The Multifactorial Effect of Digestate on the Availability of Soil Elements and Grain Yield and Its Mineral Profile—The Case of Maize
Round 1
Reviewer 1 Report
The manuscript describes how various fertilizer treatments influence P uptake by 2 different crops. The main justification of the work was to demonstrate that rock phosphate effects of delivering soluble P to crops can be enhanced by elemental S addition. This is not really a novel concept as it is well known that acidification of the soil and rock phosphate can enhance P availability.
In general, the manuscript needs to be extensively revised to improve clarity and grammar. The methods section is somewhat complex and a table showing the various treatments should be included to help the readers understand the treatments.
A more comprehensive justification of treatment selection is requested. Also, a pot study is not the ideal experimental unit to use for agronomic studies, which is a limitation of this study.
Specific comments are provided below.
The abstract should be revised to focus less on methods and more on results/discussion. As is, only 5 lines of the abstract showcase the results. It would be better to present the major findings and conclusions of the study.
Line 19. Also include the time frame of the study. Dissolution of rock phosphate in soil is a long process and not readily detectable in a short time frame used in this study.
Line 99. This is the place in manuscript were pot size and soil volume need to be mentioned. It seems that this information is also presented later in the methods, but it is best to put all information in one place.
How different are the banded and broadcast applications in a small pot? Is there really much difference in placement of fertilizers in this instance? Also, broadcast fertilizer typically entails surface application of the materials, but according to the methods section, broadcast fertilizer was then mixed into the soil, thus similar to banding?
Please provide information of the elemental S used, including granular vs. powder, particle size, etc., and application methods. elemental sulfur dissolution in soil is also a long process and generally not completable within the short time frame of this study.
What was justification for maintaining 70% soil water-holding capacity, and how was this level maintained?
Line 147 is confounding. Did the treatments receiving elemental S also receive sulfate? And if so, why?
The short time frame of the study is a limiting factor for this manuscript. It takes a long time for rock phosphate to dissolve, even in soil pH has been decreased. Thus, it is difficult to ascribe treatment effectiveness based only after 40 to 55 days?
Author Response
Agronomy 683274R1 – response
Reviewer #1
Questions/remarks are written in Calibril 10
Response are written in New Times Roman 11
The introductory statement by the reviewer No. 1 sounds as follows:
„The manuscript describes how various fertilizer treatments influence P uptake by 2 different crops. The main justification of the work was to demonstrate that rock phosphate effects of delivering soluble P to crops can be enhanced by elemental S addition. This is not really a novel concept as it is well known that acidification of the soil and rock phosphate can enhance P availability.”
The evaluation made by the Reviewer No. 1 deals with phosphorus uptake by two crops fertilized with phosphate rock with added elemental sulfur.
We could not respond to comments by the Reviewer No. 1. This specific review, in fact, does not refer to our manuscript. The content of our manuscript focuses to the evaluation of maize response to the method and the rate of applied digestate.
Reviewer 2 Report
In overall the manuscript is well written and understandable.
However, some questions rised by reading the text.
In material and methods - explain also the origin of the digestate - is it from cattle manure ect. Is the content of Pb and Cd "low", "average" or "high" compared to the average content in digestates?
Control the format of the tables - what should be in bould, where the horizontal lines should be.
The question of most of figures, except of Figure 1 and 4. The figures repeats the data already presente in tables. There is no meaning to repeat them. Next to that, that the figures repeats the data - figure one: design of figure need to be improved. Bars are not explained in the figure caption. Figure 2, 3, 5 and 6 - what is the difference with the data presented already in the tables. The small letters on the figure at the bars are not described in figure caption.
Table 2 - include to the table capture also the depth of the topsoil (0-30 cm) as all tables and figures should be understandable without reading the main text of the paper.
Discussion - I would love to have short general discussion of the rates and nutrients and quality before the conclusion or extend the conclusion.
In conclusion - "The variability of Cd …" Where? In soil?
