Utilization of Biochar for Eliminating Residual Pharmaceuticals from Wastewater Used in Agricultural Irrigation: Application to Ryegrass

Round 1

Reviewer 1 Report

I commend the authors Mayowa Akintoroye, Robert Ato Newton, Sylvie Kříženecká, Stanislave Hejda, Pavel Krystyník, Markus Ahnert, Josef Trögl, Peter Krebs, Karim Suhail Al Souki of the manuscript titled “Utilization of Biochar for Eliminating Residual Pharmaceuticals from Wastewater Used in Agricultural Irrigation: Application to Ryegrass” for their work  on  the removal of pharmaceutical contaminants from wastewater using biochar.

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I have several drawback on this study:

1-      The amount of biochar or the ratio added was still low even in the high amount of 0.5 g/L

2-      The application to ryegrass in not suffecient to get a conclusion on the efficiency of biochar in removing pharmaceutical residues from soil. Other phytoremediation crops should be used. The novel part of the study is tracking such molecules in the plant and nothing was detected in the ryegrass which is not logic or need other crops to be examined on or applying other methods of detection.

3-      If we are talking about soil microbes, its much better to focus on specific group of organisms such as bacteria or fungi, hence the detection and accuracy and application will be much more important. You may even remove the soil microbial activities from the study because its not comprehensive work.

4-      The conclusions in the discussion is not supported by the results achieved in some parts.

However, the study is still publishable but with modifications.

 In the introduction you should state why you used the ryegrass as testing crop.

1.       In the materials and methods,

-          A citation should be used for : 2.4. Pharmaceuticals concentrations in soil and ryegrass sample

-           

3-      In the results part:

-          Table 3 states the significant reduction of pharmaceutical molecules, however large amount of these molecules are still there, this finding should be highlighted clearly in the abstract and conclusion of this study, such as Iopamidol ; even at the high dose of biochar which is 0.5 g its still there and in high ratio which confirms my previously mentioned droughts.

-          Table 4, not all compounds were traced in soil as mentioned said in the abstract, you should mentioned that you tracked some of them only and not all in the soil.

4-      In the discussion part,    

-          Line 376 “ This proves the relatively low pharmaceuticals doses in the soil.” Lower the tone or remove it or further explain it.

-          You may remove the part of Impacts on plant biomass and soil microbial activities, because its not significant from my point of view

I give you major revision

Author Response

Comments and Suggestions for Authors

I commend the authors Mayowa Akintoroye, Robert Ato Newton, Sylvie Kříženecká, Stanislave Hejda, Pavel Krystyník, Markus Ahnert, Josef Trögl, Peter Krebs, Karim Suhail Al Souki of the manuscript titled “Utilization of Biochar for Eliminating Residual Pharmaceuticals from Wastewater Used in Agricultural Irrigation: Application to Ryegrass” for their work on the removal of pharmaceutical contaminants from wastewater using biochar.

Dear reviewer, we would like to thank you for accepting to review our paper and for the valuable comments which were considered within revision. We generally agree with the points that were mentioned which represent good potentials for further research within the context of biochar treatment of wastewater and application to agriculture. We would also like to trigger your attention that the current work represents the master’s internship of Mr. Akintroye, and thus the time constraints were pressuring in certain decisions that we took (among which, the choice of the plant). We tried to clarify as much as possible the points you considered as drawbacks, hoping that we managed to obtain your approval. All of the modified sentences based on your recommendations could be Tracked.

I have several drawback on this study:

• The amount of biochar or the ratio added was still low even in the high amount of 0.5 g/L

The biochar amount was based on a previous experiment done by the co-authors Sylvie Kříženecká and Stanislav Hejda who concluded that an amount of 1 g.L^-1 of the biochar are sufficient to remove more than 90% of most of the pharmaceuticals in the wastewater (2 reports were added as supplementary materials S1 and S2, giving more details about the biochar used “Biochar 4073” as well as clarifying the choice of concentration to be added, in Czech language but could be easily translated). Therefore, the decision was not to add more than 0.5 g.L^-1.

• The application to ryegrass in not suffecient to get a conclusion on the efficiency of biochar in removing pharmaceutical residues from soil. Other phytoremediation crops should be used. The novel part of the study is tracking such molecules in the plant and nothing was detected in the ryegrass which is not logic or need other crops to be examined on or applying other methods of detection.

We totally agree with the idea that ryegrass alone is not sufficient and further investigations should be done on several plant models to validate the conclusions. However, the choice of ryegrass was taken (phrases were added to the modified text to justify our choice; kindly check the modified manuscript) due to the fact that it is used as a plant model in our labs as well as (most importantly) the fact that it needs only 4 to 5 weeks for the biomass to be collected (time constraints were decisive in our choice). However, among the future perspectives of our lab, is to test and validate the obtained results on larger scale experiments (larger pots and fields), with different plant models.

