Review Reports - Seedling Establishment Test for the Comprehensive Evaluation of Compost Phytotoxicity

Round 1

Reviewer 1 Report

General comments:

The manuscript entitled “Seedling establishment test with compost water extract for the comprehensive evaluation of compost phytotoxicity” investigated the effects of compost water extract on the phytotoxicity of compost based on the dry weight and wet weight of samples and explored the feasibility of seedling establishment tests in compost phytotoxicity evaluation—without (CM treatment) and with the addition of a microbial agent (ACM treatment)—based on the addition of corn straw and spent mushroom substrate in cow manure composting. The manuscript is interesting and holds scientific potential but a few points need to be addressed before final publication to improve the overall quality of the manuscript.

English language and style are acceptable/minor spell check is required, and a few typographical errors need to be corrected.

Major comments for authors:

Introduction: It should be more informative with suitable references.

L96: Correct spelling of moisture

L96-97: In the table, mention the complete form of TOC, TN, C/N at the end.

L112: Approximately,

L114: (NH₄⁺-N)

L161: Results and Discussion

L233: Rewrite the heading “Basic parameters of composts”

Add more references to support the manuscript's findings in the Discussion part.

L384-385: In the table, mention the complete form of parameters at the end.

Conclusion: It should be more precise and highlight only the present study's significant findings.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments

Point 1: The manuscript entitled “Seedling establishment test with compost water extract for the comprehensive evaluation of compost phytotoxicity” investigated the effects of compost water extract on the phytotoxicity of compost based on the dry weight and wet weight of samples and explored the feasibility of seedling establishment tests in compost phytotoxicity evaluation—without (CM treatment) and with the addition of a microbial agent (ACM treatment)—based on the addition of corn straw and spent mushroom substrate in cow manure composting. The manuscript is interesting and holds scientific potential but a few points need to be addressed before final publication to improve the overall quality of the manuscript.

English language and style are acceptable/minor spell check is required, and a few typographical errors need to be corrected.

Response 1: Thanks for the comments. We checked the whole manuscript and revised the issues.

Point 2: Introduction: It should be more informative with suitable references.

Response 2: Thanks for the comments. In this part, we proposed that it is necessary to evaluate the compost phytotoxicity, but the previous study on evaluation methods was insufficient. Therefore, we compared different extraction methods and tried to achieve a comprehensive evaluation of phytotoxicity by seedling establishment test in this study. In addition, due to the lack of relevant research, we cited the related references in the field of toxicology. And we checked the cited references and replaced them with the more suitable ones. The revised contents were marked in blue color.

Point 3: L96: Correct spelling of moisture

Response 3: It was careless of us. We revised to “moisture”.

Point 4: L96-97: In the table, mention the complete form of TOC, TN, C/N at the end.

Response 4: Thanks for the suggestion. We added the “Note: Moisture content was calculated based on wet basis; TOC: Total Organic Carbon; TN: Total Nitrogen. TOC and TN were acquired based on dry basis”.

Point 5: L112: Approximately,

Response 5: Thanks for the suggestion. We revised to “Approximately, 300 g of fresh sample was collected and divided into two parts at each sampling time”.

Point 6: L114: (NH₄⁺-N)

Response 6: Thanks for the suggestion. We revised to “(NH₄⁺-N)”.

Point 7: L161: Results and Discussion

Response 7: Thanks for the suggestion. We revised to “Results and Discussion”.

Point 8: L233: Rewrite the heading “Basic parameters of composts”

Response 8: Thanks for the suggestion. We revised to “Physicochemical indicators of composts”.

Point 9: Add more references to support the manuscript's findings in the Discussion part.

Response 9: Thanks for the suggestion. We added references [36] [38] [39] [41] [42-43] to the discussion part.

[36] Ma, X.; Geisler-Lee, J.; Deng, Y.; Kolmakov, A. Interactions between engineered nanoparticles (ENPs) and plants: phytotoxicity, uptake and accumulation. Sci Total Environ. 2010, 408, 3053-3061.

[38] Milon, A.; Chang, S.; Ravindran, B. Biochar amended compost maturity evaluation using commercial vegetable crops seedlings through phytotoxicity germination bioassay. J. King Saud University. 2022, 34, 101770.

