Soil quality - Effects of pollutants on earthworms - Part 3: Guidance on the determination of effects in field situations (ISO 11268-3:2014)

ISO 11268-3: 2014 specifies techniques for determining the effects of substances on earthworms in the field and provides a basis for determining the effects of chemicals applied to or incorporated into soil, including soil injections or drilled pelleted formulations.

Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Regenwürmer - Teil 3: Anleitung für die Bestimmung von Wirkungen unter Freilandbedingungen (ISO 11268-3:2014)

Qualité du sol - Effets des polluants vis-à-vis des vers de terre - Partie 3: Lignes directrices relatives à la détermination des effets sur site (ISO 11268-3:2014)

ISO 11268-3:2014 décrit des techniques utilisées pour étudier les effets de substances sur les vers de terre sur le terrain et constitue une base pour la détermination des effets des produits chimiques appliqués ou incorporés au sol, y compris des injections dans le sol ou l'enfouissement de granulés par semoirs.

Kakovost tal - Učinki onesnaževal na deževnike - 3. del: Navodilo za ugotavljanje učinkov v terenskih razmerah (ISO 11268-3:2014)

Ta del standarda ISO 11268 opisuje tehnike za določanje učinkov snovi na deževnike v terenskih razmerah ter zagotavlja podlago za ugotavljanje učinkov kemikalij, uporabljenih na tleh ali vključenih v tla.

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Published
Publication Date
11-Aug-2015
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6060 - Definitive text made available (DAV) - Publishing
Due Date
12-Aug-2015
Completion Date
12-Aug-2015

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SLOVENSKI STANDARD
SIST EN ISO 11268-3:2015
01-november-2015

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Soil quality - Effects of pollutants on earthworms - Part 3: Guidance on the determination

of effects in field situations (ISO 11268-3:2014)

Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Regenwürmer - Teil 3: Anleitung

für die Bestimmung von Wirkungen unter Freilandbedingungen (ISO 11268-3:2014)

Qualité du sol - Effets des polluants vis-à-vis des vers de terre - Partie 3: Lignes

directrices relatives à la détermination des effets sur site (ISO 11268-3:2014)
Ta slovenski standard je istoveten z: EN ISO 11268-3:2015
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 11268-3:2015 en,fr,de

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

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SIST EN ISO 11268-3:2015
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SIST EN ISO 11268-3:2015
EUROPEAN STANDARD
EN ISO 11268-3
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2015
ICS 13.080.30
English Version
Soil quality - Effects of pollutants on earthworms - Part 3:
Guidance on the determination of effects in field situations (ISO
11268-3:2014)

Qualité du sol - Effets des polluants vis-à-vis des vers de Bodenbeschaffenheit - Wirkungen von Schadstoffen auf

terre - Partie 3: Lignes directrices relatives à la Regenwürmer - Teil 3: Anleitung für die Bestimmung von

détermination des effets sur site (ISO 11268-3:2014) Wirkungen unter Freilandbedingungen (ISO 11268-3:2014)

This European Standard was approved by CEN on 6 August 2015.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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: Avenue Marnix 17, B-1000 Brussels

© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11268-3:2015 E

worldwide for CEN national Members.
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SIST EN ISO 11268-3:2015
EN ISO 11268-3:2015 (E)
Contents Page

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

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SIST EN ISO 11268-3:2015
EN ISO 11268-3:2015 (E)
European foreword

The text of ISO 11268-3:2014 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 11268-3:2015 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 February 2016, and conflicting national standards shall be withdrawn

at the latest by February 2016.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece,

Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,

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

Endorsement notice

The text of ISO 11268-3:2014 has been approved by CEN as EN ISO 11268-3:2015 without any modification.

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SIST EN ISO 11268-3:2015
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SIST EN ISO 11268-3:2015
INTERNATIONAL ISO
STANDARD 11268-3
Second edition
2014-10-15
Soil quality — Effects of pollutants
on earthworms —
Part 3:
Guidance on the determination of
effects in field situations
Qualité du sol — Effets des polluants vis-à-vis des vers de terre —
Partie 3: Lignes directrices relatives à la détermination des effets sur site
Reference number
ISO 11268-3:2014(E)
ISO 2014
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SIST EN ISO 11268-3:2015
ISO 11268-3:2014(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2014

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
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved
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SIST EN ISO 11268-3:2015
ISO 11268-3:2014(E)
Contents Page

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

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

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

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

3 Units ................................................................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 1

5 Reagents and material .................................................................................................................................................................................... 2

6 Apparatus ..................................................................................................................................................................................................................... 2

7 Procedure..................................................................................................................................................................................................................... 2

