Key Factors Controlling Cadmium and Lead Contents in Rice Grains of Plants Grown in Soil with Different Cadmium Levels from an Area with Typical Karst Geology

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled „Key factors controlling cadmium and lead contents in rice grains of plants grown in soil with different cadmium levels from an area with typical karst geology” by Long Li, Lijun Ma, Lebin Tang, Fengyan Huang, Naichuan Xiao, Long Zhang and Bo Song tackles the significant topic from a scientific and socioeconomic point of view. Cadmium poses a severe threat to human and animal health and life, and for non-smokers’ plant-based food is the main route of exposure to Cd. On the other hand, the mechanisms of Cd uptake, translocation, and sequestration in plants are still elusive. Therefore, the manuscript is timely and focuses on an important crop – rice, which serves as a staple food for more than 3 billion people. The manuscript needs to be improved before it can be recommended for publication (please see details below).   

1. The abstract lacks a clear description of soil- and environment-related factors that are known to affect Cd uptake from soil and further translocation from root to shoot. The description of Cd uptake and translocation in plants is very generic.

2. Materials and methods:

- plant cultivation and sample collection should be described in the manuscript, not in supplementary data

-  please add details about ICP-OES and ICP-MS

- figure 2 does not really show metabolic pathways; this figure is also chaotic and needs to be rethought  and reorganized

3. Results:

- the caption of figure 3 should be improved

- section 3.2. – the authors wrote: “When comparing the dry weight of organs within the same rice variety across different soil groups, no significant differences were observed in stem-leaf or root weight, while notable discrepancies were found in the dry weight of the ears among the various soil types.” but there are no results for ears in Figure 4a. In this section, the authors should also mention whether there is a relationship between dry mass, soil type, and the rice cultivar (Cd-high and Cd-low accumulating).

- Figure 5 – why TF is shown as a line? This is not a trend; a line joins points showing TF for plants grown in different soil types is incorrect. What is marked with dashed lines on some graphs?

4. Discussion

- the discussion needs to be rewritten because some part of the discussion is the description of the results, e.g. lines 491-502, 528-538 (in fact, in the results section, some sentences should be moved to the discussion) and the rest of the discussion is rather a summary/conclusion without a deep enough discussion of the presented results on the background of existing literature

5. General remarks

- Ca and Mg are not heavy metals

Comments on the Quality of English Language

English needs some improvement, some statements are awkward or incorrect, e.g. “RA group”, “WP group”, and “XQ group” – why group since this is an acronym for the site of soil collection, “…heavy metals were compared…” – should be rather the contents of heavy metal were compared, “ Characterization of Data on BCF and TF at Different Phases” – should be rather BCF and TF at phase 4 and phase 5

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors study Cd and Pb uptake in three different rice varieties in three different Chinese soils using a pot experiment for 80 days, from planting to harvest. Two soils are from naturally high heavy metal karst regions (one, XQ, also has a Pb-Zn mining area), and one is a control from a non-contaminated region. An aim is not formulated, but it seems the authors want to study what controls the uptake of Cd and Pb in rice, and they have included one rice variety that usually accumulate less heavy metals. The pots contained 20 kg soil and water was added to fully saturate the soil. There were 3 soils x 3 rice varieties=9 treatments. Each 9 treatments had four pots (replicates). No negative control was performed without any rice grown, it might have been good to include but is not strictly necessary.  The pots were sampled for soil before irrigation, before transplanting, at the booting stage, full heading stage, and full ripe stage after 80 days. Both soil and different parts of rice were sampled and analyzed for soil: pH, CEC, OM, total metals including Cd, Pb, Zn, Fe, Mn, Ca, and Mg. Rice plants were harvested at the full heading stage and full ripe stage, and their heavy metal contents (Cd, Pb, Zn, Fe, Mn, Ca, and Mg) were assessed in each part of rice, including the ears, leaves, stems, and roots.

The topic is of interest and high concern, Cd and Pb content in such an important food source as rice, is of world-wide interest. It is also an enigma around the world what determines the uptake of toxic heavy metals such as Cd and Pb in all kinds of crops. Many studies show that the total content of heavy metals in soil very seldom correlate with uptake in crop. This study is therefore interesting, especially the determination of metal at the different stages of rice development and in different parts of rice plant. However, I have some major comments on the scientific design and the conclusions drawn. An aim is lacking, making it difficult to review. Experimental design is not properly described. The authors draw conclusions based on their discovered relationships, that are possible theories but they cannot claim it is the truth. In such systems there are so many factors involved. Some important parameters are even lacking, I would have liked to see the texture of the soils and also the amount of iron (hydr)oxides, determined by e.g. ammonium oxalate extraction on dry soils. Another comment is that 27 of 77 references are in Chinese, it is too many for an international journal. The language is very good, I only have some comments on typos. I suggest a major revision.

Major comments

L98. I lack a statement of the aim of this study and research questions, necessary in all scientific studies. If proper questions are written down, the results are also more easily read.  

