Ibuprofen and Ketoprofen—Inert Drugs or Potential Environmental Hazard?
, Arkadiusz Telesiński 2, Marcin Sysa 1, Agnieszka Godela 1, Radomír Ščurek 3 and Robert Biczak 1Round 1
Reviewer 1 Report
Abstract: Authors have been used lot of abbreviation in abstract without providing proper full name, which should be avoid. Please see the NSAIDs in line 17, IBU and KET in line 24, CAT, POD, SOD in line 25, AsA in line 26, DW in line 27 and MDA in line 29.
Introduction
Line 36: please put the references after the statement… in veterinary and animal medicine.
Line 40: Close the bracket of citation that is [1-3].
Line 46: please put the references after the statement….. source of NSAIDs in the environment.
Line 56: Please add the examples of other bodies in bracket
Materials and Methods
Line 111: please put the references after the statement… CFU (colony forming 111 unit)/g DW of soil.
Line 124 -125: Please use similar abbreviation for NSAIDs or NSAID in whole MS. Check line 33, 37,43, 46, 67 and so on.
Line 144: MDA stand for what???
Authors have been not providing any information on the composition of mixture of IBU+KET. Please provide that information. Also no information on the quantity of IBU and KET.
Results and Discussion
Figure 1 and 2: Put the better quality of graph and also explain in vertical axis caption * put as superfix of CFU. What its mean?.
Line 219: Please put the similar abbreviations in whole MS. Here author used IBUs for ibuprofen while in another place only IBU.
Line 229-230: This statement is not clear to me … “It should be remembered that soil is the habitat of many organisms, in 229 which plants consumed by humans and by animals grow”.
Line 235: Please check it … OstracodtoxkitFTM . particularly FTM.
Line 244: The results obtained 243 indicate that the tested drugs affect the growth of clamshells… here first-time clamshells come in discussion. There is no inform on it in materials and methods section.
Table 1: first column .. why “I” is small in NSAiDs. If typing mistake please correct it
Line 369-272: this statement is not clear to me…. “The first stage in the development of any plant, which has a tremendous impact on 269 growth, plant development and, consequently, on the yield obtained and its quality, is 270 germination. The proper course of this stage is affected by many factors, including the 271 presences of various types of stressors.”
Please check it again or rewrite it.
Table 2: first column .. ILs is stand for …?
Figure 3: In this figure authors talk about Shoot and Root length inhibition while those parameters have been not discussed in the methodology section of the MS.
Likewise, the information discussed and shown in figure 3 is not explain in the methodology section of the MS.
Line 320-321: R. sativum and L. sativa, please write the full name of genus of this plant.
Line 323: what if the finding of the study reported by “Wang et al. [47] studying the effect of KET on rice seedlings”
Line 351: PS I and PSII ???
Table 3: Eexplain the meaning of abbreviation used in this table and also put the dash between chlorophyll and it type like Chl-a or Chl-b.
In column 5 and 7: Chla/Chlb and Chl(a+b)/Car. what does mean division or ratio?
It would be good to reader if author provide summary of each drug individually for each column.
Line 388: ….ibuprofen and naproxen for Atriplex patula, spinach and lettuce….Please follow one kind of pattern, either put the botanical name or common English name of studied plants.
Line 399: MDA????
Line 408-411: There is lack of clarity in the caption of figure 5, neither in figure cation nor in graph differentiated or explain that in figure 5 which graph for which parameter, it should be discussed. Please clearly describe which graph for which parameter. Also, in caption of the figure 5 only mention proline while in graph its mentioned as free proline. Please correct it accordingly.
Line 425: correct the chemical formula of hydrogen peroxide
Line 426: ROS???
Line 444-446: where is the results of linear corelation.
Line 448-435: In whole MS authors have been not provide the data of all the parameters studied just simple discussed why?
Line 471-472: not clear
Line 481-483: which drugs a? and how much quantity of it used???
Table 4: Again, nowhere discussed the full form of abbreviation used in this table why???
Author Response
Dear Reviewer,
We are very grateful for all the comments related to our manuscript. We have taken an attitude towards the suggestions and comments of the Reviewers, which was included in the text below. According to the suggestion of Reviewer 1, the linguistic correction of the manuscript was made, which was confirmed by a relevant certificate. In accordance with the reviewers' indications, changes were made in the chapters Introduction, Results and discussion, and References. We also inform that all changes in manuscript are marked in red.
Your sincerely,
Barbara Pawłowska
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Reply to the Reviewer’s 1 Report
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Abstract: Authors have been used lot of abbreviation in abstract without providing proper full name, which should be avoid. Please see the NSAIDs in line 17, IBU and KET in line 24, CAT, POD, SOD in line 25, AsA in line 26, DW in line 27 and MDA in line 29.
