Assessment of Bioaccumulation of Heavy Metals and Their Ecological Risk in Sea Lettuce (Ulva spp.) along the Coast Alexandria, Egypt: Implications for Sustainable Management

Round 1

Reviewer 1 Report

I consider that the manuscript presented by the authors is well supported. The authors could do a better wording of the conclusions.

Author Response

Dear Reviewer,

We appreciate your comments and your time to improve our MS to be available to publish.

Comments and Suggestions for Authors

I consider that the manuscript presented by the authors is well supported. The authors could do a better wording of the conclusions.

Response: Many thanks for your support, and the conclusion has been revised.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript ID 2184472, titled: “Potential Risk Assessment of Heavy Metals in the Coastal  Aquatic Ecosystems of Alexandria, Egypt: The Need for Sustainable Management”.

 1.      The title needs to fit the content. The authors, in the aim of the work,  focused on Sea Lettuce growing in the coastal zones of Alexandria and some dominant Ulva species to provide a baseline for the studied trace elements.  It is not fit with the title.

2.      Line 268-271: (2.6. Bioaccumulation factor, BAF): “The BAF was evaluated to show how effective the algae were at accumulating trace elements from the sediment using Equation 6:

BAF = trace element concentrations in algae tissues (mg kg.1) / trace elements concentration in the sediment (mg kg.1) Equation 6. I ask the authors to explain how the sediment has a direct effect on trace elements accumulation in Ulva over the seawater, especially, in the selected study area,  most Ulva species are attached to the rocks. Please, revise ref. [59]. The nature of seaweed cells is not the same as plant cells.

 Here are some other comments in pdf.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

We appreciate your comments and your time to improve our MS to be available to publish.

Comments and Suggestions for Authors

The manuscript ID 2184472, titled: “Potential Risk Assessment of Heavy Metals in the Coastal Aquatic Ecosystems of Alexandria, Egypt: The Need for Sustainable Management”.

1. The title needs to fit the content. The authors, in the aim of the work, focused on Sea Lettuce growing in the coastal zones of Alexandria and some dominant Ulva species to provide a baseline for the studied trace elements.  It is not fit with the title.

Response: many thanks for your suggestion. We followed your comment and changed the title to be:

“Ecological Risk and Bioaccumulation Assessment in Sea Lettuce (Ulva spp.) Along the Coast of Alexandria, Egypt: Implications for Sustainable Management Applications”

2. Line 268-271: (2.6. Bioaccumulation factor, BAF): “The BAF was evaluated to show how effective the algae were at accumulating trace elements from the sediment using Equation 6:

BAF = trace element concentrations in algae tissues (mg kg.1) / trace elements concentration in the sediment (mg kg.1) Equation 6. I ask the authors to explain how the sediment has a direct effect on trace elements accumulation in Ulva over the seawater, especially, in the selected study area, most Ulva species are attached to the rocks. Please, revise ref. [59]. The nature of seaweed cells is not the same as plant cells.

Response: we found some published articles based on seawater (like Agarwal et al. 2022), and other based on both seawater and sediments (like Peng et all. 2022). So, we corrected and calculated according to both sediments and seawater as presented in Table 8

Agarwal, S.; Albeshr, M.F.; Mahboobb, S.; Atique, U.; Pramanick, P.; Mitra, A. Bioaccumulation Factor (BAF) of heavy metals in green seaweed to assess the phytoremediation potential. Journal of King Saud University - Science, 2022, 34 (5), 102078. https://doi.org/10.1016/j.jksus.2022.102078.

Peng, Z.; Guo, Z.; Wang, Z.; Zhang, R.; Wu, Q.; Gao, H.; Wang, Y.; Shen, Z.; Lek, S.; Xiao, J. Species-specific bioaccumulation and health risk assessment of heavy metal in seaweeds in tropic coasts of South China Sea. Sci Total Environ, 2022, 832, 155031. https://doi.org/10.1016/j.scitotenv.2022.155031.

Here are some other comments in pdf.

What type of algae? In all the MS, change to seaweed

Response: Thanks for your comment. We changed all in the revised MS to be seaweed, thanks!

At the first time, the scientific names should be full-written.

Response: done, thanks!

