Review Reports
by
- Mziwanda Mangwane 1,2,*,
- Ignacio Casper Madakadze 1 and
- Tlou Julius Tjelele 2
- et al.
Reviewer 1: Anonymous Reviewer 2: Barbara Wiewióra Reviewer 3: Anonymous Reviewer 4: Anonymous
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript by Mziwanda NA Mangwane and co-authors present a new study which examines the Evaluation of germination and early seedling growth of different grasses irrigated with treated mine-water. The thematic is of interest and authors did al lot of assessments and data analysis.
Comments:
The abstract deserves to be implemented with more details on results. In the abstract, write the research problem, the aim, very briefly the methodology, indicate the treatments.
The keywords are adequate, “phytoremediation” could be also included.
It is not still clear in the chapter of Introduction, why these plants have been chosen for the research. I think the authors should expand the introduction on the commercially available grasses that could be used in coal mine rehabilitation..
Materials and Methods. The approach seems correct and the methodology used is adequate.
Results. Results and discussion should be written separately.
Discussion should be improved and developed (too little analysis of the data of other authors)..
The conclusion should be rewritten and more concertized.
The English is clear and does not have any major spelling errors. But I'm not a native speaker.
On my opinion this article deserves to be published after revision.
Author Response
Dear reviewer,
I trust that you are well. Attached please find the rebuttal addressing your comments.
Thank you very much for considering our research work.
Yours sincerely,
Mziwanda
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsComments:
1. On what basis grass varieties were selected for the Seedling Growth Experiment? Why this is not a continuation of research from stage 1, i.e. seed germination?
2. Lack of analysis of the statistical significance of the difference (or lack thereof) in germination parameters in post-mining water and distilled water.
3. Lack of analysis of the chemical composition, pH and electroconductivity parameters of distilled water.
4. In the results and discussion there are no comparison to the results between the control (distilled water) and post-mining water, which in my opinion is obligatory to draw appropriate conclusions
The manuscript is not suitable for publication in Grasses journal in its present form.
Author Response
Dear reviewer,
I trust that you are well. Attached please receive the rebuttal that addresses your comments.
Thank you very much for your help and considering our research work.
Kind regards,
Mziwanda
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis work focuses on the impact of mine water on the germination and accumulation of biomass in seedlings of several grass taxa. The research topic of this paper is interesting, and the issue related to the reuse of mine waters for plant irrigation discussed in it, is relevant. Although mine water is commonly considered a source of pollution, it is also a source of essential metals and sulphates, as well as a valuable water resource for plant irrigation. The knowledge of the impact of mine water on the germination and growth of several grasses enables determining the grass species that can be used for rehabilitation of areas contaminated with mining waste.
In this study, seeds of eleven grass taxa (Eragrostis curvula ((Schrad.) Nees) cv Ermelo and cv Agpal, Digiteria eriantha (Steud.) cv Irene, Pennisetum clandestinum (Hochst. ex. Chiov.) cv Whittet, Chloris gayana (Kunth) cv Katambora, Lolium multiflorum (Lam.) cv Archie and cv AgriBoost, Lolium perenne (L.) cv Halo, cv Belay, Festuca arundinacea ((Schreb.) Darbysh.) cv Dovey and cv Feugo were allowed to germinate in treated mine water (TMW) and then germination parameters were evaluated (final germination percentage (FGP), germination rate index (GRI), corrected germination rate index (CGRI) and time to reach 50% of final germination (T50)). Five grass taxa Eragrostis curvula ((Schrad.) Nees) cv Ermelo, Lolium multiflorum (Lam.) cv Archie and cv AgriBoost, Cynodon dactylon ((L.) Pers.) cv Bermuda and Panicum maximum (Jacq.) properly Megathyrsus maximus (Jacq.) B.K. Simon & S.W.L. Jacobs cv PUK8 were used to evaluate effect of TMW irrigation for four weeks on biomass of seedling growing on mine soil.
The work includes appropriate statistical analyses of research results. The discussion of the results focuses on comparing grass taxa, rather than on differences between treated and control seeds/seedlings. This seems partially justified, especially in relation to the FGP parameter and seedling biomass, as it allows for the identification of species with the highest germination percentage and growth in an environment containing the components of this mine water. However, based on the data presented on Fig. 2, it is difficult to assess whether the use of TMW had a statistically significant effect on final seed germination percentage. The letters indicating statistically significant differences between the means should be added to this graph. Moreover, some parameters related to germination speed (T50, GRI, CGRI) have not been sufficiently discussed.
