The Importance of Biological and Ecological Properties of Phragmites Australis (Cav.) Trin. Ex Steud., in Phytoremendiation of Aquatic Ecosystems—The Review

Round 1

Reviewer 1 Report

The present  manuscript is very comprehensive and carefully written. The topic discussed, phytoremediation of aquatic ecosystems, especially the efficiency of pollutant removal, and the P. australis as one of the most effective emergent species to accumulate various nutrients, is very important and sensible. 

The findings collected regarding the reed's ability to phytoremediate aquatic ecosystems are presented very systematically. The tables are meaningful, transparent and informative.

A good point is to address the problem of medicines and pharmaceuticals in aquatic ecosystems. 

Minor correction: A different font has been used since Chapter 4. Please unify with the rest.

In my opinion the manuscript is suitable for journal Water, and could be accepted in current form.

Author Response

Dear Reviewer 1:

We are sending You the revised version of the paper entitled “THE IMPORTANCE OF BIOLOGICAL AND ECOLOGICAL PROPERTIES OF PHRAGMITES AUSTRALIS (Cav.) Trin. Ex Steud., IN PHYTOREMEDIATION OF AQUATIC ECOSYSTEMS-THE REVIEW” with identification number “WATER- 787758” for consideration to be published in Water journal.

Thank you for the evaluations you have made to add value to our work.

Reviewer #1:

Comment #1. Minor correction: A different font has been used since Chapter 4. Please unify with the rest.

Response: We have acknowledged  your valuable suggestion and we have made the appropriate correction in the text. The font used since Chapter 4 has been changed to match the rest of the text.

Yours faithfully

Justyna Milke, Małgorzata Gałczyńska, Jacek Wróbel

Author Response File: Author Response.pdf

Reviewer 2 Report

Please find below my comments and suggestions on the manuscript of a review on Phragmites australis. Although I commend the authors on their attempt to write a broad account of this species' ecology and its use in bioremediation and nutrient removal, I feel the review is somewhat unbalanced, from a very brief introduction and statement of intent, to very detailed descriptions of studies on removal of particular heavy metals. I feel the authors have discovered quite some information on the heavy metal and nutrient removal of this and comparable species, which can be of great importance when choosing species for phytoremediation and for future design of treatment wetlands. By focusing more on these findings (maybe even approaching the form of a meta-analysis), shortening the introduction (using only relevant observations, and more of a narrative structure instead of a list of properties) and structuring the manuscript in a way that there is a build up of arguments supporting the author's goal, leading to a natural conclusion, I believe the work could be fit for resubmission to this journal.

General remarks

• The introduction is very brief, and does not really explain why the review has been written. Since the goal is stated as “demonstrating the suitability of Phragmites for use in phytoremediation”, it makes more sense to combine the introduction and section 2, into an actual introduction. Now, the introduction is a list of properties, most of which are repeated later in the manuscript, whereas section 2 is a random collection of background information on reed that does not necessarily contribute to the story on how this species is useful for phytoremediation.
• Figure 3 and 4 just list the same points as mentioned in the text. I do not feel that these figures are necessary or informative.
• The section 2.5 on microbial (interactions?) is very brief and does not actually contribute to the overall story. The authors mention that the rhizosphere contains many microorganisms and name a few of these, without explaining why these species are important, what they do, how they contribute to reed’s functioning as a species in phytoremediation. The other study that is cited, describes the establishment of symbiotic bacteria in quite some detail, again without mentioning why this would be important.
• In table 3, removal of heavy metals is reported for different species. However, this data seems to have been collected from different studies, which most likely have differences in heavy metal concentration and other environmental factors. The authors should address this. In addition, there could be a methods section indicating where the authors collected these studies from, what their criteria were for including them, what search terms they used to collect these references, etc.
• Some studies or references are discussed in a lot of detail (e.g. L. 258-366) while other facts are hardly mentioned, but the authors do not always manage to convey the relevance of the cited studies for the narrative of this review.

Specific remarks

• The distance between the lines decreases towards the end of the manuscript, making it harder to read
• 85: I presume you mean photographer, instead of author?
• 106 and L. 117; These headings consist of adjectives without objects.
• In Table 2, please add the type of system (natural/mesocosm; climate conditions)
• The authors put emphasis on reed’s invasive properties, outcompeting local species, etc. It is, however, unclear if the species is actually considered invasive, or is just expanding due to changes in its environment

Author Response

Dear Reviewer 2:

We are sending You the revised version of the paper entitled “THE IMPORTANCE OF BIOLOGICAL AND ECOLOGICAL PROPERTIES OF PHRAGMITES AUSTRALIS (Cav.) Trin. Ex Steud., IN PHYTOREMEDIATION OF AQUATIC ECOSYSTEMS-THE REVIEW” with identification number “WATER- 787758” for consideration to be published in Water journal.

Thank you for the evaluations you have made to add value to our work.

            All your comments have been considered and we have modified the text following your suggestions.