Improve English style to:

Monitoring trace elements in the environment is crucial due to their potential harm to human health

Response: done, thanks! (From Eric Brevik): The change suggested by the reviewer is fine, but there was nothing wrong with the English of the previously existing sentence. The reviewer is not correcting improper English, they are advocating for their personal preferred way to write.

Macroalgal blooms, known as green tides, are linked to coastal eutrophication and are becoming increasingly common across the globe [25]

Wrong sentence:

seaweed blooms not known as a green tide, there are many tides regarding the seaweed types of red, brown, and green. Please, correct

Response: Many thanks for your comment, we corrected!

Common Ulva species include Ulva lactuca, U. armoricana, U. rigida, U. prolifera, U. pertusa, U. fasciata and U. rotundata [26]

Correct English style and grammar

Response: The entire MS was reviewed again by our co-author Prof. Eric Brevik, who is a native English speaker from the USA. (From Eric) There is nothing wrong with the sentence identified here by the reviewer, so I do not know what the reviewer wants done to it.

The largest macroalgal blooms in China were recorded during the period 2007 to 2019 [28].

Delete.

Response: deleted, thanks!

Biofertilizers derived from seaweeds have been used in agriculture to improve soil management, water efficiency, nutritional strategies, and drought tolerance [40].

Use this ref. (https://doi.org/10.3390/life13010085).

[40] Ashour, M.; Al-Souti, A.S.; Hassan, S.M.; Ammar, G.A.G.; Goda, A.M.A.-S.; El-Shenody, R.; Abomohra, A.E.-F.; El-Haroun, E.; Elshobary, M.E. Commercial Seaweed Liquid Extract as Strawberry Biostimulants and Bioethanol Production. Life, 2023, 13, 85. https://doi.org/10.3390/life13010085

Response: we added your suggested article to be in the list of refs. and the sentence has been changed to

Biofertilizers derived from seaweeds have been used in agriculture to improve soil management, water efficiency, nutritional strategies, and drought tolerance [39,40].

Microalgae, as autotrophic microscopic cells, have many applications including pharmaceutical, nutraceutical, and renewable biofuel energy [41]. As a result, algae have a unique capacity to assess metal(loid)s contamination in marine ecosystems. Algal species have been utilized as metal biomonitoring agents all over the world [13].

delete

Response: We checked these sentences and found them to be correct.

Anyway, we deleted them as you wanted!

13. Haghshenas, V.; Kafaei, R.; Tahmasebi, R.; Dobaradaran, S.; Hashemi, S.; Sahebi, S.; et. al. Potential of green/brown algae for monitoring of metal (loid) s pollution in the coastal seawater and sediments of the Persian Gulf: ecological and health risk assessment. Environ Sci Pollut Res, 2020, 27(7), 7463-7475.‏ https://doi.org/10.1007/s11356-019-07481-0
14. Sarwer, A.; Hamed, S.M.; Osman, A.I.; Jamil, F.; Al‑Muhtaseb, A.H.; Alhajeri, N.S.; Rooney, D.W. Algal biomass valorization for biofuel production and carbon sequestration: a review. Environ Chem Letter, 2022, 20, 2797–2851. https://doi.org/10.1007/s10311-022-01458-1

In this study, we focused on sea lettuce growing in the coastal zones of Alexandria and some dominant Ulva species to provide a baseline for the studied trace elements.

Correct English style and grammar

Moreover, correct to "Sea Lettuce"

Response: (from Eric Brevik, a native English speaker) There is nothing wrong with the English in this sentence, or at any of the points the reviewer specifically identified. Furthermore, “sea lettuce” should not be capitalized, it is not a proper noun. The reviewer is focused on their preferred writing style, but there is nothing improper about the English they have “corrected” or asked be corrected.

Many thanks again for your comments, which really improved the MS, thanks again!

Author Response File: Author Response.docx

Reviewer 3 Report

Figure 3: Needs to be careful with colour selection.

Sampling: The first sentence is not complete. The first and second sentences could be combined. 

Sediment analysis: Describe the method and then give the reference in brackets or state it at the end of the sentence to keep the flow of thoughts throughout the MS. 

The entire MS should be checked for the sequence of clear flow of thoughts and English. 

Lines 152-154: Not clear.

Please align all equation numbers

"DNA was extracted from algal samples that were frozen at...." instead of this sentence, you may write Algal DNA was extracted using..........

Lines 276-277: "DNA was isolated using Cetyl-trimethyl ammonium bromide..." It should be stated as DNA was extracted using the CTAB method. Did you use the original CTAB method or a modified method? If modified, what modifications were done? 

Line 280: PCR and gel electrophoresis. PCR is the first step so put it first. 

Line 281: Should start line "A PCR was done with random amplified polymorphic DNA (RAPD) primers (Table XXX). Why RAPD? It is a very old technique with very low repeatability. Also, it is a dominant marker. 

The entire methods section needs to be revised to make it clear, simple and concise. 

Table 2: How do you know the number of bands for each primer before the results? Is it from someone else's experiment? It is not clear why you selected RAPD primers.

Table 3 needs to be reformatted.

Table 4 is not needed as you have represented the information using figure 5.  

Figure 4: Red and yellow colours are not appealing. 

Figure and table titles need to be revisited. Each of them should be self-explanatory.

Table 6: The title needs to be revised. mg per kg can be mentioned in the title itself.  

What was the purpose of the correlation analysis? 

Too many tables and figures that could be meaningfully abridged into a few of them. 

Overall comments: The manuscript has tried to provide important insights into the problem using a case in Egypt. But the manuscript needs rigorous revisions to meet the standards of the journal. Special emphasis should be given to writing, English and the logical flow of thoughts.  

Author Response

Dear Reviewer,

We appreciate your comments and your time to improve our MS to be available to publish.

1- Figure 3: Needs to be careful with color selection.

Response: changes colors were made, thanks!

2- Sampling: The first sentence is not complete. The first and second sentences could be combined.

Response: done, thanks!

3- Sediment analysis: Describe the method and then give the reference in brackets or state it at the end of the sentence to keep the flow of thoughts throughout the MS.

Response: done, thanks!

4- The entire MS should be checked for the sequence of clear flow of thoughts and English.

Response: The revised MS was edited again by a native English speaker to be clear for the reader, thanks!

5- Lines 152-154: Not clear.

Response: Changed, thanks!

6- Please align all equation numbers

Response: done, thanks!

7- "DNA was extracted from algal samples that were frozen at...." instead of this sentence, you may write Algal DNA was extracted using..........

Response: Thanks a lot, the sentence was rewritten according your suggestion.

8- Lines 276-277: "DNA was isolated using Cetyl-trimethyl ammonium bromide..." It should be stated as DNA was extracted using the CTAB method. Did you use the original CTAB method or a modified method? If modified, what modifications were done?

Response: Thanks a lot, the sentence was rewritten according your suggestion. The original CTAB method was used (Doyle, J.J.; Doyle, J.L. Isolation of plant DNA from fresh tissue. Focus, 1990, 12, 13–15).

9- Line 280: PCR and gel electrophoresis. PCR is the first step so put it first.

Response: done, thanks!

10- Line 281: Should start line "A PCR was done with random amplified polymorphic DNA (RAPD) primers (Table XXX). Why RAPD? It is a very old technique with very low repeatability. Also, it is a dominant marker.

Response: Thanks a lot, the sentence was modified according your suggestion. RAPD has many advantages over the other DNA markers;

1- they are quick and easy to assay.

2- Low quantities of template DNA are required.

3- Random primers are commercially available.

4- RAPDs have a very high genomic abundance and are randomly distributed throughout the genome.

11- The entire methods section needs to be revised to make it clear, simple and concise.

Response: more improvements in the materials and methods section, thanks!

12- Table 2: How do you know the number of bands for each primer before the results? Is it from someone else's experiment? It is not clear why you selected RAPD primers.

Response: We combined Primer name and sequences which should be mentioned in the materials and methods section with the results generated by these primers (total no. of band, monomorphic band, polymorphic bands and polymorphism %) which should be represented in the Results section in one Table (Table 2). This table could be easily moved to the Results section.  

13- Table 3 needs to be reformatted.

Response: done, thanks!

14- Table 4 is not needed as you have represented the information using figure 5. 

Response: Thanks a lot for your comment, but Table 4 represents Genetic similarity percentages while figure 5 represents the phylogenetic tree and clustering analysis

15- Figure 4: Red and yellow colours are not appealing.

Response: Changed to black colors, thanks!

