Stability Evaluation of Oxyhydrogen and Hydrogen Nanobubbles Under Thermal and pH Stress Conditions

Stability Evaluation of Oxyhydrogen and Hydrogen Nanobubbles Under Thermal and pH Stress Conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Stability Evaluation of Oxyhydrogen and Hydrogen Nanobubbles Under Thermal and pH Stress Conditions Veranica Agustin, Aditya Hernowo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7269183/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 14 You are reading this latest preprint version Abstract Nanobubbles (NBs), gas-dispersed systems < 200 nm in size, are increasingly explored for therapeutic and drug delivery applications but are highly sensitive to environmental conditions. This study evaluated the stability of nanobubbles generated from oxyhydrogen (HHO) and pure hydrogen (H₂) gases under thermal and pH stress. HHO nanobubbles were subjected to heating (80°C and 100°C) and freezing (− 17°C), while H₂ nanobubbles were stored at pH 4–9 for seven days. Particle size (mode, mean, D50) and concentration were measured using Nanoparticle Tracking Analysis (NTA). HHO nanobubbles showed significant degradation at 100°C, with reduced concentration and increased particle size, whereas freezing caused moderate instability. H₂ nanobubbles were most stable at neutral pH (6–7), maintaining size and concentration, while highly acidic (pH 4) and alkaline (pH 9) conditions accelerated particle disintegration. Neutral pH (6–7) is optimal for nanobubble stability, while extreme temperatures (100°C and freezing) markedly reduce stability. These findings provide insights into designing nanobubble formulations with enhanced environmental resilience for clinical applications such as therapeutic gas infusion and advanced drug delivery. Physical sciences/Chemistry Physical sciences/Materials science Physical sciences/Nanoscience and technology Nanobubble Oxyhydrogen Hydrogen Stability pH Temperature Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nanobubbles (NBs) are ultra-small gas bubbles, typically < 200 nm in size, possessing unique physicochemical characteristics such as high internal pressure, large interfacial area, and exceptional stability in liquid media [ 1 ]. These unique properties make NBs highly promising for various biomedical and therapeutic applications, including active substance delivery systems, medical gas carriers, and infusion media for regenerative tissue stimulation [ 2 ]. However, nanobubble stability is not absolute; it largely depends on the surrounding environmental conditions, particularly solution temperature and pH. Changes in temperature can increase kinetic energy and reduce surface tension, whereas extreme pH may alter surface charge and interfacial interactions. The combination of these factors can accelerate bubble coalescence, disintegration, or gas release from nanobubble particles [ 3 ]. Understanding how nanobubbles respond to environmental stress is therefore a critical step in optimizing formulations for both clinical and pharmaceutical applications. In this study, two primary approaches were employed: temperature treatment (including heating and freezing) and pH treatment (pH 4 to 9) to evaluate the physical stability of nanobubbles formed using two different gases, namely HHO (oxyhydrogen mixture) and pure H₂. The selection of HHO gas for temperature treatment testing was based on its highly reactive nature and ease of dissociation when exposed to thermal energy. HHO is the result of water electrolysis containing fractions of O₂ and H₂ in an explosive mixture (commonly referred to as Brown’s gas), which theoretically generates higher internal pressure within the bubble structure compared to single-gas nanobubbles. This property makes HHO an ideal model system to evaluate the physical interfacial resistance of nanobubbles under extreme thermal stress such as boiling and long-term freezing. As temperature increases or water freezes, differential pressure from gases within HHO bubbles is expected to exhibit more dramatic changes, thereby revealing the physical stability limits of NB systems [ 4 ]. In contrast, pure H₂ gas was selected as the model for pH stability testing due to its inert, small nature and non-reactivity with H⁺ or OH⁻ ions. This prevents gas-related interference with chemical changes in the solution, ensuring that any observed changes in nanobubble size or concentration can be attributed solely to pH effects rather than gas reactivity. Moreover, hydrogen molecules are widely recognized in medical literature as therapeutic gases with notable antioxidant and anti-inflammatory properties and are extensively used in infusion therapy in various preclinical studies [ 2 , 5 ]. Therefore, evaluating the stability of hydrogen nanobubbles across both biological and extreme pH ranges is an important step toward validating their potential in safe and precise medical gas delivery systems. To assess the impact of temperature and pH treatments on nanobubble physical parameters, measurements were performed using Nanoparticle Tracking Analysis (NTA). This technique enables direct monitoring of particle size distribution (mode, mean, and median) as well as particle concentration in solution. Using this approach, the present study aims to provide a strong scientific foundation for identifying optimal parameters to maintain nanobubble physical stability, supporting the development of NB-based formulations resistant to environmental variations, and opening opportunities for applications in gas therapy, regenerative medicine, and nanobubble-based drug delivery systems. Methods Materials Oxyhydrogen nanobubble (HHO) solution prepared in reverse osmosis (RO) water was used for temperature treatments, while hydrogen nanobubble (H₂) solution prepared in RO water was used for pH treatments. To maintain pH stability during sample preparation and analysis, a buffer consisting of 1% ascorbic acid and 1% sodium bicarbonate was employed. Phosphate-buffered saline (PBS) was used as the diluent prior to analysis using Nanoparticle Tracking Analysis (NTA) to ensure that particle concentrations fell within the instrument’s detection range. Additionally, sodium dodecyl sulfate (SDS) and Water One were used as rinsing solutions to clean the NTA instrument before and after each measurement, thereby preventing cross-sample contamination. All materials were prepared under sterile conditions and in accordance with laboratory protocols to ensure the integrity of the research data. Experimental Design Thermal and Freezing Treatments (HHO Nanobubbles) This study utilized four types of oxyhydrogen nanobubble (HHO NB) samples subjected to different conditions to evaluate the effects of temperature treatment on the physicochemical characteristics of nanobubbles. The four sample groups were as follows: RO : Post-production sample without any additional treatment (baseline). RO T80C : HHO NB sample heated at 80°C for 10 minutes. RO T100C : HHO NB sample heated at 100°C for 10 minutes. RO FREEZE : HHO NB sample frozen in a standard freezer (approximately − 17°C) for 192 hours (8 days). pH Treatments (Hydrogen Nanobubbles) Hydrogen nanobubble (H₂ NB) samples were adjusted to target pH values (4–9) using 1% ascorbic acid and 1% sodium bicarbonate solutions. All samples were stored for 0, 1, 3, 5, and 7 days under refrigerated conditions (approximately 4°C). Table 1 pH Buffer Composition for Hydrogen Nanobubble Solution Treatments Target pH Ascorbic Acid 1% (µL) Sodium Bicarbonate 1% (µL) H₂ Volume (µL) Description 4 600 0 19,400 Strongly acidic 5 400 0 19,600 Moderately acidic 6 200 0 19,800 Slightly acidic 7 0 0 20,000 Neutral (control) 8 0 200 19,800 Slightly alkaline 9 0 400 19,600 Moderately alkaline Table 1 presents the variation in volumes of 1% ascorbic acid and 1% sodium bicarbonate used to produce solutions with specific target pH values (pH 4 to pH 9). Each solution was prepared to a fixed total volume of 20 mL using hydrogen nanobubble water as the solvent, adjusted to achieve the desired pH value. Descriptive annotations are provided to categorize the level of acidity or alkalinity of each mixture. Measurement Parameters All nanobubble characteristics, including those subjected to thermal and pH treatments, were measured using the NanoSight Pro system (Malvern Panalytical, UK), based on Nanoparticle Tracking Analysis (NTA) technology. Analyses were performed at room temperature, precisely controlled using a Peltier module, and employed a Low Volume Flow Cell (LVFC) to ensure stable flow and accurate detection. The instrument used was the NS XPLORER (Malvern Panalytical), equipped with a 405 nm violet laser for nano-sized particle detection. Prior to analysis, all samples were diluted 50-fold with phosphate-buffered saline (PBS) to ensure that particle concentrations were within the optimal detection range. Measured parameters included mode (dominant size), mean (average size), D50 (median diameter), and