Integrated Monitoring with Moss-Bag and Mussel Transplants in Reservoirs
, Deyana Georgieva 2, Vesela Genina 3, Borislava Todorova 1 and Ivelin Mollov 1Round 1
Reviewer 1 Report
The authors apply the active bio monitoring technique to study a total of 24 compounds -including heavy elements, PBDEs and SCCPs- in three standing water bodies. The study is rather descriptive but can add some important information to the existing scientific knowledge about biomonitor application in reservoirs. I raised here some main and minor concerns that hope can be addressed by the authors. Main concerns:- Introduction should be shorter, too many non essential details are given.
- Line 124 essential details about the study design are not specified. The authors says that they used 10 replicates of biomonitors, per lagoon? each of the bio monitor? where these replicates where located? how far from each other? any stream or source of contamination close? also, they are included in cages, what are the cages made of? were they cagesin contact with the bottom? which is the bottom floor compostion? I strongly suggest to include a more detailed description of this section and include a picture of the biomonitors in the cage to better understand the system. Also how many grams where located inside each bag?
- Related with the previous point: how the microenvironment will affect the bioaccumulation if the water is still? It is important to discuss this more deeply since this type of studies are commonly performed in streams or rivers.
- Sinanodonta woodiana is a exotic species originated in china, and considered invasive in a big part of Europe. The authors claim that that species of mussels are not present naturally in the study site, so I wonder which could be the ecological consequences of introducing a non native species in a new environment. I think it is required that authors discuss this in the manuscript since many readers might be having the same question.
- It is necessary to further discuss how different physiological and morphological characteristics of the different biomonitors might influence the bioaccumulation of one or other element/compound.
- Cited studies in the discussion use different moss/mussel bag preparation and exposition methods and study area seems to be very different. Comparisons needs to be made with more caution.
- Figure 1 needs to be changed it is not clear in the map where the cages were placed and where the water was collected from. Also maps could include some information from industrial or contamination source if possible. I would suggest to zoom at each of the study areas and show the map of Bulgaria in small. Maps needs to include a min of information (coordinates, north direction, etc).
- Table 1 include all the data (not only min and max) indicated in mean +- SD for each of the study areas.
Author Response
To the Reviewer 1
Thank you very much for the constructive review and for the opinion that the study can add some important information to the existing scientific knowledge. Your concerns, both major and minor, were addressed and below you can find our answers to your comments. (Reviewer’s comments are shown in Italic).
Introduction should be shorter, too many non essential details are given.
The non-essential data in the Introduction were deleted: Line 36-38; Line 41-44 and Line 46-48, as well as three not important for the study literature sources were excluded.
Line 124 essential details about the study design are not specified. The authors says that they used 10 replicates of biomonitors, per lagoon? each of the bio monitor? where these replicates where located? how far from each other? any stream or source of contamination close? also, they are included in cages, what are the cages made of? were they cagesin contact with the bottom? which is the bottom floor compostion? I strongly suggest to include a more detailed description of this section and include a picture of the biomonitors in the cage to better understand the system. Also how many grams where located inside each bag?
Thanks to your suggestion, more detailed information about the study design was provided (Please see Materials and Methods, Line 128-130, a picture is also provided – within Figure 1). Each cage contains about 100 g wet weight of the moss and 10 individuals of the mussel. As stated in the manuscript, the cages were placed at a depth of 2 m in each of the reservoirs (Line 129) in order to not have contact with the sediments which could influence the results from bioaccumulation (e.g. due to the possibility to incorporate particulate matter as pointed by Gonc¸alves et al., 1992).
Related with the previous point: how the microenvironment will affect the bioaccumulation if the water is still? It is important to discuss this more deeply since this type of studies are commonly performed in streams or rivers.
We understand the reviewer’s suggestion and the arisen question is very important. The study did not aim at comparison between bioaccumulation in standing and running waters, but rather to test if transplants could assess water quality in standing water bodies. Based on the results achieved transplanted moss and mussels confirmed their ability to detect patterns of pollution. Many laboratory experiments with aquatic bryophytes were conducted in standing water solutions (e.g. Jackson et al., 1991; Carballeira et al., 1999; Cenci, 2000; Gecheva & Yurukova, 2008) in order to enlighten field observations in running waters. The driving factor of bryomonitoring is the cation exchange (Glime, Bryophyte Ecology, eBook) and the nutrient uptake into the moss is initially dependent on available exchange sites, but then it depends on affinity of a particular nutrient for appropriate transport sites of cell membranes, presence of competing elements, and turnover rate of the uptake site.
