Citation
Gentoo penguin (pygoscelis papua) feather mercury concentrations differ between Antarctic and sub-Antarctic regions as revealed by foraging ecology and stable isotope analyses

Material Information

Title:
Gentoo penguin (pygoscelis papua) feather mercury concentrations differ between Antarctic and sub-Antarctic regions as revealed by foraging ecology and stable isotope analyses
Creator:
Schutt, David
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Master's ( Master of science)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Integrative Biology, CU Denver
Degree Disciplines:
Biology
Committee Chair:
Wunder, Mike B.
Committee Members:
Vajda, Alan M.
Roane, Timberley M.
Stricker, Craig A.

Notes

Abstract:
Numerous studies on seabird mercury loads have helped characterize bioavailability of mercury as an increasing global pollutant, however the broad and dynamic foraging ecology of seabirds has made comparisons difficult. Here, we investigate foraging ecology of Gentoo penguins (Pygoscelis papua) in relation to their feather mercury concentrations to help understand regional differences in mercury uptake by these widely-distributed seabirds. Due to logistical limitations that only allowed us to collect feather samples from live birds in the Falkland Islands and from dead birds at the remaining sites, we first investigated whether distributions of feather mercury concentrations were consistent between live birds and dead birds. Our results indicate that dead birds can be used as surrogates for live birds in the study of Gentoo penguin feather mercury concentrations. Following this analysis, we used a comparative approach with stable isotope analyses to uncover baselines in trophic level and mercury biomagnification among discrete breeding populations. Additionally, we consider differences in prey composition between populations and prey selection through energy density analysis to understand differences in foraging ecology that would likely have the largest effect on penguin mercury exposure. Our results suggest that numerous factors play a role in mercury load disparities between populations, and that higher mercury concentrations in the Falkland Islands and South Georgia Island in relation to the Antarctic Peninsula are due to differences in foraging ecology coupled with a likely increase in ambient environmental mercury.

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Auraria Library
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University of Colorado Denver
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Copyright David Schutt. Permission granted to University of Colorado Denver to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Full Text
GENTOO PENGUIN (PYGOSCELISPAPUA) FEATHER MERCURY CONCENTRATIONS
DIFFER BETWEEN ANTARCTIC AND SUB-ANTARCTIC REGIONS AS REVEALED BY FORAGING ECOLOGY AND STABLE ISOTOPE ANALYSES
by
DAVID SCHUTT B.S., University of Colorado, 1990 B.A., University of Colorado, 1998
A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Biology Program
2019


This thesis for the Master of Science degree by
David Schutt has been approved for the Biology Program by
Mike B. Wunder, Chair and Co-Advisor Alan M. Vajda, Co-Advisor Timberley M. Roane Craig A. Strieker
Date: May 18, 2019
n


Schutt, David (M.S., Biology Program)
Gentoo Penguin (Pygoscelis Papua) Feather Mercury Concentrations Vary Between Antarctic and Sub-Antarctic Regions as Revealed by Foraging Ecology and Stable Isotope Analyses Thesis directed by Associate Professors Mike B. Wunder and Alan M. Vajda
ABSTRACT
Numerous studies on seabird mercury loads have helped characterize bioavailability of mercury as an increasing global pollutant, however the broad and dynamic foraging ecology of seabirds has made comparisons difficult. Here, we investigate foraging ecology of Gentoo penguins (.Pygoscelispapua) in relation to their feather mercury concentrations to help understand regional differences in mercury uptake by these widely-distributed seabirds. Due to logistical limitations that only allowed us to collect feather samples from live birds in the Falkland Islands and from dead birds at the remaining sites, we first investigated whether distributions of feather mercury concentrations were consistent between live birds and dead birds. Our results indicate that dead birds can be used as surrogates for live birds in the study of Gentoo penguin feather mercury concentrations. Following this analysis, we used a comparative approach with stable isotope analyses to uncover baselines in trophic level and mercury biomagnification among discrete breeding populations. Additionally, we consider differences in prey composition between populations and prey selection through energy density analysis to understand differences in foraging ecology that would likely have the largest effect on penguin mercury exposure. Our results suggest that numerous factors play a role in mercury load disparities between populations, and that higher mercury concentrations in the Falkland Islands
m


and South Georgia Island in relation to the Antarctic Peninsula are due to differences in foraging ecology coupled with a likely increase in ambient environmental mercury.
The form and content of this abstract are approved. I recommend its publication.
Approved: Mike B. Wunder and Alan M. Vajda
IV


This work is dedicated to everyone who helped me through this challenging, but extremely rewarding thesis project. In particular, this work is dedicated to Heather, my understanding and supportive wife and cheerleader, and my son, Edan, who learned as much as I did throughout this journey and will likely follow a new path through life after traveling to the far corners of the world to get to “hold baby penguins.” This work is also dedicated to my good friends who I met at McMurdo so many years ago where I got to play with penguins for the first time.
v


ACKNOWLEDGEMENTS
I would like to thank Mike Wunder, Alan Vajda, Timberley Roane and Craig Strieker, my committee members, for directing me through this research and teaching me more about ecology, stable isotopes, statistics and environmental toxicology than I ever thought I’d learn. I would also like to thank Jono Handley who taught me everything I needed to know about working with penguins and, through his own doctorate work, inspired me to develop my own research. Thanks to Alan Franklin and Jeff Chandler for storing samples and for use of the lab, to Rebecka Brasso, Lisa Stoneham and Megan Faulkner for conducting mercury analysis, and to Craig Strieker for conducting stable isotope analyses. Special thanks go to logistical support from Quark Expeditions, Quixote Expeditions, and Penguins International. Additional thanks to the Falkland Islands Government, the Government of South Georgia and the South Sandwich Islands and the NSF for assistance throughout the permitting process. Special thanks go to Adrian Lowe, Jan Cheek, Derek Pettersson, Micky Reeves and John and Michelle Jones for their hospitality and assistance in the Falkland Islands. IACUC Protocol #92517(08)1C.
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TABLE OF CONTENTS
CHAPTER
I. CONSISTENCY OF FEATHER MERCURY CONCENTRATIONS IN LIVE VS.
OPPORTUNISTICALLY-FOUND CARCASSES OF GENTOO PENGUINS
(PYGOSCELIS PAPUA).................................................1
Introduction.......................................................1
Methods............................................................3
Results and Discussion.............................................8
Conclusion.........................................................9
II. SUB-ANTARCTIC GENTOO PENGUIN (PYGOSCELIS PAPUA) POPULATIONS HAVE HIGHER FEATHER MERCURY CONCENTRATIONS THAN ANTARCTIC
POPULATIONS.......................................................10
Introduction......................................................10
Materials and Methods.............................................13
Results...........................................................19
Discussion........................................................22
Conclusion........................................................35
REFERENCES..............................................................37
APPENDIX................................................................49
vii


CHAPTERI
CONSISTENCY OF FEATHER MERCURY CONCENTRATIONS IN LIVE VS.
OPPORTUNISTICALLY-FOUND CARCASSES OF GENTOO PENGUINS
(PYGOSCELIS PAPUA)
Introduction
Ecotoxicological studies on seabirds have historically relied on specific tissues that often required a freshly dead specimen or the sacrificing of a live-caught individual. For ethical and logistical reasons, contemporary sampling schemes have shifted away from destructive sampling and instead employ minimally invasive or noninvasive collection methods (Pauli et al., 2010).
For example, avian sampling methods might include feather collection from live birds, collecting molted feathers from the ground, or collecting tissues opportunistically from bird carcasses (termed “found-dead birds” hereafter). Use of found-dead birds may be the only pragmatic option in studies where capture and handling of live birds is limited or impossible, such as when restrictions on handling are imposed by regulating agencies, by working in sensitive or difficult to access habitats, for studies involving birds that are difficult to live-capture, or when disruptive handling of live birds directly or indirectly impacts the survival of individual birds (Spotswood et al., 2011; Wilson & McMahon, 2006).
Due to the stability of mercury bound within the feather keratin matrix, feathers from a dead bird should maintain similar mercury levels as when the individual was alive. In a live bird, once feather synthesis is complete, the blood supply at the calamus atrophies and the feather becomes an inert tissue (Lillie, 1940), no longer subject to structural or chemical changes through any biological processes by the bird. This durable feather tissue withstands harsh
1


environmental conditions (Appelquist et al., 1984) not only while the bird is alive, but also resisting decomposition for a time following death. Such structural stability is a result of strong intramolecular disulfide bonds in the feather keratin which associate with circulating blood mercury during feather synthesis (Aschner & Syversen, 2005; Bortolotti, 2010; Crewther et al., 1965; Furness et al., 1986), effectively locking this mercury exposure record in a medium that remains stable after development. Thus, a feather will remain chemically consistent from the time of synthesis in a live bird until long after the death of the bird.
Although the sampling of dead birds is often assumed to serve as an authentic substitute for sampling of live birds (e.g. Bekhit et al., 2011; Cipro et al., 2017; Dimitrijevic et al., 2018; Kim et al., 2015; Vasil et al., 2012; Vega et al., 2009), examples of formal evaluation of this practice in ecotoxicology are lacking. For example, mercury loads above background effect levels may affect mortality (Ackerman et al., 2016; Jackson et al., 2016), which in turn might bias high the estimates of exposure as measured from found-dead birds. Likewise, sub-lethal observable responses of mercury toxicity may indirectly affect mortality (Ackerman et al., 2016; Eagles-Smith et al., 2008; Jackson et al., 2016). The wide range of feather mercury concentrations with variable sub-lethal effect responses allows for uncertainty as to causes of mercury toxicity-related mortality in found-dead birds. Evidence demonstrating that dead birds can be substituted for live birds, therefore, is a pragmatic and efficient alternative for ecotoxicology investigations. The main objective of this study was to assess the efficacy of using found-dead birds as proxies for live birds to estimate feather mercury concentrations in Gentoo penguins (Pygoscelis papua).
2


Methods
Study Area
The study colonies were in the Falkland Islands, an archipelago located approximately 500 km east of mainland South America in the South Atlantic Ocean (Fig. 1). Throughout the Falkland Islands, there are approximately 85 known breeding colonies of Gentoo penguins, collectively averaging approximately 100,000 breeding pairs annually (Baylis et al., 2013). The study sites were chosen to span a wide range of bathymetry near the breeding colonies (Carpenter-Kling et al., 2017; Handley et al., 2017). All samples were collected during the austral summer between December 2017 and February 2018.
Fig. 1 Location of Falkland Islands field sites (stars) for the present study (QGIS Development Team, 2019). For live birds, n = 15 for each site. For found-dead birds, n = 16 for Cow Bay (51° 25’ S 57° 51’ W), n = 15 for Race Point (51° 25’ S 59° 00’ W), n = 14 for Bull Point (52° 19’ S 59° 17’ W).
3


Study Species and Tissue Samples
Gentoo penguins are a non-migratory seabird that remain near their breeding colony throughout the year (Black et al., 2017; Tanton et al., 2004; Williams & Rodwell, 1992). This species is an opportunistic feeder, foraging on various crustaceans, cephalopods, and fish (Handley et al., 2015), rarely venturing farther than 30 km from shore during foraging trips (Carpenter-Kling et al., 2017; Handley el al., 2018; Robinson & Hindell, 1996). Throughout the breeding season, adult Gentoos capture prey during these localized foraging trips and return typically on alternating days to feed their chicks the regurgitate which often contains whole or partially-digested prey items (Handley et al., 2015).
Penguin chicks undergo three stages of feather growth during development and maturation, 1) hatching with a natal down 2) plumage replacement at 1 to 2 weeks of age with a secondary down, 3) growth of adult plumage just prior to fledging. The secondary down was the focus for sampling in this study, as it is derived from prey items taken by the parents and fed to the chicks. Foraging by adults during this period occurs near the natal colony (Handley et al., 2017), reflecting a comparatively localized signal of mercury exposure within this tissue over a near-term temporal scale. Because chick down is grown simultaneously, all intraindividual feathers are expected to have low variation in mercury concentration (Brasso et al., 2013). In addition, feathers allow for a minimally invasive and easily transported method of tissue collection as compared with sampling blood or stomach contents. As such, penguin chick secondary down is well suited among avian tissues for mercury biomonitoring.
4


Live Bird Sampling Methods
Live birds were collected under permit from the Falkland Islands Government (#R22/2017), with landowner permission, and using protocols approved by University of Colorado Denver IACUC (#92517(08)1C). Each colony supported -2000+ breeding pairs of Gentoo penguins. Gentoo penguin chicks in the post-guard phase (i.e. no longer protected by the parents) were captured using a long-handled net. Down feathers (10-12) were plucked from the breast using a pair of silicone-tipped forceps (DDP Instruments young tongue forceps, Miami, FL, USA) and stored at ambient temperature in small resealable plastic bags. Each chick was marked with a small amount of feather paint to prevent duplicate sampling (Sporting Pigeons Paint - green, Pigeon Products International, Post Falls, ID, USA) and then released.
Dead Bird Sampling Methods
We found dead Gentoo penguin chicks by searching within each colony boundary and along transects extending -100 m from the colony boundary to find carcasses that may have been moved by predators or scavengers. We used only birds that had died during the study season (as evidenced by presence of blood and/or comparative lack of decomposition, bleaching and weathering). We collected secondary down from the breast of intact carcasses when available, or otherwise from the legs just above the foot, sampling only white feathers using the same methods as detailed for live birds in Section 2.3.
Mercury Analysis
All feather samples were sterilized per USD A/APHIS requirements (as detailed on the importation permit) for importation into the United States. Sterilization procedure required soaking the feather samples in 70% ethanol (LabChem, Zelienople, PA, USA) for 30 minutes.
5


Following sterilization, the feather samples were triple rinsed in deionized water and dried under a fume hood for 24-48 hours at room temperature until all external moisture was removed.
In preparation for mercury analysis the feathers were cleaned to remove exogenously deposited contaminants. The feather samples were placed in a stainless steel screen and vigorously cleaned in environmental grade acetone (Alfa Aesar, Ward Hill, MA, USA) followed by rinsing under a flowing stream of deionized water. This procedure was completed three times for each feather sample to ensure all exogenous oils and contaminates were removed. The feather samples were then dried in a fume hood at room temperature for 24 - 48 hours until all external moisture was removed.
Approximately 10 mg of whole down feathers from each individual were analyzed for total mercury via atomic absorption spectrophotometry on a Nippon MA-3000 Direct Mercury Analyzer at Weber State University (Ogden, UT, USA). Each set of 20 samples analyzed was preceded and followed by two samples of a standard reference material (TORT-3, National Resource Council Canada). Mean percent recovery for the standard reference material was 101.4% (n = 22) with relative significant differences in mercury concentration of 6.9%. Mercury concentrations in chick down are reported as mg/kg fresh weight (fw).
Feather-Equivalent Mercury Calculations
Data transformations for converting blood equivalent mercury concentrations to feather mercury concentrations were calculated to standardize our feather mercury values with previously reported blood mercury values for comparison. The equation used for the conversion was developed by Eagles-Smith et al. (2008) with A2 = 0.32 (Eq. 1).
6


In (Blood TotalHgww) = 0.673 x In (Feather TotalHgdw) -1.673
(1)
Statistical Analysis
We used a two-sample Kolmogorov-Smirnov test (ks.test, R Core Team 2018) to evaluate the null hypothesis that the mercury concentrations for feathers from live-caught and found-dead birds were drawn from the same distribution.
Fig. 2 Found-dead birds vs. live birds. Whiskers indicate 1.5*IQR, horizontal bar is the median, boxplot endpoints are at the 25th and 75th percentiles. The dashed line indicates the boundary between background mercury exposure and low mercury exposure, the upper limit of no-effect levels (Jackson et al., 2016).
7


Results and Discussion
We collected feathers from a total of 45 live chicks and 45 dead chicks from across the three different sites (Fig. 1). Mercury levels in all individuals were below the lowest reported level of effect for mercury exposure (Fig. 2; Jackson et al., 2016), and there was little support for rejecting the null hypothesis that mercury concentrations in feathers from live-caught and from found-dead birds were drawn from the same distribution (D = 0,2, p = 0.33).
Our primary goal for this study was to evaluate consistencies between distributions of feather mercury concentrations of live-caught Gentoo chicks and of opportunistically-found Gentoo chick carcasses. All individuals from both live and found-dead groups were below the mercury feather-equivalent low-effect threshold of 4.3 mg/kg, and far below the feather-equivalent mercury concentration of 288 mg/kg for mortality in seabirds (Eq. 1; Fig. 2; Ackerman et al., 2016; Eagles-Smith et al., 2008; Jackson et al., 2016). We therefore make the assumption that mercury exposure did not impact survival of the found-dead group differently than that of the live group. Our results suggest that mercury concentrations in feathers from bird carcasses are similar to those from birds living in the same breeding colonies. These results are particularly important for seabird feather mercury analyses due to the practice of substituting found-dead bird carcasses for live birds in a wide variety of studies using bird feathers as indicators of marine environmental toxicant exposure (Bekhit et al., 2011; Cipro et al., 2017;
Kim et al., 2015; Vega et al., 2010). Due to the inert and stable nature of feathers and the consistency of feather mercury distributions in both live individuals and opportunistically found carcasses, this tissue is well suited to be analyzed for mercury content regardless of the living
8


state of the bird. As such, feathers from either live or dead penguins consistently reflect dietary mercury exposure of the bird.
Conclusion
The distribution of mercury concentrations in feathers from penguin chick carcasses found opportunistically near nesting sites is similar to that for feathers from live chicks. For avian mercury studies that have logistical constraints such as permitting limitations or cost constraints associated with sampling from live birds, our results suggest that found-dead birds can be used as surrogates for live birds in the study of feather mercury concentrations. Because results from this study apply specifically to feather mercury concentrations in penguins, we recommend that additional studies be conducted to evaluate feather samples from live vs. found-dead birds in other bird taxa as well as for additional toxicants such as persistent organic pollutants (POPs) to evaluate a much wider array of ecotoxicological subjects.
9


