Influence of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) on Photosynthetic Parameters and Secondary Metabolites of Plants from Fabaceae Family
, Andreea Lupitu, Dana Maria Copolovici and Lucian Copolovici *
Round 1
Reviewer 1 Report
Dear Editor and Authors,
The manuscript is well-written and presents an important approach related to emerging contaminant impacts. I consider that after a major review the study is ready for publication in Applied Sciences.
These compounds are ionizable and their mobilization in the environment depends on the pH of the soil solution. Authors must present, at least in the introduction, information on the physicochemical specificity of these compounds and their relationship with soil transport.
I suggest an integrative analysis (e.g. principal component analysis) to clearly point out which species was most affected.
Minor comments
Line 45: rivers
Topic 2.1: how many replicas were included? How many plants were included per replicate?
Line 83: Were the experiments conducted for 6 weeks?
Topics 2.3 and 2.4: please briefly describe the methods
Figure 1: Y-axis title should be revised
Lines 232-233: I suggest including the equivalent concentration in mg L-1
Line 264: standardize mass fraction in mg L-1
Figure 9: describe Y-axis
Author Response
The manuscript is well-written and presents an important approach related to emerging contaminant impacts. I consider that after a major review the study is ready for publication in Applied Sciences.
Q: These compounds are ionizable and their mobilization in the environment depends on the pH of the soil solution. Authors must present, at least in the introduction, information on the physicochemical specificity of these compounds and their relationship with soil transport.
A: We added the phrases in the introduction of MS: "The soil and groundwater contamination level with drugs depends on soil properties (such as pH, organic matter content, and clay content) and physic-chemical properties of the medicines and their metabolites [5,6]. All NSAIDs have a low acidic character (as their pKa is 4.5 for indomethacin, 4.15 for diclofenac and naproxen, and 9.38 for paracetamol) may dissociate in solution [7]. As logkOW are ranged from 0.46 for paracetamol to 4.27 for indomethacin, the transport of those drugs in the soil is mainly ruled by the absorption-desorption mechanism [7]".
Q: I suggest an integrative analysis (e.g. principal component analysis) to point out which species was most affected clearly.
A: We add the PCA plot in the discussion part
Minor comments
Q: Line 45: rivers
A: Corrected
Q: Topic 2.1: how many replicas were included? How many plants were included per replicate?
A: We added the required information
Q: Line 83: Were the experiments conducted for 6 weeks?
A: We used 3 weeks of plant old, and the experiments were conducted for three weeks. We explain in detail the experiment setup.
Q: Topics 2.3 and 2.4: please briefly describe the methods
A: Information has been added.
Q: Figure 1: Y-axis title should be revised
A: Corrected
Lines 232-233: I suggest including the equivalent concentration in mg L-1
A: Corrected
Line 264: standardize mass fraction in mg L-1
A: Corrected
Figure 9: describe Y-axis
A: Corrected
Reviewer 2 Report
It is now known that there is a possibility of release of the active ingredient of drugs into the environment, especially diclofenac. This article investigated the effect of Nonsteroidal anti-inflammatory medications (NSAIDs) on plants of the Fabaceae family. Diclofenac, indomethacin, naproxen and paracetamol were tested. Drugs (NSAIDs) are toxic to ecosystems The parameters of photosynthesis were chosen as the subject of the study.
The authors of the study attribute the decrease in plant synthesis of chlorophyll, carotenoids, polyphenols and flavanoids, as well as increased volatile emissions to the effects of NSAIDs.
As a wish for future work, in our opinion, it would be useful to do the following.
1. A dose-response relationship on any one plant for any one indicator, with at least one of the drugs, should have been cited as evidence of this relationship.
2. As a result of this work, we should have given specific average values of decrease/increase in photosynthesis and estimates of how significant this is for plants in terms of phytoproductivity and phytomass.
In general, the study is quite interesting and the results can be published
Author Response
It is now known that there is a possibility of release of the active ingredient of drugs into the environment, especially diclofenac. This article investigated the effect of Nonsteroidal anti-inflammatory medications (NSAIDs) on plants of the Fabaceae family. Diclofenac, indomethacin, naproxen and paracetamol were tested. Drugs (NSAIDs) are toxic to ecosystems The parameters of photosynthesis were chosen as the subject of the study.
The authors of the study attribute the decrease in plant synthesis of chlorophyll, carotenoids, polyphenols and flavanoids, as well as increased volatile emissions to the effects of NSAIDs.
