Metal concentrations in coastal sharks from The Bahamas with a focus on the Caribbean Reef shark

Abstract

Over the last century anthropogenic activities have rapidly increased the influx of metals and metalloids entering the marine environment, which can bioaccumulate and biomagnify in marine top consumers. This may elicit sublethal effects on target organisms and have broad implications for human seafood consumers. We provide the first assessment of metal (Cd, Pb, Cr, Mn, Co, Cu, Zn, As, Ag, and THg) and metalloid (As) concentrations in the muscle tissue of coastal sharks from The Bahamas. A total of 36 individual sharks from six species were evaluated, spanning two regions/study areas, with a focus on the Caribbean reef shark (Carcharhinus perezi), and to a lesser extent the tiger shark (Galeocerdo cuvier), due their high relative abundance and ecological significance throughout coastal Bahamian and regional ecosystems. Caribbean reef sharks exhibited some of the highest metal concentrations compared to five other species, and peaks in the concentrations of Pb, Cr, Cu, Zn, and Ag were observed as individuals reached sexual maturity. Observations were attributed to foraging on larger, more piscivorous prey, high longevity, as well a potential slowing rate of growth. We observed correlations between some metals, which are challenging to interpret but may be attributed to trophic level and ambient metal conditions. Our results provide the first account of metal concentrations in Bahamian sharks, suggesting individuals exhibit high concentrations which may potentially cause sublethal effects. Finally, these findings underscore the potential toxicity of shark meat and have significant implications for human consumers.

Full text

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Metal concentrations in coastal
sharks from The Bahamas
with a focus on the Caribbean Reef
shark
Oliver N. Shipley
1*, Cheng‑Shiuan Lee
1,2, Nicholas S. Fisher
1, James K. Sternlicht
3,
Sami Kattan
3, Erica R. Staaterman
3, Neil Hammerschlag
4 & Austin J. Gallagher
3
Over the last century anthropogenic activities have rapidly increased the inux of metals and
metalloids entering the marine environment, which can bioaccumulate and biomagnify in marine top
consumers. This may elicit sublethal eects on target organisms, having broad implications for human
seafood consumers. We provide the rst assessment of metal (Cd, Pb, Cr, Mn, Co, Cu, Zn, As, Ag, and
THg) and metalloid (As) concentrations in the muscle tissue of coastal sharks from The Bahamas.
A total of 36 individual sharks from six species were evaluated, spanning two regions/study areas,
with a focus on the Caribbean reef shark (Carcharhinus perezi), and to a lesser extent the tiger shark
(Galeocerdo cuvier). This is due their high relative abundance and ecological signicance throughout
coastal Bahamian and regional ecosystems. Caribbean reef sharks exhibited some of the highest
metal concentrations compared to ve other species, and peaks in the concentrations of Pb, Cr, Cu
were observed as individuals reached sexual maturity. Observations were attributed to foraging on
larger, more piscivorous prey, high longevity, as well a potential slowing rate of growth. We observed
correlations between some metals, which are challenging to interpret but may be attributed to trophic
level and ambient metal conditions. Our results provide the rst account of metal concentrations in
Bahamian sharks, suggesting individuals exhibit high concentrations which may potentially cause
sublethal eects. Finally, these ndings underscore the potential toxicity of shark meat and have
signicant implications for human consumers.
Over the past century, anthropogenic activities such as rapid industrialization, smelting, and fossil fuel combus-
tion have signicantly increased the concentration of metals and metalloids (herein metals) entering marine
environments1. Many metals (e.g., Cr, Cu, and Zn) are introduced into marine systems via freshwater inputs,
euent run-o, weathering, and ocean–atmosphere interactions2,3. Although essential metals such as Cr, Cu,
and Zn are required at low concentrations to support healthy cellular processes, many can become toxic when
they exceed threshold concentrations4,5. ese eects may cause sublethal impacts in aquatic organisms such as
delayed growth, reproductive impairment, and greater incidence of disease6,7 and may have carcinogenic and
neurotoxicological impacts for humans8. Most metals bioconcentrate and a few biomagnify in marine organisms
once they enter the ocean3,9,10. Accordingly, long-lived, large-bodied marine predators that exhibit higher trophic
positions oen display potentially toxic concentrations of metals and other toxicants11–14,and can therefore be
used as environmental sentinels for regional loadings15–17. As many higher trophic-level marine shes comprise
a proportion of the global seafood demand, a need exists to monitor metal concentrations and evaluate the
potential toxicity risk for humans readily consuming sh protein8,16,17.