CHAPTER II
SUB-ANTARCTIC GENTOO PENGUIN (PYGOSCELIS PAPUA) POPULATIONS HAVE HIGHER FEATHER MERCURY CONCENTRATIONS THAN ANTARCTIC
POPULATIONS
Introduction
Mercury is a global pollutant that has been growing steadily in the atmosphere due directly to human activities. Artisanal gold mines, fossil fuel combustion and various industrial processes have led to a 3- to 5-fold increase in atmospheric mercury over the past 150 years (Driscoll et al., 2013; Streets et al., 2017). Estimates of ocean surface mercury concentrations as a result have increased by a factor of 2-3 (Driscoll et al., 2013; Fitzgerald et al., 2007) which is subsequently bioaccumulating in marine food webs.
Penguins are mesopredator species in the Southern Hemisphere and have been used as bioindicators of marine contaminants in numerous studies due to their interaction with the marine food web at higher trophic levels (Burger & Gochfeld, 2004; Furness & Camphuysen, 1997; Kahle & Becker, 1999). The study of mercury loads in higher trophic level seabirds has provided much understanding regarding bioavailability and trends in marine fauna mercury uptake, which can point to changes in global pollution dynamics. The relationship is challenging to quantify, however, due to mercury’s bioaccumulating properties, and involves complexities of foraging ecology and an organism’s interaction with the food web. Seabirds in particular are subject to large variations in mercury loading within a population due to their broad distribution in a highly variable marine environment. Intraspecific mercury exposure disparities have been recorded from individual seabirds within a single population (Catry et al., 2008; Lescroel et al.,
10


2004; Miller et al., 2009; Polito et al., 2016; Thompson et al., 1998) which is likely a function of differences in foraging strategies and extensive foraging range (Thompson et al., 1998). Understanding foraging behavior of a seabird population is therefore imperative to help explain differences in mercury uptake (Polito et al., 2016) in a dynamic marine environment.
The range in diets and its effects on mercury accumulation within seabird populations points to increased accuracy of mercury data when coupled with stable isotope analyses. Carbon isotopic differences can be readily attributed to various foraging geographies, namely benthic/ onshore vs. pelagic/offshore locations and also differences in latitude resulting in a decline in carbon isotopes at higher latitudes (Cherel & Hobson, 2007; Kelly, 2000). Nitrogen isotopic inferences of trophic level help standardize interactions within the food web (Atwell et al., 1998; Bearhop et al., 2000; Santos et al., 2005; Seixas et al., 2014), and are of particular importance when understanding mercury biomagnification and comparisons of dietary exposure (Polito et al., 2016). Trophic level approximations may therefore provide an explanation for increases in mercury that are unrelated to diet selection, and may be attributed to other factors such as environmental baseline mercury differences. To understand why intraspecific differences in trophic levels exist, neither isotopic mixing models nor stomach content dietary studies are sufficient alone to infer foraging niche of wide-ranging generalist seabirds such as Gentoo penguins (Pygoscelispapua; Polito et al., 2016). As such, we include a review of the literature to discern differences in Gentoo foraging strategy and its effects on feather mercury concentrations.
Our study focuses on three regions in the Southern Hemisphere, with a primary focus on the Falkland Islands, a region that is understudied and relatively unknown with respect to seabird mercury exposures. The Falkland Islands is an archipelago off the Patagonian coast of South
11


America with large numbers of seabirds but almost non-existent seabird mercury investigations. South Georgia Island is a remote seabird-rich island in the turbulent waters of the Scotia Sea.
The Antarctic Peninsula, like the rest of the Antarctic continent, is a region that is isolated from the global oceanic system by the massive flow of the Antarctic Circumpolar Current and its abrupt thermal and salinity gradients (Bargagli, 2005). Because previous studies on seabirds along the Antarctic Peninsula show relatively low mercury exposure (Brasso et al., 2014), we use this isolated region as a reference with which to compare mercury exposure in seabirds from the Falkland Islands and South Georgia Island.
In this study, we investigated mercury loads in relation to foraging ecology of sub-Antarctic and Antarctic populations of Gentoo penguins, a non-migratory species that inhabits both sides of the Antarctic Circumpolar Current. Based on the geography of the region and the relative isolation of Antarctica from the rest of the oceanic system, we would expect the Falkland Islands and South Georgia Island to have higher bioavailable mercury than Antarctica. For this investigation, we examined Gentoo foraging patterns throughout the study sites to understand behavioral and dietary sources of mercury exposure disparity. Different prey items consumed by Gentoos bioaccumulate mercury differently in relation to trophic level and vertical location within the water column and therefore influence dietary exposure (Monteiro et al., 1996; Thompson et al., 1998). We also used stable isotope analyses of Gentoo feathers in relation to prey items to understand differences in trophic level and potential <515N enrichment between the
regions. Finally, we compared our feather mercury data with trophic level and foraging behavior to explain differences in Gentoo feather mercury concentrations among the Falkland Islands, South Georgia Island and the Antarctic Peninsula.
12


80°W 60°W 40°W 20°W
Fig. 1 Sampling regions for the present study. Bar plot shows the relative proportion of each Gentoo prey classification for each region.
Materials and Methods
Study Areas
We collected feather samples during the austral summer of 2017-2018 from live and dead Gentoo chicks at four colonies around the Falkland Islands, from dead Gentoo chicks at four colonies on South Georgia Island, and from dead Gentoo chicks at five colonies along the Antarctic Peninsula (Fig. 1). Previous studies have demonstrated consistency between feather mercury concentrations in both live and found-dead chicks (Schutt et al., submitted manuscript),
13


allowing dead birds to be used as surrogates for live birds in feather mercury studies. Because Gentoo penguins maintain high breeding site fidelity (Williams & Rodwell, 1992) and rarely forage more than 30 km from shore (Boersma et al., 2002; Carpenter-Kling et al., 2017;
Kokubun et al., 2009; Lescroel & Bost, 2005; Robinson & Hindell, 1996; Trivelpiece et al.,
1986; Wilson et al., 1998), we consider each region to be an independent population due to the unlikelihood that they interact with each other. Samples from each region were therefore pooled together into regional groups.
The Falkland Islands are an archipelago located approximately 500 km from the coast of mainland South America in the southern Atlantic Ocean, situated north of the Antarctic Circumpolar Current on the relatively shallow Patagonian Shelf (Copello et al., 2011). Throughout the Falkland Islands, there are approximately 85 breeding colonies of Gentoo penguins, averaging approximately 100,000 breeding pairs each year (Baylis et al., 2013).
South Georgia Island is a sub-Antarctic island at the south of the Polar Front. This island was previously the site of heavy whaling activities in the early part of the 20th century, but in the past five decades has only been inhabited by temporary seasonal personnel for science and tourism duties (Moore et al., 1999). Throughout South Georgia Island, there are approximately 80,000 - 100,000 breeding pairs of Gentoos annually (Varty et al., 2008).
The Western Antarctic Peninsula is a region typically free of marine fast ice in the summer season with breeding colonies of Gentoo penguins scattered among the few ice-free areas along the coastline. The region sustained large numbers of whaling and sealing communities in the early 20th century, but now is only inhabited by seasonal science personnel at a small number of research stations. Throughout the Western Antarctic Peninsula, there are
14


approximately 100,000 breeding pairs of Gentoo penguins annually (Borboroglu & Boersma, 2013).
Study Species and Tissue Samples
Gentoo penguins are a non-migratory seabird that remain near their breeding colony year-round (Black et al., 2017; Tanton et al., 2004; Williams & Rodwell, 1992). Throughout the breeding season, adult Gentoos capture prey during localized foraging trips and return typically on alternating days to feed their chicks the regurgitate which often contains whole or partially-digested prey items (Handley et al., 2015). Penguin chicks undergo three stages of feather growth during development and maturation, 1) hatching with a natal down 2) plumage replacement at 1 to 2 weeks of age with a secondary down, 3) growth of adult plumage just prior to fledging. The secondary down was the sampling focus in this study, as it is derived from prey items taken by the parents and fed to the chicks. Once ingested, organic mercury circulating through the bloodstream associates with keratin disulfide bonds during feather synthesis which subsequently incorporates mercury directly in the feather structure (Appelquist et al., 1984; Bortolotti, 2010; Furness et al., 1986). Because parental foraging occurs near the natal colony (Handley et al., 2017), this secondary chick down reflects a comparatively localized signal of mercury exposure over a near-term temporal scale. In addition, chick down is grown simultaneously, therefore all intra-individual feathers are expected to have low variation in mercury concentration (Brasso et al., 2013). As such, penguin chick secondary down is ideally suited as a mercury biomonitoring tissue.
15


Live Bird Sampling Methods
Due to logistical constraints, live birds were only collected at three sites in the Falkland Islands and were obtained under permit from the Falkland Islands Government (#R22/2017), with landowner permission, and using protocols approved by University of Colorado Denver IACUC (#92517(08) 1C). Each colony supported -2000+ breeding pairs of Gentoo penguins. Gentoo penguin chicks in the post-guard phase (i.e. no longer protected by the parents) were captured using a long-handled net. Down feathers (10-12) were plucked from the breast using a pair of silicone-tipped forceps (DDP Instruments young tongue forceps, Miami, FL, USA) and stored at ambient temperature in small resealable plastic bags. Each chick was marked with a small amount of feather paint to prevent duplicate sampling (Sporting Pigeons Paint - green, Pigeon Products International, Post Falls, ID, USA) and then released.
Dead Bird Sampling Methods
Dead Gentoo chicks were collected at the Falkland Islands under permit from the Falkland Islands Government (#R22/2017) and with landowner permission, from South Georgia Island under permit from the Government of South Georgia and the South Sandwich Islands (#2017/044), and from the Antarctic Peninsula under permit from the National Science Foundation (#ACA 2018-003). We found dead Gentoo penguin chicks by searching within each colony boundary (Falkland Islands only) and along transects extending -100 m from the colony boundary to find carcasses that may have been moved by predators or scavengers. We used only birds that had died during the study season (as evidenced by presence of blood and/or comparative lack of decomposition, bleaching and weathering). We collected secondary down
16


from the breast of intact carcasses when available, or otherwise from the legs just above the foot, sampling only white feathers using the same methods as detailed for live birds.
Mercury Analysis
All feather samples were sterilized per USD A/APHIS requirements (as detailed on the importation permit) for importation into the United States. Sterilization procedure required soaking the feather samples in 70% ethanol (LabChem, Zelienople, PA, USA) for 30 minutes. Following sterilization, we triple rinsed feather samples in deionized water and dried the samples under a fume hood for 24-48 hours at room temperature until all external moisture was removed.
In preparation for mercury analysis, the feathers were cleaned to remove exogenously deposited contaminants. The feather samples were placed in a stainless steel screen and vigorously cleaned in environmental grade acetone (Alfa Aesar, Ward Hill, MA, USA) followed by rinsing under a flowing stream of deionized water. We completed this procedure three times for each feather sample to ensure all external oils and contaminates were removed. The feather samples were then dried in a fume hood at room temperature for 24 - 48 hours until all external moisture was removed.
Approximately 10 mg of whole down feathers from each individual was analyzed for total mercury via atomic absorption spectrophotometry on a Nippon MA-3000 Direct Mercury Analyzer at Weber State University (Ogden, UT, USA). Each set of 20 samples analyzed was preceded and followed by two samples of a standard reference material (TORT-3, National Resource Council Canada). Mean percent recovery for the standard reference material was 101.4% (n=22) with relative significant differences in mercury concentration of 6.9%. Detection limit = 0.015 ng. Mercury concentrations in chick down are reported as mg/kg fresh weight (fw).
17


Stable isotope analysis
We used feather samples which had been previously cleaned and dried during preparation for mercury analysis as detailed above. We transferred approximately 1 mg of feather down material to 4 x 6 mm tin capsules and crimp-sealed the capsules. Feather samples were analyzed by continuous flow-isotope ratio mass spectrometry using a Carlo Erba NCI500 interfaced to a Micromass Optima mass spectrometer following established methods (Fry et al., 1992). Isotopic data were expressed in delta notation after normalizing to Air and V-PDB using the primary standards USGS 40 (<515N = -4.52 %o, <513C = -26.39 %o) and USGS 41a (<515N = 47.55 %o, <513C =
36.55 %o). Accuracy was assessed by analyzing USGS 40 as an unknown (+/- 0.1 for both isotopes). Analytical error was assessed by USGS 40, 41a, and an internal standard (+/- <0.2 %o for both isotopes).
Statistical analysis
We removed any data points below detection limits decreasing our total sample size from 169 to 166. All plots and statistical analyses were completed in R (R Core Team 2018). Mercury data were log transformed where appropriate to approximate a normal distribution. We compared feather mercury concentration means among the three regions using Welch ANOVA testing for unequal variances (as tested by Fligner-Killeen test for homogeneity of variances) and followed the ANOVA model with Tukey HSD post hoc testing for pairwise comparisons. We fit linear regression of mercury on <515N (as a proxy for diet) to estimate how variance in mean mercury
changes as a function of diet (Atwell et al., 1998; Chen et al., 2008). Maps were created with QGIS (QGIS Development Team, 2019) using the Quantarctica package (Matsuoka et al., 2018).
18


