10.5061/DRYAD.KSN02V75B
Charapata, Patrick
Baylor University
Clark, Casey
Washington Department of Fish and Wildlife
Miller, Nathan
The University of Texas at Austin
Kienle, Sarah
Baylor University
Costa, Daniel
University of California, Santa Cruz
Goebel, Michael
University of California, Santa Cruz
Gunn, Heather
The University of Texas at Austin
Sperou, Emily
Baylor University
Kanatous, Shane
Colorado State University
Crocker, Daniel
Sonoma State University
Borras-Chavez, Renato
Pontifical Catholic University of Chile
Trumble, Stephen
0000-0001-6319-9633
Baylor University
Data from: Whiskers provide time-series of toxic and essential trace
elements, Se:Hg molar ratios, and stable isotope values of an apex
Antarctic predator, the leopard seal
Dryad
dataset
2021
FOS: Biological sciences
Trace elements
pinniped
Ecotoxicology
stable isotope
mercury exposure
Se:Hg molar ratio
Antarctica
Leopard Seal
National Science Foundation of Sri Lanka
https://ror.org/010xaa060
1644004
C. Gus Glasscock, Jr. Endowed Fund for Excellence in Environmental
Science at Baylor University*
2022-12-12T00:00:00Z
2022-12-12T00:00:00Z
en
https://doi.org/10.1016/j.scitotenv.2022.158651
https://doi.org/10.5281/zenodo.7336839
8180593 bytes
6
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
In an era of rapid environmental change and increasing human presence,
researchers need efficient tools for tracking contaminants to monitor the
health of Antarctic flora and fauna. Here, we examined the utility of
leopard seal whiskers as a biomonitoring tool that reconstructs
time-series of significant ecological and physiological biomarkers.
Leopard seals (Hydrurga leptonyx) are a sentinel species in the Western
Antarctic Peninsula due to their apex predator status and top-down effects
on several Antarctic species. However, there are few data on their
contaminant loads. We analyzed leopard seal whiskers (n = 18 individuals,
n = 981 segments) collected during 2018–2019 field seasons to acquire
longitudinal profiles of non-essential (Hg, Pb, and Cd) and essential (Se,
Cu, and Zn) trace elements, stable isotope (ẟ15N and ẟ13C) values and to
assess Hg risk with Se:Hg molar ratios. Whiskers provided between 46 and
286 cumulative days of growth with a mean ~125 days per whisker (n = 18).
Adult whiskers showed variability in non-essential trace elements over
time that could partly be explained by changes in diet. Whisker Hg levels
were insufficient (<20 ppm) to consider most seals being at “high”
risk for Hg toxicity. Nevertheless, maximum Hg concentrations observed in
this study were greater than that of leopard seal hair measured two
decades ago. However, variation in the Se:Hg molar ratios over time
suggest that Se may detoxify Hg burden in leopard seals. Overall, we
provide evidence that the analysis of leopard seal whiskers allows for the
reconstruction of time-series ecological and physiological data and can be
valuable for opportunistically monitoring the health of the leopard seal
population and their Antarctic ecosystem during climate change.
Whisker collection Leopard seal whiskers were collected between April–May
in 2018 and 2019 during field work conducted at the U.S. Antarctic Marine
Living Resources (AMLR) Program research station on Cape Shirreff,
Livingstone Island, Antarctic Peninsula (National Marine Fisheries Service
permit #19439 and Antarctic Conservation Act permit #2018-016) (Fig. 1).
Leopard seals were chemically immobilized using a butorphanol-midazolam
protocol administered with a jab stick following Pussini and Goebel
(2015). While sedated, morphometric data were collected (e.g., mass, kg;
length, cm; girth, cm). The longest whisker was plucked with the root
intact from the muzzle of each seal and stored in a sterilized plastic
Whirl-Pak® (Madison, WI, USA) at ambient temperature. The final study
collection consisted of 18 whiskers from 15 females (n = 1 juvenile and n
= 14 adults) and 3 males (n = 3 adults). Following shipping to Baylor
University, whiskers were stored at −80 °C until analysis. Whisker length
and mass were measured using digital calipers (± 0.01 mm, Neiko 01407A)
and a Mettler Toledo microbalance (± 0.1 mg). Scaled body mass index (SBM
index) Scaled body mass (SBM) index was calculated for each sampled
leopard seal using the equation (Peig and Green, 2009): Mp = Mi *
(Lo/Li)^bsma Where Mi was the mass (kg) of the seal, Li was the
seal's standard length (cm), Lo was the mean standard length of the
different age classes (juvenile n = 1 female) or adult (n = 17 [n = 3
males and n = 14 females]), and bsma was the scaling exponent from
plotting natural log transformed mass by standard length for all seals and
the Mp was the predicted mass of the individual seal when standardized to
Lo. This SBM index has been shown to scale more accurately with growth and
mass compared to other condition indices used in wildlife studies,
including pinnipeds (DeRango et al., 2019; Peig and Green, 2009).
