Environmental Toxicology and Chemistry, Vol. 34, No. 12, pp.

2771–2781, 2015
# 2015 SETAC
Printed in the USA

Environmental Toxicology

MERCURY CONCENTRATIONS IN DIFFERENT TISSUES OF TURTLE AND

CAIMAN SPECIES FROM THE RIO PURUS, AMAZONAS, BRAZIL

SAM EGGINS,*y LARISSA SCHNEIDER,y FRANK KRIKOWA,y RICHARD C. VOGT,z RONIS DA SILVEIRA,x
and WILLIAM MAHERy
yInstitute for Applied Ecology, University of Canberra, Bruce, Australian Capital Territory, Australia
zCoordenacao de Biodiversidade, Instituto Nacional de Pesquistas da Amaz^ onia, Manaus, Amazonas, Brazil
xInstituto de Ci^encias Biol
ogicas, Universidade Federal do Amazonas, Manaus, Amazonas, Brazil

(Submitted 22 February 2015; Returned for Revision 1 April 2015; Accepted 6 July 2015)

Abstract: Total mercury (Hg) concentrations of muscle, liver, blood, and epidermal keratin were measured in typically consumed,
economically and culturally important species of turtle (Podocnemis unifilis and Podocnemis expansa) and caiman (Melanosuchus niger
and Caiman crocodilus) from the Rio Purus in the Amazon basin, Brazil. Methylmercury (MeHg) concentrations were also measured in
muscle tissue, representing the first analysis of MeHg concentrations in Amazonian reptile species. In muscle tissues Hg was mostly
MeHg (79–96%) for all species. No correlations existed between animal size and total Hg or MeHg concentrations for any species other
than M. niger, possibly as a result of growth dilution or the evolution of efficient Hg elimination mechanisms. Significant linear
correlations were found between total Hg concentrations in all pairs of nonlethally sampled tissues (keratin and blood) and internal
tissues (muscle and liver) for M. niger and between keratin and internal tissues for P. expansa, indicating that nonlethally sampled
tissues can be analyzed to achieve more widespread and representative monitoring of Hg bioaccumulation in Amazonian reptiles.
Although mean Hg concentrations in muscle for all species were below the World Health Organization guideline for safe consumption
(500 mg kg–1), mean concentrations in caiman liver were above the safe limit for pregnant women and children (200 mg kg–1). No
significant differences were found between total Hg and MeHg concentrations in tissues from wild-caught and farm-raised P. expansa,
suggesting that farming may not reduce Hg exposure to humans. Environ Toxicol Chem 2015;34:2771–2781. # 2015 SETAC

Keywords: Mercury Methylmercury Bioaccumulation Reptile Amazon

INTRODUCTION predominantly of natural origin [9–12]. Aquatic ecosystems in

Mercury (Hg) contamination in aquatic and terrestrial
ecosystems is of worldwide concern because of its adverse
effects on ecosystem and human health [1]. In the environment
Hg is ubiquitous and bioaccumulates within the organs and
tissue of biota over time and biomagnifies into higher trophic
levels [2,3]. Methylmercury (MeHg or CH3Hg) is the principal
bioaccumulating species and is of particular interest because
of its extreme neurotoxicity, resulting in lethal and sublethal
health effects in humans and other animals [3]. Mercury is
considered to be of greatest threat to crocodilians [4], and its
toxicity to reptiles is of particular concern as reptile species
are currently experiencing population declines [5]. Previous
studies have highlighted reptiles as ideal biomonitors of Hg
contamination in aquatic environments as they are long-lived,
occupy diverse habitats, and feed across a range of trophic
levels [6].
Over the last 2 decades concern has developed over high
Hg concentrations in the Amazon basin [7]. Hg concentrations
were initially attributed to gold mining [8]; however, high Hg
concentrations have been measured in water, soil, and biota in
remote areas far removed from mining activities [9]. Despite
significant rates of atmospheric deposition and reemission of Hg
in the region, as well as the potential for short-range and long-
range atmospheric transport, the soil reservoir of Hg is so large
in comparison that Hg in the Amazon basin is thought to be
All Supplemental Data may be found in the online version of this article.
* Address correspondence to sameggins@gmail.com
Published online in Wiley Online Library
(wileyonlinelibrary.com).
DOI: 10.1002/etc.3151
2771
the Amazon basin are particularly susceptible to Hg bioaccu-
mulation because of the potential for rapid accumulation of
Hg into watercourses leached from Hg stored in sediments [13].
The Rio Purus basin is the sixth largest drainage basin
(375 000 km2) within the greater Amazon basin [14] and
contains approximately 40 000 km2 of floodplain forest, more
than any other tributary of the Amazon River [14,15]. The lower
Rio Purus is seasonally inundated with white and black waters
for as long as 6 mo of the year [14], making it a potential hot spot
for Hg methylation and bioaccumulation in aquatic food webs.
In the Rio Purus basin, Hg is likely to be of natural origin as
there are no records of gold mining in this basin [6]; however,
the magnitude of atmospheric Hg depositon in this basin
from long-range sources remains unknown. If Hg is indeed
naturally high in the Rio Purus, local biota may have evolved
efficient mechanisms for elimination of Hg from vital body
tissues [6,16].
In Amazonian waterways, Hg poses a threat to local human
populations [7,13], with elevated concentrations having been
found in both fish (>500 mg kg–1) and human hair (>20 000
mg kg–1) [13,17]. Bioaccumulation of Hg in Amazonian reptiles
is of particular concern as human consumption of crocodilians
and turtles provides an important source of protein and is part
of local culture [5,18–20]. In the Amazon basin, increased
commercialization and trade of turtles and caimans has also
resulted in their unsustainable exploitation, population declines,
and localized extinctions [5,21]. The Rio Purus basin in
particular contains some of the largest populations of caiman
in the Amazon, as well as the largest illegal caiman trade
in Latin America [16]. Farming of turtle and caiman species is
now being used in the Amazon as a potentially more sustainable
2772 Environ Toxicol Chem 34, 2015
alternative to wild capture. In American alligators, farming has
been demonstrated to protect wild populations from over-
exploitation, as well as significantly lowering Hg concentrations
in animals and reducing the risk of dietary Hg intake for
consumers [22].