8-
6-
Region
AP ] SGI
m-
0
T3
0-
2-
0
2
3
4
Feather Hg (mg kg )
Fig. 2 Gentoo chick feather mercury concentration distributions for each region in the study, Antarctic Peninsula (AP), South Georgia Island (SGI) and Falkland Islands (FI).
Results
We collected samples from a total of 105 birds from the Falkland Islands, 23 birds from South Georgia Island, and 41 birds from the Antarctic Peninsula during the sampling period. Mean feather mercury concentrations with range in parentheses (mg/kg) are as follows: Falkland Islands: 1.583 mg/kg (0.726-4.739); South Georgia Island: 0.437 mg/kg (0.236-0.751); Antarctic Peninsula: 0.106 mg/kg (0.032-0.249). We found that in general, feather mercury concentrations of Gentoo penguins were higher in sub-Antarctic populations than the Antarctic Peninsula population. Additionally, distributions of Gentoo feather mercury concentrations were different
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Region • AP a SGI FI
10.0 12.5 15.0
S15 N (%o)
Fig. 3 Carbon and nitrogen (<513C and <515N) stable isotope value (%o) relationships for Gentoo
chick feather mercury concentrations at the Antarctic Peninsula (AP), South Georgia Island
(SGI) and Falkland Islands (FI).
between each individual region (F2.i63 = 225.2, p = 2xl016; Fig. 2), with virtually no overlap in distributions between the Falkland Islands and the Antarctic Peninsula. Specifically:
FalklandIslcmdsmeanHg > South Georgia IslcmdmeanHg > Antarctic PeninsulameanHg (Falkland Islands and S. Georgia Islandp < lxlO14; S. Georgia Island and Antarctic Peninsulap = 0.0058).
Our analysis of Gentoo feather stable isotopes indicates there is no overlap within the C and N delta space between penguins at any of the regions, suggesting each population is interacting with the food web differently or with different food webs entirely (Fig. 3; Jennings & van der Molin, 2015; Kelly, 2000; Perkins et al., 2014). Delta13C follows predictable latitudinal changes among the regions (Fig. 3). Mercury concentrations at each region are distinct from each
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Region • AP a SGI FI
Fig. 4 Linear regression model of sub-Antarctic (dashed line) and Antarctic (solid line) Gentoo chick feather mercury (Hg) concentration relationship with <515N stable nitrogen isotope values
(%o) as a proxy for trophic level. Individual regions are Antarctic Peninsula (AP), South Georgia Island (SGI), and Falkland Islands (FI).
other as shown by clustering among regions in <515N:Hg space (Fig. 4). Based on similar slopes
of the two regression lines, we point to consistent mercury biomagnification in both regions (Chen et al., 2008). The Falkland Island birds are foraging at a higher trophic level than the South Georgia Birds, however both of these sub-Antarctic populations have a similar relationship between mercury loading and trophic level as evidenced by their position on the same regression line. Due to similar <515N values of primary producers at each region (Table 1),
we conclude that the <515N bases are not likely meaningfully different between the regions.
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Prey
0 Cephalopod 0 Crustacean 0 Fish
Region • AP A SGI ■ FI
Fig. 5 Carbon and nitrogen (<513C and <515N) stable isotope value (%o) relationships for
representative Gentoo penguin prey species for Antarctic Peninsula (AP), South Georgia Island (SGI) and Falkland Islands (FI).
Therefore, we provide evidence that the baseline for mercury bioavailability is higher in the sub-Antarctic than the Antarctic Peninsula as indicated by the change in y-intercept between the two regression lines (Chen et al., 2008).
Discussion
To explore differences in mercury exposure among regions, we compiled published data on Gentoo penguin foraging ecology and prey composition (Fig. 1) to examine differences in foraging ecology, and stable isotope values for representative prey species to compare baseline prey 15N enrichment (Fig. 5). We compiled prey energy densities to indicate potential prey
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Table 1 Summary of energy density (kJ g-1 dry weight) for representative Gentoo penguin prey species. Patagonia samples are substituted for Falkland Island samples for which we could not find data.
Prey Spp Region Prey Type Energy Density Author dw (kJ g-1)
Chaenocephalus aceratus South Georgia Island Fish 25.4 Vanella et al., 2005
Champsocephalus gunnari South Georgia Island Fish 22.5 Vanella et al., 2005
Electrona antarctica South Georgia Island Fish 25.5 Donnelly et al., 1990
Electrona antarctica South Georgia Island Fish 29.4 Van de Putte et al., 2006
Electrona antarctica Antarctic Peninsula Fish 31.9 Ruck etal., 2014
Electrona carlsbergi South Georgia Island Fish 22.8 Clarke & Prince, 1980
Electrona carlsbergi South Georgia Island Fish 23.5 Cherel & Ridoux, 1992
Euphausia crystallorophias Antarctic Peninsula Crustacean 21.8 Ruck etal., 2014
Euphausia lucens Patagonia Crustacean 18.2 Ciancio et al., 2007
Euphausia superba South Georgia Island Crustacean 15.2 Torres et al., 1994
Euphausia superba Antarctic Peninsula Crustacean 21.1 Ruck etal., 2014
Euphausia superba Antarctic Peninsula Crustacean 17.1 Ichii et al., 2007
Euphausia superba South Georgia Island Crustacean 22.7 Clarke, 1980
Gobionotothen gibberifrons South Georgia Island Fish 21.2 Vanella et al., 2005
Krefftichthys anderssoni South Georgia Island Fish 26.4 Cherel & Ridoux, 1992
Loligo gahi South Georgia Island Cephalopod 16.2 Croxall & Prince, 1982
Loligo gahi Patagonia Cephalopod 21.16 Ciancio et al., 2007
Micromesistius australis Patagonia Fish 21.13 Ciancio et al., 2007
Munida gregaria Patagonia Crustacean 11 Ciancio et al., 2007
Munida spinosa Patagonia Crustacean 19.96 Lovrich et al., 2005
Munida subrugosa Patagonia Crustacean 17.42 Lovrich et al., 2005
Munida subrugosa Patagonia Crustacean 20.26 Lovrich et al., 2005
Pleuragramma antarcticum Antarctic Peninsula Fish 24.6 Ruck etal., 2014
Pseudochaenichthys georgianus South Georgia Island Fish 22.75 Vanella et al., 2005
Sprattus fuegensis Patagonia Fish 24.06 Ciancio et al., 2007
Themisto gaudichaudii Antarctic Peninsula Crustacean 12.7 Torres et al., 1994
Themisto gaudichaudii Patagonia Crustacean 22.19 Ciancio et al., 2007
Thysanoessa macrura Antarctic Peninsula Crustacean 28.5 Ruck etal., 2014
Thysanoessa macrura South Georgia Island Crustacean 17.03 Torres et al., 1994
Thysanoessa macrura Antarctic Peninsula Crustacean 16.1 Torres et al., 1994
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Table 2 Summary of mercury (Hg) concentrations (mg/kg) for representative baseline prey species of Gentoo penguins. Hg concentration includes standard deviation where reported. CV = coefficient of variation. AP = Antarctic Peninsula, SGI = South Georgia Island.
Prey Spp Regio n Prey Type Hg Concentration (mg/kg) Hg CV Author
Bovallia gigantea AP Crustacean 0.0364 - Santos et al., 2005
Champsocephalus gunnari SGI Fish 0.02 ±0.01 46.02 Anderson et al., 2009
Cheirimedon femoratus AP Crustacean 0.035 - Santos et al., 2005
Dissostichus eleginoides SGI Fish 0.05 ±0.02 43.3 Anderson et al., 2009
Electrona antarctica AP Mesopelagic fish 0.031 ±0.017 53.31 Polito et al., 2016
Euphausia superba AP Crustacean 0.002 ±.001 34.74 Polito et al., 2016
Euphausia superba SGI Crustacean 0.01 ±.01 67.08 Anderson et al., 2009
Euphausia superba AP Crustacean 0.0346 - Santos et al., 2005
Geotria australis SGI Fish 0.04 ± .04 87.08 Anderson et al., 2009
Gonatus antarcticus SGI Cephalopod 0.6 ±0.02 3.57 Anderson et al., 2009
Gondogeneia antarctica AP Crustacean 0.037 - Santos et al., 2005
Lepidonotothen larseni SGI Fish 0.27 ± 0.04 14.81 Anderson et al., 2009
Lepidonotothen squamifons AP Benthic fish 0.041 ±.21 50.37 Polito et al., 2016
Parachaenichthys georgianus SGI Fish 0.08 - Anderson et al., 2009
Patagonotothen guntheri SGI Fish 0.03 ±.01 41.72 Anderson et al., 2009
Pleuragramma antarcticum AP Epipelagic fish 0.008 ± .002 22.85 Polito et al., 2016
Pseudochaenichthys georgianus SGI Fish 0.02 ±.01 49.79 Anderson et al., 2009
Psychroteuthis glacialis SGI Cephalopod 0.18 ± .11 60.61 Anderson et al., 2009
Themisto gaudichaudii SGI Crustacean 0.02 ±.01 48.45 Anderson et al., 2009
selection with respect to optimal foraging strategy (Table 1; Clarke, 2001). Additionally, we
include available prey mercury data in an attempt to quantify potential differences in baseline mercury bioavailability (Table 2). In our search of the literature, however, we were unable to find any published data on Gentoo prey species mercury loads in the Falkland Islands.
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Foraging ecology of Gentoo penguins
Penguin foraging behavior is not well understood due to the cryptic nature of this group of seabirds in the marine environment. However, details on Gentoo foraging ecology come from previous studies employing stomach sampling, data loggers and video cameras to help understand snapshots of foraging behavior (Carpenter-Kling et al., 2017; Handley et al., 2018; Lescroel & Bost, 2005; Miller et al., 2009; Robinson & Hindell, 1996; Takahashi et al., 2008; Volkman et al., 1980) and used in combination with isotopic mixing models to estimate diet mixtures (Polito et al., 2016). This diet approximation helps standardize trophic level of the birds and its subsequent effects on mercury biomagnification in each population. The chick meal consists entirely of parental prey captures that are regurgitated and fed to the chick without the addition of stomach oil (Roby et al., 1989). Therefore, chicks are only exposed to dietary contaminants which are foraged by the adults in their foraging range (Bearhop et al., 2000).
Most species of penguins appear to exploit some level of opportunism, a likely necessity when a species lives in an extreme climate to be able to take advantage of prey abundance while it is available (Kaspari & Joem, 1993). Gentoos in particular have also been noted as generalist foragers, consuming any prey item within their foraging range (Handley et al., 2017; Herman et al., 2017; Polito et al., 2015; Waluda et al., 2017). This foraging strategy is well-documented in various diet studies investigating Gentoo prey composition through stomach sampling (Croxall et al., 1988; Handley et al., 2017; Miller et al., 2009; Piitz et al., 2001; Robinson & Hindell, 1996; Volkman et al., 1980; Xavier et al., 2017). Previous studies have recorded substantial differences in primary prey types (crustacean, cephalopod, fish; Fig. 1) of Gentoos at different colonies throughout the Falkland Islands in both the same year and across years (Clausen & Piitz, 2002;
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Handley et al., 2017; Piitz et al., 2001). Generalism is also reported in multi-year diet studies along the Antarctic Peninsula as well, although with a strong preference toward krill (Miller et al., 2009; Polito et al., 2016).
Horizontal foraging range of Gentoos is reported by several GPS tracking studies that Gentoos rarely venture farther than 30 km from shore during foraging trips (Boersma et al.,
2002; Carpenter-Kling et al., 2017; Kokubun et al., 2009; Lescroel & Bost, 2005; Robinson & Hindell, 1996; Trivelpiece et al., 1986; Wilson et al., 1998). This range suggests that Gentoos preferentially remain over the continental shelf based on bathymetry near their breeding sites (Carpenter-Kling et al., 2017; Handley et al., 2015). Gentoo winter foraging patterns are still relatively unknown, although camera monitoring studies and GPS tracking suggest that Gentoos remain nearby their breeding colonies throughout the winter (Black, et al., 2017; Tanton et al., 2004). Breeding season foraging behavior in particular is of interest to our study because Gentoo chick feather tissue is derived entirely from local prey resources provisioned by the parents (Handley et al., 2015).
Vertical foraging patterns within the water column are more specific, however, with Gentoos preferring benthic zones as indicated by diving studies (Carpenter-Kling et al., 2017; Kokubun et al., 2009; Takahashi et al., 2008). Video cameras mounted on birds and stomach contents analyses confirm that Gentoo prey typically reside in the benthic zone (Handley et al., 2018; Robinson & Hindell, 1996), although stomach contents have been recovered that indicate Gentoos are also successful in mid-water and epipelagic zones as well, but in much smaller proportions (Handley et al., 2015; Lescroel et al., 2004). This foraging preference within the water column locates Gentoos generally deeper than most sympatric penguins which forage in
26


mid-water and epipelagic zones (Kokubun et al., 2009; Miller et al., 2009; Trivelpiece et al., 1987). Dive and travel distance records also suggest that penguins which forage farther offshore perform shallower dives (Ludynia et al., 2012), in contrast to the Gentoo inshore benthic preference. Gentoo penguins have the deepest recorded dives of any of the small-bodied penguins (Wilson, 1998), with numerous max recorded dives near 200 m (Carpenter-Kling et al., 2017; Lescroel & Bost, 2005; Robinson & Hindell, 1996). This deep diving behavior is suggested to be a function of the Gentoo’s larger mass and body size than sympatrics, except the large Aptenodytes spp, King and Emperor penguins which forage in deep waters far offshore (Bost et al., 1997; Ratcliffe et al., 2014; Trivelpiece et al., 1987; Volkman et al., 1980; Xavier et al., 2017). The benthic preference of Gentoos is likely a mechanism to differentiate resource exploitation by avoiding overlapping dive profiles with most sympatric penguin species and thereby decreasing competition (Cimino et al., 2016; Kokubun et al., 2009; Trivelpiece et al., 1987). This interspecific segregation combined with generalist foraging preferences likely plays a large role in the breeding success of Gentoos throughout a wide range of climates (Borboroglu & Boersma, 2013; Clucas et al., 2014; Lynch et al., 2008; Miller et al., 2009; Polito et al., 2015). Gentoo prey composition
The three general classifications of penguin prey include crustaceans, cephalopods and fish, and different patterns of prey selection can expose a predator to different levels of bioaccumulating toxicants. The opportunistic/generalist foraging strategy of Gentoos is well pronounced across the three regions in our study (Fig. 1). Although most of the data is from stomach sampling which only provides snapshots of a penguin’s foraging behavior, in general Gentoos in the Falkland Islands are eating a substantially higher proportion of fish than
27


crustaceans in their diet, while Antarctic Peninsula Gentoos are foraging on a larger proportion of krill (Fig. 1).
Diet studies in the Falkland Islands point to widely fluctuating diet compositions of Gentoos in that region. Stomach sampling conducted over multiple years in the Falkland Islands reported proportions of 30% to >95% fish by mass across a 10-year span (Putz et al., 2001) and 65% to >95% fish by mass across 4 years in a different study (Handley et al., 2015). Crustacean proportions range from <2% - 44% by mass. Cephalopods were found in similar amounts of <2% - 31% by mass (Clausen et al., 2005; Handley et al., 2015; Putz et al., 2001). Clausen et al., (2005) suggest that trawling studies of Gentoo foraging sites in the Falkland Islands demonstrate that the birds will preferentially select fish over crustaceans when both are available. The Antarctic Peninsula, alternatively, has much more consistently demonstrated Gentoos as preferentially selecting not only crustaceans as their primary prey, but more specifically krill as the most important diet component. South Georgia Island shows similar year-over-year variability to that of the Falkland Island populations, with a generally higher preference toward crustaceans than fish. South Georgia crustacean prey composition varies widely from 25-68% by mass, with fish composition ranging from 25-50% by mass. Cephalopods make up a minimal proportion of the Gentoo diet at South Georgia Island, ranging from negligible amounts in some years to 12% by mass (Fig. 1; Waluda et al., 2017; Xavier et al., 2017).
Among all prey species, Gentoos along the Antarctic Peninsula strongly prefer krill (-60-80% by mass), most commonly Antarctic krill Euphausia superba and amphipods Themisto gaudichaudii over any other prey type. Fish components, while a smaller proportion of the diet along the Antarctic Peninsula, indicate a strong preference for benthic fish over mid-water and
28


epipelagic species (Dimitrijevic et al., 2018; Kokubun et al., 2009; Miller et al., 2009; Polito et al., 2016). For diet studies at South Georgia Island, crustacean prey are overwhelmingly dominated by the amphipod T. gaudichaudii while the bulk of fish species are benthic residents. Cephalopod specimens found in stomach contents analyses are likewise largely benthic squid species, commonly juveniles (Handley et al., 2015; Waluda et al., 2017; Xavier et al., 2017). In the Falkland Islands, Gentoo crustacean prey consists of only negligible amounts of krill, comprising less than 1% of the diet with the bulk of crustaceans most commonly bottom dwelling squat lobster Munida spp (Handley et al., 2015). Reported fish prey includes mostly benthic species and temporary juvenile demersal residents. Despite the large variability in prey among regions, these diet proportions indicate a strong benthic inshore foraging preference coupled with opportunistic foraging patterns in response to prey abundance at each region (Barrera-Oro et al., 2002; Kaspari & Joern, 1993; Lescroel et al., 2004; Polito et al., 2015; Polito etal., 2016; Williams, 1991).
Energy densities and Gentoo prey selection
As generalists, Gentoo penguins can readily exploit numerous prey species within their foraging range so long as it offsets the energy required for capture (Clarke, 2001; Mori & Boyd, 2004). Although studies demonstrate that krill energy densities are typically lower than most Gentoo fish prey (Table 1), variations by season, region, age status and gravid status of the female (Hartman & Brandt, 1995; Van de Putte et al., 2006) can cause seasonal increases in krill lipid content that approach or surpass energy content of many fish. Similarly, larger prey within the same species are thought to yield higher energy content (Tierney et al., 2002), making energy transfer through the food web difficult to predict for a generalist forager. Clarke & Prince (1980)
29


noted this inconsistency in their early studies on the subject when they measured squid calorific values from the same site as ranging from 3.27 - 6.62 kJ g-1 wet weight. However, bioenergetics models on potential prey items indicate in general terms that fish prey offer the highest energy density of the reported penguin prey species (Table 1).
The benefit of higher energy fish is exploited by Gentoos throughout the Falkland Islands and South Georgia Island. Different strategies predominate throughout the Antarctic Peninsula, however, where krill make up the largest proportion of Gentoo diet by mass. For a consumer to select such a lower energy dense prey is likely attributed to abundance of krill, lack of fish prey, or competition for resources. Because the major sympatric competitors are krill specialists, we expected Gentoos as generalists to exploit the higher energy density and decreased competition associated with fish-based foraging, in particular for a consumer that reportedly prefers fish over crustacean prey when both are available (Clausen et al., 2005). However, due to the population growth of Gentoos along the Antarctic Peninsula in recent years (Clucas et al., 2014), krill abundance is the most parsimonious explanation for krill selection by Gentoos in this region. High krill abundance throughout the Southern Ocean is proposed to be a result of the massive whaling in the early 20th century removing the major krill consumers from the region (Emslie et al., 2013). Numerous seabirds subsequently fill this gap and thrive off the abundance of low energy, readily available prey. The exploitation of a low energy prey is an effective strategy of numerous baleen whales that choose quantity over quality to become some of the most massive animals on the planet (Dolphin, 1987; Goldbogen et al., 2010). Similarly, low-energy krill in the Antarctic are a desirable prey for Gentoos who benefit from the high quantities coupled with increased foraging efficiency in dense krill swarms (Hazen et al., 2015).
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Certain inshore areas along the Antarctic Peninsula specifically favor the accumulation of large-scale krill aggregations and these hotspots are believed to be the exclusive food source for some penguin populations (Bernard et al., 2017). The major krill species along the Antarctic Peninsula, E. superba, is described as the most important component of the Southern Ocean food web (Lea et al., 2002), further suggesting that fish are a secondary prey component in many vertebrate consumers in the region (Bost et al. 1997; Cherel et al. 1994; Croxall et al., 1988;
Hull, 1999; Raclot et al. 1998). The constraints placed on an inshore benthic forager, therefore, and in particular along the Antarctic Peninsula which has less biodiversity (Barrera-Oro, 2002), may subject Gentoos to a more homogeneous prey mix with fewer fish and cephalopod prey sources available, suggesting krill are more frequently encountered by the consumer. In the Falkland Islands and South Georgia Island, heterogeneous mixtures of inshore benthic prey provide Gentoos a greater choice of prey selection, allowing these consumers to reportedly bypass abundant crustaceans and select fish prey of higher energy content instead (Clausen et al., 2005), suggesting a possible deliberate choice made by Gentoos of energy density over prey abundance in some regions.
Stable isotope analyses of Gentoo feathers and prey
Our Gentoo feather stable isotope analyses indicate that Gentoo penguins interact with the food web differently at each region (Fig. 4), likely a function of their propensity for generalist foraging strategy combined with plasticity at each region. While differences in <513C
can be attributed to spatial differences in foraging, <515N approximates trophic level at which the
consumer is foraging. Delta13C values are complex in the marine environment, however. Higher latitude regions, enhanced by the cold environment around Antarctica, lead to depletion in 13C,
31


but so too does distance from shore and height in the water column (Cherel et al., 2000; Cherel & Hobson 2007; Kelly, 2000). Because 13C depletion with increasing latitude is dynamic, we point to changes in carbon isotopes among the study regions as a confounding mix of differences in foraging strategy and latitude (Anderson et al., 2009). We attribute the 2-3° increase in latitude from the Falkland Islands to South Georgia Island, and the additional 8-10° farther south latitude to the Antarctic Peninsula as a large component of our reported 13C decline across the C:N space (Fig. 3). Gentoos as benthic foragers should tend toward increased enrichment of 13C, however foraging on mid-water krill along the Antarctic Peninsula likely depletes 13C in those birds more than just from higher latitude alone. Because the Falkland Islands and South Georgia Island are at closer latitudes, we cautiously suggest that this relatively large difference between these two locations in regard to <513C (Fig. 3) is primarily due to differences in foraging strategy with a
slight latitudinal component.
Comparisons of a biomagnifying toxicant such as mercury require standardization of trophic level estimations due to differences in nitrogen assimilation (Jennings & van der Molen, 2015). As such, we analyzed <515N in Gentoo baseline prey species for each region (Fig. 5; Table
1). Isotopic values from baseline prey E. superba and T. gaudichaudii indicate these primary producers are located in similar C:N space in all three of our study regions (Anderson et al.,
2009; Ciancio et al., 2010; Dunton et al., 2001; Handley et al., 2017; Polito et al., 2016; Rey et al., 2012). This similarity in baseline prey isotopic space allows for standardized comparisons in foraging ecology of Gentoos between the regions (Chen et al., 2008; Jennings & van der Molin, 2015; Perkins et al., 2014). Nitrogen enrichment generally increases with trophic level foraging (Anderson et al., 2009; Atwell et al., 1998; Bearhop et al., 2000; Wada et al., 1987), and because
32


the bases of the food webs are assimilating 15N similarly, we conclude that the Falkland Island birds are interacting with the food web at higher trophic levels than the other two regions. This higher trophic foraging points to a higher proportion of fish consumed in the Falkland Islands in relation to South Georgia Island and, more so, than the Antarctic Peninsula at which Gentoos appear to be exploiting higher amounts of krill in their diet. Likewise, numerous studies have reported positive linear correlations between trophic level and mercury loads (Atwell et al.,
1998; Bearhop et al., 2000; Santos et al., 2005; Polito et al., 2016; Seixas et al., 2014). Due to biomagnification of mercury, the higher trophic fish in the diet of the Falkland Islands birds would therefore be expected to have increased mercury concentrations in their tissues as we report here (Fig. 3). However these biomagnifying effects of trophic level alone do not explain the regional shift in the mercury base (Fig. 4).
Mercury levels of Gentoo feathers and prey
Our regression analysis indicates that both the Falkland Islands and South Georgia Island populations have the same trophic level :mercury ratio (Fig. 4), suggesting that both of these populations have similar environmental exposure to mercury. The disparity in feather mercury concentrations between these two regions is thus a function of foraging at different trophic levels. A clear difference exists between the sub-Antarctic and the Antarctic Peninsula trophic level:mercury ratios, however, which indicates a shift in mercury uptake that is likely not accounted for by differences in dietary exposure, but instead by differences in environmental baseline mercury (Chen et al., 2008).
A clear distinction exists in our data in Gentoo feather mercury levels between each region. Mercury has been demonstrated to biomagnify across the food web as well as to
33


