ISO/FDIS 21285

ISO/DISFDIS 21285:2025(en)
ISO/TC 190/SC 4/WG 2
Date: 2026-02-13
Secretariat: AFNOR
Date: 2026-09-17
Soil quality — Inhibition of reproduction of the soil mite (Hypoaspis
(Gaeolaelaps) aculeifer) by soil contaminants
Qualité du sol — Inhibition de la reproduction de l'acarienl’acarien prédateur (Hypoaspis (Gaeolaelaps)
aculeifer) par desles contaminants duprésents dans le sol
FDIS stage
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ii
ISO/DISFDIS 21285:20252026(en)
Contents
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Reagents and material . 4
5.1 Biological material . 4
5.2 Test mixtures . 4
5.3 Reference substance . 6
6 Apparatus . 6
7 Procedure . 7
7.1 Experimental design . 7
7.2 Preparation of test mixtures . 8
7.3 Addition of the biological material . 10
7.4 Test conditions and measurements . 10
7.5 Feeding of the mites . 10
7.6 Determination of surviving predatory mites . 11
8 Calculation and expression of results . 11
8.1 Calculation . 11
8.2 Expression of results . 11
9 Validity of the test . 11
10 Statistical analysis . 12
10.1 General . 12
10.2 Single-concentration tests . 12
10.3 Multi-concentration tests . 12
11 Test report . 13
Annex A (informative) Techniques for rearing and breeding of predatory mites . 15
Annex B (informative) Basic information on the biology of Hypoaspis (Gaeolaelaps) aculeifer . 17
Annex C (normative) Determination of water-holding capacity . 18
Annex D (informative) Guidance on adjustment of pH of artificial soil . 19
Annex E (informative) Extraction and counting of predatory mites . 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
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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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO had not received notice of patents(a)
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Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
characterization, 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 second edition cancels and replaces the first edition (ISO 21285:2019), which has been technically
revised.
The main changes are as follows:
— — Refinementrefinement of the species name in the title and throughout the document to include the
genus Gaeolaelaps, and correction of this name in the text where the former version had already used it;
— Correctioncorrection of terms and definitions in Clauses 3Clauses 3 and Clause 7 to align with the ISO and
IEC terminological databases;
— Additionaddition of detailed recommendations for testing with waste materials in 5.2.15.2.1, 7.1.1, 7.1.1
and 7.1.27.1.2;;
— Refinementrefinement of the description of the methods to apply to control soil in 5.2.25.2.2;;
— Additionaddition of recommendations in 7.5Feeding and mites section of 7.5;;
— Updateupdate of the bibliographic references.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
ISO/DISFDIS 21285:20252026(en)
Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil and
[1][1] [2][2]
are proposed to complement conventional chemical analysis (see ISO 15799 and ISO 17616 ).).
ISO 15799 includes a list and short characterization of recommended and standardized test systems and
ISO 17616 gives guidance on the choice and evaluation of the bioassays. Aquatic test systems with soil eluate
are applied to obtain information about the fraction of contaminants potentially reaching the groundwater by
the water path (retention function of soils), whereas terrestrial test systems are used to assess the habitat
function of soils.
Mites (Acari) are a world-wide and diverse group of arthropods belonging to the class Arachnida with over
40 000 species recorded, divided into two super-orders (Acariformes and Parasitiformes). Due to their
relative small size (a few µm to a few cm), they occupy specific ecological niches on plants as well as in soils
(see Reference 3 [3]).).
Among soil-inhabiting mites, the role of predation is ensured by, for example, Hypoaspis (Gaeolaelaps) sp.
(Laelapidae). Because they are exposed to chemical contamination, mites are already considered in the
[4]
environmental risk assessment of pesticides, as non-target organisms (see Reference [4]).). Indeed, among
the data required for active substances of pesticides, effects on predatory mites are assessed, i.e. for the plant-
inhabitant Typhlodromus pyri (Phytoseiidae) and the soil-inhabitant Hypoaspis (Gaeolaelaps) aculeifer
[5]
(Laelapidae) (see Reference [5]).).
