Nanotechnologies — Toxicity assessment and bioassimilation of manufactured nano-objects in suspension using the unicellular organism Tetrahymena sp.

This document provides a reliable and repeatable method for simultaneous assessment of both exposure and toxicity of manufactured nano-objects (MNOs) using Tetrahymena sp. The ingested, internalized material (MNOs) indicates aquatic exposure. This document is intended to be used by all the centers working with nano(eco)toxicity of MNOs and capable of culturing of Tetrahymena sp. The method uses Tetrahymena sp. to assess exposure and effects of MNOs. In addition, the test can be used by centers (laboratories) interested in investigating the biological interaction of MNOs with living cells. This method is applicable to nano-objects such as nanoparticles, nanofibres of certain size (in a µm size range), nanoplates, as well as their aggregates and agglomerates.

Nanotechnologies — Évaluation de la toxicité et de la bioassimilation des nano-objets manufacturés en suspension à l’aide de l’organisme unicellulaire Tetrahymena sp.

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ISO/TS 4988:2022 - Nanotechnologies — Toxicity assessment and bioassimilation of manufactured nano-objects in suspension using the unicellular organism Tetrahymena sp. Released:5/25/2022
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TECHNICAL ISO/TS
SPECIFICATION 4988
First edition
2022-05
Nanotechnologies — Toxicity
assessment and bioassimilation
of manufactured nano-objects in
suspension using the unicellular
organism Tetrahymena sp.
Nanotechnologies — Évaluation de la toxicité et de la bioassimilation
des nano-objets manufacturés en suspension à l’aide de l’organisme
unicellulaire Tetrahymena sp.
Reference number
ISO/TS 4988:2022(E)
© ISO 2022
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ISO/TS 4988:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

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Published in Switzerland
© ISO 2022 – All rights reserved
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ISO/TS 4988:2022(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction .................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ..................................................................................................................................................................................... 1

3 Terms and definitions .................................................................................................................................................................................... 1

4 Abbreviated terms ............................................................................................................................................................................................. 3

5 Materials ....................................................................................................................................................................................................................... 4

5.1 Test organism and culture medium ..................................................................................................................................... 4

5.2 Chemicals .................................................................................................................................................................................................... 4

5.2.1 General chemicals ............................................................................................................................................................. 4

5.2.2 Additional chemicals for nutrient medium ................................................................................................. 4

6 Technical equipment ....................................................................................................................................................................................... 5

7 Preparation and characterization of the nano-object ................................................................................................. 5

7.1 Nano-object characterization.................................................................................................................................................... 5

7.2 Dispersion preparation ................................................................................................................................................................... 6

7.3 Dispersion characterization ....................................................................................................................................................... 6

7.4 Preparation of media for toxicity tests ............................................................................................................................. 6

8 Culture of Tetrahymena sp. .......................................................................................................................................................................6

8.1 General ........................................................................................................................................................................................................... 6

8.2 Tetrahymena culturing conditions ........................................................................................................................................ 6

8.2.1 Tetrahymena growth conditions .......................................................................................................................... 6

8.2.2 Tetrahymena conditions during exposure ................................................................................................... 7

9 Effect of nano-objects on Tetrahymena sp. .............................................................................................................................. 7

9.1 Test concentrations ............................................................................................................................................................................ 7

9.1.1 Range finding test ............................................................................................................................................................. 7

9.1.2 Definitive test ........................................................................................................................................................................ 7

9.2 Duration ........................................................................................................................................................................................................ 8

9.3 Observations ............................................................................................................................................................................................ 8

9.4 Detailed description of exposure condition ................................................................................................................. 8

9.5 Toxicity assessments ......................................................................................................................................................................... 9

9.5.1 Cell viability ............................................................................................................................................................................ 9

9.5.2 Population growth impairment tests .............................................................................................................. 9

9.5.3 ATP assay .................................................................................................................................................................................. 9

9.5.4 MTT assay ........................................................................................................................................... ...................................... 9

9.5.5 LDH assay ................................... ........................................................................................................ .................................... 10

9.6 Phagocytic activity and material bioassimilation ............................................................................................... 10

10 Data analysis .........................................................................................................................................................................................................10

11 Test report ...............................................................................................................................................................................................................10

12 Results validity with negative control.......................................................................................................................................11

Bibliography .............................................................................................................................................................................................................................12

iii
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ISO/TS 4988:2022(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 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 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.
© ISO 2022 – All rights reserved
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ISO/TS 4988:2022(E)
Introduction

In recent years, many studies have been carried out to investigate the effect of manufactured nano-

objects (MNOs) on aquatic organisms and their ecosystem. Development and more common use of

MNOs in consumer products lead to an increased exposure, and hence a higher possibility of impact on

human health and the environment, in case the MNO cause adverse effects. Nanoparticles are used for

example in various household products, industrial processes, and in products spanning applications

from construction to health and fitness, and MNOs can end up in the environment, for example, bound

to wastewater sludge, ultimately entering into the aquatic environment.