bioaccumulate with age of an organism (Fitzgerald et al., 2007). Differences in mercury loading can therefore be caused by differences in foraging behavior as well as differences in environmental mercury exposure. Studies have shown a positive correlation between mercury exposure and trophic level, in agreement with our data. Because Gentoos in the Falkland Islands are foraging at a higher trophic level than in either South Georgia Island or the Antarctic Peninsula (Fig. 4), biomagnification can be one source of increased mercury loading in Gentoos. Additionally, the benthic zone is enriched in methylmercury, the bioavailable form of mercury, due to deposition from microbial mercury methylation which takes place in the deeper, nutrient-rich benthic waters, making marine sediments a sink for methylmercury (Cossa et al., 2011; Eagles-Smith et al., 2008; Fitzgerald et al., 2007). Therefore, bioavailable mercury is more available in the food web in the mesopelagic and benthic zones, subjecting consumers in these deeper layers to higher mercury exposure (Chen et al., 2008; Fitzgerald et al., 2007; Monteiro et al., 1996). Reports concur that benthic penguin prey show higher mercury concentrations than epipelagic prey (Polito et al., 2016; Thompson et al., 1998). Falkland Islands and South Georgia Island Gentoos foraging on benthic fish and crustaceans therefore consume a larger percentage of mercury-laden prey than the krill-foraging Gentoos of the Antarctic Peninsula (Cossa & Gobeil, 2000). Krill ecology studies throughout South Georgia Island and the Antarctic Peninsula report that inshore krill remain in the epipelagic zone during the day when Gentoos primarily forage (Cleary et al., 2016; Cresswell et al., 2009), pointing to lower mercury uptake.
Because large differences in prey composition can affect mercury exposure (Thompson et al., 1998), foraging ecology cannot be denied a role in the higher mercury exposure associated with the Falkland Islands and South Georgia Island Gentoos in relation to the Antarctic Peninsula
34


birds. Our evidence points also to increased baseline mercury in those regions due to the shift in
the <515N:Hg ratios (Fig. 4). Although we were able to find baseline mercury data for prey species
at South Georgia Island and the Antarctic Peninsula, we could not find any published reports on penguin prey mercury data in the Falkland Islands with which to compare mercury baselines (Table 2). Without this data, one possible explanation for the baseline shift in mercury at South Georgia Island in particular can be attributed to its geographic location at the edge of the Antarctic Divergence along the Scotia Arc which causes high turbulence and deep ocean water upwelling (Hunt et al., 2016). This explanation, however, does not point to increased mercury availability in the Falkland Islands which is located 1500 km upstream from South Georgia Island on the shallower Patagonia Shelf. Global inputs of mercury pollution are likely impeded from entering the Antarctic due to the action of the Antarctic Circumpolar Current as a barrier to external input (Bargagli, 2005), suggesting global pollution elevations may be one cause for the mercury baseline shift outside of the Antarctic. Further, it is possible that Antarctic Peninsula Gentoos are feeding on more mid-water prey and have less mercury exposure than would benthic foragers. However, we contend that the different feather mercury concentrations in Gentoo penguins at the three regions are explained by a combination of differences in foraging behavior coupled with a likely higher level of ambient environmental mercury north of the Antarctic Circumpolar Current, potentially from global pollution.
Conclusion
Our results indicate higher feather mercury concentrations in Gentoo penguins in sub-Antarctic regions as compared to the Antarctic Peninsula. This disparity in mercury loading between regions is likely explained by both differences in foraging ecology as well as potentially
35


increased baseline mercury in the marine system around the Falkland Islands and South Georgia Island. Because our mercury levels coincide with previous mercury studies on penguins throughout the Antarctic Peninsula, it appears that mercury levels in that region are stable. These novel data from the Falkland Islands requires further analysis, however, to understand why these levels are higher than the baseline levels of Antarctica. To explore this mechanism further, we recommend future studies are needed to investigate biomagnification factors and methylation potential of mercury in the food webs of these regions. In particular, primary producer mercury concentrations are needed in these regions for baseline comparisons. Additionally, baseline isotopic studies in the Falkland Islands would help understand mercury biomagnification factors as well as species-specific discrimination factors to help improve isotopic mixing models in this understudied region.
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Vega, C. M., Siciliano, S., Barrocas, P. R., Hacon, S. S., Campos, R. C., Jacob, S. D., & Ott, P. H. (2009). Levels of Cadmium, Mercury, and Lead in Magellanic Penguins (Spheniscus magellanicus) Stranded on the Brazilian Coast. Archives of Environmental Contamination and Toxicology, 58(2), 460-468. doi:10.1007/s00244-009-9349-0
Volkman, N. J., Presler, P, & Trivelpiece, W. (1980). Diets of Pygoscelid Penguins at King George Island, Antarctica. The Condor, 82(4), 373. doi: 10.2307/1367558
Wada, E., Terazaki, M., Kabaya, Y., & Nemoto, T. (1987). 15N and 13C abundances in the Antarctic Ocean with emphasis on the biogeochemical structure of the food web. Deep Sea Research Part A. Oceanographic Research Papers, 34(5-6), 829-841. doi:
10.1016/0198-0149(87)9003 9-2
Waluda, C. M., Hill, S. L., Peat, H. J., & Trathan, P. N. (2017). Long-term variability in the diet and reproductive performance of penguins at Bird Island, South Georgia. Marine Biology,
164(3). doi: 10.1007/s00227-016-3067-8
Williams, T. D. (1991). Foraging ecology and diet of Gentoo Penguins Pygoscelispapua at South Georgia during winter and an assessment of their winter prey consumption. Ibis, 133(1), 3-13. doi:10.1111/j. 1474-919x.l99l.tb04803.x
Williams, T. D., & Rodwell, S. (1992). Annual Variation in Return Rate, Mate and Nest-Site Fidelity in Breeding Gentoo and Macaroni Penguins. The Condor, 94(3), 636-645. doi: 10.2307/1369249
47


Wilson, R. R, Alvarrez, B., Latorre, L., Adelung, D., Culik, B., & Bannasch, R. (1998). The movements of gentoo penguins Pygoscelispapua from Ardley Island, Antarctica. Polar Biology, 19(6), 407-413. doi: 10.1007/s003000050266
Wilson, R. P., & McMahon, C. R. (2006). Measuring devices on wild animals: What constitutes acceptable practice? Frontiers in Ecology and the Environment, 4(3), 147-154. doi:
10.1890/1540-9295(2006)004[0147:mdowaw]2.0.co;2
Xavier, J. C., Trathan, P. N., Ceia, R R., Tarling, G. A., Adlard, S., Fox, D., . . . Cherel, Y. (2017). Sexual and individual foraging segregation in Gentoo penguins Pygoscelis papua from the Southern Ocean during an abnormal winter. Plos One, 12(3). doi: 10.1371/journal.pone.0174850
48


APPENDIX
NATIONAL SCIENCE FOUNDATION OFFICE OF POLAR PROGRAMS
ANTARCTIC CONSERVATION ACT PERMIT
Permit Number: ACA 2018-003
Effective Dates: November 6, 2017 -June 1, 2018
Permit Holder: David Schutt, Penguins International, PO Box 100483, Denver, CO 80250
Location: West Antarctic Peninsula region including South Orkney Islands, Elephant Island, South Shetland Islands
Permitted Activities: Harmful Interference; Import Into USA. The permit holder may collect samples of feathers (molted or plucked from carcasses), guano, and fragments from discarded eggshells of gentoo penguins, Pygoscelis papua, for use in a study of pollutant exposure in penguins across a wide latitudinal gradient, as described in the attached permit application. The permit holder may collect samples opportunistically from the ground or from penguin carcasses from outside, the periphery of, and/or low-density areas of the colonies in order to minimize disturbance to the penguins. The samples may be imported into the USA and analyzed at the home institution.
Definition of "permit". The "permit" consists of the permit application dated August 1,2017, revised November 6, 2017, submitted by David Schutt, Attachment 1, as amended or supplemented by the permit and associated conditions signed by the Director, NSF, Office of Polar Programs.
PERMIT CONDITIONS: The following permit conditions are incorporated by reference.
1. Possession of permit. A copy of this permit must be carried by the permit holder when conducting any permitted activity.
2. Right of entry and inspection. Any NSF employee, contractor, or agent designated by the Director, Office of Polar Programs; an Antarctic Conservation Act Enforcement Officer; or an NSF Representative may inspect any permitted activity and records related thereto to verify compliance with permit conditions.
3. Permit Agents. The permit holder is required to submit a list of his agents to the Permit Office by September 15th of each year during the course of this permit.
4. Annual summary report. A written report on activities conducted, and on the environmental conditions at the site(s) visited under this permit, must be submitted to the Permit Officer by April 1st of each year for the preceding 12-month period ending March 31, throughout the term of the permit. For fauna and flora permits, reports must include the number of animals taken and activities performed on them, and for plants the quantities taken.
5. Sample Collection Restrictions. The permit holder shall only collect samples from areas outside of, in the periphery of, and/or in low-density areas of gentoo penguin colonies in order to minimize disturbance to the penguins, especially nesting birds. The permit holder shall avoid taking of any wildlife, including penguins.
6. Violations. Violation of the Antarctic Conservation Act, its implementing regulations, or the terms or conditions of this permit, may result in civil fines of up to $27,950 for each occurrence, criminal fines and imprisonment for up to one year under the Act, and administrative penalties up to and including debarment. Further, violations of any condition of this permit by the permit holder may result in the exercise of NSF's discretion to refuse to issue future permits under the Antarctic Conservation Act to the permit holder.
November 6, 2017
Dr. Polly A. Penhale, Environmental Officer Office of Polar Programs
Date
49


FORM APPROVED OMB NO. 3145-0034 Expires:October 31, 2019
NATIONAL SCIENCE FOUNDATION ARLINGTON, VIRGINIA 22230
ANTARCTIC CONSERVATION ACT APPLICATION AND PERMIT FORM
PROPOSAL NO.
1. TYPE OF PERMIT REQUESTED â–¡TAKE
â–¡HARMFUL INTERFERENCE
â–¡ENTER ANTARCTIC SPECIALLY PROTECTED AREA
â–¡INTRODUCE NON-INDIGENOUS SPECIES INTO ANTARCTICA
□IMPORT INTO USA—PORT OF ENTRY As Applicable □ EXPORT FROM USA
2. NAME, ADDRESS, PHONE/FAX NO. AND E-MAIL ADDRESS OF APPLICANT (IF APPLICANT IS A CORPORATION, FIRM, PARTNERSHIP, INSTITUTION, OR AGENCY, EITHER PUBLIC OR PRIVATE, COMPLETE NO. 4).
Penguins International PO Box 100483 Denver, Colorado 80250 628-400-7301
david@penguinsinternational.org
3. IF APPLICANT IS AN INDIVIDUAL, INCLUDE BUSINESS OR INSTITUTIONAL AFFILIATION (IF DIFFERENT THAN NO. 2) 4. NAME AND ADDRESS OF PRESIDENT OR PRINCIPAL OFFICER David Schutt, President
5. NAME OF PERMIT HOLDER’S AGENTS (FIELD PARTY MEMBERS), IF ANY (USE'TBA" AND INDICATE NUMBER, IF NAMES UNKNOWN) David Schutt Laura K.O. Smith 6. DESIRED EFFECTIVE DATES 10/01 /2017 through 6/01 /2018
7. LOCATION(S)—INCLUDE DESCRIPTION, TIME PERIOD, AND PROPOSED ACCESS TO THE LOCATION See attached Addendum 1.
8. SPECIMEN INFORMATION
(a) SPECIMEN/SPECIES
(scientific name, if applicable)
(b) NUMBER
PER ANNUM
(c) DESCRIPTION OF SPECIMEN(S)
(life stage, sex, sample type & size, etc.)
(d) MANNER OF TAKE OR INTERFERENCE
(e) IMPORT (Y/N)
(f) ULTIMATE DISPOSITION
Gentoo penguin (Pygoscelis papua)
Fecal samples
Spent eggshell fragments
Collected from the ground or from deceased bird carcasses
Collected from the ground Collected from the ground
Analysis at UCC
Analysis at UCD
Analysis at UCD
CERTIFICATION
I certify that the information submitted in this application for a permit is complete and accurate to the best of my knowledge and belief. Any false statement will subject me to the criminal penalties of 18 U.S.C. 1001.
SIGNATURE DATE
August 1,2017
FOR NSF USE ONLY
This application for a permit under the Antarctic Conservation Act, P.L. 95-541, as amended, and NSF regulations contained in Title 45 Part 670 of the Code of Federal Regulations is approved subject to the following conditions:
Please refer to the conditions on page 1 of this permit.
THIS PERMIT EXPIRES ON: June 1,2018
(Date)
TYPED NAME AND TITLE AND SIGNATURE OF NSF AUTHORIZING OFFICIAL Polly A. Penhale, Environmental Officer
NSF Form 1078
HAL .
^ 2$
DATE
11/06/2017
CONTINUE ON REVERSE SIDE
50


9. DESCRIPTION OF ACTIVITY FOR WHICH PERMIT IS NEEDED AND JUSTIFICATION FOR PROJECT. ALSO INCLUDE HERE ADDITIONAL INFORMATION RELATING TO THE SPECIFIC ACTION FOR WHICH THE PERMIT IS BEING SOUGHT.
The activity proposed for this requested permit is part of a larger study to quantify pollutant exposure in penguins across a wide latitudinal gradient. Target contaminants for this investigation are mercury and antimicrobial compounds. Because penguins are high trophic predators, are relatively sedentary compared to flying birds, have a very limited migratory range during the non-breeding season, and forage in a manner that efficiently samples the surrounding marine environment, penguins therefore serve as ideal subjects for this study. We will solely be opportunistically collecting abandoned feathers and fecal samples from Gentoo penguins (Pygoscelis papua) to perform a systematic single-species study across the widest possible latitudinal range.
Our investigation aims to sample Gentoo penguins at up to 11 colonies within the Antarctic Treaty area to quantify and compare contamination levels with those outside the Treaty area. Sample sites within the Treaty area may include S. Orkney Islands, Elephant Island, S. Shetland Islands and the species' entire range along the west coast of the Antarctic Peninsula.
Analyzed tissues will consist exclusively of Gentoo penguin feathers, spent eggshell fragments, and fecal samples. All feathers will be collected opportunistically from the ground as molted feathers or from deceased Gentoo adult and chick carcasses. Feathers provide a long-term signal of the bird's contaminant exposure prior to feather growth. Spent eggshell fragments will be analyzed to estimate maternal elimination of contaminants into the egg. Fecal samples will be collected from the ground to analyze penguin gut microbiota for evidence of antibiotic resistance. All collection will be performed with utmost caution outside of the colony or at the low-density periphery of the colony to prevent disruption to nesting birds. Collection will only be performed at a distance that prevents disturbance to any other wildlife.
PRIVACY ACT AND PUBLIC BURDEN STATEMENTS
The information requested in this application is solicited under the authority of the Antarctic Conservation Act (ACA), as amended, the National Science Foundation Act of 1950, as amended, and NSF regulations at 45 CFR Part 670. The information will be used in administration of the ACA, particularly to make a determination on eligibility for an ACA permit. The information requested may be disclosed to other Federal agencies or a court, administrative, or adjudicative body involved in implementing or enforcing the ACA; to Federal, state, or local agencies, or foreign governments, where necessary to obtain records in connection with an investigation, or to persons, including witnesses, who may have information, documents, or knowledge relevant to an ACA investigation or enforcement proceeding; to other Federal agencies when relevant to a decision by that agency on a security clearance, on the award of a contract or grant, on the issuance of a license or other benefit, or on a disciplinary or other administrative action concerning its employee; to government contractors, experts, volunteers and researchers as necessary to complete assigned work; to a grantee institution or contractor in connection with an investigation or enforcement proceeding where an ACA violation is alleged against it or one of its employees, researchers, or subcontractors; and to another Federal agency, court or party in a court or Federal administrative proceeding if the government is a party. See Systems of Records, NSF-56, "Antarctic Conservation Act Files," 59 Federal Register 5784 (February 8, 1994). Submission of this information is voluntary. However, failure to provide full and complete information necessary for an eligibility determination may reduce the possibility of receiving a permit.
Public reporting burden for this collection of information is estimated to average one half hour per response, including the time for reviewing instructions. Send comments regarding this burden estimate and any other aspect of this collection of information, including suggestions to reduce this burden, to the NSF Reports Clearance Officer at c/o the address directly below.
SUBMIT THIS DIVISION OF POLAR PROGRAMS (PERMIT OFFICE)
APPLICATION NATIONAL SCIENCE FOUNDATION, ROOM 755 E-MAIL: ACAPERMITS@NSF.GOV
T0: ARLINGTON, VIRGINIA22230
51


Addendum 1
Proposed dates for this expedition will be January 27, 2018 - February 27, 2018 for access to Gentoo penguin colonies at the following locations or comparable locations of nearby vicinity, to be visited at any time during these dates as weather conditions and tour operator schedules allow:
Signy Island, S. Orkneys (60° 43’ S 45° 36’ W)
Elephant Island (61° 08’ S 55° 07' W)
King George Island, S. Shetland Islands (62° 02’ S 58° 21’ W)
Yankee Harbour, S. Shetland Islands (62° 32’ S 59° 47’ W)
Trinity Island (63° 47’ S 60° 44’ W)
Cuverville Island (64° 41 ’ S 62° 38’ W)
Danco Island (64° 44’ S 62° 37’ W)
Paradise Bay (64° 49’ S 62° 52’ W)
Petermann Island (65° 10’ S 64° 10’ W)
Cape Tuxen (65° 16’ S 64° 8’ W)
Detaille Island (66° 52’ S 66° 47’ W)
No ASPAs will be on the itinerary.
Proposed access to the above locations will be made on foot following shore landings from the Ocean Tramp, a tourist yacht operated by Quixote Expeditions.
52