particle concentration (particles/mL). Camera settings such as gain, shutter speed, and laser intensity were optimized according to the particle type under observation, including adjustment for the dilution factor. Measurements were conducted using the following settings: five captures per sample, temperature at 25°C, 3,000 frames per capture, and a flow rate of 30 µL/min. Each sample was analyzed in quintuplicate to obtain mean values and standard deviations, thereby increasing data validity and minimizing measurement error. Results Thermal and Freezing Treatments on Oxyhydrogen Nanobubbles (HHO) Table 2 presents the analysis results of nanobubble size and concentration formed in reverse osmosis (RO) water following high-temperature treatments (80°C and 100°C) and freezing. The measured parameters included mode, mean, standard deviation, size distribution (D10, D50, D90), and particle concentration. The data demonstrate variations in nanobubble size and concentration as a result of temperature changes, with high-temperature treatment generally increasing the mean particle size and decreasing particle concentration. Sample Mode (nm) Mean (nm) Standard Deviation (nm) D10 (nm) D50 (nm) D90 (nm) Concentrations (p/ml) RO 85.0 249 226 74 175 502 7.41 × 10 9 RO T80C 82.5 155 156 56 107 308 7.31 × 10 9 RO T100C 122.5 262 244 94 195 473 4.83 × 10 9 RO FREEZE 112.5 213 184 86 151 401 7.12 × 10 9 Table 2. Characteristics of Nanobubbles Under Thermal and Freezing Treatments Using HHO Gas Figure 1 illustrates the changes in mean particle size and median (D50) across different temperature treatments. The data clearly indicate that heating at 100°C significantly increases particle size, whereas heating at 80°C reduces both mean and median sizes compared to the control. The freezing treatment results in moderate size alterations relative to untreated samples. Figure 2 demonstrates the concentration changes of HHO nanobubbles after exposure to different thermal conditions. It is evident that heating at 100°C markedly decreases particle concentration, whereas the 80°C heating and freezing (-17°C for 8 days) treatments maintained concentrations comparable to the control. pH Treatments on Hydrogen Nanobubbles (H₂) This section evaluates the effects of pH variation on hydrogen nanobubbles (H₂). Samples were adjusted to target pH values (4–9) and stored for up to seven days to observe changes in particle size distribution and concentration. The results highlight how acidic, neutral, and alkaline environments influence nanobubble stability, providing insights into optimal conditions for maintaining particle integrity and functional performance. PH Day Mode (nm) Mean (nm) SD (nm) D10 (nm) D50 (nm) D90 (nm) Concentration (p/ml) 4 0 112.5 220 332 74 147 377 1.29 + 10 9 4 1 112.5 267 396 81 171 500 9.55 + 10 8 4 3 118.5 289 469 86 214 764 9.25 + 10 8 4 5 127.5 372 477 84 218 817 9.18 + 10 8 4 7 127.5 365 459 82 162 645 8.05 + 10 8 Table 3. Changes in Nanobubble Size and Concentration in pH 4 Solution During 7 Days of Storage Table 4 shows that at pH 5, a gradual increase in particle size and a decrease in concentration occurred throughout the incubation period. Although the degradation was not as rapid as at pH 4, these results indicate that moderately acidic conditions still progressively affect nanobubble stability, representing a transitional phase toward a more stable state. PH Day Mode (nm) Mean (nm) SD (nm) D10 (nm) D50 (nm) D90 (nm) Concentration (p/ml) 5 0 82.5 231 341 65 139 463 1.11 + 10 9 5 1 97.5 257 461 70 137 448 8.99 + 10 8 5 3 82.5 297 465 66 136 669 8.25 + 10 8 5 5 97.5 307 647 67 133 658 7.72 + 10 8 5 7 87.5 311 590 66 146 557 6.93 + 10 8 Table 4. Changes in Nanobubble Size and Concentration in pH 5 Solution During 7 Days of Storage Table 5 shows that particle size (mode and mean) remained stable and particle concentration stayed high up to day 7 at pH 6. This indicates that pH 6 is a relatively optimal zone for maintaining nanobubble structure and population, with minimal interfacial disruption by environmental ions. PH Day Mode (nm) Mean (nm) SD (nm) D10 (nm) D50 (nm) D90 (nm) Concentration (p/ml) 6 0 82.5 160 205 58 114 290 2.16 + 10 9 6 1 82.5 177 184 68 130 318 2.13 + 10 9 6 3 82.5 147 148 55 102 299 1.78 + 10 9 6 5 82.5 214 234 67 143 429 1.71 + 10 9 6 7 82.5 225 372 63 134 422 1.73 + 10 9 Table 5. Changes in Nanobubble Size and Concentration in pH 6 Solution During 7 Days of Storage Table 6 shows that at pH 7 (neutral), nanobubble size and concentration tended to remain stable with minimal fluctuations. The mode diameter consistently remained around 82.5 nm, and the decrease in concentration occurred gradually, indicating that pH 7 is an optimal condition for short-term stability. PH Day Mode (nm) Mean (nm) SD (nm) D10 (nm) D50 (nm) D90 (nm) Concentration (p/ml) 7 0 82.5 236 345 66 131 488 1.46 + 10 9 7 1 82.5 193 276 62 123 358 1.42 + 10 9 7 3 82.5 220 282 72 139 453 1.37 + 10 9 7 5 87.5 193 219 67 130 371 1.35 + 10 9 7 7 82.5 220 241 62 142 493 1.32 + 10 9 Table 6. Changes in Nanobubble Size and Concentration in pH 7 (Baseline) Solution During 7 Days of Storage Table 7 shows that at pH 8 (slightly alkaline), particle size remained relatively constant, and particle concentration remained stable up to day 5. This indicates fairly good stability, although a declining trend began to appear toward the end of the storage period. PH Day Mode (nm) Mean (nm) SD (nm) D10 (nm) D50 (nm) D90 (nm) Concentration (p/ml) 8 0 97.5 229 395 70 142 380 1.42 + 10 9 8 1 97.5 238 393 71 139 454 1.46 + 10 9 8 3 97.5 233 255 66 159 454 1.35 + 10 9 8 5 87.5 223 326 90 156 394 1.35 + 10 9 8 7 82.5 247 298 64 135 379 1.25 + 10 9 Table 7. Changes in Nanobubble Size and Concentration in pH 8 Solution During 5 Days of Storage Table 8 shows that at pH 9 (moderately alkaline), nanobubbles exhibited slight fluctuations but remained within a stable range. Particle concentration decreased slightly from day 5 to day 7, indicating a moderate potential for long-term instability. PH Day Mode (nm) Mean (nm) SD (nm) D10 (nm) D50 (nm) D90 (nm) Concentration (p/ml) 9 0 82.5 228 360 64 129 448 1.04 + 10 9 9 1 82.5 256 594 75 142 358 1.17 + 10 9 9 3 82.5 234 420 63 127 449 1.20 + 10 9 9 5 87.5 196 254 67 139 337 1.19 + 10 9 9 7 87.5 194 211 60 128 389 1.11 + 10 9 Table 8. Changes in Nanobubble Size and Concentration in pH 9 Solution During 5 Days of Storage Figure 3 shows a trend of increasing dominant particle size, indicating agglomeration caused by interfacial disruption from H⁺ ions. In contrast, pH 6 and 7 remained stable at approximately 82.5 nm, indicating the most optimal conditions. pH 5, 8, and 9 exhibited slight fluctuations, reflecting transitional states or moderate ionic stress affecting nanobubble stability. Figure 4 shows that highly acidic environments (pH 4 and 5) caused a significant increase in the average size of nanobubble particles, indicating agglomeration or coalescence due to interfacial disruption by H⁺ ions. In contrast, pH 6 and 7 maintained relatively small and stable particle sizes, indicating optimal stability under neutral conditions. In alkaline environments (pH 8–9), fluctuations were minor but still demonstrated a gradual tendency toward reduced stability. Figure 3 shows that pH 4 experienced a significant increase in D50 up to day 5, indicating substantial agglomeration under highly acidic conditions. In contrast, pH 6 and 7 maintained low and stable D50 values, demonstrating stable particle size distribution. Meanwhile, pH 5, 8, and 9 exhibited slight fluctuations, reflecting transitional states or more moderate interfacial disturbances. Figure 6 shows that nanobubble concentration decreased most drastically at pH 4 and 5, indicating rapid disintegration under highly acidic conditions. In contrast, pH 6 and 7 maintained high and stable concentrations, demonstrating that neutral environments are most favorable for stability. At pH 8 and 9, a gradual decrease was observed, reflecting the long-term destabilizing effects of OH⁻ ions. Interestingly, the Indonesian Molecule Institute (IMI) had previously succeeded in producing HHO gas-based nanobubbles with an exceptionally high concentration of 2.99 × 10¹⁰ particles/mL, far exceeding the concentration range observed in this study. This achievement highlights the significant potential of local technology in generating high-density nanobubble formulations, which theoretically can enhance the efficiency of active substance delivery and improve particle stability in clinical and regenerative applications. Discussion The results of this study clearly demonstrate that the physical stability of nanobubbles (NBs) is strongly influenced by two primary factors: the type of gas used for their formation and external environmental conditions, particularly solution temperature and pH. These factors directly affect particle size, size distribution, and the concentration of nanobubbles retained in the solution during storage. In the group