Document ID: EPA-HQ-OPP-2012-0230-0203
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2012-06-21T04:00Z

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 analyzed 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.  

Table 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 reported
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 2009
Imm/Inf
No effects on infection rate @ 200 ppb
Summary data not reported
Qualitative lower to Invalid
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

                          Citation / ECOTOX(E)/ MRID
        Species / Source / Exposure Period (stages & test duration)
                                   TS / % ai
             Solvent  (%) / NC/SC (Y/N);       MA in CNT (Y/N/NS)
                             Test vessel material
                            Loading; Renewal Period
Reported Results
            Indentified Uncertainties / Concerns and Classification

                                       
                                       
                                       
                                       
Endpoint
NOEC
(ug/L)
LOEC (ug/L)
Effect

Allran & Karasov 2000 / E56572

Rana pipiens / Nasco(unknown 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-Lower 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; Invalid 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 96 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-Lower level of confidence  as low levels of atrazine measured at organism collection site; Invalid for growth, swimming and hemoglobin due to lack of summary data
Birge et al. 1980 / E6187
R. catesbeiana, R. pipiens, R. palustris, B. americanus / NS (purchased) / E for 4 dph
                                    F / 80
                        NS / (Y unsure if NC or SC); NS
                                     glass
                            50 -103 E / 0.5L; 2.5 h
Surv 4 dph LC50
--
--
R. catesbeiana = 410
R. pipiens = 7680
R. palustris = 17960
B. americanus >48000
LC50s were not based on mortality per se, but on abnormalities that would reportedly preclude survival under natural conditions; solvent (if used) was not identified; formulation used so uncertainty in source of toxicity

Qualitative  -  Higher level of confidence; however LC50 values are combination of both lethality and abnormal effects and formulation used
Brodeur  et al. 2009 / E112588 
R. arernarum / LP/ TP (G38/39)-M
                                     T/ 98
                                NA / (Y/NA); N
                                     glass
                              10 TP /0.04L; 48 hr
Metam
Surv (LC50) 

NDR (N)
NA

100
NA

↓ & ↑Time metamorphic stage and/or duration between stages
LC50 (stage 4) = 7150-25370
LC50 (stage 25) = 2320-27160
LC50 (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 ug/L depending on metam endpoint (100 lowest conc tested); hypothesis testing for effects on metamorphosis conducted using EC50 values 

Qualitative-Lower level of confidence based on higher loading with no water quality parameters reported
Brodkin et al 2007 / E103351
R. pipiens / Field (ANA) / A for 8 d
                                     T/ NS
                            NA / (N & N/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

Invalid 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 survival concentration and dilutions from that conc. 
Limited reporting on water quality parameters and test design (# of replicates); conducted above water solubility

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); no 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/Inf

18.4 (M)
18.4
184
184

184
184
>184
>184

↑ larval period
↓ mass & SVL
% mortality
Infection Rate

Limited reported water quality;

Qualitative-Lower 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 ug/L) ; potential stress from repetitive mass measurements; summary data for metamorphosis and growth not presented

Qualitative-Lower level of confidence 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 /  Xenopus I (adults) / E  - NF 46 
                                     T/ NS
                            DMSO (1%) / (Y/NS); NS
                                      NS
                               20 E/0.01L; 24 hr
Metam
Surv
(FETAX)
--
--
X. laevis- 
LC50 = 24500 or 25600
EC50 = 4100 or 4400
X. tropicalis
LC50 = 19400  -  31800
EC50 = 2100 - 9300 
Limited water quality reported; use of not recommended solvent and high conc (although allowed under ASTM FETAX assay guideline)

Invalid based on solvent choice and concentration
Freeman & Rayburn 2004 / E78644
B. americanus & X. laevis / Field (parents)(Bufo) & Nasco  / 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-Lower 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
                                      NS
                           NS (1 TP/3L tank); weekly
Metam
Gro
Bio/Mol

Surv

<100 (N)
800
Varied

--

100
>800
800

--

↓ metamorphic stage
Mass
↑ flow cytometric 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 of NOEC/LOEC not reported

Stats for flow cytometric 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 loading; metamorphosis and mass not dose responsive (metam-not sign at 800 ug/L in other test different age at initiation; mass- not sign at 800 in other test or 250-10000 in another test)

Qualitative- Lower level of confidence for Metamorphosis and Bio/Mol- due to loading not defined and no water quality parameters with weekly renewal; Invalid for weight due to potential confounding factor
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), NS (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 analyses for atrazine formulation based on lack of test design 
Goleman & Carr 2003 / 45867707
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 / Field / TP (NS) for 2 d
                                     T/ NS
                            IPA (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 when 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 uncertain 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
                                    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

