Disinfection of Dental Chair Water Using Aqueous Chlorine Dioxide

Round 1

Reviewer 1 Report

The submitted manuscript entitled “Disinfection of Dental Chair Water using Aqueous Chlorine Dioxide” deals with suggestion on how to improve water quality of dental chairs using simple, economical and environmentally friendly approach. In order to scientific soundness, the manuscript needs some improvements in terms of formatting, English editing etc. as well for better research and publish aims. Some of these suggestions include:

• The study objectives in Page 2, Line 85-87 should be improved.
• Why RO water is used in this disinfection process? Justify!
• The abbreviations are widely used in main text and throughout manuscript especially in methodology. Please make separate section of abbreviations and write all terms.
• Apart from biofilms formations, what are the other factors influencing the water quality in water supply networks?
• For better understanding, the methodology section should be improved. For example, what concentration of chlorine dioxide solutions were obtained, how author dilute it for their designed experiments etc. The authors need to comprehensively explain the methodology so that the readers and researchers can reproduce it for their facilities as well.
• Line 76! Rephrase the line for better understanding it.
• Line 93! What is DPD1?
• Why you chose only ORP v/s ACD?
• For better clarity, include heading 3.1 “Water Purification and Disinfectant System” in methodology section along with the process diagram.
• Improve and enhance your graphs/ Figures 02-03 in different software. If possible, make 1-line short notes for explain them.
• Line 217! Clarify the days and timing of monitoring in Table 03
• Line 223! What are the effects of chloroxyanions.
• Line 259! Place/ shift “a” and “b” with figure 07 in order to show pipeline situation before and after the ACD.
• Line 256! Table 05! Justify the sampling time and date. How you chose the mentioned date and time in 12 different clinics for dental chairs.
• You should’ve added study area and map 12 clinics dimensions by Arc GIS for better clarity. Why you chose only 12 clinics in your area?
• Include future recommendations of your work by adding additional section for good quality research paper.
• The conclusion section should be shortened in length. Normally 300-400 words are enough for fulfilling the purpose of this section.
• As a generic comment, I would like to suggest to include relevant literature and supporting information in results and discussion section. The researchers have rarely referred to other studies in this section.

Author Response

Reviewer 1

1. The study objectives in Page 2, Line 85-87 should be improved.

Answer

It has been improved in revised manuscript.

In this study, the ACD dental chair was designed, fabricated, and studied. The effective concentration of ClO2 can be maintained in the ACD dental chair system to effectively clean the water and keep it in a sterile state. In addition to the application of ACD in dental systems, this new system may also be implemented in the water control of closed pipeline systems, such as food and cosmetic factories.

2. Why RO water is used in this disinfection process? Justify!

Answer

RO water devices are installed in Taiwan dental clinics to generate the RO water for patients' treatment. Even so, after long-term use, the bacterial contamination problems are occurred. Therefore, we use RO water as the water source for research.

3. The abbreviations are widely used in main text and throughout manuscript especially in methodology. Please make separate section of abbreviations and write all terms.

Answer

Abbreviation：aqueous chlorine dioxide (ACD)；oxidation-reduction potential (ORP)；Reverse Osmosis (RO) ；chlorine dioxide (ClO₂)

The Abbreviations are inserted under Keywords.

4. Apart from biofilms formations, what are the other factors influencing the water quality in water supply networks?

Answer

In general, dental clinics routinely maintains its clean environment as use RO water as the water source and have proper sanitation management. The most serious impact on the water quality is the inaccessible deep pipelines. After long use, it often causes biofilm pollution problems. The other factors such as unclean objects or bodies cause water pollution can be managed to prevent it from happening. The aerosol and splatter generated with contaminated water by the dental chair hand pieces could also pose a risk for both dental personnel and patients [4].

5. For better understanding, the methodology section should be improved. For example, what concentration of chlorine dioxide solutions were obtained, how author dilute it for their designed experiments etc. The authors need to comprehensively explain the methodology so that the readers and researchers can reproduce it for their facilities as well.

Answer

It has been corrected in revised manuscript.

6. Line 76! Rephrase the line for better understanding it.

Answer

It has been corrected in revised manuscript.

Hence, after treatment and a specified amount of time, the gaseous chlorine dioxide molecules reduce or disappear in water and decrease its cleaning power. In order to maintain the clean water quality, it causes an increase in labor costs and clean water management issues.

