Review Reports - Anxiety in Duckweed–Metabolism and Effect of Diazepam on Lemna minor

Round 1

Reviewer 1 Report

Dear Authors,

This is an interesting study with some interesting results. Here are a few things to consider when revising your manuscript for publication in Water:

The title is overstepping the work: only diazepam (and its spontaneously generated metabolites) was investigated in this study, other benzodiazepines on the market were not, therefore "benzodiazepines" should be replaced with "diazepam" in the title of the article
Some language issues:

-"GABA" is an acronym nowhere defined in the manuscript

-line 22: "evolutionarily" not "evolutionally"

-Line 92: "over a" not "in" ... seven-day exposure

-Decimal point should be used rather than a comma, for instance in "99,9%"

-There are several instances of inappropriate first person usage, e.g. lines 157, 275, 344: "we used", "we elevated", "we present"; please rephrase in an impersonal manner

-Plants are personified in several instances, e.g. lines 242 and 318 "treated plants' biomass" should read "biomass of treated plants" / "plants' metabolic pathways" should read "plant metabolic pathways"

Author Response

Dear Reviewer,

Firstly, thank you kindly for the time you have spent on reviewing our article, we truly appreciate your comments and suggestions, which were all incorporated into the article. We are sure these changes will make it easier and clearer for the readers to grasp. Our reaction to your comments can be found below each of your points in bold.

Dear Authors,

This is an interesting study with some interesting results. Here are a few things to consider when revising your manuscript for publication in Water:

The title is overstepping the work: only diazepam (and its spontaneously generated metabolites) was investigated in this study, other benzodiazepines on the market were not, therefore "benzodiazepines" should be replaced with "diazepam" in the title of the article

Thank you for the suggestion, you are absolutely right and we have changed the title, replacing benzodiazepines by diazepam (see page 1, line 2-3: „Anxiety in duckweed – metabolism and effect of diazepam on Lemna minor“)

Some language issues:

-"GABA" is an acronym nowhere defined in the manuscript

This is very much agreed, we have added the explanation of γ-Aminobutyric acid (GABA) abbreviation at its first occurrence in the text (see page 1, line 21). To further explain the role of GABA receptors in the mechanism of action of benzodiazepines and make it clear to any reader from other fields, we have also added a sentence: „In humans, diazepam (DIA) binds to a specific benzodiazepine binding site on GABA receptors to mediate its sedative and anxiolytic effect by opening Cl^- channels and hyperpolarizing the membrane of neuronal cells [19].“ (see page 2, lines 70 – 72).

-line 22: "evolutionarily" not "evolutionally"

Thank you for noticing, we have amended the spelling to evolutionarily (see page 1, line 24).

-Line 92: "over a" not "in" ... seven-day exposure

Indeed, the expression has been edited to „… effect of three different concentrations of diazepam on Lemna minor over a 7-day exposure and to assess the products of diazepam…“ (see page 3, line 99).

-Decimal point should be used rather than a comma, for instance in "99,9%"

Decimal point was added instead of decimal comma, as it already was the case in all other uses of decimals. It was amended on page 3, lines 106 and 116.

-There are several instances of inappropriate first person usage, e.g. lines 157, 275, 344: "we used", "we elevated", "we present"; please rephrase in an impersonal manner

To keep impersonal manner, we have replaced these expressions in all instances:

We cultivated -> Lemna was cultivated (page 1, line 13)

We measured -> parameters were assessed (page 1, line 14)

To detect, we used… -> benzodiazepines were detected and quantified by… (page 4, line 167)

We evaluated -> levels were evaluated (page 8, line 293)

We have detected -> all three metabolites were detected (page 10, line 339)

We present -> findings are presented (page 10, line 364)

-Plants are personified in several instances, e.g., lines 242 and 318 "treated plants' biomass" should read "biomass of treated plants" / "plants' metabolic pathways" should read "plant metabolic pathways"

Once again, thank you very much for noticing, all instances were edited (see page 7, line 258 and page 10, line 337).

Reviewer 2 Report

The presented work is devoted to a relevant topic, a laboratory study of the possible consequences of pollution of aquatic ecosystems by the medical drug diazepam by analyzing its effect on the growth and biochemical parameters of the Lemna minor model plant. The text of the manuscript is well structured, the objectives of the study and conclusions are clearly formulated.

A number of major comment s can be made on the work.

1) There is no description of the statistical data analysis procedure in the methods section. Although the results of statistical evaluations are presented in some of the results section. For example: chapter "3.3 Fronds growth" - α = 0.008; d = 3.75; chapter "4.3 Photosynthesis" - P ≤ 0.05. It is completely unclear how these figures were obtained, what methods and programs were used for the calculations. All descriptions of statistical procedures and programs must be added into a manuscript.