Falkland Islands Government
The Environmental Planning Department
Telephone: (+500) 284S0
P.O. Box 611 Stanley
E-mail: mend ell @ ul anni nit, go v. Ik
Falkland Islands
Research Licence No: R22/2017
CONSERVATION OF WILDLIFE AND NATURE ORDINANCE 1999
SECTION 9
LICENCE TO CARRY OUT SCIENTIFIC RESEARCH
1. Licensee:
Name of the person leading the research
Affiliation
Position
Postal Address
Phone number Email
David Sclmtt
University of Colorado Denver (LISA)
Graduate Student
PO Box 100483
Denver, Colorado 80250 USA
001-720-261-8848
day id. schutt (fl'ucdenver. edit
2. Nature of licence:
2.1 This licence is issued to David Schutt under Section 9 of the Conservation of
Wildlife and Nature Ordinance 1999. It is granted to David Schutt to permit his staff and bona fide field assistants or researchers employed on their behalf or under their overall jurisdiction. It is granted only for the following activities using methods specified in the research licence application on research on ‘ Extent of mercury exposure in Gentoo penguins (Pygoscelis papua) across a wide latitudinal gradient’ approved by the Environmental Committee on 21st September 2017:
• A total of 15 adult Gentoo Penguins will be captured from each colony with the use of a long-handled net.
• Morphometric measurements will be taken along with tissue samples for analysis.
• Approximately 10 breast feathers will be collected.
• Feathers form bead birds found at colonies may be collected.
• Spent eggshells and abandoned eggs may be collected from study sites.
• 1.5 mL of blood drawn from an intcrdigital foot vein and a cloacal swab taken from each bird.
• In addition, a total of 15 Gentoo chicks >3 weeks of age and in the post-guard (creche) phase will likewise be captured from each colony with a long-handled net.
• Morphometric measurements will be taken along with approximately 10 breast feathers from each chick.
• All birds will be released immediately at the same location from which they were captured.
53


• Sample size is calculated by statistical power analysis and represents the minimum number of birds required to support the objective of our study.
• Total handling time from capture to release is expected to be <8 minutes per adult bird and <3 minutes for chicks based on previous experience with Gentoo penguin sampling.
• Extreme care will be taken to minimize disruption to nearby birds during capture and birds will not be captured if they are on a nest, guarding eggs or chicks, or nearby another bird which is guarding eggs or chicks.
2.2 This licence shall not be construed as authorising Ihe licensee to enter upon Ihe land of another w ithout the ow ner’s permission or consent.
2.3 The research protocol is approved subject to confirmation of a field assistant being provided to support the lead researcher in all fieldwork.
2.4 This licence does not constitute a permit to remove biological items from protected species from the Falkland Islands. An export licence should be sought from the Customs and Immigration Department to allow for the removal of any biological material or protected species from the Falkland Islands.
3. Period of licence
3.1 This licence is valid for the period commencing on 1st December 2017 and terminating on 31st January 2018.
3.2 This licence may be revoked at any time by the Governor, but otherwise shall be valid for the period stated in paragraph 3.1.
4. Conditions of licence
4.1 This licence is issued on condition that the licensee shall:
a! Submit to the Environmental Planning Officer, Malvina House Garden, PO Box 611, Stanley, Falkland Islands, not later than 1 June 2018, a report detailing the research w ork carried out and the methods used in that research; and
b) Deposit with the Environmental Planning Officer copies of all subsequent reports on the research work carried out.
c) Deposit with the Environmental Planning Officer copies of any data collected as part of this study. Data will be stored and will not be published or circulated without researcher approval for a period of 2 years.
5. Purpose of Research
54


The purpose of the research work carried out by the licensee is set out in the resear ch proposal approved by Environmental Committee on 21st September 2017.
Signed:
Nick Rcndcll Environmental Officer
Dated: 2l'h September 2017
55


South Georgia & the South Sandwich Islands
Office of the Commissioner Government House Stanley Falkland Islands
Regulated Activity Permit
Permit number 2017/044 - amended 15/11/17
Project title Extent of mercury exposure in Gentoo penguins
Activities Science
Permit holder David Schutt
Participant names Research assistant - TBD
Dates 01/01/2018 to 30/03/2018
Area of Operation Sites listed on ship’s visit permit
Support vessel Ocean Endeavour
Project description/permitted activities
Collection of faecal material from the ground and feathers from dead gentoo, king and macaroni penguins in order to: 1. Investigate differences in mercury levels of various tissues and ages of penguins in the region. 2. Compare mercury levels from this region with published results of penguin mercury levels at similar latitudes in other parts of the Southern Ocean. 3. Compare mercury signals in dead versus live penguins to provide support for the use of dead penguins for future biomonitoring
Conditions
1 Methods and mitigation measures should follow those described in the project application form and supporting documentation
2 Biosecurity protocols outlined in the ‘Biosecurity Handbook 2017/18’ must be adhered to at all times.
3 Upon completion of the project a ‘Report on Permitted Activities’ should be submitted to the Office of the Commissioner
4 If you remove any samples collected under this permit from the Territory, you must submit an ‘Exported Material Declaration’ by 30th April 2018
See attached annexes 1) Wildlife and Protected Areas 2) Sample Collection
Environment Date: 151
Signed /
/©/ ~
CTl
Email: permits@gov.gs
Tel: (+500) 28207 Facsimile (+500) 228811
56


Annex 1
Wildlife and Protected Areas Ordinance
Subject to the conditions outlined overleaf, the following actions that would otherwise be prohibited under the Wildlife and Protected Areas Ordinance 2011 are hereby authorised:
Samples listed in Annex 2
I am satisfied that this permit is limited so as to comply with section 21 (7)
Signed
Environment Officer Date: 15th November 2017
Email: permits@gov.gs
Tel: (+500) 28207 Facsimile (+500) 228811
57


Annex 2
Sample collection
Subject to the conditions outlined overleaf and, collection of the following samples is permitted:
Samples from species
Species Location Sample type Number
Gentoo penguin Sites listed on ship’s visit permit Feathers from dead birds 180 birds (approx. 20 feathers per bird)
Faecal samples from ground Approx. 35
Macaroni penguin Sites listed on ship’s visit permit Feathers from dead birds 90 birds (approx. 20 feathers per bird)
Faecal samples from ground Approx. 35
King penguin Sites listed on ship’s visit permit Feathers from dead birds 4 birds (approx. 20 feathers per bird)
Faecal samples from ground Approx. 35
NOTE: Providing your samples are not from CITES listed species, you may export material up to the amount shown above, providing an ‘Exported Material Declaration’ is submitted to GSGSSI by 30th April 2018.
Email: permits@.gov.gs
Tel: (+500) 28207 Facsimile (+500) 228811
58


U.S.DEPARTMENT OF AGRICULTURE ANIMAL AND PLANT HEALTH INSPECTION SERVICE VETERINARY SERVICES RIVERDALE, MARYLAND 20737 f ile: ///D :/l netpu b/w/wvrooVEpermits/i mages/
UNITED STATES VETERINARY PERMIT FOR IMPORTATION AND TRANSPORTATION OF CONTROLLED MATERIALS AND ORGANISMS AND VECTORS
PERMIT NUMBER
124197 Research
DATE ISSUED
12/12/2017
DATE EXPIRES 12/12/2018
NAME AND ADDRESS OF SHIPPER(S)
Falklands Conservation
Jubillee Villas
Ross Road
Stanley FIQQ 1ZZ
FALKLAND IS LAND S (MALVINAS)
[see attached list]
NAME AND ADDRESS OF PERMITTEE INCLUDING ZIP CODE AND TELEfljHO
David Schutt
University of Colorado Denver Dept, of Integrative Biology Rm 2071 1201 5th St.
Denver, Colorado 80204
720-261-8848
[see attached list]
CC:
Service Center, CO (Lakewood, CA)
'l/.S. PORT(S) OF ARRIVAL
'AS APPLICABLE
MODE OF TRANSPORTATION
AS REQUESTED IN YOUR APPLICATION. YOU ARE AUTHORIZED TO IMPORT OR TRANSPORT THE FOLLOWING MATERIALS
Feathers, bones, eggshell fragments, and/or blood samples (avian origin)
RESTRICTIONS AND PRECAUTIONS FOR TRANSPORTING AND HANDLING MATERIALS AND ALL DERIVATIVES
THIS PERMIT IS ISSUED UNDER AUTHORITY CONTAINED IN 9 CFR CHAPTER 1, PARTS 94,95 AND 122. THE AUTHORIZED MATERIALS OR THEIR DERIVATIVES SHALL BE USED ONLY IN
ACCORDANCE WITH THE RESTRICTIONS AND PRECAUTIONS SPECIFIED BELOW (ALTERATIONS OF RESTRICTIONS CAN BE MADE ONLY WHEN AUTHORIZED BY USDA, APHIS, VS).
o Adequate safety precautions shall be maintained during shipment and handling to prevent dissemination of disease.
oWith the use of this permit I, David Schutt, Permittee, acknowledge that the regulated material(s) will be imported/transported within the United States in accordance with the terms and conditions as are specified in the permit. The Permittee is the legal importer/recipient [as applicable] of regulated article(s) and is responsible for complying with the permit conditions. The Permittee must be at least 18 years of age and have and maintain an address in the United States that is specified on the permit; or if another legal entity, maintain an address or business office in the United States with a designated individual for service of process; and serve as the contact for the purpose of communications associated with the import, transit, or transport of the regulated article(s). **Note: Import/Permit requirements are subject to change at any time during the duration of this permit.
continued on subsequent paqe(s)
TO EXPEDITE CLEARANCES AT THE PORT OF ENTRY, BILL OF LADING, AIRBILL OR OTHER DOCUMENTS ACCOMPANYING THE SHIPMENT SHALL BEAR THE PERMIT NUMBER
signature Deborah Langford title Staff Veterinarian National Import Export Services NO. LABELS
VS FORM 16-6A (MAR 95)
Replaces VS Form 16-3A and 16-28 which are obsolete
Page 1 of 4
59


U.S.DEPARTMENT OF AGRICULTURE
APHIS / VETERINARY SERVICES, RIVERDALE, MARYLAND 20737.
ATTACH TO U.S. VETERINARY PERMIT - 124197 (DATE ISSUED: 12/12/2017)
SHIPPERS (continued from Permit Form VS 16-6) DAP
O’Higgins 891 Punta Arenas CHILE
PERMITEES (continued from Permit Form VS 16-6)
David Schutt
c/o USDA/APHIS/WS National Wildlife Research Center
4101 LaPorte Ave.
Fort Collins, CO 80521
RESTRICTIONS AND PRECAUTIONS: (continued from Permit Form VS 16-6)
o***Each shipment shall be accompanied by an ORIGINAL signed document from the producer/manufacturer confirming that the exported materials: 1) contain feathers, bones, eggshell fragments, and/or blood samples derived from birds (avian) that originated in the Falkland Islands (Malvinas), South Georgia and the South Sandwich Islands, and/or Antarctica; and 2) were not exposed to or commingled with any other animal origin material.
[This certification must CLEARLY correspond to the shipment by means of an invoice number or shipping marks or lot number or other identification method. An English translation must be provided.]
o ***Materials shall be consigned directly to the permittee at the inspected laboratory facility specified below on this permit. Materials imported under this permit may be hand carried in personal baggage from the country of origin to the port of arrival, but must be declared and made available to port officials for inspection, and must be transported directly to the permittee by someone with identification and current, signed written authority from the permittee. The permittee's authorizing document must be original, on letterhead, and specific to the particular shipment(s), and shall be valid for no more than 2 months from the date of issuance.
signature Deborah Langford
TITLE
Staff Veterinarian National Import Export
Services
Page 2 of 4
60


U.S.DEPARTMENT OF AGRICULTURE
______APHIS / VETERINARY SERVICES, RIVERDALE, MARYLAND 20737.________
ATTACH TO U.S. VETERINARY PERMIT - 124197 (DATE ISSUED: 12/12/2017)
RESTRICTIONS AND PRECAUTIONS: (continued from Permit Form VS 16-6)
o THIS PERMIT IS VALID ONLY FOR WORK CONDUCTED OR DIRECTED BY YOU OR YOUR DESIGNEE IN YOUR PRESENT U.S. FACILITY OR APPROPRIATELY INSPECTED LABORATORY. THE AUTHORIZED IMPORTED MATERIAL(S) MUST BE SHIPPED/CONSIGNED DIRECTLY TO USDA/APHIS/WS National Wildlife Research Center, 4101 LaPorte Ave., Ft. Collins, CO 80521. (MATERIALS AND/OR THEIR DERIVATIVES SHALL NOT BE MOVED TO ANOTHER U.S. LOCATION, OR DISTRIBUTED WITHIN THE U.S., WITHOUT USDA, APHIS, VS, NIES AUTHORIZATION.) ++EXCEPTION++ Materials and/or their derivatives are authorized to be distributed for further evaluation only after the imported materials have been appropriately mitigated in the BSL-2 laboratory. All samples must be accompanied by documentation verifying the treatment performed. David Schutt, University of Colorado Denver, retains responsibility for compliance to the permit. Locations shall be recorded and made available to the USDA upon request.
oAll treatments must be conducted in Biosafety Level 2 facilities located in USDA/APHIS/WS National Wildlife Research Center, 4101 LaPorte Ave., Ft. Collins, CO 80521, rooms #B102, B105, B105, B109, B118, B125, B209, and/or B211 which has/have been inspected and approved by the USDA. This facility shall be reinspected every 3 years.
oImported materials must be treated prior to any work being done with the materials. All feathers, bones, and eggshell fragments will be soaked in 70% ethanol for 30 minutes; blood samples will be mitigated with FTA cards, treated with 70% ethanol, or treated with 10% sodium hypochlorite.
oPackaging, containers, and all equipment in contact with these materials shall be sterilized or considered a biohazard and be disposed of accordingly.
oCOMMERCIAL DISTRIBUTION OF THE IMPORTED MATERIALS AND/OR THEIR DERIVATIVES IS PROHIBITED.
oThis permit DOES NOT authorize direct or indirect exposure of or inoculation into domestic or laboratory livestock (including but not limited to: birds/poultry/eggs, cattle, sheep, goats, swine, and/or horses). Work with materials and/or their derivatives shall be limited to in vitro uses only.
oOn completion of your work, all permitted materials and all derivatives therefrom shall be destroyed.
oImported material may be subject to regulations enforced by the United States Department of Interior, Fish and Wildlife Service (FWS). Importer must contact FWS, information is available at web pages http://www.FWS.gov/permits/ and/or http://www.FWS.gov/le/travelers.html
signature Deborah Langford

TITLE
Staff Veterinarian National Import Export
Services
Page 3 of 4
61


U.S.DEPARTMENT OF AGRICULTURE
APHIS / VETERINARY SERVICES, RIVERDALE, MARYLAND 20737.
ATTACH TO U.S. VETERINARY PERMIT - 124197 (DATE ISSUED: 12/12/2017)
RESTRICTIONS AND PRECAUTIONS: (continued from Permit Form VS 16-6)
oThe restrictions on this permit remain in force as long as the material is in the United States.
oThis permit does not exempt the permittee from responsibility for compliance with any other applicable federal, state, or local laws and regulations.
oAny person who VIOLATES the terms and conditions of permits, and/or who forge, counterfeit, or deface permits may be subject to criminal and civil penalties in accordance with applicable law. In addition, all current permits may be cancelled and future permit applications denied.
oA copy of this permit must be included with the shipping documents. For imported materials, these documents must be presented to CBP Agricultural Specialists upon arrival at the U.S. port of arrival.
SIGNATURE
Deborah
Langford
TITLE

Staff Veterinarian National Import Export
Services
Page 4 of 4
62


Full Text

PAGE 1

GENTOO PENGUIN ( PYGOSCELIS PAPUA ) FEATHER MERCURY CONCENTRATIONS DIFFER BETWEEN ANTARCTIC AND SUB-ANTARCTIC REGIONS AS REVEALED BY FORAGING ECOLOGY AND STABLE ISOTOPE ANALYSES by DAVID SCHUTT B.S., University of Colorado, 1990 B.A., University of Colorado, 1998 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Biology Program 2019

PAGE 2

This thesis for the Master of Science degree by David Schutt bas been approved for the Biology Program by Mike B. Wunder, Chair and Co-Advisor Alan M. Vajda, Co-Advisor Timberley M. Roane Craig A. Stricker Date: May 18, 2019 ! ii

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Schutt, David (M.S., Biology Program) Gentoo Penguin ( Pygoscelis Papua ) Feather Mercury Concentrations Vary Between Antarctic and Sub-Antarctic Regions as Revealed by Foraging Ecology and Stable Isotope Analyses Thesis directed by Associate Professors Mike B. Wunder and Alan M. Vajda ABSTRACT Numerous studies on seabird mercury loads have helped characterize bioavailability of mercury as an increasing global pollutant, however the broad and dynamic foraging ecology of seabirds has made comparisons difficult. Here, we investigate foraging ecology of Gentoo penguins ( Pygoscelis papua ) in relation to their feather mercury concentrations to help understand regional differences in mercury uptake by these widely-distributed seabirds. Due to logistical limitations that only allowed us to collect feather samples from live birds in the Falkland Islands and from dead birds at the remaining sites, we first investigated whether distributions of feather mercury concentrations were consistent between live birds and dead birds. Our results indicate that dead birds can be used as surrogates for live birds in the study of Gentoo penguin feather mercury concentrations. Following this analysis, we used a comparative approach with stable isotope analyses to uncover baselines in trophic level and mercury biomagnification among discrete breeding populations. Additionally, we consider differences in prey composition between populations and prey selection through energy density analysis to understand differences in foraging ecology that would likely have the largest effect on penguin mercury exposure. Our results suggest that numerous factors play a role in mercury load disparities between populations, and that higher mercury concentrations in the Falkland Islands ! iii

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and South Georgia Island in relation to the Antarctic Peninsula are due to differences in foraging ecology coupled with a likely increase in ambient environmental mercury. The form and content of this abstract are approved. I recommend its publication. Approved: Mike B. Wunder and Alan M. Vajda ! iv

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This work is dedicated to everyone who helped me through this challenging, but extremely rewarding thesis project. In particular, this work is dedicated to Heather, my understanding and supportive wife and cheerleader, and my son, Edan, who learned as much as I did throughout this journey and will likely follow a new path through life after traveling to the far corners of the world to get to "hold baby penguins." This work is also dedicated to my good friends who I met at McMurdo so many years ago where I got to play with penguins for the first time. ! v

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ACKNOWLEDGEMENTS I would like to thank Mike Wunder, Alan Vajda, Timberley Roane and Craig Stricker, my committee members, for directing me through this research and teaching me more about ecology, stable isotopes, statistics and environmental toxicology than I ever thought I'd learn. I would also like to thank Jono Handley who taught me everything I needed to know about working with penguins and, through his own doctorate work, inspired me to develop my own research. Thanks to Alan Franklin and Jeff Chandler for storing samples and for use of the lab, to Rebecka Brasso, Lisa Stoneham and Megan Faulkner for conducting mercury analysis, and to Craig Stricker for conducting stable isotope analyses. Special thanks go to logistical support from Quark Expeditions, Quixote Expeditions, and Penguins International. Additional thanks to the Falkland Islands Government, the Government of South Georgia and the South Sandwich Islands and the NSF for assistance throughout the permitting process. Special thanks go to Adrian Lowe, Jan Cheek, Derek Pettersson, Micky Reeves and John and Michelle Jones for their hospitality and assistance in the Falkland Islands. IACUC Protocol #92517(08)1C. ! vi

