ISO 20814:2019
(Main)Nanotechnologies — Testing the photocatalytic activity of nanoparticles for NADH oxidation
Nanotechnologies — Testing the photocatalytic activity of nanoparticles for NADH oxidation
This document specifies a method for the measurement of the photocatalytic activity (PCA) of nanoparticles (NPs), suspended in an aqueous environment in physiologically relevant conditions, by measuring the ultraviolet (UV)-induced nicotine adenine dinucleotide hydrate (NADH) oxidation. The measurement is intended to assess the potential for the photo-toxicity of nanomaterials. The method is also applicable to NP aggregates and agglomerates.
Nanotechnologies — Test de l'activité photocatalytique des nanoparticules pour l'oxydation du NADH
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INTERNATIONAL ISO
STANDARD 20814
First edition
2019-12
Nanotechnologies — Testing
the photocatalytic activity of
nanoparticles for NADH oxidation
Nanotechnologies — Test de l'activité photocatalytique des
nanoparticules pour l'oxydation du NADH
Reference number
©
ISO 2019
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms, definitions, symbols and abbreviated terms . 1
3.1 Terms and definitions . 1
3.2 Symbols and abbreviated terms. 2
4 Description of the test method . 3
5 Reagents and apparatus . 3
5.1 Reagents. 3
5.2 Apparatus . 4
6 Measurement procedure . 4
6.1 Measurement of NP suspension basic properties . 4
6.1.1 UV-Vis absorption spectrum measurement. 4
6.1.2 NP suspension stability measurement . 5
6.2 UV trans-illuminator light intensity calibration based on 2NB actinometry . 5
6.3 Measurement of NADH solution fluorescence intensity . 6
6.3.1 NADH photo-oxidation rate measurement at various NP concentrations . 6
6.3.2 Calculation of NADH photo-oxidation rate at various NP concentrations . 7
7 Test report . 8
7.1 Information . 8
7.2 Report data format . 9
7.2.1 Correction factors C(i,j) obtained by actinometry (see 7.2) with λ(max,TI) . 9
7.2.2 Calibrated slope of NADH fluorescence decrease. 9
7.2.3 Plot of k versus NP concentration .10
app
7.2.4 NADH equivalent specific PCA .10
8 Precision .10
8.1 Repeatability .10
8.2 Reproducibility .10
Annex A (normative) Schematic diagram of 96-well positioning block .11
Annex B (informative) Sample calibration of UV trans-illuminator light intensity .12
Annex C (informative) Interlaboratory comparison study of TiO NP PCA .17
Bibliography .22
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.
iv © ISO 2019 – All rights reserved
Introduction
Photocatalytic activity (PCA) is the measure of capacity of a material to promote a specific photochemical
reaction under defined conditions (as defined in ISO 20507:2014, 2.3.31). With the expanding use of
nanomaterials in various industries, the possible impacts on human health and the environment due to
the enhancement of detrimental chemical reactions in the presence of light (both natural and artificial)
is an ongoing concern. The absorption of a photon with sufficient energy generates an electron-hole
pair that can migrate to the nanoparticle (NP) surface and react with water and oxygen, thus forming
extremely reactive radicals and reactive oxygen species (ROS). Generation of the ROS by some wide-
bandgap materials, such as TiO , ZnO, WO , CeO , carbon nanotubes, quantum dots and some metal
2 3 2
NPs when illuminated by UV-VIS light, can cause oxidative stress, resulting in toxic effects in living
[5]
organisms . Therefore, measuring the nanomaterial PCA under physiological conditions allows for an
assessment of its photo-toxicity potency.
Existing standard test methods for particle and surface PCA measurement (see ISO 10676 and
ISO 10678) are not directly applicable to determine nanomaterial PCA leading to photo-toxicity, as
they require a large test volume and/or long measurement duration, while utilizing organic dyes as
indicators that are not biocompatible.
The in vitro NP PCA test for NADH oxidation is intended to evaluate the nanomaterial photo-toxicity
potency when exposed to an ultraviolet (UV) light.
INTERNATIONAL STANDARD ISO 20814:2019(E)
Nanotechnologies — Testing the photocatalytic activity of
nanoparticles for NADH oxidation
1 Scope
This document specifies a method for the measurement of the photocatalytic activity (PCA) of
nanoparticles (NPs), suspended in an aqueous environment in physiologically relevant conditions, by
measuring the ultraviolet (UV)-induced nicotine adenine dinucleotide hydrate (NADH) oxidation.
