Water clarity, maternal behavior, and physiology
combine to eliminate UV radiation risk to
amphibians in a montane landscape
Wendy J. Palena,1and Daniel E. Schindlerb
aEarth to Ocean Research Group, Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada V5A 1S6; andbSchool of Aquatic and
Fishery Sciences, University of Washington, Seattle, WA 98195-5020
Edited by Craig E. Williamson, Miami University, Oxford, OH, and accepted by the Editorial Board April 21, 2010 (received for review November 10, 2009)
Increasing UV-B radiation (UV-B; 290–320 nm) due to stratospheric
phibians for nearly 2 decades. Yet, the likelihood that UV-B can in-
declines has not yet been evaluated. A key limitation has been in
relating results from individual sites to the effect of UV-B for popu-
lations distributed across heterogeneous landscapes. We measured
critical embryonic exposures to UV-B for two species of montane
amphibians with contrasting physiological sensitivities, long-toed
salamander (Ambystoma macrodactylum) and Cascades frog (Rana
cascadae), at field sites spanning a gradient of UV-B attenuation in
portion of embryos exposed to harmful UV-B across a large number
of breeding sites. By combining surveys of the incubation timing,
incident UV-B, optical transparency of water, and oviposition depth
sive assessment of the risk posed by UV-B for montane amphibians
of the Pacific Northwest. We found that only 1.1% of A. macrodac-
tylum and no R. cascadae embryos across a landscape of breeding
sites are exposed to UV-B exceeding lethal levels. These results em-
phasize that accurately estimating the risk posed by environmental
stressors requires placing experimental results in a broader ecolog-
ulations distributed across natural landscapes.
amphibian declines|ultraviolet radiation|risk analysis|dissolved organic
sity loss. Declining amphibian populations in particular have been
heralded as sentinels of subtle environmental degradation in oth-
erwise intact systems, and have been a focal point of intense study
for more than 20 years (1–6). Although diverse causes have been
proposed, a leading hypothesis for amphibian declines in western
North America has been that increasing UV-B radiation (290–320
nm wavelengths) due to anthropogenic ozone depletion (7–9) has
increased mortality rates (10, 11). Assessments of the impact of
ambient UV-B exposure have been conducted for more than 50
larval survival under a variety of laboratory and field conditions
(reviewed in refs. 11–14). Despite this level of investigation, few if
of studies conducted at individual experimental sites to the broad
spatial and temporal scales relevant to amphibian population dy-
namics (5, 15–17). A critical limitation preventing such analyses is
the lack of quantitative data regarding actual UV-B exposures
across the range of conditions experienced by individuals within
a population, coupled to experimentally identified thresholds of
UV-B over time at multiple sites in the field is currently limited by
instrumentation and expense,site-level UV-Bexposureshave been
estimated from the primary features of the terrestrial and aquatic
pecies declines and extirpations in seemingly well-protected
habitats represent an alarming component of global biodiver-
environment that affect UV-B transmission, including geographic
location, elevation, cloud cover, topographic and vegetative shad-
ing, and the attenuation of UV-B in water (16, 18). However, an
additionallimitation ofeven these sophisticatedmodelsisthe need
to incorporatenot onlyvariation amongsites,butalsothe variation
in UV-B exposure among individuals within a site that may result
from variation in individual behaviors (19, 20).
environments is strongly influenced by the concentration of colored
dissolved organic matter (DOM), which explains 85–92% of the
variation in the rate at which UV-B is attenuated (Kd) with water
depth (21, 22). Colored DOM represents a suite of compounds de-
23) and is transported to surface waters in montane areas by runoff
associated with precipitation and snowmelt, resulting in seasonal
type of watershed vegetation and soil generates tremendous site-to-
site heterogeneity in the UV-B environment experienced by fresh-
water taxa (26–28); thus, DOM serves to link the dynamics of ter-
restrial vegetation and soils to the mosaic of exposures experienced
by amphibians in aquatic ecosystems.
Landscape-scale variation in water transparency among in-
dividual lakes and ponds is compounded with variation in expo-
sure among individuals within each site. Differences in UV-B
exposure of embryos within sites is generated by maternal ovi-
water depths and in microhabitats with differing exposure to light
surfaces). For some montane amphibians, maternal oviposition
in reduced embryonic UV-B exposures in highly transparent sites
by laying eggs deeper or in shaded microhabitats (19). As such,
simple extrapolations from laboratory-based physiological assays
or assessments of field exposures will overestimate UV-B risk if
such behaviors are not accounted for.
