The Land Snail, Eobania vermiculata, as a Bioindicator of the Heavy Metal Pollution in the Urban Areas of Sulaimani, Iraq
Round 1
Reviewer 1 Report
Overall, the science behind the experiment is sound. However, there are some grammatical errors that need to be corrected, some of which are highlighted within the attached document.
1. My main concern was the use of stainless steel tools/equipment which may contribute to metal contamination of the samples. At least blanks need to be considered to eliminate this possibility.
2. The ICP-OES parameters are not mentioned - nebulizer flow, pump rpm's, etc...
3. In section 2.6, the statistical tools should be mentioned, even though you mentioned them at a later stage.
4. Where are the metal units in tables 2 and 4?
5. In section 3.2, a negative value has to be avoided. What are the LOD and LOQ levels for each metal? Do the results go below the LOQ?
6. Person should read Pearson and R2 should be in superscript R2.
7. The last sentence of the results and discussion should be reworded
8. The reference styles vary between references. Please follow journal guidelines.
9. Having different categories for sites, I would have conducted a Principal Component Analysis (PCA) in order to determine whether there is a discrimination between groups according to their metal profiles. This might substantiate more your findings.
Comments for author File: 
Comments.pdf
Author Response
Response to the reviewer 1 comments
Overall, the science behind the experiment is sound. However, there are some grammatical errors that need to be corrected, some of which are highlighted within the attached document.
Response: Thanks for your appreciation and recommendation. We have tried our level best to correct the typos and grammatical mistakes in the whole manuscript and highlighted them as yellow
- My main concern was the use of stainless steel tools/equipment which may contribute to metal contamination of the samples. At least blanks need to be considered to eliminate this possibility.
Response: The reviewer has indicated a very important and critical point. However, we used a new, clean and normal sampling trowel instead of stainless steel tools. We have rechecked and corrected (Please see line 100)
- The ICP-OES parameters are not mentioned - nebulizer flow, pump rpm's, etc...
Response: The ICP-OES parameters have been mentioned (Please see lines 149-155)
- In section 2.6, the statistical tools should be mentioned, even though you mentioned them at a later stage.
Response: We have added the details regarding statistical analysis (Please see lines 161-168)
- Where are the metal units in tables 2 and 4?
Response: We have mentioned the units for metal concentration in soil (Table 2) and snail (Table 4) samples and highlighted them as yellow
- In section 3.2, a negative value has to be avoided. What are the LOD and LOQ levels for each metal? Do the results go below the LOQ?
Response: As per the suggestion of the reviewer, we have replaced the negative value with the minimum value detected (Please see line 281). Also, the LOD or LOQ have been mentioned below the Table 4
- Person should read Pearson and R2 should be in superscript R2.
Response: We have corrected as per the suggestion of the reviewer and highlighted
- The last sentence of the results and discussion should be reworded
Response: We have revised the last two sentences and modified (Please see lines 410-412)
- The reference styles vary between references. Please follow journal guidelines.
Response: We have corrected and made all the references as per the format of the journal and highlighted as yellow where changes made
- Having different categories for sites, I would have conducted a Principal Component Analysis (PCA) in order to determine whether there is a discrimination between groups according to their metal profiles. This might substantiate more your findings.
Response: The reviewer has pointed out a very important point. However, here our objective was to investigate the concentration and distribution of heavy metals (As, Cr, Ni, Pb, and Zn) in Sulaimani's urban soil and the native land snail, Eobania vermiculata in urban areas and to estimate its potential role as a bioindicator for soil heavy metals contamination. Regarding whether there is discrimination between groups according to their metal profiles, we will consider this point in our future studies.
Response to the comments in pdf file
One or more than one?
Response: Corrected (Please see line 45)
Sentence Construction
Response: Corrected (Please see lines 67-69)
Should be avoided for metal analysis, perhaps a pestle and mortar would have been more suitable. Since the work is already done, do you have any blanks to compensate for any possible metal release?
Response: Corrected (Please see line 108-109)
You mean submerged or treated with
Response: Corrected (Please see line 113)
What are the ICP-OES parameters: nebuliser flow, pump rpm's, etc...
Response: The details have been added (Please see lines 149-155)
What tools were used? e.g. Pearson etc should be stated here.
Response: A complete description regarding statistical analysis has been added (Please see line 161-168)
Not a sentence!
Response: Corrected (Please see line 253-254)
Avoidance of negative values
Response: We have corrected (Please see line 292)
Units with metals concentration in Table 4
Response: We have added the units
Correction in the title of Table 5
Response: We have corrected the mentioned typos and highlighted
What does this mean?
Response: We have modified the sentence (Please see line 411-412)
Where is the opening brackets?
Response: We have modified the sentence (Please see line 420)
Please check all references for consistency: 15-20, 22-40, 42, 46-47, 51-53
Response: We have cross-checked all the references and made them as per the format of the journal
Author Response File: 
Author Response.docx
Reviewer 2 Report
See attached file.
Comments for author File: Comments.pdf
Author Response
Response to Reviewer 2 comments
Manuscript ID: sustainability-1458413
Land snail as bioindicators of the heavy metal pollution in the urban areas of Sulaimani, Iraq
The submitted paper is based on passive biomonitoring which is one of the methods to use terrestrial snails as bioindicators of environmental pollution. The other way is based on active biomonitoring (by caging on site of snails from known origin). As here wild autochthonous Eobania vermiculata were sampled, this must be specified in the abstract that passive biomonitoring was performed.