of nanobubbles generated using HHO gas (oxyhydrogen), exposure to high temperatures had a significant impact on the physical integrity of the NBs. Specifically, heating to 100°C caused a substantial increase in both mean and median particle sizes, accompanied by a drastic reduction in particle concentration. This phenomenon is most likely due to increased kinetic energy, which triggers the expansion of the nanobubble’s internal volume, thereby facilitating particle coalescence and ultimately causing the rupture of trapped gas bubbles. This process accelerates gas release into the surrounding environment and reduces the number of detectable particles in the system [ 6 ]. Meanwhile, freezing treatment for eight days at − 17°C also resulted in decreased concentration and changes in particle size, which can be explained by physical disruption caused by ice crystal formation. Such crystallization likely damages the nanobubble interfacial structure, breaking its protective layer and eliminating the ability of NBs to maintain internal pressure. Conversely, nanobubbles generated using pure hydrogen gas (H₂) exhibited greater resistance to pH variations, particularly within the neutral pH range (6–7). Under these conditions, NB physical parameters—such as mode, mean, and median diameters—remained stable throughout the seven-day storage period. Particle concentration also remained relatively high, indicating that a neutral environment provides an optimal balance between interfacial tension and electrostatic repulsion, thereby maintaining nanobubble structural integrity and homogeneous distribution. These findings confirm previous reports [ 7 , 8 ], which stated that nanobubble stability is highest within the physiological pH range. Under highly acidic conditions (pH 4), particle size increased sharply over time, while NB concentration decreased drastically from the first day. This reflects a rapid disintegration process most likely triggered by the high concentration of H⁺ ions. These ions interact with the NB interfacial layer, disrupting surface stability and promoting gas release as well as particle coalescence [ 9 ]. At pH 5, although degradation was not as rapid as at pH 4, there was still a clear tendency for size fluctuations and a decline in particle concentration, indicating that NB stability was in a transitional zone toward optimal conditions. Meanwhile, in alkaline environments (pH 8–9), nanobubbles exhibited moderate stability. Particle size showed slight fluctuations, and particle concentration began to decrease from day 3 to day 7. This may be attributed to the effect of OH⁻ ions on interfacial tension and surface charge distribution, which, over time, can reduce internal NB pressure and accelerate gas diffusion out of the particles. Overall, these findings highlight that the pH range of 6–7 is optimal for maintaining NB size and concentration stability in liquid media. This is particularly important in therapeutic applications, especially when NBs are used as carriers for active substances or medical gases that require medium- to long-term stability. The reduction in stability under extreme conditions indicates that NB formulations for medical applications must take into account both storage and physiological environments. Moving forward, these results open opportunities for further exploration, such as the incorporation of stabilizers, surface coatings, or the development of core–shell nanobubbles that are more resistant to environmental variations. Furthermore, in the context of local production in Indonesia, these findings provide an important scientific foundation for the development of more standardized HHO- and H₂-based nanobubble technologies that are ready for application in clinical and regenerative therapies. This experiment evaluated the stability of nanobubbles generated from two gas types, HHO and H₂, under thermal treatment, freezing, and pH variation. Results obtained using Nanoparticle Tracking Analysis (NTA) showed that each treatment produced different impacts on size parameters (mode, mean, median/D50) and particle concentration, reflecting the physical stability of nanobubbles in aqueous media. The use of HHO gas for thermal treatment and H₂ gas for pH evaluation was based on the physical properties and availability of these gases in the laboratory system. HHO, consisting of a oxyhydrogen mixture produced through water electrolysis, can be generated in large quantities and is more stable for extreme temperature testing. In contrast, pure H₂ gas was used for pH studies due to its potential as a redox therapeutic agent sensitive to environmental conditions, making it suitable for assessing stability in varying pH environments. Overall, this study demonstrates that nanobubble stability is highly dependent on environmental treatments, with gas type selection and external conditions being key factors in maintaining their functional effectiveness. In HHO nanobubbles, exposure to high temperatures such as heating to 80°C and 100°C resulted in a significant decrease in particle concentration, particularly at 100°C. Boiling the sample for 10 minutes caused many nanobubbles to disappear, as indicated by reduced concentration and increased average particle size. This is presumed to be due to the high kinetic energy triggering gas expansion and coalescence, ultimately leading to bubble rupture and gas release into the atmosphere. Freezing for eight days at freezer temperature (approximately − 18°C) also demonstrated stability degradation, characterized by increased particle size and decreased concentration. Ice crystallization likely distorted and damaged the nanobubble interfacial layer, causing the trapped gas structure to be permanently lost. Regular RO-treated samples used as controls remained relatively stable, whereas heated RO water (without nanobubbles) still showed larger particle sizes and lower concentration, confirming that nanobubble structures are highly vulnerable to thermal and freezing stress. Conclusion This study demonstrates that the physical stability of nanobubbles (NBs) is highly dependent on the type of gas used and environmental conditions, particularly temperature and pH. HHO-based nanobubbles were shown to be sensitive to thermal and freezing treatments, exhibiting significant decreases in particle concentration and increases in particle size after heating to 100°C or freezing for eight days. These findings indicate that extreme temperatures can accelerate nanobubble structural degradation through mechanisms involving expansion, coalescence, and disintegration. In contrast, hydrogen-based nanobubbles (H₂) exhibited stable performance within a pH range of 6–7, maintaining relatively constant particle size and concentration over a seven-day incubation period. Highly acidic (pH 4) and strongly alkaline (pH 8–9) conditions reduced stability in terms of both particle size and concentration, suggesting disruptions to interfacial tension and surface charge under extreme environmental conditions. Therefore, neutral pH (6–7) can be concluded as the optimal zone for maintaining nanobubble stability in liquid media, making it an ideal condition for the development of infusion formulations or nanobubble-based active substance delivery systems. This study provides an important scientific foundation for medical nanobubble applications and opens opportunities for developing nanobubble technologies with greater resistance to environmental variation through advanced formulation engineering. Declarations Additional Information Competing Interests: The authors declare no competing financial or non-financial interests. Author Contribution V.A. conceptualized the study, performed experiments, and analyzed data. A.H. assisted with experimental design, data interpretation, and manuscript preparation. Both authors reviewed and approved the final manuscript. Acknowledgement The authors thank the Indonesian Molecule Institute for providing laboratory facilities and technical support. Data Availability The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. Funding: This research received no external funding. References Agarwal, A., Ng, W.J., Liu, Y. Principle and applications of microbubble and nanobubble technology for water treatment. Chemosphere 84, 1175–1180. https://doi.org/10.1016/j.chemosphere.2011.05.054 (2011) Ohta, S. Molecular hydrogen as a preventive and therapeutic medical gas: initiation, development and potential of hydrogen medicine. Pharmacology & Therapeutics 144, 1–11. https://doi.org/10.1016/j.pharmthera.2014.04.006 (2014) Ushikubo, F.Y., Furukawa, T., Nakagawa, R., Enari, M., Makino, Y., Kawagoe, Y., Shiina, T., Oshita, S. Evidence of the existence and the stability of nano-bubbles in water. Colloids and Surfaces A: Physicochemical and Engineering Aspects 361, 31–37. https://doi.org/10.1016/j.colsurfa.2010.03.005 (2010) Montazeri, S.M., Kalogerakis, N., Kolliopoulos, G. 