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Relative Growth Rate and Specific Absorption Rate of Nutrients in Lactuca sativa L. Under Secondary Paper Sludge Application and Soil Contamination with Lead

Agriculture 2025, 15(14), 1541; https://doi.org/10.3390/agriculture15141541

by Elena Ikkonen^*

and Marija Yurkevich

Reviewer 1: Anonymous

Reviewer 2:

Xiaoe Yang

Reviewer 3: Anonymous

Agriculture 2025, 15(14), 1541; https://doi.org/10.3390/agriculture15141541

Submission received: 13 May 2025 / Revised: 14 July 2025 / Accepted: 16 July 2025 / Published: 17 July 2025

(This article belongs to the Section Agricultural Soils)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Abstract

The year and the place of the experiment have to be mentioned in the abstract;
What was the statistical method for the experiment?
The abstract needs to be improved by adding some important numerical results;

Introduction

Because of the nature of your study, which disuses the using of secondary paper sludge in a contaminated soil and also the fate of Pb in the soil and plant, the authors have to present some information about the sources of heavy metals (here Pb) in agriculture/soil and their potential toxicity or harmful effects on public health. By this matter the continuity of the work is improved and the readers can simply understand the main questions and objectives of the study. For this matter, there are some nice and newer works on the sources of the metals in the soil/ agriculture and their impacts on public health as follows, and dear authors may use them for improving the quality of the literature reviews and discussing the measurements;

Please by citing the relevant works and rsearch, discus about the involved mechanisms of sludge for reducing the limiting effects of heavy metals on plant growth.
The novelty of the work has to be highlighted in the introduction section.

Materials and methods

There was no information about the year and the place of the study, the climatological condition and etc.,
The number of the seeds for sowing per pot, the properties of the pots in terms of the size, the weight/volume of the soil used for each pot have to be presented.
Did the sludge using in this study analyzed? Just the content of N, P, and K was presented; the organic carbons, the Pb content of the sludge, and the electrical conductivity have to be mentioned.
As you know there are more important properties for the soil which they are important and determining of the plant growth; for example organic carbon content, electrical conductivity, acidity, calcium carbonate, etc., I could not find these information in the work.

Results and discussion

The measurements have to be discussed by using the involved mechanisms and the factors that affect the functions of sludge on the growth of the plant and the behavior of Pb in the soil;

Author Response

Dear Reviewer!

Let me thank you for your high appreciation of our manuscript and useful comments, which undoubtedly improved its quality.

Let me highlight all the changes made based on your comments:

Abstract

The year and the place of the experiment have to be mentioned in the abstract;

Thank you. We added in the Abstract the year and the place of the experiment:

‘For the 46-day pot experiment, which was carried out in 2022 under controlled conditions at the Karelian Research Centre of RAS, sandy loam soil was used to which Pb was added with a rate of 0, 50 and 250 mg Pb(NO₃)₂ kg^-1’.

What was the statistical method for the experiment?

We added in the text:

‘Two-way ANOVA test was used to determine the effect of sludge application and soil Pb on plant parameters’.

The abstract needs to be improved by adding some important numerical results;

Thank you. We added numerical results:

‘For plants grown on soil with the highest Pb concentration studied, secondary sludge reduced root lead content by an average of 35%. Soil contamination with lead increased nutrient SAR by 79 and 39% when applied as 20 and 40% solutions, respectively, and application of 40% sludge increased nitrogen SAR to 51% but did not change phosphorus and potassium SAR’.

Introduction

Because of the nature of your study, which disuses the using of secondary paper sludge in a contaminated soil and also the fate of Pb in the soil and plant, the authors have to present some information about the sources of heavy metals (here Pb) in agriculture/soil and their potential toxicity or harmful effects on public health. By this matter the continuity of the work is improved and the readers can simply understand the main questions and objectives of the study. For this matter, there are some nice and newer works on the sources of the metals in the soil/ agriculture and their impacts on public health as follows, and dear authors may use them for improving the quality of the literature reviews and discussing the measurements;

Thank you. We added in the Introduction section a text related to the sources of heavy metals in agricultural soils and negative impact on crop production and human health:

‘Unfortunately, the possibility of contamination of agricultural soils with heavy metals including lead exists throughout the world [19]. Natural sources, anthropogenic activities and atmospheric deposition have been identified as potential sources of heavy metal with the primary role of human contribution to soil contamination with heavy metals [20]. Industrial production and agricultural practices such as fertilization, pesticide use, and wastewater irrigation can lead to the accumulation of high concentrations of metals in soil, posing a serious threat to food production and human health [21]’.

