August 4, 2003

MEMORANDUM

SUBJECT: Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting

Held June 17-20, 2003 

TO: James J. Jones, Director

Office of Pesticide Programs 

FROM: Paul I. Lewis, Designated Federal Official

FIFRA Scientific Advisory Panel

Office of Science Coordination and Policy

THRU: Larry C. Dorsey, Executive Secretary

FIFRA Scientific Advisory Panel

Office of Science Coordination and Policy

Joseph J. Merenda, Jr., Director

Office of Science Coordination and Policy

Please find attached the meeting minutes of the FIFRA Scientific Advisory Panel open meeting held in

Arlington, Virginia from June 17 to 20, 2003. This report addresses a set of scientific issues being considered

by the Environmental Protection Agency regarding the potential developmental effects of atrazine on

amphibians. 

Attachment
2

cc: 

Susan Hazen

Adam Sharp

Anne Lindsay

Janet Andersen

Debbie Edwards

Steven Bradbury

Jay Ellenberger

Arnold Layne

Tina Levine

Frank Sanders

Betty Shackleford

Margaret Stasikowski

William Jordan

Antonio Bravo

Douglas Parsons

David Deegan

Vanessa Vu (SAB)

OPP Docket

FIFRA Scientific Advisory Panel Members

Stephen M. Roberts, Ph.D.

Steven Heeringa, Ph.D.

Gary E. Isom, Ph.D. 

Fumio Matsumura, Ph.D. 

Mary Anna Thrall, DVM

FQPA Science Review Board Members

Joel Coats, Ph.D. 

Peter Delorme, Ph.D. 

Robert J. Denver, Ph.D. 

James Gibbs, Ph.D. 

Sherril L. Green, DVM 

Darcy B. Kelley, Ph.D. 

Werner Kloas, Ph.D. 

Gerald A. LeBlanc, Ph.D. 

Carl Richards, Ph.D. 

David Skelly, Ph.D.
3

SAP Report No. 2003-01

REPORT

FIFRA Scientific Advisory Panel Meeting, 

June 17-20, 2003, held at the Crowne Plaza Hotel

Arlington, Virginia

A Set of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding:

POTENTIAL DEVELOPMENTAL EFFECTS OF

ATRAZINE ON AMPHIBIANS 
4

NOTICE

These meeting minutes have been written as part of the activities of the Federal

Insecticide, Fungicide, and Rodenticide Act (FIFRA), Scientific Advisory Panel (SAP). These

meeting minutes represent the views and recommendations of the FIFRA SAP, not the United

States Environmental Protection Agency (Agency). The content of these meeting minutes do not

represent information approved or disseminated by the Agency. They have not been reviewed

for approval by the Agency and, hence, the contents of these meeting minutes do not necessarily

represent the views and policies of the Agency, nor of other agencies in the Executive Branch of

the Federal government, nor does mention of trade names or commercial products constitute a

recommendation for use.

The FIFRA SAP is a Federal advisory committee operating in accordance with the

Federal Advisory Committee Act and was established under the provisions of FIFRA, as

amended by the Food Quality Protection Act (FQPA) of 1996. The FIFRA SAP provides

advice, information, and recommendations to the Agency Administrator on pesticides and

pesticide-related issues regarding the impact of regulatory actions on health and the

environment. The Panel serves as the primary scientific peer review mechanism of the EPA,

Office of Pesticide Programs (OPP) and is structured to provide balanced expert assessment of

pesticide and pesticide-related matters facing the Agency. Food Quality Protection Act Science

Review Board members serve the FIFRA SAP on an ad-hoc basis to assist in reviews conducted

by the FIFRA SAP. Further information about FIFRA SAP reports and activities can be

obtained from its website at http://www.epa.gov/scipoly/sap/ or the OPP Docket at (703) 305-

5805. Interested persons are invited to contact Larry Dorsey, SAP Executive Secretary, via email at dorsey.larry@.epa.gov.
5

SAP Report No. 2003-01

REPORT:

FIFRA Scientific Advisory Panel Meeting, 

June 17-20, 2003, held at the Crowne Plaza Hotel,

Arlington, Virginia

A Set of Scientific Issues Being Considered by the

Environmental Protection Agency Regarding:

POTENTIAL DEVELOPMENTAL EFFECTS OF

ATRAZINE ON AMPHIBIANS 

Mr. Paul Lewis Stephen Roberts, Ph.D.

Designated Federal Official FIFRA SAP Session Chair

FIFRA Scientific Advisory Panel FIFRA Scientific Advisory

Panel

Date: August 4, 2003 Date: August 4, 2003
6

Federal Insecticide, Fungicide, and Rodenticide Act 

Scientific Advisory Panel Meeting

June 17-20, 2003

POTENTIAL DEVELOPMENTAL EFFECTS OF 

ATRAZINE ON AMPHIBIANS

PARTICIPANTS

FIFRA SAP Session Chair

Stephen M. Roberts, Ph.D., Professor and Program Director, University of Florida

Center for Environmental & Human Toxicology, Gainesville, Florida

FIFRA Scientific Advisory Panel Members

Steven Heeringa, Ph.D., Research Scientist and Director for Statistical Design

Institute for Social Research, University of Michigan, Ann Arbor, MI 

Gary E. Isom, Ph.D., Professor of Toxicology, School of Pharmacy and Pharmacal Sciences,

Purdue University, West Lafayette, IN 

Fumio Matsumura, Ph.D., Professor of Environmental Toxicology and Director of the Center for

Environmental Health Sciences University of California at Davis, Davis, CA 

Mary Anna Thrall, DVM., Professor, Department of Microbiology, Immunology and Pathology,

Colorado State University, Fort Collins, CO 

FQPA Science Review Board Members

Joel Coats, Ph.D., Professor and Chair, Department of Entomology, Iowa State University,

Ames, Iowa 

Peter Delorme, Ph.D., Senior Evaluation Officer, Environmental Assessment Division

Pest Management Regulatory Agency, Health Canada, Ottawa, Ontario, Canada 

Robert J. Denver, Ph.D., Associate Professor and Associate Chair for Undergraduate Studies,

Department of Molecular, Cellular and Developmental Biology; Associate Professor,

Department of Ecology and Evolutionary Biology, The University of Michigan, Ann Arbor, MI 

James Gibbs, Ph.D., Associate Professor, Faculty of Environmental and Forest Biology,

SUNY-ESF, Syracuse, NY 

Sherril L. Green, DVM, Associate Professor, Department of Comparative Medicine, Stanford

University School of Medicine, Stanford, CA 

Darcy B. Kelley, Ph.D., Professor of Biological Sciences, Columbia University, 

New York, NY 

Werner Kloas, Ph.D., Professor of Endocrinology, Department of Endocrinology, Institute of
7

Biology, Hymboldt University, Berlin; Head of Department of Inland Fisheries, Leibniz-Institute

of Freshwater Ecology and Inland Fisheries, Berlin, Germany

Gerald A. LeBlanc, Ph.D., Professor of Toxicology, Department of Environmental & Molecular

Toxicology, North Carolina State University, Raleigh, NC 

Carl Richards, Ph.D., Director and Professor, Minnesota Sea Grant College Program, University

of Minnesota Duluth, Duluth, MN 

David Skelly, Ph.D., Associate Professor of Ecology, Yale School of Forestry and

Environmental Studies, New Haven, CT 

PUBLIC COMMENTERS

Oral statements were made by:

Ronald Kendall, Ph.D., Texas Tech University, Glen Van Der Kraak, Ph.D., University of

Guelph, Canada, Ernest Smith, Ph.D., Texas Tech University, John Giesy, Ph.D., Michigan State

University, Louis Du Preez, Ph.D., Potchefstroom University, South Africa, K.R. Solomon,

Ph.D., University of Guelph, Canada, Timothy Gross, Ph.D., United States Geological Survey

and the University of Florida, Robert Sielken, Jr., Ph.D. Sielken and Associates, Inc., James

Carr, Ph.D., Texas Tech University, and Ms. Catherine Bens, United States Department of

Agriculture, on behalf of Ecorisk, Inc. 

John Ashby, Ph.D. and Charles Breckenridge, Ph.D. on behalf of Syngenta

Tyrone Hayes, Ph.D., the University of California, Berkeley, as a private citizen 

Nigel Noriega, Ph.D.,USEPA , as a private citizen 

Janis McFarland, Ph.D. on behalf of Syngenta

Angelina Duggan, Ph.D. on behalf of CropLife America

Mr. Scott Slaughter on behalf of the Center for Regulatory Effectiveness

Mr. Jere White, Kansas Corn Growers Association and Kansas Grain Sorghum Producers

Association, Ms. Stephanie Whalen, Hawaii Agriculture Research Center, Bill Kubecka, DVM,

Kubecka Farms, and Mr. Gary Marshall, Missouri Corn Merchandising Council and Missouri

Corn Growers Association, on behalf of the Triazine Network

Richard Fawcett, Ph.D., on behalf of the Iowa Corn Growers Association

Mr. Robert Hedberg on behalf of the Weed Science Society of America

Jennifer Sass, Ph.D., on behalf of the Natural Resources Defense Council

Steven Sheffield, Ph.D., George Mason University, as a private citizen
8

Mr. James Tozzi on behalf of Multinational Business Services

Mr. John Hall on behalf of the Kentucky Corn Growers Association

Mr. Daniel Botts on behalf of the Florida Fruit and Vegetable Association

Written statements were received from: 

Center for Regulatory Effectiveness

Ecorisk, Inc.

Natural Resources Defense Council

Rachel Carson Council 

Rachel Jordan, as a private citizen

Sielken & Associates Consulting, Inc.

State of New York, Office of the Attorney General 

Steven Sheffield, Ph.D., George Mason University, as a private citizen

Syngenta

Tyrone Hayes, University of California, Berkeley, as a private citizen

Triazine Network

Weed Science Society of America

INTRODUCTION

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), Scientific Advisory

Panel (SAP) has completed its review of the set of scientific issues being considered by the

Agency pertaining to the potential developmental effects of atrazine on amphibians. Advance

notice of the meeting was published in the Federal Register on February 24, 2003 and May 8,

2003. The review was conducted in an open Panel meeting held in Arlington, Virginia, from

June 17-20, 2003. The meeting was chaired by Stephen Roberts, Ph.D. Mr. Paul Lewis served

as the Designated Federal Official. Steven Bradbury, Ph.D. (Office of Pesticide Programs, EPA)

summarized the goals and objectives of the Agency’s presentation. Thomas Steeger, Ph.D.

