Remediation of Pb and Cd Polluted Soils with Fulvic Acid

Round 1

Reviewer 1 Report

Comments to Authors

Journal name:  Forests

Article Title:  Remediation of Pb and Cd Polluted Soils with Fulvic Acid

Summary:

Dear Editor,

I have thoroughly reviewed the paper and found it interesting. In my opinion phytoremediation of polluted soil and to promote plant health or clean food production has a great importance in years to come for environment protection.  I therefore, suggest minor revision for this article and recommend it for publication after some modification in the article.

There are few comments which needs to be address in the article:

Abstract

Comment 1.

Please provide data in percentage decrease or increase for Pd & Cd in shoots and roots (Line 19-20) compared to control. Lot of parameters were discussed un-necessary in abstract (line15-17), I think author should emphasis more on significant results of studied parameters (include data %age or values).

Comment 2

Why no comparisons of studied parameters were made with uncontaminated soil (control) to have clear idea of FA in HMs (Pd and Cd) remediation. I think BCFT of control should also be mention and compared in the abstract.

Introduction

Comment 3

Introduction is well written and covers key aspects. Use HM or HMs instead of Heavy Metals in the manuscript.

Material and Methods

Comment 4

Please change the expression mg/kg to mg kg^-1, dS/m to dS m^-1 and so on throughout manuscript.

Comment 5

Briefly describe incubation conditions for soil after heavy metals addition.

Results & discussion

Comment 6

This portion lacks enzymatic assays to determine the negative effect of heavy metals on plant growth especially the treatment without FA application. Add data regarding negative effect of heavy metals on enzymatic activity to assess FA role in alleviating HM stress.

Comments 7

No soil data is presented after the harvest. It is very important to determine heavy metals (Pd and Cd in this case) after harvest to draw conclusion on the efficiency of plant for its phytoremediation ability. In this study FA is applied with soil to mitigate HM stress, so, sequential extraction of heavy metal is a necessary to check the role FA in metals availability or reduction.

Discussion is good and supporting the results in more logical way.

Conclusion

Conclusion is well written and easy to understand. Overall, it is a good study and lot of parameters were analyzed. And it was found that FA is best amendment to remediate Cd/Pb polluted soil and its role in reduction of HMs to upper parts of plant.

Comments for author File: Comments.docx

Author Response

Journal name:  Forests

Article Title:  Remediation of Pb and Cd Polluted Soils with Fulvic Acid

Summary:

Dear Editor,

I have thoroughly reviewed the paper and found it interesting. In my opinion phytoremediation of polluted soil and to promote plant health or clean food production has a great importance in years to come for environment protection.  I therefore, suggest minor revision for this article and recommend it for publication after some modification in the article.

There are few comments which needs to be address in the article:

Abstract

Comment 1.

Please provide data in percentage decrease or increase for Pd & Cd in shoots and roots (Line 19-20) compared to control. Lot of parameters were discussed un-necessary in abstract (line15-17), I think author should emphasis more on significant results of studied parameters (include data %age or values).

Answer1.

Abstract: Heavy metal pollution is among the important environmental problems in the world. Many techniques have already been used to remove the heavy metals such as lead (Pb) and cadmium (Cd). Among them, the phytoremediation method is an environmentally friendly and green technology. This study was carried out to determine the efficiency of fulvic acid (FA) application in removing Pb and Cd from polluted soil using Tagetes eracta and Zinnia elegans ornamental plants. The results indicated that, FA application, number of flower per plants, and plant fresh weight of Tagetes eracta plants and Zinnia elegans plants increased 187.5%, 104.5% and 155.5%, 57.7%, respectively with application of 7000 mg L-1 FA at 100 mg kg-1 Pb pollution condition, whereas 42.85%, 16.5%, and 44.4%-36.1%with application of 7000 mg L-1 FA at 30 mg kg-1 Cd pol-lution condition, respectively. With the FA application in the Zinnia elegans plant, the root part has accumulated 51.53% more Pb than the shoot part. For Cd, the shoot part accumulated 35.33% more Cd than the root. The effect of FA application on superoxide dismutase (SOD), peroxidase (POD) and, catalase (CAT) of the Tagetes eracta were decreased as 32.7%,33.1%, and 35.1% for Pb, 21.2%,25.1%, and 26,1%, for Cd, and 15.1%,22.7%, and 37.7% for Pb, and 7.55%,18.0%, and 18.8% for Cd were in Zinnia elegans respectively. In conclusion, Tagetes eracta and Zinnia elegans can not be recommended for remediation of Pb and Cd polluted area, but FA can be recommended for Pb and Cd stabilization in polluted soil.