LA-ICP-MS analysis of trace elements along whiskers Whiskers were prepared
for laser ablation inductively coupled plasma mass spectrometry
(LA-ICP-MS) analysis by removing visible surface contaminants (e.g.,
external root sheath fragments) by wiping with a Kimwipe moistened with a
2:1 chloroform methanol solution (Keogh et al., 2021; Rea et al., 2015),
and drying for ≥24 h in a ventilation hood. Cleaned whiskers were shipped
in sealed Whirl-Pak® bags to The University of Texas at Austin, and then
stored in a desiccator until LA-ICP-MS analysis. Continuous elemental (Hg,
Pb, Cd, Se, Cu, Zn) base-to-tip whisker concentrations were measured by
LA-ICP-MS, using an ESI NWR193 excimer laser ablation system (193 nm, 4 ns
pulse width) coupled to an Agilent 7500ce ICP-MS. Whiskers, ranging from
48.3 to 99.4 mm in length, were mounted on double-sided stick tape. The
most stable mounts were achieved by allowing whiskers to best retain their
natural curvatures in 2D; whiskers mounted in a straight line were found
to move over time. Coordination of whisker transects involved establishing
long segmented lines with 1–2 nodes placed per mm, and each node adjusted
in x-y to follow the central growth axis. The z axis was adjusted to
maintain laser focus along the surface of the traverse as whiskers greatly
taper from base-to-tip. The LA-ICP-MS system was optimized for sensitivity
across the atomic mass unit (AMU) mass range and low oxide production
(ThO/Th: 0.28 ± 0.01) by tuning on a standard (NIST 612). Final parameters
where whisker ablations were obtained from trial transects on
representative areas of trial whisker samples (via iterative tests of
energy density, repetition rate, and gas flow) to obtain robust and
consistent elemental signals free from spectral skew). Following
pre-ablation (100 μm spot, 100 μm/s scan rate, 2.7 J/cm2 energy density
[fluence]) to remove shallow superficial contaminants, a single
base-to-tip transect was performed along the center of each whisker, using
a 90 μm diameter spot, 100 μm/s scan rate, 2.44 ± 0.07 J/cm2 energy
density, 10 Hz repetition rate, and carrier gas flows (L/min) of 0.85 for
Ar and 0.85 for He. The quadrupole time-resolved method measured eight
masses with integration times of 10 ms (34S, 63Cu, 64Zn, 83Zr), 205 ms
(202Hg), and 100 ms (82Se, 114Cd, 208Pb). Measured intensities were
converted to elemental concentrations (ppm) using iolite software (Paton
et al., 2011), with 34S as the internal standard and a S index value of 5
wt% for whisker unknowns (Legrand et al., 2004; Noël et al., 2016;
Rodushkin and Axelsson, 2003; Stadlbauer et al., 2005). Signals were
converted to base-to-tip distance (μm) along the whisker based on the scan
rate and duty cycle. Any data points that fell below detectable
concentrations were assigned ½ the concentration of the limit of detection
calculated for the analytical run of each whisker (Clark et al., 2021;
Gilbert, 1987, Table S1). Outliers were defined as concentrations measured
along the whisker >4 standard deviations above the mean and removed
from statistical analysis (Clark et al., 2021; Tukey, 1977). Previous
researchers have used commercially available human hair as an appropriate
reference standard (wt% S content, keratin matrix) for mammal hair and
whisker LA-ICP-MS studies (e.g., Noël et al., 2016, Noël et al., 2014).