Analyses and presentation of the results require significant revisions before publication. In particular, the description of results needs to be improved, reorganized, and clarified.
The manuscript requires significant improvements, so I cannot recommend publishing it in current form.
Abstract, lines 20-21: “Relative to distilled water, GRI and CGRI were highest when grasses were watered using the treated mine water.” - replace with seeds, - this is not true, see the data presented in table 3, the values for GRI were higher for seeds germinating in water for 5 taxa (no significant statistical differences – 6 taxa), the values for CGRI were higher for seeds in water for 2 taxa (no significant statistical differences – 8 taxa) and only for 1 taxa germinating in TMW.
Materials and Methods, lines 75-77: “The seeds were allowed to germinate in distilled water or treated mine water which has an electrical conductivity of 557 mS.m-1.” - Please briefly describe the origin of the mine water (type of deposit extracted from the mine) used in the study.
Please briefly describe how the mine water was treated before it was used in this study. In another part of the manuscript (line 157, Result and Discussion), it was suggested that mine water was treated with gypsum - this information should be included in the description of the water used in the experiment.
As the degree of environmental pollution by mine water is dependent on its composition and pH, please explain whether the water used in these studies was tested for lead, cadmium, lithium, and other toxic elements. The content of these elements was not specified in table number 2, whether because their content was not tested or because their presence was not detected in the water used for the study.
Materials and Methods, lines 77-80: “The eleven grasses × two treatment combinations were replicated three times in a completely randomized design on germination benches in an air-conditioned glasshouse with constant temperatures set at 25 to 30 oC (daytime) and 10 to 15 oC (nighttime) to mimic field conditions, with 12 hour day/night cycle.” Consider replacing with “12 hour photoperiod”.
Materials and Methods, Table 1. Please provide the correct names of grass taxa.
Materials and Methods, lines 83-84: “The petri dishes were watered twice daily which was later reduced to once daily towards the end of the experiment.” - The Petri dishes were watered twice daily for … days, which was later reduced to once daily towards the end of the experiment.
Materials and Methods, lines 85-90: “All germinated seeds were counted and removed from each petri dish daily. Towards the end of the experiments, irrigation continued according to international seed testing association (2015) using a solution containing 0.02% KNO2 to induce germination of potentially dormant seeds to ensure there was no germination for at least three consecutive days towards the end of the experiment.” - Names should be written in capital letters.
Materials and Methods, lines 91-97: “Germination performance was assessed through final germination percentage (FGP), germination rate index (GRI), corrected germination rate index (CGRI) and time taken to reach fifty percent of the final germination (T50), for each treatment combination. Germination rate index (GRI) was calculated as daily germination percentage divided by the number of days since placement on treatment solutions. Germination rate index values were corrected for final germination to obtain corrected germination rate index by dividing with the respective final germination percentage and multiplying them by hundred.” - Please present the formulas (equations) according to which the following parameters were calculated: FGP, GRI, CGRI and T50, and cite the appropriate references.
Materials and Methods, lines 99-101: “The selected grasses used for the seedling growth experiment were Eragrostis curvula (L) cv Ermelo, Lolium multiflorum (L.) cv Archie and Lolium multiflorum (L) cv AgriBoost, Cynodon dactylon (L.) cv Bermuda and Panicum maximum (L.) cv PUK8.” – Please, provide the correct names of these taxa and explain why these taxa were selected for seedling growth experiment. Explain why the germination of Cynodon dactylon ((L.) Pers.) cv Bermuda and Panicum maximum (Jacq.) properly Megathyrsus maximus (Jacq.) B.K. Simon & S.W.L. Jacobs cv PUK8 seeds was not investigated. To more precisely determine the suitability of a given plant species for growth in post-mining areas, it would be necessary to examine both seed germination in mine water and growth in mine soil.
Materials and Methods, lines 101-103: “. Twenty seeds of each of these grasses were sown in pots containing mine soil from the mine disturbed area and irrigated using distilled water until the two-leaf stage.” - The paper presents the characteristics of treated mine water, but was the chemical composition of the soil used in the experiment also examined? If so, please attach this data on water in table number 2.