Response: We have already mentioned it in the 3rd last line of the abstract.
The use of snails as bioindicators is not new, this the purpose of the study shall be modified (lines 72-73): is the main question asked this one: based on their internal concentrations of As, Cr, Ni, Pb, and Zn, are Eobania vermiculata of the urban areas of Sulaimani consumable for humans?
Response: We have modified (Please see lines 73-78).
If people of the region eat these snails, which part is eaten? Foot or all body? Often or rarely?
Response: The snail (Eobania vermiculata) has not been eaten yet in Sulaimani or even in Iraq.
Overall the references are too numerous and not always necessary; studies dealing with aquatic gastropods must be deleted. Indeed, enough publications are available with terrestrial snails as bioindicators.
Response: We have removed the mentioned sentences
Some reference are not adapted, e.g. line 55, ref 11 (de Vaufleury, 2015) does not report data on aquatic environment but on terrestrial environment. This this reference must be cited line 58. Important references are lacking, e.g. the one of Louzon et al (who published TGV in landsnails) or Pauget et al (who published CIref values in landsnails) or also Itziou et al (e.g. STOTEN 2011: Cd Pb Cu) who worked for a long time on the Mediterranean snail species.
Response: We have modified the references as per the suggestion of the reviewer
Regarding the Mat & meth section, 30 snail samples are cited line 94: how many snails par sampled pn each of the 30 plots studied?
Response: We have modified the sentence as “All 30 soil samples with 30 corresponding land snail samples, each containing five individuals were collected from the same parks that snails are habitually available and survive in all directions (North, south, east, and west) in the city.”
Did snails starved before being analysed? They well killed at 105°C?
Response: The sentence has been modified as “The other group of samples was starved for three days in the laboratory to empty their digestive tracts. Then, these were put in distilled water for one day, and whole soft tissues dead snails were dissected properly and placed in an oven to dry at 105 °C for 18 hours before milling with ceramic pestle and mortar.” (Please see lines 118-121)
Which part of snails was analysed: all (shell + soft body) or just the soft body?
Response: Please see the above response
For the metal analysis in soils and snails, did authors used certified reference material to check the accuracy of their analysis?
Response: Please see the supplementary information
Fig.2 is not necessary; the sampling locations can be added in fig 1.
Response: Yes, we agree with the reviewer, but, it may be a little bit difficult and figure 2 is clearer.
Table 1 can be moved to supplemental material.
Response: We disagree with the reviewer and have kept Table 1 as the main text as it contains the basic information about soil samples which is the main theme of the paper
As soil concentrations are very low (below the soil quality guidelines by Canadian standards, or SGV reported by Louzon et al, STOTEN 2020) excepted for Ni and Cr but with a low excess, and pH was basic, it was expected that the bioavailability and bioaccumulation of metals will be reduced. Thus, as recommended above, the focus/working hypothesis can be more on the possible risk for human of the consumption of these wild snails than on the interest of Eobania vermiculata as bioindicator of pollution.
Response: As indicated above, the snails are not consumed yet by the Kurdish and Iraqi people. Therefore, we focused on using the Eobania vermiculata as a bioindicator of heavy metals pollution.
The discussion of table 2 (1.5 pages) must be reduced to better underline the highlights of the results. Same remark of part 3.2. snail description. The permissible limits of metals in snails must appear in the Table 4. To facilitate the reading of the table, values > than these limits can appear in bold character.
Response: We have modified the discussion under sections 3.1 and 3.2 and made the values above permissible limits bold
The conclusions shall be rewritten and focused on the highlights/originalities of the paper. As the working hypothesis shall be modified, the conclusion shall also be modified according to the objective of the study. Avoid copy and paste some sentences in the abstract.
Response: We have modified the conclusion section as per the suggestion of the reviewer
Author Response File: Author Response.docx
Reviewer 3 Report
MS 1458413. This is a very basic study about the detection and quantification of heavy metals in soil and biological (snails) samples in polluted sites. Although it could be an interesting study about the potential role of land snails as bioindicators of heavy metal pollution, the presentation must be improved considerably before the manuscript could be considered for publication.
Please find below some suggestions as well as some specific recommendations for revision:
Abstract
- Please review this section, the wording is a bit confusing.
- Line 18: Please replace “objective” by “aim”.
- Line 20: “..and study whether land snails act as bio-indicator for heavy metal contamination in the urban area.” It is not consistent with the true objective of the work that is presented more clearly in the Introduction section: “….to estimate their potential role as bio-indicators for heavy metals contamination in soils.”
- Rewrite the results more clearly gathering the information regarding the snails and the soil samples.
- The last sentence sounds confusing, please separate the concepts and rewrite clearly.
Introduction
- This section reads as very fragmented. I suggest writing coherently.
- In the first paragraph, only humans are mentioned as susceptible to the effects of heavy metals, however the aim of this study is to analyze the bioaccumulation of these pollutants in molluscs. It would be convenient to introduce the risk in all the organisms that are part of the impacted environments, since in the second paragraph it refers directly to snails.
- Line 54: “….. of the aquatic environment and continuously exist in oceanic organisms through bioaccumulation [10,11]. Consequently, land snails have been recommended…” What do the authors mean?
