Ecological Characterization and Bio-Mitigation Potential of Heavy Metal Contamination in Metallurgically Affected Soil

Round 1

Reviewer 1 Report

Was reference material used to verify the quality of the results obtained? If yes, please specify which and which contents of the tested metals were determinable, and which should be.

Any statistical methods used are not described. Most studies of this type,  not have a normal distribution. For this reason, non-parametric statistical tests are used. In the text we have that statistical significance of differences, but it is not clear what test was used. This should be precisely described in the methodology. 

For what reason the As and Mo content of vegetation was not investigated when exactly these elements were in significant amounts in the environment, especially in close distances from the pollution source.

Mo and As were present in the highest concentrations at the closest locations from the source, hence it can be suspected that their transfer is probably related to large particulates, as they are the ones that fall fastest on the ground due to their weight.

Author Response

We highly appreciate the reviewer for the response on this descriptive manuscript. We want our paper to be understandable for the general community, so we have expanded the description about heavy metal pollution in soil and some explanations. Please find below our detailed responses and corresponding revisions.

Was reference material used to verify the quality of the results obtained? If yes, please specify which and which contents of the tested metals were determinable, and which should be.
The reference material (water) was used to make sure that the analyzer remained calibrated during the measurements and measured accurately.  Unfortunately, soil or biological reference materials were generally not available to verify the quality of the results.

Any statistical methods used are not described. Most studies of this type, not have a normal distribution. For this reason, non-parametric statistical tests are used. In the text we have that statistical significance of differences, but it is not clear what test was used. This should be precisely described in the methodology.

We have inserted the description of all the statistical methods used (lines 223-228), which is described as below.

Since the Geary’s test of normality showed nearly normal distribution of HMs and biomass [27], the Student’s t-test was used to determine statistical significance of differences between HM/biomass value averaged for the sites located within 50-700 m distance from the Alaverdi copper smelter and HM/biomass value measured in the reference site [28]. Relationship between HMs and between HMs and biomass was determined by the Pearson’s correlation coefficient [29].

27. Geary, R.C. The Ratio of the Mean Deviation to the Standard Deviation as a Test of Normality. Biometrika 1935, 27, 310–332. https://0-doi-org.brum.beds.ac.uk/10.2307/2332693
28. The Probable Error of a Mean. Biometrika 1908, 6, 1–25. https://0-doi-org.brum.beds.ac.uk/10.2307/2331554
29. Benesty, J., Chen, J., Huang, Y., Cohen, I. Pearson Correlation Coefficient. In Noise Reduction in Speech Processing; Springer: Berlin, Heidelberg, Germany, 2009, 1–4. https://0-doi-org.brum.beds.ac.uk/10.1007/978-3-642-00296-0_5

For what reason the As and Mo content of vegetation was not investigated when exactly these elements were in significant amounts in the environment, especially in close distances from the pollution source.

Mo and As were present in the highest concentrations at the closest locations from the source, hence it can be suspected that their transfer is probably related to large particulates, as they are the ones that fall fastest on the ground due to their weight.

Soil and biological samples were analyzed in the frame of different projects, and this difference in metal species between soil and biological samples was conditioned by the resources and capacities of the projects. Considering also the valuable information provided by you that Mo and As are mostly accumulated in close distance from the source due to their weight, it seems that, in our study, the biological samples located about 800 m far from the smelter would hardly accumulate noticeable concentrations of As and Mo, which not only supports your observation but also somewhat mitigates gap in our measurements. Thank you for this.

Author Response File: Author Response.doc

Reviewer 2 Report

Please find the attached file for comments details. 

Comments for author File: Comments.pdf

Author Response

Although this article is interesting but some corrections are needed for publication. Some of mandatory comments given below in order to process it further process.

We sincerely appreciate of your review and positive feedback on the manuscript! We want our paper to be understandable for the general community, so we have expanded the description about heavy metal pollution in soil and some explanations. Please find below our detailed responses and corresponding revisions.

1. Abstract: Line No 10: Replace the words environment when pollute soil with environmental pollution as it is more scientific.

We have replaced the words according to the reviewer's suggestion (line 19).

2. Introduction: Line No 64-65: Please separate the aims and objectives of the study. Authors should described the aims of the study in one paragraph.

We have separated the aims and objectives according to the reviewer's comment (lines 63-66).

3. Material and methods: Line: 109-112: All glassware used were pre-washed with 10% HNO₃, followed by rinsing with distilled water prior to use. To ensure that the AAS remained calibrated during the experiments, certified reference materials were analyzed for soil and biological samples. Reference is missing. Please mention appropriate reference.

We have mentioned a reference (line 113).

4. Material and methods: Line: 222-223: Please mention the statistical section as this study is based on analyses. Please mentioned appropriate references in statistical section or relevant supportive.

