# Scien tific Issues Associa te d with Pr oblem Fo rmula tion fo r the Re assessmen t of Ec ological Risk s fr om the Use of At ra zine U.S. Environmental Pr otection Agency **1**



# Opening Re mark s St even Br adbury

, Ph.D ., Dir ect or Office of P es ticide Pr ogr ams U. S. En vir onmen ta l Pr otection AgencyU.S. Environmental Pr otection Agency **2**

# Fr amew ork fo r Assessing the Po te nt ial Eco logic al Risk s P osed by

A tr azine U.S. Environmental Pr otection Agency **3**



## Dana S. Spatz, M.S. En vir onmen ta l Fa te a nd E ff ects Division Office of P es ticide Pr ogr ams U. S. En vir onmen ta l Pr otection AgencyU.S. Environmental Pr otection Agency **4**

# Re gula to ry His to ry

• 

## At ra zine fir st re gis te re d in 1958

• 

## Primarily used in corn, sor ghum, and sug ar cane pr oduction Br oadleaf and some gr ass y w eeds U.S. Environmental Pr otection Agency **5**



### • 

## Br oadleaf and some gr ass y w eeds

• 

## Interim Reregistr ation Eligibility Decision (IRED) in 2003

• 

## Memor andum of Agr eemen t (MO A) in 2003U.S. Environmental Pr otection Agency **6**

# 2003 IRED and MO A

• 

## Monitor f or atr azine in a subset of 1172 vulner able w ate rs heds

• 

## Iden tification of an appr opriate ecological Level of Concern Mitig ate e nv ir onmen ta l exposur es if EP A determines that U.S. Environmental Pr otection Agency **7**



### • 

## Mitig ate e nv ir onmen ta l exposur es if EP A determines that mitig ation is necessary

• 

## Determine potential f or eff ects on amphibian gonadal dev elopmen t

# Scien tific Advisory Pa nel Mee tings on

Ec ological Risk Issues

**June 2003**  amphibian gonadal development **December 2007**  a quatic plant community level effects **June 2012**  problem formulation: u pdate on amphibians, PAT I, monitoring, and the atrazine L OC U.S. Environmental Pr otection Agency **8**

**October 2007**  f ollow up on amphibian gonadal development **May 2009** Plant Assemblage To xicity Index and watershed vulnerability analysis**Integr ate**  **Ava ilable Information**  **PROBLEM FORMULA** 

**TION**

**Analysis FRAMEWORK FOR ECOL**  **OGICAL RISK**  **ASSESSMENT**

**) As**  **Necessary Acquire**  **Data, Iterate**  **Process, Planning**  **(Risk Assessor/ Risk Manager**  **Dialogue)** U.S. Environmental Pr otection Agency **9**

**Risk Management Risk Management**  **Communicating Results to the Risk Manager**  **Analysis Plan** 

**ANAL YSIS** 

**RISK CHARACTERIZA** 

**TION Monitor**  **Results**

# Wha t’ s New **Amphibian To**  **xicity –**  **Am y**  **Blankinship**

• 

### open liter at ur e on amphibian to xicity da ta

• 

### e va luation of test design elements

• 

### a dv er se outcome pathwa y **At ra**  **zine Level of Concern –**  **Fr ank**  **Fa rruggia**  **& Russell Erickson**  U.S. Environmental Pr otection Agency **10**



### At **ra zine Level of Concern**  **– Fr**  **ank Fa**  **rruggia & Russell**  **Erickson**

• 

### refinements to the Plant Assemblage T ox icity Inde x (P

AT I)

• 

### r e - evaluation of atr azine cosm st udies

• 

### m odifications to determining atr azine L OC f or aquatic plant communities

• 

### s ensitivity analysis **Comparing Monitoring Da**  **ta to the L OC**  **– James**  **Hetrick**

• 

### dev elopment of bias f act or s

• 

### i ntegr at ion of a va ilable monitoring data to define scope

# Ev alua tion of Amphibian To xicity Da ta fo r At ra zine U.S. Environmental Pr otection Agency **11**



## Am y Blankinship, M.S. En vir onmen ta l Fa te a nd E ff ects Division Office of P es ticide Pr ogr ams U. S. En vir onmen ta l Pr otection Agency

# Ov er view

• 

## Backgr ound on pr evious ev aluations of amphibian to xicity data

• 

## Curr en t methodology f or ev aluation of amphibian to xicity data Re sults of curr en t ev aluation U.S. Environmental Pr otection Agency **12**



### • 

## Re sults of curr en t ev aluation

• 

## Uncert ain ties

• 

## Over all conclusions

# Pr evious E va luations of Amphibian T ox icity Dat a fo r A

tr azine

• 

### 2003 SAP – Po te ntial Developmental E ff ects of A tr azine on Amphibians

• 

### EP A pr esented and the SAP P anel agr eed upon: 1. Ava ilable dat a support ed further evaluation of atr azine exposur e on amphibian gonadal development 2. A tier ed t es ting appr oach 3. Critical t es t design elements f or futur e t es ting: Te st species and stage sensitivity U.S. Environmental Pr otection Agency **13**

• Te st species and stage sensitivity

• Te st conditions



### • 

### 2004 – EP

A issued a Dat a Call
- in (DCI) fo r ev aluating amphibian developmental eff ects fr om atr azine e xposure

# Pr evious E va luations of Amphibian T ox icity Dat a fo r A

tr azine 2007 SAP – Po te ntial f or A tr azine t o Af fe ct Amphibian Gonadal Development

• 

### Reviewed DCI data (Hosmer et al. 2007 (r egistr ant - submit te d) and published as Kloas et al. 2009) 

### and other r elevant da ta

• DCI dat a wer e two studies conduct ed at two diff er ent labs, using the same pr otocol, to ex amine r epr oducibility of eff ects U.S. Environmental Pr otection Agency **14**

**Study Details**  **Study Att**  **ributes Te**  **st concentr**  **ations** Negative Control, 0.01, 0.1, 1.0, 25 and 100 μg /L and 0.2 μg /L E2 **No. of replicat**  **es/treatment** 8 f or atr azine and E2 gr oups; 16 f or negative control **No. of tadpoles/replicat**  **e** 25 **No. of tadpoles/treatment**  200 f or atr azine and E2 gr oups; 400 fo r negative control **No. of tadpoles/**  **study** 1,600 A total of 3,200 tadpoles ex posed f or both studies conducted using same pr otocol No. of fr ogs used in analyses at test termination 1,312 (Lab #1) + 1,555 (Lab #2) = 2,867

# Pr evious E va luations of Amphibian T ox icity Dat a fo r A

tr azine 2007 SAP – Po te nt ial f or A tr azine to Aff ect Amphibian Gonadal Dev elopmen t

• 

## EP A concluded that DCI study pr ovided r eliable inf ormation on amphibian to xicity U.S. Environmental Pr otection Agency **15**



### • 

## Pa nel concluded DCI s tudy pr ovided r eliable r esults f or Xe nopus laevis ; however

, had concerns r eg ar ding:

• 

### use of X. laevis as surr og ate f or indigenous species; and

• 

### sensitivity of X. laevis st ra in used in s tudy

# St atus of Amphibian T

ox icity Dat a

• 

## Additional amphibian t ox icity st udies published since 2007 SAP

• 

## New studies e xa mined man y dif fe re nt endpoin ts

• 

## An ev aluation of these additional s tudies needed t o be U.S. Environmental Pr otection Agency **16**



### • 

## An ev aluation of these additional s tudies needed t o be completed

• 

## Ther e ar e other published r eviews that e xa mine/ ev aluate re lationship between atr azine and amphibian eff ects ( i.e. , Ro hr and McCoy 2010, Ha ye s et al. 2011, Solomon et al. 2008)

# Ev alua tion Me thodology
- Objectiv es

1 • Iden tify critic al test design elements

2 • Classify each amphibian to xicity st udy U.S. Environmental Pr otection Agency **17**



## 2
3 • Ca tegoriz e endpoints in each study

4 • Compar e re sults acr oss studies f or an endpoint categor y

# Ev alua tion Me thodology
- Objectiv es

1 • Iden tify critic al test design elements

2 • Classify each amphibian to xicity st udy U.S. Environmental Pr otection Agency **18**



## 2
3 • Ca tegoriz e endpoints in each study

4 • Compar e re sults acr oss studies f or an endpoint categor y

# Open Lit er atur e Pr eliminary Scr eening Crit eria

• 

## All open liter atur e studies under go a pr eliminary scr eening f or further ev aluation
– this is f or all chemicals ev aluated by the Office of P es ticide Pr ogr ams (OPP)

• 

## A st udy was included f or further ev aluation if: U.S. Environmental Pr otection Agency **19**

• 

### tr eatments were e xposed to only at ra zine

• 

### eff ect on liv e, in tact or ga nisms, and tes t species r eported and v erified

• 

### paper is primary sour ce of data, published in English as a full article

• 

### st udy type is r eported

# Open Lit er atur e Pr eliminary Scr eening Crit eria

• 

## If study met pr eliminary scr eening crit eria it was r etained f or further ev aluation

• 

## 64 labor ator y studies included f or further ev aluation Ex cluded st udies: U.S. Environmental Pr otection Agency **20**



### • 

## Ex cluded st udies:

• 

### 11 co sm studies
- while passed screening, e xc luded f or this analysis due t o pot en tial multiple in te ra ctions resulting in both pot en tial direct and/ or indirect eff ects (still c onsidered f or further eva luation and pot en tial use in risk assessmen t)

• 

### 9 field studies
- exc luded due t o pot en tial major c onf ounding fa ctor s ( i.e. , en vir onmen ta l c onditions) and not c onsidered f or use in risk assessmen t

# Te st D esign Elemen ts (Further Ev aluation)

• 

## Curr en tly no standar diz ed guideline/methodology f or **chronic** dev elopmen ta l testing with amphibians ( e.g ., ASTM guideline)

• 

## EP A identified critical te st design elemen ts that could potentially conf ound/influence the ability to discern a cause

eff ect r ela tionship between atr azine and a biological r ecept or U.S. Environmental Pr otection Agency **21**



## eff ect r ela tionship between atr azine and a biological r ecept or

• 

## EP A identified test elements wer e based on:

• 

### ASTM guidelines

• 

### Conclusions fr om the 2003 and 2007 SAP P anels; and

• 

### U. S. EP A guidelines



### • 

## Iden tified elemen ts wer e further classified

# Te st D esign Elemen ts Te st design elemen ts that would in validat e st udy r esults
**Te st Design**  **Element -**  **Invalidate** 1 At ra zine concentr ations not r eport ed 2 Contr ol gr oup not run concurr ently 3 Te st chemical contamination of control U.S. Environmental Pr otection Agency **22**

3 Te st chemical contamination of control 4 Contr ol mort ality >30% 5 Solvent concentr ation > 0.05% 6 Presence of other str essor s (biological, chemical, ph ysical) expected to compr omise study 7 Aberr ant eff ects in contr ol 8 Insufficient r eplication (n=2) 9 Insufficient dat a to allow reviewer to under sta nd/ substantiate study r esults

# Te st D esign Elemen ts Te st design elemen ts that would raise c oncern by themselv es but not necessarily in va lidate study r esults **Te st Design**  **Element –**  **Raise concern** 1 When solvent used, both neg ative and solvent gr oup not t es te d 2 St atistically significant diff er ence between neg ative and solvent gr oup U.S. Environmental Pr otection Agency **23**

3 Solvent concentr ation >0.01% 4 DMSO used as solvent carrier choice 5 Plastic used as t es t vessels 6 Study not conduct ed using t echnical gr ade atr azine ( e.g ., f ormulation) 7 High loading ra te (pot entially coupled with longer r enew al ra te and lack of re port ed w ater quality) 8 Field collected or ga nisms

