Pillar[5]arenes as Modulators for the Glyphosate and 2,4-D Herbicidal Activity: The Effect of Self-Assembly on Phyto- and Ecotoxicity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors

Your work is interesting and presented a clear indicator about this riskly topic with use of these herbicides. My suggestion in introduction point to  add data about world application amounts Gly and 2,4, rates. Your concentration are related with rates used in the model crops of experiment?.

My comments a included in attached document. However, some suggestions are described bellow: 

Title to be more concise

Tables, figures and associated leyend must be edited. 

If methodology was included in suplementary material, this section muts be included in manuscript for a easier asociation with results. 

Figures need a better edition, particularly in those added elements (arrows, legends in graph, among other). 

Some elements from introdcution must be removed and replaced with background more associated to experiment (marked in attached file).  

Finally, authors must check englsih language with native speaker with tecnichal support in this area. 

Comments for author File: Comments.pdf

Author Response

Reviewer #1:

Dear Authors

 

Your work is interesting and presented a clear indicator about this riskly topic with use of these herbicides.

 

Response:

Dear Reviewer! Thank you very much for carefully reading and reviewing our paper! We are very pleased that you have given it such a high rating.

 

My suggestion in introduction point to add data about world application amounts Gly and 2,4, rates. Your concentration are related with rates used in the model crops of experiment?

 

Response:

 

According to literature data, the global use of Glyp and 2,4-D is estimated to be 700,000 and 150,000 tons per year, respectively [10.3390/microorganisms13071492, 10.1016/j.scitotenv.2020.137167, 10.1038/s41597-019-0169-4]. Data on the global use of Glyp and 2,4-D has been added to the manuscript.

The goal of working with minimal pesticide concentrations is to reduce the pesticide load on the environment while maintaining the desired herbicidal effect. The study aims to develop a herbicide delivery system that reduces the concentration of active ingredients, such as 2,4-D and glyphosate, while maintaining their effectiveness.

According to the manufacturer's instructions, the minimum concentration of 2,4-D in the working solution of herbicides is 2 g/L. The widely used herbicide Ballerina (August, Russia) contains 410 g/L of 2,4-D. According to the manufacturer's instructions, the working solution should be prepared by adding 1.5-3.0 liters of the product to 200-300 liters of water [https://www.pesticidy.ru/active_substance/ethylhexyl]. Since the herbicide will dilute when applied to the field and transferred to other environmental components, a concentration 10 times lower (0.2 g/L) than the working solution was used. Furthermore, sublethal concentrations of herbicides, which inhibit rather than kill plants, allow for quantitative rather than qualitative assessment of the effect (plant death/not death). Thus, the study focused on a concentration of 0.2 g/L, which is the minimum concentration for which a herbicidal effect has been demonstrated in eluate tests, according to literature.

 

Title to be more concise

 

Response:

 

The title of the manuscript has been changed to “Pillar[5]arenes as modulators for the glyphosate and 2,4-D herbicidal activity: the effect of self-assembly on phyto- and ecotoxicity”.

 

Tables, figures and associated leyend must be edited.

 

Response:

 

We have carefully checked the tables and figures, as well as their associated captions. All captions, figures and tables have been edited.

 

If methodology was included in suplementary material, this section muts be included in manuscript for a easier asociation with results.

 

Response:

 

The section 'General experimental procedures' has been moved to the 'Materials & Methods' section of the manuscript.

 

Figures need a better edition, particularly in those added elements (arrows, legends in graph, among other).

 

Response:

 

All the figures have been carefully checked. The meaning of the arrows and legends in the graph have been explained in the captions.

 

Some elements from introdcution must be removed and replaced with background more associated to experiment (marked in attached file: to large, I thing you can reduce last paragraph and more focused on study subject).

 

Response:

 

The last paragraph was shortened and rewritten to focus on the subject of the study. Some information from the last paragraph was updated and moved to previous paragraph.

 

Finally, authors must check englsih language with native speaker with tecnichal support in this area.

 

Response:

 

A professional translator has reviewed and edited the English. All changes made by the translator are highlighted in yellow

 

For what all emails, and corresponding author include a non corporative email

 

Response:

 

All the email addresses have been carefully checked. We have included the personal email addresses that the authors used for scientific correspondence. We believe it is important to use email addresses that are active and verified by the authors for future interactions with readers.