7. Line 93! What is DPD1?

Answer

DPD1 is a mixture containing sulfuric acid (5%-10%) and N.N-diethyl-14-phenylenediamine sulfate (2.5%).

8. Why you chose only ORP v/s ACD?

Answer

The various indicators of water quality are pH (acid-base value), DO (dissolved oxygen), ORP (oxidation-reduction potential), Conductivity, BOD (biochemical oxygen demand) and COD (chemical oxygen demand). The oxidation-reduction potential (ORP) value is a measure of the oxidation-reduction capacity of aqueous solutions. ORP is a comprehensive indicator of the environmental conditions of the medium (including soil, natural water, culture medium, etc.). It has been used for a long time and it characterizes the relative degree of oxidative or reductive properties of a medium.

Our goal is to keep the water in a sterile state. The effective concentration of ClO2 in the water must be maintained to achieve the state of clean water. Due to the volatility of ClO2 gas, we must design a system to monitor the concentration of ClO2 in the water and refill the ClO2 into the water to maintain the effective concentration of ClO2. At this time, the concentration of ClO2 in the water body is most appropriate to be detected by ORP.

9. For better clarity, include heading 3.1 “Water Purification and Disinfectant System” in methodology section along with the process diagram.

Answer

Materials

The water purification and disinfectant system, composed of control unit, sensing unit, dosing unit, and mixed disinfectant water supply unit were provided by Lian Medical Protection Equipment Co. Ltd.

heading 3.1

The water purification and disinfectant system for dental treatment includes a RO water filter appliance, control unit, ORP sensing unit, disinfectant dosing unit, and a mixed disinfectant water supply unit (Figure 1).

10. Improve and enhance your graphs/ Figures 02-03 in different software. If possible, make 1-line short notes for explain them.

It has been corrected in the revised manuscript.

11. Line 217! Clarify the days and timing of monitoring in Table 03

It has been corrected in the revised manuscript.

12. Line 223! What are the effects of chloroxyanions

The toxicity studies of acute exposure, short term exposure, long term exposure, reproductive and developmental toxicity, mutagenicity and related end-points, and carcinogenicity for chloroxyanions ( ClO₂⁻ and ClO₃⁻) have been investigated and described in report of Chlorite and Chlorate in Drinking-water (20). High concentration of chloroxyanions can be harmful to human health.

13. Line 259! Place/ shift “a” and “b” with figure 07 in order to show pipeline situation before and after the ACD.

Answer

It has been corrected in the manuscript.

14. Line 256! Table 05! Justify the sampling time and date. How you chose the mentioned date and time in 12 different clinics for dental chairs.

Answer

We visited and encouraged the dental clinics that can accept new equipment for water sterility. The date selection and execution time was based on the dental clinics that were willing to conduct the trial.

15. You should’ve added study area and map 12 clinics dimensions by Arc GIS for better clarity. Why you chose only 12 clinics in your area?

Answer

Two additional dentist clinic points were added in the Table 5. The total dental clinics is 14. The study areas are mapped in figure 7.  ACD is a new concept of water sterility in dental clinics. Most dental clinics were reluctant to try this new device. Therefore, only 14 dental clinics were willing to try.

16. Include future recommendations of your work by adding additional section for good quality research paper.

Answer

The future recommendations of your work have been added in last part of Results and Discussion.

4.7. Future Study

The efficacy and safety of injecting chlorine dioxide in water systems to prevent hospital-acquired Legionnaires disease has been extensively evaluated (26, 27). After the chlorine dioxide system was installed, a significant decrease of Legionella organisms was observed. However, an extended period (duration, 18 months) was needed to achieve this significant reduction. The delayed reduction in the rate of Legionella positivity is more likely caused by the low residual concentration of chlorine dioxide in the hot water. Therefore, in order to achieve quicky eradicating bacteria in water and keep the water sterile, the effective chlorine dioxide concentration require to maintain in the water body. In the future, the water purification and disinfectant system using ACD can be further investigated in Hospital water system.

26. Sidari F.P., Stout J.E., VanBriesen J.M., et al. Keeping Legionella out of water systems. J. Am. Water Works Assoc., 2004, 96, 111-119.

27. Zhang Z.; McCann C.; Stout J.E.; Piesczynski S.; S. Hawks S.; Vidic R.; Yu. L.V. Safety and Efficacy of Chlorine Dioxide for Legionella Control in a Hospital Water System. Infect. Control Hospital Epidemiol., 2007, 28, 1009-1012.

