Effects of Soil pH on the Growth and Cadmium Accumulation in Polygonum hydropiper (L.) in Low and Moderately Cadmium-Contaminated Paddy Soil

Round 1

Reviewer 1 Report

This study is devoted to the problem of soil pH effects on the growth and cadmium accumulation in Polygonum hydropiper L. in cadmium- contaminated soil. The authors found that Cd accumulation in stems and roots, as well as sub-cellular distribu- 328 tion in P. hydropiper was affected by pH. However, the manuscript can be published only after a number of shortcomings have been eliminated.

1.      In your work, you determined the main soil parameters. Why was total nitrogen not determined, which is an important factor in plant growth?

2.      Why do you think that it is pH that has a significant effect? Why was multivariate analysis not performed taking into account the content of organic C, nitrogen and phosphorus?

3.      It is not clear from the caption to Figure 1 that there are parameters a and b. Also, from the caption to Table 2, it is not clear which coefficient is represented.

4.      Was control presented in the experiment? And is it possible to talk about the absence of stimulation of low Cd content if there is no comparison with the control?

5.      Line 26-266. On what basis do you make this assumption? Are there any similar studies?

Comments for author File: Comments.pdf

Author Response

March 3, 2023

Land

Dear reviewer:

        Thank you for providing us with the opportunity to revise our manuscript “Effects of soil pH on the growth and cadmium accumulation in Polygonum hydropiper (L.) in low and moderately cadmium-contaminated paddy soil”. We greatly appreciate your thoughtful comments and revised the manuscript accordingly. Below we respond to your comments in turn, demonstrating how we changed the manuscript.

Point 1:  In your work, you determined the main soil parameters. Why was total nitrogen not determined, which is an important factor in plant growth?

Response: We determined the soil total nitrogen content and provided in the Table 1. The content of total nitrogen in low and moderately Cd-contaminated paddy soil was 0.18 and 0.27g/kg respectively.

Point 2:  Why do you think that it is pH that has a significant effect? Why was multivariate analysis not performed taking into account the content of organic C, nitrogen and phosphorus?

Response: Thank you for your suggestion! Soil organic matter, microorganisms, pH and other factors may affect the potential of phytoremediation technology^[1]. Among them, the change of soil pH will affect the bioavailability of heavy metals in soil, and then affect the ability of plants to extract Cd^[2]. This study focuses on enhancing the restoration efficiency of Polygonum hydropiper in Cd-polluted paddy soil by adjusting soil pH. The changes of soil organic carbon, nitrogen and phosphorus may also affect the ability of plants to accumulate and transport heavy metals. Further research may take into account organic C, nitrogen and phosphorus.

Point 3:  It is not clear from the caption to Figure 1 that there are parameters a and b. Also, from the caption to Table 2, it is not clear which coefficient is represented.

Response: Thank you for your valuable advice, we have rewritten the title as Different lowercase letters (a,b,c) indicate significant differences among three pH treatments. Table2. Summary of the influence of each term (F-value) in a general linear model (GLM) testing the interacting influence of soil pH and cadmium (Cd) levels on biomass accumulation of Polygonum hydropiper. Table 3. Summary of the influence of each term (F-value) in a general linear model (GLM) testing the interacting influence of soil pH and cadmium (Cd) levels on Cd accumulation by Polygonum hydropiper.

Point 4: Was control presented in the experiment? And is it possible to talk about the absence of stimulation of low Cd content if there is no comparison with the control?

Response: This experiment used two levels of Cd-polluted paddy soil in Beishan town, Changsha County. In the study site, there is no paddy soil without Cd contamination. In the discussion, we deleted the sentences about stimulation of low Cd content on plant growth.

Point 5:   Line 26-266. On what basis do you make this assumption? Are there any similar studies?

Response: In Line 26, we pointed out that P. hydropiper has the potential to remediate Cd-polluted paddy soil based on the relatively high bioconcentration factor (BCF) and translocation factor (TF). Bioconcentration and translocation factors have previously been used to evaluate the capacity of plants to absorb Cd ^[3-5]. In general, a plant can be a good accumulator when it has high BCF and TF ^[3]. Vystavna's study on grapes and Cui's study on Cardamine violifolia both confirmed this view^[3,4].

In Line 266, we pointed out that higher Cd levels would cause the imbalance between the production and removal of reactive oxygen species (ROS) in plant cells when plants are subjected to high Cd stress. Mao et al.'s research on Japanese honeysuckle is consistent with our study and corroborates this view^[6].

References

[1]          Gul I, Manzoor M, Hashim N, et al. Challenges in microbially and chelate-assisted phytoextraction of cadmium and lead - A review. Environmental Pollution, 2021, 287.

