³¹P NMR Investigations on Roundup Degradation by AOP Procedures

Query

No.

Line/ Tab./Fig.

Before correction

After correction

1

L. 2

³¹P NMR investigations on Roundup utilization by AOP procedures

³¹P NMR investigations on Roundup degradation by AOP procedures

2

L. 10

the splitting of C-P was observed in the ratios   dependent on the applied   pH of the reaction mixture.

the splitting of C-P was observed in the ratios   dependent on the pH of the reaction mixture.

3

L. 21

EC   2.5.1.19) – involved in the bio-synthesis of AAA substrates …

EC 2.5.1.19) – involved in the bio-synthesis of aromatic amino acids (AAA) substrates …

4

L. 36/37

… to investigate these process, using ³¹P   NMR monitoring …

… to investigate these process by using ³¹P   NMR monitoring …

5

L. 51

^a/Applied names were in accordance with the JUPAC rules

^a/Applied names were in accordance with the IUPAC rules

6

L. 54

Diisopropylphosphite were purchased from ACROS OrganicTM.

Diisopropylphosphite was purchased from ACROS OrganicTM.

7

L. 69

was homogeneous at 1H NMR and ³¹P NMR

we supported these data with elemental analysis; ³¹P   NMR data are listed in Table 6

8

Fig. 1

Reactor used for Roundup utilization   by

Reactor used for Roundup degradation   by

9

L. 114/

115

“samples of PMG (12 mmol), contained in Roundup   Ultra 170 SL Herbicide (16 ml)”. Does this mean that 16 mL of Roundup   solution contained 12 mmol of PMG? Please rephrase it to make it more clear.

…, samples of PMG contained in Roundup Ultra 170 SL Herbicide (16 ml; 12   mmol of PMG),..

10

L. 206/

207

“The data presented suggest that PMG are inert   toward H2O2” this has been shown only for one pH value. Please correct   appropriately.

The reactions of PMG with H₂O₂   were run under UV irradiation, at ambient temperature for 48h….. The data presented suggest   that PMG are inert toward H₂O₂ without UV irradiation,   (pH=3.5)

11

L. 207/

208

“The analysis of ³¹P NMR spectra of AA^P-H₂O₂   reaction mixtures”. Did the authors mean PMG-H2O2 reaction mixtures? Please   correct accordingly.

The analysis of ³¹P NMR spectra of PMG-H₂O₂   reaction mixtures …

12

All graphs

Please put axis description and units near axis. For   example: Time, h; Residual PMG, % etc.

OK

13

Table 3:

Please make the table more clear. What is shown in   the column between columns 10 M H₂O₂ and FeSO₄?   What is the volume indicated in PMG column entry 1?

The table 3 have been corrected

(formatting error).

14

Figure 4 and the text   asso-ciated therewith:

The reason of choosing pH value 3.5 for this   experiment is not clear. Assumption that “protonation of the amino function   in PMG efficiently decrease an interaction of PMG and H2O2” is not supported   by data. Does PMG reacts with H2O2 or H2O2/Fe(II) at higher pH?

The solutions PMG-H2O2-H2O-Fe(II) with higher   concentration of Fe(II) exhibited pH 3.5. For unifications, other applied   solutions (pH ~ 4) were acidified to this value of pH, since ³¹P  shifts of P-acids strongly depend on the pH   [see Refs. 27 and 28].

15

.., why the oxidation reaction of PMG with hydrogen   peroxide was tested only at pH =3.5 and not at other Ph.

At higher pH precipitation of some type of colloids   [presumably PMG×Fe(II) + Fe(OH)₂] appeared, which, in spite of prior   centrifugations, caused widening of ³¹P signals

16

Figure 6:

It seems that y axis here represent relative signal   intensity in 31P NMR spectra, but not %PMG. Please correct.

Figure   6. The profile of   PMG reaction with H₂O₂ in systems PMG-H₂O-H₂O₂   (UV, temp. 25°C) at pH = 12; temp. 25°C {relative P-compound contribution (Gly^P: 13.6 ppm, PMG:   10.6 ppm; P_i: -0.071)   [%] vs exposition time [h]}

17

Figures

7, 8:

NMR spectra are too little. It is hard to read.   Please zoom them and use larger font.

The figures 7 and 8 have been enlarged.

Q.

Author’s remark

Text in original version

Text after correction

1

Nevertheless, several   references should be cited in the Introduction section to elaborate about the   two forms (glyphosate and Roundup), for example: Toxics 2018, 6, 2;   doi:10.3390/toxics6010002 and Int. J. Environ. Res. Public Health 2018, 15,   1117; doi:10.3390/ijerph15061117.

Also   the World Health Organization’s International Agency for Research on Cancer   concluded that glyphosate is “probably   carcinogenic to humans.” [13] and recently Alleeva et al. [14]   demonstrated that PMG, even at low doses, induce DNA damage responses.

Also the World Health   Organization’s International Agency for Research on Cancer concluded that   glyphosate is “probably carcinogenic to   humans.” [13] and Anifandis et al. [14,15] demonstrated that   glyphosate/PMG, induce DNA fragmentation.