We have titled the statistical section (line 222) and added appropriate references (line 223-228), which is given bellow.

2.5. Statistical Analyses

Since the Geary’s test of normality showed nearly normal distribution of HMs and biomass [27], therefore, the Student’s t-test was used to determine the statistical sig-nificance of differences between HM/biomass value averaged for the sites located within 50–700 m distance from the Alaverdi copper smelter and HM/biomass value measured in the reference site [28]. Relationship between HMs and between HMs and biomass was determined by the Pearson’s correlation coefficient [29]. Statistical anal-yses were performed using Statistica 8 and Microsoft Excel 2019 software programs.

27. Geary, R.C. The Ratio of the Mean Deviation to the Standard Deviation as a Test of Normality. Biometrika 1935, 27, 310–332. https://0-doi-org.brum.beds.ac.uk/10.2307/2332693
28. The Probable Error of a Mean. Biometrika 1908, 6, 1–25. https://0-doi-org.brum.beds.ac.uk/10.2307/2331554
29. Benesty, J., Chen, J., Huang, Y., Cohen, I. Pearson Correlation Coefficient. In Noise Reduction in Speech Processing; Springer: Berlin, Heidelberg, Germany, 2009, 1–4. https://0-doi-org.brum.beds.ac.uk/10.1007/978-3-642-00296-0_5
30. Results and discussion: Line: 331-334: Noncarcinogenic and carcinogenic health risks of single and total HMs in almost all the investigated sites potentially affected by the ex-operation of the Alaverdi copper smelter were even posed through a single pathway such as the ingestion or inhalation of metals. Authors have described the heavy metals effects as it reflected the major prospects of the study. Can this study affect the biological activities of biological organisms?

Hazard index indicating noncarcinogenic and carcinogenic health risks through ingestion, dermal absorption and inhalation of HMs from soil only reflects the effects on human health. For assessing the biological effects, we investigated the biomass and the ecological risk index the results of which are described in the section 3.2.

6. Conclusion: Please summarize the comprehensive one separate paragraph of conclusion section: It is recommended to update it as separate heading as Conclusion it reflected the major study concerns.

We have updated the conclusion section according to the reviewers suggestion (lines 368-395), which is given bellow.

4. Conclusions

This study showed that the ex-operation of the copper smelting plant in Alaverdi Town negatively affected the environment which was expressed by the noticeable HM contamination in the soil even 700 m far from the smelter. Although the contamination decreased with an increase in distance from the smelter, however, the average concentrations of almost all the investigated HMs in the soil affected by the ex-operation of the smelter significantly exceeded the reference levels. Such contamination caused the substantial loss of the biomass of invertebrates occurring in the investigated soil and posed serious ecological risks as well as alarmed to have probability of non-carcinogenic (for children) and carcinogenic health effects. Investigated plant and invertebrate species inhabiting over the smelter showed different levels of HM accumulation indicating opportunities to use the plant and animal species with high capacities of HM accumulation for the selective bioremoval of HMs from the soil. The results presented can be very useful for environmental monitoring and management of soil contamination in mining and metallurgical areas.

5. Recommendations

Based on the capacities of HM accumulation by the investigated plant and invertebrate species, the plant species Fraxinus excelsior, Acer platanoides, Robinia pseudoacacia and Aesculus hippocastanum are recommended to grow in the soil around the Alaverdi copper smelter to mitigate environmental impacts in the ecologically vulnerable areas of Alaverdi Town, while the slug species Deroceras caucasicum and Limax flavus can be used for Cu removal from the soil and the earthworm species Eisenia rosea for Zn removal from the soil. For implementing such mitigation actions, it is necessary to place large and medium-sized stones or make small piles in the area around the smelter which will create ecological conditions attractive for the invertebrates. The plants and animals should be regularly removed from the area and replaced with new ones.

7. Please enlist the abbreviations used throughout the manuscript.

We highly appreciate this suggestion. Following journal’s guidelines, we have expanded all abbreviations the first time they are introduced in the text. This is also done for space considerations. Furthermore, we feel that there are only a limited abbreviations which readers are mostly familiar with. However, we will be glad to include a list of abbreviations used in the manuscript, provided as below in the text.

Agency for Toxic Substances and Disease Registry: ATSDR

Atomic Absorption Spectrometer: AAS

Federal Center for Analysis and Assessment of Technogenic Impact: FCAATI

Federal State Institution: FSI

Health-hazard Quotient: HQ

Heavy Metals: HMs

Office of Environmental Health Hazard Assessment: OEHHA

Overall hazard index: OHI

Potential Ecological Risk Index: PERI

U.S. Department of Energy: USDOE

U.S. Environmental Protection Agency: USEPA

8. References: Please follow the journal guidelines:

Done

Author Response File: Author Response.doc