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HEAVY METAL CONCENTRATIONS IN SEA TURTLES AND
THEIR PREY IN THE NORTHWEST ATLANTIC
by
YiWynn Chan
A Thesis
Submitted to the Faculty of Purdue University
In Partial Fulfillment of the Requirements for the degree of
Master of Science
Department of Biological Sciences at Purdue Fort Wayne
Fort Wayne, Indiana
May 2024
2
THE PURDUE UNIVERSITY GRADUATE SCHOOL
STATEMENT OF COMMITTEE APPROVAL
Dr. Frank Paladino, Chair
Department of Biological Sciences
Dr. Ahmed Mustafa
Department of Biological Sciences
Dr. Michael Columbia
Department of Chemistry and Biochemistry
Dr. Samir Patel
Coonamessett Farm Foundation
Approved by:
Dr. Jordan M. Marshall
3
Mum, Dad
Thank you for having Lyn, Meng, and Zhen first
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ACKNOWLEDGMENTS
These past two years have been nothing short of educational and personal growth,
adventures and forming lifelong friendships. For this, I thank Drs. Frank Paladino, Samir Patel,
and Nathan Robinson for welcoming me into the sea turtle world. Thank you for your guidance
and support throughout my project, and for sharing your amassed encyclopedia of sea turtle
knowledge with me. To Dr. Michael Columbia, I thank you for imparting your chemistry wisdom
to me, which this project would have been impossible without. To my final committee member,
Dr. Ahmed Mustafa, I thank you for steering me through the hoops and hurdles of being an
international graduate student, ensuring I always knew my available options. I thank all of you for
being more than just my academic advisors, but for also being a mentor and a friend. I would also
like to thank Drs. Na Gou, Jordan Marshall, and Chelsea Clyde-Brockway for going out of their
way and helping me with operating chemistry instrumentations, running statistical analysis and
refining my writing.
This project would not have been possible without funding from The Leatherback Trust,
Purdue University Fort Wayne (PFW) Center for Marine Conservation and Biology, PFW's Jack
W. Schrey Distinguished Professor Fund, Coonamesset Farm Foundation, Inc, the Bureau of
Ocean Energy Management and National Oceanic and Atmospheric Administration (Interagency
Agreements M14PG00005, M10PG00075, M19PG00007), The Atlantic Sea Scallop Research
Set-Aside Program, and the Massachusetts Environmental Trust. I would also like to thank the
staff and volunteers from the Mass Audubon Wellfleet Bay Wildlife Sanctuary for their rescue and
recovery of cold-stunned sea turtles annually. I also thank the captain and crew of F/V Kathy Ann
for helping capture the loggerheads and prey samples in the Mid-Atlantic Bight and the captain
and crew of F/V Salvation for helping capture the loggerheads in the North Carolina Regions. This
work was conducted under ESA permit 23639. I also extend my gratitude to Laura St. Andrews
and Sophie Mills for their help in planning the initial stages of the project, collecting the samples,
and providing me with invaluable advice throughout this project.
To my families away from home, from UWCM to St. Olaf and now PFW and TLT, thank
you for being my home away from home away from home throughout these past 7 years. Life has
been more joyous, colorful, and full of positive vibes because of you. A special shoutout goes to
Sheldon, Isa, Trevor, Chelsea, Allyssa, and Faridah for being the best lab mates one could have
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ever asked for. Thank you for sharing all the seasons of life with me, feeding me, pretending to
understand my project and above all, being my friend. To Dr. Anne Gothmann and the CEMACS
family, thank you for sparking my interest in marine research, showing me that it is possible to
combine both my intellect and sense of adventure as a career. I would also like to thank Gina,
Marley and Sheridan for their unwavering support and availability always to set the perfect
ambience for writing my thesis. After spending 4+2 years in the mid-west, I hope this master’s
degree will finally allow me to move somewhere coastal, where my heart belongs.
And last but not least, to my friends and family, my biggest cheerleaders. I would not be
here today without you. Thank you for having always cheered me on throughout all the crazy
adventures I have ever embarked on. I know my life has been a wild rollercoaster ride with random
surprises to the middle of nowhere and showing up back home unannounced. Thank you for
dealing with my spontaneous shenanigans and welcoming me home with open arms, always. I
thank you for your eternal love and support since day 1, enabling me to fly wherever the wind may
blow.
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TABLE OF CONTENTS
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LIST OF TABLES
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LIST OF FIGURES
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LIST OF ABBREVIATIONS
Abbreviation
Definition
Ag
Silver
Al
Aluminum
As
Arsenic
Cd
Cadmium
Co
Cobalt
Cr
Chromium
Fe
Iron
MA
Cape Cod Bay, Massachusetts
MAB
Mid-Atlantic Bight
Mn
Manganese
NC
North Carolina
Ni
Nickel
Pb
Lead
Se
Selenium
Zn
Zinc
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ABSTRACT
The Northwest Atlantic Ocean, which surrounds the US eastern coastline, is an area rich
in marine life. The US eastern coastline is also highly urbanized, resulting in a lot of pollutants
(like heavy metals) entering the marine environment. This is of concern for long-lived marine
species like sea turtles. Since sea turtles are long-lived and highly migratory, their tissues can often
incorporate these pollutants through environmental and dietary exposure. I collected tissue
samples from 5 different sea turtle populations in the Northwest Atlantic and analyzed them for
concentrations of silver (Ag), aluminum (Al), arsenic (As), cadmium (Cd), cobalt (Co), chromium
(Cr), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), selenium (Se) and zinc (Zn) using an
Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The first chapter looks at skin (reflects
exposure ~1 year ago) and scute (reflects exposure from 4-6 years ago) samples collected during
necropsies of juvenile green (Chelonia mydas) (n=8), Kemp’s ridley (Lepidochelys kempii) (n=30)
and loggerhead (Caretta caretta) (n=17) turtles that were found cold-stunned in Cape Cod Bay,
Massachusetts. In scute samples, the heavy metal with the highest concentration for green turtles
was iron, zinc for loggerhead turtles, and arsenic for Kemp’s ridley turtles. In skin samples, the
heavy metal with the highest concentration for green turtles was iron, arsenic for loggerhead turtles,
and aluminum for Kemp’s ridley turtles. Overall, I found scute samples to have higher heavy metal
concentrations than skin samples. The second chapter looks at scute samples collected from
loggerhead turtles of different life stages. These samples were collected during necropsies of cold-
stunned loggerhead turtles from Cape Cod Bay, Massachusetts (CCB; n=17), as well as from live
loggerhead turtles in the Mid-Atlantic Bight (MAB; n=37) and off the coast of North Carolina (NC;
n=9). We also collected commonly known loggerhead turtle prey items including whelk
(Buccinum undatum) (n=12), Atlantic scallop (Placopecten magellanicus) (n=10) and Jonah crab
(Cancer borealis) (n=5) from the Mid-Atlantic Bight region to study the occurrence of
biomagnification through trophic pathways. NC loggerhead turtles had higher heavy metal
concentrations than other locations except for cadmium and zinc, where CCB loggerhead turtles
were higher. I found that all heavy metals except silver, cadmium, and lead appear to be
biomagnified (TTF>1) in loggerhead turtles. These two chapters provided baseline information on
heavy metal concentrations in sea turtles in east coast US.
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GENERAL INTRODUCTION
1.1 Introduction
The Northwest Atlantic Ocean, off the east coast of USA, is a coastal zone rich in resources.
Among the valuable fisheries species are the Atlantic Sea Scallop and American lobster fishery
(National Marine and Fisheries Service 2017, Seidov et al. 2022). The NW Atlantic coast is also
an important recruitment area for juvenile Atlantic green turtles (Chelonia mydas), Kemp’s ridley
turtles (Lepidochelys kempii), and loggerhead turtles (Caretta caretta) after their oceanic
development stage. Other adult turtles like loggerhead and Kemp’s ridley turtles also forage in the
warmer months of summer and fall (Morreale et al. 1992) and migrate towards the Southwest
Atlantic Ocean in the colder winter months (Musick et al. 1994).
Apart from being important natural habitats for flora and fauna, the US east coast is also
home to numerous development projects and technological advancements. This has resulted in a
lot of runoff pollution from agriculture, farmland, industrialization, and roads to enter the NW
Atlantic Ocean (NRC 2000, Howarth et al. 2002, Valiela and Bowen 2002). Some of the known
pollutants are plastic, rare earth elements, and heavy metals (Herbst and Klein 1995, da Silva et al.
2014).
Heavy metals are naturally occurring inorganic elements. They can be found at low
concentrations in rocks, water, and soil in non-polluted ecosystems. However, anthropogenic
activities have led to an increase in concentration of these elements in the environment. For
example, smelting activities release cadmium and arsenic into terrestrial, aquatic, and marine
environments (ATSDR 2003, ATSDR 2004, ATSDR 2007). While they are harmless and can even
be beneficial to organisms at low concentrations, they become toxic at high concentrations
(ATSDR 2004, 2012). The release of heavy metals into marine environments is especially true in
the NW Atlantic as this area undergoes a lot of development both on land and in the coastal area.
As the NW Atlantic Ocean is an area where environmental pollution and ecological habitats
intersect, it is important to be able to measure pollution levels in the area as well as its organisms.
Previous studies have found that the long-lived and highly migratory nature of sea turtles make
them possible biomarkers for marine pollution (Bjorndal 1985, Omedes et al. 2024). This is
because they incorporate elements from their diet and environment into their body tissues
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(Seminoff et al. 2006, Vander Zanden et al. 2013, Barraza et al. 2019, Franzellitti et al. 2004). For
example, skin samples have a quicker formation rate, which is likely to reflect heavy metal
exposure of sea turtles within approximately 1 year (Seminoff et al. 2006). On the other hand,
scute samples (from the carapace) have a longer turnover rate and have been found to reflect heavy
metal exposure from 4-6 years ago (Vander Zanden et al. 2013).
We analyzed heavy metal concentrations in a few different populations of sea turtles found
in the NW Atlantic Ocean. In the second chapter of my thesis, I analyzed cold-stunned juvenile
green, loggerhead, and Kemp’s ridley turtles that were encountered in Cape Cod Bay,
Massachusetts. These are turtles that did not migrate southward early enough after foraging in the
north over summer and were cold stunned (Henwood and Ogren 1987, Keinath 1993, Still et al.
2005). These turtles were juveniles and were probably just transitioning their diet, with loggerhead
and Kemp’s ridley turtles transitioning to a predominantly carnivorous diet (Nelson 1988, Reyes-
López et al. 2021) and green turtles transitioning to a predominantly herbivorous diet (Bjorndal
1985), We collected skin and scute samples to investigate if the different tissues reflect heavy
metals from different diet types.
The third chapter of my thesis focuses on loggerhead turtle scutes sampled from different
sites within the NW Atlantic Ocean. These turtles were also of different life stages — Cape Cod
Bay turtles were the smallest juveniles, North Carolina turtles were late-stage juveniles, and Mid-
Atlantic Bight turtles were considered as sub-adults. I also collected loggerhead turtle prey to study
the occurrence of biomagnification through their trophic pathways. These preys are whelk
(Buccinum undatum), Atlantic scallop (Placopecten magellanicus) and Jonah crab (Cancer
borealis). Biomagnification was calculated as a ratio of an element in the tissue compared to the
element in the prey item (DeForest et al. 2007). Biomagnification is observed when the ration,
which is denoted as Trophic Transfer Factor (TTF), is greater than 1 (Matthews and Fisher 2008).
As loggerhead turtles are considered as high-level predators, they are susceptible to the effects of
biomagnification of heavy metals.
To date, no other studies have conducted an extensive study on heavy metal concentrations
in sea turtles from the NW Atlantic Ocean. I measured seven essential heavy metals (chromium,
cobalt, iron, manganese, nickel, selenium, and zinc) to see if the different sea turtle populations
were obtaining similar concentrations. I also measured five non-essential heavy metals (arsenic,
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aluminum, cadmium, lead, and silver) to better understand the state of our turtles’ health and
possible physiological implications of the heavy metals on our turtles.
1.2 References
Agency for Toxic Substances and Disease Registry. 2003. Toxicology Profile for Selenium.
Department of Health and Human Services - USA.
Agency for Toxic Substances and Disease Registry. 2004. Toxicology Profile for Cobalt.
Department of Health and Human Services - USA.
Agency for Toxic Substances and Disease Registry. 2007. Toxicological Profile for Arsenic.
Department of Health and Human Services - USA.
Agency for Toxic Substances and Disease Registry. 2012. Toxicological Profile for Cadmium.
Department of Health and Human Services - USA.
Barraza AD, Komoroske LM, Allen C, Eguchi T, Gossett R, Holland E, Lawson DD, LeRoux RA,
Long A, Seminoff JA. Lowe CG. 2019. Trace metals in green sea turtles (Chelonia mydas)
inhabiting two southern California coastal estuaries. Chemosphere 223:342–350.
da Silva CC, Varela AS, Barcarolli IF, Bianchini A. 2014. Concentrations and distributions of metals
in tissues of stranded green sea turtles (Chelonia mydas) from the southern Atlantic coast of
Brazil. Science of The Total Environment 466-467:109–118.
DeForest DK, Brix KV, Adams WJ. 2007 Assessing metal bioaccumulation in aquatic environments:
The inverse relationship between bioaccumulation factors, trophic transfer factors and
exposure concentrations. Aquatic Toxicology 84:236-246.
Franzellitti S, Locatelli C, Gerosa G, Vallini C, Fabbri E. 2004. Heavy metals in tissues of
loggerhead turtles (Caretta caretta) from the northwestern Adriatic Sea. Comparative
Biochemistry and Physiology C 138:187-194.
Bjorndal KA. 1985. Nutritional ecology of sea turtles. Copeia 1985(3):736-751.
Henwood TA, Ogren LH. 1987. Distribution and migrations of immature Kemp’s ridley turtles,
Lepidochelys kempi, and green turtles, Chelonia mydas, off Florida, Georgia and South
Carolina (USA). Northeast Gulf Science 9:153-159.
Herbst LH, Klein PA. 1995. Green turtle fibropapillomatosis: Challenges to assessing the role of
environmental cofactors. Environmental Health Perspectives 103:4, 27–30.
Howarth R, Walker D, Shatpley A. 2002. Sources of nitrogen pollution to coastal waters of the
United States. Estuaries 25:656-676.
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Keinath JA. 1993. Movements and behavior of wild and head-started sea turtles. Ph.D. Thesis,
College of William and Mary, Williamsburg, Virginia.
Omedes S, Crespo-Picazo JL, Robinson NJ, García-Párraga D, Sole M. 2024. Identifying
biomarkers of pollutant exposure in ocean sentinels: Characterisation and optimisation of
B-esterases in plasma from loggerhead turtles undergoing rehabilitation. Chemosphere
348:140770. https://doi.org/10.1016/j.chemosphere.2023.140770.
Matthews T, Fisher NS. 2008. Trophic transfer of seven trace metals in a four-step marine food
chain. Marine Ecology Progress Series 367: 23-33. Doi:10.3354/meps07536.
Morreale SJ, Meylan AB, Sadove SS, Standora EA. 1992. Annual occurrence and winter mortality
of marine turtles in New York waters. Journal of Herpetology 26(3): 301-308.
