ISO/TS 20787:2017

Nanotechnologies - Aquatic toxicity assessment of manufactured nanomaterials in saltwater lakes using Artemia sp. Nauplii

ISO/TS 20787:2017 specifies a test method, aiming to maximize repeatability and reliability of testing, to determine whether MNMs are toxic to aquatic organisms, specifically Artemia sp. nauplius. ISO/TS 20787:2017 is intended to be used by ecotoxicological laboratories that are capable in the hatching and culturing of Artemia sp. and the evaluation of toxicity of nanomaterials using Artemia sp. nauplius. This method uses Artemia sp. nauplii in a simulated environment, artificial seawater, to assess effects of nanomaterials. ISO/TS 20787:2017 is applicable to MNMs that consist of nano-objects such as nanoparticles, nanopowders, nanofibres, nanotubes, nanowires, as well as aggregates and agglomerates of such MNMs.

Nanotechnologies - Evaluation de la toxicité des nanomatériaux manufacturés dans les lacs d’eau salés par des Artemia sp. Nauplii

General Information

• Status: Published
• Publication Date: 03-Dec-2017
• ICS: 07.120 - Nanotechnologies
• Technical Committee: ISO/TC 229 - Nanotechnologies
• Drafting Committee: ISO/TC 229 - Nanotechnologies
• Current Stage: 9093 - International Standard confirmed
• Start Date: 22-Jul-2024
• Completion Date: 14-Feb-2026

Overview

ISO/TS 20787:2017 - Nanotechnologies - Aquatic toxicity assessment of manufactured nanomaterials in saltwater lakes using Artemia sp. nauplii - specifies a standardized test method for assessing whether manufactured nanomaterials (MNMs) are toxic to salt‑water aquatic organisms. The method uses freshly hatched Artemia sp. nauplii in artificial seawater to simulate salt lake ecosystems and generate repeatable, reliable ecotoxicity data for regulatory, research and safety assessments.

Key keywords: ISO/TS 20787:2017, nanotechnologies, aquatic toxicity assessment, Artemia sp. nauplii, manufactured nanomaterials, artificial seawater, nanoparticles.

Key topics and technical requirements

This technical specification defines the essential elements of a robust aquatic nanotoxicity bioassay:

• Test organism and culture: procedures for hatching, culturing and harvesting Artemia sp. nauplii from cysts to obtain uniform age and physiological state.
• Exposure media: use of artificial seawater to reproduce salt‑lake conditions appropriate for Artemia (noting their broad salinity tolerance).
• MNM scope: applicable to nano‑objects and their aggregates/agglomerates - e.g., nanoparticles, nanopowders, nanofibres, nanotubes, nanowires.
• Dispersion handling: preparation, physico‑chemical characterization and stability assessment of MNM dispersions in stock suspensions and in artificial seawater.
• Test design: definition of control groups (including positive control), test concentrations, exposure conditions, observation schedules and test duration to maximize repeatability.
• Analytical and biological endpoints: measurement of exposure concentrations, observations of nauplius effects, and data handling.
• Reporting and validity: standardized information to include in test reports and criteria for result validity and repeatability.

The specification references established ISO nanotechnology and ecotoxicology guidance documents to ensure alignment with broader characterization and reporting practices.

Applications and who uses it

ISO/TS 20787:2017 is intended for:

• Ecotoxicology laboratories experienced in hatching and culturing Artemia and conducting aquatic bioassays.
• Researchers studying environmental fate and effects of MNMs in saline lake ecosystems.
• Regulatory bodies and industry groups performing hazard assessment, product stewardship, or environmental risk assessments for nanomaterials.
• Test facilities developing comparative datasets for nanotoxicology, safety dossiers, or eco‑labeling.

Practical uses include screening MNMs for acute toxicity in saline environments, comparing material formulations, and supporting environmental risk assessment for products containing nanomaterials.

