Antioxidant Responses and Adaptation Mechanisms of Tilia tomentosa Moench, Fraxinus excelsior L. and Pinus nigra J. F. Arnold towards Urban Air Pollution

Round 1

Reviewer 1 Report

The manuscript presented for review concerns the topic antioxidant responses and adaptation mechanisms of Tilia tomentosa Moench., Fraxinus excelsior L. and Pinus nigra J.F. Arnold towards urban air pollution. It is a current topic, although it has been researched for a longer time.

In general, the manuscript requires changes and additions.

I put my comments below:

I'm not sure if urban forestry fits in the keywords.

Neither the map (or rather a drawing / diagram, as it does not meet the requirements of a cartographic map) nor the text contains a description of the size of the selected plots. Are they plots or rather points? What is the surface area?

There is no information on the age of the seedlings used in the experiment.

My doubts are to use only 3 individuals of each species in each plot. I do not think that it is possible to draw correct conclusions from the research on such a small number of specimens.

The charts presented in the results are not very legible and are not self-explanatory.

In the results of GPX activity (point 3.3) there is an element of discussion that should be included in the Discussion (line 261-266).

The same comments apply to point 3.4.

Author Response

Response to Reviewer 1

 

We would like to sincerely thank all the reviewers and editor for your comments and suggestions, which helped us to greatly improve our paper. All the comments and suggestions from you have been considered carefully. The detailed responses to the reviewers are given as follows.

1. I'm not sure if urban forestry fits in the keywords.

Response: Thank you for your professional comment. “Urban forestry” was replaced by “Urban trees”

 

2. Neither the map (or rather a drawing / diagram, as it does not meet the requirements of a cartographic map) nor the text contains a description of the size of the selected plots.Are they plots or rather points? What is the surface area?

Response: Thank you for this question. The surface area of each plot was added in the site’s description

 

3. There is no information on the age of the seedlings used in the experiment.

Response: Thank you for mentioning this important omission. The age of saplings (8-years old) was added in the text.

 

4. My doubts are to use only 3 individuals of each species in each plot.I do not think that it is possible to draw correct conclusions from the research on such a small number of specimens.

Response: Thank you for your professional comment. We would like to highlight that this is an experiment in a real urban environment, so to find a “free space” for planting trees along the main traffic arteries (especially in the central part) is a significant challenge. Furthermore, we have seeking for plots with similar soil conditions (Fluvisol with anthropogenic-influenced upper horizons), pH, sun exposure and a normal moistening regime, but with different extent to anthropogenic impact (traffic volume and intensity of urbanization). When planting, we followed the standard manual and we have to leave enough free space between saplings according to their specific requirements. Based on the abovementioned limitations, we decided to use 3 individuals per species per plot.

The sufficient number of analyses for drawing conclusions was assured by: i) multiple observations during the vegetation period; ii) prolonged experimental period (2015-2020); iii) big volume of samples (80–100 fully expanded leaves (needles) per tree are collected and a composite sample is prepared for analyses).

 

5. The charts presented in the results are not very legible and are not self-explanatory.

Response: Thank you for your professional comment. We have changed the chart type and indicated the statistical differences of data (according to the Reviewer 2 recommendation)

6. In the results of GPX activity (point 3.3) there is an element of discussion that should be included in the Discussion (line 261-266).
7. The same comments apply to point 3.4.

Response: Thank you for your professional comment. We have added some discussions for GPX activity.

Reviewer 2 Report

Urban ecosystem resilience is known to depend on the diversity and vitality of urban trees. The article deals with important issue of approaches for evaluating stress resistance of urban trees based on their  physiological and biochemical attributes. 

Authors cited references predominantly before 2016. This is unacceptable. You should focus on most recent data especially in such an intensively developing studies as resilience and adaptation of urban trees. Please, review your reference list. Your cited references should be mostly recent publications (within the last 5 years).

 

Comments to the text of the manuscript:

Line 19. Specify what you mean by "anthropogenic pressure". 

Lines 48-49 and 402. Statement "the elements of the ecological system of man - society - nature" is incorrect. It is not an ecosystem.

Line 79 "(°OH)": replace "°" by "•".

Line 85 "That's why": replace by "That is why" or "Therefore".

Lines 93-96. Statement "three of the widespread ornamental trees both in urban and rural areas" is not correct. Probably, stutied tree species are widespred in the southern cities but not everywhere. Please, specify the region. It is not obvious from the text.

Lines 96-99 "For this purpose ... have been performed": you told about this in 2.2. Experimental design. 

Lines 139-140. Is it correct? Prove this statement with exact data from soil investigation. Unlikely that soil conditions are the same in such different by intensity of urbanization plots. 

Table 1. You indicate that the sources of air pollution in the plots are from vehicle emissions. Is there any plants or factories near the plots? Specify this.

Lines 146-147. You state: "Periodically observations are made on their development, physiology and health status through 2015-2020". What development and healt parameters did you assess? Moreover, initial size of saplings is unknown. Please, provide the information.

It is not clear what data are presented in the study: means for six years of research (2015-2020) or for any particular year.

Line 158. "reading" raplace by "determination"

Line 181. Using of Student t-test requires the normal distribution of data. Indicate this if it was so.

Lines 185-186. Incorrect reference [32]. Exclude it from References. Indicate the version of the software, release year and developer name.

Lines 211-214 Is this your data? If it is not, provide the reference.

Figures 2-5 are without statistics. You should indicate the statistical differences of data. 

Tables 3-5 Is it Sperman or Pearson correlation? Specify number of observations (n = ?)

Line 321. How do you find "strong correlation"? By determination coefficients? The strength of relation between two factors is commonly determined by correlation coefficient (not by determination coefficient). 

Figure 6. These graphs indicate only 7-8 observations (dots). The above mentioned research design (lines 145-146) used 3 (individual trees) x 3 (tree species) x 4 (plots) = 36 samples. How does it compare?

 

Another some lexical/grammatical uncertainties to be revised:

Lines 97,144 etc. "greenery". Was it "nursery"? Replace this word.

Line 64. "temps of urbanization", line 66 "permanent cronic concentrations", line 69 "To address these problem", line 74 "It is obvious from many years", line 78 "an increase"

Author Response

Response to Reviewer 2

We would like to sincerely thank all the reviewers and editor for your comments and suggestions, which helped us to greatly improve our paper. All the comments and suggestions from you have been considered carefully. The detailed responses to the reviewers are given as follows.

 

Authors cited references predominantly before 2016. This is unacceptable. You should focus on most recent data especially in such an intensively developing studies as resilience and adaptation of urban trees. Please, review your reference list. Your cited references should be mostly recent publications (within the last 5 years).

Response: Thank you for your professional comment. We have changed the prevailing part of the references with more recent publications.

 

Line 19. Specify what you mean by "anthropogenic pressure". 

Response: Thank you for this question. We have used this term for description of the anthropogenic impact on trees, both worsened air and soil quality, higher albedo, higher temperature, etc., but now we replaced it with “impact”.

 

Lines 48-49 and 402. Statement "the elements of the ecological system of man - society - nature" is incorrect. It is not an ecosystem.

Response: Thank you for your professional comment. We clarified this using the term “socio-ecological system”.

 

Line 79 "(°OH)": replace "°" by "•".

Response: Thank you very much. We have done this correction.

 

Line 85 "That's why": replace by "That is why" or "Therefore".

Response: Thank you very much. We have done this correction.

 

Lines 93-96. Statement "three of the widespread ornamental trees both in urban and rural areas" is not correct. Probably, stutied tree species are widespred in the southern cities but not everywhere. Please, specify the region. It is not obvious from the text.

Response: Thank you for your professional comment. We specified that statement as valuable for Europe and cited a reference - Chorological maps for the main European woody species, Availlable at: https://www.genres.eu/results/content/31f4-chorological-maps-for-the-main-european-woody-species/

 

Lines 96-99 "For this purpose ... have been performed": you told about this in 2.2. Experimental design. 

Response: Thank you very much. The sentence was deleted from the aim.

 

Lines 139-140. Is it correct? Prove this statement with exact data from soil investigation. Unlikely that soil conditions are the same in such different by intensity of urbanization plots. 

Response: Thank you for your professional comment. We have added a short description of soil content of potentially toxic elements as well as some detailed results of chemical analyses in Table 2.