- Line 67: “Besides, land snails in some places can be eaten by humans 67 as these have been consumed by numerous animals (hedgehogs, carabid beetles, and 68 thrushes)” Please revise this sentence.
- It is striking that the authors do not consider the concept of biomagnification if they refer to the transfer of heavy metals through the trophic chain.
- Line 74: “present research work” I suggest selecting an appropriate term.
M&M
Please review this whole section, the wording is a bit confusing.
- Why did the authors select that time of year to collect the snails? Are there any physiological characteristics that make them more susceptible to heavy metal toxicity?
- Some important details about the snail samples should be included as part of the body analyzed, size, e.g.
- Line 110: “were properly labeled by using a permanent fabric marker pen and” Please delete.
- “2.5. Soil and snail digestion”. Please detail the methodology about heavy metals analysis in both types of samples more clearly.
Results and Discussion
Please revise all this section, as it is written in a very confusing way and some paragraphs sound redundant.
- It could have been interesting to evaluate the granulometry of the soil samples.
- If only Eobonia vermiculata was evaluated in the study, it would be convenient to mention the organism as such to facilitate comparison with other studies where other species of snails were evaluated since they could present different bioaccumulation capacity.
- It is not clear how the authors use the terms “LOI” and “SOM”.
- Line 169: “The results of the descriptive statistics showed that the mean values of all heavy metals were below the Canadian standards except for Ni. Moreover, the concentration of Cr in the soil samples from half of the location sites was greater than Canadian standards” Sounds contradictory, please rewrite more clearly.
- Line 379: “As the concentration of heavy metals (except Ni) in the current study soil was lower than the Canadian standard for soil guidelines….”?? Please see the previous comment.
- The toxic mechanism of heavy metals could be explained in terms of their ecotoxicological risk since the discussion seems very poor and only comes down to a simple quantitative analysis of these pollutants.
Author Response
Response to the reviewer 3 comments
MS 1458413. This is a very basic study about the detection and quantification of heavy metals in soil and biological (snails) samples in polluted sites. Although it could be an interesting study about the potential role of land snails as bioindicators of heavy metal pollution, the presentation must be improved considerably before the manuscript could be considered for publication. Please find below some suggestions as well as some specific recommendations for revision:
Response: Thanks for your recommendation. We have incorporated all the suggestions made by the reviewer and highlighted them as yellow
Abstract
- Please review this section, the wording is a bit confusing.
Response: The whole section was reviewed and corrected
- Line 18: Please replace “objective” by “aim”.
Response: Replaced (Please see line 18)
- Line 20: “... and study whether land snails act as bioindicator for heavy metal contamination in the urban area.” It is not consistent with the true objective of the work that is presented more clearly in the Introduction section: “….to estimate their potential role as bio-indicators for heavy metals contamination in soils.”
- Rewrite the results more clearly gathering the information regarding the snails and the soil samples.
Response: The objectives have been modified in the Introduction section. The results have been rewritten more clearly and highlighted
- The last sentence sounds confusing, please separate the concepts and rewrite clearly.
Response: The last sentence has been modified (Please see lines 35-38)
Introduction
- This section reads as very fragmented. I suggest writing coherently.
- In the first paragraph, only humans are mentioned as susceptible to the effects of heavy metals, however the aim of this study is to analyze the bioaccumulation of these pollutants in molluscs. It would be convenient to introduce the risk in all the organisms that are part of the impacted environments, since in the second paragraph it refers directly to snails.
- Line 54: “….. of the aquatic environment and continuously exist in oceanic organisms through bioaccumulation [10,11]. Consequently, land snails have been recommended…” What do the authors mean?
Response: This part that deals with aquatic was deleted as recommended by another reviewer (to delete aquatic research) “….. of the aquatic environment and continuously exist in oceanic organisms through bioaccumulation.
- Line 67: “Besides, land snails in some places can be eaten by humans 67 as these have been consumed by numerous animals (hedgehogs, carabid beetles, and 68 thrushes)” Please revise this sentence.
Response: The sentence was rewritten (Please see lines 67-69)
- It is striking that the authors do not consider the concept of biomagnification if they refer to the transfer of heavy metals through the trophic chain.
Response: We have added a sentence highlighting the biomagnification by entering into the food chain (Please see lines 50-51)
- Line 74: “present research work” I suggest selecting an appropriate term.
Response: Corrected (Please see line 74)
M&M
Please review this whole section, the wording is a bit confusing.
Response: We have modified the whole section and made it easy to understand
- Why did the authors select that time of year to collect the snails? Are there any physiological characteristics that make them more susceptible to heavy metal toxicity?
Response: The sentence was modified as “The snail and soil samples were collected from urban areas of Sulaymaniyah during autumn and early spring 2020 as the snail remain active during these seasons.” (Please see lines 88-89)
- Some important details about the snail samples should be included as part of the body analyzed, size, e.g.
Response: We have added the suggested information (Please see lines 104-105)
- Line 110: “were properly labeled by using a permanent fabric marker pen and” Please delete.
Response: Deleted
- “2.5. Soil and snail digestion”. Please detail the methodology about heavy metals analysis in both types of samples more clearly.
Response: We have added more information to clarify the methodology
Results and Discussion
Please revise all this section, as it is written in a very confusing way and some paragraphs sound redundant.
