Evaluation of Environmentally Important Elements from Glacial Ice-Water and Associated Glacial Sediments

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

This study analyzes environmentally significant elements (nutrients, heavy metals) and microbial communities in glacial ice-water and sediments from the HKH region of Pakistan, revealing their distribution patterns and microbial ecological adaptations, while noting current elemental concentrations comply with international safety standards but warrant attention to potential pollution sources. While the study provides valuable baseline data, addressing these limitations would enhance its scientific rigor and practical relevance for environmental management in vulnerable glacial regions.

Line 14: pristine——>"major" or“significant Line

17: “is forecasted”——>“has been forecasted"

Line 31: "Furthermore, cultivation techniques revealed diverse bacterial communities inhabiting the sampled glaciers.": It is suggested to specify the type of cultivation techniques used, such as “microbial cultivation techniques”.

Line 40: Literature review lacks specificity regarding microbial studies in Pakistani glaciers, weakening contextual grounding.

Line 124: Sampling limited to surface layers (0–10 cm) ignores deeper ice and seasonal variations, reducing generalizability.

Line 176: 16S rRNA sequencing lacks details on sequencing depth, quality control, and database alignment thresholds, raising concerns about result reliability.

Line 359: Arsenic/lead enrichment attributed vaguely to "geogenic/anthropogenic factors" without direct evidence (e.g., local agricultural/industrial emission data).

Line 425: It is suggested to specify the potential consequences of bioaccumulation, such as "potentially affecting aquatic life and human health".

Line 427: It is recommended to suggest specific approaches to understanding these risks, such as "through long-term monitoring and risk assessment models".

Author Response

This study analyzes environmentally significant elements (nutrients, heavy metals) and microbial communities in glacial ice-water and sediments from the HKH region of Pakistan, revealing their distribution patterns and microbial ecological adaptations, while noting current elemental concentrations comply with international safety standards but warrant attention to potential pollution sources. While the study provides valuable baseline data, addressing these limitations would enhance its scientific rigor and practical relevance for environmental management in vulnerable glacial regions.

Thank you for your comprehension and for encouraging remarks about this work.

Line 14: pristine——>"major" or “significant Line Pristine is changed with significant.

17: “is forecasted” ——> “has been forecasted" The change has been made accordingly.

Line 31: "Furthermore, cultivation techniques revealed diverse bacterial communities inhabiting the sampled glaciers.": It is suggested to specify the type of cultivation techniques used, such as “microbial cultivation techniques”. Modification has been made accordingly.

Line 40: Literature review lacks specificity regarding microbial studies in Pakistani glaciers, weakening contextual grounding. For the needful comparison with our findings, several studies on Pakistani glaciers have now been cited – in the discussion section – please see L521-615.

Line 124: Sampling limited to surface layers (0–10 cm) ignores deeper ice and seasonal variations, reducing generalizability. We agree with the reviewer and so plan our future study including the deep layers too. Currently the data is about surface dynamics, so we request to consider it accordingly.

Line 176: 16S rRNA sequencing lacks details on sequencing depth, quality control, and database alignment thresholds, raising concerns about result reliability. The 16S rRNA gene of bacterial isolates (n = 9) was amplified using universal primers (27F and 1492R). PCR reactions were performed in 25 µL volumes with initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30 sec, 55°C for 30 sec, 72°C for 1.5 min, and a final extension at 72°C for 10 min. Sanger sequencing was performed by Macrogen (Korea), each sequence was read at an average depth of 500–800 bp with double-end reads to ensure accuracy. Sequences were manually validated using Chromas software (version 2.6.5). Sequences were compared against the NCBI database using the Basic Local Alignment Search Tool (BLAST) to identify the closest matching bacterial species. Multiple sequence alignments were performed using ClustalW in MEGA 11 (version 11.0.10). A Neighbor-Joining tree was constructed with the Maximum Composite Likelihood model, and 1000 bootstrap replicates were applied to evaluate branch support. Partial sequences of isolated glacier-associated bacterial strains were submitted to NCBI under accession numbers PQ805136–PQ805144. L234-255 of the revised version.

Line 359: Arsenic/lead enrichment attributed vaguely to "geogenic/anthropogenic factors" without direct evidence (e.g., local agricultural/industrial emission data). The data is now supported with mineralogical studies of the study area, please see L429-504 of the revised manuscript.

Line 425: It is suggested to specify the potential consequences of bioaccumulation, such as "potentially affecting aquatic life and human health". Our study did not intend to evaluate the human or other biological risk, so such discussion may deviate from the focus and detract the reader from the environmental perspective of this work. Please see L668-72.

Line 427: It is recommended to suggest specific approaches to understanding these risks, such as "through long-term monitoring and risk assessment models". In continuation to the above, we fear to discuss the dedicated risks but, yet we have mentioned the study-limitations and recommended the long-term monitoring via risk assessment models. Please see L668-72.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

 Comment

 The article “Evaluation of Environmentally Important Elements from Glacial Ice-Water and Associated Glacial Sediments” submitted to Journal Eath by  Authors: Kashmala Jadoon, Syeda Fazoon Kazmi, Sidra Arshad, Noor ul Huda Sajid, Adnan Ahmad Tahir, Özgür Doğan, Alidehou Jerrold Agbankpe, Rashid
Nazir is devoted to the global problem of glacier conservation, which is especially relevant for countries located in the “dust belt”. The authors consider a number of glaciers that are important for providing clean water for the population.