We evaluated the context dependency of UV-B effects for the
embryonic survival of two montane amphibians of the Pacific
Northwest, Cascades frogs (Rana cascadae) and long-toed sala-
manders (Ambystoma macrodactylum), by experimentally esti-
variation in DOM concentrations and patterns of maternal ovi-
position behavior. Importantly, these two species exhibit con-
trasting physiological sensitivity to UV-B (10, 19), with Cascades
frogs among the least sensitive of Pacific Northwest species and
Author contributions: W.J.P. and D.E.S. designed research; W.J.P. performed research;
W.J.P. analyzed data; and W.J.P. and D.E.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. C.E.W. is a guest editor invited by the Editorial
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| May 25, 2010
| vol. 107
| no. 21
long-toed salamanders among the most sensitive. We used field
experiments in breeding sites spanning a natural gradient of water
transparency to UV-B (i.e., DOM concentration) to identify the
cumulative exposure to UV-B associated with significant embry-
onic mortality for each species. We then used the experimental
results to evaluate the risk posed by UV-B to embryos of each
species across a large number of breeding sites. Our assessment
of risk is based on estimates of the cumulative UV-B exposure
experienced by individual embryos in each of 22 breeding sites
(R. cascadae, n = 107,352; A. macrodactylum, n = 4,159). These
exposures were estimated by combining site-specific estimates of
incident UV-B, seasonal variation in the UV-B transmission
properties of water (Kd), measurement of naturally occurring
embryos and their microhabitats (depth and light exposure), and
duration of incubation. By comparing the level of UV-B–induced
mortality from the field experiments with natural field exposures,
we are better able to estimate the frequency with which lethal
conditions are experienced by individuals distributed across this
heterogeneous montane landscape.
Field Experiments. To evaluate the context dependency of UV-B
effects for R. cascadae and A. macrodactylum, we estimated the sur-
vival of embryos incubated in near-ambient and reduced–UV-B
environments at seven different breeding sites in Olympic National
Park, Washington, spanning a gradient of optical transparency to
and two sites in common). Embryos were incubated at 10 cm below
the water surface. The embryos in near-ambient UV-B exhibited
significantly higher mortality than those shielded from UV-B (6.7%
transparent sites (Table S1). More specifically, the difference in sur-
vival between UV-B–shielded and –exposed treatments was directly
related to the cumulative UV-B experienced by embryos in the ex-
posed treatment over the course of the incubation period. UV-B–
exposed embryos suffered significantly higher mortality at UV-B
for R. cascadae (Fig. 1). The higher UV-B exposures tolerated by
R. cascadae embryos compared with A. macrodactylum embryos
confirm differences in physiological sensitivity documented under
controlled laboratory exposures (10, 19).
UV-B Risk Assessment. To estimate the risk posed by UV-B expo-
sure for R. cascadae and A. macrodactylum embryos at broader
the depth distribution of embryos and their exposure to light,
seasonal timing and duration of incubation, and water trans-
parency at 22 breeding sites for each species spanning a gradient
of UV-B transparency. To accomplish this, we combined the cu-
mulative UV-B exposure distributions estimated for each survey
site with the results of the egg incubation experiments for each
of A. macrodactylum embryos encounter cumulative UV-B
exposures that exceed the levels associated with significant mor-
tality in the experiments (22,918 KJ·m−2for R. cascadae and
10,534 KJ·m−2for A. macrodactylum) (Fig. 2). To account for the
uncertainty in defining exact thresholds of exposure associated
Mean difference % survival
0 5000100001500020000 25000
Total UV-B exposure (KJ.m-2)
(Upper) and A. macrodactylum (Lower) incubation experiments. Data are
presented as effect sizes, calculated as the average difference in percent
survival between embryos incubated in reduced and near-ambient UV-B
environments at a water depth of 10 cm at each site (± SE). Asterisks denote
effect sizes significantly different from 0 (α = 0.05).
Summary of cumulative UV-B exposure estimates for R. cascadae
Total UV-B exposure (KJ*m-2)
107,352 embryos) and A. macrodactylum (Lower; 4,519 embryos) embryos sur-
veyed across 22 breeding sites for each species. Thresholds for both panels
correspond to cumulative exposures received in field experiments that had no
A. macrodactylum, these values were 11.2% and 1.1%, respectively. Ten em-
bryos of A. macrodactylum had an estimated UV-B exposure exceeding the
range of the x-axis; these are denoted by (10) just beyond the x-axis.
Total cumulative UV-B exposure estimates for all R. cascadae (Upper;
| www.pnas.org/cgi/doi/10.1073/pnas.0912970107Palen and Schindler
with significant mortality, we also estimated risk across the survey
sites using a more conservative threshold based on the highest
UV-B exposure from the experimental incubations that resulted
in no difference in survival between UV-B treatments (10,534
KJ·m−2for R. cascadae vs. 5,321 KJ·m−2for A. macrodactylum).
