Scientific Issues Associated with Problem 

Formulation for the Reassessment of 

Ecological Risks from the Use of Atrazine

U.S. Environmental Protection Agency 1

Opening Remarks

Steven Bradbury, Ph.D., Director 

Office of Pesticide Programs

U.S. Environmental Protection Agency
U.S. Environmental Protection Agency 2
Framework for Assessing the Potential 

Ecological Risks Posed by Atrazine

U.S. Environmental Protection Agency 3

Dana S. Spatz, M.S.

Environmental Fate and Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency
U.S. Environmental Protection Agency 4
Regulatory History

• Atrazine first registered in 1958

• Primarily used in corn, sorghum, and sugarcane production

Broadleaf and some grassy weeds

U.S. Environmental Protection Agency 5

• Broadleaf and some grassy weeds

• Interim Reregistration Eligibility Decision (IRED) in 2003

• Memorandum of Agreement (MOA) in 2003
U.S. Environmental Protection Agency 6
2003 IRED and MOA

• Monitor for atrazine in a subset of 1172 vulnerable watersheds

• Identification of an appropriate ecological Level of Concern

Mitigate environmental exposures if EPA determines that 

U.S. Environmental Protection Agency 7

• Mitigate environmental exposures if EPA determines that 

mitigation is necessary

• Determine potential for effects on amphibian gonadal 

development
Scientific Advisory Panel Meetings on 

Ecological Risk Issues

June 2003

amphibian 

gonadal 

development

December 2007

aquatic plant 

community level 

effects

June 2012

problem formulation:

update on amphibians, 

PATI, monitoring, and 

the atrazine LOC 

U.S. Environmental Protection Agency 8

October 2007

follow up on 

amphibian gonadal 

development

May 2009

Plant Assemblage 

Toxicity Index and 

watershed vulnerability 

analysis
Integrate Available Information

PROBLEM

FORMULATION

Analysis

FRAMEWORK FOR ECOLOGICAL RISK ASSESSMENT

)

As

Necessary

Acquire

Data,

Iterate

Process,

Planning

(Risk Assessor/

Risk Manager

Dialogue)

U.S. Environmental Protection Agency 9

Risk Management Risk Management

Communicating Results

to the Risk Manager

Analysis

Plan

ANALYSIS

RISK

CHARACTERIZATION

Monitor

Results
What’s New

Amphibian Toxicity – Amy Blankinship

• open literature on amphibian toxicity data

• evaluation of test design elements

• adverse outcome pathway

Atrazine Level of Concern – Frank Farruggia & Russell Erickson

U.S. Environmental Protection Agency 10

Atrazine Level of Concern – Frank Farruggia & Russell Erickson

• refinements to the Plant Assemblage Toxicity Index (PATI)

• re-evaluation of atrazine cosm studies

• modifications to determining atrazine LOC for aquatic plant communities

• sensitivity analysis

Comparing Monitoring Data to the LOC – James Hetrick

• development of bias factors

• integration of available monitoring data to define scope 
Evaluation of Amphibian Toxicity Data

for Atrazine

U.S. Environmental Protection Agency 11

Amy Blankinship, M.S.

Environmental Fate and Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency
Overview

• Background on previous evaluations of amphibian toxicity data

• Current methodology for evaluation of amphibian toxicity data

Results of current evaluation

U.S. Environmental Protection Agency 12

• Results of current evaluation

• Uncertainties

• Overall conclusions
Previous Evaluations of Amphibian Toxicity Data for Atrazine

• 2003 SAP – Potential Developmental Effects of Atrazine on Amphibians

• EPA presented and the SAP Panel agreed upon:

1. Available data supported further evaluation of atrazine exposure on 

amphibian gonadal development

2. A tiered testing approach 

3. Critical test design elements for future testing:

Test species and stage sensitivity

U.S. Environmental Protection Agency 13

• Test species and stage sensitivity

• Test conditions

• 2004 – EPA issued a Data Call-in (DCI) for evaluating amphibian 

developmental effects from atrazine exposure
Previous Evaluations of Amphibian Toxicity Data for Atrazine

2007 SAP – Potential for Atrazine to Affect Amphibian 

Gonadal Development

• Reviewed DCI data (Hosmer et al. 2007 (registrant-submitted) and published as 

Kloas et al. 2009) and other relevant data

• DCI data were two studies conducted at two different labs, using the same 

protocol, to examine reproducibility of effects

U.S. Environmental Protection Agency 14

Study Details Study Attributes

Test concentrations Negative Control, 0.01, 0.1, 1.0, 25 and 100 μg/L and 0.2 μg/L E2

No. of replicates/treatment 8 for atrazine and E2 groups; 16 for negative control

No. of tadpoles/replicate 25

No. of tadpoles/treatment 200 for atrazine and E2 groups; 400 for negative control

No. of tadpoles/study 1,600

A total of 3,200 tadpoles exposed for both studies conducted using same protocol

No. of frogs used in analyses at test termination 1,312 (Lab #1) + 1,555 (Lab #2) = 2,867
Previous Evaluations of Amphibian Toxicity Data for Atrazine

2007 SAP – Potential for Atrazine to Affect Amphibian 

Gonadal Development

• EPA concluded that DCI study provided reliable information 

on amphibian toxicity

U.S. Environmental Protection Agency 15

• Panel concluded DCI study provided reliable results for 

Xenopus laevis; however, had concerns regarding:

• use of X. laevis as surrogate for indigenous species; and 

• sensitivity of X. laevis strain used in study
Status of Amphibian Toxicity Data

• Additional amphibian toxicity studies published since 2007 SAP

• New studies examined many different endpoints

• An evaluation of these additional studies needed to be 

U.S. Environmental Protection Agency 16

• An evaluation of these additional studies needed to be 

completed

• There are other published reviews that examine/evaluate 

relationship between atrazine and amphibian effects (i.e., Rohr 

and McCoy 2010, Hayes et al. 2011, Solomon et al. 2008)
Evaluation Methodology - Objectives

1

• Identify critical test design elements

2

• Classify each amphibian toxicity study

U.S. Environmental Protection Agency 17

2

3

• Categorize endpoints in each study

4

• Compare results across studies for an endpoint 

category 
Evaluation Methodology - Objectives

1

• Identify critical test design elements

2

• Classify each amphibian toxicity study

U.S. Environmental Protection Agency 18

2

3

• Categorize endpoints in each study

4

• Compare results across studies for an endpoint 

category 
Open Literature Preliminary Screening Criteria 

• All open literature studies undergo a preliminary screening for 

further evaluation – this is for all chemicals evaluated by the 

Office of Pesticide Programs (OPP)

• A study was included for further evaluation if: 

U.S. Environmental Protection Agency 19

• treatments were exposed to only atrazine

• effect on live, intact organisms, and test species reported and verified

• paper is primary source of data, published in English as a full article

• study type is reported
Open Literature Preliminary Screening Criteria

• If study met preliminary screening criteria it was retained for 

further evaluation

• 64 laboratory studies included for further evaluation 

Excluded studies:

U.S. Environmental Protection Agency 20

• Excluded studies:

• 11 cosm studies - while passed screening, excluded for this 

analysis due to potential multiple interactions resulting in both 

potential direct and/or indirect effects (still considered for further 

evaluation and potential use in risk assessment)

• 9 field studies - excluded due to potential major confounding 

factors (i.e., environmental conditions) and not considered for use 

in risk assessment
Test Design Elements (Further Evaluation)

• Currently no standardized guideline/methodology for chronic

developmental testing with amphibians (e.g., ASTM guideline)

• EPA identified critical test design elements that could 

potentially confound/influence the ability to discern a causeeffect relationship between atrazine and a biological receptor

U.S. Environmental Protection Agency 21

effect relationship between atrazine and a biological receptor

• EPA identified test elements were based on:

• ASTM guidelines

• Conclusions from the 2003 and 2007 SAP Panels; and

• U.S. EPA guidelines

• Identified elements were further classified
Test Design Elements

Test design elements that would invalidate study results

Test Design Element - Invalidate

1 Atrazine concentrations not reported 

2 Control group not run concurrently

3 Test chemical contamination of control

U.S. Environmental Protection Agency 22

3 Test chemical contamination of control

4 Control mortality >30%

5 Solvent concentration > 0.05%

6

Presence of other stressors (biological, chemical, physical) expected to 

compromise study

7 Aberrant effects in control

8 Insufficient replication (n=2)

9 Insufficient data to allow reviewer to understand/substantiate study results
Test Design Elements

Test design elements that would raise concern by themselves but 

not necessarily invalidate study results

Test Design Element – Raise concern

1 When solvent used, both negative and solvent group not tested

2 Statistically significant difference between negative and solvent group

U.S. Environmental Protection Agency 23

3 Solvent concentration >0.01%

4 DMSO used as solvent carrier choice

5 Plastic used as test vessels

6 Study not conducted using technical grade atrazine (e.g., formulation)

7 High loading rate (potentially coupled with longer renewal rate and lack of 

reported water quality)

8 Field collected organisms
Test Design Element – Organismal Loading

Current recommended loading = 1 tadpole/L (≈ 1 g/L) (Based on 

2003 SAP, using X. laevis and ASTM)

• ASTM loading: 1 g/L/day for flow-through designs; 0.5-0.8 g/L for static and static-renewal

