Review Reports - Exclusion and Trapping Mechanisms of Boron in Forage Grasses Irrigated with Treated Oilfield-Produced Water

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study addresses boron dynamics in a soil–plant system irrigated with treated produced water (PW) and explores the potential of nature-based solutions (biochar and gypsum) to mitigate boron accumulation. The topic is relevant; however, the manuscript has several significant shortcomings that limit its scientific reliability and clarity. Lack of statistical transparency and incomplete assessment of plant response significantly weaken the manuscript. In particular, the absence of biomass data prevents a proper evaluation of phytotoxicity, making several conclusions speculative.

General comments: Statistical Analysis - Although statistical analysis is mentioned in the methodology (ANOVA), no supporting information is provided. The number of replicates is not stated. Tables and figures do not include any statistical indicators (e.g., standard deviation, error bars, significance levels). There is no evidence demonstrating that ANOVA was appropriately conducted or that its assumptions were met.

Assessment of Phytotoxicity - The study evaluates boron concentrations in roots, shoots, and soil, as well as BAF and TF. However, biomass production data are missing, which is essential for assessing phytotoxicity. Concentration-based indicators alone (B content, BAF, TF) are insufficient to determine plant stress or toxicity without growth/yield data.

Abstract: Line 22: “Soil” → “soil”

Introduction

Lines 113–122: Presentation of previous results is unclear; the purpose of Table 1 is not justified. If these data refer to water used in the study, they should be moved to Materials and Methods/Results.
Line 125: “Boron” → “boron”
Lines 137–139: Results should not be presented in the introduction; rephrase.

Results

- Several figures are unclear, redundant, or insufficiently explained:
  - Figure 1: Plant labels (Panicum, Buffelgrass) are missing; only Pmax and Alpha are shown; empty horizontal lines are present; it is unclear which data correspond to which species.
  - Figures 2 and 3: “Site 1” and “Site 2” are not defined.
  - Figures 4 and 5: Repeat information already presented in Figures 2 and 3.
  - Figure 6: Completely unclear; lacks explanation of what is being compared and how values were calculated.
  - Figure 7 vs text (Lines 256–264): Reported TF values in the text are inconsistent with those shown in the figure. The figure appears correct (taking into account fig 2), suggesting errors in the text interpretation.
  - Figure 8: Symbols (A, B, Pmax, etc.) are not explained in the legend.
  - Figure 9: Not necessary; only images a–f should be retained.

Discussion

- The discussion is superficial and not sufficiently grounded in the presented results.
- There is no meaningful discussion of:
  - The effects of biochar and gypsum
  - Differences between PW and other water sources (e.g., wastewater or groundwater)
- Without biomass data, conclusions regarding phytotoxicity are not supported.
- Due to inconsistencies in TF values, parts of the discussion (Lines 293–301) are incorrect. Errors in TF interpretation and lack of statistical validation undermine the reliability of the conclusions. Overall, the results are partially speculative and not fully supported by the data.

Materials and Methods

Line 356: “Gypsum” → “gypsum”, there is similar error over the entire text, which should be checked and corrected.
Lines 356–361: Do not belong in this section; either move them to Introduction or remove them (well-known information).
Lines 364–358: Redundant with Section 4.5; should be removed.

Conclusion not supported by the presented data due to: Lack of statistical validation, missing biomass data, errors in TF interpretation.

Author Response

Dear Editor and Reviewer 1,

Thank you for your thorough and constructive evaluation of our manuscript. We greatly appreciate the time you took to provide such detailed feedback, which has significantly improved the scientific rigor of our study. We have carefully revised the manuscript to address every point you raised.

Regarding your General Comments on statistical transparency and phytotoxicity: We agree that a rigorous study must be transparent. We have updated Section 4.7 to explicitly state the number of replicates (n=4), define the statistical tests used (ANOVA, Shapiro-Wilk, Levene’s test),. Furthermore, we fully accept your point regarding phytotoxicity. We have transparently reframed the study as a "targeted ion-partitioning study," acknowledging that whole-plant biomass yield was not recorded. We have scrubbed the manuscript of definitive claims of phytotoxicity, replacing them with cautious observations that concentrations exceeded safe agronomic thresholds, and explicitly stating in the Conclusion that long-term biomass tracking is required to confirm yield losses.

