯ 25 ml of potassium phosphate buffer (5.7);
and fill up to 1 000 ml with sterile water.
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 300 mg of Bengal Rose to 1 000 ml of water and stir thoroughly.
5.11 Ludox suspension.
1) 3 3
Dilute Ludox TM 50 (colloidal silica; density: 1,4 g/cm ) with water to a density of 1,13 ± 0,005 g/cm [mix
1)
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 sample, approximately
50 ml of Ludox-suspension are required.
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 (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.
TM
1) Ludox 50 is an example of a suitable product available commercially. This information is given for the convenience
of users of this International Standard and does not constitute an endorsement by ISO of this product.
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ISO 10872:2010(E)
The dry sediment is maintainable without restraint.
This sediment serves as negative control for tests in sediments.
WARNING — If a different artificial control sediment is used (e.g. OECD 218), the kaolin content of the
> 5 % mass fraction can cause deleterious effects on growth, fertility and reproduction of C. elegans.
5.13 Control soil.
2)
Standard soil St. 2.2 from LUFA :
⎯ soil type: loamy sand;
⎯ organic carbon: (2,16 ± 0,4) % mass fraction;
⎯ pH: 5,4 ± 0,1;
⎯ cation exchange capacity: (10 ± 1) mmol /100 g;
c
NOTE mmol /100 g is synonymous with meq/100 g.
c
⎯ water holding capacity: (48,2 ± 5) g/100 g;
⎯ clay content: (6,4 ± 0,9) % mass fraction particles < 0,002 mm;
⎯ silt content: (12,7 ± 2,6) % mass fraction particles 0,002 mm to 0,063 mm;
⎯ sand content: (81,2 ± 5,1) % mass fraction particles 0,063 mm to 2 mm.
This soil serves as negative control for tests in soil.
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 (CAS No.: 56-81-5).
6 Apparatus
6.1 Autoclave.
6.2 Facilities, with constant temperature for 20 °C and 37 °C, e.g. incubator or temperature-controlled
chamber.
6.3 Drigalski spatula, glass spatula for distributing bacteria on an agar plate.
6.4 Erlenmeyer flasks, e.g. volume 250 ml.
6.5 Plastic vials, autoclavable and sealed, volume 1,5 ml.
6.6 Filter gauze, 5 µm, 10 µm.
6.7 Freezer, capable of being maintained at −20 °C.
2) Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer.
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ISO 10872:2010(E)
6.8 Micropipette.
Draw a Pasteur pipette over a Bunsen burner to a thin capillary. Plug the Pasteur pipette in a section 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 resolution.
6.18 Drying oven, approximately 80 °C.
2
6.19 Multidishes, with 12 wells, 3,5 cm /well.
6.20 Centrifuge with swing-out rotor.
6.21 Piston pipettes, 10 ml to 100 ml, 100 ml to 1 000 ml.
6.22 Sieves, 1 mm and 2 mm.
6.23 Magnetic stirrer and magnetic stirring bar.
6.24 pH-meter.
6.25 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, it is necessary to test a reference substance as
a positive control in parallel with each test, using one concentration near the EC for growth. The test
50
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 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 %.
50
Additionally, the EC of the reference substance shall be determined at least every 12 months. The EC
50 50
(growth) in water shall be in the range of 8 mg to 22 mg BAC/l using an EC design as specified in
x
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 (10.1, 10.2.2, 10.3).
6 © ISO 2010 – All rights reserved
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ISO 10872:2010(E)
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 A). 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 days 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
3)
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 (3.13)
serve as stock cultures for C. elegans that should be replenished every two months. If too many males occur
(W 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) with E. coli from
a bacterial lawn on agar [as sent by the Caenorhabditis Genetic Center (CGC)] using an inoculating
loop (6.25) and incubate for 17 h at 37 °C on a shaker (6.11) (overnight culture). Transfer 200 µl glycerol
(5.15) into each 1,5 ml plastic vial (6.5) and sterilize in an autoclave (6.1) at 121 °C for 20 min. Add under
s
...
NORME ISO
INTERNATIONALE 10872
Première édition
2010-06-15
Qualité de l'eau — Détermination de
l'effet toxique d'échantillons de sédiment
et de sol sur la croissance, la fertilité et la
reproduction de Caenorhabditis elegans
(nématodes)
Water quality — Determination of the toxic effect of sediment and soil
samples on growth, fertility and reproduction of Caenorhabditis elegans
(Nematoda)
Numéro de référence
ISO 10872:2010(F)
©
ISO 2010
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ISO 10872:2010(F)
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Publié en Suisse
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ISO 10872:2010(F)
Sommaire Page
Avant-propos .iv
Introduction.v
1 Domaine d'application .1
2 Références normatives.1
3 Termes et définitions .1
4 Principe.3
5 Réactifs.3
6 Appareillage .6
7 Substance de référence.7
8 Organismes.7
8.1 Organisme d'essai.7
8.2 Source nutritive .7
9 Cultures mères et pré-cultures .7
9.1 Cultures mères .7
9.2 Pré-culture.8
10 Mode opératoire.8
10.1 Préparation du milieu nutritif .8
10.2 Préparation du matériau d'essai et des témoins.9
10.3 Essai .9
10.4 Séparation des nématodes.10
10.5 Mesurages et calculs .10
10.6 Planification de l'essai .12
11 Critères de validité .13
12 Expression des résultats.13
13 Rapport d'essai.14
Annexe A (informative) Illustrations des vers adultes C. elegans.15
Annexe B (informative) Données relatives à la fidélité .16
Bibliographie.18
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ISO 10872:2010(F)
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes nationaux de
normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est en général confiée
aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude a le droit de faire partie du
comité technique créé à cet effet. Les organisations internationales, gouvernementales et non
gouvernementales, en liaison avec l'ISO participent également aux travaux. L'ISO collabore étroitement avec
la Commission électrotechnique internationale (CEI) en ce qui concerne la normalisation électrotechnique.
