SIST EN ISO 20963:2011

SLOVENSKI STANDARD
SIST EN ISO 20963:2011
01-november-2011
.DNRYRVWWDO9SOLYRQHVQDåHYDOQDOLþLQNHåXåHON2[\WK\UHDIXQHVWD
'RORþHYDQMHDNXWQHVWUXSHQRVWL,62
Soil quality - Effects of pollutants on insect larvae (Oxythyrea funesta) - Determination of
acute toxicity (ISO 20963:2005)
Bodenbeschaffenheit - Auswirkungen von Schadstoffen auf Insektenlarven (Oxythyrea
funesta) - Bestimmung der akuten Toxizität (ISO 20963:2005)
Qualité du sol - Effets des polluants vis-à-vis des larves d'insectes (Oxythyrea funesta) -
Détermination de la toxicité aiguë (ISO 20963:2005)
Ta slovenski standard je istoveten z: EN ISO 20963:2011
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 20963:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 20963:2011

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SIST EN ISO 20963:2011


EUROPEAN STANDARD
EN ISO 20963

NORME EUROPÉENNE

EUROPÄISCHE NORM
June 2011
ICS 13.080.30
English Version
Soil quality - Effects of pollutants on insect larvae (Oxythyrea
funesta) - Determination of acute toxicity (ISO 20963:2005)
Qualité du sol - Effets des polluants vis-à-vis des larves Bodenbeschaffenheit - Auswirkungen von Schadstoffen auf
d'insectes (Oxythyrea funesta) - Détermination de la toxicité Insektenlarven (Oxythyrea funesta) - Bestimmung der
aiguë (ISO 20963:2005) akuten Toxizität (ISO 20963:2005)
This European Standard was approved by CEN on 10 June 2011.

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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20963:2011: E
worldwide for CEN national Members.

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SIST EN ISO 20963:2011
EN ISO 20963:2011 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 20963:2011
EN ISO 20963:2011 (E)
Foreword
The text of ISO 20963:2005 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 20963:2011 by
Technical Committee CEN/TC 345 “Characterization of soils” 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 December 2011, and conflicting national standards shall be withdrawn
at the latest by December 2011.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] 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, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 20963:2005 has been approved by CEN as a EN ISO 20963:2011 without any modification.

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SIST EN ISO 20963:2011

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SIST EN ISO 20963:2011


INTERNATIONAL ISO
STANDARD 20963
First edition
2005-04-01

Soil quality — Effects of pollutants on
insect larvae (Oxythyrea funesta) —
Determination of acute toxicity
Qualité du sol — Effets des polluants vis-à-vis des larves d'insectes
(Oxythyrea funesta) — Détermination de la toxicité aiguë





Reference number
ISO 20963:2005(E)
©
ISO 2005

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SIST EN ISO 20963:2011
ISO 20963:2005(E)
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©  ISO 2005
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
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ii © ISO 2005 – All rights reserved

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SIST EN ISO 20963:2011
ISO 20963:2005(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 1
4 Principle. 2
5 Test environment. 3
6 Reagents. 3
7 Apparatus. 4
8 Procedure. 4
8.1 Test design. 4
8.2 Preparation of test mixture . 5
8.3 Preparation of control container . 6
8.4 Food addition. 6
8.5 Introduction of the biological material. 6
8.6 Test conditions and measurements. 6
8.7 Reference substance. 6
9 Expression of results. 7
9.1 Calculation. 7
9.2 Expression of results. 8
10 Validity of the test . 8
11 Test report. 8
Annex A (informative) Example of breeding technique for Oxythyrea funesta . 9
Annex B (informative) Results of the collaborative trial carried out in France. 11
Bibliography . 12

© ISO 2005 – All rights reserved iii

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SIST EN ISO 20963:2011
ISO 20963:2005(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 20963 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
iv © ISO 2005 – All rights reserved

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SIST EN ISO 20963:2011
ISO 20963:2005(E)
Introduction
This International Standard describes a method for the determination of the acute toxicity of contaminated
soils and chemicals to the larvae of Oxythyrea funesta, a phytophagous coleopteran (Scarabaeidae,
Cetoniinae) with wide geographic distribution (Europe, North Africa and the Middle East).
Oxythyrea funesta has many characteristics which make it suitable for soil quality monitoring or testing effects
of chemicals:
 ecological relevance: this type of organism contributes in many ways to soil structure by stimulating soil
aeration and drainage;
 the first stages of development, i.e. incubation of eggs, larval cycle and pupation, are underground;
 the larvae of Oxythyrea funesta are tolerant to modifications of the test substrate granulometry;
 this species can be bred under controlled conditions.

