SIST EN ISO 18763:2020
(Main)Soil quality - Determination of the toxic effects of pollutants on germination and early growth of higher plants (ISO 18763:2016)
Soil quality - Determination of the toxic effects of pollutants on germination and early growth of higher plants (ISO 18763:2016)
ISO 18763:2016 describes a technique for determining the effects of soil and soil-related materials on the seed germination and early growth of higher plants. These endpoints are useful indicators for the assessment of the quality of a soil as a habitat for organisms. It is applicable to all soils in which soil organisms are active and may be used to evaluate:
- the effects on plants due to toxicity of solid or liquid chemicals contaminating soil or materials (compost, sludge, waste) and chemicals added to soil;
- the changes in the soil effect on plants after restoration measures.
Bodenbeschaffenheit - Bestimmung der toxischen Wirkung von Schadstoffen auf die Keimung und das frühe Wachstum höherer Pflanzen (ISO 18763:2016)
Die vorliegende Internationale Norm beschreibt ein Verfahren zur Bestimmung der Wirkungen von Boden und bodenähnlichen Materialien auf die Samenkeimung und das frühe Wachstum höherer Pflanzen. Diese Endpunkte sind nützliche Indikatoren für die Beurteilung der Qualität eines Bodens als Lebensraum für Organismen. Diese Internationale Norm ist auf alle Böden anwendbar, in denen Bodenorganismen aktiv sind, und darf zur Bewertung von Folgendem angewendet werden:
- der Wirkungen auf Pflanzen aufgrund der Toxizität von festen oder flüssigen Chemikalien, die den Boden oder Materialien (Kompost, Schlamm, Abfälle) verunreinigen, und Chemikalien, die dem Boden zugegeben werden;
- der Änderungen der Wirkung des Bodens auf Pflanzen nach Sanierungsmaßnahmen.
Qualité du sol - Détermination des effets toxiques des polluants sur la germination et les premiers stades de croissance des végétaux supérieurs (ISO 18763:2016)
La présente Norme internationale décrit une méthode permettant de déterminer les effets de sols et de matériaux apparentés aux sols sur la germination des semences et les premiers stades de la croissance des végétaux supérieurs. Ces critères d'effet sont des indicateurs utiles pour l'évaluation de la qualité d'un sol en tant qu'habitat pour des organismes. La présente Norme internationale est applicable à tous les sols dans lesquels des organismes sont actifs et peut être utilisée pour évaluer:
— les effets sur les végétaux dus à la toxicité des produits chimiques solides ou liquides polluant les sols ou les matériaux (compost, boues, déchets) ainsi que les produits chimiques ajoutés aux sols;
— les variations dans les effets sur les végétaux du sol après des actions de réhabilitation.
Kakovost tal - Določanje toksičnih učinkov onesnaževal na kalivost in zgodnjo rast višjih rastlin (ISO 18763:2016)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 18763:2020
01-november-2020
Kakovost tal - Določanje toksičnih učinkov onesnaževal na kalivost in zgodnjo
rast višjih rastlin (ISO 18763:2016)
Soil quality - Determination of the toxic effects of pollutants on germination and early
growth of higher plants (ISO 18763:2016)
Bodenbeschaffenheit - Bestimmung der toxischen Wirkung von Schadstoffen auf die
Keimung und das frühe Wachstum höherer Pflanzen (ISO 18763:2016)
Qualité du sol - Détermination des effets toxiques des polluants sur la germination et les
premiers stades de croissance des végétaux supérieurs (ISO 18763:2016)
Ta slovenski standard je istoveten z: EN ISO 18763:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 18763:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 18763:2020
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SIST EN ISO 18763:2020
EN ISO 18763
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Determination of the toxic effects of
pollutants on germination and early growth of higher
plants (ISO 18763:2016)
Qualité du sol - Détermination des effets toxiques des Bodenbeschaffenheit - Bestimmung der toxischen
polluants sur la germination et la croissance primaire Wirkung von Schadstoffen auf die Keimung und das
des plantes supérieures (ISO 18763:2016) frühe Wachstum höherer Pflanzen (ISO 18763:2016)
This European Standard was approved by CEN on 13 April 2020.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18763:2020 E
worldwide for CEN national Members.
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SIST EN ISO 18763:2020
EN ISO 18763:2020 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 18763:2020
EN ISO 18763:2020 (E)
European foreword
The text of ISO 18763:2016 has been prepared by Technical Committee ISO/TC 190 "Soil quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 18763:2020 by
Technical Committee CEN/TC 444 “Environmental characterization of solid matrices” the secretariat of
which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by October 2020, and conflicting national standards shall
be withdrawn at the latest by October 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 18763:2016 has been approved by CEN as EN ISO 18763:2020 without any modification.