Whereas Hg bioaccumulation in fish and humans is well
documented in the Amazon basin [13,23], few studies have
reported Hg concentrations in Amazonian reptiles. Only 5
studies have investigated Hg bioaccumulation in Amazonian
turtles [7,16,24–26], and only 2 studies have investigated
Amazonian caimans [6,25]. These studies indicate that substan-
tial Hg bioaccumulation is occurring but are largely inconclusive
as to what factors control Hg bioaccumulation in Amazonian
reptiles. To better understand Hg bioaccumulation, more data
are required, particularly for MeHg concentrations for which
no information currently exists. In this context it is further
worth noting that total Hg concentrations are not always useful
for predicting MeHg concentrations in biota [27].
The present study investigated the biological and environ-
mental factors that influence Hg bioaccumulation in turtle and
caiman species within the Amazon basin and whether Hg
concentrations in these species pose a risk to humans. It also
assessed whether farming of these reptiles can reduce Hg
exposure to consumers and whether nonlethal sampling
techniques can be used to monitor Hg concentrations in
threatened Amazonian reptile species. With the threatened
status of many reptile species [28], it is becoming increasingly
important to develop reliable nonlethal sampling techniques
to monitor Hg concentrations in their tissues [24,25,29].
Accordingly, we focused on commonly consumed species
that are of high commercial value, specifically 2 species of
turtle of the Podocnemididae family (Podocnemis unifilis and
Podocnemis expansa) and 2 species of caiman (Melanosuchus
niger and Caiman crocodilus). We determined the relation-
ships between total Hg and MeHg concentrations in a range of
Figure 1. Locations of study sites in the Amazon basin: the lower Rio Purus (arrow; 4845.6020 S, 62811.3850 W) and the city of Manaus (triangle; 038060 S,
608010 W), Brazil.
S. Eggins et al.
tissues (muscle, liver, epidermal keratin, and blood) of turtles
and caimans taking animal size and gender into account. We
assessed possible dietary and environmental influences on
Hg concentrations in Amazonian caiman and turtle species,
determined whether farming of turtles results in reduced Hg
concentrations through comparison of Hg concentrations in
farm-reared and wild-caught P. expansa, determined whether
Hg concentrations in blood and keratin can serve as nonlethal
biomonitors of Hg in internal tissues (muscle and liver),
and determined whether Hg concentrations in muscle, liver,
and blood exceeded World Health Organization (WHO)
guidelines for safe consumption.
METHODS
Sampling sites
Specimens of caiman species M. niger (n ¼ 16) and C.
crocodilus (n ¼ 17) as well as turtle species P. unifilis (n ¼ 28)
and P. expansa (n ¼ 8) were collected from the lower Rio
Purus in the Amazon basin (Amazonas, Brazil; 4845.6020 S,
62811.3850 W; Figure 1). Specimens of P. expansa were
augmented with specimens confiscated from poachers in
the same area, although it is not known precisely where the
poachers captured these specimens. Farm-reared P. expansa
specimens (n ¼ 30) were donated by various turtle farms located
around the Amazonas state capital of Manaus (038060 S,
608010 W). Specimens were collected during March 2008, after
the wet season.
Animal collection
Caiman specimens were captured at night using locking
cable snares and Ketch-All animal restraining poles (IBM), as
described by Da Silveira et al. [30]. Turtle specimens were
collected from the wild using trammel nets. Caimans were sexed
and had their snout–vent length measured as an index of size.
Mercury concentrations in reptiles from the Rio Purus
Snout–vent length was used instead of tail length, which is
susceptible to seasonal variations with change in somatic
mass [6]. Turtles were sexed visually using sexually dimorphic
patterning and features [19]. Straight carapace length, the
distance from the outside edge of the nuchal at the front
middle of the carapace to the middle of the 2 rear marginals on
the carapace, was measured as an index of size.
Tissue sampling and preparation
Body tissues targeted for sampling in turtles and caimans
were muscle tissue, liver tissue, keratin, and blood. Liver tissue
and blood were sampled as indicators of short-term dietary Hg
intake, whereas muscle and keratin were sampled as indicators
of long-term Hg bioaccumulation. Blood and keratin were
selected as potential nonlethal samples, which might be used to
predict Hg concentrations in internal tissues. Muscle is usually
targeted for analysis as it provides a reliable measure of long-
term Hg exposure and bioaccumulation [31].
Muscle tissue collected from caimans (4 cm3) was
sampled from the dorsal section of the tail as this muscle is
typically used for human consumption [6] and is assumed to
be representative of all muscle. Skinless and boneless muscle
tissue was extracted from turtles from the left hind leg of each
specimen. Carapace samples from turtles were cut from the
eighth right marginal scale of the carapace. Approximately
3 triangles of scute were also taken from the dorsal section of
the tails of caiman specimens. For all species, liver samples
were collected as far from the gallbladder as possible, to
avoid bile, and blood samples were extracted directly from the
jugular vein using a syringe immediately after animals were
euthanized.
All tissue samples had their wet mass recorded and were
sealed in labeled plastic bags. Samples were then frozen and
transported to Instituto Nacional de Pesquistas da Amaz^onia
(Manaus, Brazil), where they were freeze-dried and stored in
sealed containers until they were transported to the University
of Canberra, Australia.
Prior to Hg analysis all samples were freeze-dried again for
approximately 24 h (Labconco) and subsequently weighed to
determine their total dry mass. Wet mass and dry mass ratios for
samples were used to recalculate Hg concentrations on a weight/
weight wet mass basis following analysis. Scute fragments of
both turtle and caiman samples were washed with deionized
water for 15 min in an ultrasonic bath to remove algae and other
encrusting residues and sediment grains. They were then freeze-
dried for 48 h, allowing easy separation of the keratin layers
from the dermal bone with a scalpel. The bony dermal material
was not analyzed for Hg concentrations. All tissue samples were
ground to a homogeneous powder using an IKA A11 handheld
analytical mill.
