Evidence for Links between Feeding Behavior of Daphnia magna and Water Framework Directive Elements: Case Study of Crestuma-Lever Reservoir

Round 1

Reviewer 1 Report

This paper presents a study on the Water Framework Directive (WFD) in the European legislation on water policy, which is used to assess water quality according to time-consuming metrics and specific taxonomic needs. This study aims to evaluate the sensitivity of Daphnia magna feeding rate as says to assess/discriminate the water quality of heavily modified and artificial water bodies. Monthly, for one year, the quality of the Crestuma-Lever reservoir (in two sampling sites: Crestuma and Marina) was assessed using physical, chemical, and biological (concentration of chlorophyll-a) elements proposed by the WFD. Daphnia magna was exposed to the collected water samples and feeding rates were evaluated, including an ecosystem function evaluation in water quality assessment. The WFD metrics showed that overall, the Crestuma-Lever reservoir has a rating of Good to Moderate Ecological Potential regardless of site. Feeding rates varied with the sampling site and months, demonstrating that feeding behavior evaluation is a sensitive tool that allowing to discriminate potential effects indicative of a lower water quality. This finding was recorded by the decrease in the feeding rate (Crestuma: May, Sept; Marina: Nov, Jan, May), despite the WFD classification, and once the organisms are affected by the components present in the water samples. The methodology provides a reference for the similar water quality monitoring. The study is interesting, but there are some points to be improved as below:

1) Literature review should be addressed. Some important references were missing, such as Chen Q. et al., The role of remote sensing technology in the EU water framework directive (WFD), Environmental Science and Policy, 2004, 7(4), 267-276.

2) In Figure 1, the legend should be clear.

3) In tables 1 and 2, the decimal number should be same, not in different number of one, two or three, such as 2.0, 0.50, 0.060?

4) Discussion should be separated from Results.

Author Response

1) Literature review should be addressed. Some important references were missing, such as Chen Q. et al., The role of remote sensing technology in the EU water framework directive (WFD), Environmental Science and Policy, 2004, 7(4), 267-276.

R: Thank you for the comment. The suggestions were taken into consideration and information focused on remote sensing analysis to assess water quality was added in the introduction section.

2) In Figure 1, the legend should be clear.

R: Thank you for the suggestion, the legend was rephrased and the map clarified.

3) In tables 1 and 2, the decimal number should be same, not in different number of one, two or three, such as 2.0, 0.50, 0.060?

R: Thank you for the suggestion due to the high differences between values we try to maintain consistency and always represent 3 significant figures.

4) Discussion should be separated from Results.

R: Thank you for the comment, however, we decide on this approach to facilitate the interpretation of the results. Indeed, this format is allowed and suggested according to the journal's submission guidelines.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

Line 44 to assess the ecological potential

R: The suggestion was accepted.

Line 74 to include in a monitoring water quality program

R: The suggestion was accepted.

Line 122  as Good or …. ( Incomplete sentence)

R: Thanks for your comment. The sentence is not incomplete. It is only separated due to the page change. “According to WFD, the physical and chemical parameters are classified as Good or More and Moderate ecological potential based on the corresponding environmental quality standards (EQS, Table 1 and 2) [20]”. The words Moderate and Good or More have been written in capital letters to emphasize the classification.

Line 132 considered at that time

R: The suggestion was accepted.

Author Response File: Author Response.pdf

Reviewer 3 Report

This is a highly proffesional work for assesing the quality of water of Portoguese dam in accord with WFD of EU. A huge set of data are presented. In short: Daphnia Magna has been introduced in two water samples, taken from different places of Crestuma-Lever reservoir. A special feed assay has been applied for 2 months, thus observing the ecological response of Daphnia. The results were found very good, thus stating that the dam is ecologically good. The paper is of high quality and I suggest publication.

Author Response

This is a highly proffesional work for assesing the quality of water of Portoguese dam in accord with WFD of EU. A huge set of data are presented. In short: Daphnia Magna has been introduced in two water samples, taken from different places of Crestuma-Lever reservoir. A special feed assay has been applied for 2 months, thus observing the ecological response of Daphnia. The results were found very good, thus stating that the dam is ecologically good. The paper is of high quality and I suggest publication.

R: Thank you for your comment.