Metal concentrations are typically evaluated in higher-order, commercially important shes to mitigate
potential toxic eects on humans; this concern has led to widespread monitoring and scientic study8,18–20.
However, for higher order predators of historically low commercial value, such as sharks, assessments are much
sparser. Sharks are medium to large-bodied predators that occupy meso-to-apex trophic positions throughout
marine food-webs21–23 and as a result are intrinsic to healthy ecosystem function and resilience24,25. Despite the
OPEN
1School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794, USA. 2New York
State Center for Clean Water Technology, Stony Brook University, Stony Brook, NY 11794, USA. 3Beneath the
Waves, PO Box 126, Herndon, VA, USA. 4Rosenstiel School of Marine and Atmospheric Science, University of
Miami, Miami, FL 33149, USA. *email: Oliver.shipley@stonybrook.edu
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historically low commercial value of shark meat, relative demand as a human protein source appears to be a
growing global trend26. A need to establish baseline concentrations of metals in sharks and their relatives, as
well as potential routes of exposure is therefore required14,17,20. is is particularly true for developing nations,
where less stringent environmental regulations regarding wastewater treatment, anthropogenic emissions, and
subsequent management may lead to elevated levels of metals entering coastal waters.
e developing nation of e Bahamas houses a diversity of productive marine ecosystems such as seagrass
beds, oolitic sand banks, open ocean, coral reefs, and mangroves27. is high productivity supports biomass of
upper trophic level predators, such as sharks, and large teleost shes28,29. High shark diversity and abundance in
this region stems from over two decades of legislated protection: commercial long-lining was banned in 199330,31,
and the Bahamian EEZ was declared a ‘shark sanctuary’ in 2011, thereby prohibiting the capture, harvest, or
trade of shark products within the exclusive economic zone31,32. However, the highly migratory nature of coastal
sharks33,34 may increase sheries capture and eventual human consumption as a protein source in other neighbor-
ing regions of the Greater Caribbean. Baseline monitoring of metal concentrations is therefore necessary to exam-
ine the primary route/s of metal exposure and establish the potential toxicity of shark meat. Ultimately, this will
allow for the determination of potential hotspots that may benet from focused environmental management16.
is study provides the rst assessment of metal (Cd, Pb, Cr, Mn, Co, Cu, Zn, Ag, Hg) and metalloid (As)
concentrations in muscle tissue of large-bodied, common shark species from the coastal Bahamas. We present
preliminary concentrations for ve species: blacknose sharks (Carcharhinus acronotus), bull sharks (Carcharhinus
leucas), tiger sharks (Galeocerdo cuvier), nurse sharks (Ginglymostoma cirratum), and lemon sharks (Negaprion
brevirostris). However, we focus much of our analyses on the Caribbean Reef shark Carcharhinus perezi35–37 owing
to their high abundance, ecological signicance on Bahamian coral reefs, and high capture rate in neighboring
regions where they remain unprotected from shing and human consumption (e.g., South American sheries38).
We also examined correlations between metal concentrations across individuals and examined trends with size.
ese ndings establish the rst baseline estimates of metal concentrations in Bahamian sharks with broader
implications for the human consumption of shark meat and allow for preliminary inferences regarding the
ultimate route/s of metal exposure for these species.
Results
Metal concentrations in muscle tissue were measured for 36 individuals spanning six species (Table1). Most
sharks sampled were mature based on established size-at-maturity estimates (Compagno etal. 2005), but for
Caribbean reef sharks we were able to sample individuals across a broader size range. Total mercury (THg)
concentrations were up to 1.5 times higher in Caribbean Reef sharks (16.490 ± 8.331mg kg−1; mean ± SD) than
in any other species and these values were higher than those reported in most other sharks species that are
commonly found and sampled from e Bahamas and neighboring regions (Table2). Despite their larger size,
tiger sharks exhibited the lowest THg concentrations of all species (4.442 ± 1.619mg kg−1, Table1), and these
results were statistically signicant when comparing Caribbean Reef sharks and Tiger sharks (Wilcoxon test,
W = 6.000, p < 0.001, Fig.1).
e highest concentrations of Cd were found in the tissues of Nurse sharks (0.263 ± 0.301mg kg−1) and Lemon
sharks (0.231 ± 0.170mg kg−1) and the lowest values were found in Caribbean reef sharks (0.119 ± 0.085mg kg−1).
