ISO 14669:2026

Water quality — Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)

This document describes a method for the determination of the acute toxicity to one of three specified species of marine copepod (Copepoda, Crustacea). This document is applicable to: industrial or sewage effluents, treated or untreated, after decantation, filtration or centrifugation if necessary; marine or estuarine waters; )aqueous extracts (pore water, elutriates, eluates and leachates) from sediments; chemical substances which are soluble, or which can be maintained as a stable suspension or dispersion, under the conditions of the test.

Qualité de l'eau — Détermination de la toxicité létale aiguë vis-à-vis de copépodes marins (Copepoda, Crustacea)

La présente Norme internationale spécifie une méthode de détermination de la toxicité aiguë vis-à-vis l'une quelconque de trois espèces spécifiées de copépodes marins (Copepoda, Crustacea) a) de substances chimiques solubles, ou pouvant être maintenues en suspension stable ou en dispersion stable dans les conditions de l'essai; b) d'effluents industriels et urbains, épurés ou non, s'il y a lieu, après décantation, filtration ou centrifugation; c) des eaux de mer ou d'estuaires.

General Information

• Status: Published
• Publication Date: 15-Jun-2026
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: ISO/TC 147/SC 5 - Biological methods
• Drafting Committee: ISO/TC 147/SC 5/WG 2 - Toxicity to invertebrates
• Current Stage: 6060 - International Standard published
• Start Date: 16-Jun-2026
• Due Date: 10-Oct-2026
• Completion Date: 16-Jun-2026

Relations

• Revises: ISO 14669:1999 - Water quality — Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)
• Effective Date: 28-Oct-2023

Overview

ISO 14669:2026 – Water quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea) is an internationally recognized standard developed by ISO for assessing the acute toxicity of various substances and waters using marine copepods. This document outlines a reliable, standardized method to evaluate the effect of chemicals, industrial effluents, sewage, marine or estuarine waters, and extracts from sediments on three specific copepod species. The protocol determines the lethal concentration for 50% of tested organisms (LC50) within prescribed exposure periods, supporting environmental risk assessments for marine ecosystems.

Key Topics

• Test Organisms: The standard specifies three copepod species for toxicity testing: Acartia tonsa, Tisbe battagliai, and Nitocra spinipes. Organisms must be healthy and at specified life stages, and may be sourced from laboratory cultures or certified hatcheries.
• Scope of Application:
  • Treated or untreated industrial and sewage effluents, post-decantation, filtration, or centrifugation.
  • Marine and estuarine waters.
  • Aqueous extracts (pore water, elutriates, eluates, leachates) from marine and estuarine sediments.
  • Soluble chemicals or those suspended/dispensed stably throughout the test duration.
• Test Conditions: The acute toxicity test is performed under controlled laboratory settings, maintaining stable temperature (20 °C ± 2 °C) and 16:8 h light:dark cycles. Proper test preparation includes using natural or artificial seawater of specified salinity and quality.
• Test Procedure: The process includes:
  • Preliminary and definitive exposure stages.
  • Preparation and storage of samples as per referenced ISO sampling guidelines.
  • Use of appropriate controls and, where suitable, reference toxicants (e.g., 3,5-dichlorophenol) to validate sensitivity and test performance.
  • Measurement and documentation of test solution parameters (salinity, pH, dissolved oxygen) and detailed recording of results, including LC50 calculation and confidence intervals.

Applications

ISO 14669:2026 is fundamental to environmental monitoring and pollution management. Common uses include:

• Environmental risk assessment: Governments and regulatory agencies employ the ISO 14669 method to determine the toxicity of chemicals, industrial discharges, and wastewater before release into marine environments.
• Effluent and sediment testing: Water utilities, industries, and environmental laboratories use this standard for routine monitoring of acute toxicity in effluents and sediment extracts.
• Chemical hazard evaluation: Manufacturers conduct early-stage aquatic toxicity testing on new and existing chemical substances as part of product stewardship and compliance with safety and environmental legislation.
• Research and development: Academic and applied research projects utilize this standard method for comparative studies on marine toxicity, ecological impact assessments, and development of water treatment technologies.

