Mercury Levels in Hair of Domestic and Wild Animals

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall Assessment

The manuscript entitled “Mercury Levels in Hair of Domestic and Wild Animals” addresses an environmentally and socially important issue. The use of hair as a non-invasive biomarker of mercury (Hg) exposure in both domestic pigs and wild animals across Brazilian regions is relevant for biodiversity conservation, food safety, and public health. The study is timely and offers descriptive data across multiple regions.

However, the manuscript remains largely observational and descriptive. The statistical analysis is simplistic, the mechanistic discussion is limited, and the figures do not effectively convey the significance of findings. The manuscript would benefit from a more rigorous analytical framework and stronger connection to toxicological thresholds and biological mechanisms.

I recommend major revision before this manuscript can be considered for publication.

Major Comments. 

Abstract

1. The abstract should provide specific quantitative findings (highest vs. lowest Hg concentrations, statistical significance).

2. The statement that Hg levels are “not harmful” lacks support. Please compare with international toxicological thresholds (e.g., FAO/WHO standards).

Introduction

1. The introduction describes general Hg sources but does not contextualize sufficiently for the studied regions. Please integrate regional data on mining, industry, or agriculture.

2. Expand the rationale for using pigs as a sentinel species in food safety.

Materials and Methods

1. Clarify whether the washing procedure of hair adequately removes external contamination.

2. Provide details on handling and preservation of wild animal (roadkill) samples to ensure representativeness.

3. Statistical analysis is limited to one-way ANOVA. Given multiple influencing factors (species, location, rearing system), multivariate or mixed-effect models should be considered.

Results and Discussion

1. The differences among sows, piglets, and wild animals are presented descriptively but not mechanistically explained. Please discuss biological bases (diet, growth stage, metabolic turnover).

2. The discussion section is repetitive and lacks depth. Compare observed Hg levels with toxicological benchmarks. Explore mechanisms (oxidative stress, immune function, reproductive toxicity).

3. Compared with recent integrative approaches in trace-element studies, this work lacks exploration of how Hg exposure may link to physiological responses.

Figures and Data Presentation

1. Add statistical significance indicators (letters or bars) to Figures 2 and 3.

2. Present concentration ranges alongside mean ± SE to illustrate variability.

3. Consider summarizing data in tables for clarity.

Conclusion

1. Consider discussing how omics-based or biomarker approaches could enhance future research.

Minor Comments

1. Several references are outdated or incomplete. Update with recent toxicological and environmental monitoring literature.

2. Language is generally clear but repetitive in the discussion; consider condensing.

3. Data availability statement (“data cannot be shared”) does not meet journal transparency standards. Please provide at least summary datasets.

4. Ethics section is adequate but could further emphasize welfare considerations in the collection of roadkill samples.

Author Response

Authors' Responses to Reviewer's Comments (Reviewer 1)

Comments and Suggestions for Authors

Overall Assessment

The manuscript entitled “Mercury Levels in Hair of Domestic and Wild Animals” addresses an environmentally and socially important issue. The use of hair as a non-invasive biomarker of mercury (Hg) exposure in both domestic pigs and wild animals across Brazilian regions is relevant for biodiversity conservation, food safety, and public health. The study is timely and offers descriptive data across multiple regions.

However, the manuscript remains largely observational and descriptive. The statistical analysis is simplistic, the mechanistic discussion is limited, and the figures do not effectively convey the significance of findings. The manuscript would benefit from a more rigorous analytical framework and stronger connection to toxicological thresholds and biological mechanisms.

I recommend major revision before this manuscript can be considered for publication.

Major Comments.

