Review Reports - The Potential Benefits of Therapeutic Treatment Using Gaseous Terpenes at Ambient Low Levels

Round 1

Reviewer 1 Report

This paper presented a very interesting experimental study, the material and method are properly designed and the results are promising.

1. what is the relationship between ppb/ppm in the paper and the "pptv" in table 1 caption. If possible please use the same unit.

2. is there a "0-ppb" test (can be before or in between you apply the real ppb levels) with the same device and same measurement object included in the experiment. This kind of placebo can be useful to compare in a fair way.

3. a more objective study can be performed with wearable devices to measure like ECG, PPG, GSR etc. to evaluate the stress and other physiological and psychological parameters.

4. A practical issue, seems you did not measure the real ppb in the experiment environment, how to do sure that level of ppb is indeed applied.

Author Response

Response to Reviewer 1’s comments:

This paper presented a very interesting experimental study, the material and method are properly designed and the results are promising.

what is the relationship between ppb/ppm in the paper and the "pptv" in table 1 caption. If possible please use the same unit.

ANS] We appreciate your comments.

In general, 1 ppmv (parts per million by volume) = 1,000 ppbv (parts per billion by volume) = 1,000,000 pptv (parts per trillion by volume). Due to a different level of concentrations using in the MS, the same unit may lead to a more complicated number. For example: 2 pptv = 0.000002 ppmv; 3.123 ppmv = 3,123,000 pptv. Therefore, we used different unit to show just simple number.

is there a "0-ppb" test (can be before or in between you apply the real ppb levels) with the same device and same measurement object included in the experiment. This kind of placebo can be useful to compare in a fair way.

ANS] Yes, there is. “0 ppbv” level was used to test with the same device and the same subjects in the mixed monoterpenes case (please see Sections 2.3.2 and 3.2). These results were used as a background condition of all subjects in this MS.

a more objective study can be performed with wearable devices to measure like ECG, PPG, GSR etc. to evaluate the stress and other physiological and psychological parameters.

ANS] We really want to take into account your suggestion in the near future work. We have addressed this in the Conclusion section (1^st paragraph, page 11).

A practical issue, seems you did not measure the real ppb in the experiment environment, how to do sure that level of ppb is indeed applied.

ANS] We have tested the real concentration of monoterpene emitted from the generation system using a gas chromatograph coupled with a mass selective detector. We have also investigated the consistency of the generation system. Please refer to Section 2.1., 1^st paragraph, page 3 in the MS.

Reviewer 2 Report

The manuscript : The potential therapeutic benefits of airborne terpenes at ambient low ppb levels is quite interesting and well-written. But certain points required more attention by the authors.

Among these points:

1- The title should not contain any abbreviation

2- The abstract should give solid results with clear numerical values instead of vague sentences which could not be measured. and since we agree that the abstract might present te only source of knowledge for many researchers so the abstract should be restructured to give more results.

3-Table 1 in the introduction is not clear. the percentages are clear but the number before the brackets are not defined.

4- The approval of the ethical committee is missed .

5- The purity of the used standards should be mentioned and the vendors from where they were purchased.

6- An important question should be added to the questionnaire that the subjects were using any perfume 24 h prior to the experiment or not because it might affect the results.

7- Question 7 in the the first experiment is quite vague. the subjects should not know the concentrations used and it is requested to know they should be blind during the experiment.

8- More discussion is required to relate the effect of the components in improving the psychological and physiological performance with the published data about the individual components.

Author Response

Response to Reviewer 2’s comments:

The manuscript : The potential therapeutic benefits of airborne terpenes at ambient low ppb levels is quite interesting and well-written. But certain points required more attention by the authors.

Among these points:

1- The title should not contain any abbreviation

ANS] We appreciate your comments. We have changed the title as follows:

“The potential benefits of therapeutic treatment using gaseous terpenes at ambient low levels”

2- The abstract should give solid results with clear numerical values instead of vague sentences which could not be measured. and since we agree that the abstract might present the only source of knowledge for many researchers so the abstract should be restructured to give more results.

ANS] We have added numerical values in the abstract (page 1)

“When the MT levels increased from 0 to 20 ppbv, the mean values of brain alpha wave derived from all participants increased from 9.8 to 15.1. In contrast, the stress index values declined from 46.2 to 34.7.”

3-Table 1 in the introduction is not clear. the percentages are clear but the number before the brackets are not defined.

ANS] We have revised the Table 1 caption (page 2). The percentage is composition ratio of a compound in the total monoterpenes. The number before brackets is the concentration level of a compound in the pptv unit.

4- The approval of the ethical committee is missed .

