Review Reports - Sociodemographic and Lifestyle Factors Associated with Historical Exposure to Persistent Flame Retardant Concentrations in a Spanish Cohort

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a well-designed and carefully conducted study assessing historical exposure to PBDEs and Dechlorane Plus using adipose tissue samples from a well-characterized Spanish cohort. The topic is timely and relevant, and the use of adipose tissue as a biomarker of long-term exposure represents a clear strength of the study.

The introductory paragraph provides an appropriate general overview of persistent organic pollutants (POPs). However, the authors could slightly improve clarity by briefly specifying why PBDEs and Dechlorane Plus are of particular concern compared to other POPs already in this opening paragraph. Lines 37–41

The description of PBDEs and Dechlorane Plus is clear and informative. Nonetheless, the transition between PBDEs and DP could be smoother. Consider explicitly stating why DP is discussed together with PBDEs. Lines 42–53

The description of the geographical and socioeconomic characteristics of the recruitment areas is informative and relevant. The authors may consider briefly explaining why these differences are important in the context of PBDE/DP exposure. Lines 84–88

Sample preparation and extraction procedures are described in sufficient detail to ensure reproducibility. Minor language revisions are recommended to improve fluency (“instrumental analysis was performance” should be “performed”). Lines 116–129

The description of GC–MS conditions is detailed and technically sound. The rationale for using negative chemical ionization (NCI) could be briefly stated to reinforce methodological choices. Lines 130–141

The regression modeling strategy is generally sound. However, the description of the forward–backward variable selection procedure is relatively complex; simplifying the explanation or adding a brief schematic description could improve readability. Lines 182–195

The description of lifestyle characteristics (smoking, alcohol consumption, occupational class) is informative. However, this section could be slightly streamlined to avoid repetition with Table 1. Lines 207–213

Detection frequencies are clearly reported. The authors may consider briefly highlighting which congeners are most relevant for subsequent regression analyses. Lines 214–217

The discussion of DP isomer distribution is clear. The anti-DP fraction is consistent with commercial mixtures; however, the authors may consider explicitly stating whether this suggests primarily legacy exposure. Lines 224–227

The decision to split regression results into three separate tables improves technical organization but makes it difficult for readers to identify overarching exposure patterns. A brief summary paragraph highlighting the strongest and most consistent associations would improve readability. Lines 235–240

The additional discussion on sex-related lifestyle differences is informative. However, these variables are not explicitly modeled; the authors may consider clarifying that these are hypothesized explanatory factors. Lines 250–257

The negative association between BMI and several congeners is consistent with the dilution hypothesis. The inclusion of sensitivity analyses is commendable, although a brief summary of results in the main text would be helpful. Lines 265–271

Occupational class associations are well interpreted. The authors may consider clarifying whether “manual work” captures current or historical occupational exposure. Lines 272–276

The inverse associations with perceived exposure to toxic metals are interesting but somewhat speculative. The proposed explanation regarding risk perception and protective behavior should be framed more cautiously. Lines 294–299

The inverse association between BDE-99 and egg consumption is unexpected. The authors appropriately interpret this cautiously; however, this result could be more clearly labeled as exploratory. Lines 321–325

The association with fruit consumption is speculative. The authors correctly acknowledge uncertainty; however, this finding might be better framed as hypothesis-generating. Lines 334–336

The identification of sex, BMI, occupational class, perceived exposure, and dietary habits as key determinants is appropriate. However, the authors may consider prioritizing the most robust and consistently observed associations to avoid an overly broad conclusion. Lines 372–374

The reference to public health relevance is appropriate. This section could be strengthened by briefly specifying how these findings might inform targeted interventions or surveillance strategies. Lines 374–375

The conclusions are appropriately cautious and do not overstate the findings. Given the exploratory nature of the study, it may be useful to explicitly state that the results are hypothesis-generating and warrant confirmation in larger, prospective cohorts. Line 376

Comments on the Quality of English Language

The quality of English in the manuscript is good and does not require improvement. The text is clear and well-structured, and the scientific content is communicated effectively throughout the manuscript.

Author Response

Response to Reviewer 1

Thank you for your constructive suggestions and comments on the manuscript. They have definitely helped us improve the final version. We have addressed all of them as follows:

Thank you for the recommendation. We have rewritten the 1^st paragraph of revised Introduction section as follows (Lines 39-50):

“Persistent organic pollutants (POPs) are a myriad of chemicals that share a high resistance to degradation. As a result, they have a high potential for bioaccumulation in living organisms and biomagnification over the food chain [1,2]. In the general population, exposure to POPs occurs through multiple pathways, including dietary intake, particularly of animal-derived foods rich in lipids, ingestion and inhalation of contaminated indoor dust, inhalation of polluted indoor and outdoor air, and dermal contact with consumer products and treated materials [2–4]. Concretely, Polybrominated diphenyl ethers (PBDEs) and Dechlorane Plus (DP) merit special attention among POPs because they were extensively used as halogenated flame retardants across many products (e.g., textiles, plastics, building materials and electronic devices) [5–7], and they have been found to accumulate in indoor dust and human tissues and travel long distances in the environment [8].”