Using these lower thresholds, we estimate that 5.7% of R. cas-
cadae embryos and 11.2% of A. macrodactylum embryos experi-
We also evaluated how the median UV-B exposure received by
(Kd) at each site, and found a pattern of increasing exposure with
increasing water clarity for R. cascadae (Fig. 3 Top) and a pattern
forA.macrodactylum,because thelowest median exposuresoccur
in sites with the greatest water transparency to UV-B (Fig. 3 Bot-
R. cascadae, but not of A. macrodactlyum.
Our results demonstrate that very few R. cascadae and A. mac-
rodactylum embryos are exposed to lethal levels of UV-B across
a montane landscape of the Pacific Northwest. Whereas critical
UV-B levels are expected to be encountered by some embryos, we
estimate that the vast majority are exposed to only very low levels
of UV-B (<2,500 KJ·m−2) over the incubation period (R. casca-
dae, 53%; A. macrodactylum, 80%). This pattern of low exposure
is driven by a combination of factors that ameliorate the poten-
tially high levels of UV-B otherwise present in montane envi-
ronments, including the optical properties of water at each site
(Kd) and variation in the depth and light exposure of embryos
(maternal oviposition behavior). As UV-B transparency increases
across sites, R. cascadae embryos are exposed to higher median
cumulative UV-B, whereas A. macrodactylum median exposures
are highest at intermediate levels of UV-B transparency (Kd)
(Fig. 3). This pattern confirms an earlier observation that ovipo-
sition by adult female R. cascadae is relatively static across sites,
with egg masses generally deposited in full sunlight at a water
depth of ∼10 cm (19). In contrast, oviposition site choice by adult
female A. macrodactylum is known to correlate with site level
water transparency to UV-B, with nearly 100% of embryos laid in
shaded areas of sites with the highest UV-B penetration (small
Kd) (19). Given the high physiological sensitivity of A. macro-
dactylum embryos, this pattern suggests the possibility of behav-
ioral avoidance in sites that might otherwise be characterized as
having lethal UV-B exposures. Our results support the conclusion
that variation in water transparency combined with patterns of egg-
laying serve to reduce A. macrodactylum UV-B exposures to below
Our approach to estimating in situ cumulative UV-B expo-
sures allows us to evaluate the specific contribution of oviposi-
tion behavior and site-level water transparency (Kd) to each
species’ exposure estimates. For example, if we assumed that
oviposition was constant across sites, with all embryos exposed to
direct sunlight near the water surface, then our estimates of UV-B
risk would be 3- to 17-fold higher for A. macrodactylum (Table 1).
Similarly, if we assumed that water transparency to UV-B was
consistently high, with all embryos incubated in water equal to the
most UV-B transparent breeding site for each species, then the
percentage of embryos exposed to harmful levels of UV-B would
increase 5- to 51-fold for A. macrodactylum and 0- to 1.3-fold for
R. cascadae (Table 1). Despite the much greater physiological sen-
sitivity of A. macrodactylum embryos compared with R. cascadae
embryos (10, 19), the risk posed by UV-B measured in this study
suggests that embryos of the two species are equally unlikely to be
affected by UV-B at spatial scales that capture the landscape vari-
ation in site-level transparency to UV-B and oviposition behavior.
Our results suggest that it is exceedingly unlikely that UV-B–
induced embryonic mortality could affect the overall population
status for either of these two species. However, we are not yet
able to explicitly link patterns of embryonic mortality with adult
population dynamics for most amphibian species, due to the
limited data regarding population status and stage-specific de-
mographic rates. Despite this limitation, many amphibian species
undergo strong density-dependent growth and survival during
the aquatic larval stage (29, 30), which has been shown to temper
the effect of additional mortality in embryonic and larval stages.
These results suggest that even large reductions in embryonic
survival might not translate into changes in population size or
population growth rates (31, 32).
Because embryonic survival at natural breeding sites is gov-
erned by many factors other than UV-B, including predation,
hydroperiod, disease, and temperature, we specifically isolated
the contribution of UV-B by comparing the mean difference in
survival between embryos shaded from most UV-B and those
incubated under near-ambient conditions in each experiment. As
macrodactylum (Bottom) embryos as a function of the average UV-B trans-
parency (Kd) for each site. Shaded areas represent UV-B exposures from egg
incubation experiments corresponding to the highest incubation exposure
associated with no survival effect (light shading, R. cascadae = 10,534 KJ·m−2;
A. macrodactylum = 5,321 KJ·m−2) and the lowest exposure associated with
significantly reduced survival (dark shading, R. cascadae = 22,918 KJ·m−2; A.
macrodactylum = 10,534 KJ·m−2). Bold line represents a smoothed trend in
median cumulative UV-B exposures across transparency fit by distance-
weighted least squares. UV-B transparency decreases with larger Kdvalues.
Log median cumulative UV-B exposure of R. cascadae (Top) and A.
