ISO 24758-2:2025

International
Standard
ISO 24758-2
First edition
Fine bubble technology —
2025-10
Evaluation method for determining
the reactive oxygen species in
ultrafine bubble dispersions —
Part 2:
APF (3'-(p-aminophenyl)
fluorescein) assay
Technologie des fines bulles — Méthode d'évaluation pour
déterminer les espèces réactives de l'oxygène dans les dispersions
de bulles ultrafines —
Partie 2: Dosage de l'APF (3'-(p-aminophenyl) fluorescein)
Reference number
© ISO 2025
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Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
3.1 Terms.1
3.2 Abbreviations and chemical formulas .2
4 Principle . 2
4.1 Reaction principle of APF .2
4.2 APF principle for distinguishing different ROS .3
5 Reagents . 5
5.1 Chemicals .5
5.2 Hydroxyl radical formation .5
5.3 Superoxide anion radical formation .5
5.4 Singlet oxygen formation .5
5.5 Hydrogen peroxide formation .5
6 Apparatus and materials . 6
7 Requirements . 6
7.1 Sample .6
7.2 Measuring instruments .6
7.3 Environment .6
8 Procedure . 6
8.1 General .6
8.2 Standard curve .7
8.2.1 Fluorescence response of APF to ·OH .7
8.2.2 Fluorescence response of APF to dissolved ozone .8
8.2.3 Fluorescence response of APF to H O .9
2 2
-
8.2.4 Fluorescence response of APF to O · .10
8.2.5 Fluorescence response of APF to O .11
8.3 ROS identification . 12
8.3.1 Fluorescence response of APF in an unknown sample . 12
8.3.2 Determine whether there are ROS types other than dissolved ozone in samples . 12
8.3.3 Determination of the presence of H O in the sample . 13
2 2
−
8.3.4 Determination of the presence of O · in the sample . 13
9 Report . 14
9.1 Report of the testing results.14
9.2 Report of the testing conditions .14
Annex A (Informative) Example of test result for existence of H O in water after combining
2 2
oxygen UFB with plasma treatment .16
Annex B (Informative) Example of test result for existence of H O in oxygen UFB water . 19
2 2
Bibliography .20

iii
Foreword
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This document was prepared by Technical Committee ISO/TC 281, Fine bubble technology.
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Any feedback or questions on this document should be directed to the user’s national standards body. A
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iv
Introduction
Recently, fine bubble technology has gained extensive application in the agricultural and environmental
fields. Investigations reveal that air microbubbles (MBs) and ultrafine bubbles (UFBs) can enhance the
growth of plants, shellfish, and yeast. Furthermore, ozone UFBs are proven effective in removing residual
pesticides from vegetables, inactivating microorganisms, and reducing organic material in wastewater.
Despite these promising outcomes, the mechanisms underpinning the fine bubble process are not entirely
understood. A key factor is the generation of reactive oxygen species (ROS).
A moderate ROS level positively influences growth, while a higher concentration effectively disinfects
pathogens and degrades pollutants in water purification systems. However, detecting the physiological
level of ROS is challenging, necessitating more sensitive methodologies. Fluorescence methodology,
complemented by suitable probes, emerges as an ideal solution for ROS detection due to its high sensitivity
and ease of data collection. Therefore, this document recommends the 3’-(p-aminophenyl) fluorescein (APF)
assay for detecting and quantifying ROS in UFB solutions.
Among ROS, the most biologically significant and extensively studied are hydroxyl radical (·OH), superoxide
·− 1
anion radical (O ), singlet oxygen ( O ), and hydrogen peroxide (H O ). The 3’-(p-aminophenyl) fluorescein
2 2 2 2
(APF) assay requires strong oxidizing power for the ipsosubstitution reaction. Given the varying oxidizing
powers of these ROS, APF’s fluorescence responses to each differ. This variability allows the differentiation
of the ROS types using APF.
In ISO 24758-1, the application of probe-based kinetic models for measuring the cumulative concentrations
of various ROS over time, as well as their real-time concentrations at each treatment interval, is described.
This document outlines methods for detecting ROS in MB and UFB dispersions using the APF assay. This
technique is highly sensitive and capable of detecting ROS at submicromolar levels in MB and UFB dispersed
liquids. Its application is crucial for evaluating MB and UFB technology’s effectiveness in agriculture and
disinfection.
v
International Standard ISO 24758-2:2025(en)
Fine bubble technology — Evaluation method for determining
the reactive oxygen species in ultrafine bubble dispersions —
Part 2:
APF (3'-(p-aminophenyl) fluorescein) assay
1 Scope
·−
This document describes the procedure for testing and evaluating the generation of ROS such as OH, O ,
H O , and O in MB and UFB dispersions using the APF assay.
2 2 3
This method is suited for systems generating ROS at physiological levels, not in large quantities, and
complements the probe kinetic
...