Application of Tween 80 in the Remediation of Diesel-Contaminated Podzolic Soils Under Boreal Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is focused on the Application of Tween 80 in the Remediation of Diesel-Contaminated Podzolic Soils under Boreal Conditions. 

1. Why authors used Tween 80 in the Remediation of Diesel-Contaminated Podzolic Soils. Authors need to discuss in introduction section. I did not understand the reason of uses, as authors did not discuss in the introduction section.
2. What about the novelty of this work, authors should add if any. 
3. Why T2 samples shows the more effective than other, need to be proper explaination. 
4.  What about the effect of temperature, authours should be incorporate more data effect of temperature like lower, normal, and higher temperature for better understanding. 
5. What about the exact mechanism of removal by using Tween 80, authors should be added figure also... and discuss in details. 
6. Authors should be added comparative table in the form table for better understanding.  

Author Response

Dear Reviewer 1,

We thank you for your constructive and detailed feedback. All comments have been carefully addressed, and the revised manuscript has been updated accordingly. Specific responses and changes are outlined below.

Comment 1: Why authors used Tween 80 in the remediation of diesel-contaminated Podzolic soils. Authors need to discuss in introduction section. I did not understand the reason of uses, as authors did not discuss in the introduction section.

Response 1: Thank you for this observation. We have expanded the Introduction to explicitly explain the rationale for selecting Tween 80. A new sentence (Lines 74–84) highlights its favorable environmental profile, biodegradability, high hydrophilic–lipophilic balance, and proven performance in acidic or low-buffered soils, especially under low-temperature conditions.

Comment 2: What about the novelty of this work, authors should add if any.

Response 2: We appreciate this suggestion. The Introduction now explicitly states the novelty of our research (Lines 95–110), emphasizing that this study evaluates Tween 80 performance in highly acidic, low-buffered Albic Podzolic soils under boreal temperature constraints - conditions rarely addressed in previous research. We also note that our work uniquely combines sub-CMC dosing, dual temperature regimes, and integrated ecotoxicological assessment.

Comment 3: Why T2 samples shows the more effective than other, need to be proper explanation.

Response 3: We agree this point required clarification. The Discussion now includes a detailed mechanistic explanation (Lines 540–546) for the higher effectiveness of the T2 samples, which used a sub-CMC concentration of Tween 80. At sub-CMC levels, monomers interact more freely with hydrophobic contaminants and soil surfaces, enhancing desorption without entrapping hydrocarbons in micelles, unlike higher doses where micelle formation may reduce bioavailability and transport.

Comment 4: What about the effect of temperature, authors should be incorporate more data effect of temperature like lower, normal, and higher temperature for better understanding.

Response 4: Thank you for pointing this out. While our study was limited to two representative temperature regimes (22–24 °C and 2–3 °C), the Discussion now expands on temperature effects on surfactant behavior and hydrocarbon mobility (Lines 557–575). We also outline future research needs, including the study of freeze–thaw cycles and extended thermal gradients to better understand temperature–desorption relationships and scalability.

Comment 5: What about the exact mechanism of removal by using Tween 80, authors should be added figure also… and discuss in details.

Response 5: We appreciate this valuable suggestion. We have added a detailed explanation of the molecular-level action of Tween 80 (Lines 551–556) and included a new schematic illustration (Figure 4) that shows how Tween 80 reduces interfacial tension, enhances wettability, and mobilizes hydrocarbons at sub- and above-CMC concentrations.

Comment 6: Authors should be added comparative table in the form table for better understanding.

Response 6: We thank the reviewer for this suggestion. A new comparative table (Table 5) has been added in the Discussion section (Lines 705–713). It summarizes the performance of Tween 80 and other commonly used surfactants under different soil and environmental conditions, highlighting Tween 80’s suitability for acidic, cold-region soils where many other surfactants are less effective or destabilize soil chemistry.

Reviewer 2 Report

Comments and Suggestions for Authors

Why was the Khanty-Mansi Autonomous Okrug–Yugra region selected for study beyond being a representative cold region? Were there any specific contamination events or historical data that prompted this selection? Please provide a convincing explanation as to why you did not select other regions.

Why was Tween 80 chosen over other non-ionic surfactants? Are there comparative studies that support its superior performance in cold, acidic soils? Please support this critical choice with literature.