(Office of Pesticide Programs, EPA) and Mr. Joseph Tietge (Office of Research and

Development, EPA) provided an overview of the atrazine studies and conceptual model for

potential studies, respectively. Thomas Steeger, Ph.D. (Office of Pesticide Programs, EPA)

completed the Agency’s presentation by providing concluding remarks. 

In preparing these meeting minutes, the Panel carefully considered all information
9

provided and presented by the Agency presenters, as well as information presented by public

commenters. This document addresses the information provided and presented within the

structure of the charge by the Agency. 

CHARGE

1. In reviewing the available laboratory and field studies, the Agency used a number of criteria

to evaluate individual investigations. Criteria such as experimental design, test protocols, and

quality assurance information were used to ascertain the reliability of the generated data in terms

of its ability to adequately assess a hypothesis that atrazine elicits developmental effects in

amphibians, and if so, the nature and strength of associated dose-response relationships. 

a) Does the SAP have any comments and recommendations regarding the EPA’s approach and

criteria used to evaluate the studies? 

b) Given the evaluation criteria employed by the Agency, please comment on EPA’s overall

characterization of the currently available studies.

c) Please comment on the availability, as of February 28, 2003, of additional, relevant studies in

the open literature that were not addressed in the white paper.

d) Since February 28, 2003, is the Panel aware of any studies that would be relevant?

2. In its evaluation of existing field studies, the Agency has concluded that these investigations

are of limited value. The reasons include: (1) the high variability in environmental conditions

and uncertainties in the pre-existing status and condition of field-collected animals, (2) the

spatial and temporal aspects of atrazine exposure (i.e., spatial and temporal variability over the

course of the studies and the extent to which such aspects of atrazine exposure were empirically

measured or otherwise accounted for), and (3) the possible co-occurrence of additional chemical

and/or non-chemical stressors. 

a) To the extent that the field studies appear to indicate that atrazine may not adversely affect

development, please comment on EPA’s conclusion that the body of data from field studies does

not provide the means to ascertain whether the lack of a relationship between atrazine exposure

and developmental effects is due to the absence of a causal relationship or limitation in study

methodologies.

b) To the extent that any field studies appear to indicate that atrazine may adversely affect

development, please comment on EPA’s conclusion that these field studies do not provide

sufficient information to resolve the potential role of additional co-occurring stressors.

3. In an evaluation of the existing laboratory-based studies, the Agency concluded that there was

sufficient information to establish a hypothesis that atrazine could cause adverse gonadal

developmental effects. However, due to different experimental designs and variability in the

nature and extent of experimental conditions (e.g., level of excessive mortality, delayed

development in untreated organisms, lack of response to positive controls) it was not possible to

adequately assess the hypothesis that atrazine causes developmental effects. It was further

concluded that the current body of information did not provide the means to characterize the
10

nature of any associated dose-response relationships. 

a) Please comment on EPA’s determination that the laboratory studies provide a plausible basis

for the means to establish a hypothesis concerning the potential for atrazine to cause

developmental effects. Also, please comment on whether the overall body of available data is

adequate to demonstrate whether or not atrazine causes developmental effects under the

conditions described in these studies. 

b) Please comment on EPA’s conclusion that given the variability in the available dose-response

data across the studies (e.g., an approximately 250-fold difference in reported thresholds for

observed developmental effects as well as reports of monotonic and non-monotonic doseresponse curves), it is not possible to ascertain the relationship, if any, of atrazine exposure to

developmental effects in amphibians. 

4. Many of the available studies proposed that aromatase induction results in elevated estrogen

levels that lead to feminization (ovotestes/intersex/hermaphroditism) in genetically male

amphibians. 

a) Please comment on EPA’s conclusion that, to date, aromatase induction by atrazine has not

been demonstrated in any anuran in controlled laboratory investigations. 

b) The variability associated with plasma sex steroid concentrations and aromatase activities is

high. Is this variability normal? Please comment on any readily apparent or available

methodological improvements (e.g., changes in sampling design, analytical techniques) that

could efficiently address this variability in future studies. 

c) Please comment on whether there are additional data, other than those summarized in the

white paper, that suggest late exposure of amphibians (i.e., juveniles or adults) to estrogens or

estrogenic chemicals can induce ovotestes formation. 

d) Please comment on whether there are additional data, other than those summarized in the

white paper, that suggest alternative mechanisms that could explain the apparent feminization of

genetically-male amphibians.

5. With regard to specific endpoints, the Agency does not currently have sufficient information

to quantitatively relate gonadal/laryngeal effects to reproductive outcomes. A major underlying

uncertainty is the ecological relevance of ovotestes occurrence to the maintenance of anuran

populations. 

a) Can the Panel provide sources of data on background rates of ovotestes occurrence in

amphibian species and any associated considerations for interpreting this information in the

context of the reviewed studies? 

b) Can the Panel characterize any evidence that suggests that the presence of ovotestes in male

anurans results in reproductive impairment via reductions in fertility? 

c) The reduction of laryngeal muscle area suggests diminished testosterone in males. If this is

found to be a valid observation and if estrogen concentrations do increase as testosterone

concentrations decrease, what other endpoints (e.g., secondary sexual characteristics and
11

reproductive behavior) would likely be affected? 

6. While some of the available data indicate there may be an association between atrazine

exposure and developmental effects in amphibians, the Agency’s evaluation of the existing body

of laboratory and field studies has determined that there is not sufficient scientific evidence to

indicate that atrazine consistently produces effects across the range of amphibian species

examined. However, the current body of knowledge has deficiencies and uncertainties that limit

its usefulness in assessing potential developmental atrazine effects and the extent of any

associated cause-effect and dose-response relationships. Consequently, the Agency has

determined that there are not sufficient data to reject the hypothesis that atrazine can cause

adverse developmental effects in amphibians. 

Does the SAP concur with these conclusions? If not, what lines-of-evidence would lead to an

alternative conclusion? 

7. Assuming the Agency determined an ecological risk assessment with a greater degree of

certainty concerning developmental effects of atrazine on amphibians were needed, please

comment on EPA’s conclusion that additional information is required to evaluate potential

causal relationships between atrazine exposure and gonadal development. Please also comment

on the added utility, if any, of additional information to interpret the shape of dose-response

curves for potential developmental endpoints and the extent to which threshold or non-threshold

response relationships can be quantified. 

8. The Agency has developed a conceptual model from which to develop a set of study

protocols for evaluating the potential effects of atrazine on gonadal development in amphibians. 

The Agency has proposed a research approach using focused, empirical, laboratory studies based

on initial investigations with X. laevis followed by selective, confirmatory studies with frog

species native to North America. 

a) Please comment on the proposed sequence of study objectives.

b) Please comment on whether the Agency’s first set of proposed studies has accounted for the

major sources of uncertainty associated with the potential effects of atrazine on anuran sexual

differentiation. In addition to time to metamorphosis, gonadal abnormalities, and sex ratios in the

proposed Phase I assays, please comment on any other endpoints that should be considered in

this initial phase. 

c) Please also comment on the range, spacing and number of atrazine concentrations that should

be employed in the proposed testing sequence to resolve uncertainties in the shape and nature of

dose-response relationships for any observed developmental effects.

d) Please comment on the Agency’s recommendation that X. laevis be used as the primary

biological model in the proposed studies and whether or not the mechanisms involved in sexual

differentiation of the ranid and pipid species are sufficiently similar to predict effects and

associated dose-response curves for Rana and/or to efficiently design Rana studies. 

e) In this regard, are there important differences between the species to conclude that any

affected developmental processes observed in X. laevis would not occur in Rana? 
12

f) Alternatively, are there developmental pathways in Rana, but not in X. laevis, that raise

concerns about using X. laevis as the primary biological model in any future atrazine studies? 

g) Assuming X. laevis and Rana are sufficiently concordant from a toxicodynamic perspective

with regard to potential developmental effects of atrazine, what critical toxicokinetic processes

should be considered for extrapolating X. laevis dose-response relationships to Rana and/or for

designing subsequent studies with Rana? 

DETAILED RESPONSE TO THE CHARGE

The specific issues to be addressed by the Panel are keyed to the Agency's background

documents "White Paper on Potential Developmental Effects of Atrazine on Amphibians”, dated

May 29, 2003, and are presented as follows:

1. In reviewing the available laboratory and field studies, the Agency used a number of

criteria to evaluate individual investigations. Criteria such as experimental design, test

protocols, and quality assurance information were used to ascertain the reliability of the

generated data in terms of its ability to adequately assess a hypothesis that atrazine elicits

developmental effects in amphibians, and if so, the nature and strength of associated doseresponse relationships. 

a) Does the SAP have any comments and recommendations regarding the EPA’s approach

and criteria used to evaluate the studies? 

b) Given the evaluation criteria employed by the Agency, please comment on EPA’s overall

characterization of the currently available studies.

The Panel considered parts A and B of question 1 together. The Agency’s criteria in

evaluating the available studies included experimental design, protocols and quality assurance,

strength of cause/effect and/or concentration/response, mechanistic plausibility and ecological

relevance. These criteria reflect requirements for sound and consistent science in ecological risk

assessments. 

The Panel concluded that the review was thorough, the approaches and criteria were

appropriate and that the conclusions were valid, given the data reviewed. The Panel raised a few

minor concerns relating to the Agency’s approach. One experiment (Syngenta # 2233-02)

categorized as a field study is actually a mesocosm experiment. Mesocosms, while potentially

powerful, are not capable of providing inferences regarding the fate of natural populations. With

regard to the analysis of laboratory studies, the Panel expressed minor concerns about the

reanalysis of the data but agreed that the overall characterization of the studies was reasonable. 