Various aquatic organisms (such as fish, daphnia, artemia, algae) are currently used to predict the

potential harmful effects of chemicals, including MNOs, on the aquatic environment. Unicellular

protozoa of the genus Tetrahymena sp. are freshwater organisms with widespread distribution in

aquatic environments and are at the bottom of the aquatic food chain. Tetrahymena sp. (Protozoa,

Ciliata, Oligohymenophorea) are non-pathogenic, free-living eukaryotes and ubiquitously distributed in

nature and constituting an important connection between the highly productive and nutrient retaining

microbial loop and the metazoans of the classical food chain. This unicellular eukaryote which is bigger

than many mammalian cells (approximately 30 µm to 50 µm), can be found in temperate freshwater

environments and exhibits nuclear dimorphism (two types of cell nuclei). They have a larger, non-

germline macronucleus and a small, germline micronucleus. Tetrahymena sp. has a fast generation

time, shows a high level of complexity and it is a typical eukaryotic cell resembling cells in multicellular

organisms including humans. In addition, although it is unicellular, it possesses many core processes

conserved across a wide diversity of eukaryotes (including humans) that are not found in other single-

celled model systems (e.g. the yeasts Saccharomyces cerevisiae).

The protozoan Tetrahymena sp. is an established experimental model in biological studies and it has

been extensively used for more than six decades as a toxicological model organism to test the toxicity

[12]

of different substances using several endpoints. During the last several years, considerable effort

has been devoted to computational modelling of the toxicity of chemicals to Tetrahymena pyriformis

[27]

for medium and large sized data sets using computational modelling. It means that data from

standardized tests is highly needed. In recent years, viability of cells of Tetrahymena sp. has been

[1]-[24]

suggested also as a routine test of MNOs toxicity. There are several advantages to using

Tetrahymena sp. as a biological model for a toxicological test model system in freshwater aquatic

toxicology and in bioassimilation experiments:

— abundant information is available about using Tetrahymena sp. in cellular biology, ecology and

ecotoxicology and its role in the microbial food web;
— cells of Tetrahymena sp. can easily be cultured at high densities;

— Tetrahymena sp. possesses features of both single eukaryotic cells and whole organisms;

— Tetrahymena sp. plays an important role as grazers of microbes in aquatic environments and

balancing bacterio-plankton production;

— Tetrahymena sp. has acceptable sensitivity to exposure to different xenobiotics;

— some species of Tetrahymena possess a genetically fully sequenced macronucleus, thus facilitating

the study of changes in gene expression patterns under pollution stress (toxicogenomics);

— Tetrahymena sp. is an invertebrate, lacks the characteristic of vertebrates but can still be used to

replace the use of animals in toxicity testing at initial stages of testing;

— Tetrahymena sp. eats anything that fits into their mouth; it has a highly developed system for the

internalization of nanoscale and microscale particles which makes them an ideal model system in

nanotoxicity and material cellular internalization (bioassimilation) research.

To ensure the sustainable development of nanotechnology, there is a need for hazard identification and

risk assessment of MNOs. This document provides a standard protocol intended to generate reliable

© ISO 2022 – All rights reserved
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ISO/TS 4988:2022(E)

toxicity and bioassimilation data by using Tetrahymena sp. for evaluation of MNOs in any experimental

suspension of MNOs of interest or in samples from freshwater ecosystems.

Tetrahymena is positioned as a primary consumer in the freshwater food chain, so it is considered as a

potential vehicle of environmental contaminants. Tetrahymena phagocytic activity is a cost-effective,

suitable and rapid assessment tool towards cell internalization (uptake and possible assimilation) of

[4]

pollutants including particles. It can act as a very early and sensitive indicator for the toxic effect of

various xenobiotic compounds as well as an indication of internalization / bioassimilation of xenobiotics.