Comment #1. The introduction is very brief  and does not really explain why the review has been written. Since the goal is stated as “demonstrating the suitability of Phragmites for use in phytoremediation”, it makes more sense to combine the introduction and section 2, into an actual introduction. Now, the introduction is a list of properties, most of which are repeated later in the manuscript, whereas section 2 is a random collection of background information on reed that does not necessarily contribute to the story on how this species is useful for phytoremediation.

Response: Thank you for your valuable comments! We have reorganized the paper accordingly. Chapter 1 Introduction was thoroughly reorganized altogether and, according to the Reviewer’s suggestions, the content of Chapter 2 Ecology and Biology was implemented into Chapter 1 (Introduction). The whole description was directed at demonstrating all the properties of Phragmites australis which are vital in phytoremediation of aquatic ecosystems. Table 1 was supplemented with a description in the form of an additional paragraph, which stresses the conditions in which phytoremediation is more effective. Listing numerous biological features of common red which are not directly connected with the phytoremediation capacity of this species was eliminated from the text. The number of references quoted therein was reduced, and the aim of the paper was more thoroughly described.

In the new version of the manuscript (new or transformed content is marked in red):

1. Introduction

Phragmites australis is a perennial, an emergent a typical swamp and aquatic plant with a very wide geographical range, encompassing many climatic and ecological zones [1-3]. It inhabits both aquatic and terrestrial ecosystems [4]. It is a cosmopolitan species, widespread in temperate and tropical regions around the world except Antarctica [5, 6-7]. The natural range is difficult to determine due to the dilation of this species in many places in the world and its easy placement [8-9]. The plant is extensively distributed in North America, with the exception of Alaska it can be found in all US states and Canadian provinces and territories except Nunavut and Yukon [10]. Common reed is a native plant to Puerto Rico and nonnative to Hawaii [11]. In North America, the nonnative common reed haplotype is extensively distributed [12]. Its occurence extends from British Columbia to Quebec and in the south,  it is found throughout the contiguous United States [3]. The plant is common in Europe, North Asia, Central and South-West Asia (from the Mediterranean to Pakistan), East Asia and in Australia [3, 13-14]. P. australis is found in a belt around the dense forest zone in tropical Africa, from Senegal east to Eritrea, as well as in a southern direction from Ethiopia and Eritrea to Mozambique, Zimbabwe, Namibia, South Africa and Swaziland. It also occurs in Madagascar [15]. The lineages and genotypes of P. australis show diversity within as well as among populations. Also, genes from relatives from other phylogeographic regions and species can be incorporated into populations [1].  P. australis is a cosmopolitan wetland grass, which is classified as one species (Figure 1) but consists of three main phylogeographic groups [5, 16].

Figure 1. Main phylogeographic groups, source: [1].

Regardless of the geographical location, this species is an indicator of moderately warm, well-lit habitats, however it is also found in partial shade. It prefers wetlands, although it also grows on areas that are periodically devoid of stagnant water.  As P. australis stands usually show high intraspecific diversity, the species manifests tolerance to a rapidly changing climate, which can in fact be beneficial to the plant. It plays a key role in overgrowing water reservoirs. P. australis provides food and habitat for some organisms and serves to stabilize soils against erosion Many biological features of this species predispose it to be used as a biological pollution filter [6].  In comparison with other species of emergent aquatic plants, P. australis has  annual cane-like stems and is characterized by relatively high growth and mass (up to 6 m in height), show variations in diameter from 4 to 10 mm, and have long hollow internodes of 10–25 cm in length and has an extensive rhizome system. The perennial rhizomes have both horizontal as well as vertical components. Extension of the size of the clone is due to horizontal rhizomes, while the annual upright stems are due to vertical rhizomes. Rhizomes are characterised by an extensive aerenchymatous tissue. Roots develop from rhizomes and other submerged parts of shoots. Rhizomes form the largest densities at a depth of 0.5 m. Individual rhizomes live on average about 6 years and can grow within a radius of 10 meters at a speed of 1 meter per year. It grows well on various types of substrates, from sandy and gravel, through peat soils to various types of gyttia and mules. Its leaves are smooth, alternate with narrow-lanceolate laminae of 20–70 cm in length and 1–5 cm in width. The leaves are closely nerved, and taper to long slender points [12]. The inflorescence is a terminal panicle, often 30 cm long, lax, the colour varies from dull purple to yellow, and the main branches bear many spikelets. The smooth branches usually have scattered groups of long silky hairs [17] (Figure 2).

Figure 2. Phragmites australis (Photographer: Justyna Milke, Małgorzata Gałczyńska).