The Panel agreed that additional studies are warranted. Studies from several research

laboratories provided evidence that atrazine can cause developmental abnormalities in

amphibians. Although not considered in the Agency's White Paper, the findings are consistent

with studies on effects of atrazine exposure to other vertebrates, both aquatic (e.g. fish) [Moore,

2003] and terrestrial (some rodents). Given the conservation of many basic pathways for

endocrine regulation, these studies in other species are relevant to the issues addressed in the

Agency's White Paper. However, a range of abnormalities have been reported in amphibians and

these are not consistent from study to study (bearing in mind that the literature includes studies
13

in several anuran species). 

c) Please comment on the availability, as of February 28, 2003, of additional, relevant

studies in the open literature that were not addressed in the white paper. Since February

28, 2003, is the Panel aware of any studies that would be relevant?

The Agency appropriately identified the extant published literature relevant to atrazine

effects on the development of the amphibian gonad. The Panel recommended adding mortality

studies to the Agency’s assessment because they offer a toxicological perspective regarding the

atrazine concentrations used in developmental studies. The Panel also identified selected

additional published studies ("open literature") that do not need to be included in the Agency’s

White Paper, but are relevant to ongoing deliberations and may be useful for the Agency’s risk

assessment. These studies encompass several topics including:

1) Effects of atrazine on survival and early development of anurans

Allran JW and Karasov WH. 2001. Effects of atrazine on embryos, larvae, and adults of anuran

amphibians. Environ Toxicol Chem. Apr;20(4):769-775. 

Bevan CL, Porter DM, Prasad A, Howard MJ, and Henderson LP. 2003. Environmental

estrogens alter early development in Xenopus laevis. Environ Health Perspect. Apr;111(4):488-

496. 

Morgan MK, Scheuerman PR, Bishop CS, and Pyles RA. 1996. Teratogenic potential of atrazine

and 2,4-D using FETAX. J Toxicol Environ Health. Jun 7;48(2):151-168. 

2) Hormone effects on survival and early development of anurans

Antila E. 1977. Early steroid metabolism in Xenopus laevis, Rana temporaria and Triturus

vulgaris embryos. Differentiation. Aug 11;8(2):71-77.

Fort DJ, McLaughlin DW, Rogers RL, and Buzzard BO. 2003. Evaluation of the developmental

toxicities of ethanol, acetaldehyde, and thioacetamide using FETAX. Drug Chem Toxicol. 

Feb;26(1):23-34.

Nishimura N, Fukazawa Y, Uchiyama H, and Iguchi T. 1977. Effects of estrogenic hormones

on early development of Xenopus laevis. J Exp Zool. Jul 1;278(4):221-233.

3) Effects of atrazine on vulnerability to infection and immune function

Christin MS, Gendron AD, Brousseau P, Menard L, Marcogliese DJ, Cyr D, Ruby S, and

Fournier M. 2003. Effects of agricultural pesticides on the immune system of Rana pipiens and

on its resistance to parasitic infection. Environ Toxicol Chem. May; 22(5):1127-1133. 

Gendron AD, Marcogliese DJ, Barbeau S, Christin MS, Brousseau P, Ruby S, Cyr D, and

Fournier M. 2003. Exposure of leopard frogs to a pesticide mixture affects life history

characteristics of the lungworm Rhabdias ranae. Oecologia. May;135(3):469-476. 

4) Normative and experimental data on hormones, sex determination and sexual differentiation
14

in anurans; hormone sensitive biomarkers

Bögi C, Schwaiger J, Ferling H, Mallow U, Steineck C, Kalbfus W, Negele RD, Lutz I, and

Kloas W. 2003. Endocrine effects of environmental pollution on Xenopus laevis and Rana

temporaria. Environ Res. (in press).

Bögi C, Schwaiger J, Ferling H, Mallow U, Steineck C, Kalbfus W, Negele RD, Lutz I, and

Kloas W. 2002. Endocrine effects of environmental pollution on amphibians. Proceedings of

2nd Status-Seminar Endocrine Disrupters, 2nd - 4th April 2001, Berlin, Germany,

http://www.status-umwelthormone.de, 59-62.

Bögi C, Levy G, Lutz I, and Kloas W. 2002. Functional genomics and sexual differentiation in

amphibians. Comp Biochem Physiol B. 133 (4): 559-570.

Catz D, Fischer L, and Kelley D. 1995. Androgen regulation of a laryngeal-specific myosin

heavy chain isoform whose expression is sexually differentiated. Dev Biol. 171:448-457. 

Kang L, Marin M, and Kelley D. 1995. Androgen biosynthesis and secretion in developing

Xenopus laevis. Gen Comp Endocrinol. 100: 293 - 307. 

Kelley DB. 1982. Female sex behaviors in the South African clawed frog, Xenopus laevis:

gonadotropin-releasing, gonadotropic, and steroid hormones. Horm Behav. Jun; 16(2):158-174. 

Kelley DB and Pfaff DW. 1976. Hormone effects on male sex behavior in adult South African

clawed frogs, Xenopus laevis. Horm Behav. Jun; 7(2): 159-182.

Kloas W. 2002. Amphibians as model for the study of endocrine disruptors. Int Rev Cytol. 216,

1-57. 

Kloas W, Levy G, Bögi C, Opitz R, and Lutz I. 2002. Effects of environmental chemicals on

reproductive biology of amphibians. Proceedings of 2nd Status Seminar Endocrine Disrupters,

2nd - 4th April 2001, Berlin, Germany, http://www.status-umwelthormone.de, 55-58. 

Kloas W, Bögi C, Levy G, Würtz S, and Lutz I. 2002. Sexual differentiation in amphibians. 

Proceedings of the 21st Conference of the European Comparative Endocrinologists (Bonn,

Germany, 26-30 August, 2002), Monduzzi Editore, 87-90.

Kloas W, Lutz I, and Einspanier R. 1999. Amphibians as model to study endocrine disruptors:

II. Estrogenic activity of environmental chemicals in vitro and in vivo. Drugs and Hormones as

Pollutants of the Aquatic Environment – Determination and Ecotoxocological Impacts Sci. Total

Environ. 225, 59-68.

Levy G, Bögi C, Lutz I, Opitz R, and Kloas W. 2002. Amphibians as model to study endocrine

disruptors: I. In vivo effects on reproductive biology. Proceedings of 2nd Status-Seminar

Endocrine Disrupters 2nd - 4th April 2001, Berlin, Germany, http://www.statusumwelthormone.de, 99-102. 

Levy G, Lutz I, Opitz R, Krüger A, and Kloas W. 2002. Bisphenol A induces feminization in

Xenopus laevis tadpoles via estrogen-response systems. Proceedings of the 21st Conference of
15

the European Comparative Endocrinologists (Bonn, Germany, 26-30 August, 2002), Monduzzi

Editore, 91-94.

Levy G, Lutz I, Opitz R, Krüger A, and Kloas W. 2003. Bisphenol A induces feminization in

Xenopus laevis tadpoles. Environ Res. (in press).

Lutz I, Würtz S, Schulz A, Levy G, Bögi C, Opitz R, and Kloas W. 2002. Establishment of

estrogen receptor-mRNA as estrogenic biomarker in the amphibian Rana temporaria.

Proceedings of the 21st Conference of the European Comparative Endocrinologists (Bonn,

Germany, 26-30 August, 2002), Monduzzi Editore, 95-98.

Lutz I, and Kloas W. 1999. Amphibians as model to study endocrine disruptors: I.

Environmental pollution and estrogen receptor binding. Drugs and Hormones as Pollutants of the

Aquatic Environment – Determination and Ecotoxicological Impacts, Sci Total Environ. 225:

49-57.

Marin ML, Tobias ML, and Kelley DB. 1990. Hormone-sensitive stages in the sexual

differentiation of laryngeal muscle fiber number in Xenopus laevis. Development. 110:703-711. 

Miyashita K, Shimizu N, Osanai S, and Miyata S. 2000. Sequence analysis and expression of

the P450 aromatase and estrogen receptor genes in the Xenopus ovary. J Steroid Biochem Mol

Biol. 75: 101-107.

Miyata S, Koike S, and Kubo T. 1999. Hormonal reversal and the genetic control of sex

differentiation in Xenopus. Zool Sci. 15: 335-340.

Mosconi G, Carnevali O, Franzoni MF, Cottone E, Kloas W, Lutz I, Yamamoto K, Kikuyama

S, and Polzonetti-Magni AM. 2002. Environmental estrogens and reproductive biology in

amphibians. Gen Comp Endocrinol. 126: 125-129. 

Opitz R, Levy G, Bögi C, Lutz I, and Kloas W. 2002. Endocrine disruption in fishes and

amphibians. Transworld Research Network, Recent Res Devel Endocrinol. 3: 127-170. 

Robertson J and Kelley D. 1996. Thyroid hormone controls the onset of androgen sensitivity in

the developing larynx of Xenopus laevis. Dev Biol. 176:108-123.

Tobias M, Tomasson J, and Kelley DB. 1998. Attaining and maintaining strong vocal synapses

in female Xenopus laevis. J Neurobiol. 37: 441-448.

Tobias ML, Marin ML, and Kelley DB. 1991. Temporal constraints on androgen directed

laryngeal masculinization in Xenopus laevis. Dev Biol. 147: 260-270. 

Tobias ML, Marin ML and Kelley DB. 1991. Development of functional sex differences in the

larynx of Xenopus laevis. Dev Biol. 147: 251-259. 

van Wyk JH, Pool EJ, and Leslie AJ. 2003. The effects of anti-androgenic and estrogenic

disrupting contaminants on breeding gland (nuptial pad) morphology, plasma testosterone levels,
16

and plasma vitellogenin levels in male Xenopus laevis (African clawed frog). Arch Environ

Contam Toxicol. 44:247-256. 

Varriale B and Chieffi P. 1997. Oestrogen control of the sexual dimorphism in the Harderian

gland of Xenopus laevis. J Steroid Biochem Mol Biol. Aug;62(5-6):455-60. 

Wetzel DM and Kelley DB. 1983. Androgen and gonadotropin effects on male mate calls in

South African clawed frogs, Xenopus laevis. Horm Behav. 17: 388-404. 

5)Anuran husbandry

Bögi C, Schwaiger J, Ferling H, Mallow U, Steineck C, Kalbfus W, Negele RD, Lutz I, and

Kloas W. 2003. Endocrine effects of environmental pollution on Xenopus laevis and Rana

temporaria. Environ Res. (in press).