The highest concentrations of Pb were found in Caribbean reef sharks (0.367 ± 0.231 mg kg−1). For
Cr, the highest values were observed in Caribbean reef sharks (2.641 ± 3.272 mg kg−1) and blacknose
sharks (2.577 ± 2.156 mg kg−1). Manganese and cobalt concentrations were highest in blacknose sharks
(Mn = 1.515 ± 1.396 mg kg −1, Co = 0.042 ± 0.073 mg kg−1) and tiger sharks (Mn = 1.765 ± 1.938 mg kg−1,
Co = 0.047 ± 0.033 mg kg−1). Cu concentrations were up to five times higher in lemon sharks
(30.877 ± 25.443mg kg−1) than in any other species sampled (< 6.5mg kg−1). Zn concentrations were highest
Table 1. Mean heavy metal concentrations (± SD, mg kg−1, dw) measured in white muscle tissue of sharks
captured from coastal waters of Great Exuma and Nassau New Providence Island, e Bahamas. Sample sizes
(n) represent the total number of individuals sampled from which metal data were generated, the sample sizes
of individual metals may dier based on the removal of data due to potential contamination. a n = 4. b n = 4.
c n = 3. d n = 6. e n = 21. f n = 19. g n = 23. h n = 2.
Species Common
name Size range
(TL, cm) nCd Pb Cr Mn Co Cu Zn As Ag THg
Car-
charhinus
acronotus
Blacknose
shark 97–114 3 0.151
(0.028) 0.104
(0.056)h2.577
(2.156) 1.515
(1.396) 0.042
(0.073) 4.227
(1.686) 104.168
(114.608) 2.576
(3.860) 0.067
(0.047) 7.908
(1.747)
Carcharhi-
nus perezi Caribbean
reef shark 94–197 24 0.119
(0.085)e0.367
(0.231)f2.641
(3.272)e0.971
(0.659)e0.027
(0.040)e4.897
(3.110)e80.130
(64.833)e7.307
(19.287)e0.066
(0.068)e15.490
(8.331)g
Carcharhi-
nus leucas Bull shark 242 1 0.216 0.142 0.09 0.16 0.534 2.73 37 1.38 0.055 6.722
Galeocerdo
cuvier Tiger shark 155–320 7 0.138
(0.085)b0.099
(0.074)c0.736
(0.494)b1.765
(1.938)b0.047
(0.033)b3.459
(0.912)b41.298
(7.932)b1.001
(0.764)b0.118
(0.124)b4.442
(1.619)d
Gingly-
mostoma
cirratum Nurse shark 204–267 5 0.263
(0.301) 0.108
(0.045)a1.530
(1.112) 0.640
(0.313) 0.009
(0.019) 6.411
(4.412) 88.004
(28.582) 3.750
(3.909) 0.094
(0.126) 9.030
(2.894)
Negaprion
brevirostris Lemon
shark 240, 248 2 0.231
(0.170) 0.125
(0.108) 2.305
(2.553) 0.467
(0.217) 0.010
(0.014) 30.877
(25.443) 64.140
(37.640) 0.306
(0.432) 0.518
(0.408) 4.846
(0.332)
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in blacknose sharks (104.168 ± 114.608mg kg−1) and nurse sharks (88.004 ± 28.582mg kg−1). In some spe-
cies, As concentrations were up to two times higher in Caribbean reef sharks (7.307 ± 19.287mg kg−1) than in
the other species sampled (< 9.000mg kg−1). Ag concentrations were over four times higher in lemon sharks
(0.518 ± 0.408mg kg−1) than in the other species sampled (< 0.120mg kg−1).
