Migration and Transformation of Multiple Heavy Metals in the Soil–Plant System of E-Waste Dismantling Site
Round 1
Reviewer 1 Report
I enjoyed reading this manuscript, which describes the effect of growing two different cultivars of Chinese cabbage on heavy metal accumulation and microbial communities in soil contaminated by e-waste. While the experiments described in this manuscript appear to be appropriate and methodologically sound, the way the methods are described and the data is currently presented is extremely confusing, especially for the general-interest reader of Microorganisms.
Specific comments:
Introduction:
- The abstract contains a lot of unexplained terms (e.g. low-accumulation and high-accumulation cultivars) and unexplained abbreviations (e.g. CK) which are inappropriate for an abstract, particularly one for a general-interest audience.
- For the first paragraph, more information is necessary on how e-waste is "released" as this is not described. Is it into water, soil? How does it move from the e-waste site into the surrounding environment?
- Throughout the paper, the terms "conversion", "transformation" and "migration" are used to describe how heavy metals move between fractions, apparently interchangeably. If these terms have different technical meanings this must be defined.
- Lines 78-81: more information is needed to justify this sentence. Is there a specific site that was studied, or is this from a review of multiple heavy-metal contaminated sites?
- Lines 102-105: Bioaccumulation is mentioned here, but this is not picked up in great detail in the Results or Discussion. I would either remove this aspect or make it more prominent throughout the manuscript.
Methods:
- Line 115: "Notorious" is a very emotionally charged negative term - potentially replace with more netural wording
- Section 2.2.: this is a very confusing description of the experimental setup. I would suggest adding a diagram to help orient the reader. Also, why is "control" abbreviated CK? As far as I know this is not common and may confuse a reader.
- Section 2.6: Only bacteria and fungi are surveyed, which means other microorganisms such as heterotrophic flagellates are not included in this study. Make sure this is referenced in the text as HFs can have a considerable effect on ecosystem processes.
- Line 194: Caporaso et al. is the reference for the entire QIIME toolkit rather than a specific pipeline - make sure to include information on e.g. databases used for taxonomic assignment, algorithms used for clustering/denoising of sequences.
Results:
- Figures 1,2,3: the lettering system you've used for significance is really confusing. I suggest using the * system for denoting significance in bar charts. For Figure 1, I would suggest separate graphs for each metal so the y axis is not truncated.
- In various experiments, the control is either the control soil, the soils in which no plants are present, or the soils in which there is no biochar. Because you are testing three different presence/absence variables at the same time, it is very confusing when the manuscript switches between them. This is particularly true in Figure 3, as the abundances are relative and, by eye, the largest effects appear to be based on the type of cultivar present in the soil. This makes it very hard to link the significance percentages quoted in the text to the diagram. Also, in Line 278 the AF of Al is quoted as 142.3%, which is impossible.
- Line 333: This is confusing because of the ambiguity of which controls are being referred to in each experiment. When Bacteriodetes decrease and Chloroflexi increase, what is this relative to? This is an issue with most of the quoted statistics from the microbiome section.
Discussion
- Could any of the changes in % HM in each fraction be due to disproportionate uptake of that fraction into plants, as opposed to HM migrating from one fraction to another? How is accumulation in the plants taken into account when speculating on movement of HMs between fractions?
I suggest minor revisions because all of my comments refer to the presentation of the data, rather than the data itself. However, substantial editing is required for the overall structure, scope and conclusions of this paper to be clear.
Author Response
I enjoyed reading this manuscript, which describes the effect of growing two different cultivars of Chinese cabbage on heavy metal accumulation and microbial communities in soil contaminated by e-waste. While the experiments described in this manuscript appear to be appropriate and methodologically sound, the way the methods are described and the data is currently presented is confusing, especially for the general-interest reader of Microorganisms.
Specific comments:
Introduction:
The abstract contains a lot of unexplained terms (e.g. low-accumulation and high-accumulation cultivars) and unexplained abbreviations (e.g. CK) which are inappropriate for an abstract, particularly one for a general-interest audience.
