White Paper on the Potential for Atrazine to Affect Amphibian Gonadal Development

322 Pages·2007·8.51 MB·English

White Paper on the Potential for Atrazine to Affect
Amphibian Gonadal Development

In Support of an
Interim Reregistration Eligibility Decision
on Atrazine

Submitted to the FIFRA Scientific Advisory Panel
for Review and Comment
October 9 – 12, 2007

Office of Prevention, Pesticides, and Toxic Substances
Office of Pesticide Programs
Environmental Fate and Effects Division
Washington, D. C.

September 21, 2007
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AUTHORS

Thomas Steeger, Mary Frankenberry, and Lisa Eisenhauer, Office of Prevention,
Pesticides, and Toxic Substances, Office of Pesticide Programs, Environmental Fate and Effects
Division and Joseph Tietge, Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Mid-Continent Ecology Division were responsible
for preparation of the white paper.

ACKNOWLEDGMENTS

The authors acknowledge the input of a number of reviewers: Les Touart from the Office
of Science Coordination and Policy; Frank Gostomski from the Office of Water, and Sigmund
Degitz, Michael Hornung and Rodney Johnson of the Mid-Continent Ecology Division, Office of
Research and Development. From the Office of Pesticide Programs, reviewers included William
Jordan (Immediate Office), Steven Bradbury1, Stephanie Irene, Karen McCormack, Anita Pease,
Ingrid Sunzenauer, Arthur-Jean Williams, and Keith Sappington (Environmental Fate and Effects
Division).

1 Currently Special Review and Reregistration Division.
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TABLE OF CONTENTS
EXECUTIVE SUMMARY...........................................................................................................5
BACKGROUND......................................................................................................................................5
OVERVIEW OF LABORATORY AND FIELD STUDIES.................................................................6
LABORATORY STUDIES.........................................................................................................6
FIELD STUDIES.......................................................................................................................7
CONCLUSIONS FROM LABORATORY AND FIELD STUDIES....................................................8
CHAPTER 1................................................................................................................................10
INTRODUCTION.......................................................................................................................................10
BACKGROUND......................................................................................................................10
SUMMARY OF PESTICIDE EFFECTS CHARACTERIZATIONS..............................................14
DOCUMENT OBJECTIVE..........................................................................................................................15
CHAPTER 2................................................................................................................................17
STUDIES IN RESPONSE TO DATA CALL-IN..............................................................................................17
CHAPTER 3................................................................................................................................41
OPEN LITERATURE STUDY REVIEWS.....................................................................................................41
LABORATORY STUDIES.......................................................................................................42
FIELD STUDIES.....................................................................................................................89
ADDITIONAL OPEN LITERATURE...........................................................................................................99
CHAPTER 4..............................................................................................................................101
STUDY STRENGTHS AND LIMITATIONS..................................................................................101
LABORATORY STUDIES IN RESPONSE TO DCI.................................................................101
OPEN LITERATURE LABORATORY STUDIES....................................................................106
OPEN LITERATURE FIELD STUDIES.................................................................................109
CHAPTER 5..............................................................................................................................111
CONCLUSIONS..................................................................................................................................111
CHAPTER 6..............................................................................................................................116
CHARGE TO THE PANEL........................................................................................................................116
QUESTIONS.........................................................................................................................116
APPENDIX 1 FINAL REPORTS ON STUDIES REVIEWED AS INTERIM REPORTS IN
USEPA (2003)............................................................................................................................128
APPENDIX 2 STATISTICAL ANALYSES SUMMARY TABLES...................................195
APPENDIX 2B...........................................................................................................................198
WIL GROSS MORPHOLOGY AND HISTOPATHOLOGY STATISTICAL ANALYSES
.....................................................................................................................................................198
APPENDIX 2C..........................................................................................................................232
IGB GROSS MORPHOLOGY AND HISTOPATHOLOGY STATISTICAL ANALYSES
.....................................................................................................................................................232
APPENDIX 2D..........................................................................................................................261
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BASIC SUMMARY STATISTICS WIL AND IGB GROSS MORPHOLOGY AND
HISTOPATHOLOGY..............................................................................................................261
APPENDIX 3. DIGITAL IMAGES OF SELECTED HISTOLOGICAL ENDPOINTS..319

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EXECUTIVE SUMMARY

BACKGROUND

Under an amended consent decree entered in to conclude litigation between the
Environmental Protection Agency (EPA) and the Natural Resources Defense Council (NRDC),
the Agency issued an Interim Reregistration Eligibility Decision (IRED) for atrazine on January
31, 2003. The decree further stipulated that EPA would issue a revised IRED on October 31,
2003 that considers studies conducted prior to February 28, 2003 on the effects of atrazine on
amphibians. After developing a paper that addressed the significance of the amphibian risk data,
the Agency agreed, under the consent decree, to seek external peer review of its evaluation of
these studies from a Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) Scientific
Advisory Panel (SAP). In June 2003, the SAP reviewed a White Paper prepared by EPA
(USEPA 2003) that critically evaluated the collective available data from 17 laboratory and field
studies, discussed the nature of remaining uncertainties in evaluating the potential effects of
atrazine on amphibian development, and outlined the nature of future studies that could address
these uncertainties.