• If we are talking about soil microbes, its much better to focus on specific group of organisms such as bacteria or fungi, hence the detection and accuracy and application will be much more important. You may even remove the soil microbial activities from the study because its not comprehensive work.

We agree with your point of view. However, our objective from performing the FDHA analysis in the different used soil was to get an idea about the general state/health of the soils upon the application of biochar treated and non-treated wastewaters from one side as well as to verify if the plant itself could contribute to the soil microbial activities within this defined context.

• The conclusions in the discussion is not supported by the results achieved in some parts.

Sorry, but we did not understand this point.

However, the study is still publishable but with modifications.

In the introduction you should state why you used the ryegrass as testing crop.

Phrases were added according to your recommendation, however in the materials and methods part, section 2.3 (Kindly check the modified manuscript).

1. In the materials and methods,

-          A citation should be used for : 2.4. Pharmaceuticals concentrations in soil and ryegrass sample

The experiments were applied based on the thesis work of Mrs. Jitka Tolaszová. Citation was added (Kindly check the modified manuscript).

3-      In the results part:

-          Table 3 states the significant reduction of pharmaceutical molecules, however large amount of these molecules are still there, this finding should be highlighted clearly in the abstract and conclusion of this study, such as Iopamidol; even at the high dose of biochar which is 0.5 g its still there and in high ratio which confirms my previously mentioned droughts.

Thank you for triggering our attention to this point. In fact, we have mentioned in the results section, that with the exception of Bezafibrate, all of the other pharmaceuticals concentrations were decreasing with the increase of the biochar doses. However, the question is whether these remaining concentrations represent a risk on the plant health. In other words, could the treated wastewater be used for irrigation, in spite the remaining amounts of pharmaceuticals? According to what we obtained, there was no impacts on the plant model health, and thus it is possible to be used.

However, we have modified/clarified the aims of the work to better fit with the obtained results (Kindly check the last paragraph of the introduction). We also mentioned in the results section, that in spite the significant reduction in certain pharmaceuticals, considerable amounts still exist even at the highest biochar doses.

-          Table 4, not all compounds were traced in soil as mentioned said in the abstract, you should mentioned that you tracked some of them only and not all in the soil.

The phrase was added according to your recommendation (Kindly check the modified manuscript).

4-      In the discussion part,   

-          Line 376 “ This proves the relatively low pharmaceuticals doses in the soil.” Lower the tone or remove it or further explain it.

The phrase was removed.

-          You may remove the part of Impacts on plant biomass and soil microbial activities, because its not significant from my point of view

It is true that the results were not quite different between the different treatment, however, this outcome itself could be considered as a starting point to be compared with. Therefore, we prefer to keep presenting the results and hoping that it could be further developed in other research experiments.

I give you major revision

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript investigated the impacts of biochar application and/or concentration on the sorption of pharmaceuticals in water, showed that most pharmaceuticals concentrations were significantly but not totally reduced from the wastewater upon biochar addition. It proved that biochar could be approached as a good substrate for eliminating pharmaceuticals from wastewaters used for agricultural irrigation. I think this paper can be accepted after Minor revise.

Some comments are below:

1. The information of biochar pretreatment is needed because it affects the surface properties, and the produced organics during pyrolysis may influence the pharmaceuticals determination result.

2. Table 1: mg.l^-1 should be mg.L^-1;

3. Line 208: 0.19 ± 0.00 0.08 ng.g-1?

4. S0, S0.1, S0.25, S0.5, G0, G0.1, G0.25 and G0.5 should be defined, not in the table caption.

5. Table 3 and Table 4: a, ab, abc, etc, should be explained.

6. Table 4: “0.22 ± 0.28”, why such kind of data?

7. “DM” means what?

8. Figures and tables need to be well drawn.

9. Please carefully check the language to avoid errors.

Author Response

The manuscript investigated the impacts of biochar application and/or concentration on the sorption of pharmaceuticals in water, showed that most pharmaceuticals concentrations were significantly but not totally reduced from the wastewater upon biochar addition. It proved that biochar could be approached as a good substrate for eliminating pharmaceuticals from wastewaters used for agricultural irrigation. I think this paper can be accepted after Minor revise.

Dear reviewer, first of all, we would like to express our appreciation for accepting to review our paper and your efforts and recommendations. We would like to thank you as well for the positive feedback you gave.

Some comments are below:

1. The information of biochar pretreatment is needed because it affects the surface properties, and the produced organics during pyrolysis may influence the pharmaceuticals determination result.

We have added 2 supplementary files S1 and S2 (in Czech language, but could be translated) that describe in details the biochar processing and its characteristics as well as the choice of the concentrations added to the waste water.