Regarding the Specific Comments: We have addressed all typographical errors (e.g., capitalization of 'Soil' and 'Gypsum'). We have moved Table 1 to the Introduction to serve as background context rather than experimental data. We have completely rewritten the Discussion to provide a deep, mechanism-based explanation of the effects of biochar/gypsum and the differences between water sources. We have removed speculative claims regarding specific transporter mechanisms, focusing instead on defensible, data-supported directional trends. Finally, we have cleaned up the Results section by removing redundant figures, correcting transposed TF values in the text to match the actual figure data, explaining the factorial plots, and adding clear species legends to the experimental setup figures.

Below, we provide point-by-point responses to all specific comments.

Reviewer 1

Comments and Suggestions for Authors

Reviewer 1 Comments:

Author Response: We thank the reviewer for highlighting this important point. The Materials and Methods section has been revised to clarify the experimental design and provide the missing statistical details. These revisions ensure that the experimental replication, design structure, and statistical approach are now clearly defined and transparent.

Reviewer 1 Comments:

Author Response: We fully agree with the reviewer’s strict and correct definition of phytotoxicity. We also appreciate the opportunity to clarify the scope of this experiment.

We must transparently clarify that whole-plant biomass yield (g/pot) was not recorded during the three harvest stages. The primary objective of this experiment was to conduct a targeted ion-partitioning study—specifically to calculate the Bioaccumulation Factor (BAF) and Translocation Factor (TF)—rather than a full agronomic yield trial. The plants were dried and weighed strictly to determine the concentration of boron (mg B per kg dry tissue), not to quantify total harvestable yield per pot.

Because we lacked yield data, we agree that using the term "phytotoxicity" to describe our own results was scientifically over-reaching. We have therefore carefully revised the entire manuscript

However, we respectfully submit that the BAF and TF indices remain highly valuable to the scientific community, even in the absence of this specific experiment's biomass data. While biomass tells us if a plant suffered, BAF and TF begin to explain why and how the plant handles the ion internally (e.g., Buffelgrass trapping boron at TF < 1 versus Alfalfa translocating it at TF > 1). This mechanistic insight is crucial for crop selection in water reuse systems, even before full yield trials are conducted.

We hope the reviewer agrees that by transparently admitting this limitation and reframing the manuscript's claims to match the actual data collected, the scientific integrity of the study is maintained, and it provides a strong foundation for future yield-based research.

Reviewer 1 Comments:

Abstract: Line 22: “Soil” → “soil”

Thank you, its corrected in the new version

Reviewer 2 Comments:

Introduction

Lines 113–122: Presentation of previous results is unclear; the purpose of Table 1 is not justified. If these data refer to water used in the study, they should be moved to Materials and Methods/Results.

Author Response: Thank you for this important comment. We clarify that Table 1 does not represent the specific water used in the experimental irrigation trials. Instead, it provides background information on the overall performance of the produced water treatment system by comparing upstream and downstream water quality.

To improve clarity, we have revised the text to clearly state the purpose of the table and its contextual role. We have also ensured it is properly referenced as supporting information rather than part of the experimental dataset.

Line 125: “Boron” → “boron”

Thank you, its corrected in the new version

Lines 137–139: Results should not be presented in the introduction; rephrase.

Thank you for this comment. We agree that results should not be presented in the Introduction. The sentence has been revised to remove direct references to study data and tables, and now provides only general context regarding boron thresholds in water reuse guidelines.

Reviewer 1 Comments:

Results

Several figures are unclear, redundant, or insufficiently explained:

Figure 1: Plant labels (Panicum, Buffelgrass) are missing; only Pmax and Alpha are shown; empty horizontal lines are present; it is unclear which data correspond to which species.

Author Response: Thank you for your helpful comment. Figure 1 has been revised to improve clarity and presentation. Plant labels (Panicum and Buffelgrass) have now been added, empty horizontal lines have been removed, and the figure has been updated to clearly distinguish the data corresponding to each species.

Figures 2 and 3: “Site 1” and “Site 2” are not defined.

Thank you for your comment. The manuscript has been revised to clearly define “Site 1” and “Site 2” in the text. In addition, the figure captions for Figures 2 and 3 have been updated to ensure consistency and clarity for the reader.

