2 

White Paper 

(Review Document) Source Author(s) Date 

FIFRA 10(g) 

Protected 

Problem Formulation for the 

Environmental Fate and Ecological 

Risk Assessment for Atrazine 

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Appendix A: 

Supporting Environmental Fate Data 

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Appendix B: 

Supporting Ecological Toxicity Data 

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Appendix C: 

Open Literature Review of Amphibian 

Data 

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Appendix D: 

Bibliography of Microcosm and 

Mesocosm Studies and Criteria Used 

to Screen Studies for Analysis of 

Atrazine Risks to Aquatic Plant 

Communities 

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Appendix E: 

Proposed Methodology for Specifying 

Atrazine Levels of Concern for 

Protection of Plant Communities in 

Freshwater Ecosystems 

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Appendix F: 

Critique of Syngenta Corporation’s 

Probabilistic Implementation of 

CASMATZ for Ecological Risk 

Assessment 

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Appendix G.a.: 

The PATI Model Working Program

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Appendix G.b.: 

Microcosm and Mesocosm and 

Atrazine Ecological Exposure 

Monitoring Program Input Files 

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Appendix G.c.: 

PATI Output and CE-LOC Calculation 

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Appendix G.d.: 

Monitoring Data Processing Macro File 

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Appendix G.e.: 

Summary of Monitoring Data, Including 

Listing Water Monitoring Sites That 

Exceed the Aquatic Plant Community 

Level of Concern 

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Appendix G.f.: 

Breakdown of Species Tested in 

Ecotoxicity Studies 

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Appendix G.g.: 

Log-Linear 95% C.I. Bias Factor 

Sensitivity Analysis Using AEEMP 

Monitoring Data 

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Appendix G.h.: 

NAWQA Map Data: 60-Day Maximum 

Average 7 ppb Exceeders 

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Appendix G.i.: 

NAWQA Map Data: Bias Factor 

Adjusted 4 ppb Exceeders 

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Appendix G.j.: 

NAWQA Map Data: Bias Factor 

Adjusted 7 ppb Exceeders 

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Appendix G.k.: 

NAWQA Map Data: 60-Day Maximum 

Average 4 ppb Exceeders 

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Appendix G.l.: 

Monitoring Data Used in Bias Factor 

Estimation (Definitive Analysis) 

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Appendix G.m.: 

Preliminary Bias Factor Analysis 

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Appendix G.n.: 

Monitoring Data Used in Bias Factor 

Estimation (Preliminary Analysis) 

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Appendix G.o.: 

Crystal Ball Programs 

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Appendix G.p.: 

Crystal Ball Output Files 

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Appendix G.q.: Bias Factor Calculation CD 

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Appendix H: 

Bibliography of ECOTOX Accepted 

Papers (2003-2011) 

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Appendix I: 

Bibliography of ECOTOX Rejected 

Papers (2003-2011) 

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Appendix J: 

Bibliography of ECOTOX Excluded 

Papers (2003-2011) 

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Appendix K: 

Bias Factor Regression Equations 

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Appendix L: 

Degradation Product Structures 

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Appendix M: Reanalysis of Toxicity 

Data for Constructing PATI 

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Appendix N: Evaluation Guidelines for 

Ecological Toxicity Data in the Open 

Literature 

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4 

Charge Questions 

Evaluation of the Data on Amphibians 

There are an increasing number of literature studies examining whether there is a causal 

relationship between atrazine exposure and abnormal sexual development, metamorphosis 

and growth, and/or immune response in amphibians (Chapter II of the White Paper). In 

order to determine the degree to which these studies are appropriate for incorporation into 

risk assessment, EPA evaluated the data within the context of considering the nature and 

degree of the uncertainties in each study. 

1. Is the SAP aware of any other laboratory-based or field-based studies not included in 

this White Paper that should be considered? 

2. EPA identified test design elements that could potentially confound the ability of a study 

to discern a causal relationship between exposure to atrazine and an effect on 

amphibians (Section 7.2). Based on consideration of those test design elements, EPA 

then evaluated the available amphibian data and assigned a classification (e.g., 

Quantitative, Qualitative (high, medium, and low level of confidence), and Invalid) to 

each study indicating EPA’s confidence in the study’s conclusions (Section 7.3 and 

Appendix C). The confidence in each study was based on an evaluation of the identified 

test design elements and resulting level of uncertainty in determining a direct causal 

relationship between atrazine and potential effects to amphibians. 

a. Please comment on the completeness of EPA’s list of pertinent test design 

elements. Also, please comment on the degree to which these test design 

elements, singularly or in combination, would be expected to contribute towards 

confounding the test results. 

b. Please comment on EPA’s conclusions about the level of confidence placed on 

each study’s results. 

3. After evaluating all the available amphibian studies, one study was found to have 

accounted for all the identified test design elements (Question #2) and determined to be 

suitable for quantitative use in risk assessment for the endpoints of survival, growth and 

development (Section 7.3 and Appendix C). This study was required by an EPA Data 

Call-In (DCI) Notice following the recommendations from the 2003 SAP on atrazine and 

amphibians. The resulting study examined the effects of atrazine on Xenopus laevis at 

concentrations of 0.01 to 100 μg/L at two different laboratories. Based on the 2007 

SAP, the conclusion was, and there was agreement by the Panel, that the data from this 

study were robust and sufficient to conclude that exposure to atrazine at concentrations 

ranging from 0.01 to 100 μg/L had no effect on X. laevis development (which included 

survival, growth, metamorphosis and sexual development). 

a. Please comment on whether any new information has become available that 

leads to a different conclusion from the one which EPA reached in that the 

results of the DCI study were adequate to evaluate potential effects of atrazine 

exposure to amphibians. 
5 

b. If such information is now available, please comment on how a threshold 

determination (a concentration that is expected to cause no effect) may be 

accomplished using the identified studies.

4. After evaluation of the available amphibian toxicity data, EPA concluded that the DCI 

study mentioned in Question #3 was appropriate for quantitative use in a risk 

assessment for survival, growth and development. While the 2007 SAP Panel agreed 

that atrazine appeared to have no effect on X. laevis development at atrazine 

concentrations ranging from 0.01 to 100 μg/L, they expressed concerns about the 

suitability of X. laevis as a surrogate for native species. Review of the available toxicity 

data utilizing indigenous species suggests that suitable protocols, including adequate 

husbandry methods in particular, that would enable EPA to quantify a toxicity endpoint 

representative of a clear and consistent response from atrazine for native species, may 

not exist. Please comment on whether there are suitable methods for testing native 

amphibians with particular regard to husbandry and laboratory culturing conditions, 

consistent with the design elements recommended by the 2003 SAP. 

5. A number of studies report the potential for atrazine to modify immune function and 

infection susceptibility in amphibians (Appendix C). EPA believes the research on these 

different hypotheses does not provide sufficient data to establish causal linkages among 

different levels of biological organization to result in adverse effects. Therefore, EPA 

concluded that a mode of action or adverse outcome pathway leading to effects on 

amphibian survival, growth or development cannot be established at this time. 

a. Please comment on whether the data in the existing database reasonably 

supports the hypotheses, or demonstrates that atrazine affects immune function 

and/or infection susceptibility leading to adverse effects on survival, growth or 

development; i.e., are there sufficient data to establish an adverse outcome 

pathway for atrazine effects on immune function? Please provide a rationale for 

the Panel’s position and discuss the associated strengths and weakness with the 

data supporting the rationale. 

b. If the Panel concludes that the existing data are sufficient to formulate 

hypotheses that atrazine adversely affects immune function and infection 

susceptibility, but are not sufficient to test the hypotheses (refute or confirm), 

then please comment on specific study protocols that can be used to test these 

hypotheses with sufficient rigor to identify effects that can be directly and 

quantitatively attributed to adverse impacts on amphibian reproduction, growth 

and/or survival. 
6 

The Method for Determining the Atrazine Level of Concern 

for Aquatic Plant Communities 

Chapter I, Section 6.14 and Appendix D discuss the mesocosm and microcosm (cosm) study 

selection criteria for determining acceptability in evaluating the impact of atrazine exposure on 

the structure, function and productivity of aquatic plant communities. Chapter I, Section 6.14, 

and Appendix G(f) also provide summaries of the source of the natural communities used to 

populate the acceptable cosm studies, as well as details on species composition of the cosms. 

The acceptable cosm studies were used in the establishment of an Aquatic Plant Community 

Concentration Equivalent Level of Concern (CE-LOC) for atrazine. EPA has refined its method 

for determining an CE-LOC for atrazine based on the recommendations provided by SAPs in 

2003, 2007, and 2009 (Chapter IV, Sections 13 & 14; for a summary of changes since 2003 

please refer to Table 21). This methodology will be used for a national-scale assessment of 

atrazine concentrations in freshwater and estuarine/marine ecosystems in the upcoming 

registration review. The following questions concern the composition and biological relevance of 

the cosm studies, and the method, assumptions and uncertainties in determining a CE-LOC for 

atrazine. 

6. The cosms were comprised of natural communities of periphyton/phytoplankton; in some 

cases, vascular plants, invertebrates and vertebrates present in those communities were 

included in the study (Chapter I, Section 6.1.4). These sources were generally described 

as streams, lakes, reservoirs, and springs, and are considered to be representative of 

the structure and function of aquatic plant communities in such water bodies. Given the 

diversity of sources and the described communities, please comment on the extent to 

which these cosm studies taken together provide useful and reasonable physical models 

of the natural aquatic plant communities exposed to atrazine in the U.S. 