With its clear procedural requirements and robust validity checks, ISO 14669:2026 ensures consistent, reproducible results and supports regulatory compliance globally.

Related Standards

For a comprehensive assessment and correct implementation, users should consider related ISO standards, particularly those referenced within ISO 14669:2026:

• ISO 5667-1: Guidance on the design of sampling programmes and sampling techniques.
• ISO 5667-15: Preservation and handling of sludge, sediment, and suspended matter samples.
• ISO 5667-16: Guidance on biotesting of samples.
• ISO 5667-19: Sampling of marine sediments.
• ISO/TS 20281: Statistical methods for analyzing toxicological test data.
• ISO 21268-2: Leaching procedures for aqueous extracts from sediments.

These standards complement ISO 14669 by ensuring sample integrity, procedural consistency, and reliable analytical outcomes in aquatic toxicity testing. Following these guidelines enhances the validity of ecological risk evaluations and helps maintain global best practices in water quality management.

Standards Content (Sample)

International
Standard
ISO 14669
Second edition
Water quality — Determination
2026-06
of acute lethal toxicity to marine
copepods (Copepoda, Crustacea)
Qualité de l'eau — Détermination de la toxicité létale aiguë vis-à-
vis de copépodes marins (Copepoda, Crustacea)
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
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
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Test environment . 2
6 Reagents and materials . 2
7 Apparatus . 4
8 Sampling, treatment and preparation of samples . 4
8.1 Sampling and storage of samples .4
8.2 Preparation of solutions of substances to be tested .4
8.2.1 Preparation of stock solutions .4
8.2.2 Preparation of aqueous extracts from sediments .4
8.2.3 Preparation of test solutions .5
9 Procedure . 5
9.1 Preliminary test .5
9.2 Definitive test .5
9.3 General procedure .5
9.4 Limit test .6
9.5 Check of sensitivity of copepods and conformity with the procedure.6
10 Calculation of the LC50 . 7
11 Validity of results . 7
12 Expression of results . 7
13 Performance data . 7
14 Test report . 7
Annex A (informative) Example of determination of acute lethal toxicity of a chemical substance
to a marine copepod . 9
Annex B (informative) Culture methods for marine copepods Acartia tonsa, Tisbe battagliai
and Nitocra spinipes .11
Annex C (informative) Precision data . 16
Annex D (informative) Defined culture and test media . 17
Bibliography . 19

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 (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
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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.
This second edition cancels and replaces the first edition (ISO 14669:1999), which has been technically
revised.
The main changes are as follows:
— aqueous extracts (pore water, elutriates, eluates and leachates) have been added to the scope;
— possibility of using copepods from hatcheries has been included;
— informative Annexes A, B and C were amended;
— informative Annex D (culture and test media) was added.
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
International Standard ISO 14669:2026(en)
Water quality — Determination of acute lethal toxicity to
marine copepods (Copepoda, Crustacea)
1 Scope
This document describes a method for the determination of the acute toxicity to one of three specified
species of marine copepod (Copepoda, Crustacea). This document is applicable to:
a) industrial or sewage effluents, treated or untreated, after decantation, filtration or centrifugation if
necessary;
b) marine or estuarine waters;
c) aqueous extracts (pore water, elutriates, eluates and leachates) from sediments;
d) chemical substances which are soluble, or which can be maintained as a stable suspension or dispersion,
under the conditions of the 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 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes and sampling
techniques
ISO 5667-15, Water quality — Sampling — Part 15: Preservation and handling of samples of sludge, sediment
and suspended matter
ISO 5667-16, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 5667-19, Water quality — Sampling — Part 19: Guidance on sampling of marine sediments
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
50 % lethal concentration
LC50
concentration at which 50 % of the test organisms would die in an experiment
[SOURCE: ISO 13073-3:2016, 2.28]

