Absorption–Translocation of Veterinary Antibiotics in Rice Plants Introduced with Irrigation Water
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors1- What are the basis of selecting your parameters in this experiment. Please provide a strong evidence explaining the direct effect of those antibiotics in particular on chlorophyl.
2- I think Figure 1 is unnecessary since you already provided the GPS coordinates.
3- Please add stat analysis to the histograms in Figure 2, and all Tables.
4- I suggest using box and whisker blot rather than histograms
5- I don’t see significant variation between chlorophyll b and total Chlorophyll, so I suggest using only one and avoid redundancy.
6- in Figure 3, variation bars denote no statistical difference between the control and treatments. Please revisit the stats.
7- The results were not sufficiently discussed.
Comments for author File: 
Comments.pdf
manuscript language is clear. I suggest using past simple instead of past perfect or past perfect continuous.
Author Response
Review #1. Comments and Suggestions for Authors
- What are the basis of selecting your parameters in this experiment. Please provide a strong evidence explaining the direct effect of those antibiotics in particular on chlorophyl.
Answer: The purpose of this study was not to investigate the effect of VAs on chlorophyll. This study investigates the absorption and translocation of VAs unintentionally introduced into rice fields through irrigation water. The effects of VAs on chlorophyll are mentioned in the manuscript.
- I think Figure 1 is unnecessary since you already provided the GPS coordinates.
Answer: It was deleted according to the reviewer's recommendation.
- Please add stat analysis to the histograms in Figure 2, and all Tables.
Answer: If statistical data is inserted into Figure 2 and all tables, readers may have difficulty understanding the figures and tables. A detailed description of the statistics is provided in Results and Discussion. Therefore, statistical data was inserted in Figure 3.
- I suggest using box and whisker blot rather than histograms
Answer: The Figures were supplemented with a new drawing.
- I don’t see significant variation between chlorophyll b and total Chlorophyll, so I suggest using only one and avoid redundancy.
Answer: There were differences in the data for chlorophyll a, b, and total. Therefore, all Figure was redrawing
- in Figure 3, variation bars denote no statistical difference between the control and treatments. Please revisit the stats.
Answer: Statistical data was inserted according to the reviewer's recommendation.
7- The results were not sufficiently discussed.
Answer: Some content has been supplemented, and I think the conclusion is sufficiently explained.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe increasing use of antibiotics both in human treatment and in animal husbandry leads to negative consequences. One of them is the possibility of antibiotic residues getting into the soil and, therefore, also into the plants grown on it. Therefore, I believe that the research presented in the work is interesting and up-to-date. Thematically, the manuscript falls within the thematic scope of the journal ‘Applied Sciences’. The experiments presented in the manuscript were properly planned, it allows to explain the hypotheses put forward in the introduction. The research material is sufficient. The results were statistically analyzed and discussed and interpreted in detail. Tables and graphs are generally well prepared, understandable and legible.
I just have a few comments to consider
1. Please provide what DAT means under table 1. This is explained, but only on page 3.
2. It may be worth describing (providing the formula) in subsection 2.3 how BCF was calculated.
3. In subsection 2.4 authors write that ‘Data were analyzed by analyzing variance (ANOVA) and Tukey's test of significance at p < 0.05 to determine the differences between VA treatments’. Which data was analyzed in this way? The results of this statistical analysis are not marked in tables or figures. The significance of the differences is indicated in Figure 3, but a different test was used for rice yield – “Duncan's Multiple Range Test at 95% confidence intervals separated mean differences in rice yield…”
4. Please correct the numbering of the 'Results and Discussion' chapter and subchapters
5. Please correct the citation on page 5, second paragraph. “A decrease in chlorophyll content was reported to cause a reduced rate of photosynthesis, leading to a reduction in crop yield (Liu, Lv, Xu et al. 2018). Our results corroborate previous studies that have evaluated the effects of VAs on plants (Riaz et al. 2017; Tasho et al. 2020)”.
5. Please remove the legend in chart 3. It is already given on the X axis. Please explain under the chart what the letters a, b mean.
Author Response
Review #2. Comments and Suggestions for Authors
- Please provide what DAT means under table 1. This is explained, but only on page 3.
Answer: It was inserted according to the reviewer's recommendation (line75).
- It may be worth describing (providing the formula) in subsection 2.3 how BCF was calculated.
Answer: It was inserted according to the reviewer's recommendation (line257-259).
- In subsection 2.4 authors write that ‘Data were analyzed by analyzing variance (ANOVA) and Tukey's test of significance at p < 0.05 to determine the differences between VA treatments’. Which data was analyzed in this way? The results of this statistical analysis are not marked in tables or figures. The significance of the differences is indicated in Figure 3, but a different test was used for rice yield – “Duncan's Multiple Range Test at 95% confidence intervals separated mean differences in rice yield…”
Answer: It was inserted according to the reviewer's recommendation (Figure 2 line 276-277).
