prEN ISO 16387

Soil quality - Effects of contaminants on Enchytraeidae (Enchytraeus sp.) - Determination of effects on reproduction (ISO/DIS 16387:2022)

Bodenbeschaffenheit - Wirkungen von Verunreinigungen auf Enchytraeidae (Enchytraeus sp.) - Bestimmung der Wirkungen auf die Reproduktion (ISO/DIS 16387:2022)

Qualité du sol - Effets des contaminants sur les Enchytraeidae (Enchytraeus sp.) - Détermination des effets sur la reproduction (ISO/DIS 16387:2022)

Kakovost tal - Vpliv onesnaževal na enhitreje (Enchytraeus sp.) - Ugotavljanje vplivov na razmnoževanje (ISO/DIS 16387:2022)

General Information

• Status: Not Published
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: CEN/TC 444 - Environmental characterization
• Drafting Committee: CEN/TC 444/WG 4 - Biological characterization
• Current Stage: 4599 - Dispatch of FV draft to CMC - Finalization for Vote
• Due Date: 26-Oct-2022
• Completion Date: 26-Oct-2022
• Ref Project: oSIST prEN ISO 16387:2022 - Soil quality - Effects of contaminants on Enchytraeidae (Enchytraeus sp.) - Determination of effects on reproduction (ISO/DIS 16387:2022)

RELATIONS

• Revised: EN ISO 16387:2014 - Soil quality - Effects of contaminants on Enchytraeidae (Enchytraeus sp.) - Determination of effects on reproduction (ISO 16387:2014)
• Effective Date: 13-Nov-2019

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN ISO 16387:2022
01-april-2022
Kakovost tal - Vpliv onesnaževal na enhitreje (Enchytraeus sp.) - Ugotavljanje
vplivov na razmnoževanje (ISO/DIS 16387:2022)
Soil quality - Effects of contaminants on Enchytraeidae (Enchytraeus sp.) -
Determination of effects on reproduction (ISO/DIS 16387:2022)
Bodenbeschaffenheit - Wirkungen von Verunreinigungen auf Enchytraeidae
(Enchytraeus sp.) - Bestimmung der Wirkungen auf die Reproduktion (ISO/DIS
16387:2022)

Qualité du sol - Effets des contaminants sur les Enchytraeidae (Enchytraeus sp.) -

Détermination des effets sur la reproduction (ISO/DIS 16387:2022)
Ta slovenski standard je istoveten z: prEN ISO 16387
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 16387:2022 en,fr,de

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

---------------------- Page: 1 ----------------------
oSIST prEN ISO 16387:2022
---------------------- Page: 2 ----------------------
oSIST prEN ISO 16387:2022
DRAFT INTERNATIONAL STANDARD
ISO/DIS 16387
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2022-02-07 2022-05-02
Soil quality — Effects of contaminants on Enchytraeidae
(Enchytraeus sp.) — Determination of effects on
reproduction

Qualité du sol — Effets des contaminants sur les Enchytraeidae (Enchytraeus sp.) — Détermination des

effets sur la reproduction
ICS: 13.080.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 16387:2022(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2022
---------------------- Page: 3 ----------------------
oSIST prEN ISO 16387:2022
ISO/DIS 16387:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022

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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
© ISO 2022 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST prEN ISO 16387:2022
ISO/DIS 16387:2022(E)
Contents Page

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

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

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

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

3 Terms and definitions .................................................................................................................................................................................... 1

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

5 Reagents and material ................................................................................................................................................................................... 3

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

7 Test environment ................................................................................................................................................................................................ 6

8 Procedure ....................................................................................................................................................................................................................7

8.1 Experimental design ......................................................................................................................................................................... 7

8.1.1 General ........................................................................................................................................................................................ 7

8.1.2 Range-finding test ............................................................................................................................................................. 7

8.1.3 Definitive test ........................................................................................................................................................................ 7

8.2 Preparation of test mixtures ..................................................................................................................................................... 8

8.2.1 Testing contaminated soil .......................................................................................................................................... 8

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

8.2.3 Preparation of control container ......................................................................................................................... 9

8.3 Addition of food ..................................................................................................................................................................................... 9

8.4 Addition of the biological material ....................................................................................................................................... 9

