Contaminants as Contributing Factors to Wood Frog Abnormalities on the Kenai National Wildlife ...

257 Pages·2008·13.74 MB·English

U.S. Fish & Wildlife Service

Contaminants as Contributing Factors
to Wood Frog Abnormalities on the
Kenai National Wildlife Refuge, Alaska

Final Report: AFWFO TR-08-01

DEPARTMENT OF THE INTERIOR
U.S. FISH AND WILDLIFE USFWS
REGION 7
by
Mari K. Reeves
Contaminants Specialist
Environmental Contaminants Program
Anchorage Fish and Wildlife Field Office

and
Kimberly A. Trust
Branch Chief
Environmental Contaminants Program
Anchorage Fish and Wildlife Field Office

for
Ann Rappoport
Field Office Supervisor
Anchorage Fish and Wildlife Field Office
Anchorage, AK

Congressional District # AK/109
August 30, 2008

Suggested Citation: Reeves, M.K. and K.A. Trust. 2008. Contaminants as Contributing Factors
to Wood Frog Abnormalities on the Kenai National Wildlife Refuge, Alaska. Final Report. U.S.
Fish and Wildlife Service Technical Report. AFWFO TR#2008-01. 257 pp.

Acknowledgements: We thank the staff at the Kenai National Wildlife Refuge, especially C.
Caldes, J. Hall, J. Morton, and R. West for logistical support. We also thank Unocal/Chevron and
Marathon Oil Corporation employees, especially G. Merle, for additional help with logistics. At
the University of California at Davis, M. Holyoak, M. Johnson, A.K. Miles, N. Willits, and R.
Tjeerdema assisted with statistical design and data analysis. For field assistance thank M. Fan, P.
Jensen, S. Jensen, S. Keys, N. Maxon, E. Moreno, M. Nemeth, M. Perdue, J. Ramos, C. Schudel,
J. Stout, H. Tangermann, and C. Wall. For parasitology, we thank P. Johnson at the University of
Colorado at Boulder and D. Larson at University of Alaska, Fairbanks. For radiography, we
thank D. Green with the U.S. Geological Survey (USGS), L. Guderyahn with Ball State
University, and M. Lannoo with Indiana University. For gonad histology and analysis, we thank
C. Kersten at McNeese State University. For DNA and biomarker analysis, we thank J. Jenkins.
For sediment, water and UVB testing, we thank C. Bridges, R. Calfee, and E. Little. For
invertebrate sampling and assessment, we thank P. Jensen and S. Jensen. We honor the memory
of D. Sutherland, formerly with the University of Wisconsin, La Crosse, who performed most
parasitology for this project. The USFWS Division of Environmental Quality provided support
for this project (FFS Number: 7N23; DEC ID: 200470001).
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DISCLAIMER:
DATA PRESENTED IN THIS REPORT ARE STILL UNDERGOING ANALYSIS PENDING
PUBLICATION IN PEER REVIEWED OUTLETS.
DO NOT CITE THIS DOCUMENT WITHOUT AUTHOR PERMISSION.
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KEYWORDS: Abnormality, Alaska, Amphibian, Malformation, Rana sylvatica, Lithobates
sylvaticus, Wood Frog, National Wildlife Refuge, Metals, Invertebrate Predators, Climate
Change, FFS Number: 7N23; DEC ID: 200470001

ABBREVIATIONS:
AIC Akaike’s Information Criterion
CCC criterion continuous concentration - chronic limit for the priority pollutant in fresh
water
CERC Columbia Environmental Research Center
DO dissolved oxygen
DNA deoxyribonucleic acid
metamorph frog between Gosner stage 42 and 46
MSCL Mississippi State Chemical Laboratory
NWRC National Wetlands Research Center
OC organochlorine
OR odds ratio
PAH polycyclic aromatic hydrocarbon
PCA principal components analysis
PCB polychlorinated biphenyl
PEL probable effects level - concentration or exposure level at which significant
adverse effects become likely
pH negative log of the hydrogen ion concentration, a measure of acidity
Refuge National Wildlife Refuge
SpC specific conductivity
SPMD semipermeable membrane device
SVL snout to vent length
TDS total dissolved solids
TEL threshold effects level - the concentration or exposure level below which a
significant adverse effect is not observed
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UCR University of California at Riverside
UET Upper effects threshold
USFWS U.S. Fish and Wildlife Service
USGS U.S. Geological Survey
UVB ultraviolet-B radiation
UWL University of Wisconsin LaCrosse

