Amphibian populations around the world are

declining at alarming rates, but the causes of

most declines are unknown. Habitat destruction and alteration are most likely the largest

contributors to population declines, but other

factors such as introduction of exotic species,

emerging diseases, global climate change, and

environmental contamination likely influence

population viability (Collins and Storfer

2003; Stuart et al. 2004). The importance of

environmental contamination in population

declines is poorly understood, partly because

of the dearth of information on effects of

contaminants on responses of individuals

(e.g., reproduction) that directly influence the

viability of natural populations.

Exposure of sensitive embryonic life stages

to contaminants may be an important mechanism of impaired reproductive success in

amphibians. Developmental pathways in

embryonic amphibians can be altered when

embryos are exposed to contaminants via two

primary mechanisms: uptake from their surroundings (water) and transfer from females

to offspring. Most of what is currently known

about the effects of contaminants on amphibian development is derived from studies in

which embryos are exposed via water. In fact,

aqueous exposure has formed the foundation

for the only widely accepted standardized

amphibian toxicity test (frog embryo teratogenesis assay-Xenopus) [American Society for

Testing and Materials (ASTM) 1998; Bantle

et al. 1991]. In contrast, maternal transfer has

been an understudied mechanism of reproductive impairment in amphibians. One previous study demonstrated that amphibians

partition significant quantities of cadmium in

reproductive tissues (in follicles and eggs

before oviposition), suggesting the potential

for maternal transfer to offspring (Linder et al.

1998). Kadokami et al. (2004) went a step

further when they demonstrated that frogs can

maternally transfer organic contaminants to

their eggs. However, no studies have examined the effect of maternal transfer on reproduction and development in amphibians,

despite well-known effects of maternally

derived contaminants in all other major vertebrate lineages (e.g., Skorupa 1998). Ironically,

for many of the most toxic contaminants (e.g.,

lipophilic compounds and mercury), maternal

transfer may represent the most important

route of exposure for amphibian embryos

because water concentrations of these substances are generally low.

The purpose of the present study was to

determine whether amphibians maternally

transfer metals and metalloids to their eggs

and whether such transfer has implications for

reproductive success by affecting offspring viability. We focused on anurans inhabiting a site

contaminated by a coal-burning power plant

because these sites are a major global source of

selenium contamination (Lemly 1996; Rowe

et al. 2002). Selenium is a contaminant that is

readily transferable from mother to egg, highly

teratogenic, and associated with population

declines in other vertebrates (Lemly 1996;

Ohlendorf 2003). Previous work in the contaminated study site suggests that it is inhabited

by at least 22 species of amphibians, but

recruitment of juveniles from the contaminated wetlands appears low (Rowe and

Hopkins 2003). We hypothesized that disruption of normal development due to maternal

transfer of Se and/or other elements may partially underlie these observations.

Materials and Methods

Study species and sites. The species chosen

for study, the eastern narrow-mouth toad

(Gastrophryne carolinensis), is a common constituent of amphibian communities in the

southeastern United States. Like many other

anurans with complex lifecycles, G. carolinensis

adults are terrestrial and congregate near water

during the breeding season (May–September in

South Carolina). The species is particularly successful at breeding in ephemeral water bodies.

Adult G. carolinensis are small, highly fossorial,

and feed predominately on ants, termites, and

Environmental Health Perspectives • VOLUME 114 | NUMBER 5 | May 2006 661

Research

Address correspondence to W.A. Hopkins,

Department of Fisheries and Wildlife Sciences,

Virginia Polytechnic Institute and State University,

100 Cheatham Hall, Blacksburg, VA 24061 USA.

Telephone: (540) 231-7292. Fax: (540) 231-7580.

E-mail: hopkinsw@vt.edu

We thank J. Baionno, M. Komoroski, D. Scott,

D. Harrelson, J. Unrine, and J. Snodgrass for providing assistance. 

Support was provided by the Eppley Foundation,

the U.S. Environmental Protection Agency (STAR

R-82908701), the U.S. Department of Energy

[Environmental Remediation Sciences Division/

Office of Biological and Environmental Research,

award DE-FC09-96SR18546 to the University of

Georgia (UGA) Research Foundation], and a young

faculty award from the UGA.

The authors declare they have no competing

financial interests.

Received 30 June 2005; accepted 8 December 2005.