Please by citing the relevant works and rsearch, discus about the involved mechanisms of sludge for reducing the limiting effects of heavy metals on plant growth.

Thank you. Done. We added:

‘The positive effects of pulp and paper sludges as a rich source of organic substrates on soil properties have been found for different soil types and climatic zones [12,25,26]. In addition to increasing the organic matter content of soil, agricultural use of paper sludges has been found to have a positive effect on soil nutrient content, microbial activity, as well as immobilization of toxic elements [6,7,27]’.

The novelty of the work has to be highlighted in the introduction section.

The novelty has been added to the Introduction section:

‘Furthermore, the novelty of the study was to evaluate the effect of soil lead content on plant growth rate and the associated net uptake rate to understand whether the application of secondary sludge affects the response of lettuce to heavy metals’.

Materials and methods

There was no information about the year and the place of the study, the climatological condition and etc.,

Thank you. We added information:

‘The soil was collected in 2022 from the field site of the Korza station located in the Northwest of Russia’.

The conditions of plant growth are described here:

‘The plants were cultivated under controlled conditions of 23/20 ◦C of day/night temperature, 250 µmol m−2 s−1 of photosynthetic photon flux density, and 16−h photoperiod’.

The number of the seeds for sowing per pot, the properties of the pots in terms of the size, the weight/volume of the soil used for each pot have to be presented.

Thanks. We described pot size and soil density in pots:

‘The soil substrates were incubated for 14 days and placed in 0.8 L pots (D = 15 cm) with a soil bulk density of about 1.4 g cm⁻³’.

Number of seeds per pot (one) and number of pots per treatment (16) have also been added.

Did the sludge using in this study analyzed? Just the content of N, P, and K was presented; the organic carbons, the Pb content of the sludge, and the electrical conductivity have to be mentioned.

We showed sludge properties:

‘The secondary sludge used in this study contained 238, 64, and 9 mg L−1 of N, P, and K, respectively, and 47, 233 and 22 mg L−1 of Na, Ca, and Mg, respectively, without any Pb content. The carbon content of the sludge was 56.7% and pH was 7.16’.

As you know there are more important properties for the soil which they are important and determining of the plant growth; for example organic carbon content, electrical conductivity, acidity, calcium carbonate, etc., I could not find these information in the work.

We added in the text the properties of the collected soil:

‘..sandy loam soil characterized by a low natural fertility, with N content of 0.39%, P and K content of 0.16%, Mg and Ca content of 0.20 and 1.95 g kg-1 accordingly, and a natural Pb content of 0.13 mg kg−1 [28]. The humus content of the collected soi was 0.7% and pH was 5.46’.

Results and discussion

The measurements have to be discussed by using the involved mechanisms and the factors that affect the functions of sludge on the growth of the plant and the behavior of Pb in the soil;

Thank you. We added discussion of possible mechanisms responsible for the sludge effect on plant growth and soil Pb.

Dear Reviewer, thank you again!

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents an investigation into the use of secondary pulp and paper-mill sludge to mitigate the effects of lead (Pb) contamination on Lactuca sativa seedlings. The study addresses an important environmental and agricultural issue, especially relevant for sustainable waste management and soil remediation. The experimental design is good, and key physiological parameters (e.g., RGR, NAR, SAR) are appropriately used to assess plant performance. However, several minor issues require to be addressed before publication.

(1) Line 1-2: "Economic methods for increasing soil fertility and mitigating the negative impact of heavy metals contamination of agricultural soils are currently being studied and discussed."

The phrase “economic methods” may be not clear. It could be interpreted as cost-related or policy-driven. The phrase "heavy metals contamination" should be "heavy metal contamination"

Replace “economic” with a more specific term like “cost-effective” or “sustainable.” Also correct grammar.

Example: "Cost-effective methods for improving soil fertility and mitigating the negative impact of heavy metal contamination in agricultural soils are currently under investigation."

(2) Line 3-4: "In this study, we aimed to evaluate the impact of soil lead (Pb) contamination and of secondary pulp and paper mill sludges application on the relative growth rate (RGR) and its determinants..."

“and of...sludges application” is grammatically incorrect. “Sludges” is also typically uncountable in this context.

Example: "This study aimed to evaluate the impact of soil lead (Pb) contamination and the application of secondary pulp and paper mill sludge on the relative growth rate (RGR) and its determinants..."

(3) Line 6: "to which Pb was added with a rate of 0, 50 and 250 mg Pb(NO3)2 kg-1" “with a rate of” is not correct. Use “at the rates of”.

Example: "to which Pb was added at the rates of 0, 50, and 250 mg Pb(NO₃)₂ kg⁻¹."