Is it safe or not safe to use digestate? Any cumulative effect of Cd and Pb in soil after 3 years of use?
Include some more recent references to the introduction, discussion and list of references.
Author Response
Agronomy 683274R1 – response
Reviewer #2
Questions/remarks are written in Calibril 10
Response are written in New Times Roman 12
However, some questions rised by reading the text.
In material and methods - explain also the origin of the digestate - is it from cattle manure ect. Is the content of Pb and Cd "low", "average" or "high" compared to the average content in digestates?
The applied digestate originated from biogas plant, using cow slurry and maize silage as raw materials.
The cadmium concentration was in the frequently published data. However, it was slightly above threshold value, as required by Estonian regulation (xxx). In our study the Cd concentration was on average 1.68 mg kg-1 DW, whereas the threshold value in Estonia is 1.3 mg kg-1 DW.
Control the format of the tables - what should be in bould, where the horizontal lines should be.
It has been corrected.
The question of most of figures, except of Figure 1 and 4. The figures repeats the data already presente in tables. There is no meaning to repeat them. Next to that, that the figures repeats the data - figure one: design of figure need to be improved. Bars are not explained in the figure caption. Figure 2, 3, 5 and 6 - what is the difference with the data presented already in the tables. The small letters on the figure at the bars are not described in figure caption.
The laid out of tables was corrected following suggestion made also by the Reviewer No. 3. Figures No. 2, 3, 5 present interactional effect of years and experimental factors on respective characteristics.
Figure No. 2 present the effect of interaction of years and digestate rate on maize grain yield (Table 3).
Figure 3 shows the response of maize grain to interactional effects of years x application method x digestate rate (Y x AM x R) on the content of nitrate nitrogen (Table 4).
Figures 5 and 6 present the impact of interaction of years x application method x digestate rate (Y x AM x R) on the concentration of Cd and Pb in maize grains (Table 7).
There has been added a double standard error to Figures No. 5 and 6, which has been presented as a vertical bar, specific for each column.
Table 2 - include to the table capture also the depth of the topsoil (0-30 cm) as all tables and figures should be understandable without reading the main text of the paper.
This comments refers probably to Table 1. It has been corrected in accordance to the reviewer suggestion.
Discussion - I would love to have short general discussion of the rates and nutrients and quality before the conclusion or extend the conclusion.
The conclusion has been extended, including the basic outcomes of the study.
In conclusion - "The variability of Cd …" Where? In soil?
It refers to variability of the Cd content in soil. It has been corrected in accordance to the reviewer suggestion.
Is it safe or not safe to use digestate? Any cumulative effect of Cd and Pb in soil after 3 years of use?
The Authors opinion on digestate, originating from agricultural biogas plants is positive. The application of biogas slurry is save for soil and plant health, provided a high soil fertility level with respect to soil pH and the content of basic nutrients, like Ca, Mg, and Mg. This opinion has been clearly expressed in the conclusion chapter.
Include some more recent references to the introduction, discussion and list of references.
There has been included some references, explaining the effect of applied digestate on the content of soil available nutrients and the mineral profile of maize grain.
Reviewer 3 Report
Dear Authors,
The manuscript titled "The multifactorial effect of digestate on the availability of soil elements and grain yield and its health – a case of maize" contains interesting data and could be of interest to the scientific community. However, before it can be published, improvements must be made. I recommend that someone with English as native language review the document because it has many grammar errors and it is very difficult to follow and understand. Specific comments:
Title: "...and its health" what do you mean with health? to the soil? with which paramenters did you measure its "health"? this does not fit with the data
line 19: "in the dry 2015"? maybe in the dry season 2015
Keywords: please improve them. For example what do you mean with "soil availability"? maybe nutrient availability?
line 30: "national economy" national, to which country do you refer?
line 40: not only Ch4 and CO2. there are other gases
line 43: not clear
line 52: ammonia??? CH4 is ammonium
95, 96: please write units in all values, not only in the last one
110: crops? you only investigated one crop
114: you have two times G-good, you only need to describe it once
Table 1: improve format
table 1 and fig. 1: when was the sampling time? weather conditions on that day? this has also a big influence. Maybe you can describe it better in the methodology
Table 2: improve title and format
132: delete the first "and"
140: also delete "and"
157: which two values?