 

Line 24-25: first mentioned, complete scientific name

 

Response:

 

The abstract has been updated to include the complete names of the investigated organisms.

 

Figure 1: agregates

 

Response:

 

It was an unfortunate typo. Figure 1 has been corrected.

 

Line 235-236: scientifi names were mentiomned previously.

 

Response:

 

The necessary changes have been made in accordance with the Reviewer’s comments.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is good prepared, comprehensible, complex and with interesting data focusing to using of pillar[5]arenes as supramolecular macrocyclic molecules and for encapsulation of herbicides (2,4-dichlorophenoxyacetic acid, 2,4-D; and glyphosate, Glyp) to decrease of their ecotoxicity to freshwater algae (Chlorella vulgaris), terrestric plants (Triticum aestivum and Raphanus sativus), unicellar protist (Paramecium caudatum) and crustacea (Ceriodapnia affinis). Authors confirmed in their research that pesticide antidotes (two studied molecules decamethoxypillar[5]arene and monohydroxypillar[5]arene) could be helpful do decrease of Glyp and 2,4-D toxicity in lower food chains. Although the manuscript is well written, I have still a few recommendations and comments:

Abstract – please, avoid of abbreviated forms of used organisms. Here it should be clearly demonstrated which organisms were used in their full name. Nothing is mentioned about results on studied terrestrical plants, why?  

LID10 is not the most used lethal dose abbreviations. How did you calculate it per weight of organism that is typical reason for using dose in ecotoxicology? If you cannot calculate per weight of body than I highly recommend calculating only concentrations. In the case of study of mortality, I recommend using the standard abbreviation of lethal concentrations – LC10.

I recommend inserting the description of used methods and materials (including of origin of chemicals and their purity) in the main manuscript. It shouldn’t be completely only in Supplementary Material.

Line 64 – until here I am missing short description of basic properties of Glyp and 2,4-D about their hydrophilic properties, if they are easily dissolving in the water. It should be mentioned because then this research can be more interesting for broader part of readers due higher impact to trophic chains, aquatic organisms, etc.

Did was used some specific software to create Figure 1? If yes, it should be mentioned in the Materials and Methods in the section of e.g. Statistical analyse or similar.

I recommend inserting the reference for the sentences at lines 98-100 and 116-119.

Missing important part of manuscript that is Discussion.

Figure 3 – I recommend clarifying in the Legend for these graphs what is “additive spectrum”.

Missing statistical significance in Figure 5, Table 2, Table 3. Please, add it. Which kind of deviations are in the graphs and tables – standard deviations SD or standard error of mean SEM? Please, clarify it for all relevant values.

Table 5 – why the three numbers are in the green field in this table?

Please, carefully check the main manuscript together with Supplementary Materials and write all Latin names in italic font.

Page S7 – I highly recommend inserting also SI units together with ppm units here.  

Author Response

Reviewer #2:

The manuscript is good prepared, comprehensible, complex and with interesting data focusing to using of pillar[5]arenes as supramolecular macrocyclic molecules and for encapsulation of herbicides (2,4-dichlorophenoxyacetic acid, 2,4-D; and glyphosate, Glyp) to decrease of their ecotoxicity to freshwater algae (Chlorella vulgaris), terrestric plants (Triticum aestivum and Raphanus sativus), unicellar protist (Paramecium caudatum) and crustacea (Ceriodapnia affinis). Authors confirmed in their research that pesticide antidotes (two studied molecules decamethoxypillar[5]arene and monohydroxypillar[5]arene) could be helpful do decrease of Glyp and 2,4-D toxicity in lower food chains. Although the manuscript is well written, I have still a few recommendations and comments.

 

Response:

Dear Reviewer! Thank you very much for carefully reading and reviewing our paper! We are very pleased that you have given it such a high rating.

 

Abstract – please, avoid of abbreviated forms of used organisms. Here it should be clearly demonstrated which organisms were used in their full name. Nothing is mentioned about results on studied terrestrical plants, why?

 

Response:

 

The abstract has been updated to include the complete names of the investigated organisms.

The following sentence has been added to the abstract: “The addition of pillar[5]arenes to 2,4-D reduced the wheat (Triticum aestivum L.) germination index by 4.5-fold compared to the pure herbicide.”

