Warmer temperatures limit the effects of antidepressant pollution on life-history traits

Pharmaceutical pollutants pose a threat to aquatic ecosystems worldwide. Yet, few studies have considered the interaction between pharmaceuticals and other chronic stressors contemporaneously, even though the environmental challenges confronting animals in the wild seldom, if ever, occur in isolation. Thermal stress is one such environmental challenge that may modify the threat of pharmaceutical pollutants. Accordingly, we investigated how fluoxetine (Prozac), a common psychotherapeutic and widespread pollutant, interacts with temperature to affect life-history traits in the water flea, Daphnia magna. We chronically exposed two genotypes of Daphnia to two ecological relevant concentrations of fluoxetine (30 ng l−1 and 300 ng l−1) and a concentration representing levels used in acute toxicity tests (3000 ng l−1) and quantified the change in phenotypic trajectories at two temperatures (20°C and 25°C). Across multiple life-history traits, we found that fluoxetine exposure impacted the fecundity, body size and intrinsic growth rate of Daphnia in a non-monotonic manner at 20°C, and often in genotypic-specific ways. At 25°C, however, the life-history phenotypes of individuals converged under the widely varying levels of fluoxetine, irrespective of genotype. Our study underscores the importance of considering the complexity of interactions that can occur in the wild when assessing the effects of chemical pollutants on life-history traits.


Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author This report explores the effects of the antidepressant fluoxetine on the life history responses of two clones of Daphnia magna at two temperatures, with a fully crossed design. The main findings of the study were that the effects of fluoxetine were non-monotonic, clone and temperature dependent. Their results highlight the difficulties of extrapolating from laboratory studies to complex environments. The implementation and analysis of the study were good, including accurately validating the extremely low fluoxetine concentrations and their results do demonstrate the complexity in understanding impacts of pollutants at field-level scales. The following comment may assist in improving the paper: L54-L68. In this paragraph the authors discuss the issue of multiple stressors. However, I am not conv temperature as a variable that can affect the effects of toxicants rather than as a stressor.
L110. What were the rearing conditions of the lines? How many parents were used to produce the test generation? What brood number were they from? L116. It is more conventional to present algal concentrations as cells/mL of C/mL. Was this amount of food ad libitum? Particularly at the higher temperature was there any evidence of food limitation? What was the pH of the water? Did this vary with temperature? L119. 70% humidity. I thought it would be 100% humidity in the water! L122. I can't find mention anywhere of how many individuals/treatment? L140. Were survival and fecundity recorded daily? If it was only twice a week, significant errors can occur in the estimation of life history parameters in such a rapidly breeding taxa. L158. Please state survival in all treatments. And please include data on timing and brood size in each instar for each treatment, at least in an appendix. Were all offspring born healthy? Did offspring differ in size? worth commenting on. Fig. 2. There is too much information in graphs C and D. Either find a way to simplify or delete. I don't think they add a lot anyway.

Recommendation
Major revision is needed (please make suggestions in comments) Scientific importance: Is the manuscript an original and important contribution to its field? Excellent General interest: Is the paper of sufficient general interest? Excellent Quality of the paper: Is the overall quality of the paper suitable? Excellent Is the length of the paper justified? Yes Should the paper be seen by a specialist statistical reviewer? No

Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Comments to the Author
This manuscript is a novel contribution to unraveling the complex responses of organisms to multiple potential stressors (e.g., antidepressants and temperature). The experiments and analyses are sound and the results are well supported and of interest. The manuscript was well written and the results were presented very clearly. In general, the manuscript provides a clear description of the need for this research and a good description of the research conducted. These types of experiments are imperative for understanding how multiple stressors affect organisms in nature, where multiple stressors exist.
My only comments for revision are to request that the authors provide a bit more detail about the study organism and its thermal optimum and how the temperature treatment compares to their thermal optimum. The authors indicate that the higher temperatures represent a stressor, but do not provide adequate information in the text to ascertain how sensitive D. magna is to a 20 vs. 25C thermal regime.
Along the same lines, in the discussion the authors make some statements which I think need to be refined given thermal regimes in lakes, stratification and the potential behavior of Daphnia.
The statement "Indeed, warmer temperatures appear to negate the effects of fluoxetine on an organism's life-history, suggesting that the effects of this widespread pharmaceutical will not necessarily be made worse under common scenarios of global change." And other similar statements in the discussion could be refined because in any given lake or pond where Daphnia exist, they will be exposed to a variety of temperatures. Daphnia are also known to migrate vertically in the water column as well (thereby experiencing different temperatures).
So, although the point is certainly useful that there is a strong potential for temperature to interact with contaminants like the one studied here, how this plays out in a given ecosystem will be very complex. I would recommend that the authors avoid the word "negate" and stress the interactions of stressors and their findings demonstrate that the effects of this pollutant are strongly dependent on the temperature during exposure, at least within the rather narrow range that has been examined.