Based on the above, in this pilot study we assume that uptake of the contaminants is rapid when concentrations are high. The influence of the water velocity has to be studied further.
Due to the importance of this issue we incorporated a sentence in the Conclusion – Line 448-449.
Sinanodonta woodiana is a exotic species originated in china, and considered invasive in a big part of Europe. The authors claim that that species of mussels are not present naturally in the study site, so I wonder which could be the ecological consequences of introducing a non native species in a new environment. I think it is required that authors discuss this in the manuscript since many readers might be having the same question.
We agree with the reviewer. Sinanodonta woodiana has still (and fortunately) not been recorded at the studied reservoirs. It was used as a biomonitor for 30 days in three heavily modified water bodies (deep large reservoirs). One of the reasons to select these human-made lakes was to minimize potential threats caused by accidentally introduced species. There is also no documented serious economic impact caused by S. woodiana (e.g. in comparison with Dreissena polymorpha). – Line 140-142.
It is necessary to further discuss how different physiological and morphological characteristics of the different biomonitors might influence the bioaccumulation of one or other element/compound.
Thanks to your suggestion and those of Reviewer 2, a paragraph was added in Section 3.4., Line 392-403. Both bryophytes and mussels are in a close relationship with their immediate environment. The selected bryomonitor F. antipyretica has large biomass and high cation exchange capacity. Mussels as filter feeders were known to reflect levels of organic and inorganic pollutants occurring in the water, food or sediment and thus also raise public health concerns.
Cited studies in the discussion use different moss/mussel bag preparation and exposition methods and study area seems to be very different. Comparisons needs to be made with more caution.
We agree with the Reviewer and thank the reviewer for his/her careful thoughts. We only make comparison with previous studies with different study design because of the pilot character of our study (except for the transplanted F. antipyretica in copper contaminated Orta Lake in Italy).
Figure 1 needs to be changed it is not clear in the map where the cages were placed and where the water was collected from. Also maps could include some information from industrial or contamination source if possible. I would suggest to zoom at each of the study areas and show the map of Bulgaria in small. Maps needs to include a min of information (coordinates, north direction, etc).
Figure 1 was changed: a picture of a cage with biomonitors was incorporated (according to a previous comment to include a picture) and the locations for both cages and water samples were indicated. After discussing, we presented Bulgaria in a large format in order to ensure the readers understanding of the studied water bodies location on the Bulgarian territory. The coordinates and sources of contamination were presented in the manuscript before revision and now (Line 136-137). Scales and north direction were also added thanks to Reviewer 2 recommendation.
Table 1 include all the data (not only min and max) indicated in mean +- SD for each of the study areas.
We thank the reviewer for this suggestion. Table 1 was reorganized as recommended.
Minor concerns
Line 17: Change "contamination" by "water quality"
It was changed.
Line 22: Include the name of all the trace metals determined
They were not included in the previous version in order not to exceed the limit of 200 words maximum. The studied trace elements are now listed as you recommended.
Line 24: indicate if this happen in the 3 study areas "The trace element accumulation was significant after the exposition period"
Yes – done.
Line 29: indicate if the statement is true for the 2 types of biomonitors.
It is valid only for the moss and that is why the sentence states that “The SCCPs’ levels were alarmingly high in all plant samples.”.
Line 113: Any previous treatment to the plastic mesh? they can contain some contamination that needs to be removed before the experiment starts.
The mesh was rinsed in distilled water and air-dried – a sentence was added at Line 114.
Line 120: does biometric analysis of mussels are also performed after the exposition period?
Yes – Line 121.
Line 122: Indicate all the trace elements and organic compounds names determined in the analysis.
Implemented – Line 125-127.
Line 127: does the water samples were collected at the same depth as biomonitors were placed? please indicate this in the methods
Yes – added, Line 130.
Line 132: wrong coordinate, please fix.
Thank you – correct coordinates were provided, Line 136.
Line 133: Any previous study or public data that supports the link of different industrial activities in each of the lakes?