# Te st D esign Elemen t – Or ga nismal Loading Curr en t re commended loading = 1 tadpole/L (≈ 1 g /L) (Based on 2003 SAP , using X. laevis and ASTM)

• ASTM loading: 1 g /L/day f or flow
- thr ough designs; 0.5
- 0.8 g /L f or static and static
- re newal

• Studies below were conducted using a static
- re newal design **Species We**  **ight (gr**  **ams) per**  **or ga**  **nism Sour**  **ce** Rana pipiens 2.5 2003 SAP (Hayes et al. 2002c) U.S. Environmental Pr otection Agency **24**

Rana pipiens 2.5 2003 SAP (Hayes et al. 2002c) Rana pipiens 0.83 Allr an and Kar asov 2000 Rana pipiens 2.2 Hayes et al. 2006 Rana clamitans 2 2003 SAP (Heck er et al. 2003) Litoria raniformis 0.9 Choung et al. 2011 Hyla versicolor 0.5 LaFiandr a et al. 2008 Ambystoma barbouri 0.75 to 0.95 Ro hr et al. 2011 Ambystoma macrodactylum 0.85 Forson and Storf er 2006 Ambystoma tigrinum 11
- 16 Lar son et al. 1998

# Ev alua tion Me thodology
- Objectiv es

1 • Iden tify critic al test design elements

2 • Classify each amphibian to xicity st udy U.S. Environmental Pr otection Agency **25**



## 2
3 • Ca tegoriz e endpoints in each study

4 • Compar e re sults acr oss studies f or an endpoint categor y

# Study Classification

• 

## Ea ch st udy (n = 64) ev aluated using the list of identified potential conf ounding tes t elemen ts

• 

## The r esults f or each s tudy wer e classified accor ding to level of confidence in the s tudy r esults U.S. Environmental Pr otection Agency **26**



### • 

## Studies we re classified as:

• 

### Quantitative

• 

### Qualitative (higher

, medium or lower lev el of confidence)

• 

### In va lid

# Re sults of Study Classification

• 

## Common potential conf ounding test design elements obser ve d in a va ilable data included:

• 

### Lack of reporting /measuring atr azine in contr ol tr eatments

• 

### Use of field collected or ga nisms

• 

### Higher than r ecommended loading (1 tadpole/L (≈ 1 g /L) fr om 2003 SAP) U.S. Environmental Pr otection Agency **27**

• 

### Higher than r ecommended loading (1 tadpole/L (≈ 1 g /L) fr om 2003 SAP) coupled with lack of r eported w ater quality and infr equent test solution re newal periods (>48 hr s)



### • 

## The majority (54 out of 64) of the a va ilable labor ator y studies had a classification of Qualit ative with a lower level of confidence (n = 30) or In va lid (n = 24)

# Re sults of Study Classification

15 20 25 30**# o** **f S** **t u** **d i** **e s**  **Qualitative (Lower Confidence)** 

8 10 12 14 16 **Invalid** U.S. Environmental Pr otection Agency **28**

0 5 10 15**# o** **f S** **t u** **d i** **e s** 

0 2 4 6 8

# Re sults of Study Classification One study was classified as Quantitativ e (s yn thesiz ed in Kloas et al
. 2009)

• 

### This was the study c onduct ed as a r esult of the 2003 SAP and required by the 2004 DCI which was then submit te d by the registr an t (Hosmer et al. 2007) U.S. Environmental Pr otection Agency **29**



### registr an t (Hosmer et al. 2007)

• 

### The study design was able t o acc oun t f or pot en tial c onf ounding eff ects that limit ed the utility of previously c onduct ed studies

• 

### Re port ed NO AE C = 100 ppb f or sur viv al, metamorphosis, gr owth, beha vior al eff ects, and se xu al development, highest c oncen tr ation te sted

# Re sults of Study Classification
– Quan tit ative Study Due t o the r obustness of this study it was able t o acc oun t f or pot en tial co nf ounding t est design elemen ts **Potential Limitations**  **of Study Identified by EP A**  **How Resolved**  Measurement of atr azine in one cluster (n=4 replicates) in Neg ative Control tanks f or Lab #1 Ta nks dropped from analysis, 2007 SAP P anel concurred with EP

A on decision to omit 8 or U.S. Environmental Pr otection Agency **30**

replicates) in Neg ative Control tanks f or Lab #1 concurred with EP A on decision to omit 8 or half of the Neg ative Control tanks given the power of the test Microbial bloom in one cluster of Neg ative Control tanks fo r and one tank at 1 ppb at Lab #1 A qualitativ e rat her than quantitative assessment of certain histological endpoints Pa nel concurred with qualitativ e fo r some endpoints; data fo r other histological endpoints reanalyz ed resulting in same conclusion One pathologist fo r analysis of histological endpoints Pa nel recommended additional review; conducted and resulted in same conclusion

# Re sults of Study Classification
– Quan tit ative Study Due t o the r obustness of this study it was able t o acc oun t f or pot en tial co nf ounding t est design elemen ts **Potential Limitations**  **of Study Identified by Pa**  **nel How**  **Resolved** Sensitivity of X. laevis strain Re sponse to positive contr ol compar ed to other species shown to be sensitiv e U.S. Environmental Pr otection Agency **31**

other species shown to be sensitiv e Re sults from labs analyz ed separ ately Combined data which resulted in same conclusion
Lack of exposure to degr ada tes Tw o degr ada tes measured in study (DIA and DEA); also due to atr azine half
- lives, none of the primary degr adates lik ely to f orm within a 48
- or 72
- hour renewal time
- fr ame Lack of ev aluating reproductive fitness (gr ow - out) Currently , robust methods to examine reproduction function and fitness are not ava ilable and current evaluation of data indicate that higher tiered testing is not warr anted

# Re sults of Study Classification Nine (14%) s tudies wer e classified as Qualitativ e with either a higher or medium lev el of confidence **Study Classification**  **Ef fe**  **cts ( μg**  **/L) Species**  **Te sted**  **Acut e**  **Surviv al/T** 

**er atogenesis** 
1 Bir ge et al . 1980 Higher LC 50 (4 days post
- hatch) = 410 to >48,000 (surviv al and ter atogenesis) Rana catesbeiana, R. pipiens, R. palustris, Bufo americanus U.S. Environmental Pr otection Agency **32**

2 Howe et al. 1998 Medium 96 hr LC 50 = 14,500
- 47,600 R. pipiens, B. americanus 3 Mor gan et al. 1996 Medium 96 hr LC 50 = 40,800
- 51,408 96 hr EC 50 = <3,264 or 13,464 X. laevis 4 Wa n et al. 2006 Medium 96 hr LC 50 >16,000 R. catesbeiana **Behavior Modification** 5 Ro hr et al. 2009 Higher NOAEC = 196; L OAEC >196 (f ear cues) B. americanus 6 Storr s Mendez et al. 2009 Medium NOAEC = 1,430 μg /kg; L OAEC >1,430 (soil pr eference) B. americanus

# Re sults of Study Classification Nine studies were classified as Qualit ative with either a higher or medium level of c onfidence **Study Classification**  **Ef fe**  **cts ( μg**  **/L) Species**  **Te sted**  **Subchr onic/Chr**  **onic Gr**  **owth and Development**

7 Choung et al. Higher Metamorph
. Gr owth NOAEC=25; LOA EC > 25

NOAEC=25; LOA EC > 25 Litoria U.S. Environmental Pr otection Agency **33**

7 Choung et al. 2011 (Chr onic) Higher Gr owth Surviv al NOAEC=25; LOA EC > 25

>97.5% fo r study Litoria raniformis

8 Spoly arich et al. 2010 (Chr onic) Higher Metamorph
. Gr owth Se x develop. Surviv al NOAEC=30; LOA EC > 30

NOAEC=30; LOA EC > 30 NOAEC=30; LOA EC > 30 2.3% mortality L imnodynastes tasmaniensis

9 Fr eeman & Rayburn 2005 (Subchr onic) Medium /

Higher Metamorph
.

Gr owth Molecular Surviv al NOAEC<100; LOA EC =100

NOAEC=800; LOA EC >800 NOAEC=varied; L OAEC= 800 0
- 16.7% mortality X. Laevis64 Studies Open lit er ature & re gistr ant submit te d Apply t es t element crit eria

9 Qualitative Medium/High 30 Qualitative Low 24

In valid 

### 1 **Quantita tiv**  **e** U.S. Environmental Pr otection Agency **34**

Not f or use in risk assessment Low confidence in r esults & f or use in risk assessment Use r esults in char act erizing risk in assessment


### Use results **quantitatively in risk**  **assessmen t** 

# Ev alua tion Methodology
- Objectiv es

1 • Iden tify critic al test design elements

2 • Classify each amphibian to xicity st udy U.S. Environmental Pr otection Agency **35**



## 2
3 • Ca tegoriz e endpoints in each study

4 • Compar e re sults acr oss studies f or an endpoint categor y

# Cat egorizing Endpoin ts Endpoin ts in each s tudy (n = 64 labor ator y) we re categoriz ed accor ding to br oad endpoin t par ameter s

**Endpoint Categories**  1 Survival 2 Metamorphosis U.S. Environmental Pr otection Agency **36**

2 Metamorphosis 3 Gr owth 4 Sexual Development 5 Immune r esponse and Inf ection Susceptibility 6 Biochemical/Molecular modifications 7 Beha vior al modifications 

## Fo r each s tudy

, the NO AEC and L OA EC s were ex tr act ed fo r the abov e categories

# Ev alua tion Methodology
- Objectiv es

1 • Iden tify critic al test design elements

2 • Classify each amphibian to xicity st udy U.S. Environmental Pr otection Agency **37**



## 2
3 • Ca tegoriz e endpoints in each study

4 • Compar e re sults acr oss studies f or an endpoint categor y

# Ev aluation of Labor ato ry Studies

• 

## All labor ator y studies included (n = 64) which includes all dif fe re nt classifications

• 

## Compar ed NO AEC and L OA EC va lues f or giv en endpoin ts acr oss st udies U.S. Environmental Pr otection Agency **38**



## st udies

• 

### Not every study evaluated all identified endpoint categories

• 

### Not all s tudies had a corr esponding NOAEC/L OAEC va lue f or each endpoint

• 

### Endpoints within a s tudy ma y ha ve been giv en dif fe re nt classifications

# Ev aluation of Entir e Labor ato ry Dat aset (n = 64) Sur viv al
- Acut e Exposur e

20 40 60 80 100**P e** **r c** **e n** **t R** **a n** **k** U.S. Environmental Pr otection Agency **39**

**LC 50**  In va lid Qualitative (Lower Level of Confidence) Qualitative (Medium) Qualitative (Higher) **E C**  **50** In va lid Qualitative (Higher) 0 20
100 1000 10000 100000**P e** **r c** **e n** **t R** **a n** **k**

**At ra**  **zine concentr**  **at ion (**  **μ g/**  **L)**

# Ev aluation of Entir e Labor ato ry Dat aset (n = 64) Sur viv al
- Chr onic and Sub
- chr onic Exposur e

4 **0 60**  **80** 1 **00 P** **e r** **c e** **n t** **R a** **n k**  U.S. Environmental Pr otection Agency **40**

**0 20**  4 **0**

**0.** 1 1 1 **0** 1 **00** 1 **000** 1 **0000**

**No Ef**  **fect** In va lid Qualit ative (Lower Level of Confidence) Qualit ative (Higher) Quantitative **Ef fect**  In va lid Qualit ative (Lower)**P e** **r c** **e n** **t R** **a n** **k**

**At ra**  **zine concentr**  **at ion (**  **μ g/**  **L)**

# Ev aluation of Entir e Dat aset Met amorphosis

60 80 100**P e** **r c** **e n** **t R** **a n** **k** U.S. Environmental Pr otection Agency **41**