17. The conclusion section should be shortened in length. Normally 300-400 words are enough for fulfilling the purpose of this section.

Answer

It has been corrected in the revised manuscript.

18. As a generic comment, I would like to suggest to include relevant literature and supporting information in results and discussion section. The researchers have rarely referred to other studies in this section.

Answer

The reference 26 and 27 are relevant literatures of ClO2 disinfection in hospital were added. There are not many articles on ClO2 research. Aiming at the lack of ClO2, it is a feature of this research to propose an implementation method to increase the utilization of ClO2. This is an innovative and applied article. As far as our knowledge is concerned, this is the first article about the application of ClO2 to a closed tubular system by automatic monitoring and control of ORP to achieve a constant control environment, achieve sterility, and reduce pollution.

Author Response File: Author Response.pdf

Reviewer 2 Report

This paper includes a feasible application of aqueous chlorine dioxide (ACD) for disinfection of dental chair water. However, current version of manuscript is not acceptable for the publication. The concerns regarding on this paper are:

- Authors need to provide the details about methodology. It is hard to follow the ACD supplement in dental chair. It looks like continuous supplement system with ORP sensing unit. Also, please describe the ORP value regarding what does it mean. Since the gap between begin dosing and stop dosing is only 10 mV, how ORP sensing system to control ACD is accurate.

- Most critical point of this study is that pH of ACD. The authors claimed that acidic pH can inhibit microorganism but pH is below the critical pH of dental enamel (~5.5). This could adversely affect the tooth enamel (acid-dissolution). What would be an optimal concentration of ACD without affecting pH?

- Previous studies showed electrolyzing neutral water by sodium chloride showed microbicidal effects with pH of 5.5-7.5 and ~20 ppm chlorine (N. Horiba, et al. Bactericidal effect of electrolyzed neutral water on bacteria isolated from infected root canals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 87 (1999), pp. 83-87).

- I appreciated clinical verification but some of works should be validated in the experimental setting. Particularly, effects of ACD on the biofilm formation and colony counts should be performed in the controlled experimental setting. Please provide information if the authors have the data showing optimal concentration of ACD can inhibit the biofilm formation on the pipeline under the controlled setting (showing Fig. 7) together with it bactericidal effect using known species.

Minor: Check semicolon in Abstract whether it used appropriately.

Author Response

Reviewer 2

1. Authors need to provide the details about methodology. It is hard to follow the ACD supplement in dental chair. It looks like continuous supplement system with ORP sensing unit. Also, please describe the ORP value regarding what does it mean. Since the gap between begin dosing and stop dosing is only 10 mV, how ORP sensing system to control ACD is accurate.

Answer

More information was added in the methodology.

The ORP of 5,10 and 25 ppm ACS are 794, 846 and 902 mV, respectively.

The ORP at 10 ppm chlorine dioxide reaches 820 mV or more. The ACD at 0.1-10 ppm and 20-50 ppm can destroy most bacteria and fungi, respectively (10).

A quantitative mixing chamber shown in Figure 1 is designed to quickly stir the circulating water and the injected ClO2 to achieve a uniform state, so that the ORP value can be accurately detected and controlled.

The ORP sensing system was examined start dosing at 800 mV and stop dosing at 810 mV. The ORP was detected every 30 seconds/per time per record stored in the system storage device. In total, 13,080 records were collected and the ORP curve was graphed as shown in Figure 6. The graph shows the actual experimental curve of water ORP monitored at 800 and 810 mV in the water purification and disinfectant system for five consecutive days.

2. Most critical point of this study is that pH of ACD. The authors claimed that acidic pH can inhibit microorganism but pH is below the critical pH of dental enamel (~5.5). This could adversely affect the tooth enamel (acid-dissolution). What would be an optimal concentration of ACD without affecting pH?

Answer

The pH of 4.79, 9.76, and 23.9 ppm of ACD are 5.86, 4.91 and 3.62, respectively.

The ORP of 5,10 and 25 ppm ACS are 794, 846 and 902 mV, respectively.

The ACS concentration in purification and disinfective system was controlled between 810 and 860 mV that is correspondence to 5-10 ppm of ACS. The pH of 5-10 ppm of ACS is 5.86-4.91. The pH of RO water is about 6.0 at 25°C.

The optimal concentration of ACD is <5 ppm with little influence of pH.