Response: We have modified all the sections and made changes where necessary and highlighted
- It could have been interesting to evaluate the granulometry of the soil samples.
Response: We agree with the reviewer that it could have been interesting to evaluate the granulometry of the soil samples. Sorry, we have not done this in the present study. We will consider this important suggestion in our future studies
- If only Eobonia vermiculata was evaluated in the study, it would be convenient to mention the organism as such to facilitate comparison with other studies where other species of snails were evaluated since they could present different bioaccumulation capacity.
Response: We have compared the accumulation of heavy metals in different snail species (Please see lines 299-300, 311-312, 333-334, 351-352, and 362-363)
- It is not clear how the authors use the terms “LOI” and “SOM”.
Response: We have replaced “LOI” with “Organic matter” as per the suggestion of other reviewers as well
- Line 169: “The results of the descriptive statistics showed that the mean values of all heavy metals were below the Canadian standards except for Ni. Moreover, the concentration of Cr in the soil samples from half of the location sites was greater than Canadian standards” Sounds contradictory, please rewrite more clearly.
Response: We have modified the sentences as per the suggestion of the reviewer “The results of the descriptive statistics showed that the mean values of As, Pb and Zn were below the Canadian standards except for Ni in all and Cr in half of the soil samples.” (Please see line 184-185)
- Line 379: “As the concentration of heavy metals (except Ni) in the current study soil was lower than the Canadian standard for soil guidelines….”?? Please see the previous comment.
Response: Modified as per the suggestion of the reviewer
- The toxic mechanism of heavy metals could be explained in terms of their ecotoxicological risk since the discussion seems very poor and only comes down to a simple quantitative analysis of these pollutants.
Response: We have highlighted the mechanisms of heavy metals toxicity to different snail species and it was dependent on the species under study, their body size, location, etc. (Please see lines 379-382 and 391-398)
Author Response File: Author Response.docx
Reviewer 4 Report
Title: Land snail as bioindicators of the heavy metal pollution in the urban areas of Sulaimani, Iraq
Authors: Aso H. Saeed H. Salih, Abdulla A. Hama, Karzan A. M. Hawrami, Allah Ditta
Manuscript ID: sustainability-1458413
Comments on Manuscript Review
This study reports on some heavy metals levels (As, Cr, Ni, Pb, and Zn) in snails associated with the soil from different locations and study whether the land snail Eobania vermiculata act as a bioindicator for heavy metal pollution in the Suliamani urban areas, Iraq. A high metal pollution load especially Cr and thus residents are under threat for using snails’ soft tissues as food. I'm not sure if Iraqi Kurdistan eats snails and if so I am sure this study will be useful to consumers in that country. Overall this article is interesting and contains good information from Iraq. There are however a number of major points strongly consider in revising their paper:
1- the manuscript would need an accurate general editorial revision as some sentences are complicated in different parts of the text because the English is not clear enough.
2- There are a lot of lacking information about the reliability of methodology. i.e. accuracy, precision and validation of the analytical procedure. So the authors should perform the quality control procedure. As for the analytical accuracy of methodology- which certified reference material used for each matrix (soil or snail)? and where the certified reference material was purchased? for the analytical precision- how many replicates analyses of each soil or snail sample? and for the validation of the analytical procedure- what are the values of recoveries of all metals studied? Also, the authors should be mentioned the limit of detection (LOD) and limit of quantification (LOQ) of each element in both soil and snail due to the method used.
3- Please spell out the genus names of species in full, upon first mention in the text. Every species must at least once be spelled with its full name (lines # 287, 298, 299, 311, 312, 331, 332). The authors should be also updated the scientific names as MolluscaBase (http://www.molluscabase.org/): for example; Cornu aspersum or Cantareus aspersus (before Helix aspersa), Lissachatina fulica (before Achatina fulica), Massylaea vermiculata (before Eobania vermiculata)- Please check the other species throughout the text.
Title:
I would suggest a more specific title somewhat like "The land snail, Massylaea vermiculata as a bioindicator of heavy metal pollution in the urban areas of Sulaimani, Iraq "
Abstract:
Line # 19 Please add "the levels of some" after "to investigate" .
Line # 20 re-write the sentence as "whether the land snail, Massylaea vermiculata acts as a bioindicator for ---------".
Line # 21 re-write the sentence as "Soil and snail samples (30 per each) were taken from Sulaimani urban areas.
Line # 23 chromium (Cr) like that in Line # 28 nickel (Ni)
Lines # 29 & 30 re-write the sentence as " The snails bioaccumulate certain metals in their tissues in the following order: Cr > Zn >Ni
Line # 31 change " association" to " correlation" because association is not a statistical word.
Line # 34 Please add the scientific name of the tested snail as "the land snail, M. vermiculata is an --------".
Line # 35 change "snail" to " organism" also change "with snail" to "with this snail".
Lines # 35 &36 Change "must" to "should" also change "discovered" to "occurred".
Keywords: re-write as "land snails; Massylaea vermiculata; heavy metals; bioindicator; Sulaimani urban soil"
Introduction:
Line # 50 re-write the sentence as "Land snails inhabit in terrestrial environments are sensitive to pollutants and soil acidification".
