Fertilization Effects of Solid Digestate Treatments on Earthworm Community Parameters and Selected Soil Attributes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors investigate the effects of digestate, an organic fertilizer additive, on soil organisms, particularly earthworms, emphasizing the need for further long-term research to fully understand its impact across different soil types and cropping systems. Highlighting the limited knowledge on how both liquid and solid fractions of digestate influence soil ecosystems, the study underscores the importance of such research for optimizing organic fertilizer use and advancing sustainable agriculture. The article concludes by stressing the necessity of future research to explore digestate’s role in soil ecosystems, particularly its effects on key organisms like earthworms. The authors are advised to consider following comments in the revision.

1, The authors are suggested to proofread the article, correcting grammar errors and incomplete sentences. Meanwhile, it is recommended to optimize the paragraph structure to make the content of the article clearer and easier to read.

2, The authors are suggested to add a review of relevant literature on digestive juices as organic fertilizer additives to enhance the background and motivation of the research

3, The authors are suggested to provide a detailed description of the experimental design and methods, including experimental setup, sample size, data collection, and analysis methods. This helps readers and reviewers better understand the scientific validity and reproducibility of the research.

4, Figure 1 a, b, and Figure 6, it seems that the box-plot is adopted in this study, the authors should explain why some boxes are tall while the others are small. The authors should provide morecaptions and other image explanations in the picture.

5, The authors are suggested to provide a detailed summary of the main findings of the study in the conclusion section and discuss their significance for relevant fields. In addition, it is suggested to propose future research directions, such as further exploring the effects of digestive juices on other soil organisms or their applications under different environmental conditions.

Author Response

Response to Reviewer Comments on Ref.: Ms. No. agriculture-3696513

Fertilization Effects of Solid Digestate Treatments on Earthworm Community Parameters and Selected Soil Attributes

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

Reviewer #1

The authors investigate the effects of digestate, an organic fertilizer additive, on soil organisms, particularly earthworms, emphasizing the need for further long-term research to fully understand its impact across different soil types and cropping systems. Highlighting the limited knowledge on how both liquid and solid fractions of digestate influence soil ecosystems, the study underscores the importance of such research for optimizing organic fertilizer use and advancing sustainable agriculture. The article concludes by stressing the necessity of future research to explore digestate’s role in soil ecosystems, particularly its effects on key organisms like earthworms. The authors are advised to consider following comments in the revision.

1, The authors are suggested to proofread the article, correcting grammar errors and incomplete sentences. Meanwhile, it is recommended to optimize the paragraph structure to make the content of the article clearer and easier to read.

Thank you, some changes have been made to the manuscript.

2, The authors are suggested to add a review of relevant literature on digestive juices as organic fertilizer additives to enhance the background and motivation of the research

Thank you, added in text (lines: 84-94).

3, The authors are suggested to provide a detailed description of the experimental design and methods, including experimental setup, sample size, data collection, and analysis methods. This helps readers and reviewers better understand the scientific validity and reproducibility of the research.

Thank you for your suggestion. There are some additions to the methodology. The authors hope that this will contribute to better understanding for readers.

4, Figure 1 a, b, and Figure 6, it seems that the box-plot is adopted in this study, the authors should explain why some boxes are tall while the others are small. The authors should provide morecaptions and other image explanations in the picture.

This is a bar chart where the height of the bars is the average for a given position, and the box groups averages that are the same (the difference is statistically insignificant)

5, The authors are suggested to provide a detailed summary of the main findings of the study in the conclusion section and discuss their significance for relevant fields. In addition, it is suggested to propose future research directions, such as further exploring the effects of digestive juices on other soil organisms or their applications under different environmental conditions.

Thank you, changes have been made to the conclusions.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Your experiments were conducted continuously for three consecutive years with every year application of digestate. This is good for examining long-time effects of digestate applications, and it was in line with the last two objectives (about the dynamics). However, the Results and Discussion did not support the last two objectives. In addition, researchers conducting research for more than two years normally to examine the long-term effects of applying something. Your Results and Discussion did not support these objectives.