According to the reviewer’s recommendations, we have smoothed the transition between PBDEs - DP, and we explicitly stated why PBDEs and DP were studied together in the 2^nd paragraph of the revised Introduction section, as detailed below (Lines 58-62):

“Another halogenated flame retardant is DP, which was commercialized as a mixture of their two stereoisomers, being predominant anti-over syn-DP [11,12]. In this article, we decided to study PBDEs and DP together because, as mentioned above, they share similar applications, physicochemical properties and exposure pathways.”

The description of the geographical and socioeconomic characteristics of the recruitment areas is informative and relevant. The authors may consider briefly explaining why these differences are important in the context of PBDE/DP exposure. Lines 84–88.

Following the reviewer’s recommendation, we have included a brief explanation of the location differences related to PBDE/DP exposure as detailed in the 1^st paragraph of the revised Materials and Methods (section 2.1. Study design and population) (Lines 100-106):

“The city of Granada (238,292 inhabitants in 2004) and its metropolitan area (213.709 inhabitants in 2004) are mainly economically based on the service sector and light industry, and frequently experience atmospheric thermal inversion episodes, leading to peaks in air concentrations of industrial pollutants [44]. In contrast, Motril (55,078 inhabitants in 2004) and surroundings are characterized by a high density of intensive agriculture, including greenhouse farming, and windier conditions due to their coastal location [44,50]”

We have corrected the word “performance” by “performed” as detailed in the 1^st sentence of the 3^rd paragraph the revised Materials and methods (section 2.3. Laboratory analyses) (Lines 172-175):

As suggested, we have revised Materials and methods (section 2.3. Laboratory analyses) as follows (Lines 172-178):

“Based on a previous study [59], instrumental analysis was performed by gas chromatography (GC) (HP 7890A Series, Hewlett-Packard, Palo Alto, CA) equipped with a multimode inlet (MMI) and coupled to single quadrupole mass spectrometer in negative chemical ionization mode (NCI-MS, 5975C Agilent). This MS soft-ionization is widely used for determination of polybrominated compounds because it provides less fragmentation and high sensitivity, i.e. up to 15 times higher than electron ionization (EI-MS) [60,61].”

We agree with the reviewer that the description of the forward-backward variable selection procedure is relatively complex. To improve clarity without compromising accuracy, we have modified the 2^nd paragraph of the revised Materials and methods (section 2.4. Statistical analyses) as detailed below (Lines 219-228):

“Associations of sociodemographic, lifestyle, occupation and perceived chemical exposure (independent variables) with PBDEs and both DP isomers (dependent variables) were identified using multivariable linear regression with manual step forward-backward model variable selection. First, variables were selected based on the available literature and biological plausibility as potential factors of PBDEs/DP exposure [63–67]. Then, we used a two-stage selection procedure (Supplementary Material, Figure S1):

Forward selection stage: independent variables with a p-value ≤ 0.20 were retained in the same multivariable model.
Backward elimination stage: independent variables with p-value ≥ 0.10 and/or substantial increase of Bayesian information criterion (BIC) (ΔBIC ≥ 10, in accordance with Raftery’s approach [68]) were excluded from the model.

Additionally, to facilitate understanding of the analytical process, we have included a schematic of the statistical process in the Supplementary Material, as follows:

Figure S1. Manual step forward-backward model variables selection

¹ Bayesian information criterion

Following your suggestion, we have shortened 1^st paragraph of revised Results (section 3.1. Description of the study population and adipose tissue pollutant concentrations) to reduce repetition with Table 1, as detailed below (Lines 235-243):

“The main characteristics of the study population are summarized in Table 1. This subcohort included slightly fewer females than males. The mean BMI in our cohort was 27.9 kg/m². This was higher than the average BMI reported for the Andalusian population in 2003 [71], likely because our hospital-based cohort included participants with obesity-related conditions. In our population, approximately one third of the participants were smokers at recruitment, and 69.9% of males and 29.5% of females reported being alcohol consumers. These results are similar to those reported for the Andalusian population [71]. In our study, manual workers were predominant, while a higher proportion of females (but not males) resided in semirural areas vs urban and rural areas.”

Detection frequencies are clearly reported. The authors may consider briefly highlighting which congeners are most relevant for subsequent regression analyses. Lines 214–217

We thank the reviewer for this suggestion. All selected congeners were treated as equally relevant in the regression analyses because of the exploratory nature of the study.

We thank the reviewer for this insightful suggestion. As noted, the observed distribution of DP isomers may indeed be indicative of predominantly legacy exposure. We have revised the 3^rd paragraph of the revised Results (section 3.1. Description of the study population and adipose tissue pollutant concentrations) to explicitly acknowledge this interpretation while maintaining a cautious tone. The modified text now reads (Lines 255-262):

“No differences in bioaccumulation were observed between the two DP isomers. In our study, anti-DP was the predominant form with an average anti-DP fraction (anti-DP/total DP) of 0.75 ± 0.11 (data not shown), consistent with the reported proportions in commercial DP products ranging between (0.60-0.80) [83], and in prior studies in humans (sample size ranging from 20 to 105) ranging anti-DP fractions between 0.71 and 0.79 [11,36,84,85]. These findings may suggest no stereoselectivity in the entry into the human body or metabolism and primarily legacy exposure. However, in highly exposed occupational roles populations with lower anti-DP fractions have been reported [86,87].”

