Improvement of Physiological Features and Essential Oil Content of Thymus vulgaris after Soil Amendment with Chitosan Nanoparticles under Chromium Toxicity
, Gholamreza Mahdavinia 3, Sahar Azizi 2, Rosario Nicoletti 4,*, Otilia Cristina Murariu 5,*, Alessio Vincenzo Tallarita 6,* and Gianluca Caruso 6
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsI attach the document with some observations, please address them
Comments for author File: 
Comments.pdf
Author Response
1-The figure 1 does not show how to prepare the chitosan nanoparticles. This show results of instrumental analysis.
Answer: Dear Reviewer, thank you for your comments. We are sorry for missing the procedure related to chitosan nanoparticle preparation in Figure 1, which has been added as recommended.
2- Use, free groups instead of pendants.
Answer: "pendants" was changed to "free groups".
3- Did they not calculate the z potential with the DLS? It would be interesting to observe the charge of the nanoparticles.
Answer: We agree that it would have been interesting to perform the mentioned determination and we will plan to do that in future research about this topic.
4- Please review this foundation carefully, because FTIR only identifies functional groups and not charge interactions.
Answer: In the case of FTIR analysis, the main functional groups can indeed be identified. However, it may be noted that physical interactions such as hydrogen bonding and electrostatic interactions can lead to a shift of frequencies of the main functional groups.
5- discuss more broadly and appropriately XRD. Why the TPP decreased the crystalline form?
Answer: The discussion of XRD analysis was deepened.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript investigated chromium stress on physiological process and essential oil production of Thymus vulgaris and developed a chitosan nanoparticle to provide a strategy to alleviate the chromium toxicity. The manuscript should be reconsidered after major revision. Please see my comments to improve the manuscript.
Experimental design: the authors compared the control and 0.05-0.2 % of CS-TPP gelation NPs, but the simple physical mixture of chitosan+TPP+acetic acid also should be employed as comparations. There were also lack of only chitosan, acetic acid, TPP alone group to eliminate their effects respectively. Also, although the authors characteristic the NPs using chemical approaches, it just transformed from large particles of commercial chemical reagent to nano-size particles, the authors need to clarify the principle of the effect of nanoscale chitosan particles.
-I recommend the author provide pictures of the plant to show the morphology of the plant.
-The authors also need to study on how Cr content residue in the soil after NPs treatment.
-Section 2.17, please provide the replication information of the article.
-Figure 1, all the images have been distorted, please use the original scale.
Author Response
- Experimental design: the authors compared the control and 0.05-0.2 % of CS-TPP gelation NPs, but the simple physical mixture of chitosan+TPP+acetic acid also should be employed as comparations. There were also lack of only chitosan, acetic acid, TPP alone group to eliminate their effects respectively.
Answer: Dear Reviewer, thank you for your comments. The addition of the mentioned treatments would have further widened the experimental protocol which was arranged to include the most interesting treatments in our opinion, also referred to the following previous investigations:
Turan, V., Khan, S. A., Iqbal, M., Ramzani, P. M. A., & Fatima, M. (2018). Promoting the productivity and quality of brinjal aligned with heavy metals immobilization in a wastewater irrigated heavy metal polluted soil with biochar and chitosan. Ecotoxicology and environmental safety, 161, 409-419.
Zubair, M., Ramzani, P. M. A., Rasool, B., Khan, M. A., Akhtar, I., Turan, V., ... & Iqbal, M. (2021). Efficacy of chitosan-coated textile waste biochar applied to Cd-polluted soil for reducing Cd mobility in soil and its distribution in moringa (Moringa oleifera L.). Journal of environmental management, 284, 112047.
Qu, D. Y., Gu, W. R., Zhang, L. G., Li, C. F., Chen, X. C., Li, J., ... & Wei, S. (2019). Role of chitosan in the regulation of the growth, antioxidant system, and photosynthetic characteristics of maize seedlings under cadmium stress. Russian journal of plant physiology, 66, 140-151.
- Also, although the authors characteristic the NPs using chemical approaches, it just transformed from large particles of commercial chemical reagent to nano-size particles, the authors need to clarify the principle of the effect of nanoscale chitosan particles.
Answer: Addressed.
- recommend the author provide pictures of the plant to show the morphology of the plant.
Answer: a supplementary file has been provided.
- The authors also need to study how Cr content residue in the soil after NPs treatment.
Answer: The mentioned topic have been developed as follows:
- Lines 504-515: Chromium is a trace mineral in food that competes with similar minerals for absorption sites, from which it is transported into the cells [55-59]. CS-NP effectiveness in chelating Cr is probably due to the abundance of amino- and hydroxyl groups in its structure, which leads to a decrease in the availability of Cr in soil, thus reducing the uptake of this element in thyme plants [23,60]. Our findings are consistent with previous studies showing that CS-NP can immobilize HM in soil [61]. The toxicity of heavy metals is alleviated as they are adsorbed onto CS-NP surfaces in soil through ion exchange, precipitation, and surface complexation processes [62]. In previous studies [63,64], chitosan soil amendment increased metal phytoremediation efficiency. However, the variations revealed in experimental outcome are directly linked to the soil characteristics and the type of chitosan employed [61].
- Lines 531-535: In plants, CS-NPs can help reduce the uptake, mod-ify the translocation, and decrease Cr mobility in soil, thereby alleviating its toxicity and modifying cell wall membranes to mitigate oxidative stress [61]. These findings are con-sistent with previous research showing how chitosan can lessen the harmful influences of heavy metal toxicity and lead to improved performance [27,29,70,71].
- Section 2.17, please provide the replication information of the article.
Answer: Addressed.
- Figure 1, all the images have been distorted, please use the original scale.
Answer: Addressed.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsCompared with v1, the author did not make any changes to Figure 2. (a) to (c), the width of the DCL graph is inconsistent, and the axis numbers are too small. (e) to (f), the SEM image size is inconsistent, and the scale is indistinguishable. (g) to (h), the image scale is flattened, and all the annotated numbers are unrecognizable.
The red labeled part of Table 2 in version v2 is consistent with v1. Can the author explain what revisions were made?
Author Response
Thank you for your support in helping us to improve the quality of the manuscript. Please, find below our answers to your comments.
- Compared with v1, the author did not make any changes to Figure 2. (a) to (c), the width of the DCL graph is inconsistent, and the axis numbers are too small. (e) to (f), the SEM image size is inconsistent, and the scale is indistinguishable. (g) to (h), the image scale is flattened, and all the annotated numbers are unrecognizable.
Answer: We have addressed your comments and performed the required revisions in Figure 2, directly implementing the modifications in the uploaded version of the manuscript.
- The red labeled part of Table 2 in version v2 is consistent with v1. Can the author explain what revisions were made?
Answer: In version v2, we incorporated the revisions in response to your valuable comment inside Table 4. In the first version, we had reported the soil chromium (Cr) content before planting. However, based on your request for a more comprehensive analysis, we have added data related to the soil Cr residues after harvesting the thyme plants, providing valuable insights into the dynamics of Cr accumulation and distribution in the soil during the cultivation period. In this respect, in Table 4 of version v2 we have replaced the initial Cr content before planting with the updated data regarding Cr residues post-harvest. The latter modifications make our findings more robust and reflective of the complete cycle of thyme cultivation.