Palen and SchindlerPNAS
| May 25, 2010
| vol. 107
| no. 21
a result, this analysis accounts for the direct mortality associated
with UV-B exposure, but does not necessarily predict differences
in the overall survival of embryos between sites, or nonlethal but
potentially detrimental effects of UV-B that might not be
expressed until later life-history stages or that might interact with
other stressors (15, 33–35). For example, embryonic mortality in
UV-B–shielded treatments varied across experimental sites from
1% to 65% for A. macrodactylum and from 2% to 57% for
R. cascadae, suggesting that the importance of direct mortality by
UV-B is small relative to other sources of mortality, even in sites
with statistically significant UV-B effects (UV-B mortality, 42%
in A. macrodactylum and 7% in R. cascadae). Although esti-
mating the importance of mortality due to factors other than
UV-B was not the focus of this experiment, these values em-
phasize the variability in amphibian reproduction and embryonic
survival, and represent the broader ecological context within
which potential UV-B effects are embedded.
It is important to evaluate the assumptions made in this analy-
sis for our conclusions regarding the level of risk posed by current
UV-B. In all cases in which a simplifying assumption was required
in our calculations, we intentionally chose the assumption that
would favor overestimating UV-B exposure, and as a result we have
confidence that these estimates represent a near-maximum effect of
UV-B on embryos of these two species. This approach increases
the confidence in our conclusion that current UV-B levels are not
causing widespread mortality across this montane landscape.
We found significant reductions in the survival of embryos ex-
perimentally exposed to UV-B in breeding sites with high optical
transparency, corresponding to survival decreases of 7% for
R. cascadae embryos and 42% for A. macrodactylum embryos.
However, given the tremendous variation in UV-B transparency
among amphibian breeding sites, experiments conducted at any
one site will not accurately characterize the importance of UV-B
for amphibian populations distributed across heterogeneous land-
scapes encompassing hundreds to thousands of individual sites.
When variation in UV-B transparency of breeding sites and ovi-
position behavior are not included in our estimates of risk, we
predict that 9- to 90-fold more embryos would be exposed to po-
tentially harmful UV-B levels (Table 1). This is analogous to con-
ducting an exposure experiment at the most UV-B–transparent
breeding site, anchoring embryos at a water depth of 10 cm in full
sunlight, and extrapolating those results to the full complement of
sites used by each species. These results highlight the fundamen-
tal importance of DOM in predicting UV-B exposures in ponds
across the landscape, and underscore the fact that individual be-
haviors may further buffer the effect of otherwise harmful envi-
ronmental stressors (36). To critically evaluate threats to specific
populations, experiments must go beyond testing local conditions
in isolation of the broader ecological context within which pop-
ulations occur (37), and explicitly consider the suite of physical,
chemical, and biological features that might ameliorate or exac-
erbate impacts to individuals. By combining data from multiple
spatial scales and levels of biological organization, we are able to
improve predictions of the extent of harmful UV-B effects at the
spatial scales relevant to amphibian population dynamics.
Materials and Methods
Field Experiments. To evaluate the context dependency of UV-B effects on the
survival of A. macrodactylum and R. cascadae embryos, we monitored the
survival of embryos incubated in near-ambient and reduced UV-B environ-
ments at seven different breeding sites spanning a gradient of optical
transparency to UV-B during July–September 2002. All sites are at subalpine
elevations (1278–1500 m) within the headwaters of the Sol Duc drainage of
Olympic National Park, Washington (N 47.917°, W 123.784°), and have little
direct anthropogenic disturbance. Sites were selected to span the range of
UV-B water transparency previously documented for the area (17), and se-
lection was based specifically on seasonal water transparency and amphib-
ian breeding distribution data collected in 2001. At each site, we monitored
the timing of breeding for R. cascadae and A. macrodactylum, and collected
∼400–1,000 eggs for each experiment within 48 h of egg deposition (Table 1).
with moderate to high levels of DOM (17, 38), and as a result R. cascadae
from a preliminary incubation experiment conducted in the clearest site with
R. cascadae breeding in 2001 suggested no difference in embryonic survival
threshold cumulative exposure of UV-B associated with significant embryonic
mortality for R. cascadae, in 2002 embryos were moved to one site with much
higher UV-B penetration than the clearest R. cascadae breeding site within
experiment from the nearest natural breeding site (∼0.2 km away) and oth-
erwise handled identically to those in parallel experiments. Immediately after
collection at each site, egg masses were gently separated into small groups
(∼1–10 embryos) and randomly assigned to treatment, with a total of 25–70
eggs per replicate.
