ISO/DTS 21738

ISO/TS DIS DTS 21738:2025(E)
ISO/TC 147/SC 5
Secretariat: DIN
Date: 2026-02-24
Water quality — Active biomonitoring method with in situ caged
benthic amphipods
DTS stage
Warning for WDs and CDs
This document is not an ISO International Standard. It is distributed for review and comment. It is subject to
change without notice and may not be referred to as an International Standard.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of
which they are aware and to provide supporting documentation.

ISO #####-#:####(X)
2 © ISO #### – All rights reserved

ISO/TS DIS DTS 21738:2024(E:(en)
Published in Switzerland
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication
may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying,
or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO
at the address below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. Phone: + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail: copyright@iso.org
Website: www.iso.org
Published in Switzerland
iii
ISO/TS DIS DTS 21738:2025(E:(en)
www.iso.org
iv
ISO/TS DIS DTS 21738:2024(E:(en)
Contents
Foreword . vi
Introduction . vii
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle of the method . 2
5 Organisms and equipment . 2
5.1 Organism origin . 2
5.2 Test organism . 3
5.3 Equipment . 5
6 Test organism preparation in laboratory . 9
6.1 Acclimatization period of amphipods . 9
6.2 Test organism selection criteria . 10
6.3 Laboratory measurements and conditioning of organisms for analysis . 12
6.4 Caging of test organisms for dispatch . 12
7 Exposure of test organisms in the monitoring station . 12
7.1 Monitoring station environmental condition check . 12
7.2 Dispatch to monitoring station . 12
7.3 Test organism transplantation . 12
7.4 Retrieval of test organisms . 14
8 Test organism conditioning in the laboratory . 14
8.1 Reception of test organisms at the laboratory . 14
8.2 Placing of test organisms in vials . 14
9 Test report . 15
Annex A (informative) Application domain . 16
Annex B (informative) Acclimatization conditions . 18
Bibliography . 20

v
ISO/TS DIS DTS 21738:2025(E:(en)
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 documentsdocument 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 (a) patent(s)
which may be required to implement this document. However, implementers are cautioned that this may not
represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water Qualityquality, Subcommittee SC 5,
Biological methods.
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.
vi
ISO/TS DIS DTS 21738:2024(E:(en)
Introduction
Some chemical substances (metals or hydrophobic organic contaminants) accumulate in organisms. The
measurement of bioaccumulation using living organisms is found to be a valid approach for monitoring
chemical contamination.
Two biomonitoring strategies maycan be applied to measure chemical substances in the biota: either passive
[11]
approaches based on sampling or collecting indigenous organisms at the monitoring station[11] ,, or active
[4] ,[7]
approaches based on transplanting, by caging, organisms from a reference source population. [4, 7].
Active biomonitoring makes it possible to 1):
— overcome the lack of indigenous organisms at certain monitoring stations and 2);
— minimize the biological variability of the responses measured, associated with the impact of confounding
factors such as the exposure history and time, reproductive status, physiological state, age, or sex of the
organisms sampled, thus enabling a possible and reliable comparison of results between stations and between
sampling dates [6].
[6]
This enables a reliable comparison of results between stations and between sampling dates.
This method limits the risk of organisms escaping during caging: 1)
— enclosure chambers present holes with diameter inferior to organisms' size and are securely closed with
screwed cap; 2)
— enclosure chambers are fixed and protected from breakage by being installed inside an exposure system .
1)
.
Amphipods are relevantThis document describes a method to expose test organisms for the analysis
of(amphipods), directly on the field by a caging methodology, with the aim to measure bioaccumulation of
chemical substances [2, 18]. Moreover, amphipods represent important keystone species in aquatic
ecosystems, they play a key role in biogeochemical cycle (e.g., litter breakdown processes) and constitute an
important element in food webs by providing prey for secondary consumers.
The application domain of method is depending on the characteristics ofon a monitoring station, i.e. either the
concentrations of metals or organic compounds, or both, accumulated in the used species. A summary table is
proposed in Annex B to reference for each species the main indications about exposure time, matrix,
physicochemical parameters for optimal exposure, range of average weight per individual, organisms’ density
in cage. Further species could be added in this annex.

Around 7,000 gammarid caging experiments were carried out in rivers mainly in France, but also in
Belgium and Luxembourg. No broken or capless chamber was found during all these experiments.”
1)
Around 7,000 gammarid caging experiments were carried out in rivers mainly in France, but also in Belgium and
Luxembourg. No broken or capless chamber was found during these experiments.