16- Figure and table titles need to be revisited. Each of them should be self-explanatory.

Response: done, thanks!

17- Table 6: The title needs to be revised. mg per kg can be mentioned in the title itself. 

Response: done, thanks!

18- What was the purpose of the correlation analysis?

Response: correlation in this study has an important purpose to understand the relationship between studied factors including the negative and positive relation. Correlations are useful because they can indicate a predictive relationship that can be exploited in practice. This relationship may be significant at the 0.01 or 0.05 level

19- Too many tables and figures that could be meaningfully abridged into a few of them.

Response: thanks for your comments, some indices were deleted and figure and tables were removed, thanks!

20- Overall comments: The manuscript has tried to provide important insights into the problem using a case in Egypt. But the manuscript needs rigorous revisions to meet the standards of the journal. Special emphasis should be given to writing, English and the logical flow of thoughts. 

Response: many thanks for your comments!

Our co-author Prof. Eric (a native English speaker from the USA) has revised and edited the MS.

Thanks again!

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Accept

Author Response

Dear Reviewer 2#

Many thanks for your time and accepting our corrections

Comments and Suggestions for Authors: Accept

Response: Thanks again!

Author Response File: Author Response.docx

Reviewer 3 Report

Thanks for addressing the comments, but the MS still needs minor changes.

The title is still not clear. Suggestion: Assessment of bioaccumulation of heavy metals and its ecological risk in sea lettuce (Ulva spp.) along the coast Alexandria, Egypt: Implications for sustainable management

Line 72: "all over the world such as China" change this to "in many countries such as"

Lines 107-110: This paragraph needs to be edited and rephrased.

Lines 151-153: Please rephrase the sentence for clarity.

Line 162: Change to "Total phosphorus was determined using a modified method of John" and then mention the modification.

Lines 162-172: Needs to be edited and improved. Suggestion:

Total phosphorus was determined using a modified version of the John method [43]. First, 0.1 ml of clear digestate was diluted with 9 ml of distilled water. Two drops of a 0.1% solution of the indicator (2,4 dinitrophenol) were added, followed by the addition of 5 M NaOH drop by drop until the solution turned yellow. Next, 2 M HCl was added drop by drop until the solution became colorless. One milliliter of the combined reagents was added, and the solution was gently shaken. The absorbance was measured at 710 nm after 30 minutes.

The mixed reagent was prepared by mixing antimony molybdate acid and recently prepared 2.5% ascorbic acid in a 4:1 ratio. The mixture was stabilized for 1 day. The antimony molybdate acid was prepared by dissolving 0.75 g of (NH4)6Mo7O24·4H2O and 0.014 g of antimony potassium tartrate in 50 ml of distilled water. Then, 8.8 ml of concentrated H2SO4 was added, and the solution was cooled. The volume was adjusted to 100 ml with distilled water, and the solution was stored in a dark-colored glass bottle.

Line 184: seawater water samples??

Line 194: Change "(ratio 2:2:1) " to (2:2:1)"

Line 202: Calculation of ........

Lines 242-245: Not clear. Sugegstion: To extract algal DNA from the samples that were frozen at -20°C, a 150 mg sample was ground in liquid nitrogen using a mortar and pestle. The DNA was isolated using Cetyl-trimethyl ammonium bromide method [54], and the concentrations were determined using a Nanodrop spectrophotometer (Implen, Westlake Village, CA).

Line 251: a one random primer... a or one?; 25 ng of DNA template change to "25 ng DNA template"

Line 252: Delete "magnification".

Lines 254-255: Change the phrase "then by 35 1-min. cycles at 94°C, 30 S at 36°C, and 3 min at 72°C, for denaturation, annealing, and polymerization, respectively " to "then 35 cycles of 94°C for 1-min., 36°C for 30 S and 72°C for 3 min. for denaturation, annealing, and polymerization, respectively, "

Lines 255-256: "expansion stage" Change to "extension step"

Lines 256-259: Not clear. Needs to be edited and rephrased.

Line 261: Quantification of ........ activities??

Line 273: "Quality" change to small "q"

Lines 288-296: Needs to be revised and edited. Not clear nor in a logical sequence.

Please check the entire results and discussion sections for clarity and logical sequence.

Lines 354-355: Please mention what V02, F08 and so on in the figure title.