Les Normes internationales sont rédigées conformément aux règles données dans les Directives ISO/CEI,
Partie 2.
La tâche principale des comités techniques est d'élaborer les Normes internationales. Les projets de Normes
internationales adoptés par les comités techniques sont soumis aux comités membres pour vote. Leur
publication comme Normes internationales requiert l'approbation de 75 % au moins des comités membres
votants.
L'attention est appelée sur le fait que certains des éléments du présent document peuvent faire l'objet de
droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable de ne
pas avoir identifié de tels droits de propriété et averti de leur existence.
L'ISO 10872 a été élaborée par le comité technique ISO/TC 147, Qualité de l'eau, sous-comité SC 5,
Méthodes biologiques.
iv © ISO 2010 – Tous droits réservés
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ISO 10872:2010(F)
Introduction
Les nématodes constituent le groupe de métazoaires le plus abondant et le plus riche en espèces dans les
[1][2]
sédiments et les sols et jouent un rôle important dans les réseaux trophiques benthiques et les réseaux
[3][4]
trophiques du sol . Les nématodes sont des organismes endobenthiques que l'on trouve à différents
niveaux trophiques en raison de l'évolution des différents types d'alimentation (bactérivores, alguivores,
omnivores, prédateurs).
L'organisme d'essai Caenorhabditis elegans (Maupas, N2 var. Bristol) est un nématode bactérivore que l'on
trouve principalement dans les sols terrestres mais également parfois dans les sédiments aquatiques des
[5][6]
systèmes d'eau douce polysaprobes . C. elegans est un organisme largement étudié et très facile à
[7]
cultiver .
[8][9][10]
L'essai est conçu pour mesurer la réponse à des substances dissoutes et liées à des particules .
Il s'applique aux sédiments, aux sols, aux déchets, à l'eau interstitielle, aux élutriats et aux extraits aqueux.
© ISO 2010 – Tous droits réservés v
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NORME INTERNATIONALE ISO 10872:2010(F)
Qualité de l'eau — Détermination de l'effet toxique
d'échantillons de sédiment et de sol sur la croissance,
la fertilité et la reproduction de Caenorhabditis elegans
(nématodes)
AVERTISSEMENT — Il convient que l'utilisateur de la présente Norme internationale connaisse bien
les pratiques courantes de laboratoire. La présente Norme internationale n'a pas pour but de traiter
tous les problèmes de sécurité qui sont, le cas échéant, liés à son utilisation. Il incombe à l'utilisateur
d'établir des pratiques appropriées en matière d'hygiène et de sécurité, et de s'assurer de la
conformité à la réglementation nationale en vigueur.
IMPORTANT — Il est absolument essentiel que les essais réalisés conformément à la présente Norme
internationale soient exécutés par du personnel ayant reçu une formation adéquate.
1 Domaine d'application
La présente Norme internationale spécifie une méthode de détermination de la toxicité d'échantillons
environnementaux sur la croissance, la fertilité et la reproduction de Caenorhabditis elegans. La méthode
s'applique aux sédiments d'eau douce (salinité maximale de 5 ‰), sols et déchets contaminés, ainsi qu'à l'eau
interstitielle, aux élutriats et aux extraits aqueux obtenus à partir de sédiments, sols et déchets contaminés.
2 Références normatives
Les documents de référence suivants sont indispensables pour l'application du présent document. Pour les
références datées, seule l'édition citée s'applique. Pour les références non datées, la dernière édition du
document de référence (y compris les éventuels amendements) s'applique.
ISO 5667-16, Qualité de l'eau — Échantillonnage — Partie 16: Lignes directrices pour les essais biologiques
des échantillons
ISO 7027, Qualité de l'eau — Détermination de la turbidité
ISO 10390, Qualité du sol — Détermination du pH
ISO 10523, Qualité de l'eau — Détermination du pH
ISO 11465, Qualité du sol — Détermination de la teneur pondérale en matière sèche et en eau — Méthode
gravimétrique
3 Termes et définitions
Pour les besoins du présent document, les termes et définitions suivants s'appliquent.
3.1
boîte de gélose
boîte de Petri remplie de gélose NGM (5.8)
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ISO 10872:2010(F)
3.2
témoin aqueux
eau servant de témoin négatif pour les essais sur des échantillons aqueux
3.3
sédiment témoin artificiel
sédiment artificiel défini (5.12)
3.4
culture mère bactérienne
culture mère de bactéries nutritives
3.5
réplicat à blanc
réplicat supplémentaire ne contenant pas d'organisme d'essai mais traité de la même façon que les autres
réplicats d'un échantillon
3.6
témoin
traitement servant de témoin négatif auquel est comparé l'effet sur le matériau d'essai correspondant (3.2, 3.3,
3.7)
3.7
sol témoin
sol standard défini (5.13)
3.8
«larve dauer»
stade larvaire quiescent
stade de développement adopté par C. elegans pour supporter des périodes de pénurie alimentaire
NOTE Les «larves dauer» poursuivent leur développement normal en cas d'apport de nourriture.