vii
Water quality — Active biomonitoring method with in situ caged
benthic amphipods
1 Scope
This document describes a method to expose test organisms (amphipods), directly on the field by a caging
methodology, with the aim to measure bioaccumulation of chemical substances on a monitoring station, i.e.
either the concentrations of metals or organic compounds, or both, accumulated in the organisms. organisms.
Unlike tests carried out on water samples, the major advantage of the caging method is to be able to integrate
the temporal variability of exposure via continuous caging for several days or weeks.
This method can be used for a comparative study at large scale (including stations in several watersheds) and
for upstream/downstream studies to assess discharge impacts.
The use of invasive species should be avoided at areas where they are not already present. The introduction
of several species and the possibility to use local populations as source organisms in this document has the
advantage of being able to circumvent this issue.
A summary table is proposed in Annex AAnnex A to reference for each species its indigenous area. Further
species could be added in this annex. Freshwater or marine species could be used, while paying attention to
the cannibalistic behaviorbehaviour of certain amphipod species.
This document also describes the specifications for test organism selection and conditioning, in situ exposure,
and finally sorting and conditioning of the surviving organisms after exposure.
The organism preparation methods (freeze-drying, extraction, mineralization) and quantification of the
chemical substances do not fall within the scope of this document.
[2],[18]
Amphipods are relevant organisms for the analysis of the bioaccumulation of chemical substances.
Moreover, amphipods represent important keystone species in aquatic ecosystems, they play a key role in
biogeochemical cycle (e.g., litter breakdown processes) and constitute an important element in food webs by
providing prey for secondary consumers.
The application domain of method depends on the characteristics of the used species. A summary table is
proposed in Annex B to reference for each species the main indications about exposure time, matrix,
physicochemical parameters for optimal exposure, range of average weight per individual, organisms’ density
in cage. Further species can be added to this table.
viii
Water quality — Active biomonitoring method with in situ caged
benthic amphipods
1 Scope
This document describes a method to expose test organisms (amphipods), directly on the field by a caging
methodology, with the aim to measure bioaccumulation of chemical substances on a monitoring station, i.e.
either the concentrations of metals or organic compounds, or both, accumulated in the organisms.
This document also describes the specifications for test organism selection and conditioning, in situ exposure,
and finally sorting and conditioning of the surviving organisms after exposure.
This document does not apply to organism preparation methods (freeze-drying, extraction, mineralization)
and quantification of the chemical substances.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological 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
acclimatization period
period prior to in situ exposure and during which the organisms are kept under controlled laboratory
conditions (i.e.,
Note 1 to entry: Controlled laboratory conditions can include temperature, electric conductivity, oxygen, diet, organism
density, medium replacement frequency, and photoperiod).
3.2 3.2
organism calibration
operation consisting of selecting specimens in order to obtain test organisms of homogeneous weight and of
the same sex
Note 1 to entry: A size to weight ratio of the organisms used is measured at each experiment (detail for each species is
given in Annex AAnnex A).).
3.3 3.3
monitoring station
location in the water-body characteristic of the impacted or reference location being studied
3.4 3.4
caging point
sub-area of the monitoring station
3.5 3.5
bioaccumulation
phenomenon by which a chemical substance present in the medium accumulates in a living organism
Note 1 to entry: This phenomenon is observed when the rate of absorption exceeds the rate of elimination of the
contaminant.
(SOURCE[SOURCE: ISO 24032:2021(en),, 3.2)]
3.6 3.6
monitoring
process of repetitive observation for defined purposes of one or more elements of the environment, according
to pre-arranged schedules in space and time using comparable methods for environmental sensing and data
collection
3.7
3.8
bioconcentration
increase in concentration of the test chemical in or on an organism relative to the concentration of test
chemical in the water
4 Principle of the method
The principle of this caging method is focused on comparative study at large scale (including stations in several
watersheds) and on upstream/ and downstream studies to assess for example discharge impacts.
The method consists firstly of an acclimatization period, then calibrating the organisms in the laboratory. Only
males are selected and put in enclosure chambers (presenting holes with diameter smaller than the size of
organisms)), which are securely closed with screwed cap.
The test organisms are then exposed directly on field, with no feeding, directly at the monitoring station at the
caging point. Enclosure chambers are fixed and protected from breakage by being installed inside an exposure
system.
Finally, the organisms are recovered at the end of the exposure period, shipped to the laboratory, sorted in
order to count the survivors and calculate the survival rate, weighed, conditioned in a suitable vial for
measuring either metals and/or organic compounds, or both, and finally stored in a freezer according to
method requirements. The organisms are prepared and analysed with a view to evaluating the concentrations
of accumulated chemical substances.
5 Organisms and equipment
5.1 Organism origin
The test organisms are chosen according to their indigenous character. A summary table is proposed in
Annex AAnnex A to reference for each species its indigenous area and its application domain.
NOTE 1 According to the literature, some species maycan be proposed, e.g. Gammarus pulex and Gammarus fossarum
[16] [8],[17] ,[3] [10]
for Europe [16] ; ; Hyalella azteca for Canada and USA; [8, 17, 3]; Hyalella curvispina for Argentina; [10, 15];
,[15] [19] [14]
