## 1



# Problem Formulation for the Reassessment of Ecological Risks from the Use of Atrazine Errata Sheet for Appendix C of the White Paper

- Open Literature Review of Amphibian Data

While preparing the SAP presentation on evaluation of amphibian toxicity data, discrepancies were observed for some of the endpoint classifications as well as some clarifications needed for others. The following table represents the classification changes made to endpoints in a study . In all but three cases, the endpoints reported no effects at the highest concentration tested . In all but four cases, the reason for the classification change was due to lack of summary data ; in the other four cases, endpoints were downgraded as there was uncertainty

in whether statistical results were anal yz ed using pseudoreplication which may have changed the outcome of the results or in the number of replicates used in the study . Also, all the classification changes were from Qualitative lower level of confidence to Invalid or the endpoint was already classified as Invalid. As such, these changes do not change the overall understanding of the effects for these endpoints when considering the entire available dataset

.

T able 1. Clarifications of Endpoints Citation Endpoint Endpoint Result Reason for Change Classification Change Changes in Endpoint Classification Allran and Karasov 2000 Growth;

Bio/Mol No effect on mass or hematocrit

@ 192 ppb Summary data not reported (SVL results reported) Qualitative lower to Invalid

Allran and Karasov 2001 Growth;

Behavior; Bio/Mol No effect on mass, swimming,

hemoglobin @ 20000ppb Summary data not reported Qualitative lower to Invalid

Clements et al. 1997 Bio/Mol ↑ DNA damage (erythrocyctes) @ 4800 ppb Lack of test design (# reps)/possible pseudoreplication Qualitative lower to Invalid

Coady et al . 2005 Growth;

Metamorphosis No effect on mass, SVL or age at

metamorphosis @ 25 ppb Summary data not rep orted Stayed Invalid

Forson and Storfer 2006b Growth;

Metamorphosis No effect on time to

metamorphosis, mass or SVL @ 160 ppb Summary data not reported Qualitative lower to Invalid

Gibble et al . 2011 Bio/Mol No effect on peptide activity @ 986 ppb Limited data on test design (# reps and animals Qualitative lower to Invalid

Haridon et al . 1993 Bio/Mol No effect on micronucleated RBCs (genotox) at 300 ppb Uncertain if only one replicate used (pseudoreplication) Qualitative lower to Invalid

Kerby and Stofer Imm/Inf No effects on infection rate @ Summary data not reported Qualitative lower to Invalid

## 2

Citation Endpoint Endpoint Result Reason for Change Classification Change 2009 200 ppb Orton et al . 2006 Growth;

Metamorphosis No effect on SVL or time for

forelimb emergence @ 15 ppb Summary data not reported Stayed Invalid

Rohr et al . 2003 Growth No effect on mass @ 400 ppb Summary data not reported (SVL results reported) Qualitative lower to Invalid

Tavera

-Mendoza et al. 2002a Sexual

Development Effects on male gonadal

development @ 18 ppb Uncertain if statistical analysis used pseudoreplication Qualitative lower to Invalid

Tavera

-Mendoza et al. 2002b Sexual

Development Effects on female gonadal

development @ 18 ppb Uncertain if statistical analysis used pseudoreplication Qualitative lower to Invalid

Williams and Semlitsch 2010 Growth No effect on mass @ 1.25 ppb Summary data not reported Qualitative lower to Invalid

Other Endpoint Clarifications Allran and Karasov 2001 Behavioral Feeding LOAEC = 20000 ppb; NOAEC = 12000 ppb Need to add endpoint to study
summary in Table in Appendix C Qualitative lower

Coady et al . 2005 Metamorphosis Age at metamorphosis NOAEC = 25 ppb; LOAEC > 25 ppb Need to add endpoint to study
summary in Table in Appendix C Invalid (see above)

Forson and Storfer 2006a Imm/Inf Infection rate NOAEC = 184 ppb; LOAEC > 184 ppb Need to add endpoint to study
summary in Table in Appendix C Qualitative lower

Hecker et al . 2005 a Bio/Mol Aromatase, estradiol, CYP19 NOAEC = 258; LOAEC > 258 Need to add endpoint to study
summary in Table in Appendix C Qualitative lower

Morgan et al. 1996 Survival Need to remove results reported as a.i. Results already adjusted for percent active ingredient Qualitative medium

Villeneuve et al. 2003 Bio/Mol No effect on gonad and brain aromatase @ 25 ppb Typographical error in classification Qualitative higher to lower

## 3 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

Allran & Karasov 2000 / E56572 Rana pipiens /

Nasco(unknow n if Lab/field / TP (G25) to M (138 d total) T /

99 NA / (Y/NA); NS NS 20 TP/7L;

48 hr Metam Gro Bio/Mol Surv 192 (M)

192 192 192 >192

>192 >192 >192 % & time to metamorphosis Mass; SVL Hematocrit % mortality Metamorphosis and growth endpoints based on subset (50%) of organisms and uncertainty in ability to get a true randomization based on information presented; summary data for each treatment group for mass, mortality and hematocrit not reported (only reported mean & SE for all replicates combined); ammonia levels not reported

Qualitative

- Lo wer level of confidence for growth and metamorphosis endpoints based on sub sampling as cannot discount potential confounding factors and also for mortality and hematocrit as no treatment summary information present ; I nvalid for mass and hematocrit as summary data not presented

Allran & Karasov 2001 / E59251 R. pipiens &R.

sylvatica & Bufo americanus / Field / A (R. pipiens) for 14 d and E for 9 6 hr ph T /

99 NA / (Y/NA); NS Glass (E) /

NS (A) 50 or 30

E/0.075L (E); 24 hr Meta

Gro Behav (E) Behav (A) Behav (A) Gro (A) Bio/Mol (A) Surv 2590

20000 20000 2590 12000 20000 20000

20000 4320

>20000 >20000 4320 20000 >20000 >20000

>20000 Deformities (tail, facial edema)(E) Mass (E) Swimming (E) Buccal ventilation ↓ Feeding Mass (A) Hemoglobin

Hatch and mortality Atrazine measured at organism collection site; water quality parameters not specified

- authors state conducted according to ASTM ; Summary data growth,, swimming, hemoglobin not presented

Qualitative

- Lo wer level of confidence as low levels of atrazine measured at organism collection site ; I nvalid for growth, swimming and hemoglobin due to lack of summary data Birge et al . 1980 / E6187 R. catesbeiana,

R. pipiens, R. F /

80 NS / (Y unsure if glass 50

- 103 E

/ 0.5L ; 2.5 Surv 4

dph LC50 -- -- R. catesbeiana = 410 R. pipiens = 7680 LC 50s were not based on mortality per se, but on abnormalities that would reportedly preclude

1 

### TP = tadpole; LV = larvae; M= metamorphosis; E= embryo; G = Gosner stage; NF=Nieuwkoop and Faber; 

### SVL=snout

- vent

- length; A = adults; H = hatch; J = juvenile; if reported in study, developmental stage or some other identifier at test initiation is given, if not reported than designated as NS (not specified);
2 

### TS = Test substance; T = technical; F = f ormulation
3 

### NA = not applicable; NS = not specified; ACTN = acetone; EtOH= ethanol; DMSO=dimethylsulfoxide; MeOH=methanol; IPA = isopropyl alcohol
4 

### Negative Control (NC) and/or Solvent Control (SC) included in study design; MA in CNT = Measurable atrazine in control treatment group(s)

- Yes (Y) or No (N) or Not Specified (NS)
5 

### The values reported represent loading reported as mass (g) or number of larvae/tadpoles in a volume of water (L). If loading wa s reported as number of larvae/tadpoles in a certain test vessel size but test solution volume was not reported, then this is presented # organisms in tank (volume reported if available) as actual loading could not be determined; PE = polyethylene
6 

### Water
7 

### Metam = metamorphosis; Gro = grow th; Imm/Inf = imm

une system or infection; Bio/Mol = biochemical/Molecular; Sexdev = sexual development; Behav=behavioral; Surv = survival; N = nominal conc.; M = measured conc; NDR = not dose responsive (no significance at higher dose).