The decision to split regression results into three separate tables improves technical organization but makes it difficult for read0ers to identify overarching exposure patterns. A brief summary paragraph highlighting the strongest and most consistent associations would improve readability. Lines 235–240

Following the reviewer’s suggestion, we have included the following summary paragraph in the revised Results after the 1^st paragraph of section 3.2. Factors associated with adipose tissue PBDEs and DP concentrations as follows (Lines 275-279):

“Overall, we observed robust statistically significant inverse associations between BMI and concentrations of PBDEs and DP. We also found robust positive associations between males and PBDEs (Table 2), perceived exposure to paints and PBDEs levels, notably BDE-47 and BDE-99 (Table 3), and fish consumption and PBDEs, specifically BDE-183 and -197 (Table 4). These findings will be more extensively discussed below.”

We have clarified this section in the revised manuscript (4^th paragraph of section 3.2. “Factors associated with adipose tissue PBDEs and DP concentrations”) as a hypothesis (Lines 292-299):

“We hypothesize that the higher PBDE concentrations in males vs. females in our study population may be related, at least in part, to lifestyle differences, as previously observed for other POPs in GraMo cohort [39,41,88]. Indeed, in our study, males reported an increased consumption of fatty food from animal origin, such as pork (23.3% vs. 6.6%), while chicken consumption was predominant in females (41.0% vs. 26.0%). Additionally, 20.55% of males, and 1.64% of females acknowledge involvement in industrial activities such as operators of installations, machinery and assemblers, craftsmen and, workers in industry construction and mining.”

As suggested, a summary of the most interesting results of our sensitivity analyses has been included in the revised Results section (6^th paragraph of section 3.2. Factors associated with adipose tissue PBDEs and DP concentrations), as follows (Lines 309-313):

“Particularly, the category 25-30 kg/m² vs. < 25 kg/m² showed significant negative beta coefficients ranging from -0.37 to -0.50 for BDE-183, -197, and both syn- and anti-DP. The association for BDE-153 was also inverse but did not reach statistical significance (beta = -0.27, p-value = 0.092).”

Occupational class associations are well interpreted. The authors may consider clarifying whether “manual work” captures current or historical occupational exposure. Lines 272–276

Following the reviewer’s recommendation, we have clarified the social occupational class variable in the Results section, concretely at the end of section 2.2. Sociodemographic, lifestyle and clinical information, as follows (Lines 143-150):

“Although this classification is based on participants’ principal occupation at enrollment, participants had remained in the same role for a mean of 16 years. Thus, occupational exposure captures not only current workplace conditions, but also cumulative, long-term exposure associated with their occupation. Furthermore, occupational social class reflects the socioeconomic hierarchy and is closely linked to income, material resources, and lifestyle-related factors, including diet, physical activity, and health behaviors, which may further influence both environmental exposures and health outcomes [54,55].”

We have rewritten the referred paragraph in the revised Results section (9^th paragraph of section 3.2. Factors associated with adipose tissue PBDEs and DP concentrations) accordingly (Lines 342-347):

“We hypothesize that the negative associations observed may be explained by greater implementation or adherence to safety measures among individuals who perceive themselves as being at risk, as shown in previous studies [110-112]. This interpretation, although highly speculative, opens the door to future population-based interventions to confirm whether increasing awareness of exposure is effective for reducing the body burden.”

We agree with the reviewer and have clarified this aspect in the revised 11^th paragraph of section 3.2. (Factors associated with adipose tissue PBDEs and DP concentrations), as follows (Lines 270-274):

“Considering the limited biological plausibility and the exploratory nature of this study, we consider that this might be a chance finding or even a result of a confounding effect of a third uncontrolled variable in the models, since BDE-99 has been reported as the dominant congener in eggs [124].”

The association with fruit consumption is speculative. The authors correctly acknowledge uncertainty; however, this finding might be better framed as hypothesis-generating. Lines 334–336

We have explicitly clarified the need for future confirmation of these findings in the revised Results section (14^th paragraph of section 3.2. Factors associated with adipose tissue PBDEs and DP concentrations) as suggested (Line 385-386):

“Nevertheless, this finding has no precedent and, therefore, warrants further confirmation.”

Following the reviewer’s suggestion, we have highlighted the most robust associations in the revised Conclusion section, as detailed below (Line 428-432):

Following the reviewer’s recommendation, we clarified the referred paragraph in the revised Conclusions section, as follows (Line 433-434):

“These results might help further targeted public-health interventions by identifying modifiable factors associated with the exposure.”