Related standards

Relevant normative references cited in ISO/TS 20787:2017 include:

• ISO 10993-12 (sample preparation)
• ISO/TS 12805, 16195, 17200 (nanomaterials specifications)
• ISO/TR 13014 (physico‑chemical characterization guidance)
• ISO/TS 80004 series (nanotechnology vocabulary)
• ISO 15088 (zebrafish egg toxicity methodology) - as an example of related aquatic testing approaches

Using ISO/TS 20787:2017 helps generate comparable, repeatable data for aquatic nanotoxicology and supports responsible development and regulation of manufactured nanomaterials.

Standards Content (Sample)

TECHNICAL ISO/TS
SPECIFICATION 20787
First edition
2017-11
Nanotechnologies - Aquatic toxicity
assessment of manufactured
nanomaterials in saltwater lakes using
Artemia sp. Nauplii
Nanotechnologies - Evaluation de la toxicité des nanomatériaux en
milieu aquatique par des Artemia sp
Reference number
©
ISO 2017
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2017 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Materials . 5
4.1 Test organism . 5
4.2 Chemicals . 5
5 Technical equipment . 5
6 Preparation and characterization of dispersion of nanomaterial .6
6.1 Dispersion preparation . 6
6.2 Dispersion characterization . 7
6.3 Dispersion stability in stock suspension . 7
6.4 Dispersion stability in artificial seawater . 7
6.5 Preparation of exposure media for toxicity tests . 7
7 Hatching procedure . 7
7.1 General . 7
7.2 Dilution water . 7
7.3 Storage of cysts . 8
7.4 Disinfection of Artemia sp. cysts . 8
7.5 Hatching method of Artemia sp. cysts . 8
7.6 Harvesting of nauplii . 8
7.7 Calculation of hatching percentage . 9
8 Effect of nanomaterial on Artemia sp. nauplii . 9
8.1 Test groups and controls . 9
8.2 Test concentrations . 9
8.3 Exposure condition .10
8.4 Duration .10
8.5 Observations .10
8.6 Analytical measurements .10
9 Data analysis .10
10 Test report .10
10.1 Test procedure .10
10.2 Information to include in the report .11
10.2.1 Test nanomaterial .11
10.2.2 Test species .11
10.2.3 Test conditions.11
10.2.4 Bioassay results .11
11 Results validity .11
Bibliography .13
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 documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on 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 the following
URL: www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
iv © ISO 2017 – All rights reserved

