ISO 16387:2023

INTERNATIONAL ISO
STANDARD 16387
Third edition
2023-03
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
Reference number
© ISO 2023
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ii
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 . 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 (normative) Determination of total 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
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
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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).
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www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization, in collaboration with the European Committee for Standardization (CEN)
Technical Committee CEN/TC 444, Environmental characterization of solid matrices, in accordance with
the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This third edition cancels and replaces the second edition (ISO 16387:2014), which has been technically
revised.
The main changes 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.
iv
Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in
[38]
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 document 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
[24],[25]
Organization for Economic Co-operation and Development (OECD ).
v
INTERNATIONAL STANDARD ISO 16387:2023(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, for
example, from contaminated sites, amended soils, soils after remediation, agricultural or other sites
under concern and waste materials.
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 are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 10390, Soil, 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
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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
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 refers to hatched juvenile enchytraeids.
3.2
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 EC is a concentration estimated to cause an effect on a test end point in 50 % of an exposed
population over a defined exposure period.
Note 1 to entry: The ECx is expressed as a percentage of soil to be tested (dry mass) per soil mixture (dry mass).
When substances are tested, the ECx is expressed as mass of the test substance per dry mass of soil, in milligrams
per kilogram.
3.3
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.
3.4
NOEC
no observed effect concentration
highest test substance concentration immediately below the LOEC (3.3) 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.
3.5
test mixture
mixture of contaminated soil or test substance (e.g. chemical, biosolid, waste) with control soil (3.10)
3.6
test mixture ratio
ratio between the soil to be tested and the control soil (3.10) in a test mixture (3.5)
3.7
contaminant
substance or agent present in the soil as a result of human activity
3.8
reference soil
uncontaminated soil with comparable pedological properties (nutrient concentrations, pH, organic
carbon content and texture) to the soil being studied
3.9
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.
3.10
control soil
reference (3.8) or standard soil (3.9) 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) 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;
— a 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 offspring hatched from cocoons compared with the control
(NOEC), and the lowest concentration causing effects (LOEC).
NOTE A suitable reference soil is used to demonstrate the appropriate status of the test population and to
avoid misinterpretation of results.
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).
5 Reagents and material
5.1 Biological material, recommended test species is Enchytraeus albidus Henle 1837 (white
potworm; Enchytraeidae, subclass: Oligochaeta, phylum: Annelida). E. albidus is one of the largest
enchytraeid species, measuring 15 mm to 40 mm, and has a world-wide distribution (see, for example,
References [22] and [32]). 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, for example, References [7], [10]
and [33]). 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 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 Reference [23]) 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 in accordance with Annex C;
e) cation exchange capacity in accordance with ISO 11260;
f) organic carbon in accordance with ISO 10694.
The water holding capacity of all mixtures used in the test should also be measured.
5.2.2 Control soil, either a) reference (3.8) or b) standard soil (3.9) 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 soil (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 Taking the properties of highly non-polar (log K > 2, where K is the octanol/water coefficient) or
ow ow
ionizing substances into account, 5 % of peat have proven to be sufficient for maintaining the desired structure
[25]
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 total 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 total water holding capacity.
The total water holding capacity is determined according to Annex C; the pH is determined in accordance
with ISO 10390.
5.3 Food
Rolled oats, preferably autoclaved (heating is also possible) before use to avoid infection with other
[32]
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 rolled
oats 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
The usual laboratory equipment and the following shall be used.
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 with lids (6.1) 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.
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 in accordance with Annex C.
In some cases, for example 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) in accordance with 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.
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 the required number of replicates for the selected test design (see 8.1.3).
Determine the pH for each test mixture (one container per concentration) in accordance with 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 in accordance with 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 food
Prior to addition of the Enchytraeidae, 50 mg of ground rolled oats (5.3) are added into each container
and mixed with the test mixtures, the test soil, the reference soil, the standard soil depending on the
aim of the study.
8.4 Addition of the biological material
Into each test container and the control container, 10 Enchytraeidae (5.1) are placed carefully on the
surface of the substrate, 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).
8.5 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 twice (week 3 and week 5)
with 25 mg dry mass rolled oats per vessel. 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.
Annex D includes also one method using colloidal silica that has been successfully applied for Enchytraeus
crypticus.
8.6 Reference substance
The NOEC and/or the ECx of the reference substance shall be determined to provide assurance that
the laboratory test conditions are adequate and to verify that the change in the response of the test
organism is not statistically significant 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 or a test soil. Boric acid is recommended as a reference substance. If
the compound is mixed into the substrate, effects on reproduction (α = 0,05) should be observed 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, such as Williams, Dunnett’s 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 a
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