 

LID10 is not the most used lethal dose abbreviations. How did you calculate it per weight of organism that is typical reason for using dose in ecotoxicology? If you cannot calculate per weight of body than I highly recommend calculating only concentrations. In the case of study of mortality, I recommend using the standard abbreviation of lethal concentrations – LC10.

 

Response:

 

Since this study involved acute toxicity tests and the test function was organism mortality, the LC₁₀ parameter can replace the LID₁₀ parameter, as the former is more widely available in the literature. In this case, the calculations will be the same. The dose-response curve for concentration versus mortality will yield the concentration that causes 10% mortality. Throughout the text, LID₁₀ has been replaced with LC₁₀.

 

I recommend inserting the description of used methods and materials (including of origin of chemicals and their purity) in the main manuscript. It shouldn’t be completely only in Supplementary Material.

 

Response:

 

The section 'General experimental procedures' has been moved to the 'Materials & Methods' section of the manuscript.

 

Line 64 – until here I am missing short description of basic properties of Glyp and 2,4-D about their hydrophilic properties, if they are easily dissolving in the water. It should be mentioned because then this research can be more interesting for broader part of readers due higher impact to trophic chains, aquatic organisms, etc.

 

Response:

 

The solubility of glyphosate in water at 25°C is 12 g/L. The solubility of 2,4-D, however, depends on the form of the compound included in the herbicide. The solubility of 2,4-D in its acid form is extremely low in water (approximately 0.54 g/L). However, it is highly soluble in ethanol (130 g/L) and diethyl ether (243 g/L). The 2,4-D dimethylamine salt is highly soluble in water at concentrations of up to 4200 g/L. 2-ethylhexyl ester of 2,4-D is poorly soluble in water but highly soluble in organic solvents, such as oils. However, commercial herbicides primarily use the ether form, which ensures more effective penetration of weed tissue. The following sentences have been added to the manuscript:

1. “Glyp is one of the most widely used herbicides. Glyphosate-based herbicides have adverse effects on a broad range of terrestrial and aquatic organisms due to their relatively high solubility in water (12 g/L at 25 °C) [https://www.pesticidy.ru/active_substance/glyphosate].”
2. “Despite the low solubility of 2,4-D (approximately 0.54 g/L) and its ester form (the most widely used form in commercial herbicides due to more efficient penetration into weeds) [https://www.pesticidy.ru/active_substance/ethylhexyl], Glyp and 2,4‑D can alter the structure of phytoplankton and periphyton communities in freshwater microcosms, reducing chlorophyll content and algal abundance [15].”

 

Did was used some specific software to create Figure 1? If yes, it should be mentioned in the Materials and Methods in the section of e.g. Statistical analyse or similar.

 

Response:

 

The authors created Figure 1 themselves using templates they had previously created in the CorelDraw program.

 

I recommend inserting the reference for the sentences at lines 98-100 and 116-119.

 

Response:

 

The necessary references have been added to lines 98–100 and 116–119, and are highlighted in green.

 

Missing important part of manuscript that is Discussion.

 

Response:

 

We are very grateful for this useful remark. It was an unfortunate typo. The original draft of the article included both the results and their discussion in Section 3. Therefore, this section has now been renamed “Results & Discussion”.

 

Figure 3 – I recommend clarifying in the Legend for these graphs what is “additive spectrum”.

 

Response:

 

The additive spectrum is the sum of the spectra of the pillar[5]arene and the herbicide.

It is used to evaluate interactions between parent compounds. If there were no interactions between the mixture's components, its optical density would coincide with the sum of the densities obtained from the individual components at the same concentrations. However, a deviation in the fluorescence intensity of the mixture (A_complex) from the additive spectrum (ΣA_mixture), ΔA = A_complex – ΣA_mixture was found for the mixture of pillar[5]arenes 1 and 2 with Glyp and 2,4-D. This deviation indicates interaction between the macrocycle and herbicide molecules, which results in a hypochromic effect. The necessary information has been added to the caption and highlighted in green.

 

Missing statistical significance in Figure 5, Table 2, Table 3. Please, add it. Which kind of deviations are in the graphs and tables – standard deviations SD or standard error of mean SEM? Please, clarify it for all relevant values.