Did the authors experimentally verify the CMC of Tween 80 under the tested soil and temperature conditions, or was the CMC value sourced from the literature? Given that CMC is temperature- and matrix-dependent, this could impact interpretation. I would particularly like to provide data related to the sustainability analysis.

How were the temperature conditions maintained over the 90-day period? Were fluctuations monitored or recorded to ensure stability in ambient and refrigerated setups? Do you have any preliminary studies on this topic? Please specify.

- Were TPH reductions normalized to soil moisture content or mass loss due to evaporation? If not, how do the authors ensure that observed TPH reductions are not partially attributable to water content changes?

- Was the reduction in TPH over time fitted to any kinetic model (e.g., first-order degradation)? If so, what are the rate constants, and how do they compare across treatments?

- Given that Vibrio fischeri and C. affinis are aquatic organisms, how do their responses translate to real soil ecotoxicological risks? Would the inclusion of terrestrial test species (e.g., Eisenia fetida) strengthen the assessment? Also, let me know what standard you followed.

- The manuscript briefly mentions future research directions. Could the authors elaborate more concretely on the limitations of this study (e.g., short incubation, no microbial analysis) and how these will be addressed?

Author Response

Dear Reviewer 2,

We thank you for your thoughtful and constructive feedback. Below we provide detailed responses to each comment and describe the corresponding revisions made to the manuscript. Line numbers refer to the revised manuscript with tracked changes.

Comment 1: Why was the Khanty-Mansi Autonomous Okrug–Yugra region selected for study beyond being a representative cold region? Were there any specific contamination events or historical data that prompted this selection? Please provide a convincing explanation as to why you did not select other regions.

Response 1: Thank you for this important question. We have clarified the rationale for choosing the Khanty-Mansi Autonomous Okrug–Yugra (KMAO–Yugra) region in the revised manuscript. In addition to being a representative boreal area, KMAO–Yugra is one of Russia’s most productive oil provinces, with extensive infrastructure and a documented history of petroleum contamination in podzolic soils. This makes it particularly relevant for testing region-specific remediation approaches. The explanation has been added to the Introduction (Lines 58–63).

Comment 2: Why was Tween 80 chosen over other non-ionic surfactants? Are there comparative studies that support its superior performance in cold, acidic soils? Please support this critical choice with literature.

Response 2: We appreciate this observation. The Introduction (Lines 74–84) now contains an expanded justification for selecting Tween 80, citing comparative literature that supports its environmental safety, compatibility with acidic soils, and effectiveness under low-temperature conditions. These properties make Tween 80 particularly suitable for remediation in boreal environments.

Comment 3: Did the authors experimentally verify the CMC of Tween 80 under the tested soil and temperature conditions, or was the CMC value sourced from the literature? Given that CMC is temperature- and matrix-dependent, this could impact interpretation.

Response 3: We have clarified in Section 2.2 (Lines 302–306) that the CMC of Tween 80 was not experimentally measured in this study but selected based on published aqueous-phase values. We also note that temperature, ionic strength, and soil matrices can shift the effective CMC upward, and this limitation is discussed when interpreting the concentration-dependent effects in the Discussion.

Comment 4: How were the temperature conditions maintained over the 90-day period? Were fluctuations monitored or recorded to ensure stability in ambient and refrigerated setups? Do you have any preliminary studies on this topic? Please specify.

Response 4: We have revised Section 2.2 (Lines 309–311) to describe the temperature control methodology. Ambient conditions were maintained in a climate-controlled laboratory room with daily monitoring; refrigerated samples were stored in a temperature-regulated chamber with ±1 °C stability. No significant fluctuations were recorded. Preliminary test runs confirmed the stability of these regimes, although detailed logs are not included in this manuscript.

Comment 5: Were TPH reductions normalized to soil moisture content or mass loss due to evaporation? If not, how do the authors ensure that observed TPH reductions are not partially attributable to water content changes?

Response 5: This is an important point. As clarified in Section 2.2 (Lines 314–315), soil moisture content was determined gravimetrically before and after incubation following GOST 5180-2015. All TPH concentrations were expressed on an oven-dry mass basis. Moisture variation was minor (< 7%) and did not affect interpretation of hydrocarbon removal trends.