159: write units
160-161: write the reference number in a correct format
181-182: not clear
192-193: how do you explain this?
Table 3 and 4: you need to improve these tables. I would suggest to ask a statistician about the analysis of variance. You take the year as another factor and evaluate the interaction between the three factors, I recommend clarifying this with a statistician to see what the real factors are and what analysis to do
Table 5: this table is not necessary, you could include it in supplementary material
Fig 3 and 5 and 6: please include standard error or deviation
314: do not repeat abbreviations
318-319: "spposite trend"? in the correlation matrix this is clear but in the data you presented (Table 6) this is not clear. It will be interesting to include the sodium content in the soil analysis
Table 7: how do you explain the negative correlation between Mg and the yield?
382-401: how do you explain this? this equations only help to undertand the data but how do you explain this results?
406: check abbreviation
408-409: Why? how do you relate this increase with your soil analysis? you have also Cd in the control (Fig. 5)
Author Response
Agronomy 683274R1 – response
Reviewer #3
Questions/remarks are written in Calibril 10
Response are written in New Times Roman 12
The manuscript titled "The multifactorial effect of digestate on the availability of soil elements and grain yield and its health – a case of maize" contains interesting data and could be of interest to the scientific community. However, before it can be published, improvements must be made. I recommend that someone with English as native language review the document because it has many grammar errors and it is very difficult to follow and understand. Specific comments:
Title: "...and its health" what do you mean with health? to the soil? with which paramenters did you measure its "health"? this does not fit with the data
The title of a manuscript has been slightly corrected in accordance to the Reviewer suggestions. The present title sounds as follows:
The multifactorial effect of digestate on the availabilty of soil elements, grain yield and its mineral profile – a case of maize
line 19: "in the dry 2015"? maybe in the dry season 2015
It has been corrected in accordance to the reviewer suggestion.
Keywords: please improve them. For example what do you mean with "soil availability"? maybe nutrient availability?
Keywords have been corrected in accordance to the reviewer suggestion.
line 30: "national economy" national, to which country do you refer?
The phrase “national economy” has been changed in the test.
line 40: not only Ch4 and CO2. there are other gases
It is clear that biogas contains also other gases, but the term biogas, exactly means a mixture of CH4 and CO2 (reference: Achinas S. et al. 2017. A technological overview of biogas production from biowaste. Engineering 3, 299-307)
line 43: not clear
line 52: ammonia??? CH4 is ammonium
This sentence refers to nitrogen and its components in biogas slurry.
95, 96: please write units in all values, not only in the last one
Al required units have been inserted into the text.
110: crops? you only investigated one crop
It has been corrected.
114: you have two times G-good, you only need to describe it once
It has been corrected.
Table 1: improve format
It has been corrected.
table 1 and fig. 1: when was the sampling time? weather conditions on that day? this has also a big influence. Maybe you can describe it better in the methodology
This part of the MM chapter has been corrected in accordance to the reviewer suggestion. Soil was sampled two days after maize harvest, i.e. keeping the same meteorological conditions as that during harvest. Maize grain was harvested under constant weather conditions, i.e. dry air for a week, at least.
Table 2: improve title and format
It has been corrected.
132: delete the first "and"
It has been deleted.
140: also delete "and"
It has been deleted.
157: which two values?
There were compared even three values. This part has been corrected.
159: write units
Units have been added.
160-161: write the reference number in a correct format
This reference has been written in a right form.
181-182: not clear
It has been corrected.
“As shown in Figure 3, in 2014 the NO3-N content was 3-folds, and about 7.5-folds higher with respect to 2015 and 2016.”
192-193: how do you explain this?
The explanation is presented below.