We have included the following statement at the end of the revised Conclusion section (Line 434-435):

“However, due to the explorative nature of this study, our findings needed to be confirmed in further research.”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The aim of this study was to estimate historical exposure to a selection of Polybrominated Diphenyl Ethers (PBDEs) and Dechlorane Plus (DP) in a subsample of a Spanish adult cohort by analyzing their residues in adipose tissue samples, as well as to identify the potential sociodemographic and lifestyle factors associated.

The manuscript presents a good research work. In general, it is well organized, with potential to be useful for the future research. Before publication, manuscript needs minor revision.

General comments:

Please remove the period at the end of the title of the manuscript.
The Introduction should more clearly emphasize the novelty of the study and its contribution to the existing literature, highlighting what makes this research unique and its potential impact on this field of research. In addition, the manuscript should explicitly state the specific objectives of the study to help readers better understand the purpose and significance of the research.
The inclusion of additional visual elements, such as an image or map of the study area, schematic diagrams illustrating key methodological steps (e.g., preparation and analysis of adipose tissue samples), and graphical representations of the statistical analyses, would enhance the clarity, accessibility, and overall engagement of the presented findings.
Sample collection for this study was conducted in 2003/2004, while the analysis of PBDEs and DP was performed in 2024. Please clarify the reasons for the more than 20-year gap between sample collection and analysis.
With the exception of Table 1, the tables in the manuscript are overcrowded and complicated for interpretation. The authors are encouraged to revise and simplify the tables to ensure that the data are clearly presented.
The Conclusion section should be expanded to include more concrete recommendations and future research perspectives.

Author Response

Response to Reviewer 2

Thank you for your constructive suggestions and comments on the manuscript. They have definitely helped us improve the final version. We have addressed all of them as follows:

Please remove the period at the end of the title of the manuscript.

As kindly suggested by the reviewer, we have removed the period at the end of the title as follows (Lines 2-4):

“Sociodemographic and lifestyle factors associated with historical exposure to persistent flame retardant concentrations in a Spanish cohort”

The Introduction should more clearly emphasize the novelty of the study and its contribution to the existing literature, highlighting what makes this research unique and its potential impact on this field of research. In addition, the manuscript should explicitly state the specific objectives of the study to help readers better understand the purpose and significance of the research.

Following the reviewer’s suggestions, we highlighted the novelty of the study in the revised Introduction section, concretely in the 4^th paragraph of section 1. Introduction, as detailed below (Lines 81-87):

“Although prior literature has already studied legacy exposure to PBDEs/DP [36–41], few have measured these compounds in adipose tissue [42], and have been conducted in Spain [43]. Moreover, hardly any study has comprehensively examined sociodemographic, occupational, dietary, and lifestyle determinants of adipose tissue PBDE/DP levels. Our study addresses these gaps by quantifying PBDEs/DP in human adipose tissue from a Spanish cohort and by performing a detailed analysis of the factors associated with their concentrations.”

And we clarified our objectives in the 5^th paragraph of section 1. (Introduction), as follows (Lines 88-91):

“Based on the abovementioned, the objectives of this study were: (i) to quantify historical exposure to a selection of PBDEs and DP in a subsample of a Spanish adult cohort by measuring their concentrations in adipose tissue; and (ii) to identify sociodemographic and lifestyle factors associated with the pollutant levels.”

The inclusion of additional visual elements, such as an image or map of the study area, schematic diagrams illustrating key methodological steps (e.g., preparation and analysis of adipose tissue samples), and graphical representations of the statistical analyses, would enhance the clarity, accessibility, and overall engagement of the presented findings.

Following the reviewer’s recommendation, we have included additional visual elements, such as Figure 1 that represent the geographical distribution of the participants included in this study and Figure S1 related to the variable selection process used in this manuscript to improve reader understanding and engagement (Line 116).

Figure 1. Map of Granada province with participants’ distribution.

Figure S1. Manual step forward-backward model variables selection.

If the reviewer and editor agree, we would prefer not include a schematic diagram illustrating methodological steps because we think that it is not the main focus of this manuscript. Notwithstanding, we have improved section 2.3. (Laboratory analyses) and have included new references (60 and 61 for consultation as detailed below (Lines 172-178):

“Based on previous study [59], instrumental analysis was performed by gas chromatography (GC) (HP 7890A Series, Hewlett-Packard, Palo Alto, CA) equipped with a multimode inlet (MMI) and coupled to single quadrupole mass spectrometer in negative chemical ionization mode (NCI-MS, 5975C Agilent). This MS soft-ionization is widely used for determination of polybrominated compounds because it provides less fragmentation and high sensitivity i.e. up to 15 times higher than electron ionization (EI-MS) [60,61]”

Sample collection for this study was conducted in 2003/2004, while the analysis of PBDEs and DP was performed in 2024. Please clarify the reasons for the more than 20-year gap between sample collection and analysis.