[39] Li, C.; Li, H.; Yao, T.; Su, M.; Ra, F.; Han, B.; Li, J.; Lan, X.; Zhang, Y.; Yang, X.; Gun, S. Microbial inoculation influences bacterial community succession and physicochemical characteristics during pig manure composting with corn straw. Bioresour Technol. 2019, 289, 121653.

[41] Siles-Castellano, A.; López, M.; López-González, J.; Suárez-Estrella, F.; Jurado, M.; Estrella-González, M.; Moreno, J. Comparative analysis of phytotoxicity and compost quality in industrial composting facilities processing different organic wastes. J. Clean prod. 2020, 252, 119820.

[42] Oleszczuk, P. Phytotoxicity of municipal sewage sludge composts related to physico-chemical properties, PAHs and heavy metals. Ecotoxicol Environ Saf. 2008, 69, 496-505.

[43] Martins, G.; Correa, L.; Guidoni, L.; Lucia, T.; Gerber, M.; Silva, F.; Correa, E. Toxicity and physicochemical parameters of composts including distinct residues from agribusiness and slaughterhouse sludge. Waste Manage. 2022, 138, 75-82.

Point 10: L384-385: In the table, mention the complete form of parameters at the end.

Response 10: Thanks for the suggestion. We added the “Note: HA-C, content of humic acid carbon;

FA-C, content of fulvic acid carbon”.

Point 11: Conclusion: It should be more precise and highlight only the present study's significant findings.

Response 11: Thanks for the suggestion. We revised to “The preparation of the compost water extract based on the dry weight of the samples could reflect the phytotoxicity more accurately without the interference of moisture content. In this study, the characteristics and requirements of different stages of plant growth were considered and it was found that the chlorophyll content of cotyledons was the same as the RRG of seeds, which could characterize the evolution of phytotoxicity during composting. The seedling establishment test, therefore, facilitated a comprehensive evaluation of compost phytotoxicity”.

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

GENERAL COMMENTS:

The authors have tried to design the article in a good manner. The introduction shows a good synthesis of work, methods are clear and well defined, except the control has not been well defined. I can’t find any control treatment. The results and discussion section need thorough revision because, in my opinion, they are not up to the mark.

Specific comments

Many comments are in the attached pdf file of the original article as annotations. Some quarries require attention:

1. RRG formula what is control?

2. Page 5-Line 184-186 “which was related to…..” not agree with the statement. The low RRG has been tried to relate to the production of high NH4-N and organic acids. The NH4-N is produced in higher levels when anaerobic conditions prevail in pile which is not the suitable condition. On the other hand, if the organic acids are produced at higher levels than why pH is alkaline at day 1-3 of decomposition (Fig 4 a,b).

3. Page 5-Line 186: Are the differences between treatments statistically significant??

4. Page 5- Line 205-209: these results (% increase) are not present in Fig 2.

5. Line 213-217: The results are not clear….does not match with the chl a and b content mentioned in above lines.

6. Line 227-229: Justify?? If the chlorophyll ha been increased in all treatments than how detrimental effects are mentioned?

7. All figures must include self-explanatory captions. Error bars show what values??

8. Line 243-245 Is this true? Fig 3 shows high temperature in CM from day 20 to 50 than how ACM has been claimed to be high??

9. Line 270: “acetic acid……volatilization…….” No substantial proof for this claim

10. Line 271: NH4-N curve is decreasing continuously hence there is no stability. The claim that it stabilized at the end is not true.

11. Line 278: a different argument for increase in pH i.e. NH4 and moisture content.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2 Comments

Point 1: The authors have tried to design the article in a good manner. The introduction shows a good synthesis of work, methods are clear and well defined, except the control has not been well defined. I can’t find any control treatment. The results and discussion section need thorough revision because, in my opinion, they are not up to the mark.

Response 1: Thanks for the comment. We checked the whole manuscript and revised the issues.

Point 2: RRG formula what is control?

Response 2: Thanks for the comment. The relative radicle length of seeds cultured with deionized water was the control, which could be seen from the denominator of the calculation formula. In the seedling establishment test, 5 mL of the compost water extract or deionized water for the control was added to Chinese cabbage seeds (20 seeds per dish, replicated three times) and the seeds were incubated at 25 °C for 48 h in the dark. Therefore, the control of RRG formula is average radicle length of all seeds in deionized water.