2) In the figures of the bar charts, the “whiskers” mark the scatter of the estimated values (as far as I understood the scatter of the three measurements). It is unclear whether this is a confidence interval or a spread of values from maximum to minimum. This should be clarified in the figure captions.

3) Some of the results contain statistically unsubstantiated statements about the increase and decrease in values depending on the day of cultivation. For example, Figure 1A only obvious the difference in Superoxide dismutase concentrations from the positive control only, I don't see any statistically significant differences by days of cultivation and diazepam concentrations. All statements in the result about the fall or rise of something must be statically significant. Otherwise, it must be said that there were no significant differences.

4) If the authors used Student's t test to assess the significance of differences between means, then they must prove the assumption of a normal distribution in samples of the compared values (for example, using the Shapiro-Wilk normality test). If the normality of distribution laws is not confirmed, then it is necessary to use the Wilcoxon rank sum exact test.

5) I would recommend the authors to use the PCA (Principal Component Analysis) method to visualize the results of their work. As objects for visualization, it is necessary to use samples (repeats) characterized by the parameters Superoxide dismutase levels, catalase level, levels of QYmax, Fronds growth, Mass growth, Mass per leaf, concentration of oxazepam, TEM concentration and NOR concentration. Points on the PCA biplot should be marked by baseline diazepam concentration and culture day. Analysis can be done using the R programming language as a guide (https://www.datacamp.com/community/tutorials/pca-analysis-r) or the Python programming language as a guide (https://plotly.com/python/pca- visualization/).

Minor remarks:

1) Poor quality and resolution of figures, it is necessary to increase the resolution to 300 - 600 dpi

2) The figure with the caption Figure 1 appears in the manuscript twice in the introduction section and in the results section.

3) There are no references to figures in the text of the results section in the description of the results.

After eliminating the comments, doing additional work on data analysis and re-reviewing the manuscripts, it can be published in the journal.

Author Response

Dear Reviewer,

first of all, thank you for your extensive input on the article, it is truly appreciated, and all suggested changes were incorporated. We very much appreciate your time spent on the article reviewing. All your comments have been very much welcomed and our detailed reflections can be found written in bold below your comments.

The presented work is devoted to a relevant topic, a laboratory study of the possible consequences of pollution of aquatic ecosystems by the medical drug diazepam by analysing its effect on the growth and biochemical parameters of the Lemna minor model plant. The text of the manuscript is well structured, the objectives of the study and conclusions are clearly formulated.

A number of major comment s can be made on the work.

There is no description of the statistical data analysis procedure in the methods section. Although the results of statistical evaluations are presented in some of the results section. For example: chapter "3.3 Fronds growth" - α = 0.008; d = 3.75; chapter "4.3 Photosynthesis" - P ≤ 0.05. It is completely unclear how these figures were obtained, what methods and programs were used for the calculations. All descriptions of statistical procedures and programs must be added into a manuscript.

Thank you for noticing, we have indeed forgetfully omitted the Statistics chapter. It has been corrected by adding chapter 2.7 Statistical Analysis, which reads:

„Data were evaluated using IBM SPSS Statistics software. Normal distribution in samples was assessed using Shapiro-Wilk normality test and significance was assessed by Student’s T-test, comparing sample values against the control.“ (see page 5, lines 196 – 199).

Moreover, further information related to the statistics was added (e.g., in Table 1, page 5, line 195).

In the figures of the bar charts, the “whiskers” mark the scatter of the estimated values (as far as I understood the scatter of the three measurements). It is unclear whether this is a confidence interval or a spread of values from maximum to minimum. This should be clarified in the figure captions.

Thank you kindly for the suggestion, it should truly be well explained in the text. Following sentence was added to the page 6, lines 232 – 234, and to the page 7, lines 262 – 264.

„Data represent mean over 3 repetitions, standard deviations are indicated by whiskers. Statistical significance is marked according to the T-test p-values: * for p < 0.05, ** for p < 0.01 and *** for p < 0.001.“

Some of the results contain statistically unsubstantiated statements about the increase and decrease in values depending on the day of cultivation. For example, Figure 1A only obvious the difference in Superoxide dismutase concentrations from the positive control only, I don't see any statistically significant differences by days of cultivation and diazepam concentrations. All statements in the result about the fall or rise of something must be statically significant. Otherwise, it must be said that there were no significant differences.

Of course, this is important to be very transparent, therefore, the statement „…however, differences were not statistically significant.” was added to the Results section on page 5, lines 207 and 208.

