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Article
Peer-Review Record

Copper Toxicity and Prediction Models of Copper Content in Leafy Vegetables

Sustainability 2019, 11(22), 6215; https://doi.org/10.3390/su11226215
by Wei-Yang Chiou and Fu-Chiun Hsu *
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Sustainability 2019, 11(22), 6215; https://doi.org/10.3390/su11226215
Submission received: 15 October 2019 / Revised: 28 October 2019 / Accepted: 5 November 2019 / Published: 6 November 2019
(This article belongs to the Section Sustainable Agriculture)

Round 1

Reviewer 1 Report

The topic of your presentation is of high interest for stake holders such as farmers and bureaus ranking contamination of areas or quality of food and feed. You are correctly pointing out that you have tested, whether your method can be applied for the tested vegetables. Moreover you are stating that similar tests have to be done prior to using the method to rank other plant species. You should add that similar constraints apply with respect to plant cultivation methods: predominantly fertilizing with manure instead of nitrate is increasing copper availability to the roots and, thus is increasing uptake and toxicity, for instance.

I suggest to check the use of English language and to take care of correct use of terms. Please, clearly discriminate total amounts of a compound from the concentration of a compound. - For explanation, I have listed my comments on the first part of your manuscript:

line 10
I suggest to use the word „continuous“ instead of „sustainable“, because in current literature „sustainable“ is linked to positive aspects.

line 12
Please replace „agriculture“ by „agricultural“

line 16
… the soil copper content …

line 17
„soil copper “ - do you mean „copper content in the soil water phase“ ?

line 18
… „plant copper content“ …

line 24
… daily copper intake. …

line 31
… discharge, with further accumulates … - discharge, which further accumulates
Well, I suggest not to call industrial pollution „illegal“. There are several cases, where pollution can be traced back to years, when current regulations on pollution levels were not in place.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors,

Reviewer comments sustainability-629720

The manuscript entitled „Copper toxicity and prediction models of copper content in leafy vegetables“ represents an excellent study on determination vs prediction of copper content in four species of leafy vegetables, water spinach, amaranth, pakchoi, and garland chrysanrthemum, grown on five levels of copper-contaminated soil. The authors analysed several aspects of the experiments: the effect of copper concentration level in soil on copper content in plants as well as the fitness of several linear regression model on prediction of copper content in plants when applying correlation analyses with experimental data. In Figure 5, significant correlations between the linear regression formulae vs measured plant Cu conncetration are presented indicating significant correlations when comparing Cu concentrations determined by the regression models vs plant Cu concentration determined experimentally. Overall, the manuscript is prepared very precisely by the authors.

I have only one major comment on the manuscript:

In Table 3 as well as in the corresponding text, the authors compared two regression models for Cu concnetration in each leafy vegetable studied, i.e., aqua regia digestion vs HCl, NaNO3 or EDTA digestions resulting in the data that the second models fit better (R2 adj). In Figure 5, based on the information given in Figure 5 legend, I think that the authors compared expected plant Cu concentration determined by the second type of regression models as indicated by the equations provided in Figure 5 legend, with plant Cu concentration from aqua regia digestion?? I am not familiar with metal analyses in plant tissues thus I am not sure whether „aqua regia“ digestion and HCl, NaNO3 and EDTA extractions are different extraction ort he following steps in one extraction process. I think that the extraction process should be clearly described in Materials and methods. Similarly, clear information should be given regarding the regression models (two different regression models compared in Table 3 regarding the statistics) and in Figure 5 (if aqua regia digestion and HCl, NaNO3 or EDTA extractions represent two independent extraction protocols why the authors compare the regression model to one extraction protocol with experimental data obtained from the second extraction protocol??). I think that the authors should clearly define the extraction protocols used in their study both in Materials and methods and in results sections, namely in Figure 5 legend.

Otherwise, I have only a few formal comments on the manuscript:

Use SI units for volume, i.e., use „dm3“  instead of „l“, „cm3“ instead of „mL“ and „mm3“ instead of „μl.“

In Table 2, soil copper concnetration levels are presented. these are probably mean values from several replicate measurements. Thus, I recommend to add the information on the number of replicate measurements into table legend as well as data variability in table such as SD values.

Formal comments on the manuscript text:

Abstract, line 9: Modify a singular form of the word „area“ by a plural form „areas“ in the sentence „Copper, a toxic metal pollution found in soil and water of industrialized areas,…“

Table 2 legend, line 202: Add „4“ preceding the words „from February to March 2018“, i.e., „and 4, from February to march 2018.“

Results, line 296: Remove „a“ preceding the words „regression models“ in the sentnece „Similar approaches were used to build regression models for amaranth,…“

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors have studied the accumulation of copper by 4 leafy vegetables, and developed regression models to predict the uptake of Cu based on single soil extractions. Even if well-written, the paper fails to provide any valuable or novel insights regarding Cu phytotoxicity. Moreover, the so called prediction models have been poorly conceived and fall short to have any valid application. Please see specific comments below.

 

Since the main objective of the paper was clearly to assess Cu phytoxicity for the selected vegetables, in addition to the standard biometric (weight; height) parameters, several other measurements should have been carried out - e.g. levels of H2O2; lipid peroxidation; proteins; proline; ascorbic acid; starch; soluble sugars; antioxidant enzymes such as SOD, CAT, APX, POX; photosynthetic pigments; leaf area; to list but a few!!! Furthermore, the analysis to Cu content in plant tissue was rather poor. Concentrations were not split by root and shoot, nor indicate on a DW basis. Bioconcentration and translocation factors should have also been calculated. The data upon which the models have been developed, is based on single-extractions from spiked soil - which is not optimal, due to soil-metal equilibrium concerns. More importantly, spiked soil hardly represents a metal-polluted soil, for in a real scenario, soil presenting high levels of Cu will also feature elevated levels of other metals that will unavoidably influence Cu availability and/or uptake by the plants. Curiously, the papers cited by the authors for comparison with their results, did not use spiked soils. The choice of  NaNO3, HCl, and EDTA is also questionable, for the literature strongly suggests that these agents will not extract the phytoavailable fraction. Surprisingly, the authors did not use DTPA. The Rsquares of the obtained models are also to low to have any practical value. Lastly, this type of approach is very outdated. Due to its simplicity, this type of modelling approach has been tried since the 1990s, but without much success and impact.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

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