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Colloids and Surfaces A: Physicochemical and Engineering Aspects 495, 176–186. https://doi.org/10.1016/j.colsurfa.2016.01.050 (2016) Jing, D., Li, D., Pan, Y., Bhushan, B. Surface charge-induced EDL interaction on the contact angle of surface nanobubbles. Langmuir 32, 11123–11132. https://doi.org/10.1021/acs.langmuir.6b00976 (2016) Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 22 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 02 Sep, 2025 Reviews received at journal 01 Sep, 2025 Reviews received at journal 29 Aug, 2025 Reviews received at journal 18 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers invited by journal 14 Aug, 2025 Editor assigned by journal 13 Aug, 2025 Editor invited by journal 11 Aug, 2025 Submission checks completed at journal 09 Aug, 2025 First submitted to journal 09 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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08:38:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7269183/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7269183/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-27868-z","type":"published","date":"2025-12-22T15:58:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89658676,"identity":"10a77376-6292-47fb-a4ea-e68f780d6041","added_by":"auto","created_at":"2025-08-22 10:44:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":645644,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of mean particle size and median (D50) for HHO nanobubbles under various thermal treatments.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7269183/v1/c641f96abcd471811596d16f.jpeg"},{"id":89658673,"identity":"7f2aeb11-f801-455d-9fe5-b3782d88fe15","added_by":"auto","created_at":"2025-08-22 10:44:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":137860,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration of HHO nanobubbles after thermal treatments.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7269183/v1/2e7b6667eb48b09024932f96.png"},{"id":89659061,"identity":"f22c0588-1927-4c03-a5dc-823d2c2e7b94","added_by":"auto","created_at":"2025-08-22 10:52:39","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":238355,"visible":true,"origin":"","legend":"\u003cp\u003eGraph of Changes in Nanobubble Mode Diameter at Various pH Levels During 7 Days of Storage\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7269183/v1/be207218057f8cc4648d9325.jpeg"},{"id":89659060,"identity":"790f664e-43ee-412c-bc23-2e44210c90a4","added_by":"auto","created_at":"2025-08-22 10:52:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":134158,"visible":true,"origin":"","legend":"\u003cp\u003eGraph of Changes in Nanobubble Mean Diameter at Various pH Levels During 7 Days of Storage\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7269183/v1/dae7a632666194fedda4f62b.png"},{"id":89658678,"identity":"af01baf4-d885-4eba-a0a9-5c8447c6053c","added_by":"auto","created_at":"2025-08-22 10:44:40","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":302616,"visible":true,"origin":"","legend":"\u003cp\u003eGraph of Changes in Nanobubble Median Diameter (D50) at Various pH Levels During 7 Days of Storage\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7269183/v1/7d0213ad40a183b17ab24403.jpeg"},{"id":89659947,"identity":"3f3a625b-9674-4caa-ac0e-7c7fa40349e8","added_by":"auto","created_at":"2025-08-22 11:00:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":126375,"visible":true,"origin":"","legend":"\u003cp\u003eGraph of Changes in Nanobubble Concentration at Various pH Levels During 7 Days of Storage\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7269183/v1/06b49fdcc3c98e091c455c14.png"},{"id":99172864,"identity":"ec507157-6c4b-40a5-aca1-c37f85886535","added_by":"auto","created_at":"2025-12-29 16:11:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2446394,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7269183/v1/5899441c-9e93-4b67-a538-e4f779384f23.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stability Evaluation of Oxyhydrogen and Hydrogen Nanobubbles Under Thermal and pH Stress Conditions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanobubbles (NBs) are ultra-small gas bubbles, typically\u0026thinsp;\u0026lt;\u0026thinsp;200 nm in size, possessing unique physicochemical characteristics such as high internal pressure, large interfacial area, and exceptional stability in liquid media [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These unique properties make NBs highly promising for various biomedical and therapeutic applications, including active substance delivery systems, medical gas carriers, and infusion media for regenerative tissue stimulation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, nanobubble stability is not absolute; it largely depends on the surrounding environmental conditions, particularly solution temperature and pH. Changes in temperature can increase kinetic energy and reduce surface tension, whereas extreme pH may alter surface charge and interfacial interactions. The combination of these factors can accelerate bubble coalescence, disintegration, or gas release from nanobubble particles [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Understanding how nanobubbles respond to environmental stress is therefore a critical step in optimizing formulations for both clinical and pharmaceutical applications.\u003c/p\u003e\u003cp\u003eIn this study, two primary approaches were employed: temperature treatment (including heating and freezing) and pH treatment (pH 4 to 9) to evaluate the physical stability of nanobubbles formed using two different gases, namely HHO (oxyhydrogen mixture) and pure H₂.\u003c/p\u003e\u003cp\u003eThe selection of HHO gas for temperature treatment testing was based on its highly reactive nature and ease of dissociation when exposed to thermal energy. HHO is the result of water electrolysis containing fractions of O₂ and H₂ in an explosive mixture (commonly referred to as Brown\u0026rsquo;s gas), which theoretically generates higher internal pressure within the bubble structure compared to single-gas nanobubbles. This property makes HHO an ideal model system to evaluate the physical interfacial resistance of nanobubbles under extreme thermal stress such as boiling and long-term freezing. As temperature increases or water freezes, differential pressure from gases within HHO bubbles is expected to exhibit more dramatic changes, thereby revealing the physical stability limits of NB systems [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast, pure H₂ gas was selected as the model for pH stability testing due to its inert, small nature and non-reactivity with H⁺ or OH⁻ ions. This prevents gas-related interference with chemical changes in the solution, ensuring that any observed changes in nanobubble size or concentration can be attributed solely to pH effects rather than gas reactivity. Moreover, hydrogen molecules are widely recognized in medical literature as therapeutic gases with notable antioxidant and anti-inflammatory properties and are extensively used in infusion therapy in various preclinical studies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, evaluating the stability of hydrogen nanobubbles across both biological and extreme pH ranges is an important step toward validating their potential in safe and precise medical gas delivery systems.\u003c/p\u003e\u003cp\u003eTo assess the impact of temperature and pH treatments on nanobubble physical parameters, measurements were performed using Nanoparticle Tracking Analysis (NTA). This technique enables direct monitoring of particle size distribution (mode, mean, and median) as well as particle concentration in solution. Using this approach, the present study aims to provide a strong scientific foundation for identifying optimal parameters to maintain nanobubble physical stability, supporting the development of NB-based formulations resistant to environmental variations, and opening opportunities for applications in gas therapy, regenerative medicine, and nanobubble-based drug delivery systems.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eMaterials\u003c/p\u003e\u003cp\u003eOxyhydrogen nanobubble (HHO) solution prepared in reverse osmosis (RO) water was used for temperature treatments, while hydrogen nanobubble (H₂) solution prepared in RO water was used for pH treatments. To maintain pH stability during sample preparation and analysis, a buffer consisting of 1% ascorbic acid and 1% sodium bicarbonate was employed. Phosphate-buffered saline (PBS) was used as the diluent prior to analysis using Nanoparticle Tracking Analysis (NTA) to ensure that particle concentrations fell within the instrument\u0026rsquo;s detection range. Additionally, sodium dodecyl sulfate (SDS) and Water One were used as rinsing solutions to clean the NTA instrument before and after each measurement, thereby preventing cross-sample contamination. All materials were prepared under sterile conditions and in accordance with laboratory protocols to ensure the integrity of the research data.