Materials and Methods, lines 120-122: “All plants in each pot were gently washed free of the soil, oven dried at 65 ºC until a constant mass was achieved and the final mass was used to determine total biomass per grass.” – Was the dry weight of the entire seedlings (roots + aboveground part) determined in the experiment, or only the aboveground part of the seedlings? Were the roots not damaged when removed from the soil?
Results and Discussion, lines 140-142: “Results from the current study showed that all grasses started to germinate within 3 – 4 days when watered using the treated mine-water.” – to assess the germination onset (the lag time) the t0 parameter should be calculated. Was the time it took for the seeds placed in TMW to germinate the same or different from the time it took for the seeds to germinate in water?
Results and Discussion, Fig 1., Fig. 2, tab.3. Fig.1- Please place the charts for individual taxa in the same order as in Table 1. Similarly, in Figure 2 and Table 3, please arrange the individual taxa in the same order as in Table 1.
Results and Discussion, lines 145-146: “The highest cumulative germination was recorded for L. multiflorum cv Archie and E. curvula cv Ermelo.” Please carefully analyse cumulative germination results of seeds treated with TMW. Based on results presented on Fig. 1 it is easy to notice that the germination dynamics of seeds of these two grass taxa treated with the TMW was much lower than that of control seeds. Therefore, treating these seeds with the TMW significantly reduced their germination speed. Based on results presented on Fig. 1: On the third day after start of imbibition, the cumulative germination percentage of control seeds (both L. multiflorum cv Archie and E. curvula cv Ermelo) exceeded 80%, while the seeds treated with the TMW had not yet begun to germinate. It also follows, that for seeds of both taxa germinating in water, the t50 values should not exceed 3. However, in Table 3, these values are much higher. Therefore, the t50 calculations should be rechecked.
Results and Discussion, lines 159-161: “Despite that, some grasses e.g., Digiteria eriantha (L) cv Irene, Lolium perenne (L) cv Halo, Festuca arundinacea (L) cv Feugo still showed poor tolerance to salinity present in TMW.” – It is difficult to agree with this statement because the seeds treated with the TMW germinated in the same way (F. cv Feugo and L. perenne cv Halo) or similarly (D. eriantha cv Irene,) as the control seeds. Therefore, it is difficult to attribute this delay in onset of germination to the sensitivity of these seeds to the components contained in the TMW.
Results and Discussion, lines 161-163: “Eragrostis curvula (L) cv Ermelo and Lolium multiflorum (L) cv Archie had a strong tolerance to TMW showing a potential for phytoremediation of open-cast coal mines.” – it seems that seeds of Eragrostis curvula cv Ermelo and Lolium multiflorum cv Archie were rather moderately susceptible to TMW as they germinated much slower than control seeds. Indeed, among the analysed grasses, they achieved the highest final germination percentage alongside Eragrostis curvula cv Agpal and Pennisetum clandestinum cv Whittet.
Results and Discussion, lines 164-165: ”Final germination percentages in grasses watered with treated mine water differed across all species and cultivars (Figure 2).” - Did final germination percentages significantly differ statistically between control seeds and those treated with the TMW?
Results and Discussion, lines 169-173: “For the other grasses, the final germination percentage when watered using the treated mine water ranged from 24 – 56 %. The final germination percentage for Festuca arundinacea (L) cv Dovey, Festuca arundinacea (L) cv Feugo, Lolium multiflorum (L) cv AgriBoost and Pennisetum clandestinum (L) cv Whittet ranged from 50 – 76%.”
Results and Discussion, lines 135-175, When analysing and describing cumulative germination, it is first necessary to compare the germination in water and TMW to determine whether the components of the TMW affected the time course of cumulative germination, and only then compare taxa with each other.
The results and discussion section lacks an analysis of the following parameters related to seed germination speed: GRI, CGRI and T50.
Results and Discussion, Figure 1. Please explain, why, for seeds of the same taxa germinating in water and in a solution, were cumulative germination measurements conducted for different periods of time, especially since not all seeds had germinated before the measurements were completed, eg.: E. curvula cv Agpal, D. eriantha cv Irene, Ch. gayana cv Katambora, L. multiflorum cv Archie and cv AgriBoost.
According to ISTA rules the final germination percentage of L. multiflorum should be assessed on 14th day after start of imbibition. Why were the measurements of L. multiflorum cv Archie seed germination terminated after 7 (control) and 11 (TMW treated) days?