3.2
x % confidence interval
interval of values that theoretically covers the true value of the estimated parameter with x % confidence
Note 1 to entry: Standard confidence intervals are based on the assumption that the underlying mathematical model
is correct. It does not capture model uncertainty.
Note 2 to entry: A more precise definition is given in ISO 3534-1:2006, 1.28: interval estimator (T , T ) for the
0 1
parameter θ with the statistics T and T as interval limits and for which it holds that P[T > θ > T ] ≥ 1 − α.
0 1 0 1
Note 3 to entry: Associated with this confidence interval is the confidence level 100(1 − α) % where α is generally a
small number. The confidence level is typically 90 % or 95 %. The inequality P[T > θ > T ] ≥ 1 − α holds for any specific
0 1
but unknown population value of θ.
Note 4 to entry: A confidence interval does not reflect the probability that the observed interval contains the true
value of the parameter (it either does or does not contain it). The confidence reflects the proportion of cases that
the confidence interval would contain the true parameter value in a long series of repeated random samples under
identical conditions.
[SOURCE: ISO/TS 20281:2006, 3.5]
4 Principle
Copepods are exposed to a range of concentrations of chemical substances in seawater, effluent, water
samples or aqueous extracts from sediments. Mortality of the copepods is recorded after 24 h and 48 h.
In 48 h, the concentration at which 50 % of exposed copepods are killed, under the test conditions described
above, is determined. This concentration, known as the median lethal concentration, is designated 48 h
lethal concentration 50 % (48 h LC50).
NOTE If possible, the concentration which kills 50 % of the exposed copepods in 24 h is also determined (24 h
LC50). It is possible to extend the exposure period to 96 h (96 h LC50).
An indication of the lowest concentration tested which kills all the copepods and the highest concentration
tested which does not kill any of the copepods is desirable and provides useful information in cases where
the 48 h LC50 cannot be determined.
The test is carried out in one or two stages:
— a preliminary test which determines the range of concentrations to be tested in the definitive test and
gives an approximate value of the 48 h LC50 (and where appropriate, the 24 h LC50);
— a definitive test, if toxicity is observed, which permits calculation of the 48 h LC50 (and where appropriate,
the 24 h LC50) and determines concentrations corresponding to 0 % and 100 % mortality.
If the method described in this document is used for chemical substances, a limit test should be performed at
100 mg/l or at a lower concentration which is the maximum at which the substance is soluble or is in stable
dispersion under the conditions of the test.
5 Test environment
The procedure described in this document shall be carried out in a room, incubator or water-bath controlled
at 20 °C ± 2 °C and under a 16 h:8 h light: dark photoperiod. The atmosphere shall be free from vapours or
dusts toxic to copepods.
6 Reagents and materials
6.1 Test organism, one of the following species of marine copepod:
a) Acartia tonsa Dana;
b) Tisbe battagliai Volkmann-Rocco;
c) Nitocra spinipes Boeck.
Obtain the test organisms from laboratory cultures. Guidance on identification and culture methods for each
species are given in Annex B. After hatching of eggs, the lifecycle of copepods consists of naupliar, copepodid
and adult stages. The age and life stage at the start of the test shall be indicated in the test report and are as
follows:
— Acartia tonsa: large copepodids (Stage 5) or adults;
— Tisbe battagliai: copepodids 6 ± 2 days old;
— Nitocra spinipes: adults 3 to 4 weeks old.
Suitable organisms may be purchased from hatcheries outside the laboratory that performs the acute
toxicity test. Only healthy animals from an unpolluted source should be used.
If individuals are received at an early stage, they should be maintained in cultured conditions similar to
the hatchery (sea water, temperature) and fed with appropriate algae mix (similar to the hatchery) until
they reach the proper development stage. Organisms should be tested after confirming that the above
developmental stage has been reached.
If individuals are received at the life stage mentioned above, the test should be conducted the day of reception
or the day after in order to guarantee the developmental stage certified by the hatchery. In this case, feeding
should be added at the start of the test with the same algae mix that in the hatchery and the test conditions
should be similar to the hatchery water characteristics (water salinity and temperature).
In any case, the copepods shall have the age and life stage mentioned above and be in a good health at the
beginning of the test.
6.2 Dilution water.
A natural or an artificial seawater may be used as the dilution water. Culture and test media are prepared
from either reconstituted salt water or filtered (0,2 μm) natural marine water from an unpolluted location.