L301. pH and SOM contents decreased and then increased. I do not think it was significant? Please state if it was significant. If not, it is not a change, just normal variation due to soil sampling and analysing errors. Generally, soil chemical properties changes slowly and not as fast as 80 days. I admit that paddy soils are more unstable than non-water-saturated soils, the latter being more of my expertise. But I still do not believe that CEC, OC, and metal concentrations will change significantly on that time scale. The differences noted are most likely due to soil heterogeneity or analysing error. pH and redox potential can change though. However, in this case pH changes very little.

L480-481. “Mn in rice roots was found to inhibit Pb translocation…” The authors cannot claim that, it is a possible theory but it is not proven by the data you present, there are too many parameters involved. To discover a significant relationship is not the same as claiming that this is the mechanism.

L490-491. The authors are citing two studies in Chinese, which are unavailable to read for most. Pb binds strongly to SOM, reducing solubility in soil. If there is a lot of dissolved organic matter, Pb binds strongly to it. However, I do not know the bioavailability of Pb bound to DOM to rice. Please refer to a source in English that explains this mechanism.

L497. “Showing that SOM played a significant role in higher Pb level in grain”. I do not think that statement can be proven using a study like this. It could very well be some other parameter that is contributing. Change to “Indicating” or another word that is less certain. You only look at three soils, it is too little to draw conclusions like this. Furthermore SOM binds Pb very hard, making it less soluble and less prone to biological uptake. Another important soil parameter for Pb chemistry is iron (hydr)oxides that bind Pb very strongly, and their presence is neither studied nor discussed here. In different stages in the rice mature stage, the oxides can dissolve due to low oxidation state where Fe3+ is reduced to Fe2+. Then they could release the Pb adsorbed to the oxides to soil solution, which can then be taken up. I suggest to look closer on Fe content in soils, and preferably analyse the amount of amorphous iron (hydr)oxides with the acid ammonium oxalate method in the soils (can be done on dry soil).  

L501. “Implies” is a better word than showing, but once again, there are too few soils to make this conclusion, and it could very well be something else.

L416-434. This section belong to Material and Methods, and should not be duplicated here.

Minor comments

Line numbers in SI would have been helpful, please include next time.

Title. Too long, and cadmium written twice. Please shorten.

L 50. Here you could write the current FAO and WHO standards for Cd content in rice and soil.

L79-80. Clarify that it is Chinese soil standards.

L 84. This statement is very universal, so it should be possible to cite another source than only Chinese ones.

Fig 1. Soil Cd content. Please use the same y-axis range on all three subfigures, to make it clear to reader that the sites have different Cd concentrations. Now it looks like WP has lower Cd content than RA.

SI. Test soil section:

SI. NPK in which proportions? Water was added: Tap water? Was the water analysed for salt and Cd/Pb concentrations?

Table S1. Only show 3 significant figures in the values, the uncertainties can be shown with only 2. This is valid throughout manuscript.

L129; L 133, L138. Typo. Headlines the same.

Table S3. Use a straight – instead of tilde sign ~

L139. It would be good with e.g. a figure in SI were the sampling plan of the pots are illustrated. Was a subsample of the soil and rice plant sampled at each time interval from all pots? How large was the subsample? Or was a whole pot removed? How large were the soil samples and the rice parts?

L161-162. Refer to the brand of the various instruments here (page S3).

L181-182. Unclear. Was it duplicate samples on 10% of the number of total samples? Why 10%? How many total samples were there?

L190-191. Move up.

L220-L230. Is there a reference to Eq 4?

L232. Fig. 2 I lack iron oxides in the Figure.

L260. One decimal figure is enough for percentages.

L272- Consider changing ”Group” to ”soil” or ”site”, would make it clearer. It is unclear now throughout the ms if it is the soil/site or rice variety or both that is “group”.

L294. Fig 4. caption: Typo. Remove: Results of Heavy metal content….

Fig. S2. Change y-axes to have the same range for all three sites, otherwise it is very difficult to interpret as a reader. Also the major units are very strange, sometimes 0.2, 0.3, or 0.5 within the same figure. Harmonize to the same y-axis for all sites using the same major unit, and do similarly for all the parameters..

Fig. S2. Caption: Typo indexes

Table S4. Very difficult to read this table. Perhaps remove the TF-XX and BCF-XX and only show the elements in order. Or think of another way to show this. a) Foot note: Group, unclear if it is site or rice variety.

Reference list. About 27 references out of 77 is in Chinese, excluding many readers who cannot read these references and it makes it difficult to review. When applicable, change to an international journal.  Regarding Chinese chemical standards, write a short description of the method in Supporting Information or find a similar international standard. It would also be valuable to see WHO guidelines for Cd and Pb content in rice and in soil.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

I read your article and, unfortunately, I cannot recommend it for publication. I can understand the typical research trying to explain mechanisms of heavy metal uptake by plants, crops, microorganism, and so on (the scientific literature is plenty of this kind of articles). But here you are forcing an undesirable condition (i.e., cultivate rice in Cd-polluted soils) and modelling metal-uptake just to understand what could happen in this condition. But the common sense says that we need to claim for farmer do not try to do this. Even without any other chance than to growth rice in metal-contaminated soils (which, in fact, is something questionable for multiple points of view), the crops will need to pass all the standards and quality criteria for food consumption, commercialization, and so on. So, I don´t understand the sense of your research.