Author Response File: Author Response.pdf

Reviewer 4 Report

The authors carried out a series of analyses and assessed the possibility of daphnia as a marker to test/assess the quality of water. This study is carried out on physiological parameters. Though experimental demonstration supports the idea/hypothesis, however, this reviewer believes the author needs to include information and/or any relevant data, if any for other behavioural and biochemical parameters to strengthen the claim of the potential of daphnia as a biomarker in determining the quality of water. A few comments are listed below for consideration/clarification, and recommended for a major  revision  

·         Even a low dose/s of chemical substances may interfere with metabolism, Is there any possibility that the low feeding rate/FR of daphnia in the sampling site could be attributed to any pharmaceutical or chemical discharge?

·         Likewise as the phytoplankton profile is not done, do authors believe that Daphnia may also prefer other food rather than plankton?

·         Since, the diversity of plankton varies with season owing to different atmospheric variables, thus evaluating the impact of any undesirable chemical exposure, and/or seasonal fluctuation on the growth of Daphnia could provide further insight into the food preference of daphnia in the corresponding season.

·         As daphnia is sensitive to industrial waste. Is there any industrial site near to sampling site?

·         Add a graph of chlorovarapyll a (which represents phytoplankton biomass) with FR assay.

·         There is a correlation of dissolved oxygen (DO) with chlorophyll (representing phytoplankton biomass, increase in phytoplankton biomass (chlorophyll a) raises DO level but in the Crestuma sample site in December DO level is average despite of high Chl concentration. Please clarify.

·         What is the timing of sample /water collection, Is it day or night, as DO level increases during the day because of photosynthesis? DO levels decreases during the night owing to respiration.

·         Significance difference in Nitrate level between Crestuma and Marina clarify.

·         In general,  DO level decreases with an increase in Temp in the Marina site also (May-August) increase in Temp leads to a decrease in DO level, and chl also decreases with an increase in temp. but the feeding rate of D. magna increases from May-August. Does phytoplankton diversity/density affect the feeding of D. magna?

Author Response

The authors carried out a series of analyses and assessed the possibility of daphnia as a marker to test/assess the quality of water. This study is carried out on physiological parameters. Though experimental demonstration supports the idea/hypothesis, however, this reviewer believes the author needs to include information and/or any relevant data, if any for other behavioural and biochemical parameters to strengthen the claim of the potential of daphnia as a biomarker in determining the quality of water. A few comments are listed below for consideration/clarification, and recommended for a major  revision 

• Even a low dose/s of chemical substances may interfere with metabolism, Is there any possibility that the low feeding rate/FR of daphnia in the sampling site could be attributed to any pharmaceutical or chemical discharge?

R: Thank you for the comment. Several studies already demonstrate that chemical or pharmaceutical discharge will affect the feeding rate of the Daphnia magna. However, the study area is used as a water catchment area for multiple uses, so this type of contamination is reduced. Nevertheless, this aspect was considered and taken into account in the discussion of our results (Lines 253-254; 261-264; 266-269).

• Likewise as the phytoplankton profile is not done, do authors believe that Daphnia may also prefer other food rather than plankton?

R: Daphnia is a non-selective filter feeder, and any seston of a size capable of being consumed by Daphnia could be a potential food source. However, the nutritional value is different, and Daphnia has the ability to select the phytoplankton more nutritive and palatable (Lines 286-288, 305-307, 311-314). Additional information was added in the manuscript to clarify this issue (Lines 314-319).

• Since, the diversity of plankton varies with season owing to different atmospheric variables, thus evaluating the impact of any undesirable chemical exposure, and/or seasonal fluctuation on the growth of Daphnia could provide further insight into the food preference of daphnia in the corresponding season.

R: We agree with the reviewer. Indeed, there are already studies focused on the assessment of different stress factors (e.g. xenobiotics, physical and chemical water parameters, …) in feeding Daphnia responses. However, in this study, no water xenobiotics evaluation was done, but the potential effects of FR response were focused in the discussion section.

• As daphnia is sensitive to industrial waste. Is there any industrial site near to sampling site?

R: There is no industrial site nearby, however, one of the sites - Marina - is located within the marine Angra do Douro (a place with a high circulation of recreational boats).

• Add a graph of chlorovarapyll a (which represents phytoplankton biomass) with FR assay.

R: We agree with the reviewer that visual information ([chlorophyll a] and FR results) allow us to better assess the potential effects in FR. Since figure 2 with all information was very confusing, we decided to add a new table to the figure with the information on chlorophyll a concentration.