An example of reconstituted salt water suitable for cultivation and testing is given in Annex D (see Tables D.1,
D.2, D.3 and D.4). Reconstituted salt water media with a known composition in which the copepods show
suitable long-term survival, normal behaviour, development and fecundity may be used as culture and test
media, i.e. dilution water.
If natural seawater is used, it shall be collected from a location as distant as possible from known sources of
pollution and filtered to remove indigenous organisms. If artificial seawater is used, it shall be prepared by
dissolving reagents of recognized analytical grade, or a commercially available formulation, in distilled or
deionized water. However, for the copepod species in this document, there is insufficient information on the
use of artificial seawater to allow a particular example to be recommended.
The salinity of the dilution water shall be between 25 pratical salinity unit (PSU) and 36 PSU. The use of a
lower salinity, which is more appropriate for tests concerning estuarine or brackish water situations, shall
be justified in the test report. Nitocra spinipes can be used at salinities down to 1 PSU and Tisbe battagliai
can be used at salinities down to 20 PSU. Whichever salinity is employed, the test organisms shall be
cultured or maintained at the same salinity (±3 PSU) for at least seven days before the start of the test. The
dilution water shall have a dissolved oxygen concentration above 80 % of the air saturation value, and a pH
of 8,0 ± 0,3 before being used to prepare the test solutions.
The dilution water shall permit survival of the copepods for at least 48 h and should be from the same source
as water that has been found to support culture of the organisms through at least two generations.
6.3 Reference chemical toxicant, e.g. 3,5-dichlorophenol or a suitable alternative (9.5), of recognized
analytical grade.
7 Apparatus
Ordinary laboratory apparatus shall be used, and in particular the following.
7.1 Apparatus, for measuring dissolved oxygen, temperature, salinity and pH.
7.2 Low-power stereo microscope, preferably with darkfield illumination.
7.3 Ultrasonic device or other apparatus for the preparation of stock solutions of poorly soluble
substances (8.2.1).
7.4 Test containers, of chemically and physically inert material and of sufficient capacity (e.g. glass
beakers or disposable rigid plastic tissue-culture well-plates). Loose-fitting lids or covers are recommended
to minimize evaporation of the test solutions. Containers which are suitable for low-power microscopical
observation should be used for nauplii or copepodid stages.
Before use, the test containers shall be carefully washed and then rinsed, first with deionised water and
then with the dilution water (6.2).
8 Sampling, treatment and preparation of samples
8.1 Sampling and storage of samples
Sampling of water or effluent shall be carried out in accordance with the general procedure specified in
ISO 5667-1. Bottles shall be completely filled to exclude air (if the toxicity test is carried out within 48 h and
not frozen).
The preservation and storage of marine sediment shall be carried out in accordance with ISO 5667-15 or
ISO 5667-19. The preservation and storage of water or effluent samples shall be carried out in accordance
with ISO 5667-16; the following is only a summary. The toxicity test should be carried out as soon as possible,
ideally within 12 h of collection. If this time interval cannot be observed, cool the sample (0 °C to 4 °C) and
test the sample within 48 h. If testing cannot be carried out within 48 h, the sample may be frozen (below
−18 °C) for testing within 2 months of collection.
8.2 Preparation of solutions of substances to be tested
8.2.1 Preparation of stock solutions
Prepare stock solutions of the substance to be tested by dissolving or diluting a known quantity of the
substance in a known volume of dilution water, deionized water or distilled water in a glass container. They
shall be prepared at the moment of use unless the substance is known to be stable in solution, in which case
the stock solution may be prepared up to two days in advance.
For substances which are poorly soluble in water, ultrasonic or other suitable devices may be used in the
preparation of the stock solutions to aid solubilization or dispersion of the substance. Organic and inorganic
solvents of low toxicity to copepods (for example acetone) may be used provided that the concentration of
the solvent in the final test solution does not exceed 0,1 ml/l and that two series of control tests, one with no
solvent, the other with the maximum concentration of solvent, are carried out at the same time as the test.
No single procedure for the preparation of stock solutions of poorly soluble substances can be recommended
due to the differing nature of chemicals and physical agents.
8.2.2 Preparation of aqueous extracts from sediments
The aqueous extracts from sediments are carried out with the appropriate protocol, for example, in
accordance with the ISO 21268-2.

