Characteristics of Cadmium and Lead Accumulation and Transfer by Chenopodium Quinoa Will
Round 1
Reviewer 1 Report
This is a descriptive study on cadmium and lead accumulation and transfer by quinoa. This study is worth being publish after some revision.
1- Do not recall words from title in the keywords.
2- Give a definition of PTE, check Pourret and Hursthouse 2019
Pourret, O., Hursthouse, A., 2019. It’s Time to Replace the Term “Heavy Metals” with “Potentially Toxic Elements” When Reporting Environmental Research. International Journal of Environmental Research and Public Health, 16(22): 4446.
3- Do not use ppm units, replace by SI units (mg/kg).
4- A table with the condition would be suitable.
5- In the discussion recall the condition details, not only the number of the condition. It is difficult to follow interpretation.
6- Discussion is weak, authors should compare their results with other metals and their accumalition in other wheats or plants. Check the special issue on metal in agriculuture https://www.mdpi.com/journal/sustainability/special_issues/Environmental_Impacts
7- Authors should expand their discussion by looking at speciation/fractionation of Cd and Pb.
8- There are many typos in the reference list; carefully check it.
Author Response
Point 1
Do not recall words from title in the keywords.
Response 1
We changed Keywords to potentially toxic elements; water-to-plant transfer; bioaccumulation factor; translocation factor; metal excluders. Thank you for this comment.
Point 2
Give a definition of PTE, check Pourret and Hursthouse 2019
Pourret, O., Hursthouse, A., 2019. It’s Time to Replace the Term “Heavy Metals” with “Potentially Toxic Elements” When Reporting Environmental Research. International Journal of Environmental Research and Public Health, 16(22): 4446.
Response 2
According to your advised reference, we clarified this part in Introduction (line 27-32):
In numerous publications, the term "heavy metals" is a group term for metals that are contaminants with the toxicity defined as well know relationship between dose and effect. Therefor, the term “Potentially Toxic Element(s)” (PTEs) can implement uniform and comparative use.
Point 3
Do not use ppm units, replace by SI units (mg/kg).
Response 3
This has been corrected. Units ppm were replaced by mg/kg (line 87-90; 155; 159-161; 166-168; 212-214; 217; 223-224;229)
Point 4
A table with the condition would be suitable.
Response 4
Than you for this valuable comment. We added a table of conditions (experiment design, applied treatments) line 96.
Point 5
In the discussion recall the condition details, not only the number of the condition. It is difficult to follow interpretation.
Response 5
In line with your comment, we added condition details throughout the discussion (line 211-212; 223; 253).
Point 6
Discussion is weak, authors should compare their results with other metals and their accumalition in other wheats or plants. Check the special issue on metal in agriculuture https://www.mdpi.com/journal/sustainability/special_issues/Environmental_Impacts
Response 6
Thank you for advising us to go through this special issue. We added a reference, Zhang, A.; Cortes, V.; Phelps, B.; Ryswyk, H.V.; Srebotnjak, T. Experimental Analysis of Soil and Mandarin Orange Plants Treated with Heavy Metals Found in Oilfield-Produced Wastewater. Sustainability 2018, 10(5), 1493. The additional discussion (line 243-248) gave an overview with mandarin orange tree.
Point 7
Authors should expand their discussion by looking at speciation/fractionation of Cd and Pb.
Response 7
Thank you very much for your comment. Yes, we agree that such explanation is necessary for readers. In line with your comment, we have explained that our study covered free ions bearing in mind that bioavailability is decreasing when soil pH and organic content is increasing (lines 207-209). Also, in the Conclusion we specified that certain limitation may occur since speciation/fractionation of Cd and Pb was not fully determined (lines 283-284).
Point 8
There are many typos in the reference list; carefully check it.
Response 8
We have checked reference list and corrected (line 301 – 408).
Reviewer 2 Report
Dear editor,
I have now completed my review of the manuscript “Characteristics of cadmium and lead accumulation and transfer by Chenopodium quinoa Will”.
This study is quite interesting and within the scope of the journal.
Thus, it could have merit for publication. However, as it stands, it seems that it is preliminary and inconclusive.
Some major concerns regarding the experimental design:
A number of Heavy Metals (HM) have been identified as a hazard, but only two (Cd and Pb) have been analyzed. Is there a specific reason why only two pollutants have been inquired? Many abundant and toxic HM exist across lands and water reservoirs worldwide (i.e. Arsenic, Mercury, Nickel etc). Since the authors aim to pioneer the characterization of quinoa as a potentially toxic element accumulator, more pollutants must be taken into account.
Furthermore, only one soil type (commercial) was used and critical parameters (physical and chemical properties) like soil texture (Sandy:clay:loam), ECe (dS/m), Organic Matter (%), CaCO3 (%), pH etc were not presented. These constraints are essential and could affect the mobility of heavy metals and intake by plants. Using only one soil type cannot be used for extrapolating quinoa as an accumulator (or not). Different attributes could have a different pattern of HM build-up, hence must be determined thoroughly. Moreover, crucial parameters like the weight of the soil used (pots) and the volume of irrigation was not presented.
The number of biological replicates is low. The authors report that “A total of 60 plants were distributed in 12 treatments with 5 simultaneous repetitions”. Does this mean that in total only five plants were used for one treatment? Please clarify. 12 treatments and 5 repetitions would require hundreds of discrete pots in order to have robust statistics (you cannot test if data are normally distributed). I can understand that space/materials can be a limiting factor and an obstacle, but this should not impede the statistical significance, especially when presenting novel data. Perhaps you should reduce the number of different treatments.
The statistics used are not appropriate. One-way ANOVA is suitable for one variable only and clearly this is not the case. Moreover, one-way AMOVA does not allow the estimation of interaction effects across the levels of the two pollutants studied (Cd * Pb).