(4) Line 7: "Secondary sludge was added to each watering of plants at a concentration of 0, 20 or 40% solution." “Each watering of plants” is not clear; “at a concentration of 0, 20 or 40% solution” could be clearer. Simplify and improve flow.

Example: "Secondary sludge was applied with each watering at concentrations of 0%, 20%, and 40%."

(5) Line 8-9: "The RGR values 15 demonstrated a high degree of variability, which was mainly due to variability in net assimilation rate (NAR) rather than specific leaf area." Repetition of “variability” is not good.

Example: "RGR values varied significantly, primarily due to changes in net assimilation rate (NAR) rather than specific leaf area (SLA)."

(6) Line 14: "Soil contamination with Pb increased SAR of nutrients and while sludge application can lead to an increased SAR of N, but not P and K." (First time use element notation (N, P, K vs. full names).

(7) Lines 28–30: To increase crop yields, agricultural soils with low nutrient capacity require improvement with commercial high-cost fertilizers. New economic methods for increasing soil fertility and reducing agricultural production costs are currently being 30 studied and discussed.

“Low nutrient capacity” use “low fertility” instead.

Example: To increase crop yields, agricultural soils with low fertility are typically amended with costly commercial fertilizers. Alternative, cost-effective methods to improve soil fertility and reduce production costs are currently under investigation.

(8) Lines 33–34: Secondary sludge from the pulp and paper industry is produced by the biological treatment of primary sludges by microorganisms to reduce the charge of dissolved organic substrates. Repetitive use of "sludge(s)." improve the sentences

Example: Secondary sludge is generated through the biological treatment of primary sludge, during which microorganisms break down dissolved organic matter.

(9) Lines 35–36: To activate biological processes, nitrogen and phosphorus are added to biological treatment processes, resulting in higher nitrogen and phosphorus contents in secondary sludge. Avoid Repetitive words with “biological processes” and “biological treatment.”

Example: To stimulate microbial activity during treatment, nitrogen and phosphorus are added, which resulted in elevated concentrations of these nutrients in the secondary sludge.

(10) Lines 37–38: In addition, secondary sludge has a higher organic matter content and a lower carbon to nitrogen ratio compared to primary sludges. Improve sentence as:

Example: Secondary sludge also contains more organic matter and has a lower carbon-to-nitrogen (C:N) ratio than primary sludge.

(11) Lines 39–40: Secondary sludge application can result in increased soil pH, C, N, P and K 39 concentrations, and increased N content in plants, as found for wheat. Repetition (“increased...increased”). improve the sentence

Example: Application of secondary sludge has been shown to increase soil pH and the concentrations of carbon (C), nitrogen (N), phosphorus (P), and potassium (K), as well as plant nitrogen uptake, particularly in wheat.

(12) Lines 45–48: Nutrient status certainly has a direct impact on plant growth and yield, as macronutrients are part of numerous compounds and perform vital functions for plants, ensuring their successful development and growth. It was suggested that nutrient status has a more direct effect on plant growth rate than biomass accumulation, which is essentially a consequence of growth rate.

Overuse of general statements (“certainly,” “numerous compounds”). Simplify the sentence structure.

Example: Nutrient availability directly affects plant growth and yield, as macronutrients are essential components of biochemical compounds and physiological processes. It has been suggested that nutrient status more directly influences plant growth rate than biomass accumulation, which is a result of growth rate over time.

(13) Lines 58–60: However, there is little information on the effects of sludge on macronutrient accumulation in plants and the coupling between nutrient content and relative plant growth rate. Sentence is good but even more better as:

Example: However, limited information is available on how sludge application influences macronutrient accumulation and its relationship with plant growth rate.

(14) Unfortunately, the possibility of contamination of agricultural soils with heavy 61 metals, including lead, exists throughout the world (Sahito et al., 2023). Add more relevant reference here regarding lead contamination.

Sahito, Z. A., Zehra, A., Yu, S., Chen, S., He, Z., & Yang, X. (2023). Chinese sapindaceous tree species (Sapindus mukorosii) exhibits lead tolerance and long-term phytoremediation potential for moderately contaminated soils. Chemosphere, 338, 139376.

(15) Lines 67–68: Secondary sludges can be effective biosorbents for heavy metal ions, reducing their uptake by plants. Thus, it can be assumed that sludge applications can reduce the limiting effect of heavy metals on plant growth processes. Sentence is good but it can be more logical:

Example: Secondary sludge may act as an effective biosorbent for heavy metals, potentially reducing their uptake by plants. Consequently, sludge application might alleviate the growth-limiting effects of heavy metal contamination.