The GraMo cohort is a multipurpose cohort focused on exploring the levels of multiple chemical exposures and their health effects, funded by several competitively awarded projects over the years. Although the tissue samples were collected in 2003/2004, funding and approval for the PBDE/DP analyses were obtained at a later stage. This has now been detailed in the revised Materials and Methods section (section 2.1 “Study design and population), as follows (Lines 119-121):

“Although the data collection for this cross-sectional study was conducted in 2003-04, the analysis of PBDEs and DP was carried out in 2024 following the subsequent funding received from the Instituto de Salud Carlos III, Spain (PI20/01568).”

With the exception of Table 1, the tables in the manuscript are overcrowded and complicated for interpretation. The authors are encouraged to revise and simplify the tables to ensure that the data are clearly presented.

We agree that the tables contain a large amount of information. Following the reviewer’s suggestion, we carefully explored alternative ways of presenting them. However, we could not identify a format that would further simplify the tables without removing key information essential for interpretation. Therefore, if the reviewer and editor agree, we would prefer to retain the tables in their current form, unless a specific alternative approach is suggested. To clarify the table information, we have included the following summary paragraph in the revised Results after the 1st paragraph of section 3.2. Factors associated with adipose tissue PBDEs and DP concentrations as bellow (Lines 275-279):

“Overall, we observed robust statistically significant inverse associations between BMI and concentrations of PBDEs and DP (Table 2). We also found robust positive associations between males and PBDEs (Table 2), perceived exposure to paints and PBDEs levels, notably BDE-47 and BDE-99 (Table 3), and fish consumption and PBDEs, specifically BDE-183 and -197 (Table 4). However, all these findings will be more extensively discussed below.”

The Conclusion section should be expanded to include more concrete recommendations and future research perspectives.

Following the reviewer’s suggestion, we have expanded the Conclusion section as follows (Lines 428-435):

“Our study revealed that exposure to PBDEs and DP in the GraMo cohort, as estimated through the analysis of adipose tissue samples, was ubiquitous in the study population, ranging pollutant levels between 73.1 for BDE-28 and 1,335.2 ng/kg lipid for BDE-153. Based on the consistency and robustness of the findings, sex, BMI, self-reported exposure to paints and solvents were identified as the main factors associated with exposure. These results might help further targeted public-health interventions by identifying modifiable factors associated with the exposure. However, due to the exploratory nature of this study, our findings needed to be confirmed in further research.”

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The exposure pathways of persistent organic pollutants (POPs) can be introduced in more detail and comprehensively.
Have there been any reports on historical exposure concentrations of persistent flame retardants in studies similar to this one? If so, they should be introduced in the introduction.
Why were the data of this study collected from July 2003 to June 2004 without the latest research data and comparison? This makes the study lack value. As we all know, with more than 20 years of social development, the risk exposure of pollutants has changed greatly.
Section 2.3 should be placed before the pollutant analysis, as this is an important research design background.
In Section 2.3, a "semi-quantitative questionnaire" was used for the dietary survey, but the reliability and validity of the questionnaire, the quantification standard of food portion size, etc., were not explained.
Although the discussion mentions that some results are consistent with previous studies, it lacks a systematic comparison. The comparison between the results and existing studies should be strengthened.
The conclusion is too simple. It should present the exposure level of pollutants, which type of pollutants are the main risk factors, and how to prevent and control them. The research focus of the two should also be elaborated.

Author Response

Response to Reviewer 3

Thank you for your constructive suggestions and comments on the manuscript. They have definitely helped us improve the final version. We have addressed all of them as follows:

1. The exposure pathways of persistent organic pollutants (POPs) can be introduced in more detail and comprehensively.

Following the reviewer’s suggestion, we have rewritten the 1^st paragraph of the revised introduction as follows (Lines 39-50):

“Persistent organic pollutants (POPs) are a myriad of chemicals that share a high resistance to degradation. As a result, they have a high potential for bioaccumulation in living organisms and biomagnification over the food chain [1,2]. In the general population, exposure to POPs occurs through multiple pathways, including dietary intake, particularly of animal-derived foods rich in lipids, ingestion of contaminated indoor dust, inhalation of polluted indoor and outdoor air, and dermal contact with consumer products and treated materials [2–4]. Polybrominated diphenyl ethers (PBDEs) and Dechlorane Plus (DP) merit special attention among POPs because they were extensively used as halogenated flame retardants across many products (e.g., textiles, plastics, building materials and electronic devices) [5–7], and they have been found to accumulate in indoor dust and human tissues and travel long distances in the environment [8].”

2. Have there been any reports on historical exposure concentrations of persistent flame retardants in studies similar to this one? If so, they should be introduced in the introduction.

We have incorporated the reviewer´s suggestion into the revised 4^th paragraph of section 1. Introduction, as detailed below (Lines 81-87):

“Although prior literature has already studied legacy exposure to PBDEs/DP [36–41], few have measured these compounds in adipose tissue [42], and have been conducted in Spain [43]. Moreover, hardly any study has comprehensively examined sociodemographic, occupational, dietary, and lifestyle determinants of adipose PBDE/DP levels. Our study addresses these gaps by quantifying PBDEs/DP in human adipose tissue from a Spanish cohort and by performing a detailed analysis of the factors associated with their concentrations.”

