Review Reports - Ecotoxicological Effects of Heavy Metals on Rice (<i>Oryza sativa</i> L.) Across Its Life Cycle and Health Risk Assessment in Soils Around Pb–Zn Mine

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Here are comments:

Generally, the paper is well-written with a lot of important information about HMs risk in agricultural soil in the neighborhood of the Pb-Zn mine.

However, there is a need for the minor improvement given in the text.

Comments for author File: Comments.pdf

Author Response

Dear Reviewers:

We greatly appreciate your thorough reviews on our manuscript entitled “Health risk assessment of heavy metals in agriculture soil around large Pb-Zn mine based on life-cycle toxicity test of rice” (plants-403378), which we find to be very beneficial for strengthening our work. The questions you mentioned have been answered below with clear and concise responses. If you have any further questions, we would be delighted to provide answers and revisions to the best of our abilities. And, we are hopeful that this manuscript will be accepted by plants. The reviewer’s comments are copied in italics, our responses are in bold, and the revision made to the manuscript is marked in blue and bold.

Sincerely,

Fangyu Hu and Baoyu Wang,

On behalf of all authors.

Response: We sincerely appreciate your kind review of our work. We have made point-by-point responses to your comments as carefully as possible. The replies provided in detail are as follows:

(1) We have rewritten the Introduction, and the issues raised by the reviewer have been specifically addressed and emphasized in the revised version.

(2) The term “heavy metals” has been consistently revised to “HMs” throughout the manuscript.

(3) In line with comments from another reviewer, the term “concentration” has been uniformly replaced with “content” in the manuscript.

(4) The abbreviation “QCZ” has been revised to the full name “Qingchengzi” for clarity.

(5) Following suggestions from multiple reviewers, the experimental design section has been substantially supplemented and clarified; detailed revisions can be found in the indicated lines of the revised manuscript.

(6) Relevant parts of the Abstract have also been revised accordingly.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a relevant and data rich study on heavy metal contamination in agricultural soils near a Pb-Zn mine using a pot experiment with rice, combined with ecological and human health risk assessments. The topic fits the journal scope and the dataset is potentially useful. However, there are important problems in experimental design transparency, in how ecological and health risks are quantified and interpreted, and in the way the work is framed as a life cycle toxicity assessment. These issues affect the strength and credibility of the conclusions, so I recommend major revision before the manuscript can be considered for publication.