Musick JA, Barnard D, Keinath JA. 1994. Aerial estimates of seasonal distribution and abundance
of sea turtles near the Cape Hatteras faunal barrier. In Schroeder BA, Witherington BE
(compilers). Proceedings of the Thirteenth Annual Symposium on Sea Turtle Biology and
Conservation. NOAA Technical Memorandum NMFS-SEFSC-341, pp.121-123.
National Marine Fisheries Service. 2017. Fisheries of the United Stated, 2016. U.S. Department of
Commerce NOAA Current Fishery Statistics No. 2016.
National Research Council. 2000. Clean coastal waters: Understanding and reducing the effects of
lllltrielll pollution. Washington, DC, National Academies Press.
Nelson DA. 1988. Life history and environmental requirements of loggerhead turtles. US Fish and
Wildlife Service Biology Report 88(23). U.S. Army Corps of Engineers TR EL-86-2(Rev.).
Reyes-López MA, Camacho-Sánchez FY, Hart CE, Leal-Sepúlveda V, Zavala-Félix KA, Ley-
Quiñónez CP, Aguirre AA, Zavala-Norzagaray AA. 2021. Rediscovering Kemp’s ridley sea
turtle (Lepidochelys kempii): Molecular analysis and threats. IntechOpen. doi:
10.5772/intechopen.96655.
Seidov D, Mshonov AV, Baranova OK, Boyer TP, Nyadjro E, Bouchard C, Cross SL. 2022.
Northwest Atlantic Regional Ocean climatology version 2. NOAA Atlas NESDIS 88, Silver
Spring, MD, 75 pp. https://doi.org/10.25923/c6fz-fp67.
Seminoff JA, Jones TT, Eguchi T, Jones DR, Dutton PH. 2006. Stable isotope discrimination (δ13C
and δ15N) between soft tissues of the green sea turtle Chelonia mydas and its diet. Marine
Ecology Progress Series 308:271-278. https://doi.org/10.3354/meps308271.
Still BM, Griffin CR, Prescott R. 2005. Climatic and oceanographic factors affecting daily patterns
of juvenile sea turtle cold-stunning in Cape Cod Bay, Massachusetts. Chelonian
Conservation and Biology 4(4):870-877.
Valiela I, Bowen JL. 2002. Nitrogen sources to watersheds and estuaries: role of land cover mosaics
and losses within watersheds. Environmental Pollution 118(2):239-248.
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Vander Zanden HB, Bjorndal KA, Mustin W, Ponciano JM, Bolten AB. 2012. Inherent variation
in stable isotope values and discrimination factors in two life stages of green turtles.
Physiological and Biochemical Zoology 85:431-441. https://doi.org/10.1086/666902.
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HEAVY METAL CONCENTRATIONS IN SKIN AND SCUTE OF
COLD-STUNNED GREEN, KEMP’S RIDLEY AND LOGGERHEAD SEA
TURTLES IN CAPE COD BAY, MASSACHUSETTS
2.1 Abstract
Heavy metal pollution is a growing threat to marine life worldwide. As sea turtles are a long-
lived and migratory species, their tissues often incorporate these pollutants over vast ocean habitats.
In turn, this means that sea turtles can function as broad-scale indicators of heavy metal pollution.
To determine heavy metal concentrations in the tissues of sea turtles in the Northwest Atlantic, we
collected skin and scute samples during necropsies of green (Chelonia mydas) (n=8), Kemp’s
ridley (Lepidochelys kempii) (n=30) and loggerhead (Caretta caretta) (n=17) that were found cold-
stunned in Cape Cod Bay, Massachusetts. These sea turtle species have different diets, with their
skin reflecting heavy metal exposure within approximately 1 year and their scute reflecting
exposure from 4-6 years ago. We analyzed the concentrations of silver (Ag), aluminum (Al),
arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), iron (Fe), manganese (Mn), nickel (Ni),
lead (Pb), selenium (Se) and zinc (Zn) using an Inductively Coupled Plasma Mass Spectrometry
(ICP-MS). We found different turtle species to have different heavy metals that were of the highest
concentrations. In scute samples, the heavy metal with the highest concentration for green turtles
was iron (mean ± SD wet weight; 351.8 ± 505.1 μg g-1), zinc (202.8 ± 51.0 μg g-1) for loggerhead
turtles, and arsenic (4.68 ± 2.54 μg g-1) for Kemp’s ridley turtles. In skin samples, the heavy metal
with the highest concentration for green turtles was iron (46.2 ± 46.2 μg g-1), arsenic (5.07 ± 2.26
μg g-1) for loggerhead turtles, and aluminum (25.0 ± 38.2 μg g-1) for Kemp’s ridley turtles. Across
all species and heavy metals, scute samples had higher heavy metal concentrations compared to
skin samples. This is likely due to the accumulation of unwanted heavy metals in the keratinized
tissues and their longer turnover rate. Arsenic, cadmium, and cobalt concentrations found in tissues
of these stranded turtles are above normal levels found in most other living organisms, including
humans.
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2.2 Introduction
Modern day anthropogenic activities have resulted in an increase in pollutants in the
environment (Borrelle et al. 2020). While much media attention often focuses on plastic pollution,
other pollutants such as heavy metals that are typically invisible to the human eye are also of
increasing concern (Herbst and Klein 1995, da Silva et al. 2014). These heavy metals are often
naturally occurring in non-polluted ecosystems and the organisms that inhabit them; however, as
their concentrations rise, they can become toxic to wildlife (ATSDR 2004, 2006, 2012).
Heavy metals can be found at all trophic levels. At the base of the food chain, plants are
exposed to heavy metals from uptake of the environment. This leads to some plants, like seaweed,
having high heavy metal concentrations in their tissues (Zhou et al. 2008). Despite organisms at
the base of the food chain being exposed to heavy metals, different heavy metals biomagnify
through trophic levels differently. Some heavy metals (i.e. mercury, lead, and zinc) increase in
concentration along trophic pathways, some (i.e. arsenic and nickel) are not passed on through
trophic levels, and some (i.e. cadmium, chromium, and copper) remain constant (Sun et al. 2020).
With regards to heavy metal that biomagnify, organisms that occupy higher trophic levels are
likely to have higher heavy metal concentrations (Jakimska et al. 2011). Thus, the
biomagnification of certain heavy metals often poses a threat to high-level predators, like sea
turtles. It is therefore important to identify species that can be used as indicators to assess heavy
metal concentrations within the marine environment.
Heavy metals can be divided into essential elements (i.e. chromium, iron, selenium, and zinc)
that play key roles in physiological and biochemical pathways and non-essential elements (i.e.
cadmium and lead) that are not commonly useful to most organisms (Brown and Depledge 1985,
Nordberg et al. 2007). Most heavy metals like cobalt, arsenic, and selenium are naturally found in
low concentrations. Some of these natural sources include rocks, soil, water, and air (ATSDR 2003,
ATSDR 2004, ATSDR 2007). However, many of these elements are entering the environment as
by-products of anthropogenic activities. For example, smelting facilities and coal-fired
powerplants release cobalt (ATSDR 2004) and pesticides, farm animal feed, and electrical
conductors release arsenic (ATSDR 2007). Even heavy metals like cadmium which are not as
commonly found make its way into the environment through activities such as electroplating and
smelting (ATSDR 2008). As a result, many of these elements dissolve and are deposited into the
environmental soil, sediment and water columns (ATSDR 2007).
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Sea turtles are long-lived, migratory species (Bjorndal 1985). As they migrate over wide
geographic areas, the elements that they are exposed to may be incorporated into their bodily
tissues (Seminoff et al. 2006, Vander Zanden et al. 2013, Barraza et al. 2019, Franzellitti et al.
2004). Therefore, sea turtles can serve as possible biomarkers for pollution in the marine
ecosystem (Bjorndal 1985, Omedes Martínez et al. 2024). In the northwest Atlantic coast, juvenile
Atlantic green turtles (Chelonia mydas), Kemp’s ridley turtles (Lepidochelys kempii), and
loggerhead turtles (Caretta caretta) recruit to the Atlantic coast after their oceanic development
stage. These juvenile turtles forage in the Northwest Atlantic Ocean during the summer and fall
when the surface water is warm (Morreale et al. 1992), then, as the water temperature drops, they
migrate southward to the Southwest Atlantic Ocean for the winter (Musick et al. 1994). However,
if sea turtles do not migrate southward early enough, they are susceptible to cold-stunning
(Henwood and Ogren 1987, Keinath 1993, Still et al. 2005). Within a migratory cycle, these turtles
may therefore be exposed to pollution from most of the continental shelf of the northwestern
Atlantic Ocean.
The recruitment of these turtles to the Atlantic shelf is usually accompanied by a transition
in diet. This ontogenetic shift occurs at >20 cm straight carapace length in green and Kemp’s ridley
turtles, and >25cm in loggerhead turtles. While all three species are omnivorous in their oceanic
habitats, when recruiting to coastal habitats, green turtles transition to a predominantly herbivorous
diet (Bjorndal 1985), while loggerhead and Kemp’s ridley turtles transition to a predominantly
carnivorous diet (Nelson 1988, Reyes-López et al. 2021). As the turtles transition their diet, the
elements which they were exposed to at different life stages are reflected in different bodily tissues.
It has been demonstrated that skin samples have a quicker formation rate, reflecting exposure
within approximately 1 year (Seminoff et al. 2006), whereas scute samples reflect exposure from
4-6 years ago (Vander Zanden et al. 2013). Therefore, skin samples are likely to reflect more recent
(and possibly local to the NW Atlantic) heavy metal exposure of sea turtles to their environment
and diet, whereas scute samples are likely to reflect diet and environmental exposure from their
oceanic phase.
Numerous studies have analyzed heavy metal concentrations in sea turtle tissues, and
primarily in the organs of dead sea turtles (Sakai et al. 2000, van de Merwe et al. 2010). We
propose that skin and scute samples are suitable indicators of turtle diet and habitat at a given
period relative to the rate of tissue formation. To date, within the NW Atlantic, there has only been
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one heavy metal study conducted on cold-stunned sea turtles in Cape Cod Bay and no other studies
in the Mid-Atlantic Bight region. While Innis et al. (2008) investigated heavy metal concentrations
in Kemp’s ridley sea turtles from the region, no other studies have analyzed heavy metal
concentrations in green and loggerhead turtles even though they occupy very similar habitats
(Robinson et al. 2020).
The primary goal of our study was to analyze the heavy metal concentrations in the skin and
scute samples of green, loggerhead, and Kemp’s ridley turtles that cold-stunned in Cape Cod Bay,
Massachusetts. We measured seven essential heavy metals (chromium, cobalt, iron, manganese,
nickel, selenium, and zinc) to see if the different sea turtle species were obtaining similar
concentrations. We measured five non-essential heavy metals (arsenic, aluminum, cadmium, lead,
and silver) to better understand the state of our turtles’ health and possible physiological
implications of the heavy metals on our turtles. Apart from selecting most heavy metals to compare
NW Atlantic values to other parts of the world (Faust et al. 2014, Barraza et al. 2019, Jerez et al.
2010, Sakai et al. 2000), few studies have measured aluminum and selenium and sea turtle scute
samples (Komoroske et al. 2011, Rossi et al. 2015, Mondragón et al. 2023, Barraza et al. 2019)
and no other known studies have analyzed silver and aluminum and sea turtle skin samples.
The objectives of this study were 1) to investigate the difference in heavy metal
concentration between the different sea turtle species and 2) to investigate the changes in exposure
of sea turtles to heavy metals in recent years through comparative tissue analysis. 3) to compare
values obtained from our studies to studies conducted in other parts of the world. We predict that
green turtle skin would exhibit higher zinc and cobalt concentrations as these are heavy metals
associated with a more herbivorous diet (Smith and Carson 1981). Conversely, we predict that
loggerhead and Kemp’s ridley turtles’ skin would exhibit higher arsenic and cadmium
concentrations which are found in cephalopods (Bustamante et al. 1998, Storelli and Marcotrigiano
2003). We also predict that scute samples would have overall higher heavy metal concentrations
as keratinized tissue are often used to deposit unwanted inorganic elements (Mondragón et al. 2023)
and that heavy metals linger in keratinized tissue longer than skin tissue (Seminoff et al. 2006 and
Vander Zanden et al. 2013). We also predict that sea turtles from NW Atlantic are likely to have
higher heavy metal concentrations when compared to studies conducted in more pristine
environments.
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2.3 Methods
2.3.1 Field Sample Collection
This study took place in Cape Cod Bay, Massachusetts, USA, a 1100 km2 semi-enclosed
bay in the south of the Gulf of Maine (Figure 2-1). During the necropsies organized by
Massachusetts Audubon Wellfleet Bay Wildlife Sanctuary (WBWS) after the winters of 2019 and
2021, researchers collected skin and scute samples from cold-stunned, recently deceased green
(n=8), loggerhead (n=17), and Kemp’s ridley (n=30) sea turtles. We collected skin samples from
the right shoulder after sterilizing the area between the neck and flipper by twisting a 6mm biopsy
punch about 2mm deep to collect 0.5g of tissue (Eckert et al. 1999). We collected scute samples
(~0.5g) by scraping the biopsy punch along the rear of the first lateral scute of each turtle (Day et
al. 2005).
Figure 2-1 Map of study area in the USA. The red circle is Cape Cod Bay, highlighting the hook-
shaped bay which results in the entrapment of numerous turtles as they migrate south every winter.
2.3.2 Heavy Metal Analysis
We analyzed both skin and scute samples for silver (Ag), aluminum (Al), arsenic (As),
cadmium (Cd), cobalt (Co), chromium (Cr), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb),
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selenium (Se) and zinc (Zn) at Purdue University West Lafayette. Heavy metal concentrations
were determined using an Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Thermo
Scientific Element 2) equipped with a Teledyne Cetac Aridus II nebulizer following their standard
protocols (N. Gou, personal communication, September 15, 2022). We weighed out 0.2-0.5g of
each sample and added 2mL of ultra-high purity nitric acid and 0.5mL of ultrapure water into
borosilicate digestion vessels (Anton Paar 179436). We digested these samples along with method
blanks in a microwave digestor (Anton Paar 7000 Microwave Digestion System) using the
preconfigured ‘Organic’ program. After digestion, we diluted the samples and blanks to a final
volume of 50mL using ultrapure water and added 125 μL of 5 ppb indium as an internal standard.
We prepared standard solutions ranging from 0.01-1000ppb for all heavy metals from 10 ppm
standard solutions purchased from Inorganic Ventures. The limits of detection (LOD) of each
heavy metal were calculated as three times the standard deviation of the ten independent
measurements of the blank, divided by the slope of the calibration curve. As we were analyzing
110 samples for 12 heavy metals each, we recalibrated the ICP-MS between runs, resulting in a
range of LODs. The range of LODs (µg mL-1) of each heavy metal are as follows: Ag: 0.00001-
0.00007, Al: 0.00012-0.00246, As: 0.00001-0.00007, Cd: 0.00002-0.00008, Co: 0.00001-0.00011,
Cr: 0.00001-0.00006, Fe: 0.00894-0.01387, Mn: 0.00003-0.00014, Ni: 0.00003-0.00029, Pb:
0.00001-0.00004, Se: 0.00032-0.00153, Zn: 0.00006-0.00051. We considered heavy metal
concentrations below the LOD as undetectable (i.e. 0). We report heavy metal concentrations in
µg g⁻¹ wet weight of the tissue samples.