Our response:
Of course, we agree with the Reviewer that using abbreviations should explain them at the first use. The use of only abbreviations in the abstract resulted from the requirements of the journal as to the length of the abstract. With the large amount of research, we present in this paper, providing this information in an abstract is a problem.
As suggested by the Reviewer, all abbreviations have been explained in the abstract.
Please see the revised manuscript.
Introduction
Line 36: please put the references after the statement… in veterinary and animal medicine.
Our response:
Thank you for your suggestion. The literature source for the indicated fragment has been given. Please see the revised manuscript.
Line 40: Close the bracket of citation that is [1-3].
Our response:
Sorry for the oversight. The parenthesis has been closed. Please see the revised manuscript.
Line 46: please put the references after the statement….. source of NSAIDs in the environment.
Our response:
Thank you for your suggestion. The literature source for the indicated fragment has been given. Please see the revised manuscript.
Line 56: Please add the examples of other bodies in bracket
Our response:
Thank you for your suggestion. In parentheses, the article lists other organizations that are working on this issue. Please see the revised manuscript.
Materials and Methods
Line 111: please put the references after the statement… CFU (colony forming 111 unit)/g DW of soil.
Our response:
Thank you for your suggestion. The literature source for the indicated fragment has been given. Please see the revised manuscript.
Line 124 -125: Please use similar abbreviation for NSAIDs or NSAID in whole MS. Check line 33, 37,43, 46, 67 and so on.
Our response:
Thank you for your suggestions. The abbreviation was standardized throughout the manuscript. Please see the revised manuscript.
Line 144: MDA stand for what???
Our response:
Sorry for not explaining the abbreviation. The abbreviation MDA stands for malondialdehyde. This has been corrected in the manuscript.
Authors have been not providing any information on the composition of mixture of IBU+KET. Please provide that information. Also no information on the quantity of IBU and KET.
Our response:
Information indicated by the reviewer can be found in the manuscript in chapter 2.4. Spring barley culture, line 137-138. IBU, KET and mixture IBU+KET were applied at concentrations of 0.1, 1, 10, 50, 100, 1000 mg·kg-1 dry weight of soil. The content of NSAIDs in the mixture was 1:1.
Results and Discussion
Figure 1 and 2: Put the better quality of graph and also explain in vertical axis caption * put as superfix of CFU. What its mean?.
Our response:
Thank you for your suggestion. The quality of the charts has been improved. CFU is colony-forming units. This is also stated in the manuscript under the figures. Please see the revised manuscript.
Line 219: Please put the similar abbreviations in whole MS. Here author used IBUs for ibuprofen while in another place only IBU.
Our response:
Thank you for your suggestion. The abbreviation for ibuprofen was standardized throughout the paper. Please see the revised manuscript.
Line 229-230: This statement is not clear to me … “It should be remembered that soil is the habitat of many organisms, in 229 which plants consumed by humans and by animals grow”.
Our response:
The incomprehensibility of the indicated sentence is due to errors in its translation. It has been removed from the manuscript. Please see the revised manuscript.
Line 235: Please check it … OstracodtoxkitFTM . particularly FTM.
Our response:
We apologize for the mistake in the product name. Of course, it should be Ostracodtoxkit F. This error has been corrected throughout the manuscript.
Line 244: The results obtained 243 indicate that the tested drugs affect the growth of clamshells… here first-time clamshells come in discussion. There is no inform on it in materials and methods section.
Our response:
We apologize for the error that occurred during the translation of the text. The authors did not study mussels, but H. incongruens crustaceans. This error has been corrected in the manuscript.
Table 1: first column .. why “I” is small in NSAiDs. If typing mistake please correct it
Our response:
Sorry for the error. Of course, the "I" should be capitalized in NSAIDS for short. This has been corrected in the manuscript.
Line 369-272: this statement is not clear to me…. “The first stage in the development of any plant, which has a tremendous impact on 269 growth, plant development and, consequently, on the yield obtained and its quality, is 270 germination. The proper course of this stage is affected by many factors, including the 271 presences of various types of stressors.”
Please check it again or rewrite it.
Our response:
Thank you for your suggestions. The indicated fragment has been rewrite. View revised manuscript.
Table 2: first column .. ILs is stand for …?
Our response:
We apologize for the mistake. It should be NSAIDs, not ILS. This has been corrected in the manuscript.
Figure 3: In this figure authors talk about Shoot and Root length inhibition while those parameters have been not discussed in the methodology section of the MS. Likewise, the information discussed and shown in figure 3 is not explain in the methodology section of the MS.