Photosynthesis is the primary physiological process which determines plant growth, crop productivity and influences many other plant processes. There are three different plant systems in nature viz., C₃, C₄ and CAM, characterized by CO₂ trapping mechanisms. However, C₄ and CAM plants are generally found to employ C₃ pathway to trap CO₂ as an initial step. P. australis is a plant that exhibits photosynthetic properties for the C₃ pathway [18-19]. It was also observed that P. australis exhibits characteristics of both C₃ and C₄ pathways, because its carbon anhydrase activity is typical of a C₃ plant, while its phosphoenolpyruvate (pep) carboxylase activity ratio is indicative of a C₄ plant [20-21]. Table 1 shows P. australis survival strategy in two types of environmental conditions.

Table 1. The C₃–C₄ ecotype of Phragmites australis, source: [1, 21], modified.

The analysis of the information presented in Table 1 allows to determine that the phytoremediation process is more effective at low soil moisture than in a typical water environment (floating islands with P. australis). In conditions similar to the terrestrial environment, the plant is characterized by a greater increase in biomass associated with an increased uptake of biogenic compounds, particularly nitrogen, and better developed mycorrhiza which supports the plant in the process of decontamination of pollutants. The listed biological features of this species and its photosynthetic properties for different CO₂ trapping mechanisms are related to: relatively high growth, the possibility of obtaining high biomass in the crop and a very well developed root system - being crucially important adaptive properties of plants for phytoremediation of aquatic ecosystems.

Phragmites australis (Cav.) Trin. Ex Steud. is considered native to Europe, but the  adaptive features of this plant show its competitive character. Owing to its high intraspecific diversity and phenotypic plasticity, common reed shows an extensive ecological amplitude as well as great acclimatization capacity to adverse environmental conditions. P. australis grows in soils with various salinity [22-25], fertility [26],  textures [27], of different pH [28-29] and attains high productivity under different climatic conditions [2,30-31]. Is a highly adaptable emergent macrophyte, with a broad range of tolerance to salinity and flooding regimes [32-37]. Being a native species, P. australis shares many characteristics with invasive species [38-41], namely rapid growth, high biomass, rhizome fragmentation, and tolerance to high salinities [42]. P. australis shows high tolerance capacity towards many pollutants and environmental stress factors which can be considered as important characteristics for effective biomonitoring and phytoremediation studies [43-44].

The purpose of preparing the review article was to show the most important elements of the current knowledge about the possibilities of using P. australis for phytoremediation. In the last 25 years, the mechanisms of removing heavy metals mainly by this species have been defined quite precisely. Many research teams have increasingly undertaken research related to determining the role of common reed in the removal of specific organic compounds found in industrial and domestic sewage. The main focus of this review is to demonstrate that P. australis is good absorber of heavy metal ions and has the potential to remove nutrients, which makes it an ideal species for phytoremediation.

Comment #2. Figure 3 and 4 just list the same points as mentioned in the text. I do not feel that these figures are necessary or informative.

Response: Thank you for your valuable comments!  Figure 3 and 4 were removed as suggested by the Reviewer. 

Comment #3. The section 2.5 on microbial (interactions?) is very brief and does not actually contribute to the overall story. The authors mention that the rhizosphere contains many microorganisms and name a few of these, without explaining why these species are important, what they do, how they contribute to reed’s functioning as a species in phytoremediation. The other study that is cited, describes the establishment of symbiotic bacteria in quite some detail, again without mentioning why this would be important.

Response: Thank you for your valuable comments! The preceding subchapter 2.5 was moved to chapter 2 (The potential for heavy metal absorption by common reed compared to other aquatic plants) and now constitutes the subchapter 2.2. As rightfully suggested by the Reviewer, more attention was paid to describe the mutual relationships between microorganisms in rhizosphere and the secretory activity of common reed’s roots connected with the mechanisms of phytoremediation. New references to additionally added literature on the subject were made in the process. The reference to the article by White et al. [59] was removed from the paper as it did not concern the mechanism of bacteria influencing the phytoremediation process.

In the new version of the manuscript (new or transformed content is marked in red):

2.2. Microbial Interactions With Common Reed In Heavy Metal Uptake

To a great extent, plant growth and development depend on the activity of soil microorganisms found in the rhizosphere. Microorganisms located in this way influence the shaping of plants in various ways [56], and the mutualisms between plants and their microbiome are common and facilitate plant invasion processes [57]. P. australis is a macrophyte that is very productive, and its root zone is rich in dissolved oxygen [58] and organic carbon [59] providing suitable conditions for the colonization of microorganisms. Bacteria and mycorrhizal fungi found in the rhizosphere play an important role in phytoremediation trough degrading metals, organic pollutants, radionuclides, and xenobiotic compounds [57-65]. Soil microbes participate in the mobilization of metals for plant uptake or immobilization of metals in the rhizosphere to restrict leaching. They help in these processes through acidification, chelation and reduction of metals in the soil (for example Pseudomonas fluorescens produce citric acid but Rhodococcus sp. reduce As (VI) to As (III) [66]. For example, in the rhizosphere, P. communis - the most abundant acidophilic bacterium Gp6 and the dominant heterotrophic microorganism Gp7 were important members of soil microbes. Zhao et al. [63] pointed that As and Ni promoted the growth and reproduction of Gp6 and Gp7. In turn, the dominant bacteria such as Gp6 and Longilinea were involved in metabolizing multiple carbohydrates and amino acid in the soil. Aerobic tissues in the stems of P. australis enable the roots to release oxygen and other primary and secondary metabolites into the rhizosphere [67, 59], and consequently create an oxygen enriched sediment microhabitat. In their research, Chaturvedi et al. [60] have shown that the rhizosphere P. australis contains many aerobic microorganisms such as: Microbacterium hydrocarbonoxydans, Achromobacter xylosoxidans, Alcaligens faecalis and species that belong to the genus Bacillus and Pseudomonas.  Fifteen culturable bacterial sp. were grown on effluent supplemented medium as a sole carbon source resulting in the reduction of the levels of distillery effluent pollution with heavy metals. The latest research provides information on the impact of the winter or summer season on the diversity and composition of the microbiome [68], which in addition to slowing cane vegetation may additionally determine the pace of the phytoremediation process.

