As-Hg Compound Pollution: Rice Growth, Yield, and Environmental Safety Limits
Round 1
Reviewer 1 Report
Line 41: please define PET (like in line 92-93)
Line 73: in spite of many soil types occurring in China, you have selected a soil typical for Chinese southern lowlands (?). The control soil is a non-contaminated clay-rich soil.
Line 111: aging and drying of soil fixes soluble Hg and As species - you might explain; Longer periods ? How about mobile soil phases? You have chosen a worst case scenario. Sulfidic bound (e.g. pyrite) As and Hg are much less transferable to green plants. Trivalent As, as used in your experiment, is more mobile than pentavalent As.
You have added the most toxic form of As, and a medium toxic form of Hg (di-methyl-Hg is utmost toxic)
Line 159: the biotoxicity response coefficients seem arbitrary at a first look; but I have found the provisionable tolerable weekly intake is 4 µg/kg BW for Hg and 15 µg/kg BW for As, which means that Hg is 4 times as poisonous than As
The addition of As and Hg simultaneously is realistic, but you will not see synergistic effects; why not high As with low Hg?
Addition of Se might be antagonistic to both of them. How about sulfate addition to fix HgS?
Anyway, levels of Hg and As in rice are higher than in cereals and soybeans: we found 0,70 µg/kg Hg in composite feeds for piglets, 0,65µg/kg in composite feeds for pigs, and 0,47 µg/kg in compositge feeds for chickens. For As, it was 11 µg/kg for maize, 20 µg/kg for cereals, and 180 µg/kg as median values.
Line 173: to my information, China has set a tolerable concentration of inorganic arsenic in rice at 150 µg/kg - not 200 - please check
The FAO Codex alimentarius recommends < 200 mg/kg for polished rice
The conclusions of soil PTEs health risk limits for As are very low - this is from using the most toxic and mobile form of As.
Sager, Manfred; (2006); Occurrence and mobility of arsenic in environment and agriculture in Austria; Ecological Chemistry and Engineering; 13 (3-4) : 207-222
Chung, Eun Hye; Lee, Jin Soo; Chon, Hyo Taek; Sager, Manfred; (2005); Environmental contamination and bioaccessability of arsenic and metals around the Dongjeong Au-Ag-Cu min, Korea; Journal of Geochemistry: Exploration, Environment, Analysis; Vol. 5 (1) : 69-74
Sager, Manfred; Reichel, Günter; Grüner, Manfred; Würzner, Herbert; (1997); Quecksilbergehalte von Futtermittelproben in Österreich; Die Bodenkultur; 48 (I) : 23-32
Author Response
Point 1: Line 41: please define PET (like in line 92-93)
Response 1: Thank you for your comment.
We have now used the exact abbreviation (PTEs) for term "potentially toxic elements" instead of the term “PET” throughout the manuscript. It was a typological mistake.
Point 2: Line 73: in spite of many soil types occurring in China, you have selected a soil typical for Chinese southern lowlands (?). The control soil is a non-contaminated clay-rich soil.
Response 2: Thank you for your comment.
The soil we used in the experiments was a typical paddy soil from China, 93% of which are distributed in southeast rice regions, ranking first in grain production. Also according to the investigation and analysis of related departments in China, the arsenic and mercury in farmland soils in Longyou County（Belongs to Quzhou area） of Zhejiang Province are higher than those in most areas in China. Therefore, in this study, the typical paddy soil in this area was selected as the test soil. In order to obtain soils with different pollution concentrations, the unpolluted soil in this area was specially selected as the initial soil in order to obtain potted soil from low concentration to high concentration through external addition of toxic elements.
Point 3: Line 111: aging and drying of soil fixes soluble Hg and As species - you might explain; Longer periods ? How about mobile soil phases? You have chosen a worst case scenario. Sulfidic bound (e.g. pyrite) As and Hg are much less transferable to green plants. Trivalent As, as used in your experiment, is more mobile than pentavalent As.
You have added the most toxic form of As, and a medium toxic form of Hg (di-methyl-Hg is utmost toxic)
Response 3: We fully agree with the reviewer's comments，some studies have shown that the bioavailability of arsenic and mercury in soil will gradually decrease over time, but how arsenic and mercury migrate and transform between solid and liquid phases in soil, still needs further research.
Point 4: Line 159: the biotoxicity response coefficients seem arbitrary at a first look; but I have found the provisionable tolerable weekly intake is 4 µg/kg BW for Hg and 15 µg/kg BW for As, which means that Hg is 4 times as poisonous than As.