7.1 Sampling of earthworm populations ................................................................................................................................... 2

7.2 Preservation .............................................................................................................................................................................................. 3

7.3 Determination of biomass ............................................................................................................................................................. 3

8 Preparation for the test ................................................................................................................................................................................. 3

8.1 Test site .......................................................................................................................................................................................................... 3

9 Procedure..................................................................................................................................................................................................................... 6

9.1 Application of test substance ......... ............................................................................................................................................. 6

9.2 Sampling dates ........................................................................................................................................................................................ 6

9.3 Reference substance ........................................................................................................................................................................... 7

10 Data assessment.................................................................................................................................................................................................... 7

10.1 End points ................................................................................................................................................................................................... 7

10.2 Identification of earthworm species .................................................................................................................................... 8

10.3 Determination of biomass with gut content .................................................................................................................. 8

11 Calculation and expression of results ............................................................................................................................................. 8

12 Validity of the test ................................................................................................................................................................................................ 8

13 Test report ................................................................................................................................................................................................................... 9

Annex A (informative) Additional requirements of pesticide testing .............................................................................10

Annex B (informative) Information on specific earthworm species or communities in

different climatic or geographic regions ..................................................................................................................................11

Bibliography .............................................................................................................................................................................................................................12

© ISO 2014 – All rights reserved iii
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SIST EN ISO 11268-3:2015
ISO 11268-3:2014(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.

This second edition cancels and replaces the first edition (ISO 11268-3:1999), which has been

technically revised.

ISO 11268 consists of the following parts, under the general title Soil quality — Effects of pollutants

on earthworms:
— Part 1: Determination of acute toxicity to Eisenia fetida/Eisenia andrei

— Part 2: Determination of effects on reproduction to Eisenia fetida/Eisenia andrei

— Part 3: Guidance on the determination of effects in field situations
iv © ISO 2014 – All rights reserved
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SIST EN ISO 11268-3:2015
ISO 11268-3:2014(E)
Introduction

The earthworm field test is based on a method being developed by the German Federal Biological

[6]

Research Centre for Agriculture and Forestry for the testing of pesticides. Later, it was internationally

standardized by the International Organization for Standardization (ISO), taking into account

[7]

results and recommendations of an international workshop in 1991 in Sheffield, United Kingdom,

“Ecotoxicology of Earthworms”, as a tool for characterizing soil quality. Growing experience has shown

that the practical performance of the test can be improved. In two meetings organized by the Federal

Biological Research Centre for Agriculture and Forestry (Braunschweig, 2002) and by the German

Federal Agency for Consumer Protection and Food Safety (Lille, 2005), an ad-hoc working group of

experts from various countries and institutions proposed recommendations that should be taken into

account if revision has been approved by voting in the periodical review. A report of the discussions,

[8]
comments, and recommendations has been published.

In cases where earthworms and other organisms are used as bioindicators to assess the soil quality of a

site as a habitat for soil organisms, guidance for extraction procedures and advice for planning a survey

is given in ISO 23611-1 to ISO 23611-6.
© ISO 2014 – All rights reserved v
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SIST EN ISO 11268-3:2015
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SIST EN ISO 11268-3:2015
INTERNATIONAL STANDARD ISO 11268-3:2014(E)
Soil quality — Effects of pollutants on earthworms —
Part 3:
Guidance on the determination of effects in field situations
1 Scope

This part of ISO 11268 specifies techniques for determining the effects of substances on earthworms

in the field and provides a basis for determining the effects of chemicals applied to or incorporated into

soil, including soil injections or drilled pelleted formulations.
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 10390, Soil quality — Determination of pH

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

ISO 11274, Soil quality — Determination of the water-retention characteristic — Laboratory methods

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

sieving and sedimentation

ISO 23611-1, Soil quality — Sampling of soil invertebrates — Part 1: Hand-sorting and formalin extraction

of earthworms
3 Units

Rates of application of test substances are expressed in kilograms per hectare (kg/ha) or litres per

hectare (l/ha) of the substance applied. When this is a formulated material, the application rate is

expressed in terms of the amount of active ingredient applied.

The concentrations of test substances incorporated in the soil are given in mg active ingredient (a.i.)/

kg soil dry mass, d . The same units are used when comparing the results of this field test with those

gained in laboratory studies.
4 Principle

Species, numbers, and biomass of earthworms collected by sampling plots treated with a test substance

are compared with those collected from treated control and reference plots. Sampling is performed as

specified in ISO 23611-1. The duration of the study depends on the characteristics of the test substance

but is usually of one year’s duration. Sampling dates are chosen to lie within the periods of activity of

the earthworms.

