Effects of Vermireactor Modifications on the Welfare of Earthworms Eisenia fetida (Sav.) and Properties of Vermicomposts

Round 1

Reviewer 1 Report

This study is interesting in that this study was performed to analyze the effectiveness of modifications in the structure of vermicompost for the preservation of earthworm welfare along with  production of vermicompost using E. fetida. 

 This paper can be improved by considering the following:

• In the experimental design, more diverse agents to induce stressful conditions should be tested to simulate real situations. The additional agents would be heavy metals, salts, pesticides, synthetic organic chemicals, solvents and other hazardous compounds that could be present in the organic wastes.

• Please show the concentrations of NH4+, NO3- and PO4--- (at initial and after composting) that are potential nutrients for plants.
• Concentrations of Cu, Zn and Cd increased after composting while Mn and Pb decreased. Why? Could these concentrations of the heavy metals be allowed in the criteria of compost quality? Otherwise, how do you circumvent this problem if the compost needs to be used in field situations?
• If this composting product were used for the purpose of commercial use, please show data that should be included in terms of compost quality.

Author Response

Response to Reviewer Comments on Ref.: Ms. No. agriculture-956596

Effects of vermireactor modifications on the welfare of earthworms Eisenia fetida (Sav.) and properties of vermicomposts

Please see our responses to the points raised by the reviewer below.

This study is interesting in that this study was performed to analyze the effectiveness of modifications in the structure of vermicompost for the preservation of earthworm welfare along with  production of vermicompost using E. fetida. 

 This paper can be improved by considering the following:

1. In the experimental design, more diverse agents to induce stressful conditions should be tested to simulate real situations. The additional agents would be heavy metals, salts, pesticides, synthetic organic chemicals, solvents and other hazardous compounds that could be present in the organic wastes.

Organic waste of plant origin intended for vermicomposting may contain an increased content of e.g. heavy metals, salt or pesticides. In such a form, they can pose a serious threat to the earthworm population. In the analysis of the protective substrate application effectiveness experiment, onion extract was selected and used as a stress factor for earthworms. This stressor in the appropriate concentration used by Steffen et al. (2013) was to be irritating to these invertebrates, and at the same time should not cause their mortality. The use of onion extract was only intended to create unfavorable conditions for E. fetida in the waste. A similar situation would be achieved by adding heavy metals or pesticides to waste, but in order to avoid mortality of earthworms, it would be necessary to determine a safe dose of each of these stressors in advance (and this is the problem of other studies). The authors of the work would like to thank the reviewer for the specific remark that may contribute to further experiments.

2. Please show the concentrations of NH4+, NO3- and PO4--- (at initial and after composting) that are potential nutrients for plants.

In this study, the authors focused on the analysis of macronutrients and heavy metals in the initial waste biomass and in the vermicompost obtained. The values suggested by the reviewer were not measured in this paper. In the literature on research with the use of vermicompost, the concentration of N-NH₄⁺ and N-NO₃^- was analyzed in the outflow of water from soils fertilized with, among others, vermicompost (Jouquet P. et al. 2010. The rehabilitation of tropical soils using compost and vermicompost is affected by the presence of endogeic earthworms. Applied Soil Ecology. 46. 125-133.

In turn, Zhang et al. (2020) in their article stated (after other authors) that N-NH₄⁺ and N-NO₃^- are considered important indicators of the stabilization process in vermicomposting

(Zhang H., Li J., Zhang Y., Huang K. 2020. Quality of Vermicompost and Microbial Community Diversity Affected by the Contrasting Temperature during Vermicomposting of Dewatered Sludge. Int. J Environ. Res. Public Health. 17. 1748.)

According to the research of Eghball et al. (1997) and Martins, Dewes (1992) composting causes both weight loss and transformation of many compounds, especially nitrogen. As the biodegradation of the compost material progressed, a significant increase in the concentration of ammonium nitrogen was observed, which may lead to significant losses due to ammonia volatilization. An increase in the concentration of nitrate nitrogen was also noted, which proves the ongoing process of nitrification. The consequence of changes in carbon and nitrogen content is the narrowing of the C/N ratio in mature compost (Eghball B., Power J.F., Gilley J.E., Doran W.J. 1997. Nutrient, carbon, and mass loss during composting of beef cattle feedlot manure. Journal of Environmental Quality. 26. 1. 189-193. Martins O., Dewes T. 1992. Loss of nitrogenous compounds during composting of animal wastes. Bioresource Technology. 42. 2. 103-111.)