• Studies below were conducted using a static-renewal design

Species

Weight (grams)

per organism 

Source

Rana pipiens 2.5 2003 SAP (Hayes et al. 2002c)

U.S. Environmental Protection Agency 24

Rana pipiens 2.5 2003 SAP (Hayes et al. 2002c)

Rana pipiens 0.83 Allran and Karasov 2000

Rana pipiens 2.2 Hayes et al. 2006

Rana clamitans 2 2003 SAP (Hecker et al. 2003)

Litoria raniformis 0.9 Choung et al. 2011

Hyla versicolor 0.5 LaFiandra et al. 2008

Ambystoma barbouri 0.75 to 0.95 Rohr et al. 2011 

Ambystoma macrodactylum 0.85 Forson and Storfer 2006

Ambystoma tigrinum 11-16 Larson et al. 1998 
Evaluation Methodology - Objectives

1

• Identify critical test design elements

2

• Classify each amphibian toxicity study

U.S. Environmental Protection Agency 25

2

3

• Categorize endpoints in each study

4

• Compare results across studies for an endpoint 

category 
Study Classification 

• Each study (n = 64) evaluated using the list of identified 

potential confounding test elements

• The results for each study were classified according to level of 

confidence in the study results

U.S. Environmental Protection Agency 26

• Studies were classified as:

• Quantitative

• Qualitative (higher, medium or lower level of confidence)

• Invalid
Results of Study Classification

• Common potential confounding test design elements observed 

in available data included:

• Lack of reporting/measuring atrazine in control treatments

• Use of field collected organisms

• Higher than recommended loading (1 tadpole/L (≈ 1 g/L) from 2003 SAP) 

U.S. Environmental Protection Agency 27

• Higher than recommended loading (1 tadpole/L (≈ 1 g/L) from 2003 SAP) 

coupled with lack of reported water quality and infrequent test solution 

renewal periods (>48 hrs)

• The majority (54 out of 64) of the available laboratory studies 

had a classification of Qualitative with a lower level of 

confidence (n = 30) or Invalid (n = 24)
Results of Study Classification

15

20

25

30

#

of Studies

Qualitative (Lower Confidence)

8

10

12

14

16

Invalid

U.S. Environmental Protection Agency 28

0

5

10

15

#

of Studies

0

2

4

6

8
Results of Study Classification

One study was classified as Quantitative (synthesized in Kloas et al. 

2009)

• This was the study conducted as a result of the 2003 SAP and 

required by the 2004 DCI which was then submitted by the 

registrant (Hosmer et al. 2007)

U.S. Environmental Protection Agency 29

registrant (Hosmer et al. 2007)

• The study design was able to account for potential confounding 

effects that limited the utility of previously conducted studies

• Reported NOAEC = 100 ppb for survival, metamorphosis, growth, 

behavioral effects, and sexual development, highest concentration 

tested
Results of Study Classification –Quantitative Study

Due to the robustness of this study it was able to account for potential 

confounding test design elements

Potential Limitations of Study Identified by EPA How Resolved

Measurement of atrazine in one cluster (n=4 

replicates) in Negative Control tanks for Lab #1 Tanks dropped from analysis, 2007 SAP Panel 

concurred with EPA on decision to omit 8 or 

U.S. Environmental Protection Agency 30

replicates) in Negative Control tanks for Lab #1 concurred with EPA on decision to omit 8 or 

half of the Negative Control tanks given the 

power of the test Microbial bloom in one cluster of Negative 

Control tanks for and one tank at 1 ppb at Lab #1

A qualitative rather than quantitative assessment

of certain histological endpoints

Panel concurred with qualitative for some 

endpoints; data for other histological

endpoints reanalyzed resulting in same 

conclusion

One pathologist for analysis of histological

endpoints

Panel recommended additional review; 

conducted and resulted in same conclusion
Results of Study Classification –Quantitative Study

Due to the robustness of this study it was able to account for potential 

confounding test design elements

Potential Limitations of Study Identified by 

Panel How Resolved

Sensitivity of X. laevis strain Response to positive control compared to 

other species shown to be sensitive

U.S. Environmental Protection Agency 31

other species shown to be sensitive

Resultsfrom labs analyzed separately Combined data which resulted in same 

conclusion

Lack of exposure to degradates

Two degradates measured in study (DIA and 

DEA); also due to atrazine half-lives, none of 

the primary degradates likely to form within 

a 48- or 72-hour renewal time-frame

Lack of evaluating reproductive fitness

(grow-out)

Currently, robust methods to examine 

reproduction function and fitness are not 

available and current evaluation of data 

indicate that higher tiered testing is not 

warranted
Results of Study Classification

Nine (14%) studies were classified as Qualitative with either a higher or 

medium level of confidence

Study Classification Effects (μg/L) Species Tested

Acute Survival/Teratogenesis

1 Birge et al. 1980 Higher

LC50 (4 days post-hatch) 

= 410 to >48,000

(survival and teratogenesis)

Rana catesbeiana, R. 

pipiens, R. palustris, Bufo 

americanus

U.S. Environmental Protection Agency 32

2 Howe et al. 1998 Medium 96 hr LC50 = 14,500 - 47,600 R. pipiens, B. americanus 

3

Morgan et al. 

1996 Medium 96 hr LC50 = 40,800 - 51,40896 hr EC50 = <3,264 or 13,464 X. laevis

4 Wan et al. 2006 Medium 96 hr LC50 >16,000 R. catesbeiana

Behavior Modification

5 Rohr et al. 2009 Higher NOAEC = 196; LOAEC >196 

(fear cues) B. americanus

6

Storrs Mendez et 

al. 2009 Medium NOAEC = 1,430 μg/kg; LOAEC >1,430 (soil preference) B. americanus
Results of Study Classification

Nine studies were classified as Qualitative with either a higher or 

medium level of confidence

Study Classification Effects (μg/L) Species Tested

Subchronic/Chronic Growth and Development

7

Choung et al. Higher

Metamorph. 

Growth

NOAEC=25; LOAEC >25

NOAEC=25; LOAEC >25 Litoria 

U.S. Environmental Protection Agency 33

7

Choung et al. 

2011 (Chronic) Higher Growth

Survival

NOAEC=25; LOAEC >25

>97.5% for study

Litoria 

raniformis

8

Spolyarich et 

al. 2010 

(Chronic)

Higher

Metamorph.

Growth

Sex develop.

Survival

NOAEC=30; LOAEC >30

NOAEC=30; LOAEC >30

NOAEC=30; LOAEC >30

2.3% mortality 

Limnodynastes 

tasmaniensis 

9

Freeman & 

Rayburn 2005 

(Subchronic)

Medium / 

Higher

Metamorph.

Growth

Molecular

Survival

NOAEC<100; LOAEC=100

NOAEC=800; LOAEC>800

NOAEC=varied; LOAEC= 800

0 - 16.7% mortality

X. Laevis
64

Studies

Open literature & 

registrant submitted

Apply test element criteria

9

Qualitative 

Medium/High

30

Qualitative Low

24 

Invalid

1

Quantitative

U.S. Environmental Protection Agency 34

Not for use in 

risk assessment

Low confidence in results & for 

use in risk assessment

Use results in characterizing 

risk in assessment

Use results quantitatively in risk 

assessment
Evaluation Methodology - Objectives

1

• Identify critical test design elements

2

• Classify each amphibian toxicity study

U.S. Environmental Protection Agency 35

2

3

• Categorize endpoints in each study

4

• Compare results across studies for an endpoint 

category 
Categorizing Endpoints

Endpoints in each study (n = 64 laboratory) were categorized 

according to broad endpoint parameters

Endpoint Categories

1 Survival

2 Metamorphosis

U.S. Environmental Protection Agency 36

2 Metamorphosis

3 Growth

4 Sexual Development

5 Immune response and Infection Susceptibility

6 Biochemical/Molecular modifications

7 Behavioral modifications

For each study, the NOAEC and LOAECs were extracted for 

the above categories
Evaluation Methodology - Objectives

1

• Identify critical test design elements

2

• Classify each amphibian toxicity study

U.S. Environmental Protection Agency 37

2

3

• Categorize endpoints in each study

4

• Compare results across studies for an endpoint 

category 
Evaluation of Laboratory Studies

• All laboratory studies included (n = 64) which includes all 

different classifications

• Compared NOAEC and LOAEC values for given endpoints across 

studies

U.S. Environmental Protection Agency 38

studies

• Not every study evaluated all identified endpoint categories

• Not all studies had a corresponding NOAEC/LOAEC value for each 

endpoint

• Endpoints within a study may have been given different classifications
Evaluation of Entire Laboratory Dataset (n = 64)

Survival - Acute Exposure

20

40

60

80

100

P

e

r

c

e

n

t

R

a

n

k

U.S. Environmental Protection Agency 39

LC50

Invalid

Qualitative (Lower Level of Confidence)

Qualitative (Medium)

Qualitative (Higher)

EC50

Invalid

Qualitative (Higher)

0

20

100 1000 10000 100000

P

e

r

c

e

n

t

R

a

n

k

Atrazine concentration (μg/L)
Evaluation of Entire Laboratory Dataset (n = 64)