Comment #4. In table 3, removal of heavy metals is reported for different species. However, this data seems to have been collected from different studies, which most likely have differences in heavy metal concentration and other environmental factors. The authors should address this. In addition, there could be a methods section indicating where the authors collected these studies from, what their criteria were for including them, what search terms they used to collect these references, etc.

Response: Thank you for your valuable comments! Table 3 (in the new version of the manuscript line 303) was supplemented with the elements suggested in the Review, such as the initial metal concentration in water and reaction of water (pH). As a result of the changes, the discussion concerning the said Table was completely reorganised. Additionally, the comparison of phytoremediation capacity of Phragmites australis with other species of aquatic plants was thoroughly changed. Some references to literature items were removed and substituted with references to other sources. Paragraph have been added to the work regarding the results of the analysis related to searching scientific articles. The results of this analysis were discussed (lines 252-270).

Comment #5. Some studies or references are discussed in a lot of detail (e.g. L. 258-366) while other facts are hardly mentioned, but the authors do not always manage to convey the relevance of the cited studies for the narrative of this review.

Response: Thank you for your valuable comments! In order to demonstrate the significance of the sources referred to in this review paper, parts of the preceding chapters 3 and 4 were reorganised and substantively enriched. Some of the literature items referred to were removed, and additional ones were introduced.

In the new version of the manuscript (new or transformed content is marked in red) in chapter 3, new content is found on the following lines: 337-358; 378-389; 396-414; 417-423; 430-432, and in chapter 4, on the lines : 496-500; 511-527.

Comment #6. The distance between the lines decreases towards the end of the manuscript, making it harder to read.

Response: Thank you for your valuable comments! The distance between the lines was normalised for the whole text.

Comment #7. Line 85: I presume you mean photographer, instead of author?

Response: Thank you for your valuable comments! The change from Author to Photographer was made in Figure 1.

Comment #8. Lines 106 and 117; These headings consist of adjectives without objects.

Response: Thank you for your valuable comments! The title of the subchapter 2.4 (line 106, the old version of the manuscript), was completely removed and, having been reedited, incorporated into the redrafted Introduction. The title of the preceding subchapter 2.5 (line 117, the old version of the manuscript) was extended (Microbial interactions with common reed heavy metal uptake). As of now, this subchapter is 2.2 (lines 146-171).

Comment #9. In Table 2, please add the type of system (natural/mesocosm; climate conditions)

Response: Thank you for your valuable comments! Table 2 (line 205 in the new version of the manuscript) was supplemented with additional elements as suggested by the Reviewer. A type of aquatic ecosystem was added, as well as the column with climatic conditions under which the experiments were conducted.

Comment #10. The authors put emphasis on reed’s invasive properties, outcompeting local species, etc. It is, however, unclear if the species is actually considered invasive, or is just expanding due to changes in its environment

Response: Thank you for your valuable comments!

 The invasive species is the one that settles in the originally foreign area and is most expansive, i.e. it produces viable offspring, often in large quantities, spreads long distances from mother plants and colonizes large areas in a short time. The invasive species threatens local native species in new conditions, changes the nature of habitats, contributes to measurable economic losses, and to some extent even threatens the health of humans and animals. Phragmites australis (Cav.) Trin. Ex Steud. is considered native to Europe. However, it easily competes with other native species due to its better ability to adapt to a polluted environment. Its invasive character has been particularly apparent in North America where it has become dominant in a range of wetland habitats replacing native species and biotypes including the native North American P. australis subsp. americanus. Bird, fish and insect populations can also be affected.

In two sentences, the changes were made.

In the new version of the manuscript (new or transformed content is marked in red):

“Phragmites australis (Cav.) Trin. Ex Steud. is considered native to Europe, but the adaptive features of this plant show its competitive character.”

“Being a native species, P. australis shares many characteristics with invasive species, namely rapid growth, high biomass, rhizome fragmentation, and tolerance to high salinities.”