ANS] We have addressed the approval of this study by IRB (1^st paragraph, page 7).

“All experimental materials and procedures involved in these experiments have been reviewed and approved by the Konkuk University Institutional Review Board (reference number: 7001355-201507-HR-065, email: irb@konkuk.ac.kr)”

5- The purity of the used standards should be mentioned and the vendors from where they were purchased.

ANS] We have addressed this information in the MS (1^st paragraph of Section 2.1, page 3).

6- An important question should be added to the questionnaire that the subjects were using any perfume 24 h prior to the experiment or not because it might affect the results.

ANS] We requested all participants not to use any perfume or strong smelly cosmetics 24 h prior to the experiment. We have added more detailed information in the MS (1 paragraph of section 2.2, page 4).

7- Question 7 in the the first experiment is quite vague. The subjects should not know the concentrations used and it is requested to know they should be blind during the experiment.

ANS] We gave the questionnaire to the subjects after they had gone through all experiments. Therefore, the concentrations in the questionnaire could not affect the experimental results. We have addressed clearer information in the MS (1^st paragraph, page 5).

8- More discussion is required to relate the effect of the components in improving the psychological and physiological performance with the published data about the individual components.

ANS] We have revised this issue in the MS (Section 4, page 10)

Reviewer 3 Report

COMMENTS TO THE AUTHOR(S)

The manuscript is well written and well organized and requires minor revisions before to be accepted for publication in applied science journal.

General comments:

The authors should discuss in detail how they selected the monoterpenes concentration levels to be produced in the chamber. As reported in table 1, the total monoterpenes concentration emitted from the forests are between 107 and 19600 pptv. The data are not normally distributed and shows a median value of 295 pptv (IQR of 450), which is about ten times lower to 2 ppbv. This aspect seems to be not consistent with the aim of the work (i.e. studying the effects of monoterpenes to the human being and health care human exposure), as stated by the authors at the end of the introduction.

Specific comments:

Pag 1, L22, according to the sentence, did the authors knows the effect of MTs at high concentration levels?

Pag 1, L29, why the authors used ppb instead of ppbv?

Pag 3, L84, what is the purity of the zero-air generator?

Pag 3, L88, The contamination of room air is not a new aspect in the field of breath analysis. As example, the following article can be used for the discussion and included in the references.

DOI: 10.1016/j.jpba.2014.11.052

As consequence, this point should be considered since the experiments are performed at low concentration levels, i.e. 2 ppb. Did the authors analyzed the room air during the experiments? Did they analyzed blank samples collected from the testing room?

Pag 3, L92, what was the purpose of analyzing the mixture with GC-MS? Considering the concentration values and the sensitivity of most MS-based GC instrumentation, the analytes should be preconcentrated using sorbent tubes or needle trap device. How the authors collected gaseous samples from the outlet of the generator? Please indicate additional details of the analytical methods used to determine the concentration of the target analytes. As example, the following articles can be used for the discussion and included in the references.

DOI: 10.1088/1752-7163/aa94e7

DOI: 10.1088/1752-7155/7/1/017115

Pag 3, L 95, did the authors tested a different humidity of the mixture?

Pag 3, L100, how the authors selected the participants? Please explain the inclusion and exclusion criteria. Are they characterized in terms of olfactometric capabilities? Did the authors received the authorization from the ethical committee? Please, include the protocol number.

Pag 4, L136, how the authors estimated the break time between experiments?

Pag 7, L202, why participants preferred d-limonene instead of alpha- and beta-pinene? Is there any hypothesis?

Pag 10, conclusions, did the authors tested a placebo group? How the authors excluded any potential interferences during the experiments? Please discuss these aspects.

Author Response

Response to Reviewer 3’s comments:

COMMENTS TO THE AUTHOR(S)

The manuscript is well written and well organized and requires minor revisions before to be accepted for publication in applied science journal.

General comments:

[1] The authors should discuss in detail how they selected the monoterpenes concentration levels to be produced in the chamber. As reported in table 1, the total monoterpenes concentration emitted from the forests are between 107 and 19600 pptv. The data are not normally distributed and shows a median value of 295 pptv (IQR of 450), which is about ten times lower to 2 ppbv. This aspect seems to be not consistent with the aim of the work (i.e. studying the effects of monoterpenes to the human being and health care human exposure), as stated by the authors at the end of the introduction.

ANS] We appreciate your comments. The MTs concentration emitted from the forest are from pptv to ppbv level. However, as a result of this study, it was found that subjects could not detect the odor of MTs with concentrations less than 5 ppbv. Moreover, the objective of this study was to investigate the human psychological and physiological response in inhaling the three major MTs generated at low ppbv levels. Therefore, mixed MTs were prepared in the range of 7 to 20 ppbv. It was assumed that this MT concentration range might occur occasionally in the real forest under certain circumstances.