3.9
organismes d'essai exposés
individus de C. elegans introduits au début de l'essai
3.10
milieu nutritif
suspension bactérienne aqueuse définie (10.1)
3.11
stade J
1
premier des quatre stades juvéniles (J à J ) au cours du développement de C. elegans
1 4
3.12
culture nocturne
culture définie d'Escherichia coli dans du milieu LB (9.1.2)
3.13
boîte sans nourriture
boîte de gélose avec des «larves dauer»
3.14
matériau d'essai
portion discrète d'un échantillon environnemental contaminé (10.2) ou solution de la substance de référence
(Article 7)
2 © ISO 2010 – Tous droits réservés
---------------------- Page: 7 ----------------------
ISO 10872:2010(F)
4 Principe
Les organismes juvéniles de l'espèce C. elegans sont exposés à l'échantillon environnemental pendant une
période de 96 h. Dans les témoins, les organismes d'essai exposés peuvent accomplir un cycle de vie
complet au cours de cette période. Un effet toxique de l'échantillon environnemental est observé si l'inhibition
de la croissance, de la fertilité ou de la reproduction de C. elegans comparée au témoin (témoin aqueux,
sédiment ou sol témoin) dépasse une certaine valeur seuil. La toxicité peut être quantifiée par l'intensité de
l'effet en pourcentage d'inhibition.
5 Réactifs
Utiliser uniquement des réactifs de qualité analytique reconnue.
5.1 Eau, distillée ou déionisée ou eau d'une pureté équivalente, conductivité u 10 µS/cm.
5.2 Milieu LB.
Dissoudre
⎯ 0,5 g de peptone de caséine,
⎯ 0,25 g d'extrait de levure,
⎯ 0,5 g de chlorure de sodium (NaCl),
dans 50 ml d'eau dans une fiole de 250 ml et autoclaver pendant 20 min à 121 °C.
5.3 Solution mère de cholestérol.
Dissoudre 500 mg de cholestérol en poudre dans 100 ml d'éthanol absolu (pureté > 99 %) en agitant et en
chauffant légèrement (< 50 °C). Remplacer l'éthanol perdu par évaporation.
5.4 Solution mère de chlorure de calcium, 1 mol/l CaCl .
2
Dissoudre 147 g de CaCl , 2 H O dans 1 000 ml d'eau et autoclaver pendant 20 min à 121 °C.
2 2
5.5 Solution mère de sulfate de magnésium, 1 mol/l MgSO .
4
Dissoudre 247 g de MgSO , 7 H O dans 1 000 ml d'eau et autoclaver pendant 20 min à 121 °C.
4 2
5.6 Hydroxyde de potassium, KOH, pastilles.
5.7 Solution tampon de phosphate de potassium, 1 mol/l KH PO .
2 4
Dissoudre 136 g de KH PO dans 1 000 ml d'eau, ajuster le pH à 6,0 ± 0,2 avec du KOH (5.6) et autoclaver
2 4
pendant 20 min à 121 °C.
© ISO 2010 – Tous droits réservés 3
---------------------- Page: 8 ----------------------
ISO 10872:2010(F)
5.8 Milieu gélosé de croissance des nématodes (gélose NGM).
Dissoudre
⎯ 2,5 g de peptone de caséine,
⎯ 17 g de gélose bactériologique,
⎯ 3 g de NaCl,
dans 900 ml d'eau dans une fiole de 1 000 ml et autoclaver pendant 20 min à 121 °C. Après refroidissement à
55 °C, ajouter les solutions stériles suivantes:
⎯ 1 ml de solution mère de cholestérol (5.3);
⎯ 1 ml de solution mère de chlorure de calcium (5.4);
⎯ 1 ml de solution mère de sulfate de magnésium (5.5);
⎯ 25 ml de solution tampon de phosphate de potassium (5.7);
et compléter à 1 000 ml avec de l'eau stérile.
Transférer des portions de gélose NGM (20 ml à 25 ml environ) dans des boîtes de Petri stériles.
5.9 Milieu M9.
Dissoudre
⎯ 6 g de Na HPO ,
2 4
⎯ 3 g de KH PO ,
2 4
⎯ 5 g de NaCl,
⎯ 0,25 g de MgSO , 7 H O
4 2
dans 1 000 ml d'eau dans une fiole de 1 000 ml.
5.10 Solution mère de rose Bengale.
Ajouter environ 300 mg de rose Bengale dans 1 000 ml d'eau et agiter soigneusement.
5.11 Suspension de Ludox.
1) 3
Diluer du Ludox TM 50 (silice colloïdale; masse volumique: 1,4 g/cm ) avec de l'eau, jusqu'à une masse
3 1)
volumique de 1,13 ± 0,005 g/cm [mélanger environ 1 part de Ludox TM 50 avec 2 parts d'eau et contrôler la
masse volumique en pesant 1 ml de la suspension sur une balance; 1 ml de la suspension pèse
(1,13 ± 0,005) g]. Environ 50 ml de suspension de Ludox sont requis pour un échantillon.
TM
1) Ludox 50 est un exemple de produit approprié disponible sur le marché. Cette information est donnée à l'intention
des utilisateurs de la présente Norme internationale et ne signifie nullement que l'ISO approuve ou recommande l'emploi
exclusif du produit ainsi désigné.
4 © ISO 2010 – Tous droits réservés
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ISO 10872:2010(F)
5.12 Sédiment témoin artificiel.
Mélanger soigneusement les composés suivants dans les proportions indiquées:
⎯ Al O , 20 % (fraction massique);
2 3
⎯ CaCO , 1 % (fraction massique);
3
⎯ dolomite (argile), 0,5 % (fraction massique);
⎯ Fe O , 4,5 % (fraction massique);
2 3
⎯ sable siliceux (W4, granulométrie moyenne: 0,063 mm), 30 % (fraction massique);
⎯ sable siliceux (de 0,1 mm à 0,4 mm), 40 % (fraction massique);
⎯ tourbe (tourbe décomposée à partir de haute tourbière, non traitée, finement moulue et tamisée à
< 1 mm), 4 % (fraction massique).
Le sédiment sec peut être conservé sans restriction.
Ce sédiment sert de témoin négatif pour les essais sur sédiments.