Lines # 72-77 re-write the paragraph as "Based on the above, it has been hypothesized that the land snails may serve as bio-indicators of trace elements contamination in the terrestrial environment. The objective of the present research work was to investigate the concentration and distribution of heavy metals (As, Cr, Ni, Pb, and Zn) in Sulaimani's urban soil and the native land snail, Massylaea vermiculata in urban areas and to estimate its potential role as a bio-indicator for soil heavy metals contamination".
Materials and Methods:
Line # 99 Pl add "soil" before "samples".
Lines # 112-113 snail samples were divided into two groups: one was merged in ethanol (70%) for detecting the genome of the snail species – where, how to analyze and which method is used to characterize the genome? Give one to two lines in the results section which indicate this aspect for identifying the snail species Massylaea vermiculata.
Lines # 113-116 The other group of samples was washed by using tap water, rinsed in distilled water for final cleaning and washing, then the samples were dissected splendidly from the shell and dried in an oven at 105 °C for 18 hours before milling with ceramic pestle and mortar-which organ/tissue was used to determine the concentration of the examined metals -Is hepatopancreas, albumen gland, digestive tract, reproductive system, mantle, foot, shell or whole soft tissues. please verify?
Line # 127 & Tables 2 & 4 The authors used five replicates from the soil for each site to determine its physicochemical properties, but they did not mention the number of replicates from snails - is it one replicate? Please clarify. Also, clarify the number of determinations per each metal ?
Line # 129 Please indicate how many samples were digested for each soil site and associated snails.
Lines # 138-141 The sources of all reagents used in the study should be in an analytical grade. Please clarify
Lines # 142-145 The authors noted that metals analyzed and quantified using ICP-OES. However, still insufficient details about the instrumental (iCAP 7600 ICP-OES (Thermo Fisher)) equipment's and operating conditions such as nebulizer, autosampler, RF power, nebulizer gas pressure, and integration time for axial and radial.
Results and Discussion:
Line # 151 Please change "3.1. Soil description" to "Levels of heavy metals in soil"
Line # 270 Please change "3.2. Snail description" to "Levels of heavy metals in snail"
Lines # 271, 305 Please update the scientific name of snail as M. vermiculata
Line # 287 Please spell out the genus names of species in full (C. capucinus, L. melanostoma, C. obtusa, and N. articulata)
Lines # 289,299 the same as above T. zilli, C. gariepinus
Lines # 311, 331 Please spell out the genus names of species in full as Helix engaddensis
Lines # 312, 332 Please spell out the genus names of species in full (B. bengalensis)
Line # 366 Please write the scientific name of the Roman snail as "Helix pomatia (Roman snail) samples from eight locations and concluded that all snails originated in"
Line # 391 Please change "such as" to " as follows"
Line # 393 Please update the scientific name of snail as M. vermiculata
Line # 394 Please change "no associations" to "no correlations"
Tables:
Lines # 165-168 Please re-write the title of table (2) as " Table 2: pH, organic matter (%) and some heavy metal concentrations (mg/kg dry weight basis) in soil samples collected from thirty different locations in Sulaimani city.
Table (2): In the third column, Pl change "LOI (%)" to " Organic matter (%)" Also in the footnote under the table, Pl correct as "Each value represents mean ± SD (n=5)"
Table (3): In the first column, Pl change "LOI (%)" to " Organic matter (%)" and justify and indicate statistical indicator (* or **) super values.
Lines # 275-277 Please re-write the title of table (4) as " Table 4: Concentrations of Pb, As, Cr, Ni, and Zn (mg/kg dry weight basis) in snails collected from thirty different soil locations in Sulaimani city"
Table (4), in the heading of columns 2-6 Pl delete "in snail" - Also under the table (4), the authors refer to BDL = Below detection limit -What is the detection limit for each metal according the procedure used?
Overall I recommend the manuscript to be accepted after taking into consideration the above major comments and suggestions.
Author Response
Response to the reviewer 4 comments
Title: Land snail as bioindicators of the heavy metal pollution in the urban areas of Sulaimani, Iraq
Authors: Aso H. Saeed H. Salih, Abdulla A. Hama, Karzan A. M. Hawrami, Allah Ditta
Manuscript ID: sustainability-1458413
Comments on Manuscript Review
This study reports on some heavy metals levels (As, Cr, Ni, Pb, and Zn) in snails associated with the soil from different locations and study whether the land snail Eobania vermiculata act as a bioindicator for heavy metal pollution in the Suliamani urban areas, Iraq. A high metal pollution load especially Cr and thus residents are under threat for using snails’ soft tissues as food. I'm not sure if Iraqi Kurdistan eats snails and if so I am sure this study will be useful to consumers in that country. Overall this article is interesting and contains good information from Iraq. There are however a number of major points strongly consider in revising their paper:
Response: Thanks for the appreciation and recommendation of our manuscript
1- the manuscript would need an accurate general editorial revision as some sentences are complicated in different parts of the text because the English is not clear enough.
Response: We have tried our level best to correct any typos and/or grammatical mistakes in the whole manuscript and highlighted as yellow where changes were made
2- There are a lot of lacking information about the reliability of methodology. i.e. accuracy, precision and validation of the analytical procedure. So the authors should perform the quality control procedure. As for the analytical accuracy of methodology- which certified reference material used for each matrix (soil or snail)? and where the certified reference material was purchased? for the analytical precision- how many replicates analyses of each soil or snail sample? and for the validation of the analytical procedure- what are the values of recoveries of all metals studied? Also, the authors should be mentioned the limit of detection (LOD) and limit of quantification (LOQ) of each element in both soil and snail due to the method used.
