Kinetic and Equilibrium Studies on the Adsorption of Lead and Cadmium from Aqueous Solution Using Scenedesmus sp.

Round 1

Reviewer 1 Report

 

This work describes the application of a new kind of biosorbent, a biomass obtained form a previously little studied algae type, for sorptive removal of heavy metals Pb and Cd from aqueous solutions. The work seem to be rather minimalistic, nevertheless the presented results can be sufficient for being considered for publication in Sustainability.

Minor corrections are necessary, including careful check of the grammar.

 

Minor corrections

 

1. line 55-56, Check the grammar of the sentence

2. line 40-41, The references 4,5, are not about water remediation from metals, but from organic pollutants. As for heavy metal ions, more relevant works should be cited, e.g. the following works, dealing with inorganic sorbent materials applied for Pb and Cd removal.

https://doi.org/10.1007/s11814-019-0262-6

https://doi.org/10.3390/app10082726

line 92, “ known amounts of metals …”

line 110, “that is 0.5-2 g l-1” replace with: “in the range 0.5-2 g l-1”

line 153, heterogeneous

line 202, delete the word “correspondingly”

line 390, “with amine groups”

 

Replace figures with clean versions, which do not contain background scales visible under the vertical axes titles.

 

Author Response

 

Reviewer 1

Dear editor and reviewers,

Thank you so much for reviewing the manuscript by providing your valuable comments and recommendations that has really improved the quality and structure of our manuscript. All suggestions/recommendations have been properly addressed as the reviewers suggested/asked and now we hope that the manuscript is highly improved. We again critically reviewed the manuscript for typos errors and other mistakes. If you recommend further suggestions, let us know, we will be very happy to address.

1. line 55-56, Check the grammar of the sentence

Response: Thanks for your quality and deep review. Now this line is more appropriate in the revised manuscript according to your worthy reviewer comment. Changes made have been highlighted yellow. (Line 87-88).

2. Line 40-41, The references 4,5, are not about water remediation from metals, but from organic pollutants. As for heavy metal ions, more relevant works should be cited, e.g. the following works, dealing with inorganic sorbent materials applied for Pb and Cd removal.

https://doi.org/10.1007/s11814-019-0262-6

https://doi.org/10.3390/app10082726

Response: Now new citations are done in revised manuscript accroding to worthy reviewer suggestion. Thanks for deep review and quality time . Changes made have been highlighted yellow. (Line 45, references 5 and 6).

3. line 92, “ known amounts of metals …”

Response: Thanks for your deep review and quality time. Now this line is more appropriate in the revised MS according to your worthy reviewer comment. Changes made have been highlighted yellow. (Line 147).

4. Line 110 “that is 0.5-2 g l-1” replace with: “in the range 0.5-2 g l-1”

Response: Thanks for your comments. Now this line is more appropriate in the revised MS according to your worthy comment. Changes made have been highlighted yellow. (Line 157).

 

5. line 153, heterogeneous

 Response: Thanks for your review. Now the word has been corrected in revised manuscript and highlighted as yellow. (Line 196).

6. line 202, delete the word “correspondingly”

Response: Thanks for your comments. Now this word has been removed in the revised MS according to your worthy comment and replaced with more appropriate word (highlighted yellow). (Line 246)

7. line 390, “with amine groups”

 

Response: Thanks for your suggestion. Now this line is more appropriate in the revised MS according to your comment highlighted as yellow. (Line 455)

8. Replace figures with clean versions, which do not contain background scales visible under the vertical axis titles.

 

Response: Thanks for your deep review. The figures have been replaced (see Fig. 2,4,5,6,7,8,9).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                                   

Reviewer 2 Report

The manuscript was found to highly structured and topic will be more relevant to current scenario. However, there are some few suggestions to improve the quality of the manuscript.

Apart from the points discussed in page 2 line 66 and 67, authors can state some more reasons, on what basis they have selected the Scenedesmus sp ?

Also, why the authors specifically selected lead and cadmium ?

Mention the impact of Pd and Cd on environment.

In Graphs, error bars can be added.... Some of the graphs are not clear, So please check the graphs and fix the ordinate and absscis.

How many trials were carried out? Statistical analysis can be added.

The results of the present study can be compared with the other Pb & Cd removal methods as well as other adsorbates.