[6] [7][6][7]
The first authors introducing H. aculeifer as a test organism in ecotoxicological studies were later
proposed a two-species test system in the European project SECOFASE (Sublethal Effects of Chemicals on
Fauna in the Soil Ecosystem), including the collembolan Folsomia fimetaria as prey. In the context of the
development of an ecotoxicological test for the assessment of plant protection products on non-target
[8] [5]
arthropods (see References  [8] [5]),), a protocol on soil predatory mites using H. aculeifer was further
proposed. After that, a standard test protocol for the assessment of chemicals was developed for this species
[9] [9]
by OECD in 2008 and revised in 2016 . . The results of the associated international ring-test were published
[10]
in Reference [10].
Among mites, the predator Hypoaspis (Gaeolaelaps) aculeifer is the most studied species in the laboratory. The
reproduction end point was found in general to be more sensitive than mortality and avoidance. Compared to
other soil meso-fauna invertebrates, mites were found in general less sensitive than or as sensitive as other
test species, depending on the end points and chemicals studied. Considering semi-field studies, H. aculeifer
was used as a top predator whereas other soil invertebrates, mainly springtails, were ranked in the grazer
group. In these studies, mites showed to be quite tolerant towards anthropogenic contamination. This
statement was also corroborated by field surveys. However, the applicability of laboratory test methods for
the assessment of environmental samples (contaminated soils, wastes etc.) with mites is emphasized, as to
date a limited number of studies are available.
This document describes a method that is based on the determination of lethal and sublethal effects of
contaminated soils or waste materials to adult predatory mites of the species Hypoaspis (Gaeolaelaps)
aculeifer. This species is considered to be representative of predatory soil arthropods. Background
[11]
information on the ecology of these mites and their use in ecotoxicological testing is available in Reference
[11].
v
DRAFT International Standard ISO/DIS 21285:2025(en)

Soil quality — Inhibition of reproduction of the soil mite (Hypoaspis
(Gaeolaelaps) aculeifer) by soil contaminants
1 Scope
This document specifies a chronic test method for evaluating the habitat function of soils and determining
effects of soil contaminants and substances on the reproduction of Hypoaspis (Gaeolaelaps) aculeifer. This
method is applicable to soils and soil materials of unknown quality, e.g. from contaminated sites, amended
soils, soils after remediation, industrial, agricultural or other sites under concern and waste materials (e.g.
dredged material, municipal sludge from a wastewater treatment plant, composed material, or manure,
especially those for possible land disposal).
This document provides information on how to use this method for testing samples (soils or substances)
under temperate conditions.
This document is not applicable to substances for which the air/soil partition coefficient is greater than one,
or to substances with vapour pressure exceeding 300 Pa at 25 °C.
NOTE The stability of the test substance cannot be ensured over the test period. No provision is made in the test
method for monitoring the persistence of the substance under test.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
EN 14735, Characterization of waste — Preparation of waste samples for ecotoxicity tests
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, Sludge and solid environmental matrices — Determination of dry residue or water content and
calculation of the dry matter fraction on a mass basis
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 terminologicalterminology 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 3.1
contaminant
substance or agent present in the soil as a result of human activity
3.2 3.2
effect concentration for x % effect
EC
x
concentration (mass fraction) of a test material or test sample that causes x % of an effect on a given end point
within a given exposure period, when compared with a control
EXAMPLE An 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 EC is expressed as a percentage of test soil (dry mass) per soil mixture (dry mass). When substances
x
are tested, the EC is expressed as the mass of the test substance per dry mass of soil in milligrams per kilogram.
x
3.3 3.3
effect rate
ER
x
dilution of a soil to be tested that causes an x % of an effect on a given end point within a given exposure
period, when compared with a control
3.4 3.4
limit test
single concentration test
EXAMPLE theThe test soil without any dilution or one concentration of a test substance mixed into the control soil
(3.11) and compared with the control.