Survival - Chronic and Sub-chronic Exposure

40

60

80

100

P

e

r

c

e

n

t

R

a

n

k

U.S. Environmental Protection Agency 40

0

20

40

0.1 1 10 100 1000 10000

No Effect

Invalid

Qualitative (Lower Level of Confidence)

Qualitative (Higher)

Quantitative

Effect

Invalid

Qualitative (Lower)

P

e

r

c

e

n

t

R

a

n

k

Atrazine concentration (μg/L)
Evaluation of Entire Dataset

Metamorphosis 

60

80

100

P

erc

e

n

t

R

a

n

k

U.S. Environmental Protection Agency 41

0

20

40

0.1 1 10 100 1000 10000

Atrazine concentration (μg/L)

LOAEC

Invalid

Qualitative (Lower)

Qualitative (Higher)

NOAEC

Invalid

Qualitative (Lower Level of Confidence)

Qualitative (Higher)

Quantitative

P

erc

e

n

t

R

a

n

k
Evaluation of Entire Dataset

Growth

40

60

80

100

P

erc

e

n

t

R

a

n

k

U.S. Environmental Protection Agency 42

0

20

40

0.1 1 10 100 1000 10000 100000

Atrazine Concentration (μg/L)

NOAEC

Invalid

Qualitative (Lower Level of Confidence)

Qualitative (Higher)

Quantitative

LOAEC

Invalid

Qualitative (Lower)

P

erc

e

n

t

R

a

n

k
Evaluation of Entire Dataset

Sexual Development

60

80

100

P

erc

e

n

t

R

a

n

k

U.S. Environmental Protection Agency 43

0

20

40

0.01 0.1 1 10 100 1000

Atrazine Concentrations (μg/L) NOAEC

Invalid

Qualitative (Lower Level of Confidence)

Qualitative (Higher)

Quantitative

LOAEC

Invalid

Qualitative (Lower)

P

erc

e

n

t

R

a

n

k
Summary of Effects for Biochemical/Molecular Modifications

All available studies classified as Qualitative (lower lever of confidence) or 

Invalid except one Qualitative Medium (Freeman & Rayburn 2005)

Species Reported Effect Reported LOAEC/NOAEC (μg/L)

Xenopus laevis ↓ plasma testosterone in males

2.5 / <2.5; 

25 / <25; 

258 / 24.8; 

no effect on plasma testosterone or estradiol in 

U.S. Environmental Protection Agency 44

X. laevis no effect on plasma testosterone or estradiol in 

males >107 / 107

X. laevis no effect on aromatase (gonad and/or brain) >25 / 25; > 100 / 100; > 258 / 258

X. laevis no effect on LH, FSH, 5-alpha reductase (gonad 

and/or brain) >25 / 25

Rana pipiens no effect on hematocrit >192 / 192

R. pipiens & R. 

sylvatica & Bufo 

americanus 

no change in hemoglobin >20,000 / 20,000

Ambystoma 

tigrinum ↑plasma thyroxine;↓ corticosterone 82 / <82
Summary of Effects for Biochemical/Molecular Modifications

All available studies classified as Qualitative (lower lever of confidence) or 

Invalid

Species Reported Effect Reported LOAEC/NOAEC (μg/L)

Xenopus laevis ↑ MMP18 expression (to evaluate tissue development) 3,500 / 350

X. laevis / Bufo 

americanus no effect on nuclear heterogeneity (represent genotox) >800 / 800

U.S. Environmental Protection Agency 45

americanus

X. laevis

↑ flow cytometric analysis meant to illustrate 

metamorphosis and development; results varied based 

on test 

800 / Varied

X. laevis change in expression of multiple genes for proteolysis, 

digestion, carbohydrate metabolism, growth 400 / <400

X. laevis changes in gene expression (acyl-CoA dehydrogenase 

and glucocorticoid receptor) 400 / <400

Rana catesbeiana

no change in relative fold difference for CYP19 ; 

correlation of CYP19 and SF-1 m RNA in the brain when 

combining 1 & 10 ppb resulted in significant difference

>10 / 10
Summary of Reported effects for Behavior Modifications

All available studies classified as Qualitative (lower lever of confidence) or 

Invalid

Species Effect LOAEC / NOAEC (μg/L)

Ambystoma barbouri ↑ locomotion activity and/or ↓ refuge use 400 / 40

A. barbouri ↓ water conserving behavior; ↑ activity 40 / 4

Bufo americanus no preference for untreated or treated soil 1,430 μg /kg

U.S. Environmental Protection Agency 46

B. americanus no effect on fear cues >196 / 196

Rana pipiens ↑ buccal ventilation 4,320 / 2,590

R. pipiens ↓ Feeding 20,000 / 12,000

R. pipiens, R. sylvatica, 

B. americanus no effect on swimming >20,000 / 20,000

Xenopus laevis suppressed mating behavior 2.5 / <2.5

X. laevis abnormal swimming 19.5 / 10.3

X. laevis no effect on feeding >400 / 400
Uncertainties

1. Effects to Indigenous Species (Native Species)

• 2007 SAP Panel stated concern about suitability of Xenopus laevis 

as surrogate for native species

U.S. Environmental Protection Agency 47

• Based on current review of available data there is still an apparent 

lack of standardized/suitable laboratory husbandry protocols
Uncertainties

2. Effects on Immune System or Parasite Susceptibility

• Some data available suggest that exposure to atrazine results in 

modifications to immune response and/or infection susceptibility

U.S. Environmental Protection Agency 48

• Currently, a clear or defined adverse outcome pathway (AOP) for 

the immune system modification interaction is considered 

insufficient
Reported Effects on Immune Response and Infection 

Susceptibility

Species Effect LOAEC/NOAEC (ppb)

Ambystoma 

tigrinum

↑ viral infection rate (effect at middle concentration); 

↓ leukocytes

16 / 1.6 

A. tigrinum & A. 

macrodactylum no effect on viral infection rate >200 / 200; >184 / 184

Rana sylvatica ↑ intensity of parasite infection and mean metacercarial cysts 30 / 3

All available studies classified as Qualitative (lower level of confidence) or Invalid

U.S. Environmental Protection Agency 49

R. sylvatica ↓ eosinophils; ↑ % of encysted cercariae 3 / <3

R. pipiens ↓ white blood cells & phagocytic cells 0.01 / <0.01

R. pipiens

↓ plaques representing antibody-secreting cells; no effect on 

lymphocytes 1 / <1; >10 / 10

R. clamitans ↑ % of encysted cercariae 201 / <201

Xenopus laevis ↑ activated caspase 3 immuno (+) cells (evaluate apoptosis) 400 / <400

X. laevis no effects on peptide activity (expressed as total protein) >986 / 986

X. laevis ↑ thymic plaques 0.19 / <0.19
Overall Conclusions

• Majority of available studies contained potential confounding 

elements that limit their utility in a risk assessment

• This is based on test criteria identified in ASTM, OPP Open 

Literature Guidance and input from 2003 and 2007 SAPs

• Additional uncertainties regarding:

U.S. Environmental Protection Agency 50

• Additional uncertainties regarding:

• effects on immune response in terms of establishing use in a risk 

assessment; and 

• ability to examine effects to native species due to lack of suitable 

husbandry protocols

• Study conducted in response to 2003 SAP resulted in 

Quantitative classification and is considered reliable for use 

in estimating risk in a risk assessment
An Overview of the Methodology for 

Determining the Level of Concern for 

Atrazine Based on Aquatic Plant 

Communities

U.S. Environmental Protection Agency 51

Frank T. Farruggia, Ph.D.

Environmental Fate and Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency

Russell Erickson, Ph.D.

Mid-Continent Ecology Division

Office of Research and Development

U.S. Environmental Protection Agency
Objective: The Method for Determining the Level 

of Concern for Aquatic Plant Communities

Stage 2: Develop effects index based on 

single-species toxicity data

Stage 1. Characterize cosm exposures and effects

U.S. Environmental Protection Agency 52

Stage 4a. Apply effects index to each 

monitoring site-year chemograph 

Stage 3a. Apply effects index to each cosm 

study endpoint

Stage 3b. Define the effects index-based 

Level of Concern

Stage 4c. Convert the effects-based LOC into a 

concentration-equivalent LOC

Stage 4b. Identify the watersheds with 

monitoring chemographs that exceed the 

effects index-based LOC
Overview

• Characteristics of atrazine

• Mobile and persistent in the environment

• Photosystem II inhibitor: reduces the production of sugar, oxygen and ATP

• Concern to Aquatic Plant Communities 

• EPA is concerned with protecting biological diversity from harmful levels of 

U.S. Environmental Protection Agency 53

• EPA is concerned with protecting biological diversity from harmful levels of 

atrazine. This encompasses protection of species, assemblage, ecosystem 

and landscape levels of biological organization and its structural, 

compositional and functional components

• Available Data

• Single species as well as community level toxicity studies

53
Mesocosm and Microcosm Studies (cosms)

• Potential effects of atrazine on plant communities have been evaluated 

using available microcosm and mesocosm studies

• Cosm studies conducted with atrazine report community based 

endpoints that reflect the aggregate responses of multiple species

U.S. Environmental Protection Agency 54

• Cosm studies incorporate partitioning, degradation, and dissipation, 

factors that are not usually accounted for in laboratory toxicity studies, 

but that may influence the magnitude of ecological effects

• Recovery after atrazine exposure is defined as a return to pre-exposure 

conditions for the affected individual, population or community, not for 

a replacement population or community of more tolerant species
Cosm Review/Selection Criteria