The effect of MNOs on Tetrahymena can be induced by the ingested (phagocytosed) MNOs, but also by

the contact with MNOs (without internalization) or by the metal ions released from metal-containing

MNOs in the suspension. The effect of ingested (phagocytosed) material is measured via cell viability

(endpoint of effect) measurements. Phagocytic activity is particle internalisation by cells which, in this

case, can be measured by the number and appearance of food vacuoles. Detection of MNOs in living

cells exposed to a suspension indicates that the suspension contains MNOs that can be internalized by

living cells. This can be taken as a characteristic of biological significance of a suspension containing

NMOs. “Biological significance” in this case means that material can be internalized (phagocytosed)

by cells. In case of exposure to MNOs, the number and appearance of food vacuoles can also be used as

a measure that particles of a defined size (which fit into their mouth) are present in a suspension. This

can be used as a biological indication of exposure and in parallel the effects of ingested material can

be studied. Tetrahymena sp. possesses features of both single eukaryotic cells and whole organisms.

Several studies have highlighted their potential as models in in vitro toxicological assessment of

chemical pollutants using various endpoints. Tetrahymena based pilot ring test has been initiated by

[11]

the German Federal Environmental Agency for ecological risk assessment and further elaborated

[26]

by OECD for activated sludge. Although the OECD’s working party on manufactured nanomaterials

has recently reviewed the relevance of its various test guidelines on traditional experimental models

for the testing of MNOs (see Reference [31]), Reference [31] did not review any methods that utilize

the Tetrahymena sp. phagocytic activity, as mentioned earlier, is a cost-effective physiological endpoint,

which can act as a very early and sensitive indicator for the toxic effect of various xenobiotic compounds

as well as an indication of internalization or bioassimilation of xenobiotics. In case of MNO exposure,

this endpoint can also serve as a measure of exposure to MNOs in any suspension of MNOs where their

cellular internalization is of interest.
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TECHNICAL SPECIFICATION ISO/TS 4988:2022(E)
Nanotechnologies — Toxicity assessment and
bioassimilation of manufactured nano-objects in
suspension using the unicellular organism Tetrahymena
sp.
1 Scope

This document provides a reliable and repeatable method for simultaneous assessment of both exposure

and toxicity of manufactured nano-objects (MNOs) using Tetrahymena sp. The ingested, internalized

material (MNOs) indicates aquatic exposure.

This document is intended to be used by all the centers working with nano(eco)toxicity of MNOs and

capable of culturing of Tetrahymena sp. The method uses Tetrahymena sp. to assess exposure and

effects of MNOs. In addition, the test can be used by centers (laboratories) interested in investigating

the biological interaction of MNOs with living cells.

This method is applicable to nano-objects such as nanoparticles, nanofibres of certain size (in a µm size

range), nanoplates, as well as their aggregates and agglomerates.
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 80004(all parts), Nanotechnologies — Vocabulary — Part 1: Core terms
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 80004 (all parts) and the

following 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
agglomerate

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 1 to entry: The forces holding agglomerates together are weak forces, for example van der Waals forces, or

simple physical entanglement.

Note 2 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]
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ISO/TS 4988:2022(E)
3.2
aggregate

particle (3.7) 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
stock suspension

concentrated suspension that will be diluted to some lower concentration for actual use

[SOURCE: ISO/TS 20787:2017, 3.7]
3.4
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 80004-2:2015, 2.1]
3.5
nano-object

discrete piece of material with one, two or three external dimensions in the nanoscale (3.4)

Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each

other.
[SOURCE: ISO/TS 80004-2:2015, 2.2]
3.6
nanoparticle

nano-object (3.5) with all external dimensions in the nanoscale (3.4) 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 (3.9) may be preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
3.7
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 (3.5).

[SOURCE: ISO/TS 80004-2:2015, 3.1]
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ISO/TS 4988:2022(E)
3.8
nanofibre

nano-object (3.5) with two external dimensions in the nanoscale (3.4) 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 (3.5), note 1 to entry.
[SOURCE: ISO/TS 80004-2:2015, 4.5]
3.9
nanoplate

nano-object (3.5) with one external dimension in the nanoscale (3.4) 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 (3.5), note 1 to entry.
[SOURCE: ISO/TS 80004-2:2015, 4.6]
3.10
sample

one or more sampling items intended to provide information on the population or on the material

3.11
endpoint

recorded observation of a study conducted to determine if a substance has any associated hazards

Note 1 to entry: Endpoints in toxicity studies are measured parameters at different levels of biological complexity

(mortality, behaviour, reproductive status, physiological, biochemical changes, etc.)