At each experimental site, we compared embryonic survival in near-
ambient and reduced–UV-B environments by incubating embryos at a water
depth of 10 cm in floating clear plastic enclosures with 1-mm mesh screened
bottoms and light selective films attached to the top (13 × 13 × 9 cm). Near-
ambient conditions were maintained with 0.13-mm cellulose acetate film,
which allows an average of 84% ± 4.3% transmission of UV-B wavelengths
(10, 39, 40), and a reduced UV-B environment was created with 0.13-mm
mylar-D film (Dupont), which is known to selectively reduce the intensity of
most UV-B wavelengths, with ∼50% transmission at 315 nm. The optical
properties of mylar-D and acetate film across the UV-B, UV-A, and visible
spectrum wavelengths (290–900 nm) were confirmed by spectrophotometry
(Shimadzu UV-Vis 2100) before and after the experiments and were similar
to those published elsewhere (10, 39, 40). Each of the two experimental
treatments (acetate and mylar-D) was replicated six times at each site for
each species. Embryos were visually monitored every 2–6 days to track de-
velopment, because development time varies among sites due primarily to
water temperature, and survival to hatching was determined when all em-
bryos had either hatched or died (i.e., arrested development) (Table S1).
Data Analysis. Whereas embryonic incubations conducted at each site for
each species can be considered separate factorial experiments assessing the
impact of UV-B exposure on embryonic survival, the motivation for con-
ducting experiments at multiple sites (four sites for R. cascadae and five sites
for A. macrodactylum) was to describe how the effect of ambient UV-B
changes as a function of the transparency of water to UV-B. Therefore, to
compare the importance of UV-B across sites for each species, we calculated
the effect size of UV-B exposure by bootstrapping the difference in survival
between pairwise combinations of treatments at each site 6,000 times (41).
The statistical significance of UV-B exposure on embryonic survival was eval-
uated based on whether the 95% confidence interval (CI) of the effect size,
calculated from the bootstrapped sample [CI = sample mean ± (t1,n-1· SE)],
overlapped with 0.
Table 1. Effect of behavior and DOM on estimates of UV-B risk
No behavior, actual DOM
Actual behavior, constant DOM*
No behavior, constant DOM*
The percentage of embryos exposed to UV-B (KJ·m−2) exceeding high and
low thresholds determined experimentally for each species: for A. macro-
dactylum, high =10,534 KJ·m−2, low = 5,321 KJ·m−2; for R. cascadae, high =
22,918 KJ·m−2, low = 10,534 KJ·m−2. Scenarios represent calculations of risk
based on actual observed conditions, including behavior and landscape var-
iation in DOM, or assumptions of constant DOM across all sites (most trans-
parent site for each species: *Kd= 0.00037;†Kd= 0.00897) or of no behavioral
response to UV-B exposure (all embryos classified as “exposed”). R. cascadae
exhibited no variation in oviposition behavior relevant to UV-B; therefore,
only the constant DOM scenario is shown.
| www.pnas.org/cgi/doi/10.1073/pnas.0912970107Palen and Schindler
Embryonic Survey. To characterize the exposure of R. cascadae and A. mac-
rodactylum embryos to UV-B at broader spatial scales than could be evalu-
ated experimentally, we surveyed 22 breeding sites for each species
spanning a gradient of UV-B transparency over the course of the embryonic
incubation period in 2002 (Kd, 0.00286–0.34653 cm−1). All sites were within
the same drainage system in which the incubation experiments were con-
ducted (elevation, 1,077–1,472 m), and surveys were conducted simulta-
neously with the field experiments. The site selection was based on previous
surveys of water transparency and species occupancy (i.e., breeding), and
represent >90% of R. cascadae breeding sites and >50% of A. macro-
dactylum breeding sites within this headwater basin.
The timing of oviposition by each species was monitored every 1–4 days at
each site, and the distribution of egg depths (in cm) beneath the surface of
embryos are often laid singly or in loose clumps and can be attached to debris
or vegetation. Individual females can produce clutches of 50 to several hun-
were sampled within each site, but have confidence that the distribution of
water depths and light exposures were accurately characterized for each site
by carefully searching all oviposition habitats (under layers of loose rock, un-
dercut pond edges, emergent vegetation, woody debris, and open substrate).