However, these standards have only sub-ppm concentrations for several of
the metals assessed (Se, Hg) during this study. Thus, we made and
validated reliable standards using methods described in the Supplementary
Material. Stable isotope analysis and timestamps After whiskers were
analyzed for trace elements using LA-ICP-MS analysis they were returned to
Baylor University for sampling of bulk carbon (ẟ13C) and nitrogen (ẟ15N)
stable isotope analysis (Fig. 2). Lipids were removed by cleaning each
whisker with a 1:1 ethanol: methanol solution, following previous leopard
seal whisker stable isotope ratios studies (Botta et al., 2018; Rogers et
al., 2016). After allowing whiskers to dry in a ventilation hood (≤ 24 h),
whiskers were sectioned into 0.50 ± 0.01 mm lengths (using digital
calipers and a hand chisel) to enable fine-scale comparison with the
LA-ICP-MS trace element time-series; segment lengths were somewhat longer
near the frayed tip of whiskers to obtain the minimum required mass (~0.3
mg) for stable isotope analysis. Carbon and nitrogen stable isotope
analysis was performed in the Baylor University Stable Isotope Facility,
using an Elemental Analyzer (EA) Costech 4010 Elemental Combustion System
(ECS) paired with a Conflow IV interphase (Thermo Scientific) and Thermo
Delta V Advantage continuous flow Isotope Ratio Mass Spectrometer
(EA-IRMS). Prior to combustion and isotopic analysis, whisker segments
were placed into pre-weighed tin capsules (Costech 5 × 9 mm), tin capsules
reweighed with a Mettler Toledo XP26 digital scale (±0.001 mg). Whisker
nitrogen (ẟ15N) and carbon (ẟ13C) isotope values are reported as the ratio
of the heavy to light isotope relative to international standards;
atmospheric nitrogen and Vienna Peedee Belemnite (VPDB), respectively,
using the following equation: ẟX = [(Rsample/Rstandard)-1] *1000 where X
is the targeted isotope (nitrogen or carbon) ratio expressed in delta
notation (ẟ) with units per mil (‰), Rsample is the isotopic ratio of
heavy to light isotopes (15/14 N or 13/12C) of the sample, and Rstandard
is the isotopic ratio of heavy to light isotopes measured in the standard.
A two-point calibration curve for calculating nitrogen ẟ15N and ẟ13C
values of samples was established using USGS-40 and USGS-41A international
standards. The accuracy and precision of isotopic measurements was
calculated based on the long-term mean and standard deviation of 105
replicates of an internal lab standard (Acetanilide, reported ẟ13C =
−29.53 ± 0.01 ‰, ẟ15N = 1.18 ± 0.02 ‰) measured during each analytical run
(n = 3 replicates/run). The replicate grand averages obtained are within
(ẟ15N = 1.28 ± 0.17 ‰) or very close to (ẟ13C = −29.45 ± 0.05 ‰)
analytical uncertainty of reported values. Acceptable atomic C:N ratios of
whisker segments ranged from approximately 3.0–3.8 based on previous
leopard seal whisker isotope studies (Botta et al., 2018; Rogers et al.,
2016). Nearly all whisker segments had acceptable atomic C:N ratios (3.47
± 0.13, 3.03–4.00, mean ± standard deviation (SD), min–max, respectively
(Fig. S1); one segment with anomalously low atomic C:N ratio <2.8,
was excluded from statistical analysis. Whisker segments were assigned
approximate timestamps relative to date of collection based on leopard
seal whisker growth characteristics and the application of a discrete Von
Bertalanffy growth model (Hall-Aspland et al., 2005; Rogers et al., 2016;
von Bertalanffy, 1938). The discrete Von Bertalanffy equation can be
written as: δL/δT = K(La – Lp–1) where δL/δT is growth rate for sections
p-1 to p. This can be rearranged to δL = (Lp–Lp–1) and δT = (Tp–Tp-1)
allowing calculation of time intervals for whisker section(s) p-1 to p as
done in Hall-Aspland et al. (2005) and Rogers et al. (2016): (Tp – Tp–1) =
(Lp – Lp–1)/[K(La – Lp–1)] where Lp is the total length of the whisker,
Lp–1 is the remaining length of the whisker after sampling section p, K is
the growth coefficient, and La is the asymptotic length of leopard seal
whisker. We used the recommended K value of 0.013 and La of 101.2 mm
developed by Rogers et al. (2016) for leopard seal whiskers and applied
this equation to each section to acquire approximate segment growth in
days. Cumulative δT values over respective segment intervals were then
subtracted from the collection date of each whisker to develop an
approximate timestamp for individual segments. Aligning trace element and
stable isotope data The accurate alignment of trace element and stable
isotope data per whisker was standardized to the segment lengths submitted
for stable isotope analysis. For example, the first section of a whisker
(i.e., section “1”) with a length of 0.50 mm submitted for isotope
analysis meant a number “1” was assigned to all trace element data
obtained during the 0.0–0.50 mm of whisker sampled during LA-ICP-MS (Fig.