3.5 3.5
lowest observed effect concentration
LOEC
lowest test substance concentration that has a statistically significant effect (probability p ≤ 0,05)
Note 1 to entry: In this test, the LOEC is expressed as a mass of test substance per dry mass of the soil to be tested. All test
concentrations above the LOEC should usually show an effect that is statistically different from the control.
3.6 3.6
lowest observed effect rate
LOER
lowest dilution of a soil to be tested in a control soil (3.11) at which a statistically significant effect (p ≤ 0,05)
is observed
3.7 3.7
no observed effect concentration
NOEC
highest test substance concentration immediately below the LOEC (3.5(3.5)) at which no statistically
significant effect is observed
© ISO #### 2026 – All rights reserved
ISO/DISFDIS 21285:20252026(en)
Note 1 to entry: In this test, the concentration corresponding to the NOEC, has no statistically significant effect
(probability p ≤ 0,05) within a given exposure period when compared with the control.
3.8 3.8
no observed effect rate
NOER
lowest dilution of a soil to be tested immediately below the LOER (3.6(3.6)) which, when compared to the
control, has no statistically significant effect (probability p ≤ 0,05) within a given exposure period when
compared with the control
3.9 3.9
reference soil
uncontaminated site-specific soil with properties (nutrient concentrations, pH, organic carbon content and
texture, etc.) similar to the test soil
3.10 3.10
standard soil
field-collected soil or manufactured (artificial) soil whose main properties (e.g. pH, texture, organic matter
content) are within a known range
EXAMPLE Euro soils, artificial soil, LUFA standard soil type 2.2.
Note 1 to entry: The properties of standard soils can differ from the soil to be tested.
3.11 3.11
control soil
uncontaminated substrate or natural soil, used as a control and as medium for preparing dilution series with
test soils or chemicals to be tested, that allows fulfilling the validity criteria
Note 1 to entry: In the case of natural soil, it is advisable to demonstrate its suitability for a test and for achieving the test
validity criteria before using the soil in a definitive test.
3.12 3.12
test mixture
mixture of contaminated soil or the test substance (e.g. chemical, biosolid, waste) with control soil (3.11(3.11))
3.13 3.13
test mixture ratio
ratio between the test soil and the control soil (3.11(3.11)) in a test mixture (3.12)
4 Principle
The reproduction (= number of juveniles) is the measured parameter of the test. The test reflects the
bioavailability of a mixture of contaminants in natural soils (contaminated site soils) to a species which
represents a trophic level which is not covered by other ISO standards. This test is not intended to replace the
[12] [12] [1] [1]
earthworm (see ISO 11268-2 )) or Collembola (see ISO 11267 )) reproduction tests since this
species belongs not only to a different trophic group but also a different taxonomic group (= mites;, i.e.
arachnids) than those used usually.
Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate. For
contaminated soils, the effects are determined in the soil that is intended to be tested and in a control soil.
Depending on the objective of the study, the control and dilution substrate (dilution series of contaminated
soil or waste material) are either an uncontaminated soil comparable to the test soil (reference soil) or a
standard soil (e.g. artificial soil).
Adult females are exposed to the test soil and the effects on reproduction measured are compared to those
observed for females exposed to a control soil. If appropriate, effects based on exposure to a dilution range of
contaminated soil or waste material and control soil or a range of concentrations of a test substance mixed
into control soil are determined. Test mixtures are prepared at the start of the test and are not renewed within
the test period. The test is started with 10 adult females per test vessel. Males are not introduced in the test,
because experience has shown that females mate immediately or shortly after hatching from the deutonymph
stage, if males are present. As the females are introduced into the test approximately 7 d after they have
reached the adult stage, the females can be considered as already mated (Annex A(Annex A and
Annex BAnnex B).). The test runs until the first offspring have reached the deutonymph stage. At 20 °C the
exposure time ends at day 14 after introducing the females (day 0), followed by two days of extraction. The
number of surviving females and the number of juveniles per test vessel are determined. The reproductive
output of the mites exposed to the test mixtures is compared to that of the controls in order to determine the
dilutions/concentrations which cause no effects on mortality and reproduction (NOER/NOEC) and the
dilution/concentration resulting in x % reduction of juveniles hatched from eggs compared to the control
(ER /EC ) respectively, depending on the experimental design (see 7.1.37.1.3).).
