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

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Published
Publication Date
03-Dec-2017
Technical Committee
Current Stage
9093 - International Standard confirmed
Start Date
19-Mar-2021
Completion Date
19-Mar-2021
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ISO/TS 20787:2017 - Nanotechnologies - Aquatic toxicity assessment of manufactured nanomaterials in saltwater lakes using Artemia sp. Nauplii
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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/TS 20787:2017(E)
ISO 2017
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ISO/TS 20787:2017(E)
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ii © ISO 2017 – All rights reserved
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ISO/TS 20787:2017(E)
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

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ISO/TS 20787:2017(E)
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
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ISO/TS 20787:2017(E)
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.
© ISO 2017 – All rights reserved v
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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.
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ISO/TS 20787:2017(E)

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
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ISO/TS 20787:2017(E)

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]
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ISO/TS 20787:2017(E)
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

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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

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.
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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 concentrated suspension of the test nanomaterial. The concentration of organic

solvents, emulsifiers or dispersants should not exceed 100 mg/l.
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ISO/TS 20787:2017(E)
6.2 Dispersion characterization

The dispersion state of the nanomaterials should be characterized with DLS (dynamic light scattering)

as described in ISO 22412 or other suitable methods such as ultrasonic attenuation spectroscopy as

described in ISO 20998-1.
6.3 Dispersion stability in stock suspension

The size distribution of the nanoparticle dispersion and its stability over time shall be characterized

in relevant intervals. Also the concentration of the nanoparticle in the stock suspension should be

evaluated using an appropriate method. In the case of metal-based nanomaterials, which tend to convert

to metal ions and vice versa, the proportions of metal ions and nanoparticles should be determined.

6.4 Dispersion stability in artificial seawater

The stability of the dispersion and the actual concentration of the MNM in the artificial seawater shall

be verified at an appropriate range of concentrations during experiment (6 h, 24 h and 48 h). Since

the sea water would possibly influence the physico-chemical properties of the test nanomaterials,

further characterizations of the test nanomaterial are recommended. In this regard, degree of

aggregation/agglomeration (or changing of particle size distribution), and the amount of metal ions in

the exposure media shall be evaluated.
6.5 Preparation of exposure media for toxicity tests

All the exposure media are prepared fresh from stock suspensions (e.g. 1 000 mg/L). Appropriate

volumes of stock suspension should be added directly to sterilized artificial seawater to achieve

relevant concentrations of the MNM in exposure media. Although Artemia sp. do not need sterile

conditions for hatching and growth, efforts should be made to minimize the development of unicellular

algae and bacterial contamination. Sterile filtration is the best method of sterilizing artificial seawater

without altering its chemistry and therefore is recommended for sterilizing artificial seawater. 0,1 µm

filters can be used to yield water free of bacteria.
7 Hatching procedure
7.1 General

Thoroughly wash the hatching and culture vessels with a light chlorine solution, rinse, and allow air-

drying between uses. Avoid soap, because it will leave a slight residue that will foam from aeration

during hatching, and leave cysts stranded above the water level. Although complete sterility of the

material is not necessary, efforts should be made to minimize the development of unicellular algae and

bacterial contamination.
7.2 Dilution water

7.2.1 A standard artificial seawater with salinity of 35 ± 1 g/L should be used for the hatching as well

as for all toxicity tests. After aeration and stabilization for 24 h, the dilution water should have a pH of

8,0 ± 0,5, and the oxygen content should be at least 90 % saturation. If necessary, the pH should be adjusted

with concentrated HCl or NaOH. Before using the salt water, it should be filtered through a 1 µm filter.

7.2.2 The current international standard for making artificial seawater is ASTM D1141-98. It can be

[38]

found at ASTM international. Chemical composition of ASTM artificial seawater consists of NaCl

(24,53 g/L), MgCl2 (5,20 g/L), Na2SO4 (4,09 g/L), CaCl2 (1,16 g/L), KCl (0,695 g/L), NaHCO3 (0,201 g/L),

KBr (0,101 g/L), H3BO3 (0,027 g/L), SrCl2 (0,025 g/L), and NaF (0,003 g/L).
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ISO/TS 20787:2017(E)
7.3 Storage of cysts

Sealed cans of Artemia sp. cysts can be stored for years at room temperature, but, once opened, should

be used within two months. After each use, the can should be tightly covered with a plastic lid and

stored in a refrigerator. If the entire contents of a can will not be used in two months, the portion that is

expected to be unused should be placed in a tightly closed container and frozen until needed.

7.4 Disinfection of Artemia sp. cysts
7.4.1 Prepare 200 ppm sodium hypochlorite (NaOCl) solution.
7.4.2
...

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