Mercury exposure in an endangered seabird: long-term changes and relationships with trophic ecology and breeding success

Mercury (Hg) is an environmental contaminant which, at high concentrations, can negatively influence avian physiology and demography. Albatrosses (Diomedeidae) have higher Hg burdens than all other avian families. Here, we measure total Hg (THg) concentrations of body feathers from adult grey-headed albatrosses (Thalassarche chrysostoma) at South Georgia. Specifically, we (i) analyse temporal trends at South Georgia (1989–2013) and make comparisons with other breeding populations; (ii) identify factors driving variation in THg concentrations and (iii) examine relationships with breeding success. Mean ± s.d. feather THg concentrations were 13.0 ± 8.0 µg g−1 dw, which represents a threefold increase over the past 25 years at South Georgia and is the highest recorded in the Thalassarche genus. Foraging habitat, inferred from stable isotope ratios of carbon (δ13C), significantly influenced THg concentrations—feathers moulted in Antarctic waters had far lower THg concentrations than those moulted in subantarctic or subtropical waters. THg concentrations also increased with trophic level (δ15N), reflecting the biomagnification process. There was limited support for the influence of sex, age and previous breeding outcome on feather THg concentrations. However, in males, Hg exposure was correlated with breeding outcome—failed birds had significantly higher feather THg concentrations than successful birds. These results provide key insights into the drivers and consequences of Hg exposure in this globally important albatross population.

classified as a failed breeder, but it is a failed breeder because it was absent. Being a failed breeder to me implies the bird was present, laid an egg, but then failed to fledge young. You point out that it is natural for them to usually breed every other year, so some of these failed breeders seem to just be doing what they normally should do. It seems like you should have 2 different analyses, one for years between reproductive attempts and one for initiated a clutch that failed or successful. Or, you need to clarify your data sheet with some additional explanation on the general statement page.
When during the reproductive stage does breeding failure occur? If the nest fails prior to hatching, that suggests an issue associated with the parent's exposure to Hg or perhaps body condition. But if the nest hatches but fails prior to fledging, then doesn't that suggest a less direct relationship between the parent's Hg exposure and nest failure? Date of failure may not capture this because there will be variation in when birds initiated their clutch. Does feather mercury correlate with nest initiation?
Line 158, Seems like this should say were used to test. Line 306-308. Why is the total feather mercury showing a pattern opposite of shown by methyl mercury concentrations in ocean water?

Review form: Reviewer 2
Recommendation Accept with minor revision (please list 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? Good Quality of the paper: Is the overall quality of the paper suitable? Excellent

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.