3
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SIST EN ISO 18763:2020
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SIST EN ISO 18763:2020
INTERNATIONAL ISO
STANDARD 18763
First edition
2016-07-01
Soil quality — Determination of
the toxic effects of pollutants on
germination and early growth of
higher plants
Qualité du sol — Détermination des effets toxiques des polluants sur
la germination et la croissance primaire des plantes supérieures
Reference number
ISO 18763:2016(E)
©
ISO 2016
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SIST EN ISO 18763:2020
ISO 18763:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
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SIST EN ISO 18763:2020
ISO 18763:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Reagents, test organisms and media . 3
6 Apparatus and materials. 4
7 Treatment and preparation of samples . 6
8 Procedure. 6
9 Measurement . 8
10 Calculation of the percentage inhibition . 9
11 Reference chemical .10
12 Precision .10
13 Validity criteria .11
14 Test report .11
Annex A (informative) Application of the phytotoxicity test in transparent test plates on
natural and artificial soils and soil materials, with different plant species .12
Annex B (informative) Assemblage of test plates for the phytotoxicity test .14
Annex C (informative) International interlaboratory comparisons on the phytotoxicity test .15
Bibliography .20
© ISO 2016 – All rights reserved iii
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SIST EN ISO 18763:2020
ISO 18763:2016(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
iv © ISO 2016 – All rights reserved
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SIST EN ISO 18763:2020
ISO 18763:2016(E)
Introduction
Ecotoxicological testing of test soils or waste materials to be disposed on soil are required to assess
the potential environmental risk resulting from soil pollution or the disposal of wastes such as sewage
sludge on farmland. There is also a need to monitor the quality of soil after reclamation of industrial
sites. Therefore, a very practical and rapid germination and growth test has been developed based on
seed germination and seedling growth in controlled environmental conditions.
The assay, which does not require any pretreatment of the seeds, is performed in “transparent test plates”,
incubated vertically, to allow the roots and the shoots of the germinated seeds to be seen. After 72 h
exposure, a picture of the transparent test plates is taken and can be analysed “by image analysis” for
multiple endpoints, such as percentage of seed germination and of length of roots and shoots. To account
for the plant species variability in sensitivity, the assays are performed with the seeds of three plant
species: one monocotyl (Sorghum saccharatum) and two dicotyls (Lepidium sativum and Sinapis alba).
A major advantage of this test is that after the shooting and storing of the pictures of the test plates, the
measurements by image analysis can be postponed to any appropriate timing.
Reference or standard soils can be used as negative controls, such as, for example, the ISO standard
artificial soil according to ISO 11269-1 and ISO 11269-2.
Commercially available seeds, with a shelf life longer than one year, allow the use of this test at any time
of the year.
Two International interlaboratory comparisons demonstrated that the test provides good results.
A substantial number of studies report data on the application of this test on various types of soils and
soil materials with several types of plant species.
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SIST EN ISO 18763:2020
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SIST EN ISO 18763:2020
INTERNATIONAL STANDARD ISO 18763:2016(E)
Soil quality — Determination of the toxic effects of
pollutants on germination and early growth of higher plants
1 Scope
This International Standard describes a technique for determining the effects of soil and soil-related
materials on the seed germination and early growth of higher plants. These endpoints are useful
indicators for the assessment of the quality of a soil as a habitat for organisms. This International
Standard is applicable to all soils in which soil organisms are active and may be used to evaluate:
— the effects on plants due to toxicity of solid or liquid chemicals contaminating soil or materials
(compost, sludge, waste) and chemicals added to soil;
— the changes in the soil effect on plants after restoration measures.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 11269-1, Soil quality — Determination of the effects of pollutants on soil flora — Part 1: Method for the
measurement of inhibition of root growth
ISO 11269-2, Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of
contaminated soil on the emergence and early growth of higher plants
ISO/TS 20281, Water quality — Guidance on statistical interpretation of ecotoxicity data
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
artificial soil
mixture of sand, kaolinite, peat and calcium carbonate prepared according to ISO 11269-1 and
ISO 11269-2
3.2
control soil
reference or standard soil used as a control and as a medium for preparing dilution series with test
soils or a reference substance
3.3
reference soil
uncontaminated site-specific soil (e.g. collected in the vicinity of a contaminated site) with similar
properties (nutrient concentrations, pH, organic carbon content and texture) as the test soil
3.4
standard soil
field-collected soil or artificial soil whose main properties (e.g. pH, texture, organic matter content) are
within a known range
Note 1 to entry: The properties of standard soils can differ from the test soil.
© ISO 2016 – All rights reserved 1
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SIST EN ISO 18763:2020
ISO 18763:2016(E)
1)
[1] [2]
EXAMPLE Euro-soils , artificial soil , LUFA soil.