Authors: Thank you for your comments. “low accumulated Z1 and non-low-accumulated Z2” has been changed into “heavy metals low-accumulated variety of Z1 and non-low-accumulated Z2”. “CK” has been changed into “the blank soil (CK)”. An Abbreviation has been added in this part in order to help readers understand this paper clearly.
For the first paragraph, more information is necessary on how e-waste is "released" as this is not described. Is it into water, soil? How does it move from the e-waste site into the surrounding environment?
Authors: Thank you for your comments. “The incineration residues, broken particles, wastewater, and dust generated during the recovery of e-waste will enter the local ecosystem through atmospheric subsidence and rainwater runoff [1,2].” has been added in this part to explain the releasement of e-waste.
References:
[1] Vaccari, M.; Vinti, G.; Cesaro, A.; Belgiorno, V.; Salhofer, S.; Dias, M.I.; Jandric, A. WEEE Treatment in Developing Countries: Environmental Pollution and Health Consequences-An Overview. International Journal of Environmental Research and Public Health 2019, 16.
[2] Cesaro, A.; Belgiorno, V.; Gorrasi, G.; Viscusi, G.; Vaccari, M.; Vinti, G.; Jandric, A.; Dias, M.I.; Hursthouse, A.; Salhofer, S. A relative risk assessment of the open burning of WEEE. Environmental Science and Pollution Research 2019, 26, 11042-11052.
Throughout the paper, the terms "conversion", "transformation" and "migration" are used to describe how heavy metals move between fractions, apparently interchangeably. If these terms have different technical meanings this must be defined.
Authors: Thank you for your comments. We are sorry these words cause ambiguity, and “conversion” has been changed into “migration” or “transformation”. In addition, “migration” means the movement of heavy metals in the media and “transformation” means the change of one metal fraction to another, which has been defined in the text.
Lines 78-81: more information is needed to justify this sentence. Is there a specific site that was studied, or is this from a review of multiple heavy-metal contaminated sites?
Authors: Thank you for your comments. The site in this research located in a typical lead-zinc mining area in Hanyuan, Sichuan Province, China, which has been added in the manuscript.
Lines 102-105: Bioaccumulation is mentioned here, but this is not picked up in great detail in the Results or Discussion. I would either remove this aspect or make it more prominent throughout the manuscript.
Authors: Thank you for your comments. We are sorry that such expression may cause ambiguity. Bioaccumulation has been removed and we focus on “rhizosphere soil” here instead.
Line 115: "Notorious" is a very emotionally charged negative term - potentially replace with more netural wording
Authors: Thank you for your comments. “Notorious” has been changed into “typical”.
Section 2.2.: this is a very confusing description of the experimental setup. I would suggest adding a diagram to help orient the reader. Also, why is "control" abbreviated CK? As far as I know this is not common and may confuse a reader.
Authors: Thank you for your comments. Table 2 has been added in 2.2 to explain the different treatments. We are sorry that this sentence cause ambiguity. “control (CK)” has been changed into “blank soil (CK)”.
Section 2.6: Only bacteria and fungi are surveyed, which means other microorganisms such as heterotrophic flagellates are not included in this study. Make sure this is referenced in the text as HFs can have a considerable effect on ecosystem processes
Authors: Thank you for your comments. Heterotrophic dinoflagellates belong to protists, which is really less in the soil of this study, and their impact on soil heavy metal migration or transformation is not significant. In addition, it is reported that as the exposed metal concentration increased (Cd, Zn, Pb, Hg), the numbers of protists decreased significantly [3]. In a soil-microcosms study, Zn, Pb and Cu remarkably decreased protists number at concentrations higher than 35 mg/kg, 9 mg/kg and 8.1 mg/kg, respectively [3,4]. However, the concentrations of Pb, Zn and Cu in the soil we studied are far more higher than the concentration of heavy metals reported. Therefore, the impact on soil heavy metal migration or transformation by protists is not considered in this study. This part has been rearranged and we only focus on bacteria and fungi in the rhizosphere.
References:
[3] Amin, N.M.J.T.F.o.E. Techniques for assessment of heavy metal toxicity using Acanthamoeba sp., a small, naked and free-living amoeba. 2012, 199-212.