Point 3: Page 5-Line 184-186 “which was related to…..” not agree with the statement. The low RRG has been tried to relate to the production of high NH₄⁺-N and organic acids. The NH₄⁺-N is produced in higher levels when anaerobic conditions prevail in pile which is not the suitable condition. On the other hand, if the organic acids are produced at higher levels than why pH is alkaline at day 1-3 of decomposition (Fig 4 a,b).

Response 3: Thanks for the comment. Previous research results show that the seed germination is inhibited by phytotoxic substances such as low molecular weight organic acids and inorganic nitrogen in the aqueous extracts of composts (Chen et al., 2021). Therefore, we speculate that the decrease of RRG may be related to low molecular organic acids. However, the organic acid content was not determined in this experiment, so we revised to “which was related to the incomplete degradation of organic matter and high NH₄⁺-N content”.

Point 4: Page 5-Line 186: Are the differences between treatments statistically significant??

Response 4: Thanks for the comment. There were no significant differences in RRG between the two treatments (P>0.05). The purpose of this study was not to compare which of the two compost treatments was better, but to compare the differences in the evaluation of phytotoxicity of compost water extract based on dry weight and wet weight of samples in different compost treatments.

Point 5: Page 5- Line 205-209: these results (% increase) are not present in Fig 2.

Response 5: Thanks for the comment. By comparing to the original data, we found some errors in the Figure 2 (d) and corrected it. The following table shows the original data.

Treatment		The relative chlorophyll content (Wet basis)		Increment	The relative chlorophyll content (Dry basis)		Increment
		Day 1	Day 70		Day 1	Day 70
CM	Chlorophyll a	1.6598	2.1765	51.67%	0.9836	1.7812	79.76%
CM	Chlorophyll b	1.5710	1.9523	38.13%	0.9627	1.4116	44.89%
ACM	Chlorophyll a	1.3419	1.9706	62.87%	1.1889	2.1334	94.45%
ACM	Chlorophyll b	1.5026	1.6604	15.78%	1.1829	2.2558	107.29%

Point 6: Line 213-217: The results are not clear….does not match with the chl a and b content mentioned in above lines.

Response 6: Thanks for the comment. We revised to “At the end of composting, the relative chlorophyll content of seedlings extracted based on dry weight was lower than that under wet weight extraction in the CM treatment”.

Point 7: Line 227-229: Justify?? If the chlorophyll has been increased in all treatments than how detrimental effects are mentioned?

Response 7: Thanks for the comment. No detrimental effects of compost were detected on chlorophyll synthesis in this study, so we deleted this sentence.

Point 1: All figures must include self-explanatory captions. Error bars show what values??

Response 8: Thanks for the comment. Each sample was replicated three times. Data are presented as the mean value ± standard deviation in this study, so the error bars show standard deviations.

Point 9: Line 243-245 Is this true? Fig 3 shows high temperature in CM from day 20 to 50 than how ACM has been claimed to be high??

Response 9: Thanks for the comment. We revised to “The temperature of the ACM treatment was higher than that of the CM treatment before day 14, suggesting that the addition of the microbial agent rapidly increased the temperature of the piles”.

Point 10: Line 270: “acetic acid……volatilization…….” No substantial proof for this claim

Response 10: Thanks for the comment. Based on the results of previous studies, we speculated that the production of low-molecular-weight organic acids may reduce pH, but we did not determine the content of organic acids during this experiment. Therefore, we revised to “The rapid drop in pH in the two treatments at the initial stage of composting and then increased, which were consistent with the results of Nie et al [21]. They attributed the decrease in pH to the production of low molecular weight organic acids and the increase in pH was due to the rapid degradation of organic acids and proteins in the late stage of composting to produce a large amount of NH₃ release, while ammoniation produces a large amount of OH^-”.

Point 11: Line 271: NH₄⁺-N curve is decreasing continuously hence there is no stability. The claim that it stabilized at the end is not true.