(16) Line 78: "We used" is not formal in scientific writing

Example: Sandy loam soil with low natural fertility was used for the pot experiment.

(17) Line 80: Air drying soil................................

correction:“The air-dried soil” was passed through a 2 mm sieve…

(18) line 81: “Then the soil was mixed with Pb(NO3)2 with the rate of 0, 50, and 250 mg kg−1…” “With the rate of” is incorrect.

Example: The soil was then amended with Pb(NO₃)₂ at concentrations of 0, 50, and 250 mg kg⁻¹ (designated as 0 Pb, 50 Pb, and 250 Pb, respectively).

(19) Line 83: “Seeds of Lactuca sativa L. var. Medvezhje ushko were sowing…”

Grammar error – “were sowing” → “were sown.”

(20) Line 85: “of 23/20 ◦C of day/night temperature…”

Unclear format and extra "of." Revise it

Example: ...at a day/night temperature of 23/20 °C...

(21) Line 86: “250 µmol m−2 s−1 of photosynthetic photon flux density…”

Add “a” and simplify the sentence

Example:...a photosynthetic photon flux density (PPFD) of 250 µmol m⁻² s⁻¹...

(22) Line 87-88: “...with 0, 20, or 40% solutions of the secondary pulp and paper-mill sludges... accordingly.”

“Accordingly” should be “respectively.”

Example: .....with 0%, 20%, or 40% solutions of secondary pulp and paper mill sludge, referred to as the 0%, 20%, and 40% treatments, respectively.

(23) line 120-121:“All data were shown as mean ± SE... LSD test at a p < 0.05 level was used...”

"were shown" → "are presented."

(24) Lines 130-132: “For untreated 21– and 34–day-old plants, leaf area values tendet to decrease…” Typo error: “tendet” should be “tended”

(25) line 131 “Two-way ANOVA test did not reveal any signifcant effect (p > 0.05) of Pb on leaf area…”

“signifcant” should be “significant”

(26) line 135: “Regardless of soil Pb content no signifcant diferences…”

“signifcant” should be “significant”; “diferences” → “differences”

(27) lines 140-141: “...increase in root biomass accumilation regardless of soil Pb content but not in all cases this increase was supported by statistical test.”

“accumilation” should be “accumulation”

(28) line 142: “No strong and clear effect of sludge treatment on leaf area values were observed in this study.”

“were” should be was (singular subject)

(20) line 143: “The 17–day-old plants had the highest SLA values regardless soil Pb content and sludge doze (Table 1).”

“doze” have different meaning. It should be “dose”

(30) lines 257-267: In our study, we aimed to understand how secondary sludge application to nutrient-poor soils affect plant growth and nutrient uptake.

“affect” should be “affects” (subject-verb agreement)

(31) Temporal variability in RGR of lettuce leaves and roots found in this study confirm age-mediated changes in relative growth rate…

“confirm” should be “confirms” (subject-verb agreement)

(32) lines 277-283: The RGR variability was stated to be closely related to plant physiology and morphology. “was stated to be” is not correct.

Example: RGR variability is closely linked to plant physiological and morphological traits.

(33) lines 294-304: Pb accumulation in roots and shoots of L. sativa was found to decreased NAR at early stage…

“to decreased” → “to decrease” or “to be decreased”

“at early stage” → “at early growth stages”

(34) Close associations between SAR and RGR founded in our study and reported early suggest a strong involvement of SAR in determining RGR.

“founded” should be “found”

“reported early” should be “reported earlier”

(35) Materials and Methods (Lines 81–82): The manuscript states that Pb(NO₃)₂ was applied at rates of 0, 50, and 250 mg kg⁻¹. It would strengthen the study to explain how these levels were selected—whether based on preliminary studies or existing literature? Please clarify the rationale behind choosing these concentrations.

(36) Materials and Methods (Lines 89–94): The study mentions the sludge's N, P, K content, but does not report other critical parameters such as heavy metal content, organic pollutants, pH, electrical conductivity (EC), and the stability of the organic matter. These are essential for evaluating the safety and agronomic suitability of the sludge. Please provide this information.

(37) Scope of Physiological Assessment: While the study focuses on biomass and nutrient uptake, incorporating physiological parameters such as photosynthesis rate, chlorophyll content, or stress biomarkers (e.g., proline levels, antioxidant enzyme activities) would provide a more comprehensive understanding of the plant’s response to Pb and sludge treatments.

(38) Long-Term Implications of Sludge Application: The study does not discuss the potential long-term environmental risks associated with industrial sludge use, such as the possibility of secondary pollution or heavy metal accumulation. Please consider including a brief discussion of these aspects and suggestions for addressing them in future research.