All the bests.

Comments on the Quality of English Language

Nothing to declare.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Second review 2024-08-06 by Reviewer 2 agronomy-3077513

In general the authors have replied satisfactory to my suggestions, some minor things need to be adjusted, see below. I suggest minor revision.

L146 in new MS: I lack amount of soil sampled (g) each time.

Comment 4:

Apparently SOM can both reduce the solubility of Pb and in other soils the uptake is increased. (Heavy Metals in Agricultural Soils: Sources, Influencing Factors, and Remediation Strategies by Yanan Wan,Jiang Liu,Zhong Zhuang,Qi Wang and Huafen Li. Toxics 2024, 12(1), 63; https://doi.org/10.3390/toxics12010063

Comment 10:

Regarding FAO and WHO standards. It is well known that each region on Earth has different standards, since the soil varies that much and also other things. But it is still of interest to show some of the various standards together in a table, then there are some values to compare with. In my country the standards are sometimes lower than the heavy metal content presence in the natural soils (high background values), causing problems when natural soils are considered hazardous materials. Therefore a comparison of WHO (international) and some regional standards (e.g. India) is of interest. For example, I found this statement that I thought was interesting in Zhao, Front. Agr. Sci. Eng. 2020, 7(3): 333–338, DOI10.15302/J-FASE-2020335

 ”The maximum permissible limit of Cd in rice grain in China is half of that recommended by FAO/WHO (0.2 versus 0.4 mg/kg–1). This strict limit is deemed necessary because of the high consumption rate of rice by the Chinese population”

Some values that put your study in an international context and make it more comprehensible for an international audience. But I will not stress this further, it is not a major point.

Comment 12:

The fact that the concentration of (heavy) metals in the soil, very seldom reflect the concentrations in the plants, is a very well known fact in the international community of soil-plant researchers. I did a quick search on review articles on this, and found for example these three:

1.       Remediation techniques and heavy metal uptake by different rice varieties in metal-contaminated soils of Taiwan: New aspects for food safety regulation and sustainable agriculture Zeng-Yei Hseu,Shaw-Wei Su,Hung-Yu Lai,Horng-Yuh Guo,Ting-Chien Chen &Zueng-Sang Chen Pages 31-52 Published online: 21 Dec 2010 https://doi.org/10.1111/j.1747-0765.2009.00442.x

2.       Front. Agr. Sci. Eng. 2020, 7(3): 333–338, DOI10.15302/J-FASE-2020335

3.       Heavy Metals in Agricultural Soils: Sources, Influencing Factors, and Remediation Strategies by Yanan Wan,Jiang Liu,Zhong Zhuang,Qi Wang and Huafen Li. Toxics 2024, 12(1), 63; https://doi.org/10.3390/toxics12010063

Comment 13. Different axis. Ok, but please write ”Note the different y-axis ranges” in the Figure caption.

Comment 14. NPK-Ok. Generelly the proportions are stated when buying a product, but it should be of limited importance in this study to know the exact numbers. Tap water, likewise. I was just interested if you used deionized water, artificial water, or tap water. I agree that it is most likely not a source of Cd and Pb. I just wondered if it is similar to the water in natural paddy fields, regarding salt content, pH etc.

Comment 21

Not so important. I thought that you could move the sentence higher up in the text (formatting issue), but it is probably not important anymore.

Comments 23:

I am glad you included the BCR analysis. Regarding the figure, I thought that iron and Mn oxides should be represented as a sorbing phase in the soil.

Comment 30:

Thank you for your kind reply, I was not offended. I am glad you have been helped by my feedback. I would like to clarify myself: I do realize that you need to include some national articles/books/standards from your own country when writing about agriculture or similar topics, I do it myself all the time. Furthermore, I admit that the language barrier is not a problem anymore, I tested and could read very well some of your articles. However, I and most of the international society of researchers, have agreed that we value international journals that are peer-reviewed by an international audience of researchers more than national journals from any country in the world. It is a good check to see if researchers from various places in the world think your data is good or not. It is not a perfect system for sure, and it is annoying to write something which is not in your native tongue (English is also my second language), but it is still the system we have now. Furthermore, regarding the international journals, I know who they are and which are good, but if I were to keep track of all other countries’ journals it would be an impossible task.

Please note that I am not saying that the articles you cite are not any good, they are probably very good, but I rather trust an international journal that I have heard of. I also believe that Chinese researchers are generally very good at producing and writing about research in international journals, see the references I give above. You may think I deliberately chose those articles, but they were the best I found on this topic.

To sum up, you should continue to cite Chinese standards and local procedures, it is fine. However, when it comes to science that is of worldwide interest, I would aim to cite more international journals. Since you added 13 international references I am satisfied. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf