Soil quality - Inhibition of reproduction of the soil mite (Hypoaspis aculeifer) by soil contaminants (ISO 21285:2019)

This document specifies a chronic test method for evaluating the habitat function of soils and determining effects of soil contaminants and substances on the reproduction of Hypoaspis aculeifer by ? mainly ? alimentary uptake. This method is applicable to soils and soil materials of unknown quality, e.g. from contaminated sites, amended soils, soils after remediation, industrial, agricultural or other sites under concern and waste materials (e.g. dredged material, municipal sludge from a wastewater treatment plant, composed material, or manure, especially those for possible land disposal). The reproduction (= number of juveniles) is the measured parameter of the test. The test reflects the bioavailability of a mixture of contaminants in natural soils (contaminated site soils) to a species which represents a trophic level which is not covered by other ISO standards. This test is not intended to replace the earthworm (see ISO 11268-2) or Collembola (see ISO 11267) reproduction tests since this species belongs not only to a different trophic group but also a different taxonomic group (= mites; i.e. arachnids) than those used usually.
Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate. For contaminated soils, the effects are determined in the soil to be tested and in a control soil. Depending on the objective of the study, the control and dilution substrate (dilution series of contaminated soil) are either an uncontaminated soil comparable to the soil to be tested (reference soil) or a standard soil (e.g. artificial soil).
This document provides information on how to use this method for testing samples (soils or substances) under temperate conditions.
This document is not applicable to substances for which the air/soil partition coefficient is greater than one, or to substances with vapour pressure exceeding 300 Pa at 25 °C.
NOTE The stability of the test substance cannot be ensured over the test period. No provision is made in the test method for monitoring the persistence of the substance under test.

Bodenbeschaffenheit - Hemmung der Reproduktion von Raubmilben (Hypoaspis aculeifer) durch Bodenverunreinigungen (ISO 21285:2019)

Dieses Dokument legt ein chronisches Verfahren zur Bewertung der Lebensraumfunktion von Böden und zur Bestimmung der Wirkungen von Bodenverunreinigungen und Substanzen auf die Reproduktion von Hypoaspis aculeifer — hauptsächlich — bei Aufnahme durch die Nahrung fest. Diese Methode ist anwendbar für Böden und Bodenmaterialien unbekannter Beschaffenheit, z. B. von verunreinigten Standorten, beauf¬schlagten Böden, Böden nach der Sanierung, industriellen, landwirtschaftlichen oder anderen betroffenen Standorten und Abfallmaterialien (z. B. Baggergut, kommunaler Schlamm aus einer Kläranlage, Kompost¬material oder Stalldung, speziell solche für eine mögliche Landdeponie). Die Reproduktion (= Anzahl der Juvenilen) ist der gemessene Parameter des Tests. Die Prüfung gibt die Bioverfügbarkeit einer Mischung von Kontaminanten in natürlichen Böden (Böden von verunreinigten Standorten) für eine Art wieder, die eine trophische Ebene repräsentiert, die nicht von anderen ISO Standards abgedeckt ist. Diese Prüfung soll nicht die Reproduktionsprüfung von Regenwürmern (ISO 11268 2) oder Collembolen (ISO 11267) ersetzen, da diese Spezies nicht nur zu einer anderen trophischen Gruppe, sondern auch zu einer anderen taxonomischen Gruppe (= Milben; d. h. Arachniden) als die üblicherweise verwendeten gehört.
Die Wirkungen der Substanzen werden mit einem Standardboden, vorzugsweise mit einem festgelegten künstlichen Bodensubstrat, bewertet. Bei verunreinigten Böden werden die Wirkungen im zu prüfenden Boden und in einem Kontrollboden bestimmt. Abhängig von der Zielsetzung der Untersuchung sind die Kontrolle und das zur Verdünnung verwendete Substrat (Verdünnungsreihen des verunreinigten Bodens) entweder ein mit dem zu prüfenden Boden vergleichbarer, nicht verunreinigter Boden (Referenzboden) oder ein Standardboden (z. B. künstlicher Boden).
Dieses Dokument gibt Informationen, wie dieses Verfahren zur Prüfung von Proben (Böden oder Substanzen) unter gemäßigten Bedingungen anzuwenden ist.
Dieses Dokument gilt nicht für Substanzen, deren Luft /Boden Verteilungskoeffizient größer eins ist oder für Substanzen mit einem Dampfdruck von über 300 Pa bei 25 °C.
ANMERKUNG Es kann nicht sichergestellt werden, dass die Prüfsubstanz während der gesamten Versuchsdauer stabil bleibt. Im Prüfverfahren wird die Persistenz der Prüfsubstanz nicht überwacht.

