Assessment of Sewage Molecular Markers: Linear Alkylbenzenes in Sediments of an Industrialized Region in Peninsular Malaysia

Round 1

Reviewer 1 Report

This study utilized linear alkylbenzenes (LABs) to identify human activity sources impacting coastal environments and river ecosystems. LABs were detected in sediment samples from the Kim Kim River and the Port Dickson Coast using GC-MS. The results highlight varying LABs concentrations, significant C13-LABs homolog differences, and the importance of improving wastewater treatment systems for monitoring contamination. However, here are several questions that leave me puzzled:

1. Can the authors provide more details about the specific selection criteria for the research sites in Johor and Sembilan, Malaysia? How were these locations chosen, and what factors were considered to ensure they represent appropriate areas for the study?

2. Were there any potential challenges or limitations in maintaining the collected sediment samples at -20°C in stainless-steel containers? How did the authors address any concerns related to sample preservation during transport and storage?

3. How was the efficiency of the extraction method with two columns evaluated? Were there any potential issues related to the removal of polar compounds and the fractionation process? If yes, how were these potential issues addressed to ensure the accuracy of the results?

4. Can the authors explain in more detail how the surrogate standards were selected and their role in ensuring the accurate recovery of target LABs? Were there any specific considerations taken into account for the selection of these surrogate standards?

5. Were there any potential interferences or background noise observed during the GC-MS analysis? How did the authors validate the accuracy and sensitivity of their GC-MS method, especially when detecting compounds of LABs at m/z = 91, 92, and 105 in the selective ion monitoring (SIM) mode?

6. Can the authors provide more insight into the factors contributing to the strong Pearson correlation (r = 0.88, p < 0.05) between LABs concentrations in the investigated areas? How does this correlation support the potential use of LABs as indicators of both domestic and industrial pollution?

7. What are the possible reasons for the notable disparity in the allocation of LABs homologs among the different sampling locations, as indicated by Tables 2a-b with a statistical significance level of p < 0.05? How might regional characteristics or activities influence the distribution of LABs in these areas?

8. In reference to previous studies mentioned in the discussion, what could be the reasons for the relatively higher quantities of LABs detected in this study compared to some other regions worldwide and Malaysia? Are there specific local factors that may explain these differences?

9. Can the authors elaborate on why LC–LABs such as C13 and C14 dominate the distribution of LABs in the PDC sediments, while SC–LABs like C10 and C11 are present in low levels? Are there specific environmental or anthropogenic factors contributing to this pattern?

10. How do the findings of this study contribute to the existing knowledge of LABs distribution in riverine environments and coastal areas? What are the potential implications of the results for environmental monitoring and pollution control measures in the studied regions?

Minor editing of English language required

Author Response

Reviewer 1

• This study utilized linear alkylbenzenes (LABs) to identify human activity sources impacting coastal environments and river ecosystems. LABs were detected in sediment samples from the Kim Kim River and the Port Dickson Coast using GC-MS. The results highlight varying LABs concentrations, significant C13-LABs homolog differences, and the importance of improving wastewater treatment systems for monitoring contamination. However, here are several questions that leave me puzzled:

1. Can the authors provide more details about the specific selection criteria for the research sites in Johor and Sembilan, Malaysia? How were these locations chosen, and what factors were considered to ensure they represent appropriate areas for the study?

Response:  The authors thank very much the reviewer for the appreciation of the manuscript, which ‎was greatly improved following the reviewer’ comments and suggestions.‎ Based on your comment, more details about the specific selection criteria for study area as such’’ Seven sediment samples were collected from sites chosen for their proximity to agricultural and industrial activity, such as the Kim Kim River in Johor Bahru State, as well as urban and tourism activity, such as Port Dickson Beach in Sembilan State.’’

Lines 105-107. Page 3

‎2- Were there any potential challenges or limitations in maintaining the collected sediment samples at -20°C in stainless-steel containers? How did the authors address any concerns related to sample preservation during transport and storage?

Response: The authors thank very much the reviewer for this good observation. There were no challenges in maintaining sediment samples cool. The sentences were amended to be clearer as such’’ The collected sediment was then put into a stainless-steel container that had been cleaned, and the top 4 cm of the sediment cake was sliced, immediately kept in a double-clean Ziploc bag, labelled, and then put on ice at 4 °C in a cooler box for transport to the lab, where it was stored at -20 °C upon arrival. The samples were then put through a freeze-drying procedure to remove any remaining moisture and get them ready for more analysis.’’

