Effect of Partial Organic Fertilizer Substitution on Heavy Metal Accumulation in Wheat Grains and Associated Health Risks

Round 1

Reviewer 1 Report

Dear Authors,

I find the manuscript presented to me for evaluation interesting from the point of view of agronomy. Due to the low use of nitrogen from mineral fertilizers and environmental pressures, organic fertilizers are being used more and more frequently and in larger quantities. The manuscript presented here presents a different approach outlining the potential risks associated with this, which is overlooked in many studies in this area. In my opinion, the manuscript needs minor additions and clarifications.

In several places, the authors use the term long-term on ongoing field research. In my opinion, two-year research is not long-term. I suggest replacing it with another term.

L32 I understand that the Authors focused on the effects of HM on human health and the risks associated with it when using organic fertilizers. In my opinion, it would also be good to mention in a few sentences other sources of HM appearing in crops, such as industrial development, transportation.

L83 Due to the fact that the analysis was conducted in years, weather conditions during the field research should be given. In addition, there is no information about the HM content in the soil before the field research was established

L98-99 I suggest converting to P and K component and stating what mineral fertilizer was applied.

L101 Is it possible to provide the content of N, P, K in the organic fertilizers applied?

L111 I understand that the values given in the table are appropriate for the organic and mineral fertilizers used in the field study? Or are they general values?

L125 I suggest you state how the samples were prepared for analysis on ICPMS

In addition to the values given in the Tables, I suggest giving the standard deviation.

I also believe that consideration should be given to moving the wheat grain yield results to the main body of the manuscript. The authors discuss these results in the discussion section and refer to the concentration effect at the obtained HM concentrations, so in my opinion, adding this parameter to the body of the manuscript would be appropriate.

Author Response

I find the manuscript presented to me for evaluation interesting from the point of view of agronomy. Due to the low use of nitrogen from mineral fertilizers and environmental pressures, organic fertilizers are being used more and more frequently and in larger quantities. The manuscript presented here presents a different approach outlining the potential risks associated with this, which is overlooked in many studies in this area. In my opinion, the manuscript needs minor additions and clarifications.

Response: We appreciate the comments from the reviewer. We have carefully revised the comments/suggestions from the reviewing process. We feel the manuscript had been greatly improved.

In several places, the authors use the term long-term on ongoing field research. In my opinion, two-year research is not long-term. I suggest replacing it with another term.

Response: This study was from a long-term field experiment with six treatments as mentioned in the M&M part. This site-experiment was initiated in 2016. In wheat cropping seasons of 2021 and 2022, we collected soils and grains sample to evaluate the effects of partial organic fertilizer substitution on health risks posed by HMs. Therefore, the long-term effects of organic fertilizer substitution should be considered in this study.

L32 I understand that the Authors focused on the effects of HM on human health and the risks associated with it when using organic fertilizers. In my opinion, it would also be good to mention in a few sentences other sources of HM appearing in crops, such as industrial development, transportation.

Response: We accepted your suggestion and added other sources of HM appearing in crops a few sentences in Introduction part.

L83 Due to the fact that the analysis was conducted in years, weather conditions during the field research should be given. In addition, there is no information about the HM content in the soil before the field research was established.

Response: We accepted your suggestion and added the weather conditions information and HMs contents of the initial soil in Materials and Methods part.

L98-99 I suggest converting to P and K component and stating what mineral fertilizer was applied.

Response: Thank you for your suggestion. We have converted P₂O₅ and K₂O to P and K component, respectively. Meantime, we stated the mineral fertilizer used in the experiment.

L101 Is it possible to provide the content of N, P, K in the organic fertilizers applied?

Response: The contents of N, P, K in the organic fertilizers applied had been provided in Materials and Methods part.

L111 I understand that the values given in the table are appropriate for the organic and mineral fertilizers used in the field study? Or are they general values?

Response: The values given in Table 1 are the HMs contents of organic and mineral fertilizers used in the field study.