We observed strong, positive correlations (r > 0.4) between many of the metals measured within the muscle
tissues of Caribbean reef sharks (Table3, Fig.2). Cd concentrations were positively correlated with Co, and
negatively correlated with THg. Pb concentrations were positively correlated with Cr, Mn, Cu, Zn, As, and THg.
Cr concentrations were positively correlated with Mn and Co. Mn concentrations were positively correlated with
Cu, Zn, As, and THg. Co concentrations were positively correlated with Ag. Cu concentrations were positively
correlated with Zn. Zn concentrations were positively correlated with As and THg. Finally, signicant positive
correlations were observed between concentrations of As and THg (Table3, Fig.2). We observed a negative
correlation between THg and Cd.
Generalized additive models revealed variable trends in metal accumulation with size (Table4, Fig.3) for
Caribbean reef sharks. For three of the metals (Pb, Cr, and Cu) there appeared to be signicant increases in con-
centrations as individuals approached sexual maturity (152–168 cm45; 150–170 cm46); a relatively high percentage
of the total deviance was also explained by these models (> 39%, Table4). Trends for Mn, Co, Zn, As, and Ag
were less conspicuous, with little or no trend observed. For THg, GAMs revealed a positive, linear relationship
with size (Table4, Fig.3).
Discussion
is study represents the rst evaluation of metal concentrations in large-bodied sharks from e Bahamas.
e high and variable concentrations of metals in the muscle tissue of coastal sharks in this region exceeded
concentrations considered toxic for human consumption (e.g., THg8). Considering the demand for shark meat
worldwide47, our data provide baseline concentrations of metals and further emphasize the potential toxicity of
shark meat for human consumers8,48,49. Despite the potential implications for humans, we focus our discussion on
the potential drivers of metal concentrations in sharks, and why these may vary across and within taxa. We found
that Caribbean reef sharks exhibited the highest concentrations in four of metals (Pb, Cr, As, and THg) relative
to other larger-bodied species, some of which peaked as animals approached sexual maturity. We also found
some signicant (both positive and negative) correlations between metal concentrations in this species, which
could be attributed to foraging dynamics, longevity, physiology, and a slower growth rate in older individuals.
A notable nding was the elevated metal concentrations in Caribbean reef sharks, particularly THg, relative
to the other larger-bodied species sampled and values reported for other coastal sharks sampled from neigh-
boring regions (see Table2). Species-specic dierences in bioaccumulation trajectories of toxicants have been
Table 2. Summary of literature-derived total mercury (THg) concentrations (mg kg−1, wet weight) reported
for shark muscle tissue in species typically found throughout e Bahamas and neighboring regions (updated
and adapted from Matulik etal. 2017). Concentrations for sharks captured in this study were converted to wet
weight by multiplying dry weight concentrations by 0.3 assuming a ~ 70% moisture content reported for shark
muscle tissue44.
Species n Mean Range/SD Sampling location Study
Carcharhinus limbatus 21 0.77 0.16–2.3 Florida, US 39
Carchahinus leucas 53 0.77 0.24–1.7
Carcharhinus limbatus 5 1.9 1.44–2.73 Unknown 40
Carcharhinus spp. 9 1.61 0.46–4.08 Gulf of Mexico, US 41
Carcharhinus acronotus 11 1.76 SD: ± 0.8
Florida, US 12
Carcharhinus limbatus 28 2.65 SD: ± 0.9
Carcharhinus leucas 7 1.48 SD: ± 1.2
Nepagrion brevirostris 2 – 1.67 and 1.69
Sphyrna mokkarran 4 1.65 SD: ± 0.4
Galeocerdo cuvier 8 0.37 SD: ± 0.3
Carcharhinus acronotus 8 2.93 1.65–4.90
Florida, US 42
Carcharhinus leucas 7 3.95 1.89–7.43
Carcharhinus limbatus 23 3.22 1.20–5.99
Nepagrion brevirostris 8 1.28 0.85–2.40
Carcharhinus longimanus 24 5.04 1.86–11.20 Cat Island, Bahamas 43
Carcharhinus acronotus 3 2.37 1.84–2.89
New Providence Island and Great Exuma, Bahamas is study
Carcharhinus perezi 24 4.65 1.11–1.72
Carcharhinus leucas 1 2.02 –
Galeocerdo cuvier 7 1.33 0.73–1.93
Ginglymostoma cirratum 5 2.71 1.23–3.54
Nepagrion brevirostris 2 1.45 1.38–1.52
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reported in sharks12,50, whereby variable physiologies, trophic ecologies, and maternal ooading may inuence
the initial concentrations and subsequent bioconcentration42,51. One explanation for the generally high metal
concentrations in Caribbean reef sharks could be ascribed to a piscivorous diet in larger individuals, foraging
upon predominantly larger coral reef-associated shes (e.g., Grouper, Snapper, and Barracuda), which exhibit
high metal concentrations at other Bahamian locales (e.g., South Eleuthera52). For THg, the remarkably high
concentrations reported here exceed those in nearly all other marine animals17,53, which may be ascribed in
part to diet54,55but also to ambient oceanographic conditions specic to sub-tropical waters such as high ocean
temperatures (which increase methylation rates of inorganic Hg by marine microbes56). is may explain the
Figure1. Total mercury concentrations (mg kg−1, DW) in the tissues of tiger (n = 6) and Caribbean Reef sharks
(n = 23) sampled from the coastal waters of e Bahamas. Asterisk indicates statistical signicance at α = 0.05.