- Please correct the numbering of the 'Results and Discussion' chapter and subchapters
Answer: It was corrected according to the reviewer's recommendation (line195).
- Please correct the citation on page 5, second paragraph. “A decrease in chlorophyll content was reported to cause a reduced rate of photosynthesis, leading to a reduction in crop yield (Liu, Lv, Xu et al. 2018). Our results corroborate previous studies that have evaluated the effects of VAs on plants (Riaz et al. 2017; Tasho et al. 2020)”.
Answer: It was inserted according to the reviewer's recommendation (line153-154).
- Please remove the legend in chart 3. It is already given on the X axis. Please explain under the chart what the letters a, b mean.
Answer: It was inserted according to the reviewer's recommendation (line276-277).
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe subject of the research is quite unexpected. The veterinary antibiotics are also found in animal products and there are some rules and upper limits to be respected. Moreover, although tetracyclins are less used for treatment of human diseases, amoxicilin is still quite widely recommended as an antibacterial drug. The authors report that: “AMX residues were not detected in soil when the rice paddy field was exposed to the background level and ten-fold background level; however, at the fifty-fold background level, AMX residues were detected ”. Fifty-fold is quite a lot; from where this source of VA ?
In case of a bacterial infection responsive to AMX the recommended dosage for an adult human is about 2000 mg per day. There are reported values below 100 micrograms/kg of VA.
Typographical errors must be corrected. Tables 3, 4, 5 are too crowded.
Author Response
Review #3. Comments and Suggestions for Authors
- The subject of the research is quite unexpected. The veterinary antibiotics are also found in animal products and there are some rules and upper limits to be respected. Moreover, although tetracyclins are less used for treatment of human diseases, amoxicilin is still quite widely recommended as an antibacterial drug. The authors report that: “AMX residues were not detected in soil when the rice paddy field was exposed to the background level and ten-fold background level; however, at the fifty-fold background level, AMX residues were detected ”. Fifty-fold is quite a lot; from where this source of VA ?
Answer: It was inserted according to the reviewer's recommendation (line 24-25). This study did not discuss AMX used for human use. We only evaluated the impact of AMX on crops unintentionally exposed to the environment.
- Typographical errors must be corrected. Tables 3, 4, 5 are too crowded.
Answer: This is absolutely necessary for readers to understand this paper.
- Plants growing in these contaminated soils absorb the VA residues, disturbing both enzymatic and non-enzymatic antioxidant systems, affecting the PSII pigment system, and ulti-mately lowering the plant's productivity [3, 8, 9].
Answer: It was inserted according to the reviewer's recommendation (line 33-36).
- Among these, three antibiotics, amoxicillin (AMX, 0.1 mg L-1), chlortetracycline (CTC, 0.3 mg L-1), and oxytetracycline (OTC, 0.05 mg L-1), have been found to be persistent in soil, with frequent detection in aquatic systems and high absorption by plants.
Answer: It was inserted according to the reviewer's recommendation (line 33-36).
- I would delete this part from the table
Answer: This is absolutely necessary for readers to understand this paper.
- Table 3. Delete ND part
Answer: This is absolutely necessary for readers to understand this paper.
- What is the purpose of this section?
Answer: It's a meaningless question.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI wonder why those antibiotics are introduced to rice fields. You mentioned that they are unintentionally introduced, which does not raise a significant value for such an issue.
You assumed that this study wasn't meant to analyze chlorophyll, while the first figure in the manuscript represents the effect of chlorophyll. I think you mistakenly used this parameter because there are numerous factors that affect the chlorophyll content in plants. Similarly, the crop yield is affected by numerous other factors that have not been considered in the study.
The introduction doesn't provide enough explanation and support for the study.
I recommend converting tables into histograms.
All results were not sufficiently discussed, please revisit.
Author Response
Dear Editor-in-Chief
Thank you very much for your e-mail of 12 October, 2023, with regard to our manuscript (Applied Sciences, manuscript ID: ID: applsci-2655394).
Review #1. Comments and Suggestions for Authors
I wonder why those antibiotics are introduced to rice fields. You mentioned that they are unintentionally introduced, which does not raise a significant value for such an issue.
Answer: The sentence was inserted according to the reviewer's recommendation.
Arable lands serve as primary reservoirs of residual antibiotics. Antibiotics used in livestock enter the environment through direct application of veterinary antibiotics-laden manure to the arable lands [3]. In other words, VAs contained in manure applied to agricultural fields are unintentionally introducing into the agricultural ecosystem through the rainfall-runoff events.
