The Effect of Soil Amendments on Trace Elements’ Bioavailability and Toxicity to Earthworms in Contaminated Soils

Round 1

Reviewer 1 Report

My recommendation to reject the manuscript is based on the follow key points:

1. There are a lot of confusing sentences and whole paragraphs in manuscript.  Some examples:

„After being counted and weighted earthworms were transported into Petri dishes 205 with the filter paper. A few drops of distilled water were applied to keep earthworms 206 moister. They were kept at 25 °C for 48 hours and filter papers were changed daily to 207 allow full depuration of their gut contents. Earthworms were then killed in liquid nitrogen and further oven-dried at 100°C for 24 h. Trace elements content in earthworms was determined using dried and crushed earthworm sample digested with concentrated………..

„This effect rather cannot be exclusively attributed to metal toxicity but negative 242 change in soil conditions after fresh biosolids application. In other measured variants-??? the 243 survival rate…..”

 Line 22: “BSAF for soil  Cd in the contaminated decreased to the greatest extent after…..” -

 l.41 “… due  to changing environmental conditions or changes in soil properties, may be converted to….” There is no subject in the last part of sentence.

L. 275 “These amendments did not bring high amount 275 of potentially toxic metals to the soil or reactive organic substances,…..”

2. The discussions at the end of each part (after the results presented) are in most cases hypotheses.  The results from points 3.3 and 3.4 (about changes in the pH and solubility of trace elements after application of soil amendments) could be used in better way to explain the results from points 3.1 and 3.2 (about accumulation of TE in earthworms and survival rate and etc). It will be better if the discussion is given after presenting all of the results obtained.

3.There is a lot of redundant information and unnecessary references in the text \esspecially in points 3.1 and 3.2).

4. The cited literature sources are not up-to -date.  The 49 references are given but only 6 of them are from the latest 5-years period. 

5. Sentence on l. 280 „Numerous studies investigated the effects of metals availability on earthworms mor-280 tality [12,21,29], loss of weight [12,13,24,29,33], cocoon production and variability [28] as 281 well as growth [13,21,24].” is the same as in Ref. 12, only the references in brackets are changed.

6. similar situation with the sentece from line 442 “Usually, the longer the residence time, the greater the decrease in trace element desorption, 442 because of a rearrangement of surface complexes and/or a conversion of surface complexes into surface precipitates [11,25].”

Author Response

Response to the Reviewer #1

Dear Reviewer,

We would like to thank you for suggestions.  We have studied your comments carefully and have made corrections according to yours opinion. All the introduced changes as well as responses to the review are listed below. 

My recommendation to reject the manuscript is based on the follow key points:

1. There are a lot of confusing sentences and whole paragraphs in manuscript. Some examples:

„After being counted and weighted earthworms were transported into Petri dishes 205 with the filter paper. A few drops of distilled water were applied to keep earthworms 206 moister. They were kept at 25 °C for 48 hours and filter papers were changed daily to 207 allow full depuration of their gut contents. Earthworms were then killed in liquid nitrogen and further oven-dried at 100°C for 24 h. Trace elements content in earthworms was determined using dried and crushed earthworm sample digested with concentrated………..

„This effect rather cannot be exclusively attributed to metal toxicity but negative 242 change in soil conditions after fresh biosolids application. In other measured variants-??? the 243 survival rate…..”

 Line 22: “BSAF for soil  Cd in the contaminated decreased to the greatest extent after…..” -

 l.41 “… due  to changing environmental conditions or changes in soil properties, may be converted to….” There is no subject in the last part of sentence.

1. 275 “These amendments did not bring high amount 275 of potentially toxic metals to the soil or reactive organic substances,…..”

 Resp.: These sentences have been reworded/rephrased or supplemented. The other Reviewers comments have also been included.

2. The discussions at the end of each part (after the results presented) are in most cases hypotheses. The results from points 3.3 and 3.4 (about changes in the pH and solubility of trace elements after application of soil amendments) could be used in better way to explain the results from points 3.1 and 3.2 (about accumulation of TE in earthworms and survival rate and etc). It will be better if the discussion is given after presenting all of the results obtained.

Resp.: We partially agree with your point of view but would prefer not to separate the results and discussions. Therefore, in the search for a compromise without disturbing the overall structure of the manuscript the section 3.4 ‘Influence of amendments application and earthworm activity on soil pH’ was moved before section 3.3. ‘Solubility of trace elements in soils driven by applied amendments and earthworm activity’ to better present and explain the obtained results.

3.There is a lot of redundant information and unnecessary references in the text \esspecially in points 3.1 and 3.2).

Resp.: Redundant sentences have been removed.

4. The cited literature sources are not up-to -date. The 49 references are given but only 6 of them are from the latest 5-years period.

Resp.: Most of the references in the literature are actually older but pertain to key issues related to TE migration and their behavior in soil as well as remediation processes. This is source literature that should be cited as the primary compendium of knowledge however, more recent literature references has also been added.

5. Sentence on l. 280 „Numerous studies investigated the effects of metals availability on earthworms mor-280 tality [12,21,29], loss of weight [12,13,24,29,33], cocoon production and variability [28] as 281 well as growth [13,21,24].” is the same as in Ref. 12, only the references in brackets are changed.

Resp.: Appropriate corrections have been made in this regard. The indicated sentences have been deleted.

6. similar situation with the sentece from line 442 “Usually, the longer the residence time, the greater the decrease in trace element desorption, 442 because of a rearrangement of surface complexes and/or a conversion of surface complexes into surface precipitates [11,25].”

Resp.: Appropriate corrections have been made in this regard. The indicated sentences have been deleted.

Reviewer 2 Report

Abstract - the use of abbreviations is not advisable at the abstract stage. Generally lacks conclusions and suggestions at the end of the text. What does the reader gain after reading the abstract? In my opinion, 2-3 sentences summing up and indicating the best methods should be added at the end. What are the limitations ? Since Pb has completely different properties than other heavy metals, it is useful to show whether it behaves differently or perhaps the same as other metals and to convey this information very accurately. Usually Pb is not very mobile in the environment, while other metals have great mobility.

The introduction lacks an indication of the scientific problem in a clear way. We know that heavy metals are ubiquitous, but from an environmental cleanup perspective, small amounts of heavy metals are always more difficult to remove than large amounts. Trace amounts will always be shared among soil-forming fractions, making them more difficult to access. Are earth-worms supposed to be an indicator of bioavailability?   

Cation exchange capacity could be provided too, but still all important factors were included in the soil analysis.

I am not a fan of introducing geographic names that tell nothing to readers. Please explain the origins and differences once in the methods and then don't use them because they are empty words to the international reader.

In the methods, please emphasize clearly that the authors considered both fractions of metals, total and leachable. 

There are interesting conclusions and comments in the conclusion. I think they are even worth discussing more strongly as limitations of current methods. The authors can and even should speculate a bit based on the results presented. As much as I appreciate the analytical part and its description, I miss an interesting discussion that points out limitations and factors that should be studied in the future.

Author Response

Response to the Reviewer #2

Dear Reviewer,

We would like to thank you for comments and suggestions, which contributed very much to improve our manuscript.  We have studied your comments carefully and have made corrections which we hope meet with approval. All suggestions and recommendations have been adopted, and the introduced changes are listed in the responses to the review. 

Abstract - the use of abbreviations is not advisable at the abstract stage. Generally lacks conclusions and suggestions at the end of the text. What does the reader gain after reading the abstract? In my opinion, 2-3 sentences summing up and indicating the best methods should be added at the end. What are the limitations? Since Pb has completely different properties than other heavy metals, it is useful to show whether it behaves differently or perhaps the same as other metals and to convey this information very accurately. Usually Pb is not very mobile in the environment, while other metals have great mobility.

Resp.: The abstract has been complemented with missing phrases. The summary has been enriched with additional explanations summarizing the conducted experiment. Moreover, for clarity, the abbreviations have been accordingly explained in the first sentences of the abstract.

The introduction lacks an indication of the scientific problem in a clear way. We know that heavy metals are ubiquitous, but from an environmental cleanup perspective, small amounts of heavy metals are always more difficult to remove than large amounts. Trace amounts will always be shared among soil-forming fractions, making them more difficult to access. Are earth-worms supposed to be an indicator of bioavailability?   

Resp.: Some of the paragraphs in the introduction section have been expanded and the research problems are emphasized in more detail, e.g:

1. 91-96: Hence, the search for the most effective soil amendment is constantly being carried out, with a high potential for the immobilization of TE in the soil and characterized by low risk of metals release during their aging processes.

Recovering contaminated soils in highly polluted areas provides the necessary action to prevent the deterioration of the environment, as well as control humans and other liv-ing organism exposures to hazardous and toxic compounds.

1. 104-105: Direct responses of organisms exposed to potentially polluting elements obtained by bio-assays are indicative of the actual risk of the pollutants.
2. 110-117: For these purpose, earthworms are considered to be the most important bio-indicators due to their intimate (externally and internally) contact with the soil [31,7,13] and possibility to evaluate the potential of TE bioaccumulation and food chain transfer [31,7,13, 33,34]. Studies published in recent years pointed out that the variation of TE bioaccumulation in worms may be dependent on their bioavailability [35] as affected by soil factors infuencing metal solubility (pH and redox) and com-plexation with organic matter [33;38]. However, these factors and mechanisms are still poorly understood. Moreover, earthworm studies in contaminated areas focus mainly on the analysis of the impact of various species of earthworms on TE assimilation in artificially contaminated soils. Relatively few studies have been conducted to examine the effectiveness of soil amendments on bioavailability of TE measured as the actual level of bioaccumulation of the given element.

Cation exchange capacity could be provided too, but still all important factors were included in the soil analysis.

Resp.: Unfortunately, the obtained results are part of a larger project and, the CEC analyzes for the tested soils have not been performed. We agree with the Reviewer that CEC is an important and useful indicator of soil quality because it shows the soil's fertility and thus ability to supply three important plant nutrients: calcium, magnesium and potassium. However, in aspects of our research, it would not allow to obtain more revealing results.

I am not a fan of introducing geographic names that tell nothing to readers. Please explain the origins and differences once in the methods and then don't use them because they are empty words to the international reader.

Resp.: We not sure what names this comment refers to, but we suppose to denote soil amendments. Probably we could use the names 'amendment 1', 'amendment 2', 'amendment 3', etc, but to unify all of our publications, the uniform (the same) names have been used. Nevertheless, the Reviewer remark is very valuable and will be important advice for the future during implementing new projects and publishing their results.

In the methods, please emphasize clearly that the authors considered both fractions of metals, total and leachable. 

Resp.: The appropriate change has been applied in the materials and methods section. It was clearly emphasized that the total and leachable metal fractions were studied. (L. 156-159  “The aim of the experiment was to determine the total and bioavailable metal frac-tion that was absorbed directly by earthworms. Therefore, after the end of the bioassay period the survival and change of weight of earthworms as well as soil physicochemical properties were evaluated (section 2.2.1 and 2.2.2) )

There are interesting conclusions and comments in the conclusion. I think they are even worth discussing more strongly as limitations of current methods. The authors can and even should speculate a bit based on the results presented. As much as I appreciate the analytical part and its description, I miss an interesting discussion that points out limitations and factors that should be studied in the future.

Resp.: The research described in the presented publication is only an initial part of the research carried out as part of a large project, therefore the discussion is limited to some extent. More detailed descriptions and wider analyzes will be described in the next manuscript.

Reviewer 3 Report

The authors have a investigated the bioavailability of trace elements in earthworms after addition of different amendments in polluted soil. 

Although the manuscript has been well written, there are issues that I believe should be resolved before publication. 

On the first place Eisenia veneta is not a valid species name, and its is not given in the OECD protocol as you have written (p4 l173). What species did you have? 

Secondly, there is a lot of literature on the accumulation of metals and toxicity of metals to earthworms that  has been neglected. 

Figures are hard to follow as the explanation of abbreviations is not given in figure captions. I also do not see UP values in the figures (and values are not given as a relative to control). 

All the abbreviations in the abstract should be fully written (TE, BSAF, ZC, GC, etc.)

p6l241 This sentence should be removed

Was was the survival in your experiments? As you have given the cumulative weight of a replicate in Figure 1, have you normalize it to the number of earthworms? It is not clear. 

p6l247 I don't see data for the control to see if it is consistent.p

p7 l280 Yes, metallothoneins one of the biomarkers measured upon metal exposure, but you haven't measured any so I do not see a point of having that paragraph.

p6l263 mortal effect?

It is not Manna-Whitney's test, but a Mann-Whitney in English 

Author Response

Dear Reviewer,

We would like to thank you for comments and suggestions, which contributed very much to improve our manuscript.  We have studied your comments carefully and have made corrections which we hope meet with approval. All suggestions and recommendations have been adopted, and the introduced changes are listed in the responses to the review. 

The authors have a investigated the bioavailability of trace elements in earthworms after addition of different amendments in polluted soil. Although the manuscript has been well written, there are issues that I believe should be resolved before publication. 

On the first place Eisenia veneta is not a valid species name, and it is not given in the OECD protocol as you have written (p4 l173). What species did you have? 

Resp.: We used the Eisenia veneta (Alternative name: Dendrobaena veneta) due to its rapid growth, life cycle and high compostability parameters according to the literature data: Polak A., Kostecka J., Mazur-Pączka A., Garczyńska M., Pączka G., Szura R. 2020 Life Cycle of the Eisenia fetida and Dendrobaena veneta Earthworms (Oligohaeta, Lumbricidae). Journal of Ecological Engineering 21(1):40–45. DOI: https://doi.org/10.12911/22998993/113410

The incorrect reference has been removed from the text and properly replaced with a correct literature reference.

Secondly, there is a lot of literature on the accumulation of metals and toxicity of metals to earthworms that  has been neglected. 

Resp.: Several new literature items have been additionally cited, in particular those that have been published in recent years. All of them have been included and marked in the 'References' section

Figures are hard to follow as the explanation of abbreviations is not given in figure captions. I also do not see UP values in the figures (and values are not given as a relative to control). 

Resp.: The control in these combinations was contaminated soil (soil P) because the influence of soil amendments characterized by different properties on the potential uptake of TE by earthworms was analyzed. The application of the uncontaminated soil (soil UP) as a control in these cases would be inappropriate and would not compare the real impact of the applied amendments.

Appropriate abbreviation explanations have been added below each figure.

All the abbreviations in the abstract should be fully written (TE, BSAF, ZC, GC, etc.)

Resp.: The mistake has been corrected, all abbreviations have been accordingly explained in the first sentences of the abstract.

p6l241 This sentence should be removed

Resp.: The sentence has been removed.

Was was the survival in your experiments? As you have given the cumulative weight of a replicate in Figure 1, have you normalize it to the number of earthworms? It is not clear. 

Resp.: Yes, the weight change results were normalized to the number of survivors. Hence, (perhaps) such slight differences in the observed changes of worms weight. Nevertheless, for the clarity, the same graph shows the weight change and the  percentage of surviving earthworms. A suitable explanation was added in the "materials and methods" section and in the description below the figure 1.

p6l247 I don't see data for the control to see if it is consistent.p

Resp.: in this part of the experiment, contaminated soil (soil P)was the control, therefore the survival and body weight change in earthworms in the soil with additives was related and comparable to this combination. In uncontaminated soil (soil UP), the SR =100%  while initial weight = 2.9 and final weight = 3.4 at SD = 0.2. Comparing the obtained parameters for uncontaminated soils and contaminated soils enriched by different amendments, p was significant and lower than 0.05.

p6l263 mortal effect?

Resp.: The expression has been changed.

p7 l280 Yes, metallothoneins one of the biomarkers measured upon metal exposure, but you haven't measured any so I do not see a point of having that paragraph.

Resp.: This paragraph has been deleted from the manuscript.

It is not Manna-Whitney's test, but a Mann-Whitney in English

Resp.: The mistake has been corrected in description under figure 3 and 4 as well as ‘Statistical analysis’ section.

Round 2

Reviewer 1 Report

In the revised version the idea of the study and the results obtained are stated more logically ; the literature is updated. The rather extensive and unnecessary references to other papers have been removed. The revised manuscript is definitely more understandable to the reader.  

Below I make some suggestions related to technical or stylistic errors.