The novelty and conceptual positioning of the study are not clearly defined relative to existing soil-rice heavy metal literature: the Introduction summarizes mining related pollution and the importance of rice (lines 30–41; lines 42–66), and the stated aims focus on distribution and transport in the soil-rice system, ecotoxicological effects on rice, and ecological and health risks (lines 71–75), which are all well-established topics; the authors should clearly explain what is new compared with previous studies on soil-rice systems near Pb-Zn mines (for example Du et al. 2018 and Tang et al. 2019 already cited), and clarify how combining a pot experiment with risk indices provides added value, otherwise the contribution reads largely confirmatory rather than innovative.
The description of the study area, soil sampling, and pot experiment lacks essential detail on spatial design and replication, which undermines the statistical validity and reproducibility of the results: in Section 3.1, the QCZ mining area and climate are described (lines 273–278), and it is stated that soils were collected from farmland around the mining area and that each pot contained 1 kg of soil with five seeds, later thinned to one plant (line 279 to line 283), but there is no information on the number and location of sampling points, whether samples were composited, how many pots per treatment were established, how pots were arranged or randomized outdoors, or whether fertilization and water management simulated flooded paddy conditions; because all figures and Table 1 present means with significance testing (for example Figure 1 and Figure 2, and the note on Table 1, lines 135–136), the authors must clearly report the number of independent soil samples and the number of replicate pots per soil type, specify whether the statistical unit is the pot or the plant, and describe any blocking or randomization procedures.
The manuscript repeatedly refers to pollution and ecological risk indices but does not adequately present the underlying soil properties and contamination levels that control metal bioavailability: the ecological risk assessment section states that PI, NIPI, EI and RI were used (lines 221–222) and that Zn, Cd, Pb and As in the tested soil significantly exceeded Chinese soil quality standards (lines 223–224), yet the main text never reports basic soil physicochemical parameters such as pH, organic matter, texture, or redox status, and all detailed values are relegated to supplementary tables (Table S1 and S2 referenced, lines 222–223 and lines 367–369); given that bioaccumulation patterns (Sections 2.1.1 and 2.1.2) and EI values (lines 235–82) depend strongly on these properties, the authors should provide at least a summary table in the main text with soil pH, organic carbon, texture, and total metal concentrations for the contaminated and control soils, and explicitly state whether the contamination levels used in the pots span the range observed in the surrounding farmland.
There is a serious inconsistency between the narrative classification of health risk and the quantitative risk metrics, which currently suggests severe risks while the Abstract describes only a moderate level: the Abstract concludes that “The potential health risk reached a moderate level, with Cd identified as the dominant factor” (lines 23–25), but in Section 2.4 the total hazard index (HI) values for adults and children are reported as approximately 49 and 105 times higher than the non-carcinogenic threshold, and total carcinogenic risk (TCR) values as 423 and 892 times higher than the unacceptable level (page 8, lines 246–251), with the corresponding HI and TCR numbers in Table 2 (lines 270–269) confirming extremely high risk estimates; the authors need to recheck all calculations, clearly present the thresholds used, and then either revise the Abstract and Conclusions to acknowledge that their own metrics indicate a very high and unacceptable risk, or justify an alternative classification scheme if “moderate” is based on a different standard.
The health risk assessment methodology is not sufficiently transparent about exposure assumptions and data sources, which makes it difficult to judge whether the extremely high HI and TCR values are realistic: Section 3.6 describes the ADI, HQ, HI, CR and TCR formulae (lines 371–399) and refers to parameter values in Table S4 and S5 (lines 383–386 and 399–400), but the main text does not indicate whether rice concentrations used in the ADI food term are from the pot experiment or from field grown rice, does not state the assumed rice consumption rates for adults and children, and does not provide any sensitivity or uncertainty analysis despite reporting TCR values orders of magnitude above typical regulatory thresholds; the authors should explicitly state which concentration data are used for each pathway, summaries the key exposure parameters in the main text (for example daily rice intake, body weight, exposure duration), and discuss whether their assumptions are representative for the local population, since unrealistic parameter combinations could explain the extremely high risk indices.