Total Hg analysis
Tissue samples were digested following the procedure of
Baldwin et al. [32] with some minor alterations. Freeze-dried
samples greater than 0.07 g dry mass were subsampled so that
masses of 0.07 g were used for digestion. Samples were
weighed into acid-washed 7-mL polytetrafluoroacetate diges-
tion vessels (A.I. Scientific). Concentrated nitric acid (1.0 mL,
Suprapur; Merck) was added to each vessel, which was left
loosely capped for approximately 24 h. Polytetrafluoroacetate
vessel caps were then tightened to 2.3 N  m and placed
in pairs inside 120-mL screw-top Teflon pressure vessels,
each containing 10 mL of deionized water. The screw-top
vessels were then tightened to 16.3 N  m, and samples were
Environ Toxicol Chem 34, 2015 2773
digested in a 950-W microwave oven (CEM MDS 2100)
at 570 W for 2 min, followed by 45 min at 314 W [32].
Once cool, samples were transferred into 10-mL polypro-
pylene centrifuge tubes (Sarstedt) and diluted to 10 mL with
deionized water. An autodilutor (Gilson GX 271) was used
to achieve a further 10-fold dilution for a final concentration
of acid:tissue (1% v/v) in water.
Hg concentrations in digested samples, procedural blanks,
and certified reference material were measured using a Perkin
Elmer ELAN DRC-e inductively coupled plasma mass
spectrometer (ICPMS) following Maher et al. [33]. A
multielement internal standard containing 10 mg L–1 of 6Li,
Ho, In, Tb, Rh, and Sc in 1% HNO3 (EM Science) was added
to samples during autodilution and used to monitor and
correct for instrumental drift and matrix suppression effects.
The Hg concentration analyses were calibrated using ICPMS
standards (Merck) containing 1 mg L–1, 10 mg L–1, 100 mg L–1,
and 1000 mg L–1 of Hg. Two National Research Council of
Canada certified reference materials, DORM-2 and TORT-2,
along with 2 blanks were analyzed every 20 samples for
quality assurance purposes. These particular certified reference
materials encompass the range of expected Hg values for the
tissue samples analyzed in the present study. They are also
certified for MeHg concentrations.
MeHg analysis
MeHg concentrations were analyzed for muscle tissue
samples that had total Hg concentrations above detection
limits (>0.05 mg/g) and sufficient remaining dry mass for
total MeHg determination. The procedure for Hg species
determination followed that of Rai et al. [34]. Subsamples
(0.2 g) were taken from freeze-dried samples and weighed
into 50-mL fluorinated ethylene propylene tubes (Selby
Scientific), along with 10 mg of protease type XIV (Sigma)
and 8 mL of phosphate buffer (0.06 mol L–1) containing
0.1% w/v l-cysteine (Sigma-Aldrich), and adjusted to a pH
of 7.5 with addition of ammonia (Pronalys; May and Becker).
The tubes were sealed and incubated at 37 8C for 2 h in a
hybridization oven (XTRON HI 200; Bartlett Instruments)
with rotation of samples at 20 rpm to ensure homogeneity.
Extracts were then centrifuged for 10 min at 4500 rpm
(Eppendorf 5804-R centrifuge), and the supernatant was
filtered through Acrodisc LC 13-mm syringe filters with
20-mm poly(vinylidene difluoride) membranes (Gelman),
before being transferred to acid-washed 10-mL polypropylene
tubes (Sarstedt).
Hg species (MeHg and Hg[II]) concentrations were
determined on 100-mL aliquots of enzyme extracts using
high-performance liquid chromatography (HPLC)-ICPMS. The
HPLC-ICPMS system consisted of a Perkin Elmer series
200 HPLC pump and a Perkin Elmer 3 mm C8 column
(30 mm  3 mm) coupled to a Perkin Elmer ELAN DRC-e
ICPMS. A mobile phase consisting of 0.06 mol/L ammonium
acetate (Merck) in 3% v/v methanol and 0.1% w/w l-cysteine
was used. Hg Species were quantified by integration of
chromatogram peak areas using the chromatography software
package Turbochrom Navigator (Perkin Elmer).
MeHg concentrations were calibrated against MeHg
standards (12 mg L–1 and 120 mg L–1) and cross-referenced to
1 mg L–1, 10 mg L–1, and 100 mg L–1 inorganic Hg calibration
standards by analysis of total Hg via injection without
attachment of the HPLC column into the ICPMS. The
DORM-2 certified reference material and a procedural blank
were analyzed with every 8 samples for quality assurance.
2774 Environ Toxicol Chem 34, 2015
Quality assurance of analytical results
Total Hg concentrations measured for certified reference
materials DORM-2 and TORT-2 (4670  440 mg kg–1 and
330  20 mg kg–1, respectively) were in agreement with certi-
fied values (4640  260 mg kg–1 and 270  80 mg kg–1, respec-
tively). MeHg concentrations measured for DORM-2
(4380  420 mg kg–1) also agreed within the 95% confidence
interval of the certified value (4470  320 mg kg–1).
Statistical analysis
Statistical analyses were performed using the R Develop-
ment Core Team’s statistical software package R, Ver 2.15.2.
For all statistical tests a degree of significance was accepted if
p < 0.05. Shapiro-Wilk tests for normality and Levene’s tests
for homogeneity of variances were conducted to determine
whether Hg concentrations for each tissue type and each species
were normally distributed and homoscedastic.
A one-way analysis of variance (ANOVA) with a post hoc
pairwise Tukey’s test was used on log-normalized data to
describe interspecific differences in total Hg concentrations for
each tissue type and MeHg concentrations for muscle tissue.
Pairwise linear regressions with Pearson product–moment
correlation were used to determine whether there were
significant relationships between Hg concentrations in non-
lethally sampled tissues (blood and keratin) and internal,
lethally sampled tissues (muscle and liver).
Linear regression models with Pearson product–moment
correlation were used to determine the significance of relation-
ships between size (snout–vent length or straight carapace
length) and Hg concentrations for different tissues. Length
distributions were normalized where appropriate via log-
transformations, and a parametric one-way analysis of covari-
ance (ANCOVA) was used to test for the influence of both size
and gender on Hg concentrations in tissues. Gender was used as
a categorical predictor, and size (either snout–vent length or
straight carapace length) was used as a covariate predictor of
Hg concentration. As the comparison of adjusted means relies
on equality of regression slopes for the covariate, a post hoc F
test was used to determine homogeneity of regression slopes.