Major N and Wassersug RJ. 1998. Survey of current techniques in the care and maintenance of

the African clawed frog (Xenopus laevis). Contemp Top Lab Anim Sci. Sep;37(5):57-60.

Moore A, Scott AP, Lower N, Katsiadaki I, and Greenwood L. 2003. The effects of 4-

nonylphenol and atrazine on Atlantic salmon (Salmo salar L) smolts. Aquaculture 222: 253-263.

Wright KM. 2001. Diets for captive amphibians. In K.M. Wright and R.B. Whittaker (ed.) 

Amphibian Medicine and Captive Husbandry. Krieger Publishing Company, Malabar, FL. pp.

63-72.

2. In its evaluation of existing field studies, the Agency has concluded that these

investigations are of limited value. The reasons include: (1) the high variability in

environmental conditions and uncertainties in the pre-existing status and condition of fieldcollected animals, (2) the spatial and temporal aspects of atrazine exposure (i.e., spatial and

temporal variability over the course of the studies and the extent to which such aspects of

atrazine exposure were empirically measured or otherwise accounted for), and (3) the

possible co-occurrence of additional chemical and/or non-chemical stressors. 

a) To the extent that the field studies appear to indicate that atrazine may not adversely

affect development, please comment on EPA’s conclusion that the body of data from field

studies does not provide the means to ascertain whether the lack of a relationship between

atrazine exposure and developmental effects is due to the absence of a causal relationship

or limitation in study methodologies.

The Panel concluded that the absence of an established causal relationship derived from

laboratory studies was not critical in limiting the interpretation of the field studies. Ecological

field studies are routinely, and successfully, conducted in the absence of such information. 

However, the Panel believed strongly that all of the field studies reviewed had serious design or

methodological flaws that limit their usefulness in evaluating hypotheses related to the effects of
17

atrazine exposure on anuran developmental responses. Common, important problems in the field

studies considered included inappropriate site selection practices (e.g., designation of control

sites with concentrations of atrazine that exceeded some exposure sites) and failure to identify a

sampling frame and to choose sampling sites randomly from within it, as well as insufficient

statistical power associated with too few sampling sites to evaluate study hypotheses. These

problems render interpretation of results problematic, if not impossible.

It also was noted that the field studies focused on measurement of endpoints identified in

laboratory studies. None of the field studies measured responses for which field studies are most

revealing. Specifically, whereas abundance and age structure were measured occasionally,

highly relevant endpoints related to reproduction, recruitment and population viability were

entirely absent. It should also be noted that, aside from one mesocosm experiment, all of the

field studies were observational. While observational field studies are necessary and potentially

yield strong inference, carefully designed field experiments offer opportunities to manipulate the

natural environment, thereby controlling for some potentially confounding factors and allowing

direct interpretation of responses. Such an opportunity was unexploited in the pool of field

studies considered by the Panel.

b) To the extent that any field studies appear to indicate that atrazine may adversely affect

development, please comment on EPA’s conclusion that these field studies do not provide

sufficient information to resolve the potential role of additional co-occurring stressors.

The Panel determined that the field studies provided important information. Most

notably, multiple studies conducted by different labs have uncovered gonadal abnormalities in

wild populations of anurans. Since the Panel concluded that atrazine exposure is a plausible

hypothesis explaining gonadal abnormalities and comparable abnormalities have been described

in natural populations of native amphibians, further field studies are warranted and can be

conducted in the absence of knowledge concerning a specific mechanism(s) that cause(s)

deformities. The Panel concluded that the field studies conducted to date do not, however,

provide sufficient information to resolve the potential role of additional co-occurring stressors,

for reasons cited in the response to Question 2(a).

3. In an evaluation of the existing laboratory-based studies, the Agency concluded that

there was sufficient information to establish a hypothesis that atrazine could cause adverse

gonadal developmental effects. However, due to different experimental designs and

variability in the nature and extent of experimental conditions (e.g., level of excessive

mortality, delayed development in untreated organisms, lack of response to positive

controls) it was not possible to adequately assess the hypothesis that atrazine causes

developmental effects. It was further concluded that the current body of information did

not provide the means to characterize the nature of any associated dose-response

relationships. 

a) Please comment on EPA’s determination that the laboratory studies provide a plausible

basis for the means to establish a hypothesis concerning the potential for atrazine to cause

developmental effects. Also, please comment on whether the overall body of available data

is adequate to demonstrate whether or not atrazine causes developmental effects under the

conditions described in these studies. 

The Panel concurred with the Agency’s determination that the laboratory studies on the
18

effects of atrazine on anuran gonadal development are sufficient to hypothesize that atrazine

interferes with normal development. Available data on the effects of atrazine on gonadal

development of frogs are limited; however, existing lines of evidence support the hypothesis. 

Seven laboratory investigations were designed to evaluate the effects of atrazine exposure on

larval frog gonadal development and are described in the Agency’s White Paper. Five studies

detected abnormalities of gonadal development, including the development of ovotestis (Hayes

et al., 2003; Carr et al., 2003) and reductions in primary spermatogonia (males) and oogonia

(females) (Tavera-Mendoza et al., 2001a, 2001b). Differences among the types of effects

observed in these studies could be due to species differences, differences in exposure, and

specific endpoints selected for evaluation. The two studies that reported no effects of atrazine on

gonadal development were unpublished reports. One study suffered from very high mortality

among organisms in all treatments (Hecker et al., 2003a). Both studies (Hecker et al., 2003a and

Hecker et al., 2003b), indicated problems relating to poor water quality. The inability to detect

gonadal abnormalities with atrazine exposure in these studies should not detract from the

positive results noted in the majority of the studies. Deficiencies in all laboratory studies were

noted as related to experimental design, data analyses, or performance standards. 

 Significant data gaps exist in our understanding of the effects of atrazine on anuran

development. These gaps include a lack of understanding of the mechanism by which atrazine

might elicit developmental toxicity, the nature of the concentration-response relationship,

definition of susceptible windows of exposure, variable terminology used to describe effects, and

identification of a threshold concentration. Panel members agreed that sufficient data were

available to establish the hypothesis that atrazine interferes with normal gonadal development in

anurans but were hesitant to accept the hypothesis with the limited available data. It was agreed

that more data are necessary to properly test the hypothesis. These data should be generated

under standardized conditions and must be subject to independent verification.

Clarification was requested of the Agency as to whether agreement with the charge

statement: “...data is adequate to demonstrate ...(that)... atrazine causes developmental effects

under the conditions described in these studies” implies acceptance of the hypothesis or implies

that sufficient data exist to warrant concern. During the Panel deliberations, the Agency

expressed the latter interpretation. All Panel members agreed that sufficient data existed to

warrant concern.

b) Please comment on EPA’s conclusion that given the variability in the available doseresponse data across the studies (e.g., an approximately 250-fold difference in reported

thresholds for observed developmental effects as well as reports of monotonic and nonmonotonic dose-response curves), it is not possible to ascertain the relationship, if any, of

atrazine exposure to developmental effects in amphibians.

A major deficiency that exists among laboratory studies of the effects of atrazine on

anuran gonadal development has been the difficulty in defining the concentration-response

relationship, and accordingly, a threshold concentration. The magnitude of effects reported in

the peer-reviewed scientific literature are summarized in Table 1 below. 

Table 1. Effects of Atrazine on General Gonadal Development or Germ Cell Content of

Gonads
19

Percent Animals with Effects

Atrazine Concentration (mcg/L)

0.01 0.1 1.0 10 20-25 200

Hayes et al. (2002a) 0 ~ 18 ~ 18 ~ 18 ~ 18 ~ 18

Carr et al. (2003) – – 4.5 7 13 –

Hayes et al. (2003) – 65 – – 20 –

Tavera-Mendoza et al. (2001a) – – – – ~ 70 –

Tavera-Mendoza et al. (2001b) – – – – ~ 30 –

– not tested

From these data, the Panel could draw no conclusions regarding a concentration-response

relationship. More data are required to define the concentration-response relationship between

atrazine and gonadal development of anuran larvae. However, the Panel believes that the data

supports the hypothesis that the effect of atrazine on amphibian general gonadal development

occurs with a threshold concentration between 0.01 and 25 ug/L. 

It was noted that, in general, maximum effects on gonadal development at atrazine

exposure concentrations (when observed) averaged ~20%, irrespective of exposure

concentration. One Panel member noted that this may reflect a plateau of the concentrationresponse curve at this level of effect and that perhaps effects of greater magnitude should not be

expected in future studies. Another Panel member disagreed, suggesting that there might be a

possibility of monotonic as well as non-monotonic responses. 

Precedence was discussed for chemicals having the ability to cause intersex conditions in

only a small percentage of the exposed population in laboratory-controlled experiments. 

Tributyltin is a marine biocide known to cause intersex conditions in some snail species. 

Incidents of 100% intersex females have been noted in field populations inhabiting tincontaminated environments (Curtis and Barse, 1990; Gooding et al., 1999; Gooding, 2003;

Morcillo and Porte, 1999; and Smith, 1981). During many laboratory experiments (Gooding et

al., 2003; Oberdorster et al., 1998; and Smith, 1981), a maximum incidence of intersex of only

~30% has been observed. This discrepancy has been attributed to limitations of laboratory

experiments. For example, exposure of maternal organisms during oocyte maturation or of

embryos may be required to maximally induce intersex among the resulting organisms. In the

absence of such exposure during laboratory experiments, perhaps only the most susceptible

individuals develop intersex conditions. The limited susceptibility of anuran larvae to the

developmental effects of atrazine, coupled with variations in experimental design, could result in

low and variable responses that may mask any suggestion of a concentration-response

relationship.

4. Many of the available studies proposed that aromatase induction results in elevated

estrogen levels that lead to feminization (ovotestes/intersex/hermaphroditism) in

genetically male amphibians. 

a) Please comment on EPA’s conclusion that, to date, aromatase induction by atrazine has

not been demonstrated in any anuran in controlled laboratory investigations. 
20

The Panel agreed with the Agency’s conclusion that, to date, aromatase induction by

atrazine has not been demonstrated in anurans by controlled laboratory studies. The

experimental designs used by several investigators in order to demonstrate effects on aromatase

induction using long term exposures are inappropriate to demonstrate any influence of atrazine,

if there might be one. 

The aromatase hypothesis, as applied to the partial feminizing effects of atrazine on male

Xenopus laevis, originated with the well-established feminizing effects of estrogen in this

species. Exposure of Xenopus laevis tadpoles to sufficient estradiol during the critical period

resulted in 100% phenotypic females. When backcrossed to genetic males (zz genotype), half of

these females produce only male offspring. The results indicated that half of the phenotypic

females were genotypic males.

Indirect support for the aromatase hypothesis comes from findings in a human carcinoma

cell line (H295R) in which atrazine (and two other triazines) increased aromatase (CYP19)

enzyme activity and mRNA levels (Sanderson et al., 2000, 2001). In addition, Hayes et al.

(2002a) reported that exposure of adult male Xenopus laevis to atrazine decreased plasma

testosterone, and these investigators hypothesized that this decrease was due to the induction of

aromatase. The Hayes et al. (2002a) study did not directly address the hypothesis that atrazine

induces aromatase, and the apparent decrease in plasma testosterone might be explained by other

mechanisms (e.g., changes in hormone clearance). Studies submitted to the Agency by Ecorisk

(Giesy et al., 2003; Hecker et al., 2003b) were purportedly designed to evaluate the hypothesis

that atrazine induces aromatase in Xenopus laevis. However, the Panel identified at least two

flaws in the design of these studies that limit their usefulness. First, the investigators exposed

adult frogs to atrazine for long periods (26, 43 or 47 days). In such long term exposure

experiments, compensatory physiological adjustments could have obscured any effects that

atrazine may have had on aromatase activity/expression in the short term. Second, the

deleterious effects of atrazine on amphibians are hypothesized to occur during gonadal

development (during a sensitive premetamorphic tadpole stage). Thus, the appropriate

developmental stages were not tested, and developmental changes in sensitivity to atrazine could

confound interpretation.

Possible feminizing/demasculinizing effects of atrazine could be produced by modes of

action other than induction of aromatase (enhanced estrogen synthesis). For example, atrazine

might exert an antiandrogenic effect. Feminization effects caused by estrogen administration to

Xenopus tadpoles are dose dependent (Kloas et al., 1999; and unpublished data) and can also be

obtained at least in part by antiandrogens such as cyproterone acetate, p,p’-DDE, and vinclozolin

(Kloas, 2002; Bogi et al., 2002).

 

Several potential modes of action should be considered in addition to the aromatase

hypothesis. While the available data do not support any one mode of action, the following

hypotheses could be proposed:

(1) Estrogenic effects of atrazine:

a) the induction of aromatase resulting in the elevation in plasma and/or intragonadal estradiol

levels

b) agonist action mediated by binding to the estrogen receptor (no data available in amphibians)

(2) Antiandrogenic effects:
21

a) inhibition of 5"-reductase resulting in a decrease in the dihydrotestrosterone (DHT)/

testosterone(T) ratio 

b) interference with androgen receptor mediated actions (no data available in amphibians)

(3) Generalized effects on steroid metabolism

(4) Interference via the hypothalamus-pituitary-gonad axis (i.e., Cooper et al., 2000)

Furthermore, atrazine could exert feminizing effects through multiple modes of action. 

While focus has been placed on a causal endocrine disruptive effect of atrazine, one cannot

currently rule out the hypothesis that the feminizing effects of atrazine are due to a generalized

(or even specific) interference with genetic pathways responsible for gonadal development. 

Atrazine could act upstream of the endocrine system (endocrine changes have not been

demonstrated in tadpoles in any study) and endocrine (epigenetic) changes caused by atrazine

could be a byproduct and not the cause. In addition, atrazine could affect the thyroid system,

which is known to influence gonadal development and steroid hormone action in developing

amphibians. 

 

b) The variability associated with plasma sex steroid concentrations and aromatase

activities is high. Is this variability normal? Please comment on any readily apparent or

available methodological improvements (e.g., changes in sampling design, analytical

techniques) that could efficiently address this variability in future studies. 

Inter-individual variability in plasma sex steroid concentrations and steroid converting

enzyme activities in lower vertebrates such as fishes, amphibians and reptiles is often high. This

variability may be attributed to sex differences, age, stage of reproductive development, dietary

influences, and social dominance, among others. However, the Panel was unable to determine

whether the variability reported in the studies submitted to the EPA by ECORISK was due to

biological variation or technical limitations. The most common technique used to analyze

steroid hormones in animal tissues and plasma is radioimmunoassay (RIA). Although less

common, enzyme-linked immunosorbant assay (ELISA) is also used to measure plasma steroids,

and this technique was used in the ECORISK studies. It is noteworthy that the estradiol

measurements in male Xenopus laevis obtained by the ECORISK group by ELISA are one to

two orders of magnitude higher than those reported in the open literature in which RIA was used. 

Also, in the ECORISK studies, there is no mention of attempts to validate the ELISAs for use

with Xenopus laevis plasma (also, appropriate validations were not conducted in the original

reports that described the ELISA techniques which are cited in the ECORISK reports (Hecker et

al., 2003b). Recovery analyses (using radioactive hormone) following organic extraction were

not conducted in these studies, but are essential to control for variation in extraction efficiency

among samples. This alone could account for significant variation in the reported values. No

other standard assay validations were conducted (described below) nor were intra- or interassay

coefficients of variation reported. These omissions make it difficult to evaluate the validity of

the findings reported by ECORISK. 

In the studies published by Hayes (2002a), and as indicated during his presentation to the

FIFRA SAP, Hayes had been unable to measure plasma estradiol in Xenopus using RIA. 

However, members of the Panel noted that other researchers have successfully measured

estradiol in male Xenopus laevis (Tobias et al., 1998; Bögi et al., 2003). In addition, in the

Hayes et al. (2002a) study, measures of plasma testosterone are based on a sample size of only
22

four per treatment. The Hayes et al. (2002a) study is the only direct evidence for an endocrine

disruptive effect of atrazine in an amphibian, and as such should be replicated and extended from

adults to the appropriate developmental stages.

It is essential that the assays used for plasma sex steroid measurements be validated for

use in the amphibian species under study. Validations should include, but are not limited to: 1)

parallelism between the standard curve and dilutions of plasma/serum; 2) recovery of labeled

(i.e., radioactive) hormone added to the plasma/serum sample before extraction; 3) verification

that residual plasma sex hormone binding globulins and other plasma proteins do not interfere in

the assay; 4) verification of the specificity of the antiserum; and 5) corroboration of results by an

independent method (e.g., comparison of ELISA and RIA). As mentioned above, an important

quality control is to conduct recovery estimates (using a radiolabeled tracer) for each extracted

sample to account for differences in extraction efficiencies. 

Regarding the sampling technique for wild-caught Xenopus laevis, the ECORISK group

trapped frogs, transported them to the laboratory, and then maintained them for 48 hours in the

laboratory, purportedly to allow them to recover from capture stress. No attempt was made to

evaluate whether animals had indeed recovered from capture stress (e.g. by measuring plasma

corticosterone) or if the animals continued to suffer from confinement stress. Stress can have

profound effects on plasma sex steroid concentrations, and individuals respond differently to

stress. Thus, a major component of the variability in measurements could be due to the method

of sampling. Future studies of adults (if, in fact, this life history stage is studied) should strive to

collect plasma from animals immediately after capture (in field experiments). Also, the time of

day when plasma is collected in both field and laboratory studies should be considered in the

design of experiments and reported. There are distinct circadian rhythms in plasma hormones in

frogs and thus the choice of time of day or night to sample (it should be noted that Xenopus

laevis are nocturnal) could impact the results.

In relation to biological variability, every effort should be made to record and correlate

physiological parameters that might account for variability in plasma sex steroid concentrations

independent of exposure to atrazine or other toxicants. These parameters should include, but are

not limited to, body weight (lean vs. fat wet and dry weights), stage of reproductive development

(e.g., calculation of gonadosomatic index, plasma vitellogenin concentration in females), and

stress (e.g., plasma corticosterone).

Future mechanistic studies should focus on short term exposures in

tadpoles/juveniles/adults (e.g. 6 hr, 12 hr, 1 d, 3 d, 7 d, and 14 d). Measures should include

estradiol, the androgens T and DHT and activities of aromatase as well as 5"_-reductase. In

testing the aromatase hypothesis, biochemical assays could be included that test for direct

interference of atrazine with aromatase (or 5"-reductase) enzyme activity. Possible effects of

atrazine on aromatase gene expression should be tested using reverse transcriptase-polymerase

chain reaction (RT-PCR) techniques such as those described by Miyata et al. (1999) and

Miyashita et al. (2000). Particular emphasis should be placed on using these techniques to

analyze effects of atrazine on aromatase gene expression in larval amphibians. A

complementary approach for testing for estrogenic actions would be to determine effects of

atrazine exposure on estrogenic biomarkers now available for Xenopus such as plasma

vitellogenin (ELISA-kit, BIOSENSE, Norway or TOWA-KAGAKU, Japan) or vitellogenin

mRNA (RT-PCR, Kloas et al., 1999).
23

c) Please comment on whether there are additional data, other than those summarized in

the white paper, that suggest late exposure of amphibians (i.e., juveniles or adults) to

estrogens or estrogenic chemicals can induce ovotestes formation. 

The Panel is unaware of reports that address the possibility of ovotestis formation in

Xenopus at late exposures. For other amphibians, it is known in bufonids (Ponse, 1945) and

some ranid species (Browder, 1975) that there may arise sex reversal at later stages. 

d) Please comment on whether there are additional data, other than those summarized in

the white paper, that suggest alternative mechanisms that could explain the apparent

feminization of genetically-male amphibians.

As mentioned in response to question 4a, a mode of action other than aromatase

induction might be responsible for feminizing/demasculinizing effects on gonadal development

of genetically male amphibians. One experimental approach to address specifically such effects

could be to produce genetic male (ZZ) female phenotypes and use their progeny (all ZZ males)

specifically for such experiments. In addition, slight effects on the thyroid system cannot be

excluded by the experiments presented up to now.