Please read carefully my comments and revise this manuscript. 

Comments for author File: Comments.PDF

Author Response

Response to Reviewer Comments on Ref.: Ms. No. agriculture-3696513

Fertilization Effects of Solid Digestate Treatments on Earthworm Community Parameters and Selected Soil Attributes

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

Reviewer #2

Your experiments were conducted continuously for three consecutive years with every year application of digestate. This is good for examining long-time effects of digestate applications, and it was in line with the last two objectives (about the dynamics). However, the Results and Discussion did not support the last two objectives. In addition, researchers conducting research for more than two years normally to examine the long-term effects of applying something. Your Results and Discussion did not support these objectives.

Please read carefully my comments and revise this manuscript.

Comment [WW1]: Dynamics mean changes from time to time, and these objectives have not been proven in the Results and Discussion.

We thank the reviewer for this important comment. Perhaps we used the word "dynamics" in error. Corrected in the text (lines: 116-118).

Comment [WW2]: Methods in a research paper must be repeatable, i.e. everybody must be easy to repeat the experiment, and to make it easy to repeat, all procedures must be clearly and chronologically described or apripriately referenced. So, please revise the contents of Materials and Methods.

Thank you for your attention. Additions have been made to the methodology. The authors hope that this will contribute to better understanding for readers.

Comment [WW3]: Please explicitly describe what experimental design was used to arrange the treatment plots, and how many experimental replications was prepared.

For each experimental site and control site, one square measuring 10 x 10 m (area 100 m²) was designated, which were located in the same area. It was decided not to create, for example, three plots in each of the research groups (3 replicates), because: i) according to the authors of this manuscript, 100 m² for each site is a sufficient area to obtain reliable results, ii) there was a possibility that the digestate used could have a negative impact, among others, on earthworms (although none of the earthworm species is legally protected, preventive measures should always be taken to minimize the potentially negative impact of activities on these invertebrates). The sites were separated by a buffer zone about 5 m wide. Sampling at the sites took place three times a year (June, August, October) in three annual cycles (2021-2023). In each of the five areas, five earthworm samples and three soil samples were collected at each time, randomly selecting them but in such a way as not to duplicate sampling locations in subsequent sampling periods.

Comment [WW4]: The year of application and cropping MUST be treated as the second treatment factor to compare between years (effects of years) in each digestate dose, and between digestate doses in each year, if the interaction effect of Doses x Years was significant. In this experiment, year of application also means frequencies of digestate application, i.e. once (Y1), twice 129 made in the same designated initial plots throughout the experiment. Common pumpkin 130 was grown annually in the DG and CL monocultures; no pesticides or supplemental fer- 131 tilization was applied, and weeds were removed by hand. 132 2.3 Earthworm and Soil Sampling 133 One year prior to the experiment, i.e., before the application of different digestate 134 treatments, the qualitative and quantitative structure of Lumbricidae was analyzed in des- 135 ignated plots using the procedures later used during the experiment (the results obtained 136 were homogeneous in all plots, very similar to those shown in Table 2, stand CL). During 137 the study, earthworm sampling at the test and control plots was conducted three times 138 per year (June, August, and October) in three annual cycles (2021-2023). Five randomly 139 determined samples were taken each time within the five plots (with an area of 100 m2). 140 A method using the action of electric current was used to collect earthworms [17]. The 141 (Y2), and three times (Y3). Although the authors compared the results between years and between plots (or doses), such as those presented in Fig. 6, there is no indication of the analysis of the interaction effects. It’s not clear where are plot F(4,30) and year F (2,30) derived from. Where the error df=30 from?