Yours faithfully

Justyna Milke, Małgorzata Gałczyńska, Jacek Wróbel

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I thank the authors for acknowledging my concerns with their initial submission and for revising a substantial portion of their manuscript. I feel the revised manuscript shows clear improvement, but still lacks some clarity in terms of structure and focus. A review on the role of Phragmites in phytoremediation of heavy metals and other harmful substances can provide an interesting contribution to the journal Water. The aim of this review, from what I gather, is to show:

1. Reed is a versatile and adaptable species, and can therefore be implemented in constructed wetlands for phytoremediation in various geographical regions (Ecological background in the first section)
2. An overview of how well Reed can remove different pollutants from waste water, compared to other aquatic species (Table 3 and Section 4)
3. Present the usefulness of applying reed in different types of constructed wetlands, to remove a number of frequently occurring pollution (Fig 9)

To make these three points come across stronger, I’d recommend restructuring the review with those main goals in mind, so that they are clearly introduced, discussed/presented in the main body of the manuscript and then revisited in the conclusion. Below, I’ve written an example of how to improve structure in the introduction, but the same should be applied to the rest of the manuscript. In addition, I have listed my other concerns, separated into general/main concerns, and smaller remarks. In addition, should the paper be accepted, I would recommend the manuscript to be checked for language before publication. I have avoided listing too many language issues, as I feel the review process should be about the scientific quality, regardless of whether the writer is a native English speaker or not. Irrespective of the use of the English language, however, there is sloppiness in the writing, with words missing, species names not in italics, repetition and inconsistency in explaining used abbreviations, which should be addressed.

General remarks:

Introduction: I think it works well to present the ecological information of Phragmites in the introduction in this revised version. However, instead of diving straight into the characteristics of reed, I would recommend starting off by introducing the topic and linking it directly to phytoremediation to make the reader see why this is important. A suggestion would be:

• Increasing pollution in aquatic systems due to human activities. Polluting substances include heavy metals, ///, etc.
• In many countries, different types of constructed wetlands have been created to improve water quality.
  • There are different types, but they generally consist of ///.
  • CWs have the following advantages ///.
  • Plant species differ in their ability to extract pollutants from wastewater.
  • Reed is a species that is often used in these systems
• There are many reasons why reed is often chosen. For one, reed is a widespread species. Explain wide geographical distribution (L 28-45)
• Why is this species so widespread? Discuss the ecology of Reed (L. 48-68)
• Discuss the different adaptations of reed, including its photosynthetic adaptations, competitive character, etc (L. 71-100)
• In this review, we show ///

There is quite some repetition in the manuscript, partially also in the new inserts in this revised manuscript. Please try to stick to one topic per paragraph or section, and read the manuscript carefully to remove overlap and repetition.

Table 2; In this table, there are sometimes multiple references listed for the same heavy metal, but only one value per plant organ. Please give a range when multiple references are used for the same values. Also, if leaf tissue concentration would be derived from a different study site than root content, these values cannot be compared. For a useful comparison, a lot more studies are required for each heavy metal presented in this table. In general, when presenting values, please inform the reader whether they are presented with standard deviation or standard error.

Table 3; The concentration in the newly inserted column (please add that this is the concentration in the environment) shows that there are very large differences in initial concentration. For example, for Cu, the initial concentration for Phragmites was 0.041-0.051 mg/L, whereas for Ceratophyllum demersum, the initial concentration was a 1000 times higher! It is impressive that this species then managed to take up 79.8% (31.92 mg/L). With only 1 reference per heavy metal / plant combination, and such highly diverse concentrations it is impossible to conclude that Phragmites is the most suitable species for phytoremediation. Therefore, the removal pathways illustrated in Figure 5 and 6 (which I do like, if they would be based on more information) cannot be presented in this way. It is a very ambitious mission to present this data for this number of heavy metals. If additional data is unavailable for some plants and/or pollutants, maybe pick one metal as an example. This metal would be a common pollutant, ensuring enough publications on the uptake rate by macrophytes under different loading.

Fig 5 and 6; In addition to my comments on the data that these figures are based on, I wonder how you chose this sequence of plants? Would all of these plants grow in the same environment and system? Another minor comment on Fig 6, is that the caption mentions P. stratiotes, whereas the figure displays Lemna.

Section 3 (Constructed wetlands); I like the information added to this section to include the practical implications, but please read carefully and remove repetition. The addition of the number of studies reporting on constructed wetlands including Phragmites is useful, but could also be combined with Figure 4, as there seems to be some overlap here. Seeing how many studies you were able to find on phytoremediation in constructed wetlands with/without Phragmites, it should be possible to add more references to Table 3.

Fig 9: I like how you present these data, but up to this point, the focus on the review has been on heavy metals, and these TN/TP/TSS/BOD/COD are not really introduced. For BOD/COD, the explanation of what these abbreviations mean, also comes at the end, after already using them for a few pages.

