Effects of Combined Pollution of Tetracycline and Sulfamethazine on Tomato Growth and Antibiotic Absorption
Round 1
Reviewer 1 Report
The manuscript (agronomy-2195963) showed a combination two antibiotics and they effects for tissues in tomato plants. Introduction, material and methods, the results, discussion and conclusion for your paper is very interesting considering many questions of environment and growth and development in tomato plants. The authors have done a large amount of work, employing various analytical methods. The experimental work seems to be carried out carefully and the manuscript reports and discusses the findings in a logical way, with most of the results consistently backed up by the experimental data. The structure is adequate, the information is new and of great significance. However, suggesting to author major English language and style were needed. Please, check discussion section.
#There is a scope for improvement in the introduction section: a) additional emphasis on the significance of the study, b) scientific contribution of the paper in integration and importance to farming/productions/environment? Maybe a one paragraphs with potential economic by ecotypes selected in your manuscript; (Mill. ; not italic).
#Keyword in alphabetic order;
#Please. All standardization of nomenclature equipment/reagents/software was performed when necessary. Example: Fabricant, City, State, Country (three-letter). Check all manuscript. Its this has not been changed properly;
#Please, consider changes old references, for example before 2010 years old, and check reference following “Instructions for Authors”.
-Why 50th were selected from measure?
-Table 2. Check format; I don’t understand;
L76. Space
L183-185. Confuse, increased – increased?;
L199-202. Discussion;
L358-379. References. Please, check!
Best regards,
Author Response
Response to Reviewer 1 Comments
The manuscript (agronomy-2195963) showed a combination two antibiotics and they effects for tissues in tomato plants. Introduction, material and methods, the results, discussion and conclusion for your paper is very interesting considering many questions of environment and growth and development in tomato plants. The authors have done a large amount of work, employing various analytical methods. The experimental work seems to be carried out carefully and the manuscript reports and discusses the findings in a logical way, with most of the results consistently backed up by the experimental data. The structure is adequate, the information is new and of great significance. However, suggesting to author major English language and style were needed. Please, check discussion section.
Response :We sincerely thank the reviewer for the comments and greatly appreciate all the efforts the reviewer has made to help us improve the manuscript. The English language and style we obtained help from colleagues proficient in English to make appropriate modifications to the expression in the revised manuscript. Hope these efforts meet your expectations.
Point 1: There is a scope for improvement in the introduction section: a) additional emphasis on the significance of the study, b) scientific contribution of the paper in integration and importance to farming/productions/environment? Maybe a one paragraphs with potential economic by ecotypes selected in your manuscript; (Mill. ; not italic).
Response 1: We greatly appreciate this advice. We have added the significance of the study in the introduction section (Line 76~84). In the manuscript, we discussed how the experimental results can be applied in real production and analyzed the shortcomings of this study (Line 85~99).
Point 2: Keyword in alphabetic order.
Response 2: Thank you very much for your suggestion. We adjusted the keywords in alphabetic order in the revised manuscript according to your suggestions: Antibiotics; Absorption; Combined pollution; Physiological effect; Tomato plants.
Point 3: Please. All standardization of nomenclature equipment/reagents/software was performed when necessary. Example: Fabricant, City, State, Country (three-letter). Check all manuscript. Its this has not been changed properly.
Response 3: Thanks for pointing it out. The information has been supplemented.
Point 4: Please, consider changes old references, for example before 2010 years old, and check reference following “Instructions for Authors”.
-Why 50th were selected from measure?;
-Table 2. Check format; I don’t understand.;
-L76. Space;
-L183-185. Confuse, increased – increased?;
-L199-202. Discussion;
-L358-379. References. Please, check!
Response 4: Thank you very much for your questions. We are very sorry about those mistakes, and corrected in the revised manuscript. We checked all references carefully, and ensure that they are complete, accurate and following “Instructions for Authors”. We have replaced some of the references before 2010, cited the references of recent years.
-We selected 50th as the measurement time because about 20th for tomato plants belongs to the seedling stage, and about 100th belong to the harvest stage. During this period, a growth period is needed as the measurement index to observe the effect of soil antibiotic residues on tomato plants, whether it is continuously decreasing from the seedling stage (20th) to the harvest stage (100th), or there is a law of increasing and then decreasing during the growth period. The experimental results showed that the absorption of residual antibiotics in soil by tomato plants showed a continuous upward trend from the seedling stage (20th) to growth period(50th), and then gradually decreased until the harvest stage(100th).