e median sizes of sharks sampled was 162cm for Caribbean reef sharks* and 301cm for tiger sharks. *Note
that median length estimates for Caribbean reef sharks are based o n = 22 as a single individual that was
measured for THg was DOA and could not be accurately measured for TL.
Table 3. Correlation coecients (p value) for Spearman’s correlation tests examining relationships between
trace metal concentrations in the muscle tissue of Caribbean Reef sharks. Bold indicates statistically signicant
correlation at α = 0.05 level. Sample sizes for each specic comparison are shown in the second horizontal.
Cd Pb Cr Mn Co Cu Zn As Ag THg
Cd –n = 19 n = 21 n = 21 n = 21 n = 21 n = 21 n = 21 n = 21 n = 20
Pb −0.076 (0.758) – n = 19 n = 19 n = 19 n = 19 n = 19 n = 19 n = 19 n = 18
Cr −0.030 (0.897) 0.664 (0.002) –n = 21 n = 21 n = 21 n = 21 n = 21 n = 21 n = 20
Mn −0.187 (0.418) 0.731 (< 0.001) 0.661 (0.001) –n = 21 n = 21 n = 21 n = 21 n = 21 n = 20
Co 0.510 (0.018) 0.323 (0.178) 0.465 (0.034) 0.311 (0.170) – n = 21 n = 21 n = 21 n = 21 n = 20
Cu −0.076 (0.743) 0.732 (< 0.001) 0.768 (< 0.001) 0.503 (0.020) 0.266 (0.243) – n = 21 n = 21 n = 21 n = 20
Zn −0.141 (0.543) 0.820 (< 0.001) 0.487 (0.025) 0.548 (0.010) 0.110 (0.634) 0.669 (0.001) –n = 21 n = 21 n = 20
As −0.257 (0.261) 0.426 (0.069) 0.244 (0.286) 0.386 (0.084) −0.284 (0.213) 0.438 (0.047) 0.696 (< 0.001) –n = 21 n = 20
Ag 0.060 (0.796) 0.214 (0.379) 0.376 (0.093) 0.003 (0.991) 0.137 (0.553) 0.396 (0.076) 0.194 (0.400) 0.367 (0.102) – n = 20
THg −0.529 (0.016) 0.548 (0.018) 0.162 (0.494) 0.457 (0.043) −0.233 (0.323) 0.334 (0.150) 0.408 (0.075) 0.334 (0.150) −0.162 (0.496) –
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Figure2. (A) Box and whisker plots highlighting the distribution of measurements for metals (mg kg−1, DW).
Solid horizontal line represents the median, box limits are 1st and 3rd quantiles, whiskers are 1.5 times the
interquartile range, and circles represent outliers. (B) Correlograms highlighting Spearman’s correlation tests
assessing covariance between trace metal concentrations in Caribbean Reef sharks. Circles represent signicant
correlations at alpha = 0.05 level, size of circles scales with size of correlation (i.e., larger circles indicate higher
correlation coecient) coecient and colors ramp illustrates whether correlations are positive (blue) or negative
(red). See Table2 for sample sizes associated with each statistical comparison. Correlograms were created in the
R package “corrplot” (Wei and Simko, 2017).