## 4 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

palustris, B. americanus / NS (purchased) / E for 4 dph NC or SC); NS h R. palustris = 17960 B. americanus > 48000 survival under natural conditions ; solvent (if used) was not identified ; formulation used so uncertainty in source of toxicity

Qualitative

– Higher level o f confidence; however LC 50 values are combination of both lethality and abnormal effects and formulation used

Brodeur et al . 2009 / E112588 R. arernarum /

LP 8/ TP (G38/39)

- M T/

98 NA / (Y/NA 9 ); N glass 10 TP

/0.04L; 48 hr Metam Surv (LC50) NDR (N)

NA 100

NA ↓ & ↑Time metamorphic stage and/or duration between stages LC 50 (stage 4) = 7150

- 25370 LC 50 (stage 25) = 2320

- 27160 LC 50 (stage 38

- 39) = 12630

- 18270 (LC50s range from 4 d to 28 d) No water quality parameters reported; metamorphosis results not dose responsive (not sign. at 1000 or 5000 μg/L depending on metam endpoint (100 lowest conc tested) ; hypothesis testing for effects on metamorphosis conducted using EC50 values

Qualitative

- Lo wer level of confidence based on higher loading with no water quality parameters reported

Brodkin et al 2007 / E103351 R. pipiens /

Field (ANA 10 ) / A for 8 d T/

NS NA / (N & N 11 /NA); NS plastic NA; 24 hr Imm/inf Surv <0.01(N)

10 0.01

>10 ↓ White Blood Cells Study indicated atrazine did not cause mortality; not explicitly reported in terms of NOEC/LOEC Adult frogs cultured using Styrofoam rafts; uncertainty in how organisms were given what treatments; effects are not clearly and quantitatively linked to the assessment endpoints of growth, reproduction, and survival

Inval id based on lack dilution water only control (dilution water but subject to immune treatments used)

Choung et al

. 2011a / E153858 Litoria

raniformis / Lab / TP (G26) for 10 wks T/

>98 NA / (Y/NA); NS glass 10 TP

/10L ; 2X/wk Metam

Gro Surv 25 (N)

25

-- >25

>25

-- Time to metamorphosis Mass; SVL Study indicated survival >97.5% for study; not explicitly reported in terms of NOEC/LOEC Limited reporting of potential contaminants in food and water; number of egg masses used not specified; measured ammonium (not NH3)

Qualitative

- Higher level of confidence; however there is a lack of reported measurements in food
water and number of egg masses

Clements et al

. 1997 / E20274 R. catesbeiana /

CBSC / TP for 24 hr F /

88.2 NA / (Y/NA); NS glass NS; NA Bio/Mol

Surv <4800

(4200 ai)

-- 4800

(4200 ai) ↑ DNA damage in erythrocyctes

NS; reported testing the maximum Limited reporting on water quality parameters and test design (# of replicates); conducted above water solubility

8 

### LP = adults used to collect eggs were obtained from local provider
9 

### Test concentrations 1

- 25 mg/L prepared without solvent; 30 mg/L prepared using acetone (0.2%) so solvent control (0.2% acetone) was also included in design 10 

## ANA= Amphibians of North America (Nashville, TN, USA)
11 

### A treatment group that was only exposed to dilution water (and not injected with Ringer solution or subjected to thioglycollate stim ulation) was not included.

## 5 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

-- survival concentration and dilutions from that conc. Invalid based on lack of test design parameters

Coady et al 2004 / 46965605 R. clamitans/

Field / TP (G25)

- G42 (273 d) T/

97.1 EtOH (0.005) / (Y/Y); Y NS 30 TP/4L

then 16L; 72 hr Metam

Gro Surv NDR(M)

28 28 11

>28 >28 ↑ Time to metamorphosis at 11 but not 28 Mass; SVL % mortality Poor water quality; high control mortality; lack of responsiveness to positive control (estradiol)

Invalid based on control contamination; high control mortality

Coady et al 2005 / 46965602 Xenopus

laevis/Lab/ TP (72 h ph)

- M T/

97.1 NA / (Y/NA); Y glass 30 TP/4L ;

50% every 72 hr Gro Sexdev

Bio/Mol Surv Metam 25

25

0.1 25 25 >25

>25

1 >25 >25 Mass; SVL Gonad development; laryngeal muscle ↓ estradiol males % mortality Age at metamorphosis Higher loading with 50% renewal of solution at 72 hr may have promoted poor water quality; delayed metamorphosis in controls; statistical difference between controls (possible confounding factor); lack of responsiveness to positive control (estradiol) ; n o summary data for growth, metamorphosis

Invalid based on control contamination and for growth and metamorphosis for lack of summary data

Forson & Storfer 2006a / E82033 A.

macrodactylum / Field / LV (ca. 6 wks) for 30 d T /

86.5 NA / (Y/NA); NS PE 1 LV/

0.5L ; 3X/wk Metam

Gro Surv Imm/I nf 18.4 (M) 18.4 184 184 184

184 >184 >184 ↑ larval period ↓ mass & SVL % mortality Infection Rate Limited reported water quality ;
Qualitative

- Lo wer level of confidence due to lack of reported measurements in control with slightly higher than recommended loading with limited water quality and a renewal rate of 72hr periodically

Forson & Storfer 2006b / E103072 A. tigrinum /

Lab / LV (12wks)

- M (111 d total) T /

NS NA / (Y/NA); NS PE 1 LV

/0.5L ; every 3 days Metam Gro Imm/inf

Surv 160 (N)

160 1.6 (NDR); 16

-- >160

>160 16 ;160

-- Time to metamorphosis Mass, SVL ↑ infection rate; ↓leukocytes

Reported 31 mortalities (384 exposed, 8% mortality); results not presented in terms of NOEC/LOEC Limited reported water quality; infection rate not dose responsive (not sign at 160 μg/L) ; potential stress from repetitive mass measurements ; summary data for metamorphosis and growth not presented

Qualitative

- Lo wer level of confidenc e due to slightly higher than recommended loading with limited water quality and a renewal rate of 72hr; Invalid for metamorphosis and growth as summary data not presented Fort et al . 2004 / E81751 X. tropicalis &

X. laevis / T/

NS DMSO (1%) / NS 20

E/0.01L; Metam

Surv -- -- X. laevis

LC 50 = 24500 or 25600 Limited water quality reported; use of not recommended solvent and high conc (although

## 6 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

Xenopus I (adults) / E

–

NF 46 (Y/NS); NS 24 hr (FETAX) EC50 = 4100 or 4400 X. tropicalis LC 50 = 19400

– 31800 EC50 = 2100

- 9300 allowed under ASTM FETAX assay guideline)