• Based on the recommendations from the FIFRA SAPs in 

2007 and 2009, selection criteria were developed

• A study was included only if: 

• treatments were exposed to only atrazine, 

U.S. Environmental Protection Agency 55

• treatments were exposed to only atrazine, 

• exposure concentrations were reported, 

• measured effects were specific to aquatic plant 

communities, and

• the study report is available in English
Cosm Review/Selection Criteria

A study would be rejected as invalid, if any of the following 

acceptability criteria were not met:

1. Controls run concurrently and other stressors below potential effects 

levels

2. Quantification of Exposure - minimally, measured initial concentrations 

U.S. Environmental Protection Agency 56

2. Quantification of Exposure - minimally, measured initial concentrations 

must be provided

3. Experimental Design - at least two replicates and solvents must be 

<0.5 ml/L in static tests or <0.1 ml/L in flow-through

4. Statistical Methods and Data Interpretation

• At least 3 treatments (2 replicates/each)

• Without 2 replicates for each treatment, non-linear regression requires 

sample size of 5 including control

• Statistical methods must be described and presented
Cosm Review/Selection Criteria

• A total of 35 cosm studies were originally included in the 2003 

Interim Reregistration Eligibility Decision (IRED).

• An additional 38 cosm studies were identified by the May 2009 

SAP for a total of 73 studies.

U.S. Environmental Protection Agency 57

• After the review/selection criteria were applied: 

• 31 of the original 35 studies remained

• 15 of the 38 studies recommended for consideration in the 

May 2009 SAP report remained

• These results are outlined in Appendix D
Cosms Represent Natural Aquatic Plant 

Communities

• The majority of cosms were started from natural 

communities collected from streams, lakes, reservoirs, and 

springs

• The cosms are considered to be representative of the 

U.S. Environmental Protection Agency 58

• The cosms are considered to be representative of the 

structure and function of aquatic plant communities in 

such water bodies

• Taken together they provide useful and reasonable 

physical models of the natural aquatic plant communities 

exposed to atrazine in the U.S.
Cosm Composition

Taxonomic Group Genera Species

EUBACTERIA: CYANOBACTERIA: 14 27

EUKARYOTES

ARCHAEOPLASTIDA GREEN PLANTS:

EMBRYOPHYTA: (Vascular Land Plants) 11 20

CHLOROPHYTA and STREPTOPHYTA (Green Algae): 43 86

PRASINOPHYTA (Prasinophytes): 1 1

CHROMALVEOLATES HACROBIA: 

HAPTOPHYTA (Coccolithophorads): 2 4

CRYPTOPHYTA (Cryptomonads): 4 13

U.S. Environmental Protection Agency 59

CRYPTOPHYTA (Cryptomonads): 4 13

STRAMENOPILES: 

BACILLARIOPHYTA (DIATOMS): 24 67

CHRYSOPHYTA (Golden Algae): 7 12

XANTHOPHYTA (Yellow-Green Algae): 2 2

AVEOLATES:

PYRROPHYCOPHYTA (Dinoflagellates): 4 4

EXCAVATES EUGLENOZOA (Euglenoids): 1 1

UNIKONTS FUNGI: 2 2

CHOANOFLAGELLIDA: 3 3

ANIMALS:

VERTEBRATES: 9 15

INVERTEBRATES: 137 196
Cosm Endpoints and Duration

• A total of 87 endpoints were used in the analysis to develop the 

level of concern for atrazine

• Effects observed in the cosm studies included:

• changes in aquatic plant biomass, 

• chlorophyll a concentration, 

U.S. Environmental Protection Agency 60

• chlorophyll a concentration, 

• photosynthesis rate (14C uptake, oxygen production),

• shifts in aquatic plant community structure (e.g., species composition 

and diversity)

• The durations of these studies ranged from a few weeks to 

several years at constant or variable and declining exposure 

concentrations ranging from 0.1 μg/L to 10,000 μg/L
Effect/No-Effect Endpoint Classification

• Move away from Brock Scores

• 5-point effects scoring system: 1 and 2 are no-effects, while 

3, 4, and 5 are effects

• Binary Coding, Effects or No-effects

U.S. Environmental Protection Agency 61

• Binary Coding, Effects or No-effects

• Chosen based on the recommendations by the 2009 SAP

• An effect classification was determined based on the 

statistical significance and/or magnitude of effect
Cosm Endpoint Classification

1000

10000

g

/

L

atra

zin

e)

Effect

No Effect

Effects of atrazine on experimental ecosystems as a function of exposure 

duration and average concentration (46 studies, 87 exposures)

U.S. Environmental Protection Agency 62

0.01

0.1

1

10

100

1 10 100

A

v

era

g

e

Stu

d

y

C

o

n

c

e

ntratio

n(μ

g

/

L

Exposure Duration (days)
Goals of the LOC Methodology

The LOC must address differences in concentration time-series among cosm studies 

100

1000

10000

(μ

g

/

L

atra

zin

e)

Effect

No Effect

Concentration

30

40

50

U.S. Environmental Protection Agency 63

0.01

0.1

1

10

100

1 10 100

A

v

era

g

e

Stu

d

y

C

o

n

c

e

ntratio

n(

Exposure Duration (days)

Time from Exposure Start (d)

0 20 40 60 80

Concentration

0

10

20

30

Time from Exposure Start (d)

0 20 40 60 80

Concentration

0

2

4

6

8

10
Goals of the LOC Methodology

Examples of atrazine exposure timeseries (chemographs) for natural 

freshwater systems

• Compare the toxicity of 

different atrazine 

concentrations and length of 

exposure times

• Compare data from cosm 

studies to even more variable 

U.S. Environmental Protection Agency 64

Compare data from cosm 

studies to even more variable 

environmental monitoring 

data
The Method for Determining the Level of Concern 

for Aquatic Plant Communities

Stage 2: Develop effects index based on 

single-species toxicity data

Stage 1. Characterize cosm exposures and effects

Stage 4a. Apply effects index to each 

U.S. Environmental Protection Agency 65

Stage 4a. Apply effects index to each 

monitoring site-year chemograph 

Stage 3a. Apply effects index to each cosm 

study endpoint

Stage 3b. Define the effects index-based 

Level of Concern

Stage 4c. Convert the effects-based LOC into a 

concentration-equivalent LOC

Stage 4b. Identify the watersheds with 

monitoring chemographs that exceed the 

effects index-based LOC
• Problem: need to compare effects across 

different exposure time-series

•Quantify cumulative relative toxic severity 

over an assessment period

Stage 2: The Effects Index

U.S. Environmental Protection Agency 66

•Candidates: ppb-days, Species Sensitivity 

Distribution (SSD), Plant Assemblage Toxicity 

Index (PATI), community simulation models

•Function only of exposure time-series, not 

other system properties
The Method for Determining the Level of Concern 

for Aquatic Plant Communities

Stage 2: Develop effects index based on 

single-species toxicity data

Stage 1. Characterize cosm exposures and effects

U.S. Environmental Protection Agency 67

Stage 4a. Apply effects index to each 

monitoring site-year chemograph

Stage 3a. Apply effects index to each cosm 

study endpoint

Stage 3b. Define the effects index-based 

Level of Concern

Stage 4c. Convert the effects-based LOC into a 

concentration-equivalent LOC

Stage 4b. Identify the watersheds with 

monitoring chemographs that exceed the 

effects index-based LOC
The Method for Determining the Level of Concern 

for Aquatic Plant Communities

Effects Index

Effects Index EEF

LOC



Effects Exceedance Factor (EEF):

U.S. Environmental Protection Agency 68

Concentration Exceedance Factor (CEF):

Factor by which entire exposure series must be reduced so that 

the effects index = LOCEffects Index

Original Chemograph CEF

Chemograph Adjusted to LOC


• Comprehensive Aquatic Systems Model 

(CASM) was our initial effects index candidate

•CASM simulates biomass versus time for

inter-related species in a model community

Community Model Evaluations

U.S. Environmental Protection Agency 69

inter-related species in a model community

•Each term in the equation is a function of 

various biomasses and input variables

•In CASM, each species is described by an 

equation that addresses various biomass 

gains and losses
• For atrazine assessment, CASM formulated 

to represent a generic corn belt stream

•Preliminary version, CASMATZ1, was used for 

2003 evaluations and for the 2007 SAP 

review; problems w/realism of biomasses 

and toxicity 

Community Model Evaluations

U.S. Environmental Protection Agency 70

review; problems w/realism of biomasses 

and toxicity 

•More realistic version, CASMATZ2, was 

developed and evaluated for the 2009 SAP 

review; problems with large uncertainties

•“Probabilistic CASM” was introduced by 

Syngenta at the 2009 SAP review
Key CASM uncertainty is selection of 

toxicity relationships for growth of a few 

dominant species

Community Model Evaluations

%

R

e

d

u

c

tio

n in

S

p

e

cific

G

r

o

w

t

h

R

a

t

e100

U.S. Environmental Protection Agency 71

Atrazine Concentration (g/L)