The addition of As and Hg simultaneously is realistic, but you will not see synergistic effects; why not high As with low Hg?
Addition of Se might be antagonistic to both of them. How about sulfate addition to fix HgS?
Response 4: We fully agree with the reviewer's comments, however we can study these combinations and role of other elements on As and Hg uptake in our future research.
Point 5: Anyway, levels of Hg and As in rice are higher than in cereals and soybeans: we found 0,70 µg/kg Hg in composite feeds for piglets, 0,65µg/kg in composite feeds for pigs, and 0,47 µg/kg in compositge feeds for chickens. For As, it was 11 µg/kg for maize, 20 µg/kg for cereals, and 180 µg/kg as median values.
Response 5: We agree with the reviewers' comments. Rice is an important source of As and Hg contamination. Rice has a strong ability to absorb soil arsenic and mercury, especially rice grown in arsenic and mercury contaminated areas.
Point 6: Line 173: to my information, China has set a tolerable concentration of inorganic arsenic in rice at 150 µg/kg - not 200 - please check
The FAO Codex alimentarius recommends < 200 mg/kg for polished rice.
Response 6: Thank you for your comment.
China has set a tolerable concentration of inorganic arsenic in rice at 200 µg/kg. See details in Food safety national standard-Contaminant limits in food (GB 2762-2017) .
https://max.book118.com/html/2017/0407/99215158.shtm
Point 7: The conclusions of soil PTEs health risk limits for As are very low - this is from using the most toxic and mobile form of As.
Sager, Manfred; (2006); Occurrence and mobility of arsenic in environment and agriculture in Austria; Ecological Chemistry and Engineering; 13 (3-4) : 207-222
Chung, Eun Hye; Lee, Jin Soo; Chon, Hyo Taek; Sager, Manfred; (2005); Environmental contamination and bioaccessability of arsenic and metals around the Dongjeong Au-Ag-Cu min, Korea; Journal of Geochemistry: Exploration, Environment, Analysis; Vol. 5 (1) : 69-74
Sager, Manfred; Reichel, Günter; Grüner, Manfred; Würzner, Herbert; (1997);
Quecksilbergehalte von Futtermittelproben in Österreich; Die Bodenkultur; 48 (I) : 23-32
Response 7: We agree with the reviewers' comments. As pointed out by reviewing experts, some studies have shown that at the same total arsenic contamination concentration, exogenous arsenic preparations added to potted soil are more toxic and mobile than soils contaminated by arsenic in the field, and the corresponding health risk thresholds may be low, in addition, different soil types due to their differences in physical and chemical properties will also affect the threshold of health risks. So, in order to make the research results closer to the actual situation, we will verify the result in the field test and further modify the threshold of health risks.
Reviewer 2 Report
Point 1: heavy metal is still used (i.e Line 14, 42, 47, 48, 50 etc.....). Moreover PTEs (potential toxic elements) and not PETs...
if the authors do not agree with comments, please explain why and do not joke (pets are aninmals).
line 93-95 please use international references for threshold (i.e. https://doi.org/10.1016/j.jhazmat.2019.04.007)
Author Response
Point 1: heavy metal is still used (i.e Line 14, 42, 47, 48, 50 etc.....). Moreover PTEs (potential toxic elements) and not PETs...
if the authors do not agree with comments, please explain why and do not joke (pets are aninmals).
Response 1: Thank you for your comment.
We have now used the exact abbreviation (PTEs) for term "potentially toxic elements" instead of the term “PET” throughout the manuscript. It was a typological mistake.
Point 2: line 93-95 please use international references for threshold (i.e. https://doi.org/10.1016/j.jhazmat.2019.04.007)
Response 2: Thank you for your comment. We have replaced “the national soil environmental quality standards of China (GB15618-2018) ” with “the USEPA, Technology Alternatives for the Remediation of Soils Contaminated With As, Cd, Cr, Hg, and Pb.”(See Line 94-96) .
Reviewer 3 Report
Comments and Suggestions for Authors
Upon revision I can see that the authors have made a concerted effort to address all my comments. I still believe the presentation of data and statistical analysis needs further development and explanation in the methods. Notably the authors have added comparisons for each cultivar between treatments - which I think are additional 1 way ANOVAs (if something else was used it is not clear to me from the methods). While these tests lend statistical weight some of the statements in the manuscript they do not cover all of them (see comments below) additionally the presentation of these additional tests confuses the figure quite a bit.
line by line comments:
In figure legends the word alphabet should be replaced with ‘letters’ or deleted entirely where it follows an asterisk.
