ISO 16387:2014

INTERNATIONAL ISO
STANDARD 16387
Second edition
2014-01-15
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 survie et la
reproduction
Reference number
©
ISO 2014
© 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

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
8.2 Preparation of test mixtures . 8
8.3 Addition of the biological material . 9
8.4 Test conditions and measurements . 9
8.5 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 .10
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) .19
Bibliography .20
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 16387:2004), which has been technically
revised.
iv © ISO 2014 – All rights reserved

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 International Standard has been drawn up taking into consideration test procedures recommended
[22] [24]
by the Organization for Economic Cooperation and Development (see , ).
INTERNATIONAL STANDARD ISO 16387:2014(E)
Soil quality — Effects of contaminants on Enchytraeidae
(Enchytraeus sp.) — Determination of effects on
reproduction
1 Scope
This International Standard 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 International Standard provides information on how to use this method for testing substances
under temperate conditions.
The method is not applicable to volatile substances, i.e. substances for which H (Henry’s constant) or the
air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,013 3 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 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil under
aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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
2 © ISO 2014 – All rights reserved

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
[21] [31]
15 mm to 40 mm, and has a world-wide distribution (see e.g. , ). 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
[7] [10] [32]
verified whether such a culture is contaminated by other, usually smaller species (see e.g. , , ). 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 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). A synchronisation of the breeding
culture is not necessary. The Enchytraeidae should be acclimatised in 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.
[23]
reconstituted water as described in ) 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 10381-6 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.
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)).
4 © ISO 2014 – All rights reserved

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          10 %
remains
− Kaolinite clay containing not less than 30 % kaolinite         20 %
− Industrial quartz sand (dominant fine sand with more          69 %
than 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 peat
ow
[22]
have proven to be sufficient for maintaining the desired structure of the artificial soil.
Prepare the artificial soil at least three days prior to start the test, by mixing the dry constituents
listed above thoroughly in a large-scale laboratory mixer. A portion of the deionized water required
is added while mixing is continued. The amount of calcium carbonate required can vary, depending
on properties of the individual batch of sphagnum peat and should be determined by measuring sub-
samples immediately before the test. Store the mixed artificial soil at room temperature for at least two
days to equilibrate acidity. To determine pH and the maximum water holding capacity, the dry artificial
soil is pre-moistened one or two days before starting the test by adding deionised water to obtain half of
the required final water content of 40 % to 60 % of the maximum water holding capacity.
The total water holding capacity is determined according to Annex C, the pH is determined according to
ISO 10390.
NOTE 2 Allowance should be made for any water that is to be used for introducing the test substance into the
soil.
5.3 Food
Rolled oats, preferably autoclaved (heating is also possible) before use to avoid infection with other
[31]
organisms, were found to be suitable . The first feeding is made by mixing 50 mg of ground rolled
oats per test vessel into the soil (after application of the test substance but before adding the worms);
additional feedings (25 mg per vessel per week except after 28 d) are made only on the surface to avoid
harming the worms. Since the need for food can vary in the different vessels, feeding should be adjusted
to demand (i.e. over-feeding shall be avoided). Some soil particles should be placed on top of the flasks
in order to reduce fungal growth.
5.4 Reagents
5.4.1 Bengal red.
5.4.2 Ethanol.
5.4.3 Boric acid, suitable as reference substance.
6 Apparatus
Usual laboratory equipment and the following.
6.1 Test container, of capacity 0,20 l to 0,25 l, with a diameter (e.g. 5 cm) enabling a depth of 1,5 cm to
2 cm of soil, with lids (e.g. glass or perforated plastic film). The beakers shall be suitable as test vessels,
containing an amount of artificial soil corresponding to 20 g dry mass. The lids shall permit gaseous
exchange between the soil substrate and the atmosphere.
6.2 Drying cabinet.
6.3 Stereomicroscope.
6.4 Balances with a weighing range of 50 g to 32 kg; precision at least 1 g.
6.5 Analytical balance with a weighing range of 10 mg to 200 g; precision at least 1 mg.
6.6 pH-meter.
6.7 Temperature registration (e.g. temperature/humidity recorder).
6.8 Lux meter.
6.9 Mixer.
6.10 Incubator or small room with air-conditioner.
6.11 Jeweller’s tweezers, hooks, loops or a small brush.
6.12 Photo basins with ribbed bottoms.
7 Test environment
Cover the test vessels (6.1) with glass lids to prevent the test substrate from drying, and keep under test
conditions for two weeks (range-finding test) or six weeks (definitive test). The test temperature shall
be (20 ± 2) °C; higher temperatures can affect reproduction. Carry out testing in a controlled light-dark
cycle of long-day conditions, preferably 16 h to 8 h at 400 lx to 800 lx in the area of the test vessels, to
prevent the worms from escaping from the soil.
Weigh the vessels at the beginning of the test and thereafter once a week. Replenish the mass loss with
the appropriate amount of deionized water. This loss can be minimized by maintaining a high humidity
(>80 %) in the test incubator (6.10). Place all test vessels in the test incubator in a random order, which
should be changed every week.
At the beginning and the end of both the range-finding test and the definitive test, the water content and
the pH should be measured. To facilitate checking of the pH and water content of the test substrate, use
of additional containers (replicates) for each concentration and for the control is recommended.
6 © ISO 2014 – All rights reserved