 

Author Response

                                                                      Reviewer 2

Dear editor and reviewers,

Thank you so much for reviewing the manuscript by providing your valuable comments and recommendations that has really improved the quality and structure of our manuscript. All suggestions/recommendations have been properly addressed as the reviewers suggested/asked and now we hope that the manuscript is highly improved. We again critically reviewed the manuscript for typos errors and other mistakes. If you recommend further suggestions, let us know, we will be very happy to address.

Comment: Apart from the points discussed in page 2 line 66 and 67, authors can state some more reasons, on what basis they have selected the Scenedesmus sp ?

Response: Thanks for your review. Now the reason has been added in revised manuscript. Highly appreciated and highlighted as yellow. (Line 108-110).

Comment: Also, why the authors specifically selected lead and cadmium?

   Response: Thanks for your comments. The reason for selection of lead and cadmium has been written in manuscript and highlighted yellow. (Line 110-112)

Comment: Mention the impact of Pd and Cd on environment.

   Response: Thanks for your suggestion. The impact of Pb and Cd has been added in the revised according to your worthy reviewer comment. Changes made have been highlighted yellow. (Line 45-70, Figure 1).

Comment: In Graphs, error bars can be added.... Some of the graphs are not clear, So please check the graphs and fix the ordinate and absscis.

Response: Thanks for your comment. Now error bars have been added in all figures and graphs are clearer. (Figure 2,4,5,6,7,8,9)

Comment: How many trials were carried out? Statistical analysis can be added.

Response: The experiment was performed in triplicate. Statistical analysis has been added. (Figure 2,4,5,6,7,8,9)

Comment: The results of the present study can be compared with the other Pb & Cd removal methods as well as other adsorbates.

Response: Thanks for your suggestion. The present study has been compared with other biosorbent for the removal of lead and cadmium ions with the previous literature and highlighted as yellow (Line 408-415; table 3). Other methods for the removal of heavy metals are discussed and highlighted as yellow (Section 1; Line 71-74).

Reviewer 3 Report

Comments:

Journal ID: sustainability-2224301

I have reviewed the manuscript entitled “Kinetic and equilibrium studies on the adsorption of heavy metals from aqueous solution using Scenedesmus sp.”. Manuscript is well drafted but need following improvement before consider for publication.

Abstract

Authors should use the units for 85 and 83.

Authors should add some characterizations of the adsorbetns such as pHzpc, surface area, proton binding capacities etc.

Desorption study should be mention in terms of tangible data.

Introduction

Introduction is poorly drafted, authors should write about the Pb and Cd pollutions sources, its quantity in wastewater, what researchers currently doing for its remedy etc.

Another paragraph write about the Algal biomass, its production, current use, and why authors thinks its use as adsorbent is more important than any other use.

In second paragraph, “According to [8],   ………” what is it?

Cite these references for Cd and Pb removal doi: 10.5004/dwt.2020.26309, https://doi.org/10.1002/clen.201000185,

Cite references for algal species used in this sencentce, “There have been numerous prior studies that have used microalgal species such as Chlorella Vulgaris,…………………., as adsorbents.”

“These species have maximum adsorption capacities (qmax) exceeding 50 mg/g.” this adsorption capacity refers to which species against which metal ions?

In the last paragraph authors should clearly mention the objectives of this study.

Materials and method

Authors should give reason for keeping sample at 4 °C, what happed if kept room temperature (25 °C)?

Authors should mention which lab in university or research institute this study was conducted.

write the samples code prepared in this study.

sec 2.2, how authors now that algal adsorbent was amorphous?.

write the detection limit of the pollutants Cd and Pb analysis methods.

Section 2.6, which formula was used to calculate desorption and regenerations.

 

Results

Add standard deviation in each data point in Fig. 1, 2, 4, and 7.

Why scale of SEM images are not clear, whatever explanation written for SEM images are not clearl observed in the given images.

Conclusion

Conclusion is well written.

Author Response

Reviewer 3

Dear editor and reviewers,

Thank you so much for reviewing the manuscript by providing your valuable comments and recommendations that has really improved the quality and structure of our manuscript. All suggestions/recommendations have been properly addressed as the reviewers suggested/asked and now we hope that the manuscript is highly improved. We again critically reviewed the manuscript for typos errors and other mistakes. If you recommend further suggestions, let us know, we will be very happy to address.