In its 2003 White Paper, EPA concluded that while there was sufficient information to
formulate a hypothesis that atrazine exposure can affect amphibian gonadal development, there
was insufficient information to refute or confirm the hypothesis. Collectively, the available
studies had deficiencies and uncertainties that limited their usefulness in interpreting potential
atrazine effects. In response to these uncertainties, the Agency identified additional information
necessary to evaluate the potential causal relationship between atrazine exposure and gonadal
development in amphibians and the nature of any associated dose-response relationship. EPA
proposed that future amphibian studies be conducted in a tiered approach. After reviewing EPA’s
analysis and recommendations, the SAP concurred that the Agency’s review was thorough, the
approaches and criteria were appropriate, and the conclusions were valid, given the data
reviewed. The SAP also agreed that additional studies were warranted and that a tiered testing
approach was appropriate (SAP 2003).

In November 2004, EPA issued a Data Call-in (DCI) Notice to Syngenta Crop Protection,
Inc., and other atrazine registrants requiring the registrants to conduct a study consistent with the
first tier of testing described in the 2003 White Paper. After the Agency and the registrant agreed
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to a protocol for testing potential effects of atrazine on gonadal development of amphibians in
April 2005, the registrant submitted to EPA in June 2007 its final report entitled “Response of
Larval Xenopus laevis to Atrazine Exposure: Assessment of Metamorphosis and Gonadal
Morphology. Consistent with the Tier 1 study proposed by EPA, the registrant’s protocol and
final report focused on the effects of atrazine alone on amphibian gonadal development.

OVERVIEW OF LABORATORY AND FIELD STUDIES

Before developing the 2007 White Paper, EPA searched the open literature for any
studies published since the preparation of the 2003 White Paper (USEPA 2003). After
identifying recent laboratory and field studies related to potential effects of atrazine on gonadal
development in amphibians, EPA published a list of these studies in the Federal Register and
solicited recommendations for additional studies from the public. Based on this literature search,
a total of 19 new studies (Table 1), which were published in the open literature or submitted by
the registrant, were reviewed. Ten were laboratory-based investigations and nine were field
studies. Of the ten laboratory-based studies, two were the final versions of studies reviewed as
interim reports for the 2003 SAP (USEPA 2003). All of the field studies were final versions of
studies reviewed as interim reports for the 2003 SAP (USEPA 2003). Each study was
individually evaluated with regard to the effects of atrazine alone on amphibian gonadal
development. Furthermore, each of the studies was evaluated relative to experimental design,
protocols and data quality assurance, strength of the cause-effect and/or dose-response
relationship, and ecological relevancy of measured endpoints.
Laboratory Studies
Similar to what was discussed in the Agency’s 2003 White Paper (USEPA 2003),
laboratory studies addressed a variety of endpoints: time to metamorphosis, growth, gonadal
abnormalities, sex ratios, laryngeal muscle area, plasma steroid concentrations, and aromatase
activity. In evaluating the effects of atrazine on amphibian development, researchers tested a
variety of frog species, including African clawed frogs (Xenopus laevis), the Northern leopard
frog (Rana pipiens), and the green frog (R. clamitans). Not all species were used in every study.
All of the studies, except one that was submitted by the registrant in response to the DCI, used
static renewal exposures. Within individual studies the number of atrazine exposures ranged
from only one concentration up to five different concentrations; in one study, atrazine
concentrations spanned five orders of magnitude (0.01 to 100 ppb). In some cases, the nominal
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atrazine concentrations were confirmed analytically. Although three studies (Hayes et al. 2006b;
Orton et al. 2006; Sullivan and Spence 2003) reported effects of atrazine in combination with
other chemicals, this review focused on the effects of atrazine alone. As recommended by the
2003 SAP (SAP 2003), this review also focused on gonadal development in amphibians and not
other endpoints.
Only one of the laboratory studies, i.e., the study submitted by the registrant in response
to the DCI, incorporated all of the design elements recommended by the Agency and the FIFRA
SAP (SAP 2003) to address uncertainties identified in the Agency’s 2003 White Paper (USEPA
2003). This submission represented the results of two separate studies conducted in two
independent laboratories. Although both these studies contain limitations, e.g., contamination of
a subset of contols in one study, the overall design and conduct of the studies contain a high
degree of quality control that allows them to be used in characterizing ecological risk. The results
of these recently submitted studies show that atrazine did not have a consistent effect on time to
or size at metamorphosis or on gonadal development in X. laevis.
The studies submitted in response to the DCI were the only laboratory studies that fully
accounted for environmental and animal husbandry factors capable of influencing endpoints
which the studies were attempting to measure. Other laboratory studies reviewed by the Agency
did not fully account for environmental conditions that could influence relevant endpoints.
Field Studies
All of the field studies were reviewed as interim reports in the 2003 White Paper
(USEPA 2003). The field studies, which include a mesocosm study, evaluated growth, gonadal
deformities, sex ratios, plasma steroid concentrations, and aromatase activity in X. laevis, R.
clamitans, bullfrogs (R. catesbeiana), and cane toads (Bufo marinus) in Florida, Indiana, Iowa,
Michigan, and South Africa.
Similar to the conclusions regarding field studies discussed in the 2003 White Paper
(USEPA 2003), the Agency concludes that the currently available field studies are of limited
value because of the high variability in environmental conditions (e.g., photoperiod, temperature,
water quality) under which field-collected organisms lived, uncertainty as to amphibian
developmental status and condition at the initiation of the studies, and an inability to relate the co-
occurrence of atrazine with key developmental windows for the organisms under investigation.
Consequently, EPA cannot determine whether the failure of several studies to show any
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relationship between measured or predicted aqueous atrazine concentrations and developmental
effects reflects the absence of a causal relationship or the limitations of the study methodologies.
In addition, the actual or possible co-occurrence of additional chemical and/or non-chemical
stressors confound attempts to attribute any observed responses to atrazine exposure.

