ISO/TS 22082:2020

TECHNICAL ISO/TS
SPECIFICATION 22082
First edition
2020-05
Nanotechnologies — Assessment
of nanomaterial toxicity using
dechorionated zebrafish embryo
Nanotechnologies — Évaluation de la toxicité des nanomatériaux au
moyen d'embryons déchorionés de poisson zèbre
Reference number
©
ISO 2020
© ISO 2020
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ii © ISO 2020 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Materials . 2
6 Apparatus . 3
6.1 Technical equipment . 3
6.2 Analytical instruments . 3
7 Procedures . 4
7.1 Culture . 4
7.1.1 Zebrafish strain . 4
7.1.2 Feeding regime . 4
7.1.3 Photoperiod . 4
7.1.4 Temperature and pH . . 4
7.1.5 Tank material . 4
7.2 Spawning stimulation . 4
7.3 Dechorionation of embryos . 5
7.3.1 Methods . 5
7.3.2 Enzymatic dechorionation method . 5
7.4 Preparation of nanomaterials stock solution . 6
7.5 Testing concentrations . 6
7.6 Dispersion of nanomaterials . 6
7.7 Positive chemical: 3,4-dichloroaniline. 6
7.8 Test methods . 7
7.8.1 Water quality measurement . 7
7.8.2 Preparation of dechorionated embryos . 7
7.8.3 Exposure . 7
7.8.4 Endpoints observation . 7
7.9 Data analysis . 8
8 Test report . 8
8.1 Test procedure . 8
8.2 Information to be included in the report . 8
8.2.1 Test nanomaterials . 8
8.2.2 Test species . 8
8.2.3 Materials and apparatus . . 8
8.2.4 Maintenance . 9
8.2.5 Test design . 9
8.2.6 Test conditions. 9
8.2.7 Results . 9
Annex A (informative) Mechanical dechorionation method .10
Annex B (informative) Zebrafish embryo spawning procedures .11
Annex C (informative) Results validation .12
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 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.
The committee responsible for this document is ISO/TC 229, Nanotechnologies.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved

Introduction
Fish assays are important and widely used tools for evaluating the toxicity of chemicals in the aquatic
environment. However, there are animal welfare concerns regarding the use of vertebrate animals for
chemical testing, including fish. The use of early life stage embryos, instead of adult or juvenile fish,
is considered an alternative assay because there are animal welfare benefits to testing fish embryos
as an alternative to the clear distress caused by testing more developed juvenile fish, e.g. by using
OECD TG 203.
Nanotechnology is positively affecting many commercial sectors, but there remain concerns regarding
the potential adverse environmental effects from nano-enabled products. The OECD test guideline
using fish embryos to evaluate acute toxicity (see OECD TG 236) states that some substances having a
molecular weight ≥ 3 kDa, a very bulky molecular structure, and substances causing delayed hatching
might be inappropriate for testing using that method. The presence of the chorion could also confound
assessment of the nanomaterials biological activity. The chorion is the outmost acellular envelope of a
fish embryo and it can serve as an exposure barrier for some chemicals or nanomaterials. It is currently
not possible to predict which nanomaterials might be blocked by the chorion. Using dechorionated
embryos for toxicity assessments may not provide direct ecotoxicological information, but may help to
better identify potentially hazardous nanomaterials. Accordingly, many researchers around the world
[1]
have developed a number of methods for removing a chorion from early life stage zebrafish embryos
[2]
. There are two ways to remove chorion from embryos: by enzymatic or mechanical method. The
enzymatic dechorionation method has some advantages over the mechanical dechorionation method
(see Annex A), including time and labour efficiency by easy preparation for dechorionation, no mechanical
embryonic damage, and the ability to simultaneously prepare a large number of dechorionated embryos
for a high throughput-based approaches. On the other hand, there is a disadvantage of variability in
pronase activity that could influence the success rate of chorion removal. Numerous groups have used
[3][4][5][6]
dechorionated embryos for the assessment of chemical and nanomaterial toxicity . However,
[7][8][9][10]
these methods have not yet been fully standardized .
Dechorionated zebrafish embryos toxicity assay can serve as a surrogate system to detect potentially
hazardous nanomaterials for other vertebrate systems. As the use of higher organism animal
models for toxicity testing is being refined, there is an increasing need for alternative test methods.
Early life stage zebrafish (up to independent feeding, e.g. 120 HPF) could be an excellent alternative
[22][23][24][25][26][27]
model of in vivo toxicity . Compared with other animal models, zebrafish have a
number of advantages for assessing toxicity, including the relative ease of rearing and breeding, high
fecundity (external fertilization, 200 embryos to 300 embryos from a female), short generation time
(approximately 3 months to adulthood), availability of genomic resources (complete zebrafish genome
sequence), and genetic similarity to humans. About 70 % of human diseases have at least one zebrafish
[11]
orthologue and 84 % of the human genes associated with disease have orthologues in zebrafish .
Therefore, the use of zebrafish to assess chemical toxicity is increasing.
This document provides an optimized procedure to remove chorions along with recommendations on
how to conduct toxicity assays using dechorionated zebrafish embryos. It also discusses the advantages
of the fish toxicity assay using dechorionated embryos.
TECHNICAL SPECIFICATION ISO/TS 22082:2020(E)
Nanotechnologies — Assessment of nanomaterial toxicity
using dechorionated zebrafish embryo
1 Scope
This document specifies a method for rapidly assessing nanomaterial toxicity (fish early life stage,
0 HPF to 120 HPF). It includes information on the importance of acellular chorion removal, detailed
chorion removal procedures, and a complete protocol for the toxicity assessment of nanomaterials
using dechorionated zebrafish embryos. The focus of this document is on testing nanomaterial toxicity.
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/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/TS 17200, Nanotechnology — Nanoparticles in powder form — Characteristics and measurements
ISO/TR 18196, Nanotechnologies — Measurement technique matrix for the characterization of nano-objects
ISO 22412, Particle size analysis — Dynamic light scattering (DLS)
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
OECD. Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD Guidelines for the Testing of Chemicals.
Section 2. OECD Publishing, Paris, 2013
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/TS 12805, ISO/TR 13014,
ISO/TS 17200, ISO/TS 80004-1, ISO/TS 80004-2, OECD TG 236 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
solubilizing agent
solvent or dispersant that can disperse and stabilize nanomaterials in solution
3.2
spawning
releasing the eggs into the water for fertilization
3.3
positive control
any well-characterized material or substance that, 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
[SOURCE: ISO 10993-10:2010, 3.14]
3.4
range-finding test
abbreviated acute test that exposes test organisms to a broad range of nanomaterial testing solutions
to establish the range of concentrations to be used in the definitive test
Note 1 to entry: The test includes at least five concentrations of the nanomaterial and untreated control.
[SOURCE: OECD TG 236:2013, modified]
3.5
LC
concentration of a toxic substance that is lethal to half of a group of test organisms (50 %)
Note 1 to entry: Usually, the exposure to the substance is continuous and the LC is defined by reference to a
specified exposure period.
[SOURCE: ISO 6107-3:1993, 39, modified]
4 Abbreviated terms
DPF day post fertilization
EM embryo media
HPF hour post fertilization
NOAEL no observed adverse effect level
DO dissolved oxygen
5 Materials
5.1 Organism (zebrafish, Danio rerio).
Zebrafish (Danio rerio, see Figure 1) is a tropical freshwater teleost belonging to the Cyprinidae,
naturally distributed throughout India, Pakistan, Bangladesh, Nepal and Burma. The average size of
an adult is 2 cm to 3 cm and has blue stripes, similar to a zebra on the side of the body. The adult male
zebrafish has a thinner body, with blue and gold hue stripes. The female has a whitish belly, larger than
male and silver stripes instead of gold. This species can be kept and bred in aquaria. The lifespan of
zebrafish is approximately 2 to 3 years. Organogenesis of zebrafish is completed within 5 DPF and it is
an adult within approximately 3 months.
2 © ISO 2020 – All rights reserved

a)  Male b)  Female
Figure 1 — Zebrafish
5.2 Stock solution, freshly prepared with purified water.
5.3 Positive control, 3,4-dichloroaniline (3,4-DCA) (CAS# 95-76-1).
6 Apparatus
6.1 Technical equipment
6.1.1 Vessels.
Multi-well plates (6 wells or 12 wells) can be used. To prevent evaporation during exposure, vessels
should be sealed with a biocompatible substrate (i.e. parafilm or transparent self-adhesive foil).
6.1.2 Incubator, with controlled temperature at (27 ± 1) °C and photoperiod (10:14, light:dar k).
6.1.3 Microscope, stereo- or inverted with a capacity of at least 80-fold magnification.
6.1.4 Spawning tanks, of any dimensions and design, typically consisting of a mating cage and a slightly
larger container.
Mating cages with mesh bottom of grid size (2 ± 0,5) mm allow freshly released eggs to fall down to
avoid being eaten by the parent fish.
6.1.5 Pipettes.
A single channel pipette can be used.
6.2 Analytical instruments
Use the analytical instruments given in ISO/TR 18196 and the following.
6.2.1 Scanning electron microscopy (SEM).
Scanning electron microscope that produces magnified images of the nanomaterials by scanning the
surface with an elec
...