Abstract

Comment: Authors should use the units for 85 and 83.

Response: Thanks for quality time and deep review. Corrections have been done in revised manuscript. (Line 22).

Comment: Authors should add some characterizations of the adsorbents such as pHzpc, surface area, proton binding capacities etc.

Response: Thanks for your comments. Yes, it has been added in abstract as well as materials methodology and results sections and highlighted yellow (Line 22-23).

Comment: Desorption study should be mention in terms of tangible data.

Response:  Thanks for deep review. Yes, it has been mentioned in the revised manuscript and highlighted yellow. (Line 26).

Introduction

Comment: Introduction is poorly drafted, authors should write about the Pb and Cd pollutions sources, its quantity in wastewater, what researchers currently doing for its remedy etc.

Response.   Thanks for your comments. The introduction has been thoroughly reviewed and improved wherever needed as per reviewer suggestion. Yes, it has been added to the introduction section and highlighted as yellow (Line 35-37 and 45; see figure 1).

Comment: Another paragraph write about the Algal biomass, its production, current use, and why authors thinks its use as adsorbent is more important than any other use.

Response. Thanks for your comprehensive review. The comment has been addressed as suggested by worthy reviewer and has been added in revised manuscript and highlighted yellow. (Line 98-106).

Comment: In second paragraph, “According to [8],   ………” what is it?

Response: Thanks for your comments. Now it has been corrected in revised manuscript and highlighted as yellow. (Line 78).

Comment: Cite these references for Cd and Pb removal doi: 10.5004/dwt.2020.26309, https://doi.org/10.1002/clen.201000185,

Response: Thanks for your comments. It has been added in revised manuscript and highlighted as yellow. (See 7 and 8 references).

Comment: Cite references for algal species used in this sentence, “There have been numerous prior studies that have used microalgal species such as Chlorella Vulgaris,…………………., as adsorbents.”

Response: Thanks for your suggestions. Citations have been added in revised manuscript and highlighted as yellow. (Line 94).

Comment: In the last paragraph authors should clearly mention the objectives of this study.

Response:  Thanks for worthy comments it has been done in revised MS and highlighted as yellow. (Line 113-117).

                                           Materials and method

Comment: Authors should give reason for keeping sample at 4 °C, what happed if kept room temperature (25 °C)?

Response:  Thanks for comments. Yes, it can be place at room temperature. Because it is in powered form and not effected by temperature.

Comment: Authors should mention which lab in university or research institute this study was conducted.

Response:  Thanks for suggestions. It has been mentioned in revised manuscript and highlighted as yellow (Line 124).

Comment: write the samples code prepared in this study.

Response: Thanks for your comments. It has been written in revised manuscript and highlighted as yellow (Line 122 and 224).

Comment: sec 2.2, how authors now that algal adsorbent was amorphous?.

Response: Thanks for your comments. Algal biosorbent is in powered form and their processing are written in section 2.1. (Line 131-133). Algal biosorbent is dried and powered. Algal biosorbent is not used in living form it is used in immobilized form which has not define shape. SEM also confirms that it is amorphous form.

Comment: write the detection limit of the pollutants Cd and Pb analysis methods.

Response:  Thanks for your comments. It has been written in revised manuscript and highlighted as yellow (Line 161).

Comment: Section 2.6, which formula was used to calculate desorption and regenerations.

Response:  Thanks for your suggestions. It has been added in revised manuscript and highlighted as yellow. (Equation 6 and Equation 7; Line 219 and 220).

Results

Comment: Add standard deviation in each data point in Fig. 1, 2, 4, and 7.

Response: Thanks for deep review and quality time. Thanks for worthy comments. See figure 2, 4, 5, 6, 9.

Comment: Why scale of SEM images are not clear, whatever explanation written for SEM images are not clearly observed in the given images.

Response: Thanks for your comments. Now SEM images has improved and in Pakistan we have instruments which give images of these types. We have not any advanced instruments.

Conclusion

Comment: Conclusion is well written.

Response: Thanks for quality time and deep review. Yours suggestions are valuable to our manuscript. We critically reviewed the, revised and improved the conclusion section.

Reviewer 4 Report

Kinetic and equilibrium studies on the adsorption of heavy metals from aqueous solution using Scenedesmus sp.