The same statistical analyses were used to determine relation-
ships between MeHg content in muscle tissues (as absolute
concentrations and as a fraction of total Hg) and size and gender
for each species.
Differences between Hg concentrations in farm-reared and
wild-caught subsets of P. expansa were assessed using one-way
ANCOVA with origin (farm-reared or wild-caught) employed
as a categorical predictor and straight carapace length as a
covariate. In satisfying the assumptions of ANCOVA, a
Shapiro-Wilk test was used to confirm that length distributions
Table 1. Gender and size (mean  1 standard deviation [range] [cm]) distributions of turtle and caiman specimens collected from the Rio Purus, Brazil
n SVL or SCL (cm)
Species M F Total
Caiman crocodilus 12 5 17
Melanosuchus niger 11 5 16
Podocnemis unifilis 8 20 28
Podocnemis expansa  8 8 
Podocnemis expansaa 14 16 30
a
Specimen characteristics of farm-reared P. expansa collected near Manaus, Brazil.
SVL ¼ snout-vent length; SCL ¼ straight-carapace length.
S. Eggins et al.
were normal and F test and t tests to determine whether length
distributions for farm-reared or wild-caught origins had
significantly comparable means and were homoscedastic. As
only female wild-caught P. expansa were analyzed, ANCOVAs
were carried out with a pooled data set that included farm-reared
P. expansa and were repeated with males omitted to remove the
influence of gender. The 2 tests returned equivalent results.
RESULTS
Specimen characterization
The features used to characterize sampled reptilian species
were gender and snout–vent length for caimans and gender and
straight–carapace length for turtles. Sample size, size distribu-
tion, and range by gender are summarized in Table 1.
Total Hg concentrations across different tissues
Box plots for log10-normalized total Hg concentrations
measured in each species and tissue, including statistical
results, are presented in Figure 2. The means and ranges of
total Hg concentrations measured in muscle, liver, keratin, and
blood tissues are listed in Supplemental Data, Table S1. For
all species other than M. niger, mean total Hg concentrations
were highest in keratin, followed by liver tissue, then muscle
tissue, then blood. In M. niger the mean Hg concentration in
keratin was lower than the mean Hg concentration in liver
tissue.
Linear correlations between nonlethally sampled tissues
(blood and keratin) and internal tissues (muscle and liver) were
significant between all tissue pairs for M. niger (p < 0.05;
Figure 3). As no significant difference was found between
wild-caught and farm-reared P. expansa, the 2 data sets were
pooled, revealing a significant linear correlation only between
total Hg concentrations in muscle and keratin (p < 0.01) but not
for other tissue pairs (p  0.05; Figure 4). No significant linear
correlations were found between the total Hg concentrations of
any tissue type for C. crocodilus or for P. unifilis (p  0.05).
MeHg concentrations in muscle tissue
Means and ranges of MeHg concentrations in muscle tissue
as well as the fraction of total Hg that is MeHg (%MeHg) for
each species are provided in Supplemental Data, Table S2. A
box plot comparing %MeHg between species is presented in
Figure 5. Among all species, the majority of Hg content in
muscle tissue was present as MeHg (Figure 5).
Interspecific differences in Hg concentrations
Caiman crocodilus and M. niger did not have significantly
different log10-normalized total Hg concentrations for any tissue
types except for muscle, where C. crocodilus had significantly
M F Total
84  14 (62105) 70  5 (6577) 80  14 (62105)
100  31 (82191) 106  21 (75–129) 102  27 (75191)
30  5 (2741) 37  4 (3146) 35  5 (2746)
43  12 (2557) 43  12 (2557)
33  6 (2745) 37  13 (2364) 35  10 (2364)
Mercury concentrations in reptiles from the Rio Purus Environ Toxicol Chem 34, 2015 2775

Figure 2. Box plots (showing the median, range [whiskers], and quartiles [boxes]) for log10-transformed total mercury and methylmercury concentrations
measured in tissues of turtle and caiman species. For each separate tissue, letters above and below boxes indicate groups of species, which for a given letter have
Hg concentration distributions that are not significantly different (analysis of variance, p  0.05). MeHg ¼ methylmercury; THg ¼ total mercury.

higher log10-normalized total Hg concentrations than M. niger
(F3,46 ¼ 47.14, p ¼ 0.004; Figure 2). Log10-normalized total
Hg concentrations in all tissue types of both caiman species
were significantly higher than those in the corresponding tissues
of both turtle species (Figure 2). As only 1 keratin sample
was analyzed from wild-caught P. expansa, this result could
not be statistically analyzed. No significant differences in
log10-normalized total Hg concentrations existed between the
2 turtle species for any tissue types except for blood, in which
log10-normalized total Hg concentrations were significantly
higher for P. unifilis than for wild-caught P. expansa
(F3,37 ¼ 45.46, p ¼ 0.015; Figure 2).
Interspecific differences between MeHg concentrations
of muscle tissues followed the same pattern as those of total
Hg concentrations (Figure 2). Caiman crocodilus had
significantly higher log10-normalized MeHg concentrations
compared to M. niger (F1,17 ¼ 6.557, p ¼ 0.020), and both
caiman species had significantly higher log10-normalized

Figure 3. Linear correlations between mercury concentrations (micrograms per kilogram w/w wet wt) of internal, lethally sampled tissues (muscle and liver) and
nonlethally sampled tissues (blood and keratin) for Melanosuchus niger. Significant linear correlations (p < 0.05) are positive within 95% confidence intervals.
THg ¼ total mercury.
2776 Environ Toxicol Chem 34, 2015
Figure 4. Linear correlations between Hg concentrations (micrograms per kilogram w/w wet wt) of internal, lethally sampled tissues (muscle and liver) and
nonlethally sampled tissues (blood and keratin) for pooled (combined wild-caught and farm-raised) Podocnemis expansa. Significant linear correlations
(p < 0.05) are positive within 95% confidence intervals. THg ¼ total mercury.