0 20 40

0.1 1 10 100 1000 10000 **At ra**  **zine concentr ation**  **( μ**  **g/ L)**  **LOA EC**  Invalid Qualitative (Lower) Qualitative (Higher) **NO AE**  **C** Invalid Qualitative (Lower Level of Confidence) Qualitative (Higher) Quantitative**P e** **r c** **e n** **t R** **a n** **k**

# Ev aluation of Entir e Dat aset Gr owth

40 60 80 100**P e** **r c** **e n** **t R** **a n** **k** U.S. Environmental Pr otection Agency **42**

0 20 40

0.1 1 10 100 1000 10000 100000 **At ra**  **zine Concentr ation**  **( μ**  **g/ L)**  **NO AE**  **C** Invalid Qualitative (Lower Level of Confidence) Qualitative (Higher) Quantitative **LOA EC**  Invalid Qualitative (Lower)**P e** **r c** **e n** **t R** **a n** **k**

# Ev aluation of Entir e Dat aset Se xual Development

60 80 100**P e** **r c** **e n** **t R** **a n** **k** U.S. Environmental Pr otection Agency **43**

0 20 40

0.01 0.1 1 10 100 1000 **At ra**  **zine Concentr ations (**  **μ g/**  **L) NO**  **AE C**  Invalid Qualitative (Lower Level of Confidence) Qualitative (Higher) Quantitative **LOA EC**  Invalid Qualitative (Lower)**P e** **r c** **e n** **t R** **a n** **k**

# Summary of E ff

ects fo r Biochemical/Molecular Modifications All a va ilable st udies classified as Qualitativ e (lower lever of confidence) or In va lid e xc ept one Qualitativ e Medium (Fr eeman & Ra yburn 2005) **Species Reported**  **Ef fe**  **ct Reported**  **LOA EC**  **/NOAEC (μg /L)**  Xenopus laevis ↓ plasma testosterone in males 2.5 / <2.5; 25 / <25; 258 / 24.8; no effect on p lasma testosterone or estradiol in U.S. Environmental Pr otection Agency **44**

X. laevis no effect on p lasma 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 th yroxine;↓ corticosterone 82 / <82

# Summary of E ff

ects fo r Biochemical/Molecular Modifications All a va ilable st udies classified as Qualitativ e (lower lever of confidence) or In va lid **Species Reported**  **Ef fe**  **ct Reported**  **LOA EC**  **/NOAEC (μg /L)**  Xenopus laevis ↑ MMP18 e xpression (to evaluate tissue development ) 3,500 / 350 X. laevis / Bufo americanus no effect on n uclear heterogeneity (represent genotox ) >800 / 800 U.S. Environmental Pr otection Agency **45**

americanus X. laevis ↑ flow cytometric analysis meant to illustrate

metamorphosis and development; results varied based on test 800 / V aried
X. laevis change in expression of multiple genes for proteolysis,

digestion, carboh ydrate metabolism, growth 400 / <400
X. laevis changes in gene e xpression (acyl

- CoA deh ydrogenase

and glucocorticoid receptor) 400 / <400
Rana catesbeiana no change in relative fold diff erence for CYP19 ;

correlation of CYP19 and SF
- 1 m RNA in the brain when combining 1 & 10 ppb resulted in significant diff erence >10 / 10

# Summary of R eported eff ects fo r Beha vior Modifications All a va ilable st udies classified as Qualitativ e (lower lever of confidence) or In va lid **Species Ef**  **fe ct**  **LOA EC / NO**  **AE C (μg**  **/L)** Ambystoma barbouri ↑ locomotion activity and/ or ↓ refuge use 400 / 40 A. barbouri ↓ water conserving behavior; ↑ activity 40 / 4 Bufo americanus no preference fo r untreated or treated soil 1,430 μg /kg U.S. Environmental Pr otection Agency **46**

B. americanus no effect on f ear 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 f eeding >400 / 400

# Uncert ain ties 1. 

# Ef fe cts to Indigenous Species (Nativ e Species)

• 

### 2007 SAP P anel stated co ncern about suit ability of Xe nopus laevis as surr og ate f or native species U.S. Environmental Pr otection Agency **47**



### • 

### Based on curren t review of a va ilable data there is still an apparent lack of st andardiz ed/ suit able labor ato ry husbandry pr ot oc ols

# Uncert ain ties 2. 

# Ef fe cts on Immune Syste m or P ar asite Susceptibility

• 

### Some dat a a va ilable sugg est that e xposure t o a tr azine r esults in modific ations t o immune response and/ or inf ection susceptibility U.S. Environmental Pr otection Agency **48**



### • 

### Currently

, a clear or defined adver se out co me pathway (AOP) fo r the immune s ystem modific ation in te ra ction is c onsidered insufficient

# Re ported Ef

fe cts on Immune R esponse and Inf ection Susceptibility
**Species Ef**  **fe ct**  LOA EC/NOAEC (ppb) Am bystoma tigrinum ↑ viral inf ection rate (eff ect at middle concentration) ;

↓ leuk ocytes 16 / 1.6 A. tigrinum & A. macrodactylum n o effect on viral inf ection rate >200 / 200; >184 / 184 Rana sylvatica ↑ intensity of parasite inf ection and mean metacercarial cysts 30 / 3 All available studies classified as Qualit ative (lower level of confidence) or In valid U.S. Environmental Pr otection 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; n o effect on

lymphocytes 1 / <1; >10 / 10
R. clamit ans ↑ % of encysted cercariae 201 / < 201 Xenopus laevis ↑ activated caspase 3 immuno (+) cells (evaluate apoptosis) 400 / <400 X. laevis no effects on peptide activity (e xpressed as total protein ) >986 / 986 X. laevis ↑ th ymic plaques 0.19 / <0.19

# Ov er all Conclusions

• 

## Majority of a va ilable s tudies contained potential conf ounding elemen ts that limit their utility in a risk assessment

• 

### This is based on t est crit eria iden tified in ASTM, OPP Open Lit er ature Guidance and input fr om 2003 and 2007 SAP s

• 

### Additional uncert ain ties reg ar ding: U.S. Environmental Pr otection Agency **50**



### • 

### Additional uncert ain ties reg ar ding:

• eff ects on immune r esponse in terms of es tablishing use in a risk assessment; and

• ability to e xa mine eff ects t o native species due to lack of suitable husbandry pr otocols



### • 

## Study conducted in r esponse t o 2003 SAP r esult ed in Quan tit ative classification and is consider ed r eliable f or use in es timating risk in a risk assessmen t

# An Ov er vie w of the Methodology fo r De te rmining the Level of Concern fo r At ra zine Based on Aqua tic Plan t Communities U.S. Environmental Pr otection Agency **51**



## Fr ank T. F arruggia, Ph.D
. En vir onmental F ate and E ffe cts Division Of fice of P es ticide Pr ogr ams U. S. En vir onmental Pr otection Agency 

## Russell Erick son, Ph.D
. Mid
- Continent Ecology Division Of fice of R esear ch and Dev elopment U. S. En vir onmental Pr otection Agency

# Objectiv e: The Me thod fo r De te rmining the Level of Concern fo r Aqua tic Plan t Communities

**Stage 2:**  Develop eff ects index based on single
- species toxicity data **Stage 1.**  Characteriz e cosm ex posures and eff ects **52** U.S. Environmental Pr otection Agency

**Stage 4a.**  Apply eff ects index to each monitoring site
- year chemograph **Stage 3a.**  Apply eff ects i ndex to each cosm

study endpoint **Stage 3b.**  Define the eff ects i ndex
- based

L evel of Concern
**Stage 4c.**  Con vert the eff ects - based L OC into a concentration
- eq uivalent LO C **Stage 4b.**  Identify the watersheds with monitoring chemographs that exceed the effects index
- based L OC

# Ov er vie w

• 

## Char act eris tics of atr azine

• Mobile and per sistent in the envir onment

• Photos ystem II inhibitor: re duces the pr oduction of sug ar , o xygen and A

TP



### • 

## Concern to Aquatic Plant Communities

• EP A is concerned with pr ot ecting biological diver sity fr om harmful levels of **53** U.S. Environmental Pr otection Agency

• EP A is concerned with pr ot ecting biological diver sity fr om harmful levels of atr azine. This encompasses pr ot ection of species, assemblage, ecos ystem and landscape levels of biological or ga niz ation and its structur al, compositional and functional components



### • 

## Ava ilable Data

• 

### Single species as well as community lev el tox icity s tudies

**53**

# Mesocosm and Micr ocosm Studies (cosms)

• 

### Po tential eff ects of a tr azine on plant communities ha ve been ev aluated using a va ilable micr ocosm and mesocosm s tudies

• 

### Cosm studies conducted with atr azine r eport community based endpoints that reflect the ag gr eg ate responses of multiple species U.S. Environmental Pr otection Agency **54**

• 

### Cosm studies incorpor ate partitioning , degr ada tion, and dissipation, fa ct or s tha t ar e not usually accounted f or in labor ator y to xicity s tudies, but that ma y influence the magnitude of ecological eff ects

• 

### Re covery after atr azine e xposur e is defined as a r eturn to pr e - ex posur e conditions fo r the aff

ected individual, population or community , not f or a replacement population or community of more toler ant species

# Cosm R eview/Selection Crit eria

• 

## Based on the r ec ommendations fr om the FIFRA SAP s in 2007 and 2009, selection criteria we re dev eloped

• 

## A st udy was included only if:

• 

## tr ea tmen ts we re e xposed to only atr azine, U.S. Environmental Pr otection Agency **55**



### • 

## tr ea tmen ts we re e xposed to only atr azine,

• 

## ex posur e concentr ations wer e r eport ed,

• 

## measur ed eff ects w er e specific t o aquatic plan t communities, and

• 

## the st udy re port is a va ilable in English

# Cosm R eview/Selection Crit eria **A study would be reject ed as in**  **va lid, if**  **an y**  **of the f ollowing**  **accept ability crit**  **eria were not**  **met:** 1. Contr ols run concurr ently and other s tr essor s below potential eff ects levels 2. Quantification of Exposur e - minimally , measur ed initial concentr at ions U.S. Environmental Pr otection Agency **56**



### 2. Quantification of Exposur e - minimally , measur ed initial concentr at ions mus t be pr ovided 3. Experimental Design
- at leas t tw o replicates and solv ents mus t be <0.5 ml/L in s tat ic t es ts or <0.1 ml/L in flow

- thr ough 4. Statistical Methods and Data Interpretation

• At least 3 tr eatments (2 r eplicates/ each)

• Without 2 r eplicates f or each tr eatment, non - linear re gr ession r equir es sample siz e of 5 including control

• St atistical methods must be described and pr esent ed

# Cosm R eview/Selection Crit eria

• 

## A total of 35 cosm studies wer e originally included in the 2003 Interim Reregistr ation Eligibility Decision (IRED).

• 

## An additional 38 cosm studies wer e iden tified by the Ma y 2009 SAP f or a total of 73 studies. U.S. Environmental Pr otection Agency **57**



### • 

## Aft er the r eview/ selection crit eria we re applied:

• 

## 31 of the original 35 s tudies r emained

• 

## 15 of the 38 studies r ec ommended f or consider ation in the Ma y 2009 SAP r eport r emained

• 

## These r esults ar e outlined in Appendix D

# Cosms R epr esen t Na tur al Aqua tic Plan t Communities

• 

## The majority of cosms wer e started from natur al communities collected fr om streams, lak es, r eser voir s, and springs

• 

## The cosms ar e consider ed to be r epr esen tat iv e of the U.S. Environmental Pr otection Agency **58**



### • 

## The cosms ar e consider ed to be r epr esen tat iv e of the st ructur e and function of aquatic plant communities in such water bodies

• 

## Ta ke n together they pr ovide useful and r easonable ph ys ical models of the natur al aquatic plant communities ex posed to atr azine in the U .S.