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SIST EN ISO 20963:2011

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SIST EN ISO 20963:2011
INTERNATIONAL STANDARD ISO 20963:2005(E)

Soil quality — Effects of pollutants on insect larvae (Oxythyrea
funesta) — Determination of acute toxicity
1 Scope
This International Standard describes a method for the determination of the effects of contaminated soils and
substances on the survival of the larvae of Oxythyrea funesta. The larvae are exposed to the pollutants by
cuticular and alimentary uptake.
For contaminated soils, the effects on the survival are determined in the test soil and in a control soil.
Depending on the objectives of the study, the control and dilution substrates (dilution series of contaminated
soil) are either uncontaminated soil comparable to the soil sample to be tested or artificial soil substrate.
Effects of substances are assessed using a defined artificial soil substrate.
This International Standard is not applicable to volatile substances, i.e. substances for which Henry’s constant
or the air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,001 33 Pa at
25 °C.
NOTE This method does not take into account the possible degradation of the substances or pollutants during the
test.
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 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil for
the assessment of aerobic microbial processes in the laboratory
ISO 10390, Soil quality — Determination of pH
ISO 11268-1, Soil quality — Effects of pollutants on earthworms (Eisenia fetida) — Part 1: Determination of
acute toxicity using artificial soil substrate
1)
ISO 11269-2:— , Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of
chemicals on the emergence and growth of higher plants
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

1) To be published. (Revision of ISO 11269-2:1995)
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SIST EN ISO 20963:2011
ISO 20963:2005(E)
3.1
lethal concentration 50
LC 50
median lethal concentration of test substance or percent dilution of contaminated soil, which kills 50 % of the
test organism within the test period
3.2
lethal concentration x
LCx
concentration of the test substance or percent dilution of contaminated soil which kills x % of the test organism
within the test period
NOTE x is the percentage (10, 20, 25) of this effect.
3.3
Lowest Observed Effect Concentration
LOEC
lowest tested concentration of test substance or contaminated soil at which a statistically significant effect is
observed compared with the control
NOTE All test concentrations above the LOEC have a harmful effect equal or greater than those observed at the
LOEC.
3.4
No Observed Effect Concentration
NOEC
highest tested concentration of test substance or contaminated soil at which no statistically significant effect is
observed compared with the control
NOTE The NOEC is the test concentration immediately below the LOEC.
3.5
test mixture
mixture of the test substance with the test substrate, mixture of contaminated soil with the test substrate or
mixture of contaminated soil with an uncontaminated soil comparable to the soil sample to be tested
3.6
diapause
interruption of metabolism during egg, larva, pupa or imago development
4 Principle
Larvae of Cetoniinae (species Oxythyrea funesta) are exposed to a dilution range of contaminated soil or to a
range of concentrations of test substance. The mortality of larvae is determined after 10 days. Test mixtures
are prepared at the start of the test and are not renewed within the test period.
The results obtained from the test are compared with a control and are used to determine the concentration
which causes mortality of 50 % of the larvae (LC 50 ).
10days
The test is conducted in two steps:
 a preliminary test to determine appropriate dilution/concentration range in the final test;
 the definitive test to determine the dilutions/concentrations causing between 10 % and 90 % mortality,
which yields the test result.
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SIST EN ISO 20963:2011
ISO 20963:2005(E)
It may also be possible to determine the effects of contaminated soils or substances on the growth of larvae
(optional). The increase in mass within the test period allows this criterion to be considered as complementary
to mortality, in order to assess the effects of contaminated soils or substances.
5 Test environment
Tests shall be performed at a temperature of (26 ± 1) °C in complete darkness.
6 Reagents
6.1 Biological material
The species used in the test is Oxythyrea funesta (Scarabaeidae, Cetoniinae). Third-instar larvae with a fresh
mass within the range 100 mg to 200 mg are required to perform the test. The larvae shall be healthy, without
any bites or other visible injuries.
NOTE Depending on the breeding conditions, described in Annex A, larvae approximately two weeks old are suitable
for the test.
Larvae of similar size shall be selected. The difference in mass between the smallest and the largest larva
within a single test container shall not exceed 50 mg.
Eliminate the particles of breeding substrate stuck to the integument using, for example, a soft brush before
weighing the larvae. It is also possible to leave the animals to move along on slightly moist paper in order to
eliminate the breeding substrate stuck to the integument.
Synchronisation of breeding is necessary. An example of breeding technique for Oxythyrea funesta is given in
Annex A.
6.2 Test substrate
The mass of substrate used per glass container (7.1) shall be equivalent to 300 g (dry mass).
The substrate, called artificial soil, shall have the following composition (in accordance with ISO 11268-1):
 sphagnum peat, air-dried, finely ground and with no visible plant remains: 10 % (expressed on a dry mass
basis);
 kaolinite clay containing not less than 30 % kaolinite: 20 % (expressed on a dry mass basis);
 industrial quartz sand (dominant fine sand with more than 50 % particle size between 0,05 mm and
0,20 mm): 70 % (expressed on a dry mass basis).
Add pulverised calcium carbonate (CaCO ), of recognised analytical grade, as necessary to bring the pH of
3
the wetted substrate to 6,0 ± 0,5 (commonly between 0,5 % and 1 % of the mass of the dry ingredients).
Prepare the artificial soil by mixing the dry constituents listed above thoroughly in a large-scale laboratory
mixer. The amount of calcium carbonate required can vary, depending on properties of the individual batch of
sphagnum peat, and should be determined by weighing subsamples immediately before the test
...