Our response:
Growth inhibition is described in section 2.5. Determination of growth responses. However, the word "inhibition" was missing, which could have caused an incorrect reception. This has been corrected to be readable by everyone.
Line 320-321: R. sativum and L. sativa, please write the full name of genus of this plant.
Our response:
Thank you for your suggestions. The full names of the plants are given where indicated.
Line 323: what if the finding of the study reported by “Wang et al. [47] studying the effect of KET on rice seedlings”
Our response:
Wang et al. [47], studying the effect of KET on rice seedlings, showed that low concentrations of drugs slightly stimulate plant growth, while high concentrations can significantly inhibit growth by reducing biomass and destroying roots.
We understand that the complexity of a sentence makes it difficult to understand. This sentence has been corrected for better readability. Please see the revised manuscript.
Line 351: PS I and PSII ???
Our response:
PSI and PSII are photosystems that occur in chloroplasts. They play complementary roles. The PSI center absorbs rays with a wavelength of up to 700 nm and is located on the stroma thylakoids, while the PSII absorbs radiation with a wavelength of up to 680 nm and is located on the grana thylakoids.
In order to emphasize what exactly the manuscript is about, the term "PSI and PSII systems" has been changed to the more precise "PSI and PSII photosystems".
Table 3: Eexplain the meaning of abbreviation used in this table and also put the dash between chlorophyll and it type like Chl-a or Chl-b.
Our response:
At the suggestion of the reviewer, hyphens and explanations of all abbreviations in the table have been included in the manuscript.
In column 5 and 7: Chla/Chlb and Chl(a+b)/Car. what does mean division or ratio?
It would be good to reader if author provide summary of each drug individually for each column.
Our response:
Thank you for your suggestion. According to the Reviewer's comment, each of the tested parameters regarding the content of assimilation pigments was discussed separately. Please see the revised manuscript.
Line 388: ….ibuprofen and naproxen for Atriplex patula, spinach and lettuce….Please follow one kind of pattern, either put the botanical name or common English name of studied plants.
Our response:
Thank you for your suggestion. The method of giving plant names has been standardized. All three plant names are given as botanical names. Please see the revised manuscript.
Line 399: MDA????
Our response:
The abbreviation MDA stands for malondialdehyde. This is currently explained in the abstract, in the Materials and methods chapter on the determination of this parameter. However, as a reminder, in the first line of the chapter on the discussion of the obtained results, we explain this abbreviation again. So, it seems to me that the abbreviation itself can be used in the indicated line. Of course, if the reviewer or editor sees the need for further development, we will make this change to the manuscript immediately.
Line 408-411: There is lack of clarity in the caption of figure 5, neither in figure cation nor in graph differentiated or explain that in figure 5 which graph for which parameter, it should be discussed. Please clearly describe which graph for which parameter. Also, in caption of the figure 5 only mention proline while in graph its mentioned as free proline. Please correct it accordingly.
Our response:
According to the Reviewer's comment, all graphs were marked with letters so that it was clear from the individual graphs and the description below which graph shows which parameter. The caption under the figure for proline has also been corrected and is now consistent with what is shown in the graph. Please see the revised manuscript.
Line 425: correct the chemical formula of hydrogen peroxide
Our response:
We apologize for the mistake. The H2O2 formula has been corrected.
Line 426: ROS???
Our response:
Sorry for the oversight. ROS stands for Reactive Oxygen Species. This abbreviation has been explained in the manuscrypt in the indicated place.
Line 444-446: where is the results of linear corelation.
Our response:
These results are presented in Figure 5d. This was indicated at the end of the paragraph on the impact of individual NSAIDs on spring barley seedlings.
The graph shows that the AsA content in spring barley seedlings increases with the increase in KET concentration. At the same time, we agree that the use of the term linear correlation was rather unfortunate, so it has been removed.
Line 448-435: In whole MS authors have been not provide the data of all the parameters studied just simple discussed why?
Our response:
The results regarding the content of ascorbic acid are presented in Figure 5d. Because for each of the tested NSAIDs, this parameter changed differently with increasing concentration, which, according to the authors, required a more thorough analysis when describing these results. If the reviewer or editor deems it redundant or inappropriate, we will rewrite the passage immediately.
Line 471-472: not clear
Our response:
We apologize for the unclear wording of the sentence. It has been corrected to make it more readable. Please see the revised manuscript.
Line 481-483: which drugs a? and how much quantity of it used???
Our response:
Stuchlikova et al. [65] studied the effect of flubendazole and fenbendazole on Plantago lanceolata. Sosua et al. [64] studied the effect of diclofenac on Solanum lycopersicum L. This information was also included in the manuscript.