L125 I suggest you state how the samples were prepared for analysis on ICPMS

Response: Thanks for your suggestion! We have stated how the samples were prepared for analysis on ICPMS in M&M part.

In addition to the values given in the Tables, I suggest giving the standard deviation.

Response: We accepted your suggestion and provided SD in all tables.

I also believe that consideration should be given to moving the wheat grain yield results to the main body of the manuscript. The authors discuss these results in the discussion section and refer to the concentration effect at the obtained HM concentrations, so in my opinion, adding this parameter to the body of the manuscript would be appropriate.

Response: We accepted your suggestion and added the yield result in the body of the manuscript.

Author Response File: Author Response.DOCX

Reviewer 2 Report

The manuscript “Effect of partial organic fertilizer substitution on heavy metal accumulation in wheat grains and associated health risks” is a study of the incorporation of organic fertiliser from livestock waste as a sustainable strategy to substitute chemical fertilisers in wheat production. The main novelty is the emphasis on metal accumulation in wheat grains and the assessment of human health risks, both carcinogenic and non-carcinogenic, from heavy metal exposure via wheat consumption in a general exposure scenario. The article is well written and uses a large number of appropriate references to put into context the line of research they are addressing and the problem they hope to solve. However, I suggest the following recommendations and adjustments to improve the overall quality of the manuscript.

The authors must clarify why in the methodology section, line 85, it is stated that the experiment started in 2016, but in the S1 graphs of the supplementary material and in Table 1 of the results, the authors refer to the production of the period 2021 and the period 2022, these graphs should show the period 2016 and 2017.

The authors need to show the nutrient content of the organic fertiliser employed, as the only information given on organic fertiliser is metal concentration. This information is relevant to validate the calculations they propose in their study.

The phrasing of section 2.1 (lines 99 to 105) could be difficult to understand in some sentences, especially in relation to the proportions and amounts of fertilisers applied during the trial, it is suggested to show the amounts applied per treatment in a table, it is suggested to provide the information applied for each crop and for each year studied.

In the Materials and Methods section, the authors use the abbreviation "OF" but never define it, one intuitively assumes that they use this abbreviation to refer to an organic fertiliser, but this should be clearly stated.

For section 2.2, please give the standard method you have used for the analysis of metals in soil and cereals. Please give details of the detection limits and further details of the analytical quality control, which can be included in the supplementary material and referred to in this section.

The subtitle of section 2.3.1 should be Accumulation Factor, when we refer to translocation we use what is transferred from the root to the aerial tissue.

In section 2.4 the authors mention that they will do a one-factor ANOVA, but in table 1 of the results section it is noted that a two-way ANOVA with interactions was performed, please clarify and correct.

In section 3.1, lines 172 - 176, it is important to clarify that the concentrations refer to the grain.

The authors present the table of results of metal accumulation in wheat grain for each year, but Figure 1 of metal accumulation in soils does not follow this order and shows only the average of the two years, they should be consistent in the presentation of results and show them annually.

Figure 1 must indicate what the box plots are (mean, median, interquartile range, minimum, maximum). On the other hand, the scale in Figure 1 could be adjusted to better highlight the differences between treatments, for example by trying not to start from zero on the Y-axis. The heading of the Y-axis in Figure 1 is incorrect for the As and Cr results.

Although the organic fertiliser contains large amounts of Zn, As and Cr, it would be expected that its application to the soil would increase the concentrations of these metals in the soil and that this would be reflected in Figure 1, but this is clearly not the case. In the case of Cu and Pb, the organic fertiliser contributes large amounts of Cu and Pb to the soil, which is clearly shown in Figure 1.

Something is wrong with figure 2, in the 40%OF treatment bar, the contribution of Zn is missing and the contribution of Cr is exaggerated.

Lines 197 - 202 regarding FA do not contribute to the interpretation of the results, as well as Table 2. Please consider not including this variable in the manuscript, the same consideration for the paragraph of the discussion section.