Lines 111-116 page 3

3. How was the efficiency of the extraction method with two columns evaluated? Were there any potential issues related to the removal of polar compounds and the fractionation process? If yes, how were these potential issues addressed to ensure the accuracy of the results?

Response: The authors are thankful the reviewer for such a valuable comment. The samples were purified and fractioned by following established validation protocols described elsewhere Zakaria et al. (2002), to ensure that the method meets accuracy, precision, and recovery standards. In addition, quality control method including the surrogate standards which they performed as a quality control function on the appropriateness of the analytical method for the particular analyses to carry out that method with reasonable proficiency. The LABs surrogates in this study were successfully recovered between 60–120% range with minimal loss of the target chemicals during the analytical procedures.

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4. Can the authors explain in more detail how the surrogate standards were selected and their role in ensuring the accurate recovery of target LABs? Were there any specific considerations taken into account for the selection of these surrogate standards?

Response: The authors are thankful for such a good comment. The surrogate standards (SS), which are similar to the target analytical compounds used in the analytical method for the purpose of identification and could be recovered from the sample matrix with reasonable efficiency, were preloaded in each sample to ensure the precise recovery of the target LABs. The SS contains 50 μL of 1-Cn LABs, where 1- denotes each LAB homologue's initial isomer and n denotes its carbon number (8–14).

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5. Were there any potential interferences or background noise observed during the GC-MS analysis? How did the authors validate the accuracy and sensitivity of their GC-MS method, especially when detecting compounds of LABs at m/z = 91, 92, and 105 in the selective ion monitoring (SIM) mode?

Response: The authors are thankful for such a good observation. The method utilized for this data operated with specified parameters such signal-to-noise ratio (S/N), peak detection limits, and a 5-point calibration curve. LABs peaks in the samples were obtained by comparing the retention times of each target compound to the retention times of the LAB standards run on the same day, which were then confirmed by the m/z value (91, 92, and 105).

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6. Can the authors provide more insight into the factors contributing to the strong Pearson correlation (r = 0.88, p < 0.05) between LABs concentrations in the investigated areas? How does this correlation support the potential use of LABs as indicators of both domestic and industrial pollution?

Response: The authors are thankful for such a good observation. The significant Pearson correlation between the LAB concentrations in the investigated areas (r = 0.88, p < 0.05) is most likely caused by the fact that LAB concentrations are higher in areas with a high population density, industrial, agricultural, and tourism activities as these areas are more likely to have a high volume of wastewater and detergent discharge. Overall, the strong correlation between LAB concentrations and pollution indicates that LABs could serve as a useful measure of residential and industrial contamination.

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7. What are the possible reasons for the notable disparity in the allocation of LABs homologs among the different sampling locations, as indicated by Tables 2a-b with a statistical significance level of p < 0.05? How might regional characteristics or activities influence the distribution of LABs in these areas?

Response: The authors are thankful for such a good comment. As your comment, the geographical features of different sampling locations, such as proximity to coastlines, rivers, or urban centres, as well as industrial discharges, urban runoff, or agricultural activities and inadequate treatment, could influence the input sources of LABs and their subsequent distribution.

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8. In reference to previous studies mentioned in the discussion, what could be the reasons for the relatively higher quantities of LABs detected in this study compared to some other regions worldwide and Malaysia? Are there specific local factors that may explain these differences?

Response: The authors are thankful for such a good observation. The higher levels of LABs detected in this study compared to some other regions worldwide and Malaysia could be attributed to a combination of specific local factors and differences in pollution sources such as significant industrial, tourism and urban activities that release LABs into the environment. Port Dickson is known to be one of Malaysia’s famous recreational beaches with numerous hotels and resorts that offer a wide range of accommodation, as well as the growth in urbanization, small industries, tourism and shipping.

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9. Can the authors elaborate on why LC–LABs such as C13 and C14 dominate the distribution of LABs in the PDC sediments, while SC–LABs like C10 and C11 are present in low levels? Are there specific environmental or anthropogenic factors contributing to this pattern?