The impact of soil available Mg on the NO3-N content can be explained in two ways. Firstly, the amount of post-harvest N-NO3 showed a declined yearly trend, being in accordance with the declining content of available Mg (Table 4). Only in 2014, there was observed a linear increase in the content of N-NO3 in response to the amount of Mg added in digestate. The observed phenomena, i.e. drop in postharvest N-NO3 content in the dry season 2015 can also be explained by a shortage of water, but in 2016 by a shortage of easily available N pool (Table 1). The 2nd explanation corroborates the hypothesis on the required synchrony between N release from organic fertilizers and maize growth [20]. The lack of this synchrony in 2015 and 2016 was probably the key reason of the grain yield drop.
Table 3 and 4: you need to improve these tables. I would suggest to ask a statistician about the analysis of variance. You take the year as another factor and evaluate the interaction between the three factors, I recommend clarifying this with a statistician to see what the real factors are and what analysis to do
Tables No. 3, 4, and 5 have been prepared in accordance to Reviewer suggestions. The ANOVA has been conducted in a way to show the real effect of experimental factors in each of the studied years on examined characteristics of soil and maize grain. There has been also shown F-values for interactions of experimental factors and years. Results, concerning soil fertility characteristics have been presented separately for macro, and micronutrients (Table 4 and 5). Results about mineral profile have been also presented individually for macro, and micronutrients plus heavy metals (Tables 6 and 7).
Table 5: this table is not necessary, you could include it in supplementary material
Tables 5 and 7 have been treated as supplementary material.
Fig 3 and 5 and 6: please include standard error or deviation
All these three figures have been corrected; a double standard error has been inserted. The bar for an individual column indicates a double standard error of the mean.
314: do not repeat abbreviations
It has been corrected throughout the text.
318-319: "spposite trend"? in the correlation matrix this is clear but in the data you presented (Table 6) this is not clear. It will be interesting to include the sodium content in the soil analysis
In has been improved in the text. There is no chance to include the sodium content into, because we do not stored soil.
Table 7: how do you explain the negative correlation between Mg and the yield?
This is specific case. The content of Mg in maize grain in 2016 was high, but yield two low. This means that the supply of Mg to maize grains was sufficiently high, but their number (see table 3), but their number was too low. Simply, the sink size was too low to dilute Mg concentration in maize grains.
382-401: how do you explain this? this equations only help to understand the data but how do you explain this results?
The source of plant available heavy metals can be both digestate (Table 2) and also their inherent soil resources (31). In the past, one of the most important source of both trace elements in arable soils were phosphorous fertilizers (32). The high Fe soil content was due to a high rate of nitrification, as shown in Fig. 4. It is well documented that oxidation of NH4+ ion leads to release of H+ ions, which in turn are responsible for soil acidification (33). In consequence, this process results, as accelerated by digeatate application, in release into soil solution cations of different metals, including both Fe and heavy metals (25).
406: check abbreviation
It has been corrected throughout the text.
408-409: Why? how do you relate this increase with your soil analysis? you have also Cd in the control (Fig. 5)
There was not recorded a significant change in the Cd content in soil after maize harvest. There was recorded a significant impact of year x digestate rate on the Cd content (Table 5). The course of the soil Cd content as shown in the added Figure was very similar to that observed for the content of N-NO3 and Fe. The content of soil Cd, as results form Table A1, was significantly correlated with these two soil characters (r = 0.68; Cd x N-NO3; r = 0.86 for Cd x Fe. As shown in Table A2 concentration of Cd in maize grain was significantly correlated with N and Ca concentration. In all years as shown in Table 6, a slightly higher N concentration was recorded on plots treated with digestate; the highest increase was recorded in 2015. The same trend was recorded for Cd. Our study corroborates findings by Kurtyka et al. (2007). Thse authors based on data from the hydroponic study showed that Cd and Ca ions were taken synergistically by maize seedling over a broad ranges of Ca concentration in the solution.