Reproduction, Embryonic Development, and Maternal Transfer of

Contaminants in the Amphibian Gastrophryne carolinensis

William Alexander Hopkins,1,2 Sarah Elizabeth DuRant,1,2 Brandon Patrick Staub,1 Christopher Lee Rowe,3

and Brian Phillip Jackson1,4

1Savannah River Ecology Laboratory, University of Georgia, Aiken, South Carolina, USA; 2Department of Fisheries and Wildlife

Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA; 3Chesapeake Biological Laboratory, 

University of Maryland Center for Environmental Science, Solomons, Maryland, USA; 4Departments of Chemistry and Earth 

Sciences, Dartmouth College, Hanover, New Hampshire, USA

Although many amphibian populations around the world are declining at alarming rates, the

cause of most declines remains unknown. Environmental contamination is one of several factors

implicated in declines and may have particularly important effects on sensitive developmental

stages. Despite the severe effects of maternal transfer of contaminants on early development in

other vertebrate lineages, no studies have examined the effects of maternal transfer of contaminants on reproduction or development in amphibians. We examined maternal transfer of contaminants in eastern narrow-mouth toads (Gastrophryne carolinensis) collected from a reference site

and near a coal-burning power plant. Adult toads inhabiting the industrial area transferred significant quantities of selenium and strontium to their eggs, but Se concentrations were most notable

(up to 100 μg/g dry mass). Compared with the reference site, hatching success was reduced by

11% in clutches from the contaminated site. In surviving larvae, the frequency of developmental

abnormalities and abnormal swimming was 55–58% higher in the contaminated site relative to

the reference site. Craniofacial abnormalities were nearly an order of magnitude more prevalent in

hatchlings from the contaminated site. When all developmental criteria were considered collectively, offspring from the contaminated site experienced 19% lower viability. Although there was

no statistical relationship between the concentration of Se or Sr transferred to eggs and any measure of offspring viability, our study demonstrates that maternal transfer may be an important

route of contaminant exposure in amphibians that has been overlooked. Key words: amphibian,

coal combustion, development, embryo, maternal transfer, mercury, reproduction, selenium,

strontium. Environ Health Perspect 114:661–666 (2006). doi:10.1289/ehp.8457 available via

http://dx.doi.org/ [Online 8 December 2005]
beetles. Unlike other anurans in the southeastern United States, the larvae of G. carolinensis

filter-feed plankton from the water column

(Pechmann 1994).

Toads were collected from two sites on

the Savannah River Site, near Aiken, South

Carolina. The contaminated site was the

D-area electric power generation facility,

which has associated with it at least 126 ha of

land affected by historical and current disposal

of sluiced coal combustion wastes (CCWs).

Current disposal of CCWs occurs in large settling basins, one of which has partially filled

and become extensively revegetated. The

revegetated portion of this CCW basin is centrally located within power plant disposal

operations and was the focal sampling area for

this study. The reference site was Ginger’s

Bay, a 1.5-ha Carolina bay (i.e., isolated freshwater wetland) located approximately 13.5 km

from the polluted site. The bay has been

extensively sampled for amphibians in the past

and has no deliberate anthropogenic input of

contaminants (Hopkins et al. 2000).

Collection, breeding, and assessment of

development. Animals used in this study were

treated humanely and with regard for the alleviation of suffering according to approved protocols of the University of Georgia’s Animal

Care and Use Committee. Adult male and

female G. carolinensis were collected during the

breeding season (22 May–7 July 2003) at both

sites using drift fences. Upon collection, adults

were transported to an array of outdoor mesocosms (n = 76) up to 3,000 L in volume. One

female and one or two males collected from the

same site were placed within each mesocosm

and allowed to breed. Each mesocosm was

slightly slanted such that one end of the mesocosm contained at least 10 cm well water, but

the remaining portion of the mesocosm’s interior base was dry and covered with leaf litter

and debris collected from the reference site.

Mesocosms were checked every morning for

breeding activity, and fresh egg masses were

removed immediately upon discovery.

Egg masses (n = 20 and 37, contaminated

site and reference site, respectively) were

returned to the laboratory, where they were

enumerated and a subsample of 100 fertile eggs

were set aside for assessments of development.

These 57 subsamples were then allowed to

develop in 3.8 L dechlorinated tap water (dissolved oxygen = 9.03 ± 0.10 mg/L; pH, 8.17 ±

0.02; conductivity = 142 ± 1.96 μS/cm) for

96 hr at 25°C. After 96 hr, hatchlings from

each subsample were counted to quantify

hatching success. Hatchlings were then placed

in a 60 × 15 mm glass Petri dish filled with

dechlorinated tap water.

Each hatchling was encouraged to swim

by gently prodding with a plastic pipette.

Swimming behavior was classified as abnormal

if individuals failed to respond after repeated

prods, failed to maintain upright posture, or

swam erratically in circles. Normal and

abnormal swimmers were separated and individuals were placed in 20-mL vials containing

MEMFA (0.1 M MOPS, pH 7.4, 2.0 mM

EGTA, 1.0 mM MgSO4, 3.7% formaldehyde). After 2 hr of fixation under continual

rotation, the MEMFA solution was completely

exchanged with 100% ethanol, and samples

were stored at 4°C for subsequent morphologic

assessments. Each hatchling was examined

under 6×–25× magnification for morphologic

abnormalities according to the methods of

Bantle et al. (1991) and ASTM (1998).

Morphologic abnormalities were classified as

edema or swelling, skin blistering, craniofacial

malformations, and/or four types of axial malformations (dorsal flexure, lateral flexure, wavy

tail, and axial shortening).