Qualité du sol - Inhibition de la reproduction de l'acarien prédateur (Hypoaspis aculeifer) par des contaminants du sol (ISO 21285:2019)

Le présent document décrit une méthode d'essai chronique pour évaluer la fonction d'habitat des sols et déterminer les effets des contaminants du sol et des substances sur la reproduction de l'espèce Hypoaspis aculeifer par — principalement — absorption par voie alimentaire. Cette méthode est applicable aux sols et matériaux du sol de qualité inconnue, par exemple, provenant de sites contaminés, de sols amendés, de sols ayant fait l'objet d'une remédiation, de sites industriels, agricoles et autres, et aux déchets (par exemple, matériau de dragage, boue résiduaire des stations d'épuration des eaux usées, engrais ou fumier, notamment ceux pour épandage éventuel). La reproduction (= nombre de juvéniles) est le paramètre mesuré au cours de l'essai. L'essai reflète la biodisponibilité d'un mélange de contaminants dans les sols naturels (sols de sites contaminés) vis-à-vis d'une espèce qui représente un niveau trophique qui n'est pas couvert par les autres normes ISO. Il n'est pas prévu d'utiliser cet essai pour remplacer les essais de reproduction vis-à-vis des vers de terre (voir ISO 11268-2) ou des collemboles (voir ISO 11267) car cette espèce appartient non seulement à un autre groupe trophique, mais également à un autre groupe taxonomique (= acariens; c'est-à-dire, arachnides) que les autres espèces utilisées habituellement.
Les effets des substances sont évalués en utilisant un sol standard, de préférence un substrat défini de sol artificiel. Pour les sols contaminés, les effets sont déterminés dans le sol à analyser et dans un sol témoin. Selon l'objectif de l'étude, le substrat témoin et le substrat de dilution (séries de dilutions du sol contaminé) sont soit un sol non contaminé comparable au sol à analyser (sol de référence), soit un sol standard (par exemple, sol artificiel).
Le présent document fournit des informations sur la façon d'utiliser cette méthode pour analyser des échantillons (sols ou substances) dans des conditions tempérées.
Il n'est pas applicable aux substances pour lesquelles le coefficient de partage air/sol est supérieur à 1, ou aux substances dont la tension de vapeur dépasse 300 Pa à 25 °C.
NOTE La stabilité de la substance d'essai ne peut pas être assurée tout au long de la période d'essai. La méthode d'essai ne prévoit pas de contrôler la persistance de la substance soumise à essai.

Kakovost tal - Zaviranje razmnoževanja pršice (Hypoaspis aculeifer) zaradi onesnaževal v tleh (ISO 21285:2019)

General Information

Status
Published
Public Enquiry End Date
02-Feb-2020
Publication Date
23-Sep-2020
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
17-Aug-2020
Due Date
22-Oct-2020
Completion Date
24-Sep-2020

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SLOVENSKI STANDARD
SIST EN ISO 21285:2020
01-november-2020
Kakovost tal - Zaviranje razmnoževanja pršice (Hypoaspis aculeifer) zaradi
onesnaževal v tleh (ISO 21285:2019)

Soil quality - Inhibition of reproduction of the soil mite (Hypoaspis aculeifer) by soil

contaminants (ISO 21285:2019)
Bodenbeschaffenheit - Hemmung der Reproduktion von Raubmilben (Hypoaspis
aculeifer) durch Bodenverunreinigungen (ISO 21285:2019)

Qualité du sol - Inhibition de la reproduction de l'acarien prédateur (Hypoaspis aculeifer)

par des contaminants du sol (ISO 21285:2019)
Ta slovenski standard je istoveten z: EN ISO 21285:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 21285:2020 en,fr,de

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

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SIST EN ISO 21285:2020
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SIST EN ISO 21285:2020
EN ISO 21285
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Inhibition of reproduction of the soil mite
(Hypoaspis aculeifer) by soil contaminants (ISO
21285:2019)

Qualité du sol - Inhibition de la reproduction de Bodenbeschaffenheit - Hemmung der Reproduktion

l'acarien prédateur (Hypoaspis aculeifer) par des von Raubmilben (Hypoaspis aculeifer) durch

contaminants du sol (ISO 21285:2019) Bodenverunreinigungen (ISO 21285:2019)
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 21285:2020 E

worldwide for CEN national Members.
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SIST EN ISO 21285:2020
EN ISO 21285:2020 (E)
Contents Page

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

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SIST EN ISO 21285:2020
EN ISO 21285:2020 (E)
European foreword

The text of ISO 21285:2019 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 21285:2020 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 21285:2019 has been approved by CEN as EN ISO 21285:2020 without any modification.