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TABLE OF CONTENTS CHAPTER I I. CONSISTENCY OF FEATHER MERCURY CONCENTRATIONS IN LIVE VS. OPPORTUNISTICALLY-FOUND CARCASSES OF GENTOO PENGUINS (PYGOSCELIS PAPUA) 1 ..................................................................................................... Introduction 1 .......................................................................................................................... Methods 3 ................................................................................................................................ Results and Discussion 8 ........................................................................................................ Conclusion 9 ........................................................................................................................... II. SUB-ANTARCTIC GENTOO PENGUIN (PYGOSCELIS PAPUA) POPULATIONS HAVE HIGHER FEATHER MERCURY CONCENTRATIONS THAN ANTARCTIC POPULATIONS 10 ................................................................................................................. Introduction 10 ........................................................................................................................ Materials and Methods 13 ....................................................................................................... Results 19 ................................................................................................................................ Discussion 22 .......................................................................................................................... Conclusion 35 ......................................................................................................................... REFERENCES 37 .............................................................................................................................. APPENDIX 49 .................................................................................................................................... ! vii

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CHAPTER I I. CONSISTENCY OF FEATHER MERCURY CONCENTRATIONS IN LIVE VS. OPPORTUNISTICALLY-FOUND CARCASSES OF GENTOO PENGUINS (PYGOSCELIS PAPUA) Introduction Ecotoxicological studies on seabirds have historically relied on specific tissues that often required a freshly dead specimen or the sacrificing of a live-caught individual. For ethical and logistical reasons, contemporary sampling schemes have shifted away from destructive sampling and instead employ minimally invasive or noninvasive collection methods (Pauli et al., 2010). For example, avian sampling methods might include feather collection from live birds, collecting molted feathers from the ground, or collecting tissues opportunistically from bird carcasses (termed "found-dead birds" hereafter). Use of found-dead birds may be the only pragmatic option in studies where capture and handling of live birds is limited or impossible, such as when restrictions on handling are imposed by regulating agencies, by working in sensitive or difficult to access habitats, for studies involving birds that are difficult to live-capture, or when disruptive handling of live birds directly or indirectly impacts the survival of individual birds (Spotswood et al., 2011; Wilson & McMahon, 2006). Due to the stability of mercury bound within the feather keratin matrix, feathers from a dead bird should maintain similar mercury levels as when the individual was alive. In a live bird, once feather synthesis is complete, the blood supply at the calamus atrophies and the feather becomes an inert tissue (Lillie, 1940), no longer subject to structural or chemical changes through any biological processes by the bird. This durable feather tissue withstands harsh ! 1

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environmental conditions (Appelquist et al., 1984) not only while the bird is alive, but also resisting decomposition for a time following death. Such structural stability is a result of strong intramolecular disulfide bonds in the feather keratin which associate with circulating blood mercury during feather synthesis (Aschner & Syversen, 2005; Bortolotti, 2010; Crewther et al., 1965; Furness et al., 1986), effectively locking this mercury exposure record in a medium that remains stable after development. Thus, a feather will remain chemically consistent from the time of synthesis in a live bird until long after the death of the bird. Although the sampling of dead birds is often assumed to serve as an authentic substitute for sampling of live birds (e.g. Bekhit et al., 2011; Cipro et al., 2017; Dimitrijevi" et al., 2018; Kim et al., 2015; Vasil et al., 2012; Vega et al., 2009), examples of formal evaluation of this practice in ecotoxicology are lacking. For example, mercury loads above background effect levels may affect mortality (Ackerman et al., 2016; Jackson et al., 2016), which in turn might bias high the estimates of exposure as measured from found-dead birds. Likewise, sub-lethal observable responses of mercury toxicity may indirectly affect mortality (Ackerman et al., 2016; Eagles-Smith et al., 2008; Jackson et al., 2016). The wide range of feather mercury concentrations with variable sub-lethal effect responses allows for uncertainty as to causes of mercury toxicity-related mortality in found-dead birds. Evidence demonstrating that dead birds can be substituted for live birds, therefore, is a pragmatic and efficient alternative for ecotoxicology investigations. The main objective of this study was to assess the efficacy of using found-dead birds as proxies for live birds to estimate feather mercury concentrations in Gentoo penguins ( Pygoscelis papua ). ! 2

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Methods Study Area The study colonies were in the Falkland Islands, an archipelago located approximately 500 km east of mainland South America in the South Atlantic Ocean (Fig. 1). Throughout the Falkland Islands, there are approximately 85 known breeding colonies of Gentoo penguins, collectively averaging approximately 100,000 breeding pairs annually (Baylis et al., 2013). The study sites were chosen to span a wide range of bathymetry near the breeding colonies (Carpenter-Kling et al., 2017; Handley et al., 2017). All samples were collected during the austral summer between December 2017 and February 2018. ! 3 Fig. 1 Location of Falkland Islands field sites (stars) for the present study (QGIS Development Team, 2019). For live birds, n = 15 for each site. For found-dead birds, n = 16 for Cow Bay (51¼ 25' S 57¼ 51' W), n = 15 for Race Point (51¼ 25' S 59¼ 00' W), n = 14 for Bull Point (52¼ 19' S 59¼ 17' W).

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Study Species and Tissue Samples Gentoo penguins are a non-migratory seabird that remain near their breeding colony throughout the year (Black et al., 2017; Tanton et al., 2004; Williams & Rodwell, 1992). This species is an opportunistic feeder, foraging on various crustaceans, cephalopods, and fish (Handley et al., 2015), rarely venturing farther than 30 km from shore during foraging trips (Carpenter-Kling et al., 2017; Handley el al., 2018; Robinson & Hindell, 1996). Throughout the breeding season, adult Gentoos capture prey during these localized foraging trips and return typically on alternating days to feed their chicks the regurgitate which often contains whole or partially-digested prey items (Handley et al., 2015). Penguin chicks undergo three stages of feather growth during development and maturation, 1) hatching with a natal down 2) plumage replacement at 1 to 2 weeks of age with a secondary down, 3) growth of adult plumage just prior to fledging. The secondary down was the focus for sampling in this study, as it is derived from prey items taken by the parents and fed to the chicks. Foraging by adults during this period occurs near the natal colony (Handley et al., 2017), reflecting a comparatively localized signal of mercury exposure within this tissue over a near-term temporal scale. Because chick down is grown simultaneously, all intraindividual feathers are expected to have low variation in mercury concentration (Brasso et al., 2013). In addition, feathers allow for a minimally invasive and easily transported method of tissue collection as compared with sampling blood or stomach contents. As such, penguin chick secondary down is well suited among avian tissues for mercury biomonitoring. ! 4

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Live Bird Sampling Methods Live birds were collected under permit from the Falkland Islands Government (#R22/2017), with landowner permission, and using protocols approved by University of Colorado Denver IACUC (#92517(08)1C). Each colony supported ~2000+ breeding pairs of Gentoo penguins. Gentoo penguin chicks in the post-guard phase (i.e. no longer protected by the parents) were captured using a long-handled net. Down feathers (10-12) were plucked from the breast using a pair of silicone-tipped forceps (DDP Instruments young tongue forceps, Miami, FL, USA) and stored at ambient temperature in small resealable plastic bags. Each chick was marked with a small amount of feather paint to prevent duplicate sampling (Sporting Pigeons Paint green, Pigeon Products International, Post Falls, ID, USA) and then released. Dead Bird Sampling Methods We found dead Gentoo penguin chicks by searching within each colony boundary and along transects extending ~100 m from the colony boundary to find carcasses that may have been moved by predators or scavengers. We used only birds that had died during the study season (as evidenced by presence of blood and/or comparative lack of decomposition, bleaching and weathering). We collected secondary down from the breast of intact carcasses when available, or otherwise from the legs just above the foot, sampling only white feathers using the same methods as detailed for live birds in Section 2.3. Mercury Analysis All feather samples were sterilized per USDA/APHIS requirements (as detailed on the importation permit) for importation into the United States. Sterilization procedure required soaking the feather samples in 70% ethanol (LabChem, Zelienople, PA, USA) for 30 minutes. ! 5

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Following sterilization, the feather samples were triple rinsed in deionized water and dried under a fume hood for 24-48 hours at room temperature until all external moisture was removed. In preparation for mercury analysis the feathers were cleaned to remove exogenously deposited contaminants. The feather samples were placed in a stainless steel screen and vigorously cleaned in environmental grade acetone (Alfa Aesar, Ward Hill, MA, USA) followed by rinsing under a flowing stream of deionized water. This procedure was completed three times for each feather sample to ensure all exogenous oils and contaminates were removed. The feather samples were then dried in a fume hood at room temperature for 24 48 hours until all external moisture was removed. Approximately 10 mg of whole down feathers from each individual were analyzed for total mercury via atomic absorption spectrophotometry on a Nippon MA-3000 Direct Mercury Analyzer at Weber State University (Ogden, UT, USA). Each set of 20 samples analyzed was preceded and followed by two samples of a standard reference material (TORT-3, National Resource Council Canada). Mean percent recovery for the standard reference material was 101.4% ( n = 22) with relative significant differences in mercury concentration of 6.9%. Mercury concentrations in chick down are reported as mg/kg fresh weight (fw). Feather-Equivalent Mercury Calculations Data transformations for converting blood equivalent mercury concentrations to feather mercury concentrations were calculated to standardize our feather mercury values with previously reported blood mercury values for comparison. The equation used for the conversion was developed by Eagles-Smith et al. (2008) with R 2 = 0.32 (Eq. 1). ! 6

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ln(Blood TotalHg ww ) = 0.673 x ln(Feather TotalHg dw ) 1.673 (1) Statistical Analysis We used a two-sample Kolmogorov-Smirnov test (ks.test, R Core Team 2018) to evaluate the null hypothesis that the mercury concentrations for feathers from live-caught and found-dead birds were drawn from the same distribution. ! 7 Fig. 2 Found-dead birds vs. live birds. Whiskers indicate 1.5*IQR, horizontal bar is the median, boxplot endpoints are at the 25th and 75th percentiles. The dashed line indicates the boundary between background mercury exposure and low mercury exposure, the upper limit of no-effect levels (Jackson et al., 2016). ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Lowest Adverse Effects Level 1 2 3 4 Dead Live Feather Hg concentration (mg/kg)

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Results and Discussion We collected feathers from a total of 45 live chicks and 45 dead chicks from across the three different sites (Fig. 1). Mercury levels in all individuals were below the lowest reported level of effect for mercury exposure (Fig. 2; Jackson et al., 2016), and there was little support for rejecting the null hypothesis that mercury concentrations in feathers from live-caught and from found-dead birds were drawn from the same distribution ( D = 0.2, p = 0.33). Our primary goal for this study was to evaluate consistencies between distributions of feather mercury concentrations of live-caught Gentoo chicks and of opportunistically-found Gentoo chick carcasses. All individuals from both live and found-dead groups were below the mercury feather-equivalent low-effect threshold of 4.3 mg/kg, and far below the featherequivalent mercury concentration of 288 mg/kg for mortality in seabirds (Eq. 1; Fig. 2; Ackerman et al., 2016; Eagles-Smith et al., 2008; Jackson et al., 2016). We therefore make the assumption that mercury exposure did not impact survival of the found-dead group differently than that of the live group. Our results suggest that mercury concentrations in feathers from bird carcasses are similar to those from birds living in the same breeding colonies. These results are particularly important for seabird feather mercury analyses due to the practice of substituting found-dead bird carcasses for live birds in a wide variety of studies using bird feathers as indicators of marine environmental toxicant exposure (Bekhit et al., 2011; Cipro et al., 2017; Kim et al., 2015; Vega et al., 2010). Due to the inert and stable nature of feathers and the consistency of feather mercury distributions in both live individuals and opportunistically found carcasses, this tissue is well suited to be analyzed for mercury content regardless of the living ! 8

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state of the bird. As such, feathers from either live or dead penguins consistently reflect dietary mercury exposure of the bird. Conclusion The distribution of mercury concentrations in feathers from penguin chick carcasses found opportunistically near nesting sites is similar to that for feathers from live chicks. For avian mercury studies that have logistical constraints such as permitting limitations or cost constraints associated with sampling from live birds, our results suggest that found-dead birds can be used as surrogates for live birds in the study of feather mercury concentrations. Because results from this study apply specifically to feather mercury concentrations in penguins, we recommend that additional studies be conducted to evaluate feather samples from live vs. founddead birds in other bird taxa as well as for additional toxicants such as persistent organic pollutants (POPs) to evaluate a much wider array of ecotoxicological subjects. ! 9

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CHAPTER II II. SUB-ANTARCTIC GENTOO PENGUIN ( PYGOSCELIS PAPUA ) POPULATIONS HAVE HIGHER FEATHER MERCURY CONCENTRATIONS THAN ANTARCTIC POPULATIONS Introduction Mercury is a global pollutant that has been growing steadily in the atmosphere due directly to human activities. Artisanal gold mines, fossil fuel combustion and various industrial processes have led to a 3to 5-fold increase in atmospheric mercury over the past 150 years (Driscoll et al., 2013; Streets et al., 2017). Estimates of ocean surface mercury concentrations as a result have increased by a factor of 2-3 (Driscoll et al., 2013; Fitzgerald et al., 2007) which is subsequently bioaccumulating in marine food webs. Penguins are mesopredator species in the Southern Hemisphere and have been used as bioindicators of marine contaminants in numerous studies due to their interaction with the marine food web at higher trophic levels (Burger & Gochfeld, 2004; Furness & Camphuysen, 1997; Kahle & Becker, 1999). The study of mercury loads in higher trophic level seabirds has provided much understanding regarding bioavailability and trends in marine fauna mercury uptake, which can point to changes in global pollution dynamics. The relationship is challenging to quantify, however, due to mercury's bioaccumulating properties, and involves complexities of foraging ecology and an organism's interaction with the food web. Seabirds in particular are subject to large variations in mercury loading within a population due to their broad distribution in a highly variable marine environment. Intraspecific mercury exposure disparities have been recorded from individual seabirds within a single population (Catry et al., 2008; Lescro‘l et al., ! 10

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2004; Miller et al., 2009; Polito et al., 2016; Thompson et al., 1998) which is likely a function of differences in foraging strategies and extensive foraging range (Thompson et al., 1998). Understanding foraging behavior of a seabird population is therefore imperative to help explain differences in mercury uptake (Polito et al., 2016) in a dynamic marine environment. The range in diets and its effects on mercury accumulation within seabird populations points to increased accuracy of mercury data when coupled with stable isotope analyses. Carbon isotopic differences can be readily attributed to various foraging geographies, namely benthic/ onshore vs. pelagic/offshore locations and also differences in latitude resulting in a decline in carbon isotopes at higher latitudes (Cherel & Hobson, 2007; Kelly, 2000). Nitrogen isotopic inferences of trophic level help standardize interactions within the food web (Atwell et al., 1998; Bearhop et al., 2000; Santos et al., 2005; Seixas et al., 2014), and are of particular importance when understanding mercury biomagnification and comparisons of dietary exposure (Polito et al., 2016). Trophic level approximations may therefore provide an explanation for increases in mercury that are unrelated to diet selection, and may be attributed to other factors such as environmental baseline mercury differences. To understand why intraspecific differences in trophic levels exist, neither isotopic mixing models nor stomach content dietary studies are sufficient alone to infer foraging niche of wide-ranging generalist seabirds such as Gentoo penguins ( Pygoscelis papua ; Polito et al., 2016). As such, we include a review of the literature to discern differences in Gentoo foraging strategy and its effects on feather mercury concentrations. Our study focuses on three regions in the Southern Hemisphere, with a primary focus on the Falkland Islands, a region that is understudied and relatively unknown with respect to seabird mercury exposures. The Falkland Islands is an archipelago off the Patagonian coast of South ! 11

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America with large numbers of seabirds but almost non-existent seabird mercury investigations. South Georgia Island is a remote seabird-rich island in the turbulent waters of the Scotia Sea. The Antarctic Peninsula, like the rest of the Antarctic continent, is a region that is isolated from the global oceanic system by the massive flow of the Antarctic Circumpolar Current and its abrupt thermal and salinity gradients (Bargagli, 2005). Because previous studies on seabirds along the Antarctic Peninsula show relatively low mercury exposure (Brasso et al., 2014), we use this isolated region as a reference with which to compare mercury exposure in seabirds from the Falkland Islands and South Georgia Island. In this study, we investigated mercury loads in relation to foraging ecology of subAntarctic and Antarctic populations of Gentoo penguins, a non-migratory species that inhabits both sides of the Antarctic Circumpolar Current. Based on the geography of the region and the relative isolation of Antarctica from the rest of the oceanic system, we would expect the Falkland Islands and South Georgia Island to have higher bioavailable mercury than Antarctica. For this investigation, we examined Gentoo foraging patterns throughout the study sites to understand behavioral and dietary sources of mercury exposure disparity. Different prey items consumed by Gentoos bioaccumulate mercury differently in relation to trophic level and vertical location within the water column and therefore influence dietary exposure (Monteiro et al., 1996; Thompson et al., 1998). We also used stable isotope analyses of Gentoo feathers in relation to prey items to understand differences in trophic level and potential ! 15 N enrichment between the regions. Finally, we compared our feather mercury data with trophic level and foraging behavior to explain differences in Gentoo feather mercury concentrations among the Falkland Islands, South Georgia Island and the Antarctic Peninsula. ! 12

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Materials and Methods Study Areas We collected feather samples during the austral summer of 2017-2018 from live and dead Gentoo chicks at four colonies around the Falkland Islands, from dead Gentoo chicks at four colonies on South Georgia Island, and from dead Gentoo chicks at five colonies along the Antarctic Peninsula (Fig. 1). Previous studies have demonstrated consistency between feather mercury concentrations in both live and found-dead chicks (Schutt et al., submitted manuscript), ! 13 Fig. 1 Sampling regions for the present study. Bar plot shows the relative proportion of each Gentoo prey classification for each region. Antarctic Peninsula South Georgia Island Falkland Islands 0 25 50 75 100 Prey Percentage Cephalopod Crustacean Fish