# Cosm Composition
**Ta xo**  **nomic Group**  **Gener a**  **Species EUBACTERIA:**  CY

ANOBACTERIA: 14 27 **EUKARY OT**  **ES ARCHAEOPLASTID**  **A GREEN**  **PLANTS:**

EMBRY OPHYT

A: (V ascular Land Plants) 11 20 CHL OROPHYT

A and STREPT OPHYT

A (Green Algae): 43 86 PRASINOPHYT

A ( Prasinoph yt es ): 1 1 **CHROMAL**

**VEOLA TES**  **HACROBIA:** HAPT OPHYT

A (Coccolithophorads): 2 4 CRYPT OPHYT

A ( Cryptomonads ): 4 13 U.S. Environmental Pr otection Agency **59**

CRYPT OPHYT

A ( Cryptomonads ): 4 13 **STRAMENOPILES:** BACILLARIOPHYT

A (DIA TO MS): 24 67 CHRYSOPHYT

A (Golden Alg ae): 7 12 XANTHOPHYT

A (Y ellow
- Green Algae): 2 2 **AV EOLA**  **TES:** PYRROPHY COPHYT

A ( Dinoflagellates ): 4 4 **EX CA**  **VA TES**  **EUGLENO ZOA**  **( Euglenoids**  **):** 1 1 **UNIK ONTS**  **FUNGI:** 2 2

**CHO ANOFLAGELLID**  **A:** 3 3 **ANIMALS:** VERTEBRA

TES: 9 15 INVERTEBRA

TES: 137 196

# Cosm Endpoin ts and Dur at ion

• 

## A total of 87 endpoints wer e used in the analysis to dev elop the lev el of concern f or atr azine

• 

## Ef fe cts obser ve d in the cosm studies included:

• 

### changes in aqua tic plant biomass,

• 

### chlor oph yll a concentr at ion, U.S. Environmental Pr otection Agency **60**

• 

### chlor oph yll a concentr at ion,

• 

### phot os yn thesis r ate ( 14 

### C uptak e, o xygen pr oduction),

• 

### shifts in aqua tic plant community s tructur e ( e.g. , species composition and div er sity)



### • 

## The dur ations of these s tudies r ang ed fr om a f ew weeks to sever al year s at constant or v ariable and declining e xposur e concentr ations r anging fr om 0.1 μg /L t o 10,000 μg /L

# Ef fe ct/No

- Ef fe ct Endpoin t Classifica tion

• 

# Move awa y fr om Br ock Scor es

• 

## 5
- point eff ects scoring s yste m: 1 and 2 ar e no - eff ects, while 3, 4, and 5 ar e eff ects

• 

# Binary Coding , Ef

fe cts or No
- eff ects U.S. Environmental Pr otection Agency **61**



## • 

# Binary Coding , Ef

fe cts or No
- eff ects

• 

## Chosen based on the r ec ommendations by the 2009 SAP

• 

## An eff ect classification wa s determined based on the stat is tical significance and/ or magnitude of eff ect

# Cosm Endpoin t Classifica tion

1000 10000**g /** **L a** **t r** **a z** **i n** **e )**  Effect No Effect 

### Ef fe cts of atr azine on e xperimental ecos ystems as a function of e xposur e dur at ion and a ve ra ge concentr at ion (46 s tudies, 87 e xposur es) U.S. Environmental Pr otection Agency **62**

0.01 0.1 1 10 100

1 10 100**A v** **e r** **a g** **e S** **t u** **d y** **C o** **n c** **e n** **t r** **a t** **i o** **n (** **μ g** **/ L** 

**Exposure Dur**  **ation (days)** 

# Goals of the L OC Me thodology The L OC must addr ess diff er ences in concentr ation time
- series among cosm studies

100 1000 10000**( μ** **g /** **L a** **t r** **a z** **i n** **e )**  Ef fect No Effect**C o** **n c** **e n** **t r** **a t** **i o** **n 30**  **40 50**  U.S. Environmental Pr otection Agency **63**

0.01 0.1 1 10 100

1 10 100**A v** **e r** **a g** **e S** **t u** **d y** **C o** **n c** **e n** **t r** **a t** **i o** **n (** 

**Exposure Dur**  **ation (days)**  **Time from Exposure Start (d) 0**  **20 40**  **60 80** **C o** **n c** **e n** **t r** **a t** **i o** **n**

**0 10**  **20 30** 

**Time from Exposure Start (d) 0**  **20 40**  **60 80** **C o** **n c** **e n** **t r** **a t** **i o** **n**

**0 2**  **4 6**  **8 10** 

# Goals of the L OC Me thodology
Ex amples of atr azine exposur e time
series (chemogr aphs) fo r natur al fr eshw ater systems 

### • Compare the t ox icity of diff erent atr azine co ncentr ations and leng th of ex posure times

• Compare data fr om c osm studies t o even more v ariable U.S. Environmental Pr otection Agency **64**



### Compare data fr om c osm studies t o even more v ariable en vir onmen ta l monit oring data

# The Me thod fo r De te rmining the Level of Concern

fo r Aqua tic Plan t Communities

**Stage 2:**  Develop eff ects index based on single
- species toxicity data **Stage 1.**  Characteriz e cosm ex posures and eff ects

**Stage 4a.**  Apply eff ects i ndex to each **65** U.S. Environmental Pr otection Agency

**Stage 4a.**  Apply eff ects i ndex to each monitoring site
- year chemograph **Stage 3a.**  Apply eff ects i ndex to each cosm

study endpoint **Stage 3b.**  Define the eff ects index
- based

Level of Concern
**Stage 4c.**  Con vert the eff ects - based L OC into a concentration
- e quivalent LO C **Stage 4b.**  Identify the watersheds with monitoring chemographs that exceed the effects index
- based L OC

# • 

# Pr oblem: need t o c ompar e ef fe cts acr oss dif fe re nt e xposur e time
- series

• Quan tify cumulativ e **relative** tox ic sev erity over an assessment period 

# St ag e 2: The E ff ects Inde x U.S. Environmental Pr otection Agency **66**



# • Candidat es: p pb
- da ys, Species Sensitivity Dis tribution (SSD), Plan t Assemblage T ox icity Inde x (P AT I), c ommunity simulation models • F unction only of e xposur e time
- series, not other s yste m pr operties

# The Me thod fo r De te rmining the Level of Concern

fo r Aqua tic Plan t Communities

**Stage 2:**  Develop eff ects index based on single
- species toxicity data **Stage 1.**  Characteriz e cosm ex posures and eff ects **67** U.S. Environmental Pr otection Agency

**Stage 4a.**  Apply eff ects i ndex to each monitoring site
- year chemograph **Stage 3a.**  Apply eff ects i ndex to each cosm

study endpoint **Stage 3b.**  Define the eff ects i ndex
- based

Level of Concern
**Stage 4c.**  Con vert the eff ects - b ased L OC into a concentration
- equivalent LO C **Stage 4b.**  Identify the watersheds with monitoring chemographs that exceed the effects index
- based L OC

# The Me thod fo r De te rmining the Level of Concern

fo r Aqua tic Plan t Communities

Effects Index 

## Effects Index EEF LOC  

# Ef fe cts Ex ceedance F act or (EEF): U.S. Environmental Pr otection Agency **68**



# Concen tr ation Ex ceedance F act or (CEF): Fa ctor by which entir e exposur e series must be r educed so that the eff ects index = LO C Ef fects Index



## Original Chem ograph CE F Chem ograph Adjusted to LOC 

# • 

# Compr ehensiv e Aquatic Syste ms Model (CASM) w as our initial eff ects inde x c andidat e

• CASM simulat es biomass v er sus time f or in te r - re lat ed species in a model co mmunity 

# Community Model E va lua tions **69** U.S. Environmental Pr otection Agency



# in te r - re lat ed species in a model co mmunity

• Ea ch t erm in the equation is a function of va rious biomasses and input va riables • In CASM, e ach species is described by an equation that addr esses v arious biomass ga ins and losses

# • 

# Fo r atr azine assessmen t, CASM f ormulat ed to r epr esen t a generic corn belt s tr eam

• P re liminary ve rs ion, CASM AT Z1 

# , w as used f or 2003 ev aluations and f or the 2007 SAP re view; pr oblems w/r ealism of biomasses and t ox icity 

# Community Model E va lua tions **70** U.S. Environmental Pr otection Agency



# re view; pr oblems w/r ealism of biomasses and t ox icity

• M or e re alis tic ve rs ion, CASM AT Z2 

# , w as dev eloped and ev aluat ed f or the 2009 SAP re view; pr oblems with lar ge uncert ain ties

• “ P ro babilis tic CASM” w as in tr oduced by Sy ngent a at the 2009 SAP r eview

# Ke y CASM uncert ain ty is selection of tox icity r elationships fo r gr owth of a f ew

dominan t species 

# Community Model E va lua tions%ReductioninSpecificGrowthRate 100 **71** U.S. Environmental Pr otection Agency



### Atrazine Concentration (  g/L)

20 50 200 500 10 100 1000%ReductioninSpecificGrowthRate

0 20 40 60 80

Vascular Plants Green Algae Bluegreen Algae Diatoms

# Community Model E va lua tionsg/LAtrazine 0

50 100 150

60 90 MO 07 2 010 MO 01 2010 Me dian CEF = 0.69 (10 th -90 th = 

### 0.16-2.00 )

Me dian CEF = 1.85 **72** U.S. Environmental Pr otection Agency



### Julian Dayg/LAtrazine

120 150 180 210

0 10 20 30 0 30 60

MO 02 2010 Me dian CEF = 1.85 (10 th -90 th = 

### 0.58-4.65 )

Me dian CEF = 1.08 (10 th -90 th = 

### 0.30-3.49 )

# • 

# Giv en the v arious uncert ain ties of CASM, alt ernativ e e ff ects indices wer e co nsider ed

• C an the effects index be based on well
established processes in CASM important to the effects index, while eliminating major 

# Community Model E va lua tions **73** U.S. Environmental Pr otection Agency



# • 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?%ReductioninSpecificGrowthRate

20

40 60 80 100 

# Plan t Assemblag e T

ox icity Inde x **74** U.S. Environmental Pr otection Agency

20 50 200 500 10 100 1000SpecificGrowthRate

0

Atrazine Concentration (  g/L)

20 50 200 500 10 100 1000Average%ReductionacrossAllSpecies

0 20 40 60 80 100
Plant Assemblage To xicity Index (P

AT I)

# • 

# PAT I is a simple e xt ension of the Species Sensitivity Dis tribution co ncept

• To addr ess cumulativ e eff ect of time
- va rying co ncen tr ations, “ Cumulativ e PAT I” is the sum of daily P

AT I v alues ov er a designat ed 

# Plan t Assemblag e T

ox icity Inde x **75** U.S. Environmental Pr otection Agency



# sum of daily P

AT I v alues ov er a designat ed “a ssessmen t period” (units = %
- da ys)

• This simple summation is jus tified by the fa ct that, t o ha ve the same t ot al eff ect on gr owth ov er a period, **an y**  series of specific gr owth r ate s mus t add t o the same v alue

# Plan t Assemblag e T

ox icity Inde x Assessmen t Pe riod Selection Issues
– Cosm Dat a **76** U.S. Environmental Pr otection Agency

100 1000 10000**A v** **e r** **a g** **e S** **t u** **d y** **C o** **n c** **e n** **t r** **a t** **i o** **n (** **u g** **/ L** **a t** **r a** **z i** **n e** **)** Effect No Effect **14 -**  **Da y**  **60 -**  **Da y**  U.S. Environmental Pr otection Agency **76**

0.01 0.1 1 10 100

1 10 100**A v** **e r** **a g** **e S** **t u** **d y** **C o** **n c** **e n** **t r** **a t** **i o** **n (** **u g** **/ L** **a t** **r a** **z i** **n e** **)**