Introduction
With the increasing development and use of manufactured nanomaterials (MNMs) in consumer and
other products, concern about the possible impact of MNMs on human and environmental health is
increasing. Various aquatic organisms (such as fish, daphnia, algae, etc.) are currently used to predict
the possible adverse effects of chemicals, including nanomaterials, on the aquatic environment. Brine
[42]
shrimp (Artemia sp.) are found nearly worldwide in saline lakes and pools, and are one of the most
widespread euryhaline organisms that are suitable for ecotoxicity testing. Artemia sp. nauplii can
be used to assess the effects of nanomaterials in salt water ecosystems, primarily salt lakes. Artemia
sp. usually live in salt lakes, and are almost never found in an open sea. This species also adapts to a
wide range of salinities (5 g/L to 300 g/L) and temperatures (6 °C to 40 °C). In fact, the physiologically
optimal levels of salinity for Artemia sp. are about 30 g/L to 35 g/L. Due to predators at these salt
levels, however, Artemia sp. seldom occur in natural habitats at salinities of less than 45 g/L to 80 g/L.
Favoured for the absence of predators and food competitors in such places, Artemia sp. develop very
dense populations.
There are several advantages to using Artemia sp. as a biological model in salt water aquatic toxicology:
a) Less concern about animal welfare than for a vertebrate species;
b) There is good knowledge of Artemia sp. biology and ecology;
c) Artemia sp. have a wide geographic distribution in salt water lakes and pools;
d) Tests performed on Artemia sp. nauplii are simple and cost-effective;
e) Small body size allows accommodation of Artemia sp. nauplii in small beakers or plates;
f) Artemia sp. adapt to a wide range of water salinity and temperature;
g) Artemia sp. are simple to maintain in the laboratory;
h) The life cycle of Artemia sp. is short, so it is suitable for growth, reproduction and short-term
toxicity tests;
i) Artemia sp. cysts are commercially and readily available so that the tests can be carried out
worldwide. The cysts can be stored for years under cool and dry conditions without losing viability.
Upon immersion in sea water, the free swimming nauplii will hatch within approximately 24 h;
j) Hatching from cysts gives organisms of similar age, genotype and physiological condition.
In recent years, several researchers around the world have used Artemia sp. as a test organism in aquatic
nanotoxicology (see References [1] to [35]). The lack of a standardized protocol for testing Artemia sp.
for aquatic toxicity means that data from these studies are more likely to be non-repeatable and non-
[22]
reliable. The goal of this document is to provide a standard protocol intended to generate reliable
aquatic toxicity data by testing Artemia sp., which can be used for ecotoxicity evaluation of MNMs in salt
water lake ecosystems.
TECHNICAL SPECIFICATION ISO/TS 20787:2017(E)
Nanotechnologies - Aquatic toxicity assessment of
manufactured nanomaterials in saltwater lakes using
Artemia sp. Nauplii
1 Scope
This document specifies a test method, aiming to maximize repeatability and reliability of testing, to
determine whether MNMs are toxic to aquatic organisms, specifically Artemia sp. nauplius.
This document is intended to be used by ecotoxicological laboratories that are capable in the hatching
and culturing of Artemia sp. and the evaluation of toxicity of nanomaterials using Artemia sp. nauplius.
This method uses Artemia sp. nauplii in a simulated environment, artificial seawater, to assess effects of
nanomaterials.
This document is applicable to MNMs that consist of nano-objects such as nanoparticles, nanopowders,
nanofibres, nanotubes, nanowires, as well as aggregates and agglomerates of such MNMs.
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 10993-12, Biological evaluation of medical devices — Part 12: Sample preparation and reference
materials
ISO/TS 11931, Nanotechnologies — Nanoscale calcium carbonate in powder form — Characteristics and
measurement
ISO/TS 12805, Nanotechnologies — Materials specifications — Guidance on specifying nano-objects
ISO/TR 13014, Nanotechnologies  —  Guidance  on  physico-chemical  characterization  of  engineered
nanoscale materials for toxicologic assessment
ISO 15088, Water quality — Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio)
ISO/TS 16195, Nanotechnologies  —  Guidance  for  developing  representative  test  materials  consisting  of
nano-objects in dry powder form
ISO/TS 17200, Nanotechnology — Nanoparticles in powder form — Characteristics and measurements
ISO 26824, Particle characterization of particulate systems — Vocabulary
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 80004-4, Nanotechnologies — Vocabulary — Part 4: Nanostructured materials
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 10993-12, ISO/TS 11931,
ISO/TS 12805, ISO 15088, ISO/TS 16195, ISO/TS 17200, ISO 26824, ISO/TS 80004-1, ISO/TS 80004-2
and ISO/TS 80004-4 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
agglomerate
Note 1 to entry: collection of weakly or medium strongly bound particles where the resulting external surface
area is similar to the sum of the surface areas of the individual components
Note 2 to entry: The forces holding agglomerates together are weak forces, for example van der Waals forces, or
simple physical entanglement.
Note 3 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.4]
3.2
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area is
significantly smaller than the sum of surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example covalent or ionic bonds,
or those resulting from sintering or complex physical entanglement, or otherwise combined former primary
particles.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.5]
3.3
hatching vessel
vessel appropriate for Artemia sp. cyst hatching
Note 1 to entry: Cone should be transparent or semi-translucent (preferably colourless) for ease of harvesting
and light transmission.
Note 2 to entry: As shown in Figure 2, constant aeration from the bottom of the hatching vessel should be used to
keep cysts in suspension, and to provide sufficient oxygen levels for the cysts to hatch.
Note 3 to entry: Hatching vessels include glass or plastic cone or “V”–bottomed container as shown in Figure 1.
Figure 1 — Schematic of appropriate hatching vessel for Artemia sp
2 © ISO 2017 – All rights reserved