Glaciers are the pristine sources of fresh water on planet earth. And, Hin-dukush-Karakoram-Himalayan (HKH) glaciers provide water supply to more than a half human population of this globe, to agricultural activities, biodiversity survival and ecosystem services.

 

There are predictions that the loss of glacial ice will cause problems such as sea level rise, changes in water availability, and the release of pollutants that are on or within the surface of glaciers.  In these processes, mineralogical deposits play a significant role in glacier geochemistry and element cycling.  The authors of this paper analyzed various nutrients and elemental pollutants found in glaciers of Pakistan and studied the diverse bacterial communities inhabiting them.  The authors investigated nine elements including co-factors, heavy metals and nutrients in selected samples from Gilgit, Hunza and Swat glaciers. Using atomic absorption spectrophotometry a number of significant results were obtained. The authors found that higher concentration of these elements in the ice of Gilgit glacier and sediments of Swat glacier (except chromium and iron). The concentration of chromium and iron was found to be higher in Gilgit sediments. The amount of Cu and Cr appeared to be similar in the ice of Swat glacier and the foot of Swat glacier.  However, the concentration of some elements (As, K, Pb, Zn) is higher in the ice of the Swat glacier, and the amount of some elements (Cd, Ni) is higher in the foot of the Swat glacier. The detected elements are within the permissible limits set by WHO and USEPA. The authors used culturing techniques and identified a variety of bacterial communities inhabiting the selected glaciers. The study showed high homology (99-100%) with previously reported species.

 

1.Please specify the period of sample collection.

2.If possible, please indicate the ecological risk of arsenic, lead and cadmium according to the results of the study

3.How to explain the high concentration values of some elements (As, K, Pb, Zn) of Swat glacier ice and high concentration values of elements (Cd, Ni) at the foot of Swat glacier. Is it possible to indicate the sources of input of the mentioned elements on the studied glaciers?

4.It would be illustrative if in the table comparing the results of the present study on heavy metals for the three studied glaciers also include the results of other authors. heavy metals for the three glaciers studied, also include results of other authors, comparison with WHO and USEPA limits of permissible concentration.

5. Apart from the necessary further interdisciplinary research, what can you recommend for glacier conservation based on the results of your research?

In general, the article corresponds to the objectives of the journal, written in clear language.
The structure of the article is consistent. The actual material and the results of the study have elements of scientific novelty and practical significance. The modern precision method of research was used. The list of references is sufficient.

I recommend for publication after minor revision

 

Author Response

Dear reviewer, thank you for your time and efforts to evaluate our manuscript. The valuable comments and suggestions have significantly improved the quality of our work.

Below, we provide responses to the comments and outline the revisions made, accordingly.

Red, italicized text represents our response to the reviewers’ feedback in black non-italic text.

 

The article “Evaluation of Environmentally Important Elements from Glacial Ice-Water and Associated Glacial Sediments” submitted to Journal Eath by  Authors: Kashmala Jadoon, Syeda Fazoon Kazmi, Sidra Arshad, Noor ul Huda Sajid, Adnan Ahmad Tahir, Özgür Doğan, Alidehou Jerrold Agbankpe, Rashid Nazir is devoted to the global problem of glacier conservation, which is especially relevant for countries located in the “dust belt”. The authors consider a number of glaciers that are important for providing clean water for the population. Thank you for your comprehension and for encouraging remarks about this work.

Glaciers are the pristine sources of fresh water on planet earth. And, Hindukush-Karakoram-Himalayan (HKH) glaciers provide water supply to more than a half human population of this globe, to agricultural activities, biodiversity survival and ecosystem services. There are predictions that the loss of glacial ice will cause problems such as sea level rise, changes in water availability, and the release of pollutants that are on or within the surface of glaciers.  In these processes, mineralogical deposits play a significant role in glacier geochemistry and element cycling.  The authors of this paper analyzed various nutrients and elemental pollutants found in glaciers of Pakistan and studied the diverse bacterial communities inhabiting them.  The authors investigated nine elements including co-factors, heavy metals and nutrients in selected samples from Gilgit, Hunza and Swat glaciers. Using atomic absorption spectrophotometry a number of significant results were obtained. The authors found that higher concentration of these elements in the ice of Gilgit glacier and sediments of Swat glacier (except chromium and iron). The concentration of chromium and iron was found to be higher in Gilgit sediments. The amount of Cu and Cr appeared to be similar in the ice of Swat glacier and the foot of Swat glacier.  However, the concentration of some elements (As, K, Pb, Zn) is higher in the ice of the Swat glacier, and the amount of some elements (Cd, Ni) is higher in the foot of the Swat glacier. The detected elements are within the permissible limits set by WHO and USEPA. The authors used culturing techniques and identified a variety of bacterial communities inhabiting the selected glaciers. The study showed high homology (99-100%) with previously reported species. Thank you for your comprehension and for encouraging remarks about this work.