Table 4: Again, nowhere discussed the full form of abbreviation used in this table why???
Our response:
All the abbreviations of the tested enzymes are explained earlier in the Materials and methods section. However, as a reminder, we provide an explanation of them below the table. Please see the revised manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The article requires correction. Clamps are necessary in the methods section (see comment in the text). The main problem the article is not quite suitable scale of drag concentrations 0, 0.1, 10, 50, 100, 1000 mg/kg . Most of the consideration effects are negligible in the range 0.1 -100 mg/g and too hight at 1000 mg/kg (mortality 100 % - see table 1). The best way to improving the article to add extra concentration poins in rang 100-1000 mg/kg.
Comments for author File: Comments.pdf
Author Response
Dear Reviewer,
We are very grateful for all the comments related to our manuscript. We have taken an attitude towards the suggestions and comments of the Reviewers, which was included in the text below. According to the suggestion of Reviewer 1, the linguistic correction of the manuscript was made, which was confirmed by a relevant certificate. In accordance with the reviewers' indications, changes were made in the chapters Introduction, Results and discussion, and References. We also inform that all changes in manuscript are marked in red.
Your sincerely,
Barbara Pawłowska
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Reply to the Reviewer’s 2 Report
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The article requires correction. Clamps are necessary in the methods section (see comment in the text). The main problem the article is not quite suitable scale of drag concentrations 0, 0.1, 10, 50, 100, 1000 mg/kg . Most of the consideration effects are negligible in the range 0.1 -100 mg/g and too hight at 1000 mg/kg (mortality 100 % - see table 1). The best way to improving the article to add extra concentration poins in rang 100-1000 mg/kg.
Our response:
Thank you for all your suggestions. The article was revised according to the reviewer's comments. The concentration scale chosen by the authors is not accidental. Research on plants was conducted on the basis of the PN-EN ISO 11269-2 standard and the OECD/OCDE 208/2006 guide. There are strict conditions under which such studies should be carried out and what concentrations should be tested in order to determine the effect of compounds on plants. In order to be able to compare the results obtained for different organisms, the same concentrations were used for the remaining tested organisms.
Of course, we agree with the Reviewer that between concentrations of 100 mg/kg of soil DW and 1000 mg/kg of soil DW there is a large gap in the soil so it is not possible to observe exactly at what concentration a given drug starts to affect the test organisms, especially when only the highest concentration used is toxic. It should be noted, however, that currently NSAIDs are detected in the environment in concentrations ranging from a few nanograms to hundreds of micrograms per liter. The highest level of acetylsalicylic acid was recorded in municipal sewage and amounted to 1.407 mg/l (Sim, W.-J., Lee, J.-W., Lee, E.-S., Shin, S.-K., Hwang , S.-R., and Oh, J.-E., Occurrence and distribution of pharmaceuticals in wastewater from households, livestock farms, hospitals and pharmaceutical manufactures, Chemosphere, 2011, 82, 179–186.). Mussa et al. (2022) (Mussa, Z. H. et al., A Comprehensive Review for Removal of Non-Steroidal Anti-Inflammatory Drugs Attained from Wastewater Observations Using Carbon-Based Anodic Oxidation Process, Toxics, 2022, 10,598) report that the average concentrations of selected NSAIDs vary greatly between countries, ranging from 3992 to 27,061 µg/L (influent wastewater) and 1208 to 7943 µg/L (effluent wastewater). Therefore, most of the articles on the effects of NSAIDs on plants concerns very low concentrations of drugs that are observed in the environment. Most researchers consider it unnecessary to conduct research for such high concentrations as 1000 mg/kg of soil DW. In the studies presented by the authors, we also focused on lower concentrations of the tested drugs. A concentration of 1000 mg/kg of soil DW is also shown, which is indicated in the literature as the limit value for plant toxicity. According to the guidelines and standards, if the tested compound is not toxic at this concentration, it is considered non-toxic to plants. At the moment, there are no studies that would indicate such a huge content of NSAIDs in the environment. However, the authors wanted to draw attention and thus emphasize the fact that if the presence of drugs in the environment is not strictly controlled, their accumulation may occur and concentrations may be reached that will have a negative impact on the environment, and consequently on animals and humans.
What kind of soil? Where was sampling? Minimal characteristic: pH, C-contain?
Our response:
The soil used in the experiment was clayey sand, with about 11% fraction content of < 0.02 mm in diameter, organic carbon – 8.5 g kg−1 and pH(KCl) equal to 6.0.
Did take account of leaching of IBU and KET from pots? How you can sure that drags are not wash off from soil before palant germination?