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SIST EN ISO 21285:2020
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SIST EN ISO 21285:2020
INTERNATIONAL ISO
STANDARD 21285
First edition
2019-04
Soil quality — Inhibition of
reproduction of the soil mite
(Hypoaspis aculeifer) by soil
contaminants
Qualité du sol — Inhibition de la reproduction de l'acarien prédateur
(Hypoaspis aculeifer) par des contaminants du sol
Reference number
ISO 21285:2019(E)
ISO 2019
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SIST EN ISO 21285:2020
ISO 21285:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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SIST EN ISO 21285:2020
ISO 21285:2019(E)
Contents Page

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

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

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

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

3 Terms and definitions ..................................................................................................................................................................................... 2

4 Principle ........................................................................................................................................................................................................................ 3

5 Reagents and material .................................................................................................................................................................................... 4

5.1 Biological material ............................................................................................................................................................................... 4

5.2 Test mixtures ............................................................................................................................................................................................ 4

5.3 Reference substance ........................................................................................................................................................................... 5

6 Apparatus ..................................................................................................................................................................................................................... 6

7 Procedure..................................................................................................................................................................................................................... 6

7.1 Experimental design .......................................................................................................................................................................... 6

7.1.1 General...................................................................................................................................................................................... 6

7.1.2 Range-finding test (preliminary test) ............................................................................................................ 7

7.1.3 Definitive test ...................................................................................................................................................................... 7

7.1.4 Limit test ................................................................................................................................................................................. 7

7.2 Preparation of test mixtures ....................................................................................................................................................... 8

7.2.1 Testing contaminated soil ........................................................................................................................................ 8

7.2.2 Testing substances added to the test substrate .................................................................................... 8

7.2.3 Preparation of control containers ..................................................................................................................... 9

7.3 Addition of the biological material ........................................................................................................................................ 9

7.4 Test conditions and measurements ...................................................................................................................................... 9

7.5 Feeding of the mites............................................................................................................................................................................ 9

7.6 Determination of surviving predatory mites .............................................................................................................10

8 Calculation and expression of results ..........................................................................................................................................10

8.1 Calculation ...............................................................................................................................................................................................10

8.2 Expression of results .......................................................................................................................................................................10

9 Validity of the test .............................................................................................................................................................................................10

10 Statistical analysis ............................................................................................................................................................................................11

10.1 General ........................................................................................................................................................................................................11

10.2 Single-concentration tests ..........................................................................................................................................................11

10.3 Multi-concentration tests............................................................................................................................................................11

10.3.1 Range-finding test ........................................................................................................................................................11

10.3.2 Definitive test ...................................................................................................................................................................11

11 Test report ................................................................................................................................................................................................................12

Annex A (informative) Techniques for rearing and breeding of predatory mites .............................................14

Annex B (normative) Determination of water-holding capacity ..........................................................................................16

Annex C (informative) Guidance on adjustment of pH of artificial soil .........................................................................17

Annex D (informative) Extraction and counting of predatory mites ................................................................................18

Annex E (informative) Basic information on the biology of Hypoaspis (Geolaelaps) aculeifer .............19

Bibliography .............................................................................................................................................................................................................................20

© ISO 2019 – All rights reserved iii
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SIST EN ISO 21285:2020
ISO 21285:2019(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

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

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,

Biological characterization.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved
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SIST EN ISO 21285:2020
ISO 21285:2019(E)
Introduction

Ecotoxicological test systems are applied to obtain information about the effects of contaminants in

soil and are proposed to complement conventional chemical analysis (see ISO 15799 and ISO 17616).

ISO 15799 includes a list and short characterization of recommended and standardized test systems

and ISO 17616 gives guidance on the choice and evaluation of the bioassays. Aquatic test systems with

soil eluate are applied to obtain information about the fraction of contaminants potentially reaching

the groundwater by the water path (retention function of soils), whereas terrestrial test systems are

used to assess the habitat function of soils.

Mites (Acari) are a world-wide and diverse group of arthropods belonging to the class Arachnida with

over 40 000 species recorded, divided into two super-orders (Acariformes and Parasitiformes). Due to

their relative small size (a few µm to a few cm), they occupy specific ecological niches on plants as well

as in soils (see Reference [13]).