Figure 2 — Schematic of aeration from the bottom of a hatching vessel for Artemia sp
3.4
test vessel
vessel appropriate for Artemia sp. culture
Note 1 to entry: Test vessels and other apparatus that will come into contact with the test solutions should be
made entirely of glass or other chemically inert material.
Note 2 to entry: Test vessels include flasks or beakers.
3.5
positive control
well-characterized material and/or substance, which, when evaluated by a specific test method,
demonstrates the suitability of the test system to yield a reproducible, appropriately positive or reactive
response in the test system
Note 1 to entry: Potassium dichromate (K Cr O ) is suggested as a suitable treatment for the positive control in
2 2 7
Artemia sp. toxicity test.
3.6
test nanomaterial
manufactured nanomaterial in a dispersion that is subjected to biological or chemical testing or
evaluation
3.7
stock suspension
concentrated suspension that will be diluted to some lower concentration for actual use
3.8
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from larger sizes are predominantly exhibited in this
length range.
[SOURCE: ISO/TS 80004-1:2015, 2.1]
3.9
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each other.
[SOURCE: ISO/TS 80004-1:2015, 2.5]
3.10
nanoparticle
nano-object with all external dimensions in the nanoscale where the lengths of the longest and the
shortest axes of the nano-object do not differ significantly
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms such as
nanofibre or nanoplate may be preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
3.11
particle
minute piece of matter with defined physical boundaries
Note 1 to entry: A physical boundary can also be described as an interface.
Note 2 to entry: A particle can move as a unit.
Note 3 to entry: This general definition of particle applies to nano-objects.
3.12
nanofibre
nano-object with two external dimensions in the nanoscale and the third dimension significantly larger
Note 1 to entry: The largest external dimension is not necessarily in the nanoscale.
Note 2 to entry: The terms nanofibril and nanofilament can also be used.
Note 3 to entry: See nanoparticle, note 1 to entry.
[SOURCE: ISO/TS 80004-2:2015, 4.5]
3.13
nanoplate
nano-object with one external dimension in the nanoscale and the other two external dimensions
significantly larger
Note 1 to entry: The larger external dimensions are not necessarily in the nanoscale.
Note 2 to entry: See nanoparticle, note 1 to entry.
[SOURCE: ISO/TS 80004-2:2015, 4.6]
3.14
Artemia sp
species of the genus of aquatic crustaceans known as brine shrimp (Artemia)
3.15
nauplii
newly hatched brine shrimp larvae
Note 1 to entry: The nauplius larvae of Artemia sp. are less than 0,4 mm in length when they first hatch.
3.16
cyst
dormant Artemia sp. eggs
Note 1 to entry: The cysts may be stored for long periods and hatched on demand.
3.17
hatching
process of converting cysts to nauplii under appropriate environmental conditions
4 © ISO 2017 – All rights reserved