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allowing dead birds to be used as surrogates for live birds in feather mercury studies. Because Gentoo penguins maintain high breeding site fidelity (Williams & Rodwell, 1992) and rarely forage more than 30 km from shore (Boersma et al., 2002; Carpenter-Kling et al., 2017; Kokubun et al., 2009; Lescro‘l & Bost, 2005; Robinson & Hindell, 1996; Trivelpiece et al., 1986; Wilson et al., 1998), we consider each region to be an independent population due to the unlikelihood that they interact with each other. Samples from each region were therefore pooled together into regional groups. The Falkland Islands are an archipelago located approximately 500 km from the coast of mainland South America in the southern Atlantic Ocean, situated north of the Antarctic Circumpolar Current on the relatively shallow Patagonian Shelf (Copello et al., 2011). Throughout the Falkland Islands, there are approximately 85 breeding colonies of Gentoo penguins, averaging approximately 100,000 breeding pairs each year (Baylis et al., 2013). South Georgia Island is a sub-Antarctic island at the south of the Polar Front. This island was previously the site of heavy whaling activities in the early part of the 20th century, but in the past five decades has only been inhabited by temporary seasonal personnel for science and tourism duties (Moore et al., 1999). Throughout South Georgia Island, there are approximately 80,000 100,000 breeding pairs of Gentoos annually (Varty et al., 2008). The Western Antarctic Peninsula is a region typically free of marine fast ice in the summer season with breeding colonies of Gentoo penguins scattered among the few ice-free areas along the coastline. The region sustained large numbers of whaling and sealing communities in the early 20th century, but now is only inhabited by seasonal science personnel at a small number of research stations. Throughout the Western Antarctic Peninsula, there are ! 14

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approximately 100,000 breeding pairs of Gentoo penguins annually (Borboroglu & Boersma, 2013). Study Species and Tissue Samples Gentoo penguins are a non-migratory seabird that remain near their breeding colony yearround (Black et al., 2017; Tanton et al., 2004; Williams & Rodwell, 1992). Throughout the breeding season, adult Gentoos capture prey during localized foraging trips and return typically on alternating days to feed their chicks the regurgitate which often contains whole or partiallydigested prey items (Handley et al., 2015). Penguin chicks undergo three stages of feather growth during development and maturation, 1) hatching with a natal down 2) plumage replacement at 1 to 2 weeks of age with a secondary down, 3) growth of adult plumage just prior to fledging. The secondary down was the sampling focus in this study, as it is derived from prey items taken by the parents and fed to the chicks. Once ingested, organic mercury circulating through the bloodstream associates with keratin disulfide bonds during feather synthesis which subsequently incorporates mercury directly in the feather structure (Appelquist et al., 1984; Bortolotti, 2010; Furness et al., 1986). Because parental foraging occurs near the natal colony (Handley et al., 2017), this secondary chick down reflects a comparatively localized signal of mercury exposure over a near-term temporal scale. In addition, chick down is grown simultaneously, therefore all intra-individual feathers are expected to have low variation in mercury concentration (Brasso et al., 2013). As such, penguin chick secondary down is ideally suited as a mercury biomonitoring tissue. ! 15

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Live Bird Sampling Methods Due to logistical constraints, live birds were only collected at three sites in the Falkland Islands and were obtained under permit from the Falkland Islands Government (#R22/2017), with landowner permission, and using protocols approved by University of Colorado Denver IACUC (#92517(08)1C). Each colony supported ~2000+ breeding pairs of Gentoo penguins. Gentoo penguin chicks in the post-guard phase (i.e. no longer protected by the parents) were captured using a long-handled net. Down feathers (10-12) were plucked from the breast using a pair of silicone-tipped forceps (DDP Instruments young tongue forceps, Miami, FL, USA) and stored at ambient temperature in small resealable plastic bags. Each chick was marked with a small amount of feather paint to prevent duplicate sampling (Sporting Pigeons Paint green, Pigeon Products International, Post Falls, ID, USA) and then released. Dead Bird Sampling Methods Dead Gentoo chicks were collected at the Falkland Islands under permit from the Falkland Islands Government (#R22/2017) and with landowner permission, from South Georgia Island under permit from the Government of South Georgia and the South Sandwich Islands (#2017/044), and from the Antarctic Peninsula under permit from the National Science Foundation (#ACA 2018-003). We found dead Gentoo penguin chicks by searching within each colony boundary (Falkland Islands only) and along transects extending ~100 m from the colony boundary to find carcasses that may have been moved by predators or scavengers. We used only birds that had died during the study season (as evidenced by presence of blood and/or comparative lack of decomposition, bleaching and weathering). We collected secondary down ! 16

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from the breast of intact carcasses when available, or otherwise from the legs just above the foot, sampling only white feathers using the same methods as detailed for live birds. Mercury Analysis All feather samples were sterilized per USDA/APHIS requirements (as detailed on the importation permit) for importation into the United States. Sterilization procedure required soaking the feather samples in 70% ethanol (LabChem, Zelienople, PA, USA) for 30 minutes. Following sterilization, we triple rinsed feather samples in deionized water and dried the samples under a fume hood for 24-48 hours at room temperature until all external moisture was removed. In preparation for mercury analysis, the feathers were cleaned to remove exogenously deposited contaminants. The feather samples were placed in a stainless steel screen and vigorously cleaned in environmental grade acetone (Alfa Aesar, Ward Hill, MA, USA) followed by rinsing under a flowing stream of deionized water. We completed this procedure three times for each feather sample to ensure all external oils and contaminates were removed. The feather samples were then dried in a fume hood at room temperature for 24 48 hours until all external moisture was removed. Approximately 10 mg of whole down feathers from each individual was analyzed for total mercury via atomic absorption spectrophotometry on a Nippon MA-3000 Direct Mercury Analyzer at Weber State University (Ogden, UT, USA). Each set of 20 samples analyzed was preceded and followed by two samples of a standard reference material (TORT-3, National Resource Council Canada). Mean percent recovery for the standard reference material was 101.4% (n=22) with relative significant differences in mercury concentration of 6.9%. Detection limit = 0.015 ng. Mercury concentrations in chick down are reported as mg/kg fresh weight (fw). ! 17

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Stable isotope analysis We used feather samples which had been previously cleaned and dried during preparation for mercury analysis as detailed above. We transferred approximately 1 mg of feather down material to 4 x 6 mm tin capsules and crimp-sealed the capsules. Feather samples were analyzed by continuous flow-isotope ratio mass spectrometry using a Carlo Erba NC1500 interfaced to a Micromass Optima mass spectrometer following established methods (Fry et al., 1992). Isotopic data were expressed in delta notation after normalizing to Air and V-PDB using the primary standards USGS 40 ( ! 15 N = -4.52 ä, ! 13 C = -26.39 ä) and USGS 41a ( ! 15 N = 47.55 ä, ! 13 C = 36.55 ä). Accuracy was assessed by analyzing USGS 40 as an unknown (+/0.1 for both isotopes). Analytical error was assessed by USGS 40, 41a, and an internal standard (+/<0.2 ä for both isotopes). Statistical analysis We removed any data points below detection limits decreasing our total sample size from 169 to 166. All plots and statistical analyses were completed in R ( R Core Team 2018) . Mercury data were log transformed where appropriate to approximate a normal distribution. We compared feather mercury concentration means among the three regions using Welch ANOVA testing for unequal variances (as tested by Fligner-Killeen test for homogeneity of variances) and followed the ANOVA model with Tukey HSD post hoc testing for pairwise comparisons. We fit linear regression of mercury on ! 15 N (as a proxy for diet) to estimate how variance in mean mercury changes as a function of diet (Atwell et al., 1998; Chen et al., 2008). Maps were created with QGIS (QGIS Development Team, 2019) using the Quantarctica package (Matsuoka et al., 2018). ! 18

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Results We collected samples from a total of 105 birds from the Falkland Islands, 23 birds from South Georgia Island, and 41 birds from the Antarctic Peninsula during the sampling period. Mean feather mercury concentrations with range in parentheses (mg/kg) are as follows: Falkland Islands: 1.583 mg/kg (0.726-4.739); South Georgia Island: 0.437 mg/kg (0.236-0.751); Antarctic Peninsula: 0.106 mg/kg (0.032-0.249). We found that in general, feather mercury concentrations of Gentoo penguins were higher in sub-Antarctic populations than the Antarctic Peninsula population. Additionally, distributions of Gentoo feather mercury concentrations were different ! 19 0 2 4 6 8 0 1 2 3 4 Feather Hg (mg kg ! 1 ) density Region AP SGI FI Fig. 2 Gentoo chick feather mercury concentration distributions for each region in the study, Antarctic Peninsula (AP), South Georgia Island (SGI) and Falkland Islands (FI).

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between each individual region ( F 2,163 = 225.2 , p = 2x10 -16 ; Fig. 2), with virtually no overlap in distributions between the Falkland Islands and the Antarctic Peninsula. Specifically: Falkland Islands meanHg > South Georgia Island meanHg > Antarctic Peninsula meanHg (Falkland Islands and S. Georgia Island p < 1x10 -14 ; S. Georgia Island and Antarctic Peninsula p = 0.0058). Our analysis of Gentoo feather stable isotopes indicates there is no overlap within the C and N delta space between penguins at any of the regions, suggesting each population is interacting with the food web differently or with different food webs entirely (Fig. 3; Jennings & van der Molin, 2015; Kelly, 2000; Perkins et al., 2014). Delta 13 C follows predictable latitudinal changes among the regions (Fig. 3). Mercury concentrations at each region are distinct from each ! 20 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 27 ! 24 ! 21 ! 18 10.0 12.5 15.0 ! 15 N (ä) ! 13 C (ä) Region ! AP SGI FI Fig. 3 Carbon and nitrogen ( ! 13 C and ! 15 N) stable isotope value (ä) relationships for Gentoo chick feather mercury concentrations at the Antarctic Peninsula (AP), South Georgia Island (SGI) and Falkland Islands (FI).

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other as shown by clustering among regions in ! 15 N:Hg space (Fig. 4). Based on similar slopes of the two regression lines, we point to consistent mercury biomagnification in both regions (Chen et al., 2008). The Falkland Island birds are foraging at a higher trophic level than the South Georgia Birds, however both of these sub-Antarctic populations have a similar relationship between mercury loading and trophic level as evidenced by their position on the same regression line. Due to similar ! 15 N values of primary producers at each region (Table 1), we conclude that the ! 15 N bases are not likely meaningfully different between the regions. ! 21 Fig. 4 Linear regression model of sub-Antarctic (dashed line) and Antarctic (solid line) Gentoo chick feather mercury (Hg) concentration relationship with ! 15 N stable nitrogen isotope values (ä) as a proxy for trophic level. Individual regions are Antarctic Peninsula (AP), South Georgia Island (SGI), and Falkland Islands (FI). ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 3 ! 2 ! 1 0 1 10.0 12.5 15.0 ! 15 N (ä) Log Feather Hg (mg kg ! 1 ) Region ! AP SGI FI

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Therefore, we provide evidence that the baseline for mercury bioavailability is higher in the subAntarctic than the Antarctic Peninsula as indicated by the change in y-intercept between the two regression lines (Chen et al., 2008). Discussion To explore differences in mercury exposure among regions, we compiled published data on Gentoo penguin foraging ecology and prey composition (Fig. 1) to examine differences in foraging ecology, and stable isotope values for representative prey species to compare baseline prey 15 N enrichment (Fig. 5). We compiled prey energy densities to indicate potential prey ! 22 Fig. 5 Carbon and nitrogen ( ! 13 C and ! 15 N) stable isotope value (ä) relationships for representative Gentoo penguin prey species for Antarctic Peninsula (AP), South Georgia Island (SGI) and Falkland Islands (FI). ! ! ! ! ! ! ! ! ! ! ! 30 ! 25 ! 20 ! 15 4 8 12 16 ! 15 N (ä) ! 13 C (ä) Prey ! ! ! Cephalopod Crustacean Fish Region ! AP SGI FI

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! 23 Prey Spp Region Prey Type Energy Density dw (kJ g -1 ) Author Chaenocephalus aceratus South Georgia Island Fish 25.4 Vanella et al., 2005 Champsocephalus gunnari South Georgia Island Fish 22.5 Vanella et al., 2005 Electrona antarctica South Georgia Island Fish 25.5 Donnelly et al., 1990 Electrona antarctica South Georgia Island Fish 29.4 Van de Putte et al., 2006 Electrona antarctica Antarctic Peninsula Fish 31.9 Ruck et al., 2014 Electrona carlsbergi South Georgia Island Fish 22.8 Clarke & Prince, 1980 Electrona carlsbergi South Georgia Island Fish 23.5 Cherel & Ridoux, 1992 Euphausia crystallorophias Antarctic Peninsula Crustacean 21.8 Ruck et al., 2014 Euphausia lucens Patagonia Crustacean 18.2 Ciancio et al., 2007 Euphausia superba South Georgia Island Crustacean 15.2 Torres et al., 1994 Euphausia superba Antarctic Peninsula Crustacean 21.1 Ruck et al., 2014 Euphausia superba Antarctic Peninsula Crustacean 17.1 Ichii et al., 2007 Euphausia superba South Georgia Island Crustacean 22.7 Clarke, 1980 Gobionotothen gibberifrons South Georgia Island Fish 21.2 Vanella et al., 2005 Krefftichthys anderssoni South Georgia Island Fish 26.4 Cherel & Ridoux, 1992 Loligo gahi South Georgia Island Cephalopod 16.2 Croxall & Prince, 1982 Loligo gahi Patagonia Cephalopod 21.16 Ciancio et al., 2007 Micromesistius australis Patagonia Fish 21.13 Ciancio et al., 2007 Munida gregaria Patagonia Crustacean 11 Ciancio et al., 2007 Munida spinosa Patagonia Crustacean 19.96 Lovrich et al., 2005 Munida subrugosa Patagonia Crustacean 17.42 Lovrich et al., 2005 Munida subrugosa Patagonia Crustacean 20.26 Lovrich et al., 2005 Pleuragramma antarcticum Antarctic Peninsula Fish 24.6 Ruck et al., 2014 Pseudochaenichthys georgianus South Georgia Island Fish 22.75 Vanella et al., 2005 Sprattus fuegensis Patagonia Fish 24.06 Ciancio et al., 2007 Themisto gaudichaudii Antarctic Peninsula Crustacean 12.7 Torres et al., 1994 Themisto gaudichaudii Patagonia Crustacean 22.19 Ciancio et al., 2007 Thysanoessa macrura Antarctic Peninsula Crustacean 28.5 Ruck et al., 2014 Thysanoessa macrura South Georgia Island Crustacean 17.03 Torres et al., 1994 Thysanoessa macrura Antarctic Peninsula Crustacean 16.1 Torres et al., 1994 Table 1 Summary of energy density (kJ g -1 dry weight) for representative Gentoo penguin prey species. Patagonia samples are substituted for Falkland Island samples for which we could not find data.

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selection with respect to optimal foraging strategy (Table 1; Clarke, 2001). Additionally, we include available prey mercury data in an attempt to quantify potential differences in baseline mercury bioavailability (Table 2). In our search of the literature, however, we were unable to find any published data on Gentoo prey species mercury loads in the Falkland Islands. ! 24 Table 2 Summary of mercury (Hg) concentrations (mg/kg) for representative baseline prey species of Gentoo penguins. Hg concentration includes standard deviation where reported. CV = coefficient of variation. AP = Antarctic Peninsula, SGI = South Georgia Island. Prey Spp Regio n Prey Type Hg Concentration (mg/kg) Hg CV Author Bovallia gigantea AP Crustacean 0.0364 Santos et al., 2005 Champsocephalus gunnari SGI Fish 0.02 ± 0.01 46.02 Anderson et al., 2009 Cheirimedon femoratus AP Crustacean 0.035 Santos et al., 2005 Dissostichus eleginoides SGI Fish 0.05 ± 0.02 43.3 Anderson et al., 2009 Electrona antarctica AP Mesopelagic fish 0.031 ± 0.017 53.31 Polito et al., 2016 Euphausia superba AP Crustacean 0.002 ± .001 34.74 Polito et al., 2016 Euphausia superba SGI Crustacean 0.01 ± .01 67.08 Anderson et al., 2009 Euphausia superba AP Crustacean 0.0346 Santos et al., 2005 Geotria australis SGI Fish 0.04 ± .04 87.08 Anderson et al., 2009 Gonatus antarcticus SGI Cephalopod 0.6 ± 0.02 3.57 Anderson et al., 2009 Gondogeneia antarctica AP Crustacean 0.037 Santos et al., 2005 Lepidonotothen larseni SGI Fish 0.27 ± 0.04 14.81 Anderson et al., 2009 Lepidonotothen squamifons AP Benthic fish 0.041 ± .21 50.37 Polito et al., 2016 Parachaenichthys georgianus SGI Fish 0.08 Anderson et al., 2009 Patagonotothen guntheri SGI Fish 0.03 ± .01 41.72 Anderson et al., 2009 Pleuragramma antarcticum AP Epipelagic fish 0.008 ± .002 22.85 Polito et al., 2016 Pseudochaenichthys georgianus SGI Fish 0.02 ± .01 49.79 Anderson et al., 2009 Psychroteuthis glacialis SGI Cephalopod 0.18 ± .11 60.61 Anderson et al., 2009 Themisto gaudichaudii SGI Crustacean 0.02 ± .01 48.45 Anderson et al., 2009

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Foraging ecology of Gentoo penguins Penguin foraging behavior is not well understood due to the cryptic nature of this group of seabirds in the marine environment. However, details on Gentoo foraging ecology come from previous studies employing stomach sampling, data loggers and video cameras to help understand snapshots of foraging behavior (Carpenter-Kling et al., 2017; Handley et al., 2018; Lescro‘l & Bost, 2005; Miller et al., 2009; Robinson & Hindell, 1996; Takahashi et al., 2008; Volkman et al., 1980) and used in combination with isotopic mixing models to estimate diet mixtures (Polito et al., 2016). This diet approximation helps standardize trophic level of the birds and its subsequent effects on mercury biomagnification in each population. The chick meal consists entirely of parental prey captures that are regurgitated and fed to the chick without the addition of stomach oil (Roby et al., 1989). Therefore, chicks are only exposed to dietary contaminants which are foraged by the adults in their foraging range (Bearhop et al., 2000). Most species of penguins appear to exploit some level of opportunism, a likely necessity when a species lives in an extreme climate to be able to take advantage of prey abundance while it is available (Kaspari & Joern, 1993). Gentoos in particular have also been noted as generalist foragers, consuming any prey item within their foraging range (Handley et al., 2017; Herman et al., 2017; Polito et al., 2015; Waluda et al., 2017). This foraging strategy is well-documented in various diet studies investigating Gentoo prey composition through stomach sampling (Croxall et al., 1988; Handley et al., 2017; Miller et al., 2009; PŸtz et al., 2001; Robinson & Hindell, 1996; Volkman et al., 1980; Xavier et al., 2017). Previous studies have recorded substantial differences in primary prey types (crustacean, cephalopod, fish; Fig. 1) of Gentoos at different colonies throughout the Falkland Islands in both the same year and across years (Clausen & PŸtz, 2002; ! 25