Table 4. Deviance explained (%) by generalized additive models between size and metal concentrations for
Caribbean Reef sharks.
Metal Deviance explained (%)
Cd 8.3
Pb 42.6
Cr 39.6
Mn 37.8
Co 4.25
Cu 48.3
Zn 14.4
As 0.6
Ag 30
THg 28.8
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higher THg concentrations in less mobile sharks either known or assumed to display high residency within
Bahamian waters (e.g., Caribbean reef sharks36; blacknose sharks and nurse sharks), relative to more transient
species that move throughout much of the northern Western Atlantic ocean, such as tiger sharks33,34. Although
it remains unknown if high THg concentrations in Caribbean reef sharks elicit neurological eects, there are
human health concerns as this species is commonly consumed in certain regions of the Caribbean57, as well as
in South America38. Because THg concentrations were high across multiple species sampled in this study, fur-
ther evaluation of local euent and runo of Hg sources into Bahamian marine systems is certainly warranted.
is argument is strengthened by observations of elevated concentrations reaching 0.8ppm (WW) for THg,
which have been observed in teleost species such as king mackerel (Scomberomorus cavalla) and great barracuda
(Sphyraena barracuda) from neighboring waters of South Eleuthera52.
Very few studies have explored correlations between metal concentrations in sharks, and trends are oen
inconsistent among species possibly due to variable metabolisms14,51,58. Bosch etal.8 found no correlation among
metals in smoothound sharks (Mustelus mustelus), whereas Kim etal.59 found a signicant relationship between
Hg and Pb in copper sharks (Carcharhinus brachyurus). In this study, we found that zinc and manganese were
positively correlated with lead, arsenic and mercury in the muscle of Caribbean reef sharks. It is thus possible
that these micronutrient metals have some protective eects against heavy metal toxicity60,61. Metal competition
(e.g., competition for metal binding sites) could lead to negative correlations62,63, whereas similar accumulation
Figure3. Generalized additive models (GAMs) t for Caribbean Reef sharks investigating relationships
between size and metal concentrations (mg kg−1, dw). Blue shaded region represents standard error and blue
dotted region represents range of published size-at-maturity estimates (152–168 cm45; 150–170cm, Pikitch
etal.46). Sample sizes dier between metals owing to removal of data that were potentially contaminated.
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behaviors, detoxication processes, and similar input sources could result in positive correlations64,65. For exam-
ple, it is suggested that metallothioneins induced by elevated Zn and Cu can interact with and detoxify metal
ions such as Cd, Hg, Pb, and Ag60,61. Here, we did not observe signicant, positive correlations for Zn–Cd and
Cu–Cd, but positive correlations were found for Zn–Pb, Zn–Cu, Zn–Hg, and Cu–Pb. Similarly, we found positive
correlations between Pb and most of the metals (except Cd, Co, and Ag), implying they might be introduced
into the study area though similar geochemical pathways (e.g., dust deposition for Pb and Mn, metal-rich par-
ticulate/organic matter from runo or suspended sediment). Although the ultimate cause and implications of
metal correlations are challenging to establish for wild sampled sharks, our descriptive approach indicates that
direct experimental study assessing the biological and environmental factors that drive metal correlations, or
lack thereof is warranted.
We observed size-based shis in concentrations of Pb, Cr, Cu, and THg in Caribbean Reef sharks, which
peaked as animals reached sexual maturity (Belize: 150–170 cm46). Indeed, for Pb, Cr, and Cu. is observa-
tion could be explained by a resource-use shi from inshore habitats to deeper continuous reefs35, running
parallel to deep slopes of the Tongue of the Ocean (near our Nassau, New Providence sampling region) from
juvenile/sub-adult to mature life-history stages. is behavior may thus present a dierent prey base11, or ambi-
ent concentrations of metals in the water column (i.e., if the primary pathway of metal accumulation is through
the gills). As sharks reach sexual maturity, energetic requirements associated with reproduction may increase
overall daily energy budgets44, requiring individuals to consume a greater biomass of potentially higher trophic
position prey items. Although we were unable to denitively test this hypothesis within the connes of this
study, ecogeochemical tracer techniques such as stable isotope and fatty acid analyses may provide insight into
whether resource-use shis are in fact occurring between size classes. In other shark species, such as tope sharks
(Galeorhinus galeus) shis in metal concentrations have been attributed to habitat shis11, but it is apparent that
trends are not uniform across all metals, tissue types, and species. Combined, this suggests that factors other than
the organism’s ecology may play a role in the accumulation of metals. For example, metabolic processes, such as
reduced growth rates may lead to greater metal accumulation in Caribbean reef shark tissues as processes such
as growth dilution are signicantly reduced53.