Response: The metals concentration was detected using an inductively coupled plasma - optical emission spectrometry (ICP-OES) instrument (iCAP™ 7600, Thermo Fisher Scientific, USA). The ICP-OES parameters/conditions were as follows: Detector type = High-performance solid-state CID86 chip, View direction = Radial, Nebulizer = V-groove, Spray chamber = Glass cyclonic, UV exposure time = 15 s, UV RF power = 1150 W, UV nebulizer gas flow = 0.5 L min.-1, VIS exposure time = 5 s, VIS RF power = 1150 W, VIS nebulizer gas flow = 0.5 L min.-1, Cool gas flow rate = 12 L min.-1 and Auxiliary gas flow rate = 0.5 L min.-1 (Please see lines 149-155). We have mentioned the limit of detection (LOD) and limit of quantification (LOQ) of each element in both soil and snail due to the method used (Please see lines 329-330)
3- Please spell out the genus names of species in full, upon first mention in the text. Every species must at least once be spelled with its full name (lines # 287, 298, 299, 311, 312, 331, 332). The authors should be also updated the scientific names as MolluscaBase (http://www.molluscabase.org/): for example; Cornu aspersum or Cantareus aspersus (before Helix aspersa), Lissachatina fulica (before Achatina fulica), Massylaea vermiculata (before Eobania vermiculata)- Please check the other species throughout the text.
Response: All the genus names of species were full spelled out and the scientific names were updated as well from both of the MolluscaBase and WORM World Register of Marine Species sites.
http://www.marinespecies.org/aphia.php?p=taxlist and http://www.molluscabase.org
1- Eobania vermiculata (O. F. Müller, 1774)
Status: accepted
https://www.molluscabase.org/aphia.php?p=taxdetails&id=934402
2- Massylaea vermiculata (O. F. Müller, 1774)
Status: unaccepted
Accepted Name: Eobania vermiculata (O. F. Müller, 1774)
https://www.molluscabase.org/aphia.php?p=taxdetails&id=1035519
3- Cornu aspersum (O. F. Müller, 1774)
Status: accepted
https://www.molluscabase.org/aphia.php?p=taxdetails&id=820000
4- Cantareus aspersus (O. F. Müller, 1774)
Status: unaccepted
Accepted Name: Cornu aspersum (O. F. Müller, 1774)
https://www.molluscabase.org/aphia.php?p=taxdetails&id=1289510
5- Lissachatina fulica (Bowdich, 1822)
Status: accepted
https://www.molluscabase.org/aphia.php?p=taxdetails&id=881469
6- Tilapia zilli (Gervais, 1848)
Status: unaccepted
Accepted Name: Coptodon zillii (Gervais, 1848)
http://www.marinespecies.org/aphia.php?p=taxdetails&id=405519
7- Achatina (Achatina) achatina (Linnaeus, 1758)
Status: alternate representation
Accepted Name: Achatina achatina (Linnaeus, 1758)
Represented as Achatina Lamarck, 1799
https://www.molluscabase.org/aphia.php?p=taxdetails&id=1430683
8- Chicoreus (Rhizophorimurex) capucinus (Lamarck, 1822)
Status: alternate representation
Accepted Name: Chicoreus capucinus (Lamarck, 1822)
Represented as Chicoreus Montfort, 1810
https://www.molluscabase.org/aphia.php?p=taxdetails&id=406095
9- Nerita (Amphinerita) articulata Gould, 1847
Status: unaccepted
Accepted Name: Nerita balteata Reeve, 1855
http://www.marinespecies.org/aphia.php?p=taxdetails&id=818076
10- Cerithidea obtusa (Lamarck, 1822)
Status: accepted
http://www.marinespecies.org/aphia.php?p=taxdetails&id=215141
11- Littoraria melanostoma (Gray, 1839)
Status: accepted
https://www.molluscabase.org/aphia.php?p=taxdetails&id=445603
Title:
I would suggest a more specific title somewhat like "The land snail, Massylaea vermiculata as a bioindicator of heavy metal pollution in the urban areas of Sulaimani, Iraq "
Response: We have modified the title as “Land snail, Eobania vermiculata as a bioindicator of the heavy metal pollution in the urban areas of Sulaimani, Iraq”
Abstract:
Line # 19 Please add "the levels of some" after "to investigate".
Response: We have modified it as “The aim of the present study was to investigate the concentration of heavy metals …” (Please see lines 18-19)
Line # 20 re-write the sentence as "whether the land snail, Massylaea vermiculata acts as a bioindicator for ---------".
Response: The mentioned sentence has been modified as per the suggestion of the reviewer (Please see line 20)
Line # 21 re-write the sentence as "Soil and snail samples (30 per each) were taken from Sulaimani urban areas.
Response: The sentence has been modified as “Thirty soil and snail samples in triplicate from each sampling site were taken from the urban areas of Suliamani”
Line # 23 chromium (Cr) like that in Line # 28 nickel (Ni)
Response: The full form of Chromium has been mentioned (Please see line 24)
Lines # 29 & 30 re-write the sentence as " The snails bioaccumulate certain metals in their tissues in the following order: Cr > Zn >Ni
Response: The sentence has been modified (Please see lines 30-31)
Line # 31 change "association" to "correlation" because association is not a statistical word.