The framing of the work as a “life cycle toxicity test of rice” is not well supported by how the ecological and health risk assessments are actually conducted: the title and Abstract emphasize “life-cycle toxicity test of rice” (lines 1–3 and 8–13), and the Introduction states that health risks are assessed “based on life-cycle toxicity tests of rice” (lines 73–75), while Sections 2.2.1–2.2.3 and 3.3 describe detailed measurements of plant growth, antioxidant enzymes, MDA, and amino acid exudation across tillering, heading, and maturity (pages 5–7 and 11–12), yet the ecological (Section 2.3) and health risk (Section 2.4) indices are entirely concentration based and do not incorporate these biological endpoints in any quantitative way; to avoid a mismatch between title and content, the authors should either demonstrate how the life cycle toxicity data refine risk assessment (for example by linking specific growth or biochemical thresholds to risk categories) or adjust the title and aims so that the toxicity test is presented as a mechanistic component rather than the basis of the risk indices.
The statistical analysis and presentation of variability need clarification so that readers can properly interpret significance claims: Figures 1–3 and the amino acid figure (Figure 4, lines 219–220) all show error bars and use asterisks to indicate significant differences between control and contaminated soil, and Table 1 uses different letters to denote significant differences in bioaccumulation and translocation factors across growth stages (line 134 to line 136), while Section 3.7 simply states that SPSS 23 and Tukey’s HSD test were used to examine treatment differences (lines 403–405); the authors should specify the number of replicates contributing to each mean value, state whether error bars are standard deviation or standard error, explain whether assumptions of normality and homoscedasticity were checked (and whether any transformations were applied), and clarify within what grouping the letters in Table 1 indicate difference (within a metal across stages, or across metals within a stage).
The ecological risk assessment section has several structural and referencing problems that reduce clarity, including mislabeled figures and potentially confusing classification language: the text states “As shown in Fig. 6a, The NIPI values were all > 3 (Fig. 5a)” (lines 224–225), which appears to reference two different figure numbers for the same bar chart, and later refers to RI values shown in “Fig. 5b” (line 235), while the amino acid figure immediately before this section is captioned as “Figure 4” (lines 219–220), creating a non-sequential figure order; in addition, the RI value “exceeded 150” and is described as indicating a “moderate ecological risk” while Cd is said to pose “considerable” risk (lines 235–241), yet the classification thresholds are only given in supplementary tables (lines 367–369) and are not restated; I suggest checking and harmonizing all figure numbers and references, ensuring that each figure is called in numerical order, and briefly stating the RI and EI class boundaries in the main text so that terms such as “low,” “moderate,” and “considerable” are immediately interpretable.
The micro X ray fluorescence analysis is described in technical beamline detail but lacks basic information about sample selection and the role of these data in the study, which makes this subsection feel disconnected from the rest of the work: Section 3.2.2 gives extensive specifics on electron energy, current, monochromator, polycapillary lens, step size, detector type, live time, and data reduction software (lines 305–313), yet does not state how many rice plants and which organs were scanned, how samples were prepared (for example fresh or dried, section thickness, mounting), whether multiple biological replicates were analyzed, or how the qualitative maps in Figure S1 support the quantitative BF and TF results reported in Section 2.1.2; the authors should add information on sample numbers and preparation and briefly explain what additional mechanistic insight the μ-XRF images provide beyond the bulk concentration data, or consider moving some of the beamline technical detail to the Supplementary Materials.
There are several recurring presentation issues that should be corrected before publication, including inconsistent terminology and some confusing labels in figures and text: the section heading “Ecotoxicological toxicities of HMs on rice in the life-cycle” (line 137) is superfluous and could be simplified; in the text, the trait is called “panicle emergence” (lines 149–150) whereas the y axis label and Methods use “Head emergence number” (line 157 and line 290), and Figure 3 panels label the growth stages as “Tillering period, Flowing period, Mature period” with “Flowing” apparently a typographical error for “Heading” (lines 172–188); the amino acid figure caption calls this “Figure 4” (lines 219–220) while the text refers to “Fig. 5” (lines 196–199), and in several places “soil-rice” is written with inconsistent spacing; a careful pass to standardize trait names, correct the “Flowing period” typo, align all figure numbers between text and captions, and ensure consistent hyphenation of “soil-rice” and similar terms will improve readability.