8 Procedure
8.1 Experimental design
8.1.1 General
A sample of field-collected soil can be tested at a single concentration (typically 100 %) or evaluated
for toxicity in a multi-concentration test whereby a series of concentrations (dilutions) are prepared
by mixing measured quantities with a control soil (5.2.2). Depending on the knowledge of relevant
response levels a range-finding test may precede the definitive test. Each definitive test consists of a
series of soil mixtures (treatments). Each treatment is replicated at least four times.
8.1.2 Range-finding test
A test to find the range of mixture ratio affecting Enchytraeidae is optional. If it is necessary to determine
the range of concentrations (dilutions) for use in the definitive test, perform a range-finding (acute) test
in a large range of concentrations (dilutions) of the contaminated soil, e. g. 0 % – 1 % – 5 % – 25 % – 50 %
– 75 % – 100 %, or of the test substance, e.g. 0 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg and 1 000 mg/kg
(the concentrations being expressed in milligrams of test substance per kilogram of dried standard soil
(5.2.2) and a control using 10 worms per container.
The range-finding test is conducted without replication.
8.1.3 Definitive test
The design of the definitive test depends on the test objectives. Typically the habitat properties of
samples of a field-collected soil are characterized by a comparison of the biological effects for the soil(s)
to be tested with the effects found in a reference soil or, if not available or not appropriate due to toxicity
or atypical physicochemical characteristics, in a standard soil. Results for the standard soil assist in
distinguishing contaminant effects from non-contaminant effects caused by soil physicochemical
properties. Regardless of whether a reference soil or standard soil is used for the statistical comparisons,
[14]
the results from standard soil shall be used to judge the validity and acceptability of the test.
If, for characterization purposes, a test design including dilution series is required, three designs are
possible (the concentrations shall be spaced by a factor not exceeding 2):
— For the NOEC approach, at least five concentrations in a geometric series should be used. Four
replicates for each concentration plus eight controls are recommended.
— For the ECx approach, 12 concentrations should be used. Two replicates for each concentration
plus at least six controls are recommended. The spacing factor can be variable; smaller at low
concentrations, larger at high concentrations.
— For the mixed approach, six to eight concentrations in a geometric series should be used. Four
replicates for each concentration plus at least eight controls are recommended. This mixed approach
allows a NOEC as well as an ECx evaluation.
A limit test can be sufficient if in the range-finding test no toxic effect was observed. In the limit test only
the soil to be tested without any dilution and the control (or the soil to be tested vs. the control soil) shall
be tested with at least four replicates each.
If the soil to be tested has properties that are quite different from the usual standard control soils (e.g.
OECD artificial soil or LUFA 2.2 soil), for instance a rather low or high pH, or very low or high organic
matter or clay contents, it is essential to have a reference soil that has similar properties. In that case,
control performance of the enchytraeids can, however, be less good than ‘usual’ and quality control of
the test is not possible on the basis of the reference soil. For assessing toxicity of the soil to be tested, it
is important however to have such a reference soil, which can also be used to prepare dilution series of