Trace element analysis. Immediately after

removing the 100 egg subsets from the

clutches for developmental assessments, we

froze the remaining eggs from 10 and 18 of

the clutches (contaminated and reference site,

respectively) at –60°C for subsequent chemical

analysis. We held the females that produced

these clutches for 48 hr after oviposition to

ensure that they were postabsorptive and then

anesthetized them before freezing (–60°C).

Adult toads and their eggs were later prepared

for contaminant analysis according to the

methods of Snodgrass et al. (2004, 2005).

We digested approximately 20–180 mg

lyophilized adult toad carcasses and eggs by

adding trace metal grade nitric acid (70%

HNO3, 2.5–5.0 mL) to samples before digestion in a microwave (MDS 2000; CEM

Corp., Matthews, NC) with heating steps of

60, 60, 70, and 80% microwave power for

10, 10, 15, and 20 min, respectively. After

HNO3 digestion, we added 1.0 mL hydrogen

peroxide to the samples and microwaved

them at the same power and duration as the

HNO3 digestion. After digestion, we brought

samples to a final volume of 10 mL with

18 MΩ deionized water. Soil and water samples were also collected (n = 3/matrix/breeding

site). Soil samples (~ 200 mg) were oven dried

before digesting with 9.0 mL HNO3 and

1.0 mL H2O2 as described above, and

brought to a final volume of 50 mL with

deionized water before analysis. Water samples (10 mL) were filtered (0.44 μm) and

acidified before analysis.

We performed element analysis (arsenic,

Cd, copper, iron, Hg, nickel, Se, strontium,

vanadium, and zinc) by inductively coupled

plasma mass spectrometry (Perkin Elmer,

Norwalk, CT) on samples diluted 1:10 with

deionized water. We calibrated the instrument daily using calibration standards covering a range of 1–500 μg/L prepared by serial

dilution of NIST traceable primary standards (National Institute of Standards and

Technology, Gaithersburg, MD). For quality

control purposes, we included certified reference material (TORT-2; National Research

Council of Canada, Ottawa, Canada) and

reagent blanks in the digestion and analysis

procedure. Mean percent recoveries for elements in tissue reference material ranged from

84 to 106%. Detection limits (micrograms

per liter) in water samples were as follows: As,

0.41; Cd, 0.09; Cu, 1.59; Fe, 2.40; Hg, 0.11;

Ni, 0.25; Se, 0.13; Sr, 0.03; V, 0.06; Zn,

5.13. Detection limits (nanograms per gram)

in soil and tissue were as follows: As, 0.13;

Cd, 0.17; Cu, 0.55; Fe, 19.67; Hg, 0.11; Ni,

2.21; Se, 0.26; Sr, 0.14; V, 0.12; Zn, 5.15.

Concentrations of elements in tissues and

soils are reported on a dry mass basis.

Statistical analysis. In all statistical comparisons, significance was assessed at p < 0.05,

but in cases where multiple comparisons were

made, we adjusted p-values using a sequential

Bonferroni adjustment to maintain an experiment wide error rate of α = 0.05. We used log

or angular transformations to better approximate assumptions of parametric statistical

models.

Trace element concentrations in water,

soil, adult females, and freshly laid eggs were

compared between sites using either a series of

analyses of variance (ANOVAs) or a multivariate analysis of variance (MANOVA). For soils

and water, small sample sizes (n = 3/site) prohibited us from using multivariate analyses.

Therefore, ANOVAs were used for comparison of 10 elements between sites. Only one

soil concentration (Hg in a single reference

soil sample) was below the detection limit

(BDL) and was replaced with 50% of the analytical detection limit. For tissues from adults,

> 50% of Ni and As concentrations were

BDL; therefore these elements were not compared between sites. Hg and V concentrations

were BDL in two and eight adults, respectively, collected at the reference site; these values were replaced with 50% of the detection

limit for statistical comparisons. Adult tissue

concentrations were then compared by

MANOVA. For egg masses, > 50% of Ni, As,

Cd, and Hg concentrations were BDL and

were therefore not compared statistically. One

egg mass collected from the contaminated site

had low V concentrations and was replaced

with 50% of the detection limit before inclusion in a multivariate model. We used logistic

regression to model how Se concentration in

eggs influenced the probability of Hg transfer

to eggs at levels above our detection limits. To

examine the relationship between contaminant concentrations in female carcasses and

their eggs, we used linear regression of logtransformed data both within and between

sites. We also used Pearson correlation coefficients to describe relationships between elements that were maternally transferred.

Hopkins et al.

662 VOLUME 114 | NUMBER 5 | May 2006 • Environmental Health Perspectives
For elements that were maternally transferred, total preovipositional body burdens

(micrograms) were reconstructed using individual tissue (egg and whole body) element

concentrations and tissue dry masses. Total

body (egg + carcass) burdens (dry) were compared between sites using ANOVA. The percentages of total body burdens deposited in

eggs were then calculated and compared using

ANOVA.