Please check the entire manuscript for clarity and a logical flow of thoughts.

Author Response

Dear Reviewer 3#

Many thanks for your time and accepting our corrections

Comments and Suggestions for Authors

Thanks for addressing the comments, but the MS still needs minor changes.

Response: Many thanks again!

The title is still not clear. Suggestion: Assessment of bioaccumulation of heavy metals and its ecological risk in sea lettuce (Ulva spp.) along the coast Alexandria, Egypt: Implications for sustainable management

Response: Many thanks, done!

Assessment of Bioaccumulation of Heavy Metals and their Ecological Risk in Sea Lettuce (Ulva spp.) Along the Coast Alexandria, Egypt: Implications for Sustainable Management

Line 72: "all over the world such as China" change this to "in many countries such as"

Response: Many thanks, done!

Lines 107-110: This paragraph needs to be edited and rephrased.

Response: Many thanks, done!

Lines 151-153: Please rephrase the sentence for clarity.

Response: Many thanks, done!

Line 162: Change to "Total phosphorus was determined using a modified method of John" and then mention the modification.

Response: Many thanks, done!

Lines 162-172: Needs to be edited and improved. Suggestion:

Total phosphorus was determined using a modified version of the John method [43]. First, 0.1 ml of clear digestate was diluted with 9 ml of distilled water. Two drops of a 0.1% solution of the indicator (2,4 dinitrophenol) were added, followed by the addition of 5 M NaOH drop by drop until the solution turned yellow. Next, 2 M HCl was added drop by drop until the solution became colorless. One milliliter of the combined reagents was added, and the solution was gently shaken. The absorbance was measured at 710 nm after 30 minutes.

The mixed reagent was prepared by mixing antimony molybdate acid and recently prepared 2.5% ascorbic acid in a 4:1 ratio. The mixture was stabilized for 1 day. The antimony molybdate acid was prepared by dissolving 0.75 g of (NH4)6Mo7O24·4H2O and 0.014 g of antimony potassium tartrate in 50 ml of distilled water. Then, 8.8 ml of concentrated H2SO4 was added, and the solution was cooled. The volume was adjusted to 100 ml with distilled water, and the solution was stored in a dark-colored glass bottle.

Response: Many thanks, done!

Line 184: seawater water samples??

Response: Many thanks, corrected!

Line 194: Change "(ratio 2:2:1) " to (2:2:1)"

Response: Many thanks, corrected!

Line 202: Calculation of ........

Response: Many thanks, done!

Lines 242-245: Not clear. Sugegstion: To extract algal DNA from the samples that were frozen at -20°C, a 150 mg sample was ground in liquid nitrogen using a mortar and pestle. The DNA was isolated using Cetyl-trimethyl ammonium bromide method [54], and the concentrations were determined using a Nanodrop spectrophotometer (Implen, Westlake Village, CA).

Response: Many thanks, done!

Line 251: a one random primer... a or one?; 25 ng of DNA template change to "25 ng DNA template"

Response: Many thanks, done!

Line 252: Delete "magnification".

Response: Many thanks, done!

Lines 254-255: Change the phrase "then by 35 1-min. cycles at 94°C, 30 S at 36°C, and 3 min at 72°C, for denaturation, annealing, and polymerization, respectively " to "then 35 cycles of 94°C for 1-min., 36°C for 30 S and 72°C for 3 min. for denaturation, annealing, and polymerization, respectively, "

Response: Many thanks, done!

Lines 255-256: "expansion stage" Change to "extension step"

Response: Many thanks, done!

Lines 256-259: Not clear. Needs to be edited and rephrased.

Response: Many thanks, done!

Line 261: Quantification of ........ activities??

Response: Many thanks, done!

Line 273: "Quality" change to small "q"

Response: Many thanks, done!

Lines 288-296: Needs to be revised and edited. Not clear nor in a logical sequence.

Response: Many thanks, done!

Please check the entire results and discussion sections for clarity and logical sequence.

Response: Many thanks, done!

Lines 354-355: Please mention what V02, F08 and so on in the figure title.

Response: Many thanks, done! These are the primer number.

Please check the entire manuscript for clarity and a logical flow of thoughts.

Response: Many thanks, done!

Many thanks again, for your time and good work, thanks!

Author Response File: Author Response.docx