-We are so sorry for the format of the uploaded manuscript version is changed from dot to docx, resulting in the corruption of some images(Figure 2-5.) and the confusion of table data (Table 2.), We have made appropriate modification in the manuscript.
-L76. Space has been modified (changed to Line 100 )
-L183-185. Sorry for the unclarity. We have revisited this document and corrected inappropriate statements in the manuscript (Line 227 ~229). The levels of antibiotics in plant tissues elevated with the increases of additive antibiotics in the soil, indicating that tomato plants absorbed more antibiotics with the increase of antibiotic pollution level in the soil.
-L199-202. Thank you for pointing this out. We have modified inappropriate statements in the manuscript (Line 242~244) and summarize in the discussion section(Line 464~466) . There was no significant difference in the contents of antibiotics in roots and shoots between two kinds of antibiotic combined antibiotic contamination and single antibiotic contamination treatments.
-L358-379. References. Thank you very much for your comments. We are very sorry about this mistake, and the references were added in the discussion section of the manuscript.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This work studied the negative effect of the tetracycline and sulfamethazine which is well reported before. The work has various major points should be revised and clarified
1- what is the significance of the work as application in agriculture, the negative effect of the chemical antibiotic is well reported.
2- the authors treated the plants with the antibiotics without the presence of any infection. the antibiotics are used under certain infection and this is not achievec here
3- the results of the present study are not clear and are represented in messy form speially the figures. the figures should be reformatted and I suggested represent the results as columns not as line figures
4- the conclusion should be rewritten to summarize the significance of the work and limitations of this study which can be solved in future prospectives
Author Response
Response to Reviewer 2 Comments
Point 1: What is the significance of the work as application in agriculture, the negative effect of the chemical antibiotic is well reported.
Response 1: We sincerely thank the reviewer for the comments and greatly appreciate all the efforts the reviewer has made to help us improve the manuscript. We have added the significance and our aim of the study in the introduction section (Line 85~99).
Point 2: The authors treated the plants with the antibiotics without the presence of any infection. the antibiotics are used under certain infection and this is not achieve here.
Response 2: Thank you very much for your comments.The main objective of this study was to revealed the characteristics of the effects of soil antibiotic pollution on representative vegetable plants, analyzed the accumulation pattern of antibiotics in various organs of vegetables and the influencing factors, and revealed the existence of compound pollution effects of antibiotics on vegetable crops. In soil, the main source of these two antibiotics is livestock manure, which cannot be absorbed and metabolized by animal matrices. Therefore, our experiments simulated the effect of antibiotics introduced by livestock manure on tomato growth and the uptake of antibiotics from soil by tomato.
Point 3: The results of the present study are not clear and are represented in messy form specially the figures. the figures should be reformatted and I suggested represent the results as columns not as line figures.
Response 3: We greatly appreciate this advice. We are very sorry about those mistakes, and corrected in the revised manuscript. We are so sorry for the format of the uploaded manuscript version is changed from dot. to docx., resulting in the corruption of some images(Figure 2-5.) and the confusion of table data(Table 2.), We have made appropriate modification in the manuscript.
In the process of drawing, we drew columns figures and line figures for comparison, and finally, after discussion and deliberation, we decided that a line figures would be more reflective of the trend and columns figures would be more intuitive, so we finally decided to do a line figures. However, some trends are not obvious we use the form of columns figures(Figure 6.).
Point 4: The conclusion should be rewritten to summarize the significance of the work and limitations of this study which can be solved in future prospectives.
Response 4: Thanks for pointing it out. We revised the conclusion, added the significance and limitations of this study, and looked forward to some problems in the current study (Line 463~480). From the results of this study, the accumulation of antibiotics in vegetables should not be underestimated. Eating fresh vegetables grown in soil rich in antibiotics may cause potential risks to human health. At present, the toxicological research of antibiotics and other drugs mainly involves the direct effects on animals, plants and microorganisms; The ecotoxicological study of antibiotics on plants is mainly focused on the toxicological study of aquatic plants and terrestrial plants under the conditions of hydroponic culture under the experimental simulation conditions. The toxicological study of soil as substrate and antibiotics on major types of vegetables in China is very few. The mechanism of absorption and enrichment of antibiotics by plants and the influencing factors need further study. Since the activity of antibiotics can change greatly after they enter the soil environment, it is necessary to study the dynamic absorption characteristics of residual antibiotics in soil and their relationship with antibiotic residues in soil by vegetables and other crops.