The mass and snout–vent length (SVL) of

females was compared between sites using

MANOVA, and Pillai’s trace statistic was

used to assess statistical significance. Clutch

size of females was compared between sites

using ANOVA. Body size was not included as

a covariate in this analysis because there was

no relationship between female size and

clutch size.

The percentages of individuals from the

57 clutch subsets that hatched, exhibited

abnormal morphology, and swam abnormally

were compared between sites using a series of

Wilcoxon two-sample tests. In an effort to

estimate the overall viability of each clutch, we

then combined the number of individuals not

hatching, the number of individuals exhibiting

morphologic abnormalities, and the number

of individuals exhibiting abnormal swimming

behavior (but were otherwise morphologically

normal) to calculate a viability index. The percentage of viable hatchlings produced from

the clutch subsets were compared between

sites using a Wilcoxon two-sample test.

To determine whether there was a

relationship between maternal transfer of contaminants and developmental parameters

(hatching, swimming, malformations, and viability), we conducted a series of linear regressions. Iterations of these models were

conducted using data only for the contaminated site as well as for both study sites pooled.

Results

Contaminant concentrations. As we expected,

soils from the contaminated site had significantly higher concentrations of several trace

elements compared with the reference site

(Table 1). Cu, Ni, Se, Sr, and V concentrations were three to eight times higher in contaminated soils than in reference soils (in all cases

p ≤ 0.001). Arsenic concentrations were more

than 60 times higher in contaminated soils

compared with reference soils (p < 0.001). In

contrast, soil concentrations of Hg, Zn, and

Fe were similar between sites (p ≥ 0.08).

Although Cd concentrations were twice as

high in contaminated soils than in reference

soils (p = 0.027), the difference was not statistically significant after Bonferroni adjustment.

Water concentrations of contaminants

also varied greatly between sites (Table 1).

Cu, Fe, and Zn concentrations were significantly higher in the reference site (in all cases

p ≤ 0.004). In contrast, dissolved concentrations of As, Ni, Se, Sr, and V were higher in

the contaminated site compared with the reference site (in all cases p ≤ 0.005). Cd concentrations were similar in the water at each

site (p = 0.016), and Hg concentrations were

BDL in all water samples.

Adult females collected at the contaminated

site had significantly higher whole-body concentrations of trace elements compared with

females from the reference site (MANOVA,

F8,19 = 12.33; p < 0.001). Most notably, accumulation of V, Sr, and Se was 3, 7, and

22 times higher in females from the contaminated site compared with the reference site

(Table 2). However, the opposite was true for

Hg. Females collected from the reference site

had whole-body Hg concentrations that were

60% higher than in females from the contaminated site.

Of the three elements (Se, Sr, and V)

accumulated by females at the contaminated

site, Sr and Se were also transferred to their

eggs at greater concentrations than to reference eggs (p ≤ 0.01; Table 2). Concentrations

of Se and Sr in eggs were positively correlated

(r = 0.682). Se concentrations in eggs ranged

from 5.37 to 99.18 μg/g dry mass and were

strongly influenced by the concentration of Se

in female tissues (r2 = 0.94, p < 0.001;

Figure 1A). The relationship between Sr concentration in females and their eggs was also

Maternal transfer of contaminants in amphibians

Environmental Health Perspectives • VOLUME 114 | NUMBER 5 | May 2006 663

Table 1. Elemental composition of water and soil collected from the contaminated and reference sites, and

laboratory water used for rearing embryos for 96 hr.

Site water (μg/L) Soil (μg/g dry mass)

Element Lab water (μg/L) Reference Contaminated Reference Contaminated

As BDL 1.10 ± 0.03 117.70 ± 5.93 1.74 ± 0.23 114.96 ± 7.90

Cd 0.60 ± 0.01 0.55 ± 0.01 0.71 ± 0.04 0.58 ± 0.11 1.17 ± 0.07

Cu 2.81 ± 0.49 9.39 ± 0.76 4.61 ± 0.39 30.66 ± 4.08 100.17 ± 5.28

Fe 18.10 ± 1.14 736 ± 20.87 111 ± 5.84 11,399 ± 5,326 25,224 ± 3,084

Hg BDL BDL BDL 0.11 ± 0.03 0.20 ± 0.01

Ni 0.49 ± 0.03 1.53 ± 0.16 2.70 ± 0.09 8.94 ± 0.60 46.88 ± 1.38

Se 0.28 ± 0.04 0.19 ± 0.03 3.93 ± 0.14 1.64 ± 0.10 8.25 ± 0.64

Sr 3.38 ± 0.14 11.77 ± 0.36 407.15 ± 22.39 28.88 ± 10.12 222.50 ± 11.20

V BDL 0.26 ± 0.01 8.34 ± 0.36 13.99 ± 1.25 70.15 ± 1.76

Zn 24.83 ± 4.62 182.05 ± 12.14 41.47 ± 5.25 1047.63 ± 139.58 919.08 ± 13.25

Data are presented as mean ± 1SE; n = 3 samples/matrix/site.