513 - 530 This whole section is on mycorrhiza, and not on “Removal of other substances by common reed” as the heading says. Please check the text carefully to ensure all paragraphs fall under the correct headings. In addition, it probably makes more sense to discuss removal of other substances before discussing the different types of constructed wetlands (see my earlier remark on structure).

Detailed / small comments:

17-18 Rephrase “In general, P. australis has a higher ability to accumulate […] than other aquatic plants.

21 Replace their by its

21 Missing word “research” after “further”

28. This is probably too much information in one sentence. I’d probably change this to: “Phragmites australis is an emergent, perennial plant with a very wide […] ecological zones [1-3]. As a typical swamp and aquatic plant species, it inhabits both aquatic and terrestrial ecosystems.” Or just remove the swamp/aquatic bit, since it is implied by saying it grows in aquatic and terrestrial sites.

33 There is some repetition in this sentence, and the first part can be removed.

44 A sentence shouldn’t be started with an abbreviation, so here, the species name should be written in full. Please check the manuscript carefully for similar issues. In addition, the species name is not always presented in italics. Plus, sometimes chemicals are presented written in full, whereas other times they are only presented using their chemical formula. Please be consistent, and mention the full name of a substance first (for example L. 177), adding the abbreviation in brackets. Then use the abbreviation/formula throughout the rest of the text.

122 Replace “patterns” with “processes” or “pathways”

130 It is probably meant that plants survive heavy metals, instead of the other way around

132 - 134 This sentence doesn’t make a lot of sense

146 “The role of microbial ///”

148 Replace “microorganisms located in this way” with “These microorganisms”

202 “Higher than” what?

233 [Despite some…] - L. 236 […characteristics] This is a good introductory sentence for this section, to compare the heavy metal uptake in different plant species. I’d recommend moving it up.

245 A word seems to be missing between “leads to” and “are”

249 - 252 Because the new insertion, this sentence can be removed.

337 Add “constructed” before wetland.

464 Remove wither “substances” or “contaminants”

499-503 This new insert lacks references. Please add.

Author Response

x

Dear Reviewer 2 (round 2):

We are sending You the revised version of the paper entitled “THE IMPORTANCE OF BIOLOGICAL AND ECOLOGICAL PROPERTIES OF PHRAGMITES AUSTRALIS (Cav.) Trin. Ex Steud., IN PHYTOREMEDIATION OF AQUATIC ECOSYSTEMS-THE REVIEW” with identification number “WATER- 787758” for consideration to be published in Water journal.

Thank you for the evaluations you have made to add value to our work. We appreciate your time and will be pleased to answer your possible new comments or suggestions.

All your comments have been considered and we have modified the text following your suggestions.

We have corrected the grammar and syntax of the paper.

General comment. I’d recommend restructuring the review with main goals in mind, so that they are clearly introduced, discussed/presented in the main body of the manuscript and then revisited in the conclusion. Below, I’ve written an example of how to improve structure in the introduction, but the same should be applied to the rest of the manuscript. In addition, I have listed my other concerns, separated into general/main concerns, and smaller remarks. In addition, should the paper be accepted, I would recommend the manuscript to be checked for language before publication. I have avoided listing too many language issues, as I feel the review process should be about the scientific quality, regardless of whether the writer is a native English speaker or not. Irrespective of the use of the English language, however, there is sloppiness in the writing, with words missing, species names not in italics, repetition and inconsistency in explaining used abbreviations, which should be addressed.

Response: Thank you for your valuable comments! As recommended by the Reviewer 2, the introduction has been reconstructed in terms of its main goals:

• Reed is a versatile and adaptable species, and can therefore be implemented in constructed wetlands for phytoremediation in various geographical regions (Ecological background in the first section);
• An overview of how well Reed can remove different pollutants from wastewater, compared to other aquatic species (Table 3 and Section 3- currently);
• Present the usefulness of applying reed in different types of constructed wetlands, to remove a number of frequently occurring pollution (Figure 9).

All other sections, including conclusions, were also reconstructed. The manuscript has also been revised by a native speaker.

Comment #1. Introduction. I think it works well to present the ecological information of Phragmites in the introduction in this revised version. However, instead of diving straight into the characteristics of reed, I would recommend starting off by introducing the topic and linking it directly to phytoremediation to make the reader see why this is important. A suggestion would be:

Increasing pollution in aquatic systems due to human activities. Polluting substances include heavy metals, ///, etc.

In many countries, different types of constructed wetlands have been created to improve water quality.

There are different types, but they generally consist of ///.

CWs have the following advantages ///.

Plant species differ in their ability to extract pollutants from wastewater.

Reed is a species that is often used in these systems

There are many reasons why reed is often chosen. For one, reed is a widespread species. Explain wide geographical distribution (L 28-45)

Why is this species so widespread? Discuss the ecology of Reed (L. 48-68)

Discuss the different adaptations of reed, including its photosynthetic adaptations, competitive character, etc (L. 71-100).