Comments on the Quality of English Language

The English is generally understandable but requires thorough editing for grammar, spelling, and style. There are multiple typographical errors such as “focuse” and “uesd to reduct” in the μ XRF description (lines 309–313), “Flowing period” instead of “Heading period” in Figure 3 (lines 179–188), and inconsistent capitalization of “HMs,” “Soil samples,” and “Chinese” (lines 223–224). Spacing and hyphenation are also irregular, for example “soil-rice system” vs “soil –rice system,” and there are several long sentences that would benefit from splitting, especially in the Introduction and Discussion. I recommend careful revision by a fluent English speaker or professional editing service to correct spelling, improve sentence structure, ensure consistent terminology, and remove layout artefacts such as duplicated spaces and erratic hyphenation.

Author Response

Dear Reviewers:

Sincerely,

Fangyu Hu and Baoyu Wang,

On behalf of all authors.

Response: We sincerely appreciate your kind review of our work. We have made point-by-point responses to your comments as carefully as possible. The replies provided in detail are as follows:

1.The novelty and conceptual positioning of the study are not clearly defined relative to existing soil-rice heavy metal literature: the Introduction summarizes mining related pollution and the importance of rice (lines 30–41; lines 42–66), and the stated aims focus on distribution and transport in the soil-rice system, ecotoxicological effects on rice, and ecological and health risks (lines 71–75), which are all well-established topics; the authors should clearly explain what is new compared with previous studies on soil-rice systems near Pb-Zn mines (for example Du et al. 2018 and Tang et al. 2019 already cited), and clarify how combining a pot experiment with risk indices provides added value, otherwise the contribution reads largely confirmatory rather than innovative.

Response: We sincerely thank the reviewer for the valuable comments. The Introduction has been thoroughly revised to emphasize the novelty of this study compared with previous research. Specifically, this study dynamically elucidates the uptake, accumulation, and translocation of heavy metals from the perspective of the entire rice life cycle. Moreover, within a single controlled experimental system, we establish an integrated evidence chain linking soil contamination to growth and biochemical toxicity, metal accumulation and translocation, and ultimately human health risk assessment. This integrated framework provides stronger explanatory power than conventional risk assessments based solely on field data.

The description of the study area, soil sampling, and pot experiment lacks essential detail on spatial design and replication, which undermines the statistical validity and reproducibility of the results: in Section 3.1, the QCZ mining area and climate are described (lines 273–278), and it is stated that soils were collected from farmland around the mining area and that each pot contained 1 kg of soil with five seeds, later thinned to one plant (line 279 to line 283), but there is no information on the number and location of sampling points, whether samples were composited, how many pots per treatment were established, how pots were arranged or randomized outdoors, or whether fertilization and water management simulated flooded paddy conditions; because all figures and Table 1 present means with significance testing (for example Figure 1 and Figure 2, and the note on Table 1, lines 135–136), the authors must clearly report the number of independent soil samples and the number of replicate pots per soil type, specify whether the statistical unit is the pot or the plant, and describe any blocking or randomization procedures.

Response: We sincerely thank the reviewer for the valuable comments on the study area, soil sampling, and pot experiment design. Relevant information has been added and clarified in the revised manuscript to improve statistical reliability and reproducibility. Contaminated soils were collected from farmland surrounding the mining area (Fig. 1) using a plum-blossom (five-point) sampling method, with five sampling points composited into one representative sample. Control soils were collected from uncontaminated paddy fields located far from mining influence using the same sampling procedure. Each treatment included four parallel replicates (n = 4), and statistical analyses were conducted with the pot as the independent experimental unit. All pots were placed outdoors on an open platform to simulate field conditions and arranged in a completely randomized design (CRD), with pot positions randomly rotated weekly to minimize micro-environmental effects such as light exposure and wind direction. Water and fertilizer management followed local paddy field flooding practices: basal fertilizer was applied before sowing/transplanting (150 mg N kg⁻¹, 75 mg P₂O₅ kg⁻¹, and 100 mg K₂O kg⁻¹), with nitrogen topdressing (50 mg N kg⁻¹) applied at the tillering and panicle initiation stages. Water depth was maintained at 2–3 cm during establishment, 5–7 cm during tillering, and 3–5 cm from heading to grain filling, followed by mid-season drainage for 5–7 days and final drainage 7–10 days before harvest; all treatments received identical water management. These details have been added in the revised manuscript (see Lines 348-358).

The manuscript repeatedly refers to pollution and ecological risk indices but does not adequately present the underlying soil properties and contamination levels that control metal bioavailability: the ecological risk assessment section states that PI, NIPI, EI and RI were used (lines 221–222) and that Zn, Cd, Pb and As in the tested soil significantly exceeded Chinese soil quality standards (lines 223–224), yet the main text never reports basic soil physicochemical parameters such as pH, organic matter, texture, or redox status, and all detailed values are relegated to supplementary tables (Table S1 and S2 referenced, lines 222–223 and lines 367–369); given that bioaccumulation patterns (Sections 2.1.1 and 2.1.2) and EI values (lines 235–82) depend strongly on these properties, the authors should provide at least a summary table in the main text with soil pH, organic carbon, texture, and total metal concentrations for the contaminated and control soils, and explicitly state whether the contamination levels used in the pots span the range observed in the surrounding farmland.