Author Response File: Author Response.docx
Reviewer 3 Report
This manuscript studies the effects of single and combined pollution of tetracycline and sulfamethazine on tomato plants.
Please address the following concerns:
Please insert the aim of the study at the end of the introduction section.
Lines 74-88 - Please include only the collection and soil sample preparation. The other information provided should be redistributed in the manuscript (i.e., experimental design or a reagents section).
Lines 124-154 – Please describe all the analysis you performed in detail (i.e., please include a detailed method for the analysis of C, N, P, K, Ca). Other researchers should be able to understand properly all the methods you described and reproduce them.
Figures – Please use the caption of the figures to exactly explain the used symbols and please make the figures self-explanatory (there is a lot of overlapping regarding letters).
Please expand the Discussion section and provide comparisons to other studies by using their results.
Author Response
Response to Reviewer 3 Comments
Point 1: Please insert the aim of the study at the end of the introduction section.
Response 1: We sincerely thank the reviewer for the comments and greatly appreciate all the efforts the reviewer has made to help us improve the manuscript. We have added the aim of the study in the introduction section (Line 85~99). In China, aside from food crops, vegetables are the most important crops in terms of economic standing and planting area. Due to the high demand for vegetables in urbanized regions, vegetable farms are mostly located near populated areas. However, the production and quality of vegetables are seriously threatened by the widespread use of chemical fertilizers and animal manure as organic fertilizers. As a major fruit and vegetable crop, the production of tomato (Lycopersicon esculentum Mill.) has reached 18,000 million tons in 2020. In this study, to investigate the effects of tetracycline and sulfamethazine on plants, tomato plants were planted in a greenhouse with different concentrations of tetracycline and sulfamethazine. Furthermore, the adverse effects of combined contamination of the two antibiotics on plants were also studied in different growth stages. We thoroughly determined the accumulation patterns and influencing factors of individual and combined antibiotic contamination in tomato organs. The findings are essential for assessing the impacts of soil antibiotic contamination on the safety, yield and quality of agricultural products, and will also add to our knowledge of antibiotic migration into the food chain.
Point 2: Lines 74-88 - Please include only the collection and soil sample preparation. The other information provided should be redistributed in the manuscript (i.e., experimental design or a reagents section).
Response 2: We greatly appreciate this advice. Through our discussion and the study of other papers, we thought that this is the basic physicochemical properties of the initial soil samples, and therefore it would be more reasonable to place it directly after the collection and preparation of the soil samples.
Point 3: Lines 124-154 - Please describe all the analysis you performed in detail (i.e., please include a detailed method for the analysis of C, N, P, K, Ca). Other researchers should be able to understand properly all the methods you described and reproduce them.
Response 3: Thanks for pointing it out. We described all the analysis we were performed in detail in our manuscript (Line 149~172).
Point 4: Figures - Please use the caption of the figures to exactly explain the used symbols and please make the figures self-explanatory (there is a lot of overlapping regarding letters).
Response 4: Thank you very much for your questions. We are very sorry about those mistakes, appropriate modifications were made in the figures and added the caption of the figures to exactly explain the used symbols.
Point 5: Please expand the Discussion section and provide comparisons to other studies by using their results.
Response 5: Thank you very much for your comments. We revised the discussion section, added the strong support for our conclusions through comparison with other studies results(Line 392~420).
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors can mprove the manuscript and thus it can be published
Author Response
Dear Editor and Reviewers:
Thank you very much for your comments on our manuscript entitled “Effects of combined pollution of tetracycline and sulfamethazine on tomato growth and antibiotic absorption”. The comments listed by reviewers are all very valuable and helpful for revising and improving our paper, as well as the important guiding significance to our research. We have carefully studied the comments of the three reviewers and have completely revised the manuscript in the hope that it will be approved (marked in red).