05-Jan-2022
Dear Ms Aulsebrook I am pleased to inform you that your manuscript RSPB-2021-2701 entitled "Warmer temperatures limit the effects of antidepressant pollution on life-history traits" has been accepted for publication in Proceedings B.
The referee(s) have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the referee(s)' comments and revise your manuscript. Because the schedule for publication is very tight, it is a condition of publication that you submit the revised version of your manuscript within 7 days. If you do not think you will be able to meet this date please let us know.
To revise your manuscript, log into https://mc.manuscriptcentral.com/prsb and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision. You will be unable to make your revisions on the originally submitted version of the manuscript. Instead, revise your manuscript and upload a new version through your Author Centre.
When submitting your revised manuscript, you will be able to respond to the comments made by the referee(s) and upload a file "Response to Referees". You can use this to document any changes you make to the original manuscript. We require a copy of the manuscript with revisions made since the previous version marked as 'tracked changes' to be included in the 'response to referees' document.
Before uploading your revised files please make sure that you have: 1) A text file of the manuscript (doc, txt, rtf or tex), including the references, tables (including captions) and figure captions. Please remove any tracked changes from the text before submission. PDF files are not an accepted format for the "Main Document".
2) A separate electronic file of each figure (tiff, EPS or print-quality PDF preferred). The format should be produced directly from original creation package, or original software format. PowerPoint files are not accepted.
3) Electronic supplementary material: this should be contained in a separate file and where possible, all ESM should be combined into a single file. All supplementary materials accompanying an accepted article will be treated as in their final form. They will be published alongside the paper on the journal website and posted on the online figshare repository. Files on figshare will be made available approximately one week before the accompanying article so that the supplementary material can be attributed a unique DOI.
Online supplementary material will also carry the title and description provided during submission, so please ensure these are accurate and informative. Note that the Royal Society will not edit or typeset supplementary material and it will be hosted as provided. Please ensure that the supplementary material includes the paper details (authors, title, journal name, article DOI). Your article DOI will be 10.1098/rspb.[paper ID in form xxxx.xxxx e.g. 10.1098/rspb.2016.0049]. 4) A media summary: a short non-technical summary (up to 100 words) of the key findings/importance of your manuscript.

5) Data accessibility section and data citation
It is a condition of publication that data supporting your paper are made available either in the electronic supplementary material or through an appropriate repository. Please see our Data Sharing Policies https://royalsociety.org/journals/authors/author-guidelines/#data.
In order to ensure effective and robust dissemination and appropriate credit to authors the dataset(s) used should be fully cited. To ensure archived data are available to readers, authors should include a 'data accessibility' section immediately after the acknowledgements section. This should list the database and accession number for all data from the article that has been made publicly available, for instance: • DNA sequences: Genbank accessions F234391-F234402 • Phylogenetic data: TreeBASE accession number S9123 • Final DNA sequence assembly uploaded as online supplemental material • Climate data and MaxEnt input files: Dryad doi:10.5521/dryad.12311 NB. From April 1 2013, peer reviewed articles based on research funded wholly or partly by RCUK must include, if applicable, a statement on how the underlying research materials -such as data, samples or models -can be accessed. This statement should be included in the data accessibility section.
If you wish to submit your data to Dryad (http://datadryad.org/) and have not already done so you can submit your data via this link http://datadryad.org/submit?journalID=RSPB&amp;manu=(Document not available) which will take you to your unique entry in the Dryad repository. If you have already submitted your data to dryad you can make any necessary revisions to your dataset by following the above link. Please see https://royalsociety.org/journals/ethics-policies/data-sharing-mining/ for more details.
6) For more information on our Licence to Publish, Open Access, Cover images and Media summaries, please visit https://royalsociety.org/journals/authors/author-guidelines/.
Once again, thank you for submitting your manuscript to Proceedings B and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch.
Sincerely, Dr Locke Rowe mailto: proceedingsb@royalsociety.org Associate Editor Board Member: 1 Comments to Author: I have now obtained two expert reviews of this paper; both referees agree that this this study is very well-done and will make a strong contribution to the field -I completely agree. As you will see below, both reviewers make a number of (relatively minor but very helpful) suggestions for improvements. I call particular attention to the issues of (1) the natural thermal optimum of the study organism and whether 25C indeed represents a stressor or not -a point that needs clarification; (2) the discussion and interpretation of the interaction of fluoxetine and temperature which might be context-dependent; and, on a related issue, (3) the notion that in natural situations may be much more complex: likely only few lakes in nature will consistently experience 20 or 25C across time and space in a persistent manner, and Daphnia might avoid particular temperatures by vertical migration -in essence, a bit more ecological realism should be added to the discussion. For the detailed comments by the referees please see below. This paper should be acceptable pending some minor revisions and will make a strong and interesting contribution to the field.