Invalid based on solvent choice and concentration

Freeman & Rayburn 200 4 / E 78644 B. americanus

& X. laevis / Field (parents)(Bufo) & Na sco / TP (NF NS ) for 3

- 5 wks T /

NS NS / ( C used NS if NC, SC ); NS NS NS (2

TP/3L tank) ; weekly Bio/Mol

Surv 800

NS >800

NS Nuclei (represent genotox) Not specified; based on reported ‘N’ in tables appears 100% survival Stats for nuclei conducted on CVs ; uncertainty in sample sizes; no reported water quality; loading unknown (fill volume not reported)

Qualitative

- Lo wer level of confidence due to uncertainty in sample sizes and loading unknown with no reported water quality and relatively long renewal period

Freeman & Rayburn 2005 / E81459 X. laevis /

Nasco/Xenopus Express / TP (varied NF40 to 54) for 3

- 5 wks T /

98 NA / (Y/NA); NS 12 NS NS (1

TP/3L tank) ; weekly Metam

Gro Bio/Mol

Surv <100 (N)

800 Varied

-- 100

>800 800

-- ↓ metamorphic stage Mass ↑ f low cy tometric analysis meant to illustrate metemorphosis and development) ; results varied based on test % Mortality (0

- 16.7%) for different assays, reported as occurring randomly in treatments; mortality in
terms o f NOEC/LOEC not reported Stats for f low cy tometric conducted on CVs; uncertainty in weight analysis with regards to accounting for different stages of organisms; fill volume of test vessel (3L) not reported however water quality appears to be acceptable

Qualitative

- Medium level of confidence for molecular endpoint due to analysis using CV and other reasons described for metamorphosis; Invalid for weight due to potential confounding factor ; Qualitative

–Higher

–for metamorphosis stage ; however, lack of analysis of atrazine in control

Freeman et al . 2005 / E81460 Bufo

americanus / Field (Adults) / TP (G20 or 25) for 3 or 4 wks T/

98 NA/ (Y/NA); NS NS NS (2

TP/3L tank) ; weekly Metam

Gro Bio/Mol Surv NDR (N)

NDR 10000

-- 250

800 >10000

-- ↑ Stage at test completion ↓ Mass at test termination Mean total # of nuclei Reports no acute toxicity observed; mortality in terms of NOEC/LOEC not reported Water quality parameters in study not reported; fill volume of test vessel (3L) not reported

-unable to verify l oading ; metamorphosis and mass not dose responsive (metam

- not sign at 800 μg/L in other test different age at initiation; mass

- not sign at 800 in other test or 250

- 10000 in another test)

Qualitative

- Lo wer level of confidence for Metamorphosis and Bio/Mo l

- due to loading not defined and no water quality parameters with weekly renewal ; I

nvalid for weight due to potential confounding factor

12 

### Study reports that solutions measured from experimental tanks and atrazine concentrations were within expected range; however unsure if this included the control

## 7 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

Gibble & Baer 2011 / E153856 X. laevis /

Nasco / TP (NF49/50)

- M (90 d total) T/

98 or F/ 40.8 DMSO (0.0173) / (Y/Y); N (Tech)
, N S (Form) NS 1g/L [per day

– reviewer assume] for T / NS for F ; 7X/day or 3X/wk Metam

Gro Surv Imm --

--

-- 986 (N) --

--

-- >986 Specific parameters within these endpoints not reported for metamorphosis
, growth , survival Peptide activity (expressed as total protein) Atrazine measured in solvent control (exposed for potentially 48 hours) in atrazine technical study ; survival not reported ; study design (replicates, # organisms) not reported for the formulation ; appears frogs used as experimental unit, not tanks, for peptide assay for tech atrazine (unknown for formulation)

Invalid for growth
, survival and metamorphosis based on lack of reporting results and control contamination ; Invalid

- for peptide bioactivity analys es for atrazine formulation based on lack of test design

Goleman & Carr 2003 / 458 6 7707 X. laevis /

Xenopus Express (Adults) / TP (48 hph)

-NF66 (78 d total) T/

98.6 ethanol (0.0025)

- E2 group only / (Y/Y);N glass Varied 60

- 65 TP /0.250

– 4L ; 50% every 72 hr Metam Gro Behav Sexdev Surv 19.5 (M)

19.5 10.3 10.3 19.5 >19.5

>19.5 19.5 19.5 >19.5 Forelimb emergence, tail resorption Mass; SVL ↑abnormal swimming ↑ ovotestes % mortality Water quality concerns; lack of response to positive control and potential delayed development

Invalid based on appeared lack of response to positive control and potential overall delayed development in the study

Gunderson et al 2011 / E153613 R.catesbeiana /

Fie ld / TP (NS) for 2 d T/

NS IP A (0.001) / (N/Y); NS glass 0.63 g/L ; static (48hr exposure) Bio/Mol

Surv 10

-- >10

-- Relative fold difference for CYP19 and SF

- 1 Reported no mortalities in study not reported in terms of LOEC/NOEC Limited water quality reported; while no sign diff in CYP19 and SF

- 1 for each treatment, correlation of CYP19 and SF

- 1 m RNA in the brain w hen combining 1 & 10 ppb resulted in sign diff.

Invalid based on use on solvent choice (not recommended solvent) and no negative control used in study

Haridon et al . 1993 / E4324 Pleurodeles

waltl / LV for 12 d T /

NS DMSO (0.05) / (NS/NS); NS glass 15 LV /

1.5L ; 24 hr Bio/Mol Surv 300

NS >300

NS Micronuceated RBCs (genotox) Reported that maximum concentration was dose not to induce detectable physiological disturbances for 12 days of treatment Limited water quality reported; Used DMSO at higher than recommended conc.; uncertain in what the control was comprised of; uncertain if used
pseudoreplication in results

Invalid due to uncer

tain if only one replicate used (pseudoreplication

Hayes 2002 / E70207 X.laevis / Lab /

TP(NF46)

-

M (NF66); A for 46 d T/

NS EtOH (0.004) (N/Y) ;NS
13 plastic 30 TP /4L ;

72 hr Metam Gro Sexdel Bio/Mol Surv 200 (N)

200 0.01;0.8 <25 200 >200

>200 0.1;1 25 >200 Time to metamorphosis Mass; SVL ↑Ovotestes; ↓Laryngeal dilator ↓Plasm testosterone Mortality Limited water quality reporting; use of plastic test vessels; indirect measured of effect (laryngeal); in report results on metamorphosis, growth and survival not reported in study

13 

### Study indicates concentrations confirmed by two laboratories; however, unclear if this included the controls.

## 8 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

Qualitative

– Lo wer l evel of confidence based on higher loading with limited water quality reporting and a renewal period of every 3 days

Hayes et al . 2003 R. pipiens /

Field / (48hr post hatch)

-

M T / 98 EtOH (0.0036) / (N/Y) NS 14 NS 30 TP /

4L ; 72 hr Metam

Growth Surv Sexdev NS

NS NS <0.1 NS

NS NS 0.1 Results not presented in report for metamorphosis, growth, survival ↑ male gonadal dysgenesis (underdeveloped); Sex reversal No statistical results associated with sexual development endpoints ; study reported at metamorphosis growth measurements taken but results not presented in study

Qualitative

– Lo wer level of confidence based on higher loading with limited water quality reporting and a renewal period of every 3 days

Hayes et al 2006a X.laevis / Lab /

TP ( 4d post hatch)

- M (NF66) T/

NS MeOH (0.004) / (Y/Y); NS plastic 30 TP/4L ; 72 hr Sexdev Uncertainty if animals exposed to atrazine in this study or

if from another study. May have used different clutches to make comparisons Sex ratio of controls skewed; lack of a consistent and clear dose response; limited water quality parameters reported.