Thank you for your important suggestion. Of course, we performed a two-way analysis of variance analysis, unfortunately it was not properly described in section 2.5 Data Analysis, it has been corrected. We did not describe the results of the analysis of variance in detail, believing that in this rather simple model we will be able to explain it in the text by focusing on the main effects, after the comments - we changed it by giving the results of the analysis of variance at the beginning of section 3.2 and placing the information on the figures.

Comment [WW5]: Were the data analyzed separately in each sampling month or averaged as the year average, or how the data were analyzed? Please explicitly describe them in the Methods for clarity and repeatability.

Depending on the needs, the data were analyzed as an annual average or an average over the entire (3-year) research period.

Comment [WW6]: What are the functions of these five samples? Are they experimental replications or just sampling replications?

Comment [WW7]: Were the sites in the same position or the same quadrate in every sampling time?

Comment [WW8]: What are the functions of these samples in the design of the experiment? Are they the experimental replications, or just sampling replicates? {lease explicitly describe them.

In response to comments (WW6-WW8) – as presented in the response to comment (WW3), sampling at the sites took place three times a year (June, August, October) in three annual cycles (2021-2023). Five earthworm samples and three soil samples were collected at each of the five areas, randomly selecting them, but in such a way as not to duplicate sampling locations on subsequent dates. Taking several earthworm or soil samples is nothing more than a sampling replication aimed at obtaining the most accurate results. It should be noted here that the results presented in the work are averages, e.g. abundance, biomass per m². The methods used in this study are commonly used in studies on earthworm communities in both semi-natural and anthropogenic areas. It very often happens that different species of Lumbricidae are not distributed evenly in a given area, which is why five samples of these invertebrates were collected each time, in a random manner, from different places in a 10 x 10 m square. Any additions were made in the text of the manuscript.

Comment [WW9]: Were they obtained before or after the application of digestate?

Samples of both earthworms and soil were collected in June, August and October (line 165), i.e. after the application of digestate, which took place annually in April (line 143).

Comment [WW10]: ???

Thank you. Corrected in the text.

Comment [WW11]: It is not clear why Table 1 is placed in the Methods? Were the data obtained before application of the digestate? However, looking at the lowercase letters assigned to each mean value indicating significant differences, it seems that those data are results of the experiments, but why the position is in the Methods NOT in the Results and Discussion. This is confusing. If those data are the results of the experiment for the three years of digestate application, then where is the Year of application?

This table contains information on the characteristics of the applied digestate, the soil at the control site and the soil at the experimental sites after the application of digestate (average from three years of fertilization; added in the text, lines: 185-186). The authors of the manuscript did not want to divide the table into two parts and place information on soils after the application of digestate in the results part (moreover, Figure 6 shows changes in the described features in the subsequent years of the study). In the authors' opinion, the compilation of this data next to each other in one table for illustrative purposes increases the value of this manuscript. Therefore, the authors ask the Reviewer to accept leaving the table in its current form, in the Material and Methods chapter.

Comment [WW12]: Where are the 27 samples from? Please explicitly describe in the Design of the experiments and in data acquisition methods.

Thank you, soil sampling (n=3) at each site took place three times a year (June, August, October) in three annual cycles (2021-2023). Supplemented in the text (lines: 181-182).

Comment [WW13]: Please show in the Results the main effects of digestate rates (or doses), the main effects of years of digestate application, and their interaction effects; and please show the values of the Tukey’s HSD.

Same answer as in point WW4

After WW16's remark it was decided to abandon the Tukey HSD test, instead the Tukey-adjusted pairwise comparisons on marginal means test was performed (https://idahoagstats.github.io/mixed-models-in-R/chapters/means-and-contrasts.html?utm_source=chatgpt.com)

Comment [WW14]: The Results and Discussion MUST be revised in order to be in line with the objectives of the study and the Methods. The experiments were conducted for three years of digestate application applied every year, but the Results did not show any relevant statistical analysis describing the effects of the years of application and the interaction between the doses and years of application. The objectives or hypotheses about the dynamics must be proven using statistical changes of the data from year to year, which are not proven in the Results and Discussion.