Reviewer(s)' Comments to Author:
Referee: 1 Comments to the Author(s) This report explores the effects of the antidepressant fluoxetine on the life history responses of two clones of Daphnia magna at two temperatures, with a fully crossed design. The main findings of the study were that the effects of fluoxetine were non-monotonic, clone and temperature dependent. Their results highlight the difficulties of extrapolating from laboratory studies to complex environments. The implementation and analysis of the study were good, including accurately validating the extremely low fluoxetine concentrations and their results do demonstrate the complexity in understanding impacts of pollutants at field-level scales. The following comment may assist in improving the paper: L54-L68. In this paragraph the authors discuss the issue of multiple stressors. However, I am not temperature as a variable that can affect the effects of toxicants rather than as a stressor.
L110. What were the rearing conditions of the lines? How many parents were used to produce the test generation? What brood number were they from? L116. It is more conventional to present algal concentrations as cells/mL of C/mL. Was this amount of food ad libitum? Particularly at the higher temperature was there any evidence of food limitation? What was the pH of the water? Did this vary with temperature? L119. 70% humidity. I thought it would be 100% humidity in the water! L122. I can't find mention anywhere of how many individuals/treatment? L140. Were survival and fecundity recorded daily? If it was only twice a week, significant errors can occur in the estimation of life history parameters in such a rapidly breeding taxa. L158. Please state survival in all treatments. And please include data on timing and brood size in each instar for each treatment, at least in an appendix. Were all offspring born healthy? Did offspring differ in size? L16 worth commenting on. Fig. 2. There is too much information in graphs C and D. Either find a way to simplify or delete. I don't think they add a lot anyway.
Referee: 2 Comments to the Author(s) This manuscript is a novel contribution to unraveling the complex responses of organisms to multiple potential stressors (e.g., antidepressants and temperature). The experiments and analyses are sound and the results are well supported and of interest. The manuscript was well written and the results were presented very clearly. In general, the manuscript provides a clear description of the need for this research and a good description of the research conducted. These types of experiments are imperative for understanding how multiple stressors affect organisms in nature, where multiple stressors exist.
My only comments for revision are to request that the authors provide a bit more detail about the study organism and its thermal optimum and how the temperature treatment compares to their thermal optimum. The authors indicate that the higher temperatures represent a stressor, but do not provide adequate information in the text to ascertain how sensitive D. magna is to a 20 vs. 25C thermal regime.
Along the same lines, in the discussion the authors make some statements which I think need to be refined given thermal regimes in lakes, stratification and the potential behavior of Daphnia.
The statement "Indeed, warmer temperatures appear to negate the effects of fluoxetine on an organism's life-history, suggesting that the effects of this widespread pharmaceutical will not necessarily be made worse under common scenarios of global change." And other similar statements in the discussion could be refined because in any given lake or pond where Daphnia exist, they will be exposed to a variety of temperatures. Daphnia are also known to migrate vertically in the water column as well (thereby experiencing different temperatures).
So, although the point is certainly useful that there is a strong potential for temperature to interact with contaminants like the one studied here, how this plays out in a given ecosystem will be very complex. I would recommend that the authors avoid the word "negate" and stress the interactions of stressors and their findings demonstrate that the effects of this pollutant are strongly dependent on the temperature during exposure, at least within the rather narrow range that has been examined.