10 20 50 100 200 500 1000

%

R

e

d

u

c

tio

n in

S

p

e

cific

G

r

o

w

t

h

R

a

t

e

0

20

40

60

80

Vascular Plants

Green Algae

Bluegreen Algae

Diatoms
Community Model Evaluations

g

/

L

A

t

r

a

zin

e

0

50

100

150

60

90

MO 07 2010

MO 01 2010

Median CEF = 0.69 

(10th-90th = 0.16-2.00)

Median CEF = 1.85 

U.S. Environmental Protection Agency 72

Julian Day



g

/

L

A

t

r

a

zin

e

120 150 180 210

0

10

20

30

0

30

60

MO 02 2010

Median CEF = 1.85 

(10th-90th = 0.58-4.65)

Median CEF = 1.08

(10th-90th = 0.30-3.49)
•Given the various uncertainties of CASM, 

alternative effects indices were considered 

•Can the effects index be based on wellestablished processes in CASM important to 

the effects index, while eliminating major 

Community Model Evaluations

U.S. Environmental Protection Agency 73

•Primary CASM action is a daily reduction in 

the specific growth rate of each plant 

species, equal to a weighted reduction in 

total plant community daily production

the effects index, while eliminating major 

uncertainties and unimportant 

complexities?
%

R

e

d

u

ctio

n in

S

p

e

cific

G

r

o

wth

R

ate

20

40

60

80

100

Plant Assemblage Toxicity Index

U.S. Environmental Protection Agency 74

10 20 50 100 200 500 1000

S

p

e

cific

G

r

o

wth

R

ate

0

Atrazine Concentration (g/L)

10 20 50 100 200 500 1000

A

v

e

r

a

g

e

%

R

e

d

u

ctio

n

a

c

r

o

s

s

All S

p

e

cie

s

0

20

40

60

80

100

Plant Assemblage

Toxicity Index (PATI)
• PATI is a simple extension of the Species 

Sensitivity Distribution concept

•To address cumulative effect of time-varying 

concentrations, “Cumulative PATI” is the 

sum of daily PATI values over a designated 

Plant Assemblage Toxicity Index

U.S. Environmental Protection Agency 75

sum of daily PATI values over a designated 

“assessment period” (units = %-days)

•This simple summation is justified by the

fact that, to have the same total effect on 

growth over a period, any series of specific 

growth rates must add to the same value
Plant Assemblage Toxicity Index

Assessment Period Selection Issues – Cosm Data

76

U.S. Environmental Protection Agency

100

1000

10000

A

v

era

g

e

Stu

d

y

C

o

n

c

e

ntratio

n (u

g/L

atra

zin

e)

Effect

No Effect

14-Day 60-Day

U.S. Environmental Protection Agency 76

0.01

0.1

1

10

100

1 10 100

A

v

era

g

e

Stu

d

y

C

o

n

c

e

ntratio

n (u

g/L

atra

zin

e)

Exposure duration (days)
Plant Assemblage Toxicity Index

Assessment Period Selection Issues – Field Data

g

/

L

A

t

r

a

zin

e

0

50

100

150

90

MO 01 2010 14-Day

60-Day

U.S. Environmental Protection Agency 77

Julian Day



g

/

L

A

t

r

a

zin

e

120 150 180 210

0

10

20

30

0

30

60

90

MO 07 2010

MO 02 2010
Plant Assemblage Toxicity Index

Temporal dependence assumed by PATI is similar to 

that assumed by CASM

L

e

v

el o

f

C

o

n

c

e

r

n

fo

r

C

o

n

sta

nt

g

/

L

Atr

a

zin

e)

50

100

PATI

CASM

U.S. Environmental Protection Agency 78

Duration (d)

10 20 50

L

e

v

el o

f

C

o

n

c

e

r

n

fo

r

C

o

n

sta

nt

C

o

n

c

e

ntr

atio

n (

g

/

L

Atr

a

zin

e)

5

20

10

CASM
Plant Assemblage Toxicity Index

g

/

L

A

t

r

a

zin

e

0

50

100

150

60

90

MO 07 2010

MO 01 2010 PATI CEF = 0.65

10th-90th = 0.62-0.68

CASM=0.69

10th-90th = 0.16-2.00

PATI CEF = 2.34

10th-90th = 2.24-2.46 

U.S. Environmental Protection Agency 79

Julian Day



g

/

L

A

t

r

a

zin

e

120 150 180 210

0

10

20

30

0

30

60

MO 02 2010

10 -90 = 2.24-2.46 

CASM=1.85

10th-90th = 0.58-4.65

PATI CEF = 1.07

10th-90th = 1.02-1.13 

CASM=1.08

10th-90th = 0.30-3.49
The Method for Determining the Level of 

Concern for Aquatic Plant Communities

Stage 2: Develop effects index based on 

single-species toxicity data

Stage 1. Characterize cosm exposures and effects

U.S. Environmental Protection Agency 80

Stage 4a. Apply effects index to each monitoring 

site-year chemograph

Stage 3a. Apply effects index to each cosm 

study endpoint

Stage 3b. Define the effects index-based 

Level of Concern

Stage 4c. Convert the effects-based LOC into a 

concentration-equivalent LOC

Stage 4b. Identify the watersheds with 

monitoring chemographs that exceed the 

effects index-based LOC
Cumulative PATI for Cosm Studies

• For each cosm study endpoint a 

chemograph is created

• For each chemograph a 

cumulative PATI value is 

calculated using the PATI 

distribution 20

30

40

50

60

70

80

90

C

o

nce

ntratio

n (μ

g

/L atrazin

e)

Cosm Endpoint #7 , Effect

Cumulative PATI = 1869 %-days

U.S. Environmental Protection Agency 81

distribution

0.01

0.1

1

10

100

1000

10000

1 10 100

A

verage Study

C

oncentration

(μg/L atrazine)

Exposure duration (days)

Effect

No Effect

0

10

20

0 10 20 30 40 50 60

C

o

nce

ntratio

n

Day

A

v

erag

e

% to

xic effect

across all sp

ecies
0.5

1

1.5

2

2.5

3

3.5

C

o

nce

ntratio

n (μ

g

/L atrazin

e)

Cosm Endpoint #76, No Effect

Cumulative PATI for Cosm Studies

• For each cosm study endpoint a 

chemograph is created

• For each chemograph a 

cumulative PATI value is 

calculated using the PATI 

distribution

Cumulative PATI = 39.6 %-days

U.S. Environmental Protection Agency 82

0

0 5 10 15 20 25

C

o

nce

ntratio

n

Day

distribution

0.01

0.1

1

10

100

1000

10000

1 10 100

A

verage Study

C

oncentration

(ug/L atrazine)

Exposure duration (days)

Effect

No Effect

A

v

erag

e

% to

xic effect

across all sp

ecies
The Method for Determining the Level of 

Concern for Aquatic Plant Communities

Stage 2: Develop effects index based on 

single-species toxicity data

Stage 1. Characterize cosm exposures and effects

U.S. Environmental Protection Agency 83

Stage 4a. Apply effects index to each monitoring 

site-year chemograph

Stage 3a. Apply effects index to each cosm 

study endpoint

Stage 3b. Define the effects index-based 

Level of Concern

Stage 4c. Convert the effects-based LOC into a 

concentration-equivalent LOC

Stage 4b. Identify the watersheds with 

monitoring chemographs that exceed the 

effects index-based LOC
Setting the LOC

• Cosm study endpoints 

plotted as effect (closed 

triangle)/no-effect (open 

triangles) versus PATI

• Fitted to a logistic 

relationship for the 

U.S. Environmental Protection Agency 84

Fitted to a logistic 

relationship for the 

probability of an effect 

versus PATI

• LOC defined as the 

cumulative PATI value, 

which equals a 50% 

probability of an effect

LOCPATI = 140 %-days
Stage 2: Develop effects index based on 

single-species toxicity data

Stage 1. Characterize cosm exposures and effects

The Method for Determining the Level of 

Concern for Aquatic Plant Communities

U.S. Environmental Protection Agency 85

Stage 4a. Apply effects index to each monitoring 

site-year chemograph.

Stage 3a. Apply effects index to each cosm 

study endpoint

Stage 3b. Define the effects index-based 

Level of Concern

Stage 4c. Convert the effects-based LOC into a 

concentration-equivalent LOC

Stage 4b. Identify the watersheds with 

monitoring chemographs that exceed the 

effects index-based LOC
• Atrazine Ecological 

Exposure Monitoring 

Program (AEEMP)

• For each environmental

monitoring chemograph a 

cumulative PATI value is 

calculated using the PATI 

Cumulative PATI for Monitoring Data

40

50

60

70

80

90

100

C

o

nce

ntratio

n (p

p

b)

MO-07 2010

Maximum 60-Day 

Cumulative PATI = 

588 %-days

U.S. Environmental Protection Agency 86

calculated using the PATI 

distribution

0

10

20

30

40

0 50 100 150 200 250 300 350

C

o

nce

ntratio

n (p

p

b)

Julian Day

588 %-days

A

v

erag

e

% to

xic effect

across all sp

ecies
The Method for Determining the Level of 

Concern for Aquatic Plant Communities

Stage 2: Develop effects index based on 

single-species toxicity data

Stage 1. Characterize cosm exposures and effects

U.S. Environmental Protection Agency 87

Stage 4a. Apply effects index to each monitoring 

site-year chemograph

Stage 3a. Apply effects index to each cosm 

study endpoint

Stage 3b. Define the effects index-based 

Level of Concern

Stage 4c. Convert the effects-based LOC into a 

concentration-equivalent LOC

Stage 4b. Identify the watersheds with 

monitoring chemographs that exceed the 

effects index-based LOC
Comparing the Monitoring Data to the 

Cosm Studies

Based on the cumulative PATI value the monitoring site can be 

determined as above or below the LOC

U.S. Environmental Protection Agency 88
The Method for Determining the Level of 

Concern for Aquatic Plant Communities

Stage 2: Develop effects index based on 

single-species toxicity data

Stage 1. Characterize cosm exposures and effects

U.S. Environmental Protection Agency 89

Stage 4a. Apply Effects Index to each monitoring 

site-year chemograph.