The test is of a randomized complete block design with four replicates per treatment. Statistical analysis

of numbers of each species collected at each sampling occasion is used to determine the effects of

treatments by comparing abundance, biomass, and diversity between control and treated plots.

NOTE The test also generates samples of earthworms from treated plots for residue analysis where such

information is appropriate.
© ISO 2014 – All rights reserved 1
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SIST EN ISO 11268-3:2015
ISO 11268-3:2014(E)
5 Reagents and material
5.1 Formalin [formaldehyde solution, 4 % (volume fraction)].
5.2 Formalin [formaldehyde solution, 37 % (volume fraction)].
5.3 Ethanol, 70 % (volume fraction).

5.4 Carbendazim, e.g. applied as Derosal® formulation (360 g a.i./l) as reference substance.

6 Apparatus
Use standard laboratory equipment and the following.
6.1 Plastic vessel, (250 ml and 500 ml) for storing the worms.
6.2 Plastic hand gloves.
6.3 Forceps.
2 2
6.4 Piece of thick plastic (1 m to 2 m ).
6.5 Spade or shovel.
6.6 Dissecting microscope, with low magnification (10× to 40×).
6.7 Balance (0,01 g to 200 g).
6.8 Water-can, (preferably 20 l) with water (20 l per sampling plot).
6.9 Pencil, note book, water resistant marker, labels.
6.10 Thermometer, e.g. for measuring air temperature.
6.11 Drying cabinet, for soil water content determination.
7 Procedure
7.1 Sampling of earthworm populations

Sampling of earthworms is done by a combination of two different methods: hand-sorting and formalin

extraction. Based on several comparative studies, this combination is clearly recommended in the various

[9] [10] [13]

reviews on earthworm ecology (e.g. , , ). For details of extraction procedures, see ISO 23611-1.

Sampling should be done at times of the year where the animals are not forced by the environmental

conditions (i.e. low soil water content and/or extremely high or low temperatures) into diapause (i.e. are

not reacting to formalin).

Due to the individual size of the worms, a large plot shall be identified: a square of 50 cm × 50 cm is

often sufficient in the Holarctic where most adult earthworms have a length approximately between

1) Derosal is an example of a suitable product available commercially. This information is given for the convenience

of users of this document and does not constitute an endorsement by ISO of this product.

2 © ISO 2014 – All rights reserved
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SIST EN ISO 11268-3:2015
ISO 11268-3:2014(E)

1 cm and 20 cm. However, at places with a low density of earthworms [e.g. soils with low pH (<4,5) or

which are anthropogenically used like crop sites], larger plots (i.e. 1 m ) are recommended. On the other

hand, at sites with a high earthworm density (e.g. many meadows in temperate regions), a smaller plot

2 [12]

of 0,125 m is sufficient. The individual samples are taken randomly over the test plot.

NOTE 1 If in special situations, such as in Southern Europe, anecic species do not occur, sampling by hand-

sorting up to a soil depth of 30 cm is sufficient. When sampling conditions are not optimal, e.g. at extremely low

soil water content or dense root layer, hand-sorting can even be the only effective extraction method.

In case in a behavioural sampling method formalin alone is used, an additional check by hand-sorting

is necessary and it should be performed as described in this International Standard. Hand-sorting

combined with the formalin method usually yields satisfying results. If a combination of hand-sorting

and an extraction method is used, no additional efficiency check is necessary.
7.2 Preservation
Preservation shall be carried out according to ISO 23611-1.
7.3 Determination of biomass
Determination of biomass shall be carried out according to ISO 23611-1.
8 Preparation for the test
8.1 Test site
8.1.1 Selection and description

In general, the test site should be as homogenous as possible to improve the statistical power of the

test. Gradients in environmental conditions should be avoided (e.g. adjacent ditches), canopy influences

as woodland borders, or compacted tractor tracks on the site. The site should be on level ground and

should have the same cropping and soil characteristics throughout.

Grassland is the preferred study site for testing effects of substances on earthworms. In grassland,

earthworm density and diversity are generally higher and more stable than on arable land, which makes

it easier to detect significant effects on earthworm populations. If effects on earthworms are observed

on a grassland site, a refined risk assessment should include specific scenarios (crops and regions)

covering the intended use patterns of the test substance (e.g. pesticide). Orchards are not recommended

for testing because of the heterogeneity of the site due to tree rows and strips without trees. If an

orchard is used, it shall be ensured that the higher variability is compensated by taking more samples or

restricting sampling to specific areas. A suitable grassland test area should have an earthworm density

of at least 100 individuals per square metre. With lower population densities, more samples should be

taken than recommended in 8.1.3.