8.5 Test conditions and measurements ..................................................................................................................................... 9

8.6 Reference substance .......................................................................................................................................................................... 9

9 Calculation and expression of results ........................................................................................................................................10

9.1 Calculation ............................................................................................................................................................................................... 10

9.2 Expression of results ........................................................................................................................................... ........................... 10

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

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

11.1 General ........................................................................................................................................................................................................ 10

11.2 Range-finding test ............................................................................................................................................................................ 11

11.3 Definitive test ....................................................................................................................................................................................... 11

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

Annex A (informative) Conditions for culture of Enchytraeus sp .......................................................................................13

Annex B (informative) Test procedure using other Enchytraeus species .................................................................15

Annex C (informative) Determination of maximum water-holding capacity .......................................................17

Annex D (informative) Detailed description of extraction techniques .......................................................................18

Annex E (informative) Overview of the statistical assessment of data (NOEC determination) ........20

Bibliography .............................................................................................................................................................................................................................21

iii
© ISO 2022 – All rights reserved
---------------------- Page: 5 ----------------------
oSIST prEN ISO 16387:2022
ISO/DIS 16387:2022(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 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 the following

URL: www.iso.org/iso/foreword.html.

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

Biological characterization.

This third edition cancels and replaces the second edition (ISO 16387:2014), which has been technically

revised. The main changes compared to the previous edition are as follows:

— correction of the vapor pressure value to update Clause 1 according to the recommendations of

ECHA/OECD;

— addition in Annex D of an extraction method of enchytraeids with colloidal silica, suitable for

Enchytraeus crypticus.

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.
© ISO 2022 – All rights reserved
---------------------- Page: 6 ----------------------
oSIST prEN ISO 16387:2022
ISO/DIS 16387:2022(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. ISO 15799 includes a list and

short characterization of recommended and standardized test systems. 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. For the latter, a standardized test system using Enchytraeidae (a

chronic test with end-point reproduction) is proposed.

This International Standard describes a method that is based on the determination of acute and

sublethal effects of contaminated soils to adult Enchytraeidae of the genus Enchytraeus. Optionally, the

method can be used for testing substances added to standard soils (e.g. artificial soil) for their sublethal

hazard potential to Enchytraeidae.

Soil-dwelling annelids of the genus Enchytraeus are ecologically relevant, i.e. they are abundant in

many soils where earthworms are scarce, but can also reach high population densities in soils well

inhabited by earthworms. Enchytraeidae can be used in laboratory tests as well as in semi-field and

field studies. From a practical point of view, many Enchytraeus species are easy to handle and breed,

and their generation time is significantly shorter than that of earthworms [the test duration for a

reproduction test with Enchytraeidae is four weeks to six weeks, compared to eight weeks (12 weeks

including synchronization) with earthworms]. In addition, a much smaller volume of soil is needed in

the enchytraeid test compared to the amount needed in earthworm tests.

This document has been drawn up taking into consideration test procedures recommended by the

Organization for Economic Cooperation and Development (see [22],[25]).
© ISO 2022 – All rights reserved
---------------------- Page: 7 ----------------------
oSIST prEN ISO 16387:2022
---------------------- Page: 8 ----------------------
oSIST prEN ISO 16387:2022
DRAFT INTERNATIONAL STANDARD ISO/DIS 16387:2022(E)
Soil quality — Effects of contaminants on Enchytraeidae
(Enchytraeus sp.) — Determination of effects on
reproduction
1 Scope

This document specifies one of the methods for evaluating the habitat function of soils and determining

effects of soil contaminants and substances on the reproduction of Enchytraeus sp. by dermal and

alimentary uptake in a chronic test. It is applicable to soils and soil materials of unknown quality,

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

concern and waste materials.

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. According

to 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 substances under temperate

conditions.

The method is not applicable to substances, for which the air/soil partition coefficient is greater than 1,

or to substances for which the vapour pressure exceeds 300 Pa at 25 °C.

NOTE No provision is made in the test method for monitoring the persistence of the substance under test.