2.3.3 Statistical Analysis
We conducted statistical analyses using R (R Core Team, 2020). We executed a two-way
ANOVA with post-hoc Tukey HSD tests to compare the concentrations of each heavy metal
between species (green, loggerhead, Kemp’s ridley) and sample types (skin, scute). When the
assumption of normality was not met for ANOVA and post-hoc Tukey HSD, we log transformed
the values of non-parametric heavy metal concentrations to normalize the data. For running
correlations, we used Pearson’s correlation to investigate the significance between heavy metals
in skin and scute samples for parametric datasets, and Spearman’s correlation for non-parametric
datasets. We used Pearson’s and Spearman’s correlation to assess the relationships between heavy
metal concentrations and turtle body size. When needed, we converted the heavy metal
26
concentrations of loggerhead scutes of other studies from µg g⁻¹ dry weight to µg g⁻¹ wet weight,
using the value of 29.1% moisture content (Rodriguez et al. 2022). However, to the best of our
knowledge, there are no known moisture values for sea turtle skin samples that could help us
standardize heavy metal concentrations reported in dry weight.
2.4 Results
We sampled 55 juvenile to subadult sea turtles for skin and scute tissues (n=8 green; n=30
Kemp’s ridley; n=17 loggerhead; Table 2-1).
Table 2-1 Range, mean and standard deviation of straight carapace length (SCL) of the three
different sea turtle species collected during cold-stunned events from Cape Cod Bay,
Massachusetts, USA.
Species
n
SCL (cm)
Range
Mean ± SD
Green
Chelonia mydas
8
26.5-33.9
28.7 ± 2.33cm
Kemp’s ridley
Lepidochelys kempii
30 18.6-32.8
25.8 ± 2.84cm
Loggerhead
Caretta caretta
17 28.5-69.2
51.7 ± 9.7cm
2.4.1 Heavy metal concentrations between skin and scute samples within each species
For all species, eight out of twelve heavy metals were found in higher concentrations in
scute samples compared to skin samples (silver, aluminum, cadmium, cobalt, chromium, iron,
manganese, and zinc). On the other hand, arsenic and selenium concentrations in green turtles and
loggerhead turtles as well as nickel concentrations in green turtles and Kemp’s ridley turtles were
found to be higher in skin samples compared to scute samples. Kemp’s ridley turtles had nine
elements that were significantly different (p<0.05) between its skin and scute samples, loggerhead
turtles had four and green turtles had two elements (
24
Table 2-2). For Kemp’s ridley turtles, they were aluminum (skin=25.013±38.247 µg g-1;
scute=67.581±77.604 µg g-1, p=0.0004), cadmium (skin=0.058±0.033 µg g-1;
scute=0.322±0.178µg g-1, p<0.0001), cobalt (skin=0.027±0.029 µg g-1; scute=0.238±0.379 µg g-1,
p<0.0001), chromium (skin=0.262±0.350 µg g-1; scute=1.080±1.419 µg g-1, p<0.0001), iron
(skin=44.331±60.660 µg g-1; scute=151.801±116.620 µg g-1, p<0.0001), manganese
(skin=0.635±0.931 µg g-1; scute=3.083±3.076 µg g-1, p<0.0001), nickel (skin=3.330±12.226 µg g-
1; scute=2.290 ±1.441 µg g-1, p=0.003), lead (skin=0.061±0.077 µg g-1; scute=0.397±0.318 µg g-
1, p<0.0001), and zinc (skin=19.980±6.822 µg g-1; scute=166.972±72.265 µg g-1, p<0.0001). For
loggerhead turtles, they were arsenic (skin=5.069±2.258 µg g-1; scute=1.792±0.849 µg g-1,
p=0.0001), cadmium (skin=0.092±0.025 µg g-1; scute=0.256±0.150 µg g-1, p<0.0001), chromium
(skin=0.153±0.088 µg g-1; scute=0.854 ± 0.968 µg g-1, p=0.002) and zinc (skin=11.271±4.850 µg
g-1; scute=201.786±50.971 µg g-1, p=0). As for green turtles, they were manganese
(skin=0.529±0.655 µg g-1; scute=5.209±9.128µg g-1, p=0.04) and zinc (skin=21.561±9.226 µg g-
1; scute=108.095±33.384 µg g-1, p=0).\
25
Table 2-2 Heavy metal concentrations detected in blood, skin and scute samples of green (n=8), Kemp’s ridley (n=30) and loggerhead
(n=17) turtles from Cape Cod Bay, Massachusetts, USA.
NOTE: Skin and scute heavy metal concentration values are µg g-1 wet weight
n = number of samples that were detected for respective heavy metals
a indicates significant differences between different tissues of the same species
b indicates significant differences between tissues of different species
Skin
Scute
Elements
Species
n
mean ± SD
n
mean ± SD
Non-essential elements
Silver
Green
6
0.006 ± 0.007
1
0.034
Kemp’s ridley
11 0.009 ± 0.011
4
0.049 ± 0.014
Loggerhead
11 0.009 ± 0.006
2
0.028 ± 0.010
Aluminum Green
8
22.911 ± 33.710
7
140.273±224.762
Kemp’s ridley
30 25.013 ± 38.247 a
28 67.581 ± 77.604 a
Loggerhead
17 19.015 ± 14.860
17 43.695 ± 35.238
Arsenic
Green
8
3.614 ± 2.569
7
2.212 ± 1.606
Kemp’s ridley
30 4.580 ± 1.753
30 4.678 ± 2.536 b
Loggerhead
17 5.069 ± 2.258 a
17 1.792 ± 0.849 a, b
Cadmium
Green
8
0.075 ± 0.052
5
0.144 ± 0.046
Kemp’s ridley
29 0.058 ± 0.033 a, b
26 0.322 ± 0.178 a
Loggerhead
17 0.092 ± 0.025 a, b
17 0.256 ± 0.150 a
Lead
Green
8
0.050 ± 0.044
5
0.328 ± 0.462
Kemp’s ridley
28 0.061 ± 0.077 a
19 0.397 ± 0.318 a
Loggerhead
17 0.077 ± 0.083
12 0.197 ± 0.264
Essential elements
Cobalt
Green
8
0.051 ± 0.021 b
4
0.117 ± 0.111
Kemp’s ridley
27 0.027 ± 0.029 a, b
8
0.238 ± 0.379 a, b
Loggerhead
17 0.016 ± 0.008 b
3
0.032 ± 0.012 b
Chromium Green
8
0.291 ± 0.345
6
0.692 ± 0.869
Kemp’s ridley
30 0.262 ± 0.350 a
26 1.080 ± 1.419 a
Loggerhead
17 0.153 ± 0.088 a
16 0.854 ± 0.968 a
Iron
Green
8
46.239 ± 46.222
5
351.753±505.120
Kemp’s ridley
30 44.331 ± 60.660 a
26 151.801 ± 116.620 a
Loggerhead
17 28.849 ± 23.796
17 73.884 ± 52.190
Manganese Green
8
0.529 ± 0.655 a
7
5.209 ± 9.128 a
Kemp’s ridley
30 0.635 ± 0.931 a
28 3.083 ± 3.076 a, b
Loggerhead
17 0.510 ± 0.410
17 1.302 ± 1.480 b
Nickel
Green
8
3.743 ± 6.497
8
2.497 ± 1.799
Kemp’s ridley
30 3.330 ± 12.226 a
30 2.290 ± 1.441a
Loggerhead
17 0.589 ± 0.363
17 1.528 ± 1.691
Selenium
Green
8
2.181 ± 2.950
2
0.233 ± 0.013
Kemp’s ridley
4
0.700 ± 0.446
2
2.009 ± 1.226
Loggerhead
8
0.940 ± 0.895
2
0.257 ± 0.047
Zinc
Green
8
21.561 ± 9.226 a
8
108.095±33.384 a, b
Kemp’s ridley
30 19.980 ± 6.822 a
30 166.972 ± 72.265 a
Loggerhead
17 11.271 ± 4.850 a
17 201.786±50.971 a, b
26
In Kemp’s ridley turtles, we observed significant positive correlations between skin and
scute samples for aluminum (R=0.38, p=0.04), cadmium (R=0.66, p=0.0004), iron (R=0.46,
p=0.02), manganese (R=0.51, p=0.007), nickel (R=0.48, p=0.008) (Figure A-7). In green turtles
and loggerhead turtles, we did not observe any significant positive correlations between skin and
scute samples (Figure A-8; Figure A-9).
2.4.2 Interspecific patterns of heavy metal concentrations
When comparing interspecific patterns of heavy metal concentrations in skin samples, we
observed significantly higher cobalt concentrations in green turtles (0.0.051±0.021 µg g-1)
compared to loggerhead turtles (0.016±0.008 µg g-1, p=0.004) and Kemp’s ridley turtles
(0.027±0.029 µg g-1, p=0.04). We observed significantly higher cadmium concentrations in
loggerhead turtles (0.092±0.025 µg g-1) compared to Kemp’s ridley turtles (0.058±0.033 µg g-1,
p=0.004), but not for green turtles (0.075±0.052 µg g-1, p>0.05).
When comparing interspecific pattens of heavy metal concentrations in scute samples, we
observed significantly higher arsenic (p=0.0001), cobalt (p=0.04) and manganese (p=0.05)
concentrations in Kemp’s ridley turtles (4.68±2.54 µg g-1; 0.238±0.379 µg g-1; 3.08±3.08 µg g-1
respectively) compared to loggerhead turtles (1.79±0.85 µg g-1; 0.032±0.012 µg g-1; 1.3±1.5 µg g-
1 respectively), but not for green turtles (2.21±1.61 µg g-1; 0.117±0.111 µg g-1; 5.21±9.13 µg g-1;
p>0.0.5 respectively). We also observed significantly higher zinc in loggerhead turtles (202.8±51.0
µg g-1, p=0.004) than green turtles (108.1±33.4 µg g-1), but not for Kemp’s ridley turtles
(167.0±72.3 µg g-1, p>0.05).
2.4.3 Correlating skin, scute, and SCL
Linear correlation revealed only silver concentrations in green turtles’ skin had a
significant positive correlation with increasing carapace size (R=0.81, p=0.05) (Figure A-1).
Although there were no significant relationships for all the other heavy metals when running
correlations between green turtles’ tissues and increasing carapace size, two greens CS.CM.6 (SCL:
28.6cm) and CS.CM.8 (SCL: 28.4cm) were outliers for both skin and scute samples. CS.CM.6 had
the highest skin concentrations for aluminum, cobalt, chromium, iron, manganese, and lead (Figure
A-1) and was the only green scute sample with silver above the LOD (
26
Table 2-2). CS.CM.8 on the other hand had the highest skin concentrations for arsenic,
cadmium, and zinc (Figure A-1) and the highest scute concentrations for all heavy metals except
nickel (Figure A-2). Kemp’s ridleys had an overall more clustered heavy metal concentrations in
both its skin and scute samples (Figure A-3, Figure A-4), whereas loggerheads heavy metal
concentrations were more scattered (Figure A-5, Figure A-6).
2.5 Discussion
The cold-stunned green and Kemp’s ridley turtles in our study are of similar sizes and are still
considered as juveniles, whereas the loggerhead turtles in our study are slightly bigger, ranging
from juveniles to subadults. All our turtles were undergoing or have undergone an ontogenetic
shift where they recruit to neritic developmental waters and transition their diets — Kemp’s ridley
and loggerhead turtles towards a predominantly carnivorous diet (Nelson 1988, Reyes-López et al.
2021), green turtles towards a predominantly herbivorous diet (Bjorndal 1985). Skin samples will
reflect more recent exposure (~1 year; Seminoff et al. 2006) whereas scute samples will reflect
exposure prior to their ontogenetic shift (4-6 years ago; Vander Zanden et al. 2013). Through the
heavy metals we analyzed, we see the shifts in green turtle diet through higher cobalt and zinc
concentrations in their skin samples. Furthermore, the changes in arsenic and cadmium
concentrations of loggerhead turtles provide insights to elements in which they are exposed to
through the environment they forage in. Kemp’s ridley turtles’ scute samples have 9 (out of 12)
heavy metals that are significantly higher than their skin samples highlight the changes in their
environmental exposure as well as the possibility of different physiological reaction to unwanted
inorganic elements.
2.5.1 Interspecies comparison of heavy metal concentrations – Diet and Environment
Green turtle skin samples had higher cobalt concentration than Kemp’s ridley and loggerhead
turtles (
26
Table 2-2). As cobalt is associated with herbivorous diet, this suggests the highly herbivorous
diet of green turtles as they approach sexual maturity. In a review by Smith and Carson (1981),
they found that cobalt concentrations in algae have a wide range between 0.1ppm to 100ppm,
whereas marine shells typically have cobalt concentrations between 0.15ppm to 1.2ppm, far below
that of algae. This is supported by the fact that these green turtles’ stomachs were found to contain
mostly seagrass during necropsies (S. Patel, personal communication, September 19, 2023).
Furthermore, the lower cobalt concentrations in Kemp’s ridley and loggerhead turtles’ skin
samples are probable indications of their transition away from algae and seagrass to a more
carnivorous diet (Shaver 1991).
Figure 2-2 Boxplots comparing zinc concentration of scute and skin samples found in greens (n=8),
Kemp’s ridley (n=30) and loggerhead (n=17) sea turtles in Cape Cod Bay, Massachusetts. Please
note the values of the Y axes vary between plots. Boxes are the middle 50% quartiles, lines are
median, whiskers are the range of the minimum and maximum values.
Green turtle scute samples had lower zinc concentrations than loggerhead and Kemp’s
ridley turtle scute samples. However, green turtle skin samples had higher zinc concentrations than
26
the other two species (Figure 2-2). This inverse pattern likely reflects the dietary transition of green
turtles towards herbivory and loggerhead and Kemp’s ridley turtles towards carnivory as they
reach sexual maturity. Although Mondragón et al. (2023) also found that green turtle scute had
lower zinc concentrations compared to other species nesting along the Northeast Coast of Quintana
Roo State, Mexico, this finding is interesting as green turtle hatchlings are omnivorous and
consume mollusks which have a relatively high zinc concentration (76µg g-1 dry weight) compared
to the diets of loggerhead turtle hatchlings which consume relatively more algae (zinc: 51.3µg g-1
dry weight) (Conti et al. 2007). It is possible that these green sea turtles were consuming more
zooplankton in the pelagic waters and could result in lower zinc concentrations in their scute
samples. Achary et al. (2020) compiled global zooplankton zinc concentrations that had a wide
range of 53-5800.3 µg g-1 wet weight. Furthermore, according to Balthis et al. (2009), Cooksey et
al. (2010), Balthis et al. (2013), and Cooksey et al. (2014), zinc concentrations in the Gulf of
Mexico, South Atlantic Bight, and Mid-Atlantic Bight are similar. As these locations are the likely
migratory routes of our sampled turtles, it is unlikely that the varying zinc concentrations in the
scute samples of the different species are due to environmental exposure.