1. Please specify the period of sample collection. Modification has been made accordingly, please see L177-9 of the revised submission.
2. If possible, please indicate the ecological risk of arsenic, lead and cadmium according to the results of the study. Our study did not intend to evaluate the human or other biological risk, so such discussion may deviate from the focus and detract the reader from the environmental perspective of this work. Yet, we revised the description of our results in accordance with the minerology of the region. Please see L668-72.
3. How to explain the high concentration values of some elements (As, K, Pb, Zn) of Swat glacier ice and high concentration values of elements (Cd, Ni) at the foot of Swat glacier. Is it possible to indicate the sources of input of the mentioned elements on the studied glaciers? For the needful comparison with our findings about heavy metals, several studies on Pakistani glaciers have now been cited – in the discussion section – please see L521-615.
4. It would be illustrative if in the table comparing the results of the present study on heavy metals for the three studied glaciers also include the results of other authors. heavy metals for the three glaciers studied, also include results of other authors, comparison with WHO and USEPA limits of permissible concentration. As per literature, different studies evaluated different medium like soil, ice, meltwater, sediments, river water and/or lake water. So, the comparison would complicate the overall message. Though, we have prepared a supplementary table-1 for our ice-water samples in comparison with WHO and USEPA limits – just as an idea.
5. Apart from the necessary further interdisciplinary research, what can you recommend for glacier conservation based on the results of your research? Briefly added recommendation, please see L678-9.

In general, the article corresponds to the objectives of the journal, written in clear language.
The structure of the article is consistent. The actual material and the results of the study have elements of scientific novelty and practical significance. The modern precision method of research was used. The list of references is sufficient. I recommend for publication after minor revision. We appreciate your supportive remarks...!

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Please check attached file for details

Comments for author File: Comments.pdf

Author Response

Dear reviewer, thank you for your time and efforts to evaluate our manuscript. The valuable comments and suggestions have significantly improved the quality of our work.

Below, we provide responses to the comments and outline the revisions made, accordingly.

Red, italicized text represents our response to the reviewers’ feedback in black non-italic text.

 

The study "Evaluation of Environmentally Important Elements from Glacial Ice-Water and Associated Glacial Sediments" fills a significant gap by examining contaminants and microbial diversity in Pakistan's glaciers, an area with scarce existing data. The integration of geochemical analysis with microbiological characterization enhances the environmental evaluation. To augment scientific rigour, the authors employed conventional analytical methods, including Atomic Absorption Spectrophotometry (AAS) and 16S rRNA phylogenetic analysis. The paper appears satisfactory; nonetheless, it has weaknesses and areas that require enhancement. The paper contains several minor flaws that need revision before acceptance. The manuscript's structure is coherent; nevertheless, certain portions, particularly the introduction, are overly packed with material and might benefit from refinement for enhanced clarity. Thank you for your remark, and yes, we have improved the revised manuscript to overcome the flaws you mentioned.

• The abstract contains grammatical errors and poor phrasing (e.g., "reside on the surfaces of glaciers or within them") that require correction for improved reading. Phrase is revised for better comprehension, please see L31-2.
• The methods section is deficient in comprehensive details regarding sample collection protocols, preservation techniques, and analytical procedures. To ensure repeatability, delineate sample sizes, the quantity of repeats, and control measures. The methodology has been improved with further details, please see section 2.2 of the revised manuscript.
• The information regarding the microbial isolation technique and phylogenetic analysis is scant; including primer sequences, PCR conditions, and sequence accession numbers would enhance reproducibility. Along with literature citations, the information is further added, please see L235-255.
• The criteria for picking the nine examined elements require elucidation—are they determined by toxicity, prevalence, or additional factors? They were picked based on logistic support available. Sorry, the argument is not of the intellectual level but sometimes researchers are restricted for some reasons. Moreover, the selected elements represent a range of nutritional elements, cofactors and toxic heavy metals, so we believe to have a reasonable set of selected elements.
• The results are detailed in the text but might be enhanced by systematically arranged tables and figures depicting the element concentrations across various sites and sample types. We have 4-5 figure to systematically exhibit these results; table would just repeat the representation.
• Details regarding statistical analysis, such as significance testing and measures of variability, are absent. Incorporating them might facilitate the evaluation of the findings' strength. Please see L264-5 of the section 2.6.
• The similarities between the WHO and USEPA standards are briefly noted. A table that summarizes concentrations concerning these standards would improve understanding. we have prepared a supplementary table-1 for our ice-water samples in comparison with WHO and USEPA limits – just as an idea.

The discourse addresses microbial diversity and geochemistry but is deficient in depth about ecological ramifications, potential pollutant sources, and environmental risks. For the needful comparison with our findings about heavy metals, several studies on Pakistani glaciers have now been cited – in the discussion section – please see L521-615.