3. Previous studies showed electrolyzing neutral water by sodium chloride showed microbicidal effects with pH of 5.5-7.5 and ~20 ppm chlorine (N. Horiba, et al. Bactericidal effect of electrolyzed neutral water on bacteria isolated from infected root canals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 87 (1999), pp. 83-87).

Answer

The bactericidal effect of electrolyzed neutral water is the chlorine that is different from ClO2. The Cl2 and ClO2 can be obtained from electrolyzing neutral water with sodium chloride. The use of different ion membranes in the electrolyzer results in the production of Cl2 or ClO2. Chlorine dissolved in water will dissociate into hypochlorous acid and free chlorine, which in turn produces a sterilization effect, not as good as ClO2. Chlorine dioxide does not dissociate when dissolved in water. It exists in the water in molecular form, and its bactericidal power is mainly in the form of chlorine dioxide molecules.

4. - I appreciated clinical verification but some of works should be validated in the experimental setting. Particularly, effects of ACD on the biofilm formation and colony counts should be performed in the controlled experimental setting. Please provide information if the authors have the data showing optimal concentration of ACD can inhibit the biofilm formation on the pipeline under the controlled setting (showing Fig. 7) together with it bactericidal effect using known species.

Answer

Chlorine dioxide can remove biofilms from water pipes [16,17] and stainless steel [18]. We first confirmed that biofim has been generated with the old pipeline, and measured the colony formation number in the outlet water. After ACS treatment, the biofim in the old pipeline and the number of bacteria in the outlet water were then examined. Our results show that chlorine dioxide is effective in removing biofim and the bacterial count in the outlet water is less than 5 cfu/mL.

5. Minor: Check semicolon in Abstract whether it used appropriately.

It has been corrected in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

The content of the paper is interesting since this can be an answer to how disinfections in dental care can be avoided and both dentist and patient will be protected. The protection must be done also so that it is technically possible and economic saving instruments and water pipe systems.

In part 2.2. you used DPD. Is it chemically N,N-diethyl-p-phenylenediamine? Use this, if it is what you mean!

In part 2.6. give the medium (how many grams of all components)! Do the try tone be tryptone? Did you incubate bacteria in a normal atmosphere? You considered Petri dishes with 30 – 300 colonies. How about the dishes without any colonies? How did you get the results <5 CFU/mL?

Usually the part 2.6. is also the place where the detection limit is given. If you spread 0.2 mL water to agar in a Petri dish and you would find 1 CFU, the results would be 5 CFU/mL. If there are no colonies, the result will be <5 CFU/mL. I propose that you give the detection limit in part 2.6.

It is easy to understand that high ORP will be critical for most bacteria due to oxidative stress.

In your work, the water was safe when the pH was less than 4.

In Fig. 3 you present the dependence between ORP and Cl₂O. Your figure would be better if you would present only the concentrations between 0 and 100 ppm of Cl₂O and you could also stretch the x-axis. It would give readers a better possibility to see the optimal Cl₂O concentration near the ORP values of 800 – 900 mV.

3.4.1. How much a solution of 2000 ppm Cl₂O was used?

Table 1 is not very elegant. Omit the column showing numbers from 1 to 12, since it has no content. I would omit also the column showing that the initial value of ORP was 800 mV. In this case, add to caption text: The initial values of ORP were 800 mV and it increased by adding slowly a solution of 2000 ppm of Cl₂O.

Chemical odor → chlorine odor. Think still this! You have not described in materials and methods, how this odor was measured. In my laboratory where chlorine had been used, the odor is easy to feel – as well as in swimming pools. The odor can be weaker or stronger, but often people can feel it.

Table 2. Do you need a column for experiments 1-3? Hardly.

Fig 5. Is the white reservoir tank for Cl₂O?

4.6. Second paragraph: The pipe system can allow the bacterial densities to increase. Often this is a problem in old parts of cities – and especially if the consumption of water will reduce and the pipes are still over scaled.

Fig 7: What do you mean with different colors in different areas? What mean the white numbers on blue background (not well visible) and usually there is only one number but in some cases two. Is the Chinese text a  key for these, but sorry many readers (including me) are illiterate in this part)?

You analyzed the rapidly growing aerobic (?) heterotrophs. In some cases, Mycobacteria (cause tuberculosis, etc.) and spores of bacilli can be problematic and more resistant to chlorine. Should you study those? See still 10.3390/ijerph10062596 ! 