Is it accessible? Yes
This action has been taken on the advice of referees, who have recommended that substantial revisions are necessary. With this in mind we would be happy to consider a resubmission, provided the comments of the referees are fully addressed. However please note that this is not a provisional acceptance.
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Sincerely, Dr Locke Rowe mailto: proceedingsb@royalsociety.org Associate Editor Board Member: 1 Comments to Author: Thank you for submitting your manuscript "Mercury exposure in an endangered albatross: longterm changes and consequences for breeding succes" to Proceedings B. I've now received two reviews of your manuscript and reviewed the paper myself. Both reviewers were positive about your manuscript but suggested revisions that need to be addressed. Reviewer 1 has asked for better clarification and classification of failed breeding attempts, which should help pinpoint reproductive stages more strongly affected by Hg exposure and how Hg affects reproduction. Both reviewers felt the article was a bit too myopic for the readership of Proc B, and Reviewer 2 suggested ways to broaden the scope of the manuscript. Reviewer 2 also makes an interesting point about "toxicity thresholds" Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) This paper examines factors (sex, age, breeding experience) that could influence total feather mercury concentrations in grey-headed albatrosses and uses stable isotopes to determine if foraging latitude or trophic position influence these concentrations as well. The authors compare these results to previous years and other locations where this species and breeds and found that the concentrations at the colony on South Georgia Island are greater than those found at other colonies and that they have increased greatly over time. They also show that birds with greater total mercury were more likely to experience reproductive failure. I found the paper to be well written, the study to be well designed, and the analyses to be well done. I do believe that the paper would benefit from greater explanation in some sections. The paper shows that Hg concentrations are greater at South Georgia Island compared to several other breeding colonies. How do rates of population decline or reproductive success at South Georgia compare to these other sties? Are they declining throughout the entire range, despite differences in Hg concentrations? That would suggest that Hg is not the main contributor to population decline. Do grey-headed albatross breeding on other islands forage in similar areas to those from South Georgia while molting? How far does this species travel to forage during the breeding season? Do they reach some of the higher latitude areas that are associated with greater feather Hg while those from other breeding colonies do not? One of the key results is that failed breeders have greater total feather mercury than successful breeders, but this difference is very subtle, 2 ug/g dw. In the discussion, you need to explain how this small difference could lead to reproductive failure. Are there other studies that show small differences in feather mercury cause physiological or behavioral effects or can lead to reproductive failure?
What about the reproductive stage. Is it a failure associated with hatching or fledging the young? Date of failure may not capture this because there will be variation in when birds initiate their clutch.
You describe several negative health consequences associated with elevated Hg concentrations. How do those Hg concentrations compare to the ones you found in this study (are yours as high as some of the studies on captive birds you refer to?). When I look at your data sheet, you have a breeding history column that includes not only if they bred successfully but also if they were seen that year. It seems that any bird you don't see is classified as a failed breeder, but it is a failed breeder because it was absent. Being a failed breeder to me implies the bird was present, laid an egg, but then failed to fledge young. You point out that it is natural for them to usually breed every other year, so some of these failed breeders seem to just be doing what they normally should do. It seems like you should have 2 different analyses, one for years between reproductive attempts and one for initiated a clutch that failed or successful. Or, you need to clarify your data sheet with some additional explanation on the general statement page.
When during the reproductive stage does breeding failure occur? If the nest fails prior to hatching, that suggests an issue associated with the parent's exposure to Hg or perhaps body condition. But if the nest hatches but fails prior to fledging, then doesn't that suggest a less direct relationship between the parent's Hg exposure and nest failure? Date of failure may not capture this because there will be variation in when birds initiated their clutch. Does feather mercury correlate with nest initiation?
Line 158, Seems like this should say were used to test. Line 306-308. Why is the total feather mercury showing a pattern opposite of shown by methyl mercury concentrations in ocean water? Referee: 2 Comments to the Author(s) Title: The title highlights two of the sexier findings in the article but does not truly describe the study. The temporal trend data are based on a loose comparison with other studies, and the difference in Hg levels between successful and unsuccessful birds is suggestive at best, and does not show a "consequence" of mercury exposure, which suggests causation. A more descriptive and accurate title would be "Mercury exposure in an endangered albatross: feather concentrations with respect to diet, breeding success, and past studies." Abstract: This accurately relates the results on a clear manner. Introduction: This section is efficient and well organized. Methods: The procedure for combining the results from the two or more aliquots, and then the results for the three feathers per bird should be described. Were the six or more sample values simply averaged, or was weighted averaging used, or something else? Why was the number of aliquots variable between feathers. It is hard to evaluate what an SD of <10% "between runs" means, especially in the absence of understanding the previous step, in terms of what was being compared to what. Is a "run" an entire batch of samples? If these aliquots are considered duplicates, wouldn't it be better to present relative percent difference between pairs of duplicate samples from the same feather? That's what is normally judged to be "OK" if <10%.In general the QAQC section for mercury analysis is limited and unclear. Results: The adverse effects levels of 5-40 ppm are extremely arbitrary and have little basis in fact. I agree that the authors need to pick some level, and these may be the benchmarks to use for lack of others, but they should not be perpetuated as "toxicity thresholds" as in line 197. They are based on archaic methods and speculation and are a range within which the true threshold supposedly lies. There are several articles indicating lower effects thresholds (see recent Ackerman et al. review already cited). Discussion: In general the article is well-written and the Discussion sticks to the data. However, the overall viewpoint of the paper is rather myopically focused on albatrosses and closely related seabirds, and could benefit from bringing in some mention of the many other studies, especially effects studies, that have been done with mercury and non-albatross birds. This will broaden appeal and strengthen conclusions -as is, only two papers from non-marine birds are cited, both rather tangentially. Mercury is mercury and birds are birds, so authors should take advantage of what is known about mechanisms of reproductive failure especially. There are also numerous (hundreds) of papers on temporal trends in mercury in biota -bringing in a few of these to buttress and interpret their claim that levels are rising might also be good.