The authors thank you for the interesting idea that could be used in other research.

3. Concentrations of Cu, Zn and Cd increased after composting while Mn and Pb decreased. Why? Could these concentrations of the heavy metals be allowed in the criteria of compost quality? Otherwise, how do you circumvent this problem if the compost needs to be used in field situations?

As shown in lines 355-359 and 362-364 „Deolalikar et al. [2005] and Hait and Tare [2012] reported that the increase in heavy metal content in vermicompost may result from a reduction in the volume and weight of the final product. Results concerning the increase in the concentration of heavy metals in vermicomposts compared to the initial biomass were presented by Yuvaraj et al. [2019], Bhat et al. [2013] and Gupta and Garg [2008].” „Reduction in the concentration of these elements (Pb and Mn) in vermicompost may be, among others, caused by the fact that some metal fractions readily bio-accumulate in earthworm tissues [Li et al. 2010].”

The content of heavy metals in the vermicomposts obtained did not disqualify their suitability as ferilizers, because their level did not exceed the permissible content of these elements in composts in EU countries (Heavy metals and organic compounds from wastes used as organic fertilizers. Annex 2 Compost quality definition – legislation and standards. Env.a.2./etu/2001/0024. [Online http://ec.europa.eu/environment/waste/compost/pdf/hm_annex2.pdf] (accessed 07.08.2020).

4. If this composting product were used for the purpose of commercial use, please show data that should be included in terms of compost quality.

Vermicomposts obtained by vermicomposting various organic wastes have been used in research on plant cultivation, including:

Arancon N.Q., Edwards C.A., Bierman P., Welch C. Metzger J.D. 2004. Influences of vermicomposts on field strawberries: 1. Effects on growth and yields.   Bioresource Technology. 93. 2. 145-153.

Singh R.D., Sharma R.R., Kumar S., Gupta R.K., Patil R.T. 2008. Vermicompost substitution influences growth, physiological disorders, fruit yield and quality of strawberry (Fragaria x ananassa Duch.). Bioresource Technology. 99. 17. s. 8507-8511.

Gutierrez-Miceli F.A., Santiago-Borraz J., Molina J.A.M., Nafate C.C., Abud-Archila M., Llaven M.A.O., Rincón-Rosales R., Dendooven L. 2007. Vermicompost as a soil supplement to improve growth, yield and fruit quality of tomato (Lycopersicum esculentum). Bioresource Technology. 98. 15. s. 2781-2786.

Zaller J.G. 2007. Vermicompost as a substitute for peat in potting media: Effects on germination, biomass allocation, yields and fruit quality of three tomato varieties. Scientia Horticulturae. 112. 2. s. 191-199.

The chemical, physical and biological parameters of the vermicomposts produced are different. This is due to, inter alia, the type of processed waste and the diversity of habitat conditions of earthworms. One of the most commonly used indicators for assessing vermicompost maturity is the C/N ratio. A decrease in the C/N ratio below 20 indicates an advanced degree of organic matter stabilization and reflects the satisfactory degree of vermicompost maturity. The pH of the vermicompost (optimal 6-7) and low salinity are also important.

Author Response File: Author Response.doc

Reviewer 2 Report

The paper presents an interesting field, focusing on a way to prevent damages to E.fetida in order to obtain high quality of vermicompost, both.

The aim is clearly defined. Material and method are thoroughly investigated.

Results are clear and discussed in deph. The paper is organic and linear, with well-defined concepts. 

The argument concerns the circular economy concept that should be better highlighted.

Author Response

Response to Reviewer Comments on Ref.: Ms. No. agriculture-956596

Effects of vermireactor modifications on the welfare of earthworms Eisenia fetida (Sav.) and properties of vermicomposts

Please see our responses to the points raised by the reviewers below.

The paper presents an interesting field, focusing on a way to prevent damages to E. fetida in order to obtain high quality of vermicompost, both.