Response 11: Thanks for the comment. We revised to “The pH of the two treatments based on wet weight extraction of the sample changed substantially during the composting process. The rapid drop in pH in the two treatments at the initial stage of composting and then increased, which were consistent with the results of Nie et al [21]. They attributed the decrease in pH to the production of low molecular weight organic acids and the increase in pH was due to the rapid degradation of organic acids and proteins in the late stage of composting to produce a large amount of NH₃ release, while ammoniation produces a large amount of OH^-”.

Point 12: Line 278: a different argument for increase in pH i.e.NH₄⁺-N and moisture content.

Response 12: Thanks for the comment. We deleted the sentence. We believed that exploring the reasons for the increase or decrease in pH may not be an important part of this study, and deleting too much discussion to highlight the purpose of this article.

Many comments are in the attached pdf file of the original article as annotations.

Point 13: Line 45: the evolution of phytotoxicity

Response 13: Thanks for the suggestion. We revised to “the evaluation of phytotoxicity”.

Point 14: Line 105: Bacillus licheniformis and Bacillus subtilis should be in italics

Response 14: Thanks for the suggestion. We revised to “Bacillus licheniformis and Bacillus subtilis”.

Point 15: Line 117: Measurement and analysis of methods

Response 15: Thanks for the suggestion. We revised to “Measurement and analysis methods”.

Point 16: Line 178-180: Should be excluded or re-written.

Response 16: Thanks for the suggestion. We deleted this part.

Point 17: Line 182: Subheading is not correct. It should be made more clear by considering the compost extract rather than "during composting"

Response 17: Thanks for the suggestion. It is a common practice to use compost water extract in phytotoxicity evaluation, here is to emphasize the changes of different composting indexes with composting process.

Point 18: Line 188: effect of phytotoxicity

Response 18: Thanks for the suggestion. We revised to “phytotoxic effects”.

Point 19: Line 210-211: looks general statement..should be avoided

Response 19: Thanks for the suggestion. We deleted the sentence.

Point 20: Line 227-229: Justify????? Results show increased effects than how detrimental???

Response 20: Thanks for the comment. We revised to “As the composting temperature decreased, moisture evaporation slowed, and the moisture content of the compost stabilized in the CM treatment”.

Point 21: Line 249-250: Is this true?? The ACM curve is continuously declining..there is no stability in moisture.

Response 21: Thanks for the comment. We revised to “As the composting temperature decreased, moisture evaporation slowed, and the moisture content of the compost stabilized in the CM treatment”.

Point 22: Line 253-255: There is no significant difference in temperature under both treatments. Moreover, CM is showing higher temp from day 20 to day 50 than ACM...See fig 3 a.

Response 22: Thanks for the comment. During the thermophilic phase, the temperature peaks in the ACM treatment and CM treatments were 69.1°C (3 d) and 67.3°C (4 d), respectively. The temperature of the ACM treatment was always higher than that of the CM treatment in the thermophilic stage. Therefore, we think this may be because the addition of the microbial agent rendered the temperature of the ACM treatment higher than that of the CM treatment at the thermophilic phase, thus making the aerobic fermentation more thorough.

Point 23: Line 267: albeit

Response 23: Thanks for the comment. We revised to “The pH of the two treatments based on wet weight extraction of the sample changed substantially during the composting process. The rapid drop in pH in the two treatments at the initial stage of composting and then increased, which were consistent with the results of Nie et al [21]. They attributed the decrease in pH to the production of low molecular weight organic acids and the increase in pH was due to the rapid degradation of organic acids and proteins in the late stage of composting to produce a large amount of NH₃ release, while ammoniation produces a large amount of OH^-”.

Point 24: Line 271-272: NH₄⁺-N is decreasing during the course of decomposition…so how it increased pH.

Response 24: Thanks for the comment. We revised to “The rapid drop in pH in the two treatments at the initial stage of composting and then increased, which were consistent with the results of Nie et al [21]. They attributed the decrease in pH to the production of low molecular weight organic acids and the increase in pH was due to the rapid degradation of organic acids and proteins in the late stage of composting to produce a large amount of NH₃ release, while ammoniation produces a large amount of OH^-”.

Point 25: Line 295: were then analyzed

Response 25: Thanks for the suggestion. We revised to “were analyzed”.

Point 26: Line 303: The reference is placed at wrong place.

Response 26: Thanks for the suggestion. We placed the reference [26] behind the “As the primary source of composting phytotoxicity”.