If information on effects on bare soils is required, then arable plots may be used, provided that there are

at least 60 earthworms per square metre present at the start of the test according to the results of the

pre-sampling.
[11]

The experimental plots should support a mixed population of earthworms which are generally

representative of the type of environment selected. In agricultural areas, for example, important anecic

and endogeic species should be present at a sufficiently high density (at least 10 % of the population for

each group) that plots can be taken as representative. Care should be taken not to select plots where

uncharacteristic species predominate.

NOTE Due to natural reasons, a certain ecological group possibly does not occur in some regions (e.g. anecic

worms in parts of the Mediterranean). In such cases, expert knowledge is required in order to identify the

ecologically most important species of that region.
© ISO 2014 – All rights reserved 3
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SIST EN ISO 11268-3:2015
ISO 11268-3:2014(E)

In order to satisfy these requirements, samples should be taken from prospective plots before the start

of the study for preliminary investigation of species distribution.

Extreme soil types, e.g. very sandy, clay, or moory soils, should be avoided when selecting the test site.

A description of the test site should contain the following physicochemical and biological information:

— particle-size distribution (as specified in ISO 11277);
— organic-carbon content (as specified in ISO 10694);
— pH-value (as specified in ISO 10390);
— water-holding capacity, WHC (in the A-horizon, as specified in ISO 11274);
max
— description of vegetation.
Determination of these characteristics should be made using standard methods.

Microclimate measurements (soil and air temperature, soil water content, rainfall quantity, sunshine

duration) are particularly important for the period of chemical application and temperature, and rainfall

quantity should be recorded over the year.

The history of the test site should be known (e.g. applications of pesticides, mineral fertilizers, sewage

sludge, tillage).
8.1.2 Design of experiment

The experimental design depends on the objectives of the study and the amount and quality of

information available from the study site. Usually (i.e. in the case of testing a chemical substance),

a negative control (i.e. plots sprayed just with water) and a positive control (i.e. plots sprayed with

a reference substance, e.g. a substance known to be toxic to earthworms) are sprayed. In general, it

should be taken into consideration that a dose-response design clearly facilitates environmental risk

assessment as compared to single-dose studies. In any case, the reasons for the selected test design shall

be explained in the study report.

The test should be designed as a randomized complete block. The number of the treatment groups and

planned sampling dates determine the number of plots and, therefore, the surface area of the field site.

However, the size of the individual study plots should be at least 100 m (10 m × 10 m). The samples

are taken exclusively from the central area of the plots so that around the sampling area, there is a 1 m

to 2 m wide edge strip which is also treated (see Figure 1). The sampling area of the samples taken

per treatment, plot (replicate), and date depends on the earthworm density and distribution of the

2 2 2

selected experimental field (see 7.1) and can range between 1 m and 0,125 m , but most often, 0,25 m

is appropriate.
4 © ISO 2014 – All rights reserved
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SIST EN ISO 11268-3:2015
ISO 11268-3:2014(E)
Key
1 sampling area
2 edge strip
Figure 1 — Schematic view of a test plot

Samples taken on the same date should be at least 2 m apart and sampled areas should not be used for

sampling at subsequent sampling dates.

The required number of random samples depends, among other things, on the density and distribution

[14]
of the earthworm population over the test area.

For each test variant (control, reference substance, test substance), at least four replicates should be

used and four random samples taken per replicate (i.e. 16 individual samples per test variant).

On grassland, a sampling area of 0,25 m per individual sample is sufficient. Use of a metal or plastic

enclosure with a size of 50 cm × 50 cm (square) or a diameter of 56 cm and a height of 10 cm to 15 cm is

recommended. On arable land, the sample area shall usually be increased to 1 m due to low population

density or non-homogeneous distribution of the worms.

On grassland, the vegetation at the sampling area should be cut carefully before sampling so that all

earthworms appearing on the surface can be seen and collected.

Care should be taken that the entries of earthworm holes are not blocked and, therefore, operators

should avoid walking on sampling areas.
NOTE Guidance concerning application rates of pesticides is given in Annex A.
8.1.3 Maintenance of test fields

Grassland fields should be mulched regularly (two times to six times per year) using grass clippings of a

mulching lawn mower in order to keep the grass cover short. Mulching should be carried out one week

to two weeks before the application of the test substance to ensure that the grass on the surface, which

acts as a food source for some earthworms, has been in contact with the test material. The last mulch

before application of the test substance may only remain on the field, provided that it does not create a

coherent grass mat. In the case of mulching over the course of the year, the mulch should remain on the

field as it serves as food for some earthworm species.
If a test is carried out on arable land, usual agricultural practice sh
...

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