x x
In case there is no prior knowledge of the dilution/concentration of the test soil or the test substance likely to
have an effect, then it is useful to conduct the test in two steps:
— — A range-finding test on reproduction is carried out, to give an indication of the effect
dilution/concentration, and the dilution/concentration giving no mortality (NOER/NOEC).
Dilutions/concentrations to be used in the definitive test can then be selected;.
— — theThe definitive test on reproduction is carried out, to determine sublethal effects of (dilutions of)
contaminated soil, a waste material or the concentration of a substance which, when evenly mixed into
the standard soil, causes no significant effects on numbers of offspring hatched from eggs compared with
the control (NOER/NOEC), and the lowest dilution/concentration causing effects (LOER/LOEC).
The use of aA reference soil is an essential requirementshall be used to demonstrate the present status of the
test population, and to avoid misinterpretation of results.
5 Reagents and material
5.1 Biological material
In this test, Hypoaspis (Gaeolaelaps) aculeifer. adult female mites (7 d to 14 d after becoming adult; 28 d to 35 d
after the start of the egg laying in the synchronisation) are required to start the test. The mites shall be selected
from a synchronised cohort (see Annex BAnnex B).).
5.2 Test mixtures
5.2.1 5.2.1 Field-collected soil or waste material. The field-collected soils or the waste material used in
the test shall be passed through a sieve of 4 mm square mesh to remove coarse fragments and thoroughly
mixed. If necessary, the soil or waste material can be air-dried without heating before sieving. Storage of the
soil or waste material that is intended to be tested should be as short as possible. The test sample shall be
stored in accordance with ISO 18400--206 (soils) and EN 14735 (waste materials), using containers that
minimize losses of contaminants sorption to the container walls. If soils or test mixtures have been stored,
they should be mixed a second time immediately before use. Soil pH should not be corrected as it can influence
bioavailability of contaminants.
For interpretation of test results, the following characteristics shall be determined for each soil sampled from
a field site:
a) a) pH in accordance with ISO 10390;
b) b) texture (sand, loam or silt, clay) in accordance with ISO 11277;
© ISO #### 2026 – All rights reserved
ISO/DISFDIS 21285:20252026(en)
c) c) water content in accordance with ISO 11465;
d) d) water-holding capacity in accordance with Annex CAnnex C;;
e) e) cationic exchange capacity in accordance with ISO 11260;
f) f) organic carbon in accordance with ISO 10694;
g) g) percentage of material removed by the 4 mm sieve.
NOTE It is not practical to measure the water holding capacity of all mixtures used in the test. Therefore, soil
moisture content is checked by gently squeezing the soil in the hand; if the moisture content is correct, small drops of
water will appear between the fingers.
5.2.2 5.2.2 Control soil, either a) reference soil or b) standard soil that allows the presence of predatory
mites. Control soil and soil used for dilution shall not differ in one test [either a) or b)].
a) a) If reference soils from uncontaminated areas near a contaminated site are available, they
should be treated and characterized like the soils that are intended to be tested. If a toxic contamination
or unusual soil properties cannot be ruled out, standard control soils should be preferred.
b) b) For testing the effects of substances mixed into soil, standard soils (e.g. artificial soil, LUFA
standard soil type 2.2.) 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
— — Sphagnumsphagnum peat finely ground [a particle size 5 %
of (2 ± 1) mm is acceptable] and with no visible plant remains
— — Kaolinitekaolinite clay containing not less than 30 % 20 %
kaolinite
— — Industrialindustrial quartz sand (dominant fine sand 74 %
with more 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 obtain
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 and
ow
74 % of quartz sand have proven to be sufficient for maintaining the desired structure of the artificial soil.
