Review Reports - Fluoride Exposure Among U.S. Children Aged ≤ 19 Years: Findings from NHANES 2013–2016

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The present investigation shows an overview of the presence of fluoride in USA, considering the official information on fluoride concentrations in water. In addition, based on the NHANES 2013-2016 a biomonitory was carried out to evaluate individual fluoride concentrations in both plasma and urine. The information is important as it provides a complete picture of exposure to this pollutant in the ichildren and adolescent residents at USA.

Some suggestions and recommendations which may improve the information in this work are given below:

Since in most regions fluoride is added artificially, more information should be provided on the process of artificial fluorination, for example which fluoride concentration and which fluoride compound are added.

It should be clarified how controled the possible hydration/dehydration of the person who donates urine in the spot urine sample. Was the urinary fluoride concentration adjusted for its urinary specific gravity? The methodology used for fluorine evaluation, both in water and in biological samples should also be mentioned.

It has been recognized that both plasma fluoride assessment and urine fluoride quantification are good biomarkers of fluoride exposure. Was evaluated yhe correlation of both biomarkers?, is there a good correlation?

In line 64 it is peported that used blood samples, clariffied it.

In table 5 the units of fluoride concentrations in urine and plasma should be indicated, this information is missing. In addition it is suggested to increase another category in the information of fluoride in urine, for example range of 0.2- 1.5 mg/L, since the range of 0.2- 3.2 mg/L is too wide.

Author Response

Comment 1 : Since in most regions fluoride is added artificially, more information should be provided on the process of artificial fluorination, for example which fluoride concentration and which fluoride compound are added.

Response: Most U.S. public water systems use standardized artificial fluoridation compounds such as fluorosilicic acid (H₂SiF₆), sodium fluorosilicate (Na₂SiF₆), or sodium fluoride (NaF). These compounds are added to achieve a target fluoride concentration.
I have added this point to the manuscript.

Comment 2: It should be clarified how controled the possible hydration/dehydration of the person who donates urine in the spot urine sample. Was the urinary fluoride concentration adjusted for its urinary specific gravity? The methodology used for fluorine evaluation, both in water and in biological samples should also be mentioned.

Response: NHANES urinary fluoride concentrations are derived from spot urine samples. No adjustments for urinary specific gravity or creatinine were made, consistent with NHANES laboratory reporting procedures for fluoride.Plasma and urine fluoride were measured by the NHANES laboratory using ion-selective electrode potentiometry after microdiffusion, following CDC standard operating procedures. Household tap water fluoride was analyzed using an ion chromatography system with conductivity detection. All laboratory analyses underwent internal QC and external proficiency testing.

Comment 3: It has been recognized that both plasma fluoride assessment and urine fluoride quantification are good biomarkers of fluoride exposure. Was evaluated yhe correlation of both biomarkers?, is there a good correlation?
Response: We did not conduct a formal correlation analysis between plasma and urinary fluoride; therefore, we cannot comment on the degree of concordance between the two biomarkers in this dataset.

Comment 4: In line 64 it is peported that used blood samples, clariffied it.
Response: Plasma fluoride measurements were obtained from NHANES venous blood samples collected from participants aged 6–19 years.

Comment 5: In table 5 the units of fluoride concentrations in urine and plasma should be indicated, this information is missing. In addition it is suggested to increase another category in the information of fluoride in urine, for example range of 0.2- 1.5 mg/L, since the range of 0.2- 3.2 mg/L is too wide.
Response: These were the ranges set by CDC.

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript authors have investigated fluoride level between U.S. children (≤ 19 Years). The research topic in important from the public health perspectives. However, the manuscript needs considerable revision in many sections to enhance the overall quality.

Comments:

Abstract: Authors are suggested to explain the abbreviations which are not well-known to the broader readers as it will improve readability and understanding of the reported findings. There are some spacing related issues in the reported data. Check carefully.
Introduction: In the first paragraph of introduction, mention the typical concentrations range of fluoride found in various aquatic environments including groundwater.
Introduction: It is too short and highly descriptive without quantitative information. Moreover, strong justification should be given on the novelty and importance of this study with respect to the existing knowledge in scientific literature.
Line 62: “Groundwater fluoride data were obtained from the U.S. Geological Survey”. Provide the duration of data collection.
Line 65 – 66: “derived from the NHANES 2013–2016 among U.S. children aged 0–19 years.”. Explain the abbreviation. Why you specifically targeted to this age group? Are they most vulnerable upon exposure to elevated level of Fluoride?
Line 72: “To assess spatial patterns of naturally occurring fluoride in groundwater…” Are key groundwater qualities parameters available?
Line 73: “developed a county-wide choropleth map…”. Need more information about how the map was created.
Before conclusions, add a paragraph describing the important “implications, challenges and future perspectives of this work”
Why data were analysed in different percentiles 10^th – 90^th.
Are there any statistically significant difference between fluoride level found in different sex (male vs female) and/or age groups.

Author Response

Comment 1: Abstract: Authors are suggested to explain the abbreviations which are not well-known to the broader readers as it will improve readability and understanding of the reported findings. There are some spacing related issues in the reported data. Check carefully.

Response: I corrected it in the manuscript by mentioned the complete abbreviations and addressed the spacings.

Comment 2: Introduction: In the first paragraph of introduction, mention the typical concentrations range of fluoride found in various aquatic environments including groundwater.

Response:Fluoride concentrations vary widely across aquatic environments. Typical freshwater bodies contain <0.5 mg/L, while groundwater may range from <0.1 mg/L to >10 mg/L in regions with geologic fluoride deposits. Surface waters generally have lower concentrations than groundwater due to dilution, whereas coastal and estuarine waters average around 1.2–1.5 mg/L due to natural mineral dissolution.