The topic of the manuscript “Kinetic and equilibrium studies on the adsorption of heavy metals from aqueous solution using Scenedesmus sp.” is interesting and thoroughly investigated by the author. However, require certain clarifications/revision for improving the basics of the manuscript.       

1.               It is recommended to add an graphical abstract, which will help the readers to recognize this study.

2.               In the introduction section, examples of management strategies for heavy metals pollution should be given. There are recent reports of photocatalytic reduction of heavy metals in the literature, which are for reference (https://doi.org/10.1016/j.jclepro.2022.134589)

3.               The number of biological replicates and technical replicates of this study was not mentioned in the materials and methods.

4.               The interrelationship between the effect factors on adsorption of Pb and Cd by Scenedesmus sp was not mentioned in this study. It is recommended to optimize and model the adsorption performance of Pb and Cd using the response surface approach (RSM). The pH vaule, contact time, initial metal concentration and biomass concentration were modeled as independent factors to optimize the adsorption conditions. The analysis of RSM results has been recently reported in the literature for reference

 

5.                Added a Section 3.9 about implications of this work.

Comments for author File: Comments.pdf

Author Response

Dear editor and reviewers,

Thank you so much for reviewing the manuscript by providing your valuable comments and recommendations that has really improved the quality and structure of our manuscript. All suggestions/recommendations have been properly addressed as the reviewers suggested/asked and now we hope that the manuscript is highly improved. We again critically reviewed the manuscript for typos errors and other mistakes. If you recommend further suggestions, let us know, we will be very happy to address.

Comments and Suggestions for Authors

The topic of the manuscript “Kinetic and equilibrium studies on the adsorption of heavy metals from aqueous solution using Scenedesmus sp.” is interesting and thoroughly investigated by the author. However, require certain clarifications/revision for improving the basics of the manuscript.

1. It is recommended to add a graphical abstract, which will help the readers to recognize this study

Response: Thanks for your comment. Graphical abstract has been added in revised manuscript. Highly appreciated and highlighted as yellow. (Line 16-29 ).

2. In the introduction section, examples of management strategies for heavy metals pollution should be given. There are recent reports of photocatalytic reduction of heavy metals in the literature, which are for reference (https://doi.org/10.1016/j.jclepro.2022.134589)

   Response: Thanks for your comments. Examples of management strategies for heavy metals pollution has been written in manuscript and highlighted as yellow. (Line 70-74)

3. The number of biological replicates and technical replicates of this study was not mentioned in the materials and methods.

Response: Thanks for your review. The experiment was performed in triplicate and highlighted in yellow (Line 161). Statistical analysis has been added. (Figure 2,4,5,6,7,8,9)

4. The interrelationship between the effect factors on adsorption of Pb and Cd by Scenedesmus sp was not mentioned in this study. It is recommended to optimize and model the adsorption performance of Pb and Cd using the response surface approach (RSM). The pH value, contact time, initial metal concentration and biomass concentration were modeled as independent factors to optimize the adsorption conditions. The analysis of RSM results has been recently reported in the literature for reference.

   Response: Thanks for your suggestion. We have already addressed the comments of 4 reviewers. This paper is mandatory for degree. Your comments are of great value for us. We are working on several articles where we will analyze the data/ results through RSM to further enhance the structure and quality of our manuscript. Thanks for your deep review, suggestions and we highly appreciate your services.

5. Added a Section 3.9 about implications of this work

Response: Thanks for your comment. Implications have been written in 3.9 section and highlighted as yellow (486-492).

Author Response File: Author Response.pdf

Reviewer 5 Report

The study is a good as far as the phytoremediation aspects of heavy metals are concerned. Though, there are certain queries and confusions in the manuscript which must be addressed:

1. Language must be improved.

2. Many grammatical mistakes must be corrected

3. Title must be specific with the heavy metal uptake. As only two were taken into consideration. Introduction section needs to be improved.

4. There is confusing statements regarding biosoprtion and bioadsorption which has been used interchangeably.

5. Methodology must be revisited to avoid any confusions.

6. Sources and status of heavy metal pollution in poonch AKP.

7. Why was a test microalga taken from a stream???

8. Recent references of 2020, 2021, 2022, 2023 are missing.

9. The quality, language and the clarity of the manuscript must be improved.

 

 

 

 

Comments for author File: Comments.pdf

Author Response

Reviewer 5

Dear editor and reviewers,

Thank you so much for reviewing the manuscript by providing your valuable comments and recommendations that has really improved the quality and structure of our manuscript. All suggestions/recommendations have been properly addressed as the reviewers suggested/asked and now we hope that the manuscript is highly improved. We again critically reviewed the manuscript for typos errors and other mistakes. If you recommend further suggestions, let us know, we will be very happy to address.