 

Response:

 

The decision was made not to use the Mann-Whitney test to compare paired samples. The statistical analysis was redone. First, we tested for normality using the Shapiro-Wilk test. Then, we compared the significance of the differences using a one-way ANOVA followed by a post hoc test, p < 0.05. There were five replicates for the microalga and fifteen for the plants. Data are presented as mean ± SD. The figure now includes differences between groups, and the figure caption has been updated.

 

Table 5 – why the three numbers are in the green field in this table?

 

Response:

 

We compared the analyzed data as a whole for monocotyledonous (wheat) and dicotyledonous (radish) plants. The systems that scored highest overall are shaded green.

 

Please, carefully check the main manuscript together with Supplementary Materials and write all Latin names in italic font.

 

Response:

 

The manuscript and Supplementary materials have been carefully reviewed. All Latin names have been written in italics.

 

Page S7 – I highly recommend inserting also SI units together with ppm units here.

 

Response:

 

The chemical shift (δ) in NMR spectroscopy is typically expressed in parts per million (ppm) by frequency because it is calculated using the following formula:

 

δ = [ν_sample − ν_ref] / [ν_ref]

 

where ν_sample is the absolute resonance frequency of the sample, and ν_ref is the absolute resonance frequency of a standard reference compound (deuterated solvent), both of which are measured in the same applied magnetic field. Since the numerator is usually expressed in hertz and the denominator in megahertz, δ is expressed in ppm.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Paper “Pillar[5]arenes as a tool for modulating the herbicidal activityof glyphosate and 2,4-D: the effect of self-assembly and host- guest complexation on phyto- and ecotoxicity” He deals with an interesting and actual topic. The main value of the paper is the attempt to show that supramolecular systems can reduce the risk to non-target aquatic organisms while completely retaining herbicidal activity. The obtained results are significant for the potential development of ecotoxicologically safer pesticide (herbicide) formulations.

Comments

 1. Section "Materials and Methods"

The complete "Materials and Methods" section is given in the Supplementary Materials.

Authors should give main information on the equipment, methods, and physical–chemical characterization in the section "Materials and Methods", while some details  and figures could be given in the Supplementary Materials

2. The biological relevance of the chosen concentration of has not been sufficiently convincingly explained

The authors chose a concentration of 0.2 g/L for biological tests, with the explanation that this is the minimum concentration at which herbicidal properties were previously observed in elute experiments. At the same time, they themselves state that the recommended working concentrations of commercial formulations are significantly higher, 5 g/L for glyphosate and up to 2 g/L for 2,4-D. It is not clear enough is the selected concentration is agronomically realistic, nor whether the obtained effects can be transferred to practical application conditions.

The authors need to explain more clearly: why exactly 0.2 g/L is the most suitable concentration, whether that concentration corresponds to real residual, elution or application conditions, and how well these results can be extrapolated to field conditions.

3. The claim of "preserved efficiency" is partly exaggerated

The abstract and conclusion suggest that combining pillar[5]arene with glyphosate significantly reduces ecotoxicity while maintaining effectiveness against monocots. However, the results for wheat show that the macrocycles themselves have pronounced phytotoxicity: the GI for wheat is 13–14% and without herbicides, which means that the carrier is not biologically neutral towards this model organism. Also, for system 1 + Glyp the GI for wheat drops from 4 to 2, but for radish the GI is increased from 24 to 42, which means that the effect is not a universal "preservation" of herbicidal activity, but a very specific modulation depending on the species.

Due to that in the abstract, discussion and conclusion should be softened that claims. It would be more accurate to say that with certain test systems a reduction of ecotoxicity was achieved with partial preservation or selective modification of phytotoxicity, rather than general preservation of efficiency.

4. The interpretation of the mechanism remains too speculative

The authors conclude that there is no classic herbicide inclusion in the macrocyclic cavity, because there are no chemical shift changes in the 1H NMR spectra for 2,4-D, while for glyphosate NMR analysis was not possible due to solubility in DMSO-d6. They then propose that stabilization occurs through entropy-driven supramolecular ensembles. It is a possible hypothesis, but at the moment it is not sufficiently substantiated.

Since direct NMR evidence is lacking for glyphosate, claims about the nature of the interaction should be formulated more cautiously. It would be useful if the authors clearly separate: what has been experimentally proven, what inference is, and what a working hypothesis remains.