What data is presented on the chart in the first column in the second row? According to the information provided in Table 1 in the paper, Eragrostis curvula cv AgriBoost was not studied.
Why are all the bars representing SEM in all the graphs shown in Figure 1 of the same size, even where the seeds have not yet started germinating and the germination percentage is zero? This should be corrected by presenting the actual SEM values.
Results and Discussion, Figure 2. The graph lacks results of statistical analysis, please, add letters indicating statistically significant differences between means.
Why do the data on germination percentage of P. clandestinum cv Whittet presented in Figure 1 (GP on the last day of test) and Figure 2 (The final GP) differ from each other? The same for L. multiflorum cv Archie.
Correct the taxa name by replacing Eragrostis curvula cv Angapal with Eragrostis curvula cv Agpal.
Results and Discussion, Table 3. Provide the units of parameters: GRI, CGRI, T50. Please, check if all the letters have been entered correctly. There are mistakes, for example, the T50 values for seeds of D. eriantha cv Irene and P. clandestinum cv Whittet germinating in water are the same and equal to 8.0, but the letters indicate that these values are statistically different.
Lines 21-22: “Means within the same columns followed by different lowercase letters are significantly different at (p≤0.05) using Fishers’ protected LSD (0.05) TMH2O = Treated mine water (557 mS.m-1).” - Does this mean that the values of germination parameters for control seeds and seeds treated with TMW were not compared? For each taxa, this data is presented in the rows of the table.
Please, explain what type of ANOVA was used for the statistical analysis of germination parameters: FGP, GRI, CGRI, and T50.
Results and Discussion, lines 7 and 13: “The growth rates progressively increased with increasing number of weeks for all grasses watered with distilled water.” In this study, biomass accumulation in seedlings was examined rather than growth rate.
Results and Discussion, Fig 3. please consider analysing the data on changes in biomass accumulation in seedlings growing on mine soil irrigated with MTW using repeated measures ANOVA.
Results and Discussion, lines 13-15: “The highest growth rates in grasses irrigated with distilled water were recorded for Lolium multiflorum (L) cv AgriBoost and Cynodon dactylon (L) vs Bermuda.” The results presented on Figure 3 do not confirm this statement. However, they indicate that the highest biomass was achieved by the seedlings of P. maximum cv PUK8.
Results and Discussion, In the paper, it should be emphasized that the components of solution may limit the plant growth by interrupting numerous physiological processes and altering metabolism in plants, as well as to cite relevant publications. It is also worth mentioning that some of the studied species are characterized by high tolerance to salinity, which allows them to germinate as quickly as the control seeds, achieving a high final germination percentage eg. Pennisetum clandestinum (https://doi.org/10.1016/j.envexpbot.2012.07.009).
Conclusions, lines 2-4: “The results showed that treated mine water had no adverse effect on germination of all grasses evaluated and, therefore, could be used for irrigation during mine rehabilitation.” This sentence should be clarified. Perhaps the differences in FGP values were not statistically significant, but for several grass taxa germination was slower in TMW (see Fig3).
Conclusions, lines 6-9: “Lolium multiflorum (L) cv Archie and Eragrostis curvula (L) cv Ermelo were the first grasses to germinate and this showed that the two grasses had an ability to quickly germinate and avoid accumulation of toxic ions during the germination process.” - In the study, it was not shown that seeds avoid the accumulation of toxic components, as their content was not examined in germinating seeds.
Conclusions, lines 10-11: “Time taken to reach 50 % of the final germination was generally low across all grasses.” This suggests that all the seeds germinated quickly. However, the data presented in Table 1 indicates that the T50 parameter values ranged from 5.0 to 10.66 for control seeds and from 5.7 to 11.3 for seeds treated with TMW.
Conclusions. Recommended species for phytoremediation of mined areas should be those that germinate in mine water quickly but also achieve a high final germination percentage and significant biomass growth.
Author Response
Dear reviewer,
I hope that you are well. Attached please find the rebuttal that addresses you comments to our work.
Thank you very much for you constructive comments to our research work.
Yours sincerely,
Mziwanda
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe study addresses a serious problem, and the results could be very useful for reclamation of coal mines. The design and analysis are appropriate and adequate. The tables and graphs are good. The sentence structure and grammar are good.