NOTE 2 It has been demonstrated that Hypoaspis (Gaeolaelaps) aculeifer can conform with the validity criteria even
on reproduction when tested in field soils with lower organic carbon content (e.g. 2,7 %), and there is experience that
this can be achieved in artificial soil with 5 % peat. Therefore, it is not necessary before using such a soil in a definitive
test to demonstrate the suitability of the artificial soil for allowing the test to conform with the validity criteria unless the
peat contents lowered more than specified above.
Prepare the artificial soil at least three days prior to start the test, by mixing the dry constituents listed above,
e.g.,. in a large-scale laboratory mixer. Determine the pH and, if necessary, adjust. The amount of calcium
carbonate required can vary, depending on properties of the individual batch of sphagnum peat (see
Annex DAnnex D).). Determine the maximum water holding capacity in accordance with Annex CAnnex C. A
portion of the deionized water required is added two to seven days before starting the test to obtain
approximately half of the required final water content of 40 % to 60 % of the maximum water holding capacity.
Allowance should be made for any water that is used for introducing the test substance into the soil. Store the
mixed artificial soil at room temperature until starting the test to equilibrate acidity.
The total water-holding capacity shall be determined in accordance with Annex CAnnex C,, the pH shall be
determined according to ISO 10390.
5.3 Reference substance
5.3.1 5.3.1 To ensure the quality of the test system, tests should be performed regularly (once or twice a
year) with a reference substance.
The NOEC or the EC of a reference substance, or both, shall be determined to provide assurance that the
x
laboratory test conditions are adequate and to verify that the response of the test organisms did not change
over time. The reference substance can be tested in parallel to the determination of the toxicity of each test
sample at one concentration, which needs be demonstrated beforehand in a dose response study to result in
an effect of about 50 %. In this case, the number of replicates should be the same as that in the controls.
Alternatively, the reference substance is tested once or twice a year in a dose-response test. Depending on the
design chosen, the number of concentrations and replicates and the spacing factor differ (see 7.1.37.1.3),), but
a response of 10 % to 90 % effect should be achieved (spacing factor of 1,8). Dimethoate as well as boric acid
[10] [10]
are suitable reference substances that have shown to affect reproduction .
The EC for dimethoate based on the number of juveniles should fall in the range between 3,0 mg a.s. (active
substance)/kg soil (dry mass) and 7,0 mg a.s. (active substance)/kg soil (dry mass). Based on the results
obtained with boric acid so far, the EC based on the number of juveniles should fall in the range between
100 mg/kg (dry mass) soil and 300 mg/kg (dry mass) soil.
11) 22)
5.3.2 5.3.2 Dimethoate (CAS 60--51--5), C H NO PS , to be tested as a formulation [e.g. Perfekthion
5 12 3 2
(ca. 40 % dimethoate)].
5.3.3 5.3.3 Boric acid (CAS 10043--35--3), H BO (99 %).
3 3
WARNING — When handling these substances, appropriate precautions should be taken to avoid
ingestion or skin contact.
6 Apparatus
Use laboratory equipment and the following.
6.1 6.1 Test containers, made of glass or other chemically inert material of about 100 ml capacity and
with a diameter of about 5 cm, with lids (e.g. plastic, glass discs or parafilm, able to be closed tightly).
6.2 6.2 Apparatus to determine the dry mass of the substrate, in accordance with ISO 11465.

Chemical Abstracts Service (CAS) Registry Number® is a trademark of the American Chemical Society (ACS). This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the
product named. Equivalent products can be used if they can be shown to lead to the same results.