Response: Changed (Please see line 33)
Line # 34 Please add the scientific name of the tested snail as "the land snail, M. vermiculata is an --------".
Response: The mentioned sentence has been modified (Please see lines 35-36)
Line # 35 change "snail" to "organism" also change "with snail" to "with this snail".
Response: Changed "snail" to “organism" (Please see line 36) also changed "with snail" to "with this snail" (Please see line 37)
Lines # 35 &36 Change "must" to "should" also change "discovered" to "occurred".
Response: Changed (Please see line 37)
Keywords: re-write as "land snails; Massylaea vermiculata; heavy metals; bioindicator; Sulaimani urban soil"
Response: Keywords have been modified (Please see line 39)
Introduction:
Line # 50 re-write the sentence as "Land snails inhabit in terrestrial environments are sensitive to pollutants and soil acidification".
Response: Modified the sentence as per the suggestion of the reviewer (Please see lines 50-51)
Lines # 72-77 re-write the paragraph as "Based on the above, it has been hypothesized that the land snails may serve as bio-indicators of trace elements contamination in the terrestrial environment. The objective of the present research work was to investigate the concentration and distribution of heavy metals (As, Cr, Ni, Pb, and Zn) in Sulaimani's urban soil and the native land snail, Massylaea vermiculata in urban areas and to estimate its potential role as a bio-indicator for soil heavy metals contamination".
Response: We have the mentioned paragraph (Please see lines 73-78)
Materials and Methods:
Line # 99 Pl add "soil" before "samples".
Response: Added (Please see line 100)
Lines # 112-113 snail samples were divided into two groups: one was merged in ethanol (70%) for detecting the genome of the snail species – where, how to analyze and which method is used to characterize the genome? Give one to two lines in the results section which indicate this aspect for identifying the snail species Massylaea vermiculata.
Response: The explanation has been added (Please see lines 113-116 and 402-409)
Lines # 113-116 The other group of samples was washed by using tap water, rinsed in distilled water for final cleaning and washing, then the samples were dissected splendidly from the shell and dried in an oven at 105 °C for 18 hours before milling with ceramic pestle and mortar-which organ/tissue was used to determine the concentration of the examined metals -Is hepatopancreas, albumen gland, digestive tract, reproductive system, mantle, foot, shell or whole soft tissues. please verify?
Response: The whole soft tissues were used to determine the concentration of the examined metals (Please see lines 118-119).
Line # 127 & Tables 2 & 4 The authors used five replicates from the soil for each site to determine its physicochemical properties, but they did not mention the number of replicates from snails - is it one replicate? Please clarify. Also, clarify the number of determinations per each metal ?
Response: From every location site, three samples of soil and snail were digested by microwave acid -digestion. Therefore, the total number of digested samples (soil and snail) was equal to 180 samples (Please see lines 133-136)
Line # 129 Please indicate how many samples were digested for each soil site and associated snails.
Response: From every location site, three samples of soil and snail were digested by microwave acid -digestion. Therefore, the total number of digested samples (soil and snail) was equal to 180 samples (Please see lines 133-136)
Lines # 138-141 The sources of all reagents used in the study should be in an analytical grade. Please clarify
Response: All reagents used in the present study were of analytical grade e.g. HNO3 and H2O2 were purchased from Biochem - France (analytical grade) while ICP- standard solution (multi-elements standard QSC (27E) with CRM certificate was purchased from Chem-lab –Belgium. (Please see lines 156-160)
Lines # 142-145 The authors noted that metals analyzed and quantified using ICP-OES. However, still insufficient details about the instrumental (iCAP 7600 ICP-OES (Thermo Fisher)) equipment's and operating conditions such as nebulizer, autosampler, RF power, nebulizer gas pressure, and integration time for axial and radial.
Response: We have added the requested information (Please see lines 149-155)
Results and Discussion:
Line # 151 Please change "3.1. Soil description" to "Levels of heavy metals in soil"
Response: We have changed to “Concentration of heavy metals in soil samples” (Please see line 170)
Line # 270 Please change "3.2. Snail description" to "Levels of heavy metals in snail"
Response: We have changed to “Concentration of heavy metals in snail samples” (Please see line 289)
Lines # 271, 305 Please update the scientific name of snail as M. vermiculata
Response: We have not updated as Eobonia vermiculata is correct while M. vermiculata is not acceptable.
Line # 287 Please spell out the genus names of species in full (C. capucinus, L. melanostoma, C. obtusa, and N. articulata)
Response: We have written the full form of all the mentioned species (Please see lines 303-304)
Lines # 289,299 the same as above T. zilli, C. gariepinus
Response: We have written the full form of all the mentioned species (Please see lines 315-316)
Lines # 311, 331 Please spell out the genus names of species in full as Helix engaddensis
Response: We have written full (Please see line 336)
Lines # 312, 332 Please spell out the genus names of species in full (B. bengalensis)
Response: We have written the full genus name when appeared the first time and abbreviated it when appeared later (Please see line 337)
Line # 366 Please write the scientific name of the Roman snail as "Helix pomatia (Roman snail) samples from eight locations and concluded that all snails originated in"
Response: We have written the scientific name of the Roman snail as per the suggestion of the reviewer (Please see lines 388-389)
Line # 391 Please change "such as" to " as follows"
Response: Corrected (Please see line 419-420)
Line # 393 Please update the scientific name of snail as M. vermiculata
Response: We have not updated as Eobonia vermiculata is correct while M. vermiculata is not acceptable.