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.

Do you have any ethical concerns with this paper? No
Comments to the Author I think the authors did a very good job addressing my comments. There were just a couple of minor edits that should be made. Line 67. Fix wording Line 348. Fix wording Line 355. Explanation of wet weight abbreviation should come here Decision letter (RSPB-2020-2683.R0)

25-Nov-2020
Dear Mr Mills I am pleased to inform you that your manuscript RSPB-2020-2683 entitled "Mercury exposure in an endangered seabird: long-term changes and relationships with trophic ecology and breeding success" has been accepted for publication in Proceedings B.
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Sincerely, Dr Locke Rowe mailto: proceedingsb@royalsociety.org Associate Editor Board Member Comments to Author: Thank you for submitting your manuscript, "Mercury exposure in an endangered seabird: longterm changes and relationships with trophic ecology and breeding success" to Proceedings of the Royal Society, B. The reviewers and I have now assessed your revised manuscript and have no additional comments.

Reviewer(s)' Comments to Author:
Referee: 1 Comments to the Author(s). I think the authors did a very good job addressing my comments. There were just a couple of minor edits that should be made. Line 67. Fix wording Line 348. Fix wording Line 355. Explanation of wet weight abbreviation should come here Decision letter (RSPB-2020-2683.R1)

27-Nov-2020
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RESPONSE TO REFEREES ASSOCIATE EDITOR BOARD MEMBER -COMMENTS TO THE AUTHOR(S)
COMMENT: Thank you for submitting your manuscript "Mercury exposure in an endangered albatross: long-term changes and consequences for breeding success" to Proceedings B. I've now received two reviews of your manuscript and reviewed the paper myself. Both reviewers were positive about your manuscript but suggested revisions that need to be addressed. Reviewer 1 has asked for better clarification and classification of failed breeding attempts, which should help pinpoint reproductive stages more strongly affected by Hg exposure and how Hg affects reproduction. Both reviewers felt the article was a bit too myopic for the readership of Proc B, and Reviewer 2 suggested ways to broaden the scope of the manuscript. Reviewer 2 also makes an interesting point about "toxicity thresholds". REPLY: Thanks for your comments and the opportunity to resubmit our manuscript to Proceedings of the Royal Society B: Biological Sciences. We have addressed all of the reviewers' comments by changes to the text (see detailed reply below).

REFEREE 1 -COMMENTS TO THE AUTHOR(S)
COMMENT: This paper examines factors (sex, age and breeding experience) that could influence total feather mercury concentrations in grey-headed albatrosses and uses stable isotopes to determine if foraging latitude or trophic position influence these concentrations as well. The authors compare these results to previous years and other locations where this species and breeds and found that the concentrations at the colony on South Georgia Island are greater than those found at other colonies and that they have increased greatly over time. They also show that birds with greater total mercury were more likely to experience reproductive failure. I found the paper to be well written, the study to be well designed, and the analyses to be well done. I do believe that the paper would benefit from greater explanation in some sections. REPLY: Thanks for the positive response. We have addressed your specific comments below.
COMMENT: The paper shows that Hg concentrations are greater at South Georgia Island compared to several other breeding colonies. How do rates of population decline or reproductive success at South Georgia compare to these other sties? Are they declining throughout the entire range, despite differences in Hg concentrations? That would suggest that Hg is not the main contributor to population decline. REPLY: Grey-headed albatrosses are declining more steeply at South Georgia than at any other island group where there is a major population; numbers are increasing at Diego Ramirez and Crozet, broadly stable at the Prince Edward Islands, and declining slowly at Kerguelen (Ryan et al. 2009 African J. Mar. Sci.;Robertson et al. 2017 Polar Biol.;Weimerskirch et al. 2018 Polar Biol.). However, the differing population trends could relate to factors other than Hg burdens, particularly the relative overlap with different fishing fleets and hence bycatch rates, which vary greatly. For that reason, we agree that the steep population decline at South Georgia does not necessarily relate to the high Hg burdens, although they may be a contributing factor. We now make this