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExperimental Design\u003c/h2\u003e\u003cp\u003eThermal and Freezing Treatments (HHO Nanobubbles)\u003c/p\u003e\u003cp\u003eThis study utilized four types of oxyhydrogen nanobubble (HHO NB) samples subjected to different conditions to evaluate the effects of temperature treatment on the physicochemical characteristics of nanobubbles. The four sample groups were as follows:\u003c/p\u003e\u003cp\u003eRO : Post-production sample without any additional treatment (baseline).\u003c/p\u003e\u003cp\u003eRO T80C : HHO NB sample heated at 80\u0026deg;C for 10 minutes.\u003c/p\u003e\u003cp\u003eRO T100C : HHO NB sample heated at 100\u0026deg;C for 10 minutes.\u003c/p\u003e\u003cp\u003eRO FREEZE : HHO NB sample frozen in a standard freezer (approximately \u0026minus;\u0026thinsp;17\u0026deg;C) for 192 hours (8 days).\u003c/p\u003e\u003cp\u003epH Treatments (Hydrogen Nanobubbles)\u003c/p\u003e\u003cp\u003eHydrogen nanobubble (H₂ NB) samples were adjusted to target pH values (4\u0026ndash;9) using 1% ascorbic acid and 1% sodium bicarbonate solutions. All samples were stored for 0, 1, 3, 5, and 7 days under refrigerated conditions (approximately 4\u0026deg;C).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003epH Buffer Composition for Hydrogen Nanobubble Solution Treatments\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTarget pH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAscorbic Acid 1% (\u0026micro;L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSodium Bicarbonate 1% (\u0026micro;L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH₂ Volume (\u0026micro;L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19,400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStrongly acidic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19,600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eModerately acidic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19,800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSlightly acidic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNeutral (control)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19,800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSlightly alkaline\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19,600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eModerately alkaline\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the variation in volumes of 1% ascorbic acid and 1% sodium bicarbonate used to produce solutions with specific target pH values (pH 4 to pH 9). Each solution was prepared to a fixed total volume of 20 mL using hydrogen nanobubble water as the solvent, adjusted to achieve the desired pH value. Descriptive annotations are provided to categorize the level of acidity or alkalinity of each mixture.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMeasurement Parameters\u003c/h3\u003e\n\u003cp\u003eAll nanobubble characteristics, including those subjected to thermal and pH treatments, were measured using the NanoSight Pro system (Malvern Panalytical, UK), based on Nanoparticle Tracking Analysis (NTA) technology. Analyses were performed at room temperature, precisely controlled using a Peltier module, and employed a Low Volume Flow Cell (LVFC) to ensure stable flow and accurate detection. The instrument used was the NS XPLORER (Malvern Panalytical), equipped with a 405 nm violet laser for nano-sized particle detection.\u003c/p\u003e\u003cp\u003ePrior to analysis, all samples were diluted 50-fold with phosphate-buffered saline (PBS) to ensure that particle concentrations were within the optimal detection range. Measured parameters included mode (dominant size), mean (average size), D50 (median diameter), and particle concentration (particles/mL). Camera settings such as gain, shutter speed, and laser intensity were optimized according to the particle type under observation, including adjustment for the dilution factor. Measurements were conducted using the following settings: five captures per sample, temperature at 25\u0026deg;C, 3,000 frames per capture, and a flow rate of 30 \u0026micro;L/min. Each sample was analyzed in quintuplicate to obtain mean values and standard deviations, thereby increasing data validity and minimizing measurement error.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThermal and Freezing Treatments on Oxyhydrogen Nanobubbles (HHO)\u003c/p\u003e\n\u003cp\u003eTable 2 presents the analysis results of nanobubble size and concentration formed in reverse osmosis (RO) water following high-temperature treatments (80\u0026deg;C and 100\u0026deg;C) and freezing. The measured parameters included mode, mean, standard deviation, size distribution (D10, D50, D90), and particle concentration. The data demonstrate variations in nanobubble size and concentration as a result of temperature changes, with high-temperature treatment generally increasing the mean particle size and decreasing particle concentration.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"602\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMode (nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean (nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStandard Deviation (nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD10 (nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD50 (nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD90 (nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentrations (p/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eRO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e85.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e249\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e226\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e7.41 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eRO T80C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e7.31 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eRO T100C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e122.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e262\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e244\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e473\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e4.83 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eRO FREEZE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e112.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e401\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e7.12 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eCharacteristics of Nanobubbles Under Thermal and Freezing Treatments Using HHO Gas\u003c/p\u003e\n\u003cp\u003eFigure 1 illustrates the changes in mean particle size and median (D50) across different temperature treatments. The data clearly indicate that heating at 100\u0026deg;C significantly increases particle size, whereas heating at 80\u0026deg;C reduces both mean and median sizes compared to the control. The freezing treatment results in moderate size alterations relative to untreated samples.\u003c/p\u003e\n\u003cp\u003eFigure 2 demonstrates the concentration changes of HHO nanobubbles after exposure to different thermal conditions. It is evident that heating at 100\u0026deg;C markedly decreases particle concentration, whereas the 80\u0026deg;C heating and freezing (-17\u0026deg;C for 8 days) treatments maintained concentrations comparable to the control.\u003c/p\u003e\n\u003cp\u003epH Treatments on Hydrogen Nanobubbles (H₂)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;This section evaluates the effects of pH variation on hydrogen nanobubbles (H₂). Samples were adjusted to target pH values (4\u0026ndash;9) and stored for up to seven days to observe changes in particle size distribution and concentration. The results highlight how acidic, neutral, and alkaline environments influence nanobubble stability, providing insights into optimal conditions for maintaining particle integrity and functional performance.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"603\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMode\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD10\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD50\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD90\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(p/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e112.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e332\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 68px;\"\u003e\n \u003cp\u003e377\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e1.29 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e112.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e396\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 68px;\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e9.55 + 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e118.