2). This was repeated for subsequent sections until trace element data
were assigned a section number that directly corresponded with stable
isotope data. Mean and standard deviation values were calculated for trace
element data based on its respective section number to accurately be
paired with stable isotope data (Fig. 2). This approach worked for all but
three whiskers (n = 3), where cumulative segment lengths for stable
isotope analysis were greater than the trace element whisker length data.
This most likely was due to error when measuring and sampling individual
segments for stable isotope analysis. To correct this error in the three
whiskers, the total sampling error was calculated (i.e., total whisker
length prior to sampling minus cumulative segment lengths post sampling)
for these three whiskers and the total error was divided by the total
number of whisker segments. The resulting values ranged from 0.06 to 0.08
mm and were subtracted from section lengths for all segments. The process
of integrating trace element with stable isotope data was rerun with the
corrected segment lengths and resulted in all trace element data being
assigned to a corresponding stable isotope segment. Statistical analysis
Type II ANOVAs (F-tests for linear models) were used to determine
significant differences in mean log10 transformed whole whisker trace
elements, ẟ15N, and ẟ13C values between sexes, standard length, and SBM
index in respective linear models (Garcia-Cegarra et al., 2021). Due to
only having one juvenile, age class was not assessed among these whole
whisker trace element and stable isotope data. Whole whisker analyte
concentrations are calculated means across all whisker segments per
individual, whereas segment analyte concentrations are averaged only
across segment length (Fig. 2). Pearson correlations of log10 transformed
trace elements were performed within and among individuals of pooled sexes
(n = 15 females, n = 3 males) using segmented and whole whisker trace
element data, respectively. Among individual correlations were calculated
using average whole whisker trace element concentrations (i.e.,
correlations among mean trace element concentrations for each individual
seal) and can provide general insight into trace element associations
within leopard seal whiskers (e.g., individuals with high concentrations
of element “A” tend to have low concentrations of element “B”). In
contrast, within individual correlation coefficients were calculated using
a smoothed time series of trace element concentrations across the whisker
of each individual. The resulting Pearson correlation coefficients were
then Fisher-Z transformed, averaged across all individuals, and
transformed back to a mean Pearson correlation coefficient. This approach
reveals correlations among trace elements through time (i.e., along the
whisker) that are consistent across seals, thus are likely to represent
processes or phenomena that affect most or all leopard seals in this
study, which may include things like physiological, temporal, and spatial
intrinsic influences on trace element intake or uptake (e.g., Clark et
al., 2021). The R package SIBER was used to calculate intraindividual
standard ellipse area (SEA) corrected for small sample sizes (SEAc) across
whisker segment ẟ13C and ẟ15N values for each seal (Jackson et al., 2011;
Scholz et al., 2020). The SEAc calculates the variability among ẟ13C and
ẟ15N values across whisker segments to provide insight into range of
trophic level and foraging locations (i.e., trophic niche) of an
individual seal. We used SEAc values to understand how trophic niche width
related to whole whisker trace element concentrations. We also calculated
a “population” SEAc using whole whisker ẟ15N and ẟ13C values from all
whiskers (n = 18) to compare with a previous leopard seal whisker study
(Botta et al., 2018). Bivariate linear models of log10 transformed trace
element data and ANOVAs (F-tests for linear models) were used to determine
relationships of trace elements with intraindividual SEAc values. Linear
models and Type II ANOVAs (F-tests for linear models) were used to assess
intraindividual SEAc with sex, standard length, and SBM index. Linear
mixed models (LMMs) were constructed to determine relationships among
trace elements with changes in ẟ15N and ẟ13C over time while incorporating
sex and biometrics (standard length and SBM index) as covariates. All
trace element data were log10 transformed to approximate normal
distribution to meet LMM assumptions. Trace element data from adult
leopard seal whiskers were modeled (n = 17 adults [n = 3 males and n = 14
females]). Full models were constructed using the R Studio software
(RStudio Team, 2020) and the package lme4 (Bates et al., 2015) based on
our objectives to assess temporal relationships among trace elements with
changes in diet (ẟ15N and ẟ13C values), that also incorporated sex and
biometric data (Zuur and Ieno, 2016). A numbered “Week” of the year (1–52)
was assigned to individual segments relative to the earliest segment
timestamp and only retained “Weeks” that included a minimum of three
unique seals (total n = 17 seals, n = 834 segments, and n = 28 consecutive
weeks). The full model took the form of: log10(trace element) ~ Standard
Length (numeric) + SBM index (numeric) + Sex (factor, “Male” or “Female”)
+ Week (numeric) + Carbon (numeric, ẟ13C of segment) + Nitrogen (numeric,
ẟ15N of segment) + Carbon*Week + Nitrogen*Week + (1|FieldSeason) (random
intercept, controlling for whisker collection year) + (1|Seal.ID) (random
intercept, controlling for differences in average concentrations among
individuals). Biologically relevant permutations with the fixed effects of
the full model were constructed to compare with the full model (Table S2).