5. The bioassay design is too simple for stronger conclusions about herbicidal value

Phytotoxicity was assessed by the method on filter paper, with 10 seeds per Petri dish and measured after 72 h. It is an acceptable preliminary screening, but not sufficient to draw stronger conclusions about potential formulations for use in the field.

Missing: 1) tests on a larger range of concentrations for plants, 2) dose–response curve, 3)comparison with a commercial herbicide formulation, 4) assessment of translocation, uptake or persistence, 5) any data from the soil or substrate system. Without that, the paper should be clearly presented as a proof-of-concept study, and not as a step that is already close to a practical formulation.

6. Table 5 ("Overall score" table) is not methodologically sufficiently explained

Table 5 summarizes the changes of several parameters and derives total scores, but it is not clear enough how these scores were calculated, why all parameters are implicitly weighted equally, and whether such an approach is biologically justified. For example, it is not the same to treat GI change in a target plant and LID10 in an aquatic organism as equal contributors to a single score.

Authors should either:

to explain in detail the formula, weighting and biological logic of this scoring,

or to keep the table only as a descriptive overview without too much reliance on the "overall score".

7. Statistical reporting is insufficiently detailed

Although the Supplementary Materials state that at least two independent series with a minimum of three replicates were performed and that the Mann–Whitney U test was used, the main text lacks key information: exact n for each experiment, significance level, p-values, and clear labeling of statistically significant differences in figures and tables.

For a better quality manuscript, it would be important for the authors to:

• for each figure/table, state the number of biological and technical repetitions,
• indicate statistical significance directly on graphs,
• indicate whether they checked for multiple comparisons,
• and clarify exactly what the errors are: SD, SE or something else.

8. Terminology should be more consistent.

 In the text, the terms "complexes", "associates", "ensembles", "aggregates" and "systems" alternate. Since the authors themselves suggest that there is no classical inclusion, it would be useful to distinguish consistently:

• molecular binding/association,
• self-organization/aggregation,
• and formal host-guest complexes.

9. Table 3

In Table 3, the DMSO control for wheat and radish is not completely neutral, so it should be clarified whether all treatments are statistically compared to water or to the corresponding solvent blank.

It would be useful to point out more clearly that the DMSO control had non-trivial effects in the phytotoxicity table, as this may affect the interpretation of the results.

Author Response

Reviewer #3:

Paper “Pillar[5]arenes as a tool for modulating the herbicidal activityof glyphosate and 2,4-D: the effect of self-assembly and host- guest complexation on phyto- and ecotoxicity” He deals with an interesting and actual topic. The main value of the paper is the attempt to show that supramolecular systems can reduce the risk to non-target aquatic organisms while completely retaining herbicidal activity. The obtained results are significant for the potential development of ecotoxicologically safer pesticide (herbicide) formulations.

 

Response:

Dear Reviewer! Thank you very much for carefully reading and reviewing our paper! We are very pleased that you have given it such a high rating.

 

1. Section "Materials and Methods"

 

The complete "Materials and Methods" section is given in the Supplementary Materials.

 

Authors should give main information on the equipment, methods, and physical–chemical characterization in the section "Materials and Methods", while some details  and figures could be given in the Supplementary Materials

 

Response:

 

The section 'General experimental procedures' has been moved to the 'Materials & Methods' section of the manuscript.

 

 

2. The biological relevance of the chosen concentration of has not been sufficiently convincingly explained

The authors chose a concentration of 0.2 g/L for biological tests, with the explanation that this is the minimum concentration at which herbicidal properties were previously observed in elute experiments. At the same time, they themselves state that the recommended working concentrations of commercial formulations are significantly higher, 5 g/L for glyphosate and up to 2 g/L for 2,4-D. It is not clear enough is the selected concentration is agronomically realistic, nor whether the obtained effects can be transferred to practical application conditions.

The authors need to explain more clearly: why exactly 0.2 g/L is the most suitable concentration, whether that concentration corresponds to real residual, elution or application conditions, and how well these results can be extrapolated to field conditions.

 

Response:

 

The goal of working with minimal pesticide concentrations is to reduce the pesticide load on the environment while maintaining the desired herbicidal effect. The study aims to develop a herbicide delivery system that reduces the concentration of active ingredients, such as 2,4-D and glyphosate, while maintaining their effectiveness.