1)
Chemical Abstracts Service (CAS) Registry Number® is a trademark of the American Chemical Society (ACS). This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the
product named. Equivalent products can be used if they can be shown to lead to the same results.

Perfekthion is an example of a suitable product available commercially. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO of this product.
2)
Perfekthion is an example of a suitable product available commercially. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO of this product.

© ISO #### 2026 – All rights reserved
ISO/DISFDIS 21285:20252026(en)
6.3 6.3 Large scale laboratory mixer, for the preparation of the test mixture (5.2(5.2).).
6.4 6.4 Suitable accurate balances.
6.5 6.5 Apparatus, capable of measuring pH and water content of the substrate.
6.6 6.6 Exhauster, for transfer of mites (see ISO 11267:2023, Clause A.2).
6.7 6.7 Test environment.
6.7.1 6.7.1 Enclosure, capable of being controlled to a temperature of (20 ± 2) °C.
6.7.2 6.7.2 Light source, capable of delivering a constant light intensity of 400 lx to 800 lx at the substrate
surface at a controlled light:dark cycle of between 12 h:12 h and 16 h:8 h.
6.8 6.8 Extraction apparatus, Tullgren funnel or comparable methods such as McFadyen (see
Annex EAnnex E).).
7 Procedure
7.1 Experimental design
7.1.1 General
A sample of field-collected soil or waste material can be tested at a single concentration (typically 100 % for
field-collected soil or 25 % for waste material when ratios above 25 % are not practical, e.g. sludge or manure)
or evaluated for toxicity in a multi-concentration test whereby a series of concentrations (dilutions) is
prepared by mixing measured quantities with a control soil (5.2.2(5.2.2).). When testing substances, a series
of concentrations is prepared by mixing quantities of the test substance with a standard soil (e.g. artificial
soil). The concentrations are expressed in milligrams of test substance per kilogram of dried control soil
(5.2.2(5.2.2).). Depending on the knowledge of relevant response levels a range-finding test can precede the
definitive test. Each definitive test consists of a series of soil mixtures (treatments).
7.1.2 Range-finding test (preliminary test)
A preliminary test to find the range of mixture ratio affecting predatory mites is optional, e.g. 0 %, 1 %, 5 %,
25 %, 50 %, 75 %, 100 % soil for field-sampled soil and 0 %, 1,56 %, 3,12 %, 6,25 %, 12,5 %, 25 % for waste
material (e.g. sludge or manure), 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 control
soil (see 5.2.25.2.2)) and a control using 10 mites per container]. The preliminary test is conducted without
replication. The duration of the range finding test is 14 d (exposure time), followed by an extraction time of
two days. After a total 16 d, mortality of the adult mites and the number of juveniles is determined. Based on
the results of the range finding test, the ER /EC is roughly determined by calculating the geometric mean
50 50
of those two dilutions/concentrations showing 0 % and 100 % mortality. The concentration/dilution range in
the final test should preferably be chosen so that it includes concentrations at which juvenile numbers are
affected while survival of the maternal generation is not. This, however, cannotcan be not possible for
substances that cause lethal and sub-lethal effects at similar concentrations.
When no effects are observed, even at 100 % contaminated soil, 25 % waste material or at concentrations of
1 000 mg test substance/kg standard soil (dry mass), the definitive test can be designed as a limit test.
7.1.3 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 comparison of the biological effects found in the test soil with those
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, the results from standard soil shall be used to judge
[27] [27]
the validity and acceptability of the test. . The duration of the definitive test is 14 d (exposure time),
followed by an extraction time of two days. After a total of 16 d, the mortality of the adult mites and the number
of juveniles isare determined.
If for characterization purposesIf a test design including dilution series is required for characterization
purposes, one of the following designs should be used (the concentrations shall be spaced by a factor not
exceeding 2):
— — For the NOER/NOEC approach, at least five concentrations or test mixtures in a geometric series
should be used. Four replicates for each treatment plus eight controls are recommended.