The aim is clearly defined. Material and method are thoroughly investigated.

Results are clear and discussed in deph. The paper is organic and linear, with well-defined concepts. 

The argument concerns the circular economy concept that should be better highlighted.

The authors of the assessed work would like to thank the reviewer for analyzing the submitted manuscript. The suggestion that the concept of the circular economy should be emphasized in the manuscript is justified by the authors (see lines 414-416).

Author Response File: Author Response.doc

Reviewer 3 Report

The topic of the study is actual and several similar reports were published in various scientific journals so it can expect a wide audience in the scientific community. The study is basically well-written but its composition probably generates a lack in readers who are interested in vermicomposting.

In the abstract there is a statement: “The vermicomposts obtained were not significantly different from each other but were characterized by a higher content of C, N, P, K, Ca and Mg compared to the initial waste biomass.” What does it mean? Why and how increased the content of the above-mentioned elements in the vermicomposts? To the best of my knowledge, the elemental composition of the digestible compounds cannot be changed during the vermicomposting since earthworm are not nuclear reactors which transform elements.

The next one was: “Contents of Cu, Mn, Zn, Cd and Pb in vermicomposts did not prevent their use as fertilizers.” What does it mean?

Materials and methods

What method was used for species determination? E. fetida and E. andrei are close relatives but are characterized by distinct tolerance to environmental conditions like pH, salt concentration, etc. and they have distinct bacterial symbionts as well. Did the authors investigate morphological characteristics of the adult specimens or apply CO1 barcode assay? If authors have self-evidently identified breeding stock of E. fetida a reference is absolutely needed for.

Description of the composition of the Protective Substance is incomplete. The ratio of distinct components is absolutely missing. There is a problem with the definition of the salinity (probably NaCl concentration) since it is not self-evidently determined. NaCl concentration of the substratum significantly influence the body mass gain of the worms (Fischer E, and Molnár L, (1997): Growth and reproduction of Eisenia fetida (Oligochaeta, Lumbricidae) in various metal chlorides containing semi-natural soil. Soil Biology & Biochemistry, 29: 667-670.).

Results and discussion

Figures are incomplete (see e.g. Fig. 3). Please add dimensions to figures!

The influence of certain heavy metals on reproduction was shown in a  recent publication (Viktoria Takacs, Laszlo Molnar, Beata Klimek, Adrianna Galuszka, A. John Morgan and Barbara Plytycz (2016) Exposure of Eisenia andrei (Oligochaeta; Lumbricidea) to cadmium polluted soil inhibits earthworm maturation and reproduction but not restoration of experimentally depleted coelomocytes or regeneration of amputated segments. Folia Biologica (Kraków), 64 (4), 275-284.)

The cocoon production of the earthworms is mediated by not only heavy metals but also NaCl (Fischer E, and Molnár L, (1997): Growth and reproduction of Eisenia fetida (Oligochaeta, Lumbricidae) in various metal chlorides containing semi-natural soil. Soil Biology & Biochemistry, 29: 667-670.).

The overview and reorganization of the study are absolutely needed.

Author Response

Response to Reviewer Comments on Ref.: Ms. No. agriculture-956596

Effects of vermireactor modifications on the welfare of earthworms Eisenia fetida (Sav.) and properties of vermicomposts

Please see our responses to the points raised by the reviewers below.

The topic of the study is actual and several similar reports were published in various scientific journals so it can expect a wide audience in the scientific community. The study is basically well-written but its composition probably generates a lack in readers who are interested in vermicomposting.

1. In the abstract there is a statement: “The vermicomposts obtained were not significantly different from each other but were characterized by a higher content of C, N, P, K, Ca and Mg compared to the initial waste biomass.” What does it mean? Why and how increased the content of the above-mentioned elements in the vermicomposts? To the best of my knowledge, the elemental composition of the digestible compounds cannot be changed during the vermicomposting since earthworm are not nuclear reactors which transform elements.

Numerous studies show that in the vermicomposting process, the physical, chemical and biological properties of the waste plant mass processed by earthworms are modified:

Huang, K.; Li, F.; Wei, Y.; Chen, X.; Fu, X. Changes of bacterial and fungal community compositions during vermicomposting of vegetable wastes by Eisenia foetida. Bioresour. Technol. 2013, 150, 235-241.