Under methods ‘2.7 Data analysis and methods’ I cannot see mention of which analyses were used. (e.g. One or two-way ANOVA, t-tests?). are correlations Pearson or Spearman’s
Ln 214: “this effect varied for the different rice varieties and PTEs.” There is still no explicit test of this statement presented. The added between-treatment comparisons do not explicitly test the hypothesis that ‘varieties respond differently to the heavy metals’. The reason that between variety differences alone (which are shown) do not support this hypothesis is that there is inherent varietal variation in the control. So one needs to disentangle the impact of the heavy metals from the varietal variation. As this is the key question, a test that explicitly test and explain this needs to be thought about. A two-way anova with post hoc tests could be a solution - but i suspect there are other strategies that could work just as well.
Revisiting my previous comment:
Ln 222 “however, there were significant differences in As and Hg accumulation among different rice varieties (P < 0.05). 223” again the statistical test of among-variety comparisons are not presented in the figures. As they are key to the discussion they should be presented.
I believe now that the significant differences in AS/Hg accumulation were shown in the original figure, but the other half of the statement “it can be seen that the contents of As and Hg in the four rice varieties increased significantly with increasing soil contamination” was not statistically tested. (my apologies). Clearly this latter statement is tested now with the addition of the new tests. But the way this -and other- data is presented is confusing. For example, in figure 3a the AS content of XS 134 is marked as different between CK and T1. But not between CK and T2,T3 or T4. I assume they are different from CK but not from T1 but the main issue us that the way these graphs are presented requires the reader to make a lot of assumptions. Is there a clearer way to present this data that retains all of the necessary information?
Author Response
Point 1: In figure legends the word alphabet should be replaced with ‘letters’ or deleted entirely where it follows an asterisk.
Response 1: Thank you for your comment. We have replaced “alphabet” with “letter” and deleted where it followed asterisks.
Point 2: Under methods ‘2.7 Data analysis and methods’ I cannot see mention of which analyses were used. (e.g. One or two-way ANOVA, t-tests?). are correlations Pearson or Spearman’s
Response 2: Thank you for your comment and good suggestion, one and two-way ANOVA wre used in data analysis (See Line 178-179) .
Point 3: Ln 214: “this effect varied for the different rice varieties and PTEs.” There is still no explicit test of this statement presented. The added between-treatment comparisons do not explicitly test the hypothesis that ‘varieties respond differently to the heavy metals’. The reason that between variety differences alone (which are shown) do not support this hypothesis is that there is inherent varietal variation in the control. So one needs to disentangle the impact of the heavy metals from the varietal variation. As this is the key question, a test that explicitly test and explain this needs to be thought about. A two-way anova with post hoc tests could be a solution - but i suspect there are other strategies that could work just as well.
Response 3: Thank you for your comments and useful suggestions. In the revised manuscript, the data was re-statistically analyzed using two-factor analysis of variance (See line 211 and line 230).
Point 4: Revisiting my previous comment:
Ln 222 “however, there were significant differences in As and Hg accumulation among different rice varieties (P < 0.05). 223” again the statistical test of among-variety comparisons are not presented in the figures. As they are key to the discussion they should be presented.
Response 4: Thank you for your comment. In the revised manuscript, a two-way analysis of variance was used to re-statistically analyze the differences in arsenic and mercury accumulation between different rice varieties(See Line 252).
Point 5: I believe now that the significant differences in AS/Hg accumulation were shown in the original figure, but the other half of the statement “it can be seen that the contents of As and Hg in the four rice varieties increased significantly with increasing soil contamination” was not statistically tested. (my apologies). Clearly this latter statement is tested now with the addition of the new tests. But the way this -and other- data is presented is confusing. For example, in figure 3a the AS content of XS 134 is marked as different between CK and T1. But not between CK and T2,T3 or T4. I assume they are different from CK but not from T1 but the main issue us that the way these graphs are presented requires the reader to make a lot of assumptions. Is there a clearer way to present this data that retains all of the necessary information?
Response 5: Thank you for your comment. In order to make the comparison results clearer, in the revised manuscript, all the figures were modified based on the statistical results of the two-factor analysis of variance..
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
No further objections
Author Response
We have improved our English writing.
Reviewer 2 Report
Point 1: heavy metal is still used (i.e Line 13, 41, 46, 47, 49 etc.....).
Point 2: why are not performed the sequential extraction?
Author Response
Point 1: heavy metal is still used (i.e Line 13, 41, 46, 47, 49 etc.....).
Response 1: Thank you for your comment.
We have now used the term "potentially toxic elements" (PTEs) instead of the term “heavy metal” throughout the manuscript.
Point 2: why are not performed the sequential extraction?
Response 2: Thank you for your comment.
The objective of this study was to elucidate the relationship between total soil arsenic/mercury and rice growth, and the relationship between total soil arsenic/mercury and arsenic/mercury contents in rice grains, so sequence extraction data results were not shown in this article, as we think it was not in the scope of our objectives of the study.
The purpose of this study was to clarify the relationship between soil arsenic and mercury concentrations and rice grain absorption and accumulation of these potentially toxic elements (PTEs) in order to obtain the environmental safety limits of total concentration of soil arsenic and mercury. The bioavailability of As and Hg was not the focus of this study. However, we determined the available content according to BCR sequential extraction method. The results will be reported in the next study.
Before rice transplanting, the contents of soil available As and Hg (acid extractable and reducible) were determined and analyzed according to BCR sequential extraction method.
The results were as follows:
Table 1. Available states of As and Hg in potted soil under different treatments
|
|
CK (mg/kg) |
T1 (mg/kg) |
T2 (mg/kg) |
T3 (mg/kg) |
T4 (mg/kg) |
|
As |
0.056±0.0026 a |
0.195±0.0031 b |
0.570±0.0030 c |
1.035±0.0263 d |
1.500±0.0376 e |
|
Hg |
Not Detected (<2 ng/g) |
0.0024±0.0005 a |
0.0069±0.0028 b |
0.0126±0.0033 c |
0.0182±0.0052 d |
The extraction coefficients of As and Hg in soil were:
As: 0.0084, 0.013, 0.019, 0.023, 0.025;
Hg: /, 0.0047, 0.0069, 0.0084, 0.0091.
The results showed that with the increase of As and Hg in potted soil, the available state extraction coefficient increased. The available state content continued to increase. These results were consistent with the trend of As and Hg accumulation in rice.
Reviewer 3 Report
This article investigates the impact of Hg and As across different rice varieties. attempts to place toxicity findings in a environmental risk management context have been made via the incoorperation of Ecological risk assessment and safety limit values. this aspect is refreshing but could be clearer.
the key goal of the study - to compare the impact of the toxins on plant growth across varieties - is never explicitly tested. as far as i can see, all statistical comparisons present are made within each cultivar, not between. as such the key finding (different impacts between cultivars) is simply not supported - although it can be quite easily with the right stats comparisions.
similarly statements regarding differences in the strength of correlation in standard versus hybrid cultivars do not seem supported (see line by line comments)
line by line comments:
Ln 88: what is ‘simple’ soil and what is ‘old’ soil please clarify, this is not descriptive enough
Ln 101: what is ‘water suspension method ‘ please clarify or add reference.
Ln 135 heather -> heated
Ln 194 “These results showed that the response of different rice varieties to As and Hg stress was significantly different (p < 0.05).” the results and statistics presented in in figure 1 do not support this. They show that there was a significant decrease in plant parameters as toxicity increase within each rich variety. The data does not present statistical test of differences in response between variety, which is what text alludes to
Ln 208: “this effect varied for the different rice varieties and PTEs.” Similar to the above comment. There is no explicit test of this statement presented. All comparison made are within variety comparisons.
Ln 222> “however, there were significant differences in As and Hg accumulation among different rice varieties (P < 0.05). 223” again the statistical test of among-variety comparisons are not presented in the figures. As they are key to the discussion they should be presented.
Ln 238 “Data for ecological risk parameters are given in Table 3.” Should this be table 2?
Ln242: “the correlations between panicle weight and As and Hg concentrations in hybrid rice (CY 84 and YY 538) were relatively weak.”the correlation presented in table 2 indicate R2 values similar to the conventional varieties. Indeed the R2 for YY is higher than JH.
Ln 186 “The results of plant height (Fig. 1) showed that height of control plants (CK) of hybrid rice 186 varieties (CY 84, YY 538) were all greater than those of conventional rice (XS 134, JH 218).” this may be true but I can see no statistics comparing plant height between treatment so this statement is not supported.
Ln 294: “plant height of hybrid rice (CY 84, YY 538) was greater than that of conventional rice (XS 134, JH 218)” this may be true but I can see no statistics comparing plant height between treatment so this statement is not supported.
Round 2
Reviewer 2 Report
I do not find the scientific approach correct and usually the BCR analysis is not valid for As or Hg