Our Kemp’s ridley turtles had nine heavy metal concentrations which were significantly
higher in the scute samples compared to the skin samples — aluminum, cadmium, cobalt,
chromium, iron, manganese, nickel, lead, and zinc. This observed difference could be due to
hatchlings being exposed to elevated heavy metals in their early developmental days or due to
maternal transfer of heavy metals from nesting Kemp’s ridley turtles. Kemp’s ridley turtles are the
only species which exclusively nest and hatch on the nesting beaches in the Gulf of Mexico. The
hatchlings enter the Gulf of Mexico before entering current systems of the Gulf of Mexico or the
Northwest Atlantic’s Gulf Streams, where they enter their oceanic feeding phase (Reyes-López et
al. 2021). The heavy metal levels in the waters of the Gulf of Mexico are generally higher than
that of the Atlantic Ocean (Vázquez-Botello et al. Unpublished, Wang et al. 2023). Vázquez-
Botello et al. (Unpublished) compiled data from a few studies that revealed the seawater in the
Gulf of Mexico had chromium concentrations of 5.2-7.4ppm back in the 1990s. These
concentrations are much higher compared to that of the seawater in the Atlantic Ocean where
chromium is only found at a concentration of 0.00012ppm (Wang et al. 2023). Furthermore,
aluminum and lead levels in northwestern Gulf of Mexico sediments (3.440 ± 1.587% dry weight
and 16.622 ± 4.502 µg g-1 dry weight respectively) were found to be higher than that of the Mid-
26
Atlantic Bight (1.374 ± 0.680% dry weight and 9.348 ± 5.858 µg g-1 dry weight respectively)
(Balthis et al. 2009, Balthis et al. 2013). The elevated heavy metal levels in the Gulf of Mexico
are probably a result of the offshore oil and gas activities, which occasionally result in catastrophic
events, including the Deep Horizon Oil Spill in 2010. We postulate that the difference in heavy
metal concentrations in Kemp’s ridley turtles’ skin and scute samples indicates that they were
exposed to high levels of heavy metals as hatchlings. While this exposure could be a result of them
moving through the Gulf of Mexico for a brief period before entering the Northwest Atlantic’s
current system, it is more likely that their elevated levels of heavy metals came from maternal
transfer, where nesting females transfer the excess chemicals in their body to their eggs (Camacho
et al. 2017). The higher heavy metal concentrations in Kemp’s ridley turtles’ scute samples
possibly indicate that they deposit unnecessary elements in their scutes as a detoxifying mechanism
(Martín et al. 2021).
Kemp’s ridley turtles’ scute samples had higher arsenic concentrations than loggerhead
turtles’ scute samples. Maher and Butler (1988) noted that diet is a big contributing factor towards
the accumulation of arsenic in marine animals. Apart from crustaceans as a source of arsenic which
both loggerheads and Kemp’s ridleys have in their diet (Storelli and Marcotrigiano 2003), this
higher arsenic concentration in Kemp’s ridley turtles’ scute is likely due to the algae that the
hatchlings consume in their oceanic days. Francesconi and Edmonds (1993) reported that algae
can accumulate arsenic up to 50000 times their surrounding seawater. This, coupled with higher
arsenic concentrations in the Gulf of Mexico, likely resulted in high exposure of Kemp’s ridleys
to arsenic as hatchlings. While green and loggerhead turtles do nest in the Gulf of Mexico, it is
possible that the cohorts we sampled in the NW Atlantic came from other nesting beaches.
The cadmium and arsenic concentrations in loggerhead turtles’ skin samples are likely due
to heavy metals found in their diet as well as the environment that they forage in. Loggerhead
turtles’ skin samples had higher cadmium concentrations when compared to Kemp’s ridley turtles.
Although not statistically significant, loggerhead turtles’ skin samples also had higher arsenic
concentrations when compared to the other two species. Loggerhead turtles are likely
accumulating arsenic and cadmium from consuming crustaceans such as cephalopods and
mollusks (Bustamante et al. 1998, Clark 1992, Storelli and Marcotrigiano 2003). The overall
higher arsenic and cadmium concentrations in loggerhead turtles’ skin samples are possibly due to
loggerhead turtles foraging for benthic organisms in Massachusetts which is a highly polluted area
26
due to smelting activities (Eckel et al. 2001). Despite the more recent exposure to possibly higher
cadmium concentrations as loggerhead turtles forage in Massachusetts benthic environment,
loggerhead turtles’ skin samples (recent exposure) had lower cadmium concentrations than their
scute samples. Since cadmium is not an essential element, the accumulation of cadmium in the
scute might be a detoxifying mechanism (Martín et al. 2021).
2.5.2 Comparing heavy metal concentrations to other studies and their implications
Our green turtles’ skin samples had higher cobalt and zinc concentrations than loggerhead
and Kemp’s ridley turtles. While this is likely attributed to a predominantly herbivorous diet
(seagrass), our green turtles’ skin cobalt concentration (0.051 ± 0.021 µg g-1) was higher than that
of other studies (Table 2-3). To the best of our knowledge, Faust et al. (2014) is the only other
paper to have studied cobalt concentrations in green turtle skin samples, and all 12 of their turtles
had cobalt concentrations below detectable limits. While cobalt is necessary for the development
of numerous organisms, excessive exposure to cobalt has been found to cause negative behavioral
and physiological effects and could possibly be carcinogenic to humans (ATSDR 2004). Finlayson
et al. (2020) also found that high cobalt concentrations are cytotoxic to green turtle skin cells and
alter their Glutathione-S-transferase activity, which are enzymes that protect cellular
macromolecules. Despite the cobalt concentrations in all species’ skin samples being much higher
than normal human blood concentrations that range from 0.00005-0.0027 ppm (synonymous with
µg g-1) (Catalani et al. 2011), they are much lower than acute toxicity values (LC50) of rainbow
trout (1.343-1.704 ppm) (Stubblefield et al. 2020). This indicates that our green turtles may have
elevated cobalt levels, but it is probably still far from acute toxicity levels. However, we suggest
that future studies should collect blood samples for a more direct comparison to known human
blood cobalt concentrations.
While our green and loggerhead turtles had a much lower concentration of arsenic in their
skin samples (3.614 ± 2.569 µg g-1 and 5.069 ± 2.258 µg g-1 respectively) compared to studies
conducted in Laguna Madre, USA (Faust et al. 2014) and Murcia, Spain (Jerez et al. 2010) (Table
2-3), our loggerhead turtles’ scute samples (1.792 ± 0.849 µg g-1) had higher arsenic concentrations
than that of loggerhead turtles (0.96 ± 0.98 µg g-1) sampled in an area affected by mining tailings
in Brazil (Miguel et al. 2022). It is worth noting that while Faust et al. (2014) and Jerez et al. (2010)
also conducted their studies on juvenile turtles, their skin samples were dried prior to analysis.
26
From a mathematical perspective, the lack of moisture content makes the skin samples lighter,
resulting in the calculation of higher arsenic concentrations. To the best of our knowledge, there
are no known moisture values for sea turtle skin samples that could help us standardize these
calculations. Furthermore, since arsenic is not an essential element, it is likely that our higher
arsenic concentrations compared to that of Miguel et al.’s (2022) study is due to Mid-Atlantic
Bight having higher levels of arsenic pollutants, despite Miguel et al.’s (2022) study site being
affected by mining tailings. As arsenic is not an essential element, exposure to arsenic could result
in negative effects such as digestive irritation, decreased white and red blood cell production, and
cancer (ATSDR 2007). Finlayson et al. (2020) found arsenic to be cytotoxic to green turtle skin
cells, which might also be the case for loggerhead turtles. Other living organisms, including
humans, usually have less than 1.00 ppm of arsenic in their tissue (Eisler 1988, Gomez-Caminero
et al. 2001). Furthermore, Lian and Wu (2017) found that safe arsenic concentrations for Lanzhou
catfish is 1.288 ppm. As all species’ skin and scute samples are above these values, it is probable
that they are being exposed to concerning amounts of arsenic throughout their migratory paths and
diets as they approach adulthood.
Cadmium concentrations in our turtle skins were higher than that of other studies. Our
loggerhead turtles had (0.092 ± 0.025 µg g-1) that is double the concentration (0.04 ± 0.02 µg g-1)
of loggerhead turtles’ skin sample in Murcia, Spain (Jerez et al. 2010). As mentioned in discussing
arsenic concentrations, Jerez et al. (2010) used dried skin samples for their study. This means that
their arsenic concentrations would be much lower if they used wet samples. Our green turtles’ skin
samples also had higher cadmium concentration (0.075 ± 0.052 µg g-1) when comparing to Faust
et al. (2014) study in Texas, USA, whose cadmium concentrations were below method detection
limits. As Massachusetts is an area with high smelting activities (Eckel et al. 2001), it is likely that
these elevated cadmium concentrations are a result of environmental exposure in the area.
Cadmium is a non-essential element and has been found to cause respiratory damage, cancer, liver
disease, and neural damage in animals (ATSDR 2012). Despite higher cadmium concentrations in
our turtles, evidence of contamination is only considered when whole body tissues exceed 2ppm
in vertebrates and is only considered life-threatening at 5ppm (Eisler 1985). However, 10% of
occupationally exposed humans have shown signs of tubular damage in chronic blood
concentrations as low as 0.0056 (ATSDR 2008). This means that our turtles’ skin samples are
showing higher levels of cadmium concentration compared to occupationally exposed humans,
26
but lower than that of dangerous levels for vertebrates. As cadmium has been shown to accumulate
in human and sea turtle liver and kidneys (Sakai et al. 2000, Storelli et al. 2005), we suggest that
future studies should collect liver and kidney samples from the cold-stunned turtles to determine
if the turtles have been exposed to excessive cadmium concentrations in the environment.
Our selenium concentrations were comparable to other studies (Faust et al. 2014, Barraza
et al. 2019, Jerez et al. 2010). Despite selenium being detected in all our green turtles’ skin samples,
Kemp’s ridley and loggerhead turtles only had less than half of their skin samples which were
above detectable levels. Furthermore, selenium concentrations were detected in only 25%, 6.7%
and 11.8% of green, Kemp’s ridley and loggerhead turtles’ scute samples. This is critical as
selenium is essential for regulating seleno-aminoacid functions (Thiry et al. 2013). Furthermore,
selenium has been found to reduce mercury toxicity impacts in mammals and aquatic biota
(Raymond and Ralston 2020). One possible explanation of the lack of selenium in our scute
samples is that selenium has been used in mercury regulation mechanisms, and therefore was not
deposited in the scute. However, we did not conduct analysis on mercury concentrations.
2.5.3 Correlating Skin, Scute and SCL
We found higher concentrations of heavy metals in scute samples than skin samples across
all species. This is likely due to both the accumulation of unwanted inorganic elements in the
keratinized tissues (Mondragón et al. 2023) as well as keratinized tissue having lower turnover
rates compared to skin tissue (Seminoff et al. 2006 and Vander Zanden et al. 2013). Since
keratinized tissue (scute) have lower turnover rates and reflect exposure of 4-6 years (Vander
Zanden et al. 2013) and skin tissue only reflect exposure of ~1 year (Seminoff et al. 2006), scute
samples tend to accumulate a greater concentration of heavy metals when compared to skin
samples. Furthermore, Martín et al. 2021 described such detoxifying mechanisms in feathers of
birds and skins of amphibians and reptiles. Despite cobalt, chromium, manganese, nickel, selenium,
zinc, silver, arsenic, cadmium, and lead having found to bioaccumulate in organisms (ATSDR
1990, ATSDR 2005, ATSDR 2007, ATSDR 2023, Fatima et al. 2014, Lemly and Smith 1987),
we however only found silver to bioaccumulate in green sea turtle skin (Figure A-4). This opposes
the findings of Komoroske et al. (2011) where aluminum, manganese, copper, and lead correlated
moderately and positively with increasing CCL while mercury had a strong negative correlation.
26
We did not observe any correlation between the size of the sea turtles with increasing heavy
metal concentrations (Figure A-1, Figure A-2, Figure A-3). Larger turtles did not appear to have
higher heavy metal concentrations in their skin and scute samples. Despite the significant
differences of chromium, manganese, and zinc concentrations between skin and scute samples, we
did not find any significant differences between the skin samples of different species. As these are
essential elements, this finding indicates that our sea turtles are obtaining these elements from their
environment.
We did not find any correlations between skin and scute samples for any of the heavy
metals analyzed (Figure A-7, Figure A-8, Figure A-9). Other studies have found correlations
between different sea turtle tissues for certain heavy metals. For example, van de Merwe (2010)
found strong correlations in cobalt concentrations between blood and liver, kidney, and muscle
tissue of green turtles and Bezerra et al. (2013) found correlations in mercury concentrations
between muscles and scute samples of green turtles. These correlations indicate that blood and
scute samples are useful non-invasive methods for predicting cobalt and mercury concentrations
in the green turtles. The fact that we did not find any correlation between skin and scute samples
suggests that scute samples (which are relatively less invasive) cannot be used as a replacement
for skin biopsies in juvenile green, loggerhead, and Kemp’s ridley turtles.
The two green turtle outliers (CS.CM.6 and CS.CM.8) which had higher concentrations of
heavy metals in their skin or scute samples are indicative of these two turtles originating from
different cohorts compared to the other cold-stunned green turtles (Figure A-4, Figure A-7). The
higher levels of heavy metal concentrations tested in these two green turtles (CS.CM.6 skin - Al,
Co, Cr, Fe, Mn, Pb; CS.CM.8 skin – As, Cd, Zn; CS.CM.8 scute – Al, As, Cd, Co, Cr, Fe, Mn, Pb)
are likely due to these two turtles originating from different nesting populations. On the other hand,
the clustered data points of heavy metal concentrations in Kemp’s ridley turtle’s skin (Figure A-5)
and scute samples (Figure A-8) are indicative that most of these turtles have similar diets and have
foraged and migrated through similar environments. This contrasts the spread-out data points of
heavy metal concentrations in loggerhead turtles’ skin (Figure A-6) and scute (Figure A-9) samples,
which might be a result of loggerheads having high fidelity to their individual preferences of prey,
habitat, and geographical location (Vander Zanden et al. 2010).
35
Table 2-3 Heavy metal concentrations of blood, skin and scute samples in green, Kemp’s ridley and loggerhead sea turtles found in the
present study and other studies.
Skin
Scute
References
Species
Elements
n
mean ± SD
n
mean ± SD
Green
Silver
6 0.006 ± 0.007
1 0.034
Present Study
8 0.06 ± 0.02
Barraza et al. (2019)
Aluminum 8 22.9 ± 33.7
7 140.3 ± 224.8 Present Study
20 209.99 ± 40.18
Barraza et al. (2019)
Arsenic
8 3.61 ± 2.57
7 2.21 ± 1.61
Present Study
12 17.6 ± 1.5 1
d.w.
20 0.58 ± 0.07 2
1Faust et al. (2014)
2Barraza et al. (2019)
Cadmium 8 0.075 ± 0.052
5 0.144 ± 0.046 Present Study
0 <LOD 1
20 0.3 ± 0.06 2
1Faust et al. (2014)
2Barraza et al. (2019)
Lead
8 0.050 ± 0.044
5 0.328 ± 0.462 Present Study
0 <LOQ 1
20 3.10 ± 0.96 2
1Faust et al. (2014)
2Barraza et al. (2019)
Cobalt
8 0.051 ± 0.021
4 0.117 ± 0.111 Present Study
0 <LOQ 1
20 0.4 ± 0.09 2
1Faust et al. (2014)
2Barraza et al. (2019)
36
Table 2-3 continued
Chromium
8 0.29 ± 0.35
6 0.69 ± 0.87
Present Study
12 46.9 ± 4.7 1
d.w.