Should the corrosion be considered, since some pipes and their connectors can be sensitive to chlorine? Can you see other weak points in the system you present, as in some places it might be difficult to get Cl₂O? Add to Discuss!

Check still from guidance to authors what should be italics and what should not be underlined in references! 

Author Response

Reviewer 3

The content of the paper is interesting since this can be an answer to how disinfections in dental care can be avoided and both dentist and patient will be protected. The protection must be done also so that it is technically possible and economic saving instruments and water pipe systems.

1. In part 2.2. you used DPD. Is it chemically N,N-diethyl-p-phenylenediamine? Use this, if it is what you mean!

Answer

DPD1 is a mixture containing sulfuric acid (5%-10%) and N.N-diethyl-14-phenylenediamine sulfate (2.5%). DPD is used as colorimetric reagent. DPD can be oxidized by oxidants as peroxydisulphate (PDS), peroxymonosulphate (PMS) and ClO2 to form pink color, showed absorption maximum at 510 and 551 nm (a, b).

133. Ludvig Moberg and Bo Karlberg, An improved N,N′-diethyl-p-phenylenediamine (DPD) method for the determination of free chlorine based on multiple wavelength detection. Analytica Chimica Acta. 2000, 407, 127-133.
134. Subramanian Gokulakrishnan, Akhil Mohammed, Halan Prakash. 2016, Determination of persulphates using N,N-diethyl-p-phenylenediamine as colorimetric reagent: Oxidative coloration and degradation of the reagent without bactericidal effect in water. Chemical Engineering Journal, 2016, 286, 223-231.

2. In part 2.6. give the medium (how many grams of all components)! Do the try tone be tryptone? Did you incubate bacteria in a normal atmosphere? You considered Petri dishes with 30 – 300 colonies. How about the dishes without any colonies? How did you get the results <5 CFU/mL?

Answer

The medium ingredients are corrected 2.8. Total Bacteria Measurement in revised manuscript as follows.

a Petri dish (1 g glucose, 5 g tryptone, 2.5 g yeast extract, 15 g agar/1000mL H₂O)

Thank you for your correction. try tone is misspelled. It is tryptone.

The bacteria were cultivated in incubator set at 35 ± 1°C.

When the dishes without any colonies, the number of bacteria is counted to 1, and then 1 is multiplied by the value of the lowest dilution factor. If the calculated value is less than 5 (including 0), it is expressed as <5 CFU/mL.

3. Usually the part 2.6. is also the place where the detection limit is given. If you spread 0.2 mL water to agar in a Petri dish and you would find 1 CFU, the results would be 5 CFU/mL. If there are no colonies, the result will be <5 CFU/mL. I propose that you give the detection limit in part 2.6.

Answer

It has been added in part 2.6 of revised manuscript.

The detection limit is <5 CFU/mL.

4. It is easy to understand that high ORP will be critical for most bacteria due to oxidative stress.

Answer：Yes, it is.

5. In your work, the water was safe when the pH was less than 4.

Answer

The design of the ACD dental chair is set to control the chlorine dioxide concentration and pH value within the safe range. If a failure occurs or the set value is out, the addition of chlorine dioxide will be automatically stopped, and send a message to notify maintenance. Hence, the pH value is less than 4 will not occur in this system.

6. In Fig. 3 you present the dependence between ORP and Cl₂ Your figure would be better if you would present only the concentrations between 0 and 100 ppm of Cl₂O and you could also stretch the x-axis. It would give readers a better possibility to see the optimal Cl₂O concentration near the ORP values of 800 – 900 mV.

Answer：The Fig. 3 has been corrected in the manuscript.

7. 4.1. How much a solution of 2000 ppm Cl₂O was used?

Answer

The sentence is corrected in the 3.4.1. of revised manuscript as follows.

The 800 mV water (ORP at initiation) was prepared by adding 2.5 mL of 2000 ppm ClO2 to 1 L of RO water.

8. Table 1 is not very elegant. Omit the column showing numbers from 1 to 12, since it has no content. I would omit also the column showing that the initial value of ORP was 800 mV. In this case, add to caption text: The initial values of ORP were 800 mV and it increased by adding slowly a solution of 2000 ppm of Cl₂

Answer：It has been corrected in the manuscript.

9. Chemical odor → chlorine odor. Think still this! You have not described in materials and methods, how this odor was measured. In my laboratory where chlorine had been used, the odor is easy to feel – as well as in swimming pools. The odor can be weaker or stronger, but often people can feel it.