Response: We sincerely thank the reviewer for the valuable comments. Relevant soil physicochemical properties and heavy metal contents have been added to Table 2 in the main text. In addition, the soils used in the experiment were collected from mining-impacted farmland and therefore represent the actual contamination levels observed in the surrounding agricultural soils.

There is a serious inconsistency between the narrative classification of health risk and the quantitative risk metrics, which currently suggests severe risks while the Abstract describes only a moderate level: the Abstract concludes that “The potential health risk reached a moderate level, with Cd identified as the dominant factor” (lines 23–25), but in Section 2.4 the total hazard index (HI) values for adults and children are reported as approximately 49 and 105 times higher than the non-carcinogenic threshold, and total carcinogenic risk (TCR) values as 423 and 892 times higher than the unacceptable level (page 8, lines 246–251), with the corresponding HI and TCR numbers in Table 2 (lines 270–269) confirming extremely high risk estimates; the authors need to recheck all calculations, clearly present the thresholds used, and then either revise the Abstract and Conclusions to acknowledge that their own metrics indicate a very high and unacceptable risk, or justify an alternative classification scheme if “moderate” is based on a different standard.

Response: We sincerely thank the reviewer for pointing out this inconsistency. We acknowledge that the distinction between these two types of risk was not clearly expressed in the original manuscript. The phrase “The potential health risk reached a moderate level, with Cd identified as the dominant factor” was intended to refer to the potential ecological risk, rather than human health risk. In contrast, the total hazard index (HI) and total carcinogenic risk (TCR) specifically represent human health risks. We have corrected this misstatement in the revised manuscript and clarified the terminology throughout the Abstract and main text to clearly distinguish between ecological risk and human health risk

The health risk assessment methodology is not sufficiently transparent about exposure assumptions and data sources, which makes it difficult to judge whether the extremely high HI and TCR values are realistic: Section 3.6 describes the ADI, HQ, HI, CR and TCR formulae (lines 371–399) and refers to parameter values in Table S4 and S5 (lines 383–386 and 399–400), but the main text does not indicate whether rice concentrations used in the ADI food term are from the pot experiment or from field grown rice, does not state the assumed rice consumption rates for adults and children, and does not provide any sensitivity or uncertainty analysis despite reporting TCR values orders of magnitude above typical regulatory thresholds; the authors should explicitly state which concentration data are used for each pathway, summaries the key exposure parameters in the main text (for example daily rice intake, body weight, exposure duration), and discuss whether their assumptions are representative for the local population, since unrealistic parameter combinations could explain the extremely high risk indices.

Response: We thank the reviewer for noting that the exposure assumptions and data sources in the health risk assessment were not sufficiently transparent. Following the reviewer’s suggestions, we have systematically supplemented and clarified the revised manuscript as follows: (1) we explicitly specify the concentration data used for each exposure pathway-metal concentrations in rice grains used for the dietary intake pathway were derived from the measured concentrations in mature grains from our pot experiment, whereas soil concentrations used for soil ingestion, dermal contact, and inhalation pathways were based on the measured values in the contaminated and control soils; (2) the exposure parameter values reported in Tables S4 and S5 were taken from the guidance released by the U.S. Environmental Protection Agency (USEPA, 2011), and this source has now been clearly cited in the revised text; and (3) we further addressed the uncertainty of both carcinogenic and non-carcinogenic risk estimates using a Monte Carlo simulation in Section 2.5.