Figures 4 and 5: Repeat information already presented in Figures 2 and 3.

Thank you for your comment. Figures 4 and 5 have been revised to avoid repetition with Figures 2 and 3. The updated figures now present complementary information, and all figure captions have been clarified to highlight their distinct contributions to the study.

Figure 6: Completely unclear; lacks explanation of what is being compared and how values were calculated.

We completely agree with the reviewer. The previous text inadequately described the figure and incorrectly referenced soil layers that were not included in the factorial plot, leading to confusion.

We have entirely rewritten Section 2.5 and updated the Figure 9 caption to explicitly address both of your concerns. We believe these revisions make the statistical analysis transparent, easily interpretable, and deeply integrated with the study's conclusions.

Figure 7 vs text (Lines 256–264): Reported TF values in the text are inconsistent with those shown in the figure. The figure appears correct (taking into account fig 2), suggesting errors in the text interpretation.

Author Response: We sincerely thank the reviewer for catching this critical error. The reviewer is absolutely correct; the values reported in the text were inaccurate and did not match the actual Translocation Factor (TF) data presented in the figure. We realized that during drafting, we inadvertently transposed Bioaccumulation Factor (BAF) values and included outdated calculation numbers into the TF description.

We have completely rewritten Section 2.6 to ensure the text strictly and accurately reflects the actual TF values (the blue bars) shown in Figure 10. All erroneous numbers (e.g., 74.84, 36.23, etc.) have been removed. The text now accurately reports that Panicum reached maximum TF values of 49.05, Alfalfa ranged between 11.42 and 14.48, and Buffelgrass maintained low exclusion values ranging from 0.24 to 9.65. We apologize for this oversight and appreciate the reviewer's meticulous attention to detail.

Figure 8: Symbols (A, B, Pmax, etc.) are not explained in the legend.

Author Response: We apologize for this oversight. We have completely revised the caption for Figure 8 to explicitly define all abbreviations used in the schematic. The legend now clearly states that A represents Alfalfa, b represents Buffelgrass, p represents Panicum, and Pm represents Panicum maximum (including their scientific names). Additionally, we took this opportunity to clarify the other elements of the schematic in the legend, such as the three water sources, the soil amendment treatments, and the layout of the four replicates, to ensure the figure is entirely self-explanatory to the reader.

Figure 9: Not necessary; only images a–f should be retained.

Author Response: We respectfully prefer to retain all images (a–l) in the Figure to provide a complete chain-of-custody for our sample processing. While we agree that standard laboratory procedures (such as grinding and liquid handling) are routine, we included these images specifically to document the rigorous Quality Assurance/Quality Control protocols applied in this study. Because we were quantifying trace boron concentrations using ICP-OES—a process highly susceptible to cross-contamination—showing the distinct use of specialized digestion tubes (15 mL vs. 50 mL), the controlled heating blocks, and the clean sample preparation environment (g–l) serves as visual proof of the methodological rigor. We believe retaining these images enhances the reproducibility of our methodology for readers attempting similar trace element analyses, while still keeping the figure concise. We hope the reviewer will accept this slight expansion in the interest of full methodological transparency.

Reviewer 1 Comments:

Discussion

The discussion is superficial and not sufficiently grounded in the presented results.
There is no meaningful discussion of:

The effects of biochar and gypsum
Differences between PW and other water sources (e.g., wastewater or groundwater)

Without biomass data, conclusions regarding phytotoxicity are not supported.

Author Response: Thank you for these valuable comments. The Discussion section has been substantially revised to strengthen its connection to the presented results and to improve its scientific depth.

We agree with the reviewer that true phytotoxicity requires biomass data. We have removed definitive claims of phytotoxicity from the Discussion. We now clearly state that our study was a targeted ion-uptake study, and while concentrations exceeded agronomic thresholds, we acknowledge that future studies must integrate biomass tracking to confirm exact tipping points.

Due to inconsistencies in TF values, parts of the discussion (Lines 293–301) are incorrect. Errors in TF interpretation and lack of statistical validation undermine the reliability of the conclusions. Overall, the results are partially speculative and not fully supported by the data.