In contrast, R. cascadae lay conspicuous, often conglomerate, egg masses
ranging in size from 50 to 5,000 eggs per mass. We have high confidence that
all egg masses of R. cascadae were detected at each survey site and that for
eachegg mass,the minimumand maximumdepths(incm)wererecorded, and
dimensional volume(in mL) was estimated. Based ontherelationshipbetween
egg mass volume and number of embryos per mass for embryos of approxi-
mately the same stage (Neggs= 1.091·volumeeggmass), we estimated the total
number of embryos within each mass. To associate a water depth with each
embryo within each R. cascadae egg mass, we allocated embryos to depths (in
cm) according to the volume–depth relationship for a sphere occurring be-
tween the minimum and maximum depth of each mass. Importantly, R. cas-
overlying embryos, because weighting the relative exposures of embryos at
different positions within a mass would require understanding of the optical
properties of the jelly matrix and embryonic tissue, which is currently un-
known. Coincident with the measurement of egg depths, we described the
light exposure of each individual egg (for A. macrodactylum) or egg mass (for
R. cascadae) as either fully shaded (with no direct light reaching the eggs) or
exposed to light. The eggs in partial shade were categorized as exposed; we
intentionally applied this conservative definition to ensure that the light ex-
posure of embryos was not underestimated. Sites were continually monitored
over the course of embryonic incubation to estimate the total time that em-
bryos were exposed to UV-B radiation. In cases where either oviposition or
hatching was notdirectlyobserved,theincubationwindow was assumedtobe
the same as that at the next-nearest site, given that temporal asynchrony
generally increases with distance between sites within the study area, and
imparts at most a ±1- to 2-day level of uncertainty to our estimates.
Site UV-B Transparency. To estimate the UV-B transparency of water at each
experimental and survey site, we collected between two and eight water
samples over the course of embryonic incubation to estimate the UV-B at-
tenuation coefficient (Kd). The amount of UV-B reaching any depth (z, in cm)
in the water column can be estimated as the exponential decay of surface
UV-B (UV-B0) according to the formula UV-Bz= UV-B0·e-Kd·z(21). Water sam-
(1.2 μm: Whatman GF-C; 0.2 μm: Millipore PTFE; 47 mm diameter), and stored
in the dark at ∼5–10 °C until being transported to the laboratory. Absorbance
(A) of 440-nm light passed through a 10-cm quartz cuvette (path length, z)
containing each filtered water sample was determined using a Shimadzu UV-
2100 double-beam spectrophotometer, and was related to absorption (a) at
Based on a previously documented relationship between absorption at
440nminaspectrophotometer andinsitu UV-B attenuationprofilesfor lakes
in this drainage [Kd(cm−1) = 0.0795 · a440(cm−1); R2= 0.98; P < 0.00001] (17)
and elsewhere (16, 18), we calculated the integrated extinction coefficient
(Kd) for UV-B wavelengths. To account for the temporal variation in UV-B
transparency of individual sites in our UV-B exposure estimates, we linearly
interpolated daily estimates of Kdbetween water samples at each site.
Incident UV-B Measurements. We estimated incident daily UV-B exposure for
each experimental and survey site for each day of the incubation period for
each species. High spectral resolution (0.5 nm) incident UV flux was recorded
at a nearby site (∼20 km) in Port Angeles, Washington as part of the Envi-
ronmental Protection Agency’s network of UV spectrophotometers (Mark IV
spectrophotometer; Brewer #147; N 48.097°, W 123.426°; elevation, 9.3 m
above sea level). Cumulative daily integrated UV-B estimates (287–320 nm)
were provided by the National UV Monitoring Center (ftp://ftp.epa.gov/
nerlpb/uvnet/olympic/) according to a cosine-corrected trapezoidal in-
tegration method. To account for the increase in UV-B irradiance with alti-
tude (elevation, in m), daily cumulative exposures were scaled to the
elevation of each breeding site according to the formula UV-Belev= UV-B0·
([elevation/1,000] · 1.18) (42). No adjustments were made for site-level dif-
ferences in landscape position, topography, or aspect; thus, reported daily
UV-B estimates represent overestimates for some sites, but can be inter-
preted to be the theoretical maximum daily exposure received at each site.
UV-B Exposure Estimates. To estimate the distribution of UV-B exposures for
embryos of R. cascadae and A. macrodactylum at each survey site we com-
bined (i) cumulative daily UV-B estimates scaled to the elevation of each site,
(ii) interpolated daily estimates of Kdat each site, and (iii) the distribution of
eggs with water depth and their exposure to light (no exposure = 0 KJ·m−2
UV-B) (Fig. S1). Cumulative daily UV-B estimates were made on a per embryo
basis for each day of the observed incubation period for each species at each
site, and then these were summed across all days to arrive at a total cu-
mulative UV-B exposure received by each measured embryo. To date, no
wavelength-specific damage rates, also referred to as action spectra (43),
have been established for any amphibian species. As a result, we chose to
use unweighted cumulative exposure (KJ·m−2·day) integrated across the UV-
B range as the basis for comparing differences in exposure between sites.
Previous risk assessments for amphibian breeding sites in midwestern US
wetlands and US national parks also have used this maximum theoretical
exposure (16, 18) in the absence of a more mechanistic understanding of
wavelength-specific damage rates.