8.2.3 Preparation of test solutions
Prepare the test solutions by adding the stock solutions (see 8.2.1) or aqueous sample (see 8.1 and 8.2.2) to
the dilution water (6.2) in specified quantities to obtain the concentrations selected (see 9.1 and 9.2) for the
test.
If the stock solutions are prepared in deionized or distilled water, all the solutions, including the control,
shall receive the same quantity of distilled or deionized water and the final salinity and pH, shall be within
the range specified for the test (6.2).
It is recommended to prepare a volume of test solution sufficient to allow determination of the dissolved
oxygen concentration, pH, temperature and salinity at the start of the test (9.3) using the excess remaining
after filling the test containers.
9 Procedure
9.1 Preliminary test
The preliminary test provides an approximate value of the 48 h LC50 and enables, if necessary, a range of
concentrations to be selected for use in the definitive test. For this purpose, a wide range of concentrations
(generally chosen in geometric series) of the chemical substance, effluent or water sample is tested.
Typically, a factor of 10 or 3,2 between concentrations and a minimum of five animals per concentration,
without replication, is appropriate.
An example is given in Annex A.
9.2 Definitive test
The definitive test enables to determine the percentages of copepods which are killed by different
concentrations and the 24 h and 48 h LC50. Select a range of concentrations (at least five), based on the results
of the preliminary test (9.1), but employing a smaller factor (typically 1,8 or 2) between concentrations. It is
desirable that the concentrations selected result in three percentages of mortality between 10 % and 90 %.
An example of the selection of a range of concentrations is given in Annex A.
For each concentration and each control, use a minimum of 20 copepods (e.g. four replicates each containing
five copepods). Replicate containers are recommended to facilitate counting of the copepods.
9.3 General procedure
Place equal volumes of the test solutions (see 8.2.3) into a series of test containers (7.4). The volume per
container shall be such that, with the required number of copepods, the density of copepods does not exceed
1 per 0,5 ml of solution. For Acartia tonsa, the recommended maximum density is 1 copepod per 5 ml of
solution. For each series of tests, prepare control containers each having a volume of dilution water (6.2)
equal to the volume of the test solutions. If a solvent is used to solubilize or disperse the substance, prepare a
second set of control containers with the dilution water containing the solvent at the maximum concentration
used.
Before the start of the test determine, as a minimum, the dissolved oxygen concentration and pH of the
dilution water (or control solution) and the pH of test solutions corresponding with the lowest and highest
concentrations being tested.
Place the required number of copepods in each test container. It is recommended to transfer the copepods to
the test solutions using a pipette of sufficiently wide bore to avoid damage to the organisms. Minimize the
quantity of water transferred to the test solutions.
The copepods shall not be fed during the test if they come from a culture in the laboratory or obtained from
hatcheries and maintained in the laboratory until the appropriate life stage.

If copepods are fed for some reason (see 6.1), it should be detailed in the report (quantity and content of the
algae mix) and the positive control test should be performed in the same condition. The addition of algae mix
volume should not exceed 10 % of the total tested volume and shall be provided at the beginning of the test.
During the test, keep the vessels at a temperature of 20 °C ± 2 °C and under a 16 h:8 h light: dark photoperiod.
Nitocra spinipes is an epibenthic species and lives in dim light conditions. The vessels in the N. spinipes
bioassay are preferably placed in darkness. A photoperiod of 16:8 h light: dark may also be used but at a low
‒1 ‒2
light intensity (5 µmol to 10 µmol × s × m ).
After 24 h and 48 h, count the surviving copepods in each container. It is recommended to use a low-power
microscope (7.2) to aid observation. Copepods which are showing no swimming or appendage movements
within an observation period of 10 s are considered to be dead. Record an
...