We thank the reviewer for this critical catch. We agree that using discrete max/min TF values (e.g., 74.84 and 0.24) without standard deviation was statistically over-reaching and speculative regarding specific cellular transporters. We have completely rewritten this section. We removed the specific outlier numbers, removed definitive claims about 'BOR transporters', and instead focused on the robust directional trend (TF > 1 vs TF < 1) which is scientifically defensible

Reviewer 1 Comments:

Materials and Methods

Line 356: “Gypsum” → “gypsum”, there is similar error over the entire text, which should be checked and corrected.

Thank you, its corrected in the new version

Lines 356–361: Do not belong in this section; either move them to Introduction or remove them (well-known information).

Author Response: We agree with the reviewer. The general background information regarding the broad agricultural benefits of biochar and gypsum was misplaced in the Methods section. We have removed these sentences entirely to maintain the strict procedural focus of the Methods. Instead, we added a brief, single-sentence justification in Section 4.2 explaining that these amendments were applied specifically "to test their efficacy in mitigating boron uptake under high-salinity conditions.

Lines 364–368: Redundant with Section 4.5; should be removed.

We agree with the reviewer. This sentence was inadvertently duplicated in the earlier section. We have deleted lines 364–368 to remove the redundancy.

Reviewer 1 Comments:

Conclusion not supported by the presented data due to: Lack of statistical validation, missing biomass data, errors in TF interpretation.

Author Response: We thank the reviewer for this valuable and constructive comment. The Conclusion section has been thoroughly revised to ensure that all statements are fully aligned with the presented data and do not exceed the scope of the study.

Specifically:

The interpretation of Translocation Factor (TF) values has been corrected and reformulated using cautious, data-supported language. Overstated claims (e.g., “hyper-translocation” and definitive mechanistic assertions) have been removed.
Statements implying definitive conclusions regarding plant tolerance, forage suitability, or toxicity have been tempered. The revised text now clearly reflects that the findings are based on concentration-derived indices (BAF and TF) rather than direct productivity measures.
A limitation statement has been explicitly added to acknowledge the absence of comprehensive statistical validation and biomass data, clarifying that conclusions regarding yield performance and phytotoxicity remain preliminary.
The discussion of biochar and gypsum effects has been revised to avoid overgeneralization, emphasizing that the observed responses are context-dependent and require further validation.

These revisions ensure that the Conclusion is now fully consistent with the experimental design, analytical scope, and available data, and that all interpretations are presented in a scientifically cautious and defensible manner.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript addresses a practically relevant topic — the behavior of boron in a soil–plant system irrigated with treated oilfield-produced water — and presents an interesting finding regarding divergent boron tolerance strategies among forage species. The use of Bioaccumulation Factors (BAF) and Translocation Factors (TF) to differentiate species-specific mechanisms is a meaningful analytical contribution. However, the manuscript has substantial deficiencies in structural organization, methodological reporting, data presentation, and internal consistency that must be addressed before it can be considered for publication. Major revision is required.

1.Line 61–62 contains an incomplete sentence: "moderately tolerant crops such as barley and maize can withstand lower boron concentrations in irrigation water than more tolerant crops such as alfalfa and [9]."

2.Table 1 is presented within the Introduction section (lines 113–121), which is unconventional for a research article.The author is asked to consider moving Table 1 to another location and replacing it in the main text with a brief description of the water quality data.

3.Section 2.1 (lines 153–165) opens with "Boron concentrations in plant tissues increased progressively across harvest stages," but no statistical results (e.g., ANOVA F-values, p-values, or post-hoc test outcomes) are reported to support this claim.

4.Lines 160–162 state: "Overall, the results demonstrate that boron dynamics in soil–plant systems are governed by multiple interacting factors, emphasizing the need for integrated management approaches." This is a broad interpretive conclusion that is more appropriate for the Discussion section. The Results section should restrict itself to reporting findings without interpretation.

5.Section 2.4 (lines 210–213) states: "Biochar may influence boron bioavailability through adsorption; higher application rates or different biochar types may be required." The use of the modal verb "may" implies speculation, which is inappropriate in the Results section. If these are inferences drawn from the data, they should be moved to the Discussion. If they are direct observations, they should be stated as such with supporting data.

6.Section 4.1 (line 346) "uniformly packed into [Size/Volume, e.g., 15-L] plastic pots." The actual pot size must be provided, as it directly affects the interpretation of boron loading calculations and plant growth conditions.