The authors of the manuscript would like to thank the Reviewer for raising this issue. Currently, there are relatively few studies on the impact of digestate on the soil environment, including earthworms, which play a key role in the functioning of soil ecosystems. Lumbricidae play a very important role in soil ecosystems because through their activity they affect the availability of nutrients for plants, participate in the formation of soil structure and have a large impact on the dynamics of organic matter. Therefore, it is very important to sufficiently understand the effects of digestate fertilization on selected features of Lumbricidae and on individual species classified into specific ecological groups, which play various functions in soil ecosystems. Some of the available studies have shown that digestate adversely affects soil organisms (including earthworms). Therefore, it seems that the significant value of this study is to demonstrate that the higher doses of digestate fertilizers used in practice, all in all, not only do not negatively affect Lumbricidae (also individual ecological groups), but actually contribute to the increase in abundance and biomass (Figure 1 a,b) and biomass of endogeic earthworms. Additionally, Figure 6a-h shows that no significant differences were found in the values ​​of all analyzed soil characteristics within individual plots in the successive years of conducting the study (lines 434-435). The same figure also shows significant differences or their absence in the values ​​of the analyzed features between individual research plots. The research was conducted for three years, because in field studies on Lumbricidae, three years is the minimum period to obtain reliable results and formulate conclusions. These three years can also be accepted as the next three repetitions of the experiment. Each year, specific doses of digestate were applied, because identical treatments are used in practice, in agriculture in monoculture crops grown in the same area. Therefore, the discussed experiment was a kind of simulation of practices often used in plant production.

Comment [WW15]: The data analyzed in this Table 2 came from which year of application, and where are the effects of the years of dgiestate application, as the short- and long-term effect?

Table 2 presents average values ​​from three years of the study. The effect of digestate fertilization on selected features of Lumbricidae in subsequent years is shown in Figure 3 and Figure 5. However, the authors would like to emphasize that the main essence of this study was the assessment of the effect of applying different doses of digestate on: i) earthworm abundance, ii) earthworm biomass, iii) selected features of Lumbricidae ecological groups, iv) selected physicochemical soil properties (see changes in the text, lines: 116-118). An increasingly common fertilization practice is the use of digestate for many years in the same area, conducting monoculture cultivation. Therefore, the information obtained in this study, presenting selected average values ​​of the analyzed features of earthworm species and Lumbricidae ecological groups, is important. However, a common practice in field studies of earthworm communities in a given area is to conduct research for at least three years (where subsequent years of research are often treated as a kind of repetition of the experiment). The final results obtained (e.g. abundance or biomass) are also presented as an average for the entire period of research. This is exactly what has been shown in Table 2, in relation to specific species that have been classified into specific ecological groups and a dominance coefficient has been determined (this is important information for scientists studying Lumbricidae).

Comment [WW16]: Please revise that this types of comparison (comparison between treatment groups) CANNOT be done using Teukey’s HSD, as stated in Line 190. Group comparison is normally done using orthogonal contrast analysis. However, doses cannot be compared using the orthogonal contrast technique because doses do not have orthogonal properties. Under orthogonal contrast technique, when comparing between (DG100, DG75, and DG50) and (DG25 and CL), the software basically compares the value of DG75 and the average of (DG25 and CL). If a treatment factor is quantitative like the different doses in this experiment, the researchers normally use polynomial comparison to find out if the effect of the doses was linear, quadratic, cubic, etc, so that the optimum dode can be calculated. Please read Gomez & Gomez (1984), Statistical procedures for agricultural research.

Instead of Tukey's HSD test, the Tukey-adjusted pairwise comparisons on marginal means test was performer(https://idahoagstats.github.io/mixed-models-in-R/chapters/means-and-contrasts.html?utm_source=chatgpt.com and "An improvement of Tukey's HSD with application to ranking institutions", Al Mohamad et al., 2017, DOI: 10.48550/arXiv.1708.02428)

Comment [WW17]: In this Fig.6 the authors show the main effect of doses with plot F-value, and the effect of year with year-F value, then where are the interaction effects? I think all the bar charts are more efficient and more exact if presented in the form of numerical tables, so it becomes easier to calculate how many % increase or decrease, and if they are significant or not.