The assertion that elements are within acceptable limits is comforting but must be contextualized with possible cumulative or long-term effects. Our study did not intend to evaluate the human or other biological risk, so such discussion may deviate from the focus and detract the reader from the environmental perspective of this work. Yet, we revised the description of our results in accordance with the minerology of the region. Please see L668-72.

• The importance of microbial diversity studies, particularly concerning their ecological functions or prospective biotechnological uses, requires additional clarification. Numerous grammatical problems and typographical mistakes are evident (e.g., inconsistent tenses, punctuation discrepancies). Comprehensive language editing is advised. We have considered these points for our revised manuscript, thanks for the feedback.

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript titled “Evaluation of Environmentally Important Elements from Glacial Ice-Water and Associated Glacial Sediments” is an intriguing study that examines chemical elements in glacial water and solid phases and identifies bacterial communities capable of developing on three glaciers in Pakistan. In the 80 ice samples used in this investigation, nine chemical elements were determined. The acquired data went through statistical processing. One of the most serious criticisms is that the presented material is not consistent with the many referenced sources in the manuscript. Numerous references cited in the manuscript are not reliable. A summary of other important lacks is provided below. While many of the conclusions may be supported by the results, the manuscript in its current form necessitates major revisions before it can be recommended for publication.

Comments for author File: Comments.pdf

Author Response

Dear reviewer, thank you for your time and efforts to evaluate our manuscript. The valuable comments and suggestions have significantly improved the quality of our work.

The manuscript titled “Evaluation of Environmentally Important Elements from Glacial Ice-Water and Associated Glacial Sediments” is an intriguing study that examines chemical elements in glacial water and solid phases and identifies bacterial communities capable of developing on three glaciers in Pakistan. In the 80 ice samples used in this investigation, nine chemical elements were determined. The acquired data went through statistical processing. One of the most serious criticisms is that the presented material is not consistent with the many referenced sources in the manuscript. Numerous references cited in the manuscript are not reliable. A summary of other important lacks is provided below. While many of the conclusions may be supported by the results, the manuscript in its current form necessitates major revisions before it can be recommended for publication.