(39) Tables 1 and 2: The current table formatting is quite crowded which makes it difficult to read. Consider improving the layout, perhaps by splitting into separate tables or using clearer grouping and spacing to enhance readability.

Comments on the Quality of English Language

English should be thoroughly revised and corrected at several places

Author Response

Dear Reviewer!

Let me thank you for your high appreciation of our manuscript and useful comments, which undoubtedly improved its quality.

Let me highlight all the changes made based on your comments:

This manuscript presents an investigation into the use of secondary pulp and paper-mill sludge to mitigate the effects of lead (Pb) contamination on Lactuca sativa seedlings. The study addresses an important environmental and agricultural issue, especially relevant for sustainable waste management and soil remediation. The experimental design is good, and key physiological parameters (e.g., RGR, NAR, SAR) are appropriately used to assess plant performance. However, several minor issues require to be addressed before publication.

(1) Line 1-2: "Economic methods for increasing soil fertility and mitigating the negative impact of heavy metals contamination of agricultural soils are currently being studied and discussed."

The phrase “economic methods” may be not clear. It could be interpreted as cost-related or policy-driven. The phrase "heavy metals contamination" should be "heavy metal contamination"

Replace “economic” with a more specific term like “cost-effective” or “sustainable.” Also correct grammar.

Example: "Cost-effective methods for improving soil fertility and mitigating the negative impact of heavy metal contamination in agricultural soils are currently under investigation."

Thank you! Done.

“and of...sludges application” is grammatically incorrect. “Sludges” is also typically uncountable in this context.

Thank you! We used your correct form.

(3) Line 6: "to which Pb was added with a rate of 0, 50 and 250 mg Pb(NO3)2 kg-1" “with a rate of” is not correct. Use “at the rates of”.

Example: "to which Pb was added at the rates of 0, 50, and 250 mg Pb(NO₃)₂ kg⁻¹."

Example: "Secondary sludge was applied with each watering at concentrations of 0%, 20%, and 40%."

Thank you! Fixed!

Example: "RGR values varied significantly, primarily due to changes in net assimilation rate (NAR) rather than specific leaf area (SLA)."

We changed this sentence to:

‘Soil contamination with lead increased nutrient SAR by 79 and 39% when applied as 20 and 40% sludge, respectively, while 40% sludge increased nitrogen SAR by 51% but did not change phosphorus and potassium SAR’.

“Low nutrient capacity” use “low fertility” instead.

Thank you! Done.

Example: Secondary sludge is generated through the biological treatment of primary sludge, during which microorganisms break down dissolved organic matter.

Thank you! Done.

Example: To stimulate microbial activity during treatment, nitrogen and phosphorus are added, which resulted in elevated concentrations of these nutrients in the secondary sludge.

Thank you!

(10) Lines 37–38: In addition, secondary sludge has a higher organic matter content and a lower carbon to nitrogen ratio compared to primary sludges. Improve sentence as:

Example: Secondary sludge also contains more organic matter and has a lower carbon-to-nitrogen (C:N) ratio than primary sludge.

Thank you! Done.

Thank you!

Overuse of general statements (“certainly,” “numerous compounds”). Simplify the sentence structure.

Example: Nutrient availability directly affects plant growth and yield [12], as macronutrients are essential components of biochemical compounds and physiological processes [13,14].. It has been suggested that nutrient status more directly influences plant growth rate than biomass accumulation, which is a result of growth rate over time.

Done.

Example: However, limited information is available on how sludge application influences macronutrient accumulation and its relationship with plant growth rate.

Thank you!

Done! Thank you!

Thank you!

(16) Line 78: "We used" is not formal in scientific writing

Example: Sandy loam soil with low natural fertility was used for the pot experiment.

Done.

(17) Line 80: Air drying soil................................

correction:“The air-dried soil” was passed through a 2 mm sieve…

Thank you!

(18) line 81: “Then the soil was mixed with Pb(NO3)2 with the rate of 0, 50, and 250 mg kg−1…” “With the rate of” is incorrect.

Example: The soil was then amended with Pb(NO₃)₂ at concentrations of 0, 50, and 250 mg kg⁻¹ (designated as 0 Pb, 50 Pb, and 250 Pb, respectively).

Corrected.

(19) Line 83: “Seeds of Lactuca sativa L. var. Medvezhje ushko were sowing…”

Grammar error – “were sowing” → “were sown.”

Ok.

(20) Line 85: “of 23/20 ◦C of day/night temperature…”

Unclear format and extra "of." Revise it

Example: ...at a day/night temperature of 23/20 °C...

Corrected.