The measurement is intended to assess the potential for the photo-toxicity of nanomaterials. The
method is also applicable to NP 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/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
3 Terms, definitions, symbols and abbreviated terms
For the purposes of this document, the terms and definitions given in ISO/TS 80004-1, ISO/TS 80004-2
and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1 Terms and definitions
3.1.1
actinometry
method to measure the number of photons integrally or per unit of time
3.1.2
catalytic activity
property of a component corresponding to the catalysed substance rate of conversion of a specified
chemical reaction, in a specified measurement system
[SOURCE: ISO 18153:2003, 3.2, modified — The notes have been deleted.]
3.1.3
oxidation
chemical reaction accompanying a gain of oxygen, loss of hydrogen of an organic substrate or loss of
one or more electrons from a molecular entity
3.1.4
NADH equivalent specific PCA
PCA measured as the NADH photo-oxidation (3.1.5) rate per unit weight of nanoparticles
3.1.5
photo-oxidation
oxidation reactions induced by light
3.2 Symbols and abbreviated terms
DIW deionized water with ≥ 18 MΩ·cm resistivity
NADH nicotine adenine dinucleotide hydrate
NaOH sodium hydroxide
2NB 2-nitrobenzaldehyde
NP nanoparticle
PB phosphate buffer
PCA photocatalytic activity
ROS reactive oxygen species
TI trans-illuminator
TiO titanium dioxide
UV ultraviolet
UV-Vis ultraviolet and visible
A (i,j) phenolphthalein absorbance before exposure to trans-illuminator UV irradiation in
c
each well (i = B, C, D, E, F, G; j = 2, 3, 4, …, 10, 11)
A (i,j) phenolphthalein absorbance after exposure to trans-illuminator UV irradiation in each
e
well (i = B, C, D, E, F, G; j = 2, 3, 4, …, 10, 11)
ΔA(i,j) change in phenolphthalein absorbance after exposure to trans-illuminator UV
irradiation in each well (i = B, C, D, E, F, G; j = 2, 3, 4, …, 10, 11)
ΔA average change of phenolphthalein absorbance over all wells before and after UV
a
irradiation by using a UV trans-illuminator
C starting concentration of the NP suspension for a dilution series of test solutions;
the suspension absorbance at 310 nm or 365 nm (depending on the used UV
trans-illuminator) is 1,4 < A < 1,6
C(i,j) light intensity correction factor of each well, which accounts for the UV irradiation
intensity variation of the UV trans-illuminator at the location of each well
(i = B, C, D, E, F, G; j = 2, 3, 4, …, 8, 9)
I (i,j) NADH fluorescence intensity measured before UV irradiation in each well
F,0
(i = B, C, D, E, F, G; j = 2, 3, 4, …, 8, 9)
I (i,j) NADH fluorescence intensity measured following the UV irradiation of t duration by
F,t
using a UV trans-illuminator in each well (i =
...
INTERNATIONAL ISO
STANDARD 20814
First edition
2019-12
Nanotechnologies — Testing
the photocatalytic activity of
nanoparticles for NADH oxidation
Nanotechnologies — Test de l'activité photocatalytique des
nanoparticules pour l'oxydation du NADH
Reference number
©
ISO 2019
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms, definitions, symbols and abbreviated terms . 1
3.1 Terms and definitions . 1
3.2 Symbols and abbreviated terms. 2
4 Description of the test method . 3
5 Reagents and apparatus . 3
5.1 Reagents. 3
5.2 Apparatus . 4
6 Measurement procedure . 4
6.1 Measurement of NP suspension basic properties . 4
6.1.1 UV-Vis absorption spectrum measurement. 4
6.1.2 NP suspension stability measurement . 5
6.2 UV trans-illuminator light intensity calibration based on 2NB actinometry . 5
6.3 Measurement of NADH solution fluorescence intensity . 6
6.3.1 NADH photo-oxidation rate measurement at various NP concentrations . 6
6.3.2 Calculation of NADH photo-oxidation rate at various NP concentrations . 7
7 Test report . 8
7.1 Information . 8
7.2 Report data format . 9
7.2.1 Correction factors C(i,j) obtained by actinometry (see 7.2) with λ(max,TI) . 9
7.2.2 Calibrated slope of NADH fluorescence decrease. 9
7.2.3 Plot of k versus NP concentration .10
app
7.2.4 NADH equivalent specific PCA .10
8 Precision .10
8.1 Repeatability .10
8.2 Reproducibility .10
Annex A (normative) Schematic diagram of 96-well positioning block .11
Annex B (informative) Sample calibration of UV trans-illuminator light intensity .12
Annex C (informative) Interlaboratory comparison study of TiO NP PCA .17
Bibliography .22
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.