Based on the above comments i recommend a major revision.
Author Response
Comment 1
A number of Heavy Metals (HM) have been identified as a hazard, but only two (Cd and Pb) have been analyzed. Is there a specific reason why only two pollutants have been inquired? Many abundant and toxic HM exist across lands and water reservoirs worldwide (i.e. Arsenic, Mercury, Nickel etc). Since the authors aim to pioneer the characterization of quinoa as a potentially toxic element accumulator, more pollutants must be taken into account.
Response 1
Thank you for this comment. We fully agree that a number of Hma are hazard. However, we focused on Cd and Pb because these elements are one of the dominant metal inputs to agricultural soils and they have high ecotoxic potency to soil organisms (Sydow et al., 2017).
Sydow, M.; Chrzanowski, Ł.; Cedergreen, N.; Owsianiak, M. Limitations of experiments performed in artificially made OECD standard soils for predicting cadmium, lead and zinc toxicity towards organisms living in natural soils. J. Environ. Manag. 2017, 198, 32–40.
Also, plants have high potency to accumulate these elements.
Within this study, we aim to characterise quinoa as cadmium and lead accumulator.
In line with your comment, such a sentence is added in Conclusion regarding future research across different HMs (line 285-287). Our intention was to analyze for these two, and if promising results were reached, to expand the research on other HMs.
Comment 2
Furthermore, only one soil type (commercial) was used and critical parameters (physical and chemical properties) like soil texture (Sandy:clay:loam), ECe (dS/m), Organic Matter (%), CaCO3(%), pH etc were not presented. These constraints are essential and could affect the mobility of heavy metals and intake by plants. Using only one soil type cannot be used for extrapolating quinoa as an accumulator (or not). Different attributes could have a different pattern of HM build-up, hence must be determined thoroughly. Moreover, crucial parameters like the weight of the soil used (pots) and the volume of irrigation was not presented.
Response 2
Thank you for this suggestion. We added more information about the commercial soil type that was used for the experiment. (line 78-79)
Our experiment lasted for 90 days, and for 50 days we watered plant with contaminated water. As we explained, during the entire duration of the experiment, soil moisture was maintained at level of 60% of the water retention capacity. The volume of irrigation was determined daily based on soil moisture, which was determined using humidity meter.
Comment 3
The number of biological replicates is low. The authors report that “A total of 60 plants were distributed in 12 treatments with 5 simultaneous repetitions”. Does this mean that in total only five plants were used for one treatment? Please clarify. 12 treatments and 5 repetitions would require hundreds of discrete pots in order to have robust statistics (you cannot test if data are normally distributed). I can understand that space/materials can be a limiting factor and an obstacle, but this should not impede the statistical significance, especially when presenting novel data. Perhaps you should reduce the number of different treatments.
Response 3
Thank you very much for your comment. Yes, we agree that out explanation was not clear enough. In lines 80-84 we have clarified that durring our experiment we simulated pollution in the form of different metal concentration via irrigation water and a total of 60 plants were distributed in 12 treatments with 5 simultaneous repetitions.
The experiment had 12 treatments with 5 simultaneous repetitions, in total 60 plants.
Comment 4
The statistics used are not appropriate. One-way ANOVA is suitable for one variable only and clearly this is not the case. Moreover, one-way AMOVA does not allow the estimation of interaction effects across the levels of the two pollutants studied (Cd * Pb).
Response 4
One-way ANOVA was used in order to analyze statistical differences for Cd and Pb separately. As proposed by reviewer #3 to show statistical differences between Cd and Pb (Tables 2 and 3), line 184-187, an independent sample t-test was performed to analyze Bioaccumulation factor between Cd and Pb. In such a way we analyzed statistical difference between the two toxic metals.
Reviewer 3 Report
I revised the manuscript "Characteristics of cadmium and lead accumulation and transfer by Chenopodium quinoa Will.". The ms might be publisced after extensive improvement of the Discussuon section (see specific comments in PDF).
Comments for author File: 
Comments.pdf
Author Response
Thank you for advising us to add more references to our discussion (line 207-209; 218-219, 243-248 ), we added 5 new references.
As you proposed we performed Independent sample t-test to analyze Bioaccumulation factor between Cd and Pb. In such a way we analyzed statistical difference between the two toxic metals. (table 2 and 3, line 184-187).
Round 2
Reviewer 1 Report
My comments were considered.
Author Response
Thank you, we appreciate your time and comments that enabled us to improve our manuscript. The manuscript has certainly benefited from these insightful revision suggestions.
Reviewer 2 Report
The samples analyzed are not enough and the statistical analyses conducted cannot adequately support the conclusions
Author Response
Comment
The samples analyzed are not enough and the statistical analyses conducted cannot adequately support the conclusions.
Response
Thank you very much for your comment. We conducted a two-way ANOVA and Tukey's post hoc test to analyse the interaction between treatments and PTEs. It consisted of examining the transfer of Cd and Pb from water to plants during the formation and maturation of seed. We added a new section that describes this interaction "Interaction between treatments and PTEs" (lines 168-178).
Reviewer 3 Report
The ms has been improved. Minor revision is required before publication (see PDF)
Comments for author File: Comments.pdf
Author Response
Comment
The ms has been improved. Minor revision is required before publication (see PDF)
Response
Thank you very much for your suggestion. As you proposed we corrected figure 3 (b), line 200. Also, we corrected out typing error, line 185.
Round 3
Reviewer 2 Report
the manuscript is improved and could be accepted
Author Response
Thank you, we appreciate your time and comments that enabled us to improve our manuscript. The manuscript has certainly benefited from these insightful revision suggestions.