 

Table 1. You indicate that the sources of air pollution in the plots are from vehicle emissions. Is there any plants or factories near the plots? Specify this.

Response: Thank you for your professional comment. We have specified that there are no plants or factories near the plots. All industrial emitters are situated in the city’s outskirts which is more than 3 km distance by air from the experimental plots.

 

Lines 146-147. You state: "Periodically observations are made on their development, physiology and health status through 2015-2020". What development and healt parameters did you assess? Moreover, initial size of saplings is unknown. Please, provide the information.

Response: Thank you for mentioning this important omission. The age of saplings (8-years old) was added in the text.

When regarding the development and health status of trees, the following observations and analyses have been performed: 1) intensity of photosynthesis, intensity of respiration, stomatal conductance; 2) content of photosynthetic pigments; 3) content of antioxidant enzymes; 4) changes in the anatomical structure of leaves under the influence of air pollution; 5) growth of stem diameter and height; 6) visual damages as leaf injuries, chlorosis and necrosis; 7) bioaccumulation of potentially toxic elements in leaves (needles), etc. So, we have collected a large database but here we present a small part of the results obtained.

 

It is not clear what data are presented in the study: means for six years of research (2015-2020) or for any particular year.

Response: Thank you for this question. We have added that the commented results are average from the 6-year period

 

Line 158. "reading" raplace by "determination"

Response: Thank you very much. It is done.

 

Line 181. Using of Student t-test requires the normal distribution of data. Indicate this if it was so.

Response: Thank you for your professional comment. That was indicated in the Data processing subsection.

Lines 185-186. Incorrect reference [32]. Exclude it from References. Indicate the version of the software, release year and developer name.

Response: Thank you very much. It is done.

 

Lines 211-214 Is this your data? If it is not, provide the reference.

Response: Thank you for this question. These results are our own data collected in parallel with the observations of saplings in the experimental plots.

 

Figures 2-5 are without statistics. You should indicate the statistical differences of data. 

Response: Thank you for your professional comment. We have changed the chart type and indicated the statistical differences of data.

 

Tables 3-5 Is it Sperman or Pearson correlation? Specify number of observations (n = ?)

Response: Thank you for your professional comment. It is Pearson correlation and the number of observations is n=72, 3 individuals × 4 plots × 6 years. All this information was included in the tables captions.

 

Line 321. How do you find "strong correlation"? By determination coefficients? The strength of relation between two factors is commonly determined by correlation coefficient (not by determination coefficient).

Response: Thank you for your professional comment. We do not have in mind that this statement revealed from the determination coefficients. We just wanted to say that linear regression also highlighted the significant relationships between these parameters as was proved by the Pearson correlations. Here we have shown all relationships as significant at p<0.05, but some of them have been significant at p<0.001.

 

Figure 6. These graphs indicate only 7-8 observations (dots). The above mentioned research design (lines 145-146) used 3 (individual trees) x 3 (tree species) x 4 (plots) = 36 samples. How does it compare?

Response: Thank you for your professional comment. Yes, we agree that the number of observations is significantly higher – n= 216 as calculated by 3 individual trees x 3 species x 4 plots x 6 years.

We would like to explain the reason for viewing only 7 dots. In fact, we entered here not the measured values of all studied biomarkers but the proposed reference scale values from 1 to 4 (from low to very high, respectively) for both air pollution level (Table 1) and biomarker level (Table 3). By this way, twelve combinations could be obtained, i.e. “low air pollution level – low biochemical marker value” (1-1), “low air pollution level – medium biochemical marker value” (1-2), etc. So, all data entered revealed at 7 of these combinations and have overlapped. We have added some explanations of this in the text.

 

Lines 97,144 etc. "greenery". Was it "nursery"? Replace this word.

Response: Thank you very much. It was done.

 

Line 64. "temps of urbanization", line 66 "permanent cronic concentrations", line 69 "To address these problem", line 74 "It is obvious from many years", line 78 "an increase"

Response: Thank you very much. We have revised all of these.

 

Round 2

Reviewer 1 Report

The authors took into account the comments of the reviewers. The manuscript has been improved. In my opinion, it is worth adding photos from the research sites - goal: to show the specificity of the place due to anthropopressure. It would also be encouraging to show photos of the tested seedlings / trees at various stages of the experiments - whether there was any change in the morphology of the research objects. In further research, it would be worth taking microscopic photos.