If the authors used Student's t test to assess the significance of differences between means, then they must prove the assumption of a normal distribution in samples of the compared values (for example, using the Shapiro-Wilk normality test). If the normality of distribution laws is not confirmed, then it is necessary to use the Wilcoxon rank sum exact test.

As a response to the comment you have already mentioned above, we have added a whole paragraph on statistical analysis that was unfortunately not included in the first version of the manuscript and it also includes the description of normal distribution test. Paragraph 2.7 Statistical Analysis now reads:

Thank you very much for the suggestion. While we fully agree that PCA can clearly visualize more information in case of complicated data analyses, we believe that key note and idea of the paper is to quickly communicate the message that diazepam is not ecotoxic and that it can even have stimulating effect. We are already working on developing the idea further and will surely employ PCA when more data are available, since we believe it is very useful to help reader quickly and clearly navigate through the complicated results consisting of several parameters.

Minor remarks:

Poor quality and resolution of figures, it is necessary to increase the resolution to 300 - 600 dpi

Indeed, we have increased the quality of all figures to 600 dpi.

The figure with the caption Figure 1 appears in the manuscript twice in the introduction section and in the results section.

Thank you for noticing, it has been edited and numbered correctly.

There are no references to figures in the text of the results section in the description of the results.

This is relevant suggestion making it easier for the reader to follow the results. We have added references across the whole Results section (see page 2, line 74; page 5, line 224; page 6, lines 226, 236, 244, 251; page 7, lines 267, 270, 275; page 9, line 311).

Reviewer 3 Report

The manuscript of Lamaczová et al. refers to the effects of human drugs and their metabolites on an aquatic plant. Although the data presented is relevant and forthcoming there are some issues that require clarification. First why was this aquatic plant chosen and are there any other indicators of the presence of diazepam and metabolites in the aquatic systems? Is this problem of fresh or marine environments?

Regarding the drug analysed what are the impacts on water quality globally? Are there any regulations proposed on any country?

Finally, with these results how do the authors foresee the control of this drug, impacts on water quality and on aquatic biota? Are expected future bloom episodes of this species on aquatic environments?

Author Response

Dear reviewer,

firstly, we would like to thank you for taking time and reviewing our manuscript. Your input has been of great value, and we believe it will further enhance the transmission of the information we wish to communicate to the readers via the article. Below, you can find our detailed explanations to each of your comments, which have all been considered and incorporated into the new version of the manuscript.

Q: The manuscript of Lamaczová et al. refers to the effects of human drugs and their metabolites on an aquatic plant. Although the data presented is relevant and forthcoming there are some issues that require clarification. First why was this aquatic plant chosen and are there any other indicators of the presence of diazepam and metabolites in the aquatic systems? Is this problem of fresh or marine environments?

A: Lemna minor is cosmopolitan aquatic plant species and has been widely used for ecotoxicological tests all over the world. In 2006, OECD has published Test No. 221: Lemna sp. Growth Inhibition Test in their Guidelines for the Testing of Chemicals. We have added the information into the Introduction for the sake of clarity. It now reads:

„Lemna minor, L., commonly called duckweed, is a globally cosmopolitan species of monocotyledons which is reflected by OECD by defining Test No. 221: Lemna sp. Growth Inhibition Test in the OECD Guidelines for the Testing of Chemicals [31].” (see page 3, lines 87 – 88).

Diazepam and its metabolites have been previously detected in the aquatic systems in both fresh and saline surface waters. To further explain and support the statement, specification of fresh and saline waters has been added to the Introduction (see page 2, line 78).

Q: Regarding the drug analysed what are the impacts on water quality globally? Are there any regulations proposed on any country?

A: According to the recent literature, psychopharmaceuticals are serious problem worldwide and their occurrence in the environment is clearly reflecting growing consumption. The situation is already reflected in new European regulations, diazepam is included in the Voluntary Groundwater Watch List as a part of Common Implementation Strategy For the Water Framework Directive and the Floods Directive which intends to facilitate implementation of Directives 2000/60/EC and 2006/118/EC.

Q: Finally, with these results how do the authors foresee the control of this drug, impacts on water quality and on aquatic biota? Are expected future bloom episodes of this species on aquatic environments?

A: This paper proves that diazepam does not exhibit acute toxicity toxic for representant of aquatic plants, which is commonly spread in freshwater environment. Lemna can be often found at the WWTP effluents, and our findings proved that in contrast to other pharmaceuticals, like antibiotics and cytostatics, our chosen representant of psychopharmaceuticals (diazepam) is not acutely toxic and will not cause the collapse of the Lemna population in these localities.

Round 2

Reviewer 2 Report

The authors corrected the manuscript according to all the comments I made. The work may be published in a journal.

Reviewer 3 Report

The authors have adressed to my comments and now the manuscript warrants publication.