Author Response

The manuscript “Effect of partial organic fertilizer substitution on heavy metal accumulation in wheat grains and associated health risks” is a study of the incorporation of organic fertiliser from livestock waste as a sustainable strategy to substitute chemical fertilisers in wheat production. The main novelty is the emphasis on metal accumulation in wheat grains and the assessment of human health risks, both carcinogenic and non-carcinogenic, from heavy metal exposure via wheat consumption in a general exposure scenario. The article is well written and uses a large number of appropriate references to put into context the line of research they are addressing and the problem they hope to solve. However, I suggest the following recommendations and adjustments to improve the overall quality of the manuscript.

Response: We appreciate your valuable comments and suggestions to improve our manuscript.

The authors must clarify why in the methodology section, line 85, it is stated that the experiment started in 2016, but in the S1 graphs of the supplementary material and in Table 1 of the results, the authors refer to the production of the period 2021 and the period 2022, these graphs should show the period 2016 and 2017.

Response: This study was from a long-term field experiment with six treatments as mentioned in the M&M part. This site-experiment was initiated in 2016. In wheat cropping seasons of 2021 and 2022, we collected soils and grains sample to evaluate the effects of partial organic fertilizer substitution on health risks posed by HMs. Therefore, the results of wheat yield provided in the study was refer to the period 2021 and the period 2022.

The authors need to show the nutrient content of the organic fertiliser employed, as the only information given on organic fertiliser is metal concentration. This information is relevant to validate the calculations they propose in their study.

Response: Agree. We had provided the N, P, K and organic matter contents of organic fertilizer applied in the experiment in in M&M part.

The phrasing of section 2.1 (lines 99 to 105) could be difficult to understand in some sentences, especially in relation to the proportions and amounts of fertilisers applied during the trial, it is suggested to show the amounts applied per treatment in a table, it is suggested to provide the information applied for each crop and for each year studied.

Response: We agree with the reviewer that the experimental description was a bit confused and needed improvement. We have revised this section to make it easy to understand.

In the Materials and Methods section, the authors use the abbreviation "OF" but never define it, one intuitively assumes that they use this abbreviation to refer to an organic fertiliser, but this should be clearly stated.

Response: Editing change was made as suggestion.

For section 2.2, please give the standard method you have used for the analysis of metals in soil and cereals. Please give details of the detection limits and further details of the analytical quality control, which can be included in the supplementary material and referred to in this section.

Response: Thanks for your suggestion! We have added the analytical quality control in Materials and Methods part. The detection limits for HMs of grain and soil were presented in the note of Table2 and Fig.2, respectively.

The subtitle of section 2.3.1 should be Accumulation Factor, when we refer to translocation we use what is transferred from the root to the aerial tissue.

Response: Editing change was made as suggestion.

In section 2.4 the authors mention that they will do a one-factor ANOVA, but in table 1 of the results section it is noted that a two-way ANOVA with interactions was performed, please clarify and correct.

Response: Thanks! We have clarified and corrected the statistical analysis in M&M part.

In section 3.1, lines 172 - 176, it is important to clarify that the concentrations refer to the grain.

Response: Thanks. We have clarified and revised concentration as content in the manuscript.

The authors present the table of results of metal accumulation in wheat grain for each year, but Figure 1 of metal accumulation in soils does not follow this order and shows only the average of the two years, they should be consistent in the presentation of results and show them annually.

Response: Thanks for your suggestion! In fact, different from wheat grain, the HMs contents in soil is more stable the variation between years was less. Considering that the trend of variation in HMs contents in the soil is consistent during the two years, the box diagram with 2 years data of HMs contents was better to compare the differences between the treatments.

Figure 1 must indicate what the box plots are (mean, median, interquartile range, minimum, maximum). On the other hand, the scale in Figure 1 could be adjusted to better highlight the differences between treatments, for example by trying not to start from zero on the Y-axis. The heading of the Y-axis in Figure 1 is incorrect for the As and Cr results.