Stage 3a. Apply Effects Index to each cosm 

study endpoint.

Stage 3b. Define the Effects Index based Level of 

Concern.

Stage 4c. Convert the Effects Based LOC into a 

concentration equivalent LOC.

Stage 4b. Identify the watersheds with 

monitoring chemographs that exceed the Effects 

Index based LOC.
Converting LOCPATI

into a Concentration 

Equivalent LOC (CE-LOC)

Cumulative PATI for Each Monitoring Site-Year

LOCPATI 

= Effects Exceedance Factor

U.S. Environmental Protection Agency 90
Changes in the 60-day Mean CE-LOC Due to

Changes in the Methodology or Available Data

• The original screening concentration (2003 IRED) based on a review 

of toxicity data was 10-20 ppb over an undefined period of time.

• Based on the changes recommended by 2007 SAP the CE-LOC 

ranged from 7.2 to 8.1.

U.S. Environmental Protection Agency 91

ranged from 7.2 to 8.1.

• In 2009, EPA considered 10 ppb concentration for a 60-day 

assessment period to be above levels where adverse effects to the 

aquatic plant community would be expected. 

• Based on the changes recommended by the 2009 SAP the CE-LOC 

dropped to 4.2.
Assessment Period Effects on the CE-LOC

Effect of averaging period on the percent of AEEMP site/years exceeding the CE-LOC. 

Averaging Period 7-Day 14-Day 30-Day 60-Day 90-Day

Cumulative PATI Value (%-days) 65.5 107.4 132.7 140 141.8

CE-LOC (μg/L) Linear Regression 

(0.5-2.0 EEF Range) 18 14.9 8.3 4.2 2.7

% AEEMP Sites-years Exceeding the 

CE-LOC

39 33 44 58 60

U.S. Environmental Protection Agency 92

0.01

0.1

1

10

100

1000

10000

1 10 100

Average Study Concentration (ug/L atrazine)

Exposure duration (days)

Effect

No Effect

• Most cosm effects/no-effects data is 

captured between 7-63 days

• The 60-day assessment period better 

represents the typical atrazine 

exposure profile. 

• 60-day CE-LOC is protective of 

shorter assessment periods
Sensitivity Analysis

Brock Score “2” in 2009: 

• Endpoint #51 (Brockway et al., 

1984): 50 ppb

• 25% reduction in phytoplankton 

oxygen production

• Endpoint #59 (Pratt et al., 1988): 

32 ppb

Brock Score “3” in 2009:

• Endpoint #52 (deNoyelles et al., 

1982, 1989): 20 ppb

• 50% decline in 14C-uptake

• 50% decline in phytoplankton 

biomass. 

U.S. Environmental Protection Agency 93

32 ppb

• 35% decrease in dissolved oxygen, 

• slight reductions in magnesium 

and calcium levels

• Endpoint #60 (Pratt et al., 1988): 

110 ppb

• 35% decrease in dissolved oxygen

• 60% reduction in chlorophyll-a

• slight reductions in magnesium 

and calcium levels

Brock Score “4” in 2009:

• Endpoint #58 (Lampert et al., 

1989): 1 ppb

• 50% decrease in chlorophyll-a 

• 50% reduction in oxygen saturation
Sensitivity Analysis

75th Percentile LOCPATI = 446 %-days; CE-LOC = 15 ppb

50th Percentile LOCPATI = 140 %-days; CE-LOC = 4 ppb 

25th Percentile LOCPATI = 35 %-days; CE-LOC = 1 ppb

U.S. Environmental Protection Agency 94

Range of uncertainty around the LOCPATI calculation

• 95% confidence intervals around the 50th percentile were calculated

• These were converted into EEFs

• The linear regression equations were used to estimate concentrations for these 

intervals.

• 4 ppb CE-LOC (2-10 ppb)

• 7 ppb CE-LOC (3-15 ppb)
Conclusions

• The cosm studies provide reasonable models of ecological 

communities found throughout the primary atrazine use areas of 

North America. 

• The method for determining the Level of Concern for Aquatic Plant 

U.S. Environmental Protection Agency 95

• The method for determining the Level of Concern for Aquatic Plant 

Communities has been updated based on the recommendations of 

SAPs in 2007 and 2009. 

• The CE-LOC of 4-7 ppb over a 60-day assessment period is protective 

of aquatic plant communities and in turn protective of the entire 

aquatic ecosystem.
Method for Comparing Monitoring 

Data to the Aquatic Plant Community 

CE-LOC for Atrazine

U.S. Environmental Protection Agency 96

James A. Hetrick, Ph.D.

Environmental Fate and Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency
Introduction 

• Extensive monitoring data for atrazine

• Estimating atrazine exposure

U.S. Environmental Protection Agency 97

• Estimating atrazine exposure in the absence of monitoring 

data

• Estimating atrazine concentrations from monitoring data
Objectives

• Develop a bias factor method to address uncertainty in 

estimating atrazine concentrations from various monitoring 

data with varying sample frequency

• Illustrate a screening level process using a bias factor method 

U.S. Environmental Protection Agency 98

• Illustrate a screening level process using a bias factor method 

for assessing potential site-year exceedance of the CE-LOC from 

monitoring data

• Develop bias factors for maximum 60-day rolling mean concentration

• Explore different bias factor estimation methods

• Test performance of bias factor methods

• Illustrate application of a bias factor method in a screening level 

assessment of monitoring data
Use of Bias Factor Screening Level Process in the 

Risk Assessment Framework

Problem Formulation

Environmental Fate

Data

Monitoring Data

As

Necessary

Acquire

Data,

Iterate

Process,

Monitor

Results

FRAMEWORK FOR ECOLOGICAL RISK ASSESSMENT

Planning

(Risk Assessor/

Risk Manager

Dialogue)

Watershed 

Vulnerability 

Analysis Tools

U.S. Environmental Protection Agency 99

Risk Management

Communicating Results

to the Risk Manager to the 

Bias Factor

Screen

ANALYSIS

RISK

CHARACTERIZATION

Modeling

CE-LOC

Watershed 

Identification

Modeling
Nature of Pesticide Occurrence in Water

• Complex spatial patterns

• Complex temporal patterns

U.S. Environmental Protection Agency 100

• Complex temporal patterns
Atrazine Chemographs

• Heidelberg University 

(NCWQR) data for OH

• 17,000 km2 catchment area

• Syngenta atrazine ecological 

exposure monitoring (AEEMP)

• 30 km2 catchment area

16

Maumee River, 1995 100

U.S. Environmental Protection Agency 101

0

2

4

6

8

10

12

14

91 98 105 112 119 126 133 140 147 154 161 168 175 182 189

Atrazine C

oncentration, μg/L

Maumee River, 1995

0

10

20

30

40

50

60

70

80

90

100

91 98 105 112 119 126 133 140 147 154 161 168 175 182 189

Atrazine C

oncentration, μg/L

MO-01, 2007
Schematic for Bias Factor Estimate from a 

Single Site-Year

Atrazine Conc (ug/L)

2

4

6

8

10

12

14

16

18

Atrazine Conc (ug/L)

4

6

8

10

12

14

16

18

4, 7, 14, 28 

Day 

Sampling

Intervals

U.S. Environmental Protection Agency 102

Julian Day

100 120 140 160 180 200 220 240 260

0

Julian Day

100 120 140 160 180 200 220 240 260

0

2

Julian Day

90 100 110 120 130 140

Atrazine Concentration (ug/L)

0

2

4

6

8

10

12

14

16

18

True max 60-day mean 

5

th percentile max 60-day mean 

Random Sampling 

Distribution of 

maximum 60-day 

mean concentration
Monitoring Data Selection

• Selected site-year data from Atrazine Ecological Exposure 

Monitoring Program (AEEMP) and Heidelberg University 

National Center of Water Quality Research (NCWQR)

• High sample frequency

• 4-day sampling intervals vs. daily sampling

Type of sampling

U.S. Environmental Protection Agency 103

• Type of sampling

• Auto-sampling vs. grab samples

• Length of sampling period

• AEEMP focused on the 2nd and 3

rd quarters of the year 

• Select a range of site-year conditions

• Watershed sizes

• Hydrology (average flow)
Preliminary AEEMP and NCWQR Monitoring Data 

Site Year

Watershed Area 

(acres)