Response: The authors are thankful for such a good comment. As commented, the dominance of LC–LABs (long-chain linear alkylbenzenes) such as C₁₃ and C₁₄ over SC–LABs (short-chain linear alkylbenzenes) like C₁₀ and C₁₁ in the distribution of LABs in PDC sediments can be influenced by several environmental and chemical factors. LC–LABs, being longer-chain compounds, are generally less volatile and more hydrophobic compared to SC–LABs and this can result in differences in their environmental fate and transport. LC–LABs may have a greater tendency to adsorb onto particles and sediments due to their higher molecular weight and hydrophobicity. This enhances their sorption and partitioning into sediment matrices, making them more likely to be retained in the sediments over time. LC–LABs also might undergo slower degradation or transformation processes due to their larger size and more complex structure.

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10. How do the findings of this study contribute to the existing knowledge of LABs distribution in riverine environments and coastal areas? What are the potential implications of the results for environmental monitoring and pollution control measures in the studied regions?

Response: The authors are thankful for such a good observation. As commented, the findings of this study may provide light on the level of pollution in the studied regions as well as its distribution and sources of organic pollutants (LABs). Public knowledge of the pollution issues in the studied regions can be improved, support for pollution control measures, and environmentally responsible behaviour can be encouraged by sharing the findings of environmental monitoring studies. The information can be used to inform decisions made by the authorities about the implementation or modification of pollution control regulations. To decrease sewage contamination in the near future, more effective STPs are required in neighbouring areas, together with ongoing monitoring of the marine environment to prevent potential contamination from sewage pollution.

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11. Minor editing of English language required

Response: The authors are thankful for such a good observation. The manuscript was properly edited in English by an English expert.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have pointed a concern over the wastewater treatment system and require sustainable management. Well written and clear presentation but I think the number of samples included in this study are not sufficient to represents the Peninsular Malaysia

Comments

1. Improve methods section: Mention how many samples from each study site were collected? What was the criteria for site selection and sampling?

2. Huge variation in the LABs concentrations across the site was notices . What was reason of this variation specially among the samples from PDC (119 to 256 ng/g)?.

3. I see only one gas chromatogram in supplementary file only, why not all the chromatogram in supplementary file?? attach all the GC-MS data for all samples analyzed for review purpose only. 

 

Author Response

Reviewer 2

• The authors have pointed a concern over the wastewater treatment system and require sustainable management. Well written and clear presentation but I think the number of samples included in this study are not sufficient to represents the Peninsular Malaysia:
• Improve methods section: Mention how many samples from each study site were collected? What was the criteria for site selection and sampling?

Response: The authors thank very much the reviewer for the consideration of this manuscript, which ‎was greatly improved following your comments and suggestions.‎ As suggested, the method was improved and the samples with selection criteria were mentioned as such’’ Seven sediment samples were taken from sites chosen for their proximity to agricultural and industrial activity, such as the Kim Kim River in Johor Bahru State, as well as urban and tourism activity, such as Port Dickson Beach in Sembilan State.

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• Huge variation in the LABs concentrations across the site was notices . What was reason of this variation specially among the samples from PDC (119 to 256 ng/g)?.

Response: The authors are thankful for such a good observation. The huge variation specially among the samples from PDC are attributed to anthropogenic activities nearby sampling stations such as urbanization growth, small industries, tourism and shipping.

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• I see only one gas chromatogram in supplementary file only, why not all the chromatogram in supplementary file?? attach all the GC-MS data for all samples analyzed for review purpose only.

Response: The authors are thankful for such a good observation. As suggested, gas chromatogram for PDC1 was selected to be a typical gas chromatogram of LABs of study area, 

S1 file

Author Response File: Author Response.docx

Reviewer 3 Report

Water

Manuscript Number: water-2540279-peer-review-v1

Title: Evaluation of Sewage Molecular Markers in a Highly Industrialized Zone in Peninsular Malaysia

 

The thematic of the paper is in accordance with the aims and scopes of this journal.

It is very well written in understandable and scientific language.

However some aspects need to be discussed in more depth.

Comment 1. In Keywords section there is “Wastewater contamination”, which is not correct so please change it. We suggest “pollutants contained in wastewater” or similar.