Kurtyka, R., Małkowski, E., Kita, A., Karcz, W. Effect of calcium and cadmium on growth and accumulation of cadmium, calcium, potassium and sodium in maize seedlings. Polish J. of Environ. Study 2008, 17(1), 51-56.
Round 2
Reviewer 1 Report
Here is the review for the original manuscript submission.
This topic has been reported in the literature for different types of cropping systems, so nothing really is new about this type of research. Unless of course the specific geographic area or soil type.
Many strong conclusions were made about the data, but little discussion about causative mechanisms has been made. A significant correlation observed between 2 parameters was discussed as being a cause/effect relationship but in fact more r2 values reported were relatively low, indicating only a weak relationship that may not be consistent from year to year. So there needs to be more of a mechanistic discussion on how one factor can be controlling another factor. Specifically,
Line 10. Please define digestate here in the abstract
Line 20. Please explain the chemical or biological mechanism for how lead can reduce heavy metals in grain? Is there a metabolic process occurring within the plant, or chemical reaction occurring in the soil that leads to Pb accumulation?
Line 94. States that sufficient N is present in soil. So why add more N as described in line 104?
Line 108. What is row spacing between crop rows? How wide was digestate applied with the knife application? In other words what proportion of surface area was treated with digestate for the band application compared to the broadcast application?
Line 184. What is the mechanism for the inconsistent trends in NO3 concentrations in soil across years?
Line 300. How is P easily enriched? Were other elements also enriched in the same manner, and if not, why?
Table 6. Are there any standard guidelines for minimum nutrient concentrations that indicate a plant deficiency? And if so, how did these levels relate to the minimum standards?
Line 386. What is the chemical or biological mechanism for Fe controlling Cd concentrations?
Line 390 and 391 appears to contradict earlier statement about Fe controlling Cd.
Line 433. Where did the Pb go? Change from available to unavailable form? All taken up by plants? Leaching or runoff?
Line 437. Please explain how drought can cause the changes to Pb concentrations.
Line 452. In soil or in plants?
Line 453. Please discuss how iron controls Cd.
Line 455. Please explain how NO3 controls Pb. I think you need more than a simple significant correlation to make this statement.
Author Response
Agronomy 683274R2
Reviewer #1
Questions/remarks are written in Calibril 10
Response are written in New Times Roman 12
This topic has been reported in the literature for different types of cropping systems, so nothing really is new about this type of research. Unless of course the specific geographic area or soil type.
We do not agree with the reviewer’s opinion. There are a lot of controversies concerning application of digestate to plant crops. This study clearly showed that there is necessary to fix the digestate rate in order to:
achieve the maximum grain yield; decrease a threat of heavy metals accumulation in grain; find an appropriate production means to decrease the potential threat of heavy metal accumulation in edible plant parts; The production means were clearly defined in the study: K, Na, Zn and Fe applied as foliar fertilizer to maize foliage just before silking.
Many strong conclusions were made about the data, but little discussion about causative mechanisms has been made. A significant correlation observed between 2 parameters was discussed as being a cause/effect relationship but in fact more r2 values reported were relatively low, indicating only a weak relationship that may not be consistent from year to year. So there needs to be more of a mechanistic discussion on how one factor can be controlling another factor. Specifically,
Line 10. Please define digestate here in the abstract
Digestate is byproduct of organic material digestion in biogas plant.
Line 20. Please explain the chemical or biological mechanism for how lead can reduce heavy metals in grain? Is there a metabolic process occurring within the plant, or chemical reaction occurring in the soil that leads to Pb accumulation?
This is a mistake. This sentence should sounds as follows:
Cadmium concentration in grain was controlled by the availability of soil Fe and Pb by the availability of N-NO3, zinc and copper.
Line 94. States that sufficient N is present in soil. So why add more N as described in line 104?
It has been assumed that application of digestate would accelerate the soil organic N mineralization. Therefore, the amount of N of 140 kg ha-1 in the soil/crop system should be sufficient to produce 10 t ha-1 grain. The study clearly supported this assumption.