1. Language must be improved.

Response: Thanks for your deep review. Now the manuscript has been thoroughly reviewed for English proficiency and improved wherever needed.

2. Many grammatical mistakes must be corrected

Response: Thanks for your suggestions. Grammatical mistakes have been corrected and improved wherever needed.

3. Title must be specific with the heavy metal uptake. As only two were taken into consideration. Introduction section needs to be improved.

Response: Thanks for your comments. Tile has been changed according to your suggestion and introduction has been improved (Line 33-112).

4. There is confusing statements regarding biosoprtion and bioadsorption which has been used interchangeably.

Response: Thanks for your comments. Adsorption is the physical adherence or bonding of ions and molecules onto the surface of the solid material. Biosorption is a subcategory of adsorption, where the sorbent is a biological matrix. Both can be used. In the literature both terms are used (Abdel -Aty et al., 2012; ayub et al., 2020).

5. Methodology must be revisited to avoid any confusions.

Response: Thanks for your comment. Methodology has been revised/improved and changes made have been highlighted as yellow (Line 135-225).

6. Sources and status of heavy metal pollution in poonch AKP.

Response: Thanks for your comments. The samples are collected from the stream of Poonch AJK. The stream is contaminated with animal waste, domestic waste, detergent and untreated sewage by the local people. Domestic waste, untreated sewage and animal manure comes into the stream. Animal manure is one of the diffusion routes of heavy metals and metalloids into the environment, where the soil can accumulate them. Heavy metals and metalloids can then be released into groundwater sources, be absorbed by crops, and enter the food chain with negative effects for human and animal health.

7. Why was a test microalga taken from a stream???

Response: Thanks for your comments. Stream is polluted by animal waste, domestic waste, detergent and untreated sewage by the local people. So, test organism collected from this stream has potential to tolerate stress and remove the contamination from water.

8. Recent references of 2020, 2021, 2022, 2023 are missing.

Response: Thanks for your comments. New references have been added (see references 3, 5, 6,7, 16 17,18).

9. The quality, language and the clarity of the manuscript must be improved.

Response: Thanks for your suggestions. The authors have improved language and clarity of manuscript and all the changes are highlighted in yellow.

 

 

 

 

Round 2

Reviewer 3 Report

Authors have revised it well, can be accepted for publication

Author Response

Dear editor and reviewer,

Thank you so much once again for your comprehensive review and valuable time. Your comments/ suggestions have really improved the structure and quality of our manuscript. We are happy to hear about your positive decision.

Reviewer 4 Report

The quality of the manuscript has been further improved. It can be accepted.

Author Response

Dear editor and reviewer,

Thank you so much once again for your comprehensive review and valuable time. Your comments/ suggestions have really improved the structure and quality of our manuscript. We are happy to hear about your positive decision.

Reviewer 5 Report

The manuscript has been improved but still a lot of scope for improvement. Implications part is not up to the mark. The authors are requested to address the raised queries. Language must be improved. 

Comments for author File: Comments.pdf

Author Response

Dear editor and reviewer,

Thank you so much once again for your comprehensive review and valuable time. Your comments/ suggestions have really improved the structure and quality of our manuscript. We have once again critically reviewed the manuscript for all types of minor mistakes in grammar, sentence structure etc and made necessary changes wherever needed to further enhance the structure and quality of manuscript. We are happy to hear about your decision.

Comments and Suggestions for Authors

The manuscript has been improved but still a lot of scope for improvement. Implications part is not up to the mark. The authors are requested to address the raised queries. Language must be improved. 

L20: What is PHPZC?

Response: Thanks for your quality and in-depth review. According to your worthy reviewer's comment, PZC is point zero charge has been written in the revised manuscript. Highly appreciated and highlighted in yellow. (Line 39).

      What is Grahical?

Response: Thanks for your quality review. The Grahical word has been corrected (Graphical) in revised manuscript highlighted as yellow. (Line 16).

Line 44: Use grazing food chain instead of human food chain?