Invalid due to uncertainty if animals were exposed to atrazine in this study or were from another study

Hayes et al . 2006b / E85815 R..pipiens /

Field (Adult)/ TP (48hr post hatch)

- G46; X. laevis /Lab /A for 27 d T/

98 EtOH (0.0036) / (N/Y); N plastic 30 TP /4L ; 72 hr Metam Gro Imm/inf Surv 0.19 (M)

<0.19 <0.19 0.19 >0.19

0.19 0.19 >0.19 Time to metamorphosis ↓ Mass; SVL ↑ Thymic plaques (X. laevis) Mortality Water quality parameters not reported; reported results are inconsistent with the data presented in report; authors rely on the lack of statistical significance to draw conclusions.

Qualitative

–Lo wer level of confidence based on higher loading with limited water quality reporting and a renewal period of every 3 days as well as inconsistent reporting adding to level of uncertainty

Hayes et a l 2010 / E153853 X.laevis / Lab /

H

- A (up to 3 yrs post M) T/

NS EtOH (0.003) / (N/Y); NS plastic 30 TP/4L ; 72 hr Sexdev

Bio/Mol Behav Surv Metam Gro <2.5(N)

<2.5 <2.5 NS NS NS 2.5

2.5 2.5 NS NS NS ↓ Breeding gland, laryngeal development ↓ Testosterone ↓Mating behavior Results on survival, metamorphosis and growth of animal not reported In general, the study does not describe and/or account for all of the animals used in each treatment/replicate; uncertainty in reported results (e.g., # of phenotypic female/genotypic male compared to total exposed) ; Results for survival, metamorphosis and growth not reported

Qualitative

– Lo wer level of confidence based on higher loading with limited water quality reporting and a renewal period of every 3 days and

uncer tainty in reported results add to level of uncertainty ; I

nvalid for survival, growth and metamorphosis

14 

### Study indicates concentrations verified; however, unclear if this included control s

## 9 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

Hecker et al 2005a / E79287 X.laevis /

Xenopus Express / A for 36 d T/

97.1 NA (Y/NA) ; N NS 1

frog/10L ; 3 days Sexdev

Bio/Mol Bio/Mol Surv 258(M)

24.6 258

-- > 258

258 >258

-- Gondal abnormalities ↓ Plasma testosterone Aromatase, estradiol, CYP19 4 total mortalities (65 exposed) reported; mortality nor presented in terms of NOAEC/LOAEC Uncertainty in methods for weights (and it cascading effect) and gonad evaluation ; LOD/LOQ not reported for steroid analyses; unclear about sample size for steroid analyses

Qualitative

- Lo wer level of confidence due to uncertainty in methodology for weights
, gonad evaluation and biochemical analyses

Hecker et al 2005b / E79288 X.laevis /

Xenopus Express / A for 49 d T/

97.1 NA (Y/NA) ; N fiberglass 4.9 g/L ;3

days Sexdev Bio/Mol Surv <11.6 M

107

-- 11.6

>107

-- GS I Plasma testosterone; estradiol 1 total mortality reported; mortality not presented in terms of NOAEC/LOAEC No solvent control for positive control; Some water quality reported but no reporting of nitrates, nitrites, and ammonia; limited sensitivity for aromatase assay

Qualitative

– Lo wer level of confidence based on higher loading with limited water quality repor ting and a renewal period of every 3 days

Howe et al 1998 / MRID 45202910 R. pipiens, B.

americanus / F / TP (G29 & 40) for 96 h F /

40.8 NA (Y/NA); N glass 20 TP /

15L Surv 96 hr LC 50 -- -- LC50 values R. pipiens (early stage) = 47600 R. pipiens (late stage) = 14500 B. americanus (early) = 26500 B. americanus (late) = 1 07 00 Control survival not reported; no raw data; tested above water solubility

Qualitative

– Medium level of confidence based on lack of control data and only LC50 values reported and testing above water solubility

Houck & Sessions 2006 / E117318 R. pipiens / NS

/ A for 4 or 8 wks NS NS / (NS/NS); NS glass NA ; 3

days Imm/inf

Imm/inf Surv <1(N)

10

-- >10

>10

-- ↓ plaques representing antibody

secreting cells (4 wks) Number of Lymphocytes 1 mortality (believed 24 exposed) reported; mortality not presented in terms of NOAEC/LOAEC No information provided on culturing conditions and limited information on experimental parameters; uncertainty in whether used a solvent

or n ot as test solution preparation not reported; uncertainty in source of frogs; frogs were reported to not be feeding in trial 2; analytical measurements of treatment groups not reported; uncertainty in how animals in Trial 1 were used in study

Invalid bas ed on frogs not eating in trial 2 and overall lack of culturing and experimental parameters and uncertainty in reported results in Trial 1

Kerby and Storfer 2009a / E117314 A. tigrinum /

Lab / LV (4wks) for 14 d T/

NS MeOH (0.02); (N/Y); NS plastic 1 LV /0.5L ; 1X/we ek Imm/inf

Surv 200 (N)

200 >200

>200 Infection rate % Survival basedon atrazine alone, reported interactive effect (p=0.03) atrazine

- virus with atrazine slightly increasing survival w/o virus and slightly decrease survival w/virus Uncertainty in a ffect of dead larvae on measured viral load that was used in analyses; limited water quality reported

Qualitative

- Lo wer level of confidence given higher than recommended loading rate with limited water quality reported and a weekly

## 10 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

renewal period ; I nvalid for infection rate as summary data not presented

Kiesecker 2002 / E68326 R. sylvatica

/Field /TP (G25) for 4 wk T /

NS ACNT & DMSO (NS); (Y,Y); NS PE 1 TP/3.5L ; 48 hr Imm/inf Surv <3

NS 3

NS ↓ eosinophils ↑ trematode infections Mortality not reported; based on sample size in Figure 2 in report survival appears to be 100% Uncertain if c ercariae used for infection trial were exposed to atrazine , therefore there is some uncertainty in the environmental relevance of this scenario; dilution water only group (no exposure to cercariae not used; limited water quality reported ; uncertainty in statistical analysis

Invalid based on lack of dilution water only control, also due to environmental relevance with different exposure scenarios between tadpole and parasite
, unsure of statistical analyses adds to uncertainty

Kloas 2009 a / E112914 X. laevis /

Xenopus I

/ TP (8 dpf) for 83 d T /

NS NA / (Y/NA); Y 15 glass <1

g/L /day ; 7X/day Metam

Gro Sexdev Surv 100 (N)

100 100 100 >100

>100 >100 >100 Time to metamorphosis Mass; SVL>100 16 Sex ratio; gonadal development % Survival Atrazine contamination of controls; marked fluctuations in water quality parameters; histological endpoint comparisons not made relative to amphibian reference

Quantitative based on fact that although deficiencies, study design and reporting were robust enough to properly evaluate results

Kloas 2009b / 153612 X. laevis / Lab /

TP (NF 48 and 58) for 4 d T /

NS NS / (NS,NS); NS glass NS (25TP

/ 10L tank); 24 hr Bio/Mol

Surv 25

NS >25

NS Aromatase, LH, FSH, 5

- alpha reductase Mortality not reported Analysis based on n=8, however only two independent tanks tested (removed 4 from each tank that contained 25 tadpoles at initiation) ; limited water quality parameters reported during
test , test solution preparation and what constitutes the control was not reported