Average Flow

(m3/sec)

Maximum

Peak

Maximum

60-day Average

μg/L 

IA-01 2004 13,633 1.41 10.03 1.21 

IA-01 2005 13,633 1.86 1.21 0.31 

IA-02 2004 13,896 0.59 5.37 1.40 

IA-02 2005 13,896 0.60 5.53 0.93 

IL-01 2004 13,921 2.84 13.18 2.48 

MO-01 2007 7,439 0.02 91.60 11.61 

U.S. Environmental Protection Agency 104

MO-01 2007 7,439 0.02 91.60 11.61 

MO-04a 2008 5,382 0.86 144.69 11.84 

TN-01 2005 62,549 0.49 7.55 2.93 

IL-03 2006 20,305 1.07 2.46 0.33 

IL-09 2004 33,847 0.92 13.25 4.57 

NE-02 2006 41,615 1.08 82.00 7.26 

IN-11 2008 5,780 1.36 27.12 2.03 

IN-06 2006 19,483 5.88 24.30 2.59 

Sandusky 1995 800,621 325 15.46 6.47 

Sandusky 2007 800,621 538 19.7 3.1 

Cuyahoga 2007 452,449 137 0.5 0.2 

Grand 2005 438,364 294 0.4 0.1 

Scioto 2008 2,463,393 756 15.4 4.8 

Grand Miami 2006 1,738,139 498 17.9 5.5 
Definitive AEEMP and NCWQR Monitoring Data

Site Year

Watershed 

Area (acres)

Average Flow

(m3/sec)

Maximum

Peak

Maximum

60-day 

Average

μg/L

MO-05 2008 16,192 0.32 37.83 5.74

U.S. Environmental Protection Agency 105

MO-05b 2008 116,781 26.15 36.83 7.04

MO-02 2009 18,023 NR 155.20 16.83

MO-02 2008 18,023 0.11 56.60 7.57

MO-02 2007 18,023 0.019 16.18 3.46

MO-04a 2008 5,382 0.86 144.69 11.84

IN-11 2008 5,780 1.36 27.12 2.03

Sandusky 1995 800,621 328.48 15.46 6.47
Spatial Distribution of Monitoring Sites

IA-01

IA-02 NE-02

IN-06

IL-03

IL-01

Sandusky R

Grand R

Cuyahoga R

U.S. Environmental Protection Agency 106

MO-01 MO-04a

MO-02 MO-05

MO-05b

TN-01

IN-11

IN-06

IL-09

Gr.Miami R

Scioto R

MO-01

MO-04a

MO-05 MO-02

MO-05b
Assumptions, Limitations and Uncertainties 

with Bias Factor Estimation

• Atrazine occurrence from April 1st to September 17th is 

assumed to be equal to 2nd and 3rd quarters of the year

• Sample Number ≈ 180 days/Sample Interval

• Estimates will differ for different sampling strategies

U.S. Environmental Protection Agency 107

• Extent of data infilling increases uncertainty

• Serial correlation effects are dependent on the sampling 

interval

• Prediction capability is dependent on the underlying data
Absolute Value of Bias Factors for Preliminary Data

Site 4-day 7-day 14-day 28-day

IA-01 2004 1.02 2.81 3.67 4.48 

IA-01 2005 1.00 1.63 1.94 2.21 

IA-02 2004 1.52 1.97 2.98 4.83 

IA-02 2005 1.94 2.58 3.44 4.43 

IL-01 2004 1.01 1.71 2.18 2.88 

MO-01 2007 1.53 1.74 4.68 9.07 

MO-04a 2005 2.41 4.27 6.80 22.34 

TN-01 2005 1.23 1.49 1.92 3.05 

IL-03 2006 1.94 2.06 2.54 3.30 

U.S. Environmental Protection Agency 108

IL-09 2004 1.01 1.82 2.69 3.91 

NE-02 2006 3.80 5.81 7.19 25.93 

IN-11 2008 1.13 1.92 2.57 5.08 

IN-06 2006 1.61 1.92 3.16 5.40 

Sandusky 1995 1.17 1.21 2.00 2.13 

Sandusky 2007 1.47 2.06 2.90 11.37 

Cuyahoga 2007 1.50 NA NA NA 

Grand 2005 1.36 1.36 1.50 1.67 

Scioto 2008 1.16 1.66 2.19 6.10 

Great Miami 2006 1.23 1.61 2.44 5.34 

Descriptive Statistics

Average 1.53 2.20 3.15 6.86

Median 1.36 1.87 2.63 4.65

Maximum 3.80 5.81 7.19 25.93

Minimum 1.00 1.21 1.50 1.67
Absolute Value of Bias Factors for Definitive Data

Site

Sampling Interval

4-day 7-day 14-day 28-day

MO-05b 2008 1.47 2.02 3.89 7.91

MO-05 2008 1.89 3.48 3.36 4.67

MO-02 2008 1.94 2.64 1.94 7.81

U.S. Environmental Protection Agency 109

MO-02 2009 1.90 2.36 3.10 8.50

MO-04a 2008 2.41 4.27 6.80 22.34

IN-11 2008 1.13 1.92 2.57 5.08

Sandusky 1995 1.17 1.21 2.00 2.13

Descriptive Statistics

Mean 1.79 2.56 3.38 8.35

Median 1.89 2.46 3.23 7.86

Maximum 2.41 4.27 6.80 22.34

Minimum 1.13 1.21 1.94 2.13
Relationship of Watershed Size, Average Flow, 

Sample Number with Bias Factor

Forward Stepwise Multiple Regression

• Sample Number (180 days/Sample Interval)

• Significant (p<0.05)

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• Watershed Area

• Non-significant (p>0.05)

• Average Flow 

• Non-significant (p>0.05)
Relationship of Sampling Number

and Bias Factor 

20

25

30

Preliminary Data

Definitive Data

U.S. Environmental Protection Agency 111

Number of Samples 2nd and 3rd Quarters (180 days/sample interval)

0 10 20 30 40 50

Bia

s

F

a

ctor

0

5

10

15

20
Categorical Estimation Approach

Bia

s

F

a

ctor

8

10

12

U.S. Environmental Protection Agency 112

Number of Samples in 2nd and 3rd Quarter

10 20 30 40 50

Bia

s

F

a

ctor

0

2

4

6
Non-Linear Regression Equations

Bia

s

F

a

ctor

15

20

25

Non-Linear Equation (All Data)

Y=470.8251*exp(-0.0708*X)-458.7698*exp(-0.0708*X)

r

2

=0.3304; p=0.02

Upper 95th Percentile Confidence Interval (All Data; )

Y=1383.0681*exp(-1.0681*X)+11.6378*exp(-0.0425*X)

r

2

=0.9993; p=0.0001

U.S. Environmental Protection Agency 113

113

Number of Samples in 2nd and 3rd Quarters

0 10 20 30 40 50

Bia

s

F

a

ctor

0

5

10

15

Nonlinear Regression Equation

Upper 95th Percent Confidence Interval

Mean

Non-Linear (Mean) 

Y= 170.5297*exp(-0.5580*X)+4.1246*exp(-0.0186*X)

r

2

=1.000
Linear Regression Equations

L

o

g

Bia

s

F

a

ctor

100

Linear Regression

log Y=0.78616-0.013729*X

r2=0.4634,p=0.0001

Upper 95th Percentile Confidence Interval

log Y=0.946326366-0.007773*X

U.S. Environmental Protection Agency 114

Number of Samples in the 2nd and 3rd Quarter

5 10 15 20 25 30 35 40 45

L

o

g

Bia

s

F

a

ctor

1

10 Bias Factor 

95% Confidence Bounds
Comparison of Bias Factor Prediction 

Sampling 

Interval 

(Days)

Number of 

Samples 

Over 180 

Days

Categorical Log 

Linear

Log Linear 

Bias Factor 

Upper 95% 

Confidence 

Interval

Non-Linear

Non-Linear 

Bias Factor 

Upper 95% 

Confidence

Interval

Average 

Non-Linear

4 45 1.87 1.55 3.95 1.00 1.72 1.79

U.S. Environmental Protection Agency 115

4 45 1.87 1.55 3.95 1.00 1.72 1.79

7 26 2.94 2.79 5.58 1.95 3.90 2.56

10 18 2.94 3.53 6.40 3.37 5.42 2.96

14 13 4.01 4.13 7.02 4.85 6.74 3.38

20 9 4.01 4.65 7.52 6.37 8.03 4.61

28 6 10.83 5.02 7.88 7.65 10.30 8.38
Performance Test of Bias Factor 

Regression Equations

Percentage of the AEEMP site-years (N=190) that failed to meet or exceed the actual 

60-day maximum running mean based on various sampling intervals

Sampling 

Interval 

60-Day 

Maximum 

Running Log Linear

Log Linear Bias 

Factor Upper 

95% 

NonLinear

Non-Linear 

Bias Factor 

Upper 95% 

Mean 

Non-Linear 

U.S. Environmental Protection Agency 116

Interval 

(Days)