AVERTISSEMENT — Si un sédiment témoin artificiel différent est utilisé (de l'OECD 218, par exemple),
une teneur en kaolin > 5 % (fraction massique) peut provoquer des effets négatifs sur la croissance, la
fertilité et la reproduction de C. elegans.
5.13 Sol témoin.
2)
Sol standard St. 2.2, de l'institut LUFA :
⎯ type de sol: sable limoneux;
⎯ carbone organique: (2,16 ± 0,4) % (fraction massique);
⎯ pH: 5,4 ± 0,1;
⎯ capacité d'échange cationique: (10 ± 1) mmol /100 g;
c
NOTE mmol /100 g est synonyme de meq/100 g.
c
⎯ capacité de rétention d'eau: (48,2 ± 5) g/100 g;
⎯ teneur en argile: (6,4 ± 0,9) % (fraction massique) de particules < 0,002 mm;
⎯ teneur en limon: (12,7 ± 2,6) % (fraction massique) de particules de 0,002 mm à 0,063 mm;
⎯ teneur en sable: (81,2 ± 5,1) % (fraction massique) de particules de 0,063 mm à 2 mm.
Ce sol sert de témoin négatif pour les essais sur sols.
2) Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer.
© ISO 2010 – Tous droits réservés 5
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ISO 10872:2010(F)
5.14 Chlorure d'hexadécylbenzyldiméthylammonium monohydrate, solution mère (BAC-C16).
Dissoudre 30 mg de BAC-C16 (C H CIN, H O; numéro CAS: 122-18-9) dans 1 000 ml d'eau.
25 46 2
5.15 Glycérol (numéro CAS: 56-81-5).
6 Appareillage
6.1 Autoclave.
6.2 Installations, à température constante pour 20 °C et 37 °C, par exemple incubateur ou chambre
thermostatée.
6.3 Spatule de Drigalski, spatule en verre pour répartir les bactéries sur une boîte de gélose.
6.4 Erlenmeyers, d'un volume de 250 ml, par exemple.
6.5 Flacons en plastique, autoclavables et hermétiquement clos, d'un volume de 1,5 ml.
6.6 Gaz filtrante, 5 µm, 10 µm.
6.7 Congélateur, pouvant être maintenu à −20 °C.
6.8 Micropipette.
Étirer une pipette Pasteur sur un bec Bunsen pour former un fin capillaire. Tamponner la pipette Pasteur sur
une coupelle à son extrémité la plus épaisse.
6.9 Microscope, grossissement 100×, avec graduation.
6.10 Thermomètre, minimum-maximum.
6.11 Agitateur, pour les Erlenmeyers de 250 ml.
6.12 Microscope stéréoscopique, grossissement de 4× à 20×, à lumière transmise.
6.13 Paillasse propre.
6.14 Boîtes de Petri stériles, de 3 cm, 6 cm ou 10 cm de diamètre.
6.15 Spectromètre, capable de fonctionner à une longueur d'onde de 600 nm.
6.16 Agitateur pour tubes à essai.
6.17 Balance, ayant une résolution de 0,001 g.
6.18 Étuve, à une température d'environ 80 °C.
2
/puits.
6.19 Plaque multi-puits, avec 12 puits, 3,5 cm
6.20 Centrifugeuse à rotor oscillant.
6.21 Pipettes à piston, de 10 ml à 100 ml et de 100 ml à 1 000 ml.
6.22 Tamis, de dimensions nominales d'ouverture de 1 mm et 2 mm.
6.23 Agitateur magnétique et barreau aimanté.
6.24 pH-mètre.
6.25 Boucle d'inoculation.
6 © ISO 2010 – Tous droits réservés
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ISO 10872:2010(F)
7 Substance de référence
Afin de garantir que les conditions d'essai du laboratoire (y compris l'état et la sensibilité des organismes
d'essai exposés) sont adéquates et n'ont pas subi de variation significative, il est nécessaire de soumettre à
essai, en parallèle de chaque essai, une substance de référence en tant que témoin positif, à une
concentration proche de la CE pour le paramètre «croissance». Les paramètres d'essai «fertilité» et
50
«reproduction» ne sont pas analysés lors de l'essai de la substance de référence. Comme substance de
référence, utiliser du chlorure d'hexadécylbenzyldiméthylammonium (BAC-C16, 5.14) qui s'est avéré affecter
la croissance de C. elegans. Le témoin positif est soumis à essai dans l'eau conformément aux instructions
relatives aux essais sur des substrats aqueux (10.1, 10.2.2, 10.3). À une concentration de 15 mg/l (CE pour
50
le BAC C16, 5.4) il convient que l'inhibition de la croissance, comparée au témoin, soit comprise entre 20 % et
80 %.
En outre, la CE de la substance de référence doit être déterminée au moins tous les 12 mois. La CE
50 50
(croissance) dans l'eau doit être comprise entre 8 mg et 22 mg de BAC/l, en utilisant un dispositif de type CE ,
x
comme spécifié dans l'ISO 5667-16. Des solutions mères de BAC aux concentrations de 7,1 mg/l, 10,6 mg/l,
16 mg/l, 24 mg/l, 36 mg/l, 54 mg/l et 81 mg/l sont préparées dans l'eau et soumises à essai conformément
aux instructions pour les essais sur des substrats aqueux (10.1, 10.2.2, 10.3).
8 Organismes
8.1 Organisme d'essai
Caenorhabditis elegans (Maupas, 1899) est un nématode libre du sol très répandu qui se nourrit
principalement de bactéries. Les vers adultes mesurent environ 1,0 mm à 1,5 mm de long et sont
hermaphrodites et, dans de rares cas, des mâles (voir l'Annexe A). Les hermaphrodites se reproduisent
généralement par auto-fertilisation bien qu'ils puissent également être fécondés par des mâles. Après
l'éclosion, C. elegans se développe jusqu'à l'âge adulte en passant par quatre stades juvéniles séparés par
des mues. Dans des conditions de pénurie alimentaire, un stade quiescent, les «larves dauer», peut
remplacer le troisième stade larvaire. Le cycle de vie des vers cultivés sur E. coli est d'environ 3 jours à 20 °C.