Author Response File: Author Response.docx
Reviewer 3 Report
Most of the suggestions I made to the manuscript were addressed properly.
Please take into account the following note:
Chemical analyses section - I consider that even though the authors included the devices they used to assess several compounds and properties of the analyzed samples, they did not include the actual methods. I consider that the used methods are very important for the proper understanding of the manuscript.
Author Response
Dear Editor and Reviewers:
Thank you very much for your comments on our manuscript entitled “Effects of combined pollution of tetracycline and sulfamethazine on tomato growth and antibiotic absorption”. The comments listed by reviewers are all very valuable and helpful for revising and improving our paper, as well as the important guiding significance to our research. We have carefully studied the comments of the three reviewers and have completely revised the manuscript in the hope that it will be approved (marked in red). The responses to the reviewer’s comments are as following:
Point 1: Chemical analyses section - I consider that even though the authors included the devices they used to assess several compounds and properties of the analyzed samples, they did not include the actual methods. I consider that the used methods are very important for the proper understanding of the manuscript.
Response 1: We sincerely thank the reviewer for the comments and greatly appreciate all the efforts the reviewer has made to help us improve the manuscript. We have added the actual methods in the chemical analyses section (Line 165~199).
Soil pH was determined with a pH meter in the supernatant at the water: soil ratio of 2.5:1 (FE20-FiveEasy™ pH, Mettler Toledo, German). Sand, silt and clay concentrations in the soil samples were evaluated by the micropipette method: Add dispersant to disperse the particles, then put the suspension in the settling cylinder and measure it by specific gravity meter.[36]. SOC was determined by Element Analyzer after soil grounded through 0.149 mm sieve and wrapped in tin foil (Elementar Vario EL Cube, German)[37]. Soil available potassium (AK) was extracted with 1 mol/L ammonium acetate and determined with atomic absorption spectrometry (AAS, Analytik Jena novAA 300, German)[38]. Soil available phosphorus (AP) was determined by 0.5mol/L NaHCO3 extraction ascorbic acid/molybdate colorimetry[38].
After the fresh plant samples are ground with a small food grinder, 5g of each sample is weighed and extracted with 20ml EDTA-Mcilvine (0.05mol/L EDTA + 0.06mol/L Na2HPO4 + 0.08mol/L citric acid, pH4) for 5 minutes[39]. After extraction, the supernatant was separated under a centrifugal force of 2000 g for antibiotic determination. The sample was purified by chromatography column and determined by a UV detector (wate UV 2487370 nm). The chromatographic column is SHIM-PACK.VP-ODS (5 μm, 150 mm×4.6 mm ID), the mobile phase is 0.01 mol/L oxalic acid +acetonitrile + methanol (76+16+8), and the flow rate is 1ml/min. The detection limits of tetracycline and sulfamethazine were 2.5 and 5 μg/kg, and the results are expressed based on fresh weight. Acetonitrile and methanol used in the test were HPLC grade, and citric acid, Na2HPO4 and EDTA were analytical reagent grade[39]. The total carbon(C) and total nitrogen(N) of plant leaves were measured with CHN elemental analyzer after soil grounded and wrapped in tin foil (Flash EA 1112, Thermo Finnigan LLC., Californian, USA). After digesting 0.5g dry leaf sample with H2O-HNO3 solution, the phosphorus (P) was determined by 0.5mol/L NaHCO3 extraction ascorbic acid/molybdate colorimetry, the concentrations of calcium(Ca) and potassium(K) were determined by atomic absorption spectrometry leaching with 1 mol/L ammonium acetate (AAS, Analytik Jena novAA 300, German)[38]. At the same time, the contents of nitrate of plant fruits were measured with a UV spectrophotometer (UV-5800, Metash Intrusments Co.,Ltd., Shanghai, China), Vitamin C was measured with high -performance liquid chromatograph leaching with 20g/L oxalic acid (HPLC, Agilent-1260, Agilent Technologies Inc., Californian, USA)[40]and reducing sugar was determined by Potassium permanganate titration extraction with ethanol, determination by titration with Fehling reagent (National standard method GB5009.7-85)[41], and the results were expressed based on dry mass.
Author Response File: Author Response.docx