Running 

Average, 

Unadjusted

Log Linear 95% 

Confidence 

Interval

Linear

Upper 95% 

Confidence 

Interval

Non-Linear 

4 82 12 1 82 7 3

7 94 7 0 16 3 9

10 88 8 1 8 2 10

14 88 8 1 5 1 13

20 86 16 4 7 4 16

28 89 15 5 5 3 5
Translation of Monitoring Data with the Bias Factors

Site-year chemograph from monitoring data

Atra

zin

e

C

o

n

c

e

ntratio

n (u

g/L)

15

20

U.S. Environmental Protection Agency 117

Julian Day

0 100 200 300

Atra

zin

e

C

o

n

c

e

ntratio

n (u

g/L)

0

5

10
Translation of Monitoring Data with the Bias Factors

Stair-step interpolation of site-year chemograph

Atra

zine

C

onc

entration (ug/L)

15

20

U.S. Environmental Protection Agency 118

Julian Day

0 100 200 300

Atra

zine

C

onc

entration (ug/L)

0

5

10 Samples=38
Translation of Monitoring Data with the Bias Factors

Calculate maximum 60-day rolling mean

Atra

zin

e

C

o

n

c

e

ntratio

n (u

g/L)

15

20

U.S. Environmental Protection Agency 119

Julian Day

0 100 200 300

Atra

zin

e

C

o

n

c

e

ntratio

n (u

g/L)

0

5

10

15

Max 60 day rolling mean=3.14ug/L
Translation of Monitoring Data with the Bias Factors

• Use log-linear upper 95th percentile confidence interval 

regression equation 

Bias Factor = 10^(0.946326366-0.007773*38) = 4.48

• Multiply the maximum 60-day rolling mean by the estimated 

bias factor 

U.S. Environmental Protection Agency 120

bias factor 

4.48 * 3.14 μg/L = 14.06 μg/L

• Compare the bias factor adjusted 60-day rolling mean to the 

CE-LOC

14.06 μg/L > CE-LOC 4 μg/L and 7 μg/L

• Site-year may exceed CE-LOC; more frequent monitoring may 

reduce uncertainty in assessing atrazine concentration
Monitoring Data Sources 

• AMP Monitoring Data Finished & Raw Water 2003-2011 

• AEEMP Monitoring Data

• NCWQR Monitoring Data 

• IA DNR 2003-2006

• KS WQ 2009-2011

U.S. Environmental Protection Agency 121

• KS WQ 2009-2011

• MN MDA SW 2008-2011

• MN MDA WQ 1993-2007

• NAWQA

• NE State Monitoring

• Pesticide Data Program

• WI 2008-2011

• USEPA Reservoir Data 1999-2000 
Non-adjusted Monitoring Data

• Site-years with ≥4 samples (Julian Day 0 to 365) 

• 6,917 site-years

• Estimated number of site-years with concentrations 

U.S. Environmental Protection Agency 122

• Estimated number of site-years with concentrations 

exceeding a CE-LOC of 4 μg/L

• 2006-2011: 387 site-years (6 %); 13 states

FL, IA, IL, IN, KS, KY, LA, MO, MS, NE, OH, TX, WI

• Estimated number of site-years with concentrations 

not exceeding a CE-LOC of 4 μg/L

• 2006-2011: 6,502 (94%)
Bias Factor Adjusted Monitoring Data

• Site-Years with ≥6 samples (Julian Day 90 to 270) 

• 2,283 site-years

• Bias factors ranged from 3.36 to 7.94

• Estimated number of site-years with concentrations 

U.S. Environmental Protection Agency 123

• Estimated number of site-years with concentrations 

exceeding the CE-LOC of 4 μg/L

• 2006-2011: 1,424 site-years (62%); 22 states

AL, AR, CA, FL, IA, IL, IN, KS, KY, LA, MD, MN, MO, MS, NC, NE, OH, PA, TN, TX, VA, WI

• Estimated number of sites-years with concentrations not 

exceeding the CE-LOC of 4 μg/L

• 2006-2011: 868 (38%)
Percentage of Site-Years with Estimated Atrazine 

Concentrations Exceeding a CE-LOC of 4 μg/L for Non-Adjusted 

and Bias Factor Adjusted Monitoring Data 

P

erc

e

nt of Site-Y

e

ars

100

120

Non-Adjusted Monitoring Data 

 Bias Factor Adjusted Monitoring Data 

U.S. Environmental Protection Agency 124

Sample Number in 2nd and 3rd Quarters

0 10 20 30 40 50

P

erc

e

nt of Site-Y

e

ars

0

20

40

60

80
2006-2011 NAWQA Non-Adjusted Monitoring Data 

CE-LOC = 4 μg/L

U.S. Environmental Protection Agency 125
2006 - 2011 NAWQA Bias Factor Adjusted Monitoring Data 

CE-LOC = 4 μg/L

U.S. Environmental Protection Agency 126
Conclusions

• Sample frequency is significant

• Watershed area and average flow are not significant

• Application of bias factors to NAWQA data

• Monitoring site-years exceeding the CE-LOC are generally associated with 

high atrazine use intensity areas and regions of the country with previously 

U.S. Environmental Protection Agency 127

high atrazine use intensity areas and regions of the country with previously 

identified vulnerable watersheds

• Application of bias factors identified monitoring sites potentially 

exceeding CE-LOC outside areas with high atrazine use intensity and 

vulnerable watersheds

• The bias factor method may be an efficient screening tool to assess 

uncertainty in estimating atrazine concentration from monitoring data and 

to identify monitoring sites requiring higher resolution exposure 

assessment
Topic A: 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 

U.S. Environmental Protection Agency 128

the context of considering the nature and degree of the uncertainties 

in each study.

Charge Question 1: Is the SAP aware of any other laboratorybased or field-based studies not included in this White Paper that 

should be considered?
Charge Questions 2a and 2b: 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, 

U.S. Environmental Protection Agency 129

Topic A: Evaluation of the Data on Amphibians

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.
2a. 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.

U.S. Environmental Protection Agency 130

Topic A: Evaluation of the Data on Amphibians

2b. Please comment on EPA’s conclusions about the level of 

confidence placed on each study’s results.
Charge Questions 3a and 3b: 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. 

U.S. Environmental Protection Agency 131

Topic A: Evaluation of the Data on Amphibians

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).
3a. 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.

U.S. Environmental Protection Agency 132

Topic A: Evaluation of the Data on Amphibians

3b. 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.
Charge Question 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 

U.S. Environmental Protection Agency 133

Topic A: Evaluation of the Data on Amphibians

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.
Charge Question 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 

U.S. Environmental Protection Agency 134

Topic A: Evaluation of the Data on Amphibians

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. 
5a: 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 

U.S. Environmental Protection Agency 135

Topic A: Evaluation of the Data on Amphibians

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.
5b: 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 

U.S. Environmental Protection Agency 136

Topic A: Evaluation of the Data on Amphibians

with sufficient rigor to identify effects that can be directly and 

quantitatively attributed to adverse impacts on amphibian 

reproduction, growth and/or survival.
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 

U.S. Environmental Protection Agency 137

Topic B: The Method for Determining the Atrazine Level of 

Concern for Aquatic Plant Communities

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.
Charge Question 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 

U.S. Environmental Protection Agency 138

Topic B: The Method for Determining the Atrazine Level of 

Concern for Aquatic Plant Communities

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.
Charge Question 7: The Aquatic Plant Community CE-LOC 

methodology for atrazine is a four stage process that uses singlespecies 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 

U.S. Environmental Protection Agency 139

Topic B: The Method for Determining the Atrazine Level of 

Concern for Aquatic Plant Communities

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).
Charge Question 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 

U.S. Environmental Protection Agency 140

Topic B: The Method for Determining the Atrazine Level of 

Concern for Aquatic Plant Communities

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.
Charge Question 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 

U.S. Environmental Protection Agency 141

Topic B: The Method for Determining the Atrazine Level of 

Concern for Aquatic Plant Communities

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 CE-LOC is lower than exposures that 

result in significant effects on the growth, survival and 

reproduction of aquatic animals.
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 

U.S. Environmental Protection Agency 142

Topic C: Method for Comparing Monitoring Data to the 

Aquatic Plant Community LOC for Atrazine

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.

Charge Question 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.
Charge Question 11a and 11b: 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 

U.S. Environmental Protection Agency 143

Topic C: Method for Comparing Monitoring Data to the 

Aquatic Plant Community LOC for Atrazine

the number of samples taken in the 2 and 3 quarters of the 

year (April 1

st to September 30th). 

11a. Please comment on EPA’s prediction of bias factors from 

monitoring data using categorical or regression method 

approaches. 

11b. Please comment on any additional methods for estimating 

bias factors that would be useful in this situation.
Charge Question 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 

U.S. Environmental Protection Agency 144

Topic C: Method for Comparing Monitoring Data to the 

Aquatic Plant Community LOC for Atrazine

and average flow rate in the 2 and 3 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?
Charge Question 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 

U.S. Environmental Protection Agency 145

Topic C: Method for Comparing Monitoring Data to the 

Aquatic Plant Community LOC for Atrazine

for atrazine. This analysis showed that application of a bias 

factor, based on sample number during the 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?
Charge Question 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 

U.S. Environmental Protection Agency 146

Topic C: Method for Comparing Monitoring Data to the 

Aquatic Plant Community LOC for Atrazine

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?