La biologie de C. elegans a été largement étudiée et, à de nombreux égards, il s'agit de l'animal ayant été le
mieux caractérisé. La souche N2 de «type sauvage» est utilisée comme organisme d'essai.
8.2 Source nutritive
La bactérie E. coli (souche déficiente en uracile; OP50) est utilisée comme source nutritive pour C. elegans.
9 Cultures mères et pré-cultures
9.1 Cultures mères
9.1.1 Caenorhabditis elegans
3)
C. elegans est maintenue sur des boîtes de gélose (3.1) avec un tapis bactérien (E. coli OP 50; 8.2) à
(20 ± 2) °C. Une fois les bactéries consommées, C. elegans forme des «larves dauer» (3.8) par manque de
3) Fournisseur de Caenorhabditis elegans et Escherichia coli:
Caenorhabditis Genetic Center
Theresa Stiernagle
University of Minnesota
6-160 Jackson Hall
321 Church Street S.E.
Minneapolis, MN 55455
E-mail: cgc@umn.edu
http://www.cbs.umn.edu/CGC/
Cette information est donnée à l'intention des utilisateurs de la présente Norme internationale et ne signifie nullement que
l'ISO approuve ou recommande l'emploi exclusif des produits ainsi désignés.
© ISO 2010 – Tous droits réservés 7
---------------------- Page: 12 ----------------------
ISO 10872:2010(F)
nourriture. Ces boîtes sans nourriture (3.13) servent de cultures mères de C. elegans et il convient de les
renouveler tous les deux mois. Si une quantité trop importante de mâles apparaît (W 10 % lors des essais), il
convient de commander de nouvelles cultures mères.
9.1.2 Escherichia coli
Inoculer dans des conditions stériles 50 ml de milieu LB (5.2) dans un Erlenmeyer de 250 ml (6.4) avec E. coli
provenant d'un tapis bactérien sur de la gélose [expédié par le CGC (Caenorhabditis Genetic Center)] en
utilisant une boucle d'inoculation (6.25) et incuber pendant 17 h à 37 °C sur un agitateur (6.11) (culture
nocturne). Transférer 200 µl de glycérol (5.15) dans des flacons en plastique de 1,5 ml (6.5) et stériliser à
l'autoclave (6.1) à 121 °C pendant 20 min. Ajouter dans des conditions stériles 800 µl de la culture
nocturne (3.12) d'E. coli, agiter et congeler immédiatement à −20 °C. Les cultures mères bactériennes
peuvent uniquement être décongelées une fois et elles doivent être éliminées après usage. Il convient de
renouveler ces cultures tous les six mois.
9.2 Pré-culture
Inoculer dans des conditions stériles une boîte de gélose (3.1) avec environ 200 µl (6.4) d'une culture
nocturne (3.12) d'E. coli, répartir uniformément avec une spatule de Drigalski (6.3) et incuber pendant au
2
moins 8 h à (37 ± 2) °C. Couper deux ou trois petits morceaux (de 1 cm environ) sur une boîte sans
nourriture (3.13) et les transférer dans des conditions stériles sur une boîte de gélose avec un tapis frais
d'E. coli. Au bout d'environ 3 jours à 20 °C, un grand nombre d'hermaphrodites gravides et de juvéniles de
stades 1 et 2 sont présents sur la boîte. L'essai démarre avec des vers au premier stade juvénile. Afin
d'obtenir des vers synchronisés à ce stade, rincer la boîte avec du milieu M9 (5.9). Puis, filtrer la suspension
contenant les nématodes dans une cascade de gaze filtrante (6.6) de 5 µm et 10 µm d'ouverture de maille
afin de retenir les plus grands juvéniles et les adultes. La suspension filtrée contient uniquement des juvéniles
du premier stade (J ). Mesurer la longueur de 30 J (tués par la chaleur) pour obtenir la longueur initiale des
1 1
organismes d'essai exposés introduits (valeur moyenne).
10 Mode opératoire
10.1 Préparation du milieu nutritif
Après décongélation, remuer (6.20) un flacon contenant les cultures d'E. coli. Inoculer dans des conditions
stériles x × 50 ml de milieu LB (5.2) (selon la demande) dans des Erlenmeyers de 250 ml (6.4) avec environ
20 µl de culture d'E. coli dans chacun et incuber pendant 17 h à 37 °C sur un agitateur (6.11). Pour maîtriser
la densité bactérienne après l'incubation, diluer une aliquote de suspension bactérienne à 1→10 avec du
milieu LB et mesurer la densité optique à 600 nm par rapport au milieu LB. La turbidité de la suspension
bactérienne est exprimée, comme spécifié dans l'ISO 7027, en unités d'absorption de formazine (FAU). La
DO600 est généralement > 200 FAU. Après centrifugation de la suspension bactérienne (20 min, 2 000g),
éliminer le surnageant et remettre en suspension le culot dans du milieu M9 (5.9). Répéter la centrifugation et
éliminer le surnageant, puis remettre en suspension le culot dans environ
⎯ x × 8 ml de milieu M9 et ajuster la densité bactérienne à (12 000 ± 600) FAU pour les essais sur
sédiments, sols et déchets;
⎯ x × 100 ml de milieu M9 et ajuster la densité bactérienne à (1 000 ± 50) FAU pour les essais sur eau
interstitielle, élutriats, extraits ou solutions de substance de référence.