7. The Aquatic Plant Community CE-LOC methodology for atrazine is a four stage process 

that uses single-species plant toxicity data and cosm studies to discern atrazine 

concentrations and exposure durations that may cause adverse effects on aquatic plant 

communities. As a result, a CE-LOC for atrazine is developed which, together with 

monitoring data, can be used to identify watersheds where concentrations may result in 

adverse effects to aquatic plant community structure, function, and/or productivity. 

Please comment on the methodology EPA has used to derive the atrazine CE-LOC for 

aquatic plant communities, and in particular on EPA’s characterization of the 

uncertainties and assumptions in this methodology (Chapter IV, Sections 13 & 14). 

8. The 2009 SAP recommended using an effects index or concentration metric, rather than 

categorical LOC thresholds in order to take advantage of data from Syngenta’s Atrazine 

Ecological Exposure Monitoring Program (AEEMP). At that time the LOC threshold for 

atrazine effects to plant communities was established at 10 μg/L for a 60-day rolling 

average. The current analysis using the Plant Assemblage Toxicity Index (PATI) 

indicates the CE-LOC can range from 4 to 7 μg/L (Chapter IV, Section 14.3 & 14.4). 

Please comment on this CE-LOC and whether it reasonably represents a range below 

which permanent or irreversible change in aquatic plant community structure, function, 

and/or productivity due to atrazine exposure would not be expected. 
7 

9. Based on previous analyses of the available ecotoxicity data, EPA concluded for 

atrazine that the level of concern for effects on aquatic plant communities (CE-LOC) was 

lower than the atrazine concentrations observed to produce significant direct or indirect 

effects on invertebrates, fish and amphibians. Given the current analysis of the 

ecotoxicity data (Chapter I, Section 6) and the Aquatic Plant Community LOC 

methodology, EPA continues to believe the original conclusion still holds true. Please 

comment on how well the available database supports EPA’s conclusion that the CELOC is lower than exposures that result in significant effects on the growth, survival and 

reproduction of aquatic animals. 

Method for Comparing Monitoring Data to the 

Aquatic Plant Community LOC for Atrazine 

Chapter V and Section 16 address the issue of identifying surface water bodies with potential 

levels of atrazine that exceed the aquatic plant community levels of concern (CE-LOC). EPA 

evaluated several statistical methods (kriging, stochastic simulation, covariate analysis, and bias 

factors) to address uncertainties associated with sampling frequency as it impacts the ability to 

quantify atrazine concentrations from monitoring data (USEPA, 2011). The FIFRA SAP in 2011 

advised that use of bias factors was a viable approach for addressing uncertainty in monitoring 

data. The following questions are related to the use of bias factors in identification of surface 

water bodies exceeding the atrazine CE-LOC. 

 

10. Please comment on the strengths and limitations of EPA’s development and use of bias 

factors (Chapter V, Section 16.1) for addressing uncertainties in monitoring data. 

11. Prediction of bias factors is dependent on the selection of an appropriate model. EPA 

illustrated (Chapter V, Section 16.1) both categorical and regression methods for 

prediction of bias factors based solely on the number of samples taken in the 2nd and 3rd

quarters of the year (April 1st to September 30th). 

a) Please comment on EPA’s prediction of bias factors from monitoring data using 

categorical or regression method approaches. 

b) Please comment on any additional methods for estimating bias factors that would 

be useful in this situation.

12. EPA illustrated (Chapter V, Section 16.1) both categorical and regression methods for

estimation of bias factors as a function of the sampling frequency of monitoring data. 

Step-wise regression analysis indicates that watershed size and average flow rate in the 

2nd and 3rd quarters of the year are not significant variables for prediction of bias factors. 

However, the number of samples in the 2nd and 3rd quarters of the year was found to be 

a significant variable, accounting for 46% of the variation in the bias factor. What other 

variables, if any, should be considered in the prediction of bias factors? 

 

13. EPA examined (Chapter V, Section 16.1) the performance of various regression 

equations to assess the failure percentage for identification of monitoring site-years with 

true maximum 60-day average concentrations exceeding the CE-LOC for atrazine. This 

analysis showed that application of a bias factor, based on sample number during the 
8 

2nd and 3rd quarter of the year, substantially reduced the number of sites with 

underestimation of true maximum 60-day means. Given the EPA analysis, what other 

tests, if any, should be conducted to assess the performance of regression models for 

prediction of bias factors? 

14. An important consideration for bias factor prediction is the ability to identify monitoring 

sites that potentially exceed the CE-LOC for atrazine. EPA provided an example 

(Chapter V, Section 16.2 and 16.3) of the use of a log-linear regression equation to 

estimate bias factors from the USGS National Water Quality Assessment Program 

NAWQA data and identification of monitoring site-years exceeding the CE-LOC for 

atrazine. This analysis identified sites in both the Midwestern United States as well as 

outside the major corn growing areas with atrazine concentrations potentially above the 

CE-LOC. What are the strengths and limitations of using a single regression model for 

prediction of spatially and temporally explicit bias factors for a nationally-distributed 

monitoring data set?