MeHg concentrations than both turtle species. There was
no significant difference between the log10-normalized
MeHg concentrations in muscle tissues of both turtle species
(F3,46 ¼ 38.99, p ¼ 0.207). In addition, there was no significant
difference between %MeHg in muscle tissues of any species
Figure 5).
Influence of gender and size on Hg concentrations
Log10-normalized total Hg concentrations in all tissues
(muscle, liver, keratin, and blood) did not differ between
genders for any species. Gender also had no influence on MeHg
Figure 5. Box plot (showing the median, range [whiskers], and quartiles
[boxes]) comparing the fraction of total mercury that is methylmercury
(%MeHg) determined in caiman and turtle species. There was no significant
difference between %MeHg in any species (analysis of variance, p  0.05).
MeHg ¼ methylmercury; THg ¼ total Hg.
S. Eggins et al.
concentrations. Wild-caught P. expansa specimens were all
female, so the influence of gender could not be assessed for
this group. Given that gender showed no influence on total Hg
concentrations, male and female data were pooled together for
size regressions.
Size (snout–vent length or straight carapace length) was
not significantly linearly correlated with total Hg concen-
trations in any tissues for C. crocodilus, P. unifilis, and
both subsets of P. expansa. For M. niger, snout–vent length
predicted total Hg concentrations in muscle (b ¼ 2.22, t[9] ¼
3.13, p ¼ 0.012), liver (b ¼ 63.97, t[9] ¼ 5.90, p < 0.001), and
blood (b ¼ 0.93, t[10] ¼ 4.91, p < 0.001), but not keratin (b ¼
19.23, t[11] ¼ 2.00, p ¼ 0.07; Figure 6). There was also a
significant linear correlation between MeHg concentrations
in muscle tissue and snout–vent length for M. niger (b ¼ 0.23,
t[9] ¼ 2.92, p ¼ 0.017; Figure 6A). No significant linear
correlations were found between size and MeHg concen-
trations or size and %MeHg in muscle tissue for any other
species.
Comparison between Hg concentrations in farm-reared and
wild-caught P. expansa
Although total Hg concentrations in all tissues of farm-
reared P. expansa were higher than those in wild-caught
P. expansa (see Supplemental Data, Table S1), no significant
variation in log10-normalized total Hg concentrations of
muscle, liver, or blood was found between wild-caught and
farm-reared P. expansa. Only 1 keratin sample from wild-
caught P. expansa was analyzed, so comparisons could not
be made for this tissue type. No significant variation in
log10-normalized MeHg concentrations or %MeHg in muscle
tissue was found between wild-caught and farm-reared
P. expansa.
Mercury concentrations in reptiles from the Rio Purus
Figure 6. Relationships between snout–vent length and total mercury concentrations (black circles and solid regression lines) in muscle (A), liver (B), blood (C),
and keratin (D), as well as the relationship between snout–vent length and methylmercury concentrations (white circles and dashed regression line) in muscle
tissue, for Melanosuchus niger. Regression lines are plotted only for significant data (p < 0.05). Pearson correlations are all positive within 95% confidence
intervals. MeHg ¼ methylmercury; THg ¼ total Hg.
DISCUSSION
Total Hg and MeHg concentrations in caimans and turtles from
the Rio Purus
Total Hg concentrations measured in muscle tissues of
Amazonian turtles and caimans in the present study are
notably higher than the lowest values reported for turtles and
crocodilians worldwide but are comparable with concentrations
measured in turtles and crocodilians that have been exposed to
high levels of environmental Hg in the Amazon basin and
elsewhere (see Supplemental Data, Tables S3 and S4). Evidence
of significant bioaccumulation of Hg in turtles, caimans, and fish
has been reported in the Amazon basin previously [6,16,35,36]
and has been attributed to regionally high Hg bioavailability
from both natural and anthropogenic sources [11]. It is
important to note that size (as a proxy for age) may be a
confounding factor when assessing intraspecific and interspe-
cific differences in Hg concentrations, particularly for M. niger
for which we found a positive correlation between snout–vent
length and total Hg concentrations in muscle, liver, and blood.
As no correlation was found between size and Hg concen-
trations in any tissues from P. unifilis or P. expansa in the
present study or in previous studies [7,16], it is reasonable to
make comparisons between spatially or temporally separate
studies of these species. Muscle tissues of P. expansa and
P. unifilis from the Rio Purus measured in the present study had
lower Hg concentrations than those of the same species from the
Rio Negro basin reported by Schneider et al. [7]. This indicates
that bioavailable Hg concentrations may be significantly lower
in the Rio Purus basin than in the Rio Negro basin. As no studies
have measured Hg concentrations or speciation in water or
soil in the Rio Purus, this hypothesis cannot yet be tested. It is
Environ Toxicol Chem 34, 2015 2777
also possible that the very small sample populations (n  5)
of Podocnemididae species examined by Schneider et al. [7]
may not be representative of natural populations in the Rio
Negro basin. Total Hg concentrations in muscle tissues of
C. crocodilus and M. niger measured in the present study are
similar to those reported previously from the Rio Purus by
Schneider et al. [6]. Animals in the 2 studies are of comparable
size categories; however, they comprise relatively small
numbers of samples that span a range of Hg concentrations
and may not be fully representative of wild populations.
The present study provides the first MeHg concentration data
for Amazonian caimans and turtles. The %MeHg values
measured in muscle tissues of caimans (Figure 5) in the present
study are slightly higher than, but broadly consistent with, %
MeHg values reported for caimans (>70%) from the Pantanal
region [37]. The %MeHg values measured in turtle muscle
tissue partly overlap with a very high value (94%) reported
for Chelydra serpentina from North America. The %MeHg
in both caimans and turtles in the present study (Figure 5)
are also notable for being similar to values in predatory fish
species (Chicla spp.) that are exposed to natural Hg in the Rio
Negro (74–99%) [38]. As with the majority of other studies,
the present study showed that Hg in muscle tissue exists
predominantly as MeHg.