Table 2. Elemental composition of postovipositional female G. carolinensis and their eggs from reference and

contaminated sites in South Carolina.

Females (μg/g dry mass) Eggs (μg/g dry mass)

Element Reference Contaminated Reference Contaminated

As BDL BDL 0.74 ± 0.56 BDL

Cd 0.34 ± 0.03 0.44 ± 0.21 BDL BDL

Cu 4.09 ± 0.17 4.61 ± 0.99 9.30 ± 2.06 7.01 ± 3.47

Fe 442.93 ± 37.50 448.72 ± 109.40 278.29 ± 34.91 195.29 ± 171.35

Hg 0.31 ± 0.04 0.19 ± 0.13 0.11 ± 0.02 BDL

Ni BDL BDL BDL BDL

Se 1.85 ± 0.14 42.40 ± 38.78 1.63 ± 0.12 43.96 ± 37.62

Sr 44.22 ± 3.24 324.23 ± 303.43 2.68 ± 0.23 4.14 ± 1.73

V 0.09 ± 0.01 0.29 ± 0.22 0.49 ± 0.05 0.43 ± 0.37

Zn 173.97 ± 12.17 181.05 ± 59.72 223.49 ± 41.78 222.71 ± 138.93

Data are presented as mean ± 1SE; n = 10 and 18 contaminated and reference, respectively.

2.4 A B

2.0

1.6

1.2

0.8

0.4

0.0

–0.4

Log [Se] females Log [Sr] females

1.0

0.8

0.6

0.4

0.2

0.0

Log [Se] eggs

Log [Sr] eggs

0.0 0.4 0.8 1.2 1.6 2.0 2.4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Figure 1. Relationship between Se (A) and Sr (B) concentrations (μg/g dry mass) in female G. carolinensis

and their eggs. Females were collected from both contaminated and reference sites but bred under controlled, uncontaminated conditions. Female concentrations were determined 48 hr after oviposition. (A) y =

1.0255x – 0.0448, r

2 = 0.9415. (B) y = 0.2634x – 0.0237, r2 = 0.4026. 
strong (r2 = 0.40, p < 0.001; Figure 1B) but

was more variable than the relationship

observed for Se. In contrast, maternal transfer

of V was low and similar between sites

(p = 0.14). 

Although Hg concentrations were generally low in all samples, Hg concentrations in

eggs from contaminated females spanned

orders of magnitude, and mean concentrations were much lower than in eggs from reference females. However, 7 of 10 egg masses

from the contaminated site had Hg concentrations BDL, preventing rigorous statistical

comparisons between sites. Logistic regression

indicated that the Se concentration in eggs

strongly influenced (p < 0.01) the probability

of Hg being transferred to eggs at concentrations above our detection limits (Figure 2).

Reconstruction of preovipositional body

burdens and the percentage of this burden lost

at oviposition revealed different partitioning

patterns for Se and Sr. Preovipositional body

burdens of Se were > 25 times higher in

females from the contaminated site compared

with the reference site (hereafter all data are

presented as mean ± 1 SE: 8.45 ± 2.39 vs. 0.33

± 0.02 μg Se, respectively; F = 96.83,

p < 0.001). However, the proportion of the

total body Se burden that was transferred to

eggs was high and equivalent between sites

(53–54%, p = 0.96; Figure 3). In contrast, preovipositional body burdens of Sr were approximately 10 times higher in females from the

contaminated site compared with the reference

site (30.23 ± 9.03 vs. 3.83 ± 0.26 μg Sr, respectively; F = 30.04, p < 0.001), but the total

amount of Sr maternally transferred was similar between sites (0.40 ± 0.06 vs. 0.29 ±

0.03 μg Sr, respectively; F = 3.16, p = 0.09).

Thus, the proportion of total body Sr burden

that was transferred to eggs was low (~ 3–8%)

but was more than two times higher in females

from the reference site compared with the contaminated site (F = 16.95, p < 0.001; Figure 3).

Reproduction and development. Adult

females collected from the reference site (SVL

= 29.1 ± 0.26 mm; mass = 2.19 ± 0.07 g) were

significantly larger (Pillai’s trace = 0.246;

F2,54 = 8.83; p < 0.001) than adult females

from the contaminated site (SVL = 27.4 ±

0.39 mm; mass = 1.76 ± 0.07 g). However,

clutch size was similar between sites (F = 0.16;

p = 0.69; 674 ± 50 and 598 ± 41 eggs, reference and contaminated site, respectively). No

relationship was detected between body size

and clutch size for reference site only

(r2 < 0.001, F < 0.01, p = 0.95), contaminated

site only (r2 = 0.105, F = 2.12, p = 0.16), or

both sites combined (r2 = 0.012, F = 0.65,

p = 0.42).

Embryonic development was impaired in

eggs originating from the contaminated site.