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e289\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e469\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e214\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 68px;\"\u003e\n \u003cp\u003e764\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e9.25 + 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e127.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e372\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 68px;\"\u003e\n \u003cp\u003e817\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e9.18 + 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 49px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003e127.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e459\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 68px;\"\u003e\n \u003cp\u003e645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e8.05 + 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eChanges in Nanobubble Size and Concentration in pH 4 Solution During 7 Days of Storage\u003c/p\u003e\n\u003cp\u003eTable 4 shows that at pH 5, a gradual increase in particle size and a decrease in concentration occurred throughout the incubation period. Although the degradation was not as rapid as at pH 4, these results indicate that moderately acidic conditions still progressively affect nanobubble stability, representing a transitional phase toward a more stable state.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"609\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMode\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD10\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD50\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD90\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(p/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e341\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e463\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e1.11 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e8.99 + 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e297\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e465\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e669\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e8.25 + 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e658\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e7.72 + 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e87.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e311\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e590\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e6.93 + 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u0026nbsp;\u003c/strong\u003eChanges in Nanobubble Size and Concentration in pH 5 Solution During 7 Days of Storage\u003c/p\u003e\n\u003cp\u003eTable 5 shows that particle size (mode and mean) remained stable and particle concentration stayed high up to day 7 at pH 6. This indicates that pH 6 is a relatively optimal zone for maintaining nanobubble structure and population, with minimal interfacial disruption by environmental ions.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"607\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMode\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD10\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD50\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD90\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(p/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e2.16 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e2.13 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e148\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e1.78 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e214\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e429\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e1.71 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e372\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e422\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 99px;\"\u003e\n \u003cp\u003e1.73 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5.\u0026nbsp;\u003c/strong\u003eChanges in Nanobubble Size and Concentration in pH 6 Solution During 7 Days of Storage\u003c/p\u003e\n\u003cp\u003eTable 6 shows that at pH 7 (neutral), nanobubble size and concentration tended to remain stable with minimal fluctuations. The mode diameter consistently remained around 82.5 nm, and the decrease in concentration occurred gradually, indicating that pH 7 is an optimal condition for short-term stability.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"609\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMode\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD10\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD50\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD90\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(p/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e345\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e488\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.46 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e276\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.42 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e282\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.37 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e87.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e371\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.35 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e493\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.32 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6.\u0026nbsp;\u003c/strong\u003eChanges in Nanobubble Size and Concentration in pH 7 (Baseline) Solution During 7 Days of Storage\u003c/p\u003e\n\u003cp\u003eTable 7 shows that at pH 8 (slightly alkaline), particle size remained relatively constant, and particle concentration remained stable up to day 5. This indicates fairly good stability, although a declining trend began to appear toward the end of the storage period.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"609\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMode\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD10\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD50\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD90\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(p/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e395\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e380\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.42 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e454\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.46 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e233\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e454\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.35 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e87.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e223\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e394\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.35 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e298\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e379\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.25 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7.\u0026nbsp;\u003c/strong\u003eChanges in Nanobubble Size and Concentration in pH 8 Solution During 5 Days of Storage\u003c/p\u003e\n\u003cp\u003eTable 8 shows that at pH 9 (moderately alkaline), nanobubbles exhibited slight fluctuations but remained within a stable range. Particle concentration decreased slightly from day 5 to day 7, indicating a moderate potential for long-term instability.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"609\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMode\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD10\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD50\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD90\u0026nbsp;\u003cbr\u003e\u0026nbsp;(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(p/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e360\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.04 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e594\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.17 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e82.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e420\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.20 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e87.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e337\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.19 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 60px;\"\u003e\n \u003cp\u003e87.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 70px;\"\u003e\n \u003cp\u003e389\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.11 + 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 8.