5. With regard to specific endpoints, the Agency does not currently have sufficient

information to quantitatively relate gonadal/laryngeal effects to reproductive outcomes. A

major underlying uncertainty is the ecological relevance of ovotestes occurrence to the

maintenance of anuran populations. 

a) Can the Panel provide sources of data on background rates of ovotestes occurrence in

amphibian species and any associated considerations for interpreting this information in

the context of the reviewed studies? 

The Panel began its response to this question by defining ovotestes as follows: the

occurrence of frank testicular tissue and ovarian segments within a recognizable gonad. Witschi

(1956) reported 1 case of ovotestes. There is at least one study in which the prevalence of

ovotestes in a control population of laboratory frogs has been described (Bögi et al., 2003; Levy

et al., 2003). To the Panel’s knowledge, the background rates of ovotestes in wild amphibian

populations have not been reported. The Panel believed the frequency of occurrence of

ovotestes in normal healthy populations of amphibians is probably very low and likely varies

among species. This is based on the relatively rare occurrence of ovotestes in Panel members’

laboratory amphibians. Without objective analysis and surveys on the background rates of

ovotestes in wild populations, it is not possible to assess the impact, if any, that the presence of

ovotestes in male frogs may have on anuran populations.

b) Can the Panel characterize any evidence that suggests that the presence of ovotestes in

male anurans results in reproductive impairment via reductions in fertility? 

To the Panel’s knowledge, there are no published reports that indicate that ovotestes in

male anurans either do or do not result in reproductive impairment. In amphibians and other

species, decreases in sperm production are correlated with a decrease in testicular size. The

consequences of gonadal abnormalities for reproductive impairment of individuals or for wild

populations of anurans are entirely unknown. Three broad hypotheses exist:
24

1. Gonadal abnormalities among genetic males may have no effect on local population

dynamics. This could occur if abnormal males do not breed or participate in breeding

congresses. The breeding system of many anurans leads to a small fraction of males achieving

matings with a disproportionate fraction of females. 

2. Gonadal abnormalities among genetic males may lead to declines in wild amphibian

populations. This could occur if abnormal males participate in breeding congregations and

achieve matings with females that lead to incomplete or entire failure of fertilization. 

3. Gonadal abnormalities in genetic male frogs may lead to increases in wild amphibian

populations. This could occur if abnormal genetic males act as females in the breeding dynamics

of natural populations. 

Thus, it will be impossible to evaluate the ecological relevancy of reported gonadal

abnormalities without measuring endpoints related to reproductive behavior. The Panel

therefore recommended that feminized phenotypic males be included in “grow-out” studies for

the purpose of using them in breeding experiments to test the hypothesis that ovotestes impair

reproductive function.

c) The reduction of laryngeal muscle area suggests diminished testosterone in males. If this

is found to be a valid observation and if estrogen concentrations do increase as testosterone

concentrations decrease, what other endpoints (e.g., secondary sexual characteristics and

reproductive behavior) would likely be affected?

The Panel concluded that the following endpoints could be used. All are endocrine

biomarkers. The first six can be evaluated using noninvasive methods and would not require

sacrificing the animal:

(1) Snout to vent length, body weight (feminized males should be bigger)

(2) Nuptial pads

(3) Enlargement of the ventral folds of the cloacae

(4) Strength and pattern of the male calling signal

(5) Clasping

(6) Seminal fluid analysis (sperm count, motility evaluation, morphology)

(7) Time course examining synthesis of vitellogenin by the liver in response to estrogen

challenge

(8) Oviduct development

(9) Proteins expressed in Harderian glands around the eye (three proteins expressed by females,

1 in males)

(10) Number and size of muscle fibers in larynx, myosin expression in larynx muscle

Continuous studies (e.g., studies that follow atrazine-exposed animals through sexual

maturity) would be of great value to field studies. The animal’s gonadal development could be

assessed using minimally invasive techniques such as ultrasonography, endoscopy, or magnetic

resonance imaging.

6. While some of the available data indicate there may be an association between atrazine

exposure and developmental effects in amphibians, the Agency’s evaluation of the existing
25

body of laboratory and field studies has determined that there is not sufficient scientific

evidence to indicate that atrazine consistently produces effects across the range of

amphibian species examined. However, the current body of knowledge has deficiencies

and uncertainties that limit its usefulness in assessing potential developmental atrazine

effects and the extent of any associated cause-effect and dose-response relationships. 

Consequently, the Agency has determined that there are not sufficient data to reject the

hypothesis that atrazine can cause adverse developmental effects in amphibians. Does the

SAP concur with these conclusions? If not, what lines-of-evidence would lead to an

alternative conclusion?

The EPA expressed several conclusions in this question. The Panel examined each

conclusion separately. 

The first conclusion put forth was: “there is not sufficient scientific evidence to indicate

that atrazine consistently produces effects across the range of amphibian species examined.” 

The Panel agreed with this conclusion. The Panel previously noted in response to question # 3

that there was sufficient information to establish a hypothesis that atrazine could cause adverse

gonadal developmental effects in amphibians. Studies were available that reported results from

eight species of frog, including, Xenopus laevis (African clawed frog), Rana pipiens (northern

leopard frog), and Rana clamitans (green frogs) in laboratory or controlled exposure studies

(mesocosm studies). Field observational studies reported on effects in X. laevis, R. pipiens, Acris crepitans (cricket frogs), Bufo maninus (cane toad) and Bufo terrestris (southern toad). 

The response of the species, both in terms of the endpoints considered and the magnitude of

response, was inconsistent across the species studied and among studies which used the same

species. Comparison among the studies was difficult because of the problems identified with

respect to the design and conduct of both the laboratory and field studies, which confound their

interpretation.

The second conclusion put forth was: “the current body of knowledge has deficiencies and uncertainties

that limits its usefulness in assessing potential developmental atrazine effects...”. The Panel agreed with the

conclusions in the Agency’s White Paper that there were deficiencies and uncertainties with respect to the

methods, conduct, and results of the studies submitted, as were identified in responses to questions 2 and 3. 

Among the major factors identified were difficulties with the husbandry in laboratory studies, presence of

atrazine in control exposures and reference sites and a lack of consideration of and/or information on the

presence or potential impact of other stressors in observational field studies. Given these deficiencies and

limitations, the Panel concluded that the current data would not be suitable for ecological risk assessment. 

Further, it was recognized by the Panel that in order to conduct a scientifically sound ecological risk

assessment, the Agency needs to have results from studies where other factors can be ruled out as a cause in

either the presence or the absence of effects. 

The third conclusion put forth was that the uncertainties and deficiencies limited “the extent of (the

identification of) any associated cause-effect and concentration-response relationships.” The Panel concluded

that although they agreed that a causal relationship can be hypothesized between atrazine and effects on

gonadal development, the uncertainties and deficiencies in existing studies precluded acceptance of the

hypothesis (see response to question 3). Further, the exact nature of the response in gonadal development in

amphibians (shape of the concentration/response function, presence of a threshold) cannot be characterized at

this point for the species tested. The Panel also addressed this issue in its response to questions 3(b) and 7. 

Finally, the Panel noted that knowledge of the concentration/response function is a necessary element to
26

conduct an ecological risk assessment.

The final conclusion put forth was: “the Agency has determined that there are not

sufficient data to reject the hypothesis that atrazine can cause adverse developmental effects in

amphibians.” The Panel agreed that the available data suggest that atrazine can affect gonadal

development in amphibians. However, the available data do not allow a proper characterization

of the nature and magnitude of the response at either the organism or population level, nor do

they offer sufficient support for the identification of a plausible mechanism.

The Panel further agreed that information gained from the available studies contributes to

our knowledge and will be useful in the design and conduct of future studies. It was further

concluded by the Panel that the adverse effects, in this case the presence of abnormalities in

gonadal development described, need to be connected to the assessment endpoints of

reproductive competence (i.e., fertilization success and subsequent potential effects at the

population level).

7. Assuming the Agency determined an ecological risk assessment with a greater degree of

certainty concerning developmental effects of atrazine on amphibians were needed, please

comment on EPA’s conclusion that additional information is required to evaluate potential

causal relationships between atrazine exposure and gonadal development. Please also

comment on the added utility, if any, of additional information to interpret the shape of

dose-response curves for potential developmental endpoints and the extent to which

threshold or non-threshold response relationships can be quantified. 

The Panel agreed with the conclusion that additional information is required to evaluate

potential causal relationships between atrazine exposure and gonadal development. Several

points were made in regard to this conclusion. There is a need to confirm the causal relationship

that is suggested by the existing data, and some similarity of data, or patterns or trends, from

different labs needs to be presented to show repeatability of the effects. One of the tenets of the

scientific method is the repeatability of experiments. Further, as previously noted, it is necessary

to characterize the nature of the dose-response (or more correctly, concentration-response)

function. Finally, there is a need to identify a plausible mechanism, supported by data. The

characterization of a mechanism can, in part, aid in the extrapolation of results from surrogate

test species to species of concern in the environment.

With respect to concentration-response curves, the Panel emphasized that these are

extremely important to the question of any detrimental effects of a toxicant to an organism and

necessary for risk assessment. Regardless of whether behavior demonstrates either a monotonic

or atypical concentration-response relationships for a given endpoint, it should be possible to

ascertain the shapes of the curves, given enough concentrations, replications, and controlled

conditions. Repeatability in other laboratories should be feasible, if the same species, stage,

water, concentrations and timing are utilized.

It was further put forth that studies on the quantitative structure-activity relationships

(QSAR) can often provide information about a mechanism of action or provide a rationale for

the data that are generated from comparative testing. Experiments using a series of closely

related compounds (cyanazine, propazine, simazine, terbuthylazine, etc.) could elucidate patterns

that would help explain the interaction between the molecules and the putative receptor,

addressing the causal relationship. This approach seems to be lacking so far. It could be
27

valuable in the in vivo tests for gonadal development as well as for induction of aromatase or

expression of mRNA for aromatase.

There are few studies among those reviewed in the Agency’s White Paper that

acknowledge the existence of metabolites that may be biologically significant. Three chlorinated

metabolites are potentially bioactive in the same mode as atrazine -- deethyl atrazine,

deisopropyl atrazine, and didealkyl atrazine, which is also called diamino chlorotriazine. There

also is one major dechlorinated metabolite (hydroxy atrazine) that should be evaluated as well,

based on its presence as a primary degradate of atrazine. These have been addressed in the

context of water quality and mammalian toxicology, but scarcely in the amphibian development

studies. They should be evaluated because they are commonly occurring transformation

products, and they also could be interesting if included in the QSAR studies suggested above.