Qualitative

– Lo w level of confidence based on lack test design reporting

Koprivnikar et al 2007 / E103348 R. sylvatica /

Field / TP (G28/29) for ca. 31 d F /

40.8 NA / (N 17/NA); NS plastic 1 TP

/1.8L ; 72 hrs Imm/inf

Surv 3 (N)

30 30

>30 ↑ intensity of infection (frogs exposed but cercariae not) Mortality Control survival was 61.9%; Dilution water only
treatment (no cercariae) was not used; replicate variability not reported; effects only observed when tadpoles, not cercari a e were exposed to atrazine

– uncertainty in environmental relevance;

15 

### Atrazine detected in one out of 4 blocks of negative control tanks in one of the studies; it was excluded from analysis
16 

### Significant decrease in female weight and SVL observed at 0.01, 1 and 25 for females only but not at 0.1 and 100 ppb in one o f the two reported studies; No effect on wt and SVL reported in other study; EFED does not consider the differences in wt and SVL to be a concentration

- dependent effect
17 

### A dilution water only control was not included. A control that contained no atrazine but cercariae that were also not exposed to atrazine was used in the study

## 11 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

limited water quality reported

Invalid based on lack of dilution water only control, lack of reported replicate variability and adverse effects observed only when tadpoles, not cercariae, were exposed (unknown environmental relevance) adds to the level of uncertainty

Koprivnikar et al 2010 / E153870 R. pipiens / E

from CBSC 18 / TP (<G27)

- ~ G43 19 T/

99.2 EtOH (100ug/L) / (Y/Y); NS plastic 1 TP/L ;

weekly Metam

Gro Surv <3 (N)

<3 <3 3

3 3 Developmental stage Mass Mortality Variability in mortality across 3 ppb treatment groups; uncertain if developmental stage was accounted for in mass endpoint; uncertainty in reported results in figures; uncertainty if organisms were field collected or laboratory reared

Invalid based on uncertainty in reported results based on Figures in report and limited reported water quality with relatively long renewal period and variability in mortality across treatment groups add to uncertainty

La Fiandra et al

. 2008 / E103336 H . versicolo r /

Lab / TP (G25)

- 30 d past M F/

88.2 NA; (Y/NA); NS glass 3 TP/7L ;

50% 1X/wk Metam

Gro Sexdev Surv 195 (M)

195 195 195 >195

>195 >195 >195 Larval period Mass; SUL20 Sex ratio; gonad development % Survival Limited water quality reported; uncertainty if adults used to obtain eggs for culturing were from the field or laboratory

Qualitative

- Lo wer level of confidence, while loading was within recommended limit, limited reported water quality with relatively long renewal period(50% renewal every week) may have potential environmental condition issues

Langerveld 2009 / E121497 X. laevis / Lab /

TP (NF43) to NF46 T/

99.4 NA / (Y/NA); NS Pyrex 4 TP/1L ;

every 2

- 3 days Gro Bio/Mol Surv <400 (N)

<400 <400 400

400 400 ↓ Mass; SVL Expression of multiple genes % Survival Limited water quality reported; Uncertainty regarding overall mortality in control and 400 ppb however, substantial mortality observed at 400 ppb when approaching stage 63, therefore may not be unexpected to observe significant sublethal changes

Invalid for growth and bio/mol due to significant mortality compared to control in the only atrazine concentration tested also high loading rate with limited water quality and renewal rate periodically

18 

### CBSC = Carolina Biological Supply Company (Burlington, NC)
19 

### G43

- first observance of reabsorbed tail
20 

### SUL = snout

- urostyle length

## 12 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

>48 hours; Qualitative Lo w level of confidence for survival based on limited water quality, loading and renewal rate

.

Larson et al . 1998 / E60632 A. tigrinum /

Field / LV (SVL= 30mm) for 86 d T /

NS ACTN (NS)/ (N/Y); Y PE 1 TP/4L ; 50% at 48hr Metam Gro Bio/Mol Surv <81.8(M

81.8 <81.8

-- 81.8

>81.8 81.8

-- ↓metamorphosis Mass; SVL ↑plasma thyroxine;↓ corticosterone Reported no exposure related mortalities; mortality not reported in terms of NOAEC/LOAEC Atrazine measured in controls; lower dissolved oxygen reported

Invalid due to control contamination and solvent
concentration not reported and no negative control tested add to uncertainty

Lenkowski & McLaughlin 2010 / E153851 X. laevis / Lab /

TP (NF41) for 6

- 48 hr T/

NS DMSO (0.1

- 0.35) / (Y/Y); NS NS 13

- 18 TP

/0.01L ; 24 hr (48 hr exposure) Bio/Mol

Surv 350(N)

NS 3500

NS ↑ MMP18 expression Limited reporting on test elements (replicates, solution preparation, test vessels, analysis of test solutions, water quality parameters, loading, control survival)

Invalid due to solvent concentration and solvent choice and lack of reporting on test design and experimental parameters contributing to higher level of uncertainty

Lenkowski 2010 / E153876 X. laevis / Lab /

TP (NF41) for 48 hr T/

NS DMSO (0.1

- 0.35) / (Y/Y); NS NS 13

- 18 TP

/0.010L ; 24 hr (48

- hr exposure) Metam

Surv 350 (N)

-- 3500

-- ↑ Malformations in intestines and cranial cartilage Reported no mortality in atrazine exposures, control mortality not explicitly reported Limited reporting on test elements (solution preparation, test vessels, analysis of test solutions, water quality parameters)

Invalid due to solvent concentration and choice and lack of reporting on test design and experimental parameters contributing to higher level of uncertainty

Lenkowski et al 2008 / E103268 X. laevis / Lab

/TP (NF41

- 46) for 48 hr T /

98 DMSO (0.1

- 0.35) / (Y/Y); NS NS 15 TP

/0.01L ; 24 hr (48 hr exposure) Metam

Surv <10,000

(N)

-- 10000

-- ↑ visceral bleeding & organ malformations Mortality reported as <5% Limited reporting on test elements (test vessels, analysis of test solutions, water quality parameters)

Invalid due to solvent concentration and lack of reporting on test design and experimental parameters contributing to higher level of uncertainty

Oka 2008 / E103345 X. laevis / Lab /

TP (NF49)

- M (56 d total) T/

97.4 EtOH (0.01) / (N/Y); NS glass 30 TP /10L ; 3X/wk Metam

Bio/Mol Sexdev Surv 100 (N)

100 1

-- >100

>100 10

-- Time to metamorphosis Aromatase ↑ female ratio Reported ≥ survival in controls and some treatment groups with low survival in one of two tanks for two concentrations Estrogenic activity measured in parental food; variable replicate mortality which resulted in only one replicate for analysis for two treatment groups for sex ratio endpoint; no statistical results reported for metamorphosis; limited water quality reported

Qualitative

- Lo wer level of confidence based on higher loading with limited water quality reporting

## 13 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

and an occasional renewal period of every 3 days coupled with variable mortality in tanks for two treatment groups (not dose responsive); I nvalid for

metamorphosis as statistical results not reported.