1. The abstract needs to be edited. In the abstract, it is not necessary to state the purpose of the study, but to concentrate on the novelty of the study, the methods, the results obtained and the prospects for their application. Instead of “The purpose of this study was to analyze different nutrients and elemental pollutants found in the glaciers of Pakistan and to investigate diverse bacterial communities residing therein.” should be used - “As a result of the study...”. Instead of “The samples (ice and sediments) were collected from Gilgit, Hunza and Swat glaciers.” - “The objects of the study were glaciers...”. Lines 339-345 are good for the abstract, you could move them.
2. The introduction needs reorganization. Give a very little information on the toxicological effects of the elements in question through drinking but please avoid much information on this (e.g. the arsenic which is well known). Explain why this is important for an international audience. Add information about similar glacial water chemistry studies around the world and their results.
3. Line 20. The purpose of this study was to analyze different nutrients... What exactly are the “different nutrients”, you have data only on potassium, but its role as a “nutrient” is not considered. It's better to remove it from the keywords as well.
4. Move lines 106-114 to the Introduction.
5. Figure 1A Try to avoid political coloring in your research paper, especially since your research regions have nothing to do with it. Indicate simply the territory of Pakistan, without dividing it into states.
6. In the methodology section, it would be advisable to put the detection limits of the methods used.
7. Lines 127-131 Does not match the sampling points in Figure 1. Indicate two sampling locations for Matiltan glacier in Swat.
8. Line 130-131 “Due to site limitations, only one sampling location was possible for the other two glaciers i.e., Ghamurbam (Gilgit) and Shispare (Hunza) [33]”. What is the purpose of the reference to sites in Turkey? [33] Candaş, A., Sarikaya, M. A., Köse, O., Şen, Ö. L., & Ciner, A. (2020). Modelling Last Glacial Maximum ice cap with the Parallel Ice Sheet Model to infer palaeoclimate in south-west Turkey. Journal of Quaternary Science, 35(7), 935-950.
9. Line 132. What tools did you use to drill the ice? Were they metal? Show that metal contamination from the tool was avoided.
10. Line 136 Upon arrival, melted ice samples were immediately filtered to minimize the contamination from container surfaces.
11. Line 137 Indicate the filter brand and pore size.
12. Line 150: City and country of equipment and reagent manufacturer.
13. Figures 2, 3. Please, explain what "abcdef" means in the figure captions. The meaning of these letters is not clear. In line 195 you indicate significant differences in figures are mentioned with statistical groups shown with different alphabets [37], but this is not enough. Decipher the meaning of these groups.
14. Move line 239 to the Introduction.
15. Need to be more specific. Line 339 “This study provides a comprehensive analysis of the elemental composition and microbial diversity present in glacial ice-water and associated sediments from three distinct glaciers in northern Pakistan.” You did not conduct “a comprehensive analysis of the elemental composition” in your results you only present concentrations of a few heavy metals. You do not specify water type, salinity, pH, etc. Be consistent in describing only what you did.
16. Line 349 “ANOVA” you have not previously specified this method anywhere. Add the information in 2.6 Statistical Analyses.
17. The references cited in the manuscript are questionable. The order of references in the text is probably incorrect. Lines 347-349 Are you sure the reference is correct? «The elemental analysis revealed that potassium (K) and iron (Fe) concentrations were significantly higher in sediments compared to ice-water samples (p < 0.001, ANOVA), just like earlier studies [38]» You are referring to a work that is not obvious: 38. Tian, H., Xu, R., Canadell, J. G., Thompson, R. L., Winiwarter, W., Suntharalingam, P., & Yao, Y. (2020). A comprehensive quantification of global nitrous oxide sources and sinks. Nature, 586(7828), 248-256.
18. Same for line 196: «Statistical significance was considered at p < 0.05, and significant differences in figures are mentioned with statistical groups shown with different alphabets [37].» 37. Bhatta, B. P., Khanal, M., & Malla, S. (2023). Whole genome and 16S rRNA dataset of Pectobacterium carotovorum strain 21TX0081 isolated from a symptomatic onion foliage in Texas. Data in Brief, 46, 108823. 533
19. 19. Lines 347–348. Water redox characteristics must be studied at the very least in order to determine the source of the elemental concentration in water. The genesis of elements cannot be inferred without an in-depth examination. The sources of the components in glacier waters aren't well explained by the paper. Just standard assumptions.
20. Line 356-358 in the Conclusion.
21. Line 358-361 to Introduction.
22. Line 365 You're referring to work: [44] «Khan, M., Ellahi, A., Niaz, R., Ghoneim, M. E., Tageldin, E., & Rashid, A. (2022). Water quality assessment of alpine glacial blue water lakes and glacial-fed rivers. Geomatics, Natural Hazards and Risk, 13(1), 2597-2617» speaking about “solubility of cadmium in glacial meltwater”. But there is no such information in this work. Refer to more reliable publications when considering the hydrochemistry of cadmium.
23. In Chapter 4.1. Elemental Composition and Its Environmental Significance, there is no discussion of elemental sources, only assumptions. Strengthen the discussion.
24. Supplementary file does not contain any tables. It is strange that there are no tables in the article with data on 80 samples. It is strongly recommended to add a table with values of K, Fe, As, Cd, etc. for the three studied glaciers.
25. Line 400. Incorrect reference [57] to work on Hg, you are not considering Hg.
26. Line 402. Incorrect reference [58] this paper did not consider Pb or As.
27. Same for reference [17], [61] and [62] work.
28. In the abstract you write: The elements detected are within permissible limits set by WHO and USEPA. But this is not discussed anywhere in the paper. It is only repeated in line 271. Give reference values for K, Fe, As, Cd, Pb, Zn, Ni, Cu and Cr, and compare your findings.
29. Line 83 IPCC 6th Assessment Report, 2022 no reference in the reference list.

Please see the revised submission, we tried our best to incorporate all your suggestions, Thanks.

Reviewer 5 Report (New Reviewer)

Comments and Suggestions for Authors

An interesting study on the presence of heavy metals and microorganisms in a glacial environment. Although the measured values do not exceed drinking water standards, they help characterize the contribution of glacial systems to water sources that will be used further downstream for drinking, industrial, or agricultural purposes. The authors cite human activity as the main reason for the high metal values. However, this overlooks the role of the geochemistry of the bedrock at the base of and around glaciers. The microbial composition is consistent with the chemical environment of the water and sediments.

Suggestion: The authors could enhance their manuscript by examining the mineralogical and geochemical environment of the bedrock eroded by glaciers.

Author Response

Dear reviewer, thank you for your time and efforts to evaluate our manuscript. The valuable comments and suggestions have significantly improved the quality of our work.

Below, we provide responses to the comments and outline the revisions made, accordingly.

Red, italicized text represents our response to the reviewers’ feedback in black non-italic text.

 

An interesting study on the presence of heavy metals and microorganisms in a glacial environment. Although the measured values do not exceed drinking water standards, they help characterize the contribution of glacial systems to water sources that will be used further downstream for drinking, industrial, or agricultural purposes. Thank you for your comments about this work.

The authors cite human activity as the main reason for the high metal values. However, this overlooks the role of the geochemistry of the bedrock at the base of and around glaciers. For the needful comparison with our findings, several studies on Pakistani glaciers have now been cited – in the discussion section – please see L521-615.

The microbial composition is consistent with the chemical environment of the water and sediments. Thank you for your comments, we have further enhanced the discussion on this aspect.

Suggestion: The authors could enhance their manuscript by examining the mineralogical and geochemical environment of the bedrock eroded by glaciers. For the needful comparison with our findings, several studies on Pakistani glaciers have now been cited – in the discussion section – please see L521-615.

Round 2

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

The authors have seriously revised the manuscript. But they did not respond to my comments point by point. I still have a lot of unanswered questions and remarks.