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 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 10390, Soil, treated biowaste and sludge – 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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2022 – All rights reserved
---------------------- Page: 9 ----------------------
oSIST prEN ISO 16387:2022
ISO/DIS 16387:2022(E)
3.1
reproduction

mean number of offspring per test vessel after incubation under the specified test conditions

Note 1 to entry: The test period for the determination of the reproduction (definitive test) is six weeks.

Note 2 to entry: offspring = hatched juvenile enchytraeids
3.2
reproduction rate
mean number of offspring produced per a number of adults over the test period

Note 1 to entry: The test period for the determination of the reproduction (definitive test) is six weeks.

Note 2 to entry: offspring = hatched juvenile enchytraeids
3.3
ECx
effect concentration for x % effect

concentration (mass fraction) of a test substance that causes x % of an effect on a given endpoint within

a given exposure period when compared with a control

EXAMPLE An EC50 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 mass of the test substance per dry mass of soil, in milligrams

per kilogram.
3.4
LOEC
lowest observed effect concentration

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.
3.5
NOEC
no observed effect concentration

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

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

(p < 0,05) within a given exposure period when compared with the control.
3.6
test mixture

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

3.7
test mixture ratio
ratio between the soil to be tested and the control soil in a test mixture
3.8
contaminant
substance or agent present in the soil as a result of human activity
3.9
reference soil

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

carbon content and texture) to the soil being studied
© ISO 2022 – All rights reserved
---------------------- Page: 10 ----------------------
oSIST prEN ISO 16387:2022
ISO/DIS 16387:2022(E)
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

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 a 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.
4 Principle

The effects on survival and reproduction of adult Enchytraeidae (Enchytraeus sp.) exposed to a dilution

range of contaminated soil or range of concentrations of a test substance are determined. Test mixtures

are prepared at the start of the test and are not renewed within the test period.

The test can be divided into two distinct steps: a short (two weeks) test in which the range of toxic

effects (mainly mortality) is determined, and a long-term (six weeks) definitive test in which the

survival of parental worms and the fecundity (number of juveniles) are measured. The results obtained

from the tests are compared with a control and are used to determine the dilutions or concentrations

which cause no effects on survival and reproduction (NOEC) and the concentration (dilution) resulting

in x % reduction of juveniles hatched from cocoons compared to the control (ECx, 42 d).

All test dilutions/concentrations above the LOEC have a harmful effect equal to, or greater than that

observed at the LOEC. Where there is no prior knowledge of the dilution/concentration of the test

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

— an acute toxicity test (range-finding test) is carried out, to give an indication of the effect dilution/

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

to be used in the definitive test can then be selected;

— the definitive test on reproduction to determine sublethal effects of (dilutions of) contaminated

soil or the concentration of a substance which, when evenly mixed into the standard soil, causes

no significant effects on numbers of offsprings hatched from cocoons compared with the control

(NOEC), and the lowest concentration causing effects (LOEC).

NOTE The use of a suitable reference soil is an essential requirement to demonstrate the present status of

the test population, and to avoid misinterpretation of results.
5 Reagents and material

5.1 Biological material, recommended test species is Enchytraeus albidus Henle 1837 (white

potworm; Enchytraeidae, Oligochaeta, Annelida). E. albidus is one of the largest enchytraeid species,

measuring 15 mm to 40 mm, and has a world-wide distribution (see e.g. [21], [33]). It can be easily

recognized by two characteristics: four setae per bundle ventrally, and the very long seminal duct in

the clitellum region as well as some segments behind it. The species can be found in marine, limnic and

terrestrial habitats, mainly in decaying organic matter (seaweed, compost) and only rarely in meadows.

This broad ecological tolerance and some morphological variations indicate that the species probably

consists of several races (or ecotypes). E. albidus can be obtained commercially, since it is sold as food

for fish. It should be verified whether such a culture is contaminated by other, usually smaller species

(see e.g. [7], [10], [34]). If contamination of the culture occurs, all worms are washed in water in a Petri

dish. With the help of a stereomicroscope, large adult specimens of E. albidus are selected to start a new

© ISO 2022 – All rights reserved
---------------------- Page: 11 ----------------------
oSIST prEN ISO 16387:2022
ISO/DIS 16387:2022(E)

culture. All other worms of the original culture are discarded. E. albidus can be bred easily in a wide

range of organic materials (see Annex A) and has a short life cycle, reaching maturity between 33 d (at

18 °C) and 74 d (at 12 °C). Only cultures which have been kept in the laboratory for at least five weeks

(one generation cycle) without problems shall be used for testing purposes.