Olivier & Moon 2010 / E153629 A.maculatum /

Field / E for ~ 76 d F/

42.6 NA / (Y/NA); NS plastic 1 egg

mass /0.750L ; 3 wks Metam

Surv 50 (N)

50 100

100 Developmental stage before death or hatch % Hatch; mortality reported as egg mass mortality specific stats not reported Temperature regulator malfunction in which pathologies were observed in treatment groups (not control) shortly thereafter and mortality in atrazine treatments occurred shortly after solution

renewal; Uncertainty in number of embryos per treatment and evaluation of oxygen depletion; limited water quality

Qualitative

- Lo wer level of confidence as it appears that additional potential stressors of temperature change and solution renewal may have contributed to observed pathologies as well as limited water quality reported with long renewal period (21 d)

Orton et al 2006 / E82034 R.pipiens /

Carolina Biological / Taylor

- Kollros Stage 2/3

– 23.24 T/

98.2 NA / (Y/NA) ; N NS 20 TP /8L ;

75% at 48 hr Metam Gro Sexdev Surv 15 (M)

15 <15 <15 >15

>15 15 15 Time for forlimb emergence SVL Sex ratio; gonad develop % mortality; lower in treatment
groups High control mortality (ca. 50%); higher incidence of intersex (12.5%) in controls; sex ratio of controls appears skewed ; summary data for metamorphosis and growth not presented

Invalid based on higher incidence of reported gonadal intersex in control and higher control mortality also no summary data for metamorphosis and growth

Morgan 1996 / E63246 X. laevis /

Xenopus I (adults) / E for 96 hr F /

40.8 NA / (Y/NA) ; NS 21 plastic NS (20

E/petri dish) ; 24 hr Metam Surv -- 11000

or 1100 LC 50 = 100000 or 126000 EC50 (abnormalities) = 33000 or <8000 Limited water quality reported, test solution fill volume not reported; conducted above water solubility; formulation used so source of toxicity unknown

Qualitative

– Medium level of confidence based on limited water quality reported

Rohr et al ., 2003/ E71723 A. barbouri /

Field / E for 37 d (53 d total test) T /

80 DMSO (0.03) / (Y/Y); NS Glass 10 LV/2L ; 50% at 48 hr Gro Behav Surv 400 (N)

40 400 >400

400 >400 Mass; SVL; limb deformaties ↑Locomotor activity Total Survival Solvent control contained both acetone and DMSO whereas atrazine concentrations only contained DMSO (which is not a recommended solvent); author states organisms likely exposed to atrazine prior to test due to agricultural activities around collection site; limited water quality

21 

### Test concentrations measured but do not report re sults for control; unsure if analyzed

## 14 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

reported; uncertain if potentially different stages at test termination (stages not reporte d) influenced growth analysis ; mass summary data not presented

Qualtiative

- Lo wer level of confidence based on higher than recommended solvent concentration using a non

- recommended solvent in addition the limited reported water quality with the higher loading rate add to the level of uncertainty ; I nvalid for mass as summary data not presented

Rohr et al ., 2004 / E81748 A. barbouri /

Field / E

-M (mean 64 d) T /

80 DMSO (0.03) & ACTN (0.03) 22 / (N/Y); N NS 31 or 40 E/9.5L ; weekly Metam

Gro Behav Surv 4(N)

40 40 40 40

400 400 400 ↑ time to metamorph ↓ mass ↑ locomotion activity; ↓refuge use ↓ Survival White film on dead embryo unknown if fungus caused or fol lowe d mortality; larval survival differences between years; although pH, temp, and DO ok, nitrates and ammonia not reported;

Qualitative

- Lo wer level of confidence based on higher than recommended solvent concentration using a non

- recommended solvent in addition to uncertainty in the source of mortality in the dead embryos

Rohr and Palmer
, 2005 / E81664 A. barbouri /

Field / Metamorphs for 5 hrs T /

80 DMSO (NS) / (N/Y); NS NS NA ; 5 hr
exposure Behav

Surv 4(N)

-- 40

-- ↓ water conserving behavior; ↑ activity Reported no mortality; results not presented in NOAEC/LOAEC terms This study used metamorphs from previous study (Rohr et al 2004, E81748) in which organisms were exposed to different atrazine concentrations and test subjects in this study were not exposed to any additional atrazine during the behavioral study (water conserving behaviors). See Rohr et al 2004 (E81748) for comments

Qualitative

- Lo wer level of confidence based on identified uncertainties in the other study from which the organisms in this study were obtained

Rohr 2008b / E112912 R. clamitans /

Field / TP (G24

- 26) for 14 d T/

>98 ACTN (NS) / (Y/Y); NS glass 4 TP /0 .3L ; NS Imm/inf

Surv <201

(M) 201 201

>201 ↑ % of encyted cercariae Mortality Tadpoles exposed to atrazine before and after cercariae exposure but cercariae not

-uncertainty in environmental relevance; for cercari ae

- tadpole results, the treatments are compared to the solvent control suggesting a significant difference between the controls; limited water quality reported; control mortality not clearly indicated

Qualtiative

- Lo wer level of confidence as control

22 

### Solvent concentration was calculated by reviewer

## 15 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

mortality not clearly reported limited water quality reported with a non

- specified renewal period (tadpoles exposed for 7 days at a time) and higher than recommended loading and environmental relevancy that adds to the uncertainty

Rohr et al . 2009 / E117315 B. americanus /

Field / LV (G25

- 27) for 4 d T/

99 ACTN (0.0002) / (N/Y); NS NS 6 TP/0.5L (96hr) then 1/3.5L; static Behav Surv 196 (M)

-- >196

-- Fear queues (predator and parasite); activity Reported no effect on mortality with nearly 100% survival No negative control (authors report no effect from solvent based on previous data); limited water quality reported

Qualitative

- Higher level of confidence, however no negative control and limited water quality

Rohr et al ., 2006 / E82038 A. barbouri /

Field / A T /

80 DMSO (0.03) & ACTN (0.03) 23 / (N/Y); N NS 31 or 40 E/9.5L ; NA Surv <4 4 ↓ Long term survival (carryover effects) This study used metamorphs from previous study (Rohr et al 2004, E81748) in which organisms were exposed to different atrazine conc entrations and test subjects in this study were not exposed to any additional atrazine to observe survival due to carryover effects. See Rohr et al 2004 (E81748) for comments

Qualitative

- Lo wer level of confidence based on identified uncertainties in the other study from which the organisms in this study were obtained

Rohr et al . 2011 / E153803 A.barbouri /

Field / E

- H (49d) or LV (NS)

- M or 19 d T/

99 ACTN (8.25) / (N/Y); NS glass 12 E/2L ; weekly Gro(E)

Metam (L) Gro (L) Surv 40 (N)

NS

NS <4 400

NS

NS 4 Mass Authors indicate effect on metam and growth but conc. not reported

Embryos

– ↑ Mortality; larval experiment

- no significant for atrazine treatment group Larval survival lower across all treatments and control for study at 19°C; larval mass from embryo study conducted using preserved organisms; limited water quality reported

Invalid based on higher than recommended solvent concentration using a non

- recommended solvent in addition to not using a negative control group; also limited water quality reporting with higher than recommended loading and weekly renewal period add to the level of uncertainty

Spolyarich et al

. 2010 / E153638 L. tasmaniensis

/ Field / TP (G28) to G42 (4wks) T/

98 NA / (Y/NA); N glass 10

TP/10L ; 2X/wk Metam

Gro Sexdev Surv 30 (N)

30 30

-- >30

>30 >30

-- Rate of development Length Sex ratio; gonad development Reported 2.3% mortality in test overall; mortality not reported in terms of NOAEC/LOAEC Sex determination based on gross morphology; histology on male gonads only; Metamorphosis and length data analysis appear to be based using the individual frog as the experimental unit and not the tank as data in figures appears to be on pooled results of the three trials

23 

### Solvent concentration was calculated by reviewer

## 16 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

Qualitative

- Higher level of confidence as loading seemed within recommended levels, however, ammonia and nitrate concentrations not reported with a 2X/wk renewal period and field collected organisms (Sydney Australia suburb) so prior exposure history is unknown

Storrs and Kiesecker 2004 / E78290 Pseudacris

crucifer
, R. sylvatica, B. americanus
, R. clamitans / Field / TP ( early stage G25

- 27) & ( late stage G29

- 36) for ca. 30 d F /

85 NA / (Y/NA); NS PE 1 TP/ 0.1

or 0.5L ; every 3 days Surv NDR 2.84 Survival

- not dose responsive, for all but wood frog, report sign. effects at 2.84, but not always at 25 or 64 Survival not dose

- responsive; a nimals that metamorphosed during the experiment were eliminated from the data set ; limited water quality
repor ted

Qualitative

- Lo wer level of confidence due to survival response not dose

- responsive and limited water quality reported with loading and renewal rate

.