Enfin, ajouter le volume exact de solution mère de cholestérol (0,2 % du volume de la suspension
bactérienne; par exemple 100 µl de solution mère de cholestérol dans 50 ml de suspension bactérienne).
NOTE Les densités de bactéries diffèrent suivant que les essais sont réalisés avec des matériaux solides ou liquides,
en raison des conditions d'exposition différentes.
8 © ISO 2010 – Tous droits réservés
---------------------- Page: 13 ----------------------
ISO 10872:2010(F)
10.2 Préparation du matériau d'essai et des témoins
10.2.1 Sédiment, sol et déchets
Passer le matériau d'essai (3.14) sur un tamis de 2 mm (6.22). Déterminer la masse à sec du matériau d'essai
et du sédiment ou du sol témoin selon l'ISO 11465 en séchant une petite prise de l'échantillon pour essai.
Déterminer le pH du matériau d'essai et du sédiment ou du sol témoin conformément à l'ISO 10523 (matériau
d'essai aqueux, sédiments) et à l'ISO 10390 (sols).
Préparer au moins quatre réplicats pour chaque matériau d'essai et pour le témoin [sédiment témoin artificiel
(5.12) ou sol témoin (5.13)]. Préparer un réplicat supplémentaire à blanc (sans organismes d'essai) afin
d'estimer le nombre de nématodes déjà présents dans les échantillons. Pour les substrats artificiels, tels que
le sédiment témoin artificiel, aucun réplicat à blanc n'est nécessaire. Dans le cas d'un matériau d'essai ayant
une teneur en eau W 40 % (basée sur la masse totale), transférer (0,500 ± 0,010) g (masse à l'état frais) de
matériau d'essai dans chaque puits d'essai (6.19). Dans le cas d'un matériau d'essai ayant une teneur en eau
< 40 % (basée sur la masse totale), de sédiment témoin artificiel et de sol témoin, transférer (m ± 0,010) g
dans chaque puits d'essai, ajouter (0,500 − m) ml de milieu M9 (5.9) et agiter à l'aide d'une spatule afin
d'obtenir une suspension homogène. Calculer m, exprimé en grammes, à l'aide de l'Équation (1):
0,5 × 0,60
(1)
m =
m
o,t
où m est la masse mesurée à sec du matériau d'essai.
o,t
Conserver au réfrigérateur à (8 ± 2) °C pour éviter toute déperdition d'eau.
Agiter le milieu nutritif (3.10, 10.1, 12 000 FAU) pour garantir son homogénéité et ajouter immédiatement
...
SLOVENSKI STANDARD
oSIST ISO 10872:2011
01-maj-2011
.DNRYRVWYRGH'RORþHYDQMHXþLQNRYVWUXSHQRVWLY]RUFHYXVHGOLQLQWDOQDUDVW
SORGQRVWLQUD]PQRåHYDQMH&DHQRUKDEGLWLVHOHJDQV 1HPDWRGD
Water quality - Determination of the toxic effect of sediment and soil samples on growth,
fertility and reproduction of Caenorhabditis elegans (Nematoda)
Qualité de l'eau - Détermination de l'effet toxique d'échantillons de sédiment et de sol sur
la croissance, la fertilité et la reproduction de Caenorhabditis elegans (nématodes)
Ta slovenski standard je istoveten z: ISO 10872:2010
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST ISO 10872:2011 en,fr
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST ISO 10872:2011
---------------------- Page: 2 ----------------------
oSIST ISO 10872:2011
INTERNATIONAL ISO
STANDARD 10872
First edition
2010-06-15
Water quality — Determination of the
toxic effect of sediment and soil samples
on growth, fertility and reproduction of
Caenorhabditis elegans (Nematoda)
Qualité de l'eau — 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 10872:2010(E)
©
ISO 2010
---------------------- Page: 3 ----------------------
oSIST ISO 10872:2011
ISO 10872:2010(E)
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ii © ISO 2010 – All rights reserved
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oSIST ISO 10872:2011
ISO 10872:2010(E)
Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Normative references.1
3 Terms and definitions .1
4 Principle.3
5 Reagents.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
9.2 Pre-culture.7
10 Procedure.8
10.1 Preparation of food medium.8
10.2 Preparation of test material and controls .8
10.3 Test .9
10.4 Nematode separation.9
10.5 Measurements and calculations .9
10.6 Timetable of the test.11
11 Validity criteria.12
12 Expression of results.12
13 Test report.13
Annex A (informative) Figures of adult worms C. elegans .14
Annex B (informative) Precision data .15
Bibliography.17
© ISO 2010 – All rights reserved iii
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oSIST ISO 10872:2011
ISO 10872:2010(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 10872 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2010 – All rights reserved
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oSIST ISO 10872:2011
ISO 10872:2010(E)
Introduction
[1][2]
Nematodes are the most abundant and species-rich group of metazoans in sediments and soils and play
[3][4]
an important role in benthic and soil food webs . Nematodes are endobenthic organisms that are found at
various trophic levels due to the evolution of different feeding types (bacterivorous, algal feeder, omnivorous,
predators).
The test organism Caenorhabditis elegans (Maupas, N2 var. Bristol) is a bacterivorous nematode that is found
[5][6]
primarily in terrestrial soils but it also occurs in aquatic sediments of polysaprobial fresh-water systems .
[7]
C. elegans is a well-studied organism and very easy to cultivate .
[8][9][10]
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.
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oSIST ISO 10872:2011
INTERNATIONAL STANDARD ISO 10872:2010(E)
Water quality — Determination of the toxic effect of sediment
and soil samples on growth, fertility and reproduction of
Caenorhabditis elegans (Nematoda)
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 according to this International Standard
be carried out by suitably trained staff.