Among soil-inhabiting mites, the role of predation is ensured by, for example, Hypoaspis sp. (Laelapidae).

Because they are exposed to chemical contamination, mites are already considered in the environmental

risk assessment of pesticides, as non-target organisms (see Reference [10]). Indeed, among the data

required for active substances of pesticides, effects on predatory mites are assessed, i.e. for the plant-

inhabitant Typhlodromus pyri (Phytoseiidae) and the soil-inhabitant Hypoaspis aculeifer (Laelapidae)

(see Reference [6]).
[23][17]

The first authors introducing H. aculeifer as a test organism in ecotoxicological studies were later

proposed a two-species test system in the European project SECOFASE (Sublethal Effects of Chemicals

on Fauna in the Soil Ecosystem), including the collembolan Folsomia fimetaria as prey. In the context

of the development of an ecotoxicological test for the assessment of plant protection products on non-

target arthropods (see References [5][6]), a protocol on soil predatory mites using H. aculeifer was

further proposed. More recently, a standard test protocol for the assessment of chemicals was developed

for this species by OECD in 2008 and revised in 2016. The results of the associated international ring-

test were published in Reference [25].

Among mites, the predator Hypoaspis aculeifer is the most studied species in laboratory. The reproduction

end point was found in general to be more sensitive than mortality and avoidance. Compared to other

soil meso-fauna invertebrates, mites were found in general less sensitive than or as sensitive as other

test species, depending on the end points and chemicals studied. Considering semi-field studies,

H. aculeifer was used as a top predator whereas other soil invertebrates, mainly springtails, were

ranked in the grazer group. In these studies, mites showed to be quite tolerant towards anthropogenic

contamination. This statement was also corroborated by field surveys. However, the applicability of

laboratory test methods for the assessment of environmental samples (contaminated soils, wastes etc.)

with mites is emphasized, as to date a limited number of studies are available.

This document describes a method that is based on the determination of lethal and sublethal effects

of contaminated soils to adult predatory mites of the species Hypoaspis aculeifer. This species is

considered to be representative of predatory soil arthropods. Background information on the ecology

of these mites and their use in ecotoxicological testing is available in Reference [14].

© ISO 2019 – All rights reserved v
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SIST EN ISO 21285:2020
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SIST EN ISO 21285:2020
INTERNATIONAL STANDARD ISO 21285:2019(E)
Soil quality — Inhibition of reproduction of the soil mite
(Hypoaspis aculeifer) by soil contaminants
1 Scope

This document specifies a chronic test method for evaluating the habitat function of soils and

determining effects of soil contaminants and substances on the reproduction of Hypoaspis aculeifer by

– mainly – alimentary uptake. This method is applicable to soils and soil materials of unknown quality,

e.g. from contaminated sites, amended soils, soils after remediation, industrial, agricultural or other

sites under concern and waste materials (e.g. dredged material, municipal sludge from a wastewater

treatment plant, composed material, or manure, especially those for possible land disposal). The

reproduction (= number of juveniles) is the measured parameter of the test. The test reflects the

bioavailability of a mixture of contaminants in natural soils (contaminated site soils) to a species which

represents a trophic level which is not covered by other ISO standards. This test is not intended to

replace the earthworm (see ISO 11268-2) or Collembola (see ISO 11267) reproduction tests since this

species belongs not only to a different trophic group but also a different taxonomic group (= mites; i.e.

arachnids) than those used usually.

Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate. For

contaminated soils, the effects are determined in the soil to be tested and in a control soil. Depending

on the objective of the study, the control and dilution substrate (dilution series of contaminated soil)

are either an uncontaminated soil comparable to the soil to be tested (reference soil) or a standard soil

(e.g. artificial soil).

This document provides information on how to use this method for testing samples (soils or substances)

under temperate conditions.

This document is not applicable to substances for which the air/soil partition coefficient is greater than

one, or to substances with vapour pressure exceeding 300 Pa at 25 °C.

NOTE The stability of the test substance cannot be ensured over the test period. No provision is made in the

test method for monitoring the persistence of the substance under test.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 10390, Soil quality — Determination of pH

ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary

analysis)

ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level

using barium chloride solution

ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by

sieving and sedimentation

ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis —

Gravimetric method

ISO 18400-206, Soil quality — Sampling — Part 206: Collection, handling and storage of soil under aerobic

conditions for the assessment of microbiological processes, biomass and diversity in the laboratory

© ISO 2019 – All rights reserved 1
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SIST EN ISO 21285:2020
ISO 21285:2019(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
contaminant
substance or agent present in the soil as a result of human activity
3.2
effect concentration for x % effect

concentration (mass fraction) of a test sample that causes x % of an effect on a given end point within a

given exposure period when compared with a control

EXAMPLE An EC is a concentration estimated to cause an effect on a test end point in 50 % of an exposed

population over a defined exposure period.