According to the manufacturer's instructions, the minimum concentration of 2,4-D in the working solution of herbicides is 2 g/L. The widely used herbicide Ballerina (August, Russia) contains 410 g/L of 2,4-D. According to the manufacturer's instructions, the working solution should be prepared by adding 1.5-3.0 liters of the product to 200-300 liters of water [https://www.pesticidy.ru/active_substance/ethylhexyl]. Since the herbicide will dilute when applied to the field and transferred to other environmental components, a concentration 10 times lower (0.2 g/L) than the working solution was used. Furthermore, sublethal concentrations of herbicides, which inhibit rather than kill plants, allow for quantitative rather than qualitative assessment of the effect (plant death/not death). Thus, the study focused on a concentration of 0.2 g/L, which is the minimum concentration for which a herbicidal effect has been demonstrated in eluate tests, according to literature.

 

3. The claim of "preserved efficiency" is partly exaggerated

The abstract and conclusion suggest that combining pillar[5]arene with glyphosate significantly reduces ecotoxicity while maintaining effectiveness against monocots. However, the results for wheat show that the macrocycles themselves have pronounced phytotoxicity: the GI for wheat is 13–14% and without herbicides, which means that the carrier is not biologically neutral towards this model organism. Also, for system 1 + Glyp the GI for wheat drops from 4 to 2, but for radish the GI is increased from 24 to 42, which means that the effect is not a universal "preservation" of herbicidal activity, but a very specific modulation depending on the species.

 

Due to that in the abstract, discussion and conclusion should be softened that claims. It would be more accurate to say that with certain test systems a reduction of ecotoxicity was achieved with partial preservation or selective modification of phytotoxicity, rather than general preservation of efficiency.

 

Response:

 

The manuscript was reviewed one more time to ensure that no unsubstantiated claims remained. The abstract and conclusion has been revised according to the Reviewer’s recommendations.

 

4. The interpretation of the mechanism remains too speculative

The authors conclude that there is no classic herbicide inclusion in the macrocyclic cavity, because there are no chemical shift changes in the 1H NMR spectra for 2,4-D, while for glyphosate NMR analysis was not possible due to solubility in DMSO-d6. They then propose that stabilization occurs through entropy-driven supramolecular ensembles. It is a possible hypothesis, but at the moment it is not sufficiently substantiated.

 

Since direct NMR evidence is lacking for glyphosate, claims about the nature of the interaction should be formulated more cautiously. It would be useful if the authors clearly separate: what has been experimentally proven, what inference is, and what a working hypothesis remains.

 

Response:

 

Thank you for your comment. Indeed, further study is required to establish the mechanism of interaction between macrocycles and herbicides. Nevertheless, interactions between pillar[5]arenes and herbicides were confirmed by fluorescence spectroscopy. Effective association constants were also determined. The absence of a classical inclusion complex formation between the macrocycle and 2,4-D was demonstrated by NMR spectroscopy. However, such an assessment was impossible for glyphosate due to its low solubility in DMSO at 10 mM. Dynamic light scattering data showed that macrocycles can aggregate in the presence of herbicides, forming stable, nanometer-sized aggregates. TEM data confirmed this finding. Based on experimental data, we suggest that the main mechanism of interaction between macrocycles and herbicides is aggregation with the following formation of supramolecular associates.

 

5. The bioassay design is too simple for stronger conclusions about herbicidal value

Phytotoxicity was assessed by the method on filter paper, with 10 seeds per Petri dish and measured after 72 h. It is an acceptable preliminary screening, but not sufficient to draw stronger conclusions about potential formulations for use in the field.

 

Missing: 1) tests on a larger range of concentrations for plants, 2) dose–response curve, 3)comparison with a commercial herbicide formulation, 4) assessment of translocation, uptake or persistence, 5) any data from the soil or substrate system. Without that, the paper should be clearly presented as a proof-of-concept study, and not as a step that is already close to a practical formulation.