UV-B Risk Assessment. To assess the risk posed by current UV-B levels for
embryos occurring across breeding sites with different depth distributions,
lightexposures,andwater transparency toUV-B,wecombinedthecumulative
UV-B exposure distributions estimated for each survey site with the results of
the egg incubation experiments for each species (Fig. S1). Because all embryos
in the field experiments were incubated at a common depth of 10 cm, and
the transmission properties of the near-ambient treatment are well described
(0.13 mm cellulose acetate = 84% ± 4% ambient UV-B transmission) (10, 39,
40), we can calculate cumulative UV-B exposures over the course of in-
cubation for each species at each experimental site in the same way as was
done for each survey site (see above). Based on these estimates and the level
of statistical significance for the effect of UV-B exposure in each experiment,
we are able to bound the threshold of direct lethal UV-B effects for each
species as occurring between (i) the maximum cumulative UV-B exposure that
produced no significant difference in embryonic survival and (ii) the minimum
cumulative UV-B exposure that significantly increased mortality. By evaluat-
ing the percentage of embryos of each species exposed to both the “highest
known safe” (low) and the “lowest known lethal” (high) cumulative UV-B
exposures, we acknowledge the considerable uncertainty that exists in de-
fining an exact dose–response threshold.
ACKNOWLEDGMENTS. We thank the US National Park Service for access to
field sites and permission to conduct the experiments; Michael Kimlin, Jack
Shreffler, and Roger Hoffman for UV-B irradiance measurements from
Olympic National Park; Mike Adams for logistical support; Mike Brett for
use of his spectrophotometer; Brice Semmens, Mary Power, Sarah Kupfer-
berg, K. B. Suttle, and three anonymous reviewers for constructive feedback
on earlier drafts; and Jennifer Jones, Adam Goodwin, Kristel Dillon, Jon
Moore, Monika Winder, Justin Fox, Tessa Francis, Jackie Carter, Kemp Jones,
Anne Salomon, Laura Payne, and Eric Wagner for field assistance. This work
was supported by the US Geological Survey, US National Park Service
Inventory and Monitoring Program, Canon National Park Science Scholars
Program, and the Department of Biology, University of Washington.
1. Blaustein AR, Wake DB (1990) Declining amphibian populations: A global phenom-
enon. Trends Ecol Evol 5:203–204.
2. Wake DB (1991) Declining amphibian populations. Science 253:860.
3. Drost CA, Fellers GM (1996) Collapse of a regional frog fauna in the Yosemite area of
the California Sierra Nevada, USA. Conserv Biol 10:414–425.
5. Alford RA, Richards SJ (1999) Global amphibian declines: A problem in applied
ecology. Annu Rev Ecol Syst 30:133–165.
6. Stuart SN, et al. (2004) Status and trends of amphibian declines and extinctions
worldwide. Science 306:1783–1786.
Palen and SchindlerPNAS
| May 25, 2010
| vol. 107
| no. 21
7. Kerr JB, McElroy CT (1993) Evidence for large upward trends of ultraviolet-B radiation Download full-text
linked to ozone depletion. Science 262:1032–1034.
8. Madronich S (1994) Increases in biologically damaging UV-B radiation due to
stratospheric ozone reductions: A brief review. Archiv für Hydrobiol Beiheft Ergebnisse
9. World Meterological Organization and United Nations Environment Program (WMO/
UNEP) (2006) Scientific Assessment of Ozone Depletion: 2006. Global Ozone Research
and Monitoring Project Report No. 50. (World Meteorological Organization, Geneva).
10. Blaustein AR, et al. (1994) UV repair and resistance to solar UV-B in amphibian eggs: A
link to population declines? Proc Natl Acad Sci USA 91:1791–1795.
11. Blaustein AR, Romansic JM, Kiesecker JM, Hatch AC (2003) Ultraviolet radiation, toxic
chemicals and amphibian population declines. Divers Distrib 9:123–140.
12. Corn PS (2000) Amphibian declines: Review of some current hypotheses. Ecotoxi-
cology of Amphibians and Reptiles, eds Sparling DW, Linder G, Bishop CA (Society of
Environmental Toxicology and Chemistry, Pensacola, FL), pp 663–696.
13. Licht LE (2003) Shedding light on ultraviolet radiation and amphibian embryos.
14. Bancroft BA, Baker NJ, Blaustein AR (2007) Effects of UVB radiation on marine and
freshwater organisms: A synthesis through meta-analysis. Ecol Lett 10:332–345.
15. Tietge JE, et al. (2001) Ambient solar UV radiation causes mortality in larvae of three
species of Rana under controlled exposure conditions. Photochem Photobiol 74:
16. Diamond SA, Peterson GS, Tietge JE, Ankley GT (2002) Assessment of the risk of solar
ultraviolet radiation to amphibians, III: Prediction of impacts in selected northern
midwestern wetlands. Environ Sci Technol 36:2866–2874.