The selected model was determined based on lowest AICc and highest AIC
weight (Burnham et al., 2011; Table S3). We assessed the full and selected
models for each trace element by plotting residuals with fitted values,
residuals with all covariates, and assessed the distribution of residuals
(Zuur and Ieno, 2016). Selected LMMs for each trace element had fitted and
95 % confidence intervals constructed using the “bootpredictlme4” package
in R using n = 500 iterations to estimate the fit of selected models with
the trace element data (Clark et al., 2021; Duursma, 2021). If the
interaction terms were retained in the selected model, we predicted trace
element concentrations over time keeping ẟ15N and ẟ13C at biologically
relevant “lower”, “median”, and “upper” values, while keeping the other
isotope and/or main effects at their median values, if applicable. For
ẟ15N, the lower value was 10.15 ‰ (12.5 % range of our ẟ15N values,
between 0 and 1st quartile), median value was 10.83 ‰, and upper value was
12.47 ‰ (87.5 % range of data, between 3rd and 4th quartiles of our ẟ15N
values). For ẟ13C, the lower value was −22.74 (12.5 % range of data,
between 0 and 1st quartile of our ẟ13C values), median value = −21.98, and
upper value was = −21.41 ‰ (87.5 % range of data, between 3rd and 4th
quartiles of our ẟ13C values). We then visually assessed the fit of model
predictions with the leopard seal whisker trace element data from the four
seals that fell within those stable isotope categories (i.e., “lower”,
“median”, and “upper” ẟ13C and ẟ15N values) (Clark et al., 2021; Zuur and
Ieno, 2016). Mercury is a relatively well-studied toxin in pinnipeds with
published Hg toxicological thresholds from hair concentrations (McHuron et
al., 2019; O’Hara and Hart, 2018; Rea et al., 2020), which also correlate
with whisker Hg concentrations (Noël et al., 2016). Previous studies
suggest different toxicity thresholds for hair Hg concentrations that are
associated with deleterious effects to wildlife and humans including; 5.4
ppm [μg/g dw] in brain tissue of polar bears (Ursus maritimus) which
correlated with a reduction in genomic DNA methylation and NMDA receptors,
10 ppm in human infants was correlated with delayed development, and ~30
ppm in mink (Neogale vison) hair that had resulted in acute Hg toxicity
and death in some individuals (Van Hoomissen et al., 2015; Yates et al.,
2005). Since hair and whisker Hg thresholds are unknown for leopard seals,
we followed Rea et al. (2020) and used Hg toxicological thresholds of
<10 ppm to assign whole whisker and individual segment
concentrations as “Low” risk, 10 to 20 ppm as “Moderate”, and “High” risk
of Hg toxicity if Hg concentrations were >20 ppm (O’Hara and Hart,
2018; Rea et al., 2020). Additionally, molar Se:Hg ratios were calculated
for each segment using the formula: (Se ppm /78.96)/(Hg ppm/200.59)
following McCormack et al. (2021). An ANOVA with Tukey's Post Hoc
honestly significant difference (HSD) was used to determine overall
differences in mean Se:Hg molar ratios among risk groups (“low”,
“moderate”, and “high”). We then visually assessed how Se:Hg molar ratios
patterns changed over time with respect to Hg risk classification to
analyze the potential importance of Se to leopard seals as a Hg detoxicant
(Rea et al., 2020). An alpha level of 0.05 was used for threshold of
significance for ANOVAs (F-tests for linear models). All data presented in
figures are median ± interquartile range (IQR) due to high variability
among data.
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