Paramelita nigroculus for South Africa [19] or Melita plumulosa for Australia [14].
The organisms should be sampled from a field source population located for example in a geographic area
subject to little or no sources of contamination, specifically in headwaters for freshwater systems or in outdoor
rearing ponds. For upstream/ and downstream studies, it will beis possible to use a local field population
upstream of the site under investigation. The organisms maycan be also breed in laboratory in agreement with
the national and local regulations for animal transport.
NOTE 2 National and local regulations for animal transport can apply.
The chemical quality of the test organisms shall be checked using a sample of male specimens following an
acclimatization period, in order to check the concentrations of chemical substances without in situ exposure.
In the same way, national regulated pathogens maycan be tracked. A summary table is proposed in
Annex BAnnex B to reference for each species the main acclimatization conditions, including the availability
of chemical quality criteria for test organisms (i.e., chemical substances for which maximum concentrations
are defined). Further species couldcan be added into this annex. table.
In addition, a survival test in 48/96 hours should be realized 2 times per year with a model substance to check
the sensitivity of the organism's population.
5.2 Test organism
5.2.1 Sex
Bioaccumulation maycan be modulated by fat content. Fat content strongly varies in mature females
(according to their reproductive status) compared to males. Consequently, adult male specimens with
homogeneous weight are selected in order to getobtain more reproducible results.
Due to significant sexual dimorphism in several species of amphipods, visible to the naked eye, the sex of the
organisms is easily identifiable. However, amphipod species that have a poorly transparent or
coloredcoloured exoskeleton should be avoided because it would beis unreliable to obtain males only. It is
also possible to gently separate from a pair of amphipods in amplexus position the outer, larger specimen,
which is always the male (e.g., like two left pictures in Figure 1Figure 1).). Finally, it remains possible to ensure
the reliability of sorting to identify males by observing the presence of genital papillae, using a microscope, on
each individual; but it is (very)much more time consuming. Figure 1The Figure 1 illustrates in Gammarus
fossarum and Hyalella azteca species females having black dorsal gonads and/or embryos in a ventral brood
pouch. Gammarus fossarum individuals are shown in two photos on the top and Hyalella azteca are shown in
two photos at the bottom of the Figure 1Figure 1.
Length: 1 to 2 cm for Gammarus fossarum (on the top) and about 5 mm for Hyalella azteca (at the bottom). On the left, a
pair of amphipods with a female with black dorsal gonads and orange-coloured embryos in the marsupial pouch situated
in the ventral region. On the right, a female with black embryos in the marsupial pouch.
Length: 1 to 2 cm for Gammarus fossarum (on the top) and about 5 mm for Hyalella azteca (at the bottom). On the left, a pair of
amphipods with a female with black dorsal gonads and orange-coloured embryos in the marsupial pouch situated in the ventral
region. On the right, a female with black embryos in the marsupial pouch.
Figure 1— Sexual dimorphism in Gammarus fossarum and Hyalella azteca species
5.2.2 Weight
On the day before on-site exposure, male specimens have tomust be sorted in the laboratory so as to obtain
organisms of visually homogeneous size. From all the organisms sorted throughout the day, a batch of
specimens are sampled at random, to determine their total weight and more particularly the average weight
per individual. Here, the average weight per individual is used as a quality indicator of the lot of test organisms.
It is necessary to define forFor each species, a range of weights (minimum and maximum of average weight
per individual) shall be defined in order to guarantee obtaining adult organisms only and comparable batches
of test organisms between experiments.
5.3 Equipment
5.3.1 Sieve
Sieves are used to collect pre-sorted specimens from the field reference source population of amphipods. The
aperture size should be adapted for each species in order to obtain, at the time of sorting the test organisms,
a large part of organisms whose weight per individual is within the target weight range (5.2.2(5.2.2).).
5.3.2 Cage
The in situ exposure of the test organisms is carried out using one of more polypropylene (PP) cages. This cage
model is proposed as a reference for the caging of amphipods: easy to produce and well suited for most of
amphipods considering its large size. PP plastics are used because these are the one of the most inert plastics
available at low prices in the market. Other more inert and more expensive plastics (PET) can be used, in
which case the change should be specified in the test report.
A cage measuring about 6 cm in diameter and 10 cm in height (volume: 180 mL or 180 cm ) is recommended
(see Figure 2Figure 2).). This cage model shall be perforated to enable exchanges of water with the
environment. A unitary orifice size of 1 mm in diameter to maximize the exchanges with the environment and
suitable oxygenation; it also helps prevent the organisms from escaping. The cage should have 422 holes,
including 149 on the cap, 105 at the base of the container and 168 on the edge, where the holes are distributed
in 8 rows of 21 holes. The dimensions of this cage seem suitable for a large majority of amphipod species but
can be adapted depending on the species. If another cage design has been used (dimensions, hole diameter,
number and positioning of holes), it shall be specified in test report.

Figure 2— Recommended cage design for amphipod caging
It should be checked that, depending on the size range of test organisms, the hole diameter is sufficient to
avoid escape of test organisms escaping. It should also be checked that the density of individuals is reasonable
and shall not exceed 5,3 mg (wet weight) per cm .
The cages are secured using a fixing clip inside an exposure system (see subclause 5.3.3Subclause 5.3.3)), and
they shall not touch each other and not move inside the system.
The cages can be reused after following cleaning protocol as follows. They are placed in a 3 % acetic acid
solution for at least 2 da
...