Response: We agree with the reviewer and revised Fig. 2 to better highlight the differences between HMs contents of treatments.

Although the organic fertiliser contains large amounts of Zn, As and Cr, it would be expected that its application to the soil would increase the concentrations of these metals in the soil and that this would be reflected in Figure 1, but this is clearly not the case. In the case of Cu and Pb, the organic fertiliser contributes large amounts of Cu and Pb to the soil, which is clearly shown in Figure 1.

Response: Nice question. It is interesting to see that the application of organic fertilizer which contains large amounts of Zn (284.5 mg kg^-1) didn’t increased the total Zn content of soils. We thought that the reasons can be into: 1) The transfer and accumulation capacity of Zn from soil to straw and grain is stronger than other HMs, more Zn from organic fertilizer addition is transferred to crops. 2) The amount of Zn from organic fertilizer annually was approximate 1.7 kg Zn ha^-1 even at the highest substitution ratio. However, the background content of total Zn in the soil is the highest, and the variation between the replicates was higher than other HMs. The variation between replicates in the field environment most likely weakened the effect of organic fertilizer application on the improvement of soil total Zn content.

Something is wrong with figure 2, in the 40%OF treatment bar, the contribution of Zn is missing and the contribution of Cr is exaggerated.

Response: Thanks for your suggestion! We have replaced the right Fig.3 in the manuscript.

Lines 197 - 202 regarding FA do not contribute to the interpretation of the results, as well as Table 2. Please consider not including this variable in the manuscript, the same consideration for the paragraph of the discussion section.

Response: Agree. We have deleted this variable in M&M, Results and Discussion parts.

Author Response File: Author Response.DOCX

Reviewer 3 Report

In my opinion, the manuscript is well-conducted but it does not include any relevant information about the issue. In addition, the results are regional and not applicable to other world areas. The information about the samples is not indicated and is strongly needed.

In my opinion, the manuscript is well-conducted but it does not include any relevant information about the issue. In addition, the results are regional and not applicable to other world areas. The information about the samples is not indicated and is strongly needed.

Author Response

Reviewer #3:

In my opinion, the manuscript is well-conducted but it does not include any relevant information about the issue. In addition, the results are regional and not applicable to other world areas. The information about the samples is not indicated and is strongly needed.

Response: Thank you for the comments. We have carefully checked the journal website (https://www.mdpi.com/journal/agronomy/special_issues/1HSPGA7U18) and confirmed that the topic of our study fits well with the special issue scope in term of food safety. The North China Plain where the site experiment located, is an important wheat production base in China, with wheat yield accounting for 70% of the country's total amount. Considering that organic fertilizer application is becoming a common fertilization method in China, it is imperative to investigate the accumulation of HMs in wheat grain and assess the health risk. In addition, this study provide an assessment method that balancing the need for agricultural productivity, sustainability, and food safety in organic farming system, which was very helpful to conduct similar studies in other world areas. Meantime, we have added more details about samples analysis and quality control in M&M part.

Author Response File: Author Response.pdf

Reviewer 4 Report

All my notes were inserted in the pdf manuscript

Comments for author File: Comments.pdf

Quality of the English Language is OK 

Author Response

Reviewer #4:

Abstract: how the use of organic manures increases the concentrations of the heavy metals

Response: Thanks for your suggestion! The mechanism of how organic manures increases the HMs contents of grain was discussed in Discussion part. We feel it would be better to leave it out of the Abstract.

Introduction: please insert a paragraph on the wheat and its importance, shortage of production, composition

Response: Thanks for your suggestion! The wheat and its importance, the challenge of food safety was described in line 40-43.

Line62 should go to up to be two paragraphs on the heavy metals then a paragraph on organic fertilization

Response: Thanks for your suggestion! In fact, the second and third paragraphs contains the background of both heavy metals and organic fertilizer application. We feel that the present manuscript is a better expression of our need for conducting this study.