[4] Diaz, S.; Martin-Gonzalez, A.; Carlos Gutierrez, J. Evaluation of heavy metal acute toxicity and bioaccumulation in soil ciliated protozoa. Environment International 2006, 32, 711-717.
Line 194: Caporaso et al. is the reference for the entire QIIME toolkit rather than a specific pipeline - make sure to include information on e.g. databases used for taxonomic assignment, algorithms used for clustering/denoising of sequences.
Authors: Thank you for your comments. This part has been rearranged in the manuscript and the sequence analysis is followed by the method described by Gao [5].
References:
[5] Gao, F.; Fan, H.; Chapman, S.J.; Yao, H. Changes in soil microbial community activity and composition following substrate amendment within the MicroResp™ system. Journal of Soils and Sediments. 2022.
Figures 1,2,3: the lettering system you've used for significance is really confusing. I suggest using the * system for denoting significance in bar charts. For Figure 1, I would suggest separate graphs for each metal so the y axis is not truncated.
Authors: Thank you for your comments. The figures have been rearranged accordingly.
In various experiments, the control is either the control soil, the soils in which no plants are present, or the soils in which there is no biochar. Because you are testing three different presence/absence variables at the same time, it is very confusing when the manuscript switches between them. This is particularly true in Figure 3, as the abundances are relative and, by eye, the largest effects appear to be based on the type of cultivar present in the soil. This makes it very hard to link the significance percentages quoted in the text to the diagram. Also, in Line 278 the AF of Al is quoted as 142.3%, which is impossible.
Authors: Thank you for your comments. In this paper, “the control” means the blank soil without biochar or plants (CK), we compare CK with the treatment of biochar (RB) or the treatment of plants (Z1, Z2) to analyze the effect of a single variable on heavy metal migration in soil. “The control” has been rearranged in the manuscript accordingly. In addition, “the AF of Al is quoted as 142.3%” is the increased proportion of OF-Al percentage of Z2 to that of CK, which is the relative ratio, not the absolute concentration. In order to make the readers understand clearly, this sentence has been changed into “In addition, Compared to the CK, the OF of Al in the rhizosphere soil of Z2 significantly increased, reaching 55.0%”.
Line 333: This is confusing because of the ambiguity of which controls are being referred to in each experiment. When Bacteriodetes decrease and Chloroflexi increase, what is this relative to? This is an issue with most of the quoted statistics from the microbiome section.
Authors: Thank you for your comments. This sentence has been changed into “In addition, compared to CK, the Bacteroidetes significantly reduced from 4.1% to 1.8%, but Chloroflexi increased significantly when treated with RB+Z1 and RB+Z2.” In order to make the paper more fluent and clear, the same issue in the microbiome section has been proofed and changed.
Could any of the changes in % HM in each fraction be due to disproportionate uptake of that fraction into plants, as opposed to HM migrating from one fraction to another? How is accumulation in the plants taken into account when speculating on movement of HMs between fractions?
Authors: Thank you for your comments. The changes in % HM in each fraction are not due to disproportionate uptake of that fraction into plants. In fact, plants would not uptake conservative fractions like ResF but mainly accumulate soluble fractions like AF instead. This is also proved by other study which showed the increased bioavailability of heavy metals in soil promoted the accumulation of heavy metals in rice and brassicas [6]. It is also showed that only the mobile and potentially bioavailable heavy metals in the soil would threaten the quality of crops and enter into the food chain [7]. Actually, the change of % HM in each fraction is mainly depend on the rhizosphere environmental factors, such as the secretion of root exudates (low molecular organic acids), the microorganisms in rhizosphere and organic matters [8-10]. Therefore, we focus on the metal fractionations changes in the soil, which is equilibrium state reached by plants and rhizosphere soil.
References:
[6] Xiao, L.; Guan, D.; Peart, M.R.; Chen, Y.; Li, Q. The respective effects of soil heavy metal fractions by sequential extraction procedure and soil properties on the accumulation of heavy metals in rice grains and brassicas. Environmental Science and Pollution Research 2017, 24, 2558-2571.