Thank you, no significant interaction effect was observed in any of the analyses, so for clarity these values ​​are not reported but are added to the figure as requested.

Comment [WW18]: The contents of the Conclusion are too long. Conclusion must summarized the main finding and the implications only.

Thank you, changes have been made.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No further comments

Author Response

Response to Reviewer Comments on Ref.: Ms. No. agriculture-3696513

Fertilization Effects of Solid Digestate Treatments on Earthworm Community Parameters and Selected Soil Attributes

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

Reviewer #1

Comments and Suggestions for Authors: No further comments

The authors thank the reviewer for taking the time to review our article and for the important and insightful comments that have led to improvements in the current version of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

This revised version is getting better, but it still need to be revised. There are still many inconsistencies in relation to data analyses and the scientific interpretation on the results. As I commented before, the year of digestate application also means the frequency of application and/or total amount of digestate application, in which in year 1 there was only once application, in year 2 twice application and in year 3 it was three times. Therefore, analyzing (ANOVA) using one-way ANOVA on the mean values of 27 sampling for three years really ignores the scientific basis of the individual measurement and the treatment effects. Soil is a dynamic body; it changes with time. So, Table 1 & Table 2 really ignore these, and ignore the facts that there are different situations between those measurements. Fig.1, especially Fig.1.a &1.b, but not 1.c, already take into account these scientific basis, but the way the data analyzed was incorrect. It can be seen from the df used to calculate the F values (I think the Authors should supply us with the dataset so we can make an appropriate suggestions on the correct way to analyze the data). Line 165-166 says 5 sampling replications, which means that there were 75 experimental units or data units in each sampling time (5T x 3Y x 5R), but they were only 45 units in Fig.5, and I think this is one factor causing the non-significant interaction effect on the earthworm biomass. This is a big mistake of data analysis. Please read my further comments carefully on the manuscript.

To improve the scientific basis of your paper, please first revise the data analyses.

Comments for author File: Comments.PDF

Author Response

Response to Reviewer Comments on Ref.: Ms. No. agriculture-3696513

Fertilization Effects of Solid Digestate Treatments on Earthworm Community Parameters and Selected Soil Attributes

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

Reviewer #2

This revised version is getting better, but it still need to be revised. There are still many inconsistencies in relation to data analyses and the scientific interpretation on the results. As I commented before, the year of digestate application also means the frequency of application and/or total amount of digestate application, in which in year 1 there was only once application, in year 2 twice application and in year 3 it was three times. Therefore, analyzing (ANOVA) using one-way ANOVA on the mean values of 27 sampling for three years really ignores the scientific basis of the individual measurement and the treatment effects. Soil is a dynamic body; it changes with time. So, Table 1 & Table 2 really ignore these, and ignore the facts that there are different situations between those measurements. Fig.1, especially Fig.1.a &1.b, but not 1.c, already take into account these scientific basis, but the way the data analyzed was incorrect. It can be seen from the df used to calculate the F values (I think the Authors should supply us with the dataset so we can make an appropriate suggestions on the correct way to analyze the data). Line 165-166 says 5 sampling replications, which means that there were 75 experimental units or data units in each sampling time (5T x 3Y x 5R), but they were only 45 units in Fig.5, and I think this is one factor causing the non-significant interaction effect on the earthworm biomass. This is a big mistake of data analysis. Please read my further comments carefully on the manuscript. To improve the scientific basis of your paper, please first revise the data analyses.

The authors thank the Reviewer for taking the time to review our article and for the important and insightful comments that have led to improvements in the current version of the manuscript. The authors carefully considered the comments and endeavored to respond to each of them. We hope your revised manuscript will meet your high standards.