Storrs & Semlitsch 2008 / E103378 H. versicolor;

R. sphenocphala; B. americanus / Field / TP (G25)

- Metamorphs T/

99.2 EtOH (0.0125) / (Y/Y); N Glass &

plastic 1 TP/

0.8L ; 72 hr Metam & Sexdev (H. vers.) Metam & Sexdev (R. sphen) Metam & Sexdev (B. amer) Surv (all) 25.1 (M)

30.4

125

NS >25.1

>30.4

>125

NS Time to metamorphosis; Ovarian stage

Time to metamorphosis; Ovarian stage

Time to metamorphosis; Ovarian stage

Mortality not reported Limited water quality reported; very slight estrogenic activity measured in dilution water; Uncertainty in number of animals exposed

Qualitative

- Lo wer level of confidence based on limited water quality reported with slightly higher than recommended loading and renewal period of 72hr as well as slightly higher than recommended solvent concentration

Storrs Mendez et al . 2009/ E118898 B. americanus /

Field / Metamorphs for 2 d T/

99.2 ACTN (NS)/ (NS/NS); NS NS NA ; NA Behav

Surv 1430

μg/kg (N) NS > 1430

NS Soil choice preference (ATZ or no ATZ) Limited reporting on control group preparation

uncertainty regarding whether control contained solvent; uncertainty in measured conc. due to less than nominal and long storage time

Qualitative

- Medium l evel of confidence based on limited reporting for control preparation

Storrs

- Mendez & Semlitsch 2009/ E153841 H. versicolor;

R. sphenocphala; B. americanus / Field / TP (G25)

- Metamorphs and TP (G25)

- J (4 or 6 mnths) T/

99.2 EtOH (NS) / (N/Y); N NS 1 TP/0.8L

or 20TP/20L; 72 hr Sexdev (L) B. amer R. sphen H. versi. Sexdev (J) B. amer. R. sphen H. versi Surv All 7.55(M)

0.8 <0.92

<3 29.5 24.6

NS 124

3 0.92

3 >29.5 >24.6

NS Sex ratio for all endpoints Mortality not specified Results on larvae appear to be same as reported in Storrs & Semlitsch 2008 / E103378; Results not dose responsive between treatments and time; Uncertainties about test design for juveniles

Qualitative

- Lower level of confidence; see comments for Storrs & Semlitsch 2008 / E103378

## 17 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

Sullivan & Spence 2003 X.laevis /

Xenopus I(Adults) / TP(NF46

- 48)

- M T

/99 ACTN (0.0032) / (Y/N) ; NS glass 6 TP/0.5L starting varied

over time ;every 3

- 4 days Metam

Gro

Surv 40

<20

320 320

20

>320 ↑ Time to metamorphosis w/nitrate ↓Mass at metamorphosis; no effect on metamorphosis for this study Reported mean mortality for entire atrazine only study (p value not reported); when atrazine w/nitrite, mortality sign. greater for nitrate wi th 320 μg/L atrazine than 0 μg/L nitrate Changing exposure conditions and alterations in the number of replicates;

Qualitative

- Lo wer level of confidence based on changing exposure conditions and alterations in number of replicates

Tavera

- Mendoza et al 2002 / E60639 X.laevis/Xenopu

s I/ TP(NG56) for 48 h T/

99 NA / (Y/NA) ; N glass 16 TP/

15L ; static (48hr exposure) Sexdev

Surv <18

-- 18

-- ↓ testes size, spermatogonial nests and nurse cells Reported no mortalities in control or treatment; mortality not reported in terms of NOAEC/LOAEC Need documentation of the measurement endpoints, elaboration on the ecological relevancy of the measurement endpoints, and clarification of the number of exposed animals; appeared to use individual animals not replicates as experimental unit

Invalid based on uncertainty if results based on pseudoreplication

Tavera

- Mendoza et al 2002 / E77830 X. laevis

/Xenopus I / TP(NF56) for 48 h T /

99 NA / (Y/NA) ; N glass 16

TP/15L ; static (48 hr exposure) Sexdev

Surv <18

NS 18

NS ↓ primary oogonia ↑ secondary oogonia ↑ atresia Survival not specified but based on sample size it appears to be 100 % Only one dose; uncertainty of the nature of the measurement endpoints, and the relevancy of the endpoint to higher order measures o f organismal fitness ; used individual animals not replicates as experimental unit

Invalid based on uncertainty if results based on pseudoreplication

Villeneuve et al. 2003 / 45867708 X. laevis / / A

for 26, 43 or 47 d T /

NS NA / (Y/NA); NS fiberglass 15 A

/120L ; 50% every 3 days Bio/Mol

Surv 25 (N)

-- >25

-- Gonad and brain aromatase Mortality ranged from 3

- 19% and was not presented in terms of NOAEC/LOAEC La ck of replication in some studies; observed variability in endpoints; potential water quality issues

Invalid for studies (male) due to no replication; Qualitative

- Lo wer level of confidence in remaining study (female) due to higher variability in endpoint in control

Wan et al . 2006 / E89626 R. catesbeiana

/ Ward’s Natural Science / TP (10cm) for 96 hr T /

98 & F / 48.5 ACNT (NS); (Y/Y); NS (dilution
water analysed) glass 0.8 g/L; static Surv

-- -- Technical 96 hr LC50 > 16000

Formulation 96 hr LC50 > 480000 (232800 ai) Solvent concentration not reported; raw data not provided, control mortality not reported

Qualitative

– Medium level of confidence based on lack of summary data and no reported solvent concentration Williams & Semlitsch 2010 / E153825 B. americanus

& P. triseriata & H. versicolor F /

48.8 NA / (Y/N); N Glass

&plastic NS (1

TP/1L container); Metam

Gro (all 1.25(M)

1.25 >1.25

>1.25 Time to metamorphosis Mass Limited water quality reported ; fill volume of test vessel not reported (1 TP /1L container) ; mass summary data not presented

## 18 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

/ Field / TP (G25)

- M 72 hr species) Surv 1.25 1.25 % Surviving Qualitative

- Lo wer level of confidence based on limited water quality reported with a renewal period of 72 hrs and probably slightly higher than recommended loading ; I

nvalid for mass as summary data not presented

Zaya et al . 2011a / E153847 X. laevis / NS

(Nasco?) / TP (NF47)

-NF 58or 62 T/

NS NA / (Y/NA); NS glass 4 TP/1L ;

3X/wk Metam

Gro Behav Sexdev Imm/inf Surv 200 (N)