Point 27: Line 321: Is this statement require reference?

Response 27: Thanks for the suggestion. We deleted this reference.

Point 28: Line 333-335: Please justify.

Response 28: Thanks for the suggestion. We revised to “The TN content increased from day 28, during which the ammonia loss rate decreased and became lower than the nitrogen mineralization rate”.

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

Please avoid abbreviations in the abstract.

Ligne 37: remove "and".

page3: table 1, moisture, please correct this word.

ligne 102: CM and ACM??? what does it mean?

ligne 102: please provide more details about this agent and why the authors used this agent?

ligne 105: how can the authors know that these species are present if the authors have made a molecular identification?

ligne 178: in my opinion this part is not necessary, maybe you should delete it.

Ligne 233: This part should be placed before the phytotoxicity part.

lign 237: What is the difference between the two treatments? The authors said that the ACM treatment was added by a microbial agent but we can see that there is not a big difference between the two temperature treatments either for the other parameters?

In my opining, there is no novelty in this article, all the parameters analyzed are already valid in the literature.

Author Response

Response to Reviewer 3 Comments

Point 1: Please avoid abbreviations in the abstract.

Response 1: Thanks for the suggestion. We deleted the abbreviations and replaced them with full names.

Point 2: Line 37: remove "and".

Response 2: Thanks for the suggestion. We revised to “As a sustainable environmentally friendly method”.

Point 3: page3: table 1, moisture, please correct this word.

Response 3: Thanks for the comment. It was careless of us. We revised to “moisture”.

Point 4: line 102: CM and ACM??? what does it mean?

Response 4: Thanks for the suggestion. The raw material of compost was mainly cow manure, so CM means cow manure compost and ACM means cow manure compost with added agent (an organic matter-decomposing microbial agent).

Point 5: line 102: please provide more details about this agent and why the authors used this agent?

Response 5: Thanks for the suggestion. We revised to the “An organic matter-decomposing microbial agent (Shanxi Kaisheng Fertilizer Co., Ltd.) was added in the ACM treatment as per 10% of the dry weight of cow manure in the pile. The number of viable bacteria per gram is ≥50 million, and the effective bacteria are Bacillus licheniformis and Bacillus subtilis.”

Point 6: line 105: how can the authors know that these species are present if the authors have made a molecular identification?

Response 6: Thanks for the comment. The microbial agent used in the experiment was purchased from the local market. We learned that it is rich in different kinds of microbes for decomposing cellulose and protein. The straw and animal manure become high quality organic fertilizer by removing odor and reducing the fertilizer cost through microbial degradation. The number of viable bacteria per gram is ≥50 million, and the effective bacteria are Bacillus licheniformis and Bacillus subtilis, according to the product introduction.

Point 7: line 178: in my opinion this part is not necessary, maybe you should delete it.

Response 7: Thanks for the suggestion. We deleted it.

Point 8: Line 233: This part should be placed before the phytotoxicity part.

Response 8: Thanks for the suggestion. Because the focus of this article is on the part of phytotoxicity evaluation, we put this part of the results in the front. We think it is reasonable and does not affect the reading.

Point 9: line 237: What is the difference between the two treatments? The authors said that the ACM treatment was added by a microbial agent but we can see that there is not a big difference between the two temperature treatments either for the other parameters?

Response 9: Thanks for the comment. The temperature of the ACM treatment was always higher than that of the CM treatment in the thermophilic stage. In addition, the moisture content of the ACM treatment decreased faster than that of the CM treatment, which can be confirmed that the moisture content in the ACM treatment evaporated faster due to the high temperature.

Point 10: In my opining, there is no novelty in this article, all the parameters analyzed are already valid in the literature.

Response 10: Thanks for the comment. The innovation of the article lies in the evaluation method, the measured indicators are conventional. In this study, we compared the effects of the two extraction methods on phytotoxicity evaluation by preparing compost water extracts based on the dry and wet weight of the samples. Moreover, a comprehensive evaluation of compost phytotoxicity was performed through a seedling establishment test of Chinese cabbage seeds. Therefore, the highlights of this study are as follows:

The phytotoxicity of compost water extract was compared based on the dry weight and wet weight.