Other species of the genus Enchytraeus, especially the true soil-inhabiting but smaller species E. crypticus

Westheide and Graefe 1992 or E. buchholzi Vejdovsky 1879, are also suitable as test organisms (see

Annex B). If other species of Enchytraeus are used, they shall be clearly identified and the rationale for

the selection of the species as well as deviations of the experimental method should be reported in this

case.

The worms used in the tests should be adult with eggs (white spots) in the clitellum region and

should have approximately the same size (approximately 15 mm for E. albidus). A synchronisation of

the breeding culture is not necessary. The Enchytraeidae should be acclimatised in the reference soil,

the standard soil or the untreated artificial soil under test conditions for at least 24 h prior to testing.

During this period, the same food which is used as a food source in the test should be given in sufficient

amount.

For one test, an excess number of adult clitellate worms should be taken from the culture box without

observing them in detail in order to get enough suitable worms. At the end of the acclimatization period,

only worms with eggs and behaving as usual (e.g. not trying to leave the artificial soil) are selected

for the test. This selection is made by placing the worms in a Petri dish filled with a small amount of

water under a stereomicroscope, and discarding the animals without eggs. A freshwater medium (e.g.

reconstituted water as described in [24]) should preferably be used, since demineralized water or tap

water (risk of copper contamination) can harm the Enchytraeidae. During this process, other organisms

living in the cultures, such as mites, are also removed from the worms.
NOTE An example of culturing Enchytraeus sp. is given in Annex A.

5.2 Test mixture, which may consist of field-collected soil or control soil amended by the test

substance.
5.2.1 Field-collected soil or waste

The sample(s) can be field-collected soil from an industrial, agricultural or other site of concern, or

waste materials (e.g. dredged material, municipal sludge from a wastewater treatment plant, composed

material, or manure) under consideration for possible land disposal.

The fields soils used in the test shall be passed through a sieve of 4 mm square mesh to remove coarse

fragments and thoroughly mixed. If necessary, soil may be air-dried without heating before sieving.

Storage of soils to be tested should be as short as possible. The soil shall be stored in accordance

with ISO 18400-206 using containers that minimize losses of soil contaminants by volatilisation and

sorption to the container walls. Soil pH should not be corrected as it can influence bioavailability of soil

contaminants.

For interpretation of test results, the following characteristics shall be determined for each soil sampled

from a field site:
a) pH in accordance with ISO 10390;
b) texture (sand, loam, silt) in accordance with ISO 11277;
c) water content in accordance with ISO 11465;
d) water holding capacity according to Annex C;
e) cation exchange capacity in accordance with ISO 11260;
f) organic carbon in accordance with ISO 10694.

NOTE It is important to measure the water holding capacity of all mixtures used in the test.

© ISO 2022 – All rights reserved
---------------------- Page: 12 ----------------------
oSIST prEN ISO 16387:2022
ISO/DIS 16387:2022(E)

5.2.2 Control soil, either a) reference (3.9) or b) standard soil (3.10) that allows the presence of

Enchytraeidae (at least the validity criteria shall be fulfilled). Control soil and soil used for dilution shall

not differ in one test (either a) or b)).

a) If reference soils from uncontaminated areas near a contaminated site are available, they should

be treated and characterized like the soils to be tested. If a toxic contamination or unusual soil

properties cannot be ruled out, standard control soils should be preferred.

b) For testing the effects of substances mixed into soil, standard soils (e.g. artificial soil, LUFA) shall

be used as test substrate. The properties of the field-collected standard soil shall be reported.

The substrate called artificial soil can be used as a standard soil and 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 69 %
50 % of particle size 0,05 mm to 0,2 mm)

Approximately 0,3 % to 1,0 % calcium carbonate (CaCO , pulverised, analytical grade) are necessary to

get a pH of 6,0 ± 0,5.

NOTE 1 Taking the properties of highly non-polar (log K > 2) or ionizing substances into account, 5 % of

[22]

peat have proven to be sufficient for maintaining the desired structure of the artificial soil .

Prep
...