Response: Thank you very much for noticing good changes that were introduced in the previous version (round 1) of the manuscript. New suggestions, for which we are very grateful, were even more helpful in refining this section.

Comment #2. In this review, we show ///

There is quite some repetition in the manuscript, partially also in the new inserts in this revised manuscript. Please try to stick to one topic per paragraph or section, and read the manuscript carefully to remove overlap and repetition.

Response: Thank you for your valuable comments! We analysed the content of the entire manuscript and removed the repetitions, both from the original and from its previous version (round 1). We tried to stick to one topic per paragraph or section.

Comment #3. Table 2; In this table, there are sometimes multiple references listed for the same heavy metal, but only one value per plant organ. Please give a range when multiple references are used for the same values. Also, if leaf tissue concentration would be derived from a different study site than root content, these values cannot be compared. For a useful comparison, a lot more studies are required for each heavy metal presented in this table. In general, when presenting values, please inform the reader whether they are presented with standard deviation or standard error.

Response: Thank you for your valuable comments!  As suggested by the Reviewer 2, we did an additional research of phytoremediation scientific articles. In a situation where additional information about the concentration of metal  in the organs of Phragmites australis was found, the data in Table 2 were changed and supplemented. The authors of the articles did not always analyse the level of metal concentration in all organs. In order for the reader not to compare the concentration of metal from different sources, each citation and data was included in a separate line of the discussed table. Almost 90% of all the data of the average value of the metal concentration contained information about the standard deviation. Where the average value was given with a standard error, the symbol * was introduced.

The whole table is in the current version (round 2) of the manuscript.

Table 2. Heavy metal concentrations in the organs of P. australis [mg/kg, standard deviation SD].

Legends: standard error (SE)*

Comment #4. Table 3; The concentration in the newly inserted column (please add that this is the concentration in the environment) shows that there are very large differences in initial concentration. For example, for Cu, the initial concentration for Phragmites was 0.041-0.051 mg/L, whereas for Ceratophyllum demersum, the initial concentration was a 1000 times higher! It is impressive that this species then managed to take up 79.8% (31.92 mg/L). With only 1 reference per heavy metal / plant combination, and such highly diverse concentrations it is impossible to conclude that Phragmites is the most suitable species for phytoremediation. Therefore, the removal pathways illustrated in Figure 5 and 6 (which I do like, if they would be based on more information) cannot be presented in this way. It is a very ambitious mission to present this data for this number of heavy metals. If additional data is unavailable for some plants and/or pollutants, maybe pick one metal as an example. This metal would be a common pollutant, ensuring enough publications on the uptake rate by macrophytes under different loading.

Response: Thank you for your valuable comments!  According to the comment of  Reviewer 2, we added information in table 3 that the metal concentration was determined in the natural environment. We checked the Cu removal data by Ceratophyllum demersum because indeed there was a suspicion regarding the read concentration unit from the article. The data in the table was correct. The Reviewer 2 rightly points out the difference in the initial values ​​of metal concentration in polluted waters. The purpose of creating this table was to find information on the nearly 100% removal of specific metals from wastewater by Phragmites australis, but also to indicate the efficiency of removal of these metals by other plant species, even if the metal concentrations were different than when Phragmites australis were used. Collecting  this information can help you decide whether to choose only Phragmites australis in constructed wetlands or create a system of many plots of constructed wetlands with different species of aquatic plants on each plot. A hydrophyte system created in this way could reduce the very high concentration of metal or metals in sewage. Of course, the Reviewer 2 rightly points out that the data in this table do not indicate that Phragmites australis has always been the most effective species for use in phytoremediation. Therefore, we have carried out the search of scientific articles again. We found such studies in which the efficiency of removal by Phragmites australis and other species of emergent aquatic plants was compared at the same metal concentration. These data are presented in the new Figure 6. They indicate the general regularity that higher metal concentration in the wastewater led to decreased removal efficiency. In addition, some of the data of rate removal of metals are very similar to each other even though they show  the results from different studies. Therefore, it seems to us that the schematic diagram of mercury removal from wastewater presented in Figure 5 is an indication of the potential for Phragmites australis to remove this element to 2.05 mg/L. If there was a need for even greater wastewater purification, adding two subsequent purification stages with Lemna minor and Eichornia crassipes would reduce the concentration to 0.37 mg/L.

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   a) b)

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   c) d)

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   e)                                                                 f)

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   g) h)

Figure 6. Heavy metals removal rate % for P. australis and the others emergent aquatic plants.

Comment #5. Fig 5 and 6; In addition to my comments on the data that these figures are based on, I wonder how you chose this sequence of plants? Would all of these plants grow in the same environment and system? Another minor comment on Fig 6, is that the caption mentions P. stratiotes, whereas the figure displays Lemna.