Conclusions
e study provides the rst analysis of metal concentrations in the tissues of coastal sharks from e Bahamas.
e higher trophic position of the shark assemblage sampled in this study may partly explain why concentrations
were elevated. For Caribbean reef sharks, we found the highest levels of harmful THg compared with the other
species sampled. We also found peaks in metal concentrations as this species reached sexual maturity, which
could be associated with the known ontogenetic shi in habitat/primary prey base combined with growth dilu-
tion eects. We recognize that obtaining larger sample sizes should improve comparisons across species, and
arm that there are limitations for interpreting some of the relationships detected here due to knowledge gaps
in our understanding of metal trophodynamics in elasmobranch shes. Overall, our ndings suggest that sharks
residing within relatively pristine ecological environments may possess high levels of potentially harmful met-
als, which may have public health implications if they are consumed by local human populations. Further, our
ndings suggest that Bahamian food-webs may support elevated concentrations of toxic metals and although
e Bahamas legally protects sharks from shing, sublethal impacts may still be induced. As such, future work
should seek to determine the habitat-level sources and assimilation factors of metals in sharks and whether
overall tness is aected by high tissue concentrations.
Methods
Animal ethics statement
All research was conducted under scientic research permits issued to A. Gallagher (unnumbered) by e
Bahamian Department of Marine Resources. Animal handling and sampling protocols followed guidelines listed
by the Association for the Study of Animal Behavior65. Ethical approval for animal sampling was given by the
Canada research chair for animal care (Carelton University, Ottawa, Canada).
Animal capture and tissue sampling
Sharks were sampled from the coastal waters of Nassau, New Providence and Great Exuma between February
2018 and February 2019 (Fig.4) using standardized circle-hook research drum lines. Upon capture, animals
were secured alongside the research vessel and sex and morphometric measurements were taken. A small inci-
sion was made into the dorsal musculature using a sterilized scalpel and approximately 1–2g of white muscle
tissue was excised using a modied 10mm biopsy punch (Deglon, iers, France). All samples were frozen on
ice in 2mL microcentrifuge tubes in the eld and then stored −20°C before preparation for elemental analysis.
Samples were oven dried at 60°C for ~ 48h and ground to a ne powder using a mortar and pestle. For statisti-
cal purposes, all Caribbean reef shark samples were pooled because capture locations are consistent between
islands (e.g., lagoon and forereef habitat), and we cannot discount the movement of individuals between islands.