Interspecific differences in Hg bioaccumulation
As organisms tend to accumulate Hg primarily through
dietary exposure; trophic positions, diet composition, foraging
behaviors, and habitats are typically used to explain the
interspecies differences in Hg concentrations within food webs
[36]. In the present study, Hg concentrations measured in the
tissues of caimans and turtles should in general reflect their
2778 Environ Toxicol Chem 34, 2015
trophic positions and dietary specializations. As %MeHg did
not vary significantly between species but the majority of Hg
was in that form, the chemical properties of MeHg are what
determine total Hg bioaccumulation in muscle tissue.
Measured total Hg concentrations in all tissues of turtle
species in the present study were significantly lower than those
in caiman species. The higher total Hg concentrations found
in caiman tissues are consistent with their being carnivorous
predators [21] and occupying the highest trophic position
among Amazonian food webs. The lower values in most turtles
are consistent with relatively minor Hg bioaccumulation and
with the low trophic positions of Podocnemididae species,
which are primarily herbivorous and occasionally eat insects,
snails, and small fish [20]. The present study’s finding of
significantly higher total Hg concentrations in the blood of
P. unifilis compared with that of P. expansa suggests higher
dietary Hg exposure for P. unifilis; however, current character-
izations of diet imply no major differences in the diets of these
species [20]. We also found significantly higher total Hg
concentrations in muscle tissues of C. crocodilus compared with
M. niger, which is consistent with the findings of Schneider
et al. [6]. Total Hg concentrations measured in liver tissues and
blood in the present study did not, however, differ significantly
between M. niger and C. crocodilus, suggesting that the rate
of dietary Hg intake is similar for both species. Given that
M. niger consume smaller quantities of terrestrial prey [39] and
terrestrial animals generally bioaccumulate less Hg, it was
expected that M. niger would have a higher dietary intake of Hg
than C. crocodilus. Melanosuchus niger grow to significantly
larger sizes than C. crocodilus (up to 4.5 m vs 2 m total
length) [21], and there is evidence that growth rates are
negatively correlated with size for C. crocodilus but not for
M. niger, for which growth rates are thought to be dependant on
resource availability [40]. As the lower Rio Purus is a highly
productive area, it is conceivable that M. niger specimens
from the present study have faster growth rates relative to
C. crocodilus. This could result in a growth dilution effect and
account for lower Hg concentrations in muscle tissue of
M. niger compared to C. crocodilus as observed in the present
study. It may also be that C. crocodilus have evolved a
more efficient mechanism for sequestering Hg into muscle
tissue. Alternatively, as MeHg has a high binding affinity
to selenoproteins [41], differences in dietary selenium intake
between caiman species could result in different rates of Hg
sequestration into muscle tissue.
Feeding strategies and foraging locations within floodplain
rivers have been shown previously to be important determinants
of Hg bioaccumulation in fish species [36] and likely also
influence bioaccumulation in turtles and caimans. Significant
spatial variation in MeHg production [42] may occur with water
depth and habitat within the study areas, with MeHg production
being elevated in flooded forests and macrophyte mats (relative
to open waters) and being associated with periphyton on roots
and submerged leaf litter [42,43]. Temporal variation in MeHg
production is also pronounced in the Amazon basin, with high
influxes of dissolved organic carbon resulting from seasonal
flooding leading to enhanced Hg methylation rates [43,44]. This
effect is likely to be pronounced in the lower Rio Purus, which
receives dissolved organic carbon–rich black water contribu-
tions from the Rio Solimoes during the wet season [14].
Seasonal inundation also provides access to larger areas of
periphyton-rich flooded forest habitat, resulting in changes in
feeding strategies and in the structures and lengths of food
webs [35,36]. Turtles move into flooded forest habitats during
S. Eggins et al.
the wet season, where they feed on a wider variety of plant
material, and move out into the river channel during the dry
season [19,20]. Seasonal variation in the diets of M. niger
and C. crocodilus also suggests that they adjust their feeding
strategies to account for changing prey abundances [21,39]. As a
result of relatively short residence times for Hg in liver and
blood, it is likely that a seasonal shift in diet could temporarily
alter Hg concentrations in these tissues, whereas Hg concen-
trations in muscle and keratin would remain stable. According-
ly, a dietary shift exclusively in 1 species may have confounded
interspecific comparisons of Hg concentrations in blood and
liver tissues in the present study. The role of seasonal changes
in diet and foraging habitats, in addition to characterization of
overall diet and trophic level of species, may need to be more
adequately addressed to better understand Hg bioaccumulation
and biomagnification in Amazonian turtles and caimans. Stable
isotope (d15N and d13C) compositions of food-web compo-
nents could be employed in future for assessing trophic position
and tracking autotrophic carbon sources to particular turtle and
caiman species, while accounting for seasonal variation.
Influences of size and gender on Hg bioaccumulation
Total Hg and MeHg concentrations did not vary with gender
for any tissue types in any species. It has been suggested that
gender-related differences in Hg concentrations could arise
from altered feeding habits of females during reproductively
active periods [16,20] and/or Hg elimination by females through
transfer to eggs [45]. There is a paucity of information as to how
the diets of Amazonian reptiles vary between genders during
reproductive periods.
Vertebrates typically consume prey of greater size and
trophic level with age, so bioaccumulation of Hg typically
increases over their lifetimes [13]. Prey size has been
documented to increase with increasing size of both caiman
species M. niger and C. crocodilus [21], such that tissue Hg
concentrations could be expected to correlate positively with
size. Both the present study and Schneider et al. [6] found
positive correlations between snout–vent length and muscle
tissue Hg concentrations for M. niger but not for C. crocodilus in
the Rio Purus. In addition, significant correlations between
size and total Hg concentrations in blood as well as liver for
M. niger suggest increased dietary intake of Hg with growth,
as expected. Amazonian caimans, like many other species of
crocodilian, undergo dietary shifts with ontogenesis [21,46,47].
Significant correlations between size and muscle and liver
tissue Hg concentrations in juvenile and subadult Alligator
mississippiensis, but not in adults, occur because of an
ontogenetic shift in feeding habits for this species [48]. It is
plausible that increased terrestrial feeding behavior with size
may be exclusive to C. crocodilus in the Amazon; however, no
definitive evidence exists for this.
