Foreword

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This document was prepared by Technical Committee ISO/TC 281, Fine bubble technology.

A list of all parts in the ISO 24758 series can be found on the ISO website.

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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 anion radical (O₂^·−), singlet oxygen (¹O₂), and hydrogen peroxide (H₂O₂). The 3’-(p-aminophenyl) fluorescein (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.

1 Scope

This document describes the procedure for testing and evaluating the generation of ROS such as OH, O₂^·−, H₂O₂, and O₃ in MB and UFB dispersions using the APF assay.

This method is suited for systems generating ROS at physiological levels, not in large quantities, and complements the probe kinetic model for detecting H₂O₂ in MB and UFB dispersions.

2 Normative references

3 Terms and definitions

3.1 Terms

3.2 Abbreviations and chemical formulas

4 Principle

4.1 Reaction principle of APF

4.2 APF principle for distinguishing different ROS

5 Reagents

5.1 Chemicals

5.2 Hydroxyl radical formation

5.3 Superoxide anion radical formation

5.4 Singlet oxygen formation

5.5 Hydrogen peroxide formation

6 Apparatus and materials

7 Requirements

7.1 Sample

7.2 Measuring instruments

7.3 Environment

8 Procedure

8.1 General

8.2 Standard curve

8.3 ROS identification

9 Report

9.1 Report of the testing results

9.2 Report of the testing conditions

Bibliography