However, I was left underwhelmed by the Results and Discussion. Rather than list specific sentences, I am going to give the general concerns. These need to be addressed line by line throughout the Results and Discussion.
As written, it is just Results - no discussion. It is fine as research report but lacks the discussion to make it a published peer-reviewed paper. There is practically no literature cited in the results AND no discussion about how this research compares to other similar studies. I'm confident that these forage grasses have been grown using saline irrigation and there would be some comparisons to make that would or does not support the reported results. This would give credibility to the study and the results.
The authors also try to explain "the why" of some of the unexpected results, such as, "Treated mine water resulted in high cumulative germination in all grasses (Figure 1). This could be linked to beneficial effects of other elements in treated mine water (Table 2), regardless of the salt ions found in treated mine water." What other elements? Has that been shown before? Etc, etc. In another instance the manuscript states, "High biomass production under treated mine water might be due to the influence of other essential elements found in treated mine water such as Phosphorus, Potassium and Calcium presented in Table 2." Do other studies show this type of effect on growth from these elements?
Likewise, why does it take 3 to 4 weeks for the grasses to start to grow using distilled water, but we see immediate growth using the saline irrigation? This type of contrasting growth responses is typical for halophytic species .... so are the authors suggesting these are halophytic species (which they are not)? So there needs to be some discussion of why these elements are increasing growth/germination more than the salinity is decreasing it. These type of additions and discussion are a must to support the conclusions of the authors.
Another point of discussion that is lacking is why different cultivars of the same species responded differently. That is important to try and tease out. What are their background differences? This is particularly important as it has implications in terms of choosing species and cultivars for mineland reclamation?
Finally, I would like to see the probability listed following statements like "was high", "progressively increased", etc (e.g., p=0.05). This is important as I think some of these statements are from statistical comparison and some are of eyeball comparisons (e.g., not actually compared using a statistical test). I'm not against a few eyeball comparations (I usually call them trends) when looking at different regressions, etc, but I want to know if they are statistically different or the authors believe they are different based upon observation.
Hopefully these suggestions are valuable as I do believe the research can add to the scientific literature.
Author Response
Dear reviewer,
I trust that you are well. Attached please find the rebuttal with the responses to your comments.
Thank you very much for your comments.
Kind regards,
Mziwanda
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
The work is interesting, it indicates which grasses can be used for recultivation of post-mining areas and how they behave in less favorable conditions for their development (irrigated with treated mine water).
I lack information in the literature whether and possibly what plants, apart from grasses, can also be used for such a purpose (Page 9 lines 52-53 specifically what plants or groups of plants).
Why are the ISTA Rules from 2015 (old references), especially since changes are introduced every year, please use the newer edition.
Missing data and detailed explanations that were not available in the previous version were supplemented, which increased the value of the work. All my comments were taken into account and in my opinion, the work in this form meets the requirements of the journal and can be published.
Author Response
Dear reviewer,
I trust that you are well. Attached please find the attached document is the rebuttal to your comments.
Thank you very for your kind help and considering our research work.
Kind regards,
Mziwanda
Author Response File: 
Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
The manuscript (grasses-3008390) has been only partially improved. It still requires serious corrections. Most of the comments were not taken into account, necessary corrections were not made, and doubts were not explained. Unfortunately, many of the previous comments and suggestions for Authors have been ignored.
Please detailly revise your manuscript according to previous review report and address all the comments mentioned in the previous review.
The manuscript still does not meet the requirements for a good scientific publication, so I cannot recommend publishing it.
Author Response
Dear reviewer,
I trust that you are well. Attached please receive the rebuttal to your comments.
Thank you very much for your help and considering our research work.
Kind regards,
Mziwanda
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsAs near as I can tell the authors have addressed my concerns and have added discussion. However, some major formatting issues make it difficult to fully determine.
There appear to be cut and paste issues as the results and discussion subsections are mixed up, particularly 4.2 Total Biomass. Results section 4.1: .... Germination... starts on page 5, line 157 and appears to end on page 7, line 4 where the next subsection heading for 4.2: Total biomass ... appears. However, there are multiple paragraphs on germination within this subsection and they are often in between paragraphs on biomass. So this appears to be a formatting issue unless I'm missing something. I double checked and this was not the case in version #1. Normally formatting issues would be a 'minor revision' but since these are somewhat more than just minor, I've recommended 'major revision' with the thoughts the authors can easily sort this out.