Two of the studied reservoirs: Kardzhali and Studen kladenets were selected due to the long-term impact of the ore extraction and mining activities. Another important reasons to conduct the study in the above standing water bodies are (i) there are scarce complex literature data for the water quality and (ii) they are used for fishfarming. Thanks to your comment the article of Velcheva (2006) focused on Zn content in fish from both reservoirs was cited in the manuscript – Line 138, reference 47.
Line 140: Section 2.2. is too long, I would suggest that if the analytical procedure follows a previous paper or standard method I recommend to cite it in the text to reduce this part.
We agree with the Reviewer 1. The section contains description of methods for analyses of different matrices (water, moss, mussel) for both inorganic and organic content. Due to the lack of previous complex paper to be cited and due to the importance of the analytical procedures for further similar studies we decided not to reduce the section. Our decision was based mainly on our intention to be of methodologically support for other researchers.
Line 141: how samples where transfered to the lab to ensure the lack of cross contamination? any treatment after collection? any washing? which part of the plant/mussel was selected for chemical analysis.
All samples were transported at the laboratory on the day of the sampling (two sampling teams worked simultaneously). Samples of biomonitors for inorganic analyses were taken in plastic bags, those for organic analyses - in aluminum foil (water samples in glass containers). The plant material was rinsed on-site with the reservoir water before being transferred to the laboratory, as well as material in poor condition and foreign objects were removed. Two to three-centimeter tips of F. antipyretica were analyzed as recommended in previous cited studies (e.g. Gecheva & Yurukova, 2014). The whole tissue samples from mussels were analyzed. Please, see Line 151-154.
Line 148: remove this: "including some toxic, enzymatic or 148 essential"
Done.
Line 275: Change sentence "is related" by "could be related"
Done.
Line 283 (and others) Scientific names are sometimes given in italic and sometimes does not. Please check.
All scientific names are now given in Italic.
Line 378: Indicate which elements in the text.
Done.
Line 381 why Figure 4 does not include all the contaminants? add another figure to show the organic ones?
We did not process the data for organic contaminant with Canoco because the dataset contained a lot of values (1/3 of the data) below the limit of detection (Please see Table 1).
Line 384-385: please expand this sentence "This probably reflects different bioaccumulation properties or metabolic transformation processes of the PBDE-congeners in aquatic environment". is there any supporting data or study to that statement?
Additional reference was incorporated – Line 414-415.
Line 401: change "followed" by "applied".
Done – Line 410.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Water-818271
The topic falls into the scope of the WATER Special Issue "Biomonitors in Water Quality Control". It is very original, firstly because the simultaneous use of moss-bags and mollusks is new in water quality biomonitoring, and secondly because the types of pollutants studied that includes organic priority substances like polybrominated diphenyl ethers and short-chain chlorinated paraffins (not usually determined in bryomonitoring studies). Moreover the MS is well written and properly organized and it is interesting for an international audience; figures and tables are clear and of sufficient quality. Finally, analytical quality control is well documented, Certified Reference Materials were used and limits of detection are provided (but only for PBDEs and SCCPs not for metals and metalloids). Nevertheless, some flaws have been identified and must be corrected before the final acceptance of the article.
Specific comments
- First of all, authors must justify the short number of sampling sites sampled; I realize that it could be difficult to find suitable sites to do the experiments but this must be properly explained.
- Figure 1. The maps lacks of both scale and north position, please add them.
- Authors include in their results the determination of some nutrients (e.g. Mg, Na or P) not only pollutants; this must be justified in the MS, why are these interesting?
- Regarding the data treatment, although I agree with authors with the use of contamination factors and Metal Pollution Index, I suggest to calculate also the Enrichment Factors (i.e. ratio between pollutant levels after exposure and pollutant levels at the beginning of the experiment). The EFs allow knowing if indeed there was enrichment in pollutant levels in mosses and mussels during the experiment (independently whether the levels were higher than those from unpolluted sites, the information already provided by the contamination factors).
- Details about Limits of detection for metals and metalloids must be included because are missing in the submitted version of MS.
- Table 1. Periods and commas are used for decimal values, please unify all the data using only periods.
- Avoid using abbreviations in tables captions (e.g. Table 2, lines 277-279; do not write MPI and CFs but Metal Pollution Index and Contamination Factors).
- Table 3. Revise the number of decimal positions included in the data, in some cases are present (e.g. Cr, Mg, Mn in BAF values from Studen Kladenets) but in the rest of the table are absent.