(21) Line 86: “250 µmol m−2 s−1 of photosynthetic photon flux density…”

Add “a” and simplify the sentence

Example:...a photosynthetic photon flux density (PPFD) of 250 µmol m⁻² s⁻¹...

Thank you.

(22) Line 87-88: “...with 0, 20, or 40% solutions of the secondary pulp and paper-mill sludges... accordingly.”

“Accordingly” should be “respectively.”

Yes, you are right!

Example: .....with 0%, 20%, or 40% solutions of secondary pulp and paper mill sludge, referred to as the 0%, 20%, and 40% treatments, respectively.

(23) line 120-121:“All data were shown as mean ± SE... LSD test at a p < 0.05 level was used...”

"were shown" → "are presented."

Thank you!

(24) Lines 130-132: “For untreated 21– and 34–day-old plants, leaf area values tendet to decrease…” Typo error: “tendet” should be “tended”

Yes!

(25) line 131 “Two-way ANOVA test did not reveal any signifcant effect (p > 0.05) of Pb on leaf area…”

“signifcant” should be “significant”

(26) line 135: “Regardless of soil Pb content no signifcant diferences…”

“signifcant” should be “significant”; “diferences” → “differences”

(27) lines 140-141: “...increase in root biomass accumilation regardless of soil Pb content but not in all cases this increase was supported by statistical test.”

“accumilation” should be “accumulation”

Thank you! I'm so careless. Sorry!

(28) line 142: “No strong and clear effect of sludge treatment on leaf area values were observed in this study.”

“were” should be was (singular subject)

Yes! Thank you!

(20) line 143: “The 17–day-old plants had the highest SLA values regardless soil Pb content and sludge doze (Table 1).”

“doze” have different meaning. It should be “dose”

Thank you!!!

(30) lines 257-267: In our study, we aimed to understand how secondary sludge application to nutrient-poor soils affect plant growth and nutrient uptake.

“affect” should be “affects” (subject-verb agreement)

Thank you!

(31) Temporal variability in RGR of lettuce leaves and roots found in this study confirm age-mediated changes in relative growth rate…

“confirm” should be “confirms” (subject-verb agreement)

You are right!

(32) lines 277-283: The RGR variability was stated to be closely related to plant physiology and morphology. “was stated to be” is not correct.

Example: RGR variability is closely linked to plant physiological and morphological traits.

Yes! Done.

(33) lines 294-304: Pb accumulation in roots and shoots of L. sativa was found to decreased NAR at early stage…

“to decreased” → “to decrease” or “to be decreased”

“at early stage” → “at early growth stages”

Thank you!

(34) Close associations between SAR and RGR founded in our study and reported early suggest a strong involvement of SAR in determining RGR.

“founded” should be “found”

“reported early” should be “reported earlier”

Thank you!

We added in the text of the Materials and Methods section:

‘The concentrations studied were selected in a preliminary experiment’.

Thank you. We have added some information related to the sludge studied:

‘The secondary sludge used in this study contained 238, 64, and 9 mg L⁻¹ of N, P, and K, respectively, and 47, 233 and 22 mg L⁻¹ of Na, Ca, and Mg, respectively, without any Pb content. The carbon content of the sludge was 56.7% and pH was 7.16’.

Yes, you are right.

We added in the text:

‘However, the long-term impacts of using pulp and paper mill sludges in agriculture and the potential risk of accumulation of contaminants and heavy metals in soils with long-term sludge application deserve further study’.

Thank you! The tables are now corrected.

Let me thank you again, dear Reviewer, for your help with the manuscript!

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Manuscript ID: agriculture-3668820

Title: Relative growth rate and specific absorption rate of nutrients in Lactuca sativa L. under secondary paper sludge application and soil contamination with lead

Main Comments

This manuscript presents a relevant study that fits within the scope of Agriculture, assessing the role of secondary pulp and paper mill sludges in mitigating lead (Pb) impacts in lettuce. The results are promising. However, I have some concerns about clarity, the data presentation and its statistical analysis, and the writing quality, which need to be solved before the manuscript can be considered for publication. In my opinion, this manuscript requires a more rigorous discussion to meet the journal’s standards. I also recommend clearly emphasising the novelty in the Abstract and Introduction sections. There are some grammatical errors, typos, and strange phrasing throughout the manuscript that should be addressed before acceptance. The manuscript also would benefit from language polishing.

Abstract

This section is short and clear and provides enough information. Still, the conclusions provided by the authors need to be checked. The use of secondary pulp and paper mill sludge as soil amendment shows that this product can help remediate Pb-contaminated soils and reduce its toxicity to plants. In fact, this could also be emphasised during the introduction part – the use of sludges as remediation tools. In line 21, quantitative results should be included.