3.18
control solution
test medium without sample under test
3.19
immobilization
inability of the nauplii to swim during the 15 second following gentle agitation of the test and control
solutions, even if the nauplii can still move their appendages
[SOURCE: ISO 6341:2012, 3.3, modified — “organisms” has been replaced by “nauplii”, and “antennae”
has been replaced by “appendages”.]
3.20
EC
concentration at which there is an effect on 50 % of the organisms in line with the test criterion
[SOURCE: ISO 15088:2007, 3.3]
4 Materials
4.1 Test organism
Different species of Artemia sp. can be used, but Artemia salina and Artemia franciscana are the preferred
test species. There are many commercial sources of brine shrimp cysts. Artemia sp. nauplii (newborn
brine shrimp) should be produced by hatching high quality cysts in the laboratory.
4.2 Chemicals
4.2.1 Artificial seawater.
4.2.2 Potassium dichromate.
4.2.3 Lugol's solution (Lugol's iodine).
4.2.4 Sodium hypochlorite (5,25 % NaOCl).
4.2.5 Sodium hydroxide solution (400 g/L NaOH).
5 Technical equipment
5.1 Adequate apparatus for temperature control.
5.2 Microscope.
5.3 Binocular stereoscope.
5.4 Centrifuge.
5.5 Air pump.
5.6 Single channel pipettes.
5.7 Laboratory balance.
5.8 Laboratory water purification system.
5.9 Laboratory oven.
5.10 Laboratory autoclave.
5.11 Centrifuge.
5.12 Sonication system.
5.13 Hot plate stirrer.
5.14 Oxygen meter.
5.15 Thermometer.
5.16 pH meter.
5.17 Salt meter (salinity meter).
5.18 Multiparameter photometer.
5.19 Light source.
5.20 Adequate apparatus for the control of the lighting regime and measurement of light
intensity (lux meter).
5.21 Equipment for the determination of total organic carbon.
5.22 Equipment for the determination of chemical oxygen demand (COD).
6 Preparation and characterization of dispersion of nanomaterial
6.1 Dispersion preparation
Most nanomaterials tend to agglomerate/aggregate strongly in water, and this situation could be
exacerbated in salt water. Before assessing the toxicity of a MNM using Artemia sp., the MNM shall
be well dispersed in artificial seawater. The preparation of the MNM dispersion should be well
documented, preferably via a standard operating procedure (see References [36] and [37]), as this
step in the testing is known to impact on the tested material. Dispersion is often done in a two-step
procedure, first a stock suspension is prepared, and then an aliquot of this is further diluted when the
testing starts. Dispersion of MNMs in stock suspension can be achieved by stirring, sonication, or by
means of functionalizing groups as well as using biocompatible dispersant reagents. Sonication should
be carried out in a way that produces no other new materials and the effects of sonication should be
evaluated. Chemicals that would have a detrimental effect on Artemia sp. should be avoided. When such
vehicles are used, an additional control should be exposed to the same concentration of the vehicle as
that used in the most conce
...

TECHNICAL ISO/TS
SPECIFICATION 20787
First edition
2017-11
Nanotechnologies - Aquatic toxicity
assessment of manufactured
nanomaterials in saltwater lakes using
Artemia sp. Nauplii
Nanotechnologies - Evaluation de la toxicité des nanomatériaux en
milieu aquatique par des Artemia sp
Reference number
©
ISO 2017
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2017 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Materials . 5
4.1 Test organism . 5
4.2 Chemicals . 5
5 Technical equipment . 5
6 Preparation and characterization of dispersion of nanomaterial .6
6.1 Dispersion preparation . 6
6.2 Dispersion characterization . 7
6.3 Dispersion stability in stock suspension . 7
6.4 Dispersion stability in artificial seawater . 7
6.5 Preparation of exposure media for toxicity tests . 7
7 Hatching procedure . 7
7.1 General . 7
7.2 Dilution water . 7
7.3 Storage of cysts . 8
7.4 Disinfection of Artemia sp. cysts . 8
7.5 Hatching method of Artemia sp. cysts . 8
7.6 Harvesting of nauplii . 8
7.7 Calculation of hatching percentage . 9
8 Effect of nanomaterial on Artemia sp. nauplii . 9
8.1 Test groups and controls . 9
8.2 Test concentrations . 9
8.3 Exposure condition .10
8.4 Duration .10
8.5 Observations .10
8.6 Analytical measurements .10
9 Data analysis .10
10 Test report .10
10.1 Test procedure .10
10.2 Information to include in the report .11
10.2.1 Test nanomaterial .11
10.2.2 Test species .11
10.2.3 Test conditions.11
10.2.4 Bioassay results .11
11 Results validity .11
Bibliography .13
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 documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on 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 the following
URL: www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
iv © ISO 2017 – All rights reserved