Qualitative  -  Lower level 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
                                      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  -  Lower 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  - Lower 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 al 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  -  Lower level of confidence based on higher loading with limited water quality reporting and a renewal period of every 3 days and uncertainty in reported results add to level of uncertainty; Invalid for survival, growth and metamorphosis
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-Lower 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
--

GSI
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  -  Lower level of confidence based on higher loading with limited water quality reporting 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 LC50
--
--
LC50 values
R. pipiens (early stage) = 47600
R. pipiens (late stage) = 14500
B. americanus (early) = 26500
B. americanus (late) = 10700
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 not 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 based 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/week 
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 affect of dead larvae on measured viral load that was used in analyses; limited water quality reported

Qualitative-Lower level of confidence given higher than recommended loading rate with limited water quality reported and a weekly renewal period; Invalid 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 cercariae 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 2009a / E112914
X. laevis / Xenopus I / TP (8 dpf) for 83 d 
                                    T / NS
                                NA / (Y/NA); Y
                                     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
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  -  Low 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/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 cercariae were exposed to atrazine  - uncertainty in environmental relevance; 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 / TP (<G27)- ~ G43
                                    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. versicolor / 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; SUL
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-Lower 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 >48 hours; Qualitative Low 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- Lower level of  confidence based on higher loading with limited water quality reporting and an occasional renewal period of every 3 days  coupled with variable mortality in tanks for two treatment groups (not dose responsive); Invalid 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-Lower 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
                                    plastic
                         NS (20 E/petri dish) ; 24 hr
Metam
Surv 
--
11000 or 1100
LC50 = 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 reported; uncertain if potentially different stages at test termination (stages not reported) influenced growth analysis; mass summary data not presented

Qualtiative- Lower 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; Invalid 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) / (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 followed mortality; larval survival differences between years; although pH, temp, and DO ok, nitrates and ammonia not reported; 

Qualitative-Lower 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-Lower 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 cercariae-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- Lower level of confidence as control 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) / (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 concentrations 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-Lower 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

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;  animals that metamorphosed during the experiment were eliminated from the data set; limited water quality reported

Qualitative-Lower 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-Lower 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 ug/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 level 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
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 with  320 ug/L atrazine than 0 ug/L nitrate
Changing exposure conditions and alterations in the number of replicates; 

Qualitative-Lower level of confidence based on changing exposure conditions and alterations in number of replicates
Tavera-Mendoza et al 2002 / E60639
X.laevis/Xenopus 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 of 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
Lack of replication in some studies; observed variability in endpoints; potential water quality issues

Invalid for studies (male) due to no replication; Qualitative-Lower 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 / Field / TP (G25)-M
                                   F / 48.8
                                 NA / (Y/N); N
                              Glass &plastic
                         NS (1 TP/1L container); 72 hr
Metam
Gro
(all species)
Surv
1.25(M)
1.25

1.25
>1.25
>1.25

1.25
Time to metamorphosis
Mass

% Surviving
Limited water quality reported; fill volume of test vessel not reported (1 TP /1L container); mass summary data not presented 

Qualitative- Lower level of confidence based on limited water quality reported with a renewal period of 72 hrs and probably slightly higher than 
recommended loading; Invalid 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-Lower 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-Lower 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. 1996 / E16839
R. pipiens / Field / TP (NS) for 41 d
                                     F/ 85
                                NA / (Y/NA); Y
                             Wooden cage w/plastic
                         20 TP /cage/ flowering stream
Metam
Surv
25 (N)
NS
>25
NS
% adult appearance; hind limb length 
Not specified
Control contamination; short acclimation periods; 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/2,500L
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 
                                      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
                             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) / (Y/Y); NS
                                      PE
                                  20 TP /10L
Metam
Gro
Behav
Surv
50
50
<50
--

>50
>50
50
--

Stage at test termination
Mass
↓% of hiding
Reported no dead tadpoles found during study; mortality not presented in terms of NOAEC/LOAEC
Atrazine concentrations not monitored; unclear acclimation periods; prior exposure for tadpoles

Qualitative
Rohr et al. 2008a / E112910
R.pipiens & R. clamitans / Field/ TP for 4 wks
                                     T/ 98
                                 NS (Y/Y); NS
                                    plastic
                                65 amphibs/800L
Imm/Inf

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