Note 1 to entry: The ECx is expressed as a percentage of soil to be tested (dry mass) per soil mixture (dry mass).

When substances are tested, the ECx is expressed as the mass of the test substance per dry mass of soil in

milligrams per kilogram.
3.3
effect rate

rate of a soil to be tested that causes an x % of an effect on a given end point within a given exposure

period when compared with a control
3.4
limit test

single concentration test consisting of at least four replicates each, the soil to be tested without any

dilution or the highest concentration of test substance mixed into the control soil and the control

3.5
lowest observed effect concentration
LOEC

lowest test substance concentration that has a statistically significant effect (probability p < 0,05)

Note 1 to entry: In this test, the LOEC is expressed as a mass of test substance per dry mass of the soil to be

tested. All test concentrations above the LOEC should usually show an effect that is statistically different from

the control.
3.6
lowest observed effect rate
LOER

lowest rate of a soil to be tested in a control soil at which a statistically significant effect is observed

3.7
no observed effect concentration
NOEC

highest test substance concentration immediately below the LOEC (3.5) at which no effect is observed

Note 1 to entry: In this test, the concentration corresponding to the NOEC, has no statistically significant effect

(probability p < 0,05) within a given exposure period when compared with the control.

2 © ISO 2019 – All rights reserved
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SIST EN ISO 21285:2020
ISO 21285:2019(E)
3.8
no observed effect rate
NOER

lowest rate of a soil to be tested immediately below the LOER (3.6) which when compared to the control

has no statistically significant effect (probability p < 0,05) within a given exposure period

3.9
reference soil

uncontaminated soil with comparable pedological properties (nutrient concentrations, pH, organic

carbon content and texture) to the soil being studied
3.10
standard soil

field-collected soil or artificial soil whose main properties (pH, texture, organic matter content) are

within a known range
EXAMPLE Euro soils, artificial soil, LUFA standard soil type 2.2.

Note 1 to entry: The properties of standard soils can differ from the soil to be tested.

3.11
control soil

reference or standard soil used as a control and as medium for preparing dilution series with soils to be

tested or a reference substance, which fulfils the validity criteria

Note 1 to entry: In the case of natural soil, it is advisable to demonstrate its suitability for a test and for achieving

the test validity criteria before using the soil in a definitive test.
3.12
test mixture

mixture of contaminated soil or the test substance (e.g. chemical, biosolid, waste) with control soil

3.13
test mixture ratio
ratio between the soil to be tested and the control soil in a test mixture
4 Principle

Adult females are exposed to the soil to be tested and the effects on reproduction measured are

compared to those observed for females exposed to a control soil. If appropriate, effects based on

exposure to a dilution range of contaminated soil and control soil or a range of concentrations of a test

substance mixed into control soil are determined. Test mixtures are prepared at the start of the test

and are not renewed within the test period. The test is started with 10 adult females per test vessel.

Males are not introduced in the test, because experience has shown that females mate immediately or

shortly after hatching from the deutonymph stage, if males are present. As the females are introduced

into the test about 7 d after they have reached the adult stage, the females can be considered as already

mated (Annex A and Annex E). The test runs until the first offspring have reached the deutonymph

stage. At 20 °C the exposure time ends at day 14 after introducing the females (day 0), followed by

two days of extraction. The number of surviving females and the number of juveniles per test vessel

are determined. The reproductive output of the mites exposed to the test mixtures is compared to

that of the controls in order to determine the concentrations which cause no effects on mortality and

reproduction (NOER/NOEC) and the concentration resulting in x % reduction of juveniles hatched from

eggs compared to the control (ER /EC ) respectively, depending on the experimental design (see 7.1.3).

x x

In case there is no prior knowledge of the dilution/concentration of the soil to be tested or the test

substance likely to have an effect, then it is useful to conduct the test in two steps:

— A range-finding test on reproduction is carried out, to give an indication of the effect dilution/

concentration, and the dilution/concentration giving no mortality (NOER/NOEC). Dilutions/

concentrations to be used in the definitive test can then be selected;
© ISO 2019 – All rights reserved 3
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SIST EN ISO 21285:2020
ISO 21285:2019(E)
— the definitive test on reproduction to determine sublethal effects
...

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