See the following specific comments which all follow the subheading for 4.2, Total Biomass.
p.7, starting with line 5: This paragraph is about germination and not biomass.
p.8, starting with line 18: This paragraph is discussion of the biomass but no biomass results have been reported to this point.
p.9, line 36-54: These two paragraphs are about germination again ... wrong section. Germination was section 4.1.
p.9, line 58-65: Now this paragraph is about biomass but not sure if this is reporting or discussing the results so not sure of its order.
p.10, line 67-75: Another paragraph on germination.
p.12, line (no line numbers?): Again a paragraph on biomass but not sure of its order.
Author Response
Dear reviewer,
I hope you are well. Attached please receive the my response to your comments.
Kind regards,
Mziwanda
Author Response File: Author Response.pdf
Round 3
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
The manuscript has been improved. The corrections that were made significantly increased the value of this work. However, the manuscript still requires corrections, which should be done with due diligence.
The list of comments and suggestions:
1. Please correct the spelling of the words: gypsum, line 15 and Petri dishes, line 22, line 85, line 93, line 98, line 101. Please write the name of Petri dishes correctly, which is named after the researcher Petri J.R.
2. Please remove repeated sections of text, lines 86-90: “The seeds were allowed to germinate in distilled water or treated mine water which has an electrical conductivity of 557 mS.m-1 and it was collected from acid mine drainage Pittsburgh which was later treated with gypsum. Treated mine water was also tested and treated for lead, cadmium, lithium, and other toxic elements since it is also used for the irrigation of pastures used for grazing animals. Mine water was collected from acid mine drainage Pittsburgh which was later treated with gypsum.”
3. Please remove repeated sections of text, lines 112-114: ”Germination rate index (GRI), corrected germination rate index (CGRI) and Germination rate index (GRI) was calculated as daily germination percentage divided by the number of days since placement on treatment solutions.”
4. Please provide proper, complete formula for parameter T50; explain what each symbol (N, 𝑡𝑗, 𝑡𝑖, nj, ni) in the formula for parameter T50 means (eq 4), line 125 and add the assumption: when ni < N/2 < nj
5. Lines 187 -189; “Despite that, some grasses e.g., Digiteria eriantha (Steud.) cv Irene, Lolium multiflorum (Lam.) cv Halo, Festuca arundinacea ((Schreb.) Darbysh.) cv Feugo still showed poor tolerance to salinity present in TMW.” – Consider improving this sentence.
6. Lines 187-190 “Despite that, some grasses e.g., Digiteria eriantha (Steud.) cv Irene, Lolium multiflorum (Lam.) cv Halo, Festuca arundinacea ((Schreb.) Darbysh.) cv Feugo still showed poor tolerance to salinity present in TMW.” – data presented in Fig. 1 do not support this statement, as for Halo and Feugo germination in MTW did not differ from gemmation in water.
7. Lines 6-9: “For seeds germinated using treated mine water, the highest FGP was recorded for Lolium multiflorum (Lam.) cv Archie (91 %) and Eragrostis curvula ((Schrad.) Nees) cv Ermelo (90 %) followed by Eragrostis curvula ((Schrad.) Nees) cv Agpal (71 %).” – This sentence contains an incorrect statement. Based on results presented in fig 2 P. clandestinum cv Whittet had highest FGP than E. curvula cv Agpal.
8. Lines 9-19: “There were no significant differences (p > 0.05) between Lolium multiflorum (Lam.) cv Archie and Eragrostis curvula ((Schrad.) Nees) cv Ermelo.” – Results presented in fig 2 also suggest that: There were no significant differences (p > 0.05) between L. multiflorum cv Archie, E. curvula cv Ermelo, E. curvula cv Agpal and P. clandestinum cv Whittet.
9. Lines 41-43: “The highest growth in grasses irrigated with distilled water were recorded for Lolium multiflorum (Lam.) cv AgriBoost and Cynodon dactylon ((L.) Pers.) cv Bermuda.” This sentence is imprecise, it is not known what values are compared here, whether at the beginning of the experiment or at its end, i.e. after 4 weeks of TMW treatment. Results presented in fig 3 suggest that: At the end of experiment the highest growth in grasses irrigated with distilled water was recorded for PUK8 (total biomass surpassed above 2 g DM plant-1).