Compost water extract based on dry weight more accurately reflects phytotoxicity.

Plant skotomorphogenesis and photomorphogenesis were considered.

Seedling establishment was more comprehensive than seed germination for phytotoxicity testing.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The manuscript is improved very well but i suggest the double check on language of newly added paragraphs.

Author Response

Response to Reviewer 2 Comments

Point 1: The manuscript is improved very well but i suggest the double check on language of newly added paragraphs.

Response 1: Thanks for the comment. We checked the whole manuscript and revised as follows:

Line 57: [10-11]

We placed references [10-11] behind “composting”.

Line 104: means cow manure compost; means cow manure compost added with microbial agent

We deleted “means”.

Line 105: An organic matter-decomposing microbial agent (Shanxi Kaisheng Fertilizer Co., Ltd.) was added in the ACM treatment as per 10% of the dry weight of cow manure in the pile. The number of viable bacteria per gram is ≥50 million, and the effective bacteria are Bacillus licheniformis and Bacillus subtilis.

We revised to “An organic matter-decomposing microbial agent (Shanxi Kaisheng Fertilizer Co., Ltd.) was added in the ACM treatment with a weight ratio of 10% of the cow manure dry weight in the pile. The effective bacteria in the microbial agent are Bacillus licheniformis and Bacillus subtilis, with viable bacteria number over 5.0 x 10⁷ cfu/g”.

Line 131: or deionized water (the control)

We revised to “or deionized water for the control”.

Line 151: Determination of humic substances’ contents and E₄/E₆

We revised to “Determination of humic substances contents and E₄/E₆”.

Line 159: E₄/E₆ is the ratio of the absorbance of the humic substances at 465 nm and 665 nm.

We revised to “E₄/E₆ is the ratio of the absorbance of the humic acid at 465 nm and 665 nm”.

Line 262: The rapid drop in pH in the two treatments at the initial stage of composting and then increased, which were consistent with the results of Nie et al [21]. They attributed the decrease in pH to the production of low molecular weight organic acids and the increase in pH was due to the rapid degradation of organic acids and proteins in the late stage of composting to produce a large amount of NH₃ release, while ammoniation produces a large amount of OH^-.

We revised to “The rapid decrease in pH in the two treatments in the early stage of the composting process, followed by an increase in pH was consistent with the results of Nie et al [21]. They attributed the decrease in pH to the formation of low molecular weight organic acids and the increase in pH to the rapid degradation of organic acids and proteins in the late stage of the composting process, releasing large amounts of NH₃ and producing large amounts of OH^- through ammonification.”.

Line 378: Note: HA-C, content of humic acid carbon; FA-C, content of fulvic acid carbon.

We revised to “Note: HA-C, humic acid carbon; FA-C, fulvic acid carbon”.

Line 498-499: Chinese technical specification for animal manure composting. (NY/T 3442-2019) http://down.foodmate.net/standard/sort/5/80143.html

We revised to “Ministry of Agriculture and Rural Affairs of China. Chinese technical specification for animal manure composting (NY/T 3442-2019), Beijing, China, 2019. http://down.foodmate.net/standard/sort/5/80143.html”.

Reviewer 3 Report

line 105: remove "means"

I appreciate the efforts of the authors to increase the quality of the document. But I´m still thinking that there is no novelty. However, the manuscript has the approval of the other reviewer, so I do not object to the publication, if that is what the editor wishes.

Author Response

Response to Reviewer 3 Comments

Point 1: line 105: remove "means"

Response 1: Thanks for the comment. We revised to " CM (cow manure compost) and ACM (cow manure compost added with microbial agent)".

Point 2: I appreciate the efforts of the authors to increase the quality of the document. But I´m still thinking that there is no novelty. However, the manuscript has the approval of the other reviewer, so I do not object to the publication, if that is what the editor wishes.

Response 2: Thanks for the comment. In this study, the evaluation of compost phytotoxicity was extended from seed germination to seedling establishment. Not only the effect of compost on seed radicle, but also the effect of compost on seed cotyledon was concerned. This is a very beneficial exploration for the comprehensive evaluation of compost phytotoxicity. This content was also reflected in introduction and part 3.5.2. We will also further explore and improve upon this in future research.

Author Response File: Author Response.docx