L. 46 “Soil contamination with trace elements (TE) is a common problem in terrestrial eco systems a result of their dispersion related with…………….”  Should be “……. systems AS a result …….”

l.52 “Despite TE present in soil ls originate from …….”   Delete  “ls“

l. 110 “Moreover, earthworm studies in contaminated areasfocus mainly on the analysis TE assimilation by earthworms in artificially contaminated soils”   should be “Moreover, the earthworm studies in contaminated areas HAS mainly focusED on the analysis OF the TE assimilation ………..”.

l.156 Correct  “….iron ox-ide-IO….”

l. 186 “…as doses equivalent to approx. 4 t ha-1 of P and Fe, respectively “ Please, specify what is mean notation P - is this contaminated “soil P” or the authors have in mind  element phosphorus? (!!!  In table 1 notation K is given for phosphorus).  

l. 309 “Data on …. is scarce…..” should be “ Data on….. are scarce….”

l.362 “ …. earthworms accumulate contaminants in their bodies with  different extent….. “ correct to “…. earthworms accumulate DIFFERENT contaminants in their bodie IN different extent…..”

l. 352 It will be better if you put i) and ii) in the sentence : ”Assimilation of metals by earthworms occurs through two pathways…… .. : i) absorption following dermal contact and ingestion of organic matter and soil particles, and ii) adsorption through the gut tissues ….”

l. 401 replace “to learn” with “to estimate”

Please, re-read the text carefully and note the use of definite and indefinite articles, which are missing in many places in the text.

Author Response

Dear Reviewer,

We would like to thank you for comments and suggestions.  We have studied your comments carefully and have made corrections which we hope meet with approval. All suggestions and recommendations have been adopted, and the introduced changes are listed in the responses to the review. 

,In the revised version the idea of the study and the results obtained are stated more logically ; the literature is updated. The rather extensive and unnecessary references to other papers have been removed. The revised manuscript is definitely more understandable to the reader.  

Below I make some suggestions related to technical or stylistic errors.

l.46 “Soil contamination with trace elements (TE) is a common problem in terrestrial eco systems a result of their dispersion related with…………….” Should be “……. systems AS a result …….”

Resp.: The change has been introduced.

l.52 “Despite TE present in soil ls originate from …….”   Delete  “ls“

Resp.: The ‘ls’ has been deleted.

l.110 “Moreover, earthworm studies in contaminated areasfocus mainly on the analysis TE assimilation by earthworms in artificially contaminated soils” should be “Moreover, the earthworm studies in contaminated areas HAS mainly focusED on the analysis OF the TE assimilation ………..”.

Resp.: The sentence has been reworded.

l.156 Correct  “….iron ox-ide-IO….”

Resp.: The change has been introduced.

l.186 “…as doses equivalent to approx. 4 t ha-1 of P and Fe, respectively “ Please, specify what is mean notation P - is this contaminated “soil P” or the authors have in mind element phosphorus? (!!! In table 1 notation K is given for phosphorus). 

Resp.: In the text the “doses equivalent to approx. 4 t ha-1 of P”-  the abbreviation P stands for the symbol of the phosphorus element. The symbol 'K' has been incorrectly used in the table and has been changed to 'P' accordingly.

The designation 'soil P' is not related in any way to the determination of phosphorus and its abbreviation comes from the name of the sampling place (P as Pulawy).

l.309 “Data on …. is scarce…..” should be “ Data on….. are scarce….”

Resp.: The change has been introduced.

l.362 “ …. earthworms accumulate contaminants in their bodies with  different extent….. “ correct to “…. earthworms accumulate DIFFERENT contaminants in their bodie IN different extent…..”

Resp.: The sentence has been reworded.

l.352 It will be better if you put i) and ii) in the sentence : ”Assimilation of metals by earthworms occurs through two pathways…… .. : i) absorption following dermal contact and ingestion of organic matter and soil particles, and ii) adsorption through the gut tissues ….”

Resp.: The change has been introduced.

l.401 replace “to learn” with “to estimate”

Resp.: The sentence has been reworded

Please, re-read the text carefully and note the use of definite and indefinite articles, which are missing in many places in the text.

Resp.: During the editing process, the manuscript will additionally pass a proof prepared by the publishing. If a linguistic correction of the text will be still required, it will be additionally corrected by English proofreading company.