**Exposure duration (day s)** 

# Plan t Assemblag e T ox icity Inde x Assessmen t Pe riod Selection Issues
– Field Dat ag/LAtrazine 0 50 100 150

90 MO 01 2010 **14 -**  **Da y**  **60 -**  **Da y**  U.S. Environmental Pr otection Agency **77**



### Julian Dayg/LAtrazine

120 150 180 210 0 10 20 30 0 30 60 90 MO 07 2010

MO 02 2010

# Plan t Assemblag e T ox icity Inde x Te mpor al dependence assumed by PAT I is similar t o

that assumed by CASMLevelofConcernforConstantg/LAtrazine) 50 100 **PA**

**TI CA**  **SM** U.S. Environmental Pr otection Agency **78**



### Duration (d) 20 50 10LevelofConcernforConstantConcentration(g/LAtrazine)

5 20
10 **CA SM** 

# Plan t Assemblag e T ox icity Inde xg/LAtrazine 0 50 100 150

60 90 MO 07 2010 MO 01 2010 PATI CEF = 0.65 10 th -90 th = 0.62-0.68 CASM=0.69 10 th -90 th = 0.16-2.00
PATI CEF = 2.34 10 th -90 th = 2.24-2.46 **79** U.S. Environmental Pr otection Agency



### Julian Dayg/LAtrazine

120 150 180 210

0 10 20 30 0 30 60

MO 02 2010 10 -90 = 2.24-2.46 CASM=1.85 10 th -90 th = 0.58-4.65
PATI CEF = 1.07 10 th -90 th = 1.02-1.13 CASM=1.08 10 th -90 th = 0.30-3.49

# The Me thod fo r De te rmining the Level of Concern fo r Aqua tic Plan t Communities

**Stage 2:**  Develop eff ects index based on single
- species toxicity data **Stage 1.**  Characteriz e cosm ex posures and eff ects U.S. Environmental Pr otection Agency **80**

**Stage 4a.**  Apply eff ects index to each monitoring

site
- year chemograph **Stage 3a.**  Apply eff ects index to each cosm

study endpoint **Stage 3b.**  Define the eff ects index
- based

Level of Concern

**Stage 4c.**  Con vert the eff ects - based L OC into a concentration
- equivalent LO C **Stage 4b.**  Identify the watersheds with monitoring chemographs that exceed the effects index
- based L OC

# Cumula tive PAT I fo r Cosm Studies

• 

### For each cosm study endpoint a chemogr aph is cr eated

• 

### For each chemogr aph a cumula tiv e P

AT I v alue is calculated using the P

AT I dis tribution 20 30 40 50 60 70 80 90Concentration(μg/Latrazine) **Cosm Endpoint #7**  **, E ff**  **ect**

Cumulative PAT I = 1869 %
- days U.S. Environmental Pr otection Agency **81**



### dis tribution

0.01 0.1 1 10 100 1000 10000

1 10 100**A v** **e r** **a g** **e S** **t u** **d y** **C o** **n c** **e n** **t r** **a t** **i o** **n (** **μ g** **/ L** **a t** **r a** **z i** **n e** **)**

**Exposure duration (days)** Effect No Effect 0 10 20

0 10 20 30 40 50 60Concentration

Da y**A v** **e r** **a g** **e %** **t o** **x i** **c e** **f f** **e c** **t a** **c r** **o s** **s a** **l l** **s p** **e c** **i e** **s**0.5 1 1.5 2 2.5 3 3.5Concentration(μg/Latrazine) **Cosm Endpoint #76, No E**  **ff ect**  

# Cumula tive PAT I fo r Cosm Studies

• 

### For each cosm study endpoint a chemogr aph is cr eated

• 

### For each chemogr aph a cumula tiv e P

AT I v alue is calculated using the P

AT I dis tribution Cumulative PAT I = 39.6 %
- days U.S. Environmental Pr otection Agency **82**

0 0 5 10 15 20 25Concentration

Da y 

### dis tribution

0.01 0.1 1 10 100 1000 10000

1 10 100**A v** **e r** **a g** **e S** **t u** **d y** **C o** **n c** **e n** **t r** **a t** **i o** **n (** **u g** **/ L** **a t** **r a** **z i** **n e** **)**

**Exposure duration (days)** Effect No Effect**A v** **e r** **a g** **e %** **t o** **x i** **c e** **f f** **e c** **t a** **c r** **o s** **s a** **l l** **s p** **e c** **i e** **s**

# The Me thod fo r De te rmining the Level of Concern fo r Aqua tic Plan t Communities

**Stage 2:**  Develop eff ects index based on single
- species toxicity data **Stage 1.**  Characteriz e cosm ex posures and eff ects U.S. Environmental Pr otection Agency **83**

**Stage 4a.**  Apply eff ects index to each monitoring

site
- year chemograph **Stage 3a.**  Apply eff ects index to each cosm

study endpoint
**Stage 3b.**  Define the eff ects index
- based

Level of Concern

**Stage 4c.**  Con vert the eff ects - b ased L OC into a concentration
- equivalent LO C **Stage 4b.**  Identify the watersheds with monitoring chemographs that exceed the effects index
- based L OC

# Se tt ing the L OC

• 

## Cosm st udy endpoints plot te d as eff ect (closed triangle)/no

- eff ect (open triangles) ve rs us P

AT I

• 

## Fit te d to a logistic re lationship f or the U.S. Environmental Pr otection Agency **84**



## Fit te d to a logistic re lationship f or the pr obability of an eff ect ve rs us P AT I

• 

## LO C defined as the cumulativ e PAT I v alue, which equals a 50% pr obability of an eff ect **LO C**  **PAT I**  

### = 140 % **- da**  **ys Stage**  **2:** Develop eff ects index based on single
- species toxicity data **Stage 1.**  Characteriz e cosm ex posures and eff ects 

# The Me thod fo r De te rmining the Level of Concern fo r Aqua tic Plan t Communities U.S. Environmental Pr otection Agency **85**

**Stage 4a.**  Apply eff ects index to each monitoring

site
- year chemograph. **Stage 3a.**  Apply eff ects index to each cosm

study endpoint **Stage 3b.**  Define the eff ects index
- based

Level of Concern

**Stage 4c.**  Con vert the eff ects - b ased L OC into a concentration
- equivalent LO C **Stage 4b.**  Identify the watersheds with monitoring chemographs that exceed the effects index
- based L OC

### • At ra zine E co logic al Exposure Monit oring Pr ogr am (AEEMP)

• Fo r each en vir onmen ta l monit oring chemogr aph a cumulative P

AT I v alue is ca lculated using the P

AT I 

# Cumula tive PAT I fo r Monit oring Da ta

40 50 60 70 80 90 100**C o** **n c** **e n** **t r** **a t** **i o** **n (** **p p** **b )**  **MO -**  **07 2010**

Maximum 60
- Day Cumulative PAT I = 588 %
- days U.S. Environmental Pr otection Agency **86**



### ca lculated using the P

AT I distribution

0 10 20 30 40

0 50 100 150 200 250 300 350**C o** **n c** **e n** **t r** **a t** **i o** **n (** **p p** **b )** 

**Julian Da y**  588 %
- daysAverage%toxiceffectacrossallspecies

# The Me thod fo r De te rmining the Level of Concern fo r Aqua tic Plan t Communities

**Stage 2:**  Develop eff ects index based on single
- species toxicity data **Stage 1.**  Characteriz e cosm ex posures and eff ects U.S. Environmental Pr otection Agency **87**

**Stage 4a.**  Apply eff ects index to each monitoring

site
- year chemograph **Stage 3a.**  Apply eff ects index to each cosm

study endpoint
**Stage 3b.**  Define the eff ects index
- based

Level of Concern

**Stage 4c.**  Con vert the eff ects - based L OC into a concentration
- equivalent LO C **Stage 4b.**  Identify the watersheds with monitoring chemographs that exceed the effects index
- based L OC

# Comparing the Monit oring Da ta to the

Cosm Studies Based on the cumulativ e P AT I v alue the monit oring site can be determined as abov e or below the L OC U.S. Environmental Pr otection Agency **88**

# The Me thod fo r De te rmining the Level of Concern fo r Aqua tic Plan t Communities

**Stage 2:**  Develop eff ects index based on single
- species toxicity data **Stage 1.**  Characteriz e cosm ex posures and eff ects U.S. Environmental Pr otection Agency **89**

**Stage 4a.**  Apply E ffects Index to each monitoring

site
- year chemograph. **Stage 3a.**  Apply E ffects Index to each cosm

study endpoint.
**Stage 3b.**  Define the E ffects Index based Level of

Concern.

**Stage 4c.**  Con vert the E ffects Based L OC into a concentration equivalent LO C. **Stage 4b.**  Identify the watersheds with monitoring chemographs that exceed the E ffects

Index based L OC.

# Con ve rting LO C PAT I 

# in to a Concen tr ati on Eq uiv alen t LO C (CE

- LO C)

Cumulative P

AT I for Ea ch Monitoring Site
- Ye ar LO C PAT I 

### = E ff ects Ex ceedance Fa ctor U.S. Environmental Pr otection Agency **90**

# Chang es in the 60
- da y Mean CE
- LO C Due to Chang es in the Methodology or A va ilable Da ta

• The original screening c oncen tr ation (2003 IRED) based on a review of t ox icity dat a was 10
- 20 ppb over an undefined period of time.

• Based on the chang es r ec ommended by 2007 SAP the CE
- LO C ra nged fr om 7.2 t o 8.1. U.S. Environmental Pr otection Agency **91**



### ra nged fr om 7.2 t o 8.1.

• In 2009, EP

A c onsidered 10 ppb c oncen tr ation f or a 60
- da y assessmen t period t o be above levels where adver se e ff ects to the aquatic plant community wo uld be e xpect ed.

• Based on the chang es r ec ommended by the 2009 SAP the CE
- LO C dr opped t o 4.2.

# Assessmen t Pe riod Ef

fe cts on the CE
- LO C

**Ef fe**  **ct of averaging period**  **on the percent of AEEMP site/years**  **exc eeding**  **the CE**  **- LO**  **C .**  **Ave ra**  **ging Pe**  **riod 7**  **- Da**  **y 14**  **- Da**  **y 30**  **- Da**  **y 60**  **- Da**  **y 90**  **- Da**  **y Cumulative**  **PAT I V** 

**alue (%**  **- days)**  **65.5 107.4**  **132.7 140**  **141.8 CE**  **- LO**  **C (μg**  **/L) Linear Regression**  **(0.5 -**  **2.0 EEF Range) 18**  **14.9 8.3**  **4.2 2.7**  **% AEEMP Sites -**  **years Ex**  **ceeding the**  **CE -**  **LO C**  **39 33**  **44 58**  **60** U.S. Environmental Pr otection Agency **92**

0.01 0.1 1 10 100 1000 10000

1 10 100AverageStudyConcentration(ug/Latrazine)

Exposure duration (days) Effect No Effect • Most cosm eff ects/no

- eff ects dat a is

captured between 7
- 63 da ys

• The 60
- da y assessment period bet te r re pr esents the typical atr azine exposur e pr ofile.