1. Line 61. Incorrect—"Shen“, Liu, Wang, & Adhikari, 2019”. Change to Shen et al., 2019 (dot).

2.      Line 133 does not match the sampling points in Figure 1. Please indicate two sampling locations for the Matiltan glacier in Swat.

3. Line 136: “Due to site limitations, only one sampling location was possible for the other two glaciers, i.e., Ghamurbam (Gilgit) and Shispare (Hunza) (Candaş et al., 2020).” You haven't changed anything from last time, the figures show three selection points for each of the areas!

4. Why do you refer to Candaş, A., Sarikaya, M. A., Köse, O., Şen, Ö. L., & Ciner, A. (2020)? What does this work relate to your sampling?

5. Figure 1A Try to avoid political coloring in your research paper, especially since your research regions have nothing to do with it. Indicate simply the territory of Pakistan, without dividing it into states. For example, see Khan et al., 2024.

6.      Figures 2, 3. Please explain what "abcdef" means in the figure captions. The meaning of these letters is not clear. Decipher the meaning of these groups.

7.      Need to be more specific. Line 356 “This study provides a comprehensive analysis of the elemental composition and microbial diversity present in glacial ice water and associated sediments from three distinct glaciers in northern Pakistan.” You did not conduct “a comprehensive analysis of the elemental composition.” In your results, you only present concentrations of a few heavy metals. You do not specify water type, salinity, pH, etc. Be consistent in describing only what you did.

8.      The references cited in the manuscript are questionable. «The elemental analysis revealed that potassium (K) and iron (Fe) concentrations were significantly higher in sediments compared to ice-water samples (p < 0.001), just like earlier studies (Tian et al., 2020).» You are referring to a work that is not obvious: Tian, H., Xu, R., Canadell, J. G., Thompson, R. L., Winiwarter, W., Suntharalingam, P., & Yao, Y. (2020). A comprehensive quantification of global nitrous oxide sources and sinks. Nature, 586(7828), 248-256.

9.      Same for lines 202-204: «Statistical significance was considered at p < 0.05, and significant differences in figures are mentioned with statistical groups shown with different alphabets [Bhatta].» B. P., Khanal, M., & Malla, S. (2023). Whole genome and 16S rRNA dataset of Pectobacterium carotovorum strain 21TX0081 isolated from symptomatic onion foliage in Texas. Data in Brief, 46, 108823. 533

10. Line 365 You're referring to work: «Khan, M., Ellahi, A., Niaz, R., Ghoneim, M. E., Tageldin, E., & Rashid, A. (2022). Water quality assessment of alpine glacial blue water lakes and glacial-fed rivers. Geomatics, Natural Hazards and Risk, 13(1), 2597-2617, speaking about “solubility of cadmium in glacial meltwater.” But there is no such information in this work. Refer to more reliable publications when considering the hydrochemistry of cadmium.

11. Double reference—43 and 67.

12. Line 159. Why do you refer to Bali, E., Aradi, L. E., Zierenberg, R., Diamond, L. W., Pettke, T., Szabó, Á., & Szabó, C. (2020) Geothermal energy  and ore-forming potential of 600 C mid-ocean-ridge hydrothermal fluids. Geology, 48(12), 1221-1225.?

13. Line 287 and supplementary Table S1—Add references to WHO and US EPA documents to the reference list.

14. Line 441. Incorrect reference. Shiozaki et al., 2020, did not consider Pb or As.

15. Line 83, IPCC 6th Assessment Report, 2022 no reference in the reference list.

16. Lines 65, 66, 68, 73, etc.—нет точек в предложениях.

17.  В тексте нет ссылки на Liu et al., 2020. [59]

The manuscript in its present form still needs revision before it can be recommended for publication.

 

Author Response

earth-3648187.r2

The authors have seriously revised the manuscript. But they did not respond to my comments point by point. I still have a lot of unanswered questions and remarks.

Dear reviewer, thank you for your comprehension and for encouraging remarks. We appreciate your time and efforts to evaluate our manuscript.

Below, herein, we provide responses to your comments and outline the revisions made, accordingly.

In response to your feedback in black non-italic text, red and italicized text represents our corrections.

 

1. Line 61. Incorrect—"Shen“, Liu, Wang, & Adhikari, 2019”. Change to Shen et al., 2019 (dot).

Corrected.

2. Line 133 does not match the sampling points in Figure 1. Please indicate two sampling locations for the Matiltan glacier in Swat.

For panel ‘3. Matiltan glacier (Swat region)’ each dot represents three different locations which were geographically indistinguishable (through 10-15m away from each other). While, 2- pink dots herein represent the samples from two distinct elevations (i.e., higher and lower), which were approximately 500 m apart.

3. Line 136: “Due to site limitations, only one sampling location was possible for the other two glaciers, i.e., Ghamurbam (Gilgit) and Shispare (Hunza) (Candaş et al., 2020).” You haven't changed anything from last time, the figures show three selection points for each of the areas!