Introduction

This section is well-written. It is short and clear. Still, more information on the use of pulp and paper mill sludge in agriculture and its relevance should be added. In addition, this section did not develop the reason for choosing this type of soil amendment compared to others. The positive effects of pulp and paper mill sludge reported by other authors were explored, but not in sufficient depth. In addition, their comparison to other soil amendments used for similar purposes was not addressed. From the reader's point of view, there is also some confusion because the authors treat the sludge application as a tool to limit the effect of metals, but also as a tool for soil fertility. Be clear about this. The introduction also lacks a concise hypothesis/research question. I recommend that the authors end this section with a clear hypothesis, objective, and specific aims.

Materials & Methods

The CAS number and supplier of Pb are missing. A more detailed characterisation of the pulp and paper mill sludge is required. I also have one doubt regarding the application of the sludge. When was it first applied? At the beginning of the experiment? Was the soil pH, the electrical conductivity and the Pb content checked? The presentation of the statistical methodology is brief and poorly detailed; for example, there is no mention of normality and variance assumption checks, or how the letters that indicate the significance were assigned.

Why were these concentrations of Pb (line 82) and sludge (line 88) selected?

Results

This section requires major improvement. It lacks clarity in how the treatments differ statistically. I find it difficult to understand if the differences stated (in all tables and figures; figure 1 does not have the differences shown – must be a typo?) are among the Pb concentrations, the sludge rates, or the different time points. The statistical annotations are not well explained, and their interpretation is ambiguous. I suggest that the authors clarify the comparisons that were made. In this section, I also found that the results are generalised when they are only supported in specific conditions, which is misleading. In some sections, the authors are referring to specific results (for example, 34-day-old pants without sludge), but after they refer to the 46-day-old plants, it becomes hard for the reader to understand and follow the developed idea by the authors (e.g., lines 129 to 130).

Tables and Figures: Figure 1 lacks statistical annotation. The authors must clarify whether the values are means with standard deviation/error, and information regarding the statistical analysis should be provided (the meaning of the letters/symbols). In my opinion, Table 1 is a bit overwhelming to understand and interpret… I would recommend that the authors transform this table into two figures to improve the readability – one for the biomass parameter (root and shoot dry weight) and the other for the leaf area and specific leaf area.

Discussion

This section covered all the results. Still, the authors dedicated more to stating again the results rather than critically analysing them, which is fundamental in this part of the manuscript. The authors also did not discuss the study's limitations. There is no mention of the potential mechanisms through which the sludge affects the Pb uptake. What happens in the soil? What can be the potential long-term biological implications of sludge application in soil? What are the main challenges of using pulp and paper mill sludges?

Conclusion

The phrasing of this section should be improved for clarity and accuracy.

Author Response

Dear Reviewer!

Let me thank you for your high appreciation of our manuscript and useful comments, which undoubtedly improved its quality.

Let me highlight all the changes made based on your comments:

Title: Relative growth rate and specific absorption rate of nutrients in Lactuca sativa L. under secondary paper sludge application and soil contamination with lead

Main Comments

Abstract

Thank you! In the Abstraction section the conclusion was checked, the mechanisms of positive effect of sludges on soil contaminated with heavy metals are included. The quantitative results also are shown.

According to your comments we added in the Abstraction section:

‘Sludge-mediated reduction in root Pb content and increase NAR suggest that secondary pulp and paper mill sludge may contribute to the remediation of Pb-contaminated soils and reduce the toxicity of heavy metals to plants’.

Introduction

Thank you. According to your comments, we added in the Introduction section some text parts:

‘… the possibility of contamination of agricultural soils with heavy metals including lead exists throughout the world [19]. Natural sources, anthropogenic activities and atmospheric deposition have been identified as potential sources of heavy metal with the primary role of human contribution to soil contamination with heavy metals [20]. Industrial production and agricultural practices such as fertilization, pesticide use, and wastewater irrigation can lead to the accumulation of high concentrations of metals in soil, posing a serious threat to food production and human health [21]’.

Materials & Methods

The CAS number and supplier of Pb are missing. A more detailed characterisation of the pulp and paper mill sludge is required.

Thank you. We showed sludge properties:

I also have one doubt regarding the application of the sludge. When was it first applied? At the beginning of the experiment?

We highlighted that ‘The pots were watered every two days with 0%, 20%, or 40% solutions of secondary pulp and paper mill sludge, referred to as the 0%, 20%, and 40% treatments, respectively.

Was the soil pH, the electrical conductivity and the Pb content checked?