CONCLUSIONS FROM LABORATORY AND FIELD STUDIES
The Agency has reviewed 36 open literature and registrant-submitted studies related to
the potential effects of atrazine on gonadal development in amphibians. Overall, the weight-of-
evidence based on these studies does not show that atrazine produces consistent, reproducible
effects across the range of exposure concentrations and amphibian species tested. In laboratory
studies where environmental and animal husbandry factors were controlled, atrazine exposures
(0.01 – 100 µg/L) did not affect time to or size at metamorphosis, sex ratio, or gonadal
development. While there were several effects on secondary gross and histological endpoints that
were statistically significant, their relationship to apical endpoints of intersex and/or gonadal
development effects is not considered relevant.
Because of the uncertainties associated with all the laboratory and field studies
conducted prior to 2003, the 2003 SAP recommended that additional studies be conducted to
determine if exposure to atrazine affects amphibian gonadal development. In their report, the
SAP recommended using X. laevis as the test species as well as indigenous species. Because the
SAP report did not identify what benefits the indigenous species would provide, the Agency
concluded that testing with X. laevis would be sufficient for a Tier 1 study. Based on the recent
nineteen studies reviewed, including the recently submitted DCI studies showing no effects of
atrazine on amphibian gonadal development, the Agency has further concluded that the higher
tiers of testing proposed in the 2003 White Paper (USEPA 2003) are not needed at this time.

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Table 1. Summary of open literature and registrant-submitted studies evaluated in White Paper.
Study First Author EPA Study Number or Species Study Type
Open Literature Citation
Environmental Toxicology and
Freeman and Rayburn 2005 Chemistry 24(7) 1648 - 1653 Xenopus laevis laboratory
Environmental Health Perspectives 114
Hayes et al. 2006a Xenopus laevis laboratory
(Supplement 1): 134 - 141
Environmental Health Perspectives 114 Rana pipiens and
Hayes et al. 2006b (Supplement 1): 40 - 50 Xenopus laevis laboratory
Hecker et al. 2005a Toxicological Sciences 86(2): 273 – 280. Xenopus laevis laboratory
Hecker et al. 2005b Aquatic Toxicology 72: 383 - 396 Xenopus laevis laboratory
Environmental Toxicology and
Orton et al. 2006 Rana pipiens laboratory
Chemistry 25(1): 65 - 71
Environmental Toxicology and
Sullivan and Spence 2003. Xenopus laevis laboratory
Chemistry 22(3): 627 – 635.
Journal of Toxicology and Environmental
Health 67 (Part A): 941 – 957.
Coady et al. 2004 Rana clamitans laboratory
MRID 469656-05
Ecotoxicology and Environmental Safety
Coady et al. 2005 62: 160 – 173 Xenopus laevis laboratory
MRID 469656-02
Hosmer et al. 2007 MRID 471535-01 Xenopus laevis laboratory
Archives of Environmental Rana clamitans
Murphy et al. 2005 Contamination 51(3): 467 – 477. Rana catesbeiana field
MRID 469656-01 Rana pipiens
Murphy et al. 2006a Aquatic Toxicology 76: 230 - 245 Rana clamitans field
Murphy et al. 2006b Aquatic Toxicology 77: 153 - 166 Rana clamitans field
Environmental Science and Technology
Jooste et al. 2005 39: 5255 – 5261 Xenopus laevis mesocosm
MRID 469656-03
African Journal of Herpetology 54(1): 69
Smith et al. 2005 – 76. Xenopus leavis field
MRID 469656-04
African Journal of Herpetology 54(1): 61
Du Preez et al. 2005 Xenopus laevis field
– 68.
Hecker et al. 2004. Environmental Toxicology and Xenopus laevis field
Chemistry 23(8): 1996 – 2007.
Smith et al. 2007 MRID 472045-01 Rana catesbeiana field
Gross 2007 MRID 472044-01 Bufo marinus field

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