Analyses of metals and metalloids
Total mercury (ppm, mg kg−1) analysis was conducted on a Milestone DMA-8-Direct Mercury Analyzer. Machine
error calculated from repeat measurements of certied reference material (DORM-4) fell within expected ranges
(0.412 ± 0.036mg kg−1). e remaining trace metals (Cr, Mn, Co, Cu, Zn, As, Ag, Cd, and Pb) were analyzed
by a sector eld double focusing high-resolution inductively-coupled plasma mass spectrometer (HR-ICP-MS,
Element 2, ermo Fisher Scientic). Tissue samples were digested into liquid prior to analysis. In brief, a small
amount (10 ~ 20mg) of tissue was soaked in 1mL of nitric acid (70%, trace metal grade, Fisher Chemical) in a
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metal-free polypropylene vial at room temperature overnight. Aer adding 1mL of hydrogen peroxide (30%,
trace analysis, Supelco), the vial was sealed and heated at 90°C for 12h until complete digestion. e digest was
then combined with Milli-Q water to obtain samples in 2% nitric acid solution which were ready for ICP-MS
analysis. 114Cd and 208Pb were measured in the low-resolution mode, while 52Cr, 55Mn, 59Co, 63Cu, 66Zn, 75As, and
107Ag were measured in the medium-resolution mode. Internal standards 115In and 89Y were used for correcting
potential matrix interference. e accuracy of the standard calibration was validated with the certied reference
material, Trace Metals in Water Standard A (CRM-TMDW-A, High-Purity Standards). e mid-point standard
and the blank were checked every twelve measurements to correct instrumental dris of the background and the
slope of the calibration curve. Two random digest samples were fortied with a known quantity of elements, and
the recovery of each spiked element ranged from 74 to 97% (Supplementary TableS1). e certied reference
material of sh tissue, DORM-4 (NRCC), was used to ensure the accuracy of the digestion and analytical proce-
dure, which fell within certied ranges (Supplementary TableS2). e instrument detection limit for each metal
is listed in Supplementary TableS3. Data above the limit of detection (LOD) were presented. e few samples
that exhibited anomalous metal concentrations indicative of contamination were removed from the analyses
which are likely to have occurred randomly during subsampling or transportation in the eld. e concentration
range of each metal presented in this study was comparable to other common shark species reported in earlier
literature8,11 though the shark species may dier). Note that we reported the concentration on a dry weight basis
and the metal concentration would drop 60–80% as converted to wet weight44.
Statistical analyses
All data were analyzed in the statistical programming soware R (version 4.0.0). Statistical signicance α was
0.05. Shapiro–Wilks tests and F tests were used to examine normality and heteroscedasticity of data, respectively.
For Caribbean reef sharks, we examined potential correlation between trace metals through Spearman’s correla-
tion coecients, presented as correlograms (R package “corrplot”66. We used a rank order Spearman’s correla-
tion because some values fell below detection limits and are therefore expressed as zeros. A Wilcoxon signed
ranks test was used to examine whether mean THg concentrations statistically diered between Caribbean reef
sharks and tiger sharks; low sample sizes for other species and metals precluded comparisons between other
species. We also compiled literature derived THg concentrations for shark muscle in species found throughout
the Bahamas and neighboring waters for comparative purposes. Because relationships between size and metal
concentrations are not necessarily linear, we investigated these using generalized additive models (GAMs, R
package “mgcv”67). e smoothing parameter k was set to 9 to ensure sucient degrees of freedom to represent
a trend and this parameterization was validated using the ‘gam.check’ function (R package ‘mgcv’) for each tted
model (p > 0.90 for all models66).
Figure4. Sampling location of sharks from Great Exuma (black boxes, top le panel) and Nassau New
Providence Island (red boxes, bottom le panel), e Bahamas in relation to North Americas and wider
western Atlantic Ocean (right panel). Sources: ESRI, GEBCO, NOAA, National Geographic, DeLorme, HERE,
Geonames.org, and other.
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Received: 31 August 2020; Accepted: 10 December 2020
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Acknowledgements
We are grateful for the support from Y. Silver, K. Hetterman and S. Lindblad, E. Fullerton, M. Saylor, J. and M.
McClurg, For marine operations we thank J. Halvorsen and the crew of the M/Y Marcato, G. Allen, A. Cushwah,
J. Doyle, M. Segren, and S. Rosikov from Fleet Miami, as well as E. Quintero. Additional support was provided
by T. Gilbert and the International Seakeepers Society, J. Todd and P. Nicholson from GIV, and S. Cove. For
logistical and eld assistance we thank S. Kessel, M. Van Zinnicq Bergmann, M. Adunni, J. Pankey, S. Moorhead,
D. Camejo, B. Phillips, S. Teicher, and K. Zacarian. Funding to Beneath the Waves for this work was provided
by a handful of private donors and groups.
Author contributions
O.N.S., C.S.L., N.F., and A.J.G. devised the project concept. O.N.S., E.S., N.H., J.S., S.K., and A.J.G. conducted
eld sampling. C.S.L. and O.N.S. conducted laboratory analysis. O.N.S. and A.J.G. wrote the manuscript with
help from C.S.L., N.F., N.H. All authors approved the nal submission.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 020- 79973-w.
Correspondence and requests for materials should be addressed to O.N.S.
Reprints and permissions information is available at www.nature.com/reprints.
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