Response: Thank you for your valuable comments!  The choice of plant sequences for removing mercury from wastewater was based on Phragmites australis, because this species reduced to the greatest extent the highest concentration of mercury in wastewater documented by studies in constructed wetlands based on the literature available to us. Then, two other species of plants were added to the proposed system that could further reduce mercury levels if such a result was needed. Of course, these species would grow in adequate environmental conditions, which were additionally included in the schematic diagram in the new Figure 5. We removed Figure 6 regarding the removal of zinc from wastewater, because Phragmites australis was not present in the treatment system.

Figure 5. Plant sequence as biosorbents and the effect of reducing Hg concentration in water.

Comment #6. Section 3 (Constructed wetlands); I like the information added to this section to include the practical implications, but please read carefully and remove repetition. The addition of the number of studies reporting on constructed wetlands including Phragmites is useful, but could also be combined with Figure 4, as there seems to be some overlap here. Seeing how many studies you were able to find on phytoremediation in constructed wetlands with/without Phragmites, it should be possible to add more references to Table 3.

Response: Thank you for your valuable comments! According to the comment of Reviewer 2, we tried to remove the repetitions. We agree that the information contained in Figure 7 regarding the use of other plants in constructed wetlands partly overlaps with those related to phytoremediation (Figure 4). Therefore, Figure 8 has been changed. We left only data on the use of Phragmites australis in constructed wetlands due to two types of wastewater flow through the hydrophyte system. These information are valuable because they indicate the most common use of Phragmites australis in constructed wetlands with subsurface flow (vertical or horizontal or vertical and horizontal or general subsurface flow). Of course, Reviewer 2 is right that we still could expand Table 3 with new information from the added scientific articles, which was done and developed in the form of Figure 6.

Comment #7. Fig 9: I like how you present these data, but up to this point, the focus on the review has been on heavy metals, and these TN/TP/TSS/BOD/COD are not really introduced. For BOD/COD, the explanation of what these abbreviations mean, also comes at the end, after already using them for a few pages.

Response: Thank you for your valuable comments! This note is valuable. Of course, the shortcomings have been corrected. We have introduced a brief description of five water or wastewater quality indicators.

Comment #8. 513 - 530 This whole section is on mycorrhiza, and not on “Removal of other substances by common reed” as the heading says. Please check the text carefully to ensure all paragraphs fall under the correct headings. In addition, it probably makes more sense to discuss removal of other substances before discussing the different types of constructed wetlands (see my earlier remark on structure).

Response: Thank you for your valuable comments! Lines 513-530 were connected with a description on the role of bacteria in metal removal in the rhizosphere. Therefore, paragraph 2.2 currently fully describes The Role Of Microbial Interactions With Common Reed In Heavy Metal Uptake. As suggested by Reviewer 2, the discussion of the removal of other substances from wastewater by Phragmites australis was placed in section 3 preceding sections 4- Common reed in constructed wetlands.

Comment #9. Detailed / small comments:

1. 17-18 Rephrase “In general, P. australis has a higher ability to accumulate […] than other aquatic plants.
2. 21 Replace their by its
3. 21 Missing word “research” after “further”
4. This is probably too much information in one sentence. I’d probably change this to: “Phragmites australis is an emergent, perennial plant with a very wide […] ecological zones [1-3]. As a typical swamp and aquatic plant species, it inhabits both aquatic and terrestrial ecosystems.” Or just remove the swamp/aquatic bit, since it is implied by saying it grows in aquatic and terrestrial sites.
5. 33 There is some repetition in this sentence, and the first part can be removed.
6. 44 A sentence shouldn’t be started with an abbreviation, so here, the species name should be written in full. Please check the manuscript carefully for similar issues. In addition, the species name is not always presented in italics. Plus, sometimes chemicals are presented written in full, whereas other times they are only presented using their chemical formula. Please be consistent, and mention the full name of a substance first (for example L. 177), adding the abbreviation in brackets. Then use the abbreviation/formula throughout the rest of the text.
7. 122 Replace “patterns” with “processes” or “pathways”
8. 130 It is probably meant that plants survive heavy metals, instead of the other way around
9. 132 - 134 This sentence doesn’t make a lot of sense
10. 146 “The role of microbial ///”
11. 148 Replace “microorganisms located in this way” with “These microorganisms”
12. 202 “Higher than” what?
13. 233 [Despite some…] - L. 236 […characteristics] This is a good introductory sentence for this section, to compare the heavy metal uptake in different plant species. I’d recommend moving it up.
14. 245 A word seems to be missing between “leads to” and “are”
15. 249 - 252 Because the new insertion, this sentence can be removed.
16. 337 Add “constructed” before wetland.
17. 464 Remove wither “substances” or “contaminants”
18. 499-503 This new insert lacks references. Please add.

Response: Thank you for your valuable comments! All corrections were made in accordance with the comments and suggestions of Reviewer 2 in the last version of this manuscript.

Yours faithfully

Justyna Milke, Małgorzata Gałczyńska, Jacek Wróbel

Author Response File: Author Response.pdf