[2]          Nouri H, Hashempour Y. Phytoremediation of Cd and Pb in polluted soil: a systematic review. International Journal of Environmental Analytical Chemistry.

[3]          Vystavna Y, Rätsep R, Klymenko N, et al. Comparison of soil-to-root transfer and translocation coefficients of trace elements in vines of Chardonnay and Muscat white grown in the same vineyard. Scientia Horticulturae, 2015, 192: 89-96.

[4]          Cui L, Tian X, Xie H, et al. Cardamine violifolia as a potential Hg hyperaccumulator and the cellular responses. Science of The Total Environment, 2023, 863: 160940.

[5]    Li, X.; Yang, Y.P. Preliminary study on Cd accumulation characteristics in Sansevieria trifasciata Prain. Plant Diversity 2020, 42, 351–355.

[6]          Mao X F, Xu X B, Chen L L. Effects of heavy metal Pb and Cd stress on physiological characteristics of Japanese honeysuckle. Applied Ecology and Environmental Research, 2019, 17(3): 6415-27.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript is interesting and fits well the scopes of Land. The topic is useful from a scientific point of view. However, I have some comments.

1.     Introduction: please, more emphasis novelty of your research.

2.     Introduction: the specific objectives and hypotheses should be more precise (lines 74-85).

3.     Materials and methods: Table 1 table 1 contains some of the test results. Why was it included in the methodology? Please move it to the Results section and comment. The values contained therein are not commented anywhere.

4.     Materials and methods: I suggest the use of multidimensional statistical techniques to determine the relationships between Cd fractions in soil and individual plant parts, pH, organic matter and other.

5.     Results: this part should be re-written in a less schematic style.

Author Response

March 3, 2023

Land

Dear reviewer:

         Thank you for providing us with the opportunity to revise our manuscript “Effects of soil pH on the growth and cadmium accumulation in Polygonum hydropiper (L.) in low and moderately cadmium-contaminated paddy soil”. We greatly appreciate your thoughtful comments and revised the manuscript accordingly. Below we respond to your comments in turn, demonstrating how we changed the manuscript.

Point 1: Introduction: please, more emphasis novelty of your research.

Response: Thanks for your valuable advice! We have emphasized the novelty of our research in several places. 1) Most plants used in phytoremediation are mesophytes or xerophytes, which are difficult to apply in paddy soils ^[14] because of the fluctuating cultivation conditions. (Line 49-51). 2) However, the synergistic effects of soil pH and Cd contamination degree on efficiency of phytoremediation by wetland macrophytes have rarely been examined. (Line 68-69).

Point 2: Introduction: the specific objectives and hypotheses should be more precise (lines 74-85).

Response: Thank you for your advice! We have made the objectives more specific and the hypotheses more precise.

Our objective is to investigate the effect of soil pH and Cd contamination degree on the efficiency of P. hydropiper in the remediation of paddy soil. To this end, we manipulated three levels of soil pH (5, 6, and 7) and used two degrees of Cd-contaminated paddy soil (low and moderate) to test their effects on the growth, Cd accumulation, and translocation of P. hydropiper. We hypothesized that lower soil pH and higher soil Cd contamination would decrease plant growth, but would increase the Cd accumulation in P. hydropiper.

Point 3: Materials and methods: Table 1 table 1 contains some of the test results.  Why was it included in the methodology?  Please move it to the Results section and comment.  The values contained therein are not commented anywhere.

Response: Thanks for your suggestion. Table 1 describes the chemical characteristics of low and moderately contaminated paddy soil used in the experiment, which provided the information of experimental materials. Therefore, we included in the Materials and methods.

Point 4: Materials and methods: I suggest the use of multidimensional statistical techniques to determine the relationships between Cd fractions in soil and individual plant parts, pH, organic matter and other.

Response: We collected low and moderately Cd-contaminated soil from the same small watershed. Soil physiochemical characteristics were similar except Cd pollution level (Table 1). Therefore, it is difficult to determine the relationships between Cd fractions in soil and individual plant parts, pH, organic matter and other. Thanks for your suggestion! Our further research may take into account these factors.

Point 5: Results: this part should be re-written in a less schematic style.

Response: We have rewritten the results section in a less schematic style with the help of language editing company (https://www.editage.cn/).

Author Response File: Author Response.docx

Reviewer 3 Report

Comments for author File: Comments.pdf

Author Response

March 3, 2022

Land

Dear reviewer:

         Thank you for providing us with the opportunity to revise our manuscript “Effects of soil pH on the growth and cadmium accumulation in Polygonum hydropiper (L.) in low and moderately cadmium-contaminated paddy soil”. We greatly appreciate your thoughtful comments and revised the manuscript accordingly. Below we respond to your comments in turn, demonstrating how we changed the manuscript.