3. Why were the data of this study collected from July 2003 to June 2004 without the latest research data and comparison? This makes the study lack value. As we all know, with more than 20 years of social development, the risk exposure of pollutants has changed greatly.

The GraMo cohort is a multipurpose cohort funded by several competitively awarded projects over the years. Although the tissue samples were collected in 2003/2004, funding and approval for the PBDE/DP analyses were obtained at a later stage. We have acknowledged this aspect in the 15^th paragraph of the revised Results section concretely, section 3.2. (Factors associated with adipose tissue PBDEs and DP concentrations), as follows (Lines 405-412):

“Thirdly, the chemical exposure profile has changed over time, with some compounds now restricted and others newly introduced. Nevertheless, we consider that the exposure characterization presented in this work is highly relevant for two reasons. On the one hand, many of the compounds studied, although restricted, are still frequently detected in the population due to their high persistence and ubiquity, therefore posing public health concerns. On the other hand, this study represents a crucial first step toward longitudinal investigations of the health effects of this exposure, which are currently being conducted in the GraMo cohort.”

4. Section 2.3 should be placed before the pollutant analysis, as this is an important research design background.

Following the reviewer’s suggestion, we placed section 2.3 (Sociodemographic, lifestyle and clinical information) before section 2.2. (Laboratory analyses) in the revised Materials and Methods section.

5. In Section 2.3, a "semi-quantitative questionnaire" was used for the dietary survey, but the reliability and validity of the questionnaire, the quantification standard of food portion size, etc., were not explained.

We thank the reviewer for the suggestion. We have added more information about the semi-quantitative questionnaire used for the dietary survey in the revised Materials and Methods section, specifically, in the section 2.2 (Sociodemographic, lifestyle and clinical information) as follows (Lines 133-136):

“Dietary habits were assessed using a semi-quantitative questionnaire [51,52], including the following food groups: meat, cold meats, fats, fish, eggs, dairy products (excluding milk and cheese), milk, cheese, vegetables, legumes, fruits, bread, and pasta.”

6. Although the discussion mentions that some results are consistent with previous studies, it lacks a systematic comparison. The comparison between the results and existing studies should be strengthened.

We appreciate the reviewer’s suggestion. Although a systematic review falls outside the scope and primary objectives of the present manuscript, which are to describe PBDE/DP concentrations in adipose tissue and to identify factors associated with PBDE/DP concentrations in the GraMo cohort, we have strengthened the revised section Results and discussion, specifically, 2^nd and 3^rd paragraph of section 3.1. (Description of the study population and adipose tissue pollutant concentrations) as follows (Lines 247-249 and 255-262):

“In accordance with previous studies, BDE-153 was the predominant congener [10,30,32,72–79], while in other studies the most prevalent congener was BDE-47 [42,80,81].”

“No differences in bioaccumulation were observed between the two DP isomers. In our study, anti-DP was the predominant form with an average anti-DP fraction (an-ti-DP/total DP) of 0.75 ± 0.11 (data not shown), consistent with the reported propor-tions in commercial DP products ranging between (0.60-0.80) [83], and in prior studies in humans (sample size ranging from 20 to 105) ranging anti-DP fractions between 0.71 and 0.79 [11,36,84,85]. These findings may suggest no stereoselectivity in the entry into the human body or metabolism and primarily legacy exposure. However, in highly exposed occupational roles populations with lower anti-DP fractions have been re-ported [86,87]”

And also, in the 7^th paragraph of section 3.2. (Description of the study population and adipose tissue pollutant concentrations) as follows (Lines 318-322):

“These findings are consistent with previous studies identifying specific manual activities such as industrial manufacturing worker, construction or e-waste recycling sites have been identified as sources of PBDEs [103–105].”

7. The conclusion is too simple. It should present the exposure level of pollutants, which type of pollutants are the main risk factors, and how to prevent and control them. The research focus of the two should also be elaborated.

We thank the reviewer for their recommendations. We have modified the conclusion section as follows (Lines 428-435):

“Our study revealed that exposure to PBDEs and DP in the GraMo cohort, as estimated through the analysis of adipose tissue samples, was ubiquitous in the study population, ranging pollutant levels between 73.1 for BDE-28 and 1,335.2 ng/kg lipid for BDE-153. Based on the consistency and robustness of the findings, sex, BMI, self-reported exposure to paints and solvents were identified as the main factors associated with exposure. These results might help further targeted public-health interventions by identifying modifiable factors associated with the exposure. However, due to the explorative nature of this study, our findings needed to be confirmed in further research.”

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This study examines human exposure to persistent flame retardants. It uses stored fat samples from 134 adults in a Spanish cohort to explore how sociodemographic and lifestyle variables correlate with these pollutant levels. I have a few comments in this regard.

