ISO/TS 4988:2022

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 2022
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ii
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
Foreword
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described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
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This document was prepared by Technical Committee ISO/TC 229 Nanotechnologies.
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iv
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
...