Line74 The article is focused on only nitrogen fertilization, what about the other required nutrients

Response: Thanks for your valuable suggestion! Because it is impossible to substitute the same ratio of chemical N, P and K with organic fertilizer at the same time. Only one of the indicators can be used as the reference to calculate the amount of organic fertilizer. Considering the wide application of nitrogen fertilizer in this area, we used N as a reference to calculate the amount of organic fertilizer used to substitute chemical fertilizer.  

Line82 the aim of the study not clear and the importance of is not clear

Response: Thanks for your suggestion! We have revised the aim and importance of the study to make them clear.

M&M: the analysis of the soil should be more detailed

Response: Thanks for your suggestion! We have added more detail on the analysis of the soil samples.

What is the type of organic manure to have such heavy metals?

Response: The organic fertilizer used in the study was made of cow manure and crop straw. We have added the related detail in M&M part.

Why the authors concentrate on the substitution only on nitrogen not the other nutrients

Response: Thanks for your valuable suggestion! Because it is impossible to substitute the same ratio of chemical N, P and K with organic fertilizer at the same time. Only one of the indicators can be used as the reference to calculate the amount of organic fertilizer. Considering the wide application of nitrogen fertilizer in this area, we used N as a reference to calculate the amount of organic fertilizer used to substitute chemical fertilizer. 

Table1: is Zn from the heavy metals

Response: The Table 1 showed the results of heavy metal contents in wheat grains.

Should be rewrite and to be related to the obtained results

Response: Thanks for your suggestion! We have made appropriate changes to the Discussion section to make it clear and related to the obtained results.

What is the source of organic fertilization to have such high contents from these heavy metals?

Response: Thanks for your suggestion! We have clarified the source of organic fertilizer in M&M part.

These is no Zn as you reported

Response: Yes. As Fig.2 showed, organic fertilizer substitution increased the total contents of Cu, Cd, Pb and As in the study. There were no significant differences in the contents of Zn, Ni, and Cr in soils between the treatments.

Conclusions: Not clear. It must be rewrite again and it should be more related to the results.

Response: Thanks for your suggestion! We have revised the conclusions part to make it clear and more related to the results.

Author Response File: Author Response.pdf

Reviewer 5 Report

Dear Authors, your work is very valuable and very interesting. However, as a reviewer, I have some comments that may make it even better. First of all, I believe that the table with the composition of heavy elements from the fertilizer should not be included in the additional materials, but in the main part of the work in the methodology chapter. I also believe that the fertilizer should be characterized in more detail in terms of its origin. This is very important in the context of its composition. Organic fertilizers coming from polluted or highly industrialized places, or from animals fed with feed containing a significant content of heavy metals in the feed will also have a high content of heavy metals in the manure. In addition, soil properties are also important for the collection of heavy metals, including the content of organic matter, which is provided in the additional materials, but soil pH is also crucial here and such information should be included.

Author Response

Dear Authors, your work is very valuable and very interesting. However, as a reviewer, I have some comments that may make it even better. First of all, I believe that the table with the composition of heavy elements from the fertilizer should not be included in the additional materials, but in the main part of the work in the methodology chapter. I also believe that the fertilizer should be characterized in more detail in terms of its origin. This is very important in the context of its composition. Organic fertilizers coming from polluted or highly industrialized places, or from animals fed with feed containing a significant content of heavy metals in the feed will also have a high content of heavy metals in the manure. In addition, soil properties are also important for the collection of heavy metals, including the content of organic matter, which is provided in the additional materials, but soil pH is also crucial here and such information should be included.

Response: Yes, you are right. We accepted your suggestion and moved Table S1 into the body of the manuscript. Meantime, we provided more detail in terms of organic fertilizer origin and its composition in M&M part. We also provide soil properties data including SOM, pH, total N etc. in additional materials.

Author Response File: Author Response.DOCX

Round 2

Reviewer 3 Report

The manuscript has been improved.

Reviewer 4 Report

I accept the current manuscript to publish in agronomy journal.

English language is OK