3.5
test soil
either a natural or an artificial clean soil that is spiked with the test substance or a contaminated
[5]
natural soil (a site soil)
3.6
seeding emergence
appearance of a visible seedling above the surface of the cover material
[SOURCE: ISO 17126:2005, 3.1, modified]
3.7
germination
appearance of a root of at least 1 mm of length
3.8
pure water
grade of water, produced, for example, by single distillation, by de-ionization, by ultra-filtration or by
[5]
reverse osmosis
3.9
root length
length of the root from seed to root tip
3.10
shoot length
length of the part that grows upward, from seed to tip
3.11
water saturation
maximum water content that a soil can retain against gravity under undisturbed soil conditions,
conventionally stated as water content two days to three days after full saturation with water
[SOURCE: ISO 11074:2015, 2.1.5 field capacity, modified]
3.12
water saturated soil
soil which has reached its maximum water content
3.13
water-holding capacity
mass of water that evaporates from soil saturated with water when the soil is dried to constant mass at
[7]
105 °C, divided by the dry mass of the soil
3.14
negative control
any well-characterized material or substance that, when tested by a specific procedure, demonstrates
the suitability of the procedure to yield a reproducible, appropriately negative, non-reactive or minimal
response in the test system
[SOURCE: ISO 10993-10:2010, 3.12, modified]
3.15
effect percentage
percentage decrease of the seed germination and the growth of the plant roots and/or shoots in the test
soil in comparison to the control soil
1) Euro-soils, artificial soil and LUFA soil are examples of suitable products available commercially. This information
is given for the convenience of users of this document and does not constitute an endorsement by ISO of these
products.
2 © ISO 2016 – All rights reserved
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SIST EN ISO 18763:2020
ISO 18763:2016(E)
4 Principle
This method compares the seed germination and early growth of monocotyledonous and dicotyledonous
plants in a test soil and/or a series of mixtures with a control soil. This method may also be used for the
testing of compost, sludge or waste.
Seeds of one monocotyledonous plant, such as Sorghum saccharatum (L.) Moench, and two
dicotyledonous plants, such as Lepidium sativum L. and Sinapis alba L., are exposed to the test material
under controlled conditions. After (72 ± 1) h, the number of germinated seeds is recorded and the
length of the roots of the test plants is measured in the test soil and in the control soil.
If different seed species are used, the length of the incubation period may be adjusted, depending on the
time of germination of the seeds and the growth speed of the roots.
The test makes use of unique flat and shallow transparent test plates (6.3) composed of two
compartments, the lower one of which contains hydrated soil.
Seeds of the selected test plants are positioned at equal distance near the middle ridge of the test plate
(6.3) on a black filter paper (6.5) placed on top of the hydrated soil.
After closing the test plates (6.3) with their transparent cover, the test plates are placed vertically in a
holder (6.4) and incubated at (25 ± 1) °C for (72 ± 1) h.
At the end of the incubation period, the length of each root (and shoot, if wished) can be measured
directly with a ruler and recorded.
Alternatively, a “digital” picture is taken of the test plates (6.3) with the germinated plants (either
with a digital camera, a webcam camera or a flatbed paper scanner) for storage in a computer file. The
subsequent root length measurements are performed by image analysis. The analyses on germination
and root growth can then be made immediately or postponed to any appropriate timing.
NOTE The same procedure can be applied to also measure the shoot height, if wished. Calculation of the
shoot/root length ratio is a possible additional effect parameter.
5 Reagents, test organisms and media
5.1 Water.
Pure water having a conductivity below 10 μS/cm.
5.2 Test organisms.
The test organisms are seeds of one monocotyledonous plant, such as Sorghum saccharatum (L.)
Moench, and two dicotyledonous plants, such as Lepidium sativum L. and Sinapis alba L.
Investigations have been performed not only with the three plant species indicated in 5.2 but also with
other monocotyl and dicotyl plant species. A synthesis on these published studies is given in Annex A.
Seeds coated with insecticides and/or fungicides should be avoided.
5.3 Control soil.
Either reference or standard soils can be used as the control soil, if unhindered growth of the test plants
in these soils can be expected.
When comparing the root elongation in soils of known and unknown quality, the control soil and soil
under test should be of the same textural class, and be as similar as practicable in all respects other
than the presence of the chemical or contaminant being investigated. Indeed, significant differences in
soil characteristics other than the presence of contaminant may lead to differences in root lengths and
may induce false positive test results.
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SIST EN ISO 18763:2020
ISO 18763:2016(E)
Alternatively, artificial soil according to ISO 11269-1 and ISO 11269-2 may be used. The substrate called
“artificial soil” has the following composition:
Percentage
expressed on
dry-mass basis
Sphagnum peat finely ground and with no visible plant remains 10 %
Kaolinite clay containing not less than 30 % kaolinite 20 %
Industrial quartz sand (dominant fine sand with more than 50 % of particle size 69 %
0,05 mm to 0,2 mm)
NOTE As indicated in ISO 11269-1, 5 % peat have proven to be sufficient for maintaining the desired
structure of the artificial soil (with a corresponding increase of the sand percentage to 75 %). A lower percentage
of kaolinite clay (10 % instead of 20 %) is furthermore very close to the clay content of LUFA 2.2 and hence more
representative for a natural soil. The following composition of the artificial soil is therefore recommended: peat
5 %, kaolinite clay 10 % and sand 85 %.
Approximately 0,3 % to 1,0 % calcium carbonate (CaCO , pulverized, analytical grade) is necessary to
3
get a pH of 6,0 ± 0,5.
6 Apparatus and materials
6.1 Incubator or temperature controlled room, suitable for maintaining the specified conditions at
(25 ± 1) °C.
6.2 Digital camera, webcam camera or flatbed paper scanner, to shoot pictures of the test plates
with the germinated seeds, for storage in a computer file.
6.3 Test plates, transparent plates in polyvinylchloride (PVC).
The test plates are composed of a bottom part separated by a middle ridge into an upper part and a
lower part and a flat cover. Test plates can be “handmade” with the aid of transparent PVC sheet and
small rectangular sticks as described in Annex B.
2)
Alternatively, commercially available test plates can be used. The lower part of these test plates is
3
intended to hold approximately 90 cm of test soil. The latter test plates have on both parts on their
side small rectangular cavities for closing the plates tightly by a unique click system. The test plates
shall be provided with a label to record the specifics of each test plate (type of soil, type of seed, number
of the replicate).
2) The test plates supplied by MicroBioTests Inc. Mariakerke-Gent, Belgium are 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.
4 © ISO 2016 – All rights reserved
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SIST EN ISO 18763:2020
ISO 18763:2016(E)
Figure 1 — Test plate with cavities on the side for tight closing of the plate
6.4 Test plate holders, cardboard holders for vertical incubation of six test plates (6.3) each.
2
6.5 Black filter papers, rectangular high purity black filter papers (e.g. 85 g/m , 0,17 mm thickness,
45 s filtration speed) fitting the lower part of the test plate, to be placed on top of the soil in the lower
compartment of the test plates (6.3).
6.6 Microsieve cylinder, small plastic cylinder provided at the bottom with a nylon gauze, to be used
for determination of the water to be added to the test soil.
6.7 Wide mouth micropipette, plastic micropipette to be used with the microsieve cylinder (6.6) for
determination of the water to be added to the test soil.
6.8 Sieve, sieve of 2 mm mesh for sieving the test soil prior to use for the tests.
6.9 Thin spatula, hand tool with a thin blade used to mix the test soil with water.
6.10 Flat spatula, hand tool with a broad, flat blade that is used to spread and flatten the test soil into
the lower compartment of the test plates (6.3).
6.11 Tweezers, small pincer-like tool for handling the seeds.
© ISO 2016 – All rights reserved 5
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SIST EN ISO 18763:2020
ISO 18763:2016(E)
7 Treatment and preparation of samples
7.1 Soil samples
The assays are carried out at water saturation of the soils at the start of the tests.
7.1.1 Water-holding capacity determination
The water saturated soil can be obtained by adding to the soil mass the appropriate volume of pure
water determining the water-holding capacity.
7.1.2 Alternative procedure for determination of the volume of water to be added in the test
plates for hydration of air-dried soils
A simple and quick alternative procedure can be used to determine the volume of water to be added to
air-dried soils in the test plates (6.3).
For the artificial soil recommended in 5.3, the amount of water needed to be added for hydration has
been determined experimentally. Based on a water/soil ratio (on a vol/vol basis) of 0,39, one needs to
3
add 35 ml pure water to the 90 cm control soil in the test plate (6.3).
For other control soils, the amount of water needed to be added for hydration needs to be experimentally
determined, following the procedure indicated in the next paragraph for test soil samples.
For test soil samples, they first need to be air-dried, then the dry soil is sieved through a sieve (6.8) to
eliminate all coarse material.
NOTE Air drying is requested only on a separate soil sample for subsequent determination of the volume of
water to be added in the test plates.
3
Mix thoroughly 50 ml pure water and 90 cm test soil in a beaker with the aid of a thin spatula (6.9).
After 1 min to 2 min, two layers show up: the hydrated soil and a layer of water on top.
Lower vertically the microsieve cylinder (6.6) into the beaker, down to the surface of the hydrated soil,
and then lower it a little further down, so that it starts filling with supernatant.
With the wide mouth micropipette (6.7), suck up the water inside the microsieve and transfer it into a
graduated cylinder.
Put the microsieve cylinder (6.6) again into the beaker, and push it down a little further, so that it takes
up additional water from the soil. Transfer again the recovered water in the graduated cylinder and
repeat the former manipulations until no water comes out anymore from the soil.
Calculate how much water was needed for a complete hydration of the test soil. This volume (Vsat) is the
volume of water that has originally been added to the soil (= 50 ml) minus the volume of supernatant
water (S) which has been recovered in the graduated cylinder (Vsat = 50 − S).
8 Procedure
3
The procedure described hereafter is intended for use of 90 cm soil in the test plates (6.3).
In case of assessment of the dose-response relationship on an unknown solid sample, a full test shall be
performed comprising a number of mixtures of the control soil with the sample (e.g. compost, sludge,
waste). The dilutions shall be prepared within a geometric series with a separation factor not exceeding
2,0 (see ISO 11269-1). According to the selected dilution range, the test soil is mixed with the reference
soil or the standard soil thoroughly (either manually or by using a hand mixer). The homogeneity of the
mixture is checked visually. For each of the resulting dilutions, the volume of water to be added in the
test plates (6.3) shall be experimentally determined (7.1.2).
6 © ISO 2016 – All rights reserved
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SIST EN ISO 18763:2020
ISO 18763:2016(E)
8.1 Test procedure for determination of the effects of contaminated soils
8.1.1 Addition of control soil and test soil to the test plates and hydration of the soils
8
...
SLOVENSKI STANDARD
oSIST prEN ISO 18763:2020
01-januar-2020
Kakovost tal - Določevanje vpliva onesnaževal na kalivost in zgodnjo rast višjih
rastlin (ISO 18763:2016)
Soil quality - Determination of the toxic effects of pollutants on germination and early
growth of higher plants (ISO 18763:2016)
Qualité du sol - Détermination des effets toxiques des polluants sur la germination et la
croissance primaire des plantes supérieures (ISO 18763:2016)
Ta slovenski standard je istoveten z: prEN ISO 18763
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 18763:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST prEN ISO 18763:2020
---------------------- Page: 2 ----------------------
oSIST prEN ISO 18763:2020
INTERNATIONAL ISO
STANDARD 18763
First edition
2016-07-01
Soil quality — Determination of
the toxic effects of pollutants on
germination and early growth of
higher plants
Qualité du sol — Détermination des effets toxiques des polluants sur
la germination et la croissance primaire des plantes supérieures
Reference number
ISO 18763:2016(E)
©
ISO 2016
---------------------- Page: 3 ----------------------
oSIST prEN ISO 18763:2020
ISO 18763:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
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ISO 18763:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Reagents, test organisms and media . 3
6 Apparatus and materials. 4
7 Treatment and preparation of samples . 6
8 Procedure. 6
9 Measurement . 8
10 Calculation of the percentage inhibition . 9
11 Reference chemical .10
12 Precision .10
13 Validity criteria .11
14 Test report .11
Annex A (informative) Application of the phytotoxicity test in transparent test plates on
natural and artificial soils and soil materials, with different plant species .12
Annex B (informative) Assemblage of test plates for the phytotoxicity test .14
Annex C (informative) International interlaboratory comparisons on the phytotoxicity test .15
Bibliography .20
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ISO 18763:2016(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
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Introduction
Ecotoxicological testing of test soils or waste materials to be disposed on soil are required to assess
the potential environmental risk resulting from soil pollution or the disposal of wastes such as sewage
sludge on farmland. There is also a need to monitor the quality of soil after reclamation of industrial
sites. Therefore, a very practical and rapid germination and growth test has been developed based on
seed germination and seedling growth in controlled environmental conditions.
The assay, which does not require any pretreatment of the seeds, is performed in “transparent test plates”,
incubated vertically, to allow the roots and the shoots of the germinated seeds to be seen. After 72 h
exposure, a picture of the transparent test plates is taken and can be analysed “by image analysis” for
multiple endpoints, such as percentage of seed germination and of length of roots and shoots. To account
for the plant species variability in sensitivity, the assays are performed with the seeds of three plant
species: one monocotyl (Sorghum saccharatum) and two dicotyls (Lepidium sativum and Sinapis alba).
A major advantage of this test is that after the shooting and storing of the pictures of the test plates, the
measurements by image analysis can be postponed to any appropriate timing.
Reference or standard soils can be used as negative controls, such as, for example, the ISO standard
artificial soil according to ISO 11269-1 and ISO 11269-2.
Commercially available seeds, with a shelf life longer than one year, allow the use of this test at any time
of the year.
Two International interlaboratory comparisons demonstrated that the test provides good results.
A substantial number of studies report data on the application of this test on various types of soils and
soil materials with several types of plant species.
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INTERNATIONAL STANDARD ISO 18763:2016(E)
Soil quality — Determination of the toxic effects of
pollutants on germination and early growth of higher plants
1 Scope
This International Standard describes a technique for determining the effects of soil and soil-related
materials on the seed germination and early growth of higher plants. These endpoints are useful
indicators for the assessment of the quality of a soil as a habitat for organisms. This International
Standard is applicable to all soils in which soil organisms are active and may be used to evaluate:
— the effects on plants due to toxicity of solid or liquid chemicals contaminating soil or materials
(compost, sludge, waste) and chemicals added to soil;
— the changes in the soil effect on plants after restoration measures.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 11269-1, Soil quality — Determination of the effects of pollutants on soil flora — Part 1: Method for the
measurement of inhibition of root growth
ISO 11269-2, Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of
contaminated soil on the emergence and early growth of higher plants
ISO/TS 20281, Water quality — Guidance on statistical interpretation of ecotoxicity data
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
artificial soil
mixture of sand, kaolinite, peat and calcium carbonate prepared according to ISO 11269-1 and
ISO 11269-2
3.2
control soil
reference or standard soil used as a control and as a medium for preparing dilution series with test
soils or a reference substance
3.3
reference soil
uncontaminated site-specific soil (e.g. collected in the vicinity of a contaminated site) with similar
properties (nutrient concentrations, pH, organic carbon content and texture) as the test soil
3.4
standard soil
field-collected soil or artificial soil whose main properties (e.g. pH, texture, organic matter content) are
within a known range
Note 1 to entry: The properties of standard soils can differ from the test soil.