8. The Agency has developed a conceptual model from which to develop a set of study

protocols for evaluating the potential effects of atrazine on gonadal development in

amphibians. The Agency has proposed a research approach using focused, empirical,

laboratory studies based on initial investigations with X. laevis followed by selective,

confirmatory studies with frog species native to North America. 

a) Please comment on the proposed sequence of study objectives.

The Panel was in agreement that a logical progression of studies as proposed by the

Agency would elucidate effects and document mechanisms associated with any gonadal

abnormalities due to atrazine exposure. The Panel had a number of suggestions as to how this

model could be improved and which of the studies provided the most immediate and useful tests

of the atrazine hypothesis. There was consensus that laboratory experiments (Phase 1 of the

proposed approach) should proceed immediately. These studies would confirm whether gonadal

deformities occur with exposure to atrazine and bracket concentrations at which effects are

observed. One Panel member suggested that Phase 1 testing include characterization of a

concentration-response relationship using some cellular/molecular marker of atrazine’s effect. 

Such an endpoint would likely be sensitive and detectable in a timely fashion.

Panel members concurred that the studies identified under Phase 5 should be initiated as

early as possible within the framework of the study plan. These studies would require grow-out

of individuals with gonadal deformities to appropriate life stages to examine the effects of the

reported gonadal deformities on fecundity and fertility. The Panel concluded that these studies

were essential since true ecological effects are dependent on the hypothesis that reproduction of

populations is impaired by individual reproductive impairment.

The Panel believed that, subject to study design constraints (i.e., both logistical and the

necessity for a sound statistical design), the ideal study would address the effect of atrazine on

gonadal development/morphology and have sufficient individuals to allow for the continuation

of the study for assessment of the effects on fertility/reproduction. If such a study were not

feasible, grow-out studies under Phase 5 should begin as soon as possible as noted above.

There was consensus among the Panel that the Agency should proceed with confirmatory

studies with a North American Rana species due to potential differences in response with those

species compared to Xenopus.
28

Some Panel members believed that the importance of field studies should be more

prominent in the Agency’s approach. Field studies often alter, sometimes drastically,

conclusions derived from laboratory studies. The same study organisms under multiple,

interacting stressors in a field situation often exhibit a dramatically elevated sensitivity to a

particular compound in comparison to exposure to it in isolation in a lab situation. These Panel

members contended that field studies are vital for addressing population-level effects of atrazine

and generating insights about ecologically relevant endpoints and therefore they should not be

consigned to a second priority to Phase 1 lab studies. 

Experimental, field-based studies could be quite incisive. More specifically, a highly

revealing experimental system in the field would be a small, temporary wetland with established,

seasonal breeding populations of amphibians. Multiple wetlands can be surrounded with drift

fences such that all individuals entering (pre-breeding adults) and leaving (post-breeding adults

and metamorphs) are captured, marked, measured, and released. Many such wetlands could be

included in a given study with subsets left as controls and others experimentally treated with

various levels of atrazine. A wealth of demographic information could be gleaned from such an

experimental study (studies by KA Berven [1990] provide a useful example of this approach and

the information that can be gained from it). Moreover, high densities of such wetlands with little

or no previous exposure to atrazine are readily available, thereby limiting many of the

complications associated with contaminated controls that have arisen in many of the studies

conducted on the topic to date in agricultural areas. Additional benefits would accrue if

laboratory studies were able to identify a suite of external morphological characteristics that

were associated with feminization/demasculinization and were indices of internal gonadal

abnormalities noted during laboratory studies. Use of such indices would potentially obviate the

need to sacrifice animals in the field for time-consuming histopathological analysis and thereby

increase efficacy of field surveys.

b) Please comment on whether the Agency’s first set of proposed studies has accounted for

the major sources of uncertainty associated with the potential effects of atrazine on anuran

sexual differentiation. In addition to time to metamorphosis, gonadal abnormalities, and

sex ratios in the proposed Phase I assays, please comment on any other endpoints that

should be considered in this initial phase. 

There was Panel consensus that the Agency’s first set of proposed studies have accounted

for the major sources of uncertainty associated with the potential effects of atrazine on anuran

sexual differentiation.

The Panel had a number of suggestions on potential endpoints that could be measured in

Phase 1. Many of these endpoints have been presented in response to question 5c. The Panel

was in consensus that a clear set of definitions concerning the terminology for classifying

gonadal deformities should be developed by the Agency. This is essential for quantifying results

of past and future studies.

Regarding the major sources of uncertainty associated with the potential effects of

atrazine on anuran sexual differentiation, the Panel agreed with the Agency that the lack of

standardization of husbandry protocols for laboratory Xenopus laevis and Rana pipiens likely

played a significant role. The Panel is aware that the Agency has expertise in these areas. The

Panel concurred that ASTM guidelines for water quality should be followed. For example, pH,

conductivity, ammonia (total, ionized and unionized forms), nitrate, nitrite, dissolved oxygen,
29

chlorine or chloramine levels, copper and iron levels should be standardized among experiments. 

Alterations in any of these parameters may alter experimental results, particularly those

involving growth rates and development. With FETAX, Holtfreter’s solutions with adequate

calcium are adequate for raising tadpoles.

Animals should be loaded in flow-through tanks at a density according to ASTM

guidelines. Flow through tanks are preferable, but data collected from static renewal tanks

would be acceptable provided animals are loaded according to ASTM guidelines and water

quality is assessed on a daily basis (and maintained within the ASTM guidelines).

Xenopus laevis are carnivores. Therefore, diet should contain at least 14% protein. Diets

formulated for herbivores or for omnivorous fish or turtles are not suitable. Diets formulated

especially for Xenopus are commercially available for both tadpoles and adults. The quantity of

feed (g/animal) should be based on the manufacturer’s recommendation and adjusted as the

animal grows.

The following reference is recommended as a guide for housing and husbandry of

anurans: Amphibian Medicine and Captive Husbandry. (Eds.): KM Wright and BR Whitaker,

Krieger Publishing Co., Malabar, FL.

One Panel member felt that a larger number of male/female pairs should be used to

develop tadpole treatment groups. The current use of three pairs is minimal and may contribute

to variation in results among experiments reported to date. 

One Panel member believed that a stock colony of Xenopus animals should be developed

that researchers could draw upon. This would minimize potential variation among populations

studied in different laboratories. This stock colony should include phenotypic females with a ZZ

genotype so that sex ratios can be accurately determined.

c) Please also comment on the range, spacing and number of atrazine concentrations that

should be employed in the proposed testing sequence to resolve uncertainties in the shape

and nature of dose-response relationships for any observed developmental effects.

 The Panel recognized that it must answer this question in the context of realistic constraints on

the cost and effort that can be devoted to any single study to determine if aqueous concentration

of atrazine bears a relationship to gonadal irregularities in amphibians. The scope of any new

experimental study will be determined by the number of concentrations and controls tested, the

number of intra-laboratory replications (e.g. tanks) for each concentration level, and the number

of test animals per experimental replication. Range and spacing of the experimental

concentration levels are related to the number of feasible experimental points and may also be

governed by the hypothesized functional form of any underlying relationship of concentration

levels to response. 

 

 The Panel considered the components of the experimental design for a new study in the

following order: 1) selection of controls; 2) range of observations for experimental

concentrations; 3) number of independent replicates per treatment; 4) number of test animals per

experimental replicate; and 5) number and spacing of experimental concentrations.

 
30

1) Selection of controls. The experiment should include untreated control replicates and a

positive control treatment in which test animals are exposed to a concentration of 17$-estradiol. 

The Panel supported the Agency’s statement that a positive androgen control group is not

needed.

2) Range of observations: The experimental concentrations used in the experiment should

include an untreated control and span the range of ecologically relevant atrazine concentrations

to include at least one concentration exceeding the upper percentile bounds that have been

measured in natural, aquatic environments. 

3) Number of replications per treatment: From the Xenopus Hayes et al., (2002a) and Carr et al.

(2003) studies and Rana study (Hayes et al., 2002b), estimates of the empirical intra-replication

(intra-tank) correlation can be calculated. The estimated intra-class correlation should be used to

determine the number of replicates per treatment arm and the allocation of total sample size to

replications and test animals per replication. Since there will be uncertainty in the estimation of

intra-class correlation for a future study, the Panel advised the Agency to err in selecting values

that are near the high end of the range of values observed in the previous studies. 

Underestimation of the intra-replicate correlation in planning the sample size allocation can

seriously attenuate the true power of the tests of the hypothesis for the chosen endpoints. In

contrast, overestimation of the intra-class correlation will produce some cost inefficiency in the

design, but will not generally endanger the power to detect significant true effects in the

experimental study.

4) Number of test animals subjects per experimental replicate: Having established a working

value for the intra-replicate correlation for the class of outcomes of interest and the desired levels

of statistical power for the specific hypothesis tests, the optimal number of animal subjects per

replicate can be determined using standard sample size allocation formulas. The allocation is

obviously constrained by bio-loading and water quality considerations that are discussed in the

Agency’s White Paper. 

5) Number and spacing of experimental concentrations: Determination of the optimal number

and spacing of treatments is governed by the hypothesized shape of any underlying

concentration-response relationship. The Panel had determined that data from existing studies

support testing the hypothesis that there is a relationship between atrazine concentrations and

gonadal abnormalities in amphibians. Hayes, in his presentation to the Panel, provided data and

arguments that the relationship is not monotonic --potentially an “inverted” response. However,

Hayes’ conclusion has not been replicated in other studies. The Panel believes there is

insufficient data for an understanding of the shape of the concentration-response curve. Data are

needed to evaluate, at the low end of the curve, whether a non monotonic relationship exists. A

robust design approach would be to follow the pattern of previous studies and use multiple

concentration points to accommodate the possibility that any effect is monotonic, or alternatively

that there is a simple non-monotonic relationship between atrazine concentration and rates of

gonadal abnormalities in the amphibian test subjects. There is an advantage to retaining

concentration points that have been used in the prior research by Hayes, Carr, Hecker and others:

0,0.01,0.1, 1, 10 and 25 ug/l. The Panel also recommended adding an upper concentration level

that exceeds the 25 ug/l value at which Hayes’ and Carr’s studies have detected increases in the

number of abnormalities. Such a spacing of concentration treatments would be sufficient to test

the hypothesis of an effect of atrazine concentration on gonadal abnormalities and to secondarily

test whether any real effect is monotonic or non-monotonic across atrazine concentration levels. 
31

d) Please comment on the Agency’s recommendation that X. laevis be used as the primary

biological model in the proposed studies and whether or not the mechanisms involved in

sexual differentiation of the ranid and pipid species are sufficiently similar to predict

effects and associated dose-response curves for Rana and/or to efficiently design Rana

studies. 