the soil to be tested. For test quality assessment it is essential in such case to include both a reference
and a standard control soil.
8.2 Preparation of test mixtures
8.2.1 Testing contaminated soil
According to the selected dilution range, the soil to be tested is mixed with the reference soil or the
standard soil thoroughly (either manually or by using a hand mixer). The homogeneity of the mixture is
checked visually. The total mass of the soil to be tested and the reference soil or the standard soil shall
be equal to 20 g (dry mass) in each test container (6.1). The test mixture shall be wetted with deionised
water to reach 40 % to 60 % of the total water holding capacity determined according to Annex C. In
some cases e.g. when testing waste materials, higher percentages are required. This can be easily tested
by compressing the substrate by the fist and looking for water coming through the fingers.
Determine the pH for each test mixture (one container per concentration) according to ISO 10390 at the
beginning and end of the test (do not adjust the pH).
WARNING — Contaminated soils can contain unknown mixtures of toxic, mutagenic, or otherwise
harmful chemicals or infectious microorganisms. Occupational health risks can arise from dust
or evaporated chemicals as well as via dermal contact during handling and incubation.
8.2.2 Testing substances added to the test substrate
For each test container (6.1), the quantity of substrate used shall be equivalent to 20 g (dry mass).
Standard soil (5.2.2) is used as test substrate. Substances are added to the test substrate and mixed
thoroughly.
For the introduction of test substances use either method a), b) or c), as appropriate:
a) Water-soluble substance
Immediately before starting the test, dissolve the quantity of the test substance in the water required
for the replicates of each concentration (or that portion of it necessary to wet the soil) in order to meet
the requirements of 5.2.2, and mix it thoroughly with the soil before introducing it into a test container.
b) Substances insoluble in water but soluble in organic solvents
Dissolve the quantity of test substance required to obtain the desired concentration in a volatile solvent
(such as acetone or hexane) and mix it with a portion of the quartz sand required. After evaporating the
solvent by placing the container under a fume hood, add the remainder of the soil and the water and mix
it thoroughly before introducing it into the test containers.
NOTE Ultrasonic dispersion, organic solvents, emulsifiers or dispersants can be used to disperse substances
with low aqueous solubility. When such auxiliary substances are used, all test concentrations and an additional
control should contain the same minimum amount of auxiliary substance.
WARNING — Take appropriate precautions when dealing with solvent vapour to avoid danger
from inhalation or explosion, and to avoid damage to extraction equipment, pumps, etc.
c) Substances insoluble in water or organic solvents
For a substance insoluble in a volatile solvent, prepare a mixture of 10 g of finely ground industrial quartz
sand (see 5.2.2) and the quantity of the test substance required to obtain the desired concentration.
Place the mixture, the remainder of the soil (5.2.2) and the water into the test container (6.1) and mix
thoroughly.
Base the concentrations selected to provide the LOEC/NOEC on the results of the range-finding test.
Space the concentrations by a factor not exceeding 2.
Substances mixed into the substrate do not need to be tested at concentrations higher than 1 000 mg/kg
mass of test substrate.
Proceed simultaneously with at least four replicates per concentration and the control(s).
8 © ISO 2014 – All rights reserved