7.Section 4.5 (lines 394–395) contains yet another placeholder: "digested using a microwave-assisted acid digestion system with [HNO₃/H₂O₂, specify ratios]." The acid digestion protocol is a fundamental methodological detail that must be fully specified. Its omission undermines the reproducibility of the study.

8.References 15 and 19 are duplicates; please check for citation errors.

Author Response

Reviewer 2

Comments and Suggestions for Authors

Dear Reviewer,

We sincerely thank you for your rigorous evaluation of our manuscript. We fully agree with your assessment that the initial submission contained deficiencies in structural organization, methodological reporting, data presentation, and internal consistency. We have conducted a comprehensive Major Revision of the manuscript to address every specific limitation you identified. Specifically, we have relocated data tables to their proper methodological sections, removed speculative language from the Results section, corrected duplicate citations, filled in all missing methodological protocols (e.g., EPA Method 3051, exact pot sizes), and reorganized the text to strictly separate objective results from interpretive discussion. We believe these targeted revisions have fully resolved the structural and scientific consistency issues you highlighted, resulting in a significantly strengthened manuscript.

Reviewer 2 Comments:

Author Response: We thank the reviewer for catching this typo. The sentence has been rewritten for grammatical correctness and clarity, ensuring the reference is properly used as supporting literature rather than a noun. The corrected text now reads: "Available crop-tolerance data indicates that moderately tolerant crops, such as barley and maize, can withstand lower boron concentrations in irrigation water compared to highly tolerant crops such as alfalfa, as documented by [9]."

Reviewer 2 Comments:

Author Response: We completely agree with the reviewer. Presenting detailed data tables within the Introduction disrupts the flow of the background literature. We have removed the detailed descriptive text and relocated Table 1 out of the Introduction. In its place, we provided a concise, one-sentence summary that directs the reader to the data. Table 1 has been moved to Section 4.1 (Experimental Design and Water Characterization) to contextualize the specific water quality parameters of the irrigation sources used in the experiment. This ensures the data is presented in its proper methodological context.

Reviewer 2 Comments:

Author Response: We completely agree. Stating a progressive increase without providing the statistical evidence undermines the scientific rigor of the results. We have revised Section 2.1 to explicitly state that the temporal accumulation was statistically validated. The text now clearly states that ANOVA confirmed the differences between harvest stages were statistically significant, providing the required F-values and p-values to support this claim.

Reviewer 2 Comments:

Author Response: We completely agree with the reviewer. The Results section must remain strictly factual and objective, leaving interpretation for the Discussion. We have deleted the interpretive concluding sentence from Section 2.1 and replaced it with a purely descriptive transition sentence. We have also conducted a thorough review of the entire Results section to ensure no other subjective or concluding statements remain, ensuring all interpretive discussions regarding "interacting factors" and "management approaches" are exclusively reserved for the Discussion section.

Reviewer 2 Comments:

Author Response: We completely agree with the reviewer. The use of the modal verb "may" in the Results section inappropriately introduced speculation about biochar's theoretical mechanisms, especially since the actual data in this section showed no significant reduction in boron mobility. We have rewritten the text to remove the speculation. The revised sentence now strictly reports the direct observation (limited influence of biochar) and reframes the idea of higher application rates or different biochar types as a hypothesis generated by the null result, which is now appropriately discussed in the Discussion. We have also reviewed the rest of the Results section to ensure no other speculative modal verbs were used inappropriately.

Reviewer 2 Comments:

Author Response: We appreciate the reviewer's attention to detail. We confirm that the placeholder text has been removed in the revised manuscript. The actual experimental pot size is now explicitly stated as 15-L plastic pots

Reviewer 2 Comments:

Author Response: We agree with the reviewer that omitting the specific digestion protocol compromises reproducibility. We have removed the placeholder text. The text now explicitly states that the samples were digested using a microwave-assisted acid digestion system following the standard EPA Method 3051 protocol. This standard method ensures complete digestion of the plant tissue required for accurate trace element analysis and provides exact parameters for future reproducibility. We have reviewed the entire manuscript to ensure no other placeholders remain.

Reviewer 2 Comments:

8.References 15 and 19 are duplicates; please check for citation errors.

Author Response: We thank the reviewer for identifying this citation error. You are entirely correct; References 15 and 19 are identical.