• 60
- da y CE
- LO C is pr ot ective of short er assessment periods

# Sensitivity Analy sis
**Br ock Scor**  **e “2” in 2009:**

• 

### Endpoint #51 (Br ockw ay et al. , 1984): 50 ppb

• 25% r eduction in ph yt oplankton ox ygen production

• 

### Endpoint #59 (Pr att et al. , 1988): 32 ppb **Br ock Scor**  **e “3” in 2009:**

• 

### Endpoint #52 ( deNoyelles et al. , 1982, 1989): 20 ppb

• 50% decline in 14 C
- uptak e

• 50% decline in ph yt oplankton biomass. U.S. Environmental Pr otection Agency **93**



### 32 ppb

• 35% decr ease in dissolved o xygen,

• slight reductions in magnesium and calcium levels

• 

### Endpoint #60 (Pr att et al. , 1988): 110 ppb

• 35% decr ease in dissolved o xygen

• 60% r eduction in chlor oph yll
- a

• slight reductions in magnesium and calcium levels 

### Br **ock Scor e “4” in 2009:** 

• 

### Endpoint #58 ( Lampert et al. , 1989): 1 ppb

• 50% decr ease in chlor oph yll
- a

• 50% r eduction in o xygen satur ation

# Sensitivity Analy sis

75 th Pe rcentile L OC PAT I = 446 %
- da ys; CE
- LO C = 15 ppb 50 th Pe rcentile L OC PAT I = 140 %
- da ys; CE
- LO C = 4 ppb 25 th Pe rcentile L OC PAT I = 35 %
- da ys; CE
- LO C = 1 ppb U.S. Environmental Pr otection Agency **94**

Range of uncert ainty ar ound the L OC PAT I calculation

• 95% confidence intervals around the 50 th per centile were calculated

• These wer e converted into EEF s

• The linear re gr ession equations wer e used to estimat e concentr ations f or these intervals.

• 4 ppb CE
- LO C (2

- 10 ppb)

• 7 ppb CE
- LO C (3

- 15 ppb)

# Conclusions

• 

## The cosm st udies pr ovide r easonable models of ecological communities fo und thr oughout the primary atr azine use ar eas of North America.

• 

## The method fo r determining the Lev el of Concern fo r Aquatic Plan t U.S. Environmental Pr otection Agency **95**



### • 

## The method fo r determining the Lev el of Concern fo r Aquatic Plan t Communities has been updated based on the r ec ommendations of SAP s in 2007 and 2009.

• 

## The CE
- LO C of 4
- 7 ppb over a 60
- da y assessmen t period is pr otectiv e of aquatic plan t communities and in turn pr otectiv e of the en tir e aquatic ec os

yste m.

# Method fo r Comparing Monit oring Da ta to t he Aqua tic Plan t Community CE
- LO C fo r A

tr azine U.S. Environmental Pr otection Agency **96**



## James A. Hetrick, Ph.D
. En vir onmental F ate and E ffe cts Division Of fice of P es ticide Pr ogr ams U. S. En vir onmental Pr otection Agency

# In tr oduction

• 

# Ext ensiv e monitoring dat a f or atr azine

• 

# Es timating atr azine e xposur e U.S. Environmental Pr otection Agency **97**



### • 

## Es timating atr azine e xposur e in the absence of monitoring data

• 

## Es timating atr azine concentr ations fr om monitoring data

# Objectiv es

• 

## Dev elop a bias f act or method to addr ess uncert ain ty in es timating atr azine concentr ations fr om v arious monitoring data with v arying sample fr equency

• 

## Illus tr ate a scr eening lev el pr ocess using a bias f act or method U.S. Environmental Pr otection Agency **98**



### • 

## Illus tr ate a scr eening lev el pr ocess using a bias f act or method fo r assessing potential site
- ye ar e xc eedance of the CE
- LO C fr om monitoring data

• 

### Dev elop bias f act or s f or maximum 60
- da y ro lling mean concentr at ion

• 

### Explor e diff er ent bias f act or es timation methods

• 

### Te st perf ormance of bias f act or methods

• 

### Illus tr ate application of a bias f act or method in a scr eening lev el assessment of monitoring data

# Use of Bias F act or Scr eening Level Pr ocess in the

Risk Assessmen t Fr amework

**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 Pr otection Agency **99**

**Risk Management Communicating**  **Results to**  **the Risk Manager**  **to the**  **Bias Factor**

**Screen ANALYSIS** 

**RISK CHARACTERIZATION**  **Modeling**

**CE -**  **LOC Watershed**  **Identification Modeling** 

# Na tur e of P es ticide Occurr ence in Wa te r

• 

# Comple x spatial pat te rns

• 

# Comple x t empor al pat te rns U.S. Environmental Pr otection Agency **100**



## • 

# Comple x t empor al pat te rns

# At ra zine Chemogr aphs

• 

## Heidelber g Univ er sity (NCW QR) data f or OH

• 

## 17,000 km 2 

## catc hmen t ar ea • 

## Sy ng en ta at ra zine ecological ex posur e monitoring (AEEMP)

• 

## 30 km 2 

## catc hmen t ar ea

16 **Maumee River**  **, 1995** 100 U.S. Environmental Pr otection 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**A t** **r a** **z i** **n e** **C o** **n c** **e n** **t r** **a t** **i o** **n ,** **μ g** **/ 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**A t** **r a** **z i** **n e** **C o** **n c** **e n** **t r** **a t** **i o** **n ,** **μ g** **/ L**  **MO -**  **01, 2007** 

# Schema tic fo r Bias F act or Es tima te f ro m a Single Sit e - Ye arAtrazineConc(ug/L)

2 4 6 8 10 12 14 16 18AtrazineConc(ug/L)

4 6 8 10 12 14 16 18 4, 7, 14, 28 Day Sampling Intervals U.S. Environmental Pr otection 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 140AtrazineConcentration(ug/L)

0 2 4 6 8 10 12 14 16 18 **Tr ue max 60**  **- day**  **mean 5**  **th per**  **centile max 60 -**  **day mean**  Random Sampling Distribution of maximum 60
- da y mean concentr ation

# Monit oring Da ta S election

• 

## Selected site
- ye ar data from A tr azine Ecological Exposur e Monitoring Pr ogr am (AEEMP) and Heidelber g Univer sity National Center of W ater Quality R esear ch (NCW QR)

• 

### High sample fr equency

• 4
- da y sampling interv als vs. daily sampling 

### Ty pe of sampling U.S. Environmental Pr otection Agency **103**

• 

### Ty pe of sampling

• Auto
- sampling v s. gr ab samples

• 

### Leng th of sampling period

• AEEMP f ocused on the 2 nd and 3 rd quarter s of the year



### • 

## Select a r ang e of site
- ye ar conditions

• 

### Wa te rs hed siz es

• 

### Hydr ology (a ve ra ge flow)

# Pr eliminary AEEMP and NCW QR Monit oring Dat a

**Site Ye**  **ar Wa**  **tershed Area**  **(acres) Ave**  **ra ge**  **Flow**

**(m 3**  **/s ec)**  **Maximum**

**Pe ak**  **Maximum**

**60 -**  **day Ave**  **ra ge**  **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 Pr otection 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 Cuyahog a 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

# Definitiv e AEEMP and NCW QR Monit oring Dat a

**Sit e**  **Ye ar**  **Wa te**  **rs hed**  **Area (acres) Aver**  **age Flow**

**(m 3**  **/s ec)**  **Maximum**

**Peak Maximum** 

**60 -**  **day Aver**  **age μg**  **/L** MO
- 05 2008 16,192 0.32 37.83 5.74 U.S. Environmental Pr otection 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

# Spa tial Dis tribution of Monit oring Sit es

**IA -**  **01**

**IA -**  **02 NE**  **- 02**  **IN -**  **06 IL**  **- 03**  **IL -**  **01 Sa**  **ndusky R**  **Gr and**  **R Cu**  **ya hoga**  **R** U.S. Environmental Pr otection Agency **106**

**MO -**  **01 MO**  **- 04a** 
**MO -**  **02 MO**  **- 05**  **MO -**  **05b**

**TN -**  **01 IN**  **- 11**  **IN -**  **06**
**IL -**  **09 Gr**  **.Miam i**  **R Sc**  **ioto R** 

**MO -**  **01 MO**  **- 04a** 

**MO -**  **02 MO**  **- 05**  **MO -**  **05b**

# Assumptions, Limit at ions and Uncert ain ties with Bias F act or Es tima tion

• 

## At ra zine occurr ence fr om April 1 st 

## to September 17 th 

## is assumed to be equal to 2 nd 

## and 3 rd 

## quart er s of the year

• Sample Number ≈ 180 da ys/Sample Int erv al



### • 

## Es timates will diff er f or diff er en t sampling s tr ate gies U.S. Environmental Pr otection Agency **107**



### • 

## Ext en t of data infilling incr eases uncert ain ty

• 

## Serial corr ela tion eff ects ar e dependen t on the sampling in te rv al

• 

## Pr ediction capability is dependen t on the underlying data

# Absolut e Va lue of Bias F act or s f or Pr eliminary Dat a **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 Pr otection 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 **Cuyahog a 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** 

# Absolut e Va lue of Bias Fa ct or s fo r Definitiv e Da ta

**Sit e**  **Sampling Interval** 

**4 -**  **da y**  **7 -**  **da y**  **14 -**  **da y**  **28 -**  **da y**  **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 Pr otection 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

# Re la tionship of W ate rs hed Siz e, Av er ag e Flow , Sample Number with Bias F act or Fo rw ar d Stepwise Multiple Re gr ession

• 

## Sample Number (180 da ys/Sample Inter va l)

• Significant (p<0.05) U.S. Environmental Pr otection Agency **110**



### • 

## Wa te rs hed Ar ea

• Non
- significant (p>0.05)



### • 

## Ave ra ge Flow

• Non
- significant (p>0.05)

# Re la tionship of Sampling Number

and Bias F act or

20 25 30

Preliminary Data Definitive Data U.S. Environmental Pr otection Agency **111**

Number of Samples 2nd and 3rd Quarters (180 days/sample interval) 0 10 20 30 40 50BiasFactor

0 5 10 15 20

# Ca te go ric al Es tima tion Appr oachBiasFactor 8

10 12 U.S. Environmental Pr otection Agency **112**

Num ber o f S am ples in 2 nd a nd 3 rd Q uar te r 10 20 30 40 50BiasFactor

0 2 4 6

# Non
- Linear Re gr ession Eq ua tionsBiasFactor 15 20 25 **Non-Linear Equation (A ll 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 (A ll Data; )**  Y=1383.0681*exp(-1.0681*X)+11.6378*exp(-0.0425*X) r 2 =0.9993; p=0.0001 U.S. Environmental Pr otection Agency **113**

113 Number of Samples in 2nd and 3rd Quarters 0 10 20 30 40 50BiasFactor

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 Re gr ession Eq ua tionsLogBiasFactor 100 **Linear Regression** log Y=0.78616-0.013729*X r2=0.4634,p=0.0001 **Upper 95th Percentile Confidence Interv a**  l log Y=0.946326366-0.007773*X U.S. Environmental Pr otection Agency **114**

Number of Samples in the 2nd and 3rd Quarter 5 10 15 20 25 30 35 40 45LogBiasFactor

1 10 Bias Factor 95% Confidence Bounds

# Comparison of Bias F act or Pr ediction

**Sampling Interval**  **(Da ys)**  **Number of** 

**Samples Over 180**  **Da ys**  **Categorical Log**  **Linear Log**  **Linear**

**Bias F actor**  **Upper 95% Confidence**  **Interval Non**  **- Linear**  **Non -**  **Linear Bias F**  **actor Upper 95%**  **Confidence Interval**  **Average**

**Non -**  **Linear**

**4 45**  1.87 1.55 3.95 1.00 1.72 1.79 U.S. Environmental Pr otection 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

# Pe rf

ormance Te st of Bias F act or Re gr ession Eq ua tions

**Pe rcentage**  **of the AEEMP site**  **- years**  **(N=190) that**  **fa iled**  **to meet or ex**  **ceed the actual**  **60 -**  **day maximum**  **running mean based**  **on various sampling interv**  **als**

**Sampling Interval**  **60 -**  **Da y** 

**Maximum Running**  **Log Linear**  **Log Linear Bias Fa**  **ctor Upper 95%**  **Non**  

** Linear** **Non -**  **Linear**

**Bias F actor**  **Upper 95% Mean** 

**Non -**  **Linear** U.S. Environmental Pr otection Agency **116**

**Interval (Da**  **ys) 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

# Tr anslation of Monit oring Dat a with the Bias F act or s

Sit e - ye ar chemogr aph fr om monitoring dataAtrazineConcentration(ug/L) 15 20 U.S. Environmental Pr otection Agency **117**