As mentioned above, the pink dots represent the sampling locations in the glaciers’ surfaces. For 1 and 2, three dots mean the biological replicates of the samples collected from a location. While, for panel 3 (Matiltan glacier - Swat region)’ each dot represents three different locations which were geographically indistinguishable (through 10-15m away from each other). Figures are changes accordingly, please see the revised manuscript.

Why do you refer to Candaş, A., Sarikaya, M. A., Köse, O., Şen, Ö. L., & Ciner, A. (2020)? What does this work relate to your sampling?

Sorry for this recurrent mistake; the reference has now been removed.

5. Figure 1A Try to avoid political coloring in your research paper, especially since your research regions have nothing to do with it. Indicate simply the territory of Pakistan, without dividing it into states. For example, see Khan et al., 2024.

As suggested, the names of the states are removed from the map. Yet, the grey colour on the north side represents the HKH mountain-ranges spanning in Afghanistan, Pakistan, China and India. These demarcations have nothing to do with political boarders but are meant to represent the mountain glaciers of the region from where the study is reported – for reference to other researchers for better comprehension.

6. Figures 2, 3. Please explain what "abcdef" means in the figure captions. The meaning of these letters is not clear. Decipher the meaning of these groups.

These alphabets represent different statistical classes; the corrections have now been incorporated in the revision.

7. Need to be more specific. Line 356 “This study provides a comprehensive analysis of the elemental composition and microbial diversity present in glacial ice water and associated sediments from three distinct glaciers in northern Pakistan.” You did not conduct “a comprehensive analysis of the elemental composition.” In your results, you only present concentrations of a few heavy metals. You do not specify water type, salinity, pH, etc. Be consistent in describing only what you did.

Agreed, the more generalized statement has now been modified to specify the work we performed; please see L369 of the revised manuscript.

8. The references cited in the manuscript are questionable. «The elemental analysis revealed that potassium (K) and iron (Fe) concentrations were significantly higher in sediments compared to ice-water samples (p < 0.001), just like earlier studies (Tian et al., 2020).» You are referring to a work that is not obvious: Tian, H., Xu, R., Canadell, J. G., Thompson, R. L., Winiwarter, W., Suntharalingam, P., & Yao, Y. (2020). A comprehensive quantification of global nitrous oxide sources and sinks. Nature, 586(7828), 248-256.

Sorry for this recurrent mistake; the reference has now been revised with updated studies from the region. Please refer to L384-390.

9. Same for lines 202-204: «Statistical significance was considered at p < 0.05, and significant differences in figures are mentioned with statistical groups shown with different alphabets [Bhatta].» B. P., Khanal, M., & Malla, S. (2023). Whole genome and 16S rRNA dataset of Pectobacterium carotovorum strain 21TX0081 isolated from symptomatic onion foliage in Texas. Data in Brief, 46, 108823. 533

This inappropriate reference has been removed, thanks for the correction.

10. Line 456 You're referring to work: «Khan, M., Ellahi, A., Niaz, R., Ghoneim, M. E., Tageldin, E., & Rashid, A. (2022). Water quality assessment of alpine glacial blue water lakes and glacial-fed rivers. Geomatics, Natural Hazards and Risk, 13(1), 2597-2617, speaking about “solubility of cadmium in glacial meltwater.” But there is no such information in this work. Refer to more reliable publications when considering the hydrochemistry of cadmium.

Khan et al., (2022) reported the metals in glacial meltwater while Suska-Malawska et al., (2022) talked about spatial and in-depth distribution of heavy metals. With minor re-adjustment of the sentence text, the references are now appropriately corrected. Please see L452-4.

11. Double reference—43 and 67.

One removed, thanks for the correction.

12. Line 159. Why do you refer to Bali, E., Aradi, L. E., Zierenberg, R., Diamond, L. W., Pettke, T., Szabó, Á., & Szabó, C. (2020) Geothermal energy and ore-forming potential of 600 C mid-ocean-ridge hydrothermal fluids. Geology, 48(12), 1221-1225.?

The unnecessary reference is deleted, thanks for the notice.

13. Line 287 and supplementary Table S1—Add references to WHO and US EPA documents to the reference list.

These references have now been added in the list, please see the revised manuscript.

 

14. Line 441. Incorrect reference. Shiozaki et al., 2020, did not consider Pb or As.

The reference is corrected accordingly, please see L466 of the revised manuscript.

15. Line 83, IPCC 6th Assessment Report, 2022 no reference in the reference list.

The reference was included in the reference list of previous draft; here it is further made explicit. Please see L611-2.

16. Lines 65, 66, 68, 73, etc.—нет точек в предложениях.

All these typo mistakes are corrected; accordingly, please see the revised manuscript.

17. В тексте нет ссылки на Liu et al., 2020. [59]

The unnecessary refence is removed from the list, thanks for the notice.

 

The manuscript in its present form still needs revision before it can be recommended for publication.

We trust that the revisions outlined above satisfactorily address the reviewer's comments, and we appreciate the valuable feedback provided during the peer review process. The insightful suggestions have significantly improved the manuscript's quality, and we are thankful for the reviewer's expertise.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this paper, the authors studied the diversity and potential ecological significance of environmentally important elements and bacterial communities in glacial ice water and related glacial sediments in Gilgit, Hunza and Swat regions of northern Pakistan.