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1)
[1] [2]
EXAMPLE Euro-soils , artificial soil , LUFA soil.
3.5
test soil
either a natural or an artificial clean soil that is spiked with the test substance or a contaminated
[5]
natural soil (a site soil)
3.6
seeding emergence
appearance of a visible seedling above the surface of the cover material
[SOURCE: ISO 17126:2005, 3.1, modified]
3.7
germination
appearance of a root of at least 1 mm of length
3.8
pure water
grade of water, produced, for example, by single distillation, by de-ionization, by ultra-filtration or by
[5]
reverse osmosis
3.9
root length
length of the root from seed to root tip
3.10
shoot length
length of the part that grows upward, from seed to tip
3.11
water saturation
maximum water content that a soil can retain against gravity under undisturbed soil conditions,
conventionally stated as water content two days to three days after full saturation with water
[SOURCE: ISO 11074:2015, 2.1.5 field capacity, modified]
3.12
water saturated soil
soil which has reached its maximum water content
3.13
water-holding capacity
mass of water that evaporates from soil saturated with water when the soil is dried to constant mass at
[7]
105 °C, divided by the dry mass of the soil
3.14
negative control
any well-characterized material or substance that, when tested by a specific procedure, demonstrates
the suitability of the procedure to yield a reproducible, appropriately negative, non-reactive or minimal
response in the test system
[SOURCE: ISO 10993-10:2010, 3.12, modified]
3.15
effect percentage
percentage decrease of the seed germination and the growth of the plant roots and/or shoots in the test
soil in comparison to the control soil
1) Euro-soils, artificial soil and LUFA soil are examples of suitable products available commercially. This information
is given for the convenience of users of this document and does not constitute an endorsement by ISO of these
products.
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4 Principle
This method compares the seed germination and early growth of monocotyledonous and dicotyledonous
plants in a test soil and/or a series of mixtures with a control soil. This method may also be used for the
testing of compost, sludge or waste.
Seeds of one monocotyledonous plant, such as Sorghum saccharatum (L.) Moench, and two
dicotyledonous plants, such as Lepidium sativum L. and Sinapis alba L., are exposed to the test material
under controlled conditions. After (72 ± 1) h, the number of germinated seeds is recorded and the
length of the roots of the test plants is measured in the test soil and in the control soil.
If different seed species are used, the length of the incubation period may be adjusted, depending on the
time of germination of the seeds and the growth speed of the roots.
The test makes use of unique flat and shallow transparent test plates (6.3) composed of two
compartments, the lower one of which contains hydrated soil.
Seeds of the selected test plants are positioned at equal distance near the middle ridge of the test plate
(6.3) on a black filter paper (6.5) placed on top of the hydrated soil.
After closing the test plates (6.3) with their transparent cover, the test plates are placed vertically in a
holder (6.4) and incubated at (25 ± 1) °C for (72 ± 1) h.
At the end of the incubation period, the length of each root (and shoot, if wished) can be measured
directly with a ruler and recorded.
Alternatively, a “digital” picture is taken of the test plates (6.3) with the germinated plants (either
with a digital camera, a webcam camera or a flatbed paper scanner) for storage in a computer file. The
subsequent root length measurements are performed by image analysis. The analyses on germination
and root growth can then be made immediately or postponed to any appropriate timing.
NOTE The same procedure can be applied to also measure the shoot height, if wished. Calculation of the
shoot/root length ratio is a possible additional effect parameter.
5 Reagents, test organisms and media
5.1 Water.
Pure water having a conductivity below 10 μS/cm.
5.2 Test organisms.
The test organisms are seeds of one monocotyledonous plant, such as Sorghum saccharatum (L.)
Moench, and two dicotyledonous plants, such as Lepidium sativum L. and Sinapis alba L.
Investigations have been performed not only with the three plant species indicated in 5.2 but also with
other monocotyl and dicotyl plant species. A synthesis on these published studies is given in Annex A.
Seeds coated with insecticides and/or fungicides should be avoided.
5.3 Control soil.
Either reference or standard soils can be used as the control soil, if unhindered growth of the test plants
in these soils can be expected.