Line # 394 Please change "no associations" to "no correlations"
Response: We have changed "no associations" to "no correlations" (Please see line 423)
Tables:
Lines # 165-168 Please re-write the title of table (2) as " Table 2: pH, organic matter (%) and some heavy metal concentrations (mg/kg dry weight basis) in soil samples collected from thirty different locations in Sulaimani city.
Response: We have replaced the title as per the suggestion of the reviewer and highlighted
Table (2): In the third column, Pl change "LOI (%)" to " Organic matter (%)" Also in the footnote under the table, Pl correct as "Each value represents mean ± SD (n=5)"
Response: We have replaced “LOI” with “organic matter” and corrected the Table footnotes as per the suggestion of the reviewer
Table (3): In the first column, Pl change "LOI (%)" to " Organic matter (%)" and justify and indicate statistical indicator (* or **) super values.
Response: We have replaced “LOI” with “organic matter” in Table 3 and checked throughout the manuscript and highlighted where changes were made. The statistical indicators added were typos and removed
Lines # 275-277 Please re-write the title of table (4) as " Table 4: Concentrations of Pb, As, Cr, Ni, and Zn (mg/kg dry weight basis) in snails collected from thirty different soil locations in Sulaimani city"
Response: We have added the title of Table 4 as per the suggestion of the reviewer
Table (4), in the heading of columns 2-6 Pl delete "in snail" - Also under the table (4), the authors refer to BDL = Below detection limit -What is the detection limit for each metal according the procedure used?
Response: Thanks for your comment and suggestion. We have deleted “in snail” and written the detection limits for different metals measured in the present study (Please see Table 4)
Overall I recommend the manuscript to be accepted after taking into consideration the above major comments and suggestions.
Response: Thanks for your acceptance and recommendation. Your comments and suggestions have improved our manuscript very much
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
the revised version answers most of my comments. However some minor points need to be considered: regarding the supplemental material provided by authors, I don't understand what is ST1, ST2 ST3? Authors must explain if these standard are for soil? animal tissue? if yes which king of tissue (usually lobster hepatopancreas with know metal concentration is used). The supplemental material must be cited in the text. Lines 391-395 must be modified: the interpretation of the paper is not good. In the bibliography , all the references are now in yellow. Some were changed btu I think that some references can be deleted to reduce the number (e.g. de Wolf; Liang, Cravo; Sivaperumal; adedeji: focused on aquatic gastropod).Author Response
Response to the reviewer 2 comments
Comment: The revised version answers most of my comments. However some minor points need to be considered:
Response: Thanks for your appreciation. We have incorporated all the suggestions and comments raised
Comment: Regarding the supplemental material provided by authors, I don't understand what is ST1, ST2 ST3? Authors must explain if these standard are for soil? animal tissue? If yes which kind of tissue (usually lobster hepatopancreas with known metal concentration is used).
Response: We have explained about ST1, ST2, and ST3 in the text as “these were a standard solution with different concentrations of heavy metals as clear from the supplementary file” We have already clarified that standards used during the analysis of soil and snail samples were solutions multi-elements standard QSC (27E) (ST1, ST2 and ST3), made from chemicals with CRM certificate from Chem - lab - Belgium (Please see lines 157-190)
Comment: The supplemental material must be cited in the text.
Response: We have cited the supplementary material (Please see lines 159-160)
Comment: Lines 391-395 must be modified: the interpretation of the paper is not good.
Response: We have modified the suggested sentence as “The results of another study conducted at Metaleurop Nord, Nord-Pas-de-Calais, France, showed that the species impact was the greatest significant variable explaining the soft tissue concentrations of snails [60].” (Please see lines 392-394)
Comment: In the bibliography, all the references are now in yellow. Some were changed btu I think that some references can be deleted to reduce the number (e.g. de Wolf; Liang, Cravo; Sivaperumal; adedeji: focused on aquatic gastropod).
Response: We have deleted the suggested references
Author Response File: Author Response.docx
Reviewer 3 Report
The authors have made some modifications in the revision. This review has no further comments so the manuscript could be considered for publication.
Author Response
Response to the reviewer 3 comments
Comment: The authors have made some modifications in the revision. This review has no further comments so the manuscript could be considered for publication.
Response: Thanks for your appreciation and recommendation
Author Response File: Author Response.docx
Reviewer 4 Report
After the second revision, the manuscript ID: 1458413R1 entitled "Land snail as bioindicators of the heavy metal pollution in the urban areas of Sulaimani, Iraq" has been substantially improved and modification/answers to all comments/suggestion are acceptable.
Author Response
Response to the reviewer 4 comments
Comment: After the second revision, the manuscript ID: 1458413R1 entitled "Land snail as bioindicators of the heavy metal pollution in the urban areas of Sulaimani, Iraq" has been substantially improved and modification/answers to all comments/suggestion are acceptable.
Response: Thanks for your appreciation and recommendation
Author Response File: Author Response.docx