\u0026nbsp;\u003c/strong\u003eChanges in Nanobubble Size and Concentration in pH 9 Solution During 5 Days of Storage\u003c/p\u003e\n\u003cp\u003eFigure 3 shows a trend of increasing dominant particle size, indicating agglomeration caused by interfacial disruption from H⁺ ions. In contrast, pH 6 and 7 remained stable at approximately 82.5 nm, indicating the most optimal conditions. pH 5, 8, and 9 exhibited slight fluctuations, reflecting transitional states or moderate ionic stress affecting nanobubble stability.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Figure 4 shows that highly acidic environments (pH 4 and 5) caused a significant increase in the average size of nanobubble particles, indicating agglomeration or coalescence due to interfacial disruption by H⁺ ions. In contrast, pH 6 and 7 maintained relatively small and stable particle sizes, indicating optimal stability under neutral conditions. In alkaline environments (pH 8\u0026ndash;9), fluctuations were minor but still demonstrated a gradual tendency toward reduced stability.\u003c/p\u003e\n\u003cp\u003eFigure 3 shows that pH 4 experienced a significant increase in D50 up to day 5, indicating substantial agglomeration under highly acidic conditions. In contrast, pH 6 and 7 maintained low and stable D50 values, demonstrating stable particle size distribution. Meanwhile, pH 5, 8, and 9 exhibited slight fluctuations, reflecting transitional states or more moderate interfacial disturbances.\u003c/p\u003e\n\u003cp\u003eFigure 6 shows that nanobubble concentration decreased most drastically at pH 4 and 5, indicating rapid disintegration under highly acidic conditions. In contrast, pH 6 and 7 maintained high and stable concentrations, demonstrating that neutral environments are most favorable for stability. At pH 8 and 9, a gradual decrease was observed, reflecting the long-term destabilizing effects of OH⁻ ions.\u003c/p\u003e\n\u003cp\u003eInterestingly, the Indonesian Molecule Institute (IMI) had previously succeeded in producing HHO gas-based nanobubbles with an exceptionally high concentration of 2.99 \u0026times; 10\u0026sup1;⁰ particles/mL, far exceeding the concentration range observed in this study. This achievement highlights the significant potential of local technology in generating high-density nanobubble formulations, which theoretically can enhance the efficiency of active substance delivery and improve particle stability in clinical and regenerative applications.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study clearly demonstrate that the physical stability of nanobubbles (NBs) is strongly influenced by two primary factors: the type of gas used for their formation and external environmental conditions, particularly solution temperature and pH. These factors directly affect particle size, size distribution, and the concentration of nanobubbles retained in the solution during storage.\u003c/p\u003e\u003cp\u003eIn the group of nanobubbles generated using HHO gas (oxyhydrogen), exposure to high temperatures had a significant impact on the physical integrity of the NBs. Specifically, heating to 100\u0026deg;C caused a substantial increase in both mean and median particle sizes, accompanied by a drastic reduction in particle concentration. This phenomenon is most likely due to increased kinetic energy, which triggers the expansion of the nanobubble\u0026rsquo;s internal volume, thereby facilitating particle coalescence and ultimately causing the rupture of trapped gas bubbles. This process accelerates gas release into the surrounding environment and reduces the number of detectable particles in the system [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Meanwhile, freezing treatment for eight days at \u0026minus;\u0026thinsp;17\u0026deg;C also resulted in decreased concentration and changes in particle size, which can be explained by physical disruption caused by ice crystal formation. Such crystallization likely damages the nanobubble interfacial structure, breaking its protective layer and eliminating the ability of NBs to maintain internal pressure.\u003c/p\u003e\u003cp\u003eConversely, nanobubbles generated using pure hydrogen gas (H₂) exhibited greater resistance to pH variations, particularly within the neutral pH range (6\u0026ndash;7). Under these conditions, NB physical parameters\u0026mdash;such as mode, mean, and median diameters\u0026mdash;remained stable throughout the seven-day storage period. Particle concentration also remained relatively high, indicating that a neutral environment provides an optimal balance between interfacial tension and electrostatic repulsion, thereby maintaining nanobubble structural integrity and homogeneous distribution. These findings confirm previous reports [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], which stated that nanobubble stability is highest within the physiological pH range.\u003c/p\u003e\u003cp\u003eUnder highly acidic conditions (pH 4), particle size increased sharply over time, while NB concentration decreased drastically from the first day. This reflects a rapid disintegration process most likely triggered by the high concentration of H⁺ ions. These ions interact with the NB interfacial layer, disrupting surface stability and promoting gas release as well as particle coalescence [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. At pH 5, although degradation was not as rapid as at pH 4, there was still a clear tendency for size fluctuations and a decline in particle concentration, indicating that NB stability was in a transitional zone toward optimal conditions.\u003c/p\u003e\u003cp\u003eMeanwhile, in alkaline environments (pH 8\u0026ndash;9), nanobubbles exhibited moderate stability. Particle size showed slight fluctuations, and particle concentration began to decrease from day 3 to day 7. This may be attributed to the effect of OH⁻ ions on interfacial tension and surface charge distribution, which, over time, can reduce internal NB pressure and accelerate gas diffusion out of the particles.\u003c/p\u003e\u003cp\u003eOverall, these findings highlight that the pH range of 6\u0026ndash;7 is optimal for maintaining NB size and concentration stability in liquid media. This is particularly important in therapeutic applications, especially when NBs are used as carriers for active substances or medical gases that require medium- to long-term stability. The reduction in stability under extreme conditions indicates that NB formulations for medical applications must take into account both storage and physiological environments.\u003c/p\u003e\u003cp\u003eMoving forward, these results open opportunities for further exploration, such as the incorporation of stabilizers, surface coatings, or the development of core\u0026ndash;shell nanobubbles that are more resistant to environmental variations. Furthermore, in the context of local production in Indonesia, these findings provide an important scientific foundation for the development of more standardized HHO- and H₂-based nanobubble technologies that are ready for application in clinical and regenerative therapies.\u003c/p\u003e\u003cp\u003eThis experiment evaluated the stability of nanobubbles generated from two gas types, HHO and H₂, under thermal treatment, freezing, and pH variation. Results obtained using Nanoparticle Tracking Analysis (NTA) showed that each treatment produced different impacts on size parameters (mode, mean, median/D50) and particle concentration, reflecting the physical stability of nanobubbles in aqueous media. The use of HHO gas for thermal treatment and H₂ gas for pH evaluation was based on the physical properties and availability of these gases in the laboratory system. HHO, consisting of a oxyhydrogen mixture produced through water electrolysis, can be generated in large quantities and is more stable for extreme temperature testing. In contrast, pure H₂ gas was used for pH studies due to its potential as a redox therapeutic agent sensitive to environmental conditions, making it suitable for assessing stability in varying pH environments. Overall, this study demonstrates that nanobubble stability is highly dependent on environmental treatments, with gas type selection and external conditions being key factors in maintaining their functional effectiveness.\u003c/p\u003e\u003cp\u003eIn HHO nanobubbles, exposure to high temperatures such as heating to 80\u0026deg;C and 100\u0026deg;C resulted in a significant decrease in particle concentration, particularly at 100\u0026deg;C. Boiling the sample for 10 minutes caused many nanobubbles to disappear, as indicated by reduced concentration and increased average particle size. This is presumed to be due to the high kinetic energy triggering gas expansion and coalescence, ultimately leading to bubble rupture and gas release into the atmosphere. Freezing for eight days at freezer temperature (approximately \u0026minus;\u0026thinsp;18\u0026deg;C) also demonstrated stability degradation, characterized by increased particle size and decreased concentration. Ice crystallization likely distorted and damaged the nanobubble interfacial layer, causing the trapped gas structure to be permanently lost. Regular RO-treated samples used as controls remained relatively stable, whereas heated RO water (without nanobubbles) still showed larger particle sizes and lower concentration, confirming that nanobubble structures are highly vulnerable to thermal and freezing stress.