Comment 2. On page 4, line 150 there is abbreviation LAN, which may be the mistake.

Comment 3. In supplementary material Significant Pearson correlation is stated to be p < 0.01 and in manuscript p < 0.05. Please explain and uniformize.

Comment 4. Are there other parameters that are monitored, beside LAB, in order to assess the effectiveness of WWTS or detect fresh sewage effluent discharge in environment?

Comment 5. What are the advantages of using LAB as sewage molecular markers?

Author Response

Reviewer 3

• The thematic of the paper is in accordance with the aims and scopes of this journal. It is very well written in understandable and scientific language. However, some aspects need to be discussed in more depth.

1. In Keywords section there is “Wastewater contamination”, which is not correct so please change it. We suggest “pollutants contained in wastewater” or similar.

Response: The authors thank very much the reviewer for the consideration of this manuscript, which ‎was greatly improved following your comments and suggestions.‎ As suggested, the “Wastewater contamination” changed to “pollutants contained in wastewater”

Keywords section

2. On page 4, line 150 there is abbreviation LAN, which may be the mistake.

Response: The authors are thankful for such a good observation. This word ‘’LAN’’ was corrected and changed to LAB

Line 161 page 4

3. In supplementary material Significant Pearson correlation is stated to be p < 0.01 and in manuscript p < 0.05. Please explain and uniformize.

Response: The authors are thankful for such a good observation. The Significant Pearson correlation was stated correctly.

S1 file

4. Are there other parameters that are monitored, beside LAB, in order to assess the effectiveness of WWTS or detect fresh sewage effluent discharge in environment?

Response: The authors are thankful for such a good comment. Indeed, LABs were discovered in applications involving detergents. These substances wind up in wastewater treatment plants after being flushed down the toilet. However, LABs are hard for bacteria in wastewater treatment plants to break down, which allows them to bypass treatment and enter the aquatic environment. The different biodegradation capacities of the internal and external isomers of LABs are used as an indication of sewage treatment efficiencies and the recency of sewage contamination in the environment. LABs are rather simple to measure. This makes LABs a simpler and less costly indicator than other parameters such faecal sterols.

5. What are the advantages of using LAB as sewage molecular markers?

Response: The authors are thankful for such a good comment. In fact, using LABs as sewage molecular markers has a number of benefits, including their global ubiquity, hydrophobic and persistent properties, which tend to stick to particles and accumulate in sediments, and their ease of measurement and cost-effectiveness as indicators.

Round 2

Reviewer 1 Report

The manuscript has been modified by the author in accordance with the suggestions provided.

After careful consideration, I am pleased to state that I am satisfied with the modifications made by the author. 

Author Response

The authors thank very much the reviewer for the consideration of this manuscript, which ‎was greatly improved following the reviewer’ comments and suggestions.‎ 

Reviewer 2 Report

The authors have made substantial revisions to the manuscript; however, I was unable to locate additional GC-MS chromatograms of sample replicates in the supplementary files, as I had previously requested.

Author Response

Reviewer#2 comment

The authors have made substantial revisions to the manuscript; however, I was unable to locate additional GC-MS chromatograms of sample replicates in the supplementary files, as I had previously requested

Response:

We appreciate your careful review of our manuscript and your valuable feedback. We are pleased to hear that you found our revisions substantial, and we would like to address your concern regarding the additional GC-MS chromatograms of sample replicates in the supplementary files.

Furthermore, we understand your request for more chromatograms, and we apologize for not providing them as originally requested. As the study has spanned a considerable amount of time since its inception, it has been challenging to gather and include all the chromatograms for sample replicates. However, we acknowledge the importance of replicability and transparency in scientific research.

To address this concern, we have included a typical gas chromatogram for PDC1 in the Supplementary Information (S1 file). While we cannot provide all the chromatograms due to the limitations mentioned earlier, we believe that this representative chromatogram will offer insight into the characteristics of LABs in the study area.

We also appreciate your suggestion for considering additional chromatograms in future studies. We will certainly take this into account for future research projects to enhance the comprehensiveness of our data presentation.

Once again, we thank you for your valuable feedback, and we hope that the inclusion of the representative chromatogram in the Supplementary Information addresses your concerns. If you have any further questions or require additional information, please do not hesitate to reach out.

Author Response File: Author Response.docx