1 Scope
This International Standard 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.
2 Normative references
The following referenced documents are indispensable for the application 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, Water quality — Determination of turbidity
ISO 10390, Soil quality — Determination of pH
ISO 10523, Water quality — Determination of pH
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric
method
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
agar plate
Petri dish filled with NGM agar (5.8)
3.2
aqueous control
water that serves as negative control for tests in aqueous samples
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3.3
artificial control sediment
defined artificial sediment (5.12)
3.4
bacterial stock culture
stock culture of food bacteria
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 (3.2,
3.3, 3.7)
3.7
control soil
defined standard soil (5.13)
3.8
dauer larva
developmental stage adopted by C. elegans to endure periods of lack of food
NOTE 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 (10.1)
3.11
J stage
1
first of four juvenile stages (J to J ) in the development of C. elegans
1 4
3.12
overnight culture
defined culture of Escherichia coli in LB-medium (9.1.2)
3.13
starved plate
agar plate with dauer larvae
3.14
test material
discrete portion of a contaminated environmental sample (10.2) or solution of the reference substance
(Clause 7)
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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. 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. Toxicity
can by quantified by the intensity of the effect as percentage inhibition.
5 Reagents
Use only reagents of recognized analytical grade.
5.1 Water, distilled or deionized water or water of equivalent purity, conductivity u 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 147 g of CaCl ·2H O in 1 000 ml water and autoclave for 20 min at 121 °C.
2 2
5.5 Magnesium sulfate stock solution, 1 mol/l MgSO .
4
Dissolve 247 g of MgSO ·7H O in 1 000 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 136 g of KH PO in 1 000 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;
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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 (5.3);
⎯ 1 ml of calcium chloride stock solution (5.4);
⎯ 1 ml of magnesium sulfate stock solution (5.5);
⎯ 25 ml of potassium phosphate buffer (5.7);
and fill up to 1 000 ml with sterile water.
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 300 mg of Bengal Rose to 1 000 ml of water and stir thoroughly.
5.11 Ludox suspension.
1) 3 3
Dilute Ludox TM 50 (colloidal silica; density: 1,4 g/cm ) with water to a density of 1,13 ± 0,005 g/cm [mix
1)
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 sample, approximately
50 ml of Ludox-suspension are required.
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 (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.
TM
1) Ludox 50 is an example of a suitable product available commercially. This information is given for the convenience
of users of this International Standard and does not constitute an endorsement by ISO of this product.
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The dry sediment is maintainable without restraint.
This sediment serves as negative control for tests in sediments.
WARNING — If a different artificial control sediment is used (e.g. OECD 218), the kaolin content of the
> 5 % mass fraction can cause deleterious effects on growth, fertility and reproduction of C. elegans.
5.13 Control soil.
2)
Standard soil St. 2.2 from LUFA :
⎯ soil type: loamy sand;
⎯ organic carbon: (2,16 ± 0,4) % mass fraction;
⎯ pH: 5,4 ± 0,1;
⎯ cation exchange capacity: (10 ± 1) mmol /100 g;
c
NOTE mmol /100 g is synonymous with meq/100 g.
c
⎯ water holding capacity: (48,2 ± 5) g/100 g;
⎯ clay content: (6,4 ± 0,9) % mass fraction particles < 0,002 mm;
⎯ silt content: (12,7 ± 2,6) % mass fraction particles 0,002 mm to 0,063 mm;
⎯ sand content: (81,2 ± 5,1) % mass fraction particles 0,063 mm to 2 mm.
This soil serves as negative control for tests in soil.
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 (CAS No.: 56-81-5).
6 Apparatus
6.1 Autoclave.
6.2 Facilities, with constant temperature for 20 °C and 37 °C, e.g. incubator or temperature-controlled
chamber.
6.3 Drigalski spatula, glass spatula for distributing bacteria on an agar plate.
6.4 Erlenmeyer flasks, e.g. volume 250 ml.
6.5 Plastic vials, autoclavable and sealed, volume 1,5 ml.
6.6 Filter gauze, 5 µm, 10 µm.
6.7 Freezer, capable of being maintained at −20 °C.
2) Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer.
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oSIST ISO 10872:2011
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6.8 Micropipette.
Draw a Pasteur pipette over a Bunsen burner to a thin capillary. Plug the Pasteur pipette in a section 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 resolution.
6.18 Drying oven, approximately 80 °C.
2
6.19 Multidishes, with 12 wells, 3,5 cm /well.
6.20 Centrifuge with swing-out rotor.
6.21 Piston pipettes, 10 ml to 100 ml, 100 ml to 1 000 ml.
6.22 Sieves, 1 mm and 2 mm.
6.23 Magnetic stirrer and magnetic stirring bar.
6.24 pH-meter.
6.25 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, it is necessary to test a reference substance as
a positive control in parallel with each test, using one concentration near the EC for growth. The test
50
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 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 %.
50
Additionally, the EC of the reference substance shall be determined at least every 12 months. The EC
50 50
(growth) in water shall be in the range of 8 mg to 22 mg BAC/l using an EC design as specified in
x
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 (10.1, 10.2.2, 10.3).
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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 A). 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 days 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
3)
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 (3.13)
serve as stock cultures for C. elegans that should be replenished every two months. If too many males occur
(W 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) with E. coli from
a bacterial lawn on agar [as sent by the Caenorhabditis Genetic Center (CGC)] using an inoculating
loop (6.25) and incubate for 17 h at 37 °C on a shaker (6.11) (overnight culture). Transfer 200 µl glycerol
(5.15) into each 1,5 ml plastic vial (6.5) and sterilize in an autoclave (6.1) at 121 °C for 20 min. Add under
sterile conditions 800 µl of the E. coli overnight culture (3.12), vortex and freeze immediately at −20 °C.