- Scientific names must be always written in italics throughout the MS, please review the document carefully because in some cases this is not done (e.g. , line 283; Figure 2 caption, line 301; Figure 3 caption, line 346).
- Any reference in discussion about non-significant results (e.g. lines 390-391) must be removed.
Author Response
Reviewer 2
We sincerely thank the Reviewer 2 for the evaluation of the manuscript and valuable comments. We appreciate the inputs given, which definitely helped to improve our manuscript. Below you can find our answers to your specific comments. (Reviewer’s comments are shown in Italic).
First of all, authors must justify the short number of sampling sites sampled; I realize that it could be difficult to find suitable sites to do the experiments but this must be properly explained.
We thank the reviewer for pointing out this issue and we agree that a limited number of cages were positioned – this is due to the difficulty of finding a proper and safe site to place the transplants. All three studied reservoirs belong to the type Large deep reservoirs with concrete walls surrounding the prevailing part of their area. The only possible stations for positioning of the cages were shallow areas which are used for fish farming. The above creates a serious problem because most of the owners do not agree to take samples (fearing of the results). We hope that the added explanation (Line 142-145) could be considered as a proper.
Figure 1. The maps lacks of both scale and north position, please add them.
Scales and north direction were added. Following recommendations of the Reviewer 1, a picture of a cage with biomonitors was incorporated, as well as the locations for both cages and water samples were indicated.
Authors include in their results the determination of some nutrients (e.g. Mg, Na or P) not only pollutants; this must be justified in the MS, why are these interesting?
The reviewer brings up an important point. The mentioned essential nutrients were analyzed due to their relationship with the trace element uptake, as well as potential nutrient deficiencies to be examined. We added a paragraph focused on amounts of macroelements – please, see Line 391-403.
Regarding the data treatment, although I agree with authors with the use of contamination factors and Metal Pollution Index, I suggest to calculate also the Enrichment Factors (i.e. ratio between pollutant levels after exposure and pollutant levels at the beginning of the experiment). The EFs allow knowing if indeed there was enrichment in pollutant levels in mosses and mussels during the experiment (independently whether the levels were higher than those from unpolluted sites, the information already provided by the contamination factors).
We thank the reviewer for this valuable suggestion. We have calculated the enrichment in moss tissues based on contamination factor (CF) - calculated as the ratio between the metal content in the moss from a given reservoir (after exposure) and from the unpolluted stream (at the beginning of the experiment) – Please see Table 2. For the assessment of the results of CF we applied scale based on the geometric progression adopted by Mouvet – Line 224-230.
We use the term CF because in the literature the enrichment factor (EF) is applied to indicate if the accumulated by a terrestrial moss element originate from the soil (Kłos et al., 2011). The factor of enrichment is sometimes presented as a proportion between the content of trace element in the plant and the concentration in the water (Jenkins, 1972; Hellawell, 1986; García-Álvaro et al., 2000). It is also known as bioconcentration factor (BCF) or bioaccumulation factor. The last was calculated for the mussel (Table 3).
Details about Limits of detection for metals and metalloids must be included because are missing in the submitted version of MS.
The limits of detection for elements under the limits of detection were presented: Al, As, Cd, Co, Hg and Fe (Line 189-191). Because Reviewer 1 recommended reducing the length of Section 2.2 we added information only for these elements, but if needed the LOD for all analyzed inorganic content will be pointed out.
Table 1. Periods and commas are used for decimal values, please unify all the data using only periods.
Table 1 was totally reorganized as recommended Reviewer 1. Commas were replaced by periods.
Avoid using abbreviations in tables captions (e.g. Table 2, lines 277-279; do not write MPI and CFs but Metal Pollution Index and Contamination Factors).
Done.
Table 3. Revise the number of decimal positions included in the data, in some cases are present (e.g. Cr, Mg, Mn in BAF values from Studen Kladenets) but in the rest of the table are absent.
Decimal points were removed.
Scientific names must be always written in italics throughout the MS, please review the document carefully because in some cases this is not done (e.g. , line 283; Figure 2 caption, line 301; Figure 3 caption, line 346).
Thank you – it was corrected.
Any reference in discussion about non-significant results (e.g. lines 390-391) must be removed.
They were removed – please see Line 423-428.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Dear authors,
Thank you very much for your detailed response and all the additional information provided. From my point of view the MS is now ready to be accepted for publishing.
Best regards
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