Comment 6: Was the reduction in TPH over time fitted to any kinetic model (e.g., first-order degradation)? If so, what are the rate constants, and how do they compare across treatments?

Response 6: We appreciate this suggestion. Given the limited number of time points in our dataset, robust kinetic modeling was not feasible within the scope of this work. The primary aim was to compare treatment outcomes under different conditions. We acknowledge the value of kinetic modeling and note that it will be incorporated in future studies with more frequent sampling.

Comment 7: Given that Vibrio fischeri and C. affinis are aquatic organisms, how do their responses translate to real soil ecotoxicological risks? Would the inclusion of terrestrial test species (e.g., Eisenia fetida) strengthen the assessment? Also, let me know what standard you followed.

Response 7: We have revised the Discussion (Lines 578–590) to note that while Vibrio fischeri and Ceriodaphnia affinisare aquatic organisms, they are widely used in standardized environmental bioassays (ISO 11348-3:2007; OECD 202) for their sensitivity and relevance to groundwater exposure pathways. We agree that the inclusion of terrestrial test species such as Eisenia fetida would enhance ecological relevance and will incorporate such species in future work.

Comment 8: The manuscript briefly mentions future research directions. Could the authors elaborate more concretely on the limitations of this study (e.g., short incubation, no microbial analysis) and how these will be addressed?

Response 8: We agree on the importance of clearly stating the limitations. In the Discussion and Conclusions, we explicitly note that the study did not assess microbial dynamics, chronic toxicity, or long-term environmental effects. We also outline plans for extended-duration field trials, microbial community analysis, and terrestrial ecotoxicity testing to address these gaps in future research.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents a valuable contribution to the field of environmental remediation, particularly concerning petroleum-contaminated soils in cold-climate regions. The authors investigate the efficacy of Tween 80 as a surfactant in reducing total petroleum hydrocarbons (TPH) under controlled laboratory conditions simulating boreal temperature regimes. The study is well-designed and integrates both chemical and ecotoxicological analyses, supporting the potential environmental applicability of the proposed method.

The manuscript is scientifically sound and methodologically rigorous, though several areas require clarification and expansion to improve clarity, reproducibility, and contextual impact.

1. The manuscript refers to the lower concentration as being below the critical micelle concentration (CMC), which is a key point. However, the CMC value of Tween 80 under the tested conditions (e.g., temperature, ionic strength, soil type) is not stated. This value should be provided and briefly discussed, ideally with a supporting reference.
2. While the abstract mentions TPH reduction of up to 21%, a comprehensive comparison across all treatment groups is missing. Please include a table or figure summarizing TPH values before and after treatment for each group, with statistical significance annotations.
3. Clarify whether control samples (T₀ and X₀) showed any natural attenuation over 90 days. The acute toxicity data is presented clearly, but the tested concentrations (≤0.3 mg/dm³) are significantly lower than the concentrations applied in soil (up to ~41.6 mg/dm³). Clarify whether the tested concentrations represent expected leachate/pore water concentrations after soil application. If not, the implications for environmental safety should be reassessed or discussed.
4. The manuscript would benefit from a short discussion on scalability and field application challenges. How might soil heterogeneity, weather variability, or surfactant degradation affect field performance?
5. Are the findings consistent with previous field or pilot-scale studies using Tween 80 or other non-ionic surfactants?
6. The fate of Tween 80 in the soil environment is not discussed. Was any attempt made to quantify residual surfactant in the soil after 90 days? If not, please mention this as a limitation and suggest future investigation.
7. Specify if soils were incubated under static conditions or periodically mixed.
8. Indicate if moisture was monitored or adjusted during the 90-day period.
9. Ensure consistent use of concentration units (e.g., mg/L vs. mg/dm³).
10. Replace “TPH content was confirmed at 50.01 ± 1.14 mg/kg” with “...was determined to be...”, as “confirmed” implies a reference value.
11. Consider including a schematic or graphical abstract illustrating the experimental workflow.
12. Improve readability of Table 1 by aligning columns clearly and reducing redundancy (e.g., repeating the same TPH and soil mass values).
13. The discussion could be enriched with comparison to recent literature on surfactant-enhanced remediation, especially studies under cold conditions or involving biosurfactants.
14. What is the CMC of Tween 80 under the tested soil conditions?
15. Did control (non-treated) soils show any reduction in TPH during the 90-day incubation?
16. How do the tested concentrations in the toxicity assays compare to estimated environmental exposure levels post-application?
17. Was surfactant leaching or biodegradation monitored or estimated? If not, can the authors comment on potential implications?