As a wish for future work, in our opinion, it would be useful to do the following.
Q1. A dose-response relationship on any one plant for any one indicator, with at least one of the drugs, should have been cited as evidence of this relationship.
A: In our previous studies, we have been shown the relationship between the dose and response (Monica Tăşchină, Dana Maria Copolovici, Simona Bungău, Andreea Lupitu, Lucian Copolovici, Ciprian Iovan, The influence of residual acetaminophen on beans (Phaseolus vulgaris L.) plants secondary metabolites, Farmacia, 2017, 65, 709-713; 56. Lucian Copolovici, Daniela Timiş, Monica Tăşchină, Simona Bungău, Gabriela Cioca, Dana Copolovici, Diclofenac influence on photosynthetic parameters and volatile organic compounds emission from Phaseolus vulgaris L. plants, Revista de chimie, 2017, 68, 2076-2078)
- As a result of this work, we should have given specific average values of decrease/increase in photosynthesis and estimates of how significant this is for plants in terms of phytoproductivity and phytomass.
In general, the study is quite interesting and the results can be published
A: Thank you for your comments and recommendations. In our future work, we will try to assess the influence of photosynthesis on mass plant production.
Reviewer 3 Report
My comments are below.
line 3 title: on plants, changed to, on the growth and secondary metabolites of plants. That is, a more meaningful title may be: Influence of nonsteroidal anti-inflammatory drugs (NSAIDs) on photosynthetic parameters and secondary metabolites of plants from Fabaceae family.
line 37: In contract, changed to, Recently.
line 54: diclofenac, changed to, paracetamol.
lines 57-58: remove whole sentence "These .... [24]".
line 61: at the end of sentence, please add a reference [xx]. Or if it is [25] then remove "In contract" in line 62, and continue the paragraph to [25] in line 63.
line 71: 10-3 mol. changed to, 10-3 mol
line 76: references [29,30] are for the effect of diclofenac on Phaseolus vulgaris L only. Please cite a reference for the effect of paracetamol on Phaseolus vulgaris L. If not, please remove paracetamol.
line 76: "low drug concentrations", please specify how low, what are the concentrations.
line 80: diclofenac was studied by the authors in references [29,30], please explain why it was included here, or remove it.
lines 86-87: please explain "field capacity".
line 87: "The treatments were started 21 days after sowing the seeds". So the plants came from seeds? but line 83 stated, "Experiments were carried out ... using six weeks of plants". Which one is correct? Please clearly explain to the reader the day NSAIDs exposure began, and the day samples were collected for experimental measurements. Please explain too why the concentration 0.5 mg/L solution (100 ml) used to water plant was reported as the drug concentration in the results section? How many pot plants of each species were used for each group measurement (replicates).
lines 88-89: four NSAIDs (diclofenac, paracetamol, naproxen, and indomethacin) here are different from that in line 80. please explain the discrepancy. Why was paracetamol was used here, as a positive control? Why was ketoprofen in line 79 dropped and not studied and reported here?
line 144: Pisum sativum (a), Lens culinaris (b), Vicia faba (c), and Cicer arietinum (c), changed to, Pisum sativum (i), Lens culinaris (ii), Vicia faba (iii), and Cicer arietinum (iv). The same for all other figures.
lines 144-145: treated with 0.5 mg/L NSAIDs, changed to, treated with watering 100 ml of 0.5 mg/L NSAIDs every second day to 0.5 L plant growing pots for xxx days. Please change the same in all other figures. Please show the replicates used for each treatment. Please provide all original experimental data in a table form as supplemental data. Same for all other figures.
lines 229-300, Discussion section: Plesae revise accordingly based on the changes above.
line 302: three significant organic contaminants. please specify the names of three significant organic contaminants. Please explain why the fourth was not included.
Author Response
line 3 title: on plants, changed to, on the growth and secondary metabolites of plants. That is, a more meaningful title may be: Influence of nonsteroidal anti-inflammatory drugs (NSAIDs) on photosynthetic parameters and secondary metabolites of plants from Fabaceae family.
A: Thank you very much for the suggestion. We changed the title.
line 37: In contract, changed to, Recently.
A: Corrected
line 54: diclofenac, changed to, paracetamol.
A: Corrected
lines 57-58: remove whole sentence "These .... [24]".