Bacterial stock cultures are thawed only once and discarded after use. Bacterial stock cultures should be
replenished after six months.
9.2 Pre-culture
Inoculate under sterile conditions an agar plate (3.1) with approximately 200 µl of an overnight culture (3.12)
of E. coli, distribute equally using a Drigalski spatula (6.3) and incubate for at least 8 h at (37 ± 2) °C. Cut two
2
or three small pieces (approximately 1 cm ) out of a starved plate (3.13) and transfer them under sterile
3) Supplier for Caenorhabditis elegans and Escherichia coli.
Caenorhabditis Genetic Center
Theresa Stiernagle
University of Minnesota
6-160 Jackson Hall
321 Church Street S.E.
Minneapolis, MN 55455
E-mail: cgc@umn.edu
http://www.cbs.umn.edu/CGC/
This information is given for the convenience of users of this International Standard and does not constitute an
endorsement by ISO of these products.
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conditions on to an agar plate with a fresh lawn of E. coli. After about 3 days at 20 °C, a lot of gravid
hermaphrodites as well as juveniles of stages 1 and 2 are found on the plate. The test starts with worms in the
first juvenile stage. In order to obtain worms synchronized to this life stage, rinse the plate with M9-medium
(5.9). Then filter the suspension containing the nematodes through a cascade of filter gauze (6.6) of 5 µm and
10 µm mesh size to retain larger juveniles and adults. The filtered suspension contains only first-stage
juveniles (J ). Measure the length of 30 J (killed with heat) to obtain the initial length of the introduced
1 1
exposed test organisms (mean value).
10 Procedure
10.1 Preparation of food medium
After thawing, vortex (6.20) a vial with the E. coli stock vigorously. Inoculate under sterile conditions x × 50 ml
of LB-medium (5.2) (depending on the demand) in 250 ml Erlenmeyer flasks (6.4) with approximately 20 µl of
the E. coli stock each and incubate for 17 h at 37 °C on a shaker (6.11). To control the bacterial density after
incubation, dilute an aliquot of the bacterial suspension 1→10 with LB-medium and measure the optical
density at 600 nm against the LB-medium. The turbidity of the bacterial suspension is given as specified in
ISO 7027 in FAU (formazine absorption units). OD600 is usually > 200 FAU. After centrifugation of the
bacterial suspension (20 min, 2 000g), remove the supernatant and resuspend the pellet in M9-medium (5.9).
After repeated centrifugation and removing of the supernatant, resuspend the pellet in approximately
⎯ x × 8 ml of M9-medium and adjust the bacterial density to (12 000 ± 600) FAU for testing sediment, soil
and waste;
⎯ x × 100 ml of M9-medium and adjust the bacterial density to (1 000 ± 50) FAU for testing pore water,
elutriates, extracts or solutions of reference substance.
Finally, the accurate volume of cholesterol stock solution is added (0,2 % of volume of bacterial suspension;
e.g. 100 µl of cholesterol stock solution in 50 ml bacterial suspension).
NOTE Densities of bacteria differ between tests with solid and liquid test material due to different exposure conditions.
10.2 Preparation of test material and controls
10.2.1 Sediment, soil and waste
Pass the test material (3.14) through a 2 mm sieve (6.22). Determine the dry mass of the test material and
control sediment or control soil in accordance with ISO 11465 by drying a small portion of the test sample.
Determine the pH of the test material and control sediment or control soil in accordance with ISO 10523
(aqueous test material, sediments) and ISO 10390 (soils).
Prepare at least four replicates for each test material and the control [artificial control sediment (5.12) or
control soil (5.13)]. Prepare one additional blank replicate (without test organisms) to estimate the number of
indigenous nematodes in the samples. For artificial substrates, such as the artificial control sediment, it is not
necessary to set up a blank replicate. For test material with W 40 % water content (based on total mass),
transfer (0,500 ± 0,010) g (wet mass) of test material into each test well (6.19). For test material with < 40 %
water content (based on total mass), artificial control sediment and control soil, transfer m ± 0,010 g into each
test well, add (0,500 − m) ml of M9-medium (5.9), and stir with a spatula to achieve a homogenous suspension.
Calculate m, expressed in grams, as given in Equation (1):
0,5 × 0,60
m = (1)
m
o,t
where m is the measured dry mass of the test material.
o,t
Store in a refrigerator at (8 ± 2) °C to avoid loss of moisture.
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Stir the food medium (3.10, 10.1; 12 000 FAU) to ensure homogeneity and add, immediately before the start
of the test, 0,5 ml of homogenized food medium (3.10) to each test well. Mix artificial control sediment or
control soil and test material with the added food medium thoroughly with a spatula.
10.2.2 Pore water, elutriate, extract
Prepare at least four replicates for each test material (3.14) and the control (aqueous control; 3.2). Transfer
0,5 ml of test material and aqueous control into each test well (6.19). Stir the food medium (3.10, 10.1;
1 000 FAU) to ensure homogeneity and add, immediately before the start of the test, 0,5 ml of homogenized
food medium to each test well.
10.2.3 Solution of reference substance
Prepare at least four replicates for each solution of the reference substance (5.14, Clause 7) and the control
(aqueous control; 3.2). Transfer 0,5 ml of the solution of reference substance and aqueous control into each
test well (6.19). Stir the food medium (3.10, 10.1; 1 000 FAU) to ensure homogeneity and add, immediately
before the start of the test, 0,5 ml of homogenized food medium to each test well.
10.3 Test
At the start of the test, transfer ten first-stage juveniles (J ; exposed test organisms) from the filtrate by
1
micropipette (6.8) to each of the test wells containing test material (3.14) and food medium (3.10) (after
allowing temperature to equilibrate to room temperature). Non-
...
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