Comment 2

Why no comparisons of studied parameters were made with uncontaminated soil (control) to have clear idea of FA in HMs (Pd and Cd) remediation. I think BCFT of control should also be mention and compared in the abstract.

Answer2. I have already mention BCFT in abstract section in the manuscript.

Introduction

Comment 3

Introduction is well written and covers key aspects. Use HM or HMs instead of Heavy Metals in the manuscript.

Answer 3.

We have changed heavy metals as HM in all manuscript.

Material and Methods

Comment 4

Please change the expression mg/kg to mg kg^-1, dS/m to dS m^-1 and so on throughout manuscript.

Answer 4. I have changed mg/kg to mg kg^-1, dS/m to dS m^-1 in all manuscript.

Comment 5

Briefly describe incubation conditions for soil after heavy metals addition.

Answer 5.

The incubation process continued as a 8-week process. During the incubation period, the water level was kept 30% below the soil field capacity and the ambient temperature was 29 degrees. The soils in the pots were mixed homogeneously every week.

Results & discussion

Comment 6

This portion lacks enzymatic assays to determine the negative effect of heavy metals on plant growth especially the treatment without FA application. Add data regarding negative effect of heavy metals on enzymatic activity to assess FA role in alleviating HM stress.

Answer 6

Effects of Fulvic Acid Applied under Heavy Metal Stress on Enzymatic Activity

It is known that the enzyme activities of plants grown in soils contaminated with various heavy metals are affected by stress (Han et al. 2015; Goswami and Das 2016). In this study, the application of FA significantly affected enzyme activity of Tagetes eracta and Zinnia elegans at Pb, and Cd pollution. The effect of FA application on Superoxide dis-mutase (SOD), peroxidase (POD) and, Catalase (CAT) of the Tagetes eracta were decreased as 32.7%, 33.1%, and 35.1% for Pb, 21.2%,25.1%, and 26,1%, for Cd, and Zinnia elegans SOD, POD and CAT activity were 15.1%, 22.7%, and 37.7% for Pb, and 7.55%,18.0%, and 18.8% for Cd, respectively (Table 7). Obtained results showed that, the activities of SOD, CAT and POD were reduced after treatment with FA. This result indicates that FA may serve directly as an antioxidant to eliminate excess ROS, or as a signal molecule to promote antioxidant production. In consistent with our results, Yildirim et al. (2021) reported that FA treatment in garden cress under cadmium stress reduced SOD and CAT activity.

Table 7. Antioxidant enzyme activity of Tagetes eracta and Zinnia elegans

EU: Enzyme unite, SOD: Superoxide dismutase, POD: peroxidase, CAT: Catalase

Comments 7

No soil data is presented after the harvest. It is very important to determine heavy metals (Pd and Cd in this case) after harvest to draw conclusion on the efficiency of plant for its phytoremediation ability. In this study FA is applied with soil to mitigate HM stress, so, sequential extraction of heavy metal is a necessary to check the role FA in metals availability or reduction.

Answer 7.

After harvest soil Cd and Pb contentration was 0.4 mg kg^-1 for Pb, and 2.1 mg kg^-1 for Tagetes eracta  and 0,05 mg mg kg^-1 for Pb and 1.1 mg kg^-1for Zinnia  elegans..

