ISO/TR 23016-5:2024

Technical
Report
ISO/TR 23016-5
First edition
Fine bubble technology —
2024-08
Agricultural applications —
Part 5:
Practical data collection to promote
the germination of typical vegetable
seeds using ultrafine bubbles
Technologie des fines bulles — Applications agricoles —
Partie 5: Collecte de données pratiques pour favoriser la
germination de graines de légumes types par l'utilisation de
bulles ultrafines
Reference number
© ISO 2024
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Test subjects . 1
5 Judgement of significant difference in T . 2
50in
6 Seeds and measurement device . . 2
6.1 Seeds for germination test .2
6.2 Measurement device for UFB size and concentration .2
6.3 Examination range of UFB number concentration for vegetable seeds and adjustment
of dissolved oxygen concentration (DO) .2
7 Test data . 3
7.1 General .3
7.2 Carrot seeds of positive photoblastic .3
7.3 Tomato seeds of negative photoblastic .4
7.4 Spinach seeds of neutral photoblastic .5
7.5 Effect of DO (Dissolved oxygen concentration) .6
7.6 Effect of UFB number concentration . .7
7.7 Presence or absence of seed size effect on the promotion by UFB .8
7.7.1 General .8
7.7.2 Carrot seed of comparatively large size .8
7.7.3 Carrot seed of comparatively small size .9
8 Suggestion . 10
Annex A (informative) Example of measured UFB data of size distribution and number
concentration of UFB used in this document .11
Annex B (informative) 95 % confidence intervals of T values corresponding to Figures 1 to 7. 14
Bibliography .21

iii
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
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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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
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rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
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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
The market for technologies using fine bubbles has been rapidly growing in many applications throughout
the industrial, domestic, and academic sectors. Especially, the application of ultrafine bubble (UFB)
technology to the agricultural area is arousing great interest. It is thought to be one of the advanced
technologies improving the productivity and efficiency of agriculture, thereby contributing to the United
Nations Sustainable Development Goals (SDGs) by means of providing sufficient food and maintaining water
resources on land, as an example.
In this context, ISO 23016-2:2019 has been published, describing the test method for promoting barley seed
germination by application of a UFB water generation system. Although the promotion of germination of
barley seed is closely related to UFB number concentration, effects of the concentration were not specified
at that time.
Thus, in order to provide users with a guideline for selecting an appropriate UFB generation system,
ISO/TR 23016-3:2021 has been published to indicate the minimum viable number concentration of ultrafine
bubbles that promotes the germination of barley seeds.
Furthermore, based on the continual accumulation of data revealing positive and negative effects of UFB on
germination depending on the variety of barley seeds, ISO 23016-4 has been published to provide a method
to evaluate the UFB number concentration ensuring the promotion of germination of the barley seeds
without taking into account their varieties.
This document describes the data collected from the experimental observations on applying UFB water to
promote the germination of vegetable seeds, based on ISO 23016-2, ISO/TR 23016-3 and ISO 23016-4. It was
developed to support the application of the UFB technology to vegetables specially grown in hydroponic
culture system, which market is quite large and still growing.

v
Technical Report ISO/TR 23016-5:2024(en)
Fine bubble technology — Agricultural applications —
Part 5:
Practical data collection to promote the germination of
typical vegetable seeds using ultrafine bubbles
1 Scope
This document provides practical data collection of promoting the germination of typical vegetable seeds by
applying ultrafine bubbles (UFBs) within the effective number concentrations for barley seeds specified in
ISO 23016-2, ISO/TR 23016-3 and ISO 23016-4. While the application of UFB to barley seeds is systematically
standardized, reports on UFB application to vegetable seeds germination are scattered worldwide.
Therefore, this document intends to illustrate the effectiveness of UFBs to promote the germination of
vegetable seeds depending on their response to light, i.e.:
a) require light to germinate (positive photoblastic),
b) require darkness to germinate (negative photoblastic), and
[1]
c) neutral to light .
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, 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 23016-4, 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
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 23016-2, ISO/TR 23016-3 and
ISO 23016-4 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 Test subjects
The items subjected to the test are air UFB water stored in bottles or other containers for preservation and
transport in accordance with ISO 21255, and the UFB generating system used to generate the UFB water.