When comparing the root elongation in soils of known and unknown quality, the control soil and soil
under test should be of the same textural class, and be as similar as practicable in all respects other
than the presence of the chemical or contaminant being investigated. Indeed, significant differences in
soil characteristics other than the presence of contaminant may lead to differences in root lengths and
may induce false positive test results.
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Alternatively, artificial soil according to ISO 11269-1 and ISO 11269-2 may be used. The substrate called
“artificial soil” has the following composition:
Percentage
expressed on
dry-mass basis
Sphagnum peat finely ground and with no visible plant remains 10 %
Kaolinite clay containing not less than 30 % kaolinite 20 %
Industrial quartz sand (dominant fine sand with more than 50 % of particle size 69 %
0,05 mm to 0,2 mm)
NOTE As indicated in ISO 11269-1, 5 % peat have proven to be sufficient for maintaining the desired
structure of the artificial soil (with a corresponding increase of the sand percentage to 75 %). A lower percentage
of kaolinite clay (10 % instead of 20 %) is furthermore very close to the clay content of LUFA 2.2 and hence more
representative for a natural soil. The following composition of the artificial soil is therefore recommended: peat
5 %, kaolinite clay 10 % and sand 85 %.
Approximately 0,3 % to 1,0 % calcium carbonate (CaCO , pulverized, analytical grade) is necessary to
3
get a pH of 6,0 ± 0,5.
6 Apparatus and materials
6.1 Incubator or temperature controlled room, suitable for maintaining the specified conditions at
(25 ± 1) °C.
6.2 Digital camera, webcam camera or flatbed paper scanner, to shoot pictures of the test plates
with the germinated seeds, for storage in a computer file.
6.3 Test plates, transparent plates in polyvinylchloride (PVC).
The test plates are composed of a bottom part separated by a middle ridge into an upper part and a
lower part and a flat cover. Test plates can be “handmade” with the aid of transparent PVC sheet and
small rectangular sticks as described in Annex B.
2)
Alternatively, commercially available test plates can be used. The lower part of these test plates is
3
intended to hold approximately 90 cm of test soil. The latter test plates have on both parts on their
side small rectangular cavities for closing the plates tightly by a unique click system. The test plates
shall be provided with a label to record the specifics of each test plate (type of soil, type of seed, number
of the replicate).
2) The test plates supplied by MicroBioTests Inc. Mariakerke-Gent, Belgium are 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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Figure 1 — Test plate with cavities on the side for tight closing of the plate
6.4 Test plate holders, cardboard holders for vertical incubation of six test plates (6.3) each.
2
6.5 Black filter papers, rectangular high purity black filter papers (e.g. 85 g/m , 0,17 mm thickness,
45 s filtration speed) fitting the lower part of the test plate, to be placed on top of the soil in the lower
compartment of the test plates (6.3).
6.6 Microsieve cylinder, small plastic cylinder provided at the bottom with a nylon gauze, to be used
for determination of the water to be added to the test soil.
6.7 Wide mouth micropipette, plastic micropipette to be used with the microsieve cylinder (6.6) for
determination of the water to be added to the test soil.
6.8 Sieve, sieve of 2 mm mesh for sieving the test soil prior to use for the tests.
6.9 Thin spatula, hand tool with a thin blade used to mix the test soil with water.
6.10 Flat spatula, hand tool with a broad, flat blade that is used to spread and flatten the test soil into
the lower compartment of the test plates (6.3).
6.11 Tweezers, small pincer-like tool for handling the seeds.
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7 Treatment and preparation of samples
7.1 Soil samples
The assays are carried out at water saturation of the soils at the start of the tests.
7.1.1 Water-holding capacity determination
The water saturated soil can be obtained by adding to the soil mass the appropriate volume of pure
water determining the water-holding capacity.
7.1.2 Alternative procedure for determination of the volume of water to be added in the test
plates for hydration of air-dried soils
A simple and quick alternative procedure can be used to determine the volume of water to be added to
air-dried soils in the test plates (6.3).
For the artificial soil recommended in 5.3, the amount of water needed to be added for hydration has
been determined experimentally. Based on a water/soil ratio (on a vol/vol basis) of 0,39, one needs to
3
add 35 ml pure water to the 90 cm control soil in the test plate (6.3).
For other control soils, the amount of water needed to be added for hydration needs to be experimentally
determined, following the procedure indicated in the next paragraph for test soil samples.
For test soil samples, they first need to be air-dried, then the dry soil is sieved through a sieve (6.8) to
eliminate all coarse material.
NOTE Air drying is requested only on a separate soil sample for subsequent determination of the volume of
water to be added in the test plates.
3
Mix thoroughly 50 ml pure water and 90 cm test soil in a beaker with the aid of a thin spatula (6.9).
After 1 min to 2 min, two layers show up: the hydrated soil and a layer of water on top.
Lower vertically the microsieve cylinder (6.6) into the beaker, down to the surface of the hydrated soil,
and then lower it a little further down, so that it starts filling with supernatant.