3.12
median effective concentration

concentration at which there is an effect on 50 % of the organisms in line with the test criterion

[SOURCE: ISO 15088:2007, 3.3, modified — Note 1 to entry has been deleted.]
3.13
50 % impairment growth concentration

concentration of a substance that inhibits 50 % of the growth of the test population (i.e. Tetrahymena

sp.) within a designated period (i.e. 24h)
3.14
bioassimilation

absorption or adsorption and digestion of food or nutrients by an organism, which is the state or

condition of being absorbed or adsorbed into the organism
4 Abbreviated terms
ATP Adenosine triphosphate
CCD Charge-coupled device
DDW Double distilled water
DLS Dynamic light scattering
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ISO/TS 4988:2022(E)
EC Median effective concentration
EDTA Ethylenediaminetetraacetic acid
IGC 50 % impairment growth concentration
LDH Lactate dehydrogenase
MIAN Minimal information about nanomaterials
MNO Manufactured nano-object
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
NADH Nicotinamide adenine dinucleotide (NAD) + hydrogen (H)
PCC Physicochemical characterization
SEM Scanning electron microscope
TEM Transmission electron microscope
5 Materials
5.1 Test organism and culture medium

Tetrahymena is a genus of free-living ciliates, common in freshwater ponds and used as model organisms

in biomedical research. There are different species of Tetrahymena sp. used as model organisms in

biomedical research such as T. thermophila and T. pyriformis. Different species respond differently

towards various toxicants because of differences in their uptake and metabolic processes. Tetrahymena

thermophila is the more common species, which has been most commonly used in toxicity tests. This

pear-shaped freshwater microorganism (30 μm × 50 μm) grows easily to high density in the laboratory.

Axenic cultures of T. thermophila from the Protoxkit FTM (MicroBioTests Inc.) grow for 24 h in the

[19]

dark at 32 °C in a semi-defined proteose-peptone based medium a nutrient rich medium (detailed

information is provided in 8.2). The cell density obtained in these culture conditions is approximately

5 3 [19]

10 cells/cm . The cells are then processed according to method described by Schultz (1997) in

a nutrient poor medium. All experiments are performed in batch cultures of 100 cm in Erlenmeyer

flasks and aerated by shaking (90 rpm) in darkness.
5.2 Chemicals
5.2.1 General chemicals
— Potassium dichromate (K Cr O ).
2 2 7
— Hydrogen peroxide (H O ).
2 2
— Milli-Q water.
— DDW.
5.2.2 Additional chemicals for nutrient medium
— Proteose-peptone (bacteriological peptone).

1) Protoxkit FTM (MicroBioTests Inc.) is an example of a suitable product available commercially. This

information is given for the convenience of users of this document and does not constitute an endorsement by ISO

of this product.
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ISO/TS 4988:2022(E)
— D-glucose (C H O ).
6 12 6
— Yeast extract (for microbiology).
— Trizma-base ® [Tris Hydroxymethyl Aminomethane Base, (HOCH ) CNH ].
2 3 2
— Calcium chloride dihydrate (CaCl ·2H O).
2 2
— Copper(II) chloride dihydrate (CuCl ·2H O).
2 2
— Iron(III) chloride hexahydrate (FeCl ·6H O).
3 2
— Magnesium sulfate heptahydrate (MgSO ·7H O).
4 2
— Ammonium iron(II) sulfate hexahydrate (Fe(NH )2(SO ) ·6H O).
4 4 2 2
— Magnesium chloride hexahydrate (MgCl ·6H O).
2 2
— Zinc chloride (ZnCl ).
— EDTA.
— 37 % aqueous solution of hydrogen chloride (HCl).
— Cell proliferation kit I (MTT) .
— ATP bioluminescent assay kit.
— Trypan blue.
6 Technical equipment
— Adequate apparatus for temperature control.
— Light microscope equipped for imaging.
— Centrifuge.
— Pipettes.
— Laboratory oven.
— Autoclave.
— Sonicator (ultrasonic device).
— Plate stirrer.
— Spectrophotometer.
7 Preparation and characterization of the nano-object
7.1 Nano-object characterization

The complete physical-chemical characteristics of test nano-object (e.g. shape, purity, size) should be

determined according to ISO/TR 13014. Particle morphology of test nano-object should be determined

using TEM or SEM.

2) Trizma-base ® is an example of a suitable product available commercially. This information is given for the

convenience of users of this document and does not constitute an endorsement by ISO of this product.

3) Cell Proliferation Kit I (MTT) is an example of a suitable product available commercially. This information is

given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.

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ISO/TS 4988:2022(E)
7.2 Dispersion preparation

The preparation of the MNO dispersion should be well documented, preferably via a standard operating

procedure 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 MNOs in stock suspension can be achieved by stirring or

sonication at least 15 min (depends on type of MNOs) using ultrasonic device where appropriate. Stock

suspensions shall be vortexed first and then sonicated for at least 15 min (depending on the type of

MNO) using an ultrasonic device fo
...

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