Line 108. What is row spacing between crop rows? How wide was digestate applied with the knife application? In other words what proportion of surface area was treated with digestate for the band application compared to the broadcast application?
Row spacing between maize rows was 70 cm. The digestate application path was 15 cm wide. The surface area treated with digestate constituted 15% of the total surface area.
Line 184. What is the mechanism for the inconsistent trends in NO3 concentrations in soil across years?
Data presented in Figure 3 refers to the post-harvest N-NO3 soil status. The much lower values in 2015 were due to extended drought in July and August. It is well documented that a severe water shortage results in a drastic reduction in the activity of microorganisms (Schimel, 2018). At the same time, N concentration in maize grain was the same as in the optimal 2014. Consequently, the soil N-NO3 resources were extensively exploited by maize. In 2016, the initial resources of N-NO3 were lower by 12% and 16% as compared to 2014 and 2015, respectively. As a consequence grain yield in 2016 was by 7% and 19% lower as compared to 2014 and 2015, respectively. At the same time the maximum achievable yield in 2016 of 9.2 t ha -1 was lower by 20% (2.3 t ha-1), and 15% (1. 6 t ha-1) compared to 2014 and 2015, respectively. Assuming that the Nitrogen Harvest Index for maize is about 65% and N concentration in grain is of 15 kg t-1, the extra amount of N accumulated in maize at harvest was 53 kg ha-1 in 2014 and 42 kg ha-1 in 2015. These two figures indirectly show that in 2016 conditions for soil N mineralization were much worse than in both previous years.
Line 300. How is P easily enriched? Were other elements also enriched in the same manner, and if not, why?
Generally each nutrient incorporated into soil with digestate enriched its internal resources. The amount of incorporated P as compared to K was low. It also refers to heavy metals. In contrast to Mg and K (except 2014), the content of soil available in the top soil P was high.
Table 6. Are there any standard guidelines for minimum nutrient concentrations that indicate a plant deficiency? And if so, how did these levels relate to the minimum standards?
There are any standard values for minimum nutrient concentration in maize grain that indicate a plant deficiency. The value obtained in our study was considered as low, but only for Polish conditions. The Grain Nitrogen Concentration (GNC) in the US North Central region, as reported by Tenorio et al. (2019), ranged from 7.6 to 16.6 g kg-1. In 2019 a year with a deep drought in many areas of Europe, including Poland, GNC was frequently below 10 g kg-1 DW, which resulted in a deep yield depression.
Line 386. What is the chemical or biological mechanism for Fe controlling Cd concentrations?
One of the key processes leading to soil acidification is oxidation of ammonium ions:
2NH4+ + 3O2 --> 4H+ + 2NO3-
The net outcomes of this process is release of protons (H+).
Line 390 and 391 appears to contradict earlier statement about Fe controlling Cd.
The content of the soil available Fe was significantly correlated with N-NO3, N-NH4, P, K, Mg and with heavy metals, i.e. Cd (r = 0.86***) and Pb (r = 0.34**). The increasing concentration of protons in soil solution leads to dissolution of several compounds, for example iron (Lindsay, 1979):
1) phosphorus, taken by plants in the form of H2PO4-
2CaHPO4 + 2H+ --> Ca(H2PO4)2 + Ca2+
iron – solubility of Strengite:
FePO4 + 2H+ --> Fe3+ + H2PO4 log K = -6.85
Cations presented in soil body (Cation Exchange Complex) undergo exchange with H+ ions present in the soil solution in accordance to the simplified exchange model:
2Haq+ + Mgsoil --> Mgaq2+ + 2Hsoil+
The analysis of Table 7 shows that any increase in Fe concentration in maize grain resulted in a simultaneous decrease in Cd concentration. This was most visible comparing 2015 with those obtained in 2016.
Lindsay W. 1979. Chemical equilibria in soils. J. Wiley & Sons, New York, 450 p.