• There is a correlation of dissolved oxygen (DO) with chlorophyll (representing phytoplankton biomass, increase in phytoplankton biomass (chlorophyll a) raises DO level but in the Crestuma sample site in December DO level is average despite of high Chl concentration. Please clarify.

R: Yes, it is normal to expect high oxygen levels with high phytoplankton biomass. However, dissolved oxygen is also dependent on temperature (e.g., high temperature and low oxygen values recorded on august 13 in Crestuma and also in Marina) and water residence time. Crestuma is the last of a series of reservoirs and water stagnation may occur depending on decisions made by hydroelectric managers. Adding this information to the manuscript it is a speculative exercise that we doubt would benefit the reader.

• What is the timing of sample /water collection, Is it day or night, as DO level increases during the day because of photosynthesis? DO levels decreases during the night owing to respiration.

R: All the sampling campaigns were carried out during the morning period. This information was now included in the manuscript.

• Significance difference in Nitrate level between Crestuma and Marina clarify.

R: The nitrate values never exceeded the reference values established by the WFD (≤ 25 mg/L) in both sampling sites during the sampling period. In Crestuma nitrates showed low levels and were only detected in quantifiable concentrations between Feb and Aug, while in Marina in almost sampling periods nitrate concentrations were measured. These differences were explained in the manuscript and are related to the characteristics of each site (e.g., boat circulation, depth, lixiviation’s water residence time, increase water temperature) and the impacts from lixiviation from surrounding areas (with agricultural crops) (Line 218-225).

• In general, DO level decreases with an increase in Temp in the Marina site also (May-August) increase in Temp leads to a decrease in DO level, and chl also decreases with an increase in temp. but the feeding rate of D. magna increases from May-August. Does phytoplankton diversity/density affect the feeding of D. magna?

R: We understand the doubt of the reviewer, and several studies already demonstrate that phytoplankton diversity and/or density can affect Daphnia magna feeding. Although Daphnia is a non-selective filter feeder if phytoplankton is not palatable and has no nutritional quality Daphnia reduces the food intake, resorting to survival strategies (e.g., reducing the number of neonates). This fact is already mentioned in the manuscript (Lines 286-288, 305-307, 311-319).

Author Response File: Author Response.pdf

Reviewer 5 Report

The manuscript is well presented and written, but it needs more scientific innovation from an ecological point of view. There is only one sentence on the possible repercussions. I recommend expanding this concept.

In this case, the discussions written with the results don’t help in understanding the implications of this work. I recommend separating them and, at most, merging them to the conclusions if you want to reduce the number of paragraphs.

About the bibliography, on only 38 works, the self-citations are too many. Reduce them.

All the bibliography is recent. The reading is smooth.

Author Response

1) The manuscript is well presented and written, but it needs more scientific innovation from an ecological point of view. There is only one sentence on the possible repercussions. I recommend expanding this concept.

R: We are very grateful for the reviewer's comment, as indeed the article now presents more scientific innovation from an ecological point of view, with added information and more relevant bibliography. All information included is duly marked in the manuscript, especially in the final part of section 3.2. Feeding rate assays.

2) In this case, the discussions written with the results don’t help in understanding the implications of this work. I recommend separating them and, at most, merging them to the conclusions if you want to reduce the number of paragraphs.

R: We understand the point of view of the reviewer, but we decide on this approach to facilitate the interpretation of the results. Indeed, this format is allowed and suggested according to the journal's submission guidelines. However, in terms to explore the results obtained an additional paragraph was included to focus the biologic and ecological implications of feeding rate changes in the environmental ecosystems.

 3) About the bibliography, on only 38 works, the self-citations are too many. Reduce them.

R: We believe that previous studies of our team are important to show the different sensitivities of this ecological tool. However, we understand that explore the scientific innovation and possible repercussions is a significant improve of the manuscript. So a new set of references were included and discussed in Discussion section.

 4) All the bibliography is recent. The reading is smooth.

R: Thank you for your comment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The revised version has been greatly improved and is agreed to be accepted for publication.

Reviewer 4 Report

The authors have satisfactorily responded & clarified the raised queries. Revised article is now acceptable for publication.

Reviewer 5 Report

good changes,

the manuscript can be accepted.