- You assumed that this study wasn't meant to analyze chlorophyll, while the first figure in the manuscript represents the effect of chlorophyll. I think you mistakenly used this parameter because there are numerous factors that affect the chlorophyll content in plants. Similarly, the crop yield is affected by numerous other factors that have not been considered in the study.
Answer: We will make up for any shortcomings through additional research.
- The introduction doesn't provide enough explanation and support for the study.
Answer: This is the same as Comment 1.
- I recommend converting tables into histograms.
Answer: The table was converted to a figure according to the reviewer’s recommendation. BCF was maintained.
- All results were not sufficiently discussed, please revisit.
Answer: The sentence was inserted according to the reviewer's recommendation.
The continual introduction of VA's in soil, through repeated manure application, may eventually build up their concentration high enough to enter the terrestrial environment as a potent threat [3]. Kumar et al. [37] demonstrated to determine whether or not plants grown in manure-applied soil absorb VAs present in manure. The study shows that onions, cabbage, and corn take up chlortetracycline from soils mixed with VAs containing manure. Small concentrations of chlortetracycline (2–17ng g−1 fresh weight), accumulated in plant tissues in a dose-dependent manner.
I believe the manuscript has been improved satisfactorily and hope it will be accepted for publication in Applied Sciences.
Sincerely yours,
Professor, Jae-Young CHO
Author Response File: Author Response.pdf
Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsI do not think your answers support the research problem, and the research design isn't sufficient to prove your assumptions. I recommend you submit this research work to another journal that deals with toxicity, and environmental pollution.
Author Response
Dear Editor-in-Chief
Thank you very much for your e-mail of 31 October, 2023, with regard to our manuscript (Applied Sciences, manuscript ID: ID: applsci-2655394).
Academic Editor Notes: Comments and Suggestions for Authors
Q1. In view of the favourable decisions of two of the reviewers and after having read the article, I consider that the article can be published with a minor revision. I think that the problem of the accumulation of antibiotics through the use of manure can be a problem in certain circumstances, although in my opinion, the authors should provide bibliographic data in the introduction on concentrations of antibiotics in manure/water that would justify the highest doses / (50 x) that they have used, since it could indeed be an unrealistic dose, although this does not nullify the main conclusions of the article.
Answer: The sentence was inserted according to the recommendation. (line 25-29)
Wei et al. [8] investigated that antibiotics distribution in animal wastewaters, pond waters, and animal farm-effluents in Jiangsu Province, China. The most frequently detected antibiotics were sulfamethazine (75%), oxytetracycline (64%), tetracycline (60%), sulfadiazine (55%) and sulfamethoxazole (51%) which were detected with a maximum concentration of 211, 72.9, 10.3, 17.0 and 63.6 µg L-1, respectively.
Q2. The Methods section should include information on the characterization of the soil, its main physical-chemical characteristics and texture.
Answer: The sentence and Table was inserted according to the recommendation. (line 56-61)
The fundamental soil properties are listed in Table 1. The test plot soil used in this study is the Songsan series (coarse loamy, mesic family of Typic Dystrudepts) and soil texture is sandy loam.
Table 1. Physical and chemical properties of the test plot soil.
|
Item |
Chemical properties |
Particle size fraction (%) |
|
|
Soil organic matter (g 100g-1) |
2.59 |
Sand |
50.3 |
|
pH (1:5H2O) |
5.68 |
Silt |
40.2 |
|
Total-N (mg kg-1) |
921.4 |
Clay |
9.5 |
|
Total-P (mg kg-1) |
336.2 |
|
|
|
CEC (cmol+ kg-1) |
8.6 |
|
|
|
Electrical conductivity (µS cm-1) |
0.3 |
|
|
|
Ca++ (cmol+ kg-1) |
1.6 |
|
|
|
Mg++ (cmol+ kg-1) |
1.2 |
|
|
|
K+ (cmol+ kg-1) |
1.5 |
|
|
Q3. I also think the authors should delete the phrase. Since the structure and physicochemical properties of VAs and ex-changeable Ca2+ are compatible, antibiotic-Ca2+ complexes can be formed, resulting in reduced Ca2+ uptake in plants. I find it doubtful that the small amounts of antibiotics provided (compared to the amounts of nutrients that plants take in) could pose a problem for sufficient absorption of Ca, which is a very abundant element in most soils and is a macroelement in terms of to its abundance and use by crops.
Answer: The sentence was deleted according to the recommendation. (line 285-286)
More details:
- A new reference (No. 8) was inserted and an unnecessary reference (No. 39) was deleted.
- Changed content is marked in yellow in the manuscript text.
I believe the manuscript has been improved satisfactorily and hope it will be accepted for publication in Applied Sciences.
Sincerely yours,
Professor, Jae-Young CHO