Response: Thanks for your valuable comment. Human food chain has been replaced by grazing food chain in the revised MS according to your worthy reviewer's comment. Highly appreciated and highlighted in yellow. (Line 53).

 Line 54: I don’t agree with the only single reference regarding density it is more than 5 gr cm-3…….?

Response: We appreciate your comment. This sentence has been corrected and more citations are added and highlighted as yellow (Line 54).

Line 53: It is not correct to start the sentence with numeric. Rewrite the sentence.

Response: Thank you for your suggestion. Now this sentence is more appropriate in the revised MS according to your worthy reviewer comment. Highly appreciated and highlighted in yellow. (Line 61).

Line 54: HM where denoted in the text?

Response: Thanks for the comment. HM means heavy metals and denotes in line 52 and highlighted in yellow. (Line 52).

Line 61-62: Rewrite the sentence……..

Response: Thank you for the kind comment. Now this line is more appropriate in the revised MS according to your worthy reviewer comment. Highly appreciated and highlighted in yellow. (Line 67-68)

Line 83: It is copy pasted text. Recently microalgae are regarded?

Response: Thank you for the comment. Now this sentence is more appropriate in the revised MS according to your worthy reviewer comment. Highly appreciated and highlighted as yellow. (Line 85-86).

Line 83-106: No continuity in sentence. Seems it is taken from where else…Line 96-97 no reference in the support.

Response: Thanks for your review. Now these lines are connected in the revised MS according to your worthy reviewer comment. Highly appreciated and highlighted as yellow. (Line 95-110). In Line 96-97 citation has been added now these lines are105-107.

Line 104-109: Botanical names are not following scientific protocol.

Response: Thanks for your deep review. Now species names have been written in italic and highlighted as yellow. (111-112).

 Line 111-121: Not continuity with previous paragraph?

Response: Thanks for your in-depth review. Now this paragraph is added in appropriate place and show continuity with the previous paragraph in the revised MS according to your worthy reviewer comment. Highly appreciated and highlighted as yellow. (Line 86-94).

Line 118: Green algae are found grammatical mistake.

Response: Thanks for your kind review. The sentence has been modified as per your suggestion and now, this line is more appropriate. Please refer to the revised manuscript file. (Line 92-93)

Line 122: A few places Ld and Pb are used. Please follow single protocol.

Response: Thanks for your in-depth review. Now lead and cadmium has been written in revised manuscript and highlighted as yellow.

Line 123: What is AJK. Elaborate it

Response: Thanks for your deep review. AJK is Azad Kashmir and has been elaborated in in the revised MS according to your worthy reviewer comment. Highly appreciated and highlighted as yellow. (Line 118, 132 and 525).

Line 124: It showed or shows?

Response: Thanks for your review. Now this word has been corrected in revised manuscript and highlighted as yellow (Line 119).

Line 131-138: Seems copy pasted from the thesis.

Response: Thanks for your comment. Now these lines are rephrased in revised manuscript and highlighted as yellow (Line 124-129).

Line 161-167: Huge grammatical mistake

Response: Thanks for your review. Now these lines have been corrected and highlighted as yellow (Line 149-154).

Line 173: under of with?

Response: Thanks for comments. Now conditions have been added highlighted as yellow (Line 159-160).

Line 180: At certain places Pb and Cd are used. While other places lead and cadmium are used

Response: Thanks for comments. Now lead and cadmium have been added in revised manuscript and highlighted as yellow.

Line 215: This?

Response: Thanks for review. This has been deleted in revised manuscript (Line 196).

Line 287: Again cadmium and lead

Response: Thanks for comments. Now lead and cadmium have been added (uniform pattern) in revised manuscript and highlighted as yellow.

Line 364: Again cadmium and lead

Response: Thanks for comments. Now lead and cadmium are in uniform pattern in revised manuscript and highlighted as yellow.

Line 368: Follow one protocol. Either use full form of metal or short form

Response: Thanks for deep review.  Now lead and cadmium are in full form in revised manuscript and highlighted as yellow.

Line 399: Again

Response: Thanks for deep review.  Now lead and cadmium are in full form in revised manuscript and highlighted as yellow.

Line 539-546: What is molecular aspect? Molecular studies is based on DNA…..Please remove unnecessary stuffing?

Response: Thanks for deep review.  Now unnecessary line has been deleted in revised manuscript (Line 502).