Soil pH and Pb content was checked, but not electrical conductivity. We added in the text:

‘The soil was characterized by a low natural fertility, with N content of 0.39%, P and K content of 0.16%, Mg and Ca content of 0.20 and 1.95 g kg^-1 accordingly, and a natural Pb content of 0.13 mg kg⁻¹ [29]. The humus content of the collected soil was 0.7% and pH was 5.46. The soil was collected from the field site of the Korza station located in the Northwest of Russia in 2022’.

The presentation of the statistical methodology is brief and poorly detailed; for example, there is no mention of normality and variance assumption checks, or how the letters that indicate the significance were assigned.

Thank you! The statistic section is now extended:

The two-way ANOVA test was used to determine the effect of sludge application and soil Pb, as well as their interaction on plant parameters. The linear function was used to describe a relation between the parameters.

Why were these concentrations of Pb (line 82) and sludge (line 88) selected?

We added in the text information that the concentrations studied were selected in a preliminary experiment.

Results

Thank you! The text of the Results section has been carefully revised and improved in accordance with your recommendations. Statistical annotations have been added, and comparisons have been clarified. All changes to the text are highlighted in yellow.

We added the statistical annotation to Figure 1. Thank you. In the Materials and Methods section, we explained that ‘All data are presented as mean ± SE…’ The meaning of letters has been added.

Discussion

Thank you. We added discussion of possible mechanisms responsible for the sludge effect on plant parameters and soil Pb:

‘Sludge-mediated stabilization of the ratio of respiration to photosynthesis can be responsible for the enhanced NAR in lettuce plants. Moreover, since NAR was positively related to plant nitrogen content (Figure 4), the increase in leaf nitrogen content may be one of the factors causing the increase in NAR and providing its positive relationship with RGR (Figure 6).

Nitrogen, phosphorus, and potassium are often the primary limiting factors for plant growth [45]. Secondary sludge applications resulted in an increase in root P content (Figure 3b), which may be related to sludge-mediated increase in soil phosphorus content [10,12] due to the introduction of phosphorus into the biological treatment processes of paper sludges [46]. Nunes et al. [9] also showed a positive effect of secondary sludge on the content of some macronutrients, including phosphorus, in agricultural soils. Increased soil phosphorus content may partially contribute to the restoration of photosynthetic activity and stabilization of the respiration to photosynthesis ratio and, consequently, NAR of lettuce plants. Paper sludge application can be responsible for the changes not only chemical but also physical properties of soils [11]. Butilkina and Ikkonen [11] showed an increase in the proportion of aeration pores, heat transfer and water-holding capacity of the soil under conditions of sludge addition, which could have a positive effect on the physiological characteristics of the plants. The results of our study displayed sludge-mediated decrease in Pb contents in both the roots and leaves (Table 2), which may be associated with an increase in organic matter content and a decrease in Pb mobility in the soil due to the formation of organo-mineral complexes [47]. Moreover, Pb phytoavailability can be reduced by phosphate application into soil [48]. Both organic matter and additional phosphorus may be responsible for decreased Pb uptake by plants, thereby helping to reduce the heavy metal load on plants’.

What can be the potential long-term biological implications of sludge application in soil? What are the main challenges of using pulp and paper mill sludges?

Thank you! According to the comments we added to the text:

Conclusion

The phrasing of this section should be improved for clarity and accuracy.

We have rewritten the Conclusion section. Thank you! All changes are highlighted in yellow.

Dear Reviewer, thank you again!

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Author have thoroughly revised the manuscript and addressed the key concerns; no further comments are required. Manuscript is recommended for acceptance.

Author Response

First, we would like thank the Reviewer for attentive revision of our manuscript and constructive comments and suggestions.

Let me indicate the modifications made in the manuscript in the light of yours comments.

We used ‘heavy metal and metalloids’ instead of ‘heavy metals’;
According to you comment related to the content of toxic elements in sludges we added in the text:

‘Although paper sludge may contain traces of potentially toxic elements, their concentrations are generally low and similar to those in organic fertilizers [8]. It was therefore concluded that application of paper sludge to soil at rates consistent with agricultural practice does not pose a significant risk of heavy metal accumulation in plants [8]’.

We added site coordinate:

‘The soil was collected from a field site of the research station of the Karelian Scientific Centre of the Russian Academy of Sciences (61°84´71.23”N, 33°21´12.28”W) in 2022’.

We added in the text:

‘The content of lead, cadmium, mercury, and arsenic in the sludge under study was 0.4, 0.02, 0.002, and 0.06 mg L−1, respectively’.

Best regards,

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Relative Growth Rate and Specific Absorption Rate of Nutrients in Lactuca sativa L. Under Secondary Paper Sludge Application and Soil Contamination with Lead

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