The Panel concluded that Rana should be used as a collaborating species. The choice of

X. laevis as a primary biological model is well justified since developmental and sexual

differentiation of Xenopus laevis have been extensively studied, the published literature is

extensive, and this is not the case for any North American species. However, the primary

ecological concern for the Agency remains effects, if any, on North American anurans. Rana is

the best studied North American genus and is the best choice for study. Sexual differentiation in

ranids has not been as extensively studied as in Xenopus but, given strong conservation of basic

developmental mechanisms and no known dissimilarities, there is no reason at this time to

believe that mechanisms will differ substantively.

e) In this regard, are there important differences between the species to conclude that any

affected developmental processes observed in X. laevis would not occur in Rana? 

Several Panel members stated that there is little or no evidence to demonstrate that there

are significant differences between Rana or Xenopus developmental pathways that would

preclude the Agency from using Xenopus as a model in future studies. However, some Panel

members noted that there are significant differences between the two groups of species in timing

of life cycle events such that concerns about differences in developmental pathways cannot be

eliminated.

f) Alternatively, are there developmental pathways in Rana, but not in X. laevis, that raise

concerns about using X. laevis as the primary biological model in any future atrazine

studies? 

No differences have been identified to date that would raise concerns about differences in

developmental pathways.

g) Assuming X. laevis and Rana are sufficiently concordant from a toxicodynamic

perspective with regard to potential developmental effects of atrazine, what critical

toxicokinetic processes should be considered for extrapolating X. laevis dose-response

relationships to Rana and/or for designing subsequent studies with Rana?

To the Panel’s knowledge, there are no pharmacokinetic studies on Xenopus

laevis or Rana pipiens that would allow direct comparisons between uptake, metabolism,

or depuration. However, given that Xenopus is a fully aquatic species and Rana is semiterrestrial, there are bound to be differences. The significance of such differences, if any,

is uncertain. Measuring whole body burden and/or residues in specific tissues will yield

information on this topic.
32

REFERENCES

Bögi C, Schwaiger J, Ferling H, Mallow U, Steineck C, Kalbfus W, Negele RD, Lutz I,

and Kloas W. 2003. Endocrine effects of environmental pollution on Xenopus laevis and

Rana temporaria. Environ Res. (in press).

Berven KA. 1990. Factors affecting fluctuations in larval and adult stages of the wood

frog (Rana sylvatica). Ecology 71: 1599-1608.

Carr JA, Gentles A, Smith EE, Goleman WL, Urquidi LJ, Thuett K, Kendall RJ, Giesy

JP, Gross TS, Solomon KR, and Van Der Kraak G. 2003. Response of larval Xenopus

laevis to atrazine: assessment of growth, metamorphosis, and gonadal and laryngeal

morphology. Environ. Toxicol. and Chem. 22(2): 396 - 405.

Chang and Witschi. 1956. Genic control and Hormonal Reversal of Sex Differentiation

in Xenopus. Proc Soc Exp Biol Med. 93: 140 - 144.

Cooper RL, Stoker TE, Tyrey L, Goldman JM, and McElroy WK. 2000. Atrazine

disrupts the hypothalamic control of pituitary-ovarian function. Toxicol Sci. 53: 297-307.

Curtis LA and Barse AM. 1990. Sexual anomalies in the estuarine snail Ilyanassa

obsoleta: imposex in females and associated phenomena in males. Oecologia 84: 371-

375.

Giesy JP, Hecker M, and Jones PD. 2003. South African analytical support – hormone

and aromatase analysis (SA- 01C). Aquatic Toxicology Laboratory, Michigan State

University, National Food Safety and Toxicology Center, E. Lansing, MI. Sponsor:

Syngenta Crop Protection, Inc., Laboratory Study ID ECORISK Number MSU-07.

Gooding MP, Wilson VS, Folmar LC, Marcovich DT, and LeBlanc GA. 2003. The

biocide tributyltin reduces the retention of testosterone as fatty acid esters in the mud

snail (Ilyanassa obsoleta). Environ Health Perspect. 111: 426-430.

Gooding M, Gallardo C, and LeBlanc GA. 1999. Imposex in three marine gastropod

species in Chile and potential impact on muriciculture. Mar Pollut Bull. 38: 1127-1231.

Hayes T, Haston K, Tsui M, Hoang A, Haeffele C, and Vonk A. 2003. Atrazine-induced

hermaphroditism at 0.1 ppb in American leopard frogs (Rana pipiens): laboratory and

Field Evidence. Enviro. Health Perspect. 111 (4), 568-575.

Hayes T, Collins BA, Lee M, Mendoza M, Noriega N, Stuart AA, and Vonk A. 2002a. 

Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low

ecologically relevant doses. Proc Nat Acad Sci 99 (8): 5476-5480.

Hayes T, Haston K, Tsui M, Hoang A, Haeffele C and Vonk A. 2002b. Atrazineinduced hermaphroditism at 0.1 ppb in American leopard frogs (Rana pipiens):

laboratory and field evidence. Enviro Health Perspect. http://dx.doi.org [Online 23

October 2002].
33

Hecker M, Coady KK, Villeneuve DL, Murphy MB, Jones PD and Giesy JP. 2003a. A

pilot study of response of larval Rana clamitans to atrazine exposure: assessment of

metamorphosis and gonadal and laryngeal morphology and selected hormones and

enzyme activities. Aquatic Toxicology Laboratory, Michigan State University, National

Food Safety and Toxicology Center, E. Lansing, MI. Sponsor: Syngenta Crop

Protection, Inc., Laboratory Study ID ECORISK Number MSU-03. (MRID No.

45867703).

Hecker M, Coady KK, Villeneuve DL, Murphy MB, Jones PD, and Giesy JP. 2003b. 

Response of Xenopus laevis to atrazine exposure: assessment of the mechanism of action

of atrazine. Aquatic Toxicology Laboratory, Michigan State University, National Food

Safety and Toxicology Center, E. Lansing, MI. Sponsor: Syngenta Crop Protection, Inc.,

Laboratory Study ID ECORISK Number MSU-04. (MRID No. 45877-04). 

Kloas W. 2002. Amphibians as model for the study of endocrine disruptors. Int Rev

Cytol. 216, 1-57. 

Kloas W, Lutz I, and Einspanier R. 1999. Amphibians as model to study endocrine

disruptors: II. Estrogenic activity of environmental chemicals in vitro and in vivo. Drugs

and Hormones as Pollutants of the Aquatic Environment – Determination and

Ecotoxicological Impacts ,Sci Total Environ. 225: 59-68.

Levy G, Lutz I, Opitz R, Krüger A, and Kloas W. 2003. Bisphenol A induces

feminization in Xenopus laevis tadpoles. Environ. Res. (in press).

Miyata S, Koike S, and Kubo T. 1999. Hormonal reversal and the genetic control of sex

differentiation in Xenopus. Zool Sci. 15: 335-340.

Miyashita K, Shimizu N, Osanai S, and Miyata S. 2000. Sequence analysis and

expression of the P450 aromatase and estrogen receptor genes in the Xenopus ovary. J

Steroid Biochem Mol Biol. 75: 101-107.

MorcilloY and C Porte. 1999. Evidence of endocrine disruption in the imposex-affected

gastropod Bolinus brandaris. Environ Res. 81: 349-354.

Moore A, Scott AP, Lower N, Katsiadaki I, and Greenwood L. 2003. The effects of 4-

nonylphenol and atrazine on Atlantic salmon (Salmo salar L) smolts. Aquaculture 222:

253-263.

Oberdorster E, Rittschof D, and McCleelan-Green P. 1998. Testosterone metabolism in

imposex and normal Ilyanassa obsoleta: A comparison of field and TBT Cl-induced

imposex. Mar Pollut Bull. 36: 144-151.

Sanderson JT, Seinen W, Giesy JW, and Van den Berg M. 2001. 2-chloro-S-triazine

herbicides induce aromatase activity in H295R human adrenocortical carcinoma cells. A

novel mechanism for estrogenicity. Toxicol Sci. 53:121-127.

Sanderson JT, Letcher RJ, Heneweer M, Giesy JW, and Van den Berg M. 2000. Effects
34

of chloro-S-triazine herbicides and metabolites on aromatase activity in various human

cell lines and on vitellogenin production in male carp hepatocytes. Environ Health

Perspect. 109: 1-6.

Smith BS. 1981. Tributyltin compounds induce male characteristics on female mud snails

Nassarius obsoletus = Ilynassa obsoleta. J Appl Toxicol. 1: 141-144.

Tavera-Mendoza L, Ruby S, Brousseau P, Fournier M, Cyr D, and Marcogliese D. 

2001a. Response of the amphibian tadpole Xenopus laevis to atrazine during sexual

differentiation of the ovary. Environ Tox and Chem. 21 (6): 1264 - 1267.

Tavera-Mendoza L, Ruby S, Brousseau P, Fournier M, Cyr D, and Marcogliese D. 

2001b. Response of the amphibian tadpole Xenopus laevis to atrazine during sexual

differentiation of the testes. Environ Tox and Chem. 21 (3): 527 - 531.

Tobias M, Tomasson J, and Kelley DB. 1998. Attaining and maintaining strong vocal

synapses in female Xenopus laevis, J Neurobiol. 37: 441-448.

Wright KM. 2001. Diets for captive amphibians. In K.M. Wright and R.B. Whittaker

(ed.) Amphibian Medicine and Captive Husbandry. Krieger Publishing Company,

Malabar, FL. pp. 63-72.