— — For the ER /EC approach, 12 concentrations or test mixtures should be used. Two replicates for each
x x
concentration plus six controls are recommended. The spacing factor can be variable;: smaller at low
concentrations, larger at high concentrations.
— — For the mixed approach, 6 to 8 concentrations or test mixtures in a geometric series should be used.
Four replicates for each treatment plus eight controls are recommended. This mixed approach allows a
NOER/NOEC as well as an ER /EC evaluation.
x x
To facilitate checking of the pH and humidity of the test sample, use of additional containers for each
concentration and for the control is recommended.
Each test container (replicate) is filled with 20 g dry mass of the test sample. To ensure easy migration of mites
the substrate in the test container should not be compressed.
7.1.4 Limit test
If no effects are observed at the highest concentration in the range-finding test (i.e. 1 000 mg/kg or 100 %),
the reproduction test can be performed as a limit test, using a test concentration of 1 000 mg/kg or undiluted
soil. A limit test will provide the opportunity to demonstrate that the NOEC/NOER or the EC ER for
10/ 10
reproduction is greater than the limit concentration while minimising the number of mites used in the test.
Eight replicates should be used for both the treated soil and the control.
7.2 Preparation of test mixtures
7.2.1 Testing of contaminated soil and waste materials
According to the selected dilution range, the soil or waste material that is intended 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 test mixture and the reference soil or the standard soil
shall be 20 g (dry mass) in each test container (6.1(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 CAnnex C. In some cases, e.g. when testing waste materials, higher or lower percentages are required.
A rough check of the test mixture moisture content can be obtained by gently squeezing the test mixture in
the hand, if the moisture content is correct small drops of water will appear between 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 (when acid or basic samples are tested, do not adjust the pH).
Prepare the appropriate number of replicates per test mixture and the control(s) according to the selected
approach (see 7.1.37.1.3).).
© ISO #### 2026 – All rights reserved
ISO/DISFDIS 21285:20252026(en)
WARNING — Contaminated soils and waste materials can contain unknown mixtures of toxic,
mutagenic, or otherwise harmful substances or infectious microorganisms. Occupational health risks
can arise from dust or evaporated substances as well as via dermal contact during handling and
incubation.
7.2.2 Testing substances added to the test substrate
Standard soil (5.2.2(5.2.2)) is used to prepare the test sample. For each test container (6.1(6.1),), the mass of
the substrate used shall be 20 g (dry mass). 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) a) Water-soluble substance
— — Immediately before starting the test, dissolve the quantity of the test substance in the water or
a portion of it required to wet the soil samples for the replicates of one concentration in order to reach
a final water content of 40 % to 60 % of the maximum water holding capacity, and mix it thoroughly
with the soil before introducing it into the test containers.
b) 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) mix it with a portion (up to 10 %) 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 are intended to 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) c) Substances insoluble in water or organic solvents
— — For a substance insoluble in a volatile solvent, separate a portion (up to 10 %) of the quartz
sand required (see 5.2.25.2.2).). Prepare a mixture of an appropriate quantity of quartz sand and the
quantity of the test substance required to obtain the desired concentration. Add that mixture and any
unused quartz sand to the remainder of the soil and the water and mix thoroughly before introducing
it into a test container.
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 sample.
Proceed simultaneously with all replicates per concentration and the control(s) required according to the
selected approach.
Determine the pH for each test mixture (one container per concentration) in accordance with ISO 10390 at
the beginning and end of the test.
7.2.3 Preparation of control containers
The control container contains the control soil (5.2.2(5.2.2)) wetted with deionised water to reach 40 % to
60 % of the total water holding capacity (determined in accordance with Annex CAnnex C).).
Perform one control container for the range-finding test and at least six control containers for the definitive
test.
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 7.2.27.2.2),), use at least six additional control containers p
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