Hatching success was significantly reduced in

eggs from females collected in the contaminated site compared with the reference site

(percent hatching = 83 ± 6.8 vs. 93 ± 2.6,

respectively; p = 0.003). The frequency of

developmental abnormalities in hatchlings

was significantly higher in the contaminated

site than in the reference site (clutch mean

percent abnormal = 16.7 ± 5.7 vs. 10.5 ± 3.6,

respectively; p = 0.019). Axial malformations

were the most commonly observed abnormalities in both sites (Table 3). Craniofacial

abnormalities were rare in reference hatchlings but were quite common among abnormal larvae from the contaminated site. Many

individuals that exhibited morphologic

abnormalities also displayed impaired swimming behavior. Of the 574 malformed hatchlings from the 57 clutches examined, 88%

exhibited abnormal swimming behavior. In

contrast, only 3% of 4,528 individuals with

normal morphology swam abnormally.

Frequency of abnormal swimming was higher

among hatchlings from the contaminated site

compared with the reference site (percent

abnormal swimming: 19.1 ± 6.3 vs. 12.4 ±

4.2, respectively; p = 0.009). When hatching,

morphology, and swimming were considered

together as a viability index, the percentage of

viable offspring was significantly higher in the

reference site (82.2 ± 4.8) compared with the

contaminated site (66.6 ± 8.2; p = 0.003).

Based on our statistical models, there was

no relationship between Se concentration in

eggs and hatching success, malformation frequency, percentage of individuals exhibiting

abnormal swimming behavior, or total viability, regardless of whether the contaminated

site was considered independently or if the

sites were pooled (in all cases r2 < 0.20,

p > 0.19). Similarly, Sr concentration in eggs

was not statistically related to any of the

measured developmental parameters (in all

cases r2 < 0.08, p > 0.14).

Discussion

Our study definitively demonstrated that

amphibians, like other vertebrates, are capable

of transferring potentially harmful concentrations of inorganic contaminants to their eggs.

Most notably, we described maternal transfer

of high concentrations of Se that were strongly

related to the concentration of Se accumulated

in female tissues. In addition, we documented

a significant reduction in offspring viability in

frogs collected near a coal-burning power

plant compared with a reference site. Our

findings highlight the importance of further

research on maternal transfer of contaminants

in amphibians, especially as it relates to viability of natural populations.

Hopkins et al.

664 VOLUME 114 | NUMBER 5 | May 2006 • Environmental Health Perspectives

0

Contaminated

Reference

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Egg Se concentration (μg/g dry mass)

Egg Hg concentration (μg/g dry mass)

40 60 80 100

1.0

0.8

0.6

0.4

0.2

0.0

Probability of Hg transfer

20

Figure 2. Relationship between Se and Hg concentrations maternally transferred to eggs. The solid

line represents the probability (logistic regression

model) of transferring Hg to eggs at concentrations

above detection limits (0.11 ng/g).

9

8

7

6

5

4

3

2

1

0

Contaminated Reference Contaminated Reference

35

30

25

20

15

10

5

0

Se burden (μg)

Sr burden (μg)

53%

54%

3%

8%

Eggs

Females

A B

Figure 3. Total body burden (μg) of Se (A) and Sr (B) in female G. carolinensis collected from both contaminated and reference sites. Total body burden is partitioned between eggs and the postovipositional female

carcass. Sr in reference eggs is not visible on the graph because these eggs only contained an average of

0.29 μg Sr. Percentages above bars signify the proportion of the total body burden that was transferred to

eggs at oviposition.

Table 3. Comparison of specific morphologic abnormalities among abnormal G. carolinensis hatchlings

collected from females from contaminated and reference sites.

Percent abnormal individualsa

Site Edema/swelling Blisters Craniofacial Axial

Reference (n = 37 clutches) 17.5 1.8 4.6 84.0

Contaminated (n = 20 clutches) 22.8 0 38.8 61.6

aCumulative percentage of abnormalities exceeds 100% because many individuals displayed multiple morphologic

abnormalities.
Maternal transfer. Although females

inhabiting the contaminated site accumulated

several trace elements in their tissues, only Se

and Sr were maternally transferred to eggs in

appreciable quantities. Unlike some lipophilic

compounds, Se and Sr were likely transferred

from mother to offspring due to their substitution for sulfur and calcium (respectively) in

egg components (e.g., Kroll and Doroshov

1991; Wysolmerski 2002). Because Se is also

an essential micronutrient, small additional

quantities of this element may have been

transferred to eggs as an active constituent of

important seleno-proteins. Maternal transfer

of Sr to eggs was much more conservative

than Se transfer, resulting in egg Sr concentrations 20–80 times lower than Sr concentrations in the female carcass. In contrast,

maternal transfer of Se resulted in egg concentrations similar to concentrations in the

female carcass. Functional relationships

between Se concentrations in female carcass

and eggs have not been well described in the

literature for fish or other wildlife. However,

comparison of Se concentrations in whole

postpartum female mosquitofish and their fry

indicated that offspring had higher concentrations of Se than their mothers (Saiki et al.