Answer

The chemical order measurement is added in materials and methods in the revised manuscript.

2.6. Chemical Odor Measurement

The characteristic pungent chlorine-like odor of ACD comes from ClO₂. The testing subject took 10 mL of different pipeline water sources containing various concentrations of ClO2 into the mouth for 30 seconds. The subject then immediately indicated the chemical odor on a scale from 0 to 3; 1- very weak chemical odor, 2- slight chemical odor and 3- strong chemical odor.

10. Table 2. Do you need a column for experiments 1-3? Hardly.

Answer

The column for experiments 1-3 is deleted in the revised manuscript.

11. Fig 5. Is the white reservoir tank for Cl₂O?

Answer：Yes, it is.

12. 6. Second paragraph: The pipe system can allow the bacterial densities to increase. Often this is a problem in old parts of cities – and especially if the consumption of water will reduce and the pipes are still over scaled.

Answer

It can be considered to use aqueous chlorine dioxide (ACS) to clean water and kill bacteria.

13. Fig 7: What do you mean with different colors in different areas? What mean the white numbers on blue background (not well visible) and usually there is only one number but in some cases two. Is the Chinese text a  key for these, but sorry many readers (including me) are illiterate in this part)?

Answer

The Fig 7 is corrected to be more readable in the revised manuscript.

14. You analyzed the rapidly growing aerobic (?) heterotrophs. In some cases, Mycobacteria (cause tuberculosis, etc.) and spores of bacilli can be problematic and more resistant to chlorine. Should you study those? See still 10.3390/ijerph10062596 ! 

Answer

Many microbes were tested in our previous report (10). The test organisms were as follows: Escherichia coli (BCRC 11634/ATCC 8739), Staphylococcus aureus (BCRC 10451/ATCC 6538P), Pseudomonas aeruginosa (BCRC 11633/ATCC 9027), S. aureus subsp. aureus (BCRC 15211/ATCC 33591), Bacillus subtilis subspecies. (BCRC 10447/ATCC 6633), Listeria monocytogenes (BCRC 14848/ATCC 19114), Acinetobacter baumannii (BCRC 10591/ATCC 19606), Salmonella enterica subspecies. (BCRC 12947/ATCC 13311), Klebsiella pneumoniae (BCRC 16082/ATCC 4352), Penicillium funiculosum (BCRC 30438/ATCC 11797), and Candida albicans (BCRC 21538/ATCC10231).

15. Should the corrosion be considered, since some pipes and their connectors can be sensitive to chlorine? Can you see other weak points in the system you present, as in some places it might be difficult to get Cl₂O? Add to Discuss!

Answer

The following paragraph is added to the discussion of 4.6. Application of the ACD dental chair in the revised manuscript as follows.

The chlorine dioxide produced by acidifying the sodium chlorite solution is corrosive due to the existence of excess acid. Chlorine dioxide will not hydrolyze in water to form acidic compounds. Thus, the ACS, pure chlorine dioxide in water, is not corrosive. Pure chlorine dioxide in water will not corrode stainless steel (26). In our experience, the application of 4-5 ppm ClO2 to the pipes made of carbon, iron, polyvinyl chloride (PVC), and copper did not cause corrosion. For pipes made of stainless and PE were used in the ACD dental chair, no corrosion was observed.

16. Check still from guidance to authors what should be italics and what should not be underlined in references! 

Answer：It is corrected.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

1. There are inconsistent explaination about the ACD conc. and pH. Following the author's response, <5 ppm would be optimal concentration (minimal impact on pH level of RO water). However, it is supplied between 5-10 ppm, right? Line 301-305, the author claimed that acidic pH via application of ACD (pH 2.65 with~200 ppm chlorine dioxide ) can inhibit the microorganisms growth. This is confusing and inconsitent with author's response; if this is what the authors want to argue here, that cause adverse impact on the tooth.
2. Regarding 'chemical odor' (in Table 2), please describe the way to sensing the chemical odor. If it depends on the human sense (smell), it is subject and not scientific measurement (not acceptable).
3. Insufficient evidance on the biofilm inhibition; it is not standarized methodology to confirm the biofilm eradication via ACD here. If the author want to claim this effect, need to prove sufficient evidance under the controlled exprimental condition. Just comparison before and after is not a scienfic evidance.