20 0.61 ± 0.1 2
1Faust et al. (2014)
2Barraza et al. (2019)
Iron
8 46.2 ± 46.2
5 351.8 ± 505.1 Present Study
20 340.53 ± 56.92 Barraza et al. (2019)
Manganese
8 0.53 ± 0.66
7 5.21 ± 9.1
Present Study
12 1.15 ± 0.23 1
d.w.
20 15.81 ± 4.3
1Faust et al. (2014)
2Barraza et al. (2019)
Nickel
8 3.74 ± 6.50
8 2.50 ± 1.80
Present Study
0 <LOD 1
20 2.37 ± 0.442
1Faust et al. (2014)
2Barraza et al. (2019)
Selenium
8 2.18 ± 2.95
2 0.23 ± 0.01
Present Study
12 2.01 ± 0.18 1
d.w.
20 0.48 ± 0.06 2
1Faust et al. (2014)
2Barraza et al. (2019)
Zinc
8 21.6 ± 9.2
8 108.1 ± 33.4
Present Study
12 43.8 ± 4.6 1
d.w.
20 225.39 ± 19.15 2
1Faust et al. (2014)
2Barraza et al. (2019)
37
Table 2-3 continued
Loggerhead
Arsenic
17 5.07 ± 2.26
17 1.79 ± 0.85
Present Study
2 52.13 ± 6.1
d.w.
Jerez et al. (2010)
Cadmium
17 0.092 ± 0.025
17 0.256 ± 0.15
Present Study
2 0.04 ± 0.02 1
d.w.
6 0.129 ± 0.034 2
1Jerez et al. (2010)
2Sakai et al. (2000)
Lead
17 0.077 ± 0.083
12 0.197 ± 0.264 Present Study
2 0.02 ± 0.03 1
d.w.
6 2.42 ± 0.52 2
1Jerez et al. (2010)
2Sakai et al. (2000)
Iron
17 28.8 ± 23.8
17 73.9 ± 52.2
Present Study
6 26.2 ± 19.1
Sakai et al. (2000)
Manganese
17 0.51 ± 0.41
17 1.3 ± 1.
Present Study
6 7.01 ± 4.49
Sakai et al. (2000)
Nickel
17 0.59 ± 0.36
17 1.53 ± 1.69
Present Study
6 0.0094 ± 0.022 Sakai et al. (2000)
Selenium
8 0.94 ± 0.90
2 0.26 ± 0.05
Present Study
2 2.25 ± 0.93
d.w.
Jerez et al. (2010)
Zinc
17 11.3 ± 4.9
17 202.8 ± 51.0
Present Study
2 13.9 ± 8.6 1
d.w.
6 198 ± 37.2
1Jerez et al. (2010)
2 Sakai et al. (2000)
38
Table 2-3 continued
NOTE: Skin and scute heavy metal concentration values are µg g-1 wet weight, unless stated otherwise.
* were converted from dry weight to wet weight using the value of 29.1% moisture content as seen in Rodriguez et al. 2022
d.w. = dry weight; <LOQ = below limit of quantification; <LOD = below method detection limit
Kemp’s ridley Silver
11 0.009 ± 0.011
4 0.049 ± 0.014 Present Study
61 0.102 ± 0.113* Wang (2005)
Cadmium 29 0.058 ± 0.033
26 0.322 ± 0.178 Present Study
61 0.092 ± 0.087* Wang (2005)
Lead
28 0.061 ± 0.077
19 0.397 ± 0.318 Present Study
61 0.865 ± 1.219* Wang (2005)
Chromium 30 0.26 ± 0.3
26 1.08 ± 1.42
Present Study
61 0.23 ± 0.25*
Wang (2005)
Zinc
30 20.0 ± 6.8
30 167.0 ± 72.3
Present Study
61 233.3 ± 208.5* Wang (2005)
39
2.6 Conclusion
As indicated by the number of heavy metal concentrations that are significantly different
between different sea turtle species and their tissue types, it is important to assess this population
for essential and non-essential heavy metals that they are exposed to as they transition into
adulthood. As there is potential to use skin and scute samples to indicate physiological impacts of
elevated heavy metal concentrations on sea turtles, it would be beneficial to also collect organ
samples from these cold-stunned turtles to determine if there is a correlation between heavy metal
loads that internal organs deal with before they are deposited into skin and scute tissue.
The ability to use skin and scute samples as bioindicators of sea turtle health is of great
importance considering the environmental stressors that they are exposed to throughout their lives.
Climate change is a pressing issue that is of concern for the turtles in the NW Atlantic. According
to thermal models by Patel et al. (2021), thermal windows in the NW Atlantic are expected to
increase. This will likely increase the period in which loggerheads (and potentially other turtles)
would spend foraging further up north, where we know they are potentially being overexposed to
arsenic, cadmium, and cobalt concentrations. If it is true that turtles are accumulating higher heavy
metal levels in their scute samples as a way of disposing of unwanted inorganic materials, perhaps
this projected prolonged foraging period in the Mid-Atlantic Bight is not of that great a concern.
However, this assumption is based off studies on amphibian and avian species (Martín et al. 2021)
and it is very likely that the elevated heavy metals are impacting physiological functions of sea
turtles in the region negatively.
Determining baseline heavy metal concentrations in Mid-Atlantic Bight turtles allows us
to better understand their exposure to pollutants via diet and environment throughout the past 6
years of their lives. Such information is useful in monitoring the health status of the turtles which
could be useful information for implementing sustainable development efforts. It is however
important to note that different species may metabolize different heavy metals differently
(Swarthout et al. 2010) and that different tissues accumulate different heavy metals differently too
(Camacho et al. 2017). Therefore, to be able to confidently use sea turtle tissue as indicators of the
environment, future analysis would have to compare same tissues from turtles of the same species,
similar size and from the same site to our baseline heavy metal concentrations.
40
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Wang HC. 2005. Trace metal uptake and accumulation pathways in Kemp's ridley sea turtles
(Lepidochelys kempii). Doctoral dissertation, Texas A&M University. Texas A&M
University. Available electronically from https://hdl.handle .net /1969 .1 /2413.
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2023. Biogeochemical cycling of chromium and chromium isotopes in the sub-tropical
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10.3389/fmars.2023.1165304
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Gullivan Bay, Ten Thousand Islands, southwest Florida. Bulletin of Marine Science
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KJ, Musick J. Boca Raton, FL: CRC Press.
Zhou Q, Zhang J, Fu J, Shi J, Jiang G. 2008. Biomonitoring: an appealing tool for assessment of
metal pollution in the aquatic ecosystem. Analytica Chimica Acta 606:135-150.
47
COMPARING HEAVY METAL CONCENTRATIONS OF
LOGGERHEAD TURTLES AND THEIR PREY ALONG THE US EAST
COAST
3.1 Abstract
The eastern coastline of the USA is highly urbanized, which has contributed to a significant
anthropogenic output of pollutants (such as heavy metals) entering the environment and washing
out to the ocean. This is of particular concern for long-lived marine species like sea turtles. Sea
turtles, therefore, make useful indicator species because they incorporate environmental and
dietary heavy metals as they migrate through marine habitats. Scute samples (from the carapace)
can be collected in a relatively non-invasive manner and can reflect the environment and diet of
sea turtles within 4-6 years of their life. To better understand trophic accumulation of heavy metals
in loggerhead sea turtles (Caretta caretta), we collected scute samples during necropsies of cold-
stunned loggerhead turtles from Cape Cod Bay, Massachusetts (CCB; n=17), as well as from live
loggerhead turtles in the Mid-Atlantic Bight (MAB; n=37) and off the coast of North Carolina (NC;
n=9). The three loggerhead turtle groups are of different life stages and exposure duration, with
CCB having the smallest loggerhead turtles and MAB having the largest loggerhead turtles.
Therefore, the heavy metal concentrations in their scutes act as indicators of what these sea turtles
were exposed to in the environments they experienced and their diet at different stages of their
lives. We also collected several commonly known prey items of loggerhead turtles including whelk
(Buccinum undatum) (n=12), Atlantic scallop (Placopecten magellanicus) (n=10) and Jonah crab
(Cancer borealis) (n=5) from the Mid-Atlantic Bight region. The concentrations of silver (Ag),
aluminum (Al), arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), iron (Fe), manganese
(Mn), nickel (Ni), lead (Pb), selenium (Se) and zinc (Zn) were analyzed using an Inductively
Coupled Plasma Mass Spectrometry (ICP-MS). NC loggerhead turtles had higher heavy metal
concentrations than other locations except for cadmium (mean ± SD μg g-1 wet weight; CCB 0.256
± 0.150; NC=0.103 ± 0.042; MAB=0.095 ± 0.040) and zinc (CCB=201.79 ± 0.50.97; NC=184.66
± 70.85; MAB=172.92 ± 52.16), where CCB loggerhead turtles were higher. As NC and CCB
loggerhead turtles’ scute samples are probably still reflecting heavy metal concentrations from
their juvenile omnivorous diets, the higher NC heavy metal concentrations are likely indicative of
the heavy metals bioaccumulating in the larger NC turtles. On the other hand, NC turtles having
48
higher heavy metal concentrations than MAB turtles indicates that MAB turtles’ scute samples are
probably reflecting heavy metal concentrations from their carnivorous adult loggerhead diet. We
found that all heavy metals except silver, cadmium, and lead appear to be biomagnified (TTF>1)
in loggerhead turtles. This study provided baseline information on heavy metal concentrations in
loggerhead scute samples and their prey in east coast US.
3.2 Introduction
Environmental pollution has been an issue of growing concern in recent years.
Advancements in technology, especially in the realms of industrialization, agriculture, and
medicine, have resulted in a lot of pollutants entering the marine ecosystem. Heavy metals are one
of the concerning groups of pollutants that enter the environment. Most heavy metals like arsenic,
selenium, and cobalt are found naturally in rocks, water, and soil at low concentrations (ATSDR
2003, ATSDR 2004, ATSDR 2007). However, these elements are also entering the environment
due to anthropogenic activities such as smelting facilities, agriculture, and electroplating (ATSDR
2003, ATSDR 2004, ATSDR 2007). At high concentrations, these elements can become toxic to
flora and fauna (ATSDR 2004, ATSDR 2012).
Caretta caretta (loggerhead) turtle is a widely distributed sea turtle species. While it is found
abundantly in the Mediterranean Sea, it can also be found in other basins including the Atlantic,
Indian, and Pacific Ocean (Caurant et al. 1999, Day et al. 2005, Mingozzi et al. 2007, Nagelkerken
et al. 2003). As loggerhead turtles are long-lived and highly migratory, it is likely that their tissue
accumulates heavy metals that they are exposed to through diet and the environment (Bjorndal
1985, Vander Zanden et al. 2013, Barraza et al. 2019). While heavy metals can be found at all
trophic levels, some elements are biomagnified through trophic levels differently. For example,
mercury, lead, and zinc are known to increase along trophic pathways whereas arsenic and nickel
do not get passed on (Sun et al. 2020). Since loggerheads are generalist predators and occupy a
higher trophic level, they are more susceptible to the biomagnification of heavy metals (Jakimska
et al. 2011). The trophic transfer factor (TTF) is used as an indicator of biomagnification in an
organism’s diet and is the ratio of the specific elemental concentration in an organism’s tissue
compared to the concentration of the food items (DeForest et al. 2007). Thus, a TTF value >1
suggests biomagnification is occurring (Matthews and Fisher 2008).
49
The northwest Atlantic Ocean, off the east coast of the USA, is a popular foraging and
migratory route for loggerhead turtles (Patel et al. 2016, TEWG 2009, Winton et al. 2018). In
summer and early fall, loggerhead turtles are found in regions spanning from Cape Hatteras, North
Carolina to Long Island, New York. In late fall to spring, they inhabit more southerly waters along
the coast of Florida (TEWG 2009). Loggerhead turtles in this region have been found to feed on
gelatinous prey (e.g., Lion’s mane jellies, comb jellies and salps) in the pelagic waters, and on
benthic crustaceans (e.g., rock crabs and Atlantic Sea scallops) in the coastal shelves (Smolowitz
et al. 2015). As we know the diets of loggerhead turtles in this area, they are good target species
for understanding the occurrence of heavy metal biomagnification through their diets.
Several studies have assessed heavy metal concentrations in the tissues of loggerhead turtles
(Sakai et al. 2000, van de Merwe et al. 2010), including those using scute samples (Casini et al.
2018, Miguel et al. 2022, Mondragón et al. 2023); however, no studies have analyzed heavy metal
concentrations in loggerhead turtles from the NW Atlantic. Scute samples have become an
increasingly popular tool to study trace metals in sea turtles as it is a relatively easy and non-
invasive process. (Seminoff et al. 2006). Furthermore, scute samples have slower isotopic turnover
rates that reflect the environment and diet of loggerhead turtles for their last 4-6 years (vander
Zanden et al. 2013).
The main goal of our study was to determine the differential exposure to heavy metals as
reflected in scute samples and prey items of loggerhead turtles in the NW Atlantic Ocean. We
collected scute samples of loggerhead turtles from 3 different locations (Cape Cod Bay
Massachusetts, Mid-Atlantic Bight, North Carolina) within the northwestern Atlantic Ocean, as
well as loggerhead prey — whelk (Buccinum undatum), Atlantic scallop (Placopecten
magellanicus) and Jonah crab (Cancer borealis). To investigate if our loggerhead turtles were
accumulating similar concentrations of essential heavy metals, we measured chromium, cobalt,
iron, manganese, nickel, selenium, and zinc. To obtain an overview of our loggerhead turtles’
health and possible physiological implications of non-essential heavy metals, we measured the
concentrations of arsenic, aluminum, cadmium, lead, and silver.
The objectives of this study were 1) to investigate the difference in heavy metal
concentrations between loggerhead turtles sampled from different locations and life stages within
the NW Atlantic 2) to investigate if heavy metals are biomagnified through trophic pathways of
loggerhead sea turtles. 3) to compare heavy metal concentrations in our loggerhead turtles with
50
studies conducted in other regions of the world. We predict that the bigger sized turtles from Mid-
Atlantic Bight would have higher concentrations of arsenic and cadmium which are found in
cephalopods associated with adult diets (Bustamante et al. 1998, Storelli and Marcotrigiano 2003).
We also predict that lead and zinc would have a TTF>1 as these are heavy metals associated with
biomagnification (Sun et al. 2020). Lastly, we predict that our loggerhead turtles in the NW
Atlantic would have higher heavy metal concentrations when compared to other studies conducted
in more pristine environments.
3.3 Methods
3.3.1 Field Sample Collection
This study took place in three areas off the east coast of the United States of America Cape
Cod Bay in Massachusetts (CCB), Mid-Atlantic Bight (MAB) and the coast of North Carolina
(NC) (Figure 3-1). CCB is a 1100 km2 semi-enclosed bay where cold-stunned sea turtles wash up
in the winter. During the necropsies organized by Massachusetts Audubon Wellfleet Bay Wildlife
Sanctuary (WBWS) after the winters of 2019 and 2021, researchers collected scute samples from
cold-stunned, recently deceased loggerhead sea turtles (n=17). MAB is a coastal shelf located 40-
100km off the shores of New Jersey through Virginia, USA (latitudinal range = 37.0° to 40.0°;
longitudinal range = -75.5° to -73.0°) and NC is on the coastal shelf less than 10km off the shores
of North Carolina (latitudinal range = 35.10° to 35.14°; longitudinal range = -75.71° to -75.66°).