2004). More directly analogous comparisons

have been made for Hg; strong linear relationships have been observed between Hg

concentrations in female fish and their eggs

(Hammerschmidt and Sandheinrich 2005;

Hammerschmidt et al. 1999).

Partitioning of Se and Sr burden (micrograms) between somatic tissues and eggs was

different for each element. Regardless of Se

exposure history, female frogs partitioned

approximately 53% of their total Se burden

into their eggs at oviposition. In contrast, the

percentage of Sr body burden transferred to

eggs was much lower and dependent upon

exposure history. Frogs from the reference site

proportionally transferred more than twice as

much of their Sr burden to eggs compared

with frogs from the contaminated site (8% vs.

3%, respectively). The observation that Sr

transfer only ranged from 0.29 to 0.40 μg

despite a 10-fold range in total body burdens

between sites suggests that the upper limit of Sr

transfer to eggs may have been approached. In

contrast, there was no evidence that maternal

transfer of Se was asymptotic. Such partitioning relationships have not been well described

for Se or Sr in wildlife. However, a recent

study provided a detailed description of Se partitioning among organs in male and female

fence lizards (Sceloporus occidentalis) fed low

(1 μg/g) and high (15 μg/g) Se concentrations

(Hopkins et al. 2005). Regardless of the

amount of Se fed to lizards, females partitioned

approximately 33% of their total Se burden to

ovaries, most of which had produced enlarged,

yolked follicles at the time they were examined.

Because lizards were not permitted to oviposit,

it is not known what the total partitioning of

Se would have ultimately been to eggs.

However, the facts that both G. carolinensis

and S. occidentalis transferred a constant proportion of their total Se burden to eggs or follicles (~ 53% and 33%, respectively) and that

the relationship between Se concentrations in

female G. carolinensis and their eggs was nearly

1:1 suggest that the amount of Se ultimately

transferred to young is largely governed by the

mass of reproductive tissue produced. Indeed,

the dry mass of frog eggs (present study) and

lizard follicles (Hopkins et al. 2005) represented 51 ± 0.03% and 25 ± 0.02% of total

body dry mass. This observation suggests that

the reproductive strategy employed by organisms (e.g., large vs. small clutches) may have

important implications for the quantity of Se

transferred during each reproductive event.

Se concentrations in female G. carolinensis

greatly exceeded concentrations measured in

other amphibian species previously studied at

the same contaminated site, but Sr concentrations were similar among adults of all species

examined heretofore. For example, adult southern toads (Bufo terrestris) sampled near the

power plant had mean Se and Sr concentrations

of 17 and 387 μg/g, respectively (Hopkins et al.

1998). More recently, adult B. terrestris and

leopard frogs (Rana sphenocephala) sampled

from a contaminated downstream area had

mean Se and Sr concentrations up to 7 and

325 μg/g, respectively (Roe et al. 2005). It is

unclear why G. carolinensis accumulates such

high concentrations of Se (42 μg/g) while

simultaneously accumulating comparable concentrations of Sr (324 μg/g) compared with

other species studied. The most parsimonious

explanation may involve the unusual ecology

of G. carolinensis, which is highly fossorial and

feeds primarily on ants, termites, and beetles. It

is possible that these habits result in greater

trophic exposure to Se than that experienced

by adult B. terrestris or R. sphenocephala.

Regardless of the reason for the observed differences in Se accumulation among various

species, high interspecies variability highlights

the importance of sampling multiple species of

amphibians with diverse ecologies when trying

to assess risk in contaminated habitats.

Maternal transfer of Se by G. carolinensis

also greatly exceeded the Se concentrations

maternally transferred by female reptiles,

birds, and fish studied at the same site.

American alligators (Alligator mississippiensis)

and common grackles (Quiscalus quiscala)

both transferred Se concentrations of 6–7 μg/g

to their eggs, but neither of these studies measured female Se concentrations or rigorously

addressed the effects of transfer on reproduction and development (Bryan et al. 2003; Roe

et al. 2004). Adult female mosquitofish

(Gambusia holbrooki) had Se concentrations of

12 μg/g in their carcasses after giving birth to

offspring with Se concentrations of 16 μg/g

(Staub et al. 2004). Reproductive success and

offspring viability in mosquitofish were not

affected by maternal transfer of Se, an observation consistent with the known tolerance of

mosquitofish to Se (Cherry et al. 1976; Lemly

1985; Saiki et al. 2004).