Our response:
Watering was applied in such a way as to maintain the field water capacity at about 70%. Each of the pots stood on a separate stand and at no stage of the research (even during watering) water was not observed on the stands. This is controlled during the trials so as not to lead to leaching of the study drugs. The flow of water from the pot to the trays could leach the drugs and thus distort the test results.
The method of watering the tested plants is the result of many years of research experience. The tested NSAIDs are relatively poorly soluble in water, which additionally hinders their leaching from the soil. With complete certainty, the authors can therefore certify that there was no leaching of the tested compounds from the soil. As proof, below is an example photo of one of the pots during cultivation.
In the all cases a decrease of CFU on 14th day can be explained by leaching the drags
Our response:
As stated in the previous commentary, there was no leaching of drugs during the study, so this cannot be a justification for the obtained test results.
The problem with leaching of drugs or other tested compounds exists in field studies and is difficult to control and may affect the results obtained, especially in the case of compounds that are highly soluble in water. Research conducted in pots can be very strictly controlled, thanks to which we can completely exclude leaching of compounds from the soil.
How did you distinguish degradation from leaching?
Our response:
In order to distinguish degradation from leaching, a detailed study of the soil in which the plants grew and the plants themselves (both above-ground parts of plants and roots) should be carried out to determine how much drugs were taken up by them and how many drugs remained in the soil. When conducting this type of research, it is also necessary to take into account and determine the content of compounds that are formed as a result of the degradation of the tested drugs.
That is why it is so important to conduct research under strictly controlled conditions, and not only in field crops. In natural conditions or in field crops, we have no influence on many natural phenomena that have a huge impact on the cultivation of plants (i.e. leaching of compounds from the soil, temperature, lighting or various types of biotic factors). In controlled conditions, we have an impact on temperature, humidity and many other factors that we cannot control in natural conditions. If we carry out the research in pots and conduct them in such a way that there is no leaching, we can focus on what happens to the tested compounds and what effect they have on plants, for example.
Unsuccessfully selected the scale at 100 mg kg-1 effect negligible or low but at 1000 very hight. Need corrected the expirement.
Our response:
The response to this Reviewer's suggestion is provided under the reviewer's first comment on this paper.
Numbers should be adequately rounded 10.401 +/-0.044 => 10.4 +/-0.04
Our response:
Thank you for your suggestion. Numbers have been rounded as suggested by the Reviewer. Based on this method of rounding the results, the authors also rounded the results regarding POD and SOD activity (Table 4). Please see the revised manuscript.
Dimensions?
Our response:
Sorry for the oversight. In fact, we did not specify the units in which the activity of individual enzymes is expressed. The activity of catalase, guaiacol peroxidase and ascorbate peroxidase is expressed in units of U mg-1 protein min-1, while the activity of superoxide dismutase is expressed in U mg-1 protein. This was also stated in the manuscript. Please see the revised manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Hello Author's
Appreciate your effect to pay attention to reviewer comments. I would be in case of authors briefly elaborate some of the important parameters methodologies under the materials and methods sections.
Also, improve some of the data interpretation within the text such as the authors mentioned in the first reviewer's response that " The results regarding the content of ascorbic acid are presented in Figure 5d. Because for each of the tested NSAIDs, this parameter changed differently with increasing concentration, which, according to the authors, required a more thorough analysis when describing these results. If the reviewer or editor deems it redundant or inappropriate, we will rewrite the passage immediately."
Also, suggest if possible provide a table based on your fining and previous studies reported by various researchers around the globe. it would be good for readers.
Author Response
Thank you very much for your valuable suggestions. The materials and methods chapter was prepared in such an abbreviated version due to problems with plagiarism. It is very difficult to describe the methodology in detail for the twentieth or thirtieth time so that the description is not a self-plagiarism of earlier own works and at the same time does not constitute a copy of the methodology of other authors' works, especially if generally known and used methodologies of individual assays are used.
However, in accordance with the reviewer's recommendation, the methodology for determining the factors that have been described very briefly has been extended.
We've also made some changes to the Results and Discussion section.
All changes made are marked in green. Please see the revised manuscript.
In this article, we did not want to include any aggregate information on our own research or research from other authors on the effect of drugs, e.g. about AsA. We wanted readers to focus in this work on the results we obtained and their analysis. This is intended to be a research paper, not a review. A collective summary of our own and other authors' results regarding the effect of drugs on plant growth and development and the oxidative stress they cause will be presented in a review article that we are currently preparing. So please be patient and we invite you to read our next article.
Author Response File: Author Response.pdf
Reviewer 2 Report
Accept in present form