For MAB and NC sites, researchers from the Coonamessett Farm Foundation collected samples
from live loggerheads (n=37; n=9 respectively) (under ESA permit 23639). See Patel et al. 2018
for details on loggerhead captures at-sea. These scute samples (~0.5g) were collected by scraping
the biopsy punch along the rear of the first lateral scute of each turtle (Day et al. 2005). Scute
samples from live turtles were collected only if the turtles exhibited no external injuries (Barraza
et al. 2019, Bean and Logan 2019, Day et al. 2005).
Commercial scallop fishermen in the area where C. caretta are known to forage (Patel et
al. 2016) provided samples of several prey taxa for loggerhead turtles, including scallops, whelks,
and crabs – from the Mid-Atlantic Bight region. To prepare the prey samples for heavy metal
analysis, we separated the scallop and whelk meat from the shell, and the operculum of the whelk
was also processed separately. All samples were weighed and placed in the oven at 60°C until
51
constant weight was obtained. Upon samples being completely dried, we crushed the samples
using a mortar and pestle until the samples were in powder form.
Figure 3-1 Map of study area in the USA. The red circle is Cape Cod Bay, highlighting the hook-
shaped bay which results in the entrapment of numerous turtles as they migrate south every winter.
The orange shape is the mid-Atlantic bight region, 40-100km from the shore, and the green circle
is the coastal area off the shores of North Carolina, less than 10km from shore. The red cross is
the commercial scallop fishing site where our prey samples were collected from.
3.3.2 Heavy Metal Analysis
We analyzed both skin and scute samples for silver (Ag), aluminum (Al), arsenic (As),
cadmium (Cd), cobalt (Co), chromium (Cr), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb),
selenium (Se) and zinc (Zn) at Purdue University West Lafayette. Heavy metal concentrations
were determined using an Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Thermo
Scientific Element 2) equipped with a Teledyne Cetac Aridus II nebulizer following their standard
protocols (N. Gou, personal communication, September 15, 2022). We weighed out 0.2-0.5g of
each sample and added 2mL of ultra-high purity nitric acid and 0.5mL of ultrapure water into
borosilicate digestion vessels (Anton Paar 179436). We digested these samples along with method
blanks in a microwave digestor (Anton Paar 7000 Microwave Digestion System) using the
preconfigured ‘Organic’ program. After digestion, we diluted the samples and blanks to a final
52
volume of 50mL using ultrapure water and added 125 μL of 5 ppb indium as an internal standard.
We prepared standard solutions ranging from 0.01-1000ppb for all heavy metals from 10 ppm
standard solutions purchased from Inorganic Ventures. Due to the different sample preparation
methods, the heavy metal concentrations of scute samples were expressed in µg g⁻¹ wet weight,
whereas the prey samples were expressed in µg g⁻¹ dry weight. The limits of detection (LOD) of
each heavy metal were calculated as three times the standard deviation of the ten independent
measurements of the blank, divided by the slope of the calibration curve. As we were analyzing
90 samples for 12 heavy metals each, we recalibrated the ICP-MS between runs, resulting in a
range of LODs. The range of LODs (µg mL-1) of each heavy metal are as follows: Ag: 0.000007-
0.00027, Al: 0.00012-0.03014, As: 0.000007-0.0001, Cd: 0.000008-0.00009, Co: 0.00001-
0.00011, Cr: 0.000008-0.00056, Fe: 0.00168-0.0168, Mn: 0.00003-0.00063, Ni: 0.00003-0.00153,
Pb: 0.00001-0.00008, Se: 0.00016-0.00153, Zn: 0.00006-0.00072. We considered heavy metal
concentrations below the LOD as undetectable (i.e. 0). We report heavy metal concentrations in
µg g⁻¹ wet weight of the tissue samples.
3.3.3 Statistical Analysis
We conducted statistical analyses using R (R Core Team, 2020). We executed a one-way ANOVA
to compare the concentrations of each heavy metal between loggerhead turtles from different
locations (CCB, MAB, NC). When the assumption of normality was not met for ANOVA, we log
transformed the values of non-parametric heavy metal concentrations to normalize the data. For
running correlations, we used Pearson’s correlation to investigate the significance between heavy
metals in skin and scute samples for parametric datasets, and Spearman’s correlation for non-
parametric datasets. We used Pearson’s and Spearman’s correlation to assess the relationships
between heavy metal concentrations and turtle body size. When needed, we converted the heavy
metal concentrations of loggerhead scutes of other studies from µg g⁻¹ dry weight to µg g⁻¹ wet
weight, using the value of 29.1% moisture content (Rodriguez et al. 2022).
For the prey samples, we ran two subsets of crushed prey samples per prey. When the
difference between heavy metal concentrations of both subsets were greater than 1 magnitude, we
assumed that the crushed samples were not well mixed and ran a third subset for analysis. We also
converted the heavy metal concentrations of loggerhead scutes from µg g⁻¹ wet weight to µg g⁻¹
53
dry weight, using the value of 29.1% moisture content (Rodriguez et al. 2022). We calculated the
TTF as a ratio of the median concentration of each heavy metal in the loggerhead scute per location
to the median concentration of each prey sample (DeForest et al. 2007).
3.4 Results
We sampled 63 loggerhead sea turtles for scute tissue (n=17 MA; n=9 NC; n=37 MAB).
TEWG (2009) classifies loggerhead life stages based on their straight carapace length (SCL) —
Stage II juveniles range between 41-82cm SCL, Stage III juveniles range between 63-100cm SCL,
and adults are greater than 82cm SCL. Due to the overlap in the groupings, our loggerhead turtles
from CCB and NC are classified as Stage II or Stage III juveniles, and MAB loggerhead turtles
are classified as Stage III juvenile or adults (Table 3-1). It is unlikely that the CCB and NC turtles
have reached sexual maturity while some of the MAB turtles have possibly reached sexual
maturity. Heavy metal concentrations of the scute samples are reported in
54
Table 3-1 Range, mean and standard deviation of straight carapace length (SCL) of the loggerheads
collected during cold-stunned events from Cape Cod Bay in Massachusetts, Mid-Atlantic Bight
and North Carolina coast in the USA.
Location
n
SCL (cm)
Range
Mean ± SD
Cape Cod Bay (CCB)
17 28.5-69.2
51.7 ± 9.7cm
North Carolina Coast (NC) 9 53.2-82
68.7 ± 10.3cm
Mid-Atlantic Bight (MAB) 37 64.6-95.6
82.9 ± 8.4cm
55
Table 3-2 Heavy metal concentrations detected in scute samples of loggerheads from Cape Cod
Bay, Massachusetts (n=17), North Carolina (n=9) and Mid-Atlantic Bight (n=37).
NOTE: Scute heavy metal concentration values are reported both in µg g-1 wet weight.
*indicates significant differences between different locations
<LOD = below method detection limit
3.4.1 Heavy metal concentrations of scute samples from loggerhead turtles in different
locations
We found scute samples collected from turtles in different locations to differ statistically
in aluminum (p<0.0001), arsenic (p<0.0001), cadmium (p<0.0001), chromium (p=0.002), iron
(p<0.0001), manganese (p<0.0001), lead (p=0.008) and selenium (p=0.004). Across the different
locations, we found iron (CCB=73.88±52.19 µg g-1; NC=425.17±361.99 µg g-1;
MAB=245.65±271.21 µg g-1) and selenium (CCB=0.257±0.047 µg g-1; NC=63.357 (n=1) µg g-1;
MAB=11.383±11.066 µg g-1) to vary significantly. We also found aluminum to be much higher in
NC samples (CCB=43.70±35.24 µg g-1; NC=246.67±229.80 µg g-1; MAB=68.21±68.79 µg g-1)
and chromium to be much lower in CCB samples (CCB=0.854±0.968 µg g-1; NC=4.187±2.517 µg
g-1; MAB=3.189±3.977 µg g-1).
Cape Cod, MA
North Carolina
Mid-Atlantic Bight
Elements
n
mean ± SD
n
mean ± SD
n
mean ± SD
Essential elements
Silver
2 0.028 ± 0.010
0 <LOD
3 0.030 ± 0.015
Aluminum* 17 43.70 ± 35.24
9 246.67 ± 229.79
37 68.21 ± 68.79
Arsenic*
17 1.79 ± 0.85
9 2.78 ± 1.52
32 0.83 ± 0.56
Cadmium* 17 0.256 ± 0.150
4 0.103 ± 0.042
20 0.095 ± 0.040
Lead*
12 0.197 ± 0.264
7 0.509 ± 0.350
16 0.125 ± 0.100
Non-essential elements
Cobalt
3 0.032 ± 0.012
8 0.124 ± 0.130
10 0.095 ± 0.059
Chromium* 16 0.854 ± 0.968
7 4.187 ± 2.517
35 3.189 ± 3.977
Iron*
17 73.89 ± 52.19
9 425.17 ± 361.99
37 245.65 ± 271.21
Manganese* 17 1.302 ± 1.480
9 5.392 ± 4.589
37 2.962 ± 3.038
Nickel
17 1.528 ± 1.691
1 3.258
37 1.097 ± 0.511
Selenium*
2 0.257 ± 0.047
1 63.357
6 11.383 ± 11.066
Zinc
17 201.79 ± 50.97
9 184.66 ± 70.85
37 172.92 ± 52.16
56
We did not observe any strong correlations between heavy metals and increasing carapace
size (Figure B-1). However, there were a few heavy metals that did have significant relationships
but weak correlations when heavy metal concentrations and increasing carapace size were
compared. Arsenic (R=-0.5, p<0.0001) and cadmium (R=-0.55, p<0.0001) decreased with
increasing carapace size, whereas chromium (R=0.37, p=0.0058), iron (R=0.34, p=0.0079) and
manganese (R=0.27, p=0.0037) increased with increasing carapace size (Figure B-1). We found
that zinc concentrations across the three loggerhead locations were not consistent, and the data was
varied over a wide range (87.42-304.69 µg g-1).
It is of interest to note that one turtle from North Carolina, NC.CC.2 (SCL: 60.0cm) was
an outlier for numerous heavy metals across all sites. When we compared this turtle to other NC
turtles, we found it to have the highest concentrations for aluminum, cobalt, iron, manganese, and
selenium. When compared to turtles from all sites, we found it to have the highest concentrations
for aluminum (CCB=43.70±35.24 µg g-1; NC=246.67±229.79 µg g-1; MAB=68.21±68.79 µg g-1)
and selenium (CCB=0.257±0.047 µg g-1; NC=63.357 µg g-1; MAB=11.383±11.066 µg g-1), and
second highest concentrations for manganese (CCB=1.302±1.480 µg g-1; NC=5.392±4.589 µg g-
1; MAB=2.962±3.038 µg g-1), iron (CCB=73.89±52.19 µg g-1; NC=425.17±361.99 µg g-1;
MAB=245.65±271.21 µg g-1), and cobalt (CCB=0.032±0.012 µg g-1; NC=0.124±0.130 µg g-1;
MAB=0.095±0.059 µg g-1).
3.4.2 Heavy metal concentrations in loggerhead turtles and their prey
We analyzed a total of 27 prey samples for heavy metals (n=5 crab; n=10 scallop; n=13
whelk) (Table 3-3). However, one of the whelk samples was empty and so it’s flesh or operculum
could not sampled, only the shell (Table 3-4).
57
Table 3-3 Range, mean and standard deviation of prey dry weight collected off the coast of New
Jersey by commercial scallop fishermen.
Prey
n Weight (g)
Range
Mean ± SD
Crab
Cancer borealis
5 53.0-158.7
90.8 ± 40.4cm
Scallop
Placopecten
magellanicus
10 61.2-131.7
91.5 ± 23.2cm
Whelk
Buccinum undatum
12 16.8-35.5
23.9 ± 45.36cm
58
Table 3-4 Heavy metal concentrations (µg g-1 dry weight) detected in whole crabs (n=5), scallop flesh (n=10) and shell (n=10), whelk
flesh (n=12), shell (n=13) and operculum (n=12) collected from the coast of New Jersey, USA.
Crab
Scallop
Whelk
Flesh
Shell
Flesh
Shell
Operculum
Elements n mean ± SD n mean ± SD n mean ± SD n mean ± SD n mean ± SD n mean ± SD
Non-essential elements
Silver
5 0.842 ±
0.37
5
0.286 ±
0.097
2
0.358 ±
0.255
12 2.922 ±
1.471
4
0.447 ±
0.200
1
0.395
Aluminum 5 592.14 ±
401.49
10 1607.503 ±
661.01
10
87.47 ±
43.99
12 76.59 ±
44.81
13 169.76 ±
119.98
12 795.10 ±
489.42
Arsenic
5 18.31 ±
13.19
10 6.22 ± 0.68 10 1.68 ± 0.68 12 31.86 ±
12.82
13 1.63 ± 1.28 12 4.66 ± 3.90
Cadmium
5 4.69 ± 4.86 10
29.47 ±
6.16
10 0.30 ± 0.20 12 22.00 ±
15.64
13 1.10 ± 1.84 11 2.39 ± 2.88
Lead
5 1.59 ± 0.87 10
2.196 ±
1.004
10
0.252 ±
0.175
12 0.182 ±
0.047
13
0.332 ±
0.280
12 1.95 ± 1.45
Essential elements
Cobalt
5 0.446 ±
0.269
10
0.781 ±
0.223
4
0.301 ±
0.379
12 0.133 ±
0.051
8
0.145 ±
0.217
11 0.44 ± 0.24
Chromium 5 1.77 ± 1.34 10 3.17 ± 1.42 1
1.33
0
<LOD
5 0.58 ± 0.39 4 2.97 ± 1.35
Iron
5 1242.07 ±
1047.27
10 2336.57 ±
964.94
10 105.71 ±
53.86
12 113.33 ±
31.52
13 174.13 ±
163.13
12 968.12 ±
756.34
Manganese 5 246.70 ±
131.17
10
51.70 ±
19.86
10
20.34 ±
7.06
12 6.71 ± 1.94 13
23.19 ±
14.26
12 153.92 ±
12.72
Nickel
4 0.956 ±
0.401
6
1.587 ±
0.380
0
<LOD
0
<LOD
0
<LOD
3
3.073 ±
0.872
Selenium
5 8.80 ± 5.64 10 8.61 ± 8.99 10
19.37 ±
4.81
10 1.54 ± 0.65 13
10.42 ±
9.20
12
11.79 ±
5.91
Zinc
5 27.64 ±
22.66
10
43.86 ±
7.48
3 2.36 ± 1.87 12 178.38 ±
68.89
7 4.22 ± 4.82 12
38.19 ±
38.22
NOTE: <LOD = below detection limit
59
When comparing median heavy metal concentrations between loggerhead turtle groups and
prey samples (Table B-1), we found CCB turtles consistently to have one of the lowest
concentrations for all heavy metals, whereas MAB was ranked in the middle for all heavy metals.
The exception to this is zinc, where all loggerhead groups had higher concentrations than any prey
samples.