Author Response

Dear Reviewer #3,

We sincerely thank you for your thorough and constructive review of our manuscript. Your comments have greatly helped to improve the clarity, scientific rigor, and completeness of the work. Below, we address each comment in detail. Line numbers refer to the revised manuscript with tracked changes.

Comment 1: The manuscript refers to the lower concentration as being below the critical micelle concentration (CMC), which is a key point. However, the CMC value of Tween 80 under the tested conditions (e.g., temperature, ionic strength, soil type) is not stated. This value should be provided and briefly discussed, ideally with a supporting reference.

Response 1: We have now included a discussion of Tween 80’s CMC in the soil–water context in the Discussion section (Lines 540–547). Reported aqueous-phase CMC values at 20–25 °C range from ~10 to 160 μM (≈15–200 mg L⁻¹), depending on method and ionic strength. Adsorption to mineral and organic phases can elevate the effective CMC in soils. Based on these data, we interpret the 1.5 × 10⁻⁴ mol L⁻¹ treatment as a near-/sub-CMC dose.

Comment 2: While the abstract mentions TPH reduction of up to 21%, a comprehensive comparison across all treatment groups is missing. Please include a table or figure summarizing TPH values before and after treatment for each group, with statistical significance annotations.

Response 2: We have added Table 3 (Section 3.3, Lines 462–470) summarizing initial and final TPH concentrations, percentage reductions, and statistical significance groupings for all treatments.

Comment 3: Clarify whether control samples (T₀ and X₀) showed any natural attenuation over 90 days.

Response 3: This has been clarified in Section 3.3 (Lines 435–439). Control samples exhibited ~6 % TPH reduction, likely due to volatilization, sorption, and limited natural degradation.

Comment 4: The acute toxicity data is presented clearly, but the tested concentrations (≤0.3 mg/dm³) are significantly lower than the concentrations applied in soil (up to ~41.6 mg/dm³). Clarify whether the tested concentrations represent expected leachate/pore water concentrations after soil application.

Response 4: We have clarified this in the Discussion (Lines 633–643). Tested concentrations were selected to represent environmentally realistic surfactant levels in leachate or porewater after soil application, accounting for sorption and dilution.

Comment 5: The manuscript would benefit from a short discussion on scalability and field application challenges.

Response 5: We have added a new paragraph (Lines 650–670) discussing field-scale factors such as soil heterogeneity, seasonal temperature fluctuations, freeze–thaw cycles, and Tween 80 biodegradation, which may shorten effective treatment duration but also limit long-term persistence.

Comment 6: Are the findings consistent with previous field or pilot-scale studies using Tween 80 or other non-ionic surfactants?

Response 6: The Discussion now includes a comparison with previous field and pilot-scale studies (Lines 650–670), showing agreement in contaminant mobilization trends and highlighting Tween 80’s performance in acidic, cold-region soils.

Comment 7: The fate of Tween 80 in the soil environment is not discussed. Was any attempt made to quantify residual surfactant in the soil after 90 days?

Response 7: While residual Tween 80 was not measured, we have added a discussion of its fate and biodegradation in soils (Lines 691–703), supported by literature data, and suggested that future studies quantify residual surfactant and transformation products.

Comment 8: Specify if soils were incubated under static conditions or periodically mixed.

Response 8: We have clarified in Section 2.2 (Lines 316–317) that soils were incubated under static conditions without periodic mixing.

Comment 9: Indicate if moisture was monitored or adjusted during the 90-day period.

Response 9: We have added details in Section 2.2 (Lines 311–313) stating that soil moisture content was determined gravimetrically before and after incubation. Variations were <7 %.

Comment 10: Ensure consistent use of concentration units (e.g., mg/L vs. mg/dm³).

Response 10: The manuscript was reviewed and updated to use mg L⁻¹ consistently for aqueous concentrations and mol L⁻¹ for molar units, per MDPI guidelines.

Comment 11: Replace “TPH content was confirmed at 50.01 ± 1.14 mg/kg” with “…was determined to be…”.

Response 11: This change has been made in Section 2.2.