A: Corrected
line 61: at the end of sentence, please add a reference [xx]. Or if it is [25] then remove "In contract" in line 62, and continue the paragraph to [25] in line 63.
A: Corrected
line 71: 10-3 mol. changed to, 10-3 mol
A: Corrected
line 76: references [29,30] are for the effect of diclofenac on Phaseolus vulgaris L only. Please cite a reference for the effect of paracetamol on Phaseolus vulgaris L. If not, please remove paracetamol.
A: Corrected
line 76: "low drug concentrations", please specify how low, what are the concentrations.
A: Corrected
line 80: diclofenac was studied by the authors in references [29,30], please explain why it was included here, or remove it.
A: The references in which reported studies of two of the NSAIDs (diclofenac and acetaminophen (paracetamol)) are considered important for the present study, which continuous our research with different plants species.
Q: lines 86-87: please explain "field capacity".
A: corrected with pot capacity
Q: line 87: "The treatments were started 21 days after sowing the seeds". So the plants came from seeds? but line 83 stated, "Experiments were carried out ... using six weeks of plants". Which one is correct? Please clearly explain to the reader the day NSAIDs exposure began, and the day samples were collected for experimental measurements. Please explain too why the concentration 0.5 mg/L solution (100 ml) used to water plant was reported as the drug concentration in the results section? How many pot plants of each species were used for each group measurement (replicates).
A: We added more information, and now it should be clearly
Q: lines 88-89: four NSAIDs (diclofenac, paracetamol, naproxen, and indomethacin) here are different from that in line 80. please explain the discrepancy. Why was paracetamol was used here, as a positive control? Why was ketoprofen in line 79 dropped and not studied and reported here?
A: We corrected the mistake from L 79. Thank you. The study drugs have been diclofenac, naproxen, indomethacin and paracetamol.
Q: lines 144-145: treated with 0.5 mg/L NSAIDs, changed to, treated with watering 100 ml of 0.5 mg/L NSAIDs every second day to 0.5 L plant growing pots for xxx days. Please change the same in all other figures. Please show the replicates used for each treatment. Please provide all original experimental data in a table form as supplemental data. Same for all other figures.
A: We added the require information. The raw data is presented in S1.
Q: lines 229-300, Discussion section: Plesae revise accordingly based on the changes above.
A: Revised.
Q: line 302: three significant organic contaminants. please specify the names of three significant organic contaminants. Please explain why the fourth was not included.
A: The fourth contaminant has been added.
Round 2
Reviewer 1 Report
No comments.
Author Response
No comments.
A: Thank you very much.
Reviewer 3 Report
Line 39, ) may, changed to ) and may
Line 39, logkow, changed to log Kow
line 42, For example, delete it
line 43, have, changed to has
line 53, also, delete it
lines 97-98, “Three plants in different pots were used for every experimental group. All experiments have been performed in triplicates.” Does it mean that in each drug treatment group or control group, 3 plants of the same species were used, that is, 3 independent measurements and each independent measurement was quantified 3 times (3 replicates)? However, in all your figures, those data were all from 4 independent measurements and the number of replicates in each independent measurement was not stated. This is why the original review requested the authors to submit all original data to be published as supplemental data. The author did submit a table of supplemental data, but this table of data was used for PCA analysis in Figure 10. Was the data in the table also used for Figures 1, 2, 5, 6 and 7? If so, why did not provide the data in a table for Figures 3, 4, 8 and 9?
Line 151, 2.1, changed to 3.1. Likewise, please use 3.2 to 3.9 for the results in Figures 2 to 9, respectively.
Line 154, 0.5 g/L NSAIDs, this is 1000 times of 0.5 mg/L in the method section. Which concentration is correct and was actually used?
Lines 161-162, Figure 1, “Plants treated with 0.5 mg L-1 NSAIDs every second day. The plants have been treated for ten days.”, changed to “Plants in 0.5 L pot filled with commercial garden soil were treated by watering 100 ml of 0.5 mg/L NSAIDs every second day over a total of 20 days.” Please change the same for Figures 2-10.
Line 255, Discuss section. It would be good to discuss if the concentration of NSAIDs affects the results in Figures 1-9 or not. Considering the vast concentration difference reported in the literature, the discussion on the concentration factor is important. And the authors also need to justify why only a single point of concertation 0.5 mg/L was selected and used for 4 different NSAIDs. By specifically stating that “Plants in 0.5 L pot filled with commercial garden soil were treated by watering 100 ml of 0.5 mg/L NSAIDs every second day over a total of 20 days”, readers can readily work out the actual amounts of drugs which were exposed to the plants. This is very important to be stated in Figures 1-10.