We found six heavy metals in MAB scutes to have a TTF>1 (Table 3-5). They are
chromium in crab; whelk shell and scallop shell; nickel in crab; iron and cobalt in whelk flesh,
whelk shell and scallop shell; zinc in all prey samples; selenium in crab, whelk flesh and scallop
flesh. We found nine heavy metals in NC loggerhead turtles to have a TTF>1 (Table 3-5). They
are lead in whelk flesh, whelk shell and scallop shell; arsenic in whelk shell and scallop shell;
aluminum in whelk flesh, whelk shell and scallop shell; chromium in crab, whelk operculum,
whelk shell, scallop flesh and scallop shell; nickel in crab, whelk operculum and scallop flesh; iron
in whelk flesh, whelk shell and scallop shell; cobalt in whelk shell; zinc and selenium in all prey
components.
We found zinc to be the only heavy metal to have a TTF>1 across all prey samples and
locations (Table 3-5). Selenium was the second most with TTF>1 in all prey samples in the NC
turtles, and crab, whelk flesh and scallop flesh in MAB turtles. We found that arsenic only had a
TTF>1 in whelk shell and scallop shell in NC turtles. On the other hand, we found manganese,
silver, and cadmium to have a TTF<1 across all prey samples and locations. In MAB turtles, lead
and aluminum had TTF<1 across all prey samples.
60
Table 3-5 Trophic transfer values for loggerheads from Cape Cod Bay, Massachusetts, Mid-Atlantic Bight and North Carolina.
Heavy Metals
Mid-Atlantic Bight
North Carolina
C
rab
S
callop F
lesh
S
callop S
hell
W
helk
F
lesh
W
helk
S
hell
W
helk
Operculu
m
C
rab
S
callop F
lesh
S
callop S
hell
W
helk
F
lesh
W
helk
S
hell
W
helk
Operculu
m
Silver
0.03 0.13 0.09 0.01 0.07 0.08
NA NA NA NA NA NA
Aluminum
0.09 0.03 0.57 0.79 0.43 0.07
0.42 0.15 2.61 3.60 1.98 0.32
Arsenic
0.06 0.17 0.71 0.04 0.83 0.29
0.17 0.49 2.04 0.11 2.39 0.83
Cadmium
0.05 0.00 0.47 0.01 0.31 0.09
0.05 0.00 0.51 0.01 0.33 0.09
Lead
0.08 0.06 0.57 0.66 0.47 0.06
0.53 0.39 3.65 4.24 3.03 0.42
Cobalt
0.28 0.18 1.18 1.07 3.81 0.33
0.24 0.15 0.99 0.90 3.19 0.28
Chromium
1.63 0.62 1.53 NA 4.55 0.72
3.35 1.27 3.13 NA 9.32 1.47
Iron
0.25 0.11 2.32 2.02 1.65 0.30
0.40 0.17 3.76 3.27 2.67 0.48
Manganese
0.01 0.05 0.13 0.36 0.12 0.02
0.02 0.09 0.24 0.66 0.21 0.04
Nickel
1.29 0.78 NA NA NA 0.38
4.12 2.48 NA NA NA 1.20
Selenium
1.06 1.73 0.44 5.55 0.81 0.73
10.43 17.07 4.30 54.60 7.95 7.19
Zinc
10.20 5.35 152.25 1.38 129.02 9.68
8.21 4.31 122.48 1.11 103.79 7.79
Trophic transfer factors >1 are bolded, indicating potential risk of biomagnification.
NAs are heavy metal concentration ratios which were not calculated due to one or both values being <LOD.
61
3.5 Discussion
Our CCB and NC loggerhead turtles are still considered to be in their juvenile stages
whereas some of our MAB loggerhead turtles are considered adults. While we can be quite certain
that most of our MAB loggerhead turtles have recruited to neritic developmental waters and
transitioned to a predominantly carnivorous diet, our CCB and NC turtles are probably undergoing
or have just undergone this ontogenetic shift (Nelson 1988). Since scute samples reflect exposure
from 4-6 years ago, it is likely that CCB and NC turtles’ scute samples may have yet to reflect
heavy metals from a predominantly carnivorous diet (Vander Zanden et al. 2013). Through
analyzing heavy metal concentrations, we see shifts in loggerhead turtle diet through higher
chromium, manganese, iron, and selenium concentrations in the MAB turtles, despite them having
mean SCL measurements greater than CCB but smaller than NC turtles. We also see effects of
environmental exposure in CCB turtles as they have the highest cadmium concentrations despite
being the smallest sized turtles. As for comparing heavy metal concentrations in loggerhead turtle
scute samples to their prey, zinc is the only heavy metal to show signs of biomagnification across
all samples and prey, whereas silver, cadmium and manganese do not show any signs of
biomagnification across all samples and prey.
3.5.1 Inter-site comparison of heavy metal concentrations – Environment
Despite CCB (0.256 ± 0.150 µg g-1) loggerhead turtles being the smallest in size, they had
the highest cadmium concentrations when compared to NC (0.103 ± 0.042 µg g-1) and MAB (0.095
± 0.040 µg g-1) samples (
62
Table 3-2). We postulate that the higher cadmium concentrations in the CCB samples likely
stem from Massachusetts smelting sites (Eckel et al. 2001) that releases cadmium as a by-product
(Bradl 2005). As cadmium is a non-essential element, it is likely that the cadmium is accumulating
in the loggerhead turtles’ carapace as a detoxifying mechanism (Martín et al. 2021). This is
supported by the fact that cadmium concentrations have a negative correlation with increasing
carapace size, indicating that cadmium does not bioaccumulate in the loggerheads (Figure B-1).
We also did not find MAB loggerhead turtles to have a TTF>1 when compared to any prey items,
indicating that cadmium does not biomagnify up trophic levels (Table 3-5). Sun et al. (2020) also
found cadmium to not biomagnify in higher trophic levels through marine food webs.
3.5.2 Inter-site comparison of heavy metal concentrations - Diet
Although NC loggerhead turtles are bigger than CCB turtles and smaller than MAB turtles,
we found NC turtles to have a significantly higher concentration of chromium, manganese (CCB:
1.302 ± 1.480 µg g-1; NC: 5.329 ± 4.589 µg g-1; MAB: 2.962 ± 3.038 µg g-1), iron (CCB: 73.89 ±
52.19 µg g-1; NC: 425.17 ± 361.99 µg g-1; MAB: 245.65 ± 271.21 µg g-1), and selenium (CCB:
0.257 ± 0.047 µg g-1; NC: 63.357 µg g-1; MAB: 11.383 ± 11.066 µg g-1) compared to the other two
locations. We postulate that the higher heavy metal concentrations when comparing NC samples
to the smaller CCB turtles is likely due to bioaccumulation in the turtles. This is supported by the
weak but significant positive correlation in chromium (R=0.37, p-value=0.0058), iron (R=0.34, p-
value=0.0079), and manganese (R=0.27, p-value=0.037) with increasing carapace size (Figure B-
1). On the other hand, we found that MAB turtles that are bigger than NC turtles have lower
chromium, manganese, iron, and selenium concentrations in their scute. This is likely due to the
transition of loggerhead turtle diets from juveniles having an omnivorous diet to adults having a
predominantly carnivorous diet (Shaver 1991). It is likely that NC loggerhead turtles might still be
consuming algae and seagrass that have high manganese concentrations (Shaver 1991) and have
the potential to bioaccumulate iron (Andreani et al. 2008). Even if the NC turtles have transitioned
to a carnivorous diet, scute samples show longer term diet and environmental exposure (Vander
Zanden et al. 2013). This means that scute samples from NC turtles might still reflect remnants of
their omnivorous diet, whereas MAB scute samples could have begun reflecting elements of a
carnivorous diet.
63
We found NC (201.79 ± 50.97 µg g-1) and MAB (172.92 ± 52.16 µg g-1) loggerhead turtles to have
comparable zinc concentrations, although they were lower than CCB (184.66 ± 70.85 µg g-1)
samples (
64
Table 3-2). The difference in zinc concentrations is likely due to CCB loggerhead turtles
being smaller than NC and MAB loggerhead turtles. According to Hatase and Tsukamoto (2008),
smaller adult loggerhead female turtles had higher reproductive energy costs compared to larger
turtles. This also meant that smaller turtles needed to consume more prey to meet this higher
reproductive energy cost. While Hatase and Tsukamoto’s (2008) study focused on reproductive
energy cost, the theory of smaller loggerhead turtles having higher energy cost and requiring
greater prey biomass could possibly be applied to other forms of energy consumption as well (i.e.
growth). It is likely that our smaller CCB loggerhead turtles consume a greater quantity as part of
their diet, contributing to higher zinc concentrations in their scute.
We found nickel concentrations in CCB (1.528 ± 1.691 µg g-1) and MAB (1.097 ± 0.511
µg g-1) turtles to be comparable, despite NC (3.258 µg g-1) having a higher concentration outlier.
Interestingly, we only detected nickel in one sample from NC loggerhead turtles, at higher
concentrations than that of any CCB and MAB samples. Nickel being detected in fish samples in
the Mid-Atlantic Bight (Balthis et al. 2009) and South Atlantic Bight (Cooksey et al. 2010)
indicates that there is not a lack of nickel in these environments. While the reason behind the lack
of nickel in the NC samples is unclear, this occurrence could possibly be related to their difference
in behavior and habitat use. Although loggerhead turtles do forage in the MAB, the NC turtles
behave slightly differently than those sampled directly in that foraging ground. Patel et al. (2022)
found that turtles tagged in NC foraged more inshore and some travelled farther north than those
that were tagged directly in offshore MAB.
3.5.3 Heavy metal concentrations in loggerhead turtles and their prey
Comparing prey samples to other studies conducted on the same species (Table 3-6), I
found that whole crab samples had higher concentrations of cadmium, lead, and chromium but
lower concentration of zinc when compared to leg muscle, leg and hepatopancreas on crab from
the New England Coast in the 1980s (Pecci 1987). Our scallop flesh had slightly higher silver and
lead concentrations, but much higher chromium and zinc concentrations when compared to scallop
muscle collected from Eastern United States in the 1970s (Greig et al. 1978). These differences in
concentration might be due to different crab and scallop parts having different concentrations of
heavy metals, or the fact that heavy metal concentrations have drastically changed over the past
40-50 years. Comparing whelk flesh to a study on the same species collected in France, our whelk
65
flesh had higher concentrations of silver, cadmium, and zinc but lower lead levels (Amiard et al.
2008). Despite our samples being measured in dry weight and other studies being measured in wet
weight, all heavy metals in all samples except for silver and lead in scallops are different by
magnitudes big enough for the comparisons to remain true. The differences in heavy metal
concentrations are therefore probably due to regional environmental differences.
66
Table 3-6 Heavy metal concentrations (µg g-1 wet weight) of same-species preys (crab leg muscle, crab leg, crab hepatopancreas, whelk
flesh, scallop muscle, scallop male gonad and scallop female gonad) conducted in other studies.
Crab
(Pecci 1987)
Scallop
(Greig et al. 1978)
Whelk
(Amiard et al.
2008)
Leg Muscle
Leg
Hepatopan-
creas
Muscle
Male Gonad Female Gonad
Flesh
Elements n
mean ±
SD
n
mean ±
SD
n
mean ±
SD
n
mean ±
SD
n
mean ±
SD
n
mean ±
SD
n
mean ±
SD
Non-essential elements
Silver
13
0.15 ±
0.04
24
0.33 ±
0.15
23
0.30 ±
0.13
6
0.63 ±
0.05
Cadmium
6
0.11 ±
0.08
5
0.53 ±
0.39
1 17.3 0 <LOD 26
1.30 ±
0.75
25
1.56 ±
0.80
6
1.70 ±
1.00
Lead
6
0.09 ±
0.06
5
5
0.35 ±
0.39
1 0.92 2
1.30 ±
0.57
2
1.15 ±
0.49
4
0.86 ±
0.21
6
0.37 ±
0.14
Essential elements
Chromium 6
0.08 ±
0.06
5
0.35 ±
0.39
1 0.92 10
0.49 ±
0.12
4
1.51 ±
1.52
2
0.43 ±
0.08
Nickel
0 <LOD 9
0.87 ±
0.67
10
0.55 ±
0.16
Zinc
6
71.30 ±
7.62
89.42 ±
9.91
1 56.5 40
3.98 ±
1.63
26
15.83 ±
7.72
25
43.46 ±
15.96
16
61.00 ±
25.00
67
Zinc was the only heavy metal that had TTF>1 across loggerhead groups and the different
prey samples. This indicates that there is a high potential for zinc to biomagnify up the loggerhead
sea turtle food chain. Despite zinc being an essential element, it can be toxic at high concentrations
(Wang 2005). Like cadmium, zinc is probably accumulating in the carapace as a detoxifying
mechanism (Martín et al. 2021). In a global marine food web meta-analysis, Sun et al. (2020)
found that zinc was transferred between trophic levels inconsistently. They suggested that this
occurrence is likely due to different seasonal bioavailability of zinc and varying metabolic
regulation mechanisms in different organisms.
The TTF<1 in silver, cadmium and manganese in all loggerhead groups indicate that these
elements probably do not biomagnify in loggerhead turtles on east coast USA. While silver was
detected in <12% of the loggerhead turtle samples, cadmium and manganese were detected in
majority (65%) of the loggerhead turtle samples. In a heavy metal study on hawksbill, green, and
loggerhead sea turtles, Mondragón et al. (2023) found manganese to biodilute with higher trophic
levels. Wang (2005) also suggested that silver may not bioaccumulate in carapace tissue, which
explains the lack of silver in our scute samples. This however does mean that scute samples are
probably not useful indicators of silver concentrations in sea turtles.
3.5.4 Comparing heavy metal concentrations to other studies and their implications
Our NC loggerhead turtles had higher aluminum (246.67 ± 229.79 µg g-1), arsenic (2.78 ±
1.52 µg g-1), and lead (0.509 ± 0.350 µg g-1) concentrations than CCB (Al: 43.70 ± 35.24 µg g-1;
As: 1.79 ± 0.85 µg g-1; Pb: 0.197 ± 0.264 µg g-1) and MAB (Al: 68.21 ± 68.79 µg g-1; As: 0.83 ±
0.56 µg g-1; Pb: 0.125 ± 0.100 µg g-1) samples. As our arsenic concentrations did not increase with
increasing carapace size, this indicates that arsenic does not bioaccumulate in sea turtles (Figure
B-1). It is therefore likely that our smaller sized loggerhead turtles from CCB and NC had higher
arsenic concentrations because of their more recent omnivorous diet which contains algae and sea
grass (Shaver 1991). This is supported by the fact that our sub-adult MAB turtles had similar
arsenic concentrations to Miguel et al.’s (2022) adult loggerhead turtles (Location 1: 0.96 ± 0.98
µg g-1; Location 2: 1.01 ± 0.84 µg g-1) (Table 3-7). Despite arsenic being a non-essential element
that could result in negative physiological effects like reduced red and white blood cell count, as
well as cancer (ATSDR 2007), our larger MAB loggerhead turtles having lower arsenic
concentrations is reassuring as it shows that arsenic concentrations in loggerhead turtle scute
68
samples are likely to decrease as the turtles transition to a carnivorous diet. Although CCB and
NC loggerhead turtles have higher arsenic concentrations than the safe levels for Lanzhou catfish
is 1.288ppm (synonymous with µg g-1), our MAB loggerhead turtles’ levels are lower (Lian and
Wu 2017).