We found no significant linear correlations between size
and total Hg or MeHg concentrations in any tissues of turtle
species. The diets of Amazonian Podocnemididae remain
primarily herbivorous throughout their life cycles, such that
their dietary exposure to Hg should not change substantially
with size. Increased herbivory in grizzly bears has been shown
to reduce bioaccumulation of persistent organic pollutants
through fugacity-driven excretion [49]. It may be the case that
the same elimination mechanism allows Podocnemididae to
eliminate organic Hg at relatively high rates (cf. fish), reaching
equilibrium with rates of Hg intake. This follows the suggestion
of Golet and Haines [31], who found no correlation between
size and Hg concentrations in the North American turtle
Mercury concentrations in reptiles from the Rio Purus
C. serpentina. Given that Hg in Amazonian environments is
naturally high, it is possible that Amazonian turtles have
evolved efficient Hg elimination mechanisms. Changes in
growth rate with age could also affect any systematic linear
relationship with size [50].
Given that MeHg bioavailability varies with water depth and
location in floodplain rivers [36,42] variations in habitat and
foraging locations could also be important determinants of
Hg bioaccumulation differences between individuals and/or
size classes within species. The physiology, foraging habitat,
diet, and migratory and nonmigratory movements of individuals
within a species’ population can also vary seasonally and with
ontogenesis. Ontogenetic habitat shifts have been observed
in North American A. mississippiensis populations [47] and
may also be significant for Amazonian caimans as caimans
have been observed to migrate >30 km in only a few
weeks [51]. In addition, some species of Amazonian turtle,
such as Podocnemis erythrocephala, have been observed to
travel long distances between tributaries [16]. These factors
could obscure any systematic exposure of individual animals
to bioavailable Hg and mask correlations between Hg
bioaccumulation and environmental or biological variables.
Accounting for the confounding effects of such uncontrolled
factors creates one of the largest sources of uncertainty in
ecological studies, which commonly assume uniform behavior
across a species [51].
Recent studies have measured d13C and d15N along growth
layers in the scutes of sea turtles to determine ontogenetic
variations in diet, habitat, and geographical location [52,53].
Also, the potential for the direct measurement of Hg
concentrations in carapace growth rings via laser ablation has
been demonstrated and may enable the analysis of ontogenetic
variation in Hg bioaccumulation in turtle species [25].
Distribution of Hg concentrations between tissues and the
potential for nonlethal monitoring
Differences in total Hg concentrations between different
tissue types in caimans and turtles measured in the present
study conform to patterns widely observed in reptiles that
reflect specific tissue affinities. In general, liver tissues had the
highest Hg concentrations, followed by keratin, muscle, and
then blood. Different tissue types tend to have different
proportions of Hg(II) and MeHg that, if accounted for, could
improve correlations between tissue pairs [29,31]. Reptile
muscle, keratin, and blood contain up to 100% MeHg,
whereas the liver contains much greater fractions of Hg(II)
[54]. As MeHg is generally in equilibrium between tissue
types and Hg(II) is not [55], the measurement of MeHg
concentrations in different tissues may improve between-
tissue correlations and, thus, the potential for nonlethal
monitoring; however, %MeHg is typically consistent within
particular types of tissue.
No correlations were found between Hg concentrations in
tissues of C. crocodilus and P. unifilis, suggesting that blood and
keratin cannot be used as reliable predictors of Hg concen-
trations of internal tissues for these species. The significant
correlation with good predictability (99% of variation pre-
dicted) between Hg concentrations in keratin and muscle for
P. expansa suggests that nonlethal keratin sampling may have
the potential to be used to infer Hg concentrations of internal
muscle and liver tissues for this species. This result corroborates
Schneider et al. [25], who found that total Hg concentrations of
keratin reliably predicted total Hg concentrations of muscle
when data for both turtle species were amalgamated. Also,
Environ Toxicol Chem 34, 2015 2779
positive correlations between total Hg concentrations in all
tissue type pairs for M. niger indicate that nonlethally sampled
blood and keratin has the potential to be used for inferring
internal Hg concentrations of muscle and liver tissues of
M. niger. For this species, internal tissue concentrations were
predicted most reliably based on blood concentrations, which
were able to predict 72% of variation in muscle tissues and 61%
of variation in liver tissues. Far poorer predictability was
obtained using keratin, which was able to predict 47% of
variation in muscle tissues and 32% of variation in liver tissues
and, therefore, is unreliable. Although Schneider et al. [25]
found that total Hg in keratin of both caiman species did not
predict total Hg concentrations in muscle, the poor predictabili-
ty indicated for M. niger combined with lack of any correlation
for C. crocodilus in the present study remain broadly consistent
with that result. Our results suggest that total Hg concentrations
in blood are better than keratin at predicting internal tissue total
Hg concentrations. The small sample sizes in the present study’s
data set, however, may be inadequate to rigorously assess the
feasibility of using nonlethally sampled keratin or blood to
monitor overall Hg burdens in our study species, though there is
sufficient evidence to suggest its plausibility. Variations in
growth rate and diet between individuals of a species may also
play a role in obscuring correlations between Hg concentrations
in different tissues.
Human health consequences
The WHO recommends a safety guideline for the consump-
tion of fish flesh of 500 mg kg–1 w/w wet weight of Hg and
a limit of 200 mg kg–1 for vulnerable groups, such as pregnant
women and children [56]. Mean total Hg concentrations
in muscle tissue for all turtle and caiman species in the
present study were below the WHO guideline. Only 1 C.
crocodilus specimen contained a muscle total Hg concentra-
tion (841 mg kg–1) above the safety guideline, whereas the
mean Hg concentration of C. crocodilus muscle tissue
(389  76 mg kg–1) and a number of individual M. niger
muscle samples exceeded the 200 mg kg–1 limit. This indicates
that pregnant women and children in the Rio Purus basin
may be at risk of unsafe levels of MeHg accumulation if
they consume caiman meat regularly. Given that MeHg
constitutes most of the total Hg in muscle tissue, the risk of
accumulation of dietary Hg by humans is heightened as this
is the most bioavailable and toxic Hg species.