iv © ISO 2019 – All rights reserved
Introduction
Photocatalytic activity (PCA) is the measure of capacity of a material to promote a specific photochemical
reaction under defined conditions (as defined in ISO 20507:2014, 2.3.31). With the expanding use of
nanomaterials in various industries, the possible impacts on human health and the environment due to
the enhancement of detrimental chemical reactions in the presence of light (both natural and artificial)
is an ongoing concern. The absorption of a photon with sufficient energy generates an electron-hole
pair that can migrate to the nanoparticle (NP) surface and react with water and oxygen, thus forming
extremely reactive radicals and reactive oxygen species (ROS). Generation of the ROS by some wide-
bandgap materials, such as TiO , ZnO, WO , CeO , carbon nanotubes, quantum dots and some metal
2 3 2
NPs when illuminated by UV-VIS light, can cause oxidative stress, resulting in toxic effects in living
[5]
organisms . Therefore, measuring the nanomaterial PCA under physiological conditions allows for an
assessment of its photo-toxicity potency.
Existing standard test methods for particle and surface PCA measurement (see ISO 10676 and
ISO 10678) are not directly applicable to determine nanomaterial PCA leading to photo-toxicity, as
they require a large test volume and/or long measurement duration, while utilizing organic dyes as
indicators that are not biocompatible.
The in vitro NP PCA test for NADH oxidation is intended to evaluate the nanomaterial photo-toxicity
potency when exposed to an ultraviolet (UV) light.
INTERNATIONAL STANDARD ISO 20814:2019(E)
Nanotechnologies — Testing the photocatalytic activity of
nanoparticles for NADH oxidation
1 Scope
This document specifies a method for the measurement of the photocatalytic activity (PCA) of
nanoparticles (NPs), suspended in an aqueous environment in physiologically relevant conditions, by
measuring the ultraviolet (UV)-induced nicotine adenine dinucleotide hydrate (NADH) oxidation.
The measurement is intended to assess the potential for the photo-toxicity of nanomaterials. The
method is also applicable to NP 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/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
3 Terms, definitions, symbols and abbreviated terms
For the purposes of this document, the terms and definitions given in ISO/TS 80004-1, ISO/TS 80004-2
and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1 Terms and definitions
3.1.1
actinometry
method to measure the number of photons integrally or per unit of time
3.1.2
catalytic activity
property of a component corresponding to the catalysed substance rate of conversion of a specified
chemical reaction, in a specified measurement system
[SOURCE: ISO 18153:2003, 3.2, modified — The notes have been deleted.]
3.1.3
oxidation
chemical reaction accompanying a gain of oxygen, loss of hydrogen of an organic substrate or loss of
one or more electrons from a molecular entity
3.1.4
NADH equivalent specific PCA
PCA measured as the NADH photo-oxidation (3.1.5) rate per unit weight of nanoparticles
3.1.5
photo-oxidation
oxidation reactions induced by light
3.2 Symbols and abbreviated terms
DIW deionized water with ≥ 18 MΩ·cm resistivity
NADH nicotine adenine dinucleotide hydrate
NaOH sodium hydroxide
2NB 2-nitrobenzaldehyde
NP nanoparticle
PB phosphate buffer
PCA photocatalytic activity
ROS reactive oxygen species
TI trans-illuminator
TiO titanium dioxide
UV ultraviolet
UV-Vis ultraviolet and visible
A (i,j) phenolphthalein absorbance before exposure to trans-illuminator UV irradiation in
c
each well (i = B, C, D, E, F, G; j = 2, 3, 4, …, 10, 11)
A (i,j) phenolphthalein absorbance after exposure to trans-illuminator UV irradiation in each
e
well (i = B, C, D, E, F, G; j = 2, 3, 4, …, 10, 11)
ΔA(i,j) change in phenolphthalein absorbance after exposure to trans-illuminator UV
irradiation in each well (i = B, C, D, E, F, G; j = 2, 3, 4, …, 10, 11)
ΔA average change of phenolphthalein absorbance over all wells before and after UV
a
irradiation by using a UV trans-illuminator
C starting concentration of the NP suspension for a dilution series of test solutions;
the suspension absorbance at 310 nm or 365 nm (depending on the used UV
trans-illuminator) is 1,4 < A < 1,6
C(i,j) light intensity correction factor of each well, which accounts for the UV irradiation
intensity variation of the UV trans-illuminator at the location of each well
(i = B, C, D, E, F, G; j = 2, 3, 4, …, 8, 9)
I (i,j) NADH fluorescence intensity measured before UV irradiation in each well
F,0
(i = B, C, D, E, F, G; j = 2, 3, 4, …, 8, 9)
I (i,j) NADH fluorescence intensity measured following the UV irradiation of t duration by
F,t
using a UV trans-illuminator in each well (i =
...
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