ISO 23016-4:2023

Fine bubble technology - Agricultural applications - Part 4: Test method for evaluating the number concentration of ultrafine bubbles (UFB) achieving the promotion of barley seed germination

This document specifies a method to assess the ultrafine bubble (UFB) number concentration in order to find whether the number concentration of UFB generated by users is in the adequate range for promoting the barley seed germination stably irrespective of seed variety conforming to ISO 23016-2 and ISO/TR 23016-3.

Titre manque — Partie 4: Titre manque

General Information

• Status: Published
• Publication Date: 19-Dec-2023
• ICS: 07.030 - Physics. Chemistry
• ICS: 65.020.20 - Plant growing
• Technical Committee: ISO/TC 281 - Fine bubble technology
• Drafting Committee: ISO/TC 281 - Fine bubble technology
• Current Stage: 6060 - International Standard published
• Start Date: 20-Dec-2023
• Due Date: 10-Nov-2025
• Completion Date: 20-Dec-2023

Overview

ISO 23016-4:2023 - Fine bubble technology - Agricultural applications - Part 4 defines a standardized test method to evaluate ultrafine bubble (UFB) number concentration needed to stably promote barley seed germination irrespective of variety. The standard helps users verify that UFB water produced by their generation system falls within an effective concentration range for accelerating germination, reducing variability between seed varieties and ensuring reproducible outcomes.

Key topics and technical requirements

• Scope: Assessment of air UFB water and the UFB generating system to confirm number concentrations that promote barley seed germination in line with ISO 23016‑2 and ISO/TR 23016‑3.
• Sample and water quality: Use of distilled raw water meeting ISO 23016‑2 quality (A2) and UFB water stored/transported per ISO 21255.
• Measurement of UFBs: Size, quantity and concentration must be measured. The document references particle tracking analysis (PTA) instrumentation examples (measuring range ~50 nm–1 000 nm) and analysis software settings for reproducible counts.
• Test approach: Generate two UFB waters with different number concentrations (UFB1 > UFB2). Measure germination kinetics by fitting S‑curve regression and determining median germination time (T50) for each treatment and control.
• Decision logic: Three outcome cases guide selection:
  • Case 1: UFB1 produces greater acceleration than UFB2 and both beat control - choose UFB2 to avoid potential negative effects at higher concentrations.
  • Case 2: UFB1 and UFB2 give similar promotion - both reach upper effective range; choose UFB2.
  • Case 3: Higher UFB1 suppresses germination versus UFB2 - indicates excessive concentration; select the lower concentration.
• Statistical validation: Use regression-derived T50 and 95% confidence intervals (sum of squared residuals) to determine significant differences between treatments.

Applications and users

Practical applications include:

• Optimization and validation of UFB generation systems used in seed priming and nursery operations.
• Research on fine bubble agricultural efficacy and dose–response across barley varieties.
• Quality assurance for agri‑tech providers, seed companies and agronomists integrating UFB treatments into production.
• Regulatory bodies and certification laboratories developing reproducible test results.

Who will use it:

• UFB generator manufacturers, agronomists, seed technologists, agricultural researchers, and QA labs seeking standardized methods to quantify UFB number concentration for consistent barley germination outcomes.

Related standards

• ISO 23016‑2:2019 - Test method for evaluating promotion of barley seed germination
• ISO/TR 23016‑3 - Guidelines for minimum viable UFB number concentration for promotion
• ISO 21255 - Storage and transportation of UFB dispersion in water

Keywords: ISO 23016-4:2023, fine bubble technology, ultrafine bubbles, UFB number concentration, barley seed germination, test method, agricultural applications, particle tracking analysis (PTA).