10. Lines 49-57; “The decline in final germination with increase in salinity could be ascribed to continuous uptake of Na+ and Cl ions at high amounts [16]. Accumulation of these ions could have reduced the enzymatic and hormonal activities including mobilization and synthesis of the embryonic sugars and proteins of the seeds [13]. Amongst these enzymes is alpha amylase (α amylase) that helps in degrading the starch reserves during imbibition. Therefore, impaired water uptake by salt ions means the reduction in performance of α amylase enzyme. As in Halo, Dovey, Feugo and Belay in this study, noted a decline in germination percentage of Lolium perenne [20]. [3] reported that grass seeds tend to lose viability when subjected to high saline conditions.” – Please move these sentences to the section describing the impact of TWM on germination.
11. Line 140-142; „Lolium perenne (L.) cv Halo, Festuca arundinacea ((Schreb.) Darbysh.) cv Dovey, Festuca arundinacea ((Schreb.) Darbysh.) cv Feugo and Lolium perenne (L.) cv Belay in this study, noted a decline in germination percentage of Lolium perenne. “- The sentence is unclear.
12. Lines 52-54 and 136-140: “Amongst these enzymes is alpha amylase (α amylase) that helps in degrading the starch reserves during imbibition. Therefore, impaired water uptake by salt ions means the reduction in performance of α amylase enzyme.” and: “Amongst these enzymes is alpha amylase (α amylase) that helps in degrading the starch reserves during imbibition. Therefore, impaired water uptake by salt ions means the reduction in performance of α amylase enzyme.” – The sentences are repeated in the text of the work.
13. Abstract, line 30-31: “Relative to distilled water, GRI and CGRI were highest when seeds were watered using the treated mine water.” – I reiterate the comment presented in the first review: this sentence is true only for one taxon (L. multiflorum cv AgriBoost) but is not for all other taxa. See the data presented in table 3, the values for GRI were higher for seeds germinating in water for 5 taxa (no significant statistical differences – 6 taxa), the values for CGRI were higher for seeds in water for 2 taxa (no significant statistical differences – 8 taxa) and only for 1 taxa germinating in TMW. Thus, I recommend removing the sentence or correcting it to accurately describe the results.
14. Line 122 Equation 3, as a reference for this equation the work of Sheoran et al [22] was cited. However, this study did not address germination coefficient calculations. Please provide the appropriate reference.
15. Results and Discussion, Fig 1, and Table 3. Based on results presented in Fig. 1: On the fourth day after start of imbibition, the cumulative germination percentage of control seeds (both L. multiflorum cv Archie and E. curvula cv Ermelo) exceeded 80%. It also follows, that for seeds of both taxa germinating in water, the t50 values should not exceed four. However, in Table 3, these values are much higher, 5.0 for Archie and 5.7 for Ermelo. The T50 values for both taxa presented in the table 3 appear to be miscalculated. Because the results shown in the figure do not align with those presented in Table 3. Therefore, the t50 calculations should be rechecked.
16. Lines 187-190: “Despite that, some grasses e.g., Digiteria eriantha (Steud.) cv Irene, Lolium multiflorum (Lam.) cv Halo, Festuca arundinacea ((Schreb.) Darbysh.) cv Feugo still showed poor tolerance to salinity present in TMW.” I reiterate the comment presented in the first review: It is difficult to agree with this statement because the seeds treated with the TMW germinated in the same way (F. arundinacea cv Feugo and L. perenne cv Halo) or similarly (D. eriantha cv Irene,) as the control seeds. Therefore, it is difficult to attribute this delay in onset of germination to the sensitivity of these seeds to the components contained in the TMW. Authors comment: “Page 5: Line 183 – 187: Comment noted, however, for Digiteria eriantha cumulative germination started earlier than Lolium perenne (L) cv Halo, Festuca arundinacea (L) cv Feugo. Furthermore, the had different cumulative germination % and the cumulative curve is also different.” To determine the tolerance of a given species to the influence of a certain factor, one must compare parameter values between control plants and plants treated with that factor. Comparing parameter values across different species does not justify claims about tolerance or sensitivity to factor if there is no reference to control plants. Since their germination in water was the same as in TMW, this means that the salinity of TMW did not affect germination, and the seeds showed good tolerance to TMW salinity. The claim that these three taxa showed low tolerance to TMW salinity is unfounded.