Comment [WW1]: Please provide the ref(s) for this sentence.

Thank you for your attention. Done.

Comment [WW2]: Please provide the ref(s) for this clause.

Thank you for your attention. Done.

Comment [WW3]: Please also  explicitly state the objective(s) of conducting the experiment for 3 years? Otherwise, there is no need  to conduct the experiment for 3 years. Was it to assess the long-term effects of ….?

The research was conducted for three years, because in field research on Lumbricidae (as well as in other faunal studies), three years is the minimum period to obtain reliable results and formulate conclusions. These three years can also be considered as three subsequent repetitions of the experiment. Each year, specific doses of digestate were used, because identical treatments are used in practice, in agriculture in monoculture crops grown in the same area. Therefore, the discussed experiment was a kind of simulation of practices often used in plant production. The methods used in this research are commonly used in research on earthworm communities in both semi-natural and anthropogenic areas.

Comment [WW4]: What was  the size of the sampling area each  of the 5 samples?

As shown in 2.3 Earthworm and Soil Sampling, the sampling area size is 0.22 m² (lines 181-182).

Comment [WW5]: Please describe what were the spatial position of these 3 sampling replications relative to the 5 sampling sites for extracting the earthworms in Line 166. This is very important to determine whether the sampling sites for extracting earthworms and for measuring soil properties are related or independent each other. It will be better also if the number of replicates are the same (i.e. 3 sites for more reliable data), especially in relation to the interpretation of the relationships between earthworm data and soil properties.

Soil sampling was each time related to the places where earthworms were collected.

As presented in Methodology 2.3 Earthworm and Soil Sampling, lines 173-178 "During the study, earthworm sampling at the experimental and control plots was conducted three times per year (June, August, and October) in three annual cycles (2021-2023). Five randomly determined samples were taken each time within the five plots (CL, DG25, DG50, DG75 and DG100; with an area of 100 m² each), but in such a way as not to duplicate the places where earthworms are extracted on subsequent dates.” It may have been imprecise in the description of the methodology, but each time 5 random earthworm collections were used in an octet with a circle area (earthworm collection area) of 0.22 m² to obtain the total values ​​of the analyzed features of Lumbricidae species (abundance and biomass) per m² (5 earthworm collections x 0.22 m²). That is, these 5 collections gave one specific value concerning, for example, the biomass of a specific species per m². Therefore, in total, in 5 fields, for 3 years, earthworms were collected 3 times each year (June, August, and October). Supplemented in the text (lines: 184-187).

Comment [WW6]: Soil is a dynamic body; it changes with time. So, analyzing (ANOVA) the mean values of data obtained from 27 sampling times for three years means that the author(s) really ignore that soil is a dynamic body. Soil is alive. In addition, as my previous comments, in this study, different year means different frequency and different dose of digestate (fertilizer) application. So, analyzing (ANOVA) the mean values of 27 sampling times in 3 years is a very BIG mistake, meaning that the author(s) ignore the scientific basis of the experiment. It is most likely impossible that organic fertilization did not change soil properties from once to twice then three times of digestate application. Therefore, as I recommended before, Year must be treated as the second factor consisting of three levels: 1 year, 2 years & 3 years, because the plots received different amount of digestate between 1 year, 2 years and 3 years, using mean values from 3 samplings in yeas 1, 3 sampling in year 2 and 3 sampling in year 3. Please also remember that the replications from 3 sampling sites per sampling time should be source of variation in the ANOVA with 2 df, so that the error df should be = 28 not 30. Therefore, Table 1 must be extended with 3 columns to the right to contain mean values in each year. Please also remember to indicate whether the interaction effects are significant or not. If the interaction effect was mot significant, presentation of the mean values cannot be done like in Fig.6.

We have a fairly clear design on 5 fields, for 3 years we take 3 samples (replicates) every year, and we want to check how two factors affect selected soil parameters. First, different dose of digestate (Research site) and second year. And having 5 x 3 x 3 = 45 samples, we performed a two-way analysis of variance, also checking the interaction effect (which does not occur anywhere in this data).