Author Response

Dear Reviewer,

We want to thank you again for your valuable comments and suggestion in order to enhance the scientific significance of our manuscript. 

We have also tried to select and add in the text some photos both of the experimental plots and of the saplings. Unfortunately, we revealed that this task needs more than 12 pictures as the details should be noticed and compared. 

Thank you for this recommendation and we will follow it in the next manuscript which will concern the macroscopic traits of trees - changes in the morphology, phenology, growing rate, leaf injuries, etc. We will take some microscopic photos too. Till now, we have found some changes at the anatomy of stomata (firstly pronounced on the 3th year after planting) but we will keep regularly monitoring up to 10th year.

Reviewer 2 Report

Generally, the authors have answered to all suggestions and the manuscript is now substantially improved.

I think that this manuscript needs some minor revisions. Some points should be corrected and explained by the Authors.

 

Line 142-144 Why do you consider Mg, Fe and Mn as "potentially toxic"? Mg is major mineral constituents of the soil as well as Fe and Mn are minor elements and they do not have toxic effect for living organisms at such concentarions. Toxicity is possible at severe industrial pollution and due to specific soil conditions providing their mobility. I sugget you include in table 2 Maximum permissible concentrations (MPC) in urban soils for each element. It will be more informative.

Line 143 Underlined What was the sampling soil depth? Was it topsoil (0-30 cm) or deeper?

 

Table 2 and table 3 have the same title. Write the correct title for the table 2.

What is RSD? Specify this abbreviation. How did you calculate this? It is not clear from the text.

 

Subsection 2.3 Reagents should be described in this section (specify producer, etc.)

 

Line 491 Delete "Please add:"

 

Manuscript parts for corrections/explanations are highlighted in the pdf-file.

Author Response

Dear Reviewer,

We want to thank you again about your valuable comments and suggestions in order to enhance the scientific soundness of our manuscript.

We have made the following corrections according your review:

Line 142-144 Why do you consider Mg, Fe and Mn as "potentially toxic"? Mg is major mineral constituents of the soil as well as Fe and Mn are minor elements and they do not have toxic effect for living organisms at such concentarions. Toxicity is possible at severe industrial pollution and due to specific soil conditions providing their mobility. I sugget you include in table 2 Maximum permissible concentrations (MPC) in urban soils for each element. It will be more informative.

Thank you for this note. Firstly, we have followed the prerequisite that each element in deficit or excess could be potentially toxic. But now, based on the results obtained, we corrected as "chemical elements".

The MPC according to Bulgarian legislation was added in the table.

 

Line 143 Underlined What was the sampling soil depth? Was it topsoil (0-30 cm) or deeper?

Thank you for this professional comment. We have sampled the topsoil layer (0-30 cm) and that is now mentioned in the text. 

 

Table 2 and table 3 have the same title. Write the correct title for the table 2.

Thank you very much. We have corrected this technical error.

 

What is RSD? Specify this abbreviation. How did you calculate this? It is not clear from the text.

 Thank you for this question. RSD (Relative Standard Deviation) value was calculated as follows: RSD = 100 * Stand Dev / Mean. We prefer to use this parameter as it is more indicative for the analytical procedure accuracy. 

A short description of this parameter: The RSD tells you whether the “regular” std dev is a small or large quantity when compared to the mean for the data set. For example, you might find in an experiment that the std dev is 0.1 and your mean is 4.4. Your RSD for this set of numbers is: 100 x 0.1 / |4.4| = 2.3%.
This result tells you that your standard deviation is 2.3% of the mean of 4.4, which is pretty small. On the other hand, if your percentage was large, say, 55%--this would indicate your data is more spread out. The RSD is sometimes used for convenience but it can also give you an idea about how precise your data is in an experiment. The more precise your data, the smaller the RSD.

 

 

Subsection 2.3 Reagents should be described in this section (specify producer, etc.)

Thank you very much. Such information was added in the text.

 

Line 491 Delete "Please add:"

 Thank you very much. That was corrected. 

 

Manuscript parts for corrections/explanations are highlighted in the pdf-file.

Thank you very much. We have not found such pdf-file, so we apologize if there are some points missing.