The study relies on samples collected nearly two decades ago (2003–2004).
The sample size is small.
The paper offers speculative physiological explanations for certain findings without supporting data. For instance, it suggests women’s lower PBDE levels might result from “a clearance process during pregnancy and breastfeeding”, yet the study did not collect any information on maternity or lactation history to verify this hypothesis.
The study documents associations between personal factors and chemical concentrations without linking these exposures to any health outcomes or risk metrics.
The manuscript inconsistently reports pollutant concentrations using different units.
The presentation of regression results across three separate tables by predictor category (Tables 2 - 4) is cumbersome.
The article would be more appealing if 1-2 figures were added.
“Secondly, although our hospital-based design may limit generalizability; there is no clear that our participants’ characteristics differ substantially from those of the broader population” - it should be rephrased.
The manuscript contains several grammatical errors that impede readability, e.g. “stablished”
Please do not start sentences with abbreviations.

Author Response

Response to Reviewer 4

Thank you for your constructive suggestions and comments on the manuscript. They have definitely helped us improve the final version. We have addressed all of them as follows:

1. The study relies on samples collected nearly two decades ago (2003–2004).

The GraMo cohort is a multipurpose cohort funded by several competitively awarded projects over the years. Although the tissue samples were collected in 2003/2004, funding and approval for the PBDE/DP analyses were obtained at a later stage. We have acknowledged this aspect in the revised Results section, concretely in the 15^th paragraph of section 3.2. (Factors associated with adipose tissue PBDEs and DP concentrations), as follows (Lines 405-412):

2. The sample size is small.

We acknowledge that the sample size is modest. However, this study is exploratory in nature. Therefore, our findings should be interpreted with caution and will require confirmation in future studies, as we have stated in the 15^th paragraph of section 3.2. (Factors associated with adipose tissue PBDEs and DP concentrations), as follows (Lines 394-400):

“This study has potential limitations to take into account in the interpretation of the results. Firstly, the study size, and especially the sex-stratified subsamples, was relatively small, although it was sufficient to detect trends warranting confirmation in larger cohorts. Secondly, although our hospital-based design may limit generalizability, the study population resembles the general adult population of the study area in several important aspects like sex distribution and prevalence of major risk factors such as smoking and alcohol consumption [50]”

This study also has important strengths such as the use of adipose tissue as biological matrix as stated in the 16^th paragraph of the same section, as follow (Lines 413-417).

“As previously mentioned, the use of adipose tissue for exposure assessment provides a highly accurate measure of long-term PBDE accumulation. This matrix also reduces intra-sample variability compared to other biological specimens such as serum, thereby minimizing random error and lowering the sample size needed to detect associations [33].”

3. The paper offers speculative physiological explanations for certain findings without supporting data. For instance, it suggests women’s lower PBDE levels might result from “a clearance process during pregnancy and breastfeeding”, yet the study did not collect any information on maternity or lactation history to verify this hypothesis.

As stated in the manuscript, this study is exploratory in nature and should be considered hypothesis-generating, in line with prior literature on persistent organic pollutants, including PBDEs and DP [1–5]. In revised results, specifically in section 3.2. (Factors associated with adipose tissue PBDEs and DP concentrations), we have clearly stated the explorative nature of our findings as follows (Lines 342-347, 368-374 and 383-386):

“We hypothesize that the negative associations observed may be explained by greater implementation or adherence to safety measures among individuals who perceive themselves as being at risk, as shown in previous studies [110–112]. This interpretation, although highly speculative, opens the door to future population-based interventions to confirm whether increasing awareness of exposure is effective for reducing the body burden."

“This is consistent with previous epidemiological studies that have associated meat consumption with PBDE levels [30,67,75]. However, in our population, an inverse association was found for BDE-99 and eggs (Table 4). Considering the limited biological plausibility and the exploratory nature of this study, we consider that this might be a chance finding or even a result of a confounding effect of a third uncontrolled variable in the models, since BDE-99 has been reported as the dominant congener in eggs [124].”

“Nonetheless, higher BDE-197 concentrations were positively linked with fruit consumption. Other congeners have been related to fruit through industrial processes such as “hydro-cooling” which involves the use of BDE-209 coated pallets [7,28]. Nevertheless, this finding has no precedent and, therefore, warrants further confirmation.”

We also have performed sensitivity analyses to assess the robustness of the findings on breastfeeding and PBDEs/DP in section 3.2. (Factors associated with adipose tissue PBDEs and DP concentrations), as follows (Lines 281-284):

“Furthermore, sensitivity analyses examining the association between PBDEs/DP and breastfeeding (dichotomized) showed inverse, although non-significant, trends, except of BDE-28 (data not shown).”

Additionally, we have performed sensitivity analyses for BMI-related findings in the section 3.2. (Factors associated with adipose tissue PBDEs and DP concentrations) as detailed below (Lines 307-313):

“The BMI was negatively associated with BDE-153, -183, -197 and syn- and anti-DP (Table 2). A sensitivity analysis entering BMI categorized in three categories as < 25 kg/m², 25-30 kg/m² and ≥ 30 kg/m², corroborated this negative trend (data not shown). Particularly, the category 25-30 kg/m² vs. < 25 kg/m² showed significant negative β coefficients ranging from -0.37 to -0.50 for BDE-183 and -197 and both syn- and anti-DP. The association for BDE-153 was also inverse but did not reach statistical significance (β = -0.27, p-value = 0.092).”

