ISO/TS 21738:2026

Water quality — Active biomonitoring method with in situ caged benthic amphipods

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.

Qualité de l'eau — Méthode de biosurveillance active avec des amphipodes benthiques en cage in situ

General Information

• Status: Published
• Publication Date: 17-Jun-2026
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: ISO/TC 147/SC 5 - Biological methods
• Drafting Committee: ISO/TC 147/SC 5 - Biological methods
• Current Stage: 6060 - International Standard published
• Start Date: 18-Jun-2026
• Due Date: 08-Dec-2026
• Completion Date: 18-Jun-2026

Overview

ISO/TS 21738: Water Quality - Active Biomonitoring Method with In Situ Caged Benthic Amphipods is a technical specification developed by the International Organization for Standardization (ISO). This document outlines a standardized approach for active biomonitoring in aquatic environments using caged benthic amphipods. The method enables direct, field-based exposure of amphipods to measure the bioaccumulation of chemical substances-such as metals and organic compounds-in organisms deployed at monitoring stations. The procedure includes detailed recommendations on organism selection, caging, exposure, and retrieval, ensuring consistent and reliable monitoring of water quality across different geographic locations and aquatic systems.

Key Topics

• Scope and Purpose: The standard provides methodologies to assess chemical accumulation in aquatic environments by monitoring test organisms (amphipods) in situ using caging techniques. This addresses both freshwater and marine habitats.
• Organism Selection: It emphasizes the use of indigenous, non-invasive amphipod species, referencing Annex A for region-specific guidance. The use of invasive species is discouraged where not already established.
• Preparation and Acclimatization: Guidelines include the selection, acclimatization, and conditioning of test organisms under controlled laboratory conditions before field deployment.
• Caging Methodology: Amphipods are placed in specially designed cages to allow continuous exposure while preventing escape, minimizing stress, and ensuring valid comparisons between sites.
• Field Exposure and Retrieval: The standard covers best practices for in situ deployment at monitoring stations, environmental compatibility checks, and retrieval procedures post-exposure for subsequent analysis.
• Parameters and Reporting: It details key parameters to be measured (e.g., survival rate, weight, water chemistry) and specifies reporting requirements for reproducibility.

Applications

ISO/TS 21738 provides valuable procedures for a variety of practical water quality assessment and environmental monitoring scenarios, including:

• Regulatory Monitoring: Used by regulatory agencies to track and compare environmental contaminant levels across time and locations, especially for assessing compliance near discharges or industrial activities.
• Impact Assessment: Supports studies on upstream/downstream differences and before-after control-impact (BACI) analysis to assess the effects of specific discharges or remediation measures.
• Comparative Environmental Studies: Facilitates comparative studies across multiple watersheds or regions by standardizing organism selection, caging, and exposure conditions.
• Research and Ecological Studies: Enables researchers to study bioaccumulation trends, amphipod response to chemical stressors, and aquatic ecosystem health.
• Site Assessments: Useful for evaluating the biological quality of aquatic habitats, supporting decisions on water resource management, habitat restoration, and conservation.

Related Standards

Several closely related standards and guidelines can complement the implementation of ISO/TS 21738:

• ISO 24032:2021: Provides definitions and principles relating to bioaccumulation in aquatic organisms.
• ISO/TC 147 - Water Quality Standards: Offers a comprehensive suite of methods for the biological and chemical assessment of water quality.
• Regional Guidance: Many countries have additional protocols for the selection and transport of test organisms, and regulated methodologies for aquatic toxicology and environmental analysis.

By implementing ISO/TS 21738, laboratories, regulatory bodies, and researchers ensure that water quality biomonitoring with caged amphipods is robust, scientifically sound, and comparable across a wide range of environmental conditions. This supports effective environmental protection and sustainable water management worldwide.

Standards Content (Sample)

Technical
Specification
ISO/TS 21738
First edition
Water quality — Active
2026-06
biomonitoring method with in situ
caged benthic amphipods
Qualité de l'eau — Méthode de biosurveillance active avec des
amphipodes benthiques en cage in situ
Reference number
© ISO 2026
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
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or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
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Phone: [phone]
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Website: www.iso.org
Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
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
5.2.1 Sex .3
5.2.2 Weight .4
5.3 Equipment .5
5.3.1 Sieve .5
5.3.2 Cage .5
5.3.3 Vials used for storage prior to chemical analysis .7
5.3.4 Spin dryer .7
5.3.5 Balance .7
5.3.6 Physico-chemical probes . . .7
6 Test organism preparation in laboratory . 7
6.1 Acclimatization period of amphipods .7
6.2 Test organism selection criteria .8
6.3 Laboratory measurements and conditioning of organisms for analysis .9
6.4 Caging of test organisms for dispatch .9
7 Exposure of test organisms in the monitoring station . 9
7.1 Monitoring station environmental condition check .9
7.2 Dispatch to monitoring station .10
7.3 Test organism transplantation .10
7.4 Retrieval of test organisms .11
8 Test organism conditioning in the laboratory .11
8.1 Reception of test organisms at the laboratory .11
8.2 Placing of test organisms in vials .11
9 Test report .12
Annex A (informative) Application domain .13
Annex B (informative) Acclimatization conditions .15
Bibliography . 17

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 organizations,
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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 document 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)
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this may not represent the latest information, which may be obtained from the patent database available at
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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 quality, 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.

iv
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 can be applied to measure chemical substances in the biota: either passive
[11]
approaches based on sampling or collecting indigenous organisms at the monitoring station, or active
[4],[7]
approaches based on transplanting, by caging, organisms from a reference source population.
Active biomonitoring makes it possible to:
— overcome the lack of indigenous organisms at certain monitoring stations;
— 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.
[6]
This enables a reliable comparison of results between stations and between sampling dates.
This method limits the risk of organisms escaping during caging:
— enclosure chambers present holes with diameter inferior to organisms' size and are securely closed with
screwed cap;
— enclosure chambers are fixed and protected from breakage by being installed inside an exposure
1)
system.
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. 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 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 behaviour 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,
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.

v
physicochemical parameters for optimal exposure, range of average weight per individual, organisms’
density in cage. Further species can be added to this table.

vi
Technical Specification ISO/TS 21738:2026(en)
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
acclimatization period
period prior to in situ exposure and during which the organisms are kept under controlled laboratory
conditions
Note 1 to entry: Controlled laboratory conditions can include temperature, electric conductivity, oxygen, diet,
organism density, medium replacement frequency, and photoperiod.
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 A).
3.3
monitoring station
location in the water-body characteristic of the impacted or reference location being studied
3.4
caging point
sub-area of the monitoring station

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: ISO 24032:2021, 3.2]
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
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 studies at a 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 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 A to reference for each species its indigenous area and its application domain.
NOTE 1 According to the literature, some species can be proposed, e.g. Gammarus pulex and Gammarus fossarum for
[16] [8],[17],[3] [10],[15]
Europe; Hyalella azteca for Canada and USA; Hyalella curvispina for Argentina; Paramelita nigroculus
[19] [14]
for South Africa or Melita plumulosa for Australia .
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 is possible to use a local field population
upstream of the site under investigation. The organisms can be also breed in laboratory.
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 can be tracked. A summary table is proposed in Annex 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 can be added to this 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 can 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 obtain 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 coloured
exoskeleton should be avoided because it is 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 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 much more time consuming. Figure 1 illustrates 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 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 mar-
supial 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 must 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 is 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. For 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).
5.3.2 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
amphipods considering its large size. PP plastics are used because these are 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 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 test organisms escaping. It should also be checked that the density of individuals is reasonable and
shall not exceed 5,3
...