The WHO recommended guidelines and limits for Hg
intake are based on typical consumption rates of fish muscle,
whereas consumption rates of turtle and caiman muscle among
Amazonian populations are likely to be different. Moreover
turtle blood and liver are regularly consumed by Amazonian
communities. Assessment of the full risk to Amazonian
populations requires revision of existing guidelines in consid-
eration of consumption rates of liver and muscle tissues of
different turtle and caiman species. Although MeHg bioaccu-
mulation can be toxic to wildlife, a lack of knowledge on the
health effects of bioaccumulation on Amazonian turtles and
caimans precludes any meaningful speculation as to the
toxicological significance of tissue Hg concentrations. No
evidence of health effects has been observed either on capture
or during postmortem examinations of caimans in the Rio
Purus [6]. A targeted study of neurological effects following
laboratory-controlled Hg dosages, however, is needed to
determine whether Amazonian turtle and caiman species are
experiencing adverse health effects from the measured Hg
bioaccumulation levels.
2780 Environ Toxicol Chem 34, 2015
Can farming reduce Hg concentrations in Amazonian reptiles?
The lack of significant differences between the Hg
concentrations in muscle and liver tissues of farm-raised versus
wild-caught turtles indicates that farming of Amazonian turtle
species does not reduce Hg concentrations in their tissues
or reduce health risks to consumers. In Florida, farm-reared
A. mississippiensis have much lower muscle Hg concentrations
than wild-caught alligators as a result of lower dietary Hg intake
by farm-reared animals because their food is sourced from
terrestrial locations with lower environmental Hg concentra-
tions [22]. Farm-reared turtles in the Amazon are generally fed
with terrestrially sourced plant material, so it was anticipated
that they would have lower muscle tissue Hg concentrations
than wild turtles. However, P. expansa prefer to consume
meat in captivity [20], and depending on its origin, meat could
provide a significant dietary source of Hg to farm-reared
P. expansa. It is also a possibility that plant material fed to these
animals is being grown in industrialized areas that may be
contaminated with Hg. Without full knowledge of the diets of
farm-reared turtles, there is no basis for understanding and
predicting differences in Hg bioaccumulation between farm-
reared and wild-caught animals. Characterizing the Hg
concentrations of typical food items of wild-caught and farm-
reared turtles could be a simple experiment that would clarify
whether farming of turtles could reduce the risk of Hg intake to
human consumers.
CONCLUSIONS
The present study is the first conducted on Amazonian
reptiles that has measured MeHg concentrations in their tissues,
revealing that Hg in the muscle tissue of these animals exists
mostly as MeHg and does not vary significantly between
species. This is consistent with the majority of studies on aquatic
biota. As with previous studies in the Amazon basin, we found
in the present study that turtles and caimans in the Rio Purus are
bioaccumulating significant concentrations of Hg within their
tissues. Differences between the total Hg concentrations in
tissues of Amazonian turtle and caiman species can generally be
reconciled through current knowledge of their respective diets
and growth rates; but without proper characterization of diet and
its change with ontogenesis and season, as well as trophic
structure of the entire system, it is impossible to provide a sound
explanation for interspecific differences in Hg concentrations.
It is imperative that future studies on Hg concentrations in
Amazonian reptiles focus on the development and application of
a whole–food web Hg bioaccumulation model, including
analysis of N and C stable isotopic compositions of food-web
components. This would provide a deeper understanding of
the sources, trophic transfer dynamics, and fate of MeHg in
Amazonian aquatic ecosystems and would allow for improved
ecotoxicological assessment and risk management.
Significant correlations between size and Hg concentrations
in muscle, liver, and blood of M. niger found in the present study
are typical for carnivores as they have a relatively high intake
of dietary Hg. The overall paucity of correlations between Hg
concentrations and body size for other species in the present
study corroborates previous findings in the Amazon and
elsewhere, suggesting that caimans and turtles are possibly
eliminating Hg more rapidly than they are accumulating it, a
trait that may have evolved as a response to naturally high Hg
concentrations in the environment.
Total Hg concentrations of keratin and blood significantly
predicted those of muscle and liver tissues in M. niger, and
S. Eggins et al.
keratin is a good predictor of muscle total Hg concentrations in
P. expansa. This information contributes to a growing body
of evidence that nonlethally sampled keratin or blood could
be used for widespread monitoring of Hg body burdens in
Amazonian caiman and turtle species. To develop a sound
mathematical relationship, however, future studies should
employ larger sample sizes.
Mean Hg concentrations in muscle tissues of all turtle and
caiman species were below the WHO guideline for fish
consumption (500 mg kg–1); however, they exceeded WHO
guidelines for children and pregnant women (200 mg kg–1), and
liver total Hg concentrations in caiman species were well in
excess of WHO consumption guidelines. Given that Amazonian
peoples consume both liver and muscle, it is recommended that
existing fish-based Hg concentration guidelines be revised to
accommodate dietary intake rates for Hg present in turtle and
caiman liver and muscle tissues. The lack of difference between
Hg concentrations in tissues of farm-reared and wild-caught
P. expansa indicates that farming does not decrease Hg
concentrations in P. expansa and does not decrease risk to
consumers.
SUPPLEMENTAL DATA
Tables S1–S4 (364 KB PDF).
Acknowledgment—We thank the Conselho Nacional de Desenvolvimento
Cientıfico e Tec- nol
ogico, which funded the BAJAQUEL Project (408760/
2006-0 granted to R.D. Silveira). Laboratory analyses were funded by the
Ecochemistry Laboratory at the University of Canberra and performed with
the assistance of F. Krikowa. Turtle and caiman samples were collected in
conjunction with Instituto Nacional de Pesquistas da Amaz^ onia and
Universidade Federal do Amazonas, Brazil, under the auspices of IBAMA
collection permit 13347-1, IBAMA/CITES export permit 11BR006659/DF,
CITES import permit 2011-AU-635980, and AQUAS quarantine permit
NA11102340. All procedures were conducted in accordance with the
Brazilian government permit requirements for the BAJAQUEL project.
The veterinarian A. Kluczkovski, Jr. assisted with sample extraction from
and euthanasia of live animals.
Data availability—All data, including raw data and statistical results, are
publicly available on request by contacting the corresponding author,
S. Eggins (sameggins@gmail.com).
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