Discussion is good and supporting the results in more logical way.

Conclusion

Conclusion is well written and easy to understand. Overall, it is a good study and lot of parameters were analyzed. And it was found that FA is best amendment to remediate Cd/Pb polluted soil and its role in reduction of HMs to upper parts of plant.

Reviewer 2 Report

The paper addresses an important topic but requires major improvements.
The paper is written carelessly and must first be finished.

Units of measurement are not unitary???.

Decimals are separated sometimes by a point, sometimes by a comma ???.
All methods used for analysis must be presented.
The abbreviations used in the tables must be explained in footnotes.

Author Response

Comment 1.The article has been enriched in terms of grammar.

Answer 1. The article was grammatically checked by a native linguist.

Comment 2. Methods section should be clarify..

Answer 2. The method part has been corrected and made more understandable.

. Materials and Methods

This study to evaluate efficiency of fulvic acid on Pb and Cd uptake from Pb and Cd contaminated soil, and the phytoremediation capacity was conducted using a randomized complete block design

2.1. Plant Growth Parameters

Harvest took place 90 days after seedling planting. Plant height (mm), plant fresh weight (gr), plant dry weight (gr), stem diameter (mm), plant flower number, flower stem length (mm), flower diameter (mm), flower stem thickness (mm), flower fresh weight (gr), flower dry weight (gr), and chlorophyll content (SPAD) were measured. At the end of the experiment, all plants were harvested. The fresh weights of the plants were measured with an electronic balance (CX-600), incubated at 68 °C for 48 hours, then measured again for dry weight. Leaf chlorophyll readings of Zinnia elegans L. and Tagetes Erecta plants were determined with a portable chlorophyll meter (SPAD-502; Konica Minolta Sensing, Inc., Japan).

.

2.2. Plant and Soil Analyses

For mineral analysis, samples from the roots and leaves of Zinnia elegans L. and Tagetes Erecta plants were dried in an oven at 68 °C for 48 hours and then ground. The Kjeldahl method with a Vapodest 10 Rapid Kjeldahl Distillation Unit (Gerhardt, Konigswinter, Germany) was used to determine the total N. An inductively coupled plasma spectrophotometer (Optima 2100 DV, ICP/OES; Perkin-Elmer, Shelton, CT) was used to determine tissue K, P, Mg, Na, Fe, Zn, B, Ca, S, Pb, Cd ,Mn, B and Cl

After 8 weeks of incubation with HM, soil samples from each plot were taken from a 0- to 10-cm depth to determine baseline soil properties, heavy metal pollution degree, and soil element fractions using sequential extraction.

Physical and chemical analyses were performed after soil samples were air dried, crumbled, and passed through a 2-mm sieve [34].

Soil pH was determined according to McLean [35]. The method of Rhoades (1996) [36] was used to measure electrical conductivity (EC). Kjeldahl method [37] and the sodium bicarbonate method of Olsen et al. [38] were used to determine the organic N and plant-available P contents, respectively. Soil organic matter was determined using the Smith–Weldon method [39]. The exchangeable cations were determined according to Thomas [40]. Diethylenetriaminepentaacetic acid (DTPA) extraction methods were used to determine the microelements in the soils [41].

Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) activities were determined based on the method given by Sahin et al.

Comment 3 Are the result clearly descript.

Answer 3. Result and discussion section has been corrected.

Comments 4. Unit of measurement are not unitary.

Answer 4. All units of measurement in the article are given in a certain format.

Comment 5. Decimals are separated sometimes by a point sometimes a comma.

Answer 5. Fixed numeric data in the article as a dot instead of a comma.

Comment 6. All methods for analysis must be presented.

Answer 6. Analysis methods of all analyzed parameters are given.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The paper is improved in the process of reviews and at this stage I recommend acceptance of the paper for publication.