 

Response:

 

This work is indeed one of the preliminary and important stages that allows to evaluate the influence of the macrocycle on the herbicidal properties and ecotoxicity of herbicides. The study developed a screening methodology for pillararene-herbicide systems, incorporating physicochemical and ecotoxicological characterizations of the resulting associates. However, we recognize that conducting the rather complex experiments outlined by the Reviewer is only feasible for the top compounds. The chemical structures will undergo further optimization, and the most promising compounds will be tested in greenhouse experiments with model weeds, such as Arabidopsis thaliana.

The following sentence has been added in the conclusion: “This study is an important step toward evaluating how pillar[5]arenes affect the herbicidal properties and ecotoxicity of herbicides.”

 

6. Table 5 ("Overall score" table) is not methodologically sufficiently explained

Table 5 summarizes the changes of several parameters and derives total scores, but it is not clear enough how these scores were calculated, why all parameters are implicitly weighted equally, and whether such an approach is biologically justified. For example, it is not the same to treat GI change in a target plant and LID10 in an aquatic organism as equal contributors to a single score.

 

Authors should either:

 

to explain in detail the formula, weighting and biological logic of this scoring,

 

or to keep the table only as a descriptive overview without too much reliance on the "overall score".

 

Response:

 

“The ratio of the value of the macrocycle-herbicide system to the value of the same parameter for the pure herbicide was calculated for each parameter. This calculation allowed to determine the effect of the herbicide binding to pillar[5]arene on changes in parameters (increases or decreases), eliminating the need for units of measurement and enabling comparisons between different parameters. A score was calculated by summing the parameters that positively characterize the complex and subtracting the parameters that negatively characterize it. A weighting system was not used. This was partly because seven parameters characterized herbicidal properties in this summary assessment (three for each plant species and one for microalgae inhibition), while only two parameters assessed ecotoxicity. This allowed for a greater overall weighting of herbicidal properties.” – this explanation has been added in the manuscript.

 

7. Statistical reporting is insufficiently detailed

Although the Supplementary Materials state that at least two independent series with a minimum of three replicates were performed and that the Mann–Whitney U test was used, the main text lacks key information: exact n for each experiment, significance level, p-values, and clear labeling of statistically significant differences in figures and tables.

 

For a better quality manuscript, it would be important for the authors to:

 

for each figure/table, state the number of biological and technical repetitions,

indicate statistical significance directly on graphs,

indicate whether they checked for multiple comparisons,

and clarify exactly what the errors are: SD, SE or something else.

 

Response:

 

The decision was made not to use the Mann-Whitney test to compare paired samples. The statistical analysis was redone. First, we tested for normality using the Shapiro-Wilk test. Then, we compared the significance of the differences using a one-way ANOVA followed by a post hoc test, p < 0.05. There were five replicates for the microalga and fifteen for the plants. Data are presented as mean ± SD. The figure now includes differences between groups, and the figure caption has been updated.

 

8. Terminology should be more consistent.

 In the text, the terms "complexes", "associates", "ensembles", "aggregates" and "systems" alternate. Since the authors themselves suggest that there is no classical inclusion, it would be useful to distinguish consistently:

 

molecular binding/association,

self-organization/aggregation,

and formal host-guest complexes.

 

Response:

 

We do agree that establishing the mechanism of interaction between macrocycles and herbicides requires further study. We suggest that the main mechanism of interaction between macrocycles and herbicides is aggregation with the following formation of supramolecular associates. We limited ourselves to using the terms “molecular binding” and “association” to explain the interactions studied. The manuscript has been revised according to the Reviewer’s recommendations.

 

9. Table 3

In Table 3, the DMSO control for wheat and radish is not completely neutral, so it should be clarified whether all treatments are statistically compared to water or to the corresponding solvent blank.

 

It would be useful to point out more clearly that the DMSO control had non-trivial effects in the phytotoxicity table, as this may affect the interpretation of the results.

 

Response:

 

According to the Reviewer's comment, the materials and methods section states: “Filter paper discs moistened with a dimethyl sulfoxide (DMSO) solution were used as a control, as well as distilled water, which was used as a negative control”.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors

I have readed your responses to my comments being satisfactiry for me. This version, include relevant information as background aimed to develop the investigation. 

Greetings 

Author Response

Dear Reviewer! Thank you very much for carefully reading and reviewing our paper! We are very pleased that you have given it such a high rating.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript was enough improved, and I have no other suggestions.