Author Response

Reply to the referee 3:

Thanks for the referees’ valuable comments and suggestions. I have carefully revised my manuscript and answer the questions as follows:

1. There are inconsistent explaination about the ACD conc. and pH. Following the author's response, <5 ppm would be optimal concentration (minimal impact on pH level of RO water). However, it is supplied between 5-10 ppm, right? Line 301-305, the author claimed that acidic pH via application of ACD (pH 2.65 with~200 ppm chlorine dioxide ) can inhibit the microorganisms growth. This is confusing and inconsitent with author's response; if this is what the authors want to argue here, that cause adverse impact on the tooth.

Answer

The content of manuscript is described as followed.

「When the ACD is added to RO water, the ORP increases and pH decreases. Above 25 ppm chlorine dioxide, the rate of increase in the ORP tends to slow down. Above 200 ppm chlorine dioxide, its pH value is 2.65, which is highly acidic. The pH of RO water is around 6.0 at 25°C. Gaseous chlorine dioxide dissolves in the water to cause a low pH value, which can inhibit the microorganism growth.」

Due to the main antibacterial effect of ACS solution is ClO2 molecule, not pH. But, it is well known that low pH can prevent the microbial growth.

This sentence is rewrited in the revised manuscript as followed

The ACD was controlled between 810 and 860 mV which corresponds to 5-10 ppm of ClO₂. The pH of 5-10 ppm ClO₂ is 5.86-4.91, which is also a factor inhibiting the microorganism growth.

This ACS seems to cause adverse impact on people as reviewer mentioned. However, the pH of RO water is about 6.0 at 25°C. The pH of 5-10 ppm of ACS is 5.86-4.91 which is limited hurt to people. Since the treatment of teeth is short-time contact and contact period is only during dental problems, it is infrequent contact. Considering the risk and benefit assessment, it is feasible. In fact, through the actual treatment of dental clinics, no patients were found to complain or have caused the dental problems.

1. Regarding 'chemical odor' (in Table 2), please describe the way to sensing the chemical odor. If it depends on the human sense (smell), it is subject and not scientific measurement (not acceptable).

Answer

The test method for chemical odor is to ingest pipe water containing chlorine dioxide into the mouth to feel the chemical smell.

The ACS is composed of RO water and ClO2. RO water is tasteless. The main smell is from ClO2. The concentration of ClO2 is a scientific measurement, and the ORP value is a scientific measurement. Human perception of smell system is sensing measurement. The human smell data were at least 20 replicates in test. There are a lot of data accumulation in Dental clinic after installation of ACD. Therefore, I remain question that it is unscientific measurement that a specific ClO2 concentration causes an unpleasant sense with human perception odor system.

1. Insufficient evidance on the biofilm inhibition; it is not standarized methodology to confirm the biofilm eradication via ACD here. If the author want to claim this effect, need to prove sufficient evidance under the controlled exprimental condition. Just comparison before and after is not a scienfic evidance.

Answer

Compared with other disinfectants, many studies have shown that chlorine dioxide (ClO2) is an environmentally friendly disinfectant that is relatively less harmful to the human body [13-15] and quickly decomposes into chlorine and oxygen in the atmosphere [6]. The main problem of using chlorine dioxide is that it does not last long in the environment, which makes it difficult to maintain cleanliness continuously.

Chlorine dioxide is a powerful oxidant, and many studies have confirmed that it can kill bacteria, fungi, and viruses [7-12], and remove biofilm [16-18]. The main subject of this study is to design a system to continuously retain the effective ClO2 concentration to eradicate the microbes and biofilm in an environment, but do not study the growth of a specific bacteria into biofilm, prove that chlorine dioxide kills the specific bacteria and its biofilm.

1. English language and style are fine/minor spell check required

Answer

The spell of Manuscript English was checked with Software word.

The Manuscript English was checked and revised by a native English-speaking colleague who is also author, James R. Carey.

Author Response File: Author Response.pdf

Reviewer 3 Report

Still, there is a need to correct it.

In part 2.2. Describe all chemicals in DPD-1!

Add to Fig 2 caption text “in water”

2.6. How many members were in the odor panel? Still, I would speak about odor -  not about chemical   

Please see the guidance about references

in ref. 7 Laszio is a given name.

See https://www.mdpi.com/journal/water/instructions

Consider all instructions as!

. after the last author! 

There is no et al. Give all authors!   There is no and between names.