References

Wu, Z.; He, C.; Han, W.; Song, J.; Li, H.; Zhang, Y.; Jing, X.; Wu, W. Exposure Pathways, Levels and Toxicity of Polybrominated Diphenyl Ethers in Humans: A Review. Environ. Res. 2020, 187, 109531, doi:10.1016/j.envres.2020.109531.
Wu, N.; Herrmann, T.; Paepke, O.; Tickner, J.; Hale, R.; Harvey, E.; La Guardia, M.; McClean, M.D.; Webster, T.F. Human Exposure to PBDEs: Associations of PBDE Body Burdens with Food Consumption and House Dust Concentrations. Environ. Sci. Technol. 2007, 41, 1584–1589, doi:10.1021/es0620282.
Kim, J.; Son, M.; Shin, E.; Choi, S.-D.; Chang, Y.-S. Occurrence of Dechlorane Compounds and Polybrominated Diphenyl Ethers (PBDEs) in the Korean General Population. Environ. Pollut. 2016, 212, 330–336, doi:10.1016/j.envpol.2016.01.085.
Bramwell, L.; Harrad, S.; Abou-Elwafa Abdallah, M.; Rauert, C.; Rose, M.; Fernandes, A.; Pless-Mulloli, T. Predictors of Human PBDE Body Burdens for a UK Cohort. Chemosphere 2017, 189, 186–197, doi:10.1016/j.chemosphere.2017.08.062.
Estill, C.F.; Slone, J.; Mayer, A.; Chen, I.-C.; LaGuardia, M. Worker Exposure to Flame Retardants in Manufacturing, Construction and Service Industries. Environ. Int. 2020, 135, 105349, doi:10.1016/j.envint.2019.105349.

4. The study documents associations between personal factors and chemical concentrations without linking these exposures to any health outcomes or risk metrics.

We thank the reviewer for this observation. This paper represents the first step toward subsequent studies that will investigate associations with health outcomes and risk metrics. Indeed, the present paper aims to describe levels of PBDEs/DP and their associated factors, which will help identify confounders to be used in further research linking exposure to health outcomes. We have acknowledged that this work is the first step towards future investigations in the revised Results, concretely in the 15^th paragraph of section 3.2. (Factors associated with adipose tissue PBDEs and DP concentrations), as follows (Lines 405-412):

5. The manuscript inconsistently reports pollutant concentrations using different units.

We appreciate this comment and have revised all pollutant concentrations from manuscript to improve consistency and clarity. We acknowledge that we have used the same units (ng/g lipid) in the whole paper, except for table 1 (ng/Kg lipid). The decision was made in order to improve the readability of tables, avoiding the use of multiple “0” before or after the dot.

In the revised Materials and methods (section 2.4. Statistical analyses), we included this clarification (Lines 206-209):

“Chemical concentrations were reported in ng/Kg lipid for clarity in the descriptive analysis, whereas these variables were entered as ng/g lipid in the regression models to improve the interpretability of the model´s coefficients.”

In addition, we included a clarification in the footnote of Table 1 (Kubes 267-268).

“Pollutant concentrations were expressed as ng/Kg lipid in order to improve readability”

6. The presentation of regression results across three separate tables by predictor category (Tables 2 - 4) is cumbersome.

We agree that the tables contain a large amount of information. Following the reviewer’s suggestion, we carefully explored alternative ways of presenting them. However, we did not identify a format that would further simplify the tables without removing key information essential for interpretation. Therefore, if the reviewer and editor agree, we would prefer to retain the tables in their current form, unless a specific alternative approach is suggested. To clarify the table information, we have included the following summary paragraph in the Results section 3.2 (Factors associated with adipose tissue PBDEs and DP concentrations), as detailed below (Lines 275-279):

7. The article would be more appealing if 1-2 figures were added.

Following the reviewer’s recommendation, we have included additional visual elements such as the Figure 1 that represent the geographical distribution of the participants included in this study and Figure S1 related to the variable selection process used in this manuscript to improve reader understanding and engagement (Line 115).

Figure 1. Map of Granada province with participants’ distribution.

Figure S1. Manual step forward-backward model variables selection.

8. “Secondly, although our hospital-based design may limit generalizability; there is no clear that our participants’ characteristics differ substantially from those of the broader population” - it should be rephrased.

We thank the reviewer for pointing this out. We have made a correction in the 15^th paragraph of section 3.2. (Factors associated with adipose tissue PBDEs and DP concentrations). The sentence has been rephrased as follows (Lines 397-400):

“Secondly, although our hospital-based design may limit generalizability; the study population resembles the general adult population of the study area in several important aspects like sex distribution and prevalence of major lifestyle factors such as smoking and alcohol consumption [50].”

9. The manuscript contains several grammatical errors that impede readability, e.g. “stablished”.

We have carefully proofread the manuscript and corrected typographical and grammatical errors, including replacing “stablished” with “established” and fixing other instances.