Comment 12: Consider including a schematic or graphical abstract illustrating the experimental workflow.

Response 12: A new schematic diagram (Figure 1) was added in Section 2.1, illustrating soil sampling, contamination, Tween 80 dosing, incubation, and analytical procedures.

Comment 13: Improve readability of Table 1 by aligning columns clearly and reducing redundancy.

Response 13: Table 1 was reformatted for improved readability by aligning columns, grouping repeated values, and using footnotes for repeated parameters.

Comment 14: The discussion could be enriched with comparison to recent literature on surfactant-enhanced remediation, especially studies under cold conditions or involving biosurfactants.

Response 14: The Discussion was expanded (Lines 650–670) with references to recent studies, including those on biosurfactants and remediation under cold conditions.

Comment 15: What is the CMC of Tween 80 under the tested soil conditions?

Response 15: Addressed together with Comment 1, noting literature ranges and matrix effects.

Comment 16: Did control (non-treated) soils show any reduction in TPH during the 90-day incubation?

Response 16: Addressed together with Comment 3 in Section 3.3 (Lines 435–439).

Comment 17: Was surfactant leaching or biodegradation monitored or estimated?

Response 17: Addressed together with Comment 7, noting no direct measurements but discussing biodegradation based on literature and its implications for persistence and reapplication.

Reviewer 4 Report

Comments and Suggestions for Authors

Comments and suggestions for the authors are presented in the attached file.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 4,

We sincerely thank you for the careful reading of our manuscript and for the positive overall evaluation of its scientific quality, novelty, and practical relevance. We are grateful for your specific suggestions aimed at improving the clarity and contextual depth of the work. In response, we have revised the manuscript to address each point in detail, making targeted additions to the Abstract, Introduction, Results, and Discussion. These changes include clarifying the novelty of the study, expanding contextual comparisons with prior research, explicitly stating statistical significance, and highlighting limitations and future research needs. All modifications have been carefully incorporated into the revised manuscript and are indicated in the responses below.

Comment 1: The Abstract could briefly mention the broader implications of the study, for example, relevance to cold-region remediation policies or field applications.

Response 1: The Abstract has been revised to include a statement on broader implications, highlighting the relevance of our findings to cold-region remediation strategies and field-scale applications (Lines 26–30).

Comment 2: The Introduction could briefly summarise the novelty of the study.

Response 2: A novelty statement has been added to the end of the Introduction (Lines 95–110), emphasizing that this work uniquely combines sub-CMC dosing, dual temperature regimes, and integrated ecotoxicological assessment for remediation of acidic, low-buffered soils under boreal climate conditions.

Comment 3: How do the TPH reductions compare to other surfactants or soils?

Response 3: A new comparative table (Table 5) has been added in the Discussion (Lines 705–713), summarizing TPH reductions achieved with Tween 80 and other surfactants under different soil types and environmental conditions.

Comment 4: Would you explain the choice of Tween 80 concentrations? Why are CMC levels and sub-CMC levels selected?

Response 4: The Discussion has been expanded to clarify the rationale for concentration selection (Lines 540–547), noting literature-based CMC values, expected matrix effects in acidic soils, and the mechanistic advantages of sub-CMC dosing.

Comment 5: A brief comparison of results to prior studies is needed. This would contextualise the findings.

Response 5: The Discussion now includes expanded comparison with prior work, citing relevant laboratory, pilot-, and field-scale studies on non-ionic surfactants and biosurfactants.

Comment 6: Statistical significance, as indicated by p-values, could be explicitly mentioned in the text.

Response 6: The Results section has been updated to explicitly state p-values for significant treatment effects.

Comment 7: The study focuses on surfactant-enhanced remediation, but it does not explore microbial degradation mechanisms. This could further explain TPH reduction.

Response 7: The Discussion now acknowledges the potential contribution of microbial degradation (Lines 691–703) and outlines plans for incorporating microbial community analysis in future studies.

Comment 8: Acute toxicity tests are useful, but chronic effects of residual Tween 80 or diesel byproducts are not assessed.

Response 8: The limitations section in the Discussion has been expanded (Lines 591–596) to acknowledge that chronic ecotoxicological effects were not assessed and to recommend their inclusion in future research.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept

Reviewer 3 Report

Comments and Suggestions for Authors

Agree with the revised manuscript.