25 400 400 200 400 400

200 >400 >400 400 >400 Time to metamorphosis Mass; fat bodies Feeding Sex ratio (gross morphology) ↑ Activated caspase 3 immuno(+) cells Survival Potential effects of stress during weighing and staging during study; uncertainty about protocol for feeding endpoint; variability in fat body and liver size endpoints; limited water quality reported

Qualitative

- Lo wer level of confidence based on limited water quality reported with a higher than recommended loading and a renewal period of 72 hours

Zaya et al 2011b / E153848 X. laevis / NS

(Nasco?) / TP (NF47)

-NF 58or 62 T/

NS NA / (Y/NA); NS glass 4 TP/1L ;

3X/wk Bio/Mol

Surv <400 (N)

400 400

>400 Changes in gene expression (actyl

CoA ehydrogenase and glucocorticoid receptor) Used organisms from Zaya et al 2011a (E153847) etiology of the effects on gene expression reported in this article are uncertain; potential bias in sampling; limited water quality reported

Qualitative

- Lo wer level of confidence based on potential biasing and limited water quality reported with a higher than recommended loading and a renewal period of 72 hours
Cosms
Boone & Bridges

Britton 2006 / E88290 Hyla. versicolor

/ Field / T (G25)

- M (35 d) F /

40.8 NA / (Y/NA); NS PE 30 TP

/1000L Metam

Gro Surv 20 (N)

20 20 >20

>20 >20 Days to metamorphosis Mass Survival Low power to detect differences;

Qualitative

Boone & James 2003 / E81455 R.

Sphenocephala B. americanus A. maculatum & A. texanum / Field / LV for 39

-41 or 80 d F/

40.8 NA / (Y/NA) ; NS PE 20 or 60

TP/1000L Metam

Gro Surv 197 &

<197

< 197 197 >197 &

197 197 >197 ↑ time to metamorphosis for A. texanum; no effect other species ↓Mass (all species) Survival Limited data to interpret atrazine only effects for some endpoints/scenarios; lack of analytical measurements in dilution water

Qualitative

DeNoyelles 1989 / 47543608 R. catesbiana /

NS/ Stage NS for 805 d F /

41 & T/ 97 NA / (Y/NA); NS NS NS Gro Surv <20 (N) 20 ↓ overall biomass Bullfrogs immigrated to ponds

-uneven distribution likely; potential chemicals in ponds prior to testing unknown

Qualitative
Detenbeck et al

. R. pipiens / F/ 85 NA / Wooden 20 TP Metam 25 (N) >25 % adult appearance; hind limb length Control contamination; short acclimation periods;

## 19 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

1996 / E16839 Field / TP (NS)

for 41 d (Y/NA); Y cage w/plastic /cage/

f lowering stream Surv NS NS Not specified higher variability ( lower power)

Qualitative based on review for plants

Diana et al 2000 / E59818 H.

versicolor/ Field / TP to metamorphs T/

NS ACTN (0.04% )/ (N/Y) ; Y plastic 40 LV /90L Metam Gro Surv 2000

20 2000 >2000

200 >2000 Time to metamorphosis ↓Mass; SVL % Survival Control contamination; pre

- metamorphosis weight and length were not determined

Qualitative based on review for plants

Du Preez et al 2008 (lab and cosm) / E103271 X laevis / Field

/ TPto A (2 yrs post metam) T /

NS MeOH (0.025) / (N/Y) ; N NS 75

frogs/2,50 0L Metam Sexdev Surv 24.8

24.8 24.8 >24.8

>24.8 >24.8 Time to metamorphosis Gonadal development; sex ratio % Survival Continuation of Jooste 2005 (46965603); Temp concerns; uncertainty about adding tadpoles at different times effect on metamorphosis endpoint; higher occurance in testicular oocytes in controls

Invalid based on control oocytes

Jooste et al 2005 / MRID 46965603 X.laevis / Field

(Adults) / TP

- M T/ MeOH (0.025%) / (N/Y) ; NS 24 PE 800 TP /

1100L Metam

Sexdev Surv 30.8 (M)

30.8 NS >30.8

>30.8 NS Time to metamorphosis Testicular oocytes Preliminary to DuPreez 2008 (E103271); Unusually long development times; feeding and temperatures may have limited development. Higher incidence of testicular oocytes in controls

Invalid based on control oocytes

Langlois 2010 / E120046 R.pipiens /

Field / TP(G27)

- M(G42) F /

(NS) NA / (Y/NA) ; Y HDPE 0.1 g/L Metam Sexdev Bio/Mol Surv <0.1

0.1 0.1 0.1 0.1

1.8 1.8 1.8 ↓ Metamorphic success ↑ Sex ratio (female) ↑ and ↓Brain eralph and tail doi3 Total survival Control contamination and atrazine detected in area where organisms collected ; uncertainty in biochemical responses on gross endpoints ; metamorphic success and sex ratio based on total exposed, no replicate variability reported

Qualitative

Relyea 2009 / E114296 H.versicolor &

R. pipiens / NS (collected as eggs) / TP

-

M T/ 99 EtOH (0.003) / (Y/Y) ; NS 25 cattle

watering tanks 9 TP/m2 H. vers

Metam Gro Surv R. pipiens Metam Gro Surv 6.4

< 6.4 6.4

6.4 6.4 6.4 >6.4

6.4 >6.4

>6.4 >6.4 >6.4 Time to metamorphosis ↑Mass Survival

Time to metamorphosis Mass Survival Atrazine concentrations were not monitored throughout the experiment, although an initial measured value was reported. Control tanks were not analyzed for atrazine and the other nine pesticides tested. Additionally, it is unclear how much ethanol was added to atrazine

- only treatment tanks.

Qualitative Rohr and Crumine 2005 / E117319 R.sylvatica/

Field / TP

-

M F / ACTN

(0.00246) PE 20 TP /10L Metam Gro 50

50 >50

>50 Stage at test termination Mass Atrazine concentrations not monitored; unclear acclimation periods; prior exposure for tadpoles

24 

### Atrazine measured in treatment tanks; unclear if analyzed in control
25 

### Controls not analyzed; however, dilution water prior to addition to tanks was analyzed and found to be free of atrazine

## 20 **Citation / ECOTOX(E)/**  **MRID Species /** 

**Source / Exposure**  **Period (stages & test**  **duration) 1**  **TS 2**  **/**

**% ai Solvent**  **(%) 3**  **/ NC/SC**  **(Y/N); MA in**  **CNT (Y/N/NS)**  **4 Test** 

**vessel**

**ma terial** **5 Loading;** 

**Re newal**  **Period 6**  **Reported Results 7**  **Indentified Uncertainties / Concerns and Classification**  **Endpoint NOEC** 

**(μg/L) LOEC** 

**(μg/L) Effect** 

/ (Y/Y) ; NS 26 Behav Surv <50

-- 50

-- ↓% of hiding Reported no dead tadpoles found during study; mortality not presented in terms of NOAEC/LOAEC Qualitative

Rohr et al . 2008a / E112910 R.pipiens &R.

clamitans / Field/ TP for 4 wks T/

98 NS (Y/Y) ; NS 27 plastic 65

amphibs/8 00L Imm/I

nf

Surv <117

117 &<117 117

117 &>117 ↓ eosinophils ↑ trematode cycts (R. clam); ↓Melanomacrophage ↓ % survival for R. pipiens and no effect for R. clamitans Unknown solvent and concentration. Atrazine concentrations were not monitored during test. Unknown if in algal communities similar at test initiation .

Qualitative

26 

### Atrazine measured in treatment tanks; unclear if analyzed in control
27 

### Atrazine measured in treatment tanks; unclear if analyzed in control