Author Response

Dear Reviewer! Thank you very much for carefully reading and reviewing our paper! We are very pleased that you have given it such a high rating.

Reviewer 3 Report

Comments and Suggestions for Authors

The revised version has been significantly improved, especially in the Materials and Methods section, the softening of the claims in the abstract, the statistical presentation and the explanation of concentration. However, there remain 2-3 poorly resolved points that, as a reviewer, I would mark as a minor revision before acceptance.

 

1. The authors stated that both DMSO control and distilled water were used as a control, and Table 3 shows that DMSO has a significant effect, e.g. GI for wheat 24%, for radish 55%. However, the authors did not discuss it sufficiently. A sentence or two should be added in the discussion that the DMSO control shows a non-trivial effect and that therefore the results must be interpreted in relation to the corresponding solvent blank.
2. The claim of preserving of herbicidal activity has not yet been sufficiently mitigated everywhere. The abstract is good, but the conclusion still contains the wording that the Glyp system reduces ecotoxicity while preserving activity against monocotyledons. It would be better: "while partially preserving phytotoxic effects toward wheat under the applied laboratory screening conditions".
3. Table 5 / Overall score is still methodologically debatable. The authors explained the calculation, but did not convincingly justify why different biological parameters can be added to a single score. I would recommend that they clearly write that the score is only a descriptive comparative index and not a validated biological indicator.

Author Response

The revised version has been significantly improved, especially in the Materials and Methods section, the softening of the claims in the abstract, the statistical presentation and the explanation of concentration. However, there remain 2-3 poorly resolved points that, as a reviewer, I would mark as a minor revision before acceptance.

 

Response:

Dear Reviewer! Thank you very much for pointing out the inaccuracies in the wording and suggesting ways to correct them.

 

1. The authors stated that both DMSO control and distilled water were used as a control, and Table 3 shows that DMSO has a significant effect, e.g. GI for wheat 24%, for radish 55%. However, the authors did not discuss it sufficiently. A sentence or two should be added in the discussion that the DMSO control shows a non-trivial effect and that therefore the results must be interpreted in relation to the corresponding solvent blank.

 

Response:

 

We are very grateful to the reviewer for pointing out an important comment that we had missed when describing the results. Indeed, DMSO negatively affected the growth and development of wheat and radish plants. Therefore, all calculations for the experimental samples were performed using DMSO as a control. This was not clearly stated in the Materials and Methods section. We added the relevant information to both the text of the article and Table 3.

 

“It is important to note that the original solvent, dimethyl sulfoxide (DMSO), had a negative impact on the growth and development of both wheat and radish seedlings. In wheat, DMSO reduced root length and the germination index (GI) by 76%. The inhibitory effect was less pronounced in radishes, with root length and GI decreasing by 28% and 45%, respectively. Nevertheless, DMSO also negatively affected radish germination. Therefore, all parameters for the experimental samples were calculated relative to the DMSO control to evaluate the effects of the tested compounds exclusively.”

 

2. The claim of preserving of herbicidal activity has not yet been sufficiently mitigated everywhere. The abstract is good, but the conclusion still contains the wording that the Glyp system reduces ecotoxicity while preserving activity against monocotyledons. It would be better: "while partially preserving phytotoxic effects toward wheat under the applied laboratory screening conditions".

 

Response:

 

The sentence “Thus, the association of pillar[5]arenes with Glyp made it possible to reduce the ecotoxicity of the herbicide while preserving its target activity against monocotyledonous plants.” The conclusion was rewritten as follows: “Thus, the association of pillar[5]arenes with Glyp made it possible to reduce the ecotoxicity of the herbicide while partially preserving its phytotoxic effects toward wheat under the applied laboratory screening conditions.”

 

3. Table 5 / Overall score is still methodologically debatable. The authors explained the calculation, but did not convincingly justify why different biological parameters can be added to a single score. I would recommend that they clearly write that the score is only a descriptive comparative index and not a validated biological indicator.

 

Response:

 

The relevant information has been added to the text of the article:

“However, it's important to note that this score should be viewed as a descriptive, comparative index, not a validated biological indicator. Future studies will further refine the proposed evaluation system, including determining weighting coefficients for each parameter and incorporating phytotoxicity data for the tested compounds against model weed species such as Arabidopsis thaliana.”

Author Response File: Author Response.pdf