Introduction
With the increasing development and use of manufactured nanomaterials (MNMs) in consumer and
other products, concern about the possible impact of MNMs on human and environmental health is
increasing. Various aquatic organisms (such as fish, daphnia, algae, etc.) are currently used to predict
the possible adverse effects of chemicals, including nanomaterials, on the aquatic environment. Brine
[42]
shrimp (Artemia sp.) are found nearly worldwide in saline lakes and pools, and are one of the most
widespread euryhaline organisms that are suitable for ecotoxicity testing. Artemia sp. nauplii can
be used to assess the effects of nanomaterials in salt water ecosystems, primarily salt lakes. Artemia
sp. usually live in salt lakes, and are almost never found in an open sea. This species also adapts to a
wide range of salinities (5 g/L to 300 g/L) and temperatures (6 °C to 40 °C). In fact, the physiologically
optimal levels of salinity for Artemia sp. are about 30 g/L to 35 g/L. Due to predators at these salt
levels, however, Artemia sp. seldom occur in natural habitats at salinities of less than 45 g/L to 80 g/L.
Favoured for the absence of predators and food competitors in such places, Artemia sp. develop very
dense populations.
There are several advantages to using Artemia sp. as a biological model in salt water aquatic toxicology:
a) Less concern about animal welfare than for a vertebrate species;
b) There is good knowledge of Artemia sp. biology and ecology;
c) Artemia sp. have a wide geographic distribution in salt water lakes and pools;
d) Tests performed on Artemia sp. nauplii are simple and cost-effective;
e) Small body size allows accommodation of Artemia sp. nauplii in small beakers or plates;
f) Artemia sp. adapt to a wide range of water salinity and temperature;
g) Artemia sp. are simple to maintain in the laboratory;
h) The life cycle of Artemia sp. is short, so it is suitable for growth, reproduction and short-term
toxicity tests;
i) Artemia sp. cysts are commercially and readily available so that the tests can be carried out
worldwide. The cysts can be stored for years under cool and dry conditions without losing viability.
Upon immersion in sea water, the free swimming nauplii will hatch within approximately 24 h;
j) Hatching from cysts gives organisms of similar age, genotype and physiological condition.
In recent years, several researchers around the world have used Artemia sp. as a test organism in aquatic
nanotoxicology (see References [1] to [35]). The lack of a standardized protocol for testing Artemia sp.
for aquatic toxicity means that data from these studies are more likely to be non-repeatable and non-
[22]
reliable. The goal of this document is to provide a standard protocol intended to generate reliable
aquatic toxicity data by testing Artemia sp., which can be used for ecotoxicity evaluation of MNMs in salt
water lake ecosystems.
TECHNICAL SPECIFICATION ISO/TS 20787:2017(E)
Nanotechnologies - Aquatic toxicity assessment of
manufactured nanomaterials in saltwater lakes using
Artemia sp. Nauplii
1 Scope
This document specifies a test method, aiming to maximize repeatability and reliability of testing, to
determine whether MNMs are toxic to aquatic organisms, specifically Artemia sp. nauplius.
This document is intended to be used by ecotoxicological laboratories that are capable in the hatching
and culturing of Artemia sp. and the evaluation of toxicity of nanomaterials using Artemia sp. nauplius.
This method uses Artemia sp. nauplii in a simulated environment, artificial seawater, to assess effects of
nanomaterials.
This document is applicable to MNMs that consist of nano-objects such as nanoparticles, nanopowders,
nanofibres, nanotubes, nanowires, as well as aggregates and agglomerates of such MNMs.
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 10993-12, Biological evaluation of medical devices — Part 12: Sample preparation and reference
materials
ISO/TS 11931, Nanotechnologies — Nanoscale calcium carbonate in powder form — Characteristics and
measurement
ISO/TS 12805, Nanotechnologies — Materials specifications — Guidance on specifying nano-objects
ISO/TR 13014, Nanotechnologies  —  Guidance  on  physico-chemical  characterization  of  engineered
nanoscale materials for toxicologic assessment
ISO 15088, Water quality — Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio)
ISO/TS 16195, Nanotechnologies  —  Guidance  for  developing  representative  test  materials  consisting  of
nano-objects in dry powder form
ISO/TS 17200, Nanotechnology — Nanoparticles in powder form — Characteristics and measurements
ISO 26824, Particle characterization of particulate systems — Vocabulary
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 80004-4, Nanotechnologies — Vocabulary — Part 4: Nanostructured materials
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 10993-12, ISO/TS 11931,
ISO/TS 12805, ISO 15088, ISO/TS 16195, ISO/TS 17200, ISO 26824, ISO/TS 80004-1, ISO/TS 80004-2
and ISO/TS 80004-4 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
agglomerate
Note 1 to entry: collection of weakly or medium strongly bound particles where the resulting external surface
area is similar to the sum of the surface areas of the individual components
Note 2 to entry: The forces holding agglomerates together are weak forces, for example van der Waals forces, or
simple physical entanglement.
Note 3 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.4]
3.2
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area is
significantly smaller than the sum of surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example covalent or ionic bonds,
or those resulting from sintering or complex physical entanglement, or otherwise combined former primary
particles.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.5]
3.3
hatching vessel
vessel appropriate for Artemia sp. cyst hatching
Note 1 to entry: Cone should be transparent or semi-translucent (preferably colourless) for ease of harvesting
and light transmission.
Note 2 to entry: As shown in Figure 2, constant aeration from the bottom of the hatching vessel should be used to
keep cysts in suspension, and to provide sufficient oxygen levels for the cysts to hatch.
Note 3 to entry: Hatching vessels include glass or plastic cone or “V”–bottomed container as shown in Figure 1.
Figure 1 — Schematic of appropriate hatching vessel for Artemia sp
2 © ISO 2017 – All rights reserved