This paper provides valuable insights into the distribution of elements and bacterial diversity of glaciers in northern Pakistan, with comprehensive methods and detailed data, which are important for understanding the biogeochemical cycles and environmental risks of glacial ecosystems.    However, there are some deficiencies in the depth and breadth of the study, and the derivation of some conclusions lacks stronger evidence support.

I think this is just a survey and analysis of the article, the depth of the article is not enough.

First of all, the research depth is not enough.    This is reflected in the lack of in-depth analysis of element sources and limited microbial function analysis.

1) While the article mentions natural and anthropogenic sources of elements, there is no detailed analysis of the proportion and contribution of specific sources.    For example, for sources of arsenic and lead, only possible links to mining activities and coal burning are mentioned, but no quantitative analysis or specific chains of evidence are provided.

2) Although the diversity of bacterial communities was analyzed by 16S rRNA gene sequence, the specific functions of these bacteria in glacial ecosystems (such as nutrient cycling, metal tolerance mechanism, etc.) have not been thoroughly studied.    For example, while it is mentioned that certain bacteria have metal tolerance, its tolerance mechanism and specific role in a glacial environment are not detailed.

Second, the study sample and time are limited.

In this paper, only three glaciers were selected for sampling, and the representativeness of the samples may be insufficient.    There are numerous glaciers in northern Pakistan, and inferring the glacial environment of the entire region from samples of just three glaciers may be biased.    In addition, this paper only provides the sample analysis results of the current time point, and lacks long-term monitoring data to evaluate the changing trend of the glacier environment.    This is particularly important for understanding the environmental impact of glacial retreat.

Specific comments:

Line 15: I don't quite understand the “pristine sources of fresh water” statement, please check whether there is such a statement.

Line 19: The transition was a little abrupt.    Perhaps the authors should clarify how mineralogical sediments affect the mass balance of glaciers.    Or glacier changes/mass balance affecting the mineralogical sediments.

Lines 37-39: The first two sentences mainly describe the changes of glaciers, and the last sentence focuses on ecology.    It doesn't match the beginning and the end.

Line 82-89: This paragraph is not very logically related to the previous one.    At this point, I would like to see the current research status, what is the problem that the current research has not solved?

Lines 104-109: I don't think the purpose of the research is very clear, and I can't see the expected goal here.

Line 209: study-effort——>study

Line 212: You shouldn't end a sentence with a colon.

Line 319: The discussion section can be divided into several sub-headings to make it easier for readers to read.

Lines 365-377: In comparison with elemental concentrations in Svalbard and Arctic glaciers, only concentration differences are mentioned, but the specific environmental and human factors that lead to these differences are not analyzed.

Line 452: It is possible to be more specific about which contaminants and potential risks.

Reviewer 2 Report

Comments and Suggestions for Authors

This is a good and in my opinion qualitative study that provides new data on the status of glacial meltwater in terms of heavy metals and microbial communities. This is an important study because global warming and melting of glaciers around the world will be a source of potential toxic elements and possibly potagenic bacterial strains. In order to predict the ecological state in mountain glacier areas, it is necessary to have information on pollutants and their sources in glaciers. The authors of this manuscript have carried out such studies on some glaciers in Pakistan. I have no formal comments on the manuscript and it can be accepted for publication after correction of some editorial deficiencies. In terms of scientificity, the manuscript is well constructed. Perhaps the English language should be revised. Also important is the need to expand the conclusion section. 

Reviewer 3 Report

Comments and Suggestions for Authors

This paper is not great because it is just very confused, I don’t think the authors have a clear research question that they are investigating. All of the sampling and things that were measured are quite random and uncoordinated. The authors need to think much more carefully about what they are actually doing here. It is really not clear how glacial sediments are relevant here and you do not describe anywhere how these samples were processed in order to extract biological materials, this makes no sense, it is not even clear WHERE the samples were taken from or even how many samples there are. There is just no information.

There has been a lot of work on bacterial communities on glaciers in the Himalaya and in Antarctica, none of this is mentioned or reviewed. This needs to go in the introduction. Aims are not well described, be SPECIFIC. Study are section is not good at all, I have no idea what this area looks like, why it was investigated, or how. The methods are very poorly justified there is nothing here at all, logic is completely missing. 2.6 says there was stat analysis but no results are presented at all. It is unclear how or why glacial sediments were looked at, where they were sampled from, or how they relate to underlying geology (which is not even mentioned). Results are not well presented, there is very little here, including no statistics on anything. Discussion is long but is not related to any EVIDENCE, this is just very general statements from the literature that say nothing at all.

Overall this is not great, there is no clear idea of what this study is about, how the data were collected and analysed, or why. The results are pretty much missing throughout. This paper needs to be looked at again and put together in a much more focused and coherent way. Please see the attached pdf.

Comments for author File: Comments.pdf