Julian Day 0 100 200 300AtrazineConcentration(ug/L)

0 5 10

# Tr anslation of Monit oring Dat a with the Bias F act or s

St air
- ste p in te rpolation of site
- ye ar chemogr aphAtrazineConcentration(ug/L) 15

20 U.S. Environmental Pr otection Agency **118**

Julian Day 0 100 200 300AtrazineConcentration(ug/L)

0 5 10 Samples=38

# Tr anslation of Monit oring Dat a with the Bias F act or s

Calculate ma ximum 60
- da y r olling meanAtrazineConcentration(ug/L) 15 20 U.S. Environmental Pr otection Agency **119**

Julian Day 0 100 200 300AtrazineConcentration(ug/L)

0 5 10 15

Max 60 day rolling mean=3.14ug/L

# Tr anslation of Monit oring Dat a with the Bias F act or s

• 

## Use log
- linear upper 95 th 

## per cen tile confidence inter va l re gr ession equation Bias F act or = 10^(0.946326366

- 0.007773* **38** ) = **4.48**



### • 

## Multiply the maximum 60
- da y r olling mean by the es timated bias f act or U.S. Environmental Pr otection Agency **120**



## bias f act or 4.48 * 3.14 μg /L = **14.06 μg**  **/L**



### • 

## Compar e the bias f act or adjus te d 60
- da y r olling mean to the CE
- LO C **14.06 μg**  **/L > CE -**  **LO C 4 μg**  **/L and 7 μg /L** 



### • 

## Sit e - ye ar ma y e xc eed CE
- LO C; mor e fr equen t monit oring ma y re duce uncert ain ty in assessing atr azine concen tr ation

# Monit oring Da ta S our ces

• 

## AMP Monitoring Data Finished & Raw W ate r 2003
- 2011

• 

## AEEMP Monitoring Data

• 

## NCW QR Monitoring Data

• 

## IA DNR 2003
- 2006

• 

## KS WQ 2009
- 2011 U.S. Environmental Pr otection Agency **121**



### • 

## KS WQ 2009
- 2011

• 

## MN MD A SW 2008
- 2011

• 

## MN MD A WQ 1993
- 2007

• 

## NA WQ A

• 

## NE State Monitoring

• 

## Pe st icide Data Progr am

• 

## WI 2008
- 2011

• 

## USEP A Re ser voir Data 1999
- 2000

# Non
- adjus te d Monit oring Da ta

• 

# Site
- ye ar s with ≥4 samples (Julian Da y 0 t o 365)

• 

# 6,917 site
- ye ar s

• 

# Es timated number of site
- ye ar s with concen tr ations U.S. Environmental Pr otection Agency **122**



## • 

# Es timated number of site
- ye ar s with concen tr ations exc eeding a CE
- LO C of 4 μg /L

• 

## 2006
- 2011: 387 site
- ye ar s (6 %); 13 states FL, IA, IL, IN, KS, KY

, LA, MO , MS, NE, OH, TX, WI



## • 

# Es timated number of site
- ye ar s with concen tr ations not e xc eeding a CE
- LO C of 4 μg /L

• 

## 2006
- 2011: 6,502 (94%)

# Bias F act or Adjus te d Monit oring Da ta

• 

## Site
- Ye ar s with ≥6 samples (Julian Da y 90 to 270)

• 

## 2,283 site
- ye ar s

• 

## Bias fa ctor s r ang ed fr om 3.36 to 7.94

• 

## Estimated number of site
- ye ar s with concentr at ions U.S. Environmental Pr otection Agency **123**



### • 

## Estimated number of site
- ye ar s with concentr at ions ex ceeding the CE
- LO C of 4 μg /L

• 

### 2006
- 2011: 1,424 sit e - year s (62%); 22 st ates AL, AR, CA, FL, IA, IL, IN, KS, KY , LA, MD , MN, MO , MS, NC, NE, OH, P A, TN, TX, V

A, WI



### • 

## Estimated number of sites
- ye ar s with concentr at ions not ex ceeding the CE
- LO C of 4 μg /L

• 

### 2006
- 2011: 868 (38%)

# Pe rc en ta ge of Site
- Ye ar s with Es timated A tr azine Concen tr ations Ex ceeding a CE
- LO C of 4 μg /L f or Non
- Adjus te d and Bias F act or Adjus te d Monit oring Dat aPercentofSite-Years 100 120 Non-Adj usted M onitoring Data

Bias Factor Adj usted M onitor ing Data U.S. Environmental Pr otection Agency **124**

Sample Num ber in 2nd and 3rd Quarters 0 10 20 30 40 50PercentofSite-Years

0 20 40 60 80

# 2006
- 2011 NA

WQ A Non
- Adjus te d Monitoring Dat a CE
- LO C = 4 μg /L U.S. Environmental Pr otection Agency **125**

# 2006
- 2011 NA

WQ A Bias F act or Adjus te d Monit oring Dat a CE
- LO C = 4 μg /L U.S. Environmental Pr otection Agency **126**

# Conclusions

• 

### Sample fr equency is significant

• 

### Wa ter shed ar ea and a ve ra ge flow are not significant

• 

### Application of bias f act or s to NA WQA data

• 

### Monitoring site
- year s exc eeding the CE
- LO C ar e gener ally associated with high at ra zine use intensity ar eas and r egions of the country with pr eviously U.S. Environmental Pr otection Agency **127**



### high at ra zine use intensity ar eas and r egions of the country with pr eviously identified vulner able w atersheds

• Application of bias f act or s identified monitoring sites pot entially exc eeding CE
- LO C outside ar eas with high atr azine use intensity and vulner able w atersheds

• 

### The bias f act or method ma y be an efficient scr eening tool to assess uncert ainty in estimating at ra zine concentr at ion fr om monitoring data and to identify monitoring sites re quiring higher r esolution e xposur e assessment

# To **pic A: E**

**va luation of the Data on Amphibians**  There are an increasing number of lit er ature studies e xa mining whether there is a c ausal relationship between atr azine e xposure and abnormal se xu al development, metamorphosis and gr owth, and/ or immune response in amphibians (Chapt er II of the Whit e P aper). In or der to determine the degree t o which these studies are appr opriat e fo r inc orpor ation into risk assessmen t, EP A ev aluated the data within the c on tex t of c onsidering the nature and degree of the uncert ain ties U.S. Environmental Pr otection Agency **128**



### the c on tex t of c onsidering the nature and degree of the uncert ain ties in each study
. 

## Charge Question 1 : Is the SAP aware of an y other laborat ory based or field
- based studies not included in this Whit e P aper that should be c onsidered?

## Charge **Questions 2a and**  **2b** : EP A iden tified t es t design elemen ts that could potentially conf ound the ability of a s tudy t o discern a causal r ela tionship between e xposur e t o atr azine and an eff ect on amphibians (Section 7.2). Based on consider ation of those te st d esign elemen ts, EP

A then ev aluated the a va ilable amphibian data and assigned a classification ( e.g ., Quantitative, U.S. Environmental Pr otection Agency **129** 

# To **pic A: E va**  **luation of the Data on Amphibians**

amphibian data and assigned a classification ( e.g ., Quantitative, Qualit ative (high, medium, and low lev el of confidence), and In va lid) to each study indicating EP A’ s confidence in the s tudy ’s conclusions (Section 7.3 and Appendix C). The confidence in each st udy was based on an ev aluation of the identified tes t design elemen ts and r esulting lev el of uncert ain ty in determining a dir ect causal r ela tionship between atr azine and potential eff ects to amphibians.

## 2a. Please c omment on the c ompleteness of EP A’s list of pertinen t te st design elements. Also , please c omment on the degree to which these test design elemen ts, singularly or in co mbination, would be e

xpect ed to contribute towards co nf ounding the t es t results. U.S. Environmental Pr otection Agency **130** 

# To **pic A: E va**  **luation of the Data on Amphibians**

**2b.** Please c omment on EP A’s co nclusions about the level of co nfidence placed on each study ’s results.

### Charge Ques **tions 3a and 3b** : Aft er ev aluating all the a va ilable amphibian studies, one study was f ound t o ha ve accounted fo r all the iden tified t est design elemen ts (Question #2) and det ermined to be suit able f or quan tit ative use in risk assessmen t fo r the endpoin ts of sur viv al, gr owth and developmen t (Section 7.3 and Appendix C). This study was required by an EP A Data Call
- In (DCI) Notice f ollowing the recommendations fr om the 2003 SAP on atr azine and amphibians. U.S. Environmental Pr otection Agency **131** 

# To **pic A: E va**  **luation of the Data on Amphibians**

recommendations fr om the 2003 SAP on atr azine and amphibians. The resulting study e xa mined the e ff ects of a tr azine on Xe nopus laevis at concentr ations of 0.01 t o 100 μg /L at two diff erent labor ato ries. Based on the 2007 SAP

, the c onclusion was, and there was agreement by the P anel, that the dat a fr om this study were r obust and sufficient to c onclude that e xposure t o a tr azine at c oncen tr ations r anging fr om 0.01 t o 100 μg /L had no e ff ect on X. laevis development (which included sur viv al, gr owth, met amorphosis and se xu al development).

## 3a. Please c omment on whether an y new inf ormation has bec ome available that leads t o a diff eren t c onclusion from the one which EP

A reached in that the results of the DCI study were adequat e to evaluate potential eff ects of atrazine e

xposure t o amphibians. U.S. Environmental Pr otection Agency **132** 

# To **pic A: E va**  **luation of the Data on Amphibians**

**3b.** If such inf

ormation is now available, please c omment on how a threshold determination (a c oncentration that is e xpect ed to cause no eff

ect) may be ac co mplished using the identified studies.

### Charge Ques **tion 4** : Aft er ev aluation of the a va ilable amphibian tox icity dat a, EP

A c oncluded that the DCI study men tioned in Question #3 was appr opriat e f or quan tit ative use in a risk assessmen t f or sur viv al, gr owth and developmen t. While the 2007 SAP P anel agreed that atr azine appeared t o ha ve no eff ect on X. laevis development at atr azine c oncen tr ations r anging fr om 0.01 t o 100 μg /L, they e xpressed co ncerns about the suit ability of X. laevis as a surr og ate f or native species. R eview of the a va ilable tox icity dat a utilizing indig enous U.S. Environmental Pr otection Agency **133** 

# To **pic A: E va**  **luation of the Data on Amphibians**

species. R eview of the a va ilable tox icity dat a utilizing indig enous species sugg ests that suit able pr ot oc ols, including adequat e husbandry methods in particular

, that would enable EP

A t o quan tify a t ox icity endpoin t representative of a clear and c onsist en t r esponse fr om atr azine f or native species, ma y not e xist. 

## Please c omment on whether there are suit able methods f or te sting native amphibians with particular regard to husbandry and laborat ory culturing c onditions, c onsis te nt with the design elemen ts rec ommended by the 2003 SAP
.

## Charge **Question 5**  : A number of s tudies r eport the potential f or atr azine to modify immune function and inf ection susceptibility in amphibians (Appendix C). EP A believ es the r esear ch on these dif fe re nt h ypotheses does not pr ovide sufficient data to establish causal link ag es among diff er en t lev els of biological or ga niz ation to re sult in adv er se eff ects. Ther ef or e, EP A concluded that a U.S. Environmental Pr otection Agency **134** 

# To **pic A: E va**  **luation of the Data on Amphibians**

to re sult in adv er se eff ects. Ther ef or e, EP A concluded that a mode of action or adv er se outcome pathwa y leading to eff ects on amphibian sur viv al, gr owth or development cannot be es ta blished at this time.