The framing of the work as a “life cycle toxicity test of rice” is not well supported by how the ecological and health risk assessments are actually conducted: the title and Abstract emphasize “life-cycle toxicity test of rice” (lines 1–3 and 8–13), and the Introduction states that health risks are assessed “based on life-cycle toxicity tests of rice” (lines 73–75), while Sections 2.2.1–2.2.3 and 3.3 describe detailed measurements of plant growth, antioxidant enzymes, MDA, and amino acid exudation across tillering, heading, and maturity (pages 5–7 and 11–12), yet the ecological (Section 2.3) and health risk (Section 2.4) indices are entirely concentration based and do not incorporate these biological endpoints in any quantitative way; to avoid a mismatch between title and content, the authors should either demonstrate how the life cycle toxicity data refine risk assessment (for example by linking specific growth or biochemical thresholds to risk categories) or adjust the title and aims so that the toxicity test is presented as a mechanistic component rather than the basis of the risk indices.

Response: We sincerely thank the reviewer for the valuable comment. After careful consideration, we have revised the title to: “Ecotoxicological effects of heavy metals on rice across its life cycle and health risk assessment in agricultural soils around a Pb-Zn mine.”

The statistical analysis and presentation of variability need clarification so that readers can properly interpret significance claims: Figures 1–3 and the amino acid figure (Figure 4, lines 219–220) all show error bars and use asterisks to indicate significant differences between control and contaminated soil, and Table 1 uses different letters to denote significant differences in bioaccumulation and translocation factors across growth stages (line 134 to line 136), while Section 3.7 simply states that SPSS 23 and Tukey’s HSD test were used to examine treatment differences (lines 403–405); the authors should specify the number of replicates contributing to each mean value, state whether error bars are standard deviation or standard error, explain whether assumptions of normality and homoscedasticity were checked (and whether any transformations were applied), and clarify within what grouping the letters in Table 1 indicate difference (within a metal across stages, or across metals within a stage).

Response: We sincerely thank the reviewer for the professional suggestions regarding the statistical analysis and presentation of variability. We have supplemented the data analysis description in Section 3.8 of the revised manuscript. In addition, we added a note to Table 1 to clarify that different letters indicate significant differences in the bioaccumulation and translocation factors of heavy metals in rice among different growth stages (p < 0.05)

The ecological risk assessment section has several structural and referencing problems that reduce clarity, including mislabeled figures and potentially confusing classification language: the text states “As shown in Fig. 6a, The NIPI values were all > 3 (Fig. 5a)” (lines 224–225), which appears to reference two different figure numbers for the same bar chart, and later refers to RI values shown in “Fig. 5b” (line 235), while the amino acid figure immediately before this section is captioned as “Figure 4” (lines 219–220), creating a non-sequential figure order; in addition, the RI value “exceeded 150” and is described as indicating a “moderate ecological risk” while Cd is said to pose “considerable” risk (lines 235–241), yet the classification thresholds are only given in supplementary tables (lines 367–369) and are not restated; I suggest checking and harmonizing all figure numbers and references, ensuring that each figure is called in numerical order, and briefly stating the RI and EI class boundaries in the main text so that terms such as “low,” “moderate,” and “considerable” are immediately interpretable.

Response: We sincerely thank the reviewer for the valuable comments. We have carefully checked and harmonized all figure numbers and in-text references throughout the manuscript. In addition, the classification criteria for the ecological risk index (RI) have been explicitly added to the main text (Lines 270) to improve clarity and interpretability

The micro X ray fluorescence analysis is described in technical beamline detail but lacks basic information about sample selection and the role of these data in the study, which makes this subsection feel disconnected from the rest of the work: Section 3.2.2 gives extensive specifics on electron energy, current, monochromator, polycapillary lens, step size, detector type, live time, and data reduction software (lines 305–313), yet does not state how many rice plants and which organs were scanned, how samples were prepared (for example fresh or dried, section thickness, mounting), whether multiple biological replicates were analyzed, or how the qualitative maps in Figure S1 support the quantitative BF and TF results reported in Section 2.1.2; the authors should add information on sample numbers and preparation and briefly explain what additional mechanistic insight the μ-XRF images provide beyond the bulk concentration data, or consider moving some of the beamline technical detail to the Supplementary Materials.