UFB water is generated by supplying raw water to the UFB generating system. Distilled water with a quality
of Grade 2 according to ISO 3696 or greater is used as raw water that is distilled water supplied as a raw
material for both UFB water and control water according to ISO 23016-2:2019, 3.6.
The size, quantity and concentration of UFB in UFB water were measured. Examples of measured data are
given in Annex A.
5 Judgement of significant difference in T
50in
After determining the correlation curve using ISO 23016-2:2019, Formula (2), the sum squared of residual
(SSR) and standard error (SE) around the time T are calculated. From this curve, the 95 % confidence
interval of T is determined. The results are shown in Figures B.1 to B.7 in Annex B.
6 Seeds and measurement device
6.1 Seeds for germination test
The following vegetable seeds of different responses to light were used as the seeds for the germination test:
a) require light to germinate (positive photoblastic),
b) require darkness to germinate (negative photoblastic), and
[1]
c) neutral to light (neutral photoblastic) .
It is not necessary to rinse the seeds in water before use; the test is initiated using the dry seeds as supplied.
The germination test was conducted at the constant temperature of 25 °C.
6.2 Measurement device for UFB size and concentration
The UFB generating system is capable of supplying the UFB water described in ISO 23016-2. The air UFB
water samples had their size and concentration measured using a particle tracking analysis instrument (see
ISO 19430:2016). The size, quantity and concentration of UFB in UFB water were measured. Measurements
were carried out at a room temperature around 22 °C.
6.3 Examination range of UFB number concentration for vegetable seeds and adjustment of
dissolved oxygen concentration (DO)
As the UFB generation system given in ISO 23016-2 stably generates UFB in the number concentration
7 8 7 8
ranging from 10 /ml, 10 /ml and greater, the UFB in the range from 10 /ml to 10 /ml was applied to
vegetable seeds to ensure the effect of germination promotion. The air UFB number concentration in this
range is known to be within the range that can be measured by the commercially available measurement
device in 6.2.
Although an example of size distribution and number concentration of UFB used for barley seed germination
is given in ISO/TR 23016-3:2021, Annex A, this case illustrates small-scale fluctuation. In this document,
examples with fluctuation to some extent are shown in Figure A.1 for UFB number concentration of 1,5 × 10 /
ml and Figure A.2 for UFB number concentration of 1,1 × 10 /ml.
The DO of water containing UFBs was adjusted to be the same as that of control water by introducing air
[2]
and nitrogen gases through a mixed-gas flow regulator in order to observe only the effect of UFBs on
vegetable seed germination by cutting out the effect of DO.

7 Test data
7.1 General
This document covers the following studies:
— The promotion effect on the germination for each of three types of seeds, which is described in 7.2 to 7.4.
— The effects of DO and number concentration of UFB on the germination promotion, which are described
in 7.5 and 7.6.
— An effect of the seed size on the germination with using UFB is described in 7.7. In order to confirm the
significant differences of T values between UFB and control sections, 95 % confidence intervals of T
50 50
of UFB and control section were calculated and shown as Figures B.1 to B.7, each of those is relating to
Figures 1 to 7.
7.2 Carrot seeds of positive photoblastic
The analysis result of germination processes of carrot seeds is shown in Figure 1 indicating the
germination promotion effect of UFB. The number concentration of UFB was 4,4 × 10 /ml. Dissolved oxygen
concentration (DO) was adjusted to 8,0 mg/l for both UFB and control water. A remarkable improvement at
the final germination ratio of 59,5 % in UFB section was observed compared to 38,5 % for control section.
Furthermore, 95 % confidence intervals of T of UFB and control section were not overlapped as shown in
Figure B.1. Thus, the promotion effect of UFB was verified on the germination of carrot seed which requires
light for germination (positive photoblastic).

Key
data at control section
data at UFB section
T measurement time, expressed in hours
G germination ratio, expressed in per cent
A regression curve of germination ratio of seeds in UFB water
B regression curve of germination ratio of seeds in control water
T crossing time with 50 % of maximum germination ratio and the curve of control section, expressed in hours
50, Control
T crossing time with 50 % of maximum germination ratio and the curve of UFB section, expressed in hours
50, UFB
Figure 1 — Promotion effect of UFB with number concentration of 4,4 × 10 /ml on germination of
carrot seeds when DO of both control and UFB water is adjusted to 8,0 mg/l
7.3 Tomato seeds of negative photoblastic
The analysis result of germination processes of tomato seeds is shown in Figure 2 indicating the germination
promotion effect of UFB. The number concentration of UFB was 1,1 × 10 /ml. Dissolved oxygen concentration
(DO) was adjusted to 7,6 mg/l to 7,7 mg/l for both UFB and control water. A remarkable improvement in the
final germination ratio of 97,0 % for UFB section was observed compared to 79,0 % for control section.
Furthermore, 95 % confidence intervals of T of UFB and control section were not overlapped as shown in
Figure B.2. Thus, the promotion effect of UFB was verified on the germination of tomato seed which requir
...