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Handley et al., 2017; PŸtz et al., 2001). Generalism is also reported in multi-year diet studies along the Antarctic Peninsula as well, although with a strong preference toward krill (Miller et al., 2009; Polito et al., 2016). Horizontal foraging range of Gentoos is reported by several GPS tracking studies that Gentoos rarely venture farther than 30 km from shore during foraging trips (Boersma et al., 2002; Carpenter-Kling et al., 2017; Kokubun et al., 2009; Lescro‘l & Bost, 2005; Robinson & Hindell, 1996; Trivelpiece et al., 1986; Wilson et al., 1998). This range suggests that Gentoos preferentially remain over the continental shelf based on bathymetry near their breeding sites (Carpenter-Kling et al., 2017; Handley et al., 2015). Gentoo winter foraging patterns are still relatively unknown, although camera monitoring studies and GPS tracking suggest that Gentoos remain nearby their breeding colonies throughout the winter (Black, et al., 2017; Tanton et al., 2004). Breeding season foraging behavior in particular is of interest to our study because Gentoo chick feather tissue is derived entirely from local prey resources provisioned by the parents (Handley et al., 2015). Vertical foraging patterns within the water column are more specific, however, with Gentoos preferring benthic zones as indicated by diving studies (Carpenter-Kling et al., 2017; Kokubun et al., 2009; Takahashi et al., 2008). Video cameras mounted on birds and stomach contents analyses confirm that Gentoo prey typically reside in the benthic zone (Handley et al., 2018; Robinson & Hindell, 1996), although stomach contents have been recovered that indicate Gentoos are also successful in mid-water and epipelagic zones as well, but in much smaller proportions (Handley et al., 2015; Lescro‘l et al., 2004). This foraging preference within the water column locates Gentoos generally deeper than most sympatric penguins which forage in ! 26

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mid-water and epipelagic zones (Kokubun et al., 2009; Miller et al., 2009; Trivelpiece et al., 1987). Dive and travel distance records also suggest that penguins which forage farther offshore perform shallower dives (Ludynia et al., 2012), in contrast to the Gentoo inshore benthic preference. Gentoo penguins have the deepest recorded dives of any of the small-bodied penguins (Wilson, 1998), with numerous max recorded dives near 200 m (Carpenter-Kling et al., 2017; Lescro‘l & Bost, 2005; Robinson & Hindell, 1996). This deep diving behavior is suggested to be a function of the Gentoo's larger mass and body size than sympatrics, except the large Aptenodytes spp, King and Emperor penguins which forage in deep waters far offshore (Bost et al., 1997; Ratcliffe et al., 2014; Trivelpiece et al., 1987; Volkman et al., 1980; Xavier et al., 2017). The benthic preference of Gentoos is likely a mechanism to differentiate resource exploitation by avoiding overlapping dive profiles with most sympatric penguin species and thereby decreasing competition (Cimino et al., 2016; Kokubun et al., 2009; Trivelpiece et al., 1987). This interspecific segregation combined with generalist foraging preferences likely plays a large role in the breeding success of Gentoos throughout a wide range of climates (Borboroglu & Boersma, 2013; Clucas et al., 2014; Lynch et al., 2008; Miller et al., 2009; Polito et al., 2015). Gentoo prey composition The three general classifications of penguin prey include crustaceans, cephalopods and fish, and different patterns of prey selection can expose a predator to different levels of bioaccumulating toxicants. The opportunistic/generalist foraging strategy of Gentoos is well pronounced across the three regions in our study (Fig. 1). Although most of the data is from stomach sampling which only provides snapshots of a penguin's foraging behavior, in general Gentoos in the Falkland Islands are eating a substantially higher proportion of fish than ! 27

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crustaceans in their diet, while Antarctic Peninsula Gentoos are foraging on a larger proportion of krill (Fig. 1). Diet studies in the Falkland Islands point to widely fluctuating diet compositions of Gentoos in that region. Stomach sampling conducted over multiple years in the Falkland Islands reported proportions of 30% to >95% fish by mass across a 10-year span (Putz et al., 2001) and 65% to >95% fish by mass across 4 years in a different study (Handley et al., 2015). Crustacean proportions range from <2% 44% by mass. Cephalopods were found in similar amounts of <2% 31% by mass (Clausen et al., 2005; Handley et al., 2015; PŸtz et al., 2001). Clausen et al., (2005) suggest that trawling studies of Gentoo foraging sites in the Falkland Islands demonstrate that the birds will preferentially select fish over crustaceans when both are available. The Antarctic Peninsula, alternatively, has much more consistently demonstrated Gentoos as preferentially selecting not only crustaceans as their primary prey, but more specifically krill as the most important diet component. South Georgia Island shows similar year-over-year variability to that of the Falkland Island populations, with a generally higher preference toward crustaceans than fish. South Georgia crustacean prey composition varies widely from 25-68% by mass, with fish composition ranging from 25-50% by mass. Cephalopods make up a minimal proportion of the Gentoo diet at South Georgia Island, ranging from negligible amounts in some years to 12% by mass (Fig. 1; Waluda et al., 2017; Xavier et al., 2017). Among all prey species, Gentoos along the Antarctic Peninsula strongly prefer krill (~60-80% by mass), most commonly Antarctic krill Euphausia superba and amphipods Themisto gaudichaudii over any other prey type. Fish components, while a smaller proportion of the diet along the Antarctic Peninsula, indicate a strong preference for benthic fish over mid-water and ! 28

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epipelagic species (Dimitrijevi" et al., 2018; Kokubun et al., 2009; Miller et al., 2009; Polito et al., 2016). For diet studies at South Georgia Island, crustacean prey are overwhelmingly dominated by the amphipod T. gaudichaudii while the bulk of fish species are benthic residents. Cephalopod specimens found in stomach contents analyses are likewise largely benthic squid species, commonly juveniles (Handley et al., 2015; Waluda et al., 2017; Xavier et al., 2017). In the Falkland Islands, Gentoo crustacean prey consists of only negligible amounts of krill, comprising less than 1% of the diet with the bulk of crustaceans most commonly bottom dwelling squat lobster Munida spp (Handley et al., 2015). Reported fish prey includes mostly benthic species and temporary juvenile demersal residents. Despite the large variability in prey among regions, these diet proportions indicate a strong benthic inshore foraging preference coupled with opportunistic foraging patterns in response to prey abundance at each region (Barrera-Oro et al., 2002; Kaspari & Joern, 1993; Lescro‘l et al., 2004; Polito et al., 2015; Polito et al., 2016; Williams, 1991). Energy densities and Gentoo prey selection As generalists, Gentoo penguins can readily exploit numerous prey species within their foraging range so long as it offsets the energy required for capture (Clarke, 2001; Mori & Boyd, 2004). Although studies demonstrate that krill energy densities are typically lower than most Gentoo fish prey (Table 1), variations by season, region, age status and gravid status of the female (Hartman & Brandt, 1995; Van de Putte et al., 2006) can cause seasonal increases in krill lipid content that approach or surpass energy content of many fish. Similarly, larger prey within the same species are thought to yield higher energy content (Tierney et al., 2002), making energy transfer through the food web difficult to predict for a generalist forager. Clarke & Prince (1980) ! 29

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noted this inconsistency in their early studies on the subject when they measured squid calorific values from the same site as ranging from 3.27 6.62 kJ g -1 wet weight. However, bioenergetics models on potential prey items indicate in general terms that fish prey offer the highest energy density of the reported penguin prey species (Table 1). The benefit of higher energy fish is exploited by Gentoos throughout the Falkland Islands and South Georgia Island. Different strategies predominate throughout the Antarctic Peninsula, however, where krill make up the largest proportion of Gentoo diet by mass. For a consumer to select such a lower energy dense prey is likely attributed to abundance of krill, lack of fish prey, or competition for resources. Because the major sympatric competitors are krill specialists, we expected Gentoos as generalists to exploit the higher energy density and decreased competition associated with fish-based foraging, in particular for a consumer that reportedly prefers fish over crustacean prey when both are available (Clausen et al., 2005). However, due to the population growth of Gentoos along the Antarctic Peninsula in recent years (Clucas et al., 2014), krill abundance is the most parsimonious explanation for krill selection by Gentoos in this region. High krill abundance throughout the Southern Ocean is proposed to be a result of the massive whaling in the early 20th century removing the major krill consumers from the region (Emslie et al., 2013). Numerous seabirds subsequently fill this gap and thrive off the abundance of low energy, readily available prey. The exploitation of a low energy prey is an effective strategy of numerous baleen whales that choose quantity over quality to become some of the most massive animals on the planet (Dolphin, 1987; Goldbogen et al., 2010). Similarly, low-energy krill in the Antarctic are a desirable prey for Gentoos who benefit from the high quantities coupled with increased foraging efficiency in dense krill swarms (Hazen et al., 2015). ! 30

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Certain inshore areas along the Antarctic Peninsula specifically favor the accumulation of large-scale krill aggregations and these hotspots are believed to be the exclusive food source for some penguin populations (Bernard et al., 2017). The major krill species along the Antarctic Peninsula, E. superba , is described as the most important component of the Southern Ocean food web (Lea et al., 2002), further suggesting that fish are a secondary prey component in many vertebrate consumers in the region (Bost et al. 1997; Cherel et al. 1994; Croxall et al., 1988; Hull, 1999; Raclot et al. 1998). The constraints placed on an inshore benthic forager, therefore, and in particular along the Antarctic Peninsula which has less biodiversity (Barrera-Oro, 2002), may subject Gentoos to a more homogeneous prey mix with fewer fish and cephalopod prey sources available, suggesting krill are more frequently encountered by the consumer. In the Falkland Islands and South Georgia Island, heterogeneous mixtures of inshore benthic prey provide Gentoos a greater choice of prey selection, allowing these consumers to reportedly bypass abundant crustaceans and select fish prey of higher energy content instead (Clausen et al., 2005), suggesting a possible deliberate choice made by Gentoos of energy density over prey abundance in some regions. Stable isotope analyses of Gentoo feathers and prey Our Gentoo feather stable isotope analyses indicate that Gentoo penguins interact with the food web differently at each region (Fig. 4), likely a function of their propensity for generalist foraging strategy combined with plasticity at each region. While differences in ! 13 C can be attributed to spatial differences in foraging, ! 15 N approximates trophic level at which the consumer is foraging. Delta 13 C values are complex in the marine environment, however. Higher latitude regions, enhanced by the cold environment around Antarctica, lead to depletion in 13 C, ! 31

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but so too does distance from shore and height in the water column (Cherel et al., 2000; Cherel & Hobson 2007; Kelly, 2000). Because 13 C depletion with increasing latitude is dynamic, we point to changes in carbon isotopes among the study regions as a confounding mix of differences in foraging strategy and latitude (Anderson et al., 2009). We attribute the 2-3¼ increase in latitude from the Falkland Islands to South Georgia Island, and the additional 8-10¼ farther south latitude to the Antarctic Peninsula as a large component of our reported 13 C decline across the C:N space (Fig. 3). Gentoos as benthic foragers should tend toward increased enrichment of 13 C, however foraging on mid-water krill along the Antarctic Peninsula likely depletes 13 C in those birds more than just from higher latitude alone. Because the Falkland Islands and South Georgia Island are at closer latitudes, we cautiously suggest that this relatively large difference between these two locations in regard to ! 13 C (Fig. 3) is primarily due to differences in foraging strategy with a slight latitudinal component. Comparisons of a biomagnifying toxicant such as mercury require standardization of trophic level estimations due to differences in nitrogen assimilation (Jennings & van der Molen, 2015). As such, we analyzed ! 15 N in Gentoo baseline prey species for each region (Fig. 5; Table 1). Isotopic values from baseline prey E. superba and T. gaudichaudii indicate these primary producers are located in similar C:N space in all three of our study regions (Anderson et al., 2009; Ciancio et al., 2010; Dunton et al., 2001; Handley et al., 2017; Polito et al., 2016; Rey et al., 2012). This similarity in baseline prey isotopic space allows for standardized comparisons in foraging ecology of Gentoos between the regions (Chen et al., 2008; Jennings & van der Molin, 2015; Perkins et al., 2014). Nitrogen enrichment generally increases with trophic level foraging (Anderson et al., 2009; Atwell et al., 1998; Bearhop et al., 2000; Wada et al., 1987), and because ! 32

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the bases of the food webs are assimilating 15 N similarly, we conclude that the Falkland Island birds are interacting with the food web at higher trophic levels than the other two regions. This higher trophic foraging points to a higher proportion of fish consumed in the Falkland Islands in relation to South Georgia Island and, more so, than the Antarctic Peninsula at which Gentoos appear to be exploiting higher amounts of krill in their diet. Likewise, numerous studies have reported positive linear correlations between trophic level and mercury loads (Atwell et al., 1998; Bearhop et al., 2000; Santos et al., 2005; Polito et al., 2016; Seixas et al., 2014). Due to biomagnification of mercury, the higher trophic fish in the diet of the Falkland Islands birds would therefore be expected to have increased mercury concentrations in their tissues as we report here (Fig. 3). However these biomagnifying effects of trophic level alone do not explain the regional shift in the mercury base (Fig. 4). Mercury levels of Gentoo feathers and prey Our regression analysis indicates that both the Falkland Islands and South Georgia Island populations have the same trophic level:mercury ratio (Fig. 4), suggesting that both of these populations have similar environmental exposure to mercury. The disparity in feather mercury concentrations between these two regions is thus a function of foraging at different trophic levels. A clear difference exists between the sub-Antarctic and the Antarctic Peninsula trophic level:mercury ratios, however, which indicates a shift in mercury uptake that is likely not accounted for by differences in dietary exposure, but instead by differences in environmental baseline mercury (Chen et al., 2008). A clear distinction exists in our data in Gentoo feather mercury levels between each region. Mercury has been demonstrated to biomagnify across the food web as well as to ! 33

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bioaccumulate with age of an organism (Fitzgerald et al., 2007). Differences in mercury loading can therefore be caused by differences in foraging behavior as well as differences in environmental mercury exposure. Studies have shown a positive correlation between mercury exposure and trophic level, in agreement with our data. Because Gentoos in the Falkland Islands are foraging at a higher trophic level than in either South Georgia Island or the Antarctic Peninsula (Fig. 4), biomagnification can be one source of increased mercury loading in Gentoos. Additionally, the benthic zone is enriched in methylmercury, the bioavailable form of mercury, due to deposition from microbial mercury methylation which takes place in the deeper, nutrientrich benthic waters, making marine sediments a sink for methylmercury (Cossa et al., 2011; Eagles-Smith et al., 2008; Fitzgerald et al., 2007). Therefore, bioavailable mercury is more available in the food web in the mesopelagic and benthic zones, subjecting consumers in these deeper layers to higher mercury exposure (Chen et al., 2008; Fitzgerald et al., 2007; Monteiro et al., 1996). Reports concur that benthic penguin prey show higher mercury concentrations than epipelagic prey (Polito et al., 2016; Thompson et al., 1998). Falkland Islands and South Georgia Island Gentoos foraging on benthic fish and crustaceans therefore consume a larger percentage of mercury-laden prey than the krill-foraging Gentoos of the Antarctic Peninsula (Cossa & Gobeil, 2000). Krill ecology studies throughout South Georgia Island and the Antarctic Peninsula report that inshore krill remain in the epipelagic zone during the day when Gentoos primarily forage (Cleary et al., 2016; Cresswell et al., 2009), pointing to lower mercury uptake. Because large differences in prey composition can affect mercury exposure (Thompson et al., 1998), foraging ecology cannot be denied a role in the higher mercury exposure associated with the Falkland Islands and South Georgia Island Gentoos in relation to the Antarctic Peninsula ! 34

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birds. Our evidence points also to increased baseline mercury in those regions due to the shift in the ! 15 N:Hg ratios (Fig. 4). Although we were able to find baseline mercury data for prey species at South Georgia Island and the Antarctic Peninsula, we could not find any published reports on penguin prey mercury data in the Falkland Islands with which to compare mercury baselines (Table 2). Without this data, one possible explanation for the baseline shift in mercury at South Georgia Island in particular can be attributed to its geographic location at the edge of the Antarctic Divergence along the Scotia Arc which causes high turbulence and deep ocean water upwelling (Hunt et al., 2016). This explanation, however, does not point to increased mercury availability in the Falkland Islands which is located 1500 km upstream from South Georgia Island on the shallower Patagonia Shelf. Global inputs of mercury pollution are likely impeded from entering the Antarctic due to the action of the Antarctic Circumpolar Current as a barrier to external input (Bargagli, 2005), suggesting global pollution elevations may be one cause for the mercury baseline shift outside of the Antarctic. Further, it is possible that Antarctic Peninsula Gentoos are feeding on more mid-water prey and have less mercury exposure than would benthic foragers. However, we contend that the different feather mercury concentrations in Gentoo penguins at the three regions are explained by a combination of differences in foraging behavior coupled with a likely higher level of ambient environmental mercury north of the Antarctic Circumpolar Current, potentially from global pollution. Conclusion Our results indicate higher feather mercury concentrations in Gentoo penguins in subAntarctic regions as compared to the Antarctic Peninsula. This disparity in mercury loading between regions is likely explained by both differences in foraging ecology as well as potentially ! 35

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increased baseline mercury in the marine system around the Falkland Islands and South Georgia Island. Because our mercury levels coincide with previous mercury studies on penguins throughout the Antarctic Peninsula, it appears that mercury levels in that region are stable. These novel data from the Falkland Islands requires further analysis, however, to understand why these levels are higher than the baseline levels of Antarctica. To explore this mechanism further, we recommend future studies are needed to investigate biomagnification factors and methylation potential of mercury in the food webs of these regions. In particular, primary producer mercury concentrations are needed in these regions for baseline comparisons. Additionally, baseline isotopic studies in the Falkland Islands would help understand mercury biomagnification factors as well as species-specific discrimination factors to help improve isotopic mixing models in this understudied region. ! 36

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