Response: We sincerely thank the reviewer for the valuable comments. We have added details on the number of μ-XRF samples and the sample preparation procedure in the revised manuscript, and we have moved the highly technical beamline/instrumental details to the Supplementary Materials (Text S1)

There are several recurring presentation issues that should be corrected before publication, including inconsistent terminology and some confusing labels in figures and text: the section heading “Ecotoxicological toxicities of HMs on rice in the life-cycle” (line 137) is superfluous and could be simplified; in the text, the trait is called “panicle emergence” (lines 149–150) whereas the y axis label and Methods use “Head emergence number” (line 157 and line 290), and Figure 3 panels label the growth stages as “Tillering period, Flowing period, Mature period” with “Flowing” apparently a typographical error for “Heading” (lines 172–188); the amino acid figure caption calls this “Figure 4” (lines 219–220) while the text refers to “Fig. 5” (lines 196–199), and in several places “soil-rice” is written with inconsistent spacing; a careful pass to standardize trait names, correct the “Flowing period” typo, align all figure numbers between text and captions, and ensure consistent hyphenation of “soil-rice” and similar terms will improve readability.

Response: We sincerely thank the reviewer for the valuable comments. We have addressed each issue accordingly: (1) the section heading has been simplified to “Toxicities of HMs on rice growth”; (2) the term “panicle emergence” has been revised to “Head emergence number” for consistency with the y-axis label and Methods; (3) the typo “Flowing” in Figure 3 has been corrected to “Heading”; (4) all figure numbers and in-text citations have been carefully checked and harmonized between the main text and figure captions; and (5) the hyphenation of compound terms such as “soil-rice” has been standardized throughout the manuscript.

Comments on the Quality of English Language

Response: We sincerely thank the reviewer for the valuable comments. We have carefully checked the manuscript for errors and thoroughly polished the language and sentence structure to meet the journal’s requirements.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors

I read with great interest the manuscript entitled "Health Risk Assessment of Heavy Metals in Agricultural Soil Around a Large Pb-Zn Mine Based on a Life-Cycle Toxicity Test of Rice." The topic of the study is highly relevant. New data has been obtained that may have practical implications. They are important for assessing the negative impact of heavy metals on the environment in areas where mining operations are located. Moreover, the authors used soil from fields located around such operations, which is crucial. It is also significant that the authors analyzed a wide range of heavy metals in the soil and rice plants. Heavy metal levels and their effects on certain physiological parameters were determined at different stages of development and in various plant organs and parts. The authors assessed the bioaccumulation of heavy metals and their translocation within the plant. Unlike many similar studies, this study assessed the pollution index and potential risks of such mining operations to the environment and human health. I believe this manuscript is of undoubted interest and can be recommended for publication after considering a number of comments.

1. The authors collected soil samples from agricultural lands surrounding a mining area. This requires greater specificity, as the word "around" is completely uninformative. It would be preferable to indicate the distance from the source of contamination where the soil was collected.

2. How should the phrase "soil from rice roots..." be understood? "see Soil from the rice roots was collected carefully with a brush to measure the amino acid content."

3. For biochemical analysis, samples are typically stored at -70-80 degrees Celsius, not -20 degrees Celsius.

4. Section 3.3.1 describes methods for analyzing the activity of a number of antioxidant enzymes. The methods should be described in more detail, with mandatory references to the literary sources from which these methods were borrowed. In this case, the authors did not provide a single reference to the methods used. Centrifugation speed should be specified in g, not rpm. Typically, the models of the instruments used (centrifuges, spectrophotometers, etc.) are specified.

5. All enzyme activities are expressed in mg/kg, which is absolutely unacceptable. Typically, enzyme activity is expressed in units of activity per unit of protein. It is important to specify for each enzyme what is considered to be a unit of enzymatic activity.

6. To assess lipid peroxidation, the authors measured TBARS (thiobarbituric acid-reactive substances) rather than MDA. Measuring MDA would require using a slightly different method that ensures specificity of the interaction of thiobarbituric acid with MDA. In this case, TBARS should be specified in the manuscript instead of MDA.