2. Table 1: mg.l-1 should be mg.L-1;

It was corrected (Kindly check the modified manuscript).

3. Line 208: 0.19 ± 0.00 0.08 ng.g-1?

It was corrected (Kindly check the modified manuscript).

4. S0, S0.1, S0.25, S0.5, G0, G0.1, G0.25 and G0.5 should be defined, not in the table caption.

The symbols were defined in the materials and methods, section 2.3 (Kindly check the modified manuscript).

5. Table 3 and Table 4: a, ab, abc, etc, should be explained.

The different letters refer to significant differences between the values (Tukey HSD test, p ≤ 0.05). The explanation is present in the table caption.

6. Table 4: “0.22 ± 0.28”, why such kind of data?

It is 0.028, and it was rounded to 0.03. Mistake was corrected (Kindly check the modified manuscript).

7. “DM” means what?

DM corresponds to dry mass. It was defined in the modified text. (Kindly check the modified manuscript).

8. Figures and tables need to be well drawn.

The figures were re-drawn with better resolutions (Kindly check the modified manuscript).

9. Please carefully check the language to avoid errors.

The paper was revised for English language by an expert (Mrs. Layal El Wattar) who holds a Bachelor degree in English Language and Literature (kindly find a copy of her certificate).

Author Response File: Author Response.docx

Reviewer 3 Report

Author Response

Response to reviewer 3:

Dear reviewer, we would like to thank you for the time you spent on reading and evaluating the current work. Your comments were taken into consideration in the modified version of the manuscript.

• 1: in which way biochar can be used? I suggest to the author to add this part before line 43, when they start again to talk about biochar.

The sentence was shifted based on the recommendation and the reference order as well (Kindly check the modified manuscript).

• 2: change with steps

It was changed based on the recommendation (Kindly check the modified manuscript).

• 2: please add the references of the several words at the end of the sentence

In fact, the references are 12-14. The localization of the numbers was modified (Kindly check the modified manuscript).

• 2: An amount of the sample (50 mL) was

It was changed based on the recommendation (Kindly check the modified manuscript).

• 2: what kind of acid?

It was formic acid (Kindly check the modified manuscript).

• 3: i suggest the authors to put the unit after the name of the parameter: BET surface area (m2.g-1)

It was changed based on the recommendation (Kindly check the modified manuscript).

• 3: please add some comma in this sentence, to many and

It was changed based on the recommendation (Kindly check the modified manuscript).

• 3: I can't find this article and a doi is not present in the references section. Because the experimental design was adapted from this article it's important to see the article cited. Kříženecká, S.; Hejda, S.; Kaule, P. Analysis of Wastewater with a Specific Focus Using Sorbents Based on Biochar and/ 436 or Its Modifications; Jan Evangelista Purkyně University in Ústí nad Labem; 2020

In fact, there was a mistake in citation. The reference is a research report written by 2 of the co-authors based on an experiment containing details about the different biochar concentrations used. The report was added as a supplementary material (Kindly check the modified manuscript).

• 4: temperature? humidity? light intensity? please add the parameters inside the greenhouse

In fact, the phrase was corrected to semi-controlled, since the temperature and humidity were the only factors under control. As the greenhouse was transparent, the day/night and light intensity were dependent on the weather factors at the time of the experiment (Kindly check the modified manuscript).

• 4: What were the characteristics of the soil used?

Soil characteristics were added in Table S1 supplementary material (Kindly check the modified manuscript).

• 4: why 1 g of seeds and not an equal number of seeds? Weighing the seeds does not give you confidence that you have put the same number of seeds in all the pots.

We think that both point of views are accepted. However, as a justification, due to the small size of the ryegrass seeds, and based on published papers, the choice was to weigh the seeds rather than adding a precise number of them at the beginning.

Kindly check the links of papers I added in which the same manner was used (seeds mass rather than number was followed for cultivating ryegrass in pot experiments)

https://doi.org/10.1016/j.scienta.2020.109314

https://doi.org/10.1016/j.scitotenv.2019.04.349

https://doi.org/10.1016/j.ecoenv.2020.110654

• 4: At what percentage of moisture were the pots kept?

We studied the water holding capacity of the soil before starting the experiment, and the decision was to maintain the soil wet at 70% of the soil WHC. That’s why we added 100 mL per irrigation. The phrase was added (Kindly check the modified manuscript).

• 4: The final weight is dependent on the seeds germinated, but now it's impossible that the authors record this value without count the number of the seeds sown.

Your point of will be taken into consideration in future experiments. Unfortunately, it is not possible to modify the outcome of the current work.

• 7: grams for pots without the amount of seeds sown and the % of germination of the seeds is not a comparable way between the treatments.

What we did is that at the end of experiment we just cut the aboveground biomass of the samples and weighed it.

• 7: i suggest the authors to spend more time in making graphs, at least the title can be put on the y-axis.