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 23016-4
First edition
2023-12
Fine bubble technology — Agricultural
applications —
Part 4:
Test method for evaluating the
number concentration of ultrafine
bubbles (UFB) achieving the
promotion of barley seed germination
Reference number
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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ISO copyright office
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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
4 Requirements for UFB water used . 1
5 Basic characteristics of UFB water relevant to promotion of barley seed
[1]
germination . 2
[1]
6 Assessment of the adequacy of UFB number concentration . 3
6.1 General . 3
6.2 Case 1: UFB1 water results in greater promotion effect than UFB2 water . 3
6.3 Case 2: UFB1 and UFB2 waters result in similar promotion effect . 5
6.4 Case 3: UFB1 water results in suppression of germination comparing to UFB2
water . 7
7 Test report . 9
Annex A (informative) Example of measured data of UFB’s promotion effect on another
variety of barley seed (Ichibanboshi) than that of ‘Yumesakiboshi’ .10
Annex B (informative) Attributes (storage duration and storage temperature) of seeds
that can affect the degree of seed germination .11
Bibliography .12
iii
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 23016 series can be found on the ISO website.
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
Introduction
Fine bubble (FB) technology is increasingly attracting attention in numerous different fields. In the
agriculture and aquaculture fields, ultrafine bubbles (UFBs) have been widely and practically used to
accelerate the growth of plants and fish (air or oxygen UFBs). However, a lot of cases reported in recent
years are field data, some of which are not supported statistically.
In the light of this situation of scientifically insufficient information on fine bubble technology in
agriculture, this document has been developed to establish standards in this area focusing on a
method to find an adequate number concentration of UFBs demonstrating the effect for promoting the
germination of barley seeds.
A test method for the promotion of the germination of barley seeds has been published as ISO 23016-2
and a guideline of the minimum viable number concentration of UFBs for promoting the germination
of barley seeds has been published as ISO/TR 23016-3. Successive accumulation of data, however,
revealed that a positive/negative effect on germination appears depending on the variety of barley
seed. Therefore, a method to assess the UFB number concentration which assures the promotion of
germination irrespective of variety is needed for popularizing this technology.
v
INTERNATIONAL STANDARD ISO 23016-4:2023(E)
Fine bubble technology — Agricultural applications —
Part 4:
Test method for evaluating the number concentration of
ultrafine bubbles (UFB) achieving the promotion of barley
seed germination
1 Scope
This document specifies a method to assess the ultrafine bubble (UFB) number concentration in order
to find whether the number concentration of UFB generated by users is in the adequate range for
promoting the barley seed germination stably irrespective of seed variety conforming to ISO 23016-2
and ISO/TR 23016-3.
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 23016-2:2019, Fine bubble technology — Agricultural applications — Part 2: Test method for evaluating
the promotion of the germination of barley seeds
ISO/TR 23016-3, Fine bubble technology — Agricultural applications — Part 3: Guidelines for the minimum
viable number concentration of ultrafine bubbles for promoting the germination of barley seeds
ISO 21255, Fine bubble technology — Storage and transportation of ultrafine bubble dispersion in water
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 23016-2 and ISO/TR 23016-3
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/
4 Requirements for UFB water used
The items subject to the test shall be air UFB water stored in bottles or other containers for preservation
and transport in accordance to ISO 21255, and the UFB generating system used to generate UFB water.
UFB water shall be generated by supplying raw water to the UFB generating system. Distilled water
1)
with a quality of A2 or greater shall be used as raw water according to ISO 23016-2.
The size, quantity and concentration of UFB in UFB water shall be measured. For the generation of air
UFB in water, a pressure dissolution system, whose pressure just after the pressurizing pump is around
700 kPa and that at the saturator is around 300 kPa was used. For measurement, a commercial device
1) Electrical conductivity 0,1 mS/S (25 °C), total organic carbon (TOC) 0,5 mgC/l or less, zinc 0,5 µgZn/l or less,
- 2-
silica 50 µgSiO /l or less, chloride ions µgCl /l, sulfide ions µgSO /l.
2 4
using particle tracking analysis (PTA) was used, whose measuring range is from 50 nm to 1 000 nm,
wave length of laser light source is 635 nm, power is 40 mW, mounted camera is black and white CCD
and analysis software is NTA 3.1 Build 3.1.46. Setting parameters are given in Annex A. Measuring
temperature was at a room temperature around 22 °C.
The UFB generating system shall be capable of supplying the UFB water described in ISO 23016-2.
5 Basic characteristics of UFB water relevant to promotion of barley seed
[1]
germination
Barley seeds of the variety ‘Kobinkatagi’ are used as a baseline example and they are selected according
to the test procedure in ISO 23016-2.
Hollow circles in Figure 1 show the observed germination ratio at each observation time of seeds in
control water (distilled water) and its regression curve (S-curve) is drawn in dashed line by using
ISO 23016-2:2019, Formula (2). In this case, median germination time, T -control, was 16,5 h.
Hollow rectangle, solid diamond, hollow triangle and cross symbols in Figure 1 show the observed
germination ratios at each observation time of which number concentrations of UFB1, UFB2, UFB3 and
8 8 8 8
UFB4 are 8,5×10 /ml, 5,0×10 /ml, 3,8×10 /ml and 1,4×10 /ml and T -UFB1, T -UFB2, T -UFB3 and
50 50 50
T -UFB4 are 13,1 h, 13,7 h, 14,1 h and 14,3 h, respectively. Each of the four regression curves is drawn
in solid line in Figure 1.
These experimental observations indicate that the relationship between T -UFB and T -control is
50 50
given as T -UFB 50 50
when number concentration of UFB is in the adequate range for promoting seed germination. Thus, the
basic characteristics of UFB water appears in the way that the effect o
...