17. Palen WJ, et al. (2002) Optical characteristics of natural waters protect amphibians
from UV-B in the US Pacific Northwest. Ecology 83:2951–2957.
18. Diamond SA, et al. (2005) Estimated ultraviolet radiation doses in wetlands in six
national parks. Ecosystems 8:462–477.
19. Palen WJ, Williamson CE, Clauser AA, Schindler DE (2005) Impact of UV-B exposure on
amphibian embryos: Linking species physiology and oviposition behaviour. Proc R Soc
B Biol Sci 272:1227–1234.
20. Belden LK, Wildy EL, Blaustein AR (2000) Growth, survival and behaviour of larval
long-toed salamanders (Ambystoma macrodactylum) exposed to ambient levels of
UV-B radiation. J Zool 251:473–479.
21. Morris DP, et al. (1995) The attentuation of solar UV radiation in lakes and the role of
dissolved organic carbon. Limnol Oceanogr 40:1381–1391.
22. Scully NM, Lean DRS (1994) The attenuation of ultraviolet radiation in temperate
lakes. Arch Hydrobiol 43:135–144.
23. McKnight DM, et al. (2001) Spectrofluorometric characterization of dissolved organic
matter for indication of precursor organic material and aromaticity. Limnol Oceanogr
24. Brooks PD, et al. (2005) Spatial and temporal variability in the amount and source of
dissolved organic carbon: Implications for ultraviolet exposure in amphibian habitats.
25. Boyer EW, Hornberger G, Bencala KE, McKnight DM (1997) Response characteristics of
DOC flushing in an alpine catchment. Hydrol Process 11:1635–1647.
26. Williamson CE, et al. (2001) Ultraviolet radiation and zooplankton community
structure following deglaciation in Glacier Bay, Alaska. Ecology 82:1748–1760.
27. Hope D, Billett MF, Milne R, Brown TAW (1997) Exports of organic carbon in British
rivers. Hydrol Process 11:325–344.
28. Gergel SE, Turner MG, Kratz TK (1999) Dissolved organic carbon as an indicator of the
scale of watershed influence on lakes and rivers. Ecol Appl 9:1377–1390.
29. Wilbur HM (1980) Complex life-cycles. Annu Rev Ecol Syst 11:67–93.
30. Alford RA (1999) Tadpoles: The Biology of Anuran Larvae, eds McDiarmid RW, Altig R
(Univ Chicago Press, Chicago), pp 240–278.
31. Vonesh JR, De la Cruz O (2002) Complex life cycles and density dependence: Assessing
the contribution of egg mortality to amphibian declines. Oecologia 133:325–333.
32. Biek R, Funk WC, Maxell BA, Mills LS (2002) What is missing in amphibian decline
research: Insights from ecological sensitivity analysis. Conserv Biol 16:728–734.
33. Pahkala M, Laurila A, Merila J (2001) Carry-over effects of ultraviolet-B radiation on
larval fitness in Rana temporaria. Proc R Soc Lond B Biol Sci 268:1699–1706.
34. Belden LK, Blaustein AR (2002) Exposure of red-legged frog embryos to ambient UV-B
radiation in the field negatively affects larval growth and development. Oecologia
35. Smith MA, Kapron CM, Berrill M (2000) Induction of photolyase activity in wood frog
(Rana sylvatica) embryos. Photochem Photobiol 72:575–578.
36. Huey RB, Hertz PE, Sinervo B (2003) Behavioral drive versus behavioral inertia in
evolution: A null model approach. Am Nat 161:357–366.
37. Gonzalez MJ, Frost TM (1994) Comparisons of laboratory bioassays and a whole-lake
experiment: Rotifer responses to experimental acidification. Ecol Appl 4:69–80.
38. Adams MJ, Schindler DE, Bury RB (2001) Association of amphibians with attenuation
of ultraviolet-B radiation in montane ponds. Oecologia 128:519–525.
39. Nozais C, Desrosiers G, Gosselin M, Belzile C, Demers S (1999) Effects of ambient UVB
radiation in a meiobenthic community of a tidal mudflat. Mar Ecol Prog Ser 89:
40. Blaustein AR, Kiesecker JM, Chivers DP, Anthony RG (1997) Ambient UV-B radiation
causes deformities in amphibian embryos. Proc Natl Acad Sci USA 94:13735–13737.
41. Efron B, Tibshirani RJ (1994) An Introduction to the Bootstrap (Chapman & Hall,
42. Blumthaler M, Ambach W, Ellinger R (1997) Increase in solar UV radiation with
altitude. J Photochem Photobiol B 39:130–134.
43. McKinlay AF, Diffey BL (1987) Human Exposure to Ultraviolet Radiation: Risks
and Regulations, eds Passchler WR, Bosnajokovic BFM (Elsevier, Amsterdam), pp
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