The figures were re-drawn with better resolutions (Kindly check the modified manuscript).

• 8: like figure 1

The figures were re-drawn with better resolutions (Kindly check the modified manuscript).

• 8: this increase is significant?

According to the authors, the increase is not significant, unlike our obtained results where we had a significant decrease.

Response to reviewer 3:

Dear reviewer, we would like to thank you for the time you spent on reading and evaluating the current work. Your comments were taken into consideration in the modified version of the manuscript.

• 1: in which way biochar can be used? I suggest to the author to add this part before line 43, when they start again to talk about biochar.

The sentence was shifted based on the recommendation and the reference order as well (Kindly check the modified manuscript).

• 2: change with steps

It was changed based on the recommendation (Kindly check the modified manuscript).

• 2: please add the references of the several words at the end of the sentence

In fact, the references are 12-14. The localization of the numbers was modified (Kindly check the modified manuscript).

• 2: An amount of the sample (50 mL) was

It was changed based on the recommendation (Kindly check the modified manuscript).

• 2: what kind of acid?

It was formic acid (Kindly check the modified manuscript).

• 3: i suggest the authors to put the unit after the name of the parameter: BET surface area (m2.g-1)

It was changed based on the recommendation (Kindly check the modified manuscript).

• 3: please add some comma in this sentence, to many and

It was changed based on the recommendation (Kindly check the modified manuscript).

• 3: I can't find this article and a doi is not present in the references section. Because the experimental design was adapted from this article it's important to see the article cited. Kříženecká, S.; Hejda, S.; Kaule, P. Analysis of Wastewater with a Specific Focus Using Sorbents Based on Biochar and/ 436 or Its Modifications; Jan Evangelista Purkyně University in Ústí nad Labem; 2020

In fact, there was a mistake in citation. The reference is a research report written by 2 of the co-authors based on an experiment containing details about the different biochar concentrations used. The report was added as a supplementary material (Kindly check the modified manuscript).

• 4: temperature? humidity? light intensity? please add the parameters inside the greenhouse

In fact, the phrase was corrected to semi-controlled, since the temperature and humidity were the only factors under control. As the greenhouse was transparent, the day/night and light intensity were dependent on the weather factors at the time of the experiment (Kindly check the modified manuscript).

• 4: What were the characteristics of the soil used?

Soil characteristics were added in Table S1 supplementary material (Kindly check the modified manuscript).

• 4: why 1 g of seeds and not an equal number of seeds? Weighing the seeds does not give you confidence that you have put the same number of seeds in all the pots.

We think that both point of views are accepted. However, as a justification, due to the small size of the ryegrass seeds, and based on published papers, the choice was to weigh the seeds rather than adding a precise number of them at the beginning.

Kindly check the links of papers I added in which the same manner was used (seeds mass rather than number was followed for cultivating ryegrass in pot experiments)

https://doi.org/10.1016/j.scienta.2020.109314

https://doi.org/10.1016/j.scitotenv.2019.04.349

https://doi.org/10.1016/j.ecoenv.2020.110654

• 4: At what percentage of moisture were the pots kept?

We studied the water holding capacity of the soil before starting the experiment, and the decision was to maintain the soil wet at 70% of the soil WHC. That’s why we added 100 mL per irrigation. The phrase was added (Kindly check the modified manuscript).

• 4: The final weight is dependent on the seeds germinated, but now it's impossible that the authors record this value without count the number of the seeds sown.

Your point of will be taken into consideration in future experiments. Unfortunately, it is not possible to modify the outcome of the current work.

• 7: grams for pots without the amount of seeds sown and the % of germination of the seeds is not a comparable way between the treatments.

What we did is that at the end of experiment we just cut the aboveground biomass of the samples and weighed it.

• 7: i suggest the authors to spend more time in making graphs, at least the title can be put on the y-axis.

The figures were re-drawn with better resolutions (Kindly check the modified manuscript).

• 8: like figure 1

The figures were re-drawn with better resolutions (Kindly check the modified manuscript).

• 8: this increase is significant?

According to the authors, the increase is not significant, unlike our obtained results where we had a significant decrease.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Accepted for me 

Author Response

Dear reviewer, 

We would like to thank you again for your remarks and recommendations. They have really enhanced the quality of the manuscript.

Reviewer 3 Report

Thank you for the responses, I think it is the case to make explicit the choice of biochar concentrations and the experimental design as given in the attached file.

Comments for author File: Comments.pdf

Author Response

Supplementary file S2 is not written in English, so it is the case that the choice of biochar concentrations and the experimental design in the text should be explained clearly. 

Dear reviewer,

Thank you again for your feedback and recommendations. A brief explanatory text was added to section 2.2 to justify the choice of biochar, doses and time of mixing (kindly check the modified version 2).