Italic for the name of the journal!

Bold for the year.

Italic for volume.

References should be described as follows, depending on the type of work:

• Journal Articles:
  1. Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal NameYear, Volume, page range.
• Books and Book Chapters:
  2. Author 1, A.; Author 2, B. Book Title, 3rd ed.; Publisher: Publisher Location, Country, Year; pp. 154–196.
  3. Author 1, A.; Author 2, B. Title of the chapter. In Book Title, 2nd ed.; Editor 1, A., Editor 2, B., Eds.; Publisher: Publisher Location, Country, Year; Volume 3, pp. 154–196.

Author Response

Reply to the referee:

Thanks for the referees’ valuable comments and suggestions. I have carefully revised my manuscript and answer the questions as follows:

Still, there is a need to correct it.

1. In part 2.2. Describe all chemicals in DPD-1!

Answer

The following sentence is added to the revised manuscript.

DPD1 is a mixture containing sulfuric acid (5%-10%) and N.N-diethyl-14-phenylenediamine sulfate (2.5%).

2. Add to Fig 2 caption text “in water”

Answer

The Fig 2 caption text is corrected in the revised manuscript.

The relationship between the ClO₂ concentration of ACD and pH in water.

3. 6. How many members were in the odor panel? Still, I would speak about odor -  not about chemical   

Answer

Each test has 20 replicates.

The chemical odor is changed to chlorine-like Odor.

4. Please see the guidance about references

in ref. 7 Laszio is a given name.

Answer：It is corrected in the revised manuscript.

Kaly-Kullai, K.; Wittmann, M.; Noszticzius, Z.; and Rosivall, L. Can chlorine dioxide prevent the spreading of coronavirus or other viral infections? Medical hypotheses. AK J. Physiol. Int. 2020, 107, 1-11.

5. See https://www.mdpi.com/journal/water/instructions

Consider all instructions as! . after the last author! 

Answer

Thanks for your correction. As your comments, all are corrected in the revised manuscript.

There is no et al. Give all authors!   There is no and between names.

Italic for the name of the journal!

Bold for the year.

Italic for volume.

References should be described as follows, depending on the type of work:

• Journal Articles:
  1. Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal NameYear, Volume, page range.
• Books and Book Chapters:
  2. Author 1, A.; Author 2, B. Book Title, 3rd ed.; Publisher: Publisher Location, Country, Year; pp. 154–196.
  3. Author 1, A.; Author 2, B. Title of the chapter. In Book Title, 2nd ed.; Editor 1, A., Editor 2, B., Eds.; Publisher: Publisher Location, Country, Year; Volume 3, pp. 154–196.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

This version is still not acceptable for the publication following reasons:

I understand that this paper describes a feasible application of ACD for the dental chair. However, some of methodology are unstandarized (too subjective) and also not technically sound (confirmation of biofilm removal).

Because this is an academic paper, the authors need to use reliable (not subjective) and reproducible (with techically sound methods/precedures) methodology.

Suggestion would be following;

Remove the data based on human sense.

Add biofilm assessment under the laboratory setting.

one more thing, if "there are a lot of data accumulation in Dental clinic after installation of ACD", please include the date here.

Author Response

Reivewer 2

This version is still not acceptable for the publication following reasons:

I understand that this paper describes a feasible application of ACD for the dental chair. However, some of methodology are unstandarized (too subjective) and also not technically sound (confirmation of biofilm removal).

Because this is an academic paper, the authors need to use reliable (not subjective) and reproducible (with techically sound methods/precedures) methodology.

Suggestion would be following;

1. Remove the data based on human sense.

Answer

Thank you for your suggestion. Chemical odor is the key factor in this study. In practice, dental water disinfection uses appropriate ClO2 concentration to inhibit microorganisms without chemical odor. Let users not feel the irritating smell of disinfection is the focus of the investigation. Once we leave the human sensory test, the application of this article in real life is meaningless.

2. Add biofilm assessment under the laboratory setting.

Answer

Thank you for your suggestion.

3. one more thing, if "there are a lot of data accumulation in Dental clinic after installation of ACD", please include the date here.

Answer

4.6. Application of the ACD dental chair

Paragraph 2, the content「In 2018 and 2019, 14 dental clinics installed water purification and disinfectant systems incorporating them into dental chairs shown in Figure 7.」describes the set date of ACD dental chair.

Author Response File: Author Response.pdf