Finally, patterns of accumulation and

maternal transfer of Hg were noteworthy. In

contrast to other elements, concentrations of

Hg were higher in females and eggs from the

reference site compared with the contaminated site. Logistic regression suggested that

females that transferred concentrations of Se

≥ 20 μg/g to their eggs had very little (i.e.,

concentrations BDL) Hg in their eggs. We

hypothesize that high concentrations of Se in

females from the contaminated site may have

inhibited Hg accumulation in female tissues,

resulting in low concentrations of Hg available

for subsequent transfer to offspring. Although

many studies have demonstrated antagonistic

bioaccumulation patterns between Se and Hg

(Cuvin-Aralar and Furness 1991; Southworth

et al. 2000), we know of few studies that have

highlighted the important implications of

Se–Hg antagonism for maternal transfer of Hg

(Heinz and Hoffman 1998).

Reproductive and developmental effects. In

ecologic terms, maternal transfer of contaminants is important because of effects on reproductive success and ultimately, population

viability. We found that breeding adults at the

contaminated site experienced a 19% reduction in the percentage of their offspring that

were viable compared with adults from the

reference site. The abnormalities that contributed to this reduction were consistent with

those in fish exposed to seleniferous discharge

from other power plants (Lemly 1996).

A perplexing finding from this study was

the lack of a relationship between concentrations of Se or Sr transferred to eggs and

indices of offspring viability. It is not known

whether the concentrations of Sr observed in

eggs should be expected to cause abnormalities because little is known about the effects of

Sr on early embryonic development in

wildlife, although injection studies suggest

that high concentrations of Sr can be embryotoxic (Ridgway and Karnofsky 1952). In contrast, Se in the contaminated site was

transferred at levels known to be embryotoxic

to fish and birds (4–16 μg/g dry mass) (Lemly

1996; Ohlendorf 2003). At the highest Se

concentrations observed in frog eggs (i.e.,

80–100 μg/g dry mass) offspring viability was

quite variable but as high as 95%. There are

several possible explanations for not detecting

a relationship between Se transfer and offspring viability. From a methodologic perspective, we measured contaminant concentrations

in 28 clutches of eggs, but only five of these

Maternal transfer of contaminants in amphibians

Environmental Health Perspectives • VOLUME 114 | NUMBER 5 | May 2006 665
Hopkins et al.

666 VOLUME 114 | NUMBER 5 | May 2006 • Environmental Health Perspectives

clutches contained Se concentrations > 20 μg/g

dry mass. Thus, our statistical power for

detecting functional relationships between concentrations and effects was probably limited

within the range of concentrations where

effects should be most predominant. From a

biologic perspective, it is possible that G. carolinensis is more tolerant of Se than most fish

and birds studied to date. Such tolerance

would not be unprecedented; mosquitofish

and cutthroat trout appear extraordinarily

tolerant of Se embryotoxicity, remaining

asymptomatic even after transferring Se concentrations of approximately 20–80 μg/g dry

mass to their young (Kennedy et al. 2000;

Saiki et al. 2004). Alternatively, Se partitioning

among egg components could have obfuscated

our results. Frogs produce a gelatinous jelly

coat around their eggs that contains a variety of

sulfur-containing mucoproteins and mucopolysaccharides that provide important structural barriers around the embryo essential for

fertilization and hatching but are not believed

to be nutritive (Duellman and Trueb 1986).

Our contaminant analysis of eggs included this

jelly coat and may therefore include some Secontaining compounds that were not bioavailable to the developing embryo. Last, we

cannot rule out the possibility that other contaminants may have been present at the industrial site and contributed to our findings.

Although metals and metalloids are believed to

be the key toxic constituents of CCW (Rowe

et al. 2002), organic contaminants such as

polycyclic aromatic hydrocarbons could occur

in this complex waste stream. However, available information suggests that the concentrations of these organic materials in CCWs are

extremely low [U.S. Environmental Protection

Agency (EPA) 1999].

Finally, our findings shed further insight

into the ecologic and human health hazards

posed by disposal of solid wastes from coalfired power plants. Many previous studies

have demonstrated that amphibian larvae

developing in sites contaminated by CCWs

bioaccumulate high concentrations of contaminants and exhibit developmental, physiologic,

and behavioral abnormalities (reviewed by

Rowe et al. 2002). Laboratory studies and

transplantation of larvae from reference sites

to CCW-contaminated sites confirm these

accumulation patterns and reveal additional

effects on growth, survival, and key life history

characteristics (Rowe et al. 2002; Snodgrass

et al. 2004, 2005). Taken together, these

experiments also suggest that the severity of

these effects may be exacerbated by confounding ecologic factors (e.g., overwintering,

per capita resource availability) in the field.

The observations that adult female anurans

inhabiting these sites can accumulate high

concentrations of Se and Sr in their tissues,

transfer Se and Sr concentrations to their offspring, and exhibit reductions in offspring viability raises many new questions regarding the

impact of aquatic CCW-disposal practices on

amphibians. For example, because our study

only tracked development for the first 96 hr,

the possibility remains that latent effects of

maternal transfer may become evident later in

development (e.g., Heinz et al. 1989). Further

studies designed to track amphibian development through metamorphosis are clearly

needed to assess the consequences of maternal

transfer to reproductive success and ultimately

as one of many important contributing factors

to amphibian declines.

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