Lines 318-319: oxidative short-time stress, changed to short-time oxidative stress.
Line 331, Figure 10: The advantage of PCA is the inclusion of all data. Please state why only data on Stomatal Conductance to water vapour, Assimilation rate, Chlorophyll a, Chlorophyll b, and Beta-carotene were included. What would be the outcome if all data were included in the analysis as Figure 11? Please discuss.
Author Response
Line 39, ) may, changed to ) and may
A: Corrected
Line 39, logkow, changed to log Kow
A: Corrected
line 42, For example, delete it
A: Corrected
line 43, have, changed to has
A: Corrected
line 53, also, delete it
A: Corrected
lines 97-98, “Three plants in different pots were used for every experimental group. All experiments have been performed in triplicates.” Does it mean that in each drug treatment group or control group, 3 plants of the same species were used, that is, 3 independent measurements and each independent measurement was quantified 3 times (3 replicates)? However, in all your figures, those data were all from 4 independent measurements and the number of replicates in each independent measurement was not stated. This is why the original review requested the authors to submit all original data to be published as supplemental data. The author did submit a table of supplemental data, but this table of data was used for PCA analysis in Figure 10. Was the data in the table also used for Figures 1, 2, 5, 6 and 7? If so, why did not provide the data in a table for Figures 3, 4, 8 and 9?
A: We used four pots with plants for every drug treatment. Because the plants have a limited number of leaves with the same ages, we made some determinations with three replicates or with four replicates, as it is written under the figures. For clarity, after every figure, we added the number of replicates used for the experiments.
We added in Table S1 all raw data. For figure 10 and figure 11 the data presented in Figure 1-9 were used.
Line 151, 2.1, changed to 3.1. Likewise, please use 3.2 to 3.9 for the results in Figures 2 to 9, respectively.
A: Corrected
Line 154, 0.5 g/L NSAIDs, this is 1000 times of 0.5 mg/L in the method section. Which concentration is correct and was actually used?
A: Corrected
Lines 161-162, Figure 1, “Plants treated with 0.5 mg L-1 NSAIDs every second day. The plants have been treated for ten days.”, changed to “Plants in 0.5 L pot filled with commercial garden soil were treated by watering 100 ml of 0.5 mg/L NSAIDs every second day over a total of 20 days.” Please change the same for Figures 2-10.
A: Corrected
Line 255, Discuss section. It would be good to discuss if the concentration of NSAIDs affects the results in Figures 1-9 or not. Considering the vast concentration difference reported in the literature, the discussion on the concentration factor is important. And the authors also need to justify why only a single point of concertation 0.5 mg/L was selected and used for 4 different NSAIDs. By specifically stating that “Plants in 0.5 L pot filled with commercial garden soil were treated by watering 100 ml of 0.5 mg/L NSAIDs every second day over a total of 20 days”, readers can readily work out the actual amounts of drugs which were exposed to the plants. This is very important to be stated in Figures 1-10.
A: In our previous studies, we have been shown the relationship between the dose and response (Monica Tăşchină, Dana Maria Copolovici, Simona Bungău, Andreea Lupitu, Lucian Copolovici, Ciprian Iovan, The influence of residual acetaminophen on beans (Phaseolus vulgaris L.) plants secondary metabolites, Farmacia, 2017, 65, 709-713; 56. Lucian Copolovici, Daniela Timiş, Monica Tăşchină, Simona Bungău, Gabriela Cioca, Dana Copolovici, Diclofenac influence on photosynthetic parameters and volatile organic compounds emission from Phaseolus vulgaris L. plants, Revista de chimie, 2017, 68, 2076-2078). Anyway, in these papers, we used very high concentrations of drugs. In the present study, only one concentration was chosen, but the drugs are at the environmental level.
Lines 318-319: oxidative short-time stress, changed to short-time oxidative stress.
A: Corrected
Line 331, Figure 10: The advantage of PCA is the inclusion of all data. Please state why only data on Stomatal Conductance to water vapor, Assimilation rate, Chlorophyll a, Chlorophyll b, and Beta-carotene were included. What would be the outcome if all data were included in the analysis as Figure 11? Please discuss.
A: We added all data and made figure 11.