Determine the pH for each test mixture (one container per concentration) according to ISO 10390 at the
beginning and end of the test (do not adjust the pH).
8.2.3 Preparation of control container
The control container contains the control soil (5.2.2) wetted with deionised water to reach 40 % to
60 % of the total water holding capacity (determined according to Annex C).
Use one control container for the range-finding test and four to eight control containers, depending on
the test design (see 8.1.3).
Prepare the control containers in the same way as the test containers. If the preparation of the test
requires the use of a solvent (see 8.2.2), use an additional control prepared with solvent but without the
test substance. Cover the containers as indicated in 6.1.
8.3 Addition of the biological material
Into each test container and the control container 10 Enchytraeidae (5.1) are placed carefully on the
test mixture surface, using a suitable device (6.11). The selection of the individual worms and their
assignment to batches of 10 should be made in a randomized fashion.
Cover the container as indicated in 6.1 and place it in the incubator or small room with air-conditioner
(6.10).
8.4 Test conditions and measurements
The duration of the first part of the test is 21 d (assessment of mortality). The adult worms are fed once a
week with 50 mg at the beginning of the test and afterwards with 25 mg dry mass rolled oats per vessel.
If food consumption is low, reduce feeding to a minimum to avoid fungal growth or moulding. After 21 d,
the soil to be tested is carefully searched manually (e.g. using a jeweller’s tweezers, a hook or loop, or a
small brush with a hook) for the adult worms, which are then removed and counted. Morphological and
behavioural changes of the adult worms are recorded. If mortality is the main end point of the test, the
whole procedure is stopped at this point.
The same soil to be tested to which the adult worms were exposed, including cocoons deposited during
the first three weeks of the test, is incubated under the same test conditions for another three weeks.
The juvenile worms hatched in the second half of the definitive test are fed with 25 mg dry mass rolled
oats per vessel per week (except after four weeks). Again, over-feeding shall be avoided (see 5.3). After
total test duration of six weeks, the juveniles hatched in the meantime are counted by staining with
Bengal red (5.4). Wet (but not heat) extraction techniques have also proved to be suitable (see Annex D).
The first method is recommended, since wet extraction is difficult to use with artificial soil because the
clay particles make the water turbid.
NOTE Annex D gives examples of two suitable methods, including one which allows counting of cocoons.
8.5 Reference substance
The NOEC and/or the ECx of a reference substance shall be determined to provide assurance that
the laboratory test conditions are adequate and to verify that the response of the test organism does
not change statistically over time. It is advisable to test a reference substance at least twice a year
or, when testing is carried out in lower frequency, in parallel to the determination of the toxicity of
a test substance. Boric acid is recommended as a reference substance. If the compound is mixed into
the substrate, observe the effects on reproduction (α = 0,05) at concentrations of between 400 mg and
600 mg boric acid per kilogram dry mass of substrate.
9 Calculation and expression of results
9.1 Calculation
For each dilution or concentration, determine the percent mortality, of the adults and number of offspring
produced in the definitive test.
Compare means by suitable statistical methods, e.g. Williams, Dunnetts or Student-t-test and test for
significance (α = 0,05) of differences from control(s).
9.2 Expression of results
A graphical presentation of the mean values of the end points including standard deviation of the
measured values against the soil(s) to be tested, control soil(s) or test mixture ratio should be prepared.
This comparison or curve gives an impression of the quality of effects and their magnitudes. Express the
mixture ratio as based on soil dry mass.
If dilution series were performed indicate:
— in % soil to be tested based on dry mass or in milligrams per kilogram of dried soil substrate, the
median percent dilution of contaminated soil or median lethal concentration of the test substance,
which reduces the number of juvenile worms to 50 % compared to the control within the test period
and
— the soil mixture ratio immediately below the LOEC or highest tested concentration of a test substance
which when compared to the control has no statistically significant lethal or other effect such as
reproduction (p < 0,05).
10 Validity of the test
The results are considered to be valid if the following conditions are met in the control:
a) The mortality of the adult worms should not exceed 20 % on average at the end of the range-finding
test and after the first three weeks of the definitive test.
b) In the definitive test, the average number of juveniles should be higher than 25 per test vessel at the
end of the definitive test, assuming that 10 adult worms (with eggs in the clitellum region) per test
vessel were introduced at the beginning of the test.
c) The coefficient of variation calculated for the reproduction data should be not higher than 50 % at
the end of the definitive test.
11 Statistical analysis
11.1 General
Analyses of the results differ according to the purposes and particular designs of the test. Standard
statistical procedures (e.g. Analysis of variance and multiple comparisons) generally are sufficient for
analysing the results.
11.2 Range-finding test
If a clear dose-response is obvious, ECx-values can be estimated by using regression techniques like
logistic regression function or probit analysis. In other cases the effect range should be determined by
expert knowledge.
10 © ISO 2014 – All rights reserved

11.3 Definitive test
Analysis of variance (ANOVA) involving multiple comparisons of end-point data derived for undiluted
soils to be tested (single-concentration test) including field replicates of field-collected soil from more
than one sampling location is commonly used for statistical interpretation of the significance of findings
from soil toxicity tests (see Annex E). This is a hypothesis-testing approach, and is subject to appreciable
[7]
weaknesses. The parametric analyses (e.g. ANOVA and multiple comparisons) for such data assume
that the data are normally distributed, that the treatments are independent, and that the variance is
homogenous among the different treatments. These assumptions shall be tested. If the data satisfy these
assumptions, analysis may proceed. If not, data may be transformed and tested again. As parametric
tests are reasonably robust in the face of moderate deviations from normality and equality of vari
...