Abstract

1. The abstract should provide specific quantitative findings (highest vs. lowest Hg concentrations, statistical significance). The Abstract has been duly adjusted as per the requests of reviewer 1

2. The statement that Hg levels are “not harmful” lacks support. Please compare with international toxicological thresholds (e.g., FAO/WHO standards). Rewrite in introduction (line 48 at 103)

Introduction

1. The introduction describes general Hg sources but does not contextualize sufficiently for the studied regions. Please integrate regional data on mining, industry, or agriculture. Rewrite in introduction (line 48 at 103)

2. Expand the rationale for using pigs as a sentinel species in food safety. (line 48 at 102)

Materials and Methods

1. Clarify whether the washing procedure of hair adequately removes external contamination. See on the text

2. Provide details on handling and preservation of wild animal (roadkill) samples to ensure representativeness. See on the text

3. Statistical analysis is limited to one-way ANOVA. Given multiple influencing factors (species, location, rearing system), multivariate or mixed-effect models should be considered. See on the text

The analysis of mercury levels in the hair of domestic and wild animals did not include multivariate or mixed-effect models in the methodology of this study, as such procedures were not planned in the original proposal. Considering the analyses performed and the results presented, it is understood that the information obtained is sufficient to justify and meet the objectives and scope defined by the project, according to its title.

Results and Discussion

1. The differences among sows, piglets, and wild animals are presented descriptively but not mechanistically explained. Please discuss biological bases (diet, growth stage, metabolic turnover). The farms did not provide feed for nutrient assessment. And there was no way to determine the dead animals.

2. The discussion section is repetitive and lacks depth. Compare observed Hg levels with toxicological benchmarks. Explore mechanisms (oxidative stress, immune function, reproductive toxicity). Redone. See on the text.

3. Compared with recent integrative approaches in trace-element studies, this work lacks exploration of how Hg exposure may link to physiological responses. This wasn't done because the farm owners wouldn't allow nutritional assessment. This information wasn't available for the dead animals either. Therefore, it wasn't done.

Figures and Data Presentation

1. Add statistical significance indicators (letters or bars) to Figures 2 and 3. On the text

2. Present concentration ranges alongside mean ± SE to illustrate variability. On the text

3. Consider summarizing data in tables for clarity. On the text

Conclusion

1. Consider discussing how omics-based or biomarker approaches could enhance future research. Yes, it's possible. However, it will depend on new proposals and the interests of other researchers in the field.

Minor Comments

1. Several references are outdated or incomplete. Update with recent toxicological and environmental monitoring literature. Now I hope it´s ok.

2. Language is generally clear but repetitive in the discussion; consider condensing. Now I hope it´s ok.

3. Data availability statement (“data cannot be shared”) does not meet journal transparency standards. Please provide at least summary datasets. We can´t now do it. May be on the future.

4. Ethics section is adequate but could further emphasize welfare considerations in the collection of roadkill samples. The animals were found on Highway 262. In the morning, those found were collected. The collection methodology followed the same as for farm animals. Samples were taken from the scapular region.

Reviewer 2 Report

Comments and Suggestions for Authors

This is a good and interesting review. Please look at these points:

1. Please make the sign 1 (Pato Bragada) in Figure 1 more clear.
2. Please use PR, MG, GO, MS in the Figure 1
3. Please explain the different colours in Figure 1
4. Some of countries name is not readable ibn Figure 1
5. Please give more news about collection season and weather conditions such as rainy days
6. How did you prevent the risk of mercury contamination to those conducting the study?
7. Please give more news about to collect hair samples. Did you collect same places and same weight for all animals?
8. Did you measure mercury on soil, plants and/or water in this field? Please use some sentences about the source of mercury contamination.
9. Please use more new literatures such as below:

• Determination of Heavy Metal Levels and Assessment of L. monocytogenes and Salmonella spp. Presence in Fishery Products and Mussels from the Marmara …(2025)   E Akkaya, K Muratoglu, D Tarhan, NP Ozsobaci, AM Ercan, H Colak, ...Toxics 13 (3), 153
• Human exposure to heavy metals and possible public health risks via consumption of mussels M. galloprovincialis from the Albanian sea cost (2024) E Ozuni, E Andoni, M Castrica, CM Balzaretti, G Brecchia, S Agradi, ... Chemosphere 368, 143689
• Use of hair as matrix for trace elements biomonitoring in cattle and roe deer sharing pastures in northern Italy (2024) S Draghi, NE Fehri, F Ateş, NP Özsobacı, D Tarhan, B Bilgiç, ... Animals 14 (15), 2209
• Goat hair as a bioindicator of environmental contaminants and adrenal activation during vertical transhumance (2023) S Agradi, A Munga, O Barbato, R Palme, D Tarhan, B Bilgiç, B Dokuzeylül, ... Frontiers in Veterinary Science 10, 1274081
• Roe Deer (Capreolus capreolus) Hair as a Bioindicator for the Environmental Presence of Toxic and Trace Elements (2023) S Draghi, S Agradi, F Riva, D Tarhan, B Bilgiç, B Dokuzeylül, AM Ercan, ... Toxics 11 (1), 49

Author Response

Authors' Responses to Reviewer's Comments (Reviewer 2)
Comments and Suggestions for Authors
This is a good and interesting review. Please look at these points:
1. Please make the sign 1 (Pato Bragada) in Figure 1 more clear. Change the map
2. Please use PR, MG, GO, MS in the Figure 1 Change the map
3. Please explain the different colours in Figure 1 Change the map
4. Some of countries name is not readable ibn Figure 1 Change the map
5. Please give more news about collection season and weather conditions such as rainy days – increase on the text
6. How did you prevent the risk of mercury contamination to those conducting the study?
All wore personal protective equipment (PPE). They had no contact with mercury. They only collected hair samples from the animals.
7. Please give more news about to collect hair samples. Did you collect same places and same weight for all animals? – increase on the text
8. Did you measure mercury on soil, plants and/or water in this field? Please use some sentences about the source of mercury contamination. – increase on the text
9. Please use more new literatures such as below:
• Determination of Heavy Metal Levels and Assessment of L. monocytogenes and Salmonella spp. Presence in Fishery Products and Mussels from the Marmara …(2025) E Akkaya, K Muratoglu, D Tarhan, NP Ozsobaci, AM Ercan, H Colak, ...Toxics 13 (3), 153 – used
• The others not used.
• Human exposure to heavy metals and possible public health risks via consumption of mussels M. galloprovincialis from the Albanian sea cost (2024) E Ozuni, E Andoni, M Castrica, CM Balzaretti, G Brecchia, S Agradi, ... Chemosphere 368, 143689
• Use of hair as matrix for trace elements biomonitoring in cattle and roe deer sharing pastures in northern Italy (2024) S Draghi, NE Fehri, F Ateş, NP Özsobacı, D Tarhan, B Bilgiç, ... Animals 14 (15), 2209
• Goat hair as a bioindicator of environmental contaminants and adrenal activation during vertical transhumance (2023) S Agradi, A Munga, O Barbato, R Palme, D Tarhan, B Bilgiç, B Dokuzeylül, ... Frontiers in Veterinary Science 10, 1274081
• Roe Deer (Capreolus capreolus) Hair as a Bioindicator for the Environmental Presence of Toxic and Trace Elements (2023) S Draghi, S Agradi, F Riva, D Tarhan, B Bilgiç, B Dokuzeylül, AM Ercan, ... Toxics 11 (1), 49

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all comments raised in the previous review round.

The revised manuscript shows clear improvements in structure, data presentation, and clarity of interpretation. The abstract and introduction have been strengthened, the discussion section is now more concise, and figures include statistical annotations and variability indicators as requested.

Although multivariate statistical models were not incorporated, the authors provided a reasonable justification based on the scope of the original study design. The response regarding mechanistic discussion and data availability is acceptable given the constraints described.

Overall, the manuscript is now scientifically sound and well organized. Minor language or phrasing issues can be refined during copy-editing.

I have no further major concerns and recommend the manuscript for publication in its current form.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for all revisions.