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that the physical stability of nanobubbles (NBs) is highly dependent on the type of gas used and environmental conditions, particularly temperature and pH. HHO-based nanobubbles were shown to be sensitive to thermal and freezing treatments, exhibiting significant decreases in particle concentration and increases in particle size after heating to 100\u0026deg;C or freezing for eight days. These findings indicate that extreme temperatures can accelerate nanobubble structural degradation through mechanisms involving expansion, coalescence, and disintegration.\u003c/p\u003e\u003cp\u003eIn contrast, hydrogen-based nanobubbles (H₂) exhibited stable performance within a pH range of 6\u0026ndash;7, maintaining relatively constant particle size and concentration over a seven-day incubation period. Highly acidic (pH 4) and strongly alkaline (pH 8\u0026ndash;9) conditions reduced stability in terms of both particle size and concentration, suggesting disruptions to interfacial tension and surface charge under extreme environmental conditions.\u003c/p\u003e\u003cp\u003eTherefore, neutral pH (6\u0026ndash;7) can be concluded as the optimal zone for maintaining nanobubble stability in liquid media, making it an ideal condition for the development of infusion formulations or nanobubble-based active substance delivery systems. This study provides an important scientific foundation for medical nanobubble applications and opens opportunities for developing nanobubble technologies with greater resistance to environmental variation through advanced formulation engineering.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cb\u003eAdditional Information\u003c/b\u003e\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003cp\u003eThe authors declare no competing financial or non-financial interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eV.A. conceptualized the study, performed experiments, and analyzed data. A.H. assisted with experimental design, data interpretation, and manuscript preparation. Both authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank the Indonesian Molecule Institute for providing laboratory facilities and technical support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eFunding: This research received no external funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgarwal, A., Ng, W.J., Liu, Y. Principle and applications of microbubble and nanobubble technology for water treatment. Chemosphere 84, 1175\u0026ndash;1180. https://doi.org/10.1016/j.chemosphere.2011.05.054 (2011)\u003c/li\u003e\n\u003cli\u003eOhta, S. Molecular hydrogen as a preventive and therapeutic medical gas: initiation, development and potential of hydrogen medicine. Pharmacology \u0026amp;amp; Therapeutics 144, 1\u0026ndash;11. https://doi.org/10.1016/j.pharmthera.2014.04.006 (2014)\u003c/li\u003e\n\u003cli\u003eUshikubo, F.Y., Furukawa, T., Nakagawa, R., Enari, M., Makino, Y., Kawagoe, Y., Shiina, T., Oshita, S. Evidence of the existence and the stability of nano-bubbles in water. Colloids and Surfaces A: Physicochemical and Engineering Aspects 361, 31\u0026ndash;37. https://doi.org/10.1016/j.colsurfa.2010.03.005 (2010)\u003c/li\u003e\n\u003cli\u003eMontazeri, S.M., Kalogerakis, N., Kolliopoulos, G. Effect of chemical species and temperature on the stability of air nanobubbles. Scientific Reports 13. https://doi.org/10.1038/s41598-023-43803-6 (2023)\u003c/li\u003e\n\u003cli\u003eOhsawa, I., Ishikawa, M., Takahashi, K., Watanabe, M., Nishimaki, K., Yamagata, K., Katsura, K., Katayama, Y., Asoh, S., Ohta, S. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nature Medicine 13, 688\u0026ndash;694. https://doi.org/10.1038/nm1577 (2007)\u003c/li\u003e\n\u003cli\u003eMeegoda, J.N., Aluthgun Hewage, S., Batagoda, J.H. Stability of Nanobubbles. Environmental Engineering Science 35, 1216\u0026ndash;1227. https://doi.org/10.1089/ees.2018.0203 (2018)\u003c/li\u003e\n\u003cli\u003eOhgaki, K., Khanh, N.Q., Joden, Y., Tsuji, A., Nakagawa, T. Physicochemical approach to nanobubble solutions. Chemical Engineering Science 65, 1296\u0026ndash;1300. https://doi.org/10.1016/j.ces.2009.10.003 (2010)\u003c/li\u003e\n\u003cli\u003eSun, Y., Xie, G., Peng, Y., Xia, W., Sha, J. Stability theories of nanobubbles at solid\u0026ndash;liquid interface: A review. Colloids and Surfaces A: Physicochemical and Engineering Aspects 495, 176\u0026ndash;186. https://doi.org/10.1016/j.colsurfa.2016.01.050 (2016)\u003c/li\u003e\n\u003cli\u003eJing, D., Li, D., Pan, Y., Bhushan, B. Surface charge-induced EDL interaction on the contact angle of surface nanobubbles. Langmuir 32, 11123\u0026ndash;11132. https://doi.org/10.1021/acs.langmuir.6b00976 (2016)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nanobubble, Oxyhydrogen, Hydrogen, Stability, pH, Temperature","lastPublishedDoi":"10.21203/rs.3.rs-7269183/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7269183/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNanobubbles (NBs), gas-dispersed systems\u0026thinsp;\u0026lt;\u0026thinsp;200 nm in size, are increasingly explored for therapeutic and drug delivery applications but are highly sensitive to environmental conditions. This study evaluated the stability of nanobubbles generated from oxyhydrogen (HHO) and pure hydrogen (H₂) gases under thermal and pH stress. HHO nanobubbles were subjected to heating (80\u0026deg;C and 100\u0026deg;C) and freezing (\u0026minus;\u0026thinsp;17\u0026deg;C), while H₂ nanobubbles were stored at pH 4\u0026ndash;9 for seven days. Particle size (mode, mean, D50) and concentration were measured using Nanoparticle Tracking Analysis (NTA). HHO nanobubbles showed significant degradation at 100\u0026deg;C, with reduced concentration and increased particle size, whereas freezing caused moderate instability. H₂ nanobubbles were most stable at neutral pH (6\u0026ndash;7), maintaining size and concentration, while highly acidic (pH 4) and alkaline (pH 9) conditions accelerated particle disintegration. Neutral pH (6\u0026ndash;7) is optimal for nanobubble stability, while extreme temperatures (100\u0026deg;C and freezing) markedly reduce stability. These findings provide insights into designing nanobubble formulations with enhanced environmental resilience for clinical applications such as therapeutic gas infusion and advanced drug delivery.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Stability Evaluation of Oxyhydrogen and Hydrogen Nanobubbles Under Thermal and pH Stress Conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 10:44:35","doi":"10.21203/rs.3.rs-7269183/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-02T16:08:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-01T05:28:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-29T10:14:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T22:42:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298676315818106989284231642330948031280","date":"2025-08-15T23:04:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259234981245921297549986831512743415100","date":"2025-08-15T05:52:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279999854909051112254838156000697638422","date":"2025-08-15T04:59:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102988411018184430006829571901049103186","date":"2025-08-15T02:51:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248456571973952415901775259686824623095","date":"2025-08-15T02:11:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-15T02:08:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-13T13:46:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-11T13:57:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-09T14:30:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-09T14:28:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bf4faf24-1c0c-4e43-b0b9-223d05792a31","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53343424,"name":"Physical sciences/Chemistry"},{"id":53343425,"name":"Physical sciences/Materials science"},{"id":53343426,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2025-12-29T16:08:46+00:00","versionOfRecord":{"articleIdentity":"rs-7269183","link":"https://doi.org/10.1038/s41598-025-27868-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-22 15:58:15","publishedOnDateReadable":"December 22nd, 2025"},"versionCreatedAt":"2025-08-22 10:44:35","video":"","vorDoi":"10.1038/s41598-025-27868-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-27868-z","workflowStages":[]},"version":"v1","identity":"rs-7269183","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7269183","identity":"rs-7269183","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}