Reviewer: "...because the plots received a different amount of digestate between 1 year, 2 years and 3 years..."

No, the amount in a given field is the same every year. Lines 143-146.

Reviewer: "Please also remember that the replications from 3 sampling sites per sampling time should be a source of variation in the ANOVA with 2 df"

In our experimental design, three replicate soil samples were collected within each combination of year and fertilization dose. These replicates were treated as independent observations and contributed to the residual variance in the ANOVA model. Since they represent random sampling within treatment combinations and not a structured block or repeated measure, we did not include replication as a fixed or random factor. Nevertheless, the use of replicates increased the reliability of estimated means and enabled testing of interaction effects with appropriate residual degrees of freedom.”

Reviewer: “Therefore, Table 1 must be extended with 3 columns to the right to contain mean values ​​in each year.”

Adding three additional columns to this table, which is already very large, is quite difficult, and as can be seen in Fig. 6, for no soil parameter there is no interaction, and only in the case of C/N ratio the year effect is significant, in the remaining ones it has no effect.

Comment [WW7]: Similar comments to Table 1 also apply to Table 2. In addition, Line 166 says that there were 5 sampling replications, which means that for earthworm data, there were 5 treatments (plots) x 3 years x 5 replications, resulting in 75 experimental units (from 15 treatment combinations). This means that total df = 74, replications df = 4, treatment df = 4, year df = 2, interaction df = 8; then error df = 56 (Not 30 as shown in Fig. 1 a&b. Since replication variance will reduce error variance, these will increase the calculated F values, especially for the interaction.

Similarly to the above, we have a 5-field design, for 3 years we collect earthworms 3 times a year (repeats), and we want to check how two factors affect the number, biomass and biodiversity index. The first is a different dose of digestate (Research site) and the second year. And having 5 x 3 x 3 = 45 samples, we performed a two-way analysis of variance, also checking the interaction effect.

In our experimental design, three replicate soil samples were collected within each combination of year and fertilization dose. These replicates were treated as independent observations and contributed to the residual variance in the ANOVA model. Since they represent random sampling within treatment combinations and not a structured block or repeated measure, we did not include replication as a fixed or random factor. Nevertheless, the use of replicates increased the reliability of estimated means and enabled testing of interaction effects with appropriate residual degrees of freedom.”

This means that total df = 44, treatment df = 4, year df = 2, interaction df = 8; then error df = 30.

Comment [WW8]: These values are not in line with the statement in Line 166, saying that the sampling replications were 5, which means that the experiments were in 5 blocks. Please read the comments for Table 2.

Same answer as to Comment [WW5]

Comment [WW9]: This figure must not show differences between treatments but also between years, and a figure or table containing analysis of the interaction effects, which are more important than just the main effects of treatments, because when the interaction effect was significant, it means that the effects of the 5 treatments are different between years, which also means different between different frequencies of fertilization. The length of the error bars between treatments, such as in Fig.1.c looks exactly the same, which is almost impossible. Please use the treatments SE (normally SE is used).

To illustrate the differences between treatments and years, we have used Figure 3 in this chapter.

Reviewer: The length of the error bars between treatments, such as in Fig.1.c looks exactly the same, which is almost impossible. Please use the treatments SE (normally SE is used).

Corrected.

Comment [WW10]: Why Fig.1.c has different experimental units from Fig.1.a and Fig.1.b? And there is no interaction effect is shown for the data in Fig.1.c?

Figure 1c has different experimental units, because it shows the calculated Shannon-Wiener diversity index (H') determined (as shown in the methodology) from H' = Σpi× log(n) pi where pi - the ratio of the number of organisms of a given species to the total number of all organisms. This index is a popular measure used in ecology. The F-test results were performed on raw data by species, corrected, and the interaction effect was added.