Figure 2 — Schematic of aeration from the bottom of a hatching vessel for Artemia sp
3.4
test vessel
vessel appropriate for Artemia sp. culture
Note 1 to entry: Test vessels and other apparatus that will come into contact with the test solutions should be
made entirely of glass or other chemically inert material.
Note 2 to entry: Test vessels include flasks or beakers.
3.5
positive control
well-characterized material and/or substance, which, when evaluated by a specific test method,
demonstrates the suitability of the test system to yield a reproducible, appropriately positive or reactive
response in the test system
Note 1 to entry: Potassium dichromate (K Cr O ) is suggested as a suitable treatment for the positive control in
2 2 7
Artemia sp. toxicity test.
3.6
test nanomaterial
manufactured nanomaterial in a dispersion that is subjected to biological or chemical testing or
evaluation
3.7
stock suspension
concentrated suspension that will be diluted to some lower concentration for actual use
3.8
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from larger sizes are predominantly exhibited in this
length range.
[SOURCE: ISO/TS 80004-1:2015, 2.1]
3.9
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each other.
[SOURCE: ISO/TS 80004-1:2015, 2.5]
3.10
nanoparticle
nano-object with all external dimensions in the nanoscale where the lengths of the longest and the
shortest axes of the nano-object do not differ significantly
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms such as
nanofibre or nanoplate may be preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
3.11
particle
minute piece of matter with defined physical boundaries
Note 1 to entry: A physical boundary can also be described as an interface.
Note 2 to entry: A particle can move as a unit.
Note 3 to entry: This general definition of particle applies to nano-objects.
3.12
nanofibre
nano-object with two external dimensions in the nanoscale and the third dimension significantly larger
Note 1 to entry: The largest external dimension is not necessarily in the nanoscale.
Note 2 to entry: The terms nanofibril and nanofilament can also be used.
Note 3 to entry: See nanoparticle, note 1 to entry.
[SOURCE: ISO/TS 80004-2:2015, 4.5]
3.13
nanoplate
nano-object with one external dimension in the nanoscale and the other two external dimensions
significantly larger
Note 1 to entry: The larger external dimensions are not necessarily in the nanoscale.
Note 2 to entry: See nanoparticle, note 1 to entry.
[SOURCE: ISO/TS 80004-2:2015, 4.6]
3.14
Artemia sp
species of the genus of aquatic crustaceans known as brine shrimp (Artemia)
3.15
nauplii
newly hatched brine shrimp larvae
Note 1 to entry: The nauplius larvae of Artemia sp. are less than 0,4 mm in length when they first hatch.
3.16
cyst
dormant Artemia sp. eggs
Note 1 to entry: The cysts may be stored for long periods and hatched on demand.
3.17
hatching
process of converting cysts to nauplii under appropriate environmental conditions
4 © ISO 2017 – All rights reserved