[7] Barac, N.; Skrivanj, S.; Mutic, J.; Manojlovic, D.; Bukumiric, Z.; Zivojinovic, D.; Petrovic, R.; Corac, A. Heavy Metals Fractionation in Agricultural Soils of Pb/Zn Mining Region and Their Transfer to Selected Vegetables. Water Air and Soil Pollution 2016, 227.
[8] Ma, J.F.; Shen, R.; Zhao, Z.; Wissuwa, M.; Takeuchi, Y.; Ebitani, T.; Yano, M. Response of rice to Al stress and identification of quantitative trait Loci for Al tolerance. Plant & cell physiology 2002, 43: 652-659.
[9] Shetty, R. & Prakash, N. B. Effect of different biochars on acid soil and growth parameters of rice plants under aluminium toxicity. Scientific Reports 2020, 10, 12249.
[10] Qian, L.; Chen, B.; Chen, M. Novel Alleviation Mechanisms of Aluminum Phytotoxicity via Released Biosilicon from Rice Straw-Derived Biochars. Scientific Reports 2016, 6.
I suggest minor revisions because all of my comments refer to the presentation of the data, rather than the data itself. However, substantial editing is required for the overall structure, scope and conclusions of this paper to be clear.
Authors: We thank the reviewer for his/her valuable comments and suggestions for improving our manuscript. We have invited a native English speaker to help to proof and revise the paper to make it clearer.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The article "Migration and transformation of multiple heavy metals in the soil-plant system of e-waste dismantling site: based on continuous 13CO2 labeling combined with high-throughput sequencing" is interesting and of great importance. The problem of environmental pollution with heavy metals is indeed very important and such studies are very relevant. The study was carried out at a good methodological level, interesting and well presented. Nevertheless, the article is quite difficult to read due to the large number of abbreviations and symbols. It would be good to simplify the presentation or summarize the abbreviations in a separate list at the beginning of the article. In addition, in Figure 1 it is not clear what is meant by letter designations.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
In the presented manuscript complex tasks are set, but their solution looks as if the authors are doing it first in the world. Schemes of sequential selective extractions (speciation) able to extract different amounts of metal fractions associated with various soil components. For example, the scheme of Tessier et al. (1979) recovers 5 fractions. The species of metals are conditional, however, the first isolated fractions are characterized by a weak bond with soil components, therefore they are mobile, and the last fractions, especially the residual, are conservative. In this regard, the increase in the content of copper and zinc in the residual fraction when biochar was added (line 272), as well as copper, zinc and lead when growing Beijing cabbage (line 274) for a short time are doubtful. It is also unlikely that the total content of heavy metals and aluminum in the studied volume of soils will decrease after growing Beijing cabbage, especially since most of the metals were in conservative fractions. It is necessary to provide convincing data from other researchers, confirming each of the conclusions made in this manuscript.
Notes to the text.
Line 48. It is categorically stated that 4 forms of heavy metals are released in the soil. According to what method?
Line 53. Migration of conservative fractions is not possible. Transformation takes place over a long period of time.
Line 58 The use of biochar has become more common in recent times, but it is not a traditional agricultural practice.
Line 100. This is a "uptake", not an "absorption".
Line 104. Studying the relationship between the speciation of metals and the composition of the microbial community is a complex (including methodological) task. It is not solved as simply as the authors try to make it.
Line 109. What is “non-low”? High?
Table 1. Is there no potassium in biochar?
Line 126-127. Perhaps authors are talking about varieties of cabbage “New Beijing” etc?
Line 215. Must be "Heavy metals and Al"
Figure 1. The amount of aluminum is reduced by 2 times. The data is highly questionable. Literary evidence is required.
Figure 2(d). Is there no lead in the leaves?
It is necessary to give a scheme of BCR selective extractions in the "Methods" section and characterize the physical meaning of the isolated fractions.
Figures 4, 6. There are no statistical data.
Table S1 and elsewhere. It is necessary to decipher the variants of the experiment.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
I do not agree with some of the conclusions made by the authors in the manuscript, as well as with a number of the results obtained, but since the authors corrected the manuscript in accordance with my comments and cited literature data to prove their conclusions, the paper may be published.
Author Response
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Author Response File: 
Author Response.docx