7. I believe that referring to the heavy metal content per kg of dry weight as a concentration is not entirely accurate, since the term "concentration" is usually used for solutions. In this case, it is better to refer to the metal content.

8. In the Plants journal, Results and Discussion are usually presented in separate sections.

Kind regards

Author Response

Dear Reviewers:

Sincerely,

Fangyu Hu and Baoyu Wang,

On behalf of all authors.

Response: We sincerely appreciate your kind review of our work. We have made point-by-point responses to your comments as carefully as possible. The replies provided in detail are as follows:

The authors collected soil samples from agricultural lands surrounding a mining area. This requires greater specificity, as the word "around" is completely uninformative. It would be preferable to indicate the distance from the source of contamination where the soil was collected.

Response: We sincerely thank the reviewer for this valuable comment. We have supplemented the information on sampling locations and sampling strategy in Figure 1, providing clearer details on the sampling sites and methods

How should the phrase "soil from rice roots..." be understood? "see Soil from the rice roots was collected carefully with a brush to measure the amino acid content."

Response: We sincerely thank the reviewer for this comment. Here, “soil from rice roots” refers to rhizosphere soil, i.e., soil tightly adhering to the rice root surface and collected gently using a brush. We have revised the manuscript to clearly specify “rhizosphere soil” to avoid ambiguity

For biochemical analysis, samples are typically stored at -70-80 degrees Celsius, not -20 degrees Celsius.

Response: We thank the reviewer for pointing this out. This was an error in the original description, and the storage temperature has been corrected accordingly in the revised manuscript.

Section 3.3.1 describes methods for analyzing the activity of a number of antioxidant enzymes. The methods should be described in more detail, with mandatory references to the literary sources from which these methods were borrowed. In this case, the authors did not provide a single reference to the methods used. Centrifugation speed should be specified in g, not rpm. Typically, the models of the instruments used (centrifuges, spectrophotometers, etc.) are specified.

Response: We sincerely thank the reviewer for this important suggestion. Detailed descriptions of antioxidant enzyme assays have now been added. Due to length considerations, the full methodological details and literature references have been moved to Supplementary Text S2. In addition, centrifugation speeds have been uniformly converted from rpm to ×g.

All enzyme activities are expressed in mg/kg, which is absolutely unacceptable. Typically, enzyme activity is expressed in units of activity per unit of protein. It is important to specify for each enzyme what is considered to be a unit of enzymatic activity.

Response: We thank the reviewer for this valuable comment. Enzyme activity units have been revised in Figure 4, and all enzyme activities are now uniformly expressed (U/g).

To assess lipid peroxidation, the authors measured TBARS (thiobarbituric acid-reactive substances) rather than MDA. Measuring MDA would require using a slightly different method that ensures specificity of the interaction of thiobarbituric acid with MDA. In this case, TBARS should be specified in the manuscript instead of MDA.

Response: We sincerely thank the reviewer for this clarification. In this study, MDA (malondialdehyde) content was measured. The previous description may not have been sufficiently clear. We have now provided a detailed and explicit description of the MDA determination method in Supplementary Text S2.

I believe that referring to the heavy metal content per kg of dry weight as a concentration is not entirely accurate, since the term "concentration" is usually used for solutions. In this case, it is better to refer to the metal content.

Response: We thank the reviewer for this helpful suggestion. The term “concentration” has been consistently replaced with “metal content” throughout the manuscript.

In the Plants journal, Results and Discussion are usually presented in separate sections.

Kind regards

Response: We thank the reviewer for this comment. In the present study, toxicity responses of rice across different growth stages, heavy metal accumulation characteristics, and ecological/health risk assessments are highly interrelated. Presenting the results and discussion together allows immediate mechanistic interpretation and risk implication following each set of results, thereby improving overall coherence and readability. Therefore, we have chosen to retain the combined “Results and Discussion” structure.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors make changes according to the comments. And I am satisfied now from my end.
I have no further comments.

Thank you and good luck to the authors.