Point 1: Change the writing style for all latin plant names across the text.

Response: Thanks for your valuable advice! We have italicized the latin names of the plants throughout the manuscript.

Point 2: Line 72 # This clearly indicates that P. hydropiper is tolerant to Cd stress and has potential to remediate Cd-contaminated paddy soil.”As can be seen from the previous two sentences, the statement should be opposite or be clarified by specifying the range of Cd contamination.  I've checked reference [16] and unfortunately cannot find reference [31].  Nevertheless, it can already be stated that Polygonum has limited potential for remediation of Cd-contaminated paddy soils, since the main indicator (BCF) drops already at a Cd concentration of 2 mg/L, and drastically drops to 0.68 and 2 for above-ground shoots and roots,respectively, in the case of 100 mg/kg.  Therefore, the statement should be reformulated.

Response: Thanks you for your valuable advice. Reference [16] is a hydroponic experiment. Under 1 mg·L^-1 cadmium solution for 30 days of treatment, the bioconcentration factor of the aboveground and belowground parts of P. hydropiper was 45.6 and 111.7 respectively, and the transport factor was 0.41. Although the bioconcentration factor of cadmium in the underground and aboveground parts of P. hydropiper increased first and then decreased with the cadmium concentration, the bioconcentration factor and transport factor are still higher in comparison with common wetland plants. Reference [31] is a pot experiment, which shows that among the four plants studied, P. hydropiper had the highest Cd absorption in the aboveground part, which was 68mg/kg. Therefore, we assumed that P. hydropiper has the potential to restore polluted paddy soil based on above references.

We reformulate this statement as “The above studies demonstrated that P. hydropiper is tolerant to Cd stress and has the potential to be used in remediation of Cd-polluted paddy soil.”

Point 3: Line 79 # Please check the value as 0.2 mg/kg was mentioned in line 40.

Response: Line 40 mentions that 0.2 mg/kg is the critical value of Cd exceeding the standard for rice, while line 79 mentions that 0.3 mg/kg is for soil.

Point 4: Line 79 # Better to provide real concentrations ranges for each type of contamination.

Response: We provided real concentrations ranges for each type of contamination. Paddy fields are defined as lightly polluted when total Cd concentration within the range 0.3–0.6 mg/kg and moderately polluted when total Cd concentrations within the range of 0.9–1.5 mg/kg. (Line 83-85).

Point 4: Line 93 # Please provide SE for average values.

Response: We provided SE for average values. We mixed each type of contaminated soils evenly, so SE is small. The Cd content of the two paddy soils was 0.52±0.03 mg/kg and 0.92±0.01 mg/kg.

Point 5: 2.4 Some formatting problems with words likes “CO2 , NH4F–0.025 mol/L HCL , NH4OAc”

Response: We solved these formatting problems.

Point 6: Line144 # Perform one-way ANOVA to be sure that these soil parameters did not influence the results obtainted.

Response: We sampled each type of contaminated soil from one patch of rice field and mixed each type of soil evenly before the experiment. Theoretically, the Cd content and other chemical characteristics was same for each type of soil. Therefore, these soil parameters did not influence the results obtained.

Point 7: Line 159 # “2400 r/min” is rpm?

Response: 2400 r/min expresses the unit of rotational speed, which has been modified to the international unit "rpm" in the manuscript.

Point 8: Line 168 # What do you mean by the "content", the concentration?

Response: The “content” here refers to an amount of Cd in per mass of plant organs or soil.

Point 9: Line 178 # Please provide all values for biomass as supplementary table in the form of "mean & se"

Response: We provided all values for biomass in the form of mean ± se as supplementary table S1.

Point 10: Line 186 # Figure should be re-drawn since accoridng to Table 2, pH had no infuence on the parameters studied, therefore, statistical groups could no differ within one Cd contamination level between different pHs, except for root biomass.

Response: We re-drawn Fig. 1. We assessed the effect of soil pH value and Cd level on shoot mass, root mass, and total mass using a genera linear model. We performed multiple comparisons of the means using Tukey’s test at a significance level of 0.05 (p < 0.05).The differences among treatments in the Figure 1 were based on statistical results.

Point 11: Line 193 # Please provide all values for Cd concentration in plant organs as supplementary table in the form of "mean & se"

Response: We provided all values for Cd concentration in plant organs in the form of mean ± se as supplementary Table S2.

Point 12: Line 202 # Figure 2a should be re-drawn as only pH significantly affected the Cd content in leaves, therefore statistical groups should be shown only for pHs without separation by contamination.