Line 433. Where did the Pb go? Change from available to unavailable form? All taken up by plants? Leaching or runoff?
No significant differences were found between Pb content in the digestate control plot and in plots fertilized with increased Pb rates. The simple balance between the Pb content in the soil and its concentration in grain indicates a net uptake by maize. This element cannot be leached because it is strongly fixed by organic matter or other compounds. As reported by Brennan et al. (1999) Pb in soil rich in phosphorus undergoes precipitation as Pb-phosphate.
Line 437. Please explain how drought can cause the changes to Pb concentrations.
The observed phenomenon can be explained in two completely different ways. Three elements, i.e. Ca, Mn, and Pb showed higher accumulation in the dry 2015 season as compared to other years. As reported by Rose et al. (2015) over 90% of Ca accumulates in wheat grain during the first 14 days of grain growth. Ca concentration in a plant cell increases significantly in response to water stress (White, 2003). At the same time, drought stress reduces the accumulation of starch in the developing grain (Yi et al., 2014). As a result of these two processes, the concentration of Ca in grain increases. In our study concentration of Ca and Pb were significantly correlated (r = 0.48***) and Thousand Grain Weight was significantly lower in 2015 as compared to 2014, indirectly corroborating the divagations presented above.
The observed phenomenon can be also explain by the fact that plants under drought increase the amount of released exudates to rhizosphere. Plant root exudates consist of numerous organic compounds, including phytosidorephores, which solubilize unavailable soil minerals, including Ca, Fe, and others (Dakora and Phillips, 2002). In addition digestate also consists of a wide range of natural ligands, containing minerals in plant available forms (Carbonell et al., 2011; Lavado et al., 2007).
Line 452. In soil or in plants?
In fact it refers to Fe concentration in grain.
Line 453. Please discuss how iron controls Cd.
The analysis of Table 7 shows that any increase in Fe concentration in maize grain resulted in a simultaneous decrease in Cd concentration. This was the most visible when comparing 2015 and 2016. He et al. (2017) showed that Cd uptake by a model plant, i.e. Arabidopsis thaliana L., was governed by two mechanisms:
1) an enhanced Fe --> Cd antagonism: an increased exogenous supply of Fe results in decreased Cd uptake by plant roots;
2) an inhibition IRT1 , a divalent cation transporter of numerous cations, including Fe and Cd à an increased Fe supply in the growth medium inhibits IRT1 expression, lowering Cd uptake by plant roots.
Maize as an mocotydenelous plant takes Fe as Fe3+ ions from soil solution (Marschner and Römhold, 1994). It is probable that similar mechanisms to those proposed by He et al. (2017) for Arabidopsis thaliana L. are also present in maize.
Line 455. Please explain how NO3 controls Pb. I think you need more than a simple significant correlation to make this statement.
The control of Pb concentration in maize grain through the content of N-NO3 can be explained by the enhanced content of the soil available Fe (Table A1; Figure 4). An mechanism as proposed by He et al. (2017) for Cd.
Reviewer 3 Report
Dear Authors,
The manuscript has been considerably improved, most of my suggestions were incorporated. I still recommend checking some errors in writing before publication. Specific comments:
line 12: add "in the year 2014..." and not only "in 2014"
line 19-20: this is still not clear, check grammar.
Keywords: the word "maize" should be incorporated in one of the keywords
Fig. 5 and 6: improve these figures, some letters are on the error bars and it is difficult to read
Author Response
Agronomy 683274R2
Reviewer #3
Questions/remarks are written in Calibril 10
Response are written in New Times Roman 12
Specific comments:
line 12: add "in the year 2014..." and not only "in 2014"
line 19-20: this is still not clear, check grammar.
It has been corrected.
Keywords: the word "maize" should be incorporated in one of the keywords
It has been introduced.
Fig. 5 and 6: improve these figures, some letters are on the error bars and it is difficult to read
Both Figures have been corrected in accordance to the reviewer’s comment.
Author Response File: 
Author Response.docx