12-Jan-2022
Dear Ms Aulsebrook I am pleased to inform you that your manuscript entitled "Warmer temperatures limit the effects of antidepressant pollution on life-history traits" has been accepted for publication in Proceedings B.
You can expect to receive a proof of your article from our Production office in due course, please check your spam filter if you do not receive it. PLEASE NOTE: you will be given the exact page length of your paper which may be different from the estimation from Editorial and you may be asked to reduce your paper if it goes over the 10 page limit.
If you are likely to be away from e-mail contact please let us know. Due to rapid publication and an extremely tight schedule, if comments are not received, we may publish the paper as it stands.
If you have any queries regarding the production of your final article or the publication date please contact procb_proofs@royalsociety.org Your article has been estimated as being 8 pages long. Our Production Office will be able to confirm the exact length at proof stage.
Data Accessibility section Please remember to make any data sets live prior to publication, and update any links as needed when you receive a proof to check. It is good practice to also add data sets to your reference list.
Open Access You are invited to opt for Open Access, making your freely available to all as soon as it is ready for publication under a CCBY licence. Our article processing charge for Open Access is £1700. Corresponding authors from member institutions (http://royalsocietypublishing.org/site/librarians/allmembers.xhtml) receive a 25% discount to these charges. For more information please visit http://royalsocietypublishing.org/open-access.
Paper charges An e-mail request for payment of any related charges will be sent out shortly. The preferred payment method is by credit card; however, other payment options are available.
Electronic supplementary material: All supplementary materials accompanying an accepted article will be treated as in their final form. They will be published alongside the paper on the journal website and posted on the online figshare repository. Files on figshare will be made available approximately one week before the accompanying article so that the supplementary material can be attributed a unique DOI.
You are allowed to post any version of your manuscript on a personal website, repository or preprint server. However, the work remains under media embargo and you should not discuss it with the press until the date of publication. Please visit https://royalsociety.org/journals/ethicspolicies/media-embargo for more information.
Thank you for your fine contribution. On behalf of the Editors of the Proceedings B, we look forward to your continued contributions to the Journal.

Dear Dr Rowe,
We are delighted by the positive response to our submission and take the opportunity to thank you, the editorial board member, and reviewers for the constructive input. As you will see below, we have carefully considered the editor and reviewer comments in revising our manuscript. We hope that the paper is now acceptable for publication, but are, of course, happy to make further changes as required.
Yours sincerely, Lucinda Aulsebrook, Bob Wong and Matthew Hall BOARD MEMBER's COMMENTS TO THE AUTHOR BOARD MEMBER COMMENT: I have now obtained two expert reviews of this paper; both referees agree that this this study is very well-done and will make a strong contribution to the field -I completely agree. As you will see below, both reviewers make a number of (relatively minor but very helpful) suggestions for improvements. I call particular attention to the issues of (1) the natural thermal optimum of the study organism and whether 25C indeed represents a stressor or not -a point that needs clarification; (2) the discussion and interpretation of the interaction of fluoxetine and temperature which might be context-dependent; and, on a related issue, (3) the notion that in natural situations may be much more complex: likely only few lakes in nature will consistently experience 20 or 25C across time and space in a persistent manner, and Daphnia might avoid particular temperatures by vertical migration -in essence, a bit more ecological realism should be added to the discussion. For the detailed comments by the referees please see below. This paper should be acceptable pending some minor revisions and will make a strong and interesting contribution to the field.

RESPONSE:
We have attended to all of the three main points raised by the editorial board member. First, we can confirm that 25°C does, indeed, represent a stressor. We have now clarified this point and provided details on the established responses to 25°C in the manuscript. Second, we have now adjusted the discussion and interpretation, as requested. Lastly, on the point about ecological complexity, we agree with the reviewer and have adjusted the discussion to highlight this complexity. For details of how we have specifically addressed these (and other) reviewer comments, please see our responses to reviewers below.