2

The authors may also   elaborate in the Discussion section and not just to mention what they found   in bullets.

The Conclusion part   has been extended. In this new form it answers the Referee query.

3

The authors may state   about the aim of their study more clearer and to mention in the Discussion   section what exactly they found and what is the interpretation of these   findings

4

Last some figures are   very hard to follow and need more explanations.

All figures have been   corrected.

Sugg.#

Line

Suggestions

After   correction

1

L.   2. &

Fig.   1

…the term “utilization” can be replaced by   “degradation” or equivalent, ….

The term the term “utilization” has been replaced by   degradation (see   line 2 and Fig. 1)

2

20/21

Full terms should be given when abbreviations are   used the first time, for instance, line 20, EPSP, line 21 - AAA

…. – 5-enolpyruvylshikimate-3-phosphate (EPSP) synthetase (EC   2.5.1.19) – involved in the bio-synthesis of aromatic amino acids (AAA) substrates

3

Table   1

In the title of table 1,  Glyp is a term that   was not defined prior to its appearance at this point.

Explanation is given as the footnote of the Table: Gly^P –  see Scheme 1.

4

Lines:   111, 118, 148;

Fig.   3; Sch. 3, 4

The name used for the same compound should be also   consistent, for example, hydrogen   peroxide vs hydrogen dioxide.

We unified the names of H₂O₂   as: dihydrogen peroxide

5

It would be in a  more logical order if   representative ³¹P NMR spectra were presented first to show how   oxidation is observed and studied (Figure 8) before comparing reactions under   different conditions (Figures 4, 5, and 6)

Figures 7 and 8 are ³¹P NMR illustrations   - supplemental to Figures 5 and Fig. 6.

6

How did the authors determine the percent   degradation of PMG by ³¹P NMR quantitatively? It is not described   anywhere in the manuscript.

These are taken from the spectra:

Where S_(PMG), S_(R-P) and S(_Pi)   present

the areas of the ³¹P signal corresponding   to PMG; phosphonic acids, and inorganic phosphates, respectively.

7

In Figure 6, what do the three values/curves   indicate? The information can’t be found in the caption or the main text.

Figure 6. The profile of PMG   reaction with H₂O₂ in systems PMG-H₂O-H₂O₂  (UV, temp. 25°C) at pH = 12; temp.   25°C {relative   P-compounds contribution (Gly^P: 13.6 ppm, PMG:   10.6 ppm; P_i: -0.071) [%] vs exposition time [h]}

8

The resolution of NMR images in Figure 7 and Figure   8 needs to be improved. 

The spectra have been enlarged

9

The caption for figure 1 should be revised and   contains complete sentences, except the title.

The title of Fig. 1 has been corrected

Q

L

Suggestion

Before   correction

After correction

1

16

Please correct diapasons like   "2>pH>12" to "2<pH<12" throughout the paper

2 ≥ pH ≥ 12

2 ≤ pH ≤ 12

193

2 ≥ pH ≥12

2 ≤ pH ≤ 12

198

pH2 ≥ pH ≥12

2 ≤ pH ≤ 12

226

2≥pH≥10

2 ≤ pH ≤ 10

227

2≥pH≥10

2 ≤ pH ≤ 10

237

2≥pH≥8

2 ≤ pH ≤ 10

Q

L

Suggestion

Before correction

After correction

1

12

5÷10 excess of hydrogen dioxide…,

 I believe the authors meant    5~10 or 5-10

5÷10 excess of hydrogen dioxide

5-10 excess of hydrogen   dioxide

2

16-17

the splitting of C-P 16 was   observed in the ratios dependent on the applied pH.., ratios of what?

 It   should be described clearly.

In this mode of PMG oxidation, the splitting of C-P bond was   observed in the ratios dependent on the applied pH of the reaction mixture.

3

Sch. 2

has two figures between line 142 and 145 that   are identical but of different sizes/arrangements.

Only one image is needed.

We enlarged the size of   Scheme 2 to size of other figures.

We have chosen the scheme of   enlarged size.

4

Figure 7

and

Figure 8 

Figure 7 and Figure 8 show the ³¹P-NMR    spectra of the degradation mixtures of PMG-H2O-H2O2-UV after 6 hours at pH 10   and pH 12. The two sets of images areexactly the same. The degradation   percentages of PMG, therefore, would be the same at pH 10 and pH 12, which is   very different from what’s shown in Figure 5 (where more than 60%   degradationat pH 12 compared to less than 10% at pH 10).

I believe the images in Figure 7 are not   representative of the reaction at pH 10.

Figures 7 & 8 (obviously the same) have been   substituted by the set of representative figures, assigned now as Figure(s)   7.

5

232

The grammar in the text between line 232 to   line 235 should be checked and corrected. 

OK

6

233,234

Also in line 233 and 234, the word ”stability   “should have an adjective, it that “high stability” or “low stability”?

·           Stability   of PMG in reaction with H₂O₂ (48 h);

·           Full   stability of PMG in reaction with H₂O₂ (48 h);

234

·           Stability   of PMG in reaction with H₂O₂/Fe²⁺ (48 h).

·           Full   stability of PMG in reaction with H₂O₂/Fe²⁺   (48 h).