Response: We re-drawn Figure 2. We assessed the effect of soil pH value and Cd level on leaf Cd content, stem Cd content, and root Cd content using a genera linear model. We performed multiple comparisons of the means using Tukey’s test at a significance level of 0.05 (p < 0.05). The differences among treatments in the Figure 2 were based on statistical results.

Point 13: Line 219 # Statistical groups for soluble fraction should be changed as only Cd contamination level had singificant influence on this parameter.

Response: We changed the statistical groups for soluble fraction.

Point 14: Line 220 # Should be re-written in accordance to shown statistical groups.

Response: We re-written the figure legend in according to shown statistical groups. Subcelluar cadmium (Cd) distribution (3A) and the distribution of Cd in the soluble fractions (3B) under three soil pH values with two Cd levels. Different lowercase letters indicate significant differences between different pH treatments.  * and ns indicates differences between two soil Cd levels (* p < 0.05, ** p < 0.01, *** p < 0.001，ns p > 0.05).

Point 15: Please provide all BCF and TF values for each plant organ as supplementary table in the form of "mean & se"

Response: We provided all BCF and TF values for each plant organ in the form of mean ± se as supplementary table S3.

Point 16: Line 235 # How did you calculate the TF? If you have separate contaminant concentrations in leaves and stems, you should normally get 2 TF values. Please provide a detailed explanation.

Response: We calculated the translocation factor (TF) from Cd content in the aboveground parts of plants divided by the Cd content in the roots. We used the average Cd content of leaves and stems as aboveground Cd content, which has been applied in many references. Therefore, we got only one TF values.

Point 17: Line239 # How were BCF values calculated? Did you use available soil Cd concentrations for the calculation?

Response: We calculated the BCF using the Cd content in each plant organ divided by the total Cd content in the soil (Line178). We used the soil total Cd concentration for the calculation. We made this clear in the manuscript.

Point 18: Line 243 # Please provide values of available soil Cd as a supplementary table in form of "mean & SE".

Response: We provided values of available soil Cd in the form of mean ± se as supplementary S4.

Point 19:Line 244 # When were these concentrations measured? Before the experiment or after the plants were grown in the soil?

Response: We measured these concentrations after the plants were grown in the soil. We revised the subtitle, table caption and figure legend.

Point 20: Line 257 # “However, we did not observe a stimulatory effect of low soil Cd content on the growth of P. hydropiper.”It seems that this statement can not be made as you don't have a control plants (grown in non-contaminated soil). Please remove this sentence.

Response: Thanks for your great advice! We removed this sentence.

Author Response File: Author Response.docx

Reviewer 4 Report

The article entitled: Effects of soil pH on the growth and cadmium accumulation in Polygonum hydropiper L. in low and moderately cadmium-contaminated paddy soil is well structured and written. It has an average-to-high level of originality. However, in the Introduction Section, I recommend inserting 2-3 sentences to highlight this study's originality and/or novelty.

I add some comments and observations directly in the manuscript.

After these corrections/modifications, the article can be published in Land Journal.

Comments for author File: Comments.pdf

Author Response

March 3, 2023

Land

Dear reviewer:

         Thank you for providing us with the opportunity to revise our manuscript “Effects of soil pH on the growth and cadmium accumulation in Polygonum hydropiper (L.) in low and moderately cadmium-contaminated paddy soil”. We greatly appreciate your thoughtful comments and revised the manuscript accordingly. Below we respond to your comments in turn, demonstrating how we changed the manuscript.

Point 1: Line 16 # Persicaria hydropiper L. (it alic) The authors must revise the entire manuscript for this

Response: Thanks for your suggestion! We revised throughout the manuscript

Point 2: Line21 # The authors must rewrite this sentence clarity

Response: We rewrote this sentence for clarity. The Cd content in the roots and stems of P. hydropiper significantly decreased with the increase of soil pH in the moderate Cd contamination soil.

Point 3: Line 35 # if the authors use cadium, must keep style and insert the symbols for nickel copper and arsenic

Response: Change made. Thank you! In China, 19.4% of the arable land is polluted by heavy metals, such as cadmium (Cd), nickel (Ni), copper (Cu), and arsenic (As).

Point 4: Line 37 # All scientific names must be written it alic font

Response: Thanks for your advice! All the plant names in the manuscript have been written in italics.

Point 5: Line 59 # Remediation efficiency can be replace with remediation potential

Response: Change made. Thank you! Several studies have shown that plants have the highest remediation potential in acidic environments.

Point 6: Line 96 113 118# 10-mm, 12g, 4℃

Response: Thank you for your suggestion! we have reformatted all the units in the manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Reviewer 3 Report

Thank you for considering my remarks and improving the manuscript!