Bhat, S.A.; Singh, J.; Vig, A.P. Effect on Growth of Earthworm and Chemical Parameters During Vermicomposting of Pressmud Sludge Mixed with Cattle Dung Mixture. Procedia Environ. Sci. 2016, 35, 425–434.

The vermicomposts obtained by the authors of this study were characterized by similar changes in the content of the macronutrients described, for example, by the above authors. The decrease in the mass and volume of organic matter caused by its decomposition during vermicomposting may be responsible for the increase in the concentration of macronutrients in the obtained fertilizers.

2. The next one was: “Contents of Cu, Mn, Zn, Cd and Pb in vermicomposts did not prevent their use as fertilizers.” What does it mean?

The content of heavy metals in the obtained vermicomposts does not disqualify their suitability as fertilizers, because the level of their content does not exceed the permissible standards for these elements in composts in EU countries (Heavy metals and organic compounds from wastes used as organic fertilizers. Annex 2 Compost quality definition legislation and standards. Env.a.2./etu/2001/0024. [Online http://ec.europa.eu/environment/waste/compost/pdf/hm_annex2.pdf] (accessed 07.08.2020).

Materials and methods

3. What method was used for species determination?  E. fetida and E. andrei are close relatives but are characterized by distinct tolerance to environmental conditions like pH, salt concentration, etc. and they have distinct bacterial symbionts as well. Did the authors investigate morphological characteristics of the adult specimens or apply CO1 barcode assay? If authors have self-evidently identified breeding stock of E. fetida a reference is absolutely needed for.

Thank you for this important question.
Apart from the differences in body pigmentation, both closely related species are morphologically similar. E. fetida is characterized by pale or yellow transverse lines between the segments without pigmentation. E. andrei is uniformly dark red. These features were taken into account in the diagnosis of E. fetida species. Additionally, as shown in lines 81-83, "E. fetida earthworms used in the experiment originated from our own long-term breeding line run at the Department of the Basis of Agriculture and Waste Management at the University of Rzeszów. " The initial population was purchased 10 years ago from a company specializing in large-scale earthworm farming (EKAGRO Słupsk, Poland). The DNA barcode has not been used so far, because the research conducted is not based on ecotoxicological tests, where the use of the CO1 barcode test would be necessary. Perhaps it would be worthwhile, for the sake of accuracy in future research, to determine the species of earthworms with the above-mentioned test.

4. Description of the composition of the Protective Substance is incomplete. The ratio of distinct components is absolutely missing. There is a problem with the definition of the salinity (probably NaCl concentration) since it is not self-evidently determined. NaCl concentration of the substratum significantly influence the body mass gain of the worms (Fischer E, and Molnár L, (1997): Growth and reproduction of Eisenia fetida(Oligochaeta, Lumbricidae) in various metal chlorides containing semi-natural soil. Soil Biology & Biochemistry, 29: 667-670.).

The authors want to thank the reviewer for the valuable attention. Corrections were made in the text of the manuscript (see lines 108-111).

Results and discussion

5. Figures are incomplete (see e.g. Fig. 3). Please add dimensions to figures!

The corrections were made in accordance with the reviewer's suggestion. The authors hope that this has improved the legibility of the figures.

6. The influence of certain heavy metals on reproduction was shown in a  recent publication (Viktoria Takacs, Laszlo Molnar, Beata Klimek, Adrianna Galuszka, A. John Morgan and Barbara Plytycz (2016) Exposure of Eisenia andrei(Oligochaeta; Lumbricidea) to cadmium polluted soil inhibits earthworm maturation and reproduction but not restoration of experimentally depleted coelomocytes or regeneration of amputated segments. Folia Biologica (Kraków), 64 (4), 275-284.)

Thank you for the advice (see lines 246-247).

7. The cocoon production of the earthworms is mediated by not only heavy metals but also NaCl (Fischer E, and Molnár L, (1997): Growth and reproduction of Eisenia fetida(Oligochaeta, Lumbricidae) in various metal chlorides containing semi-natural soil. Soil Biology & Biochemistry, 29: 667-670.).

Thank you for the advice (see lines 255-256).

Author Response File: Author Response.doc