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TABLE OF CONTENTS

LIST OF TABLES AND LIST OF FIGURES...............................................................................8
EXECUTIVE SUMMARY..........................................................................................................10
INTRODUCTION........................................................................................................................11
OBJECTIVES...............................................................................................................................12
FIELD METHODS........................................................................................................................13
Study Area and Selection of Sites..........................................................................................13
UVB.......................................................................................................................................16
Basic Water Quality..............................................................................................................16
Water Sampling - Organics..................................................................................................16
Water Sampling - Inorganics................................................................................................16
Sediment Sampling - Organics and Inorganics....................................................................16
Temperature..........................................................................................................................17
Invertebrate Predator Assessments.......................................................................................17
Species Selection...................................................................................................................17
Abnormality Assessment.......................................................................................................17
ABNORMALITY CLASSIFICATION...............................................................................................18
ADDITIONAL FROG DIAGNOSTICS: PARASITES, GONAD STRUCTURE, AND BIOMARKERS..........18
CONTROLLED STUDIES: TOXICITY TESTING AND PREDATOR EXCLUSION..................................19
STATISTICAL ANALYSIS AND HYPOTHESIS EVALUATION...........................................................20
Predictor Variables...............................................................................................................20
Selection of Assessment Endpoints.......................................................................................24
Model Building......................................................................................................................24
Experimental Data Analysis.................................................................................................24
RESULTS – FIELD ASSESSMENTS.........................................................................................25
ABNORMALITY TYPES AND PREVALENCE..................................................................................25
INTERSEX...................................................................................................................................29
PARASITES.................................................................................................................................30
ORGANIC CONTAMINANTS IN SEDIMENT...................................................................................30
INORGANIC CONTAMINANTS IN SEDIMENT................................................................................31
ORGANIC CONTAMINANTS IN WATER........................................................................................32
INORGANIC CONTAMINANTS IN WATER.....................................................................................33
RESULTS – STATISTICAL ASSESSMENT AND CONTROLLED EXPERIMENTS...........34
SKELETAL ABNORMALITIES AND MALFORMATIONS..................................................................34
EYE ABNORMALITIES.................................................................................................................38
DISEASE.....................................................................................................................................39
INTERSEX...................................................................................................................................40
DISCUSSION...............................................................................................................................41
SKELETAL ABNORMALITIES AND MALFORMATIONS..................................................................41
EYE ABNORMALITIES.................................................................................................................45
DISEASE.....................................................................................................................................45
INTERSEX...................................................................................................................................46
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HUMAN DISTURBANCE..............................................................................................................46
CONCLUSIONS...........................................................................................................................47
MANAGEMENT RECOMMENDATIONS AND FUTURE DIRECTIONS.............................48
REFERENCES.............................................................................................................................50

Appendix A - Invertebrate Study Reports……………………………………………………….57

Appendix B - DNA and Biomarker Report…………………………………………………….127

Appendix C – Sediment/Water/UVB Testing…………………………………………....…….175

Appendix D – Study Plan for USFWS Sediment and Water Toxicity Study………………….226

Appendix E – Study Plan for Heritable Abnormality Study……………………………...……233

Appendix F – Environmental Health Perspectives Paper………………………………………235

Appendix G – Disease Papers………………………………………………………………….242

Appendix H – Contaminants Data (Tables 3-6)………………………………………………..248
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LIST OF TABLES

Table 1. Summary of abnormalities in wood frog populations at the Kenai Refuge.
Table 2. Summary of intersex frogs in study sites at the Kenai Refuge.
Table 3. Polycyclic aromatic hydrocarbons in study site sediments.
Table 4. Organochlorines in study site sediments.
Table 5. Inorganic contaminants in study site sediments.
Table 6. Inorganic contaminants in study site water.

LIST OF FIGURES

Figure 1. Map of Study Site Locations.
Figure 2. Pictures of the four most common abnormalities in Alaskan wood frogs. A.
Micromelia, B. Ectromelia, C. Amelia, and D. Unpigmented Iris.
Figure 3. Skeletal abnormalities and malformations versus distance to the nearest road.
Symbols are prevalence of frogs with skeletal abnormalities during single collection events at
different refuges: Arctic (□) Innoko (○) Kenai (Δ) Tetlin( ) and Yukon Delta (◊).
Figure 4. Skeletal abnormalities and malformations versus size. Values are proportion of
frogs abnormal in each category. Error bars are calculated based on the underlying binomial
pˆ(1− pˆ)
distribution: s(pˆ) = where s(pˆ)is the standard error estimate, pˆ is the proportion
n
abnormal in that category, and n is the number sampled in each category.

Figure 5. Skeletal abnormalities and malformations versus developmental stage. Values are
proportion of frogs abnormal in each category. Error bars are calculated based on the
pˆ(1− pˆ)
underlying binomial distribution: s(pˆ) = where s(pˆ)is the standard error
n
estimate, pˆ is the proportion abnormal in that category, and n is the number sampled in each
category.