## 5a : Please c omment on whether the dat a in the e xis ting dat abase reasonably supports the h ypotheses, or demons trat es that atrazine aff

ects immune function and/ or inf ection susceptibility leading to adverse eff ects on sur vival, growth or development; i.e., are there sufficient dat a to est ablish an adverse out co me path way for atrazine eff ects on immune U.S. Environmental Pr otection Agency **135** 

# To **pic A: E va**  **luation of the Data on Amphibians**

adverse out co me path way for atrazine eff ects on immune function? Please provide a rationale f or the P anel’

s position and discuss the associat ed strengths and weakness with the dat a supporting the rationale.

## 5b : If the P anel c oncludes that the e xis ting dat a are sufficient t o fo rmulat e hy

potheses that atrazine adversely aff ects immune function and inf

ection susceptibility

, but are not sufficient to test the hy potheses (refut e or confirm), then please c omment on specific study protoc ols that can be used to test these h ypotheses with sufficient rigor t o identify eff ects that can be directly and U.S. Environmental Pr otection Agency **136** 

# To **pic A: E va**  **luation of the Data on Amphibians**

with sufficient rigor t o identify eff ects that can be directly and quantit atively attributed to adverse impacts on amphibian reproduction, growth and/ or sur vival.

### Chapt er I, Section 6.14 and Appendix D discuss the mesocosm and micr ocosm ( cosm ) study selection crit eria f or det ermining accept ability in evaluating the impact of atr azine e xposure on the structure, function and pr oductivity of aquatic plant communities. Chapt er I, Section 6.14, and Appendix G(f) also pr ovide summaries of the source of the natur al communities used t o populat e the accept able cosm studies, as well as details on species composition of the cosms . The accept able cosm studies U.S. Environmental Pr otection Agency **137** 

# To **pic B: The Me thod f** 

**or De te**  **rmining the A**

**tr azine Lev**  **el of Concern f**  **or Aqua tic Plant Communities** 

details on species composition of the cosms . The accept able cosm studies were used in the establishment of an Aquatic Plant Community Concentr ation Equivalent Level of Concern (CE

- LO C) f or atr azine. EP A has refined its method f or det ermining an CE
- LO C f or atr azine based on the recommendations pr ovided by SAP s in 2003, 2007, and 2009 (Chapt er IV , Sections 13 & 14; f or a summary of changes since 2003 please ref er to T able 21). This methodology will be used f or a national
- scale assessment of atrazine concentr ations in freshwater and estuarine/marine ecos yste ms in the upcoming registration review . The f ollowing questions concern the composition and biological relevance of the cosm studies, and the method, assumptions and uncert ainties in det ermining a CE
- LO C f or atr azine.

## Charge **Question 6**  : The cosms we re comprised of natur al communities of periph yt on/ph yt oplankton; in some cases, va scular plan ts, in ve rt ebr ate s and v ert ebr ate s pr esen t in those communities we re included in the s tudy (Chapter I, Section 6.1.4). These sour ces w er e g ener ally described as s tr eams, lak es, re ser voirs, and springs, and ar e consider ed t o be r epr esen tative U.S. Environmental Pr otection Agency **138** 

# To **pic B: The Me thod f** 

**or De te**  **rmining the A**

**tr azine Lev**  **el of Concern f**  **or Aqua tic Plant Communities** 

re ser voirs, and springs, and ar e consider ed t o be r epr esen tative of the s tructur e and function of aquatic plant communities in such water bodies. Given the diversity of sources and the described c ommunities, please c omment on the e xt en t to which these co sm studies t ak en to gether provide useful and reasonable ph ysical models of the natural aquatic plant c ommunities e xposed t o atrazine in the U. S.

## Charge **Question 7**  : The Aquatic Plant Community CE
- LO C methodology fo r atr azine is a f our s ta ge pr ocess that uses single
species plan t to xicity data and cosm st udies to discern atr azine concentr ations and e xposur e dur ations that ma y cause adv er se eff ects on aquatic plant communities. As a r esult, a CE
- LO C f or atr azine is dev eloped which, t ogether with monitoring data, can U.S. Environmental Pr otection Agency **139** 

# To **pic B: The Me thod f** 

**or De te**  **rmining the A**

**tr azine Lev**  **el of Concern f**  **or Aqua tic Plant Communities** 

atr azine is dev eloped which, t ogether with monitoring data, can be used to identify water sheds wher e concentr ations ma y r esult in adv er se eff ects t o aquatic plant community structur e, function, and/ or pr oductivity
. Please c omment on the methodology EP A has used t o derive the atrazine CE
- LO C f

or aquatic plant c ommunities, and in particular on EP A’s charact eriz ation of the uncert ainties and assumptions in this methodology (Chapter IV , Sections 13 & 14).

## Charge **Question 8**  : The 2009 SAP r ec ommended using an eff ects inde x or concentr ation metric, r ather than categorical LO C thr esholds in or der t o tak e adv an ta ge of data fr om Sy ng en ta ’s At ra zine E cological Exposur e Monitoring Pr ogr am (AEEMP). At that time the L OC thr eshold f or atr azine eff ects t o plan t communities wa s established at 10 μg /L f or a 60
- da y r olling U.S. Environmental Pr otection Agency **140** 

# To **pic B: The Me thod f** 

**or De te**  **rmining the A**

**tr azine Lev**  **el of Concern f**  **or Aqua tic Plant Communities** 

plan t communities wa s established at 10 μg /L f or a 60
- da y r olling ave ra ge . The curr en t analysis using the Plant Assemblage T ox icity Inde x (P AT I) indicates the CE

- LO C can r ang e fr om 4 to 7 μg /L (Chapter IV

, Section 14.3 & 14.4). Please c omment on this CE
- LO C and whether it reasonably represen ts a range below which permanent or irreversible change in aquatic plant c ommunity s tructure, function, and/ or productivity due to atrazine e xposure would not be e

xpect ed.

## Charge **Question 9**  : Based on pr evious analyses of the a va ilable ec oto xicity data, EP

A concluded f or atr azine that the lev el of concern fo r eff ects on aquatic plant communities (CE

- LO C) wa s lower than the atr azine concentr ations obser ve d t o pr oduce significant dir ect or indir ect eff ects on in ve rt ebr ate s, fish and amphibians. Giv en the curr en t analysis of the ecoto xicity data U.S. Environmental Pr otection Agency **141** 

# To **pic B: The Me thod f** 

**or De te**  **rmining the A**

**tr azine Lev**  **el of Concern f**  **or Aqua tic Plant Communities** 

amphibians. Giv en the curr en t analysis of the ecoto xicity data (Chapter I, Section 6) and the Aquatic Plan t Community LO C methodology

, EP A continues to believe the original conclusion st ill holds true. Please c omment on how well the available dat abase supports EP A’s co nclusion that the CE
- LO C is lower than e xposures that result in significant eff

ects on the growth, sur vival and reproduction of aquatic animals.

### Chapt er V and Section 16 address the issue of iden tifying surf ace water bodies with pot en tial levels of atr azine that e xc eed the aquatic plan t co mmunity levels of c oncern (CE

- LO C). EP A ev aluated sever al statistical methods ( kriging , s to chastic simulation, c ov ariat e analy sis, and bias fa ctor s) t o addr ess uncert ain ties associat ed with sampling frequency as it impacts the ability t o quan tify atr azine c oncen tr ations fr om monit oring dat a (USEP

A, 2011). The FIFRA SAP in 2011 advised that U.S. Environmental Pr otection Agency **142** 

# To **pic C: Me**  **thod f**

**or Comparing Monitoring Data to the Aquatic Plant Community L**  **OC f or A**  **tr azine** 

monit oring dat a (USEP

A, 2011). The FIFRA SAP in 2011 advised that use of bias f act or s w as a viable appr oach f or addressing uncert ain ty in monit oring dat a. The f ollowing questions are relat ed t o the use of bias fa ctor s in iden tific ation of surf ace water bodies e xc eeding the atr azine CE
- LO C. **Charge Ques tion 10:**  Please c ommen t on the s trengths and limit ations of EP A’s development and use of bias fact ors (Chapt er V , Section 16.1) for addressing uncertainties in monitoring dat a.

## Charge **Question 11a and**  **11b** : Pr ediction of bias f act or s is dependen t on the selection of an appr opriate model. EP A illus tr ate d (Chapter V

, Section 16.1) both categorical and re gr ession methods f or pr ediction of bias f act or s based solely on the number of samples tak en in the 2 nd 

## and 3 rd 

## quart er s of the U.S. Environmental Pr otection Agency **143** 

# To **pic C: Me**  **thod f**

**or Comparing Monitoring Data to the Aquatic Plant Community L**  **OC f or A**  **tr azine** 

the number of samples tak en in the 2 and 3 quart er s of the ye ar (April 1 st 

## to September 30 th 

## ). **11a.** Please c omment on EP A’s prediction of bias f

actors from monit oring dat a using cat egorical or regression method approaches. **11b.** Please c omment on an y additional methods f or estimating bias factors that would be useful in this situation.

## Charge **Question 12**  : EP A illus tr ate d (Chapter V

, Section 16.1) both categorical and r egr ession methods f or es timation of bias fa ct or s as a function of the sampling fr equency of monit oring data. Step
- wise r egr ession analysis indicates that water shed siz e and a ve ra ge flow r ate in the 2 nd 

## and 3 rd 

## quart er s of the year ar e U.S. Environmental Pr otection Agency **144** 

# To **pic C: Me**  **thod f**

**or Comparing Monitoring Data to the Aquatic Plant Community L**  **OC f or A**  **tr azine** 

and a ve ra ge flow r ate in the 2 and 3 quart er s of the year ar e not significant v ariables f or pr ediction of bias f act or s. However , the number of samples in the 2 nd 

## and 3 rd 

## quart er s of the year wa s f ound t o be a significant v ariable, accoun ting f or 46% of the va riation in the bias f act or . What other variables, if an y, should be c onsidered in the prediction of bias f

actors?

## Charge **Question 13:**  EP A e xa mined (Chapter V, Section 16.1) the perf ormance of v arious r egr ession equations to assess the f ailur e per cen ta ge fo r iden tification of monitoring site
- ye ar s with true ma ximum 60
- da y a ve ra ge concentr ations ex ceeding the CE
- LO C fo r atr azine. This analysis showed that application of a bias U.S. Environmental Pr otection Agency **145** 

# To **pic C: Me**  **thod f**

**or Comparing Monitoring Data to the Aquatic Plant Community L**  **OC f or A**  **tr azine** 

fo r atr azine. This analysis showed that application of a bias fa ct or , based on sample number during the 2 nd 

## and 3 rd 

## quart er of the year , substantially r educed the number of sit es with under es timation of true maximum 60
- da y means. Given the EP

A analysis, what other t es ts, if an y, should be co nduct ed t o assess the perf ormance of regression models f or prediction of bias f

actors?

### Charge Ques **tion 14:** An import an t c onsider ation f or bias f act or prediction is the ability t o iden tify monit oring sit es that pot en tially exc eed the CE
- LO C fo r atr azine. EP A pr ovided an e xa mple (Chapt er V , Section 16.2 and 16.3) of the use of a log

- linear regression equation t o estimate bias f act or s fr om the USGS National W ater Quality Assessmen t Pr ogr am NA WQA data and identification of monit oring U.S. Environmental Pr otection Agency **146** 

# To **pic C: Me**  **thod f**

**or Comparing Monitoring Data to the Aquatic Plant Community L**  **OC f or A**  **tr azine** 

Assessmen t Pr ogr am NA WQA data and identification of monit oring sit e - year s e xc eeding the CE
- LO C f or atr azine. This analysis iden tified sit es in both the Midwest ern Unit ed St ates as well as outside the major c orn gr owing areas with atr azine c oncen tr ations pot en tially above the CE
- LO C. What are the s trengths and limit ations of using a single regression model for prediction of spatially and t emporally e xplicit bias fact ors for a nationally
- dis tribut ed monit oring dat a set ?