3.18
control solution
test medium without sample under test
3.19
immobilization
inability of the nauplii to swim during the 15 second following gentle agitation of the test and control
solutions, even if the nauplii can still move their appendages
[SOURCE: ISO 6341:2012, 3.3, modified — “organisms” has been replaced by “nauplii”, and “antennae”
has been replaced by “appendages”.]
3.20
EC
concentration at which there is an effect on 50 % of the organisms in line with the test criterion
[SOURCE: ISO 15088:2007, 3.3]
4 Materials
4.1 Test organism
Different species of Artemia sp. can be used, but Artemia salina and Artemia franciscana are the preferred
test species. There are many commercial sources of brine shrimp cysts. Artemia sp. nauplii (newborn
brine shrimp) should be produced by hatching high quality cysts in the laboratory.
4.2 Chemicals
4.2.1 Artificial seawater.
4.2.2 Potassium dichromate.
4.2.3 Lugol's solution (Lugol's iodine).
4.2.4 Sodium hypochlorite (5,25 % NaOCl).
4.2.5 Sodium hydroxide solution (400 g/L NaOH).
5 Technical equipment
5.1 Adequate apparatus for temperature control.
5.2 Microscope.
5.3 Binocular stereoscope.
5.4 Centrifuge.
5.5 Air pump.
5.6 Single channel pipettes.
5.7 Laboratory balance.
5.8 Laboratory water purification system.
5.9 Laboratory oven.
5.10 Laboratory autoclave.
5.11 Centrifuge.
5.12 Sonication system.
5.13 Hot plate stirrer.
5.14 Oxygen meter.
5.15 Thermometer.
5.16 pH meter.
5.17 Salt meter (salinity meter).
5.18 Multiparameter photometer.
5.19 Light source.
5.20 Adequate apparatus for the control of the lighting regime and measurement of light
intensity (lux meter).
5.21 Equipment for the determination of total organic carbon.
5.22 Equipment for the determination of chemical oxygen demand (COD).
6 Preparation and characterization of dispersion of nanomaterial
6.1 Dispersion preparation
Most nanomaterials tend to agglomerate/aggregate strongly in water, and this situation could be
exacerbated in salt water. Before assessing the toxicity of a MNM using Artemia sp., the MNM shall
be well dispersed in artificial seawater. The preparation of the MNM dispersion should be well
documented, preferably via a standard operating procedure (see References [36] and [37]), as this
step in the testing is known to impact on the tested material. Dispersion is often done in a two-step
procedure, first a stock suspension is prepared, and then an aliquot of this is further diluted when the
testing starts. Dispersion of MNMs in stock suspension can be achieved by stirring, sonication, or by
means of functionalizing groups as well as using biocompatible dispersant reagents. Sonication should
be carried out in a way that produces no other new materials and the effects of sonication should be
evaluated. Chemicals that would have a detrimental effect on Artemia sp. should be avoided. When such
vehicles are used, an additional control should be exposed to the same concentration of the vehicle as
that used in the most conce
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