Figure 6. Metamorphic wood frog infected with the perkinsus-like protozoan organism.
Figure 7. Histological slide of an intersex wood frog’s gonad.
Figure 8. Total PCBs in study site sediment. Red line is PEL. Orange line is TEL.
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Figure 9. Sediment concentrations of As, Cd, Cu, and Se plotted against PCA vector 2, a
significant predictor of skeletal abnormalities. Red lines are PELs. Orange lines are TELs for
each element.
Figure 10. Sediment concentrations of Ni and Fe plotted against PCA vector 1, not a
predictor of skeletal abnormalities. Orange lines are TEL for Ni and the UET for Fe.
Figure 11. Inorganic contaminants in water plotted against PCA vector used to represent
them in statistical analysis. Orange lines are CCCs.
Figure 12. Skeletal abnormalities versus the PCA vectors for metals in sediment (As, Cd, Cu,
Se) and water (Ba, Fe, K, Pb).
Figure 13. Skeletal abnormalities versus average pond water temperature and early season
abundance of dragonfly larvae.
Figure 14. Skeletal malformations versus average pond water temperature and the PCA
vector for metals in sediment (As, Cd, Cu, Se).
Figure 15. Differences in hatching success for controlled sediment and water toxicity
experiment.
Figure 16. Prevalence of the Perkinsus-like organism in metamorphic wood frogs assessed
for abnormalities versus average pond temperature.
Figure 17. Prevalence of the Perkinsus -like organism in metamorphic wood frogs assessed
for abnormalities versus average pond pH.
Figure 18. Prevalence of the Perkinsus -like organism in metamorphic wood frogs assessed
for abnormalities versus average pond TDS and the PCA vector found to be associated with a
decreased incidence of the disease (Al, Be, Co, Cr, Fe, K, Mg, Mn, Ni, Ti, Vd, and Zn).
Figure 19. Pictures of R. pipiens limbs amputated early in development with original caption
(from Fry 1966).
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EXECUTIVE SUMMARY

BACKGROUND: Amphibian abnormalities and diseases are not well understood, and appear to be
increasing while global populations decline.

OBJECTIVES: The goals of this study were to identify stressors associated with amphibian abnormalities
on the Kenai Refuge and assess whether anthropogenic factors contributed to these abnormalities.

METHODS: Between 2004 and 2006, we assessed 38 breeding sites for prevalence of abnormal wood
frogs. We chose 21 ponds for more intensive study, measuring the following variables known to cause
abnormalities in amphibians: UVB, temperature, basic water quality, contaminants, and abundance of
predatory invertebrates. On a subset of frogs, we assessed gonadal structure, DNA integrity, and
biomarkers of genetic damage, and identified and enumerated parasites. We analyzed field data with
logistic regression, using AIC to compare competing models.

RESULTS: Of 5,716 metamorphic wood frogs examined, 450 (7.9%) had skeletal or eye abnormalities. We
documented 558 abnormalities in these 450 abnormal frogs because frogs often had more than one
abnormality. Over 25 abnormality types were seen. The four most common were micromelia (small
limb), ectromelia (truncated limb), amelia (no limb), and unpigmented iris. We found evidence for two
diseases of conservation concern, Batrachochytrium dendrobatidis, a fungal pathogen responsible for
global amphibian population declines, and an undescribed protozoan, quite virulent in Kenai study
populations. We also observed intersex frogs, 41 of 163 frogs (25%) examined had abnormal gonadal
morphology. None of the 448 frogs assessed for parasites were infected with the abnormality-inducing
trematode, Ribeiroia ondatrae. We quantified predatory invertebrates in study sites, including dragonfly
larvae, damselfly larvae, water beetles, leeches, and spiders. Organic and inorganic contaminants
exceeded toxic thresholds in study site sediments. PCBs were found in every pond, and DDT was higher
than toxic thresholds in four sites. Arsenic, iron, selenium, cadmium, copper, and nickel were all higher
than toxic thresholds in sediments, and barium, iron, cadmium, and copper surpassed thresholds in water.
In statistical analyses, we identified dragonflies, toxic metals, and temperature as predictors for skeletal
abnormalities and malformations. Metals that correlated with skeletal abnormalities included arsenic,
cadmium, copper, and selenium in sediment and barium, iron, potassium, and lead in water.
Environmental factors predictive of disease were temperature, acidity, metals, and total dissolved solids.
Controlled experiments showed toxicity but not teratogenicity from abiotic site media.

DISCUSSION: We propose the ultimate cause of skeletal abnormalities in Kenai wood frogs is amputation
injury, probably by dragonfly larvae. The significant effects of metals and temperature in our statistical
analyses suggest one or both of these factors may be disrupting the normal predator-prey relationship
between dragonflies and wood frogs. Contaminants in sediment may slow development or interfere with
normal predator detection and avoidance strategies. Warmer temperatures may increase abundance of
dragonfly larvae or change the timing of dragonfly presence relative to tadpole growth. Higher
temperatures and poor water quality were positively associated with disease. Two initial hypotheses for
the intersex condition are high temperatures and PCBs, both previously shown to cause endocrine
disruption in amphibians. Anthropogenic disruption of climate and consequent high temperatures appear
linked to three of the four abnormality types we documented. These temperature effects may be
particularly significant in the face of further predicted global change.

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