17. Results and Discussion, lines 5-6: ”Final germination percentages in grasses watered with treated mine water differed across all species and cultivars (Figure 2).” - Did final germination percentages significantly differ statistically between control seeds and those treated with the TMW? Authors comment: “There were no statistical differences within treatments thus this was not reported in the results.” There were no statistically significant differences, which is a very important result that should be reported in the Results section. This means that the FGP of all taxa was not influenced by TMW. This implies that the seeds of all examined taxa were not very sensitive to TMW salinity.
18. Figure 1. I reiterate the comment presented in the first review: Why are all the bars representing SEM in all the graphs shown in Figure 1 of the same size, even where the seeds have not yet started germinating and the germination percentage is zero? This should be corrected by presenting the actual SEM values. Authors comment: “The comment by the reviewer has been accepted.” The answer is unsatisfactory. The charts should display accurate, actual SEM values.
19. Why do the data on germination percentage of P. clandestinum cv Whittet presented in Figure 1 (GP on the last day of test) and Figure 2 (The final GP) differ from each other? The same for L. multiflorum cv Archie. Authors comment: “Page 7: Figure 2: Final germination percentage of P. clandestinum cv Whittet presented in Figure 2 has been corrected and now it is the same for both figures.” – What about data on germination of L. multiflorum cv Archie?
20. Figure 2. Correct the taxa name by replacing Eragrostis curvula cv Angapal with Eragrostis curvula cv Agpal.
21. Table 3. Please provide proper unit of GRI. For details of coefficients characterizing the course of the seed germination process (and GRI unit) please see work of Kader 2005 “A Comparison of Seed Germination Calculation Formulae and the Associated Interpretation of Resulting Data” Journal & Proceedings of the Royal Society of New South Wales, Vol. 138, p. 65–75.
22. Table 3. Please, check if all the letters have been entered correctly. There are mistakes, for example, the T50 values for seeds of D. eriantha cv Irene and P. clandestinum cv Whittet germinating in water are the same and equal to 8.0, but the letters indicate that these values are statistically different.
23. Comment from the first review: “The highest growth rates in grasses irrigated with distilled water were recorded for Lolium multiflorum (L) cv AgriBoost and Cynodon dactylon (L) vs Bermuda.” The results presented on Figure 3 do not confirm this statement. However, they indicate that the highest biomass was achieved by the seedlings of P. maximum cv PUK8. Authors comment: Page 11: Line 80 – 81: Yes, this is true, and the comments has been considered and corrected on the revised manuscript. However, the sentence has not been corrected, lines 41-43: “The highest growth in grasses irrigated with distilled water were recorded for Lolium multiflorum (Lam.) cv AgriBoost and Cynodon dactylon ((L.) Pers.) cv Bermuda.” – Please correct the sentence to include accurate descriptions of the results.
24. Lines 148-155: “The results showed that treated mine water had no adverse effect on germination of few forage grasses evaluated and, therefore, could be used for irrigation during mine rehabilitation using these high tolerant forage grasses. These grasses showed a differential response on the cumulative germination patterns with some grasses germinating within the first few days after placement in distilled water and treated mine-water. Therefore, these grasses can be used for mine rehabilitation under irrigation by treated mine water.” The above sentences should be clarified. Perhaps the differences in FGP values were not statistically significant, but for several grass taxa germination was slower in TMW (see Fig3). Please consider the following corrections to this text: The results showed that treated mine water had no adverse effect on final germination percentage of all forage grasses evaluated and, …….. . However, these grasses showed a different cumulative germination patterns, with some grasses germinating equal within the first few days after placement both in distilled water and treated mine-water. Therefore, the grasses with faster germination can be used for …………………. .
25. Line 161-163: “Time taken to reach 50 % of the final germination was generally differed across all grasses where some grasses to longer to reach FGP.” – this sentence requires linguistic correction.
26. There is no need to write the full taxon name every time.
Author Response
Dear Reviewer,
I trust that you doing well. Attached please find the rebuttal that addresses you comments in our research work.
Kind regards,
Mziwanda
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsAuthors have addressed my previous comments/concerns.
Author Response
Dear reviewer,
I hope you doing well. Attached please find the rebuttal that addresses your comments in our research work.
Kind regards,
Mziwanda
Author Response File: Author Response.pdf