Comment 3 Introduction: It is too short and highly descriptive without quantitative information. Moreover, strong justification should be given on the novelty and importance of this study with respect to the existing knowledge in scientific literature.
Response, added to the maniscript: Despite decades of water fluoridation policy in the United States, there is limited contemporary evidence assessing fluoride exposure using multiple biomarkers (tap water, urine, plasma) within the same nationally representative cohort. Previous studies have typically examined single biomarkers or focused on adults. The novelty of this study lies in its multi-matrix approach, integration of geospatial groundwater data, and evaluation of fluoride exposure specifically in children. Children and the younger population overall are more suceptible due to greater intake relative to body weight and developing renal systems.This study aims to provide an updated, quantitative evidence needed to suppport the need for reevaluation of fluoride policies.
Comment4 : Groundwater fluoride data were obtained from the U.S. Geological Survey”. Provide the duration of data collection.
Response: Groundwater fluoride data were obtained from the U.S. Geological Survey (USGS), using national monitoring datasets collected between 2010 and 2020.

Comment5: “derived from the NHANES 2013–2016 among U.S. children aged 0–19 years.”. Explain the abbreviation. Why you specifically targeted to this age group? Are they most vulnerable upon exposure to elevated level of Fluoride?
Response, the NHANES only has data avaialbe for these age ranges. Furthermore, as I corrected in comment 3, younger age groups are more susceptible to these exposures.

Comment 6 : Line 72: “To assess spatial patterns of naturally occurring fluoride in groundwater…” Are key groundwater qualities parameters available?
Response: Only groundwater fluoride concentration data were used in this analysis. Other groundwater quality parameters (e.g., pH, hardness, TDS, alkalinity) were not available at matched national coverage and were therefore not included.

Comment 7: “developed a county-wide choropleth map…”. Need more information about how the map was created.

Response: We developed a county-level choropleth map using ArcGIS Pro 3.2. Groundwater fluoride concentrations were spatially joined to county boundaries using Federal Information Processing Series (FIPS) codes. Data were categorized into four concentration intervals (<0.2 mg/L, 0.2–<0.7 mg/L, 0.7–1.5 mg/L, >1.5 mg/L) and symbolized using a sequential color gradient. Public water fluoridation data from CDC were overlaid as a second layer to visualize alignment between natural fluoride levels and fluoridation policy. (I added this to hte manuscript

Comment 8. Add paragraph before Conclusions – Implications, challenges, future perspectives
Response: added to the manuscript.

Comment 9 Why data were analysed in different percentiles 10^th – 90^th.
Response : to capture the full distribution of fluoride exposure and identify potential subgroups at both low and high ends of exposure, rather than relying solely on mean values which may obscure variability.
Comment 10.: Are there any statistically significant difference between fluoride level found in different sex (male vs female) and/or age groups
Response: Significant sex differences were observed in urinary fluoride concentrations, with males showing higher median and 90th percentile values (p < 0.0001). Age-related differences were also statistically significant, with younger age groups (6–11 years) consistently showing higher urinary fluoride than adolescents (p < 0.05). Plasma fluoride differences by sex were small and not statistically significant, whereas modest but significant age-related differences were present (p < 0.05).

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

The article is very interesting and addresses the key issue of the risks of fluoride exposure for children. However, in general terms, the article should be improved in the following sections:

A) More references are needed, especially in the introduction, methodology, and discussion sections.

B) Statistical techniques and graphical support should be implemented to better understand the research results.

C) Table 2 could be reduced in size and supplemented with statistically analyzed graphs or figures.

D) The discussion is correct, but there is no conclusions section. It is important to add general and specific conclusions that address the study objectives stated in the conclusions.

E)This work covers a very large area, the USA, so I advise you to clearly define the study area in the introduction. It is also unclear how many samples were analyzed, who analyzed them, and over what period.

Given the scope of the study and its social interest, this work can be published if these sections are improved. Once improved, a more thorough review will be conducted.

Author Response

Comments

A) More references are needed, especially in the introduction, methodology, and discussion sections.

B) Statistical techniques and graphical support should be implemented to better understand the research results.

Response: I have addressed these and have made the changes to the manuscript. In response to the request for additional references, we have expanded the Introduction, methodology and discussion sections by adding new citations. A total of 11 new references have been added throughout the revised manuscript to meet this requirement.

C) Table 2 could be reduced in size and supplemented with statistically analyzed graphs or figures.
Response: We need the table to show the complete distribution.

D and E :

D) The discussion is correct, but there is no conclusions section. It is important to add general and specific conclusions that address the study objectives stated in the conclusions.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

In the rebuttal, authors have addressed and incorporated the comments adequately.

Author Response

In the rebuttal, authors have addressed and incorporated the comments adequately.

Response: Thank you for your review and positive feedback. We appreciate the opportunity to revise our manuscript and are glad that the changes addressed the concerns adequately. Please let us know if any further clarification or adjustments are needed.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

The review was done correctly, but I believe the conclusions section could be separated from the discussion, or you should add a separate conclusions section. A paper of this type needs to end with clear and concise conclusions. You should also review the journal's rules to correct the reference citation's format.

Author Response

Response Thank you for your valuable feedback and careful review. We appreciate your suggestion regarding the Conclusions section. In the revised manuscript, we have separated the Conclusions from the Discussion to provide a clearer and more concise closing to the paper. We have also reviewed the journal’s guidelines and corrected the reference citation format accordingly. Please let us know if any further adjustments are needed.