Specific comments:

[2] Pag 1, L22, according to the sentence, did the authors knows the effect of MTs at high concentration levels?

ANS] Yes, we did. We already addressed the effect of MTs at high concentration levels in the Introduction section (pages 2 and 3) and Discussion section (page 10)

[3] Pag 1, L29, why the authors used ppb instead of ppbv?

ANS] We have changed all ppm, ppb, and ppt to ppmv, ppbv, and ppbv.

[4] Pag 3, L84, what is the purity of the zero-air generator?

ANS] The generation system was clean. You can see the chromatogram of the chamber blank in the following figure A. We have added more detailed information in the MS (page 3)

“The outlet air of the zero-air generator did not contain any quantifiable levels of VOCs.”

Figure A. Chromatogram of chamber blank.

[5] Pag 3, L88, The contamination of room air is not a new aspect in the field of breath analysis. As example, the following article can be used for the discussion and included in the references.

DOI: 10.1016/j.jpba.2014.11.052

ANS] We have discussed and cited this reference in the MS (Last paragraph, page 4).

[6] As consequence, this point should be considered since the experiments are performed at low concentration levels, i.e. 2 ppb. Did the authors analyzed the room air during the experiments? Did they analyzed blank samples collected from the testing room?

ANS] Sample gases were dispensed from a funnel close to the nose with a flow rate of 5 L/min. Thus, the total volume of MTs was 12.5 L at each test. Moreover, subjects could not detect monoterpenes with the concentration less than 5 ppbv. In addition, the room was fully ventilated after each experiment for 15 minutes. After each cycle it was ventilated for 1 hour. The room volume was 147 m³. The ventilation flow rate was 30 m³/min. Hence, it was three revolutions of ventilation for 15 minutes. Therefore, the effect of contaminated room air was negligible.

We have added more information in the MS (last paragraph, page 4)

[7] Pag 3, L92, what was the purpose of analyzing the mixture with GC-MS? Considering the concentration values and the sensitivity of most MS-based GC instrumentation, the analytes should be preconcentrated using sorbent tubes or needle trap device. How the authors collected gaseous samples from the outlet of the generator? Please indicate additional details of the analytical methods used to determine the concentration of the target analytes. As example, the following articles can be used for the discussion and included in the references.

DOI: 10.1088/1752-7163/aa94e7

DOI: 10.1088/1752-7155/7/1/017115

ANS] We have added more detailed information and cited these references in the MS (section 2.1., 1^st paragraph, page 3)

[8] Pag 3, L 95, did the authors tested a different humidity of the mixture?

ANS] Since we used zero-air to dilute the target gases, humidity was considered negligible.

[9] Pag 3, L100, how the authors selected the participants? Please explain the inclusion and exclusion criteria. Are they characterized in terms of olfactometric capabilities? Did the authors received the authorization from the ethical committee? Please, include the protocol number.

ANS] We have addressed more detailed information about participant the selection of participants in the MS (1^st paragraph of section 2.2, page 3).

“A total of 59 healthy and non-asthmatics volunteer subjects were recruited for the test. Among the volunteers, those who had problem with olfactometric capabilities were excluded.”

We have addressed the approval of ethical committee in the MS (1^st paragraph, page 7).

[10] Pag 4, L136, how the authors estimated the break time between experiments?

ANS] As we have mentioned above, the room volume was about 147 m³. Therefore, air circulation for 15 min was three revolutions while the total volume of MTs was only 12.5L. Thus, 15 minutes break time was enough.

[11] Pag 7, L202, why participants preferred d-limonene instead of alpha- and beta-pinene? Is there any hypothesis?

ANS] We have added a hypothesis in the MS (2^nd paragraph of section 3.1, page 7).

“Most of the participants preferred d-limonene as it is preferably used in many commercial and popular products (as orange, lime, lemon, etc.)”

[12] Pag 10, conclusions, did the authors tested a placebo group? How the authors excluded any potential interferences during the experiments? Please discuss these aspects.

ANS] We tested 0 ppbv of mixed monoterpenes as a placebo. These results were used as a background condition of all subjects in the study. This placebo can be also useful to compare in a fair way. We excluded the effect of perfume or cosmetic odor. To investigate more interferences during the experiments, more kinds of monitors would be used in the future works. We have addressed these issues in the Method (1^st paragraph of section 2.2, page 4) and Conclusion section (pages 10-11).