With the wide mouth micropipette (6.7), suck up the water inside the microsieve and transfer it into a
graduated cylinder.
Put the microsieve cylinder (6.6) again into the beaker, and push it down a little further, so that it takes
up additional water from the soil. Transfer again the recovered water in the graduated cylinder and
repeat the former manipulations until no water comes out anymore from the soil.
Calculate how much water was needed for a complete hydration of the test soil. This volume (Vsat) is the
volume of water that has originally been added to the soil (= 50 ml) minus the volume of supernatant
water (S) which has been recovered in the graduated cylinder (Vsat = 50 − S).
8 Procedure
3
The procedure described hereafter is intended for use of 90 cm soil in the test plates (6.3).
In case of assessment of the dose-response relationship on an unknown solid sample, a full test shall be
performed comprising a number of mixtures of the control soil with the sample (e.g. compost, sludge,
waste). The dilutions shall be prepared within a geometric series with a separation factor not exceeding
2,0 (see ISO 11269-1). According to the selected dilution range, the test soil is mixed with the reference
soil or the standard soil thoroughly (either manually or by using a hand mixer). The homogeneity of the
mixture is checked visually. For each of the resulting dilutions, the volume of water to be added in the
test plates (6.3) shall be experimentally determined (7.1.2).
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8.1 Test procedure for determination of the effects of contaminated soils
8.1.1 Addition of control soil and test soil to the test plates and hydration of the soils
8.1.1.1 Control soil
3
If the artificial soil (5.3) is used as control, put 90 cm control soil (5.3) in the lower compartment of a test
plate (6.3), then slowly drop 35 ml pure water over the whole surface of the control soil in the test plate.
If another control soil is used, add the amount of water experimentally determined (7.1.2).
Wait 1 min to 2 min so that the water hydrates the soil totally.
With the flat blade spatula (6.10), flatten the wet soil evenly over the total surface of the bottom
compartment of the test plate (6.3), in order to obtain a layer of uniform depth.
Repeat the former operations for all the negative control test plates (6.3) (= nine test plates).
8.1.1.2 Test soil
3
Put 90 cm of sieved test soil into the bottom compartment of a test plate (6.3), then hydrate the test
soil as indicated above for the control soil by dropping a volume of pure water equal to Vsat (7.1.2) on
the surface of the soil in the test plate and flattening it with the flat blade spatula (6.10).
Repeat the former operations for all the other plates with test soil (= nine test plates).
3
NOTE An alternative procedure to measuring repeatedly a volume of 90 cm of soil in a graduated vessel
3
is to determine the weight of test soil corresponding to 90 cm and prepare such weights for transfer into the
corresponding test plates (6.3).
8.1.2 Placing of the seeds
Place on top of the hydrated soil one black filter paper (6.5) in all the test plates (6.3), to avoid trapping
air bubbles under the filter, and wait 1 min to 2 min until the filter is totally wet.
The assay is performed in three replicates with three different seeds. This means that three test plates
(6.3) with control soil, and three test plates with test soil, shall be inoculated with each type of seed.
With the aid of tweezers (6.11), take 10 seeds of the same test plant and put them in one row on the
black filter paper (6.5) at equal distance of each other, and at about 1 cm from the middle ridge of the
test plate (6.3).
Put the cover part of the test plate on the bottom part, close the test plate tightly and label it indicating
the specifics of the test (type of soil, type of seed, number of the replicate).
Repeat the former operation for each seed for the three control test plates and the three replicates with
test soil.
8.1.3 Incubation of the test plates
Place the six test plates (6.3) inoculated with the same seed (three with test soil and three with control
soil) vertically in one test plate holder (6.4).
Repeat this operation for the two other seeds.
Put the three test plate holders (6.4) with their test plates (6.3) in the incubator (6.1) and incubate at
(25 ± 1) °C for (72 ± 1) h.
NOTE Light does not seem to have an influence on the germination of the seeds nor the growth of the roots
during the short (three days) incubation time. It is therefore advised not to provide illumination in the incubator
during the test period.
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8.1.4 Image recording
The picture of the test plates at the end of the exposure period can be taken either with a webcam
camera, a digital camera or a flatbed paper scanner. The picture shall then be transferred to a file in a
computer.
Any type of image analysis programme can be used for the subsequent analyses, provided it allows for
length measurements.
NOTE A convenient and practical image analysis programme is “Image J” which can be downloaded directly
from the Internet (http://rsb.info.nih.gov/ij/download.html).
9 Measurement
All the measurements should be made within (60 ± 10) min after the end of exposure period or can be
postponed to any appropriate timing by image recording (8.1.4) of the test plates (6.3).
9.1 Counting the number of germinated seeds
Count the number of germinated seeds, N , in the test plates (6.3) and record the figure on the results
s
sheet “Individual data”.
9.2 Measurement of the root lengths of the germinated seeds
9.2.1 Measurement of all the root lengths in each test plate
9.2.1.1 Procedure for direct visual length meas
...
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