Despite CCB loggerhead turtles being the smallest in size, we found scute samples
collected from CCB loggerhead turtles (0.256± 0.150 µg g-1) to have higher cadmium
concentrations than NC (0.103 ± 0.042 µg g-1) and MAB (0.095 ± 0.040 µg g-1) samples. Across
all loggerhead scute samples, we found that cadmium concentrations decreased with increasing
carapace size. This indicates that cadmium is not likely to bioaccumulate in sea turtles (Figure B-
1). Furthermore, we also found that none of the distinct loggerhead scute origins had a TTF>1
when compared to any sampled prey (Table 3-5). We postulate that cadmium does not biomagnify
up trophic levels which is in agreement with Sun et al.’s (2020) finding that cadmium does not
biomagnify up marine food webs. As scute samples collected from CCB also had higher cadmium
concentrations compared to other similar studies in Japan (0.129 ± 0.034 µg g-1; Sakai et al. 2000)
and Brazil (Location 1: 0.004 ± 0.004 µg g-1; Location 2: 0.008 ± 0.01 µg g-1; Miguel et al. 2022),
it is likely that these higher cadmium concentrations stem from Massachusetts smelting sites
(Eckel et al. 2001) that releases cadmium as a by-product (Bradl 2005). As cadmium is a non-
essential element, it is likely that the cadmium is accumulating in the loggerhead carapace as a
detoxifying mechanism (Martín et al. 2021). While cadmium concentrations in our CCB
loggerhead turtles were higher, this level is still lower than 2ppm, which is the level at which
contamination is only considered in whole body tissue.
69
Table 3-7 Heavy metal concentrations found in loggerhead scute samples in the present study compared to other studies.
NOTE: Scute heavy metal concentration values are reported in µg g-1 wet weight
Cape Cod, MA
North Carolina
Mid-Atlantic Bight
References
Elements
n
mean ± SD
n
mean ± SD
n
mean ± SD
Non-essential elements
Arsenic
17 1.79 ± 0.85
9
2.78 ± 1.52
32 0.83 ± 0.56
Present Study
66 0.96 ± 0.98 b
37 1.01 ± 0.84 c
b Miguel et al. (2022)
c Miguel et al. (2022)
Cadmium
17 0.256 ± 0.150
4
0.103 ± 0.042
20 0.095 ± 0.040
Present Study
6
0.129 ± 0.034 a
66 0.004 ± 0.004 b
37 0.008 ± 0.01c
a Sakai et al. (2000)
b Miguel et al. (2022)
c Miguel et al. (2022)
Lead
12 0.197 ± 0.264
7
0.509 ± 0.350
16 0.125 ± 0.100
Present Study
6
2.42 ± 0.52 a
66 0.05 ± 0.08 b
37 0.05 ± 0.08 c
a Sakai et al. (2000)
b Miguel et al. (2022)
c Miguel et al. (2022)
Essential elements
Chromium
16 0.854 ± 0.968
7
4.187 ± 2.517
35 3.189 ± 3.977
Present Study
66 0.5 ± 0.9 b
37 0.39 ± 0.46 c
b Miguel et al. (2022)
c Miguel et al. (2022)
Iron
17 73.89 ± 52.19
9
425.17 ± 361.99
37 245.65 ± 271.21 Present Study
6
26.2 ± 19.1a
66 358 ± 411b
37 247 ± 201c
a Sakai et al. (2000)
b Miguel et al. (2022)
c Miguel et al. (2022)
Manganese
17 1.302 ± 1.480
9
5.392 ± 4.589
37 2.962 ± 3.038
Present Study
6
7.01 ± 3.49 a
66 8.44 ± 5.21b
37 7.16 ± 4.91c
a Sakai et al. (2000)
b Miguel et al. (2022)
c Miguel et al. (2022)
Nickel
17 1.528 ± 1.691
1
3.258
37 1.097 ± 0.511
Present Study
6
0.0094 ± 0.022
a
a Sakai et al. (2000)
Zinc
17 201.79 ± 50.97
9
184.66 ± 70.85
37 172.92 ± 52.16 Present Study
6
198 ± 37.2 a
66 33.7 ± 16.3 b
37 34.1 ± 25.7 c
a Sakai et al. (2000)
b Miguel et al. (2022)
c Miguel et al. (2022)
70
Zinc was the only heavy metal that had TTF>1 across loggerhead groups and the different
prey samples. This indicates that there is a high potential for zinc to biomagnify up the loggerhead
sea turtle food chain. Despite zinc being an essential element, it can be toxic at high concentrations
(Wang 2005). Like cadmium, zinc is probably accumulating in the carapace as a detoxifying
mechanism (Martín et al. 2021). In a global marine food web meta-analysis, Sun et al. (2020)
found that zinc was transferred between trophic levels inconsistently. They suggested that this
occurrence is likely due to different seasonal bioavailability of zinc and varying metabolic
regulation mechanisms in different organisms.
The TTF<1 in silver, cadmium and manganese in all loggerhead groups indicate that these
elements probably do not biomagnify in loggerheads on east coast USA. While silver was detected
in <12% of the loggerhead samples, cadmium and manganese were detected in majority (65%) of
the loggerhead samples. In a heavy metal study on hawksbill, green, and loggerhead sea turtles,
Mondragón et al. (2023) found manganese to biodilute with higher trophic levels. Wang (2005)
also suggested that silver may not bioaccumulate in carapace tissue, which explains the lack of
silver in our scute samples. This however does mean that scute samples are probably not useful
indicators of silver concentrations in sea turtles.
Despite heavy metals in crabs not correlating with any loggerhead groups, numerous heavy
metals still had TTF>1 for the different loggerhead groups. MA loggerheads had TTF>1 for nickel
and zinc, MAB and NC loggerheads had TTF>1 for chromium, nickel, zinc and selenium. This
shows that loggerheads are possibly still consuming crabs but not all the heavy metals are being
incorporated into the scute. It is also important to note that our crab sample size was rather small
(n=5) and varied greatly in size (SD: 40.42g d.w.). A larger sample size is needed to determine if
any heavy metals in crabs biomagnifies in loggerhead carapace.
3.6 Conclusion
We see that heavy metal concentrations between loggerhead turtles from different locations
within the same region differ significantly. As we postulate that these differences are probably due
to their different life stages, it is important to analyze the heavy metal concentrations in these
populations as they make their ontogenetic shifts into adulthood. It is especially important to
monitor zinc concentrations as this is the only heavy metal we found to biomagnify through trophic
levels regardless of prey items and the sampling site of the loggerhead turtles. Knowing the
71
difference in heavy metal concentrations between juvenile and adult loggerhead turtles would help
us better understand if the heavy metals are entering the turtles through diet or the environment.
This is especially important as the east coast US continues to undergo more development.
The NW Atlantic, a known foraging ground for green, loggerhead, and Kemp’s ridley sea
turtles as they recruit to neritic habitats, is susceptible to even more pollution in years to come.
The Atlantic Ocean is polluted by many non-point sources such as runoff from agriculture and
farmland, roads, atmospheric deposition, and septic tank discharge (NRC 2000, Howarth et al.
2002, Valiela and Bowen 2002). The ever-increasing development in northeastern US will only
cause more pollutants to enter the NW Atlantic. Furthermore, The Executive Office of Energy and
Environmental Affairs (EEA) are working on initiatives to develop offshore wind projects in
Massachusetts waters (Mass.gov). While these windmills are meant to decarbonize energy supply
(Mass.gov), their galvanic anodes erode and release aluminum, zinc, and indium to the ocean.
There is also a possibility that they might release cadmium, lead, and copper too (BSH and Hereon
2022). Establishing baseline heavy metal concentrations in scute samples of loggerhead turtles in
the Mid-Atlantic Bay will facilitate future studies on pollution in the NW Atlantic.
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77
GENERAL CONCLUSIONS
Our study shows that heavy metal concentrations do indeed differ based on species, tissue,
and location of turtle populations sampled. We found that turtle species believed to be associated
with a heavier algae/ seagrass diet (i.e. green turtles skin samples, juvenile loggerhead turtles) tend
to reflect higher concentrations of manganese, iron, cobalt, and selenium; whereas turtle
populations associated with more polluted environments (i.e. Cape Cod Bay Massachusetts) tend
to reflect higher concentrations of cadmium and arsenic, among other pollutants. Overall, we also
saw higher heavy metal concentrations in scute samples compared to skin samples, which is likely
due to scute samples having a slower turnover rate.
While we do not know the exact toxic concentration of these heavy metals in sea turtles,
this study provides a baseline knowledge of heavy metal concentrations in skin and scute samples
of sea turtles in the NW Atlantic Ocean. Such information is especially important with plans for
offshore windfarm development soon and projected increase in thermal windows in the NW
Atlantic. Collecting skin and scute samples from turtles is a relatively non-invasive process.
Therefore, future studies would be able to collect skin and scute samples from turtles in the area
to monitor local environmental pollution as well as the health of the turtle populations.
78
APPENDIX A. CAPE COD TURTLES
Figure A-1. Relationship between heavy metal concentrations in green sea turtle skin samples with
increasing carapace size.
NOTE: SCL = Straight Carapace Length, Ag = silver, Al = aluminum, As = arsenic, Cd =
cadmium, Co = cobalt, Cr = chromium, Fe = iron, Mn = manganese, Ni = nickel, Pb = lead, Se
= selenium, Zn = zinc.
79
Figure A-2. Relationship between heavy metal concentrations in green sea turtle scute samples
with increasing carapace size.
NOTE: SCL = Straight Carapace Length, Al = aluminum, As = arsenic, Cd = cadmium, Co =
cobalt, Cr = chromium, Fe = iron, Mn = manganese, Ni = nickel, Pb = lead, Zn = zinc.
80
Figure A-3. Relationship between heavy metal concentrations in Kemp’s ridley sea turtle skin
samples with increasing carapace size.
NOTE: SCL = Straight Carapace Length, Ag = silver, Al = aluminum, As = arsenic, Cd =
cadmium, Co = cobalt, Cr = chromium, Fe = iron, Mn = manganese, Ni = nickel, Pb = lead, Se
= selenium, Zn = zinc.
81
Figure A-4. Relationship between heavy metal concentrations in Kemp’s ridley sea turtle scute
samples with increasing carapace size.
NOTE: SCL = Straight Carapace Length, Ag = silver, Al = aluminum, As = arsenic, Cd =
cadmium, Co = cobalt, Cr = chromium, Fe = iron, Mn = manganese, Ni = nickel, Pb = lead, Zn
= zinc.
82
Figure A-5. Relationship between heavy metal concentrations in loggerhead sea turtle skin samples
with increasing carapace size.
NOTE: SCL = Straight Carapace Length, Ag = silver, Al = aluminum, As = arsenic, Cd =
cadmium, Co = cobalt, Cr = chromium, Fe = iron, Mn = manganese, Ni = nickel, Pb = lead, Se
= selenium, Zn = zinc.
83
Figure A-6. Relationship between heavy metal concentrations in loggerhead sea turtle scute
samples with increasing carapace size.
NOTE: SCL = Straight Carapace Length, Al = aluminum, As = arsenic, Cd = cadmium, Cr =
chromium, Fe = iron, Mn = manganese, Ni = nickel, Pb = lead, Zn = zinc.
84
Figure A-7. Relationship between heavy metal concentrations in skin and scute samples of Kemp’s
ridley sea turtles.
NOTE: Ag = silver, Al = aluminum, As = arsenic, Cd = cadmium, Co = cobalt, Cr = chromium,
Fe = iron, Mn = manganese, Ni = nickel, Pb = lead, Zn = zinc.
85
Figure A-8. Relationship between heavy metal concentrations in skin and scute samples of green
sea turtles.
NOTE: Al = aluminum, As = arsenic, Cd = cadmium, Co = cobalt, Cr = chromium, Fe = iron,
Mn = manganese, Ni = nickel, Pb = lead, Zn = zinc.
86
Figure A-9. Relationship between heavy metal concentrations in skin and scute samples of
loggerhead sea turtles.
NOTE: Al = aluminum, As = arsenic, Cd = cadmium, Cr = chromium, Fe = iron, Mn = manganese,
Ni = nickel, Pb = lead, Zn = zinc.
87
APPENDIX B. LOGGERHEADS AND PREY
Figure B-1. Relationship between heavy metal concentrations in loggerhead sea turtle scute
samples with increasing carapace size.
NOTE: SCL = Straight Carapace Length, Grey area = 95% Confidence Interval, Ag = silver, Al
= aluminum, As = arsenic, Cd = cadmium, Co = cobalt, Cr = chromium, Fe = iron, Mn =
manganese, Ni = nickel, Pb = lead, Se = selenium, Zn = zinc.
88
Table B-1 Median heavy metal concentrations (µg g-1 dry weight) of all loggerhead groups (MA, NC, MAB) and different prey samples,
ranked in order from lowest to highest concentration (left to right) for each heavy metal.
Lead
MAB
Whelk
Flesh
Scallop
Shell
Whelk
Shell
NC
Crab
Whelk
Operculum
Scallop
Flesh
0.123
0.186
0.216
0.26
0.789
1.481
1.897
2.007
Arsenic
MAB
Scallop
Shell
MA
NC
Whelk
Operculum
Scallop
Flesh
Crab
Whelk
Flesh
1.029
1.452
1.985
2.965
3.588
6.11
17.413
27.394
Aluminum
MA
Whelk
Flesh
Scallop
Shell
Whelk
Shell
NC
Crab
Whelk
Operculum
Scallop
Flesh
35.999
65.449
90.498
119.275
235.8
561.496
741.492
1606.833
Chromium
Whelk
Shell
Crab
Scallop
Shell
MAB
Whelk
Operculum
Scallop
Flesh
NC
Whelk
Flesh
0.447
1.245
1.333
2.035
2.84
3.272
4.168
NA
Manganese
MA
NC
Whelk
Flesh
Scallop
Shell
Whelk
Shell
Scallop
Flesh
Whelk
Operculum
Crab
1.087
4.502
6.802
19.142
21.109
48.436
128.344
258.03
Nickel
Crab
MAB
Scallop
Flesh
Whelk
Operculum
NC
Whelk
Flesh
Scallop
Shell
Whelk
Shell
1.021
1.32
1.698
3.514
4.206
NA
NA
NA
Iron
MA
Whelk
Flesh
Whelk
Shell
MAB
NC
Whelk
Operculum
Crab
Scallop
Flesh
87.615
112.54
137.582
227.216
367.986
767.812
909.366
2163.578
Cobalt
Whelk
Shell
NC
Scallop
Shell
Whelk
Flesh
MAB
Whelk
Operculum
Crab
Scallop
Flesh
0.036
0.115
0.116
0.128
0.137
0.413
0.482
0.779
Zinc
Scallop
Shell
Crab
Whelk
Operculum
Scallop
Flesh
Whelk
Flesh
NC
MA
MAB
1.599
23.86
25.157
45.469
176.15
195.849
230.746
243.455
Selenium
MA
Scallop
Flesh
Crab
MAB
Whelk
Shell
Whelk
Operculum
Scallop
Shell
NC
0.332
4.793
7.842
8.311
10.29
11.375
19.032
81.794
Silver
MAB
Scallop
Flesh
Scallop
Shell
Whelk
Operculum
Whelk
Shell
Crab
Whelk
Flesh
NC
0.032
0.252
0.358
0.395
0.435
0.946
2.644
NA
Cadmium
MAB
MA
Scallop
Shell
Whelk
Shell
Whelk
Operculum
Crab
Whelk
Flesh
Scallop
Flesh
0.126
0.258
0.266
0.41
1.451
2.556
15.595
29.716